Cell population production method

Abstract

The purpose of the present invention is to efficiently proliferate a cell population containing cells that are CD3-negative and CD56-positive without using a feeder cell line and without using a genet

Description

Problem to be solved by the invention

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

[0010] Formulations using NK cells derived from ES cel 1 s / iPS cells, such as one described in Non-patent document 3, show insufficient activity against, in particular, solid tumors. Further, the inventions described in Patent document 2 or Patent document 3 relate to T cell proliferation and have not been shown to be useful for NK cell proliferation. Methods using genetic recombination techniques or feeder cell lines, such as those of Non-patent document 5 or Patent document 4, raise hurdles for quality control, in addition, complicate manufacturing / purification processes (Non-patent document 6), and may also pose further challenges relating to increased medical costs.

[0011] Therefore, an object of the present invention is to efficiently proliferate a cell population containing CD3-negative and CD56-positive cells without using feeder cell lines and without employing genetic recombination techniques. Means for solving the object

[0012] The inventors of the present invention diligently investigated to solve the above problem, as a result, found that a method comprising a) the 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 medium containing IL-2, IL-21 and 4-1BB agonist, wherein IL-21 and 4-1BB agonist are humoral factors, enables amplification and production of a cell population containing CD3-negative and CD56-positive cells in larger quantities compared with conventional production methods, and accomplished the present invention.

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

[0014] According to the present invention, a cell population containing CD3-negative and CD56-positive cells can be amplified and obtained in larger quantities compared with conventional production methods, without using feeder cell lines and without employing genetic recombination techniques. Brief Description of the Drawings

[0015] [Fig. 1] Fig. 1 shows the results of NK cell culture tests conducted under different conditions for culture medium and additives (changes in cell number). [Fig. 2] Fig. 2 shows the results of culture tests conducted under different conditions for cells used as raw materials (changes in cell amplification rate). [Fig. 3] Fig. 3 shows the results of flow cytometry performed for each cell surface marker. [Fig. 4] Fig. 4 shows the results of culture tests comparing different concentrations of anti-4-IBB antibody and IL-21 (changes in cell number). [Fig. 5] Fig. 5 shows the results of culture tests comparing different concentrations of anti-4-IBB antibody and IL-21 (changes in cell amplification rate). [Fig. 6] Fig. 6 shows the results of flow cytometry performed for each cell surface marker. [Fig. 7] Fig. 7 shows the results of flow cytometry performed by using cell populations of day 14 for CD3 / CD56 or CXCR3, and the results of measuring cytotoxic activity of the cell populations of day 17 against K562 cells. [Fig. 8] Fig. 8 shows the results of verifying the antitumor effect of administering the collected cell populations on tumor-bearing mice produced with SK-OV-3 / CMV-Luc (luciferase-expressing human ovarian cancer cell line) (change in SK-OV-3 / CMV-Luc cell number). [Fig. 9] Fig. 9 shows the results of verifying the antitumor effect of administering the collected cell populations on tumor-bearing mice produced with SK-OV-3 / CMV-Luc (results of IVIS (in vivo imaging system) on day 69). [Fig. 10] Fig. 10 shows the results of flow cytometry for CD3 / CD56 or CXCR4. [Fig. 11] Fig. 11 shows the results of measuring cytotoxic activity of the collected cell population against K562 and Raji cells. [Fig. 12] Fig. 12 shows the results of the culture tests performed with adding Simulect and tacrolimus to the medium (change in cell amplification rate). [Fig. 13] Fig. 13 shows the results of the culture tests performed with using a recombinant 4-1BB ligand instead of anti-4-lBB antibody (change in cell amplification rate). [Fig. 14] Fig. 14 shows the results of flow cytometry performed for each cell surface marker. [Fig. 15] Fig. 15 shows the results of flow cytometry performed for each cell surface marker. Mode for Carrying out the Invention

[0016] [Method for producing cell population] <Step a> As one aspect of the present invention, there is provided a method for producing a cell population containing CD3-negative and CD56-positive cells, comprising the following step: a) the 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.

[0017] <Cell population> In the present invention, the term cell population refers to a group consisting of a plurality of cells, for example, 1 x 105 cells or more. The cell population provided by the present invention can be prepared at various cell densities. For example, it can be prepared at a density of 1 x 105 cells / mL or higher.

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

[0019] The method for producing a cell population of the present invention can use a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells. Such a cell population containing mononuclear cells may contain various lymphocytes such as T cells, B cells, NK cells, monocytes, and dendritic cells. The cell population containing mononuclear cells may be peripheral blood mononuclear cells obtained by isolating mononuclear cells from blood cells collected from a donor. Blood cells can be collected from a donor by using methods known to those skilled in the art, for example, by collecting them from the donor's peripheral blood by the apheresis method. Blood cells may also be collected from donor's umbilical cord blood, bone marrow, lymph nodes, etc. Mononuclear cells can be separated from blood cells using such a method as density gradient centrifugation. Blood cells may also be obtained by differentiating embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) and used. Methods for differentiating into blood cells may be methods known to those skilled in the art, and for example, the methods described in to H. Melichar et al. Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines. PLoS One. 2011;6(5):el9854. doi:10.1371 / journal.pone.0019854 etc. can be referred to. The removal of CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells may be performed according to methods known to those skilled in the art, for example, by isolating CD3-positive cells or CD34-positive cells using magnetic beads conjugated with antibodies against CD3 or CD34.

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

[0021] The cell population containing mononuclear cells may be cryopreserved and thawed upon use. Cryopreservation may be performed by, for example, suspending the cell population containing mononuclear cells at an appropriate cell density in a medium suitable for cryopreservation, for example, a complete medium containing a cryoprotectant such as dimethyl sulfoxide (DMSO), placing the suspension in a cryovial or other cryopreservation containers, and storing it at -80°C or lower. Thawing can be performed by, for example, transferring the cry opreservation container containing the cell population containing mononuclear cells to a 37°C water bath and gently swirling it.

[0022] <Medium> The method for producing a cell population of the present invention comprises a) the 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. The medium may further contain additives, reagents, etc. suitable for culture. Hereinafter, the terms "medium used in the method for producing a cell population of the present invention", "medium used in the present invention", "medium" not specified as "the medium used in the step a)", "the medium used in the step b)" or the like shall refer to a medium used in one or more steps selected from the steps a), b), and c).

[0023] In this specification, concentrations of cytokines, polypeptides, additives, etc. in the medium described hereafter may be those in the medium at the start of each step, e.g., the step a), b), or c), and do not define concentrations throughout the entire duration of each step.

[0024] (IL-2, IL-21, and 4-IBB agonist) The medium used in the method for producing a cell population of the present invention may contain cytokines, polypeptides, etc., suitable for amplifying the cell population. Suitable cytokines include, for example, one or more types of interleukins (IL), preferably IL-2 and IL-21. Suitable polypeptides are preferably 4-IBB agonists. 4-1BB belongs to the tumor necrosis factor receptor superfamily (TNFRSF) and is a transmembrane receptor primarily expressed on cell surfaces of lymphocytes. 4-IBB is also sometimes referred to as CD137, TNFRSF9, etc. In the present invention, for example, the term 4-1BB agonist means a substance that can bind to 4-1BB to promote its functions. Functions of 4-1BB include, for example, enhancing the production of IL-8 from human peripheral blood mononuclear cells and the production of interferon (IFN)-y from human T lymphocytes. Enhancement of the production of IL-8 and IFN-y by 4-1BB can be measured by methods known to those skilled in the art. The 4-1BB agonist may be, for example, one that can bind to 4-IBB and promote the production of IL-8 or IFN-y. Examples of the 4-1BB agonist include, for example, anti-4-lBB antibodies, 4-1BB ligands (4-1BBL), etc.

[0025] In this specification, the term anti-4-IBB antibody refers to an antibody that can recognize one or more specific epitopes on 4-1BB, and bind thereto to multimerize 4-1BB. Examples thereof include urelumab, utomilumab, and antibodies capable of recognizing the same epitopes as those that are recognized by the foregoing antibodies.

[0026] In this specification, the term "4-1BB ligand (4-1BBL) "refers to a polypeptide or protein capable of binding to 4-IBB (excluding antibody). 4-1 BBL may be one synthesized by methods known to those skilled in the art or may be one artificially produced by genetic recombination techniques. 4-1BBL artificially produced by genetic recombination techniques is referred to as recombinant 4-1BBL. In one embodiment, 4-1BBL may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 shown below, a polypeptide containing the extracellular domain of 4-1 BBL (SEQ ID NO: 3), or a polypeptide consisting of an amino acid sequence showing an identity of, at least, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher to the sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and having an activity of promoting proliferation of CD3-negative and CD56-positive cells. The term identity of amino acid sequences refers to an identity between amino acid sequences of polypeptides in question, which is expressed as percentage (%) of amino acid residues that match between the amino acid sequences optimally aligned. Identity of amino acid sequences can be calculated by using homology search programs known to those skilled in the art, such as BLAST or FASTA. The term polypeptide having an activity of promoting proliferation of CD3-negative and CD56-positive cells refers to a polypeptide that can amplify the number of cells in a cell population containing CD3-negative and CD56-positive cells twice or more, preferably three times or more, when used in the cell culture method described in the section of Examples of this specification,.

[0027] (SEQ ID NO: 1) MREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQL TQGATVLGLFRVTPEIPAGLPSPRSE

[0028] (SEQ ID NO: 2) MDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLT VDKSRWQQGNVF SC S VM HEALHNHYTQKSLSLSPGKIEGRREGPELSPDDPAGLLDLRQGMFAQLVAQNVL LIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0029] (SEQIDNO: 3) ALACYSPROTRPALAVALSERGLYALAARGALASERPROGLYSERALAALASER PROARGLEUARGGLUGLYPROGLULEUSERPROASPASPPROALAGLYLEULEU ASPLEUARGGLNGLYMETPHEALAGLNLEUVALALAGLNASNVALLEULEUILE ASPGLYPROLEUSERTRPTYRSERASPPROGLYLEUALAGLYVALSERLEUTHRG LYGLYLEUSERTYRLYSGLUASPTHRLYSGLULEUVALVALALALYSALAGLYVA LTYRTYRVALPHEPHEGLNLEUGLULEUARGARGVALVALALAGLYGLUGLYSE RGLYSERVALSERLEUALALEUHISLEUGLNPROLEUARGSERALAALAGLYAL AALAALALEUALALEUTHRVALASPLEUPROPROALASERSERGLUALAARGA SNSERALAPHEGLYPHEGLNGLYARGLEULEUHISLEUSERALAGLYGLNARGL EUGLYVALHISLEUHISTHRGLUALAARGALAARGHISALArRPGLNLEUTHRG LNGLYALATHRVALLEUGLYLEUPHEARGVALTHRPROGLUILEPROALAGLYLE UPROSERPROARGSERGLU

[0030] 4-1BBL may be either a monomer or a multimer. The term multimer refers to an assembly of multiple subunits associated through intermolecular interactions, non-covalent bonds, covalent bonds, or the like. In one embodiment, the 4-1BB agonist used in the present invention is selected from the group consisting of urelumab, utomilumab, and 4-IBB ligands. In a preferred embodiment of the production method of the present invention, the 4-1BB agonist comprises one or more selected from urelumab and polypeptides consisting of an amino acid sequence having an identity of 90% or higher to the sequence of SEQ ID NO: 1 and exhibiting an activity of promoting proliferation of CD3-negative and CD56-positive cells. It is preferred that the cytokines, polypeptides, etc., to be used have human amino acid sequences thereof.

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

[0032] The concentration of IL-2 in the medium used in the present invention is not particularly limited, and it can be 50 lU / mL or higher, and is preferably 100 lU / mL or higher, more preferably 150 lU / mL or higher, further preferably 200 lU / mL or higher, particularly preferably 250 lU / mL or higher, most preferably 281 lU / mL or higher. The concentration of IL-2 in the medium used in the step a) may be, for example, 3,000 lU / mL or lower, 2,810 lU / mL or lower, 2,500 lU / mL or lower, 2,000 lU / mL or lower, 1,500 lU / mL or lower, 1,000 lU / mL or lower, 500 lU / mL or lower, or 300 lU / mL or lower. The concentration range of IL-2 may also be expressed by combining the maximum and minimum concentrations of IL-2 described above.

[0033] The concentration of IL-21 in the culture medium used in the step a) of the production method of the present invention is not particularly limited, and it can be 0.01 ng / mL or higher, and is preferably 0.05 ng / mL or higher, more preferably 0.1 ng / mL or higher, further preferably 0.5 ng / mL or higher, particularly preferably 1 ng / mL or higher. The concentration of IL-21 in the medium used in the step a) can be, for example, 1000 ng / mL or lower, may be 500 ng / mL or lower, and is preferably 100 ng / mL or lower, more preferably 50 ng / mL or lower, further preferably 10 ng / mL or lower, and it may be 9 ng / mL or lower, 8 ng / mL or lower, 7 ng / mL or lower, 6 ng / mL or lower, 5 ng / mL or lower, 4 ng / mL or lower, 3 ng / mL or lower, or 2 ng / mL or lower. The concentration range of IL-21 may also be expressed by combining the maximum and minimum concentrations of IL-21 described above.

[0034] The concentration of the 4-1BB agonist in the medium used in the step a) of the production method of the present invention is not particularly limited, and it can be 0.01 pg / mL or higher, and is preferably 0.05 pg / mL or higher, more preferably 0.1 pg / mL or higher, further preferably 0.5 pg / mL or higher, particularly preferably 1 pg / mL or higher. Further, the concentration of the 4-IBB agonist in the medium used in the step a) is not particularly limited, and it can be, for example, 1000 pg / mL or lower, and is preferably 500 pg / mL or lower, more preferably 100 pg / mL or lower, further preferably 50 pg / mL or lower. The concentration range of the 4-1 BB agonist may also be expressed by combining the maximum and minimum concentrations of the 4-IBB agonist described above.

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

[0036] When 4-1BBL is used as the 4-1BB agonist, the concentration of 4-1BBL in the medium used in step a) of the production method of the present invention can be 0.1 pg / mL or higher, and is preferably 0.5 pg / mL or higher, more preferably 1 pg / mL or higher, further preferably 2 pg / mL or higher, 3 pg / mL or higher, 4 pg / mL or higher, or 5 pg / mL or higher, particularly preferably 10 pg / ml or higher. The concentration of 4-1BBL in the medium used in the step a) can be, for example, 1000 pg / mL or lower, and is preferably 500 pg / mL or lower, more preferably 100 pg / mL or lower, further preferably 50 pg / mL or lower.

[0037] In the medium used in the present invention, IL-21 and 4-IBB agonist are humoral factors. The term humoral means that the substances of interest such as proteins and cytokines are not immobilized, and can be dissolved in the medium. The humoral factors refer to such soluble proteins and so forth. Contrasting concepts include, for example, such a state that the target protein etc. are immobilized on beads as described in Non-patent document 4, or such a state that the target protein etc. are expressed on the cell membranes of feeder cells, obtained by transforming the feeder cells, as described in Non-patent document 5. The medium containing humoral factors is preferably a liquid.

[0038] (Medium) The medium (culture medium) used in the present invention is not particularly limited. Examples include KBM501 medium (KOHJIN BIO), KBM502 medium (KOHJIN BIO), NTI medium (FUKOKU), Cosmedium 008 (Cosmo Bio), FKCM101 medium (FUKOKU), CellGro SCGM medium (CellGenix, Iwai Chemical), X-VIVO15 medium (Lonza, Takara Bio), Gibco (registered trademark) CTS (registered trademark) AIM V (registered trademark) Medium (Thermo Fisher Scientific, serum-free medium of known composition for growth and manipulation of T cells and dendritic cells), CTS OpTimizer T Cell Expansion Basal Medium (Thermo Fisher Scientific, for growth and proliferation of human T lymphocytes), IMDM, MEM, DMEM, and R.PM1-1640. For the present invention, the expression that culture of cells (cells are cultured) means to maintain cells in a medium or a similar solution for a certain period of time for any purpose selected from the group consisting of maintaining cell viability, amplifying cells, and activating cells, unless especially stated. To carry out a treatment at a specific temperature for a certain period of time may be sometimes referred to as incubation (to incubate).

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

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

[0041] Culture vessels used for the culture include, but are not limited to, commercially available dishes, flasks, plates, and multi-well plates. In addition to culture vessels, culture may also be performed by using various culture materials such as supports. Culture conditions are not particularly limited, so long as the culture effect is not impaired, but culture conditions of 37°C, 5% CO2, and saturated water vapor atmosphere are generally used.

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

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

[0044] The medium may further contain appropriate proteins, cytokines, antibodies, compounds, or other ingredients on condition that the solution of the problems according to the present invention is not inhibited. Cytokines may be, in addition to IL-2 and IL-21 mentioned above, 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 should preferably have human amino acid sequences thereof, and be produced by recombinant DNA technique for safety reasons.

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

[0046] The medium used in the present invention may contain one or more types of immunosuppressants. If the medium contains an immunosuppressant, proliferation of CD3-positive cells can be suppressed. The immunosuppressant may be, for example, a calcineurin inhibitor such as tacrolimus or cyclosporine, cytokine inhibitor such as basiliximab, etc., 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, and is preferably 0.1 to 50 ng / mL, more preferably 1 to 10 ng / mL. The concentration of basiliximab in the medium may be, for example, 0.005 to 50 pg / mL, and is preferably 0.05 to 30 pg / mL, more preferably 0.5 to 10 pg / mL. Tacrolimus may be obtained as, for example, Prograf Injection available from Astellas Pharma. Basiliximab may be obtained as, for example, Simulect IV Injection available from Novartis Pharma.

[0047] In a preferred embodiment, the culture method of the present invention does not use feeder cells. In this specification, the term feeder cells refers to cells other than the target cells (cells obtained by the present invention), which play an auxiliary role, such as producing cytokines that act as growth factors, to establish the culture conditions for the target cells. Feeder cells refer to those commonly used in the technical field of the present invention, such as cells of transgenic animal cell lines, leukocyte cells treated with various cytokines etc., T cells, B cells, monocytes, antigen-presenting cells, macrophages, etc. Specific examples of feeder cells include such genetically engineered antigen-presenting cells that express membrane-bound IL-21 as described in Non-patent document 5. Feeder cells may be cells whose proliferative ability has been largely lost through treatments with gamma irradiation or antibiotics, or the like, but they are not limited to cells with reduced or lost proliferative ability.

[0048] (Culture period) The step a) may comprise culturing a cell population obtained by removing CD3-positive cells and CD34-positive cells from a cell population containing mononuclear cells for a predetermined period. The predetermined period is 1 day or longer, and may be or 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, 11 days or longer, 12 days or longer, 13 days or longer, 14 days or longer, or a further longer period, and may be 30 days or shorter, 25 days or shorter, 20 days or shorter, 15 days or shorter, or a further shorter period, and it is not particularly limited. The culture period in the step a) may be, for example, 1 to 20 days, preferably 3 to 18 days, and may be 4 to 16 days, 5 to 15 days, or 6 to 14 days. During this period, the same medium may be replenished, or the medium may be replaced (in this case, the step a) can be considered to be continued as long as the medium meets the requirements). By adjusting the cell density at the time of seeding, maintenance (such as medium replenishment or replacement) during the culture period may be avoided.

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

[0050] In this specification, the term subculturing means transferring the cell population to a fresh medium, and the method is not particularly limited. Transferring the cell population to a fresh medium may be, for example, taking a portion of the medium containing dispersed cells and adding it to fresh medium. Although a portion of the medium itself used in the culture previous to the subculture may sometimes be added to the fresh medium at the time of the subculture, in this specification, the composition of the medium used in the culture previous to the subculture may be disregarded in the composition of the fresh medium into which the cell population is sub cultured.

[0051] The medium used in the step b) of the method for producing a cell population of the present invention contains IL-2. The medium used in the step b) may further contain a 4-1BB agonist and may further contain IL-21. For specific descriptions of IL-2, 4-IBB agonist, and IL-21, reference may be made to the foregoing descriptions concerning them. In a preferred embodiment, the medium used in the step b) of the method for producing a cell population of the present invention contains IL-2 and a 4-1BB agonist, and does not contain IL-21.

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

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

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

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

[0056] The step b) may comprise culturing the cell population cultured in the step a) for a predetermined period. The predetermined period is 1 day or longer, also may be 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, 11 days or longer, 12 days or longer, 13 days or longer, 14 days or longer, 15 days or longer, or a further longer period, and may be 30 days or shorter, 25 days or shorter, 20 days or shorter, or a further shorter period, and it is not particularly limited. The culture period in the step b) may be, for example, 1 or more days, preferably 2 to 21 days, more preferably 3 to 19 days, further preferably 4 to 17 days, particularly preferably 8 to 15 days. During this period, the same medium may be replenished, or the medium may be replaced (also in this case, the step b) is considered to be continued as long as the medium meets the requirements).

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

[0058] The medium used in the step c) of the method for producing a cell population of the present invention contains IL-2. The medium used in the step c) may further contain a 4-1BB agonist and may further contain IL-21. For specific descriptions of IL-2, 4-1BB agonist, and IL-21, reference may be made to the foregoing descriptions concerning them. In one preferred embodiment, the medium used in the step c) of the method of the present invention contains IL-2 and does not contain IL-21 and 4-IBB agonist. In one more preferred embodiment, the medium used in the step c) of the method of the present invention contains IL-2, IL-21, and a 4-1BB agonist.

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

[0060] When the medium used in the step c) contains a 4-1BB agonist, the concentration of the 4-IBB agonist in the medium can be, for example, 0.01 pg / mL or higher, 0.1 pg / mL or higher, or 1 pg / mL or higher, and 100 pg / mL or lower, 10 pg / mL or lower, or 5 pg / mL or lower.

[0061] When an anti-4-lBB antibody is used as the 4-1BB agonist, the concentration of the anti-4-lBB antibody in the medium used in the step c) can be 0.01 pg / mL or higher, and it is preferably 0.05 pg / mL or higher, more preferably 0.1 pg / mL or higher, further preferably 0.5 pg / mL or higher, particularly preferably 1 pg / mL or higher, and may be 100 pg / mL or lower, 10 pg / mL or lower, 5 pg / mL or lower, 4 pg / mL or lower, 3 pg / mL or lower, or 2 pg / mL or lower.

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

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

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

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

[0066] [Cell population obtainable by the production method of the present invention] The cell population obtained by the production method of the present invention contains cells that are at least CD3-negative and CD56-positive, and preferably have one or more cell surface marker phenotypes selected from the group consisting of being CD3-negative, CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1 (integrin al)-positive, and ITGA3 (integrin a3)-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, ITGAl-positive, and ITGA3-positive. The CCR and CXCR used as cell surface marker are types of chemokine receptors, and ITG (integrin) is a type of cell adhesion molecules.

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

[0068] Further, in the present invention, in such a cell population containing cells that are CD3-negative and CD56-positive, and CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGAl-positive, or ITGA3-positive, the proportion of cells exhibiting such cell surface marker phenotypes within the cell population may be, for example, 10% or higher, 20% or higher, 30% or higher, 40% or higher, or 50% or higher, is preferably 60% or higher, more preferably 70% or higher, further preferably 80% or higher, and may be particularly preferably 90% or higher. A cell population in which the proportion of cells that are CD3-negative and CD56-positive, and also CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, or ITGA3-positive is lower than any of the aforementioned proportions is distinguished from the cell population obtained by the present invention containing cells that are CD3-negative and CD56-positive, and also 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, more preferably CD34-negative, CCR5-positive, CCR6-positive, CXCR3-positive, ITGA1-positive, and ITGA3-positive.

[0069] [Cytotoxic activity] Cells contained in the cell population obtained by the present invention can exhibit high cytotoxic activity. Cytotoxic activity can be evaluated by, for example, measuring an ability of cells of interest (effector cells (E)) to lyse target cells (T), such as tumor cells. As the tumor cells, cells of tumor cell lines derived from, for example, leukemia, glioma, breast cancer, colorectal cancer, ovarian cancer, prostate cancer etc. can be used, but they are not limited to these. As the target cells, spheroids prepared from tumor cell lines can also be used. The ratio of effector cells to target cells (E:T) is not particularly limited, and may be, for example, 1:1,2:1,5:1, etc. Cytotoxic activity can be represented as the percentage (%) of target cells killed by the effector cells, and is calculated in accordance with the following equation.

[0070] Cytotoxicity activity rate (% Lysis) = (Target-cell death rate - Negative control cell death rate (natural cell death rate)) / (Positive control cell death rate (maximum cell death rate) - Negative control death rate) x 100

[0071] Cytotoxic activity can be measured by, for example, distinguishing effector cells from target cells and live cells from dead cells using reagents such as antibodies labeled with radioactive substances or fluorescent dyes, and quantifying these cells using such methods as flow cytometry to determine the respective dead cell rates used in the above equation. When the cell population of the present invention is used as effector cells, incubation time with target cells can be appropriately set depending on the type of target cells used. For example, when K562 cells, cells of a leukemia cell line, are used as the target cells, the incubation time can be set to be 2 hours.

[0072] Regarding the present invention, the term "high cytotoxic activity" means that cytotoxic activity measured after mixing effector cells with K562 cells as the target cells at an E:T ratio of 1 to 5:1 and co-culturing them for 2 hours is 50% or higher, unless especially specified. Cytotoxic activity is preferably 60% or higher, more preferably 70% or higher.

[0073] Further, when cytotoxic activity of the cell population obtained by the present invention as the effector cells is measured, it can also be determined that the desired cytotoxic activity can be obtained by detecting CD107a-positive cells within the effector cells. This is because CD 107a is present within NK cell granules and migrates to the NK cell membrane surfaces upon degranulation, and therefore being CD 107a-positive indirectly indicates that cytotoxic activity, i.e., attack on target cells by cells contained in the cell population obtained by the present invention, has been achieved. Detection of CD107a-positive cells can be performed by, for example, incubating target cells with a fluorescently labeled anti-CD107a antibody and then measuring luminescence. When the cell population obtained by the present invention is used as effector cells and cells of any tumor cell line as target cells at a cell number ratio (E:T) of 1 to 2:1, the proportion of CD 107a-positive cells in the cell population obtained by the present invention is at least 20% or higher, preferably 30% or higher, more preferably 40% or higher, further preferably 50% or higher. The tumor cell line may be, for example, a leukemia cell line, such as K562 cells, or the like.

[0074] [Antitumor effect] The cell population obtained by the present invention exhibits antitumor effects also when administered in vivo. Such in vivo antitumor effects can be evaluated by, for example, administering the cell population obtained by the present invention to a tumor-bearing model animal and measuring changes in tumor volume or tumor cell number. The preparation of tumor-bearing model animals and the measurement of changes in tumor volume or tumor cell number can be performed by using methods known to those skilled in the art, for example, a method comprising transplanting fluorescently labeled tumor cells into animals and observing them. As a specific example, there can be mentioned a method using an in vivo imaging system (IVIS), wherein luciferase-expressing SKOV cells (human ovarian cancer cell line) are transplanted into immunodeficient mice, luciferin is administered to the mice, and tumor luminescence is observed under anesthesia. In this method, the method for administering the cell population obtained by the present invention is not particularly limited. The administration may be performed once or multiple times, and the administration interval for multiple administrations can be appropriately set. The time of the administration can be any point within a period of, for example, 1 to 30 days after tumor transplantation. The administration dose can be, for example, 1.0 x 104 to 1.0 x 1010 cells / body, preferably 1.0 x 105 to 1.0 x 109 cells / body, more preferably 1.0 x 106 to 1.0 x 108 cells / body, particularly preferably 1.5 x 107 to 2.7 x 107 cells / body, per administration. The administration route is not particularly limited, and the administration may be intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, but intraperitoneal administration is preferably selected. IL-2 may be administered simultaneously to maintain the activity of the cell population obtained by the present invention in vivo. For this purpose, the dose of IL-2 can be 1,000 to 10,000 lU / body, preferably 5,000 lU / body. In one embodiment, administration of the cell population obtained by the present invention can reduce the number of tumor cells in a tumor-bearing model animal to one-tenth or less compared with that observed without the administration.

[0075] The cell population obtained by the present invention can be cryopreserved. Cryopreservation can be performed by suspending the cell population at an appropriate cell density in a medium suitable for cryopreservation, such as a complete medium containing cryoprotectant such as dimethyl sulfoxide (DMSO), placing the suspension in cryovials or other cryopreservation containers, and storing it at -80°C or lower. The appropriate cell density may be, for example, 1 x 103 to 1 x 1010 cells / mL, preferably 2 x 108 cells / 5 mL. The cryoprotectant may optionally contain inorganic salts, pH adjusters, etc.

[0076] Further, by thawing the cryopreserved cell population by 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 cry opreservation container containing the cell population to a water bath at 37°C and gently swirling it.

[0077] [Pharmaceutical composition] As one aspect of the present invention, a pharmaceutical composition containing a cell population obtained by the present invention is provided. The pharmaceutical composition may contain additives acceptable as pharmaceuticals. Examples of additives acceptable as pharmaceuticals include, for example, isotonic agents, pH adjusters, buffers, stabilizers, cryoprotectants, dissolution aids, soothing agents, antibiotics, etc.

[0078] The pharmaceutical composition is typically in the form of a suspension of the cell population obtained by the present invention in a solution. The solution for suspending the cell population obtained by the present invention is generally, for example, a cryoprotective solution containing DMSO, physiological saline, phosphate-buffered saline (PBS), culture medium, serum, or the like. The solution may contain pharmaceutically acceptable carriers for pharmaceuticals and quasi-drugs.

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

[0080] The pharmaceutical composition of the present invention may be administered to patients having an HLA genotype different from that of the cell population of the present invention.

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

[0082] The pharmaceutical composition of the present invention may be used in combination with antibody drugs. Specific examples of antibodies that can be used in combination with the pharmaceutical composition of the present invention include ibritumomab tiuxetan, iodine-131, catumaxomab, blinatumomab, muromonab-CD3, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, siltuximab, dinutuximab, obiltoxaximab, daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab, certolizumab pegol, mogamulizumab, pertuzumab, trastuzumab, obinutuzumab, vedolizumab, pembrolizumab, idarucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.

[0083] Antibodies that can be used in combination with the pharmaceutical composition of the present invention are preferably those with high affinity for CD 16. Further, in the pharmaceutical composition of the present invention, at least a part of the antibodies may be bound to cells contained in the cell population of the present invention.

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

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

[0086] The following examples are provided to further specifically explain the present invention, but the present invention should not be interpreted as limited thereby.

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

[0088] A) Example 1 (Donor Mix A) NK cell culture tests were performed under various conditions differing in culture medium and additives.

[0089] -Day 0 NK cells (CD37CD56+, >80%) were isolated from thawed PBMCs using EasySep Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055). The isolated NK cells were prepared at a density of 2 x 105 cells / mL in the culture media of the following 18 groups, the suspensions were seeded in a volume of 0.65 mL each per well on 12-well plates, and culture was started.

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

[0091] - Day 7 For all the groups, the cell suspensions in the wells were thoroughly mixed by pipetting, and cell numbers were counted. For (8) and (14), 0.5 mL each per well of the cell suspensions were taken on 6-well plates and 1 mL each per well of the following media were added.

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

[0093] -Day 11 For all the groups, the cell suspensions in the wells were thoroughly mixed by pipetting, and cell numbers were counted.

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

[0095] [Table 1] 0 7 11 RPMI CTS+ 1.30E+05 9.75.E+03 3.25.E+03 UG+ 1.30E+05 3.80.E+05 4.39.E+05 FBS+ 1.30E+05 6.50.E+03 1.E+01 CTS 1.30E+05 6.50.E+03 1.E+01 UG 1.30E+05 2.60. E+04 2.60.E+04 FBS 1.30E+05 1.30.E+04 1.E+01 KBM CTS+ 1.30E+05 1.63.E+05 8..78.E+04 UG+ 1.30E+05 4.10.E+05 1.06.E+06 FBS+ 1.30E+05 2.02.E+05 3.02.E+05 CTS 1.30E+05 1.30.E+04 2.93.E+04 UG 1.30E+05 2.93. E+04 5.53.E+04 FBS 1.30E+05 0.00.E+00 6..18..E+04 NTI CTS+ 1.30E+05 4.23. E+04 9..75.E+04 UG+ 1.30E+05 8.09.E+05 4.52.E+06 FBS+ 1.30E+05 1.59.E+05 4.29.E+05 CTS 1.30E+05 5.20.E+04 5.53.E+04 UG 1.30E+05 6.83.E+04 3.02.E+05 FBS 1.30E+05 6.50.E+04 9.75.E+04

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

[0097] B) Example 2 (Donor Mix B, Single Donor A) Culture tests were performed by varying the conditions of the cells used as culture raw materials.

[0098] - Day 0 Cryopreserved PBMCs were thawed by a predetermined method, and cell suspensions prepared for the following 4 groups were seeded in a volume of 0.65 mL each per well on 12-well plates, and culture was started. (1): PBMC (CD3‘, CD34) 4 x 105 cells / mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (2): PBMC (CD3‘, CD34) 4 x 105 cells / mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3): PBMC (CD3+, CD34 ) 4 x 105 cells / mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4): PBMC (CD3+, CD34+) 4 x 105 cells / mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0099] -Day 6 The cell suspensions in the wells were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions of the respective groups were seeded in a volume of 1 mL each per well on 12-well plates under the following conditions for subculture, and cultured. (1)-1: Cell suspension of (1) 4 x 105 cells / 1 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (2)-1: Cell suspension of (2) 4 x 105 cells / 1 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-1: Cell suspension of (3) 4 x 105 cells / 1 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4)-1: Cell suspension of (4) 4 x 105 cells / 1 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0100] - Day 7 The cell suspensions in the wells of the respective groups were thoroughly mixed by pipetting, seeded in a volume of 2.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (1)-2: Cell suspension of (1)-1 1 mL, NTI medium*1 supplemented with urelumab [1 pg / mL] 1.0 mL (2)-2: Cell suspension of (2)-1 1 mL, RPMI medium*2 supplemented with urelumab [1 pg / mL] 1.0 mL (3)-2: Cell suspension of (3)-1 1 mL, NTI medium*1 supplemented with urelumab [1 pg / mL] 1.0 mL (4)-2: Cell suspension of (4)-1 1 mL, RPMI medium*2 supplemented with urelumab [1 pg / mL] 1.0 mL

[0101] - Day 10 The cell suspensions in the wells were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions of the respective groups were seeded in a volume of 3.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (1)-3: Cell suspension of (1)-2 5 x 103 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (2)-3: Cell suspension of (2)-2 5 x 105 cells / 3 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-3: Cell suspension of (3)-2 5 x 105 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4)-3: Cell suspension of (4)-2 5 x 103 cells / 3 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0102] -Day 12 The cell suspensions of (1)-3, (2)-3, (3)-3, and (4)-3 in the wells were thoroughly mixed by pipetting, and the cell numbers were counted.

[0103] - Day 14 The cell suspensions in the wells were thoroughly mixed by pipetting, and the cell numbers were counted. For the groups (1), (3), and (4), the cell suspensions were seeded in a volume of 3.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (1)-4: Cell suspension of (1)-3 5 x 103 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-4: Cell suspension of (3)-3 5 x 105 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4)-4: Cell suspension of (4)-3 5 x 105 cells / 3 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0104] -Day 17 The cell suspensions in the wells were thoroughly mixed by pipetting, and the cell numbers were counted. The cell suspensions of the groups (1), (3), and (4) were seeded in a volume of 3.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (1)-5: Cell suspension of (1)-4 5 x 10' cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-5: Cell suspension of (3)-4 5 x 105 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4)-5: Cell suspension of (4)-4 5 x 105 cells / 3 mL, RPMI medium*2, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0105] - Day 19 The cell suspensions in the wells of (1)-5, (3)-5, and (4)-5 were thoroughly mixed by pipetting, and cell numbers were counted.

[0106] -Day 21 The cell suspensions in the wells of (1)-5, (3)-5, and (4)-5 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspension of (3)-5 was seeded in a volume of 3.0 mL on 6-well plates under the following conditions for subculture, and cultured. (3)-5-1: Cell suspension of (3)-5 5 x 105 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-5-2: Cell suspension of (3)-5 1.5 x 106 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0107] - Day 24 The cell suspensions in the wells of (3)-5-1 and (3)-5-2 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions of (3)-5-1 and (3)-5-2 were seeded in a volume of 3.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (3)-5-1: Cell suspension of (3)-5-1 5 x 105 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-5-2-a: Cell suspension of (3)-5-2 5 x 10? cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3)-5-2-b: Cell suspension of (3)-5-2 1.5 x 106 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0108] - Day 28 The cell suspensions in the wells of (3)-5-1, (3)-5-2-a, and (3)-5-2-b in the wells were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspension of (3)-5-2-b was seeded in a volume of 3.0 mL on a 6-well plate under the following conditions for subculture, and cultured. (3)-5-2-b': Cell suspension of (3)-5-2-b 1.5 x 106 cells / 3 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0109] -Day 31 The cell suspension in the well of (3)-5-2-b' was thoroughly mixed by pipetting, and cell number was counted.

[0110] Fig. 2 shows the changes in cell proliferation rate. It was found that use of the NTI medium resulted in better cell amplification efficiency compared with use of the RPMI medium.

[0111] Fig. 3 shows the results of flow cytometry for each cell surface marker for (1), (3), and (4). The resulting cell populations contained many cells exhibiting phenotypes similar to those of the cells with high cytotoxic activity described in Patent document 1 etc., but CD3-positive cells were also amplified.

[0112] C) Example 3 (Donor Mix B) Culture tests were performed with different concentrations of anti-4-IBB antibody and IL-21.

[0113] -Day 0 Cryopreserved PBMCs were thawed by a predetermined method. Cell suspensions prepared for the following 12 groups were seeded in a volume of 0.65 mL each per well on 12-well plates, and culture was started. (1): PBMC (CD3; CD34) 4 x 105 cells / mL, KBM502 medium*3 (2): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, IL-21 [1 ng / mL] (3): PBMC (CD3\ CD34 ) 4 x 105 cells / mL, KBM502 medium*3, IL-21 [10 ng / mL] (4): PBMC (CD3; CD34 ) 4 x 105 cells / mL, KBM502 medium*3, IL-21 [100 ng / mL] (5): PBMC (CD3; CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [1 pg / mL] (6): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [1 pg / mL], IL-21 [1 ng / mL] (7): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [1 pg / mL], IL-21 [10 ng / mL] (8): PBMC (CD3; CD34 ) 4 x 105 cells / mL, KBM502 medium*3, urelumab [1 pg / mL], IL-21 [100 ng / mL] (9): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [10 pg / mL], (10): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [10 pg / mL], IL-21 [1 ng / mL] (11): PBMC (CD3‘, CD34) 4 x 105 cells / mL, KBM502 medium*3, urelumab [lOpg / mL], IL-21 [10 ng / mL] (12): PBMC (CD3; CD34 ) 4 x 105 cells / mL, KBM502 medium*3, urelumab [lOpg / mL], IL-21 [100 ng / mL]

[0114] Additionally, a cell suspension prepared at the following density was seeded in a volume of 13.5 mL into a T75 flask, and culture was started. (13) PBMC (CD3; CD34) 5 x 105 cells / mL, KBM502 medium*3

[0115] - Day 6 The cell suspensions in the wells of (6), (7), and (8) were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 0.65 mL each per well on 12-well plates under the following conditions for subculture, and cultured. To the wells of (6), (7), and (8), 0.6 mL each per well of fresh KBM502 medium*3 was further added. (6)-1: Cell suspension of (6) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL] (6)-2: Cell suspension of (6) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL], IL-21 [1 ng / mL] (7)-1: Cell suspension of (7) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL] (7)-2: Cell suspension of (7) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL], IL-21 [10 ng / mL] (8)-1: Cell suspension of (8) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL] (8)-2: Cell suspension of (8) 0.25 mL, KBM502 medium*3 0.4 mL, urelumab [1 pg / mL], IL-21 [100 ng / mL]

[0116] -Day 7 The cell suspensions in the wells of (1) to (12) were thoroughly mixed by pipetting, and cell numbers were counted. Further, the cell suspensions in the wells of (10), (11), and (12) were seeded in a volume of 0.65 mL each per well on 12-wells plate under the following conditions for subculture, and cultured. (10)-1: Cell suspension of (10) 0.1 mL, KBM502 medium*3 0.55 mL (10)-2: Cell suspension of (10) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL] (10)-3: Cell suspension of (10) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [1 ng / mL] (10)-4: Cell suspension of (10) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [10 ng / mL] (10)-5: Cell suspension of (10) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [100 ng / mL]

[0117] (11)-1: Cell suspension of (11) 0.1 mL, KBM502 medium*3 0.55 mL (11)-2: Cell suspension of (11) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL] (11)-3: Cell suspension of (11) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [1 ng / mL] (11)-4: Cell suspension of (11) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [10 ng / mL] (11)-5: Cell suspension of (11) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [100 ng / mL]

[0118] (12)-1: Cell suspension of (12) 0.1 mL, KBM502 medium*3 0.55 mL (12)-2: Cell suspension of (12) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL] (12)-3: Cell suspension of (12) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [1 ng / mL] (12)-4: Cell suspension of (12) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [10 ng / mL] (12)-5: Cell suspension of (12) 0.1 mL, KBM502 medium*3 0.55 mL, urelumab [1 pg / mL], IL-21 [100 ng / mL]

[0119] - Day 8 The cell suspensions in the wells of (6)-1, (6)-2, (7)-1, (7)-2, (8)-1, and (8)-2 subcultured on day 6 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 1.8 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (6)-1-1: Cell suspension of (6)-1 0.6 mL, KBM502 medium*3 1.2 mL (6)-2-1: Cell suspension of (6)-2 0.6 mL, KBM502 medium*3 1.2 mL (7)-1-1: Cell suspension of (7)-1 0.6 mL, KBM502 medium*3 1.2 mL (7)-2-1: Cell suspension of (7)-2 0.6 mL, KBM502 medium*3 1.2 mL (8)-1-1: Cell suspension of (8)-1 0.6 mL, KBM502 medium*3 1.2 mL (8)-2-1: Cell suspension of (8)-2 0.6 mL, KBM502 medium*3 1.2 mL

[0120] - Day 10 The cell suspensions in the wells of (6)-1-1, (6)-2-1, (7)-1-1, (7)-2-1, (8)-1-1, and (8)-2-1 subcultured on day 8 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 10 mL each per well on 10-cm dishes under the following conditions for subculture, and cultured. (6)-1-2: Cell suspension of (6)-1-1 1.5 mL, KBM502 medium*3 8.5 mL (6)-2-2: Cell suspension of (6)-2-1 1.5 mL, KBM502 medium*3 8.5 mL (7)-1-2: Cell suspension of (7)-1-1 1.5 mL, KBM502 medium*3 8.5 mL (7)-2-2: Cell suspension of (7)-2-1 1.5 mL, KBM502 medium*3 8.5 mL (8)-1-2: Cell suspension of (8)-1-1 1.5 mL, KBM502 medium*3 8.5 mL (8)-2-2: Cell suspension of (8)-2-1 1.5 mL, KBM502 medium*3 8.5 mL

[0121] The cell suspensions in the wells of (1), (2), (3), (4), (5), and (9) seeded on day 0 were thoroughly mixed by pipetting, and cell numbers were counted. To the well of (1), 0.7 mL of fresh KBM502 medium*3 was further added.

[0122] The cell suspensions in the wells 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 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 2.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (10)-1-1: Cell suspension of (10)-1 0.5 mL, KBM502 medium*3 1.5 mL (11)-1-1: Cell suspension of (11)-1 0.5 mL, KBM502 medium*3 1.5 mL (12)-1-1: Cell suspension of (12)-1 0.5 mL, KBM502 medium*3 1.5 mL

[0123] (10)-2-1: Cell suspension of (10)-2 0.5 mL, KBM502 medium*3 1.5 mL (11)-2-1: Cell suspension of (11)-2 0.5 mL, KBM502 medium*3 1.5 mL (12)-2-1: Cell suspension of (12)-2 0.5 mL, KBM502 medium*3 1.5 mL

[0124] (10)-3-1: Cell suspension of (10)-3 0.5 mL, KBM502 medium*3 1.5 mL (11)-4-1: Cell suspension of (11)-4 0.5 mL, KBM502 medium*3 1.5 mL (12)-5-1: Cell suspension of (12)-5 0.5 mL, KBM502 medium*3 1.5 mL

[0125] - Day 13 The cell suspensions in the wells 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 subcultured on day 10 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 2.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (10)-1-2: Cell suspension of (10)-1-1 5 x 103 cells / 2 mL, KBM502 medium*3 supplemented with urelumab [1 pg / mL]*3 (10)-2-2: Cell suspension of (10)-2-1 5 x 103 cells / 2 mL, KBM502 medium*3 supplemented with urelumab [1 pg / mL]*3 (10)-3-2: Cell suspension of (10)-3-1 5 x 105 cells / 2 mL, KBM502 medium*3 supplemented with urelumab [1 pg / mL]*3 (11)-2-2: Cell suspension of (11)-2-1 5 x 105 cells / 2 mL, KBM502 medium*3 supplemented with urelumab [1 pg / mL]*3

[0126] (10)-1-3: Cell suspension of (10)-1-1 5 x 103 cells / 2 mL, KBM502 medium*3 (10)-2-3: Cell suspension of (10)-2-1 5 x 105 cells / 2 mL, KBM502 medium*3 (10)-3-3: Cell suspension of (10)-3-1 5 x 105 cells / 2 mL, KBM502 medium*3 (11)-2-3: Cell suspension of (11)-2-1 5 x 105 cells / 2 mL, KBM502 medium*3

[0127] - Day 14 The cell suspension of (13) in the T75 flask from in which culture was started on day 0 was thoroughly mixed by pipetting, and cell number was counted.

[0128] The cell suspensions in the 10-cm dishes of (6)-1-2, (6)-2-2, (7)-1-2, (7)-2-2, (8)-1-2, and (8)-2-2 subcultured on day 10 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 2.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (6)-a: Cell suspension of (6)-1-2 5 x 103 cells / 2 mL, KBM502 medium*3 (6)-b: Cell suspension of (6)-1-2 5 x 103 cells / 2 mL, KBM502 medium*3, urelumab [1 pg / mL] (6)-c: Cell suspension of (6)-1-2 5 x 105 cells / 2 mL, KBM502 medium*3, urelumab [1 pg / mL], IL-21 [1 ng / mL] (6)-d: Cell suspension of (6)-1-2 5 x 105 cells / 2 mL, KBM501 medium*4

[0129] -Day 15 The cell suspensions in the wells 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 subcultured on day 13 were thoroughly mixed by pipetting, and cell numbers were counted.

[0130] -Day 17 The cell suspensions in the wells 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 subcultured on day 13 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 2.0 mL each per well on 6-well plate under the following conditions for subculture, and cultured. (10)-1 -3': Cell suspension of (10)-1-3 1.0 mL, KBM502 medium*3 1.0 mL (11)-2-21: Cell suspension of (11)-2-2 1.0 mL, KBM502 medium*3 1.0 mL

[0131] The cell suspensions in the wells of (6)-a, (6)-b, (6)-c, and (6)-d subcultured on day 14 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions were seeded in a volume of 2.0 mL each per well on 6-well plates under the following conditions for subculture, and cultured. (6)-a': Cell suspension of (6)-a 1.0 mL, KBM502 medium*3 1.0 mL (6)-c': Cell suspension of (6)-c 1.0 mL, KBM502 medium*3 1.0 mL

[0132] - Day 20 The cell suspensions in the wells of (10)-1-2, (10)-2-2, (10)-3-2, (10)-2-3, (10)-3-3, and (11)-2-3 subcultured on day 13 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions in the wells of (6)-b and (6)-d subcultured on day 14 were thoroughly mixed by pipetting, and cell numbers were counted.

[0133] - Day 21 The cell suspensions in the wells of (10)-1 -3', (11)-2-2( (6)-a( and (6)-c' subcultured on day 17 were thoroughly mixed by pipetting, and cell numbers were counted.

[0134] The results are shown in Table 2 (fold change of overall cells), Fig. 4 (change in cell number), and Fig. 5 (fold change). In Table 2, the results of cell amplification were evaluated by classifying relative values of fold change in cell number of last count (on day 10 or thereafter) based on the value of day 0, which is taken as 1, into the following categories A to D. The results classified as C or higher indicate that efficient cell amplification has been obtained, and the object has been achieved. A: Fold change is 100 or higher. B: Fold change is 10 or higher. C: Fold change is 2 or higher. D: Fold change is lower than 2.

[0136] 0 6 7 8 10 13 14 15 17 20 21 Evaluation (1) 0.61 1,65 D (2) 1.00 q 7A c (3) 1.00 c (4) 1.00 3,66 c (5) 1)11.00 1 3.36 c ¢6) -a l|ii.oo|)| 1.62 1133.231 109.51 162.30 194.76 A (6) -b ; ,1.62) D|33.23| 109.51 140.40 157.70 A ¢6) -c 1.00 1.62 33.23 109.51 206.10 187.37 A (6) -d 1.00 1.62 33.23 109.51 ni.72 178.73 A (6) -2 ||11.00)U 1.62 21.30 B (7) 111100)11 2.62 24.99 oo 70 B (7) -2 1.00 2.62 21.98 - B (8) -1 1.00 2.54 20.42 B (8) -2 liji.ooll 2.54) ))D18.92| d8.22 B (9) liii.oo;l 3.10 C (10) -1-3 ■ llli.oo) ■1|D 5.2311 14.23 2n '0 27.66 33.20 B (10) -1-2 || l||i.oo'l 5.23 XU, XU 39.62 B (10) -2-1 ■ 1.00 5.73 16.23 gl: T 7 27.27 B (10) -2-2 |||1.00| 5.73 12 53 ■1 7 25.97 B (10) -3-1 Jlll.00) 6.24 10.62 12.53 19.49 23.40 B (10) -3-2 1.00 6.24 12.10 12.23 18.34 B (10) -4 1.00 3,13 C (10) -5 111)1.00)) 6.04 C (11) -1 lijiwl 8.69 C (ID -2-3 1.00 1.32 10.08 16 53 x w <U v 35.07 B (11) -2-2 1.00 4.32 13 50 19.95 22.85 B (11) -3 1.00 3,58 c (11) -4 1.00 3.00 o 77 c (11) -5 11111661 7 77 c (12) -1 1.00 4.50 5.-6 c (12) -2 llli.00) 4.78 5.66 c (12) -3 1111.001 5.69 c (12) -4 UflOOi 4.03 c (12) -5 1.00 1.46 2.35 c

[0135] [Table 2] The results of (2) to (4) showed that when cells were cultured with addition of IL-21 alone, the amplification efficiency was poor. The results of (5) and (9) showed that when cultured without IL-21, the initial proliferation was significantly poor. The results of (6) to (8) and (10) to (12) showed that when cultured with addition of IL-21 and urelumab, the initial proliferation was favorable.

[0137] Fig. 6 shows the results of flow cytometry performed for each cell surface marker in the cell populations of (6)-a, (6)-c, (10)-1-3, and (11)-2-2.

[0138] D) Example 4 (Donor Mix B) Cytotoxic activities of obtained cell populations were confirmed.

[0139] - Day 0 Cryopreserved PBMCs (CD3‘, CD34') were thawed by a predetermined method. A cell suspension prepared at 4 x 105 cells / mL in NTI medium*1 supplemented with IL-18 [100 ng / mL], urelumab [1 ug / mL], and IL-21 [1 ng / mL] was seeded in a volume of 1.5 mL per well on a 6-well plate, and culture was started. Further, a cell suspension prepared at 4 x 105 cells / mL in NTI medium*1 supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL] was seeded in a volume of 12.5 mL each into two T75 flasks, and culture was started.

[0140] - Day 7 The cell suspensions in the wells of the 6-well plate were thoroughly mixed by pipetting, and cell numbers were counted. A cell suspension was prepared at a density of 2 x 105 cells / mL in fresh NTI medium*1, IL-18 [100 ng / mL], urelumab [1 pg / mL], and IL-21 [1 ng / mL] were added to the cell suspension, and the cell suspension was seeded in a volume of 14.4 mL into T75 flask A for subculture, and cultured.

[0141] The cell suspensions in the two T75 flasks were thoroughly mixed by pipetting, and cell numbers were counted. A cell suspension was prepared at a density of 2 x 105 cells / mL in fresh NTI medium*1, urelumab [1 pg / mL] and IL-21 [1 ng / mL] were added to the cell suspension, and the cell suspension was seeded in a volume of 45 mL each into two T225 flasks ((1) and (2)), and 64 mL each into two T225 flasks ((3) and (4)) for subculture, and cultured.

[0142] -Day 10 The cell suspension in the T75 flask A was thoroughly mixed by pipetting, and cell number was counted. A cell suspension was prepared at a density of 2 x 105 cells / mL in fresh NTI medium*1, and seeded in a volume of 15 mL into T75 flask B for subculture, and cultured. After the collection of cells, 15 mL of NTI medium*1 was added to the T75 flask A for subculture, and cultured. The cell suspensions in the T225 flasks of (1) and (2) were thoroughly mixed by pipetting, and cell numbers were counted. Cell suspensions were prepared for the following two groups, and each seeded into two separate T225 flasks for subculture. (1)-1: The cell suspension of (1) was taken for 9 x 106 cells, and centrifuged at 500 g for 5 minutes, and then the cells were suspended in 45 mL of fresh NTI medium*1 (2 x 105 cells / mL). (1)-2: Cell suspension of (1) 9 x 106 cells / 45 mL, NTI medium*1.

[0143] The cell suspensions in the T225 flasks of (3) and (4) were thoroughly mixed by pipetting, and cell numbers were counted. After the collection of cells, 50 mL each of NTI medium*1 was added to the T225 flasks of (1), (2), (3), and (4) for subculture, and cultured.

[0144] - Day 14 The cell suspensions in the T225 flasks of (1)-1 and (1)-2 were thoroughly mixed by pipetting, and cell numbers were counted. Cell suspensions were prepared for the following two groups, seeded into two separate T225 flasks for subculture, and cultured. (1)-1-1: The cell suspension of (1)-1 was taken for 2.5 x 106 cells, and centrifuged at 500 g for 5 minutes, and then the cells were suspended in 50 mL of fresh NTI medium*1. (1)-2-1: Cell suspension of (1)-1 2.5 x 106 cells / 50 mL, NTI medium*1

[0145] The cell suspensions in the T75 flasks A and B were thoroughly mixed by pipetting, and cell numbers were counted. Cell suspensions were prepared for the following two groups, seeded on 6-well plates for subculture, and cultured. A-l: Cell suspension of A 1.0 x 106 cells / 2 mL, NTI medium*1 B-l: The cell suspension of A was taken for 1.0 x 106 cells, and centrifuged at 500 g for 5 minutes, and then the cells were suspended in fresh NTI medium*1 2 mL.

[0146] -Day 15 The cell suspensions in the T225 flasks of (1), (2), (3), and (4) were thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell numbers were counted. Cell suspensions were prepared at a density of 1.18 x 106 cells / mL in fresh NTI medium*1, seeded in a volume of 50 mL each into two T75 flasks ((5) and (6)) for subculture, and cultured.

[0147] - Day 16 The cell suspensions in one each of the T225 flasks of (1)-1-1 and (1)-2-1 were thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell numbers were counted.

[0148] -Day 17 The cell suspensions in one each of the T225 flasks of (1)-1-1 and (1)-2-1 were thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell numbers were counted. The cell suspensions in the wells of A-1 and B-1 were thoroughly mixed by pipetting, and cell numbers were counted.

[0149] [Cytotoxicity assay for tumor cells] For cytotoxicity assay, a group of the cells collected on day 17 and reacted with K562 cells, a group of K562 cells alone as a negative control, and a group of K562 cells treated with 10% formalin as a positive control were prepared.

[0150] «Cells collected on day 17» The cells collected on day 17 were prepared at a density of 1 x 106 cells / ml in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution).

[0151] <<K562 cells» K562 cells were suspended in RPMI 1640 medium (serum-free), stained by using PKH26 Red Fluorescent Cell Linker Kit, and then prepared at a density of 2 x 106 cells / mL in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillinstreptomycin mixed solution).

[0152] The cells collected on day 17 and K562 cells were added to wells of a 96-well plate (IWAKI, 4870-800SP) at a cell ratios of 1:1 and 2:1, mixed, and reacted at 37°C and 5% CO2 for 2 hours. After the reaction, the cell suspension was centrifuged at 500 g for 5 minutes, the supernatant was removed, then a 7-AAD solution diluted in PBS was added to suspend the cells, and the cell suspension was incubated at room temperature for 20 minutes. Measurement was performed by using a flow cytometer, and the results were analyzed with FlowJo software to calculate the cytotoxic activity rate (% Lysis).

[0153] Cytotoxic activity rate (% Lysis) = (K562 cell dead cell rate - Negative control dead cell rate) / (Positive control dead cell rate - Negative control dead cell rate) x 100

[0154] Cytotoxic activity rates calculated in accordance with the above equation is evaluated by classifying them into the following categories A to D. Results classified as C or higher are judged to indicate that high cytotoxic activity has been obtained, and the object has been achieved. A: 70% or higher B: 60% or higher C: 50% or higher D: Lower than 50%

[0155] The results are shown in Fig. 7. For both E (effector cells (collected cells)):T (target cells (K562 cells)) ratios of 1:1 and 2:1, the cytotoxic activity rate of the collected cell population was evaluated as A, indicating particularly high cytotoxic activity against tumor cells.

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

[0157] -Day 0 Cryopreserved PBMCs (CD3\ CD34 ) were thawed by a predetermined method. A cell suspension prepared at 4 x 105 cells / mL in NTI medium*1 supplemented with urelumab [1 pg / mL as final concentration] and IL-21 [1 ng / mL as final concentration] was seeded into two T75 flasks in a volume of 12.5 mL each, and culture was started.

[0158] -Day 6 The cell suspensions in the two T75 flasks were collected in a volume of 25 mL each, and cell numbers were counted. The cell suspensions were supplemented with 12.5 mL of fresh NTI medium*1, and then with urelumab [1 pg / mL] and IL-21 [1 ng / mL] for subculture, seeded into three T75 flasks in a volume of 12.5 mL each, and cultured.

[0159] - Day 8 The cell suspensions in the T75 flasks were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions in the flasks were transferred to T225 flasks of a larger volume, and supplemented with 37.5 mL of fresh NTI medium*1, and then with urelumab [1 pg / mL] and IL-21 [1 ng / mL] for subculture, and cultured.

[0160] - Day 10 The cell suspensions in the T225 flasks were thoroughly mixed by pipetting, collected, and centrifuged at 500 g for 5 minutes, and the cell pellets were suspended in 60 mL of the culture supernatant. Fresh NTI medium*1 was added in a volume of 240 mL each for subculture, and the cell suspensions were seeded into four T225 flasks in a volume of 75 mL each, and cultured.

[0161] - Day 13 The cell suspension in one T225 flask was thoroughly mixed by pipetting, collected, and centrifuged at 500 g for 5 minutes, and cell number was counted. A cell suspension was prepared at a density of 9 x 10' cells / mL in fresh NTI medium*1, seeded into four T225 flasks in a volume of 50 mL each for subculture, and cultured.

[0162] - Day 14 The cell suspension in one T225 flask was thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell number was counted.

[0163] - Day 15 The cell suspension in one T225 flask was thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell number was counted.

[0164] -Day 16 The cell suspension in one T225 flask was thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTAZPBS, and cell number was counted.

[0165] -Day 17 The cell suspension in one T225 flask was thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell number was counted.

[0166] [Animal experiments] Animals used: Female NOD.Cg-PrkdcscldI12rgtalSug / ShiJic (NOG) mice (6 weeks old, purchased from Japan Clea / Central Institute for Experimental Medicine and Life Science) were acclimated for up to 5 days, and used in the experiments at 6 to 7 weeks old. The animals were fed in a number of 3 or 4 animals per cage in a climate-controlled animal facility maintained at a temperature of 21 to 25°C, humidity of 40 to 60% (controlled by air conditioning), and lighting of 12 hours (7:00-19:00), and fed CRF-1 solid feed (gamma-irradiated, Oriental Yeast Co., Ltd.) ad libitum. As drinking water, the animals were fed autoclaved tap water ad libitum.

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

[0168] [Table 3] Group 1 Treatment Transplanted cells and transplanted cell number (spheres / 0.5 mL / head) Administration dose of collected cells (cefls / head) IL-2 (unit / head) Administration number Administration route Number of animals 1. Control group SK-OV-3 / CMV- Luc 450 - i.p. 3 2. Test substance administration a roup j Collected I cells 1 IL-2 Collected cells** 50,000 5 Times 3 ♦♦Administration dose of collected cells First administration: 1.8 x 107 cells / 0.4 mL Second administration: 1.5 x 107 cells / 0.4 mL Third administration: 1.9 x 107 cells / 0.4 mL Fourth administration: 2.2 x 107 cells / 0.4 mL Fifth administration: 2.7 x 107 cells / 0.4 mL

[0169] Preparation of tumor-bearing mice «Preparation of SK-0V-3 / CMV-Luc cell spheres» SK-0V-3 / CMV-Luc cells were prepared at 1 x 106 cells / 3 ml per well in RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin mixed solution) and seeded on EZSPHERE (registered trademark) 6-well plates (IWAKI, 4810-900SP). After culture of 72 hours, the formed spheres (2700 spheres / well) were collected, centrifuged at 100 g for 1 minute, and after the supernatant was removed, gently suspended in PBS at 2700 spheres / 3 mL.

[0170] SK-OV-3 / CMV-Luc (luciferase-expressing human ovarian cancer cell line) was intraperitoneally transplanted into mice (450 spheres / 0.5 mL / head) using a 27G needle to establish a peritoneal seeded model.

[0171] Test Substances: Collected cells: Manufactured in-house Human interleukin-2 (IL-2) preparation: Imunace (generic name, teceleukin (recombinant)), Shionogi.

[0172] Administration of test substance: SK-OV-3 / CMV-Luc transplantation day was defined as day 0, and the collected cells were intraperitoneally administered on day 3 (1st dose), day 4 (2nd dose), day 5 (3rd dose), day 6 (4th dose), and day 7 (5th dose).

[0173] Follow-up observation: Fluorescence observation of intraperitoneal tumors using in vivo imaging system (IVIS) For IVIS observation, luciferin (3 mg / head in 200 pL PBS) was subcutaneously administered into the dorsal regions of the mice, and luminescence in tumors was confirmed. The observation was performed under isoflurane anesthesia (using an animal inhalation anesthetic machine, and air as carrier gas, induced by 4% isoflurane concentration and maintained by 2% concentration). Thereafter, observation was conducted over time 1 or 2 times / week.

[0174] Weight measurement and visual inspection: After SK-OV-3 / CMV-Luc transplantation, weight measurements and visual inspections were performed 1 or 2 times / week.

[0175] Humane endpoint: Difficulty eating or drinking

[0176] The results are shown in Fig. 8 (changes in SK-OV-3 / CMV-Luc cell number) and Fig. 9 (I VIS results on day 69). Antitumor effects were observed in the group administered with the collected cell population.

[0177] F) Example 6 (Donor Mix C) - Day 0 Cryopreserved PBMCs (CD3‘, CD34) were thawed by a predetermined method, and culture was started under the following conditions: (1): 1.3 x 105 cells / 0.65 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL], 24-well plate (2): 1.6 x 105 cells / 0.65 mL, KBM501 medium*4, 24-well plate (3): 1.3 x 105 cells / 0.65 mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL], G-Rex 24-well plate (4): 1.6 x 105 cells / 0.65 mL, KBM501 medium*4, G-Rex 24-well plate A: 7.5 x 105 cells / 15 mL, KBM501 medium*4, T75 flask B: 7.5 x 105 cells / 15 mL, KBM501 medium*4, T75 flask

[0178] - Day 7 The cell suspension in the well of (1) was thoroughly mixed by pipetting, and cell number was counted. The cells were subcultured and cultured under the following conditions. (1)-1: Cell suspension of (1) 0.65 mL, NTI medium*1 1.5 mL, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0179] - Day 10 Fresh NTI medium*1 (35 mL) was added to the T75 flask A, and culture was performed. Fresh NTI medium*1 (35 mL) supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL] was added to the T75 flask B, and culture was performed. The suspension in the well of (2) was thoroughly mixed by pipetting, and cell number was counted. Fresh KBM501 medium*4 (1 mL) was added.

[0180] -Day 11 The suspension in the well of (1)-1 was thoroughly mixed by pipetting, and cell number was counted. The suspension was made 6 mL with fresh NTI medium*1, and supplemented withurelumab [1 pg / mL] and IL-21 [1 ng / mL] for subculture, and cultured for the following three groups. (1)-2: Cell suspension of (1)-1 2 mL (1)-3: Cell suspension of (1)-1 2 mL (5): G-Rex 24-well plate, cell suspension of (1)-1 2 mL Further, the cell suspension of (1)-1 was also subcultured in 2 mL of fresh NTI medium*1 contained in an empty well.

[0181] -Day 13 The cells were subcultured in 135 mL of fresh NTI medium*1 in T75 flask A. The cell suspension in the well of (1)-2 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of the G-Rex 24-well plate was thoroughly mixed by pipetting, and cell number was counted.

[0182] - Day 14 The cell suspension in the well of (1)-2 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension was made 6 mL with fresh NTI medium*1, seeded on a 6-well plate in a volume of 2 mL per well for subculture, and cultured. (1)-2-1: Cell suspension of (1)-2 2 mL (1)-2-2: Cell suspension of (1)-2 2 mL (1)-2-3: Cell suspension of (1)-2 2 mL The cell suspension in the well of (1)-3 was thoroughly mixed by pipetting, and cell number was counted. The cells were subcultured and cultured on a 6-well plate under the following conditions. (1)-3-1: The cell suspension of (1)-3 was centrifuged at 500 g for 5 minutes, and the cells were thoroughly suspended in 3 mL of fresh NTI medium*1 supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and seeded.

[0183] (5): The cell suspension in the well of the G-Rex 24-well plate was thoroughly mixed by pipetting, and cell number was counted. Then, 4 mL of fresh NTI medium*1 was added for subculture, and culture was performed.

[0184] The cell suspensions in the T75 flasks A and B were thoroughly mixed by pipetting, the cells were collected by using 1 mM EDTA / PBS, and cell numbers were counted.

[0185] -Day 17 The cell suspension in the well of (1)-2-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of (1)-3-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of the G-Rex 24-well plate of (5) was gently withdrawn in a volume of 4.7 mL, and 4 mL of fresh NTI medium*1 was added. The cell suspension was thoroughly mixed by pipetting, and cell number was counted. Further, 1 mL of fresh NTI medium*1 was added.

[0186] - Day 20 The cell suspension in the well of (1)-2-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of (1)-3-1 was thoroughly mixed by pipetting, and cell number was counted.

[0187] -Day 21 The cell suspension in the well of (1)-2-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of (1)-3-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspension in the well of the G-Rex 24-well plate of (5) was thoroughly mixed by pipetting, and cell number was counted.

[0188] - Day 24 The cell suspension in the well of (1)-1 was thoroughly mixed by pipetting, and cell number was counted. The cell suspensions in the wells of (1)-2 and (1)-3 were thoroughly mixed by pipetting, and cell numbers were counted.

[0189] Further, the numbers of live cells were counted, 2 x 108 of the cells were suspended in 5 mL of HSC-BANKER (ZENOAQ, CB071), and the cell suspensions were frozen at -80°C. The frozen cells were thawed on a water bath at 37°C and then diluted 10-fold with Plasma-Lyte A. Cytotoxic activity of the thawed cells against K562 cells or Raji cells was measured according to the same method as in Example 4. Cytotoxic activity rates were evaluated by classifying them into the following categories A to D. Results classified as C or higher are judged to indicate that high cytotoxic activity has been obtained, and the object has been achieved. A: 70% or higher B: 60% or higher C: 50% or higher D: Lower than 50%

[0190] The results are shown in Fig. 11. For both E (effector cells (collected cel 1 s)) T (target cells (K562 cells)) ratios of 1:1 and 5:1, the cytotoxic activity rates of the collected cell population for K562 cells and Raji cells were evaluated as A, indicating particularly high cytotoxic activity against both tumor cells.

[0191] G) Example 7 (Donor Mix C, Single Donor A) -Day 0 Cryopreserved PBMCs were thawed by a predetermined method. Cell suspensions prepared for the following 5 groups were seeded in a volume of 1.8 mL each per well on 6-well plates, and culture was started.

[0192] (1): PBMC (CDS', CD34) 4 x 105 cells / mL, KBM501 medium*4, urelumab [1 pg / mL], IL-21 [1 ng / mL] (2): PBMC (CDS', CD34') 4 x 105 cells / mL, NTI medium*1, urelumab [1 pg / mL], IL-21 [1 ng / mL] (3): PBMC (CD3', CD34') 4 x 105 cells / mL, KBM501 medium*5 containing Simulect and Prograf*5, urelumab [1 pg / mL], IL-21 [1 ng / mL] (4): PBMC (CDS', CD34') 4 x 105 cells / mL, NTI medium*6 containing Simulect and Prograf, urelumab [1 pg / mL], IL-21 [1 ng / mL] (5): PBMC (CDS , CD34 ) 4 x IO? cells / mL, NTI medium*6 containing Simulect and Prograf, urelumab [1 pg / mL], IL-21 [1 ng / mL]

[0193] - Day 7 The cell suspensions in the wells of (1) to (5) were thoroughly mixed by pipetting, and cell numbers were counted. The cells were then subcultured and cultured on 6-well plates under the following conditions. (1)-1: The cell suspension was made 5 x 105 cells / 3 mL with fresh KBM501 medium*4, supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded. (1)-2: The cell suspension was made 5 x 105 cells / 3 mL with fresh KBM501 medium*4, and then seeded. (1)-3: The cell suspension was made 5 x 105 cells / 1.83 mL with fresh KBM501 medium*4, and then seeded. (1)-4: The cell suspension was taken for 5 x 105 cells, and centrifuged at 500 g for 5 minutes, and the cells were thoroughly suspended in 3 mL of fresh KBM501 medium*4 supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded.

[0194] (2)-1: The cell suspension was made 5 x 105 cells / 3 mL with fresh NTI medium*1, supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded. (2)-2: The cell suspension was made 5 x 105 cells / 3 mL with fresh NTI medium*1, and then seeded. (2)-3: The cell suspension was made 5 x 105 cells / 1.83 mL with fresh NTI medium*1, and then seeded. (2)-4: The cell suspension was taken for 5 x 105 cells, and centrifuged at 500 g for 5 minutes, and the cells were thoroughly suspended in 3 mL of fresh NTI medium*1 supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded.

[0195] (5)-1: The cell suspension was made 5 x 105 cells / 3 mL with fresh NTI medium*1, supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded. (5)-2: The cell suspension was made 5 x 10’ cells / 3 mL with fresh NTI medium*1, and then seeded. (5)-3: The cell suspension was made 5 x 105 cells / 1.83 mL with fresh NTI medium*1, and then seeded. (5)-4: The cell suspension was taken for 5 x 105 cells, and centrifuged at 500 g for 5 minutes, the cells were thoroughly suspended in 3 mL of fresh NTI medium*1 supplemented with urelumab [1 pg / mL] and IL-21 [1 ng / mL], and then seeded.

[0196] (3)-5: The cell suspension 1.5 mL was seeded with 2.5 mL of fresh KBM501 medium*6 containing Simulect and Prograf. (4)-5: The cell suspension 1.5 mL was seeded with 2.5 mL of fresh NTI medium*6 containing Simulect and Prograf.

[0197] (1): Fresh KBM501 medium*4 3.5 mL was added for subculture and culture. (2): Fresh NTI medium*1 3.5 mL was added for subculture and culture. (3): Fresh KBM501 medium*5 3.5 mL was added for subculture and culture. (4): Fresh NTI medium*1 3.5 mL was added for subculture and culture. (5): Fresh NTI medium*1 3.5 mL was added for subculture and culture.

[0198] - Day 9 The cell suspensions in all the wells subcultured on day 7 were thoroughly mixed by pipetting, and cell numbers were counted. The cell suspensions in the wells of (1)-3, (2)-3, (5)-3, and (4)-5 were diluted 5 times (cell suspension 0.4 mL + fresh medium 1.6 mL) or 10 times (cell suspension 0.2 mL + fresh medium 1.8 mL) for subculture, and cultured in a volume of 2 mL per well on 6-well plates.

[0199] (l)-3-5x: Cell suspension of (1)-3 0.4 mL, KBM501 medium*4 1.6 mL (2)-3-5x: Cell suspension of (2)-3 0.4 mL, NTI medium*1 1.6 mL (5)-3-5x: Cell suspension of (5)-3 0.4 mL, NTI medium*6 containing Simulect and Prograf 1.6 mL (4)-5-5x: Cell suspension of (4)-3 0.4 mL, NTI medium*6 containing Simulect and Prograf 1.6 mL

[0200] (l)-3-10x: Cell suspension of (1)-3 0.2 mL, KBM501 medium*4 1.8 mL (2)-3-1 Ox: Cell suspension of (2)-3 0.2 mL, NTI medium*1 1.8 mL (5)-3-1 Ox: Cell suspension of (5)-3 0.2 mL, NTI medium*6 containing Simulect and Prograf 1.8 mL (4)-5-1 Ox: Cell suspension of (4)-3 0.2 mL, NTI medium*6 containing Simulect and Prograf 1.8 mL

[0201] -Day 11 The cell suspensions in all the wells of (3)-5, (3), and (4) subcultured on day 9 were thoroughly mixed by pipetting, and cell numbers were counted. Cell suspensions were prepared at 2 x 105 cells / 2 mL from the cell suspensions in the wells of (1 )-3-5x, (2)-3-5x, (5)-3-5x, and (5)-3-5-10x with fresh medium for subculture, and cultured on 6-well plates. (l)-3-5x-2E5: Cell suspension 2 x 105 cells / 2 mL, KBM501 medium*4 (2)-3-5x-2E5: Cell suspension 2 x 105 cells / 2 mL, NTI medium*1 (5)-3-5x-2E5: Cell suspension 2 x 105 cells / 2 mL, NTI medium*6 containing Simulect and Prograf (5)-3- 10x-2E5: Cell suspension 2 x 105 cells / 2 mL, NTI medium*6 containing Simulect and Prograf

[0202] - Day 14 The cell suspensions in all the wells of (2)-3-5x and (2)-3-1 Ox subcultured on day 11 were thoroughly mixed by pipetting, and cell numbers were counted. Cell suspensions were prepared at 4 x 105 cells / 2 mL from the cell suspensions in the wells of (2)-3-5x and (2)-3-10x with fresh medium on 6-well plates for subculture, and cultured.

[0203] The results are shown in Fig. 12.

[0204] H) Example 8 (Donor Mix) Cryopreserved PBMCs were completely thawed in a 37°C water bath and diluted 10 times with KBM501 / 5% UG medium*7. The cell suspension was centrifuged at 500 x g for 5 minutes, then 4x10’ cells / mL cell suspensions were prepared in the media of the following 6 groups, and seeded in a volume of 0.8 mL each per well on 12-well plates, and culture was started.

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

[0206] -Day 7 The KBM501 / 5% UG medium (800 pL each) was added to the cell suspensions of (2) to (5).

[0207] - Day 9 The KBM501 / 5% UG medium (1.6 mL each) was added to the cell suspensions of (1) and (6). The cell suspensions in the wells of (2) to (5) were thoroughly mixed by pipetting, and cell numbers were counted. Then, the cell suspensions were taken from the wells, and 2 x 105 cells / mL cell suspensions were prepared with KBM501 medium / 5% UG medium on 6-well plates for subculture.

[0208] -Day 11 The cell suspensions of in the wells of (6) and (2) to (5) subcultured on day 9 on 6-well plates were thoroughly mixed by pipetting, and cell numbers were counted. For (3), the culture was continued as was. For the other groups, the cell suspensions were collected from the wells, and made at densities of 2.35 x 105 cells / mL for (2), (4), and (5) and 1.84 x 105 cells / mL for (6) with KBM501 / 5% UG medium on 6-well plates for subculture.

[0209] - Day 14 From the cell suspensions of all the groups ((1) seeded on 12-well plates on day 0, and (2) to (6) subcultured on 6-well plates on day 9 or day 11), the cells were collected by using 1 mM EDTA / PBS, and cell numbers were counted. Further, 1 x 105 cells of the collected cells were stained with the antibodies listed below (concentration of each antibody 1 pg / mL).

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

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

[0212] Even when rhs-4-lBBL was used, more efficient cell proliferation was achieved compared with (1) control group (without rhs-4-lBBL or without anti-4-IBB and IL-21) and (6) IL-21 group (without rhs-4-lBBL or anti-4-lBB, but with IL-21) (Fig. 13).

[0213] *1: NTI (FUKOKU, T2108251) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *2: RPMI 1640 (Nacalai, 30264-85) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10), 2 U / mL heparin sodium (Nipro), and 100 units / mL Imunace *3: KBM502 (KOHJIN BIO, 16025020) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *4: KBM501 (KOHJIN BIO, 16025015) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) *5: KBM501 (KOHJIN BIO, 16025015) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10), 2 U / mL heparin sodium (Nipro), 0.5 pg / mL Simulect and 1 ng / mL Prograf *6: NTI (FUKOKU, T2108251) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10), 2 U / mL heparin sodium (Nipro), 0.5 pg / mL Simulect, and 1 ng / mL Prograf

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

[0215] [Reagents and equipments used] - RPMI 1640 (Nacalai Tesque, 30264-85) - KBM501 (KOHJIN BIO, 16025015) (containing IL-2 2810 lU / mL) - KBM502 (KOHJIN BIO, 16025020) (containing IL-2 281 lU / mL) - NTI (FUKOKU) - CTS Immune Cell SR (Gibco, A25961-01) - UltraGRO (AventaCell, HPCPLCRL10) -FBS (\ IC Hl REI, 174012, 19C00A) - Heparin sodium (Nipro) - Urelumab (Creative Biolabs, TAB-179) - IL-21 (PEPROTECH, AF-200-21-10pg) - IL-18 (Biolegend, 592102) - Imunace (Shionogi) - Simulect (NOVARTIS) - Prograf (Astellas) - Penicillin-streptomycin mixed solution (Nacalai Tesque, 26253-84) - 0.5 M EDTA (Invitrogen, 15575-038) - PBS (Nacalai Tesque, 14249-24) - 7-AAD (Beckman Coulter, A07704) - APC anti-human CD107a antibody (Biolegend, 328620) - APC Mouse IgGl, k Isotype Control Antibody (Biolegend, 400120) - EasySep Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055) - 6-Well plate (Thermo, 140675) - 12-Well plate (Thermo, 150628) - 24-Well plate (Thermo, 142475) - G-Rex 24-Well plate (BMBio, BMA-GP1024) - T75 Flask (Thermo Fisher, 156499) - T225 Flask (Thermo Fisher, 159933) - 10-cm Dish (Falcon, 3530003) - 96-Well plate (IWAKI, 4870-800SP) - EZSPHERE (registered trademark) 6-well plate (IWAKI, 4810-900SP)

[0216] - Alexa Fluor (registered trademark) 700-labeled anti-human CD56 antibody (Biolegend, 318316) - APC-labeled anti-human NKp30 antibody (Biolegend, 325210) - PerCP / Cy5.5-labeled anti-human CD3 antibody (Biolegend, 300430) - PE-Cy7-labeled anti-human CD 16 antibody (Biolegend, 302016) - PE-labeled anti-human CD 19 antibody (Biolegend, 302208) - FITC-labeled anti-human CD14 antibody (Biolegend, 325604) - APC-Cy7-labeled anti-human CD34 antibody (Biolegend, 343514)

[0217] - FITC-labeled anti-human CD49a antibody (Biolegend, 328307) - PE-labeled anti-human CD49c antibody (Biolegend, 343803) -APC-labeled anti-human CD61 antibody (Biolegend, 336412) - PE-Dazzle 594-labeled anti-human NKp30 antibody (Biolegend, 325231) - PE-Cy7-labeled anti-human CCR5 antibody (Biolegend, 359107) - Pacific Blue™-labeled anti-human CCR6 antibody (Biolegend, 353438) - APC-Cy7-labeled anti-human CXCR3 antibody (Biolegend, 353721)

[0218] - KBM 501 (KOHJIN BIO) - UltraGRO (AventaCell, HPCPLCLR10) - Heparin sodium (Nipro) - Simulect (Novartis Pharma) - Prograf (Astellas Pharma) - Recombinant Human 4-IBB Ligand Animal-Free manufactured (PREPROTECH)

[0219] [Sequences listed in 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 4-1 BBL

Claims

1. A method for producing a cell population containing CD3’negative and CD56-positive cells, which comprises the following step:a) the 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 medium containing IL-2, IL-21 and a 4-1BB agonist for one day or longer, wherein the IL-21 and 4-IBB agonists are humoral factors.

2. The production method according to claim 1, which further comprises the following step:b) the step of subculturing the cell population cultured in the step a) in a medium containing IL-2 and a 4-1BB agonist but not containing IL-21, and culturing it for one day or longer.

3. The production method according to claim 1, wherein concentration of the 4-1BB agonist in the medium used in the step a) is 1 pg / mL or higher, and concentration of IL-21 in the medium used in step a) is 1 ng / mL or higher.

4. The production method according to claim 2, which further comprises the following step:c) the step of subculturing the cell population cultured in the step b) in a medium containing IL-2 and culturing it for one day or longer.

5. The method according to claim 1, wherein the 4-IBB agonist is selected from an anti-4-IBB antibody and a 4-IBB ligand.

6. The method according to claim 1 or 2, wherein the medium contains a platelet lysate.INTERNATIONAL SEARCH REPORT International application No. PCT / IB2024 / 059123A. CLASSIFICATION OF SUBJECT MATTERC12N5 / 078(2010.01)1; C12N1 / 00(2006.01)1; C12N5 / 0783(2010.01)1FI: C12N5 / 078; C12N1 / 00 G; C12N5 / 0783According to International Patent Classification (IPC) or to both national classification and IPCB.FIELDS SEARCHEDMinimum documentation searched (classification system followed by classification symbols)C12N5 / 078; C12N1 / 00; C12N5 / 0783Documentation searched other than minimum documentation to the extent that such documents are included in the fields searchedPublished examined utility model applications of Japan 1922-1996Published unexamined utility model applications of Japan 1971-2024Registered utility model specifications of Japan 1996-2024Published registered utility model applications of Japan 1994-2024Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)JSTPlus / JMEDPlus / JST7580 (JDreamlll); CAplus / REGISTRY / MEDLINE / EMBASE / BIOSIS (STN)C. DOCUMENTS CONSIDERED TO BE RELEVANTCategory* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. Y A Y A JP 2022-520871 A (RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY) 01 April 2022 (2022-04-01) paragraphs [0168]-[0172], fig. 8-1, 8-2 JP 2022-548861 A (THE RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL) 22 November 2022 (2022-11-22) paragraphs [0212]-[0214] 1, 3, 5-6 2,4 1,3, 5-6 2,4 Y A JP 2013-027385 A (KYUSHU UNIVERSITY) 07 February 2013 (2013-02-07) paragraphs [0065]-[0069], fig. 17 1, 3, 5-6 2,4 Y A JP 2021-534835 A (HCW BIOLOGICS, INC.) 16 December 2021 (2021-12-16) paragraphs [ 1111 ]-[ 1112], [1218]-[ 1220], fig. 120-121, 182 1, 3, 5-6 2,4| | Further documents are listed in the continuation of Box C.annex.* Special categories of cited documents:“A" document defining the general state of the art which is not considered to be of particular relevance“D" document cited by the applicant in the international application“E" earlier application orpatent but published on or after the international filing date“L" document which may throw doubts on priority claim(s) or which is cited to establish the publication date of another citation or other special reason (as specified)“O” document referring to an oral disclosure, use, exhibition or other means“P” document published prior to the international filing date but later than the priority date claimed“T”‘Y’later document published after the international filing date or priority date and not in conflict with the application but cited to understand the principle or theory underlying the inventiondocument of particular relevance; the claimed invention cannot be considered novel or cannot be considered to involve an inventive step when the document is taken alonedocument of particular relevance; the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents, such combination being obvious to a person skilled in the aitdocument member of the same patent familyDate of the actual completion of the international searchDate of mailing of the international search report25 November 2024Name and mailing address of the ISA / JPJapan Patent Office (ISA / JP)3-4-3 Kasumigaseki, Chiyoda-ku, Tokyo 100-8915JapanAuthorized officer10 December 2024Telephone No.

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