Transforming growth factor beta-resistant natural killer cells

Abstract

To provide a method of treating cancer or infection, using an NK cell with altered gene expression profiles, resulting in high cytokine expression and TGF-β superfamily resistance.SOLUTION: The present invention provides a method of treating cancer or infection in a subject in need thereof, wherein the method includes administering to the subject a therapeutically effective number of transforming growth factor-β (TGF-β) superfamily-imprinted Natural Killer (TGFβiNK) cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 623,682, filed January 30, 2018. No. 6,399,413, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Clinicians can recognize tumor cells without the need for any specific tumor cell marker. Natural killer (NK) cells are used for adoptive transfer because of their ability to recognize and kill Alizadeh et al., Clin Cancer Res. 16(13):3399-3408(2 010). However, the development of NK cell adoptive transfer procedures has been hampered by the limited supply of viable cells. NK cells represent only a small proportion of cells in the blood, and their isolation from a typical blood draw Furthermore, NK cells were not obtained by CD3 and CD19 depletion, respectively. These cells must be purified to avoid contaminating PBMCs such as T and B cells. Childs et al., American Society of Hematology (ASH) Education Book, vol.2013 no.1 234-246(2013). This is where the presence of T and B cells increases the risk of graft-versus-host disease (GVHD). Although a necessary step for allogeneic transplantation, it further reduces NK cell yield.

[0003] NK cells are less potent in vitro compared to other cell types, mainly due to early senescence. Moreover, by using the most effective methods, NK cells can be cultured for only a few passages. are susceptible to telomere shortening and senescence after aging. Denman et al.,PLoS ONE 7(1): e30264(20 12) The most effective method to increase NK cell survival and proliferation in vitro is The most commonly used feeder cells for NK cell expansion are The cells were irradiated peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transduced EBV-transfected lymphoblastoid cell lines (EBV-LCL), constitutively expressing IL-15 or IL-21 genes These include modified K562 cells, and other irradiated tumor cell lines. Berg et al., Cyto therapy, 11(3): 34l-55(2009). Co-culturing with feeder cells increases the number of cells by 1,000 to 50,000 times. Increasing the population significantly increases NK cell survival and proliferation.

[0004] Immunotherapy is a promising treatment for cancers such as osteosarcoma (OS), where survival has not improved over the past 30 years. OS, including chemotherapy-resistant OS, are highly promising candidates for self-activation. are readily killed in vitro by both NK cells and allogeneic activated NK cells. However, the tumor microenvironment in OS and other solid tumors is a highly immunosuppressive site. kinin and transforming growth factor-β (TGF-β) levels. Lamora et al. al., Clin Cancer Res 20:5097-5112(2014);Xu et al., DNA Cell Biol,33:802-806(2014) The addition of a blocking antibody against TGF-β improved the efficacy of dendritic cell vaccines in OS (Ka Wano et al., Clin Orthop Relat Res., 470: 2288-2294 (2012)), TGF-β is an immune factor in OS Providing proof of principle that is actively inhibiting therapy.

[0005] TGF-β inhibition of NK cell function is multifaceted. TGF-β regulates NK cell development and TGF-β mediates the NK cell proliferation and function after it reaches maturity. TGF-β promotes the immature NK cell lineage by inhibiting the progression of cells. CD16+ NK cells can also be induced to become CD16-. Keskin et al., Proc Natl Acad Sci USA, 104: 3378-3383 (2007). In addition, dominant-negative TGFβR Mice expressing NK cells had increased numbers of mature NK cells compared to wild-type mice. Science signaling 9:ral9(2016).

[0006] In mature NK cells, TGF-β inhibits antitumor activity through multiple mechanisms. , reduces IL-2 and IL-15 induces NK cell proliferation (Wilson et al., PloS one 6:e228 42(2011)), and IL-15 induced mTOR activation. Viel et al.,Sci Signal.,16;9(4l5) :ral9(2016). TGF-β also inhibits IFNγ secretion, which is required to stimulate the adaptive immune system. TGF-β is important and can render tumors sensitive to NK cell lysis. Inhibits IFNγ both directly and indirectly. SMAD3 directly binds to the IFNγ promoter. by binding to and reducing expression of the IFNγ-promoting transcription factors, T-bet, and E4BP4. It can also directly inhibit IFNγ. Tang et al., Nat Commun, 8:14677(2017). However, TGF-β-mediated inhibition of IFNγ secretion was observed following pre-inhibition of NK cells with IL-12, IL-15 or IL-18. This may be partially alleviated by incubation. Yu et al., Immunity, 24:575-59 0 (2006). In addition, TGF-β also inhibits TNFα and GM-CSF secretion and inhibits chemokine receptors. Regulating expression of NK cells in the bone marrow promotes retention of NK cells. Castriconi et al., J Immunol, 190: 5321-5328(2013).

[0007] Specifically, TGF-β promotes granzyme and perforin secretion, as well as the activation of the following receptors: By reducing the expression of NKG2D, NKp30, KIRs, DNAM-1, NKp44, TRAIL and CD16 These NK cells mediate their inhibition of NK cytotoxicity by inhibiting NK cytotoxicity in malignant cells that express their cognate ligands. Inhibits NK cell recognition of cells.

[0008] There are several approaches to generating TGF-β-resistant NK and T cells. These include cells expressing dominant-negative TGFβRII and immune-based therapies. Both involve combination therapy using TGF-β small molecule inhibitors. Importantly, all of these approaches demonstrated improved efficacy of NK and T cell therapy in vitro and in vivo. Ta. Wallace et al., Clin Cancer Res.,14(12):3966-74(2008);Bollard, C.,Blood,99:317 9-3187(2002). However, broad-spectrum inhibition of TGF-β has the potential for adverse side effects. TGF-β signaling is context-dependent and exerts both tumor-promoting and tumor-suppressing effects. For example, inhibition of TGF-β in mouse models can result in circulating Increases the number of ring tumor cells (Wrzesinski et al., Clin Cancer Res., 13:5262-5270 (2007) ), and broad-spectrum inhibition of TGF-β results in severe autoimmune disease. Li et al., Immunology nity 25:455-471(2006). Thus, inhibition of TGF-β should be done with caution and with a strong anti-TGF-β response. Therefore, the use of TGF-β as a marker for tumor reactivity should be considered only after determining the reactivity of the tumor. Nonsystemic methods of generating resistance are promising for circumventing the potentially adverse effects of systemic TGF-β inhibition. It is an alternative. Summary of the Invention

[0009] Many types of cancer reduce NK cell killing through the release of TGF-β. A non-genetic approach to imprint NK cells to become resistant to TGF-β We developed TGF-β imprinted NK (TGFβi NK) cells, which are NK cells stimulated with IL-12 / 15 / 18. This was generated by adding TGF-β during stimulation of the cells. TGFβi NK cells are a type of NK cell that is more sensitive to TGFβ than conventional NK cells. In addition, TGs cultured with TGF-β maintained their cytotoxicity after stimulation. Fβi NK cells increase secretion of IFN-γ, TNF-α, and GM-CSF, which increases tumor killing Increased cytokine secretion can stimulate a broad range of immune responses. Resistance to TGF-β persists for at least one week in vitro. and mediated by SMAD3 downregulation.

[0010] The present invention may be more readily understood with reference to the following drawings. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 provides graphs showing that NK cell activation with parental (unmodified) K562 in the presence of TGFβ induces TGFβi NK cells with cytokine hypersecretion in response to tumor targets. NK cells were stimulated weekly with K562 and cultured in medium containing IL-2 (control) or IL-2 and 10 ng / mL TGFβ (TGFβi) for 14 days. After culture, IFNγ and TNFα secretion was assessed in the supernatants after coculture with MG63 tumor targets. Lines and bars represent mean ± standard deviation. Statistical significance was determined by two-way repeated measures ANOVA with Holm-Sidak's multiple comparison test for all other graphs. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Diagram 2] Figure 2 provides a graph showing that NK cell activation with proinflammatory cytokines in the presence of TGFβ induces TGFβi NK cells with cytokine hypersecretion in response to tumor targets. NK cells were activated overnight with IL-12, -15, and -18 (10 ng / mL, 50 ng / mL, and 50 ng / mL, respectively) with or without IL-2 and TGFβ, followed by culture in IL-15 (1 ng / mL) with or without IL-2 and TGFβ. After 7-14 days of culture, antitumor IFNγ and TNFα production in response to MG63 was measured by intracellular flow cytometry (n-4). Percent IFNγ+ and TNFα+ NK cells were normalized to no targets. Individual data points are shown for all. Statistical significance was determined by two-way repeated measures ANOVA with Holm-Sidak's multiple comparison test for all other graphs. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. [Diagram 3] FIG. 3 provides a graph showing that NK cell expansion with K562 mbIL-l5 (clone 4) feeder cells in the presence of TGFβ induces TGFβi NK cells with cytokine hypersecretion in response to tumor targets. After 14 days of expansion with feeder cells with (TGFβi) or without (control) TGFβ, NK cells were rested overnight in 50 IU / mL IL-2 with or without 10 ng / mL TGFβ. NK cells were then co-cultured with tumor targets in the same medium and supernatants were collected to measure cytokine secretion. Control, black; TGFβi, red. Statistical significance was determined by two-way repeated measures ANOVA with Holm-Sidak's multiple comparison test for all others. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 4] FIG. 4 provides a graph showing that NK cell expansion with K562 mbIL-21 feeder cells in the presence of TGFβ induces TGFβi NK cells with cytokine hypersecretion in response to tumor targets. After 14 days of expansion with feeder cells with (TGFβi) or without (control) TGFβ, NK cells were rested overnight in 50 IU / mL IL-2 with or without 10 ng / mL TGFβ. NK cells were then co-cultured with tumor targets in the same medium and supernatants were collected to measure cytokine secretion. Black, control; red, TGFβi. Statistical significance was determined by two-way repeated measures ANOVA with Holm-Sidak's multiple comparison test for all others. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Diagram 5]Figure 5 provides a graph showing that addition of TGFβ to generate TGFβi NK cells does not reduce proliferation capacity in expansion cultures with K562 mbIL-2l (CSTX002) feeder cells. NK cells were expanded for 14 days on feeder cells with (TGFβi) or without (control) TGFβ in paired cultures from 5 donors starting with the same number of cells on day 0. The total number of viable cells after 14 days is shown and normalized to control NK cells for each paired expansion. Not significant by Student's paired t-test. [Figure 6] FIG. 6 provides graphs showing that TGFβi NK cells maintain cytokine hypersecretion against tumor cells for several weeks. A) TGFβi and control NK cells expanded with parental K562 feeder cells for 14 days (expansion) or expanded and rested for an additional 7 days (rest) were co-cultured with MG63 target cells and supernatants were assessed for cytokine secretion. B) In a similar experiment, cytokine secretion against MG63 with K562mbIL-21 expanded control and TGFβi NK cells was assessed at days 7 and 14 of expansion and again after 21, 35, and 47 days of rest. Medians are shown with minimum to maximum whiskers. Black, control, red, TGFβi. Statistical significance was determined by paired t-test. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001 [Figure 7] Figure 7 provides a graph showing that TGFβi NK cells exhibit cytokine hypersecretion against multiple cancer types. After 14 days of growth under control conditions or with TGFβ present to induce TGFβi NK cells, NK cells were rested overnight with or without IL-2 or TGFβ. NK cells were then co-cultured with tumor targets in the same fresh medium for 3 hours, and supernatants were collected to measure IFNγ and TNFα cytokine secretion. Individual data points are shown for DAOY (medulloblastoma, n=12) and CHLA-255 (neuroblastoma, n=5). Lines and bars represent the mean ± standard deviation. [Figure 8]8 provides a graph showing that TGFβi NK cells show dramatic downregulation of the TGFβi-signaling protein, SMAD3. After 14 days of expansion under control conditions or with TGFβ present to induce TGFβi NK cells, NK cells were assessed for protein expression of SMAD3 and E4BP4 by Western blot (n=4). [Figure 9] Figure 9 provides a graph showing that TGFβi NK cells hypersecrete GM-CSF, TNFα, and IFNγ. Control and TGFβi NK cells were stimulated with 10 μg / mL PHA at 2×10e6 NK cells / mL for 4 hours and cytokine secretion was measured. Lines and bars represent mean ± standard deviation. Statistical significance was determined by paired t-test. [Figure 10] FIG. 10 provides a graph showing that TGFβi NK cells grown with parental K562 have similar cytotoxicity to control NK cells. Control and TGFβi NK cytotoxicity was measured using a 4-hour calcein-release cytotoxicity assay after overnight treatment with IL-2 alone or IL-2 and TGFβ. Lines and bars represent mean values ​​± standard deviation. Statistical significance was determined by two-way repeated measures ANOVA with Holm-Sidak's multiple comparison test. *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 11] FIG. 11 provides a graph showing that expanded TGFβi NK cells control tumor growth better than control expanded NK cells in a mouse model of osteosarcoma. Treatment of mice with TGFβi NK cells expanded on K562mb IL-21 feeder cells significantly reduces the growth of the 143b osteosarcoma cell line in the lungs of NSG mice, but not control NK cells. [Figure 12]FIG. 12 provides a graph showing that TGFβi NK cells have a gene expression profile that distinguishes them from standard expanded NK cells. Standard and TGFβi NK cells were grown in pairs on CSTX002 from four donors. mRNA was isolated and subjected to RNA sequencing. The top 100 differentially expressed genes were identified. A) Principal component analysis was performed on the 1n(x+1) transformed original values. Unit variance scaling was applied to the columns; SVD with imputation is used to calculate the principal components. The X and Y axes show Principal Component 1 and Principal Component 2, which explain 95.4% and 1.6% of the total variance, respectively. TGFβi NK cells are identified with red boxes. >95% of the variance is due to TGFβ imprinting, while <2% is a result of donor variation. B) Clustering analysis was performed on the same log-transformed data. Columns are centered; unit variance scaling was applied to the columns. Both the rows and columns are clustered using correlation distance and average linkage methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description The present invention relates to a method for the treatment of cancer cells that are highly cytotoxic, produce high levels of cytokines, and are immunosuppressive. TGF-β imprinted natidine kinase inhibitors that are resistant to the TGF-β superfamily of cytokines These cells are involved in the regulation of the TGF-β superfamily. - Chronic in vitro activation of natural killer cells in the presence of cytokines The present invention also provides a method for administering a therapeutically effective number of TGFβi NK cells to a subject. Methods of Treating Cancer or Infectious Disease in a Subject in Need thereof by Administering - Patent application Also provided.

[0013] definition For clarity of understanding and ease of reference, the brief description and the remainder of this application have been omitted. A list of terms used throughout the remainder of this document is compiled here. Some of the terms are: Although well known throughout the art and defined herein for clarity, However, some of the terms are unique to this application and therefore may be difficult to understand properly. must be defined in order to

[0014] "A" or "an" as used herein means one or more than one; at least When the plural is used herein, it generally includes the singular.

[0015] Also herein, the recitation of numerical ranges by endpoints includes every amount subsumed within that range. includes all numbers (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0016] As used herein, the term "subject" refers to a human, rat, mouse, dog, goat, sheep, etc. , horses, monkeys, apes, pigs, rabbits, cows, etc. The term may refer to any warm-blooded organism. When used in the context of a request, the term means "someone who needs The subject may be referred to as a "subject" and is clinically diagnosed as being in need of the compositions of the present application (e.g., For example, by a medical professional, such as a physician, a subject in need of the compositions of the present application. The present invention relates to a subject suspected of having a disease, a subject at risk for a disease or condition, and a subject administering to the subject a composition of the present application. Subjects who may benefit, as well as those who are already suffering from a disease or condition and who are This includes subjects who would benefit from the composition.

[0017] As used herein, the term "pharmaceutically acceptable" means any compound that is within the scope of sound medical judgment. within a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reactions or For use in contact with human and animal tissues without other problems or complications "Compounds, materials, compositions and / or dosage forms" refers to those compounds, materials, compositions and / or dosage forms that are suitable for

[0018] The term "therapeutically effective" refers to a compound that achieves the goal of reducing disease severity, while typically Refers to the number or amount of medication that would avoid harmful side effects such as those associated with alternative therapies. It is intended that a therapeutically effective amount may be administered in one or more doses. Treatments that are therapeutically effective are those that do not themselves improve disease outcome. Includes treatments that improve the quality of life of a subject.

[0019] In general, an "effective amount" includes achieving a specific desired effect as described in this application. , providing a desired local or systemic effect, e.g., effective to stimulate cytokine formation. For example, an effective amount means an amount that provides a beneficial or desired clinical result. It is enough for.

[0020] The terms "treat," "treating," or "treatment" are used broadly in the present invention and include, Each of these terms refers, among other things, to what interferes with therapy and / or what is to prevent, alleviate, or prevent a defect, dysfunction, disease or other deleterious process, including those resulting from In various embodiments, the present invention relates to a method for treating, preventing, or curing a disease or Symptoms of the disorder are at least 5%, at least 10%, at least 20%, at least 30%, or Both are mitigated by 40% or at least 50%.

[0021] As used herein, the term "administration" refers to the administration of at least a portion of a composition at a desired site. Incorporation into a subject by a method or route that results in localization of the compound to a desired effect. The term refers to an arrangement of a composition (e.g., a cellular composition) of a resistant natural killer as described herein. - The cells or compositions may be administered orally, or intravenously, intramuscularly, subcutaneously, transdermally, or via the respiratory tract (aerosol route). Parenteral routes include intravenous, pulmonary, nasal, rectal and topical (including buccal and sublingual) administration. By any suitable route known in the art, including, but not limited to, It can be administered.

[0022] As used herein, the term "cytokine" refers to a cytokine that plays a role in cell signaling and It refers to small proteins (approximately 5-20 kDa) that are important in certain immune regulation. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and and tumor necrosis factor.

[0023] Therapy using TGFβi NK cells In one embodiment, the present invention provides a method for treating cancer or an infectious disease in a subject in need thereof. The present invention provides a method of treating a subject with a therapeutically effective number of TGF-β imprinted genes. The method includes administering transformed growth factor beta (TGFβi) NK cells to the patient. Thus, typical expanded natural responses to cytokines of the TGF-β superfamily TGFβi NK cells are known to be involved in the regulation of immune function, as the resistance of killer cells protects them from suppression by the immune system. Compared to these, they are more effective and can last longer in vivo.

[0024] As used herein, "natural killer cells" ("NK cells") are cells of the immune system. NK cells are a type of cytotoxic lymphocyte that rapidly responds to virus-infected cells. Typically, immune cells react with the surface of infected cells. Detects peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the , triggering cytokine release, resulting in lysis or apoptosis. However, NK cells Whether or not peptides from pathogens are present on MHC molecules, they may be involved in stress responses. They are unique because they have the ability to recognize cells that have been dosed with a target. It is the first of its kind that does not require prior activation to kill the host, making it a NK cells are large granular lymphocytes (LGLs) and are secreted by the bone marrow. It is known that endothelial cells differentiate and mature within the endothelium, from which they then enter the circulation.

[0025] The present invention relates to a cytokine of the transforming growth factor β (TGF-β) superfamily. NK cells that are resistant to inflammatory cytokines, and a method for producing and using such resistant NK cells The TGF-β superfamily is a family of structurally related cellular regulatory proteins. TGF-β is a large group of cytokines that mediate proliferation, differentiation and other functions in many cell types. TGF-β-1 is a multifunctional peptide that controls the function of the These proteins are peptides of 112 amino acid residues derived from the proteolytic cleavage of The protein is a conserved phagocytic protein that mediates cell surface serine / threonine-specific protein kinase receptors. They interact with myri and use a conserved family of proteins called SMADs. As a major subfamily of the TGF-β superfamily, The TGF-β subfamily (including TGF-β 1 to 4 isoforms), the decapentaplegic DVR-related proteins (e.g., bone morphogenetic proteins), growth differentiation factors ( Examples include GDF-1 through GDF-15) and the activin and inhibin subfamilies. In some embodiments, the TGFβi NK cells are resistant to TGF-β.

[0026] TGFβi NK cells can be used to treat cancer or an infectious disease in a subject. Typically, TGFβi NK cells are administered by adoptive transfer of cells. In one embodiment, the subject has been diagnosed with cancer. Cancer is defined herein as A disease based on the development of cells that contain genetic lesions that result in relatively uncontrolled proliferation of cells. Genetic damage present in cancer cells is inherited in subsequent generations of cancer cell lines. The cancers to be treated by the methods of the present invention are known to those skilled in the art, and The cancer may be any of the forms of cancer described herein. Solid tumors and typically leukemias and treating cancers that manifest as both solid tumors and cancers that form instead of non-solid tumors as seen in The present invention can be used to treat a variety of different types of cancers, including carcinomas, sarcomas and lymphomas. A method for treating a subject afflicted with cancer is provided.

[0027] In some embodiments, the cancer being treated is leukemia (e.g., acute lymphoblastic leukemia). acute myeloid leukemia; chronic myeloid leukemia, chronic lymphocytic leukemia), myelodysplastic syndrome, lymphoma (e.g., B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoblastic lymphoma) lymphoma, anaplastic large cell lymphoma), solid tumors (e.g., breast cancer, prostate cancer, gastric cancer, large intestinal cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, neuroblastoma, high-grade glioma), sarcoma (e.g., Eugene In a further aspect, the present invention relates to a method for treating sarcoma, such as sarcoma of the ovarian tumor, sarcoma of the thyroid gland ... Preferably, the cancer is selected from the group consisting of leukemia, lymphoma, rhabdomyosarcoma, brain cancer and bone cancer.

[0028] The efficacy of cancer treatment is characterized by a reduction in tumor burden or tumor size in a subject in response to administration of TGFβi NK cells. The reduction in tumor burden may be measured by assessing the reduction in tumor growth. or this may be measured in terms of tumor growth delay; This is the average time it takes for a control tumor to grow to a certain volume compared to the average time it takes for a treated tumor to grow to the same volume. It is calculated by subtracting from the time required for proliferation.

[0029] In other embodiments, the subject being treated has an infection. TGFβi NK cells are It has a potent systemic effect and can be used to treat infections caused by a variety of different microorganisms. As used herein, the term "infectious disease" refers to a disease caused by a virus, a pathogenic bacterium, or It is caused by infection with a fungus and is transmitted through the respiratory tract, blood, or skin contact. Non-limiting examples of such infectious diseases are: These include Hepatitis B, Hepatitis C, Human Papillomavirus (HPV) infection, and Human Immunodeficiency Virus (HIV). , cytomegalovirus infection, viral respiratory diseases, and influenza. However, it is not limited to these.

[0030] TGF-β imprinted natural killer cells Another aspect of the present invention is the use of TGF-β superphages, referred to herein as TGFβi NK cells. Natural killer (NK) cells or NK cells cultured in the presence of cytokines The present invention provides an NK cell or cell line produced by the methods described herein. and compositions comprising the NK cells provided herein. is a pharmaceutical composition comprising one or more of the NK cells or cell lines provided herein. In some embodiments, TGFβi NK cells exhibit increased resistance to TGF-β.

[0031] The TGFβi NK cells can be allogeneic or autologous. In the present specification, the NK cells are mammalian NK cells. Examples of "mammals" or "mammals" include Examples include primates (e.g., humans), dogs, cats, rodents, pigs, and ruminants. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, and guinea pigs. In certain embodiments, the mammalian NK cells are human NK cells. It is a cell.

[0032] TGFβi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells. In some embodiments, the NK cells or cell lines exhibit increased resistance to TGF-β. In other embodiments, the NK cells are stimulated with increased amounts of interferon-γ (IFN-γ ), and / or tumor necrosis factor-α (TNF-α), and / or granulocyte-macrophage In a further embodiment, the NK cells produce reduced levels of GM-CSF. Bell's SMAD family member 3 (SMAD3) protein and / or transformin Transforming growth factor beta receptor III (TGFBR3) protein. SMAD proteins are expressed in the nematode C. elegans ( ans)Sma and Drosophila Mad (Derynck et al., Cell, 95(6), p737 -740, 1998) and received their name as a contracted form of the name of the transcriptional activator of TGF-β response. It is a factor.

[0033] TGFβi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells. In some embodiments, the NK cells have a gene expression profile substantially similar to that shown in FIG. Gene expression profiles that are substantially similar indicate that gene expression is In some embodiments, the TGFβi NK cells are within 10% of those that are IFN-γ. , producing increased amounts of one or more of the TNF-α and GM-CSF proteins. In some embodiments, the NK cells or cell lines express SCUBE1, MY07A, KLF3, WIPF3 and EPHA1. 4 shows increased expression of

[0034] TGFβi NK cells exhibit a number of characteristics that distinguish them from naturally occurring NK cells. In some embodiments, the TGFβi NK cells have reduced levels of SMAD3 protein and / or In some embodiments, the NK cells or cell lines express CD300A or TGFBR3 protein. , showing reduced expression of SGSM1, SMAD3, TBX21 and GZMK, TGFBR3 and GZMA.

[0035] TGF-β-superfamily imprinted natural killer (TGFBi NK) cell lines Preparation of Another aspect of the present invention is a method for the production of TGF-β-superfamily imprinted natural killer A method for producing a TGF-βi NK cell line, comprising: The method includes the in vitro activation of natural killer cells in the presence of a cytochrome P450 inhibitor. Additionally, the method includes administering to the subject patient one or more TGFβi The method may further include isolating or separating the NK cells. In some embodiments, the method may include culturing one or more TGFβi NK cells. In some embodiments, a TGFβi NK cell line is produced. The cell line can be maintained in cell culture. There are multiple cells.

[0036] Expansion (i.e., activation) refers to the ex vivo expansion of NK cells, thereby increasing the number of NK cell populations. NK cells can be expanded, for example, from peripheral blood mononuclear cells. However, NK cells can also be expanded from other cell types, such as hematopoietic stem or progenitor cells. The first blood or stem cells can be derived from the placenta, umbilical cord blood, placental blood, peripheral blood, spleen, or liver. The cells can be isolated from a variety of different sources, such as: Suitable cell culture media are known to those skilled in the art and include Basal Eagle's Medium (BME), Darmstadt's Medium (Dallas), and others. Dulbecco's modified Eagle's medium (DMEM), Glasgow minimal essential medium (GMEM), Dulbecco's modified Eagle's medium (DMEM), Ham's Medium / Nutrient Mixture F-12 (DMEM / F-12), Minimal Essential Medium (MEM), Iscove's Modified Rubecco's medium (IMDM), Nutrient mixture F-10 Ham (Ham F-10), Nutrient mixture F-12 Ham (Ham F- 12), RPMI-1640 medium, William's medium E, STEMSPAN® (Cat. No. Stem Cell l Technologies, Vancouver, Canada), Glycostem Basal Growth Medium (GBGM®), AI MV® medium (Invitrogen), X-VIVO™ 10 (Lonza), X-VIVO™ 15 ( Lonza), OPTMIZER (Invitrogen), STEMSPAN® H3000 (STEMCELL Technology s), CELLGRO COMPLETE™ (Mediatech) or any modified variant thereof The above-mentioned methods may be used in combination.

[0037] As used herein, the term "feeder cells" refers to cells that have the ability to divide and grow. Although they do not possess the ability to express NK cells, they do have metabolic activity and therefore produce a variety of metabolic products to stimulate the target NK cells. These refer to cells that support the growth of cells. Examples of feeder cells that may be used in the present invention are: These include transfected animal cell lines, cells treated with various cytokines or compounds, and Peripheral blood leukocytes (PBLs), autologous or allogeneic peripheral blood leukocytes (PBLs), T cells, B cells, monocytes In some embodiments, the feeder cells include, but are not limited to, In a further embodiment, the K562 feeder cells are The cells are selected from clone 4 cells, clone 9 cells and CSTX002 cells.

[0038] In some embodiments, the in vitro activation of natural killer cells is by NK-stimulating exon 1 ( ... The stimulation is carried out in the presence of exosomes or NIC nanoparticles. Tumor-derived extracellular vesicles are small extracellular vesicles derived from endosomes with diameters between 100 and 200 mm. The endothelial cell transports many molecules and factors from tumor cells and stimulates natural killer cells. Li et al., Exp Cell Res., 363(2):141-150 (2018) Nanoparticles can also be used to stimulate natural killer cells. Nanoparticles are particles between 1 and 2500 nm in size with a surrounding interfacial layer. These include ultrafine nanoparticles with sizes of 1-100 nm and nanoparticles with sizes of 100-2500 nm. These include tiny nanoparticles. Nanoparticles can be made of polymers or minerals such as graphene oxide. In some embodiments, the nanoparticles can be prepared using natural The antibody may be functionalized to include additional groups, such as antibodies that help stimulate L cells. See, for example, Loftus et al., Nano Lett., 18(5):3282-3289 (2018).

[0039] Dosage and Administration TGFβi NK cells are administered according to the site and method of administration, administration schedule, patient age, and sex. The type, weight, nature and severity of the disorder being treated or prevented, and other factors known to the physician. The cells should be administered and taken in accordance with good medical practice, taking into account the following factors: The compounds may be administered in a single dose or in divided doses. A pharma- ceutically "effective amount" for a given purpose is understood to be within the scope of such considerations as are known in the art. The amount will be determined by the objective of the study, such as improved survival or more rapid recovery, or The improvement or elimination of symptoms and other indicators as selected by the skilled artisan as appropriate. The measures must be effective to achieve the improvement, including but not limited to:

[0040] Typically, the dose is about 10×10 6 Approximately 9 x 10 cells / kg of subject weight 6 cells / kg or less, Approximately 8×10 6 Cells / kg or less, approximately 7×10 6 Cells / kg or less, approximately 6×10 6 Cells / kg or less, approximately 5×10 6 cells / kg or more In an alternative embodiment, the dose is about 0.25×10 6 cells / kg ~ approx. 5×10 6 Thin cells / kg or about 1×10 6 cells / kg ~ approx. 5×10 6 cells / kg is preferred. Thus, in a further alternative embodiment, the dose is about 0.25×10 6 cells / kg, 0.5×10 6 cells / kg, 0.6×10 6 cells / kg, 0.7×10 6 cells / kg; 0.8×10 6 cells / kg; 0.9×10 6 cells / k g; 1.1×10 6 cells / kg; 1.2×10 6 cells / kg; 1.3×10 6 cells / kg; 1.4×10 6 cells / kg; 1.5×10 6 cells / k g; 1.6×10 6 cells / kg; 1.7×10 6 cells / kg; 1.8×10 6 cells / kg; 1.9×10 6 cells / kg, or 2 x 10 6 The dose may be in the range of 0.1 to 1 million cells / kg; or in other embodiments, the dose may be in the range of 0.1 to 1 million cells / kg; is 1-2 million cells / kg; or 2-3 million cells / kg; or 3-4 million cells / kg; or 4-5 million cells / kg / kg; or 5-6 million cells / kg; or 6-7 million cells / kg; or 7-8 million cells / kg; or 8-9 million cells / kg; or 9-10 million cells / kg.

[0041] Exemplary modes of administration include, but are not limited to, injection, infusion, drip, inhalation or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, and intravesicular. , intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, spinal These include, but are not limited to, intramedullary, intracerebrospinal and intrasternal injections and infusions. In a preferred embodiment, the compositions are administered by intravenous infusion or injection.

[0042] The TGFβi NK cells can be provided in the form of a pharmaceutical composition and can be sufficiently formulated for human administration. The composition may be sterile. The formulation should be suitable for the mode of administration. The reader is advised of the general principles of pharmaceutical formulation. Cell Therapy:Stem Cell Transplantation,Gene Therapy,and Cellular Immunotherapy,b y G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic See Stem Cell Therapy, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The choice of cell excipient and any of the compositions containing the TGFβi NK cell populations are The accessory elements will be adapted according to the route and device used for administration.

[0043] In some embodiments, the TGFβi NK cells are administered with a pharma- ceutically acceptable carrier. Suitable pharma- ceutically acceptable carriers include water, salt solutions (e.g., NaCl), saline, Water, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, Carbohydrates such as benzyl alcohol, polyethylene glycol, gelatin, and lactose, Amylose or starch, dextrose, magnesium stearate, talc, acid, viscous paraffin, perfume oil, fatty acid ester, hydroxymethylcellulose, poly Vinylpyrrolidone, and the like, as well as combinations thereof. The preparations may optionally contain auxiliary substances which do not adversely react with the active compounds, such as lubricants, protective agents, etc. Preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, coloring agents, flavorings and / or may be mixed with aromatic substances, etc.

[0044] The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed herein have been discovered by the inventors to work well in the practice of the present invention. This is in accordance with the representative techniques described herein and therefore constitutes a preferred mode for its practice. However, it should be recognized by those skilled in the art that the present invention can be considered to be a method for producing a high-quality product. Many changes may be made in the disclosed specific embodiments in light of this disclosure. still without departing from the spirit and scope of the present invention. You should get the same or similar results. EXAMPLES

[0045] Example 1: Imprinting of NK cells to become TGF-β resistant by TGF-β The inventors have demonstrated that loss of one of the key TGF-β signaling proteins, SMAD3, mediates Most likely, the TGF-β cultures have reduced sensitivity to TGF-β. The inventors have demonstrated that: a) TGFβi NK cells target tumors and and b) TGFβi NK cell secretion was significantly increased in mice. We demonstrate that cells retain cytokine hypersecretion for at least one month after activation.

[0046] method Cell culture: NK cells were cultured in RPMI 1640 medium supplemented with Glutamax, 10% FBS, and antibiotics. All cells were routinely cultured using Lonza MycoAlert (Lonza, LT027-58). were tested for mycoplasma contamination and found to be negative at all time points. K562 feeder cells were purchased from ATCC (CCL-243) and irradiated at 100 Gy.

[0047] NK cell expansion: Purified primary human NK cells were cultured 1:2 with irradiated K562 on day 0 and The K562 cell lines used were unmodified (parental), expressing 4-1BBL, and The cells expressing 4-1BBL and membrane-bound IL-15 (clone 4), or 4-1BBL and membrane-bound IL-21 (clone 5), The standard expanded NK cell dose is 50 IU / ml L of recombinant human IL-2 and expanded resistant (TGFβi NK) NK cells were treated with 50 IU / ml The mice received 100 μL of IL-2 and 10 ng / mL of TGF-β (Biolegend, 580706). Inhibition of NK cell proliferation was measured by CD3 - / CD56 + Calculated based on cell percentage did.

[0048] Flow cytometry: Intracellular flow cytometry was performed using GolgiStop (BD Biosciences). Fixation / permeabilization was performed using the BD Cytofix / Cytoperm fixation / permeabilization kit (554715). Antibodies for proteins were used to assess NK phenotype and function: CD3 PeCy7 / APC-H7, CD56 FITC / BV421, NKG2D Pe-CF594 / BV5lO, TRAIL PE / APC / BV421, FasL PE, NKp30 PE / Ale xa Fluor 647 / PE-Vio6l5, Granzyme A APC, Granzyme B BV510, Perforin BV42 1, DNAM-1 BV711, CD 107a BV510, IFN-γ APC, TNF-α BV421, CD16 PE, and Tonbo G Host Dye 510 / 780. Cell events were acquired on an LSR Fortessa. Flow cytometry gated Viability was determined using cells stained with viability dye alone and single-color controls using FlowJo 7 .6.5 / 10 Analysis was performed using SMAD3 flow.

[0049] Cytotoxicity assay: NK cells were cultured in the presence of human IL-2 alone or 10 ng / mL soluble TGF-β (Biolegend Cytotoxicity assay by overnight incubation in either IL-2 (50 IU / mL) or IL-2 (50 IU / mL) A cytotoxicity assay using a calcein-AM-based method was performed. Perform at least two replicates using 3 μg Calcein AM / mL / 1,000,000 target cells in culture medium. Calcein assays were performed in the same cytokines as those used in overnight incubation of NK cells. It was. Somachi et al., Journal of visualized experiments: JoVE 48, 2540 (2011).

[0050] Intracellular functional flow cytometry: CD 107a expression and intracellular cytokines in response to tumors To determine degranulation by insulin production, 300,000 NK cells were cultured in 96-well round-bottom plates. , with 60,000 tumor cells (E:T ratio of 5:1) or without target for control. The cells were co-cultured in 200 μl of medium as described for the cytotoxicity assay. Antibodies were added to each sample along with CD 107a at the beginning of the assay. The plates were rotated for 3 min to promote cell-cell contact and then placed in a 37°C incubator for 3 hours. After 2 h, the medium was removed and staining was performed for cell surface and intracellular proteins as detailed. It all started with quality.

[0051] Cytometric Bead Array (CBA): Determining NK cell release of IFNγ and TNFα To identify NK cells, we performed intracellular functional flow cytometry analysis by excluding monensin and CD107a antibodies. The cells were cultured as described for the 3-h coculture with tumor targets or for 10 h. After 4 h of stimulation with 1 μg / mL PHA, the supernatants were collected and frozen at -75°C until use. On the day of the assay, the supernatant was thawed and 50 μL of undiluted supernatant was added to BD CBA Soluble Protein Master Kit (BD Biosciences, Cat#:558265), and IFNγ and TNFα Flex Set (BD Biosciences, Cat: 558269, 560112), or MACSPlex Cytokine 12 Kit (Miltenyi, Cat: The BD LSR II or MACSQ was used according to the manufacturer's instructions. The geometric mean for each sample was determined using Flow Jo v.10.1. The unknown samples were then compared to known standards for samples acquired with the BD LSR II. 2 ≧0.9 The standard curve was used for interpolation. Analysis of the samples obtained was performed using the MACSQuantify software. The analysis was performed using the software (version 2.8, Bergisch GladbacK, Germany). The mean APC value of the MACSPlex standard was used, and the cytokine counts in each sample were calculated. Calculate the inconcentration.

[0052] NK cell activation using cytokines: NK cell stimulation with IL-12, IL-15, and IL-18 For the study, primary NK cells were cultured as described, with 10 ng / mL IL-12 (Biolegend, 573002), 5 0 ng / mL IL-15 (Biolegend, 570302) and 50 ng / mL IL-18 (Biolegend, 592102). overnight stimulation, and then with IL-12, IL-15, and IL-18 at 1 ng / mL for 7–14 days. To determine the effects of IL-2 and TGFβ on cytokine production, NK cells were treated as described, but with overnight stimulation with IL-12, IL-15, and IL-18. As shown in , IL-2 and / or TGF-β were added, together with 1 ng / mL IL-15. To measure cytokine production, NK cells were treated with 1 ng / mL IL-15. Only overnight and throughout the assay, and at a ratio of 5:1 or target As a control without treatment, the same number of NK cells were co-cultured with MG63 and subjected to intracellular flow staining. The procedure was as described.

[0053] RT-PCR / qPCR: from fresh, never frozen, day 14 expanded human NK cells RNA was purified using the RNAeasy kit, QiaShredder Columns and RNAase-Free DNase set (all Qia cDNA was isolated using a PCR product (Agen, 74104, 79654, 79254) and the cDNA was transfected using the High Capacity cDNA Reverse Transcription Kit. PCR for the TGF-β pathway was performed using Applied Biosystems. 7900HT with Taqman Fast PCR Mastermix and Human Fast 96-well Pathway Array ( The analysis was performed using ThermoFisher (4418742).

[0054] Statistical analysis: Statistical analyses were performed using GraphPad Prism 6.0 or 7.0 (La Jolla, CA, USA). P values ​​less than 0.05 were considered significant. Ta.

[0055] Results and Discussion IFNγ and TNFα are two proinflammatory cytokines that are important in antitumor responses. It has been reported that their production is inhibited by TGF-β. To determine the production of proinflammatory cytokines, NK cells were cultured using parental (unmodified K562 ) plus or minus TGFβ for 2 weeks. At the end of the 2 weeks, control and TGFβ i NK cells were incubated with tumor targets as described and the supernatants were collected. The cells were collected and IFN-γ and TNF-α secretion was measured by cytometric bead array. In both the presence and absence of TGF-β, IFN-γ and TNFα secretion was significantly greater than that in control NK cells. A significant increase in the IL-1 expression was observed (Figure 1).

[0056] Second, stimulation with K562 or other tumor cells results in TGFβi cytokine overproduction. To this end, we determined whether NK cells were activated in the absence of tumor stimulation. We used IL-12, IL-15, and IL-18, which are well established as inhibitors of TGFβ in culture. When added, NK cells produced increased IFNγ and TNFα in response to tumor stimulation ( Figure 2).

[0057] Other K562 feeder cells enhance cytokine production by TGFβi NK cells To determine whether the cells were able to produce mbIL-15 (Figure 3) or mbIL-21 (Figure 4). K562 expressing TGFβ was co-cultured with NK cells for 2 weeks with or without TGFβ. The lead cells induced TGFβi NK cells with increased cytokine production.

[0058] TGFβ has been reported to inhibit NK cell proliferation, and therefore, K562mb may be involved in the response to IL-21 stimulation. To this end, TGFβ did not significantly affect proliferation. (Figure 5).

[0059] Next, we determined the persistence of TGFβi NK cell phenotype and function. After the completion of the 2-week activation, the NK cells were treated with low-dose IL-2 alone (e.g., TGFβi NK cells). TGFβi NK cells were incubated at 4°C for 1 h at which point proliferation and activity The secretion of IFN-γ and TNF-α was evaluated 7 to 33 days after activation. The results showed that IFN-γ and TNF-α secretion was significantly increased both at baseline and with TGF-β treatment. These increases were maintained (Figure 6).

[0060] In addition, we investigated whether TGFβi NK cells sustain cytokine overproduction in response to various stimuli. To this end, we investigated whether TGFβi NK cells could mediate the proliferation and proliferation of medulloblastoma and neuroblastoma cells. We found that the tumor cell lines produced increased IFNγ and TNFα in response to the cytokines This enhanced production was induced by PHA stimulation and was associated with increased production of antitumor cytokines. We found that the results suggest the potential of the IL-10 inhibitors for the treatment of IL-10A and IL-10B (Figs. 7 and 9).

[0061] The function of SMAD3 as a suppressor of NK cell antitumor function has been elucidated. Inhibits IFNγ expression by directly binding to the Nγ promoter and also inhibits SMAD3 - / - Mice NK cells Thus, the present inventors demonstrated that SMAD3 enhances the function of IL-1 and reduces tumor growth. The protein level was determined to be significantly reduced by ELISA using a tandem blot. We found TGFβi NK with reduced SMAD3 protein (Figure 8).

[0062] Furthermore, the cytotoxicity of TGFβi NK cells was evaluated in vitro using NK cells cultured with parental K562. NK cells grown with IL-1 and mbIL-2l were tested in vivo. Cytotoxicity was not affected. Indeed, in vivo, TGFβi NK cells were not able to upregulate the NK cells in the lungs of NSG mice. The compound significantly reduced the proliferation of osteosarcoma cells (Figure 11).

[0063] In addition, using RNA-seq analysis, we demonstrated that TGFβi NK cells are transcriptionally distinct from control NK cells and functionally We found that these results suggested broad changes in cell phenotype in addition to function (Figure 12).

[0064] In summary, human NK cells expanded with TGF-β culture (TGFβi NK) express important TGF-β receptors. TGF-β, most likely via a decrease in one of the β signaling proteins, SMAD3 TGFβi NK cells secrete IFN-γ, TNF-α, and GM-CSF. Surprisingly, and in contrast to previous studies, TGF-β significantly increased TGFβi N It did not inhibit K cell proliferation over a 2-week period. Bellone et al., J Immunol 155:1 066-1073 (1995). The stimulatory conditions used in the previous paper to induce proliferation (IL-2 and IL-15) and time point may be key to the observed differences in proliferation. Previous measurements of the effect of TGF-β on cell proliferation have examined short-term proliferation and a 2-week Proliferation was not tested. Viel et al., Science signaling 9:ral9(2016). Chronic TGF-β stimulation with tumor activation potentially upregulates SMAD3 for survival neg The characteristics of NK cells We propose that these SMAD3s drive the activation of heterologous subsets of SMAD3s. neg NK cells are cells Resistant to TGF-β-induced phosphorylation of SMAD3 in culture medium, allowing for increased proliferation Oida et al., Journal of immunological method 362:195-198(2010).

[0065] The ability of TGFβi NK cells to produce IFN-γ and TNF-α was evaluated, which indicates that these Cytokines inhibit TGF-β, and conversely, TGF-β stimulates the production of IFN-γ and TNF-α. Surprisingly, the canonical NK cell proliferation following TGFβ treatment was not observed in the control group. Significantly increased in TGFβi NK cells compared with both TGFβi and standard NK cells without TGFβi We found that SMAD3 deletion reduced tumor cell proliferation and secretion of antitumor IFN-γ and TNF-α. have demonstrated that SMAD3 increases IFN-γ production in mice, suggesting that a similar mechanism may be involved. It is highly likely that this also occurs in TGFβi NK cells that do not express TGFβi. n 8:14677(2017). TGF-β has been reported to inhibit TNFα production in primary NK cells. (Bellone et al., J Immunol 155:1066-1073 (1995)). Unexpectedly, TGFβi inhibited NK cell proliferation and proliferation. The cells were cultured with or without TGF-β in the assay medium compared to standard NK cells. The regulation of TNF-α production is less well understood, but TGF-α secretion was significantly increased. It is also known that TGFβi is inhibited by TNF-α, so the increased TNF-α production in NK cells The discovery of secretion was unexpected.

[0066] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, the form and details may vary. Various modifications of the present invention may be made without departing from the scope of the invention as encompassed by the appended claims. It will be understood by those skilled in the art that the same may be done in the same manner as described above. All patents, publications and references used are incorporated herein by reference in their entirety. can be.

Claims

1. 1. A method of treating cancer or an infectious disease in a subject in need thereof, comprising administering to said subject a trans Transforming growth factor-β (TGF-β) superfamily imprinted natural killer administering to said subject a therapeutically effective number of Ra (TGFβi NK) cells.

2. The method of claim 1 , wherein the subject has an infectious disease.

3. The method of claim 2 , wherein the infectious disease is a viral infection.

4. The method of claim 1 , wherein the subject has cancer.

5. The method of claim 4, wherein the cancer is a solid tumor.

6. The cancer is selected from the group consisting of leukemia, lymphoma, rhabdomyosarcoma, brain cancer and bone cancer. The method according to claim 4.

7. The method of claim 4, wherein the TGFβi NK cells are resistant to TGF-β.

8. The TGFβi NK cells produce increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF. The method of claim 1 .

9. The TGFβi NK cells have reduced levels of SMAD3 protein and / or TGFBR3 protein. The method of claim 1 , further comprising:

10. The TGFβi NK cells exhibited a gene expression profile substantially similar to that shown in FIG. The method of claim 1 , comprising:

11. 2. The method of claim 1, wherein the TGFβi NK cells are administered with a pharma- ceutically acceptable carrier. method.

12. Natural killer exhibits increased resistance to TGF-β superfamily cytokines - (NK) cells or NK cell lines.

13. 13. The method of claim 12, wherein the NK cells or cell lines exhibit increased resistance to TGF-β. of NK cells or cell lines.

14. The NK cells produce increased amounts of one or more of IFN-γ, TNF-α, and GM-CSF. The NK cell or cell line of claim 12.

15. The NK cells have reduced levels of SMAD3 protein and / or TGFBR3 protein. The NK cell or cell line of claim 12,

16. the NK cells have a gene expression profile substantially similar to that shown in FIG. The NK cell or cell line of claim 12.

17. The NK cells are produced by in vitro activation of natural killer cells in the presence of TGF-β. The NK cell or cell line of claim 12, prepared by:

18. The NK cell or cell line of claim 12, wherein the NK cell is a human NK cell.

19. The NK cell or cell line of claim 12, wherein the NK cell is a canine NK cell.

20. TGF-β superfamily imprinted natural killer (TGFβi NK) cell lines A method for producing a nattovirus comprising the steps of: The method comprises the in vitro activation of neural killer cells.

21. 21. The method of claim 20, wherein the TGFβi NK cell line is activated in the presence of TGF-β. method.

22. In vitro activation of natural killer cells in the presence of leukemic feeder cells The method of claim 20, further comprising:

23. The method of claim 22, wherein the feeder cells are K562 feeder cells.

24. The K562 feeder cells express costimulatory proteins and / or cytokines. The method of claim 23, wherein the gene is genetically modified such that

25. Inhibition of natural killer cell proliferation in the presence of NK-stimulating exosomes or NK-stimulating nanoparticles 21. The method of claim 20, further comprising in vitro activation.

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