WO2020152661A1

WO2020152661A1 - Production method for cell population including nk cells

Info

Publication number: WO2020152661A1
Application number: PCT/IB2020/052499
Authority: WO
Inventors: 米満　吉和; 結原田
Original assignee: 株式会社ガイアバイオメディシン
Priority date: 2019-01-21
Filing date: 2020-03-19
Publication date: 2020-07-30

Abstract

The present invention addresses the problems of providing an effective method for producing a population of NK cells for cell therapy, improving NK cell expansion efficiency in vitro, and flexibly increasing the signalling necessary for NK cell licensing. Specifically provided is a production method for a cell population including NK cells by preparing a monocyte population that is derived from multiple donors and includes NK cells, incubating and culturing the prepared monocyte population under conditions effective for the processing and proliferation of NK cells, and inducing the proliferation of the NK cells.

Description

Method for producing cell population containing NK cells

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 number one cause of death among the people, and countermeasures against them are urgently needed. In particular, the development of new treatment methods for advanced-stage intractable malignant tumors that are resistant to existing treatment methods consisting of surgery, radiotherapy and chemotherapy is extremely important and significant. In recent years, as a fourth treatment method, immunotherapy such as an immune checkpoint inhibitor or a therapy using chimeric antigen receptor (CAR) gene-modified T cells (CAR-T therapy) has been attracting attention. However, since many of them use T cells that are activated by recognizing an antigen as effectors, there is a fundamental barrier that they are restricted to specific antigens.

As 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 patients is attracting attention as a therapeutic method with relatively few side effects. However, the number of NK cells obtained from peripheral blood and the like is relatively small, and in vitro proliferation is low. Therefore, techniques for culturing and expanding NK cells have been investigated. For example, Patent Document 1 discloses 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 the remaining remaining cells as a cytokine containing only IL-2. And a step of culturing without using feeder cells, and a method for expanding NK cells, and a pharmaceutical composition for cell therapy containing a cell population containing NK cells obtained by the expansion. suggest. Further, in Patent Document 2, a step of amplifying hematopoietic progenitor cells under a single culture condition containing IL-15, SCF, IL-7 and Flt3L, and a cell obtained in the step of amplifying IL-2, A method for preparing NK cells, which comprises the step of inducing differentiation into NK cells under culture conditions comprising 5, 6, 7, 8 or 9 days, and a cell therapy containing the prepared cell population containing the NK cells. The pharmaceutical composition of

In addition, the inventors of the present invention have shown that, as cultured cells having enhanced tumor cytotoxicity, NK cells that are CD16-positive, CD56 high-expressing, CD57-negative, NKG2C-positive, NKG2A-negative-low-expressing, and CD94-positive. , 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 is as diverse as HLA. In hematopoietic stem cell transplantation, HLA is not matched as much as possible, and recently, an intentional KIR between a donor and a recipient, such as selecting a donor so that the recipient does not have a ligand for the KIR of the donor NK cell, has been proposed. /HLA mismatch is being effectively used because it can bring about more desirable results from the viewpoint of recurrence, GVHD (graft versus host disease, graft-versus-host disease) and prognosis. However, for the anti-tumor activity of NK cells, NK cells were cultured with feeder cells either matched or mismatched for inhibitory KIR ligands (lacking one or more ligands present in the NK cell donor). From a report (Non-patent Document 1) investigating to what extent the HLA class I-KIR interaction influences human NK cell proliferation in allogeneic environment (Non-patent Document 1), NK cell hyperactivation is indicated using an antitumor effect as an index. If desired, less signal from the KIR is suggested to be better. In addition, a recent report (Non-patent Document 2) on allogeneic responsiveness of HLA heterozygous NK cells (in the case of unidirectional matching) to tissues derived from HLA haplotype homozygous (HLA homo) iPS cells is related to culture of NK cells. , Strongly suggests that mixed culture containing HLA/KIR mismatch is not established.

JP, 2013-27385, A (patent No. 5728863, patent No. 5989016). JP, 2014-226079, A (patent No. 5511039, patent No. 6164650). JP, 2018-193303, A Japanese Patent Application No. 2018-059624 Application specification (not disclosed on the priority date of the present application)

The present inventors are developing 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 are limited, it is not possible to convert the therapeutic NK cell population into an off-the-shelf in preparation for future treatment.

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

Furthermore, in the body, immature NK cells that have undergone binding with a ligand (HLA class I) are “licensed” in the process of differentiating from hematopoietic stem cells in the bone marrow, and the expression of autologous HLA class I is reduced after maturation. It is thought to have the ability of "missing-self response" to detect NK, but NK cells differentiated and matured from iPS cells or ES cells may not have sufficient signals required for license to obtain an antitumor effect. There is.

The present invention provides the following.
[1] Including NK cells, comprising preparing a population of mononuclear cells derived from a plurality of donors and containing NK cells, and incubating the prepared population of mononuclear cells under conditions effective for treating NK cells Method for producing cell population.
[2] The production method according to 1, wherein the step of preparing a population of mononuclear cells includes the 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 the step of removing CD34-positive cells.
[4] The production method according to any one of 1-3, 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 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] Any of the step of preparing 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 a plurality of donors The method for production according to any one of 1-3, which is for preparing a population of mononuclear cells derived from sea urchin.
[7] The production method according to any one of 1 to 6, wherein the plurality of donors include one donor and another donor having a genotype different from that of 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) at a mixing ratio (E:T) of 1:1.
[9] A pharmaceutical composition for cell therapy, comprising a cell population produced by the production method according to any one of 1-7.
[10] A pharmaceutical composition for cell therapy, comprising the cell population according to item 8.
[11] The pharmaceutical composition according to 9 or 10 for treating infectious disease and/or cancer.

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

NK cells can be stably grown regardless of the donor, and the growth rate can be improved.

▽ Support for diversification of HLA-KIR matching and stable activation can be expected.

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

Culture test: CD3 positive cells were removed from PBMCs of a plurality of persons, and the cells were cultured for 14 days using KBM-501 medium. 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 cell cytotoxicity test: The cytotoxic activity of NK cells prepared by mixed culture and culture derived from a single donor was evaluated using the damage to SKOV3 (human ovarian cancer cell line) as an index. Culture with 500 cm ² culture bag. After 30 minutes from the start of culturing and on the 9th day of culturing, the bag was flipped over. Culture test. Frozen apheresis blood (for 2 donors) was used as a material, and after removing CD3 positive cells and CD34 positive cells using an automatic closed cell processing device, using a T75 flask or a 500 cm ² culture bag, using KBM-501 medium Cultured for a day. Monocyte/NK cell swapping experiment: The population of CD16 ^high increased under the condition that one or both of the signals from KIR3DL1 and KIR3DS1 entered. Higher ADCC activity can be expected for the CD16 ^high population.

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

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

(Preparation of 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, 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).

The population of mononuclear cells derived from a plurality of donors and containing NK cells includes a population of mononuclear cells (containing NK cells and may contain monocytes) obtained from one donor, and another population. The population of mononuclear cells (including NK cells and may include monocytes) obtained from a donor may be a mixed one, and NK cells obtained from one donor and another donor may be mixed. It may be a mixture with monocytes obtained from.

Regarding the donor, plural means two or more, and is not particularly limited as long as the above conditions are satisfied. For example, mononuclear cells derived from three or more, four or more, seven or more, and nine or more donors were mixed. One can be used. The plurality of donors may include the patient himself/herself who is administered the cell population containing NK cells, or a relative of the patient.

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

Regarding NK cells, licensing refers to the process of acquiring the "missing-self response (detection of lack of self HLA)" characteristic of NK cells in the process of cell differentiation and maturation. Regarding humans and mice, only immature NK cells that have undergone binding of HIR class I molecule with KIR or NKG2A in the process of differentiation of NK cells from hematopoietic stem cells in the bone marrow are licensed, and expression of self HLA class I after maturation is licensed. It is believed to acquire the ability to detect depressed cells. Examples of KIRs known to be involved in licenses are KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1 and NKG2A/CD94. Among classical HLA, HLA types that can be recognized by NK cells include HLA-C allotypes, about 1/3 of HLA-B allotypes, a part of HLA-A allotypes (those having a Bw4 motif), and non-classical HLA-E and HLA-G of HLA. In the present invention, when selecting a plurality of donors so as to include one donor and another donor having a genotype different from that of at least one of HLA and KIR, the KIR and HLA related to the license as described above are selected. Especially, it can be considered.

In one of the preferred embodiments of the present invention, donors having different genotypes can be selected so as to increase the repertoire of molecules used for licensing NK cells. Alternatively, the inhibitory KIR of NK cells may be selected not to be stimulated, or the inhibitory KIR may be selected to be stimulated.

When anticipating antibody-dependent cellular cytotoxicity (ADCC) activity by NK cells, the selection of donors is such that NK cells that are CD16 ^high are increased when a mixed mononuclear cell population is cultured. You may go to ADCC is known as one of the mechanisms of cytotoxicity by NK cells. NK cells bind to an antibody bound to the target cell via CD16, which is an Fc receptor on the cell surface, Injuries. Therefore, it can be expected that the ADCC activity becomes higher by increasing the number of NK cells that are CD16 ^high . An example of such a donor combination condition is to select and mix donors so that NK cells are licensed by a signal from one or both of KIR3DL1 and KIR3DS1.

Mixing is performed before administering NK cells to the subject. The mixing can be carried out in various stages as long as the desired effects can be obtained. For example, blood taken from multiple donors may be mixed, and then the mixture may be used to obtain a population of mononuclear cells. Alternatively, blood collected from each donor may be used to obtain a population of mononuclear cells, respectively. , The populations may be mixed. Alternatively, the populations of mononuclear cells collected from a plurality of donors may be cultured and then the populations may be mixed. 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 set appropriately. For example, cells from multiple donors can be included in approximately equal proportions, and cells from a particular donor can be included in greater or lesser proportion. When mixing NK cells derived from one donor and monocytes derived from another donor, the mixing ratio can be set appropriately. For example, 1 to 9 times as many monocytes as NK cells 1 can be mixed (NK cells:monocytes=1:1-9), and 2 to 4 times as many monocytes as NK cells 1 can be mixed. The spheres can be mixed (NK cells:monocytes=1:2-4). In the context of the present invention, the mixing ratio of cells is based on the number of cells, unless otherwise stated.

As a raw material for obtaining a population of mononuclear cells, blood cells collected from peripheral blood, cord blood, bone marrow and/or lymph node can be used. One of the preferable raw materials is peripheral blood, which may be collected by the apheresis method. 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 a plurality of 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 a plurality of donors.

A population of mononuclear cells, including NK cells and monocytes, can be prepared using various procedures known to those of skill in the art. For example, from blood such as peripheral blood and umbilical cord blood, red blood cells can be removed and a mononuclear cell fraction can be collected by performing density centrifugation at room temperature. Peripheral Blood Mononuclear Cells (PBMC) isolated from peripheral blood include various lymphocytes such as T cells, B cells, NK cells, monocytes and dendritic cells. In the context of 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 of skill in the art. For example, a method for separating NK cells by removing unnecessary cells from whole blood or PBMC is known. Monocytes can be prepared using various 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, etc. 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, It may be selectively separated from the cell population using an anti-KIR2DS1 antibody, 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. The 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 the step of removing CD3-positive cells or the step of removing CD34-positive cells.

The removal of CD3-positive cells and the removal of CD34-positive cells can be performed, for example, by using Dynabeads manufactured by Invitrogen, Dynabeads manufactured by Invitrogen, or immunomagnetic beads such as CliniMACS manufactured by Miltenyi Biotech, and the cell surface antigen CD3 and/or It can be carried out by separating and removing cells expressing CD34. Such a step is preferably performed so as not to exhaust NK cells and prevent monocytes from exerting an extra phagocytic function. Specifically, using relatively small magnetic beads (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 low temperature. Examples of preferable operations include setting the time relatively short, removing unreacted beads, and then using a column to obtain cells 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 population of mononuclear cells is prepared from hematopoietic stem cells derived from any one selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Good. 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.2015.0264, and Zeng J. et al., Generation of Shelf "Natural berrelloller Celloller Killer". Derived Induced Pluripotent Stem Cells, Stem Cell Reports, 2017; 9: 1796-1812). Stem cells may also be modified so that highly active NK cells can be obtained. For example, high-affinity CD16 (158V: 158th amino acid is valine) is known, and such findings can be applied. it can.

(Culturing a population of mononuclear cells)
The prepared population of mononuclear cells is incubated in a state in which cells derived from a plurality of donors are mixed 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, the incubating is accompanied by growth. Incubation with proliferation can be read as culturing or proliferation.

“Effective conditions for NK cell treatment” means conditions suitable for mixed cells derived from a plurality of donors to exert the desired mixing effect. The medium or isotonic solution used for cell suspension, time, temperature, environment, etc. are not particularly limited as long as the desired effects are exhibited.

The condition effective for NK cell treatment includes the condition effective for activation of NK cell. Such conditions include, for example, incubation in the presence of appropriate concentrations of activation factors such as IL-2 and IL-15 at 37° C., 5% CO ₂ and saturated steam atmosphere for 4-18 hours. For example.

Effective conditions for NK cell treatment include effective conditions for NK cell proliferation. Effective conditions for the growth of NK cells include using a medium suitable for the growth of NK cells. Examples of such a medium include KBM501 medium (Kojin Bio Co., Ltd.), CellGro SCGM medium (Celgenics, Iwai Chemical Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Inc.), IMDM, MEM, DMEM. , RPMI-1640 and the like, but are not limited thereto.

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 be greater than 2000 IU/mL and can be 2500-2815 IU/mL. IL-2 preferably has a human amino acid sequence, and is preferably produced by recombinant DNA technology for safety. The concentration of IL-2 may be indicated 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 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 may be added to the medium. Autologous serum and human AB-type serum are preferably added at a concentration of 1-10%, and donated human serum albumin is preferably added at a concentration of 1-10%. The test subject may be a healthy person or a patient suffering from a disease. Further, a composition formulated for the growth of immune cells may be added to the medium instead of or together with the serum. Such compositions are commercially available. For example, UltraGro series (AventaCell), CTS Immune Cell SR (Thermo Fisher Scientific) can be used for the present invention.

The culture medium may contain appropriate proteins, cytokines, antibodies, compounds and other components, provided that the amplification effect of NK cells is not impaired. Cytokines 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 a human amino acid sequence, and are preferably produced by recombinant DNA technology for safety.

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

Culture vessels include, but are not limited to, commercially available dishes, flasks, plates, multiwell plates. In order to obtain a cell population containing NK cells for treatment, it is preferable to use a culture container in which many cells can be cultured and which is easy to handle. An example of such a culture container 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 volume greatly influences the culture efficiency including cell viability and growth rate. By inverting the bag during the culture period, it is possible to move some NK cells and monocytes to the surface where cell adhesion was relatively small because it was on the upper side until then, so that the culture 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 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 the longer the culture period is, the more NK cells can be obtained. The culture period is not particularly limited, provided that the NK cells are expanded to a desired cell number. For example, it may be performed for 7 to 28 days, may be 10 to 18 days, may be 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 need not be carried out immediately after the population of mononuclear cells is prepared. The prepared cell population containing mononuclear cells may be cryopreserved, thawed depending on the time of administration to a patient, and then provided for NK cell culture. The population of mononuclear cells is frozen during or after amplification by the NK cell expansion method of the present invention, thawed according to the time of transplantation into a patient, and then thawed. May be offered. Freezing and thawing may use any method 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 culture may contain NK cell precursors, T cells, NKT cells, hematopoietic progenitor cells, etc. in addition to the target NK cells. After culturing, the NK cells of interest 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 using anti-KIR2DL5 antibody, anti-NKp46 antibody, anti-NKp30 antibody, anti-NKG2D antibody or the like. The antibody may be a monoclonal antibody, a polyclonal antibody, or the like. The NK cell of interest or a population thereof may be selected by selectively removing T cells, NKT cells, hematopoietic progenitor cells and other cells.

(Effect of mixed culture)
According to the study by the present inventors, NK cell proliferation 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 variations of the HLA/KIR combination resulted in more licensing signals entering each other. Good growth is compared with the case of culturing cells derived from any one of a plurality of donors in mixed culture, and the proliferation rate of cells (the number of cells after culture/the number of cells before 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 may be NK cells.

Further, 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. It can be equal or better. Further, the proportion of NK cells that highly express CD16 may be equal to or higher than that of NK cells derived from a single donor. The cytotoxic activity refers to the lytic ability of target cells (effector cells, E) to target cells (T), unless otherwise specified. The cytotoxic activity can be expressed as a percentage (%) of target cells that have been killed by effector cells, and is calculated by the following equation, for example.

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

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

Further, the population of NK cells of the present invention derived from a plurality of donors of the present invention is not suppressed by MDSC (Myeloid-delivered suppressor cells), or its suppression is extremely low. From the previous studies, 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 there is no or extremely low expression of receptors for humoral factors (TGF-β, IL-10). According to the present studies by the present inventors, it has been found that the above advantages are not impaired even when cells from a plurality of donors are mixed.

[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) 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) 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:
The cytotoxic activity is 50% or more when (2′) SKOV3 cells are co-cultured with target cells (T) at a mixing ratio (E:T) of 3:1.

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 specifically 90% or more.
(4) It may include both a population of NK cells having high CD16 expression and a population of NK cells having low CD16 expression. The proportion of NK cells that highly express CD16 is 50% or more.

The cytotoxic activity of NK cells can be measured and calculated by a method well known to those skilled in the art. The cytotoxic activity (%) is usually based on the viable cell number of the target cell (T) after the action of the effector cell (E), for example, by the formula: (1-viable cell number/negative control viable cell number)×100. Can be calculated.
The cytotoxic activity of NK cells as effector cells (E) and K562 cells as target cells (T) at a mixing ratio (E:T) of 1:1 is preferably 60% or more, 70 % Or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more.
The cytotoxic activity of NK cells as effector cells (E) and SKOV3 cells as target cells (T) at a mixing ratio (E:T) of 3:1 is preferably 60% or more, 70 % Or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more.

Regarding markers such as CD16, a positive result may be represented by + and a negative result may be represented by-. For example, CD16 positive may be designated as CD16 ⁺ and CD16 negative may be designated as CD16 ⁻ . Positive cases include cases of high expression and cases of low expression. High expression may be expressed as high or bright. For example, high CD16 expression may be expressed as CD16 ^high or CD16 ^bright . Low expression may be expressed as low and dim. For example, CD16 low expression may be represented as CD16 ^low , CD16 ^dim .

Positive, negative, high expression, low expression can be judged based on the chart obtained by the flow cytometry method. The position appearing on the chart may vary depending on the voltage setting of the instrument, the sensitivity setting, the antibody clone used, the staining condition, the dye used, etc., but those skilled in the art can select the cell population recognized as a group in the obtained chart. It can be appropriately drawn so as not to be divided.

To determine whether the expression of the target marker is positive or negative, it is possible to use the isotype control (Isotype control) antibody as a negative control. The Isotype control antibody is an antibody that does not react with a specific antigen. In general, in experiments using antibodies, background may occur due to nonspecific binding to proteins other than the target or binding to Fc receptors on the cell surface. By comparing with a system using an antibody as a negative control, it becomes clear that the reaction of the primary antibody against the antigen of interest is specific. In addition, the influence of background is eliminated, and the strength of the signal can be accurately interpreted.

The degree of expression of the target marker (whether it is low expression or high expression) can be judged 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 CD16 expression in a certain NK cell population was determined by using flow cytometry, and the CD16 expression level in the NK cell population was determined from the peripheral blood-derived NK cell population that was not substantially cultured (control). In comparison with the CD16 expression level in the above, it can be judged that the expression is high if the expression is similar to that of the control, and that it is low when the expression is lower than that of the control cells. The control NK cells are known to highly express 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 cells treated ex vivo to the subject, which includes immune cell therapy.

The pharmaceutical composition includes, in addition to the cell population, which is an active ingredient, 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, into a vein, an artery, subcutaneously, intraperitoneally or the like. Cell therapy with a pharmaceutical composition can be performed alone or in combination with surgery, chemotherapy, radiation therapy and the like.

Cell populations including NK cells are expected to be applied to cancer treatment and infectious disease treatment (Dahlberg CM et al., Natural Killer Cell-Based Therapeutic Targeting Cancer: Possible Strategies Anesthetics). -Tormor Activity, Front. status and future perspectives, Oncotarget, 2018;9(29):20891-20907), and the pharmaceutical composition of the present invention can be used for treating such cancer or infectious disease. More specifically, it includes, but is not limited to, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, colon cancer, kidney cancer, bladder cancer, leukemia; infectious diseases caused by viruses, bacteria and the like. In the cell therapy using the pharmaceutical composition of the present invention, NK cells may be administered, for example, into veins, arteries, subcutaneously, intraperitoneally and 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 an antitumor effect due to ADCC activity after intravenous administration. On the other hand, monocytes/macrophages and neutrophils are recruited in addition to NK cells when exerting ADCC activity. The effectors other than NK cells show ADCC activity without distinguishing between normal cells and cancer cells, which is considered to lead to the development of side effects. In the present invention, NK cells and antibody may be mixed before administration to equip the NK cells with the antibody in advance. As a result, the effector is limited to NK cells, the amount of antibody to be administered can be reduced, and it is considered to be extremely effective in reducing side effects. That is, the pharmaceutical composition of the present invention may be prepared by mixing the NK cell population and the antibody, and then removing the antibody not bound to the NK cell. That is, one of the preferred embodiments of the pharmaceutical composition of the present invention comprises NK cells and an antibody, but the antibody is bound to NK cells and substantially free of antibodies not bound to NK cells. Is. (See the above-mentioned Patent Document 3).

The pharmaceutical composition of the present invention is manufactured under the conditions (good manufacturing practice, GMP) conforming to the manufacturing control and quality control rules for drugs and quasi-drugs, and the manufacturing control and quality control standards for products such as regenerative medicine (Good Gene). , Cellular, and Tissue-based Products, Manufacturing Structure, GCTP).

The embodiments of the present invention described below are for illustrative purposes 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 features of the present invention, can be made on the condition that the gist of the present invention is not deviated.

[Example 1: Mixed culture 1 of NK cells obtained from fresh peripheral blood]
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 isolated humans were mixed at approximately the same rate, CD3 beads ^*1 was added and suspended, incubated at 4°C for 15 minutes, and then well suspended by adding 1 mL ^*2 of separation buffer to 300 xg. Centrifugation was performed for 10 minutes. The supernatant was removed, and the suspension was suspended in 0.5 mL of separation buffer, and 2 mL of the separation buffer was added to a previously moistened LD column (Miltenyi Biotech, 130-042-901) to elute from the LD column. The liquid was collected. Further, 1 mL of the separation buffer was added to the LD column, and the eluate was collected. Then, the column was washed with 1 mL of the separation buffer, the number of cells in the collected liquid was counted, and the total number of cells was calculated. After centrifugation at 500×g for 5 minutes and removing the supernatant, the cells were suspended in KBM501 medium ^*3 at 5×10 ⁵ cells ^/ mL.

Some cells were collected for flow cytometer measurement, 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) (37). ℃, 5% _CO 2). On the 5th and 9th day of culture, a part of the culture solution was taken and the number of cells was counted. On the 9th day, the final volume was 6 mL per well in the case of 6-well plate, and in the case of T-75 flask. KBM501 medium was added to 50 mL per flask. The cells were collected on the 14th day, the number of cells was counted, and a part of the cells was used to measure the cell surface antigen with a flow cytometer.

*1: CliniMACS CD3, Miltenyi Biotech, 130-017-601 (5 μL per 1×10 ⁷ cells)
*2: PBS containing 0.5% human type AB serum (Cosmobio, 12181301, 56°C inactivated for 30 minutes), 2mM EDTA (Thermo Fisher Scientific, 15575-020) ((Wako Jun Pharmaceutical industry, 045-29795)
*3: KBM 501 (Kojin Bio, 16025015) supplemented with 5% human AB type serum (Cosmo Bio, 12181301, inactivated at 56°C for 30 minutes)

The results are shown in Fig. 1. The cell growth was good in any 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 any of 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 the cell growth 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 persons) were cultured using a 6-well plate. In the culture using the 500 cm ² culture bag, the bag was turned inside out as described in Example 4 below.

Further, comparing the cell growth between the mixed culture of 7 persons 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). On the other hand, in the mixed culture, the cells grew from 7.8×10 ⁶ cells to 3.7×10 ⁷ cells (about 4.7 times), and the mixed culture showed better cell growth (C in FIG. 1). )).

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

The results are shown in Figure 2. The proliferation rate in the case of culturing cells derived from a single donor (total cell number after proliferation/total cell number before proliferation) is 3.10±0.56, whereas the proliferation rate in mixed culture is 6 0.56±1.24, and the growth rate was statistically significantly higher in the mixed culture (P<0.01).

[Example 3: Tumor cytotoxicity test]
<<Preparation of NK cells>>
Four mixed culture NK cells obtained by the method described in Example 1 from healthy volunteers and cultured NK cells from a single donor (one of four) were collected and washed, and then washed with 10% FBS (Nichirei). Bioscience, 171012-500 ML), 100 units of penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, 26253-84) in RPMI1640 medium (Wako Pure Chemical Industries, 189-02025) (hereinafter referred to as 10% FBS/RPMI1640). ), and was adjusted to a concentration of 1×10 ⁶ cells/mL with the same medium.

<<Preparation of SKOV3>>
SKOV3 cells (human ovarian cancer cell line) were prepared at a concentration of 1×10 ⁶ cells/mL in RPMI1640 medium (Wako Pure Chemical Industries, 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 at 2×10 ⁶ cells/mL and 2×10 ⁵ cells/mL. did.

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

<Cytotoxicity test>
A group of mixed cultured NK cells and SKOV3 cells derived from a plurality of donors, a group of cultured NK cells derived from a single donor and SKOV3 cells, a group in which MDSC was added to each of these two groups, a total of 4 groups, and only SKOV3 cells as a negative control A group was prepared.

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

*4: Cytotoxic activity rate=(1-live SKOV3 live cells/negative control SKOV3 live cells)×100
Number of live cells: actual number of SKOV3 cells x number of beads in added bead solution/actual number of beads Actual number of SKOV3 cells: FSC/SSC gated (debris exclusion), PKH26 ⁺ gated after double exclusion.

The results are shown in Figure 3. Mixed culture NK cells derived from multiple donors showed equivalent or higher cytotoxic activity as compared to NK cells derived from a single donor.

[Example 4: Mixed culture of NK cells prepared from frozen apheresis blood]
Frozen apheresis blood of two donors (HemaCare, PB001CLP) was 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 solution was used for CliniMACS Prodity TS 310 (Miltenyi Biotec, 130-097-183), CliniMACS CD3 MicroBeads (Miltenyi Biotec, 130-017-601), CliniMACS CD30Bi-oIcient Reactant (MINITENS). Were used to remove CD3-positive cells and CD34-positive cells, followed by washing and elution. 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, the cells were suspended in KBM501 medium at 1 x 10 ⁶ cells/mL. The culture 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 ). Regarding the culture of the bag, the bag was turned over 30 minutes after the start of the culture, and was turned over again on the 9th day of culture (see FIG. 4-1). On the 9th day of culture, a part of the culture solution was taken and the number of cells was counted, and KBM 501 medium was added so that the final solution volume was 50 mL per flask for the T-75 flask and 500 mL per bag for the culture bag. did. The cells were collected on the 14th day, the total number of cells was counted, and a part of the cells was used to measure the cell surface antigen with a flow cytometer.

Harvested cells were stained with antibody as follows:
Alexa Fluor (registered trademark) 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) at 1 μg/ After staining at a concentration of mL for 30 minutes at 4°C, centrifugation (500 xg, 5 minutes, 4°C) was performed, the supernatant was removed, and the cells were suspended in PBS(-) (Wako Pure Chemical Industries, Ltd.) and then flow cytometer ( The measurement was carried out using BD LSR Fortessa (BD Bioscience) and analyzed by FlowJo software (FLOWJO, LLC).

The results are shown in Figure 4-2. Even when NK cells obtained from frozen apheresis blood were used, the cells were sufficiently grown in 14 days by the mixed culture. Specifically, in the case of T-75 flask culture, it grew from 1.50×10 ⁸ cells to 5.72×10 ⁸ cells (about 3.81 times), and in the case of 500 cm ² culture bag culture, 1.50×10 ⁸ cells to 5. It proliferated in 60×10 ⁸ cells (about 3.73 times) (FIG. 4-2, left).

The purity of NK cells in the obtained population of cultured NK cells was 90.5% for T-75 flask culture and 91.7% for 500 cm ² culture bag culture. Regarding the expression of CD16, the population of mixed culture NK cells was bimodal, as was the population of culture NK cells derived from a single donor (see JP-A-2018-193303). Specifically, the proportions of low CD16 expression and high CD16 expression were 50.9% and 49.1% in the case of T-75 flask culture, and 46 in the case of 500 cm ² culture bag culture. It was 0.7% and 53.3% (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 the isolated peripheral blood mononuclear cells, primary NK cells were used using the EasySep ^™ Human NK Cell Enrichment Kit (STEMCELL, 19055), and EasySep ^™ Human Monocyte Enrichment Kit with ELEMCT 58 (CD16DST) was used. The cells were separated and the number of cells was counted. The primary NK cells were suspended in KBM 501 medium at 1×10 ⁵ cells/mL and monocytes at 3×10 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 in which spheres were combined were mixed so that the cell ratio of primary NK cells and monocytes was 1:3, and the culture was started in a 6-well plate (Thermo Fisher Scientific, 140675). ℃, 5% _CO 2). Similar to the method described in Example 1, KBM501 medium was added on the 9th day of culture, and the cells were collected on the 14th day. The obtained cells were used to measure the cell surface antigen and the cytotoxic activity rate against K562 (human chronic myelogenous leukemia cell line).

The measurement of cell surface antigen was performed by staining the collected cells with an antibody and analyzing them as follows:
Alexa Fluor (registered trademark) 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, and then centrifuged (500×g, 5 minutes, 4° C.) to remove the supernatant, and PBS ( -) (Wako Pure Chemical Industries, Ltd.), followed by measurement using a flow cytometer (BD LSR Fortessa, BD Bioscience) and analysis with FlowJo software (FLOWJO, LLC).

To measure the cytotoxic activity, 4 groups were prepared by reacting cultured NK cells and K562 cells, each of which was formed by combining primary NK cells of two donors and monocytes, K562 cells alone as a negative control, and K562 as a positive control. A group was prepared in which cells were fixed with 10% formalin.

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

<<Preparation of cultured NK cells>>
After collecting and washing the 4 groups of cultured NK cells obtained by culturing by combining the primary NK cells of the two donors obtained by the above method and monocytes, they were suspended in 10% FBS/RPMI1640 and 1×10 6 in the same medium. The concentration was adjusted to ⁶ cells/mL.

<Cytotoxicity test>
NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) at 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, 7-AAD solution diluted with PBS (Beckman Coulter, A07704) was added and suspended, and the mixture was incubated at room temperature for 20 minutes. The measurement was performed using a flow cytometer (BD LSR Fortessa, BD Bioscience) and analyzed by FlowJo software (FLOWJO, LLC) to calculate the cytotoxic activity rate (% Lysis) ^*5 .

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

Donor typing information is shown in the table below and the results are shown in Figure 5.

When the signal from KIR3DL1 or KIR3DS1 enters one or both of them, the population of CD16 ^high increases, and thus higher ADCC activity can be expected.

Claims

Preparing a population of mononuclear cells from multiple donors, including NK cells,
A method for producing a cell population containing NK cells, which comprises incubating the prepared population of mononuclear cells under conditions effective for treating NK cells.
The method according to claim 1, wherein the step of preparing a population of mononuclear cells includes the step of removing CD3-positive cells.
The method according to claim 1 or 2, wherein the step of preparing a population of mononuclear cells includes the step of removing CD34-positive cells.
The 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 peripheral blood collected from a plurality of donors.
The production method according to any one of claims 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.
The step of preparing a population of mononuclear cells is derived from any one selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells derived from multiple donors. The method according to any one of claims 1 to 3, wherein a population of mononuclear cells to be prepared is prepared.
The production method according to any one of claims 1 to 6, wherein the plurality of donors include one donor and another donor having a genotype different from that of at least one of HLA and KIR.
A cell population including NK cells, which 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) at a mixing ratio (E:T) of 1:1.
A pharmaceutical composition for cell therapy, comprising a cell population produced by the production method according to any one of claims 1 to 7.
A pharmaceutical composition for cell therapy, comprising the cell population according to claim 8.
The pharmaceutical composition according to claim 9 or 10 for treating infectious disease and/or cancer.