Method for producing memory-like natural killer cells and the anti-cancer applications of memory-like natural killer cells produced using the same.

Abstract

The present invention relates to a method for producing memory-like natural killer cells (NK cells) and anti-cancer use of the memory-like natural killer cells produced using the same. The method for producing memory-like natural killer cells of the present invention is an optimal autoimmune cell culture method for producing memory-like natural killer cells with increased cancer cell killing ability while maintaining cell proliferation. The memory-like natural killer cells produced through the method have superior cancer cell killing ability compared to natural killer cells (NK cells) and have memory-like ability, so when a second stimulation is given, they have the function of being reactivated and re-proliferating, and have the advantage of being able to survive in the body for more than two months, and can be used as an immune cell therapeutic agent or for the prevention or treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to a method for producing memory-like natural killer cells and to the anti-cancer applications of memory-like natural killer cells produced using this method. [Background technology]

[0002] Natural killer cells (NK cells), which belong to the lymphocytes within the human immune system, are responsible for innate immunity. They play a role in early recognition and elimination of external pathogens or internal abnormal cells such as cancer cells. In particular, while the activation of T cells, which are responsible for acquired immunity, requires a complex process in which antigen-presenting cells must first degrade target cells and present antigens to the MHC, NK cells do not require antigen presentation, which is why they are attracting attention as an immunotherapy agent.

[0003] On the other hand, NK cells are a subtype of lymphocyte found in the bone marrow, lymph nodes, and peripheral blood, accounting for approximately 10-15% of all lymphocytes, and are the third most common subtype after T cells and B cells. As immune cells capable of killing cancer cells or virus-infected cells, they play an important role in innate immune responses. The function of NK cells is regulated by the interaction between receptors on the cell surface and their corresponding target cell ligands, without stimulation by specific antigens. These receptors are broadly classified into activating receptors and inhibitory receptors. Commonly known activating receptors that transmit activation signals to NK cells and regulate the function of natural killer cells include natural cytotoxicity receptors (NCRs; NKp30, NKp44, NKp46) and NKG2D. NK cells sense target cells through a variety of activating receptors with different ligand specificities, and these receptors mainly induce activation through phosphorylation, but their signaling characteristics are very different. NCRs (NKp30, NKp44, NKp46) and CD16 are known to form complexes with FcRγ, CD3γ, and DAP12, which possess the ITAM motif, and transmit activation signals similar to the TCRs and BCRs of T cells and B cells. NKG2D binds to the DAP10 adaptor, which possesses the YINM motif, and transmits signals via the PI3K or Grb2-Vav1 complex. In particular, it has been shown that natural killer cells with high NCR expression have higher cytotoxicity than cells with low expression, and the degree of natural killer cell activation can be confirmed according to the degree of NCR expression. Activated natural killer cells can synthesize various granules and secrete them extracellularly to destroy target cells. Among the representative proteins contained in granules, perforin and granzyme play major roles in destroying target cells.Perforins accumulate on the cell membrane of target cells, forming complexes that create holes in the cell membrane and cause cell lysis. Granzymes then enter the cell through these holes, activating caspases and inducing cell death through a variety of mechanisms.

[0004] On the other hand, T cells recognize antigens presented on MHC and distinguish between self and non-self cells. When they recognize non-self cells, they recognize them as abnormal cells and kill them immediately. Therefore, cancer cells have significantly reduced MHC expression on their surface to evade the immune system. However, when NK cells recognize the MHC of other cells, an inhibitory signal is activated, and if the target cell lacks MHC, an activation signal is activated, allowing them to eliminate the target cell. Therefore, NK cells have a slight advantage over T cells in terms of cancer cell killing ability. Furthermore, NK cells induce apoptosis in cancer cells by binding to death receptors on cancer cells using death ligands such as FasL and TRAIL. This receptor binding activates caspase-8 and -10 in cancer cells, which in turn activates caspase-3 and -7, ultimately leading to apoptosis. Furthermore, natural killer cells secrete a variety of cytokines and chemokines, playing a bridgehead role in activating adaptive immune responses through direct interaction with antigen-presenting cells. IFN-α secreted from activated NK cells is known to play a crucial role in activating and maturing mononuclear and dendritic cells, increasing the innate immune response against pathogens, and is particularly important in the early stages of the Th1 immune response.

[0005] For NK cells to possess such capabilities, priming is necessary before they encounter target cells. Therefore, precursor natural killer cells isolated from rats and humans exhibit significantly reduced cancer cell-killing ability and IFN-γ production capacity. Some priming cytokines, such as IL-2 and IL-15, have been reported to maximize the capabilities of NK cells. For example, IL-2 has been reported to promote the proliferation and activation of mature NK cells. IL-15 is known to be involved in NK cell differentiation. IL-21 is a cytokine secreted by activated CD4+ T cells, and its receptor (IL-21R) is known to be expressed in lymphocytes such as dendritic cells, NK cells, T cells, and B cells.

[0006] In cancer patients, the overall immune system is not functioning properly, so using the immune cells already present in the body results in impure cancer cell-killing capabilities. Recently, immunotherapy drugs have been actively developed that isolate and culture NK cells from the patient's peripheral blood, activate their cancer cell-killing ability, and then inject them back into the patient. However, since typical NK cells enter the bone marrow and disappear 1-2 weeks after injection, the NK cell therapies developed so far require patients to receive NK cell injections once every 1-2 weeks, which places a burden on the patient. Recently, it has been confirmed that if NK cells are stimulated initially, cultured in a resting phase, and then restimulated, NK cells with memory-like cell function similar to T cells, good cancer cell killing ability, and long-term survival in the body can be produced. As a result, related research is actively progressing. However, the production process does not involve normal proliferation, and a large amount of blood is required to secure a sufficient number of NK cells, which is a burden on patients and donors, making it difficult to develop therapeutic agents / treatments (Matteo Tanzi, et al., Cancers, 13; 1577, 2021).

[0007] Therefore, there is a need to produce more active memory-like natural killer cells (NK cells) to replace the less active natural killer cells present in the body, and to use these for anti-cancer purposes. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a method for producing memory-like natural killer cells (NK cells).

[0009] Another object of the present invention is to provide memory-like natural killer (NK) cells.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer.

[0011] Another object of the present invention is to provide an immunotherapy agent for cancer treatment.

[0012] Another object of the present invention is to provide an anticancer adjuvant for enhancing the activity of anticancer agents.

[0013] Another object of the present invention is to provide a method for preventing or treating cancer. [Means for solving the problem]

[0014] To solve the aforementioned problems, the present invention provides a method for producing memory-like natural killer cells (NK cells).

[0015] Furthermore, the present invention provides memory-like natural killer cells (NK cells) produced by the method described above.

[0016] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising memory-like natural killer cells (NK cells) as an active ingredient.

[0017] Furthermore, the present invention provides an immunotherapy agent for cancer treatment that contains the memory-like natural killer cells (NK cells) as an active ingredient.

[0018] Furthermore, the present invention provides an anticancer adjuvant for enhancing the activity of anticancer agents, which contains the aforementioned memory-like natural killer cells (NK cells) as an active ingredient.

[0019] Furthermore, the present invention provides a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition or the cell therapy agent to an individual. [Effects of the Invention]

[0020] The present invention provides an optimal autoimmune cell culture method for producing memory-like natural killer cells (NK cells) that maintain cell proliferation while also increasing cancer cell killing ability. The memory-like natural killer cells produced by this method have superior cancer cell killing ability compared to natural killer cells (NK cells) and possess memory-like capabilities. They can be reactivated and proliferate again upon a second stimulation and have the advantage of being able to survive in the body for more than two months. They can be used as an immunotherapy agent or for the prevention or treatment of cancer. [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the purity (distribution) of NK cells measured in one embodiment of the present invention: control group: negative control cells obtained by culturing peripheral blood mononuclear cells in a culture medium containing 2-10% autologous plasma and 1 ng / ml of IL-15; and memory-like natural killer cells: memory-like natural killer cells produced by the method of the present invention. [Figure 2]This is a figure showing the activity (cancer cell killing ability) of memory-like natural killer cells in one embodiment of the present invention: Control group: Negative control group cells obtained by culturing peripheral blood mononuclear cells in a culture medium containing 2 - 10% autologous plasma and 1 ng / ml of IL-15; and Memory-like natural killer cells: Memory-like natural killer cells produced by the method of the present invention. [Figure 3] This is a figure analyzing the expression on the cell surface of memory-like natural killer cell-specific markers in one embodiment of the present invention: Control group: Negative control group cells obtained by culturing peripheral blood mononuclear cells in a culture medium containing 2 - 10% autologous plasma and 1 ng / ml of IL-15; and Memory-like natural killer cells: Memory-like natural killer cells produced by the method of the present invention. [Figure 4] This is a figure comparing the total number of cells at the start of culture with the total number of cells finally harvested in one embodiment of the present invention, and confirming the cumulative growth rate. [Figure 5] This is a figure comparing the total cell growth rates of the negative control group (control) and the experimental group (memory-like natural killer cells, Memory-like NK cell) in one embodiment of the present invention: Control group: Negative control group cells obtained by culturing peripheral blood mononuclear cells in a culture medium containing 2 - 10% autologous plasma and 1 ng / ml of IL-15; and Memory-like natural killer cells: Memory-like natural killer cells produced by the method of the present invention. [Figure 6] This is a figure comparing the number of NK cells at the start of culture with the number of NK cells finally produced in one embodiment of the present invention, and comparing the cell growth rates. [Figure 7] This is a figure comparing the initial culture NK cell growth rates with the final NK cell growth rates in the negative control group (control) and the experimental group (memory-like natural killer cells, Memory-like NK cell) in one embodiment of the present invention: Control group: Negative control group cells obtained by culturing peripheral blood mononuclear cells in a culture medium containing 2 - 10% autologous plasma and 1 ng / ml of IL-15; and Memory-like natural killer cells: Memory-like natural killer cells produced by the method of the present invention.

Mode for Carrying Out the Invention

[0022] The present invention will be described in detail below with reference to the attached drawings and based on examples of its implementation. However, the following examples are presented as illustrations of the present invention, and if it is determined that a specific description of a well-known technology or configuration to those skilled in the art would unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention will not be limited thereby. The present invention is subject to various modifications and applications within the scope of equivalents described in the claims below and interpreted therefrom.

[0023] Furthermore, the terminology used herein is intended to appropriately describe preferred embodiments of the present invention and may vary depending on the intent of the user, operator, or the conventions of the art to which the invention pertains. Therefore, definitions of these terms should be based on the overall content of this specification. When a part of the specification "includes" a component, this does not exclude other components, unless otherwise stated, but rather means that it further includes other components.

[0024] In one aspect, the present invention relates to a method for producing memory-like natural killer cells (Memory-like NK cells), comprising the steps of: 1) separating peripheral blood mononuclear cells (PBMCs) from peripheral blood; 2) treating and culturing the cells with plasma, anti-NKp46 antibody, IL-2, and IL-18; 3) transferring the cells to a new culture vessel and treating and culturing the cells with plasma and IL-2; 4) transferring the cells to a new culture vessel and culturing the cells with plasma, IL-12, IL-15, and IL-18; and 5) transferring the cells to a new culture vessel and culturing the cells with plasma, IL-2, and IL-15.

[0025] In one embodiment, in step 2), cells can be cultured in a culture vessel coated with fibronectin, γ-globulin, and anti-CD56 antibody.

[0026] In one example, the plasma in step 2) can be treated with 1-20% (w / w), 0.01-10 μg / ml of anti-NKp46 antibody, 10-5000 IU / ml of IL-2, and 2-500 ng / ml of IL-18.

[0027] In one embodiment, step 2) can be performed two or more times, in which plasma, anti-NKp46 antibody, IL-2, and IL-18 are used for primary treatment, and the cells are cultured until the cell density reaches 80% or more, and then plasma, anti-NKp46 antibody, IL-2, and IL-18 are used for secondary treatment, and the cells are cultured until the cell density reaches 80% or more.

[0028] In one example, the plasma in step 3) can be treated with 1-20% (w / w) and IL-2 at 10-5000 IU / ml.

[0029] In one embodiment, step 3) can be performed two or more times, in which plasma and IL-2 are applied as a primary treatment, and the cells are cultured until the cell density reaches 80% or more, and then plasma and IL-2 are applied as a secondary treatment, and the cells are cultured until the cell density reaches 80% or more.

[0030] In one example, the plasma in stage 4) can be treated with 1-20% (w / w), IL-12 at 0.5-200 ng / ml, IL-15 at 0.1-100 ng / ml, and IL-18 at 2-500 ng / ml.

[0031] In one embodiment, the culture in step 4) can be carried out for 10 to 20 hours, and more preferably for 12 to 16 hours.

[0032] In one example, plasma in stage 5) can be treated with 1-20% (w / w), IL-2 at 10-5000 IU / ml, and IL-15 at 0.1-100 ng / ml.

[0033] In one embodiment, the step after step 5) may further include processing and culturing in a new medium containing IL-2, and the IL-2 can be administered at a concentration of 10 to 5000 IU / ml.

[0034] In one embodiment, the plasma may be plasma separated from the peripheral blood, and the peripheral blood mononuclear cells (PBMCs) and plasma may be autologous to the subject.

[0035] The present invention provides an optimal autoimmune cell culture method for producing memory-like natural killer cells (Memory-like NK cells) that overcome the disadvantages of memory-like natural killer cells (Memory-like NK cells) that do not proliferate normally. This method involves isolating peripheral blood mononuclear cells, initiating culture for NK cell proliferation using the whole cells, stimulating the memory-like natural killer cells (Memory-like NK cells) during the process of active proliferation, and producing memory-like natural killer cells (Memory-like NK cells) with increased cancer cell killing ability while maintaining cell proliferation. This method has the effect of culturing high-purity NK cells without the process of removing CD3-positive T cells or feeder cells, and proliferating a sufficient number of memory-like natural killer cells (Memory-like NK cells) suitable for patient administration.

[0036] In one aspect, the present invention relates to memory-like natural killer cells produced by the manufacturing method of the present invention.

[0037] In one embodiment, memory-like natural killer cells produced by the manufacturing method of the present invention may show increased expression of CD94 or CD25 and decreased expression of NKp80 compared to NK cells.

[0038] In one embodiment, memory-like natural killer cells produced by the manufacturing method of the present invention may have an increased cell proliferation rate compared to NK cells.

[0039] Memory-like natural killer cells produced by the manufacturing method of the present invention have superior cancer cell-killing ability compared to NK cells and can be used as an immunotherapy agent. Furthermore, because they possess memory-like capabilities, they can be reactivated and proliferate again when stimulated a second time, and have the advantage of being able to survive in the body for more than two months.

[0040] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising the memory-like natural killer cells of the present invention as an active ingredient.

[0041] In one aspect, the present invention relates to an immunotherapy agent for cancer treatment that contains the memory-like natural killer cells of the present invention as an active ingredient.

[0042] In one example, cancer may be one or more selected from the group consisting of brain tumors, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, trumpet duct cancer, cancer near the anus, endometrial cancer, vaginal carcinoma, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic cancer, central nervous system tumors, spinal cord tumors, brainstem gliomas, and pituitary adenomas.

[0043] The terms "cancer" and "tumor" used in this invention may be used interchangeably.

[0044] As used in this invention, the term "prevention" means all actions that suppress or delay the onset, development, and recurrence of cancer by administering the composition according to the present invention.

[0045] As used in this invention, the term "treatment" means all actions that improve or favorably alter the symptoms of cancer and its associated complications by administering the composition according to the present invention. A person with ordinary skill in the art to which this invention belongs should be able to determine the precise criteria for diseases in which the composition of this application is effective and the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association, etc.

[0046] The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the individual's condition.

[0047] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. As used in this invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, without causing side effects. The effective dose level may be determined by factors including the individual's health status, the type and severity of cancer, the activity and sensitivity of the drug, the method of administration, the timing of administration, the route of administration and elimination ratio, the duration of treatment, the drugs used in combination or concurrently, and other factors widely known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and in single or multiple doses. Considering all the aforementioned factors, it is important to administer an amount that provides the maximum effect with the minimum amount without adverse effects, which can be readily determined by those skilled in the art.

[0048] In one embodiment, the pharmaceutical composition may be one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injection solutions, and sprays.

[0049] The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of these commonly used in biological formulations. The pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for intra vivo delivery of the composition. For example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be used in combination, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. In addition, the compositions may be preferably formulated according to each disease or component using methods appropriate to the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0050] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, of the protein based on the total weight of the composition.

[0051] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives, in which case the following can be used: starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, corn syrup, acacia gum, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opa-dry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

[0052] The compositions of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally according to the intended method, with oral administration being most preferred. The dosage ranges widely depending on the individual's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

[0053] Liquid formulations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, a variety of excipients, such as humectants, sweeteners, fragrances, and preservatives, may also be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories.

[0054] In one aspect, the present invention relates to an anticancer adjuvant for enhancing the activity of anticancer agents, which contains memory-like natural killer cells as an active ingredient.

[0055] In one specific case, the anticancer drugs used were eribulin, carboplatin, cisplatin, Halaven, 5-fluorouracil (5-FU), gleevec, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etiolus. It may also be etoposide, topotecan, irinotecan, dactinomycin, doxorubicin, daunorubicin, valrubicin, flutamide, gemcitabine, mitomycin, or bleomycin.

[0056] In one aspect, the present invention relates to a method for treating liver cancer, comprising the step of administering the pharmaceutical composition or the cell therapy agent to an individual.

[0057] In the present invention, "individual" is not particularly limited as long as it is an individual for the purpose of preventing or treating cancer, and refers to animals including humans, for example, non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, for example, cynomolgous monkeys and chimpanzees) and other mammals.

[0058] The pharmaceutical composition or cell therapy agent of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0059] In this invention, the term "therapeutably effective amount" means a pharmaceutically acceptable amount of salt of a composition effective in preventing or treating a target disease. The therapeutically effective amount of the composition of this invention may vary depending on various factors, such as the method of administration, the site of administration, and the patient's condition. Therefore, the dosage for use in humans must be determined at an appropriate level, taking both safety and efficiency into consideration. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. Such considerations when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0060] In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, without causing side effects. The level of the effective dose may be determined by factors including the patient's health status, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the timing of administration, the route of administration and elimination ratio, the duration of treatment, the drugs that are compounded or used concurrently, and other factors widely known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and in single or multiple doses. Taking all of the aforementioned factors into consideration, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0061] The present invention will be described in more detail below based on examples. These examples are provided to illustrate the present invention more concretely, and the scope of the present invention is not limited to these examples. [Examples]

[0062] Memory-like natural killer (NK) cell production

[0063] 1-1. Flask Coating

[0064] One day before blood collection, a flask to be used was filled with a solution of 5-10 μg / ml fibronectin (FC010, Sigma), 1-2 mg / ml γ-globulin (Green Cross), and 5-10 μg / ml anti-CD56 antibody (559043, BD Biosciences), diluted in DPBS, spread evenly, and coated at 2-8°C for at least 16 hours. At this time, the size of the flask was 25 cm². 2 In that case, a total of 3-5 ml, 75 cm 2 In that case, the flask was coated with 5-10 ml of the solution. After coating was complete, the flask was cleaned by removing any remaining coating solution, washing with 10 ml of DPBS, and then the DPBS was removed.

[0065] 1-2. Isolation of peripheral blood mononuclear cells (PBMCs)

[0066] Blood was collected from the veins of five healthy donors using heparin-coated vacuum blood collection tubes (367874, BD), and carefully transferred to 50 ml tubes containing 15 ml of Histopaque 1077 (10771, Sigma-Aldrich). The tubes containing the blood were centrifuged at 1000 xg at room temperature for 10 minutes with the brake off. The tubes were then carefully removed, the supernatant was transferred to a new 50 ml tube, and the tubes were inactivated in a water bath set to 56°C for 30 minutes. The tubes were then centrifuged at 1400 xg for 10 minutes. The tubes were removed, and only the supernatant was transferred to a new 50 ml tube to complete the separation of autologous plasma. The autologous plasma was stored refrigerated (2-8°C) before use. After separating the plasma, the mononuclear cell layer was carefully separated from the remaining tube and transferred to a new 50 ml tube. The tube was filled with DPBS (17-512F, Lonza) and centrifuged at 910 x g for 10 minutes. After centrifugation, the supernatant was discarded, and the precipitated cells were resuspended in 30 ml of DPBS and centrifuged at 910 x g for 10 minutes. After discarding the supernatant, the precipitated cells were resuspended in 5 ml of red blood cell lysis buffer (00-4333-57, Thermo Fisher Scientific) and allowed to stand at room temperature for 5 minutes. Then, 30 ml of DPBS was added to the cell suspension to terminate the reaction, and the cell suspension was centrifuged at 910 x g for 10 minutes, after which the supernatant was removed. The precipitated cells were resuspended in 10 ml of DPBS and centrifuged at 910 x g for 10 minutes. The supernatant was discarded, and the remaining precipitated cells were resuspended in 10 ml of culture medium (NK MACS medium, 130-114-429, Miltenyi biotec). A small amount of cells was then taken, diluted 10-fold, and counted.

[0067] 1-3. Primary culture of NK cells

[0068] Take the cell suspension prepared in Example 1-2 and add the 25 cm³ prepared in Example 1-1. 2Place the mixture in a coated flask and add 2-10% (v / v) autologous plasma, 0.1-1 μg / ml anti-NKp46 antibody (130-094-271, Miltenyi Biotec), 100-1000 IU / ml IL-2 (146545, Boehringer Ingelheim), and 5-100 ng / ml IL-18 (9124IL-500-CF, R&D Systems). Then, coat the flask at 37°C with 5% CO2. 2 The cells were cultured in an incubator until the cell density reached 80% or higher. 2.5 ml of fresh culture medium containing 10% autologous plasma, 100-1000 IU / ml of IL-2, 5-100 ng / ml of IL-18, and 0.1-1 μg / ml of anti-NKp46 antibody was added to the flask during cell culture, and the cells were continued to culture until the cell density reached 80% or higher. The cells were then cultured in a 25 cm³ incubator. 2 After removing cells from the bottom of the flask using a cell scraper, pour 7.5 ml of cell suspension into a 75 cm flask. 2 The cells were transferred to a flask. 8 ml of fresh medium containing 2-10% autologous plasma and 100-1000 IU / ml of IL-2 was added, and the cells were further cultured until the cell density reached 80% or higher. 15 ml of fresh medium containing 2-10% autologous plasma and 100-1000 IU / ml of IL-2 was added to the flask during cell culture, and the cells were further cultured until the cell density reached 80% or higher. After removing the cells adhering to the bottom of the flask using a cell scraper, 30 ml of the cell suspension was transferred to a tube and centrifuged at 910xg for 10 minutes.

[0069] 1-4. Stimulation by memory-like natural killer cells (NK cells)

[0070] The supernatant of the cells centrifuged in Examples 1-3 was removed, and after resuspending with 10 ml of DPBS, it was centrifuged again at 910 x g for 10 minutes to wash the cells. The washing was repeated twice. The washed cells were resuspended in 30 ml of a new medium containing 2-10% (v / v) autologous plasma, 2-50 ng / ml of IL-12, 1-10 ng / ml of IL-15, and 10-100 ng / ml of IL-18, and then placed in a new 75 cm 2 flask and cultured and stimulated for 12-16 hours. The cells stimulated for 12-16 hours were collected, transferred into a 50 ml tube, and centrifuged at 910 x g for 10 minutes. The supernatant was removed, resuspended with 10 ml of DPBS, and then centrifuged again at 910 x g for 10 minutes for washing. The washing was repeated twice.

[0071] 1-5. Secondary culture of NK cells

[0072] The cells washed in Examples 1-4 were resuspended in 30 ml of a new medium containing 2-10% (v / v) autologous plasma, 100-1000 IU / ml of IL-2, and 1-10 ng / ml of IL-15, and then cultured in a new 75 cm 2 flask until the cell density reached 80% or more. When the cell density in the flask reached 80% or more, it was transferred to a 175 cm 2 flask, a new medium containing 100-1000 IU / ml of IL-2 was added, and the culture was continued to produce memory-like natural killer cells (Memory-like NK cell).

Example

[0073] Analysis of the purity of NK cells

[0074] To measure the purity of the memory-like natural killer (NK) cells produced in Example 1, 1 to 5 x 10⁵ cells were taken per sample, placed in a 1.5 ml tube as a cell suspension, mixed well with 1 ml of DPBS, and then centrifuged at 910 x g for 5 minutes. The supernatant was removed and washed twice with 500 μl of DPBS. The washed cell precipitate was resuspended in 98 μl of FACS staining buffer (554657, BD Biosciences), mixed well with 2 μl of human FcR blocking reagent (130-059-901, Miltenyi Biotec), and then reacted in the dark at 0 to 4°C for 20 minutes. Subsequently, the cells were washed three times with 500 μl of DPBS. After washing, the cell precipitate was resuspended in 94 μl of FACS staining buffer. Then, 2 μl each of anti-CD3 antibody (48-0038-82, eBioscience), anti-CD56 antibody (318303, BioLegend), and anti-CD19 antibody (555415, BD Biosciences) were added and mixed thoroughly. The mixture was then incubated in the dark at 0-4°C for 30 minutes. After washing three times with 500 μl of DPBS, the cell precipitate was resuspended in 200 μl of FACS staining buffer, and the purity of CD3-negative but CD56-positive NK cells was analyzed using flow cytometry. Furthermore, immediately after obtaining peripheral blood mononuclear cells from blood samples taken from five donors, the distribution of NK cells was analyzed to obtain the distribution at day 0. The distribution of memory-like natural killer cells (12-15 days) obtained through the process described in Example 1 was then analyzed. The control group consisted of peripheral blood mononuclear cells cultured in a culture medium containing 2-10% (v / v) autologous plasma and 1 ng / ml of IL-15.

[0075] [Table 1]

[0076] Table 1 above shows the distribution of NK cells obtained from a total of five healthy donors. The distribution at day 0 was obtained by analyzing the distribution of NK cells immediately after blood collection to obtain peripheral blood mononuclear cells. After completing the culture for 12 to 15 days (days 12 to 15), the distribution of NK cells was analyzed, and the final distribution of NK cells is shown. Cells that were CD3-negative but CD56-positive were considered NK cells. The control group consisted of peripheral blood mononuclear cells cultured in a culture medium containing 2-10% autologous plasma and 1 ng / ml of IL-15 as a negative control group. As a result, it was shown that the memory-like natural killer cells were significantly increased by the method of the present invention (Table 1 and Figure 1). [Examples]

[0077] Analysis of the anti-cancer activity of NK cells

[0078] The activity of memory-like natural killer (NK) cells produced in Example 1 was measured. Specifically, the target human cancer cell line, K562, was washed twice with its culture medium, complete IMDM (IMDM containing 10% FBS). After washing, the cell precipitate was resuspended in 1 ml of complete IMDM, and then Calcein-AM (C1430, Invitrogen) at a concentration of 0.01 μM was added and mixed well. The cells were then stained at 37°C in the dark for 10 minutes. The tubes containing the stained K562 cells were centrifuged at 910xg for 5 minutes, the supernatant was removed, and the cells were washed three times with NK cell culture medium. After washing the NK cells twice with NK cell culture medium, they were placed in a 12-well plate in a 5:1 ratio (5 x 10⁵ NK cells and 1 x 10⁵ K562 cells per sample) along with the Calcein-AM stained K562 cells and 5% CO₂. 2Cells were co-cultured for 4 hours in an incubator at 37°C. During this time, 1 ml of culture medium containing 2-10% (v / v) autologous plasma and 100-1000 IU / ml of IL-2 was added to each well. Cells were harvested from the plate, transferred to a 1.5 ml tube, and centrifuged at 910xg for 5 minutes. The supernatant was removed, washed twice with 500 μl of DPBS, resuspended in 99 μl of FACS staining buffer, and then 1 μl of 7-AAD viability staining solution (420403, BioLegend) was added and mixed well. The cells were stained at room temperature in the dark for 5-10 minutes. After staining, the cells were analyzed by flow cytometry, and among the Calcein-AM positive cells, those positive for 7-AAD were considered to be cells killed by NK cells. Furthermore, as a negative control group, cells cultured in a culture medium containing 2-10% (v / v) autologous plasma and 1 ng / ml of IL-15 were used.

[0079] [Table 2]

[0080] Table 2 above shows the cancer cell-killing ability (hereinafter referred to as "cytotoxicity") of Memory-like NK cells cultured from a total of five healthy donors. As a negative control group, cells cultured in a medium containing 2-10% (v / v) autologous plasma and 1 ng / ml of IL-15 were used. As a result, it was shown that the cytotoxicity of the negative control group versus the comparison group (Memory-like natural killer cells, Memory-like NK cells) was significantly increased in cells from all donors (Table 2 and Figure 2). [Examples]

[0081] Analysis of the expression of recognition markers in memory-like natural killer cells (NK cells).

[0082] Memory-like natural killer (NK) cells show increased expression of CD94 and CD25 and decreased expression of NKp80 compared to typical NK cells (Margery Gang, et al., Semin Hematol. 57(4):185-193, 2020). Therefore, the expression of memory-like NK cell-specific identification labels in the memory-like NK cells produced in Example 1 was analyzed. Specifically, 2-10% (v / v) of autologous plasma and 1 ng / ml of IL-15 were added to the culture medium, and negative control cells were cultured for 12-15 days. Memory-like natural killer (NK) cells produced by the method of Example 1 for 12-15 days were harvested, 5 x 10⁵ cells were taken per sample, washed three times with DPBS, and then mixed thoroughly with 98 μl of FACS staining buffer and 2 μl of human FcR blocking reagent. The mixture was then incubated at 0-4°C for 20 minutes. After the reaction, the cells were washed three times with 500 μl of DPBS. The washed cell precipitate was then resuspended in 90 μl of FACS staining buffer, and 2 μl each of anti-CD3 antibody (48-0038-82, eBioscience), anti-CD56 antibody (47-0567-42, eBioscience), anti-CD94 antibody (305508, BioLegend), anti-CD25 antibody (562661, BD Biosciences), and anti-NKp80 antibody (130-125-238, Miltenyi Biotech) were added and mixed well. The mixture was then incubated at 0-4°C for 30 minutes. After the reaction was complete, the cells were washed three times with 500 μl of DPBS. The washed cell precipitate was then resuspended in 200 μl of FACS staining buffer, and NK cells that were CD3-negative but CD56-positive were selected using flow cytometry. The expression levels of each identification marker were then analyzed.

[0083] Figure 3 shows the results of comparing receptor expression in memory-like natural killer (NK) cells compared to the negative control group. It was shown that CD94 and CD25 were increased and NKp80 was decreased in memory-like NK cells (Figure 3). [Examples]

[0084] Analysis of the proliferative capacity of NK cells

[0085] 5-1. Comparison of the number of memory-like natural killer cells (NK cells) produced relative to the total number of cells.

[0086] To confirm the proliferation ability of the memory-like natural killer (NK) cells produced in Example 1, the total number of cells harvested at the end was compared to the total number of cells initially cultured. The day the culture started was set as day 0 (day 0) as the baseline, and the total number of cells was counted day by day as the culture progressed. The cumulative proliferation rate was then calculated to determine how many times the cells proliferated compared to day 0. In addition, the degree of proliferation of all cells produced in Example 1 of the present invention was confirmed compared to a negative control group (control) cultured for 12 to 15 days with 2-10% (v / v) of autologous plasma and 1 ng / ml of IL-15 added to the culture medium.

[0087] [Table 3]

[0088] [Table 4]

[0089] The results showed that, although there was some variability among donors, cells proliferated by at least 35 times and an average of 77 times compared to the start of culture (Table 3 and Figure 4). In addition, all donors showed an average cell proliferation rate 12 times higher than the negative control group (Table 4 and Figure 5).

[0090] 5-1. Comparison of the number of memory-like natural killer cells (Memory-like NK cells) produced compared to the number of NK cells at the start of culture.

[0091] To confirm the proliferative capacity of the memory-like natural killer (NK) cells produced in Example 1, the number of cells that proliferated was calculated as the culture progressed, using the initial number of NK cells at the start of culture (day 0) as a baseline, and the number of cells that proliferated was analyzed. In addition, the degree of NK cell proliferation of the memory-like natural killer (NK) cells produced in Example 1 of the present invention (experimental group) was confirmed compared to a negative control group (control) cultured for 12 to 15 days with 2-10% (v / v) autologous plasma and 1 ng / ml of IL-15 added to the culture medium.

[0092] [Table 5]

[0093] [Table 6]

[0094] The results showed that, although there was variability among donors, NK cells proliferated by a minimum of 98 times and an average of 309 times (Table 5 and Figure 6). Furthermore, in all donors, the negative control group showed an average NK cell proliferation rate of 9.5 times, while the experimental group showed an average NK cell proliferation rate of 308.8 times (approximately 32 times), indicating significantly greater NK cell proliferation in the experimental group compared to the negative control group (Table 6 and Figure 7).

Claims

1. 1) The step of separating peripheral blood mononuclear cells (PBMCs) from peripheral blood, 2) A step of treating plasma with anti-NKp46 antibody, IL-2 and IL-18 and culturing it, 3) The step of transferring the cells to a new culture vessel, treating them with plasma and IL-2, and culturing them, 4) The step of transferring the cells to a new culture vessel and treating them with plasma, IL-12, IL-15, and IL-18, and culturing them, 5) The step of transferring the cells to a new culture vessel and treating them with plasma, IL-2 and IL-15, and culturing them, In a method for producing memory-like natural killer cells (Memory-like NK cells), A method for producing memory-like natural killer cells, characterized in that the memory-like natural killer cells have increased expression of CD94 and CD25 and decreased expression of NKp80 compared to NK cells.

2. The method for producing memory-like natural killer cells according to claim 1, wherein in step 2), the cells are cultured in a culture vessel coated with fibronectin, γ-globulin, and anti-CD56 antibody.

3. The method for producing memory-like natural killer cells according to claim 1, wherein the plasma in step 2) is treated with 1-20% (w / w), 0.01-10 μg / ml of anti-NKp46 antibody, 10-5000 IU / ml of IL-2, and 2-500 ng / ml of IL-18.

4. A method for producing memory-like natural killer cells according to claim 1, wherein step 2) is performed two or more times.

5. The method for producing memory-like natural killer cells according to claim 1, wherein the plasma in step 3) is treated with 1-20% (w / w) and IL-2 at 10-5000 IU / ml.

6. A method for producing memory-like natural killer cells according to claim 1, wherein step 3) is performed two or more times.

7. The method for producing memory-like natural killer cells according to claim 1, wherein the plasma in step 4) is treated with 1-20% (w / w), IL-12 at 0.5-200 ng / ml, IL-15 at 0.1-100 ng / ml, and IL-18 at 2-500 ng / ml.

8. The method for producing memory-like natural killer cells according to claim 1, wherein step 4) involves culturing for 10 to 20 hours.

9. The method for producing memory-like natural killer cells according to claim 1, wherein the plasma in step 5) is treated with 1-20% (w / w), IL-2 at 10-5000 IU / ml, and IL-15 at 0.1-100 ng / ml.

10. The method for producing memory-like natural killer cells according to claim 1, further comprising step 5) processing and culturing in a new medium containing IL-2.

11. The method for producing memory-like natural killer cells according to claim 10, wherein IL-2 is treated at a concentration of 10 to 5000 IU / ml.

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