Tumor-sensitized natural killer cells and preparation method therefor

Abstract

The present invention relates to tumor-sensitized natural killer cells and a preparation method therefor. The tumor-sensitized natural killer cells and the preparation method therefor according to one aspect provide tumor-sensitized natural killer cells having enhanced tumor cell killing activity compared to conventional natural killer cells, and thus can be usefully employed as an anticancer immune cell therapeutic

Description

Tumor-sensitized natural killer cells and methods for producing the same

[0001] This invention relates to tumor-sensitized natural killer cells and a method for manufacturing the same.

[0002] Cancer accounts for the highest proportion of disease-related deaths worldwide and is an intractable disease in which more than 50% of patients eventually die, even when various treatments such as surgery, radiation therapy, and chemotherapy are performed in combination. In particular, for cancers that have metastasized, radiation therapy and chemotherapy are combined with surgery; however, these treatments have significant side effects on normal cells, and their effectiveness is extremely limited due to the multidrug resistance of cancer cells.

[0003] The development of immunotherapies is actively underway to overcome the limitations of these existing treatments. Immunotherapy is utilized in cancer treatment by enhancing the body's cellular immune function to activate the patient's immune system to attack cancer cells.

[0004] Among immune cells, Natural Killer (NK) cells are attracting attention as a fourth-generation anticancer treatment. As core cells of the innate immune system, NK cells possess tumor-suppressing capabilities that effectively inhibit cancer stem cells—which play a crucial role in the development, proliferation, and metastasis of cancer cells as well as cancer recurrence. Thanks to these characteristics, they offer many advantages over other immune cells as anticancer immunotherapies. Furthermore, unlike other immune cells, NK cells rarely cause immune rejection during allogeneic transplantation, making them highly promising as a safe immunotherapy.

[0005] However, NK cells have a short lifespan in the body and are rapidly inactivated by the immunosuppressive environment of cancer patients, which can lead to reduced efficacy. Therefore, for NK cells to be used as an effective immunotherapy, they must be capable of mass proliferation, maintain high anticancer ability, and effectively improve their survival rate in the body.

[0006] To solve these problems, the inventors developed a method for producing tumor-priming natural killer cells (Tumor Priming NK Cells) that maximize cytotoxicity and functional specificity by exposing NK cells to specific tumor antigens or the tumor microenvironment.

[0007] Tumor-sensitized natural killer cells produced by the method for producing tumor-sensitized natural killer cells according to the present invention exhibit a significantly enhanced cytotoxic effect against cancer cells and demonstrate excellent anticancer efficacy, thereby maximizing the therapeutic effect on cancer patients and serving as an effective anticancer immunotherapeutic agent that complements the limitations of existing treatments.

[0008] One aspect provides a method for producing tumor priming natural killer cells, comprising the steps of: (a) obtaining cord blood mononuclear cells (CBMC); and (b) co-culturing the obtained cord blood mononuclear cells and feeder cells in a medium containing cytokines.

[0009] Another aspect provides a composition for producing tumor-sensitized natural killer cells comprising IL-2, IL-15, and supporting cells irradiated with ascites cells of a cancer patient.

[0010] Another aspect is to provide tumor-sensitized natural killer cells or a population of such cells produced by the above-described method for producing tumor-sensitized natural killer cells.

[0011] Another aspect is to provide a composition for treating cancer comprising, as an active ingredient, tumor memory killer cells or a population thereof, prepared by the method for preparing tumor-sensitized natural killer cells described above.

[0012] Another aspect is to provide a cell therapeutic agent comprising, as an active ingredient, tumor memory killer cells or a population thereof, produced by the method for producing tumor-sensitized natural killer cells described above.

[0013] Another aspect is to provide a use of tumor-sensitized natural killer cells or a population of such cells produced by the above method for producing tumor-sensitized natural killer cells for use in the manufacture of pharmaceutical preparations for the prevention or treatment of cancer.

[0014] Another aspect provides a method for the prevention or treatment of cancer comprising the step of administering an effective amount of tumor memory killer cells or a population thereof, produced by the method for producing tumor-sensitized natural killer cells, to an individual in need thereof.

[0015] Another aspect is to provide a use of tumor memory killer cells or a population thereof produced by the above-described method for producing tumor-sensitized natural killer cells for preventing or treating cancer.

[0016] One aspect provides a method for producing tumor priming natural killer cells, comprising the steps of: (a) obtaining cord blood mononuclear cells (CBMC); and (b) co-culturing the obtained cord blood mononuclear cells and feeder cells in a medium containing cytokines.

[0017] In this specification, the term “Natural Killer Cell (NK cell)" refers to a cytotoxic lymphocyte belonging to the innate immune system that directly attacks and kills abnormal cells (e.g., cancer cells, virus-infected cells) without antigen-specific recognition. Natural killer cells have a CD56+CD3- phenotype, and their activation plays an important role in eliminating tumor cells or infected cells.

[0018] In this specification, the term “tumor priming NK cell” refers to a natural killer cell that is activated and primed by exposure to a specific tumor antigen or tumor microenvironment, and refers to an NK cell whose cytotoxic ability and tumor cell killing ability are increased through this process.

[0019] In one embodiment, the tumor-sensitized natural killer cells may be derived from any one selected from the group consisting of umbilical cord, cord blood, placenta, amniotic fluid, and amniotic membrane.

[0020] The above priming refers to a process in which immune cells are exposed to specific stimuli (e.g., tumor antigens, cytokines, drugs, etc.) to induce an activated state of the corresponding cells and enhance their functional characteristics (e.g., cytotoxicity, cytokine secretion ability). Through the priming process, NK cells become able to recognize and eliminate target cells more effectively.

[0021] In this specification, terms such as "manufactured cells," e.g., "manufactured tumor-sensitized natural killer cells" or "isolated activated lymphocytes," etc., refer to cells substantially manufactured or isolated from the tissue from which the cells originate, e.g., umbilical cord blood.

[0022] In one embodiment, the tumor-sensitized natural killer cells may be activated natural killer cells. The activated natural killer cells may refer to cells in which cytotoxicity, the inherent immunomodulatory ability of killer cells, or tumor memory ability is activated compared to progenitor cells, e.g., umbilical cord blood cells, or lymphocytes before activation.

[0023] In this specification, the term “positive or +” may mean, in relation to a cell marker, that the marker is present in a greater amount or at a higher concentration compared to another reference cell. That is, a cell is positive for a marker if the marker is present inside or on the surface of the cell and can be distinguished from one or more other cell types using that marker. It may also mean that the cell possesses the marker in an amount sufficient to produce a signal, for example, a signal from a cell measurement device, at a value greater than the background value.

[0024] In this specification, the term "cord blood mononuclear cell (CBMC)" refers to a cell having a spherical nucleus present in cord blood, and such cord blood mononuclear cells include immune cells such as B cells, T cells, macrophages, dendritic cells, and natural killer cells (NK cells).

[0025] In one embodiment, the cord blood mononuclear cells may be derived from any one selected from the group consisting of umbilical cord, cord blood, placenta, amniotic fluid, and amniotic membrane, and preferably may be derived from cord blood.

[0026] In this specification, the term "feeder cells" (also referred to as culture-assisted cells or feeder cells) refers to cells that do not have the ability to divide and proliferate, but possess metabolic activity and produce various metabolites to aid in the proliferation of activated lymphocytes. The feeder cells that may be used in the present invention include genetically modified animal cell lines, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, or one's own or another person's peripheral blood solid tumor cells, blood cancer cells, leukocyte cells, T cells, B cells, or monocytes. Preferably, they may be one's own solid tumor cells or one's own blood cancer cells, but are not limited thereto. Other feeder cells known to be ordinarily available in the technical field to which the present invention belongs may also be used without limitation if they are suitable for the purpose of the present invention. For example, the feeder cells may be solid tumor cells or blood cancer cells, and more specifically, the feeder cells may be derived from ascites cells of a cancer patient.

[0027] The term "ascites cells" in this specification refers to cells present in ascites fluid, which are cells isolated from body fluid accumulated mainly within the abdominal cavity and may consist of cancer cells, immune cells, inflammatory cells, and various other cells.

[0028] In one embodiment, the type of cancer having the cancer patient may be any one selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, stomach cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, lipoma, fibrocarcinoma, hematological cancer, lymphoma, and multiple myeloma, but is not limited thereto.

[0029] In one embodiment, the supporting cells may be selected from leukemia, blood cancer, lymphoma, or multiple myeloma, but are not limited thereto.

[0030] In one embodiment, the supporting cell may be irradiated with radiation. Specifically, the radiation may be gamma rays, and the preferred radiation dose may be 20 Gy to 200 Gy, more preferably 20 Gy to 100 Gy, and most preferably 50 Gy, but is not limited thereto.

[0031] In one embodiment, the co-culture may proceed in several stages to maximize the proliferation of the tumor-sensitized natural killer cells. At this time, the culture medium components, culture vessels, and culture periods may be the same or different in each culture stage, and the proliferation of natural killer cells can be ultimately maximized by setting the optimal culture period for each stage. For example, in the co-culture stage, a culture composition (or culture medium) and / or cytokines may be added to the flask at intervals of 2 to 3 days during the co-culture, and this serves to help the culture of natural killer cells to be carried out effectively.

[0032] In one embodiment, the co-culturing step may include an initial culture step and a post-initial culture step, wherein the initial culture step is performed for 1 to 10 days and the post-initial culture step is performed for 10 to 20 days. For example, the co-culturing step may include an initial culture step of 2 to 7 days and a post-initial culture step of 10 to 20 days.

[0033] In one embodiment, the composition of cytokines contained in the culture medium may be different between the initial culture stage and the subsequent culture stage.

[0034] The term "cytokine" in this specification refers to a general term for proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes, and macrophages, which play a role in regulating intercellular signaling, immune response regulation, inflammatory response induction, cell growth, and differentiation. Cytokines are classified according to their function into interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), chemokine, etc.

[0035] In one embodiment, the cytokine that may be included in the medium may be one or more selected from the interleukin class.

[0036] The term "interleukin" in this specification refers to a type of cytokine responsible for signal transduction primarily between immune cells, and means a proteinaceous bioactive substance produced mainly by T cells, B cells, monocytes, and macrophages, which is involved in the regulation of immune responses, cell proliferation, and differentiation.

[0037] The interleukins available for use in the present invention may be one or more selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and IL-23. For example, IL-2 or IL-2 and IL-15 may be used, and it is obvious to those skilled in the art that cytokines other than those mentioned above may also be utilized without limitation as long as they are suitable for the purpose of the present invention.

[0038] It has been reported that the above IL-2 has the function of promoting the proliferation and activation of mature NK cells. While it has been reported that the number of NK cells is significantly reduced in humans and mice deficient in IL-2, there are also research results indicating that IL-2 and IL-2Ra deficiency indirectly affects the number and activation of NK cells, and it is known that the IL-2R chain is involved in forming the IL-15 receptor.

[0039] Regarding the above IL-15, it has been revealed that NK cells are not found in mice deficient in IL-15 or IL-15Rα, and that NK cells are deficient in mice deficient in transcription factor interferon (IFN)-regulatory factor 1, which is required for IL-15 production. Thus, it is known that IL-15 is involved in NK cell differentiation and that IL-15 directly promotes the growth and differentiation of NK cells through IL15 receptors expressed in NK cells.

[0040] In one embodiment, the initial culture step may be co-cultured in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-18, and IL-21. For example, the initial culture step may include IL-2; IL-2 and IL-21; or IL-2, IL-21, and IL-18 in the medium, but is not limited thereto.

[0041] In one embodiment, the step after the initial culture may be co-culture in a medium containing IL-2 and IL-15.

[0042] In one embodiment, the co-culture step may involve co-culture of umbilical cord blood mononuclear cells and supporting cells in a ratio of 1:1 to 8:1.

[0043] In one embodiment, the ratio of umbilical cord blood mononuclear cells and supporting cells included in the medium may be different in the initial culture stage and the subsequent culture stage.

[0044] Specifically, the initial culture step may involve co-culturing cord blood mononuclear cells and supporting cells in a ratio of 5:1 to 8:1, and the subsequent culture step may involve co-culturing cord blood mononuclear cells and supporting cells in a ratio of 1:1 to 8:1, but is not limited thereto.

[0045] For example, the ratio of cord blood mononuclear cells to supporting cells in the initial stage of culture may be 5:1, 6:1, 7:1, or 8:1, and the ratio of cord blood mononuclear cells to supporting cells in the stage after the initial stage of culture may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1, but is not limited thereto.

[0046]

[0047] Another aspect provides a composition for producing tumor-sensitized natural killer cells comprising IL-2, IL-15, and supporting cells irradiated with multiple cells of a cancer patient.

[0048] In one embodiment, the cancer of the cancer patient may be a solid tumor or a blood cancer, and may be, for example, any one selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, stomach cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, lipoma, fibrocytic carcinoma, blood cancer, lymphoma, and multiple myeloma, but is not limited thereto.

[0049] In one embodiment, the concentration of IL-2 included in the composition may be 5 to 50 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, or 5 to 15 ng / ml, but is not limited thereto. Specifically, the IL-2 can promote the differentiation of umbilical cord blood mononuclear cells into tumor-sensitized natural killer cells.

[0050] In one embodiment, the concentration of IL-15 included in the composition may be 5 to 50 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, or 5 to 15 ng / ml, but is not limited thereto. Specifically, the IL-15 can promote the differentiation of umbilical cord blood mononuclear cells into tumor-sensitized natural killer cells.

[0051] In one embodiment, the ratio of IL-2 to IL-15 included in the composition may be 0.5 to 2, for example, 0.5:1, 1:1, 1:1.5 or 1:2, but is not limited thereto.

[0052] In one embodiment, the composition may include appropriate proteins, cytokines, antibodies, compounds, and other components, provided that the effect of producing tumor-sensitized natural killer cells is not impaired.

[0053] In one embodiment, the composition is 2.0 x 10 per 1 ml 5 Up to 2.0x10 6 It may include cells. Specifically, the number of tumor-sensitized natural killer cells included per 1 ml may be appropriately adjusted and modified by a person skilled in the art to prevent depletion of nutrients in the cell culture medium and to achieve maximum expansion and survival.

[0054] The definitions of natural killer cells, tumor sensitized natural killer cells, sensitization, cytokines, ascites cells, etc., are as previously stated.

[0055]

[0056] Another aspect provides tumor memory killer cells or a population of such cells produced by the above-described method for producing tumor sensitized natural killer cells.

[0057] In one embodiment, the tumor-sensitized natural killer cells cultured by the above method for producing tumor-sensitized natural killer cells may have increased cytotoxicity compared to ordinary natural killer cells.

[0058] In this specification, the term "general natural killer cell" refers to natural killer cells produced under standard culture conditions. Specifically, the general natural killer cell refers to natural killer cells (NK cells) produced using cord blood mononuclear cells (CBMC) and cancer cell lines that are standardly used in immune cell research and culture as supporting cells. The cancer cell lines that are standardly used include, but are not limited to, K562 (leukemia-derived cell line), Raji (Burkitt lymphoma-derived cell line), Daudi (Burkitt lymphoma-derived cell line), Jurkat (T-cell leukemia-derived cell line), HL-60 (acute myeloid leukemia-derived cell line), U937 (monocytic lymphoma-derived cell line), and THP-1 (monocyte-derived cell line).

[0059] Another aspect provides a pharmaceutical composition for treating cancer comprising, as an active ingredient, tumor memory killer cells or a population thereof, prepared by the method for preparing tumor-sensitized natural killer cells described above.

[0060] In one embodiment, the cancer may be selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, lipoma, fibrocytic carcinoma, hematological cancer, lymphoma, and multiple myeloma, but is not limited thereto.

[0061] In this specification, the term “treatment” refers to, or includes, the alleviation, inhibition of progression, or prevention of a disease, disorder, or condition, or one or more of its symptoms, and “active ingredient” or “pharmaceutical effective amount” may mean any amount of a composition used in the course of practicing the invention provided herein that is sufficient for the alleviation, inhibition of progression, or prevention of a disease, disorder, or condition, or one or more of its symptoms.

[0062] In one embodiment, the method of administration of the pharmaceutical composition is not particularly limited, but may be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation, or by topical application, or orally, depending on the intended method. The dosage varies depending on the patient's body weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dosage refers to the amount of therapeutic substance according to one embodiment that is sufficient for treating the disease state alleviated by administration to an individual requiring treatment. The effective amount of therapeutic substance depends on the specific compound, the disease state and its severity, and the individual requiring treatment, and can be ordinarily determined by a person skilled in the art. As a non-limiting example, the dosage of the composition according to one embodiment to the human body may vary depending on the patient's age, body weight, sex, form of administration, health status, and degree of disease. Based on an adult patient weighing 70 kg, for example, 1,000 to 10,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 10,000,000, 1,000 to 100,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, 1,000 to 10,000,000,000 cells / dose, or 1,000 to 100,000,000,000 cells / dose, may be administered in divided doses once or several times a day at regular intervals, or may be administered multiple times at regular intervals.

[0063] In this specification, the terms “administering,” “introducing,” and “implanting” are used interchangeably and may mean the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. The composition according to one embodiment may be administered by any suitable route that delivers at least a portion of the cells or cellular components to a desired location within a living individual. The viability of the cells after administration to the individual may be as short as a few hours, for example, from 24 hours to several days, or as long as several years.

[0064] The above administration may be administered in combination with additional anticancer agents or cytokine agents. Specifically, the additional anticancer agents may be, but are not limited to, alkylating agents, antimetabolites, spindle inhibitors, plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of the above anticancer agents may include compounds used in targeted therapy and conventional chemotherapy. In addition, examples of the above antibodies include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, vivatuzumab mertansine, cantuzumab mertansine, cedelizumab, sertolizumab pegol, sidfucituzumab, sidtuzumab, daclizumab, eculizumab, epalizumab, efratuzumab, erlizumab, felbizumab, pontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, rabetuzumab, lintuzumab, matuzumab, mepolizumab, motabizumab, motobizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, It may be peckfucituzumab, pectuzumab, pertuzumab, pexelizumab, ralibizumab, ranibizumab, reslibizumab, reslibizumab, resaibizumab, lovelizumab, luplizumab, cibrotuzumab, ciplizumab, sontuzumab, tacatuzumab, tetraxetane, tadosizumab, talizumab, tepivazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab, selmoleukin, tucucituzumab, umavizumab, urtoxazumab, and bicilizumab, but is not limited thereto.

[0065] A pharmaceutical composition according to one embodiment may include pharmaceutically acceptable carriers and / or additives. For example, it may include sterile water, physiological saline, a common buffer (phosphate, citric acid, other organic acids, etc.), a stabilizer, a salt, an antioxidant (ascorbic acid, etc.), a surfactant, a suspending agent, an isotonic agent, or a preservative. For topical administration, it may also include combinations with organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When a pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the immune cell, or the substance that increases its activity, may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0066] A pharmaceutical composition according to one embodiment may appropriately include, if necessary depending on the method of administration or dosage form, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, a reducing agent, an antioxidant, etc. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. A pharmaceutical composition according to one embodiment may be prepared in a unit dose form or contained in a multi-dose container by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granule, tablet, or capsule.

[0067]

[0068] Another aspect provides a cell therapy agent comprising tumor-sensitized natural killer cells or a population of such cells produced by the above method as an active ingredient.

[0069] In this specification, the term "cell therapy agent" refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions, such as proliferating or selecting living autologous, allogenic, or xenogenic cells in vitro or altering the biological characteristics of cells by other methods, in order to restore the function of cells and tissues.

[0070] In one embodiment, the cell therapy agent may be for the prevention or treatment of cancer.

[0071]

[0072] Another aspect provides a use for the above-mentioned tumor-sensitized natural killer cells or their cell populations in the manufacture of medicines for treating cancer.

[0073] Another aspect provides a method for treating cancer comprising the step of administering the tumor-sensitized natural killer cells or a population of such cells to an individual in need.

[0074] In this specification, the term "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0075]

[0076] Another aspect provides the use of tumor-sensitized natural killer cells or a population thereof, prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines for use in the manufacture of pharmaceutical preparations for the prevention or treatment of cancer.

[0077] Another aspect provides a method for preventing or treating cancer comprising the step of administering tumor-sensitized natural killer cells or a population thereof, prepared by co-culturing an effective amount of cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines, to an individual in need thereof.

[0078] Another aspect provides the use of tumor-sensitized natural killer cells or their cell populations prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines for preventing or treating cancer.

[0079]

[0080] The terms and methods, etc. described for the above inventions apply equally among the inventions.

[0081] According to a tumor priming NK cell according to one aspect and a method for producing the same, by providing a tumor priming NK cell that enhances the tumor cell death-inducing effect compared to existing NK cells, the same can be usefully used as an anticancer immune cell therapy.

[0082] FIG. 1 is a schematic diagram illustrating a method for producing tumor priming natural killer cells according to one embodiment of the present invention.

[0083] Figure 2 is a graph showing the results of analyzing the CD3 and CD56 phenotypes of tumor-sensitized natural killer cells prepared according to one embodiment of the present invention and normal natural killer cells induced under standard culture conditions.

[0084] Figure 3 is a graph showing the results of analyzing the CD3 and CD56 phenotypes of tumor-sensitized natural killer cells according to the culture medium.

[0085] Figure 4 is a graph showing the results of a comparison of cytotoxicity between normal natural killer cells induced under standard culture conditions for blood cancer cells and tumor-sensitized natural killer cells prepared according to one embodiment of the present invention.

[0086] Figure 5 shows the results of analyzing the phenotypes of tumor-sensitized natural killer cells derived from various cord blood donors using a flow cytometer.

[0087] Figure 6 is a graph showing the cytotoxicity of tumor-sensitized natural killer cells derived from various cord blood donors.

[0088] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since the embodiments are subject to various modifications, they are not limited to the embodiments disclosed below but can be implemented in various forms.

[0089] Example 1. Preparation of Activated Lymphocytes

[0090] 1.1. Isolation of Cord Blood Mononuclear Cells (CBMCs)

[0091] To prepare the activated lymphocytes used in the present invention, 80 to 100 mL of umbilical cord blood collected at delivery was used. The collected blood was transferred to a 50 mL test tube and diluted with phosphate buffer solution (PBS) containing 2% FBS at a 1:1 ratio. Then, 30 mL of the diluted blood was dispensed into a 50 mL test tube containing lymphoprep without mixing the layers, and centrifugation was performed at a rotation speed of 1,200 xg at room temperature for 20 minutes without a break.

[0092] The supernatant plasma layer was slowly removed to prevent the lymphocyte layer from being sucked in, and the separated monocyte layer was transferred to a 50 mL test tube. The separated monocyte layer was diluted with 50 mL of phosphate buffer solution (PBS) containing 2% FBS, centrifuged at 1,500 rpm at room temperature for 10 minutes, the supernatant was removed, and the collected cells were thawed. The cells in the test tube were washed twice with phosphate buffer solution (PBS) containing 2% FBS. After centrifuging at 1,500 rpm at room temperature for 5 minutes to collect the cells, the supernatant was removed, and the cells were thawed by suspension using a pipette.

[0093]

[0094] 1.2. Preparation of supporting cells

[0095] To produce tumor-sensitized natural killer cells, leukocyte components collected from patients with hematological cancer were used as scaffolding cells to be co-cultured with cord blood mononuclear cells (CBMCs). Patient-derived specimens and cancer cells were irradiated at 50 Gy to be used as scaffolding cells.

[0096]

[0097] Example 2. Culture of activated lymphocytes

[0098] 2.1. Culture of Activated Lymphocytes

[0099] Tumor priming natural killer cells were prepared using umbilical cord blood mononuclear cells and supporting cells prepared by the method of Example 1 above, and the specific preparation method is as shown in Fig. 1.

[0100] Specifically, tumor priming natural killer cells were cultured in a 24-well plate on day 0 with monocytes and irradiated patient-derived support cells in a 6:1 ratio using CTS TM AIM V TMThey were co-cultured in culture medium and cultured with the addition of IL-2 (1 ng / mL), IL-18 (25 ng / ml), and IL-21 (5 ng / ml). IL-2 (1 ng / mL) was supplemented every 2-3 days until the 7th day of culture.

[0101] After the 7th day of culture, lymphocytes and irradiated patient-derived support cells were re-inoculated into T-75 or T-175 flasks in a 2:1 ratio, and IL-2 (10 ng / mL) and IL-15 (10 ng / mL) were added and cultured for 7 days. Until the 14th day of culture, IL-2 (10 ng / mL) and IL-15 (10 ng / mL) were supplemented every 2-3 days.

[0102] To prevent nutrient depletion and achieve maximum expansion and survival, the number of cells per 1 mL was maintained at 2 million or less.

[0103] FIG. 1 is a schematic diagram illustrating a method for producing tumor priming natural killer cells according to one embodiment of the present invention.

[0104] 2.2. Cell Collection

[0105] After the 21st day of culture, the culture was terminated, and 250 mL of cell culture medium from one T-225 flask was transferred to five 50 mL test tubes. The cells were collected by centrifuging at a rotation speed of 1,500 rpm at room temperature for 5 minutes, the supernatant was removed, and the cells were suspended using a pipette to loosen them. The cell culture medium from the remaining flask was also collected in five identical test tubes and centrifuged under the same conditions, after which the supernatant was removed and the cells were loosened.

[0106] The cells in the test tube were washed twice with phosphate buffer solution and centrifuged at a rotation speed of 1,500 rpm at room temperature for 5 minutes. After removing the supernatant from the cell pellet collected through centrifugation, the cells were suspended with a pipette to loosen them, thereby finally completing the activated lymphocytes.

[0107]

[0108] Experimental Example 1. Analysis of the cell phenotype of manufactured activated lymphocytes

[0109] Samples of cells cultured according to the method of Example 2 were taken and the phenotype of tumor-sensitized natural killer cells was analyzed using a flow cytometry analyzer (Fluorescence-activated cell sorting, FACS). At this time, the phenotype of normal natural killer cells was also analyzed as a comparative example. The normal natural killer cells refer to tumor-sensitized natural killer cells prepared by the same method as Example 2, except that the feeder cells were K562 cell lines. The normal natural killer cells were described as "normal natural cells induced under standard culture conditions."

[0110] Specifically, IgG1-FITC and IgG1-pe-cy7 were prepared as negative control antibodies, and anti-CD3-FITC and anti-CD56 Pe-cy7 were prepared as experimental group antibodies. The buffer solution used was a phosphate buffer solution (phosphate saline, PBS) containing 0.5% albumin.

[0111] Measure the number of tumor-sensitized natural killer cells contained in the culture medium after completion, and the cell count is 0.5 x 10 6 It was prepared to a cell / mL concentration. After centrifuging at 1,500 rpm at room temperature for 5 minutes, the supernatant was removed and 0.5 x 10 6 Buffer solution was added to bring the concentration to cells / mL. After preparing a 5mL test tube, 0.5 x 10 6100 µL of cell suspension (cells / mL) was added to each sample, along with the control antibody and the experimental group antibody. After thorough mixing, the mixture was reacted at room temperature for 30 minutes under light protection. Subsequently, 1 mL of buffer solution was added, and the mixture was centrifuged at 1,500 rpm at room temperature for 5 minutes. After removing the supernatant, 500 µL of buffer solution was added, and the cell phenotype was analyzed using a flow cytometer. The results of the flow cytometer analysis of the cell phenotype are shown in Figure 2.

[0112] Figure 2 is a graph showing the results of analyzing the CD3 and CD56 phenotypes of tumor-sensitized natural killer cells prepared according to one embodiment of the present invention and normal natural killer cells induced under standard culture conditions.

[0113] As shown in Fig. 2, the CD56+CD3- cell ratio of tumor-sensitized natural killer cells produced by the method of one embodiment of the present invention was 88.5%, which was similar to 87.3% of normal natural killer cells induced under standard culture conditions (using K562 strain as support cell). This shows that the method of producing tumor-sensitized natural killer cells of the present invention does not differ significantly in the purity of NK cells compared to the generally used NK cell culture method.

[0114] In addition, the proportion of CD56+CD3+ cells was observed to be 7.15% in tumor-sensitized natural killer cells produced by the method for producing tumor-sensitized natural killer cells of the present invention, which is similar to 8.82% in normal natural killer cells induced under standard culture conditions.

[0115] These results indicate that the method for producing tumor-sensitized natural killer cells according to the present invention can improve the specific cytotoxic activation and tumor-attacking ability of NK cells while maintaining the incorporation of NK-T cells at a level similar to existing methods.

[0116] Therefore, the method for producing tumor-sensitized natural killer cells according to the present invention maintains purity similar to that of conventional NK cell culture methods while providing efficient tumor sensitization ability.

[0117]

[0118] Experimental Example 2. Phenotypic analysis of prepared activated lymphocytes according to the type of culture medium

[0119] To analyze the phenotypes of tumor-sensitized natural killer cells according to the type of culture medium, the type of culture medium was CTS TM AIM V TM Tumor priming natural killer cells were prepared in the same manner as in Example 1, except that X-VIVO 10 was used instead of culture medium.

[0120] In addition, the phenotype of tumor-sensitized natural killer cells cultured using X-VIVO 10 culture medium was analyzed by a flow cytometry analyzer (Fluorescence-activated cell sorting, FACS) in the same manner as in Experimental Example 1, and the results are shown in Fig. 3.

[0121] Figure 3 is a graph showing the results of analyzing the CD3 and CD56 phenotypes of tumor-sensitized natural killer cells according to the culture medium.

[0122] As shown in Figure 3, tumor-sensitized natural killer cells cultured in X-VIVO 10 culture medium showed a tendency for increased CD3 expression, and CTS TM AIM V TM When the culture medium was used, the proportion of CD56+ CD3- cells was found to be relatively higher.

[0123] These results indicate that the type of culture medium affects the phenotype and activation of tumor-sensitized NK cells. Furthermore, in the method for tumor-sensitizing natural killer cells according to the present invention, CTS TM AIM V TM This means that the culture medium is more effective in inducing a specific phenotype of tumor-sensitized natural killer cells and improving purity.

[0124]

[0125] Experimental Example 3. Comparison of cytotoxicity of tumor-sensitized natural killer cells and ordinary natural killer cells against blood cancer cells

[0126] To confirm the utility of tumor-sensitized natural killer cells produced by the method for producing tumor-sensitized natural killer cells of the present invention as an anticancer cell therapy, the degree of cancer cell killing was analyzed by flow cytometry when treated with ascites cells from a blood cancer patient.

[0127] Specifically, a comparative cytotoxicity experiment was conducted between tumor-priming NK cells according to one embodiment of the present invention and normal NK cells induced under the standard culture conditions of Experimental Example 1 (using strain K562 as the support cell) against blood cancer cells. To evaluate cytotoxicity, a CFSE (carboxyfluorescein diacetate succinimidyl ester) staining procedure was performed. Each 100 μg vial of CFSE was reconstituted with 36 μL of anhydrous DMSO to obtain a stock concentration of 5 mM.

[0128] A single-cell suspension was prepared using blood cancer cells (acute myeloid leukemia cells) as the target cell (T). The single-cell suspension was 1-10x10 6The cells were resuspended in PBS preheated to room temperature at a concentration of cells / mL. Subsequently, CFSE (500 nM) dissolved in PBS was added to the cells to perform staining. Immediately after mixing, the target cells were cultured at 37°C under light-blocking conditions for 30 minutes. After culture, the cells were centrifuged to pellet and washed once with fresh complete medium. The washed cells were cultured in a 96-well plate, and tumor-sensitized natural killer cells (double primed NK cells) prepared according to one embodiment of the present invention or normal natural killer cells induced under standard culture conditions (induced in the same manner as one embodiment of the present invention, except for using strain K562 as a support cell) were added with an E:T ratio set to 1:1 to 10:1. The plates were centrifuged at 1,500 rpm for 1 minute and cultured in a 37°C CO2 incubator for 4 to 24 hours. The plates after culture were centrifuged again at 1,000 rpm for 5 minutes at 37°C. Subsequently, FVD was prepared in DPBS (Dulbecco's Phosphate-Buffered Saline) (1 μL diluted in 1 mL), and 100 μL of FVD (Fixable Viability Dye) was added to each well. After addition, the mixture was incubated at 4°C for 30 minutes, and the cells were washed twice with DPBS after the reaction was complete. Finally, a flow cytometry (FACS) analyzer was used to analyze the cytotoxicity results.

[0129] The results of comparing the cytotoxicity of tumor-sensitized natural killer cells prepared according to one embodiment of the present invention and ordinary natural killer cells induced under standard culture conditions, analyzed using a flow cytometry analyzer (FACS), are shown in Figure 4.

[0130] Figure 4 is a graph showing the results of a comparison of cytotoxicity between normal natural killer cells (indicated as "NK cells" in Figure 4) induced under standard culture conditions for blood cancer cells and tumor-sensitized natural killer cells (double primed NK cells) prepared according to one embodiment of the present invention.

[0131] As shown in Figure 4, in the case of ordinary natural killer cells induced under standard culture conditions, cytotoxicity is maintained at the lowest level and there is almost no change depending on the E:T ratio. In addition, it was confirmed that NK cells (double primed NK cells) produced by the method for producing tumor-sensitized natural killer cells of the present invention have significantly higher cytotoxicity compared to ordinary natural killer cells induced under standard culture conditions.

[0132] Meanwhile, it was confirmed that the cytotoxicity of tumor-sensitized natural killer cells (double primed NK cells) prepared according to one embodiment of the present invention was significantly higher than that of NK cells sensitized once with monocytes and patient-derived support cells that had undergone radiation treatment only on day 0 of culture (once primed NK cells). In particular, NK cells (double primed NK cells) prepared by the method of the present invention using CTS-AIM culture medium exhibited the highest cytotoxicity and were confirmed to have excellent anticancer effects against blood cancer cells (AML-#5).

[0133] These results indicate that when natural killer cells are double-primed according to the method for preparing tumor-sensitized natural killer cells of the present invention and CTS-AIM culture medium is used, cytotoxicity can be maximized and it is most effective for anticancer activation.

[0134]

[0135] Experimental Example 4. Comparison of Cytotoxicity of Tumor-Sensitized Natural Killer Cells According to Umbilical Cord Blood Donor

[0136] 4.1. Analysis of Cell Distribution According to Cord Blood Donor

[0137] Activated lymphocytes were prepared and cultured from several cord blood donors according to the methods of Example 1 and Example 2, and the cell phenotype of the activated lymphocytes prepared by the method of Experimental Example 1 was analyzed.

[0138] Specifically, the cord blood donors were D122, D304, and D309, and the phenotypes of tumor-sensitized natural killer cells derived from them were analyzed using a flow cytometer and are shown in Figure 5.

[0139] Figure 5 shows the results of analyzing the phenotypes of tumor-sensitized natural killer cells derived from various cord blood donors using a flow cytometer.

[0140] As shown in Figure 5, it was confirmed that for donor D122, the proportion of CD56+ CD3- was 97.8% and the proportion of CD56+ CD3+ was 1.57%, for donor D304, the proportion of CD56+ CD3- was 92.5% and the proportion of CD56+ CD3+ was 6.50%, and for donor D309, the proportion of CD56+ CD3- was 99.2% and the proportion of CD56+ CD3+ was 0.59%.

[0141] These results indicate that while there may be differences in the purity of NK cells and the mixing ratio of NK-T cells depending on the umbilical cord blood donor, the NK cells obtained through the method for producing tumor-sensitized natural killer cells according to one embodiment of the present invention consistently maintain high purity. The high purity of the NK cells suggests the possibility that the cytotoxic ability of the NK cells has been optimized, which indicates that the method for producing tumor-sensitized natural killer cells of the present invention can consistently produce effective NK cells. Accordingly, the tumor-sensitized NK cells produced by the method for producing tumor-sensitized natural killer cells of the present invention provide effective anticancer efficacy.

[0142]

[0143] 4.2. Comparison of Cytotoxicity of Tumor-Sensitized Natural Killer Cells According to Umbilical Cord Blood Donor

[0144] To determine whether there is a difference in the cytotoxic ability of NK cells depending on the cord blood donor, the cytotoxicity of tumor-sensitized natural killer cells derived from several cord blood donors (D122, D304, D309) of Example 4.1 was compared and analyzed using the method of Experimental Example 3.

[0145] Specifically, cytotoxicity was evaluated by dividing the cells into normal natural killer cells prepared in the same manner as in Example 2 except that the K562 cell line was used (indicated as "nbs NK cell" in Fig. 6), natural killer cells primed once with monocytes and patient-derived support cells irradiated only on day 0 of culture (once), and natural killer cells primed twice according to one embodiment of the present invention (twice), and the results are shown in Fig. 6.

[0146] Figure 6 is a graph showing the cytotoxicity of tumor-sensitized natural killer cells derived from various cord blood donors.

[0147] As shown in Figure 6, the cytotoxicity of normal natural killer cells (nbs NK cells) induced under standard culture conditions was similar in the D122, D304, and D309 donors, at levels of approximately 10-12%. Once-primed natural killer cells showed increased cytotoxicity in all three donor-derived NK cells, with the NK cells from the D122 donor exhibiting relatively high cytotoxicity (D122: approximately 20%, D304 and D309: approximately 15%). Twice-primed natural killer cells showed a significant increase in cytotoxicity (D122 donor: approximately 55%, D304 donor: approximately 40%, D309 donor: approximately 35%).

[0148] These results indicate that the cytotoxic capacity of natural killer cells increases stepwise during the priming process, and that cytotoxicity improves rapidly, particularly in twice-sensitized natural killer cells.

[0149] Furthermore, the differences in the cytotoxic capacity of natural killer cells depending on the cord blood donor suggest that they may be affected by KIR (Killer-cell Immunoglobulin-like Receptor) mismatch. KIR mismatch significantly influences how a donor's natural killer cells recognize and attack recipient cells, which can provide a basis for the selective use of natural killer cells from specific donors in the development of cell therapies.

[0150]

[0151] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments and experimental examples described above should be understood as illustrative in all respects and not restrictive.

Claims

1. (a) a step of obtaining cord blood mononuclear cells (CBMC); and (b) A method for producing tumor priming natural killer cells comprising the step of co-culturing the obtained cord blood mononuclear cells and feeder cells in a medium containing cytokines.

2. A method for producing tumor-sensitized natural killer cells according to claim 1, wherein the co-culturing step comprises an initial culture step and a subsequent culture step, wherein the initial culture step is performed for 1 to 10 days and the subsequent culture step is performed for 10 to 20 days.

3. A method for producing tumor-sensitized natural killer cells according to claim 2, wherein the composition of cytokines contained in the medium during the initial culture stage and the subsequent culture stage is different.

4. A method for producing tumor-sensitized natural killer cells according to claim 2, wherein the initial culture step is co-culture in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-18, and IL-21.

5. A method for producing tumor-sensitized natural killer cells according to claim 2, wherein the step after the initial culture is co-culture in a medium containing IL-2 and IL-15.

6. A method for producing tumor-sensitized natural killer cells according to claim 1, wherein the supporting cells are solid tumor cells or blood cancer cells.

7. A method for producing tumor-sensitized natural killer cells according to claim 1, wherein the supporting cells are derived from ascites cells of a cancer patient.

8. A method for producing tumor-sensitized natural killer cells according to claim 1, wherein the co-culturing step involves co-culturing umbilical cord blood mononuclear cells and supporting cells in a ratio of 1:1 to 8:

1.

9. A method for producing tumor-sensitized natural killer cells according to claim 2, wherein the initial culture step involves co-culturing umbilical cord blood mononuclear cells and supporting cells in a ratio of 5:1 to 8:

1.

10. A method for producing tumor-sensitized natural killer cells according to claim 2, wherein the step after the initial culture is to co-culture umbilical cord blood mononuclear cells and supporting cells in a ratio of 1:1 to 8:

1.

11. Tumor-sensitized natural killer cells or their cell populations produced by the method of Claim 1.

12. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, tumor-sensitized natural killer cells or a cell population thereof, prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines.

13. A pharmaceutical composition for the prevention or treatment of cancer according to claim 12, wherein the cancer is any one selected from the group consisting of glioma, gastrointestinal stromal tumor, leukemia, breast cancer, uterine cancer, cervical cancer, stomach cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, laryngeal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, bone cancer, muscle cancer, lipoma, fibrocytic carcinoma, blood cancer, lymphoma, and multiple myeloma.

14. A cell therapeutic agent comprising, as an active ingredient, tumor-sensitized natural killer cells or a population thereof, prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines.

15. Use of tumor-sensitized natural killer cells or a population thereof prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines for use in the manufacture of pharmaceutical preparations for the prevention or treatment of cancer.

16. A method for the prevention or treatment of cancer comprising the step of administering tumor-sensitized natural killer cells or a population thereof, prepared by co-culturing an effective amount of cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines, to an individual in need thereof.

17. Use of tumor-sensitized natural killer cells or a population thereof prepared by co-culturing cord blood mononuclear cells (CBMC) and feeder cells in a medium containing cytokines for the prevention or treatment of cancer.

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