JP2021035400A - Method for producing cell population comprising nk cells - Google Patents

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

The present invention relates to a method for producing a cell population containing natural killer cells (NK cells) and its use.

Malignant tumors are the leading cause of death in the nation, and countermeasures are urgently needed. In particular, the development of new treatments for advanced refractory malignancies that are resistant to existing treatments consisting of surgery, radiation therapy and chemotherapy is extremely important and significant. In recent years, as a fourth therapeutic method, immunotherapy such as an immune checkpoint inhibitor or a therapy using chimeric antigen receptor (CAR) gene-modified T cells (CAR-T therapy) has attracted attention. However, since most of them use T cells that are activated by recognizing an antigen as an effector, there is a fundamental barrier of binding to a specific antigen.

As an immunotherapy using NK cells that act as a major factor of innate immunity, NK cell therapy, in which NK cells are proliferated in vitro and then administered to a patient, is attracting attention as a treatment method having relatively few side effects. However, the number of NK cells obtained from peripheral blood and the like is relatively small, and the proliferation in vitro is low. Therefore, techniques for culturing and proliferating NK cells have been studied. For example, Patent Document 1 describes a step of preparing a cell population containing NK cells, a step of removing T cells from the cell population containing NK cells, and a medium containing only IL-2 as a cytokine in the remaining removed cells. A method for amplifying NK cells, which comprises a step of culturing without using feeder cells, and a pharmaceutical composition for cell therapy, which comprises a cell population containing NK cells obtained by amplification. suggest. Further, Patent Document 2 describes a step of amplifying hematopoietic progenitor cells under a single culture condition containing IL-15, SCF, IL-7 and Flt3L, and a step of amplifying the cells obtained in the amplification step, IL-2. For cell therapy, which comprises a method of preparing NK cells, including a step of inducing differentiation into NK cells in 5, 6, 7, 8 or 9 days, and a cell population containing the prepared NK cells. To propose a pharmaceutical composition of.

In addition, as cultured cells with enhanced tumor cell injury activity, the present inventors are CD16-positive, CD56-highly expressive, CD57-negative, NKG2C-positive, NKG2A-negative to low-expressive, and CD94-positive NK cells. , And a cell population containing NK cells thereof (Patent Document 3), and a CD3-negative cell expressing a chemokine receptor and a cell adhesion molecule (Patent Document 4).

On the other hand, a group of receptors that recognize HLA class I is expressed on the cell surface of NK cells. Among these receptors, the KIR (Killer cell Immunoglobulin-like Receptor) family has diversity similar to HLA. In hematopoietic stem cell transplantation, instead of matching HLA as much as possible, recently, intentional KIR between donors and recipients, such as selecting donors so that recipients do not have a ligand for KIR of donor NK cells. / HLA mismatch is being effectively utilized as it can give more desirable results in terms of recurrence, GVHD (graft versus host disease) and prognosis. However, with respect to the antitumor activity of NK cells, NK cells are cultured with either matching or mismatched feeder cells for inhibitory KIR ligands (lacking one or more ligands present in the NK cell donor). From a report investigating to what extent the HLA class I-KIR interaction affects human NK cell proliferation in an allogeneic environment (Non-Patent Document 1), high activation of NK cells was performed using the antitumor effect as an index. It is suggested that less signal from KIR is better if desired. In addition, a recent report on the allogeneic responsiveness of HLA heterozygous NK cells (in the case of unidirectional adaptation) to tissues derived from HLA haplotype homozygous (HLA homozygous) iPS cells (Non-Patent Document 2) relates to culturing NK cells. , HLA / KIR mismatch strongly suggests that mixed culture is not established.

Japanese Unexamined Patent Publication No. 2013-27385 (Patent No. 5572863, Patent No. 5890016) Japanese Unexamined Patent Publication No. 2014-226079 (Patent No. 5511039, Patent No. 6164650) Japanese Unexamined Patent Publication No. 2018-193303 Japanese Patent Application No. 2018-059624 Application Specification (Unpublished at the time of filing this application)

Mingus J. J. Rose et al., Killer Ig-Like Receptor Ligand Mismatch Directs NK Cell Expansion In Vitro, The Journal of Immunology, 2009, 183: 4502-4508. Hiroshi Ichise et al., NK Cell Alloreactivity against KIR-Ligand-Mismatched HLA-Haploidentical Tissue Derived from HLA Haplotype-Homozygous iPSCs, Stem Cell Reports, 2017, 9: 853-867,

The present inventors are proceeding with the development of a cell population containing highly active NK cells. However, since the amount of peripheral blood as a raw material and the amount of blood obtained by the apheresis method is limited, it is not possible to off-the-shelf the therapeutic NK cell population for future treatment.

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

Furthermore, in the process of differentiating from hematopoietic stem cells in the bone marrow in the body, immature NK cells that have experienced binding to a ligand (HLA class I) are "licensed" and the expression of autologous HLA class I is reduced after maturation. Although it is thought to have the ability of "missing-self response" to detect, NK cells differentiated and matured from iPS cells and ES cells may not have sufficient signals required for licensing to obtain antitumor effects. There is.

The present invention provides:
[1] Prepare a population of mononuclear cells derived from multiple donors and containing NK cells.
A method for producing a cell population containing NK cells, which comprises incubating the prepared mononuclear cell population under conditions effective for NK cell treatment.
[2] The production method according to 1, wherein the step of preparing a population of mononuclear cells includes a step of removing CD3-positive cells.
[3] The production method 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 production method according to any one of 1 to 3, wherein the step of preparing a population of mononuclear cells includes a step of obtaining a population of mononuclear cells from peripheral blood collected from a plurality of donors.
[5] The production method according to any one of 1 to 4, wherein the step of preparing a population of mononuclear cells includes a step of obtaining a population of mononuclear cells from apheresis blood collected from a plurality of donors.
[6] The step of preparing a population of mononuclear cells is selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells derived from multiple donors. The production method according to any one of 1-3, which prepares a population of mononuclear cells derived from crab.
[7] The production method according to any one of 1 to 6, wherein the plurality of donors comprises one donor and another donor having a different genotype in at least one of HLA and KIR.
[8] A cell population containing NK cells having the following characteristics:
(1) Derived from multiple donors.
(2) The cytotoxic activity is 50% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixing ratio (E: T) of 1: 1.
[9] A pharmaceutical composition for cell therapy, which comprises a cell population produced by the production method according to any one of 1 to 7.
[10] A pharmaceutical composition for cell therapy comprising the cell population according to 8.
[11] The pharmaceutical composition according to 9 or 10, for treating infectious diseases and / or cancer.

Since blood or PBMC as a raw material can be mixed for a plurality of people, the amount of raw material can be increased, and off-the-shelfization can be expected.

NK cells can be stably proliferated regardless of the donor, and the proliferation rate can be improved.

It is possible to respond to the diversification of HLA-KIR matching, and stable activation can be expected.

It is possible to flexibly increase the signals required for NK cell licensing, and it is expected that the antitumor effect of the NK cell population derived from iPS cells and ES cells will be improved.

Culturing test: CD3 positive cells were removed from PBMCs of a plurality of individuals, and the cells were cultured in KBM-501 medium for 14 days. Summary of experimental results (left) and statistical analysis (right): When mixed culture was performed, the growth rate was significantly improved as compared with the culture derived from a single donor. Tumor cytotoxic activity test: The injury activity of NK cells prepared by the mixed culture and the culture derived from a single donor was evaluated using the injury to SKOV3 (human ovarian cancer cell line) as an index. Culture in a 500 cm ^{2 culture bag.} Thirty minutes after the start of the culture and on the 9th day of the culture, the bag was turned inside out. Culture test. Using frozen apheresis blood (for 2 donors) as a material, after removing CD3 positive cells and CD34 positive cells using an automatic closed system cell processing device, use a T75 flask or a 500 cm ² culture bag, and use KBM-501 medium 14 Incubated for days. ^{Monocyte / NK cell swapping experiment: The population of CD16 high} increased under the condition that one or both signals from KIR3DL1 and KIR3DS1 were input. The CD16 ^high population can be expected to have higher ADCC activity.

The present invention relates to a cell population containing NK cells.

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

(Preparation of a population of mononuclear cells 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 here includes NK cells, and may include mononuclear cells other than NK cells, for example, monocytes. The NK cells may be primary NK cells (NK cells collected from a living body and not passaged).

Populations of mononuclear cells derived from multiple donors and containing NK cells include a population of mononuclear cells (containing NK cells and may contain monocytes) obtained from one donor and the other. It may be a mixture of populations of mononuclear cells (including NK cells, which may contain monocytes) obtained from a donor, and NK cells obtained from one donor and another donor. It may be a mixture of monocytes obtained from.

With respect to donors, plural means 2 or more, and is not particularly limited as long as the above conditions are satisfied. For example, mononuclear cells derived from 3 or more, 4 or more, 7 or more, and 9 or more donors are mixed. Can be used. The plurality of donors may include the patient himself or herself to whom the cell population containing NK cells is administered, as well as relatives of the patient.

Multiple donors can be selected to include one donor and another donor of different genotypes in at least one of HLA and KIR. KIR includes KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR3DL3, KIR2DL5A, KIR2DL5B.

With respect to NK cells, licensing refers to the process of acquiring the ability of "missing-self response (detection of lack of self-HLA)" peculiar to NK cells in the process of cell differentiation and maturation. For humans and mice, in the process of NK cell differentiation from hematopoietic stem cells in bone marrow, only immature NK cells that have experienced binding of HRA class I molecule to KIR or NKG2A are licensed and self-HLA class I expression is expressed after maturation. It is believed to acquire the ability to detect degraded cells. Examples of KIRs known to be involved in licensing are KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1 and NKG2A / CD94. The HLA types that can be recognized by NK cells are all HLA-C allotypes, about 1/3 of HLA-B allotypes, some HLA-A allotypes (having Bw4 motif), and non-classical HLA types. HLA-E and HLA-G of HLA. In the present invention, when selecting a plurality of donors to include one donor and another donor having a different genotype in at least one of HLA and KIR, the KIR and HLA related to the above license are used. Especially can be considered.

In one preferred embodiment of the invention, donors of different genotypes can be selected to increase the repertoire of molecules used for NK cell licensing. Further, it may be selected so that the inhibitory KIR of NK cells is not stimulated or the inhibitory KIR is stimulated.

The choice of donor is to increase the number of ^{CD16 high} NK cells when culturing a mixed mononuclear cell population if antibody-dependent cellular cytotoxicity (ADCC) activity by NK cells is expected. You may go to. ADCC is known as one of the mechanisms of cell damage caused by NK cells, and NK cells bind to an antibody bound to the target cell via CD16, which is an Fc receptor on the cell surface, and the target cell. Injure. Therefore, it can be expected that ADCC activity will be further increased by increasing the number of NK cells having ^{CD16 high.} An example of such a donor combination condition is to select and mix donors so that NK cells are licensed by signals from one or both of KIR3DL1 and KIR3DS1.

Mixing is done prior to administration of NK cells to the subject. The mixing can be carried out at various stages as long as the desired mixing effect is obtained. For example, blood collected from multiple donors may be mixed and then a population of mononuclear cells may be obtained from the mixture, or after each population of mononuclear cells has been obtained from blood collected from each donor. , Each population may be mixed. In addition, each population may be mixed after culturing each population of mononuclear cells collected from a plurality of donors. From the viewpoint of good growth, mixing is preferably performed before culturing.

The mixing ratio of the population of mononuclear cells derived from multiple donors can be adjusted as appropriate. For example, cells from a plurality of donors can be contained in approximately equal proportions, and cells from a particular donor can be contained in large or small amounts. When mixing NK cells derived from one donor and monocytes derived from another donor, the mixing ratio can be appropriately adjusted. For example, NK cells 1 can be mixed with 1 to 9 times more monocytes (NK cells: monocytes = 1: 1 to 9), and NK cells 1 can be mixed with 2 to 4 times more monocytes. The spheres can be mixed (NK cells: monocytes = 1: 2-4). In the present invention, when expressing the mixing ratio of cells, unless otherwise specified, it is based on the number of cells.

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

Populations of mononuclear cells, including NK cells and monocytes, can be prepared using a variety of procedures known to those of skill in the art. For example, from blood such as peripheral blood and umbilical cord blood, erythrocytes can be removed and mononuclear cell fractions can be recovered by performing density centrifugation at room temperature. Mononuclear cells PBMCs (Peripheral Blood Mononuclear Cells) isolated from peripheral blood include a variety of lymphocytes such as T cells, B cells, NK cells, monocytes and dendritic cells. With respect to the present invention, mononuclear cells that are both CD3 negative and CD56 positive can be referred to as NK cells. NK cells can be prepared using a variety of procedures known to those of skill in the art. For example, a method for separating NK cells from whole blood or PBMC by removing unnecessary cells is known. Monocytes can be prepared using a variety of procedures known to those of skill in the art. For example, a method for separating monocytes by removing unnecessary cells from whole blood or PBMC is known.

(Removal of CD3 positive cells, removal of CD34 positive cells)
The population of mononuclear cells may include NK cell precursors, T cells, NKT cells, hematopoietic progenitor cells, etc., in addition to monocytes and NK cells. The desired NK cells may be selected after amplification using, for example, specific gravity centrifugation, immunomagnetic beads, FACS, flow cytometry and the like. For example, NK cells include anti-CD3 antibody, anti-CD16 antibody, anti-CD34 antibody, anti-CD56 antibody, anti-CD69 antibody, anti-CD94 antibody, anti-CD107a antibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR2DL3 antibody, anti-KIR2DL1 antibody, Anti-KIR2DS1 antibody, anti-KIR2DL5 antibody, anti-NKp46 antibody, anti-NKp30 antibody, anti-NKG2D antibody and the like may be used to selectively separate from the cell population. The antibody may be a monoclonal antibody, a polyclonal antibody, or the like. Selection of NK cells may be performed by selectively removing T cells, NKT cells, hematopoietic progenitor cells and other cells.

The step of preparing a population of mononuclear cells may include the step of removing T cells, which may be accomplished by removing CD3 positive cells. The step of preparing a population of mononuclear cells may also include the step of removing hematopoietic progenitor cells, which may be accomplished by removing CD34-positive cells. That is, the step of preparing a population of mononuclear cells may include a step of removing CD3-positive cells, or may include a step of removing CD34-positive cells.

For the removal of CD3-positive cells and CD34-positive cells, for example, using immunomagnetic beads such as Dynabeads manufactured by Dynamic Biotec sold by Invitrogen and CliniMACS manufactured by Miltenyi Biotec, the cell surface antigen CD3 and / or This can be done by separating and removing the cells expressing CD34. It is preferable that such a step is performed so as not to exhaust the NK cells and to prevent the monocytes from exerting an extra phagocytic function. Specifically, relatively small magnetic beads are used (unreacted beads that are not bound to cells can be removed together with the supernatant by centrifugation), and the beads and cell population are incubated at a low temperature. Preferred operations include setting the time relatively short, removing unreacted beads, and using a column to obtain cells that are not bound to the beads as a target cell population. The cells may be removed using an automatic closed cell processing apparatus.

(Use of iPS cells, etc.)
The mononuclear cell population was prepared from hematopoietic stem cells derived from one selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. May be good. A method for inducing mononuclear cell cells including NK cells from these stem cells is 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 give highly active NK cells, for example, high affinity CD16 (158V: amino acid 158 is valine) is known and such findings can be applied. it can.

(Culturing a population of mononuclear cells)
The prepared mononuclear cell population is incubated with a mixture of cells derived from multiple donors under conditions effective for NK cell treatment. Incubation may or may not be accompanied by cell proliferation. In one of the preferred embodiments of the invention, incubation involves proliferation. Incubation with proliferation can be read as culturing (doing) or proliferation (doing).

The conditions effective for NK cell treatment are conditions suitable for mixed cells derived from a plurality of donors to exert the effect of the desired mixing. As long as the desired effect is achieved, the medium or isotonic solution used for cell suspension, time, temperature, environment, etc. are not particularly limited.

Conditions that are effective for NK cell treatment include conditions that are effective for activating NK cells. Such conditions are, for example, incubated in the presence of factors required for activation at appropriate concentrations, such as IL-2 and IL-15, at 37 ° C. in a 5% CO ₂ and saturated steam atmosphere for 4-18 hours. For example.

Conditions that are effective for NK cell processing include conditions that are effective for NK cell proliferation. Effective conditions for NK cell proliferation include using a medium suitable for NK cell proliferation. Examples of such media are KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Cergenics, Iwai Chemical Co., Ltd.), X-VIVO15 medium (Ronza, Takara Bio Co., Ltd.), IMDM, MEM, DMEM. , RPMI-1640 and the like, but not limited to these.

Interleukin 2 (IL-2) may be added to the medium at a concentration that can achieve the object of the present invention. The concentration of IL-2 can exceed 2000 IU / mL and can be 2500-2813 IU / mL. IL-2 preferably has a human amino acid sequence and is preferably produced by recombinant DNA technology for safety reasons. The concentration of IL-2 may be expressed in national standard units (JRU) and international units (IU). Since 1 IU is about 0.622 JRU, 1750 JRU / mL is about 2813 IU / mL.

The medium may be supplemented with the subject's autologous serum, human AB type serum available from BioWhittaker or others, or blood donated human serum albumin available from the Japanese Red Cross Society. Autologous serum and human ABO blood group serum are preferably added at a concentration of 1 to 10%, and blood donated human serum albumin is preferably added at a concentration of 1 to 10%. Subjects may be healthy individuals and patients with the disease. In addition, a composition formulated for the proliferation of immune cells may be added to the medium in place of or together with serum. Such compositions are commercially available. For example, UltraGro series (AventaCell) and CTS Immune Cell SR (Thermo Fisher Scientific) can be used for the present invention.

The medium may contain suitable proteins, cytokines, antibodies, compounds and other components, provided that the amplification effect of NK cells is not impaired. Cytokines are interleukin 3 (IL-3), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 21 (IL-21), and 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 a human amino acid sequence and are preferably produced by recombinant DNA technology for safety reasons.

The medium may be changed at any time after the start of culturing, provided that the desired number of NK cells can be obtained, but it is preferably every 3 to 5 days.

Culture vessels include, but are not limited to, commercially available dishes, flasks, plates, multi-well plates. In order to obtain a cell population containing therapeutic NK cells, it is preferable to use a culture vessel in which many cells can be cultured and easy to handle. An example of such a culture vessel is a cell culture bag made of a material having high gas permeability.

When using a bag, it is preferable to use it while turning it upside down during the culture period. During culturing, both NK cells and monocytes adhere to the culture surface. In the case of adherent cells, the cell density per unit volume as well as the cell density per unit area have a great influence on the culture efficiency including the cell viability and the growth rate. By inverting the bag during the culturing period, some NK cells and monocytes can be moved to a surface where the cells have relatively little adhesion because they were on the upper side, so that the culturing is performed more efficiently. be able to.

The culture conditions are not particularly limited as long as the amplification effect of NK cells is not impaired, but _{the culture conditions are generally 37 ° C., 5% CO 2} and a saturated steam atmosphere. Since one of the objects of the present invention is to prepare a large amount of NK cells, it is advantageous that more NK cells can be obtained as the period of culturing in the medium is longer. The culture period is not particularly limited, provided that the NK cells are amplified to a desired number of cells. For example, it can be performed for 7 to 28 days, 10 to 18 days, 12 to 16 days, for example 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 above-mentioned Patent Document 1).

Culturing does not have to be performed immediately after the population of mononuclear cells has been prepared. The cell population containing the prepared mononuclear cells may be cryopreserved, thawed depending on the time of administration to the patient, and used for culturing NK cells. The mononuclear cell population is frozen during amplification by the NK cell amplification method of the present invention or after amplification is completed, thawed according to the time of transplantation to a patient, and used for transplantation to a patient. May be served. Freezing and thawing may be carried out by any method well known to those skilled in the art. Any commercially available cell cryopreservation solution may be used for freezing the cells.

The predetermined NK cell population obtained by culturing may include NK cell precursors, T cells, NKT cells, hematopoietic precursor cells, etc. in addition to the target NK cells. After culturing, the target NK cells or a population thereof may be selected using, for example, specific gravity centrifugation, immunomagnetic beads, FACS, flow cytometry, or the like. For example, anti-CD3 antibody, anti-CD16 antibody, anti-CD34 antibody, anti-CD56 antibody, anti-CD69 antibody, anti-CD94 antibody, anti-CD107a antibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR2DL3 antibody, anti-KIR2DL1 antibody, anti-KIR2DS1 antibody, The target NK cells or a population thereof may be selectively isolated by using an anti-KIR2DL5 antibody, an anti-NKp46 antibody, an anti-NKp30 antibody, an anti-NKG2D antibody, or the like. The antibody may be a monoclonal antibody, a polyclonal antibody, or the like. Selection of the target NK cells or a population thereof may be performed by selectively removing T cells, NKT cells, hematopoietic progenitor cells and other cells.

(Effect of mixed culture)
According to the studies by the present inventors, the proliferation of NK cells is improved by culturing a population of mononuclear cells derived from a plurality of donors and containing NK cells. It is considered that this is because more licensing signals are input to each other due to the increase in the variation of the HLA / KIR combination. Good proliferation means cell proliferation rate (number of cells after culturing / number of cells before culturing) as compared with the case of culturing cells derived from a single donor of any one of a plurality of donors in a mixed culture. Is improving.

Further, in the cell population obtained by the mixed culture, 70% or more, preferably 80% or more, more preferably 90% or more can be NK cells.

According to the study by the present inventors, the cytotoxic activity of NK cells obtained by mixed culture of a population of mononuclear cells containing NK cells derived from a plurality of donors is higher than that of NK cells derived from a single donor. Can be equal or better. Furthermore, the proportion of NK cells highly expressing CD16 can be equal to or higher than that of NK cells derived from a single donor. The cytotoxic activity refers to the lytic ability of a target cell (effector cell, E) to a target cell (T) unless otherwise specified. The cytotoxic activity can be expressed as a percentage (%) of the target cells that have died by the effector cells, and is calculated by, for example, the following formula.

(Cell death when co-cultured with effector cells-Natural cell death (negative control)) / (Maximum cell death (positive control) -Natural cell death (negative control)) x 100

When measuring cytotoxic activity, generally, the mixing ratio of effector cells and target cells (E: T) and the co-culture time of effector cells and target cells are determined according to the degree of cytotoxic activity of effector cells. , It can be appropriately adjusted according to the type of cells used and the strength of activity. 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 myelogenous leukemia cells, and chronic myelogenous leukemia cells. Effector cells and target cells, live cells and dead cells can be distinguished and quantified by reagents such as antibodies labeled with radioactive substances and fluorescent dyes.

In addition, the population of NK cells of the present invention derived from a plurality of donors according to the present invention is not suppressed by MDSC (Myeloid-derived suppressor cells), or the suppression thereof is extremely low. From the studies so far, the present inventors have confirmed that the population of NK cells derived from a single donor obtained by the above-mentioned culture method is not suppressed by MDSC. One of the reasons for this is that the expression of receptors for humoral factors (TGF-β, IL-10) is absent or significantly low. According to our recent studies, we have also found that the above advantages are not compromised when mixing cells from multiple donors.

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

Such a cell population may further have the following characteristics:
(3) The proportion of NK cells is 70% or more, more specifically 80% or more, and further specific 90% or more.
(4) Both a population of NK cells having a high expression of CD16 and a population of NK cells having a low expression of CD16 may be included. The proportion of NK cells with high expression of CD16 is 50% or more.

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 based on the number of viable cells of the target cell (T) after the action of the effector cell (E), for example, by the formula: (1-number of viable cells / number of negative control viable cells) × 100. Can be calculated.
When NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixing ratio (E: T) 1: 1, the cytotoxic activity is preferably 60% or more, preferably 70. % Or more, more preferably 80% or more, further preferably 90% or more, still more preferably 95% or more.
When NK cells are used as effector cells (E) and SKOV3 cells are used as target cells (T) and co-cultured at a mixing ratio (E: T) 3: 1, the cytotoxic activity is preferably 60% or more, and is 70. % Or more, more preferably 80% or more, further preferably 90% or more, still more preferably 95% or more.

For markers such as CD16, positive may be represented by + and negative may be represented by-. For example, CD16 positive, expressed as CD16 ^+, CD16 negative, CD16 ^- sometimes denoted. Positives include those with high expression and those with low expression. High expression may be expressed as high or bright. For example, CD16 high expression may be expressed as ^{CD16 high} , CD16 ^bright. Low expression may be expressed as low or dim. For example, CD16 low expression may be expressed as ^{CD16 low} , CD16 ^dim.

Positive, negative, high expression, and low expression can be determined based on the chart obtained by the flow cytometry method. The position appearing on the chart may vary depending on the voltage setting, sensitivity setting, antibody clone used, staining conditions, dye used, etc. of the device, but those skilled in the art can use a cell population recognized as a group in the obtained chart. Lines can be drawn as appropriate so as not to separate them.

Whether the expression of the target marker is positive or negative can be determined by using an Isotype control antibody as a negative control. Isotype control antibody is an antibody that does not react with a specific antigen. In general, in experiments with antibodies, background may occur due to non-specific binding to proteins other than the target or binding to the Fc receptor on the cell surface. By comparing with a system using an antibody that serves as a negative control, it becomes clear that the reaction of the primary antibody to the target antigen is specific. In addition, the influence of the background is eliminated, and the signal strength can be interpreted accurately.

The degree of expression of the marker of interest (whether it is low expression or high expression) can be determined by comparison with the results of control cells measured under the same conditions. Examples of control cells are NK cells obtained from peripheral blood that have not been substantially cultured, such as the primary NK cells described in the Examples section of the present specification.

For example, the degree of expression of CD16 in a population of NK cells is determined by using flow cytometry, and the expression level of CD16 in the population of NK cells is obtained from peripheral blood. When the expression level equivalent to that of the control is observed, it can be determined that the expression level is high, and when the expression level is lower than that of the control cell, it can be determined that the expression level is low. It is known that control NK cells are highly expressive of CD16.

[Pharmaceutical use]
The present invention also provides a pharmaceutical composition for cell therapy, which comprises the above-mentioned cell population containing NK cells as an active ingredient. Cell therapy refers to a method of treating a disease or condition in a subject by administering the treated cells in vitro to the subject, including immuno-cell therapy.

In addition to the cell population that is the active ingredient, the pharmaceutical composition contains a solution capable of suspending NK cells, for example, physiological saline, phosphate buffered saline (PBS), and the like. The pharmaceutical composition or solution may also contain pharmaceutically acceptable additives. The pharmaceutical composition can be administered, for example, intravenously, arterial, subcutaneously, intraperitoneally or the like. Cell therapy with the pharmaceutical composition can be performed alone or in combination with surgery, chemotherapy, radiation therapy and the like.

The cell population, including NK cells, is 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, it includes, but is not limited to, oral cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, liver cancer, colon cancer, kidney cancer, bladder cancer, leukemia; infections caused by viruses, bacteria and the like. In cell therapy using the pharmaceutical composition of the present invention, NK cells may be administered, for example, intravenously, arterial, subcutaneously, intraperitoneally or the like. Cell therapy may be performed alone or in combination with surgery, chemotherapy, radiation therapy, antibody drugs and the like.

Most antibody drugs are said to show antitumor effects due to ADCC activity after intravenous administration. On the other hand, when ADCC activity is exerted, monocytes / macrophages and neutrophils are also mobilized in addition to NK cells. Effectors other than NK cells show ADCC activity without distinguishing between normal cells and cancer cells, which is considered to lead to the occurrence of side effects. In the present invention, the NK cells and the antibody may be mixed before administration, and the NK cells may be preliminarily equipped with the antibody. As a result, the effector is limited to NK cells, the amount of antibody to be administered can be reduced, and it is considered that it is extremely effective in reducing side effects. That is, the pharmaceutical composition of the present invention may be prepared through a step of mixing an NK cell population and an antibody and then removing an antibody that is not bound to NK cells. That is, one of the preferred embodiments of the pharmaceutical composition of the present invention contains NK cells and an antibody, but the antibody is bound to NK cells and does not substantially contain an antibody that is not bound to NK cells. Is. (See Patent Document 3 above).

The production of the pharmaceutical composition of the present invention includes conditions (good manufacturing practice, GMP) conforming to the manufacturing control and quality control rules for pharmaceuticals and non-pharmaceutical products, and manufacturing control and quality control standards for products such as regenerative medicine (Good Gene). , Cellular, and Tissue-based Products Manufacturing Practice (GCTP).

The examples of the present invention described below are for purposes of illustration only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention, can be made on condition that the gist of the present invention is not deviated.

[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). Mix the isolated peripheral blood mononuclear cells for multiple people at almost the same ratio ^{, add and suspend CD3 beads * 1} , incubate at 4 ° C for 15 minutes, ^{then add 1 mL * 2 of} separation buffer and suspend well. Centrifugation was performed at 300 xg for 10 minutes. Remove the supernatant, suspend in 0.5 mL separation buffer, add 2 mL of separation buffer to a pre-moistened LD column (Miltenyi Biotec, 130-042-901) and elute from the LD column. The liquid was recovered. Further, 1 mL of separation buffer was added to the LD column, and the eluate was collected. Then, the column was washed with 1 mL of separation buffer, the number of cells in the collected solution was counted, and the total number of cells was calculated. Centrifugation was performed at 500 xg for 5 minutes, and after removing the supernatant, the cells were suspended in ^{KBM501 medium * 3} so as to have ^{5 x 10 5 cells / mL.}

Some cells were harvested for flow cytometer measurements and the remaining cells were cultured. Culturing was performed in a _{CO 2} incubator 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). ℃, 5% CO ₂ ). Take a portion of the culture medium on the 5th and 9th days of culture, count the number of cells, and on the 9th day, the final volume is 6 mL per well in the case of a 6-well plate, in a T-75 flask. In this case, KBM501 medium was added so as to be 50 mL per flask. On the 14th day, cells were collected, the number of cells was counted, and cell surface antigens were measured with a flow cytometer using some cells.

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

The results are shown in FIG. Cell proliferation was good in all of the 9-person mixture (A in FIG. 1), the 8-person mixture (B in FIG. 1), and the 7-person mixture (C in FIG. 1). When the purity was confirmed, 90% or more of the CD3 ^- CD56 ⁺ NK cells were found in all the mixed cultures. In the 8-person mixed culture, the cells were cultured in a T-75 flask and a 500 cm ² culture bag, and cell proliferation was good in both cases. The 9-person mixed culture, the 7-person culture, and the culture derived from a single donor (1 of 7 people) were cultured using a 6-well plate. In the culture using the 500 cm ² culture bag, the work of turning the bag over was performed as described in Example 4 described later.

Comparing the cell proliferation between the 7-person mixed culture and the culture derived from a single donor, the culture derived from a single donor ^{grew from 6.8 × 10 6} cells to 1.9 × 10 ⁷ cells (about 2.8 times), whereas the mixed culture Then, the ^{cells proliferated from 7.8 × 10 6} cells to 3.7 × 10 ⁷ cells (about 4.7 times), and the cell proliferation was better in the mixed culture (C in FIG. 1).

[Example 2: Mixed culture of NK cells obtained from fresh peripheral blood 2]
NK cells obtained by adding up the data obtained by culturing from a single donor (n = 17) and mixed culture of 4 or more people (n = 10) in a T-75 flask by the method described in Example 1. Statistical analysis was performed on the cell proliferation rate of the number. Wilcoxon rank sum test was performed using JMP Pro13 as the analysis software.

The results are shown in FIG. The proliferation rate (total number of cells after proliferation / total number of cells before proliferation) in the case of culturing cells derived from a single donor is 3.10 ± 0.56, whereas the proliferation rate in the case of mixed culture is 6.56 ± 1.24. , The mixed culture had a statistically significantly higher growth rate (P <0.01).

[Example 3: Tumor cytotoxic activity test]
<< Preparation of NK cells >>
After collecting and washing four mixed-cultured NK cells obtained from healthy volunteers by the method described in Example 1 and cultured NK cells from a single donor (one of four), 10% FBS (Nichirei) was used for each. RPMI1640 medium (Wako Pure Chemical Industries, 189-02025) (hereinafter referred to as 10% FBS / RPMI1640) containing bioscience, 171012-500ML) and 100 units of penicillin, 100 μg / mL streptomycin (Nakalitesk, 26253-84). ), And prepared in the same medium to ^{a concentration of 1x10 6 cells / mL.}

<< Preparation of SKOV3 >>
SKOV3 cells (human ovarian cancer cell line) were prepared at a concentration of ^{1x10 6} cells / mL in RPMI1640 medium (Wako Pure Chemical Industries, Ltd., 189-02025) containing no serum component. The prepared SKOV3 cells were stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT), and finally prepared with 10% FBS / RPMI1640 to 2x10 ⁶ cells / mL and 2x10 ⁵ cells / mL. did.

<< Preparation of MDSC (Myeloid-derived suppressor cells) >>
Peripheral blood mononuclear cells were isolated from healthy volunteers (separate from NK cell donors) using Ficoll and 10 ng / mL IL-6 (PEPROTECH, 200-06-5UG) and 10 ng / mL GM-CSF. MDSC was induced by culturing in 10% FBS / RPMI1640 containing (CellGenix, 1012-050) for 7-10 days. MDSC was suspended in RPMI1640 medium containing no serum components (Wako Pure Chemical Industries, Ltd., 189-02025), stained with PKH Green Fluorescent Cell Linker Kit (Sigma, MINI67-1KT), and finally 10% FBS / ^{Prepared to 8x10 5} cells / mL with RPMI 1640.

《Cytotoxic activity test》
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, a total of 4 groups in which MDSC was added to each of these 2 groups, and only SKOV3 cells as a negative control. I prepared a group.

NK cells and SKOV3 cells were seeded and mixed in a 96-well plate (IWAKI, 4870-800SP) so as to have a cell ratio of 3: 1 and _{reacted at 37 ° C. under 5% CO 2} for 4 hours. In the MDSC-added group, NK cells and MDSC were first mixed in a 96-well plate so that the cell ratio of NK cells to SKOV3 to MDSC was 5: 1: 4, and then 12 at _{37 ° C. under 5% CO 2.} Reacted for ~ 18 hours. Then, the cells were centrifuged (500 xg, 5 minutes), the supernatant was removed, SKOV3 cells were added and mixed, and the cells were _{reacted at 37 ° C. under 5% CO 2} for 4 hours. After the reaction, the mixture was centrifuged (500 xg, 5 minutes), the supernatant was removed, a PBS-diluted Zombie solution (Biolegend, 423105) was added, mixed, and incubated at room temperature for 30 minutes in the dark. After centrifugation and removal of the supernatant, the mixture was suspended in PBS and AccuCheck Counting Beads (Thermo Fisher Scientific, PCB100) was added. The cytotoxic activity rate (% Lysis) was calculated by measuring with a flow cytometer (BD LSR Fortessa, BD Bioscience) and analyzing with FlowJo software (FLOWJO, LLC) ^{* 4} .

* 4: Cytotoxic activity rate = (1-SKOV3 viable cell number / negative control SKOV3 viable cell number) x 100
Number of viable cells: Number of SKOV3 cells actually measured x Number of beads in the added bead solution / Number of beads actually measured
^{Actual number of SKOV3 cells: PKH26 +} gated after FSC / SSC gated (debris exclusion), doublet exclusion

The results are shown in FIG. Mixed-cultured NK cells derived from multiple donors showed equivalent or better cytotoxic activity than NK cells derived from a single donor.

[Example 4: Mixed culture of NK cells prepared from frozen apheresis blood]
2 Donor frozen apheresis blood (HemaCare, PB001CLP) is thawed, mixed, diluted with HBSS (-) solution (Nacalai Tesque, 17461-05), and automatically closed cell processing system Lovo Cell Processing System (FRESENIUS KABI). Was washed with PBS / EDTA solution (Miltenyi Biotec, 130-021-201) and concentrated. Next, the concentrated cell fluid was subjected to CliniMACS Prodigy TS 310 (Miltenyi Biotec, 130-097-183), CliniMACS CD3 MicroBeads (Miltenyi Biotec, 130-017-601), CliniMACS CD34 Reagent (Miltenyi Biotec, 130-017-501). CD3-positive cells and CD34-positive cells were removed using, and washed and eluted. The total number of cells was calculated by counting the number of cells in the eluate. Centrifugation was performed at 500 xg for 5 minutes, and after removing the supernatant, the cells were suspended in KBM501 medium to 1x10 ^{6 cells / mL.} _{Culturing was performed in a CO 2} incubator using a T-75 flask (Thermo Fisher Scientific, 156499) or a 500 cm ² culture bag (Nipro) (37 ° C, 5% CO ₂ ). The bag was cultured 30 minutes after the start of the culture, the bag was turned over, and on the 9th day of the culture, the bag was turned over again (see FIG. 4-1). In addition, on the 9th day of culture, a part of the culture solution was taken and the number of cells was counted so that the final amount of the solution was 50 mL per flask for the T-75 flask and 500 mL per bag for the culture bag. Was added. On the 14th day, cells were collected, the total number of cells was counted, and cell surface antigens were measured with a flow cytometer using some cells.

The recovered cells were stained with antibody as follows:
Alexa Fluor ^(R) 700 labeled anti-human CD56 antibody (Biolegend, 318316), PerCP / Cy5.5 labeled anti-human CD3 antibody (Biolegend, 300430), PE-Cy7 labeled anti-human CD16 antibody (Biolegend, 302016) 1 μg / mL After staining at 4 ° C for 30 minutes at the concentration of, centrifuge (500 xg, 5 minutes, 4 ° C), remove the supernatant, suspend in PBS (-) (Wako Pure Chemical Industries, Ltd.), and then flow cytometer ( Measurements were performed using BD LSR Fortessa (BD BioScience) and analyzed with FlowJo software (FLOWJO, LLC).

The results are shown in FIG. 4-2. Even when NK cells obtained from frozen apheresis blood were used, the cells proliferated sufficiently in 14 days by mixed culture. Specifically, T-75 flask cultures ^{grew from 1.50 x10 8} cells to 5.72 x10 ⁸ cells (approximately 3.81 times), and 500 cm ² culture bag cultures ^{grew from 1.50 x10 8} cells to 5.60 x10 ⁸ cells. (Approximately 3.73 times) (Fig. 4-2, left).

The purity of NK cells in the obtained population of cultured NK cells was 90.5% in the case of T-75 flask culture and 91.7% in the case of ^{500 cm 2 culture bag culture.} Regarding the expression of CD16, the population of mixed-cultured NK cells was bimodal, similar to the population of cultured NK cells derived from a single donor (see JP-A-2018-193303). Specifically, the percentages of CD16 low expression and CD16 high expression were 50.9% and 49.1% in order for T-75 flask culture, 46.7% and 53.3% in order for ^{500 cm 2 culture bag culture.} It was% (Fig. 4-2, right).

[Example 5: Monocyte / NK cell swapping experiment]
<< Preparation of primary NK cells and monocytes >>
Blood was collected from two healthy volunteers (donors 1 and 2), and peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll. From isolated peripheral blood mononuclear ^{cells, use EasySep TM} Human NK Cell Enrichment Kit (STEMCELL, 19055) to generate primary NK cells, and use EasySep ^TM Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL, 19058) to generate monocytes. The cells were separated and the number of cells was counted for each. Primary NK cells 1x10 ⁵ cells / mL, monocytes were suspended in KBM 501 medium so as to 3x10 ⁵ cells / mL.

《Swapping culture》
Donor 1 primary NK cells and donor 1 monocytes, donor 1 primary NK cells and donor 2 monocytes, donor 2 primary NK cells and donor 2 monocytes, donor 2 primary NK cells and donor 1 monocytes. A total of 4 groups, each of which was a combination of spheres, were mixed so that the cell ratio of primary NK cells and monocytes was 1: 3, and culture was started on a 6-well plate (Thermofisher Scientific, 140675) (37). ℃, 5% CO ₂ ). KBM501 medium was added on the 9th day of culture in the same manner as in Example 1, and cells were collected on the 14th day. Using the obtained cells, the cytotoxic activity rate against the cell surface antigen and K562 (human chronic myelogenous leukemia cell line) was measured.

For the measurement of cell surface antigens, the collected cells were stained with an antibody and analyzed 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), PerCP / Cy5.5-labeled anti-human KIR3DL1 antibody (Biolegend, 312718) was stained at a concentration of 1 μg / mL at 4 ° C for 30 minutes, centrifuged (500 xg, 5 minutes, 4 ° C), the supernatant was removed, and PBS (PBS) ( -) After suspension in (Wako Pure Chemical Industries, Ltd.), measurement was performed using a flow cytometer (BD LSR Fortessa, BD Bioscience), and analysis was performed using FlowJo software (FLOWJO, LLC).

For the measurement of cytotoxic activity, 4 groups in which each cultured NK cell formed by combining 2 donor primary NK cells and monocytes was reacted with K562 cells, a group of only K562 cells as a negative control, and K562 as a positive control. A group in which cells were fixed with 10% formalin was prepared.

《Preparation of K562》
K562 cells (human chronic myelogenous leukemia cell line) were prepared at a concentration of ^{1x10 6} cells / mL in RPMI1640 medium (Wako Pure Chemical Industries, Ltd., 189-02025) containing no serum component. The prepared K562 cells were stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT), and finally ^{prepared to 2x10 6} cells / mL with 10% FBS / RPMI1640.

<< Preparation of cultured NK cells >>
Four groups of cultured NK cells obtained by culturing a combination of two donor primary NK cells and monocytes obtained by the above method were collected and washed, suspended in 10% FBS / RPMI1640, and 1x10 in the same medium. Prepared to a concentration of ^{6 cells / mL.}

《Cytotoxic activity test》
NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) so as to have a cell ratio of 1: 1 and mixed, and _{reacted at 37 ° C. under 5% CO 2} for 2 hours. After the reaction, the mixture was centrifuged (500 xg, 5 minutes), the supernatant was removed, and a 7-AAD solution (Beckman Coulter, A07704) diluted with PBS was added and suspended, and the mixture was incubated at room temperature for 20 minutes. The cytotoxic activity rate (% Lysis) was calculated by measuring with a flow cytometer (BD LSR Fortessa, BD Bioscience) and analyzing with FlowJo software (FLOWJO, LLC) ^{* 5} .

* 5: Cell damage activity rate = (K562 cell dead cell rate-negative control dead cell rate) / (positive control dead cell rate-negative control dead cell rate) x 100

The donor typing information is shown in the table below, and the results are shown in FIG.

Higher ADCC activity can be expected because the population of ^{CD16 high} increases under the condition that one or both signals from KIR3DL1 and KIR3DS1 enter.

Claims (9)

Prepare a population of mononuclear cells, derived from multiple donors and containing NK cells,
Incubating the prepared mononuclear cell population under conditions effective for NK cell treatment, including
The step of preparing a population of mononuclear cells is derived from one selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells derived from multiple donors. A method for producing a cell population containing NK cells, which prepares a population of mononuclear cells. The production method according to claim 1, wherein the step of preparing a population of mononuclear cells includes a step of removing CD3-positive cells. The production 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. The production method according to any one of claims 1 to 3, wherein the step of preparing a population of mononuclear cells includes a step of obtaining a population of mononuclear cells from apheresis blood collected from a plurality of donors. The production method according to any one of claims 1 to 4, wherein the plurality of donors comprises one donor and another donor having a different genotype in at least one of HLA and KIR. A cell population containing NK cells for treating a disease with the following characteristics:
(1) It is derived from any one selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells collected from a plurality of cells.
(2) The cytotoxic activity is 50% or more when NK cells are used as effector cells (E) and K562 cells are used as target cells (T) and co-cultured at a mixing ratio (E: T) of 1: 1. A cell population produced by the production method according to any one of claims 1 to 5.
A cell containing a cell population having a cytotoxic activity of 50% or more 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. Pharmaceutical composition for therapy. A pharmaceutical composition for cell therapy comprising the cell population according to claim 7. The pharmaceutical composition according to claim 7 or 8, for treating an infectious disease and / or cancer.