Subsets of human natural killer cells with enhanced antibody-directed immune responses

Abstract

To provide compositions containing natural killer cells with improved responses to antibody therapy.SOLUTION: The present invention provides a composition comprising a natural killer cell subset surface positive for CD16 158V+, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+NK cell subset. In some embodiments, the subset does not express FcRγ chain, is surface positive for a KIR and / or is surface negative or low expression for Nkp30 and / or Nkp46.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 671,358, entitled "SUBSETS OF HUMAN NATURAL KILLER CELLS WITH ENHANCED ANTIBODY DIRECTED IMMUNE RESPONSES," filed May 14, 2018, the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with an electronic sequence listing, which is provided as a file entitled 776032000340SeqList.txt, created on May 14, 2019, and is 7.04 kilobytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides compositions containing a specialized subset of natural killer (NK) cells with enhanced antibody-directed immune response. The compositions are useful for treating diseases and conditions such as cancer, including in combination with antibodies capable of binding to disease-associated tissues or disease-associated cells, such as tumor cells or infected cells. [Background technology]

[0004] background Antibody-based therapies are becoming more frequently used in the treatment of cancer and other disease indications. Responses to antibody therapy have typically focused on the direct inhibitory effect of these antibodies on tumor cells (e.g., inhibition of growth factor receptors and subsequent induction of apoptosis), but the in vivo effects of these antibodies appear to be more complex, which may involve the host immune system. Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity upon binding of Fc receptors (CD16; FcγRIII) to the Fc portion of antibodies bound to antigen-bearing cells. Improved therapies are needed to improve responses to antibody therapy. Embodiments that fulfill such needs are provided herein. Summary of the Invention

[0005] overview Provided herein is a specialized subset of NK cells that exhibits enhanced antibody-directed immune responses. In some embodiments, compositions enriched for said NK cell subsets are provided. In some embodiments, the compositions can be administered to a subject in combination with an antibody or other Fc protein, such as an antibody or Fc protein for treating a disease or condition, or an antibody or Fc protein that binds to or recognizes an antigen or protein associated with a disease or condition, to treat the disease or condition. In some embodiments, the methods provided achieve an improved response compared to methods in which normal NK cells are administered, or compared to methods in which only an antibody or Fc is administered. In some embodiments, the increased response is due to increased affinity of the Fc fragment of an IgG antibody by the specialized NK cells, which can result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), and thus increased killing of cells targeted by the antibody or protein, such as tumor cells. In some embodiments, the specialized NK cells are surface positive for CD16 with a 158V+ polymorphism, and / or lack expression of FcRγ chain.

[0006] Provided herein is a composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ NK cell subset.In some embodiments, the CD16 158V+ NK cell subset does not express FcRγ chain (CD16 158V+ / g-), is surface positive for KIR, and / or is surface negative or low expressing for Nkp30 and / or Nkp46.

[0007] Provided herein is a composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+ and lacks expression of the FcR gamma chain (g-) (CD16 158V+ / g-), wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ / g- NK cell subset.

[0008] In some of any of the provided embodiments, CD16 158V+ is a surface receptor that comprises a sequence as set forth in SEQ ID NO:11, or a sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:11 and includes the 158V+ polymorphism.

[0009] In some of any of the embodiments provided, the NK cell subset of the composition is surface positive for KIR, and optionally for KIR2DL2 / 3. In some of any of the embodiments provided, the NK cell subset of the composition is negative for or has low expression of surface expression of Nkp30 and / or Nkp46. In some of any of the embodiments provided, the NK cell subset of the composition is surface positive for KIR, and optionally for KIR2DL2 / 3, and is negative for or has low expression of surface expression of Nkp30 and / or Nkp46.

[0010] In some of any of the embodiments provided, the NK cell subset of the composition is surface positive for NKG2C.

[0011] In some of the provided embodiments, the composition comprises 10 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 In some embodiments, the volume of the composition is at or about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, and / or the composition comprises 1×10 cells in which the NK cell subset is present. 5 From 1×10 8 Contains NK cell subsets at a concentration of 10 cells / mL.

[0012] In some of any such embodiments, the composition comprises a pharma- ceutically acceptable carrier, hi some embodiments, the composition comprises a cryoprotectant.

[0013] In some of any such embodiments, the NK cell subset present in the composition comprises primary NK cells obtained from a subject. In some embodiments, the subject is a human.

[0014] Provided herein is a method for enriching NK cells, the method comprising: (a) isolating NK cells or a subset thereof from a subject identified as having a CD16 158V+ NK cell genotype; and (b) culturing the isolated NK cells or a subset thereof under conditions for expanding the cells, thereby enriching the NK cells or a subset thereof from the subject. In some embodiments, the method comprises screening or identifying the subject for the presence of the CD16 158V+ NK cell genotype prior to step (a).

[0015] In some of any such embodiments, the NK cells or a subset thereof are isolated from a population of cells that includes lymphocytes. In some embodiments, the population of cells comprises a peripheral blood mononuclear cell (PBMC) sample, an apheresis product, or a leukapheresis product.

[0016] In some of any such embodiments, the method includes isolating an NK cell subset from the subject that includes cells that do not express FcRγ chain (CD16 158V+ / g-) or is enriched for cells that do not express FcRγ chain (CD16 158V+ / g-). In some embodiments, the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3. In some embodiments, the NK cell subset is negative for or has low expression of surface expression of Nkp30 and / or Nkp46. In some embodiments, the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3, and negative for or has low expression of surface expression of Nkp30 and / or Nkp46. In some embodiments, the NK cell or a subset thereof is surface positive for NKG2C.

[0017] In some of any of such embodiments, the method includes administering to the subject IL-12, IL-15, IL-18, IL-2 and / or CCL5 prior to isolating the NK cells or a subset thereof.

[0018] In some of any such embodiments, the cells are cultured in the presence of feeder cells or in the presence of one or more cytokines, optionally IL-2, IL-15 and / or IL-7. In some embodiments, the NK cells are cultured for a period of time to achieve at least a 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or more expansion of cells compared to the number of isolated NK cells prior to culture. In some embodiments, the isolated NK cells are cultured for 7-28 days, optionally for about or at least about 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the isolated cells are cultured to achieve a therapeutically effective amount of NK cells or a subset thereof. In some embodiments, the isolated cells are cultured for 10-28 days, optionally for about or at least about 14, 15, 16, 17, 18, 19, 20, or 21 days. 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 The isolated cells are cultured to achieve a number of enriched NK cells or a subset thereof.

[0019] In some of such embodiments, the method further comprises performing HLA typing of the isolated or enriched cells. In some embodiments, the method further comprises cryopreserving the cells and / or formulating the cells in the presence of a cryoprotectant. In some embodiments, the resulting enriched cells are administered to a subject to treat a disease or disorder. In some of such embodiments, the subject is a human. In some embodiments, the subject is healthy.

[0020] Also provided is a composition containing the enriched NK cells or a subset thereof produced by any of the above methods. In some embodiments, the composition contains a pharma- ceutically acceptable excipient. In some embodiments, the composition contains a cryoprotectant.

[0021] In some of any such embodiments, the composition is sterile.

[0022] Provided herein is a kit containing the composition of any of the above aspects and an additional agent for the treatment of a disease. In some embodiments, the kit further comprises instructions for administration of the composition and the additional agent for the treatment of a disease or condition.

[0023] Also provided herein are kits containing a composition of any of the provided embodiments and instructions for administering said composition in combination therapy with an additional agent for the treatment of a disease.

[0024] In some of any of such embodiments, the additional agent is an antibody or an Fc fusion protein. In some embodiments, the antibody recognizes or specifically binds to a tumor-associated antigen. In some embodiments, the antibody recognizes or specifically binds to a tumor-associated antigen. In some embodiments, the antibody recognizes or specifically binds to a tumor-associated antigen, such as CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, alpha-fetoprotein, mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate binding protein, scatter factor (Scatter factor). The antibody recognizes or binds to (Fc) receptor kinase, gangliosides, cytokerains, frizzled receptors, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-α, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some embodiments, the antibody is a full-length antibody and / or comprises an Fc domain.

[0025] Provided herein is a method of treating a disease or condition by administering the composition of any of the above embodiments to an individual or subject in need thereof. In some embodiments, the method comprises administering 1×10 8 or about 1 x 10 8 From 1×10 10 cells / m 2 or 1×10 6 Or about 1 x 10 6 From 1×10 10 The method includes administering NK cells / kg.

[0026] In some of any such embodiments, the method further comprises administering an additional agent. In some embodiments, the additional agent is an antibody or an Fc fusion protein. In some embodiments, the antibody recognizes a tumor-associated antigen. In some embodiments, the antibody recognizes a tumor-associated antigen. In some embodiments, the antibody recognizes CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, alpha-fetoprotein, mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate binding protein, scatter factor receptor kinase, ganglioside, cytokeline, frizzled receptor, VEGF, Recognizes or specifically binds to VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-α, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some embodiments, the antibody comprises an Fc domain and / or is a full-length antibody.

[0027] In some of any such embodiments, the additional agent and the composition are administered sequentially. In some embodiments, the additional agent is administered prior to administration of the composition. In some embodiments, the additional agent and the composition are administered simultaneously.

[0028] In some of any such embodiments, the disease or condition is selected from the group consisting of an inflammatory condition, an infectious disease, and a cancer. In some embodiments, the infectious disease is a viral infection or a bacterial infection. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a solid tumor.

[0029] In some of such embodiments, the individual is a human. In some of such embodiments, the NK cells in the composition administered are autologous to the subject. In some of such embodiments, the NK cells in the composition administered are allogeneic to the individual. In some embodiments, the cells are administered to a subject of the same HLA type. In some embodiments, cells from an HLA-matched donor are administered to the subject.

[0030] In some embodiments, the method includes thawing the cells prior to administering the cells to the subject. [Brief description of the drawings]

[0031] [Figure 1] Figure 1 shows the ADCC activity of g-NK, conventional NK cells, and 158V g-NK against SW-480 / cetuximab, SKOV3 / cetuximab, SKOV3 / trastuzumab, MM.1S / daratumumab, and MM.1S / elotuzumab at an E:T ratio of 5:1. Values ​​are means ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description Compositions containing a subset of human natural killer (NK) cells with enhanced antibody-directed immune response are provided herein, including compositions enriched for such subsets. In some embodiments, the provided compositions contain or are enriched for NK cells carrying a special gene polymorphism in which phenylalanine (F) is substituted with valine (V) at the 158th amino acid position of CD16 (FcγRIII) gene (referred to as 158V+). In some embodiments, the provided compositions contain or are enriched for 158V+ and are enriched for FcRγ-deficient NK cells (referred to as g-NK), thereby providing compositions containing or enriched for CD16 158V+ / g-NK cells. Methods are also provided for administering the provided compositions to a subject in combination with an antibody for treating a disease or condition.

[0033] In some embodiments, the compositions provided comprise human NK cells, and greater than or about 40%, greater than or about 50%, greater than or about 60%, greater than or about 70%, greater than or about 80%, greater than or about 90%, or more of the NK cells in the composition, or of the total cells of the composition, are CD16 158V+ NK cells or CD16 158V+ / g- NK cells.

[0034] Natural killer (NK) cells are innate lymphocytes important for mediating antiviral and anticancer immunity by secreting cytokines and chemokines and by releasing cytotoxic granules (Vivier et al. Science 331(6013):44-49(2011); Caligiuri, Blood 112(3):461-469(2008); Roda et al., Cancer Res.66(1):517-526(2006)). NK cells are effector cells that constitute the third largest population of lymphocytes and are important for host immune surveillance against tumor cells and pathogen-infected cells. However, unlike T and B lymphocytes, NK cells are thought to have only a limited capacity for target recognition using germline-encoded activating receptors (Bottino et al., Curr Top Microbiol Immunol. 298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21 (2006)).

[0035] NK cell activation can occur either through direct binding of the NK cell receptor to a ligand on the target cell, as seen in direct tumor cell killing, or through cross-linking of the Fc receptor (also known as CD16, CD16a or FcγRIIIa) by binding to the Fc portion of an antibody bound to an antigen-bearing cell. Upon activation, NK cells produce abundant cytokines and chemokines and simultaneously exhibit potent cytolytic activity. NK cells can kill tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when a receptor on the NK cell surface (such as CD16) recognizes IgG1 or IgG3 antibodies bound to the surface of the cell. This triggers the release of cytoplasmic granules containing perforin and granzymes, which lead to the death of the target cell. NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells, thus broadening target recognition (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001); Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long, Curr Opin Immunol. 20(3):344-352 (2008)). ADCC and antibody-dependent cytokine / chemokine production are primarily mediated by NK cells.

[0036] In addition to CD16 being expressed on NK cells and involved in antibody-dependent responses (such as NK cell-mediated ADCC), CD16 also exists as a glycosylphosphatidylinositol-anchored form (also known as FcγRIIIB or CD16B). CD16 as used herein refers to the CD16a form expressed on NK cells and not the glycosylphosphatidylinositol-anchored form. The CD16 receptor can associate with the adaptor ζ chain of the TCR-CD3 complex (CD3ζ) and / or the FcRγ chain to signal through immunoreceptor tyrosine-based activation motifs (ITAMs). In certain aspects, CD16 engagement (CD16 cross-linking) initiates NK cell responses via intracellular signals generated by one or both of the CD16-associated adaptor chains FcRγ or CD3ζ. CD16 triggering leads to phosphorylation of the γ or ζ chains, which then recruits the tyrosine kinases, syk and ZAP-70, initiating a cascade of signaling that leads to rapid and potent effector functions. The most well-known effector function is the release of cytoplasmic granules carrying toxic proteins to kill nearby target cells by the process of antibody-dependent cellular cytotoxicity. CD16 crosslinking also leads to the production of cytokines and chemokines, which then activate and orchestrate a range of immune responses.

[0037] This release of cytokines and chemokines may play a role in the anti-cancer activity of NK cells in vivo. NK cells also have small granules in their cytoplasm that contain perforin and proteases (granzymes). Upon release from NK cells, perforin creates small pores in the plasma membrane of targeted cells through which granzymes and related molecules can enter and induce apoptosis. The fact that NK cells induce apoptosis and not necrosis of target cells is important - necrosis of virus-infected cells would result in the release of virions, whereas apoptosis leads to the destruction of the virus within the cell.

[0038] The majority of humans express CD16, which has a relatively low affinity for IgG1 antibodies. However, NK cells from individuals carrying a single nucleotide polymorphism (SNP rs396991) in the CD16 gene, i.e., nucleotide 526 [thymidine (T) → guanine (G)] resulting in an amino acid (aa) substitution of phenylalanine (F) at position 158 of the mature (processed) protein with valine (V) (F158V, also referred to herein as 158V+), have been found to have the ability to associate with substantially higher affinity to IgG1 antibodies and 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).

[0039] It has also been found that a subset of FcR gamma chain-deficient NK cells (also referred to as g-NK) can mediate robust ADCC responses (see, e.g., patent application US2013 / 0295044). The mechanism of increased response may be due to altered epigenetic modifications affecting the expression of FcRgamma. g-NK cells abundantly express the signaling adaptor ζ chain, but lack expression of the signaling adaptor γ chain. Compared to normal NK cells, these gamma-deficient g-NK cells exhibit dramatically enhanced activity when activated by antibodies. For example, g-NK cells can be activated by antibody-mediated cross-linking of CD16 or by antibody-coated tumor cells. In some aspects, g-NK cells produce greater amounts of cytokines (e.g., IFN-γ or TNF-α) and chemokines (e.g., MIP-1α, MIP-1β, and RANTES) and / or exhibit a more advanced degranulation response than normal NK cells expressing the gamma chain. In g-NK cells, the expression of granzyme B, a component of the cytotoxicity mechanism of natural killer cells, is increased. Moreover, g-NK cells have a longer life span than normal NK cells, and their existence is maintained for a long period of time. In some embodiments, g-NK cells are functionally and phenotypically stable.

[0040] In some embodiments, CD16 158V+ NK cells and CD16 158V+ / g-NK cells are more effective at inducing ADCC responses than normal NK cells, e.g., NK cells that do not contain the 158V+ polymorphism and / or are not deficient in the gamma chain. In some cases, ADCC is the mechanism of action of therapeutic antibodies, including anti-cancer antibodies. In some aspects, cell therapy by administering NK cells can be used in conjunction with antibodies for therapeutic and related purposes. Provided herein are methods involving the combined administration of the provided compositions enriched for CD16 158V+ NK cells or CD16 158V+ / g-NK cells and antibodies, e.g., anti-cancer antibodies. In some embodiments, antibody-directed targeting of CD16 158V+ NK cells or CD16 158V+ / g- NK cells leads to improved outcomes for patients due to improved affinity, cytotoxicity and / or cytokine-mediated effector function of the CD16 158V+ NK cell or CD16 158V+ / g- NK cell subsets.

[0041] In some embodiments, g - NK cells produce significantly more cytokines than natural killer cells that express FcRγ. In another embodiment, the cytokine is interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), or a combination thereof. In one embodiment, - NK cells produce significantly higher amounts of chemokines. In one embodiment, the chemokines are MIP-1α, MIP-1β, or a combination thereof. In another embodiment, - NK cells produce cytokines or chemokines when stimulated via the Fc receptor CD16.

[0042] All references mentioned in this specification, including patent applications, patent publications, and scientific literature and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0043] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0044] I. Definition Unless otherwise defined, all terminology, notations, and other technical and scientific terms and terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. In some cases, terms with commonly understood meanings are defined herein for the purposes of clear description and / or ready reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0045] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules.

[0046] The term "about" as used herein refers to the normal error range for each value that is readily apparent to one of ordinary skill in the art. When "about" is used herein with respect to a value or parameter, the embodiment directed to the value or parameter itself is included (and described).

[0047] It is understood that the aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0048] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description encompasses instances when the event or circumstance occurs and instances when it does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0049] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, e.g., recombinantly produced, including any fragment thereof that contains at least a portion of the variable heavy and / or variable light chain region of the immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, sufficient to specifically bind to an antigen. Thus, an antibody encompasses any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Typically, an antibody will contain, at a minimum, a variable heavy (V H ) chain and / or variable light (V L ) chain. Generally, V H and V L pair with each other to form the antigen-binding site, but in some cases, a single V H Domain or V L A domain is sufficient for antigen binding. An antibody can also include all or a portion of a constant region. As used herein, antibodies include full length antibodies and antigen-binding fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0050] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, not an antibody fragment. Full-length antibodies are typically antibodies having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies produced by antibody-secreting B cells from mammalian species (e.g., human, mouse, rat, rabbit, non-human primates, etc.), and synthetically produced antibodies having the same domains. In particular, whole antibodies are those having heavy and light chains, including the Fc region. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies can have one or more effector functions.

[0051] An "antibody fragment" includes a portion of an intact antibody, the antigen-binding and / or variable regions of the intact antibody. Antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, diabodies, linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10) Antibody fragments include, but are not limited to, single chain antibody molecules such as single chain Fv (scFv) or single chain Fab (scFab), antigen-binding fragments of any of the above, and multispecific antibodies formed from antibody fragments. For purposes of this specification, an antibody fragment typically includes one sufficient to engage or crosslink CD16 on the surface of a NK cell.

[0052] The term "autologous" refers to cells or tissues that originate within or are taken from an individual's own tissues, e.g., in autologous transfer or transplantation of NK cells, the donor and recipient are the same person.

[0053] The term "allogeneic" refers to cells or tissues that belong to or are obtained from the same species, but that are genetically distinct and, in some cases, therefore immunologically incompatible. Typically, the term "allogeneic" is used to define cells that are transplanted from a donor to a recipient of the same species.

[0054] The term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. Collectively, a transcript and the encoded polypeptide may be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0055] The term "heterologous" with respect to a protein or nucleic acid refers to a protein or nucleic acid that is derived from a different genetic source. For example, a protein or nucleic acid that is heterologous to a cell is derived from a different organism or individual than the cell that expresses it.

[0056] The term "introducing" as used herein encompasses various methods of introducing DNA into cells, either in vitro or in vivo, including transformation, transduction, transfection (e.g., electroporation) and infection. Vectors serve to introduce DNA encoding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors, such as adenoviral vectors or adeno-associated vectors.

[0057] The term "composition" refers to any mixture of two or more products, substances or compounds, including cells or antibodies. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof. The preparation is generally in a form that allows the biological activity of the active ingredient (e.g., an antibody) to be effective.

[0058] The term "enriched" with respect to a cell composition refers to a composition in which the number or percentage of a cell type or cell population is increased compared to the number or percentage of that cell type in the same volume of a starting composition, e.g., a starting composition obtained or isolated directly from a subject. The term does not require the complete removal of other cells, cell types or cell populations from the composition, nor does it require that such enriched cells be 100% present, or even close to 100%, in the enriched composition.

[0059] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0060] As used herein, combination refers to any association between two or more items.Combination can be two or more separate items, such as two compositions or two collections, or it can be a mixture thereof, such as a single mixture of two or more items, or any variation thereof.The elements of a combination are generally functionally related or associated.

[0061] As used herein, a kit refers to a packaged combination, optionally including other components, such as additional agents, and instructions for use of the combination or components thereof, for purposes such as, but not limited to, therapeutic use.

[0062] The term "treatment" or "treating" as used herein refers to a clinical intervention designed to change the natural course of the treated individual or cell during the course of clinical pathology. The desired effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. An individual is successfully "treated" if, for example, one or more symptoms associated with a disorder (e.g., an eosinophil-mediated disease) are alleviated or eliminated. For example, an individual is successfully "treated" if the treatment improves the quality of life of the person suffering from the disease, reduces the dosage of other medications required to treat the disease, reduces the frequency of disease recurrence, reduces the severity of the disease, delays the onset or progression of the disease, and / or extends the individual's survival time.

[0063] An "effective amount" refers to an amount effective, at least at the dosage and for the period of time necessary, to achieve a desired or indicated effect, including a therapeutic or preventive result. An effective amount can be provided in one or more doses. A "therapeutically effective amount" is at least the minimum dose of cells necessary to achieve a measurable improvement of a particular disorder. In some embodiments, a therapeutically effective amount is an amount of a composition that reduces the severity, duration and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. A therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of a patient. A therapeutically effective amount may also be an amount in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects of the antibody. A "prophylactically effective amount" refers to an amount effective, at the dosage and for the period of time necessary, to achieve a desired prophylactic result. Because a prophylactic dose is used in pre-diseased subjects or subjects at an early stage of disease, typically, but not necessarily, a prophylactically effective amount may be less than a therapeutically effective amount.

[0064] As used herein, an "individual" or "subject" is a mammal. In the context of treatment, "mammals" include humans, domestic and farm animals, as well as zoo animals, sport animals, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0065] II. METHODS FOR OBTAINING AND PREPARING COMPOSITIONS CONTAINING NATURAL KILLER SUBSETS Methods are provided for preparing compositions containing or enriched for primary CD16 158V+ NK cells or CD16 158V+ / g- NK cells. The resulting compositions containing or enriched for primary CD16 158V+ or CD16 158V+ / g- NK cell subsets are also provided. In some embodiments, the primary cells provided are enriched from a subject, e.g., a mammalian subject, such as a human subject. In some embodiments, compositions containing or enriched for primary human CD16 158V+ NK cells or CD16 158V+ / g- NK cells are provided.

[0066] In some embodiments, the subject is a healthy subject. In some embodiments, the subject is a subject with a disease condition such as cancer. In some embodiments, the composition containing or enriched for primary CD16 158V+ NK cells or CD16 158V+ / g- NK cells can be administered for allogeneic or autologous adoptive cell therapy.

[0067] In some embodiments, the method for enriching NK cells comprises (a) isolating NK cells or a subset thereof from a subject identified as having a CD16 158V+ NK cell genotype, and (b) culturing the isolated NK cells or a subset thereof under conditions to expand the cells, thereby enriching the NK cells or a subset thereof from the subject. In some embodiments, the method comprises screening the subject for the presence of the CD16 158V+ NK cell genotype prior to step (a).

[0068] In some embodiments, the subject is screened for CD16 158V+, i.e., for the presence of the V158F polymorphism (SNP rs396991) in CD16. In some embodiments, genomic DNA is extracted from a biological sample from the subject, such as a blood sample or a bone marrow sample. In some embodiments, the sample is or comprises blood cells, such as peripheral blood mononuclear cells. In some embodiments, the sample is from a healthy donor subject. Any method for extracting DNA from a sample can be used. For example, nucleic acid can be easily isolated from a sample, such as cells, using standard techniques such as guanidinium thiocyanate-phenol-chloroform extraction (Chomocyznski et al. (1987) Anal. Biochem. 162:156). Commercial kits for extracting genomic DNA, such as Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.), are also readily available.

[0069] Genotyping can be carried out on any suitable sample.In any of the embodiments described herein, genotyping reaction can be, for example, pyrosequencing reaction, DNA sequencing reaction, MassARRAY MALDI-TOF, RFLP, allele-specific PCR, real-time allele discrimination, or microarray.In some embodiments, PCR-based techniques, such as RT-PCR, of genomic DNA are carried out using primers that are allele-specific for polymorphism. PCR methods for amplifying a target nucleic acid sequence in a sample are well known in the art and are described, for example, in Innis et al. (eds.) PCR Protocols (Academic Press, NY 1990), Taylor (1991) "Polymerase chain reaction: basic principles and automation" in "PCR: A Practical Approach" (McPherson et al. (eds.) IRL Press, Oxford), Saiki et al. (1986) Nature 324:163, and U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,889,818, all of which are incorporated herein by reference in their entireties.

[0070] Primers for detecting the 158V+ polymorphism are known or can be easily designed by those skilled in the art. For example, see International Publication WO2012 / 061814, Kim et al. (2006) Blood, 108:2720-2725, Cartron et al. (2002) Blood, 99:754-758, Koene et al. (1997) Blood, 90:1109-1114. In some embodiments, PCR can be performed using nested primers followed by allele-specific restriction enzyme digestion. In some embodiments, the first PCR primer is a nucleic acid sequence TIFF2024096956000002.tif12131, while the second PCR primer TIFF2024096956000003.tif12163. This in some cases will generate a 94 bp fragment depending on the nature of the allele. In some embodiments, the primer pair is: In some embodiments, the primer pair comprises the nucleic acid sequence set forth in TIFF2024096956000004.tif19140. In some embodiments, the primer pair comprises the nucleic acid sequence set forth in TIFF2024096956000005.tif12131. It contains the nucleic acid sequence shown in TIFF2024096956000006.tif11163.

[0071] The genomic sequence of CD16a is available at NCBI database NG_009066.1. The gene ID for CD16A is 2214. Sequence information for CD16, including gene polymorphisms, is available at UniProt accession number P08637. The sequence of CD16(F158) is shown in SEQ ID NO:9 (residue F158 is bolded and underlined). In some embodiments, CD16(F158) is It further contains a signal peptide, shown as TIFF2024096956000007.tif4128. TIFF2024096956000008.tif31158

[0072] The sequence of CD16 158V+ (polymorphism resulting in F158V) is known as VAR_003960 and has the sequence shown in SEQ ID NO:11 (the 158V+ polymorphism is bolded and underlined). In some embodiments, CD16(158V+) is It further contains a signal peptide, shown as TIFF2024096956000009.tif5128. TIFF2024096956000010.tif31153

[0073] In some embodiments, the cells provided comprise or are enriched for CD16 158V+ NK cells having a sequence of amino acids set forth in SEQ ID NO:11 or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:11 and containing a 158V+ polymorphism. In some embodiments, CD16 158V+ NK cells exhibit increased binding affinity for Fc, such as IgG Fc region, and / or are associated with increased tumor cell killing, compared to NK cells expressing CD16(F158) set forth in SEQ ID NO:9. In some embodiments, the increase is more than 1.5-fold, more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold or more.

[0074] In some embodiments, SNP analysis is used on genomic deoxyribonucleic acid (DNA) samples using allele-specific probes containing a fluorescent dye label (e.g., FAM or VIC) at the 5' end and a minor groove binder (MGB) and a nonfluorescent quencher (NFQ) at the 3' end, and unlabeled PCR primers to detect specific single nucleotide polymorphism (SNP) targets. In some embodiments, the assay measures or detects the presence of SNPs by a change in fluorescence of a dye associated with the probe. In such embodiments, the probe hybridizes to the target DNA between two unlabeled primers, and the signal from the fluorescent dye at the 5' end is quenched by the NFQ at its 3' end through fluorescence resonance energy transfer (FRET). During PCR, Taq polymerase extends the unlabeled primers using the template as a guide, and when the polymerase reaches the labeled probe, it cleaves the molecule to separate the dye from the quencher. In some aspects, the qPCR instrument is capable of detecting fluorescence from unquenched labels, such as commercially available SNP assays such as code C_25815666_10 for rs396991 (Applied Biosystems, catalog number 4351379, for SNP genotyping of V158F in CD16).

[0075] In some embodiments, a subject is identified as heterozygous or homozygous for CD16 158V+(V158F) polymorphism. In some embodiments, a subject is identified as homozygous for CD16 158V+(V158F) polymorphism. In some embodiments, a NK cell or a subset of NK cells is isolated from a subject identified as heterozygous or homozygous for CD16 158 V+ polymorphism. In some embodiments, a NK cell or a subset of NK cells is isolated from a subject identified as homozygous for CD16 158 V+ polymorphism. Methods for isolating NK cells are well known to those skilled in the art.

[0076] In some embodiments, natural killer cells are first isolated and then selected using available procedures. For example, a population of lymphocytes or peripheral blood mononuclear cells (PBMCs) can be obtained. Natural killer cells expressing one or more natural killer cell specific markers as described above are isolated from the cell population. It is within the level of one of ordinary skill in the art to choose a particular marker or combination of surface markers. In some embodiments, the surface marker is any one or more of the following surface antigens: CD11a, CD3, CD7, CD14, CD16, CD19, CD25, CD27, CD56, CD57, CD161, CD226, NKB1, CD62L, CD244, NKG2D, NKp30, NKp44, NKp46, NKG2A, NKG2C, KIR2DL1 and / or KIR2DL3. Agents for detecting such surface antigens are well known and available to those skilled in the art, including fluorochrome-conjugated antibodies.

[0077] In some embodiments, non-NK cells are excluded based on positive surface expression of CD19 and / or CD14.

[0078] In some embodiments, the NK cells are CD3 - CD56 dim In some embodiments, the isolated NK cells are isolated as present in a subpopulation that is gated as a population. - CD56 dim CD14 - CD19 - are present in the subpopulation gated as

[0079] In some embodiments, g-NK cell subsets are isolated or enriched from a subject. In some embodiments, g-NK cells are isolated or enriched from a subject identified as carrying the CD16 158V+ polymorphism, e.g., from a subject identified as heterozygous or homozygous for said polymorphism. In some embodiments, g-NK cells are isolated or enriched from a subject identified as carrying the CD16 158V+ polymorphism, e.g., from a subject identified as heterozygous or homozygous for said polymorphism.- A method for isolating NK cells, comprising identifying a subset of natural killer cells in a population of lymphocytes from a subject that does not express substantial FcRγ, thereby isolating NK cells from the subject. - Methods are provided that include identifying NK cells. In one embodiment, lymphocytes are obtained or isolated from a subject. In some embodiments, cells that do not express substantial FcRγ but express at least one marker of natural killer cells are identified.

[0080] The amino acid sequence of the FcR gamma chain (Homo sapiens, also known as high affinity immunoglobulin gamma Fc receptor I) is available in the NCBI database under accession number NP_004097.1 (GI:4758344) and is reproduced below as SEQ ID NO:12. TIFF2024096956000011.tif11149

[0081] In some embodiments, the isolation or enrichment of g-NK cells is carried out by immunoaffinity-based methods. In some embodiments, positive and / or negative strategies can be used to enrich for cells that are surface positive for one or more markers and / or surface negative or low expressing for one or more markers, respectively. In some embodiments, the isolation or enrichment is carried out by collecting cells that are gated or positive for surface expression of one or more specific markers (positive enrichment) and / or excluding cells that are positive for surface expression of one or more specific markers (negative enrichment) by fluorescence activated cell sorting (FACs). In some embodiments, the isolation or enrichment is carried out by contacting lymphocytes with a solid surface to which one or more antibodies specific for the markers are bound, and recovering cells that are not bound to the antibodies (negative enrichment) or cells that are bound to the antibodies (positive enrichment).

[0082] In some aspects, g-NK cells are identified by the presence, absence, or level of surface expression of one or more various markers that distinguish NK cells from other lymphocytes or immune cells and that distinguish g-NK cells from conventional NK cells. In some embodiments, g-NK cells can be isolated as described in patent application publication US2013 / 0295044 or Zhang et al. (2013) J. Immunol., 190:1402-1406.

[0083] In some embodiments, a cell (e.g., an NK cell subset) is positive for a particular marker if there is a detectable presence of that particular marker, typically a surface marker, on or in the cell. In embodiments, surface expression can be determined by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting binding of the antibody to the marker, where surface expression is positive if staining is detectable at a level substantially greater than that detected using an isotype-matched control performing the same procedure under otherwise identical conditions, and / or at a level substantially similar to, or in some cases, at a higher level than, and / or higher than, the level of cells known to be negative for that marker.

[0084] In some embodiments, a cell (e.g., an NK cell subset) is negative for a particular marker if there is no detectable presence of that particular marker, typically a surface marker, on or in the cell. In embodiments, surface expression can be determined by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting binding of the antibody to the marker, where surface expression is negative if the staining is not detectable at a level substantially greater than that detected using an isotype-matched control performing the same procedure under otherwise identical conditions, and / or is at a level substantially lower than cells known to be positive for that marker, and / or is at a level substantially similar to cells known to be negative for that marker.

[0085] In some embodiments, a cell (e.g., an NK cell subset) is underexpressing for a particular marker if the detectable presence of the particular marker, typically a surface marker, on or in the cell is at a lower level compared to cells known to be positive for that marker. In embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting binding of the antibody to the marker, where surface expression is underexpressed if the staining is at a lower level than cells known to be positive for that marker.

[0086] In some embodiments, NK cells known to be positive for a surface marker can be conventional NK cells and / or a subset of NK cells that represent the majority of NK cells present in a subject, or that represent the majority of NK cells present on average in a population of controls.

[0087] In some embodiments, the g-NK cell subset of NK cells can be detected by observing whether FcRγ is expressed by a population of NK cells or a subpopulation of NK cells. NK cells that do not express FcRγ are g-NK cells. In some embodiments, it may be useful to detect the expression of any of the other NK cell markers mentioned above as a positive identifier that the cells are natural killer cells, while also confirming that the cells do not express FcRγ. Expression of natural killer cell surface markers that correlate with expression (or lack thereof) of FcRγ can be detected by any available procedure that does not damage the cells. For example, g - NK cells can be detected, identified and / or isolated by flow cytometry through the use of such cell surface markers that correlate with FcRγ expression.

[0088] CD3ζ and / or FcRγ proteins are intracellular proteins that are not readily detected unless the cells are treated, e.g., by fixation and permeabilization, so that the intracellular proteins can be detected. Although such treatments can be used to confirm the identity of a substantially pure cell population, - When identifying and isolating NK cells, cell surface markers that can be detected without damaging the cells can often be used. In some embodiments, g-NK cells can be identified based on positive surface expression of KIR (Killer cell immunoglobulin-like receptor) and / or based on low or negative surface expression of Natural cytotoxicity receptor (NCR).

[0089] In some embodiments, the g-NK cells are positive for surface expression of KIR (e.g., KIR2DL1, KIR2DL2 / 3 and / or KIR3DL1) and can be isolated by positive selection methods or the like. -It can be used to identify and isolate NK cells. In some embodiments, the g-NK cells are surface positive for one or more of KIR2DL1, KIR2DL2 / 3 and KIR3DL1.

[0090] In some embodiments, NCRs normally expressed on NK cells (e.g., Nkp30 and / or Nkp46) are - They are not expressed or are expressed at low levels in NK cells and can be used to, for example, - NK cells can be identified and isolated. In some embodiments, g-NK cells are surface negative or low express NkP30 and / or Nkp46. In some embodiments, g-NK cells are surface positive for NKG2C. In some embodiments, g-NK cells express CD57 and low express NKG2A.

[0091] Antibodies and other binding entities are used to detect the expression levels of marker proteins (e.g., KIR and / or NCR), - NK cells can be isolated. For example, in some embodiments, an antibody specific for FcRγ expressing cells (e.g., an antibody specific for a cell surface marker such as NCR that correlates with FcRγ expression) can be bound to a solid substrate, natural killer cells can be contacted with the antibody-substrate, and cells that do not bind to the antibody can be collected. Other methods available to those skilled in the art can also be used. Suitable antibodies can include polyclonal, monoclonal, fragments (e.g., Fab fragments), single-chain antibodies, and other forms of specific binding molecules.

[0092] In some embodiments, natural killer cells can be identified based on surface expression of typical human natural killer cell markers, such as KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, CD16, CD56 and CD161. In some embodiments, a subset of natural killer cells in a population of lymphocytes is identified, comprising contacting a sample of lymphocytes with an antibody that binds to a natural killer cell-specific antigen. In one embodiment, the marker for natural killer cells is CD16, KIR2DL1, KIR2DL2 / 3, KIR3DL1, NKp30, NKp46 or a combination thereof. NK cells, including g-NK cells, can be isolated based on positive or negative / low surface expression of any of the above markers. In one approach, the method involves isolating a substantially pure population of natural killer cells, for example, based on surface expression of KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, CD16, CD56 and / or CD161.

[0093] In some embodiments, g - NK cells are identified as those with no or low expression of NCR protein surface expression. In some embodiments, the NCR is Nkp30 and / or Nkp46. For example, a population of cells containing natural killer cells can be sorted by flow cytometry using antibodies specific for NCR proteins (e.g., Nkp30 and / or Nkp46). Cells labeled by anti-NCR antibodies are discarded, and cells not labeled by anti-NCR antibodies are sorted by gating. - As cells that are NK cell-containing cells, or g - The cells containing NK cells are set aside as enriched cells. Alternatively, the population of cells containing natural killer cells can be contacted with a solid substrate to which anti-NCR antibodies are bound. Unbound cells are removed by ELISA. - NK cells or g -In another embodiment, a population of cells containing natural killer cells can be contacted with magnetic beads having anti-NCR antibodies attached thereto, and after incubation, the magnetic beads are removed from the cell population to yield a NK cell-enriched population. - NK cells or g - The NK cell enriched cells remain as a substantially pure population of cells.

[0094] In some embodiments, g - NK cells are identified as expressing one or more KIRs. In some embodiments, the KIRs are KIR2DL1, KIR2DL2 / 3 and / or KIR3DL1. For example, a population of cells containing natural killer cells can be sorted by flow cytometry using antibodies specific for KIR proteins (e.g., KIR2DL1, KIR2DL2 / 3 and / or KIR3DL1). Cells that are labeled by anti-KIR antibodies are set aside as a subset of NK cells that are g-NK cells or a subset of NK cells enriched for g-NK cells, and cells that are not labeled by anti-KIR antibodies are discarded. Alternatively, a population of cells containing natural killer cells can be contacted with a solid substrate to which anti-KIR antibodies are bound. The cells that bind are selected from the group consisting of g-NK cells, g-NK cells, and g-NK cells. - NK cells or g - In another embodiment, a population of cells containing natural killer cells can be contacted with magnetic beads to which anti-KIR antibodies are attached, and after incubation, the magnetic beads are collected to isolate or obtain cells that are g-NK cells or are enriched in g-NK cells from the cell population.

[0095] In some embodiments, g-NK cells that are surface positive for CD16 and KIR (e.g., KIR2DL1, KIR2DL2 / 3 and / or KIR3DL1) and / or surface negative or low expressing NCR (e.g., NKp30 and / or NKp46), e.g., CD16 + , K.I.R. + and N.C.R. - / lo(For example, Nkp30 - / lo and / or Nkp46 - / lo In some embodiments, g-NK cells are identified that are CD3 - CD56 dim CD14 - CD19 - They also exhibit a surface phenotype similar to that of

[0096] In some embodiments, after isolation or enrichment, CD16 158V+ NK cells or CD16 158V+ / g - The NK cell enriched cells are cultured in cell culture medium, e.g., to expand the isolated or enriched cells. In some cases, it may be necessary or desirable to culture and expand the NK cells before formulating the NK cells into a composition for administration. In some embodiments, the method of producing a composition comprising engineered NK cells includes culturing CD16 158V+ NK cells or CD16 158V+ / g NK cells prior to administering the NK cells to an individual in need thereof, e.g., to expand the cells to a therapeutically effective amount. - The method includes a step of culturing or incubating the isolated or enriched NK cells.

[0097] Suitable methods for culturing and expanding NK cells are known. For example, NK cells may be cultured using feeder cells or in the presence of cytokines to enhance their growth and / or activation. As used herein, "culturing" includes providing the chemical and physical conditions (e.g., temperature, gas) and growth factors necessary for the maintenance of NK cells. In one embodiment, culturing NK cells includes providing the NK cells with conditions for proliferation. Examples of chemical conditions that may support the proliferation of NK cells include, but are not limited to, buffers, nutrients, serum, vitamins and antibiotics, as well as cytokines and other growth factors that are typically provided in growth (i.e., culture) media. In one embodiment, the NK culture media comprises MEMα with 10% FCS or CellGro SCGM (Cell Genix) with 5% human serum / LiforCell® FBS Replacement (Lifeblood Products). Other media suitable for use in the present invention include, but are not limited to, Glascow's medium (Gibco, Carlsbad, Calif.), RPMI medium (Sigma-Aldrich, St. Louis, Mo.) or DMEM (Sigma-Aldrich, St. Louis, Mo.). It should be noted that many culture media contain nicotinamide as a vitamin supplement, for example, MEMα (8.19 μM nicotinamide), RPMI (8.19 μM nicotinamide), DMEM (32.78 μM nicotinamide) and Glascow's medium (16.39 μM nicotinamide).

[0098] In some embodiments, such as in applications where cells are introduced (or reintroduced) into a human subject, the culture is carried out using a serum-free formulation, such as AIM V™ Serum-Free Medium or MARROWMAX™ Bone Marrow Medium for Lymphocyte Culture. Such media formulations and supplements are available from commercial sources, such as Invitrogen (GIBCO) (Carlsbad, Calif.). Cultures can be supplemented with amino acids, antibiotics, and / or cytokines to promote optimal viability, growth, functionality, and / or survival.

[0099] In some embodiments, the culture of the population of cells comprising engineered NK cells is accomplished without a feeder layer or feeder cells. In some of these embodiments, the engineered NK cells can be cultured with growth factors. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12 and IL-21. According to some embodiments, the at least one growth factor is IL-2 or IL-7 and IL-15. According to some embodiments, the at least one growth factor is IL-2 alone. In some embodiments, the cells are cultured for 3 to 30 days, e.g., 7 to 28 days, e.g., at least or at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 days or more.

[0100] In some embodiments, CD16 158V+ NK cells or CD16 158V+ / g - Prior to isolation or enrichment of NK cells, IL-12, IL-15, IL-18, IL-2 and / or CCL5 are administered to the subject.

[0101] In some embodiments, cells can be isolated from a subject selected to express the desired MHC (HLA)-restricted HLA allele. In some embodiments, HLA typing of cells can be performed before or after isolation. In some embodiments, HLA typing of cells, including primary cells obtained from a subject, can be determined using procedures well known in the art, such as by performing tissue typing using molecular haplotype assays (BioTest ABC SSPtray, BioTest Diagnostics Corp., Denville, NJ; SeCore kit, Life Technologies, Grand Island, NY). In some cases, it is well within the level of a person skilled in the art to perform standard typing of cells to determine HLA genotype, such as by using sequence-based typing (SBT) (Adams et al., (2004) J. Transl. Med., 2:30; Smith (2012) Methods Mol Biol., 882:67-86).

[0102] III. Compositions and Kits Compositions comprising cells enriched for CD16 158V+ NK cells or CD16 158V+ / g-NK cells are provided herein. In some embodiments, the compositions comprise about 5-99% of CD16 158V+ NK cells or CD16 158V+ / g-NK cells, or any percentage of CD16 158V+ NK cells or CD16 158V+ / g-NK cells between 5-99%, inclusive. In some embodiments, the compositions can comprise an increased or greater percentage of CD16 158V+ NK cells or CD16 158V+ / g-NK cells relative to total NK cells or total cells compared to the percentage of CD16 158V+ NK cells or CD16 158V+ / g-NK cells relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased by at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold or more.

[0103] In some embodiments, the composition can comprise 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or substantially 100% CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In some embodiments, the composition comprises more than 50% CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In another embodiment, the composition comprises more than 70% CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In another embodiment, the composition comprises greater than 80% CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In some embodiments, the compositions provided include those in which CD16 158V+ NK cells or CD16 158V+ / g- NK cells constitute at least or at least about 60%, 70%, 80%, 85%, 90%, 95% or more of the cells in the composition, or of the NK cells in the composition.

[0104] Compositions include pharmaceutical compositions and formulations for administration, e.g., for adoptive cell therapy. In some embodiments, the engineered cells are formulated with a pharma- ceutically acceptable carrier.

[0105] Pharmaceutically acceptable carriers can include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. Supplementary active compounds can also be incorporated into the composition. Pharmaceutical carriers should be suitable for NK cells, such as saline solution, dextrose solution, or solutions containing human serum albumin.

[0106] In some embodiments, the pharma- ceutically acceptable carrier or vehicle for the above-mentioned CD16 158V+ NK cells or CD16 158V+ / g-NK cells is any non-toxic aqueous solution in which the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be maintained or in which the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can remain viable for a sufficient time to allow for administration of live CD16 158V+ NK cells or CD16 158V+ / g-NK cells. For example, the pharma-ceutically acceptable carrier or vehicle can be a saline solution or a buffered saline solution. The pharma-ceutically acceptable carrier or vehicle can also include various biological substances that can increase the potency of the CD16 158V+ NK cells or CD16 158V+ / g-NK cells. Cell culture media and cell carriers can be, for example, polysaccharides such as methylcellulose (MC Tate, DA Shear, SW Offman, DG Stein, MC LaPlaca, Biomaterials 22, 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (Suh JKF, Matthew HW T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000, each of which is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P (NIPAM-co-AA) (YH Bae, B. Vernon, CK Han, SW Kim, J. Control. Release 53, 249, 1998; H. Gappa, M. Baudys, JJ Koh, SW Kim, YH Bae, Tissue Eng. 7, 35, 2001, each of which is incorporated herein by reference in its entirety), and poly(oxyethylene) / poly(D,L-lactic-co-glycolic acid) (B. Jeong, KM Lee, A. Gutowska, YHAn, Biomacromolecules 3,865,2002, which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs LJ, Mikos A G. Cell Trans,8,345,1999, which is incorporated herein by reference in its entirety), PEO / PEG (Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West J L., Biomaterials,22,3045,2001; Bryant SJ, Anseth K S. Biomaterials,22,619,2001, each of which is incorporated herein by reference in its entirety), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymer,61,348,2005, which is incorporated herein by reference in its entirety), collagen (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001, which is incorporated herein by reference in its entirety), alginate (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990, each of which is incorporated herein by reference in its entirety).

[0107] In some embodiments, CD16 158V+NK cells or CD16 158V+ / g-NK cells can be present in the composition in an effective amount.The effective amount of activated CD16 158V+NK cells or CD16 158V+ / g-NK cells can vary depending on the patient and the type, severity and extent of disease.Therefore, the doctor can determine what is an effective amount after considering the subject's health condition, the extent and severity of disease, and other variables.

[0108] In certain embodiments, the number of cells in the composition provides a therapeutically effective amount of CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In some embodiments, the amount is an amount that reduces the severity, duration and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. In certain other embodiments, a therapeutically effective amount is a dose of cells that reduces the growth or spread of cancer in a patient or animal administered a composition described herein by at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the growth or spread of cancer in a patient (or animal) or group of patients (or animals) not administered the composition. In some embodiments, a cytotoxically effective amount is defined as the amount of NK cells that can inhibit or reduce the growth of cancer, viral and microbial cells.

[0109] In some embodiments, the composition comprises 10 5 Or about 10 5 About 10 12 cells, or approximately 10 5 About 10 8 cells, or approximately 10 6 About 10 12 cells, or approximately 10 8 About 10 11 cells, or approximately 10 9 About 10 10 In some embodiments, the composition comprises a dose of CD16 158V+ NK cells or CD16 158V+ / g- NK cell-enriched or CD16 158V+ NK cells or CD16 158V+ / g- NK cell-containing composition, the dose being 10 5 More than or about 10 5 Super, 10 6 More than or about 10 6 Super, 10 7 More than or about 10 7 Super, 10 8More than or about 10 8 Super, 10 9 More than or about 10 9 Super, 10 10 More than or about 10 10 Super, 10 11 More than or about 10 11 More than or 10 12 More than or about 10 12 In some embodiments, such amounts can be administered to a cancer patient.

[0110] In some embodiments, the volume of the composition is at least 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, inclusive, for example, 10 mL to 500 mL or about 10 mL to 500 mL, 10 mL to 200 mL or about 10 mL to 200 mL, 10 mL to 100 mL, or about 1 In some embodiments, the composition comprises at least or at least about 1×105 cells / mL, 5×106 cells / mL, 5×107 cells / mL, 5×108 cells / mL, 5×109 cells / mL, 5×101 cells / mL, 5×102 cells / mL, 5×103 cells / mL, 5×104 cells / mL, 5×105 cells / mL, 5×106 cells / mL, 5×107 cells / mL, 5×108 cells / mL, 5×109 ... 5 Cells / mL, 1 x 10 6 Cells / mL, 5 x 10 6 Cells / mL, 1 x 10 7 Cells / mL, 5 x 10 7 cells / mL or 1 x 10 8 In some embodiments, the composition has a cell density of 1×10 cells / mL, inclusive. 5 Cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 5 Cells / mL ~ 1 x 10 8Cells / mL, 1 x 10 5 Cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 5 Cells / mL ~ 1 x 10 7 Cells / mL, 1 x 10 5 Cells / mL ~ 1 x 10 6 cells / mL or approximately 1 x 10 5 Cells / mL ~ 1 x 10 6 Cells / mL, 1 x 10 6 Cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 6 Cells / mL ~ 1 x 10 7 Cells / mL, 1 x 10 6 Cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 6 Cells / mL ~ 1 x 10 8 Cells / mL, 1 x 10 6 Cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 6 Cells / mL ~ 1 x 10 7 cells / mL, or 1 x 10 7 Cells / mL ~ 1 x 10 8 cells / mL or approximately 1 x 10 7 Cells / mL ~ 1 x 10 8 cells / mL.

[0111] In some embodiments, the composition, including pharmaceutical compositions, is sterile.In some embodiments, cell isolation or concentration is carried out in a closed or sterile environment, for example, to minimize errors, user handling and / or contamination.In some embodiments, sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0112] Also provided herein is a composition suitable for cryopreserving the provided NK cells. In some embodiments, the composition comprises NK cells, which are or are enriched for CD16 158V+ NK cells or CD16 158V+ / g- NK cells, and a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s-glycerol. In some embodiments, the composition formulated for cryopreservation can be stored at low temperatures, e.g., cryogenic temperatures, e.g., at temperatures ranging from -40°C to -150°C, e.g., at or about 80°C±6.0°C.

[0113] In some embodiments, the composition can be cryogenically stored before administration to a patient. The composition can also be cryogenically stored after isolation from a mammalian subject, expansion and / or administration to a subject. For example, CD16 158V+ NK cells or CD16 158V+ / g- NK cells can be isolated or enriched, cryogenically stored, and then processed or expanded and harvested before administration to a subject. Alternatively, CD16 158V+ NK cells or CD16 158V+ / g- NK cells can be isolated, processed or expanded, and then cryogenically stored before administration to a subject.

[0114] A typical method for cryogenic storage on a small scale is described, for example, in U.S. Patent No. 6,0168,991. For small scale, cells can be cryogenically stored by low density suspension (e.g., at a concentration of about 200 x 106 / ml) in pre-chilled 5% human albumin serum (HAS). An equal volume of 20% DMSO can be added to the HAS solution. Aliquots of the mixture can be placed in vials and frozen overnight in a cryogenic chamber at about -80°C.

[0115] In some embodiments, the cryopreserved NK cells are prepared for administration by thawing. In some cases, the NK cells can be administered to a subject immediately after thawing. In such embodiments, the composition is ready for use without any further processing. In other cases, the NK cells are thawed and then further processed or activated, for example by resuspension in a pharma- ceutically acceptable carrier, incubation with an activating or stimulating agent, etc., prior to administration to a subject, washed, and resuspended in a pharma-ceutically acceptable buffer.

[0116] Also provided herein are kits that contain the provided compositions enriched with CD16 158V+ NK cells or CD16 158V+ / g-NK cells or that contain CD16 158V+ NK cells or CD16 158V+ / g-NK cells. For example, in some embodiments, provided herein are kits that contain CD16 158V+ NK cells or CD16 158V+ / g-NK cells and additional agents. In some embodiments, the additional agents include an Fc domain. In some embodiments, the additional agents are Fc fusion proteins or antibodies. In some embodiments, the additional agents are human antibodies, humanized antibodies, or chimeric antibodies. In some of these embodiments, the additional agents are full-length antibodies. Exemplary antibodies are described below.

[0117] IV. Method of Treatment In some embodiments, provided herein is a method of treating a condition in an individual comprising administering to an individual in need thereof a composition comprising or enriched in CD16 158V+ NK cells or CD16 158V+ / g- NK cells, such as any of the compositions described.

[0118] In some embodiments, the method comprises administering to the individual an effective amount of a composition containing CD16 158V+ NK cells or CD16 158V+ / g- NK cells. 5 Or about 10 5 About 10 12cells, or 10 5 Or about 10 5 About 10 8 cells, or 10 6 Or about 10 6 About 10 12 cells, or 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 In some embodiments, about 10 cells are administered to the subject. 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 or about 10 12 cells are administered to an individual. In some embodiments, the number of cells is based on the number of CD16 158V+ NK cells / kg or the number of CD16 158V+ / g- NK cells in the composition administered. In some embodiments, 10 6 Or about 10 6 ~10 10 CD16 158V+ NK cells / kg or CD16 158V+ / g- NK cells / kg are administered to the subject.

[0119] In some embodiments, the CD16 158V+ NK cells or CD16 158V+ / g- NK cells are administered to the individual immediately after isolation. In some embodiments, the CD16 158V+ NK cells or CD16 158V+ / g- NK cells are administered to the individual within 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days of isolation and engineering.

[0120] In another embodiment, the CD16 158V+ or CD16 158V+ / g- NK cells are stored or expanded by growth in culture prior to administration, e.g., by the methods described above. For example, the NK cells can be stored for more than 6 months, more than 12 months, more than 18 months, or more than 24 months prior to administration to the individual.

[0121] In some embodiments, the provided CD16 158V+ NK cells or CD16 158V+ / g- NK cells and compositions can be administered to a subject by any convenient route, including parenteral routes, such as subcutaneous, intramuscular, intravenous, and / or epidural routes of administration.

[0122] The provided CD16 158V+ NK cells or CD16 158V+ / g-NK cells and compositions can be used in methods of treating individuals with tumors or hyperproliferative disorders or microbial infections, such as viral infections, yeast infections, fungal infections, protozoan infections and / or bacterial infections. The disclosed methods of treating subjects with the provided CD16 158V+ NK cells or CD16 158V+ / g-NK cells and compositions can be combined with therapeutic monoclonal antibodies, such as anti-tumor antigen antibodies or anti-cancer antibodies, anti-viral antibodies or anti-bacterial antibodies. The provided CD16 158V+ NK cells or CD16 158V+ / g-NK cells and compositions can be administered for the treatment of animals, such as mammalian animals, such as human subjects.

[0123] In some instances, the methods include treating a hyperproliferative disorder, such as a hematological malignancy or a solid tumor. Examples of types of cancers and proliferative disorders that can be treated with the compositions described herein include, but are not limited to, leukemias (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic myelocytic (granulocytic) leukemia, and chronic lymphocytic leukemia), lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endothelial sarcoma, Ewing's tumor, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and hyperplasia. Treating and / or preventing cancer includes, but is not limited to, alleviating one or more symptoms associated with cancer, inhibiting or reducing the progression of cancer, promoting regression of cancer, and / or enhancing the immune response.

[0124] In some examples, the method includes treating a viral infection, such as an infection caused by the presence of a virus in the body. A viral infection includes a chronic or persistent viral infection. This is a viral infection that can infect a host and replicate in the host's cells for an extended period of time, usually weeks, months, or years, before becoming fatal. Viruses causing chronic infections that can be treated according to the invention include, for example, human papillomavirus (HPV), herpes simplex virus and other herpes viruses, hepatitis B and C viruses and other hepatitis viruses, human immunodeficiency virus, and measles virus, all of which can result in significant clinical disease. Long-term infection can ultimately lead to the induction of disease that is fatal to the patient, such as liver cancer in the case of hepatitis C virus. Other chronic viral infections that can be treated according to the invention include Epstein Barr virus (EBV), as well as other viruses, such as those that can be associated with tumors.

[0125] Examples of viral infections that can be treated or prevented by the compositions and methods described herein include retroviruses (e.g., human T-lymphotropic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpesviruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Ban virus, and cytomegalovirus), arenaviruses (e.g., Lassa fever virus), paramyxoviruses (e.g., viruses of the genus Morbillivirus, human respiratory syncytial virus, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantaviruses), cornaviruses, filoviruses (e.g., These include, but are not limited to, viral infections caused by viruses such as Ebola virus, flaviviruses (e.g., Hepatitis C virus (HCV), Yellow Fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., Hepatitis B virus (HBV)), orthomyoviruses (e.g., Sendai virus and influenza viruses A, B, and C), papovaviruses (e.g., papillomavirus), picornaviruses (e.g., rhinovirus, enterovirus, and Hepatitis A virus), poxviruses, reoviruses (e.g., rotavirus), togaviruses (e.g., rubella virus), and rhabdoviruses (e.g., rabies virus). Treating and / or preventing a viral infection includes, but is not limited to, alleviating one or more symptoms associated with the infection, inhibiting, reducing, or suppressing viral replication, and / or enhancing the immune response.

[0126] In some embodiments, the provided CD16 158V+ NK cells or CD16 158V+ / g- NK cells and compositions are used in methods of treating a yeast or bacterial infection. For example, the provided CD16 158V+ NK cells or CD16 158V+ / g- NK cells and compositions and methods described herein can be used to treat or inhibit Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinosclerotis, Klebsiella pneumoniae ... rhinoscleromotis, Staphylococcus aureus, Vibrio cholera, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri flexneri, Shigella sonnei, Salmonella typhimuriumtyphimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella aborts, Brucella suis, Brucella melitensis, Mycoplasma Infections associated with Rickettsia spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., Helicobacter pylori, or combinations thereof can be treated.

[0127] Combination treatment In some embodiments, the provided CD16 158V+ NK cells or CD16 158V+ / g- NK cells exhibit enhanced activity when activated or contacted with an antibody or Fc-containing protein. For example, CD16 158V+ NK cells or CD16 158V+ / g- NK cells can be activated by antibody-mediated cross-linking of CD16 by antibody-coated tumor cells.

[0128] In some embodiments, provided herein is a method of treating a condition in an individual, comprising administering to the subject CD16 158V+NK cells or CD16 158V+ / g-NK cells or compositions thereof and an antibody. One skilled in the art can select an appropriate therapeutic (e.g., anti-cancer) monoclonal antibody to administer to a subject together with the provided CD16 158V+NK cells or CD16 158V+ / g-NK cells and compositions described herein, for example, depending on the particular disease or condition of the individual. Suitable antibodies can include polyclonal, monoclonal, fragments (e.g., Fab fragments), single chain antibodies, and other forms of specific binding molecules.

[0129] In some embodiments, the antibody may further include a humanized antibody or a human antibody. A humanized form of a non-human antibody is a chimeric Ig, a chimeric Ig chain, or a chimeric Ig fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human Ig. In some embodiments, the antibody comprises an Fc domain.

[0130] Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and they are usually adopted from an "import" variable domain. Humanization is achieved by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). Such "humanized" antibodies are chimeric antibodies (1989) in which significantly less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some Fc residues are substituted with residues from analogous sites in rodent antibodies. Humanized antibodies include human antibodies (recipient antibodies) in which residues of the recipient's complementarity determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, corresponding non-human residues replace Fv framework residues of the human antibody. A "humanized antibody" may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, with most if not all of the CDR regions corresponding to those of a non-human Ig and most if not all of the FR regions being those of a human antibody consensus sequence. A humanized antibody optimally also comprises at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0131] Human antibodies can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al., 1991; Marks et al., 1991) and preparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell, 1985). Similarly, introduction of human Ig genes into transgenic animals in which the endogenous antibody genes have been partially or completely inactivated can also be used to synthesize human Abs. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire (1997a;1997b;1997c;1997d;1997;1997;Fishwild et al.,1996;1997;1997;2001;1996;1997;1997;1997;Lonberg and Huszar,1995;Lonberg et al.,1994;Marks et al.,1992;1997;1997;1997).

[0132] Specifically, the cells of the present invention can be targeted to tumors by administration together with an antibody that recognizes a tumor-associated antigen. Those skilled in the art will appreciate that the CD16 158V+ NK cells or CD16 158V+ / g- NK cells are suitable for use with a wide variety of antibodies that recognize tumor-associated antigens. Non-limiting examples of tumor-associated antigens include CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, mucin, PDGFR-α, TAG-72, CAIX, PSMA, folate binding protein, scatter factor receptor kinase, ganglioside, cytokeline, frizzled receptor, and the like. receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-α, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some cases, the antibody is an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, and an anti-CD38 antibody. Exemplary antibodies include rituximab, trastuzumab, aletuzumab, certuximab, daratumumab, veltuzumab, ofatumumab, ublituximab, and okaratuzumab. Antibodies specific to the selected cancer type can be selected, including any antibody approved for the treatment of cancer. Examples include trastuzumab (Herceptin) for breast cancer, rituximab (Rituxan) for lymphoma, and cetuximab (Erbitux) for head and neck squamous cell carcinoma. Those skilled in the art are familiar with FDA-approved monoclonal antibodies that can bind to specific tumor or disease antigens, any of which can be used according to the provided methods for treating tumors or diseases.

[0133] The CD16 158V+ NK cells or CD16 158V+ / g-NK cells and the additional agent can be administered sequentially or simultaneously. In some embodiments, the additional agent can be administered prior to administration of the CD16 158V+ NK cells or CD16 158V+ / g-NK cells. In some embodiments, the additional agent can be administered after administration of the CD16 158V+ NK cells or CD16 158V+ / g-NK cells. For example, the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be administered at a selected time point different from when the antibody specific for a selected cancer type is administered.

[0134] In certain examples, an effective amount of an antibody is administered to a subject before, after, or substantially simultaneously with a population of CD16 158V+ NK cells or CD16 158V+ / g- NK cells. In some examples, about 0.1 mg / kg to about 100 mg / kg (e.g., about 0.5 to 10 mg / kg, about 1 to 20 mg / kg, about 10 to 50 mg / kg, about 20 to 100 mg / kg, e.g., about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 24 mg / kg, about 36 mg / kg, about 48 mg / kg, about 60 mg / kg, about 75 mg / kg, or about 100 mg / kg) of an antibody is administered to a subject. A skilled clinician can select an effective amount of the antibody, taking into consideration the particular antibody, the particular disease or condition (e.g., tumor or other disorder), the general condition of the subject, any additional treatments the subject is undergoing or has undergone in the past, and other relevant factors. The subject is also administered a population of CD16 158V+ NK cells or CD16 158V+ / g- NK cells as described herein. The antibody and population of CD16 158V+ NK cells or CD16 158V+ / g- NK cells are typically administered parenterally, e.g., intravenously, although injection or infusion into or near the tumor (local administration) or administration into the peritoneal cavity can also be used. Those skilled in the art can determine the appropriate route of administration.

[0135] The CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be administered simultaneously or sequentially with antimicrobial, antiviral and other therapeutic agents. In some embodiments provided herein, the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be administered to an individual in combination with cytokines and / or growth factors. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12 and IL-21. In some embodiments, the CD16 158V+ NK cells or CD16 158V+ / g-NK cells and the cytokines or growth factors are administered sequentially. For example, the CD16 158V+ NK cells or CD16 158V+ / g-NK cells can be administered first, followed by the cytokines and / or growth factors. In some embodiments, the CD16 158V+ NK cells or CD16 158V+ / g- NK cells are administered simultaneously with a cytokine or growth factor.

[0136] In some embodiments, the subject is administered one or more cytokines (e.g., IL-2, IL-15, IL-21, and / or IL-12) to support survival and / or growth of the NK cells. The cytokines can be administered before, after, or substantially simultaneously with the NK cells. In some examples, the cytokines can be administered after the NK cells. In one specific example, the cytokines are administered to the subject within about 1-8 hours (e.g., within about 1-4 hours, within about 2-6 hours, within about 4-6 hours, or within about 5-8 hours) of administration of the NK cells.

[0137] In some embodiments, the methods provided can also include administering the CD16 158V+ NK cells or the CD16 158V+ / g- NK cells to the individual in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent can include cyclophosphamide, fludarabine, methyl prednasone. In some embodiments, the chemotherapeutic agent can include thalidomide; cisplatin (cis-DDP), oxaliplatin, carboplatin, anthracenedione, mitoxantrone; hydroxyurea, methylhydrazine derivatives, procarbazine (N-methylhydrazine, MM), adrenal cortex suppressants, mitotane (.omicron.,.rho.'-DDD), aminoglutethimide, RXR agonists, bexarotene, tyrosine kinase inhibitors, imatinib, mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), chlorambucil, ethylenimine, methylmelamine, hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl-CCNTJ), lomustine (CCNU), streptozotocin (streptozotocin), DNA synthesis antagonists, estramustine phosphate, triazines, dacarbazine (OTIC, dimethyl-triazenoimidazolecarboxamide), temozolomide, folic acid analogs, methotrexate (amethopterin), pyrimidine analogs, fluorouracil (5-fluorouracil, 5-FU, 5FTJ), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), gemcitabine, purine analogs, mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), ), pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine, topoisomerase inhibitors, amsacrine, vinca alkaloids, vinblastine (VLB), vincristine, taxanes, paclitaxel, protein-bound paclitaxel (Abraxane®), docetaxel (Taxotere®); epipodophyllotoxins, etoposide, teniposide, camptothecin, topotecan, irinotecan, dactinomycin (actinomycin D), daunorubicin The medicamentous steroids are selected from the group consisting of methadone (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin, buserelin, corticosteroids, prednisone, progestins, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, anastrozole; testosterone propionate, fluoxymesterone, flutamide, bicalutamide, and leuprolide.

[0138] In some embodiments, the cancer drug is thalidomide or a derivative thereof. In some embodiments, the cancer drug is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin. In certain embodiments, the cancer drug is selected from the group consisting of paclitaxel, Abraxane®, and Taxotere®. In one embodiment, the chemotherapeutic agent is selected from the group consisting of asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine, cyclophosphamide, paclitaxel, and gemcitabine.

[0139] V. Illustrative Embodiments The provided aspects include the following: 1. A composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ NK cell subset. 2. The composition of embodiment 1, wherein the CD16 158V+ NK cell subset does not express FcRγ chain (CD16 158V+ / g-), is surface positive for KIR, and / or is surface negative or has low expression of Nkp30 and / or Nkp46. 3. A composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+ and lacks expression of the FcRγ chain (CD16 158V+ / g-), wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ / g- NK cell subset. 4. The composition of any of embodiments 1-3, wherein CD16 158V+ comprises a sequence as set forth in SEQ ID NO:11, or a sequence exhibiting at least 85%, 90% or 95% sequence identity to SEQ ID NO:11 and comprising the 158V+ polymorphism. 5. The composition of any of aspects 1-4, wherein the NK cell subset is surface positive for KIR, and optionally for KIR2DL2 / 3. 6. The composition of any of aspects 1-5, wherein the NK cell subset is negative for or has low expression of surface expression of Nkp30 and / or Nkp46. 7. The composition of any of embodiments 1-6, wherein the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3, and negative or has low expression of surface expression of Nkp30 and / or Nkp46. 8. The composition of any of aspects 1-7, wherein the NK cell subset is surface positive for NKG2C. 9. 10 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 The composition of any of embodiments 1 to 8, comprising the cells. 10. The volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, and / or the composition is 1×10 5 From 1×10 8 The composition of any of embodiments 1-9, comprising cells / mL. 11. The composition of any of aspects 1-10, further comprising a pharma- ceutically acceptable carrier. 12. The composition of any of aspects 1-11, further comprising a cryoprotectant. 13. The composition of any of aspects 1-12, wherein the NK cell subset is primary NK cells obtained from the subject. 14. The composition of embodiment 13, wherein the subject is a human. 15. A method for enriching NK cells, comprising: (a) isolating NK cells or a subset thereof from a subject identified as having a CD16 158V+ NK cell genotype; and (b) culturing the isolated NK cells or a subset thereof under conditions to expand the cells, thereby enriching for NK cells or a subset thereof from said subject. The method comprising: 16. The method of embodiment 15, comprising, prior to step (a), screening the subject for the presence of a CD16 158V+ NK cell genotype. 17. The method of embodiment 16, wherein the NK cells or a subset thereof are isolated from a population of cells comprising lymphocytes. 18. The method of embodiment 17, wherein the population of cells comprises a peripheral blood mononuclear cell (PBMC) sample, an apheresis product, or a leukapheresis product. 19. The method of any of aspects 15 to 18, wherein an NK cell subset comprising cells that do not express the FcRγ chain (CD16 158V+ / g-) or enriched in cells that do not express the FcRγ chain (CD16 158V+ / g-) is isolated from the subject. 20. The method of embodiment 19, wherein the NK cell subset is surface positive for KIR, and optionally for KIR2DL2 / 3. 21. The method of any of aspects 15-20, wherein the NK cell subset is negative for or has low expression of surface expression of Nkp30 and / or Nkp46. 22. The method of any of aspects 15-21, wherein the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3, and negative or low expressing surface expression of Nkp30 and / or Nkp46. 23. The method of any of aspects 15-22, wherein the NK cells or a subset thereof are surface positive for NKG2C. 24. The method of any of embodiments 15-23, wherein the cells are cultured in the presence of feeder cells or in the presence of one or more cytokines, optionally IL-2, IL-15 and / or IL-7. 25. The method of any of embodiments 15-24, wherein the NK cells are cultured for a period of time to achieve at least a 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or more expansion of cells compared to the number of isolated NK cells prior to culture. 26. The method of any of aspects 15-25, wherein the isolated NK cells are cultured for 7 to 28 days, optionally for about 14, 15, 16, 17 or 18, 19, 20, or 21 days. 27. The method of any of aspects 15-26, wherein the isolated cells are cultured to achieve a therapeutically effective amount. 28. 10 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 The method of any of embodiments 15-27, wherein the isolated cells are cultured to achieve an enriched NK cell number of 10 cells. 29. The method of any of aspects 15-28, further comprising the step of HLA typing the isolated or enriched cells. 30. The method of any of aspects 15-29, further comprising the step of cryopreserving and / or formulating the cells in the presence of a cryoprotectant. 31. The method of any of aspects 15-30, comprising the step of administering IL-12, IL-15, IL-18, IL-2 and / or CCL5 to the subject prior to isolating NK cells or a subset thereof. 32. The method of any one of aspects 15 to 31, wherein the subject is a human. 33. The method of any one of aspects 15 to 31, wherein the subject is healthy. 34. A composition comprising enriched NK cells produced by the method of any one of aspects 15 to 33. 35. The composition of embodiment 34, comprising a pharma- ceutically acceptable excipient. 36. The composition of embodiment 34 or embodiment 35, comprising a cryoprotectant. 37. The composition of any of aspects 1 to 14 and aspects 34 to 36, which is sterile. 38. A kit comprising the composition of any of embodiments 1-14 and embodiments 34-37, and an additional agent for the treatment of a disease. 39. The kit of embodiment 38, further comprising instructions for administering the composition and an additional agent for the treatment of a disease or condition. 40. A composition according to any one of embodiments 1 to 14 and embodiments 34 to 37; Instructions for administering the composition in combination therapy with an additional agent for the treatment of a disease. Including the kit. 41. The kit of any of aspects 38-40, wherein the additional agent is an antibody or an Fc fusion protein. 42. The kit of embodiment 41, wherein the antibody recognizes or specifically binds to a tumor-associated antigen. 43. Tumor-associated antigens include CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, mucin, PDGFR-α, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, gangliosides, cytochrome P450, frizzled receptor, VEGF, 43. The kit of embodiment 42, wherein the integrin is VEGFR, integrin alphaVbeta3, integrin alpha5beta1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. 44. The kit of any one of aspects 41-43, wherein the antibody is a full-length antibody and / or comprises an Fc domain. 45. A method for treating a disease or condition, comprising administering to an individual in need thereof a composition according to any of embodiments 1-14 and embodiments 34-37. 46. ​​The step of administering comprises administering 1 x 10 8 Or about 1 x 10 8 From 1×10 10 cells / m 2 , or 1 × 10 6 Or about 1 x 10 6 From 1×10 10 The method of embodiment 45, comprising administration of NK cells / kg. 47. The method of embodiment 45 or embodiment 46, further comprising the step of administering an additional agent. 48. The method of embodiment 47, wherein the additional agent is an antibody or an Fc fusion protein. 49. The method of embodiment 48, wherein the antibody recognizes a tumor-associated antigen. 50. Antibodies against CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, mucin, PDGFR-α, TAG-72, CAIX, PSMA, folate binding protein, scatter factor receptor kinase, gangliosides, cytokines, frizzled receptors, VEGF, VEGFR, and integrin αV The method of embodiment 48 or embodiment 49, wherein the antibody recognizes or specifically binds to β3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-α, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. 51. The method of any one of aspects 48 to 50, wherein the antibody comprises an Fc domain and / or is a full-length antibody. 52. The method of any one of aspects 47-51, wherein the additional agent and the composition are administered sequentially. 53. The method of embodiment 52, wherein an additional agent is administered prior to administration of the composition. 54. The method of any one of aspects 47-53, wherein the additional agent and the composition are administered simultaneously. 55. The method of any one of aspects 45 to 54, wherein the disease or condition is selected from the group consisting of an inflammatory condition, an infectious disease, and a cancer. 56. The method of embodiment 55, wherein the infection is a viral infection or a bacterial infection. 57. The method of embodiment 55, wherein the disease or condition is cancer, and the cancer is leukemia or lymphoma. 58. The method of embodiment 55, wherein the disease or condition is cancer, and the cancer comprises a solid tumor. 59. The method of any one of aspects 45 to 58, wherein the individual is a human. 60. The method of any one of aspects 45-59, wherein the NK cells in the composition are allogeneic to the individual. 61. The method of any one of aspects 45-59, wherein the NK cells in the composition are autologous to the subject. EXAMPLES

[0140] VI. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0141] Example 1: Isolation and enrichment of g-158V+ NK cell subsets from human donors To identify subjects carrying the V158+ polymorphism, DNA is extracted from peripheral blood samples obtained from healthy donors. Genomic DNA is extracted from blood samples using Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Genotyping for CD16 158V+ (V158V polymorphism) is carried out using nested PCR followed by allele-specific restriction enzyme digestion, essentially as previously described (e.g., Cartron et al. (2002) Blood, 99:754-758; Koene et al. (1997) Blood, 90:1109-1114).

[0142] Briefly, the first PCR used specific primers to amplify a 1.2 kilobase fragment containing the polymorphic site: Use TIFF2024096956000012.tif12134. The first PCR assay is performed with approximately 1.25 μg of genomic DNA, 200 ng of each primer, 200 μM of each deoxyribonucleoside triphosphate (dNTP), and 1 U of Taq DNA polymerase. This first PCR is performed at 95°C for 10 min, followed by 35 cycles (each cycle includes steps of 95°C for 1 min, 57°C for 1.5 min, and 72°C for 1.5 min), and then 72°C for 8 min to achieve complete extension. In the second PCR, the primers Using TIFF2024096956000013.tif12140, a 94 base pair (bp) fragment is amplified, generating an NlaIII restriction site only in the 158V+ allele. This nested PCR is performed with 1 μL of amplified DNA, 150 ng of each primer, 200 μM of each dNTP, and 1 U of Taq DNA polymerase. The cycles include 95°C for 5 min, followed by 35 cycles (each including steps at 95°C for 1 min, 64°C for 1 min, and 72°C for 1 min), followed by 72°C for 9.5 min to complete the extension.

[0143] Amplified DNA (10 μL) is then digested with 10 U of NlaIII for 12 h at 37 °C and separated by electrophoresis on an 8% polyacrylamide gel. After staining with ethidium bromide, DNA bands are visualized under UV light. In the case of homozygous 158F donors, only one undigested band (94 bp) is visible. In heterozygous individuals, three bands (94 bp, 61 bp and 33 bp) are seen, whereas in the case of homozygous 158V+ patients, only two digested bands (61 bp and 33 bp) are obtained.

[0144] Peripheral blood mononuclear cells (PBMCs) from donors homozygous for 158V+ were obtained and stained for surface markers of PBMCs with antibodies specific for CD56 (N901), CD3 (UCHT1), CD14 (HCD14) and CD19 (HIB19) as described in Hwang et al. (2012) Int. Immunol., 24:793-802. dim CD3 - CD14 - CD19 -Non-NK cells were excluded from freshly isolated PBMCs by fluorescence-activated cell sorting by gating on cells that have a phenotype of FcRγ-deficient (g) and are surface positive for any of the KIRs KIR2DL1 (CD158a; HP-MA4), KIR2DL2 / 3 (CD158b; CH-L) or KIR3DL1 (CD158e; DX9) and / or surface negative for the NCRs Nkp46 (CD335; 9E2) or NKp30 (CD337; p30-15). - NK) are concentrated to a purity of about 70% or higher. Antibodies are obtained from commercial suppliers such as Beckman Coulter (CA, USA), BD Biosciences (CA, USA), Biolegend (CA, USA) or eBiosciences (CA, USA).

[0145] For expansion, the resulting enriched cells were cultured in NK cloning medium [5% pooled human AB serum (Cellgro, Virginia, USA), 10% fetal bovine serum (Hyclone, Utah, USA), 1 μg / ml PHA (Roche, Indiana, USA), 100 U ml -1 IL-2 and 10ng ml -1 Stimulation with PHA (1 μg ml ) was performed by limiting dilution in RPMI 1640 (Invitrogen, New York, USA) supplemented with IL-15 (Peprotech, New Jersey, USA). -1 Allogeneic PBMCs pretreated with PBS (1 hr at 37°C) and RPMI8866 cells (Sigma-Aldrich, MO, USA) pretreated with mitomycin C for 2 hr at 37°C are plated. Feeder cells are added at a density of 25,000 per well every 10-14 days for up to 2 months.

[0146] To assess the antibody-directed activity of enriched CD16 158V+ / g-NK cells, we injected approximately 100 μCi of multiple myeloma MM1S target cells. 51Label with Cr for 1 h at 37 °C, wash twice with PBS, and resuspend in RPMI medium. A total of 3 × 10 4 Target cells are plated in triplicate in V-bottom 96-well plates in the presence or absence of 10 μg / mL of the anti-CD38 antibody daratumumab. Tumor cells are incubated for approximately 20 min at room temperature and then washed twice to remove the antibody. Cells are resuspended in 100 μL of medium and an equal volume of CD16 158V+ / g- NK cells is added at various effector:target ratios ranging from 1:3 to 10:1, and the cells are incubated for 4 h. An aliquot of the supernatant is counted using a Packard Cobra Auto-Gamma 5000 series counting system (Meriden, CT, USA). Specific 51 The Cr release percentage is calculated according to the formula: Percent specific release = [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100. As a control, experiments are performed with CD56+ conventional NK cells or in the absence of anti-CD38 antibody daratumumab. The results show that CD16 158V+ / g-NK cells exhibit stronger ADCC activity than that of conventional NK cells in the presence of anti-CD38-coated tumor cells.

[0147] Similar experiments are performed using rituximab-coated CD20+ B-cell lymphoma cells. The results show that CD16 158V+ / g-NK cells exhibit a stronger ADCC-mediating ability when co-cultured with rituximab-coated EBV-transformed B-cell lymphoma cells than normal NK cells or controls.

[0148] Example 2: Evaluation of antibody-dependent cellular cytotoxicity (ADCC) of NK cell subsets 158V is a genetic polymorphism of CD16 in which the amino acid valine (V) is present instead of the common phenylalanine (F) at the 158th amino acid position of the protein (Koene et al., 1997, Blood 90:1109-14). This leads to increased CD16 expression and increased antibody affinity, which results in enhanced ADCC by CD16 158V+ NK cells (Hatjiharissi et al., 2007, Blood 110:2561-4). Specifically, 158V / V and 158V / F donors killed ARH-77 and Daudi cells by ADCC much more potently than 158F / F donors, with 158V / V donors showing the highest ability (Hatjiharissi et al., 2007).

[0149] Forty CMV seropositive donors from BloodWorks NW were screened to determine the 12 donors with the highest proportion of g-NK. g-NK cells from these donors were enriched from PBMCs by magnetic bead separation (CD3- / CD57+). Bulk NK cells from normal donors were enriched from PBMCs from donors without g-NK by magnetic bead separation (CD3- / CD57+). The proportion of g-NK cells was determined using flow cytometry with fluorescent antibodies against the following extracellular markers: CD45, 7-AAD, CD3, CD56, CD16, CD57, CD7, and CD161. After incubation with antibodies for 20 min at room temperature in the dark, cells were washed with PBS and incubated with 7-AAD. neg / CD45 pos The number of leukocytes was quantified by flow cytometry. Cells were washed, fixed with 4% paraformaldehyde, washed, and resuspended in 0.2% saponin to permeabilize the cell membrane. Fluorescently conjugated anti-FceRI antibodies were then added. After a further 15 min incubation at room temperature in the dark, cells were analyzed by flow cytometry and the percentage of g-NK was determined to be CD45 pos / 7-AAD neg / CD3 neg / CD56 pos / FceRI negIt was evaluated as a percentage of lymphocytes.

[0150] The enriched cells were tested for ADCC against the following tumor / antibody combinations: 1) SW-480 / cetuximab; 2) SKOV3 / trastuzumab; 3) SKOV3 / cetuximab; 4) MM.1S / daratumumab; and 5) MM.1S / elotuzumab.

[0151] For ADCC cytotoxicity assays against the ovarian cancer cell line SKOV3 (ATCC, Manassas, VA, USA) as targets, NK cells were incubated with CD71-labeled SKOV3 target cells (1.0 × 10 4 NK cells were co-cultured with 10% FBS-supplemented McCoy's 5A medium containing 1 μg / mL trastuzumab (anti-HER2) or 5 μg / mL cetuximab (anti-EGFR) at NK cell:target cell ratios of 0.5:1, 1:1, 2.5:1 and 5:1 (Bigley et al., 2014, Brain Behav Immuno., 39:160-71). The medium used for the ADCC assays was 10% FBS-supplemented McCoy's 5A medium containing 1 μg / mL trastuzumab (anti-HER2) or 5 μg / mL cetuximab (anti-EGFR). In each case, basal cytotoxicity was also measured in the absence of treatment antibodies. Tubes with target cells only were used to take into account spontaneous cell death (less than 10% for all assays). After 4 h of incubation at 37°C, cells were washed and stained with anti-CD3 and anti-CD56 antibodies to quantify the number of NK cells in the tubes. After a final wash, propidium iodide (PI) was added and the numbers of NK cells, live target cells, and dead target cells were determined using four-color flow cytometry (Bigley et al., 2018, J Appl Physiol, 126:842-853).

[0152] For ADCC cytotoxicity assays against multiple myeloma cell line MM.1S (ATCC, Manassas, VA, USA) as targets, NK cells were incubated with CD71-labeled MM.1S target cells (1.0 × 10 4NK cells were co-cultured with 10% FBS-supplemented RPMI-1640 medium containing 1 μg / mL daratumumab (anti-CD38) or 1 μg / mL elotuzumab (anti-CD319) at NK cell:target cell ratios of 0.5:1, 1:1, 2.5:1 and 5:1. The medium used for the ADCC assay was 10% FBS-supplemented RPMI-1640 medium containing 1 μg / mL daratumumab (anti-CD38) or 1 μg / mL elotuzumab (anti-CD319). In all cases, basal cytotoxicity was also measured in the absence of treatment antibodies. Tubes with target cells only were used to take into account spontaneous cell death (less than 10% for all assays). After 4 h of incubation at 37°C, cells were washed and stained with anti-CD3 and anti-CD56 antibodies to quantify the number of NK cells in the tubes. After the final wash, propidium iodide (PI) was added and the number of NK cells, live target cells and dead target cells was determined using four-color flow cytometry (Bigley et al., 2018).

[0153] For ADCC cytotoxicity assays against the CRC cell line SW-480 (ATCC, Manassas, VA, USA) as targets, NK cells were incubated with CD71-labeled SW-480 target cells (1.0 × 10 4 NK cells were co-cultured with 10% FBS-supplemented Leibovitz L-15 medium containing 5 μg / mL cetuximab (anti-EGFR) at NK cell:target cell ratios of 0.5:1, 1:1, 2.5:1, and 5:1. The medium used for the ADCC assay was 10% FBS-supplemented Leibovitz L-15 medium containing 5 μg / mL cetuximab (anti-EGFR). Basal cytotoxicity was also measured in the absence of cetuximab. Tubes with target cells only were used to take into account spontaneous cell death (<10% for all assays). After 4 h of incubation at 37°C, cells were washed and stained with anti-CD3 and anti-CD56 antibodies to quantify the number of NK cells in the tubes. After the final wash, propidium iodide (PI) was added and the number of NK cells, live target cells, and dead target cells was determined using four-color flow cytometry (Bigley et al., 2018).

[0154] The ADCC of g-NK was compared to conventional NK cells (c-NK) from donors without g-NK (n=4), as shown in Figure 1. Of these 12 donors, five were "super donors," meaning their ADCC values ​​were consistently high.

[0155] These donors were then evaluated to determine which subgroup each donor belonged to with respect to the CD16 158V polymorphism (V / V, V / F and F / F). The expected distribution was 35% V / V, 25% V / F and 40% F / F (Hatjiharissi et al., 2007; Somboonyosdech et al., 2012, Asian Biomedicine, 6:883-89), so any deviation from this expectation would be consistent with the finding that the 158V polymorphism may play a role in the high ADCC seen in these donors. For polymorphism testing, frozen NK cells were thawed, washed with PBS and centrifuged at 100g. After the washing steps, the supernatant was discarded and the NK cells were resuspended in 10 mL for cell counting by flow cytometry. Briefly, 50 μL of cell suspension was collected in a flow tube and stained with 2 μL of fluorescent antibody against CD45 (to distinguish white blood cells from remaining red blood cells) and 2 μL of 7-AAD (a viability dye). After 10 min of incubation at room temperature in the dark, cells were diluted with 500 μL of PBS and stained with 7-AAD. neg / CD45 pos The number of leukocytes was quantified by flow cytometry. After cell counting, CD16 pos To isolate the NK cell population, magnetic bead separation was performed. In this protocol, magnetic bead separation was performed by using Miltenyi MACS™ CD16 MicroBeads according to the manufacturer's instructions. After magnetic bead separation, 10X Genomics single-cell RNA sequencing was used to determine which CD16 polymorphism group (V / V, V / F, or F / F) the donor belonged to. As shown in Figure 1, the 158V (V / V or V / F) donor has the highest ADCC.

[0156] The disclosed specific embodiments are provided for purposes of illustrating, for example, various aspects of the invention, and the scope of the invention is not limited to those specific embodiments. Various modifications of the described compositions and methods will become apparent from the descriptions and teachings herein. Such variations can be made without departing from the true scope and spirit of the disclosure, and are intended to be encompassed within the scope of the invention.

[0157] Sequence information SEQUENCE LISTING <110> Indapta Therapeutics, Inc. <120> SUBSETS OF HUMAN NATURAL KILLER CELLS WITH ENHANCED ANTIBODY-DIRECTED IMMUNE RESPONSES <150> US 62 / 671,358 <151> 2018-05-14 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> 158V Primer <400> 1 atatttacag aatggcacag g 21 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> 158V Primer <400> 2 gacttggtac ccaggttgaa 20 <210> 3 <211> 32 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 3 atcagattcg atcctacttc tgcagggggc at 32 <210> 4 <211> 32 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 4 acgtgctgag cttgagtgat ggtgatgttc ac 32 <210> 5 <211> 24 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 5 cccaactcaa cttcccagtg tgat 24 <210> 6 <211> 30 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 6 gaaatctacc ttttcctcta atagggcaat 30 <210> 7 <211> 29 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 7 gaaatctacc ttttcctcta atagggcaa 29 <210> 8 <211> 28 <212> DNA <213> artificial sequence <220> <223> 158V Primer <400> 8 gaaatctacc ttttcctcta atagggca 28 <210> 9 <211> 238 <212> PRT <213> artificial sequence <220> <223> CD16 (F158) <400> 9 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 1 5 10 15 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 20 25 30 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 35 40 45 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 50 55 60 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 65 70 75 80 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 85 90 95 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 100 105 110 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 115 120 125 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 130 135 140 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 145 150 155 160 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 165 170 175 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 180 185 190 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 195 200 205 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 210 215 220 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 10 <211> 16 <212> PRT <213> artificial sequence <220> <223> Signal peptide <400> 10 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 <210> 11 <211> 238 <212> PRT <213> artificial sequence <220> <223> CD16 (158V+) <400> 11 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 1 5 10 15 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 20 25 30 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 35 40 45 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 50 55 60 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 65 70 75 80 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 85 90 95 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 100 105 110 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 115 120 125 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 130 135 140 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 145 150 155 160 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 165 170 175 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 180 185 190 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 195 200 205 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 210 215 220 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 12 <211> 86 <212> PRT <213> artificial sequence <220> <223> FcRgamma chain <400> 12 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85

Claims

1. A composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ NK cell subset.

2. The composition of claim 1, wherein the CD16 158V+ NK cell subset does not express FcRγ chain (CD16 158V+ / g-), is surface positive for KIR, and / or is surface negative or has low expression of Nkp30 and / or Nkp46.

3. 1. A composition comprising a natural killer (NK) cell subset that is surface positive for CD16 158V+ and lacks expression of the FcR gamma chain (CD16 158V+ / g-), wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition comprise the CD16 158V+ / g- NK cell subset.

4. The composition of any one of claims 1 to 3, wherein CD16 158V+ comprises a sequence as shown in SEQ ID NO:11 or a sequence exhibiting at least 85%, 90% or 95% sequence identity to SEQ ID NO:11 and comprising the 158V+ polymorphism.

5. The composition of any one of claims 1 to 4, wherein the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3.

6. The composition of any one of claims 1 to 5, wherein the NK cell subset is negative or has low expression of surface expression of Nkp30 and / or Nkp46.

7. The composition of any one of claims 1 to 6, wherein the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3, and negative or has low expression of surface expression of Nkp30 and / or Nkp46.

8. The composition of any one of claims 1 to 7, wherein the NK cell subset is surface positive for NKG2C.

9. 10 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 The composition of any one of claims 1 to 8, comprising cells.

10. The volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL or 500 mL, and / or the composition is 1×10 5 From 1×10 8 The composition of any one of claims 1 to 9, comprising cells / mL.

11. The composition of any one of claims 1 to 10, further comprising a pharma- ceutically acceptable carrier.

12. The composition of any one of claims 1 to 11, further comprising a cryoprotectant.

13. The composition of any one of claims 1 to 12, wherein the NK cell subset is a primary NK cell obtained from the subject.

14. The composition of claim 13, wherein the subject is a human.

15. 1. A method for enriching NK cells, comprising: (a) isolating NK cells or a subset thereof from a subject identified as having a CD16 158V+ NK cell genotype; and (b) culturing the isolated NK cells or a subset thereof under conditions to expand the cells, thereby enriching for NK cells or a subset thereof from said subject. The method comprising:

16. 16. The method of claim 15, further comprising, prior to step (a), screening the subject for the presence of a CD16 158V+ NK cell genotype.

17. The method of claim 15 or claim 16, wherein the NK cells or a subset thereof are isolated from a population of cells comprising lymphocytes.

18. 20. The method of claim 17, wherein the population of cells comprises a peripheral blood mononuclear cell (PBMC) sample, an apheresis product, or a leukapheresis product.

19. The method of any one of claims 15 to 18, wherein a NK cell subset comprising cells that do not express the FcRγ chain (CD16 158V+ / g-) or enriched for cells that do not express the FcRγ chain (CD16 158V+ / g-) is isolated from the subject.

20. 20. The method of claim 19, wherein the NK cell subset is surface positive for KIR, and optionally for KIR2DL2 / 3.

21. The method of any one of claims 15 to 20, wherein the NK cell subset is negative or has low expression of surface expression of Nkp30 and / or Nkp46.

22. The method of any one of claims 15 to 21, wherein the NK cell subset is surface positive for KIR, optionally for KIR2DL2 / 3, and negative or has low expression of surface expression of Nkp30 and / or Nkp46.

23. The method of any one of claims 15 to 22, wherein the NK cells or a subset thereof are surface positive for NKG2C.

24. The method of any one of claims 15 to 23, wherein the cells are cultured in the presence of feeder cells or in the presence of one or more cytokines, optionally IL-2, IL-15 and / or IL-7.

25. 25. The method of any one of claims 15-24, wherein the NK cells are cultured for a period of time to achieve at least a 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or more expansion of cells compared to the number of isolated NK cells prior to culture.

26. The method of any one of claims 15 to 25, wherein the isolated NK cells are cultured for 7 to 28 days, optionally for about 14, 15, 16, 17 or 18, 19, 20, or 21 days.

27. The method of any one of claims 15 to 26, wherein the isolated cells are cultured to achieve a therapeutically effective amount.

28. 10 5 Or about 10 5 About 10 12 Cells, 10 5 Or about 10 5 About 10 8 Cells, 10 6 Or about 10 6 About 10 12 Cells, 10 8 Or about 10 8 About 10 11 cells, or 10 9 Or about 10 9 About 10 10 The method of any one of claims 15 to 27, wherein the isolated cells are cultured to achieve an enriched NK cell number of cells.

29. The method of any one of claims 15 to 28, further comprising the step of HLA typing the isolated or enriched cells.

30. 30. The method of any one of claims 15 to 29, further comprising the step of cryopreserving and / or formulating the cells in the presence of a cryoprotectant.

31. The method of any one of claims 15 to 30, comprising administering to the subject IL-12, IL-15, IL-18, IL-2 and / or CCL5 prior to isolating NK cells or a subset thereof.

32. The method of any one of claims 15 to 31, wherein the subject is a human.

33. The method of any one of claims 15 to 31, wherein the subject is healthy.

34. A composition comprising enriched NK cells produced by the method of any one of claims 1 to 33.

35. 35. The composition of claim 34, comprising a pharma- ceutically acceptable excipient.

36. 36. The composition of claim 34 or claim 35, comprising a cryoprotectant.

37. The composition of any one of claims 1 to 14 and claims 34 to 36, which is sterile.

38. A kit comprising a composition according to any one of claims 1 to 14 and claims 34 to 37 and an additional agent for the treatment of a disease.

39. 40. The kit of claim 38, further comprising instructions for administering the composition and an additional agent for the treatment of a disease or condition.

40. A composition according to any one of claims 1 to 14 and claims 34 to 37; Instructions for administering the composition in combination therapy with an additional agent for the treatment of a disease. Including the kit.

41. The kit of any one of claims 38 to 40, wherein the additional agent is an antibody or an Fc fusion protein.

42. 42. The kit of claim 41, wherein the antibody recognizes or specifically binds to a tumor-associated antigen.

43. Antibodies against CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, mucin, PDGFR-α, TAG-72, CAIX, PSMA, folate binding protein, and scatter factor 43. The kit of claim 41 or claim 42, which recognizes or binds to a HER2 (HER3) receptor kinase, ganglioside, cytokerain, frizzled receptor, VEGF, VEGFR, integrin alphaVbeta3, integrin alpha5beta1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, syndecan-1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin.

44. The kit of any one of claims 41 to 43, wherein the antibody is a full-length antibody and / or comprises an Fc domain.

45. 38. A method of treating a disease or condition comprising administering to an individual in need thereof a composition according to any one of claims 1 to 14 and claims 34 to 37.

46. The step of administering comprises administering to the individual 1×10 8 Or about 1 x 10 8 From 1×10 10 cells / m 2 , or 1 × 10 6 Or about 1 x 10 6 From 1×10 10 46. ​​The method of claim 45, comprising administration of NK cells / kg.

47. 47. The method of claim 45 or claim 46, further comprising administering an additional agent.

48. 48. The method of claim 47, wherein the additional agent is an antibody or an Fc fusion protein.

49. 49. The method of claim 48, wherein the antibody recognizes a tumor-associated antigen.

50. Antibodies include CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, mucin, PDGFR-α, TAG-72, CAIX, PSMA, folate binding protein, scatter factor receptor kinase, gangliosides, cytochrome P450, frizzled receptor, VEGF, VEGFR, and integrin αVβ3.

50. The method of claim 48 or claim 49, wherein the antibody recognizes or specifically binds to integrin alpha5beta1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin or tenascin.

51. The method of any one of claims 48 to 50, wherein the antibody comprises an Fc domain and / or is a full-length antibody.

52. The method of any one of claims 47-51, wherein the additional agent and the composition are administered sequentially.

53. 53. The method of claim 52, wherein the additional agent is administered prior to administration of the composition.

54. The method of any one of claims 47-53, wherein the additional agent and the composition are administered simultaneously.

55. 55. The method of any one of claims 45 to 54, wherein the disease or condition is selected from the group consisting of an inflammatory condition, an infectious disease, and a cancer.

56. 56. The method of claim 55, wherein the disease or condition is an infectious disease, and the infectious disease is a viral infection or a bacterial infection.

57. 56. The method of claim 55, wherein the disease or condition is cancer, and the cancer is leukemia or lymphoma.

58. 56. The method of claim 55, wherein the disease or condition is cancer, and the cancer comprises a solid tumor.

59. The method of any one of claims 45 to 58, wherein the individual is a human.

60. The method of any one of claims 45 to 59, wherein the NK cells in said composition are allogeneic to said individual.

61. The method of any one of claims 45 to 59, wherein the NK cells in said composition are autologous to the subject.

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