Methods for selecting NK cell donor sources

Abstract

The present invention relates to a method for selecting a donor source for NK cells with improved proliferation capacity, which is characterized by identifying a specific single nucleotide polymorphism in the CISH gene. The present invention solves the problem of NK cell efficacy varying depending on the donor source in cell therapy agents containing NK cells, and enables the production of consistent NK cells with excellent proliferation capacity.

Description

[Technical Field]

[0001] The present invention relates to a method for selecting a donor source from which NK cells with improved cell proliferation ability can be obtained, and more particularly, to a method for selecting a donor source from which NK cells with improved proliferation ability can be obtained, characterized by checking a specific single nucleotide polymorphism in the CISH gene.

[0002] [Background technology]

[0003] Among the cells that make up the immune system, natural killer cells (NK cells) are known to have the ability to kill virus-infected cells and cancer cells through a comprehensive signal transduction balance between activating and inhibitory receptors, even without being sensitized to a specific antigen. The killing ability of NK cells has led to their use in the treatment of various hematologic malignancies and solid tumors, either alone or in combination with other therapies. While one method achieves anti-cancer effects by activating NK cells in vivo through the injection of drugs such as cytokines into the patient's body, another method involves activating and amplifying NK cells or genetically engineered NK cells ex vivo and then injecting them into the patient's body.

[0004] In order to effectively use NK cells in anti-cancer immune cell therapy, it is necessary to secure a large number of NK cells. However, NK cells account for 10% to 15% of lymphocytes in the blood, and in cancer patients, the number, differentiation, and function of NK cells are often reduced, making it difficult to secure a sufficient number of cells. Therefore, there is an urgent need to secure a large number of NK cells through NK cell proliferation or differentiation.

[0005] NK cells are generally derived from hematopoietic stem cells (HSCs) in the bone marrow and are known to be distributed in various tissues and blood, most commonly in the spleen, liver, bone marrow, peripheral blood, and umbilical cord blood. In vitro, various methods have been reported for isolating hematopoietic stem cells from umbilical cord blood and differentiating them into NK cells by treating them with appropriate cytokines and culturing them.

[0006] Previously reported methods for expanding NK cells have involved the use of high concentrations of various expensive cytokines or the use of peripheral blood mononuclear cells or cancer cell lines, which are not only expensive but also have limited expansion rates. However, recently, methods utilizing genetically engineered cancer cell lines (Imai C et al., Blood. 2005;106:376-83; Denman CJ et al., PLoS One. 2012;7:e30264; Thangaraj JL et al., Cancer Immunol Immunother. 2022;71:613-625) have been introduced, overcoming many of these limitations. However, to improve NK cell expansion rates, optimal expansion can be achieved by optimizing many culture conditions, including cytokine composition, trophoblast characteristics, medium composition, and the ratio of trophoblasts to mononuclear cells (peripheral blood, umbilical cord blood). Furthermore, many studies have shown that in addition to NK cells, other lymphocytes such as T cells are also expanded, which may indicate that NK cell-based immune cell therapy is inappropriate. Therefore, a method for selecting only NK cells with a high proliferation rate and high cancer cell killing ability is needed.

[0007] Therefore, in the present invention, we have made extensive efforts to develop a method for selecting individual samples from which NK cells with improved cell proliferation ability can be isolated. As a result, we have confirmed that NK cells isolated from sources with the TT allele type of rs414171, a single nucleotide polymorphism (SNP) in the CISH (cytokine-inducible SH2-containing protein) gene, have a significantly higher proliferation rate than those with other genotypes, thereby completing the present invention.

[0008]

[0009] Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a method for selecting a donor source for isolating NK cells with high cell proliferation capacity. [Means for solving the problem]

[0011]

[0012] To achieve the above object, the present invention provides a method for selecting a donor source of NK cells, which includes the following steps:

[0013] (a) determining the genotype of a single nucleotide polymorphism site (rs414171) in the CISH gene in the genome of a donor-derived sample; and

[0014] (b) selecting the donor source for NK cells if rs414171 is of the AT genotype or the TT genotype;

[0015] The present invention also provides a method for culturing NK cells, comprising the steps of:

[0016] (a) isolating NK cells from a donor source selected by the method; and

[0017] (b) A step of co-culturing NK cells with feeder cells and culturing the NK cells.

[0018] The present invention also provides NK cells cultured by the above method.

[0019] [Brief explanation of the drawings]

[0020] [Figure 1]FIG. 1 shows the results of genotyping a CISH single nucleotide polymorphism site (rs414171) using umbilical cord blood.

[0021] [Figure 2] FIG. 1 shows the results of confirming the proliferation rate and purity of NK cells based on CISH genotypes.

[0022]

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0025] In this invention, to solve the problem of NK cell efficacy varying depending on the donor source, we identified the genotype of the donor's single nucleotide polymorphism (SNP) and examined the proliferative potential of isolated NK cells according to the genotype. We then confirmed that there was a difference in the proliferative potential of isolated NK cells depending on the genotype of the rs414171 SNP derived from the CISH (cytokine-inducible SH2-containing protein) gene in umbilical cord blood.

[0026] Therefore, the present invention relates to a method for selecting a donor source of NK cells, comprising the following steps:

[0027] (a) determining the genotype of a single nucleotide polymorphism site (rs414171) in the CISH gene in the genome of a donor-derived sample; and

[0028] (b) selecting the donor source for NK cells if rs414171 is of the AT genotype or the TT genotype;

[0029] The CISH gene is one of the SOCS (suppressor of cytokine signaling) proteins and acts as a negative regulator of IL-15-mediated signal transduction in NK cells. It has been reported that CISH-deficient NK cells have improved proliferation, viability, and cytotoxicity (Huang Zhu, Cell Stem Cell, 6;27(2):224-237, 2020).

[0030] It has been reported that the rs414171 SNP, a single nucleotide polymorphism in the CISH gene, is associated with the incidence of infectious diseases such as tuberculosis and sepsis due to its mutation (Alizada, Azad. University of Toronto (Canada) ProQuest Dissertations Publishing, 2017. 10634550).

[0031] In the present invention, peripheral blood or umbilical cord blood can be used as the donor-derived sample.

[0032] In the present invention, the step (b) may be characterized by selecting the TT genotype as a donor source for NK cells.

[0033] In one embodiment of the present invention, the genotype of the CISH single nucleotide polymorphism site (rs414171) was analyzed using Tetra-primer amplification refractory mutation system (T-ARMS-PCR). Generally, any method that can confirm the SNP sequence can be used without limitation.

[0034] In one embodiment of the present invention, it was confirmed that NK cells derived from a sample having the T / T genotype at the single nucleotide polymorphism site (rs414171) had a significantly higher proliferation rate than those derived from a sample having the A / A or A / T genotype (Figure 2).

[0035] As used herein, the term "natural killer cells" or "NK cells" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system and are defined as large granular lymphocytes (LGLs). The term "natural killer cells" or "NK cells" includes natural killer cells derived from any tissue source without further transformation, including not only mature natural killer cells but also natural killer progenitor cells. Natural killer cells are activated in response to interferon or macrophage-derived cytokines and contain two types of surface receptors, labeled "activating receptors" and "inhibitory receptors," that control the cytotoxic activity of the cells. Natural killer cells can be generated from hematopoietic cells, e.g., hematopoietic stem cells or progenitors, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, or liver.

[0036] In one embodiment, the natural killer cells may be activated natural killer cells, which may refer to cells in which the cytotoxicity or inherent immunomodulatory ability of natural killer cells is activated when compared to parent cells, such as hematopoietic cells or natural killer progenitor cells.

[0037] In one embodiment, non-limiting examples of NK cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. Thus, in a specific embodiment, the NK cells can be any one selected from the group consisting of macrophages, B lymphocytes, T lymphocytes (CD8+ CTLs), mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. In a specific embodiment, the NK cells can be natural killer cells or T lymphocytes. The mixed NK cells can include one or more selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils.

[0038] In specific examples, activated innate killer cells or enriched populations of activated innate killer cells can be assessed by detecting one or more functionally associated markers, such as CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIR (e.g., KIR2DL1, KIR2DL2 / 3, KIR3DL1), and the NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D).

[0039] In another aspect, the present invention relates to a method for culturing NK cells, comprising the steps of:

[0040] (a) isolating NK cells from a donor source selected by the method; and

[0041] (b) A step of co-culturing NK cells with feeder cells and culturing the NK cells.

[0042] In the present invention, the feeder cells can be characterized as being K562 cells that express membrane-bound IL-18 and membrane-bound IL-21 on their surface.

[0043] In yet another aspect, the present invention relates to NK cells cultured by the above method.

[0044] As used herein, the term "stimulation of NK cells" may mean increasing the activity, e.g., cytotoxic activity, of natural killer cells in vitro or in vivo, or generating, increasing, amplifying, or proliferating activated natural killer cells.

[0045] In one aspect of the present invention, a cell line genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21) is used as a feeder cell.

[0046] As used herein, the term "feeder cells (also referred to as support cells or culture auxiliary cells)" refers to cells that are not capable of dividing and proliferating when irradiated but are metabolically active and produce various metabolic substances to support the proliferation of target NK cells. Examples of feeder cells that can be used herein include transgenic animal cell lines, such as human chronic myeloid leukemia cell lines (e.g., K562 cells), RPMI8866, EBV_LCL, 721221, and HFWT. In one embodiment of the present invention, K562 cells were used.

[0047] As used herein, the terms "genetic engineering" or "genetically engineered" refer to the act of introducing one or more genetic modifications into a cell or the cell produced thereby.

[0048] More specifically, the genetically engineered cell can include an exogenous gene encoding the referenced gene. The term "exogenous" means that the referenced molecule or activity has been introduced into the host cell. The molecule can be introduced into the host genetic material as an encoding nucleic acid, such as by insertion into a host chromosome, or as non-chromosomal genetic material, such as a plasmid. In the context of expression of an encoding nucleic acid, the term "exogenous" indicates that the encoding nucleic acid has been introduced into an individual in an expressible form. In the context of a biosynthetic activity, the term "exogenous" refers to an activity introduced into a host parent cell. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity after being introduced into the host parent cell. Thus, the term "endogenous" refers to a referenced molecule or activity present in the host cell. Similarly, in relation to expression of an encoding nucleic acid, the term "endogenous" refers to expression of an encoding nucleic acid contained within an individual. The term "heterologous" refers to a molecule or activity from a source other than the referenced species, and the term "homologous" refers to a molecule or activity from the host parent cell. Thus, exogenous expression of an encoding nucleic acid can use either or both heterologous and homologous encoding nucleic acids.

[0049] Thus, the genetically engineered feeder cells of the present invention can contain nucleic acids encoding mbIL-18 and mbIL-21. More specifically, the cells can be transformed with vectors containing nucleic acids encoding mbIL-18 and mbIL-21.

[0050] As used herein, the term "vector" refers to a genetic construct capable of expressing a target protein in a suitable host cell, comprising regulatory elements operably linked to a gene insert for expression. In one embodiment, the vector may comprise expression regulatory elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and / or enhancer. The promoter of the vector may be constitutive or inducible. The vector may also be an expression vector capable of stably expressing the fusion protein in host cells. The expression vector may be a conventional vector used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by various methods known in the art. For example, the vector may comprise a selectable marker for selecting host cells containing the vector, and, in the case of a replicable vector, may comprise a replication origin. The vector may be autonomously replicating or may be integrated into host DNA. The vector may be selected from the group consisting of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus, and a vaccinia virus.

[0051] In addition, in the vector, the polynucleotide sequence encoding the above-mentioned fusion protein may be operably linked to a promoter. As used herein, the term "operably linked" refers to the functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0052] As used herein, "membrane-bound interleukin" refers to an interleukin that is bound to the cell membrane, and may be distinguished from an interleukin that is secreted outside the cell.

[0053] In one embodiment, the co-culture may be performed in the presence of cytokines.

[0054] In one embodiment, the cytokine is an interleukin or a mutant thereof. Many interleukins are synthesized by monocytes, macrophages, and endothelial cells, as well as auxiliary CD4 T lymphocytes. Each interleukin can promote the development and differentiation of T and B lymphocytes and various hematopoietic cells. Non-limiting examples of each interleukin include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, and IL36. Thus, in certain embodiments, the cytokine is an interleukin or mutant thereof, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36.

[0055] In another specific example, the cytokine added to the co-culture may be IL-2. More specifically, the IL-2 may be used at a concentration of 1 U / ml to 200 U / ml, preferably 1 U / ml to 100 U / ml, more preferably 1 U / ml to 50 U / ml, and most preferably 1 U / ml to 20 U / ml.

[0056] In another specific example, the cytokine added for the restimulation may be IL-2. More specifically, the IL-2 may be used at a concentration of 10 U / ml to 500 U / ml, preferably 10 U / ml to 300 U / ml, and more preferably 10 U / ml to 250 U / ml.

[0057]

[0058] [Example]

[0059] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0060]

[0061]

[0062] Example 1. Genotyping of CISH single nucleotide polymorphism site (rs414171)

[0063] Approximately 200 μl of umbilical cord blood was collected from a healthy donor (Samsung Seoul Medical Center), and genomic DNA (gDNA) was extracted using a QIAamp DNA mini kit (QIAGEN, Hilden, Germany). The extracted gDNA was diluted in nuclease-free distilled water to a concentration of 10 ng / μl and used as template DNA.

[0064] The genotype of the CISH single nucleotide polymorphism site (rs414171) was analyzed through T-ARMS-PCR (Tetra-primer amplification refractory mutation system-PCR).

[0065] Two external primers were used as a positive control for the CISH gene, and two internal primers were used to detect the A or T allele of the -292nd genotype of CISH (rs414171). The specific base sequences are as follows:

[0066] [Table 1]

[0067] 50 ng of gDNA, 0.5 μM of each of the four primers, and nuclease-free distilled water were mixed to a total volume of 20 μL. This was then mixed with AccuPower PCR PreMix (Bioneer, Korea) and amplified by PCR using a VeritiPro Thermal Cycler (Applied Biosystems, CA, USA). The PCR reaction consisted of 30 cycles of pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 68°C for 30 seconds, and extension at 72°C for 30 seconds. The PCR product was diluted 1 / 10 in nuclease-free distilled water and analyzed by E-Gel. TM 20 μL of each sample was loaded onto EX Agarose Gels, 2% (Invitrogen, CA, USA). TM The results were detected through electrophoresis and gel imaging using a Power Snap Electrophoresis System (Invitrogen, CA, USA).

[0068] [Table 2]

[0069] In all samples, a 452-bp band representing the CISH control gene is detected. If the CISH genotype at position -292 (rs414171) is A, an additional 290-bp band is detected. If the genotype is T, an additional 208-bp band is detected. Depending on the homozygous or heterozygous genotype, bands can be detected in a total of three cases: A / A, A / T, and T / T (Figure 1).

[0070]

[0071] Example 2. Selection of donors with the T / T genotype at the CISH single nucleotide polymorphism site (rs414171) and isolation of blood-derived mononuclear cells (MNC)

[0072]

[0073] 2-1: Selection of T / T homozygotes of rs414171

[0074] Using the method described in Example 1, umbilical cord blood samples (Samsung Seoul Medical Center) collected from healthy donors were selected for those with T / T homozygotes at CISH −292 genotype (rs414171), and Dulbecco's phosphate buffered saline (DPBS, WELGENE) containing 2% (v / v) FBS (Fetal bovine serum, ThermoFisher, USA) was added to the samples at a 1:1 ratio.

[0075] 2-2: Isolation of mononuclear cells

[0076] Mononuclear cells were isolated from cord blood using Lymphoprep (Serumwerk, Bernburg AG, Oslo, Norway) density gradient centrifugation and suspended in NK MACS medium (MiltenyiBiotec, Bergisch Gladbach, FR1) containing 20 U / mL IL-2 (Peprotech, Rocky Hill, NJ), 1% (v / v) NK MACS supplements, and 10% (v / v) human AB serum (Sigma, USA). Cell counts were measured using a LUNA-FX7 automated cell counter (Aligned Genetics, Korea).

[0077] To determine the number of feeder cells to be added, we co-cultured NK cells (NK cells) with genetically engineered feeder cells at a 1:5 ratio. Specifically, we confirmed the ratio of NK cells to mononuclear cells. Specifically, we used 2.0 × 10 mononuclear cells. 5 Cells were harvested, washed with DPBS, and stained with Fixable Viability Stain 780 (BD Biosciences, San Jose, CA) for 15 minutes. Then, after washing with DPBS buffer containing 2% (v / v) FBS, they were additionally stained with anti-human CD3-FITC and anti-human CD56-APC (BD Biosciences, San Jose, CA) for 30 minutes at 4°C. The cells were washed again with DPBS containing 2% (v / v) FBS, and data were acquired using CytoFLEX (Beckman Coulter, Brea, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR).

[0078] 2-3: Preparation of genetically engineered feeder cells

[0079] K562 cells expressing membrane-bound IL-18 and membrane-bound IL-21 on their surface were prepared using the method described in Korean Patent Application Publication No. 10-2021-0152934. The genetically engineered K562-mbIL-18 / 21 cell line was cultured in an appropriately sized T-flask containing RPMI 1640 (Gibco, USA) medium supplemented with 10% (v / v) FBS in a 37°C, 5% CO2 incubator. The cells were then collected in a culture flask, centrifuged at 400 g for 3 minutes, suspended in RPMI 1640 medium supplemented with 10% (v / v) FBS, and inactivated by irradiating at 100 Gy using a Gammacell 3000 Elan gamma irradiator. The cells were centrifuged again at 400 g for 3 minutes and suspended in NK MACS medium containing 20 U / mL IL-2, 1% (v / v) NK MACS supplement, and 10% (v / v) human AB serum.

[0080]

[0081] Example 3: Cultivation of natural killing cells using mononuclear cells with the T / T genotype at the CISH single nucleotide polymorphism site (rs414171)

[0082] On day 0 of culture, 1.25 × 10 cells of the umbilical cord blood-derived mononuclear cells were cultured in a G-Rex24 culture vessel (Wilson Wolf, Saint Paul, MN). 5 cells / cm 2 The genetically engineered feeder cells were dispensed at a ratio of 1:5 to the naturally killed cell number of the umbilical cord blood-derived mononuclear cells. NK-MACS medium containing 20 U / mL IL-2, 1% (v / v) NK-MACS supplement, and 10% (v / v) human AB serum was added to a total volume of 8 mL and cultured at 37°C in a 5% CO2 incubator for 7 days.

[0083] During the 7-day culture period, approximately 20 μL of culture supernatant was collected every 3 to 5 days, and the glucose concentration was measured using a CareSens N Voice blood glucose meter (i-Sens, Korea). When the glucose concentration was 50 to 100 mg / dL, 4 mL of culture supernatant was carefully removed and replaced with 4 mL of NK-MACS medium containing 20 U / mL IL-2, 1% (v / v) NK-MACS supplement, and 10% (v / v) human AB serum, followed by half of the medium volume.

[0084] On day 7 of culture, the culture supernatant was carefully removed from the G-Rex24 culture vessel for restimulation of the genetically engineered feeder cells, and the cell number was measured using a LUNA-FX7 automated cell counter. 2.0 × 10 cultured cells were counted as described in Example 2. 5 The purity of the naturally killed cells was confirmed. Based on the total number of cells, the cell density was 1.25 × 10 5 cells / cm 2 ~4.0×10 7 cells / cm 2 The cells were maintained at 1:5 and restimulated with genetically engineered feeder cells prepared as described in Example 2. NK MACS medium containing 200 U / mL IL-2, 10 ng / mL IL-15, 1% (v / v) NK MACS supplement, and 10% (v / v) human AB serum was added to the medium up to a total volume of 8 mL, and the cells were cultured.

[0085] During the culture period up to day 21, the glucose concentration was measured every 3 to 5 days, and half of the culture medium was replaced at appropriate times. The cell count was measured every 5 to 7 days, and the time for subculture was determined. The cell density was 1.25 × 10 based on the total number of cells. 5 cells / cm 2 ~4.0×10 7 cells / cm 2The cells were diluted with NK MACS medium containing 200 U / mL IL-2, 10 ng / mL IL-15, 1% (v / v) NK MACS supplement, and 10% (v / v) human AB serum to obtain a total cell count of 1000 cells / mL.

[0086]

[0087] Example 4: Confirmation of the purity and amplification rate of spontaneously killed cells cultured from mononuclear cells with the T / T genotype at the CISH single nucleotide polymorphism site (rs414171)

[0088] In the culture according to Example 3, the total number of cells was measured every 7 and 14 days of culture using a LUNA-FX7 automated cell counter, and the purity of the naturally killed cells was analyzed using Fixable Viability Stain 780, anti-human CD3-FITC, and anti-human CD56-APC according to Example 2. The total number of naturally killed cells calculated every 7 days was compared with the number of naturally killed cells on day 0 of culture to evaluate the expansion rate.

[0089] As a result, as shown in Figure 2, it was confirmed that NK cells derived from samples with the allele type of the single nucleotide polymorphism site (rs414171) T / T genotype had a significantly higher proliferation rate than those with the allele types A / A or A / T.

[0090] [Industrial Applicability]

[0091] According to the present invention, the problem of the efficacy of NK cells varying depending on the donor source in cell therapy agents containing NK cells is solved, and NK cells with excellent proliferation capacity and consistency can be produced.

[0092]

[0093] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

[0094] [Sequence List Free Text]

[0095] Electronic file attached.

Claims

1. A method for selecting a donor source of NK cells, comprising the steps of: (a) determining the genotype of rs414171, a single nucleotide polymorphism site in the CISH gene, in the genome of a donor-derived sample; and (b) selecting the subject as a donor source for NK cells if rs414171 is of the AT genotype or the TT genotype;

2. The method of claim 1, wherein the donor-derived sample is peripheral blood or umbilical cord blood.

3. The method according to claim 1, wherein in step (b), if the patient has the TT genotype, the patient is selected as a donor source for NK cells.

4. A method for culturing NK cells, comprising the steps of: (a) isolating NK cells from a donor source selected by the method of any one of claims 1 to 3; and (b) co-culturing NK cells with feeder cells and culturing the NK cells;

5. The method according to claim 4, wherein the feeder cells are K562 cells that express membrane-bound IL-18 and membrane-bound IL-21 on their surface.

6. NK cells cultured by the method of claim 4.

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