Manipulated natural killer (NK) cells, their composition and method

Abstract

This invention provides engineered natural killer (NK) cells and methods for producing engineered NK cells. [Solution] The present invention provides engineered natural killer (NK) cells, which are genetically modified to reduce the expression, activity, and / or signaling of the FcRγ chain in the cells. The engineered NK cells and compositions comprising the engineered NK cells are useful for treating diseases such as cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority from U.S. Provisional Patent Application No. 62 / 457,098, filed on 9 February 2017, entitled "ENGINEERED NATURAL KILLER (NK) CELLS AND COMPOSITIONS AND METHODS THEREOF," and U.S. Provisional Patent Application No. 62 / 484,350, filed on 11 April 2017, whose contents are incorporated in their entirety by reference.

[0002] Inclusion by referencing sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a file named 776032000140SeqList.txt, created on February 6, 2018, and has a size of 12,126 bytes. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] Field of Invention This invention provides engineered natural killer (NK) cells and methods for producing engineered NK cells. Engineered NK cells and compositions containing engineered NK cells are useful for treating diseases such as cancer. [Background technology]

[0004] Background of the Invention NK cells were discovered 40 years ago due to their ability to recognize and kill tumor cells without the need for prior antigen exposure. Since then, NK cells have been considered a promising agent for cell-based cancer therapy. Most cancers are unaffected by antigen-specific cytotoxic T lymphocytes because, from an HLA perspective, they lack identifiable tumor-specific antigens. One of the main advantages of using NK cells is that, because a wide range of cancer cells are susceptible to NK cytotoxicity, natural killer (NK) cell transplantation can be utilized for cancer cells in an allogeneic setting without the risk of graft-versus-host disease. The therapeutic potential of NK cells has been limited because it is difficult to obtain theoretically effective quantities of NK cells, particularly those exhibiting cytotoxic effector function to kill malignant tumor cells or infected cells. Therefore, engineered NK cells for therapeutic use are needed. Embodiments that satisfy such needs are provided herein. [Overview of the project]

[0005] Engineered natural killer (NK) cells in which the expression, activity, and / or signaling of the FcRγ chain are reduced are provided herein. In some embodiments, the provided engineered cells exhibit an enhanced immune response, including in the case of treatment of cancer, microbial infection, and / or viral infection in a subject. Methods for treating a subject by administering a therapeutically effective dose of the provided engineered NK cells are also provided. In some embodiments, engineered NK cells are useful for enhancing the therapeutic response to administered therapeutic antibodies, such as anti-cancer monoclonal antibodies, anti-viral monoclonal antibodies, or anti-microbial monoclonal antibodies.

[0006] Engineered NK cells are provided herein, in some embodiments, in which NK cells are genetically engineered to reduce the expression, activity, and / or signaling of FcRγ chains in cells. In some of these embodiments, engineered NK cells include genetic disruption of a gene resulting in reduced expression, activity, and / or signaling of FcRγ chains in cells. In some cases, the genetic disruption may result in gene deletion or mutation. In some embodiments, engineered NK cells include inhibitory nucleic acids that reduce the expression of a gene resulting in reduced expression, activity, and / or signaling of FcRγ chains in cells.

[0007] In some embodiments, the engineered NK cells include genetic disruption resulting in genetic disruption of a gene encoding the FcRγ chain and / or reduced expression of the FcRγ chain in the engineered NK cells. In some embodiments, the engineered NK cells include genetic disruption resulting in genetic disruption of a gene encoding a protein that regulates the expression or activity of the FcRγ chain and / or reduced expression of a protein that regulates the expression or activity of the FcRγ chain. In some embodiments, the engineered NK cells include genetic disruption resulting in genetic disruption of a gene encoding a protein involved in FcRγ chain-dependent signaling and / or reduced expression of a protein involved in FcRγ chain-dependent signaling. In some embodiments, the engineered cells may include one or more of the above-described genetic disruptions. In some embodiments, the genetic disruption includes deletions, mutations, and / or insertions resulting in immature stop codons or frameshifts in the gene's read frame. In some embodiments, both alleles of the gene encoding the FcRγ chain, the gene encoding a protein that regulates the expression or activity of the FcRγ chain, and / or the gene encoding a protein involved in FcRγ chain-dependent signaling are disrupted in the engineered NK cells.

[0008] In some embodiments, the engineered NK cells contain inhibitory nucleic acid molecules that target genes in the NK cells, resulting in reduced expression of FcRγ chains, reduced expression of proteins that regulate FcRγ chain expression or activity, and / or reduced expression of proteins involved in FcRγ chain-dependent signaling. In some embodiments, the inhibitory nucleic acid interferes with or reduces the expression of genes encoding FcRγ chains, genes encoding proteins that regulate FcRγ chain expression or activity, and / or genes encoding proteins involved in FcRγ chain-dependent signaling. In some embodiments, the inhibitory nucleic acid includes RNA interferants. In some embodiments, the inhibitory nucleic acid includes siRNA, shRNA, or miRNA.

[0009] In some of these embodiments, the expression of FcRγ chains, proteins that regulate the expression or activity of FcRγ chains, and / or proteins involved in FcRγ chain-dependent signaling is reduced by 50%, 60%, 70%, 80%, 90%, or more than 95% or approximately 50%, 60%, 70%, 80%, 90%, or more than 95% in engineered NK cells compared to the expression of proteins in unengineered NK cells.

[0010] In some of these embodiments, the expression of a protein that modulates the expression or activity of the FcRγ chain is reduced in the engineered NK cells. In some of these embodiments, the protein that modulates the expression or activity of the FcRγ chain is a transcription factor. In some embodiments, the transcription factor is PLZF (ZBTB16) or HELIOS (IKZF2).

[0011] In some of these embodiments, the expression of proteins involved in FcRγ chain-dependent signaling is reduced in engineered NK cells. In some embodiments, the proteins involved in FcRγ chain-dependent signaling are downstream signaling molecules of FcRγ. In some of these embodiments, the downstream signaling molecule is SYK, DAB2, or EAT-2.

[0012] In some of these embodiments, FcRγ chain expression is reduced in engineered NK cells. In some embodiments, engineered NK cells include genetic disruption in the gene encoding the FcRγ chain. In some of these embodiments, genetic disruption includes deletions, mutations, and / or insertions resulting in immature stop codons or frameshifts in the gene's read frame. In some embodiments, both alleles of the gene encoding the FcRγ chain are disrupted in the genome of the engineered NK cell. In some embodiments, engineered NK cells include an inhibitory nucleic acid molecule that targets the gene encoding the FcRγ chain, thereby reducing FcRγ chain expression in the cell. In some embodiments, the inhibitory nucleic acid molecule includes a sequence complementary to the gene encoding the FcRγ chain. In some embodiments, the inhibitory nucleic acid includes an RNA interference agent. In some embodiments, the inhibitory nucleic acid includes siRNA, shRNA, or miRNA. In some embodiments, FcRγ chain expression is reduced by 50%, 60%, 70%, 80%, 90%, or more than 95% in engineered NK cells compared to expression in unengineered NK cells, or by approximately 50%, 60%, 70%, 80%, 90%, or more than 95%.

[0013] In some embodiments of the provided model, the reduction in FcRγ chain expression, activity, and / or signaling in the engineered NK cells is permanent, transient, or inducible. In some embodiments, FcRγ chain expression, activity, and / or signaling are reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to expression, activity, and / or signaling in unengineered NK cells, or by approximately 50%, 60%, 70%, 80%, 90%, or more than 95%. In some embodiments, the expression of FcRγ chain expressed in cells is undetectable by immunoblotting assays.

[0014] In some embodiments, the manipulated NK cells are derived from primary cells obtained from a subject. In some embodiments, the subject is human.

[0015] In some embodiments, the manipulated NK cells are derived from a clonal cell line. In some embodiments, the clonal cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

[0016] In some embodiments, the engineered NK cells express CD16 on their surface. In some embodiments, the engineered NK cells express a CD3-zeta (CD3ζ) chain. In some embodiments, the expressed CD16 and / or CD3ζ chain is endogenous to the NK cells. In some embodiments, the engineered NK cells are further engineered to express recombinant or heterologous CD16 and / or CD3ζ chain in the NK cells. In some embodiments, the engineered NK cells contain a recombinant or heterologous CD16 gene and / or a recombinant or heterologous CD3-zeta (CD3ζ) chain. In some embodiments, the recombinant or heterologous CD16 contains a CD16-activating mutation. In some embodiments, the CD16-activating mutation is a mutation that results in a higher affinity for IgG1. In some embodiments, CD16 contains the 158V mutation. In some embodiments, CD16 contains the 158F mutation.

[0017] Engineered NK cells with reduced surface expression of NK inhibitory receptors are also provided. In some embodiments, any of the provided engineered NK cells with reduced FcRγ chain expression, activity, and / or signaling can further reduce surface expression of NK inhibitory receptors. In some embodiments, the engineered NK cells include genetic disruption of the gene encoding the NK inhibitory receptor and / or resulting in reduced expression of the NK inhibitory receptor. In some embodiments, the engineered NK cells include inhibitory nucleic acids targeting the gene encoding the NK inhibitory receptor and / or resulting in reduced expression of the NK inhibitory receptor in the cell. In some embodiments, the inhibitory receptors are NKG2A and / or KIR2DL1. In some embodiments, the expression of inhibitory receptors such as NKG2A and / or KIR2DL1 is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% or approximately 50%, 60%, 70%, 80%, 90%, or more than 95% compared to expression, activity, and / or signaling in unengineered NK cells.

[0018] In some embodiments, engineered NK cells show increased activity when stimulated with CD16 compared to unengineered NK cells. In some embodiments, increased activity is observed after CD16 ligation via CD16 crosslinking, which can occur in the presence of the antibody due to the binding of the Fc portion of the antibody to CD16.

[0019] In some embodiments, engineered NK cells exhibit reduced surface expression of NKp46, NKp30, and / or NKp44 compared to unengineered NK cells. In some embodiments, the expression of NKp46, NKp30, and / or NKp44 is reduced in cells by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or by approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, compared to expression in unengineered NK cells.

[0020] Methods for producing engineered NK cells are also provided herein, comprising the step of genetically modifying NK cells to reduce the expression, activity, and / or signaling of FcRγ chains in the cells. In some of these embodiments, the method involves a step of disrupting or suppressing gene expression to result in reduced FcRγ chain expression, activity, and / or signaling in the cells. In some cases, genetic disruption may result in gene deletion or mutation. In some embodiments, the method involves introducing inhibitory nucleic acids into cells that reduce the expression of genes resulting in reduced FcRγ chain expression, activity, and / or signaling in the cells.

[0021] In some embodiments, the method includes a step of disrupting a gene encoding an FcRγ chain in engineered NK cells, and / or a step of disrupting a gene that results in reduced expression of the FcRγ chain. In some embodiments, the method includes a step of disrupting a gene encoding a protein that regulates the expression or activity of the FcRγ chain, and / or a step of disrupting a gene that results in reduced expression of a protein that regulates the expression or activity of the FcRγ chain. In some embodiments, the method includes a step of disrupting a gene encoding a protein involved in FcRγ chain-dependent signaling, and / or a step of disrupting a gene that results in reduced expression of a protein involved in FcRγ chain-dependent signaling. In some embodiments, the method of disrupting a gene results in a step of introducing a deletion, mutation, or insertion into the gene.

[0022] In some embodiments, the method for disrupting a gene includes the step of introducing an endonuclease engineered to target the gene into NK cells. In some embodiments, the endonuclease is a TAL nuclease, a meganuclease, a zinc finger nuclease, CRISPR-related protein 9 (Cas9), or Argonaut. In some embodiments, the endonuclease is a fusion of Cas9 with at least one guide RNA complementary to the target domain or region of the gene, or Cas9 complexed with such a guide RNA. In some embodiments, Cas9 is a Staphylococcus aureus (S. aureus) Cas9 molecule. In some embodiments, Cas9 is a Streptococcus pyogenes (S. pyogenes) Cas9 molecule. In some embodiments, the method involves introducing a Cas9-like endonuclease into cells that targets the gene encoding the FcRγ chain in NK cells.

[0023] In some embodiments, a method for suppressing gene expression includes the step of introducing an inhibitory nucleic acid into cells that targets a gene to result in a reduction in the expression, activity, and / or signaling of the FcRγ chain in the cell. In some embodiments, one or more inhibitory nucleic acid molecules are introduced into cells that target one or more genes to result in a reduction in the expression of a gene encoding the FcRγ chain, a gene encoding a protein that modulates the expression or activity of the FcRγ chain, and / or a gene encoding a protein involved in FcRγ chain-dependent signaling. In some embodiments, the inhibitory nucleic acid includes a sequence complementary to the gene encoding the FcRγ chain. In some embodiments, the inhibitory nucleic acid includes a sequence complementary to the gene encoding a protein that modulates the expression or activity of the FcRγ chain. In some embodiments, the inhibitory nucleic acid includes a sequence complementary to the gene encoding a protein involved in FcRγ chain-dependent signaling. In some embodiments, the inhibitory nucleic acid includes an RNA interference agent. In some embodiments, the nucleic acid is siRNA, shRNA, or miRNA.

[0024] In some of these embodiments, the method is performed to reduce the expression of proteins that regulate the expression or activity of FcRγ chains in NK cells. In some of these embodiments, the proteins that regulate the expression or activity of FcRγ chains are transcription factors. In some embodiments, the transcription factor is PLZF (ZBTB16) or HELIOS (IKZF2).

[0025] In some of these embodiments, the method is performed to reduce the expression of proteins involved in FcRγ chain-dependent signaling in NK cells. In some embodiments, the proteins involved in FcRγ chain-dependent signaling are downstream signaling molecules of FcRγ. In some of these embodiments, the downstream signaling molecule is SYK, DAB2, or EAT-2.

[0026] In some of these embodiments, the method is performed to reduce the expression of FcRγ chains in NK cells.

[0027] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo from a subject, such as cells isolated from a human patient. In some embodiments, the method is performed such that the reduction in expression is permanent, transient, or inducible.

[0028] In some embodiments, a method for producing engineered NK cells involves a step of performing genetic manipulation on primary NK cells obtained from a subject, such as disrupting or suppressing the expression of described genes. In some embodiments, the subject is a mammal, such as a human.

[0029] In some embodiments, the method includes (i) isolating NK cells from a sample derived from a mammalian subject, before disrupting or repressing gene expression. In some embodiments, the mammalian subject is human. In some of these embodiments, the sample includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the step of isolating NK cells includes selecting NK cells based on the surface expression of NK cell markers. In some embodiments, the NK cell markers are one or more of CD56, CD161, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, 2B4, NTB-A, CRACC, DNAM-1, CD69, and / or CD25. In some embodiments, the method includes selecting NK cells from other lymphocytes by selecting cells that do not express surface CD3, T cell antigen receptor (TCR), and / or surface immunoglobulin (Ig) B cell receptor. In some embodiments, NK cells are selected to have surface expression of CD16, or further selected. In some embodiments, NK cells express CD3ζ.

[0030] In some embodiments, the method for producing engineered NK cells involves a step of performing genetic engineering on an NK cell line. In some embodiments, the cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

[0031] In some embodiments, the method includes the step of manipulating an NK cell line to express recombinant or heterologous CD16 and / or CD3ζ. In some embodiments, the method includes the step of introducing nucleic acids encoding CD16 and / or CD3ζ into NK cells. In some embodiments, the recombinant or heterologous CD16 includes an activating mutation. In some embodiments, the activating mutation increases the affinity of CD16 to IgG. In some embodiments, CD16 includes a 158V mutation. In some embodiments, CD16 includes a 158F mutation.

[0032] In some embodiments, the method includes the step of virally transfecting NK cells with nucleic acids encoding the CD16 and / or CD3ζ genes. In some embodiments, the method includes the step of transfecting NK cells with nucleic acids encoding the CD16 and / or CD3ζ genes. In some embodiments, the method includes the step of transiently, inducibly, or persistently expressing CD16 or CD3ζ in NK cells.

[0033] In some embodiments, the method is performed such that the expression, signaling, and / or activity of the FcRγ chain is reduced by 50%, 60%, 70%, 80%, 90%, or more than 95% or approximately 50%, 60%, 70%, 80%, 90%, or more than 95% in NK cells that have not been genetically modified by the method. In some embodiments, the expression level of the FcRIγ adapter protein is undetectable by immunoblotting assays.

[0034] Methods are also provided for producing engineered NK cells with reduced surface expression of NK inhibitory receptors. In some embodiments, any of the methods provided for producing engineered NK cells with reduced FcRγ chain expression, activity, and / or signaling may further involve a step of reducing surface expression of NK inhibitory receptors. In some embodiments, such engineered NK cells are produced by a method that includes a step of disrupting the gene encoding the NK inhibitory receptor. In some embodiments, such engineered NK cells are produced by introducing inhibitory nucleic acids that target the gene encoding the NK inhibitory receptor into NK cells. In some embodiments, the inhibitory receptor is NKG2A and / or KIR2DL1. In some embodiments, such a method is carried out so that the expression of an NK inhibitory receptor such as NKG2A and / or KIR2DL1 is reduced in NK cells by 50%, 60%, 70%, 80%, 90%, or more than 95% or about 50%, 60%, 70%, 80%, 90%, or more than 95% compared to the expression of the inhibitory receptor in NK cells not genetically engineered by this method.

[0035] In some embodiments, a method provided for producing genetically modified NK cells may further include a step of growing the modified NK cells. In some embodiments, the step of growing the modified NK cells involves a step of culturing or incubating the modified NK cells in the presence of feeder cells or cytokines. In some embodiments, the step of growing the modified NK cells is carried out in vitro. In some embodiments, the step of growing the modified NK cells is carried out in vivo.

[0036] Manipulated NK cells produced by any of the above methods are also provided.

[0037] Compositions comprising any of the engineered NK cells provided herein are also provided herein. In one embodiment, the composition comprises a therapeutically effective amount of the engineered NK cells provided herein and a pharmaceutically acceptable carrier. In some embodiments, the carrier is physiological saline solution, dextrose solution, or 5% human serum albumin. In some embodiments, the composition comprises 1 × 10⁻⁶ 5 ~1 × 10 8 Contains individual cells / mL.

[0038] In some embodiments, the composition is a cryopreserved composition and / or comprises manipulated NK cells and an antifreeze agent.

[0039] Kits comprising engineered NK cells and further active agents are also provided herein. In some embodiments, the kit further includes instructions for use, e.g., instructions for administering engineered NK cells and further active agents for the treatment of disease. In some embodiments, the further active agent is an antibody or an Fc fusion protein. In some embodiments, the antibody recognizes tumor-associated antigens, viral antigens, or microbial antigens. In some embodiments, the antibody is selected from the group consisting of anti-CD20 antibodies, anti-HER2 antibodies, anti-CD52 antibodies, anti-EGFR antibodies, and anti-CD38 antibodies. In some embodiments, the antibody contains an Fc domain.

[0040] Methods for treating a condition are also provided herein, comprising the step of administering one of the provided engineered NK cells to an individual in need thereof. In some embodiments, the individual is a mammalian subject, such as a human subject. In some embodiments, the subject is a subject having cancer or an infection, such as a viral or microbial infection. In some embodiments, the method is 1 × 10⁻⁶ 8 ~1 × 10 10 individual cells / m 2 This includes the step of administering the substance to the individual.

[0041] In some embodiments, the method provided further includes a step of administering a further active agent. In some embodiments, the further active agent is an antibody or an Fc fusion protein. In some embodiments, the antibody recognizes tumor-associated antigens, viral antigens, or microbial antigens. In some embodiments, the antibody is an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, or an anti-CD38 antibody. In some embodiments, the antibody contains an Fc domain.

[0042] In some embodiments, the additional active agent and the engineered NK cells are administered sequentially. In some embodiments, the additional active agent is administered before the administration of the engineered NK cells. In some embodiments, the additional active agent and the engineered NK cells are administered simultaneously.

[0043] In some embodiments, the method includes a step of administering NK cells to treat an inflammatory condition, an infection, and / or cancer. In some embodiments, the infection is a viral or bacterial infection. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the individual expresses low-affinity FcγRIIIA. In some embodiments, the individual is human.

[0044] In some embodiments, the manipulated NK cells are allogeneic to the subject. In some embodiments, the manipulated NK cells are autologous to the subject. [Brief explanation of the drawing]

[0045] [Figure 1] This represents the activity of g-NK cells and conventional NK cells in antibody-dependent cytotoxicity (ADCC) assays in the absence or presence of the anti-CD20 antibody rituximab. [Modes for carrying out the invention]

[0046] Detailed description of the invention Engineered natural killer (NK) cells are provided herein that are genetically engineered to reduce the expression, activity, and / or signaling of FcRγ (also known as FcεRIγ). In some aspects, the provided engineered NK cells are genetically engineered to knock out (e.g., by gene disruption) or knock down (e.g., by gene silencing or repression) genes that encode FcRγ, genes that encode proteins that regulate FcRγ expression, such as transcription factors, or genes that encode proteins involved in FcRγ-dependent signaling, such as downstream signaling molecules of FcRγ.

[0047] Natural killer (NK) cells are innate lymphocytes that play a crucial role in mediating antiviral and anti-cancer immunity through cytokine and chemokine secretion, as well as through the release of cytotoxic granules (Vivier et al. Science 331(6013):44-49 (2011); Caligiuri, Blood 112(3):461-469 (2008); Roda et al., Cancer Res. 66(1):517-526 (2006)). NK cells are the third largest lymphocyte population and are important for host immune surveillance against tumor and pathogen-infected cells.

[0048] However, unlike T lymphocytes and B lymphocytes, NK cells are thought to have very limited target recognition capabilities 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)). Instead, NK cells express the activated Fc receptor CD16, which recognizes IgG-coated target cells, thereby expanding 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)). In some cases, when receptors on the surface of NK cells (such as CD16) recognize IgG1 or IgG3 antibodies bound to the cell surface, antibody-dependent cytotoxicity (ADCC) is induced. This triggers the release of cytoplasmic granules containing perforin and granzyme, leading to targeted cell death. ADCC and antibody-dependent cytokine / chemokine production are primarily mediated by NK cells.

[0049] In some cases, ADCC is the mechanism of action of therapeutic antibodies, including anti-cancer antibodies. While the use of antibodies against cancer antigens has brought significant advances in cancer treatment, patient responses to such antibodies vary. Studies of such variable responses typically focus on the direct inhibitory effects 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 can be more complex and may involve the host immune system, such as through ADCC. In some aspects, cell therapy involving the administration of NK cells can be used in conjunction with antibodies for therapeutic and related purposes.

[0050] Upon activation, NK cells produce abundant cytokines and chemokines and simultaneously exhibit potent cytolytic activity. NK cell activation can occur through direct binding of the NK cell receptor to ligands on target cells, as seen in direct tumor cell death, or through cross-linking of the Fc receptor (CD16; FcγRIII) by binding to the Fc portion of antibodies bound to antigen-carrying cells. The expression and signaling activity of several NK cell-activating receptors require physically associated adapters that transmit signals via immunoreceptor tyrosine-based activation motifs (ITAMs). Among these adapters, the FcRγ and CD3ζ chains can bind to CD16 and the innate cytotoxic receptor (NCR) as either disulfide-bonded homodimers or heterodimers, and these chains are thought to be expressed by all mature NK cells.

[0051] In some aspects, CD16 binding (CD16 crosslinking) initiates an NK cell response via intracellular signals generated through one or both of the CD16-associated adapter chains FcRγ or CD3ζ. CD16 induction leads to phosphorylation of the γ or ζ chain, which in turn recruits tyrosine kinases SYK and ZAP-70, initiating a signaling cascade resulting in rapid and potent effector function. The best-known effector function is the release of cytoplasmic granules carrying toxic proteins, which kill nearby target cells through an antibody-dependent cytotoxic process. CD16 crosslinking also triggers the production of cytokines and chemokines, which in turn activate and modulate a range of immune responses.

[0052] 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 containing perforin and proteases (granzymes) in their cytoplasm. Release from NK cells allows perforin to form pores in the cell membrane of target cells, through which granzymes and related molecules can enter and induce apoptosis. The fact that NK cells induce apoptosis rather than necrosis of target cells is significant. Necrosis of virus-infected cells releases virions, while apoptosis leads to the destruction of the virus within the cell.

[0053] The engineered NK cells provided include cells engineered to have lower FcRγ signaling adapter activity or expression. In some cases, the engineered cells are rich in the signaling adapter CD3ζ chain but lack expression of the signaling adapter FcRγ chain. In some embodiments, compared to NK cells expressing the signaling adapter FcRγ chain, these engineered NK cells exhibit dramatically enhanced activity when activated by CD16 binding, as may occur in the presence of an antibody. For example, engineered cells may be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells. Methods for producing engineered cells are also provided. The engineered NK cells and methods provided address issues related to the selection or identification of specific phenotypic NK cells, which may exist as only a small fraction of all NK cells in a subject and / or may not normally exist in the entire subject population. In some aspects, the embodiments provided allow for the engineering of NK cells with enhanced activity compared to similarly phenotypic NK cells isolated directly from a subject, but provide improved NK cell therapy that can be more readily obtained in sufficient quantities for therapeutic use, including in the case of co-administration with an antibody.

[0054] All references cited herein, including patent applications, patent publications, and scientific literature and databases, are incorporated herein by reference in whole for all purposes to the same extent as if each individual reference were specifically and individually indicated.

[0055] For the purpose of clarifying the disclosure, and not for the purpose of limitation, the detailed description is divided into the following subsections. The headings of the sections used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0056] I. Definition Unless otherwise defined, all techniques, notations, and all forms of other technical and scientific or specialized terms used herein are intended to have the same meaning as that commonly understood by those skilled in the art to which the claimed subject matter pertains. In some cases, terms are defined herein for clarity and / or for immediate reference, having a commonly understood meaning, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from that commonly understood in the art.

[0057] As used herein and in the appended claims, the singular forms (a, an, the) refer to multiple objects unless otherwise explicitly indicated. For example, a reference to "molecule" may include combinations of two or more such molecules.

[0058] As used herein, the term “approximately” means the normal range of error for each value, which is readily known to those skilled in the art. References to “approximately” values ​​or parameters herein include (and describe) aspects relating to the value or parameter itself.

[0059] As used herein, aspects and embodiments of the invention described herein are to be understood to include aspects and embodiments "comprising", aspects and embodiments "consisting of", and aspects and embodiments "consisting essentially of".

[0060] As used herein, "any" or "任选" means that the event or situation described thereafter may or may not occur, and that the description includes instances where the event or situation occurs and instances where it does not occur. For example, a group that may be optionally substituted means that the group is either unsubstituted or substituted.

[0061] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, including any fragment thereof, that contain at least a portion of the variable heavy chain and / or light chain regions of an immunoglobulin molecule that are sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen, whether natural or produced, in part or completely, by synthesis, e.g., recombinantly. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody-binding site). Typically, an antibody minimally includes all or at least a portion of the variable heavy (V H ) chain and / or variable light (V L ) chain. Generally, the pairing of V H and V L together forms an antigen-binding site, although in some cases a single V H or V L domain may be sufficient for antigen binding. An antibody can also include all or a portion of the constant region. References to antibodies herein include full-length antibodies and antigen-binding fragments. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

[0062] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antibody fragment. A full-length antibody is typically an antibody produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.) that has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4), two full-length light chains (VL-CL), and a hinge region, as well as antibodies produced by synthesis that have the same domains. Whole antibodies, in particular, include those with heavy and light chains containing an Fc region. The constant domain can be a native sequence constant domain (e.g., the human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may possess one or more effector functions.

[0063] An "antibody fragment" includes a portion of an intact antibody, the antigen-binding region of an intact antibody, and / or the variable region. Antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-bonded Fv (dsFv), Fd fragments, Fd' fragments; diabodies; and linear antibodies (Example 2 of U.S. Patent No. 5,641,870; Zapata et al., Protein Eng.). 8(10) See 1057-1062

[1995] ); single-chain antibody molecules comprising single-chain Fv (scFv) or single-chain Fab (scFab); including, but not limited to, multispecific antibodies from either of the above antigen-binding fragments and antibody fragments. For the purposes of this specification, the antibody fragment typically contains sufficient material to bind or crosslink CD16 on the surface of NK cells.

[0064] The term "autologous" refers to cells or tissues that originate within or are taken from an individual's tissues. For example, in autologous NK cell transplantation, the donor and recipient are the same person.

[0065] The term "allogeneic" refers to cells or tissues that belong to the same species or originate from the same species but are genetically different, and therefore, in some cases, immunologically incompatible. Typically, the term "allogeneic" is used to define cells transplanted from a donor of the same species to a recipient.

[0066] The term "expression" refers to the process by which polynucleotides are transcribed from a DNA template (e.g., to mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides can collectively be called "gene products." If the polynucleotides originate from genomic DNA, expression may include mRNA splicing in eukaryotic cells.

[0067] With respect to proteins or nucleic acids, the term "heterogeneous" refers to proteins or nucleic acids that originate from a different genetic source. For example, a protein or nucleic acid heterogeneous to a cell originates from an organism or individual other than the cell in which it is expressed.

[0068] As used herein, the term “introduce” encompasses a variety of methods for introducing DNA into cells, either in vitro or in vivo, including transformation, transduction, transfection (e.g., electroporation), and infection. Vectors are useful for introducing DNA-coding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors such as adenovirus vectors or adeno-associated vectors.

[0069] The term "composition" refers to any mixture of two or more products, substances, or compounds, including cells or antibodies. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. Preparations are generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

[0070] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0071] As used herein, a combination means any association between two articles or more articles. A combination may be two or more distinct articles, such as two compositions or two collectibles; a mixture thereof, such as a single mixture of two or more distinct articles; or any variation thereof. The elements of a combination are generally functionally related or associated.

[0072] As used herein, a kit is a packaged combination that may optionally include other elements, such as instructions for use of further active substances and their combinations or elements, for purposes including but not limited to therapeutic use.

[0073] As used herein, the terms “treatment” or “to treat” mean a clinical intervention designed to alter the natural course of an individual or cell being treated during the course of clinicopathology. Desired effects of treatment include a decrease in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with a disorder (e.g., eosinophil-mediated disease) are reduced or eliminated. For example, an individual is successfully “treated” if the treatment results in an improvement in the quality of life of the person with the disease, a reduction in the dosage of other drugs required to treat the disease, a decrease in the frequency of disease relapses, a decrease in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual’s survival.

[0074] "Effective dose" means the minimum effective dose and duration required to achieve a desired or demonstrated effect, including therapeutic or prophylactic outcomes. An effective dose may be provided in one or more doses. "Therapeutic effective dose" is the minimum dose of cells required to produce a measurable improvement in a particular disorder. The therapeutic effective dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight. A therapeutic effective dose may be one in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the antibody. "Prophylactic effective dose" means the effective dose and duration required to achieve a desired prophylactic outcome. Typically, but not always, a prophylactic effective dose may be less than a therapeutic effective dose because prophylactic doses are used in subjects before or in the early stages of disease.

[0075] As used herein, “individual” or “subject” refers to a mammal. “Mammals” for therapeutic purposes include humans, domesticated and livestock animals, as well as zoo animals, sporting animals, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, and cats. In some embodiments, the individual or subject is a human.

[0076] II. Manipulated NK Cells Provided herein are engineered NK cells that have been genetically modified to reduce the expression, activity, and / or signaling of the FcRγ chain in the cells. Methods for manipulating the cells are also provided. In some embodiments, the method includes the step of introducing a genetically disrupted or inhibited nucleic acid that disrupts or represses a gene, resulting in reduced FcRγ chain expression, activity, and / or signaling in the NK cells.

[0077] In some embodiments, engineered NK cells are genetically engineered to directly reduce or eliminate the expression or activity of the FcRγ chain. In some embodiments, engineered NK cells are genetically engineered to indirectly reduce or eliminate the expression or activity of the FcRγ chain, such as by reducing or eliminating the expression of proteins that regulate the expression or activity of FcRγ, for example, transcription factors that control the expression of FcRγ. In some embodiments, engineered NK cells are genetically engineered to reduce or eliminate the expression or activity of molecules involved in downstream signaling of FcRγ. In some aspects, engineered cells are retained or further engineered to transmit signals via CD3ζ, such as during CD16 binding or crosslinking.

[0078] Target for operation In some embodiments, NK cells are genetically engineered to reduce or eliminate the expression or activity of human FcRγ chain protein. In some embodiments, the engineered NK cells include genetic disruption of a gene encoding FcRγ chain or genetic disruption of a gene encoding a protein that modulates the expression or activity of FcRγ chain protein. In some embodiments, the genetic disruption results in an insertion, deletion, or mutation in the gene, such as a frameshift mutation and / or immature stop codon within the read frame. In some embodiments, one allele is disrupted. In some embodiments, both alleles are disrupted. In some embodiments, the engineered NK cells include inhibitory nucleic acids, such as siRNA or other inhibitory nucleic acid molecules, that reduce the expression of a gene encoding FcRγ chain or a gene encoding a protein that modulates the expression or activity of FcRγ chain protein.

[0079] The amino acid sequence of the FcRγ chain (also known as human (Homo sapiens) high-affinity immunoglobulin gamma Fc receptor I) is available in the NCBI database as accession number NP_004097.1 (GI:4758344) and is reproduced below as SEQ ID NO:1. TIFF2026074087000002.tif18128

[0080] The genome reference sequence for the FcRγ chain in the NCBI database is NG_029043.1 RefSeqGene. The mRNA reference sequence is 1.NM_004106.1.

[0081] Manipulated NK cells may contain mutations, disruptions, or deletions in the genes encoding the FcRγ signaling adapter, such as inactivating mutations in the exons of the FcRγ chain gene. Mutations, disruptions, or deletions may also be located in the regulatory elements of the FcRγ chain gene, such as the promoter.

[0082] Various inactivating mutations or deletions can be used to reduce the expression of the FcRγ chain. For example, mutations in the immune receptor tyrosine-based activation motif (ITAM) can be used. In some embodiments, engineered NK cells may contain an inactivating mutation in the ITAM chief. In other embodiments, the transmembrane region of the FcRγ chain can be mutated to inactivate the protein.

[0083] In some embodiments, it is also possible to indirectly reduce the expression or activity of the FcRγ chain by increasing or decreasing the expression of proteins that regulate the FcRγ chain. In some embodiments, engineered NK cells are genetically engineered to reduce or eliminate the expression of proteins that regulate the expression or activity of the FcRγ chain, such as transcription factors, e.g., PLZF or HELIOS. In some embodiments, engineered NK cells include genetic disruption of a gene encoding a protein that regulates the expression or activity of the FcRγ chain protein. In some embodiments, the genetic disruption results in an insertion, deletion, or mutation in the gene, such as a frameshift mutation and / or immature stop codon within the read frame. In some embodiments, one allele is disrupted. In some embodiments, both alleles are disrupted. In some embodiments, engineered NK cells include inhibitory nucleic acids that reduce the expression of a gene encoding a protein that regulates the expression or activity of the FcRγ chain protein, such as a transcription factor, e.g., PLZF or HELIOS.

[0084] In some embodiments, engineered NK cells may have genetic disruption, deletion, or mutation in genes that promote the transcription of FcRγ signaling adapters. In some embodiments, engineered NK cells may have genetic disruption, mutation, or deletion in genes encoding transcription factors that promote the transcription of FcRγ chains. For example, engineered NK cells may have genetic disruption in genes encoding PLZF or HELIOS transcription factors. In some embodiments, one allele of PLZF and / or HELIOS is disrupted. In some embodiments, both alleles of PLZF or both alleles of HELIOS are disrupted. In some embodiments, engineered NK cells may contain inhibitory nucleic acid molecules, such as siRNA or other inhibitory nucleic acid molecules, that reduce the expression of genes that promote the transcription of FcRγ signaling adapters. In some embodiments, engineered NK cells may contain inhibitory nucleic acid molecules that target transcription factors that promote the transcription of FcRγ chains, such as inhibitory nucleic acid molecules that target genes encoding PLZF or HELIOS transcription factors.

[0085] The amino acid sequence of PLZF is available in the NCBI database as accession number NP_001018011.1 and is reproduced below as SEQ ID NO:3. The genome reference sequence in the NCBI database is NG_012140.1. The mRNA reference sequence is NM_001018011.1. TIFF2026074087000003.tif100135

[0086] The amino acid sequence of HELIOS (IKZF2) is available in the NCBI database as accession numbers NP_057344.2 (isoform 1) and NP_001072994.1 (isoform 2). The NCBI gene identifier is NC_000002.12. The mRNA reference sequences are NM_016260.2 (isoform 1) and NM_001079526.1 (isoform 2). The gene ID is 22807.

[0087] In some embodiments, engineered NK cells are genetically engineered to reduce or eliminate proteins involved in FcRγ chain-dependent signaling. In some embodiments, engineered NK cells include a reduction in FcRγ chain-dependent signaling. For example, engineered NK cells may have reduced expression of downstream molecules involved in FcRγ chain-dependent signaling. For example, Lee and Schlums describe members of the FcRγ signaling pathway that may be suitable targets for modification and / or deletion (Lee et al., Immunity 42:431-42 (2015); Schlums et al. Immunity, 42:443-56 (2015)). In some of these embodiments, the downstream molecules include SYK, DAB2, or EAT2. The gene ID for SYK is 6850. The NCBI gene ID for DAB2 is 1601. The genomic reference sequence for DAB2 is NG_030312.1. The mRNA and protein sequences of DAB2 isoform 2 are 001244871.1 and NP_001231800.1. The mRNA and protein sequences of DAB2 isoform 1 are 2.NM_001343.3 and NP_001334.2. The NCBI gene ID for EAT2 is 175072.

[0088] In some embodiments, engineered NK cells may have genetic disruption, mutation, or deletion in genes encoding proteins involved in FcRγ chain-dependent signaling. For example, engineered NK cells may have genetic disruption in genes encoding SYK, DAB2, and / or EAT2. In some embodiments, one allele of SYK, DAB2, and / or EAT2 is disrupted. In some embodiments, both alleles of SYK, both alleles of DAB2, and / or both alleles of EAT2 are disrupted. In some embodiments, engineered NK cells contain inhibitory nucleic acid molecules, such as siRNA or other inhibitory nucleic acid molecules, that reduce the expression of genes involved in FcRγ chain-dependent signaling. In some embodiments, engineered NK cells may have inhibitory nucleic acid molecules that target signaling molecules, such as inhibitory nucleic acid molecules that target genes encoding SYK, DAB2, and / or EAT2.

[0089] Modified NK cells are also provided, genetically engineered to reduce or eliminate the expression of NK inhibitory receptors. In some aspects, the expression of NK inhibitory receptors can be reduced or eliminated by further engineering any of the provided cells, which are engineered to have reduced expression, signaling, and / or activity of the FcRγ chain as described above. In some cases, the engineered cells include disruption of the gene encoding the NK inhibitory receptor. In some embodiments, the engineered cells include an inhibitory nucleic acid molecule, such as siRNA or other inhibitory nucleic acid molecules, which reduces the expression of the gene encoding the NK inhibitory receptor. Inhibitory NK receptors are known in the art and include NKG2A (also known as KLRC1; NCBI gene ID 3821) and KIR2DL1 (NCBI gene ID 3802). NK cells may be engineered to reduce the expression of inhibitory NK receptors using interfering RNA or genetic disruption, including, for example, gene insertions, mutations, or deletions that result in stop codons or frameshifts, using any of the methods provided herein.

[0090] In the embodiments described above, such manipulated NK cells include those containing genetic disruption resulting from nucleotide substitutions, deletions, or additions. Substitutions, deletions, or additions may involve one or more nucleotides. Variations may be altered in coding regions, non-coding regions, or both. Alterations in coding regions may result in non-conservative amino acid substitutions, deletions, or additions. In some embodiments, genetic disruption includes insertions, deletions, or mutations that result in immature stop codons in a gene or frameshifts in the gene's read frame.

[0091] In some embodiments, the expression of a specific gene product, such as any of the above, in the engineered cells is reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more than 99% compared to the expression of the gene product in unengineered NK cells. In some embodiments, the expression of a specific gene product in the engineered cells is reduced to an undetectable level. In some embodiments, the expression of a specific gene product in the engineered cells is completely eliminated. In some of these embodiments, the gene is an FcRγ chain. In other embodiments, the gene may encode a protein that modulates the FcRγ chain, such as a transcription factor that promotes FcRγ chain expression, such as PLZF or HELIOS. In other embodiments, the gene may encode a protein involved in FcRγ chain-mediated signaling, such as a downstream signaling molecule SYK, DAB2, or EAT2.

[0092] In some embodiments, the expression, activity, and / or signaling of the FcRγ chain in the provided engineered NK cells are reduced by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the gene expression in unengineered NK cells. In some of these embodiments, the level of FcRγ chain expression is reduced to an undetectable level using immunoblotting assays.

[0093] In some embodiments, engineered NK cells may express CD16 (also known as CD16A or FcyRIIIa). Therefore, references to CD16 herein are not intended to refer to the glycosylphosphatidylinositol-fixed form (FcγRIIIB or CD16B). In some of these embodiments, CD16 is human CD16, either in humans or as the glycosylphosphatidylinositol-fixed form (FcγRIIIB or CD16B). Typically, the engineered cells provided express a polypeptide-fixed CD16 form that can bind to the ζ chain of the TCR-CD3 complex. CD16 binds to the antibody Fc region and initiates ADCC. In some embodiments, expression of other Fc receptor proteins is maintained in the engineered NK cells.

[0094] The genome sequence of CD16A is available in the NCBI database under the name NG_009066.1. The gene ID for CD16A is 2214. Sequence information for CD16, including genetic polymorphisms, is available under UniProt accession number P08637. The nucleic acid and protein sequences of CD16a are publicly available. For example, GenBank accession numbers NM_000569 (SEQ ID NO: 1), NM_001127596, NM_001127595, NM_001127593, and NM_001127592 disclose exemplary human CD16a nucleic acid sequences, and GenBank accession numbers NP_000560 (SEQ ID NO: 2), NP_001121068, NP_001121067, NP_00112065, and NP_001121064 disclose exemplary human CD16a protein sequences. Those skilled in the art can identify further CD16a nucleic acid and amino acid sequences that, while different from those provided herein, retain at least one activity of CD16a, such as Fc-binding activity.

[0095] CD16 is most commonly found in a form with relatively low binding affinity to the Fc portion of the IgG molecule. Another form exhibiting higher binding affinity is found in some individuals. The low-affinity and high-affinity forms of CD16 differ only in the substitution of valine (high-affinity) for phenylalanine (low-affinity) at position 158 in the matured (processed) form of the polypeptide chain. The sequence of CD16 (158F) is described in SEQ ID NO:4 (residue 158F is shown in bold and underlined). In some embodiments, CD16 (158F) is, It further includes the signal peptide shown in TIFF2026074087000004.tif4128. TIFF2026074087000005.tif33147

[0096] The sequence of CD16 158V (the polymorphism resulting in F158V) is known as VAR_003960 and has the sequence described in SEQ ID NO:6 (the 158V polymorphism is shown in bold and underlined). In some embodiments, CD16 (158V) is, It further includes the signal peptide shown in TIFF2026074087000006.tif4128. TIFF2026074087000007.tif33154

[0097] In some embodiments, the manipulated NK cells may contain a CD16 gene with an activating mutation. In some embodiments, the mutation results in a higher affinity for the IgG region. In some of these embodiments, the CD16 mutation results in a higher affinity of CD16 for IgG1, IgG2, or IgG4. In some embodiments, the CD16 mutation results in a higher affinity of CD16 for IgG1. In some embodiments, CD16 contains a 158V mutation. In some embodiments, CD16 has an amino acid sequence described in SEQ ID NO:6, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:6 and contains the 158V polymorphism. In some embodiments, the CD16 mutation is a 158F mutation. In some embodiments, CD16 has an amino acid sequence described in SEQ ID NO:4, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:4 and includes the 158F polymorphism.

[0098] In some aspects, engineered NK cells can be engineered to express recombinant CD16 or heterologous CD16. In some embodiments, even if NK cells can express CD16, they can be engineered to express higher levels of CD16 than those expressed by NK in the absence of genetic engineering and / or to express modified forms of CD16. In such examples, cells can be engineered to contain nucleic acids comprising a potent or constitutive promoter followed by the CD16 gene.

[0099] Engineered NK cells may also express other signaling adapter molecules, such as signaling molecules with an ITAM domain other than FcRγ. In some embodiments, engineered NK cells express a CD3ζ adapter chain. In some of these embodiments, CD3ζ is human CD3ζ.

[0100] The amino acid sequence of human CD3ζ (human) is available in the NCBI database under accession number ABQ28690.1 (GI:146399947), and is reproduced below as SEQ ID NO:2. TIFF2026074087000008.tif17132

[0101] The NCBI gene ID for CD3ζ (CD247) is 919.

[0102] In some aspects, engineered NK cells can be engineered to express recombinant CD3ζ or heterologous CD3ζ. In some embodiments, even if NK cells can express CD3ζ, they can be engineered to further express higher levels of CD3ζ than those expressed by NK in the absence of genetic engineering and / or to express modified forms of CD3ζ. In such examples, cells can be engineered to contain nucleic acids comprising a potent or constitutive promoter followed by the CD3ζ gene. In some embodiments, engineered cells may contain a CD3ζ gene with an activating mutation. In other embodiments, engineered NK cells may express CD3ζ at even higher levels after engineering.

[0103] In some embodiments, engineered NK cells may have reduced expression of NK cell surface receptors, including innate cytotoxic receptors. Engineered NK cells may have reduced expression of NKp46, NKp30, and / or NKp44 compared to unmodified NK cells. In some of these embodiments, cell surface receptor expression is reduced by more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 10-fold, 100-fold, or 1000-fold. In some of these embodiments, NK cell surface receptors may be undetectable in engineered NK cells. In other embodiments, NK cells may be negative for NK cell surface receptors such as NKp46, NKp30, and / or NKp44.

[0104] cell type Those skilled in the art will understand that both primary cells obtained from human tissues and existing cell lines are suitable for manipulation. For example, in some embodiments, the manipulated NK cells may be derived from primary cells obtained from subjects such as humans.

[0105] primary cells Primary cells can be manipulated using any of the methods provided herein. According to some embodiments, a population of cells including NK cells is obtained from a sample from a mammalian subject, such as a human subject. The sample or source may be umbilical cord blood, bone marrow, or peripheral blood.

[0106] In some cases, natural killer cells expressing one or more natural killer cell-specific markers can be isolated from a cell population. Methods for isolating and identifying NK cells are well known in the art, including those discussed in Dahlberg et al, Frontiers in Immunology, vol. 6 article 605 pp. 1-18 (2015).

[0107] Techniques for in vitro isolation and large-scale proliferation of NK cells are known. Exemplary procedures are described in U.S. Patent Application Publication No. 2014 / 0086890, which is incorporated herein by reference in its entirety. Those skilled in the art can identify further methods for proliferation of NK cells, for example, as described in Childs et al., Hematol. The Education Program 2013:234-246, 2013, which is incorporated herein by reference in its entirety.

[0108] In some embodiments, mononuclear cells are collected from subjects (such as donor subjects or subjects with tumors or hyperproliferative diseases). In some cases, mononuclear cells are collected by apheresis procedures. Mononuclear cells are enriched for NK cells, for example, by negative depletion using an immunomagnetic bead strategy. In some cases, NK cells are enriched by depleting mononuclear T cell samples, B cells, monocytes, dendritic cells, platelets, macrophages, and erythrocytes using a mixture of biotinylated monoclonal antibodies. Non-NK cells in the sample are removed with streptavidin-coupled magnetic beads, resulting in an enriched NK cell preparation. An exemplary commercially available kit for this method is the Dynabeads® Untouched® Human NK Cells kit (Thermo Fisher Scientific, Waltham, MA). In another example, NK cells utilize magnetic beads conjugated with anti-CD56 antibodies (e.g., CD56 MicroBeads, Miltenyi Biotec, Inc., Auburn, CA) to target CD56 + NK cells are enriched by positive selection. In other cases, CD56 is used to enrich NK cells. + A two-step method is used, which includes negative selection of NK cells (e.g., T cell depletion) followed by positive selection.

[0109] In some embodiments, natural killer cells may be identified as those expressing typical human natural killer cell markers such as KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, CD56, and CD161. In studies involving mice, natural killer cells can be identified and / or isolated using typical mouse markers such as NK1.1, CD122, the LY49 family (Ly49A, Ly49C, Ly49D, Ly49E, Ly49F, Ly49G, Ly49H, and Ly49I), or NKG2A / C / E. In some embodiments, cell staining or FACS may be used to identify cells expressing certain markers.

[0110] In some embodiments, NK cells can be selectively enriched using either positive or negative selection. For example, NK cells that do not express CD3 can be selected by exposing a mixture of cells to an immobilized anti-CD3 antibody and removing the unbound cells. According to some embodiments of the present invention, NK cells include CD56+CD3- cells. According to some embodiments of the present invention, NK cells include CD56+CD16+CD3- cells.

[0111] In one embodiment, cytokines can be targeted and administered before isolation of primary NK cells. For example, IL-12, IL-15, IL-18, IL-2, and / or CCL5 can be targeted and administered before isolation of primary NK cells.

[0112] Furthermore, it may be beneficial to enrich isolated primary NK cells for those expressing CD16 and CD3ζ. Those skilled in the art will understand that there are many methods for selectively enriching cells expressing certain markers from an existing population, such as fluorescent cell sorting or selective depletion of cells expressing certain markers using solid-phase-bound antibodies.

[0113] In some embodiments, this method may be carried out under or in accordance with Current Good Manufacturing Practice (cGMP). Those skilled in the art may identify other methods that can be used to prepare enriched NK cell populations.

[0114] In some embodiments, enriched NK cells (typically over 99% CD3-negative and over 85% CD56-positive) are grown in vitro before or after genetically engineering the cells.

[0115] In one non-limiting example, enriched NK cells were cultured for up to 21 days with irradiated EBV-LCL feeder cell lines (SMI-LCL) in X-VIVO® 20 medium (Lonza, Basel, Switzerland) containing 10% human AB serum and 500 IU / ml interleukin-2 (IL-2). Using this technique, NK cell proliferation can be achieved in the range of 200-1000-fold (the proliferated NK cells are typically over 99% CD3-negative and over 90% CD56-positive). In some examples, the initial population of enriched NK cells was approximately 0.8–1.6 × 10⁶. 8 These are total NK cells, which are grown in vitro up to 1000-fold or more over 2-4 weeks. Similar numbers of NK cells have been grown in scale-up experiments using GMP conditions. In some cases, NK cells are grown in G-Rex® containers (Wilson Wolf, New Brighton, MN). A G-Rex® 100 container holds 2.5 × 10⁶ cells. 8 Supports NK proliferation to doses of 100 NK cells / kg or more. NK cells cultured in G-Rex® 100 containers reach up to 4 × 10⁶ 6 They can be cultured at a concentration of NK cells / ml.

[0116] In some embodiments, bulk NK cells or NK cell subsets isolated by further enrichment procedures, such as the use of immunomagnetic beads or fluid sorting, may be grown in cell culture media, e.g., Cellgro SCGM serum-free medium (CellGenix, Gaithersburg, MD) containing 10% human AB serum, 50 U / mL penicillin, 50 μg / mL streptomycin, and 500 IU / mL IL-2, or in X-VIVO® 20 medium containing 10% thermo-inactivated human AB serum or 10% autologous serum.

[0117] Non-proliferating and proliferating NK cells can be analyzed by flow cytometry for the expression of markers such as CD56, CD16, TRAIL, FasL, NKG2D, LFA-1, perforin, and granzymes A and B. In some cases, the expression of one or more markers is measured at baseline and after 10 days of proliferation in vitro. Chromium-releasing assays can be used to evaluate the cytotoxicity of fresh NK cells versus proliferating NK cells against cancer cell targets. Those skilled in the art can identify other methods for evaluating NK cell populations (e.g., purity), viability, and / or activity.

[0118] In some embodiments, the proliferated primary cells derived from the subject may be proliferated and / or cultured prior to the genetic manipulation. In some embodiments, the manipulated primary cells are cultured and / or proliferated after the manipulation and prior to administration to the patient.

[0119] NK cell line In some embodiments, NK cell lines can be manipulated as described herein. In some embodiments, manipulated NK cells include manipulated NK cell lines. In some aspects, manipulated cell lines enable the production of a larger number of cells without the need to proliferate a small number of NK cells derived from the subject. Manipulated cell lines also have the advantage of being well-characterized.

[0120] In some embodiments, the cell line is a clonal cell line. In some embodiments, the cell line is derived from a patient with NK cell leukemia or lymphoma. In some embodiments, the NK cell line includes NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

[0121] NK-92 is an NK-like cell line initially isolated from the blood of subjects with large granular lymphoma and subsequently propagated in cell culture. The NK-92 cell line has been described (Gong et al., 1994; Klingemann, 2002). NK-92 cells possess the CD3- / CD56+ phenotype characteristic of NK cells. They express all known NK cell activating receptors except CD16, but lack all known NK cell inhibitory receptors except NKG2A / CD94 and ILT2 / LIR1, which are expressed at low levels. Furthermore, unlike polyclonal NK cells isolated from blood, NK-92 is a clonal cell line that expresses these receptors consistently in terms of both type and cell surface concentration. Similarly, NK-92 cells are not immunogenic and do not induce immune rejection when administered therapeutically to human subjects. In fact, NK-92 cells are well-tolerated in humans, and no adverse effects on normal tissues are known.

[0122] Some existing cell lines, such as NK-92, do not express CD16 in nature. In some embodiments, the methods provided herein include a step of manipulating an NK cell line, such as NK-92, to express CD16. Klingemann et al. also discuss the advantages of the NK92 cell line. Klingemann et al, Frontiers in Immunology, vol 7, article 91 pp 1-7 (2016). In some embodiments, the methods provided herein include a step of manipulating a cell line to express CD3ζ. In some embodiments, CD16 and / or CD3ζ can be expressed inductively or transiently. In such embodiments, the cell line is not manipulated to heterologously or recombinantly express FcRγ chains, or is manipulated to reduce or eliminate FcRγ chain expression in cells.

[0123] Method for producing manipulated NK cells Provided herein are methods for producing engineered NK cells that are genetically engineered to reduce the expression, activity, and / or signaling of the FcRγ chain in cells as described. For example, the method may include introducing genetic disruption of a gene encoding the FcRγ chain, a gene encoding a protein that modulates the expression or activity of the FcRγ signaling adapter (a transcription factor such as PLZF or HELIOS), and / or a gene encoding a protein involved in FcRγ-mediated signaling (a downstream signaling molecule such as SYK, DAP2, or EAT2). In some embodiments, the method may include introducing inhibitory nucleic acid molecules that target a gene encoding the FcRγ chain, a gene encoding a protein that modulates the expression or activity of the FcRγ signaling adapter (a transcription factor such as PLZF or HELIOS), and / or a gene encoding a protein involved in FcRγ-mediated signaling (a downstream signaling molecule such as SYK, DAP2, or EAT2).

[0124] In some embodiments, the methods provided herein include the steps of isolating NK cells from a subject, for example, by the methods described above or known to those skilled in the art, and reducing the expression, activity, and / or signaling of FcRγ chains in the cells according to the methods provided. In some embodiments, the methods provided herein include the steps of obtaining an NK cell line such as any of the methods described herein, and manipulating the cells to reduce the expression, activity, and / or signaling of FcRγ in the cells according to the methods provided.

[0125] Those skilled in the art will understand that there are many ways to reduce the expression or activity of FcRγ. For example, the transcription level can be reduced. One method of reducing gene expression, such as FcRγ chain expression, involves modifying an endogenous gene to reduce transcription. For example, the FcRγ chain gene may be deleted, disrupted, or mutated. In addition to targeting FcRγ RNA, mutating, or modifying the FcRγ gene, FcRγ protein levels can be reduced by acting on molecules that increase FcRγ gene expression or activity, such as transcription factors that regulate FcRγ transcription. In some embodiments, a gene that regulates the transcription or translation of the FcRγ chain gene may be deleted, disrupted, or mutated. In some of these embodiments, the gene is a transcription factor that regulates the expression of the FcRγ chain gene. Specifically, inhibition of a transcription factor that positively regulates FcRγ expression results in a decrease in FcRγ expression. Transcription factors that regulate FcRγ transcription include HELIOS and PLZF.

[0126] Those skilled in the art will understand that there are many suitable methods for disrupting the FcRγ chain gene or other genes, such as those described herein. For example, an entire locus, such as the FcRγ locus, may be deleted. In some cases, it may also be appropriate to delete a portion of the gene, such as an exon or domain. Specifically, the ITAM signaling domain of FcRγ may be deleted. Alternatively, the methods provided include the step of introducing one or more amino acid substitutions into a locus, such as the FcRγ locus, such as an inactivating mutation. In some embodiments, a stop codon may be introduced into mRNA, such as FcRγ mRNA, to produce a cleaved and / or inactivated version of an expressed gene, such as an FcRγ signaling adapter. In some embodiments, regulatory elements of a gene, such as the FcRγ gene, may also be mutated or deleted to reduce the expression, activity, and / or signaling of the FcRγ signaling adapter.

[0127] In some embodiments, gene disruption can be carried out in mammalian cells using site-specific endonucleases. Endonucleases that enable site-specific deletion of genes are well known in the art and may include TAL nucleases, meganucleases, zinc finger nucleases, Cas9, and Argonaut. Methods for producing engineered site-specific endonucleases are known in the art. Site-specific endonucleases can be engineered to recognize and delete or modify specific genes, such as the FcRγ chain gene.

[0128] In one embodiment, zinc finger nucleases (ZFNs) can be engineered to recognize and cleave specific sites in the genome. ZFNs are chimeric proteins containing a zinc finger DNA-binding domain fused to the nuclease domain of a Fokl restriction enzyme. The zinc finger domain can be redesigned by rational or experimental means to produce a protein that binds to a specific DNA sequence of approximately 18 base pairs in length. By fusing this engineered protein domain to a Fokl nuclease, it is possible to target DNA cleavage with genome-level specificity. ZFNs have been widely used to target gene addition, removal, and substitution in a wide range of eukaryotes (as outlined in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)).

[0129] In another embodiment, TAL effector nucleases (TALENs) can be constructed to cleave specific sites in genomic DNA. Similar to ZFNs, TALENs contain a genetically engineered site-specific DNA-binding domain fused to a Fok1 nuclease domain (as outlined in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9). However, in this case, the DNA-binding domain contains a tandem array of TAL effector domains, each specifically recognizing a single DNA base pair. Since ZFNs and TALENs are heterodimers, and co-expression of two protein monomers is required for the production of a single functional nuclease in the cell, compact TALENs provide an alternative endonuclease configuration that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4: 1762). Compact TALENs contain an engineered site-specific TAL effector DNA-binding domain fused to the nuclease domain from I-TevI ​​homing endonuclease. Unlike Fokl, I-TevI ​​does not need to dimerize to produce double-strand DNA breaks, so compact TALENs function as monomers.

[0130] In some embodiments, manipulated endonucleases based on the CRISPR / Cas9 system are also known in the art and can be used to manipulate cells in the provided methods (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al. (2013) Nat Methods. 10:957-63). A CRISPR endonuclease consists of two components: (1) a caspase effectanuclease, typically microbial Cas9; and (2) a short “guide RNA” that directs the nuclease to a site of interest in the genome. In some embodiments, the guide RNA contains a targeting sequence of approximately 20 nucleotides. By expressing multiple guide RNAs, each with a different targeting sequence, in the same cell, it is possible to simultaneously target multiple sites in the genome for DNA cleavage. Methods using CRISPR-Cas9 are well known in the art.

[0131] In some contexts, the guide sequence is any polynucleotide sequence that contains at least a segment of the target polynucleotide sequence that is sufficiently complementary to the target sequence, such as a gene encoding FcRγ, PLZF, HELIOS, SYK, DAB2, or EAT2, in order to hybridize with the target sequence and direct the CRISPR complex to sequence-specific binding to the target sequence. Typically, in the context of CRISPR complex formation, the “target sequence” generally refers to the sequence in which the guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and facilitate CRISPR complex formation. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher, or exceeds approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher, when optimally aligned using a suitable alignment algorithm. In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm.

[0132] In some embodiments, a CRISPR enzyme (e.g., Cas9 nuclease) combined with (and optionally complexed with) a guide sequence is delivered to the cell. In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus.

[0133] In one embodiment of the present invention, the DNA cleavage inducer is an engineered homing endonuclease (also known as “meganucleases”). Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base pair cleavage sites commonly found in plant and fungal genomes. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They spontaneously promote homologous recombination or gene insertion at specific locations in the host genome by inducing double-strand breaks in chromosomes that mobilize cellular DNA repair mechanisms (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that influence catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). LAGLIDADG homing endonucleases with a single copy of the LAGLIDADG motif form homodimers, while members with two copies of the LAGLIDADG motif are found as monomers.

[0134] Another method for reducing FcRγ chain expression, activity, and / or signaling involves introducing an inhibitory nucleic acid, such as an inhibitory RNA, into cells that targets a target gene transcript, such as the FcRγ PLZF, HELIOS, SYK, DAB2, or EAT2 gene transcript, and is complementary to the target gene transcript, thereby reducing the expression of the gene product. For example, the nucleic acid may target FcRγ chain mRNA. In other embodiments, the inhibitory nucleic acid may target the mRNA of a transcription factor, such as PLZF or HELIOS mRNA, a gene that regulates the transcription or translation of the FcRγ chain gene. In some embodiments, the nucleic acid targets the mRNA of a gene that encodes a protein involved in FcRγ-mediated signaling, such as SYK, DAB2, or EAT-2 mRNA.

[0135] The subject matter of this disclosure is the use of RNAi techniques (e.g., shRNA, siRNA, and miRNA molecules, as well as ribozymes) to induce downregulation of cellular genes, a process known as RNA interference (RNAi). As used herein, “RNA interference” (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing mediated by small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules, miRNA molecules, or synthetic hammerhead ribozymes. See Fire et al., Nature 391:806-811, 1998 and U.S. Patent No. 6,506,559 for more details. The process of post-transcriptional gene silencing via RNA interference (RNAi) is considered an evolutionarily conserved cellular defense mechanism that has evolved to prevent the expression of foreign genes (Fire, Trends Genet 15:358-363, 1999).

[0136] In some embodiments, recombinant viruses containing RNA-encoding nucleic acids can be produced. The manipulation of retroviral vectors is known to those skilled in the art. Such persons will readily understand, without needing further detailed discussion herein, several factors involved in the selection of appropriate viruses and vector components required to optimize recombinant virus production for use in the subject matter of this disclosure. As one non-limiting example, retroviruses containing shRNA-encoding DNA, including siRNA, can be manipulated.

[0137] Gene expression can be permanently, transiently, or inductively reduced. Suitable induction systems are well known and include heavy metal-responsive eukaryotic promoters, Lac / VP16, and tetracycline repressor systems.

[0138] On the other hand, it may be beneficial to permanently reduce gene expression by, for example, producing cell lines that have deletions, substitutions, or insertions that cause gene inactivation.

[0139] cDNA can be introduced into NK cells using retroviral systems. Methods for transfection of eukaryotic cells and transformation of prokaryotic cells are well known in the art. The choice of host cell determines the preferred technique for introducing the polynucleotide of interest. The introduction of polynucleotides into organisms can also be performed in specific organisms using ex vivo techniques with in vitro transfection methods, if any, as well as established genetic techniques.

[0140] Other vectors and packaging cell lines have been used in the preparation of genetically modified variants of NK cells and may be used similarly herein. Retroviral transduction systems have also been successfully used to transduce various genes into NK cells. Examples of these alternative methods include, but are not limited to, the p-JET vector combined with FLYA13 packaging cells (Gerstmayer et al., 1999), the plasmid-based kat retroviral transduction system, and DFG-hIL-2-neo / CRIP (Nagashima et al., 1998). Electroporation of vectors into packaging cells and "gene gun" transduction have also been practiced. The use of the pBMN-IRES-EGFP vector in combination with the Phoenix-Amphotropic packaging cell line is advantageous for the purposes of this and the following examples in that it provides high efficiency of Phoenix-Amphotropic cell transfection; the use of the Moloney LTR promoter results in high levels of CD16 expression; the virus is produced at high titers; the efficiency of NK transduction is improved compared to other vectors used to transduce NK cells; and the vector provides sufficient space to accommodate CD16 cDNA or alternative insertion fragments. The pBMN-IRES-EGFP vector further incorporates the gene for enhanced green fluorescent protein (EGFP), which can be used as an endogenous surrogate marker of gene expression. The Phoenix cell line stably expresses this vector in an episomal form along with the production of other viral components, thus allowing the cells to stably produce the virus over a long period.

[0141] Methods for introducing and expressing genes in cells are well known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0142] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0143] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents No. 5,350,674 and No. 5,585,362.

[0144] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0145] When nonviral delivery systems are used, an exemplary delivery medium is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another context, nucleic acids can be bound to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that bind to both liposomes and oligonucleotides, encapsulated in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, containing or complexed with micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or with "disintegrated" structures. They can also simply be dispersed in solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds including long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0146] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various single and multi-membrane lipid media formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, encapsulating water and dissolved solute between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with solution structures different from the usual vesicle structure are also included. For example, lipids can take on micelle structures or simply exist as heterogeneous aggregates of lipid molecules.

[0147] Various assay methods can be used to confirm the presence of recombinant DNA sequences within host cells, regardless of the method used to introduce exogenous nucleic acids into host cells or to manipulate cells by the otherwise provided method. Such assay methods include, for example, “molecular biological” assay methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, or “biochemical” assay methods, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blotting).

[0148] Characteristics of manipulated cells The expression of gene products associated with the described gene manipulation can be evaluated following or in connection with the manipulation and / or culture of NK cells. The gene products can be detected using any available procedure known to those skilled in the art, provided that the procedure does not damage the cells. For example, NK cells can be detected, identified, and / or isolated by flow cytometry for the detection of cell surface markers that correlate with gene product expression, such as FcRγ expression. Targets for which expression is regulated by the described gene manipulation, such as the FcRγ protein, are intracellular proteins that are not readily detectable unless the cells are treated, for example, by fixation and permeabilization, to allow for the detection of intracellular proteins.

[0149] In some embodiments, the methods provided herein result in the expression of a specific gene product in engineered cells that is reduced by more than approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the expression of the gene product in unengineered NK cells. In some embodiments, the methods provided herein reduce FcRγ chain expression by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the expression of FcRγ chain in unengineered NK cells. In some of these embodiments, the level of FcRγ chain expression is reduced to an undetectable level using an immunoblotting assay.

[0150] The expression of described gene products, such as FcRγ chain expression, can be measured by various methods. For example, RNA expression can be measured by Northern blotting, qPCR, and FISH. Protein expression can also be measured using methods such as flow cytometry, Western blotting, immunohistochemistry, and ELISA.

[0151] In some aspects, the provided engineered NK cells exhibit enhanced activity upon activation by antibodies, such as by antibody-mediated crosslinking of CD16 or by antibody-coated cells, e.g., antibody-coated tumor cells. In some embodiments, the provided engineered NK cells are particularly responsive in the presence of antibodies or other Fc-containing proteins and can be used in combination with administered monoclonal antibodies or other Fc-containing proteins specific to tumor, viral, or microbial cells. In some cases, the provided engineered NK cells are g -These cells exhibit the same or similar properties or characteristics as NK cells, and are a specific subset of NK cells lacking FcRγ, which are present in small numbers in individuals but in only about one-third of the population (see, for example, published patent application number US2013 / 0295044; also see Hwang et al. (2012) Int. Immunol., 24:793-802 and Lee et al. (2015) Cell Immunity, 42:431-442).

[0152] In some embodiments, an increase in activity is observed after CD16 ligation by CD16 crosslinking, which may occur in the presence of the antibody due to binding of the antibody's Fc portion to CD16 and initiation of ADCC. In some embodiments, the increase in activity can be determined by monitoring of CD3ζ chain phosphorylation, signaling molecules, CA2+ efflux, expression or secretion of cytokines (e.g., IFN-gamma or TNF-α) by cells, expression or secretion of chemokines (MIP-1α, MIP-1β, or RANTES) by engineered cells, downregulatory responses, granzyme B expression, and / or cytotoxicity / death responses. Engineered NK cells can be characterized or their activity evaluated using any of several well-known assay methods (see, e.g., Hwang et al. (2012) Int. Immunology, 24:793-802; published patent application number US2013 / 0295044). In some embodiments, the activity of engineered NK cells after CD16 crosslinking or ligation increases by 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more compared to the activity of engineered NK cells in the same assay but in the absence of CD16 crosslinking or ligation, or by approximately 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

[0153] In vitro assays are commonly used to evaluate antibody-dependent cytotoxicity (ADCC). In one example, target cells (e.g., cells expressing an antigen appropriate to the antibody being evaluated) are loaded with an indicator material (such as 51Cr), and the indicator-loaded target cells are treated with the antibody being evaluated. The resulting cells are then exposed to NK effector cells as described herein. Lysis of target cells is indicated by the release of the indicator material into the assay supernatant, where its concentration can be measured by appropriate methods such as scintillation counting (51Cr) or fluorescence intensity or lifetime determination. Similarly, efficacy can be evaluated by measuring surrogate indicators such as cytokine release by NK cells; upregulation of NK cell activation markers such as CD25, CD69, and / or CD95L; activation of NK cell transcription factors such as NF-AT or NF-κB; or activation of caspases or other markers of apoptosis in target cells. Parental NK cells (such as unmodified NK cells) are useful as a control because they allow for differentiation between ADCC-mediated cytotoxicity and other cytolytic effects that NK cells exert on target cells.

[0154] In some embodiments, the manipulated NK cells can persist in an individual for an extended period, thus reducing the number of times the cells need to be administered to achieve a therapeutic effect. In some embodiments, the manipulated cells provided herein persist for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or at least several months after administration.

[0155] III. Compositions and Kits Containing Manipulated NK Cells Compositions comprising engineered NK cells are provided herein. These compositions include pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. In some embodiments, the engineered cells are formulated with a pharmaceutically acceptable carrier.

[0156] Pharmacoherent carriers can include all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders suitable for pharmacodynamic 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 media such as liposomes and fixative oils may also be used. Auxiliary active compounds may also be incorporated into the composition. The pharmacoherent carrier must be suitable for NK cells, such as a saline solution, dextrose solution, or a solution containing human serum albumin.

[0157] In certain embodiments, the number of cells in the composition provides a therapeutically effective amount of engineered NK cells. In some embodiments, this amount is the amount that reduces the severity, duration, and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in animals. In some other embodiments, the therapeutically effective amount is the amount of cells that results in a reduction of cancer growth or spread of at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% in patients or animals administered with the composition compared to patients or animals not administered with the composition. In some embodiments, the cytotoxic effective amount is defined as the amount of engineered NK cells that can inhibit or reduce the growth of cancer, viral, and microbial cells. In some embodiments, the composition is 10 5 ~10 12 individual cells, or 10 5 ~10 8 individual cells, or 10 6~10 12 individual cells, or 10 8 ~10 11 individual cells, or 10 9 ~10 10 Individual cells or about 10 5 ~about 10 12 A single cell, or about 10 5 ~about 10 8 A single cell, or about 10 6 ~about 10 12 A single cell, or about 10 8 ~about 10 11 A single cell, or about 10 9 ~about 10 10 The composition contains a dose of engineered NK cells, which are individual cells. In some embodiments, the composition contains 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 1 or 10 12 More than 10 5 pieces, about 10 6 pieces, about 10 7 pieces, about 10 8 pieces, about 10 9 pieces, about 10 10 pieces, about 10 11 pieces or about 10 12 It contains more than one cell.

[0158] In some embodiments, the volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, for example, including both extreme values, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL, or 200 mL to 500 mL, or about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL, or 200 mL to 500 mL. In some embodiments, the composition comprises at least or at least about 1 × 10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, or 1 × 10⁶ 8 It has a cell density of cells / mL. In some embodiments, the cell density of the composition is 1 × 10⁻¹⁶, including the values ​​at both ends. 5 cells / mL~1×10 8 cells / mL, 1×10 5 cells / mL~1×10 7 cells / mL, 1×10 5 cells / mL~1×10 6 cells / mL, 1×10 6 cells / mL~1×10 7 cells / mL, 1×10 6 cells / mL~1×10 8 cells / mL, 1×10 6 cells / mL~1×10 7 cells / mL, or 1 × 10⁶ 7 cells / mL~1×10 8 cells / mL, or approximately 1 × 10⁶ 5 cells / mL~1×10 8 cells / mL, 1×105 cells / mL~1×10 7 cells / mL, 1×10 5 cells / mL~1×10 6 cells / mL, 1×10 6 cells / mL~1×10 7 cells / mL, 1×10 6 cells / mL~1×10 8 cells / mL, 1×10 6 cells / mL~1×10 7 cells / mL, or 1 × 10⁶ 7 cells / mL~1×10 8 The concentration is cells / mL.

[0159] Depending on the method of manipulating NK cells, it may be necessary or desirable to culture the NK cells to increase their size and then formulate them into a composition for administration. In some embodiments, a method for preparing a composition containing manipulated NK cells includes the step of culturing or incubating the manipulated NK cells, for example, to increase the cells to a therapeutically effective amount before administering them to an individual in need.

[0160] 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 required 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 typically provided in growth (i.e., culture) media. In one embodiment, the NK medium includes MEMα containing 10% FCS or CellGro SCGM (Cell Genix) containing 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.). Many of these media contain nicotinamide as a vitamin supplement, such as MEMα (8.19 μM nicotinamide), RPMI (8.19 μM nicotinamide), DMEM (32.78 μM nicotinamide), and Glascow's medium (16.39 μM nicotinamide).

[0161] In some applications, such as when cells are introduced (or reintroduced) into human subjects, culture is performed using serum-free preparations such as AIM V® serum-free medium or MARROWMAX® bone marrow medium for lymphocyte culture. Such medium preparations and supplements are available from commercial sources such as Invitrogen (GIBCO) (Carlsbad, Calif.). Amino acids, antibiotics, and / or cytokines may be supplemented to promote optimal viability, proliferation, functionality, and / or survival.

[0162] In some embodiments, the culture of a cell population containing engineered NK cells is achieved without a feeder layer or feeder cells. In some of these embodiments, engineered NK cells can be cultured with growth factors. According to some embodiments, at least one growth factor is selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, and IL-21. According to some embodiments, at least one growth factor is IL-2 or IL-2 and IL-15. According to some embodiments, at least one growth factor is IL-2 alone.

[0163] In some embodiments, the provided composition includes genetically engineered NK cells, such as engineered NK cells in which the expression, activity, and / or signaling of the FcRγ chain in the cell is reduced, which constitute at least or at least about 60%, 70%, 80%, 85%, 90%, 95%, or more of the NK cells in the cell or in the composition.

[0164] Compositions suitable for cryopreserving engineered NK cells are also provided herein. In some embodiments, the composition comprises engineered NK cells and a cryotherapy agent. In some embodiments, the cryotherapy agent is DMSO and / or glycerol, or comprises DMSO and / or glycerol. In some embodiments, the composition formulated for cryopreservation can be stored at low temperatures such as ultracold temperatures, for example, storage temperatures ranging from -40°C to -150°C, for example, or about 80°C ± 6.0°C.

[0165] In some embodiments, engineered NK cells can be stored at cryogenic temperatures before administration to a patient. Engineered NK cells can also be stored at cryogenic temperatures after isolation from a mammalian subject and before genetic engineering. For example, another source of lymphocytes or engineered NK cells can be isolated, stored at cryogenic temperatures, and then processed to obtain engineered NK cells. Alternatively, another source of lymphocytes or engineered NK cells can be isolated, processed to obtain engineered NK cells, and then stored at cryogenic temperatures.

[0166] A typical method for small-scale cryogenic preservation is described, for example, in U.S. Patent No. 6,0168,991. For small-scale preservation, cells can be cryogenically preserved in a low-density suspension of pre-cooled 5% human albumin serum (HAS) (e.g., at a concentration of approximately 200 × 10⁶ / ml). 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 approximately -80°C.

[0167] In some embodiments, cryopreserved NK cells are prepared for administration by thawing. In some cases, NK cells can be administered to a subject immediately after thawing. In such embodiments, the composition is readily available without further processing. In other embodiments, after thawing, NK cells are further processed, for example, by resuspension with a pharmaceutically acceptable carrier, incubation with an activator or stimulant, or activated and washed, and then resuspended in a pharmaceutically acceptable buffer before administration to the subject.

[0168] Kits containing the engineered cells are also provided herein. For example, in some embodiments, kits containing the engineered cells and additional agents are provided herein. In some embodiments, the additional agent comprises an Fc domain. In some embodiments, the additional agent is an Fc fusion protein or an antibody. In some embodiments, the additional agent is a human antibody, a humanized antibody, or a chimeric antibody. In some of these embodiments, the additional agent is a full-length antibody. Exemplary antibodies are described below.

[0169] IV. Methods of Treatment In some embodiments, provided herein is a method of treating a condition in an individual, comprising administering the engineered NK cells to an individual who needs it. <QQ

[0170] In some embodiments, the method comprises administering an effective amount of the engineered cells to the individual. In some embodiments, 10 5 ~10 12 cells, or 10 5 ~10 8 cells, or 10 6 ~10 12 cells, or 10 8 ~10 11 cells, or 10 9 ~10 10 cells or about 10 5 ~about 10 12 cells, or about 10 5 ~about 10 8 cells, or about 10 6 ~about 10 12 cells, or about 10 8 ~about 10 11 cells, or about 10 9 ~about 10 10 cells. In some embodiments, the composition is about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 1010 , about 10 11 , or about 10 12 It contains individual cells, which are administered to an individual. In some embodiments, 10 6 ~10 10 Individual manipulated NK cells / kg or approximately 10 6 ~10 10 Individual manipulated NK cells / kg are administered to the subjects.

[0171] In some embodiments, the manipulated NK cells are administered to the individual immediately after isolation and manipulation of the NK cells. In some embodiments, the manipulated NK cells are administered to the individual within 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days after isolation and manipulation.

[0172] In other embodiments, manipulated NK cells are stored or grown in culture prior to administration and / or manipulation, as in the methods described above. For example, NK cells may be stored for more than 6, 12, 18, or 24 months prior to manipulation and / or administration to an individual.

[0173] In some cases, NK cell clones originate from cancer cells and can thus divide uncontrollably upon administration to a patient. In some embodiments, engineered NK cells, such as those derived from clone cell lines, may be irradiated before administration to the target to prevent uncontrollable division.

[0174] The manipulated NK cells can be administered to the subject via any convenient route, including parenteral routes such as subcutaneous, intramuscular, intravenous, and / or epidural administration routes.

[0175] The provided engineered NK cells and compositions can be used in methods for treating individuals having tumors or hyperproliferative disorders or microbial infections, such as viral infections, yeast infections, fungal infections, protozoan infections, and / or bacterial infections. The disclosed methods for treating subjects with the engineered cells can be combined with therapeutic monoclonal antibodies, such as antitumor antigens or anticancer antibodies, antiviral antibodies, or antibacterial antibodies. The engineered NK cells can be administered for the treatment of animals, such as mammals, such as human subjects.

[0176] In some cases, this method includes a step of treating hyperproliferative disorders such as hematological malignancies or solid tumors. Examples of cancers and proliferative disorders that can be treated with the compositions described herein include, but are not limited to, leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic myeloid (granulocytic) leukemia, and chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endosarcoma, Ewing's tumor, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, liver cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and hyperplasia. Treatment and / or prevention of cancer includes, but is not limited to, the alleviation of one or more symptoms associated with cancer, the inhibition or reduction of cancer progression, the promotion of cancer regression, and / or the promotion of an immune response.

[0177] In some cases, the method includes a step of treating viral infections, such as infections caused by the presence of viruses in the body. Viral infections include chronic or persistent viral infections, which are viral infections that can infect a host and replicate within the host's cells for a long period of time, usually weeks, months, or years, before proving fatal. Viruses causing chronic infections that can be treated by the present invention include, for example, human papillomavirus (HPV), herpes simplex virus, and other herpesviruses, hepatitis B and C viruses, as well as other hepatitis viruses, human immunodeficiency virus, and measles virus, all of which can cause significant clinical disease. Long-term infection can ultimately lead to the induction of disease, which can be fatal to the patient, for example, in the case of hepatitis C virus-induced liver cancer. Other chronic viral infections that can be treated by the present invention include Epstein-Barr virus (EBV), as well as other viruses that may be associated with tumors.

[0178] Examples of viral infections that can be treated or prevented by the compositions and methods described herein include retroviruses (e.g., human T-cell lymphotropic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpesviruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Barr virus and cytomegalovirus), arenaviruses (e.g., Lassa fever virus), paramyxoviruses (e.g., measles virus, human respiratory syncytial virus, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (hepatitis C virus (HCV), yellow fever virus, Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B virus (HBV)), and orthomyxoviruses. Examples of viral infections include, but are not limited to, those caused by viruses such as Sendai virus and influenza viruses A, B, and C, papovavirus (e.g., papillomavirus), picornavirus (e.g., rhinovirus, enterovirus, and hepatitis A virus), poxvirus, reovirus (e.g., rotavirus), togavirus (e.g., rubella virus), and rhabdovirus (e.g., rabies virus). Treatment and / or prevention of viral infections include, but are not limited to, alleviating one or more symptoms associated with the infection, inhibiting, reducing, or suppressing viral replication, and / or enhancing the immune response.

[0179] In some embodiments, the composition is used in a method for treating a yeast infection or a bacterial infection. For example, the compositions and methods described herein include those of Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholera, Escherichia coli, and Pseudomonas aeruginosa. Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum pallidum), Treponema pallidum, Treponema carateneum, Borrelia vincentii, Lyme disease BorreliaIt can treat infections associated with burgdorferi, leptospira icterohemorrhagiae (which causes jaundice), Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki (which causes typhus), Rickettsia tsutsugumushi (which causes oriental scrub typhus), Chlamydia spp., Helicobacter pylori, or combinations thereof.

[0180] V. Combination Therapy In some embodiments, these manipulated NK cells exhibit enhanced activity when activated by antibodies or Fc-containing proteins. For example, the manipulated cells can be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells.

[0181] In some embodiments, methods for treating conditions in an individual are provided herein, comprising the step of administering engineered NK cells and antibodies. Those skilled in the art can select appropriate therapeutic (e.g., anti-cancer) monoclonal antibodies and administer them to the target together with engineered NK cells as described herein, depending on the specific disease or condition of the individual. Suitable antibodies may include polyclonal, monoclonal, fragment (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules.

[0182] Ab may further include humanized Ab or human Ab. Humanized forms of non-human Ab are chimeric Ig, Ig chains, or fragments (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of Ab) that contain a minimal sequence derived from non-human Ig. In some embodiments, the antibody contains an Fc domain.

[0183] Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “import” residues, and are usually derived from the “import” variable domain. Humanization is achieved by replacing a rodent CDR or CDR sequence with the corresponding sequence of the human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). Such “humanized” antibodies are chimeric antibodies (1989), in which a substantially less-than-intact human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which several CDR residues and possibly several Fc residues are replaced with residues from similar sites in the rodent antibody. Humanized antibodies (ABs) contain human Ig (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, to possess the desired specificity, affinity, and capability. In some cases, the corresponding non-human residues replace the Fv framework residues of human Ig. Humanized antibodies may contain residues not found in either the recipient antibody or the imported CDR or framework sequence. Generally, humanized antibodies contain substantially all of the variable domains, at least one, typically two, with most, if not all, of the CDR region corresponding to that of non-human Ig, and most, if not all, of the FR region corresponding to the human Ig consensus sequence. Humanized antibodies also optimally contain at least a portion of the Ig constant region (Fc), typically that of human Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0184] Human ABs can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al., 1991; Marks et al., 1991) and human mAb preparation (Boerner et al., 1991; Reisfeld and Sell, 1985). Similarly, the introduction of human Ig genes into transgenic animals in which the endogenous Ig gene is partially or completely inactivated can be utilized to synthesize human ABs. Antigen administration has been observed to induce the production of human antibodies, which closely resemble those found in humans in every respect, 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).

[0185] Specifically, the cells of the present invention can target tumors when administered together with antibodies that recognize tumor-associated antigens. Those skilled in the art will understand that the manipulated NK cells of the present invention 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-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGF2, EPHA3, FR-alpha, phosphatidylserine, syndecane 1, and SLAMF7. Examples include (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some cases, antibodies may be anti-CD20 antibodies, anti-HER2 antibodies, anti-CD52 antibodies, anti-EGFR antibodies, and anti-CD38 antibodies. Exemplary antibodies include rituximab, trastuzumab, aretuzumab, sartuximab, daratumumab, vertuzumab, ofatumumab, ubrituximab, and okalatuzumab. Antibodies specific to the selected cancer type can be chosen, including antibodies 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 capable of binding to specific tumor or disease antigens, any of which can be used in accordance with the methods provided for treating tumors or diseases.

[0186] Manipulated NK cells and further active agents can be administered sequentially or simultaneously. In some embodiments, further active agents can be administered before the administration of manipulated NK cells. In some embodiments, further active agents can be administered after the administration of NK cells. For example, manipulated NK cells can be administered simultaneously with antibodies specific to a selected cancer type. Alternatively, manipulated NK cells can be administered at a selected time different from the time when antibodies specific to a selected cancer type are administered.

[0187] In certain cases, subjects are administered an effective dose of antibody before, after, or substantially simultaneously with the manipulated population of NK cells. In some cases, subjects are administered antibody doses ranging from approximately 0.1 mg / kg to approximately 100 mg / kg (approximately 0.5–10 mg / kg, approximately 1–20 mg / kg, approximately 10–50 mg / kg, approximately 20–100 mg / kg, e.g., approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 8 mg / kg, approximately 10 mg / kg, approximately 16 mg / kg, approximately 20 mg / kg, approximately 24 mg / kg, approximately 36 mg / kg, approximately 48 mg / kg, approximately 60 mg / kg, approximately 75 mg / kg, or approximately 100 mg / kg, etc.). A skilled clinician can select an effective dose of antibody, taking into account the specific antibody, the specific disease or condition (e.g., tumor or other disorder), the subject's general condition, any further treatment the subject is receiving or has received previously, and other relevant factors. The subject is also administered a population of modified NK cells as described herein. Both the antibody and the modified NK cell population are typically administered parenterally, for example, intravenously; however, injection or infusion into or near the tumor (local administration) or intraperitoneal administration may also be used. Those skilled in the art can determine the appropriate route of administration.

[0188] Manipulated NK cells can be administered simultaneously with or sequentially with antibacterial agents, antiviral agents, and other therapeutic agents. In some embodiments of the present invention, manipulated cells can be administered to an individual in combination with cytokines and / or growth factors. According to some embodiments of the present invention, at least one growth factor is selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, and IL-21. In some embodiments, manipulated NK cells and cytokines or growth factors are administered sequentially. For example, manipulated NK cells may be administered first, followed by the administration of cytokines and / or growth factors. In some embodiments, manipulated NK cells are administered simultaneously with cytokines or growth factors.

[0189] In some embodiments, one or more cytokines (such as IL-2, IL-15, IL-21, and / or IL-12) are administered to the subject to support the survival and / or growth of NK cells. Cytokines can be administered before, after, or substantially concurrently with NK cells. In some examples, cytokines can be administered after NK cells. In one particular example, cytokines are administered to the subject within approximately 1 to 8 hours of NK cell administration (e.g., within approximately 1 to 4 hours, 2 to 6 hours, 4 to 6 hours, or 5 to 8 hours).

[0190] In some embodiments, the method provided may also include a step of administering engineered NK cells to an individual in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may include cyclophosphamide, fludarabine, and methylprednisone. In some embodiments, the chemotherapeutic agent may include thalidomide, cisplatin (cis-DDP), oxaliplatin, carboplatin, anthracendione, mitoxantrone; hydroxyurea, methylhydrazine derivatives, procarbazine (N-methylhydrazine, MM), adrenocortical inhibitors, and mitotane (.omicron., rho.'-DDD), aminoglutethimide, RXR agonist, bexarotene, tyrosine kinase inhibitor, imatinib, mechloretamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolicin), chlorambucil, ethyleneimine, methylmelamine, hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine (methyl-CCNTJ), lomustine (CCNU), streptozosin (streptozotocin), DNA synthesis antagonist, estramustine phosphate, triazine, dacarbazine (OTIC, dimethyl-triazenoimidazole carboxamide), temozolomide, folic acid analog, methotrexate (ametopterin), pyrimidine analog, fluorouracin (fiuorouracin) (5-Fluorouracil, 5-FU, 5FTJ), floxuridine (Fluorodeox 'Uridine, 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, amsacrin, vinca alkaloids, vinblastine (VLB), vincristine, taxanes, paclitaxel, protein-bound paclitaxel (Abraxane®), docetaxel (Taxotere®); Selected from the group consisting of epipodophyllotoxin, etoposide, teniposide, camptothecin, topotecan, irinotecan, dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin, buserelin, adrenocorticosteroids, prednisone, progestin, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinylestradiol, tamoxifen, anastrozole; testosterone propionate, fluoxymesterone, flutamide, bicalutamide, and leuprolide.

[0191] 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.

[0192] VI. Exemplary Examples The embodiments provided herein include the following: 1. Manipulated natural killer (NK) cells, wherein the manipulated NK cells are genetically engineered to reduce the expression, activity, and / or signaling of the FcRγ chain in the cells. 2. Genetic disruption of the gene encoding the FcRγ chain and / or genetic disruption resulting in reduced expression of the FcRγ chain in manipulated NK cells; Genetic disruption of a gene encoding a protein that regulates the expression or activity of the FcRγ chain, and / or genetic disruption resulting in a reduction in the expression of a protein that regulates the expression or activity of the FcRγ chain; and / or Genetic disruption resulting from genetic disruption of genes encoding proteins involved in FcRγ-dependent signaling and / or reduced expression of proteins involved in FcRγ-dependent signaling. The manipulated NK cells of embodiment 1, including the modified NK cells of embodiment 1. 3. Manipulated NK cells of aspect 2, wherein the genetic disruption includes deletions, mutations, and / or insertions resulting in immature stop codons or frameshifts in the gene's reading frame. 4. Manipulated NK cells of embodiment 2 or embodiment 3, wherein both alleles of the gene encoding the FcRγ chain, the gene encoding a protein that regulates the expression or activity of the FcRγ chain, and / or the gene encoding a protein involved in FcRγ chain-dependent signaling are disrupted in the manipulated NK cells. 5. Manipulated NK cells according to Embodiment 1, comprising a gene-targeting inhibitory nucleic acid molecule in NK cells that results in reduced expression of FcRγ chains, reduced expression of proteins that regulate FcRγ chain expression or activity, and / or reduced expression of proteins involved in FcRγ chain-dependent signaling. 6. Manipulated NK cells in any of embodiments 2-5, wherein the expression of FcRγ chains, proteins that regulate the expression or activity of FcRγ chains, and / or proteins involved in FcRγ chain-dependent signaling is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression of proteins in unmanipulated NK cells, or by approximately more than 50%, 60%, 70%, 80%, 90%, or 95%. 7. Manipulated NK cells in which the expression of a protein that modulates the expression or activity of the FcRγ chain is reduced, and the protein is a transcription factor, as described in any of embodiments 2 to 6. 8. Manipulated NK cells of embodiment 7, wherein the transcription factor is PLZF (ZBTB16) or HELIOS (IKZF2). 9. Manipulated NK cells in any of embodiments 2 to 6, wherein the expression of a protein involved in FcRγ chain-dependent signaling is reduced in the manipulated NK cells, and the protein is a downstream signaling molecule. 10. Engineered NK cells of aspect 9, wherein the downstream signaling molecule is SYK, DAB2, or EAT-2. 11. Manipulated NK cells in any of embodiments 2 to 6, wherein the expression of FcRγ chains is reduced in the manipulated cells. 12. Manipulated NK cells comprising genetic disruption in a gene encoding the FcRγ chain, wherein the expression of FcRγ is reduced in the cells. 13. Manipulated NK cells of aspect 12, wherein the genetic disruption includes deletions, mutations, and / or insertions resulting in immature stop codons or frameshifts in the gene's reading frame. 14. Manipulated NK cells of embodiment 12 or embodiment 13, wherein both alleles of the gene encoding the FcRγ chain are disrupted in the genome of the manipulated NK cells. 15. Manipulated NK cells in any of embodiments 1, 5, or 6, comprising an inhibitory nucleic acid molecule that targets a gene encoding an FcRγ chain. 16. Modified NK cells according to embodiment 15, wherein the inhibitory nucleic acid molecule contains a sequence complementary to the gene encoding the FcRγ chain. 17. Manipulated NK cells in any of embodiments 11-16, wherein the expression of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression in unmanipulated NK cells, or by approximately more than 50%, 60%, 70%, 80%, 90%, or 95%. 18. Manipulated NK cells in any of embodiments 5-6 and 15-17, wherein the inhibitory nucleic acid comprises an RNA interferant. 19. Manipulated NK cells in any of embodiments 5-6 and 15-18, wherein the inhibitory nucleic acid comprises siRNA, shRNA, or miRNA. 20. Manipulated NK cells in any of embodiments 1 to 19, wherein the reduction in FcRγ expression, activity, and / or signaling is permanent, transient, or inducible. 21. Manipulated NK cells of any of aspects 1 to 20, in which the expression, activity, and / or signaling of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression, activity, and / or signaling in unmanipulated NK cells, or by approximately more than 50%, 60%, 70%, 80%, 90%, or 95%. 22. Modified NK cells according to any of embodiments 1 to 21, wherein the expression of FcRγ chains expressed in the cells is undetectable by immunoblotting assay. 23. Manipulated NK cells in any of embodiments 1 to 22, wherein CD16 is expressed on the surface of the manipulated NK cells. 24. Modified NK cells expressing CD3-zeta (CD3ζ) chains, as described in any of embodiments 1 to 23. 25. Modified NK cells derived from primary cells obtained from the subject, according to any of the embodiments 1 to 24. 26. Manipulated NK cells according to embodiment 25, wherein the subject is human. 27. Modified NK cells derived from a clonal cell line, according to any of the embodiments 1 to 24. 28. Manipulated NK cells of embodiment 27, wherein the clonal cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1. 29. Manipulated NK cells in any of embodiments 1 to 28, comprising a recombinant or heterologous CD16 gene and / or a recombinant or heterologous CD3-zeta (CD3ζ) chain. 30. Manipulated NK cells of aspect 29, wherein CD16 contains a CD16 activating mutation, a mutation resulting in a higher affinity for IgG1, a 158V mutation and / or a 158F mutation. 31. Manipulated cells of any of embodiments 1 to 30, wherein the manipulated NK cells show increased activity when stimulated via CD16 compared to unmodified NK cells. 32. Manipulated NK cells in any of embodiments 1 to 31, wherein surface expression of NKp46, NKp30, and / or NKp44 is reduced compared to unmodified NK cells. 33. A method for producing engineered NK cells, comprising the step of genetically modifying NK cells to reduce the expression, activity, and / or signaling of the FcRγ chain in said cells. 34. The step of reducing expression is Disrupting or suppressing genes encoding FcRγ chains, and / or genes that result in reduced FcRγ chain expression in NK cells; Disrupting or suppressing genes encoding proteins that regulate the expression or activity of FcRγ chains, and / or disrupting or suppressing genes that result in reduced expression of proteins that regulate the expression or activity of FcRγ chains in NK cells; and / or Disrupting or suppressing genes encoding proteins involved in FcRγ chain-dependent signaling, and / or disrupting or suppressing genes that result in reduced expression of proteins involved in FcRγ chain-dependent signaling in NK cells. The method of embodiment 33, including the method of embodiment 33. 35. The method of embodiment 33 or embodiment 34, comprising the step of introducing a deletion, mutation, or insertion into the gene. 36. Any method according to embodiment 33 to 35, wherein the gene encodes the FcRγ chain. 37. Any method according to embodiment 33 to 35, wherein the gene encodes a protein that regulates the expression or activity of the FcRγ chain, which is a transcription factor. 38. The method of embodiment 37, wherein the transcription factor is PLZF or HELIOS. 39. Any method according to embodiment 33 to 35, wherein the gene encodes a protein involved in FcRγ chain-dependent signaling, which is a downstream signaling molecule. 40. The method of embodiment 38, wherein the downstream signaling molecule is SYK, DAB2, or EAT2. 41. Any method according to embodiment 33 to 40, wherein gene disruption or suppression is carried out by introducing a gene-targeting endonuclease in NK cells under conditions that enable gene disruption or suppression. 42. The method according to embodiment 41, wherein the endonuclease is selected from the group consisting of TAL nuclease, meganuclease, zinc finger nuclease, Cas9, and argonaut. 43. Any method according to embodiment 33 to 40, wherein the disruption or suppression is carried out by introducing a gene-targeting inhibitory nucleic acid into NK cells under conditions that result in gene suppression. 44. The method of embodiment 43, wherein the inhibitory nucleic acid molecule contains a sequence complementary to the gene encoding the FcRγ chain. 45. The method according to embodiment 43 or embodiment 44, wherein the inhibitory nucleic acid comprises an RNA interferant. 46. ​​The method according to any one of embodiments 43 to 45, wherein the nucleic acid is siRNA, shRNA, or miRNA. 47. A method according to any of embodiments 33 to 46, wherein the reduction in expression is permanent, transient, or inducible. 48. Any method according to aspect 33 to 47, wherein the expression, activity, and / or signaling of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to expression in unmodified NK cells, or by approximately more than 50%, 60%, 70%, 80%, 90%, or 95%. 49. Any method according to embodiment 33 to 48, wherein the expression level of the FcRγ chain is undetectable by immunoblotting assay. 50. Any method of embodiment 33 to 49, comprising the step of isolating NK cells from a human subject before the genetic manipulation. 51. The method of embodiment 50, comprising the step of isolating NK cells from peripheral blood mononuclear cells (PBMCs). 52. The method according to embodiment 50 or 51, wherein the step of isolating NK cells includes selecting NK cells from PBMCs using an NK cell marker. 53. The method of embodiment 52, wherein the NK cell marker is CD56, Cd161, KIR, NKG2A, NKG2D, NKp30, NKp44, and / or NKp46. 54. Any method according to any one of embodiments 50 to 53, further comprising the step of selecting NK cells expressing CD16 and / or CD3ζ. 55. Any method according to embodiment 50 to 54, further comprising the step of selecting NK cells that do not express surface CD3. 56. Any method according to embodiments 33 to 49, wherein the NK cells are an NK cell line. 57. The method according to embodiment 56, wherein the cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1. 58. The method of embodiment 56 or embodiment 57, further comprising the step of manipulating an NK cell line to recombinate or express heterologous CD16 and / or CD3ζ. 59. The method of embodiment 58, comprising the step of introducing nucleic acids encoding CD16 and / or CD3ζ into NK cells. 60. The method of embodiment 58 or embodiment 59, comprising a mutation in the CD16 gene that increases the activating mutation and / or the affinity of CD16 to IgG. 61. Any method according to embodiments 58 to 60, comprising a mutation in which CD16 is 158V. 62. Any method according to embodiments 58 to 60, comprising a mutation in which CD16 is 158F. 63. A method according to any one of embodiments 58 to 62, comprising the step of introducing a nucleic acid encoding CD16 and / or CD3ζ into NK cells via viral transduction. 64. A method according to any one of embodiments 58 to 62, comprising the step of transfecting NK cells with nucleic acids encoding CD16 and / or CD3ζ. 65. Any method according to embodiment 58 to 64, comprising the step of transiently, inducibly, or persistently expressing CD16 or CD3ζ in the NK cells. 66. Any method according to embodiment 33 to 65, further comprising the step of culturing or growing manipulated NK cells. 67. The method of embodiment 66, comprising the step of culturing manipulated NK cells together with feeder cells. 68. The method of embodiment 66 or embodiment 67, comprising the step of culturing manipulated NK cells with cytokines. 69. Manipulated NK cells produced by any of the methods described in aspects 33 to 68. 70. A composition comprising an effective amount of manipulated NK cells as described in any of embodiments 1 to 32 or 69. 71. A composition according to embodiment 70, further comprising a pharmaceutically acceptable carrier. 72. The composition according to embodiment 71, wherein the carrier is physiological saline solution, dextrose solution, or 5% human serum albumin. 73. 1 × 10 5 ~1 × 108 A composition according to any one of embodiments 70 to 72, containing 1 cell / mL. 74. A composition according to any of embodiments 70 to 73, comprising an antifreeze agent. 75. A kit comprising manipulated cells from any of embodiments 1 to 32 or 69 or a composition from any of embodiments 70 to 74, and further active substances for the treatment of a disease. 76. A kit according to embodiment 75, wherein the further active agent is an antibody or an Fc fusion protein. 77. A kit according to embodiment 76, wherein the antibody recognizes or specifically binds to a tumor-associated antigen. 78. 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-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGF2, EPHA3, FR-alpha, phosphatidylserine, syndecane 1, and SLAMF7. A kit according to embodiment 77, wherein (CD319) TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. 79. A kit according to any of embodiments 76 to 78, wherein the antibody is a full-length antibody and / or contains an Fc domain. 80. A method for treating a condition, comprising the step of administering manipulated NK cells from any of embodiments 1 to 32 or 69 or a composition from any of embodiments 70 to 74 to an individual in need thereof. 81. The method of embodiment 80, wherein the engineered NK cells are produced by any of the methods of embodiments 33 to 68 prior to the step of administering the engineered NK cells. 82. 1 × 10⁻⁶ individuals 8 ~1 × 10 10individual cells / m 2 Or approximately 1 x 10 8 ~1 × 10 10 individual cells / m 2 The stage of administering or 1 x 10 6 ~1 × 10 10 NK cells / kg or approximately 1 × 10⁶ 6 ~1 × 10 10 A method according to embodiment 80 or embodiment 81, comprising the step of administering NK cells / kg. 83. Any method according to embodiment 80 to 82, further comprising the step of administering an additional active substance. 84. The method of embodiment 83, wherein the further active substance is an antibody or an Fc fusion protein. 85. The method according to aspect 84, wherein an antibody recognizes a tumor-associated antigen. 86. 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-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scattering factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGF2, EPHA3, FR-alpha, phosphatidylserine, syndecane 1, and SLAMF7. The method of embodiment 84 or embodiment 85, wherein (CD319) is TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. 87. Any method according to embodiment 84 to 86, wherein the antibody comprises an Fc domain and / or is a full-length antibody. 88. Any method according to embodiment 83 to 87, wherein further active substances and manipulated NK cells are administered sequentially. 89. The method of aspect 88, wherein an additional active substance is administered before the administration of the manipulated NK cells. 90. Any method according to embodiment 83 to 87, wherein an additional active substance and manipulated NK cells are administered simultaneously. 91. Any method according to embodiment 80 to 90, wherein the condition is selected from the group consisting of inflammatory conditions, infections, and cancer. 92. The method according to aspect 91, wherein the infectious disease is a viral infection or a bacterial infection. 93. The method of aspect 92, wherein the cancer is leukemia or lymphoma. 94. The method of embodiment 92, wherein the cancer includes a solid tumor. 95. Any method according to aspects 80 to 94, wherein the individual is human. 96. Any method according to aspects 80 to 95, wherein the manipulated NK cells are homogeneous with respect to the organism. 97. Any method according to embodiment 80 to 95, wherein the manipulated NK cells are of autologous origin to the subject. 98. Modified NK cells in any of embodiments 1 to 32, which are genetically engineered to reduce the expression of NK inhibitory receptors. 99. Genetic disruption resulting in genetic disruption of the gene encoding the NK inhibitor receptor and / or reduced expression of the NK inhibitor receptor; or The inhibitory nucleic acids include those that target genes encoding NK inhibitor receptors and / or reduce the expression of NK inhibitor receptors. Manipulated NK cells in aspect 98. 100. Manipulated NK cells of embodiment 98 or embodiment 99, wherein the inhibitory receptor is NKG2A or KIR2DL1. 101. Any method according to any one of embodiments 32 to 68, further comprising the step of genetically modifying NK cells to reduce the expression or activity of inhibitory receptors. 102. The method of embodiment 101, wherein the step of reducing expression or activity includes disrupting or suppressing the expression of a gene encoding an NK inhibitory receptor or a gene resulting in reduced expression of an NK inhibitory receptor. 103. The method of embodiment 102, wherein the inhibitory receptor is NKG2A or KIR2DL1. [Examples]

[0193] VII. Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0194] Example 1: Evaluation of cell death in proliferated g-NK cells and conventional NK cells 25 x 10 from a healthy human donor 6 We obtained approximately 2 × 10⁶ peripheral blood mononuclear cells (PBMCs) and used fluorescence activation sorting based on specific cell surface phenotypes. 5 Individual FcRγ deficiencies (g - NK cells were enriched. g-NK cells were fed into 0.5 mL of irradiated autologous PBMCs (10 ng / mL OKT3-stimulated) feeder cells (1 × 10⁶). 6 Selected g-NK cells (2 × 10) in 0.5 mL (2 × 10) 5 By mixing with (10 ng / mL), the cells proliferated 250-fold in 14 days. The proliferated cells were spiked with 10 ng / mL IL-2 every two days, and on day 7, the proliferated g-NK cells were resupplied with an irradiated feeder (pre-activated with 10 ng / mL OKT3) at a feeder:g-NK cell ratio of 5:1. Conventional NK cells were also obtained and proliferated.

[0195] To evaluate the antibody-mediated activity of proliferated g-NK cells, serial dilutions of concentrated g-NK cells (E / T ratio ranging from 50:1 to 1:1) were tested in the presence or absence of the anti-CD20 antibody rituximab (5 μg / mL). 51 Cr-labeled Raji lymphoma cells were incubated in a 96-well plate. After 4 hours of incubation, the amount per well was 51 Antibody-dependent cytotoxicity (ADCC) was evaluated by determining Cr activity. As shown in Figure 1, the results indicated that g-NK cells had a higher ability to mediate ADCC than conventional NK cells when co-cultured with rituximab and Raji lymphoma cells.

[0196] The present invention is not intended to be limited in scope to the specific disclosed embodiments provided, for example, to illustrate various aspects of the invention. Various modifications to the described engineered cells, compositions, and methods will become apparent from the description and teachings herein. Such variations can be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the disclosure.

[0197] Sequence Information SEQUENCE LISTING <110> INDAPTA THERAPEUTICS, INC. <120> ENGINEERED NATURAL KILLER (NK) CELLS AND[[ID=·11]] COMPOSITIONS AND METHODS THEREOF <150> US 62 / 457,098 <151> 2017-02-09 <150> US 62 / 484,350 <151> 2017-04-11 <160> 6 <170> FastSEQ for Windows Version 4.0 <210> 1<00009·01><211> 86 <212> PRT <213> Homo sapiens <220> <223> High affinity immunoglobulin gamma Fc receptor I <300> <308> NCBI NP-004097.1 <309> 2017-12-27 <400> 1 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Pathway Leu Gly Glu Pro Gln Leu Cys Tyr To Leu Asp Path To 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 Pathway To 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 <210> 2 <211> 82 <212> PRT <213> Homo sapiens <220> <223> CD3zeta <300> <308> NCBI ABQ28690.1 <309> 2016-07-23 <400> 2 Path To Leu Gln Path Gln Leu Pro To Thr Glu Path Gln Ser Phe Gly 1 5 10 15 Leu Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile 20 25 30 Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg 35 40 45 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 50 55 60 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 65 70 75 80 Arg Gly <210> 3 <211> 673 <212> PRT <213> Homo sapiens <220> <223> PLZF <300> <308> NCBI NP_001018011.1 <309> 2017-12-28 <400> 3 Met Asp Leu Thr Lys Met Gly Met Ile Gln Leu Gln Asn Pro Ser His 1 5 10 15 Pro Thr Gly Leu Leu Cys Lys Ala Asn Gln Met Arg Leu Ala Gly Thr 20 25 30 Leu Cys Asp Val Val Ile Met Val Asp Ser Gln Glu Phe His Ala His 35 40 45 Arg Thr Val Leu Ala Cys Thr Ser Lys Met Phe Glu Ile Leu Phe His 50 55 60 Arg Asn Ser Gln His Tyr Thr Leu Asp Phe Leu Ser Pro Lys Thr Phe 65 70 75 80 Gln Gln Ile Leu Glu Tyr Ala Tyr Thr Ala Thr Leu Gln Ala Lys Ala 85 90 95 Glu Asp Leu Asp Asp Leu Leu Tyr Ala Ala Glu Ile Leu Glu Ile Glu 100 105 110 Tyr Leu Glu Glu Gln Cys Leu Lys Met Leu Glu Thr Ile Gln Ala Ser 115 120 125 Asp Asp Asn Asp Thr Glu Ala Thr Met Ala Asp Gly Gly Ala Glu Glu 130 135 140 Glu Glu Asp Arg Lys Ala Arg Tyr Leu Lys Asn Ile Phe Ile Ser Lys 145 150 155 160 His Ser Ser Glu Glu Ser Gly Tyr Ala Ser Val Ala Gly Gln Ser Leu 165 170 175 Pro Gly Pro Met Val Asp Gln Ser Pro Ser Val Ser Thr Ser Phe Gly 180 185 190 Leu Ser Ala Met Ser Pro Thr Lys Ala Ala Val Asp Ser Leu Met Thr 195 200 205 Ile Gly Gln Ser Leu Leu Gln Gly Thr Leu Gln Pro Pro Ala Gly Pro 210 215 220 Glu Glu Pro Thr Leu Ala Gly Gly Gly Arg His Pro Gly Val Ala Glu 225 230 235 240 Val Lys Thr Glu Met Met Gln Val Asp Glu Val Pro Ser Gln Asp Ser 245 250 255 Pro Gly Ala Ala Glu Ser Ser Ile Ser Gly Gly Met Gly Asp Lys Val 260 265 270 Glu Glu Arg Gly Lys Glu Gly Pro Gly Thr Pro Thr Arg Ser Ser Val 275 280 285 Ile Thr Ser Ala Arg Glu Leu His Tyr Gly Arg Glu Glu Ser Ala Glu 290 295 300 Gln Val Pro Pro Pro Ala Glu Ala Gly Gln Ala Pro Thr Gly Arg Pro 305 310 315 320 Glu His Pro Ala Pro Pro Pro Glu Lys His Leu Gly Ile Tyr Ser Val 325 330 335 Leu Pro Asn His Lys Ala Asp Ala Val Leu Ser Met Pro Ser Ser Val 340 345 350 Thr Ser Gly Leu His Val Gln Pro Ala Leu Ala Val Ser Met Asp Phe 355 360 365 Ser Thr Tyr Gly Gly Leu Leu Pro Gln Gly Phe Ile Gln Arg Glu Leu 370 375 380 Phe Ser Lys Leu Gly Glu Leu Ala Val Gly Met Lys Ser Glu Ser Arg 385 390 395 400 Thr Ile Gly Glu Gln Cys Ser Val Cys Gly Val Glu Leu Pro Asp Asn 405 410 415 Glu Ala Val Glu Gln His Arg Lys Leu His Ser Gly Met Lys Thr Tyr 420 425 430 Gly Cys Glu Leu Cys Gly Lys Arg Phe Leu Asp Ser Leu Arg Leu Arg 435 440 445 Met His Leu Leu Ala His Ser Ala Gly Ala Lys Ala Phe Val Cys Asp 450 455 460 Gln Cys Gly Ala Gln Phe Ser Lys Glu Asp Ala Leu Glu Thr His Arg 465 470 475 480 Gln Thr His Thr Gly Thr Asp Met Ala Val Phe Cys Leu Leu Cys Gly 485 490 495 Lys Arg Phe Gln Ala Gln Ser Ala Leu Gln Gln His Met Glu Val His 500 505 510 Ala Gly Val Arg Ser Tyr Ile Cys Ser Glu Cys Asn Arg Thr Phe Pro 515 520 525 Ser His Thr Ala Leu Lys Arg His Leu Arg Ser His Thr Gly Asp His 530 535 540 Pro Tyr Glu Cys Glu Phe Cys Gly Ser Cys Phe Arg Asp Glu Ser Thr 545 550 555 560 Leu Lys Ser His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys 565 570 575 Asn Gly Cys Gly Lys Lys Phe Ser Leu Lys His Gln Leu Glu Thr His 580 585 590 Tyr Arg Val His Thr Gly Glu Lys Pro Phe Glu Cys Lys Leu Cys His 595 600 605 Gln Arg Ser Arg Asp Tyr Ser Ala Met Ile Lys His Leu Arg Thr His 610 615 620 Asn Gly Ala Ser Pro Tyr Gln Cys Thr Ile Cys Thr Glu Tyr Cys Pro 625 630 635 640 Ser Leu Ser Ser Met Gln Lys His Met Lys Gly His Lys Pro Glu Glu 645 650 655 Ile Pro Pro Asp Trp Arg Ile Glu Lys Thr Tyr Leu Tyr Leu Cys Tyr 660 665 670 Val <210> 4 <211> 238 <212> PRT <213> Homo sapiens <220> <223> CD16 (158F) <400> 4 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> 5 <211> 16 <212> PRT <213> Homo sapiens <220> <223> CD16 (158F) signal peptide <400> 5 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 <210> 6 <211> 238 <212> PRT <213> Homo sapiens <220> <223> CD16 (158V) <400> 6 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

Claims

1. Engineered natural killer (NK) cells, wherein the engineered NK cells are genetically modified to reduce the expression, activity, and / or signaling of the FcRγ chain in the cells.

2. Genetic disruption resulting from genetic disruption of the gene encoding the FcRγ chain and / or reduction of FcRγ chain expression in manipulated NK cells; Genetic disruption of a gene encoding a protein that regulates the expression or activity of the FcRγ chain, and / or genetic disruption resulting in a reduction in the expression of a protein that regulates the expression or activity of the FcRγ chain; and / or Genetic disruption resulting from genetic disruption of genes encoding proteins involved in FcRγ-dependent signaling and / or reduced expression of proteins involved in FcRγ-dependent signaling. The manipulated NK cells according to claim 1, comprising:

3. The manipulated NK cell according to claim 2, wherein the genetic disruption includes deletions, mutations, and / or insertions resulting in a frameshift of an immature stop codon or a gene reading frame in the gene.

4. The engineered NK cell according to claim 2 or 3, wherein both alleles of the gene encoding the FcRγ chain, the gene encoding a protein that modulates the expression or activity of the FcRγ chain, and / or the gene encoding a protein involved in FcRγ chain-dependent signaling are disrupted in the engineered NK cell.

5. The engineered NK cells according to claim 1, comprising a gene-targeting inhibitory nucleic acid molecule in NK cells that results in reduced expression of FcRγ chains, reduced expression of proteins that regulate FcRγ chain expression or activity, and / or reduced expression of proteins involved in FcRγ chain-dependent signaling.

6. The engineered NK cells according to any one of claims 2 to 5, wherein the expression of FcRγ chains, proteins that regulate the expression or activity of FcRγ chains, and / or proteins involved in FcRγ chain-dependent signaling is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression of proteins in unengineered NK cells, or by about 50%, 60%, 70%, 80%, 90%, or 95%.

7. The engineered NK cells according to any one of claims 2 to 6, wherein the expression of a protein that modulates the expression or activity of the FcRγ chain is reduced in the engineered NK cells, and the protein is a transcription factor.

8. The engineered NK cells according to claim 7, wherein the transcription factor is PLZF (ZBTB16) or HELIOS (IKZF2).

9. The engineered NK cells according to any one of claims 2 to 6, wherein the expression of a protein involved in FcRγ chain-dependent signaling is reduced in the engineered NK cells, and the protein is a downstream signaling molecule.

10. The engineered NK cell according to claim 9, wherein the downstream signaling molecule is SYK, DAB2, or EAT-2.

11. The manipulated NK cells according to any one of claims 2 to 6, wherein the expression of the FcRγ chain is reduced in the manipulated cells.

12. Engineered NK cells comprising genetic disruption in a gene encoding the FcRγ chain, wherein FcRγ expression is reduced in the cells.

13. The manipulated NK cell according to claim 12, wherein the genetic disruption includes deletions, mutations, and / or insertions resulting in a frameshift of an immature stop codon or a gene reading frame in the gene.

14. The engineered NK cell according to claim 12 or 13, wherein both alleles of the gene encoding the FcRγ chain are disrupted in the genome of the engineered NK cell.

15. Manipulated NK cells according to any one of claims 1, 5, or 6, comprising an inhibitory nucleic acid molecule that targets a gene encoding an FcRγ chain.

16. The engineered NK cell according to claim 15, wherein the inhibitory nucleic acid molecule contains a sequence complementary to the gene encoding the FcRγ chain.

17. The engineered NK cells according to any one of claims 11 to 16, wherein the expression of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression in unengineered NK cells, or by approximately more than 50%, 60%, 70%, 80%, 90%, or 95%.

18. The engineered NK cells according to any one of claims 5-6 and 15-17, wherein the inhibitory nucleic acid comprises an RNA interferant.

19. The engineered NK cells according to any one of claims 5-6 and 15-18, wherein the inhibitory nucleic acid comprises siRNA, shRNA, or miRNA.

20. The engineered NK cells according to any one of claims 1 to 19, wherein the reduction in FcRγ expression, activity, and / or signaling is permanent, transient, or inducible.

21. The engineered NK cells according to any one of claims 1 to 20, wherein the expression, activity, and / or signaling of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to the expression, activity, and / or signaling in unengineered NK cells, or by about 50%, 60%, 70%, 80%, 90%, or 95%.

22. The manipulated NK cells according to any one of claims 1 to 21, wherein the expression of the FcRγ chain expressed in the cells is undetectable by an immunoblot assay.

23. The manipulated NK cells according to any one of claims 1 to 22, wherein CD16 is expressed on the surface of the manipulated NK cells.

24. A modified NK cell according to any one of claims 1 to 23, expressing a CD3-zeta (CD3ζ) chain.

25. Manipulated NK cells according to any one of claims 1 to 24, derived from primary cells obtained from the subject.

26. The manipulated NK cells according to claim 25, wherein the subject is human.

27. Manipulated NK cells according to any one of claims 1 to 24, derived from a clonal cell line.

28. The manipulated NK cell according to claim 27, wherein the clonal cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

29. Manipulated NK cells according to any one of claims 1 to 28, comprising a recombinant or heterologous CD16 gene and / or a recombinant or heterologous CD3-zeta (CD3ζ) chain.

30. The engineered NK cells according to claim 29, wherein CD16 comprises a CD16 activating mutation, a mutation resulting in a higher affinity for IgG1, a 158V mutation and / or a 158F mutation.

31. The manipulated cells according to any one of claims 1 to 30, wherein the manipulated NK cells show increased activity when stimulated via CD16 compared to unmodified NK cells.

32. Manipulated NK cells according to any one of claims 1 to 31, wherein surface expression of NKp46, NKp30, and / or NKp44 is reduced compared to unmodified NK cells.

33. A method for producing engineered NK cells, comprising the step of genetically modifying NK cells to reduce the expression, activity, and / or signaling of the FcRγ chain in said cells.

34. The step of reducing expression is Disrupting or suppressing genes encoding FcRγ chains, and / or genes that result in reduced FcRγ chain expression in NK cells; Disrupting or suppressing genes encoding proteins that regulate the expression or activity of FcRγ chains, and / or disrupting or suppressing genes that result in reduced expression of proteins that regulate the expression or activity of FcRγ chains in NK cells; and / or Disrupting or suppressing genes encoding proteins involved in FcRγ chain-dependent signaling, and / or disrupting or suppressing genes that result in reduced expression of proteins involved in FcRγ chain-dependent signaling in NK cells. The method according to claim 33, including the method described in claim 33.

35. The method according to claim 33 or claim 34, further comprising the step of introducing a deletion, mutation, or insertion into the gene.

36. The method according to any one of claims 33 to 35, wherein the gene encodes an FcRγ chain.

37. The method according to any one of claims 33 to 35, wherein the gene encodes a protein that regulates the expression or activity of the FcRγ chain, which is a transcription factor.

38. The method according to claim 37, wherein the transcription factor is PLZF or HELIOS.

39. The method according to any one of claims 33 to 35, wherein the gene encodes a protein involved in FcRγ chain-dependent signaling, which is a downstream signaling molecule.

40. The method according to claim 38, wherein the downstream signaling molecule is SYK, DAB2, or EAT2.

41. The method according to any one of claims 33 to 40, wherein the disruption or suppression of the gene is carried out by introducing a gene-targeting endonuclease in NK cells under conditions that allow for the disruption or suppression of the gene.

42. The method according to claim 41, wherein the endonuclease is selected from the group consisting of TAL nuclease, meganuclease, zinc finger nuclease, Cas9, and argonaut.

43. The method according to any one of claims 33 to 40, wherein the disruption or suppression is carried out by introducing a gene-targeting inhibitory nucleic acid into NK cells under conditions that result in gene suppression.

44. The method according to claim 43, wherein the inhibitory nucleic acid molecule contains a sequence complementary to the gene encoding the FcRγ chain.

45. The method according to claim 43 or claim 44, wherein the inhibitory nucleic acid comprises an RNA interferant.

46. The method according to any one of claims 43 to 45, wherein the nucleic acid is siRNA, shRNA, or miRNA.

47. The method according to any one of claims 33 to 46, wherein the reduction in expression is permanent, transient, or inducible.

48. The method according to any one of claims 33 to 47, wherein the expression, activity, and / or signaling of the FcRγ chain is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95% compared to expression in non-genetically engineered NK cells, or by about 50%, 60%, 70%, 80%, 90%, or 95%.

49. The method according to any one of claims 33 to 48, wherein the expression level of the FcRγ chain is undetectable by an immunoblot assay.

50. The method according to any one of claims 33 to 49, further comprising the step of isolating NK cells from a human subject before the aforementioned genetic manipulation.

51. The method according to claim 50, further comprising the step of isolating NK cells from peripheral blood mononuclear cells (PBMCs).

52. The method according to claim 50 or 51, wherein the step of isolating NK cells includes selecting NK cells from PBMCs using an NK cell marker.

53. The method according to claim 52, wherein the NK cell marker is CD56, Cd161, KIR, NKG2A, NKG2D, NKp30, NKp44, and / or NKp46.

54. The method according to any one of claims 50 to 53, further comprising the step of selecting NK cells that express CD16 and / or CD3ζ.

55. The method according to any one of claims 50 to 54, further comprising the step of selecting NK cells that do not express surface CD3.

56. The method according to any one of claims 33 to 49, wherein the NK cells are an NK cell line.

57. The method according to claim 56, wherein the cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

58. The method according to claim 56 or claim 57, further comprising the step of manipulating an NK cell line to recombinate or express heterologous CD16 and / or CD3ζ.

59. The method according to claim 58, comprising the step of introducing nucleic acids encoding CD16 and / or CD3ζ into NK cells.

60. The method according to claim 58 or claim 59, wherein the CD16 gene includes an activating mutation and / or a mutation that increases the affinity of CD16 to IgG.

61. The method according to any one of claims 58 to 60, comprising a mutation in which CD16 is 158V.

62. The method according to any one of claims 58 to 60, comprising a mutation in which CD16 is 158F.

63. The method according to any one of claims 58 to 62, comprising the step of virally transducing NK cells with nucleic acids encoding CD16 and / or CD3ζ.

64. The method according to any one of claims 58 to 62, comprising the step of transfecting NK cells with nucleic acids encoding CD16 and / or CD3ζ.

65. The method according to any one of claims 58 to 64, comprising the step of transiently, inducibly, or persistently expressing CD16 or CD3ζ in the NK cells.

66. The method according to any one of claims 33 to 65, further comprising the step of culturing or growing manipulated NK cells.

67. The method according to claim 66, further comprising the step of culturing manipulated NK cells together with feeder cells.

68. The method according to claim 66 or claim 67, comprising the step of culturing manipulated NK cells with cytokines.

69. Engineered NK cells produced by the method described in any one of claims 33 to 68.

70. A composition comprising an effective amount of manipulated NK cells according to any one of claims 1 to 32 or 69.

71. The composition according to claim 70, further comprising a pharmaceutically acceptable carrier.

72. The composition according to claim 71, wherein the carrier is physiological saline solution, dextrose solution, or 5% human serum albumin.

73. 1 x 10 5 ~1 x 10 8 A composition according to any one of claims 70 to 72, comprising 1 cell / mL.

74. A composition according to any one of claims 70 to 73, comprising an antifreeze agent.

75. A kit comprising manipulated cells according to any one of claims 1 to 32 or 69 or a composition according to any one of claims 70 to 74, and a further active substance for the treatment of a disease.

76. The kit according to claim 75, wherein the further active substance is an antibody or an Fc fusion protein.

77. The kit according to claim 76, wherein the antibody recognizes or specifically binds to a tumor-associated antigen.

78. 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-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGF2, EPHA3, FR-alpha, phosphatidylserine, syndecane 1, and SLAMF7. The kit according to claim 77, wherein (CD319) TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin.

79. The kit according to any one of claims 76 to 78, wherein the antibody is a full-length antibody and / or comprises an Fc domain.

80. A method for treating a condition, comprising the step of administering manipulated NK cells according to any one of claims 1 to 32 or 69 or a composition according to any one of claims 70 to 74 to an individual in need thereof.

81. The method according to claim 80, wherein, prior to the step of administering the manipulated NK cells, the manipulated NK cells are produced by the method of any one of claims 33 to 68.

82. administering to the subject 1×10 8 to 1×10 10 cells / m 2 or approximately 1×10 8 to 1×10 10 cells / m 2 or administering 1×10 6 to 1×10 10 NK cells / kg or approximately 1×10 6 to 1×10 10 NK cells / kg, the method according to claim 80 or claim 81, comprising this step of administration.

83. The method according to any one of claims 80 to 82, further comprising the step of administering a further active substance.

84. The method according to claim 83, wherein the further active substance is an antibody or an Fc fusion protein.

85. The method according to claim 84, wherein the antibody recognizes a tumor-associated antigen.

86. 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-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scattering factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGF2, EPHA3, FR-alpha, phosphatidylserine, syndecane 1, and SLAMF7. The method according to claim 84 or claim 85, wherein (CD319) is TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin.

87. The method according to any one of claims 84 to 86, wherein the antibody comprises an Fc domain and / or is a full-length antibody.

88. The method according to any one of claims 83 to 87, wherein further active substances and manipulated NK cells are administered sequentially.

89. The method according to claim 88, wherein an additional active agent is administered before the administration of the manipulated NK cells.

90. The method according to any one of claims 83 to 87, wherein an additional active substance and manipulated NK cells are administered simultaneously.

91. The method according to any one of claims 80 to 90, wherein the condition is selected from the group consisting of inflammatory conditions, infections, and cancer.

92. The method according to claim 91, wherein the infectious disease is a viral infection or a bacterial infection.

93. The method according to claim 92, wherein the cancer is leukemia or lymphoma.

94. The method according to claim 92, wherein the cancer includes a solid tumor.

95. The method according to any one of claims 80 to 94, wherein the individual is a human.

96. The method according to any one of claims 80 to 95, wherein the manipulated NK cells are homogeneous with respect to the individual.

97. The method according to any one of claims 80 to 95, wherein the manipulated NK cells are of autologous origin to the subject.

98. The engineered NK cells according to any one of claims 1 to 32, which are genetically modified to reduce the expression of NK inhibitory receptors.

99. This includes genetic disruption of the gene encoding the NK inhibitor receptor and / or genetic disruption resulting in reduced expression of the NK inhibitor receptor; or The inhibitory nucleic acids include those that target genes encoding NK inhibitor receptors and / or reduce the expression of NK inhibitor receptors. The manipulated NK cells according to claim 98.

100. The engineered NK cell according to claim 98 or claim 99, wherein the inhibitory receptor is NKG2A or KIR2DL1.

101. The method according to any one of claims 32 to 68, further comprising the step of genetically modifying NK cells to reduce the expression or activity of inhibitory receptors.

102. The method according to claim 101, wherein the step of reducing expression or activity includes disrupting or suppressing the expression of a gene encoding an NK inhibitor receptor or a gene resulting in reduced expression of an NK inhibitor receptor.

103. The method according to claim 102, wherein the inhibitory receptor is NKG2A or KIR2DL1.

Images