Method for producing cytotoxic t cells

Abstract

Disclosed is a method for producing activated cytotoxic T cells derived from iPS cells, the method including the following step (1): (1) a step for activating unactivated cytotoxic T cells derived from iPS cells in a container having excellent gas exchange properties. Also disclosed is a method for proliferating activated cytotoxic T cells derived from iPS cells, the method including the following step (1A): (1A) a step for activating unactivated cytotoxic T cells derived from iPS cells in a container having excellent gas exchange properties. Also disclosed are: a cell mass containing activated cytotoxic T cells produced by the method; and a drug containing a cell mass containing the activated cytotoxic T cells.

Description

Method for producing cytotoxic T cells

[0001] The present invention relates to a method for producing cytotoxic T cells, a method for expanding cytotoxic T cells, a cell population containing cytotoxic T cells obtained by the method, and a pharmaceutical comprising the cell population containing cytotoxic T cells. (Background of the Invention)

[0002] One known cancer treatment method is chimeric antigen receptor (CAR)-T cell therapy, which uses T cells (sometimes referred to as "CAR-T cells" herein) that express a chimeric antigen receptor (also referred to as "CAR" herein). CAR-T cells are produced, for example, using autologous T cells collected from the patient themselves, but the high production costs and risk of production failure pose business challenges. In particular, many cancer patients who undergo CAR-T cell therapy have T cells that are damaged compared to healthy individuals due to treatment with anticancer drugs, and therefore the production efficiency tends to be low in the process of producing CAR-T cells from patient-derived T cells.

[0003] One approach to solving these problems is to develop a source of allogeneic T cells. Furthermore, technology for producing T cells from pluripotent stem cells may provide a platform for producing allogeneic T cells. As one such technology, Non-Patent Document 1 reports improved T cell differentiation efficiency using induced pluripotent stem cells (sometimes referred to herein as "iPS cells") derived from antigen-specific cytotoxic T cell clones or iPS cells transfected with T cell receptors (referred to herein as "TCRs") as starting materials.

[0004] Furthermore, Non-Patent Document 2 reports that iPS cells into which a foreign TCR gene has been introduced (TCR-iPSCs) were prepared, and CD8αβ cytotoxic T cells were regenerated from these cells, and that these regenerated CD8αβ cytotoxic T cells exhibited cytotoxic activity equivalent to that of CD8αβ cytotoxic T cells regenerated from iPS cells derived from T cells.

[0005] When producing CAR-T cells, a T cell activation step using stimulatory substances (e.g., anti-CD3 antibodies and anti-CD28 antibodies) is generally carried out. In the production of autologous CAR-T cells, containers with excellent gas exchange properties have traditionally been used in the activation step. For example, Non-Patent Documents 3 and 4 describe the use of gas-permeable culture bags and gas exchange in the expansion culture of the autologous CAR-T cell production step. Additionally, Non-Patent Documents 5 and 6 also describe the use of gas-permeable culture bags in the activation step of autologous T cells.

[0006] On the other hand, the activation step of iPS cell-derived cytotoxic T cells has conventionally been performed using T flasks or well plates, and there have been no reports of using a gas-permeable container such as the above-mentioned culture bag in the activation step of iPS cell-derived cytotoxic T cells.

[0007] Nature Communications, 2021, 12, 430; Molecular Therapy Methods & Clinical Development, 2020, 19, 250-260; Cytotherapy, 2019, 21, 327-340; J. Immunother, 2009, 32(2), 169-180; Dynabeads CD3 / CD28 Catalog no. 4020 3D package insert; Takara Bio Inc. website: "Expansion of T lymphocytes (T cells) by RetroNectin Costimulation," [online], [searched September 6, 2023], Internet <URL : https: / / catalog.takara-bio.co.jp / com / tech_info_detail.php?mode=3&masterid=M100004690&unitid=U100002954>

[0008] Therefore, an object of the present invention is to provide a method for producing cytotoxic T cells derived from iPS cells that have excellent cytotoxic activity against cancer cells, a method for expanding cytotoxic T cells derived from iPS cells, and the like.

[0009] As a result of extensive research to achieve the above-mentioned object, the present inventors investigated the scaling up of the activation step and discovered that by activating iPS-derived cytotoxic T cells using a vessel with excellent gas exchange, proliferation in the expansion step is equivalent to that of activation using a T flask, and iPS-derived cytotoxic T cells with excellent cell proliferation and cancer cell cytotoxicity after freezing and thawing can be obtained after the expansion step.

[0010] The present invention was completed based on these findings and through further investigation, and provides the following methods for producing cytotoxic T cells, methods for expanding cytotoxic T cells, cell populations containing cytotoxic T cells, pharmaceuticals, etc.

[0011] [1] A method for producing activated cytotoxic T cells derived from iPS cells, comprising the following step (1): (1) activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange. [2] The method according to [1], wherein the unactivated cytotoxic T cells contain a foreign gene encoding a receptor. [3] The method according to [2], wherein the receptor is a chimeric antigen receptor. [4] The method according to any of [1] to [3], wherein the container is a bag. [4a] The method according to any of [1] to [3], wherein the container has a bottom surface made of a material with excellent gas permeability. [5] A method for expanding activated cytotoxic T cells derived from iPS cells, comprising the following step (1A): (1A) activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange. [6] The method according to [5], wherein the unactivated cytotoxic T cells contain a foreign gene encoding a receptor. [7] The method according to [6], wherein the receptor is a chimeric antigen receptor. [8] The method according to any of [5] to [7], wherein the container is a bag. [8a] The method according to any one of [5] to [7], wherein the container has a bottom surface made of a material with excellent gas permeability. [9] A cell population containing activated cytotoxic T cells obtained by the method according to any one of [1] to [8a].

[10] A pharmaceutical comprising the cell population containing activated cytotoxic T cells according to [9].

[11] The pharmaceutical according to

[10] , which is a preventive or therapeutic drug for cancer.

[12] A method for preventing or treating cancer, comprising administering the cell population containing activated cytotoxic T cells according to [9] to a subject in need thereof.

[13] The cell population containing activated cytotoxic T cells according to [9], for use in the prevention or treatment of cancer.

[14] Use of the cell population containing activated cytotoxic T cells according to [9] in the manufacture of a pharmaceutical for preventing or treating cancer.

[0012] According to the present invention, it is possible to produce iPS-derived cytotoxic T cells that have excellent cytotoxic activity against cancer cells.

[0013] 1 is a graph showing the cell count (relative value when day 0 is set to 1) (upper graph) and viability (%) (lower graph) of iPS cell-derived activated CAR-T cells after 3 days of activation culture and 3 days of expansion culture. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture after freeze-thawing. The vertical axis represents the relative value when the proliferation fold of standard iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture is set to 100%. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture after freeze-thawing, when the cytokine concentration is increased in the activation step. The vertical axis represents the relative value when the proliferation fold of standard iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture is set to 100%. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture after freeze-thawing, when the cytokine concentration is increased in the activation step. The vertical axis represents the relative value when the proliferation fold of standard iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture is set to 100%. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture after freezing and thawing, when the cytokine concentration in the activation step was increased. The vertical axis represents the relative value when the proliferation fold of standard iPS cell-derived activated CAR-T cells from day 0 to day 7 of culture is set to 100%. This graph shows the in vivo antitumor effect of iPS cell-derived activated CAR-T cells. The vertical axis represents the tumor size (mm ) of NSG mice bearing GSU cells, a cell line derived from human gastric cancer. ３). The values ​​on the graph are mean ± standard deviation, *p<0.001, significance between groups was tested by two-way analysis of variance, N=5. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from days 0 to 6 of culture after freeze-thawing, when iPS cell-derived activated CAR-T cells recognizing different antigens were used. The vertical axis represents the relative value when the proliferation fold from days 0 to 6 of culture of cells activated in a T225 flask is set to 100%. This graph shows the results of a proliferation test of iPS cell-derived activated CAR-T cells from days 0 to 7 of culture after freeze-thawing, when different containers are used in the activation step. The vertical axis represents the relative value when the proliferation fold from days 0 to 7 of culture of standard iPS cell-derived activated CAR-T cells is set to 100%. This graph shows the cell number of iPS cell-derived activated CAR-T cells (relative value when day 0 is set to 1) over 3 days of expansion culture, when the container used in the activation step was scaled up. Condition 1: N = 1, Condition 2: N = 1, Condition 3: N = 3 (values ​​are mean ± standard deviation), Condition 4: N = 1, Condition 5: N = 1

[0014] Hereinafter, embodiments of the present invention will be described in detail.

[0015] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" or "consisting essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0016] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0017] As used herein, "positive (+)" means that the protein or gene is expressed in an amount that can be detected by a method known in the art. In addition, in the case of a protein that is expressed intracellularly and not present on the cell surface (e.g., a transcription factor or a subunit thereof), the protein of interest can be detected by expressing a reporter protein together with the protein and detecting the reporter protein. Gene detection can be carried out using, for example, nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0018] As used herein, "negative (-)" means that the expression level of a protein or gene is below the lower limit of detection by all or any of the above-mentioned known techniques, or that the level of expression is low. The lower limit of detection for protein or gene expression may differ depending on the technique. The level of protein or gene expression (whether low or high expression) can be determined by comparing with the results of control cells measured under the same conditions.

[0019] Detection of cell surface markers can be carried out using immunological assays, such as ELISA, immunostaining, and flow cytometry, using antibodies specific to the cell surface marker. As used herein, a cell surface marker refers to a protein expressed on the cell surface that can be labeled (stained) with a fluorescent substance and that facilitates the detection, enrichment, isolation, etc. of cells expressing the cell surface marker. The cell surface marker refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a specific cell type, specifically a substance that is produced (positive marker) or not produced (negative marker) as mRNA by transcription of the gene in the genome or as a protein by translation of the mRNA.

[0020] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0021] As used herein, the term "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification).

[0022] As used herein, "hematopoietic progenitor cells" (HPCs) are cells that have the ability to differentiate into blood cells but do not have the self-renewal ability of stem cells. In the human body, hematopoietic progenitor cells are primarily present in the bone marrow. As used herein, hematopoietic progenitor cells (HPCs) are CD34-positive and may be CD43-positive (CD34 ＋ / CD43 ＋ Additionally, HPCs may be positive for CD24, CD62L, CD90, CD143, CD263, Notch3, CD32, CD39, CD49a, CD164, CD317, CD200, CD218a, CD7, CD144, CD56, CD226, CD262, and CD325, and negative for CD49f, CD51, CD102, CD42b, CD61, CD62P, CD69, CD102, and CD156c, as described in WO 2018 / 199186.

[0023] As used herein, "hemogenic endothelial cells (HECs)" refer to cells that express CD34 but do not express CD43, CD184, or CD73 (CD34 ＋ / CD43 － / CD184 － / CD73 － ) cells (Note: CD34 ＋ / CD43 － / CD184 － / CD73 － The cells do not express CD7, so CD34 ＋ / CD43 － / CD184 － / CD73 － The cells are CD34 ＋ / CD7 － / CD43 － / CD184 － / CD73 － (also referred to as cells).

[0024] As used herein, the term "cytotoxic T cells" refers to T cells that are positive for the surface antigen CD8 and negative for CD4 (CD4 － / CD8 ＋, CD8 single positive (SP)) and have cytotoxic activity. Cytotoxic T cells recognize antigen peptides derived from viruses, tumors, etc., presented together with class I major histocompatibility complex antigens (MHC class I, HLA class I) on antigen-presenting cells via T cell receptors (TCRs) present on their cell surfaces, and exert cytotoxic activity specifically against cells presenting the antigen peptides, which are foreign substances. Cytotoxic activity can be confirmed, for example, using the secretion or production of granzymes, perforin, etc. as indicators.

[0025] As used herein, the term "cell population" refers to two or more cells of the same or different types. The term "cell population" also refers to a mass of cells of the same or different types.

[0026] As used herein, "cytotoxic T cell proliferation" or "activated cytotoxic T cell proliferation" refers to an increase in the number (absolute number) of cytotoxic T cells in a cell population compared to before culture, and means, for example, an increase of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1200%, 1500%, 2000%, 2500%, or 3000% compared to before culture or a control. In certain embodiments of the present invention, the number (absolute number) of cytotoxic T cells in a cell population produced by the present invention is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1200%, 1500%, 2000%, 2500%, or 3000% compared to before culture.

[0027] From the viewpoint of therapeutic application, the various cells used in the present invention are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0028] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872), and Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open Publication No. 2008-307007), and the like can also be used.

[0029] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholeer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, U.S. Patent Application Publication No. 2008 / 2336610, U.S. Patent Application Publication No. 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) Any of the induced pluripotent stem cells known in the art can be used.

[0030] As induced pluripotent stem cell lines, various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain, and Kyoto University's Ff-I01s04 strain, QHJI strain, RWMH strain, DRXT strain, RJWI strain, YZWJ strain, ILCL strain, GLKV strain, 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain.

[0031] The term "nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules having functions equivalent to those nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analogue. Nucleic acids may also be single-stranded or double-stranded nucleic acids. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0032] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acid (BNA), locked artificial nucleic acid (LNA), ethylene bridged artificial nucleic acid (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0033] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0034] The method for producing activated cytotoxic T cells derived from iPS cells of the present invention (sometimes referred to herein simply as the "production method of the present invention") is characterized by comprising the following step (1): (1) a step of activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange (also referred to herein as the "activation step").

[0035] The unactivated cytotoxic T cells used in step (1) are differentiated from iPS cells, and can be differentiated into cytotoxic T cells according to known methods. For example, one such method involves inducing iPS cells to differentiate into hemogenic endothelial cells or hematopoietic progenitor cells, and then inducing the hematopoietic stem cells or hematopoietic progenitor cells to differentiate into cytotoxic T cells. The differentiation of iPS cells into hemogenic endothelial cells or hematopoietic progenitor cells can be performed, for example, as described in Nature Communications, 2021, 12, 430 and Molecular Therapy Methods & Clinical Development, 2020, 19, 250-260. In addition, differentiation of hemogenic endothelial cells or hematopoietic progenitor cells into cytotoxic T cells can be carried out according to the description in Nature Communications, 2021, 12, 430 and WO 2017 / 221975.

[0036] The unactivated cytotoxic T cells used in step (1) may be CCR7 (CD197) and / or CD25 negative, and the activated cytotoxic T cells activated in step (1) may be CCR7 and / or CD25 positive.

[0037] The iPS cells and cells for producing iPS cells used in the present invention may be derived from humans or mammals other than humans (non-human mammals), preferably humans. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys. Universalized iPS cells (i.e., cells with a specific HLA type or knockout HLA that do not cause immune rejection in many patients) can also be used.

[0038] Substances used to activate non-activated cytotoxic T cells include, for example, CD3 agonists, CD28 agonists, and CD30 agonists. Examples of CD3 agonists include anti-CD3 antibodies, examples of CD28 agonists include anti-CD28 antibodies, and examples of CD30 agonists include anti-CD30 antibodies. These can be used alone or in combination of two or more, and substances other than antibodies that have similar effects can also be used. These antibodies may be monoclonal or polyclonal, but monoclonal antibodies are preferred. In the activation step, RetroNectin (trade name) (Recombinant Human Fibronectin Fragment, Takara Bio Inc.) may be used simultaneously with the CD3 agonist, CD28 agonist, CD30 agonist, etc. The substances used to activate non-activated cytotoxic T cells and RetroNectin can also be used by binding them to a culture vessel.

[0039] The concentration of a substance used to activate non-activated cytotoxic T cells is not particularly limited, as long as it is a concentration that can stimulate the surface molecules of cytotoxic T cells, transduce a signal into the cytotoxic T cells, and induce activation. For example, when an anti-CD3 antibody, an anti-CD28 antibody, or an anti-CD30 antibody is used, the antibody can be used so that the final concentration of each antibody is 0.1 to 20 μg / mL.

[0040] The time for the activation step is not particularly limited as long as it is a time that can induce activation of cytotoxic T cells, and is, for example, 10 to 120 hours, preferably 15 to 100 hours, and more preferably 20 to 90 hours.

[0041] The activation step can be terminated by replacing the medium containing the substance used for activating the unactivated cytotoxic T cells with a medium containing no substance, or by diluting the medium containing the substance used for activating the unactivated cytotoxic T cells.

[0042] In the activation step of the present invention, except for the use of a vessel with excellent gas exchange and unactivated cytotoxic T cells derived from iPS cells, other culture embodiments, such as the culture method, culture medium, culture medium volume, and other culture conditions (temperature, atmosphere, cell density, etc.), may be in accordance with known or general methods for activating cytotoxic T cells, or may be appropriately adjusted based on such methods to suit the present invention. The cell density at the time of seeding in the activation step is, for example, 1.0 x 10 per volume. ４ ~1.0 x 10 ７ cells / mL, preferably 2.0 x 10 ４ ~6.0 x 10 ６ cells / mL, per area, for example, 5.0 x 10 3 ~5.0 x 10 6 cells / cm ２ , more preferably 2.5 × 10 4 ~2.5 x 10 6 cells / cm ２ The amount of medium in the activation step can be, for example, 0.05 to 20 mL / cm ２ and preferably 0.1 to 10 mL / cm ２ It can be said that:

[0043] A container with excellent gas exchange is used as the culture vessel in the activation step. A container with excellent gas exchange refers to a container with a structure that allows gas (gas) to be exchanged between the inside of the container or culture medium and the outside of the container or culture medium, and has excellent gas exchange efficiency. Examples of containers with excellent gas exchange include containers with a structure that allows gas outside the container to be taken into the container and gas inside the container to be discharged outside the container (e.g., a bioreactor with a structure that allows gas outside the container to be taken into the container and gas inside the container to be discharged outside the container), and containers whose bottom surface uses a material with excellent gas permeability (e.g., a bag using a material with excellent gas permeability (e.g., fluoroethyl polymer) and a container whose bottom surface uses a material with gas permeability (e.g., silicone elastomer and gas-permeable polystyrene)). In this specification, gas refers to, for example, air, oxygen, carbon dioxide, nitrogen, etc.

[0044] A container with excellent gas exchange is oxygen (O ２ In another embodiment, the culture vessel used in the activation step is a vessel having a permeability to carbon dioxide (CO ２ ) permeability is, for example, 10 to 20,000 ml / 24 hours (25°C), preferably 50 to 15,000 ml / 24 hours (25°C), and nitrogen (N ２ ) is, for example, 5 to 5000 ml / 24 hours (25°C), preferably 100 to 2500 ml / 24 hours (25°C).

[0045] The type of container can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture tanks), bioreactors, etc., depending on the culture scale. Examples of containers with excellent gas exchange include containers made of a material with excellent gas permeability (e.g., bags made of a material with excellent gas permeability (e.g., fluoroethyl polymer), and containers made of a material having gas permeability on the bottom surface (e.g., silicone elastomer and gas-permeable polystyrene)), and bioreactors that can be ventilated (e.g., bioreactors equipped with a gas supply device). Commercially available containers with excellent gas exchange include PL07-2G, PL30-2G, PL70-2G, PL120-2G, PL240-2G, PL325-2G, PL500P-2G, PL750-2G, PL1000P-2G, and PL2000P2-2G from OriGen Biomedical, Culture bag 215 from Takara Bio Inc., Culture bag GT-T610 (CultiLife Eva), and G-Rex (trade name) 10, G-Rex 10M, G-Rex 10M-CS, G-Rex 100, and G-Rex from Wilson Wolf. G-Rex 100M, G-Rex 100-CS, G-Rex 24 Well Plate, G-Rex 6 Well Plate, G-Rex 6M Well Plate, and other Miltenyi Biotec MACS (trade name) GMP Cell Culture Bags, Saint-Gobain VueLife (trade name) "C" Series Bags, VueLife (trade name) "AC" Series Bags, VueLife (trade name) "HP" Series Bags, and Nipro Corporation NIPRO Cell Culture Examples of such products include HYPERFLASK (trade name) and HYPERSTACK (trade name) from Corning.

[0046] In the activation step, a known or common medium used for culturing cytotoxic T cells can be used, and the medium can be supplemented with necessary components as appropriate.

[0047] Examples of media used in the activation step include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, Fisher's medium, and various other commercially available products for T cell culture (e.g., CTS OpTmizer (trade name) T-Cell Expansion Basal Medium (Thermo Fisher Scientific), CTS OpTmizer (trade name) Pro Serum Free Medium (Thermo Fisher Scientific), CTS Examples of suitable media include OpTmizer (trade name) Pro (Thermo Fisher Scientific), CTS OpTmizer (trade name) T-Cell Expansion Supplement (Thermo Fisher Scientific), and 4CELL (trade name) Nutri-T medium (Sartorius). Any one of these media may be used alone, or two or more may be used in combination.

[0048] The medium may be serum-containing or serum-free, or xeno-free. To prevent contamination with components derived from different species, the serum may be derived from the same animal as the cells being cultured. Serum-free medium refers to a medium that does not contain raw or unpurified serum, and thus may include media with purified blood-derived components or animal tissue-derived components (e.g., growth factors). The medium may or may not contain any substitute for serum. Serum substitutes may include materials that appropriately contain albumin (e.g., albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin, or humanized albumin, plant starch, dextran, protein hydrolysates, etc.), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thioglycerol (α-monothioglycerol, MTG), or equivalents thereof. Commercially available materials such as Knockout Serum Replacement (KSR), Chemically-defined Lipid Concentrated (Thermo Fisher Scientific), and GlutaMAX (Thermo Fisher Scientific) can also be used.

[0049] The medium may contain one or more compounds selected from the group consisting of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamins such as vitamin A (acetate), bovine serum albumin (BSA) or human albumin, fatty acid-free fraction V, catalase, human recombinant insulin, human transferrin, proteins such as superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, T3 (triiodo-L-thyronine), PSG (penicillin, streptomycin, and L-glutamine), and caspase inhibitors. The medium may also contain exogenously added ascorbic acid or a derivative thereof (e.g., ascorbic acid 2-phosphate: PAA). The medium may contain one or more substances selected from the group consisting of fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antibiotics, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and inorganic salts, each of which is added from the outside.

[0050] The medium may contain exogenously added cytokines. Examples of cytokines include FLT3 ligand (FLT3L), interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-3, IL-4, IL-6, IL-12, IL-15, IL-18, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and midkine. These cytokines may be used alone or in combination of two or more. In certain embodiments, IL-2, IL-7, IL-15, IL-18, and IL-21 are preferred cytokines to be added to the medium, and it is more preferred to use these cytokines in combination. When these cytokines are used in combination, their preferred concentrations are as follows: IL-7: 0.3 to 100 ng / mL (preferably 1.0 to 30 ng / mL, more preferably 3.0 to 20 ng / mL) IL-15: 0.3 to 100 ng / mL (preferably 1.0 to 30 ng / mL, more preferably 3.0 to 20 ng / mL) IL-2: 3 to 5000 IU / mL (preferably 10 to 3000 IU / mL, more preferably 30 to 1500 IU / mL) IL-18: 2 to 1000 ng / mL (preferably 10 to 500 ng / mL, more preferably 30 to 300 ng / mL, even more preferably 50 to 200 ng / mL, still more preferably 100 to 180 ng / mL) IL-21: 1 to 1000 ng / ml (preferably 3 to 300 ng / mL, more preferably 10 to 150 ng / mL, even more preferably 20 to 100 ng / mL, and even more preferably 50 to 80 ng / mL)

[0051] In one embodiment of the present invention, the non-activated cytotoxic T cells to be activated may be non-activated cytotoxic T cells containing a foreign gene encoding a receptor. The "foreign gene" is a gene introduced from the outside to cause the cytotoxic T cells to express a desired protein, and can be selected appropriately depending on the intended use of the cytotoxic T cells. The foreign gene may be one or more types. Examples of the receptor include a chimeric antigen receptor (CAR), a CCR (chimeric costimulatory receptor), a STAR (synthetic TCR and antigen receptor), a TCR, and a chimeric TAC (T cell antigen coupler) receptor.

[0052] The foreign gene can be, for example, a gene for expressing a chimeric antigen receptor (CAR), which can further include a gene for expressing a cytokine and / or a chemokine. Similar to common or known CARs, the CAR expressed by cytotoxic T cells is basically composed of peptides from each of the following sites linked via a spacer as needed: (i) an antigen recognition site (e.g., a single-chain antibody) that recognizes a cell surface antigen of a cancer cell; (ii) a cell membrane-spanning domain; and (iii) a signal transduction domain that induces T cell activation. The foreign gene can also be, for example, a gene for expressing an exogenous T cell receptor (TCR). The term "exogenous TCR" refers to a TCR that is exogenous to the cytotoxic T cells into which a nucleic acid encoding the exogenous TCR is introduced. The amino acid sequence of the exogenous TCR may be the same as or different from that of the endogenous TCR of the T cells. One or more types of foreign genes can be introduced (e.g., a CAR and an exogenous TCR).

[0053] The means for introducing a foreign gene into a cell is not particularly limited, and various known or common means can be used. Typically, the foreign gene is introduced into a cell using an expression vector and expressed. The expression vector may be linear or cyclic, and may be a non-viral vector such as a plasmid, or a transposon-based vector. The cells into which the foreign gene is introduced are not particularly limited, and may be at any stage of differentiation, such as iPS cells, cytotoxic T cells, hematopoietic progenitor cells, and hematopoietic endothelial cells.

[0054] The technique for introducing an expression vector into cells can be an appropriate one depending on the embodiment. For example, the expression vector can be introduced into cells by known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or using a commercially available kit (according to its instructions).

[0055] The expression vector can be introduced into cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a vector containing a nucleic acid for expressing the receptor and a packaging vector (plasmid) for each virus can be transfected into host cells using a corresponding commercially available kit to produce a recombinant virus, and then the resulting recombinant virus can be used to infect cells.

[0056] When there are multiple foreign genes, all of the foreign genes may be contained in a single expression vector, all of the foreign genes may be contained in separate expression vectors, or some of the multiple foreign genes may be contained in a single expression vector and the rest may be contained in separate expression vectors.When multiple foreign genes are contained in a single expression vector, the order in which the foreign genes are arranged from upstream to downstream is not particularly limited.

[0057] The foreign gene can be composed of a nucleic acid (polynucleotide) having a base sequence encoding the amino acid sequence of the desired receptor. Those skilled in the art can design and prepare an expression vector capable of expressing the desired receptor in cells. The nucleic acid contained in the expression vector can be prepared by chemical DNA synthesis or prepared as cDNA (cloning).

[0058] In addition to the nucleic acid encoding the desired receptor, the expression vector may contain sequences such as a promoter, terminator, enhancer, initiation codon, termination codon, polyadenylation signal, nuclear localization signal (NLS), and multiple cloning site (MCS), as necessary. The expression vector may further comprise a reporter gene (e.g., a gene encoding a fluorescent protein of each color), a drug selection gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene), a suicide gene (e.g., a diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inducible caspase 9 (inducible caspase 9)), or a gene encoding a cytochrome P450 (cyt-450). The nucleic acid sequence may include a nucleic acid (base sequence) encoding a "functional gene" such as a gene encoding a nucleotide sequence of ...

[0059] The production method of the present invention may further comprise the following step (2) and / or step (3): (2) a step of expanding and culturing the cytotoxic T cells activated in the activation step, followed by cryopreservation (referred to herein as the "cryopreservation step"); and (3) a step of thawing and culturing the activated cytotoxic T cells cryopreserved in the cryopreservation step (referred to herein as the "culture step").

[0060] The cryopreservation step is a step of cryopreserving the activated cytotoxic T cells obtained in the activation step after expansion culture. The culture period for expansion culture is not particularly limited, as long as it is a period sufficient to obtain the desired number of cells, and is, for example, 1 to 20 days, preferably 2 to 7 days.

[0061] The culture vessel, medium, added components, and other culture conditions used in the expansion culture in the cryopreservation step can be the same as those described for the activation step and can be appropriately selected from those exemplified. However, the culture vessel, medium, added components, and other conditions used in the expansion culture in the cryopreservation step do not necessarily have to be the same as those used in the activation step and can be changed as appropriate. It is not necessary to use a vessel with excellent gas exchange; a culture vessel that does not allow gas exchange or has low gas exchange efficiency may also be used. Furthermore, the substance used to activate unactivated cytotoxic T cells does not need to be included in the culture medium.

[0062] Known methods can be used to cryopreserve expanded cytotoxic T cells. Cytotoxic T cells can be stored at an appropriate optimal temperature, for example, about −10°C or below, about −30°C or below, or about −80°C or below, and can be cryopreserved in a liquid nitrogen tank, deep freezer, or the like. The temperature during cryopreservation may fluctuate somewhat during storage, and it is preferable to maintain the temperature within the aforementioned range from freezing until the cells are ready for use. The cryopreservation period for cytotoxic T cells is not particularly limited, and they can be stored for several months to several decades. Aqueous solutions such as buffer solutions, isotonic solutions, and media containing cryoprotectants, which are commonly used for cryopreservation of animal cells, can be used as storage solutions for cytotoxic T cells. Examples of such cryoprotectants include dimethyl sulfoxide, ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), and glycerin. The content of the cryoprotectant is not particularly limited and can be, for example, 2 to 10% by volume. Examples of containers for cryopreserving cytotoxic T cells include tubes, vials, well plates, and cell freezing containers.

[0063] The culture step is a step of thawing and culturing the activated cytotoxic T cells cryopreserved in the cryopreservation step. The culture period is not particularly limited as long as it is a period sufficient to obtain the desired number of cells, and is, for example, 1 to 20 days, preferably 2 to 10 days.

[0064] The method for thawing cryopreserved activated cytotoxic T cells is not particularly limited, and known methods can be used, such as rapid thawing in a warm bath at 37°C or gentle thawing at room temperature.

[0065] The culture vessel, medium, added components, other culture conditions, etc. used in the culture step can be the same as those described for the activation step and can be appropriately selected from those exemplified. However, the culture vessel, medium, added components, etc. used in the culture step do not necessarily have to be the same as those used in the activation step and can be changed as appropriate. It is not particularly necessary to use a vessel with excellent gas exchange; a culture vessel that does not allow gas exchange or has low gas exchange efficiency may also be used. Furthermore, the substance used to activate unactivated cytotoxic T cells does not need to be contained in the culture medium.

[0066] The activated cytotoxic T cells obtained in the culture step have higher cell proliferation (after cryopreservation) than unactivated cytotoxic T cells derived from iPS cells activated in a container that does not allow gas exchange or has low gas exchange efficiency (e.g., a T flask) (control group).Furthermore, the activated cytotoxic T cells obtained after the culture step have higher cancer cell cytotoxic activity (after cryopreservation) than unactivated cytotoxic T cells derived from iPS cells activated in a container that does not allow gas exchange or has low gas exchange efficiency (control group).

[0067] Furthermore, the method for expanding activated cytotoxic T cells derived from iPS cells of the present invention (sometimes referred to simply as the "expansion method of the present invention" in this specification) is characterized by comprising the following step (1A): (1A) activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange.

[0068] Step (1A) can be carried out in the same manner as step (1). The explanation regarding the production method of the present invention is applied to the proliferation method of the present invention.

[0069] The production method and expansion method of the present invention can be applied not only to iPS cell-derived cytotoxic T cells but also to iPS cell-derived NK (natural killer) cells. That is, a method for producing iPS cell-derived activated NK cells using iPS cell-derived unactivated NK cells, or a method for expanding iPS cell-derived activated NK cells, can be provided. Unactivated NK cells are differentiated from iPS cells, and differentiation into NK cells can be induced according to known methods. For example, iPS cells can be differentiated by CD34 ＋ After inducing differentiation into a cell population containing HPCs, CD4 － For example, differentiation of iPS cells into NK cells can be induced according to the method described in WO 2022 / 264033. NK cells include CD56 ＋ cells, and further CD56 ＋ / CD16 ＋ cells or CD56 ＋ / CD3 － Examples of substances used to activate unactivated NK cells include substances (ligands, antibodies, ligand-fused Fc proteins, etc.) that stimulate molecules having an immunoreceptor tyrosine-based activation motif (ITAM), such as IL-2, IL-7, IL-15, IL-21, IL-18, and NKp46, and substances (ligands, antibodies, ligand-fused Fc proteins, etc.) that stimulate molecules such as 4-1BB, OX40, CD28, ICOS, and CD30.

[0070] The use of the cell population containing cytotoxic T cells obtained by the production method and expansion method of the present invention is not particularly limited, and the cell population can be used for any desired purpose, for example, for producing a pharmaceutical (cell preparation). The percentage (cell number) of activated cytotoxic T cells contained in the cell population containing activated cytotoxic T cells obtained by the production method and expansion method of the present invention is, for example, 5% or more, preferably 15% or more, more preferably 25% or more, even more preferably 35% or more, and still more preferably 45% or more, and the upper limit is, for example, 100% or less.

[0071] The pharmaceutical (cell preparation) contains cytotoxic T cells and may further contain other components as necessary. Those skilled in the art can appropriately prepare such pharmaceuticals using cytotoxic T cells, taking into consideration the intended use (disease to be treated, recipient, etc.) and dosage form.

[0072] The pharmaceutical can be, for example, a pharmaceutical (anticancer drug) that treats and prevents cancers that correspond to a cell surface antigen (cancer-specific antigen) of cancer cells that is targeted by a CAR expressed by cytotoxic T cells. Therefore, the type of cancer that the pharmaceutical targets is not particularly limited, as long as cancer cells expressing the antigen targeted by the CAR are contained in the cancer tissue and a certain level of therapeutic and preventive effect is observed by the CAR-T cells. Examples of cancers that the pharmaceutical can target include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated cancer, large cell carcinoma, small cell carcinoma, skin cancer (e.g., melanoma, Merkel cell carcinoma), breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, non-small cell lung cancer, tracheal cancer, bronchial cancer, colon cancer, rectal cancer, small intestine cancer, colon cancer, stomach cancer, and gallbladder cancer. Examples of cancers include cancers of the esophagus, gallbladder, testicular, ovarian, fallopian tube, and nasopharyngeal cancer; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, rhabdomyosarcoma, malignant hemangioendothelioma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumors; lymphoma; leukemia, acute myeloid leukemia, and multiple myeloma.

[0073] Components other than cytotoxic T cells that can be contained in the pharmaceutical include, for example, pharmaceutically acceptable additives, more specifically, saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonicity agents, fillers, lubricants, etc.

[0074] The pharmaceutical can be administered to subjects (cancer patients, cancer-bearing animals, etc.) in need of cancer treatment and prevention in the same manner as known cytotoxic T cells (e.g., CAR-T cells). Examples of the administration method include intratumoral, intravenous, intraarterial, intramuscular, subcutaneous, and intraperitoneal injections.

[0075] The amount of cytotoxic T cells contained in the pharmaceutical can be appropriately adjusted depending on the application, dosage form, and intended therapeutic and preventive effects, taking into consideration, for example, the type, location, and severity of cancer, as well as the age, weight, and condition of the subject to be treated. For example, the pharmaceutical typically contains 1 x 10 CAR-T cells per administration. ４ ~1 x 10 １０ pieces, preferably 1 x 10 ５ ~5 x 10 ９ pieces, more preferably 5×10 ６ ~2 x 10 ９ It can be formulated for individual administration.

[0076] The administration interval of the pharmaceutical agent is not particularly limited and can be adjusted as appropriate taking into consideration the amount of the T cells of the present invention administered per administration, etc., but can be, for example, independently administered four times, three times, twice or once a day, every other day, every two days, every three days, every four days, every five days, once a week, every seven days, every eight days, every 9 days, twice a week, once a month, or twice a month.

[0077] When the pharmaceutical is for cancer treatment and prevention, it can be used in combination with known anticancer drugs, such as alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecular targeted drugs such as rituximab, cetuximab, and trastuzumab; kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; Examples of suitable anticancer drugs include proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; hormonal therapy drugs such as tamoxifen and bicaludamide; and immunosuppressants such as interferon, nivolumab, and pembrolizumab. When the target recognition site of the CAR is an antibody that recognizes the constant region of an antibody that specifically binds to a cell surface antigen of a cancer cell, the cytotoxic T cells can be used as a pharmaceutical for treating and preventing cancers corresponding to the cell surface antigen. In this case, the pharmaceutical containing the cytotoxic T cells can be used in combination with a pharmaceutical containing an antibody that specifically binds to the cell surface antigen.

[0078] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

[0079] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature, etc. Furthermore, any material having the same effect or action can be substituted.

[0080] (List of Abbreviations) BMP-4: bone morphogenetic protein-4 bFGF: basic fibroblast growth factor VEGF: vascular endothelial growth factor SCF: stem cell factor TPO: thrombopoietin FLT3L: Fms-related tyrosine kinase 3 ligand Fc-DLL4: Recombinant Human DLL4 Fc Chimera Protein DLL4: Delta-like protein 4 IL7: Interleukin-7 SDF1α: Stromal cell-derived factor 1α αMEM: alpha Modified Eagle Minimum Essential Medium TCR: T-cell receptor

[0081] Example 1: Examination of the Effect of Containers Used in the Activation Process on the Proliferation Efficiency of iPS Cell-Derived Activated CAR-T Cells After Freezing and Thawing (1) Preparation of iPS Cell-Derived CAR-T Cells The TCR gene was introduced into the QHJI01S04 iPS cell line provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, using a lentiviral vector. The TCR-transduced iPS cells were differentiated into hematopoietic progenitor cells according to the method described in Nature Communication 2021;12:430. Specifically, the iPS cells were cultured for 4 days in the presence of CHIR99021, SB431542, BMP-4, bFGF, and VEGF to induce differentiation into mesoderm. Furthermore, the iPS cells were differentiated into hematopoietic progenitor cells using the hematopoietic cytokines SCF, TPO, and FLT3L. The differentiation of the obtained hematopoietic progenitor cells into cytotoxic T cells (CTL; ​​Cytotoxic T Lymphocyte) was performed according to WO 2017 / 221975 and Nature Communication 2021;12:430. Specifically, among the obtained hematopoietic progenitor cells, CD34-positive cells purified using magnetic beads (Myltenyi Biotec) were cultured for 3 weeks on a plate immobilized with Fc-DLL4 and RetroNectin (Recombinant Human Fibronectin Fragment, Takara Bio Inc.) in α-MEM medium containing SCF, TPO, FLT3L, IL7, SDF1α, and SB203580. A CAR gene that recognizes mesothelin (using the base sequence described in WO 2023 / 009700) was introduced into the resulting TCR-positive CTLs using a retroviral vector. These cells were cryopreserved in liquid nitrogen and used below as iPS cell-derived CAR-T cells.

[0082] (2) Recovery culture of iPS cell-derived CAR-T cells iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% fetal bovine serum (FBS) and the additives listed in the "Recovery culture medium" column in Table 1, and seeded onto G-Rex 10M or G-Rex 6M (Wilson Wolf). The cells were incubated at 5°C for 1 hour at 5% CO ２The cells were cultured at 37°C for 3 days.

[0083]

[0084] (3) Reagents and Antibodies Anti-CD3 agonist antibodies used were anti-CD3 mAb GMP grade, anti-CD3 monoclonal antibody (Clone: ​​OKT3) purchased from Takara Bio Inc.

[0085] (4) Immobilization of anti-CD3 agonist antibody on culture plate An anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) dissolved in PBS at the required concentration was added to a T25 flask and a PermaLife Cell Culture Bag PL120 (OriGen Biomedical), and then left to stand overnight at 4° C. After washing with PBS, the plate was subjected to the test.

[0086] (5) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded in a medium prepared by adding the additives shown in the "Activation culture medium" column in Table 1 to IMDM medium containing 15% FBS into T25 flasks and PL120 flasks onto which an anti-CD3 agonist antibody (OKT3) had been immobilized, using the medium volume and cell volume shown in Table 2. After seeding, the cells were incubated at 5% CO ２ The iPS cell-derived CAR-T cells were cultured at 37°C for 3 days. The standard iPS cell-derived CAR-T cells used in the post-freeze-thaw proliferation test were activated and cultured in a T225 flask, and the other culture steps were performed in a similar manner. The same applies to the cells referred to as standard iPS cell-derived CAR-T cells in the following examples.

[0087]

[0088] (6) Expansion culture of iPS cell-derived activated CAR-T cells After activation culture, the iPS cell-derived activated CAR-T cells were suspended in IMDM medium containing 15% FBS to which the additives shown in the “Expansion culture medium” column in Table 1 were added at a concentration of 40,000 cells / mL. The cells were then cultured at 40,000 cells / mL using G-Rex 6M (Wilson Wolf) at 5% CO ２The cells were cultured at 37°C for 3 days. On the second day of culture, some of the cells were recovered from G-Rex 6M, the cell number was counted, and the medium was replaced with expansion culture medium. On the third day of culture, some of the cells were recovered from G-Rex (trade name) 6M, the cell number was counted, and then all the cells were collected, centrifuged, and cryopreserved in a cryopreservation solution containing 5% DMSO.

[0089] The cell number and viability of iPS cell-derived activated CAR-T cells were measured on days 0, 2, and 3 of the expansion culture described above in (6). The cell proliferation fold and viability after 3 days of activation culture and 3 days of expansion culture are shown in Figure 1. This study revealed that iPS cell-derived activated CAR-T cells cultured with PL120 proliferated during expansion culture.

[0090] (7) Proliferation Test of iPS Cell-Derived Activated CAR-T Cells After Freezing and Thawing Cryopreserved iPS cell-derived activated CAR-T cells were thawed and suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) at a concentration of 125,000 cells / mL. The cells were then cultured in a G-Rex 24-well plate (Wilson Wolf) at 5% CO ２ The cells were cultured at 37°C for 1 hour. After that, on days 1 and 3 of culture, some of the cells were collected from the G-Rex 24-well plate and the cell number was counted. On day 3 of culture, the cells were again suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) to a concentration of 125,000 cells / mL, and the cells were cultured in a G-Rex 24-well plate at 37°C for 1 hour at 5% CO. ２ The cells were cultured at 37°C. On day 7 of culture, some of the cells were recovered from the G-Rex 24-well plate and counted, and the proliferation fold from day 0 to day 7 of culture was calculated. Standard iPS cell-derived CAR-T cells were also tested at the same time, and the proliferation fold from day 0 to day 7 of culture was set at 100%, and the relative value was calculated for each condition and compared.

[0091] The results of the proliferation test from day 0 to day 7 of culture after freeze-thawing are shown in Figure 2. It was found that iPS cell-derived activated CAR-T cells activated and cultured with PL120 had excellent proliferation ability after freeze-thawing.

[0092] Example 2: Investigation of the effect of cytokine concentration in the activation step on the proliferation efficiency of activated iPS cell-derived CAR-T cells after freeze-thawing (1) Recovery culture of iPS cell-derived CAR-T cells Recovery culture of iPS cell-derived CAR-T cells was carried out by the method described in Example 1 (2). The medium used for recovery culture was an IMDM medium containing 15% fetal bovine serum (FBS) to which the additives shown in the "Recovery culture medium" column in Table 3 were added.

[0093]

[0094] (2) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded in IMDM medium containing 15% FBS to which the additives shown in the "Activation culture medium," "Activation culture medium (2x cytokines)," and "Activation culture medium (3x cytokines)" columns in Table 3 were added, in the medium volumes and cell volumes shown in Tables 4 and 5, into T25 flasks and PL120 flasks onto which an anti-CD3 agonist antibody (OKT3) had been immobilized. After seeding, the cells were incubated at 5% CO ２ The cells were cultured for 3 days at 37° C. The T25 flasks and PL120 flasks to which the anti-CD3 agonist antibody (OKT3) was immobilized were prepared by the methods described in Example 1 (3) and (4).

[0095]

[0096] (3) Expansion of iPS cell-derived activated CAR-T cells The iPS cell-derived activated CAR-T cells were expanded by the method described in Example 1 (6), and the cells were collected on day 3 of culture and cryopreserved.

[0097] (4) Test for proliferation of iPS cell-derived activated CAR-T cells after freezing and thawing The proliferation of iPS cell-derived activated CAR-T cells after freezing and thawing was measured by the method described in Example 1(7).

[0098] The results of the proliferation test from day 0 to day 7 of culture after freeze-thawing are shown in Figures 3 and 4. It was found that the proliferation ability of iPS cell-derived activated CAR-T cells cultured in PL120 was enhanced by increasing the cytokine concentration in the activation culture (Figure 3). On the other hand, the proliferation of iPS cell-derived activated CAR-T cells cultured in a T25 flask was not enhanced after freeze-thawing, even when the cytokine concentration in the activation culture was increased (Figure 4).

[0099] Example 3: Investigation of the influence of the container used in the activation step on the antitumor effect of iPS cell-derived activated CAR-T cells after freeze-thawing (1) Recovery culture of iPS cell-derived CAR-T cells Recovery culture of iPS cell-derived CAR-T cells was carried out using G-Rex 100M (Wilson Wolf) according to the method described in Example 1 (2). The medium used for recovery culture was IMDM medium containing 15% fetal bovine serum (FBS) to which the additives shown in the "Recovery culture medium" column in Table 6 were added.

[0100]

[0101] (2) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded in IMDM medium containing 15% FBS to which the additives shown in the "Activation culture medium" and "Activation culture medium (3x cytokines)" columns in Table 6 were added, at the cell numbers and medium volumes shown in Table 7, into T225 flasks and PL120 flasks onto which an anti-CD3 agonist antibody (OKT3) had been immobilized. After seeding, the cells were cultured under 5% CO ２ The cells were cultured at 37°C for 3 days.

[0102] The T225 flasks and PL120 cells to which the anti-CD3 agonist antibody (OKT3) was immobilized were prepared by the methods described in Example 1 (3) and (4).

[0103]

[0104] (3) Expansion culture of iPS cell-derived activated CAR-T cells After activation culture, the iPS cell-derived activated CAR-T cells were suspended in IMDM medium containing 15% FBS to which the additives shown in the “Expansion culture medium” column in Table 6 were added at a concentration of 40,000 cells / mL. The cells were then cultured at 40,000 cells / mL using G-Rex 100M (Wilson Wolf) at 5% CO ２ The cells were cultured at 37°C for 3 days. On day 2 of culture, the medium was replaced with expansion culture medium. On day 3 of culture, the cells were collected from G-Rex 100M, centrifuged, and then cryopreserved in a cryopreservation solution containing 5% DMSO.

[0105] (4) Proliferation Test of iPS Cell-Derived Activated CAR-T Cells After Freezing and Thawing Cryopreserved iPS cell-derived activated CAR-T cells were thawed and suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) at a concentration of 125,000 cells / mL. The cells were then cultured in a G-Rex 24-well plate (Wilson Wolf) at 5% CO ２ The cells were cultured at 37°C for 1 hour. After that, on days 1 and 3 of culture, some of the cells were collected from the G-Rex 24-well plate and the cell number was counted. On day 3 of culture, the cells were again suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) to a concentration of 125,000 cells / mL, and the cells were cultured in a G-Rex 24-well plate at 37°C for 1 hour at 5% CO. ２ The cells were cultured at 37° C. On day 7 of the culture, some of the cells were collected from the G-Rex 24-well plate and the number of cells was counted.

[0106] The proliferation fold increase on day 7 of culture after freeze-thawing is shown in Figure 5. It was found that iPS cell-derived activated CAR-T cells activated and cultured in PL120 had superior proliferation ability after freeze-thawing compared to iPS cell-derived activated CAR-T cells activated and cultured in a T225 flask.

[0107] (5) In vivo antitumor effect of iPS cell-derived activated CAR-T cells after freeze-thawing in a human tumor-bearing mouse model. NSG mice were subcutaneously implanted with GSU cells, a human gastric cancer-derived cell line, at a cell count of 2,000,000 cells / mouse. Seven days later, iPS cell-derived activated CAR-T cells that had been cryopreserved after expansion culture were intravenously administered at a cell count of 1,000,000 cells / mouse. Tumor size was measured every week using a 3D scanner until week 6 after administration. The test was performed with N=5, and the mean and standard deviation of tumor size were calculated. A two-way analysis of variance was used to test for significant differences between groups.

[0108] The in vivo antitumor effect of iPS cell-derived activated CAR-T cells is shown in Figure 6. The iPS cell-derived activated CAR-T cells activated and cultured in PL120 had a significantly improved antitumor effect compared to iPS cell-derived activated CAR-T cells activated and cultured in a T225 flask. Furthermore, the antitumor effect was further improved by increasing the cytokine concentration during activation culture.

[0109] Example 4: Investigation of the effect of freeze-thawing of CAR-T cells transfected with a CAR gene that recognizes a different antigen, CD19, on the proliferation efficiency of activated iPS cell-derived CAR-T cells (1) Recovery culture of iPS cell-derived CAR-T cells iPS cell-derived CAR-T cells transfected with a CAR gene that recognizes CD19 were prepared using a method similar to that described in Example 1(1), and the cryopreserved cells were used as iPS cell-derived CAR-T cells below. The iPS cell-derived CAR-T cells were seeded at 666,666 cells / mL into a T225 flask (Thermo Fisher Scientific) in IMDM medium containing 15% fetal bovine serum (FBS) and the additives listed in the "Recovery culture medium" column of Table 1, and the cells were incubated at 5% CO ２Similarly, iPS cell-derived CAR-T cells were seeded onto G-Rex 6M (Wilson Wolf) at 500,000 cells / mL in IMDM medium containing 15% fetal bovine serum (FBS) and the additives listed in the "Recovery culture medium" column in Table 1, excluding streptomycin sulfate, and incubated at 37°C for 4 days. ２ The cells were cultured at 37°C for 4 days.

[0110] (2) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded into T225 flasks and PL120 flasks onto which an anti-CD3 agonist antibody (OKT3) had been immobilized, at the cell numbers and medium volumes shown in Table 8. For the T225 flasks, a medium containing 15% FBS and IMDM medium supplemented with the additives shown in the "Activation culture medium" column of Table 1 was used, while for the PL120 flasks, a medium containing 15% FBS and IMDM medium supplemented with the additives shown in the "Activation culture medium" column of Table 1, except for streptomycin sulfate, was used. After seeding, the cells were incubated at 5% CO ２ The cells were cultured for 3 days at 37° C. The T225 flask and PL120 flasks to which the anti-CD3 agonist antibody (OKT3) was immobilized were prepared by the methods described in Example 1 (3) and (4).

[0111]

[0112] (3) Expansion of iPS cell-derived activated CAR-T cells Expansion of iPS cell-derived activated CAR-T cells was performed by the method described in Example 1 (6), and the cells were collected and cryopreserved on day 3 of culture. Note that the cells activated with PL120 were expanded using a medium containing 15% FBS and IMDM medium to which the additives listed in the "Expansion medium" column of Table 1, excluding streptomycin sulfate, were added.

[0113] (4) Proliferation Test of iPS Cell-Derived Activated CAR-T Cells After Freezing and Thawing Cryopreserved iPS cell-derived activated CAR-T cells were thawed and suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) at a concentration of 125,000 cells / mL. The cells were then cultured in a G-Rex 24-well plate (Wilson Wolf) at 5% CO ２ On the third day of culture, some of the cells were collected from the G-Rex 24-well plate and the cell number was counted. On the third day of culture, the cells were again suspended in IMDM medium (containing 15% FBS, 2 mM L-Glutamine, 100 μg / mL Streptomycin, 1x ITS, and 50 μg / mL Ascorbic Acid 2-phosphate) at a concentration of 125,000 cells / mL, and cultured in a G-Rex 24-well plate at 5% CO ２ The cells were cultured at 37°C. On day 6 of culture, some of the cells were recovered from the G-Rex 24-well plate and counted, and the proliferation fold from day 0 to day 6 of culture was calculated. The proliferation fold of the cells activated in the T225 flask from day 0 to day 6 of culture was set at 100%, and the relative values ​​were calculated and compared.

[0114] The results of the proliferation test after freeze-thawing from day 0 to day 6 of culture are shown in Figure 7. It was found that the proliferation ability of iPS cell-derived activated CAR-T cells that recognize different antigens was also enhanced after freeze-thawing when activated with PL120 (Figure 7).

[0115] Example 5: Investigation of the effect of the container used in the activation step on the proliferation efficiency of iPS cell-derived activated CAR-T cells after freeze-thawing (1) Recovery culture of iPS cell-derived CAR-T cells Recovery culture of iPS cell-derived CAR-T cells was carried out by the method described in Example 1 (2). The medium used for recovery culture was an IMDM medium containing 15% fetal bovine serum (FBS) to which the additives shown in the "Recovery culture medium" column in Table 3 were added.

[0116] (2) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded in a medium prepared by adding the additives shown in the "Activation culture medium" column in Table 1 to IMDM medium containing 15% FBS into T25 flasks, PL120 flasks, and G-Rex6M flasks onto which an anti-CD3 agonist antibody (OKT3) had been immobilized, at the cell numbers and medium volumes shown in Table 9. After seeding, the cells were incubated at 5% CO ２ The cells were cultured for 3 days at 37°C. The T25 flasks, PL120, and G-Rex6M flasks on which the anti-CD3 agonist antibody (OKT3) was immobilized were prepared in the same manner as described in Example 1 (3) and (4).

[0117]

[0118] (3) Expansion of iPS cell-derived activated CAR-T cells The iPS cell-derived activated CAR-T cells were expanded by the method described in Example 1 (6), and the cells were collected on day 3 of culture and cryopreserved.

[0119] (4) Test of proliferation of iPS cell-derived activated CAR-T cells after freezing and thawing The proliferation of iPS cell-derived activated CAR-T cells after freezing and thawing was evaluated by the method described in Example 1 (7). In this example, cells were cultured in a T25 flask (80,000 cells / cm). ２ , 0.6 mL / cm ２ The proliferation rate of cells activated with ) from day 0 to day 7 of culture was set at 100%, and the relative values ​​were calculated and compared.

[0120] The results of the proliferation test after freeze-thawing from day 0 to day 7 of culture are shown in Figure 8. Not only PL120 but also activation with G-Rex6M enhanced the proliferation ability after freeze-thawing (Figure 8).

[0121] Example 6: Investigation of the effect of scaling up the vessel used in the activation step on the proliferation efficiency of iPS cell-derived activated CAR-T cells after freeze-thawing (1) Recovery culture of iPS cell-derived CAR-T cells Recovery culture of iPS cell-derived CAR-T cells was performed by the method described in Example 1 (2), except for the use of G-Rex 100M as the culture vessel, the recovery culture medium, and the recovery culture period. The medium used for recovery culture was IMDM medium containing 15% fetal bovine serum (FBS) to which the additives shown in the "Recovery culture medium 1," "Recovery culture medium 2," and "Recovery culture medium 3" columns in Table 10 were added. The recovery culture period was under the conditions shown in Table 11.

[0122]

[0123] (2) Activation culture of iPS cell-derived CAR-T cells After recovery culture, the iPS cell-derived CAR-T cells were seeded onto PL2000 and PL1000 plates with immobilized anti-CD3 agonist antibody (OKT3) in the amounts of medium and cells shown in Table 12, using IMDM medium containing 15% FBS to which the additives shown in the "Activation culture medium 1," "Activation culture medium 2," and "Activation culture medium 3" columns in Table 10 were added. After seeding, the plates were incubated at 5% CO ２ The cells were cultured for 3 days at 37° C. PL2000 and PL1000, on which the anti-CD3 agonist antibody (OKT3) was immobilized, were prepared by the same method as described in Example 1 (3) and (4).

[0124]

[0125] (3) Expansion culture of iPS cell-derived activated CAR-T cells After activation culture, the iPS cell-derived activated CAR-T cells were suspended in a 3 L bioreactor to a concentration of 1,800,000 cells / mL, and incubated under 5% CO ２The cells were cultured at 37°C for 3 days. During the culture, the medium was replaced as appropriate using "Expansion Culture Medium 1," "Expansion Culture Medium 2," and "Expansion Culture Medium 3" in Table 13, as shown in Table 14, and the culture was continued for 3 days while maintaining dissolved oxygen and pH. After the culture, the cells were collected, centrifuged, and then cryopreserved in a cryopreservation solution containing 5% DMSO. The medium composition of BM301 in Table 13 is shown in Table 15.

[0126]

[0127] The proliferation rate of iPS cell-derived activated CAR-T cells over 3 days of expansion culture is shown in Figure 9. It was confirmed that iPS cell-derived activated CAR-T cells activated using the scale-up PL2000 and PL1000 showed a high proliferation rate even when seeded into a 3 L bioreactor and expanded (Figure 9).

[0128] This application is based on Japanese Patent Application No. 2023-185541 filed on October 30, 2023, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing activated cytotoxic T cells derived from iPS cells, comprising the following step (1): (1) activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange.

2. The method of claim 1, wherein said unactivated cytotoxic T cells contain an exogenous gene encoding a receptor.

3. The method of claim 2, wherein said receptor is a chimeric antigen receptor.

4. The method of claim 1, wherein the container is a bag.

5. A method for expanding activated cytotoxic T cells derived from iPS cells, comprising the following step (1A): (1A) activating unactivated cytotoxic T cells derived from iPS cells in a container with excellent gas exchange.

6. The method of claim 5, wherein said unactivated cytotoxic T cells contain an exogenous gene encoding a receptor.

7. The method of claim 6, wherein said receptor is a chimeric antigen receptor.

8. The method of claim 5, wherein the container is a bag.

9. A cell population containing activated cytotoxic T cells obtained by the method according to claim 1 or 5.

10. A medicine comprising a cell population containing the activated cytotoxic T cells according to claim 9.

11. The pharmaceutical composition according to claim 10, which is a preventive or therapeutic drug for cancer.

12. A method for preventing or treating cancer, comprising administering a cell population containing the activated cytotoxic T cells according to claim 9 to a subject in need thereof.

13. A cell population comprising the activated cytotoxic T cells according to claim 9 for use in the prevention or treatment of cancer.

14. Use of a cell population comprising the activated cytotoxic T cells according to claim 9 in the manufacture of a medicament for preventing or treating cancer.

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