Methods of Treatment and Administration of Natural Killer Cell Compositions

Abstract

Methods and uses for treatment involving administration of compositions containing NK cells are provided herein, including for treating cancers such as multiple myeloma or lymphoma, including in combination with antibody therapeutics against cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 177,956, filed April 21, 2021, entitled "METHODS OF TREATMENT AND DOSING OF NATURAL KILLER CELL COMPOSITIONS," the contents of which are incorporated by reference in their entirety for all purposes.

[0002] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 776032001240_SEQLIST.Txt, created on April 20, 2022, and 7,852 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides therapeutic methods and uses involving the administration of compositions containing NK cells, including for treating cancers such as multiple myeloma or lymphoma, including in combination with antibody therapeutics against cancer. [Background technology]

[0004] background Antibody-based therapies are becoming increasingly used to treat cancer and other diseases. While responses to antibody therapy have typically focused on the direct inhibitory effects of these antibodies on tumor cells (e.g., inhibition of growth factor receptors and subsequent induction of apoptosis), the in vivo effects of these antibodies can be more complex and involve the host immune system. Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity when Fc receptors (CD16, FcγRIII) bind to the Fc portion of an antibody bound to an antigen-bearing cell. NK cells, including certain specialized subsets thereof, can be used in therapeutic methods, including improving responses to antibody therapy. There is a need for improved methods for therapeutic use involving NK cells, including in combination with antibodies. Provided herein are embodiments that meet this need. Summary of the Invention

[0005] overview Provided herein is a method for treating multiple myeloma, comprising administering to a subject with multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain, wherein the composition of g-NK cells can be administered once a week in a predetermined number of doses. Provided herein is a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain, for use in a method for treating a subject with multiple myeloma (MM), wherein the composition can be administered once a week in a predetermined number of doses.

[0006] In some embodiments, the method can be a monotherapy without concomitant administration of an exogenous antibody for treating multiple myeloma. In some embodiments, the method further comprises administering to the subject an antibody directed against a multiple myeloma antigen. In some embodiments, the multiple myeloma antigen comprises an antigen selected from the group consisting of CD38, SLAMF7, and BCMA. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an anti-SLAMF7 antibody. In some embodiments, the antibody is an anti-BCMA antibody. In some embodiments, the antibody is an anti-CD38 antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody is directed against CD16 and BCMA. In some embodiments, the bispecific antibody is directed against CD16 and SLAMF7. In some embodiments, the bispecific antibody is directed against CD16 and CD38. In some embodiments, at least one dose of an anti-CD38 antibody has been administered to the subject prior to administration of a dose of a g-NK cell composition.

[0007] In some embodiments, the antibody is administered once every four weeks. In some embodiments, the antibody is administered once every three weeks. In some embodiments, the antibody is administered once every two weeks. In some embodiments, the antibody is administered once per week. In some embodiments, the antibody is administered twice per week. In some embodiments, the antibody is administered more than twice per week.

[0008] Also provided herein is a method for treating multiple myeloma (MM), comprising administering to a subject with multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain, wherein the g-NK cell composition may be administered once weekly in a predetermined number of doses, and the subject has been previously administered at least one dose of an anti-CD38 antibody.Also provided herein is a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in a method for treating a subject with multiple myeloma (MM), wherein the g-NK cell composition may be administered once weekly in a predetermined number of doses, and the subject has been previously administered at least one dose of an anti-CD38 antibody.

[0009] In some embodiments, the g-NK cell composition may be administered as two doses in a 14-day cycle, and the 14-day cycle may be repeated 1 to 3 times. In some embodiments, the g-NK cell composition may be administered as a total of six doses. In some embodiments, the anti-CD38 antibody is daratumumab. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0010] In some embodiments, the anti-CD38 antibody may be administered intravenously. In some embodiments, the anti-CD38 antibody may be administered as a weekly dose, optionally over one or two 28-day cycles. In some embodiments, each dose of the anti-CD38 antibody (e.g., daratumumab) may be administered in an amount that may be at or about 8 mg / kg to about 32 mg / kg, optionally at or about 16 mg / kg. In some embodiments, the anti-CD38 antibody may be administered subcutaneously. In some embodiments, the anti-CD38 antibody (e.g., daratumumab) may be administered in an anti-CD38 antibody composition comprising hyaluronidase, optionally the anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (e.g., hyaluronidase-fihj).

[0011] In some embodiments, the anti-CD38 antibody composition may be administered as a weekly dose, optionally over one or two 28-day cycles. In some embodiments, each dose of the anti-CD38 antibody composition comprises 1200 mg, or about 1200 mg to about 2400 mg, of an anti-CD38 antibody (e.g., daratumumab) and 15,000 units (U), or about 15,000 U to about 45,000 U of hyaluronidase (e.g., hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of an anti-CD38 antibody (e.g., daratumumab) and about 30,000 U of hyaluronidase (e.g., hyaluronidase-fihj). In some embodiments, the method comprises administering an anti-CD38 antibody, optionally an anti-CD38 antibody composition, once weekly for a total of eight doses, and administering a g-NK cell composition once weekly for a total of six doses, wherein one or two doses of the anti-CD38 antibody may be administered prior to administration of the composition comprising g-NK cells. In some embodiments, the multiple myeloma may be relapsed / refractory multiple myeloma.

[0012] In some embodiments, the g-NK cells have low or no expression of CD38, optionally, less than 25% of the cells in the g-NK cell composition are positive for surface CD38. In some embodiments, the cells in the g-NK cell composition have not been engineered to reduce or eliminate CD38 expression. In some embodiments, the g-NK cell composition exhibits minimal anti-CD38-induced fratricide, optionally, less than 10% of the cells in the g-NK cell composition exhibit anti-CD38-induced fratricide.

[0013] Provided herein are methods for treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain, wherein the g-NK cell composition is administered once weekly in a predetermined number of doses. Provided herein are compositions of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in methods for treating a subject having lymphoma, wherein the g-NK cell composition can be administered once weekly in a predetermined number of doses. In some embodiments, the method can be a monotherapy without concomitant administration of an exogenous antibody for treating lymphoma. In some embodiments, the method further comprises administering to the subject an antibody against a lymphoma antigen. In some embodiments, the lymphoma antigen comprises an antigen selected from the group consisting of CD19, CD20, and CD30. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an anti-CD19 antibody. In some embodiments, the antibody is an anti-CD30 antibody. In some embodiments, the antibody is an anti-CD20 antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody is directed against CD16 and a second antigen selected from the group consisting of CD19, CD20, and CD30. In some embodiments, the bispecific antibody is directed against CD16 and CD19. In some embodiments, the bispecific antibody is directed against CD16 and CD20. In some embodiments, the bispecific antibody is directed against CD16 and CD30. In some embodiments, at least one dose of an anti-CD20 antibody has been administered to the subject prior to administration of a dose of a g-NK cell composition.

[0014] In some embodiments, the antibody is administered once every four weeks. In some embodiments, the antibody is administered once every three weeks. In some embodiments, the antibody is administered once every two weeks. In some embodiments, the antibody is administered once per week. In some embodiments, the antibody is administered twice per week. In some embodiments, the antibody is administered more than twice per week.

[0015] Also provided herein is a method for treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the g-NK cell composition can be administered once a week in a predetermined number of doses, and the subject has been previously administered at least one dose of an anti-CD20 antibody.Also provided herein is a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, for use in a method for treating a subject having lymphoma, wherein the g-NK cell composition can be administered once a week in a predetermined number of doses, and the subject has been previously administered at least one dose of an anti-CD20 antibody.

[0016] In some embodiments, the lymphoma may be non-Hodgkin's lymphoma (NHL). In some embodiments, the g-NK cell composition may be administered as two doses in a 14-day cycle, and the 14-day cycle may be repeated 1 to 3 times. In some embodiments, the g-NK cell composition may be administered as a total of 6 doses. In some embodiments, the anti-CD20 antibody may be rituximab.

[0017] In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within two weeks prior to administration of the g-NK cell composition. In some embodiments, the anti-CD20 antibody may be administered intravenously. In some embodiments, the anti-CD20 antibody may be administered as a weekly dose, optionally in four or eight doses. In some embodiments, each dose of the anti-CD20 antibody may be administered in an amount that may be at or about 250 mg / m to 500 mg / m, optionally at or about 375 mg / m. In some embodiments, the anti-CD20 antibody may be administered subcutaneously.

[0018] In some embodiments, an anti-CD20 antibody (e.g., rituximab) may be administered in an anti-CD20 antibody composition comprising hyaluronidase, optionally wherein the anti-CD20 antibody composition comprises rituximab and human recombinant hyaluronidase PH20. In some embodiments, the anti-CD20 antibody composition may be administered as a weekly dose, optionally in four or eight doses, or optionally in three or seven doses following a weekly intravenous dose of anti-CD20 antibody. In some embodiments, each dose of the anti-CD20 antibody composition comprises, at or about 1200 mg to about 2400 mg of an anti-CD20 antibody (e.g., rituximab) and 15,000 units (U), or about 15,000 U to about 45,000 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1400 mg of an anti-CD20 antibody (e.g., rituximab) and about 23,400 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1600 mg of an anti-CD20 antibody (e.g., rituximab) and about 26,800 U of hyaluronidase.

[0019] In some embodiments, the method comprises administering an anti-CD20 antibody, optionally an anti-CD20 antibody composition, once weekly for a total of eight doses, and administering a g-NK cell composition once weekly for a total of six doses, wherein one or two doses of the anti-CD20 antibody can be administered prior to administration of the composition comprising g-NK cells. In some embodiments, of the cells in the g-NK cell composition, greater than or greater than about 60% of the cells are g-NK cells, greater than or greater than about 70% of the cells are g-NK cells, greater than or greater than about 80% of the cells are g-NK cells, greater than or greater than about 90% of the cells are g-NK cells, or greater than or greater than about 95% of the cells are g-NK cells.

[0020] In some embodiments, at least 50%, or about 50%, of the cells in the g-NK cell composition are FcRγ-deficient (FcRγ) NK cells (g-NK), and greater than 70%, or about 70%, of the g-NK cells are positive for perforin and greater than 70%, or about 70%, of the g-NK cells are positive for granzyme B. In some embodiments, (i) greater than 80%, or about 80%, of the g-NK cells are positive for perforin and greater than 80%, or about 80%, of the g-NK cells are positive for granzyme B, (ii) greater than 90%, or about 90%, of the g-NK cells are positive for perforin and greater than 90%, or about 90%, of the g-NK cells are positive for granzyme B, or (iii) greater than 95%, or about 95%, of the g-NK cells are positive for perforin and greater than 95%, or about 95%, of the g-NK cells are positive for granzyme B.

[0021] In some embodiments, among the cells positive for perforin, the cells express an average level of perforin, based on mean fluorescence intensity (MFI), as measured by intracellular flow cytometry, that is at least two-fold or about two-fold the average level of perforin expressed by cells that are FcRγpos, and / or among the cells positive for granzyme B, the cells express an average level of granzyme B, based on mean fluorescence intensity (MFI), as measured by intracellular flow cytometry, that is at least two-fold or about two-fold the average level of granzyme B expressed by cells that are FcRγpos. In some embodiments, greater than 10% of the cells in the g-NK cell composition are capable of degranulation against tumor target cells, optionally as measured by CD107a expression, and optionally, degranulation can be measured in the absence of an antibody against the tumor target cells.

[0022] In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the cells in the g-NK cell composition exhibit degranulation, optionally as measured by CD107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody).

[0023] In some embodiments, greater than 10% of the cells in a g-NK cell composition can produce interferon-gamma or TNF-alpha against tumor target cells; optionally, interferon-gamma or TNF-alpha can be measured in the absence of antibodies against tumor target cells. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the cells in a g-NK cell composition produce effector cytokines in the presence of cells expressing a target antigen (target cells) and antibodies against the target antigen (anti-target antibodies). In some embodiments, the effector cytokines can be IFN-gamma or TNF-alpha. In some embodiments, the effector cytokines can be IFN-gamma and TNF-alpha. In some embodiments, the g-NK cell composition is produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, and the CD3- / CD57+ cells are enriched from a biological sample from a donor subject.

[0024] In some embodiments, the donor subject may be CMV-seropositive. In some embodiments, the donor subject has a CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype, and optionally, the biological sample may be derived from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype. In some embodiments, at least 20% or about 20% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2C, NKG2Cpos), and at least 70% of the NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2A, NKG2Aneg). In some embodiments, the irradiated feeder cells are HLA class I and HLA class II deficient. In some embodiments, the irradiated feeder cells are 221.AEH cells.

[0025] In some embodiments, the culture may be performed in the presence of two or more recombinant cytokines, where at least one recombinant cytokine may be interleukin (IL)-2 and at least one recombinant cytokine may be IL-21. In some embodiments, the recombinant cytokines are IL-21 and IL-2. In some embodiments, the recombinant cytokines are IL-21, IL-2, and IL-15. In some embodiments, the g-NK cells in the composition are derived from a single donor subject expanded from the same biological sample. In some embodiments, the g-NK cell composition may be formulated in a serum-free cryopreservation medium containing a cryoprotectant; optionally, the cryoprotectant may be DMSO and the cryopreservation medium may be 5%-10% DMSO (v / v).

[0026] In some embodiments, the g-NK cells are not engineered with an antigen receptor, and optionally, the antigen receptor can be a chimeric antigen receptor. In some embodiments, the g-NK cells are not engineered with a secreted cytokine, and optionally, a cytokine receptor fusion protein, such as an IL-15 receptor fusion (IL-15RF). In some embodiments, the method does not include administering an exogenous cytokine to the subject to support the survival or expansion of the NK cells, and the exogenous cytokine can be one or more of IL-2, IL-7, IL-15, or IL-21.

[0027] In some embodiments, each dose of g-NK cells can be from 1 x 10 cells or about 1 x 10 cells to 50 x 10 cells or about 50 x 10 cells of the g-NK cell composition. In some embodiments, each dose of g-NK cells can be 5 x 10 cells or about 5 x 10 cells of the g-NK cell composition. In some embodiments, each dose of g-NK cells can be 5 x 10 cells or about 5 x 10 cells of the g-NK cell composition. In some embodiments, each dose of g-NK cells can be 10 x 10 cells or about 10 x 10 cells of the g-NK cell composition. In some embodiments, prior to administration of the dose of g-NK cells, the subject undergoes lymphocyte depletion therapy. In some embodiments, the lymphocyte depletion therapy comprises fludarabine and / or cyclophosphamide. In some embodiments, lymphocyte depletion comprises administration of fludarabine at or about 20-40 mg / m2 of the subject's body surface area, optionally at or about 30 mg / m2, daily for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m2 of the subject's body surface area, optionally at or about 300 mg / m2, daily for 2-4 days.

[0028] In some embodiments, lymphocyte depletion therapy comprises fludarabine and cyclophosphamide, hi some embodiments, lymphocyte depletion therapy comprises administration of fludarabine at or about 30 mg / m2 of a subject's body surface area daily and cyclophosphamide at or about 300 mg / m2 of a subject's body surface area daily, each for 2 to 4 days, optionally for 3 days.

[0029] In some embodiments, administration of a dose of g-NK cells may begin within, at, or about 2 weeks after initiation of lymphocyte depletion therapy. In some embodiments, administration of a dose of g-NK cells may begin within, at, or about 7 days after initiation of lymphocyte depletion therapy. In some embodiments, administration of a dose of g-NK cells may begin within, at, or about 7 days after initiation of lymphocyte depletion therapy. In some embodiments, the individual may be a human. In some embodiments, the NK cells in the composition are allogeneic to the individual. In some embodiments, the method further includes administering exogenous cytokine support to facilitate expansion or persistence of g-NK cells in vivo in the subject, and optionally, the exogenous cytokine may be or include IL-15. [Brief explanation of the drawings]

[0030] [Figure 1A] Figures 1A and 1B illustrate the expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 included in the NK cell culture medium. Figure 1A shows the total NK cell count. Figure 1B shows the g-NK cell count after 21 days of expansion. [Figure 1B] See legend to Figure 1A. [Figure 2A] Figures 2A and 2B illustrate the daratumumab- and elotuzumab-mediated cytotoxic activity 21 days after expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 included in the NK cell culture medium. Figure 2A shows the cytotoxicity of g-NK cells against the LP1 cell line. Figure 2B shows the cytotoxicity of g-NK cells against the MM.1S cell line. [Figure 2B] See legend to Figure 2A. [Figure 3A]Figures 3A-3D illustrate the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 in the NK cell culture medium. Figure 3A shows the degranulation levels of g-NK cells 13 days after expansion using the LP1 cell line. Figure 3B shows the degranulation levels of g-NK cells 13 days after expansion using the MM.1S cell line. Figure 3C shows the degranulation levels of g-NK cells 21 days after expansion using the LP1 cell line. Figure 3D shows the degranulation levels of g-NK cells 21 days after expansion using the MM.1S cell line. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A] Figures 4A-4D illustrate the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 included in the NK cell culture medium. Figure 4A shows perforin and granzyme B expression as a percentage of g-NK cells 13 days after expansion. Figure 4B shows total perforin and granzyme B expression 13 days after expansion. Figure 4C shows perforin and granzyme B expression as a percentage of g-NK cells 21 days after expansion. Figure 4D shows total perforin and granzyme B expression 21 days after expansion. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A]Figures 5A-5D illustrate daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 included in the NK cell culture medium. Figure 5A shows g-NK cell interferon-γ expression levels 13 days after expansion for the LP1 cell line. Figure 5B shows g-NK cell interferon-γ expression levels 13 days after expansion for the MM.1S cell line. Figure 5C shows g-NK cell interferon-γ expression levels 21 days after expansion for the LP1 cell line. Figure 5D shows g-NK cell interferon-γ expression levels 21 days after expansion for the MM.1S cell line. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6D illustrate daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 included in the NK cell culture medium. Figure 6A shows g-NK cell TNF-α expression levels 13 days after expansion for the LP1 cell line. Figure 6B shows g-NK cell TNF-α expression levels 13 days after expansion for the MM.1S cell line. Figure 6C shows g-NK cell TNF-α expression levels 21 days after expansion for the LP1 cell line. Figure 6D shows g-NK cell TNF-α expression levels 21 days after expansion for the MM.1S cell line. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7] Illustrates g-NK cell expansion of NK cells expanded for 15 days in the presence of various cytokine mixtures and concentrations. [Figure 8A]Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8A and 8B illustrate the daratumumab- and elotuzumab-mediated cytotoxic activity of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8A shows the cytotoxicity of g-NK cells against the LP1 cell line. Figure 8B shows the cytotoxicity of g-NK cells against the MM.1S cell line. [Figure 8B] See legend to Figure 8A. [Figure 8C] Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8C and 8D illustrate the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8C shows the g-NK cell degranulation levels for the LP1 cell line. Figure 8D shows the g-NK cell degranulation levels for the MM.1S cell line. [Figure 8D] See legend to Figure 8C. [Figure 8E] Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8E and 8F illustrate the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8E shows perforin and granzyme B expression as a percentage of g-NK cells. Figure 8F shows total perforin and granzyme B expression. [Figure 8F] See legend to Figure 8E. [Figure 8G] Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8G and 8H illustrate the daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8G shows the g-NK cell interferon-γ expression levels for the LP1 cell line. Figure 8H shows the g-NK cell interferon-γ expression levels for the MM.1S cell line. [Figure 8H] See legend to Figure 8G. [Figure 8I] Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8I and 8J illustrate daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8I shows g-NK cell TNF-α expression levels for the LP1 cell line. Figure 8J shows g-NK cell TNF-α expression levels for the MM.1S cell line. [Figure 8J] See legend to Figure 8H. [Figure 9A] Figures 9A-9L show the expansion and cellular effector function of g-NK cells expanded for 14 days in the presence of IL-21 compared to g-NK cells expanded without IL-21 (n=6). Figures 9A and 9B illustrate the expansion of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9A shows the g-NK cell percentage before and after expansion. Figure 9B shows the number of expanded g-NK cells per 10 million NK cells. Values ​​are mean ± SE. #p<0.001 for CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. ^p<0.05 for CD3neg / CD57pos expansion versus other CMVpos expansions. *p<0.001 for CMVpos expansion versus CMVnegCD3neg expansion. [Figure 9B] See legend to Figure 9A. [Figure 9C]Figures 9A-9L show the expansion and cell effector function of g-NK cells expanded for 14 days in the presence of IL-21 compared to g-NK cells expanded without IL-21 (n=6). Figure 9C illustrates a comparison of the proportion of g-NK (% of total NK cells) from CMV+ donors (n=8) and CMV- donors (n=6) before and after expansion. Figure 9D illustrates a comparison of the n-fold expansion rate of g-NK from CMV+ and CMV- donors. Figure 9E provides a representative flow plot of FcεR1γ versus CD56 for CMV+ donors. Figure 9F provides a representative histogram of FcεR1γ expression on CD3- / CD56+ NK cells for CMV+ and CMV- donors. An independent samples t-test was used to determine differences between CMV+ and CMV- donors before and after expansion (Figures 9C and 9D). Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001. [Figure 9D] See legend to Figure 9C. [Figure 9E] See legend to Figure 9C. [Figure 9F] See legend to Figure 9C. [Figure 9G] Figures 9A-9L show the expansion and cellular effector function of g-NK cells expanded for 14 days in the presence of IL-21 compared to g-NK cells expanded without IL-21 (n=6). Figures 9G and 9H illustrate the daratumumab- and elotuzumab-mediated cytotoxic activity of g-NK cells expanded in the presence of IL-21 14 days after expansion compared to g-NK cells expanded without IL-21. Figure 9G shows the cytotoxicity of g-NK cells against the LP1 cell line. Figure 9H shows the cytotoxicity of g-NK cells against the MM.1S cell line. Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. [Figure 9H] See legend to Figure 9G. [Figure 9I]Figures 9A-9L show the expansion and cell effector function of g-NK cells expanded for 14 days in the presence of IL-21 compared to g-NK cells expanded without IL-21 (n=6). Figures 9G and 9H illustrate the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9I shows the g-NK cell degranulation levels 14 days after expansion for the LP1 cell line. Figure 9J shows the g-NK cell degranulation levels 14 days after expansion for the MM.1S cell line. Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. [Figure 9J] See legend to Figure 9I. [Figure 9K] Figures 9A-9L show the expansion and cellular effector function of g-NK cells expanded for 14 days in the presence of IL-21 compared to g-NK cells expanded without IL-21 (n=6). Figures 9K and 9L illustrate the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9K shows perforin and granzyme B expression as a percentage of NK cells 14 days after expansion. Figure 9L shows total perforin and granzyme B expression 14 days after expansion. Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. [Figure 9L] See legend to Figure 9K. [Figure 9M]Baseline expression of perforin (left) and granzyme B (right) in expanded g-NK cells compared with cNK cells is depicted (n=5). An independent samples t-test was used to compare the expression of effector perforin and granzyme B between g-NK and cNK. Values ​​are means ± SE. Statistically significant differences from cNK cells are indicated by ***p<0.001. [Figure 9N] Representative histograms of perforin and granzyme B expression for g-NK and cNK cells are shown. [Figure 9O] Figures 9O and 9P illustrate daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9O shows g-NK cell interferon-γ expression levels 14 days after expansion for the LP1 cell line. Figure 9P shows g-NK cell interferon-γ expression levels 14 days after expansion for the MM.1S cell line. Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. [Figure 9P] See legend to Figure 9O. [Figure 9Q] Figures 9Q and 9R illustrate daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9Q shows g-NK cell TNF-α expression levels 14 days after expansion for the LP1 cell line. Figure 9R shows g-NK cell TNF-α expression levels 14 days after expansion for the MM.1S cell line. Values ​​are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. [Figure 9R] See legend to Figure 9Q. [Figure 9S]Illustrates daratumumab- and elotuzumab-mediated interferon-γ expression levels of expanded g-NK cells compared to cNK cells against the MM.1S cell line between different donors. [Figure 9T] Illustrates daratumumab- and elotuzumab-mediated TNF-α expression levels of expanded g-NK cells compared to cNK cells against the MM.1S cell line between different donors. [Figure 10] Illustrates the expansion of g-NK cells expanded in the presence of IL-21 / anti-IL-21 complexes (n=4). Values ​​are mean ± SE. #p<0.001 for comparison of expansion by IL-21 vs. expansion by IL-21 / anti-IL-21 complexes. [Figure 11A] Figures 11A-11H show the NK cell effector function of previously cryopreserved g-NK cells compared with that of freshly enriched g-NK cells (n=4). Values ​​are means ± SE. #p<0.05 for the comparison of freshly enriched g-NK cells vs. previously cryopreserved g-NK cells. Figures 11A and 11B illustrate the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11A shows the g-NK cell degranulation levels for the LP1 cell line. Figure 11B shows the g-NK cell degranulation levels for the MM.1S cell line. [Figure 11B] See legend to Figure 11A. [Figure 11C] Figures 11A-11H show the NK cell effector function of previously cryopreserved g-NK cells compared to that of freshly enriched g-NK cells (n=4). Values ​​are means ± SE. #p<0.05 for the comparison of freshly enriched vs. previously cryopreserved g-NK cells. Figures 11C and 11D illustrate the levels of perforin and granzyme B expression in previously cryopreserved g-NK cells compared to freshly enriched g-NK cells. Figure 11C shows total perforin expression in g-NK cells. Figure 11D shows total granzyme B expression in g-NK cells. [Figure 11D] See legend to Figure 11C. [Figure 11E] Figures 11A-11H show the NK cell effector function of previously cryopreserved g-NK cells compared with that of freshly enriched g-NK cells (n=4). Values ​​are means ± SE. #p<0.05 for the comparison of freshly enriched g-NK cells vs. previously cryopreserved g-NK cells. Figures 11E and 11F illustrate the daratumumab- and elotuzumab-mediated interferon-γ expression levels of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11E shows the g-NK cell interferon-γ expression levels for the LP1 cell line. Figure 11F shows the g-NK cell interferon-γ expression levels for the MM.1S cell line. [Figure 11F] See legend to Figure 11E. [Figure 11G] Figures 11A-11H show the NK cell effector function of previously cryopreserved g-NK cells compared with that of freshly enriched g-NK cells (n=4). Values ​​are means ± SE. #p<0.05 for the comparison of freshly enriched g-NK cells vs. previously cryopreserved g-NK cells. Figures 11G and 11H illustrate the daratumumab- and elotuzumab-mediated TNF-α expression levels of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 17G shows the g-NK cell TNF-α expression levels for the LP1 cell line. Figure 11H shows the g-NK cell TNF-α expression levels for the MM.1S cell line. [Figure 11H] See legend to Figure 11G. [Figure 12A]Figures 12A-12C illustrate the persistence of cNK (cryopreserved) and g-NK (cryopreserved or fresh) cells in NSG mice after injection of a single dose of 1 x 107 expanded cells. Figure 12A shows the number of cNK and g-NK cells in peripheral blood collected on days 6, 16, 26, and 31 post-injection. Figure 12B shows the number of NK cells present in the spleen at the time of sacrifice 31 days post-injection. Figure 12C shows the number of NK cells present in the bone marrow at the time of sacrifice. N=3 for all three treatment groups. Values ​​are mean ± SE. *p<0.05, and ***p<0.001 for the comparison of cryopreserved cNK cells with fresh or cryopreserved g-NK cells. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A] Figures 13A-13D illustrate the expression of CD20 (target for rituximab), CD38 (target for daratumumab), and SLAMF7 (target for elotuzumab) on g-NK and cNK. Figure 13A shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and MM.1S cells that express CD20. Figure 13B shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and MM.1S cells that express CD38. Figure 13C shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and MM.1S cells that express SLAMF7. Figure 13D shows the percentage of cNK and g-NK cells that express CD38 before and after expansion. N=3 for all treatment groups. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E] The mean fluorescence intensity (MFI) of CD38posNK cells before and after expansion is shown (n=4). [Figure 13F]Representative histograms illustrating reduced CD38 expression in g-NK cells relative to cNK and MM.1S cells are provided. Values ​​are mean ± SE. #p<0.001 for comparison of g-NK cells versus all other cells. [Figure 13G] Illustrates a comparison of daratumumab-induced fratricide by expanded g-NK and cNK cells. [Figure 14A] Figures 14A-14F show the effects of treatment with cNK and daratumumab (cNK+Dara) or g-NK and daratumumab (g-NK+Dara) on tumor burden and survival in a mouse model of multiple myeloma. 5 × 10 luciferase-labeled MM.1S human myeloma cells were injected intravenously (IV) into the tail vein of female NSG mice. Once weekly for a duration of 5 weeks, NSG mice received IV expanded NK cells (6.0 × 10 cells / mouse) and IP injections of daratumumab (10 μg / mouse). Figure 14A shows BLI imaging of mice twice weekly on days 20, 27, 37, 41, 48, and 57 after tumor inoculation (left). The corresponding post-treatment days are indicated on the right side of the figure. Color indicates BLI intensity (blue, lowest; red, highest). Figure 14B shows tumor BLI (photons / second) over time in the g-NK + Dara group versus the control and cNK + Dara groups. *p<0.05 for comparisons of the g-NK and control or cNK groups. Figure 14C shows survival over time, with arrows indicating administration of treatment with either cNK + Dara or g-NK + Dara. Figure 14D presents the weight change over time for mice in the control, cNK + Dara, and g-NK + Dara groups. Figure 14E illustrates the number of CD138+ tumor cells present in the bone marrow at the time of sacrifice in cNK + Dara- and g-NK + Dara-treated mice. ***p<0.001 for comparisons of g-NK and cNK cells. Values ​​are mean ± SE. Figure 14F shows representative flow plots using a gating strategy to elucidate the presence of NK cells and tumor cells in the control group and in mice treated with either cNK + Dara or g-NK + Dara. N=8 for the control group and N=7 for the g-NK or cNK groups. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] All BLI images collected throughout the study for all control, cNK+Dara, and g-NK+Dara treated mice are presented. Color indicates BLI intensity (blue, lowest; red, highest). [Figure 14H] X-ray images obtained for all mice in the control, cNK+Dara, and g-NK+Dara groups prior to sacrifice are shown. Arrows indicate fractures and deformities. The date of sacrifice is indicated below each mouse. [Figure 15A] Figures 15A-15C present comparative data on persistent NK cells in NSG mice after treatment with cNK+Dara or g-NK+Dara. All data present the amount of cells detected using flow cytometry at the time of sacrifice. Figure 15A shows the number of cNK and g-NK cells in the blood. Figure 15B shows the number of NK cells present in the spleen. Figure 15C shows the number of NK cells present in the bone marrow. Values ​​are mean ± SE. ***p<0.001 for the comparison of g-NK and cNK cells. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 16A]Figures 16A-16C show the effect of cNK and rituximab or g-NK and rituximab treatment on the presence and survival of Raji cells in a mouse model of lymphoma. 5 x 10 luciferase-labeled Raji lymphoma cells were injected intravenously (IV) into the tail vein of female NSG mice. Once weekly for a duration of 7 weeks, NSG mice received IV expanded NK cells (15 x 10 cells / mouse) and IP injections of rituximab (200 μg / mouse). Figure 16A shows weekly BLI imaging at days 0, 7, 14, 21, 28, and 35 after tumor inoculation. Figure 16B shows survival over time. Figure 16C shows percent body weight change over time. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description Provided herein are methods for treating multiple myeloma, comprising administering to a subject with cancer a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain. In some embodiments, the provided methods relate to the use of the method and composition containing g-NK cells for treating multiple myeloma (MM). In some embodiments, the provided methods relate to the use of the method and composition containing g-NK cells for treating lymphoma. In some embodiments, the g-NK cell composition may be administered weekly in a predetermined number of doses. In some embodiments, compositions of g-NK cells may be administered in combination with antibody therapeutics for treating cancer, such as anti-CD38 antibodies (e.g., daratumumab), anti-SLAMF7 antibodies (e.g., elotuzumab), or anti-BCMA antibodies (e.g., belantamab) for treating multiple myeloma, or anti-CD20 antibodies (e.g., rituximab), anti-CD19 antibodies (e.g., tafasitamab or loncastuximab), or anti-CD30 antibodies (e.g., brentuximab) for treating lymphoma.

[0032] Natural killer (NK) cells are innate lymphocytes important for mediating antiviral and anticancer immunity 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)). NK cells are effector cells comprising the third largest population of lymphocytes and are important for host immune surveillance against tumor and pathogen-infected cells. However, unlike T and B lymphocytes, NK cells use germline-encoded activating receptors and are thought to have only a limited capacity for target recognition (Bottino et al., Curr Top Microbiol Immunol. 298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21 (2006)).

[0033] NK cell activation can occur through direct binding of the NK cell receptor to a ligand on the target cell, as seen in direct tumor cell killing, or through cross-linking of the Fc receptor (also known as CD16, CD16a, or FcγRIIIa) by binding to the Fc portion of an antibody bound to an antigen-bearing cell. Upon activation, NK cells abundantly produce cytokines and chemokines and simultaneously exhibit potent cytolytic activity. NK cells can kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when receptors on the NK cell surface (such as CD16) recognize IgG1 or IgG3 antibodies bound to the cell surface. This triggers the release of cytoplasmic granules containing perforin and granzymes, leading to target cell death. Target recognition is expanded because NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells (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.

[0034] CD16 also exists in a glycosylphosphatidylinositol-anchored form (also known as FcγRIIIB or CD16B). It will be understood that references to CD16 herein are to the CD16a form, which is expressed on NK cells and is involved in antibody-dependent responses (such as NK cell-mediated ADCC), and are not meant to refer to the glycosylphosphatidylinositol-anchored form.

[0035] The CD16 receptor associates with the adaptor, the zeta chain of the TCR-CD3 complex (CD3ζ) and / or the FcRγ chain, and can transduce signals through immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, CD16 binding (CD16 cross-linking) initiates NK cell responses via intracellular signals generated through one or both of the CD16-associated adaptor chains, FcRγ or CD3ζ. Triggering of CD16 leads to phosphorylation of the γ or ζ chain, which in turn recruits tyrosine kinases, syk, and ZAP-70, initiating a signal transduction cascade 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. CD16 cross-linking also results in the production of cytokines and chemokines, which then activate and orchestrate a series of immune responses.

[0036] This release of cytokines and chemokines may play a role in the anti-cancer activity of NK cells in vivo. NK cells also possess small granules containing perforin and proteases (granzymes) in their cytoplasm. Upon release from NK cells, perforin forms pores in the plasma membrane of target cells, through which granzymes and related molecules can enter and induce apoptosis. The fact that NK cells induce apoptosis but not necrosis in target cells is important because necrosis of virus-infected cells releases virions, whereas apoptosis leads to the destruction of the intracellular virus.

[0037] A specialized subset of NK cells lacking the FcRγ adaptor protein, also known as g-NK cells, can mediate robust ADCC responses (see, e.g., published U.S. Patent Application Publication No. 2013 / 0295044). The mechanism for the increased response may be due to changes in epigenetic modifications that affect the expression of FcRγ. g-NK cells abundantly express the signaling adaptor ζ chain but lack expression of the signaling adaptor γ chain. Compared to conventional NK cells, these γ-deficient g-NK cells exhibit dramatically enhanced activity when activated by antibodies. For example, g-NK cells can be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells. In some aspects, g-NK cells produce greater amounts of cytokines (e.g., IFN-γ or TNF-α) and chemokines (e.g., MIP-1α, MIP-1β, and RANTES) and / or exhibit a greater degranulation response than gamma-chain-expressing conventional NK cells. g-NK cells provide high expression of granzyme B, a component of the natural killer cytotoxicity machinery. Furthermore, g-NK cells have an extended lifespan compared to conventional NK cells, and their existence is maintained for extended periods of time. In some embodiments, g-NK cells are functionally and phenotypically stable.

[0038] In some embodiments, g-NK cells are more effective at inducing ADCC responses than conventional NK cells, e.g., NK cells that are not gamma-chain deficient. In some embodiments, g-NK cells are more effective at inducing cell-mediated cytotoxicity than conventional NK cells, even in the absence of antibodies. In some cases, ADCC is the mechanism of action of therapeutic antibodies, including anti-cancer antibodies. In some aspects, cell therapy by administering NK cells can be used in conjunction with antibodies for therapeutic and related purposes.

[0039] Certain therapeutic monoclonal antibodies, such as daratumumab, which targets CD38; elotuzumab, which targets SLAMF7; and belantamab, which targets BCMA, have been FDA-approved to treat diseases such as multiple myeloma (MM). Other therapeutic monoclonal antibodies, such as rituximab, which targets CD20; tafasitamab or loncastuximab, which target CD19; and brentuximab, which targets CD30, have been FDA-approved to treat diseases such as lymphoma. While clinical responses to therapeutic antibodies have been promising, they are often not ideal. For example, with daratumumab in particular, initial clinical responses have generally been encouraging, but essentially all patients eventually develop progressive disease. Thus, there is a great need for new strategies to either promote deeper remissions or overcome resistance to these agents. The provided embodiments, including compositions, address these needs.

[0040] Provided herein are methods involving the combined administration of compositions containing g-NK cells, e.g., as produced by the provided methods, and antibodies, e.g., anti-cancer antibodies. In some embodiments, antibody-directed targeting of g-NK cells leads to improved outcomes for patients due to improved affinity, cytotoxicity, and / or cytokine-mediated effector function of the g-NK cell subset.

[0041] In some embodiments, the potential mechanism of action of monoclonal antibodies as therapeutic agents is through antitumor effects resulting from complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and / or antibody-dependent cellular cytotoxicity. It is contemplated that in some cases, ADCC mediated by NK cells can potently eliminate antibody-bound tumor cells, particularly in the case of multiple myeloma (MM) tumors.

[0042] NK cells are activated when the Fc portion of an antibody binds to its Fc receptor (FcγRIIIa or CD16a), triggering activation and degranulation through a process involving the adaptor proteins CD3ζ and FcεR1γ. Efforts to enhance clinical ADCC responses to antibodies, including MM antibodies, have been difficult because NK cells also express CD38 and SLAMF7 (targets of, e.g., daratumumab and elotuzumab, respectively). High CD38 expression results in rapid depletion of NK cells, especially early in the course of daratumumab treatment, largely eliminating this source of innate immune cells that could potentially drive more complete tumor eradication.

[0043] The provided g-NK cells and compositions containing them, as produced by the provided methods, exhibit several features that overcome these problems. g-NK cells are a relatively rare subset, being detectable at levels of approximately 3-10% of total NK cells in only 25-30% of CMV-seropositive individuals. The provided methods are particularly robust in their ability to expand and enrich g-NK cells, thus enabling the sufficient expansion required for in vivo use.

[0044] In some embodiments, g - NK cells produce significantly greater amounts of cytokines than natural killer cells, which express FcRγ. In another embodiment, the cytokine is interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), or a combination thereof. In one embodiment, - NK cells produce significantly greater amounts of chemokines. In one embodiment, the chemokines are MIP-1α, MIP-1β, or a combination thereof. In another embodiment, - NK cells produce cytokines or chemokines upon stimulation through the Fc receptor CD16.

[0045] g-NK cells represent a relatively small percentage of NK cells in peripheral blood, thereby limiting the ability to use these cells in therapeutic methods. Specifically, because g-NK cells are generally a rare population, a high preferential expansion rate is necessary for clinical use of g-NK cells. While other methods for expanding NK cells can achieve 1000-fold 14-day NK cell expansion rates, they result in poorly differentiated, NKG2C cells. neg , FceRIγ pos (FcRγ pos ) NK cells (Fujisaki et al. (2009) Cancer Res., 69:4010-4017; Shah et al. (2013) PLoS One, 8:e76781). Furthermore, it is found herein that optimized expansion to expand NK cells that phenotypically overlap with g-NK cells does not preferentially expand g-NK cells to amounts that support therapeutic use. Specifically, NKG2C cells that exhibit phenotypic overlap with g-NK cells are pos It has previously been reported that NK cells can be preferentially expanded using HLA-E transfected 221.AEH cells and the inclusion of IL-15 in the culture medium (Bigley et al. (2016) Clin. Exp. Immunol., 185:239-251). Culturing with such HLA-expressing cells, which constitutively express HLA-E, selectively directs NK cells to NKG2C. pos / NKG2A neg (NKG2C is an activating receptor for HLA-E, while NKG2A is an inhibitory receptor for HLA-E.) Because such cells contain g-NK within them, such a method was thought to be sufficient to expand g-NK cells. However, this method does not achieve robust expansion of g-NK cells.

[0046] The methods described herein can produce NK cell compositions enriched for g-NK cells that overcome these limitations. The provided methods utilize a greater ratio of HLA-E+ feeder cells, e.g., 221.AEH cells, that are HLA class I and HLA class II deficient, to NK cells compared to previous methods. Specifically, previous methods have used lower ratios of 221.AEH cells, such as a 10:1 NK cell to 221.AEH ratio. It has been found herein that a greater ratio of HLA-E-expressing feeder cells, such as 221.AEH cells, results in an overall expansion that is larger and more skewed toward the g-NK phenotype. In some embodiments, a greater ratio of HLA-E+ feeder cells, e.g., 221.AEH cells, is possible by irradiating the feeder cells. In some aspects, the use of irradiated feeder cell lines is also advantageous because it provides a method that is GMP-compatible. During expansion, the inclusion of any of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof has also been found to support robust expansion. In certain embodiments of the provided methods, at least one recombinant cytokine is IL-2. In some embodiments, two or more recombinant cytokines are present, and at least one recombinant cytokine is IL-2 and at least one recombinant cytokine is IL-21.

[0047] The methods provided herein are based on the discovery that culturing NK cells for expansion in the presence of IL-21 supercharges the NK cells to produce cytokines or effector molecules such as perforin and granzyme B. Compositions containing NK cells produced by the expansion processes herein are highly functional, exhibit robust proliferation, and function well even after they are cryogenically frozen without rescue. For example, NK cells produced by the provided processes not only exhibit strong ADCC activity when expanded in the presence of IL-21, but also antibody-independent cytotoxicity. For example, effector molecules (e.g., perforin and granzyme) are spontaneously present in NK cells expanded by the provided methods, thereby providing cells that exhibit high cytotoxic potential. As shown herein, NK cell compositions produced by the provided processes that include IL-21 (e.g., IL-2, IL-15, and IL-21) not only exhibit a higher percentage of NK cells positive for perforin or granzyme B than NK cell compositions produced by processes that include only IL-2 without the addition of IL-21, but also exhibit a higher average level or degree of expression of the molecules in the cells. Furthermore, NK cell compositions produced by the methods provided herein that include IL-21 (e.g., IL-2, IL-15, and IL-12) also result in g-NK cell compositions that, when combined with an antibody (e.g., daratumumab) against a target antigen (e.g., CD38), exhibit substantial effector activity, including the ability to degranulate and express greater amounts of IFN-gamma and TNF-alpha in response to target cells. This functional activity is highly preserved even after cryopreservation and thawing of the expanded NK cells. The significant increase in cytolytic enzymes, as well as a more robust activation phenotype, supports the enhanced ability of expanded g-NK cells to induce apoptosis of tumor targets when coupled with antibodies via CD16 cross-linking. The prominent antibody-independent effector phenotype also supports the potential utility of g-NK cells as monotherapy.

[0048] Furthermore, the findings herein also demonstrate the potential for provided NK cells expanded in the presence of IL-21 to persist and proliferate better for extended periods than, for example, cells expanded only in the presence of IL-2 without the addition of IL-21. Furthermore, the results showed that cryopreserved g-NK cells persisted at levels comparable to fresh g-NK cells. This significantly improved persistence highlights the potential utility of fresh or cryopreserved g-NK as an off-the-shelf cell therapy for enhancing antibody-mediated ADCC. This finding of improved persistence is advantageous, as the clinical utility of many NK cell therapies has been hindered by limited NK cell persistence.

[0049] Furthermore, the results herein demonstrate the surprising discovery that g-NK cells express low levels of CD38, the target of therapeutic antibodies such as daratumumab. A problem with many existing NK cell therapies directed against specific target antigens, such as CD38, is that NK cells can express the target antigen, resulting in "fratricide," whereby ADCC activity leads to the elimination of NK cells in addition to the tumor. Indeed, other reported NK cell compositions have been reported to express a high percentage (e.g., >90%) of CD38 high NK cells. In contrast, the findings herein demonstrate that the percentage of CD38 positive cells was significantly lower in donor-isolated g-NK cells and g-NK cells expanded therefrom than in conventional NK cells or MM-targeted cell lines. Lower CD38 expression leads to significantly reduced anti-CD38 (e.g., daratumumab)-mediated fratricide by g-NK cells relative to conventional NK cells. These results support the utility of the provided g-NK cell compositions to confer enhanced antibody antitumor activity in MM without suffering from fratricide-associated depletion. The results further suggest that g-NK cell compositions may be optimal for daratumumab-refractory patients, as expanded g-NK cells are resistant to daratumumab-induced fratricide and enhance daratumumab-specific cytotoxicity against even faintly CD38-expressing myeloma cells.

[0050] Furthermore, the above-mentioned activities demonstrated by g-NK cells can be achieved without the need for further cell manipulation to enhance antibody efficacy. For example, CD38-knockout NK cell lines have been created to avoid daratumumab fratricide, and NK cell lines with non-cleavable CD16 have been developed to enhance anti-tumor ADCC. However, potential drawbacks to clinical use include the need for genetic manipulation and irradiation of immortalized cell lines.

[0051] The superiority of the provided g-NK cell compositions, including those produced by the provided methods, was further demonstrated in studies evaluating the in vivo activity of g-NK cells. Activity in an exemplary mouse model of MM showed that g-NK cells combined with an antibody (e.g., daratumumab) eliminated the myeloma tumor burden in the majority of mice, with sustained and significant tumor regression. These results highlight the superiority of g-NK cells compared to conventional NK cells, particularly those that are FcεR1γ+, for enhancing antibody effects in vivo and support the therapeutic potential of this NK cell therapy. The high persistence and enhanced survival of NK cells in this model, as well as their resistance to fratricide, may support the superior antitumor efficacy and persistence of g-NK cells.

[0052] It has also been found that enriching NK cells from the cell sample prior to expansion, such as by enrichment for CD16 or CD57 cells prior to expansion, further substantially increases the amount of g-NK cell expansion that can be achieved compared to methods that initially enrich for NK cells based solely on CD3 depletion. In another embodiment, another enrichment that can be performed prior to expansion is enrichment for NK cells by positive selection for CD56 and negative selection or depletion for CD38. In a further embodiment, another enrichment that can be performed prior to expansion is enrichment for NK cells by positive selection for CD56 followed by NKG2A depletion. neg Negative selection or depletion against CD161 negIn another embodiment, another enrichment that may be performed before expansion is enrichment for NK cells by positive selection for CD57 followed by negative selection or depletion for NKG2A and / or positive selection for NKG2C. In another embodiment, another enrichment that may be performed before expansion is enrichment for NK cells by positive selection for CD56 followed by negative selection or depletion for NKG2A and / or positive selection for NKG2C. In any of such embodiments, pos and / or NKG2A neg Enrichment for NK cells can be performed after expansion.

[0053] In any of such embodiments, the enriched NK cells may be enriched from a cell sample containing NK cells, such as from peripheral blood mononuclear cells (PBMCs). In some embodiments, prior to enrichment of NK cells from a cell sample, T cells may be removed by negative selection or depletion against CD3. In any of such embodiments, the enriched NK cells may be enriched from a biological sample from a human subject containing NK cells (e.g., PBMCs) with a relatively high proportion of g-NK cells, e.g., a human subject selected to have a high percentage of g-NK cells among their NK cells. In any of such embodiments, the enriched NK cells may be enriched from a biological sample from a human subject containing NK cells, e.g., PBMCs, the sample having a relatively high proportion of NKG2C cells. pos NK cells (e.g., 20% or about 20% or more than 20% NKG2C pos NK cells), and / or NKG2A neg NK cells (e.g., 70% or about 70% or more than 70% NKG2A neg In any of such embodiments, the enriched NK cells may be enriched from a biological sample from a human subject containing NK cells, e.g., PBMCs, the sample containing a relatively high percentage of NKG2C posNK cells (e.g., 20% or about 20% or more than 20% NKG2C pos NK cells), and NKG2A neg NK cells (e.g., 70% or about 70% or more than 70% NKG2A neg Contains NK cells.

[0054] In summary, the provided approaches for expanding g-NK cells can achieve expansion of more than 1 billion cells, and in some cases up to 8 billion or more NK cells, from an initial 10 million enriched NK cells at the initiation of culture. Specifically, the provided methods can result in high-yield (>1000-fold) expansion rates that maintain or in some cases increase the functionality of g-NK cells after expansion. In some embodiments, the provided methods can result in g-NK cell populations that express high levels of perforin and granzyme B. Furthermore, the provided methods have been found to be sufficient to expand previously frozen NK cells, a feasibility typically not achieved by many existing methods involving the rescue of thawed NK cells. In some embodiments, this is achieved by increasing the duration of the expansion protocol. In some embodiments, this is achieved by decreasing the ratio of HLA-E+ feeder cells to NK cells, for example, to about 1:1 221.AEH to NK cells. In some embodiments, this is achieved by the inclusion of any of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof during expansion. In particular embodiments, at least one recombinant cytokine is IL-2. In some embodiments, expansion is carried out in the presence of two or more recombinant cytokines, at least one of which is recombinant IL-21 and at least one of which is recombinant IL-2. As demonstrated herein, the methods provided result in g-NK cells that exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated cytotoxicity, supporting the utility of such cells for therapeutic applications.

[0055] As demonstrated herein, the provided g-NK cells and compositions containing same, as produced by the provided methods, can be used in cancer therapy. In some aspects, the provided studies demonstrate that g-NK cells have significantly enhanced ADCC / effector function when combined with a targeted antibody against a tumor antigen (e.g., antimyeloma), and that adoptive transfer of expanded g-NK cells eliminates tumor burden in vivo when combined with a therapeutic antibody (e.g., daratumumab). Importantly, adoptive transfer of allogeneic NK cells does not result in severe graft-versus-host (GVHD), and therefore, such cell therapies, including combinations with antibodies as antibody-directed NK cell therapies, can be given in an "off-the-shelf" manner for clinical use.

[0056] All references cited herein, including patent applications, patent publications, and scientific literature and databases, are incorporated by reference in their entirety for all purposes as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0057] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0058] I. Definition Unless otherwise defined, all terminology, notation, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural of the referent unless the singular is clearly dictated by the context. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0060] The term "about" as used herein refers to a normal error range for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0061] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0062] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur. For example, an optionally substituted group means that the group is either unsubstituted or substituted.

[0063] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic, such as recombinantly produced, and includes any fragment thereof that contains at least a portion of the variable heavy and / or light chain region of an immunoglobulin molecule sufficient to form an antigen-binding site and that, when assembled, specifically binds to an antigen. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Typically, an antibody minimally comprises a variable heavy chain (VHC), a VHC, ... H ) chain and / or variable light (VL ) in whole or in part. H and V L The pairing of V together forms the antigen-binding site, but in some cases, a single V H or V L The domain is sufficient for antigen binding. An antibody may also include all or a portion of a constant region. Reference to an antibody herein includes full-length antibodies and antigen-binding fragments. As used herein, the term "immunoglobulin" (Ig) is used interchangeably with "antibody."

[0064] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to a substantially complete antibody, as opposed to an antibody fragment. Full-length antibodies typically have two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4), two full-length light chains (VL-CL), and a hinge region, such as antibodies produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.) and synthetically produced antibodies with the same domains. Specifically, whole antibodies include those having heavy and light chains comprising an Fc region. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0065] "Antibody fragments" include portions of an intact antibody, including the antigen-binding and / or variable regions of the intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fd' fragments, diabodies, linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ), single-chain antibody molecules, including single-chain Fvs (scFvs) or single-chain Fabs (scFabs), and multispecific antibodies derived from antigen-binding fragments and antibody fragments of any of the above. For purposes herein, antibody fragments typically include those sufficient to bind to or cross-link CD16 on the surface of NK cells.

[0066] The term "autologous" refers to cells or tissues that originate within or are taken from an individual's own tissue. For example, in autologous transfer or transplantation of NK cells, the donor and recipient are the same person.

[0067] The term "allogeneic" refers to cells or tissues that belong to or are obtained from the same species, but that are genetically different and therefore, in some cases, immunologically incompatible. Typically, the term "allogeneic" is used to define cells that are transplanted from a donor into a recipient of the same species.

[0068] The term "enriched" with respect to a cell composition refers to a composition in which there is an increase in the number or percentage of a cell type or population compared to the number or percentage of that cell type in the same volume of a starting composition, such as a starting composition obtained or isolated directly from a subject. The term does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells so enriched be present at or near 100% in the enriched composition.

[0069] The term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0070] The term "heterologous" with respect to a protein or nucleic acid refers to a protein or nucleic acid that is derived from a different genetic source. For example, a protein or nucleic acid that is heterologous to a cell comes from an organism or individual other than the cell in which it is expressed.

[0071] As used herein, the term "introducing" encompasses various methods of introducing DNA into cells, either in vitro or in vivo, including transformation, transduction, transfection (e.g., electroporation), and infection. Vectors are useful for introducing DNA encoding a molecule into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors such as adenoviral vectors or adeno-associated vectors.

[0072] The term "composition" refers to any mixture of two or more products, substances, or compounds, including cells or antibodies. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. The preparation is generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

[0073] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0074] As used herein, a combination refers to any association between or among two or more items. The combination may be two or more separate items, such as two compositions or two collections, or may be a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0075] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional agents, and instructions for use of the combination or elements, for a purpose including, but not limited to, therapeutic use.

[0076] As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to alter the natural history of an individual or cell being treated during a clinical pathological process. Desirable effects of treatment include a reduction in the rate of disease progression, remission or alleviation of the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a disorder (e.g., an eosinophil-mediated disease) are reduced or eliminated. For example, an individual is successfully "treated" if the treatment results in an increase in the quality of life of an individual suffering from a disease, a reduction in the dose of other drugs required to treat the disease, a reduction in the frequency of disease recurrences, a reduction in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual's survival.

[0077] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary, to achieve a desired or indicated effect, including a therapeutic or preventative result. An effective amount may be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum dose of cells required to produce a measurable improvement in a particular disorder. In some embodiments, a therapeutically effective amount is an amount of a composition that reduces the severity, duration, and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight. A therapeutically effective amount may also be an amount in which the beneficial effects of treatment outweigh any toxic or detrimental effects of the antibody. A "prophylactically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount may be less than a therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0078] As used herein, an "individual" or "subject" is a mammal. For purposes of treatment, "mammals" include humans, domestic and farm animals, and zoo, sport, or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0079] II. Treatment Methods Provided herein are compositions and methods relating to the provided cell compositions, including g-NK cells described herein, for use in treating a disease or condition in a subject. In some embodiments, provided herein are methods of treating a condition in an individual, comprising administering any of the provided compositions, such as compositions comprising g-NK cells, to an individual in need thereof. In certain embodiments, the compositions are produced by the methods provided herein. Such methods and uses include, for example, therapeutic methods and uses involving administration of therapeutic cells or compositions containing same to a subject having a disease, condition, or disorder. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the disease or disorder is a viral infection. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to effect treatment of the disease or disorder. Uses include the use of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods thereby treat a disease, condition, or disorder in a subject.

[0080] In one aspect, disclosed herein is a method of treating multiple myeloma (MM), comprising administering to a subject having multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells can be administered once a week in a predetermined number of doses.

[0081] In one aspect, disclosed herein is a method of treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells is administered once a week in a predetermined number of doses.

[0082] In some embodiments, the predetermined number of weekly doses is 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, or 12 doses. In some embodiments, the weekly doses are administered for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, or more weeks. In some embodiments, 6 doses of the g-NK cell composition are administered once a week. In some embodiments, the weekly doses are administered on consecutive weeks.

[0083] In some embodiments, the weekly dose is administered in a cycling regimen. In some embodiments, the cycling regimen is a 14-day cycle. In some embodiments, the weekly dose is administered twice in a 14-day cycle. In some embodiments, the 14-day cycle is repeated twice. In some embodiments, the 14-day cycle is repeated three times.

[0084] In some embodiments, the methods of treatment or uses involve administering to an individual an effective amount of a composition containing a composition of expanded NK cells produced by the provided methods. 5 Pieces or about 10 5 pieces ~ about 10 12 Pieces or 10 5 Pieces or about 10 5 ~10 pieces 8 Pieces or about 10 8 Pieces or 10 6 Pieces or about 10 6 ~10 pieces 12 Pieces or about 10 12 Pieces or 10 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 Pieces or 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 10 such expanded NK cells are administered to an individual subject. In some embodiments, 105 More than or about 10 5 More than 10 pieces 6 More than or about 10 6 More than 10 pieces 7 More than or about 10 7 More than 10 pieces 8 More than or about 10 8 More than 10 pieces 9 More than or about 10 9 More than 10 pieces 10 More than or about 10 10 More than 10 pieces 11 More than or about 10 11 More than 10 12 More than or about 10 12 A dose of cells containing such expanded NK cells or more is administered to an individual. In some embodiments, 10 per kg or more of the cells are administered. 6 Pieces or about 10 6 ~10 pieces 10 A number of such expanded NK cells are administered to a subject.

[0085] In some embodiments, methods of treatment or use involve administering to an individual an effective amount of any of the provided NK cell compositions, including any described herein. 5 Pieces or about 10 5 pieces ~ about 10 12 Pieces or 10 5 Pieces or about 10 5 ~10 pieces 8 Pieces or about 10 8 Pieces or 10 6 Pieces or about 10 6 ~10 pieces 12 Pieces or about 10 12 Pieces or 10 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 Pieces or 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10NK cells derived from any of the provided compositions are administered to an individual subject. 5 More than or about 10 5 More than 10 pieces 6 More than or about 10 6 More than 10 pieces 7 More than or about 10 7 More than 10 pieces 8 More than or about 10 8 More than 10 pieces 9 More than or about 10 9 More than 10 pieces 10 More than or about 10 10 More than 10 pieces 11 More than or about 10 11 More than 10 12 More than or about 10 12 In some embodiments, NK cells derived from any of the provided compositions are administered to an individual. 6 Pieces or about 10 6 ~10 pieces 10 The NK cells of any of the provided compositions are administered to a subject.

[0086] In some embodiments, each dose of g-NK cells comprises 1 x 10 of the g-NK cell composition. 8 Pieces or approximately 1 x 10 8 cells ~ 50 x 10 9 or approximately 50 x 10 9 In some embodiments, each dose of g-NK cells may be 5 x 10 cells of the g-NK cell composition. 8 cells, or about 5 x 10 8 In some embodiments, each dose of g-NK cells may be 5 x 10 cells of the g-NK cell composition. 9 cells, or about 5 x 10 9 In some embodiments, each dose of g-NK cells may be 10 x 10 of the g-NK cell composition. 9 cells, or about 10 x 10 9 It can be a cell.

[0087] In some embodiments, the method of treatment comprises administering to an individual an effective amount of a composition containing g-NK cells. 5 Pieces or about 10 5 pieces ~ about 10 12 g-NK cells, or 10 5 Pieces or about 10 5 ~10 pieces 8 Pieces or about 10 8 g-NK cells, or 10 6 Pieces or about 10 6 ~10 pieces 12 Pieces or about 10 12 g-NK cells, or 10 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 g-NK cells, or 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 g-NK cells. In some embodiments, 10 5 More than or about 10 5 g-NK cells, 10 6 More than or about 10 6 g-NK cells, 10 7 More than or about 10 7 g-NK cells, 10 8 More than or about 10 8 g-NK cells, 10 9 More than or about 10 9 g-NK cells, 10 10 More than or about 10 10 g-NK cells, 10 11 More than or about 10 11 g-NK cells or more, or 10 12 More than or about 10 12 A dose of cells containing 10 or more g-NK cells is administered to an individual. 6 Pieces or about 10 6 ~10 pieces 10 g-NK cells / kg are administered to the subject.

[0088] In some embodiments, the dose for administration according to any of the methods of treatment or uses provided is 1×10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, e.g., 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 7.5 x 10 6 or about 7.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 2.5 x 10 6 or about 2.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 5 x 10 5 cells / kg or approximately 5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 5 x 10 5 cells / kg or approximately 5 x 10 5 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 105 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 106 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, or 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 In some embodiments, the dose for administration is 1 x 10 cells / kg. 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, e.g., 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 106 cells / kg or approximately 1 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 7 cells / kg or approximately 2.5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 7 cells / kg or approximately 5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, or 7.5 x 10 7 cells / kg or approximately 7.5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg.

[0089] In some embodiments, the dose is given as the number of g-NK cells, or an NK cell subset associated with or comprising a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or the number of viable cells of any of the above. 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 methods, or the number of viable cells of any of the above.

[0090] In some embodiments, the dose for administration according to any of the methods of treatment or uses is 5×10 7 Or about 5 x 10 7 ~10×10 9 Or about 10 x 10 9 , e.g., 5 x 10 7 Or about 5 x 10 7 ~5×10 9 Or about 5 x 10 9 , about or about 5 x 10 7 ~1×10 9 or about 1 x 10 9 , 5×10 7 Or about 5 x 10 7 ~5×10 8 Or about 5 x 10 8 , about or about 5 x 10 7 ~1×10 8 or about 1 x 10 8 , 1×10 8 ~10×10 9 Or about 10 x 10 9 , 1×10 8 or about 1 x 10 8 ~5×10 9 or about 5 x 10 9 , about or about 1 x 10 8 ~1×10 9 or about 1 x 10 9 , 1×10 8 or about 1 x 10 8 ~5×10 8 Or about 5 x 10 8 , 5×10 8 Or about 5 x 10 8 ~10×10 9Or about 10 x 10 9 , 5×10 8 Or about 5 x 10 8 ~5×10 9 Or about 5 x 10 9 , about or about 5 x 10 8 ~1×10 9 or about 1 x 10 9 , 1×10 9 or about 1 x 10 9 ~10×10 9 Or about 10 x 10 9 , 1×10 9 or about 1 x 10 9 ~5×10 9 Or about 5 x 10 9 , or 5 x 10 9 Or about 5 x 10 9 ~10×10 9 Or about 10 x 10 9 In some embodiments, the dose for administration is 5×10 8 or approximately 5 x 10 8 In some embodiments, the dose for administration is 1 x 10 cells. 9 or approximately 1 x 10 9 In some embodiments, the dose for administration is 5 x 10 cells. 9 or approximately 5 x 10 9 In some embodiments, the dose for administration is 1 x 10 cells. 10 or approximately 1 x 10 10 In some embodiments, the dose is given as the number of g-NK cells, or an NK cell subset associated with or including a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or the number of viable cells of any of the above. 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 methods, or the number of viable cells of any of the above.

[0091] In some embodiments, compositions containing expanded NK cells are administered to an individual immediately after expansion by the methods provided. In other embodiments, the expanded NK cells are stored or expanded by growth in culture prior to administration, such as by the methods described above. For example, NK cells can be stored for more than 6, 12, 18, or 24 months before administration to an individual.

[0092] In some embodiments, 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 administration routes.

[0093] In certain embodiments, provided compositions are administered by intravenous infusion. 6 or approximately 10 x 10 6 cells ~ 10 x 10 9 In some embodiments, 50 x 10 cells are administered by intravenous infusion in a volume of 1 mL to 100 mL. 6 or approximately 50 x 10 6 In some embodiments, 1 x 10 cells are administered. 9 Pieces or approximately 1 x 10 9 In some embodiments, 5×10 cells are administered. 9 or approximately 5 x 10 9 In some embodiments, 10 x 10 cells are administered. 9 Pieces or approximately 10 x 10 9 Determining the volume of cells to inject to administer the number of cells is within the level of ordinary skill in the art. In one example, 0.5 x 10 cells are administered. 9 2.5 x 10 cells 7 or approximately 2.5 x 10 7 A concentration of cells / mL (e.g., 5 x 10 in 200 mL) 9 or approximately 5 x 10 9 The composition is administered by intravenous infusion in a volume of about 20 mL, such as a thawed cryopreserved composition formulated with 1000 cells (1000 cells).

[0094] In any of the above embodiments, the provided g-NK cells and compositions thereof may be used as a monotherapy for the treatment of a disease or disorder.

[0095] A. Compositions and Pharmaceutical Formulations In some embodiments, compositions for use in the provided methods contain g-NK cells. Specifically, among the compositions provided are compositions of cells enriched for g-NK cells. In some embodiments, compositions for use in the provided methods contain g-NK cells that are expanded NK cells, such as those produced by any of the provided methods. In some embodiments, the compositions contain NKG2C pos In some embodiments, the composition comprises NKG2A cells or a subset thereof. neg In some embodiments, the composition comprises NKG2C cells or a subset thereof. pos / NKG2A neg Contains cells or a subset thereof.

[0096] In some embodiments, the composition comprises about 5-99% NKG2C pos NKG2C cells or a subset thereof, or any percentage between 5% and 99% (inclusive) pos In some embodiments, the composition comprises NKG2C cells or a subset thereof relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. pos NKG2C relative to total NK cells or total cells, compared to the percentage of cells or subsets thereof pos It may include an increased or greater percentage of cells or subsets thereof, hi some embodiments, the percentage is increased by 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.

[0097] In some embodiments, the composition comprises at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 65%, at least or about 70%, at least 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%. , at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, or substantially 100% NKG2C pos In some embodiments, the composition may comprise more than 50% NKG2C cells or a subset thereof. pos In another embodiment, the composition comprises more than 60% NKG2C cells or a subset thereof. pos In another embodiment, the composition comprises more than 70% NKG2C cells or a subset thereof. pos In another embodiment, the composition comprises more than 80% NKG2C cells or a subset thereof. pos In some embodiments, the compositions provided comprise NKG2C cells or a subset thereof. pos The cells or subsets thereof include compositions in which the cells or subsets thereof constitute at least or about 60%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95% or more of the cells in the composition or NK cells in the composition.

[0098] In some embodiments, the composition comprises about 5-99% NKG2A neg NKG2A cells or a subset thereof, or any percentage between 5% and 99% (inclusive) neg In some embodiments, the composition comprises NKG2A cells or a subset thereof relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. neg NKG2A to total cells or to total cells compared to the percentage of cells or subsets thereof neg It may include an increased or greater percentage of cells or subsets thereof, hi some embodiments, the percentage is increased by 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.

[0099] In some embodiments, the composition comprises at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 65%, at least or about 70%, at least 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%. , at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, or substantially 100% NKG2A neg In some embodiments, the composition may comprise more than 50% NKG2A cells or a subset thereof. negIn another embodiment, the composition comprises more than 60% NKG2A cells or a subset thereof. neg In another embodiment, the composition comprises more than 70% NKG2A cells or a subset thereof. neg In another embodiment, the composition comprises more than 80% NKG2A cells or a subset thereof. neg In some embodiments, the compositions provided comprise NKG2A cells or a subset thereof. neg The cells or subsets thereof include compositions in which the cells or subsets thereof constitute at least or about 60%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95% or more of the cells in the composition or NK cells in the composition.

[0100] In some embodiments, the composition comprises about 5-99% NKG2C pos NKG2A neg NKG2C cells or a subset thereof, or any percentage between 5% and 99% (inclusive) pos NKG2A neg In some embodiments, the composition comprises NKG2C cells or a subset thereof relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. pos NKG2A neg NKG2C relative to total NK cells or total cells, as compared to the percentage of cells or subsets thereof pos NKG2A neg It may include an increased or greater percentage of cells or subsets thereof, hi some embodiments, the percentage is increased by 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.

[0101] In some embodiments, the composition comprises at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 65%, at least or about 70%, at least 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%. , at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%, or substantially 100% NKG2C pos NKG2A neg In some embodiments, the composition may comprise more than 50% NKG2C cells or a subset thereof. pos NKG2A neg In another embodiment, the composition comprises more than 60% NKG2C cells or a subset thereof. pos NKG2A neg In another embodiment, the composition comprises more than 70% NKG2C cells or a subset thereof. pos NKG2A neg In another embodiment, the composition comprises more than 80% NKG2C cells or a subset thereof. pos NKG2A neg In some embodiments, the compositions provided comprise NKG2C cells or a subset thereof. pos NKG2A negThe cells or subsets thereof include compositions in which the cells or subsets thereof constitute at least or about 60%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95% or more of the cells in the composition or NK cells in the composition.

[0102] In some embodiments, the composition comprises about 5-99% g-NK cells, or any percentage between 5-99%, inclusive, of g-NK cells. In some embodiments, the composition may comprise an increased or greater percentage of total NK cells or g-NK cells relative to total cells compared to the percentage of g-NK cells 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 by 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.

[0103] In some embodiments, the composition comprises at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, The composition may comprise at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% g-NK cells. In some embodiments, the composition comprises more than 50% g-NK cells. In another embodiment, the composition comprises more than 70% g-NK cells. In another embodiment, the composition comprises more than 80% g-NK cells. In some embodiments, provided compositions include compositions in which g-NK cells comprise at least or about 60%, at least or about 70%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95% or more of the cells in the composition or the NK cells in the composition.

[0104] In some embodiments, the composition comprises a population of natural killer (NK) cell subsets, wherein at least 40% or about 40%, at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of the cells in the composition are CD57 pos In some embodiments, 70% or about 70% to 90% or about 90% of the cells in the composition have the phenotype CD57 pos In some embodiments, at least or about 72%, at least 74% or about 74%, at least 76% or about 76%, at least 78% or about 78%, at least 80% or about 80%, at least 82% or about 82%, at least 84% or about 84%, at least 86% or about 86%, at least 88% or about 88%, at least 90% or about 90%, at least 92% or about 92%, at least 94% or about 94%, at least 96% or about 96%, or at least 98% or about 98% of the cells in the composition have the phenotype CD57 pos In some of any of the provided embodiments, at least 60% or about 60% of the cells in the composition have the phenotype CD57 pos In some of any of the provided embodiments, at least 70% or about 70% of the cells in the composition are of the phenotype CD57 pos In some embodiments, the phenotype comprises the surface phenotype CD3 neg In some embodiments, the phenotype further comprises the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, more than 50% of the cells having such a phenotype are FcRγ neg and optionally, 50% or about 50%-90% are FcRγneg In some of any of the provided embodiments, more than 70% of the cells with such a phenotype are FcRγ neg and optionally, 70% or about 70%-90% are FcRγ neg is.

[0105] In some embodiments, the composition comprises a population of natural killer (NK) cell subsets, wherein at least 40% or about 40%, at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of the cells in the composition are CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, 70% or about 70% to 90% or about 90% of the cells in the composition have a g-NK cell surrogate marker profile of the phenotype CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, at least or about 72%, at least 74%, or about 74%, at least 76%, or about 76%, at least 78%, or about 78%, at least 80%, or about 80%, at least 82%, or about 82%, at least 84%, or about 84%, at least 86%, or about 86%, at least 88%, or about 88%, at least 90%, or about 90%, at least 92%, or about 92%, at least 94%, or about 94%, at least 96%, or about 96%, or at least 98% or about 98% of the cells in the composition have the phenotype CD16 pos / CD57 pos / CD7 dim / neg / CD161 negIn some of any of the provided embodiments, at least 60% or about 60% of the cells in the composition have the phenotype CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some of any of the provided embodiments, at least 70% or about 70% of the cells in the composition are of the phenotype CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, the phenotype comprises the surface phenotype CD3 neg In some embodiments, the phenotype further comprises the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, more than 50% of the cells having such a phenotype are FcRγ neg and optionally, 50% or about 50%-90% are FcRγ neg In some of any of the provided embodiments, more than 70% of the cells with such a phenotype are FcRγ neg and optionally, 70% or about 70%-90% are FcRγ neg is.

[0106] In some embodiments, the composition comprises a population of natural killer (NK) cell subsets, wherein at least 40% or about 40%, at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of the cells in the composition are CD38 pos In some embodiments, 70%, or about 70% to 90%, or about 90% of the cells in the composition have a phenotype of CD38 negIn some embodiments, at least or about 72%, at least 74%, or about 74%, at least 76%, or about 76%, at least 78%, or about 78%, at least 80%, or about 80%, at least 82%, or about 82%, at least 84%, or about 84%, at least 86%, or about 86%, at least 88%, or about 88%, at least 90%, or about 90%, at least 92%, or about 92%, at least 94%, or about 94%, at least 96%, or about 96%, or at least 98% or about 98% of the cells in the composition have the phenotype CD38 neg In some of any of the provided embodiments, at least 60% or about 60% of the cells in the composition have the phenotype CD38 neg In some of any of the provided embodiments, at least 70% or about 70% of the cells in the composition are of the phenotype CD38 neg In some embodiments, the phenotype comprises the surface phenotype CD3 neg In some embodiments, the phenotype further comprises the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, more than 50% of the cells having such a phenotype are FcRγ neg and optionally, 50% or about 50%-90% are FcRγ neg In some of any of the provided embodiments, more than 70% of the cells with such a phenotype are FcRγ neg and optionally, 70% or about 70%-90% are FcRγ neg is.

[0107] In some embodiments, the composition comprises a population of natural killer (NK) cell subsets, wherein at least 40% or about 40%, at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of the cells in the composition are CD16 neg In some embodiments, 70%, or about 70% to 90%, or about 90% of the cells in the composition have a phenotype of CD16 pos In some embodiments, at least or about 72%, at least 74%, or about 74%, at least 76%, or about 76%, at least 78%, or about 78%, at least 80%, or about 80%, at least 82%, or about 82%, at least 84%, or about 84%, at least 86%, or about 86%, at least 88%, or about 88%, at least 90%, or about 90%, at least 92%, or about 92%, at least 94%, or about 94%, at least 96%, or about 96%, or at least 98% or about 98% of the cells in the composition have the phenotype CD16 pos In some of any of the provided embodiments, at least 60% or about 70% of the cells in the composition have the phenotype CD16 pos In some of any of the provided embodiments, at least 70% or about 70% of the cells in the composition are of the phenotype CD16 pos In some embodiments, the phenotype comprises the surface phenotype CD3 neg In some embodiments, the phenotype further comprises the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, more than 50% of the cells having such a phenotype are FcRγ neg and optionally, 50% or about 50%-90% are FcRγ negIn some of any of the provided embodiments, more than 70% of the cells with such a phenotype are FcRγ neg and optionally, 70% or about 70%-90% are FcRγ neg is.

[0108] In some embodiments, the composition comprises a population of natural killer (NK) cell subsets, wherein at least 40% or about 40%, at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% of the cells in the composition are NKG2A neg / CD161 neg In some embodiments, 70% or about 70% to 90% or about 90% of the cells in the composition have the phenotype NKG2A neg / CD161 neg In some embodiments, at least or about 72%, at least 74% or about 74%, at least 76% or about 76%, at least 78% or about 78%, at least 80% or about 80%, at least 82% or about 82%, at least 84% or about 84%, at least 86% or about 86%, at least 88% or about 88%, at least 90% or about 90%, at least 92% or about 92%, at least 94% or about 94%, at least 96% or about 96%, or at least 98% or about 98% of the cells in the composition have the phenotype NKG2A neg / CD161 neg In some of any of the provided embodiments, at least 60% or about 60% of the cells in the composition have the phenotype NKG2A neg / CD161 neg In some of any of the provided embodiments, at least 70% or about 70% of the cells in the composition are of the phenotype NKG2Aneg / CD161 neg In some embodiments, the phenotype comprises the surface phenotype CD3 neg In some embodiments, the phenotype further comprises the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, more than 50% of the cells having such a phenotype are FcRγ neg and optionally, 50% or about 50%-90% are FcRγ neg In some of any of the provided embodiments, more than 70% of the cells with such a phenotype are FcRγ neg and optionally, 70% or about 70%-90% are FcRγ neg is.

[0109] In some embodiments, the composition comprises more than or about more than 50% of the NK cells in the composition being g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 55% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 60% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 65% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 70% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 75% of the NK cells in the composition are g-NK cells (FcRγ negIn some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 80% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 85% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 90% of the NK cells in the composition are g-NK cells (FcRγ neg In some embodiments, the composition comprises a population of NK cells, wherein greater than or greater than about 95% of the NK cells in the composition are g-NK cells (FcRγ neg ) or a surrogate marker profile thereof. The surrogate marker profile can be any of those described herein. For example, the surrogate marker profile can be CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In other examples, the surrogate marker profile may be NKG2A neg / CD161 neg In a further example, the g-NK cell surrogate marker profile can be CD38 neg The surrogate surface marker profile is the phenotype CD45 pos / CD3 neg / CD56 pos It may further include:

[0110] 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 those skilled in the art. Methods include, for example, intracellular flow cytometry. In one example, the percentage or number of cells positive for perforin or granzyme B can be determined by permeabilizing the cells before staining with antibodies against perforin and granzyme B, e.g., using an Inside Stain Kit from Miltenyi Biotec. Cell staining can then be resolved, e.g., using flow cytometry.

[0111] In some embodiments, greater than or about 70% of the g-NK cells of the composition are positive for perforin, and greater than or about 70% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than or about 75% of the g-NK cells of the composition are positive for perforin, and greater than or about 75% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than or about 80% of the g-NK cells of the composition are positive for perforin, and greater than or about 80% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than or about 85% of the g-NK cells of the composition are positive for perforin, and greater than or about 85% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than or about 90% of the g-NK cells of the composition are positive for perforin and greater than or about 90% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than or about 95% of the g-NK cells of the composition are positive for perforin and greater than or about 95% of the g-NK cells of the composition are positive for granzyme B.

[0112] In some embodiments, the expression levels of perforin and granzyme B by NK cells, e.g., g-NK cells, can be measured by intracellular flow cytometry and the levels measured based on the level of mean fluorescence intensity (MFI). In some embodiments, the expression levels of perforin and granzyme B based on MFI can be measured by measuring the expression levels of g-NK cells and FcRγ pos In some embodiments, g-NK cells of the composition that are positive for perforin will differ between cells that are positive for FcRγ based on MFI levels. pos In some embodiments, g-NK cells of the composition that are positive for perforin express an average level of perforin that is at least two times or about two times the average level of perforin expressed by NK cells. ... pos In some embodiments, g-NK cells of the composition that are positive for perforin express an average level of perforin that is at least three times or about three times the average level of perforin expressed by NK cells. ... pos In some embodiments, the g-NK cells of the composition that are positive for granzyme B express an average level of perforin that is at least four times or about four times the average level of perforin expressed by NK cells. ... the average level of perforin expressed by NK cells pos In some embodiments, g-NK cells of the composition that are positive for granzyme B express an average level of granzyme B that is at least two times or about two times the average level of granzyme B expressed by NK cells. ... pos In some embodiments, g-NK cells of the composition that are positive for granzyme B express an average level of granzyme B that is at least three times or about three times the average level of granzyme B expressed by NK cells. ... pos They express an average level of granzyme B that is at least four times or about four times the average level of granzyme B expressed by NK cells.

[0113] In some embodiments, at least 50% or about 50% of the cells in the composition are FcRγ-deficient NK cells (g-NK), and greater than 70% or about 70% of the g-NK cells are positive for perforin and greater than 70% or about 70% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 80% or about 80% of the g-NK cells are positive for perforin and greater than 80% or about 80% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 90% or about 90% of the g-NK cells are positive for perforin and greater than 90% or about 90% of the g-NK cells are positive for granzyme B. In some embodiments, greater than 95% or about 95% of the g-NK cells are positive for perforin and greater than 95% or about 95% of the g-NK cells are positive for granzyme B. In some embodiments, the g-NK cells are FcRγ-deficient neg is.

[0114] In some of any of the embodiments, among the cells that are positive for perforin, the cells are selected based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry to identify FcRγ pos In some of the optional embodiments, among the cells that are positive for granzyme B, the cells express an average level of perforin that is at least two-fold or about two-fold the average level of perforin expressed by cells that are positive for FcRγ based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry. pos The cells express an average level of granzyme B that is at least two-fold or about two-fold the average level of granzyme B expressed by cells that are

[0115] In some of any of the embodiments, more than 10% of the cells in the composition are capable of degranulation against tumor target cells, optionally as measured by CD107a expression, and optionally the degranulation is measured in the absence of an antibody against the tumor target cells. In some of any of the embodiments, more than or about 15%, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% of the cells in the composition exhibit degranulation, optionally as measured by CD107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody). In some of any of such embodiments, more than 10% of the cells in the composition are capable of producing interferon-gamma or TNF-alpha against tumor target cells, optionally the interferon-gamma or TNF-alpha is measured in the absence of an antibody against the tumor target cells. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the cells in the composition produce effector cytokines in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody). In some embodiments, for example, the target cells can be a tumor cell line expressing CD38, and the antibody is an anti-CD38 antibody (e.g., daratumumab). In some embodiments, for example, the target cells can be a tumor cell line expressing SLAMF7, and the antibody is an anti-SLAMF7 antibody (e.g., elotuzumab). In some embodiments, for example, the target cells can be a tumor cell line expressing BCMA, and the antibody is an anti-BCMA antibody (e.g., belantamab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD20 and the antibody is an anti-CD20 antibody (e.g., rituximab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD19 and the antibody is an anti-CD19 antibody (e.g., tafasitamab or loncastuximab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD30 and the antibody is an anti-CD30 antibody (e.g., brentuximab).

[0116] In some embodiments, at least 50% or about 50% of the cells in the composition are FcRγ deficient (FcRγ negThe target cells are NK cells (g-NK), and more than or about 15% of the cells in the composition produce effector cytokines in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody). In some embodiments, more than or about 20%, more than or about 30%, more than or about 30%, more than or about 40%, or more than or about 50% produce effector cytokines in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody). In some embodiments, for example, the target cells can be a tumor cell line expressing CD38, and the antibody is an anti-CD38 antibody (e.g., daratumumab). In some embodiments, for example, the target cells can be a tumor cell line expressing SLAMF7, and the antibody is an anti-SLAMF7 antibody (e.g., elotuzumab). In some embodiments, for example, the target cells may be a tumor cell line that expresses BCMA and the antibody is an anti-BCMA antibody (e.g., belantamab). In some embodiments, for example, the target cells may be a tumor cell line that expresses CD20 and the antibody is an anti-CD20 antibody (e.g., rituximab). In some embodiments, for example, the target cells may be a tumor cell line that expresses CD19 and the antibody is an anti-CD19 antibody (e.g., tafasitamab or loncastuximab). In some embodiments, for example, the target cells may be a tumor cell line that expresses CD30 and the antibody is an anti-CD30 antibody (e.g., brentuximab).

[0117] In some of any of the embodiments, the effector cytokine is IFN-gamma or TNF-alpha.In some of any of the embodiments, the effector cytokine is IFN-gamma and TNF-alpha.

[0118] In some of the embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the cells in the composition 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). In some embodiments, for example, the target cells may be a tumor cell line expressing CD38, and the antibody is an anti-CD38 antibody (e.g., daratumumab). In some embodiments, for example, the target cells may be a tumor cell line expressing SLAMF7, and the antibody is an anti-SLAMF7 antibody (e.g., elotuzumab). In some embodiments, for example, the target cells may be a tumor cell line expressing BCMA, and the antibody is an anti-BCMA antibody (e.g., belantamab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD20 and the antibody is an anti-CD20 antibody (e.g., rituximab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD19 and the antibody is an anti-CD19 antibody (e.g., tafasitamab or loncastuximab). In some embodiments, for example, the target cells may be a tumor cell line expressing CD30 and the antibody is an anti-CD30 antibody (e.g., brentuximab).

[0119] In some embodiments, at least 50% or about 50% of the cells in the composition are FcRγ deficient (FcRγ neg) NK cells (g-NK), wherein greater than or about 15% of the cells in the composition exhibit degranulation when in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody), optionally as measured by CD107a expression. In some embodiments, greater than or about 20%, greater than or about 30%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit degranulation when in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody), optionally as measured by CD107a expression. In some embodiments, for example, the target cells can be a tumor cell line expressing CD38, and the antibody is an anti-CD38 antibody (e.g., daratumumab). In some embodiments, for example, the target cells can be a tumor cell line expressing SLAMF7, and the antibody is an anti-SLAMF7 antibody (e.g., elotuzumab). In some embodiments, for example, the target cells can be a tumor cell line expressing BCMA, and the antibody is an anti-BCMA antibody (e.g., belantamab). In some embodiments, for example, the target cells can be a tumor cell line expressing CD20, and the antibody is an anti-CD20 antibody (e.g., rituximab). In some embodiments, for example, the target cells can be a tumor cell line expressing CD19, and the antibody is an anti-CD19 antibody (e.g., tafasitamab or loncastuximab). In some embodiments, for example, the target cells can be a tumor cell line expressing CD30, and the antibody is an anti-CD30 antibody (e.g., brentuximab).

[0120] In some of any of the provided embodiments, greater than 60% or greater than about 60% of the cells in the composition are g-NK cells. In some of any of the provided embodiments, greater than 70% or greater than about 70% of the cells in the composition are g-NK cells. In some of any of the provided embodiments, greater than 80% or greater than about 80% of the cells in the composition are g-NK cells. In some of any of the provided embodiments, greater than 90% or greater than about 90% of the cells in the composition are g-NK cells. In some of any of the provided embodiments, greater than 95% or greater than about 95% of the cells in the composition are g-NK cells.

[0121] In some embodiments, the g-NK cells exhibit a g-NK cell surrogate marker profile. In some embodiments, the g-NK cell surrogate marker profile is CD16pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, the g-NK cell surrogate marker profile is NKG2A neg / CD161 neg In some embodiments, the g-NK cell surrogate marker profile is CD38 neg In some embodiments, the g-NK cell surrogate surface marker profile further comprises CD45 pos / CD3 neg / CD56 pos is.

[0122] In some of any of the above embodiments, greater than 60%, or greater than about 60%, of the cells are g-NK cells. In some of any of the above embodiments, greater than 70%, or greater than about 70% of the cells are g-NK cells. In some of any of the above embodiments, greater than 80%, or greater than about 80% of the cells are g-NK cells. In some of any of the above embodiments, greater than 90%, or greater than about 90% of the cells are g-NK cells. In some of any of the above embodiments, greater than 95%, or greater than about 95% of the cells are g-NK cells.

[0123] In some of any of the above embodiments, greater than or about greater than 80% of the cells are positive for perforin. In some of any of the above embodiments, greater than or about greater than 90% of the cells are positive for perforin. In some of the above embodiments, of the cells that are positive for perforin, the cells are positive for FcRγ based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry. pos The cells express an average level of perforin that is at least two-fold or about two-fold the average level of perforin expressed by cells that are

[0124] In some of any of the above embodiments, greater than or greater than about 80% of the cells are positive for Granzyme B. In some of the above embodiments, greater than or greater than about 90% of the cells are positive for Granzyme B. In some of any of the embodiments, of the cells that are positive for Granzyme B, the cells are more positive for FcRγ based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry. pos The cells express an average level of granzyme B that is at least two-fold or about two-fold the average level of granzyme B expressed by cells that are

[0125] In some of the provided embodiments, the composition comprises 10 6 Pieces or about 10 6 cells ~ 10 12 Pieces or about 10 12 In some of the embodiments provided, the composition comprises 10 6 Pieces or about 10 6 ~10 pieces 11 Pieces or about 10 11 cells, 10 6 Pieces or about 10 6 ~10 pieces 10 Pieces or about 10 10 cells, 10 6 Pieces or about 10 6 ~10 pieces 9 Pieces or about 10 9 cells, 10 6 Pieces or about 10 6 ~10 pieces 8 Pieces or about 10 8 cells, 10 6 Pieces or about 10 6 ~10 pieces 7 Pieces or about 10 7 cells, 10 7 Pieces or about 10 7 ~10 pieces 12 Pieces or about 10 12 cells, 10 7 Pieces or about 10 7 ~10 pieces 11Pieces or about 10 11 cells, 10 7 Pieces or about 10 7 ~10 pieces 10 Pieces or about 10 10 cells, 10 7 Pieces or about 10 7 ~10 pieces 9 Pieces or about 10 9 cells, 10 7 Pieces or about 10 7 ~10 pieces 8 Pieces or about 10 8 cells, 10 8 Pieces or about 10 8 ~10 pieces 12 Pieces or about 10 12 cells, 10 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 cells, 10 8 Pieces or about 10 8 ~10 pieces 10 Pieces or about 10 10 cells, 10 8 Pieces or about 10 8 ~10 pieces 9 Pieces or about 10 9 cells, 10 9 Pieces or about 10 9 ~10 pieces 12 Pieces or about 10 12 cells, 10 9 Pieces or about 10 9 ~10 pieces 11 Pieces or about 10 11 cells, 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 cells, 10 10 Pieces or about 10 10 ~10 pieces 12 Pieces or about 10 12 cells, 10 10 Pieces or about 10 10 ~10 pieces 11 Pieces or about 10 11 cells, or 10 11 Pieces or about 1011 ~10 pieces 12 Pieces or about 10 12 Contains cells.

[0126] In some of any of the provided embodiments, the composition comprises at least or approximately at least 10 6 In some of the embodiments provided, the composition comprises 10 6 Pieces or about 10 6 ~10 pieces 10 Pieces or about 10 10 cells, 10 6 Pieces or about 10 6 ~10 pieces 9 Pieces or about 10 9 cells, 10 6 Pieces or about 10 6 ~10 pieces 8 Pieces or about 10 8 cells, 10 6 Pieces or about 10 6 ~10 pieces 7 Pieces or about 10 7 cells, 10 7 Pieces or about 10 7 ~10 pieces 10 Pieces or about 10 10 cells, 10 7 Pieces or about 10 7 ~10 pieces 9 Pieces or about 10 9 cells, 10 7 Pieces or about 10 7 ~10 pieces 8 Pieces or about 10 8 cells, 10 8 Pieces or about 10 8 ~10 pieces 10 Pieces or about 10 10 cells, 10 8 Pieces or about 10 8 ~10 pieces 9 Pieces or about 10 9 cells, 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 Contains cells.

[0127] In some of any of the provided embodiments, the composition comprises at least or approximately at least 10 8 In some of the embodiments provided, the composition comprises at least 10 9 Pieces or about 10 9 In some of the embodiments provided, the composition comprises at least 10 10 Pieces or about 10 10 In some of the embodiments provided, the composition comprises at least 10 11 Pieces or about 10 11 In some of the provided embodiments, the composition comprises 10 cells. 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 In some of the provided embodiments, the composition comprises 10 cells. 8 Pieces or about 10 8 ~10 pieces 10 Pieces or about 10 10 In some of the provided embodiments, the composition comprises 10 cells. 8 Pieces or about 10 8 ~10 pieces 9 Pieces or about 10 9 In some of the provided embodiments, the composition comprises 10 cells. 9 Pieces or about 10 9 ~10 pieces 11 Pieces or about 10 11 In some of the provided embodiments, the composition comprises 10 cells. 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 In some of the provided embodiments, the composition comprises 10 cells. 10 Pieces or about 10 10 ~10 pieces 11 Pieces or about 10 11 Contains cells.

[0128] In some of the provided embodiments, the composition comprises at least 10 6 Pieces or about 10 6 In some of the provided embodiments, the composition comprises 10 g-NK cells. 6 Pieces or about 10 6 ~10 pieces 10 Pieces or about 10 10 g-NK cells, 10 6 Pieces or about 10 6 ~10 pieces 9 Pieces or about 10 9 g-NK cells, 10 6 Pieces or about 10 6 ~10 pieces 8 Pieces or about 10 8 g-NK cells, 10 6 Pieces or about 10 6 ~10 pieces 7 Pieces or about 10 7 g-NK cells, 10 7 Pieces or about 10 7 ~10 pieces 10 Pieces or about 10 10 g-NK cells, 10 7 Pieces or about 10 7 ~10 pieces 9 Pieces or about 10 9 g-NK cells, 10 7 Pieces or about 10 7 ~10 pieces 8 Pieces or about 10 8 g-NK cells, 10 8 Pieces or about 10 8 ~10 pieces 10 Pieces or about 10 10 g-NK cells, 10 8 Pieces or about 10 8 ~10 pieces 9 Pieces or about 10 9 g-NK cells, 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 In some of the provided embodiments, the g-NK cells are FcRγ negIn some 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 of the provided embodiments, the g-NK cells or cells having a g-NK surrogate marker profile have the surface phenotype CD45 pos / CD3 neg / CD56 pos In some of any of the provided embodiments, the g-NK cells or cells having a g-NK surrogate marker profile have the surface phenotype CD38 neg Further includes:

[0129] In certain embodiments of any of the provided compositions, the cells in the composition are derived from the same donor. Thus, the composition does not contain a mixed population of cells from one or more different donors. As provided herein, the expansion methods result in high-yield expansion of 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, or 1000-fold or more of a specific NK cell subset, particularly a g-NK cell subset, or an NK cell subset associated with or including a surrogate marker for g-NK cells, such as any of the NK cell subsets described above. In some of any of the embodiments, the increase is greater than or about 1000-fold. In some of any of the embodiments, the increase is greater than or about 2000-fold. In some of any of the embodiments, the increase is greater than or about 2500-fold. In some of any of the embodiments, the increase is greater than or about 3000-fold. In some of the embodiments, the increase is greater than or about 5,000-fold. In some of the embodiments, the increase is greater than or about 10,000-fold. In some of the embodiments, the increase is greater than or about 10,000-fold. In some of the embodiments, the increase is greater than or about 15,000-fold. In some of the embodiments, the increase is greater than or about 20,000-fold. In some of the embodiments, the increase is greater than or about 25,000-fold. In some of the embodiments, the increase is greater than or about 30,000-fold. In some of the embodiments, the increase is greater than or about 35,000-fold. In certain embodiments, expansion results in a 1,000-fold or about a 1,000-fold increase in the number of a particular NK cell subset, particularly the g-NK cell subset, or an NK cell subset associated with or comprising a surrogate marker for g-NK cells, such as any of the NK cell subsets described above.In certain embodiments, the expansion results in a 3,000-fold or about a 3,000-fold increase in the number of NK cell subsets, particularly the g-NK cell subset, or NK cell subsets associated with or comprising surrogate markers for g-NK cells, such as any of the NK cell subsets described above. In certain embodiments, the expansion results in a 35,000-fold or about a 35,000-fold increase in the number of NK cell subsets, particularly the g-NK cell subset, or NK cell subsets associated with or comprising surrogate markers for g-NK cells, such as any of the NK cell subsets described above.

[0130] 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 cells to provide multiple individual doses for administration to a subject in need. Thus, the provided methods are particularly suitable for allogeneic methods. In some cases, a single expansion from a starting population of NK cells isolated from a single donor by the provided methods can result in more than about 20 individual doses, e.g., 30 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 any value between the above, for administration to a subject in need. In some embodiments, an individual dose is greater than or equal to 1×10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, e.g., 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 105 cells / kg or approximately 1 x 10 5 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 5 x 10 5 cells / kg or approximately 5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 5 x 10 5 cells / kg or approximately 5 x 10 5 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 6 cells / kg or approximately 1 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6cells / kg, or 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg ~ 1 x 10 7 cells / kg or approximately 1 x 10 7 In some embodiments, the individual dose is 1 x 10 cells / kg. 5 cells / kg or approximately 1 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, e.g., 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 107 cells / kg or approximately 1 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 7 cells / kg or approximately 2.5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 7 cells / kg or approximately 5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 7 cells / kg or approximately 7.5 x 10 7 cells / kg ~ 1 x 10 8 cells / kg or approximately 1 x 10 8 In some embodiments, the individual dose is 5 x 10 cells / kg. 7 Or about 5 x 10 7 ~10×10 9 Or about 10 x 10 9 , e.g., 5 x 10 7 Or about 5 x 10 7 ~5×10 9 Or about 5 x 10 9 , about or about 5 x 10 7 ~1×10 9 or about 1 x 10 9 , 5×10 7 Or about 5 x 10 7 ~5×10 8 Or about 5 x 10 8 , about or about 5 x 10 7 ~1×10 8 or about 1 x 10 8 , 1×10 8 ~10×10 9 Or about 10 x 10 9 , 1×10 8 or about 1 x 10 8 ~5×10 9 Or about 5 x 10 9 , about or about 1 x 10 8 ~1×10 9or about 1 x 10 9 , 1×10 8 or about 1 x 10 8 ~5×10 8 Or about 5 x 10 8 , 5×10 8 Or about 5 x 10 8 ~10×10 9 Or about 10 x 10 9 , 5×10 8 Or about 5 x 10 8 ~5×10 9 Or about 5 x 10 9 , about or about 5 x 10 8 ~1×10 9 or about 1 x 10 9 , 1×10 9 or about 1 x 10 9 ~10×10 9 Or about 10 x 10 9 , 1×10 9 or about 1 x 10 9 ~5×10 9 Or about 5 x 10 9 , or 5 x 10 9 Or about 5 x 10 9 ~10×10 9 Or about 10 x 10 9 In some embodiments, the individual dose is 5 x 10 8 or approximately 5 x 10 8 In some embodiments, an individual dose is 1 x 10 cells. 9 or approximately 1 x 10 9 In some embodiments, the individual doses are 5 x 10 cells. 9 or approximately 5 x 10 9 In some embodiments, an individual dose is 1 x 10 cells. 10 or approximately 1 x 10 10 In any of the above embodiments, the dose is given as a number of g-NK cells, or an NK cell subset associated with or including 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 above. It is given as the number of cells in the composition of expanded cells produced, or the number of viable cells in any of the above.

[0131] Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy, hi some embodiments, the engineered cells are formulated with a pharmaceutically acceptable carrier.

[0132] Pharmaceutically acceptable carriers may include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., 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 solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. Supplementary active compounds may also be incorporated into the composition. Pharmaceutical carriers should be suitable for NK cells, such as saline, dextrose solution, or solutions containing human serum albumin.

[0133] In some embodiments, a 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 sufficient time to allow for administration of viable NK cells. For example, the pharmaceutically acceptable carrier or vehicle can be a saline solution or a buffered saline solution. The pharmaceutically acceptable carrier or vehicle can also include various biomaterials that can increase the efficiency of the NK cells. Cell vehicles and carriers include, for example, methylcellulose (MCTate, DA Shear, SW Offman, DG Stein, MC LaPlaca, Biomaterials 22, 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (Suh JKF, Matthew HW T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000, which is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P(NIPAM-co-AA) (YH 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, YH Bae, Tissue Eng. 7, 35, 2001, each of which is incorporated herein by reference in its entirety), and poly(oxyethylene) / poly(D,L-lactic-co-glycolic acid) (B. Jeong, KM Lee, A. Gutowska, YH An, Biomacromolecules 3, 865, 2002, each of which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs LJ, Mikos A G. Cell Trans, 8, 345, 1999, each of which is incorporated herein by reference in its entirety), PEO / PEG (Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West J L., Biomaterials, 22, 3045, 2001, Bryant SJ, 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, each of which is incorporated herein by reference in its entirety), collagen (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001, each of which is incorporated herein by reference in its entirety), and polysaccharides such as alginate (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, 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).

[0134] In some embodiments, NKG2C pos The NK cells, such as cells or a subset thereof, may be present in the composition in an effective amount. neg The present invention relates to a method for treating cancer, therapies, and methods for treating cancer, and therapies for ...

[0135] 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, duration, and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. In some embodiments, a therapeutically effective amount is a dose of cells that results in a reduction in cancer growth or spread by at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% in a patient or animal administered a composition described herein compared to the growth or spread of cancer in a patient (or animal) or group of patients (or animals) that are not administered the composition. In some embodiments, a therapeutically effective amount is an amount that results in cytotoxic activity that results in activity that inhibits or reduces the growth of cancer, viral, and microbial cells.

[0136] In some embodiments, the composition comprises 10 5 Pieces or about 10 5 ~10 pieces 12 pcs or 10 12 NKG2C pos cells or a subset thereof, or 10 5 Pieces or about 10 5 ~10 pieces 8 pcs or 10 8 NKG2C pos cells or a subset thereof, or 10 6 Pieces or about 10 6 ~10 pieces 12 pcs or 10 12 NKG2C pos cells or a subset thereof, or 10 8 Pieces or about 10 8 ~10 pieces 11 pcs or 10 11 NKG2C pos cells or a subset thereof, or 10 9 Pieces or about 10 9 ~10 pieces 10 pcs or 1010 NKG2C pos A quantity of NKG2C cells or a subset thereof pos In some embodiments, the composition comprises 10 5 More than or about 10 5 Over 100 NKG2C pos cells or subsets thereof, 10 6 Pieces or about 10 6 NKG2C pos cells or subsets thereof, 10 7 Pieces or about 10 7 NKG2C pos cells or subsets thereof, 10 8 Pieces or about 10 8 NKG2C pos cells or subsets thereof, 10 9 Pieces or about 10 9 NKG2C pos cells or subsets thereof, 10 10 Pieces or about 10 10 NKG2C pos cells or subsets thereof, 10 11 Pieces or about 10 11 NKG2C pos cells or a subset thereof, or 10 12 Pieces or about 10 12 NKG2C pos In some embodiments, such amounts may be administered to a subject having a disease or condition, such as a cancer patient.

[0137] In some embodiments, the composition comprises 10 5 Pieces or about 10 5 ~10 pieces 12 Pieces or about 10 12 g-NK cells, or 10 5 Pieces or about 10 5 ~10 pieces 8 Pieces or about 10 8 g-NK cells, or 10 6 Pieces or about 10 6 ~10 pieces 12Pieces or about 10 12 g-NK cells, or 10 8 Pieces or about 10 8 ~10 pieces 11 Pieces or about 10 11 g-NK cells, or 10 9 Pieces or about 10 9 ~10 pieces 10 Pieces or about 10 10 In some embodiments, the composition comprises an amount of g-NK cells that is 10 g-NK cells. 5 More than or about 10 5 More than 10 g-NK cells 6 More than or about 10 6 More than 10 g-NK cells 7 More than or about 10 7 More than 10 g-NK cells 8 More than or about 10 8 More than 10 g-NK cells 9 More than or about 10 9 More than 10 g-NK cells 10 More than or about 10 10 More than 10 g-NK cells 11 More than or about 10 11 More than 10 g-NK cells or 10 12 More than or about 10 12 In some embodiments, such amounts may be administered to a subject with a disease or condition, such as a cancer patient.

[0138] 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, e.g., 10 mL or about 10 mL to 500 mL, 10 mL or about 10 mL to 200 mL, 10 mL or about 10 mL to 100 mL, 10 mL or about 10 mL to 50 mL, 50 mL or about 50 mL to 500 mL, 50 mL or about 50 mL to 200 mL, 50 mL or about 50 mL to 100 mL, 100 mL or about 100 mL to 500 mL, 100 mL or about 100 mL to 200 mL, or 200 mL or about 200 mL to 500 mL (inclusive). In some embodiments, the volume of the composition is at least or at least about 1 x 10 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 cells / mL, or 1 x 10 8 In some embodiments, the composition has a cell density of 1 x 10 cells / mL. 5 cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 5 cells / mL ~ approx. 1 x 10 8 cells / mL, 1 x 10 5 cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 5 cells / mL ~ approx. 1 x 10 7 cells / mL, 1 x 10 5 cells / mL ~ 1 x 10 6 cells / mL or approximately 1 x 10 5 cells / mL ~ approx. 1 x 10 6 cells / mL, 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 6 cells / mL ~ approx. 1 x 10 7 cells / mL, 1 x 10 6 cells / mL ~ 1 x 10 8cells / mL or approximately 1 x 10 6 cells / mL ~ approx. 1 x 10 8 cells / mL, 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 6 cells / mL ~ approx. 1 x 10 7 cells / mL, or 1 x 10 7 cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 7 cells / mL ~ approx. 1 x 10 8 cells / mL (both inclusive).

[0139] In some embodiments, compositions, including pharmaceutical compositions, are sterile. In some embodiments, cell isolation, enrichment, or culture is performed in a closed or sterile environment, e.g., a sterile culture bag, to minimize error, user handling, and / or contamination. In some embodiments, sterility can be easily achieved, e.g., by filtration through a sterile filtration membrane. In some embodiments, culture is performed using a gas-permeable culture vessel. In some embodiments, culture is performed using a bioreactor.

[0140] Also provided herein are compositions suitable for cryopreserving the provided NK cells. In some embodiments, the NK cells are cryopreserved in a serum-free cryopreservation medium. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or glycerol. In some embodiments, the cryopreservation medium is 5% or about 5% to 10% or about 10% DMSO (v / v). In some embodiments, the cryopreservation medium is 5% or about 5% DMSO (v / v). In some embodiments, the cryopreservation medium is 6% or about 6% DMSO (v / v). In some embodiments, the cryopreservation medium is 7% or about 7% DMSO (v / v). In some embodiments, the cryopreservation medium is 8% or about 8% DMSO (v / v). In some embodiments, the cryopreservation medium is 9% or about 9% DMSO (v / v). In some embodiments, the cryopreservation medium is 10% 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 cryopreservation can be stored at low temperatures, such as ultra-low temperatures, for example, at temperatures ranging from -40°C to -150°C, such as at or about 80°C ± 6.0°C.

[0141] In some embodiments, the compositions can be stored at ultra-low temperatures before administration to a patient. In some aspects, NK cell subsets, such as g-NK cells, can be isolated, processed, and expanded, such as by the methods provided, and then stored at ultra-low temperatures before administration to a subject.

[0142] Typical methods for small-scale cryogenic storage are described, for example, in U.S. Patent No. 6,0168,991. For small-scale storage, cells can be cryogenically stored in a low-density suspension (e.g., at a concentration of about 200 x 10 / ml) in pre-chilled 5% human albumin serum (HAS). An equal volume of 20% DMSO can be added to the HAS solution. Aliquots of the mixture can be placed in vials and frozen overnight in a cryogenic chamber at about -80°C.

[0143] In some embodiments, 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 embodiments, the composition can be used immediately without any further processing. In other cases, the NK cells are further processed after thawing, such as by resuspension in a pharmaceutically acceptable carrier, incubation with an activating or stimulating agent, or activated, washed, and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

[0144] B. Combination Therapy In some embodiments, the compositions containing g-NK cells provided herein can be administered in combination therapy with one or more other agents for treating a disease or condition in a subject. In such embodiments, the compositions containing g-NK cells provided herein can be administered prior to, simultaneously with, or subsequently to (after) the administration of one or more other agents. For example, g-NK cells can be administered simultaneously with or sequentially with antimicrobial agents, antiviral agents, and other therapeutic agents. Exemplary combination therapies are described in the following subsections.

[0145] In some embodiments, compositions containing g-NK cells provided herein exhibit enhanced activity when activated by or contacted with an antibody or an Fc-containing protein, e.g., compared to conventional NK cells. For example, g-NK cells can be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells.

[0146] In some embodiments, provided herein are methods for treating a condition in an individual, comprising administering g-NK cells or compositions thereof and an antibody to the subject. One skilled in the art can select an appropriate therapeutic (e.g., anti-cancer) monoclonal antibody for administration to a subject along with the provided g-NK cells and compositions described herein, depending, for example, on the individual's particular disease or condition. Suitable antibodies may include polyclonal antibodies, monoclonal antibodies, fragments (e.g., Fab fragments), single-chain antibodies, and other forms of specific binding molecules.

[0147] In some embodiments, antibodies may further comprise humanized or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains, or fragments (such as Fv, Fab, Fab', F(ab')2, or antigen-binding subsequences of other antibodies) that contain minimal sequence derived from non-human Ig. In some embodiments, antibodies comprise an Fc domain.

[0148] Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization is achieved by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). Such "humanized" antibodies are chimeric antibodies (1989) in which substantially less than an entire human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some Fc residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies include human antibodies (recipient antibodies) in which residues from the recipient's complementary determining region (CDR) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, corresponding non-human residues replace Fv framework residues of the human antibody. Humanized antibodies may also comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with most, if not all, of the CDR regions corresponding to those of a non-human Ig and most, if not all, of the FR regions being of human antibody consensus sequences. Humanized antibodies also optimally comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0149] Human antibodies can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al., 1991; Marks et al., 1991) and preparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell, 1985). Similarly, introducing human Ig genes into transgenic animals in which the endogenous antibody genes have been partially or completely inactivated can be used to synthesize human Abs. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire (1997a, 1997b, 1997c, 1997d, 1997, 1997; Fishwild et al., 1996, 1997, 1997, 2001, 1996, 1997, 1997, 1997; Lonberg and Huszar, 1995; Lonberg et al., 1994; Marks et al., 1992, 1997, 1997, 1997).

[0150] 1. Multiple myeloma a. Anti-CD38 antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, CD38. In some embodiments, the method further comprises administering an anti-CD38 antibody to the subject. In some embodiments, the method is for treating multiple myeloma. In some embodiments, the antibody is daratumumab (e.g., Darzalex™).

[0151] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. In some embodiments, the additional agent may be administered after administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0152] In some embodiments, at least one dose of an anti-CD38 antibody has been administered to the subject prior to administration of a dose of the g-NK cell composition. Also disclosed herein in one aspect is a method of treating multiple myeloma, comprising administering to a subject having multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells may be administered once weekly in a predetermined number of doses, and wherein the subject has been previously administered at least one dose of an anti-CD38 antibody.

[0153] In some embodiments, the anti-CD38 antibody is daratumumab. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-CD38 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0154] In certain instances, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One of ordinary skill in the art can determine the appropriate route of administration.

[0155] In some embodiments, the anti-CD38 antibody may be administered as a weekly dose. In some embodiments, the anti-CD38 antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered for one or two 28-day cycles. In some embodiments, the antibody is administered once weekly for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once weekly for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, eight doses of the antibody are administered weekly. In some embodiments, the weekly doses are administered on consecutive weeks.

[0156] In some embodiments, the anti-CD38 antibody may be administered intravenously.

[0157] In some embodiments, each dose of anti-CD38 antibody (e.g., daratumumab) can be administered in an amount that can be at or about 8 mg / kg to about 32 mg / kg, hi some embodiments, each dose is at or about 16 mg / kg.

[0158] In some embodiments, the anti-CD38 antibody may be administered subcutaneously. In some embodiments, the anti-CD38 antibody (e.g., daratumumab) may be administered in an anti-CD38 antibody composition comprising hyaluronidase. For example, the antibody may be administered as an anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (e.g., hyaluronidase-fihj). Examples of such compositions are described in U.S. Patent Application Publication No. 2017 / 0121414. In some embodiments, each dose of the anti-CD38 antibody composition comprises 1200 mg, or about 1200 mg to about 2400 mg, of the anti-CD38 antibody (e.g., daratumumab) and 15,000 units (U), or about 15,000 U to about 45,000 U of hyaluronidase (e.g., hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of an anti-CD38 antibody (e.g., daratumumab) and about 30,000 U of hyaluronidase (e.g., hyaluronidase-fihj).

[0159] In some embodiments, the method comprises administering an anti-CD38 antibody once per week for a total of eight doses and administering a g-NK cell composition once per week for a total of six doses, wherein one or two doses of the anti-CD38 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0160] In some embodiments, the multiple myeloma can be relapsed / refractory multiple myeloma.

[0161] In some embodiments, the g-NK cells have low or no expression of CD38, e.g., less than 25% of the cells in the g-NK cell composition are positive for surface CD38. In some embodiments, the cells in the g-NK cell composition have not been engineered to reduce or eliminate CD38 expression. In some embodiments, the g-NK cell composition exhibits minimal anti-CD38-induced fratricide, optionally less than 10% of the cells in the g-NK cell composition exhibit anti-CD38-induced fratricide.

[0162] b. Anti-SLAMF7 antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, SLAMF7. In some embodiments, the method further comprises administering an anti-SLAMF7 antibody to the subject. In some embodiments, the method is for treating multiple myeloma. In some embodiments, the antibody is elotuzumab (e.g., EMPLICITI®).

[0163] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0164] In some embodiments, at least one dose of an anti-SLAMF7 antibody has been administered to the subject prior to administration of a dose of a g-NK cell composition. Also disclosed herein in one aspect is a method of treating multiple myeloma, comprising administering to a subject having multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells may be administered weekly in a predetermined number of doses, and wherein the subject has been previously administered at least one dose of an anti-SLAMF7 antibody.

[0165] In some embodiments, the anti-SLAMF7 antibody may be elotuzumab. In some embodiments, administration of at least one dose of the anti-SLAMF7 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-SLAMF7 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-SLAMF7 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0166] In certain instances, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One of ordinary skill in the art can determine the appropriate route of administration.

[0167] In some embodiments, the anti-SLAMF7 antibody may be administered as a weekly dose. In some embodiments, the anti-SLAMF7 antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered over one or two 28-day cycles. In some embodiments, the antibody is administered once weekly for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once weekly for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, or more weeks. In some embodiments, eight doses of the antibody are administered weekly. In some embodiments, the weekly doses are administered on consecutive weeks.

[0168] In some embodiments, the anti-SLAMF7 antibody may be administered intravenously. In some embodiments, the anti-SLAMF7 antibody may be administered subcutaneously.

[0169] In some embodiments, each dose of anti-SLAMF7 antibody (e.g., elotuzumab) may be administered weekly for two cycles, then every two weeks, in an amount that may be at or about 10 mg / kg. In some embodiments, the anti-SLAMF7 antibody is administered with lenalidomide and dexamethasone. In some embodiments, the anti-SLAMF7 antibody is administered after dexamethasone, diphenhydramine, ranitidine, and acetaminophen.

[0170] In some embodiments, the method comprises administering an anti-SLAMF7 antibody once weekly for a total of eight doses and administering a g-NK cell composition once weekly for a total of six doses, wherein one or two doses of the anti-SLAMF7 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0171] In some embodiments, the multiple myeloma can be relapsed / refractory multiple myeloma.

[0172] In some embodiments, the g-NK cells have low or no expression of SLAMF7, e.g., less than 25% of the cells in the g-NK cell composition are positive for surface SLAMF7. In some embodiments, the cells in the g-NK cell composition have not been engineered to reduce or eliminate SLAMF7 expression. In some embodiments, the g-NK cell composition exhibits minimal anti-SLAMF7-induced fratricide, optionally less than 10% of the cells in the g-NK cell composition exhibit anti-SLAMF7-induced fratricide.

[0173] c. Anti-BCMA antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, BCMA. In some embodiments, the method further comprises administering an anti-BCMA antibody to the subject. In some embodiments, the method is for treating multiple myeloma. In some embodiments, the antibody is belantamab (e.g., Blenrep).

[0174] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0175] In some embodiments, at least one dose of an anti-BCMA antibody has been administered to the subject prior to administration of a dose of the g-NK cell composition. Also disclosed herein in one aspect is a method of treating multiple myeloma, comprising administering to a subject having multiple myeloma (MM) a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells may be administered once weekly in a predetermined number of doses, and wherein the subject has been previously administered at least one dose of an anti-BCMA antibody.

[0176] In some embodiments, the anti-BCMA antibody may be belantamab. In some embodiments, administration of at least one dose of the anti-BCMA antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-BCMA antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the anti-BCMA antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0177] In certain instances, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One of ordinary skill in the art can determine the appropriate route of administration.

[0178] In some embodiments, the anti-BCMA antibody may be administered as a weekly dose. In some embodiments, the anti-BCMA antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered over one or two 28-day cycles. In some embodiments, the antibody is administered once weekly for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once weekly for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, eight doses of the antibody are administered weekly. In some embodiments, the weekly doses are administered on consecutive weeks.

[0179] In some embodiments, the anti-BCMA antibody may be administered intravenously. In some embodiments, the anti-BCMA antibody may be administered subcutaneously. In some embodiments, the anti-BCMA antibody (e.g., Blenrep) may be administered at or about 2.5 mg / kg as an intravenous infusion over 30 minutes or about 30 minutes. In some embodiments, the anti-BCMA antibody (e.g., Blenrep) is administered once every three weeks.

[0180] In some embodiments, the method comprises administering an anti-BCMA antibody once weekly for a total of eight doses and administering a g-NK cell composition once weekly for a total of six doses, wherein one or two doses of the anti-BCMA antibody may be administered prior to administration of the composition comprising g-NK cells.

[0181] In some embodiments, the multiple myeloma can be relapsed / refractory multiple myeloma.

[0182] In some embodiments, the g-NK cells have low or no expression of BCMA, e.g., less than 25% of the cells in the g-NK cell composition are positive for surface BCMA. In some embodiments, the cells in the g-NK cell composition have not been engineered to reduce or eliminate BCMA expression. In some embodiments, the g-NK cell composition exhibits minimal anti-BCMA-induced fratricide, optionally less than 10% of the cells in the g-NK cell composition exhibit anti-BCMA-induced fratricide.

[0183] 2. Lymphoma a. Anti-CD20 antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, which is CD20. In some embodiments, the method further comprises administering an anti-CD20 antibody to the subject. In some embodiments, the method is for treating lymphoma, such as non-Hodgkin's lymphoma. In some embodiments, the antibody is rituximab (e.g., Rituxan®).

[0184] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. In some embodiments, the additional agent may be administered after administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0185] In some embodiments, at least one dose of an anti-CD20 antibody has been administered to the subject prior to administration of a dose of the g-NK cell composition. Also disclosed herein in one aspect is a method of treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells may be administered once weekly in a predetermined number of doses, and wherein the subject has been previously administered at least one dose of an anti-CD20 antibody.

[0186] In some embodiments, the anti-CD20 antibody can be rituximab.

[0187] In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD20 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0188] In certain instances, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One of ordinary skill in the art can determine the appropriate route of administration.

[0189] In some embodiments, the anti-CD20 antibody may be administered as a weekly dose. In some embodiments, the anti-CD20 antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered over one or two 28-day cycles. In some embodiments, the antibody is administered once weekly for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once weekly for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, eight doses of the antibody are administered weekly. In some embodiments, the weekly doses are administered on consecutive weeks.

[0190] In some embodiments, the anti-CD20 antibody may be administered intravenously.

[0191] In some embodiments, each dose of anti-CD20 antibody is 250 mg / m or about 250 mg / m to about 500 mg / m 2 In some embodiments, each dose may be 375 mg / m 2 or approximately 375 mg / m 2 It is administered at .

[0192] In some embodiments, the anti-CD20 antibody may be administered subcutaneously. In some embodiments, the anti-CD20 antibody (e.g., rituximab) may be administered in an anti-CD20 antibody composition comprising hyaluronidase. For example, the antibody may be administered as an anti-CD20 antibody composition comprising rituximab and recombinant human hyaluronidase PH20. Illustrative examples of such compositions are described in published PCT publication WO 2011 / 029892.

[0193] In some embodiments, each dose of the anti-CD20 antibody composition comprises 1200 mg, or about 1200 mg to about 2400 mg, of an anti-CD20 antibody (e.g., rituximab) and 15,000 units (U), or about 15,000 U to about 45,000 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1400 mg of an anti-CD20 antibody (e.g., rituximab) and about 23,400 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1600 mg of an anti-CD20 antibody (e.g., rituximab) and about 26,800 U of hyaluronidase.

[0194] In some embodiments, the anti-CD20 antibody composition may be administered as a weekly dose. In some embodiments, the anti-CD20 antibody is administered as four or eight doses. In some embodiments, the antibody is administered subcutaneously in three or seven doses, followed by weekly intravenous administration of the anti-CD20 antibody. In some embodiments, the method comprises administering the anti-CD20 antibody once weekly for a total of eight doses and administering the g-NK cell composition once weekly for a total of six doses, wherein one or two doses of the anti-CD20 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0195] b. Anti-CD19 antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, CD19. In some embodiments, the method further comprises administering an anti-CD19 antibody to the subject. In some embodiments, the method is for treating lymphoma, such as non-Hodgkin's lymphoma. In some embodiments, the antibody is tafasitamab (e.g., MONJUVI®). In some embodiments, the antibody is loncastuximab (e.g., ZYNLONTA®).

[0196] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. In some embodiments, the additional agent may be administered after administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0197] In some embodiments, at least one dose of an anti-CD19 antibody has been administered to the subject prior to administration of a dose of the g-NK cell composition. Also disclosed herein in one aspect is a method of treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells can be administered once weekly in a predetermined number of doses, and wherein the subject has received prior administration of an anti-CD19 antibody.

[0198] In some embodiments, the CD19 antibody can be tafasitamab. In other embodiments, the CD19 antibody can be loncastuximab.

[0199] In some embodiments, administration of at least one dose of anti-CD19 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD19 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD19 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0200] In one particular example, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One skilled in the art can determine the appropriate route of administration.

[0201] In some embodiments, the anti-CD19 antibody may be administered as a weekly dose. In some embodiments, the anti-CD19 antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered once a week for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once a week for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, the antibody is administered once a week for eight doses. In some embodiments, the weekly doses are administered on consecutive weeks.

[0202] In some embodiments, the anti-CD19 antibody may be administered intravenously. In some embodiments, the anti-CD19 antibody may be administered subcutaneously. In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered at or about 12 mg / kg. In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered for four cycles. In some embodiments, the first cycle comprises administration on days 1, 4, 8, 15, and 22 of a 28-day cycle. In some embodiments, the second and third cycles comprise administration on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, the fourth cycle and beyond comprises administration on days 1 and 15 of a 28-day cycle. In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles.

[0203] In some embodiments, the anti-CD19 antibody (e.g., loncastuximab) is administered at or about 0.15 mg / kg every three weeks for two cycles. In some embodiments, the anti-CD19 antibody (e.g., loncastuximab) is administered at or about 0.075 mg / kg every three weeks for subsequent cycles. In some embodiments, dexamethasone is administered prior to administration of the anti-CD19 antibody (e.g., loncastuximab).

[0204] In some embodiments, the anti-CD19 antibody composition may be administered as a weekly dose. In some embodiments, the anti-CD19 antibody is administered as four or eight doses. In some embodiments, the antibody is administered subcutaneously in three or seven doses, followed by weekly intravenous administration of the anti-CD19 antibody. In some embodiments, the method comprises administering the anti-CD19 antibody once per week for a total of eight doses and administering the g-NK cell composition once per week for a total of six doses, wherein one or two doses of the anti-CD19 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0205] Illustrative examples are described in WO 2020 / 249528(A1) and U.S. Pat. No. 8,524,867.

[0206] c. Anti-CD30 antibody In some embodiments, the cells of the present invention can be targeted to tumors by administering them with an antibody that recognizes a tumor-associated antigen, which is CD30. In some embodiments, the method further comprises administering an anti-CD30 antibody to the subject. In some embodiments, the method is for treating lymphoma, such as non-Hodgkin's lymphoma. In some embodiments, the antibody is brentuximab (ADCETRIS®).

[0207] The g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered prior to administration of the g-NK cells. In some embodiments, the additional agent may be administered after administration of the g-NK cells. For example, the g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the g-NK cells may be administered at a selected time that is different from the time the antibody specific for a selected cancer type is administered.

[0208] In some embodiments, at least one dose of an anti-CD30 antibody has been administered to the subject prior to administration of a dose of the g-NK cell composition. Also disclosed herein in one aspect is a method of treating lymphoma, comprising administering to a subject having lymphoma a composition of natural killer (NK) cells (g-NK cells) that are deficient in expression of the FcRγ chain, wherein the composition of g-NK cells may be administered once weekly in a predetermined number of doses, and wherein the subject has received prior administration of an anti-CD30 antibody.

[0209] In some embodiments, the CD30 antibody can be brentuximab.

[0210] In some embodiments, administration of at least one dose of anti-CD30 antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD30 antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of anti-CD30 antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0211] In one particular example, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the administration of the population of g-NK cells. The 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 subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, e.g., intravenously; however, injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One skilled in the art can determine the appropriate route of administration.

[0212] In some embodiments, the anti-CD30 antibody may be administered as a weekly dose. In some embodiments, the anti-CD30 antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered once a week for at least one cycle, such as each cycle. In some embodiments, the antibody is administered once a week for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, eight doses of the antibody are administered once a week. In some embodiments, the weekly doses are administered on consecutive weeks.

[0213] In some embodiments, the anti-CD30 antibody may be administered intravenously. In some embodiments, the anti-CD30 antibody may be administered subcutaneously. In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered at or about 1.8 mg / kg. In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered up to 180 mg. In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered every three weeks.

[0214] In some embodiments, the anti-CD30 antibody composition may be administered as a weekly dose. In some embodiments, the anti-CD30 antibody is administered as four or eight doses. In some embodiments, the antibody is administered subcutaneously in three or seven doses, followed by weekly intravenous administration of the anti-CD30 antibody. In some embodiments, the method comprises administering the anti-CD30 antibody once per week for a total of eight doses and administering the g-NK cell composition once per week for a total of six doses, wherein one or two doses of the anti-CD30 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0215] An illustrative example is described in US Pat. No. 7,659,241.

[0216] 3.Bi-Specific Antibody (BsAb) In some embodiments provided herein, g-NK cells may be administered to an individual in combination with a bispecific antibody (BsAb). BsAbs are designed to recognize and bind to two different antigens or epitopes. Examples of BsAbs are bispecific T cell engagers (BiTEs) and bispecific natural killer cell engagers (BiKEs). BiKEs have been generated to bind CD16 on natural killer cells and a second tumor antigen; various examples of BiKEs targeting CD16 and a second tumor antigen have been described in the literature (Felices, et al. (2018) Methods Mol. Biol., 1441:333-346). For example, BiKE has been shown to target CD16 with CD19 or CD20 in B-cell non-Hodgkin's lymphoma (Glorius, et al. (2013) Leukemia, 27:190-201; Kipriyanov, et al. (2002) J. Immunol., 169:137-144; Portner, et al. (2012) Cancer immunology, immunotherapy: CII 61:1869-1875), CD16 with CD19 or CD33 in mixed phenotype leukemia (Schubert, et al. (2011) MAbs, 3:21-30), and CD16 with CD19 / CD22 in B-cell non-Hodgkin's lymphoma (Gleason, et al. (2012) Mol. Cancer Ther., 11:2674-2684), for Hodgkin's lymphoma, for CD16 in combination with CD30 (Hombach, et al. (1993), Int. J. Cancer, 55:830-836), and for multiple myeloma, for CD16 in combination with BCMA (Kakiuchi-Kiyota, et al. (2022), Leukemia, 36:1006-1014).

[0217] In some embodiments, the bispecific antibody is a bispecific T cell enhancer. In some embodiments, the bispecific antibody is a bispecific NK cell enhancer. In some embodiments, a first tumor target of a bispecific NK cell enhancer (BiKE) is CD16 and a second tumor target of BiKE is a tumor antigen. In some embodiments, a first tumor target of BiKE is CD16 and a second tumor target of BiKE is CD19. In some embodiments, a first tumor target of BiKE is CD16 and a second tumor target of BiKE is CD20. In some embodiments, a first tumor target of BiKE is CD16 and a second tumor target of BiKE is CD30. In some embodiments, a first tumor target of BiKE is CD16 and a second tumor target of BiKE is CD38. In some embodiments, a first tumor target of BiKE is CD16 and a second tumor target of BiKE is SLAMF7. In some embodiments, the first tumor target of BiKE is CD16 and the second tumor target of BiKE is BCMA.

[0218] 4. Cytokines or growth factors In some embodiments provided herein, g-NK cells may 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, and IL-27. In certain embodiments, recombinant IL-2 is administered to the subject. In other specific embodiments, recombinant IL-15 is administered to the subject. In other specific embodiments, recombinant IL-21 is administered to the subject. In some embodiments, the g-NK cells and cytokines or growth factors are administered sequentially. For example, the g-NK cells are 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.

[0219] In some embodiments, the subject is administered one or more cytokines (e.g., IL-2, IL-15, IL-21, IL-27, and / or IL-12) to support the survival and / or growth of the NK cells. The cytokines may be administered before, after, or substantially simultaneously with the NK cells. In some examples, the cytokines may be administered after the NK cells. In one specific example, the cytokines are administered to the subject within about 1-8 hours (e.g., within about 1-4 hours, about 2-6 hours, about 4-6 hours, or about 5-8 hours) of administration of the NK cells.

[0220] 5. Lymphocyte depletion therapy In some embodiments, the provided methods may also include administering the g-NK cells in conjunction with another treatment, such as a chemotherapeutic or cytotoxic agent or other treatment.

[0221] In some aspects, the provided methods may further comprise administering one or more lymphocyte depleting therapies, e.g., prior to or concurrently with the initiation of administration of the g-NK cell composition. In some embodiments, the lymphocyte depleting therapy comprises administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymphocyte depleting therapy may comprise administration of fludarabine.

[0222] In some aspects, preconditioning a subject with immunoablative (e.g., lymphocyte-depleting) therapy can improve the efficacy of adoptive cell therapy (ACT). In some embodiments, the lymphocyte-depleting therapy comprises a combination of cyclosporine and fludarabine.

[0223] Such preconditioning may be performed with the goal of reducing the risk of one or more of a variety of outcomes that may compromise the effectiveness of treatment. These include the phenomenon known as "cytokine sink," in which T cells, B cells, and 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; and the influence of negative regulators in the tumor microenvironment. (Muranski et al., Nat Clin Pract Oncol. December; 3(12):668-681 (2006)).

[0224] Thus, in some embodiments, provided methods further involve administering lymphocyte depletion therapy to the subject. In some embodiments, the methods involve administering lymphocyte depletion therapy to the subject prior to administration of the dose of cells. In some embodiments, the lymphocyte depletion therapy comprises a chemotherapeutic agent, such as fludarabine and / or cyclophosphamide. In some embodiments, administration of the cells and / or lymphocyte depletion therapy is performed via outpatient delivery.

[0225] In some embodiments, the method includes administering a preconditioning agent, such as a lymphocyte depleting agent or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administration of a dose of cells. For example, the subject may be administered a preconditioning agent, such as a lymphocyte depleting agent or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, at least two days, e.g., at least three, four, five, six, or seven days, prior to the first dose or a subsequent dose. In some embodiments, the subject is administered a preconditioning agent, such as a lymphocyte depleting agent or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, within seven days, e.g., within six, five, four, three, or two days, prior to administration of a dose of cells. In some embodiments, the subject is administered a preconditioning agent, such as a lymphocyte-depleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, within 14 days, e.g., within 13, 12, 11, 10, 9, or 8 days, prior to administration of a dose of cells.

[0226] In some embodiments, subjects are preconditioned with cyclophosphamide at a dose of 20 mg / kg to 100 mg / kg or about 20 mg / kg to about 100 mg / kg, e.g., 40 mg / kg to 80 mg / kg or about 40 mg / kg to about 80 mg / kg. In some aspects, subjects are preconditioned with 60 mg / kg or about 60 mg / kg of cyclophosphamide. In some embodiments, fludarabine can be administered in a single dose or in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once daily for one or two days.

[0227] In some embodiments, when the lymphocyte depleting agent comprises fludarabine, the subject receives fludarabine at a dose of 1 mg / m 2 ~100mg / m 2 , or about 1 mg / m 2 ~about 100mg / m 2 , e.g., 10 mg / m 2~75mg / m 2 , or about 10 mg / m 2 ~about 75mg / m 2 , 15 mg / m 2 ~50mg / m 2 , or about 15 mg / m 2 ~about 50mg / m 2 , 20 mg / m 2 ~30mg / m 2 , or about 20 mg / m 2 ~about 30mg / m 2 , or 24 mg / m 2 ~26mg / m 2 , or about 24 mg / m 2 ~about 26mg / m 2 In some instances, the subject is administered a dose of 25 mg / m 2 In some embodiments, fludarabine is administered in a single dose, or in multiple doses, such as daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, for example, for 1 to 5 days, for example, for 3 to 5 days.

[0228] In some embodiments, the lymphocyte depleting agent comprises a combination of drugs, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of drugs can 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 receives 60 mg / kg (about 2 g / m) of cyclophosphamide prior to administration of the cells. 2 ) cyclophosphamide and 25 mg / m in 3 to 5 doses. 2 Fludarabine is administered.

[0229] In some embodiments, prior to administration of a dose of g-NK cells, the subject undergoes lymphocyte depletion therapy. In some embodiments, the lymphocyte depletion therapy comprises fludarabine and / or cyclophosphamide. In some embodiments, the lymphocyte depletion comprises 20-40 mg / m 2 daily for 2-4 days. 2 or about 20-40 mg / m2 (body surface area of ​​subject), optionally 30 mg / m 2 or about 30 mg / m 2 of fludarabine, and / or 200–400 mg / m daily for 2–4 days. 2 or approximately 200-400 mg / m 2 (body surface area of ​​subject), optionally 300 mg / m 2 or about 300 mg / m 2 This includes the administration of cyclophosphamide.

[0230] In some embodiments, the lymphocyte depletion therapy comprises fludarabine and cyclophosphamide. In some embodiments, the lymphocyte depletion therapy comprises 30 mg / day 2 or about 30 mg / 2 (subject's body surface area) of fludarabine and 300 mg / m daily. 2 or approximately 300 mg / m 2 (subject's body surface area) with cyclophosphamide for 2 to 4 days each, optionally for 3 days.

[0231] In some embodiments, administration of a preconditioning agent prior to infusion of a dose of cells improves the outcome of the treatment. For example, in some aspects, lymphocyte-depleting or chemotherapeutic agents, such as cyclophosphamide, fludarabine, or a combination thereof, preconditioning improves the efficacy of the treatment or increases the persistence of NK cells in the subject. In some embodiments, preconditioning treatment increases disease-free survival, such as the percentage of subjects who survive and do not exhibit minimal residual or molecularly detectable disease after a given period of time after administration of the cells. In some embodiments, the time to median disease-free survival is increased.

[0232] Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell population in some embodiments is measured by any of several known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to antigen in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of NK cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells can also be measured by assaying the expression and / or secretion of specific cytokines or other effector molecules, such as CD107a, IFNγ, and TNF. In some embodiments, biological activity is measured by assessing clinical outcomes, such as reduction in tumor burden or tumor burden, hi some embodiments, toxicity outcomes, cell persistence and / or expansion, and / or the presence or absence of a host immune response are assessed.

[0233] III. Methods for Expanding Natural Killer Cell Subsets In some embodiments, the g-NK cell compositions 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 g-NK cell compositions include expanding FcRγ-deficient NK cells (g-NK cells) from a biological sample from a human subject. - In some embodiments, the method may include expanding a subset of cells that are NKG2C (NKG2C) from a biological sample from a human subject. pos In some embodiments, the method may include expanding a subset of NK cells that are NKG2A from a biological sample from a human subject. negIn 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 for preferential growth and / or expansion of NK cell subsets that overlap or share extracellular surface markers with the g-NK cell subject and / or 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 NK cell subsets that overlap or share extracellular surface markers with the g-NK cell subject and / or g-NK cell subset. In some aspects, the provided methods also include 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 for preferential growth and / or expansion of NK cell subsets that overlap or share extracellular surface markers with the g-NK cell subject and / or g-NK cell subset. pos and / or NKG2A neg Other subsets of NK cells may be expanded, such as any NK cell that is a

[0234] In some embodiments, the sample, e.g., biological sample, contains multiple cell populations, including an NK cell population. In some embodiments, the biological sample is or includes 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 peripheral blood mononuclear cell (PBMC) sample. Thus, in some embodiments of the provided methods, a population of peripheral blood mononuclear cells (PBMCs) can be obtained. A sample containing multiple cell populations, including an NK cell population, can be used to enrich or select NK cell subsets for expansion by the provided methods.

[0235] In some embodiments, the biological sample is from a subject who is a healthy subject. In some embodiments, the biological sample is from a subject who has a disease or condition, for example, cancer.

[0236] In some embodiments, cells are isolated or selected from a sample, such as a biological sample, e.g., a sample obtained from or derived from a subject with a particular disease or condition, or a subject in need of cell therapy, or a subject to whom cell therapy will be administered. In some aspects, the subject is a human, such as a patient in need of a particular therapeutic intervention, such as adoptive cell therapy, from which cells are isolated, processed, and / or manipulated. Thus, the cells in some embodiments are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples that are 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.

[0237] In some examples, cells from a subject's circulating blood are obtained. The sample contains lymphocytes, including, in some aspects, NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed to remove the plasma fraction and place the cells in a buffer or medium appropriate 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 the blood cell sample are removed and the cells are directly resuspended in culture medium. In some embodiments, the method includes a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient, such as by using Histopaque® density centrifugation.

[0238] In some embodiments, the biological sample is derived from an enriched leukoreduction product collected from normal peripheral blood. In some embodiments, the enriched leukoreduction product may contain fresh cells. In some embodiments, the enriched leukoreduction product is a cryopreserved sample that is thawed for use in the provided methods.

[0239] In some embodiments, the source of biological cells is 5×10 5 or approximately 5 x 10 5 pieces~5×10 8 or approximately 5 x 10 8 In some embodiments, the number of NK cells, or g-NK cell subsets, or NK cell subsets associated with or including surrogate markers for g-NK cells in the biological sample is greater than or equal to 5×10 5or approximately 5 x 10 5 pieces~1×10 8 or approximately 1 x 10 8 pieces, 5×10 5 or approximately 5 x 10 5 pieces~5×10 7 or approximately 5 x 10 7 pieces, 5×10 5 or approximately 5 x 10 5 pieces~1×10 7 or approximately 1 x 10 7 pieces, 5×10 5 or approximately 5 x 10 5 pieces~5×10 6 or approximately 5 x 10 6 pieces, 5×10 5 or approximately 5 x 10 5 pieces~1×10 6 or approximately 1 x 10 6 pieces, 1×10 6 or approximately 1 x 10 6 pieces~1×10 8 or approximately 1 x 10 8 pieces, 1×10 6 or approximately 1 x 10 6 pieces~5×10 7 or approximately 5 x 10 7 pieces, 1×10 6 or approximately 1 x 10 6 pieces~1×10 7 or approximately 1 x 10 7 pieces, 1×10 6 or approximately 1 x 10 6 pieces~5×10 6 or approximately 5 x 10 6 pieces, 5×10 6 or approximately 5 x 10 6 pieces~1×10 8 or approximately 1 x 10 8 pieces, 5×10 6 or approximately 5 x 10 6 pieces~5×10 7 or approximately 5 x 10 7 pieces, 5×10 6 or approximately 5 x 10 6 pieces~1×10 7 or approximately 1 x 10 7 pieces~1×108 or approximately 1 x 10 8 pieces, 1×10 7 or approximately 1 x 10 7 pieces~5×10 7 or approximately 5 x 10 7 pieces, or 5 x 10 7 or about 5 x 10 7 pieces~1×10 8 or approximately 1 x 10 8 There are individuals.

[0240] In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 3%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 5%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 10%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 12%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 14%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 16%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 18%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 20%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells, or an NK cell subset associated with or comprising surrogate markers for g-NK cells, is greater than or greater than about 22%.In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 24%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 26%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 28%. In some embodiments, the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or comprising surrogate markers for g-NK cells is greater than or greater than about 30%.

[0241] In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than 3% or greater than about 3%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than 5% or greater than about 5%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than 10% or greater than about 10%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than 12% or greater than about 12%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 14%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 16%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 18%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 20%.In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 22%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 24%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 26%. In some embodiments, a subject is selected when the percentage of NK cells in a biological sample that are g-NK cells or NK cell subsets associated with or including surrogate markers for g-NK cells is greater than or about 28%. In some embodiments, the subject is selected if the percentage of NK cells in the biological sample that are g-NK cells, or an NK cell subset associated with or comprising a surrogate marker for g-NK cells, is greater than or greater than about 30%.

[0242] In some embodiments, the biological sample is derived from a CMV-seropositive subject. CMV infection can result in phenotypic and functional differentiation of NK cells, including the development of a higher fraction of NKG2C-expressing NK cells, which exhibit enhanced antiviral activity. CMV-associated NK cells that express NKG2C exhibit altered DNA methylation patterns and reduced expression of signaling molecules such as FcRγ (Schlums et al., Immunity (2015) 42:443-56). These NK cells are associated with stronger antibody-dependent activation, expansion, and function compared to conventional NK cell subsets. In some cases, the biological sample can be derived from a CMV-seronegative subject, as NK cells with reduced expression of FcRγ can also be detected in CMV-seronegative individuals, although generally at lower levels. In some cases, the biological sample can be derived from a CMV-seropositive individual.

[0243] In some embodiments, subjects are selected based on the percentage of NK cells in a peripheral blood sample that are positive for NKG2C. In some embodiments, subjects are selected when at least 20% or at least about 20% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, subjects are selected when at least 25% or at least about 25% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, subjects are selected when at least 30% or at least about 30% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, subjects are selected when at least 35% or at least about 35% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, subjects are selected when at least 40% or at least about 40% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, subjects are selected when at least 45% or at least about 45% of the NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, a subject is selected if at least 50% or at least about 50% of the NK cells in a peripheral blood sample are positive for NKG2C. In some embodiments, a subject is selected if at least 55% or at least about 55% of the NK cells in a peripheral blood sample are positive for NKG2C. In some embodiments, a subject is selected if at least 60% or at least about 60% of the NK cells in a peripheral blood sample are positive for NKG2C.

[0244] In some embodiments, subjects are selected based on the percentage of NK cells in a peripheral blood sample that are negative or low for NKG2A. In some embodiments, subjects are selected if at least 70% or about 70% of the NK cells in a peripheral blood sample are negative or low for NKG2A. In some embodiments, subjects are selected if at least 75% or about 75% of the NK cells in a peripheral blood sample are negative or low for NKG2A. In some embodiments, subjects are selected if at least 80% or about 80% of the NK cells in a peripheral blood sample are negative or low for NKG2A. In some embodiments, subjects are selected if at least 85% or about 85% of the NK cells in a peripheral blood sample are negative or low for NKG2A. In some embodiments, subjects are selected if at least 90% or about 90% of the NK cells in a peripheral blood sample are negative or low for NKG2A.

[0245] In some embodiments, a subject is selected based on both the percentage of NK cells in the 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, a subject is selected if at least 20% or about 20% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 70% or about 70% of the NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a subject is selected if at least 30% or about 30% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 75% or about 75% of the NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a subject is selected if at least 40% or about 40% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 80% or about 80% of the NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a subject is selected if at least 50% or about 50% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 85% or about 85% of the NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a subject is selected if at least 60% or about 60% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 90% or about 90% of the NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a subject is selected if at least 60% or about 60% of the NK cells in the peripheral blood sample are positive for NKG2C and at least 95% or about 95% of the NK cells in the peripheral blood sample are negative or low for NKG2A.

[0246] In some embodiments, the subject is diagnosed with NKG2C if the subject is CMV seropositive and the percentage of g-NK cells among NK cells in a peripheral blood sample from the subject is greater than or greater than about 30% and the NKG2C pos The percentage of cells is greater than or greater than about 20% and NKG2Aneg If the percentage of cells is greater than or about greater than 70%, the cells are selected for expansion by the methods provided.

[0247] In some embodiments, NK cells from a subject have a single nucleotide polymorphism (SNP rs396991) at nucleotide 526 (thymidine (T) → guanine (G)) of the CD16 gene, 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, the NK cells have the CD16 158 V polymorphism (referred to herein as 158V / V) on both alleles. In some embodiments, the NK cells have the CD16 158 V polymorphism (referred to herein as 158V / F) on a single allele. Reference herein to the 158V+ genotype is understood to refer to both the 158V / V genotype and the 158V / F genotype. The CD16 F158V polymorphism has been found to be associated with substantially higher affinity for IgG1 antibodies and 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 improved affinity, cytotoxicity, and / or cytokine-mediated effector function of the CD16 158V+ / g- NK cell subset.

[0248] In some embodiments, provided methods include enriching or isolating NK cells or subsets thereof from a biological sample of a subject identified as having the CD16 158V+ NK cell genotype. In some embodiments, the methods include screening the subject for the presence of the CD16 158V+ NK cell genotype. In some embodiments, genomic DNA is extracted from a sample from the subject that is or contains NK cells, such as a blood sample or bone marrow sample. In some embodiments, the sample is or contains blood cells, e.g., peripheral blood mononuclear cells. In some embodiments, the sample is or contains isolated NK cells. In some embodiments, the sample is from a healthy donor subject. Any method for extracting DNA from a sample can be used. For example, nucleic acids can be readily isolated from a sample, e.g., cells, using standard techniques such as guanidine thiocyanate-phenol-chloroform extraction (Chomocyznski et al. (1987) Anal. Biochem. 162:156). Commercially available kits, such as the Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.), are also readily available for extracting genomic DNA.

[0249] 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, a DNA sequencing reaction (MassARRAY MALDI-TOF), RFLP, allele-specific PCR, real-time allele discrimination, or a microarray. In some embodiments, a PCR-based technique, such as RT-PCR of genomic DNA, is performed using allele-specific primers for the polymorphism. PCR methods for amplifying target nucleic acid sequences in a sample are well known in the art and are described, for example, in 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, and U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,889,818, all of which are incorporated herein by reference in their entireties.

[0250] Primers for detecting the 158V+ polymorphism are known or can be easily designed by those skilled in the art, see, e.g., published PCT application WO 2012 / 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 performed using nested primers, followed by allele-specific restriction enzyme digestion. In some embodiments, the first PCR primer comprises 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 NO:3), while the second PCR primer is 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 NO:5), which in some cases generates a 94 bp fragment depending on the nature of the allele. In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO:6 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO:7 (GAAATCTACC TTTTCCTCTA ATAGGGCAAT). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO: 6 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO: 8 (GAAATCTACC TTTTCCTCTA ATAGGGCAA). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NO: 6 (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO: 9 (GAAATCTACC TTTTCCTCTA ATAGGGCA).In some embodiments, genotyping may be performed by quantitative real-time RT-PCR after extraction of RNA using the primer sequences CD16 sense (5'-CCAAAAGCCACACTCAAAGAC-3') set forth in SEQ ID NO: 10, and antisense (5'-ACCCAGGTGGAAAGAATGATG-3') set forth in SEQ ID NO: 11, and a TaqMan probe (5'-AACATCACCATCACTCAAGGTTTGG-3') set forth in SEQ ID NO: 12.

[0251] To confirm 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').

[0252] The genomic sequence of CD16a is available in the NCBI database under NG_009066.1. The gene ID for CD16A is 2214. Sequence information for CD16, including polymorphisms, is available under UniProt accession number 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). TIFF2024516619000002.tif35160

[0253] The sequence of CD16 158V+ (the polymorphism resulting in F158V) is known as VAR_003960 and has the sequence set forth in SEQ ID NO: 18 (the 158V+ polymorphism is bold and underlined). In some embodiments, CD16(158V+) further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO: 17). TIFF2024516619000003.tif35156

[0254] In some embodiments, SNP analysis is used on genomic deoxyribonucleic acid (DNA) samples using an allele-specific probe containing a fluorescent dye label (e.g., FAM or VIC) at the 5' end, a minor groove binder (MGB) and a nonfluorescent quencher (NFQ) at the 3' end, and unlabeled PCR primers to detect specific single nucleotide polymorphism (SNP) targets. In some embodiments, the assay measures or detects the presence of SNPs by a change in fluorescence of a dye associated with the probe. In such embodiments, the probe hybridizes to the target DNA between two unlabeled primers, and the 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 primer 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 embodiments, the qPCR instrument can detect the fluorescence from the unquenched label. An exemplary reagent is a commercially available SNP assay, for example, code C_25815666_10 for rs396991 (Applied Biosystems, catalog number 4351379 for SNP genotyping of F158V in CD16).

[0255] 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 NK cell subsets are isolated or enriched from a biological sample from a subject identified as heterozygous or homozygous for the CD16 158V polymorphism. In some embodiments, NK cells or NK cell subsets are isolated or enriched from a biological sample from a subject identified as homozygous for the CD16 158V polymorphism.

[0256] In some embodiments, the method includes enriching NK cells from a biological sample, such as a population of PBMCs isolated or obtained from a 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. Selecting a particular marker or combination of surface markers is within the level of ordinary skill in the art. In some embodiments, the surface marker is any one or more of the following surface antigens: CD11a, 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 is any one or more of the following surface antigens: CD11a, 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 specific embodiments, the one or more surface antigens comprise CD3 and one or more of the following surface antigens: CD16, CD56, or CD57. In some embodiments, the one or more surface antigens are CD3 and CD57. In some embodiments, the one or more surface antigens are CD3, CD56, and CD16. In other embodiments, the one or more surface antigens are CD3, CD56, and CD38. In further embodiments, the one or more surface antigens are CD3, CD56, NKG2A, and CD161. In some embodiments, the one or more surface antigens are CD3, CD57, and NKG2C. In some embodiments, the one or more surface antigens are CD3, CD57, and NKG2A. In some embodiments, the one or more surface antigens are CD3, CD57, NKG2C, and NKG2A. In some embodiments, the one or more surface antigens are CD3 and CD56. In some embodiments, the one or more surface antigens are CD3, CD56, and NKG2C.In some embodiments, the one or more surface antigens are CD3, CD56, and NKG2A. In some embodiments, the one or more surface antigens are CD3, CD56, NKG2C, and NKG2A. Reagents, including fluorochrome-conjugated antibodies, for detecting such surface antigens are well known and available to those skilled in the art.

[0257] In some embodiments, the NK cell population isolated or enriched, e.g., by selection, from a sample by the methods provided may be positive for one or more particular markers, e.g., surface markers (e.g., marker + or marker pos ) or expressing a high level thereof (marker high ) or are negative for or express relatively low levels of one or more markers (marker- or marker neg ) cells. It is therefore understood that the terms positive, pos, or + with respect to markers or proteins expressed on or in cells are used interchangeably herein. It is therefore understood that the terms negative, neg, or - with respect to markers or proteins expressed on or in cells are used interchangeably herein. Furthermore, the markers herein neg It is understood that reference to cells that are NK cells can refer to cells that are negative for the marker and to cells that express a relatively low level of the marker, such as a low level that is not readily detectable compared to control or background levels. In some cases, such a marker will be absent or expressed at a relatively low level on a particular population of NK cells, but present or expressed at a relatively high level on certain other populations of lymphocytes (such as T cells). In some cases, such a marker will be present or expressed at a relatively high level on a particular population of NK cells, but absent or expressed at a relatively low level on certain other populations of lymphocytes (such as T cells or a subset thereof).

[0258] In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, isolation in some aspects involves separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, generally followed by a washing step and separation of cells that bound to the antibody or binding partner from cells that did not. In some embodiments, the incubation is static (without mixing). In some embodiments, the incubation is dynamic (with mixing).

[0259] Such separation steps can be based on positive selection, in which cells that bound to the reagent are retained for further use, and / or negative selection, in which cells that did not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. Separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in the complete removal of such cells. For example, in some embodiments, negative selection for CD3 may be used to increase the number or percentage of CD3. neg The method enriches for a population of cells that are CD3 pos In some instances, the cells may contain a small percentage of cells that are CD57, and may also contain some residual or small percentage of other unselected cells. pos or CD16 pos Positive selection of one of the populations was performed by targeting the CD57 pos or CD16pos Either of the populations will be enriched, but in some cases may also contain some residual or small percentage of other unselected cells, which may include the other of the CD57 or CD16 populations that are still present in the enriched population.

[0260] In some instances, multiple separation steps are performed, with the positively or negatively selected fraction from one step being subjected to another separation step, such as a subsequent positive or negative selection. In some instances, a single separation step may deplete cells that simultaneously express multiple markers, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted for negative selection. Similarly, multiple cell types may be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0261] In some aspects, the selection comprises a positive and / or negative selection step based on the expression of one or more surface antigens, such as in cells from a PBMC sample. In some embodiments, the isolation comprises a positive selection for cells expressing CD56, CD16, or CD57, and / or a negative selection for cells expressing CD38, and / or a negative selection for cells expressing non-NK cell markers, such as T cell markers, e.g., a negative selection for cells expressing CD3 (CD3 neg For example, in some embodiments, isolation involves 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, e.g., negative selection for cells expressing CD3 (CD3 neg In some embodiments, isolation involves positive selection for cells expressing CD56, CD16, or CD57, and / or CD38 (CD38 neg ), CD161(CD161 neg ), NKG2A(NKG2A neg) and / or negative selection against cells expressing CD3 (CD3 neg In some embodiments, the selection comprises CD3 negative cells (CD3 neg ) is isolated.

[0262] In some embodiments, the isolation is performed by negative selection against cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD56 (CD56 pos In some embodiments, the selection comprises negative selection against cells expressing CD38 (CD38 neg In certain embodiments, the isolated or selected cells may further comprise CD3 neg CD56 pos CD38 neg is.

[0263] In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), followed by NKG2A (NKG2A neg ) and negative selection for cells expressing CD161 (CD161 neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD56 pos NKG2A neg CD161 neg is.

[0264] In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD57 (CD57 pos In certain embodiments, the isolated or selected cells comprise CD3 neg CD57 pos is.

[0265] In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ) and positive for cells expressing CD16 (CD16pos In certain embodiments, the isolated or selected cells comprise CD3 neg CD16 pos is.

[0266] In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD57 (CD57 pos In certain embodiments, the isolated or selected cells comprise CD3 neg CD57 pos For example, NK cells are CD3 pos Cell depletion (CD3 pos negative selection for CD57 cells), followed by pos Cell selection, thereby CD57 pos NK cells can be enriched by isolating and enriching them. Separation can be performed by immunoaffinity-based methods, such as using MACS™ microbeads. For example, CD3 microbeads can be used to enrich for CD3 neg CD3 in negative selection of cells pos Subsequently, CD57 microbeads can be used for CD57 enrichment of CD3 cell-depleted PBMCs. neg / CD57 pos The enriched NK cells can be used in expansion in the provided methods.

[0267] In some embodiments, the selection is a positive selection against cells expressing NKG2C (NKG2C pos ) and / or negative selection against cellular NKG2A (NKG2A neg In some embodiments, the selection may further comprise negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD57 (CD57 pos ), and positive selection for cells expressing NKG2C (NKG2C pos In certain embodiments, the isolated or selected cells comprise CD3 neg CD57 pos NKG2Cpos In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD57 (CD57 pos ), and negative selection against cells expressing NKG2A (NKG2A neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD57 pos NKG2A neg In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD57 (CD57 pos ), positive selection for cells expressing NKG2C (NKG2C pos ), and negative selection against cells expressing NKG2A (NKG2A neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD57 pos NKG2C pos NKG2A neg is.

[0268] In some of any of the provided embodiments, the selection comprises negative selection against cells expressing CD38 (CD38 neg In certain embodiments, the isolated or selected cells may further comprise CD3 neg CD57 pos CD38 neg In certain embodiments, the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2C pos In certain embodiments, the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2A neg In certain embodiments, the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2C pos NKG2A neg is.

[0269] In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD56 (CD56 pos In certain embodiments, the isolated or selected cells comprise CD3 neg CD56 pos In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), and positive selection for cells expressing NKG2C (NKG2C neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD56 pos NKG2C pos In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), and negative selection against cells expressing NKG2A (NKG2A neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD56 pos NKG2A neg In some embodiments, the selection is a negative selection against cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), positive selection for cells expressing NKG2C (NKG2C pos ), and negative selection against cells expressing NKG2A (NKG2A neg In certain embodiments, the isolated or selected cells comprise CD3 neg CD56 pos NKG2C pos NKG2A neg is.

[0270] In some of any of the provided embodiments, the selection comprises negative selection against cells expressing CD38 (CD38 negIn certain embodiments, the isolated or selected cells may further comprise CD3 neg CD56 pos CD38 neg In certain embodiments, the isolated or selected cells are CD3 neg CD56 pos CD38 neg NKG2C pos In certain embodiments, the isolated or selected cells are CD3 neg CD56 pos CD38 neg NKG2A neg In certain embodiments, the isolated or selected cells are CD3 neg CD56 pos CD38 neg NKG2C pos NKG2A neg is.

[0271] In some 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 comprises an NK cell surrogate surface marker profile. In some of any such embodiments, the g-NK cell surrogate surface marker profile further comprises an NK cell surrogate surface marker profile. pos / CD3 neg / CD56 posIn certain embodiments, the g-NK cell surrogate surface marker profile further comprises CD45 pos / CD3 neg / CD56 pos / CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In other specific embodiments, the g-NK cell surrogate surface marker profile comprises CD45 pos / CD3 neg / CD56 pos / NKG2A neg / CD161 neg In other specific embodiments, the g-NK cell surrogate surface marker profile comprises CD45 pos / CD3 neg / CD56 pos / CD38 neg Includes.

[0272] In some embodiments, methods for isolating, selecting, and / or enriching cells, such as by positive or negative selection based on expression of a cell surface marker, can include immunoaffinity-based selection. In some embodiments, immunoaffinity-based selection involves contacting a sample containing cells, such as PBMCs, with an antibody or binding partner that specifically binds to the cell surface marker. In some embodiments, the antibody or binding partner is attached to a solid support or matrix, such as spheres or beads, e.g., microbeads, nanobeads, including agarose, magnetic beads, or paramagnetic beads, to enable separation of cells for positive and / or negative selection. In some embodiments, the spheres or beads can be packed into a column to perform immunoaffinity chromatography, and a sample containing cells, such as PBMCs, is contacted with the matrix of the column and subsequently eluted or released therefrom.

[0273] Incubation is generally carried out under conditions in which an antibody or binding partner that specifically binds to the antibody or binding partner attached to the magnetic particle or bead, if present on cells in the sample, will specifically bind to the cell surface molecule.

[0274] In some embodiments, the sample is placed in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are attracted to the magnet and separated from unlabeled cells. Positive selection retains cells that are attracted to the magnet, while negative selection retains cells that are not attracted (unlabeled cells). In some embodiments, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained and either further processed or subjected to additional separation steps.

[0275] In some embodiments, the magnetically responsive particles remain attached to the cells, which are subsequently incubated and / or cultured; in some aspects, the particles remain 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, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0276] In some embodiments, affinity-based selection is by magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic-activated cell sorting (MACS) systems are capable of high-purity selection of cells to which magnetized particles are attached. In certain embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to the magnetized particles are held in place, while unattached species are eluted. Then, after this first elution step is complete, species captured within the magnetic field and prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0277] In some of any such embodiments, the method includes 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.

[0278] In embodiments of the provided methods, the enriched NK cells are incubated or cultured in the presence of feeder cells, such as under conditions that support the proliferation and expansion of NK cell subsets, specifically the g-NK cell subset.

[0279] In certain embodiments, the feeder cells are NKG2C pos stimulate or promote the expansion of NKG2A posThe feeder cells include cells that inhibit cell expansion. In some embodiments, the feeder cells are cells that express or are transfected with a hybrid HLA-E containing an HLA-E or HLA-A2 signal sequence. For example, an example of such a hybrid is an AEH hybrid gene containing an MHC class I, such as HLA-A2, promoter and signal sequence, and an HLA-E mature protein sequence, which in some cases can result in a mature protein that is identical to the mature protein 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 cells are LCL721.221, K562, or RMA-S cells transfected to express an MHC class I, such as HLA-A2, MHC-E molecule stabilized in the presence of a leader sequence. Cell lines engineered to express MHC class I, such as HLA-A2, cell surface HLA-E stabilized in the presence of a 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 an irradiated HLA-E-expressing cell line that is otherwise HLA-negative, such as K562. In some embodiments, cell lines can be transfected to express HLA-E. In some embodiments, K562 cells expressing membrane-bound IL-15 (K562-mb15) or membrane-bound IL-21 (K562-mb21) can be used as feeder cells. An example of such a cell line for use in the methods provided herein is 221-AEH cells.

[0280] In embodiments, HLA-expressing feeder cells are cryopreserved and thawed prior to use. In some embodiments, if the cells are transfected to express HLA-E, such as 221.AEH cells, the cells may be grown in the presence of appropriate nutrients, including, for example, serum or other suitable serum replacement, and a selection agent, prior to their use in the method. For example, in the case of 221.AEH cells, the cells may be cultured in cell culture medium supplemented with hygromycin B (e.g., 0.1% to 10%, e.g., 1% or about 1%) to maintain selection pressure on the cells and maintain high levels of plasmid HLA-E. The cells may be cultured at 1 x 10 cells / ml until use. 5 cells / mL~1×10 6 The cells can be maintained at a density of 1000 cells / mL.

[0281] In certain 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 immediately 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 1,000 to 10,000 rad, e.g., 1,000 to 5,000 rad, 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, e.g., 10 to 50 Gy, to prevent cell division. In some embodiments, the cells are irradiated with 100 Gy. In other embodiments, the irradiation is performed by X-ray irradiation. In some embodiments, HLA-E-expressing feeder cells are irradiated with about 10 Gy to 100 Gy of X-rays, e.g., in the range of 10 to 50 Gy, to prevent cell division. In some embodiments, 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, and as a result of irradiation, the NK cells become directly cytotoxic to the feeder cells, causing them to die during culture.

[0282] 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), e.g., an irradiated population thereof, at a ratio of feeder cells to enriched NK cells of greater than or greater than about 1:10 HLA-E feeder cells (e.g., 221.AEH cells), e.g., an irradiated population thereof to enriched NK cells, e.g., 10:1 or about 10:1 to 10:1 or about 10:1 such feeder cells to enriched NK cells.

[0283] In some embodiments, the ratio of HLA-E-expressing feeder cells (e.g., 221.AEH cells), e.g., irradiated populations thereof, is 1:10 or about 1:10 to 10:1 or about 10:1, 1:10 or about 1:10 to 5:1 or about 5:1, 1:10 or about 1:10 to 2.5:1 or about 2.5:1, 1:10 or about 1:10 to 1:1 or about 1:1, 1:10 or or about 1:10 to 1:2.5 or about 1:2.5, 1:10 or about 1:10 to 1:5 or about 1:5, 1:5 or about 1:5 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, 1:5 or about 1:5 to 2.5:1 or about 2.5:1, 1:5 or about 1:5 to 1:1 or about 1:1, 1:5 or about 1:5 to 1:2.5 or about 1 1:2.5, 1:2.5 or about 1:2.5 to 10:1 or about 10:1, 1:2.5 or about 1:2.5 to 5:1 or about 5:1, 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1, 1:2.5 or about 1:2.5 to 1:1 or about 1:1, 1:1 or about 1:1 to 10:1 or about 10:1, 1:1 or about 1:1 to 5:1 or about 5:1, 1: The ratio of such feeder cells to enriched NK cells is 1 or about 1:1 to 3:1 or about 3:1, 1:1 or about 1:1 to 2.5:1 or about 2.5:1, 2.5:1 or about 2.5:1 to 10:1 or about 10:1, 2.5:1 or about 2.5:1 to 5:1 or about 5:1, or 5:1 or about 5:1 to 10:1 or about 10:1 (each inclusive).

[0284] In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), e.g., in an irradiated population thereof, is 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 about 1. The ratio of such feeder cells to enriched NK cells is about 25:1, about 1.5:1, about 1.75:1, about 2.0:1, about 2.25:1, about 2:5:1, about 2.75:1, about 3.0:1, about 3.25:1, about 3.5:1, about 3.75:1, about 4.0:1, about 4.25:1, about 4.5:1, about 4.75:1, or about 5:1, or any value between any of the above. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), e.g., irradiated populations thereof, is less than or less than about 5:1. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), e.g., irradiated populations thereof, is 1:1 or about 2.5:1 or about 2.5:1, inclusive. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), e.g., irradiated populations thereof, is 2.5:1 or about 2.5:1. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), e.g., irradiated populations thereof, is 2:1 or about 2:1.

[0285] In some cases, if the starting NK cell population has been cryopreserved prior to expansion, i.e., subjected to freeze / thawing, a lower 221.AEH to NK cell ratio may be used than in methods using fresh NK cells. Here, it has been found that a 1:1 ratio of 221.AEH to frozen / thawed NK cells resulted in equivalent expansion to cultures containing a 2.5:1 ratio of 221.AEH to fresh NK cells. In some aspects, a lower ratio ensures a higher number of NK cells in culture, allowing for more cell-to-cell contact, which may play a role in promoting initial growth and expansion. In some embodiments, if the initial enriched population of NK cells from the sample has been subjected to freeze / thawing, a ratio of 2:1 to 1:2 or about 2:1 to about 1:2 221.AEH to frozen / thawed NK cells is used. In certain embodiments, the ratio is 1:1. Higher ratios, such as 2.5:1 221.AEH to frozen / thawed NK cells, may be used, but it is understood that this may require longer culture, for example, at or about 21 days, to reach the desired threshold density or number.

[0286] In some embodiments, the NK cells are expanded by further adding non-dividing peripheral blood mononuclear cells (PBMCs) to the culture. 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 PBMCs are irradiated with gamma rays in the range of about 1000-10000 rad, e.g., 1000-5000 rad, to prevent cell division. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 10 Gy-100 Gy, e.g., 10-50 Gy, to prevent cell division. In some aspects, the irradiated feeder cells are present in the culture medium simultaneously with the non-dividing (e.g., irradiated) HLA-E-expressing feeder cells during at least a portion of the incubation. In some embodiments, the non-dividing (e.g., irradiated) PBMC feeder cells, HLA-E-expressing feeder cells, and enriched NK cells are added to the culture on the same day, such as the day of initiation of incubation, e.g., simultaneously or approximately simultaneously or nearly simultaneously.

[0287] 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 derived from the same subject from which the enriched NK cells were isolated or selected. In certain embodiments, the PBMCs are obtained from the same biological sample used to enrich for NK cells, e.g., whole blood or leukapheresis or apheresis product. Once obtained, a portion of the PBMCs are stored for irradiation prior to NK cell enrichment as described above.

[0288] In some embodiments, irradiated PBMCs are present as feeder cells, and the ratio of such feeder cells to enriched NK cells is 1:10 or about 1:10 to 10:1 or about 10:1, 1:10 or about 1:10 to 5:1 or about 5:1, 1:10 or about 1:10 to 2.5:1 or about 2.5:1, 1:10 or about 1:10 to 1:1 or about 1:1, 1:10 or about 1:10 to 1:2.5 or about 1:2.5, 1:10 or about 1:10 to 1:5 or about 1:5, 1:5 or about 1:5 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, 1:5 or about 1:5 to 2.5:1 or about 2.5:1, 1:5 or about 1:5 to 1:1 or about 1:1, 1:5, or about 1:5 to 1:2.5, or about 1:2.5, 1:2.5, or about 1:2.5 to 10:1, or about 10:1, 1:2.5, or about 1:2.5 to 5:1, or about 5:1, 1:2.5, or about 1:2.5 to 2.5:1, or about 2.5:1, 1:2.5, or about 1:2.5 to 1:1, or about 1:1, 1:1 or is about 1:1 to 10:1 or about 10:1, 1:1 or about 1:1 to 5:1 or about 5:1, 1:1 or about 1:1 to 2.5:1 or about 2.5:1, 2.5:1 or about 2.5:1 to 10:1 or about 10:1, 2.5:1 or about 2.5:1 to 5:1 or about 5:1, or 5:1 or about 5:1 to 10:1 or about 10:1.

[0289] In some embodiments, irradiated PBMCs are present as feeder cells, and the ratio of such feeder cells to enriched NK cells is 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, 4.85:1, 4.95:1, 5.95:1, 6.05:1, 6.25:1, 6.55:1, 6.75:1, 7.05:1, 7.25:1, 7.55:1, 7.75:1, 8.05:1, 8.25:1, 8.5 5:1, or 5:1, or about 1.25:1, about 1.5:1, about 1.75:1, about 2.0:1, about 2.25:1, about 2:5:1, about 2.75:1, about 3.0:1, about 3.25:1, about 3.5:1, about 3.75:1, about 4.0:1, about 4.25:1, about 4.5:1, about 4.75:1, or about 5:1, or any value in between any of the above. In some embodiments, the irradiated PBMCs are present at a ratio of such feeder cells to enriched cells that is 5:1 or about 5:1.

[0290] In certain embodiments, during at least a portion of the incubation or culture, one or more cells or cell types, such as T cells of irradiated PBMCs, are activated, and / or the incubation or culture is performed in the presence of at least one stimulatory agent capable of stimulating activation of one or more T cells of the PBMC feeder cells. In some embodiments, the at least one stimulatory agent specifically binds to a member of the TCR complex. In some embodiments, the at least one stimulatory agent specifically binds to CD3, optionally CD3 epsilon. In some aspects, the at least one stimulatory agent is an anti-CD3 antibody or antigen-binding fragment. Exemplary anti-CD3 antibodies include mouse anti-human CD3 (OKT3).

[0291] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is present during at least a portion of the incubation with irradiated PBMC feeder cells. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is added to the culture or incubation at or near the same time as the irradiated PBMCs. For example, the anti-CD3 antibody or antigen-binding fragment is added at or near the beginning of the incubation or culture. In certain 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 changing or washing the culture medium. In certain embodiments, the method does not include adding back or replenishing the anti-CD3 antibody or antigen-binding fragment to the culture medium after changing or washing.

[0292] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is added or present during at least a portion of the culture or incubation, and has a concentration of 10 ng / mL or about 10 ng / mL to 5 μg / mL or about 5 μg / mL, e.g., 10 ng / mL or about 10 ng / mL to 2 μg / mL or about 2 μg / mL, 10 ng / mL or about 10 ng / mL to 1 μg / mL or about 1 μg / mL, 10 ng / mL or about 10 ng / mL to 500 ng / mL or about 500 ng / mL, / mL, 10ng / mL or about 10ng / mL to 100ng / mL or about 100ng / mL, 10ng / mL or about 10ng / mL to 50ng / mL or about 50ng / mL, 50ng / mL or about 50ng / mL to 5μg / mL or about 5μg / mL, for example, 50ng / mL or about 50ng / mL to 2μg / mL or about 2μg / mL, 50ng / mL or about 50ng / mL to 1μg / mL or about 1μg / mL, 50ng / mL or about 50ng / mL to 500ng / mL L or about 500ng / mL, 50ng / mL or about 50ng / mL to 100ng / mL or about 100ng / mL, 100ng / mL or about 100ng / mL to 5μg / mL or about 5μg / mL, 100ng / mL or about 100ng / mL to 2μg / mL or about 2μg / mL, 100ng / mL or about 100ng / mL to 1μg / mL or about 1μg / mL, 100ng / mL or about 100ng / mL to 500ng / mL or about 500ng / mL, 500ng / mL or less or about 500 ng / mL to 5 μg / mL or about 5 μg / mL, 500 ng / mL or about 500 ng / mL to 2 μg / mL or about 2 μg / mL, 500 ng / mL or about 500 ng / mL to 1 μg / mL or about 1 μg / mL, 1 μg / mL or about 1 μg / mL to 5 μg / mL or about 5 μg / mL, 1 μg / mL or about 1 μg / mL to 2 μg / mL or about 2 μg / mL, or 2 μg / mL or about 2 μg / mL to 5 μg / mL or about 5 μg / mL (both inclusive).In some embodiments, the concentration of the anti-CD3 antibody or antigen-binding fragment is 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 about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL, or any value therebetween. In some embodiments, the concentration of the anti-CD3 antibody or antigen-binding fragment is 50 ng / mL or about 50 ng / mL.

[0293] In some embodiments, the term "antibody" refers to immunoglobulin molecules and antigen-binding 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 intact polyclonal or monoclonal antibodies as well as fragments thereof, such as dAbs, Fabs, Fab's, F(ab')2, Fvs), single chains (scFvs), or single domain antibodies (sdAbs). 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 an antigen of interest. In this regard, an antigen-binding fragment can generally contain one, two, three, four, five, or all six CDRs of the VH and VL sequences from an antibody that binds the antigen, such as six CDRs ("CDR1," "CDR2," and "CDR3" of each of the heavy and light chains) for antibodies containing a variable heavy chain (VH) and a variable light chain (VL), or three CDRs for antibodies containing a single variable domain.

[0294] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to 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'), diabodies, linear antibodies, variable heavy chain (V), and the like. H ) domains, scFv and single domain V H These include single-chain antibody molecules such as monoclonal antibodies, as well as multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0295] In some embodiments, the incubation or culture is performed in the presence of such enriched NK cells, such as selected and / or isolated NK cells, at a concentration of 0.05 x 10 6 or approximately 0.05 x 10 6 or at least 0.05 x 10 6 or approximately 0.05 x 10 6 0.1 x 10 enriched NK cells / mL 6 or approximately 0.1 x 10 6 0.2 x 10 enriched NK cells / mL 6 or approximately 0.2 x 10 6 0.5 x 10 enriched NK cells / mL 6 or approximately 0.5 x 10 6 enriched NK cells / mL, or 1.0 x 10 6 or approximately 1.0 x 10 6 In embodiments of the provided methods, incubation or culturing is performed in the presence of such enriched NK cells, such as selected and / or isolated NK cells, starting at a concentration of 0.05 x 10 enriched NK cells / mL. 6 or approximately 0.05 x 10 6 Number of concentrated NK cells / mL ~1.0 x 10 6 or approximately 1.0 x 10 6 Number of enriched NK cells / mL, e.g., 0.05 x 10 6 or approximately 0.05 x 10 6Number of enriched NK cells / mL ~0.75 x 10 6 or approximately 0.75 x 10 6 Enriched NK cells / mL, 0.05 x 10 6 or approximately 0.05 x 10 6 ~0.5 x 10 concentrated NK cells / mL 6 or approximately 0.5 x 10 6 Enriched NK cells / mL, 0.05 x 10 6 or approximately 0.05 x 10 6 Number of enriched NK cells / mL ~0.20 x 10 6 pieces or approximately 0.20 x 10 6 Enriched NK cells / mL, 0.05 x 10 6 or approximately 0.05 x 10 6 Number of concentrated NK cells / mL ~0.1 x 10 6 or approximately 0.1 x 10 6 0.1 x 10 enriched NK cells / mL 6 or approximately 0.1 x 10 6 Number of concentrated NK cells / mL ~1.0 x 10 6 or approximately 1.0 x 10 6 0.1 x 10 enriched NK cells / mL 6 or approximately 0.1 x 10 6 Number of enriched NK cells / mL ~0.75 x 10 6 or approximately 0.75 x 10 6 0.1 x 10 enriched NK cells / mL 6 or approximately 0.1 x 10 6 ~0.5 x 10 concentrated NK cells / mL 6 or approximately 0.5 x 10 6 0.1 x 10 enriched NK cells / mL 6 or approximately 0.1 x 10 6 Number of enriched NK cells / mL ~0.20 x 10 6 pieces or approximately 0.20 x 10 6 Enriched NK cells / mL, 0.20 x 10 6 pieces or approximately 0.20 x 10 6 Number of concentrated NK cells / mL ~1.0 x 10 6or approximately 1.0 x 10 6 Enriched NK cells / mL, 0.20 x 10 6 pieces or approximately 0.20 x 10 6 Number of enriched NK cells / mL ~0.75 x 10 6 or approximately 0.75 x 10 6 Enriched NK cells / mL, 0.20 x 10 6 pieces or approximately 0.20 x 10 6 ~0.5 x 10 concentrated NK cells / mL 6 or approximately 0.5 x 10 6 0.5 x 10 enriched NK cells / mL 6 or approximately 0.5 x 10 6 Number of concentrated NK cells / mL ~1.0 x 10 6 or approximately 1.0 x 10 6 0.5 x 10 enriched NK cells / mL 6 or approximately 0.5 x 10 6 Number of enriched NK cells / mL ~0.75 x 10 6 or approximately 0.75 x 10 6 0.75 x 10 enriched NK cells / mL 6 or approximately 0.75 x 10 6 Number of concentrated NK cells / mL ~1.0 x 10 6 or approximately 1.0 x 10 6 In some embodiments, the incubation or culture is initiated at a concentration of 0.2 x 10 enriched NK cells / mL (inclusive). In some embodiments, the incubation or culture is initiated at a concentration of 0.2 x 10 enriched NK cells / mL (inclusive) in the presence of such enriched NK cells, such as selected and / or isolated NK cells. 6 or approximately 0.2 x 10 6 The starting concentration is 100 NK cells / mL.

[0296] In some of any such embodiments, the amount of enriched NK cells, such as those selected or isolated from PBMCs as described above, added or present at the start of incubation or culture is at least 1 x 10 5 or at least about 1 x 10 5 cells, at least approximately 2 x 105 or at least about 2 x 10 5 cells, at least 3 x 10 5 or at least about 3 x 10 5 cells, at least 4 x 10 5 or at least about 4 x 10 5 cells, at least 5 x 10 5 or at least about 5 x 10 5 cells, at least 6 x 10 5 or at least about 6 x 10 5 cells, at least 7 x 10 5 or at least about 7 x 10 5 cells, at least 8 x 10 5 or at least about 8 x 10 5 cells, at least 9 x 10 5 or at least about 9 x 10 5 cells, at least 1 x 10 6 or at least about 1 x 10 6 In certain embodiments, the amount of enriched NK cells, such as those selected or isolated from PBMCs as described above, is at least 1 x 10 cells or more. 6 or at least about 1 x 10 6 pieces, or 1 x 10 6 pieces, or approximately 1 x 10 6 Each cell is an individual cell.

[0297] In some embodiments, the enriched population of NK cells comprises at least 2.0 x 10 6 or at least about 2.0 x 10 6 Enriched NK cells, at least 3.0 x 10 6 or at least about 3.0 x 10 6 Enriched NK cells, at least 4.0 x 10 6 or at least about 4.0 x 10 6 Enriched NK cells, at least 5.0 x 10 6 or at least about 5.0 x 10 6 Enriched NK cells, at least 6.0 x 10 6or at least about 6.0 x 10 6 Enriched NK cells, at least 7.0 x 10 6 or at least about 7.0 x 10 6 Enriched NK cells, at least 8.0 x 10 6 or at least about 8.0 x 10 6 Enriched NK cells, at least 9.0 x 10 6 or at least about 9.0 x 10 6 Enriched NK cells, at least 1.0 x 10 7 or at least about 1.0 x 10 7 Enriched NK cells, at least 5.0 x 10 7 or at least about 5.0 x 10 7 Enriched NK cells, at least 1.0 x 10 8 or at least about 1.0 x 10 8 Enriched NK cells, at least 5.0 x 10 8 or at least about 5.0 x 10 8 enriched NK cells, or at least 1.0 x 10 9 or at least about 1.0 x 10 9 In some embodiments, the enriched population of NK cells comprises at least 2.0 x 10 5 or at least about 2.0 x 10 5 In some embodiments, the enriched population of NK cells comprises at least 1.0 x 10 6 or at least about 1.0 x 10 6 In some embodiments, the enriched population of NK cells comprises at least 1.0 x 10 7 or at least about 1.0 x 10 7 Contains enriched NK cells.

[0298] In some embodiments, the enriched population of NK cells comprises 2.0 x 10 5 or approximately 2.0 x 10 5 Enriched NK cells ~1.0 x 10 9or approximately 1.0 x 10 9 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~1.0 x 10 8 or approximately 1.0 x 10 8 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~5.0 x 10 7 or approximately 5.0 x 10 7 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~1.0 x 10 7 or approximately 1.0 x 10 7 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~5.0 x 10 6 or approximately 5.0 x 10 6 2.0 x 10 enriched NK cells 5 or approximately 2.0 x 10 5 Enriched NK cells ~1.0 x 10 6 or approximately 1.0 x 10 6 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~1.0 x 108 or approximately 1.0 x 10 8 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~5.0 x 10 7 or approximately 5.0 x 10 7 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~1.0 x 10 7 or approximately 1.0 x 10 7 1.0 x 10 enriched NK cells 6 or approximately 1.0 x 10 6 Enriched NK cells ~5.0 x 10 6 or approximately 5.0 x 10 6 5.0 x 10 enriched NK cells 6 or approximately 5.0 x 10 6 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 5.0 x 10 enriched NK cells 6 or approximately 5.0 x 10 6 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 5.0 x 10 enriched NK cells 6 or approximately 5.0 x 10 6 Enriched NK cells ~1.0 x 10 8 or approximately 1.0 x 10 8 5.0 x 10 enriched NK cells 6 or approximately 5.0 x 10 6 Enriched NK cells ~5.0 x 10 7 or approximately 5.0 x 10 7 5.0 x 10 enriched NK cells 6 or approximately 5.0 x 10 6 Enriched NK cells ~1.0 x 10 7 or approximately 1.0 x 10 7 1.0 x 10 enriched NK cells 7 or approximately 1.0 x 10 7Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 1.0 x 10 enriched NK cells 7 or approximately 1.0 x 10 7 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 1.0 x 10 enriched NK cells 7 or approximately 1.0 x 10 7 Enriched NK cells ~1.0 x 10 8 or approximately 1.0 x 10 8 1.0 x 10 enriched NK cells 7 or approximately 1.0 x 10 7 Enriched NK cells ~5.0 x 10 7 or approximately 5.0 x 10 7 5.0 x 10 enriched NK cells 7 or approximately 5.0 x 10 7 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 5.0 x 10 enriched NK cells 7 or approximately 5.0 x 10 7 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 5.0 x 10 enriched NK cells 7 or approximately 5.0 x 10 7 Enriched NK cells ~1.0 x 10 8 or approximately 1.0 x 10 8 1.0 x 10 enriched NK cells 8 or approximately 1.0 x 10 8 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 1.0 x 10 enriched NK cells 8 or approximately 1.0 x 10 8 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 enriched NK cells, or 5.0 x 10 8 or approximately 5.0 x 108 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 In some embodiments, the enriched population of NK cells comprises 2.0 x 10 5 or approximately 2.0 x 10 5 Enriched NK cells ~5.0 x 10 7 or approximately 5.0 x 10 7 In some embodiments, the enriched population of NK cells comprises 1.0 x 10 6 or approximately 1.0 x 10 6 Enriched NK cells ~1.0 x 10 8 or approximately 1.0 x 10 8 In some embodiments, the enriched population of NK cells comprises 1.0 x 10 7 or approximately 1.0 x 10 7 Enriched NK cells ~5.0 x 10 8 or approximately 5.0 x 10 8 In some embodiments, the enriched population of NK cells comprises 1.0 x 10 7 or approximately 1.0 x 10 7 Enriched NK cells ~1.0 x 10 9 or approximately 1.0 x 10 9 Contains enriched NK cells.

[0299] In some embodiments, the percentage of g-NK cells in the enriched population of NK cells is 20%, or about 20% to 90%, or about 90%, 20%, or about 20% to 80%, or about 80%, 20%, or about 20% to 70%, or about 70%, 20%, or about 20% to 60%, or about 60%, 20%, or about 20% to 50%, or about 50%, 20%, or about 20% to 40%, or or about 40%, 20%, or about 20% to 30%, or about 30%, 30%, or about 30% to 90%, or about 90%, 30%, or about 30% to 80%, or about 80%, 30%, or about 30% to 70%, or about 70%, 30%, or about 30% to 60%, or about 60%, 30%, or about 30% to 50%, or about 50%, 30%, or about 30% to 40%, or about 40%, 40% or less or about 40% to 90% or about 90%, 40% or about 40% to 80% or about 80%, 40% or about 40% to 70% or about 70%, 40% or about 40% to 60% or about 60%, 40% or about 40% to 50% or about 50%, 50% or about 50% to 90% or about 90%, 50% or about 50% to 80% or about 80%, 50% or about 50% to 70% or or about 70%, 50%, or about 50% to 60%, or about 60%, 60%, or about 60% to 90%, or about 90%, 60%, or about 60% to 80%, or about 80%, 60%, or about 60% to 70%, or about 70%, 70%, or about 70% to 90%, or about 90%, 70%, or about 70% to 80%, or about 80%, or 80%, or about 80% to 90%, or about 90%. In some embodiments, the percentage of g-NK cells in the enriched population of NK cells is 20%, or about 20% to 90%, or about 90%. In some embodiments, the percentage of g-NK cells in the enriched population of NK cells is 40%, or about 40% to 90%, or about 90%. In some embodiments, the percentage of g-NK cells in the enriched population of NK cells is at or about 60% to 90% or about 90%.

[0300] In some of these embodiments, the NK cells may be cultured with growth factors. 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 recombinant IL-2, recombinant IL-7, recombinant IL-21, or recombinant IL-15.

[0301] 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 include any of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In some embodiments, the one or more recombinant cytokines include any of IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination 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 include IL-2, IL-7, IL-15, IL-12, IL-18, or IL-27, or a combination 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 are 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, IL-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.

[0302] In certain embodiments, provided methods include incubating or culturing the enriched NK cells and feeder cells in the presence of recombinant IL-2. In some embodiments, recombinant IL-2 is added during at least a portion of the incubation, e.g., at the beginning of the culture, and optionally one or more times during the culture, at a concentration of 1 IU / mL or about 1 IU / mL to 500 IU / mL or about 500 IU / mL, e.g., 1 IU / mL or about 1 IU / mL to 250 IU / mL or about 250 IU / mL, 1 IU / mL or about 1 IU / mL to 100 IU / mL or about 100 IU / mL, 1 IU / mL or about 1 IU / mL to 50 IU / mL or about 50 IU / mL, 50 IU / mL or about 50 IU / mL. or about 500 IU / mL or 500 IU / mL, 50 IU / mL or about 50 IU / mL to 250 IU / mL or about 250 IU / mL, 50 IU / mL or about 50 IU / mL to 100 IU / mL or about 100 IU / mL, 100 IU / mL or about 100 IU / mL to 500 IU / mL or about 500 IU / mL, 100 IU / mL or about 100 IU / mL to 250 IU / mL or about 250 IU / mL, or 250 IU / mL or about 250 IU / mL to 500 IU / mL or about 500 IU / mL (both inclusive). In some embodiments, the concentration of IL-2 during at least a portion of the incubation, e.g., at the beginning of the culture and, optionally, added one or more times during the culture, is 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 125 IU / mL, 150 IU / mL, 200 IU / mL, or about 50 IU / mL, about 60 IU / mL, about 70 IU / mL, about 80 IU / mL, about 90 IU / mL, about 100 IU / mL, about 125 IU / mL, about 150 IU / mL, about 200 IU / mL, or any value between any of the above. In certain embodiments, the concentration of recombinant IL-2 at the beginning of the culture and, optionally, added one or more times during the culture is 100 IU / mL or about 100 IU / mL. In certain embodiments, the concentration of recombinant IL-2 added at the initiation of the culture, and optionally one or more times during the culture, is at or about 500 IU / mL.

[0303] In certain embodiments, provided methods include incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-21. In some embodiments, recombinant IL-21 is added during at least a portion of the incubation, e.g., at the beginning of the culture, and optionally one or more times during the culture, at a concentration of 1 IU / mL or about 1 IU / mL to 500 IU / mL or about 500 IU / mL, e.g., 1 IU / mL or about 1 IU / mL to 250 IU / mL or about 250 IU / mL, 1 IU / mL or about 1 IU / mL to 100 IU / mL or about 100 IU / mL, 1 IU / mL or about 1 IU / mL to 50 IU / mL or about 50 IU / mL, 50 IU / mL or about 50 IU / mL. and about 500 IU / mL or 500 IU / mL, 50 IU / mL or about 50 IU / mL to 250 IU / mL or about 250 IU / mL, 50 IU / mL or about 50 IU / mL to 100 IU / mL or about 100 IU / mL, 100 IU / mL or about 100 IU / mL to 500 IU / mL or about 500 IU / mL, 100 IU / mL or about 100 IU / mL to 250 IU / mL or about 250 IU / mL, or 250 IU / mL or about 250 IU / mL to 500 IU / mL or about 500 IU / mL (both inclusive). In some embodiments, the concentration of IL-21 during at least a portion of the incubation, e.g., at the beginning of the culture, and optionally added one or more times during the culture, is 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 125 IU / mL, 150 IU / mL, 200 IU / mL, or about 50 IU / mL, about 60 IU / mL, about 70 IU / mL, about 80 IU / mL, about 90 IU / mL, about 100 IU / mL, about 125 IU / mL, about 150 IU / mL, about 200 IU / mL, or any value between any of the above. In a specific embodiment, the concentration of recombinant IL-21 at the beginning of the culture, and optionally added one or more times during the culture, is 100 IU / mL or about 100 IU / mL.

[0304] In certain embodiments, provided methods involve incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-21. In certain embodiments, the concentration of recombinant IL-21 during at least a portion of the culture, e.g., at the initiation of the culture and optionally added one or more times during the culture, is about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 90 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 10 ng / mL to about 20ng / mL, about 20ng / mL to about 100ng / mL, about 20ng / mL to about 90ng / mL, about 20ng / mL to about 80ng / mL, about 20ng / mL to about 70ng / mL, about 20ng / mL to about 60ng / mL, about 20ng / mL to about 50n g / mL, approximately 20ng / mL to approximately 40ng / mL, approximately 20ng / mL to approximately 30ng / mL, approximately 30ng / mL to approximately 100ng / mL, approximately 30ng / mL to approximately 90ng / mL, approximately 30ng / mL to approximately 80ng / mL, approximately 30ng / mL to approximately 70ng / mL , about 30ng / mL to about 60ng / mL, about 30ng / mL to about 50ng / mL, about 30ng / mL to about 40ng / mL, about 40ng / mL to about 100ng / mL, about 40ng / mL to about 90ng / mL, about 40ng / mL to about 80ng / mL, about 4 0ng / mL to about 70ng / mL, about 40ng / mL to about 60ng / mL, about 40ng / mL to about 50ng / mL, about 50ng / mL to about 100ng / mL, about 50ng / mL to about 90ng / mL, about 50ng / mL to about 80ng / mL, about 50ng / mL mL to about 70ng / mL, about 50ng / mL to about 60ng / mL, about 60ng / mL to about 100ng / mL, about 60ng / mL to about 90ng / mL, about 60ng / mL to about 80ng / mL, about 60ng / mL to about 70ng / mL, about 70ng / mL to about 100ng / mL, about 70ng / mL to about 90ng / mL, about 70ng / mL to about 80ng / mL, about 80ng / mL to about 100ng / mL, about 80ng / mL to about 90ng / mL, or about 90ng / mL to about 100ng / mL (inclusive).In certain embodiments, the concentration of recombinant IL-21 during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, added one or more times during the culture, is about 10 ng / mL to about 100 ng / mL, inclusive. In certain embodiments, the concentration of recombinant IL-21 during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, added one or more times during the culture, is 25 ng / mL or about 25 ng / mL.

[0305] In certain embodiments, the concentration of recombinant IL-15 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, one or more times during the culture, 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 In certain embodiments, the concentration of recombinant IL-15 added during at least a portion of the culture, e.g., at the initiation of the culture, and optionally, one or more times during the culture, is about 1 ng / mL to about 50 ng / mL. In certain embodiments, the concentration of recombinant IL-15 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is at or about 10 ng / mL.

[0306] In certain embodiments, the methods include culturing in the presence of IL-2, IL-15, and IL-21. In embodiments of the provided methods, for example, the concentrations of recombinant cytokines at the initiation of the culture, and optionally added one or more times during the culture, are 50 IU / mL or about 50 IU / mL to 500 IU / mL or about 500 IU / mL of IL-2, e.g., 100 IU / mL or about 100 IU / mL or 500 IU / mL of IL-2, 1 ng / mL or about 1 ng / mL to 50 ng / mL of IL-15, e.g., 10 ng / mL or about 10 ng / mL, and 10 ng / mL or about 10 ng / mL to 100 ng / mL or about 100 ng / mL of IL-21, e.g., 25 ng / mL or about 25 ng / mL. In certain embodiments, 500 IU / mL of IL-2, 10 ng / mL of IL-15, and 25 ng / mL of IL-21 are added at the beginning of the culture and, optionally, during at least a portion of the culture, such as one or more times during the culture. In certain embodiments, 100 IU / mL of IL-2, 10 ng / mL of IL-15, and 25 ng / mL of IL-21 are added at the beginning of the culture and, optionally, during at least a portion of the culture, such as one or more times during the culture.

[0307] In some embodiments, provided methods include incubating or culturing enriched NK cells and feeder cells in the presence of recombinant IL-21, where the recombinant IL-21 is added as a complex with an anti-IL-21 antibody. In some embodiments, prior to culturing, 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 some embodiments, contacting the recombinant IL-21 with the anti-IL-21 antibody to form the IL-21 / anti-IL-21 complex is performed under conditions, including temperature and time, suitable for complex formation. In some embodiments, culturing is performed at 37°C ± 2°C for 30 minutes.

[0308] In some embodiments, the anti-IL-21 antibody has a concentration of 100 ng / mL or about 100 ng / mL to 500 ng / mL or about 500 ng / mL, 100 ng / mL or about 100 ng / mL to 400 ng / mL or about 400 ng / mL, 100 ng / mL or about 100 ng / mL to 300 ng / mL or about 300 ng / mL, 100 ng / mL or about 100 ng / mL to 200 ng / mL or about 200 ng / mL, 200 ng / mL or about 200 ng / mL to 500 ng / mL or about 500 In some embodiments, the anti-IL-21 antibody is added at a concentration of 100 ng / mL, or about 100 ng / mL to 500 ng / mL or about 500 ng / mL. In some embodiments, the anti-IL-21 antibody is added at a concentration of 250 ng / mL.

[0309] In certain embodiments, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is from about 10 ng / mL to about 100 ng / mL, from about 10 ng / mL to about 90 ng / mL, from about 10 ng / mL to about 80 ng / mL, from about 10 ng / mL to about 70 ng / mL, from about 10 ng / mL to about 60 ng / mL, from about 10 ng / mL to about 50 ng / mL, from about 10 ng / mL to about 40 ng / mL, from about 10 ng / mL to about 30 ng / mL, from about 10 ng / mL to about 20 ng / mL, or from about 20 ng / mL. L ~ about 100ng / mL, about 20ng / mL - about 90ng / mL, about 20ng / mL - about 80ng / mL, about 20ng / mL - about 70ng / mL, about 20ng / mL - about 60ng / mL, about 20ng / mL - about 50ng / mL, about 20ng / mL ~40ng / mL, approximately 20ng / mL~approximately 30ng / mL, approximately 30ng / mL~approximately 100ng / mL, approximately 30ng / mL~approximately 90ng / mL, approximately 30ng / mL~approximately 80ng / mL, approximately 30ng / mL~approximately 70ng / mL, approximately 30ng / mL~ Approximately 60ng / mL, approximately 30ng / mL to approximately 50ng / mL, approximately 30ng / mL to approximately 40ng / mL, approximately 40ng / mL to approximately 100ng / mL, approximately 40ng / mL to approximately 90ng / mL, approximately 40ng / mL to approximately 80ng / mL, approximately 40ng / mL to approximately 70ng / mL, about 40ng / mL to about 60ng / mL, about 40ng / mL to about 50ng / mL, about 50ng / mL to about 100ng / mL, about 50ng / mL to about 90ng / mL, about 50ng / mL to about 80ng / mL, about 50ng / mL to about 7 In a specific embodiment, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is about 10 ng / mL to about 100 ng / mL, inclusive.In a specific embodiment, the concentration of recombinant IL-21 used to form a complex with an anti-IL-21 antibody is at or about 25 ng / mL.

[0310] In certain embodiments, the concentration of recombinant IL-12 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, one or more times during the culture, 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, In certain embodiments, the concentration of recombinant IL-12 added during at least a portion of the culture, e.g., at the initiation of the culture, and optionally, one or more times during the culture, is about 1 ng / mL to about 50 ng / mL. In certain embodiments, the concentration of recombinant IL-12 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is at or about 10 ng / mL.

[0311] In certain embodiments, the concentration of recombinant IL-18 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, one or more times during the culture, 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, In certain embodiments, the concentration of recombinant IL-18 added during at least a portion of the culture, e.g., at the initiation of the culture, and optionally, one or more times during the culture, is about 1 ng / mL to about 50 ng / mL. In certain embodiments, the concentration of recombinant IL-18 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is 10 ng / mL or about 10 ng / mL.

[0312] In certain embodiments, the concentration of recombinant IL-27 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally, one or more times during the culture, 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, In certain embodiments, the concentration of recombinant IL-27 added during at least a portion of the culture, e.g., at the initiation of the culture, and optionally, one or more times during the culture, is about 1 ng / mL to about 50 ng / mL. In certain embodiments, the concentration of recombinant IL-27 added during at least a portion of the culture, e.g., at the beginning of the culture, and optionally one or more times during the culture, is at or about 10 ng / mL.

[0313] In some embodiments, the methods include changing the culture medium, and in some aspects, washing the cells. For example, during at least...

Claims

1. A composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in a method for treating multiple myeloma (MM), wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses.

2. Use of a composition of natural killer (NK) cells (g-NK cells) lacking expression of the FcRγ chain for the manufacture of a pharmaceutical for treating multiple myeloma (MM), wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses.

3. The composition of claim 1, wherein the g-NK cell composition is used in monotherapy without concomitant administration of an exogenous antibody to treat multiple myeloma.

4. 10. The composition of claim 1 used in combination with an antibody directed against a multiple myeloma antigen, optionally comprising: (a) the antibody is a full-length antibody, and optionally the multiple myeloma antigen comprises an antigen selected from the group consisting of CD38, SLAMF7, and BCMA; or (b) the antibody is a bispecific antibody, optionally wherein the bispecific antibody is directed against CD16 and a second multiple myeloma antigen selected from the group consisting of BCMA, SLAMF7, and CD38; composition.

5. 5. The composition of claim 4, wherein the antibody is administered once every four weeks, once every three weeks, once every two weeks, once a week, or twice a week.

6. at least one dose of an anti-CD38 antibody has been administered to the subject prior to administration of a dose of the composition of g-NK cells, and optionally, the administration of the at least one dose of the anti-CD38 antibody comprises: (a) initiated within one month prior to administration of the g-NK cell composition; (b) initiated within three weeks prior to administration of the g-NK cell composition; or (c) initiated within two weeks prior to administration of the g-NK cell composition; The composition of claim 4.

7. A composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in a method for treating multiple myeloma (MM), wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses, and the subject has previously been administered at least one dose of an anti-CD38 antibody.

8. Use of a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for the manufacture of a pharmaceutical for treating multiple myeloma (MM), wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses, and the subject has previously been administered at least one dose of an anti-CD38 antibody.

9. 8. The composition of any one of claims 1 and 3-7, wherein the g-NK cell composition is administered as two doses in a 14-day cycle, the 14-day cycle being repeated 1 to 3 times, optionally for a total of 6 doses of the g-NK cell composition.

10. The composition of claim 7 , wherein the anti-CD38 antibody is administered intravenously or subcutaneously. (a) the anti-CD38 antibody is administered as a weekly dose, optionally over one or two 28-day cycles; and / or (b) each dose of the anti-CD38 antibody is administered in an amount that is at or about 8 mg / kg to about 32 mg / kg, optionally at or about 16 mg / kg; The composition of claim 7.

12. the anti-CD38 antibody is administered in an anti-CD38 antibody composition comprising hyaluronidase, optionally the anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (e.g., hyaluronidase-fihj), and optionally (a) the anti-CD38 antibody composition is administered as a weekly dose, optionally over one or two 28-day cycles; and / or (b) each dose of the anti-CD38 antibody composition comprises 1200 mg, or about 1200 mg to about 2400 mg, of an anti-CD38 antibody and 15,000 units (U), or about 15,000 U to about 45,000 U of a hyaluronidase (e.g., hyaluronidase-fihj); The composition of claim 7.

13. The composition of claim 12, wherein the anti-CD38 antibody composition is administered once a week for a total of eight doses, the g-NK cell composition is administered once a week for a total of six doses, and one or two doses of the anti-CD38 antibody are administered prior to the administration of the g-NK cell composition.

14. The composition of any one of claims 1, 3 to 7 and 10 to 13, wherein the multiple myeloma is relapsed / refractory multiple myeloma. (a) the cells in the g-NK cell composition have low or no expression of CD38, and optionally, less than 25% of the cells in the g-NK cell composition are positive for surface CD38. (b) the cells in said g-NK cell composition have not been engineered to reduce or eliminate CD38 expression, and / or (c) cells in the g-NK cell composition exhibit minimal anti-CD38-induced fratricide, optionally, less than 10% of cells in the g-NK cell composition exhibit anti-CD38-induced fratricide; The composition according to any one of claims 1, 3 to 7, and 10 to 13.

16. A composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in a method for treating lymphoma, wherein the composition of g-NK cells is administered once a week in a predetermined number of doses.

17. Use of a composition comprising natural killer (NK) cells (g-NK cells) lacking expression of the FcRγ chain for the manufacture of a medicament for treating lymphoma, wherein the g-NK cell composition is administered once a week in a predetermined number of doses.

18. The composition of claim 16, wherein the g-NK cell composition is used in monotherapy without concomitant administration of an exogenous antibody to treat the lymphoma.

19. 17. The composition of claim 16 used in combination with an antibody against a lymphoma antigen, optionally comprising: (a) the antibody is a full-length antibody, and optionally the lymphoma antigen comprises an antigen selected from the group consisting of CD19, CD20, and CD30; or (b) the antibody is a bispecific antibody, optionally the bispecific antibody being directed against CD16 and a second antigen selected from the group consisting of CD19, CD20, and CD30; composition.

20. 20. The composition of claim 19, wherein the antibody is administered once every four weeks, once every three weeks, once every two weeks, once a week, or twice a week.

21. at least one dose of an anti-CD20 antibody has been administered to the subject prior to administration of a dose of the composition of g-NK cells, and optionally, administration of the at least one dose of the anti-CD20 antibody comprises: (a) initiated within one month prior to administration of the g-NK cell composition; (b) initiated within three weeks prior to administration of the g-NK cell composition; or (c) initiated within two weeks prior to administration of the g-NK cell composition; 20. The composition of claim 19.

22. A composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for use in a method for treating lymphoma, wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses, and the subject has previously been administered at least one dose of an anti-CD20 antibody.

23. Use of a composition of natural killer (NK) cells (g-NK cells) deficient in expression of the FcRγ chain for the manufacture of a pharmaceutical for treating lymphoma, wherein the composition of g-NK cells is administered to a subject once a week in a predetermined number of doses, and the subject has previously been administered at least one dose of an anti-CD20 antibody.

24. The composition of any one of claims 16 and 18 to 22, wherein the lymphoma is non-Hodgkin's lymphoma (NHL).

25. 23. The composition of any one of claims 16 and 18-22, wherein the g-NK cell composition is administered as two doses in a 14-day cycle, the 14-day cycle being repeated 1 to 3 times, optionally for a total of 6 doses of the g-NK cell composition.

26. 23. The composition of claim 22, wherein the anti-CD20 antibody is rituximab.

27. 23. The composition of claim 22, wherein the anti-CD20 antibody is administered intravenously or subcutaneously.

28. (a) the anti-CD20 antibody is administered as a weekly dose, optionally in four or eight doses; and / or (b) each dose of the anti-CD20 antibody is administered in an amount that is at or about 250 mg / m 2 to 500 mg / m 2 , optionally at or about 375 mg / m 2 ; 23. The composition of claim 22.

29. the anti-CD20 antibody is administered in an anti-CD20 antibody composition comprising hyaluronidase, optionally the anti-CD20 antibody composition comprising rituximab and human recombinant hyaluronidase PH20, and optionally (a) the anti-CD20 antibody composition is administered as a weekly dose, optionally in four or eight doses, or optionally in three or seven doses following an intravenous weekly dose of the anti-CD20 antibody; and / or (b) each dose of the anti-CD20 antibody composition comprises 1200 mg, or about 1200 mg to about 2400 mg, of anti-CD20 antibody and 15,000 units (U), or about 15,000 U to about 45,000 U of hyaluronidase; 23. The composition of claim 22.

30. The composition of claim 29, wherein the anti-CD20 antibody composition is administered once a week for a total of eight doses, the g-NK cell composition is administered once a week for a total of six doses, and one or two doses of the anti-CD20 antibody are administered prior to administration of the g-NK cell composition. (a) of the cells in the composition of g-NK cells, more than or about 60% of the cells are g-NK cells. (b) at least 50% or about 50% of the cells in said composition of g-NK cells are FcRγ-deficient (FcRγ neg ) NK cells (g-NK); (c) greater than or about 70% of the g-NK cells are positive for perforin and greater than or about 70% of the g-NK cells are positive for granzyme B; (d) greater than 10% of cells in the composition of g-NK cells are capable of degranulation against tumor target cells, optionally as measured by CD107a expression, and optionally, said degranulation is measured in the absence of an antibody against said tumor target cells; (e) more than or about more than 15% of the cells in the composition of g-NK cells 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); (f) more than 10% of the cells in said composition of g-NK cells are capable of producing interferon-gamma or TNF-alpha against tumor target cells, optionally wherein said interferon-gamma or TNF-alpha is measured in the absence of antibodies against said tumor target cells; and / or (g) more than or about more than 15% of the cells in the composition of g-NK cells produce an effector cytokine in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody), and optionally the effector cytokine is (i) IFN-gamma or TNF-alpha, or (ii) IFN-gamma and TNF-alpha; The composition according to any one of claims 1, 3 to 7, 10 to 13, 16, 18 to 22, and 26 to 30.

32. (a) The composition of g-NK cells is produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, and the CD3- / CD57+ cells are enriched from a biological sample derived from a donor subject; or (b) the composition of g-NK cells is produced by ex vivo expansion of CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, and the CD3- / CD56+ cells are enriched from a biological sample derived from a donor subject; The composition of any one of claims 1, 3 to 7, 10 to 13, 16, 18 to 22, and 26 to 30. (a) the donor subject is CMV-seropositive, and / or (b) the donor subject has a CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype, and optionally the biological sample is derived from a human subject selected for a CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype; 33. The composition of claim 32.

34. 33. The composition of claim 32, wherein at least 20% or about 20% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2C), and at least 70% of NK cells in the peripheral blood sample are negative or have low NKG2A (NKG2A).

35. The irradiated feeder cells (a) HLA class I and HLA class II deficient; and / or (b) 221.AEH cells; 33. The composition of claim 32.

36. (a) the culturing is performed in the presence of two or more recombinant cytokines; (i) at least one recombinant cytokine is interleukin (IL)-2 and at least one recombinant cytokine is IL-21; (ii) the recombinant cytokines are IL-21 and IL-2, or (iii) the recombinant cytokines are IL-21, IL-2, and IL-15; or (b) the expansion is (i) including exogenous cytokine support to facilitate expansion or persistence of said g-NK cells in vivo in said administered subject, optionally wherein said exogenous cytokine is or includes IL-15; or (ii) does not include administration of exogenous cytokines to the subject to support the survival or expansion of NK cells, wherein the exogenous cytokines are one or more of IL-2, IL-7, IL-15, or IL-21; 33. The composition of claim 32. (a) the cells in the composition of g-NK cells are derived from a single donor subject expanded from the same biological sample; and / or (b) 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); The composition of any one of claims 1, 3 to 7, 10 to 13, 16, 18 to 22, and 26 to 30. (a) the g-NK cells are not engineered with an antigen receptor, and optionally, the antigen receptor is a chimeric antigen receptor; or (b) the g-NK cells have not been engineered with a secreted cytokine, optionally with a cytokine receptor fusion protein such as an IL-15 receptor fusion (IL-15RF); The composition of any one of claims 1, 3 to 7, 10 to 13, 16, 18 to 22, and 26 to 30.

39. Each dose of g-NK cells was 1 × 10 ８ or about 1 x 10 ８ cells ~ 50 x 10 ９ or about 50 x 10 ９ and / or a composition of said g-NK cells of 100 cells. Prior to said administration of said dose of g-NK cells, said subject has undergone lymphocyte depletion therapy, and optionally, administration of a dose of g-NK cells is initiated within, at or about 2 weeks after initiation of said lymphocyte depletion therapy; and / or optionally, (a) the lymphocyte depletion therapy comprises fludarabine and / or cyclophosphamide, optionally comprising administration of fludarabine at or about 20-40 mg / m 2 of a subject's body surface area, optionally at or about 30 mg / m 2 of a subject's body surface area, daily for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m 2 of a subject's body surface area, optionally at or about 300 mg / m 2 of a subject's body surface area, daily for 2-4 days; (b) the lymphocyte depletion therapy comprises fludarabine and cyclophosphamide, optionally wherein the lymphocyte depletion therapy comprises daily administration of fludarabine at or about 30 mg / m 2 of a subject's body surface area, and cyclophosphamide at or about 300 mg / m 2 of a subject's body surface area, each for 2 to 4 days, optionally for 3 days; The composition of any one of claims 1, 3 to 7, 10 to 13, 16, 18 to 22, and 26 to 30.

40. 31. The composition of any one of claims 1, 3-7, 10-13, 16, 18-22, and 26-30, wherein the individual is a human, and optionally the cells in the composition of NK cells are allogeneic to the individual.

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