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WO2025142807A1 - Procédé de culture cellulaire - Google Patents

Procédé de culture cellulaire Download PDF

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Publication number
WO2025142807A1
WO2025142807A1 PCT/JP2024/045351 JP2024045351W WO2025142807A1 WO 2025142807 A1 WO2025142807 A1 WO 2025142807A1 JP 2024045351 W JP2024045351 W JP 2024045351W WO 2025142807 A1 WO2025142807 A1 WO 2025142807A1
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Prior art keywords
cells
medium
serum
salt
cell
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Japanese (ja)
Inventor
忍 桑江
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This method also uses serum-containing medium in the process of producing iPS cell-derived CAR-T cells, particularly in the expansion culture process, to obtain sufficient cell proliferation effects.
  • the medium is xeno-free, i.e., does not contain heterologous components, and serum-free or low-serum is preferable.
  • iPS cell-derived T cells are particularly vulnerable to washing stress compared to primary T cells, there has been a strong demand for serum-free or low-serum medium that does not require strong washing.
  • the present inventors prepared various factors to be added to serum-free or low-serum medium and investigated their effects on cell culture or proliferation. As a result, it was confirmed that the addition of imidazole dipeptide or a salt thereof (e.g., carnosine), taurine or a precursor thereof or a salt thereof (e.g., taurine), and lysophosphatidic acid or a derivative thereof (e.g., lysophosphatidylcholine (LPC)) was effective in maintaining and promoting cell proliferation, comparable to that of culture in a serum-containing medium, even when a serum-free or low-serum medium was used.
  • imidazole dipeptide or a salt thereof e.g., carnosine
  • taurine or a precursor thereof or a salt thereof e.g., taurine
  • lysophosphatidic acid or a derivative thereof e.g., lysophosphatidylcholine (LPC)
  • [14a] The method according to any one of [1] to [14], wherein the cell is a cell into which a chimeric antigen receptor (CAR) has been introduced.
  • [14b] The method described in [14a], wherein the cell is a T cell into which a chimeric antigen receptor has been introduced (CAR-T cell) or a natural killer cell into which a chimeric antigen receptor has been introduced (CAR-NK cell).
  • a method for producing a cell population in which the number of desired cells is expanded comprising a step of culturing a cell population containing the desired cells in a serum-free or low-serum medium containing: (ia) an imidazole dipeptide or a salt thereof, or vitamin E; (ii) taurine or a precursor thereof, or a salt thereof; and (iii) lysophosphatidic acid or a derivative thereof.
  • a method for producing a cell population in which the number of desired cells is expanded comprising the step of culturing a cell population containing the desired cells in a serum-free or low-serum medium containing (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, according to [24].
  • composition according to [33] comprising (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.
  • a medium additive comprising (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, wherein the medium is a serum-free medium or a low-serum medium.
  • a medium additive comprising (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, wherein the medium is a serum-free medium or a low-serum medium.
  • the present invention even when a serum-free or low-serum medium is used, it is possible to obtain an effect of maintaining and promoting cell proliferation comparable to that achieved by culturing in a serum-containing medium.
  • cells that are sensitive to stress from operations such as cell washing are preferably cultured in a serum-free or low-serum medium that does not require extensive washing, and are suitable for culturing under such conditions.
  • a method for culturing cells (hereinafter also referred to as the "culturing method of the present invention") and a method for growing cells (hereinafter also referred to as the “growing method of the present invention”)
  • the culture or growth method of the present invention comprises a step of culturing cells in a serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.
  • the serum-free or low-serum medium may further contain, as desired, (iv) vitamin E and/or (v) boric acid or a salt thereof.
  • the culture or growth methods of the present invention may optionally use vitamin E in addition to or instead of (i) the imidazole dipeptide or salt thereof, in which case component (iv) is not required. Therefore, the culture method or proliferation method of the present invention may be a method comprising the step of culturing cells in a serum-free or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.
  • Cells The cell types to be cultured by the culture method and proliferation method of the present invention are not particularly limited. Examples of such cell types include germ cells such as sperm and eggs, somatic cells that constitute an organism, stem cells (e.g., pluripotent stem cells) and cells induced to differentiate from stem cells, progenitor cells, cancer cells isolated from an organism, cells (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have had their nuclei artificially exchanged.
  • germ cells such as sperm and eggs
  • stem cells e.g., pluripotent stem cells
  • progenitor cells e.g., cancer cells isolated from an organism
  • cells cells (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have had their nuclei artificially exchanged.
  • iPS cells are preferred as pluripotent stem cells. Identification of iPS cells can be performed using undifferentiated markers due to the undifferentiated nature of iPS cells as indicators. Examples of undifferentiation markers include alkaline phosphatase, Oct3/4, Sox2, Nanog, ERas, Esgl, etc. Methods for detecting these undifferentiation markers include methods for detecting mRNA (using primers and probes) and immunological detection methods (using antibodies and labels).
  • Cells induced to differentiate from stem cells are any cells that have been induced to differentiate from stem cells into a specific type of cell.
  • Cell lines are cells that have acquired the ability to proliferate indefinitely through artificial manipulation outside of the body. Examples include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human fetal kidney cells), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7/TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0/1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (product name), Vero, etc.
  • CHO Choinese hamster ovary cell line
  • HCT116 human fetal kidney cells
  • HeLa human uterine cancer cell line
  • HepG2 human liver cancer cell line
  • UT7/TPO human leukemia cell line
  • MDCK MDBK
  • BHK C-33A
  • HT-29
  • the cells to be cultured are immunocompetent cells.
  • Immunocompetent cells refer to cells involved in various immune responses in the body. Examples of immunocompetent cells include natural killer (NK) cells, macrophages, monocytes, mast cells, dendritic cells, Langerhans cells, neutrophils, eosinophils, basophils, B cells, and T cells, but are preferably selected from dendritic cells, B cells, T cells, and natural killer (NK) cells. More preferably, the immunocompetent cells are T cells.
  • T cells include CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, ⁇ -T cells, ⁇ -T cells, regulatory T cells, and NKT cells.
  • T cells may be subsets such as naive T cells, effector T cells, or memory T cells.
  • the immunocompetent cells may be cells isolated from humans (primary cells) or cells obtained by differentiation from stem cells such as pluripotent stem cells (e.g., iPS cells, ES cells), hematopoietic stem cells, and mesenchymal stem cells, and are preferably primary cells and cells obtained by differentiation from pluripotent stem cells (particularly iPS cells).
  • Primary cells are preferably human-derived primary T cells.
  • Stem cells can be induced to differentiate into immunocompetent cells by methods known in the art, depending on the type of stem cells used and the type of immunocompetent cells of interest. As an example, iPS cells can be induced to differentiate into T cells and NK cells can be induced to differentiate into NK cells by the methods described in the Examples.
  • the target cells may be either autologous or allogeneic cells.
  • autologous cells refers to cells obtained from a subject receiving a cell population (described below) produced by the culture method of the present invention or the proliferation method of the present invention, or cells derived from the obtained cells
  • allogeneic cells refers to cells that are not the aforementioned “autologous cells.”
  • the target cells may be cells that have been artificially genetically modified.
  • examples include immunocompetent cells into which a CAR gene has been introduced (T cells into which a CAR gene has been introduced and natural killer cells into which a CAR gene has been introduced), T cells into which an exogenous T cell receptor (TCR) has been introduced, and immunocompetent cells into which genes for expressing cytokines and/or chemokines have been introduced.
  • CAR is a structure that contains, from the N-terminus to the C-terminus of a protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signal domain for the effector function of immune cells, and the CAR gene is a gene that encodes this receptor.
  • the extracellular domain contains an antigen recognition site that exhibits specific binding to the target.
  • the transmembrane domain is located between the extracellular domain and the intracellular signal domain.
  • the intracellular signal domain transmits signals necessary for the immune cell to exert its effector function. In other words, when the extracellular domain binds to the target antigen, an intracellular signal domain is used that is capable of transmitting signals necessary to activate the immune cell.
  • Preferable examples of cells to which the culture method and proliferation method of the present invention are applied include T cells into which a CAR gene has been introduced (CAR-T cells) and NK cells into which a CAR gene has been introduced (CAR-NK cells), more preferably CAR-T cells and CAR-NK cells derived from pluripotent stem cells, even more preferably CAR-T cells and CAR-NK cells derived from iPS cells, and even more preferably CAR-T cells derived from iPS cells.
  • CAR-T cells and CAR-NK cells can be obtained by introducing a CAR gene into T cells and NK cells, respectively, or their precursor cells such as pluripotent stem cells.
  • CAR-T cells may be cells obtained by introducing a CAR gene into cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into T cells, or cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into which a CAR gene has been introduced into T cells.
  • CAR-T cells derived from iPS cells obtained by differentiating iPS cells into which a CAR gene has been introduced into T cells and CAR-T cells derived from iPS cells obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells, and these may be collectively referred to as "iPS cell-derived CAR-T cells". More preferred are CAR-T cells derived from iPS cells obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells.
  • the cells may be cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into NK cells and by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into which a CAR gene has been introduced into NK cells, or cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into which a CAR gene has been introduced into NK cells.
  • the target gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected.
  • Each viral vector can be prepared according to a conventional method or using a commercially available dedicated kit.
  • Non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors.
  • the CAR gene can be introduced into cells by the method described in the Examples.
  • the concentration of imidazole dipeptide or its salt is the concentration converted to carnosine, a representative imidazole dipeptide, unless otherwise specified. If the concentration is too high, it will affect cell viability, and if it is too low, the cell growth promoting effect will be weak.
  • the method of producing lysophosphatidic acids is not particularly limited, and those produced by known methods can be used.
  • the lysophosphatidic acids can be produced by enzymatically converting lecithin containing phosphatidic acids.
  • commercially available lysophosphatidic acids can be used simply.
  • the concentration of lysophosphatidic acids in the medium is not particularly limited as long as the desired effect is obtained, and may be increased or decreased as appropriate depending on the type of compound used, but is usually 0.1 to 50 mg/L, preferably 0.5 to 25 mg/L, more preferably 1 to 15 mg/L, and particularly preferably 2 to 10 mg/L.
  • Vitamin E is a fat-soluble antioxidant vitamin, and is mainly classified into two main groups, tocopherols and tocotrienols. These compounds have a common phenolic ring and a long fatty chain bonded to it. Tocopherols have four isomers ( ⁇ , ⁇ , ⁇ , ⁇ ) that differ depending on the position of the methyl group, and tocotrienols also have similar isomers. These may be used alone or in combination of two or more.
  • the preferred vitamin E is tocopherol or a salt thereof, and more preferably ⁇ -tocopherol or a salt thereof. Vitamin E may be in the form of a salt.
  • the salt of vitamin E is not particularly limited as long as it is pharma- ceutically acceptable.
  • it may be a salt with an organic base (e.g., a salt with an organic amine such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, picoline, etc.), or a salt with an inorganic base (e.g., an ammonium salt; an alkali metal salt such as a sodium salt or a potassium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt; or a metal salt such as an aluminum salt).
  • Vitamin E may also be in the form of a derivative.
  • the concentration of boric acid or a salt thereof in the medium is not particularly limited as long as the desired effect is obtained, but is usually 0.1 to 0.9 mM, preferably 0.2 to 0.8 mM, more preferably 0.2 to 0.7 mM, and particularly preferably 0.3 to 0.6 mM.
  • the concentration of boric acid or a salt thereof is the concentration converted to boric acid unless otherwise specified. If the concentration is too high, it affects the cell viability, and if the concentration is too low, the cell growth promoting effect is weak.
  • basal media examples include, but are not limited to, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM ZincOption medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, F12 medium, and mixed media thereof (e.g., Advanced DMEM/F12 medium, etc.).
  • Perfusion culture is a culture method in which a culture medium is continuously supplied to a culture system containing cells, and an equal amount of culture supernatant not containing cells is continuously removed from the culture system to keep the culture system in a steady state.
  • a "low-serum medium” refers to a medium in which serum has been added to a basal medium, characterized in that the serum concentration is reduced compared to that of a commonly used serum-containing medium (serum concentration: 5 to 20% (herein, serum concentration is expressed as v/v%)).
  • the culture vessel may be either cell-adhesive or cell-non-adhesive, and is selected appropriately depending on the purpose.
  • a cell-adhesive culture vessel may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), for the purpose of improving adhesion of the surface of the culture vessel to cells.
  • ECM extracellular matrix
  • the cell-supporting substrate may be any substance intended for cell adhesion.
  • the cells to be cultured in the medium of the present invention are primary T cells and T cells derived from pluripotent stem cells (particularly iPS cells), more preferably CAR-T cells derived from primary T cells and pluripotent stem cells (particularly iPS cells), and even more preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells).
  • the cells to be cultured in the medium of the present invention are primary NK cells and pluripotent stem cell (particularly iPS cell)-derived NK cells, more preferably primary NK cell-derived and pluripotent stem cell (particularly iPS cell)-derived CAR-NK cells, and even more preferably pluripotent stem cell (particularly iPS cell)-derived CAR-NK cells.
  • the culture includes an activation step and an expansion step. When cryopreserved cells are used, a recovery step may be included.
  • the culture of the cell is a concept including any one, two, or all three of recovery culture consisting of a recovery step, activation culture consisting of an activation step, and expansion culture consisting of an expansion step.
  • the culture is directed to expansion culture.
  • Another embodiment of the present invention is a culture of CAR-T cells and CAR-NK cells, preferably pluripotent stem cell (particularly iPS cell)-derived CAR-T cells and pluripotent stem cell (particularly iPS cell)-derived CAR-NK cells, which includes an activation step and an expansion step, and optionally includes a recovery step (when cryopreserved cells are used, etc.).
  • This step is carried out after thawing the cryopreserved cells. Since prolonged exposure of cells to a cryoprotectant can cause damage, the cryoprotectant is promptly removed from the cells after thawing. Then, cell damage and cell dysfunction caused by freezing and thawing are restored.
  • This step/culture method varies depending on the target cells, the cryoprotectant used, the thawing method, etc., but most cells usually recover normally by culturing them in a medium not containing a cryoprotectant for several days, preferably 1 to 5 days, more preferably about 3 days.
  • the method of this step is not particularly limited as long as the desired effect on the cells can be obtained, and for example, it can be carried out by contact with a stimulating substance.
  • the cells are cultured in the presence of a stimulating substance, specifically in a medium containing the stimulating substance, for several days, preferably 1 to 5 days, more preferably about 3 days.
  • the stimulating substance is a signal molecule that controls the activity of cells by a method such as autocrine, paracrine, or endocrine, and may be a substance that can be secreted from all cells contained in the culture system, or may be a substance that is added from the outside.
  • the step is carried out by contact with a substrate to which a ligand is bound, or by culturing in a medium containing a ligand, or the like.
  • the ligand used in the activation culture of CAR-T cells is not particularly limited as long as it is a molecule that interacts with a surface molecule of CAR-T cells and promotes their activation.
  • Examples include CD3, which conjugates with TCR and transmits signal via TCR, and molecules that specifically bind to surface molecules known as costimulatory factors for T cell activation, such as CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L, and have the function of transmitting an activation signal into the T cell.
  • Such molecules may be physiological ligands (or receptors) for the above-mentioned T cell surface molecules, or non-physiological ligands (or receptors) that have agonistic activity.
  • a preferred example of a non-physiological ligand is an agonist antibody.
  • the T cell activation ligand used in the present invention includes an antibody against CD3.
  • the antibody against CD3 may be a complete antibody or a fragment thereof (e.g., Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.) as long as it has the ability to specifically bind to CD3 expressed in target T cells that induce activation, stimulate the surface molecules of these T cells, and transmit a signal into the T cells.
  • the molecule used in the activation culture of CAR-NK cells is not particularly limited as long as it is a molecule that interacts with a surface molecule of CAR-NK cells to promote their activation.
  • it may be a physiological ligand (or receptor) for the above-mentioned NK cell surface molecule, or a non-physiological ligand (or receptor) having agonistic activity. It may also be a cytokine.
  • a preferred example of the non-physiological ligand is an antibody against CD3.
  • the antibody may be a complete antibody or a fragment thereof (e.g., Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.).
  • cytokines include IFN- ⁇ / ⁇ , IFN- ⁇ , IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, etc.
  • This step is a step of growing the cells activated as described above.
  • the method of expansion culture is not particularly limited as long as the desired effect on the activated cells can be obtained, and a person skilled in the art can appropriately adjust the method while monitoring the number of cells, etc.
  • the cells can be cultured in a medium containing the above-mentioned additive components, for example, for 2 days or more, preferably 3 days or more, more preferably 4 days or more, even more preferably 5 days or more, and even more preferably 6 days or more, and the cell number can be appropriately adjusted while monitoring the number of cells, etc., for example, for 7 days or more, 9 days or more, or 11 days or more.
  • the culture can be continued and the number of cells can be exponentially increased.
  • the upper limit of the culture period is not particularly limited, and is, for example, 28 days or less, preferably 21 days or less.
  • expansion culture can be performed in a medium containing cytokines such as IL-7, IL-15, and IL-2, for example, for 2 days or more, preferably 3 days or more.
  • the target cells are primary T cells (particularly CAR-T cells) or NK cells (preferably pluripotent stem cell (particularly iPS cell-derived)-derived NK cells (particularly CAR-NK cells)), and the purpose is to increase the number of cells
  • the cells can be expanded in a medium containing cytokines such as IL-7, IL-15, IL-21, and IL-2, for example, for 2 days or more, preferably 4 days or more, more preferably 6 days or more, even more preferably 8 days or more, even more preferably 10 days or more, and even more preferably 11 days or more.
  • cytokines such as IL-7, IL-15, IL-21, and IL-2
  • the production method of the present invention is a method for producing a cell population in which the number of desired cells is expanded, and the method comprises a step of culturing a cell population containing the desired cells in a serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and/or (v) boric acid or a salt thereof.
  • the production method may be a method comprising a step of culturing a cell population containing desired cells in a serum-free or low-serum medium containing (ia) imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.
  • the "serum-free medium or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and/or (v) boric acid or a salt thereof" and the "serum-free medium or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof" have the same meanings as those described in the above (1) culture method of the present invention and proliferation method of the present invention.
  • the desired cells are cells the proliferation of which is the objective, and include those exemplified in the section "1-1.
  • Cells in (1) above, but are preferably immunocompetent cells, more preferably T cells and NK cells, and particularly preferably T cells into which a CAR gene has been introduced, i.e., CAR-T cells and NK cells into which a CAR gene has been introduced, i.e., CAR-NK cells.
  • the desired cells are CAR-T cells and CAR-NK cells derived from pluripotent stem cells, preferably iPS cells, and even more preferably, CAR-T cells derived from pluripotent stem cells (particularly iPS cells).
  • the origin of the "cell population containing the desired cells” is not particularly limited as long as it contains the desired cells, and may be naturally derived or artificially prepared.
  • the cell population may be a cell population containing peripheral blood mononuclear cells (PBMC), blood cells, hematopoietic stem cells, cord blood mononuclear cells, etc., collected, isolated, purified, or induced from body fluids such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, etc.
  • PBMC peripheral blood mononuclear cells
  • blood cells hematopoietic stem cells
  • cord blood mononuclear cells etc.
  • body fluids such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, etc.
  • These cells may be collected from a living body or obtained through ex vivo culture, for example, a cell population obtained by the manufacturing method of the present invention may be directly or cryopreserved.
  • the cell population is not limited to a single-cell cell population, and may be a slice of a tissue mass obtained by biopsy, but is preferably a single-cell cell population.
  • a liquid cell population it is not limited to a cell population derived from a single tissue, and may be a mixed system of cell populations derived from different tissues.
  • the administration schedule is appropriately determined according to the age, weight, body surface area, symptoms, etc. of the subject, and may be a single administration, or continuous or regular multiple administrations.
  • the pharmaceutical agent of the present invention may be used for autotransplantation or allotransplantation. It may also be used in combination with other pharmaceutical agents.
  • the culture medium additive of the present invention contains (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further contains (iv) vitamin E and/or (v) boric acid or a salt thereof.
  • the culture medium additive of the present invention may contain (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.
  • the medium additive of the present invention is used to add to a serum-free medium or a low-serum medium.
  • the above components (i) to (iii) and, optionally, components (iv) and/or (v) are the same as those described in the above (1) of the culture method of the present invention and the growth method of the present invention.
  • Imidazole dipeptides usually 1 to 50 mM, preferably 3 to 40 mM, more preferably 5 to 30 mM, particularly preferably 10 to 25 mM.
  • Vitamin E usually 10 to 1000 ⁇ M, preferably 20 to 500 ⁇ M, more preferably 40 to 300 ⁇ M, particularly preferably 100 to 250 ⁇ M.
  • Taurines usually 0.1 to 20 mM, preferably 0.5 to 15 mM, more preferably 1 to 10 mM, particularly preferably 2 to 5 mM.
  • Lysophosphatidic acids usually 0.1 to 50 mg/L, preferably 0.5 to 25 mg/L, more preferably 1 to 15 mg/L, particularly preferably 2 to 10 mg/L.
  • hematopoietic cytokines SCF, TPO, and FLT3L were used to differentiate into hematopoietic progenitor cells.
  • the differentiation of the obtained hematopoietic progenitor cells into cytotoxic T lymphocytes (CTLs) was performed according to a known patent method (WO2017/221975) and literature information (Nature Communication 2021; 12: 430).
  • CD34-positive cells were purified from the obtained hematopoietic progenitor cells using magnetic beads (Myltenyi Biotec), and cultured for 3 weeks in ⁇ -MEM medium containing SCF, TPO, FLT3L, IL-7, SDF1 ⁇ , and SB203580 on a plate on which Fc-DLL4 and RetroNectin (Recombinant Human Fibronectin Fragment, TakaraBio) were immobilized.
  • Fc-DLL4 and RetroNectin Recombinant Human Fibronectin Fragment, TakaraBio
  • a CAR gene that recognizes a specific antigen was introduced into the obtained TCR-positive CTLs using a retroviral vector.
  • the cells were cryopreserved in liquid nitrogen and used as iPS cell-derived CAR-T cells.
  • iPS cell-derived CAR-T cells were suspended at 500,000 cells/mL in IMDM medium containing 15% FBS to which the additives shown in the "Recovery culture medium” column in Table 1 were added, seeded on G-Rex (registered trademark) 6M (Wilson Wolf), and cultured for 3 days under 5% CO2 /37°C.
  • Activation culture and recovery culture of iPS cell-derived CAR-T cells The iPS cell-derived CAR-T cells after the recovery culture were suspended in IMDM medium containing 15% FBS to which the additives shown in the "Activation culture medium” column in Table 1 were added, at a concentration of 133,333 cells/mL, and seeded into a T225 flask onto which an anti-CD3 agonist antibody (OKT3) had been immobilized, and cultured for 3 days under 5% CO 2 /37°C.
  • Test Example 1-2 Test for confirming proliferation ability of cryopreserved cells in serum-containing medium
  • cells were collected on the third day of expansion culture of iPS cell-derived CAR-T cells, and the number of cells was counted.
  • a suspension containing a predetermined number of cells was centrifuged at 300 xg for 5 minutes, and the supernatant was removed.
  • the cell mass was suspended at 8,000,000 cells/mL in MEM medium to which Albuminar 25% I.V. Injection 12.5g/50mL (product name) was added so that the final albumin concentration was 5%, and an equal amount of CryoStor (registered trademark) CS10 (STEMCELL Technologies) was added to this suspension and cryopreserved.
  • CryoStor registered trademark
  • the cells were recovered from the T225 flask, suspended in each of the three types of expansion culture media to a concentration of 40,000 cells/mL, and cultured at 5% CO 2 /37° C. using G-Rex (registered trademark) 100M (Wilson Wolf). Then, on the second day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M, the cell count was counted, and the medium was replaced with each medium. On the second day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M, the cell count was counted. On the third day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M, the cell count was counted.
  • G-Rex registered trademark
  • Test Example 2-1 Proliferation test verifying additive effect of carnosine and ⁇ -tocopherol in serum-free medium
  • the cell number and viability of iPS cell-derived CAR-T cells were measured on days 2 and 3 of culture in the expansion culture described in item 5 of Example 2.
  • the cell proliferation fold from day 0 of expansion culture is shown in Table 7.
  • the iPS cell-derived CAR-T cells had a higher proliferation fold in all conditions in serum-free medium than in a medium containing 15% FBS (Table 7).
  • the cell viability from day 0 of expansion culture is shown in Table 8.
  • the iPS cell-derived CAR-T cells had a viability of 90% or more in all conditions in serum-free medium (Table 8).
  • Test Example 2-2 Test for confirming proliferation ability of cryopreserved cells in serum-containing medium
  • cells were collected on the third day of expansion culture of iPS cell-derived CAR-T cells, and the cell number was counted.
  • a suspension containing a predetermined number of cells was centrifuged at 300 xg for 5 minutes, and the supernatant was removed.
  • the cell mass was suspended at 8,000,000 cells/mL in MEM medium to which Albumin 25% I.V. Injection 12.5g/50mL (product name) was added so that the final albumin concentration was 5%, and an equal amount of CS10 was added to this suspension and cryopreserved.
  • the frozen cells After thawing the frozen cells, they were suspended in IMDM medium containing 15% FBS, 2 mM glutamine, ITS (x1), and 50 ⁇ g/mL ascorbic acid 2-phosphate sesquimagnesium salt hydrate to a concentration of 125,000 cells/mL, and cultured at 5% CO 2 /37°C using a G-Rex 24-well plate. Thereafter, the number of live cells and cell viability were measured on days 0, 3, and 7 of culture. The results of the measurement are shown in Table 9.
  • the cells cultured and then cryopreserved after adding 20 mM carnosine and 200 ⁇ M ⁇ -tocopherol to the expansion culture medium showed improved proliferation rates in the proliferation ability confirmation test, and had higher proliferation rates than the cells cultured and then cryopreserved in an expansion culture medium containing 15% FBS.
  • the additive effect of carnosine and ⁇ -tocopherol in expansion culture was observed (Table 9).
  • Example 3 Activation culture of human-derived primary T cells
  • Human-derived primary T cells (Leukopak-SoloPak, Charles River Laboratories Cell Solutions) were suspended at 1,000,000 cells/mL in OpTmizer medium (Thermo Fisher) containing 2% CTS Immune Cell SR (Thermo Fisher) with the additives shown in the activation culture medium in Table 10, seeded in a PL240 bag (OriGen), and cultured for 2 days under 5% CO2 /37°C.
  • OpTmizer medium Thermo Fisher
  • CTS Immune Cell SR Thermo Fisher
  • the reagent RetroNectin (product name) was purchased from Takara Bio.
  • Expansion culture of human-derived primary T cell-derived CAR-T cells in three types of serum-free media A total of three types of expansion culture media were prepared: an expansion culture medium obtained by adding the additives shown in Expansion Culture Medium 1 in Table 10 to an OpTmizer medium containing 2% CTS Immune Cell SR, an expansion culture medium obtained by adding the additives shown in Expansion Culture Medium 2 in Table 10 to a BM220720 medium, and an expansion culture medium further obtained by adding 0.3 mM boric acid (Sigma-Aldrich), 3 mM taurine (Sigma-Aldrich), 5 mg/L lysophosphatidylcholine (Sigma-Aldrich), 20 mM carnosine (Sigma-Aldrich), and 200 ⁇ M ⁇ -tocopherol (Sigma-Aldrich).
  • an expansion culture medium obtained by adding the additives shown in Expansion Culture Medium 1 in Table 10 to an OpTmizer medium containing 2% CTS Immune Cell SR an expansion
  • the cells were reseeded in the same medium at 1.25 x 105 cells/mL onto a G-Rex (registered trademark) 24-well plate (Wilson Wolf) every 2 to 3 days, and culture was continued in a 5% CO2 /37°C incubator until the 17th day of culture.
  • G-Rex registered trademark
  • the present invention even when a serum-free or low-serum medium is used, it is possible to obtain a cell growth maintenance and promotion effect comparable to that obtained by culturing in a serum-containing medium.
  • a serum-free or low-serum medium that does not require intensive washing, and the present invention is suitable for culture under such conditions.

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Abstract

La présente invention a pour but de procurer : un procédé permettant une prolifération suffisante de cellules, plus particulièrement de cellules immunocompétentes, même dans un milieu sans sérum ou un milieu à faible teneur en sérum ; et une composition de milieu destinée à être utilisée dans ce procédé. En incorporant le dipeptide imidazole ou un de ses sels, la taurine ou un de ses précurseurs, ou un de leurs sels, et l'acide lysophosphatidique ou un de ses dérivés, il est possible d'obtenir un effet de maintien/promotion de la prolifération cellulaire comparable à celui d'une culture dans un milieu contenant du sérum, même dans un milieu sans sérum ou un milieu à faible teneur en sérum.
PCT/JP2024/045351 2023-12-25 2024-12-23 Procédé de culture cellulaire Pending WO2025142807A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070110743A1 (en) * 2005-10-24 2007-05-17 Wyeth Methods of protein production using anti-senescence compounds
JP2014521337A (ja) * 2011-07-29 2014-08-28 セルラー ダイナミクス インターナショナル， インコーポレイテッド 幹細胞由来の組織細胞中の代謝成熟
CN105462912A (zh) * 2016-01-21 2016-04-06 四川百诺吉科技有限公司 适用于二倍体细胞培养的无蛋白无血清培养基及应用
CN113025564A (zh) * 2021-03-17 2021-06-25 金华市人民医院 一种卵母细胞体外成熟培养液的制备方法
WO2024135853A1 (fr) * 2022-12-23 2024-06-27 Ｃｅｌｌ Ｅｘｏｓｏｍｅ Ｔｈｅｒａｐｅｕｔｉｃｓ株式会社 Utilisation de cellules souches mésenchymateuses ou d'un surnageant de culture de celles-ci

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070110743A1 (en) * 2005-10-24 2007-05-17 Wyeth Methods of protein production using anti-senescence compounds
JP2014521337A (ja) * 2011-07-29 2014-08-28 セルラー ダイナミクス インターナショナル， インコーポレイテッド 幹細胞由来の組織細胞中の代謝成熟
CN105462912A (zh) * 2016-01-21 2016-04-06 四川百诺吉科技有限公司 适用于二倍体细胞培养的无蛋白无血清培养基及应用
CN113025564A (zh) * 2021-03-17 2021-06-25 金华市人民医院 一种卵母细胞体外成熟培养液的制备方法
WO2024135853A1 (fr) * 2022-12-23 2024-06-27 Ｃｅｌｌ Ｅｘｏｓｏｍｅ Ｔｈｅｒａｐｅｕｔｉｃｓ株式会社 Utilisation de cellules souches mésenchymateuses ou d'un surnageant de culture de celles-ci

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EBERHARDT FRANZISKA, HüCKELHOVEN€‘KRAUSS ANGELA, KUNZ ALEXANDER, JIANG GENQIAO, SAUER TIM, REICHMAN AVINOAM, NEUBER BRIGITTE, BÃ: "Impact of serum‑free media on the expansion and functionality of CD19.CAR T‑cells", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 52, no. 1, GR , XP093329945, ISSN: 1107-3756, DOI: 10.3892/ijmm.2023.5261 *
HYLAND PAUL, DUGGAN ORLA, HIPKISS ALAN, BARNETT CHRISTOPHER, BARNETT YVONNE: "The effects of carnosine on oxidative DNA damage levels and in vitro lifespan in human peripheral blood derived CD4+T cell clones", MECHANISMS OF AGEING AND DEVELOPMENT., ELSEVIER SEQUOIA, LAUSANNE,, CH, vol. 121, no. 1-3, 1 January 2001 (2001-01-01), CH , pages 203 - 215, XP093329948, ISSN: 0047-6374, DOI: 10.1016/S0047-6374(00)00211-6 *
PARK MEEYOUNG, LI QIN, SHCHEYNIKOV NIKOLAY, ZENG WEIZONG, MUALLEM SHMUEL: "NaBC1 Is a Ubiquitous Electrogenic Na ؉ -Coupled Borate Transporter Essential for Cellular Boron Homeostasis and Cell Growth and Proliferation ", MOLECULAR CELL, vol. 16, 5 November 2004 (2004-11-05), pages 331 - 341, XP093329951, DOI: 10.1016/j.molcel.2004.09.030 *
SAITO YOSHIRO ET AL: "Cell death caused by selenium deficiency and protective effect of antioxidants.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 278, no. 41, 10 October 2003 (2003-10-10), US , pages 39428 - 39434, XP002542537, ISSN: 0021-9258, DOI: 10.1074/jbc.M305542200 *
YANG MIN; SUN LUCHUANYANG; KAWABATA YASUNOSUKE; MAEGAWA TAKAHIRO; TANIYAMA SHIGETO; TACHIBANA KATSUYASU; HIRASAKA KATSUYA: "Balenine, imidazole dipeptide, induces activation of superoxide dismutase in myotubes", FISHERIES SCIENCE, JAPANESE SOCIETY OF SCIENTIFIC FISHERIES, JP, vol. 87, no. 3, 23 April 2021 (2021-04-23), JP , pages 403 - 409, XP037451192, ISSN: 0919-9268, DOI: 10.1007/s12562-021-01516-7 *

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