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WO2025213127A1 - Traitement de maladies ou d'affections auto-immuns avec des cellules tueuses naturelles - Google Patents

Traitement de maladies ou d'affections auto-immuns avec des cellules tueuses naturelles

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Publication number
WO2025213127A1
WO2025213127A1 PCT/US2025/023300 US2025023300W WO2025213127A1 WO 2025213127 A1 WO2025213127 A1 WO 2025213127A1 US 2025023300 W US2025023300 W US 2025023300W WO 2025213127 A1 WO2025213127 A1 WO 2025213127A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
composition
subject
antibody
autoreactive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023300
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English (en)
Inventor
Stefanie MANDL-CASHMAN
Austin BIGLEY
Mark Frohlich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Indapta Therapeutics Inc
Original Assignee
Indapta Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Indapta Therapeutics Inc filed Critical Indapta Therapeutics Inc
Publication of WO2025213127A1 publication Critical patent/WO2025213127A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)

Definitions

  • 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).
  • NK Natural Killer
  • g-NK cells FcRy chain
  • 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 an antigen-bearing cell.
  • NK cells including specific specialized subsets thereof, can be used in therapeutic methods. Improved methods involving NK cells are needed for therapeutic uses related to the treatment of autoimmune diseases or disorders, including methods of using the NK cells as a monotherapy. Provided herein are embodiments that meet such needs.
  • a method of killing autoreactive cells includes contacting autoreactive cells with a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells).
  • the autoreactive cells include cells that express HLA-E and/or have upregulated HLA-E expression.
  • 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).
  • the autoreactive cells are present in a subject having an autoimmune disease or disorder.
  • a method of treating an autoimmune disease or disorder 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.
  • 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.
  • a method of treating an autoimmune disease or disorder 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.
  • the method may further include administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder.
  • the target antigen is a B cell antigen.
  • a method of prophylactically treating an autoimmune disease or disorder 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.
  • 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.
  • 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.
  • NK Natural Killer
  • 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.
  • the autoreactive cells are autoreactive B cells and/or T cells.
  • the autoreactive cells express HLA-E and/or have upregulated HLA-E expression.
  • the autoreactive cells are central nervous system (CNS)-autoreactive cells.
  • the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.
  • the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having radiologically isolated syndrome (RIS).
  • RIS radiologically isolated syndrome
  • the subject who is likely or suspected to develop an autoimmune disease or disorder has or is selected for having clinically isolated syndrome (CIS).
  • CIS clinically isolated syndrome
  • a method of treating an autoimmune disease or disorder 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.
  • the NK cells are positive for NKG2C (NKG2C pos ) and/or negative or low for NKG2A (NKG2A neg ).
  • at least 8% of the NK cells are positive for NKG2C (NKG2C pos ).
  • at least 8% of the NK cells are positive for NKG2C (NKG2C pos ) and/or negative or low for NKG2A (NKG2A neg ).
  • the autoimmune disease or disorder is systemic lupus erythematosus (SLE), systemic sclerosis (SSc), myositis (IIM), rheumatoid arthritis (RA), or multiple sclerosis (MS).
  • the autoimmune disease or disorder is a kidney or renal disease.
  • the kidney or renal disease is systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).
  • pathogenesis of the autoimmune or disorder is associated with a viral infection.
  • the autoimmune disease or condition is characterized by autoreactive B cells or autoreactive T cells with upregulated HLA-E expression.
  • the upregulation of HLA-E expression is caused by a viral infection.
  • the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).
  • the viral infection is an Epstein-Barr virus (EBV).
  • a method of treating a disease or disorder associated with an Epstein- Barr virus 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.
  • the NK cells are positive for NKG2C (NKG2C pos ) and/or negative or low for NKG2A (NKG2A neg ).
  • at least 8% of the NK cells are positive for NKG2C (NKG2C pos ).
  • at least 8% of the NK cells are positive for NKG2C (NKG2C pos ) and/or negative or low for NKG2A (NKG2A neg ).
  • the methods may include administering to the subject an antibody directed against a target antigen associated with the autoimmune disease or disorder.
  • the target antigen is a B cell antigen.
  • 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).
  • NK Natural Killer
  • the method does not comprise administering an antibody to the subject in combination with the composition of g-NK cells.
  • 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.
  • a method of treating an autoimmune disease or disorder 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.
  • NK Natural Killer
  • MS multiple sclerosis
  • the subject with multiple sclerosis has Epstein-Barr virus (EBV)-infected cells.
  • the EBV-infected cells are EBV-infected B cells.
  • the subject is seropositive for an EBV infection.
  • the subject with multiple sclerosis is seropositive for Epstein-Barr nuclear antigen 1 (EBNA).
  • EBNA Epstein-Barr nuclear antigen 1
  • the subject with multiple sclerosis has a high level of Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNA)386-405 antibodies.
  • the subject has autoreactive cells.
  • the autoreactive cells are autoreactive B cells and/or T cells.
  • the autoreactive cells express HLA-E and/or have upregulated HLA-E expression.
  • the autoreactive cells are central nervous system (CNS)-autoreactive cells.
  • CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.
  • the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and/or T cells.
  • the subject is characterized by an HLA-E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-l)-derived peptide.
  • LMP-l EBV latent membrane protein 1
  • the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20.
  • the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.
  • 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.
  • NKG2C pos at least at or about 15% of the NK cells of the composition are positive for NKG2C
  • NKG2A neg at least about 70% of NK cells of the composition are negative or low for NKG2A
  • the antibody is a full-length antibody.
  • the B cell antigen is expressed on a cell of a B cell lineage.
  • the B cell antigen is antigen selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.
  • the cell of the B cell lineage is an autoreactive B cell.
  • 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.
  • the antibody is an anti-CD19 antibody.
  • the antibody is inebilizumab, tafasitamab-cxix or obexelimab.
  • the antibody is an anti-CD20 antibody.
  • a method of treating an autoimmune disease or disorder 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.
  • the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab. In certain embodiments, the antibody is ocrelizumab.
  • 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.
  • 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.
  • the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously.
  • 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.
  • 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.
  • NK Natural Killer
  • MS multiple sclerosis
  • a method of treating an autoimmune disease or disorder 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 10 8 cells to at or about 50 x 10 9 cells 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.
  • NK Natural Killer
  • MS multiple sclerosis
  • a method of treating an autoimmune disease or disorder 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 10 8 cells to at or about 50 x 10 9 cells and each dose is administered one time daily at a frequency of every other day (Q2D) in the 7- day cycle.
  • NK Natural Killer
  • the method does not comprise administering to the subject an antibody.
  • the method further comprises: (c) administering to the subject an antibody, wherein the antibody is an anti-CD20 antibody.
  • the 7-day cycle is repeated twice, and each 7-day cycle is the same.
  • the anti-CD20 antibody is ocrelizumab.
  • 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.
  • the composition of g-NK cells is administered in a 7-day cycle.
  • the 7-day cycle is repeated one to three times.
  • the 7-day cycle is repeated one time.
  • the 7-day cycle is repeated two times.
  • 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 (FcRy neg ) NK cells (g-NK).
  • g-NK FcRy-deficient NK cells
  • FcRy neg FcRy neg NK cells
  • 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.
  • (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
  • 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.
  • the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRy pos ; and/or among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRy pos .
  • MFI mean fluorescence intensity
  • 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.
  • 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).
  • the effector cytokine is IFN-gamma or TNF-alpha. In particular embodiments, the effector cytokine is IFN-gamma and TNF-alpha.
  • 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.
  • 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.
  • 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.
  • 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).
  • the irradiated feeder cells are deficient in HLA class I and HLA class II.
  • the irradiated feeder cells are 221.
  • 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.
  • the recombinant cytokines are IL-21 and IL-2.
  • the recombinant cytokines are IL-21, IL-2, and IL- 15.
  • the g-NK cells in the composition are from a single donor subject that have been expanded from the same biological sample.
  • 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).
  • the g-NK cells are not engineered with an antigen receptor, optionally wherein the antigen receptor is a chimeric antigen receptor.
  • 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).
  • 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.
  • any of the provided methods further comprises administering IFN-P to the subject.
  • the IFN- is administered three times a week.
  • each dose of IFN-P is about 22 pg to 44 pg, optionally wherein each dose of IFN-P is 44 pg.
  • 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.
  • the methods include administering an exogenous cytokine that is or comprises IL- 15.
  • the methods include administering an exogenous cytokine that is or comprises IL-2.
  • the method comprises administering IL-2 to the subject.
  • the IL-2 is administered once a week, two times a week or three times a week.
  • the IL-2 is administered at a frequency of every other day (Q2W).
  • the IL-2 is administered five times a week, six times a week, or seven times a week.
  • the IL-2 is administered at a frequency of every day (QD).
  • the IL-2 is administered at a frequency of every week day.
  • for each day of administration the IL-2 is administered once daily.
  • 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.
  • Q2D every other day
  • 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.
  • 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.
  • the composition of g-NK cells is administered as at least one dose.
  • each dose of g-NK cells is from at or about from at or about 1 x 10 8 cells to at or about 50 x 10 9 cells of the composition of g-NK cells.
  • each dose of g-NK cells is from at or about from at or about 5 x 10 9 cells to at or about 20 x 10 9 cells of the composition of g-NK cells.
  • each dose of g-NK cells is or is about 5 x 10 8 cells of the composition of g-NK cells.
  • each dose of g-NK cells is or is about 5 x 10 9 cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 10 x 10 9 cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells is or is about 20 x 10 9 cells of the composition of g-NK cells.
  • the method does not comprise administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells.
  • the subject prior to the administration of the composition of g-NK cells, the subject has received a lymphodepleting therapy.
  • the method further comprises administering to the subject a lymphodepleting therapy prior to administering the composition of g-NK cells.
  • 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.
  • 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.
  • the lymphodepleting therapy comprises fludarabine and/or cyclophosphamide. In some embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide.
  • the lymphodepleting comprises the administration of fludarabine at or about 20-40 mg/m 2 body surface area of the subject, optionally at or about 30 mg/m 2 , daily, for 2-4 days, and/or cyclophosphamide at or about 200-400 mg/m 2 body surface area of the subject, optionally at or about 300 mg/m 2 , daily, for 2-4 days.
  • the lymphodepleting therapy further comprises administration of mesna at or about 200-400 mg/m 2 body surface area of the subject, optionally at or about 300 mg/m 2 , daily, for 2-4 days.
  • the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg/m 2 body surface area of the subject, daily, and cyclophosphamide at or about 400 mg/m 2 body surface area of the subject and mesna at or about 300 mg/m 2 , daily, each for 2-4 days, optionally 3 days.
  • the subject is a human subject. In some embodiments, the subject is aged 18 to 65 years old.
  • the subject has Epstein-Barr virus (EBV)- infected cells.
  • EBV-infected cells are EBV-infected B cells.
  • the subject is seropositive for an EBV infection.
  • the subject is seropositive for Epstein-Barr nuclear antigen 1 (EBNA).
  • the subject has a high level of Epstein-Barr virus (EBV) Epstein-Barr nuclear antigen 1 (EBNA)386-405 antibodies.
  • the subject has autoreactive cells.
  • the autoreactive cells are autoreactive B cells and/or T cells.
  • the autoreactive cells express HLA-E and/or have upregulated HLA-E expression.
  • the autoreactive cells are central nervous system (CNS)-autoreactive cells.
  • CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81.
  • the subject has GlialCAM-specific autoreactive cells, optionally autoreactive B cells and/or T cells.
  • the subject is characterized by an HLA-E molecule that is stabilized by an EBV latent membrane protein 1 (LMP-l)-derived peptide.
  • LMP-l EBV latent membrane protein 1
  • the EBV LMP-1 has the peptide sequence GGDPHLPTL set forth in SEQ ID NO: 20.
  • the EBV LMP-1 has the peptide sequence GGDPPLPTL set forth in SEQ ID NO: 21.
  • the multiple sclerosis is a treatment refractory progressive multiple sclerosis.
  • 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.
  • SPMS primary or non-active secondary progressive MS
  • the subject does not have relapsing remitting MS.
  • the subject has relapsing remitting MS.
  • the subject does not have active SPMS.
  • the subject has not received a last treatment of a B-cell depletion therapy in the past 48 weeks.
  • the subject has received a last treatment of a B-cell depletion therapy in the past 48 weeks.
  • 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.
  • FcRy FceRly
  • 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.
  • surface-bound monoclonal antibody e.g., ocrelizumab
  • 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.
  • NK cells Natural Killer 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.
  • the g-NK cells also are high in expression of NKG2C and low or negative in expression of NKG2A.
  • the g-NK cells are NKG2C pos /NKG2A neg NK cells.
  • the provided methods relate to treating an autoimmune disease.
  • 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).
  • the provided methods relate to a kidney or a renal disease.
  • 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).
  • SLE systemic lupus erythematosus
  • PMN primary membranous nephropathy
  • IgA immunoglobulin A nephropathy
  • 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.
  • 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.
  • 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.
  • NK cells 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.
  • CD16 also known as CD16a or FcyRIIIa
  • NK cells 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.
  • 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).
  • 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
  • NP_ 004097.1 accession number NP_ 004097.1 (GL4758344)
  • the mechanism for increased responses of g-NK cells may be due to changes in epigenetic modification that influence the expression of the FcRy chain as well are other factors such as Syk. These epigenetic modifications are promoted at least in part by response to CMV infection where this subset of NK cells arise in about 25% of CMV exposed individuals. This special subset is relatively rare because g-NK cells are detectable at levels of ⁇ 3% to 10% of total NK cells in only 25% to 30% of cytomegalovirus (CMV)-seropositive individuals; thus, expansion is generally required for in vivo use (see e.g., Hwang et al.
  • CMV cytomegalovirus
  • HLA-E molecules by binding to CD94/NKG2A receptors expressed by NK cells, can provide protection to cells seeking to evade NK cell killing.
  • autoreactive B cells and T cells can normally avoid NK cell lysis through upregulation of HLA-E.
  • 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.
  • 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.
  • the g-NK cells may deplete B cells that produce autoreactive antibodies and also deplete T cells to regulate autoreactive T cell responses.
  • the g-NK cells may directly inhibit and/or lyse autoreactive B cells as well as autoreactive T cells.
  • the above studies support the inventor’s recognition that the advantageous features g-NK cells, which are generally NKG2C hlgh , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a CD 19 CAR therapy or a monoclonal antibody therapy (e.g., CD20 antibody, such as ocrelizumab)
  • CD20 antibody such as ocrelizumab
  • 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.
  • NK cell therapy 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.
  • 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.
  • 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.
  • 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.
  • the above embodiments are based on the unique NKG2C+/NKG2A- phenotype of g-NK cells.
  • the g-NK cells also are able to promote ADCC killing that can further potentiate responses and treatment of autoimmune diseases and conditions.
  • g-NK cells also exhibited anti-viral properties by enhanced plasma-mediated ADCC against virally infected cells (Lee et al. Immunity, 2015).
  • g-NK 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).
  • NK cells are capable of killing cells via antibody dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody dependent cell-mediated cytotoxicity
  • 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.
  • 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.
  • CD16 also known as CD 16a or FcyRIIIa
  • Fc receptor CD 16a or FcyRIIIa
  • 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.
  • 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).
  • CD16 engagement CD16 crosslinking
  • 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 or chain, which in turn recruits tyrosine kinases, SYK and ZAP-70, initiating a cascade of signal transduction leading to rapid and potent effector functions.
  • 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.
  • 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.
  • g-NK cells are more effective in eliciting cell-mediated cytotoxicity than are conventional NK cells even in the absence of antibody.
  • y-deficient g-NK cells 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.
  • CD 16 when CD 16 is engaged by the Fc region of an antibody, the signaling is mediated by solely the £ chain of the TCR-CD3 complex (CD3Q, which transduces signals through three immunoreceptor tyrosine-based activation motifs (IT AMs).
  • 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.
  • the g-NK cells have a prolonged lifespan, compared to conventional NK cells, and their presence is maintained long-term.
  • 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.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 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.
  • 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.
  • 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.
  • g-NK cells 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).
  • g-NK cells 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.
  • 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).
  • CRS cytokine release syndrome
  • ICANs immune effector cell-associated neurotoxicity syndrome
  • 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.
  • g-NK cells 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).
  • 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.
  • the autoreactive cells are autoreactive B cells and/or T cells.
  • the contacting is carried out ex vivo or in vitro.
  • 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.
  • a composition of g-NK cells such as a subject suspected of or known to have autoreactive cells or an autoimmune disease or condition.
  • 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.
  • 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.
  • the B cells and/or T cells are autoreactive B cells and/or T cells.
  • the B cells or T cells are HLA-E positive.
  • the provided compositions comprising g-NK cells 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.
  • 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).
  • 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.
  • 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.
  • the autoimmune disease or disorder is caused by or is exacerbated by a virus infection.
  • the cells or pharmaceutical composition thereof is administered in an effective amount to effect treatment of the autoimmune disease or disorder.
  • the methods thereby treat the autoimmune disease or disorder in the subject.
  • 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.
  • 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).
  • 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.
  • the cells or pharmaceutical composition thereof is administered in an effective amount to prevent development of the autoimmune disease or disorder.
  • the methods thereby prophylactically treat the autoimmune disease or disorder in the subject.
  • 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.
  • the composition comprises about 5-99% g-NK cells, or any percentage of g-NK cells between 5 and 99%, inclusive.
  • the composition can comprise about 5-99% g-NK cells, inclusive, prior to expansion.
  • most of the NK cells in a composition, prior to expansion can be g-NK cells.
  • 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.
  • the composition can comprise about 5-99% g-NK cells, inclusive, after expansion.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition prior to expansion, 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.
  • the composition prior to expansion, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • after expansion, of the total NK cells in the composition greater than at or about 50% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.
  • cells of the composition that are g-NK cells also are characterized as NKG2C pos , NKG2A neg and CD16 pos .
  • cells of the composition that are g-NK cells are characterized as being CD57 pos , CD7 dim/neg , CD161 neg and/or CD38 neg .
  • cells of the composition of g-NK cells are NKG2A neg /CD161 neg .
  • cells of the composition of g-NK cells are CD38 neg .
  • cells of the composition of g- NK cells has the phenotype CD45 pos /CD3 neg /CD56 pos .
  • the composition contains NKG2C pos cells. In some embodiments, the compositions contain NKG2A neg cells. In some embodiments, the composition contains NKG2C pos /NKG2A neg cells. In some embodiments, g-NK cells of the composition are NKG2C pos cells. In some embodiments, g-NK cells of the composition contain NKG2A neg cells. In some embodiments, g- NK cells of the composition contain NKG2C pos /NKG2A neg cells.
  • the composition comprises about 5-99% NKG2C pos cells.
  • the composition can include an increased or greater percentages of NKG2C pos cells relative to total NK cells or total cells compared to the percentage of NKG2C pos cells naturally present in the subject from which the cells were isolated.
  • 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.
  • 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 or
  • the composition can include at least at or about 8% NKG2C pos cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C pos cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at least at least at
  • the composition can include at least at or about 8% NKG2C pos cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C pos cells of the total NK cells in the composition.
  • the provided compositions include those in which the NKG2C pos cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.
  • the total cells in the composition greater than at or about 8% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2C pos .
  • the total cells in the composition greater than at or about 40% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2C pos . In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2C pos .
  • the total NK cells in the composition greater than at or about 50% of the cells are NKG2C pos .
  • of the total NK cells in the composition greater than at or about 60% of the cells are NKG2C pos .
  • of the total NK cells in the composition greater than at or about 70% of the cells are NKG2C pos .
  • of the total NK cells in the composition greater than at or about 80% of the cells are NKG2C pos .
  • of the total NK cells in the composition greater than at or about 90% of the cells are NKG2C pos .
  • of the total NK cells in the composition greater than at or about 95% of the cells are NKG2C pos .
  • the composition comprises about 5-99% NKG2A neg cells.
  • the composition can include an increased or greater percentages of NKG2A neg cells relative to total NK cells or total cells compared to the percentage of NKG2A neg cells naturally present in the subject from which the cells were isolated.
  • 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.
  • 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% N
  • the composition can include at least at or about 15% NKG2A neg cells of the total cells in the composition.
  • 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 least at least at least at or
  • the provided compositions include those in which the NKG2A neg cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.
  • the total cells in the composition greater than at or about 10% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2A neg .
  • the total cells in the composition greater than at or about 50% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2A neg .
  • the total NK cells in the composition greater than at or about 50% of the cells are NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2A neg .
  • the composition comprises about 5-99% NKG2C pos /NKG2A neg cells.
  • the composition can include an increased or greater percentages of NKG2C pos /NKG2A neg cells relative to total NK cells or total cells compared to the percentage of NKG2C pos /NKG2A neg cells naturally present in the subject from which the cells were isolated.
  • 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.
  • 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 or
  • the composition can include at least at or about 8% NKG2C pos /NKG2A neg cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C pos /NKG2A neg cells of the total cells in the composition.
  • 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%,
  • the composition can include at least at or about 8% NKG2C pos /NKG2A neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C pos /NKG2A neg cells of the total NK cells in the composition.
  • the provided compositions include those in which the NKG2C pos /NKG2A neg cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.
  • the total cells in the composition greater than at or about 8% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2C pos /NKG2A neg .
  • the total cells in the composition greater than at or about 30% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2C pos /NKG2A neg .
  • the total cells in the composition greater than at or about 80% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2C pos /NKG2A neg .
  • the total NK cells in the composition greater than at or about 50% of the cells are NKG2C pos /NKG2A neg .
  • of the total NK cells in the composition greater than at or about 60% of the cells are NKG2C pos /NKG2A neg .
  • of the total NK cells in the composition greater than at or about 70% of the cells are NKG2C pos /NKG2A neg .
  • of the total NK cells in the composition greater than at or about 80% of the cells are NKG2C pos /NKG2A neg .
  • the total NK cells in the composition greater than at or about 90% of the cells are NKG2C pos /NKG2A neg . In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2C pos /NKG2A neg .
  • the composition comprises about 5-99% g-NK cells that are NKG2C pos /NKG2A neg cells.
  • the composition can include an increased or greater percentages of g-NK cells that are NKG2C pos /NKG2A neg cells relative to total NK cells or total cells compared to the percentage of g-NK cells that are NKG2C pos /NKG2A neg cells naturally present in the subject from which the cells were isolated.
  • 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.
  • 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 or
  • the composition can include at least at or about 8% g-NK cells that are NKG2C pos /NKG2A neg cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% g-NK cells that are NKG2C pos /NKG2A neg cells of the total cells in the composition.
  • 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%,
  • the provided compositions include those in which the g-NK cells that are NKG2C pos /NKG2A neg cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.
  • the total cells in the composition greater than at or about 8% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are g- NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are g-NK cells that are NKG2C pos /NKG2A neg .
  • the total cells in the composition greater than at or about 25% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are g-NK cells that are NKG2C pos /NKG2A neg .
  • the total cells in the composition greater than at or about 60% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are g- NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are g-NK cells that are NKG2C pos /NKG2A neg .
  • the total NK cells in the composition greater than at or about 50% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells that are NKG2C pos /NKG2A neg . In some embodiments, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells that are NKG2C pos /NKG2A neg .
  • the g-NK cells are CD16 pos .
  • 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).
  • the NK cells bear the CD16 158V polymorphism in both alleles (called 158V/V herein).
  • the g-NK cells comprise CD16 158V/V (V158).
  • the g-NK cells are CD16 158V/F.
  • the g-NK cells comprise CD16 158 F/F (F158).
  • the g-NK cells of the composition 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.
  • 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.
  • 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.
  • 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.
  • perforin and granzyme B expression levels by NK cells can be measured by intracellular flow cytometry and levels measured based on levels of mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • perforin and granzyme B expression levels based on MFI will differ between g-NK cells and cells that are FcRy pos .
  • the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about two times the mean level of perforin expressed by FcRy pos NK cells.
  • the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about three times the mean level of perforin expressed by FcRy pos NK cells. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about four times the mean level of perforin expressed by FcRy pos NK cells.
  • the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about four times the mean level of granzyme B expressed by FcRy pos NK cells.
  • the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRy pos .
  • MFI mean fluorescence intensity
  • the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRy pos .
  • a cell e.g., NK cell subset
  • 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.
  • 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.
  • a cell e.g., NK cell subset
  • a cell is low (lo or min) for a particular marker if there is a lower level of detectable presence on or in the cell of a particular marker compared to a cell known to be positive for the marker.
  • 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.
  • the composition comprises from at or about 10 6 cells to at or about 10 12 cells.
  • the composition comprises from at or about 10 6 to at or about 10” cells, from at or about 10 6 to at or about IO 10 cells, from at or about 10 6 to at or about 10 9 cells, from at or about 10 6 to at or about 10 8 cells, from at or about 10 6 to at or about 10 7 cells, from at or about 10 7 to at or about 10 12 cells, from at or about 10 7 to at or about 10” cells, from at or about 10 7 to at or about 10 10 cells, from at or about 10 7 to at or about 10 9 cells, or from at or about 10 7 to at or about 10 8 cells, from at or about 10 8 to at or about 10 12 cells, from at or about 10 8 to at or about 10” cells, from at or about 10 8 to at or about 10 10 cells, from at or about 10 8 to at or about 10 9 cells, from at or about 10 9 to at or about 10 12 cells.
  • the composition comprises from at or about 10 9 to at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 10 9 to at or about 10 10 cells. In some of any of the provided embodiments, the composition comprises from at or about 10 10 to at or about 10” cells. [0169] In some of any of the provided embodiments, the composition comprises at least at or about 10 6 g-NK cells.
  • the cells in the described composition of g-NK cells are for allogenic cell therapy.
  • 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.
  • the donor or donors are not known to have the autoimmune disease or condition.
  • 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.
  • the g-NK cells are primary g-NK cells from a subject.
  • 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.
  • 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.
  • the g-NK cells are selected and expanded such as by methods described in Section II.
  • the composition comprises greater than or greater than at or about 10 5 NKG2C pos cells or a subset thereof, at or about 10 6 NKG2C pos cells or a subset thereof, at or about 10 7 NKG2C pos cells or a subset thereof, at or about 10 8 NKG2C pos cells or a subset thereof, at or about 10 9 NKG2C pos cells or a subset thereof, at or about 10 10 NKG2C pos cells or a subset thereof, at or about 10” NKG2C pos cells or a subset thereof, or at or about 10 12 NKG2C pos cells or a subset thereof.
  • 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.
  • the composition has a cell density of at least or at least about 1 x 10 5 cells/mL, 5 x 10 5 cells/mL, 1 x 10 6 cells/mL, 5 x 10 6 cells/mL, 1 x 10 7 cells/mL, 5 x 10 7 cells/mL, or 1 x 10 8 cells/ mL.
  • 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).
  • 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.
  • 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.
  • 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.
  • the NK cells such as NKG2C pos cells or a subset thereof can be present in the composition in an effective amount.
  • the composition contains an effective amount of g-NK cells, such as FcRy neg cells or cells having a g-NK surrogate marker profile thereof.
  • An effective amount of cells can vary depending on the patient, as well as the type, severity and extent of disease. Thus, a physician can determine what an effective amount is after considering the health of the subject, the extent and severity of disease, and other variables.
  • the composition is sterile.
  • 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.
  • sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • culturing is carried out using a gas permeable culture vessel.
  • culturing is carried out using a bioreactor.
  • compositions that are suitable for cryopreserving the provided NK cells.
  • the NK cells are cryopreserved in a serum-free cryopreservation medium.
  • the composition comprises a cryoprotectant.
  • the cryoprotectant is or comprises DMSO and/or s glycerol.
  • the cryopreservation medium is between at or about 5% and at or about 10% DMSO (v/v).
  • the cryopreservation medium is at or about 5% DMSO (v/v).
  • the cryopreservation medium is at or about 6% DMSO (v/v).
  • 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 (CryoStorTM CS10). CryoStorTM CS10 is a cry opreservation medium containing 10% dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • 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.
  • compositions can be preserved at ultra-low temperature before the administration to a patient.
  • 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.
  • 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.
  • 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.
  • HAS human albumin serum
  • An equivalent amount of 20% DMSO can be added into the HAS solution. Aliquots of the mixture can be placed into vials and frozen overnight inside an ultra-low temperature chamber at about -80° C.
  • the cryopreserved NK cells are prepared for administration by thawing.
  • the NK cells can be administered to a subject immediately after thawing.
  • the composition is ready-to-use without any further processing.
  • 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.
  • cytokines can be administered to a subject prior to isolating primary NK cells.
  • IL-12, IL-15, IL-18, IL-2, and/or CCL5 can be administered to a subject prior to isolating the primary NK cells.
  • compositions in which the g-NK cells are engineered g-NK cells are provided.
  • 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.
  • 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.
  • GvHD graft vs. host disease
  • 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.
  • the g-NK cells described herein can be gene edited to reduce FcRy chain expression, activity and/or signaling in the cell.
  • 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.
  • FcRy signaling adaptor e.g., a transcription factor, such as PEZF or HEEIOS
  • a gene encoding a protein that is involved in FcRy-mediated signaling e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2
  • method of engineering may comprise introducing an inhibitory nucleic acid molecule that targets a gene encoding FcRy chain, a gene encoding a protein that regulates expression or activity of FcRy signaling adaptor (e.g., a transcription factor, such as PLZF or HELIOS) and/or a gene encoding a protein that is involved in FcRy-mediated signaling (e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2) as described.
  • FcRy signaling adaptor e.g., a transcription factor, such as PLZF or HELIOS
  • a gene encoding a protein that is involved in FcRy-mediated signaling e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2
  • 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.
  • FcRy chain expression such as knockout or disruption of FcRy chain in NK cells
  • cells are gene edited to knockout the FcRy chain using a CRISPR-Cas9 system.
  • 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.
  • 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.
  • primary cells derived from a subject may be expanded and/or cultured before gene editing.
  • the gene edited primary cells are cultured and/or expanded following gene editing and prior to administration to a patient.
  • the g-NK cell can further comprise nucleic acid encoding a heterologous CD16.
  • the heterologous CD 16 can comprise a CD16-activating mutation, wherein the mutation can result in higher affinity to IgGl.
  • the heterologous CD16 can comprise a 158V mutation.
  • the engineered g-NK cells can be derived from a primary cell obtained from a human subject.
  • FcRy 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.
  • the FcRy chain gene may be deleted, disrupted, or mutated.
  • FcRy protein level can be decreased by effecting a molecule that increases FcRy gene expression or activity, such as a transcription factor that regulates transcription of FcRy.
  • a gene that regulates transcription or translation of the FcRy chain gene may be deleted, disrupted, or mutated.
  • 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.
  • FcRy chain gene or other gene there are many suitable methods for disrupting FcRy chain gene or other gene, such as those described herein.
  • the entire gene locus such as FcRy locus
  • FcRy locus may be deleted.
  • the IT AM signaling domain of FcRy may be deleted.
  • 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.
  • 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.
  • 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.
  • 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.
  • provided g-NK cells are engineered by editing the genome of the g- NK cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene editing is carried out after the selection of cells from a subject (e.g. selecting or enriching cells that are CD3 neg CD57 pos or CD3 neg CD56 pos ) and prior to incubating or culturing the selected or enriched cells with feeder cells (e.g. HLA-E-expressing feeder cells) for proliferation or expansion of the NK cells.
  • 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.
  • ZFNs 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)).
  • TAE-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • 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).
  • the DNA binding domain comprises a tandem array of TAE-effector domains, each of which specifically recognizes a single DNA base pair.
  • 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.
  • 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.
  • the guide RNA comprises an approximately 20 nucleotide targeting sequence.
  • RNA-guided nuclease is an RNA-guided DNA endonuclease.
  • 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).
  • the RNA-guided nuclease is a Cas9 or Casl2 nuclease.
  • the RNA- guided nuclease is Cpfl (Casl2a).
  • Cpfl is Acidaminococcus sp. Cpfl (AsCpfl).
  • 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.
  • the gRNA includes a crRNA and, optionally, a tracrRNA.
  • the RNA-guided nuclease e.g., Cas9 or a Casl2
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.”
  • PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”).
  • 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.
  • 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.
  • the NK cells express a CAR directed against CD38 and CD38 expression is reduced or eliminated in the NK cells.
  • 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).
  • the gRNA targets a molecule involved in immunosuppression of the NK cell activity.
  • engineered NK cells comprise reduced or absent checkpoint inhibitory receptor function.
  • 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.
  • the NK cell cells comprise reduced or absent checkpoint inhibitory receptor function for two or more checkpoint inhibitory receptors.
  • the two or more checkpoint inhibitory receptors comprise CD96 (TACTILE), CD 152 (CTLA4), or CD328 (SIGLEC7) or CD279 (PD-1).
  • the gRNA for use in the disclosure is a gRNA targeting TIGIT (see e.g., W02020/168300 for exemplary gRNA targeting TIGIT).
  • 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).
  • 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).
  • 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).
  • 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).
  • CISH cytokine-inducible SH2-containing protein
  • 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.
  • vectors e.g., viral or non-viral vectors
  • non-vector based methods e.g., using naked DNA or DNA complexes
  • 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.
  • molecules e.g., N-acetylgalactosamine
  • the RNA-guided nuclease and gRNA are delivered into cells as a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the Cas and gRNA are separately purified and then assembled to form the RNP.
  • one or more RNP complexes are delivered to the cell sequentially in any order, or simultaneously.
  • the RNP complex is delivered into cells by electroporation.
  • the RNP complex is delivered into cells using lipid nanoparticles.
  • 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).
  • 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
  • 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).
  • a Cas endonuclease such as, for example, Cas9
  • 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.
  • gRNA guide RNA
  • RNP ribonucleoprotein
  • 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.
  • 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.
  • 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.
  • 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.
  • a CRISPR enzyme e.g., Cas9 nuclease
  • a guide sequence is delivered to the cell.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • 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.
  • 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.
  • Homing endonucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved 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.
  • 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.
  • the nucleic acid may target FcRy chain mRNA.
  • 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.
  • the nucleic acid targets the mRNA of gene encoding a protein involved in FcRy-mediated signaling, such as SYK, DAB2 or EAT-2 mRNA.
  • 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.
  • 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.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • 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).
  • an immunomodulator e.g., cytokine, either secreted or membrane-bound as described.
  • 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).
  • 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.
  • EBV-specific HLA-E- restricted T and B cells e.g., GlialCam-specific autoreactive T cells and B cells
  • 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).
  • a subject in need of a treatment as described herein can be a subject exhibiting symptoms of an autoimmune disease or condition.
  • the subject having the autoimmune disease or disorder is selected after diagnosis for the autoimmune disease or disorder using various types of diagnostic tests.
  • 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).
  • 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.
  • Addison disease e.g., celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, inflammatory bowel disease (e.g., Crohn disease, ulcerative colitis), multiple sclerosis, systemic sclerosis (SSc), my
  • 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.
  • steroids such as corticosteroids
  • non-steroid drugs such as azathioprine, cyclophosphamide, mycophenolate mofetil (MMF)/mycophenolic acid, sirolimus, or tacrolimus.
  • MMF mycophenolate mofetil
  • the subject is still receiving immunosuppressive medicines.
  • subject with the autoimmune disease or disorder may have received one or more prior treatments to treat the autoimmune disease or disorder.
  • NK Natural Killer
  • the autoimmune disease or disorder may be multiple sclerosis.
  • Multiple sclerosis 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.
  • CNS central nervous system
  • the diagnosis 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).
  • MRI magnetic resonance image
  • CSF cerebrospinal fluid
  • 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).
  • RRMS relapsing-remitting multiple sclerosis
  • SPMS secondary progressive multiple sclerosis
  • PRMS progressive relapsing multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • 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).
  • PPMS 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. L 2001; Polman et al., Ann Neurol., 2005).
  • 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).
  • MS Multiple sclerosis
  • RRMS relapsing remitting MS
  • PPMS primary progressive MS
  • SPMS secondary progressive MS
  • 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.
  • 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 vertigo
  • mild emotional disturbances 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.
  • MS 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).
  • CAR-T chimeric antigen receptor T
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • 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).
  • 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).
  • NK Natural Killer
  • g-NK cells FcRy chain
  • 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).
  • NK Natural Killer
  • g-NK cells FcRy chain
  • the prior therapy to treat the MS comprises B-cell depletion therapy.
  • the B-cell depletion therapy is treatment with an anti-CD20 therapy (e.g., administration of an anti-CD20 antibody, e.g., ocrelizumab).
  • 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).
  • a composition of Natural Killer (NK) cells deficient in expression of FcRy chain g-NK cells
  • MS multiple sclerosis
  • the subject received a last treatment of the B-cell depletion therapy in the past 48 weeks.
  • the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.
  • 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.
  • MS multiple sclerosis
  • examples of antibodies in such a provided combination therapy include any described in Section I.F.
  • the combination of g-NK cells and an anti-CD20 antibody e.g., ocrelizumab
  • an anti-CD20 antibody e.g., ocrelizumab
  • 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.
  • anti-CD20 antibody Among monoclonal antibodies for use in a provided combination therapy with g-NK cells is an anti-CD20 antibody.
  • anti-CD20 antibodies are described in Section I.F.
  • the anti-CD20 antibody is ocrelizumab.
  • 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.
  • 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.
  • CD20+ autoreactive B 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).
  • CD8+CD20+ T cells are associated with white matter injury and thalamic atrophy in PPMS (Von Essen et al., Neurol. Neuroimmunol., Neuroinflamm., 2023). Also, CD20 dim CD8+ 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 CD20 dim T cells or HLA-expressing plasma cells or HLA-E expressing autoreactive CD4 and CD8 T cells.
  • MS disease modifying therapies
  • DMTs disease modifying therapies
  • 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).
  • 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
  • cortisones prednisolone or methyl prednisolone
  • the prior therapy may be referred to as a disease modifying therapy (DMT).
  • DMT disease modifying therapy
  • 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).
  • 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.
  • RRMS worsening relapsing-remitting MS
  • PRMS progressive -relapsing MS
  • SPMS secondary-progressive MS
  • the provided methods described herein may reduce the frequency and/or severity of relapses.
  • 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.
  • 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.
  • EDSS Expanded Disability Status Scale
  • 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.
  • 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.
  • 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.
  • 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.
  • the presently described g- NK cells may be used treat RRMS and/or SPMS so it does not develop into PPMS.
  • the provided methods herein may also be used to prevent the onset of multiple sclerosis.
  • the selected subject is a subject who has experienced a first clinical episode and have MRI features consistent with MS.
  • the selected multiple sclerosis subject can have a clinically isolated syndrome (CIS).
  • CIS clinically isolated syndrome
  • 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, Lancet Neurol., 17:162-173).
  • 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).
  • the selected multiple sclerosis subject can have a radiologically isolated syndrome (RIS).
  • RIS radiologically isolated syndrome
  • incidental imaging findings suggest inflammatory demyelination in the absence of clinical signs or symptoms.
  • 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).
  • RRMS relapse-remitting multiple sclerosis
  • 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.
  • 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).
  • the subject can be evaluated, e.g., for indicia of responsiveness, prior to, during, or after receiving the composition of g-NK cells.
  • the subject may be evaluated for indicia of responsiveness prior to receiving the composition of g-NK cells.
  • the subject may be evaluated for indicia of responsiveness concurrently or with the administration of the composition of g-NK cells.
  • the subject may be evaluated for indicia of responsiveness after receiving the composition of g-NK cells.
  • 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.
  • 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.
  • 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).
  • 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.
  • an exemplary primary and/or secondary biomarker endpoint is a change in autoreactive immune cells (e.g., reactivity to EBNA-1, GlialCAM, etc.).
  • an exemplary primary and/or secondary biomarker endpoint is neuro-injury and/or inflammatory cytokines (e.g., NfL, MBP, MOG, and/or interleukins).
  • an exemplary primary and/or secondary biomarker endpoint is an IgG titer and autoreactive antibodies (e.g., EBNA-1, GlialCAM, etc.).
  • an exemplary primary and/or secondary biomarker endpoint is EBV viral reactivity.
  • 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.
  • 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.
  • 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).
  • EDSS score 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a decreased rate of relapse e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%
  • 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
  • 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.
  • the methods provided herein result in clinical improvement of MS symptoms or clinical remission of MS in the subject.
  • 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, 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 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.
  • 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.
  • 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.
  • 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 provided herein 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject 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.
  • the subject 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.
  • the autoimmune disease or disorder may be a lupus disorder.
  • the lupus disorder can be, but are not limited to, systemic lupus erythematous (SLE), discoid lupus, drug-induced lupus, and neonatal lupus.
  • the selected subject with the autoimmune disease or disorder can have SLE.
  • SLE 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.
  • 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
  • HLA-E-expressing immune cells including HLA-E-expressing B cells and HLA-E-expressing T cells
  • HLA-E-expressing B 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).
  • the HLA-expression is assessed by quantitative RT-PCR.
  • the subject with the autoimmune disease or disorder described in the methods provided herein has increased HLA-E polynucleotide expression level compared to a reference level corresponding to a healthy individual.
  • the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and/or is within one or two standard deviation(s) below the median or mean value of HLA-E in samples obtained from a group of healthy individuals.
  • the subject with the autoimmune disease or disorder described in the methods provided herein has a similar HLA-E polynucleotide expression level compared to that of a reference level corresponding to that of another subject with the same autoimmune disease or disorder benefitting from administered g-NK cells as described herein.
  • the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and/or is within one or two standard deviation(s) below the median or mean value of HLA-E in samples obtained from a group of subjects with the same autoimmune disease or disorder benefitting from administered g-NK cells as described herein.
  • the autoimmune disease or condition is associated with a viral infection, and in particular a viral infection that produce HLA-E stabilizing peptides to increase HLA-E infection on infected T and B cells that may otherwise exacerbate the disease or condition.
  • a viral infection that produce HLA-E stabilizing peptides to increase HLA-E infection on infected T and B cells that may otherwise exacerbate the disease or condition.
  • certain viral infections result in a restricted set of virus-specific peptides that can be presented on the surface of HLA-E present on the surface of virally-infected cells.
  • the result is that the peptides can act to stabilize HLA-E on the surface of the cells, which is a viral infection strategy that commonly protects target cells from lysis by engagement of the HLA-E with the inhibitory receptor CD94/NKG2A on NK cells. This then can drive infection and disease.
  • the described g-NK cells compositions are enriched for cells that have reduced expression of the inhibitory receptor NKG2A (e.g., CD94/NKG2A- NK cells) and increased expression of the activating receptor NKG2C (e.g., CD94/NKG2C+ NK cells).
  • the g-NK cells described herein can effectuate potent killing of HLA-E expressing virally infected cells because of the low expression of the CD94/NKG2A inhibitory receptor and the high expression of the CD94/NKG2C activating receptor.
  • the infected target cells are not protected by lysis from the described g-NK cells.
  • the viral infection is one that is implicated in the pathogenesis and/or known to increase the likelihood of a subject developing an autoimmune disease or condition.
  • the autoimmune disease or condition is one in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the autoimmune disease or condition.
  • the viral infection can be caused by, but is not limited to, a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).
  • the subject is identified to have a viral infection associated with the autoimmune disease or condition.
  • the viral infection associated with the autoimmune disease or condition can be, but is not limited to, a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).
  • the viral infection is a cytomegalovirus (CMV) infection.
  • the autoimmune disease or condition is one that is associated with a cytomegalovirus (CMV) infection, in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the autoimmune disease or condition.
  • the subject for treatment is or has been selected as having CMV infected cells.
  • the viral infection is a Human papillomavirus (HPV).
  • the autoimmune disease or condition is one that is associated with a Human papillomavirus (HPV), in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the autoimmune disease or condition.
  • the subject for treatment is or has been selected as having HPV infected cells.
  • the viral infection is an influenza virus.
  • the autoimmune disease or condition is one that is associated with an influenza virus, in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the autoimmune disease or condition.
  • the subject for treatment is or has been selected as having influenza virus infected cells.
  • the viral infection is an Epstein-Barr virus (EBV) infection.
  • EBV infection has been linked with various autoimmune disorders that might arise as immunopathologic consequences of long-term virus carriage.
  • autoimmune diseases associated with EBV infection include multiple sclerosis (MS), lupus such as systemic lupus erythematosus (SLE), type I diabetes (T1 D), Sjogren’s Syndrome, rheumatoid arthritis (RA), dermatomyositis (DM), and other autoimmune diseases.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • T1 D type I diabetes
  • Sjogren’s Syndrome rheumatoid arthritis
  • RA rheumatoid arthritis
  • DM dermatomyositis
  • the provided methods are for treating an EBV associated disease or condition.
  • an EBV associated disease or condition is characterized by an EBV infection in the subject.
  • an EBV infection can be a primary EBV infection, a latent EBV infection or a latent EBV infection with a lytic EBV component.
  • the provided methods relate to prevention or reduction of latent EBV infection of B cells, and thus the treatment of diseases associated with EBV infection.
  • an EBV associated disease or condition is a disease associated with any one or more of the following: a) ill-controlled or uncontrolled EBV infection in a subject; b) latent EBV infection with a lytic EBV component in a subject; and c) uncontrolled proliferation of B cell lymphocytes latently infected with EBV in a subject.
  • an EBV associated disease or condition is an autoimmune disease or condition.
  • the autoimmune disease or condition is multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, or inflammatory bowel disease.
  • observations implicate EBV in the pathogenesis of MS as indicated by universal EBV seropositivity, high anti-EBV antibody levels, alterations in EBV-specific CD8+ T-cell immunity, increased spontaneous EBV-induced transformation of peripheral blood B cells, increased shedding of EBV from saliva and accumulation of EBV-infected B cells and plasma cells in the brain (Pender and Burrow, 2014).
  • the provided methods relate to treating MS, which is an autoimmune disease or condition in which EBV is known to be involved in the pathogenesis.
  • a subject to be treated has an EBV infection.
  • a subject is selected for treatment by identifying a subject that has an EBV infection.
  • An EBV infection in a subject can be determined using methods known in the art.
  • a subject has a longterm EBV infection.
  • a subject can have an EBV infection for about 6 months or longer, about 9 months or longer, about 1 year or longer, about 2 years or longer, about 3 years or longer.
  • the EBV infection is asymptomatic.
  • active EBV infection is detected in peripheral B cell populations. In some embodiments, active EBV infection is detected in CSF B cell populations. Methods for detection of active EBV infection can include, without limitation, detection of EBV proteins on the surface of B cells, where such markers include, without limitation: BILF-1, LMP1 and LMP2. In some embodiments, methods for detection of active EBV infection can include determining the presence of transcripts associated with active infection. In some embodiments, latent infection is characterized by limited expression of viral proteins, apart from, for example, EBNA1, LMP1 and LMP2. In some embodiments, active infection can result in expression of a broader range of viral proteins, including, for example, BILF-1, LMP1 , LMP2, etc. Detection of such proteins or transcripts can be indicative of an EBV-driven autoimmune disease or condition, such as MS pathology.
  • the antibodies detected are IgG antibodies. In some embodiments, the antibodies detected are IgM antibodies. In some embodiments, the antibodies are anti-VCA IgM, anti-VCA IgG or anti-EBNA-1 IgG. The determination is optionally combined with detection of active EBV infection. A variety of methods may be utilized for the detection of antibodies. In some embodiments, any of a variety of immunoassays can be used to detect antibodies, such as by using ELISA.
  • the subject has detectable EBV-viremia.
  • EBV load can be determined by assessing viral DNA from plasma samples and detected and quantified by PCR-based methods (see e.g., Aberle et al. J Clin Cirol., 25: S79-85, 2002).
  • a subject determined to have an EBV infection has an EBV DNA load of greater than or equal to about 5,000 copies/pg DNA in blood, such greater than or equal to about 10000 copies/pg, 25000 copies/pg, 50000 copies/pg, 75000 copies/pg, 100000 copies/pg, 125000 copies/pg or 150000 copies/pg, or any value between any of the foregoing.
  • a subject determined to have an EBV infection has greater than or equal to about 1,000 copies/100 pl plasma, such as greater than or equal to about 1500 copies/100 pl plasma, 2000 copies/100 pl plasma, 2500 copies/100 pl plasma, 3000 copies/100 pl plasma, 3500 copies/100 pl plasma, 4000 copies/100 pl plasma, or 4500 copies/100 pl plasma, or any value between any of the foregoing.
  • the EBV DNA load in a subject in need of a treatment as described herein can be increasing over time. EBV DNA load can be measured using techniques known in the art.
  • the subject is infected with an EBV strain encoding for a peptide variant that results in stable upregulation of HLA-E on the surface of immune cells, such as B cells.
  • Cell surface stabilization of HLA-E requires loading with peptides, which can be derived from the signal sequences of MHC class I molecules or other proteins such as HSP60 at steady state.
  • the subject described in the provided methods herein may be infected by a virus (e.g., EBV), wherein the virus may give rise to HLA-E stabilizing peptides.
  • the peptide is an LMP-1 peptide.
  • peptides can be detected by PCR-based methods, such as by gene amplification using nested PCR, followed by sequencing (see e.g., Mbiribindi et al. Scientific Reports, 10:19973, 2020 for detection of LMP-1 variants and Lorenzetti et al., Clin Microb Infec, 20:0861-0869, 2014 for detection of BZLF1 peptides).
  • the autoimmune disease or disorder is any autoimmune disease or disorder described herein in Section I.B or Section I.C. In some embodiments, the autoimmune disease or disorder is multiple sclerosis. In some embodiments, the autoimmune disease or disorder is associated with a viral infection. In some embodiments, the viral infection is an Epstein-Barr virus (EBV) infection. In some embodiments, the autoimmune disease or condition is one that is associated with EBV infection.
  • EBV Epstein-Barr virus
  • the EBV associated disease or condition is multiple sclerosis (MS).
  • a subject to be treated in accord with the provided methods is EBV seropositive.
  • the subject is infected with an EBV strain that results in high-level EBNA-specific antibody responses against specific EBNA-derived epitopes associated with ⁇ 1,400-fold increased risk for the development of MS (Vietzen et al., JCI, 2024).
  • Each specific EBNA-derived epitope is cross-reactive with specific epitopes in central nervous system (CNS)-derived proteins, thus resulting in autoreactive immune responses and the development of MS.
  • CNS central nervous system
  • the subject described in the provided methods herein may be infected by a virus (e,g, EBV), wherein the virus may give rise to the cells or immune cells reactive to epitopes set forth in Table 4.
  • a virus e.g., EBV
  • cells or immune cells reactive to epitopes set forth in Table 4 may be described herein as “autoreactive” or “CNS-autoreactive.”
  • the subject may be infected by a virus (e.g., EBV), wherein the virus may give rise to CNS-autoreactive cells.
  • the CNS-autoreactive cells are reactive to CNS proteins, such as epitopes from CNS proteins.
  • the CNS proteins include, but are not limited to, GlialCAM, CRYAB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to GlialCAM, CRYAB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to epitopes or sequences from CNS proteins, such as epitopes or sequences from GlialCAM, CRY AB, MBP, and/or ANO2.
  • the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 74-77. In some embodiments, the CNS-autoreactive cells are reactive to at least two epitopes set forth in any one of SEQ ID NOs: 74-77. In some embodiments, the CNS- autoreactive cells are reactive to at least three epitopes set forth in any one of SEQ ID NOs: 74-77. In some embodiments, the CNS-autoreactive cells are reactive to each epitope set forth in SEQ ID NOs: 74- 77.
  • the CNS-autoreactive cells are reactive to at least one epitope set forth in any one of SEQ ID NOs: 78-81. In some embodiments, the CNS-autoreactive cells are reactive to at least two epitopes set forth in any one of SEQ ID NOs: 78-81. In some embodiments, the CNS- autoreactive cells are reactive to at least three epitopes set forth in any one of SEQ ID NOs: 78-81. In some embodiments, the CNS-autoreactive cells are reactive to each epitope set forth in SEQ ID NOs: 78- 81.
  • the CNS-autoreactive cells are B and/or T cells. In some embodiments, the CNS-autoreactive cells are B cells. In some embodiments, the CNS-autoreactive cells are T cells. In some embodiments, the T cells are CD8+ T cells and CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells.
  • the subject to be treated in accord with the provided methods is selected for the presence of CNS-autoreactive cells.
  • the CNS-autoreactive cells are B cells, CD4+ cells, and CD8+ T cells.
  • the CNS-autoreactive cells are reactive to at least three epitopes set forth in any one of SEQ ID NOs: 74-77 or at least three epitopes set forth in any one of SEQ ID NOs: 87-81.
  • how reactive CNS-autoreactive immune cells are can be detected by quantifying peptide-specific IgG antibodies using ELISA, as previously described in Vietzen et al., J CI, 2024.
  • the subject to be treated in accord with the provided methods is selected for the presence of demyelinating-appearing lesions, even in the absence of clinical symptoms present in an autoimmune disease or disorder.
  • Radiologically isolated syndrome is characterized by the presence of demyelinating-appearing lesions visible on MRI of the central nervous system (CNS), such as brain or spinal cord, suggestive of MS but prior to the presence of any clinical symptoms of MS being present (Lebrun-Frenay et al., Lancet Neurol., 2023).
  • CNS central nervous system
  • RIS is suggested to be a pre-symptomatic stage of MS.
  • subjects with RIS are likely or suspected to develop an autoimmune disease or disorder, specifically MS.
  • a subject to be treated in accord with the provided method has or is selected for having RIS.
  • the subject has asymptomatic white matter lesions in the CNS present on MRI.
  • the white matter lesions are ovoid, well circumscribed, at least 3 mm in length, and hyperintense on T2-weighted images with or without the involvement of the corpus callosum.
  • the subject selected for treatment in accord with the provided methods has RIS comprising white matter lesions, wherein the white matter lesions meet at least one, at least two, at least three, or all the following criteria: (a) one gadolinium-enhancing lesion or nine T2- hyperintense lesions; (b) one or more infratentorial lesion; (c) one or more juxtacortical lesion; and (d) three or more periventricular lesions. In some embodiments, the white matter lesions meet at least three of the aforementioned criteria.
  • the subject to be treated in accord with the provided methods has or is selected for the presence of demyelinating-appearing lesions, but has not yet fulfilled the criteria for diagnosis of multiple sclerosis (MS).
  • MS multiple sclerosis
  • Subjects with Clinically Isolated Syndrome (CIS) have experienced a first clinical episode of a disease that shows characteristics of inflammatory demyelination known in MS but cannot be diagnosed with MS due to not fulfilling the diagnostic criteria of dissemination in time (Lubin et al., Neurology, 2014). As CIS is thought to be the first sign of MS in approximately 85% of cases, subjects with CIS are likely or suspected to develop an autoimmune disease or disorder, specifically MS.
  • a subject to be treated in accord with the provided method has or is selected for having CIS.
  • 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 is likely or suspected to develop an autoimmune disease or disorder, e.g., multiple sclerosis.
  • the provided methods do not include a chemotherapeutic agent or cytotoxic agent (e.g., lymphodepleting therapy) for prophylactic treatment.
  • the provided methods include a chemotherapeutic agent or cytotoxic agent (e.g., lymphodepleting therapy), such as any described in Section I.H, for prophylactic treatment.
  • a single dose of g-NK cells is administered to the subject.
  • multiple doses of g-NK cells are administered to the subject in a predetermined number of doses.
  • the composition of g-NK cells is administered as a plurality of doses.
  • the doses of the plurality are for a predetermined number of doses.
  • the g-NK cells are administered once a week, twice a week, three times a week, once every two weeks, once every three weeks or once a month.
  • the g-NK cells are administered once a week.
  • the g-NK cells are administered once every 1 day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days. In some embodiments, the g-NK cells are administered once every 2 days. In some embodiments, the g-NK cells are administered once every 7 days. In some embodiments, the number of doses of the g- NK cells is two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, 9 doses, 10 doses, 11 doses or 12 doses. In some embodiments, the number of doses is 2 doses of g-NK cells. In some embodiments, the number of doses is 3 doses of g-NK cells.
  • the number of doses is 4 doses of g-NK cells. In some embodiments, the number of doses is 5 doses. In some embodiments, the number of doses is 6 doses. In some embodiments, the number of doses is 7 doses. In some embodiments, the number of doses is 8 doses. In some embodiments, the number of doses is 9 doses. In some embodiments, all doses of the g-NK cells are administered within a month of the first dose.
  • a dose of g-NK cells is administered once weekly (Q1W or QW). In some embodiments, a dose of g-NK cells is administered twice weekly. In some embodiments, a dose of g-NK cells is administered three times weekly (or thrice weekly), which can be administered every other day (Q2D).
  • a dose of g-NK cells is dosed at a frequency of every two days (i.e., every other day, Q2D) for a predetermined number of doses.
  • the number of doses is 3 doses in a cycle, which may be repeated.
  • a dose of g-NK cells is administered on Day 0 (first dose), Day 2 and Day 4.
  • the cycle is a 7-day cycle.
  • the cycle is repeated at least one time.
  • a second dose of g-NK cells is administered at or about at 24 hours after a first dose of g-NK cells.
  • a third dose of g-NK cells is administered at or about at 24 hours after a second dose of g-NK cells.
  • the doses of g-NK cells are administered as part of a 7-day cycle.
  • the 7-day cycle is repeated one to three times.
  • the 7-day cycle is repeated one time (for a total of two 7-day cycles).
  • doses of the composition of g-NK cells are administered as two doses in a 7-day cycle. In some embodiments, doses of the composition of g-NK cells are administered as three doses in a 7-day cycle.
  • the composition of g-NK cells is administered from two total doses to six total doses. In some embodiments, the composition of g-NK cells is administered as two or four total doses. In some embodiments, the composition of g-NK cells is administered as three or six total doses.
  • the g-NK cells are administered once weekly. In some embodiments, the number of once weekly doses is two doses. In some embodiments, the number of once weekly doses is three doses. In some embodiments, the number of once weekly doses is four doses. In some embodiments, the once weekly doses are administered in consecutive weeks. For example, the g-NK cells may be administered in a cycling regimen involving more than one 7-day cycle carried out consecutively, each with once weekly administration of the g-NK cells. In some embodiments, the number of consecutive weeks (or consecutive 7-days cycles) is 2, 3, 4 or 5. In some embodiments, the g- NK cells are administered on Day 0 (first dose), Day 7 and Day 14.
  • the g-NK cells are administered on Day 0 (first dose), Day 7, Day 14 and Day 21. In some embodiments, the g-NK cells are administered on Day 0 (first dose), Day 7, Day 14, Day 21 and Day 28. In some embodiments, the g- NK cells are administered on Day 0 (first dose), Day 7, Day 14, Day 21, Day 28 and Day 35. [0439] In some embodiments, the g-NK cells are administered twice a week (twice weekly). In some embodiments, the predetermined number of twice weekly doses is two doses. In some embodiments, the predetermined number of twice weekly doses is four doses. In some embodiments, the twice weekly doses are administered for 1 week, 2 weeks, 3 weeks, 4 weeks or more.
  • one (1) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, two (2) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, three (3) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, four (4) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, the twice weekly doses are administered in consecutive weeks.
  • the g-NK cells are administered three times a week (thrice weekly). In some embodiments, the g-NK cells are administered every other day (Q2D), such as on Day 0 (first dose), Day 2 and Day 4 of a week (such as a 7-day cycle). In some embodiments, the predetermined number of thrice weekly doses is three doses. In some embodiments, the predetermined number of thrice weekly doses is six doses. In some embodiments, the thrice weekly doses, such as administered Q2D, are administered in consecutive weeks.
  • the g-NK cells may be administered in a cycling regimen involving more than one 7-day cycle carried out consecutively, each with thrice weekly, such as Q2D, administration of the g-NK cells.
  • the thrice weekly doses are administered for 1 week, 2 weeks, 3 weeks, 4 weeks or more.
  • one (1) thrice weekly dose of the composition of g-NK cells is administered (e.g bland Day 0, Day 2 and Day 4 of the first week).
  • two (2) thrice weekly dose of the composition of g-NK cells is administered (e.gnton Day 0, Day 2 and Day 4 of the first week and Day 0, Day 2 and Day 4 of the second week).
  • three (3) thrice weekly dose of the composition of g-NK cells is administered (e.gnati Day 0, Day 2 and Day 4 of the first week; Day 0, Day 2 and Day 4 of the second week; Day 0, Day 2 and Day 4 of the third week).
  • four (4) thrice weekly dose of the composition of g-NK cells is administered (e.gnati Day 0, Day 2 and Day 4 of the first week; Day 0, Day 2 and Day 4 of the second week; Day 0, Day 2 and Day 4 of the third week; and Day 0, Day 2 and Day 4 of the fourth week).
  • the thrice weekly doses are administered in consecutive weeks.
  • the twice weekly dose is administered in a cycling regimen.
  • the cycling regimen is a 7 day cycle.
  • the twice weekly dose is administered two times in the 7 day cycle.
  • the 7 day cycle is repeated twice.
  • the 7 day cycle is repeated three times.
  • the cycling regimen is a 14 day cycle.
  • the twice weekly dose is administered four times in the 14 day cycle.
  • the 14 day cycle is repeated twice.
  • the 14 day cycle is repeated three times.
  • the thrice weekly dose is administered in a cycling regimen.
  • the cycling regimen is a 7 day cycle.
  • the thrice weekly dose is administered three times in the 7 day cycle. In some embodiments, the 7 day cycle is repeated twice. In some embodiments, the 7 day cycle is repeated three times. In some embodiments, the cycling regimen is a 14 day cycle. In some embodiments, the thrice weekly dose is administered six times in the 14 day cycle. In some embodiments, the 14 day cycle is repeated twice. In some embodiments, the 14 day cycle is repeated three times.
  • the methods provided herein result in clinical improvement or clinical remission of the autoimmune disease or disorder in the subject.
  • administration of g-NK cells is repeated multiple times.
  • administration of g-NK cells is repeated at a time when the autoimmune disease or disorder reoccurs, such as after remission or after the disease or disorder becomes more severe after evidence of improvement following a prior administration of g-NK cells.
  • administration of g-NK cells does not include combination therapy, such as any described herein in Section I.F, and/or does not include lymphodepleting therapy, such as any described herein in Section I.H.
  • administration of g-NK cells, such as a cycling regimen of g-NK cells further includes combination therapy, such as any described herein in Section I.F, administration of cytokines, such as any described herein in Section I.G, and does not include lymphodepleting therapy, such as any described herein in Section I.H.
  • administration of g-NK cells such as a cycling regimen of g-NK cells, further includes combination therapy, such as any described herein in Section I.F, administration of cytokines, such as any described herein in Section I.G, and lymphodepleting therapy, such as any described herein in Section I.H.
  • administration of g-NK cells, such as a cycling regimen of g-NK cells does not include combination therapy, such as any described herein in Section I.F, or lymphodepleting therapy, such as ny described herein in Section I.H, and further includes administration of cytokines, such as any described herein in Section I.G.
  • administration of g-NK cells does not include combination therapy, such as any described herein in Section I.F, and further includes administration of cytokines, such as any described herein in Section I.G, and lymphodepleting therapy, such as any described herein in Section I.H.
  • administration of g-NK cells such as a cycling regimen of g-NK cells, further includes combination therapy, such as any described herein in Section I.F, administration of cytokines, such as any described herein in Section LG, or lymphodepleting therapy, such as any described herein in Section I.H.
  • g-NK cells may be administered once a year (e.g., annually), once every two years, once every three years, once every four years, or once every five years.
  • regular administration of g-NK cells may reduce the risk of relapse to improve duration of remission and/or severity of the symptoms of the autoimmune disease or disorder.
  • g-NK cells may be administered in the absence of symptoms or prior to symptoms of the autoimmune disease or disorder. In some embodiments, g-NK cells may be administered following the reappearance of antibodies against any of the epitopes set forth in Table 4 or autoreactive cells, including autoreactive cells reactive to any one of the epitopes set forth in Table 4. In some embodiments, g-NK cells may be administered following the recurrence of the autoimmune disease or disorder.
  • the methods of treatment or uses involve administration of an effective amount of a composition containing a composition of expanded NK cells produced by the provided method to an individual.
  • a composition containing a composition of expanded NK cells produced by the provided method to an individual.
  • from at or about 10 5 to at about 10 12 , or from at or about 10 5 and at or about 10 8 , or from at or about 10 6 and at or about 10 12 , or from at or about 10 8 and at or about 10” or from at or about 10 9 and at or about 10 10 of such expanded NK cells is administered to an individual subject.
  • a dose of cells containing at or greater than at or about 10 5 , at or greater than at or about 10 6 , at or greater than at or about 10 7 , at or greater than at or about 10 8 , at or greater than at or about 10 9 , at or greater than at or about IO 10 , at or greater than at or about 10”, or at or greater than at or about 10 12 of such expanded NK cells are administered to the individual.
  • from or from about 10 6 to 10 10 of such expanded NK cells per kg are administered to the subject.
  • the methods of treatment or uses involve administration of an effective amount of any of the provided NK cell compositions, including any as described herein, to an individual.
  • an effective amount of any of the provided NK cell compositions including any as described herein, to an individual.
  • from at or about 10 5 to at about 10 12 , or from at or about 10 5 and at or about 10 8 , or from at or about 10 6 and at or about 10 12 , or from at or about 10 8 and at or about 10” or from at or about 10 9 and at or about 10 10 of NK cells from any of the provided compositions is administered to an individual subject.
  • a dose of cells containing at or greater than at or about 10 5 , at or greater than at or about 10 6 , at or greater than at or about 10 7 , at or greater than at or about 10 8 , at or greater than at or about 10 9 , at or greater than at or about 10 10 , at or greater than at or about 10”, or at or greater than at or about 10 12 of NK cells from any of the provided compositions are administered to the individual.
  • from or from about 10 6 to 10 10 of NK cells of any of the provided compositions per kg are administered to the subject.
  • the composition of g-NK cells may be administered as at least one dose of the composition of g-NK cells.
  • the autoreactive cells may be contacted with at least one dose of the composition of g-NK cells.
  • each dose of g-NK cells may be from at or about from at or about 1 x
  • each dose of g-NK cells may be from at or about from at or about 1 x 10 8 cells to at or about 20 x 10 9 cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be from at or about from at or about 1 x 10 9 cells to at or about 20 x 10 9 cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be from at or about from at or about 5 x 10 9 cells to at or about 20 x 10 9 cells of the composition of g-NK cells.
  • the methods of treatment comprises administering an effective amount of a composition containing g-NK cells to an individual.
  • a composition containing g-NK cells comprises administering an effective amount of a composition containing g-NK cells to an individual.
  • a dose of cells containing at or greater than at or about 10 5 g-NK cells, at or greater than at or about 10 6 g-NK cells, at or greater than at or about 10 7 g- NK cells, at or greater than at or about 10 8 g-NK cells, at or greater than at or about 10 9 g-NK cells, at or greater than at or about 10 10 g-NK cells, at or greater than at or about 10” g-NK cells, or at or greater than at or about 10 12 g-NK cells are administered to the individual. In some embodiments, from or from about 10 6 to 10’°g-NK cells /kg are administered to the subject.
  • expansion achieved by the provided methods from an initial source of NK cells obtained from a single donor can produce a composition of g-NK cells to provide a plurality of individual doses for administration to a subject in need.
  • the provided methods are particularly suitable for allogeneic methods.
  • a single expansion from a starting population of NK cells isolated from one donor in accord with the provided methods can result in greater than or greater than about 20 individual doses for administration to a subject in need, such as at or about 30 individual doses, 40 individual doses, 50 individual doses, 60 individual doses, 70 individual doses, 80 individual doses, 90 individual doses, 100 individual doses, or an individual dose that is a value between any of the foregoing.
  • the individual dose is from at or about 1 x 10 5 cells/kg to at or about 1 x 10 7 cells/kg, such as from at or about 1 x 10 5 cells/kg to at or about 7.5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 2.5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 1 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 7.5 x 10 5 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 5 x 10 5 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 2.5 x 10 5 cells/kg, from at or about 2.5 x 10 5 cells/kg to at or about 1 x 10 7 cells/kg, from at or about 2.5 x 10 5 cells/kg to at or about x 10 7 cells
  • the individual dose is from at or about 1 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, such as from at or about 2.5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 7.5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 1 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 2.5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 7.5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 1 x 10 7 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 2.5 x 10 7 cells/kg to at or about 1 x 10 8
  • the individual dose is from at or about 5 x 10 7 to at or about 10 x 10 9 , such as from at or about 5 x 10 7 to at or about 5 x 10 9 , from about or about 5 x 10 7 to at or about 1 x 10 9 , from at or about 5 x 10 7 to at or about 5 x 10 8 , from about or about 5 x 10 7 to at or about 1 x 10 8 , 1 x 10 8 to at or about 10 x 10 9 , from at or about 1 x 10 8 to at or about 5 x 10 9 , from about or about 1 x 10 8 to at or about 1 x 10 9 , from at or about 1 x 10 8 to at or about 5 x 10 8 , from at or about 5 x 10 8 to at or about 10 x 10 9 , from at or about 5 x 10 8 to at or about 10 x 10 9 , from at or about 5 x 10 8 to at or about 10 x 10 9 , from at or about 5 x 10 8 to at or
  • the individual dose is or is about 5 x 10 8 cells. In some embodiments, the individual dose is or is about 1 x 10 9 cells. In some embodiments, the individual dose is or is about 5 x 10 9 cells. In some embodiments, the individual dose is or is about 1 x 10 10 cells. In any of the above embodiments, the dose is given as the number of cells g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described above, or a number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of expanded cells produced by the method, or a number of viable cells of any of the foregoing.
  • the dose for administration in accord with any of the provided methods of treatment or uses is from at or about 1 x 10 5 cells/kg to at or about 1 x 10 7 cells/kg, such as from at or about 1 x 10 5 cells/kg to at or about 7.5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 2.5 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 1 x 10 6 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 7.5 x 10 5 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 5 x 10 5 cells/kg, from at or about 1 x 10 5 cells/kg to at or about 2.5 x 10 5 cells/kg, from at or about 2.5 x 10 5 cells/kg to at or about 1 x 10 7 cells/kg,
  • the dose for administration is from at or about 1 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, such as from at or about 2.5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 7.5 x 10 5 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 1 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 2.5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 7.5 x 10 6 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 1 x 10 7 cells/kg to at or about 1 x 10 8 cells/kg, from at or about 2.5 x 10 7 cells/kg to at or about
  • the dose is given as the number of g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or a number of viable cells of any of the foregoing.
  • the dose is given as the number of cells in a composition of expanded cells produced by the provided method, or a number of viable cells of any of the foregoing.
  • the dose for administration is at or about 5 x 10 8 cells. In some embodiments, the dose for administration is at or about 1 x 10 9 cells. In some embodiments, the dose for administration is at or about 5 x 10 9 cells. In some embodiments, the dose for administration is at or about 1 x 10 10 cells. In some embodiments, the dose is given as the number of g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or a number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of expanded cells produced by the provided method, or a number of viable cells of any of the foregoing.
  • administration of at least one dose of anti-CD20 antibody is initiated about one day prior to the first administration of the composition of g-NK cells.
  • administration of the first dose of anti-CD20 antibody (e.g., ocrelizumab) of the combination therapy is initiated at or about one day prior to the first administration of the composition of g-NK cells.
  • the g-NK cells that is administered in combination with anti-CD20 antibody is administered every other day (Q2D).
  • the g-NK cell composition is administered from two to six total doses.
  • the g-NK cell composition is administered in two total doses.
  • the g-NK cell composition is administered in three total doses.
  • the g-NK cell composition is administered in four total doses.
  • the g-NK cell composition is administered in five total doses.
  • the g-NK cell composition is administered in six total doses.
  • the g-NK cell composition is administered on Days 0, 2, and 4, and anti-CD20 antibody (e.g., ocrelizumab) is administered on Days -1 and at 6 months.
  • ocrelizumab is used for treating a subject with multiple sclerosis. In some embodiments, ocrelizumab is used in combination with a cell composition including g-NK cells for treating a subject with multiple sclerosis. In some embodiments, ocrelizumab is used for beating a subject with relapsing multiple sclerosis. In some embodiments, ocrelizumab is used in combination with a cell composition including g-NK cells for treating a subject with relapsing multiple sclerosis. In some embodiments, ocrelizumab is used for treating a subject with primary progressive multiple sclerosis. In some embodiments, ocrelizumab is used in combination with a cell composition including g-NK cells for treating a subject with primary progressive multiple sclerosis.
  • ocrelizumab used in combination with a cell composition including g- NK cells is administered as a once every 6 months (e.g., every 24 weeks) dose. In some embodiments, ocrelizumab is administered once every 6 months for at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. In some embodiments, ocrelizumab is administered for at least 30 months (e.g., at least 120 weeks). In some embodiments, each dose of ocrelizumab used in combination with a cell composition including g-NK cells is administered at a dose of about 100-1000 mg. In some embodiments, each dose of ocrelizumab is administered at a dose of about 600 mg. In some embodiments, ocrelizumab is administered intravenously (IV).
  • IV intravenously
  • the first dose of ocrelizumab is divided into two doses of 300 mg dosed two weeks apart.
  • the g-NK cell composition is administered on Days 0, 2, and 4, and ocrelizumab is administered at a dose of 300 mg on Days -14 and 0.
  • the first dose of ocrelizumab is divided into two doses of 600 mg dosed six months apart.
  • the g-NK cell composition is administered on Days 0, 2, and 4, and ocrelizumab is administered at a dose of 600 mg on Day -1 and around 6 months after Day - 1.
  • the g-NK cell composition is administered on Days 0, 2, and 4, and ocrelizumab is administered at a dose of 600 mg on Day -6 and around 6 months after Day -6.
  • the anti-CD20 antibody is rituximab.
  • rituximab is used for beating a subject with multiple sclerosis.
  • rituximab is used in combination with a cell composition including g-NK cells for treating a subject with multiple sclerosis.
  • rituximab is used for treating a subject with relapse/remitting multiple sclerosis (Hauser et al., N Engl J Med (2008)). In some embodiments, rituximab is used in combination with a cell composition including g-NK cells for heating a subject with relapse/remitting multiple sclerosis.
  • rituximab used in combination with a cell composition including g- NK cells is administered once every 2 weeks.
  • each dose of rituximab used in combination with a cell composition including g-NK cells is administered at a dose of about 200-2000 mg.
  • each dose of rituximab is administered at a dose of about 1000 mg.
  • the g-NK cell composition is administered on Days 0, 2, and 4, and rituximab is administered at a dose of 1000 mg on Days 1 and 15.
  • the claimed g-NK cell therapy is able to not only deeply deplete CD20 producing B cells and T cells but also deplete autoreactive T cells and B cells; as well as eliminate the latent viral reservoir.
  • the antibody is an anti-CD19 antibody. Any anti-CD19 antibody with means for binding CD 19 and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, inebilizumab (Uplizna®; also called MEDI-551), tafasitamab (tafasitamab- cxix; Monjuvi®; also called MOR208 or XmAb 5574), obexelimab (also known as XmAb 5871), MDX- 1342, DI-B4 or LY3541860. In some embodiments, the antibody is inebilizumab, tafasitamab, or obexelimab.
  • inebilizumab Uplizna®; also called MEDI-551
  • tafasitamab tafasitamab- cxix
  • Monjuvi® also called MOR208 or XmAb 5574
  • the subject is a subject with an autoimmune kidney disease.
  • the anti-CD19 antibody is used for treating a subject with autoimmune kidney disease.
  • the anti-CD19 antibody is used in combination with a cell composition including g- NK cells for heating a subject with autoimmune kidney disease.
  • the anti-CD19 antibody is inebilizumab.
  • inebilizumab is used for treating a subject with autoimmune kidney disease.
  • inebilizumab is used in combination with a cell composition including g-NK cells for treating a subject with autoimmune kidney disease.
  • the anti-CD19 antibody is tafasitamab.
  • tafasitamab is used for beating a subject with autoimmune kidney disease.
  • tafasitamab is used in combination with a cell composition including g-NK cells for treating a subject with autoimmune kidney disease.
  • autoimmune kidney disease is systemic lupus erythematosus (SLE), lupus nephritis, primary membranous nephropathy (PMN), or immunoglobulin A (IgA) nephropathy (IgAN).
  • SLE systemic lupus erythematosus
  • PMN primary membranous nephropathy
  • IgA immunoglobulin A nephropathy
  • tafasitamab is used for heating a subject with systemic lupus erythematosus (SLE).
  • tafasitamab is used in combination with a cell composition including g-NK cells for treating a subject with systemic lupus erythematosus (SLE).
  • the anti-CD19 antibody is used in combination with a cell composition including g-NK cells for treating a subject with primary membranous nephropathy (PMN).
  • PMN primary membranous nephropathy
  • tafasitamab is used for heating a subject with primary membranous nephropathy (PMN).
  • PMN primary membranous nephropathy
  • tafasitamab is used in combination with a cell composition including g-NK cells for treating a subject with primary membranous nephropathy (PMN).
  • inebilizumab is used for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • inebilizumab is used in combination with a cell composition including g-NK cells for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • the anti-CD38 antibody is isatuximab.
  • isatuximab is used for heating a subject with autoimmune kidney disease.
  • isatuximab is used in combination with a cell composition including g-NK cells for treating a subject with autoimmune kidney disease.
  • the anti-CD38 antibody is used in combination with a cell composition including g-NK cells for treating a subject with systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • isatuximab is used for treating a subject with systemic lupus erythematosus (SLE).
  • isatuximab is used in combination with a cell composition including g-NK cells for treating a subject with systemic lupus erythematosus (SLE).
  • the anti-CD38 antibody is used in combination with a cell composition including g-NK cells for treating a subject with primary membranous nephropathy (PMN).
  • PMN primary membranous nephropathy
  • the claimed g-NK cells can be used in combination with, for example a CD38 antibody, which has been show by research groups to deplete B cells in PMN. Rocatello et al., Nature Medicine (2023) 29:2041- 2047.
  • daratumumab is used for treating a subject with primary membranous nephropathy (PMN). In some embodiments, daratumumab is used in combination with a cell composition including g-NK cells for treating a subject with primary membranous nephropathy (PMN). [0518] In particular embodiments, isatuximab is used for treating a subject with primary membranous nephropathy (PMN). In some embodiments, isatuximab is used in combination with a cell composition including g-NK cells for treating a subject with primary membranous nephropathy (PMN).
  • the anti-CD38 antibody is used in combination with a cell composition including g-NK cells for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • IgA immunoglobulin A
  • IgAN immunoglobulin A nephropathy
  • daratumumab is used for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • daratumumab is used in combination with a cell composition including g-NK cells for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • isatuximab is used for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • isatuximab is used in combination with a cell composition including g-NK cells for treating a subject with immunoglobulin A (IgA) nephropathy (IgAN).
  • the antibody is an anti-BAFF-R antibody.
  • Any anti-BAFF-R antibody with means for binding BAFF-R and engaging CD16 via the Fc domain can be used.
  • Exemplary antibodies include, but are not limited to, belimumab and ianalumab (VAY-736).
  • the antibody is an anti-TACI antibody. Any anti-TACI antibody with means for binding TACI and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, GenSci-X002.
  • the antibody is an anti-BCMA antibody.
  • Any anti-BCMA antibody with means for binding BCMA and engaging CD 16 via the Fc domain can be used.
  • Exemplary antibodies include, but are not limited to, belantamab.
  • the subject is administered a population of g-NK cells described herein and an effective dose of an additional agent.
  • the additional agent is a bispecific antibody.
  • the subject is administered a population of g-NK cells described herein and an effective dose of a bispecific antibody.
  • the bispecific antibody comprises a first binding domain and a second binding domain, the first binding domain specifically binding to a surface antigen on an NK cell.
  • the first binding domain specifically binds to an activating receptor, for instance CD16 (CD16a), on an NK cell.
  • the second binding domain specifically binds to a B cell antigen.
  • the first binding domain specifically binds to an activating receptor, for instance CD16 (CD16a), on an NK cell. In some embodiments, the first domain specifically binds to NKG2D. In some embodiments, the second binding domain specifically binds to any target antigen expressed by cells that express HLA-E. In some embodiments, the second binding domain specifically binds to a T cell antigen. In some embodiments, the second binding domain specifically binds to a B cell antigen. In certain embodiments, the B cell antigen is BCMA. In some embodiments, the second binding domain specifically targets a B cell, for instance, by targeting BCMA. In any of the preceding embodiments, any bispecific NK cell engager (BiKE) with means for targeting a B cell and engaging CD 16 via the Fc domain can be used.
  • BiKE bispecific NK cell engager
  • the g-NK cells can be administered to an individual in combination with cytokines and/or growth factors.
  • the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL- 15, IL-12, IL-21, and IL-27.
  • the cytokines and/or growth factors are the cytokine IL-2.
  • recombinant IL-2 is administered to the subject.
  • recombinant IL-15 is administered to the subject.
  • recombinant IL-21 is administered to the subject.
  • the g-NK cells and the cytokines or growth factors are administered sequentially.
  • the g-NK cells may be administered first, followed by administration of the cytokines and/or growth factors.
  • the g-NK cells are administered simultaneously with the cytokines or growth factors.
  • the cytokines and/or growth factors are administered first, followed by administration of the g-NK cells.
  • the subject is administered one or more cytokines (such as IL-2, IL- 15, IL-21, IL-27, and/or IL- 12) to support survival and/or growth of NK cells.
  • the administered cytokines are recombinant cytokines that are administered to the subject exogenously.
  • the cytokine(s) can be administered before, after, or substantially simultaneously with the NK cells.
  • the cytokine(s) can be administered after the NK cells.
  • the cytokine(s) is administered to the subject within about 1-8 hours (such as within about 1-4 hours, about 2-6 hours, about 4-6 hours, or about 5-8 hours) of the administration of the NK cells.
  • IL-2 is administered to subjects in accord with provided methods.
  • the dosing of IL-2 in conjunction with adoptive NK cell therapy supports in vivo expansion and augments NK cell persistence.
  • the administered IL-2 is the recombinant IL-2 aldesleukin (also known as Proleukin), which is a recombinant form of human IL-2.
  • the N-terminal alanine is deleted and the sequence contains a cysteine substituted with serine at amino acid position 125 (C125S mutation) to prevent cysteine mispairing in E. coli, but this does not affect biological activity.
  • the doses of IL-2 in provided methods can typically be much lower than approved doses (e.g., approved dose of aldesleukin is 100 million lU/day).
  • the lower dose can be efficacious for NK cell activity while also reducing or eliminating the SAEs of CRS, which is the most significant safety issue with IL-2 therapy.
  • 6 million (M) IU dose of IL-2 is administered every other day (Q2D).
  • the cytokine (e.g., IL-2) is administered once every three days. In some embodiments, the cytokine (e.g., IL-2) is administered once a week. In some embodiments, the cytokine (e.g., IL-2) is administered at the same frequency as the dose of g-NK cells. In some embodiments, the cytokine (e.g., IL-2) is administered on the same day as the g-NK cells, such as typically within 12 hours of administering the g-NK cells.
  • the cytokine (e.g., IL-2) is administered 0-6 hours prior to administering each dose of the g-NK cells, such as 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour, 1-4 hours, 1-3 hours or 1-2 hours prior to administering the g-NK cells. In some embodiments, the cytokine (e.g., IL-2) is administered about or within 1 hour prior to administering each dose of the g-NK cells.
  • the cytokine is administered in a cycling regimen involving administering a dose of the cytokine one or more times in a cycle, in which the cycle may be optionally repeated.
  • the cycle is a 7 day cycle.
  • the cycle is a 14 days cycle.
  • the cycle is a 21 day cycle.
  • the cycle is a 28 day cycle.
  • a dose of the cytokine is administered one or more times in a first cycle, which is then repeated one or two times (for a total of 2 or 3 cycling regimens).
  • each dose of one or more cytokines is between at or about 2 million IU and at or about 20 million IU, between at or about 3 million IU and at or about 20 million IU, between at or about 4 million IU and at or about 20 million IU, between at or about 5 million IU and at or about 20 million IU, or between at or about 6 million IU and at or about 20 million IU.
  • each dose of the one or more cytokines is between at or about 0.5 million IU and at or about 12 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 1 million IU and at or about 12 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 0.25 million IU and at or about 6 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 0.5 million IU and at or about 6 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 0.25 million IU and at or about 3 million IU.
  • each dose of the one or more cytokines is between at or about 0.5 million IU and at or about 3 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 0.25 million IU and at or about 2 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 0.5 million IU and at or about 2 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 5 million IU and at or about 10 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 4 million IU and at or about 8 million IU.
  • each dose of the one or more cytokines is at or about 0.25 million IU, is at or about 0.5 million IU, is at or about 1 million IU, is at or about 1.5 million IU, is at or about 2 million IU, is at or about 2.5 million IU, is at or about 3 million IU, is at or about 3.5 million IU, is at or about 4 million IU, is at or about 4.5 million IU, is at or about 5 million IU, is at or about 5.5 million IU, is at or about 6 million IU, is at or about 6.5 million IU, is at or about 7 million IU, is at or about 7.5 million IU, is at or about 8 million IU, is at or about 8.5 million IU, is at or about 9 million IU, is at or about 9.5 million IU, is at or about 10 million IU, is at or about 10.5 million IU, is at or about 11 million IU, is at or about 11.5 million IU, is at or about 12 million IU
  • each dose of the one or more cytokines is at or about 1 million IU. In some embodiments, each dose of the one or more cytokines is at or about 5 million IU. In some embodiments, each dose of the one or more cytokines is at or about 6 million IU. In some embodiments, each dose of the one or more cytokines is at or about 10 million IU.
  • the one or more cytokines is IL-2, IL- 15, IL-21, IL-27, and/or IL-12. In some embodiments, the one or more cytokines is IL-2.
  • the subject is administered IL -2 once a week, two times a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In some embodiments, the subject is administered IL-2 once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.
  • each dose of IL-2 is between at or about 1 million IU and at or about 50 million IU, such as between at or about 1 million IU and at or about 25 million IU, between at or about 1 million IU and at or about 20 million IU, between at or about 1 million IU and at or about 15 million IU, between at or about 1 million IU and at or about 14 million IU, between at or about 1 million IU and at or about 13 million IU, between at or about 1 million IU and at or about 12 million IU, between at or about 1 million IU and at or about 11 million IU, between at or about 1 million IU and at or about 10 million IU.
  • each dose of IL-2 is between at or about 2 million IU and at or about 20 million IU, between at or about 3 million IU and at or about 20 million IU, between at or about 4 million IU and at or about 20 million IU, between at or about 5 million IU and at or about 20 million IU, or between at or about 6 million IU and at or about 20 million IU.
  • each dose of IL-2 is between at or about 5 million IU and at or about 12 million IU, between at or about 5 million IU and at or about 11 million IU, between at or about 5 million IU and at or about 10 million IU, between at or about 5 million IU and at or about 9 million IU, between at or about 5 million IU and at or about 8 million IU, between at or about 5 million IU and at or about 7 million IU, between at or about 5 million IU and at or about 6 million IU, or between at or about 6 million IU and at or about 7 million IU. In some embodiments, each dose of IL-2 is between at or about 0.25 million IU and at or about 12 million IU.
  • each dose of IL-2 is between at or about 0.5 million IU and at or about 12 million IU. In some embodiments, each dose of IL-2 is between at or about 1 million IU and at or about 12 million IU. In some embodiments, each dose of IL-2 is between at or about 0.25 million IU and at or about 6 million IU. In some embodiments, each dose of IL-2 is between at or about 0.5 million IU and at or about 6 million IU. In some embodiments, each dose of IL-2 is between at or about 0.25 million IU and at or about 3 million IU. In some embodiments, each dose of IL-2 is between at or about 0.5 million IU and at or about 3 million IU.
  • each dose of IL-2 is between at or about 0.25 million IU and at or about 2 million IU. In some embodiments, each dose of IL-2 is between at or about 0.5 million IU and at or about 2 million IU. In some embodiments, each dose of IL-2 is between at or about 5 million IU and at or about 10 million IU. In some embodiments, each dose of IL-2 is between at or about 4 million IU and at or about 8 million IU.
  • each dose of IL-2 is at or about 0.25 million IU, is at or about 0.5 million IU, is at or about 1 million IU, is at or about 1.5 million IU, is at or about 2 million IU, is at or about 2.5 million IU, is at or about 3 million IU, is at or about 3.5 million IU, is at or about 4 million IU, is at or about 4.5 million IU, is at or about 5 million IU, is at or about 5.5 million IU, is at or about 6 million IU, is at or about 6.5 million IU, is at or about 7 million IU, is at or about 7.5 million IU, is at or about 8 million IU, is at or about 8.5 million IU, is at or about 9 million IU, is at or about 9.5 million IU, is at or about 10 million IU, is at or about 10.5 million IU, is at or about 11 million IU, is at or about 11.5 million IU, is at or about 12 million IU, is at or about
  • each dose of IL-2 is at or about 1 million IU. In some embodiments, each dose of IL-2 is at or about 5 million IU. In some embodiments, each dose of IL- 2 is at or about 6 million IU. In some embodiments, each dose of IL-2 is at or about 10 million IU.
  • the IL-2 is administered before the administration of the g-NK cells. In some embodiments, the IL-2 is administered within about one hour of the administration of the g-NK cells. In some embodiments, each dose of IL-2 is about 6 million IU. [0541] In some embodiments provided herein, the IL-2 may be administered in a cycling regimen.
  • the cycling regimen is a 7 day cycle.
  • the IL-2 is administered as three doses in a 7-day cycle.
  • the IL-2 can be administered on consecutive days of the 7-day cycle.
  • the IL-2 can be administered only one time each day it is administered in the cycle.
  • the IL-2 is administered one time daily every other day (i.e., Q2D) in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4.
  • the IL-2 is administered one time daily once a week (e.g., QW), in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells).
  • the 7-day cycle is repeated twice.
  • the 7-day cycle is repeated three times.
  • the IL-2 is administered one time daily every day (i.e., QD) in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 1, day 2, day 3, day 4, day 5, and day 6.
  • the IL-2 is administered one time daily every weekday (e.g., Q2D). In some embodiments, administration of IL-2 on weekends is optional. For example, in some embodiments, day 0 (same day as the first administration of the g-NK cells), occurs on a Monday. Accordingly, in some embodiments, the IL-2 is administered on day 0 (Monday; same day as the first administration of the g- NK cells), day 1 (Tuesday), day 2 (Wednesday), day 3 (Thursday), and day 4 (Friday). In some embodiments, the IL-2 is not administered on day 5 (Saturday) or day 6 (Sunday). In some embodiments, the 7-day cycle is repeated twice.
  • the IL-2 is administered once time daily every weekday (e.g., Q2D) for two 7-day cycles, e.g., on day 0 (same day as the first administration of the g- NK cells), occurs on a Monday. Accordingly, in some embodiments, the IL-2 is administered on day 0 (Monday; same day as the first administration of the g-NK cells), day 1 (Tuesday), day 2 (Wednesday), day 3 (Thursday), day 4 (Friday), day 7 (Monday), day 8 (Tuesday), day 9 (Wednesday), day 10 (Thursday), and day 11 (Friday).
  • the g-NK cells and IL-2 are administered to an individual in a 7 day cycle.
  • both the g-NK cells and IL-2 are administered one time daily every other day (i.e., Q2D) in a 7 day cycle, e.g., g-NK cells and IL-2 are administered on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4.
  • the Q2D administration is a thrice weekly dose.
  • the cycling regimen includes more than one 7-day cycle, which may be the same or different.
  • the 7-day cycle is repeated two times (e.g., for a total of three 7-day cycles).
  • each 7-day cycle is the same, e.g., g- NK cells and IL-2 are administered one time daily every other day (i.e., Q2D) in the first and second 7- day cycles, and optionally also a third 7-day cycle.
  • the IL-2 is administered before the administration of the g-NK cells.
  • the IL-2 is administered within about one hour of the administration of the g-NK cells.
  • each dose of IL-2 is about 6 million IU.
  • the g-NK cells are administered on time daily every other day (i.e., Q2D) and IL-2 is administered one time daily every day (i.e., QD) for a 7 day cycle, e.g., g-NK cells are administered on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4, while IL- 2 is administered on day 0, day 1, day 2, day 3, day 4, day 5, and day 6.
  • the 7-day cycle is repeated one more time (e.g., for a total of two 7-day cycles).
  • the 7-day cycle is the same for IL-2 administration and different for g-NK administration.
  • the g-NK cells are not administered during the second 7-day cycle while the IL-2 is administered one time daily every day (i.e., QD) for the second 7-day cycle.
  • g-NK cells are administered on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4, while IL-2 is administered on day 0, day 1, day 2, day 3, day 4, day 7, day 8, day 9, day 10, and day 11.
  • the g-NK cells are administered on time daily every other day (i.e., Q2D) and IL-2 is administered one time daily every weekday (i.e., QD) for a 7 day cycle, e.g., g e.g., g- NK cells are administered on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4, while IL-2 is administered on day 0, day 1, day 2, day 3, and day 4.
  • the 7- day cycle is repeated one more time (e.g., for a total of two 7-day cycles).
  • the 7- day cycle is the same for IL-2 administration and different for g-NK administration.
  • the g-NK cells are not administered during the second 7-day cycle while the IL-2 is administered one time daily every weekday (i.e., QD) for the second 7-day cycle.
  • g-NK cells are administered on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4, while IL-2 is administered on day 0, day 1, day 2, day 3, day 4, day 7, day 8, day 9, day 10, and day 11.
  • IFN-P is administered to subjects in accord with provided methods.
  • the dosing of IFN-P supports selective anti-inflammatory action in autoimmune diseases, e.g., multiple sclerosis via downregulation of the chronic overexpression of proinflammatory cytokines (see, e.g., Zettl U.K. et al., Expert Review of Clinical Immunology, 2023).
  • the administered IFN-P is IFN-P- la.
  • a 44 pg dose of IFN- P is administered three times a week (TIW) subcutaneously.
  • the subject is administered IFN-P once a week, two times a week, three times a week (TIW), four times a week, five times a week, six times a week, or seven times a week.
  • the subject is administered IFN-P three times a week (TIW).
  • the subject is administered IFN- once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.
  • each dose of IFN-P is between at or about 20 pg and at or about 50 pg. In some embodiments, each dose of IFN-P is between at or about 20 pg and at or about 45 pg.
  • each dose of IFN-P is at or about 20 pg, or is at or about 22 pg, or is at or about 25 pg, or is at or about 30 pg, or is at or about 35 pg, or is at or about 40 pg, or is at or about 44 pg, or is at or about 45 pg, or is at or about 50 pg.
  • each dose of IFN-P is at or about 22 pg. In some embodiments, each dose of IFN-P is at or about 44 pg.
  • the IFN-P is administered subcutaneously. ff. Lymphodep/eting Therapy
  • the provided methods also can include administering g-NK cells with another treatment, such as with a chemotherapeutic agent or cytotoxic agent or other treatment.
  • preconditioning subjects with immunodepleting can improve the effects of adoptive cell therapy (ACT).
  • the lymphodepleting therapy includes combinations of cyclosporine and fludarabine.
  • Such preconditioning can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy.
  • These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL-2, IL-7, and/or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the microenvironment.
  • cytokine sink by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL-2, IL-7, and/or IL-15
  • suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system
  • impact of negative regulators in the microenvironment Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).
  • the provided method further involves administering a lymphodepleting therapy to the subject.
  • the method involves administering the lymphodepleting therapy to the subject prior to the administration of the dose of cells.
  • the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and/or cyclophosphamide.
  • the administration of the cells and/or the lymphodepleting therapy is carried out via outpatient delivery.
  • the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the administration of the dose of cells.
  • a preconditioning agent such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof
  • the subject may be administered a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose.
  • the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide. In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 200 mg/m 2 and 600 mg/m 2 , such as between or between about 200 mg/m 2 and 400 mg/m 2 . In some aspects, the subject is preconditioned with or with about 300 mg/m 2 of cyclophosphamide.
  • the subject is administered fludarabine at a dose between or between about 1 mg/m 2 and 100 mg/m 2 , such as between or between about 10 mg/m 2 and 75 mg/m 2 , 15 mg/m 2 and 50 mg/m 2 , 20 mg/m 2 and 30 mg/m 2 , or 24 mg/m 2 and 26 mg/m 2 .
  • the subject is administered 25 mg/m 2 of fludarabine.
  • the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days.
  • fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days.
  • the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine.
  • the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above.
  • the subject is administered 60 mg/kg ( ⁇ 2 g/m 2 ) of cyclophosphamide and 3 to 5 doses of 25 mg/m 2 fludarabine prior to the dose of cells.
  • the subject is administered 30 mg/m 2 fludarabine and 300 mg/m 2 or 400 mg/m 2 cyclophosphamide.
  • the subject prior to the administration of the dose of g-NK cells, the subject has received a lymphodepleting therapy.
  • the lymphodepleting therapy includes fludarabine and/or cyclophosphamide.
  • the lymphodepleting includes the administration of fludarabine at or about 20-40 mg/m 2 body surface area of the subject, optionally at or about 30 mg/m 2 , daily, for 2-4 days, and/or cyclophosphamide at or about 200-400 mg/m 2 body surface area of the subject, optionally at or about 300 mg/m 2 , daily, for 2-4 days.
  • the subject is administered a conditioning chemotherapy as a lymphodepleting therapy that includes fludarabine 30 mg/m2 per day, cyclophosphamide 400 mg/m2 per day and mesna 300 mg/m 2 per day on Days -5, -4, and -3 prior to the first dose of the g-NK cells.
  • a conditioning chemotherapy as a lymphodepleting therapy that includes fludarabine 30 mg/m2 per day, cyclophosphamide 400 mg/m2 per day and mesna 300 mg/m 2 per day on Days -5, -4, and -3 prior to the first dose of the g-NK cells.
  • the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment.
  • preconditioning such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, improves the efficacy of treatment with the dose or increases the persistence of the NK cells in the subject.
  • preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the dose of cells. In some embodiments, the time to median disease-free survival is increased.
  • the method includes isolating a population of cells enriched for natural killer (NK) cells from a biological sample from a human subject and culturing the cells under conditions in which preferential growth and/or expansion of the g-NK cell subject and/or an NK cell subset that overlaps or shares extracellular surface markers with the g-NK cell subset.
  • the NK cells may be cultured using feeder cells, or in the presence of cytokines to enhance the growth and/or expansion of g-NK cell subject and/or an NK cell subset that overlaps or shares extracellular surface markers with the g-NK cell subset.
  • the provided methods also can expand other subsets of NK cells, such as any NK cell that is NKG2C pos and/or NKG2A neg .
  • cells from the circulating blood of a donor subject are obtained.
  • the samples contain lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood cells collected from the donor subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • the biological sample is from an enriched leukapheresis product collected from normal peripheral blood.
  • the enriched leukapheresis product can contain fresh cells.
  • the enriched leukapheresis product is a cryopreserved sample that is thawed for use in the provided methods.
  • the number of NK cells, or a g-NK cell subset or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, in the biological sample is from at or about 5 x 10 5 to at or about 1 x 10 8 , from at or about 5 x 10 5 to at or about 5 x 10 7 , from at or about 5 x 10 5 to at or about 1 x 10 7 , from at or about 5 x 10 5 to at or about 5 x 10 6 , from at or about 5 x 10 5 to at or about 1 x 10 6 , from at or about 1 x 10 6 to at or about 1 x 10 8 , from at or about 1 x 10 6 to at or about 5 x 10 7 , from at or about 1 x 10 6 to at or about 1 x 10 7 , from at or about 1 x 10 6 to at or about 5 x 10 6 , from at or about 5 x 10 6 to at or about 1 x 10 8 , from at or about 5
  • the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 12%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 14%. In some embodiments, the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 16%.
  • the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 24%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 26%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 28%. In some embodiments, the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 30%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 3%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 5%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 10%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 12%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 14%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 16%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 18%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 20%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 22%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 24%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 26%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 28%.
  • a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 30%.
  • the biological sample is from a donor subject that is CMV seropositive.
  • CMV infection can result in phenotypic and functional differentiation of NK cells, including development of high fractions of NK cells expressing NKG2C that exhibit enhanced antiviral activity.
  • CMV-associated NK cells expressing NKG2C display altered DNA methylation patterns and reduced expression of signaling molecules, such as FcRy (Schlums et al., Immunity (2015) 42:443-56). These NK cells are linked to more potent antibody-dependent activation, expansion, and function relative to conventional NK-cell subsets.
  • the biological sample can be from a donor subject that is CMV seronegative as NK cells with reduced expression of FcRy can also be detected in CMV seronegative individuals, albeit generally at lower levels. In some cases, the biological sample can be from CMV seropositive individuals.
  • a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 20% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 25% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 30% of NK cells in the peripheral blood sample are positive for NKG2C.
  • the donor subject is selected if at least at or about 35% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 40% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 45% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 50% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 55% of NK cells in the peripheral blood sample are positive for NKG2C.
  • the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 65% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 70% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 75% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 80% of NK cells in the peripheral blood sample are positive for NKG2C.
  • the donor subject is selected if at least at or about 85% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 90% of NK cells in the peripheral blood sample are positive for NKG2C.
  • a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 70% of NK cells in the peripheral blood sample are negative or low for NKG2A.
  • a donor subject is selected if at least at or about 75% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 80% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 85% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 90% of NK cells in the peripheral blood sample are negative or low for NKG2A.
  • a donor subject is selected based on both the percentage of NK cells in a peripheral blood sample that are positive for NKG2C and the percentage of NK cells in the peripheral blood sample that are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A.
  • the donor subject is selected if at least at or about 40% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 80% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 50% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 85% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 90% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 95% of NK cells in the peripheral blood sample are negative or low for NKG2A.
  • a donor subject is selected for expansion of cells in accord with the provided methods if the donor subject is CMV seropositive, and if among NK cells in a peripheral blood sample from the donor subject, the percentage of g-NK cells is greater than at or about 30%, the percentage of NKG2C pos cells is greater than at or about 20%, and the percentage of NKG2A neg cells is greater than at or about 70%.
  • NK cells from the donor subject bear a single nucleotide polymorphism (SNP rs396991) in the CD16 gene, nucleotide 526 [thymidine (T) guanine (G)] resulting in an amino acid (aa) substitution of valine (V) for phenylalanine (F) at position 158 in the mature (processed) form of the protein (F158V).
  • NK cells bear the CD16 158V polymorphism in both alleles (called 158V/V herein).
  • NK cells bear the CD16 158V polymorphism in a single allele (called 158V/F herein).
  • 158V+ genotype herein refers to both the 158V/V genotype and the 158V/F genotype. It has been found that the CD 16 F158V polymorphism is associated with substantially higher affinity for IgGl antibodies and have the ability to mount more robust NK cell-mediated ADCC responses (Mellor et al. (2013) Journal of Hematology & Oncology, 6:1; Musolino et al. (2008) Journal of Clinical Oncology, 26:1789-1796 and Hatjiharissi et al. (2007) Blood, 110:2561-2564). In some embodiments, antibody-directed targeting of CD16 158V+/g-NK cells leads to improved outcomes for patients due to the improved affinity, cytotoxic and/or cytokine-mediated effect functions of the CD16 158V+/g-NK cell subset.
  • the provided methods include enriching or isolating NK cells or a subset thereof from a biological sample of a donor subject identified as having the CD16 158V+ NK cell genotype.
  • the method includes screening donor subjects for the presence of the CD16 158V+ NK cell genotype.
  • genomic DNA is extracted from a sample from a donor subject that is or includes NK cells, such as blood sample or bone marrow sample.
  • the sample is or comprises blood cells, e.g., peripheral blood mononuclear cells.
  • the sample is or comprises isolated NK cells.
  • the sample is a sample from a healthy donor subject.
  • nucleic acids can be readily isolated from a sample, e.g., cells, using standard techniques such as guanidium thiocyanate -phenol-chloroform extraction (Chomocyznski et al. (1987) Anal. Biochem. 162: 156).
  • kits also are readily available for extracting genomic DNA, such as the Wizard genomic DNA purification kit (Promega, Madison, WI).
  • Genotyping can be performed on any suitable sample.
  • the genotyping reaction can be, for example, a pyrosequencing reaction, DNA sequencing reaction, MassARRAY MALDI- TOF, RFLP, allele-specific PCR, real-time allelic discrimination, or microarray.
  • a PCR-based technique such as RT-PCR, of genomic DNA is carried out using allele-specific primers for the polymorphism.
  • the PCR method for amplifying target nucleic acid sequences in a sample is well known in the art and has been described in, e.g., Innis et al.
  • PCR can be carried out using nested primers followed by allele-specific restriction enzyme digestion.
  • the first PCR primers comprise nucleic acid sequences 5’ -ATA TTT ACA GAA TGG CAC AGG -3’ (SEQ ID NO:2) and 5’-GAC TTG GTA CCC AGG TTG AA-3’ (SEQ ID NOG), while the second PCR primers are 5’-ATC AGA TTC GAT CCT ACT TCT GCA GGG GGC AT-3’ (SEQ ID NO:4) and 5’-ACG TGC TGA GCT TGA GTG ATG GTG ATG TTC AC-3’ (SEQ ID NOG), which, in some cases, generates a 94-bp fragment depending on the nature of allele.
  • the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO:7 (GAAATCTACC TTTTCCTCTA ATAGGGCAAT). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NOG (GAAATCTACC TTTTCCTCTA ATAGGGCAA). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO:9 (GAAATCTACC TTTTCCTCTA ATAGGGCA).
  • genotyping can be carried out by quantitative real-time RT-PCR following extraction of RNA using primer sequences as follows: CD16 sense set forth in SEQ ID NO: 10 (5'- CCAAAAGCCACACTCAAAGAC-3') and antisense set forth in SEQ ID NO: 11 (5'- ACCCAGGTGGAAAGAATGATG-3') and TaqMan probe set forth in SEQ ID NO: 12 (5'- AACATCACCATCACTCAAGGTTTGG-3 ').
  • allele specific amplification can be used with a set of V allele specific primers (e.g., forward primer set forth in SEQ ID NO: 13, 5’-CTG AAG ACA CAT TTT TAC TCC CAAA-3’; and reverse primer set forth in SEQ ID NO:14, 5’-TCC AAA AGC CAC ACT CAA AGA C-3’) or a set of F allele specific primers (e.g., forward primer set forth in SEQ ID NO:15, 5’-CTG AAG ACA CAT TTT TAC TCC CAAC-3’; and reverse primer set forth in SEQ ID NO: 14, 5’-TCC AAA AGC CAC ACT CAA AGA C-3’).
  • V allele specific primers e.g., forward primer set forth in SEQ ID NO: 13, 5’-CTG AAG ACA CAT TTT TAC TCC CAAA-3’; and reverse primer set forth in SEQ ID NO:14, 5’-TCC AAA AGC CAC ACT CAA AGA C-3’
  • CD16a The genomic sequence for CD16a is available in the NCBI database at NG_009066.1.
  • the gene ID for CD16A is 2214.
  • Sequence information for CD16, including gene polymorphisms, is available at UniProt Acc. No. P08637.
  • the sequence of CD16 (F158) is set forth in SEQ ID NO: 16 (residue F158 is bold and underlined).
  • CD16 (F158) further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO: 17).
  • CD16 158V+ polymorphism resulting in F158V
  • VAR_003960 The sequence of CD16 158V+ (polymorphism resulting in F158V) is known as VAR_003960 and has the sequence set forth in SEQ ID NO:18 (158V+ polymorphism is in bold and underline).
  • CD16 (158V+) further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO:
  • single nucleotide polymorphism (SNP) analysis is employed on genomic deoxyribonucleic acid (DNA) samples using allele-specific probes containing a fluorescent dye label (e.g., FAM or VIC) on the 5’ end and a minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3’ end and unlabeled PCR primers to detect a specific SNP target.
  • a fluorescent dye label e.g., FAM or VIC
  • MGB minor groove binder
  • NFQ nonfluorescent quencher
  • the assay measures or detects the presence of an SNP by a change in fluorescence of the dyes associated with the probe.
  • probes hybridize to the target DNA between the two unlabeled primers and signal from the fluorescent dye on the 5’ end is quenched by the NFQ on its 3’ end by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • Taq polymerase extends the unlabeled primers using the template as a guide and when the polymerase reaches the labeled probe, it cleaves the molecule separating the dye from the quencher.
  • a qPCR instrument can detect fluorescence from the unquenched label.
  • Exemplary reagents are commercially available SNP Assays, e.g., code C_25815666_10 for rs396991 (Applied Biosystems, Cat No. 4351379 for SNP genotyping of F158V in CD16).
  • subjects heterozygous or homozygous for the CD16 158V (F158V) polymorphism are identified.
  • subjects homozygous for the CD16 158V (F158V) polymorphism are identified.
  • NK cells or an NK cell subset are isolated or enriched from a biological sample from a subject identified as being heterozygous or homozygous for the CD16 158V polymorphism.
  • NK cells or an NK cell subset are isolated or enriched from a biological sample from a subject identified as being homozygous for the CD16 158V polymorphism.
  • the method includes enriching NK cells from the biological sample, such as from a population PBMCs isolated or obtained from the subject.
  • the population of cells enriched for NK cells is enriched by isolation or selection based on one or more natural killer cell-specific markers. It is within the level of a skilled artisan to choose particular markers or combinations of surface markers.
  • the surface marker(s) is any one or more of the from the following surface antigens CDl la, CD3, CD7, CD14, CD16, CD19, CD25, CD27, CD56, CD57, CD161, CD226, NKB1, CD62L; CD244, NKG2D, NKp30, NKp44, NKp46, NKG2A, NKG2C, KIR2DL1 and/or KIR2DL3.
  • the surface marker(s) is any one or more of the from the following surface antigens CDl la, CD3, CD7, CD14, CD16, CD19, CD25, CD27, CD38, CD56, CD57, CD161, CD226, NKB1, CD62L; CD244, NKG2D, NKp30, NKp44, NKp46, NKG2A, NKG2C, SLAMF7 (CD319), KIR2DL1 and/or KIR2DL3.
  • the one or more surface antigen includes CD3 and one or more of the following surface antigens CD16, CD56 or CD57.
  • the one or more surface antigen is CD3 and CD57.
  • the one or more surface antigen is CD3, CD56 and CD16. In other embodiments, the one or more surface antigen is CD3, CD56 and CD38. In further embodiments, the one or more surface antigen is CD3, CD56, NKG2A and CD161. In some embodiments, the one or more surface antigen is CD3, CD57, and NKG2C. In some embodiments, the one or more surface antigen is CD3, CD57, and NKG2A. In some embodiments, the one or more surface antigen is CD3, CD57, NKG2C, and NKG2A. In some embodiments, the one or more surface antigen is CD3 and CD56. In some embodiments, the one or more surface antigen is CD3, CD56, and NKG2C.
  • the one or more surface antigen is CD3, CD56, and NKG2A. In some embodiments, the one or more surface antigen is CD3, CD56, NKG2C, and NKG2A.
  • Reagents, including fluorochrome-conjugated antibodies, for detecting such surface antigens are well known and available to a skilled artisan.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.
  • the separation need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type, such as those expressing a marker refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • the selection includes positive selection for cells expressing NKG2C (NKG2C pos ) and/or negative selection for cells NKG2A (NKG2A neg ).
  • the isolation includes positive selection for cells expressing CD56, cells expressing CD 16 or cells expressing CD57 and/or negative selection for cells expressing CD38 and/or negative selection for cells expressing non-NK cell markers, such as T cell markers, for example, negative selection for cells expressing CD3 (CD3 neg ).
  • the isolation includes positive selection for cells expressing CD56, cells expressing CD16 or cells expressing CD57 and/or negative selection for cells expressing non-NK cell markers, such as T cell markers, for example, negative selection for cells expressing CD3 (CD3 neg ).
  • the isolation includes positive selection for cells expressing CD56, cells expressing CD16 or cells expressing CD57, and/or negative selection for cells expressing CD38 (CD38 neg ), CD161 (CD161 neg ), NKG2A (NKG2A neg ), and/or negative selection for cells expressing CD3 (CD3 neg ).
  • the selection includes isolation of cells negative for CD3 (CD3 neg ).
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), followed by negative selection for cells expressing NKG2A (NKG2A neg ) and CD161 (CD161 neg ).
  • the isolated or selected cells are CD3 neg CD56 pos NKG2A neg CD161 neg .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD57 (CD57 pos ).
  • the isolated or selected cells are CD3 neg CD57 pos .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ) and positive for cells expressing CD16 (CD16 pos ).
  • the isolated or selected cells are CD3 neg CD16 pos .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD57 (CD57 pos ).
  • the isolated or selected cells are CD3 neg CD57 pos .
  • the NK cells may be enriched by depletion of CD3 pos cells (negative selection for CD3 pos cells) followed by CD57 pos cell selection, thereby isolating and enriching CD57 pos NK cells.
  • the separation can be carried out by immunoaffinity-based methods, such as using MACSTM Microbeads.
  • CD3 microbeads can be used to deplete CD3 pos cells in a negative selection for CD3 neg cells.
  • CD57 MicroBeads can be used for CD57 enrichment of CD3 cell-depleted PBMCs.
  • the CD3 neg /CD57 pos enriched NK cells can then be used in expansion in the provided methods.
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD57 (CD57 pos ), and positive selection for cells expressing NKG2C (NKG2C pos ).
  • the isolated or selected cells are CD3 neg CD57 pos NKG2C pos .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD57 (CD57 pos ), and negative selection for cells expressing NKG2A (NKG2A neg ).
  • the isolated or selected cells are CD3 neg CD57 pos NKG2A neg .
  • the selection can further include negative selection for cells expressing CD38 (CD38 neg ).
  • the isolated or selected cells are CD3 neg CD57 pos CD38 neg .
  • the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2C pos .
  • the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2A neg .
  • the isolated or selected cells are CD3 neg CD57 pos CD38 neg NKG2C pos NKG2A neg .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ) and positive selection for cells expressing CD56 (CD56 pos ).
  • the isolated or selected cells are CD3 neg CD56 pos .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), and positive selection for cells expressing NKG2C (NKG2C pos ).
  • the isolated or selected cells are CD3 neg CD56 pos NKG2C pos .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), and negative selection for cells expressing NKG2A (NKG2A neg ).
  • the isolated or selected cells are CD3 neg CD56 pos NKG2A neg .
  • the selection includes negative selection for cells expressing CD3 (CD3 neg ), positive selection for cells expressing CD56 (CD56 pos ), positive selection for cells expressing NKG2C (NKG2C pos ), and negative selection for cells expressing NKG2A (NKG2A neg ).
  • the isolated or selected cells are CD3 neg CD56 pos NKG2C pos NKG2A neg .
  • the g-NK cells are cells having a g-NK surrogate surface marker profile.
  • the g-NK cell surrogate surface marker profile is CD16 pos /CD57 pos /CD7 dim/neg /CD161 neg .
  • the g-NK cell surrogate surface marker profile is NKG2A neg /CD161 neg .
  • the g-NK cell surrogate surface marker profile is CD38 neg .
  • CD45 pos /CD3 neg /CD56 pos is used as a surrogate surface marker profile for NK cells.
  • the g-NK cell surrogate surface marker profile further includes an NK cell surrogate surface marker profile. In some of any such embodiments, the g-NK cell surrogate surface marker profile further includes CD45 pos /CD3 neg /CD56 pos . In particular embodiments the g-NK cell surrogate surface marker profile includes CD45 pos /CD3 neg /CD56 pos/ CD16 pos /CD57 pos /CD7 dim/neg /CD161 neg . In other particular embodiments, the g- NK cell surrogate surface marker profile includes CD45 pos /CD3 neg /CD56 pos/ NKG2A neg /CD161 neg . In other particular embodiments, the g-NK cell surrogate surface marker profile includes CD45 pos /CD3 neg /CD56 pos /CD38 neg .
  • the methods of isolating, selecting and/or enriching for cells can include immunoaffinity-based selections.
  • the immunoaffinity-based selections include contacting a sample containing cells, such as PBMCs, with an antibody or binding partner that specifically binds to the cell surface marker or markers.
  • the antibody or binding partner is bound to a solid support or matrix, such as a sphere or bead, for example microbeads, nanobeads, including agarose, magnetic bead or paramagnetic beads, to allow for separation of cells for positive and/or negative selection.
  • the spheres or beads can be packed into a column to effect immunoaffinity chromatography, in which a sample containing cells, such as PBMCs, is contacted with the matrix of the column and subsequently eluted or released therefrom.
  • a sample containing cells such as PBMCs
  • the incubation generally is carried out under conditions whereby the antibodies or binding partners, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • positive selection cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.
  • a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated and/or cultured; in some aspects, the particles are left attached to the cells for administration to a patient.
  • the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc.
  • the magnetizable particles are biodegradable.
  • the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA).
  • MACS Magnetic Activated Cell Sorting
  • MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered.
  • the non-target cells are labelled and depleted from the heterogeneous population of cells.
  • the method comprises administering IL-12, IL-15, IL- 18, IL-2 and/or CCL5 to the subject prior to enriching, such as selecting and/or isolating, the NK cells or subset thereof.
  • the enriched NK cells are incubated or cultured in the presence of feeder cells, such as under conditions to support the proliferation and expansion of NK cell subsets, and in particular the g-NK cell subset.
  • the feeder cells include cells that stimulate or promote expansion of NKG2C pos and/or inhibit expansion of NKG2A pos cells.
  • the feeder cells are cells that express or are transfected with HLA-E or a hybrid HLA-E containing the HLA-A2 signal sequence.
  • a hybrid is an AEH hybrid gene containing an MHC class I, such as HLA-A2, promoter and signal sequence and the HLA-E mature protein sequence, which, in some cases, can result in a mature protein identical to that encoded by the HLA-E gene but that can be stably expressed on the cell surface (see e.g., Lee et al. (1998) Journal of Immunology, 160:4951-4960).
  • the cell is an LCL 721.221, K562 cell or RMA-S cell that is transfected to express an MHC-E molecule stabilized in the presence of an MHC class I, such as HLA-A2, leader sequence.
  • MHC class I such as HLA-A2
  • leader sequence peptide are known in the art (Lee et al. (1998) Journal of Immunology, 160:4951-4960; Zhongguo et al. (2005) 13:464-467; Garcia et al. (2002) Eur J. Immunol., 32:936-944).
  • the HLA-E-expressing feeder cells e.g., 221.AEH cells
  • added to the culture are non-dividing, such as by X-ray irradiation or gamma irradiation.
  • the HLA-E-expressing feeder cells e.g., 221. AEH
  • the HLA-E-expressing feeder cells are irradiated with gamma rays in the range of about 1000 to 10000 rad, such as 1000-5000, rads to prevent cell division.
  • AEH to freeze/thaw NK-cells is used.
  • the ratio is 1:1. It is understood that higher ratio, such as 2.5:1 221.
  • AEH to freeze/thaw NK-cells can be used, but this may require a longer culture, e.g., at or about 21 days, to reach a desired threshold density or number.
  • the NK cells are expanded by further adding to the culture nondividing peripheral blood mononuclear cells (PBMC).
  • the non-dividing feeder cells can comprise X-ray-irradiated PBMC feeder cells.
  • the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells.
  • the PBMC are irradiated with gamma rays in the range of about 1000 to 10000 rad, such as 1000-5000, rads to prevent cell division.
  • the PBMC are irradiated with gamma rays in the range of about 10 Gy to 100 Gy, such as 10-50 Gy to prevent cell division.
  • the irradiated feeder cells are present in the culture medium at the same time as the non-dividing (e.g., irradiated) HLA-E-expressing feeder cells.
  • the non-dividing (e.g., irradiated) PBMC feeder cell, HLA-E-expressing feeder cells and enriched NK cells are added to the culture on the same day, such as on the day of the initiation of the incubation, e.g., at or about or near the same time.
  • the incubation or culture is further carried out in the presence of irradiated PBMCs as feeder cells.
  • irradiated PBMCs are present as feeder cells at a ratio of such feeder cells to enriched NK cells that is from at or about 1:10 to at or about 10:1, from at or about 1:10 to at or about 5:1, from at or about 1:10 to at or about 2.5:1, from at or about 1:10 to at or about 1:1, from at or about 1 : 10 to at or about 1 :2.5, from at or about 1 : 10 to at or about 1 :5, from at or about 1 :5 to at or about 10:1, from at or about 1:5 to at or about 5:1, from at or about 1:5 to at or about 2.5:1, from at or about 1:5 to at or aboutl:l, from at or about 1:5 to at or about 1:2.5, from at or about 1:2.5 to at or about 10:1, from at or about 1:2.5 to at or about 5:1, from at or about 1:2.5 to at or about 2.5:1, from at or about 1:2.5 to at or about 1:1, from at or about
  • the anti-CD3 antibody or antigen-binding fragment is present during at least a portion of the incubation that includes irradiated PBMC feeder cells.
  • the anti-CD3 antibody or antigen-binding fragment is added to the culture or incubation at or about the same time as the irradiated PBMCs.
  • the anti-CD3 antibody or antigen-binding fragment is added at or about at the initiation of the incubation or culture.
  • the anti-CD3 antibody or antigen-binding fragment may be removed, or its concentration reduced, during the course of the culture or incubation, such as by exchanging or washing out the culture medium.
  • the methods do not include adding back or replenishing the culture media with the anti-CD3 antibody or antigen-binding fragment.
  • the anti-CD3 antibody or antigen-binding fragment is added, or is present during at least a portion of the culture or incubation, at a concentration that is between at or about 10 ng/mL and at or about 5 pg/mL, such as between at or about 10 ng/mL and at or about 2 pg/mL, between at or about 10 ng/mL and at or about 1 pg/mL, between at or about 10 ng/mL and at or about 500 ng/mL, between at or about 10 ng/mL and at or about 100 ng/mL, between at or about 10 ng/mL and at or about 50 ng/mL, between at or about 50 ng/mL and at or about 5 pg/mL, such as between at or about 50 ng/mL and at or about 2 pg/mL, between at or about 50 ng/mL and at or about 1 pg/mL, between at or about 50 ng/
  • the concentration of the anti-CD3 antibody or antigen-binding fragment is at or about 10 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL, or any value between any of the foregoing. In some embodiments, the concentration of the anti-CD3 antibody or antigen-binding fragment is or is about 50 ng/mL.
  • the term “antibody” refers to immunoglobulin molecules and antigenbinding portions or fragments of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof, such as dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv) or single domain antibody (sdAb).
  • an “antigen-binding fragment” contains at least one CDR of an immunoglobulin heavy and/or light chain that binds to at least one epitope of the antigen of interest.
  • an antigen-binding fragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and variable light chain (VL) sequence from antibodies that bind the antigen, such as generally six CDRs for an antibody containing a VH and a VL (“CDR1,” “CDR2” and “CDR3” for each of a heavy and light chain), or three CDRs for an antibody containing a single variable domain.
  • VH variable heavy chain
  • VL variable light chain
  • an “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and singledomain VH single antibodies; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • the incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration that is at or about, or at least at or about, 0.05 x 10 6 enriched NK cells/mL, at or about 0.1 x 10 6 enriched NK cells/mL, at or about 0.2 x 10 6 enriched NK cells/mL, at or about 0.5 x 10 6 enriched NK cells/mL or at or about 1.0 x 10 6 enriched NK cells/mL.
  • enriched NK cells such as selected and/or isolated NK cells
  • the incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration that is between at or about 0.05 x 10 6 enriched NK cells/mL and at or about 1.0 x 10 6 enriched NK cells/mL, such as between at or about 0.05 x 10 6 enriched NK cells/mL and at or about 0.75 x 10 6 , between at or about 0.05 x 10 6 enriched NK cells/mL and at or about 0.5 x 10 6 , between at or about 0.05 x 10 6 enriched NK cells/mL and at or about 0.20 x 10 6 enriched NK cells/mL, between at or about 0.05 x 10 6 enriched NK cells/mL and at or about 0.1 x 10 6 enriched NK cells/mL, between at or about 0.1 x 10 6 enriched NK cells/mL and at or about 1.0 x 10 6 enriched NK cells/mL, between
  • the amount of enriched NK cells, such as selected or isolated from PBMCs as described above, added or present at the initiation of the incubation or culture is at least or at least about 1 x 10 5 cells, at least or at least about 2 x 10 5 cells, at least or at least about 3 x 10 5 cells, at least or at least about 4 x 10 5 cells, at least or at least about 5 x 10 5 cells, at least or at least about 6 x 10 5 cells, at least or at least about 7 x 10 5 cells, at least or at least about 8 x 10 5 cells, at least or at least about 9 x 10 5 cells, at least or at least about 1 x 10 6 cells or more.
  • the population of enriched NK cells comprises at least at or about 2.0 x 10 5 enriched NK cells. In some embodiments, the population of enriched NK cells comprises at least at or about 1.0 x 10 6 enriched NK cells. In some embodiments, the population of enriched NK cells comprises at least at or about 1.0 x 10 7 enriched NK cells.
  • the population of enriched NK cells comprises between at or about 2.0 x 10 5 enriched NK cells and at or about 1.0 x 10 9 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 5.0 x 10 8 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 1.0 x 10 8 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 5.0 x 10 7 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 1.0 x 10 7 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 5.0 x 10 6 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 1.0 x 10 6 enriched NK cells, between at or about 2.0 x 10 5 enriched NK cells and at or about 1.0 x 10 6 enriched NK cells, between at or about 2.0
  • the population of enriched NK cells comprises between at or about 2.0 x 10 5 enriched NK cells and at or about 5.0 x 10 7 enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10 6 enriched NK cells and at or about 1.0 x 10 8 enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10 7 enriched NK cells and at or about 5.0 x 10 8 enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10 7 enriched NK cells and at or about 1.0 x 10 9 enriched NK cells.
  • the percentage of g-NK cells among the population of enriched NK cells is between at or about 20% and at or about 90%, between at or about 20% and at or about 80%, between at or about 20% and at or about 70%, between at or about 20% and at or about 60%, between at or about 20% and at or about 50%, between at or about 20% and at or about 40%, between at or about 20% and at or about 30%, between at or about 30% and at or about 90%, between at or about 30% and at or about 80%, between at or about 30% and at or about 70%, between at or about 30% and at or about 60%, between at or about 30% and at or about 50%, between at or about 30% and at or about 40%, between at or about 40% and at or about 90%, between at or about 40% and at or about 80%, between at or about 40% and at or about 70%, between at or about 40% and at or about 60%, between at or about 40% and at or about 50%, between at or about 50% and at or about 90%, between at or about 50% and at or about 80%, between at or about
  • the percentage of g-NK cells among the population of enriched NK cells is between at or about 20% and at or about 90%. In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 40% and at or about 90%. In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 60% and at or about 90%.
  • the NK cells can be cultured with a growth factor.
  • the at least one growth factor comprises a growth factor selected from the group consisting of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18 and IL-21.
  • the at least one growth factor is IL-2 or IL-7 and IL-15.
  • the at least one growth factor is IL-2, IL-21 or IL-7 and IL-15.
  • the growth factor is a recombinant cytokine, such as a recombinant IL-2, recombinant IL-7, recombinant IL-21 or recombinant IL- 15.
  • the NK cells are cultured in the presence of one or more recombinant cytokines.
  • the one or more recombinant cytokines comprise any of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof.
  • the one or more recombinant cytokines comprise any of IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-21. In some embodiments, the one or more recombinant cytokines further comprises IL-2, IL-7, IL-15, IL-12, IL-18, or IL-27, or combinations thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-2. In some embodiments, the one or more recombinant cytokines is at least IL-2 and IL-21.
  • the one or more recombinant cytokines are IL-21 and IL-2. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, and IL-15. In some embodiments, the one or more recombinant cytokines are IL-21, IL-12, IL-15, and IL-18. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, 11-12, IL-15, and IL- 18. In some embodiments, the one or more recombinant cytokines are IL-21, IL- 15, IL- 18, and IL-27.
  • the one or more recombinant cytokines are IL-21, IL-2, IL-15, IL-18, and IL-27. In some embodiments, the one or more recombinant cytokines are IL-2 and IL- 15.
  • the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-2.
  • the recombinant IL-2 is present at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/m
  • the concentration of the IL-2 is at or about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 125 lU/mL, 150 lU/mL, 200 lU/mL, or any value between any of the foregoing.
  • the concentration of the recombinant IL-2 added at the initiation of the culturing and optionally one or more times during the culturing is or is about 100 lU/mL. In particular embodiments, the concentration of the recombinant IL-2 added at the initiation of the culturing and optionally one or more times during the culturing is or is about 500 lU/mL.
  • the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21.
  • the recombinant IL-21 is present at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/m
  • the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21.
  • the concentration of recombinant IL-21 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL,
  • the concentration of recombinant IL- 15 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about
  • 500 lU/mL of IL-2, 10 ng/mL of IL-15, and 25 ng/mL of IL-21 are added during at least a portion of the culturing, such as added at the initiation of the culturing and optionally one or more times during the culturing.
  • 100 lU/mL of IL-2, 10 ng/mL of IL-15, and 25 ng/mL of IL-21 are added during at least a portion of the culturing, such as added at the initiation of the culturing and optionally one or more times during the culturing.
  • the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21 and the recombinant IL-21 is added as a complex with an anti-IL-21 antibody.
  • anti-IL-21 antibody prior to the culturing, is contacted with the recombinant IL-21, thereby forming an IL-21 /anti-IL-21 complex, and the IL-21/anti- IL-21 complex is added to the culture medium.
  • contacting the recombinant IL-21 and the anti-IL-21 antibody to form an IL-21 /anti-IL-21 complex is carried out under conditions that include temperature and time suitable for the formation of the complex. In some embodiments, the culturing is carried out at 37 °C + 2 for 30 minutes.
  • anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/mL, between at or about 100 ng/mL and at or about 400 ng/mL, between at or about 100 ng/mL and at or about 300 ng/mL, between at or about 100 ng/mL and at or about 200 ng/mL, between at or about 200 ng/mL and at or about 500 ng/mL, between at or about 200 ng/mL and at or about 400 ng/mL, between at or about 200 ng/mL and at or about 300 ng/mL, between at or about 300 ng/mL and at or about 500 ng/mL, between at or about 300 ng/mL and at or about 400 ng/mL, or between at or about 400 ng/mL and at or about 500 ng/mL.
  • anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/m
  • the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL,
  • the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is at or about 25 ng/mL.
  • the concentration of recombinant IL- 12 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about
  • the concentration of recombinant IL-12 during at least a portion of the culturing is between about 1 ng/mL and about 50 ng/mL.
  • the concentration of recombinant IL-12 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is at or about 10 ng/mL.
  • the concentration of recombinant IL- 18 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about
  • the concentration of recombinant IL- 18 during at least a portion of the culturing is between about 1 ng/mL and about 50 ng/mL.
  • the concentration of recombinant IL- 18 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is at or about 10 ng/mL.
  • the concentration of recombinant IL-27 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about
  • the methods include exchanging the culture medium, which, in some aspects includes washing the cells.
  • the culture medium can be exchanged or washed out intermittently, such as daily, every other day, every three days, or once a week.
  • the culture medium is exchanged or washed out beginning within or within about 3 days to 7 days after initiation of the culture, such as at or about at day 3, day 4, day 5, day 6 or day 7.
  • the culture medium is exchanged or washed out at or about beginning at day 5. For example, media is exchanged on day 5 and every 2-3 days afterwards.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as any as described above.
  • the one or more growth factor or cytokine such as recombinant IL-2, IL- 15 and/or IL-21, is added intermittently during the incubation or culture.
  • the one or more growth factor or cytokine such as recombinant IL-2, IL-15 and/or IL-21, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out.
  • the one or more growth factor or cytokine such as recombinant IL-2, IL-15 and/or IL-21, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the one or more growth factor or cytokine, such as recombinant IL-2, IL- 15 and/or IL-21.
  • the methods include adding the one or more growth factor or cytokine, e.g., recombinant IL-2, IL- 15 and/or IL-21, at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the one or more growth factor or cytokine e.g., recombinant IL-2, IL- 15 and/or IL-21
  • the culturing is carried out in the presence of at least one of IL-2, IL- 15 and IL-21 and the culture medium is replenished to include at least one of IL-2, IL- 15 and IL-21. In some embodiments, the culturing is carried out in the presence of IL-2 and IL-21 and the culture medium is replenished to include IL-2 and IL-21. In some embodiments, the culturing is carried out in the presence of IL-2 and IL- 15 and the culture medium is replenished to include IL-2 and IL- 15.
  • the culturing is carried out in the presence of IL- 15 and IL-21 and the culture medium is replenished to include IL- 15 and IL21. In some embodiments, the culturing is carried out in the presence of IL-2, IL- 15 and IL-21 and the culture medium is replenished to include IL-2, IL- 15 and IL-21. In some embodiments, one or more additional cytokines can be utilized in the expansion of the NK cells, including but not limited to recombinant IL-18, recombinant IL-7, and/or recombinant IL-12.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-2.
  • the growth factor or cytokine such as recombinant IL-2
  • the growth factor or cytokine is added intermittently during the incubation or culture.
  • the growth factor or cytokine such as recombinant IL-2
  • the growth factor or cytokine such as recombinant IL-2
  • the methods include adding recombinant IL-2 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the recombinant IL-2 is added to the culture or incubation at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/mL and at or about 100 lU/mL, between at or about 100 lU/mL and at or about 500 lU/mL, between at or about 100 lU/mL and at or about 250 lU/mL or between at or about 250 lU/mL and at or about 500 lU/mL
  • the recombinant IL-2 is added to the culture or incubation at a concentration that is at or about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 125 lU/mL, 150 lU/mL, 200 lU/mL, or any value between any of the foregoing.
  • the concentration of the recombinant IL-2 is or is about 100 lU/mL.
  • the concentration of the recombinant IL-2 is or is about 500 lU/mL.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-21.
  • the growth factor or cytokine such as recombinant IL-21
  • the growth factor or cytokine is added intermittently during the incubation or culture.
  • the growth factor or cytokine, such as recombinant IL-21 is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out.
  • the growth factor or cytokine such as recombinant IL-21
  • the methods include adding recombinant IL-21 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the recombinant IL-21 is added to the culture or incubation at a concentration of between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL, between about 20 ng/mL and about
  • the recombinant IL-21 is added to the culture or incubation at a concentration of between about 10 ng/mL and about 100 ng/mL, inclusive.
  • the recombinant IL-21 is added to the culture or incubation at a concentration of at or about 25 ng/mL.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-21, added as a complex with an antibody, such as an anti-IL- 21 antibody.
  • the complex such as an IL-21 /anti-IL-21 antibody complex, is added intermittently during the incubation or culture.
  • the complex such as an IL- 21/anti-IL-21 antibody complex, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out.
  • the complex such as an IL-21 /anti-IL-21 antibody complex
  • the methods include adding the complex, such as an IL-21 /anti-IL-21 antibody complex, at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the anti-IL-21 antibody is contacted with the recombinant IL-21, thereby forming an IL-21 /anti-IL-21 complex, and the IL-21 /anti-IL-21 complex is added to the culture medium.
  • contacting the recombinant IL-21 and the anti-IL-21 antibody to form an IL-21 /anti-IL-21 complex is carried out under conditions that include temperature and time suitable for the formation of the complex. In any of such embodiments, the culturing is carried out at 37 °C + 2 for 30 minutes.
  • the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is at or about 25 ng/mL.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-15.
  • the growth factor or cytokine such as recombinant IL-15
  • the growth factor or cytokine is added intermittently during the incubation or culture.
  • the growth factor or cytokine, such as recombinant IL- 15 is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out.
  • the growth factor or cytokine such as recombinant IL- 15 is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL- 15.
  • the methods include adding recombinant IL- 15 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the recombinant IL- 15 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and
  • the recombinant IL-15 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 15 is added to the culture or incubation at a concentration of at or about 10 ng/mL. In particular embodiments, 500 lU/mL of IL-2, 10 ng/mL of IL-15, and 25 ng/mL of IL-21 are added to the culture or incubation.
  • the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-12.
  • the growth factor or cytokine such as recombinant IL-12
  • the growth factor or cytokine is added intermittently during the incubation or culture.
  • the growth factor or cytokine, such as recombinant IL-12 is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out.
  • the growth factor or cytokine such as recombinant IL-12
  • the methods include adding recombinant IL- 12 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.
  • the recombinant IL- 12 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and
  • the recombinant IL-12 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 12 is added to the culture or incubation at a concentration of at or about 10 ng/mL.
  • culturing is carried out using serum-free formulations, such as AIM VTM serum free medium for lymphocyte culture, MARROWMAXTM bone marrow medium or serum-free stem cell growth medium (SCGM) (e.g., CellGenix® GMP SCGM).
  • serum-free formulations such as AIM VTM serum free medium for lymphocyte culture, MARROWMAXTM bone marrow medium or serum-free stem cell growth medium (SCGM) (e.g., CellGenix® GMP SCGM).
  • SCGM serum-free stem cell growth medium
  • the cultures can be supplemented with amino acids, antibiotics, and/or with other growth factors cytokines as described to promote optimal viability, proliferation, functionality and/or and survival.
  • the culturing includes incubation that is carried out under GMP conditions.
  • the incubation is in a closed system, which in some aspects may be a closed automated system.
  • the culture media containing the one or more recombinant cytokines or growth factors is a serum-free media.
  • the incubation is carried out in a closed automated system and with serum-free media.
  • the expansion is carried out using a cell expansion system by transfer of the cells to gas permeable bags, such as in connection with a bioreactor (e.g., Xuri Cell Expansion System W25 (GE Healthcare)).
  • the cell expansion system includes a culture vessel, such as a bag, e.g., gas permeable cell bag, with a volume that is about 50 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L, or any value between any of the foregoing.
  • cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.
  • the expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium.
  • any one or more of the steps of the method can be carried out in a closed system or under GMP conditions.
  • all process operations are performed in a GMP suite.
  • a closed system is used for carrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy.
  • one or more or all of the processing steps e.g., isolation, selection and/or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self-contained system, and/or in an automated or programmable fashion.
  • the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
  • the culturing is carried out until a time at which the method achieves expansion of at least or at least about 2.50 x 10 8 g-NK cells. In some of any of the provided embodiments, the culturing is carried out until a time at which the method achieves expansion of at least or at least about 5.0 x 10 8 g-NK cells. In some of any of the provided embodiments, the culturing is carried out until the method achieves expansion of at least or at least about 1.0 x 10 9 g-NK cells. In some of any of the provided embodiments, the culturing is carried out until a time at which the method achieves expansion of at least or at least about 5.0 x 10 9 g-NK cells.
  • the culturing is carried out for at or about or at least at or at least about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 day, 21 days, 22 days, 23 days, 24 days or 25 days. In some embodiments, the culturing is carried out for at or about or at least at or about 14 days. In some embodiments the culturing is carried out for at or about or at least at or about 21 days.
  • the cells are harvested. Collection or harvesting of the cells can be achieved by centrifugation of the cells from the culture vessel after the end of the culturing. For example, cells are harvested by centrifugation after approximately 14 days of culture. After harvesting of the cells, the cells are washed. A sample of the cells can be collected for functional or phenotypic testing. Any other cells not used for functional or phenotypic testing can be separately formulated. In some cases, the cells are formulated with a cryoprotectant for cryopreservation of cells.
  • the provided methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, selection and/or enrichment. In some embodiments, the provided methods include steps for freezing, e.g., cryopreserving, the cells, either before or after incubation and/or culturing. In some embodiments, the method includes cryopreserving the cells in the presence of a cryoprotectant, thereby producing a cryopreserved composition. In some aspects, prior to the incubating and/or prior to administering to a subject, the method includes washing the cryopreserved composition under conditions to reduce or remove the cyroprotectant. Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • the cells are frozen, e.g., cryofrozen or cryopreserved, in media and/or solution with a final concentration of or of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA.
  • PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
  • HSA human serum albumin
  • the cells are generally then frozen to or to about -80° C. at a rate of or of about 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • the cells are frozen in a serum-free cry opreservation medium comprising a cryoprotectant.
  • the cryoprotectant is DMSO.
  • the cryopreservation medium is between at or about 5% and at or about 10% DMSO (v/v).
  • the cryopreservation medium is at or about 5% DMSO (v/v).
  • the cry opreservation medium is at or about 6% DMSO (v/v).

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Abstract

L'invention concerne des méthodes et des utilisations permettant de traiter une maladie ou une affection auto-immune avec des cellules tueuses naturelles (NK), comprenant le dosage de compositions contenant des cellules NK déficientes en chaîne FcRγ (cellules g-NK). Parmi les méthodes et les utilisations de l'invention, on distingue des méthodes et des utilisations permettant de traiter diverses maladies ou affections auto-immunes.
PCT/US2025/023300 2024-04-05 2025-04-04 Traitement de maladies ou d'affections auto-immuns avec des cellules tueuses naturelles Pending WO2025213127A1 (fr)

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US202463575607P 2024-04-05 2024-04-05
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US202463651387P 2024-05-23 2024-05-23
US63/651,387 2024-05-23
US202463663678P 2024-06-24 2024-06-24
US63/663,678 2024-06-24
US202463716715P 2024-11-05 2024-11-05
US63/716,715 2024-11-05
US202463728108P 2024-12-04 2024-12-04
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