Method for treating highly active NK cells

Abstract

To provide a method which is applicable to highly active NK cells, and is for cryopreserving and thawing highly active NK cells while maintaining a high survival rate and high activity.SOLUTION: Provided is a method for treating cells, the method comprising the steps for: (1) recovering cells activated in vitro from a cell culture medium; (2) suspending the recovered cells in a liquid for cryopreservation; and (3) freezing the suspended cells. Step (1) preferably includes a treatment using a medium into which any one selected from the group consisting of a bile acid and a phenylbutyric acid is added.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for cryopreserving cells having high cytotoxic activity.

Background Art

[0002] NK cells are important in the rejection of tumor cells and virus-infected cells. In August 2017 in the United States, chimeric antigen receptor (CAR) T cell therapy was approved for the treatment of relapsed / refractory B-cell acute lymphoblastic leukemia (B-ALL) in children and young adults. In recent years, the clinical application of CAR-NK is expanding instead of CAR-T (Non-Patent Document 1).

[0003] When attempting to administer cells to a patient, it is first considered to use cells collected from the patient himself / herself so that no rejection reaction occurs. However, depending on the patient's condition, it may be difficult to collect the amount of cells required for treatment. In addition, there are individual differences in the degree to which cells can be activated and proliferated in vitro, and there are cases where proliferation activation is difficult. In addition, since activation and proliferation of cells require a certain period of time, there is a problem that treatment cannot be started immediately. In this regard, it is desirable to activate the cells in advance and store them in preparation for administration.

[0004] As a method for storing cells, a method of storing cells in a suspended state without freezing for short-term storage is known (for example, Patent Document 1), and a method of freezing for long-term storage is known (for example, Patent Document 2). Furthermore, it has been studied to use a solution for freezing containing sodium salts, potassium salts, sugars, cryoprotective agents, and bicarbonates and / or carbonates, assuming that cells with high survival rates can be obtained even after freezing and thawing (Patent Document 3).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] [Non-Patent Document 1] Liu E, et al. N Engl J Med. 2020;382:545-53 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] However, conventional freezing methods using protective agents such as dimethyl sulfoxide can preserve T cell lines and primary NK cells, but are insufficient for highly cytotoxic NK cells, and their activity and viability are significantly reduced by the freeze-thaw process. This problem could not be solved by programmed freezers, cell density adjustments, or the addition of dextran, albumin, or carboxylated poly-L-lysine. Furthermore, if cells for administration are packaged assuming that a certain amount of cells will die, a reactivation culture process is required after thawing, and the resulting cells must be washed further. As a result, currently, frozen and stocked highly active NK cells can only be used in facilities that meet the standards of cell culture processing facilities (CPCs) that handle clinically-grade cells.

[0008] The object of this invention is to provide a method for cryopreserving and thawing highly active NK cells while maintaining high viability and activity, applicable to highly active NK cells. [Means for solving the problem]

[0009] The present invention provides the following: [1] A method for processing cells, comprising the following steps: (1) The cells activated in vitro are collected in a cell culture medium; (2) Suspend the collected cells in a solution for cryopreservation; (3) Freeze the suspended cells. [2] The method according to 1, wherein step (1) includes treatment with a culture medium to which any of the following are selected from the group consisting of bile acids and phenylbutyric acid is added. [3] The method according to 1 or 2, further comprising the following steps: (4) Thaw the frozen cells and suspend them in thawing solvent I. [4] The method according to any one of items 1 to 3, wherein the thawing solvent I is an aqueous solution containing the following: • Sodium chloride 9.00 ~ 108 mM, • Sodium gluconate 2.30~27.7 mM Sodium acetate 2.70~32.5 mM, • Potassium chloride 0.496~5.96 mM, and Magnesium chloride 0.148~1.78 mM. [5] The method according to any one of items 1 to 4, wherein the thawing solvent I satisfies at least one of the following conditions: • Does not contain calcium ions at a concentration of 0.423 mM or higher. • Does not contain glucose at a concentration of 5.55 mM or higher, • Does not contain lactate at concentrations of 27.7 mM or higher. [6] A solution for suspending highly active NK cells, including the following: • Sodium chloride 9.00 ~ 108 mM, • Sodium gluconate 2.30~27.7 mM Sodium acetate 2.70~32.5 mM, • Potassium chloride 0.496~5.96 mM, and Magnesium chloride 0.148~1.78 mM. [7] The solution according to claim 6, for suspending highly active NK cells that have been suspended and frozen in a solution for cryopreservation. [8] A pharmaceutical composition comprising highly active NK cells, a solution for cryopreservation, and the solution described in [6]. [9] A method for producing a cell-containing pharmaceutical composition, comprising the following steps: (1) The cells activated in vitro are collected in a cell culture medium; (2) Suspend the collected cells in a solution for cryopreservation; (3) Freeze the suspended cells.

[10] The method for producing a product according to [9], wherein step (1) is treatment with a culture medium to which any of the group consisting of bile acids and phenylbutyric acid is added. [Brief explanation of the drawing]

[0010] [Figure A] Flow cytometry results of various cells after the Mito-FerroGreen reaction. Highly active NK cells have a high iron content, suggesting the possibility of ferroptosis induction. [Figure B] Light microscope images of highly active NK cells after freezing and thawing. MP: Methylprednisolone, Dex: Dexamethasone. It was confirmed that the viability of highly active NK cells significantly decreased after freezing and thawing. [Figure 1] Viability of highly active NK cells after thawing. Dilution with Plasma-Lyte A during thawing improved viability. However, no improvement was observed when gluconic acid was added to KBM501 medium. Lacto = Lactol Ringer [Figure 2] Viability of highly active NK cells upon thawing. Dilution with Plasma-Lyte A during thawing improved viability, but dilution with Plasma-Lyte A containing 40% Serum (human type AB serum) worsened viability. [Figure 3] Number of viable cells and viability (right) and cytotoxic activity (left) of highly active NK cells after being allowed to stand for a certain period of time. Plasma = Plasma-Lyte A [Figure 4] Effects of pre-freezing treatment on highly active NK cells. Treatment with KBM501 supplemented with 4-PBA before freezing improved viability upon thawing. [Figure 5]Recovery rate of highly active NK cells after 2 hours of pretreatment, followed by freezing and thawing. Treatment with KBM501 medium supplemented with 4-PBA or TUDCA before freezing improved viability after thawing. [Figure 6] The cytotoxic activity of highly active NK cells that were frozen and thawed after 2 hours of pretreatment was examined. When the suspension solution for the thawed cells was Plasma-Lyte A, an improvement in cytotoxic activity was observed in a TUDCA concentration-dependent manner at both 1 hour and 3 hours. [Figure 7-1] Cytotoxic activity of highly active NK cells after 2 hours of pretreatment, followed by freezing and thawing. The TUDCA concentration range during pretreatment was widened. The target cell type was K562, and the procedure was performed under conditions of E:T = 2:1 and 2 hours. The number of NK cells was calculated based on the pre-freezing count. Each graph shows: 1. Immediately after thawing, 2. Plasma-Lyte A 1 hour, 3. Plasma-Lyte A 3 hours, 4. Plasma-Lyte A 1 hour + KBM501 2 hours. [Figure 7-2] Results of evaluation (3D killing assay) using highly active NK cells after thawing in a solid tumor model. [Figure 8-1] Flow cytometry analysis of cells stained with 7-ADD. After thawing, the cells were diluted 10-fold with the specified solvent and incubated at room temperature for 2 hours (right) or at 37°C for 3 hours (left). [Figure 8-2] Flow cytometry analysis of cells stained with 7-ADD. Upon collection, cells were washed with PBS (left) or KBM501 medium (right). After thawing, they were diluted 10-fold with the specified solvent and incubated at 37°C for 3 hours. [Figure 8-3] Flow cytometry analysis of cells stained with 7-ADD. After thawing, cells were diluted 10-fold with various solvents and incubated at 37°C for 3 hours. [Figure 8-4] Flow cytometry analysis of cells stained with 7-ADD. Upon collection, cells were washed with PBS (left) or KBM501 medium (right). After thawing, they were diluted 10-fold with the specified solvent and incubated at 37°C for 3 hours. [Figure 8-5]Flow cytometry analysis of cells stained with 7-ADD. Upon collection, cells were washed with PBS (left) or KBM501 medium (right). After thawing, they were diluted 10-fold with the specified solvent and incubated at 37°C for 3 hours. [Figure 9-1] Viability changes (A) and cytotoxic activity (B): Target=K562, E:T=1:2~4:1,2 hours after washing, freezing, and thawing of highly active NK cells in the administered form at room temperature. Cytotoxic activity (C): Target=K562, E:T=1:1,2~8 hours. [Figure 9-2] Cytotoxic activity of highly active NK cells after washing, freezing, and thawing. The cells were left standing at room temperature for each time period in their administered form. Target = K562, E:T = 1:1, 2 hours [Figure 10] Viability changes. Highly active NK cells, after being washed and frozen in KBM501 medium or PBS, were thawed at room temperature, allowed to stand at room temperature for each time without dilution, then diluted 10-fold in KBM501 medium or Plasma-Lyte A, stained with 7-AAD, and analyzed using FlowJo software (left). Highly active NK cells, after being washed and frozen in KBM501 medium or PBS, were thawed at 37°C, allowed to stand at room temperature for each time without dilution, then diluted 10-fold in KBM501 medium or Plasma-Lyte A, stained with 7-AAD, and analyzed using FlowJo software (right). [Figure 11] Viability changes. Highly active NK cells, after being washed and frozen in KBM501 medium or PBS, were thawed at 37°C, diluted 10-fold with KBM501 medium or Plasma-Lyte A, allowed to stand at room temperature for 0-6 hours, then stained with 7-AAD and analyzed using FlowJo software. [Figure 12-1] Viability changes. Effects of dilution with Plasma-Lyte A serially diluted with sterile distilled water, and standing at 37°C. Cells after each treatment were stained with 7-AAD and analyzed using FlowJo software (the same applies to the figures below). [Figure 12-2]Viability changes. Effects of dilution with Plasma-Lyte A serially diluted with physiological saline, and standing at room temperature or 37°C. [Figure 12-3] Viability changes. Effects of serial dilution of Plasma-Lyte A with physiological saline, or dilution with physiological saline, and standing at room temperature or 37°C. [Figure 12-4] Viability changes. Effects of dilution in each solvent and standing at room temperature. [Figure 12-5] Viability changes. Effects of dilution in each solvent and standing at 37°C. [Figure 12-6] Viability changes. Effects of dilution in each solvent and standing at room temperature when washed with PBS during recovery. [Figure 12-7] Viability changes. Effects of dilution with each solvent and standing at 37°C when washed with PBS during recovery. [Modes for carrying out the invention]

[0011] In this invention, mM is used interchangeably with mmol / L unless otherwise specified. When a numerical range is expressed as x~y, that range includes the values ​​x and y at both ends.

[0012] The present invention relates to a method for cryopreserving cells having high cytotoxic activity. [Applicable cells] The present invention can be applied to various types of cells. One type of cell to which the present invention is preferably applied is a cell that has undergone activation in vitro using some kind of cytokine, and such cells include highly cytotoxic NK cells (highly active NK cells), etc. The activation procedure typically involves incubating the cells in a medium containing interleukin (IL)-2.

[0013] Generally, NK cells are large granular lymphocytes that do not express the T cell receptor (TCR), the universal T cell marker CD3, or the membrane immunoglobulin B cell receptor. In humans, they are usually CD16-positive and CD56-positive. A person skilled in the art can easily determine whether a cell is an NK cell based on the expression pattern of cell surface markers. NK cells have cytotoxic activity, and the presence and degree of this cytotoxic activity can be measured by various known methods. NK cells can include peripheral blood NK cells, umbilical cord blood NK cells, primary NK cells, cultured NK cells, and highly active NK cells.

[0014] (raw materials) The raw materials to which the present invention is preferably applied, such as highly active NK cells, may be peripheral blood, umbilical cord blood, bone marrow and / or lymph nodes, or blood collected by apheresis (apheresis blood). The raw materials may also be prepared from at least one type of cell selected from the group consisting of hematopoietic stem cells derived from any stem cell selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem (iPS) cells, hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem cells derived from peripheral blood, hematopoietic stem cells derived from bone marrow blood, umbilical cord blood mononuclear cells, and peripheral blood mononuclear cells. The donor of the raw materials may be the patient receiving immunotherapy with highly active NK cells, a close relative of the patient, or a healthy person unrelated by blood to the patient. There may be multiple donors.

[0015] (culture medium) Culture media used for culturing highly active NK cells and the like include, but are not limited to, KBM501 medium (Kojin Bio, Inc., containing 1,750 JRU / mL of IL-2), Cosmedium 008 (Cosmo Bio, containing 1,750 JRU / mL of IL-2), FKCM101 (Fukoku, IL-2-free or containing 175 IU / mL of IL-2), CellGro SCGM medium (Celgenics, Iwai Chemicals Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Inc.), Gibco® CTS® AIM V® Medium (Thermo Fisher Scientific, a serum-free medium with a known composition for the proliferation and manipulation of T cells and dendritic cells), CTS OpTmizer T Cell Expansion Basal Medium (Thermo Fisher Scientific, for the growth and proliferation of human T lymphocytes), IMDM, MEM, DMEM, RPMI-1640, etc. Preferred examples include KBM501 medium, FKCM101, or Cosmedium 008. In relation to the present invention, when referring to culturing cells, unless otherwise specified, it means maintaining the cells in a medium or similar solution for a certain period of time for any purpose selected from the group consisting of cell survival, cell amplification, and cell activation. When a process is carried out at a specific temperature for a certain period of time, it is sometimes referred to as incubating.

[0016] The culture medium may contain IL-2 at a concentration sufficient to achieve the objectives of the present invention. The concentration of IL-2 may range from 2500 IU / mL to 2813 IU / mL. Preferably, IL-2 has a human amino acid sequence, and for safety reasons, it is preferably produced using recombinant DNA technology. The concentration of IL-2 may be expressed in Japanese Standard Units (JRU) and International Units (IU). Since 1 IU is approximately 0.622 JRU, 1750 JRU / mL of an existing culture medium corresponds to approximately 2813 IU / mL.

[0017] Simultaneously with or in place of IL-2, one of the cytokines selected from the group consisting of IL-12, IL-15, and IL-18 may be added at a concentration sufficient to achieve the objectives of the present invention (Non-patent Literature 2: Leong JW et al. Biol Blood Marrow Transplant 20 (2014) 463-473). The concentration of each cytokine may be 1 pg / mL to 1 μg / mL, regardless of the presence or concentration of other cytokines. IL-2 preferably has a human amino acid sequence, and for safety reasons, it is preferably produced using recombinant DNA technology.

[0018] The culture medium may contain the subject's autologous serum, human type AB serum available from BioWhittaker and others, or donated human serum albumin available from the Japanese Red Cross Society. Autologous serum and human type AB serum are preferably added at a concentration of 1 to 10%, and donated human serum albumin is also preferably added at a concentration of 1 to 10%. Human platelet lysate (HPL) may be added along with or in place of serum. HPL is commercially available, such as the UltraGRO™ series (AventaCell BioMedical). When using HPL, heparin sodium may be further added to the culture medium.

[0019] The culture medium may contain appropriate proteins, cytokines, antibodies, compounds, and other components, provided that they do not impair the culture effect of NK cells. Cytokines may include IL-2, IL-12, IL-15, and IL-18 as mentioned above, as well as IL-3, IL-7, IL-21, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (Flt3L). These preferably have human amino acid sequences and, for safety reasons, are preferably produced using recombinant DNA technology.

[0020] The culture medium is preferably a serum-free medium. The serum-free medium preferably contains serum albumin, transferrin, and insulin. Serum-free media for culturing lymphocytes have been developed and are commercially available, and these can be used in the present invention. One preferred example of a serum-free medium is a basal medium to which CTS Immune Cell SR (Thermo Fisher Scientific), a commercially available composition that supports the proliferation of human T cells, has been added.

[0021] The culture medium can be replaced or replenished at any time after the start of cultivation, provided that the desired culture effect is achieved, but it is preferable to do so every 3 to 5 days.

[0022] The culture vessels used during cultivation include, but are not limited to, commercially available dishes, flasks, plates, and multiwell plates. Culture conditions are not particularly limited, provided they do not impair the culture effect of NK cells, but conditions of 37°C, 5% CO2, and a saturated water vapor atmosphere are common. The culture period is not particularly limited, provided that the desired culture effect is achieved.

[0023] The highly active NK cells to which the present invention can be preferably applied include the following [1], [2], [3], and [4].

[0024] [1] NK cells having the following characteristics (1) and (2): (1) CD16 positive, CD56 highly expressive, and CD57 negative. (2) NKG2C positive, NKG2A negative to low expression, and CD94 positive.

[0025] The highly active NK cells in [1] may also be CD16 highly expressing. Furthermore, the highly active NK cells in [1] may have the following characteristics regardless of whether or not they are CD16 highly expressing. (3) When the NK cells are co-cultured as effector cells (E) and K562 cells as target cells (T) in a mixing ratio (E:T) of 1:1, the cytotoxic activity is 50% or more.

[0026] [1] Highly active NK cells can also be represented as follows: NK cells obtained by removing CD3-positive cells from peripheral blood mononuclear cells derived from healthy individuals using CD3 beads (e.g., CliniMACS CD3, Milteny Biotech, catalog number 130-017-601), an LD column (e.g., Milteny Biotech, catalog number 130-042-901), and a separation buffer (e.g., PBS containing 0.5% inactivated human type AB serum and 2 mM EDTA), and culturing the resulting cell population in a suitable medium (e.g., Cosmedium 008 supplemented with 5% inactivated human type AB serum) for 14 days, possessing the following characteristics (1) and (3): (1) CD16 positive, CD56 highly expressive, and CD57 negative. (3) When the NK cells are co-cultured as effector cells (E) and K562 cells as target cells (T) in a mixing ratio (E:T) of 1:1, the cytotoxic activity is 50% or more.

[0027] For more details on the characteristics of the highly active NK cells described in [1] and for more specific manufacturing methods, please refer to Japanese Patent Publication No. 2018-193303.

[0028] [2] The following cells: Cells that are CCR5-positive, CCR6-positive, and CXCR3-positive, and CD3-negative.

[0029] The cells in [2] may also be high in CD11c expression.

[0030] The cells in [2] can also be represented as follows: Cells that are CCR5-positive, CCR6-positive, CXCR3-positive, Integrin α1-positive, Integrin α3-positive, and Integrin β3-negative, and also CD3-negative. Alternatively, cells that are CCR5-positive, CCR6-positive, CXCR3-positive, highly CD11a-expressing, highly CD11c-expressing, and CD3-negative, with high expression determined by comparison with expression in a population of virtually uncultured NK cells obtained from peripheral blood.

[0031] According to the inventors' studies, the cells in [2] exhibit extremely high cytotoxic activity against solid tumors that have formed tumor masses. For details on the characteristics of the cells in [2] and a more specific method of production, please refer to Japanese Patent Application Publication No. 2019-170176.

[0032] [3] Highly active NK cells that can be obtained by the following methods: CD3 beads (e.g., CliniMACS CD3, Milteny Biotech, 130-017-601) are used to add CD3 beads to mononuclear cells obtained from fresh peripheral blood or frozen apheresis blood. 7 5 μL per cell), and if frozen apheresis blood is used, additional CD34 beads (e.g., CliniMACS CD34, Milteny Biotech, 130-017-501 (1x10)) 7 Add 2.5 μL per cell and suspend, incubate at 4°C for 15 minutes, then add separation buffer (e.g., PBS containing 0.5% human type AB serum (inactivated at 56°C for 30 minutes) and 2 mM EDTA), suspend well, and centrifuge. Remove the supernatant and centrifuge on an LD column (e.g., Milteny Biotech, 130-042-901) up to 1 x 10⁶ cells per column. 8 Suspend the cells in 0.5 mL of separation buffer to the desired cell count. Add 2 mL of separation buffer beforehand, then add the cell suspension to the LD column and collect the eluate from the LD column. Add another 1 mL of separation buffer to the LD column and collect the eluate. Centrifuge the collected solution, remove the supernatant, and if peripheral blood was used, eluate 5x10°C. 5cells / mL, 1x10 if frozen apheresis blood is used. 6 Suspend the cells in an appropriate medium (for example, KBM501 medium containing either 5% human type AB serum (inactivated at 56°C for 30 minutes) or 5% UltraGRO (AventaCell, HPCPLCRL10) supplemented with 2 U / mL heparin sodium) to a cell / mL ratio, and culture until day 14, changing the medium as needed.

[0033] For specific methods of producing the highly active NK cells described in [3], please refer to the Examples section of this specification.

[0034] [4] Cells obtained by culturing cells according to [1]~[3], in which any of the group selected from IL-12, IL-15, and IL-18 is added simultaneously with or in place of IL-2, at a concentration that can achieve the objective of the present invention. For specific methods of producing such cells, please refer to Non-Patent Document 2 cited above.

[0035] In the following description, the present invention may be explained using the example of highly active NK cells, but those skilled in the art will be able to understand the present invention in accordance with that description, even when using other cells that have undergone activation in vitro using some kind of cytokine.

[0036] (cytotoxic activity) In relation to the present invention, when referring to highly active NK cells, etc., activity or cytotoxic activity refers to the lytic ability of the target cells (T) of the target cells (effector cells, E), unless otherwise specified. Cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells, and can be calculated by the following formula.

[0037] (Cell death when co-cultured with effector cells - Spontaneous cell death (negative control)) / (Maximum cell death (positive control) - Spontaneous cell death (negative control)) × 100

[0038] When measuring cytotoxic activity, the mixing ratio (E:T) of effector cells and target cells, and the co-culture time of effector cells and target cells can generally be appropriately adjusted according to the type of cells used and the strength of their activity, depending on the degree of cytotoxic activity of the effector cells. When NK cells are used as effector cells, the target cells may be K562 cells, acute myeloid leukemia cells, or chronic myeloid leukemia cells, but are not limited to these. Effector cells and target cells, and living cells and dead cells, can be distinguished and quantified using reagents such as antibodies labeled with radioactive substances or fluorescent dyes. When NK cells are used as effector cells, cytotoxic activity can be measured, for example, with K562 cells as the target cells, with an E:T ratio of 1:0.05 to 10, preferably 1:0.1 to 5, and an incubation time of 0.5 to 18 hours, preferably 1 to 12 hours.

[0039] In relation to the present invention, when the activity of NK cells, etc., is described as high, unless otherwise specified, it means that the cytotoxic activity is 50% or more when the target cells are K562 cells, mixed in an E:T ratio of 2:1, and co-cultured for 1 to 3 hours, more specifically for 2 hours. The activity is preferably 60% or more, and more preferably 70% or more.

[0040] [collect] In this invention, prior to the freezing step described later, highly active NK cells and the like to be frozen are recovered from the culture system. Recovery can be performed by separating the culture medium and cells by centrifugation. If necessary, an appropriate concentration of EDTA may be added to the culture system to detach adhered cells from the surface of the culture vessel. Alternatively, the surface of the culture vessel after adding the culture medium may be washed with an appropriate solution to obtain the remaining cells. The obtained cells may be washed with an appropriate solution as necessary and suspended in an appropriate solution.

[0041] In the recovery process, solutions such as culture media, isotonic solutions, and buffers can be used for cell detachment and washing. Examples of usable culture media include KBM501 medium, Cosmedium 008, FKCM101, CellGro SCGM medium, X-VIVO15 medium, Gibco® CTS® AIM V® Medium, CTS OpTmizer T Cell Expansion Basal Medium, IMDM, MEM, DMEM, and RPMI-1640. An isotonic solution is a solution with an osmotic pressure approximately equal to that of body fluid (plasma) (285±5 mOsm / L), and in the context of this invention, it refers to a solution with an osmotic pressure of 285±13 mOsm / L. For example, the osmotic pressure of Plasma-Lyte A is 294 mOsm / L, and the osmotic pressure of PBS(-) is 280±4 mOsm / L (freezing point depression method). Examples of usable isotonic solutions include Plasma-Lyte A (Baxter), physiological saline, Ringer's solution (lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc.), and 5% glucose aqueous solution. Examples of usable buffer solutions include phosphate-buffered saline (PBS), Tris hydrochloride buffer, Tris acetate buffer, and HEPES buffer.

[0042] One preferred example of a solution used in the recovery process is a culture medium, more preferably a human lymphocyte culture medium. The human lymphocyte culture medium may contain human serum albumin, human transferrin, recombinant human insulin, and recombinant human IL-2. Preferred examples of such media are KBM501 medium, FKCM101, or Cosmedium 008. KBM501 medium contains human serum albumin, human transferrin, recombinant human insulin, and recombinant human IL-2, but no other proteins. KBM501 medium also contains an antibiotic (kanamycin), NaHCO3, L-Glutamine, and a pH adjuster.

[0043] In the recovery process, using PBS(-) may be undesirable as it can reduce cell viability upon thawing. PBS(-) typically contains 136.9 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate, and 1.47 mM potassium hydrogen phosphate.

[0044] [Pre-processing] In the present invention, highly active NK cells, etc., to be frozen may be pre-treated prior to the freezing step described later. Pre-treatment involves suspending the recovered cells in a solution containing an additive. Pre-treatment includes recovery in a solution containing an additive.

[0045] As additives used for pretreatment, any of the following can be selected from the group consisting of bile acids and phenylbutyric acid. Examples of bile acids include tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolitocholic acid, lithocholic acid, iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid or its analogs and derivatives. Examples of phenylbutyric acid include 4-phenylbutyric acid (4-PBA), glyceryl (tri-4-PBA), phenylacetic acid, 2-POAA-OMe, 2-POAA-NO2, 2-NOAA or its pharmaceutically acceptable salts, analogs, derivatives or prodrugs. Particularly preferred examples of additives used in pretreatment are selected from the group consisting of TUDCA and 4-PBA.

[0046] When bile acids are used as additives for pretreatment, the concentration can be adjusted as appropriate, but is preferably 100 to 5000 μM, more preferably 200 to 2500 μM, and even more preferably 400 to 1000 μM. This range is particularly suitable when using TUDCA. When phenylbutyric acid is used as additives for pretreatment, the concentration can be adjusted as appropriate, but is preferably 1 to 1000 μM, more preferably 5 to 500 μM, and even more preferably 10 to 100 μM. This range is particularly suitable when using 4-PBA.

[0047] Another example of an additive for pretreatment is dimethyl sulfoxide (DMSO). The concentration can be adjusted as appropriate, but is preferably 0.5 to 15%, more preferably 1 to 12.5%, and even more preferably 2 to 10%.

[0048] The solution used for pretreatment may be a culture medium, isotonic solution, buffer solution, etc., similar to the solution used during recovery. One preferred example of the solution used in pretreatment is a culture medium, more preferably a human lymphocyte culture medium, and even more preferably KBM501 medium, FKCM101, or Cosmedium 008. The culture medium used for pretreatment may also contain human serum albumin, human transferrin, recombinant human insulin, and recombinant human IL-2, and may also contain an antibiotic (kanamycin), NaHCO3, L-Glutamine, and a pH adjuster.

[0049] The time required for pretreatment is not particularly limited. After suspending the cells for pretreatment, the suspension may be allowed to stand for several minutes to several hours, for example, 5 minutes to 4 hours, more preferably 30 minutes to 3 hours. This standing may be carried out at ambient temperature (e.g., 1 to 30°C, typically 15 to 25°C) or in a CO2 incubator (e.g., 36 to 42°C, typically 37°C).

[0050] The cell density during pretreatment can be adjusted as needed, but it is best to set it to a density suitable for cell maintenance. Specifically, 1x10 5 ~1x10 7cells / mL, preferably 2x10 5 ~5x10 6 cells / mL, more preferably 5x10 5 ~2x10 6 cells / mL.

[0051] In a particularly preferred embodiment, the pretreatment is carried out by suspending the cells in KBM501 medium, FKCM101 or Cosmedia 008 supplemented with 400 - 1000 μM of TUDCA or 10 - 100 μM of 4-PBA at a cell density of 5x10 5 ~2x10 6 cells / mL. At this time, incubation at 37 °C under 5% CO2 for 30 minutes to 3 hours is advisable.

[0052] Although the pretreatment is not essential in the present invention, by pretreating highly active NK cells or the like with KBM501 medium supplemented with 4-PBA or TUDCA before freezing, the survival rate (which can also be referred to as the recovery rate) when the cells are thawed can be improved compared to the case without pretreatment.

[0053] [Freezing] In the present invention, the recovered and preferably pretreated cells are frozen by a normal procedure. Specifically, after checking the cell number and survival rate as necessary and centrifuging to remove the supernatant, the cells are suspended in a cryopreservation solution to reach an appropriate cell density. After dispensing the cell suspension into a container for cryopreservation, it is frozen in a -80 °C deep freezer and stored. If necessary, it can be cryopreserved in a liquid nitrogen tank.

[0054] The cryopreservation solution that can be used in the present invention may contain sodium salts, potassium salts, sugars, bicarbonates, carbonates, and cryoprotective agents.

[0055] The usable sodium salts are not particularly limited as long as they produce sodium ions when dissolved in the solvent, and may include oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts. One type of sodium salt or a combination of several types may be used. In the present invention, sodium chloride is preferably used as one type, and sodium chloride and sodium citrate are preferably used as multiple types. The sodium salt content is not particularly limited, but the final concentration of total sodium ions contained in the cryopreservation solution is preferably 0.01 to 5000 mM, more preferably 0.1 to 1000 mM, and even more preferably 1 to 300 mM.

[0056] The potassium salts that can be used are not particularly limited as long as they produce potassium ions when dissolved in the solvent, and may include oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts. One type of potassium salt or a combination of several types may be used. In the present invention, potassium chloride is preferably used. The potassium salt content is not particularly limited, but the final concentration of total potassium ions contained in the cryopreservation solution is preferably 0.01 to 5000 mM, more preferably 0.1 to 1000 mM, and even more preferably 1 to 100 mM.

[0057] The bicarbonate salts that can be used are not particularly limited as long as they produce bicarbonate ions when dissolved in the solvent, and salts with various cations can be used. Examples include ammonium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium bicarbonate, and magnesium bicarbonate. The carbonate salts that can be used are not particularly limited as long as they produce carbonate ions when dissolved in the solvent, and salts with various cations can be used. Examples include ammonium carbonate, potassium carbonate, calcium carbonate, sodium carbonate, barium carbonate, and magnesium carbonate. These bicarbonate salts and / or carbonate salts may be used individually or in combination. In the present invention, sodium bicarbonate is preferably used. The content of the bicarbonate salt and / or carbonate salt is not particularly limited, but the final total concentration of bicarbonate ions and carbonate ions contained in the cryopreservation solution is preferably 0.01 to 1000 mM, more preferably 0.1 to 500 mM, and even more preferably 1 to 100 mM.

[0058] The concentration ratio of sodium ions to potassium ions (sodium ions / potassium ions) in the cryopreservation solution is preferably 1 / 1000 to 1000 / 1, more preferably 1 / 100 to 100 / 1, even more preferably 1 / 10 to 100 / 1, even more preferably 1 / 1 to 100 / 1, and even more preferably 10 / 1 to 50 / 1.

[0059] The usable sugars are monosaccharides, oligosaccharides, or sugar alcohols. For example, monosaccharides include glucose, galactose, fructose, mannose, xylose, and arabinose; oligosaccharides include trehalose, sucrose, maltose, lactose, and cellobiose; and sugar alcohols include xylitol and sorbitol. These sugars may be used individually or in combination, but in the present invention, preferably, at least one sugar selected from the group consisting of glucose, galactose, fructose, mannose, xylose, and arabinose is used, and more preferably, glucose. The sugar content in the cryopreservation solution is preferably 0.01 to 100 g / L, more preferably 0.1 to 100 g / L, and even more preferably 0.25 to 50 g / L.

[0060] Examples of usable frost protection agents include dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol, and glycerol. One or more types of frost protection agents may be used. In the present invention, either selected from the group consisting of DMSO and hydroxyethyl starch is preferably used. When DMSO and hydroxyethyl starch are used in combination as frost protection agents, the total content is preferably within the above range, and the concentrations of each are preferably such that the DMSO concentration is preferably 0.01 to 50%, more preferably 1 to 30%, and even more preferably 2 to 15%, and the hydroxyethyl starch concentration is preferably 0.01 to 50%, more preferably 1 to 30%, and even more preferably 2 to 15%.

[0061] In preferred embodiments of the present invention, in addition to the essential components of the solution used in the cell cryopreservation method of the present invention described above, the solution may further contain components selected from the group consisting of proteins, magnesium salts, and calcium salts. Specific examples of usable proteins include serum albumin and serum globulin. Examples of serum albumin include human serum albumin or bovine serum albumin. In the present invention, human serum albumin is preferred. The protein content in the cryopreservation solution is preferably 0.01 to 50%, more preferably 1 to 30%, and even more preferably 2 to 15%. The usable magnesium salt is not particularly limited as long as it produces magnesium ions when dissolved in the solvent; oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts can be used. One or more types of magnesium salts may be used. In the present invention, magnesium chloride is preferably used. The magnesium salt content is not particularly limited, but the final concentration of total magnesium ions in the cryopreservation solution is preferably 0.01 to 10 mM, more preferably 0.1 to 5 mM. The calcium salts that can be used are not particularly limited as long as they produce calcium ions when dissolved in the solvent, and oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts can be used. One type of calcium salt or a combination of several types may be used. In the present invention, calcium chloride is preferably used. The calcium salt content is not particularly limited, but the final concentration of total calcium ions contained in the cryopreservation solution is preferably 0.01 to 10 mM, and more preferably 0.1 to 5 mM. In addition to the above components, the cryopreservation solution may further contain substances that do not harm cells, such as vitamins and amino acids. In addition to the above components, the cryopreservation solution may also contain phosphate ions from the viewpoint of pH adjustment and buffering effect.

[0062] The osmotic pressure of the cryopreservation solution is preferably within a range that does not damage cells during freezing. From the viewpoint of increasing the permeability of components to cells during freezing and inhibiting ice crystal formation, it is preferably, for example, 500-8000 mOsm / L, but may also be 1000-7500 mOsm / L, 1500-7000 mOsm / L, or 1800-5000 mOsm / L. The pH of the cryopreservation solution is preferably within a range that does not damage cells. For example, it is preferably 3.0-10.0, and more preferably 4.5-9.0.

[0063] In this invention, commercially available cryopreservation solutions may be used. Examples of usable products include the Cellbanker series of cryopreservation solutions for cells and tissues (STEM-CELLBANKER®), more specifically STEM-CELLBANKER (ZENOAQ, CB045).

[0064] It is preferable that the cells used for freezing are in the logarithmic growth phase.

[0065] The cell density during freezing can be adjusted as needed, but specifically, 1x10 6 ~2x10 8 The cell density is cells / mL, preferably 2 x 10 6 ~1x10 8 cells / mL, more preferably 1x10 7 ~5x10 7 The ratio is cells / mL. In one preferred embodiment, the cells are placed in a 5 mL container in a 4x10 7 The cells are stored at a concentration of cells / mL. The ability to freeze highly active NK cells at high density during frozen shipment allows for a more compact product and contributes to reduced transportation costs.

[0066] [Defrost] In this invention, cryopreserved cells can be thawed by various procedures. For example, the cryopreservation container holding the cells can be rapidly thawed in a 37°C bath, shaking as needed. Alternatively, after removing from the freezer, the cells can be left at room temperature without active heating to allow for natural thawing. After thawing, the cells are mixed with an appropriate thawing solvent I. If necessary, the supernatant can be removed by centrifugation, and the cells can be suspended in an appropriate amount of thawing solvent II for culturing or activation treatment.

[0067] (Solvent I during thawing) In the present invention, the solution used to thaw cells that have been frozen suspended in a cryopreservation solution and then diluted with the cryopreservation solution is called thawing solvent I. Various solutions can be used as thawing solvent I for cells recovered according to the present invention and pre-treated as necessary before cryopreservation.

[0068] In one preferred embodiment, the thawing solvent I may contain sodium salts, potassium salts, gluconates, and acetates. It may also contain magnesium salts.

[0069] The sodium salts that can be used as solvent I during thawing may be oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts. One type of sodium salt or a combination of several types may be used. Solvent I during thawing preferably contains any of the group selected from sodium chloride, sodium gluconate, and sodium acetate, and more preferably contains sodium chloride, sodium gluconate, and sodium acetate. The sodium salt content of solvent I during thawing is preferably 14.0 to 200 mM, more preferably 28.0 to 182 mM, and even more preferably 70.0 to 168 mM as the final concentration of total sodium ions. Alternatively, it is preferable to contain 9.00 to 108 mM of sodium chloride, 2.30 to 27.7 mM of sodium gluconate, and 2.70 to 32.5 mM of sodium acetate.

[0070] Potassium salts that can be used as solvent I during thawing may be oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts. Potassium salts may be one type or a combination of several types. Solvent I during thawing preferably contains one selected from the group consisting of potassium chloride, potassium gluconate, and potassium acetate, and more preferably contains potassium chloride. The potassium salt content of solvent I during thawing is preferably 0.50 to 8.0 mM, more preferably 1.0 to 7.0 mM, and even more preferably 2.5 to 6.0 mM as the final concentration of total potassium ions. Alternatively, it is preferable to contain 0.496 to 5.96 mM of potassium chloride.

[0071] The gluconate salts that can be used as solvent I during thawing are not particularly limited as long as they produce gluconate ions when dissolved in the solvent, and salts with various cations can be used. Examples include sodium gluconate and potassium gluconate. Solvent I during thawing preferably contains sodium gluconate. The gluconate salt content of solvent I during thawing is preferably 2.3 to 32.3 mM, more preferably 4.6 to 29.9 mM, and even more preferably 12.5 to 27.7 mM, as the final concentration of total gluconate ions.

[0072] The acetate salt that can be used as thawing solvent I is not particularly limited as long as it produces acetate ions when dissolved in the solvent, and salts with various cations can be used. Examples include sodium acetate and potassium acetate. Thawing solvent I preferably contains sodium acetate. The acetate content of thawing solvent I is preferably 2.7 to 37.8 mM, more preferably 5.4 to 35.1 mM, and even more preferably 13.5 to 32.5 mM, as the final concentration of total acetate ions.

[0073] A commercially available isotonic solution may be used as the thawing solvent I. Examples of usable products include Plasma-Lyte A and a solution of Plasma-Lyte A diluted with water. Specifically, the solution contains the following: Sodium chloride 9.00 ~ 108 mM Sodium gluconate 2.30-27.7 mM Sodium acetate 2.70~32.5 mM • Potassium chloride 0.496~5.96 mM Magnesium chloride 0.148~1.78 mM

[0074] In another preferred embodiment, the thawing solvent I may contain only sodium salts. Sodium salts usable in such thawing solvent I may be oxo salts, halides, oxides, hydroxides, inorganic salts, or organic salts. The sodium salt may be one type or a combination of several types. The thawing solvent I preferably contains only sodium chloride. The sodium salt content of the thawing solvent I is preferably 15.4 to 216 mM, more preferably 30.8 to 200 mM, and even more preferably 70.0 to 185 mM, as a final concentration of total sodium ions.

[0075] As the thawing solvent I, physiological saline or a solution of physiological saline diluted with water may be used.

[0076] Cells suspended in thawing solvent I along with the cryopreservation solution can be used directly for administration. From the viewpoint of administration, it is preferable that the mixture after mixing the cryopreservation solution and thawing solvent I is isotonic (having an osmotic pressure approximately equal to that of body fluids, specifically 285 ± 13 mOsm / L). In a preferred embodiment, the cryopreservation solution is a hypertonic solution (e.g., 1500-7000 mOsm / L), so thawing solvent I may be a solution with a low osmotic pressure. Those skilled in the art can appropriately determine the component concentrations of thawing solvent I by considering the dilution ratio of the cryopreservation solution with thawing solvent I.

[0077] Furthermore, when the dilution ratio is high (for example, 15 times or more), the influence of the composition of the liquid used for cryopreservation on the osmotic pressure of the mixed solution is small, so it is preferable to use an isotonic solution as the thawing solvent I. From this viewpoint, the thawing solvent I specifically contains the following: Sodium chloride 85.5 ~ 94.5 mM Sodium gluconate 21.8-24.2 mM Sodium acetate 25.6~28.5 mM • Potassium chloride 4.71~5.21 mM Magnesium chloride 1.40~1.55 mM Alternatively, the solvent I used during thawing is a liquid containing the following: Sodium chloride 146.3 ~ 161.7 mM

[0078] Regardless of the composition, it is preferable that thawing solvent I does not contain calcium ions at a concentration of 0.423 mM or higher. Furthermore, regardless of the composition, it is preferable that thawing solvent I does not contain glucose at a concentration of 5.55 mM or higher. Additionally, regardless of the composition, it is preferable that thawing solvent I does not contain lactate at a concentration of 27.7 mM or higher, because this may reduce the viability or cytotoxic activity of thawed cells. For these reasons, RPMI medium, Hanks' Balanced Salt Solution (HBSS)(+), and lactated Ringer's solution may not be suitable for use as thawing solvent I.

[0079] Regardless of the composition, it is preferable that solvent I at thawing concentration of 40% or higher does not contain serum, and it is even more preferable that it does not contain any serum at all. This is because the presence of serum may reduce the viability of cells.

[0080] The cell density when suspending in solvent I during thawing can be adjusted as appropriate, but it is preferable to set it to a cell density suitable for cell maintenance or a cell density suitable for administration. Specifically, 1 x 10 5 ~1x10 7 The cell density is cells / mL, preferably 2 x 10 5 ~5x10 6 cells / mL, more preferably 5x10 5 ~2x10 6 The value is cells / mL.

[0081] Cells can be maintained in solvent I during thawing for a relatively long time. After suspending the cells in solvent I during thawing, the suspension may be left to stand for several minutes to several hours, for example, 5 minutes to 6 hours, more preferably 30 minutes to 4 hours. This standing may be carried out at ambient temperature (e.g., 1 to 30°C, typically 15 to 25°C) or in a CO2 incubator (e.g., 36 to 42°C, typically 37°C).

[0082] (Solvent II during thawing) In this invention, cells frozen using thawing solvent I can be thawed, and then the cells can be cultured or activated using thawing solvent II. In this invention, thawing solvent II is the solution used to recover cells suspended in thawing solvent I and to resuspend them for culture or activation. Various types of thawing solvent II can be used depending on the purpose.

[0083] [Use in pharmaceutical compositions] The present invention provides a pharmaceutical composition comprising highly active NK cells, etc., which are recovered by an appropriate method, pretreated as necessary, and cryopreserved.

[0084] The pharmaceutical compositions provided by the present invention can be applied to the treatment and / or prevention of various diseases that are sensitive to highly active NK cells, etc. Examples of such diseases include cancer or infectious diseases, specifically including, but not limited to, skin cancer, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, prostate cancer, neuroblastoma, leukemia, and infectious diseases caused by viruses, bacteria, etc. The inventors have confirmed the effect of the present invention on a colorectal cancer model animal that would die within 30 days without treatment, using cells frozen and thawed by the present invention.

[0085] Cell therapy using the pharmaceutical composition of the present invention may be performed alone or in combination with surgery, chemotherapy, radiotherapy, antibody drugs, etc.

[0086] The features of one embodiment of the pharmaceutical composition provided by the present invention are shown below.

[0087] (Dosage form) Injectable drug (cell suspension)

[0088] (Ingredients / Content) Constituent cells: Highly active NK cells, etc. Content: 6×10 6 pieces~4.8×10 9 cells / 60kg

[0089] (Minor component) Complex electrolyte solution 10~45% Sodium chloride solution 10-45% 20-30% human serum albumin solution 5-30% Dimethyl sulfoxide 2-15% others or, 100% cryopreservation solution acceptable as a pharmaceutical additive

[0090] (Preparation method) Thaw the frozen composition in a constant temperature water bath at 37°C until completely thawed. Immediately after thawing, aseptically suspend it in a separately prepared isotonic solution that is acceptable as a pharmaceutical additive.

[0091] (Stability after thawing) The shelf life after thawing is 6 hours, preferably 4 hours, when stored at room temperature. [Examples]

[0092] [Methods common to both reference examples and embodiments] A) Method for culturing highly active NK cells Raw material 1: When using peripheral blood Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll (GE Healthcare, 17144002).

[0093] Raw material 2: In the case of frozen apheresis blood Frozen apheresis blood (HemaCare, PB001CLP) was thawed, washed, and concentrated using the Lovo Cell Processing System (FRESENIUS KABI) to obtain PBMCs.

[0094] The obtained PBMCs are fitted with CD3 beads* 1 CD34 beads* 2 (If using frozen apheresis blood) Add and suspend, incubate at 4°C for 15 minutes, then separate into buffer* 3 Add and suspend well, then centrifuge at 300 xg for 10 minutes. Remove the supernatant and fill with LD columns (Milteny Biotech, 130-042-901) up to 1 x 10⁶ per column. 8 The cells were suspended in 0.5 mL of separation buffer to the desired cell count. After adding 2 mL of separation buffer to the LD column, the cell suspension was added and the eluate from the LD column was collected. Another 1 mL of separation buffer was added to the LD column and the eluate was collected. The column was then washed with 1 mL of separation buffer, and the number of cells in the collected eluate was counted to calculate the total cell count. The cells were centrifuged at 500 xg for 5 minutes, and after removing the supernatant, the raw material 1: peripheral blood (5x10) was used. 5 cells / mL, Raw material 2: 1x10 if using frozen apheresis blood 6 KBM501 medium* to achieve a cells / mL ratio. 4 The culture was suspended in [a specific solution]. Culture was performed in a CO2 incubator (37°C, 5% CO2) using 6-well plates (Thermo Fisher Scientific, 140675), T-75 flasks (Thermo Fisher Scientific, 156499), or adhesive culture bags (Nipro). On day 9 of culture, KBM501 medium was added so that the final volume was 6 mL per well for 6-well plates, 50 mL per flask for T-75 flasks, or 500 mL per bag for bags, and the culture was incubated until day 14. In the following, cells obtained from PBMCs through this culture process will be referred to as "highly activated NK cell-like CD3-negative cells" or simply "highly activated NK cells."

[0095] *1: CliniMACS CD3, Milteny Biotech, 130-017-601 (1x10 7 (5 μL per cell) *2: CliniMACS CD34, Milteny Biotech, 130-017-501 (1x10 7 (2.5 μL per cell) *3: PBS containing 0.5% human type AB serum (Cosmo Bio, 12181301, inactivated at 56°C for 30 minutes) and 2 mM EDTA (Thermo Fisher Scientific, 15575-020) (Nacalai Tesque, 14249-24) *4: KBM501 (Kojin Bio, 16025015) contains either 5% human type AB serum (Cosmo Bio, 12181301, inactivated at 56°C for 30 minutes) or 5% UltraGRO (AventaCell, HPCPLCRL10) with 2U / mL heparin sodium (Nipro) added.

[0096] B) Method for harvesting highly active NK cells Collection Method 1: Conventional Method On day 14 of culture, the culture medium was collected, and 1 mM EDTA was added to the culture vessel to detach the adhered cells. After collecting the detached cells, the culture vessel was washed with PBS (Nacalai Tesque, 14249-24). All the collected cells were centrifuged, washed with PBS, and resuspended.

[0097] Recovery Method 2: The Method of the Present Application On day 14 of culture, the culture medium was collected, and 1 mM EDTA was added to the culture vessel to detach the adhered cells. The culture vessel, after collecting the detached cells, was washed with KBM501 medium. All the collected cells were centrifuged, washed with KBM501 medium, and resuspended.

[0098] C) Staining method using 7-AAD 1x10⁻¹⁰ highly active NK cells thawed under various conditions 5 Cells / well were centrifuged in a 96-well plate (IWAKI, 4870-800SP), the supernatant was removed, and 7-Amino-Actinomycin D (7-AAD) solution (Beckman Coulter, A07704) diluted in PBS was added and the cells were suspended. The cells were incubated at room temperature (15-25°C; the same applies to the following experiments) for 20 minutes. After staining, the cells were measured using a flow cytometer (BD LSR Fortessa, BD Bioscience). Room temperature refers to... [Reference example] Highly active NK cells, PBMCs, K562 (human chronic myeloid leukemia cell line), and THP-1 (human acute monocytic leukemia cell line) obtained using the procedure described in Method 1 for culturing and harvesting highly active NK cells were reacted with Mito-FerroGreen (Dojin Chemical Laboratories, M489) and measured using a flow cytometer (BD LSR Fortessa, BD Biosciences).

[0099] As shown in Figure A, highly active NK cells have a high iron content. Based on morphological observations and the time to death, five morphologies were hypothesized to significantly reduce the viability of highly active NK cells upon freeze-thaw cycles: Ferroptosis (oxidative stress), Apoptosis, Autophagy, Necrosis, and Necroptosis. Inhibitors for each morphology are listed below.

[0100] [Table 1]

[0101] The number of viable highly active NK cells obtained by the procedure described in Method 1 for culturing and harvesting highly active NK cells is counted, and 1 x 10 7Cells were suspended in 1 mL of STEM-CELLBANKER (ZENOAQ, CB045) and frozen at -80°C. After freezing for 48 hours or more, NK cells were thawed at 37°C in a water bath, then diluted 10-fold with the solvents described below. After standing at each temperature and time, the cells were observed and photographed using a light microscope.

[0102] <1> Dilute in KBM501 medium and incubate at 37°C for 3 hours. <2> Dilute in KBM501 medium containing Z-Vad and ferment at 37°C for 3 hours. <3> Dilute in KBM501 medium containing methylprednisolone and ferment at 37°C for 3 hours. <4> Dilute in KBM501 medium containing dexamethasone and ferment at 37°C for 3 hours. <5> Dilute in KBM501 medium and incubate at 4°C for 3 hours. <6> Dilute in KBM501 medium containing methylprednisolone, stand at 4°C for 3 hours, then at 37°C for 3 hours. <7> Dilute in KBM501 medium containing dexamethasone, stand at 4°C for 3 hours, then at 37°C for 3 hours.

[0103] The results are shown in Figure B. It was confirmed that highly active NK cells showed a significant decrease in viability after freeze-thaw cycles. Furthermore, this was not improved by the addition of methylprednisolone, dexamethasone, or Z-Vad after freeze-thaw cycles.

[0104] The highly active NK cells obtained using the procedure described in Method 1 for culturing and harvesting highly active NK cells were harvested and then subjected to freeze-thawing as indicated.

[0105] The freeze-thaw conditions and their results are summarized in the table below. Under the experimental conditions, freezing after washing with PBS resulted in no response to any viability improvement methods. It was considered difficult to obtain the desired effect with commonly known cell protection methods.

[0106] [Table 2] JPEG2023126695000003.jpg228170JPEG2023126695000004.jpg229170

[0107] [Example 1] (1-1) The number of viable highly active NK cells obtained from each of two healthy volunteers using the procedure described in Method 1 for culturing and harvesting highly active NK cells was counted, and each was divided into 1x10⁻¹⁰ cells. 7 cells, 8x10 6 Cells were suspended in 1 mL of STEM-CELLBANKER (ZENOAQ, CB045) and frozen at -80°C. After freezing for more than 48 hours, NK cells were thawed at 37°C in a water bath, and 1 x 10⁶ cells were collected immediately after thawing. 5 Cells were divided into 96-well plates (IWAKI). They were then suspended in KBM501 medium, lactate Ringer containing 3500 units / mL IL-2 (Ceroic® for Injection 40, Takeda Pharmaceutical Co.), 139.9 mM maltose (Fuso Pharmaceutical Co.), Plasma-Lyte A (Baxter) containing 3500 units / mL IL-2, and KBM501 medium containing 5.02 mg / mL sodium gluconate (Nacalai, 16720-22) to a 10-fold dilution, and incubated at 37°C under 5% CO2 for 3 hours. Cells immediately after thawing were centrifuged after division, while the other four groups were centrifuged after 3 hours of incubation. Measurements were performed using the method described for staining with 7-AAD, and the viability was calculated using FlowJo software (FLOWJO, LLC).

[0108] (1-2) In addition, 5x10 of highly active NK cells obtained from one healthy volunteer using the procedure described in Method 1 for culturing and harvesting highly active NK cells. 6 The cells were similarly frozen, thawed, processed, and measured, then analyzed using FlowJo software to calculate the viability rate.

[0109] The results are shown in Figure 1. Frozen highly active NK cells showed improved viability when diluted with Plasma-Lyte A during thawing. When diluted with KBM501 medium during thawing, no improvement was observed even when gluconic acid (sodium gluconate is one of the components contained in Plasma-Lyte A) was added.

[0110] [Example 2] The viable cells of highly active NK cells obtained by the procedure described in Method 1 for culturing and harvesting highly active NK cells were counted to 5x10 6 cells, 1x10 7 Cells were suspended in 1 mL of STEM-CELLBANKER and frozen at -80°C. After freezing for more than 48 hours, NK cells were thawed at 37°C in a water bath, and 1 x 10⁶ cells were collected immediately after thawing. 5 Cells were divided into 96-well plates. They were then suspended in KBM501 medium, lactated Ringer containing 3500 units / mL IL-2, Plasma-Lyte A containing 3500 units / mL IL-2, and Plasma-Lyte A containing 40% Serum (human type AB serum) to a 10-fold dilution, and incubated at 37°C under 5% CO2 for 3 hours. Cells immediately after thawing were centrifuged immediately after division, while the other four groups were centrifuged after 3 hours of incubation. Measurements were performed using the method described for staining with 7-AAD, and the viability was calculated by analyzing the data with FlowJo software.

[0111] The results are shown in Figure 2. Frozen, highly active NK cells showed improved viability when diluted with Plasma-Lyte A upon thawing, but worsened viability when diluted with Plasma-Lyte A containing 40% Serum (human type AB serum).

[0112] [Example 3] The number of viable cells of highly active NK cells obtained by the procedure described in Method 1 for culturing and harvesting highly active NK cells was counted in 1x10⁻¹⁰ 7Cells were suspended in 1 mL of STEM-CELLBANKER and frozen at -80°C. After freezing for 48 hours or more, NK cells were thawed in a water bath at 37°C, and then diluted with Plasma-Lyte A (solvent (1)) containing 3500 units / mL IL-2 (Imunase, Shionogi & Co., Ltd.) or KBM501 medium (solvent (2)). The number of viable cells, viability rate, and cytotoxic activity rate (%Lysis) after dilution were calculated for each group as described below.

[0113] <0> Taken from STEM-CELLBANKER immediately after thawing. <1> Dilute 10-fold with solvent (1), stand at 37°C for 1 hour, then add an equal volume of KBM501 medium and incubate at 37°C for 3 hours. <2> Dilute 10-fold with solvent (1), stand at 37°C for 1 hour, then add lactate Ringer containing an equal volume of IL-2 and incubate at 37°C for 3 hours. <3> Dilute 10-fold with solvent (1), stand at 37°C for 1 hour, then add an equal volume of KBM501 medium and incubate overnight at 37°C. <4> Dilute 10-fold with solvent (1), stand at 37°C for 1 hour, then add lactated Ringer gel containing an equal volume of IL-2 and incubate overnight at 37°C. <5> Dilute 10-fold with solvent (1) and let stand at 37°C for 3 hours. <6> Dilute 10-fold with solvent (2) and let stand at 37°C for 3 hours. <7> Dilute 10-fold with solvent (2) and let stand overnight at 37°C.

[0114] (Calculation of cytotoxic activity rate) To measure cytotoxic activity, we prepared a group in which NK cells and K562 cells were reacted, a negative control group consisting only of K562 cells, and a positive control group consisting of K562 cells fixed in 10% formalin.

[0115] 《NK cells》 After incubation for the time specified for each group, the cells were harvested and fermented in 10% FBS / RPMI1640 in 2x10⁻¹⁶ solutions. 6 The concentration was adjusted to cells / ml.

[0116] 《K562 cells》 K562 cells (human chronic myeloid leukemia cell line) were suspended in serum-free RPMI1640 medium, stained using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT), and finally stained 2x10⁶ cells in 10% FBS / RPMI1640. 6 The solution was prepared to achieve a concentration of cells / mL.

[0117] NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) in a 1:1 cell ratio, mixed, and reacted at 37°C under 5% CO2 for 2 hours. After reaction, the cells were centrifuged (500 xg, 5 minutes), the supernatant was removed, and 7-AAD solution diluted in PBS was added and the cells were resuspended. The cells were incubated at room temperature for 20 minutes. The cytotoxicity rate (%Lysis) was calculated by measuring with a flow cytometer and analyzing the data using FlowJo software.* 5 . *5: Cytotoxic activity rate = (SKOV3 cell death rate - negative control cell death rate) / (positive control cell death rate - negative control cell death rate) × 100

[0118] The results are shown in Figure 3. For highly active NK cells that were frozen and thawed, viability correlated with the number of viable cells. Furthermore, there was a tendency for an inverse correlation between the number of viable cells or viability and cytotoxic activity. Additionally, switching the treatment solution for the frozen and thawed cells from Plasma-Lyte A to another solution resulted in decreased viability.

[0119] [Example 4] The number of viable cells of highly active NK cells obtained by the procedure described in Method 1 for culturing and harvesting highly active NK cells was counted in 1x10⁻¹⁰ 6The cells were suspended in the following six solvents to a concentration of cells / mL and then seeded onto a low-adsorption 6-well plate (IWAKI, 4810-800SP) for pre-freezing treatment. For pretreatment, the following solvents were used: (1) KBM501 medium containing 10 μM 4-phenylbutyric acid (4-PBA, Tokyo Chemical Industry Co., Ltd., P0643), (2) KBM501 medium containing 100 μM 4-phenylbutyric acid, (3) Plasma-Lyte A, (4) Plasma-Lyte A containing 3000 units / mL IL-2 (Imunase, Shionogi & Co., Ltd.), (5) Plasma-Lyte A containing 10 μM Salubrinal (TOCRIS, 2347), and (6) PBS containing 23 mM sodium gluconate. Cells suspended in each solvent were incubated for 2 hours at 37°C under 5% CO2 for groups (1) and (2), and at room temperature for groups (3) to (6). After pretreatment, 1 x 10⁶ cells were incubated based on the cell count at the time of treatment. 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Pre-treated NK cells, frozen for at least 48 hours, were thawed at 37°C in a water bath and diluted as described below. All dilutions and incubations after thawing were performed in low-adsorption 6-well plates.

[0120] Pretreatment (1): KBM501 medium containing 10 μM 4-phenylbutyrate. Each solution was diluted 11-fold in Plasma-Lyte A containing 1000 units / mL IL-2 or in Plasma-Lyte A containing 1000 units / mL IL-2 and 100 μM 4-PBS, incubated at room temperature for 1 hour, and then diluted 5-fold in KBM501 medium.

[0121] Pretreatment (2): KBM501 medium containing 100 μM 4-phenylbutyric acid. Dilute 11-fold in Plasma-Lyte A, incubate at room temperature for 1 hour, then transfer to RPMI medium*. 6 Alternatively, each was diluted five-fold in RPMI medium containing 2400 units / mL of IL-2.

[0122] *6: RPMI medium (Nacalai Tesque, 30264-56) supplemented with 10% FBS (Sigma, 172012-500ML, inactivated at 56°C for 30 minutes).

[0123] Pretreatment (3) Plasma-Lyte A Each solution was diluted 11-fold in Plasma-Lyte A containing 1000 units / mL IL-2 or in Plasma-Lyte A containing 1000 units / mL IL-2 and 100 μM 4-PBA, incubated at room temperature for 1 hour, and then diluted 5-fold in KBM501 medium.

[0124] Pretreatment (4) Plasma-Lyte A containing IL-2 at 3000 units / mL After diluting 11-fold in Plasma-Lyte A and incubating for 1 hour at room temperature or 37°C under 5% CO2, the solutions were further diluted 5-fold in RPMI medium and RPMI medium containing 2400 units / mL of IL-2, respectively.

[0125] Pretreatment (5) Plasma-Lyte A containing 10 μM Salubrinal Each solution was diluted 11-fold in Plasma-Lyte A containing 1000 units / mL IL-2, and in Plasma-Lyte A containing 1000 units / mL IL-2 and 100 μM 4-PBA, respectively. After incubation at room temperature for 1 hour, the solutions were diluted 5-fold in KBM501 medium.

[0126] Pretreatment (6): PBS containing 23 mM gluconic acid. After diluting 11-fold in Plasma-Lyte A and incubating for 1 hour at room temperature or 37°C under 5% CO2, the samples were further diluted 5-fold in RPMI medium and RPMI medium containing 2400 units / mL of IL-2, respectively.

[0127] After incubating each group of cells diluted in culture medium overnight at 37°C under 5% CO2, the number of viable cells was counted. The experimental conditions are summarized below.

[0128] [Table 3]

[0129] The results are shown in Figure 4. Highly active NK cells showed improved viability after thawing when treated with KBM501 supplemented with 4-PBA before freezing.

[0130] [Example 5] The number of viable highly active NK cells obtained by the procedures described in the culture method and recovery method 1 and recovery method 2 of highly active NK cells was counted in 1x10⁻¹⁰ 6 NK cells were suspended in various solvents to a cell / mL concentration and seeded into low-adsorption 6-well plates for freeze pretreatment. NK cells obtained using recovery method 1 were suspended in PBS and incubated at room temperature for 1 hour. NK cells obtained using recovery method 2 were suspended in (1) KBM501 medium, (2) KBM501 medium containing 30 μM 4-phenylbutyrate, (3) KBM501 medium containing 30 μM Tauroursodeoxycholic Acid Dihydrate (TUDCA, Tokyo Chemical Industry Co., Ltd., T1567), and (4) KBM501 medium containing 30 μM 4-phenylbutyrate and 30 μM TUDCA, and incubated at 37°C under 5% CO2 for 2 hours. After pretreatment, 1 x 10⁶ cells were collected based on the cell count at the time of treatment. 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Pre-treated, highly active NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath, and each group was diluted 11-fold in Plasma-Lyte A and incubated at room temperature for 1 hour. After 1 hour, the cells were gently suspended and the number of viable cells was counted. Further dilution was performed in KBM501 medium, incubated at 37°C under 5% CO2 for 2 hours, and the number of viable cells was counted again to calculate the recovery rate relative to the number of cells at freezing.

[0131] The results are shown in the table below and in Figure 5. Pretreatment of highly active NK cells with KBM501 medium supplemented with 4-PBA or TUDCA before freezing improved the recovery rate after thawing.

[0132] [Table 4]

[0133] [Example 6] The number of viable highly active NK cells obtained by the procedure described in Method 2 for culturing and harvesting highly active NK cells was counted in 1x10⁻¹⁰ 6 Cells were suspended in KBM501 medium alone or in KBM501 medium containing TUDCA (10 μM, 30 μM, 90 μM, 270 μM, 810 μM) to a concentration of cells / mL. The seeds were then seeded into a low-adsorption 6-well plate and incubated at 37°C under 5% CO2 for 2 hours for freeze pretreatment. After pretreatment, 1 x 10⁶ cells were selected based on the cell count at the time of treatment. 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and frozen at -80°C. Pre-treated NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath, and 10 times the volume of Plasma-Lyte A was added to each group. The cells were incubated at room temperature for up to 3 hours. After 1 hour and 3 hours, the cells were gently suspended and the number of viable cells was counted. After 1 hour, the cells were also diluted 5-fold in KBM501 medium and incubated at 37°C under 5% CO2 for 2 hours, after which the number of viable cells was counted again. The recovery rate relative to the number of cells at the time of freezing was calculated from the number of viable cells at each point after thawing. In addition, the cytotoxic activity of highly active NK cells was measured at each time point.

[0134] (Calculation of cytotoxic activity rate) To measure cytotoxic activity, we prepared a group in which NK cells and K562 cells were reacted, a negative control group consisting only of K562 cells, and a positive control group consisting of K562 cells fixed in 10% formalin.

[0135] 《NK cells》 After thawing and diluting the cells using the method described, take the required amount based on the number of viable cells at the time of freezing, and then perform 2x10⁶ fermentation in 10% FBS / RPMI1640. 6 The solution was prepared to a concentration of cells / ml.

[0136] 《K562 cells》 K562 cells were suspended in serum-free RPMI1640 medium, stained using the PKH26 Red Fluorescent Cell Linker Kit, and then incubated in 10% FBS / RPMI1640 for 2x10⁻¹⁶ cells. 6 The solution was prepared to achieve a concentration of cells / mL.

[0137] NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) in a cell ratio of 2:1, mixed, and reacted at 37°C under 5% CO2 for 2 hours. After reaction, the cells were centrifuged (500 xg, 5 minutes), the supernatant was removed, and 7-AAD solution diluted in PBS was added and the cells were resuspended. The cells were incubated at room temperature for 20 minutes. The cytotoxicity rate (%Lysis) was calculated by measuring with a flow cytometer and analyzing the data with FlowJo software.

[0138] The recovery rates obtained under pretreatment and thawing conditions are shown in the table below. Pretreatment of highly active NK cells with KBM501 medium supplemented with TUDCA at various concentrations improved the recovery rate after thawing.

[0139] [Table 5]

[0140] The results of the cytotoxic activity measurement are shown in Figure 6. Improvement in cytotoxic activity was observed in a TUDCA concentration-dependent manner during pretreatment.

[0141] [Example 7] The number of viable highly active NK cells obtained by the procedure described in Method 2 for culturing and harvesting highly active NK cells was counted in 1x10⁻¹⁰ 6 Cells were suspended in KBM501 medium alone, KBM501 medium containing 266 μM, 810 μM, and 2400 μM TUDCA, or KBM501 medium containing 2.4% DMSO (Nacalai Tesque, 13445-74) to achieve a cell / mL ratio. The seeds were then seeded into low-adsorption 6-well plates and incubated at 37°C under 5% CO2 for 2 hours for freeze pretreatment. After pretreatment, 1 x 10⁶ cells were selected based on the cell count at the time of treatment.7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and cryopreserved at -80°C. Pre-treated NK cells, frozen for at least 48 hours, were thawed at 37°C in a water bath. Each group was then diluted 11-fold in either Plasma-Lyte A or Plasma-Lyte A containing 3000 units / mL of IL-2 and incubated at room temperature for up to 3 hours. After 1 hour and 3 hours, the cells were gently suspended and the number of viable cells was counted. After 1 hour, the cells were also diluted 5-fold in KBM501 medium and incubated at 37°C under 5% CO2 for 2 hours, after which the number of viable cells was counted again. The recovery rate relative to the number of cells at the time of freezing was calculated from the number of viable cells at each point after thawing.

[0142] Furthermore, the cytotoxic activity of highly active NK cells against K562 cells was measured at each thawed and diluted time point using the method described in Example 6.

[0143] Furthermore, highly active NK cells at 1 hour and 3 hours after thawing were evaluated in a solid tumor model (3D killing assay). Specifically, based on the cell count at the time of freezing, highly active NK cells were evaluated at 1x10⁶. 6 KBM501 medium was prepared to a concentration of cells / mL. 50 μL of the prepared NK cells were added to one SKOV3 sphere pre-seed in a 384-well plate, and the mixture was incubated at 37°C under 5% CO2 for 4 days. Subsequently, the cells were detached and harvested using Accutase, and expanded in RPMI medium in a 24-well plate. After 17 days of expanded culture at 37°C under 5% CO2, the cells were fixed with 4% PFA, stained with DAPI, and the area of ​​SKOV3 undamaged by NK cells was quantified using a fluorescence microscope (BZ-9000, KEYENCE) and the BZ-II observation application.

[0144] 《SKOV3 sphere》 SKOV3 cells (human ovarian cancer cell line) were incubated in 10% FBS / RPMI1640 in a 3x10⁶ solution. 4 Prepare the solution to a concentration of cells / mL, and place 3 x 10 cells per well in a 96-well plate. 3100 μL of seeds were seeded. The cultures were incubated at 37°C under 5% CO2 for 3 days to create spheres.

[0145] The recovery rates obtained under pretreatment and thawing conditions are shown in the table below. Pretreatment of highly active NK cells with KBM501 medium supplemented with TUDCA at various concentrations improved the recovery rate after thawing.

[0146] [Table 6]

[0147] Furthermore, the results of the cytotoxic activity measurement are shown in Figure 7-1, and the results of the evaluation in a solid tumor model (3D killing assay) are shown in Figure 7-2. In Figure 7-1, SKOV3 spheres without added NK cells are labeled as "nega".

[0148] By broadening the concentration range of TUDCA and adding evaluations using solid tumor models, it was found that TUDCA added before freezing was preferably used at a concentration between 2,400 and 267 μM, and that using 800 μM in particular resulted in high recovery rates and high toxicity activity against solid tumor models.

[0149] [Example 8] (8-1) Method for culturing and harvesting highly active NK cells: Count the number of viable highly active NK cells obtained by the procedure described in 2 and 1x10 7The cells were suspended in a STEM-CELLBANKER to a concentration of cells / mL and frozen at -80°C. Highly active NK cells frozen for more than 48 hours were thawed at 37°C in a water bath and then subjected to the following conditions: (1) Plasma-Lyte A, (2) PBS(-) containing 1.48 mM MgCl2·6H2O (Nacalai Tesque, 20909-55), (3) PBS(-), (4) KBM501 medium, (5) Plasma-Lyte A containing 62.2 mg / L CaCl2·2H2O (Nacalai Tesque, 08895-15), and (6) Plasma-Lyte A containing 0.1% glucose (50% glucose, Terumo). A, (7) glucose, (8) RPMI medium without serum components (Nacalai Tesque, 09892-15), and (9) RPMI medium containing glucose (Nacalai Tesque, 30264-85) were diluted 10-fold. (1) to (4) were incubated at room temperature for 2 hours, and (1) and (5) to (9) were incubated at 37°C for 3 hours. Subsequently, measurements were performed using the method described for staining with 7-AAD, and the results were analyzed using FlowJo software.

[0150] (8-2) Count the number of viable cells of highly active NK cells obtained by the procedure described in Method for Culture and Recovery of Highly Active NK Cells 1 or Method for Recovery 2, and 1 x 10 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and frozen at -80°C. Each highly active NK cell, frozen for at least 48 hours, was thawed in a water bath at 37°C and diluted 10-fold in (1) Plasma-Lyte A, (2) PBS(-) containing 1.48 mM MgCl2 (Nacalai Tesque, 95812-85), (3) PBS(-), (4) KBM501 medium, (5) Lactated Ringer's solution, or (6) HBSS(+) (Nacalai Tesque, 09735-75), and incubated at 37°C for 3 hours. Subsequently, measurements were performed using the method described for staining with 7-AAD, and the data was analyzed using FlowJo software.

[0151] (8-3) Similar to (8-1), however, the solvent used to dilute the cells after thawing was (1) PBS, (2) Plasma-Lyte A, (3) Plasma-Lyte A containing 27.7 mEq / L lactate (Nacalai Tesque, 20006-62), (4) Plasma-Lyte A containing 48.84 mg / L MgSO4·7H2O (Nacalai Tesque, 21002-85), (5) Lactoringer's solution, (6) Veen F (Fuso Pharmaceutical Co., Ltd.), (7) Solita-T1 (AY Pharma), (8) Solita-T3 (AY Pharma), (9) 5% glucose injection (Terumo), (10) RPMI medium, and incubated at 37°C for 3 hours. Regarding (3), lactate was added in accordance with the lactate concentration of Lactoringer's solution, and neutrality was confirmed with litmus paper. Regarding (4), MgSO4 was added in accordance with the MgSO4 concentration of HBSS. Subsequently, measurements were performed using the method described in the 7-AAD staining method and analyzed with FlowJo software.

[0152] (8-4) Similar to (8-2), however, the cells after thawing were treated with (1) PBS, (2) Plasma-Lyte A, (3) physiological saline (Otsuka Pharmaceutical), (4) bicarnate (Otsuka Pharmaceutical), or (5) Soluacet-F (Terumo) as the solvents used for dilution. Subsequently, measurements were performed using the method described in the 7-AAD staining method and analyzed with FlowJo software.

[0153] (8-5) Count the number of viable cells of highly active NK cells obtained by the procedures described in the culture method and recovery method 1 and recovery method 2 of highly active NK cells, and 1 x 10 7The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Highly active NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath. Immediately after complete thawing, the cells were diluted 10-fold with (1) Plasma-Lyte A, (2) physiological saline, (3) RPMI1640 medium, (4) lacto-Ringer's solution, and (5) 0.423 mM CaCl2 and (6) 1.36 mM CaCl2 prepared with Plasma-Lyte A, and each solution was allowed to stand at 37°C for 3 hours. Note that the CaCl2 in (5) was added to the RPMI1640 medium, and the CaCl2 in (6) was added to the lacto-Ringer's solution. Subsequently, measurements were performed using the method described for staining with 7-AAD, and the data was analyzed using FlowJo software.

[0154] (Results) The results of the analysis using FlowJo software are shown in Figures 8-1 to 8-5. Viability was improved by using the culture medium as the solvent before freezing and by using Plasma-Lyte A after thawing. After thawing and dilution, the samples could be maintained at room temperature (8-1).

[0155] [Example 9] (9-1) Method for culturing and harvesting highly active NK cells: Count the number of viable highly active NK cells obtained by the procedure described in 2 and 4x10 7 Cells were suspended in a STEM-CELLBANKER to a concentration of cells / mL and frozen at -80°C. Highly active NK cells frozen for more than 48 hours were thawed at 37°C in a water bath, diluted 10-fold in Plasma-Lyte A containing 3000 units / mL of IL-2, and allowed to stand at room temperature for up to 6 hours. Cytotoxic activity was analyzed at 2, 5, and 6 hours after thawing. Cytotoxic activity was analyzed by adding NK cells and K562 cells in a 1:1 cell ratio to a 96-well plate (IWAKI, 4870-800SP) using the method described in Example 9-1(b), mixing them, and reacting at 37°C under 5% CO2 for 2 hours.

[0156] The number of viable cells of highly active NK cells obtained by the procedure described in Method 2 for culturing and harvesting highly active NK cells was counted in 4x10⁻¹⁰ 7The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Highly active NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath, diluted 10-fold in Plasma-Lyte A containing 3000 units / mL of IL-2, and allowed to stand at room temperature for up to 4 hours. Viability changes and cytotoxic activity (fixed at 2 hours or E:T=1:1) were analyzed at points immediately after thawing, 1 hour, 2 hours, 3 hours, and 4 hours later.

[0157] a) Viability trends Mix the thawed cells well at each point, 1x10 5 Cells were sorted, measured using the method described for staining with 7-AAD, and analyzed with FlowJo software.

[0158] b) Cytotoxic activity (1) To measure cytotoxic activity, we prepared a group in which NK cells and K562 cells were reacted, a negative control group consisting only of K562 cells, and a positive control group consisting of K562 cells fixed in 10% formalin.

[0159] 《NK cells》 After dividing the required amount of NK cells based on the number of viable cells at the time of freezing, 1x10⁶ cells were froze in 10% FBS / RPMI1640. 6 The concentration was adjusted to cells / mL.

[0160] 《K562 cells》 K562 cells were suspended in serum-free RPMI1640 medium, stained using the PKH26 Red Fluorescent Cell Linker Kit, and then incubated in 10% FBS / RPMI1640 for 2x10⁻¹⁶ cells. 6 The solution was prepared to achieve a concentration of cells / mL.

[0161] NK cells and K562 cells were added and mixed in 96-well plates (IWAKI, 4870-800SP) in cell ratios of 1:2, 1:1, 2:1, and 4:1, and reacted at 37°C under 5% CO2 for 2 hours. After the reaction, measurements were performed using the method described for staining with 7-AAD, and the cytotoxicity rate (%Lysis) was calculated by analyzing the data with FlowJo software.

[0162] c) Cytotoxic activity (2) NK cells and K562 cells were prepared using the method described in b) Cytotoxic activity (1) above. The NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) in a cell ratio of 1:1, mixed, and then reacted at 37°C under 5% CO2 for 2 hours, 4 hours, 6 hours, and 8 hours. After the reaction was complete, measurements were taken using the method described in the 7-AAD staining method, and the cytotoxic activity rate (%Lysis) was calculated by analyzing the results with FlowJo software.

[0163] (9-2) Similar to (9-1), however, after thawing and dilution, the mixture was allowed to stand for up to 6 hours, and cytotoxic activity was measured at 2 hours, 5 hours, and 6 hours using the method described in b) above. Specifically, NK cells and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) in a cell ratio of 1:1, mixed, and then reacted at 37°C under 5% CO2 for 2 hours. After the reaction was complete, measurements were taken using the method described for staining with 7-AAD, and the cytotoxic activity rate (%Lysis) was calculated by analyzing the results with FlowJo software.

[0164] The results are shown in Figures 9-1 and 9-2. When highly active NK cells were pretreated, frozen, thawed, and diluted using an appropriate method, their viability and activity could be maintained for 4 hours even when left standing at room temperature in the administered form. Furthermore, it was confirmed that activity could be maintained for up to 6 hours. For example, the CAR-T cell preparation Kymriah is known to only maintain its viability and activity for 30 minutes in its administered form (after thawing). This invention significantly improves the handling of cells used in immunotherapy.

[0165] [Example 10] (10-1) Count the number of viable highly active NK cells obtained by the procedures described in Method for Culture and Recovery of Highly Active NK Cells 1 and Method for Recovery 2, and 1 x 10 7 cells / mL (1.5 ml tube. When using recovery method 1, i.e., when washing with PBS during recovery), 4 x 10 7 Cells were suspended in a STEM-CELLBANKER at a concentration of cells / mL (5 ml vial; using recovery method 2, i.e., when washing with KBM501 medium during recovery) and frozen at -80°C. Highly active NK cells that had been frozen for 48 hours or more were thawed at room temperature. The thawed cells were diluted 10-fold in Plasma-Lyte A or KBM501 medium at points of 5, 10, 15, 20, 25, 30, 60, 120, and 180 minutes immediately after complete thawing, and allowed to stand at room temperature for 180 minutes. After dilution to 180 minutes, measurements were performed using the method described for staining with 7-AAD, and the data was analyzed using FlowJo software.

[0166] (10-2) Count the number of viable cells of highly active NK cells obtained by the procedures described in Method for Culture and Recovery of Highly Active NK Cells 1 and Method for Recovery 2, and 1 x 10 7 cells / mL (1.5 ml tube. When using recovery method 1, i.e., when washing with PBS during recovery), 4 x 10 7 Cells were suspended in a STEM-CELLBANKER at a concentration of cells / mL (5 ml vial; using recovery method 2, i.e., when washing with KBM501 medium during recovery) and frozen at -80°C. Highly active NK cells frozen for more than 48 hours were thawed at 37°C in a water bath. The thawed cells were diluted 10-fold in Plasma-Lyte A or KBM501 medium at points of 5, 10, 15, 20, 25, 30, 60, 120, and 180 minutes immediately after complete thawing, and allowed to stand at room temperature for 180 minutes. After dilution to 180 minutes, measurements were performed using the method described for staining with 7-AAD, and the data was analyzed using FlowJo software.

[0167] The results are shown in Figure 10. When washed with PBS during recovery before freezing, the thawing time at room temperature was short (approximately 15 minutes) due to the small volume, but the viability was significantly low. On the other hand, when washed with KBM501 medium, the viability was maintained even after being left to stand at room temperature for 2-3 hours after natural thawing at room temperature (approximately 27 minutes). This method has an extremely wide tolerance range for thawing operations and can be said to have excellent handling characteristics as a pharmaceutical product.

[0168] Furthermore, even when thawing was performed at 37°C, the viability was maintained even after being left at room temperature for 2-3 hours after thawing, provided that the samples were washed with KBM501 medium during retrieval.

[0169] [Example 11] The number of viable highly active NK cells obtained by the procedures described in the culture method and recovery method 1 and recovery method 2 of highly active NK cells was counted in 1x10⁻¹⁰ 7 cells / mL (1.5ml tube), 4x10 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL (5 ml vial) and frozen at -80°C. Highly active NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath. Immediately after complete thawing, they were diluted 10-fold in Plasma-Lyte A or KBM501 medium and allowed to stand at room temperature for up to 6 hours. Immediately after dilution, and at 1, 2, 3, and 6 hours, 1 x 10⁶ cells were collected. 5 Cells were isolated, measured using the method described for staining with 7-AAD, and analyzed with FlowJo software.

[0170] It was found that when samples were washed with KBM501 medium during recovery, diluting them with Plasma-Lyte A after thawing sometimes helped maintain high viability. In addition, the freezing conditions could be either 5 ml vials or 1.5 ml tubes, but 5 ml vials (4x10) were preferred. 7 It was found that (cells / ml) is better. The ability to freeze highly active NK cells at high density during frozen shipment makes the product more compact and contributes to reducing transportation costs.

[0171] [Example 12] (12-1) Method for culturing and harvesting highly active NK cells: Count the number of viable highly active NK cells obtained by the procedure described in 2 and 4x10 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Highly active NK cells, frozen for more than 48 hours, were thawed at 37°C in a water bath. Immediately after complete thawing, Plasma-Lyte A was diluted 10-fold with 80%, 50%, and 10% Plasma-Lyte A diluted with UltraPure Distilled Water (invitrogen, 10977-015), and allowed to stand at room temperature and 37°C for up to 3 hours, respectively. Immediately after dilution, and at 1, 2, and 3 hours, 1x10⁶ cells were collected. 5 Cells were isolated, measured using the method described for staining with 7-AAD, and analyzed with FlowJo software.

[0172] (12-2) Cells were processed and analyzed in the same manner as in (12-1). However, instead of UltraPure Distilled Water (invitrogen, 10977-015), Plasma-Lyte A was diluted 10-fold with 80%, 50%, and 10% Plasma-Lyte A diluted with physiological saline, and allowed to stand at room temperature and 37°C for up to 3 hours, respectively.

[0173] (12-3) Cells were processed and analyzed in the same manner as in (12-1). However, instead of UltraPure Distilled Water (invitrogen, 10977-015), Plasma-Lyte A was diluted with physiological saline, or diluted 10-fold with physiological saline, and allowed to stand at room temperature and 37°C for up to 3 hours, respectively.

[0174] (12-4) The cells were processed and analyzed in the same manner as in (12-1). However, immediately after the cells were completely lysed, they were diluted 10-fold with 230 mM NaCl2, 150 mM KCl, 100 mM MgCl2, 11% trehalose, 10% sucrose, 5% sucrose, and 10% and 20% UltraGRO prepared with Plasma-Lyte A, physiological saline, and UltraPure Distilled Water, and each solution was allowed to stand at room temperature for up to 3 hours.

[0175] The cells were processed and analyzed in the same manner as in (12-5) and (12-4). However, the cells were diluted 10-fold in each solution and allowed to stand at 37°C for up to 3 hours.

[0176] (12-6) Method for culturing and harvesting highly active NK cells: Count the number of viable highly active NK cells obtained by the procedure described in 1 and 1x10 7 The cells were suspended in STEM-CELLBANKER to a concentration of cells / mL and stored frozen at -80°C. Highly active NK cells that had been frozen for more than 48 hours were thawed at 37°C in a water bath. Immediately after complete thawing, the cells were diluted 10-fold with 230mM NaCl2, 150mM KCl, 100mM MgCl2, 100mM CaCl2, 11% trehalose, 10% sucrose, 5% sucrose, and 10%, 20% UltraGRO, and 10%, 20%, 30%, and 40% AB Serum (CELLect, 2938249) prepared in Plasma-Lyte A, and allowed to stand at room temperature for up to 3 hours. Immediately after dilution, 1x10⁶ cells were measured at 1, 2, and 3 hour points. 5 Cells were isolated, measured using the method described for staining with 7-AAD, and analyzed with FlowJo software.

[0177] The cells were processed and analyzed in the same manner as in (12-7) and (12-6). However, the cells were diluted 10-fold with each solution and allowed to stand at 37°C for up to 3 hours.

[0178] (Results) The results of the analysis using FlowJo software are shown in Figures 12-1 to 12-7. Under conditions 12-1 to 12-3, the Plasma-Lyte A component used during thawing could be diluted to 1 / 10 of its original concentration, and the dilution solution could be either physiological saline or distilled water. In other words, it was found that the solution used during thawing could be either a hypotonic solution or physiological saline.

[0179] Under conditions 12-4 and 12-5, washing with KBM501 medium during recovery maintained viability as long as the solution used for thawing was NaCl, regardless of whether it was hypotonic or hypertonic. Viability was also maintained with isotonic solutions containing monosaccharides or polysaccharides, at room temperature or for short periods. On the other hand, viability was poor with 100 mM MgCl2.

[0180] Under conditions 12-6 and 12-7, it was found that maintaining viability was difficult when washed with PBS during recovery.

[0181] [List of solution compositions during thawing (units: mM)] [Table 7] [Summary of Examples 1-12] The above information is summarized in the table below. In the table below, unless otherwise stated as "xx hours", the unit for time is minutes. Also, regarding temperature, "RT" represents room temperature, and in all other cases it is 37°C. For viability, A represents 70% or more, B represents 50% or more but less than 70%, C represents less than 50%, and - represents not evaluated. For activity, A represents 70% or more, B represents 50% or more but less than 70%, C represents less than 50%, and - represents not evaluated. If the evaluation is A or B, the objective can be said to have been achieved.

[0182] [Table 8] JPEG2023126695000011.jpg169170JPEG2023126695000012.jpg236170JPEG2023126695000013.jpg220170JPEG2023126695000014.jpg172170JPEG2023126695000015.jpg232170

Claims

1. A method for protecting in vitro activated NK cells from freezing, characterized by treating the in vitro activated NK cells with a protective agent containing one of the following as an active ingredient: bile acids and phenylbutyric acid.

2. A protective agent for freezing in vitro activated NK cells, comprising one of the following as an active ingredient: bile acids and phenylbutyric acid.

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