Treatment of autoimmune diseases or conditions with natural killer cells

Abstract

Provided are methods and uses for treating an autoimmune disease or condition with Natural Killer (NK) cells, involving dosing of compositions containing NK cells deficient in FcRγ chain (g-NK cells). Among the provided methods and uses are methods and uses for treating various autoimmune diseases or conditions.

Description

TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLSCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 575,607, filed April 5, 2024, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 651,387, filed May 23, 2024, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 663,678, filed June 24, 2024, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 716,715, filed November 5, 2024, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 728,108, filed December 4, 2024, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” and U.S. provisional application No. 63 / 744,780, filed January 13, 2025, entitled “TREATMENT OF AUTOIMMUNE DISEASES OR CONDITIONS WITH NATURAL KILLER CELLS,” the contents of which are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 776032002040SeqList, created April 3, 2025, which is 118,465 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure provides methods and uses for treating an autoimmune disease or condition with Natural Killer (NK) cells, involving dosing of compositions containing NK cells deficient in FcRy chain (g-NK cells). Among the provided methods and uses are methods and uses for treating various autoimmune diseases or conditions.Background

[0004] Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity when the Fc receptor (CD16; FcyRIII) binds to the Fc portion of antibodies bound to anantigen-bearing cell. NK cells, including specific specialized subsets thereof, can be used in therapeutic methods. Improved methods involving NK cells are needed for therapeutic uses related to the treatment of autoimmune diseases or disorders, including methods of using the NK cells as a monotherapy. Provided herein are embodiments that meet such needs.Summary

[0005] Provided herein is a method of killing autoreactive cells, wherein the method includes contacting autoreactive cells with a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells). In the preceding embodiment, the autoreactive cells include cells that express HLA-E and / or have upregulated HLA-E expression. Also provided herein is a method of killing autoreactive cells, wherein the method includes contacting autoreactive cells that have upregulated HLA-E expression with a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells).

[0006] In any of the preceding embodiments, the autoreactive cells are autoreactive B and / or autoreactive T cells. In any of the preceding embodiments, the autoreactive cells express HLA-E and / or have upregulated HLA-E expression. In any of the preceding embodiments, the upregulated HLA-E expression is caused by a viral infection. In the preceding embodiment, the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV). In the preceding embodiment, the viral infection is an Epstein-Barr virus (EBV).

[0007] In any of the preceding embodiments, the autoreactive cells are central nervous system (CNS)-autoreactive cells. In the preceding embodiment, the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and / or ANO2. In the preceding embodiment, the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

[0008] In any of the preceding embodiments, the autoreactive cells are present in a subject having an autoimmune disease or disorder.

[0009] In any of the preceding embodiments, the autoreactive cells are present in a subject who is likely or suspected to develop an autoimmune disease or disorder.

[0010] Provided herein is a method of treating an autoimmune disease or disorder, wherein the method includes administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder. In the preceding embodiment, the method may further include selecting a subject with the autoimmune disease or disorder for treatment prior to administering the composition of g-NK cells.

[0011] Additionally provided herein is a method of treating an autoimmune disease or disorder, wherein the method includes (a) selecting a subject with an autoimmune disease or disorder; and (b)administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the autoimmune disease or disorder. In any of the preceding embodiments, the method may further include administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder. In some embodiments, the target antigen is a B cell antigen.

[0012] Also provided herein is a method of prophylactically treating an autoimmune disease or disorder, wherein the method includes administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject who is likely or suspected to develop an autoimmune disease or disorder. In the preceding embodiment, the method may further include selecting a subject who is likely or suspected to develop an autoimmune disease or disorder for treatment prior to administering the composition of g-NK cells.

[0013] Also provided herein is a method of prophylactically treating an autoimmune disease or disorder, wherein the method includes: (a) selecting a subject who is likely or suspected to develop an autoimmune disease or disorder; and (b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to the subject who is likely or suspected to develop an autoimmune disease or disorder.

[0014] In some aspects, the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for the presence of autoreactive cells prior to administering the composition of g-NK cells. In some embodiments, the autoreactive cells are autoreactive B cells and / or T cells. In some embodiments, the autoreactive cells express HLA-E and / or have upregulated HLA-E expression. In some embodiments, the autoreactive cells are central nervous system (CNS)-autoreactive cells. In some embodiments, the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and / or ANO2. In some embodiments, the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

[0015] In some embodiments, the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having radiologically isolated syndrome (RIS).

[0016] In some embodiments, the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having clinically isolated syndrome (CIS).

[0017] Also provided herein is a method of treating an autoimmune disease or disorder, wherein the method includes (a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the autoimmune disease or disorder; and (b) administering to the subject an antibody that is directed against a B cell antigen. In some embodiments, the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg). In some embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos). In someembodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

[0018] In some aspects, the autoimmune disease or disorder is systemic lupus erythematosus (SLE), systemic sclerosis (SSc), myositis (IIM), rheumatoid arthritis (RA), or multiple sclerosis (MS). In some aspects, the autoimmune disease or disorder is a kidney or renal disease. In certain embodiments, the kidney or renal disease is systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).

[0019] In some aspects, pathogenesis of the autoimmune or disorder is associated with a viral infection. In some embodiments, the autoimmune disease or condition is characterized by autoreactive B cells or autoreactive T cells with upregulated HLA-E expression. In some embodiments, the upregulation of HLA-E expression is caused by a viral infection. In some embodiments, the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV). In particular embodiments, the viral infection is an Epstein-Barr virus (EBV).

[0020] Also provided herein is a method of treating a disease or disorder associated with an Epstein- Barr virus (EBV), wherein the method includes administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder. In some embodiments, the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg). In some embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos). In some embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

[0021] In some aspects, the methods may include administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder. In some embodiments, the target antigen is a B cell antigen.

[0022] In any of the preceding embodiments, the autoimmune disease or disorder is multiple sclerosis (MS). Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS).

[0023] In some aspects, the method does not comprise administering an antibody to the subject in combination with the composition of g-NK cells. In the preceding embodiment, the antibody is an antibody directed against a target antigen associated with an autoimmune disease or disorder, optionally wherein the target antigen is a B cell antigen.

[0024] Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising: (a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS); and (b) administering to the subject an antibody that is directed against a B cell antigen.

[0025] In some aspects, the subject with multiple sclerosis has Epstein-Barr virus (EBV)-infected cells. In some embodiments, the EBV-infected cells are EBV-infected B cells. In some embodiments, the subject is seropositive for an EBV infection.

[0026] In some embodiments, the subject with multiple sclerosis is seropositive for Epstein-Barr nuclear antigen 1 (EBNA). In some embodiments, wherein the subject with multiple sclerosis has a high level of Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNA)386-405 antibodies.

[0027] In some embodiments, the subject has autoreactive cells. In some embodiments, the autoreactive cells are autoreactive B cells and / or T cells. In some embodiments, the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

[0028] In some embodiments, the autoreactive cells are central nervous system (CNS)-autoreactive cells. In some embodiments, the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and / or ANO2. In some embodiments, the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

[0029] In some embodiments, the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and / or T cells. In some embodiments, the subject is characterized by an HLA-E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-l)-derived peptide. In certain embodiments, the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20. In certain embodiments, the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.

[0030] In some embodiments, among cells in the composition of g-NK cells, greater than at or about 20% of the cells are g-NK cells. In some embodiments, among cells in the composition of g-NK cells, greater than at or about 30% of the cells are g-NK cells, greater than at or about 40% of the cells are g- NK cells, greater than at or about 50% of the cells are g-NK cells, greater than at or about 60% of the cells are g-NK cells, greater than at or about 70% of the cells are g-NK cells, greater than at or about 80% of the cells are g-NK cells, greater than at or about 90% of the cells are g-NK cells, or greater than at or about 95% of the cells are g-NK cells. In some embodiments, at least at or about 15% of the NK cells of the composition are positive for NKG2C (NKG2Cpos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2Aneg).

[0031] In some embodiments, the antibody is a full-length antibody. In some embodiments, the B cell antigen is expressed on a cell of a B cell lineage. In some embodiments, the B cell antigen is antigen selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI. In some embodiments, the cell of the B cell lineage is an autoreactive B cell. In particular embodiments, the cell of the B cell lineage is selected from the group consisting of pro-B cells, pre-B cells, immature B cells, naive B cells, germinal center B cells, memory B cells, plasmablasts and plasma cells.

[0032] In some embodiments, the antibody is an anti-CD19 antibody. In certain embodiments, the antibody is inebilizumab, tafasitamab-cxix or obexelimab.

[0033] In some embodiments, the antibody is an anti-CD20 antibody. Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising: (a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS); and (b) administering to the subject an anti-CD20 antibody. In certain embodiments, the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab. In certain embodiments, the antibody is ocrelizumab.

[0034] In some embodiments, the antibody is an anti-CD22 antibody. In certain embodiments, the antibody is epratuzumab. In some embodiments, the antibody is an anti-BAFF-R antibody. In certain embodiments, the antibody is belimumab.

[0035] In some embodiments, the antibody is an anti-CD38 antibody. In certain embodiments, the antibody is epratuzumab. In some embodiments, the antibody is an anti-BAFF-R antibody. In certain embodiments, the antibody is daratumumab or is isatuximab. In certain embodiments, less than 25% of the cells in the composition of g-NK cells are positive for surface CD38. In certain embodiments, the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression.

[0036] In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously.

[0037] In some embodiments, the composition of g-NK cells is administered once weekly for a predetermined number of doses. In some embodiments, the composition of g-NK cells is administered twice weekly for a predetermined number of doses.

[0038] In some embodiments, the composition of g-NK cells is administered three times weekly for a predetermined number of doses. In some embodiments, the composition of g-NK cells is dosed at a frequency of every other day (Q2D). Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising: (a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and (b) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

[0039] Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising: (a) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily at a frequency of every other day (Q2D) in a 7-day cycle; (b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is administered at a dose of from at or about 1 x 108cells to at or about 50 x 109cells and each dose is administered one time daily at a frequency of every other day (Q2D) in the 7-day cycle and on the sameday as the IL-2; and (c) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

[0040] Also provided herein is a method of treating an autoimmune disease or disorder, the method comprising: (a) administering a dose of IL-2 to the subject, wherein the dose is 0.25 million IU to 6 million IU and each dose is administered one time daily at a frequency of every day or every week day in a 7-day cycle; and (b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is administered at a dose of from at or about 1 x 108cells to at or about 50 x 109cells and each dose is administered one time daily at a frequency of every other day (Q2D) in the 7- day cycle.

[0041] In some embodiments, the method does not comprise administering to the subject an antibody.

[0042] In some embodiments, the method further comprises: (c) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

[0043] In some embodiments, the 7-day cycle is repeated twice, and each 7-day cycle is the same.

[0044] In some embodiments, the anti-CD20 antibody is ocrelizumab.

[0045] In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously. In some embodiments, the antibody is administered about once every six months. In some embodiments, the antibody is ocrelizumab and is administered once every six months at a dose at or about 600 mg. In some embodiments, a first dose of the antibody is initiated within one month prior to the administration of the composition of g-NK cells. In some embodiments, a first dose of the antibody is initiated within one week prior to the administration of the composition of g-NK cells. In some embodiments, a first dose of the antibody is initiated about six days prior to the administration of the composition of g-NK cells. In some embodiments, a first dose of the antibody is initiated about one day prior to the administration of the composition of g-NK cells.

[0046] In some embodiments, the composition of g-NK cells is administered in a 7-day cycle. In some embodiments, the 7-day cycle is repeated one to three times. In some embodiments, the 7-day cycle is repeated one time. In some embodiments, the 7-day cycle is repeated two times.

[0047] In some embodiments, the composition of g-NK cells is administered from two total doses to six total doses. In some embodiments, the composition of g-NK cells is administered as two total doses. In some embodiments, the composition of g-NK cells is administered as three total doses.

[0048] In some embodiments, the composition of g-NK cells is administered on Day 0, Day 2 and Day 4. In some embodiments, the composition of g-NK cells is administered as four total doses. In some embodiments, the composition of g-NK cells is administered as six total doses.

[0049] In some embodiments, at least at or about 20% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK). In some embodiments, wherein at least at or about 40% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

[0050] In some embodiments, greater than at or about 70% of the g-NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B. In some embodiments, (i) greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B, (ii) greater than at or about 90% of the g- NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B, or (iii) greater than at or about 95% of the g-NK cells are positive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B. In some embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos; and / or among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0051] In some embodiments, greater than 10% of the cells in the composition of g-NK cells are capable of degranulation against target cells, optionally as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of an antibody against the target cells. In some embodiments, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% exhibit degranulation, optionally as measured by CD 107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

[0052] In some embodiments, greater than 10% of the cells in the composition of g-NK cells are capable of producing interferon-gamma or TNF-alpha against target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of an antibody against the target cells. In some embodiments, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce an effector cytokine in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody). In particular embodiments, the effector cytokine is IFN-gamma or TNF-alpha. In particular embodiments, the effector cytokine is IFN-gamma and TNF-alpha.

[0053] In some embodiments, the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD56+ cells are enriched from a biological sample from a donor subject. In some embodiments, the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells are enriched from a biological sample from a donor subject.

[0054] In some embodiments, the donor subject is CMV-seropositive. In some embodiments, the donor subject has the CD16 F / F NK cell genotype. In some embodiments, the donor subject has the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype.

[0055] In some embodiments, at least at or about 15% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2Cpos) and at least 70% of NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2Aneg).

[0056] In some embodiments, the irradiated feeder cells are deficient in HLA class I and HLA class II. In particular embodiments, the irradiated feeder cells are 221. AEH cells. In some embodiments, the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is interleukin (IL) -2 and at least one recombinant cytokine is IL-21. In particular embodiments, the recombinant cytokines are IL-21 and IL-2. In particular embodiments, the recombinant cytokines are IL-21, IL-2, and IL- 15.

[0057] In some embodiments, the g-NK cells in the composition are from a single donor subject that have been expanded from the same biological sample. In some embodiments, 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 cry opreservation medium is 5% to 10% DMSO (v / v).

[0058] In some embodiments, the g-NK cells are not engineered with an antigen receptor, optionally wherein the antigen receptor is a chimeric antigen receptor.

[0059] In some embodiments, the g-NK cells are not engineered with a secreted cytokine, optionally a cytokine receptor fusion protein, such as IL-15 receptor fusion (IL-15RF). In some embodiments, the method does not include exogenous cytokine administration to the subject to support NK cell survival or expansion, wherein the exogenous cytokine is one or more of IL-2, IL-7, IL-15 or IL-21.

[0060] In some embodiments, any of the provided methods further comprises administering IFN-P to the subject. In some embodiments, the IFN- is administered three times a week. In some embodiments, each dose of IFN-P is about 22 pg to 44 pg, optionally wherein each dose of IFN-P is 44 pg.

[0061] In some embodiments, any of the provided methods further comprise administering exogenous cytokine support to facilitate expansion or persistence of the g-NK cells in vivo in the subject.In some of any of the provided embodiments, the methods include administering an exogenous cytokine that is or comprises IL- 15. In some of any of the provided embodiments, the methods include administering an exogenous cytokine that is or comprises IL-2.

[0062] In some of any of the provided embodiments, the method comprises administering IL-2 to the subject. In some embodiments, the IL-2 is administered once a week, two times a week or three times a week. In some embodiments, the IL-2 is administered at a frequency of every other day (Q2W). In some embodiments, the IL-2 is administered five times a week, six times a week, or seven times a week. In some embodiments, the IL-2 is administered at a frequency of every day (QD). In some embodiments, the IL-2 is administered at a frequency of every week day. In some embodiments, for each day of administration the IL-2 is administered once daily. In some embodiments, the IL-2 is administered in a cycling regimen of one or more 7-day cycles. In some embodiments, the IL-2 is administered in three 7- day cycles, optionally wherein the three 7-day cycles are in consecutive weeks. In some embodiments, each 7-day cycle is the same. In some embodiments, the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in one or more 7-day cycles. In some embodiments, the IL-2 is administered one time daily at a frequency of every day (QD) on day 0, day 1, day 2, day 3, day 4, day 5, and day 6 in one or more 7-day cycles. In some embodiments, the IL-2 is administered one time daily at a frequency of every day (QD) on day 0, day 1, day 2, day 3, and day 4, in one or more 7-day cycles. In some embodiments, the IL-2 is administered to the subject within about 1 hour of the administration of the g-NK cells. In some embodiments, each dose of the IL-2 is 0.25 million to 6 million IU. In some embodiments, wherein each dose of the IL-2 is at or about 1 million IU. In some embodiments, each dose of the IL-2 is 1 million to 12 million IU, In some embodiments, each dose of IL- 2 is 4 million IU to 8 million IU. In some embodiments, each dose of IL-2 is at or about 6 million IU. In some embodiments, the IL-2 is administered subcutaneously. In some embodiments, administration of the IL-2 is administered on the same day as a first dose of the composition of g-NK cells.

[0063] In some of any of the provided embodiments, the composition of g-NK cells is administered as at least one dose. In some of any of the provided embodiments, each dose of g-NK cells is from at or about from at or about 1 x 108cells to at or about 50 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is from at or about from at or about 5 x 109cells to at or about 20 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 5 x 108cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 5 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 10 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 20 x 109cells of the composition of g-NK cells.

[0064] In some of any of the provided embodiments, the method does not comprise administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells.

[0065] In some of any of the provided embodiments, prior to the administration of the composition of g-NK cells, the subject has received a lymphodepleting therapy. In some of any of the provided embodiments, the method further comprises administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells. In some embodiments, administration of a dose of the composition of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphodepleting therapy. In some embodiments, administration of a dose of the composition of g-NK cells is initiated within 7 days or at or about 7 days after initiation of the lymphodepleting therapy. In some embodiments, the lymphodepleting therapy comprises fludarabine and / or cyclophosphamide. In some embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some embodiments, the lymphodepleting comprises the administration of fludarabine at or about 20-40 mg / m2body surface area of the subject, optionally at or about 30 mg / m2, daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days. In some embodiments, the lymphodepleting therapy further comprises administration of mesna at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days. In some embodiments, the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at or about 400 mg / m2body surface area of the subject and mesna at or about 300 mg / m2, daily, each for 2-4 days, optionally 3 days.

[0066] In some of any of the provided embodiments, the subject is a human subject. In some embodiments, the subject is aged 18 to 65 years old.

[0067] In some of any of the provided embodiments, the subject has Epstein-Barr virus (EBV)- infected cells. In some embodiments, the EBV-infected cells are EBV-infected B cells. In some embodiments, the subject is seropositive for an EBV infection. In some embodiments, the subject is seropositive for Epstein-Barr nuclear antigen 1 (EBNA). In some embodiments, the subject has a high level of Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNA)386-405 antibodies.

[0068] In some embodiments, the subject has autoreactive cells. In some embodiments, the autoreactive cells are autoreactive B cells and / or T cells. In some embodiments, the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

[0069] In some embodiments, the autoreactive cells are central nervous system (CNS)-autoreactive cells. In some embodiments, the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and / or ANO2. In some embodiments, the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

[0070] In some embodiments, the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and / or T cells. In some embodiments, the subject is characterized by an HLA-E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-l)-derived peptide. In someembodiments, the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20. In some embodiments, the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.

[0071] In some embodiments, the multiple sclerosis is a treatment refractory progressive multiple sclerosis. In some embodiments, the multiple sclerosis is a subject has primary or non-active secondary progressive MS (SPMS), where non-active SPMS is defined by the absence of clinical relapse in previous two years. In some embodiments, the subject does not have relapsing remitting MS. In some embodiments, the subject has relapsing remitting MS. In some embodiments, the subject does not have active SPMS. In some embodiments, the subject has not received a last treatment of a B-cell depletion therapy in the past 48 weeks. In some embodiments, the subject has received a last treatment of a B-cell depletion therapy in the past 48 weeks. In some embodiments, the subject has multiple sclerosis is characterized by one or more, optionally all, of the following: (a) Expanded Disability Status Scale (EDSS) at screening is from 3.0 to 6.5 points; (b) score of >2.0 on the Functional Systems (FS) scale for the pyramidal system that is due to lower extremity findings; (c) disease duration from the onset of MS symptoms that is either less than 15 years in subjects with an EDSS at screening >5.0 or less than 10 years in subjects with an EDSS at screening <5.0; (d) history or presence in a cerebrospinal fluid (CSF) specimen of: (1) elevated immunoglobulin G index, or (2) two or more immunoglobulin G oligoclonal bands detected by isoelectric focusing; or (e) neurologic stability >30 days prior to treatment. In some embodiments, the subject selected for treatment has hematology values of ANC >1000 cells / mm3, platelet count >75,000 cells / mm3, and hemoglobin > 9.0 g / dL. In some embodiments, the subject has been dosed with a dosing regimen of the antibody within the prior 6 months before initiation of the administration of the composition of g-NK cells in combination with the antibody.Brief Description of the Drawings

[0072] FIG. 1 depicts the preferential expansion of g-NK cells when starting with >10% preexpansion NKG2C+ / NKG2A- NK-cells. Values are mean + SE (N=8). p < 0.05.

[0073] FIGs. 2A and 2B depict representative flow cytometry results, of conventional NK cells (FIG. 2A) and g-NK cells (FIG. 2B), prior to expansion, for intracellular expression of FceRly (abbreviated FcRy) chain and surface expression of NKG2A and NKG2C.

[0074] FIG. 3 shows the correlation between the percentage of g-NK cells and the percentage of NKG2C+ / NKG2A- expanded NK cells.

[0075] FIG. 4 depicts representative flow cytometry histograms of expanded conventional NK cells (Expansion A and Expansion B) and g-NK cells (Expansion C and Expansion D). The percentages of NKG2C+ / NKG2A- expression as well as FceRly expression are shown.

[0076] FIGs. 5A and 5B depicts the ADCC activity, after 4 hours, of g-NK cells with or without an anti-CD19 antibody against negatively enriched B cells from a healthy donor (FIG. 5 A) or a subject with systemic lupus erythematosus (SLE; FIG. 5B) across a 5-log titration. (N = 1 donor tested in technical triplicate).

[0077] FIG.6A demonstrates the ADCC activity, after 24 hours, of g-NK cells with 1 pg / mL of anti-CD19 ADCC antibody against B cells from either a healthy donor (N=l donor - tested in duplicate) or patient with SLE (N=2 patients). The effectortarget (E:T) ratio was 5:1. FIG. 6B shows representative dot plots of NK and SLE subject B cells co-cultures (5:1, E:T) after 24 hours gated on PI (live cells). FIG. 6C (*p<0.05, **p<0.005, two-way ANOVA with Sidak correction for multiple comparisons) demonstrates percent cytotoxicity, after 4 hours or 24 hours, of g-NK cells with or without 1 pg / mL of anti-CD19 ADCC antibody against B cells from patients with SLE (N=4 patients). The effectortarget (E:T) ratio was 2:1. FIG. 6D (*p<0.05, unpaired t test, two-tailed) demonstrates the ADCC activity, after 4 hours or 24 hours, of g-NK cells with or without 1 pg / mL of anti-CD19 ADCC antibody against B cells from each of four unique patients with SLE. Average + / - SEM ADCC activity of all four donors is also shown. The effector Parget (E:T) ratio was 2:1.

[0078] FIG. 7A depicts the percentage of B cell death in the presence of g-NK cells and an anti- CD19 antibody, after 4 hours, after 24 hours, or after 48 hours of incubation. B cell death is calculated as PI+ fraction of CD20+ cells. FIG. 7B shows representative dot plots of NK and SLE subject B cells cocultures (5:1, E:T) after 24 hours gated on PI (live cells). Data shown are of a healthy donor (HD; N=1 donor - tested in duplicate) or subjects with SLE (N=2 patients).

[0079] FIG. 8A depicts the percentage of g-NK cells bound antibody across time (minutes). The data is plotted at % cells with surface-bound mAh normalized to time = 0 (The percentage at t = 0 was about 32%).

[0080] FIG.8B is a representative schematic depicting the transport of surface-bound monoclonal antibody (e.g., ocrelizumab) by g-NK cells into CNS and lymph nodes.

[0081] FIG. 9 depicts three orthogonal mechanisms of action (MOA) of g-NK cells for treating of autoimmune disease, such as multiple sclerosis (MS), either mAh dependent mechanism (left box) or mAh independent mechanisms (center and right box). The mAh dependent mechanism in the left box shows the deep depletion of B cell antigen-positive (e.g., CD20) B cells and CD20+ T cells via ADCC for immune reset. Of relevance, auto-antibody producing long-lived plasma cells are poorly targeted by a B cell antigen-targeting antibody (e.g., anti-CD20 mAh). See Bosiki et al., Cells 2022, 11, 1959. The mAh independent mechanism in the center box shows depletion of HLA-E expressing autoreactive T and plasma B cells. Of relevance, auto-antibody producing plasma cells, plasma blast, and autoreactive CD4 and CD8 T cells are targeted by this mechanism. Finally, the mAh independent mechanism in the right box shows elimination of cells latently infected with EBV virus.

[0082] FIG. 10 depicts surface HLA-E expression by mean fluorescence intensity (MFI) via flow cytometry on central nervous system (CNS)-specific and control non-CNS-responsive B and T cells derived from 18 patients with multiple sclerosis (pools of 3). CNS-specific B cells, CD4+ T cells, and CD8+ T cells proliferated in response to a pool of CNS peptides set forth in Table 4.

[0083] FIG. 11A depicts degranulation (CD 107a) measured by flow cytometry in g-NK cells (left) or primary NK (pNK; right) following co-culture of CNS-specific and non-CNS-specific immune cells subsets (CD19+ B cells, CD4+ T cells and CD8+ T cells) with either g-NK or primary CD56dimCD94+NKG2C+ (pNK) effector cells at a 1:1 E:T ratio. Each data point represents a single NK cell donor co-culture. Data are shown as mean + SD. Two-way ANOVA with Tukey post-hoc test (**p<0.001, ****p<0.0001).

[0084] FIG. 11B depicts Granzyme B (GranB) measured by flow cytometry in g-NK cells (left) or primary NK (pNK; right) following co-culture of CNS-specific and non-CNS-specific immune cells subsets (CD19+ B cells, CD4+ T cells and CD8+ T cells) with either g-NK or primary CD56dimCD94+NKG2C+ (pNK) effector cells at a 1:1 E:T ratio. Each data point represents a single NK cell donor co-culture. Data are shown as mean + SD. Two-way ANOVA with Tukey post-hoc test (**p<0.001, ****p<0.0001).

[0085] FIG. 11C depicts target cell killing through percentage of Annexin-positive target cells following co-culture (8 hours) of g-NK or primary NK (pNK) cells with B and T cells that were CNS- specific or non-CNS-specific.

[0086] FIG. 12 depicts target cell lysis as measured by LDH release follow co-culture of g-NK or primary NK (pNK) cells co-cultured with pooled target cells from 24 unique patient-derived CNS- specific T and B cells. Each dot represents one individual pools patient target cells. Data are shown as mean + SD. F-Test was used to compare standard deviations (****p<0.0001). Abbreviations: RLU, relative light units.

[0087] FIG. 13 shows the experimental schema for generation of target cells that were used to test the impact of cytokines and NK cell subsets on CNS-autoreactive immune cells. Specifically, PBMCs were isolated from blood samples collected from 30 patients with MS. These PBMCs were stimulated with CNS-derived autoantigens and then sorted into CD19+ B cells, CD4+ T cells, or CD8+ T cells which were then pooled into three pools of 10 patients each. These pooled cells were then used as target cells in subsequent experiments.

[0088] FIGs. 14A-14B show the surface expression of the NKG2C ligand HLA-E (FIG. 14A) or NKG2D ligands (FIG. 14B) on individual donor samples (each dot on graphs to left) as well as donor pools (10 donors per pool, each dot on graphs to right) on CNS-specific and non-CNS-specific CD19+ B cells and CD4+ and CD8+ T cells, as analyzed by flow cytometry. One-way ANOVA with Sidak’s testfor multiple comparisons. Data are shown as mean + SD. ****p<0.0001. Abbreviations: g-NK, g minus natural killer cell; pNK, primary natural killer cell; RLU, relative light units.

[0089] FIGs. 15A-15B show NK cell degranulation, as measured by CD 107a via flow cytometry, on pooled CNS-specific CD19+ cells (left), CD4+ cells (middle), or CD8+ cells (right) which have been cultured overnight in the absence (FIG. 15A) or presence (FIG. 15B) of IL-2. Each dot represents one NK cell donor. Data are shown as mean + SD. RM one-way ANOVA (with the Geisser-Greenhouse correction and Tukey post-hoc test) was used for statistical comparison (*p<0.01, ***p<0.001, ****p<0.0001). Of relevance, only g-NK cells were active against autoreactive target cells in the absence of cytokine activation.

[0090] FIGs. 16A-16C show NK cell degranulation, as measured by CD107a via flow cytometry, on pooled CNS-specific CD19+ cells (left), CD4+ cells (middle), or CD8+ cells (right) which have been cultured overnight in either IL- 12 (FIG. 16A), IL- 15 (FIG. 16B), or IL- 18 (FIG. 16C). Each dot represents one NK cell donor. Data are shown as mean + SD. RM one-way ANOVA (with the Geisser- Greenhouse correction and Tukey post-hoc test) was used for statistical comparison (*p<0.01, ***p<0.001, ****p<0.0001) (*p<0.01, ***p<0.001, ****p<0.0001).

[0091] FIGs. 17A-17D show NK cell degranulation, as measured by CD107a via flow cytometry, on pooled CNS-specific T and B cells that were co-cultured with either g-NK cells (FIG. 17A), sorted primary NK cell subset expressing NKG2C+ / NK2GD+(FIG. 17B), sorted primary NK cell subset expressing NKG2C 7NK2GD+(FIG. 17C), or sorted primary NK cell subset expressing NKG2C / NK2GD (FIG. 17D). Each dot represents one NK cell donor that was tested against CNS-specific CD8+ T cells, CNS-specific CD4+ T cells and CNS-specific B cells (three independent experiments for each NK cell donor). Data are shown as mean + SD. RM one-way ANOVA (with the Geisser-Greenhouse correction and Tukey post-hoc test) was used for statistical comparison (*p<0.01, ***p<0.001, ****p<0.0001) Abbreviations: IL, interleukin; NK, natural killer cell; ns, non-significant.Detailed Description

[0092] Provided herein are methods of treating autoimmune diseases and conditions, wherein the method includes administering a dose of cells of a composition of Natural Killer (NK) cells deficient in expression of the signaling adaptor Fc.epsilon.RI.gamma (FceRly; also called FcRy or gamma) chain (this subset of NK cells referred to as “g-NK cells”) to a subject having an autoimmune disease or condition. In some embodiments, the g-NK cells also are high in expression of NKG2C and low or negative in expression of NKG2A. In some embodiments, the g-NK cells are NKG2Cpos / NKG2AnegNK cells. In some embodiments, the provided methods relate to treating an autoimmune disease. In some embodiments, the autoimmune disease include, but are not limited to, as systemic lupus erythematosus(SLE), systemic sclerosis (SSc), multiple sclerosis (MS), idiopathic inflammatory myopathies (IIM), or rheumatoid arthritis (RA). In some embodiments, the provided methods relate to a kidney or a renal disease. In some embodiments, the kidney or renal disease include, but are not limited to, systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).

[0093] The provided embodiments are based on the exploitation of unique features of g-NK cells that the inventors have discovered that make g-NK cells particularly suitable for cell therapy methods for treatment of autoimmune diseases and conditions, including as a monotherapy. A problem with many existing treatments for autoimmune diseases or conditions, including by existing cell therapy approaches, is that they target only autoreactive B cells. Moreover, many existing treatments are not specific to cells of the disease or condition and act to deplete all B cells. The provided embodiments provide for advantageous methods that are more specific to killing cells associated with the autoimmune disease or condition. In addition, in some embodiments, g-NK cells provide a unique approach in that they are able to exhibit a dual mechanism to deplete autoreactive B cells and autoreactive T cells.

[0094] Natural killer (NK) cells are innate lymphocytes important for mediating immunity responses through cytokine and chemokine secretion, and through the release of cytotoxic granules (Vivier et al. Science 331(6013):44-49 (2011); Caligiuri, Blood 112(3):461-469 (2008); Roda et al., Cancer Res. 66(l):517-526 (2006)). Activation of NK cells can occur through the direct binding of NK cell receptors to ligands on the target cell, or through the crosslinking of the Fc receptor (CD16; also known as CD16a or FcyRIIIa) by binding to the Fc portion of antibodies bound to an antigen-bearing cell. Upon activation, NK cells produce cytokines and chemokines abundantly and at the same time exhibit potent cytolytic activity. This release of cytokines and chemokines can play a role in the cytolytic activity of NK cells in vivo. NK cells also have small granules in their cytoplasm containing perforin and proteases (granzymes). Upon release from the NK cell, perforin forms pores in the cell membrane of targeted cells through which the granzymes and associated molecules can enter, inducing apoptosis.

[0095] g-NK cells are a specialized subset of NK cells lacking the FcRy adaptor protein, also known as g-NK cells, that are able to mediate robust ADCC responses (see e.g., published Patent Appl. No. US2013 / 0295044). In some embodiments, g-NK cells are cells that do not express substantial FcRy but do express at least one marker for Natural Killer cells. An amino acid sequence for FcRy chain (Homo sapiens, also called the high affinity immunoglobulin gamma Fc receptor I) is available in the NCBI database as accession number NP_ 004097.1 (GL4758344), and is reproduced below as SEQ ID NO:1.MIPAVVEEEEEEVEQAAAEGEPQECYIEDAIEFEYGIVET EEYCREKIQVRKAAITSYEK SDGVYTGESTRNQETYETEKHEKPPQ (SEQ ID NO:1)

[0096] The mechanism for increased responses of g-NK cells may be due to changes in epigenetic modification that influence the expression of the FcRy chain as well are other factors such as Syk. Theseepigenetic modifications are promoted at least in part by response to CMV infection where this subset of NK cells arise in about 25% of CMV exposed individuals. This special subset is relatively rare because g-NK cells are detectable at levels of ~3% to 10% of total NK cells in only 25% to 30% of cytomegalovirus (CMV)-seropositive individuals; thus, expansion is generally required for in vivo use (see e.g., Hwang et al. Int Immunol, 24:793-802, 2012; Zhang et al., J Immunol., 190:1402-1406, 2013; Bigley et al., Blood Adv 5:3021-3021, 2021). The g-NK cells are preferentially expanded from healthy donors, e.g., using methods as described in Section II, including in some cases from healthy donors screened for increased percentage of g-NK cells. Epigenetic changes in g-NK cells lead to differences in gene expression relative to conventional NK (cNK) cells, including down-modulation of FceRly, so that the more active CD3 (zeta) is used for CD 16 signaling.

[0097] The g-NK cells express the signaling adaptor CD3 (zeta) chain abundantly, but are deficient in the expression of the signaling adaptor FceRly (gamma). This means that, in some cases, all signaling activity upon their activation goes through the CD3^ chain, which contains 3 IT AM motifs (versus 1 IT AM for FcRy). g-NK cells show markedly higher cytokine and cytolytic enzyme production, and target cell killing compared to conventional NK cells (cNK cells). g-NK cells bind to Fc receptor engaging antibodies bound to target cells and kill target cells via antibody dependent cytotoxicity (ADCC). The result is that g-NK cells have been shown to exhibit stronger cell proliferation, more cytokine secretion, more cytolytic enzymes (e.g., perforin and granzyme B) and better antibody-dependent cellular cytotoxicity (ADCC) compared to conventional NK cells (see e.g., International published PCT Application Nos. W02020 / 107002 and WO2021 / 216790). Results herein demonstrate robust depletion of B cells from patients with autoimmune disease in vitro when combined with B-cell-targeting monoclonal antibodies. In some embodiments, the unique activity of g-NK cells, compared to conventional NK cells and other cell therapy platforms, is due to their high expression of CD94 / NKG2C and HEA-E targeted activity. In some embodiments, g-NK cells are more effective in eliciting cell- mediated cytotoxicity than are conventional NK cells even in the absence of antibody.

[0098] Human leukocyte antigen (HEA)-E is a nonclassical major histocompatibility complex (MHC) class I (lb) molecule. Immune cells, such as B cells, T lymphocytes, monocytes, and macrophages, basally express HEA-E. Coupel et al., Blood 109:2806-2814 (2007). In particular, HEA-E is a ligand for receptors CD94 / NKG2A and CD94 / NKG2C receptors, which are receptors expressed on NK cells and bind to HLA-E. Between the two receptors, binding of HLA-E to the inhibitory receptor NKG2A is typically favored. Specifically, interaction and binding of HLA-E with the inhibitory CD94 / NKG2A receptor results in inhibition of NK cell dependent lysis. As such, HLA-E molecules, by binding to CD94 / NKG2A receptors expressed by NK cells, can provide protection to cells seeking to evade NK cell killing. Siemanszko et al., Arch Immunol Ther Exp (Warsz) 71 ( 1):9 (2023). For example, autoreactive B cells and T cells can normally avoid NK cell lysis through upregulation of HLA-E.

[0099] Provided embodiments are based on recognition that high NKG2C expression and low NKG2A expression on g-NK cells may circumvent the NK cell evasion strategy by autoreactive T and B cells while also providing for NK cell lysis of cells expressing HLA-E, including autoreactive B and T cells associated with many autoimmune diseases and conditions. Specifically, among the provided embodiments, g-NK cells can effectuate potent killing of HLA-E expressing autoimmune reactive cells because the g-NK cells have low expression of the CD94 / NKG2A inhibitory receptor. This means that unlike conventional NK cells, g-NK cells are not susceptible to the inhibitory effect of the HLA- E / NKG2A axis that typically has been established in cancer and autoimmune diseases (see e.g., Martmez- Rodriguez et al., Mult Scler. 22(6):741-52 (2016); Vietzen et al., Cell 196(26):5705-5718 (2023)).

[0100] In embodiments of provided methods, g-NK cells also may exhibit HLA-E-targeted killing activity via the high NKG2C expression on g-NK cells. Indeed, the inventors have recognized that the high NKG2C expression on g-NK cells render these cells particularly ideal for treating autoimmune diseases due at least in part to observations that NKG2C expression has been associated with good prognosis in, for example, multiple sclerosis (see e.g., Martinez-Rodriguez et al., Mult Scler. 22(6):741- 52 (2016) and Vietzen et al., Cell 196(26):5705-5718 (2023)). Specifically, it has been observed that control of autoimmunity, specifically by NKG2C NK cell responses, is severely impaired in multiple sclerosis subjects. In some embodiments, by virtue of the HLA-E targeted killing activity, the g-NK cells may deplete B cells that produce autoreactive antibodies and also deplete T cells to regulate autoreactive T cell responses. In some embodiments, the g-NK cells may directly inhibit and / or lyse autoreactive B cells as well as autoreactive T cells.

[0101] In some embodiments, provided embodiments are based on the inventor’s recognition that the g-NK cells exhibit features that have potent anti-viral properties and can help drive responses against autoimmunity. In many cases, viral targets play important roles in autoimmune disease. This is true, for example, in multiple sclerosis (MS), which is a demyelinating disease of the CNS. Epstein-Barr virus (EBV) contributes to the MS pathogenesis because high levels of EBV EBN A w> wspccific antibodies cross react with the CNS-derived GlialCAM370-389- Also, patients with multiple sclerosis (MS) are predominantly infected with EBV variants that highly upregulate HLA-E and inhibit NKG2A+ cells, in which EBV isolates carrying GGDPHLPTL (SEQ ID NO:20) and GGDPPLPTL (SEQ ID NO:21) LMP- 1 peptide variants are particularly associated with HLA-E upregulation and inhibition of NKG2A+ cells (Vietzen et al., Cell 196(26):5705-5718 (2023)). Specifically, in the Vietzen et al. report, only NKG2C+ and NKG2D+ NK cells showed significant activation by autoimmune GlialCAM-specific B and T cells, which was observed to be higher in patients with a higher HCMV infection. It was observed by Vietzen et al. that MS subjects, who are predominately infected with EBV variants, often have highly upregulated HLA-E. In other words, MS-subject-derived GlialCAM-specific cells are able to evade immune control via the inhibitory HLA-E / NKG2A axis. This result is consistent with normal immune evasion strategiesin which the infected autoreactive cells evade NK cell recognition via the inhibitory HLA-E / NKG2A axis. Data in a murine model of MS (EAE) suggests that disrupting NKG2A / HLA-E signaling can reverse MS-like disease (Lu et al., Immunity, 2007, 26:593-604). A separate study by Martinez- Rodriguez et al. has observed that subjects who are CMV positive (i.e., positive CMV serostatus) and additionally have high number of NKG2C+ NK cells are able to delay progression in MS. That is, NKG2ChlghHCMV+ individuals may be protected, delaying disability progression in MS (Martinez - Rodriguez et al., (2016) Mult Scler. 22(6):741-52).

[0102] In some embodiments, the above studies support the inventor’s recognition that the advantageous features g-NK cells, which are generally NKG2Chlgh, may be particularly effective for adoptive transfer as an improved cell therapy for treatment of autoimmune disease, such as MS, particularly in subjects in virally (e.g., EBV) infected subjects. Moreover, g-NK cells are primed by HCMV for potent killing of virally infected cells irrespective of virus. g-NK cells thus can eradicate virally infected cells mediated by anti-viral antibodies as well as by targeting of viral peptides presented on HLA-E via high levels of NKG2C and low NKG2A expression.

[0103] While other existing NK cell therapies may in some cases be able to deplete B cells, existing NK cell therapies are not able to specifically kill autoreactive T and B cells. Further, the ability to deplete B cells by other existing NK cell therapies is non-selective; existing NK cell therapies indiscriminately kill all B cells. Further, if B cells are infected with a virus, such as EBV, as is often the case in autoimmune diseases, the B cells may exhibit peptide induced HLA-E expression. The enhanced HLA-E expression results in HLA-E / NKG2A inhibitory evasion of NK cell responses mounted by most other NK cell therapies, including in combination with an antibody via ADCC-mediated killing. Similarly, activity of CAR-T cells for treating autoimmune diseases or conditions requires engineering cells with a CAR directed against a B cell antigen to mediate B cell depletion (Schett et al., ASH 2023). However, CAR-T cell strategies, including autologous and allogeneic CAR-T cell therapies, also are not always ideal because the CAR-T cell therapy also does not exhibit HLA-E targeting. As such CAR-T cell therapies do not have the ability to kill autoreactive T and B cells, and also exhibit only non-selective or indiscriminate B cell killing based on CAR-directed targeting of B cell antigens.

[0104] The provided g-NK cells have differentiated activity from, for example, a CD 19 CAR therapy or a monoclonal antibody therapy (e.g., CD20 antibody, such as ocrelizumab), including for the treatment of autoimmune diseases. Table 1 highlights differences of exemplary modes of actions of g- NK cells in combination with the exemplary anti-CD20 antibody ocrelizumab from other therapies, demonstrating the potential for a better clinical outcome of g-NK cell therapy.Table 1. Modes of Actions of g-NK Cells with Ocrelizumab Compared to Other Therapies

[0105] Further, another advantage of NK cell therapy is that multiple dosing cycles of NK cells is feasible. In contrast, with CAR T therapy, multiple dosing cycles are not feasible at least in part because there is a risk of immune reactions against the chimeric antigen receptor. In sum, the provided embodiments employing g-NK cells for treating autoimmune diseases and conditions are thus highly differentiated from other cell therapy approaches because they provide NKG2C and anti-viral mechanisms due to low expression of NKG2A inhibitory receptor as well as robust killing by ADCC.

[0106] The provided approaches thus allow for multiple mechanisms in which the provided g-NK cells can be used to treat autoimmune diseases and conditions, including inhibition and direct lysis of autoreactive B-and T-cells that have an upregulation of HLA-E and / or by enhanced control of a latent virus that drives autoimmunity which, in some aspects, is driven by EBV infection that upregulates HLA- E on infected cells. In particular, the ability of provided g-NK cells to eradicate virally infected cells is particularly important in MS for elimination of latent viral (e.g., EBV) reservoir. In some aspects, the above embodiments are based on the unique NKG2C+ / NKG2A- phenotype of g-NK cells. Moreover, in addition to the above mechanisms, the g-NK cells also are able to promote ADCC killing that can further potentiate responses and treatment of autoimmune diseases and conditions. In particular, in addition to potent anti-viral properties due to NKG2C+ / NKG2A- phenotype, g-NK cells also exhibited anti-viral properties by enhanced plasma-mediated ADCC against virally infected cells (Lee et al. Immunity, 2015). Also, targeted ADCC killing of autoreactive cells can be achieved by g-NK cells by administering the g- NK cells in combination with an antibody (e.g., an antibody targeting a B cell antigen, such as CD19, CD20, CD22 and others as described) or by engineering the g-NK cells with a CAR directed against a target antigen, such as a B cell antigen (such as a CAR directed against CD19, CD20, CD22 and others as described).

[0107] NK cells are capable of killing cells via antibody dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when receptors on the NK cell surface (such as CD16) recognize IgGl or IgG3 antibodies bound to the surface of a cell. In addition to activation of NK cells that can occur through the direct binding of NK cell receptors to ligands on the target cell, as seen with direct HLA-E recognition, ADCC can be initiated through the crosslinking of the Fc receptor (CD16; also known as CD 16a or FcyRIIIa) by binding to the Fc portion of antibodies bound to an antigen-bearing cell. This triggers release of cytoplasmic granules containing perforin and granzymes, leading to target cell death. Because NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells, target recognition is broadened (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001); Eanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Fong, Curr Opin Immunol. 20(3):344-352 (2008)). ADCC and antibody-dependent cytokine / chemokine production are primarily mediated by NK cells.

[0108] In conventional NK cells, the CD16 receptor is able to associate with adaptors, the chain of the TCR-CD3 complex (CD3Q and / or the FcRy chain, to transduce signals through immunoreceptor tyrosine-based activation motifs (IT AMs). In some aspects, CD16 engagement (CD16 crosslinking) initiates NK cell responses via intracellular signals that are generated through one, or both, of the CD16- associated adaptor chains, FcRy or CD3^. Triggering of CD16 leads to phosphorylation of the y orchain, which in turn recruits tyrosine kinases, SYK and ZAP-70, initiating a cascade of signal transduction leading to rapid and potent effector functions. The most well-known effector function is the release of cytoplasmic granules carrying toxic proteins to kill nearby target cells through the process of antibody-dependent cellular cytotoxicity. CD 16 crosslinking also results in the production of cytokines and chemokines that, in turn, activate and orchestrate a series of immune responses. CD16 also exists in a glycosylphosphatidylinositol-anchored form (also known as FcyRIIIB or CD16B). It is understood that reference to CD 16 herein is with reference to the CD 16a form that is expressed on NK cells and that is involved in antibody-dependent responses (such as NK cell-mediated ADCC), and it is not meant to refer to the glycosylphosphatidylinositol-anchored form.

[0109] The specialized subset of g-NK cells that lack the FcRy adaptor protein are able to mediate robust ADCC responses (see e.g., published Patent Appl. No. US2013 / 0295044). The mechanism for increased responses may be due to changes in epigenetic modification that influence the expression of the FcRy. The g-NK cells express the signaling adaptor CD3 chain abundantly, but are deficient in the expression of the signaling adaptor FceRly chain. In some embodiments, g-NK cells are more effective in eliciting cell-mediated cytotoxicity than are conventional NK cells even in the absence of antibody. When activated by antibodies, y-deficient g-NK cells exhibit dramatically enhanced activity when activated by antibodies, compared to conventional NK cells, e.g., NK cells that are not deficient in the y chain. In particular, when CD 16 is engaged by the Fc region of an antibody, the signaling is mediated bysolely the £ chain of the TCR-CD3 complex (CD3Q, which transduces signals through three immunoreceptor tyrosine-based activation motifs (IT AMs). In some aspects, the g-NK cells produce greater amounts of cytokines (e.g., IFN-y or TNF-a) and chemokines (e.g., MIP-la, MIP-ip, and RANTES) and / or display higher degranulation responses than conventional NK cells expressing the y chain, and thus have a higher capacity to release cytoplasm containing perforin and proteases (granzymes). The g-NK cells provide high expression of Granzyme B, a component of natural killer cell cytotoxic machinery. Moreover, the g-NK cells have a prolonged lifespan, compared to conventional NK cells, and their presence is maintained long-term. In some embodiments, g-NK cells are functionally and phenotypically stable. The provided embodiments thus allow for approaches in which the g-NK cells exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC) as well as antibody-independent cell-mediated cytotoxicity, supporting the utility of such cells for therapeutic applications for treating autoimmune diseases and conditions. Importantly, adoptive transfer of allogeneic NK-cells does not result in severe graft-versus-host (GVHD), and thus such a cell therapy can be given in an “off-the-shelf’ manner for clinical use.

[0110] Together, the g-NK cell as provided herein for use in combination with a monoclonal antibody provide for a multifactorial mechanism of action that goes beyond B cell depletion. In some embodiments, g-NK cells in combination with a monoclonal antibody result in (1) deep depletion of antibody-targeted autoreactive T and B cells via ADCC for immune reset, such as via CD20 or other targeted antigen; (2) depletion of HLA-E expressing autoreactive T cells, plasma B cells and / or plasma blasts; and (3) elimination of viral reservoir through killing of virally infected cells that may be responsible for disease initiation and maintenance. FIG. 9 depicts a schematic of this multifactorial mechanism of action. In some embodiments, administration of g-NK cells alone can result in therapeutic activity by mechanisms (2) and (3) but combination with an antibody would have the additional advantage of directly targeting for depletion of antigen-targeted B cells and T cells. The above multiple mechanisms of action allow the potential to treat a larger spectrum of autoimmune diseases such as MS and other autoimmune diseases described herein.

[0111] While antibodies can facilitate ADCC-mediated killing of B cells, a problem with their utility in treating certain autoimmune diseases is that monoclonal antibodies are not able to efficiently cross the blood-brain barrier. As a result, administration of monoclonal antibodies are not able to efficiently reach B cells deep in lymph nodes or in ectopic germinal centers of the CNS. However, B cells contribute to pathology of certain autoimmune diseases, such as MS pathology, both in the periphery and the CNS. Among provided embodiments, results herein such as shown in Example 4 demonstrate that g-NK cells are able to retain bound antibodies on their surface for periods of time and thus are able to facilitate transport and tissue distribution of antibodies. A model depicted in FIG. 8B depicts the ability of g-NK cells to facilitate tissue distribution of antibodies across the blood-brain barrier by transport of a CD 16-bound monoclonal antibody to lymph nodes and to the brain via the lymphatic system (“glymphatics”) to mediate cytolysis of autoreactive cells in CNS (see e.g., Licastro et al. Communications Biology, 2024, 7:229). For example, co-administration of g-NK cells and a monoclonal antibody (e.g., anti-CD20 antibody, such as ocrelizumab) into the periphery by intravenous administration or subcutaneous injection can result in binding of the mAh on the surface of g-NK cells where it then can be transported to the lymph node and CNS for cytolysis of autoreactive T and B cells (e.g., CD20+ cells) in the CNS. The ability to increase tissue distribution of mAbs into lymph nodes an CNS has potential for translational significance.

[0112] Examples of properties of g-NK cells that differentiate them from other cell therapy approaches for autoimmune diseases are highlighted below in Table 2. Many other cell therapies are associated with a variety of safety risks and may not always be efficacious for treating autoimmune diseases. For instance, provided g-NK cells have a low risk of prolonged B-cell aplasia, cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANs). The superior safety compared to autologous CAR-T cells and other cell therapies allow for the potential for the provided methods to be used for treatment of a broader patient population. Alternative cellular approaches for the treatment of autoimmune disease without these safety risks are needed, particularly if such treatments are to be applied to patients with less severe disease. Moreover, the provided g-NK cells provide for an allogenic drug product, and without requiring engineering. The ability of an allogenic therapy, along with the ability of the g-NK cells to be cryopreserved and still retain potent activity, means that the product can be provided on-demand for use in the provided methods. This means there is no delay in the treatment and manufacturing can be carried out in advance from a master cell bank of cells from a healthy donor to treat multiple patients.Table 2. Modes of Actions of g-NK Cells Compared to Other Cell Therapies

[0113] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0114] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS OF TREATMENT AND METHODS OF KILLING OF AUTOREACTIVE CELLS

[0115] Provided herein are methods of killing of autoreactive cells by contacting such cells with NK cells from a composition of NK cells enriched for cells deficient in expression of FcRy chain (g-NK cells). In some embodiments, the composition of cells comprises NK cells, including g-NK cells, that are NKG2C+. Uses include uses of the cells or pharmaceutical compositions thereof in such methods, and in the preparation of a medicament in order to carry out such methods. In some embodiments, the autoreactive cells are autoreactive B cells and / or T cells. In some embodiments, the contacting is carried out ex vivo or in vitro. In some embodiments, the contacting is carried out in vivo, such as after administering a composition of g-NK cells to a subject such as a subject suspected of or known to have autoreactive cells or an autoimmune disease or condition. In some embodiments, such methods and uses of killing autoreactive cells involve targeted cytotoxic killing of such cells by g-NK cells or cells from compositions enriched for g-NK cells, for example due to the expression of HLA-E on certain autoreactive cells as described herein. In some cases, targeted killing of such cells can be potentiated by combination methods involving the compositions comprising g-NK cells in combination with an antibody targeted against B cells and / or T cells. In some embodiments, the B cells and / or T cells are autoreactive B cells and / or T cells. In some embodiments, the B cells or T cells are HLA-E positive. In particular, amongthe provided embodiments, it is recognized that the provided compositions comprising g-NK cells, even as a monotherapy without a co-administered antibody, can preferentially target or kill autoreactive B cells / and or T cells since HLA-E is a differentiator of autoreactive cells in that it is typically expressed or upregulated on autoreactive cells but not normal cells. As a result, the provided NK cell therapy and compositions can uniquely target autoreactive cells and minimize killing of normal cells (e.g., B cells and / or T cells). In some embodiments, the methods and uses of killing autoreactive cells, including autoreactive B cells and / or T cells, results in the treatment of an autoimmune disease or disorder in a subject.

[0116] Provided herein are compositions and methods relating to cell compositions comprising g- NK cells for use in treating an autoimmune disease or disorder in a subject. In some embodiments, the cell compositions comprise NK cells, including g-NK cells, that are NKG2C+. In some embodiments, provided herein is a method of treating an autoimmune disease or disorder in an individual, comprising administering a composition comprising g-NK cells, to an individual in need thereof. Uses include uses of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. The composition comprising g-NK cells can include any of the provided compositions. In some embodiments, the composition is produced by the methods provided herein. Such methods and uses include therapeutic methods and uses, for example, involving administration of the therapeutic cells, or compositions containing the same, to a subject having an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is caused by or is exacerbated by a virus infection. In some embodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to effect treatment of the autoimmune disease or disorder. In some embodiments, the methods thereby treat the autoimmune disease or disorder in the subject.

[0117] In some embodiments, the provided methods also include prophylactic treatment involving administering a composition of NK cells enriched for g-NK cells to a subject prior to the onset or likely onset of an autoimmune disease or disorder. In some embodiments, the composition of cells comprises NK cells, including g-NK cells, that are NKG2C+. Uses include uses of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. The composition comprising g-NK cells can include any of the provided compositions. In some embodiments, the composition is produced by the methods provided herein. Such methods and uses include therapeutic methods and uses, for example, involving administration of therapeutic cells, or compositions containing the same, to a subject at risk of developing an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is caused by or is exacerbated by a virus infection, and a subject is at risk of developing the autoimmune disease or disorder based on detection of the virus infection or of an immune response against the viral infection (e.g., detection of an antibody response to one or more viral epitopes in the subject). In someembodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to reduce the risk of developing or reduce onset of the autoimmune disease or disorder. In some embodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to prevent development of the autoimmune disease or disorder. In some embodiments, the methods thereby prophylactically treat the autoimmune disease or disorder in the subject.A. G-. A Ceii Compositions

[0118] In some embodiments, the compositions for use in the provided methods contain g-NK cells. In some embodiments, the compositions of g-NK cells for use in the provided methods contain a plurality of g-NK cells. In some embodiments, the compositions are pharmaceutical compositions for use in treating an autoimmune disease or conditions. Also provided herein are uses of any of the provided pharmaceutical compositions for manufacture of a medicament for use in treating an autoimmune disease or condition in a subject.

[0119] In some embodiments, the composition comprises about 5-99% g-NK cells, or any percentage of g-NK cells between 5 and 99%, inclusive. In some embodiments, the composition can comprise about 5-99% g-NK cells, inclusive, prior to expansion. In specific embodiments, most of the NK cells in a composition, prior to expansion, can be g-NK cells. In specific embodiments, the composition, prior to expansion, can comprise about 30% g-NK cells, 40% g-NK cells, 50% g-NK cells, 60% g-NK cells, 70% g-NK cells, 80% g-NK cells, 90% g-NK cells, or up to 99% g-NK cells. In some embodiments, the composition can comprise about 5-99% g-NK cells, inclusive, after expansion. In specific embodiments, the composition, after expansion, can comprise about 30% g-NK cells, 40% g-NK cells, 50% g-NK cells, 60% g-NK cells, 70% g-NK cells, 80% g-NK cells, 90% g-NK cells, or up to 99 % g-NK cells.

[0120] In some embodiments, the composition can include an increased or greater percentages of g- NK cells relative to total NK cells or total cells compared to the percentage of g- NK relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0121] In some embodiments, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total NK cells in the composition.

[0122] In some embodiments, prior to expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition.

[0123] In some embodiments, prior to expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total NK cells in the composition.

[0124] In some embodiments, after expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at orabout 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition. In some embodiments, after expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total NK cells in the composition.

[0125] In some embodiments, the provided compositions include those in which the g-NK cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, prior to expansion, the provided compositions include those in which the g-NK cells make up at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, after expansion, the provided compositions include those in which the g-NK cells make up at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, after expansion, the provided compositions include those in which the g-NK cells make up at least at or about 20% or more of the cells in the composition or of the NK cells in the composition.

[0126] In some embodiments, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, of the total cells in the composition. Greater than at or about 90% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0127] In some embodiments, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition,greater than at or about 70% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0128] In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition. Greater than at or about 90% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0129] In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0130] In some embodiments, after expansion, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0131] In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at orabout 80% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0132] In some of any embodiments, cells of the composition that are g-NK cells also are characterized as NKG2Cpos, NKG2Anegand CD16pos. In some embodiments, cells of the composition that are g-NK cells are characterized as being CD57pos, CD7dim / neg, CD161negand / or CD38neg. In some embodiments, cells of the composition of g-NK cells are NKG2Aneg / CD161neg. In some embodiments, cells of the composition of g-NK cells are CD38neg. In some embodiments, cells of the composition of g- NK cells has the phenotype CD45pos / CD3neg / CD56pos.

[0133] In some embodiments, the composition contains NKG2Cposcells. In some embodiments, the compositions contain NKG2Anegcells. In some embodiments, the composition contains NKG2Cpos / NKG2Anegcells. In some embodiments, g-NK cells of the composition are NKG2Cposcells. In some embodiments, g-NK cells of the composition contain NKG2Anegcells. In some embodiments, g- NK cells of the composition contain NKG2Cpos / NKG2Anegcells.

[0134] In some embodiments, the composition comprises about 5-99% NKG2Cposcells. In some embodiments, the composition can include an increased or greater percentages of NKG2Cposcells relative to total NK cells or total cells compared to the percentage of NKG2Cposcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0135] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%,at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cposcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cposcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cposcells of the total NK cells in the composition.

[0136] In some embodiments, the provided compositions include those in which the NKG2Cposcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0137] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Cpos.

[0138] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Cpos. In someembodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Cpos.

[0139] In some embodiments, the composition comprises about 5-99% NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages of NKG2Anegcells relative to total NK cells or total cells compared to the percentage of NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0140] In some embodiments, the composition can include at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Anegcells of the total NK cells in the composition.

[0141] In some embodiments, the provided compositions include those in which the NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0142] In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater thanat or about 20% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Aneg.

[0143] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Aneg.

[0144] In some embodiments, the composition comprises about 5-99% NKG2Cpos / NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages of NKG2Cpos / NKG2Anegcells relative to total NK cells or total cells compared to the percentage of NKG2Cpos / NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0145] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cpos / NKG2Anegcells of the total cells in the composition. Insome embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition.

[0146] In some embodiments, the provided compositions include those in which the NKG2Cpos / NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0147] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Cpos / NKG2Aneg. In someembodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Cpos / NKG2Aneg.

[0148] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Cpos / NKG2Aneg.

[0149] In some embodiments, the composition comprises about 5-99% g-NK cells that are NKG2Cpos / NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages of g-NK cells that are NKG2Cpos / NKG2Anegcells relative to total NK cells or total cells compared to the percentage of g-NK cells that are NKG2Cpos / NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0150] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at leastat or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegg-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegg-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition.

[0151] In some embodiments, the provided compositions include those in which the g-NK cells that are NKG2Cpos / NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0152] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are g- NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are g- NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg.

[0153] In some embodiments, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 70% of the cellsare g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg.

[0154] In some embodiments, the g-NK cells are CD16pos. In some embodiments, the genotype of the CD 16 protein is one in which there is a substitution of valine (V) for phenylalanine (F) at position 158 in the mature (processed) form of the protein (F158V). In some embodiments, the NK cells bear the CD16 158V polymorphism in both alleles (called 158V / V herein). In some embodiments, the g-NK cells comprise CD16 158V / V (V158). In some embodiments, the g-NK cells are CD16 158V / F. In some embodiments the g-NK cells comprise CD16 158 F / F (F158).

[0155] In some embodiments, the g-NK cells of the composition, or a certain percentage thereof, e.g., greater than about 70%, are positive for perforin and / or granzyme B. Methods for measuring the number of cells positive for perforin or granzyme B are known to a skilled artisan. Methods include, for example, intracellular flow cytometry. In an example, the percentage or number of cells positive for perforin or granyzme B may be determined by the permeabilization of cells, for instance using the Inside Stain Kit from Miltenyi Biotec, prior to staining with antibodies against perforin and granzyme B. Cell staining can then be resolved for instance using flow cytometry.

[0156] In some embodiments, greater than at or about 70% of the g-NK cells of the composition are positive for perforin, and greater than at or about 70% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 75% of the g-NK cells of the composition are positive for perforin, and greater than at or about 75% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 80% of the g- NK cells of the composition are positive for perforin, and greater than at or about 80% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 85% of the g-NK cells of the composition are positive for perforin, and greater than at or about 85% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 90% of the g-NK cells of the composition are positive for perforin, and greater than at or about 90% of the g- NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 95% of the g-NK cells of the composition are positive for perforin, and greater than at or about 95% of the g-NK cells of the composition are positive for granzyme B.

[0157] In some embodiments, perforin and granzyme B expression levels by NK cells, for instance g-NK cells, can be measured by intracellular flow cytometry and levels measured based on levels of mean fluorescence intensity (MFI). In some embodiments, perforin and granzyme B expression levels based on MFI will differ between g-NK cells and cells that are FcRypos. In some embodiments, the g-NK cells ofthe composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about two times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about three times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about four times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about two times the mean level of granzyme B expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about three times the mean level of granzyme B expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about four times the mean level of granzyme B expressed by FcRyposNK cells.

[0158] In some embodiments, at least at or about 50% of the cells in the composition are FcRy- deficient NK cells (g-NK), wherein greater than at or about 70% of the g-NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 90% of the g-NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 95% of the g-NK cells are positive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B. In some embodiments, the g-NK cells are FcRyneg.

[0159] In some of any embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos. In some of any embodiments, among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0160] In some of any of the preceding embodiments, greater than at or at about 80% of the cells are positive for perforin. In some of any of the preceding embodiments, greater than at or at about 90% of the cells are positive for perforin. In some of any of the preceding embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos.

[0161] In some of any of the preceding embodiments, greater than at or at about 80% of the cells are positive for granzyme B. In some of any of the preceding embodiments, greater than at or at about 90% of the cells are positive for granzyme B. In some of any of the preceding embodiments, among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0162] In some of any of the provided embodiments, it is understood that the terms positive, pos or + with reference to a marker or protein expressed on or in a cell are used interchangeably herein. Likewise, it is understood that the terms negative, neg or - with reference to a marker or protein expressed on or in a cell are used interchangeably herein. Further, it is understood that reference to cells that are markernegherein may refer to cells that are negative for the marker as well as cells expressing relatively low levels of the marker, such as a low level that would not be readily detectable compared to control or background levels. In some aspects, expression of any of the provided markers can be determined by their expression on the surface of the cells (surface expression) or in the cells (intracellular expression). In some embodiments, the expression can be determined by flow cytometry, for example, by staining with an antibody that specifically bind to the marker and detecting the binding of the antibody to the marker. Similar methods can be carried out to assess expression of intracellular markers, except that such methods typically include methods for fixation and permeabilization before staining to detect intracellular proteins by flow cytometry.

[0163] In some embodiments, a cell (e.g., NK cell subset) is positive (pos) for a particular marker if there is detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In embodiments, surface expression is positive if staining is detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to, or in some cases higher than, a cell known to be positive for the marker and / or at a level higher than that for a cell known to be negative for the marker.

[0164] In some embodiments, a cell (e.g., NK cell subset) is negative (neg) for a particular marker if there is an absence of detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In some embodiments, surface expression is negative if staining is not detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially lower than a cell known to be positive for the marker and / or at a level substantially similar to a cell known to be negative for the marker.

[0165] In some embodiments, a cell (e.g., NK cell subset) is low (lo or min) for a particular marker if there is a lower level of detectable presence on or in the cell of a particular marker compared to a cellknown to be positive for the marker. In embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically bind to the marker and detecting the binding of the antibody to the marker, wherein expression, either surface or intracellular depending on the method used, is low if staining is at a level lower than a cell known to be positive for the marker.

[0166] In some of any of the provided embodiments, the composition comprises from at or about 106cells to at or about 1012cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about 10” cells, from at or about 106to at or about IO10cells, from at or about 106to at or about 109cells, from at or about 106to at or about 108cells, from at or about 106to at or about 107cells, from at or about 107to at or about 1012cells, from at or about 107to at or about 10” cells, from at or about 107to at or about 1010cells, from at or about 107to at or about 109cells, or from at or about 107to at or about 108cells, from at or about 108to at or about 1012cells, from at or about 108to at or about 10” cells, from at or about 108to at or about 1010cells, from at or about 108to at or about 109cells, from at or about 109to at or about 1012cells, from at or about 109to at or about 10” cells, from at or about 109to at or about 1010cells, from at or about 1010to at or about 1012cells, from at or about 1010to at or about 10” cells, or from at or about 10” to at or about 1012cells.

[0167] In some of any of the provided embodiments, the composition comprises at least or about at least 106cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about 1010cells, from at or about 106to at or about 109cells, from at or about 106to at or about 108cells, from at or about 106to at or about 107cells, from at or about 107to at or about 1010cells, from at or about 107to at or about 109cells, from at or about 107to at or about 108cells, from at or about 108to at or about 1010cells, from at or about 108to at or about 109cells, or from at or about 109to at or about 1010cells.

[0168] In some of any of the provided embodiments, the composition comprises at least or about at least 108cells. In some of any of the provided embodiments, the composition comprises at least at or about 109cells. In some of any of the provided embodiments, the composition comprises at least at or about 1010cells. In some of any of the provided embodiments, the composition comprises at least at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 1010cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 109cells. In some of any of the provided embodiments, the composition comprises from at or about 109to at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 109to at or about 1010cells. In some of any of the provided embodiments, the composition comprises from at or about 1010to at or about 10” cells.

[0169] In some of any of the provided embodiments, the composition comprises at least at or about 106g-NK cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about IO10g-NK cells, from at or about 106to at or about 109g-NK cells, from at or about 106to at or about 108g-NK cells, from at or about 106to at or about 107g-NK cells, from at or about 107to at or about IO10g-NK cells, from at or about 107to at or about 109g-NK cells, from at or about 107to at or about 108g-NK cells, from at or about 108to at or about 10’°g-NK cells, from at or about 108to at or about 109g-NK cells, or from at or about 109to at or about 1010g-NK cells.

[0170] In some embodiments, the cells in the described composition of g-NK cells are for allogenic cell therapy. In some embodiments, the cells in the described compositions of g-NK cells are from a donor or donors that are different from the subject to be treated. In some embodiments, the donor or donors are not known to have the autoimmune disease or condition. In particular embodiments of any of the provided compositions, the cells in the composition are from the same donor. As such, the compositions do not include a mixed population of cells from one or more different donors.

[0171] In some embodiments, the g-NK cells are primary g-NK cells from a subject. According to some embodiments, the primary g-NK cells can be obtained from a sample from a mammalian subject, such as a human subject. The sample or source can be, for example, but not limited to, cord blood, bone marrow or peripheral blood. In particular, among the provided compositions are compositions of cells that are enriched for g-NK cells. In some embodiments, the compositions for use in the provided methods contain g-NK cells that are expanded NK cells such as produced by any of the provided methods. In some embodiments, the g-NK cells are selected and expanded such as by methods described in Section II.

[0172] In some embodiments, the composition of g-NK cells is produced by an ex vivo expansion method that enriches and expands for g-NK cells from a donor subject. In some embodiments, the method of expansion include those as described in Section II. As provided here, the methods of expansion result in high yield expansion of at or greater than 500-fold, at or greater than 600-fold, at or greater than 700-fold, at or greater than 800-fold, at or greater than 900-fold, at or greater than 1000-fold or more of g-NK cells. In some of any embodiments, the increase is at or about 1000-fold greater. In some of any embodiments, the increase is at or about 2000-fold greater. In some of any embodiments, the increase is at or about 2500-fold greater. In some of any embodiments, the increase is at or about 3000- fold greater. In some of any embodiments, the increase is at or about 5000-fold greater. In some of any embodiments, the increase is at or about 10000-fold greater. In some of any embodiments, the increase is at or about 15000-fold greater. In some of any embodiments, the increase is at or about 20000-fold greater. In some of any embodiments, the increase is at or about 25000-fold greater. In some of any embodiments, the increase is at or about 30000-fold greater. In some of any embodiments, the increase is at or about 35000-fold greater.

[0173] In certain embodiments, the number of such cells in the composition is a therapeutically effective amount. In some embodiments, the amount is an amount that reduces the severity, the duration and / or the symptoms associated with an autoimmune disease or disorder.

[0174] In some embodiments, the composition comprises an amount of g-NK cells that is from at or about 105and at or about 1012g-NK cells, or from at or about 105to at or about 108g-NK cells, or from at or about 106and at or about 1012g-NK cells, or from at or about 108and at or about 10” g-NK cells, or from at or about 109and at or about IO10g-NK cells. In some embodiments, the composition comprises greater than or greater than at or about 105g-NK cells, at or about 106g-NK cells, at or about 107g-NK cells, at or about 108g-NK cells, at or about 109g-NK cells, at or about IO10g-NK cells, at or about 10” g- NK cells, or at or about 1012g-NK cells. In some embodiments, such an amount can be administered to a subject having a disease or condition, such as to a subject with an autoimmune disease or disorder.

[0175] In some embodiments, the composition comprises an amount of NKG2Cposcells or a subset thereof that is from at or about 105and at or about 1012NKG2Cposcells or a subset thereof, or from at or about 105to at or about 108NKG2Cposcells or a subset thereof, or from at or about 106and at or about 1012NKG2Cposcells or a subset thereof, or from at or about 108and at or about 10” NKG2Cposcells or a subset thereof, or from at or about 109and at or about 1010NKG2Cposcells or a subset thereof. In some embodiments, the composition comprises greater than or greater than at or about 105NKG2Cposcells or a subset thereof, at or about 106NKG2Cposcells or a subset thereof, at or about 107NKG2Cposcells or a subset thereof, at or about 108NKG2Cposcells or a subset thereof, at or about 109NKG2Cposcells or a subset thereof, at or about 1010NKG2Cposcells or a subset thereof, at or about 10” NKG2Cposcells or a subset thereof, or at or about 1012NKG2Cposcells or a subset thereof. In some embodiments, such an amount can be administered to a subject having a disease or condition, such as to a subject with an autoimmune disease or disorder.

[0176] In some embodiments, the volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL or 500 mL, such as is from or from about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL, each inclusive. In some embodiments, the composition has a cell density of at least or at least about 1 x 105cells / mL, 5 x 105cells / mL, 1 x 106cells / mL, 5 x 106cells / mL, 1 x 107cells / mL, 5 x 107cells / mL, or 1 x 108cells / mL. In some embodiments, the cell density of the composition is between or between about 1 x 105cells / mL to 1 x 108cells / mL, 1 x 105cells / mL to 1 x 107cells / mL, 1 x 105cells / mL to 1 x 106cells / mL, 1 x 106cells / mL to 1 x 107cells / mL, 1 x 106cells / mL to 1 x 108cells / mL, 1 x 106cells / mL to 1 x 107cells / mL, or 1 x 107cells / mL to 1 x 108cells / mL, each inclusive.

[0177] Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. In some embodiments, the engineered cells are formulated with a pharmaceutically acceptable carrier.

[0178] A pharmaceutically acceptable carrier can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical carrier should be one that is suitable for NK cells, such as a saline solution, a dextrose solution or a solution comprising human serum albumin.

[0179] In some embodiments, the pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which the NK cells can be maintained, or remain viable, for a time sufficient to allow administration of live NK cells. For example, the pharmaceutically acceptable carrier or vehicle can be a saline solution or buffered saline solution. The pharmaceutically acceptable carrier or vehicle can also include various bio materials that may increase the efficiency of NK cells. Cell vehicles and carriers can, for example, include polysaccharides such as methylcellulose (M. C. Tate, D. A. Shear, S. W. Hoffman, D. G. Stein, M. C. LaPlaca, Biomaterials 22, 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (Suh J K F, Matthew H W T. Biomaterials, 21, 2589, 2000; Eahiji A, Sohrabi A, Hungerford D S, et al., J Biomed Mater Res, 51, 586, 2000, each of which is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P(NIPAM- co-AA) (Y. H. Bae, B. Vernon, C. K. Han, S. W. Kim, J. Control. Release 53, 249, 1998; H. Gappa, M. Baudys, J. J. Koh, S. W. Kim, Y. H. Bae, Tissue Eng. 7, 35, 2001, each of which is incorporated herein by reference in its entirety), as well as Poly(oxyethylene) / poly(D,E-lactic acid-co-glycolic acid) (B. Jeong, K. M. Fee, A. Gutowska, Y. H. An, Biomacromolecules 3, 865, 2002, which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs E J, Mikos A G. Cell Trans, 8, 345, 1999, which is incorporated herein by reference in its entirety), PEO / PEG (Mann B K, Gobin A S, Tsai A T, Schmedlen R H, West J L., Biomaterials, 22, 3045, 2001; Bryant S J, Anseth K S. Biomaterials, 22, 619, 2001, each of which is incorporated herein by reference in its entirety), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005, which is incorporated herein by reference in its entirety), collagen (Lee C R, Grodzinsky A J, Spector M., Biomaterials 22, 3145, 2001, which is incorporated herein by reference in its entirety), alginate (Bouhadir K H, Lee K Y, Alsberg E, Damm K L, Anderson K W, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990, each of which is incorporated herein by reference in its entirety).

[0180] In some embodiments, the NK cells such as NKG2Cposcells or a subset thereof can be present in the composition in an effective amount. In some embodiments, the composition contains an effective amount of g-NK cells, such as FcRynegcells or cells having a g-NK surrogate marker profile thereof. An effective amount of cells can vary depending on the patient, as well as the type, severity and extent of disease. Thus, a physician can determine what an effective amount is after considering the health of the subject, the extent and severity of disease, and other variables.

[0181] In some embodiments, the composition, including pharmaceutical composition, is sterile. In some embodiments, isolation, enrichment, or culturing of the cells is carried out in a closed or sterile environment, for example and for instance in a sterile culture bag, to minimize error, user handling and / or contamination. In some embodiments, sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. In some embodiments, culturing is carried out using a gas permeable culture vessel. In some embodiments, culturing is carried out using a bioreactor.

[0182] Also provided herein are compositions that are suitable for cryopreserving the provided NK cells. In some embodiments, the NK cells are cryopreserved in a serum-free cryopreservation medium. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, the cryopreservation medium is between at or about 5% and at or about 10% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 5% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 6% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 7% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 8% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 9% DMSO (v / v). In some embodiments, the cry opreservation medium is at or about 10% DMSO (v / v). In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor™ CS10). CryoStor™ CS10 is a cry opreservation medium containing 10% dimethyl sulfoxide (DMSO). In some embodiments, compositions formulated for cry opreservation can be stored at low temperatures, such as ultra-low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0183] In some embodiments, the compositions can be preserved at ultra-low temperature before the administration to a patient. In some aspects, NK cell subsets, such as g-NK cells, can be isolated, processed and expanded, such as in accord with the provided methods, and then stored at ultra-low temperature prior to administration to a subject.

[0184] A typical method for the preservation at ultra-low temperature in small scale is described, for example, in U.S. Pat. No. 6,0168,991. For small-scale, cells can be preserved at ultra-low temperature by low density suspension (e.g., at a concentration of about 200x106 / ml) in 5% human albumin serum (HAS) which is previously cooled. An equivalent amount of 20% DMSO can be added into the HASsolution. Aliquots of the mixture can be placed into vials and frozen overnight inside an ultra-low temperature chamber at about -80° C.

[0185] In some embodiments, the cryopreserved NK cells are prepared for administration by thawing. In some cases, the NK cells can be administered to a subject immediately after thawing. In such an embodiment, the composition is ready-to-use without any further processing. In other cases, the NK cells are further processed after thawing, such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activating or stimulating agent, or are activated washed and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

[0186] In one embodiment, cytokines can be administered to a subject prior to isolating primary NK cells. For example, IL-12, IL-15, IL-18, IL-2, and / or CCL5 can be administered to a subject prior to isolating the primary NK cells.1. Gene Editing

[0187] Among the provided composition of g-NK cells are compositions in which the g-NK cells are engineered g-NK cells.

[0188] In some embodiments, the g-NK cells described herein may be genetically engineered by gene editing to alter (e.g., reduce) expression of one or more genes by the g-NK cells, thereby altering one or more properties or activities of the NK cells. For instance, strategies for gene editing can include one or more strategy that reduced fratricide (self-killing) due to expression of target antigen on g-NK cells; reduces undesired immunoreactivity that may result in graft vs. host disease (GvHD) particularly when infused into immune-compromised HLA-matched or, in some cases, also when infused into HLA mis-matched recipients; or reduces immunosuppression by host factors, particularly in the tumor microenvironment. In some embodiments, the engineered g-NK cells, including those engineered by one or more gene editing strategy, exhibit enhanced NK cell response characteristics as compared to similar NK cells without the gene editing, e.g., enhanced target recognition, enhanced NK cell response level and / or duration, improved NK cell survival, delayed NK cell exhaustion, and / or enhanced target recognition.

[0189] In some embodiments, the g-NK cells described herein can be gene edited to reduce FcRy chain expression, activity and / or signaling in the cell. For example, methods of gene editing may comprise introducing a genetic disruption of a gene encoding FcRy chain, a gene encoding a protein that regulates expression or activity of FcRy signaling adaptor (e.g., a transcription factor, such as PEZF or HEEIOS) and / or a gene encoding a protein that is involved in FcRy-mediated signaling (e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2) as described. In some embodiments, method of engineering may comprise introducing an inhibitory nucleic acid molecule that targets a gene encoding FcRy chain, a gene encoding a protein that regulates expression or activity of FcRy signalingadaptor (e.g., a transcription factor, such as PLZF or HELIOS) and / or a gene encoding a protein that is involved in FcRy-mediated signaling (e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2) as described. In some embodiments, the g-NK cells described herein are gene edited to be deficient in or reduced in FcRy chain expression, activity and / or signaling in the cell. Methods for reduction of FcRy chain expression, such as knockout or disruption of FcRy chain in NK cells, are described in PCT. Pub. No. WO2018 / 148462 and Eiu et al. iScience, 2020; 23:101709, the disclosures of each of which are incorporated by reference in their entireties. For example, in some embodiments, cells are gene edited to knockout the FcRy chain using a CRISPR-Cas9 system. In some embodiments, cells are gene edited to knockout the FcRy chain by introducing a caspase effector nuclease, such as a Cas9, and a guide RNA, such as a guide RNA comprising the sequence set forth in SEQ ID NO: 82 and / or a guide RNA comprising the sequence set forth in SEQ ID NO: 83. In some embodiments, the caspase effector nuclease and guide RNA are introduced by delivering a ribonucleoprotein (RNP) complex comprising the caspase effector nuclease and guide RNA to the cell by electroporation of the RNP.

[0190] In some embodiments, the method provided herein comprises obtaining a primary NK cell or an NK cell line and gene editing the cell to reduce expression of FcRy expression, activity and / or signaling in the cell in accord with the provided methods. In some embodiments, the methods provided herein comprises isolating an NK cell from a subject, such as by the methods as described above or known to a skilled artisan, and reducing the expression of FcRy chain expression, activity and / or signaling in the cell in accord with the provided methods. In some embodiments, primary cells derived from a subject may be expanded and / or cultured before gene editing. In some embodiments, the gene edited primary cells are cultured and / or expanded following gene editing and prior to administration to a patient.

[0191] In some of any of the preceding embodiments, the g-NK cell can further comprise nucleic acid encoding a heterologous CD16. In some of any of the preceding embodiments, the heterologous CD 16 can comprise a CD16-activating mutation, wherein the mutation can result in higher affinity to IgGl. In some of any of the preceding embodiments, the heterologous CD16 can comprise a 158V mutation. In some of any of the preceding embodiments, the engineered g-NK cells can be derived from a primary cell obtained from a human subject.

[0192] One of ordinary skill in the art will appreciate that there are many ways of decreasing the expression or activity of FcRy. For example, the level of transcription can be decreased. One method of decreasing gene expression, such as FcRy chain expression, involves modifying an endogenous gene to decrease transcription. For example, the FcRy chain gene may be deleted, disrupted, or mutated. In addition to targeting the FcRy RNA, mutating, or modifying the FcRy gene, FcRy protein level can be decreased by effecting a molecule that increases FcRy gene expression or activity, such as a transcriptionfactor that regulates transcription of FcRy. In some embodiments a gene that regulates transcription or translation of the FcRy chain gene may be deleted, disrupted, or mutated. In some of these embodiments, the gene is a transcription factor that regulates expression of the FcRy chain gene. Specifically, inhibition of a transcription factor that positively regulates FcRy expression will result in decreased FcRy expression. Transcription factors that regulate FcRy transcription include HELIOS and PLZF.

[0193] One of ordinary skill in the art will understand that there are many suitable methods for disrupting FcRy chain gene or other gene, such as those described herein. For example, the entire gene locus, such as FcRy locus, may be deleted. In some cases, it is also suitable to delete a portion of the gene, for example an exon, or a domain. Specifically, the IT AM signaling domain of FcRy may be deleted. Alternatively, the provided methods also include introducing one or more amino acid substitutions into the gene locus, such as FcRy locus, such as an inactivating mutation. In some embodiments, a stop codon can be introduced into the mRNA, such as FcRy mRNA, to produce a truncated and / or inactivated form of the expressed gene, such as FcRy signaling adaptor. In some embodiments, regulatory elements of the gene, such as FcRy gene, can also be mutated or deleted in order to reduce expression, activity and / or signaling of FcRy signaling adaptor.

[0194] In some embodiments, gene disruption can be carried out in mammalian cells using sitespecific endonucleases. Endonucleases that allow for site-specific deletion of a gene are well known in the art and may include TAL nucleases, meganucleases, zinc-finger nucleases, Cas9, and Argonaute. Methods for producing engineered, site-specific endonucleases are known in the art. The site-specific endonuclease can be engineered to recognize and delete or modify a specific gene, such as the FcRy chain gene.

[0195] In some embodiments, provided g-NK cells are engineered by editing the genome of the g- NK cells. In some embodiments, the editing of the genome may be carried out in a method that enriches for g-NK cell subset from a starting sample of NK cells. Thus, it is understood that the provided methods do not require selecting editing the genome only of g-NK cells that have been selected for NK cells that are deficient in the FcRy chain (or only that have been selected or identified by a g-NK surrogate marker profile), but may involve gene editing of a composition of NK cells that are to be, or that have been, preferentially expanded or enriched in g-NK cells. As such, the final composition of cells that are enriched in g-NK cells include g-NK cells that have been gene edited. Exemplary methods for preparing and expanding a composition enriched in g-NK cells is provided in Section II.

[0196] In some embodiments, the editing of the genome may take place at any suitable time during the methods of expanding the g-NK cells, such as described in Section II. In some embodiments, the gene editing is carried out after the selection of cells from a subject (e.g. selecting or enriching cells that are CD3negCD57posor CD3negCD56pos) and prior to incubating or culturing the selected or enriched cells withfeeder cells (e.g. HLA-E-expressing feeder cells) for proliferation or expansion of the NK cells. In some embodiments, the gene editing is carried out after the incubation or culture with the feeder cells (e.g. HLA-E-expressing feeder cells) and thus after selected or enriched cells have proliferated or expanded.

[0197] Methods for knocking out (e.g., deleting) a target gene expression include, but not limited to, a zinc finger nuclease (ZFN), a Tale-effector domain nuclease (TALEN), and CRIPSR / Cas system. Such methods typically comprise administering to the cell one or more polynucleotides encoding one or more nucleases such that the nuclease mediates modification of the endogenous gene, for example in the presence of one or more donor sequence, such that the donor is integrated into the endogenous gene targeted by the nuclease. Integration of one or more donor molecule(s) occurs via homology-directed repair (HDR) or by non-homologous end joining (NHEJ) associated repair. In certain embodiments, one or more pairs of nucleases are employed, which nucleases may be encoded by the same or different nucleic acids.

[0198] In one embodiment, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA- binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence, about or approximately 18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)).

[0199] In other embodiments, TAE-effector nucleases (TAEENs) can be generated to cleave specific sites in genomic DNA. Eike a ZFN, a TAEEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak, et al. (2013) Curr Opin Struct Biol. 23:93- 9). In this case, however, the DNA binding domain comprises a tandem array of TAE-effector domains, each of which specifically recognizes a single DNA base pair. Because ZFNs and TAEENs are heterodimeric so that the production of a single functional nuclease in a cell requires co-expression of two protein monomers, compact TAEENs provide an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4: 1762). A compact TAEEN comprises an engineered, site-specific TAE-effector DNA-binding domain fused to the nuclease domain from the I- TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TAEEN is functional as a monomer.

[0200] In some embodiments, engineered endonucleases based on the CRISPR / Cas9 system are also known in the art and can be employed in the provided methods to gene edit the cells (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al. (2013) Nat Methods. 10:957- 63). A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short"guide RNA" that directs the nuclease to a location of interest in the genome. In some embodiments, the guide RNA comprises an approximately 20 nucleotide targeting sequence. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in in the genome. Methods of using CRISPR-Cas9 are well known in the art.

[0201] In some embodiments, gene editing is carried out using an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is an RNA-guided DNA endonuclease. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non- limiting examples of RNA-guided nucleases include any as described in PCT publication No. W02020 / 168300 (e.g., Table 2 therein). In some embodiments, the RNA-guided nuclease is a Cas9 or Casl2 nuclease. In some embodiments, the RNA- guided nuclease is Cpfl (Casl2a). In some embodiments, Cpfl is Acidaminococcus sp. Cpfl (AsCpfl).

[0202] In some embodiments, gene editing is carried out with an RNA-guided nuclease and a guide RNA (gRNA). These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation. In some embodiments, the gRNA includes a crRNA and, optionally, a tracrRNA. In some embodiments, the RNA-guided nuclease (e.g., Cas9 or a Casl2) and one or more gRNAs form ribonucleoprotein (RNP) complexes that associate with (i.e., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the gRNA (e.g., crRNA). In some embodiments, the Cas is a Cas9 nuclease, such as from Streptococcus pyogenes. It is understood that the endonuclease used herein is not limited to the Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9. In one aspect, the Cas9 can come from a different bacterial source. Substitution of the Cas9 can also be used to increase the targeting specificity so less gRNA needs to be used. Thus, for example, the Cas can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus sp. (AsCpfl), Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpfl) derived from Lachnospiracase bacterium (LbCpfl), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacter jejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HFl, Fokl-Fused dCas9, or an expanded Cas9 (xCas9). Additionally other Cas endonucleases can be used in place of a Cas9 system such as, for example, CasX, CasY, Casl4, Cas4, Csn2, Casl3a, Casl3b, Casl3c, Casl3d, C2cl, or C2c3 or using any other type of engineered Cas protein including prime editing.

[0203] In some embodiments, a genome editing system containing an RNA-guided nucleases (e.g., a Cas) and a gRNA is implemented, in certain embodiments, as a protein / RNA complex (a ribonucleoprotein, or RNP) that is introduced into the cell to be edited. In some embodiments, the RNP complex is introduced into the cells in an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, or liposome. In certain embodiments, a genome editing system containing an RNA-guided nucleases (e.g., a Cas) and a gRNA is implemented as one or more nucleic acids encoding the RNA-guided nuclease and guide RNA components. For instance, in certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus.

[0204] In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM.” The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA-guided nuclease / gRNA combinations. Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. For example, Cas9 nucleases recognize PAM sequences that are 3’ of the protospacer, while Cpfl, on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer. In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA- guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. F. novicida Cpfl recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov el al, 2015, Molecular Cell 60, 385-397, November 5, 2015.

[0205] It is understood and herein contemplated that the use of a particular Cas can change the PAM sequence which the Cas endonuclease (or alternative) uses to screen for targets. As used herein, suitable PAM sequences comprises NGG (SpCas9 PAM) NNGRRT (SaCas9 PAM) NNNNGATT (NmCAs9 PAM), NNNNRYAC (CjCas9 PAM), NNAGAAW (St), TTTV (LbCpfl PAM and AsCpfl PAM); TYCV (LbCpfl PAM variant and AsCpfl PAM variant); where N can be any nucleotide; V = A, C, or G; Y = C or T; W = A or T; and R = A or G.

[0206] In some embodiments, the gRNA promotes the specific association (or “targeting”) of an RNA-guided nuclease (e.g., a Cas, such as a Cas9 or a Cpfl) to a target sequence such as a genomic sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA) and a tracrRNA, which are usually associated with one another, for instance by duplexing). Guide RNAs, whether unimolecular or modular, include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. For instance, in connection with a Cas9the crRNA is the guide RNA that provides the targeting domain that is a nucleotide sequence complementary to the target DNA, and also can include a tracrRNA that serves as a binding scaffold for the Cas nuclease. In connection with Cpfl, which induces double stranded DNA breaks under the guidance of a single crRNA, a tracrRNA is not required and instead the crRNA includes a 5 '-handle engaging Cpfl recognition and a guide segment interacting with targeted DNA sequences through complementary binding. Targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length).

[0207] In some embodiments, the gRNA, in some cases the crRNA, is any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity, when optionally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal 1W, Clustal X, BLAT, and others known to a skilled artisan. The ability of a guide sequence (within a nucleic-acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence. Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.

[0208] Methods for designing gRNAs are known to a skilled artisan (see e.g., Cui et al. (2018) Interdisciplinary Sciences: Computational Life Sciences, 10:455-465; PCT publication No. W02019 / 010384). Methods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al, 2014 Nat Biotechnol 32(3): 279- 84, Heigwer et al, 2014 Nat Methods 11(2): 122-3 ; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. As a non-limiting example, gRNA design may involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s targetsequence, e.g., to minimize total off-target activity across the genome. While off-target activity is not limited to cleavage, the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.

[0209] For example, a guide RNA comprising a targeting sequence of RNA nucleotides would include the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and this contains uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence of RNA nucleotides, and described by a DNA sequence that includes thymidine molecules would have a targeting domain of the corresponding RNA sequence that is the same but including uracil instead of thymidine. As will be apparent to the skilled artisan, such a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA / tracerRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For Cpfl, for example, a suitable scaffold sequence comprises the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO:84), added to the 5’- terminus of the targeting domain.

[0210] In some embodiments, the NK cells are edited to reduce expression of a target antigen that is known or suspected of also being expressed at some level by the NK cells. In some embodiments, gene editing is carried out with a gRNA that targets the target antigen known or suspected of being expressed at some level by the NK cells. In some embodiments, the NK cells express a CAR directed against CD38 and CD38 expression is reduced or eliminated in the NK cells. In some embodiments, the gRNA for use in the disclosure is a gRNA targeting CD38 (see e.g., WO2019 / 222503, WO2021 / 087466 and WO2021 / 113853 for exemplary gRNA targeting CD38).

[0211] In some embodiments, the gRNA targets a molecule involved in immunoreactivity of the NK cell. In some embodiments, HLA class I expression on the surface of the engineered g-NK cell is reduced. The human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. The HLA class I proteins all have a long alpha chain and a short beta chain, B2M. Little HLA class I can be expressed in the absence of B2M and the expression of B2M is required for HLA class I proteins to present peptides from inside the cell. The present disclosure provides g-NK cells engineered to reduce expression of B2M. Thus, these cells avoid the immune surveillance and attach by cytotoxic T cells. In some embodiments, the gRNA for use in the disclosure is a gRNA targeting beta 2 microglobulin (B2M) (see e.g., W02020 / 168300,WO2018 / 064694, WO2015 / 161276, or W02017 / 152015) for exemplary gRNA targeting B2M).

[0212] In some embodiments, the gRNA targets a molecule involved in immunosuppression of the NK cell activity. Suitably, engineered NK cells comprise reduced or absent checkpoint inhibitory receptor function. Suitably, the checkpoint inhibitory receptors with reduced or absent function comprise one or more or all of CD96 (TACTILE), CD 152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, and / or TIM-3. Suitably, the NK cell cells comprise reduced or absentcheckpoint inhibitory receptor function for two or more checkpoint inhibitory receptors. Suitably, the two or more checkpoint inhibitory receptors comprise CD96 (TACTILE), CD 152 (CTLA4), or CD328 (SIGLEC7) or CD279 (PD-1).

[0213] In some embodiments the gRNA for use in the disclosure is a gRNA targeting TIGIT (see e.g., W02020 / 168300 for exemplary gRNA targeting TIGIT). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting PD-1 (see e.g., WO2015 / 161276, or W02017 / 152015) for exemplary gRNA targeting PD-1).

[0214] In some embodiments the gRNA for use in the disclosure is a gRNA targeting an adenosine receptor, such as adenosine A2a receptor (ADORA2a) (see e.g., W02020 / 168300 for exemplary gRNA targeting ADORA2a). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting a TGF beta receptor, such as TGFbetaR2 (see e.g., W02020 / 168300 for exemplary gRNA targeting TGFbetaR2). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting the gene encoding cytokine-inducible SH2-containing protein (CISH) (see e.g., W02020 / 168300 for exemplary gRNA targeting CISH).

[0215] In some embodiments, RNA-guided nuclease-encoding and / or gRNA encoding DNA, can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof. In some embodiments the nucleic acid encoding the RNA-guided nuclease (e.g., a Cas) and / or gRNA is delivered by AAV. Nucleic acids for gene editing can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells.

[0216] In some embodiments the RNA-guided nuclease and gRNA are delivered into cells as a ribonucleoprotein (RNP) complex. In some embodiments, the Cas and gRNA are separately purified and then assembled to form the RNP. In some embodiments, one or more RNP complexes are delivered to the cell sequentially in any order, or simultaneously. In some embodiments the RNP complex is delivered into cells by electroporation. In some embodiments the RNP complex is delivered into cells using lipid nanoparticles.

[0217] In one non-limiting example, to make the RNP complex, crRNA and tracrRNA can be mixed at a 1:1, 2:1, or 1:2 ratio of concentrations between about 50 pM and about 500pM (for example, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 35, 375, 400, 425, 450, 475, or 500pM), preferably between 100 pM and about 300 pM, most preferably about 200 pM at 95C for about 5 min to form a crRNA:tracrRNA complex (i.e., the guide RNA). The crRNA:tracrRNA complex can then be mixed with between about 20pM and about 50pM (for example 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 48,49, or 50pM) final dilution of a Cas endonuclease (such as, for example, Cas9).

[0218] In particular embodiments, introduction of an RNP complex into NK cells, such as expanded NK cells enriched for g-NK cells as described in Section II, is by electroporation. Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane. The application of the electric filed cause a charge gradient across the membrane which draws the charged molecules such as, nucleic acid, across the cell membrane. Thus, in one aspect, disclosed herein are methods of genetically modifying an NK cell comprising obtaining guide RNA (gRNA) specific for a target DNA sequence in the NK cell; and b) introducing via electroporation into a target NK cell, a ribonucleoprotein (RNP) complex comprising a Cas endonuclease (e.g., Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the target sequence within the genomic DNA of the NK cell.

[0219] In some aspects, the guide sequence is any polynucleotide sequence comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as a gene encoding FcRy, PLZF, HEEIOS, SYK, DAB2 or EAT2, to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. Typically, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0220] In some embodiments, a CRISPR enzyme (e.g., Cas9 nuclease) in combination with (and optionally complexed with) a guide sequence is delivered to the cell. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus.

[0221] In one embodiment of the invention, the DNA break-inducing agent is an engineered homing endonuclease (also called a "meganuclease"). Homing endonucleases are a group of naturally-occurring nucleases which recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA- repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases arecommonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757- 3774). The LAGLIDADG homing endonucleases with a single copy of the LAGLIDADG motif form homodimers, whereas members with two copies of the LAGLIDADG motif are found as monomers.

[0222] Another method of decreasing FcRy chain expression, activity and / or signaling involves introducing an inhibitory nucleic acid, such as an inhibitory RNA, into the cell that targets, e.g., is complementary to, a target gene transcript, such as an FcRy, PLZF, HELIOS, SYK, DAB2 or EAT2 gene transcript, thereby reducing expression of the gene product. For example, the nucleic acid may target FcRy chain mRNA. In other embodiments, the inhibitory nucleic acid may target the mRNA of a gene that regulates transcription or translation of the FcRy chain gene, such as a transcription factor, for example PLZF or HELIOS mRNA. In some embodiments the nucleic acid targets the mRNA of gene encoding a protein involved in FcRy-mediated signaling, such as SYK, DAB2 or EAT-2 mRNA.

[0223] The presently disclosed subject matter takes advantage of RNAi technology (for example shRNA, siRNA and miRNA molecules and ribozymes) to cause the down regulation of cellular genes, a process referred to as RNA interference (RNAi). As used herein, “RNA interference” (RNAi) refers to a process of sequence-specific post-transcriptional gene silencing mediated by a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules, miRNA molecules or synthetic hammerhead ribozymes. See generally Fire et al., Nature 391:806-811, 1998, and U.S. Pat. No. 6,506,559. The process of RNA interference (RNAi) mediated post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism that has evolved to prevent the expression of foreign genes (Fire, Trends Genet 15:358-363, 1999).

[0224] In some embodiments, a recombinant virus comprising nucleic acid encoding the RNA can be produced. Engineering retroviral vectors is known to those having ordinary skill in the art. Such a skilled artisan would readily appreciate the multiple factors involved in selecting the appropriate virus and vector components needed to optimize recombinant virus production for use with the presently disclosed subject matter without the necessity of further detailed discussion herein. As one non-limiting example, a retrovirus can be engineered comprising DNA encoding an shRNA comprising an siRNA.

[0225] The gene expression may be reduced permanently, transiently, or inducibly. Suitable inducible systems are well known and include eukaryotic promoters responsive to heavy metals, Lac / VP16, and the tetracycline repressor system.

[0226] On the other hand, it may be beneficial to permanently reduce expression of the gene, for example by producing a cell line with a deletion, substitution, or insertion that causes inactivation of the gene.

[0227] Retroviral systems can be used to introduce cDNAs into NK cells. Methods of eukaryotic cell transfection and prokaryotic cell transformation are well known in the art. The choice of host cell dictates the preferred technique for introducing the polynucleotide of interest. Introduction of polynucleotides into an organism may also be done with ex vivo techniques that use an in vitro method of transfection, as well as established genetic techniques, if any, for that particular organism.

[0228] Other vectors and packaging cell lines have been used in the preparation of genetically modified variants of NK cells and can be used equivalently herein. Retroviral transduction systems have also been successfully used to transduce a variety of genes into NK cells. By way of example, these alternative methods include, but are not limited to, the p-JET vector in conjunction with FLYA13 packaging cells (Gerstmayer et al., 1999), the plasmid-based kat retroviral transduction system, and DFG- hIL-2-neo / CRIP (Nagashima et al., 1998). Electroporation and “gene gun” introduction of the vector into the packaging cells is also practiced. Use of the pBMN-IRES-EGFP vector in combination with the Phoenix- Amphotropic packaging cell line is convenient in that it provides high efficiencies of Phoenix - Amphotropic cell transfection. The use of Moloney ETR promoters results in a high level of CD 16 expression; the virus is produced at high titers. The efficiency of NK transduction is improved over other vectors that have been used to transduce NK cells; and the vector provides adequate space to accommodate the CD16 cDNA or alternative inserts. The pBMN-IRES-EGFP vector further incorporates genes for enhanced green fluorescent protein (EGFP), which can be used as an endogenous surrogate marker for gene expression. The Phoenix cell line stably expresses this vector in episomal form along with producing other viral components, thus allowing the cells to stably produce virus for an extended period of time.

[0229] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0230] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Eaboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0231] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0232] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0233] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0234] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 deg. C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueousmedium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0235] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise gene edit the NK cell in accord with the provided methods, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR or "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots.

[0236] In some embodiments, the gene edited g-NK cells can be further selected and expanded such as by methods described in Section II.

[0237] Among the provided compositions of g-NK cells are compositions in which the g-NK cells are engineered g-NK cells.

[0238] In some embodiments, the engineered g-NK cells of the composition express a CAR. In some embodiments, the g-NK cell is engineered with a bispecific CAR or multiple different CARs. In some embodiments, the CAR or CARs are directed to target antigens expressed by cells of the autoimmune disease or conditions. In particular embodiments, the CAR or CARs are directed to a B cell antigen. Exemplary CARs and methods for engineering cells are described in Section III. A.

[0239] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR.

[0240] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR.

[0241] In some embodiments, the engineered g-NK cells of the composition express one or more other additional heterologous protein agent. In some embodiments, the engineered g-NK cells express an immunomodulator, such as a cytokine. In some embodiments, the engineered g-NK cells also express a secreted antibody. In some embodiments, the immunomodulator is an agent that is capable of regulating immune function of the NK cell. In some embodiments, an immunomodulator may be an immunoactivator. In other embodiments, an immunomodulator may be an immunosuppressant. In some embodiments, the immunomodulator is an exogenous cytokine, such as an interleukin or a functional portion thereof. Exemplary immunomodulators and methods for engineering cells are described in Section III.B.

[0242] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound asdescribed). In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described).

[0243] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described).

[0244] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologousnucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described).

[0245] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described).B. A utoimmune Diseases or Disorders

[0246] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder. In some embodiments, the subject with an autoimmune disease or disorder has a condition in which the immune system mistakenly attacks healthy cells. In some embodiments, an autoimmune cell or disorder can result in the attack of one or more types of cells, inflammation of one or more types of body tissues, abnormal growth of an organ, and / or changes in organ function. In particular embodiments, the subject with the autoimmune or disorder may experience symptoms impacting blood vessels, connective tissues, endocrine gland (e.g., thyroid or pancreas), joints, muscles, red blood cells, and skin.

[0247] In embodiments of any of the provided methods, the subject is characterized by autoreactive cells, such as any described in Section I.C or Section I.D. In some embodiments, the autoreactive cells are cells that mistakenly attack healthy cells, leading to the symptoms described above. In some embodiments, the autoreactive cells are autoreactive immune cells. In some embodiments, the autoreactive cells are autoreactive B and / or T cells. In some embodiments, the autoreactive cells are autoreactive B cells. In some embodiments, the autoreactive cells are autoreactive T cells. In some embodiments, the autoreactive cells express human leukocyte antigen-E (HLA-E) or are HLA-E expressing, as described in Section I.C. In some embodiments, the autoimmune disease or condition is characterized by autoreactive cells with upregulated HLA-E expression.

[0248] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a selected subject having an autoimmune disease or disorder. In some embodiments, the composition of g- NK cells is administered as a monotherapy without co-administration of an antibody. In other embodiments, the methods include administering a composition of g-NK cells and further include administering to the subject an antibody directed against a target antigen expressed by cells of the autoimmune disease, for example to promote ADCC by the co-administered g-NK cells. Examples of antibodies in such a provided combination therapy include any described in Section I.F. The provided therapeutic approaches facilitate treatment of the autoimmune disease by depleting EBV-specific HLA-E- restricted T and B cells (e.g., GlialCam-specific autoreactive T cells and B cells) as well as eliminating the latent viral reservoir. When further used in combination with a monoclonal antibody, provided therapeutic approaches also can enhance ADCC to more fully deplete potential disease autoreactive T and B cells (e.g., CD20+ cells targeted by combination with an anti-CD20 antibody, such as ocrelizumab).

[0249] In any of the methods described herein, a subject in need of a treatment as described herein can be a subject exhibiting symptoms of an autoimmune disease or condition. In particular embodiments,the subject having the autoimmune disease or disorder is selected after diagnosis for the autoimmune disease or disorder using various types of diagnostic tests. For example, autoimmune disease or disorder diagnosis tests can include, but are not limited to, antinuclear antibody tests, autoantibody tests, complete blood count (CBC), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR).

[0250] In some embodiments, the subject with the autoimmune disease or disorder can be prescribed medicines to control or reduce the subject’s own immune system. In particular embodiments, the medicines can involve immunosuppressive medicines. In some embodiments, the methods provided herein comprise the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder after the subject has received immunosuppressive medicines. In particular embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), the subject is still receiving immunosuppressive medicines. In some embodiments, the subject with the autoimmune disease or disorder can be currently receiving immunosuppressive medicines. In particular embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), the subject has stopped receiving immunosuppressive medicines. In some embodiments, the subject with the autoimmune disease or disorder has previously received immunosuppressive medicines. In specific embodiments, the immunosuppressive medicines may include, but are not limited to steroids, such as corticosteroids, or non-steroid drugs, such as azathioprine, cyclophosphamide, mycophenolate mofetil (MMF) / mycophenolic acid, sirolimus, tacrolimus. In particular embodiments, the medicines can involve nonsteroidal anti-inflammatory drugs (NSAIDs).

[0251] In some embodiments, the selected subject with the autoimmune disease or disorder has autoreactive cells (e.g., autoreactive T and / or B cells). In some embodiments, the autoreactive cells are HLA-E expressing and / or have upregulation of HLA-E.

[0252] In some embodiments, the selected subject with the autoimmune disease or disorder can have, but are not limited to: Addison disease, celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease (e.g., Crohn disease, ulcerative colitis), multiple sclerosis, systemic sclerosis (SSc), myositis, myasthenia gravis, pernicious anemia, arthritis, Sjogren syndrome, lupus, chronic inflammatory demyelinating polyneuropathy (CIDP), pemphigus, antisynthetase syndrome, antiphospholipid syndrome (APLS), neuromyelitis optica soectrum disorder (NMOSD), and Type I diabetes. In some embodiments, the selected subject with the autoimmune disease or disorder can have psoriasis. In some embodiments, the selected subject with the autoimmune disease or disorder can have ankylosing spondylitis. In some embodiments, the selected subject with the autoimmune disorder can have juvenile idiopathic arthritis (JIA). In some embodiments, the selected subject with the autoimmune disease or disorder can have a renal or kidney disease. In specific embodiments, the kidneyor renal disease include, but are not limited to, systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN). In some embodiments, the selected subject with the autoimmune disease or disorder can have a liver disease.

[0253] In particular embodiments, the kidney or renal disease can be systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN). Short term readouts to assess clinical proof of concept can include performing autoimmune antibody titers, assessing proteinuria, or examining infiltrates on kidney biopsies. In some embodiments, the short-term clinical readout is proteinuria. In some embodiments, the short-term clinical readout is autoimmune antibody titers. In some embodiments, the short-term clinical readout is infiltrates on kidney biopsy.

[0254] In particular embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), the subject is still receiving immunosuppressive medicines. In some embodiments, the subject with the autoimmune disease or disorder can be currently receiving immunosuppressive medicines. In particular embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), the subject has stopped receiving immunosuppressive medicines. In some embodiments, the subject with the autoimmune disease or disorder has previously received immunosuppressive medicines. In specific embodiments, the immunosuppressive medicines may include, but are not limited to steroids, such as corticosteroids, or non-steroid drugs, such as azathioprine, cyclophosphamide, mycophenolate mofetil (MMF) / mycophenolic acid, sirolimus, or tacrolimus. In some embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), the subject is still receiving immunosuppressive medicines.

[0255] In some embodiments, at the time of administering the dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), subject with the autoimmune disease or disorder may have received one or more prior treatments to treat the autoimmune disease or disorder.1. Multiple Sclerosis

[0256] In particular embodiments, the autoimmune disease or disorder may be multiple sclerosis. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system with both autoimmune and neurodegenerative features. More than 2.3 million people worldwide suffer from MS, with 400,000 people in the U.S. alone having the disease. The disease of MS presents itself through neurological impairments due to damage to various parts of the central nervous system (CNS), including the spinal cord, brainstem, optic nerves, cerebellum, and cerebrum. These impairments can lead to symptoms such as weakness, pain, vision impairment, dysfunction of the bowel / bladder, and cognitive issues. Thediagnosis of MS is typically made using a comprehensive set of diagnostic criteria that include clinical observation, neurological examination, magnetic resonance image (MRI) scans of the brain and spinal cord, evoked potential tests, and studies of the cerebrospinal fluid (CSF) (McDonald et al., Ann Neurol., 2001; Polman et al., Ann Neurol., 2005).

[0257] The multiple sclerosis can be further categorized as, but are not limited to relapsing-remitting multiple sclerosis (RRMS); secondary progressive multiple sclerosis (SPMS); progressive relapsing multiple sclerosis (PRMS); and primary progressive multiple sclerosis (PPMS). The most frequent form of MS is relapsing-remitting multiple sclerosis (RRMS), which is clinically characterized by recurring episodes of neurological symptoms. PPMS is a less common variant of MS, representing about 10% to 15% of all MS cases. PPMS is defined by a steady progression of the disease from its onset, without distinct clinical attacks or relapses (Ebers, Mult Scler., 2004; Miller & Leary, Lancet Neurol., 2007). In contrast to RRMS, PPMS typically begins at a later age, around 40 years, and affects men nearly as frequently as women (Cottrell et al., Brain, 1999). The lack of relapses presents unique diagnostic challenges, necessitating clinical proof of disease progression for at least one year from the onset of symptoms (McDonald et al., Ann Neurol.L2001; Polman et al., Ann Neurol., 2005). In some embodiments, MS can be further characterized as progression independent of relapse activity (PIRA) MS. PIRA MS may occur in subjects with RRMS or early relapsing MS, wherein subjects have worsening disability or disability progression independent of relapses (Sharrad et al., Mult Scler Relat Disord. , 2023).

[0258] Multiple sclerosis (MS) is characterized by various symptoms and signs of CNS dysfunction, with remissions and recurring exacerbations. Classifications of interest for treatment by the provided methods include relapsing remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS). In some embodiments, the provided methods include progression independent of relapse activity (PIRA) MS. The most common presenting symptoms are paresthesias in one or more extremities, in the trunk, or on one side of the face; weakness or clumsiness of a leg or hand; or visual disturbances, e.g., partial blindness and pain in one eye (retrobulbar optic neuritis), dimness of vision, or scotomas. Other common early symptoms are ocular palsy resulting in double vision (diplopia), transient weakness of one or more extremities, slight stiffness or unusual fatigability of a limb, minor gait disturbances, difficulty with bladder control, vertigo, and mild emotional disturbances. In some embodiments, any of such symptoms indicate scattered CNS involvement and often occur months or years before the disease is recognized. Excess heat can accentuate symptoms and signs.

[0259] Despite significant advances in the treatment of progressive MS, effective therapies for both PPMS and non-active SPMS remain limited. Most disease-modifying therapies (DMTs) approved for RRMS are ineffective in slowing the progression of disability in PPMS and non-active SPMS (Bayas et al., Ther Adv Neurol Disord., 2023; Hollen et al., Fed Pract., 2020). A large Phase 3 randomizedcontrolled trial with glatiramer acetate (Wolinsky et al., Ann Neurol., 2007), along with smaller randomized controlled clinical trials evaluating mitoxantrone (Stiive et al., Mult Scler., 2004), intramuscular interferon (IFN)-pia (Leary et al., Neurology, 2003), and IFN-pib (Montalban, Mult Scler., 2004), did not show a significant effect on the clinical progression in the PPMS population. An earlier randomized controlled PPMS clinical trial, the 439-patient Phase 2 / 3 OLYMPUS study (Study U2786g), compared rituximab to a placebo over a 96-week treatment period. This study did not show a significant treatment effect overall. However, subgroup analyses suggested that a younger, more active subset of PPMS patients (defined by the presence of gadolinium-enhancing lesions seen on MRI at baseline and higher lifetime rates of disability progression) may have seen clinical benefits from anti-CD20 therapy (Hawker et al., Ann Neurol., 2009). Currently, ocrelizumab is the only FDA-approved medication for the treatment of PPMS, based on the results of the ORATORIO trial, that demonstrated a modest reduction in the risk of disability progression (Montalban et al., New England Journal of Medicine, 2017). However, many PPMS patients treated with ocrelizumab continue to experience worsening of their condition despite treatment (Weinstock-Guttman et al., Mult Scler., 2022).

[0260] Treatment refractory progressive forms of MS are a significant unmet medical need. Patients with this disease develop a relentless accumulation of disability, leading to a loss of independence and a reduced quality of life (Watson et al., Neurol Ther., 2023). The progressive nature of the disease also places a substantial burden on caregivers and healthcare systems (Benini et al., PLoS One, 2023).

[0261] Current treatments for chronic autoimmune diseases, such as immunosuppressive drugs and biologies, often fail to achieve complete disease control and can cause significant side effects (Fugger, Jensen, & Rossjohn, Cell, 2020). Because B cells play a central role in the pathogenesis of autoimmune diseases by producing autoantibodies and presenting autoantigens to T cells, CAR-T cells that target B cells expressing the CD 19 antigen have been used as a treatment (Muller et al., New England Journal of Medicine, 2024). Recent clinical trials demonstrated the potential for cell therapies in autoimmune diseases. A novel approach using chimeric antigen receptor T (CAR-T) cell therapy treated advanced autoimmune diseases including systemic lupus erythematosus, systemic sclerosis and idiopathic inflammatory myositis with apparent success (Muller et al., New England Journal o / Medicine, 2024). Chronic autoimmune diseases are characterized by the production of autoantibodies and immune complexes that lead to inflammation and damage in multiple organ systems (Pisetsky, Nat Rev Nephrol. , 2023). CAR-T cell therapy appears to have induced long-term remission in autoimmune disease patients while potentially avoiding the broad immunosuppression associated with current treatments (Muller et al., New England Journal of Medicine, 2024).

[0262] CAR-T cell therapy is associated with a range of significant side effects. The most common and well-characterized is cytokine release syndrome (CRS), that occurs due to the rapid activation and proliferation of CAR-T cells, leading to a systemic inflammatory response (Wei et al., Signal TransductTarget Ther., 2020;5(l):143). CRS can manifest with fever, hypotension, hypoxia, and organ dysfunction, and in severe cases, can be life-threatening (Murthy et al., Immunotargets Ther., 2019). Another significant side effect is immune effector cell-associated neurotoxicity syndrome (ICANS), that can cause neurological symptoms such as confusion, aphasia, seizures, and cerebral edema (Rees, The EBMT / EHA CAR-T Cell Handbook, 2022). Long-term side effects, such as the potential for insertional oncogenesis due to the use of viral vectors in CAR-T cell production, are still under investigation (Bonifant et al., Mol Ther Oncolytics, 2016).

[0263] Thus, there remains a need for effective therapies for treating MS. Provided embodiments address these needs. Interestingly, g-NK cells arise in response to HCMV in some individuals, and the development of g-NK cells in HCMV seropositive individuals was associated with a significant delay in the time from disease onset to the assignment of sustained EDSS endpoints (Martmez-Rodriguez et al., Mult Scler., 2016). More recent data further support the role of g-NK cells in MS and suggest that the development of g-NK cells in response to HCMV infection may play an important role in preventing MS in individuals at high risk for developing MS (Vietzen et al., Cell, 2023).

[0264] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis who has not previously received a prior therapy to treat the multiple sclerosis (MS).

[0265] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis who has previously received a prior therapy to treat the multiple sclerosis (MS).

[0266] In some embodiments, the prior therapy to treat the MS comprises B-cell depletion therapy. In some embodiment, the B-cell depletion therapy is treatment with an anti-CD20 therapy (e.g., administration of an anti-CD20 antibody, e.g., ocrelizumab).

[0267] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis who has not previously received B-cell depletion therapy to treat the multiple sclerosis (MS). In some embodiments, the subject has not received a last treatment of the B-cell depletion therapy in the past 48 weeks.

[0268] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis who has previously received B-cell depletion therapy to treat the multiple sclerosis (MS). In some embodiments, the subject received a last treatment of the B-cell depletion therapy in the past 48 weeks.

[0269] In some embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.

[0270] In other embodiments, the methods include administering g-NK cells and further includes administering to the subject an antibody directed against a target antigen expressed by cells of the multiple sclerosis (MS), for example to promote ADCC by the co-administered g-NK cells. Examples of antibodies in such a provided combination therapy include any described in Section I.F. Based on the enhanced ADCC that offers the potential to more fully deplete potential disease causing CD20+ cells (B and T) the combination of g-NK cells and an anti-CD20 antibody (e.g., ocrelizumab) may be able to provide additional therapeutic benefit to patients with MS. The claimed g-NK cell therapy is not only able to deeply deplete EBNA-auto-antibody producing B cells (or CD20 expressing B & T cells) but also deplete GlialC AM-specific autoreactive T cells and B cells; as well as eliminate the latent viral reservoir. This is an improvement from, for example, a monotherapy with rituximab only. Research groups have shown that monotherapy with rituximab led to “shallow” depletion of B cells in the periphery. Anolik et al. Arthritis Rheum. (2007). 56(9):3044-3056.

[0271] Among monoclonal antibodies for use in a provided combination therapy with g-NK cells is an anti-CD20 antibody. Exemplary anti-CD20 antibodies are described in Section I.F. In some embodiments, the anti-CD20 antibody is ocrelizumab. In some embodiments, the g-NK cells, and their combination with an anti-CD20 antibody, are able to target autoreactive B cells and HLA-E expressing plasma B cells. Moreover, the g-NK cells, and their combination with an anti-CD20 antibody, are able to target autoreactive T cells and HLA-E expressing T cells. Specifically, not only are CD20+ autoreactive B cells associated with multiple sclerosis, but in some aspects CD20dimT cells are pathogenic autoreactive cells that play a role in MS. It has been reported that T cells may acquire CD20 from B cells via trogocytosis, and can obtain CD20 by engaging B cells in the presence of antigen (Ochs et al., Sci. Trans. Med., 2022). CD20+ T cells are associated with disease severity in patients with MS, particularly in the CNS in which reports indicate a positive correlation between CD20+ T cells in CNS and MS disease severity (Von Essen et al., 2019). Studies indicate CD8+CD20+ T cells are associated with white matter injury and thalamic atrophy in PPMS (Von Essen et al., Neurol. Neuroimmunol., Neuroinflamm., 2023). Also, CD20dimCD8+ T cells in circulation in patients with MS are inversely correlated with pretreatment MRI gadolinium lesion counts, and may be involved in MS relapse development. CD20+ T cells are believed to be pathogenic and autoreactive. CD20+ T cells have been reported to have a proinflammatory phenotype, proliferate on CNS antigen encounter, display a pathogenic phenotype in patients with MS, and associated with an increase in CD20+myelin-specific CD8+ memory T cells in patients with MS (Von Essen et al., Brain 2019; Quendt et al., Ann. Neurol., 2021; and Sabatino et al., PNAS, 2019).

[0272] Among provided embodiments include methods of administering provided g-NK cells to a subject that has autoreactive T cells, such as CD20+ (e.g., CD20dim) T cells. In particular, g-NK cells are effective killers of target cells expressing low surface level expression, i.e. “dim” levels, of antigen (see e.g., WO2021 / 216790, incorporated by reference herein, such as FIGs. 21A-B and FIGS. 22A-E therein, in which it is shown that g-NK cells exhibit cytolytic activity of target cells expressing only very low antigen, whereas conventional NK cells exhibit only very low cytotoxicity).

[0273] Among provided embodiments include methods of administering provided g-NK cells to a subject that has PPMS. In some embodiments, the provided methods of treating a subject that has PPMS include administering a composition of g-NK cells in combination with a monoclonal antibody directed against autoreactive B cells and T cells, such as an anti-CD20 antibody (e.g., ocrelizumab).

[0274] In some embodiments, the g-NK cells in combination with an anti-CD20 antibody (e.g., ocrelizumab) provide for a multifactorial mechanism of action that goes beyond B cell depletion. In some embodiments, the multifactorial actions may provide for curative treatment for MS. In some embodiments, g-NK cells in combination with an anti-CD20 antibody (e.g., ocrelizumab) result in (1) deep depletion of CD20+ B cells and CD20+ T cells via ADCC for immune reset; (2) depletion of HLA- E expressing autoreactive T cells, plasma B cells and / or plasma blasts; and (3) elimination of latent viral reservoir (e.g., EBV) that may cause flares due to disease initiation. FIG. 9 provides depicts a schematic of this multifactorial mechanism of action. In some embodiments, administration of g-NK cells alone can result in therapeutic activity by mechanisms (2) and (3) but combination with an CD20-directed antibody would have the additional advantage of directly targeting for depletion CD20+ B cells and CD20+ T cells. Notably, the multifactorial mechanism of action by the combination therapy addresses deficiencies of any one mechanism. For instance, while combination of g-NK cells and anti-CD20 antibody can result in depletion of CD20+ B cells and CD20+ T cells via ADCC, anti-CD20 antibodies (e.g., ocrelizumab) are not able to target autoantibody producing long-lived plasma cells or plasma blasts. However, g-NK cells via their NKG2C-dependent HLA-E-specific activity are able to target autoantibody producing plasma B cells, plasma blasts, and autoreactive T cells that express HLA-E. The activities of g-NK cells, alone or in combination with a monoclonal antibody, are differentiated from antibody alone therapies or from CD19 CAR therapies. For instance, antibody alone (e.g., ocrelizumab alone would not be able to deplete HLA-E expressing cells, such as plasma blasts, plasma B cells, or autoreactive CD4+ and CD8+ T cells. CD19 CAR T cells are would not be effective in targeting CD20dimT cells or HLA-expressing plasma cells or HLA-E expressing autoreactive CD4 and CD8 T cells.

[0275] In some embodiments, a subject selected for treatment in accord with the provided methods has treatment refractory progressive MS, wherein the subject meets at least one or all of the following criteria: (a) have a confirmed diagnosis of primary of non-active secondary progressive MS (SPMS) based on the 2017 revisions of the McDonald criteria (Thompson 2018, Lancet Neurol., 17:162-173),where non-active SPMS is defined by the absence of clinical relapse in the two years prior to study entry; (b) has been dosed with ocrelizumab within the prior 6 months; (c) Expanded Disability Status Scale (EDSS) at screening is from 3.0 to 6.5 points; (d) score of >2.0 on the Functional Systems (FS) scale for the pyramidal system that is due to lower extremity findings; (e) disease duration from the onset of MS symptoms that is either less than 15 years in subjects with an EDSS at screening >5.0 or less than 10 years in subjects with an EDSS at screening <5.0; (f) documented history or presence at screening of at least 1 of the following laboratory findings in a cerebrospinal fluid (CSF) specimen: (1) elevated immunoglobulin G index, or (2) two or more immunoglobulin G oligoclonal bands detected by isoelectric focusing; (g) aged 18 to 65 years old at time of enrolment; and (h) neurologic stability >30 days prior to screening and baseline.

[0276] Current pharmacological treatments for MS, including disease modifying therapies (DMTs), either modify or suppress the body’ s immune system. Many of the current pharmacological agents for MS are limited by incomplete efficacy, side effects and medical risks. These treatments have been shown to modestly reduce neurological relapses of the disease and, in some instances, incompletely slow the progression of neurological disability. Therapies for multiple sclerosis can also involve the use of systemic drugs (e.g., high dose systemic steroids), parenteral medicaments (e.g., beta-interferons, glatiramer acetate, natalizumab), oral medicaments (e.g., fingolimod (dimethyl fumarate), teriflunomide), and cortisones (prednisolone or methyl prednisolone).

[0277] In some embodiments, the prior therapy may be referred to as a disease modifying therapy (DMT). The provided methods described herein may be used in a method of treatment of relapsing forms of MS in subjects who have had an inadequate response to (or are refractory to) one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten or more disease modifying therapies (DMTs). In certain embodiments, the selected subject may have signs of or be further diagnosed with worsening relapsing-remitting MS (RRMS), progressive -relapsing MS (PRMS) or secondary-progressive MS (SPMS) to reduce neurologic disability and / or the frequency of clinical exacerbations. The provided methods described herein may reduce the frequency and / or severity of relapses.

[0278] In some embodiments, a clinical relapse, which may also be used herein as “relapse,” “confirmed relapse,” or “clinically defined relapse,” is the appearance of one or more new neurological abnormalities or the reappearance of one or more previously observed neurological abnormalities. This change in clinical state must last at least 48 hours and be immediately preceded by a relatively stable or improving neurological state of at least 30 days. In certain embodiments, an event is counted as a relapse when the subject’s symptoms are accompanied by observed objective neurological changes, consistent with an increase of at least 1.00 in the Expanded Disability Status Scale (EDSS) score or one grade in the score of two or more of the seven FS or two grades in the score of one of FS as compared to the previous evaluation.

[0279] The provided methods described herein may be used in a method of treatment of relapsing forms of MS, for example, to slow the accumulation of physical disability and / or reduce the frequency of clinical exacerbations. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis, who has experienced relapse after having previously received a prior therapy to treat the multiple sclerosis. In particular embodiments, the selected subject may suffer from relapsing-remitting multiple sclerosis. Subjects suffering from RRMS experience sporadic exacerbations or relapses, as well as periods of remission. Lesions and evidence of axonal loss may or may not be visible on MRI for subjects with RRMS.

[0280] RRMS may include subjects with RRMS as well as subjects with secondary progressive multiple sclerosis (SPMS) and superimposed relapses. In particular embodiments, the selected subject may suffer from secondary progressive multiple sclerosis (SPMS). SPMS may evolve from RRMS. Subjects afflicted with SPMS have relapses, a diminishing degree of recovery during remissions, less frequent remissions and more pronounced neurological deficits than RRMS subjects. Enlarged ventricles, which are markers for atrophy of the corpus callosum, midline center and spinal cord, are visible on MRI of subjects with SPMS. In various embodiments, the presently described g-NK cells may be used treat RRMS so it does not develop into SPMS.

[0281] In particular embodiments, the selected subject may suffer from primary progressive multiple sclerosis (PPMS). PPMS is characterized by a steady progression of increasing neurological deficits without distinct attacks or remissions. Cerebral lesions, diffuse spinal cord damage and evidence of axonal loss are evident on the MRI of subjects with PPMS. PPMS has periods of acute exacerbations while proceeding along a course of increasing neurological deficits without remissions. Lesions are evident on MRI of subjects suffering from PRMS. In various embodiments, the presently described g- NK cells may be used treat RRMS and / or SPMS so it does not develop into PPMS.

[0282] The provided methods herein may also be used to prevent the onset of multiple sclerosis. In certain embodiments, the selected subject is a subject who has experienced a first clinical episode and have MRI features consistent with MS.

[0283] In particular embodiments, the selected multiple sclerosis subject can have a clinically isolated syndrome (CIS). A clinically isolated syndrome (CIS) is a single monosymptomatic attack compatible with MS, such as optic neuritis, brain stem symptoms, and partial myelitis. Subjects with CIS who subsequently experience a second clinical attack are generally considered to have clinically definite multiple sclerosis (CDMS). Over 80 percent of subjects with CIS and MRI lesions go on to develop MS, while approximately 20 percent have a self- limited process. Subjects with CIS may show lesion dissemination on subsequent MRI scans according to McDonald’s criteria (Thompson 2018, LancetNeurol., 17:162-173). In various embodiments, the presently described g-NK cells are used to treat CIS so it does not develop into MS, including, for example relapse-remitting multiple sclerosis (RRMS).

[0284] In particular embodiments, the selected multiple sclerosis subject can have a radiologically isolated syndrome (RIS). In RIS, incidental imaging findings suggest inflammatory demyelination in the absence of clinical signs or symptoms. In various embodiments, the presently described g-NK cells are used to treat RIS so it does not develop into MS, including, for example relapse-remitting multiple sclerosis (RRMS).

[0285] In particular embodiments, the selected multiple sclerosis subject can have benign multiple sclerosis. Benign multiple sclerosis is a retrospective diagnosis which is characterized by 1-2 exacerbations with complete recovery, no lasting disability and no disease progression for 10-15 years after the initial onset. Benign multiple sclerosis may, however, progress into other forms of multiple sclerosis. In various embodiments, the presently described g-NK cells are used to treat benign multiple sclerosis so it does not develop into MS, including, for example relapse-remitting multiple sclerosis (RRMS).

[0286] In some embodiments, the subject can be evaluated, e.g., for indicia of responsiveness, prior to, during, or after receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness prior to receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness concurrently or with the administration of the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness after receiving the composition of g-NK cells.

[0287] In certain embodiments, the claimed g-NK cell therapy is able to not only deeply deplete CD20 producing B cells and T cells but also deplete GlialCAM-specific autoreactive T cells and B cells; as well as eliminate the latent viral reservoir. To evaluate for indicia of responsiveness after receiving the composition of g-NK cells, there can be several primary and / or secondary biomarker endpoints. In some embodiments, an exemplary primary and / or secondary biomarker endpoint is pharmacokinetics of g-NK cells, the anti-CD20 antibody (e.g., ocrelizumab), and / or IL-2. In some embodiments, an exemplary primary and / or secondary biomarker endpoint is immune cell frequency and / or phenotype (e.g., B cells, T cells, and / or NK cells). In some embodiments, an exemplary primary and / or secondary biomarker endpoint is change in number of oligoclonal bands. Number of oligoclonal bands are a common biomarker for diagnosing multiple sclerosis, as described in Miller et al., Ann Neurol., 1983, which is incorporated by reference in its entirety. Oligoclonal bands may indicate the presence of immunoglobulin kappa free light chain. In some embodiments, an exemplary primary and / or secondary biomarker endpoint is change in number of immunoglobulin kappa free light chain. In some embodiments, an exemplary primary and / or secondary biomarker endpoint is a change in autoreactive immune cells (e.g., reactivity to EBNA-1, GlialCAM, etc.). In some embodiments, an exemplary primary and / or secondarybiomarker endpoint is neuro-injury and / or inflammatory cytokines (e.g., NfL, MBP, MOG, and / or interleukins). In some embodiments, an exemplary primary and / or secondary biomarker endpoint is an IgG titer and autoreactive antibodies (e.g., EBNA-1, GlialCAM, etc.). In some embodiments, an exemplary primary and / or secondary biomarker endpoint is EBV viral reactivity. In any of the preceding embodiments, the assessment can be via blood, cerebrospinal fluid, or lymph node. Examples of primary and secondary biomarker endpoints and specific mechanisms of action associated with each endpoint are highlighted below in Table 3.Table 3. Mechanism-Driven Biomarkers for Autoimmune DiseaseB = blood; C = CSF / cerebrospinal fluid; L = lymph node. *These mechanisms are unique to g-NK cells

[0288] In some embodiments, the methods described herein are effective to reduce MS disease activity. The subject’s symptoms may be assessed quantitatively, such as by EDSS, or decrease in the frequency of relapses, or increase in the time to sustained progression, or improvement in the magnetic resonance imaging (MRI) behavior in frequent, serial MRI studies and compare the subject’s status measurement before and after treatment. In a successful treatment, the subject’s status will have improved (e.g., the EDSS measurement number or frequency of relapses will have decreased, or the time to sustained progression will have increased, or the MRI scans will show less pathology).

[0289] Various clinical or other indicia of effectiveness of treatment can include, but are not limited to, EDSS score; MRI scan; relapse number, rate, or severity; multiple sclerosis functional composite (MSEC); multiple sclerosis quality of life inventory (MSQLI); Paced Serial Addition Test (PASAT); symbol digit modalities test (SDMT); 25-foot walk test; 9-hole peg test; low contrast visual acuity; Modified Fatigue Impact Scale; expanded disability status score (EDSS); multiple sclerosis functional composite (MSEC); Beck Depression Inventory; 36-item Short Form Survey (SF-36); Eeroqol-5D; and 7 / 24 Spatial Recall Test can be used.

[0290] In various embodiments, the administration of the described composition of g-NK cells may cause an improvement in one or more of these measures. Further, the subject can be monitored at various times during a regimen. In various embodiments, the administration of the described composition of g- NK cells may cause a disease improvement as assessed by MacDonald dissemination in space and time. For example, for dissemination in space, lesion imaging, such as, by way of illustration, Barkhof-Tintore MR imaging criteria, may be used, including, but not limited to, at least one gadolinium-enhancing lesion or 9 T2 hyperintense lesions; at least one infratentorial lesion; at least one juxtacortical lesion; at least about three periventricular lesions; and a spinal cord lesion. For dissemination in time, MRI can also be used; for example, if an MRI scan of the brain performed at >3 months after an initial clinical event demonstrates a new gadolinium-enhancing lesion, this may indicate a new CNS inflammatory event, because the duration of gadolinium enhancement in MS is usually less than 6 weeks. If there are no gadolinium-enhancing lesions but a new T2 lesion (presuming an MRI at the time of the initial event), arepeat MR imaging scan after another 3 months may be needed with demonstration of a new T2 lesion or gadolinium-enhancing lesion.

[0291] Additional disease effects may be assessed using any of the measures described in Lavery, et al. Multiple Sclerosis International, Vol 2014 (2014), Article ID 262350.

[0292] In some embodiments, the administration of the composition of g-NK cells may result in one or more of: (a) prevention of worsening in disability defined as deterioration by 1.0 point on EDSS, (b) increase in time to relapse, (c) reduction or stabilization of number and / or volume of gadolinium enhancing lesions, (d) decreased annualized relapse rate, (e) increased relapse duration and severity by NRS score, (f) decrease in disease activity as measured by MRI (annual rate of new or enlarging lesions), (g) lower average number of relapses at 1 year, or 2 years, (h) sustained disease progression as measured by the EDSS at 3 months, (i) prevention of conversion to CDMS, (j) no or few new or enhancing T2 lesions, (k) minimal change in hyperintense T2 lesion volume, (1) increased time to McDonald defined MS, (m) prevention of progression of disability as measured by sustained worsening of EDSS at 12 weeks, (n) reduction in time to relapse at 96 weeks, and (o) reduction or stabilization of brain atrophy (e.g., percentage change from baseline). The Clec9A binding agents may be administered and is effective to result in a decreased rate of relapse (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%) or greater reduction in rate of relapse) compared to the rate of relapse before administration (e.g., compared to the rate of relapse following administration for 12 months or for less than 12 months, e.g., about 10, or about 8, or about 4, or about 2 or less months) of treatment, or before commencement of treatment, when measured between 3-24 months (e.g., between 6-18 months, e.g., 12 months) after a previous relapse.

[0293] In other embodiments, the described composition of g-NK cells may be administered and may be effective to result in a prevention of an increase in EDSS score from a pre-treatment state. The Kurtzke Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis. The EDSS replaced the previous Disability Status Scales which used to bunch people with MS in the lower brackets. The EDSS quantifies disability in eight Functional Systems (FS) and allows neurologists to assign a Functional System Score (FSS) in each of these. The Functional Systems are: pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual and cerebral.

[0294] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms or clinical remission of MS in the subject.

[0295] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18months, 24 months, 36 months, 48 months or 60 months In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0296] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for greater than 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for greater than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0297] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 18 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 24 months.

[0298] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of MS symptoms that is maintained for greater than 5 years.

[0299] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months In some embodiments, after the administration of the described g-NK cells, the methods providedherein result in clinical remission of MS in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0300] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS in the treated subject for greater than 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS in the treated subject for greater than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0301] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 18 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 24 months.

[0302] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of MS that is maintained for greater than 5 years.

[0303] In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year, two years, three years, four years, or five years or more after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of two years after initiation of the administration of the g- NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of three years after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of four years afterinitiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of five years after initiation of the administration of the g-NK cells.2. Lupus Disorder

[0304] In specific embodiments, the autoimmune disease or disorder may be a lupus disorder. In particular embodiments, the lupus disorder can be, but are not limited to, systemic lupus erythematous (SLE), discoid lupus, drug-induced lupus, and neonatal lupus. In particular embodiments, the selected subject with the autoimmune disease or disorder can have SLE.

[0305] Systemic lupus erythematosus (SLE) has been classified as an autoimmune disease that may involve many organ systems, as an inflammatory multisystem rheumatic disorder, or as a collagen vascular disease. SLE is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary between people and may be mild to severe. Common symptoms include painful and swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, feeling tired, and a red rash which is most commonly on the face. Often there are periods of illness, called flares, and periods of remission during which there are few symptoms. SLE is a chronic inflammatory autoimmune disease that can affect any organ or organ system. The mechanism involves an immune response by autoantibodies against a person’s own tissues. The presence of auto-antibodies, such as antinuclear antibodies especially those directed to double-stranded DNA, is characteristic for the disease. SLE is not a homogeneous disease, but a group of related syndromes, with widely varying presentations, degrees of body system involvement, and clinical course. There are a number of other kinds of lupus erythematosus including discoid lupus erythematosus, neonatal lupus, and subacute cutaneous lupus erythematosus.

[0306] Clinical features commonly seen in SLE are blood and lymphatic disorders (lymphadenopathy), cardiac disorders (e.g., cardiomyopathy, pericardial effusion, and / or pericarditis), eye disorders (e.g., keratoconjunctivitis sicca), gastrointestinal disorders (e.g., mouth ulceration, pancreatitis, peritonitis, and / or pharyngitis), general disorders (e.g., malaise, fatigue, pyrexia, and / or weight decrease), nervous system disorders (e.g., cerebrovascular accident, cognitive disorder, migraine, headache, and / or peripheral neuropathy), musculoskeletal and connective tissue disorders (e.g., arthralgia, arthritis (not erosive or destructive), fibromyalgia, fracture, myositis, osteonecrosis, osteoporosis, and / or osteopenia), psychiatric disorders (e.g., affective disorder, anxiety, depression, neurosis, mental disorder due to a general medical condition, and / or psychotic disorder), renal and urinary disorders (e.g., lupus nephritis, and / or nephrotic syndrome), respiratory, thoracic, and mediastinal disorders (e.g., pleurisy, pneumonitis, and / or pulmonary hypertension), skin and subcutaneous tissue disorders (e.g., alopecia, cutaneous lupus erythematosus, dermatitis, generalized erythema, livedo reticularis, panniculitis, rash maculo-papular,systemic lupus erythematosus rash, and / or urticaria) and vascular disorders (e.g., hypertension, Raynud’s phenomenon, telangiectasis, thrombocytopenia, thrombophlebitis, and / or vasculitis). Additionally, most SLE subjects present with abnormal antibody patterns, including the presence of anti-nuclear- (ANA) and anti-double stranded DNA (anti-dsDNA) antibodies.

[0307] Systemic lupus erythematosus (SLE or “lupus”) is an autoimmune disease in which symptoms are extremely heterogeneous. In some embodiments, the selected subject with SLE has at least four of the eleven criteria: (1) malar "butterfly" rash, (2) discoid rash, (3) photosensitivity, (4) oral ulcers, (5) arthritis, (6) serositis, (7) renal disorder, (8) neurologic disorder, (9) hematologic disorder, (10) immunologic disorder, and (11) presence of anti-nuclear antibody. These criteria are explained in more detail in Tan et al, (1982) Arthritis Rheum. 25:1271-1277; and Hochberg et al, Arthritis Rheum. (1997) 40:1725, which are hereby incorporated by reference in their entireties. Because of SLE’s widely varied symptoms, SLE is often mistaken for other disorders. In certain embodiments, the subject with SLE exhibits common symptoms of SLE including, but not limited to, rashes and arthritis, often accompanied by fatigue and fever. In some embodiments, a subject with SLE may exhibit one or more signs of lupus that overlap with those symptoms observed in other autoimmune diseases, such as, but not limited to the presence of autoantibodies, including anti-nuclear antibodies and anti-dsDNA antibodies, joint pain, swelling, skin rashes, and organ involvement.

[0308] Definitive diagnosis of SLE renal disease is currently established on the basis of a combination of clinical, laboratory, and pathologic findings, and often requires a renal biopsy. Three of these validated measures are the Systemic Lupus Erythematosus Disease Activity Index (SLED Al), the British Isles Lupus Assessment Group index (BILAG), and the Systemic Lupus Activity Measure (SLAM).

[0309] SLE (as well as lupus nephritis) subjects have been reported to have decreased NK cell numbers. (Muller et al. N Engl J. Med 389(8):687-700 (2024); Flesher et al., ASN Kidney Week 2023). Research groups have shown that there is a negative correlation of NK cell counts with disease activity — that is, the more sever the SLE, the lesser the NK cell count. (Spada et al., J Leukoc Biol 98(4):479-487 (2015)).

[0310] It has been observed that SLE subjects exhibit both impaired differentiation and cytotoxicity of NK cells. Park et al., Arthritis Rheum. 60(6): 1753-1763 (2009). In particular, NK cells, and their subpopulations of CD56 (+) and CD16 (+) cells have been observed to be decreased in SLE subjects, as compared to control subjects. (Thangjam et al., Cureus 15(10):e46885 (2023)). Moreover, other studies have found that SLE subjects exhibited deficient NK cytotoxicity, as compared to control subjects, and that such SLE subjected also exhibited impaired response to IL-15 (Lin et al., PLoS One 12(10):e0186223 (2017)).

[0311] The present embodiments relate to a unique subset of NK cells (g-NK cells) that are envisioned to exploit otherwise inhibitory mechanisms.

[0312] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having SLE who has previously received a prior therapy to treat the SLE.

[0313] Therapies for SLE can involve the use immunosuppressants or immunomodulators such as NSAIDs, hydroxychloroquin, systemic steroids (glucocorticoids), mycophenolat mofetil (MME), azathioprine, leflunomide, methotrexate, cyclosporine or cyclophosphamide, frequently in combination and as interval / maintenance therapy. Other therapies for SLE can include inhibitors of Type I interferons (SLE). Monoclonal antibodies can also be applied parenterally. In specific embodiments, the subject with systemic lupus erythematosus (SLE) has received a prior treatment involving the use of an anti- CD20 antibody. In specific embodiments, the anti-CD20 antibody is rituximab. In specific embodiments, the subject with systemic lupus erythematosus (SLE) has received a prior treatment involving the use of an anti-BAFF antibody. In particular embodiments, the anti-BAFF antibody is belimumab.

[0314] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having SLE, who has received immunosuppressants to manage the SLE. Corticosteroids are the cornerstone of treatment but are associated with an extensive number of side effects most frequently seen during long-term use. Other drugs used in the setting of lower-level activity include analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), local steroids, and antimalarial drugs (e.g., chloroquine or hydroxychloroquine), with common supportive medications including vasodilators (calcium channel blockers, angiotensin-converting enzyme [ACE] inhibitors) for renal hypertension or Raynaud’s syndrome, local treatments for rashes or sicca syndromes, transfusions, intravenous (i.v.) globulin for cytopenias, anticonvulsants, antimigraine medications, anticoagulants for recurrent thromboses, and antidepressants.

[0315] The provided methods described herein may be used in a method of treatment a subject experiencing in a “flare episode,” SLE in remission, or SLE in relapse. Although SLE can be a fatal disease, the clinical course typically varies from mild to severe, and involves alternating periods of remission and relapse. The clinical course of SLE is episodic, with flares recurring upon increasing underlying disability and organ damage. In some embodiments, high-dose corticosteroids, e.g., 0.5 to 1.0 mg / kg / day oral prednisone (or equivalent) or 500 mg to 1 g daily pulse i.v. methylprednisolone, are used to manage acute SLE flares, with immunosuppressants (e.g., azathioprine, cyclophosphamide, methotrexate, mycophenolate mofetil, and / or leflunomide) generally used in moderate and severe caseswhen other treatments are ineffective or to limit or prevent long-term major organ damage from the disease or corticosteroid use (“steroid-sparing”).

[0316] In certain embodiments, the subject with SLE will manifest symptoms of SLE with different severity at different times. A subject with SLE does not develop symptoms rapidly. Instead, a subject with SLE gradually accumulate and exhibit symptoms over time. In particular embodiments, the selected subject with SLE may not express symptoms because the SLE is “quiescent”. In other embodiments, the selected subject with SLE will experience an increase in the number and / or severity of their symptoms, in a “flare” episode.

[0317] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having SLE during a “flare” episode. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having SLE during a period of remission. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having SLE during a period of relapse.

[0318] In some embodiments, the subject can be evaluated, e.g., for indicia of responsiveness, prior to, during, or after receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness prior to receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness concurrently or with the administration of the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness after receiving the composition of g-NK cells.

[0319] In some embodiments, clinical effect of the methods described herein are determined by several parameters including the British Isles Lupus Assessment Group (BILAG) score for SLE. The term “BILAG score” or “BILAG” index refers to the British Isles Lupus Assessment Group score and index, respectively. The BILAG index is a comprehensive index for measuring SLE disease activity. Some of the questions are based on the subject’s history, some on examination findings, and others on laboratory results.

[0320] The BILAG index considers parameters from 8 body systems (general, mucocutaneous, neurological, musculoskeletal, cardiovascular and respiratory, vasculitis, renal, and hematological). Scoring is based on a letter system, but weighted numerical scores can also be assigned to each letter, making it possible to calculate a BILAG score in the range of 0-72 (Griffiths, et al., Assessment of Patients with Systemic Lupus Erythematosus and the use of Lupus Disease Activity Indices).

[0321] Each body system score ranges from E to A, with A being the most severe disease activity. The interpretation of body system scores are as follows: A (“Active”") = severely active disease(sufficient to require disease-modifying treatment, for example, greater than 20 mg / day of prednisone, immunosuppressants, cytoxics); B (“"Beware”") = moderately active disease (requires only symptomatic therapy, for example, less than or equal to 20 mg / day of prednisone or antimalarial drugs; C (“"Contentment”") = mild stable disease (no indication for changes in treatment); D = previously active disease - but none currently; and E = no prior disease activity. When the BILAG alphabetic organ body system scores are converted to numeric values and summed (using the rule where each BILAG A=9, each BILAG B=3, each BILAG C=l, and each BILAG D or E is worth 0), this is referred to as a Total BILAG score.

[0322] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject has a lower total BILAG score than prior to the administration of the g-NK cells.

[0323] In some embodiments, clinical effect of the methods described herein are determined by several parameters including the Systemic Lupus Erythematosus Disease Activity score / index, also referred to as SLED Al score or SLED Al index (Hawker et al., 1993). The clinical score known as SLED Al index is an index of SLE disease activity as measured and evaluated within the last 10 days (Bombardier C, Gladman D D, Urowitz M B, Caron D, Chang C H and the Committee on Prognosis Studies in SLE: Derivation of the SLED Al for Lupus Patients. Arthritis Rheum 35:630-640, 1992.). Disease activity under the SLED Al scoring system can range from 0 to 105.

[0324] The following categories of SLED Al activity have been defined: no activity (SLED Al = 0); mild activity (SLED Al = 1-5); moderate activity (SLED Al = 6-10); high activity (SLED Al = 11 -19); and very high activity (SLED Al = 20 or higher). (Griffiths, et al., Assessment of Patients with Systemic Lupus Erythematosus and the use of Lupus Disease Activity Indices). In certain instances, severe SLE is defined as patients with SLED Al scores of between and including 11-19. Alternatively, severe SLE may be defined as patients with SLED Al scores of between and including 12-20, or even 13-20. In certain instances, very severe SLE is defined as patients with SLED Al scores of greater than 20. In certain instances, moderate SLE is defined as patients with SLED Al scores of between and including 6-10. Alternatively, moderate SLE may be defined as patients with SLED Al scores of between and including 5- 10, 5-11, 5-12, 6-11, or even 6-12.

[0325] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject’s SLED Al score is considered “mild.” In certain embodiments, after the administration of the g-NK cells, the treated subject’s SLED Al score is considered “mild.”

[0326] In particular embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject’s SLED Al score is considered “no activity.”

[0327] In some embodiments, clinical effect of the methods described herein are determined the Physicians Global Assessment (PGA) Score. The PGA is the physicians’ overall evaluation of a patients’ disease activity. It is performed by the physician marking their assessment of a patients’ overall disease activity on a 3 inch visual analogue scale with anchors at 0 (none), 1 inch (mild), 2 inches (moderate), and 3 inches (severe). Improvement is measured by reduction in the PGA score from visit to visit.

[0328] In particular embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject’s PGA score is considered “mild” or “moderate.”

[0329] In particular embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject’s PGA score is considered “none.”

[0330] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0331] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for greater than 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for greater than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0332] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methodsprovided herein result in clinical improvement of SLE symptoms that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 18 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 24 months.

[0333] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of SLE symptoms that is maintained for greater than 5 years.

[0334] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0335] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for greater than 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for greater than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0336] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 18 months. In certain embodiments, after the administration ofthe described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 24 months.

[0337] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of SLE that is maintained for greater than 5 years.

[0338] In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year, two years, three years, four years, or five years or more after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of two years after initiation of the administration of the g- NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of three years after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of four years after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of five years after initiation of the administration of the g-NK cells.3. Arthritis

[0339] In particular embodiments, the autoimmune disease or disorder may be arthritis. Arthritis is to be understood as meaning an inflammatory joint disease or disorder. In certain embodiments, the subject with arthritis may have axial arthritis, which is characterized by an inflammation of the spinal joints (e.g., Bechterew disease). In certain embodiments, the subject with arthritis may have peripheral arthritis, which is characterized by inflammation of joints of the extremities such as toes, ankles, knee, fingers, hands or else elbows. In particular embodiments, the peripheral arthritis can be asymmetrical, i.e., inflamed joints are distributed unevenly over both sides of the body (e.g., psoriatic arthritis). In particular embodiments, the peripheral arthritis can be symmetrical, i.e., the same joints of both sides of the body are both affected (e.g., rheumatoid arthritis).

[0340] In particular embodiments, the arthritis can be, but are not limited to psoriatic arthritis, rheumatoid arthritis, reactive arthritis, or systemic juvenile idiopathic arthritis. In specific embodiments,the selected subject with the autoimmune disease or disorder can have psoriatic arthritis (PA). In specific embodiments, the selected subject with the autoimmune disease or disorder can have rheumatoid arthritis (RA).

[0341] In certain embodiments, the subject with arthritis experiences painful restriction of the mobility of the inflamed joints, along with reddening and increased hypothermia of the skin surrounding or near the inflamed joint. The arthritic disorders are characterized by episodic progredient progression which may result in destruction of the joints and serious disability up to invalidity. In certain embodiments, the arthritis is characterized by symmetric synovitis leading to cartilage damage and joint destruction and can be complicated by numerous extra-articular manifestations. Arthritis is generally a progressive disease with functional status decline, significant morbidity and premature mortality. Arthritis can occur at any age, with a peak incidence between the fourth and sixth decades.

[0342] In certain embodiments, a subject with arthritis may test positive for autoantibodies, such as rheumatoid factor (RF) and / or anti-cyclic citrullinated peptide (CCP) IgG antibodies. In particular embodiments, the selected subject to be administered the g-NK cells described herein is RF positive. In particular embodiments, the selected subject to be administered the g-NK cells described herein is anti- CCP antibody positive.

[0343] In certain embodiments, autoreactive B cells, Thl cells and Thl7 cells and also proinflammatory cytokines such as IFN-y, TNF, IL-6, IL-12, IL-23 and IL-17 play a central role in induction, but also progression, of the pathological processes of arthritis. In particular embodiments, immune cells also induce the production of metalloproteinases as well as maturation and activation of osteoclasts, which then results in destruction of the cartilage and the bone in the joint affected (Raychaudhuri et al., Clin Rheumatol, 2015; Burmester et al., Ann Rheum Dis, 2015; Furst and Emery, Rheumatol, 2014; Mclnnes and Schett, Engl J Med, 2011).

[0344] Research groups have shown that monotherapy with, for example, rituximab led to variable tissue response — some subjects experienced rapid decrease in synovial B cell numbers while others did not (Vos et al., Arthritis Rheum. (2007) 56(3):772-778).

[0345] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having arthritis who has previously received a prior therapy to treat the arthritis. Therapies for arthritis can involve the use immunosuppressants or immunomodulators, such as but not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), hydroxychloroquine and steroids (e.g., prednisone), and chemical disease-modifying drugs (DMARDS), such as methotrexate, sulfasalazine and leflunomide. Additional therapies to treat arthritis can also involve the use of biologies such as, but not limited to TNF blockers (infliximab, adalimumab, golimumab and certolizumab pegol, and also etanercept), monoclonalantibodies (e.g., rituximab), modified antibodies (e.g., abatacept), or interleukin-inhibiting monoclonal antibodies (e.g., ustekinumab, tocilizumab and secukinumab), or Jak / STAT inhibitors (e.g., tofacitinib).

[0346] In some embodiments, the prior therapy may be referred to as a disease modifying therapy antirheumatic drug. Disease-modifying antirheumatic drugs (DMARDs), a heterogenous collection of agents grouped by use and convention, are usually the first line of treatment for subjects with arthritis. DMARDs can be used to reduce joint swelling and pain, decrease acute-phase markers, and limit the progression of joint damage and to improve joint function. In specific embodiments, DMARDs, most often methotrexate (MTX), are prescribed upon disease diagnosis (i.e., early signs of arthritis), usually before the development of erosive disease and the deformities seen in established arthritis. In certain embodiments, MTX therapy is initiated if pain and synovitis persist (especially if function is compromised). Subsequently, additional DMARDs (with or without steroids) may be added to achieve disease control. DMARDS, however, have many adverse effects (e.g., liver damage, bone marrow suppression and severe lung infection) that limit their prolonged use. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having arthritis who has previously received a DM ARD to achieve control of the arthritis.

[0347] Unfortunately, only about 2 / 3 of patients respond to DMARDS. Once the arthritis is an established arthritis disease, DMARDs only partially control the arthritis. In certain embodiments, the provided methods described herein may be used in a method of treatment of progressing arthritis in subjects who have had an inadequate response to one or more DMARDs. In certain instances, even for the 5-20% DMARD-treated subjects who achieve remission or clinical improvement that approaches remission eventually continues to exhibit radiological disease progression.

[0348] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having arthritis, who has experienced disease progression after having previously received a prior therapy to treat the arthritis. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g- NK cells) to a subject having multiple sclerosis, who has experienced disease progression after having previously received a prior DM ARD to treat or manage the arthritis.

[0349] In particular embodiments, the subject is referred to as a DMARD-failure and / or a DMARD- inadequate responder subject. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject with arthritis who is a DMARD-failure and / or a DMARD- inadequate responder subject.

[0350] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having arthritis, who has experienced relapse after having previously received a prior therapy to treat the arthritis. Due to the inadequate responses and dangers associated with prolonged DMARD treatment, biologies have been introduced as a second line treatment. In certain embodiments, the subject with arthritis may additionally have received a second line therapy. Second line therapies can include, but are not limited to, an anti-TNF agent (Cimzia®, Enbrel®, Humira®, Remicade®, Simponi®) or a TNF inhibitor, used in combination with methotrexate to aggressively treat the established arthritis. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis, who has received one or two prior lines of treatment.

[0351] Unfortunately, 30-40% of subjects with established arthritis fail to respond to TNF antagonists and the majority of those subject who do respond initially do not achieve complete remission or lose response over time. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g- NK cells) to a subject having multiple sclerosis, who does not achieve complete remission after having previously received a biologic, such as a TNF inhibitor.

[0352] Concerns have also been raised about the short and long-term tolerability and safety of chronic biologic treatment, most notably the reactivation of serious infections (e.g., tuberculosis infections), liver toxicity, increased cardiovascular disease, induction (or exacerbation of) demyelinating conditions, and increased incidence of malignancy due to TNF-alpha antagonism. M. Khraishi (2009) J. Rheumatol Suppl. 82:25-32; Salliot et al. (2009) Ann. Rheum. Dis. 68:25- 32. However, a TNF inhibitor is usually continued unless it becomes ineffective or an adverse event arises, at which point a clinician may switch to either a different TNF inhibitor or a biological with a different mechanism of action. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having arthritis, who has experienced relapse after having previously received a biologic, such as a TNF inhibitor.

[0353] In some embodiments, the subject can be evaluated, e.g., for indicia of responsiveness, prior to, during, or after receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness prior to receiving the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness concurrently or with the administration of the composition of g-NK cells. In particular embodiments, the subject may be evaluated for indicia of responsiveness after receiving the composition of g-NK cells.

[0354] In some embodiments, the methods described herein are effective to control arthritis disease progression. The subject’s symptoms may be assessed quantitatively, for example, by using the 2010 American College of Rheumatology (ACR) classification criteria (hereinafter “2010 ACR / EULAR” criteria). The classification system focuses on features of earlier stages of arthritis that are associated with persistent and / or erosive disease. Exemplary ACR / EULAR criteria include, but are not limited to, tender or swollen joint counts, stiffness, pain, and radiographic indications and measurement of serum rheumatoid factor. An ACR 20 is indicative of a 20% improvement in the measurement ACR / EULAR criteria. An ACR 50 is indicative of a 50% improvement in the measurement ACR / EULAR criteria. An ACR 70 is indicative of a 70% improvement in the measurement ACR / EULAR criteria.

[0355] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis in the subject. In certain embodiments, after the administration of the g-NK cells, an ACR 20 is observed in the treated subject. In certain embodiments, after the administration of the g-NK cells, an ACR 50 is observed in the treated subject. In certain embodiments, after the administration of the g-NK cells, an ACR 70 is observed in the treated subject.

[0356] The subject’s symptoms may also be assessed by a Disease Activity Score (DAS) (Fransen & van Riel Clin Exp Rheumatol 23:S93-S99 2005). In some embodiments, a reduction in DAS reflects a reduction in arthritis severity. A DAS of less than 2.6 may be indicative of disease remission. A DAS between 2.6 and 3.2 may indicate low disease activity. A DAS greater than 3.2 may indicate increased disease activity. In specific embodiment, a DAS greater than 5.1 indicates severe disease activity.

[0357] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject has a DAS of between 2.6 and 3.2. In certain embodiments, after the administration of the g-NK cells, the treated subject has a DAS of less than 2.6.

[0358] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the subject. In certain embodiments, after the administration of the g-NK cells, the treated subject has a DAS of less than 2.6.

[0359] In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms or clinical remission of arthritis in the subject.

[0360] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0361] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for greater than3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for greater than4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0362] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 18 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 24 months.

[0363] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical improvement of arthritis symptoms that is maintained for greater than 5 years.

[0364] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the treated subject for at least 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the treated subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methodsprovided herein result in clinical remission of arthritis in the treated subject for at least 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0365] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the treated subject for greater than 3 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the treated subject for greater than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, 48 months or 60 months. In some embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis in the treated subject for greater than 0.5 year, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years or more.

[0366] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 6 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 12 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 18 months. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 24 months.

[0367] In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 3 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 4 years. In certain embodiments, after the administration of the described g-NK cells, the methods provided herein result in clinical remission of arthritis that is maintained for greater than 5 years.

[0368] In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year, two years, three years, four years, or five years or more after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of one year after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of two years after initiation of the administration of the g- NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of three years after initiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of four years afterinitiation of the administration of the g-NK cells. In some embodiments, after being administered the described g-NK cells, the subject is not administered the immunosuppressive agent for at least a period of five years after initiation of the administration of the g-NK cells.4. Autoimmune Kidney Diseases

[0369] In particular embodiments, the autoimmune disease or disorder may be an autoimmune kidney disease. In certain embodiments, autoimmune kidney disease is lupus nephritis (LN), primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).

[0370] In certain embodiments, the autoimmune kidney disease is lupus nephritis (LN).

[0371] Lupus nephritis (LN) is a clinical-pathological condition characterized by an alteration of the renal structure and function which can affect subjects suffering from systemic lupus erythematosus (SLE). In particular, a relevant percentage of patients affected by SLE develops such condition which, if not recognized and treated in time, can lead to renal insufficiency and death. On the contrary, a quick diagnose of lupus nephritis, and if possible, its prediction, opens to the use of effective and potentially resolving drugs and therapies if used upon the disease's beginning.

[0372] The pathogenesis of lupus nephritis is that an increased immune complex, which could be occurred due to the inappropriate overproduction of auto-antibodies such as anti-nuclear antibodies, is accumulated in the systemic organs to cause inflammatory responses. About 40-70% of lupus patients have renal involvement, and about 30% of the patients develop lupus nephritis, which is known as a bad prognostic factor in lupus patients. Although methods of treating lupus nephritis using immunosuppressive agents are attempted, remission is not necessarily induced in all patients who receive the immunosuppressive agents. Moreover, even when remission was induced, treated patients can still relapse when the use of immunosuppressive agents was reduced. Approximately 5-10% of patients with serious lupus nephritis (WHO class III and IV) die after 10 years, and 5-15% of the patients lead to endstage renal stage.

[0373] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having lupus nephritis. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having lupus nephritis, who has experienced lupus nephritis after first having SLE.

[0374] Immunoglobulin a kidney disease (IgA nephraphy, igAN) and membranous kidney disease (membranous nephropathy, MN) are both clinically common glomerulonephritis (GN).

[0375] In certain embodiments, the autoimmune kidney disease is primary membranous nephropathy (PMN).

[0376] Membranous nephropathy is a GN characterized by deposition of IgG and complement immune complexes on glomerular basement membrane (glomerular basement membrane, GBM). Membranous nephropathy is a common cause of nephrotic syndrome. About 15% of membranous nephropathy cases are secondary membranous nephropathy, caused by drugs, infections, tumors, immune diseases. The remaining 85% of membranous nephropathy cases are idiopathic, also called autoimmune primary membranous nephropathy. Its origin remains unknown. About one half of patients with idiopathic membranous nephropathy who did not receive treatments will develop end-stage renal disease requiring dialysis or renal transplantation. Among the kidney transplants, about 40% of them will relapse.

[0377] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having primary membranous nephropathy. In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having primary idiopathic membranous nephropathy.

[0378] In certain embodiments, the autoimmune kidney disease is immunoglobulin A (IgA) nephropathy (IgAN).

[0379] In certain embodiments, the autoimmune kidney disease is immunoglobulin A (IgA) nephropathy (IgAN). Immunoglobulin a nephropathy is a GN characterized by the deposition of IgA immune complexes on glomerular membranes (glomerular mesangial). In particular, IgA nephropathy is a disease characterized by being clinically poor in clinical symptoms in addition to continuous proteinuria and hematuria. Histology of kidney samples show deposits mainly composed of IgA in the glomerular mesangial region.

[0380] In some embodiments, the methods provided herein comprise administering at least one dose of a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having immunoglobulin a nephropathy.C LA -E Expressing and / or Viralfy Associate A utoimniune Disease or Disorders

[0381] In some embodiments, any of the above autoimmune disease or disorder is associated with expression of human leukocyte antigen-E (HLA-E). In some embodiments, HLA-E is also referred to as MHC-E. HLA-E (or MHC-E) is a major histocompatibility complex lb (MHC lb) cell surface protein which performs an essential role in the adaptive immune system. There are several non-classical MHC molecules (including HLA-E, HLA-F, and HLA-G), which have immune regulatory functions. HLA-E, which is encoded by an HLA-E gene (i.e., NCBI Gene ID: 3133), is a heterodimer class lb molecule that primarily functions as a ligand for the NK cell receptors CD94 / NKG2A (NKG2A) and CD94 / NKG2C(NKG2C). Specifically, HLA-E enables NK cells to monitor other MHC class I molecule expression and to tolerate self-expression. A peptide that binds to HLA-E is one which associates with HLA-E on a cell surface, forming a complex that is capable of interacting with a specific cell receptor on an immune cell. In some aspects, HLA-E can bind to peptides also recognized by MHC class I, albeit with lower affinity (Pietra et al. (2010) Journal of Biomedicine and Biotechnology, 1-8). The expression of other class I MHC molecules can regulate the expression of HLA-E, thereby allowing NK cells to monitor the state of the MHC class I dependent antigen presentation pathway in potential target cells. The level of cell surface HLA-E can regulate the NK cell cytotoxicity towards autoreactive immune cells and virally infected cells.

[0382] In particular, it has been found that HLA-E-expressing immune cells, including HLA-E- expressing B cells and HLA-E-expressing T cells, are associated with autoimmune diseases. Expression of HLA-E, which is expressed on T cells to provide HLA / peptide restricted responses, has been shown to be increased in T cells from subjects with MS and contribute to the autoimmune disease (Laroni et al. J of Autoimmunity, 72:8-18, 2016).

[0383] Natural killer cells modulate their activity through cell-surface receptors such as CD94 / NKG2A (NKG2A) and CD94 / NKG2C (NKG2C). NKG2A and NKG2C bind to the non-classical MHC-Ib HLA-E protein: peptide complexes. In their classical role, NK cells bind HLA-E in complex with a constrained set of peptides (largely resembling VMX1PRTX2X3L (SEQ ID NO: 19), wherein Xi is A or P, X2 is L or V and X3 is I, L, F, or V), derived signal peptides from signal peptides of MHC class la molecules. NKG2A binding of HLA-E inhibits NK cells whereas NKG2C binding activates NK cells. NKG2A typically possess higher peptide binding affinity that the NKG2C.

[0384] In some embodiments the HLA-E-peptide complex interacts with a NKG2A receptor, a NKG2C receptor, or both. The g-NK cells described herein are superior because they have higher expression of NKG2C and lower expression of NKG2A, effectively skewing NKG2A / NKG2C regulation, and enabling the described g-NK cells to be activated as opposed to be inhibited upon NKG2C and HLA-E binding.

[0385] In certain embodiments, surface expression of HLA-E is sufficient to protect target cells from lysis by CD94 / NKG2A+ NK cells. In particular embodiments, the described g-NK cells in the provided methods herein are CD94 / NKG2A- NK cells. In certain embodiments, surface expression of HLA-E does not protect target cells from lysis by CD94 / NKG2C+ NK cells. In particular embodiments, the described g-NK cells in the provided methods herein are CD94 / NKG2C+ NK cells. In particular embodiments, the target cells are not protected by lysis from the described g-NK cells.

[0386] In some embodiments, the target cells are autoreactive cells. In certain embodiments, the target cells are autoreactive B cells. In certain embodiments, the target cells are autoreactive T cells. Insome embodiments, the g-NK cells described herein can effectuate potent killing of HLA-E expressing autoreactive cells because the g-NK cells have low expression of the CD94 / NKG2A inhibitory receptor.

[0387] In some embodiments, the methods provided herein involve a determination, detection, quantification, or other assessment of the HLA-E expression. A subject to be treated in accord with the provided methods can be treated with or without a prior detection step to assess expression of HLA-E on the surface of cells, such as B cells or T cells. In some embodiments, the provided methods include a step of detecting an HLA-E nucleic acid or polypeptide in a biological sample from the subject (e.g., on an autoreactive cell) from an individual. A determination that a biological sample expresses HLA-E (e.g., prominently expresses; expresses HLA-E at a high level, high intensity of staining with an anti-HLA-E antibody, compared to a reference) indicates that the patient has an autoimmune disease or condition that may have a strong benefit from treatment in accord with provided methods. In one embodiment, the method comprises determining the level of expression of an HLA-E nucleic acid or polypeptide in a biological sample and comparing the level to a reference level (e.g., a value, weak cell surface staining, etc.) corresponding to a healthy individual or to an individual that does not have a virus infection (e.g., EBV infection). ...

Claims

CLAIMS1. A method of killing autoreactive cells, the method comprising contacting autoreactive cells with a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g- NK cells).

2. The method of claim 1 , wherein the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

3. A method of killing autoreactive cells, the method comprising contacting autoreactive cells with upregulated HLA-E expression with a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells).

4. The method of any one of claims 1-3, wherein the autoreactive cells are autoreactive B and / or autoreactive T cells.

5. The method of any one of claims 2-4, wherein the upregulated HLA-E expression is caused by a viral infection.

6. The method of claim 5, wherein the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

7. The method of claim 5 or claim 6, wherein the viral infection is an Epstein-Barr virus (EBV).

8. The method of any one of claims 1-7, wherein the autoreactive cells are central nervous system (CNS)-autoreactive cells.

9. The method of claim 8, wherein the CNS-autoreactive cells are reactive to GlialCAM, CRY AB, MBP, and / or AN02.

10. The method of claim 8 or claim 9, wherein the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

11. The method of any one of claims 1-10, wherein the autoreactive cells are present in a subject having an autoimmune disease or disorder.

12. The method of any one of claims 1-10, wherein the autoreactive cells are present in a subject who is likely or suspected to develop an autoimmune disease or disorder.

13. A method of treating an autoimmune disease or disorder, the method comprising administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder.

14. The method of claim 11 or claim 13, further comprising selecting a subject with the autoimmune disease or disorder for treatment prior to administering the composition of g-NK cells.

15. A method of treating an autoimmune disease or disorder, the method comprising:(a) selecting a subject with an autoimmune disease or disorder; and(b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the autoimmune disease or disorder.

16. A method of prophylactically treating an autoimmune disease or disorder, the method comprising administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject who is likely or suspected to develop an autoimmune disease or disorder.

17. The method of claim 12 or claim 16, further comprising selecting a subject who is likely or suspected to develop an autoimmune disease or disorder for treatment prior to administering the composition of g-NK cells.

18. A method of prophylactically treating an autoimmune disease or disorder, the method comprising:(a) selecting a subject who is likely or suspected to develop an autoimmune disease or disorder; and(b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to the subject who is likely or suspected to develop an autoimmune disease or disorder.

19. The method of any one of claims 12 and 16-18, wherein the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for the presence of autoreactive cells prior to administering the composition of g-NK cells.

20. The method of claim 19, wherein the autoreactive cells are autoreactive B cells and / or T cells.

21. The method of claim 19 or claim 20, wherein the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

22. The method of any one of claims 19-21, wherein the autoreactive cells are central nervous system (CNS)-autoreactive cells.

23. The method of claim 22, wherein the CNS-autoreactive cells are reactive to GlialCAM, CRY AB, MBP, and / or ANO2.

24. The method of claim 22 or claim 23, wherein the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

25. The method of any one of claims 12 and 16-24, wherein the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having radiologically isolated syndrome (RIS).

26. The method of any one of claims 12 and 16-24, wherein the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having clinically isolated syndrome (CIS).

27. The method of any one of claims 11-26, further comprising administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder.

28. The method of claim 27, wherein the target antigen is a B cell antigen.

29. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the autoimmune disease or disorder; and(b) administering to the subject an antibody that is directed against a B cell antigen.

30. The method of any one of claims 1-29, wherein the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

31. The method of any one of claims 1-30, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos).

32. The method of any one of claims 1-31, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

33. The method of any one of claims 11-32, wherein the autoimmune disease or disorder is systemic lupus erythematosus (SLE), systemic sclerosis (SSc), myositis (IIM), rheumatoid arthritis (RA), or multiple sclerosis (MS).

34. The method of any one of claims 11-32, wherein the autoimmune disease or disorder is a kidney or renal disease.

35. The method of claim 34, wherein the kidney or renal disease is systemic lupus erythematosus (SLE), lupus nephritis, primary memberanous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).

36. The method of any one of claim 11-35, wherein pathogenesis of the autoimmune or disorder is associated with a viral infection.

37. The method of claim 36, wherein the autoimmune disease or condition is characterized by autoreactive B cells or autoreactive T cells with upregulated HLA-E expression.

38. The method of claim 37, wherein the upregulation of HLA-E expression is caused by a viral infection.

39. The method of claim 38, wherein the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

40. The method of claim 38 or claim 39, wherein the viral infection is an Epstein-Barr virus(EBV).

41. A method of treating a disease or disorder associated with an Epstein-Barr virus (EBV), the method comprising administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an autoimmune disease or disorder.

42. The method of any of claims 1-41, wherein the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

43. The method of any of claims 1-42, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos).

44. The method of any one of claims 1-43, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

45. The method of any one of claims 41-44, further comprising administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder.

46. The method of claim 45, wherein the target antigen is a B cell antigen.

47. The method of any one of claims 11-46, wherein the autoimmune disease or disorder is multiple sclerosis (MS).

48. A method of treating an autoimmune disease or disorder, the method comprising administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS).

49. The method of any one of claims 1-26, 30-44, 47, and 48 wherein the method does not comprise administering an antibody to the subject in combination with the composition of g-NK cells.

50. The method of claim 49, wherein the antibody is an antibody directed against a target antigen associated with an autoimmune disease or disorder, optionally wherein the target antigen is a B cell antigen.

51. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS); and(b) administering to the subject an antibody that is directed against a B cell antigen.

52. The method of any one of claims 47-51, wherein the subject with multiple sclerosis hasEpstein-Barr virus (EBV)-infected cells.

53. The method of claim 52, wherein the EBV-infected cells are EBV-infected B cells.

54. The method of any one of claims 47-53, wherein the subject is seropositive for an EBV infection.

55. The method of any one of claims 47-54, wherein the subject with multiple sclerosis is seropositive for Epstein-Barr nuclear antigen 1 (EBNA).

56. The method of any of claims 47-55, wherein the subject with multiple sclerosis has a high level of Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNAjsswos antibodies.

57. The method of any one of claims 47-56, where the subject has autoreactive cells.

58. The method of claim 57, wherein the autoreactive cells are autoreactive B cells and / or T cells.

59. The method of claim 57 or claim 58, wherein the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

60. The method of any one of claims 57-59, wherein the autoreactive cells are central nervous system (CNS)-autoreactive cells.

61. The method of claim 60, wherein the CNS-autoreactive cells are reactive to GlialCAM, CRY AB, MBP, and / or ANO2.

62. The method of claim 60 or claim 61, wherein the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

63. The method of any one of claims 47-62, wherein the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and / or T cells.

64. The method of any one of claims 47-63, wherein the subject is characterized by an HLA- E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-1) -derived peptide.

65. The method of claim 64, wherein the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20.

66. The method of claim 64, wherein the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.

67. The method of any one of claims 1-66, wherein, among cells in the composition of g-NK cells, greater than at or about 20% of the cells are g-NK cells.

68. The method of any one of claims 1-67, wherein, among cells in the composition of g-NK cells, greater than at or about 30% of the cells are g-NK cells, greater than at or about 40% of the cells are g-NK cells, greater than at or about 50% of the cells are g-NK cells, greater than at or about 60% of the cells are g-NK cells, greater than at or about 70% of the cells are g-NK cells, greater than at or about 80% of the cells are g-NK cells, greater than at or about 90% of the cells are g-NK cells, or greater than at or about 95% of the cells are g-NK cells.

69. The method of any one of claims 1-68, wherein at least at or about 15% of the NK cells of the composition are positive for NKG2C (NKG2Cpos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2Aneg).

70. The method of any one of claims 27-40, 45-47, and 51-69, wherein the antibody is a full- length antibody.

71. The method of any one of claims 28-40 and 46-67, wherein the B cell antigen is expressed on a cell of a B cell lineage.

72. The method of any one of claims 28-40, 46, 47, and 51-71, wherein the B cell antigen is antigen selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.

73. The method of claim 71 or claim 72, wherein the cell of the B cell lineage is an autoreactive B cell.

74. The method of any one of claims 71-73, wherein the cell of the B cell lineage is selected from the group consisting of pro-B cells, pre-B cells, immature B cells, naive B cells, germinal center B cells, memory B cells, plasmablasts and plasma cells.

75. The method of any one of claims 27-40, 45-47, and 51-74, wherein the antibody is an anti-CD19 antibody.

76. The method of claim 75, wherein the antibody is inebilizumab, tafasitamab-cxix or obexelimab.

77. The method of any one of claims 27-40, 45-47, and 51-74, wherein the antibody is an anti-CD20 antibody.

78. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS); and(b) administering to the subject an anti-CD20 antibody.

79. The method of claim 77 or claim 78, wherein the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

80. The method of any one of claims 77-79, wherein the antibody is ocrelizumab.

81. The method of any one of claims 27-40, 45-47, and 51-74, wherein the antibody is an anti-CD22 antibody.

82. The method of claim 81 , wherein the antibody is epratuzumab.

83. The method of any one of claims 27-40, 45-47, and 51-74, wherein the antibody is an anti-BAFF-R antibody.

84. The method of claim 83, wherein the antibody is belimumab.

85. The method of any one of claims 27-40, 45-47, and 51-74, wherein the antibody is an anti-CD38 antibody.

86. The method of claim 85, wherein the anti-CD38 antibody is daratumumab or is isatuximab.

87. The method of claim 85 or claim 86, wherein less than 25% of the cells in the composition of g-NK cells are positive for surface CD38.

88. The method of any one of claims 85-87, wherein the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression.

89. The method of any one of claims 27-40, 45-47, and 51-88, wherein the antibody is administered intravenously.

90. The method of any one of claims 27-40, 45-47, and 51-88, wherein the antibody is administered subcutaneously.

91. The method of any one of claims 1-90, wherein the composition of g-NK cells is administered once weekly for a predetermined number of doses.

92. The method of any one of claims 1-90, wherein the composition of g-NK cells is administered twice weekly for a predetermined number of doses.

93. The method of any one of claims 1-90, wherein the composition of g-NK cells is administered three times weekly for a predetermined number of doses.

94. The method of any one of claims 1-90, wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

95. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and(b) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

96. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily at a frequency of every other day (Q2D) in a 7-day cycle;(b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is administered at a dose of from at or about 1 x 108cells to at or about 50 x 109cells and each dose is administered one time daily at a frequency of every other day (Q2D) in the 7-day cycle and on the same day as the IL-2; and(c) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

97. A method of treating an autoimmune disease or disorder, the method comprising:(a) administering a dose of IL-2 to the subject, wherein the dose is 0.25 million IU to 6 million IU and each dose is administered one time daily at a frequency of every day or every week day in a 7-day cycle; and(b) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple sclerosis (MS), wherein the composition of g-NK cells is administered at a dose of from at or about 1 x 108cells to at or about 50 x 109cells and each dose is administered one time daily at a frequency of every other day (Q2D) in the 7-day cycle.

98. The method of claim 97, wherein the method does not comprise administering to the subject an antibody.

99. The method of claim 97, wherein the method further comprises:(c) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.

100. The method of any one of claims 96-99, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

101. The method of any one of claims 95, 96, 99, and 100, wherein the anti-CD20 antibody is ocrelizumab.

102. The method of any one of claims 95, 96, and 99-101, wherein the antibody is administered intravenously.

103. The method of any one of claims 95, 96, and 99-101, wherein the antibody is administered subcutaneously.

104. The method of any one of claims 27-40, 45-47, 51-96, and 99-103, wherein the antibody is administered about once every six months.

105. The method of any one of claims 27-40, 45-47, 51-96, and 99-104, wherein the antibody is ocrelizumab and is administered once every six months at a dose at or about 600 mg.

106. The method of any one of claims 27-40, 45-47, 51-96, and 99-105, wherein a first dose of the antibody is initiated within one month prior to the administration of the composition of g-NK cells.

107. The method of any one of claims 27-40, 45-47, 51-96, and 99-106, wherein a first dose of the antibody is initiated within one week prior to the administration of the composition of g-NK cells.

108. The method of any one of claims 27-40, 45-47, 51-96, and 99-107, wherein a first dose of the antibody is initiated about six days prior to the administration of the composition of g-NK cells.

109. The method of any one of claims 27-40, 45-47, 51-96, and 99-107, wherein a first dose of the antibody is initiated about one day prior to the administration of the composition of g-NK cells.

110. The method of any one of claims 1-95 and 101-109, wherein the composition of g-NK cells is administered in a 7-day cycle.

111. The method of any one of claims 96-100 and 110, wherein the 7-day cycle is repeated one to three times.

112. The method of claim 111, wherein the 7-day cycle is repeated one time.

113. The method of claim 111, wherein the 7-day cycle is repeated two times.

114. The method of any one of claims 1-113, wherein the composition of g-NK cells is administered from two total doses to six total doses.

115. The method of any one of claims 1-95 and 101-114, wherein the composition of g-NK cells is administered as two total doses.

116. The method of any one of claims 1-95 and 101-114, wherein the composition of g-NK cells is administered as three total doses.

117. The method of claim 1-114 and 116, wherein the composition of g-NK cells is administered on Day 0, Day 2 and Day 4.

118. The method of any one of claims 1-95 and 101-114, wherein the composition of g-NK cells is administered as four total doses.

119. The method of any one of claim 1-95 and 101-114, wherein the composition of g-NK cells is administered as six total doses.

120. The method of any one of claim 1-119, wherein at least at or about 20% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

121. The method of any one of claims 1-120, wherein at least at or about 40% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

122. The method of any one of claims 1-121, wherein greater than at or about 70% of the g- NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B.

123. The method of claim 122, wherein (i) greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B, (ii) greater than at or about 90% of the g-NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B, or (iii) greater than at or about 95% of the g-NK cells are positive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B.

124. The method of claim 122 or claim 123, wherein: among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos; and / or among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

125. The method of any one of claims 1-124, wherein greater than 10% of the cells in the composition of g-NK cells are capable of degranulation against target cells, optionally as measured by CD 107a expression, optionally wherein the degranulation is measured in the absence of an antibody against the target cells.

126. The method of any one of claims 1-125, wherein, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% exhibit degranulation, optionally as measured by CD107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

127. The method of any one of claims 1-126, wherein greater than 10% of the cells in the composition of g-NK cells are capable of producing interferon-gamma or TNF-alpha against target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of an antibody against the target cells.

128. The method of any one of claims 1-127, wherein, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce an effector cytokine in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (antitarget antibody).

129. The method of claim 128, wherein the effector cytokine is IFN-gamma or TNF-alpha.

130. The method of claim 128, wherein the effector cytokine is IFN-gamma and TNF-alpha.

131. The method of any one of claims 1-130, wherein the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD56+ cells are enriched from a biological sample from a donor subject.

132. The method of any one of claims 1-130, wherein the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells are enriched from a biological sample from a donor subject.

133. The method of claim 131 or claim 132, wherein the donor subject is CMV-seropositive.

134. The method of claim 132 or claim 133, wherein the donor subject has the CD16 F / F NK cell genotype.

135. The method of claim 132 or claim 133, wherein the donor subject has the CD16 158V / V NK cell genotype or the CD 16 158 V / F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype.

136. The method of any one of claims 132-135, wherein at least at or about 15% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2Cpos) and at least 70% of NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2Aneg).

137. The method of any one of claims 132-136, wherein the irradiated feeder cells are deficient in HEA class I and HEA class IE138. The method of any one of claims 132-136, wherein the irradiated feeder cells are 22EAEH cells.

139. The method of any one of claims 132-138, wherein the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is interleukin (IL)-2 and at least one recombinant cytokine is IL-21.

140. The method of claim 139, wherein the recombinant cytokines are IL-21 and IL-2.

141. The method of claim 139, wherein the recombinant cytokines are IL-21, IL-2, and IL-15.

142. The method of any one of claims 1-141, wherein the g-NK cells in the composition are from a single donor subject that have been expanded from the same biological sample.

143. The method of any one of claims 1-142, wherein the composition of g-NK cells is formulated in a serum-free cryopreservation medium comprising a cryoprotectant, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v / v).

144. The method of any one of claims 1-143, wherein the g-NK cells are not engineered with an antigen receptor, optionally wherein the antigen receptor is a chimeric antigen receptor.

145. The method of any one of claims 1-144, wherein the g-NK cells are not engineered with a secreted cytokine, optionally a cytokine receptor fusion protein, such as IL-15 receptor fusion (IL-15RF).

146. The method of any one of claims 1-145, wherein the method does not include exogenous cytokine administration to the subject to support NK cell survival or expansion, wherein the exogenous cytokine is one or more of IL-2, IL-7, IL-15 or IL-21.

147. The method of any one of claims 1-146, further comprising administering IFN-P to the subject.

148. The method of claim 147, wherein the IFN- is administered three times a week.

149. The method of claim 147 or claim 148, wherein each dose of IFN-P is about 22 pg to 44 pg, optionally wherein each dose of IFN-P is 44 pg.

150. The method of any one of claims 1-145 and 147-149, further comprising administering exogenous cytokine support to facilitate expansion or persistence of the g-NK cells in vivo in the subject, optionally wherein the exogenous cytokine is or comprises IL- 15 or IL-2.

151. The method of any one of claims 1-95, 101-145, and 147-150, wherein the method comprises administering IL-2 to the subject.

152. The method of claim 151, wherein the IL-2 is administered once a week, two times a week or three times a week.

153. The method of claim 151 or claim 152, wherein the IL-2 is administered at a frequency of every other day (Q2W).

154. The method of claim 151, wherein the IL-2 is administered five times a week, six times a week, or seven times a week.

155. The method of claim 151 or claim 154, wherein the IL-2 is administered at a frequency of every day (QD).

156. The method of claim 151 or claim 154, wherein the IL-2 is administered at a frequency of every week day.

157. The method of any one of claims 151-156, wherein for each day of administration the IL- 2 is administered once daily.

158. The method of any one of claims 96-157, wherein the IL-2 is administered in a cycling regimen of one or more 7-day cycles.

159. The method of claim 158, wherein the IL-2 is administered in three 7-day cycles, optionally wherein the three 7-day cycles are in consecutive weeks.

160. The method of claim 158 or claim 159, wherein each 7-day cycle is the same.

161. The method of any one of claims 151-160, wherein the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in one or more 7-day cycles.

162. The method of any one of claims 151 and 154-160, wherein the IL-2 is administered one time daily at a frequency of every day (QD) on day 0, day 1, day 2, day 3, day 4, day 5, and day 6 in one or more 7-day cycles.

163. The method of any one of claims 151 and 154-160, wherein the IL-2 is administered one time daily at a frequency of every day (QD) on day 0, day 1, day 2, day 3, and day 4, in one or more 7- day cycles.

164. The method of any of claims 96-163, wherein the IL-2 is administered to the subject within about 1 hour of the administration of the g-NK cells.

165. The method of any one of claims 151-164, wherein each dose of the IL-2 is 0.25 million to 6 million IU.

166. The method of any one of claims 97-165, wherein each dose of the IL-2 is at or about 1 million IU.

167. The method of any one of claims 151-164, wherein each dose of the IL-2 is 1 million to 12 million IU.

168. The method of any one of claims 96, 100-164, and 167, wherein each dose of IL-2 is 4 million IU to 8 million IU.

169. The method of any one of claims 96, 100-164, 167, and 168, wherein each dose of IL-2 is at or about 6 million IU.

170. The method of any one of claims 96-169, wherein the IL-2 is administered subcutaneously.

171. The method of any one of claims 151-170, wherein administration of the IL-2 is administered on the same day as a first dose of the composition of g-NK cells.

172. The method of any one of claims 1-95 and 101-171, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is from at or about from at or about 1 x 108cells to at or about 50 x 109cells of the composition of g-NK cells.

173. The method of any one of claims 1-172, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is from at or about from at or about 5 x 109cells to at or about 20 x 109cells of the composition of g-NK cells.

174. The method of any one of claims 1-173, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is or is about 5 x 108cells of the composition of g-NK cells.

175. The method of any one of claims 1-173, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is or is about 5 x 109cells of the composition of g-NK cells.

176. The method of any one of claims 1-173, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is or is about 10 x 109cells of the composition of g-NK cells.

177. The method of any one of claims 1-173, wherein the composition of g-NK cells is administered as at least one dose and wherein each dose of g-NK cells is or is about 20 x 109cells of the composition of g-NK cells.

178. The method of any one of claims 1-177, wherein the method does not comprise administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells.

179. The method of any one of claims 1-177, wherein: prior to the administration of the composition of g-NK cells, the subject has received a lymphodepleting therapy.

180. The method of any one of claims 1-179, wherein the method further comprises administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells.

181. The method of claim 179 or claim 180, wherein administration of a dose of the composition of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphodepleting therapy.

182. The method of any one of claims 179-181, wherein administration of a dose of the composition of g-NK cells is initiated within 7 days or at or about 7 days after initiation of the lymphodepleting therapy.

183. The method of any one of claims 179-182, wherein the lymphodepleting therapy comprises fludarabine and / or cyclophosphamide.

184. The method of any one of claims 179-183, wherein the lymphodepleting therapy comprises fludarabine and cyclophosphamide.

185. The method of any of claims 179-184, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 20-40 mg / m2body surface area of the subject.

186. The method of any one of claims 179-185, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg / m2, daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m2body surface area of the subject.

187. The method of any one of claims 179-186, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 300 mg / m2body surface of the subject, daily, for 2-4 days.

188. The method of any one of claims 185-187, wherein the lymphodepleting therapy further comprises administration of mesna at or about 200-400 mg / m2body surface area of the subject.

189. The method of claim 188, wherein the lymphodepleting therapy further comprises administration of mesna at or about 300 mg / m2body surface area of the subject, daily, for 2-4 days.

190. The method of any one of claims 179-189, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at or about 400 mg / m2body surface area of the subject and mesna at or about 300 mg / m2, daily, each for 2-4 days, optionally 3 days.

191. The method of any one of claims 11-190, wherein the subject is a human subject.

192. The method of any one of claims 11-191, wherein the subject is aged 18 to 65 years old.

193. The method of any one of claims 11-192, wherein the subject has Epstein-Barr virus (EBV)-infected cells.

194. The method of claim 193, wherein the EBV-infected cells are EBV-infected B cells.

195. The method of any one of claims 11-194, wherein the subject is seropositive for an EBV infection.

196. The method of any one of claims 11-195, wherein the subject is seropositive for Epstein- Barr nuclear antigen 1 (EBNA).

197. The method of any one of claims 11-196, wherein the subject has a high level of Epstein- Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNA)386-405 antibodies.

198. The method of any one of claims 11-197, where the subject has autoreactive cells.

199. The method of claim 198, wherein the autoreactive cells are autoreactive B cells and / or T cells.

200. The method of claim 198 or claim 199, wherein the autoreactive cells express HLA-E and / or have upregulated HLA-E expression.

201. The method of any one of claims 198-200, wherein the autoreactive cells are central nervous system (CNS)-autoreactive cells.

202. The method of claim 201, wherein the CNS-autoreactive cells are reactive to GlialCAM, CRY AB, MBP, or ANO2.

203. The method of claim 201 or claim 202, wherein the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.

204. The method of any one of claims 11-203, wherein the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and / or T cells.

205. The method of any one of claims 11-204, wherein the subject is characterized by an HLA-E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-1 )-derived peptide.

206. The method of claim 205, wherein the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20.

207. The method of claim 205, wherein the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.

208. The method of any one of claims 33 and 47-207, wherein the multiple sclerosis is a treatment refractory progressive multiple sclerosis.

209. The method of any one of claims 33 and 47-207, wherein the multiple sclerosis is a primary or non-active secondary progressive MS (SPMS), where non-active SPMS is defined by the absence of clinical relapse in previous two years.

210. The method of any one of claims 33 and 47-209, wherein the subject does not have relapsing remitting MS.

211. The method of any one of claims 33 and 47-209, wherein the subject has relapsing remitting MS.

212. The method of any one of claims 33 and 47-211, wherein the subject does not have active SPMS.

213. The method of any one of claims 33 and 47-212, wherein the subject has not received a last treatment of a B-cell depletion therapy in the past 48 weeks.

214. The method of any one of claims 33 and 47-212, wherein the subject has received a last treatment of a B-cell depletion therapy in the past 48 weeks.

215. The method of any one of claims 33 and 47-214, wherein the multiple sclerosis is characterized by one or more, optionally all, of the following:(a) Expanded Disability Status Scale (EDSS) at screening is from 3.0 to 6.5 points;(b) score of >2.0 on the Functional Systems (FS) scale for the pyramidal system that is due to lower extremity findings;(c) disease duration from the onset of MS symptoms that is either less than 15 years in subjects with an EDSS at screening >5.0 or less than 10 years in subjects with an EDSS at screening <5.0;(d) history or presence in a cerebrospinal fluid (CSF) specimen of: (1) elevated immunoglobulin G index, or (2) two or more immunoglobulin G oligoclonal bands detected by isoelectric focusing; or(e) neurologic stability >30 days prior to treatment.

216. The method of any one of claims 33 and 47-215, wherein the subject selected for treatment has hematology values of ANC >1000 cells / mm3, platelet count >75,000 cells / mm3, and hemoglobin > 9.0 g / dL.

217. The method of any one of claims 27-40, 45-47, 51-96, 99-216, wherein the subject has been dosed with a dosing regimen of the antibody within the prior 6 months before initiation of the administration of the composition of g-NK cells in combination with the antibody.

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