Method for producing cell population comprising NK cells

Abstract

To provide an effective method for producing a population of NK cells for cell-based therapy, to improve the in vitro amplification efficiency of NK cells, and to flexibly increase signals required for NK cell licensing.SOLUTION: Provided is a method for producing a cell population comprising NK cells, which comprises: preparing a population of mononuclear cells that are derived from multiple donors and comprise NK cells; incubating and culturing the prepared mononuclear cell population under conditions effective for NK cell treatment and proliferation; and growing NK cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing a cell population including natural killer cells (NK cells) and to its use. [Background technology]

[0002] Malignant tumors are the leading cause of death in Japan, making countermeasures an urgent necessity. In particular, the development of new treatment methods for advanced, refractory malignant tumors that are resistant to existing treatments consisting of surgery, radiation therapy, and chemotherapy is extremely important and significant. In recent years, immunotherapy, such as immune checkpoint inhibitors and chimeric antigen receptor (CAR) gene-modified T cell therapy (CAR-T therapy), has attracted attention as a fourth treatment method. However, since most of these methods use T cells that are activated by recognizing antigens as effectors, they suffer from a fundamental limitation: restriction to specific antigens.

[0003] As an immunotherapy utilizing NK cells, which act as a major factor in innate immunity, NK cell therapy, in which NK cells are proliferated in vitro and then administered to patients, is attracting attention as a treatment method with relatively few side effects. However, the number of NK cells that can be obtained from peripheral blood etc. is relatively small, and their proliferative capacity in vitro is low. For this reason, techniques for culturing and proliferating NK cells have been investigated. For example, Patent Document 1 proposes a method for amplifying NK cells, characterized by comprising the steps of preparing a cell population containing NK cells, removing T cells from the cell population containing NK cells, and culturing the remaining cells without using feeder cells in a medium containing only IL-2 as a cytokine, and a pharmaceutical composition for cell therapy containing a cell population containing NK cells obtained by amplification. Furthermore, Patent Document 2 proposes a method for preparing NK cells, comprising the steps of amplifying hematopoietic progenitor cells under a single culture condition containing IL-15, SCF, IL-7, and Flt3L, and differentiating the cells obtained in the amplification step into NK cells under a culture condition containing IL-2 for 5, 6, 7, 8, or 9 days, as well as a pharmaceutical composition for cell therapy containing a cell population including the prepared NK cells.

[0004] Furthermore, the present inventors have reported NK cells that are CD16-positive, highly expressive of CD56, negative for CD57, positive for NKG2C, negative to low-expressing for NKG2A, and positive for CD94, as well as a cell population containing such NK cells (Patent Document 3), as cultured cells with enhanced tumor cytotoxic activity. They have also reported on CD3-negative cells that express chemokine receptors and cell adhesion molecules (Patent Document 4).

[0005] On the other hand, NK cells express a group of receptors that recognize HLA class I on their cell surface. Among these receptors, the KIR (Killer cell Immunoglobulin-like Receptor) family exhibits diversity similar to HLA. In hematopoietic stem cell transplantation, rather than matching HLA as much as possible, intentional KIR / HLA mismatch between donor and recipient, such as selecting donors so that the recipient does not possess ligands for the KIRs of donor NK cells, is increasingly being utilized as it can lead to more desirable results in terms of relapse, GVHD (graft-versus-host disease), and prognosis. However, regarding the antitumor activity of NK cells, a report (Non-Patent Literature 1) that investigated the extent to which HLA class I-KIR interaction affects human NK cell proliferation in an allogeneic environment by culturing NK cells with feeder cells that are either matched or mismatched (lacking one or more ligands present in the NK cell donor) in terms of inhibitory KIR ligands suggests that if high activation of NK cells is desired with antitumor effect as an indicator, less signaling from KIR is preferable. Furthermore, recent reports (Non-Patent Literature 2) on the allogeneic response of HLA heterozygous NK cells (in the case of unidirectional matching) to tissues derived from HLA haplotype homozygous (HLA homo) iPS cells strongly suggest that mixed cultures including HLA / KIR mismatches are not feasible when culturing NK cells. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2013-27385 (Japanese Patent No. 5572863, Japanese Patent No. 5989016) [Patent Document 2] Japanese Patent Publication No. 2014-226079 (Japanese Patent No. 5511039, Japanese Patent No. 6164650) [Patent Document 3] Japanese Patent Publication No. 2018-193303 [Patent Document 4] Specification of Japanese Patent Application No. 2018-059624 (not published at the time of filing of the present application)

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Disclosure of the Invention

Problems to be Solved by the Invention

[0008] The inventors are working on the development of a cell population containing highly active NK cells. However, since there is a limit to the amount of peripheral blood or blood obtained by apheresis, which is the raw material, it is not possible to make the therapeutic NK cell population off-the-shelf for future treatment.

[0009] On the other hand, according to the study by the inventors, the in vitro amplification efficiency of NK cells may be extremely poor depending on the donor.

[0010] Furthermore, in the body, immature NK cells that have experienced binding to a ligand (HLA class I) during the differentiation process from hematopoietic stem cells in the bone marrow are thought to be "licensed," and after maturation, they are thought to possess the ability of a "missing-self response" to detect cells with reduced expression of the original HLA class I. However, NK cells differentiated and matured from iPS cells or ES cells may not have sufficient signals necessary for licensing to obtain an antitumor effect. [Means for solving the problem]

[0011] The present invention provides the following: [1] Prepare a population of mononuclear cells, including NK cells, derived from multiple donors. The prepared mononuclear cell population is incubated under conditions effective for NK cell treatment. A method for producing a cell population including NK cells, including the following. [2] The method for producing mononuclear cells according to claim 1, wherein the step of preparing a population of mononuclear cells includes the step of removing CD3-positive cells. [3] The method of production according to 1 or 2, wherein the step of preparing a population of mononuclear cells includes a step of removing CD34-positive cells. [4] The manufacturing method according to any one of items 1 to 3, wherein the step of preparing a population of mononuclear cells includes the step of obtaining a population of mononuclear cells from peripheral blood collected from multiple donors. [5] The manufacturing method according to any one of items 1 to 4, wherein the step of preparing a population of mononuclear cells includes the step of obtaining a population of mononuclear cells from apheresis blood collected from multiple donors. [6] The method of manufacture according to any one of items 1 to 3, wherein the step of preparing a population of mononuclear cells is to prepare a population of mononuclear cells selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells derived from multiple donors. [7] A method of manufacturing according to any one of paragraphs 1 to 6, wherein the multiple donors include one donor and other donors whose genotype differs from that of at least one of HLA and KIR. [8] A cell population including NK cells that has the following characteristics: (1) Derived from multiple donors. (2) When NK cells are used as effector cells (E) and K562 cells are used as target cells (T) in a mixed ratio (E:T) of 1:1, the cytotoxic activity is 50% or more. [9] A pharmaceutical composition for cell therapy comprising a cell population produced by any one of the manufacturing methods described in items 1 to 7.

[10] A pharmaceutical composition for cell therapy comprising the cell population described in 8.

[11] A pharmaceutical composition according to 9 or 10 for the treatment of infectious diseases and / or cancer. [Effects of the Invention]

[0012] Since it is possible to mix blood or PBMC from multiple individuals as raw materials, the amount of raw materials can be increased, and off-the-shelf storage can be expected.

[0013] This method allows for stable proliferation of NK cells regardless of the donor, and also improves the proliferation rate.

[0014] This allows for adaptation to the diversification of HLA-KIR matching, and stable activation can be expected.

[0015] This makes it possible to flexibly increase the signals necessary for NK cell licensing, which is expected to improve the antitumor effect of NK cell populations derived from iPS cells and ES cells. [Brief explanation of the drawing]

[0016] [Figure 1] Culture test: CD3-positive cells were removed from PBMCs from multiple individuals and cultured in KBM-501 medium for 14 days. [Figure 2] Summary of experimental results (left) and statistical analysis (right): Mixed culture resulted in a significantly improved growth rate compared to cultures derived from single donors. [Figure 3] Tumor cytotoxicity test: The cytotoxic activity of NK cells prepared from mixed cultures and single-donor cultures was evaluated using damage to SKOV3 (human ovarian cancer cell line) as an indicator. [Figure 4-1] Culture was performed using 500 cm² culture bags. The bags were turned inside out 30 minutes after the start of culture and again on the 9th day of culture. [Figure 4-2] Culture test. Frozen apheresis blood (from two donors) was used as the material. After removing CD3-positive and CD34-positive cells using an automated closed cell processing system, the cells were cultured for 14 days in KBM-501 medium using a T75 flask or a 500 cm² culture bag. [Figure 5] Monocyte / NK cell swapping experiment: The CD16-high population increased when signals from either KIR3DL1 or KIR3DS1 were present, or both. The CD16-high population is expected to have higher ADCC activity. [Modes for carrying out the invention]

[0017] This invention relates to a cell population including NK cells.

[0018] [Production of cell populations] The cell population containing NK cells of the present invention can be produced by a method comprising the following steps: We prepared a population of mononuclear cells, including NK cells, derived from multiple donors. The prepared mononuclear cell population is incubated under conditions effective for NK cell treatment.

[0019] (Preparation of a population of mononuclear cells derived from multiple donors) The cell population of the present invention is obtained from a population of mononuclear cells derived from multiple donors. The population of mononuclear cells referred to herein includes NK cells, but may also include other mononuclear cells, such as monocytes. The NK cells may be primary NK cells (NK cells collected from a living organism and not passaged).

[0020] A population of mononuclear cells derived from multiple donors, including NK cells, may be a mixture of a population of mononuclear cells (including NK cells and possibly monocytes) obtained from one donor and a population of mononuclear cells (including NK cells and possibly monocytes) obtained from another donor, or a mixture of NK cells obtained from one donor and monocytes obtained from another donor.

[0021] Regarding donors, "multiple" means two or more, and is not particularly limited as long as the above conditions are met. For example, a mixture of mononuclear cells derived from three or more, four or more, seven or more, or nine or more donors can be used. Multiple donors may include the patient themselves to whom the cell population containing NK cells is administered, or close relatives of the patient.

[0022] Multiple donors can be selected to include one donor and other donors whose genotype differs from that donor in at least one of the HLA and KIR categories. KIRs include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR3DL3, KIR2DL5A, and KIR2DL5B.

[0023] In relation to NK cells, licensing refers to the process by which NK cells acquire the ability to perform a "missing-self response" (detection of the absence of their own HLA) during the differentiation and maturation of the cells. In humans and mice, it is believed that only immature NK cells that have experienced the binding of KIR or NKG2A to HLA class I molecules during the differentiation of hematopoietic stem cells in the bone marrow are licensed and acquire the ability to detect cells with reduced expression of their own HLA class I after maturation. Examples of KIRs known to be involved in licensing are KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A / CD94. The HLA types that NK cells can recognize are the classical HLA types: all HLA-C allotypes, about one-third of the HLA-B allotypes, some of the HLA-A allotypes (those with the Bw4 motif), and the non-classical HLA types HLA-E and HLA-G. In the present invention, when selecting a plurality of donors to include one donor and other donors whose genotype differs from that donor in at least one of HLA and KIR, particular consideration can be given to the KIR and HLA related to the licensing as described above.

[0024] In one preferred embodiment of the present invention, donors with different genotypes can be selected to increase the repertoire of molecules available for licensing NK cells. Alternatively, the selection may be made so as not to stimulate the inhibitory KIRs of NK cells, or so as to stimulate them.

[0025] When selecting a donor, if you expect antibody-dependent cellular cytotoxicity (ADCC) activity by NK cells, choose a donor that has CD16 cells when culturing a mixed population of mononuclear cells. high It may also be performed to increase the number of NK cells. ADCC is known as one of the mechanisms of cytotoxicity by NK cells, and NK cells bind to antibodies bound to target cells via the Fc receptor CD16 on their cell surface, thereby damaging the target cells. Therefore, CD16 highAn increase in NK cells is expected to lead to higher ADCC activity. An example of such a donor combination is to select and mix donors so that NK cells are licensed by signals from one or both KIR3DL1 and KIR3DS1.

[0026] Mixing is performed before administering NK cells to the target. Mixing can be carried out at various stages, as long as the desired effect of the mixture is achieved. For example, blood collected from multiple donors may be mixed, and then a population of mononuclear cells may be obtained from the mixed mixture. Alternatively, a population of mononuclear cells may be obtained from the blood collected from each donor, and then these populations may be mixed. Alternatively, the populations of mononuclear cells collected from multiple donors may be cultured individually, and then these populations may be mixed. From the viewpoint of promoting good proliferation, it is preferable to perform the mixing before culturing.

[0027] The mixing ratio of mononuclear cell populations derived from multiple donors can be adjusted as appropriate. For example, cells from multiple donors can be included in approximately equal proportions, or cells from a specific donor can be included in larger or smaller proportions. When mixing NK cells from one donor with monocytes from another donor, the mixing ratio can also be adjusted as appropriate. For example, 1 to 9 times the number of monocytes can be mixed with 1 NK cell (NK cell:monocyte = 1:1 to 9), or 2 to 4 times the number of monocytes can be mixed with 1 NK cell (NK cell:monocyte = 1:2 to 4). In this invention, when expressing the mixing ratio of cells, it is based on the number of cells unless otherwise specified.

[0028] As raw materials for obtaining a population of mononuclear cells, peripheral blood, umbilical cord blood, bone marrow, and / or blood cells collected from lymph nodes can be used. Peripheral blood is one preferred raw material, and peripheral blood may be collected by apheresis. That is, in a preferred embodiment of the present invention, the step of preparing a population of mononuclear cells includes the step of obtaining a population of mononuclear cells from peripheral blood collected from multiple donors. In another preferred embodiment, the step of preparing a population of mononuclear cells includes the step of obtaining a population of mononuclear cells from apheresis blood collected from multiple donors.

[0029] A population of mononuclear cells, including NK cells and monocytes, can be prepared using various procedures known to those skilled in the art. For example, from blood such as peripheral blood and umbilical cord blood, red blood cells can be removed and mononuclear cell fractions recovered by density centrifugation at room temperature. Peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood contain a variety of lymphocytes, including T cells, B cells, NK cells, monocytes, and dendritic cells. In relation to the present invention, mononuclear cells that are CD3-negative and CD56-positive can be called NK cells. NK cells can be prepared using various procedures known to those skilled in the art. For example, methods are known for separating NK cells from whole blood or PBMCs by removing unwanted cells. Monocytes can be prepared using various procedures known to those skilled in the art. For example, methods are known for separating monocytes from whole blood or PBMCs by removing unwanted cells.

[0030] (Removal of CD3-positive cells, removal of CD34-positive cells) A population of mononuclear cells may include not only monocytes and NK cells, but also NK cell precursors, T cells, NKT cells, hematopoietic progenitor cells, etc. Desired NK cells may be selected after amplification using methods such as gravity centrifugation, immunomagnetic beads, FACS, or flow cytometry. For example, NK cells may be selectively isolated from a cell population using anti-CD3 antibodies, anti-CD16 antibodies, anti-CD34 antibodies, anti-CD56 antibodies, anti-CD69 antibodies, anti-CD94 antibodies, anti-CD107a antibodies, anti-KIR3DL1 antibodies, anti-KIR3DL2 antibodies, anti-KIR2DL3 antibodies, anti-KIR2DL1 antibodies, anti-KIR2DS1 antibodies, anti-KIR2DL5 antibodies, anti-NKp46 antibodies, anti-NKp30 antibodies, anti-NKG2D antibodies, etc. Antibodies may be monoclonal or polyclonal. NK cell selection may also be performed by selectively removing T cells, NKT cells, hematopoietic progenitor cells, and other cells.

[0031] The process of preparing a population of mononuclear cells may include a step of removing T cells, which may be achieved by removing CD3-positive cells. The process of preparing a population of mononuclear cells may also include a step of removing hematopoietic progenitor cells, which may be achieved by removing CD34-positive cells. In other words, the process of preparing a population of mononuclear cells may include a step of removing CD3-positive cells, or a step of removing CD34-positive cells.

[0032] The removal of CD3-positive cells and CD34-positive cells can be carried out by separating and removing cells expressing the cell surface antigens CD3 and / or CD34 using immunomagnetic beads such as Dynabeads from Dynal Biotech (sold by Invitrogen) or CliniMACS from Milteny Biotech. It is preferable to carry out such a process in a way that does not exhaust NK cells and does not cause monocytes to exert excessive phagocytic function. Specifically, preferred procedures include using relatively small magnetic beads (unreacted beads that are not bound to cells can be removed along with the supernatant by centrifugation), setting a relatively short incubation time between the beads and the cell population at low temperature, and obtaining the target cell population of cells that are not bound to beads using a column after removing the unreacted beads. Cell removal may also be carried out using an automated closed-system cell processing device.

[0033] (Use of iPS cells, etc.) The mononuclear cell population may be prepared from hematopoietic stem cells derived from any of the following groups: embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Methods for inducing mononuclear cells, including NK cells, from these stem cells are known and can be applied to the present invention by those skilled in the art (Domogala A. et al., Natural killer cell immunotherapy: from bench to bedside, Frontiers in Immunology, 2015;6, doi: 10.3389 / fimmu.2015.00264, and Zeng J. et al., Generation of "Off-the-Shelf" Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells, Stem Cell Reports, 2017;9:1796-1812). Stem cells may also be modified to obtain highly active NK cells; for example, high-affinity CD16 (158V: amino acid 158 is valine) is known, and this knowledge can be applied.

[0034] (Culturing a population of mononuclear cells) The prepared population of mononuclear cells, mixed with cells from multiple donors, is incubated under conditions effective for NK cell treatment. Incubation may or may not involve cell proliferation. In one preferred embodiment of the present invention, incubation involves proliferation. Incubation with proliferation can be rephrased as culture or proliferation.

[0035] Effective conditions for NK cell treatment refer to conditions suitable for a mixture of cells derived from multiple donors to produce the desired effect. As long as the desired effect is achieved, there are no particular limitations on the culture medium or isotonic solution used for cell suspension, time, temperature, environment, etc.

[0036] Conditions effective for NK cell treatment include conditions effective for NK cell activation. Such conditions include, for example, incubation for 4 to 18 hours at 37°C, in a 5% CO2 and saturated water vapor atmosphere, in the presence of activation factors such as IL-2 and IL-15 at appropriate concentrations.

[0037] Conditions effective for NK cell treatment include conditions effective for NK cell proliferation. Conditions effective for NK cell proliferation include using a culture medium suitable for NK cell proliferation. Examples of such media include, but are not limited to, KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Celgenics, Iwai Chemicals Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Inc.), IMDM, MEM, DMEM, RPMI-1640, etc.

[0038] Interleukin-2 (IL-2) may be added to the culture medium at a concentration sufficient to achieve the objectives of the present invention. The concentration of IL-2 may exceed 2000 IU / mL and may range from 2500 to 2813 IU / mL. IL-2 preferably has a human amino acid sequence and, for safety reasons, is preferably produced using recombinant DNA technology. The concentration of IL-2 may be expressed in Japanese Standard Units (JRU) and International Units (IU). Since 1 IU is approximately 0.622 JRU, 1750 JRU / mL is approximately 2813 IU / mL.

[0039] The culture medium may contain the subject's autologous serum, human type AB serum available from BioWhittaker and others, or donated human serum albumin available from the Japanese Red Cross Society. Autologous serum and human type AB serum are preferably added at a concentration of 1-10%, and donated human serum albumin is also preferably added at a concentration of 1-10%. Subjects may include healthy individuals and patients suffering from diseases. In addition, the culture medium may contain compositions formulated for the proliferation of immune cells, either in place of or in conjunction with serum. Such compositions are commercially available. For example, the UltraGro series (AventaCell) and CTS Immune Cell SR (Thermo Fisher Scientific) can also be used for this invention.

[0040] The culture medium may contain appropriate proteins, cytokines, antibodies, compounds, and other components, provided that they do not impair the amplification effect of NK cells. Cytokines may include interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (Flt3L). IL-3, IL-7, IL-12, IL-15, IL-21, SCF, and Flt3L preferably have human amino acid sequences and, for safety reasons, are preferably produced using recombinant DNA technology.

[0041] The culture medium can be changed at any time after the start of culture, provided that the desired number of NK cells is obtained, but it is preferable to change it every 3 to 5 days.

[0042] Culture vessels include, but are not limited to, commercially available dishes, flasks, plates, and multiwell plates. To obtain cell populations containing therapeutic NK cells, it is preferable to use culture vessels that can culture a large number of cells and are easy to handle. An example of such a culture vessel is a cell culture bag made of a material with high gas permeability.

[0043] When using a bag, it is preferable to invert it during the culture period. During culture, both NK cells and monocytes exhibit adhesion to the culture surface. For adherent cells, both the cell density per unit volume and the cell density per unit area greatly affect the culture efficiency, including cell viability and proliferation rate. By inverting the bag during the culture period, some NK cells and monocytes can be moved to the side that was previously the upper side and therefore has relatively less cell adhesion, thus allowing for more efficient culture.

[0044] The culture conditions are not particularly limited, provided that they do not impair the amplification effect of NK cells, but culture conditions at 37°C, 5% CO2, and a saturated water vapor atmosphere are common. Since one of the objectives of the present invention is to prepare a large quantity of NK cells, it is advantageous to culture for a longer period in the culture medium, as this will yield more NK cells. The culture period is not particularly limited, provided that it amplifies the NK cells to the desired number of cells. For example, it can be carried out for 7 to 28 days, 10 to 18 days, 12 to 16 days, or even 14 days (Saito S. et al., Ex vivo generation of highly purified and activated natural killer cells from human peripheral blood. Hum Gene Ther Methods. 2013;24(4):241-252, and the aforementioned Patent Document 1).

[0045] Culturing does not have to be performed immediately after the mononuclear cell population is prepared. The prepared cell population, including mononuclear cells, may be cryopreserved and thawed according to the timing of administration to the patient for use in NK cell culture. Alternatively, the mononuclear cell population may be frozen either during or after amplification by the NK cell amplification method of the present invention, thawed according to the timing of transplantation to the patient for use in transplantation. Freezing and thawing may be performed using any method well known to those skilled in the art. Any commercially available cell cryopreservation solution may be used for freezing the cells.

[0046] A population of NK cells obtained by culture may contain, in addition to the target NK cells, NK cell precursors, T cells, NKT cells, hematopoietic progenitor cells, etc. After culturing, the target NK cells or population may be selected using methods such as gravity centrifugation, immunomagnetic beads, FACS, or flow cytometry. For example, the target NK cells or population may be selectively isolated using anti-CD3 antibodies, anti-CD16 antibodies, anti-CD34 antibodies, anti-CD56 antibodies, anti-CD69 antibodies, anti-CD94 antibodies, anti-CD107a antibodies, anti-KIR3DL1 antibodies, anti-KIR3DL2 antibodies, anti-KIR2DL3 antibodies, anti-KIR2DL1 antibodies, anti-KIR2DS1 antibodies, anti-KIR2DL5 antibodies, anti-NKp46 antibodies, anti-NKp30 antibodies, anti-NKG2D antibodies, etc. Antibodies may be monoclonal antibodies, polyclonal antibodies, etc. The selection of target NK cells, or their population, may be carried out by selectively removing T cells, NKT cells, hematopoietic progenitor cells, or other cells.

[0047] (Effects of mixed culture) According to our research, culturing a population of mononuclear cells, including NK cells, derived from multiple donors, results in improved NK cell proliferation. This is thought to be due to an increased variation in HLA / KIR combinations, which allows for more licensing signals to interact with each other. Improved proliferation means that the cell proliferation rate (number of cells after culture / number of cells before culture) is higher compared to culturing cells derived from a single donor among the multiple donors in the mixed culture.

[0048] Furthermore, in the cell population obtained by mixed culture, 70% or more, preferably 80% or more, and more preferably 90% or more may be NK cells.

[0049] Furthermore, our studies have shown that the cytotoxic activity of NK cells obtained by mixed culture of mononuclear cell populations, including NK cells derived from multiple donors, may be equivalent to or greater than that of NK cells derived from a single donor. Moreover, the proportion of NK cells with high CD16 expression may also be equivalent to or greater than that of NK cells derived from a single donor. Unless otherwise specified, cytotoxic activity refers to the lytic ability of target cells (T) by effector cells (E). Cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells, and can be calculated, for example, by the following formula.

[0050] (Cell death when co-cultured with effector cells - Spontaneous cell death (negative control)) / (Maximum cell death (positive control) - Spontaneous cell death (negative control)) × 100

[0051] When measuring cytotoxic activity, the mixing ratio (E:T) of effector cells to target cells and the co-culture time of effector cells and target cells can generally be adjusted appropriately depending on the type of cells used and the strength of their activity, according to the degree of cytotoxic activity of the effector cells. When NK cells are used as effector cells, the target cells may be, but are not limited to, K562 cells, SKOV3 cells (human ovarian cancer cell line), acute myeloid leukemia cells, or chronic myeloid leukemia cells. Effector cells and target cells, as well as living and dead cells, can be distinguished and quantified using reagents such as antibodies labeled with radioactive materials or fluorescent dyes.

[0052] Furthermore, the population of NK cells according to the present invention derived from multiple donors is not suppressed by MDSCs (Myeloid-derived suppressor cells), or the suppression is significantly low. From previous studies, the inventors have confirmed that a population of NK cells derived from a single donor, obtained by the culture method described above, is not suppressed by MDSCs. One reason for this is the absence or significantly low expression of receptors for humoral factors (TGF-β, IL-10). The inventors' current investigations have shown that even when cells from multiple donors are mixed, the above advantages are not diminished.

[0053] [Cell population including NK cells] The cell population containing NK cells obtained by the present invention has the following characteristics: (1) Derived from multiple donors. (2) When NK cells are used as effector cells (E) and K562 cells are used as target cells (T) in a mixed ratio (E:T) of 1:1, the cytotoxic activity is 50% or more. Instead of, or in addition to, the feature of (2) above, the following feature may also be present: (2') When SKOV3 cells are co-cultured as target cells (T) in a mixing ratio (E:T) of 3:1, the cytotoxic activity is 50% or more.

[0054] Such cell populations may also possess the following characteristics: (3) The proportion of NK cells is 70% or more, more specifically 80% or more, and even more specifically 90% or more. (4) The population may include both a population of NK cells that express CD16 highly and a population of NK cells that express CD16 low. The proportion of NK cells that express CD16 highly is 50% or more.

[0055] The cytotoxic activity of NK cells can be measured and calculated by methods well known to those skilled in the art. The cytotoxic activity (%) is usually calculated based on the number of viable target cells (T) after the action of effector cells (E), for example, by the formula: (1 - number of viable cells / number of viable negative control cells) × 100. When NK cells are used as effector cells (E) and K562 cells are used as target cells (T) in a co-culture with a mixing ratio (E:T) of 1:1, the cytotoxic activity is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more. When NK cells are used as effector cells (E) and SKOV3 cells are used as target cells (T) in a co-culture with a mixing ratio (E:T) of 3:1, the cytotoxic activity is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more.

[0056] Regarding markers such as CD16, a positive result is sometimes represented by a +, and a negative result by a -. For example, a CD16 positive result is represented by a CD16 + This is expressed as, and a CD16 negative result means CD16 - It is sometimes expressed as follows. A positive result includes both high and low expression. High expression is sometimes expressed as high or bright. For example, high CD16 expression is expressed as CD16 high CD16 bright It is sometimes expressed as such. Low expression is sometimes expressed as low or dim. For example, CD16 low expression is expressed as CD16 low CD16 dim It is sometimes expressed as follows.

[0057] Positive, negative, high, and low expression can be determined based on a chart obtained by flow cytometry. The position of cells on the chart may vary depending on the instrument's voltage settings, sensitivity settings, antibody clone used, staining conditions, dyes used, etc. However, a person skilled in the art can appropriately draw lines on the obtained chart so as not to separate cell populations that are recognized as a single group.

[0058] To determine whether the expression of a target marker is positive or negative, an isotype control antibody can be used as a negative control. An isotype control antibody is an antibody that does not react with a specific antigen. In general, in antibody experiments, background noise can occur due to nonspecific binding to proteins other than the target or binding to Fc receptors on the cell surface. By comparing the system with one using a negative control antibody, the specificity of the primary antibody's reaction to the target antigen becomes clear. Furthermore, the influence of background noise is eliminated, allowing for accurate interpretation of the signal strength.

[0059] The degree of expression of the target marker (whether it is low or high expression) can be determined by comparing it with the results of control cells measured under the same conditions. An example of control cells is NK cells obtained from peripheral blood that have not undergone substantial culture, such as the primary NK cells described in the Examples section of this specification.

[0060] For example, the degree of CD16 expression in a population of NK cells can be determined using flow cytometry. By comparing the CD16 expression level in that cell population with that of a population of NK cells obtained from peripheral blood that have not undergone substantial culture (control), it can be determined that the population has high expression if the expression is similar to that of the control, and low expression if it is lower than that of the control cells. It should be noted that control NK cells are known to have high CD16 expression.

[0061] [Medical Use] The present invention also provides a pharmaceutical composition for cell therapy comprising the aforementioned NK cell population as an active ingredient. Cell therapy refers to a method of treating a disease or condition in a subject by administering cells that have been processed outside the body, and this includes immunotherapy.

[0062] The pharmaceutical composition includes a cell population, which is the active ingredient, as well as a solution capable of suspending NK cells, such as physiological saline or phosphate-buffered saline (PBS). The pharmaceutical composition or solution may also contain pharmaceutically acceptable additives. The pharmaceutical composition can be administered, for example, intravenously, arterially, subcutaneously, or intraperitoneally. Cell therapy using the pharmaceutical composition can be performed alone or in combination with surgery, chemotherapy, radiotherapy, etc.

[0063] Cell populations, including NK cells, are expected to be applied to the treatment of cancer and infectious diseases (Dahlberg CM et al., Natural Killer Cell-Based Therapies Targeting Cancer: Possible Strategies to Gain and Sustain Anti-Tumor Activity, Front. Immunol., 2015;30 :https: / / doi.org / 10.3389 / fimmu.2015.00605, and Schmidt S. et al, Natural killer cells as a therapeutic tool for infectious diseases-current status and future perspectives, Oncotarget, 2018;9(29): 20891-20907). The pharmaceutical compositions of the present invention can be used to treat such cancers or infectious diseases. More specifically, this includes, but is not limited to, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, colorectal cancer, kidney cancer, bladder cancer, leukemia; and infectious diseases caused by viruses, bacteria, etc. In cell therapy using the pharmaceutical composition of the present invention, NK cells may be administered, for example, intravenously, arterially, subcutaneously, or intraperitoneally. Cell therapy may be performed alone or in combination with surgery, chemotherapy, radiotherapy, antibody drugs, etc.

[0064] Many antibody drugs are said to exhibit antitumor effects through ADCC activity after intravenous administration. However, when ADCC activity is exerted, monocytes / macrophages and neutrophils are also recruited in addition to NK cells. These effectors other than NK cells exhibit ADCC activity without distinguishing between normal cells and cancer cells, which is thought to lead to the occurrence of side effects. In the present invention, NK cells and antibodies may be mixed before administration, and the NK cells may be pre-equipped with antibodies. This limits the effectors to NK cells, reduces the amount of antibody administered, and is considered to be extremely effective in reducing side effects. That is, the pharmaceutical composition of the present invention may be prepared by mixing an NK cell population and antibodies, and then removing antibodies that are not bound to NK cells. In other words, one preferred embodiment of the pharmaceutical composition of the present invention is one in which NK cells and antibodies are included, but the antibodies are bound to NK cells, and substantially no antibodies that are not bound to NK cells are included (see Patent Document 3 above).

[0065] The pharmaceutical composition of the present invention is preferably manufactured under conditions that conform to the manufacturing control and quality control regulations for pharmaceuticals and quasi-drugs (good manufacturing practice, GMP) and the manufacturing control and quality control standards for regenerative medicine products (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice, GCTP).

[0066] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0067] [Example 1: Mixed culture of NK cells obtained from fresh peripheral blood 1] Blood was collected from healthy volunteers, and peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll (GE Healthcare, 17144002). Peripheral blood mononuclear cells from multiple individuals were mixed at approximately the same ratio, and CD3 beads※ 1 were added and suspended. After incubation at 4°C for 15 minutes, 1 mL of separation buffer※ 2 was added and suspended well, and centrifuged at 300 xg for 10 minutes. The supernatant was removed, suspended in 0.5 mL of separation buffer, added to an LD column (Miltenyi Biotec, 130-042-901) pre-wetted by adding 2 mL of separation buffer, and the eluate from the LD column was collected. Further, 1 mL of separation buffer was added to the LD column, and the eluate was collected. Thereafter, the column was washed with 1 mL of separation buffer, the number of cells in the collected liquid was counted, and the total number of cells was calculated. Centrifuged at 500 xg for 5 minutes, after removing the supernatant, suspended in KBM501 medium※ 5 to a concentration of 5x10 3 cells / mL.

[0068] A part of the cells was collected for flow cytometer measurement, and the remaining cells were cultured. The culture was carried out in a CO2 incubator (37°C, 5% CO2) using a 6-well plate (Thermo Fisher Scientific, 140675), a T-75 flask (Thermo Fisher Scientific, 156499), or a 500 cm 2 culture bag (Nipro). On the 5th and 9th days of culture, a part of the culture medium was taken, the number of cells was counted, and on the 9th day, KBM501 medium was added so that the final liquid volume was 6 mL per well in the case of a 6-well plate and 50 mL per flask in the case of a T-75 flask. On the 14th day, the cells were collected, the number of cells was counted, and cell surface antigens were measured using a flow cytometer with a part of the cells.

[0069] ※1: CliniMACS CD3, Miltenyi Biotec, 130-017-601 (5 μL per 1x10 7 cells) *2: PBS containing 0.5% human type AB serum (Cosmo Bio, 12181301, inactivated at 56°C for 30 minutes) and 2 mM EDTA (Thermo Fisher Scientific, 15575-020) (Wako Pure Chemical Industries, 045-29795) *3: KBM 501 (Kojin Bio, 16025015) to which 5% human type AB serum (Cosmo Bio, 12181301, inactivated at 56°C for 30 minutes) has been added.

[0070] The results are shown in Figure 1. Cell proliferation was good in all three mixed cultures: 9-person (Figure 1A), 8-person (Figure 1B), and 7-person (Figure 1C). Upon checking the purity, more than 90% of the cells in all mixed cultures were CD3. - CD56 + These were NK cells. In the 8-person mixed culture, T-75 flasks and 500 cm³ were used. 2 Cell culture was performed using culture bags, and cell proliferation was good in both cases. Mixed cultures from 9 donors, cultures from 7 donors, and cultures from a single donor (one of the 7 donors) were cultured using 6-well plates. 500 cm² 2 In culturing using culture bags, the bags were turned inside out as described in Example 4 below.

[0071] Furthermore, when comparing cell proliferation in a mixed culture of 7 donors with that of a single donor, the single donor culture showed a proliferation of 6.8 × 10⁶. 6 From the cells 1.9 × 10 7 Cells proliferated (approximately 2.8 times), while mixed culture yielded 7.8 × 10⁶ cells. 6 From cells 3.7 × 10 7 Cells proliferated (approximately 4.7 times), and cell proliferation was better in mixed culture (Figure 1C).

[0072] [Example 2: Mixed culture of NK cells obtained from fresh peripheral blood 2] Data from single-donor cultures (n=17) and mixed cultures of four or more donors (n=10) were collected in T-75 flasks using the method described in Example 1. Statistical analysis was then performed on the cell proliferation rate of the resulting NK cells. JMP Pro 13 was used for the analysis, and the Wilcoxon rank-sum test was performed.

[0073] The results are shown in Figure 2. The proliferation rate (total number of cells after proliferation / total number of cells before proliferation) for cells cultured from a single donor was 3.10 ± 0.56, while the proliferation rate for mixed culture was 6.56 ± 1.24, indicating that mixed culture had a statistically significantly higher proliferation rate (P < 0.01).

[0074] [Example 3: Tumor cell cytotoxicity test] Preparation of NK cells Mixed cultured NK cells from four healthy volunteers obtained using the method described in Example 1, and cultured NK cells from a single donor (one of the four) were collected and washed. These cells were then suspended in RPMI1640 medium (Wako Pure Chemical Industries, 189-02025) (hereinafter referred to as 10%FBS / RPMI1640) containing 10% FBS (Nichirei Bioscience, 171012-500ML), 100 units of penicillin, and 100 μg / mL of streptomycin (Nacalai Tesque, 26253-84), respectively, and 1x10⁶ cells were added in the same medium. 6 The concentration was adjusted to cells / mL.

[0075] Preparation of SKOV3 SKOV3 cells (human ovarian cancer cell line) were incubated in RPMI1640 medium (Wako Pure Chemical Industries, Ltd., 189-02025) without serum components at a rate of 1 x 10⁶ cells. 6 The cells were prepared to a concentration of cells / mL. The prepared SKOV3 cells were stained using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) and finally 2x10⁶ of the cells in 10% FBS / RPMI1640. 6 cells / mL and 2x10 5 The solution was prepared to achieve a concentration of cells / mL.

[0076] Preparation of MDSCs (Myeloid-derived suppressor cells) Peripheral blood mononuclear cells were isolated from healthy volunteers (separate from NK cell donors) using Ficol, and MDSCs were induced by culturing them for 7-10 days in 10% FBS / RPMI1640 containing 10 ng / mL IL-6 (PEPROTECH, 200-06-5UG) and 10 ng / mL GM-CSF (CellGenix, 1012-050). The MDSCs were suspended in serum-free RPMI1640 medium (Wako Pure Chemical Industries, 189-02025), stained using the PKH Green Fluorescent Cell Linker Kit (Sigma, MINI67-1KT), and finally incubated in 10% FBS / RPMI1640 for 8x10⁶ cells. 5 The solution was prepared to achieve a concentration of cells / mL.

[0077] 《Cytotoxic activity test》 We prepared four groups: a group of mixed cultured NK cells and SKOV3 cells derived from multiple donors, a group of cultured NK cells and SKOV3 cells derived from a single donor, and groups of these two groups with MDSC added, as well as a negative control group consisting of SKOV3 cells alone.

[0078] NK cells and SKOV3 cells were seeded and mixed in a 96-well plate (IWAKI, 4870-800SP) in a cell ratio of 3:1, and incubated at 37°C and 5% CO2 for 4 hours. For the group with added MDSCs, NK cells and MDSCs were first mixed in a 96-well plate in a cell ratio of 5:1:4, and incubated at 37°C and 5% CO2 for 12-18 hours. After centrifugation (500 xg, 5 minutes), the supernatant was removed, SKOV3 cells were added and mixed, and incubated at 37°C and 5% CO2 for 4 hours. After the reaction, centrifugation (500 xg, 5 minutes), the supernatant was removed, and Zombie solution (Biolegend, 423105) diluted in PBS was added and mixed, and incubated at room temperature in the dark for 30 minutes. After centrifugation and removal of the supernatant, the sample was suspended in PBS and AccuCheck Counting Beads (Thermo Fisher Scientific, PCB100) were added. Measurements were performed using a flow cytometer (BD LSR Fortessa, BD Biosciences), and the cytotoxicity rate (%Lysis) was calculated using FlowJo software (FLOWJO, LLC).* 4 .

[0079] *4: Cytotoxicity rate = (1 - number of viable SKOV3 cells / number of viable negative control SKOV3 cells) × 100 Number of viable cells: Actual number of SKOV3 cells × Number of beads in the added bead solution / Actual number of beads SKOV3 cell count measured: FSC / SSC gated (debris exclusion), doublet exclusion, followed by PKH26 + gated

[0080] The results are shown in Figure 3. Mixed cultured NK cells derived from multiple donors showed cytotoxic activity equivalent to or greater than that of NK cells derived from a single donor.

[0081] [Example 4: Mixed culture of NK cells prepared from frozen apheresis blood] After thawing, frozen apheresis blood from two donors (HemaCare, PB001CLP) was mixed, diluted with HBSS(-) solution (Nacalai Tesque, 17461-05), and washed and concentrated with PBS / EDTA solution (Miltenyi Biotec, 130-021-201) using the Lovo Cell Processing System (FRESENIUS KABI). Next, the concentrated cell saturation was washed and eluted to remove CD3-positive and CD34-positive cells using CliniMACS Prodigy TS 310 (Miltenyi Biotec, 130-097-183), CliniMACS CD3 MicroBeads (Miltenyi Biotec, 130-017-601), and CliniMACS CD34 Reagent (Miltenyi Biotec, 130-017-501). The total number of cells was calculated by counting the number of cells in the eluate. After centrifugation at 500 xg for 5 minutes and removal of the supernatant, 1 x 10⁻¹⁴ cells were obtained. 6 The cells were suspended in KBM501 medium to a concentration of cells / mL. Culture was performed in a T-75 flask (Thermo Fisher Scientific, 156499) or 500 cm³. 2 Culture bags (Nipro) were used and the culture was performed in a CO2 incubator (37°C, 5% CO2). For the bag culture, the bag was turned over 30 minutes after the start of culture and then turned over again on day 9 of culture (see Figure 4-1). Also, on day 9 of culture, a portion of the culture medium was taken and the number of cells was counted, and KBM 501 medium was added so that the final volume was 50 mL per T-75 flask and 500 mL per bag for the culture bags. On day 14, the cells were harvested, the total number of cells was counted, and cell surface antigens were measured using a flow cytometer on a portion of the cells.

[0082] The collected cells were stained with antibodies as follows: Alexa Fluor (R)700-labeled anti-human CD56 antibody (Biolegend, 318316), PerCP / Cy5.5-labeled anti-human CD3 antibody (Biolegend, 300430), and PE-Cy7-labeled anti-human CD16 antibody (Biolegend, 302016) were stained at a concentration of 1 μg / mL at 4°C for 30 minutes. After centrifugation (500 xg, 5 minutes, 4°C), the supernatant was removed, and the samples were suspended in PBS(-) (Wako Pure Chemical Industries). The results were measured using a flow cytometer (BD LSRFortessa, BD Biosciences) and analyzed with FlowJo software (FLOWJO, LLC).

[0083] The results are shown in Figure 4-2. Even when using NK cells obtained from frozen apheresis blood, the cells proliferated sufficiently within 14 days through mixed culture. Specifically, in the case of T-75 flask culture, the cell size was 1.50 x 10⁶. 8 From cells 5.72 x 10 8 It proliferates into cells (approximately 3.81 times), reaching 500 cm 2 For culture bag culture, 1.50 x 10 8 From cells 5.60 x 10 8 The cells proliferated (approximately 3.73 times) (Figure 4-2, left).

[0084] The purity of NK cells in the resulting cultured NK cell population was 90.5% in the T-75 flask culture, at 500 cm³. 2 In the case of culture bag culture, the expression rate was 91.7%. Regarding CD16 expression, the mixed culture NK cell population showed a bimodal pattern, similar to the single-donor culture NK cell population (see Japanese Patent Publication No. 2018-193303). Specifically, the proportions of low-CD16 expression and high-CD16 expression were 50.9% and 49.1%, respectively, in the T-75 flask culture, at 500 cm³. 2 In the case of culture bag culture, the rates were 46.7% and 53.3%, respectively (Figure 4-2, right).

[0085] [Example 5: Monocyte / NK cell swapping experiment] Preparation of Primary NK Cells and Monocytes Blood samples were collected from two healthy volunteers (referred to as Donors 1 and 2), and peripheral blood mononuclear cells were isolated by density gradient centrifugation using a Ficol. EasySep was then used to isolate the isolated peripheral blood mononuclear cells. TM Using the Human NK Cell Enrichment Kit (STEMCELL, 19055), primary NK cells were extracted using EasySep. TM Monocytes were isolated using the Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL, 19058), and the number of cells in each cell was counted. Primary NK cells were counted as 1 x 10⁶. 5 cells / mL, monocytes 3 x 10 5 The cells were suspended in KBM 501 medium to a concentration of cells / mL.

[0086] 《swapping culture》 Four groups were created by combining primary NK cells from donor 1 with monocytes from donor 1, primary NK cells from donor 1 with monocytes from donor 2, primary NK cells from donor 2 with monocytes from donor 2, and primary NK cells from donor 2 with monocytes from donor 1. These groups were mixed so that the cell ratio of primary NK cells to monocytes was 1:3, and culture was started in a 6-well plate (Thermo Fisher Scientific, 140675) (37°C, 5% CO2). KBM501 medium was added on day 9 of culture, as in the method described in Example 1, and the cells were harvested on day 14. The obtained cells were used to measure cell surface antigens and cytotoxic activity against K562 (human chronic myeloid leukemia cell line).

[0087] Cell surface antigens were measured by staining the collected cells with antibodies and analyzing them as follows: Alexa Fluor (R)700-labeled anti-human CD56 antibody (Biolegend, 318316), APC / Cy7-labeled anti-human CD3 antibody (Biolegend, 300426), FITC-labeled anti-human KIR2DL1 antibody (Miltenyi Biotec, 130-103-966), and PerCP / Cy5.5-labeled anti-human KIR3DL1 antibody (Biolegend, 312718) were stained at a concentration of 1 μg / mL at 4°C for 30 minutes. The samples were then centrifuged (500 xg, 5 minutes, 4°C), the supernatant was removed, and the samples were suspended in PBS(-) (Wako Pure Chemical Industries). The samples were then measured using a flow cytometer (BD LSRFortessa, BD Biosciences) and analyzed using FlowJo software (FLOWJO, LLC).

[0088] To measure cytotoxic activity, four groups were prepared: one in which cultured NK cells (created by combining primary NK cells and monocytes from two donors) were reacted with K562 cells; one group consisting only of K562 cells as a negative control; and one group consisting of K562 cells fixed in 10% formalin as a positive control.

[0089] Preparation of K562 K562 cells (human chronic myeloid leukemia cell line) were incubated in RPMI1640 medium (Wako Pure Chemical Industries, Ltd., 189-02025) without serum components at a rate of 1 x 10⁶ 6 The cells were prepared to a concentration of cells / mL. The prepared K562 cells were stained using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) and finally 2x10⁶ of the cells in 10% FBS / RPMI1640. 6 The solution was prepared to achieve a concentration of cells / mL.

[0090] Preparation of cultured NK cells The primary NK cells and monocytes obtained from the two donors using the method described above were combined and cultured to obtain four groups of cultured NK cells. After harvesting and washing, each group was suspended in 10% FBS / RPMI1640 and cultured in the same medium in 1x10⁶ units. 6 The concentration was adjusted to cells / mL.

[0091] 《Cytotoxic activity test》 NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) in a 1:1 cell ratio, mixed, and reacted at 37°C under 5% CO2 for 2 hours. After reaction, the cells were centrifuged (500 xg, 5 minutes), the supernatant was removed, and 7-AAD solution (Beckman Coulter, A07704) diluted in PBS was added and the cells were resuspended. The cells were incubated at room temperature for 20 minutes. Measurements were taken using a flow cytometer (BD LSR Fortessa, BD Biosciences), and the cytotoxicity rate (%Lysis) was calculated using FlowJo software (FLOWJO, LLC).* 5 .

[0092] *5: Cytotoxicity rate = (K562 cell death rate - negative control cell death rate) / (positive control cell death rate - negative control cell death rate) × 100

[0093] The donor typing information is shown in the table below, and the results are shown in Figure 5.

[0094] [Table 1]

[0095] CD16 high As the population of this group increases, higher ADCC activity can be expected.

Claims

1. A population of mononuclear cells, including NK cells, is prepared, derived from one of the following sources selected from multiple donors: peripheral blood, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. The prepared mononuclear cell population is incubated under conditions effective for NK cell treatment. A method for culturing a cell population including NK cells, including the following.

2. The culture method according to claim 1, wherein the step of preparing a population of mononuclear cells includes the step of removing CD3-positive cells.

3. The culture method according to claim 1 or 2, wherein the step of preparing a population of mononuclear cells includes a step of removing CD34-positive cells.

4. The culture method according to any one of claims 1 to 3, wherein the step of preparing a population of mononuclear cells includes the step of obtaining a population of mononuclear cells from apheresis blood collected from multiple donors.

5. The culture method according to any one of claims 1 to 4, wherein the multiple donors include one donor and other donors whose genotype differs from that donor in at least one of HLA and KIR.

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