WO2025022372A1 - Cell population production method - Google Patents

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 population

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

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

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 to be a new generation therapeutic technique to replace CAR-T therapy (Non-Patent Document 1). Methods for producing cell populations that exhibit high cytotoxicity against solid tumors are known (Patent Document 1, Non-Patent Document 2).

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

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 tumor necrosis factor receptor superfamily agonists such as 4-1BB agonists. Patent Document 3 describes a liquid composition for expanding T cells, which contains IL-2, IL-15, and IL-21 at predetermined concentrations.

On the other hand, Non-Patent Document 4 describes that under the 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, a means for realizing high NK cell proliferation efficiency by using a genetically modified feeder cell line or a synthetic lipid membrane has been developed. For example, Non-Patent Document 5 describes a method for co-culturing NK cells with genetically modified 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 when the peripheral blood mononuclear cells reach a specific cumulative division level, the cells are restimulated with feeder cells.

Patent Publication 2019-170176 Special table 2020-514289 Patent Publication No. 2021-113200 Special table 2021-518122

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-w Saito S. et al. Ex vivo generation of highly purified and activated d 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. etal. 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. LiX. etal. 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 C.J. 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.

While cells obtained by conventional methods show high cytotoxicity against solid tumors, their supply is limited.

In preparations using NK cells derived from ES cells/iPS cells such as those in Non-Patent Document 3, activity against solid tumors is particularly insufficient. Furthermore, the inventions described in Patent Documents 2 and 3 relate to the proliferation of T cells, and have not been shown to be useful for the proliferation of NK cells. In methods using gene recombination techniques or feeder cell lines such as those in Non-Patent Document 5 and Patent Document 4, the hurdles of quality control are increased, the manufacturing/purification processes become complicated (Non-Patent Document 6), and the burden of medical expenses may also increase.

The present invention aims to efficiently grow a cell population containing CD3-negative and CD56-positive cells without using a feeder cell line or genetic recombination techniques.

The inventors conducted extensive research to solve the above problems and discovered that a method including 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, where the IL-21 and the 4-1BB agonist are humoral factors, can mass-produce and amplify a cell population containing CD3-negative and CD56-positive cells, as compared to conventional manufacturing methods, and thus completed the present invention.

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 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.
[2] The method according to [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.
[3] The method for production 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 method according to [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.
[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.

According to the present invention, a cell population containing CD3-negative and CD56-positive cells can be mass-multiplied and obtained without using a feeder cell line or genetic recombination techniques, as compared to conventional manufacturing methods.

FIG. 1 shows the results (changes in cell number) of a NK cell culture test carried out under different conditions of culture medium and additives. FIG. 2 shows the results of a culture test in which the conditions for the cells used as the culture material were changed and compared (changes in the amplification fold of the cells). FIG. 3 shows the results of flow cytometry performed for each cell surface marker. FIG. 4 shows the results (changes in cell number) of a culture test comparing different concentrations of anti-4-1BB antibody and IL-21. FIG. 5 shows the results of a culture test comparing different concentrations of anti-4-1BB antibody and IL-21 (changes in cell amplification fold). FIG. 6 shows the results of flow cytometry performed for each cell surface marker. FIG. 7 shows the results of flow cytometry for CD3/CD56 or CXCR3 performed using the Day 14 cell population, and the results of measuring the cytotoxic activity against K562 cells using the Day 17 cell population. FIG. 8 shows the results of examining the antitumor effect of administration of the recovered cell population using cancer-bearing mice generated using SK-OV-3/CMV-Luc (a luciferase-expressing human ovarian cancer cell line) (changes in the number of SK-OV-3/CMV-Luc cells). Figure 9 shows the results of verifying 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). FIG. 10 shows the results of flow cytometry for CD3/CD56 or CXCR4. FIG. 11 shows the results of measuring the cytotoxic activity against K562 cells and Raji cells using the recovered cell population. FIG. 12 shows the results of a culture test (changes in cell amplification fold) when Simulect and tacrolimus were added to the medium. FIG. 13 shows the results of a culture test (changes in cell amplification fold) when recombinant 4-1BB ligand was used instead of anti-4-1BB antibody. FIG. 14 shows the results of flow cytometry performed for each cell surface marker. FIG. 15 shows the results of flow cytometry performed for each cell surface marker.

[Method of producing cell population]
<Step a>
In one aspect of the present invention, there is provided a method for producing a cell population containing CD3-negative and CD56-positive cells, the method comprising the steps of:
a) a step 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 medium.

<Cell population>
In the present invention, a cell population refers to a group of cells consisting of a plurality of cells, for example, 1×10 ⁵ cells or more. The cell population provided by the present invention can be prepared to various cell densities. For example, it can be prepared to 1×10 ⁵ cells/mL or more.

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 the cell surface marker. 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 by a similar method.

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

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.

The cell population containing mononuclear cells may be used after being thawed from cryopreservation. 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 it in a cryopreservation container such as a cryovial, and storing it 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 it.

<Culture medium>
The method for producing a cell population of the present invention includes a 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 medium. The medium may further contain additives, reagents, etc. suitable for culture. Hereinafter, when referring to "medium in the method for producing a cell population of the present invention", "medium of the present invention", "medium" or the like, and not particularly specifying "medium in step a)" or "medium in step b"), etc., it refers to the medium in one or more selected from step a), step b) and step c).

In this specification, when the concentration of cytokines, polypeptides, additives, etc. in the medium is described below, the concentration refers to 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.

(IL-2, IL-21, 4-1BB agonists)
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 are, 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 that is mainly expressed on the cell surface of lymphocytes. 4-1BB is also called CD137, TNFRSF9, etc. In the present invention, for example, a 4-1BB agonist refers to one that can bind to 4-1BB and promote its function. Examples of the function of 4-1BB include the production of IL-8 from human peripheral blood mononuclear cells and the production of interferon (IFN)-γ from human T lymphocytes, and the production of IL-8 and IFN-γ by 4-1BB can be measured by a method known to those skilled in the art. The 4-1BB agonist may be, for example, one that can bind to 4-1BB and promote the production of IL-8 or IFN-γ. Examples of the 4-1BB agonist include anti-4-1BB antibodies and 4-1BB ligand (4-1BBL).

In this specification, anti-4-1BB antibodies refer to antibodies that can recognize and bind to one or more specific antigenic determinants in 4-1BB and can multimerize 4-1BB, such as urelumab, utomilumab, or antibodies that can recognize the same antigenic determinants as these.

In this specification, the term 4-1BB ligand (4-1BBL) refers to a polypeptide or protein that can bind to 4-1BB (excluding antibodies). 4-1BBL may be synthesized by a method known to those skilled in the art, or may be artificially produced by recombinant gene technology, and 4-1BBL artificially produced by recombinant gene technology is called 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 with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and having proliferation activity of CD3-negative and CD56-positive cells. Amino acid sequence identity refers to the identity of the amino acid sequences between the target polypeptides, and is expressed as the percentage (%) of amino acid residues that match in the optimal alignment of the amino acid sequences. Amino acid sequence identity can be calculated using, for example, a homology search program known to those skilled in the art, such as BLAST or FASTA. A polypeptide having proliferation activity of CD3-negative and CD56-positive cells refers to a polypeptide that, when used in the cell culture method described in the Examples of this specification, can amplify the number of cells in a cell population containing CD3-negative and CD56-positive cells by at least two-fold, preferably three-fold.

(SEQ ID NO: 1)

(SEQ ID NO:2)

(SEQ ID NO:3)

4-1BBL may be a monomer or a multimer. A multimer refers to a combination of multiple subunits through 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, utomirumab, and a 4-1BB ligand. In a preferred embodiment of the manufacturing method of the present invention, the 4-1BB agonist is one or more selected from urelumab and a polypeptide having an amino acid sequence with 90% or more identity to SEQ ID NO: 1 and having proliferation activity of CD3-negative and CD56-positive cells. It is preferable that the cytokine, polypeptide, etc. used have a human amino acid sequence.

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.

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. 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 concentration range of IL-2.

The concentration of IL-21 in the medium in step a) of the production method of the present invention is not particularly limited, and may 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) may be, for example, 1000 ng/mL or less, 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, and 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 the concentration range of IL-21.

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. 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 upper and lower limits of any of the above-mentioned concentrations of the 4-1BB agonist may be combined to express the concentration range of the 4-1BB agonist.

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. In addition, 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.

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, even 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. In addition, 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.

In the medium of the present invention, IL-21 and 4-1BB agonists are humoral factors. "Hydrologic" means that the target protein, cytokine, or other substance is not immobilized and can dissolve in the medium, and humoral factors refer to proteins, etc. that are liquid. Opposite concepts include, for example, a state in which the target protein, etc. is immobilized on beads as described in Non-Patent Document 4, and a state in which the target protein, etc. is expressed on the cell membrane of the feeder cells after transformation as described in Non-Patent Document 5. The medium containing humoral factors is preferably liquid.

(Culture medium)
The 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 (CellGenics, 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), CTS OpTmizer T Cell Expansion Basal Medium (Thermo Fisher Scientific, for growth and proliferation of human T lymphocytes), IMDM, MEM, DMEM, 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, expanding cells, and activating cells. "Incubating" may be used when a process is carried out at a specific temperature for a certain period of time.

The medium may contain IL-2. Examples of media containing IL-2 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.

　The medium may be replaced or replenished at any time after the start of culture, provided that the desired culture effect is obtained, but it is preferable to do so every 2 to 5 days.

The culture vessels used for the culture include, but are not limited to, commercially available dishes, flasks, plates, and multi-well plates. In addition to the culture vessels, various culture materials such as supports may be used for the culture. The culture conditions are not particularly limited as long as they do not impair the culture effect, but the culture conditions are generally 37°C, 5% _CO2, and saturated water vapor atmosphere.

(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 to 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 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, and the like.

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.

The medium may further contain appropriate proteins, cytokines, antibodies, compounds and other components to the extent that the solution of the problems of the present invention is not hindered. In addition to the above-mentioned IL-2 and IL-21, the 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). All of these preferably have human amino acid sequences and are preferably produced by recombinant DNA technology for safety reasons.

The medium of the present invention may contain one or more suitable additives. Suitable additives include, for example, human platelet lysate (HPL), human serum substitute, fetal bovine serum (FBS), etc. The additives contained in the medium of the present invention are preferably HPL or FBS, and particularly preferably HPL. As HPL, for example, UltraGRO (AventaCel) can be used. 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. As heparin, sodium heparin, calcium heparin, etc. can be used. The concentration of heparin in the medium is preferably 1 to 5 U/mL, and particularly preferably 2 U/mL. 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%.

The medium of the present invention may contain one or more immunosuppressants. When the medium contains an immunosuppressant, the proliferation of CD3 positive cells can be suppressed. The immunosuppressant is, for example, a calcineurin inhibitor such as tacrolimus or cyclosporine, or a cytokine inhibitor such as basiliximab, and is 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. As the tacrolimus, for example, Prograf injection available from Astellas Pharma can be used. Basiliximab can be, for example, Simulect for intravenous injection, available from Novartis Pharma.

In a preferred embodiment, the culture method of the present invention does not use feeder cells. In this specification, feeder cells refer to cells that are different from the target cells (cells obtained by the present invention) and play an auxiliary role, for example, by producing cytokines that serve as growth factors, in order to prepare the culture conditions for the target cells. Feeder cells refer to cells that are commonly used in the technical field to which the present invention belongs, and can be, for example, animal cell lines into which genes have been introduced, white blood cells 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 can be cells that have almost completely lost their proliferation ability due to gamma irradiation, treatment with antibiotics, etc., but are not limited to cells that have reduced or lost their proliferation ability.

(Culture period)
Step a) may include 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 of time. 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, or more, and may be 30 days or less, 25 days or less, 20 days or less, 15 days or less, or less, and is not particularly limited. The culture 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 the period, the cells may be added to the same medium, or the medium may be replaced (in this case, it can be said that step a) is continued as long as the medium meets the requirements.), and if the cell density at the time of seeding is adjusted, it is possible to avoid maintenance (addition, replacement, etc. of the medium) during the culture period.

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

In this specification, passaging refers to transferring a cell population to a new medium, and the method 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 passaging may be added to the new medium, but in this specification, the composition of the medium used for the culture before passaging can be ignored in the composition of the new medium after passaging.

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, and may further contain IL-21. For a specific description of IL-2, 4-1BB agonist, and IL-21, please refer to the above. 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.

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, while 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.

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 greater than the concentration of the 4-1BB agonist in the medium in step a).

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).

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 greater than the concentration of 4-1BBL in the medium in step a).

Step b) may include culturing the cell population cultured in step a) for a predetermined period of time. 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 more, and may be 30 days or less, 25 days or less, 20 days or less, or less, and is not particularly limited. The culture period in step b) may be, for example, 1 to 2 days, 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 the period, the cells may be added to the same medium, or the medium may be replaced (in this case, it can be said that step b) is continued as long as the medium meets the requirements).

<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.

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 a specific description of IL-2, 4-1BB agonist, and IL-21, please refer to the above. In one 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 one more preferred embodiment, the medium in step c) of the present invention contains IL-2, IL-21, and a 4-1BB agonist.

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.

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.

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 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, and 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.

When 4-1BBL is used as the 4-1BBL agonist, the concentration of 4-1BBL in the 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.

Step c) may include culturing the cell population cultured in step b) for a predetermined period of time. The predetermined period is 1 day or more, and 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, 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, and is not particularly limited. 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 the period, the cells may be added to the same medium, or the medium may be replaced (in this case, it can be said that step c) is continued as long as the medium meets the requirements).

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, with no limit on the number of times, as long as cell proliferation is achieved.

By the manufacturing 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.

[Cell population obtained by the production method of the present invention]
The cell population obtained by the production method of the present invention is at least CD3 negative and CD56 positive, and preferably contains cells having a phenotype of one or more cell surface markers selected from the group consisting of CD3 negative, CD34 negative, CCR5 positive, CCR6 positive, CXCR3 positive, ITGA1 (Integrin α1) positive, and ITGA3 (Integrin α3) positive. More preferably, the cell population obtained 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.

In the present invention, when a cell population containing CD3-negative and CD56-positive cells is mentioned, 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.

Furthermore, in the present invention, when a cell population is referred to that contains cells that are CD3 negative, CD56 positive, and CD34 negative, CCR5 positive, CCR6 positive, CXCR3 positive, ITGA1 positive, or ITGA3 positive, the percentage 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 cells that are CD3 negative and CD56 positive, and CD34 negative, CCR5 positive, CCR6 positive, CXCR3 positive, ITGA1 positive, or ITGA3 positive is less than any of the above proportions, is distinguished from a cell population obtained by the present invention that contains cells that are CD3 negative and CD56 positive, and CD34 negative, CCR5 positive, CCR6 positive, CXCR3 positive, ITGA1 positive, or ITGA3 positive. The CD3 negative and 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.

[Cytotoxic activity]
The cells contained in the cell population obtained by the present invention can exhibit high cytotoxic activity. The cytotoxic activity can be evaluated, for example, by measuring the lysis ability of target cells (effector cells (E)) against target cells (T) such as tumor cells. For example, tumor cell lines of leukemia, glioma, breast cancer, colon cancer, ovarian cancer, prostate cancer, etc. can be used as tumor cells, but are not limited thereto. In addition, 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. The cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells, and is calculated by the following formula.

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

Cytotoxic activity can be measured, for example, by distinguishing between effector cells and target cells, and between live and dead cells, using reagents such as antibodies labeled with radioactive substances or fluorescent dyes, quantifying these cells using flow cytometry or the like, and determining the cell death rate 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 set appropriately depending on the type of target cells used; for example, when K562 cells, a leukemia cell line, are used as target cells, it can be set to 2 hours.

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

In addition, 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 in the effector cells. Since CD107a is present in granules in NK cells and migrates to the cell membrane surface of NK cells upon degranulation, CD107a positivity indirectly indicates that cytotoxic activity, i.e., attack of target cells by cells contained in the cell population obtained by the present invention, has been obtained. 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 is used as target cells at a cell number ratio of 1 to 2:1 (E:T), 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, a leukemia cell line, such as K562 cells.

[Antitumor effect]
The cell population obtained by the present invention exerts an antitumor effect even when administered in vivo. The antitumor effect in vivo can be evaluated, for example, by administering the cell population obtained by the present invention to a cancer-bearing model animal and measuring the change in tumor volume or the number of tumor cells. The preparation of the cancer-bearing model animal and the measurement of the change in tumor volume or the number of tumor cells can be performed using methods known to those skilled in the art, for example, a method of transplanting fluorescently labeled tumor cells into the animal and observing them. A specific example is a method using an in vivo imaging system (IVIS) in which luciferase-expressing SKOV cells (human ovarian cancer cell line) are transplanted into an immunodeficient mouse, luciferin is administered to the mouse, and the tumor luminescence is observed under anesthesia. In this method, the administration method of the cell population obtained by the present invention is not particularly limited, and the number of administrations may be single or multiple, and the administration interval when multiple administrations are performed can be appropriately set. The timing of administration can be any time point, for example, 1 to 30 days after tumor transplantation. The dosage may be, for example, 1.0×10 ⁴ to 1.0×10 ¹⁰ cells/body per administration, preferably 1.0×10 ⁵ to 1.0×10 ⁹ cells/body, more preferably 1.0×10 ⁶ to 1.0×10 ⁸ cells/body, and particularly preferably 1.5×10 ⁷ to 2.7×10 ⁷ cells/body. The route of administration may be any, including intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, with intraperitoneal administration being preferred. IL-2 may be administered simultaneously in order to maintain the activity of the cell population obtained by the present invention in vivo. The dosage of IL-2 in this case may be 1,000 to 10,000 IU/body, and preferably 5,000 IU/body. In one embodiment, administration of the cell population obtained by the present invention can reduce the number of tumor cells in a cancer-bearing model animal to 1/10 or less, compared to the case where the cell population is not administered.

The cell population obtained by the present invention can be cryopreserved. Cryopreservation can be performed, for example, 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 suspension in a cryopreservation container such as a cryovial, and storing the suspension at −80° C. or lower. The appropriate cell density may be, for example, 1×10 ³ to 1×10 ¹⁰ cells/mL, and preferably 2×10 ⁸ cells/5 mL. The cryoprotective agent may contain inorganic salts, a pH adjuster, and the like.

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

Pharmaceutical Compositions
According to one aspect of the present invention, there is provided a pharmaceutical composition comprising the cell population obtained by the present invention. The pharmaceutical composition may contain a medicament acceptable additive. Examples of the medicament acceptable additive include an isotonicity agent, a pH adjuster, a buffer, a stabilizer, a cryoprotectant, a dissolution aid, a pain-relieving agent, and an antibiotic.

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, cryoprotectants containing DMSO, saline, phosphate-buffered saline (PBS), culture medium, serum, etc. The solution may contain a pharma- ceutical acceptable carrier as a drug or quasi-drug.

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 sensitive 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 sensitive 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, into a vein, an artery, subcutaneously, or intraperitoneally.

The pharmaceutical composition of the present invention may be administered to a patient having an HLA genotype different from that of the cell population of the present invention.

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 (recombinant, product name: Immunace (Shionogi Pharmaceuticals)).

The pharmaceutical composition of the present invention may be used in combination with an antibody drug. Specific examples of antibodies that may 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, diclofenac, and ribacilaximab. nutuximab, obiltoxaximab, daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, omal izumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab, certolizum abpegol, mogamulizumab, pertuzumab, trastuzumab, obinutuzumab, vedolizumab, pembrolizuma, ida rucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumum Examples of such agents include ribacumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.

Antibodies that can be used in combination with the pharmaceutical composition of the present invention preferably have high affinity for CD16. In addition, 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.

The cells contained in the pharmaceutical composition of the present invention may express a chimeric antigen receptor (CAR). The method for expressing the CAR may be according to a method known to those skilled in the art, for example, a method in which a vector containing a gene encoding the desired CAR is used to introduce the gene into cells contained in the cell population of the present invention. The CAR may have a structure containing, for example, a short chain fragment (scFv) derived from any antibody and an intracellular signaling domain.

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

The present invention will be explained in more detail below with reference to examples, but the present invention should not be construed as being limited thereto.

[Culture method]
Thawing PBMC
Cryopreserved PBMCs (Peripheral Blood Mononuclear Cells) were completely thawed in a water bath at 37° C. 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 each condition.

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

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

(1) RPMI1640 medium, 5% CTS, IL-2 [100 IU/mL], IL-21 [100 ng/mL, Urelumab [10 μg/mL]
(2) RPMI 1640 medium, 5% UltraGRO, sodium heparin [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, sodium heparin [2 U/mL], IL-2 [100 IU/mL]
(6) RPMI 1640 medium, 5% FBS, IL-2 [100 IU/mL]
(7) KBM501 medium, 5% CTS, IL-21 [100 ng/mL], Urelumab [10 μg/mL]
(8) KBM501 medium, 5% UltraGRO, sodium heparin [2 U/mL], IL-21 [100 ng/mL], Urelumab [10 μg/mL]
(9) KBM501 medium, 5% FBS, IL-21 [100 ng/mL], Urelumab [10 μg/mL]
(10) KBM501 medium, 5% CTS
(11) KBM501 medium, 5% UltraGRO, sodium heparin [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, sodium heparin [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, sodium heparin [2 U/mL]
(18) NTI medium, 5% FBS

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.

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

・Day 11
In all groups, the wells were thoroughly pipetted and the number of cells was counted.

The results are shown in Table 1 and Figure 1.

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

B). Example 2 (Donor Mix B, Single Donor A)
Cultivation tests were conducted by changing the conditions of the cells used as the culture material.

・Day 0
Cryopreserved PBMCs were thawed by a prescribed method, and 0.65 mL of the cell suspension prepared for the following four groups was seeded onto a 12-well plate to initiate culture.
(1) PBMC (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 ⁵ cells/mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

Day 6
Each well was thoroughly pipetted and the number of cells was counted. Each group was seeded in 1 mL of a 12-well plate under the following conditions and subcultured.
(1)-1 (1) Cell suspension 4x10 ⁵ cells/1 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(2)-1 (2) Cell suspension 4x10 ⁵ cells/1 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(3)-1 (3) Cell suspension 4x10 ⁵ cells/1 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(4)-1 (4) Cell suspension 4x10 ⁵ cells/1 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

Day 7
Each well was thoroughly pipetted, and 2.0 mL of each group was inoculated into a 6-well plate under the following conditions and subcultured.
(1)-2 (1)-1 Cell suspension 1 mL, 1.0 mL Urelumab [1 μg/mL]-supplemented NTI medium ^*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

・Day 10
Each well was thoroughly pipetted and the number of cells was counted. Each group was seeded in 3.0 mL of a 6-well plate under the following conditions and subcultured.
(1)-3 (1)-2 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(2)-3 (2)-2 Cell suspension 5x10 ⁵ cells/3 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(3)-3 (3)-2 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(4)-3 (4)-2 Cell suspension 5x10 ⁵ cells/3 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

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

・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 portions onto 6-well plates under the following conditions and subcultured.
(1)-4 (1)-3 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(3)-4 (3)-3 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(4)-4 (4)-3 Cell suspension 5x10 ⁵ cells/3 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

・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 portions onto 6-well plates under the following conditions and subcultured.
(1)-5 (1)-4 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(3)-5 (3)-4 Cell suspension 5x10 ⁵ cells/3 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(4)-5 (4)-4 Cell suspension 5x10 ⁵ cells/3 mL, RPMI medium ^*2 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

・Day 19
The cells in each of wells (1)-5, (3)-5, and (4)-5 were thoroughly pipetted and counted.

・Day 21
The number of cells in each of the wells (1)-5, (3)-5, and (4)-5 was counted by pipetting thoroughly. 3.0 mL of each of the wells (3)-5 was seeded in a 6-well plate under the following conditions and subcultured.
(3)-5-1 (3)-5 Cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(3)-5-2 (3)-5 cell suspension 1.5x10 ⁶ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]

・Day 24
The number of cells in each well of (3)-5-1 and (3)-5-2 was counted by pipetting thoroughly. (3)-5-1 and (3)-5-2 were each seeded in 3.0 mL portions into a 6-well plate under the following conditions, and subcultured.
(3)-5-1 (3)-5-1 Cell suspension 5x10 ⁵ cells/3 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]
(3)-5-2-a (3)-5-2 cell suspension 5x10 ⁵ cells/3mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(3)-5-2-b (3)-5-2 cell suspension 1.5x10 ⁶ cells/3 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

・Day 28
Each well of (3)-5-1, (3)-5-2-a, and (3)-5-2-b was thoroughly pipetted and the number of cells was counted. (3)-5-2-b was seeded in 3.0 mL in a 6-well plate under the following conditions and subcultured.
(3)-5-2-b' (3)-5-2-b cell suspension 1.5x10 ⁶ cells/3 mL, NTI medium ^*1 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

・Day 31
(3)-5-2-b' well was thoroughly pipetted and the number of cells was counted.

Figure 2 shows the change in cell proliferation rate. It was found that the cell proliferation efficiency was better when NTI medium was used than when RPMI medium was used.

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 cytotoxicity described in Patent Document 1, etc., but CD3-positive cells were also amplified.

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

・Day 0
Cryopreserved PBMCs were thawed by a prescribed method, and 0.65 mL of the cell suspension prepared for the following 12 groups was seeded onto a 12-well plate to initiate culture.
(1) PBMC ( ^CD3- , ^CD34- ) ^4x105 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- ) ^4x105 cells/mL, KBM502 medium ^*3 , Urelumab [10μg/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]

Furthermore, 13.5 mL of the cell suspension prepared to the following concentration was seeded in a T75 flask to initiate culture.
(13) PBMC ( ^CD3- , ^CD34- ) ^5x105 cells/mL, KBM502 medium ^*3

Day 6
The number of cells was counted by pipetting each of wells (6), (7), and (8). 0.65 mL of the cells were seeded in a 12-well plate under the following conditions and subcultured. In addition, 0.6 mL of fresh KBM502 medium ^*3 was added to each of wells (6), (7), and (8).
(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]

Day 7
The number of cells was counted by pipetting each of wells (1) to (12). Furthermore, 0.65 mL of each of wells (10), (11), and (12) was seeded in a 12-well plate under the following conditions and subcultured.
(10)-1 (10) Cell suspension 0.1 mL, 0.55 mL 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]

(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]

(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]

・Day 8
The number of cells was counted by thoroughly pipetting each of the wells (6)-1, (6)-2, (7)-1, (7)-2, (8)-1, and (8)-2 that 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 (6)-1 Cell suspension 0.6 mL, 1.2 mL 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

・Day 10
The number of cells was counted by thorough pipetting in each well of (6)-1-1, (6)-2-1, (7)-1-1, (7)-2-1, (8)-1-1, and (8)-2-1 that had been subcultured on Day 8. 10 mL of the cells were seeded in a 10 cm dish and subcultured under the following conditions.
(6)-1-2 (6)-1-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3
(6)-2-2 (6)-2-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3
(7)-1-2 (7)-1-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3
(7)-2-2 (7)-2-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3
(8)-1-2 (8)-1-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3
(8)-2-2 (8)-2-1 Cell suspension 1.5mL, 8.5mL KBM502 medium ^*3

The cells seeded on Day 0 in each of wells (1), (2), (3), (4), (5), and (9) were thoroughly pipetted and the number of cells was counted.
0.7 mL of fresh KBM502 medium ^*3 was added to well (1).

The number of cells was 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 seeded on Day 7. 2.0 mL was seeded in a 6-well plate and subcultured under the following conditions.
(10)-1-1 (10)-1 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(11)-1-1 (11)-1 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(12)-1-1 (12)-1 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3

(10)-2-1 (10)-2 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(11)-2-1 (11)-2 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(12)-2-1 (12)-2 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3

(10)-3-1 (10)-3 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(11)-4-1 (11)-4 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3
(12)-5-1 (12)-5 Cell suspension 0.5mL, 1.5mL KBM502 medium ^*3

・Day 13
The number of cells was counted by thoroughly pipetting each well of (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 that had been subcultured on Day 10. 2.0 mL of each well was seeded in a 6-well plate and subcultured under the following conditions.
(10)-1-2 (10)-1-1 Cell suspension ^5x105 cells/2mL, KBM502 medium supplemented with Urelumab [1μg/mL] ^*3
(10)-2-2 (10)-2-1 Cell suspension ^5x105 cells/2mL, KBM502 medium supplemented with Urelumab [1μg/mL] ^*3
(10)-3-2 (10)-3-1 Cell suspension ^5x105 cells/2mL, KBM502 medium supplemented with Urelumab [1μg/mL] ^*3
(11)-2-2 (11)-2-1 Cell suspension ^5x105 cells/2mL, KBM502 medium supplemented with Urelumab [1μg/mL] ^*3

(10)-1-3 (10)-1-1 Cell suspension ^5x105 cells/2mL, KBM502 medium ^*3
(10)-2-3 (10)-2-1 Cell suspension ^5x105 cells/2mL, KBM502 medium ^*3
(10)-3-3 (10)-3-1 Cell suspension ^5x105 cells/2mL, KBM502 medium ^*3
(11)-2-3 (11)-2-1 Cell suspension ^5x105 cells/2mL, KBM502 medium ^*3

・Day 14
On Day 0, the T75 flask in which the culture had been started (13) was thoroughly pipetted and the number of cells was counted.

The number of cells was counted by thoroughly pipetting each of the 10 cm dishes of (6)-1-2, (6)-2-2, (7)-1-2, (7)-2-2, (8)-1-2, and (8)-2-2 that had been subcultured on Day 10. 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 ^5x105 cells/2mL, KBM502 medium ^*3
(6)-b (6)-1-2 Cell suspension ^5x105 cells/2mL, KBM502 medium ^*3 , Urelumab [1μg/mL]
(6)-c (6)-1-2 cell suspension ^5x105 cells/2mL, KBM502 medium ^*3 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(6)-d (6)-1-2 Cell suspension ^5x105 cells/2mL, KBM501 medium ^*4

・Day 15
On Day 13, each of the wells (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 was thoroughly pipetted and the number of cells was counted.

・Day 17
The number of cells was counted by thorough pipetting in each well 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. 2.0 mL of the cells were seeded in a 6-well plate and subcultured under the following conditions.
(10)-1-3' (10)-1-3 Cell suspension 1.0 mL, 1.0 mL KBM502 medium ^*3
(11)-2-2' (11)-2-2 Cell suspension 1.0 mL, 1.0 mL KBM502 medium ^*3

The number of cells was counted by thoroughly pipetting each of the wells (6)-a, (6)-b, (6)-c, and (6)-d that had been subcultured on Day 14. 2.0 mL of the cells were 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

・Day 20
On Day 13, each of the wells (10)-1-2, (10)-2-2, (10)-3-2, (10)-2-3, (10)-3-3, and (11)-2-3 that had been subcultured was thoroughly pipetted and the number of cells was counted.
On Day 14, the cells in each of the wells (6)-b and (6)-d that had been subcultured were thoroughly pipetted and the number of cells was counted.

・Day 21
On Day 17, the cells in each of the wells (10)-1-3', (11)-2-2', (6)-a', and (6)-c' that had been subcultured were thoroughly pipetted and the number of cells was counted.

The results are shown in Table 2 (change in total cell fold), Fig. 4 (change in cell number) and Fig. 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 or later, with Day 0 being set as 1. When the evaluation is C or higher, it can be determined that efficient cell amplification has been obtained and the purpose has been achieved.
A: Fold change is 100 or more B: Fold change is 10 or more C: Fold change is 2 or more D: Fold change is less than 2

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

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

D). Example 4 (Donor Mix B)
The cytotoxic activity of the resulting cell population was confirmed.

・Day 0
Cryopreserved PBMC ( ^CD3- , ^CD34- ) were thawed by a prescribed method, and the cell suspension was adjusted to 4x105 cells/mL in NTI medium ^*1 supplemented with IL-18 [100ng/mL], Urelumab [1μg/mL], and IL-21 [1ng/mL], and 1.5mL of the cell suspension was seeded in a 6-well plate to start culture. In addition, ^12.5mL of the cell suspension was seeded in two T75 flasks to start culture, and ^4x105 cells/mL in NTI medium ^*1 supplemented with Urelumab [1μg/mL] and IL-21 [1ng/mL], and 12.5mL of the cell suspension was seeded in two T75 flasks to start culture.

Day 7
The 6-well plate was pipetted thoroughly and the number of cells was counted. IL-18 [100 ng/mL], Urelumab [1 μg/mL], and IL- ²¹ [1 ng/mL] were added to the cell suspension prepared at 2 x 10 ⁵ cells/mL in fresh NTI medium*1, and 14.4 mL of the mixture was seeded in a T75 flask A and subcultured.

The two T75 flasks were pipetted thoroughly and the cells were counted. A cell suspension was prepared at 2 x ¹⁰⁵ cells/mL in fresh NTI medium ^*1, to which Urelumab [1 μg/mL] and IL-21 [1 ng/mL] were added. The cells were then seeded in two T225 flasks ((1) and (2)) at 45 mL each and in two T225 flasks ((3) and (4)) at 64 mL each, and subcultured.

・Day 10
The T75 flask A was pipetted thoroughly and the cells were counted. 15 mL of the cell suspension adjusted to 2 x 10 ⁵ cells/mL with fresh NTI medium ^*1 was seeded into T75 flask B and subcultured. 15 mL of NTI medium ^*1 was added to the recovered T75 flask A and subcultured.
The T225 flasks (1) and (2) were thoroughly pipetted and the cells were counted. The following two groups were prepared, seeded in two T225 flasks each, and subcultured.
(1)-1 (1) Cell suspension ^9x106 cells were taken, centrifuged at 500g for 5 minutes, and then suspended in 45mL ( ^2x105 cells/mL) of fresh NTI medium ^*1. (1)-2 (1) Cell suspension ^9x106 cells/45mL, NTI medium ^*1

The T225 flasks from (3) and (4) were thoroughly pipetted, and the cells were counted and recovered.
After recovery from (1), (2), (3), and (4), 15 mL of 50 mL of NTI medium ^*1 was added to each T225 flask and subcultured.

・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, and each was seeded in two T225 flasks and subcultured.
(1)-1-1 (1)-1 Cell suspension ^2.5x106 cells were taken, centrifuged at 500g for 5 minutes, and then suspended in 50mL of fresh NTI medium ^*1. (1)-2-1 (1)-1 Cell suspension ^2.5x106 cells/50mL, NTI medium ^*1

The T75 flasks A and B were thoroughly pipetted and the number of cells was counted. The following two groups were prepared, seeded on 6-well plates, and subcultured.
A-1 A cell suspension ^1.0x106 cells/2mL, NTI medium ^*1
B-1: Take ^1.0x106 cells from the cell suspension A, centrifuge at 500g for 5 minutes, and then suspend in 2mL of fresh NTI medium ^*1.

・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 the cell numbers were counted.
A cell suspension adjusted to 1.18 x 10 ⁶ cells/mL in fresh NTI medium ^*1 was seeded at 50 mL each into two T75 flasks ((5) and (6)) and subcultured.

・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.

・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 the number of cells was counted.
Wells A-1 and B-1 were thoroughly pipetted, and the number of cells was counted.

[Measurement of cytotoxicity 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.

<<Day 17 Recovered Cells>>
The cells collected on Day 17 were adjusted to a concentration of 1 x 10 ⁶ cells/ml in RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution).

《K562》
K562 cells were suspended in RPMI1640 medium (serum-free) and stained using PKH26 Red Fluorescent Cell Linker Kit, and then adjusted to 2 x 10 ⁶ cells/mL in RPMI1640 medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution).

Day 17 collected cells 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 reacted for 2 hours at 37°C under 5% CO2. _After the reaction, the cells were centrifuged at 500g for 5 minutes, the supernatant was removed, and then 7-AAD solution diluted with PBS was added, suspended, and incubated at room temperature for 20 minutes. Measurement was performed using a flow cytometer, and analysis was performed using FlowJo software to calculate the cytotoxic activity rate (% Lysis).

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

The cytotoxic activity rate calculated by the above formula is rated as follows, from A to D. When the rating is C or higher, it is determined 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%

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.

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

・Day 0
Cryopreserved PBMCs (CD3 ^- , CD34 ^- ) were thawed using a prescribed method, and a cell suspension adjusted to 4 x 10 ⁵ cells/mL in NTI medium ^*1 supplemented with final concentrations of Urelumab [1 μg/mL] and IL-21 [1 ng/mL] was seeded at 12.5 mL per flask into two T75 flasks to initiate culture.

Day 6
25 mL of cell suspension was collected from two T75 flasks and the cells were counted. 12.5 mL of fresh NTI medium ^*1 was added, and Urelumab [1 μg/mL] and IL-21 [1 ng/mL] were further added, and 12.5 mL each was subcultured in three T75 flasks.

・Day 8
The T75 flask was pipetted thoroughly and the number of cells was counted. Each flask was expanded to a T225 flask, and 37.5 mL of fresh NTI medium ^*1 was added, followed by the addition of Urelumab [1 μg/mL] and IL-21 [1 ng/mL] for subculture.

・Day 10
The T225 flask was pipetted thoroughly to recover the cell suspension. It was centrifuged at 500g for 5 min, and the pellet was suspended in 60 mL of culture supernatant. 240 mL of fresh NTI medium ^*1 was added, and 75 mL each was subcultured in four T225 flasks.

・Day 13
One T225 flask was thoroughly pipetted to recover the cell suspension, which was then centrifuged at 500 g for 5 minutes, and the number of cells was counted.
The cells were adjusted to 9 x 10 ⁵ cells/mL in fresh NTI medium ^*1 and subcultured in 50 mL each in four T225 flasks.

・Day 14
One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA/PBS, and the cell number was counted.

・Day 15
One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA/PBS, and the cell number was counted.

・Day 16
One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA/PBS, and the cell number was counted.

・Day 17
One T225 flask was thoroughly pipetted, and the cells were recovered using 1 mM EDTA/PBS, and the cell number was counted.

[Animal experiments]
Animals used:
Female NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /ShiJic (NOG) mice (6 weeks old, Japan CLEA/Central Institute for Experimental Animals) were purchased and used for the study at 6-7 weeks of age after acclimation for up to 5 days. The animals were housed in cages of 3-4 animals in an animal breeding room with an regulated environment of temperature: 21-25°C, humidity: 40-60% (air conditioning control), and lighting time: 12 hours (7:00-19:00), and were allowed to freely consume solid feed CRF-1 (γ-ray sterilized, Oriental Yeast Co., Ltd.). Autoclaved tap water was allowed to be consumed freely.

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

Creation of tumor-bearing mice:
<<Sphere creation of SK-OV-3/CMV-Luc cells>>
SK-OV-3/CMV-Luc cells were prepared in RPMI1640 medium (containing 10% FBS and 1% penicillin-streptomycin mixed solution) at ^1x106 cells/3mL per well and seeded on an EZSPHERE (registered trademark) 6-well plate (IWAKI, 4810-900SP). After 72 hours of culture, the prepared spheres (2700 spheres/well) were collected and centrifuged at 100g for 1 minute to remove the supernatant, and gently suspended in PBS to prepare 2700 spheres/3mL.

SK-OV-3/CMV-Luc (a luciferase-expressing human ovarian cancer cell line), 450 spheres/0.5 mL/head, was implanted into the abdominal cavity of mice using a 27G needle to create a peritoneal dissemination model.

Test substance:
Recovered cells: Produced in-house Human interleukin-2 preparation (IL-2): Imunace (generic name: Teceleukin (genetic recombinant)), Shionogi Pharmaceuticals

Test substance administration:
The day of SK-OV-3/CMV-Luc transplantation was designated as Day 0, and the recovered cells were intraperitoneally administered on Day 3 (1st), Day 4 (2nd), Day 5 (3rd), Day 6 (4th), and Day 7 (5th).

Observation:
Fluorescence observation of intraperitoneal tumors using an in vivo imaging system (IVIS) For observation using IVIS, Luciferin 3 mg/head in 200 μL of PBS was administered subcutaneously to the back of the mouse, and the tumor was confirmed to emit light. Observation was performed under isoflurane anesthesia (using an animal inhalation anesthesia machine, with air as the carrier gas and isoflurane concentration 4% induced and maintained at 2%). After that, observation was performed 1-2 times/week over time.

Body Weight Measurement and Observation of Appearance After transplantation of SK-OV-3/CMV-Luc, body weight measurement and observation of appearance were performed 1-2 times a week.

Humane Endpoints:
Difficulty eating and drinking

The results are shown in Figure 8 (changes in SK-OV-3/CMV-Luc cell count) and Figure 9 (IVIS results on Day 69). An antitumor effect was observed in the group administered the collected cell population.

F). Example 6 (Donor Mix C)
・Day 0
Cryopreserved PBMC (CD3 ⁻ , CD34 ⁻ ) were thawed by a predetermined method, and culture was initiated under the following conditions.
(1) ^1.3x105 cells/0.65mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL], 24-well plate (2) ^1.6x105 cells/0.65mL, KBM501 medium ^*4 , 24-well plate (3) ^1.3x105 cells/0.65mL, NTI medium ^*1 , Urelumab [1μg/mL], IL-21 [1ng/mL], G-Rex 24-well plate (4) ^1.6x105 cells/0.65mL, KBM501 medium ^*4 , G-Rex 24-well plate A ^7.5x106 cells/15mL, KBM501 medium ^*4 , T75 flask B 7.5x10 ⁶ cells/15mL, KBM501 medium ^*4 , T75 flask

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) Cell suspension 0.65 mL, NTI medium ^*1 1.5 mL, Urelumab [1 μg/mL], IL-21 [1 ng/mL]

・Day 10
35 mL of fresh NTI medium ^*1 was added to T75 flask A and cultured.
^{35 mL of fresh NTI medium*1} supplemented with Urelumab [1 μg/mL] and IL-21 [1 ng/mL] was added to T75 flask B and cultured.
(2) Well was pipetted thoroughly and the number of cells was counted. 1 mL of fresh KBM501 medium ^*4 was added.

・Day 11
(1)-1 well was pipetted thoroughly and the number of cells was counted. Fresh NTI medium ^*1 was added to 6 mL, and Urelumab [1 μg/mL] and IL-21 [1 ng/mL] were added, and the cells were 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
Furthermore, 2 mL of fresh NTI medium ^*1 was added to the empty well of (1)-1 and subcultured.

・Day 13
35 mL of fresh NTI medium ^*1 was added to T75 flask A and subcultured.
The wells in (1)-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.

・Day 14
(1)-2 wells were thoroughly pipetted and the number of cells was counted. The cells were then diluted to 6 mL with fresh NTI medium ^*1 and subcultured in 2 mL portions onto 6-well plates.
(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 The suspension of the thin rod was centrifuged at 500 g for 5 minutes, and then thoroughly suspended in ^{3 mL of NTI medium*1} containing fresh Urelumab [1 μg/mL] and IL-21 [1 ng/mL] and then inoculated.

(5) The G-Rex 24-well plate was thoroughly pipetted, the number of cells was counted, and 4 mL of fresh NTI medium ^*1 was added and the cells were subcultured.

T75 flasks A and B were thoroughly pipetted, and the cells were harvested using 1 mM EDTA/PBS and counted.

・Day 17
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.
(5) 4.7 mL of medium was gently removed from the G-Rex 4 wells, and 4 mL of fresh NTI medium ^*1 was added. The cells were thoroughly pipetted and counted. An additional 1 mL of fresh NTI medium ^*1 was added.

・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.

・Day 21
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.
(5) The G-Rex 24-well plate was thoroughly pipetted, and the cells were recovered using 1 mM EDTA/PBS, and the cell number was counted.

・Day 24
(1) The wells in -1 were thoroughly pipetted, and the number of cells was counted.
The cells in wells (1)-2 and (1)-3 were thoroughly pipetted, and the number of cells was counted.

The number of viable cells was counted, and ^2x108 cells were suspended in 5mL 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 by the same method as in Example 4. The cytotoxic activity rate is evaluated as A to D as follows. When the evaluation is C or higher, it is determined that high cytotoxic activity is obtained and the purpose is achieved.
A: 70% or more B: 60% or more C: 50% or more D: Less than 50%

The results are shown in Figure 11. In either case 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, and high cytotoxic activity was observed against both types of cells.

G). Example 7 (Donor Mix C, Single Donor A)
・Day 0
Cryopreserved PBMCs were thawed by a prescribed 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.

(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 [1μg/mL], IL-21 [1ng/mL]
(4) PBMC ( ^CD3- , ^CD34- ) ^4x105 cells/mL, Simulect, NTI medium containing Prograf ^*6 , Urelumab [1μg/mL], IL-21 [1ng/mL]
(5) PBMC (CD3 ⁺ , CD34 ⁺ ) 4 x 10 ⁵ cells/mL, Simulect, NTI medium containing Prograf ^*6 , Urelumab [1 μg/mL], IL-21 [1 ng/mL]

Day 7
Each well from (1) to (5) was thoroughly pipetted, and the number of cells was counted. The cells were then subcultured in 6-well plates under each of the following conditions.
(1)-1: Dilute to ^5x105 cells/3mL with fresh KBM501 medium ^*2 , add Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow. (1)-2: Dilute to ^5x105 cells/3mL with fresh KBM501 medium ^*4 and sow. (1)-3: Dilute to ^5x105 cells/1.83mL with fresh KBM501 medium ^* 4 and sow. (1)-4: Take ^5x105 cells, centrifuge at 500g for 5 minutes, then suspend in 3mL of fresh KBM501 medium ^*4 containing Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow.

(2)-1: Dilute to ^5x105 cells/3mL with new NTI medium ^*4 , add Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow. (2)-2: Dilute to ^5x105 cells/3mL with new NTI medium ^*1 and sow. (2)-3: Dilute to ^5x105 cells/1.83mL with new NTI medium ^*1 and sow. (2)-4: Take ^5x105 cells, centrifuge at 500g for 5 minutes, then suspend well in 3mL of new NTI medium ^*1 containing Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow.

(5)-1: Dilute to ^5x105 cells/3mL with new NTI medium ^*1 , add Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow. (5)-2: Dilute to ^5x105 cells/3mL with new NTI medium ^*1 and sow. (5)-3: Dilute to ^5x105 cells/1.83mL with new NTI medium ^*1 and sow. (5)-4: Take ^5x105 cells, centrifuge at 500g for 5 minutes, then suspend well in 3mL of new NTI medium ^*1 containing Urelumab [1μg/mL] and IL-21 [1ng/mL] and sow.

(3)-5 Seed 2.5 mL of new Simulect and Prograf-containing KBM501 medium ^*5 and 1.5 mL of cell suspension. (4)-5 Seed 2.5 mL of new Simulect and Prograf-containing NTI medium ^*6 and 1.5 mL of cell suspension.

(1) Add 3.5 mL of new KBM501 medium ^{*4 and subculture} . (2) Add 3.5 mL of new NTI medium ^*1 and subculture. (3) Add 3.5 mL of new KBM501 medium ^{*5 and} subculture. (4) Add 3.5 mL of new NTI medium ^*1 and subculture. (5) Add 3.5 mL of new NTI medium ^*1 and subculture.

・Day 9
All wells subcultured on Day 7 were thoroughly pipetted and the number of cells was counted. Each of the 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 new medium) and 10-fold dilution (0.2 mL of cell suspension and 1.8 mL of new medium) onto a 6-well plate.

(1)-3-5x (1)-3 cell suspension 0.4mL, KBM501 medium ^*4 1.6mL
(2)-3-5x (2)-3 Cell suspension 0.4mL, NTI medium ^*1 1.6mL
(5)-3-5x (5)-3 cell suspension 0.4mL, Simulect, Prograf NTI medium ^*6 1.6mL
(4)-5-5 times (4)-3 Cell suspension 0.4 mL, Simulect, Prograf-containing NTI medium ^*6 1.6 mL

(1)-3-10x (1)-3 cell suspension 0.2mL, KBM501 medium ^*4 1.8mL
(2)-3-10x (2)-3 Cell suspension 0.2mL, NTI medium ^*1 1.8mL
(5)-3-10x (5)-3 cell suspension 0.2mL, Simulect, Prograf NTI medium ^*6 1.8mL
(4)-5-10x (4)-3 Cell suspension 0.2mL, Simulect, Prograf NTI medium ^*6 1.8mL

・Day 11
All wells subcultured on Day 9, (3)-5, (3), and (4) were thoroughly pipetted and the number of cells was counted. From wells (1)-3-5x, (2)-3-5x, (5)-3-5x, and (5)-3-5-10x, fresh medium was added to each well to prepare 2 ^{x 105} cells/2 mL, and the cells were subcultured in a 6-well plate.
(1)-3-5x-2E5 cell suspension ^2x105 cells/2mL, KBM501 medium ^*4
(2)-3-5x-2E5 cell suspension ^2x105 cells/2mL, NTI medium ^*1
(5)-3-5x-2E5 cell suspension ^2x105 cells/2mL, Simulect, Prograf-containing NTI medium ^*6
(5)-3-10x-2E5 cell suspension ^2x105 cells/2mL, Simulect, Prograf-containing NTI medium ^*6

・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. From the (2)-3-5x and (2)-3-10x wells, fresh medium was added to each well to prepare 4 x ¹⁰⁵ cells/2 mL, and the cells were subcultured in a 6-well plate.

The results are shown in Figure 12.

H). Example 8 (Donor Mix)
Cryopreserved PBMCs were completely thawed in a 37°C water bath and diluted 10-fold with KBM501/5% UG medium ^*7 . After centrifugation at 500×g for 5 minutes, the cells were adjusted to 4×10 ⁵ cells/mL with the following 6 groups of medium, and 0.8 mL was seeded on a 12-well plate to start culture.

・Day 0
(1) Control: KBM501/5% UG medium containing Simulect and tacrolimus ^*8
(2) Urelumab: KBM501/5% UG ^medium containing Simulect and tacrolimus, 1 ng/mL IL-21, 1 μg/mL Urelumab
(3) 1 μg/mL: KBM501/5% UG ^medium containing Simulect and tacrolimus, 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, 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)

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

・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, a cell suspension was taken from the well, adjusted to 2 x 10 ⁵ cells/mL in KBM501 medium/5% UG, and subcultured in a 6-well plate.

・Day 11
For (6) and (2) to (5) that were passaged to a 6-well plate on Day 9, the wells of each group were thoroughly pipetted and the number of cells was counted. (3) was maintained in culture as is, and for the other groups, cell suspensions were taken from the wells and adjusted to 2.35 x ¹⁰⁵ cells/mL for (2), (4), and (5), and 1.84 x ¹⁰⁵ cells/mL for (6) in KBM501 medium/5% UG, and passaged to a 6-well plate.

・Day 14
For all groups ((1) seeded onto 12-well plates on Day 0, (2) to (6) passaged onto 6-well plates on Day 9 or Day 11), cells were harvested using 1 mM EDTA/PBS and the cell numbers were counted. In addition, 1 x 10 ⁵ cells out of the harvested cells were stained with the following antibodies (each antibody concentration 1 μg/mL).

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

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

Even when rhs-4-1BBL was used, efficient cell proliferation was obtained compared to (1) the control group (when neither rhs-4-1BBL, anti-4-1BB, nor IL-21 was added) and (6) the IL-21 group (when rhs-4-1BBL or anti-4-1BB was not added but IL-21 was added) (Figure 13).

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

*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.

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

Alexa Fluor® 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)

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)

・KBM 501 (Kohjin Bio)
・UltraGRO (AventaCell, HPCPLCRL10)
・Heparin sodium (Nipro)
・Simlect (Novartis Pharma)
・Prograf (Astellas Pharma)
・Recombinant Human 4-1BB Ligand Animal-Free manufactured (PREPROTECH)

[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 (6)

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 contains a platelet lysate.