Cell population production method

Abstract

The purpose of the present invention is to efficiently proliferate a cell population containing cells that are CD3-negative and CD56-positive without using a feeder cell line and without using a genetic modification technique. The present invention provides a method for producing a cell population containing cells that are CD3-negative and CD56-positive, the method including a step a). Step a) is a step for culturing, for one day or more, a cell population containing mononuclear cells, from which CD3-positive cells and CD34-positive cells have been removed, in a medium containing IL-2, IL-21, and 4-1BB agonist, wherein the IL-21 and 4-1BB agonist are liquid factors.

Description

Method for producing cell populations

[0001] The present invention relates to a method for producing or expanding and activating a cell population containing CD3-negative and CD56-positive cells.

[0002] Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that have a surface marker phenotype such as CD3 negative and CD56 positive, and act as a key factor in innate immunity.

[0003] CAR-T therapy using chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells) has been approved for the treatment of relapsed or refractory CD19-positive B-cell acute lymphoblastic leukemia, etc., but the application of highly active NK cells to therapeutic preparations for malignant tumors is considered a new generation therapeutic technique that can replace CAR-T therapy (Non-Patent Document 1). Methods for producing populations of cells that exhibit high cytotoxic activity against solid tumors have been known (Patent Document 1, Non-Patent Document 2).

[0004] Candidate NK cell preparations derived from embryonic stem cells (ES cells) / induced pluripotent stem cells (iPS cells) have also been reported. For example, Non-Patent Document 3 describes NK cells derived from human iPS cells that express a CAR containing a transmembrane domain of NK2GD, a 2B4 (CD244) costimulatory domain, and a CD3ζ signaling domain.

[0005] On the other hand, methods using 4-1BB ligand (4-1BBL), OX40 ligand, interleukin (IL)-18, IL-21, etc. are known as means for amplifying lymphocytes from peripheral blood mononuclear cells (Non-Patent Document 4). Patent Document 2 describes a method for expanding tumor-infiltrating lymphocytes using a tumor necrosis factor receptor superfamily agonist such as a 4-1BB agonist. Patent Document 3 describes a liquid composition for proliferating T cells, which contains IL-2, IL-15, and IL-21 at predetermined concentrations.

[0006] On the other hand, Non-Patent Document 4 describes that under experimental conditions, when 4-1BBL or IL-21 is used as a humoral factor, the NK cell expansion effect is inferior compared to when these are immobilized on beads, suggesting that using factors such as 4-1BBL and IL-21 as humoral factors is inefficient. Therefore, means for achieving high NK cell proliferation efficiency by using recombinant feeder cell lines or synthetic lipid membranes have been developed. For example, Non-Patent Document 5 describes a method for co-culturing NK cells with recombinant artificial antigen-presenting cells expressing membrane-bound IL-21. Patent Document 4 describes a method for producing NK cells, in which peripheral blood mononuclear cells from which CD3-positive cells have been removed are grown together with feeder cells, and then restimulated with feeder cells when the peripheral blood mononuclear cells reach a specific cumulative division level.

[0007] JP 2019-170176, JP 2020-514289, JP 2021-113200, JP 2021-518122

[0008] Liu S.et al.NK cell−based cancer immunotherapy:from basic biology to clinical development.J Hematol Oncol.(2021)14:7 doi.org / 10.1186 / s13045−020−01014−wSaito S.et al.Ex vivo generation of highly purified and activated natural killer cells from human peripheral blood.Hum Gene Ther Methods.2013 Aug;24(4):241−52.doi:10.1089 / hgtb.2012.183.Li Y.et al.Human iPSC−Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti−tumor Activity.Cell Stem Cell.2018 Aug 2;23(2):181−192.e5.doi:10.1016 / j.stem.2018.06.002.Epub 2018 Jun 28.Li X.et al.Expansion of NK cells from PBMCs using immobilized 4−1BBL and interleukin−21.Int J Oncol.2015 Jul;47(1):335−42.doi:10.3892 / ijo.2015.3005.Epub 2015 May 14.Denman CJ.et al.Membrane−Bound IL−21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells.PLoS One.2012;7(1):e30264. doi:10.1371 / journal. bone. 0030264. Epub 2012 Jan 18. Gong Y. et al. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021 May 1;14(1):73. doi:10.1186 / s13045-021-01083-5. David L. Waning et al. Molecular mechanisms of bone metastasis and associated muscle weakness. Clin Cancer Res. 2014 Jun 15;20(12):3071-7. doi:10.1158 / 1078-0432. CCR-13-1590. Epub 2014 Mar 27. ;

[0009] While cells obtained by conventional methods exhibit high cytotoxic activity against solid tumors, their supply is limited.

[0010] Preparations using NK cells derived from ES cells / iPS cells, such as those described in Non-Patent Document 3, do not have sufficient activity, particularly against solid tumors. Furthermore, the inventions described in Patent Document 2 or Patent Document 3 relate to the proliferation of T cells, and have not been shown to be useful for the proliferation of NK cells. Methods using genetic recombination techniques or feeder cell lines, such as those described in Non-Patent Document 5 and Patent Document 4, not only increase the hurdles for quality control, but also complicate the production / purification processes (Non-Patent Document 6), and may also pose an issue of increased medical costs.

[0011] Therefore, an object of the present invention is to efficiently grow a cell population containing CD3-negative and CD56-positive cells without using a feeder cell line or genetic recombination techniques.

[0012] The present inventors have conducted extensive research to achieve the above object and have found that a cell population containing CD3-negative and CD56-positive cells can be mass-produced and amplified by a method comprising the steps of: a) removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells; and culturing the resulting cell population in a medium containing IL-2, IL-21, and a 4-1BB agonist, wherein the IL-21 and the 4-1BB agonist are humoral factors. This finding led to the completion of the present invention.

[0013] That is, the present invention relates to the following: [1] A method for producing a cell population containing CD3-negative and CD56-positive cells, comprising the following steps: a) culturing a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells in a medium containing IL-2, IL-21, and a 4-1BB agonist for one day or more, wherein the IL-21 and 4-1BB agonist are humoral factors; [2] The production method according to [1], which further comprises the following steps: b) passage the cell population cultured in step a) in a medium containing IL-2 and a 4-1BB agonist but not IL-21, and culturing the cell population for one day or more; [3] The production method according to [1] or [2], wherein the concentration of the 4-1BB agonist in the medium in step a) is 1 μg / mL or more and the concentration of IL-21 in the medium in step a) is 1 ng / mL or more; [4] The production method according to [2], which further comprises the following steps: [c] a step of subculturing the cell population cultured in step b) in a medium containing IL-2 and culturing for one day or more. [5] The method according to any one of [1] to [4], wherein the 4-1BB agonist is selected from an anti-4-1BB antibody and a 4-1BB ligand. [6] The method according to any one of [1] to [5], wherein the medium contains a platelet lysate.

[0014] According to the present invention, a cell population containing CD3-negative and CD56-positive cells can be amplified and obtained in large quantities, compared to conventional production methods, without using a feeder cell line or genetic recombination technology.

[0015] Figure 1 shows the results of NK cell culture tests conducted under different culture medium and additive conditions (changes in cell count). Figure 2 shows the results of culture tests comparing different conditions for the cells used as culture materials (changes in cell amplification fold). Figure 3 shows the results of flow cytometry performed for each cell surface marker. Figure 4 shows the results of culture tests comparing different concentrations of anti-4-1BB antibody and IL-21 (changes in cell count). Figure 5 shows the results of culture tests comparing different concentrations of anti-4-1BB antibody and IL-21 (changes in cell amplification fold). Figure 6 shows the results of flow cytometry performed for each cell surface marker. Figure 7 shows the results of flow cytometry performed using the Day 14 cell population for CD3 / CD56 or CXCR3, and the results of measuring the cytotoxic activity against K562 cells using the Day 17 cell population. Figure 8 shows the results of examining the antitumor effect of administration of the recovered cell population using tumor-bearing mice generated using SK-OV-3 / CMV-Luc (a luciferase-expressing human ovarian cancer cell line) (changes in SK-OV-3 / CMV-Luc cell count). Figure 9 shows the results of examining the antitumor effect of administration of the recovered cell population using tumor-bearing mice generated using SK-OV-3 / CMV-Luc (IVIS (in vivo imaging system) results on Day 69). Figure 10 shows the results of flow cytometry for CD3 / CD56 or CXCR4. Figure 11 shows the results of measuring the cytotoxic activity of the recovered cell population against K562 cells and Raji cells. Figure 12 shows the results of a culture test (changes in cell amplification fold) when Simulect and tacrolimus were added to the medium. Figure 13 shows the results of a culture test (changes in cell amplification fold) when a recombinant 4-1BB ligand was used instead of an anti-4-1BB antibody. Figure 14 shows the results of flow cytometry performed for each cell surface marker. Figure 15 shows the results of flow cytometry performed for each cell surface marker.

[0016] [Method for producing a cell population] <Step a> One aspect of the present invention provides a method for producing a cell population containing CD3-negative and CD56-positive cells, the method comprising the following steps: a) culturing, in a predetermined medium, a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells.

[0017] <Cell Population> In the present invention, a cell population refers to a plurality of cells, for example, 1 × 10 ５ The cell population provided by the present invention can be prepared at various cell densities. For example, 1 x 10 ５ It can be prepared to a concentration of 1000 cells / mL or more.

[0018] In the present invention, a cell population containing at least CD3-negative and CD56-positive cells is obtained. Whether or not a cell is CD3-negative and CD56-positive can be determined by analyzing the expression pattern of cell surface markers. In the present invention, whether or not a cell surface marker is positive can be determined, for example, by flow cytometry, by comparing the signal from the cell surface marker with that of a control cell population that does not express or expresses the target cell surface marker at a low level. Whether or not a cell surface marker is negative can also be determined using a similar method.

[0019] The method for producing a cell population of the present invention can use a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells. The cell population containing mononuclear cells can contain various lymphocytes, such as T cells, B cells, NK cells, monocytes, and dendritic cells. The cell population containing mononuclear cells may be peripheral blood mononuclear cells obtained by separating mononuclear cells from blood cells collected from a donor. Blood cells can be collected from a donor by methods known to those skilled in the art, for example, by apheresis from the donor's peripheral blood. Blood cells may also be collected from the donor's umbilical cord blood, bone marrow, lymph nodes, etc. Mononuclear cells can be separated from blood cells using, for example, density gradient centrifugation. Blood cells obtained by differentiating embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) can also be used. Differentiation into blood cells can be performed by methods known to those skilled in the art, for example, by methods described in H. Melichar et al. Reference can be made to the techniques described in Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines. PLoS One. 2011; 6(5): e19854. doi: 10.1371 / journal.pone.0019854. CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells may be removed according to methods known to those skilled in the art, for example, by a method of separating CD3-positive cells or CD34-positive cells using magnetic beads bound to an antibody against CD3 or CD34.

[0020] The cell population containing mononuclear cells may be derived from a single donor or from multiple donors. When the cell population containing mononuclear cells is derived from multiple donors, the proportion of the cell population derived from each donor in the cell population is not particularly limited.

[0021] The cell population containing mononuclear cells may be used after being thawed after being cryopreserved. Cryopreservation can be performed, for example, by suspending the cell population containing mononuclear cells to an appropriate cell density in a medium suitable for cryopreservation, such as a complete medium containing a cryoprotective agent such as dimethyl sulfoxide (DMSO), placing the cell population in a cryopreservation container such as a cryovial, and storing the cell population at −80° C. or below. Thawing can be performed, for example, by transferring the cryopreservation container containing the cell population containing mononuclear cells to a 37° C. water bath and gently swirling the container.

[0022] <Culture Medium> The method for producing a cell population of the present invention includes step a) of culturing a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells in a predetermined culture medium. The culture medium may further contain additives, reagents, etc. suitable for culture. Hereinafter, when referring to "culture medium in the production method of a cell population of the present invention," "culture medium of the present invention," "culture medium," etc., and not specifically specifying "culture medium in step a)" or "culture medium in step b)," etc., this refers to the culture medium in one or more selected from step a), step b), and step c).

[0023] In the present specification, when the concentration of a cytokine, polypeptide, additive, or the like in a medium is described below, the concentration may be the concentration in the medium at the start of each step, such as step a), step b), step c), etc., and does not specify the concentration throughout the entire period of each step.

[0024] (IL-2, IL-21, 4-1BB agonist) The medium in the method for producing a cell population of the present invention may contain a cytokine, polypeptide, etc. suitable for expanding the cell population. Suitable cytokines include, for example, one or more interleukins (IL), preferably IL-2 and IL-21. Suitable polypeptides are preferably 4-1BB agonists. 4-1BB belongs to the tumor necrosis factor receptor superfamily (TNFRSF) and is a transmembrane receptor expressed mainly on the cell surface of lymphocytes. 4-1BB is also referred to as CD137, TNFRSF9, etc. In the present invention, for example, a 4-1BB agonist refers to a substance that can bind to 4-1BB and promote its function. Functions of 4-1BB include, for example, the production of IL-8 from human peripheral blood mononuclear cells and the production of interferon (IFN)-γ from human T lymphocytes. IL-8 and IFN-γ production by 4-1BB can be measured by methods known to those skilled in the art. The 4-1BB agonist may be, for example, one that binds to 4-1BB and promotes the production of IL-8 or IFN-γ. Examples of 4-1BB agonists include anti-4-1BB antibodies and 4-1BB ligands (4-1BBL).

[0025] As used herein, the term "anti-4-1BB antibody" refers to an antibody that can recognize and bind to one or more specific antigenic determinants in 4-1BB and multimerize 4-1BB, and examples thereof include urelumab, utomilumab, or antibodies that can recognize the same antigenic determinants as these.

[0026] As used herein, the term "4-1BB ligand (4-1BBL)" refers to a polypeptide or protein capable of binding to 4-1BB (excluding antibodies). 4-1BBL may be synthesized by methods known to those skilled in the art, or may be artificially produced by genetic recombination technology. 4-1BBL artificially produced by genetic recombination technology is referred to as recombinant 4-1BBL. In one embodiment, 4-1BBL may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 shown below, a polypeptide containing the extracellular domain of 4-1BBL (SEQ ID NO: 3), or a polypeptide consisting of an amino acid sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and having the activity of proliferating CD3-negative and CD56-positive cells. "Amino acid sequence identity" refers to the identity of the amino acid sequences between target polypeptides, and is expressed as the percentage (%) of identical amino acid residues in optimal alignment of the amino acid sequences. Amino acid sequence identity can be calculated using homology search programs known to those skilled in the art, such as BLAST and FASTA. A polypeptide having the activity of proliferating CD3-negative and CD56-positive cells refers to a polypeptide that, when used in the cell culture method described in the Examples herein, can expand the number of cells in a cell population containing CD3-negative and CD56-positive cells by at least two-fold, preferably three-fold.

[0027] (SEQ ID NO: 1)

[0028] (SEQ ID NO: 2)

[0029] (SEQ ID NO: 3)

[0030] 4-1BBL may be a monomer or a multimer. A multimer refers to a structure in which multiple subunits are associated via intermolecular interactions, non-covalent bonds, covalent bonds, or the like. In one embodiment, the 4-1BB agonist of the present invention is selected from the group consisting of urelumab, utomilumab, and a 4-1BB ligand. In a preferred embodiment of the production method of the present invention, the 4-1BB agonist is one or more selected from urelumab and polypeptides consisting of an amino acid sequence having 90% or more identity to SEQ ID NO: 1 and having the activity of proliferating CD3-negative, CD56-positive cells. It is preferable that the cytokines, polypeptides, etc. used have a human amino acid sequence.

[0031] In a preferred embodiment, the medium in step a) of the method for producing a cell population of the present invention contains IL-2, IL-21 and a 4-1BB agonist.

[0032] The concentration of IL-2 in the medium of the present invention is not particularly limited and can be 50 IU / mL or more, preferably 100 IU / mL or more, more preferably 150 IU / mL or more, even more preferably 200 IU / mL or more, particularly preferably 250 IU / mL or more, and most preferably 281 IU / mL or more. Furthermore, the concentration of IL-2 in the medium in step a) may be, for example, 3,000 IU / mL or less, 2,810 IU / mL or less, 2,500 IU / mL or less, 2,000 IU / mL or less, 1,500 IU / mL or less, 1,000 IU / mL or less, 500 IU / mL or less, or 300 IU / mL or less. The upper and lower limits of any of the above-mentioned IL-2 concentrations may be combined to represent the IL-2 concentration range.

[0033] The concentration of IL-21 in the medium in step a) of the production method of the present invention is not particularly limited and can be 0.01 ng / mL or more, preferably 0.05 ng / mL or more, more preferably 0.1 ng / mL or more, even more preferably 0.5 ng / mL or more, and particularly preferably 1 ng / mL or more. The concentration of IL-21 in the medium in step a) can be, for example, 1000 ng / mL or less, or may be 500 ng / mL or less, preferably 100 ng / mL or less, more preferably 50 ng / mL or less, even more preferably 10 ng / mL or less, or may be 9 ng / mL or less, 8 ng / mL or less, 7 ng / mL or less, 6 ng / mL or less, 5 ng / mL or less, 4 ng / mL or less, 3 ng / mL or less, or 2 ng / mL or less. The upper and lower limits of any of the above-mentioned IL-21 concentrations may be combined to represent an IL-21 concentration range.

[0034] The concentration of the 4-1BB agonist in the medium in step a) of the production method of the present invention is not particularly limited, and can be 0.01 μg / mL or more, preferably 0.05 μg / mL or more, more preferably 0.1 μg / mL or more, even more preferably 0.5 μg / mL or more, and particularly preferably 1 μg / mL or more. In addition, the concentration of the 4-1BB agonist in the medium in step a) is not particularly limited, and can be, for example, 1000 μg / mL or less, preferably 500 μg / mL or less, more preferably 100 μg / mL or less, and even more preferably 50 μg / mL or less. The concentration range of the 4-1BB agonist may be expressed by combining any of the upper and lower limits of the above-mentioned 4-1BB agonist concentrations.

[0035] When an anti-4-1BB antibody is used as the 4-1BBL agonist, the concentration of the anti-4-1BB antibody in the medium in step a) of the production method of the present invention can be 0.01 μg / mL or more, preferably 0.05 μg / mL or more, more preferably 0.1 μg / mL or more, even more preferably 0.5 μg / mL or more, and particularly preferably 1 μg / mL or more. Also, the concentration of 4-1BBL in the medium in step a) is preferably 100 μg / mL or less, more preferably 50 μg / mL or less, even more preferably 10 μg / mL or less, and may be 9 μg / mL or less, 8 μg / mL or less, 7 μg / mL or less, 6 μg / mL or less, 5 μg / mL or less, 4 μg / mL or less, 3 μg / mL or less, or 2 μg / mL or less.

[0036] When 4-1BBL is used as the 4-1BBL agonist, the concentration of anti-4-1BBL in the medium in step a) of the production method of the present invention can be 0.1 μg / mL or more, preferably 0.5 μg / mL or more, more preferably 1 μg / mL or more, more preferably 2 μg / mL or more, 3 μg / mL or more, 4 μg / mL or more, or 5 μg / mL or more, and particularly preferably 10 μg / mL or more. Also, the concentration of 4-1BBL in the medium in step a) can be, for example, 1000 μg / mL or less, preferably 500 μg / mL or less, more preferably 100 μg / mL or less, and even more preferably 50 μg / mL or less.

[0037] In the medium of the present invention, IL-21 and 4-1BB agonists are humoral factors. The term "humid" refers to a substance such as a target protein or cytokine that is soluble in the medium without being immobilized, and humoral factors refer to proteins and the like that are humoral. Opposite concepts include, for example, a state in which a target protein or the like is immobilized on beads, as described in Non-Patent Document 4, and a state in which feeder cells are transformed and a target protein or the like is expressed on the cell membrane of the feeder cells, as described in Non-Patent Document 5. The medium containing humoral factors is preferably liquid.

[0038] (Culture Medium) The culture medium of the present invention is not particularly limited, and examples thereof include KBM501 medium (KOHJIN BIO), KBM502 medium (KOHJIN BIO), NTI medium (FUKOKU), Cosmedium 008 (Cosmo Bio), FKCM101 (FUKOKU), CellGro SCGM medium (CellGenix, Iwai Chemicals Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Inc.), Gibco (registered trademark) CTS (registered trademark) AIM V (registered trademark) Medium (Thermo Fisher Scientific, a chemically defined serum-free medium for growing and manipulating T cells and dendritic cells), and CTS OpTmizer T Cell Expansion Basal Medium (Thermo Fisher Scientific, for the growth and proliferation of human T lymphocytes), IMDM, MEM, DMEM, and RPMI-1640. In the present invention, unless otherwise specified, the term "culturing" cells refers to maintaining cells in a medium or a liquid equivalent thereto for a certain period of time for any purpose selected from the group consisting of maintaining cell survival, cell expansion, and cell activation. The term "incubating" may be used when a treatment is carried out at a specific temperature for a certain period of time.

[0039] The medium may contain IL-2. Examples of IL-2-containing media include KBM501 medium (containing 2,810 IU / mL (1,750 JRU / mL) of IL-2), KBM502 medium (containing 281 IU / mL (175 JRU / mL) of IL-2), Cosmedium 008 medium (containing 1,750 JRU / mL of IL-2), FKCM101-L300 medium (FUKOKU, containing 300 IU / mL of IL-2), and FKCM101-L13T medium (FUKOKU, containing 1,300 IU / mL of IL-2). In a preferred embodiment of the production method of the present invention, the medium may contain KBM502 medium or KBM501 medium.

[0040] The medium may be replaced or replenished at any time after the start of culture, provided that the desired culture effect is obtained, but is preferably replaced or replenished every 2 to 5 days.

[0041] Culture vessels used for culturing include, but are not limited to, commercially available dishes, flasks, plates, and multi-well plates. In addition to culture vessels, culturing may be performed using various culture materials such as supports. Culture conditions are not particularly limited as long as they do not impair the culture effect, and include, but are not limited to, 37°C, 5% CO ２ Culture conditions are generally under saturated water vapor atmosphere.

[0042] (Additives, etc.) The medium of the present invention may contain human serum albumin. When human serum albumin is added to the medium, for example, a 5-25% human serum albumin preparation available from the Japanese Red Cross Society or the like can be used. The concentration of human serum albumin in the medium is preferably 2,000 mg / L or less. The medium of the present invention may also contain an antibiotic. For example, kanamycin sulfate can be used as the antibiotic. When kanamycin sulfate is added to the medium, for example, kanamycin sulfate injection available from Meiji Seika Pharma can be used. The concentration of kanamycin sulfate in the medium is preferably 60 mg / L or less. The medium of the present invention may further contain human transferrin, recombinant human insulin, etc.

[0043] The medium of the present invention may further contain IL-18. In this case, the concentration of IL-18 in the medium is preferably 10 to 1000 ng / mL, and more preferably 100 ng / mL.

[0044] The medium may further contain appropriate proteins, cytokines, antibodies, compounds, and other components to the extent that the achievement of the object of the present invention is not impaired. In addition to the above-mentioned IL-2 and IL-21, cytokines may be IL-3, IL-7, IL-12, IL-15, IL-18, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (Flt3L). These preferably have human amino acid sequences and are preferably produced by recombinant DNA technology for safety reasons.

[0045] The medium of the present invention may contain one or more appropriate additives. Examples of suitable additives include human platelet lysate (HPL), human serum substitute, and fetal bovine serum (FBS). The additive contained in the medium of the present invention is preferably HPL or FBS, with HPL being particularly preferred. Examples of HPL that can be used include UltraGRO (AventaCel). The concentration of HPL in the medium is preferably 1 to 10%, more preferably 3 to 8%, and particularly preferably 5%. When HPL is used, it is preferable to further add heparin to the medium. Examples of heparin that can be used include heparin sodium and heparin calcium. The concentration of heparin in the medium is preferably 1 to 5 U / mL, with 2 U / mL being particularly preferred. As the FBS, for example, an FBS preparation available from Nichirei can be used. The concentration of FBS in the medium is preferably 1 to 10%, more preferably 3 to 8%, and particularly preferably 5%. As the human serum substitute, for example, CTS Immune Cell SR (Gibco) can be used. The concentration of the human serum substitute in the medium is preferably 1 to 10%, more preferably 3 to 8%, and particularly preferably 5%.

[0046] The medium of the present invention may contain one or more immunosuppressants. By including an immunosuppressant in the medium, the proliferation of CD3-positive cells can be suppressed. Examples of immunosuppressants include calcineurin inhibitors such as tacrolimus and cyclosporine, and cytokine inhibitors such as basiliximab, and are preferably one or more selected from tacrolimus and basiliximab. The concentration of tacrolimus in the medium may be, for example, 0.01 to 100 ng / mL, preferably 0.1 to 50 ng / mL, and more preferably 1 to 10 ng / mL. The concentration of basiliximab in the medium may be, for example, 0.005 to 50 μg / mL, preferably 0.05 to 30 μg / mL, and more preferably 0.5 to 10 μg / mL. Tacrolimus may be, for example, Prograf Injection, available from Astellas Pharma. Basiliximab may be, for example, Simulect Injection, available from Novartis Pharma.

[0047] In a preferred embodiment, the culture method of the present invention does not use feeder cells. As used herein, feeder cells refer to cells other than the target cells (cells obtained by the present invention) that play a supporting role, for example, by producing cytokines that serve as growth factors, in order to establish culture conditions for the target cells. Feeder cells refer to cells commonly used in the technical field to which the present invention pertains, and may include, for example, transfected animal cell lines, leukocytes treated with various cytokines, T cells, B cells, monocytes, antigen-presenting cells, macrophages, etc. Specific examples of feeder cells include genetically modified antigen-presenting cells that express membrane-bound IL-21, as described in Non-Patent Document 5. Feeder cells may be cells that have almost completely lost their proliferation ability due to gamma irradiation, antibiotic treatment, etc., but are not limited to cells with reduced or lost proliferation ability.

[0048] (Culturing Period) Step a) may involve culturing a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells for a predetermined period. The predetermined period may be 1 day or more, or may be 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, or even 30 days or less, 25 days or less, 20 days or less, 15 days or less, or less. The culturing period in step a) may be, for example, 1 to 20 days, preferably 3 to 18 days, 4 to 16 days, 5 to 15 days, or 6 to 14 days. During this period, the cells may be added to the same medium, or the medium may be replaced (in this case, step a) can be considered to be continued as long as the medium meets the requirements). ), by adjusting the cell density at the time of seeding, it is possible to avoid maintenance (addition, replacement, etc. of the medium) during the culture period.

[0049] <Step b> The method for producing a cell population according to the present invention may further comprise a step b) of subculturing and culturing the cell population cultured in step a) in a predetermined medium.

[0050] As used herein, "passaging" refers to transferring a cell population to a new medium, and the method for doing so is not particularly limited. Transferring a cell population to a new medium may involve, for example, collecting a portion of the medium in which the cells are dispersed and adding it to the new medium. When passaging, a portion of the medium used for the culture before the passaging may be added to the new medium, but as used herein, the composition of the medium used for the culture before the passaging can be ignored in the composition of the new medium after the passaging.

[0051] The medium in step b) of the method for producing a cell population of the present invention contains IL-2. The medium in step b) may further contain a 4-1BB agonist, or may further contain IL-21. For specific descriptions of IL-2, a 4-1BB agonist, and IL-21, please refer to the above contents. In a preferred embodiment, the medium in step b) of the method for producing a cell population of the present invention contains IL-2 and a 4-1BB agonist, but does not contain IL-21.

[0052] When the medium in step b) contains IL-21, the concentration of IL-21 in the medium may be, for example, 0.01 ng / mL or more, 0.1 ng / mL or more, or 1 ng / mL or more; on the other hand, it is preferably 100 ng / mL or less, more preferably 50 ng / mL or less, even more preferably 10 ng / mL or less, and may be 9 μg / mL or less, 8 μg / mL or less, 7 μg / mL or less, 6 μg / mL or less, 5 μg / mL or less, 4 μg / mL or less, 3 μg / mL or less, or 2 μg / mL or less.

[0053] When the medium in step b) contains a 4-1BB agonist, the concentration of the 4-1BB agonist in the medium can be, for example, 0.01 μg / mL or more, 0.1 μg / mL or more, 1 μg / mL or more, 5 μg / mL or more, 10 μg / mL or more, or 100 μg / mL or more. The concentration of the 4-1BB agonist in the medium in step b) is preferably equal to or higher than the concentration of the 4-1BB agonist in the medium in step a).

[0054] When an anti-4-1BB antibody is used as the 4-1BBL agonist, the concentration of the anti-4-1BB antibody in the medium in step b) can be 0.01 μg / mL or more, preferably 0.05 μg / mL or more, more preferably 0.1 μg / mL or more, even more preferably 0.5 μg / mL or more, and particularly preferably 1 μg / mL or more. When an anti-4-1BB antibody is used as the 4-1BBL agonist, the concentration of the anti-4-1BB antibody in the medium in step b) is preferably equal to or higher than the concentration of the anti-4-1BB antibody in the medium in step a).

[0055] When 4-1BBL is used as the 4-1BBL agonist, the concentration of 4-1BBL in the medium in step b) can be 0.1 μg / mL or more, preferably 0.5 μg / mL or more, more preferably 1 μg / mL or more, even more preferably 5 μg / mL or more, and particularly preferably 10 μg / mL or more. When 4-1BBL is used as the 4-1BBL agonist, the concentration of 4-1BBL in the medium in step b) is preferably equal to or higher than the concentration of 4-1BBL in the medium in step a).

[0056] Step b) may involve culturing the cell population cultured in step a) for a predetermined period. The predetermined period may be 1 day or more, or 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or even 30 days or less, 25 days or less, 20 days or less, or less. The culture period in step b) may be, for example, 1 to 1 day, preferably 2 to 21 days, more preferably 3 to 19 days, even more preferably 4 to 17 days, and particularly preferably 8 to 15 days. During this period, the cells may be added to the same medium, or the medium may be replaced (in this case, step b) can be considered to be continued as long as the medium meets the requirements).

[0057] <Step c> The method for producing a cell population according to the present invention may include a step c) of subculturing and culturing the cell population cultured in step b) in a predetermined medium.

[0058] The medium in step c) of the method for producing a cell population of the present invention contains IL-2. The medium in step c) may further contain a 4-1BB agonist or may further contain IL-21. For specific descriptions of IL-2, a 4-1BB agonist, and IL-21, please refer to the above. In a preferred embodiment, the medium in step c) of the present invention contains IL-2 but does not contain IL-21 or a 4-1BB agonist. In a more preferred embodiment, the medium in step c) of the present invention contains IL-2, IL-21, and a 4-1BB agonist.

[0059] When the medium in step c) contains IL-21, the concentration of IL-21 in the medium can be, for example, 0.01 ng / mL or more, 0.1 ng / mL or more, or 1 ng / mL or more, or 100 ng / mL or less, 10 ng / mL or less, or 5 ng / mL or less.

[0060] When the medium in step c) contains a 4-1BB agonist, the concentration of the 4-1BB agonist in the medium can be, for example, 0.01 μg / mL or more, 0.1 μg / mL or more, or 1 μg / mL or more, or 100 μg / mL or less, 10 μg / mL or less, or 5 μg / mL or less.

[0061] When an anti-4-1BB antibody is used as the 4-1BBL agonist, the concentration of the anti-4-1BB antibody in the culture medium in step c) can be 0.01 μg / mL or more, preferably 0.05 μg / mL or more, more preferably 0.1 μg / mL or more, even more preferably 0.5 μg / mL or more, particularly preferably 1 μg / mL or more, or may be 100 μg / mL or less, 10 μg / mL or less, 5 μg / mL or less, 4 μg / mL or less, 3 μg / mL or less, or 2 μg / mL or less.

[0062] When 4-1BBL is used as the 4-1BBL agonist, the concentration of 4-1BBL in the culture medium in step c) can be 0.1 μg / mL or more, preferably 0.5 μg / mL or more, more preferably 1 μg / mL or more, even more preferably 5 μg / mL or more, and particularly preferably 10 μg / mL or more.

[0063] Step c) may involve culturing the cell population cultured in step b) for a predetermined period. The predetermined period is not particularly limited and may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, or 30 days or less, 25 days or less, 20 days or less, 15 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, or less. The culture period in step c) is preferably 4 to 10 days, more preferably 5 to 9 days, even more preferably 6 to 8 days, and particularly preferably 7 days. During this period, the cells may be added to the same medium, or the medium may be replaced (in this case, step c) can be considered to be continued as long as the medium meets the requirements).

[0064] The method for producing a cell population according to the present invention may include a step of further culturing the cell population cultured in step c) under the same conditions as in step c), i.e., repeating step c). Step c) may be repeated multiple times, and the number of times is not limited, as long as cell proliferation is achieved.

[0065] According to the production method of the present invention, the number of cells contained in the cell population obtained by removing CD3-positive cells and CD34-positive cells from the cell population containing mononuclear cells used in step a) is amplified at least 2-fold, preferably 10-fold, and more preferably 100-fold.

[0066] [Cell Population Obtained by the Production Method of the Present Invention] The cell population obtainable by the production method of the present invention is at least CD3-negative and CD56-positive, and preferably contains cells having a phenotype of cell surface markers selected from the group consisting of CD3-negative, CD34-negative, and CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1 (Integrin α1)-positive, and ITGA3 (Integrin α3)-positive. More preferably, the cell population obtainable by the production method of the present invention contains cells that are CD3-negative, CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, and ITGA3-positive. CCR or CXCR used as a cell surface marker is a type of chemokine receptor, and ITG (Integrin) is a type of cell adhesion factor.

[0067] In the present invention, when a cell population containing CD3-negative and CD56-positive cells is referred to, the proportion of CD3-negative and CD56-positive cells in the cell population may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. A cell population in which the proportion of CD3-negative and CD56-positive cells in the cell population is lower than any of the above proportions is distinguished from a cell population containing CD3-negative and CD56-positive cells obtained by the present invention.

[0068] Furthermore, in the present invention, when a cell population containing cells that are CD3-negative, CD56-positive, and CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, or ITGA3-positive is referred to, the proportion of cells having each phenotype of cell surface marker in the cell population may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. A cell population in which the proportion of CD3-negative, CD56-positive, and CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, or ITGA3-positive cells is lower than any of the above proportions is distinguished from a cell population obtained by the present invention that contains CD3-negative, CD56-positive, and CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, or ITGA3-positive cells. The CD3-negative, CD56-positive cells in the cell population obtained by the present invention are preferably CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, and ITGA3-positive, and more preferably CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, and ITGA3-positive.

[0069] [Cytotoxic Activity] Cells contained in the cell population obtained by the present invention can exhibit high cytotoxic activity. Cytotoxic activity can be evaluated, for example, by measuring the lytic ability of target cells (effector cells (E)) against target cells (T), such as tumor cells. Examples of tumor cells that can be used include, but are not limited to, tumor cell lines from leukemia, glioma, breast cancer, colon cancer, ovarian cancer, and prostate cancer. Spheroids prepared from tumor cell lines can also be used as target cells. The ratio of effector cells to target cells (E:T) is not particularly limited and may be, for example, 1:1, 2:1, 5:1, etc. Cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells, and is calculated using the following formula:

[0070] Cytotoxic activity rate (% Lysis) = (target cell death rate - negative control cell death rate (spontaneous cell death rate)) / (positive control cell death rate (maximum cell death rate) - negative control cell death rate) x 100

[0071] Cytotoxic activity can be measured, for example, by distinguishing between effector cells and target cells, and between live and dead cells, using a reagent such as an antibody labeled with a radioactive substance, a fluorescent dye, etc., quantifying these cells using flow cytometry or the like, and determining the cell death rate for each cell according to the above formula. When the cell population of the present invention is used as effector cells, the incubation time with target cells can be appropriately set depending on the type of target cells used; for example, when K562 cells, a leukemia cell line, are used as target cells, the incubation time can be set to 2 hours.

[0072] In the present invention, unless otherwise specified, "high cytotoxic activity" refers to a cytotoxic activity of 50% or more when the target cells are K562 cells, mixed at an E:T ratio of 1 to 5:1, and co-cultured for 2 hours. The cytotoxic activity is preferably 60% or more, and more preferably 70% or more.

[0073] Furthermore, when measuring cytotoxic activity using the cell population obtained by the present invention as effector cells, the desired cytotoxic activity can also be determined by detecting CD107a-positive cells among the effector cells. Because CD107a is present in granules within NK cells and translocates to the cell membrane surface of NK cells upon degranulation, CD107a positivity indirectly indicates cytotoxic activity, i.e., attack of target cells by cells contained in the cell population obtained by the present invention. CD107a-positive cells can be detected, for example, by measuring luminescence after incubating target cells with a fluorescently labeled anti-CD107a antibody. When the cell population obtained by the present invention is used as effector cells and any tumor cell line as target cells at a cell number ratio (E:T) of 1 to 2:1, the proportion of CD107a-positive cells in the cell population obtained by the present invention is at least 20% or more, preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. The tumor cell line may be, for example, K562 cells, a leukemia cell line.

[0074] [Anti-tumor Effect] The cell population obtained by the present invention exerts anti-tumor effect even when administered in vivo. The in vivo anti-tumor effect can be evaluated, for example, by administering the cell population obtained by the present invention to a cancer-bearing animal model and measuring changes in tumor volume or tumor cell count. Preparation of cancer-bearing animal models and measurement of changes in tumor volume or tumor cell count can be performed using methods known to those skilled in the art, including, for example, a method in which fluorescently labeled tumor cells are transplanted into the animals and observed. Specific examples include a method using an in vivo imaging system (IVIS) in which luciferase-expressing SKOV cells (a human ovarian cancer cell line) are transplanted into immunodeficient mice, luciferin is administered to the mice, and tumor luminescence is observed under anesthesia. In this method, the method of administering the cell population obtained by the present invention is not particularly limited. The number of administrations may be single or multiple, and the administration intervals for multiple administrations can be appropriately set. The timing of administration can be any time point, for example, between 1 and 30 days after tumor transplantation. The dosage is, for example, 1.0 x 10 ４ ~1.0 x 10 １０ cells / body, preferably 1.0×10 ５ ~1.0 x 10 ９ cells / body, more preferably 1.0 × 10 ６ ~1.0 x 10 ８ cells / body, and particularly preferably 1.5×10 ７ ~2.7×10 ７The administration route may be any, including intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, with intraperitoneal administration being preferred. IL-2 may be administered simultaneously to maintain the activity of the cell population obtained by the present invention in vivo. In this case, the dose of IL-2 may be 1,000 to 10,000 IU / body, preferably 5,000 IU / body. In one aspect, administration of the cell population obtained by the present invention can reduce the number of tumor cells in a cancer-bearing animal model to 1 / 10 or less compared to when the cell population is not administered.

[0075] The cell population obtained by the present invention can be cryopreserved. Cryopreservation can be performed by suspending the cell population to an appropriate cell density using a medium suitable for cryopreservation, such as a complete medium containing a cryoprotective agent such as dimethyl sulfoxide (DMSO), placing the suspended cell population in a cryopreservation container such as a cryovial, and storing the suspended cell population at −80° C. or below. An appropriate cell density is, for example, 1×10 ３ ~1×10 １０ cells / mL, preferably 2 x 10 ８ The cryoprotective reagent may contain inorganic salts, pH adjusters, and the like.

[0076] Furthermore, the cytotoxic activity of the cryopreserved cell population can be maintained even after freezing and thawing by thawing the cell population using an appropriate method, such as transferring the cryopreservation container containing the cell population to a 37°C water bath and gently swirling it.

[0077] [Pharmaceutical Composition] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising a cell population obtained by the present invention. The pharmaceutical composition may contain a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additive include an isotonicity agent, a pH adjuster, a buffer, a stabilizer, a cryoprotectant, a solubilizing agent, a soothing agent, an antibiotic, etc.

[0078] The pharmaceutical composition is typically in the form of a suspension of the cell population obtained by the present invention in a solution. Typical solutions for suspending the cell population obtained by the present invention include, for example, a cryoprotectant solution containing DMSO, saline, phosphate-buffered saline (PBS), culture medium, serum, etc. The solution may contain a pharmaceutically acceptable carrier for use as a drug or quasi-drug.

[0079] The pharmaceutical composition of the present invention may be used to treat infectious diseases or cancer. The pharmaceutical composition of the present invention may also be applied to the treatment of various diseases susceptible to the cell population of the present invention. The pharmaceutical composition of the present invention may also be applied to the prevention of various diseases susceptible to the cell population of the present invention. Diseases include, but are not limited to, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, colon cancer, kidney cancer, bladder cancer, leukemia, and infectious diseases caused by viruses, bacteria, etc. The cell therapy of the present invention may be performed alone or in combination with surgical therapy, chemotherapy, radiation therapy, antibody drugs, etc. In cell therapy using the pharmaceutical composition of the present invention, the cell population of the present invention may be administered, for example, intravenously, arterially, subcutaneously, intraperitoneally, etc.

[0080] The pharmaceutical compositions of the present invention may be administered to patients with an HLA genotype that differs from that of the cell population of the present invention.

[0081] The pharmaceutical composition of the present invention can be administered together with an IL-2 preparation. The IL-2 preparation may be a recombinant type, such as teceleukin (genetical recombination, product name: Imunace (Shionogi Pharmaceuticals)).

[0082] The pharmaceutical composition of the present invention may be used in combination with an antibody drug. Specific examples of antibodies that can be used in combination with the pharmaceutical composition of the present invention include ibritumomabtiuxetan, iodine131, catumaxomab, blinatumomab, muromonab-CD3, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, siltuximab, and d inutuximab, obiltoxaximab, daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, oma lizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab, certolizu mabpegol, mogamulizumab, pertuzumab, trastuzumab, obinutuzumab, vedolizumab, pembrolizuma, id arucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumu mab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.

[0083] Antibodies that can be used in combination with the pharmaceutical composition of the present invention preferably have high affinity for CD 16. Furthermore, in the pharmaceutical composition of the present invention, at least a portion of the antibody may be bound to cells contained in the cell population of the present invention.

[0084] The cells contained in the pharmaceutical composition of the present invention may express a chimeric antigen receptor (CAR). The CAR may be expressed according to a method known to those skilled in the art, for example, by introducing a gene encoding the CAR of interest into cells contained in the cell population of the present invention using a vector containing the gene. The CAR may have a structure comprising, for example, a short-chain fragment (scFv) derived from any antibody and an intracellular signaling domain.

[0085] The pharmaceutical composition of the present invention is preferably produced under conditions that comply with the regulations for manufacturing control and quality control of drugs and quasi-drugs (good manufacturing practice, GMP) and the standards for manufacturing control and quality control of regenerative medicine products (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice, GCTP).

[0086] The present invention will be explained in more detail below by way of examples, but the present invention should not be construed as being limited thereto.

[0087] Cryopreserved PBMCs (Peripheral Blood Mononuclear Cells) were completely thawed in a 37°C water bath and diluted 10-fold with RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution). After centrifugation at 500 g for 5 minutes, the cells were used under the respective conditions.

[0088] A). Example 1 (Donor Mix A) NK cell culture tests were carried out under different conditions of culture medium and additives.

[0089] Day 0: NK cells (CD3- / CD56+, ≥80%) were isolated from thawed PBMCs using EasySep Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055). The isolated NK cells were cultured at 2 × 10 in the following 18 groups of media. ５ The culture medium was adjusted to 0.65 cells / mL, and 0.65 mL of the medium was seeded on a 12-well plate to initiate culture.

[0090] (1) RPMI 1640 medium, 5% CTS, IL-2 [100 IU / mL], IL-21 [100 ng / mL], urelumab [10 μg / mL] (2) RPMI 1640 medium, 5% UltraGRO, heparin sodium [2 U / mL], IL-2 [100 IU / mL], IL-21 [100 ng / mL], urelumab [10 μg / mL] (3) RPMI 1640 medium, 5% FBS, IL-2 [100 IU / mL], IL-21 [100 ng / mL], urelumab [10 μg / mL] (4) RPMI 1640 medium, 5% CTS, IL-2 [100 IU / mL] (5) RPMI 1640 medium, 5% UltraGRO, heparin sodium [2 U / mL], IL-2 [100 IU / mL] (6) RPMI 1640 medium, 5% FBS, IL-2 [100 IU / mL] (7) KBM 501 medium, 5% CTS, IL-21 [100 ng / mL], urelumab [10 μg / mL] (8) KBM 501 medium, 5% UltraGRO, heparin sodium [2 U / mL], IL-21 [100 ng / mL], urelumab [10 μg / mL] (9) KBM 501 medium, 5% FBS, IL-21 [100 ng / mL], urelumab [10 μg / mL] (10) KBM501 medium, 5% CTS (11) KBM501 medium, 5% UltraGRO, heparin sodium [2 U / mL] (12) KBM501 medium, 5% FBS (13) NTI medium, 5% CTS, IL-21 [100 ng / mL], urelumab [10 μg / mL] (14) NTI medium, 5% UltraGRO, heparin sodium [2 U / mL], IL-21 [100 ng / mL], urelumab [10 μg / mL] (15) NTI medium, 5% FBS, IL-21 [100 ng / mL], urelumab [10 μg / mL] (16) NTI medium, 5% CTS (17) NTI medium, 5% UltraGRO, heparin sodium [2 U / mL] (18) NTI medium, 5% FBS

[0091] Day 7: For all groups, the wells were thoroughly pipetted and the number of cells was counted. For (8) and (14), 0.5 mL was dispensed into a 6-well plate, and 1 mL of the medium shown below was added.

[0092] (8) KBM501 medium, 5% UltraGRO, sodium heparin [2 U / mL] (14) NTI medium, 5% UltraGRO, sodium heparin [2 U / mL]

[0093] Day 11: For all groups, the wells were thoroughly pipetted and the number of cells was counted.

[0094] The results are shown in Table 1 and FIG.

[0095]

[0096] The results of culturing NK cells using RPMI, KBM, or NTI medium showed that the expansion efficiency varied depending on the culture medium and additives. However, when NK cells isolated using the EasySep Human NK Cell Enrichment Kit were used, sufficient expansion efficiency was not obtained.

[0097] B). Example 2 (Donor Mix B, Single Donor A) A culture test was carried out by changing the conditions of the cells used as the culture material.

[0098] Day 0: Cryopreserved PBMCs were thawed using a prescribed method, and 0.65 mL of the cell suspension prepared for the following four groups was seeded into a 12-well plate to initiate culture. (1) PBMCs (CD3 − , CD34 − ) 4x10 ５ cells / mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (2) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3) PBMC (CD3 ＋ , CD34 ＋ ) 4x10 ５ cells / mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4) PBMC (CD3 ＋ , CD34 ＋ ) 4x10 ５cells / mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0099] Day 6: Each well was thoroughly pipetted and the number of cells was counted. 1 mL of each group was seeded in a 12-well plate under the following conditions and subcultured: (1)-1 (1) Cell suspension 4 x 10 ５ cells / 1mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (2)-1 (2) Cell suspension 4 × 10 ５ cells / 1mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-1 (3) Cell suspension 4 × 10 ５ cells / 1mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4)-1 (4) Cell suspension 4 × 10 ５ cells / 1mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0100] Day 7: Each well was thoroughly pipetted, and 2.0 mL of each group was seeded into a 6-well plate and subcultured under the following conditions: (1)-2: 1 mL of (1)-1 cell suspension, 1.0 mL of NTI medium supplemented with urelumab [1 μg / mL] ※１ (2)-2 (2)-1 Cell suspension 1 mL, 1.0 mL Urelumab [1 μg / mL]-supplemented RPMI medium ※２ (3)-2 (3)-1 Cell suspension 1 mL, 1.0 mL Urelumab [1 μg / mL]-supplemented NTI medium ※１ (4)-2 (4)-1 Cell suspension 1 mL, 1.0 mL Urelumab [1 μg / mL]-supplemented RPMI medium ※２

[0101] Day 10: Each well was thoroughly pipetted and the number of cells was counted. Each group was seeded in 3.0 mL of the cell suspension in a 6-well plate under the following conditions and subcultured: (1)-3 (1)-2 cell suspension 5 x 10５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (2)-3 (2)-2 Cell suspension 5 × 10 ５ cells / 3mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-3 (3)-2 Cell suspension 5 × 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4)-3 (4)-2 Cell suspension 5 × 10 ５ cells / 3mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0102] Day 12: The cells in each of the wells (1)-3, (2)-3, (3)-3, and (4)-3 were pipetted thoroughly and counted.

[0103] Day 14: Each well was thoroughly pipetted and the number of cells was counted. Groups (1), (3), and (4) were seeded in 3.0 mL of each cell suspension into a 6-well plate under the following conditions and subcultured: (1)-4 (1)-3 cell suspension 5 x 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-4 (3)-3 Cell suspension 5 × 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4)-4 (4)-3 Cell suspension 5 × 10 ５ cells / 3mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0104] Day 17: Each well was thoroughly pipetted and the number of cells was counted. Groups (1), (3), and (4) were seeded in 3.0 mL of each cell suspension into a 6-well plate under the following conditions and subcultured: (1)-5 (1)-4 cell suspension 5 x 10 ５ cells / 3mL, NTI medium ※１, Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-5 (3)-4 Cell suspension 5 × 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4)-5 (4)-4 Cell suspension 5 × 10 ５ cells / 3mL, RPMI medium ※２ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0105] Day 19: The cells in each of the wells (1)-5, (3)-5, and (4)-5 were thoroughly pipetted and counted.

[0106] Day 21: (1)-5, (3)-5, and (4)-5 were thoroughly pipetted into each well and the number of cells was counted. (3)-5 was seeded in 3.0 mL of each well into a 6-well plate under the following conditions and subcultured. (3)-5-1: (3)-5 cell suspension 5 x 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-5-2 (3)-5 Cell suspension 1.5 × 10 ６ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0107] Day 24: (3)-5-1 and (3)-5-2 were pipetted thoroughly in each well and the number of cells was counted. (3)-5-1 and (3)-5-2 were seeded in 3.0 mL of each well into a 6-well plate under the following conditions and subcultured: (3)-5-1 (3)-5-1 cell suspension 5 x 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-5-2-a (3)-5-2 cell suspension 5 × 10 ５ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3)-5-2-b (3)-5-2 cell suspension 1.5 × 10 ６ cells / 3mL, NTI medium※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0108] Day 28: (3)-5-1, (3)-5-2-a, and (3)-5-2-b were thoroughly pipetted into each well and the number of cells was counted. (3)-5-2-b was seeded in 3.0 mL into a 6-well plate under the following conditions and subcultured. (3)-5-2-b' (3)-5-2-b cell suspension 1.5 x 10 ６ cells / 3mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0109] Day 31: (3)-5-2-b' wells were thoroughly pipetted and the number of cells was counted.

[0110] The change in cell proliferation fold is shown in Figure 2. It was found that when NTI medium was used, the cell proliferation efficiency was better than when RPMI medium was used.

[0111] The results of flow cytometry performed on each cell surface marker for (1), (3), and (4) are shown in Figure 3. The obtained cell population contained many cells with a phenotype similar to the cells exhibiting high cytotoxic activity described in Patent Document 1, etc., but CD3-positive cells were also expanded.

[0112] C) Example 3 (Donor Mix B) A culture test was carried out using varying concentrations of anti-4-1BB antibody and IL-21.

[0113] Day 0: Cryopreserved PBMCs were thawed using a prescribed method, and 0.65 mL of cell suspensions prepared for the following 12 groups were seeded onto a 12-well plate to initiate culture. (1) PBMCs (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ (2) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , IL-21 [1 ng / mL] (3) PBMC (CD3 −, CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , IL-21 [10 ng / mL] (4) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , IL-21 [100 ng / mL] (5) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [1 μg / mL] (6) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (7) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [10 ng / mL] (8) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [100 ng / mL] (9) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [10 μg / mL], (10) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [10 μg / mL], IL-21 [1 ng / mL] (11) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３, Urelumab [10 μg / mL], IL-21 [10 ng / mL] (12) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM502 medium ※３ , Urelumab [10 μg / mL], IL-21 [100 ng / mL]

[0114] In addition, 13.5 mL of a cell suspension prepared to the following concentration was seeded into a T75 flask and culture was initiated. (13) PBMC (CD3 − , CD34 − ) 5x10 ５ cells / mL, KBM502 medium ※３

[0115] Day 6: The cells were counted by pipetting well in each of the wells (6), (7), and (8). 0.65 mL of the cells were seeded in a 12-well plate under the following conditions and subcultured. Furthermore, fresh KBM502 medium was added to each of the wells (6), (7), and (8). ※３ (6)-1 (6) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], (6)-2 (6) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (7)-1 (7) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], (7)-2 (7) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [10 ng / mL] (8)-1 (8) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], (8)-2 (8) Cell suspension 0.25 mL, 0.4 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [100 ng / mL]

[0116] Day 7: Each well from (1) to (12) was thoroughly pipetted and the number of cells was counted. Furthermore, 0.65 mL of each well from (10), (11), and (12) was seeded in a 12-well plate under the following conditions and subcultured: (10)-1 0.1 mL and 0.55 mL of (10) cell suspension in KBM502 medium ※３ , (10)-2 (10) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL] (10)-3 (10) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (10)-4 (10) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [10 ng / mL] (10)-5 (10) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [100 ng / mL]

[0117] (11)-1 (11) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ (11)-2 (11) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL] (11)-3 (11) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (11)-4 (11) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [10 ng / mL] (11)-5 (11) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [100 ng / mL]

[0118] (12)-1 (12) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ (12)-2 (12) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３, Urelumab [1 μg / mL] (12)-3 (12) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (12)-4 (12) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [10 ng / mL] (12)-5 (12) Cell suspension 0.1 mL, 0.55 mL KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [100 ng / mL]

[0119] Day 8: The cells were counted by pipetting thoroughly in each well of (6)-1, (6)-2, (7)-1, (7)-2, (8)-1, and (8)-2, which had been subcultured on Day 6. 1.8 mL of the cells were seeded in a 6-well plate and subcultured under the following conditions: (6)-1-1 0.6 mL and 1.2 mL of (6)-1 cell suspension in KBM502 medium ※３ (6)-2-1 (6)-2 cell suspension 0.6 mL, 1.2 mL KBM502 medium ※３ (7)-1-1 (7)-1 cell suspension 0.6 mL, 1.2 mL KBM502 medium ※３ (7)-2-1 (7)-2 cell suspension 0.6 mL, 1.2 mL KBM502 medium ※３ (8)-1-1 (8)-1 cell suspension 0.6 mL, 1.2 mL KBM502 medium ※３ (8)-2-1 (8)-2 cell suspension 0.6 mL, 1.2 mL KBM502 medium ※３

[0120] Day 10: The cells were counted by pipetting thoroughly in each well of (6)-1-1, (6)-2-1, (7)-1-1, (7)-2-1, (8)-1-1, and (8)-2-1, which had been subcultured on Day 8. 10 mL of the medium was seeded in a 10 cm dish and subcultured under the following conditions: (6)-1-2 (6)-1-1 cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３ (6)-2-2 (6)-2-1 Cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３(7)-1-2 (7)-1-1 Cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３ (7)-2-2 (7)-2-1 Cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３ (8)-1-2 (8)-1-1 Cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３ (8)-2-2 (8)-2-1 Cell suspension 1.5 mL, 8.5 mL KBM502 medium ※３

[0121] The number of cells in each of the wells (1), (2), (3), (4), (5), and (9) seeded on Day 0 was counted by pipetting thoroughly. ※３ 0.7 mL of was added.

[0122] The cells were counted by pipetting thoroughly in each well of (10)-1, (10)-2, (10)-3, (10)-4, (10)-5, (11)-1, (11)-2, (11)-3, (11)-4, (11)-5, (12)-1, (12)-2, (12)-3, (12)-4, and (12)-5, which were seeded on Day 7. 2.0 mL of the cell suspension was seeded in a 6-well plate and subcultured under the following conditions: (10)-1-1 (10)-1 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３ (11)-1-1 (11)-1 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３ (12)-1-1 (12)-1 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３

[0123] (10)-2-1 (10)-2 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３ (11)-2-1 (11)-2 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３ (12)-2-1 (12)-2 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３

[0124] (10)-3-1 (10)-3 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３(11)-4-1 (11)-4 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３ (12)-5-1 (12)-5 cell suspension 0.5 mL, 1.5 mL KBM502 medium ※３

[0125] Day 13: The cells in (10)-1-1, (11)-1-1, (12)-1-1, (10)-2-1, (11)-2-1, (12)-2-1, (10)-3-1, (11)-4-1, and (12)-5-1, which had been subcultured on Day 10, were thoroughly pipetted and counted. 2.0 mL of each cell suspension was seeded in a 6-well plate and subcultured under the following conditions: (10)-1-2 (10)-1-1 cell suspension 5 x 10 ５ cells / 2mL, KBM502 medium supplemented with urelumab [1μg / mL] ※３ (10)-2-2 (10)-2-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium supplemented with urelumab [1μg / mL] ※３ (10)-3-2 (10)-3-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium supplemented with urelumab [1μg / mL] ※３ (11)-2-2 (11)-2-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium supplemented with urelumab [1μg / mL] ※３

[0126] (10)-1-3 (10)-1-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium ※３ (10)-2-3 (10)-2-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium ※３ (10)-3-3 (10)-3-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium ※３ (11)-2-3 (11)-2-1 Cell suspension 5x10 ５ cells / 2mL, KBM502 medium ※３

[0127] Day 14: The T75 flask (13) in which the culture had been started on Day 0 was thoroughly pipetted and the number of cells was counted.

[0128] On Day 10, the cells in each 10 cm dish of (6)-1-2, (6)-2-2, (7)-1-2, (7)-2-2, (8)-1-2, and (8)-2-2 were subcultured and counted by pipetting. 2.0 mL of each was seeded in a 6-well plate and subcultured under the following conditions: (6)-a (6)-1-2 cell suspension 5 x 10 ５ cells / 2mL, KBM502 medium ※３ (6)-b (6)-1-2 cell suspension 5x10 ５ cells / 2mL, KBM502 medium ※３ , Urelumab [1 μg / mL] (6)-c (6)-1-2 cell suspension 5 × 10 ５ cells / 2mL, KBM502 medium ※３ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (6)-d (6)-1-2 Cell suspension 5 × 10 ５ cells / 2mL, KBM501 medium ※４

[0129] Day 15: The cells of (10)-1-2, (10)-2-2, (10)-3-2, (11)-2-2, (10)-1-3, (10)-2-3, (10)-3-3, and (11)-2-3 that had been subcultured on Day 13 were thoroughly pipetted in each well, and the number of cells was counted.

[0130] Day 17: The cells of (10)-1-2, (10)-2-2, (10)-3-2, (11)-2-2, (10)-1-3, (10)-2-3, (10)-3-3, and (11)-2-3, which had been subcultured on Day 13, were thoroughly pipetted into each well and counted. 2.0 mL of each was seeded into a 6-well plate and subcultured under the following conditions: 1.0 mL of (10)-1-3' (10)-1-3 cell suspension, 1.0 mL of KBM502 medium ※３ (11)-2-2' (11)-2-2 cell suspension 1.0 mL, 1.0 mL KBM502 medium ※３

[0131] On Day 14, the cells in each well of (6)-a, (6)-b, (6)-c, and (6)-d were subcultured and counted by pipetting. 2.0 mL of each well was seeded in a 6-well plate and subcultured under the following conditions: (6)-a' (6)-a cell suspension 1.0 mL, 1.0 mL KBM502 medium ※３ (6)-c' (6)-c Cell suspension 1.0 mL, 1.0 mL KBM502 medium ※３

[0132] Day 20: The number of cells was counted in each well of (10)-1-2, (10)-2-2, (10)-3-2, (10)-2-3, (10)-3-3, and (11)-2-3, which had been subcultured on Day 13. The number of cells was counted in each well of (6)-b and (6)-d, which had been subcultured on Day 14, which had been pipetted.

[0133] Day 21: The cells in each well of (10)-1-3', (11)-2-2', (6)-a', and (6)-c' that had been subcultured on Day 17 were thoroughly pipetted, and the number of cells was counted.

[0134] The results are shown in Table 2 (change in total cell fold), Figure 4 (change in cell number), and Figure 5 (amplification fold). In Table 2, the cell amplification results were evaluated as A to D as follows, based on the fold change value of each cell number at the final count on Day 10 and thereafter, assuming Day 0 to be 1. Note that when the evaluation is C or higher, it can be determined that efficient cell amplification has been obtained and the objective has been achieved. A: Fold change of 100 or more B: Fold change of 10 or more C: Fold change of 2 or more D: Fold change of less than 2

[0135]

[0136] The results of (2) to (4) show that the cell expansion efficiency was poor when cells were cultured with the addition of IL-21 alone. The results of (5) and (9) show that the initial proliferation was significantly poor when cells were cultured without the addition of IL-21. The results of (6) to (8) and (10) to (12) show that the initial proliferation was good when cells were cultured with the addition of IL-21 and urelumab.

[0137] The results of flow cytometry performed on the (6)-a, (6)-c, (10)-1-3, or (11)-2-2 cell populations for each cell surface marker are shown in Figure 6.

[0138] D) Example 4 (Donor Mix B) The cytotoxic activity of the obtained cell population was confirmed.

[0139] ・Day 0 Cryopreserved PBMC (CD3 − , CD34 − ) was thawed by a predetermined method, and then cultured in NTI medium supplemented with IL-18 (100 ng / mL), urelumab (1 μg / mL), and IL-21 (1 ng / mL). ※１ 4x10 at ５ The cell suspension was prepared at 1.5 mL / mL and seeded in a 6-well plate to initiate the culture. The cells were cultured in NTI medium supplemented with urelumab (1 μg / mL) and IL-21 (1 ng / mL). ※１ 4x10 at ５ The cell suspension adjusted to 12.5 cells / mL was seeded in two T75 flasks at a volume of 12.5 mL each, and culture was initiated.

[0140] Day 7: The 6-well plate was thoroughly pipetted and the cells were counted. Fresh NTI medium was added. ※１ So 2 x 10 ５ IL-18 [100 ng / mL], urelumab [1 μg / mL], and IL-21 [1 ng / mL] were added to the cell suspension prepared at 14.4 cells / mL, and 14.4 mL of the suspension was seeded into T75 flask A and subcultured.

[0141] Two T75 flasks were pipetted thoroughly and the cells were counted. Fresh NTI medium was added. ※１ So 2 x 10 ５ Urelumab [1 μg / mL] and IL-21 [1 ng / mL] were added to the cell suspension prepared at 100 cells / mL, and the cells were seeded in two T225 flasks ((1) and (2)) at 45 mL each and in two T225 flasks ((3) and (4)) at 64 mL each, followed by subculture.

[0142] Day 10: T75 flask A was thoroughly pipetted and the cells were counted. Fresh NTI medium was added. ※１So 2 x 10 ５ 15 mL of the cell suspension prepared at 1000 cells / mL was seeded in T75 flask B and subcultured. After collection, T75 flask A was filled with NTI medium. ※１ The T225 flasks (1) and (2) were pipetted thoroughly and the cells were counted. The following two groups were prepared, each seeded in two T225 flasks, and subcultured. (1)-1 (1) Cell suspension 9 x 10 ６ The cells were separated, centrifuged at 500g for 5 minutes, and then resuspended in fresh NTI medium. ※１ 45 mL (2 x 10 ５ cells / mL) (1)-2 (1) Cell suspension 9x10 ６ cells / 45mL, NTI medium ※１

[0143] The T225 flasks (3) and (4) were thoroughly pipetted, the number of cells was counted, and the cells were recovered. After recovery, 50 mL of NTI medium was added to each of the T225 flasks (1), (2), (3), and (4). ※１ 15 mL was added and subcultured.

[0144] Day 14: The T225 flasks (1)-1 and (1)-2 were thoroughly pipetted and the number of cells was counted. The following two groups were prepared, each seeded in two T225 flasks, and subcultured: (1)-1-1 (1)-1 cell suspension 2.5 x 10 ６ The cells were separated, centrifuged at 500g for 5 minutes, and then resuspended in fresh NTI medium. ※１ Suspend in 50 mL (1)-2-1 (1)-1 cell suspension 2.5 x 10 ６ cells / 50mL, NTI medium ※１

[0145] The T75 flasks A and B were thoroughly pipetted and the cells were counted. The following two groups were prepared, seeded in 6-well plates, and subcultured: A-1: ​​Cell suspension A 1.0 x 10 ６ cells / 2mL, NTI medium ※１ B-1 A cell suspension 1.0 x 10 ６ The cells were separated, centrifuged at 500g for 5 minutes, and then resuspended in fresh NTI medium. ※１ Suspension in 2 mL

[0146] Day 15: The T225 flasks from (1), (2), (3), and (4) were thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS and counted. Fresh NTI medium was added. ※１ 1.18 x 10 ６ The cell suspension adjusted to 500 cells / mL was seeded in two T75 flasks ((5), (6)) at 50 mL each and subcultured.

[0147] Day 16: One T225 flask each of (1)-1-1 and (1)-2-1 was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS and the number of cells was counted.

[0148] Day 17: One T225 flask each of (1)-1-1 and (1)-2-1 was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS and counted. Wells A-1 and B-1 were thoroughly pipetted, and the cells were counted.

[0149] [Measurement of cytotoxic activity against tumor cells] For the measurement of cytotoxic activity, a group in which the cells collected on day 17 were reacted with K562 cells, a group containing only K562 cells as a negative control, and a group in which K562 cells were treated with 10% formalin as a positive control were prepared.

[0150] Day 17 harvested cells were cultured at a density of 1 x 10 in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution). ６ The concentration was adjusted to 1000 cells / ml.

[0151] K562 cells were suspended in RPMI 1640 medium (serum-free), stained using the PKH26 Red Fluorescent Cell Linker Kit, and then cultured at 2 × 10 cells / well in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution). ６ The concentration was adjusted to 100 cells / mL.

[0152] The cells collected on Day 17 and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) at a cell ratio of 1:1 and 2:1, mixed, and incubated at 37°C, 5% CO ２ The cells were incubated for 2 hours at 500g for 5 minutes. After the incubation, the cells were centrifuged at 500g for 5 minutes, and the supernatant was removed. 7-AAD solution diluted with PBS was added and suspended, and the cells were incubated at room temperature for 20 minutes. Measurements were performed using a flow cytometer, and the cytotoxic activity rate (% Lysis) was calculated using FlowJo software.

[0153] Cytotoxic activity rate (% Lysis) = (K562 cell death rate - negative control cell death rate) / (positive control cell death rate - negative control cell death rate) x 100

[0154] The cytotoxic activity rate calculated by the above formula is rated as A to D as follows. When the rating is C or higher, it is judged that high cytotoxic activity is obtained and the objective is achieved. A: 70% or more B: 60% or more C: 50% or more D: Less than 50%

[0155] The results are shown in Figure 7. In both cases where the ratio of E (effector cells (recovered cells)):T (target cells (K562 cells)) was 1:1 or 2:1, the cytotoxic activity rate of the recovered cell population was evaluated as A, indicating particularly high cytotoxic activity against tumor cells.

[0156] E) Example 5 (Donor Mix B) The antitumor effect of the obtained cell population was confirmed.

[0157] ・Day 0 Cryopreserved PBMC (CD3 − , CD34 − ) was thawed by a predetermined method, and then cultured in NTI medium supplemented with urelumab (final concentrations: 1 μg / mL) and IL-21 (1 ng / mL). ※１ 4x10 at ５ The cell suspension adjusted to 12.5 cells / mL was seeded into two T75 flasks at 12.5 mL each, and culture was initiated.

[0158] Day 6: 25 mL of cell suspension was collected from two T75 flasks and the cells were counted. ※１Then, urelumab [1 μg / mL] and IL-21 [1 ng / mL] were added, and the mixture was subcultured in 12.5 mL portions in three T75 flasks.

[0159] Day 8: The T75 flasks were thoroughly pipetted and the cell numbers were counted. Each flask was expanded to a T225 flask and filled with fresh NTI medium. ※１ Then, 37.5 mL of the above was added, and urelumab [1 μg / mL] and IL-21 [1 ng / mL] were further added, followed by subculture.

[0160] Day 10: The T225 flask was thoroughly pipetted to recover the cell suspension. It was centrifuged at 500 g for 5 minutes, and the pellet was suspended in 60 mL of culture supernatant. Fresh NTI medium was added. ※１ 240 mL of the mixture was added, and subcultured in 75 mL each in four T225 flasks.

[0161] Day 13: One T225 flask was thoroughly pipetted to recover the cell suspension. It was centrifuged at 500g for 5 minutes and the number of cells was counted. Fresh NTI medium was added. ※１ 9x10 ５ The culture medium was adjusted to 50 cells / mL and subcultured in four T225 flasks at 50 mL each.

[0162] Day 14: One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS, and the number of cells was counted.

[0163] Day 15: One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS, and the number of cells was counted.

[0164] Day 16: One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS, and the number of cells was counted.

[0165] Day 17: One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS, and the number of cells was counted.

[0166] [Animal experiment] Animals used: Female NOD. Cg-Prkdc ｓｃｉｄ Il2rgｔｍ１Ｓｕｇ ShiJic (NOG) mice (6 weeks old, CLEA Japan / Central Institute for Experimental Animals) were purchased and used at 6-7 weeks of age after a maximum of 5 days of acclimation. Animals were housed 3-4 per cage in an animal care room with an environmentally controlled temperature of 21-25°C, humidity of 40-60% (air-conditioned), and lighting of 12 hours (7:00-19:00). They were fed a solid diet of CRF-1 (γ-irradiation sterilized, Oriental Yeast Co., Ltd.) ad libitum. Autoclaved tap water was provided ad libitum.

[0167] Group composition: As shown in the table below, the test was conducted in two groups: a control (untreated) group and a test substance administration group.

[0168]

[0169] Preparation of tumor-bearing mice: <<Preparation of SK-OV-3 / CMV-Luc cell spheres>> SK-OV-3 / CMV-Luc cells were cultured in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution) at 1 x 10 cells per well. ６ After 72 hours of culture, the resulting spheres (2700 spheres / well) were collected and centrifuged at 100 g for 1 minute to remove the supernatant. The cells were then gently suspended in PBS to a concentration of 2700 spheres / 3 mL.

[0170] SK-OV-3 / CMV-Luc (a luciferase-expressing human ovarian cancer cell line) was transplanted into the abdominal cavity of a mouse at 450 spheres / 0.5 mL / head using a 27 G injection needle to create a peritoneal dissemination model.

[0171] Test substance: Recovered cells: Produced in-house Human interleukin-2 preparation (IL-2): Imunace (generic name: Teceleukin (genetical recombination)), Shionogi Pharmaceuticals

[0172] Administration of test substance: The day of transplantation of SK-OV-3 / CMV-Luc was set as Day 0, and the recovered cells were intraperitoneally administered on Day 3 (first administration), Day 4 (second administration), Day 5 (third administration), Day 6 (fourth administration), and Day 7 (fifth administration).

[0173] Follow-up observation: Fluorescence observation of intraperitoneal tumors using an in vivo imaging system (IVIS). For IVIS observation, 3 mg / head of luciferin was administered subcutaneously in 200 μL of PBS on the back of the mouse, and tumor luminescence was confirmed. Observation was performed under isoflurane anesthesia (using an animal inhalation anesthesia machine, with air as the carrier gas and an isoflurane concentration of 4% induced and maintained at 2%). Thereafter, observations were performed 1-2 times per week over time.

[0174] Body Weight Measurement and Observation of Appearance After transplantation of SK-OV-3 / CMV-Luc, body weight measurement and observation of appearance were carried out once or twice a week.

[0175] Humane endpoint: Difficulty in eating and drinking

[0176] The results are shown in Figure 8 (changes in the number of SK-OV-3 / CMV-Luc cells) and Figure 9 (IVIS results on Day 69). An antitumor effect was observed in the group administered with the recovered cell population.

[0177] F). Example 6 (Donor Mix C) Day 0 Cryopreserved PBMC (CD3 − , CD34 − ) was thawed by a predetermined method and culture was initiated under the following conditions: (1) 1.3 x 10 ５ cells / 0.65mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] 24-well plate (2) 1.6 x 10 ５ cells / 0.65mL, KBM501 medium ※４ , 24-well plate (3) 1.3 x 10 ５ cells / 0.65mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] G-Rex 24-well plate (4) 1.6 x 10 ５ cells / 0.65mL, KBM501 medium ※４ , G-Rex 24-well plate A 7.5x10 ６ cells / 15mL, KBM501 medium ※４ , T75 flask B 7.5x10 ６cells / 15mL, KBM501 medium ※４ , T75 flask

[0178] Day 7: The wells in (1) were thoroughly pipetted and the number of cells was counted. The cells were subcultured under the following conditions: (1)-1 (1) 0.65 mL of cell suspension, NTI medium ※１ 1.5mL, Urelumab [1μg / mL], IL-21 [1ng / mL]

[0179] Day 10: Add fresh NTI medium to T75 flask A. ※１ T75 flask B was cultured in fresh NTI medium supplemented with urelumab (1 μg / mL) and IL-21 (1 ng / mL). ※１ 35 mL of fresh KBM501 medium was added and cultured. The wells in (2) were thoroughly pipetted and the number of cells was counted. ※４ 1 mL of was added.

[0180] Day 11: (1)-1 well was thoroughly pipetted and the number of cells was counted. Fresh NTI medium was added. ※１ The cells were diluted to 6 mL with urelumab (1 μg / mL) and IL-21 (1 ng / mL) and subcultured into the following three groups: (1)-2 (1)-1 cell suspension 2 mL (1)-3 (1)-1 cell suspension 2 mL (5) G-Rex 24-well plate (1)-1 cell suspension 2 mL. Also, new NTI medium was added to the empty wells of (1)-1. ※１ 2 mL was added and subcultured.

[0181] Day 13: Add fresh NTI medium to T75 flask A. ※１ 35 mL of the solution was added and subcultured. (1) The wells in -2 were thoroughly pipetted and the number of cells was counted. (5) The G-Rex 24-well plate was thoroughly pipetted and the number of cells was counted.

[0182] Day 14: Well pipetted wells (1)-2 and counted the number of cells. Fresh NTI medium was added. ※１The solution was diluted to 6 mL with the syringe and subcultured in 2 mL portions in a 6-well plate. (1)-2-1 (1)-2 cell suspension 2 mL (1)-2-2 (1)-2 cell suspension 2 mL (1)-2-3 (1)-2 cell suspension 2 mL The wells in (1)-3 were thoroughly pipetted and the number of cells was counted. The cells were subcultured in a 6-well plate under the following conditions: (1)-3-1 (1)-3 fine rod suspension was centrifuged at 500 g for 5 minutes, and then subcultured in fresh NTI medium supplemented with Urelumab [1 μg / mL] and IL-21 [1 ng / mL]. ※１ Suspend well in 3 mL and inoculate

[0183] (5) Pipette the G-Rex 24-well well thoroughly, count the number of cells, and add fresh NTI medium. ※１ 4 mL of the solution was added and the mixture was subcultured.

[0184] The T75 flasks A and B were thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS, and the cell numbers were counted.

[0185] Day 17: (1)-2-1 wells were thoroughly pipetted and the number of cells was counted. (1)-3-1 wells were thoroughly pipetted and the number of cells was counted. (5) 4.7 mL of medium was gently removed from the 4 G-Rex wells and replaced with fresh NTI medium. ※１ 4 mL of fresh NTI medium was added. The mixture was pipetted thoroughly and the cell number was counted. ※１ 1 mL of was added.

[0186] Day 20: The wells in (1)-2-1 were thoroughly pipetted and the number of cells was counted. The wells in (1)-3-1 were thoroughly pipetted and the number of cells was counted.

[0187] Day 21: (1)-2-1 wells were thoroughly pipetted and the number of cells was counted. (1)-3-1 wells were thoroughly pipetted and the number of cells was counted. (5) G-Rex 24 wells were thoroughly pipetted, and the cells were recovered using 1 mM EDTA / PBS and the number of cells was counted.

[0188] Day 24: The cells in the well (1)-1 were pipetted thoroughly, and the number of cells was counted. The cells in the wells (1)-2 and (1)-3 were pipetted thoroughly, and the number of cells was counted.

[0189] The number of viable cells was also counted, and 2 x 10 ８ The cells were suspended in 5 mL of HSC-BANKER (ZENOAQ, CB071) and frozen at -80°C. The frozen cells were thawed in a water bath at 37°C and then diluted 10-fold with Plasma-Lyte A. Using the thawed cells, the cytotoxic activity against K562 cells or Raji cells was measured using the same method as in Example 4. The cytotoxic activity rate was evaluated as follows, from A to D. If the evaluation was C or higher, it was determined that high cytotoxic activity was obtained and the objective was achieved. A: 70% or more B: 60% or more C: 50% or more D: less than 50%

[0190] The results are shown in Figure 11. In both cases where the ratio of E (effector cells (recovered cells)):T (target cells (K562 cells)) was 1:1 or 5:1, the cytotoxic activity rate of the recovered cell population against K562 cells and Raji cells was evaluated as A, demonstrating high cytotoxic activity against both cells.

[0191] G) Example 7 (Donor Mix C, Single Donor A) Day 0: Cryopreserved PBMCs were thawed by a predetermined method, and 1.8 mL of the cell suspension prepared for the following five groups was seeded onto a 6-well plate to initiate culture.

[0192] (1) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, KBM501 medium ※４ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (2) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, NTI medium ※１ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (3) PBMC (CD3 − , CD34 − ) 4x10５ cells / mL, Simulect, KBM501 medium containing Prograf ※５ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (4) PBMC (CD3 − , CD34 − ) 4x10 ５ cells / mL, Simulect, NTI medium with Prograf ※６ , Urelumab [1 μg / mL], IL-21 [1 ng / mL] (5) PBMC (CD3 ＋ , CD34 ＋ ) 4x10 ５ cells / mL, Simulect, NTI medium with Prograf ※６ , Urelumab [1 μg / mL], IL-21 [1 ng / mL]

[0193] Day 7: Each well (1) to (5) was thoroughly pipetted and the number of cells was counted. The cells were then subcultured in 6-well plates under the following conditions: (1)-1 Fresh KBM501 medium ※２ 5 x 10 ５ The cells were diluted to 3 mL, and urelumab (1 μg / mL) and IL-21 (1 ng / mL) were added and seeded. (1)-2 Fresh KBM501 medium ※４ 5 x 10 ５ Dilute to 3 mL and sow in fresh KBM501 medium. ※４ So 5x10 ５ Diluted to 1.83 mL and seeded (1)-4 5 x 10 cells ５ The cells were collected and centrifuged at 500 g for 5 minutes, and then resuspended in fresh KBM501 medium supplemented with urelumab (1 μg / mL) and IL-21 (1 ng / mL). ※４ Suspend well in 3 mL and inoculate

[0194] (2)-1 New NTI medium ※４ 5 x 10 ５ The cells were diluted to 3 mL, and urelumab (1 μg / mL) and IL-21 (1 ng / mL) were added and the cells were seeded. (2)-2 New NTI medium ※１ 5 x 10 ５Dilute to 3 mL and sow in fresh NTI medium. ※１ 5 x 10 ５ Diluted to 1.83 mL and seeded (2)-4 5 x 10 cells ５ The cells were collected and centrifuged at 500 g for 5 minutes, and then resuspended in fresh NTI medium supplemented with urelumab (1 μg / mL) and IL-21 (1 ng / mL). ※１ Suspend well in 3 mL and inoculate

[0195] (5)-1 New NTI medium ※１ 5 x 10 ５ The cells were diluted to 3 mL, and urelumab (1 μg / mL) and IL-21 (1 ng / mL) were added and the cells were seeded. (5)-2 New NTI medium ※１ 5 x 10 ５ Dilute to 3 mL and sow in fresh NTI medium. ※１ 5 x 10 ５ Diluted to 1.83 mL and seeded (5)-4 5 x 10 cells ５ The cells were collected and centrifuged at 500 g for 5 minutes, and then resuspended in fresh NTI medium supplemented with urelumab (1 μg / mL) and IL-21 (1 ng / mL). ※１ Suspend well in 3 mL and inoculate

[0196] (3)-5 New Simulect and Prograf-containing KBM501 medium ※５ (4)-5 New Simulect and Prograf-containing NTI medium ※６ Seed 2.5 mL and 1.5 mL of cell suspension

[0197] (1) New KBM501 medium ※４ Add 3.5 mL and subculture (2) New NTI medium ※１ Add 3.5 mL of fresh KBM501 medium and subculture. ※５ Add 3.5 mL and subculture (4) New NTI medium ※１ Add 3.5 mL and subculture (5) New NTI medium ※１ Add 3.5 mL and subculture

[0198] Day 9: All wells subcultured on Day 7 were thoroughly pipetted and the number of cells was counted. Each of wells (1)-3, (2)-3, (5)-3, and (4)-5 was subcultured in 2 mL of 5-fold dilution (0.4 mL of cell suspension and 1.6 mL of fresh medium) and 10-fold dilution (0.2 mL of cell suspension and 1.8 mL of fresh medium) onto a 6-well plate.

[0199] (1)-3-5x (1)-3 cell suspension 0.4 mL, KBM501 medium ※４ 1.6 mL (2)-3-5x (2)-3 cell suspension 0.4 mL, NTI medium ※１ 1.6 mL (5)-3-5x (5)-3 cell suspension 0.4 mL, Simulect, NTI medium with Prograf ※６ 1.6 mL (4)-5-5x (4)-3 cell suspension 0.4 mL, Simulect, NTI medium with Prograf ※６ 1.6 mL

[0200] (1)-3-10x (1)-3 cell suspension 0.2 mL, KBM501 medium ※４ 1.8 mL (2)-3-10x (2)-3 cell suspension 0.2 mL, NTI medium ※１ 1.8 mL (5)-3-10x (5)-3 cell suspension 0.2 mL, Simulect, NTI medium with Prograf ※６ 1.8 mL (4)-5-10x (4)-3 cell suspension 0.2 mL, Simulect, NTI medium with Prograf ※６ 1.8 mL

[0201] Day 11: All wells subcultured on Day 9, (3)-5, (3), and (4) were thoroughly pipetted and the number of cells was counted. 2 x 10 cells were cultured with fresh medium from each of the wells (1)-3-5x, (2)-3-5x, (5)-3-5x, and (5)-3-5-10x. ５ The cell suspension was adjusted to 2 x 10 cells / 2 mL and subcultured in a 6-well plate. ５ cells / 2mL, KBM501 medium ※４ (2) 3-5x 2E5 cell suspension 2x10 ５ cells / 2mL, NTI medium ※１(5) -3-5x-2E5 cell suspension 2x10 ５ cells / 2mL, Simulect, NTI medium with Prograf ※６ (5)-3-10x-2E5 Cell suspension 2x10 ５ cells / 2mL, Simulect, NTI medium with Prograf ※６

[0202] Day 14: All wells subcultured on Day 11, (2)-3-5x, and (2)-3-10x were thoroughly pipetted, and the number of cells was counted. 4 x 10 cells were removed from each of the (2)-3-5x and (2)-3-10x wells with fresh medium. ５ The cells were then cultured in a 6-well plate at a concentration of 2 mL.

[0203] The results are shown in Figure 12.

[0204] H) Example 8 (Donor Mix) Cryopreserved PBMCs were completely thawed in a 37°C water bath and resuspended in KBM501 / 5% UG medium. ※７ After centrifugation at 500 × g for 5 minutes, the cells were diluted 10-fold with 4 × 10 ５ The culture medium was adjusted to 0.8 cells / mL, and 0.8 mL of the medium was seeded on a 12-well plate to initiate culture.

[0205] Day 0 (1) Control: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８ (2) Urelumab: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８ , 1 ng / mL IL-21, 1 μg / mL Urelumab (3) 1 μg / mL: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８ , 1 ng / mL IL-21, 1 μg / mL rhs4-1BBL (4) 5 μg / mL: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８ , 1 ng / mL IL-21, 5 μg / mL rhs4-1BBL (5) 10 μg / mL: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８, 1 ng / mL IL-21, 10 μg / mL rhs4-1BBL (6) IL-21: KBM501 / 5% UG medium containing Simulect and tacrolimus ※８ , 1 ng / mL IL-21 rhs4-1BBL = recombinant human soluble 4-1BB (CD137) ligand (polypeptide consisting of the amino acid sequence of SEQ ID NO: 1)

[0206] Day 7: 800 μL of KBM501 / 5% UG medium was added to (2) to (5).

[0207] Day 9: 1.6 mL of KBM501 / 5% UG medium was added to each of (1) and (6). For (2) to (5), the wells were thoroughly pipetted and the number of cells was counted. Then, the cell suspension was taken from the wells and 2 x 10 ５ The cells were adjusted to 1000 cells / mL in KBM501 medium / 5% UG and subcultured in a 6-well plate.

[0208] On Day 11 (6) and Day 9, the wells of (2) to (5) were subcultured in 6-well plates, and the number of cells was counted by pipetting thoroughly. (3) was maintained in culture, while the cell suspension was taken from the wells of the other groups. (2), (4), and (5) were counted at 2.35 x 10 ５ cells / mL, (6) is 1.84 × 10 ５ The cells were adjusted to 1000 cells / mL in KBM501 medium / 5% UG and subcultured in a 6-well plate.

[0209] Day 14: All groups ((1) seeded on 12-well plates on Day 0, (2) to (6) subcultured on 6-well plates on Day 9 or Day 11) were harvested using 1 mM EDTA / PBS and the number of cells was counted. ５ The cells were stained with the following antibodies (each antibody concentration 1 μg / mL).

[0210] After staining at 4°C for 30 minutes, the cells were centrifuged (500 × g, 5 minutes), the supernatant was removed, and the cells were suspended in PBS. Measurement was then performed using a flow cytometer (BD LSRFortessa, BD Biosciences) and analyzed using FlowJo software (FLOWJO, LLC).

[0211] The results of the amplification changes in the cell population are shown in Figure 13. The results of flow cytometry for each cell surface marker are shown in Figures 14 and 15.

[0212] Even when rhs-4-1BBL was used, efficient cell proliferation was obtained compared with (1) the control group (where neither rhs-4-1BBL, anti-4-1BB, nor IL-21 was added) and (6) the IL-21 group (where IL-21 was added but neither rhs-4-1BBL nor anti-4-1BB was added) ( FIG. 13 ).

[0213] *1: NTI (FUKOKU, T2108251) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *2: RPMI1640 (Nacalai, 30264-85) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10), 2 U / mL heparin sodium (Nipro) and 100 Units / mL Immunase *3: KBM502 (KOHJIN BIO, 16025020) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *4: 5% KBM501 (KOHJIN BIO, 16025015) supplemented with UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *5: 5% KBM501 (KOHJIN BIO, 16025015) supplemented with UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro), Simulect 0.5 μg / mL, and Prograf 1 ng / mL *6: 5% UltraGRO (AventaCell, HPCPLCRL10) and NTI (FUKOKU, T2108251) supplemented with 2 U / mL heparin sodium (Nipro), 0.5 μg / mL Simulect, and 1 ng / mL Prograf.

[0214] *7: KBM 501 supplemented with 5% UltraGRO and 2 U / mL heparin sodium. *8: KBM 501 supplemented with 5% UltraGRO, 2 U / mL heparin sodium, 0.5 μg / mL Simulect, and 1 ng / mL Prograf.

[0215] [Reagents and equipment used] RPMI1640 (Nacalai Tesque, 30264-85) KBM501 (KOHJIN BIO, 16025015) (containing IL-2 2810 IU / mL) KBM502 (KOHJIN BIO, 16025020) (containing IL-2 281 IU / mL) NTI (FUKOKU) CTS Immune Cell SR (gibco, A25961-01) UltraGRO (AventaCell, HPCPLCRL10) FBS (NICHIREI, 174012, 19C00A) Heparin sodium (Nipro) Urelumab (Urelumab) (Creative Biolabs, TAB-179) IL-21 (PEPROTECH, AF-200-21-10 μg) IL-18 (Biolegend, 592102) Imunace (Shionogi & Co., Ltd.) Simulect (NOVARTIS) Prograf (Astellas) Penicillin-streptomycin mixed solution (Nacalai Tesque, 26253-84) 0.5 M EDTA (Invitrogen, 15575-038) PBS (Nacalai Tesque, 14249-24) 7-AAD (Beckman Coulter, A07704) APC Anti-human CD107a Antibody (Biolegend, 328620) APC Mouse IgG1,κ Isotype Ctrl Antibody (Biolegend, 400120) EasySep Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055) 6-well plate (Thermo, 140675) 12-well plate (Thermo, 150628) 24-well plate (Thermo, 142475) G-Rex 24-well plate (BMBio, BMA-GP1024) T75 flask (ThermoFisher, 156499) T225 flask (ThermoFisher, 159933) 10 cm dish (Falcon, 3530003) 96-well plate (IWAKI, 4870-800SP) EZSPHERE (registered trademark) 6-well plate (IWAKI, 4810-900SP)

[0216] Alexa Fluor (registered trademark) 700-labeled anti-human CD56 antibody (Biolegend, 318316) APC-labeled anti-human NKp30 antibody (Biolegend, 325210) PerCP / Cy5.5-labeled anti-human CD3 antibody (Biolegend, 300430) PE-Cy7-labeled anti-human CD16 antibody (Biolegend, 302016) PE-labeled anti-human CD19 antibody (Biolegend, 302208) FITC-labeled anti-human CD14 antibody (Biolegend, 325604) APC-Cy7-labeled anti-human CD34 antibody (Biolegend, 343514)

[0217] FITC-labeled anti-human CD49a antibody (Biolegend, 328307) PE-labeled anti-human CD49c antibody (Biolegend, 343803) APC-labeled anti-human CD61 antibody (Biolegend, 336412) PE-Dazzle594-labeled anti-human NKp30 antibody (Biolegend, 325231) PE-Cy7-labeled anti-human CCR5 antibody (Biolegend, 359107) Pacific Blue™-labeled anti-human CCR6 antibody (Biolegend, 353438) APC-Cy7-labeled anti-human CXCR3 antibody (Biolegend, 353721)

[0218] KBM 501 (Kohjin Bio) UltraGRO (AventaCell, HPCPLCRL10) Heparin Sodium (Nipro) Simulect (Novartis Pharma) Prograf (Astellas Pharma) Recombinant Human 4-1BB Ligand Animal-Free Manufactured (PREPROTECH)

[0219] [Regarding the sequences listed in the sequence listing] SEQ ID NO: 1 Recombinant Human 4-1BB Ligand Animal-Free manufactured (PREPROTECH) SEQ ID NO: 2 Recombinant Human 4-1BB Ligand (N-Fc) #AP76391 (Signalway Antibody LLC) SEQ ID NO: 3 Extracellular domain of 4-1BBL

Claims

A method for producing a cell population containing CD3-negative and CD56-positive cells, comprising the steps of: a) culturing a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells in a medium containing IL-2, IL-21 and a 4-1BB agonist for one day or more, wherein the IL-21 and the 4-1BB agonist are humoral factors. 　 The method of claim 1 further comprising the steps of: b) Passaging the cell population cultured in step a) in a medium containing IL-2 and a 4-1BB agonist but not containing IL-21, and culturing the medium for one day or more. 　 The method according to claim 1, wherein the concentration of the 4-1BB agonist in the medium in step a) is 1 μg / mL or more, and the concentration of IL-21 in the medium in step a) is 1 ng / mL or more. 　 The method of claim 2 further comprising the steps of: c) Passaging the cell population cultured in step b) in a medium containing IL-2 and culturing for one day or more. 　 The method of claim 1 , wherein the 4-1BB agonist is selected from an anti-4-1BB antibody and a 4-1BB ligand. 　 The method of claim 1 or 2, wherein the medium comprises a platelet lysate.

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