Liquid for suspensing cells and use thereof

Abstract

The present invention addresses the problem of providing a thawing solution capable of maintaining or improving the toxic activity of highly active NK cells or the like after thawing. A liquid for suspending cells to be administered to a human fulfills the following conditions: (1) containing potassium ions, (2) pH 4.9 or more, (3) containing no chloride ions at a concentration of 135 mEq / L or more, (4) containing no glucose at a concentration of 5.55 mM or more, (5) containing no calcium ions at a concentration of 0.423 mM or more, (6) having a permeation pressure of 200-396 mOsm, and (7) containing any one organic acid selected from the group consisting of succinic acid, malic acid, lactic acid, citric acid, and acetic acid.

Description

Technical Field

[0001] The present invention relates to a method for treating cells such as highly active NK cells for administration to humans. Background Art

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

[0003] When administering cells to a patient, it is first necessary to study the use of cells collected from the patient himself so that no rejection reaction occurs. However, depending on the patient's condition, it is sometimes difficult to collect the cells required for treatment. In addition, there are individual differences in the degree of activation and proliferation in vitro, and there are situations where proliferation activation is difficult. In addition, since the activation and proliferation of cells require a certain amount of time, there is a problem of not being able to start treatment immediately. In this regard, it is desirable to be able to activate the cells in advance and preserve them for administration.

[0004] As methods for preserving cells, methods for preserving cells in a suspended state without freezing for short-term storage are known (e.g., Patent Document 1), and methods for preserving cells for long-term storage are known (e.g., Patent Document 2). As a method for further freezing cells and maintaining a high survival rate even after thawing, the use of freezing solutions containing sodium salts, potassium salts, sugars, cryoprotectants, and bicarbonates and / or carbonates has been studied (Patent Document 3).

[0005] In addition, a cell preservation method is known for preserving cells such as NK cells that are difficult to preserve in a solution containing sodium salts, potassium salts, sugars, and proteins as active ingredients (Patent Document 4). Furthermore, a cell preservation solution for cold storage containing potassium ions and lactate ions, an osmotic pressure of 200 to 350 mOsm / kg, and a pH of 6.0 to 8.0 is known (Patent Document 5), wherein the sugar content of the preservation solution is 0.5 to 150 mM and the calcium ion concentration is 1 to 4 mM. Furthermore, a cell preservation solution containing potassium ions and lactate ions, an osmotic pressure of 270 to 450 mOsm / l, a pH of 7 to 8, and containing K +, cell and tissue preservation fluid containing an organic acid anion (Patent Document 6). Lactic acid is exemplified as an organic acid. Furthermore, it is known that a physiological aqueous solution is used as a suspension of mammalian cells such as NK cells, and it is known that an isotonic (250-380 mOsm / l) aqueous solution such as Ringer's solution (Lactated Ringer's solution) can be used as a physiological aqueous solution (Patent Document 7). In the examples herein, lactated Ringer's solution ("Lactec Injection" manufactured by Otsuka Pharmaceutical Factory, Inc.) (paragraph 0038) is used, which contains potassium and lactic acid, has a pH of 6.0-7.5, and does not contain glucose.

[0006] The present inventors have developed a method for treating frozen highly active NK cells and a liquid containing sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride as a liquid to be used when thawing frozen highly active NK cells (Patent Document 8).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 2006-230396

[0010] Patent Document 2: Japanese Patent Application Laid-Open No. 2002-233356

[0011] Patent Document 3: WO2011 / 021618

[0012] Patent Document 4: WO2013 / 115322

[0013] Patent Document 5: Patent No. 4947948

[0014] Patent Document 6: WO2002 / 001952

[0015] Patent Document 7: Japanese Patent Application Laid-Open No. 2013-233102

[0016] Patent Document 8: WO2021 / 177279

[0017] Patent Document 9: PCT / JP2022 / 033487

[0018] Non-patent literature

[0019] Non-patent literature 1: Liu E, et al. N Engl J Med. 2020;382:545-53 Summary of the Invention

[0020] Problems to be solved by the invention

[0021] If there is a thawing liquid with a more general composition, which can maintain or improve the toxic activity of the thawed high-activity NK cells, then it is desirable. In addition, if such a liquid is also suitable as a drip, then it is more desirable.

[0022] Means for solving problems

[0023] The present invention provides the following contents.

[0024] [1] A liquid for suspending cells for administration to humans, which satisfies the following:

[0025] (1) Contains potassium ions

[0026] (2) pH 4.9 or above

[0027] (3) Does not contain chloride ions at a concentration of 135 mEq / L or more

[0028] (4) Does not contain glucose at a concentration of 5.55 mM or higher

[0029] (5) Does not contain calcium ions at a concentration of 0.423 mM or more

[0030] (6) Osmotic pressure is 200-396 mOsm

[0031] (7) Contains any one organic acid selected from succinic acid, malic acid, lactic acid, citric acid and acetic acid.

[0032] [2] The liquid described in 1, wherein (7) contains succinic acid.

[0033] [3] The liquid according to 1 or 2, further comprising (8) L-cysteine ​​or acetylcysteine.

[0034] [4] The liquid according to any one of 1 to 3, further comprising (9) albumin.

[0035] [5] The liquid according to any one of 1 to 4, which is used for diluting a frozen product containing cells for administration to humans or a thawed product thereof.

[0036] [6] The liquid described in any one of 1 to 4,

[0037] (1) Contains potassium ions at a concentration of 4.00 mEq / L or higher

[0038] (4) Does not contain glucose

[0039] (5) Does not contain calcium ions.

[0040] [7] The liquid according to any one of 1 to 6, which is a pharmaceutical composition.

[0041] [8] A pharmaceutical composition comprising the liquid according to any one of items 1 to 4 and a cell population for administration to a human, wherein the cells for administration to a human are lymphocytes.

[0042] [9] A pharmaceutical composition comprising a population of cells for administration to a human suspended in the liquid according to any one of 1 to 7, wherein the cells for administration to a human are highly active NK cell-like allogeneic CD3-negative cells.

[0043]

[10] The pharmaceutical composition described in 9, wherein the population of highly active NK cell-like allogeneic CD3-negative cells has undergone a freezing step.

[0044]

[11] A method for providing a pharmaceutical composition for infusion containing cells for administration to humans, comprising the steps of thawing frozen cells and suspending the thawed cells in a liquid according to any one of items 1 to 7 to form a pharmaceutical composition for infusion.

[0045] Effects of the Invention

[0046] According to the present invention, the cytotoxic activity of frozen highly active NK cells and the like after thawing can be enhanced.

[0047] Although a secondary effect, the use of specific organic acids in the thawing solution can maintain or enhance the high cytotoxic activity of highly active NK cells even in the low pH range. Considering the range of specifications for existing intravenous infusions, the ability to use the product in the low pH range of 6 is believed to expand its clinical usefulness. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] [ Figure 1-1 ] Toxic activity against tumor cells (Example 1)

[0049] [ Figure 1-2 ] Toxic activity against tumor cells (corrected value; Example 1)

[0050] [ Figure 2 Evaluation of pH Adjusters (Malic Acid, Succinic Acid; Example 2)

[0051] [ Figure 3 Evaluation of pH Adjusters (Citric Acid, Lactic Acid, Acetic Acid; Example 3)

[0052] [ Figure 4-1 Evaluation of pH Adjusters (Ascorbic Acid; Example 4)

[0053] [ Figure 4-2 Evaluation of pH Adjusters (Immediately after Thawing; Example 4)

[0054] [ Figure 4-3 Evaluation of pH Adjusters (4 hours after thawing and dilution, Example 4)

[0055] [ Figure 5-1 Activity after thawing (succinic acid, pH 5.9-8.0; Example 5)

[0056] [ Figure 5-2 Activity after thawing (succinic acid, pH 5.9-8.0; Example 5)

[0057] [ Figure 6-1 Activity after thawing (succinic acid, pH 4.9-5.9; Example 6)

[0058] [ Figure 6-2 Activity after thawing (succinic acid, pH 4.9-5.9; Example 6)

[0059] [ Figure 7 Summary of Examples 5 and 6. Relative % lysis (E:1 = 1 correction) and 7-AAD-4 gate (%). PlasmaLyte-A was used as 1.0. Columns are Mean + SD. DETAILED DESCRIPTION

[0060] In the present invention, unless otherwise specified, mM is used to have the same meaning as mmol / L. When a numerical range is expressed as x to y, the range includes both end values ​​x and y.

[0061] The present invention relates to methods for thawing frozen cells for administration to humans. In the present invention, unless otherwise specified, thawing refers to melting the frozen material. Thawing may involve adding a liquid for dilution. Converting a frozen solid into a liquid is sometimes referred to as melting.

[0062] [Applicable cells]

[0063] The present invention can be applicable to various cells. One of the cells to which the present invention can be preferably applied is a cell administered to a human, preferably a cell that has undergone an activation operation in vitro for a cell administered to a human, such a cell comprising NK cells (highly active NK cells) with high cytotoxic activity, etc. In addition, the cells to which the present invention can be preferably applied include GAIA-102 (highly active NK cell-like allogeneic CD3 negative cells prepared by amplification and activation culture technology of peripheral blood mononuclear cells). Other examples of cells to which the present invention can be preferably applied are CAR-T and CAR-NK. The activation operation is typically performed by incubating cells in a culture medium containing interleukin (IL) -2. It should be noted that, in the following, sometimes the case of applying the present invention to NK cells or highly active NK cells is used as an example for explanation, but if it is a person skilled in the art, based on its description, it can also be appropriately understood that the present invention is applicable to the case of cells other than it.

[0064] Generally, NK cells are large granular lymphocytes that do not express T cell receptor (TCR), CD3 as T cell universal markers and as the B cell receptor of membrane immunoglobulin, and are usually CD16 positive and CD56 positive in people. If it is a person skilled in the art, it can be easily judged whether it is a NK cell based on the expression pattern of cell surface markers etc. NK cells have cytotoxic activity, and the presence or absence of the cytotoxic activity, degree can be measured with known various methods. NK cells can include peripheral blood NK cells, cord blood NK cells, primary NK cells, cultured NK cells, and highly active NK cells.

[0065] (raw materials)

[0066] The raw materials of highly active NK cells, highly active NK cell-like allogeneic CD3-negative cells, etc. that the present invention can preferably apply can be peripheral blood, umbilical cord blood, bone marrow and / or lymph nodes, blood collected by apheresis (apheresis blood). In addition, the raw materials can be prepared from at least one cell selected from the following: hematopoietic stem cells of any stem cell origin selected from embryonic stem cells, adult stem cells and artificial 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, peripheral blood mononuclear cells. The donor of the raw material can be the patient himself who receives immunotherapy with highly active NK cells, etc., a close relative of the patient, or a healthy person who has no blood relationship with the patient. There can be multiple donors.

[0067] (Culture medium)

[0068] Culture media for culturing highly active NK cells include, but are not limited to, KBM501 medium (Kohjin Bio Co., Ltd. Contains 1,750 JRU / mL of IL-2), COS-008 (Cosmo Bio. Contains 1,750 JRU / mL of IL-2), FKCM101 (Fukoku. Does not contain IL-2 or contains 175 IU / mL of IL-2), CellGro SCGM medium (Cellgenix, Iwai Chemicals Co., Ltd.), X-VIVO15 medium (Lonza, Takara Bio Co., Ltd.), Gibco (registered trademark) CTS (registered trademark) AIM V (registered trademark) Medium (Thermo Fisher Scientific. A serum-free medium of 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, and the like. Preferred examples are KBM501 culture medium, FKCM101 or COS-008. It should be noted that, in the present invention, except for the cases specifically described, when culturing cells, it means that the cells are maintained in a culture medium or a liquid based thereon for a certain period of time for any purpose selected from the group consisting of cell survival maintenance, cell expansion, and cell activation. When a certain period of time is treated at a specific temperature, it is sometimes referred to as incubation.

[0069] Sometimes IL-2 is added to the culture medium at a concentration that can achieve the purpose of the present invention. The concentration of IL-2 may be 2500 IU / mL to 2813 IU / mL. IL-2 preferably has a human amino acid sequence and is preferably produced using recombinant DNA technology for safety. The concentration of IL-2 is sometimes expressed in Japanese standard units (JRU) and international units (IU). Since 1 IU is approximately 0.622 JRU, 1750 JRU / mL of existing culture medium is equivalent to approximately 2813 IU / mL.

[0070] In some cases, any one of IL-12, IL-15, and IL-18 may be added simultaneously with or in place of the aforementioned IL-2 at a concentration sufficient to achieve the objectives of the present invention (Non-Patent Document 2: Leong JW et al. Biol Blood Marrow Transplant 20 (2014) 463-473). The concentration of each cytokine is independent of the presence or concentration of other cytokines and may range from 1 pg / mL to 1 μg / mL. IL-2 preferably has a human amino acid sequence and, for safety reasons, is preferably produced using recombinant DNA technology.

[0071] The culture medium may be supplemented with the subject's own serum, human AB serum available from BioWhittaker and other companies, or human serum albumin from donated blood available from the Japanese Red Cross Society. Autologous serum and human AB serum are preferably added at a concentration of 1 to 10%, and human serum albumin from donated blood is preferably added at a concentration of 1 to 10%. Human platelet lysate (HPL) may be added in addition to or in place of serum. HPL is commercially available, including the UltraGRO™ series (AventaCell BioMedical). When HPL is used, heparin sodium may be added to the culture medium.

[0072] In the culture medium, under the condition that the culture effect of NK cells is not damaged, appropriate proteins, cytokines, antibodies, compounds and other components may be included. In addition to the above-mentioned IL-2, IL-12, IL-15, and IL-18, cytokines may be IL-3, IL-7, IL-21, stem cell factor (SCF), and / or FMS-like tyrosine kinase 3 ligand (Flt3L). They all preferably have human amino acid sequences and are preferably produced using recombinant DNA technology for safety.

[0073] The culture medium is preferably a serum-free medium. The serum-free medium preferably contains serum albumin, transferrin, and insulin. Serum-free culture media for culturing lymphocytes have been developed and are commercially available, and these can be utilized in the present invention. One preferred example of a serum-free culture medium is a serum-free medium obtained by adding a component to a basal medium to support the proliferation of human T cells, commercially available CTSImmune Cell SR (Thermo Fisher Scientific).

[0074] The medium may be replaced or supplemented at any time after the start of culture, provided that the desired culture effect is achieved, but is preferably performed every 3 to 5 days.

[0075] Culture vessels used for culture 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 NK cell culture effect, but are typically cultured at 37°C, 5% CO₂, and a saturated water vapor atmosphere. The culture time is not particularly limited, provided the desired culture effect is achieved.

[0076] Highly active NK cells and the like to which the present invention can be preferably applied include the following [1], [2], [3], and [4].

[0077] [1] NK cells having the following characteristics (1) and (2):

[0078] (1) CD16 positive, high expression of CD56, and CD57 negative.

[0079] (2) NKG2C positive, NKG2A negative to low expression, and CD94 positive.

[0080] The highly active NK cells of [1] may be CD16-high expressing cells. In addition, regardless of whether the highly active NK cells of [1] are CD16-high expressing cells, they may further have the following characteristics.

[0081] (3) When the NK cells were used as effector cells (E) and K562 cells were used as target cells (T) in a co-culture at a mixing ratio (E:T) of 1:1, the cytotoxic activity was 50% or more.

[0082] [1] Highly active NK cells can also be expressed as follows:

[0083] NK cells having the following characteristics (1) and (3) are obtained by culturing a cell population obtained by removing CD3-positive cells from peripheral blood mononuclear cells derived from healthy individuals using CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, catalog number 130-017-601), LD columns (e.g., Miltenyi Biotec, catalog number 130-042-901), and a separation buffer (e.g., PBS containing 0.5% human AB type serum (inactivated) and 2 mM EDTA) for 14 days in an appropriate medium (e.g., COS-008 supplemented with 5% human AB type serum (inactivated)):

[0084] (1) CD16 positive, high expression of CD56, and CD57 negative.

[0085] (3) When the NK cells were used as effector cells (E) and K562 cells were used as target cells (T) in a co-culture at a mixing ratio (E:T) of 1:1, the cytotoxic activity was 50% or more.

[0086] For details on the characteristics of highly active NK cells [1] and a more specific preparation method, please refer to Japanese Patent Application Laid-Open No. 2018-193303.

[0087] [2] The following cells:

[0088] Cells that are CCR5-positive, CCR6-positive, and CXCR3-positive and CD3-negative.

[0089] [2] The cells may further be CD11c highly expressed.

[0090] The cells of [2] can also be represented as follows:

[0091] Cells that are CCR5-positive, CCR6-positive, CXCR3-positive, integrin α1-positive, integrin α3-positive, integrin β3-negative, and CD3-negative. Alternatively, cells that are CCR5-positive, CCR6-positive, CXCR3-positive, highly express CD11a and CD11c, and are CD3-negative, wherein high expression is determined by comparison with expression in a population of NK cells obtained from peripheral blood and not subjected to parenchymal culture.

[0092] According to the research conducted by the present inventors, the cells of [2] exhibited extremely high cytotoxic activity against solid cancers that form tumor masses. For details on the characteristics of the cells of [2] and a more specific preparation method, please refer to Japanese Patent Application Laid-Open No. 2019-170176.

[0093] [3] Highly active NK cells can be obtained by the following method:

[0094] Suspended CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, 130-017-601) were added to mononuclear cells obtained from fresh peripheral blood or frozen apheresis blood (1×10 7 cells 5 μL)), and in the case of using frozen apheresis blood, further add suspended CD34 beads (e.g., CliniMACS CD34, Miltenyi Biotec, 130-017-501 (per 1×10 7 2.5 μL of cells) were incubated at 4°C for 15 minutes, and then separation buffer (e.g., PBS containing 0.5% human AB serum (inactivated at 56°C for 30 minutes) and 2 mM EDTA) was added to fully suspend the cells and centrifuged. The supernatant was removed and a maximum of 1 × 10 cells were added to each LD column (e.g., Miltenyi Biotec, 130-042-901). 8The cells were suspended in 0.5 mL of separation buffer according to the number of cells. After adding 2 mL of separation buffer in advance, the cell suspension was added to the LD column and the eluate from the LD column was recovered. 1 mL of separation buffer was further added to the LD column and the eluate was recovered. The recovered liquid was centrifuged and the supernatant was removed. In the case of peripheral blood, 5 × 10 5 cells / mL, and 1×10 6 The cells are suspended in an appropriate culture medium (e.g., KBM501 medium containing 5% human AB serum (inactivated at 56°C for 30 minutes) or 5% UltraGRO (AventaCell, HPCPLCRL10) supplemented with 2 U / mL heparin sodium) at a concentration of 1 cell / mL, the culture medium is replaced as appropriate, and the cells are cultured until day 14.

[0095] For the specific method of preparing highly active NK cells [3], please refer to the examples in this specification.

[0096] [4] When the cells of [1] to [3] are obtained, any one of IL-12, IL-15, and IL-18 is added simultaneously with or instead of IL-2 at a concentration sufficient to achieve the purpose of the present invention, and the cells are cultured. For a specific method for preparing such cells, reference can be made to the aforementioned Non-Patent Document 2.

[0097] In addition, the present invention is sometimes described below using the case of using highly active NK cells as an example. However, those skilled in the art will also understand, based on this description, that the present invention can also be applied to other cells that have undergone activation operations using certain cytokines in vitro.

[0098] (Cytotoxic activity)

[0099] In the present invention, when referring to highly active NK cells, etc., the term "activity" or "cytotoxic activity" refers to the ability of the subject cells (effector cells, E) to lyse target cells (T), unless otherwise specified. Cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells and is calculated using the following formula.

[0100] (Cell death in the case of co-culture with effector cells - natural cell death (negative control)) / (maximum cell death (positive control) - natural cell death (negative control)) × 100

[0101] In the determination of cytotoxic activity, usually, the mixing ratio (E:T) of effector cells and target cells and the time of co-culture of effector cells and target cells are appropriately set according to the degree of cytotoxic activity of effector cells, etc., and the intensity of activity can be corresponding to the type of cells used. When NK cells are used as effector cells, target cells are sometimes K562 cells, acute myeloid leukemia cells, chronic myeloid leukemia cells, but are not limited to these. Effector cells and target cells, live cells and dead cells can be distinguished and quantified by reagents such as antibodies labeled with radioactive substances, fluorescent dyes, etc. The cytotoxic activity when NK cells are used as effector cells is measured, for example, under the following conditions: using K562 cells as target cells, setting E:T=1:0.05~10, preferably setting it to 1:0.1~5, and setting the incubation time to 0.5~18 hours, preferably setting it to 1~12 hours.

[0102] In the present invention, when the activity of NK cells, etc. is high, unless otherwise specified, it means that the cytotoxic activity is 50% or higher when the target cells are K562 cells, mixed at an E:T ratio of 2:1, and co-cultured for 1 to 3 hours, more specifically 2 hours. The activity is preferably 60% or higher, and more preferably 70% or higher.

[0103] [Recycle]

[0104] In the present invention, before the freezing step described later, the highly active NK cells etc. to be frozen are recovered from the culture system. Recovery can be carried out by separating the culture medium and the cells by centrifugation. As needed, EDTA of appropriate concentration can be added to the culture system to peel off the adhered cells from the culture vessel surface. In addition, the culture vessel surface after the culture medium is recovered can be cleaned with an appropriate solution to obtain residual cells. The obtained cells are cleaned with an appropriate solution as needed and suspended in an appropriate solution.

[0105] In the recovery step, for the peeling and washing of cells, solutions such as culture medium, isotonic solution, buffer solution can be applied. As examples of usable culture medium, KBM501 culture medium, COS-008, FKCM101, CellGro SCGM culture medium, X-VIVO15 culture medium, Gibco (registered trademark) CTS (registered trademark) AIM V (registered trademark) Medium, CTS0pTmizer T Cell Expansion Basal Medium, IMDM, MEM, DMEM, RPMI-1640 are listed. Isotonic solution refers to a liquid with an osmotic pressure roughly equal to the osmotic pressure (285 ± 5mOsm / L) of body fluid (plasma). In the present invention, it refers to a liquid with an osmotic pressure of 285 ± 13mOsm / L. For example, the osmotic pressure of Plasma-Lyte A is 294mOsm / L, and the osmotic pressure of PBS (-) is 280 ± 4mOsm / L (freezing point depression method). Examples of usable isotonic solutions include Plasma-Lyte A (Baxter), physiological saline, Ringer's solution (lactated Ringer's, acetic Ringer's, bicarbonate Ringer's, etc.), and 5% aqueous glucose solution. Examples of usable buffers include phosphate-buffered saline (PBS), Tris-HCl buffer, Tris-acetate buffer, and HEPES buffer.

[0106] One of preferred embodiments of the solution used in the recovery step is culture medium, more preferably human lymphocyte culture medium. Human lymphocyte culture medium can include human serum albumin, human transferrins, recombinant human insulin and recombinant human IL-2. Preferred embodiments of such culture medium are KBM501 culture medium, FKCM101 or COS-008. KBM501 culture medium includes human serum albumin, human transferrins, recombinant human insulin, recombinant human IL-2, does not include protein beyond it. In addition, KBM501 culture medium includes antibiotic (kantlex), NaHCO , L-glutamine, pH adjusting agent.

[0107] When PBS(-) is used in the recovery step, the survival rate of cells upon thawing may be reduced and poor. 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.

[0108] [Preprocessing]

[0109] In the present invention, before the freezing step described below, the highly active NK cells to be frozen may be pretreated. Pretreatment is to suspend the recovered cells in a solution containing additives. Pretreatment includes recovery using a solution containing additives.

[0110] As the additive used in pretreatment, can use be selected from any one among bile acid and phenylbutyric acid.The example of bile acid is tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid, lithocholic acid, iodideoxycholic acid, hyodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid or its analog, derivative.The example of phenylbutyric acid is 4-phenylbutyric acid (4-PBA), glyceryl (three-4-PBA), phenylacetic acid, 2-POAA-OMe, 2-POAA-NO , 2-NOAA or its pharmaceutically allowed salt, analog, derivative or prodrug.The particularly preferred example of the additive used in pretreatment is selected from any one among TUDCA and 4-PBA.

[0111] When bile acid is used as a pretreatment additive, the concentration can be appropriately set, 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 TUDCA is used. When phenylbutyric acid is used as a pretreatment additive, the concentration can be appropriately set, 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 4-PBA is used.

[0112] Another example of the additive used for pretreatment is dimethyl sulfoxide (DMSO). The concentration can be appropriately set, but is preferably 0.5 to 15%, more preferably 1 to 12.5%, and further preferably 2 to 10%.

[0113] The solution used for pretreatment is the same as the solution used during recovery, and can be solutions such as culture medium, isotonic solution, buffer solution. One of preferred embodiments of the solution used in pretreatment is culture medium, more preferably human lymphocyte culture medium, further preferably KBM501 culture medium, FKCM101 or COS-008. In addition, the culture medium used in pretreatment can include human serum albumin, human transferrins, recombinant human insulin and recombinant human IL-2, and can include antibiotic (kantlex), NaHCO , L-glutamine, pH adjusting agent.

[0114] The time for pretreatment is not particularly limited. After suspending the cells for pretreatment, the suspension can be allowed to stand for several minutes to several hours, for example, 5 minutes to 4 hours, more preferably 30 minutes to 3 hours. Standing can be performed 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).

[0115] The cell density during pretreatment can be appropriately set to a cell density suitable for cell maintenance. Specifically, 1×10 5 ~1×10 7 cells / mL, preferably 2×10 5 ~5×10 6 cells / mL, more preferably 5×10 5 ~2×10 6 cells / mL.

[0116] In a particularly preferred embodiment, the pretreatment is performed in KBM501 medium, FKCM101 or COS-008 medium supplemented with TUDCA at 400 to 1000 μM or 4-PBA at 10 to 100 μM, at a cell density of 5×10 5 ~2×10 6 At this point, the cells can be suspended at 10 cells / mL and incubated at 37°C, 5% CO2 for 30 minutes to 3 hours.

[0117] Pretreatment is not essential in the present invention, but pretreatment of highly active NK cells with KBM501 medium supplemented with 4-PBA or TUDCA before freezing can improve the survival rate (also called recovery rate) of cells upon thawing compared to the case without pretreatment.

[0118] [freeze]

[0119] In the present invention, the recovered, preferably pretreated, cells are frozen using conventional procedures. Specifically, the cell count and viability are confirmed as needed, the supernatant is removed by centrifugation, and the cells are suspended in a freezing medium to an appropriate cell density. The cell suspension is dispensed into a freezing container and then frozen and stored in a -80°C deep freezer. If necessary, freezing and storage can be performed in a liquid nitrogen tank.

[0120] The cryopreservation solution that can be used in the present invention may contain sodium salts, potassium salts, sugars, bicarbonates, carbonates, and cryoprotectants.

[0121] The sodium salt that can be used is not particularly limited as long as it produces sodium ions when dissolved in a solvent, and can be an oxoacid salt, a halide, an oxide, a hydroxide, an inorganic salt, or an organic acid salt. One or more sodium salts can be combined. In the present invention, sodium chloride is preferably used as one, and sodium chloride and sodium citrate are preferably used as multiple. The sodium salt content is not particularly limited, but is preferably 0.01 to 5000 mM, more preferably 0.1 to 1000 mM, and even more preferably 1 to 300 mM, based on the final concentration of the total sodium ions contained in the freezing preservation solution.

[0122] The potassium salt that can be used is not particularly limited as long as it produces potassium ions when dissolved in a solvent, and can be an oxoacid salt, a halide, an oxide, a hydroxide, an inorganic salt, or an organic acid salt. One or more potassium salts can be combined. In the present invention, potassium chloride is preferably used. The potassium salt content is not particularly limited, but is preferably 0.01 to 5000 mM, more preferably 0.1 to 1000 mM, and even more preferably 1 to 100 mM, based on the final concentration of the total potassium ions contained in the freezing preservation solution.

[0123] The bicarbonate salt that can be used is not particularly limited as long as it produces bicarbonate ions when dissolved in a solvent, and salts with various cations can be used. For example, ammonium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium bicarbonate, magnesium bicarbonate, etc. are listed. The carbonate salt that can be used is not particularly limited as long as it produces carbonate ions when dissolved in a solvent, and salts with various cations can be used. For example, ammonium carbonate, potassium carbonate, calcium carbonate, sodium carbonate, barium carbonate, magnesium carbonate, etc. are listed. These bicarbonates and / or carbonates can be combined into one or more. In the present invention, sodium bicarbonate is preferably used. The content of the bicarbonate and / or carbonate is not particularly limited, and is preferably 0.01 to 1000 mM, more preferably 0.1 to 500 mM, and even more preferably 1 to 100 mM, based on the final concentration of the total bicarbonate ions and carbonate ions contained in the freezing preservation solution.

[0124] 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, further preferably 1 / 10 to 100 / 1, further preferably 1 / 1 to 100 / 1, further preferably 10 / 1 to 50 / 1.

[0125] Usable sugars include monosaccharides, oligosaccharides, and 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. One or more of these sugars may be used in combination. In the present invention, at least one sugar selected from glucose, galactose, fructose, mannose, xylose, and arabinose is preferred, with glucose being more preferred. The sugar content in the cryopreservative 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.

[0126] As examples of usable frost protection agents, dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), ethylene glycol, glycerol, etc. are listed. One or more frost protection agents can be combined. In the present invention, it is preferred to use any one selected from DMSO and hydroxyethyl starch. In the case of using DMSO and hydroxyethyl starch in combination as frost protection agents, the total content is preferably included in the aforementioned range, and with respect to their respective concentrations, the DMSO concentration is preferably 0.01 to 50%, more preferably 1 to 30%, and further preferably 2 to 15%, and the hydroxyethyl starch concentration is preferably 0.01 to 50%, more preferably 1 to 30%, and further preferably 2 to 15%.

[0127] In a preferred embodiment of the present invention, in addition to the essential components of the solution used in the cell cryopreservation method of the present invention, it may further contain a component selected from proteins, magnesium salts, and calcium salts. Specific examples of proteins that can be used include serum albumin, serum globulin, and the like. In addition, examples of serum albumin include human serum albumin and 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 magnesium salt that can be used is not particularly limited as long as it produces magnesium ions when dissolved in a solvent. Oxygen-containing salts, halides, oxides, hydroxides, inorganic salts, or organic acid salts can be used. One or more magnesium salts can be used in combination. In the present invention, magnesium chloride is preferably used. The magnesium salt content is not particularly limited, but is preferably 0.01 to 10 mM, and more preferably 0.1 to 5 mM, based on the final concentration of the total magnesium ions contained in the cryopreservation solution. The calcium salt that can be used is not particularly limited as long as it produces calcium ions when dissolved in a solvent, and oxyacid salts, halides, oxides, hydroxides, inorganic salts or organic acid salts can be used. One or more calcium salts can be combined. In the present invention, calcium chloride is preferably used. The calcium salt content is not particularly limited, and is preferably 0.01 to 10 mM, more preferably 0.1 to 5 mM, based on the final concentration of total calcium ions contained in the freezing preservation solution. In addition, in addition to the above-mentioned components, the freezing preservation solution may further contain substances that are non-toxic to cells, for example, vitamins, amino acids, etc. In addition, in addition to the above-mentioned components, the freezing preservation solution may also contain phosphate ions from the viewpoint of pH adjustment and buffering.

[0128] The osmotic pressure of the cryopreservation solution is preferably within a range that does not damage cells during freezing. From the perspective of improving the permeability of components into cells during freezing and inhibiting ice crystal formation, the range is, for example, 500 to 8000 mOsm / L, 1000 to 7500 mOsm / L, 1500 to 7000 mOsm / L, or 1800 to 5000 mOsm / L. The pH of the cryopreservation solution is preferably within a range that does not damage cells, for example, 3.0 to 10.0, more preferably 4.5 to 9.0.

[0129] In the present invention, commercially available cryopreservation solutions can be used. Examples of usable products include the Cell Banker series of cryopreservation solutions for cells and tissues (STEM-CELLBANKER (registered trademark)), specifically STEM-CELLBANKER (ZENOAQ, CB045).

[0130] The cells are preferably in the logarithmic growth phase when frozen.

[0131] The cell density during freezing can be appropriately set, specifically, 1×10 6 ~2×10 8 cells / mL, preferably 2×10 6 ~1×10 8 cells / mL, more preferably 1×10 7 ~5×10 7 In one preferred embodiment, cells are grown at a rate of 4×10 7 Cells / mL are stored in 5mL containers. High-density freezing is possible for frozen shipments of highly active NK cells, making the product more compact and contributing to reduced shipping costs.

[0132] [Thaw and dilute]

[0133] In the present invention, frozen cells can be thawed using various procedures. For example, the frozen cell storage container can be rapidly thawed in a 37°C warm bath, for example, with shaking as needed. Alternatively, cells can be removed from the freezer and left at room temperature to thaw naturally without active heating. The conversion of a frozen solid into a liquid is sometimes referred to as thawing. During or after thawing, the frozen cell material can be diluted with an appropriate liquid.

[0134] (Liquid for dilution)

[0135] The present invention relates to a liquid for suspending cells for administration to humans, and more particularly to a liquid for diluting frozen cells (sometimes referred to as a diluent or a thawing liquid).

[0136] (1) Contains potassium ions

[0137] (2) pH 4.9 or above

[0138] (3) Does not contain chloride ions at a concentration of 135 mEq / L or more

[0139] (4) Does not contain glucose at a concentration of 5.55 mM or higher

[0140] (5) Does not contain calcium ions at a concentration of 0.423 mM or more

[0141] (6) Osmotic pressure is 200-396 mOsm

[0142] (7) Contains any one organic acid selected from succinic acid, malic acid, lactic acid, citric acid and acetic acid.

[0143] In the present invention, when a liquid is said to contain no component, it means that the component is not detected by a normal measurement method, unless otherwise specified.

[0144] The diluent contains any one organic acid selected from the group consisting of succinic acid, malic acid, lactic acid, citric acid, and acetic acid. These acids adjust the pH of the diluent and may be utilized by cells.

[0145] In the present invention, when a liquid contains any one of the organic acids selected from succinic acid, malic acid, lactic acid, citric acid, and acetic acid, this also includes the case where a pharmaceutically acceptable salt of any of these acids is added during the preparation of the diluent. Furthermore, the diluent may contain only one or more of these organic acids.

[0146] In a preferred embodiment, the liquid comprises an organic acid selected from the group consisting of succinic acid, lactic acid, citric acid, and acetic acid. In a particularly preferred embodiment, the diluent comprises succinic acid.

[0147] The content of any one organic acid selected from succinic acid, malic acid, lactic acid, citric acid, and acetic acid in the dilution (or the total content thereof when multiple types are present) is not particularly limited as long as the intended effect is achieved. The content of any one of these organic acids in the dilution can be, for example, 10 to 10,000 mg per 200 mL, preferably 50 to 5,000 mg, more preferably 200 to 3,000 mg, and even more preferably 400 to 1,000 mg.

[0148] In one embodiment, the pH of the dilution solution may be 4.7 or higher, preferably 4.9 or higher, more preferably 5.9 or higher, and even more preferably 6.3 or higher. Alternatively, the pH may be 8.3 or lower, preferably 8.0 or lower, more preferably 7.9 or lower, and even more preferably 7.8 or lower.

[0149] The potassium ion concentration of the dilution solution 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. Alternatively, it preferably contains 0.496 to 5.96 mM of potassium chloride.

[0150] In a preferred embodiment, the potassium ion concentration of the dilution solution is 4.00 mEq / L or higher. The upper limit is not particularly limited, but is, for example, 18.0 mEq / L or lower, preferably 15.0 mEq / L or lower, more preferably 10.0 mEq / L or lower, and even more preferably 7.50 mEq / L or lower.

[0151] In another preferred embodiment, the diluent does not contain calcium ions regardless of the presence or concentration of other components. In another preferred embodiment, the diluent does not contain glucose regardless of the presence or concentration of other components.

[0152] The ions contained in the diluent may be ions from a salt source that can be used as a drug. Such salts may be carbonates, bicarbonates, oxoacid salts, halides, oxides, hydroxides, inorganic salts, or organic acid salts. The salt may be a single salt or a combination of multiple salts.

[0153] The diluent may contain sodium salt, gluconate, and acetate. It may further contain magnesium salt. The sodium ion concentration of the diluent is preferably 14.0 to 200 mM, more preferably 28.0 to 182 mM, and even more preferably 70.0 to 168 mM. Alternatively, it preferably contains 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. The gluconate ion concentration of the diluent 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. The acetate ion concentration of the diluent 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.

[0154] The diluent can be made up of approved pharmaceuticals. Examples of such pharmaceuticals are shown below.

[0155] Generic name: Amizet

[0156] Trade Name: Amizet B Infusion

[0157] Pharmacological classification name: Comprehensive amino acid preparation

[0158] Composition (1 bag 200mL)

[0159] Active Ingredients: L-Isoleucine 1,700mg, L-Leucine 2,700mg, Lysine Malate (as L-lysine) 2,432mg (1,600mg), L-Methionine 780mg, L-Phenylalanine 1,540mg, L-Threonine 960mg, L-Tryptophan 320mg, L-Valine 1,800mg, Cysteine ​​Malate (as L-cysteine) 310mg (200mg), L-Tyrosine 100mg, L-Arginine 2,220mg, L-Histidine 940mg, L-Alanine 1,720mg, L-Aspartic Acid 100mg, L-Glutamic Acid 100mg, Glycine 1,100mg, L-Proline 1,280mg, L-Serine 840mg

[0160] Additives: Succinic acid (pH adjuster) appropriate amount

[0161] Electrolyte: Does not contain Na ＋ 、Cl -

[0162] General name: Meylon

[0163] Trade name: Meylon intravenous injection 8.4%

[0164] Common name: sodium bicarbonate

[0165] Composition (in 20 mL)

[0166] Sodium bicarbonate: 1.68 g (8.4%)

[0167] Electrolyte concentration: Na ＋ 1000 mEq / L, HCO3 - 1000 mEq / L

[0168] General name: K. C. L.

[0169] Trade Name: K.C.L. Intravenous Solution 15%

[0170] Common name: potassium chloride

[0171] Preparation name: Potassium chloride preparation

[0172] composition

[0173] Ingredients: Potassium chloride 3g (15w / v%, 2 molar solution) [Potassium (K) amount: 40mEq (1573.36mg)]

[0174] Additives: Contains riboflavin sodium phosphate 6mg

[0175] Generic name: blood donation albumin

[0176] Trade name: Blood donation albumin 25% intravenous injection 12.5g / 50mL

[0177] Pharmacological classification name: Plasma fractionation preparation (human serum albumin preparation)

[0178] Composition (in 50 mL)

[0179] Active ingredient: Human serum albumin 12.5g

[0180] Additives: Acetyltryptophan 250.97 mg, Sodium hydroxide 43.44 mg, Sodium octanoate 169.75 mg

[0181] pH 6.4-7.4, osmotic pressure ratio (relative to normal saline) 0.5-1.0

[0182] (Preferred method)

[0183] One of the particularly preferred forms of the diluent is as follows:

[0184] Donated blood albumin 25% 20.0 mL

[0185] 111.12 mL normal saline

[0186] Amizet 19.08 mL

[0187] 56.68 mL of distilled water

[0188] Meylon 1.6 mL

[0189] KCl 0.4 mL

[0190] pH=7.2

[0191] Another preferred composition is as follows.

[0192] Donated blood albumin 25% 11.112 mL

[0193] 111.12 mL normal saline

[0194] Amizet 19.08 mL

[0195] Water 56.68 mL

[0196] Meylon 1.6 mL

[0197] KCl 0.4 mL

[0198] pH=7.2

[0199] Another preferred composition is as follows.

[0200] Donated blood albumin 25% 20.0 mL

[0201] 111.12 mL normal saline

[0202] Amizet 19.08 mL

[0203] Water 56.68 mL

[0204] Meylon 1.6 mL

[0205] KCl 0.4 mL

[0206] pH=7.2

[0207] Another preferred composition is as follows.

[0208] 11.7657 mL of normal saline

[0209] ※ 2.0202 mL

[0210] Water 6.0014mL

[0211] Meylon 0.1694 mL

[0212] KCl 0.0423 mL

[0213] Donated blood albumin 25% 2.000 mL

[0214] pH = 4.9 ~ 8.0

[0215] ※ A liquid containing L-cysteine ​​or acetylcysteine ​​and any one organic acid selected from succinic acid, lactic acid, citric acid, and acetic acid, and optionally other amino acids. This liquid is prepared by adding any one organic acid selected from succinic acid, lactic acid, citric acid, and acetic acid to the formulations A to F described in the Examples.

[0216] The cell that is suspended in diluent together with the liquid for freezing preservation can be used as it is.From the viewpoint for administration, the mixed solution after the liquid for freezing preservation and the diluent are preferably mixed becomes isotonic (having an osmotic pressure roughly equal to body fluid, specifically 285 ± 13mOsm / L). Since the liquid for freezing preservation is a hypertonic solution (for example, 1500~7000mOsm / L) in a preferred embodiment, the diluent can be a liquid with a low osmotic pressure. If it is a person skilled in the art, it can be considered that the liquid for freezing preservation passes through the dilution ratio of the diluent, and the component concentration of the diluent is appropriately determined.

[0217] Regardless of the composition, the diluent preferably does not contain serum at a concentration of 40% or more, and more preferably does not contain serum at all, because the presence of serum may reduce the survival rate of cells.

[0218] The cell density when suspended in the diluent can be appropriately set to a cell density suitable for cell maintenance or a cell density suitable for administration. Specifically, 1×10 5 ~1×10 7 cells / mL, preferably 2×10 5 ~5×10 6 cells / mL, more preferably 5×10 5 ~2×10 6 cells / mL.

[0219] Cells can be maintained in the diluent for a relatively long period of time. After suspending the cells in the diluent, the suspension can be allowed to stand for several minutes to several hours, for example, 5 minutes to 6 hours, more preferably 30 minutes to 4 hours. This can be done at ambient temperature (e.g., 1-30°C, typically 15-25°C) or in a CO2 incubator (e.g., 36-42°C, typically 37°C).

[0220] By using such diluent, the survival rate of frozen highly active NK cells etc. can be maintained high. In addition, the cytotoxic activity of frozen highly active NK cells etc. can be maintained at a high state. About survival rate, sometimes the ratio of viable cell number is set to A: more than 70%, B: more than 50% and less than 70%, C: less than 50%. About cytotoxic activity, sometimes the ratio of target cells killed by effector cells is expressed as A: more than 70%, B: more than 50% and less than 70%, C: less than 50%. No matter which situation, as long as it is A or B, then it can be said that the purpose has been achieved.

[0221] [Use in pharmaceutical compositions]

[0222] The present invention provides a pharmaceutical composition comprising highly active NK cells recovered by an appropriate method, pretreated as needed, and frozen for preservation.

[0223] The pharmaceutical composition provided according to 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 are cancer or infections, specifically, including skin cancer, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, prostate cancer, neuroblastoma, leukemia, infections caused by viruses, bacteria, etc., but not limited to these. In addition, the present inventors, etc., applied cells frozen / thawed by the method of the present application, and confirmed the effect on colorectal cancer model animals that would die within 30 days if not treated.

[0224] Cell therapy using the pharmaceutical composition of the present invention may be performed alone or in combination with surgical therapy, chemotherapy, radiotherapy, antibody drugs, and the like.

[0225] The characteristics of one embodiment of the pharmaceutical composition provided by the present invention are shown below.

[0226] (Dosage Form)

[0227] Injection (cell suspension) (sometimes also called infusion, infusion drip, drip, infusion pharmaceutical composition)

[0228] (Ingredients and Content)

[0229] Constituent cells: Highly active NK cells, etc.

[0230] Content: 6×10 6 ~4.8×10 9 cells / 60kg

[0231] (Sub-component)

[0232] Composite electrolyte solution 10~45%

[0233] Sodium chloride solution 10-45%

[0234] 20-30% human serum albumin solution 5-30%

[0235] Dimethyl sulfoxide 2-15%

[0236] other

[0237] or

[0238] 100% of the cryopreservative solution is acceptable as a drug additive

[0239] (Preparation method)

[0240] Thaw the frozen composition in a 37°C constant temperature water bath until it is completely thawed. After thawing, the composition is immediately and aseptically suspended in a separately prepared isotonic solution that is acceptable as a pharmaceutical additive.

[0241] (Stability after thawing)

[0242] The effective time after thawing is 6 hours, preferably 4 hours when stored at room temperature.

[0243] Example

[0244] [Methods common among the embodiments]

[0245] A). GAIA-102 culture method

[0246] Frozen apheresis blood (Cellero) was used as the raw material. After thawing, PBMCs were washed and concentrated using the Lovo CellProcessing System (FRESENIUS KABI).

[0247] CD3-positive cells and CD34-positive cells were removed from the obtained PBMCs using CliniMACS Prodigy (registered trademark) (Miltenyi Biotec), and the cells were cultured with KBM501 medium. ※1 Count the number of cells in the eluate and calculate the total number of cells. 2 , to become 5×10 5200 mL of a cell suspension prepared in KBM501 medium containing Simulect (Novartis Pharma) and Prograf (Astellas Pharmaceuticals) was inoculated per bag at a concentration of 10 cells / mL and cultured in a CO2 incubator (37°C, 5% CO2). On the 9th day of culture, additional KBM501 medium containing Simulect was added to a final volume of 650 mL per bag, and incubation continued until the 14th day.

[0248] ※1: KBM 501 (Kohjin Bio) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2U / mL heparin sodium (Nipro)

[0249] B) Recovery Methods for GAIA-102

[0250] On the 14th day of culture, the culture medium was recovered, and 1 mM EDTA was further added to the culture bag to detach the adhered cells. The cell recovery liquid including the detached cells was centrifuged, washed with KBM501 medium, and resuspended.

[0251] C). Freezing method of GAIA-102

[0252] The number of viable GAIA-102 cells obtained by the procedures described in the GAIA-102 culture method and recovery method was counted, and 2×10 8 cells and frozen at -80°C.

[0253] D) Viability determination by 7-AAD staining

[0254] GAIA-102 cells thawed under each condition were plated in a 96-well plate (IWAKI, 4870-800SP) at a concentration of 1×10 5 Cells were prepared at 100 cells / well and centrifuged. After removing the supernatant, a 7-AAD solution (Beckman Coulter, A07704) diluted in PBS (Nacalai Tesque, 14249-95) was added, suspended, and incubated at room temperature for 20 minutes. The stained cells were measured using a flow cytometer (BD LSRFortessa, BD Biosciences) and analyzed using FlowJo software. The survival rate was calculated based on the 7-AAD positive rate.

[0255] E). Method for determining cytotoxic activity against tumor cells

[0256] For the measurement of cytotoxic activity, a group in which thawed GAIA-102 was reacted with K562 cells, a group in which K562 cells alone were used as a negative control, and a group in which K562 cells were treated with 10% formalin as a positive control were prepared.

[0257] GAIA-102

[0258] The necessary amount of GAIA-102 cells thawed and diluted under each condition was obtained based on the number of viable cells at the time of freezing, and then diluted to 1×10 cells using 10% FBS / RPMI1640. 6 The concentration of cells / ml.

[0259] K562

[0260] K562 cells were suspended in serum-free RPMI1640 medium and stained with PKH26 Red Fluorescent CellLinker Kit (Sigma-Aldrich). Then, 10% FBS / RPMI1640 was used to make 2×10 6 The cells were prepared at 10 cells / mL.

[0261] GAIA-102 and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) at a cell-to-cell ratio of 1:1, 2:1, and 8:1, mixed, and incubated at 37°C, 5% CO2 for 2 hours. Following the reaction, the cells were centrifuged (500 × g, 5 minutes), the supernatant removed, and the suspension was added with a 7-AAD solution diluted in PBS and incubated at room temperature for 20 minutes. Flow cytometry was used for analysis and FlowJo software was used to calculate the cytotoxic activity (% lysis). ※2 .

[0262] ※2: Cytotoxicity activity rate = (K562 cell death rate - negative control cell death rate) / (positive control cell death rate - negative control cell death rate) × 100

[0263] F) Method for calculating the corrected value by statistical analysis of toxicity activity rate

[0264] From the cytotoxic activity calculated at ET ratios of 4 points (including 0) or more, calculated values ​​at ET ratios = 1 and 2 were determined by nonlinear regression analysis using JMP (registered trademark) Pro statistical analysis software.

[0265] About the reagents and drips used

[0266] In the verification of thawed solutions, the following procedures should be used for reagents and drips.

[0267] Plasma-Lyte A (Baxter)

[0268] Otsuka Normal Saline Injection (Otsuka Pharmaceutical Co., Ltd.)

[0269] Water (Nacalai Tesque, 06442-95)

[0270] Meylon intravenous injection 8.4% (Otsuka Pharmaceutical Co., Ltd.)

[0271] KCL intravenous solution 15% (Maruishi Pharmaceutical Co., Ltd.)

[0272] Donated blood albumin 25% intravenous injection (Nippon Pharmaceutical Co., Ltd.)

[0273] L-(-)-Malic acid (Nacalai Tesque, 21030-44)

[0274] Succinic acid (Nacalai Tesque, 32402-92)

[0275] Citric acid monohydrate (Nacalai Tesque, 09106-02)

[0276] Lactic acid (Nacalai Tesque, 20006-62)

[0277] Acetic acid (Nacalai Tesque, 00212-85)

[0278] L(+)-Ascorbic acid (Nacalai Tesque, 03420-52)

[0279] [Example 1]

[0280] A solution with the composition shown in the table below was prepared as thawing solution (1). Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with thawing solution (1). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and then the toxicity activity was measured.

[0281]

[0282] [Table 1-2]

[0283] <Composition of Thawing Solution (1)>

[0284]

[0285] ※3: 4 groups: prescription A only, prescription B only, prescription C only, and prescription D only

[0286] The results are shown in Figure 1. When Formulations A and B were used, high cytotoxic activity against tumor cells was observed, comparable to that observed when Plasma-Lyte A was used.

[0287] [Example 2]

[0288] Malic acid, succinic acid, malic acid, and succinic acid were added to Formulations A and B to adjust the pH to 6.9-7.4. The solution with the composition shown in the table below was used as the thawing solution (2), and the pH after preparation was measured. Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with the thawing solution (2). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and the toxicity activity was measured.

[0289]

[0290] [Table 2-2]

[0291] <Composition of Thawing Solution (2)>

[0292]

[0293] ※4: 8 sets: Formula A only (pH 7.9), Formula A + malic acid (pH 7.3), Formula A + succinic acid (pH 7.2), Formula A + malic acid + succinic acid (pH 7.3), Formula B only (pH 7.9), Formula B + malic acid (pH 7.4), Formula B + succinic acid (pH 7.2), Formula B + malic acid + succinic acid (pH 7.4)

[0294] In brackets: pH of the thawing solution after preparation

[0295] The results are shown in Figure 2 In the case of application of malic acid and succinic acid, higher toxic activity towards tumor cells was seen.

[0296] [Example 3]

[0297] Citric acid monohydrate, lactic acid, acetic acid, and succinic acid were added to Formulation B to adjust the pH to 6.9-7.7. The solution with the composition shown in the table below was used as the thawing solution (3), and the pH after preparation was measured. Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with the thawing solution (3). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and the toxicity activity was measured.

[0298]

[0299] [Table 3-2]

[0300] <Composition of Thawing Solution (3)>

[0301]

[0302] ※5: Five groups: Formula B only (pH 8.0), Formula B + citric acid monohydrate (pH 7.4), Formula B + lactic acid (pH 7.4), Formula B + acetic acid (pH 7.5), and Formula B + succinic acid (pH 7.4)

[0303] In brackets: pH of the thawing solution after preparation

[0304] The results are shown in Figure 3 In the case of using citric acid, lactic acid, and acetic acid, higher toxic activity against tumor cells was seen as in the case of using succinic acid.

[0305] [Example 4]

[0306] Ascorbic acid and succinic acid were added to Formulation B to adjust the pH to 7.5 and 7.8. Then, a solution with the composition shown in the table below was used as a thawing solution (4), and the pH after preparation was measured. Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with the thawing solution (4). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and then the toxicity activity was measured and the survival rate was measured by 7-AAD staining.

[0307]

[0308] [Table 4-2]

[0309] <Composition of Thawing Solution (4)>

[0310]

[0311] ※6: 5 groups: prescription B only (pH 8.2), prescription B + ascorbic acid (pH 7.6), prescription B + succinic acid (pH 7.9), prescription E only (pH 7.7), and prescription F only (pH 7.8)

[0312] In brackets: pH of the thawing solution after preparation

[0313] The results are shown in Figure 4-1 In the case of using ascorbic acid, high tumor cell cytotoxic activity was not observed as in the case of using succinic acid. In addition, the results of evaluating the survival rate by 7-AAD staining are shown in FIG. Figure 4-2 and Figure 4-3

[0314] [Example 5]

[0315] Succinic acid was added to Formula F to adjust the pH to 5.9-7.1. The solution with the composition shown in the table below was used as the thawing solution (5), and the pH after preparation was measured. Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with the thawing solution (5). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and the toxicity activity was measured and the survival rate was measured by 7-AAD staining.

[0316]

[0317] [Table 5-2]

[0318] <Composition of Thawing Solution (5)>

[0319]

[0320] ※7: Four groups: Formula F only (pH 7.8), Formula F + succinic acid (pH 7.5), Formula F + succinic acid (pH 7.4), and Formula F + succinic acid (pH 7.3)

[0321] In brackets: pH of the thawing solution after preparation

[0322] The results are shown in Figure 5-1 、 5-2 When succinic acid was used, good results were achieved across a wide pH range. The survival rates were 87.3% for Formulation F alone (pH 7.8), 85.1% for Formulation F + succinic acid (pH 7.5), 85.7% for Formulation F + succinic acid (pH 7.4), and 85.8% for Formulation F + succinic acid (pH 7.3). The survival rate for PlasmaLyte-A (pH 7.9) was 92.3%.

[0323] [Example 6]

[0324] Succinic acid was added to Formula F to adjust the pH to 4.9-5.9. Then, a solution with the composition shown in the table below was used as a thawing solution (6), and the pH after preparation was measured. Frozen GAIA-102 was thawed in a water bath at 37°C and diluted 45-fold with the thawing solution (6). As a control, the solution was diluted 41-fold with Plasma-Lyte A. After thawing, the solution was allowed to stand at room temperature for 3 hours, and then the toxicity activity was measured and the survival rate was measured by 7-AAD staining.

[0325]

[0326] [Table 6-2]

[0327] <Composition of Thawing Solution (6)>

[0328]

[0329] ※8: Four groups: Formula F only (pH 7.7), Formula F + succinic acid (pH 7.2), Formula F + succinic acid (pH 6.9), and Formula F + succinic acid (pH 6.1)

[0330] In brackets: pH of the thawing solution after preparation

[0331] The results are shown in Figure 6-1 、 6-2 The survival rates were, in order, 85.0% for Formula F alone (pH 7.7), 82.5% for Formula F + succinic acid (pH 7.2), 82.8% for Formula F + succinic acid (pH 6.9), and 66.6% for Formula F + succinic acid (pH 6.1). The survival rate for PlasmaLyte-A (pH 7.9) was 84.7%.

[0332] 7ADD-4

[0333] [Summary of Examples 5 and 6]

[0334] exist Figure 7 The relative % lysis (E:1=1 corrected value) and 7-AAD-4 gate (%) when the case of applying PlasmaLyte-A is set to 1.0 are summarized.

Claims

1. A liquid for suspending cells for administration to humans, which satisfies the following: (1) Contains potassium ions (2) pH 4.9 or above (3) Does not contain chloride ions at a concentration of 135 mEq / L or more (4) Does not contain glucose at a concentration of 5.55 mM or higher (5) Does not contain calcium ions at a concentration of 0.423 mM or more (6) Osmotic pressure is 200-396 mOsm (7) Contains any one organic acid selected from succinic acid, malic acid, lactic acid, citric acid and acetic acid.

2. The liquid according to claim 1, wherein (7) comprises succinic acid.

3. The liquid according to claim 1, further comprising (8) L-cysteine ​​or acetylcysteine.

4. The liquid according to claim 1, further comprising (9) albumin. 5 . The liquid according to claim 1 , which is used for diluting a frozen product containing cells for administration to humans or a thawed product thereof.

6. The liquid according to any one of claims 1 to 4, (1) Contains potassium ions at a concentration of 4.00 mEq / L or higher (4) Does not contain glucose (5) Does not contain calcium ions.

7. The liquid according to any one of claims 1 to 4, which is a pharmaceutical composition.

8. A pharmaceutical composition comprising the liquid according to any one of claims 1 to 4 and a cell population for administration to humans, wherein The cells for administration to humans are lymphocytes.

9. A pharmaceutical composition comprising the liquid according to any one of claims 1 to 4 and a cell population for administration to humans, wherein The cells for administration to humans are highly active NK cell-like allogeneic CD3-negative cells.

10. The pharmaceutical composition according to claim 8, wherein The population of highly active NK cell-like allogeneic CD3-negative cells has undergone a freezing step.

11. A method for providing a pharmaceutical composition for infusion comprising cells for administration to a human, comprising the steps of: The frozen cells are thawed, and the thawed cells are suspended in the liquid according to any one of claims 1 to 4 to form a pharmaceutical composition for infusion.

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