Methods for producing natural killer cells and compositions for treating cancer - Patents.com

Abstract

To provide a method for producing a natural killer cell.SOLUTION: A method for producing a natural killer cell including: isolating a peripheral blood mononuclear cell (PBMC) from a blood sample; isolating at least one of CD56+ cell and / or CD3- / CD56+ cell from the PBMC; and co-culturing at least one of CD56+ cell and / or CD3- / CD56+ cell together with a combination of a feeder cell under the presence of cytokine. A composition for treating cancer including the CD56+ natural killer cell produced by the method and cytokine.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Korean Patent Application No. KR-10-2018-0012938, filed February 1, 2018, Korean Patent Application No. KR-10-2018-0012942, filed February 1, 2018, Korean Patent Application No. KR-10-2019-0001981, filed January 7, 2019, and Korean Patent Application No. KR-10-2019-0001983, filed January 7, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to a method for producing highly purified natural killer cells. [Background technology]

[0003] The human body is protected from pathogens by an immune response coordinated by an immune system that is composed of many immune-related cells, chemical mediators such as cytokines, and the like. White blood cells, especially lymphocytes, play an important role in this immune system. Lymphocytes are involved in both innate and adaptive immunity.

[0004] Natural killer cells (NK cells) are a type of innate immune cell that are known to nonspecifically kill cancer cells, recognize and kill viruses, bacteria, etc., and kill pathogens with enzymes such as perforin and granzymes, or through Fas-FasL interactions. In cancer patients, the reduced cancer cytotoxicity of these NK cells is associated with the development of lung cancer (Carrega P,et al.,Cancer,2008:112:863-875), liver cancer (Jinushi M,et al.,J Hepatol.,2005:43;1013-1020), breast cancer (Bauernhofer T,et al.,Eur J Immunol.,2003:33:119-124), uterine cancer (Mocchegiani NK cells are associated with the development of various types of cancer, such as hematological cancers (Tajima F.,et al.,Br j Cancer.,1999:79:244-250), hematological cancers (Tajima F.,et al,Lekemia 1996:10:478-482), and the like. Therefore, it is desirable to increase the cytotoxicity of NK cells to cancer cells for cancer therapy.

[0005] To obtain the therapeutic effect of NK-mediated killing of cancer cells, a large amount of highly purified NK cells is required, but it is not easy to obtain a large amount of blood from cancer patients, and the proportion of NK cells in the blood is small, only about 5-20%, so it has been difficult to use NK cells as an immunotherapy drug.

[0006] As a result, it is desirable to efficiently expand and proliferate only NK cells, but conventional methods of NK cell proliferation require the use of various expensive cytokines at high concentrations, and therefore the corresponding treatment is only available to some economically stable patients. Furthermore, according to conventional methods of NK cell proliferation, other types of immune cells (e.g., T cells, B cells, etc.) may exist together with NK cells, and allogeneic administration of NK cells containing T cells may cause graft-versus-host disease (GVHD), and allogeneic administration of NK cells containing B cells to blood type incompatible subjects may cause passenger B cell syndrome, and therefore the anti-cancer effect is not maximized.

[0007] In addition to the expansion and proliferation of NK cells, it is desirable to maintain the function of the expanded and proliferated NK cells at a high level until the cells are actually used. As a result, it is desirable to promote NK cell proliferation and to increase the production of cytokines such as TNF-, INF-, and GM-CSF derived from NK cells. The development of compositions that can increase production and enhance the cytotoxicity of NK cells to cancer cells is being sought. Summary of the Invention

[0008] The present application relates to a method for producing high-purity natural killer cells and a cell therapy composition for treating cancer, comprising high-purity natural killer cells and cytokines. Any feature, structure, or step disclosed herein may be replaced, combined, or omitted with other features, structures, or steps disclosed herein. Moreover, for purposes of summarizing the disclosure, certain aspects, advantages, and features of the invention are described herein. It should be understood that not necessarily any or all such advantages will be achieved by any particular embodiment of the invention disclosed herein. No individual aspect of the present disclosure is essential or essential.

[0009] In one embodiment, a method for producing natural killer cells is disclosed, comprising isolating peripheral blood mononuclear cells (PBMCs) from a blood sample, isolating at least one of CD56+ cells and / or CD3- / CD56+ cells from the PBMCs, and co-culturing at least one of the CD56+ cells and / or CD3- / CD56+ cells with a combination of feeder cells in the presence of cytokines.

[0010] In certain embodiments, isolating at least one of CD56+ cells and / or CD3- / CD56+ cells from PBMCs is performed by using at least one of CD56 microbeads and CD3 microbeads. In certain embodiments, the cytokine is selected from the group consisting of IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferon, GM-CSF, IGF 1, and combinations thereof. In certain embodiments, the cytokine may be added at a concentration of 50-1000 IU / mL.

[0011] In certain embodiments, the feeder cell combination comprises irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte line (EBV-LCL) cells. In a variation, the ratio of irradiated Jurkat cells to irradiated EBV-LCL cells may be about 1:0.1-5. Each of the irradiated Jurkat cells and irradiated EBV-LCL cells may be obtained by irradiation of 50-500 Gy (gray).

[0012] In certain embodiments, the co-cultivation may comprise co-cultivation for 1 to 50 days.

[0013] In certain embodiments, the method may further comprise co-culturing at least one of the CD56+ cells and / or CD3- / CD56+ cells with a combination of feeder cells in the presence of a first cytokine for a first period of time, and thereafter co-culturing at least one of the CD56+ cells and / or CD3- / CD56+ cells with the combination of feeder cells in the presence of a second cytokine for a second period of time. In a variation, the second cytokine may be added one or more times during days 0-6 of the second period. The second cytokine may be added one or more times during the first 6 days of every 14-day cycle for the second period of time. The first cytokine may be IL-2. The second cytokine may be IL-21. The second cytokine may be added at a concentration of 10-100 ng / mL.

[0014] In certain embodiments, a combination of at least one of CD56+ cells and / or CD3- / CD56+ cells and feeder cells are co-cultured at a ratio of CD56+ cells and / or CD3- / CD56+ cells to feeder cells of about 1:1-100.

[0015] In certain embodiments, compositions produced by the methods are disclosed.

[0016] In one embodiment, a composition for treating cancer in a patient in need thereof is disclosed. The composition comprises: an effective amount of peripheral blood derived CD56+ natural killer cells, IL-2 having a concentration of 50-50,000 IU / mL, and a pharma- ceutically acceptable carrier, the effective amount being about 1×10 per kg of patient body weight. 6 ~5×10 8 of cells and the CD56+ natural killer cells are at least about 90% pure.

[0017] In certain embodiments, the cytokine may be selected from the group consisting of IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferon, GM-CSF, IGF 1, and combinations thereof. In variations, the cytokine may be IL-2. The cytokine may have a concentration of 50-50,000 IU / mL.

[0018] In certain embodiments, the cancer is selected from the group consisting of hematological cancer, gastric cancer, pancreatic cancer, bile duct cancer, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, renal cancer, prostate cancer and neuroblastoma.

[0019] In certain embodiments, the composition comprises less than about 1% T cells.

[0020] Various embodiments are shown in the accompanying drawings for purposes of illustration, and should in no way be construed as limiting the scope of the embodiments. Furthermore, various features of the various disclosed embodiments can be combined to produce further embodiments, which are also part of this disclosure. [Brief description of the drawings]

[0021] [Figure 1A] FIG. 1A illustrates a graph showing the cell proliferation rate of NK cells produced from PBMCs, CD56+ cells, and CD3- / CD56+ cells. [Figure 1B]FIG. 1B depicts a graph showing the cell proliferation rate of NK cells generated from PBMCs and CD56+ cells with or without treatment with IL-21. [Diagram 2] FIG. 2 depicts a graph showing the purity of CD3− / CD56+ NK cells produced from PBMCs or CD56+ cells with or without treatment with IL-21. [Diagram 3] FIG. 3 shows the plate design for analyzing the anti-cancer activity of NK cells. [Figure 4A] FIG. 4A illustrates a graph showing the short-term cytotoxicity of NK cells generated from PBMCs and CD56+ cells for various effector:target cell (E:T) ratios. [Figure 4B] FIG. 4B depicts a graph showing the long-term cytotoxicity of NK cells generated from PBMCs and CD56+ cells against AGS, A549, and MDA-MB-231 cells. [Figure 5A] FIG. 5A depicts a graph showing the cell proliferation rate of NK cells produced by treatment with IL-21 for various periods of time. [Figure 5B] FIG. 5B depicts a graph showing the cell proliferation rate of NK cells produced by treatment with IL-21 for various periods of time. [Figure 6] FIG. 6 depicts a graph showing the cell proliferation rate of NK cells produced by treatment with various concentrations of IL-21. [Figure 7A] FIG. 7A depicts a graph showing the short-term cytotoxicity of NK cells produced by treatment with IL-21 for various periods of time against various E:T ratios. [Figure 7B] FIG. 7B depicts a graph showing the long-term cytotoxicity of NK cells produced by treatment with IL-21 for various periods of time against AGS, A549, and MDA-MB-231 cells. [Figure 7C] FIG. 7C depicts a graph showing the long-term cytotoxicity of NK cells produced by treatment with IL-21 for various periods of time against AGS, A549, and MDA-MB-231 cells. [Figure 8A]FIG. 8A depicts a graph showing the short-term cytotoxicity of NK cells produced by treatment with IL-21 for various periods of time against various E:T ratios. [Figure 8B] FIG. 8B depicts a graph showing the long-term cytotoxicity of NK cells produced by treatment with various concentrations of IL-21 against AGS, A549, and MDA-MB-231 cells. [Figure 9] FIG. 9 illustrates a graph showing the cell proliferation rate of NK cells in the presence of feeder cell stimulation. [Figure 10A] FIG. 10A depicts a graph showing the cell proliferation rate of NK cells produced from PBMCs of cancer patients with or without treatment with IL-21. [Figure 10B] FIG. 10B depicts a graph showing the purity of CD3- / CD56+ NK cells produced from PBMCs of cancer patients with or without treatment with IL-21. [Figure 10C] FIG. 10C depicts a graph showing short-term cytotoxicity of NK cells generated from PBMCs with or without IL-21 treatment against K562 cells. [Figure 10D] FIG. 10D depicts a graph showing the long-term cytotoxicity of NK cells generated from PBMCs with or without IL-21 treatment against AGS, A549, and MDA-MB-231 cells. [Figure 11] FIG. 11 depicts a graph showing NK cell viability with or without treatment with IL-2. [Figure 12] FIG. 12 depicts a graph showing NK cell cytotoxicity against various cancer cells at various E:T ratios, with or without treatment with IL-2. [Figure 13] FIG. 13 shows photographs of remaining NIH:OVCAR-3 cells treated with NK cells with or without treatment with IL-2. [Figure 14] FIG. 14 shows photographs of remaining AGS cells treated with NK cells with or without treatment with IL-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present inventors have developed a method for producing highly pure NK cells without using expensive cytokines. The present inventors have found that highly pure CD56+ NK cells can be produced when CD56+ cells isolated from peripheral blood mononuclear cells are co-cultured with feeder cells in the presence of cytokines after isolating CD56+ cells from peripheral blood mononuclear cells. Furthermore, the present inventors have developed a cell therapy composition for treating cancer containing NK cells that can be effectively used for allogeneic therapy. As a result, the present inventors have found that when specific cytokines are added to CD56+ NK cells isolated from peripheral blood mononuclear cells, the cells exhibit high survival rates and high anti-cancer activity. Therefore, the present inventors have sought to develop a method for expanding NK cells and provide a cell therapy composition for cancer treatment containing peripheral blood-derived CD56+ NK cells expanded with cytokines.

[0023] According to some embodiments, a method for producing highly purified NK cells may include: isolating peripheral blood mononuclear cells (PBMCs) from a blood sample ("first isolation step"), isolating cells selected from the group consisting of CD56+ cells and CD3- / CD56+ cells from the PBMCs ("second isolation step"), and co-culturing the cells selected from the group consisting of CD56+ cells and CD3- / CD56+ cells with feeder cells in the presence of cytokines ("culture step"). Each step is described in further detail herein. The CD3- / CD56+ cells produced by the disclosed method have higher purity and higher anti-cancer activity as well as higher expression of various surface markers or activating receptors, such as CD16, CD25, CD27, CD28, CD69, CD94 / NKG2C, CD94 / NKG2E, CD266, CD244, NKG2D, KIR2S, KIR3S, Ly94D, NCRs, IFN-a, IFN-b, CXCR3, CXCR4, CX3CR, CXCR5, CXCR6, CXCR7, CXCR8, CXCR9, CXCR10, CXCR11, CXCR12, CXCR13, CXCR14, CXCR15, CXCR16, CXCR17, CXCR18, CXCR19, CXCR20, CXCR21, CXCR22, CXCR23, CXCR24, CXCR25, CXCR26, CXCR28, CXCR29, CXCR30, CXCR31, CXCR32, CXCR33, CXCR34, CXCR35, CXCR36, CXCR37, CXCR38, CXCR39, CXCR41, CXCR39, CXCR42, CXCR39, CXCR43, CXCR44, CXCR39, CXCR45, CXCR39, CXCR46, CXCR39, CXCR47, CXCR39, CXCR48, CXCR49, CXCR49, CXCR49, CXCR41, CXCR41, CXCR42, CXCR43, CXCR41, CXCR42, CXCR43, CXCR41, CXCR42, CXCR43, CXCR41 1, CD62L and CD57.

[0024] First Isolation Step As used herein, a "blood sample" may be, but is not limited to, whole peripheral blood or leukocytes isolated from peripheral blood using leukapheresis. Furthermore, peripheral blood may be obtained from, but is not limited to, a healthy individual, a patient at risk for cancer, or a patient with cancer.

[0025] As used herein, the term "leukapheresis" can refer to a method of selectively removing (isolating) white blood cells from collected blood and then re-administering the blood to a patient, and in some embodiments, the white blood cells isolated by the method may be used without additional methods, such as Ficoll-Hypaque density gradient methods.

[0026] As used herein, the term "peripheral blood mononuclear cells" may be used interchangeably with "PBMCs," "mononuclear cells," or "monocytes," and may refer to mononuclear cells isolated from peripheral blood that are commonly used in anti-cancer immunotherapy. Peripheral blood mononuclear cells may be obtained from collected human blood using known methods, such as Ficoll-Hypaque density gradient methods.

[0027] In some embodiments, the peripheral blood mononuclear cells may be autologous, however, allogeneic peripheral blood mononuclear cells may be used to produce highly purified NK cells for anti-cancer immunotherapy by the methods described herein.Furthermore, in some embodiments, the peripheral blood mononuclear cells may be obtained from healthy individuals, however, the peripheral blood mononuclear cells may be obtained from patients at risk for cancer and / or patients with cancer.

[0028] As used herein, the term "CD56+ cells" may be used interchangeably with "CD56+ NK cells" or "CD56+ natural killer cells," and the term "CD3- / CD56+ cells" may be used interchangeably with "CD3- / CD56+ NK cells." CD56+ cells or CD3- / CD56+ cells may include cells expressing CD56 glycoprotein on the cell surface, or further cells expressing CD56 glycoprotein but not CD3 glycoprotein. Even immune cells of the same type may have differences in CD types and expression rates bound to the cell surface, and thus may have different functions.

[0029] Second Isolation Step In some embodiments, isolation of CD56+ natural killer cells from a blood sample may be performed by an isolation method using at least one selected from the group consisting of CD56 microbeads and CD3 microbeads, or an isolation method using an apparatus such as CliniMACS, a flow cytometric cell sorter, and others.

[0030] For example, isolation methods using CD56 microbeads and / or CD3 microbeads may be performed by adding CD56 microbeads to PBMCs and then removing non-specific binding, or by adding CD3 microbeads to PBMCs to remove specific binding and then adding CD56 microbeads again to remove non-specific binding. In some embodiments, T cells or other non-natural killer cells may be removed by isolating CD56+ cells and / or CD3- / CD56+ cells from PBMCs.

[0031] Cultivation step As used herein, the term "cytokine" can refer to an immunoactive compound that can be used to induce peripheral blood mononuclear cells to differentiate into NK cells.

[0032] In some embodiments, the cytokine may be, but is not limited to, interleukin-2 (IL-2), IL-15, IL-21, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL-18, IL-4, type I interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF), and insulin-like growth factor 1 (IGF 1).

[0033] In some embodiments, cytokines may be used at concentrations of 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550 IU / mL. Conventional methods of expanding NK cells utilize high concentrations of various cytokines. Conversely, in some embodiments of the methods of expanding NK cells described herein, two types of feeder cells may be used in conjunction with high purity CD56+ cells, so that NK cells with high yield and purity may be expanded using only a low concentration of one cytokine.

[0034] As used herein, the term "feeder cells" can refer to cells that do not divide and proliferate, but are metabolically active and produce various metabolic products, and thus are supportive of the growth of target cells.

[0035] In some embodiments, the feeder cells may be at least one selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte line (EBV-LCL) cells, and PBMC, HFWT, RPMI 1866, Daudi, MM-170, K562, or cells genetically modified by targeting K562 (e.g., K562-mbIL-15-41BB ligand). For example, in one embodiment, the feeder cells may be irradiated Jurkat cells and EBV-LCL cells.

[0036] As used herein, the term "Jurkat cells" or "Jurkat cell line" can refer to a hematological cancer (immortalized acute T-cell leukemia) cell line developed by Dr. Arthur Weiss at the University of California, San Francisco, USA. Jurkat cells, which express various chemokine receptors and can produce IL-2, have not usually been considered as a possible candidate for feeder cells for anti-cancer immunotherapy because MHC class I, an inhibitor of natural killer cell activation, is highly expressed on its cell surface. Jurkat cells can be obtained from ATCC (ATCC TIB-152).

[0037] As used herein, the term "EBV-LCL cells" or "EBV-LCL cell line" refers to Epstein-Barr virus transformed lymphocyte subline (EBV-LCL) (DM Koelle et al., J Clin Invest, 1993:91:961-968), which is a B cell produced by infecting human B cells with Epstein-Barr virus in a test tube. EBV-LCL cells may be directly produced and used in a general laboratory by adding cyclosporine A in the method of infecting EBV in PBMC. In some embodiments, EBV-LCL cells may be produced by the following steps: 30×10 6 Add 100 mL of PBMCs and add the mixture into a T25 culture flask followed by 9 mL of EBV supernatant. Add 80 μL of cyclosporine A (50 μg / mL) and then culture at 37°C. After 7 days of culture, remove half of the supernatant and add fresh culture medium followed by 40 μL of cyclosporine A. The same method may be repeated once every 7 days until 28 days of culture. After 28 days of culture, the cell line may be usable, and from this point on, the cell line may be cultured in culture medium without the addition of cyclosporine A.

[0038] Jurkat cells and EBV-LCL cells may be used as feeder cells after irradiation.

[0039] In some embodiments, the irradiated Jurkat cells and the irradiated EBV-LCL cells may be present in a content ratio of 1:0.1-5, 1:0.1-4, 1:0.1-3, 1:0.1-2, 1:0.1-1.5, 1:0.5-1.5, 1:0.75-1.25, 0.1-5:1, 0.1-4:1, 0.1-3:1, 0.1-2:1, 0.1-1.5:1, 0.5-1.5:1, or 0.75-1.25:1. For example, the irradiated Jurkat cells and the irradiated EBV-LCL cells may be present in a content ratio of 1:1.

[0040] In some embodiments, the irradiated Jurkat cells and EBV-LCL cells may be obtained by treatment with 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120, or 90-110 Gy of radiation. For example, the irradiated Jurkat cells and / or EBV-LCL cells may be obtained by treating Jurkat cells and / or EBV-LCL cells with 100 Gy of radiation.

[0041] In some embodiments, the culture may be performed for 1 to 50, 1 to 42, 1 to 40, 1 to 35, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, or 1 to 14 days.

[0042] In some embodiments, the culturing step may further comprise the following steps: co-culturing feeder cells and a first cytokine (the "first culturing step"), and further co-culturing after addition of a second cytokine (the "second culturing step").

[0043] The second culture step may include the addition of the second cytokine one or more times between culture days 0 and 6. For example, the second culture step may include the addition of the second cytokine once each on culture days 0 and 3.

[0044] The second culture step may include the addition of a second cytokine and feeder cells during the first 6 days of a 14-day culture cycle. For example, the second culture step may include the addition of feeder cells during the 14-day cycle and the addition of a second cytokine once per cycle, on days 3 and 6 of each cycle.

[0045] In some embodiments, the first cytokine may be IL-2. In some embodiments, the second cytokine may be IL-21. In some embodiments, the second cytokine may be used at a concentration of 10-1000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100, or 10-50 ng / mL. In some embodiments, cultures in which the second cytokine is added one or more times on days 0-6 may exhibit superior proliferation and / or anti-cancer activity. In some embodiments, cultures in which feeder cells and the second cytokine are added for 6 days during a 14-day cycle may exhibit superior proliferation and / or anti-cancer activity.

[0046] In some embodiments, co-culture may be performed by including peripheral blood mononuclear cells and feeder cells (e.g., Jurkat cells and EBV-LCL cells) at a mixing ratio of 1:1-100, 1:1-90, 1:1-80, 1:1-70, 1:10-65, 1:20-65, 1:30-65, 1:40-65, 1:50-65, or 1:55-65.

[0047] The co-culture can be performed in a medium, and any suitable medium commonly used in the art for inducing and expanding peripheral blood mononuclear cells to NK cells may be used as such medium, without being limited thereto. For example, RPMI-1640, DMEM, x-vivo 10, x-vivo20, or cellgro SCGM medium may be used as such a medium. In addition, the culture conditions, such as temperature, may be according to any suitable culture conditions for peripheral blood mononuclear cells known in the art.

[0048] In some embodiments, within the NK cells produced, the ratio or purity of CD56+ NK cells may be 85% or more, 90% or more, or 95% or more, or 98% or more relative to total cells. In some embodiments, within the NK cells produced, the ratio of T cells to total cells may be 15% or less, 10% or less, 5% or less, 2% or less, 1% or less.

[0049] Cell therapy composition for cancer treatment According to some embodiments, a cell therapy composition for the treatment of cancer may comprise peripheral blood derived CD56+ NK cells and cytokines.

[0050] As used herein, the term "peripheral blood-derived" means that the cells are derived from "peripheral whole blood" or "leukocytes isolated from peripheral blood by leukapheresis." Peripheral blood-derived CD56+ NK cells may be used interchangeably with peripheral blood mononuclear cell (PBMC)-derived CD56+ NK cells.

[0051] In some embodiments, the cytokine may be used at a concentration of 18 to 180,000, 20 to 100,000, 50 to 50,000, 50 to 1,000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 550, 100 to 550, 150 to 550, 200 to 550, 250 to 550, 300 to 550, 350 to 550, 400 to 550, or 450 to 550 IU / mL. When the cytokine is used within these ranges, apoptosis of NK cells contained in the cancer treatment composition can be suppressed, and the anti-cancer activity of NK cells can be increased.

[0052] In some embodiments, the composition may include IL-2 as a cytokine.

[0053] In some embodiments, CD56+ NK cells may be obtained as described elsewhere herein. For example, CD56+ NK cells may be obtained by co-culturing with feeder cells (e.g., irradiated Jurkat cells and irradiated EBV-LCL cells). In some embodiments, the ratio (purity) of CD56+ NK cells to total cells may be 85% or more, 90% or more, 95% or more, or 98% or more.

[0054] In some embodiments, the cancer may be, but is not limited to, blood cancer, gastric cancer, pancreatic cancer, bile duct cancer, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, renal cancer, prostate cancer, or neuroblastoma.

[0055] In some embodiments, the composition may not contain T cells or may contain only trace amounts of T cells, for example, the ratio of T cells to total cells in the composition may be less than 15%, less than 10%, less than 5%, less than 2%, less than 1% or less.

[0056] As used herein, the term "T cells" refers to lymphocytes derived from the thymus that "remember" previously encountered antigens and provide information to B cells, thereby promoting the production of antibodies and playing an important role in the cellular immune system. These T cells can distinguish very small differences among various antigens to induce immune responses against alloantigens, allowing for autologous therapy, but may be limited in their use for allogeneic therapy. Therefore, cell therapy compositions that do not contain T cells may be suitable for allogeneic transplantation.

[0057] As used herein, the term "cell therapy" refers to a therapeutic, diagnostic, and Cell therapy represents medicines used for screening autologous, allogeneic, and xenogeneic live cells in vitro for prevention and treatment, and for restoring cell and tissue functions or modifying cell biological properties by other methods. Cell therapy has been regulated as a drug since 1993 in the United States and since 2002 in Korea. These cell therapy can be broadly classified into two fields: the first is stem cell therapy for tissue regeneration or organ function restoration, and the second is immune cell therapy for regulating immune responses, such as inhibiting or enhancing immune responses in vivo.

[0058] The route of administration of the cell therapy compositions described herein may be any suitable route so long as the composition reaches the target tissue. Administration may be parenteral, such as, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, or intradermal.

[0059] The cell therapy compositions described herein may be formulated in a suitable form with a pharma- tically acceptable carrier suitable or normally used for cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, or the like, when administered to the human body. Pharmaceutically acceptable carriers can include, for example, parenteral carriers such as water, suitable oils, saline, aqueous glucose and glycols, and the like, and can further include stabilizers and preservatives. Suitable stabilizers include sodium bisulfite, sodium sulfite, or ascorbic acid, sucrose, albumin, and the like. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, and the like.

[0060] The cell therapy composition may be administered by any device capable of transferring the cellular therapy agent to the target cells.

[0061] The cell therapy composition may include a therapeutically effective amount of a cell therapy agent for the treatment of a disease. The term "therapeutically effective amount" refers to an amount of an active ingredient or cell therapy composition, including an amount that induces a biological or medical response in a tissue system, animal, or human as deemed by a researcher, veterinarian, physician, or other clinician, and induces alleviation of the symptoms of the disease or disorder to be treated. It will be apparent to those skilled in the art that the cell therapy agent contained in the cell therapy composition may be changed depending on the desired effect. Therefore, the optimal content of the cell therapy agent may be easily determined by those skilled in the art, and may be adjusted depending on various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, as well as the age, body weight, general health condition, sex, and diet of the patient, the time of administration, the route of administration, the secretion rate of the composition, the duration of treatment, and drugs used simultaneously. It is important to include an amount that can obtain the maximum effect with the minimum amount without side effects by considering all factors. For example, the cell therapy composition may be administered in an amount of 1×10 per kg of body weight. 6 ~5×10 8 The cells may include a cellular therapeutic agent.

[0062] Methods for preventing or treating cancer Further, according to another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering to a subject an anti-cancer cell therapy composition comprising peripheral blood-derived CD56+ natural killer cells and cytokines. The term "subject" refers to a mammal, preferably a human, that is the subject of treatment, observation, or testing. The subject may be, but is not limited to, a patient with blood cancer, gastric cancer, pancreatic cancer, bile duct cancer, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, renal cancer, prostate cancer, or neuroblastoma.

[0063] In some embodiments, for adults, the cell therapy composition may be administered once to several times a day. The cell therapy composition may be administered daily or at intervals of 2 to 180 days, and the composition The cell therapy contained 1 × 10 6 ~1×10 11 of peripheral blood-derived CD56+ natural killer cells, e.g., approximately 1 × 10 per kg of body weight6 ~1×10 8 In some embodiments, the peripheral blood derived CD56+ natural killer cells in the cell therapy composition are at least about 90% pure. In some embodiments, the cytokine is IL-2 at a concentration ranging from about 50 to 50,000 IU / ml.

[0064] In some embodiments, the cell therapy compositions of the present invention may be administered by any suitable method, such as by rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes. In some embodiments, the NK cells contained in the composition may be allogeneic, i.e., obtained from a human other than the subject being treated. In some embodiments, the human may be a healthy individual or a cancer patient. In some embodiments, the NK cells contained in the composition may be autologous, i.e., obtained from the subject being treated.

[0065] In some embodiments, the NK cells disclosed herein and cell therapy compositions comprising the NK cells disclosed herein may be used to treat diseases or conditions other than cancer. NK cells play an important role in regulating the immune system, for example, by regulating T cells, and thus, it has been reported that cell therapy compositions having NK cells can be administered to treat conditions related to the immune system. For example, cell therapy compositions can be administered to treat neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease) or autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, spondyloarthropathy, SLE, Sjogren's syndrome, systemic sclerosis).

[0066] Beneficial Effects The features and advantages of the present invention are summarized as follows:

[0067] (a) The present invention relates to a method for producing natural killer cells.

[0068] (b) According to the method for producing natural killer cells, highly purified natural killer cells from which T cells and the like have been removed can be produced without using various expensive cytokines, making it possible to enhance the effects of cancer prevention and treatment, particularly allogeneic therapy using natural killer cells.

[0069] (c) The present invention relates to a composition for cell therapy for anti-cancer comprising peripheral blood-derived CD56+ NK cells and cytokines.

[0070] (d) The compositions of the present invention contain highly purified natural killer cells with minimal (e.g., less than about 1%) T cells, and therefore the compositions may be effectively used for allogeneic as well as autologous therapy. EXAMPLES

[0071] The following examples are provided to illustrate certain specific features and / or embodiments, and should not be construed as limiting the disclosure to the specific features or embodiments described.

[0072] Example 1. Production of CD56+ Natural Killer (NK) Cells CD56+ cells and CD3- / CD56+ cells were isolated from PBMCs by the following methods: First, PBMCs were isolated from blood using Ficoll-Hypaque density gradient method, and then the cells were counted.

[0073] Example 1-1. Preparation for producing CD56+ cells The counted PBMCs were added with MACS buffer (1x PBS + 0.5% HSA), suspended, and added with CD56 microbeads (Miltenyi Biotec) at 1.0 x 10 7The mixture was diluted to 1–20 μL per PBMC and incubated at 2–8°C for 5–30 min. After incubation, MACS buffer was added, mixed, and the mixture was centrifuged (600 × g) to pellet the cells. After centrifugation, the supernatant was removed, and the cells were suspended by adding MACS buffer, which was then added to a column connected to a MACS separator. The MACS buffer passed through the column to remove nonspecific binding. The column was separated from the MACS separator, transferred to a 15 mL conical tube, and then added with MACS buffer to isolate CD56+ cells bound to the column.

[0074] Example 1-2. Preparation for producing CD3- / CD56+ cells The counted PBMCs were added to MACS buffer (1x PBS ± 0.5% HSA), suspended, and added with CD3 microbeads (Miltenyi Biotec) at 1.0 x 10 7 The CD3- cells were collected by adding 1-20 μL per PBMC and then incubated at 2-8°C for 5-30 min. After incubation, MACS buffer was added and mixed, and the mixture was centrifuged (600 × g) to precipitate the cells. After centrifugation, the supernatant was removed, and the cells were suspended by adding MACS buffer and added to a column connected to a MACS separator. The MACS buffer passed through the column, and CD3- cells were collected. The counted CD3- cells were added with MACS buffer (1 × PBS + 0.5% HSA), suspended, and added with CD56 microbeads (Miltenyi Biotec) at 1.0 × 10 7The mixture was diluted with 1-20 μL of 100% CD3- cells and incubated at 2-8°C for 5-30 min. After incubation, MACS buffer was added, mixed, and the mixture was centrifuged (600 × g) to precipitate the cells. After centrifugation, the supernatant was removed, and the cells were suspended by adding MACS buffer, which was then added to a column connected to a MACS separator. The MACS buffer passed through the column to remove nonspecific binding. The column was separated from the MACS separator, transferred to a 15 mL conical tube, and then added with MACS buffer to isolate the CD3- / CD56+ cells bound to the column.

[0075] Examples 1-3. Production of NK cells using CD56+ cells and CD3- / CD56+ cells CD56+ cells or CD3- / CD56+ cells isolated from PBMC as in Examples 1-1 and 1-2 were added with the prepared combination of 100Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) in RPMI-1640 medium containing 10% FBS supplemented with IL-2 at a concentration of 500IU / mL, and then co-cultured in an incubator at 37°C and 5% CO2. The ratio of (CD56+ cells and / or CD3- / CD56+ cells):(Jurkat cells):(EBV-LCL cells) was about 1:30:30.

[0076] Meanwhile, Jurkat cells may be obtained from ATCC (ATCC TIB-152), and EBV-LCL cells were prepared by the following method: 30 × 10 6 PBMCs were added to 9mL of culture medium, the mixture was added to a T25 culture flask, followed by the addition of 9m of EBV supernatant. 80μL of cyclosporine A was added, and the cells were cultured at 37°C. After 7 days of culture, half of the supernatant was removed, fresh culture medium was added, followed by the addition of 40μL of cyclosporine A. The same procedure was repeated on the 7th day, once every 7 days, until the 28th day of culture. After 28 days of culture, the cell line was usable, and from this point on, the cell line was cultured in culture medium without the addition of cyclosporine A.

[0077] Example 2. Production of CD56+ Natural Killer (NK) Cells (IL-2 / IL-21 Treated) NK cells were produced using the same method as in Example 1 (1-1 to 1-3), except that IL-2 (500 IU / mL) and IL-21 (50 ng / mL) were added instead of IL-2 (500 IU / mL).

[0078] Comparative Example 1. Production of Natural Killer (NK) Cells without a CD56+ Cell Isolation Step (IL-2 Treatment) PBMCs were isolated from blood using Ficoll-Hypaque density gradient method. PBMCs were added to RPMI-1640 medium containing 10% FBS supplemented with IL-2 at a concentration of 500 IU / mL together with prepared feeder cells (Jurkat cells and EBV-LCL cells) irradiated with 100 Gy, and then co-cultured in an incubator at 37°C and 5% CO2.

[0079] Comparative Example 2. Production of Natural Killer (NK) Cells without a CD56+ Cell Isolation Step (IL-2 / IL-21 Treatment) NK cells were produced using the same method as in Comparative Example 1, except for the addition of IL-2 (500 IU / mL) and IL-21 (50 ng / mL) instead of IL-2 (500 IU / mL).

[0080] Comparative Examples 3 and 4. Production of Natural Killer (NK) Cells without a CD56+ Cell Isolation Step NK cells were produced using the same methods as in Comparative Examples 1 and 2, respectively, except that the ratio of PBMC:(Jurkat cells):EBV-LCL cells) was 1:0.5:0.5.

[0081] Experimental example 1. Confirmation of NK cell proliferation ability Regarding each of the NK cells cultured in a CO incubator according to Examples 1 and 2 and Comparative Examples 1 and 2, on the sixth day of culture in a T25 culture flask, 1.0 × 10 5 ~2.0×10 6Cells were inoculated into 350 mL bags based on cell counts in 1000 mL / mL and cultured for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 Cells were inoculated into 1 L bags based on cell counts in 1000 cells / mL and cultured for an additional 4 days. On day 14 of culture, 1.0 × 10 5 ~2.0×10 6 Cells were inoculated into 1 L bags based on cell counts / mL and then cultured for an additional 3 to 6 days.

[0082] FIG. 1A illustrates the fold increase of NK cells in culture. As shown in Table 1, the CD56+ NK cells (CD56+ and CD3- / CD56+) of Example 1 expanded 2675-fold and 1903-fold, respectively, on day 17 compared to day 0, whereas the PBMC cells of Comparative Example 1 expanded 1768-fold on day 17 compared to day 0. [Table 1]

[0083] Figure 1B illustrates the fold increase and population doubling number (PDL) of NK cells. Additionally, as shown in Figure 1B, PBMCs of Comparative Example 1 (PBMCs without IL-21) and Comparative Example 2 (PBMCs with IL-21) expanded 243-fold and 1248-fold, respectively, compared to day 0, while CD56+NK cells of Example 1 (CD56 without IL-21) and Example 2 (CD56 with IL-21) expanded 2990-fold and 20434-fold, respectively, compared to day 0.

[0084] Experimental example 2. Confirmation of purity of CD56+ NK cells The NK cells of Examples 1 and 2 and Comparative Examples 1 and 2 were washed once with FACS staining buffer, suspended in 100 μL, mixed with monoclonal antibodies conjugated to fluorescent dyes, and stored at 2-8°C for 20-30 minutes in the dark. After one further wash, the cells were suspended in 300-500 μL of FACS staining buffer, and 10,000-100,000 cells per tube were obtained and analyzed by using a CD56-FITC / CD3-PE / CD20-PerCP5 / CD14-APC panel on a flow cytometer. The purity of CD56+ NK cells was defined as the ratio of cells introduced into the CD3- / CD56+ region after FSC / SSC gating, and it was further confirmed that CD20 and CD14 were not expressed in cells in the CD3- / CD56+ region.

[0085] As shown in FIG. 2, the purity of NK cells in Comparative Example 1 (PBMC without IL-21) and Comparative Example 2 (PBMC with IL-21) was 84.2% and 84.7%, respectively, while the purity of NK cells in Example 1 (CD56 without IL-21) and Example 2 (CD56 with IL-21) was 98.6% and 99.2%, respectively.

[0086] Experimental example 3. Confirmation of NK cell cytotoxicity to cancer cells First, the cytotoxicity against K562 cells (blood cancer, ATCC® CCL-243™), a chronic myeloid leukemia cell line, was confirmed.

[0087] Before being used in the experiments, K562 cells were prepared by subculturing K562 cells suspended in RPMI 1640 medium containing 10% FBS at 37±1° C. at 3-day intervals for more than 7 days.

[0088] 1.0 x 10 K562 cells were prepared 6K562 cells were suspended in RPMI-1640 medium at a concentration of 1.0 × 10 cells / mL and co-added with a fluorescent substrate (calcein-AM) at a concentration of 4 μM. K562 cells were stained at 37 ± 1°C for 30 min and then inverted at 10 min intervals. Fluorescently stained K562 cells were centrifuged at 3,300 rpm for 3 min, washed three times, and then added to 1.0 × 10 6 K562 cells were suspended in SNK medium containing 10% FBS at a ratio of 1.0 × 10 cells / mL per well. 4 The amount of cells was seeded into round-bottom microwell plates (96 wells).

[0089] NK cells (effector cells) from Experimental Example 1 on days 14 to 20 of culture were suspended and each was diluted to 1.0 × 10 6 cells / mL, 3.0×10 5 cells / mL, 1.0×10 5 cells / mL and 0.5 x 10 5 The cells were diluted in RPMI-1640 medium containing 10% FBS at a ratio of cells / mL.

[0090] The diluted effector cells were inoculated into the plate inoculated with target cells (K562 cells) at a concentration of 100 μL per well for each of three wells (triplicates), respectively, with the effector and target cell ratios shown in Table 2 below. [Table 2]

[0091] The plate design used in this experiment is shown in Figure 3. In the negative control group (natural), fluorescently stained live K562 cells were added, and in the positive control group (maximum release), K562 cells were completely killed using TX-100 to show maximum fluorescence.

[0092] The plate seeded with the target cells and effector cells was centrifuged at 1000 rpm for 5 minutes, incubated at 37±1°C for 3-4 hours, and then centrifuged again at 1000 rpm for 5 minutes. After centrifugation, 80 μL of the supernatant was transferred to a black plate (96 wells), and the amount of fluorescence was measured using a fluorescence microplate reader, and the cytotoxicity against cancer cells was calculated using the following formula 1.

number

[0093] Figure 4A and Table 3 below show the lysis % of K562 cells at various E:T ratios. As shown in Figure 4A and Table 2 below, CD56+ cells cultured by Example 1 (CD56+ without IL-21) and Example 2 (CD56+ with IL-21) showed higher anti-cancer activity when compared with Comparative Example 3 (PBMC without IL-21) and Comparative Example 4 (PBMC with IL-21). [Table 3]

[0094] Next, we confirmed the cytotoxicity against solid tumor cells, which are known to have greater resistance to NK cells. AGS (gastric cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumor cell lines.

[0095] Each solid tumor cell was tagged with a green fluorescent marker using the CYTO-ID® Green long-term tracer kit (Enzo Life Sciences Inc.), inoculated into a plate, and cultured for 24 hours. The next day, NK cells and cancer cells were reacted at a ratio of 0.5:1 for 48 hours. After 48 hours, cytotoxicity was confirmed by measuring the number of cells showing green fluorescence using a flow cytometer.

[0096] As shown in FIG. 4B, CD56+ cells cultured according to Example 1 (CD56+ without IL-21) and Example 2 (CD56+ with IL-21) showed higher anti-cancer activity when compared with Example 3 (PBMCs without IL-21) and Comparative Example 2 (PBMCs with IL-21).

[0097] Experimental Example 4. Comparison of NK cell proliferation ability depending on timing and number of IL-21 treatment To evaluate the effect of timing of IL-21 treatment on the proliferation of NK cells, an experiment was carried out as outlined below.

[0098] CD56+ NK cells were produced by the method of Example 1, and treated with IL-21 (50 ng / mL) on days 0 to 6 (group D0-6), days 6 to 10 (group D6-10), days 10 to 14 (group D10-14), or days 14 to 17 (group D14-17). The proliferation ability of CD56+ NK cells was compared using the method of Experimental Example 1.

[0099] NK cells were treated with IL-21: twice on days 0 and 3 for the D0-6 group, once on day 6 for the D6-10 group, once on day 10 for the D10-14 group, and once on day 14 for the D14-17 group. For the control group, NK cells were not treated with IL-21.

[0100] As shown in FIG. 5A and Table 4, the D10-14 and D14-17 groups did not show significant differences in proliferation ability compared to the control group, while the D0-6 and D6-10 groups showed increased proliferation ability compared to the control group. In particular, the D0-6 group showed the largest increase in proliferation fold. [Table 4]

[0101] To evaluate the proliferation potential of NK cells depending on the number of IL-21 treatments, an experiment was carried out as outlined below.

[0102] CD56+ NK cells were produced by the method of Example 1, and treated with IL-21 (50 ng / mL) on days 0 to 3 (group D0-3), days 3 to 6 (group D3-6), or days 0 to 6 (group D0-6). The proliferation ability of CD56+ NK cells was compared using the method of Experimental Example 1.

[0103] NK cells were treated with IL-21: once on day 0 for the D0-3 group, once on day 3 for the D3-6 group, and twice on days 0 and 3 for the D0-6 group. For the control group, NK cells were not treated with IL-21.

[0104] As shown in Figure 5B and Table 5, all groups treated with IL-21 at the early stage of culture showed increased proliferation folds compared to the control group. In particular, the D0-6 groups showed the greatest increase in proliferation folds. [Table 5]

[0105] Experimental Example 5. Comparison of NK cell proliferation ability depending on the concentration of IL-21 treatment CD56+ NK cells were prepared by the method of Example 1 and treated twice with IL-21 having concentrations of 0 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL, and the proliferation ability of the CD56+ NK cells was compared using the method of Experimental Example 1.

[0106] As shown in Figure 6, even when treated with IL-21 having a concentration of 10 ng / mL, NK cells showed a larger fold proliferation compared to NK cells not treated with IL-21, and the fold proliferation of NK cells increased with increasing IL-21 concentration between 10 ng / mL and 50 ng / mL. However, when treated with IL-21 having a concentration of 100 ng / mL, NK cells did not show a significant difference in proliferation compared to NK cells treated with IL-21 having a concentration of 50 ng / mL.

[0107] Experimental Example 6. Comparison of NK cell cytotoxicity depending on timing and number of IL-21 treatment To evaluate the cytotoxicity of NK cells against cancer cells depending on the timing of IL-21 treatment, an experiment was performed as outlined below.

[0108] CD56+ NK cells were produced by the method of Example 1, and treated with IL-21 (50 ng / mL) on days 0-6 (D0-6 group), days 6-10 (D6-10 group), days 10-14 (D10-14 group), or days 14-17 (D14-17 group). The cytotoxicity of the CD56+ NK cells against blood cancer cells (K562 cells, CCL-243 (trademark)) was compared using the method of Experimental Example 3.

[0109] NK cells were treated with IL-21: twice on days 0 and 3 for the D0-6 group, once on day 6 for the D6-10 group, once on day 10 for the D10-14 group, and once on day 14 for the D14-17 group. For the control group, NK cells were not treated with IL-21.

[0110] As shown in FIG. 7A and Table 6, all groups of IL-21-treated NK cells, except for group D14-17, showed greater anti-cancer activity compared to the control group. [Table 6]

[0111] Furthermore, for each of the prepared groups of CD56+ NK cells, the cytotoxicity of CD56+ NK cells against solid tumor cells was compared using the method according to Experimental Example 3. AGS (gastric cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumor cell lines.

[0112] As shown in Figure 7B, NK cells treated with IL-21 at the early stage of culture (D0-6 groups) showed the greatest anti-cancer activity against all three types of solid tumor cells.

[0113] To evaluate the proliferation potential of NK cells depending on the number of IL-21 treatments, an experiment was carried out as outlined below.

[0114] CD56+ NK cells were produced by the method of Example 1, but were treated with IL-21 (50 ng / mL) between days 0-3 (D0-3 group), days 3-6 (D3-6 group), or days 0-6 (D0-6 group), and the cytotoxicity of CD56+ NK cells against solid tumor cells was compared using the method of Experimental Example 3. AGS (gastric cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumor cell lines.

[0115] NK cells were treated with IL-21: once on day 0 for the D0-3 group, once on day 3 for the D3-6 group, and twice on days 0 and 3 for the D0-6 group. For the control group, NK cells were not treated with IL-21.

[0116] As shown in Figure 7C, all groups treated with IL-21 at the early stage of culture showed greater anti-cancer activity compared to the control group.

[0117] Experimental Example 7. Comparison of NK cell cytotoxicity depending on the concentration of IL-21 treatment CD56+ NK cells were produced by the method of Example 1 and treated twice with IL-21 having concentrations of 0 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL, and the cytotoxicity of the CD56+ NK cells against blood cancer cells (K562 cells, CCL-243 (trademark)) was compared using the method of Experimental Example 3.

[0118] As shown in FIG. 8A, most NK cells treated with IL-21 exhibited greater cytotoxicity compared to NK cells not treated with IL-21, and when treated with IL-21 having a concentration of 100 ng / mL, NK cells did not show significant differences in proliferation compared to NK cells not treated with IL-21.

[0119] CD56+ NK cells were prepared by the method of Example 1, and treated twice with IL-21 having concentrations of 0 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, or 100 ng / mL, and the cytotoxicity of CD56+ NK cells against solid tumor cells (K562 cells, CCL-243™) was compared using the method of Experimental Example 3. AGS (gastric cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumor cell lines.

[0120] As shown in FIG. 8B, NK cells treated with IL-21 having a concentration of 50 ng / mL exhibited the greatest anti-cancer activity.

[0121] Experimental Example 8. Comparison of NK cell proliferation activity depending on the number of feeder cells treated To analyze whether multiple treatments with feeder cells sustained NK cell expansion, NK cells in culture were treated with feeder cells at 14-day intervals and NK cell proliferation was monitored for 42 days.

[0122] To further analyze the IL-21 treatment-dependent increase in NK cell proliferation, NK cells were treated with IL-21 (50 ng / mL) twice at 3-day intervals for 6 days (days 0–6, 14–20, and 28–34) following each treatment with feeder cells.

[0123] As shown in FIG. 9, NK cells showed a significant increase in fold expansion when treated with feeder cells and IL-21 more than once, with IL-21-treated NK cells showing a greater fold expansion at day 42 compared to NK cells not treated with IL-21 (approximately 3.4×10 10 vs. about 5.3×10 8 ).

[0124] Experimental Example 9. Confirmation of the effectiveness of NK cell culture using blood from specific cancer patients Except for using PBMCs from a colon cancer patient, CD56+ NK cells were produced in 17 days by the method of Example 1. The proliferation ability and purity of the produced NK cells were measured using the methods of Experimental Examples 1 and 2.

[0125] For some groups, NK cells were treated twice (on days 0 and 3 of culture) with IL-21 having a concentration of 50 ng / mL to confirm the effect of IL-21 treatment.

[0126] As shown in Figure 10A, the number of NK cells not treated with IL-21 increased 8-fold from day 0, whereas the number of NK cells increased 1461-fold from day 0 when treated with IL-21.

[0127] Furthermore, as shown in FIG. 10B, the purity of NK cells not treated with IL-21 was only 84.2%, whereas the purity of NK cells was 99.19% when treated with IL-21.

[0128] Furthermore, the cytotoxicity of IL-21-treated and non-IL-21-treated NK cells against blood cancer cells (K562 cells, CCL-243™) was compared using the method according to Experimental Example 3. As shown in Figure 10C, IL-21-treated NK cells exhibited greater anti-cancer activity compared to non-IL-21-treated NK cells.

[0129] Furthermore, the cytotoxicity of NK cells against solid tumor cells was evaluated using the method of Experimental Example 3 for NK cells treated with IL-21 and NK cells not treated with IL-21. AGS (gastric cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB-231 (breast cancer, ATCC® HTB-26™) were used as solid tumor cell lines. As shown in FIG. 10D, NK cells treated with IL-21 showed greater anti-cancer activity compared to NK cells not treated with IL-21.

[0130] Thus, by using the methods described herein, it may be possible to produce NK cells for certain cancer patients who do not normally exhibit sufficient NK cell proliferation.

[0131] Experimental Example 10. Confirmation of the viability of NK cells in a therapeutic composition On the sixth day of the culture, 1.0×10 5 ~2.0×10 6 Cells were inoculated into 350 mL bags based on cell counts in 1000 mL / mL and cultured for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 Cells were inoculated into 1 L bags based on cell counts in 1000 cells / mL and cultured for an additional 4 days. On day 14 of culture, 1.0 × 10 5 ~2.0×10 6 Cells were inoculated into 1 L bags based on cell counts / mL and then cultured for an additional 3 to 6 days.

[0132] CD56+ NK cells on days 14–20 of culture were washed three times and suspended in a base solution (physiological saline and Hartmann's solution) containing 1% albumin at 2 × 10 7 Cells were stored at 4°C for 48 hours before measuring cell viability.

[0133] To compare the effect of IL-2, CD56+ NK cells were washed and suspended in a base solution (physiological saline, Hartmann's solution, or phosphate-buffered saline) containing 1% albumin, and then added with IL-2 at a concentration of 500 IU / mL. After incubation at 4°C for 48 hours, cell viability was measured.

[0134] Add 100 μL of each composition for a total of 2 × 10 6 CD56+ NK cells were harvested, washed once with 1 mL of FACS staining buffer, centrifuged, and suspended in 100 μL of Annexin V binding buffer. 5 μL of Annexin V-FITC and 5 μL of 7-AAD (Biolegend) were added to the suspension, mixed well, stored in the dark, reacted at room temperature for 15 min, and then added with 400 μL of Annexin V binding buffer and mixed for 5 s before flow cytometry. Then, 10,000 to 100,000 cells per tube were harvested and analyzed. The cutoff was determined by setting a test tube that was not stained with Annexin V-FITC and 7-AAD as a negative control, and the viability was expressed by the percentage of the cell fraction that was negative for Annexin V-FITC or 7-AAD.

[0135] As shown in FIG. 11, with IL-2 treatment (with IL2), apoptosis of CD56+ NK cells was inhibited.

[0136] Experimental Example 11. Confirmation of cytotoxicity of NK cells in a therapeutic composition On the sixth day of the culture, 1.0×10 5 ~2.0×10 6 Cells were inoculated into 350 mL bags based on cell counts in 1000 mL / mL and cultured for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 Cells were inoculated into 1 L bags based on cell counts in 1000 cells / mL and cultured for an additional 4 days. On day 14 of culture, 1.0 × 10 5 ~2.0×10 6Cells were inoculated into 1 L bags based on cell counts / mL and then cultured for an additional 3 to 6 days.

[0137] Before being used in the experiments, the cancer cell lines were prepared by suspending them under the following conditions and subculturing them at 37±1° C. for at least one week at 3-day intervals.

[0138] CCRF-SB (blood cancer, ATCC® CCL-120™), AGS (gastric cancer, ATCC® CRL-1739™) and MIA-PACA2 (pancreatic cancer, ATCC® CRL-1420™): RPMI medium + 10% FBS;

[0139] SNU245 (cholangiocarcinoma, KCLB No. 00245), HCT15 (colon cancer, ATCC® CCL-225™) and NIH: OVCAR-3 (ovarian cancer, ATCC® HTB-161™): RPMI medium + 10% FBS + 25 mM HEPES, and

[0140] MDA-MB-231 (Breast Cancer, ATCC® HTB-26™): DMEM medium + 10% FBS.

[0141] Cancer cell lines (excluding blood cancer cell lines) in culture were detached from the culture dish using trypsin and suspended in medium at 5 × 10 4 The cells were then plated at 1 mL per well in 24 wells and allowed to attach for 1 day. To distinguish them from NK cells, the blood cancer cell lines, which are suspension culture cells, were labeled with green fluorescence and suspended in medium at 5 × 10 4 / mL, and 24 wells were inoculated with 1 mL per well.

[0142] First, in 24-well plates inoculated with cancer cell lines AGS (gastric cancer, ATCC® CRL-1739™), MIA-PACA2 (pancreatic cancer, ATCC® CRL-1420™), SNU245 (cholangiocarcinoma, KCLB No. 00245), HCT15 (colon cancer, ATCC® CCL-225™) and MDA-MB-231 (breast cancer, ATCC® HTB-26™), CD56+ NK cells were added one day later to observe cytotoxicity. The effector cells (CD56+ NK cells) and target cells (cancer cell lines) are shown in Table 7 below. [Table 7]

[0143] The plates inoculated with effector and target cells were cultured at 37±1℃ for 1-3 days, at which point 500IU / ml of IL-2 was further added to the experimental group to observe whether the anti-cancer activity was increased by IL-2. In the negative control group, no CD56+ NK cells (effector cells) were added, and there was no anti-cancer activity response.

[0144] After 1 to 3 days of culture, the cells were washed three times with RPMI to remove CD56+ NK cells that were present in suspension, and the cancer cell lines remaining in the wells were detached using trypsin, stained with trypan blue, and counted. Target cells and effector cells were then grafted. The seeded plates were cultured at 37±1° C. for 1-3 days, and then the green fluorescently labeled cells present in the 24 wells were counted using a flow cytometer.

[0145] Cytotoxicity against cancer cell lines was calculated using the following formula 2.

number

[0146] As a result, as shown in FIG. 12, it was confirmed that cancer cytotoxicity was increased when IL-2 was treated together (with IL2) compared to when CD56+ NK cells were treated alone (without IL2).

[0147] Next, CD56+NK cells were added to a 24-well plate, to which NIH:OVCAR-3 (ovarian cancer, ATCC® HTB-161™) cells were attached in a cancer cell line to observe cytotoxicity, and the ratio of target cells (cancer cell line) and effector cells (CD56+NK cells) is shown in Table 8 below. [Table 8]

[0148] The plates inoculated with effector cells and target cells were cultured at 37±1℃ for 1 day, and 500IU / ml IL-2 was further added to the experimental group to observe whether the anti-cancer activity was enhanced by IL-2. In the negative control group, CD56+ NK cells were not added, and there was no anti-cancer activity response.

[0149] After one day of culture, the cells were washed three times with RPMI to remove CD56+ NK cells present in a suspended form, and the cancer cell lines remaining in the wells were photographed using a camera.

[0150] As a result, as shown in Figure 13, cancer cytotoxicity was increased when cancer cell lines were treated with IL-2 (+IL2) compared to when they were treated with CD56+ NK cells alone (-IL2).

[0151] Next, CD56+NK cells were added to a 24-well plate, and AGS (gastric cancer, ATCC® CRL-1739™) cells were attached to the cancer cell line to observe cytotoxicity. The ratio of target cells (cancer cell line) and effector cells (CD56+NK cells) is shown in Table 9 below. [Table 9]

[0152] The plates inoculated with effector cells and target cells were cultured at 37±1℃ for 1 day, and 500IU / ml IL-2 was further added to the experimental group to observe whether the anti-cancer activity was enhanced by IL-2. In the negative control group, CD56+ NK cells were not added, and there was no anti-cancer activity response.

[0153] After one day of culture, the cells were washed three times with RPMI to remove CD56+ NK cells present in a suspended form, and the cancer cell lines remaining in the wells were photographed using a camera.

[0154] As a result, as shown in FIG. 14, cancer cytotoxicity was increased when cancer cell lines were treated with IL-2 (+IL2) compared to when they were treated with CD56+ NK cells alone (-IL2).

[0155] Experimental Example 12. Confirmation of anti-cancer effect of NK cells in animal models Except for using PBMCs from a colon cancer patient, CD56+ NK cells were produced in 17 days by the methods of Examples 1 and 2 and Comparative Examples 1 and 2. For each of the NK cells cultured in a CO2 incubator according to Examples 1 and 2 and Comparative Examples 1 and 2, on the 6th day of culture in a T25 culture flask, 1.0 × 10 5 ~2.0×10 6 Inoculate cells into 350 mL bags based on cell count / mL and culture for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 The cells are inoculated into a 1 L bag based on the cell count in 100 ml / mL and cultured for an additional 4 days. Then, on day 14 of culture, 1.0 × 10 5 ~2.0×10 6 Cells are inoculated into 1 L bags based on cell count / mL and then cultured for an additional 3-6 days.

[0156] An animal model of human cancer is constructed by xenografting human cancer cell lines into mice. The following human cancer cell lines are used: AGS (gastric cancer), MIA-PACA2 (pancreatic cancer), SNU245 (cholangiocarcinoma), HCT15 (colon cancer) and NIH: OVCAR-3 (ovarian cancer), and MDA-MB-231 (breast cancer). After cancer xenografting, the mice are randomly grouped and marked. For the control group, 200 μL of Hartmann's solution is injected into the tail vein. For the NK cell treatment (+IL-2) group, 1 × 10 7 NK cells / 200μL and 500IU / mL IL-2 were injected into the tail vein 5 times at 2-3 day intervals starting 1 week after cancer xenografting. For the NK cell treatment (-IL-2) group, 1×10 7 NK cells / 200 μL are injected into the tail vein five times at 2-3 day intervals starting one week after cancer xenograft transplantation.

[0157] Mice are examined for body weight and tumor volume three times a week to follow tumor growth during the study. The length of the major and minor axes are measured using calipers and tumor volume is determined according to the following formula (Equation 3): formula 3 Tumor volume (mm 3 ) = (major axis length (mm)) x (minor axis length (mm)) 2 ×0.5

[0158] The NK cell therapy (-IL-2) group shows a reduction in tumor growth after 8 weeks of treatment of approximately 50% compared to the control group based on luciferase imaging for each cancer type. The NK cell therapy (+IL-2) group shows a further reduction in tumor growth of approximately 60% compared to the control group for each cancer type.

[0159] Experimental Example 13. Confirmation of the anti-cancer effect of NK cells from cancer patients Except for using PBMCs from a colon cancer patient, CD56+ NK cells were produced in 18 days by the methods of Examples 1 and 2 and Comparative Examples 1 and 2. For each of the NK cells cultured in a CO2 incubator according to Examples 1 and 2 and Comparative Examples 1 and 2, on the 6th day of culture in a T25 culture flask, 1.0 × 10 5 ~2.0×106 Inoculate cells into 350 mL bags based on cell count / mL and culture for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 The cells are inoculated into a 1 L bag based on the cell count in 100 ml / mL and cultured for an additional 4 days. Then, on day 14 of culture, 1.0 × 10 5 ~2.0×10 6 Cells are inoculated into 1 L bags based on cell count / mL and then cultured for an additional 3-6 days.

[0160] Colon cancer patients were randomly grouped and marked. For the control group, no NK cells were injected. For the NK cell therapy (+IL-2) group, 1–3 × 10 NK cells were injected per kg of body weight. 7 of NK cells and 500 IU / mL of IL-2 were injected intravenously three times at intervals of 6 weeks. For the NK cell treatment (-IL-2) group, 1–3 × 10 7 The NK cells will be injected intravenously six times at six-week intervals.

[0161] Tumor growth is monitored at 1, 3, 6 and 12 months. After 12 months, the NK cell therapy (-IL-2) group shows an overall decrease in tumor size, and the NK cell therapy (+IL-2) group shows a further overall decrease in tumor size.

[0162] Experimental Example 14. Confirmation of the anti-cancer effect of NK cells from Alzheimer's disease patients Except for using PBMCs from a colon cancer patient, CD56+ NK cells were produced in 18 days by the methods of Examples 1 and 2 and Comparative Examples 1 and 2. For each of the NK cells cultured in a CO2 incubator according to Examples 1 and 2 and Comparative Examples 1 and 2, on the 6th day of culture in a T25 culture flask, 1.0 × 10 5 ~2.0×10 6 Inoculate cells into 350 mL bags based on cell count / mL and culture for an additional 4 days. On day 10 of culture, 1.0 × 10 5 ~2.0×10 6 The cells are inoculated into a 1 L bag based on the cell count in 100 ml / mL and cultured for an additional 4 days. Then, on day 14 of culture, 1.0 × 10 5~2.0×10 6 Cells are inoculated into 1 L bags based on cell count / mL and then cultured for an additional 3-6 days.

[0163] Randomly group and mark the Alzheimer's disease patients. For the control group, do not inject NK cells. For the NK cell treatment group, inject 1–3 × 10 NK cells per kg of body weight. 7 of NK cells and 500 IU / mL of IL-2 will be injected intravenously at weekly intervals for six doses.

[0164] Patients' cognitive function will be monitored at 1, 3, 6, and 12 months. After 12 months, the NK cell therapy group will show improved cognitive function.

[0165] Experimental Example 15. Confirmation of anti-cancer effect of NK cells from patients with autoimmune diseases Except for using PBMCs from a colon cancer patient, CD56+ NK cells were produced in 18 days by the methods of Examples 1 and 2 and Comparative Examples 1 and 2. For each of the NK cells cultured in a CO2 incubator according to Examples 1 and 2 and Comparative Examples 1 and 2, on the 6th day of culture in a T25 culture flask, 1.0 × 10 5 ~2.0×10 6 3 based on cell count / mL The cells are inoculated into a 50 mL bag and cultured for an additional 4 days. On the 10th day of culture, 1.0 × 10 5 ~2.0×10 6 The cells are inoculated into a 1 L bag based on the cell count in 100 ml / mL and cultured for an additional 4 days. Then, on day 14 of culture, 1.0 × 10 5 ~2.0×10 6 Cells are inoculated into 1 L bags based on cell count / mL and then cultured for an additional 3-6 days.

[0166] Multiple sclerosis patients were randomly grouped and marked. For the control group, no NK cells were injected. For the NK cell treatment group, 1–3 × 10 NK cells were injected per kg of body weight. 7 of NK cells and 500 IU / mL of IL-2 will be injected intravenously at weekly intervals for six doses.

[0167] Patients' cognitive function will be monitored at 1, 3, 6, and 12 months. After 12 months, the NK cell therapy group will show improved cognitive function.

[0168] Terminology The foregoing description of exemplary embodiments has been presented for purposes of illustration and description only and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teachings. Various combinations or subcombinations of the specific features and aspects of the above disclosed embodiments may be made and still be within one or more of the present invention. Furthermore, any particular feature, aspect, method, characteristic, property, quality, property, element, or the like disclosed herein in connection with an embodiment may be used in all other embodiments described herein. Thus, various features and aspects of the disclosed embodiments may be combined with or substituted for one another to form various modes of the disclosed invention. Thus, it is not intended that the spirit of the invention disclosed herein should be limited by the specific disclosed embodiments. Moreover, the present invention is susceptible to various modifications, alternatives of which, specific examples have been shown in the drawings and are described in detail herein. However, the invention is not intended to be limited to the particular forms or methods disclosed, but on the contrary, the invention is to be understood to encompass all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein need not be performed in the order described. The methods disclosed herein include specific acts performed by a practitioner, but may also include, explicitly or implicitly, third-party direction of any of these acts. Ranges disclosed herein also encompass any and all overlapping portions, subranges, and combinations thereof.

[0169] The embodiments have been chosen and described to illustrate the principles of the invention and their practical applications so as to encourage others skilled in the art to utilize the invention with various embodiments and various modifications suitable for the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art of the invention as defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

[0170] Conditional language such as "can," "could," "might," or "may" is typically intended to convey that a particular embodiment includes certain features, elements, and / or steps, but not other embodiments, unless specifically specified otherwise or understood otherwise within the context in which it is used. Thus, such conditional language is not typically intended to imply that the features, elements, and / or steps are in any way required for one or more embodiments.

[0171] The terms "comprising," "including," "having," and the like are synonymous and are used inclusively in an open-ended manner and do not exclude additional elements, features, acts, operations, etc. The term "or" refers, for example, to one or more elements. When used to connect a list, the term "or" is used in its inclusive sense (and not its exclusive sense) to mean one or all of the elements in the list.

[0172] Ranges disclosed herein encompass any and all overlapping portions, subranges, and combinations thereof. Language such as "up to," "at least," "greater than," "less than," "between," and the like, is inclusive of the recited numbers.

[0173] As used herein, numbers preceded by terms such as "approximately," "about," and "substantially" refer to an amount that is inclusive of the stated number (e.g., about 10%=10%) and yet approximates a stated amount that performs a desired function or achieves a desired result. For example, the terms "approximately," "about," and "substantially" refer to an amount that is within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount.

[0174] As used herein, the term "generally" refers to values, amounts, or characteristic values ​​that predominantly include or tend to be around a particular value, amount, or characteristic value. By way of example, in certain embodiments, the term "generally uniform" refers to values, amounts, or characteristic values ​​that deviate from precisely uniform by less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01%.

[0175] Ranges disclosed herein encompass any and all overlapping portions, subranges, and combinations thereof. Words such as "up to," "at least," "greater than," "less than," "between," and the like, include the recited number. Numbers preceded by terms such as "about" or "approximately" include the recited number. For example, "about 5.0 cm" includes "5.0 cm."

[0176] Some embodiments are described in conjunction with schematic diagrams. However, it should be understood that the schematic diagrams are not drawn to scale. Distances are merely illustrative and do not necessarily bear a precise relationship to the actual dimensions and layout of the devices depicted.

[0177] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. It is to be understood that not necessarily all such advantages can be achieved by any particular embodiment. Thus, for example, one skilled in the art will recognize that the present disclosure may be embodied or carried out in a manner that achieves one advantage or group of advantages taught herein without necessarily achieving other advantages as taught or suggested herein.

[0178] Furthermore, although illustrative embodiments are described herein, the scope of any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., of aspects across various embodiments), adaptations and / or alterations will be understood by those skilled in the art based on this disclosure. The limitations in the claims should be interpreted broadly based on the language used in the claims, and not limited to the examples described herein or during the prosecution of this application, which examples should be interpreted as non-exclusive. Furthermore, the acts of the disclosed processes and methods may be modified in any manner, including reordering and / or inserting and / or deleting acts. Thus, with the true scope and spirit of the claims and the full scope of equivalents, the specification and examples should be understood as merely illustrative.

Claims

1. A method for producing natural killer cells, comprising: The method comprises co-culturing PBMCs with a combination of irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte line (EBV-LCL) cells in the presence of IL-2 and IL-21.

2. The method of claim 1, wherein the PBMCs comprise at least one of CD56+ cells, CD3- / CD56+ cells, and / or CD3- cells isolated by using at least one of CD56 microbeads and CD3 microbeads.

3. The method of claim 1 or 2, wherein the ratio of the irradiated Jurkat cells to the irradiated EBV-LCL cells is 1:0.1-5 or 0.1-5:

1.

4. The method according to any one of claims 1 to 3, wherein each of the irradiated Jurkat cells and the irradiated EBV-LCL cells is obtained by irradiation of 50 to 500 Gy.

5. The method of any one of claims 1 to 4, wherein the co-culturing comprises co-culturing for 1 to 50 days.

6. co-culturing the PBMCs with a combination of feeder cells in the presence of IL-2 for a first period of time; and The method of any one of claims 1 to 5, further comprising subsequently co-culturing the PBMCs with a combination of feeder cells in the presence of IL-21 for a second period of time.

7. The method of claim 6, wherein IL-21 is added one or more times during days 0-6 of the second period.

8. The method of claim 7, wherein the combination of IL-21 and the feeder cells is added one or more times during days 0-6 of the second period.

9. A composition produced by the method of any one of claims 1 to 8.

10. 1. A composition in a form suitable for administration to a patient in need thereof, an effective amount of peripheral blood derived CD56+ natural killer cells, said effective amount being 1×10 per kg of patient body weight; ６ ~5×10 ８ and the CD56+ natural killer cells are at least 90% pure; IL-2 having a concentration of 50 to 50,000 IU / mL; and Pharmaceutically acceptable carriers A composition comprising:

11. 1. A therapeutic composition for use in the manufacture of a medicament for treating a patient in need of cancer treatment, comprising: Natural killer cells produced by co-culturing PBMCs with a combination of irradiated Jurkat cells and irradiated Epstein-Barr Virus transformed lymphocyte line (EBV-LCL) cells in the presence of IL-2 at a concentration of 50-1000 IU / mL and IL-21 at a concentration of 10-100 ng / mL. A therapeutic composition comprising:

12. 1. A method for expanding human natural killer cells in culture, comprising: isolating at least one of human CD56+ cells and / or human CD3- / CD56+ cells from human PBMCs; and co-culturing at least one of human CD56+ cells and / or human CD3- / CD56+ cells with a combination of human feeder cells in the presence of at least two human cytokines; the feeder cell combination comprises irradiated Jurkat cells and irradiated Epstein-Barr Virus transformed lymphocyte line (EBV-LCL) cells; the at least two cytokines include IL-2 and IL-21; How to enlarge.

13. The method of any one of claims 1 to 8, wherein IL-2 is present at a concentration of 50 to 1000 IU / mL and IL-21 is present at a concentration of 10 to 100 ng / mL.

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