Method for producing natural killer cells and composition thereof

Abstract

A method for producing natural killer cells is disclosed. The method includes 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. The method may further include freezing and thawing the CD56+ cells and / or CD3- / CD56+ cells. A composition for treating cancer is also disclosed. The composition includes CD56+ natural killer cells and cytokines produced by the disclosed method.

Description

[Technical Field]

[0001] This disclosure relates to the manufacture, storage, and / or natural killer cells themselves. [Background technology]

[0002] Natural killer cells (NK cells) are a type of innate immune cell that is known to kill cancer cells nonspecifically using enzymes such as perforin and granzymes, or through Fas-FasL interactions, and to recognize and kill viruses and bacteria, as well as other pathogens. In cancer patients, a decrease in the cytotoxicity of these NK cells against cancer cells is observed in various types of cancer, such as 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 E., It has been reported to be associated with the onset of hematological malignancies (e.g., Br j Cancer., 1999: 79: 244-250) and hematological malignancies (e.g., Tajima F., et al, Lekemia 1996: 10: 478-482). [Overview of the project] [Problems that the invention aims to solve]

[0003] This application relates to a method for producing high-purity natural killer cells, and to a cell therapy composition for treating cancer comprising high-purity natural killer cells and cytokines. Any feature, structure, or process disclosed herein may be substituted, combined with, or omitted using any other feature, structure, or process disclosed herein. Furthermore, for the purpose of summarizing this disclosure, certain aspects, advantages, and features of the invention are described herein. It should be understood that not necessarily any or all such advantages are achieved according to any particular embodiment of the invention disclosed herein. Individual aspects of this disclosure are not essential or indispensable. [Means for solving the problem]

[0004] In some embodiments, a method for expanding and growing natural killer cells in culture is disclosed. The method comprises: isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 (and feeder cells) for a first period; freezing the co-culturised CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 (and feeder cells) for a second period.

[0005] Any embodiment of a method provided herein and / or any method disclosed herein may include one or more of the following features: The method may further include storing frozen CD56+ cells at a temperature below -100°C. The method may further include storing frozen CD56+ cells for longer than one day before thawing. Isolated CD56+ cells may be co-cultured for 13–16 days (or 9–25 days) before freezing. Isolated CD56+ cells may be co-cultured with one or more irradiated feeder cells in the presence of IL-21. Thawed CD56+ cells may be co-cultured with one or more irradiated feeder cells in the presence of IL-21. One or more feeder cells may be one or more selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells, K562 cells, and PBMCs (e.g., autologous PBMCs). CD56+ cells can be co-cultured in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. In some embodiments, any feeder cell can be used for the first, second, or first and second expansion (e.g., with the use of Il-21). CD56+ cells can be co-cultured in a ratio of approximately 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, or 1:100 CD56+ cells to feeder cells. In some embodiments, these ratios are for KL1 / EBVLCL, and for other feeder cells, for example, 1:1 to 1:10 may be used. IL-21 can be added at a concentration of 10 to 100 ng / mL during the first and / or second period. IL-21 can be added at a concentration of 20 to 80 ng / mL during the first and / or second period. IL-21 may be added at a concentration of 30-70 ng / mL during the first and / or second period. IL-21 may be added more than once during the first and / or second period.

[0006] In some embodiments, a method is provided for expanding and growing natural killer cells in culture. The method comprises: isolating CD56+ from a blood sample (e.g., PBMC, fresh or frozen, umbilical cord blood, and / or from blood from which CD56+, CD56+CD3-, and CD3- cells have been isolated from a blood sample); co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and expanding and growing the thawed CD56+ cells (again with suitable feeder cells of any kind).

[0007] A method of any embodiment and / or any method disclosed herein may include one or more of the following features: Freezing of CD56+ cells may be carried out at a temperature lower than -100°C. The method may further include storing the frozen CD56+ cells for a period longer than one day and shorter than 10 years. CD56+ cells may be co-cultured for 13 to 16 (or 9 to 25) days prior to freezing. One or more feeder cells may be selected from the group consisting of at least one of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and / or PBMCs, and are not limited to all embodiments. CD56+ cells may be co-cultured in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. IL-21 may be added at concentrations of 10-100 ng / mL. IL-21 may be added more than once. In some embodiments, NK cells may be used with any feeder cell type, provided that IL-21 is used before freezing, followed by a restimulation process after thawing from freezing.

[0008] In some embodiments, a method for increasing the cytotoxicity of natural killer cells is disclosed. The method comprises providing the natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21. Optionally, before freezing the natural killer cells, the natural killer cells may be co-culturified (grown) with feeder cells and IL-21.

[0009] Any method in any preceding paragraph and / or any method disclosed herein may include one or more of the following features: The method may further include storing frozen natural killer cells at a temperature below -100°C; The method may further include storing frozen natural killer cells for a longer period than one day before thawing. One or more feeder cells are selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell lines, irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL), K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and / or PBMCs. Thawed natural killer cells can be co-cultured in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. IL-21 can be added at a concentration of 10 to 100 ng / mL. IL-21 may be added more than once.

[0010] In some embodiments, a method for treating a subject is disclosed. The method comprises: collecting CD56+ cells from a subject; co-culturing the CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-culturized CD56+ cells for at least one day; thawing the frozen CD56+ cells; expanding the thawed CD56+ cells; and administering the expanded CD56+ cells to a subject, wherein the cytotoxicity of the cells from the second expansion is at least X% of the cytotoxicity of the co-culturized CD56+ before freezing.

[0011] Any method in any preceding paragraph and / or any method disclosed herein may include one or more of the following features: The method may further include storing frozen CD56+ cells at a temperature below -100°C. The method may further include storing frozen CD56+ cells for longer than one day before thawing. Isolated CD56+ cells may be co-cultured for 13–16 (or 9–25) days before freezing. Expanding thawed CD56+ cells may include co-culturing thawed CD56+ with one or more irradiated feeder cells in the presence of IL-21. One or more feeder cells may be one or more selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and / or PBMCs. CD56+ cells can be co-cultured in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. IL-21 can be added at a concentration of 10 to 100 ng / mL during the first and / or second period. IL-21 may be added more than once during the first and / or second period.

[0012] In some embodiments, a composition is provided which comprises an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient. The CD56+ cells are prepared by isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating CD56+ cells from the PBMCs; co-culturing the CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines.

[0013] In some embodiments, a cell composition is disclosed. The cell composition comprises an effective amount of CD56+ cells; IL-2; and IL-21 derived from peripheral blood mononuclear cells (PBMCs) from a patient.

[0014] In some embodiments, a composition is disclosed. The composition comprises a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; IL-2; and IL-21. When thawed, the CD56+ cells have at least 80% of the cytotoxicity of a second population of CD56+ cells, the second population of CD56+ cells being unfrozen.

[0015] In some embodiments, a method is provided for expanding and growing natural killer cells in culture. This method may include: isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 for a first period; freezing the co-culturized CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 for a second period.

[0016] In some embodiments, a method is provided for expanding and growing natural killer cells in culture. The method comprises isolating CD56+ from a blood sample; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and expanding and growing the thawed CD56+ cells.

[0017] In some embodiments, a method is provided for increasing the cytotoxicity of natural killer cells, the method comprising: providing the natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21.

[0018] In some embodiments, a method is provided for treating a subject, the method comprising: collecting CD56+ cells from the subject; co-culturing the CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-culturated CD56+ cells for at least one day; thawing the frozen CD56+ cells; expanding the thawed CD56+ cells; and administering the expanded CD56+ cells to the subject, wherein the cytotoxicity of the cells from the second expansion is at least 80% of the cytotoxicity of the co-culturated CD56+ before freezing.

[0019] In some embodiments, a composition is provided which comprises an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient, the CD56+ cells being prepared by isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating CD56+ cells from the PBMCs; co-culturing the CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines.

[0020] In some embodiments, a cell composition is provided which comprises an effective amount of CD56+ cells; IL-2; and IL-21 derived from peripheral blood mononuclear cells (PBMCs) from a patient.

[0021] In some embodiments, a composition is provided which comprises a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; and IL-2, IL-21. When thawed, the CD56+ cells have at least 80% of the cytotoxicity of a second population of CD56+ cells, the second population of CD56+ cells being unfrozen.

[0022] In some embodiments, a method is provided for expanding and growing natural killer cells in culture, the method comprising: providing PBMCs; co-culturing the PBMCs in the presence of IL-21 for a first period; freezing the co-culturised PBMCs after the first period; thawing the frozen PBMCs; and thawing the thawed PBMCs in the presence of IL-21. This includes co-culturing over a second period.

[0023] In some embodiments, the composition comprises IL-2; 5-10% DMSO; 90-95% FBS; and optionally NK cells, which are CD56+ cells. In some embodiments, it further comprises CryoStor solution. In some embodiments, the composition is for frozen cells before regrowth.

[0024] In some embodiments, the composition comprises IL-2; 5-10% DMSO; 80-95% Hartman solution; 1-10% human serum albumin; and NK cells. In some embodiments, it further comprises CryoStor solution. In some embodiments, the composition is for cells frozen before injection.

[0025] Various embodiments are depicted in the accompanying drawings for illustrative purposes, but should not be construed as limiting the scope of the embodiments. Furthermore, various features of different disclosed embodiments can be combined to form additional embodiments which constitute part of this disclosure. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 illustrates the design and embodiment of a re-expansion experiment to expand and proliferate cells according to IL21 treatment (before freezing and optionally after freezing). [Figure 2A] Figures 2A and 2B depict a comparison of population doubling levels (PDL) between cell expansion and proliferation with and without IL21. (Figure 2A) Donor 1, (Figure 2B) Donor 2. [Figure 2B] Figures 2A and 2B depict a comparison of population doubling levels (PDL) between cell expansion and proliferation with and without IL21. (Figure 2A) Donor 1, (Figure 2B) Donor 2. [Figure 3A] Figures 3A and 3B depict a comparison of the expansion proliferation ratio between cell expansion proliferation with and without IL21. (Figure 3A) Donor 1, (Figure 3B) Donor 2. [Figure 3B]Figures 3A and 3B depict a comparison of the expansion proliferation ratio between cell expansion proliferation with and without IL21. (Figure 3A) Donor 1, (Figure 3B) Donor 2. [Figure 4A] Figures 4A and 4B depict a comparison of population doubling levels (PDL) between cell restimulation methods with and without IL21. (Figure 4A) Donor 1, (Figure 4B) Donor 2. [Figure 4B] Figures 4A and 4B depict a comparison of population doubling levels (PDL) between cell restimulation methods with and without IL21. (Figure 4A) Donor 1, (Figure 4B) Donor 2. [Figure 5A] Figures 5A and 5B depict a comparison of expanded proliferation levels between cell restimulation methods with and without IL21. (Figure 5A) Donor 1, (Figure 5B) Donor 2. [Figure 5B] Figures 5A and 5B depict a comparison of expanded proliferation levels between cell restimulation methods with and without IL21. (Figure 5A) Donor 1, (Figure 5B) Donor 2. [Figure 6] Figure 6 illustrates the cytotoxic activity of NK cells (IL-21+) that have been expanded and proliferated with IL-21 against K562 cells. [Figure 7] Figure 7 illustrates the cytotoxic activity of NK cells (IL-21-) that have been expanded and proliferated without IL-21 against K562 cells. [Figure 8] Figure 8 illustrates the cytotoxic activity of NK cells (IL-21+ / +) that have been expanded and restimulated with IL-21 against K562 cells. [Figure 9] Figure 9 illustrates the cytotoxic activity of NK cells (IL-21+ / -) that were expanded and proliferated with IL-21 and then restimulated without IL-21 against K562 cells. [Figure 10] Figure 10 illustrates the cytotoxic activity of NK cells (IL-21- / +) that were expanded and proliferated without IL-21 and then restimulated with IL-21 against K562 cells. [Figure 11]Figure 11 depicts the cytotoxic activity of NK cells (IL-21- / -) that were expanded and restimulated without IL-21 against K562 cells. [Figure 12A] Figure 12A illustrates a comparison of the phenotypes of NK cell activation receptors. [Figure 12B] Figure 12B illustrates the comparison of NK cell inhibition and chemokine receptor phenotypes. [Figure 13A] Figure 13A shows graphs of the population doubling levels (PDL) of NK cells that proliferated in the presence of IL-21 and then re-proliferated in the presence of IL-21 (IL-21+ / +), and NK cells that proliferated without IL-21 and then re-proliferated without IL-21 (IL-21- / -). [Figure 13B] Figure 13B shows graphs of the population doubling level (PDL) of NK cells that proliferated in the presence of IL-21 and then re-proliferated in the presence of IL-21 (IL-21+ / +), and NK cells that proliferated in the presence of IL-21 and then re-proliferated without IL-21 (IL-21+ / -). [Figure 14A] Figure 14A shows a graph of the population doubling level (PDL) of NK cells that were expanded and proliferated in the presence of IL-21 (first stimulation) and re-expanded and proliferated at least twice (second and third stimulation). [Figure 14B] Figure 14B shows a graph of the corresponding expansion multiplication factor for the results in Figure 14A. [Modes for carrying out the invention]

[0027] Incorporation by reference to any priority application This application claims the interests of U.S. Provisional Application No. 63 / 062694 filed on 7 August 2020 and Korean Patent Application No. 10-2019-0157727 filed on 29 November 2019, the disclosures of each of these applications being incorporated herein by reference in their entirety.

[0028] A method has been developed to assist NK cells in undergoing cryopreservation and subsequent recovery, and is provided herein. It has been found that CD56+ cells can successfully expand after freezing and thawing if they are initially co-cultured with feeder cells in the presence of IL-21 (during initial expansion). By using IL-21, high-purity CD56+ NK cells can be obtained as a result, and surprisingly, they retain particularly high cytotoxicity. Furthermore, after thawing, the NK cells can expand further (without IL-21, or more favorably, in the presence of additional IL-21). Thus, IL-21 in the pre-freezing process (e.g., first expansion) can enable superior regrowth at later time points. Moreover, the resulting product retains surprisingly high cytotoxicity even after two expansions and one freeze, which is further enhanced when IL-21 is used not only during the first expansion but also during the second expansion. In some embodiments, this freezing and re-expanding growth process may be repeated multiple times (each time optionally accompanied by another round of IL-21 during re-expanding growth).

[0029] In some embodiments, a method is provided for expanding and growing natural killer cells in culture. The method comprises providing PBMCs; co-culturing the PBMCs in the presence of IL-21 for a first period; freezing the co-culturated PBMCs after the first period; thawing the frozen PBMCs; and co-culturing the thawed PBMCs in the presence of IL-21 for a second period. In some embodiments, the ratio of PBMCs to feeder cells is 1:0.5:0.5. In some embodiments, the ratio may be about 1:0.5:0.5 to 1:10:10 for PBMCs (e.g., as a substitute for CD56+). In some embodiments, for CD56+ cells, the ratio is multiplied by, for example, 10 or 20 (e.g., 1:1 to 100 for CD56+ cells and feeder cells). In some embodiments, the expansion and growth is performed on CD56+ or CD56+ / CD3- cells. It is done from.

[0030] According to some embodiments, a method for producing high-purity NK cells may include co-culturing cells selected from CD56+ cells and / or CD3- / CD56+ cells with feeder cells in the presence of a first cytokine, such as IL-21 ("first culture step" or "first expansion step"); freezing the co-cultured cells ("freezing step"); thawing the frozen cells ("thawing step"); and optionally co-culturing the thawed cells further with IL-21 and added feeder cells ("second culture step" or "second expansion step"). Each step is described in more detail herein. CD3- / CD56+ cells produced according to the disclosed method may exhibit not only higher purity and higher anticancer activity, but also other distinguishing features, such as having different surface markers or activated receptors, e.g., one or more from CD16, CD25, CD27, CD28, CD69, CD94 / NKG2C, CD94 / NKG2E, CD266, CD244, NKG2D, KIR2S, KIR3S, Ly94D, NCRs, IFN-a, IFN-b, CXCR3, CXCR4, CX3CR1, CD62L, and CD57.

[0031] As used herein, “Process” is a part of a process and does not require that one “Process” be completed before the next “Process” may begin. Unless otherwise specified, processes may be provided in overlapping time periods or simultaneously, as appropriate. Naturally, there is no overlap if one process occurs before an event (e.g., freezing) and another process occurs after the same event (e.g., a first IL-21 incubation and a second IL-21 incubation).

[0032] In this specification, 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 their cell surface, or further, cells expressing CD56 glycoprotein but not CD3 glycoprotein. Even among immune cells of the same type, there may be differences in the type and expression rate of CD bound to the cell surface, and therefore their functions may differ.

[0033] In some embodiments, CD56+ cells or CD3- / CD56+ cells are obtained by the following steps: isolating peripheral blood mononuclear cells (PBMCs) from a blood sample ("first isolation step"); and isolating cells selected from the group consisting of CD56+ cells and CD3- / CD56+ cells from the peripheral blood mononuclear cells ("second isolation step").

[0034] In this specification, “blood sample” may be, but is not limited to, whole peripheral blood or leukocytes isolated from peripheral blood using leukocyte apheresis. Furthermore, the source of peripheral blood may be, but is not limited to, a normal human, a patient at risk of cancer, or a cancer patient.

[0035] In this specification, the term "leukocyte apheresis" may refer to a method of selectively removing (isolating) leukocytes from collected blood and then re-administering the blood to a patient, and in some embodiments, the leukocytes isolated by this method may be used without additional methods, such as the Ficoll-Hypaque density gradient method.

[0036] In this specification, the term “peripheral blood mononuclear cells” may be used interchangeably with “PBMCs” and “mononuclear cells,” which are commonly used from peripheral blood for anti-cancer immunotherapy. This can refer to separated mononuclear cells. Peripheral blood mononuclear cells may be obtained from collected human blood using known methods, such as the Ficoll-Hypaque density gradient method.

[0037] In some embodiments, peripheral blood mononuclear cells may be autologous, but allogeneic peripheral blood mononuclear cells may also be used to produce highly purified NK cells for anti-cancer immunotherapy according to the methods described herein. Furthermore, in some embodiments, peripheral blood mononuclear cells may be obtained from a normal human, but peripheral blood mononuclear cells may also be obtained from a patient at risk of cancer and / or a cancer patient.

[0038] In some embodiments, a second isolation step for isolating CD56+ natural killer cells from a blood sample may be performed using at least one selected from the group consisting of CD56 microbeads and CD3 microbeads, or an isolation method using equipment such as CliniMACSs, flow cytometry cell sorters, or MACS Separator, magnetic sorting systems.

[0039] For example, isolation methods using CD56 microbeads and / or CD3 microbeads can be performed by adding CD56 microbeads to PBMCs and then removing nonspecific binding, or by adding CD3 microbeads to PBMCs to remove specific binding and then adding CD56 microbeads again to remove nonspecific binding. In some cases, T cells or other non-natural killer cells can be removed by isolating CD56+ cells and / or CD3- / CD56+ cells from PBMCs.

[0040] In this specification, the term “feeder cell” may refer to a cell that does not divide or proliferate but has metabolic activity that produces various metabolites, and thus helps the proliferation of target cells.

[0041] 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 lymphoblastoid cell lines (EBV-LCL) cells, PBMCs, HFWTs, 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. In some embodiments, as provided herein, any feeder cell type may be used, insofar as it allows for regrowth when NK cells are first expanded in the presence of IL-21, then frozen, thawed, and then subjected to regrowth.

[0042] In this specification, the terms “Jurkat cells” or “Jurkat cell line” may refer to a hematological cancer (immortalized acute T-cell leukemia) cell line developed by Dr. Arthur Weiss at the University of California, San Francisco. Jurkat cells, which express various chemokine receptors and have the ability to produce IL-2, are not generally considered as a possible candidate for feeder cells for anti-cancer immunotherapy because they highly express MHC class I, a natural killer cell activation inhibitor, on their cell surface. Jurkat cells are available from ATCC (ATCC TIB-152).

[0043] In this specification, the terms “EBV-LCL cells” or “EBV-LCL cell line” refer to the Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) (DM Koelle et al., J Clin Invest, 1993:91: 961-968), a B cell line produced by infecting human B cells with Epstein-Barr virus in vitro. EBV-LCL cells can be prepared and used directly in a general laboratory by adding cyclosporine A during the process of infecting PBMCs with EBV. In some embodiments, EBV-LCL cells can be prepared by the following steps: 30 × 10 6 Add one PBMC to 9 mL of culture medium, add the mixture to a T25 culture flask, then add 9 mL of EBV supernatant. Add 80 μL of cyclosporine A (50 μg / mL), then culture at 37°C. After 7 days of culture, remove half of the supernatant, add fresh culture medium, then add 40 μL of cyclosporine A. This process can be repeated once every 7 days until 28 days of culture. The cell line may be ready after 28 days of culture, and after this point, the cell line can be cultured in culture medium without adding cyclosporine A.

[0044] Jurkat cells and EBV-LCL cells can be used as feeder cells after irradiation.

[0045] In some embodiments, prior to administration, the method may further include subjecting the enlarged and proliferated cells to a second freeze in a ready-to-inject solution.

[0046] In some embodiments, irradiated Jurkat cells and irradiated EBV-LCL cells may be present in content ratios 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, irradiated Jurkat cells and irradiated EBV-LCL cells may be present in a 1:1 content ratio.

[0047] In some embodiments, irradiated Jurkat cells and irradiated EBV-LCL cells can be obtained by treating them with irradiation at 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120, or 90-110 Gy. For example, irradiated Jurkat cells and / or irradiated EBV-LCL cells can be obtained by treating Jurkat cells and / or EBV-LCL cells with irradiation at 100 Gy.

[0048] In this specification, the term “cytokine” may be used interchangeably with “first cytokine” or “second cytokine” and may refer to an immunoactive compound that can be used to induce peripheral blood mononuclear cells to differentiate into NK cells. In some embodiments, the cytokine is IL-21 (for both the first and second expansion growth, as well as for any further rounds of expansion growth).

[0049] Where used herein, the terms “co-culture” and “extended proliferation” are interchangeable and refer to the cultivation of NK cells to result in an extended proliferation population of cells. The term “re-extended proliferation” refers to the fact that one round of co-culture or extended proliferation has already been performed on the NK cells. In some embodiments, co-culture or extended proliferation is performed in the presence of feeder cells and cytokines, such as IL-21. In some contexts herein, the term “culture” is used as an abbreviation for “co-culture.”

[0050] In some embodiments, cytokines include interleukin-2 (IL-2), IL-15, IL-21, FMS-like tyrosine kinase 3 ligand (Flt3-L), and stem cell factors. This may include, but is not limited to, subcutaneous follicular cell stimulant (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).

[0051] In some embodiments, the first cytokine may be IL-2, IL-21, IL-15, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL-18, IL-4, type I interferon, GM-CSF, insulin-like growth factor 1 (IGF 1), or any combination thereof. In some embodiments, the second cytokine may be IL-2, IL-21, IL-15, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL-18, IL-4, type I interferon, GM-CSF, insulin-like growth factor 1 (IGF 1), or any combination thereof. For example, the second cytokine may be IL-21. In some embodiments, more than one cytokine may be present throughout one or more of the steps provided herein. In some embodiments, both IL-21 and IL-2 are present in at least one of the first and second rounds of expansion growth (or any subsequent rounds). Additionally, if the various embodiments provided herein also involve the repeated use of a cytokine (e.g., IL-21) in each of the various rounds of expansion growth, such cytokine may be used for the first time (or during the first round or process) and the second time (e.g., during the re-expansion growth process). Thus, in some embodiments, the first cytokine (e.g., IL-21) may be used for both the first and second rounds of co-culture. This may also be described alternatively as the first cytokine and the second cytokine, for example, both of which are IL-21.

[0052] Method for producing NK cells Figure 1 is a flowchart illustrating some methods for expanding and growing NK cells using various exemplary feeder cells. In some embodiments, CD56+ cells or CD3- / CD56+ cells are expanded and grown by co-culturing them with feeder cells in the presence of IL-21. The feeder cells may be any type of feeder cell, e.g., Jurkat cells and EBV-LCL cells ("Type 1"), K562 cells ("Type 2"), or PBMCs ("Type 3"). In some embodiments, cells are collected on day 17 or about day 17 of culture (or at any point between days 16–21 or 9–25), and the cells produced may be referred to herein as "IL21+." Such cell expansion and growth processes that do not involve cryopreservation or a second culture step may be referred to herein as the "original process." In some embodiments, cells are collected on day 14 or about day 14 (or at any point between days 14–18 or 9–25) and subjected to cryopreservation. The culture prior to cryopreservation may be referred to herein as the “first culture step,” “first expansion growth step,” or “first co-culture step.” The cryopreserved cells may be thawed and co-cultured again with feeder cells in the presence of IL-21 (“IL-21+ / +”) or in the absence of IL-21 (“IL-21+ / -”) for expansion growth. Such a second expansion growth process may be referred to as the “second culture step,” “restimulation step,” “second co-culture step,” or “second expansion growth step.” The cells may be collected on day 17 or approximately day 17 of culture (or at any point between days 16–21 or 9–25). In some embodiments, further re-expansion growth or restimulation steps or cycles may be performed. As shown in Figure 14B, in some embodiments, there may be a first stimulation followed by two or more restimulation steps.

[0053] In some embodiments, CD56+ cells or CD3- / CD56+ cells are expanded by co-culturing them with feeder cells in the absence of IL-21. Typically, this is applied in a culture step following an initial culture step with IL-21. The cells may be any type of feeder cells for NK cells, including, for example, Jurkat cells and EBV-LCL cells ("Type 1"), K562 cells ("Type 2"), or PBMCs ("Type 3"). In some embodiments, cells are collected on day 17 or about day 17 (or day 16–21) of culture, and the produced cells may be referred to herein as "IL21-." In some embodiments, cells are collected on day 14 or about day 14 (or day 14–18) and subjected to cryopreservation. The cryopreserved cells may be thawed and co-cultured again with feeder cells in the presence of IL-21 ("IL-21- / +") or in the absence of IL-21 ("IL-21- / -"). Such a second expansion process may be referred to as the "second culture process" or "restimulation process." Cells can be collected on day 17 or approximately day 17 (or days 16–21 or 9–25) of culture. As detailed herein, the use of IL-21 in the first expansion allows for the freezing and thawing of NK cells, as well as subsequent superior expansion with optional additional IL-21 (which has further benefits, such as improved cytotoxicity).

[0054] First culture (expansion and growth or co-culture) step The first culture step may include adding cytokines once or multiple times between days 0 and 6 of the culture. More than one cytokine may be used (for example, IL-2 may also be used). For example, the first culture step may include adding one or both cytokines once on days 0 and 3 of the culture, respectively.

[0055] When co-cultured with feeder cells and a first cytokine, culture with one or more additional additions of another cytokine between days 0 and 6 may exhibit superior proliferation and / or anticancer activity. In some embodiments, culture with feeder cells and additional cytokines for 6 days during a 14-day cycle may exhibit superior proliferation and / or anticancer activity. In some embodiments, IL-21 is used at least once, and optionally before and after freezing. In some embodiments, IL-2 may be included as an additional cytokine.

[0056] In some embodiments, the first cytokine (e.g., IL-21) may be used at concentrations of 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100, or 10-50 ng / mL. In some embodiments, additional 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, or 450-550 IU / mL. In some embodiments, the concentration is approximately 50 ng / mL.

[0057] Conventional methods for proliferating NK cells utilize high concentrations of various cytokines. Conversely, in some embodiments of the methods for proliferating NK cells described herein, NK cells with high yield and high purity can be proliferated using only a single cytokine at a low concentration.

[0058] In some embodiments, co-culture (culturing, expansion (including re-expansion)) may be carried out by including peripheral blood mononuclear cells and feeder cells (e.g., Jurkat cells and EBV-LCL cells) in mixed ratios 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. In some embodiments, co-culture may be carried out by including peripheral blood mononuclear cells and feeder cells (e.g., Jurkat cells and EBV-LCL cells) in various mixed ratios. In some embodiments, the ratio may be about 1:0.5:0.5-1:10:10 for PBMCs (e.g., as a substitute for CD56+). In some embodiments, for CD56+ cells, the ratio is multiplied by, for example, 10 or 20 (e.g., 1:1 to 100 for CD56+ cells and feeder cells).

[0059] Co-culturing can be carried out in a culture medium, and any suitable medium commonly used in the art for the induction and proliferation of peripheral blood mononuclear cells into NK cells may be used as such a medium without limitation. For example, RPMI-1640, DMEM, x-vivo10, x-vivo20, or cellgro SCGM medium may be used as such a medium. In addition, culture conditions, such as temperature, may follow any suitable culture conditions for peripheral blood mononuclear cells known in the art.

[0060] In some embodiments, the first culture step may be carried out over 0-45, 0-42, 0-40, 0-30, 0-20, 0-19, 0-18, 0-17, 0-16, 0-15, or 0-14 days.

[0061] Freezing process The cultured and provided natural killer cells from the first culture step can be collected and suspended in a culture medium, and subsequently frozen and cryopreserved. In some embodiments, the culture medium may contain FBS and / or DMSO. For example, the medium may contain 90% FBS and 10% DMSO, or 90-95% FBS and 5-10% DMSO. In some embodiments, other acceptable cryopreserving agents, such as CryoStor solutions (CS10, CS5), or other components, such as sucrose or glycerol, may be included. In some embodiments, preferred preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, or polyvinylpyrrolidone. In some embodiments, IL-2 and / or human serum albumin may be present.

[0062] In some embodiments, cryopreservation may involve transferring the provided natural killer cells to a cryopreservation container containing isopropyl alcohol, freezing the natural killer cells in the cryopreservation container overnight in a cryogenic refrigerator, and storing the natural killer cells at -192°C or lower. In some embodiments, the frozen natural killer cells may be frozen and stored at -10°C or lower, -20°C or lower, -50°C or lower, -70°C or lower, -100°C or lower, -150°C or lower, -192°C or lower, or -200°C or lower. In some embodiments, the frozen natural killer cells may be stored for 1 day or longer, 2 days or longer, 3 days or longer, 7 days or longer, 14 days or longer, 30 days or longer, 60 days or longer, or 180 days or longer (including any range between any two of the preceding values). In some embodiments, the temperature is -135°C to -196°C. In some embodiments, the cells are stored for 0.5, 1, 2, 3, 4, or 5 years (including any range between any two of the preceding values).

[0063] In some embodiments, the natural killer cells provided may be cooled and / or frozen using a controlled-rate freezer (CRF). In some embodiments, the frozen natural killer cells may be stored under liquid nitrogen.

[0064] In some embodiments, the natural killer cells provided may be cooled and / or frozen using a rate-controlled cryotherapy (CRF). In some embodiments, this slow This can be done at a rapid rate (e.g., 1-8 hours, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 hours or longer). It can also be manually frozen slowly using isopropyl alcohol, in which case the vial of NK cells is placed in a cryo-container (e.g., Nalgene Mr. Frosty) and stored overnight at -70°C. The next day, the cells are transferred to liquid nitrogen (LN2).

[0065] Thawing process Frozen natural killer cells can be thawed after cryopreservation using any preferred method. In some embodiments, frozen / cryopreserved natural killer cells can be thawed using a water bath, for example at 37°C. In some embodiments, frozen natural killer cells can be thawed over 1 hour or longer, 2 hours or longer, 5 hours or longer, or 10 hours. In some embodiments, the process is carried out in water or a bead bath. In some embodiments, the thawing process is carried out as quickly as possible or immediately after removal from the frozen state (e.g., liquid nitrogen).

[0066] In some embodiments, frozen natural killer cells can be thawed in a 37°C water bath within 10 minutes, in which case the frozen vial or bag may be shaken to accelerate the thawing process. In some embodiments, the cells may also be thawed using equipment such as a heat block, an automated cell thawing device for vials (e.g., ThawStar, Biocision), or a thawing device for bags (e.g., VIA Thaw, GE Healthcare).

[0067] Second culture (second co-culture, second expansion growth, or re-expansion growth, or any subsequent culture step) The cells are first treated with IL-21 in a first culture step, which then enables a second expansion growth step. Therefore, the method may include not just one expansion growth step, but two expansion growth steps (e.g., one or more re-expansion growth steps). Preferably, the second expansion growth step is performed after the sample has been frozen, stored for some period of time, and then thawed.

[0068] During the second culture step, the thawed natural killer cells can be cultured with one or more feeder cell additions.

[0069] In some embodiments, feeder cells may be added once or multiple times during a 14-day culture cycle (or a 9-25 day cycle).

[0070] In some embodiments, culture with one or more feeder cell additions during a 14-day cycle can not only exhibit excellent proliferation and / or anticancer activity after freezing and thawing, but can also maintain sustained cell proliferation, resulting in the production of natural killer cells in sufficient quantities for clinical use.

[0071] In some embodiments, the second culture step may include adding a second round of cytokines (e.g., additional IL-21 or IL-21 plus other cytokines).

[0072] In some embodiments, the second culture step may include adding subsequent rounds of cytokines one or more times during days 0 to 6 of the culture.

[0073] Any other description of cytokines in this specification is for the second culture step. It can be applied to itokines. For example, in some embodiments, the second cytokine may be used at concentrations of 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100, or 10-50 ng / mL, and / or additional 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, or 450-550 IU / mL. The cytokine used for the second expansion and proliferation is preferably IL-21.

[0074] In some embodiments, the composition is a composition of frozen cells before regrowth and may comprise IL-2; 5-10% DMSO; 90-95% FBS; and optionally NK cells, which are CD56+ cells. In some embodiments, the composition is a frozen solid. In some embodiments, NK cells constitute at least 90% of the cell population of the composition. In some embodiments, it further comprises a CryoStor solution. In some embodiments, the composition is for frozen cells before regrowth.

[0075] In some embodiments, the composition is for frozen cells before regrowth and may comprise IL-2; 5-10% DMSO; 80-95% Hartman solution; 1-10% human serum albumin; and NK cells. In some embodiments, it further comprises CryoStor solution. In some embodiments, the composition is for frozen cells before injection.

[0076] In some embodiments, a method for expanding and growing natural killer cells in culture may include: isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 for a first period; freezing the co-culturized CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 for a second period.

[0077] In some embodiments, the method may further include storing frozen CD56+ cells at a temperature below -100°C. In some embodiments, frozen CD56+ cells may be stored at -10°C or lower, -20°C or lower, -50°C or lower, -70°C or lower, -150°C or lower, -192°C or lower, or -200°C or lower. In some embodiments, frozen CD56+ cells may be stored for longer than one day before thawing. In some embodiments, frozen CD56+ cells may be stored for two days or longer, three days or longer, seven days or longer, fourteen days or longer, thirty days or longer, sixty days or longer, or 180 days or longer (including any range between any two of the preceding values). In some embodiments, cells may be frozen for as long as they are viable when thawed.

[0078] In some embodiments, isolated CD56+ cells may be co-cultured for 13–16 days before freezing. For example, isolated CD56+ cells may be co-cultured for 14 or 15 days before freezing. In some embodiments, co-culture or expansion growth may be carried out for any appropriate time. In some embodiments, co-culture or expansion growth (including re-expansion growth) may be carried out for 9–25 days, e.g., 10–24, 11–23, 13–22, 14–21, 14–18, 14–16 days, etc. These timeframes may apply to any expansion growth and / or re-expansion growth periods provided herein (including embodiments relating to other cells).

[0079] In some embodiments, isolated CD56+ cells may be co-cultured with one or more irradiated feeder cells in the presence of IL-21. In some embodiments, thawed CD56+ cells may be co-cultured with one or more irradiated feeder cells in the presence of IL-21. The one or more feeder cells may include, but are not limited to, one or more selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cells (EBV-LCL), K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and / or PBMCs. In some embodiments, expansion is carried out using PBMCs, CD56+, and / or CD56+CD3- cells. In some embodiments, CD56+ cells may be co-cultured in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. For example, CD56+ cells can be co-cultured with feeder cells in ratios of approximately 1:2, 1:5, 1:10, 1:30, or 1:100.

[0080] In some embodiments, IL-21 may be added during the first and / or second period at a concentration of 10–100 ng / mL. For example, IL-21 may be added during the first and / or second period at concentrations of 20–80 ng / mL, 30–70 ng / mL, or 40–60 ng / mL. In some embodiments, IL-21 may be added more than once during the first and / or second period.

[0081] In some embodiments, a method for expanding and growing natural killer cells in culture may include: isolating CD56+ from a blood sample; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and expanding and growing the thawed CD56+ cells.

[0082] In some embodiments, CD56+ cells may be frozen at temperatures below -100°C. In some embodiments, CD56+ cells may be frozen at -10°C or lower, -20°C or lower, -50°C or lower, -70°C or lower, -150°C or lower, -192°C or lower, or -200°C or lower. In some embodiments, frozen CD56+ cells may be stored for longer than one day before thawing. In some embodiments, frozen CD56+ cells may be stored for 2 days or longer, 3 days or longer, 7 days or longer, 14 days or longer, 30 days or longer, 60 days or longer, or 180 days or longer (including any range between any two of the preceding values). For example, frozen CD56+ cells may be stored for a period longer than one day and shorter than 10 years.

[0083] In some embodiments, CD56+ cells may be co-cultured for 13–16 days before freezing. For example, CD56+ cells may be co-cultured for 14 or 15 days before freezing. In some embodiments, co-culture or expansion growth (including re-expansion growth) may be carried out over 9–25 days, for example, 10–24, 11–23, 13–22, 14–21, 14–18, 14–16 days, etc. These timeframes may apply to any expansion growth and / or re-expansion growth periods provided herein (including embodiments relating to other cells).

[0084] In some embodiments, one or more feeder cells may be selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cells (EBV-LCL), K562 cells, and PBMCs. CD56+ cells are present in a ratio of approximately 1:1 to 100 CD56+ cells and They can be co-cultured in ratios with feeder cells. For example, CD56+ cells can be co-cultured in ratios of approximately 1:2, 1:5, 1:10, 1:30, or 1:100 CD56+ cells to feeder cells.

[0085] In some embodiments, IL-21 may be added at a concentration of 10–100 ng / mL. For example, IL-21 may be added at concentrations of 20–80 ng / mL, 30–70 ng / mL, or 40–60 ng / mL. In some embodiments, IL-21 may be added more than once.

[0086] In some embodiments, a method for increasing the cytotoxicity of natural killer cells may include providing the natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21.

[0087] In some embodiments, the method may further include storing frozen natural killer cells at a temperature below -100°C. In some embodiments, frozen natural killer cells may be stored at -10°C or lower, -20°C or lower, -50°C or lower, -70°C or lower, -150°C or lower, -192°C or lower, or -200°C or lower. In some embodiments, frozen natural killer cells may be stored for longer than one day before thawing. In some embodiments, frozen natural killer cells may be stored for two days or longer, three days or longer, seven days or longer, fourteen days or longer, thirty days or longer, sixty days or longer, or 180 days or longer (including any range between any two of the preceding values). In some embodiments, the cells are stored for as long as any of the cells remain viable upon thawing.

[0088] In some embodiments, one or more feeder cells may be selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cells (EBV-LCL), K562 cells, and PBMCs. In some embodiments, thawed natural killer cells may be co-cultured in a ratio of approximately 1:1 to 100 natural killer cells to feeder cells.

[0089] In some embodiments, IL-21 may be added at a concentration of 10–100 ng / mL. For example, IL-21 may be added at concentrations of 20–80 ng / mL, 30–70 ng / mL, or 40–60 ng / mL. In some embodiments, IL-21 may be added more than once.

[0090] In some embodiments, a method for producing natural killer cells may include repeating the following steps: a freezing step; a thawing step; and a second culture step, which includes co-culture with the addition of feeder cells.

[0091] In some embodiments, one or more cycles of restimulation or re-expansion growth may be applied, as shown in Figure 14A (time points of the bars above the data curve). In some embodiments, a first stimulation, followed by cell culture, followed by freezing (optionally), followed by a second stimulation (restimulation), followed by a second cell culture step, followed by freezing (optionally), followed by a third stimulation (second restimulation), followed by another culture step. IL-21 may be used in each of the stimulation steps as provided herein. The optional freezing step may be applied to whole cells or fractions of cells. In some embodiments, two, three, four, five, six, seven, eight, nine, ten or more rounds of stimulation are performed (e.g., one round of stimulation followed by one, two, three, four, five, six, seven, eight, nine or more rounds of restimulation). Another cell culture / expansion growth step may be performed after each restimulation. In some embodiments, each of the cell culture or re-expansion growth may be performed over a period of nine to 25 days. A freezing step may be performed following each cell culture step. In some embodiments, the process may be a cycle of a) stimulation (or restimulation), followed by b) cell culture, followed by c) freezing (optionally), followed by d) thawing (optionally), which may be repeated as many times as desired. In some embodiments, the amount of IL-21 is 10 to 100 ng / mL, for example, 50 ng / mL. Stimulation / restimulation in Figure 14A represents the addition of IL-21 to the cells.

[0092] In some embodiments, any process provided herein may include one or more freezing steps.

[0093] In some embodiments, any embodiment relating to NK cells provided herein may include genetically modified NK cells in addition to natural NK cells.

[0094] Cell therapy compositions for treating cancer According to some embodiments, a cell therapy composition for treating cancer may comprise peripheral blood-derived CD56+ NK cells. The cells are, or are the result of, at least two rounds of expansion, the first of which is carried out in the presence of IL-21.

[0095] In this specification, the term “peripheral blood-derived” may mean that the cells are derived from “whole peripheral blood” or “leukocytes isolated from peripheral blood using leukocyte apheresis.” Peripheral blood-derived CD56+ NK cells may be used interchangeably with peripheral blood mononuclear cell (PBMC)-derived CD56+ NK cells.

[0096] In some embodiments, cytokines may be used at concentrations of 18-180,000, 20-100,000, 50-50,000, 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, and 450-550 IU / mL. When cytokines are used within these ranges, they may suppress apoptosis of NK cells contained in cancer treatment compositions and increase the anticancer activity of NK cells.

[0097] In some embodiments, the composition may include IL-2 as an additional cytokine (for example, in addition to IL-21).

[0098] In some embodiments, CD56+ NK cells may be obtained as described elsewhere in this specification. 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 higher, 90% or higher, 95% or higher, or 98% or higher.

[0099] In some embodiments, the cancer may be, but is not limited to, hematological cancers, gastric cancer, pancreatic cancer, cholangiocarcinoma, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, or neuroblastoma. In some embodiments, the method may be applied to allogeneic NK cell therapy, for example, in neurodegenerative diseases and acute infections.

[0100] 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 lower.

[0101] In this specification, the term "T cell" refers to lymphocytes derived from the thymus that can "remember" previously encountered antigens and provide information to B cells, thereby stimulating antibody production and playing a crucial role in the cellular immune system. These T cells can distinguish very small differences between different antigens and induce an immune response to allogeneic antigens, making them suitable for autotherapy, although their use for allogeneic therapy may be limited. Therefore, cell therapy compositions that do not contain T cells may be suitable for allogeneic transplantation.

[0102] In this specification, the term “cell therapy” refers to pharmaceuticals used for treatment, diagnosis, and prevention through a series of actions, such as the proliferation and screening of autologous, allogeneic, and heterologous living cells in vitro to restore the function of cells and tissues or otherwise alter the biological characteristics of cells. Cell therapy has been regulated as a medical product in the United States since 1993 and in South Korea since 2002. These cell therapy agents can be broadly classified into two areas: firstly, stem cell therapy agents for tissue regeneration or restoration of organ function; and secondly, immunotherapy agents for modulating the immune response, such as inhibiting or enhancing the immune response in vivo.

[0103] The administration route of the cell therapy compositions described herein may be any preferred route, as long as the composition reaches the target tissue. Administration may be, but is not limited to, parenteral administration, such as intraperitoneal, intravenous, intramuscular, subcutaneous, or intradermal administration.

[0104] The cell therapy compositions described herein may be formulated in suitable forms with pharmaceutically acceptable carriers suitable for or commonly used for cell therapy. “pharmaceutically acceptable” means a composition that, when administered to the human body, is physiologically acceptable and does not generally cause allergic reactions, such as gastrointestinal disorders or dizziness, or similar reactions. pharmaceutically acceptable carriers may include, for example, parenteral carriers such as water, suitable oils, saline solutions, and aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, sucrose, or albumin. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, or polyvinylpyrrolidone.

[0105] Cell therapy compositions may also be administered by any device that allows the cell therapy agent to move to target cells.

[0106] A cell therapy composition may contain a therapeutically effective amount of a cell therapy agent for the treatment of a disease. The term "therapeutically effective amount" means the amount of the active ingredient or cell therapy composition that induces a biological or medical response in a tissue system, animal, or human, as considered by researchers, veterinarians, physicians, or other clinicians, and includes the amount that induces relief of the symptoms of the disease or disorder to be treated. It will be apparent to those skilled in the art that the amount of cell therapy agent contained in a cell therapy composition may vary depending on the desired effect. Therefore, the optimal content of the cell therapy agent can be readily determined by those skilled in the art and may be adjusted according to various factors, such as the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, as well as the patient's age, weight, overall health, sex, and diet, administration time, route of administration, secretion ratio of the composition, duration of treatment, and drugs used concurrently. By considering all factors, it is possible to include an amount that has the ability to achieve the maximum effect with the minimum amount without side effects. This is important. For example, a cell therapy composition is 1 × 10 6 ~5×10 8 May contain cell-based therapeutic agents at a concentration of cells / kg body weight.

[0107] In some embodiments, NK cells of the cell therapy composition may have cytotoxicity of 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 93% or higher, 95% or higher, or 98% or higher compared to their pre-freezing population.

[0108] In some embodiments, the composition may contain an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient. CD56+ cells can be prepared by isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating CD56+ cells from the PBMCs; co-culturing the CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines.

[0109] In some embodiments, the effective amount of CD56+ cells is 1 × 10⁶ 6 ~5×10 8 It can be cells / kg body weight.

[0110] In some embodiments, the cell composition may contain effective amounts of CD56+ cells, IL-2, and IL-21 derived from peripheral blood mononuclear cells (PBMCs) from a patient.

[0111] In some embodiments, the composition may comprise a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; and IL-2 and IL-21. When thawed, the CD56+ cells have at least 80% of the cytotoxicity of a second population of CD56+ cells, the second population of CD56+ cells being unfrozen. In some embodiments, the cytotoxicity is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0112] In some embodiments, when comparing expansion without freezing (with and without IL21, IL21+ or IL21-) with expansion with freezing and co-culture with IL21+ in the first step, the average cytotoxicity of expansion with freezing was 97.7% of that without freezing (Table C: range of 83% to 131%). In some embodiments, when comparing expansion without freezing (with and without IL21, IL21+ or IL21-) with expansion with freezing and without co-culture with IL21+ in the first step (IL21--, IL21-+), the average cytotoxicity of expansion with freezing could be at least 81.4% of that with expansion without freezing (Table D, range of 61% to 83%). When IL21 was added in both steps of expansion with freezing (IL21++), the average cytotoxicity was 114% of that with expansion without freezing (Table C, values ​​of 98% to 131%). Therefore, in some embodiments, IL21+ / + (72%) versus IL21- / + (45%) indicates that if IL21 is present in the first expansion and also in the second step, the cytotoxicity is 60% higher than if IL21 were absent in the first step. Therefore, in some embodiments, the presence of IL21 in the first expansion enables a second, post-freezing expansion that is 60% higher. Regarding IL21+ / - (62%) versus IL21- / - (46.1%), this means that if IL21 is present in the first expansion but not in the second step, the cytotoxicity can be at least 35% higher than if IL21 were absent in the first step.

[0113] In some embodiments, cytotoxicity is measured as a ratio between expanded growth without freezing (with and without IL21) and expanded growth with freezing and co-culture with IL21+ in the first step. It is a comparison, and the average cytotoxicity of expansion and growth with freezing is 97.7% of that without freezing. In some embodiments, the cytotoxicity is a comparison between expansion and growth without freezing (with and without IL21) and expansion and growth without co-culture with IL21+ in the first step with freezing, and the average cytotoxicity of expansion and growth with freezing is 81.4% of that of expansion and growth without freezing. In some embodiments, IL21 is added both before and after freezing, and the average cytotoxicity is 114% of that of expansion and growth without freezing.

[0114] In some embodiments, the excellent properties of the initial IL21 treatment during co-expansion that allow subsequent re-expansion may be consistent with the results in Tables A - D below: [Table 1] [Table 2] [Table 3] [Table 4]

[0115] In any embodiment of culturing, expanding (including re-expanding) NK cells, the number of peripheral blood mononuclear cells in the culture at the start of co-culture ( culturing, expanding (including re-expanding)) is in the range of 1×10 4 ~1×10 15 cells. In some embodiments, the number of peripheral blood mononuclear cells in the culture at the start of co-culture is 1×10 4 ~5×10 4 cells, 5×10 4 ~1×10 5 cells, 1×10 5 ~5×10 5 cells, 5×10 5 ~1×10 6 cells, 1×106 ~1 × 10 7 Individual cells, 1 × 10 7 ~1 × 10 8 Individual cells, 1 × 10 8 ~1 × 10 9 Individual cells, 1 × 10 9 ~1 × 10 10 Individual cells, 1 × 10 11 ~1 × 10 12 Individual cells, 1 × 10 12 ~1 × 10 13 Individual cells, 1 × 10 13 ~1 × 10 14 individual cells, or 1 × 10⁶ 14 ~1 × 10 15 It is within the range of a few cells. In some embodiments, the number of peripheral blood mononuclear cells in the culture at the start of the first or initial expansion is 1 × 10⁶ 4 ~5×10 4 Individual cells, 5 x 10 4 ~1 × 10 5 Individual cells, 1 × 10 5 ~5×10 5 Individual cells, 5 x 10 5 ~1 × 10 6 Individual cells, 1 × 10 6 ~1 × 10 7 Individual cells, 1 × 10 7 ~1 × 10 8 Individual cells, 1 × 10 8 ~1 × 10 9 Individual cells, 1 × 10 9 ~1 × 10 10 Individual cells, 1 × 10 11 ~1 × 10 12 Individual cells, 1 × 10 12 ~1 × 10 13 Individual cells, 1 × 10 13 ~1 × 10 14 individual cells, or 1 × 10⁶ 14 ~1 × 10 15 This is within the range of a single cell. In some embodiments, the number of peripheral blood mononuclear cells in the culture at the start of re-expansion is 1 × 10⁶ 4 ~5×10 4 Individual cells, 5 x 10 4 ~1 × 10 5 Individual cells, 1 × 10 5 ~5×105 Individual cells, 5 x 10 5 ~1 × 10 6 Individual cells, 1 × 10 6 ~1 × 10 7 Individual cells, 1 × 10 7 ~1 × 10 8 Individual cells, 1 × 10 8 ~1 × 10 9 Individual cells, 1 × 10 9 ~1 × 10 10 Individual cells, 1 × 10 11 ~1 × 10 12 Individual cells, 1 × 10 12 ~1 × 10 13 Individual cells, 1 × 10 13 ~1 × 10 14 individual cells, or 1 × 10⁶ 14 ~1 × 10 15 This is within the range of a single cell. The method of the present invention may provide greater expansion and proliferation of NK cells than conventional approaches. Therefore, in any of the embodiments described above, the number of peripheral blood mononuclear cells in the culture at the start of co-culture (culturing, expansion and proliferation (including re-expansion and proliferation)) is a number of cells that would be difficult to use in conventional approaches to expand and proliferate NK cells (e.g., expand and proliferate without cytokines, e.g., IL-21 and / or IL-2) in order to provide a similar number of NK cells for therapeutic use and / or cryopreservation.

[0116] As disclosed herein, the methods of the present disclosure provide, in some embodiments, NK cells suitable for therapeutic use, for example, immunotherapy. In some embodiments, the methods of the present disclosure provide cryopreservation of NK cells that are effectively subsequently thawed and expanded for therapeutic use. Thus, in any embodiment for culturing or expanding (including refloating) NK cells, the NK cells are expanded to produce one population of expanded NK cells for therapeutic use and another population for cryopreservation. In some embodiments, the cryopreserved NK cells are later thawed and refloated for therapeutic use and / or further cryopreservation.

[0117] In any embodiment for culturing or expanding (including re-expanding) NK cells, the number of NK cells at the end of expansion or re-expanding is greater than the number of NK cells available for therapeutic use. In some embodiments, the excess NK cells are cryopreserved for future use, e.g., future thawing, expansion, and administration to patients in need. In some embodiments, NK cells are expanded or re-expanded to a degree where the number of cells is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or greater than the number of NK cells used or to be used for therapy, e.g., immunotherapy, by a percentage within the range of any two of the preceding values.

[0118] In any embodiment for culturing or expanding (including re-expanding) NK cells, the method may include repeating a freeze-thaw-expanding cycle. In some embodiments, the method may include repeating the freeze-thaw-expanding cycle 1, 2, 3, 4, 5, 6, 7, This includes repeating 8 or more times.

[0119] Compositions of cryopreserved NK cells are also provided herein, wherein the NK cells retain their biological activity, e.g., cytotoxicity, after thawing. In some embodiments, cryopreserved NK cells retain their biological activity, e.g., cytotoxicity, after thawing and regrowth. In some embodiments, the cryopreserved NK cell composition is prepared by any of the methods disclosed herein for culturing or regrowing (including regrowth) NK cells. In some embodiments, the composition comprises a population of immune cells that are at least 80%, 85%, 90%, 95%, 97%, or more NK cells. The composition may contain a suitable cryopreservation medium. In some embodiments, the composition comprises dimethyl sulfoxide (DMSO) and serum (e.g., FBS, human serum). In some embodiments, the composition comprises 1-15%, 2-15%, 5-15%, 5-10%, or about 10% DMSO. In some embodiments, the composition comprises or contains a population of cryopreserved immune cells comprising at least 90% NK cells, 10% DMSO, and 90% FBS. In some embodiments, NK cells are derived from PBMCs obtained from a subject. In some embodiments, the composition consists of or includes a population of cryopreserved immune cells containing at least 90% NK cells, 5-10% DMSO, and 90-95% FBS. In some embodiments, NK cells are derived from PBMCs obtained from a subject. In some embodiments, it further comprises a CryoStor solution.

[0120] Methods to prevent or treat cancer In some embodiments, a method for preventing or treating cancer is provided. The method involves administering an anti-cancer cell therapy composition comprising peripheral blood-derived CD56+ natural killer cells and cytokines to a target. In some embodiments, the cells are the result of two expansion processes, at least one of which is carried out in the presence of IL-21.

[0121] The term “subject” refers to a mammal, preferably a human, that is the subject of treatment, observation, or testing. A subject may be, but is not limited to, a patient with blood cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, or neuroblastoma.

[0122] In some embodiments, in adults, the cell therapy composition may be administered once or several times a day. The cell therapy composition may be administered daily or at intervals of 2 to 180 days. The cell therapy agent contained in the composition is 1 × 10⁶ per kg of body weight. 6 ~1 × 10 11 Individual peripheral blood-derived CD56+ natural killer cells, e.g., approximately 1 × 10⁶ 6 ~1 × 10 8 It may contain NK cells. 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 in the range of about 50 to 50,000 IU / ml.

[0123] In some embodiments, cell therapy compositions may be formulated in a suitable form with a pharmaceutically acceptable carrier suitable or commonly used for cell therapy. "pharmaceutically acceptable" means a composition that, when administered to the human body, is physiologically acceptable and does not generally cause allergic reactions, such as gastrointestinal disturbances or dizziness, or similar reactions. A pharmaceutically acceptable carrier may include, for example, parenteral carriers such as water, a suitable oil, saline solution, aqueous glucose, glycol, a basic compound such as Hartman solution, or a saline solution and an alternative such as plasmalyte A, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, sucrose, albumin, or human serum albumin. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, or polyvinylpyrrolidone.

[0124] In some embodiments, the cell therapy composition may be administered by any preferred method, such as rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal route. 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 normal human 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.

[0125] In some embodiments, the NK cells and cell therapy compositions comprising the NK cells disclosed herein may be used to treat diseases or conditions other than cancer. NK cells have been reported to play a crucial role in regulating the immune system, for example, by regulating T cells; therefore, cell therapy compositions containing NK cells may be administered to treat conditions associated with the immune system. For example, cell therapy compositions may be administered to treat neurodegenerative disorders (e.g., Alzheimer's disease and Parkinson's disease) or autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, spondyloarthritis, SLE, Sjögren's syndrome, systemic sclerosis).

[0126] In some embodiments, a method for treating a subject may include: collecting CD56+ cells from the subject; co-culturing the CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-culturated CD56+ cells for at least one day; thawing the frozen CD56+ cells; expanding the thawed CD56+ cells; and administering the expanded CD56+ cells to the subject, wherein the cytotoxicity of the cells from the second expansion is at least 80% (e.g., at least 80, 85, 90, 95, or 97%) of the cytotoxicity of the co-culturated CD56+ before freezing.

[0127] In some embodiments, the method may further include storing frozen CD56+ cells at a temperature below -100°C. In some embodiments, frozen CD56+ cells may be stored at -10°C or lower, -20°C or lower, -50°C or lower, -70°C or lower, -150°C or lower, -192°C or lower, or -200°C or lower. In some embodiments, frozen CD56+ cells may be stored for longer than one day before thawing. In some embodiments, frozen CD56+ cells may be stored for two days or longer, three days or longer, seven days or longer, fourteen days or longer, thirty days or longer, sixty days or longer, or 180 days or longer (including any range between any two of the preceding values).

[0128] In some embodiments, CD56+ cells may be co-cultured for 13–16 days before freezing. For example, CD56+ cells may be co-cultured for 14 or 15 days before freezing. In some embodiments, co-culture or expansion growth (including re-expansion growth) may be carried out over 9–25 days, for example, 10–24, 11–23, 13–22, 14–21, 14–18, 14–16 days, etc. These timeframes may apply to any expansion growth and / or re-expansion growth periods provided herein (including embodiments relating to other cells).

[0129] In some embodiments, thawed CD56+ cells are expanded and proliferated in the presence of IL-21 along with one or more irradiated feeder cells. This includes co-culturing. One or more feeder cells are selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cells (EBV-LCL), K562 cells, and PBMCs. In some embodiments, CD56+ cells may be co-culturned in a ratio of approximately 1:1 to 100 CD56+ cells to feeder cells. For example, CD56+ cells may be co-culturned in a ratio of approximately 1:2, 1:5, 1:10, 1:30, or 1:100 CD56+ cells to feeder cells.

[0130] In some embodiments, IL-21 may be added during the first and / or second period at a concentration of 10–100 ng / mL. For example, IL-21 may be added during the first and / or second period at concentrations of 20–80 ng / mL, 30–70 ng / mL, or 40–60 ng / mL. In some embodiments, IL-21 may be added more than once during the first and / or second period.

[0131] In some embodiments, IL-21 is human IL-21 for human NK cells.

[0132] Some embodiments provided herein include the following features and advantages: (a) A method for producing natural killer cells. (b) Natural killer cells can be produced in sufficient quantities for clinical use even after cryopreservation, making it possible to enhance the effects of cancer prevention and treatment, particularly allogeneic therapies using natural killer cells. (c) The cytotoxicity of cells resulting from two expansions (at least one with IL-21, and optionally a second expansion) is remarkably superior to that of cells that have undergone expansion without IL-21 (one and even more so with two). In some embodiments, the cells are in 1–50 mL cryovials or 10–100 mL cryobags. [Examples]

[0133] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting this disclosure to any specific features or embodiments described.

[0134] A comparative study of high-purity NK cell production methods was conducted using LCL+KL-1 type feeder cells with and without IL-21 treatment. Any other feeder cells discussed in this example are provided as prophetic examples.

[0135] The effectiveness of IL-21 in two manufacturing methods was examined: 1) the original method; this involved culturing isolated CD56+ cells with different types of feeder cells for 14–17 days under or without IL-21 treatment (without subsequent re-expansion); 2) the re-stimulation method; this involved culturing cryopreserved, expanded NK cells from the original method with different types of feeder cells under or without IL-21 treatment. Different types of feeder cells and manufacturing methods are shown in Table 1 and Figure 1, and are understood to be representative of common feeder cells for NK cell expansion. [Table 5]

[0136] The results provided by this embodiment are generally understood to be representative of the range of feeder cells, storage time, and additional components (e.g., additional cytokines) in the presence of Il-21, across a given ratio.

[0137] Example 1: Isolation of starting materials and original method Peripheral blood mononuclear cells (PBMCs) were obtained from human blood. Isolated PBMCs were used for CD56+ cell selection. PBMCs were isolated by density gradient centrifugation using Ficoll at 1.077 g / mL, washed several times with PBS, and resuspended with CD56 microbead reagent using AutoMACS Rinsing Solution (Miltenyi Biotec, Germany). CD56+ cells were selected using a magnetically activated cell sorting (MACS) system according to the manufacturer's instructions for use (Miltenyi Biotec, Germany). Selected CD56+ cells were resuspended in initial NK cell culture medium with or without 50 ng / mL of IL-21. The suspended cells were seeded into culture flasks after adding 100 Gy irradiated feeder cells, LCL+KL-1, K562, or PBMCs, and then cultured at 37°C and 5% CO2 for 6 or 7 days. Specific conditions for cell culture using each feeder type are shown in the table below. [Table 6]

[0138] On day 6 or 7 of culture, cells were collected from the culture flask by centrifugation and the cell count was assessed. The cells were resuspended in NK cell culture medium, seeded into culture bags, and then cultured at 37°C and 5% CO2 for 17 or 18 days. The cells were subcultured in fresh medium every 3 or 4 days.

[0139] Example 2: Cryopreservation of D14 cells On day 14 or 15 of culture, cells were harvested from the culture bag by centrifugation and the cell count was assessed. The cells were resuspended in a medium containing 90% FBS and 10% DMSO, with or without IL-2 at 500 IU / mL, and placed in vials in quantities of 5.0–10.0 × 10⁶. 6 The cells were aliquoted to a concentration of cells / mL, and then cryopreserved in a -196°C liquid nitrogen tank for 1 week, 1, 3, 6, 12, 24 months, and longer periods.

[0140] Example 3: Thawing and cell culture of frozen cells using the restimulation method Cells cryopreserved from the original method were thawed in a 37°C water bath and resuspended in initial NK cell culture medium with or without IL-21 at 50 ng / mL, containing supplements for each feeder cell condition. The suspended cells were seeded into culture flasks after adding 100 Gy irradiated feeder cells, LCL+KL-1, K562, or autologous PBMCs, and then cultured at 37°C and 5% CO2 for 6 or 7 days. This process was called the restimulation method. On day 6 or 7 of culture, cells were collected from the culture flask by centrifugation and the cell count was assessed. The cells were resuspended in NK cell culture medium, seeded into culture bags, and then cultured at 37°C and 5% CO2 for 17 or 18 days. Cells were subcultured in fresh medium every 3 or 4 days. The total cell culture period from the original method (D0) through the restimulation process to final harvest was 31–33 days.

[0141] Example 4: Population doubling level and cell proliferation NK cell cultures under six different conditions were performed using CD56+ cells based on the experimental designs shown in Figure 1 and Table 3. It shows the results for experimental design type 1, with NK cells co-cultured with irradiated LCL and KL-1 as feeder cells. The specific conditions for the process are as shown in Table 3. [Table 7]

[0142] The cell expansion and proliferation rate of NK cells was assessed by the expansion and proliferation ratio compared to the seeded cell number, and by the population doubling level (PDL) at each passage day, calculated as 3.32(logN - logNo) (where N is the number of cells at the end of each passage and No is the number of cells initially plated). The number of NK cells at every stage of culture was assessed by staining the cells with trypan blue.

[0143] We compared production batches from two different donors for PDL and expanded proliferation ratios. As shown in Tables 4, 5, and Figures 2A–3B, the PDL and expanded proliferation ratios of NK cells from one donor using the original method with IL-21 were shown to be higher than those without IL-21. However, batches from the other donor showed similar proliferation rates under both conditions. [Table 8] [Table 9]

[0144] Cryopreserved cells from the original method at D14 were restimulated with feeder cells by either IL-21 treatment or untreatment and cultured for 17 or 18 days. The total cell culture period from the original method at D0 through the restimulation process to final harvest was 31–33 days (shown in Figure 1).

[0145] Several production batches from two donors were compared for PDL and expanded proliferation ratios. As shown in Tables 6, 7, and Figures 4A–5B, the PDL and expanded proliferation ratios of NK cells in the restimulation method with IL-21 provided higher proliferation rates than under conditions without IL-21. Therefore, IL-21 is very useful to enable more effective subsequent expansion processes, particularly in the first round of expansion.

[0146] NK cells from donor 2 did not demonstrate a difference in proliferation rate between those with and without IL-21 using the original method, but the proliferation rate under IL-21 conditions using the restimulation method was On day 31, it was shown to be higher than the condition without IL-21. [Table 10] [Table 11]

[0147] Example 5: Purity of NK cells It is known that NK cells express CD56 and lack CD3. Flow cytometry analysis was applied to examine the purity of NK cells before expansion and after expansion by the original method and the restimulation method. NK cells were then subjected to anti-CD56-FITC and anti-CD3- The samples were stained with a fluorescently labeled antibody against PE, and then analyzed using flow cytometry.

[0148] As NK cell culture progressed under both conditions (with and without IL-21 treatment) using the original and restorative methods, the percentage of NK cells (CD56+CD3-) rapidly increased in expanded NK cells from two different donors, exceeding 99% at day 31 (Table 7). Cell surface markers for other cell types, such as CD3+, CD20+, and CD14+, were shown to be present in very low populations at the final culture stage (Table 8). [Table 12] [Table 13]

[0149] Example 6: Cytotoxic function of NK cells The cytotoxicity of NK cells against tumor target cell lines was evaluated by a fluorescence quantitative cytotoxicity assay. NK cells were co-cultured with K-562 cells stained with Calcein AM at E:T ratios of 10:1, 3:1, 1:1, and 0.5:1 for 4 hours under photoprotection. RPMI1640 containing 10% FBS or 2% triton X100 was added to target cells to induce spontaneous and maximal release. For the Calcein release assay, the supernatant after incubation of NK cells with target cells was collected, and its fluorescence was measured using a SpectraMax M2 microplate reader (Molecular devices, Sa Evaluate using n Jose (CA). Calculate the specific solubility percentage using the formula [(test release - spontaneous release) / (maximum release - spontaneous release)] × 100.

[0150] The cytotoxicity of NK cells cultured using the original method and the restimulation method was tested using a standard K-562 cell line, an NK-sensitive target. Cells expanded from the original method under conditions with and without IL-21 exhibited strong cytotoxic activity against K-562, even at low E:T ratios (1:1 and 0.5:1) (Figures 6-7). However, the cytotoxic activity of NK cells under the restimulation method showed different levels under different conditions with and without IL-21 treatment. NK cells treated with IL-21 under both the original method and the restimulation method exhibited stronger cytotoxic activity than those under the other conditions (Figures 8-11).

[0151] However, NK cells under the IL-21-free condition, both under the original and restimulated conditions, showed reduced cytotoxic activity with an E:T ratio of 0.05:1 to 3:1. Furthermore, they exhibited lower levels of cytotoxicity than those under the IL-21-treated condition at an E:T ratio of 10:1.

[0152] NK cells grown with IL-21 treatment exhibited very potent cytotoxicity against the K-562 cell line, and the cytotoxicity was similar in both NK cells produced by the original method and the restimulation method (Figures 6-11). Figure 6 shows the cytotoxic activity of NK cells grown with IL-21 (IL-21+) against K562 cells. Figure 7 shows the cytotoxic activity of NK cells grown without IL-21 (IL-21-) against K562 cells. Figure 8 shows the cytotoxic activity of NK cells grown with IL-21 and restimulated with IL-21 (IL-21+ / +) against K562 cells. Figure 9 shows the cytotoxic activity of NK cells grown with IL-21 and restimulated without IL-21 (IL-21+ / -) against K562 cells. Figure 10 shows the cytotoxic activity of NK cells (IL-21- / +) that were expanded and proliferated without IL-21 and then restimulated with IL-21 against K562 cells. Figure 11 shows the cytotoxic activity of NK cells (IL-21- / -) that were expanded and proliferated without IL-21 and then restimulated without IL-21 against K562 cells. Figure 12A shows a comparison of the phenotypes of NK cell activation receptors. Figure 12B shows a comparison of the phenotypes of NK cell inhibition and chemokine receptors.

[0153] Surface marker expression NK cell function is finely regulated by the balance between activating and inhibitory receptors expressed on their surface. To phenotypically characterize the expression levels of activating [CD16, NKp30, NKp46, NKp44, NKG2D, 2B4 (CD244), NKG2C, CRACC] or inhibitory NK receptors [NKG2A, KIR:CD158a (KIR2DL1), CD158b (KIR2DL2 / L3), CD158e (KIR3DL1)], chemokine receptors (CXCR3, CXCR4) or adhesion molecules (CD62L) were analyzed in gated CD56+ NK cells before expansion (day 0; D0) and after 17-18 days of expansion induced by the original (old) process and restimulation processes with IL-21 treatment. Surface receptor expression levels were calculated as the percentage of receptor-positive subsets of NK cells in the sample. NK cells at each stage of the original and restorative processes are stained with fluorescently labeled antibodies for each marker, and then analyzed using flow cytometry.

[0154] Surface receptor expression levels were analyzed for cells cultured from the initial and final stages (D0, D17, and D32) of a Type 1 experiment using IL-21 treatment, both in the original and restimulation processes, from four different donors. Surface receptor expression levels were calculated as the percentage of receptor-positive subsets of NK cells in the sample.

[0155] Among the activating receptors, the expression levels of CD16, NKp30, NKp46, NKp44, NKG2D, and CRACC increased during the expansion and proliferation of NK cells produced by the original process (old) and were similar to those of NK cells expanded and proliferated by the restimulation process (new), while the expression levels of NKG2C and 2B4 remained unchanged after culture expansion and proliferation by both methods (Figure 6). The expression of inhibitory receptors, CD158a and CD158e, remained generally unchanged in culture by both processes (old and new), but the proportions of NKG2A and CD158b increased significantly (Figure 12A-B). The expression levels of all inhibitory receptors analyzed were similar in NK cells produced by both methods. The expression of chemokine receptors, e.g., CXCR3 and CXCR4, was also evaluated.

[0156] The frequency of CXCR3+ NK cells significantly increased during NK cell expansion and proliferation by both methods (old and new), along with similar expression levels, while the frequency of CXCR4+ NK cells decreased after culture expansion and proliferation by both methods, with a slightly greater decrease in NK cells produced by the new method (Figure 12A-B). CD62L expression levels were slightly increased in NK cells expanded and proliferated by both methods.

[0157] As these results demonstrate, despite the fact that one set underwent a re-expansion procedure, the properties of cells expanded with IL21+ and IL21+ / + are similar.

[0158] Example 7: IL-21 concentration For various IL-21 concentrations, isolated CD56+ cells were resuspended in initial NK cell culture medium containing 10% FBS, 500 IU / mL IL-2, and 20 μg / mL gentamicin in RPMI medium with 10, 30, 50, and 100 ng / mL IL-21, using a Type 1 experimental design (IL-21+ only, no restimulation).

[0159] Example 8: IL-21 concentration Type 1 experiments were performed using feeder cells in different ratios: 1:10:10 (CD56+ cells:LCL:KL-1), 1:20:20, and 1:30:30 (IL21+ only, no restimulation).

[0160] Example 9 This non-limiting example demonstrates that IL-21 enhances the expansion and proliferation of CD56+ and CD3- / CD56+ NK cells with freeze-thaw cycles.

[0161] (1) Preparation of CD56+ natural killer cells (NK cells) - 1 First, blood PBMCs were isolated using the Ficoll density gradient (Ficoll-Hypaque density gradient method). The PBMCs were further processed according to 1-1 or 1-2 below.

[0162] 1-1. Isolation of CD56+ cells PBMCs are suspended in MACS buffer (1×PBS + 0.5% HSA), and CD56 microbeads (Miltenyi Biotec) are added in 1-20 μL / 1.0×10⁶ 7 Individual PBMCs were obtained and incubated at 2-8°C for 5-30 minutes. 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, the cells were resuspended with MACS buffer, and the cells were added to a MACS separator connected to a column. MACS buffer was passed through the column to remove nonspecific binding. The column was separated from the MACS separator, transferred to a 15 mL conical tube, and MACS buffer was added to isolate the CD56+ cells attached to the column.

[0163] 1-2. Isolation of CD3- / CD56+ cells CD3- / CD56+ cells were isolated as follows: PBMCs were suspended in MACS buffer (1×PBS + 0.5% HSA), and CD3 microbeads (Miltenyi Biotec) were added in 1-20 μL / 1.0×10⁶ solutions. 7Individual PBMCs were obtained and incubated at 2 - 8°C for 5 - 30 minutes. 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, MACS buffer was added to resuspend the cells, and the cells were added to a MACS separator connected to a column. MACS buffer was passed through the column to recover CD3− cells.

[0164] MACS buffer (1×PBS + 0.5% HSA) was added to the recovered CD3− cells to resuspend the CD3− cells, and CD56 microbeads (Miltenyi Biotec) were added at 1 - 20 μL / 1.0×10 7 Individual CD3− cells were obtained and incubated at 2 - 8°C for 5 - 30 minutes. 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, MACS buffer was added to resuspend the cells, and the cells were added to a MACS separator connected to a column. MACS buffer was passed through the column to remove non - specific binding. The column was separated from the MACS separator, transferred to a 15 - mL conical tube, and MACS buffer was added to isolate the CD3− / CD56+ cells attached to the column.

[0165] 1 - 3. Primary culture The CD56+ cells or CD3− / CD56+ cells separated from 1 - 1 and 1 - 2 were each co - cultured with feeder cells (Jurkat cells and EBV - LCL cells) previously prepared by irradiation with 100 Gy in an incubator at 37°C, 5% CO 2 in RPMI - 1640 medium containing 10% FBS in the presence of IL - 2 and IL - 21 at concentrations of 500 IU / mL and 50 ng / mL, respectively.

[0166] On day 6, the cells were inoculated at 1.0×10 5 ~2.0×10 6 / mL into a 350 - mL standard bag and cultured for an additional 4 days. On day 10, the cells were at 1.0×10 5 ~2.0×10 6Inoculated into a 1 L bag at cells / mL and cultured for an additional 4 days. At this time, the ratio (CD56+ cells or CD3- / CD56+ cells):(Jurkat cells):(EBV-LCL cells) is 1:30:30 during incubation.

[0167] 1-4. Secondary culture after freezing and thawing On the 14th day of Culture 1(1-3), the cultured cells were suspended in a solution containing 90% FBS and 10% DMSO, frozen and stored at -192 °C or lower temperature, and thawed in a constant temperature water bath at 37 °C according to the culture schedule.

[0168] Next, IL-2 and IL-21 at concentrations of 500 IU / mL and 50 ng / mL, respectively, were added to RPMI-1640 containing 10% FBS, together with feeder cells (Jurkat cells and EBV-LCL cells) irradiated with 100 Gy. After placing it in the medium, it was co-cultured in a 37 °C, 5% CO2 incubator.

[0169] On the 6th day after thawing and culturing, the cells were inoculated into a 350 mL bag at (1.0×10 5 ~2.0×10 6 / mL and incubated for an additional 4 days, and on the 10th day, the cells were inoculated into a 1 L standard bag at 1.0×10 5 ~2.0×10 6 cells / mL and cultured for an additional 4 days.

[0170] To sustain cell growth in the culture until the 14th day after thawing, the cells were co-cultured with feeder cells (Jurkat cells and EBV-LCL cells) irradiated with 100 Gy in the presence of IL-2 and IL-21 at concentrations of 500 IU / mL and 50 ng / mL, respectively. The cells were cultured in RPMI-1640 medium containing 10% added FBS in an incubator at 37 °C, 5% CO 2. 2 cultured.

[0171] On the 20th day after thawing, place the cells in a 1L bag in a 1.0 x 10 5 ~2.0×10 6 Inoculate cells at a concentration of 1 / mL, then culture for an additional 4 days. On day 24 of the culture after thawing, transfer the cells to a 1L bag in a 1.0 × 10⁶ container. 5 ~2.0×10 6 The cells were inoculated at a concentration of 100 cells / mL and cultured for an additional 4 days.

[0172] Finally, on the 28th day of culture after thawing, the cells were placed in a 1L bag in a 1.0 × 10⁶ size. 5 ~2.0×10 6 The cells were inoculated at a concentration of 100 cells / mL and subsequently cultured for 3–6 days. During this time, (CD56+ cells or CD3- / CD56+ cells):(Jurkat cells):(EBV-LCL cells) were cultured in a ratio of 1:30:30.

[0173] (2) Preparation of CD56+ natural killer cells - 2 Natural killer cells were prepared using the same method as in (1), except for the step of adding cytokines in steps 1-4.

[0174] (3) Comparative example. Preparation of natural killer cells without cytokine treatment step. Natural killer cells were prepared in the same manner as in (1), except for the step of adding cytokine (IL-21) in steps 1-3 and 1-4.

[0175] (4) Confirmation of NK cell proliferation ability The proliferative capacity of NK cells cultured by methods (1) to (3) was measured. As can be seen in Figure 13A, it was found that when cytokine treatment was not performed during primary culture (IL-21 - / -) (see (3) above), a sufficient number of NK cells for clinical application were not produced after the freeze-thaw process (Figure 13A). On the other hand, when cells were treated with cytokines (IL-21 + / +; see (1) above), a sufficient number of NK cells for clinical application were produced even after the freeze-thaw process, and these results were not limited to the case where cytokine treatment was performed after the freeze-thaw process. When cells were not treated with cytokines after the freeze-thaw process (IL-21 + / -; see (2) above), NK cells proliferated and expanded well, similar to the case where they were treated with cytokines after the freeze-thaw process (Figure 13B).

[0176] Example 10 Further results of some embodiments of various expansion and freezing and refreezing processes are shown in Figures 14A and 14B. Figure 14A shows the resulting PDL (Population Doubling Level) and depicts some embodiments of different periods during expansion. Figure 14B depicts the resulting expansion multiplication factor for the embodiments.

[0177] In this example, we analyze whether NK cells can be stimulated or proliferated multiple times after at least one freezing, and whether NK cell proliferation can be stopped and restarted rather than simply maintained. NK cells were initially cultured for 14 days. NK cells were treated twice with IL-21 (50 ng / mL) and feeder cells at 3-day intervals during the first 6-day period (days 0-6).

[0178] After this first 14-day expansion, the cells were frozen for 90 days, then thawed and cultured for another 14 days. The NK cells were again treated with IL-21 (50 ng / mL) and feeder cells twice, at 3-day intervals during the first 6-day period (days 0-6) of this second expansion.

[0179] The cells were then re-stimulated for a third time with IL-21 (50 ng / mL) and feeder cells, twice at 3-day intervals during the first 6 days (days 0-6) of this second expansion, and finally re-cultured for 18 days.

[0180] The expansion and proliferation of NK cells were monitored over a 46-day culture period. As shown in Figures 14A and 14B, NK cells exhibited significant expansion and proliferation after each stimulation, even after cell freezing, when treated twice or more with feeder cells and IL-21.

[0181] term The above description of exemplary embodiments is presented for illustrative and explanatory purposes only and is not intended to be exhaustive or to limit the invention to the exact forms disclosed. Many modifications and variations are possible in light of the above teachings. Various combinations or partial combinations of the distinctive features and aspects of the embodiments disclosed above may be made and are still expected to fall within one or more of the invention. Furthermore, any particular features, aspects, methods, characteristics, features, qualities, attributes, or elements disclosed herein in relation to the embodiments may be used in all other embodiments described herein. Thus, it should be understood that various features and aspects of the disclosed embodiments may be combined or substituted for each other to form various aspects of the disclosed invention. For this reason, the scope of the invention disclosed herein is not intended to be limited to the specific disclosed embodiments described above. Furthermore, various modifications and alternative forms of the invention are possible, specific examples of which are shown in the drawings and described in detail herein. However, it should be understood that the invention is not limited to any particular form or method disclosed, but rather, conversely, covers all modifications, equivalents, and alternatives that fall within the spirit and scope of the various embodiments and appended claims described herein. Any method disclosed herein does not have to be performed in the order described herein. The methods disclosed herein include certain actions performed by the practitioner, but they may also include any third-party instructions, either express or implicit, for such actions. The scope disclosed herein also includes any and all overlaps, partial scopes, and combinations thereof.

[0182] The embodiments have been selected and described to illustrate the principles of the invention and their practical applications so as to facilitate the use of the invention and various embodiments by those skilled in the art, and various modifications are envisioned to suit specific uses. Alternative embodiments of the invention defined by the appended claims, other than those described herein and the exemplary embodiments described therein, will be apparent to those skilled in the art.

[0183] Conditional expressions, such as "can," "could," "might," or "may," are generally intended to convey that a particular embodiment includes a particular feature, element, and / or process, while other embodiments do not, unless otherwise specifically stated or understood in the context in which they are used. Therefore, such conditional expressions are generally not intended to imply that the feature, element, and / or process is required in any way for one or more embodiments.

[0184] Terms such as "comprising," "including," and "having" are synonymous and are used in an inclusive, open-ended manner, not excluding additional elements, features, actions, and operations. The term "or" is also used in an inclusive (not exclusive) sense; therefore, for example, when used to connect a list of elements, "or" refers to one, some, or all of the elements in the list. It means "to".

[0185] The scope disclosed herein also includes any and all overlaps, partial scopes, and combinations thereof. Expressions such as “up to,” “at least,” “more than,” “less than,” and “between” include the numbers described.

[0186] Numbers preceded by terms such as “approximately,” “about,” and “substantially,” as used herein, include the stated number (e.g., approximately 10% = 10%) and also represent values ​​close to the stated quantity that still perform the desired function or achieve the desired result. For example, the terms “approximately,” “about,” and “substantially” may refer to quantities within 10%, 5%, 1%, 0.1%, and 0.01% of the stated quantity.

[0187] When used herein, the term “generally” refers to a value, quantity, or characteristic that primarily includes or tends to include a particular value, quantity, or characteristic. For example, in a particular embodiment, the term “generally uniform” refers to a value, quantity, or characteristic that deviates from exactly 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%.

[0188] The scope disclosed herein also includes any and all overlaps, partial scopes, and combinations thereof. Expressions such as “up to,” “at least,” “more than,” “less than,” and “between” include the number described. Numbers preceded by terms such as “about” or “approximately” include the number described. For example, “about 5.0 cm” includes “5.0 cm.”

[0189] Some embodiments are described in relation to schematic diagrams. However, it should be understood that the schematic diagrams are not drawn to scale. Distances are merely illustrative and do not necessarily have an accurate relationship to the actual dimensions and placement of the illustrated devices.

[0190] For the purposes of this disclosure, certain aspects, advantages, and novel features are described herein. It should be understood that not all such advantages can necessarily be achieved according to any particular embodiment. For example, those skilled in the art will recognize that this disclosure can be embodied or performed in a manner that achieves one advantage or group of advantages taught herein without necessarily achieving other advantages that can be taught or suggested herein.

[0191] Furthermore, while exemplary embodiments are described herein, the scope of any and all embodiments having equivalent elements, modifications, omissions, combinations (e.g., combinations of aspects across various embodiments), adaptations and / or changes will be recognized by those skilled in the art based on this disclosure. Limitations in the claims should be interpreted broadly based on the wording used in the claims and not limited to the examples described herein or during the filing process, and these examples should be interpreted as non-exclusive. Furthermore, the acts of the disclosed processes and methods may be modified in any way, including rearranging the acts and / or inserting additional acts and / or deleting acts. Accordingly, this specification and the examples should be considered exemplary only, and the true scope and spirit are intended to be indicated by the entire scope of the claims and their equivalents.

Claims

1. A method for expanding and proliferating natural killer cells in culture, Isolating CD56+ cells from a blood sample; The isolated CD56+ cells are co-cultured with one or more feeder cells in the presence of IL-21 for a first period of time; Freeze the cultured CD56+ cells after the first period; Thawing the frozen CD56+ cells; and A method comprising co-culturing the thawed CD56+ cells with one or more feeder cells for a second period in the presence of IL-21.

2. The method according to claim 1, further comprising storing the frozen CD56+ cells at a temperature lower than -100°C.

3. The method according to claim 1, further comprising storing the frozen CD56+ cells for more than one day before thawing.

4. The method according to claim 1, wherein the isolated CD56+ cells are cultured for 13 to 16 days before freezing.

5. The method according to claim 1, wherein the isolated CD56+ cells are co-cultured with one or more irradiated feeder cells in the presence of IL-21.

6. The method according to claim 5, wherein the thawed CD56+ cells are co-cultured with one or more irradiated feeder cells in the presence of IL-21.

7. The method according to claim 6, wherein one or more irradiated feeder cells are one or more selected from the group consisting of Jurkat cells, Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells, K562 cells, and PBMCs.

8. The CD56+ cells are such that the ratio of CD56+ cells to the feeder cells is 1:1 to 100. The method according to claim 1, wherein the culture is co-cultured within the range.

9. The method according to claim 8, wherein the CD56+ cells are co-cultured in a ratio of 1:2 CD56+ cells to feeder cells.

10. The method according to claim 8, wherein the CD56+ cells are co-cultured in a ratio of CD56+ cells to feeder cells of 1:5 to 1:

30.

11. The method according to claim 8, wherein the CD56+ cells are co-cultured in a ratio of 1:10 CD56+ cells to feeder cells.

12. The method according to claim 8, wherein the CD56+ cells are co-cultured in a ratio of 1:30 CD56+ cells to feeder cells.

13. The method according to claim 8, wherein the CD56+ cells are co-cultured in a ratio of 1:1 to 100 CD56+ cells to feeder cells.

14. The method according to any one of claims 1 to 13, wherein IL-21 is added at a concentration of 10 to 100 ng / mL during the first and / or second period.

15. The method according to claim 14, wherein IL-21 is added at a concentration of 20 to 80 ng / mL during the first and / or second period.

16. The method according to claim 14, wherein IL-21 is added at a concentration of 30 to 70 ng / mL during the first and / or second period.

17. The method according to any one of claims 1 to 13, wherein IL-21 is added more than once during the first and / or second period.

18. A method for expanding and proliferating natural killer cells in culture, (i) Isolating CD56+ cells from a blood sample; (ii) Co-culturing the CD56+ cells with one or more feeder cells in the presence of IL-2 and IL-21; (iii) Freeze the CD56+ cells after co-culturing as described in (ii); (iv) Thawing the frozen CD56+ cells; and (v) A method comprising co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of IL-2 and IL-21 to expand and grow the thawed CD56+ cells.

19. The method according to claim 18, wherein the CD56+ cells are frozen at a temperature lower than -100°C.

20. The method according to claim 18, further comprising storing the frozen CD56+ cells for a period of time longer than one day and shorter than ten years.

21. The method according to claim 18, wherein the CD56+ cells are co-cultured for 13 to 16 days before freezing.

22. One or more feeder cells irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) The method according to any one of claims 18 to 21, wherein one or more cells are selected from the group consisting of cells, K562 cells, and PBMCs.

23. The method according to claim 21, wherein the CD56+ cells are co-cultured in a ratio of CD56+ cells to feeder cells in the range of 1:1 to 100.

24. The method according to any one of claims 18 to 21, wherein IL-21 is added at a concentration of 10 to 100 ng / mL.

25. The method according to any one of claims 18 to 21, wherein IL-21 is added more than once.

26. A method for increasing the cytotoxicity of natural killer cells, To provide natural killer cells co-cultured with one or more feeder cells in the presence of IL-21; Freezing the aforementioned natural killer cells; Thawing the frozen natural killer cells; and The thawed natural killer cells are co-cultured with one or more feeder cells in the presence of IL-21. Methods that include...

27. The method according to claim 26, further comprising storing the frozen natural killer cells at a temperature lower than -100°C.

28. The method according to claim 26, further comprising storing the frozen natural killer cells for more than one day before thawing.

29. The method according to any one of claims 26 to 28, wherein one or more feeder cells are selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cells (EBV-LCL), K562 cells, and PBMCs.

30. The method according to any one of claims 26 to 28, wherein the thawed natural killer cells are co-cultured in a ratio of natural killer cells to feeder cells in the range of 1:1 to 1:

100.

31. The method according to any one of claims 26 to 28, wherein IL-21 is added at a concentration of 10 to 100 ng / mL.

32. The method according to any one of claims 26 to 28, wherein IL-21 is added more than once.

33. A method for preparing a pharmaceutical composition, (i) Isolating CD56+ cells from the target PBMC; (ii) Co-culturing the CD56+ cells with one or more irradiated feeder cells in the presence of IL-21 for a first period of time; (iii) Freeze the CD56+ cells co-cultured in (ii) above for at least one day; (iv) Thawing the frozen CD56+ cells; (v) The thawed CD56+ cells are expanded and proliferated for a second period, Expanding and growing thawed CD56+ cells includes co-culturing the thawed CD56+ cells with one or more irradiated feeder cells in the presence of IL-21; and A method comprising generating a pharmaceutical composition comprising the expanded and proliferated CD56+ cells for administration to the subject, wherein the cytotoxicity of the expanded and proliferated CD56+ cells is at least 80% of the cytotoxicity of the co-cultured CD56+ cells before freezing.

34. The method according to claim 33, further comprising storing the frozen CD56+ cells at a temperature lower than -100°C.

35. The method according to claim 33, further comprising storing the frozen CD56+ cells for more than one day before thawing.

36. The method according to claim 33, wherein the isolated CD56+ cells are co-cultured for 13 to 16 days before freezing.

37. The method according to claim 33, wherein one or more irradiated feeder cells include Jurkat cells and Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells.

38. The method according to any one of claims 33 to 37, wherein the CD56+ cells are co-cultured in a ratio of 1:1 to 100 CD56+ cells to feeder cells.

39. The method according to any one of claims 33 to 38, wherein IL-21 is added at a concentration of 10 to 100 ng / mL during the first and / or second period.

40. The method according to any one of claims 33 to 39, wherein IL-21 is added more than once during the first and / or second period.

41. A method for expanding and proliferating natural killer cells in culture, To provide PBMC; The PBMC is co-cultured with one or more feeder cells in the presence of IL-21 for a first period of time; Freeze the cultured PBMCs after the first period; Thawing the frozen PBMC; and A method comprising co-culturing the thawed PBMC with one or more feeder cells in the presence of IL-21 for a second period.

42. The method according to claim 41, wherein the ratio of PBMCs to feeder cells is 1:0.

5.

43. The method according to claim 41, further comprising storing the frozen PBMC at a temperature lower than -100°C.

44. The method according to claim 41, further comprising storing the frozen PBMC for more than one day before thawing.

45. The method according to claim 41, wherein the isolated PBMC is cultured for 13 to 16 days before freezing.

46. The isolated PBMC is subjected to one or more irradiated particles in the presence of IL-21. The method according to claim 41, wherein the cells are co-cultured with leader cells.

47. The method according to claim 46, wherein the thawed PBMC is co-cultured with one or more irradiated feeder cells in the presence of IL-21.

48. The method according to claim 47, wherein one or more feeder cells are one or more selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells, K562 cells, and PBMCs.

49. The method according to claim 48, wherein the PBMC is co-cultured in a ratio of PBMC to feeder cells in the range of 1:1 to 100.

50. The method according to claim 49, wherein the PBMCs are co-cultured in a ratio of 1:2 PBMCs to feeder cells.

51. The method according to claim 49, wherein the PBMCs are co-cultured in a ratio of 1:5 to 1:30 PBMCs to feeder cells.

52. The method according to claim 49, wherein the PBMCs are co-cultured in a ratio of 1:10 PBMCs to feeder cells.

53. The method according to claim 49, wherein the PBMC is co-cultured in a ratio of 1:30 PBMC to feeder cells.

54. The method according to any one of claims 41 to 53, wherein IL-21 is added at a concentration of 10 to 100 ng / mL during the first and / or second period.

55. The method according to claim 54, wherein IL-21 is added at a concentration of 20 to 80 ng / mL during the first and / or second period.

56. The method according to claim 54, wherein IL-21 is added at a concentration of 30 to 70 ng / mL during the first and / or second period.

57. The method according to any one of claims 41 to 53, wherein IL-21 is added more than once during the first and / or second period.

58. The method according to any one of claims 33 to 37, further comprising freezing in a solution prepared for injection.

59. The method according to any one of claims 33 to 37, wherein the cytotoxicity is a comparison between expanded growth without freezing (with and without IL-21) and expanded growth with freezing and co-cultured with IL-21+ in the first step, and the average cytotoxicity of expanded growth with freezing is at least 97.7% of that without freezing.

60. The method according to any one of claims 33 to 37, wherein the cytotoxicity is a comparison between expanded growth without freezing (with and without IL-21) and expanded growth with freezing and not co-cultured with IL-21+ in the first step, and the average cytotoxicity of expanded growth with freezing is at least 81.4% of that of expanded growth without freezing.

61. The method according to claim 33 or 34, wherein IL-21 is added both before and after freezing, and the average cytotoxicity is at least 114% of the expanded growth without freezing.

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