Method for producing improved leukocytes

Abstract

Provided is a method for efficiently producing heterologous T cell receptor or chimeric antigen receptor gene-introduced leukocytes.　The present invention relates to: a method for producing modified leukocytes, such as T cells to which heterologous a T-cell receptor or a chimeric antigen receptor gene has been introduced, by adding a ferroptosis inhibitor during the production step; and modified leukocytes produced by the method according to the present invention.

Description

Improved method for producing white blood cells

[0001] This patent application claims priority and the benefit of Japanese Patent Application No. 2023-188044 (filed November 1, 2023) and Japanese Patent Application No. 2024-088216 (filed May 30, 2024) under the Paris Convention, as well as the priority and benefit provided for in Article 41 of the Japanese Patent Act, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for reducing lipid peroxides produced during the production of chimeric antigen receptor gene-transduced T cells. More specifically, the present invention uses a ferroptosis inhibitor to reduce lipid peroxides and improve the antitumor effect of the produced chimeric antigen receptor gene-transduced T cells.

[0003] Immune functions can be broadly divided into functions due to "antibodies" secreted by plasma cells that are generated when B cells differentiate, and functions due to T cells and natural killer cells. T cells are a type of white blood cell, also known as T lymphocytes, and are classified as CD4 (cluster of differentiation 4) positive T cells (hereinafter referred to as "CD4 ＋ T cells) and CD8 (cluster of differentiation 8)-positive T cells (hereinafter referred to as "CD8 ＋ T cells). ＋ When T cells recognize antigens from pathogens or tumors, they activate effector CD4 ＋ Effector CD4 cells are activated by the immune system and produce cytokines. ＋ Most of the cells die, but some remain as memory CD4 cells in preparation for the next infection. ＋ On the other hand, CD8 ＋ T cells are effector CD4 ＋ Cytotoxic killer CD8 is activated by cytokines produced by the cells. ＋ They become T cells and destroy and kill pathogens and tumor cells.

[0004] However, there are refractory malignant tumors that are difficult to treat with normal immune function alone. Examples include solid cancers such as pancreatic cancer, biliary tract cancer, and ovarian cancer, as well as blood cancers such as leukemia. In 2019, chimeric antigen receptor gene-transduced T cell (CAR-T cell) therapy was approved in the United States as a cutting-edge treatment for blood cancers such as leukemia, malignant lymphoma, and multiple myeloma. It is now rapidly expanding as a treatment that aims to cure not only blood cancers but also refractory malignant tumors that are difficult to treat with normal immune function alone. A chimeric antigen receptor (CAR) is a receptor that combines an antibody-derived moiety that specifically recognizes the antigen of a target malignant tumor cell with an intracellular domain that transmits an activation signal derived from a T cell receptor. By genetically transferring this CAR into T cells collected from a patient, T cells (i.e., CAR-T cells) that can specifically recognize the antigen of the target malignant tumor cell are produced.

[0005] However, the activity and persistence of CAR-T cells after their introduction depend on the condition of patient-derived T cells. In particular, it has been suggested that a decline in mitochondrial function of T cells impairs the effectiveness of CAR-T cell therapy (Non-Patent Document 1). It has also been shown that CAR-T cells with stronger stem cell (or memory stem cell) properties can be maintained in vivo for a long period of time and exert antitumor effects, and various methods have been attempted to produce CAR-T cells with these properties (Non-Patent Document 2).

[0006] van Bruggen JAC, et al. Blood, 2019 Jul 4;134(1), pp.44-58Zhang X: Cancer Cell 2022, Volume 40, Issue 11, pp.1407-1422Dixon, SJ., et al., Cell, 2012, 149(5), pp.1060-1072

[0007] It is known that cell death occurs due to the accumulation of reactive oxygen species (ROS) and the progression of lipid peroxidation in cells. ２＋Iron-dependent programmed cell death (ferroptosis) has been reported, a phenomenon in which lipid peroxidation is strongly promoted by iron-dependent ferroxenic acid (RIF) and the resulting oxidative stress from ROS damages cellular lipids (Non-Patent Document 3). Suppression of lipid peroxidation via the glutathione-dependent hydroperoxidase GPX4 has been proposed to inhibit ferroptosis (Non-Patent Document 4). Figure 1 shows an overview of the mechanism of ferroptosis and its inhibition by GPX4. Ferrostatin-1 (hereinafter abbreviated as "Fer-1"), a free radical scavenger, is also known as a ferroptosis inhibitor (Non-Patent Document 5). Furthermore, the introduction of iron chelators such as deferoxamine (DFO) is also useful for removing intracellular iron ions that cause ferroptosis (Non-Patent Document 3).

[0008] Based on the above findings regarding ferroptosis, the present inventors hypothesized that inhibiting ferroptosis during the production of CAR-T cells would prevent the accumulation of ROS and the progression of lipid peroxidation. However, we were unable to find any prior art literature relating to the effect of ferroptosis inhibition on the antitumor activity of T cells.

[0009] The present inventors have succeeded in preventing ROS accumulation and the progression of lipid peroxidation in the resulting CAR-T cells by introducing a substance effective in inhibiting ferroptosis during the production of CAR-T cells. That is, the present invention includes the following aspects. <Method for producing modified leukocytes> [1] A method for producing modified leukocytes in the presence of a ferroptosis inhibitor. [2] The method according to [1], wherein the method for producing modified leukocytes comprises the step of (b) modifying the leukocytes to express at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR) by gene transfer into the leukocytes using a nucleic acid or vector encoding at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR). [3] The method of [2], further comprising at least one of the following steps: (a) activating leukocytes; (c) expanding and / or proliferating the activated or modified leukocytes; (d) culturing the activated or modified leukocytes or a population thereof; and (e) recovering and / or cryopreserving the activated or modified leukocytes or a population thereof. [4] The method of [3], wherein at least one of the steps involves producing modified leukocytes in the presence of a ferroptosis inhibitor. [5] The method of [4], wherein a ferroptosis inhibitor is present in the (a) activating leukocytes step and / or the (b) modifying leukocytes step. [6] The method of any one of [1] to [5], wherein the ferroptosis inhibitor is a lipid peroxidation inhibitor and / or an iron chelator. [7] The method according to any one of [1] to [5], wherein the ferroptosis inhibitor is ferrostatin-1 and / or deferoxamine. [8] The method according to any one of [1] to [7], wherein the leukocyte is a T cell or a NK cell. [9] The method according to any one of [1] to [7], wherein the leukocyte is a T cell.

[10] The method according to any one of [1] to [7], wherein the leukocyte is a monocyte or a granulocyte. <Modified leukocytes>

[11] Modified leukocytes produced by the method according to any one of [1] to [9].<Kit>

[12] A kit for producing modified leukocytes according to

[11] , comprising at least a ferroptosis inhibitor.

[0010] According to the present invention, when a ferroptosis inhibitor effective in inhibiting ferroptosis was added during the production of modified leukocytes such as CAR-T cells, lipid peroxidation in the produced leukocytes was suppressed, resulting in improved in vitro antitumor activity. Furthermore, when CAR-T cells produced under conditions that inhibit ferroptosis were administered to a mouse lymphocytic leukemia model, they were maintained in vivo for a longer period of time and were able to more strongly suppress tumor growth.

[0011] FIG. 1 is a schematic diagram showing the mechanism of ferroptosis and the inhibition of ferroptosis by GPX4. FIG. 2 is a scheme for producing mouse CAR-T cells using a conventional production method. FIG. 3 shows the results of flow cytometry analysis to verify the gene transfer efficiency of mouse CAR-T cells using a conventional production method. FIG. 4 is a graph evaluating (a) the amount of ROS accumulation and (b) lipid peroxidation of mouse CAR-T cells produced using a conventional production method. FIG. 5 is a scheme for producing mouse CAR-T cells treated using the production method of the present invention. FIG. 6 shows the results of flow cytometry analysis to verify the gene transfer efficiency of Fer-1-treated mouse CAR-T cells produced using the production method of the present invention. FIG. 7 is a graph evaluating (a) the amount of ROS accumulation and (b) lipid peroxidation of mouse CAR-T cells produced using a control method with DMSO treatment and the production method of the present invention with Fer-1 treatment. 8 shows the results of flow cytometry analysis of TCF1 / Tim3-stained mouse CAR-T cells produced by a control method using DMSO treatment and by the production method of the present invention using Fer-1 treatment. ＋ Time 3 － CD8 ＋ 9 shows the results of flow cytometry analysis of IFNγ / TNFα-stained mouse CAR-T cells produced by a control method using DMSO treatment and by the production method of the present invention using Fer-1 treatment. The graph on the right shows the percentage of the T cell population. ＋ TNF-α＋ CD8 ＋ 10 shows the results of flow cytometry analysis of CD8 / Granzyme B stained mouse CAR-T cells produced by the control method (DMSO treatment) and the production method of the present invention (Fer-1 treatment). ＋ Granzyme B ＋ FIG. 11 is a diagram showing the coculture of ferroptosis-inhibited mouse CAR-T cells with A20. FIG. 12 is a graph showing the evaluation of (a) cell proliferation activity and (b) in vitro cytotoxic activity of mouse CAR-T cells treated with ferroptosis inhibition using Fer-1. In (a), the vertical axis "CTVMFI" represents the mean fluorescence intensity of CellTrace (registered trademark) Violet fluorescent staining (CTV). FIG. 13 is a diagram showing the inoculation of ferroptosis-inhibited mouse CAR-T cells into a leukemia model mouse. FIG. 14 is a graph showing the evaluation of (a) the in vivo antitumor effect and (b) the in vivo viability of mouse CAR-T cells treated with ferroptosis inhibition using Fer-1. FIG. 15 shows the results of flow cytometry analysis verifying the gene transfer efficiency of DFO-treated mouse CAR-T cells produced by the production method of the present invention. Figure 16 is a graph evaluating (a) the amount of ROS accumulation and (b) lipid peroxidation in mouse CAR-T cells produced by a control method using DMSO treatment and by the production method of the present invention using DFO treatment. Figure 17 shows the results of flow cytometry analysis of TCF1 / Tim3-stained mouse CAR-T cells produced by a control method using DMSO treatment and by the production method of the present invention using DFO treatment. The graph on the right shows Tcf1 ＋ Time 3 － CD8 ＋ 18 shows the results of flow cytometry analysis of IFNγ / TNFα-stained mouse CAR-T cells produced by a control method using DMSO treatment and a production method of the present invention using DFO treatment. The graph on the right shows the percentage of the T cell population. ＋ TNF-α ＋ CD8 ＋18 shows the results of flow cytometry analysis of CD8 / Granzyme B stained mouse CAR-T cells produced by the control method (DMSO treatment) and the production method of the present invention (DFO treatment). ＋ Granzyme B ＋ Figure 20 shows the in vitro evaluation of the cell killing function of mouse CAR-T cells treated with DFO to inhibit ferroptosis. Figure 21 shows the in vivo evaluation of the antitumor effect of mouse CAR-T cells treated with DFO to inhibit ferroptosis. Figure 22 shows a scheme for producing human CAR-T cells treated by the production method of the present invention. Figure 23 shows the results of flow cytometry analysis verifying the gene transfer efficiency of Fer-1-treated human CAR-T cells produced by the production method of the present invention. Figure 24 shows a graph evaluating lipid peroxidation in human CAR-T cells produced by a control method using DMSO treatment and a production method of the present invention using Fer-1 treatment. Figure 25 shows the results of flow cytometry analysis of CD8 / granzyme B-stained human CAR-T cells produced by a control method using DMSO treatment and a production method of the present invention using Fer-1 treatment. The graph on the right shows the results of flow cytometry analysis of CD8 ＋ Granzyme B ＋ 26 shows the results of flow cytometry analysis of TCF1 / Tim3 stained human CAR-T cells produced by a control method using DMSO treatment and by the production method of the present invention using Fer-1 treatment. The graph on the right shows the percentage of TCF1 / Tim3 stained human CAR-T cells. ＋ Time 3 － CD8 ＋ Figure 27 is a graph showing the percentage of the T cell population. Figure 27 is a scheme of inoculation of ferroptosis-inhibited CAR-T cells into leukemia model mice. Figure 28 is an evaluation of the in vivo viability of human CAR-T cells treated with ferroptosis inhibition using Fer-1.

[0012] <Method for producing modified leukocytes> In one aspect, the present invention relates to a method for producing modified leukocytes in the presence of a ferroptosis inhibitor. The inventors have produced CAR-T cells using T cells derived from BALB / c mice, but have not been able to achieve the expected antitumor effect. After investigating the cause, they found that CAR-T cells produced by conventional methods had accumulated a large amount of reactive oxygen species (ROS), leading to the progression of lipid peroxidation, and thus completed the present invention.

[0013] In the present invention, the term "ferroptosis inhibitor" refers to any substance that can inhibit ferroptosis, regardless of its mechanism of action (Cell Death Discov. 2022 Dec 29;8(1):501). Ferroptosis inhibitors include lipid peroxidation inhibitors, antioxidants, iron level reducers (e.g., iron chelators), and GSH / GPX4 axis inhibitors (e.g., glutathione peroxidase 4 (GPX4) expression enhancers). These agents can be small molecules, nucleic acid molecules, or peptides.

[0014] Lipid peroxidation inhibitors are substances that suppress the progression of lipid peroxidation, and examples thereof include, but are not limited to, ferrostatin-1 and lipostatin-1. Ferrostatin-1 is preferred. Antioxidants serving as ferroptosis inhibitors are water-soluble or fat-soluble substances that suppress the progression of oxidation, and examples thereof include, but are not limited to, vitamin C, vitamin E, and vitamin K. Note that antioxidants serving as ferroptosis inhibitors do not include antioxidants or reducing agents used in general cell culture media. Iron chelators are a type of active radical-scavenging antioxidant that suppresses iron-dependent lipid peroxidation by removing iron. Examples include, but are not limited to, deferoxamine, deferasirox, 2,2-bipyridyl, and ciclopirox olamine. Deferoxamine is preferred. A GPX4 expression enhancer is a substance that enhances the expression of GPX4, a central enzyme in the anti-ferroptosis system, and inhibits ferroptosis. Examples of such an enhancer include selenium compounds such as sodium selenite and selenium-L-methionine, but are not limited to these.

[0015] In one embodiment of the present invention, the ferroptosis inhibitor can be present in any step in the method for producing modified leukocytes, for example, in at least one step, such as the step of culturing leukocytes isolated from a living organism, the step of activating the isolated leukocytes, the step of modifying the isolated leukocytes, and the step of culturing the modified leukocytes to expand and / or grow.

[0016] In an embodiment of the present invention, a ferroptosis inhibitor can be added in the step of activating leukocytes or the step of introducing the plasmid.

[0017] In the present invention, the term "leukocyte" in "modified leukocyte" refers to lymphocytes, such as T cells, NK cells (natural killer cells), and B cells; monocytes, such as macrophages and microglia; and monocytes, such as neutrophils, eosinophils, and basophils. Preferably, the leukocyte is a T cell. T cells may be embryonic stem cells and their precursors, including pluripotent stem cells (e.g., from which lymphoid cells can be differentiated). T cells mature in the thymus and are lymphocytes primarily responsible for cell-mediated immunity, but can also participate in the adaptive immune system. According to the present invention, T cells include, but are not limited to, helper T cells, cytotoxic T cells (CTLs), memory T cells, such as TEM cells and TEMRA cells, and regulatory T cells (suppressor T cells).

[0018] In the present invention, "white blood cells" also includes artificial cells, such as induced pluripotent stem cells (iPS cells). iPS cells are cells that, by introducing four types of genes into somatic cells, are endowed with the pluripotency to differentiate into a large number of cells, like embryonic stem cells, and the ability to self-replicate, maintaining this ability even after division and proliferation.

[0019] In the present invention, "modified leukocytes" refers to leukocytes that have been modified to express at least one xenogeneic T cell receptor (TCR) and / or at least one chimeric antigen receptor (CAR). The modification can be carried out by transducing / transfecting the leukocytes with a nucleic acid molecule or vector containing a nucleic acid encoding at least one xenogeneic T cell receptor (TCR) and / or at least one chimeric antigen receptor (CAR). In the present invention, there is no limitation on the type of chimeric antigen receptor (CAR).

[0020] In one embodiment, the present invention relates to a method for producing modified leukocytes in the presence of a ferroptosis inhibitor, the method comprising the step of: (b) modifying the leukocytes to express at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR) by gene transfer into the leukocytes using a nucleic acid or vector encoding at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0021] As described above, the leukocytes may be naturally derived or artificially derived. Naturally derived leukocytes can be isolated from a donor subject. In the present invention, the donor subject is a vertebrate having an adaptive immune system, including a mammal. For example, Primates include humans, gorillas, chimpanzees, ring-tailed lemurs, mandrills, Japanese macaques, and crab-eating macaques; Carnivora include cats, dogs, civets, lions, tigers, leopards, cheetahs, hyenas, mongooses, bears, weasels, and raccoons; Perissodactyla include horses, zebras, tapirs, and rhinoceroses; Artiodactyla include camels, pigs, wild boars, deer, giraffes, hippos, goats, and sheep; Diprodonts include kangaroos, koalas, wombats, and flying squirrels; Rodents include dormice, squirrels, porcupines, mice, rats, hamsters, guinea pigs, beavers, and rabbits; and Cetaceans include blue whales, sperm whales, and dolphins.

[0022] According to the present invention, the method includes a step (b) of modifying leukocytes. According to the present invention, the leukocytes are modified to express at least one xenogeneic T cell receptor (TCR) or at least one chimeric antigen receptor (CAR). The modification can be by transducing the T cells with a nucleic acid molecule or vector comprising a nucleic acid encoding at least one xenogeneic T cell receptor (TCR) and / or at least one chimeric antigen receptor (CAR). The leukocytes can also be modified by incorporating a nucleic acid encoding at least one xenogeneic T cell receptor (TCR) and / or at least one chimeric antigen receptor (CAR) into the genome of the leukocyte, for example, into the genome of a precursor of the T cell, such as an induced pluripotent stem cell or a lymphoid lineage cell derived therefrom.

[0023] In the present invention, the modified leukocytes can express at least one heterologous T cell receptor (TCR) and / or chimeric antigen receptor (CAR) that binds or specifically binds to a cancer and / or tumor antigen or a peptide antigen thereof. Preferably, the peptide antigen is associated with a cancerous condition, cancer, and / or tumor, and / or presented by a cancer cell or tumor in a tissue, optionally presented by HLA / MHC. Upon antigen binding, the modified T cell or population of T cells can exhibit T cell effector function and / or cytolytic activity against antigen-bearing cells and / or undergo proliferation and / or cell division. In certain embodiments, the modified T cell or population of T cells comprising a TCR exhibits comparable or better therapeutic efficacy compared to cells comprising a chimeric antigen receptor (CAR) targeting the same cancer and / or tumor antigen and / or peptide (antigenic peptide). Activated modified T cells or populations of T cells containing a heterologous TCR or CAR can secrete anti-tumor cytokines, which may include, but are not limited to, TNFα, IFNγ, and IL2.

[0024] In the present invention, the chimeric antigen (CAR) is selected from the group consisting of CD19, ERBB2 / HER2, B-cell mutant antigen (BCMA), CEA-CAM5, carbonic anhydrase IX, CD7, CD22, CD5, GPRC5D, and EGFR-IL13Rα2.

[0025] Cancers or tumors to which the present invention can be applied include solid cancers selected from cancers such as colon cancer, lung cancer, gastric cancer, breast cancer, prostate cancer, pancreatic cancer, and hepatocellular carcinoma; brain tumors such as glioblastoma; sarcomas such as osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma; and hematopoietic tumors selected from leukemia, malignant lymphoma, and multiple myeloma.

[0026] In another embodiment, the present invention relates to a method for producing modified leukocytes in the presence of a ferroptosis inhibitor, the method comprising: (b) modifying the leukocytes to express at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR) by gene transfer of the leukocytes using a nucleic acid or vector encoding at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR); and / or (a) activating the leukocytes, and further comprising at least one of the following steps: (c) expanding and / or proliferating the activated or modified leukocytes; (d) culturing the activated or modified leukocytes or a population thereof; and (e) recovering and / or cryopreserving the activated or modified leukocytes or a population thereof.

[0027] According to the present invention, a step (a) of activating leukocytes is included. Activation of leukocytes can be achieved by various methods. For example, it can be achieved by contacting leukocytes with an anti-CD3 antibody or a CD3-binding fragment thereof, or by contacting leukocytes with an anti-CD28 antibody or a CD28-binding fragment thereof, or by contacting leukocytes with B7 protein (B7 is a type of peripheral membrane protein found on activated antigen-presenting cells, which can generate a costimulatory signal when paired with CD28 or CD152 (CTLA-4) surface protein on T cells) or a CD28-binding fragment thereof, e.g., B7-1 or B7-2 or a CD28-binding fragment thereof. The activation means can be attached to a solid and optionally releasable surface or substrate, such as beads or magnetic beads, for example, magnetic beads coated with anti-CD3 and / or anti-CD28. Preferably, activation is carried out by adding an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof attached to beads, which can be, for example, magnetic beads, thereby allowing isolation from the cell culture medium, optionally removable beads. Activation of the leukocytes can be carried out simultaneously with or after modification of the leukocytes. Preferably, activation of the leukocytes is carried out prior to modification of the leukocytes.

[0028] In the present invention, the modified leukocytes are cultured to expand and / or grow [(c) and (d)]. The modified leukocytes can be cultured using any convenient means, technique, vessel, container, or system to produce an expanded population. Suitable culture systems include stirred tank fermentors, airlift fermentors, roller bottles, culture bags or dishes, and other bioreactors, particularly hollow fiber bioreactors. The use of such systems is well known in the art.

[0029] In one embodiment, the present invention relates to a method of the present invention, in which modified leukocytes are produced in the presence of a ferroptosis inhibitor in at least one of steps (a), (b), (c), (d), and (e). A specific example can be mentioned of the method of the present invention, in which a ferroptosis inhibitor is present in the step (a) of activating leukocytes and / or the step (b) of modifying leukocytes.

[0030] <Modified Leukocytes> In another aspect, the present invention relates to modified leukocytes, preferably modified T cells, produced by the method of the present invention. The modified leukocytes produced by the method of the present invention have suppressed lipid peroxidation compared to conventional leukocyte production methods that do not add a ferroptosis inhibitor, resulting in improved antitumor activity. Furthermore, the modified leukocytes produced by the method of the present invention can be maintained in vivo for a longer period of time and can more strongly suppress tumor growth.

[0031] <Method for Reducing Lipid Peroxides> In yet another aspect, the present invention provides a method for reducing lipid peroxides in modified leukocytes, which method uses a ferroptosis inhibitor. In one embodiment, the present invention provides a method for reducing lipid peroxides in T cells introduced with a chimeric antigen receptor gene, comprising the steps of: culturing T cells isolated from a subject in vitro with an anti-CD3 antibody and an anti-CD28 antibody in a chimeric antigen receptor medium for 24 to 72 hours to activate them in vitro; introducing an anti-chimeric antigen receptor gene plasmid one or more times in vitro into the T cells activated in the activation step; and recovering the T cells introduced with the plasmid in the plasmid introduction step, wherein the method reduces lipid peroxides in the presence of a ferroptosis inhibitor. In this aspect, the terms "modified leukocyte," "ferroptosis inhibitor," and "chimeric antigen receptor (CAR)" have the same definitions as above.

[0032] An example of a medium for producing a chimeric antigen receptor that can be used in the present invention is RPMI-1640 medium containing 10% fetal calf serum (FCS), 50 μM 2-mercaptoethanol (2ME), 100 U / mL interleukin-2 (IL-2), 100 U / mL penicillin, and 100 U / mL streptomycin. In an embodiment of the present invention, the activation step lasts for 24 to 72 hours, preferably 48 hours. In an embodiment of the present invention, the plasmid introduction step lasts for 24 to 72 hours, preferably 48 hours.

[0033] <Kit> In yet another aspect, the present invention relates to a kit for producing modified leukocytes, the kit comprising at least a ferroptosis inhibitor. Preferably, in one embodiment, the present invention provides a kit for producing CAR-T cells, the kit comprising at least: a medium for producing a chimeric antigen receptor; an anti-CD3 antibody and an anti-CD28 antibody; an anti-chimeric antigen receptor gene plasmid; a ferroptosis inhibitor, an iron chelator, or a combination thereof. In this aspect, the terms "modified leukocyte," "ferroptosis inhibitor," and "chimeric antigen receptor (CAR)" have the same meanings as defined above.

[0034] An example of a medium for producing a chimeric antigen receptor that can be used in the present invention is RPMI-1640 medium containing 10% fetal calf serum (FCS), 50 μM 2-mercaptoethanol (2ME), 100 U / mL interleukin-2 (IL-2), 100 U / mL penicillin, and 100 U / mL streptomycin.

[0035] According to the present invention, the use of a ferroptosis inhibitor during the production of CAR-T cells prevents the progression of lipid peroxides generated during the production process, thereby improving the antitumor effect of the resulting CAR-T cells. Therefore, in another aspect, the present invention relates to a pharmaceutical composition for treating or preventing malignant tumors, particularly refractory malignant tumors that are difficult to treat with normal immune function alone, comprising modified leukocytes produced by the production method of the present invention. In a further embodiment, the present invention relates to a method for treating or preventing malignant tumors, particularly refractory malignant tumors that are difficult to treat with normal immune function alone, comprising administering modified leukocytes produced by the production method of the present invention to a subject in need of such treatment, preferably a method comprising administering an effective amount of a lyophilized formulation of the present invention to such a subject. In yet another aspect, the present invention relates to modified leukocytes produced by the production method of the present invention for treating or preventing malignant tumors, particularly refractory malignant tumors that are difficult to treat with normal immune function alone. In yet another aspect, the present invention relates to the use of modified leukocytes produced by the production method of the present invention to manufacture a pharmaceutical for treating or preventing malignant tumors, particularly refractory malignant tumors that are difficult to treat using normal immune function alone.

[0036] All references cited herein, including publications, patent documents, and the like, are hereby incorporated by reference to the same extent as if each was individually and specifically incorporated by reference and the contents thereof were specifically set forth in their entirety.

[0037] The present invention will be described in more detail below with reference to examples and conventional examples. However, it should be noted that these examples do not limit the scope of the present invention and are merely illustrative.

[0038] [Conventional Example] 1. Isolation of Mouse T Cells and Production of Mouse CAR-T Cells T cells were isolated from the lymph nodes of 7- to 12-week-old female BALB / c mice using a Pan T Cell isolation Kit II (Miltenyi Biotec) and a MidiMACS Separator (Miltenyi Biotec) (Day 0). The process for producing CAR-T cells from the isolated T cells is described below (Figure 2). The isolated T cells were seeded (Day 0) in RPMI-1640 medium containing 10% fetal calf serum (FCS), 50 μM 2-mercaptoethanol (2ME), 100 U / mL interleukin-2 (IL-2), 100 U / mL penicillin, and 100 U / mL streptomycin (hereinafter referred to as "CAR-T medium"). This medium was then cultured on anti-CD3 antibody (Biolegend) and anti-CD28 antibody (Biolegend) adherent plates for 48 hours to activate the T cells. Separately, Platinum-E cells (Cosmo Bio) were transfected with a CD19 chimeric antigen receptor (CAR) plasmid to prepare a "retroviral vector." This retroviral vector was seeded on a non-tissue culture plate coated with RetroNectin (Takara), centrifuged, and allowed to adhere to create a "retrovirus-treated non-tissue culture plate."

[0039] The MSGV1-1D3-28Z.1-3 mut plasmid (addgene plasmid #107227; mouse CD19 CAR with a CD3ITAM mutation) was used as the antibody for T cell activation. This MSGV1-1D3-28Z.1-3 mut plasmid was deposited by Kochenderfer and Steven Rosenberg [http: / / n2t.net / addgene:107227]. The same plasmid was used in the following examples.

[0040] Next, 1.0 × 10 ５The T cells activated for 48 hours and adjusted to 1 mL / mL were seeded onto a separately prepared "retrovirus-treated non-tissue culture plate," followed by centrifugation, and the CD19CAR gene was introduced into the T cells (Day 2). After another 24 hours, the "retrovirus supernatant" was seeded again, followed by centrifugation, and the CD19CAR gene was introduced into the T cells (Day 3). After another 24 hours, the T cells into which the CD19CAR gene had been introduced (CAR-T cells) were collected (Day 4). The CAR-T cells obtained at this point were used in the following Examples.

[0041] 2. Verification of gene transfer efficiency The CAR-T cells collected on Day 4 were stained with anti-TCR-β antibody, a T cell population marker, and biotin anti-protein L antibody, a CAR-expressing population marker, and the gene transfer efficiency was verified by flow cytometry. The gene transfer efficiency was calculated from the ratio of protein L-positive cell population within the TCR-β-positive cell population, and was found to be 57.3% (Figure 3).

[0042] 3. Evaluation of intracellular reactive oxygen species levels and lipid peroxidation Using T cells immediately after isolation from BALB / c mice as a reference, the accumulation of ROS and the progression of lipid peroxidation in CAR-T cells collected on Day 4 were evaluated. The CAR-T cell suspension produced by the method described in 1 above was incubated at 37°C and 5% CO in the presence of CellROX® Green (Invitrogen) to evaluate the amount of ROS accumulation, or in the presence of the Lipid Peroxidation Sensor included in the Lipid Peroxidation Assay Kit (cell-based) (ab243377) (Abcam) to evaluate lipid peroxidation. ２ CAR-T cells were fluorescently stained by culturing them for 30 minutes in an incubator under the specified conditions. The fluorescently stained conventional CAR-T cells were then washed and analyzed by flow cytometry within two hours of staining. Regarding lipid peroxidation, the fluorescence of the Lipid Peroxidation Sensor changes from PE (non-peroxidized lipids) to FITC (peroxidized lipids) as lipid peroxidation progresses, so the PE / FITC ratio was used for evaluation. The results are shown in Figure 4.

[0043] CD4 ＋ T cells and CD8 ＋ The fluorescence intensity of all the T cells was higher than that of isolated T cells. This indicates that the conventional method for producing CAR-T cells increases the amount of ROS accumulated (Figure 4a). ＋ T cells and CD8 ＋ The PE / FITC ratio of all T cells was lower than that of isolated T cells, indicating that the conventional method for producing CAR-T cells increases lipid peroxides (Figure 4b).

[0044] Example 1: The antitumor effect of mouse CAR-T cells treated with ferrostatin-1, a ferroptosis inhibitor, during the manufacturing process was evaluated. In this example, ferrostatin-1 (Fer-1; Funakoshi) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 32 μM DMSO solution, and DMSO was used as a control.

[0045] 1. T Cell Isolation and Production of Ferrostatin-1-Treated CAR-T Cells (1) Production of Control CAR-T Cells T cells were isolated from the lymph nodes of 7- to 12-week-old female BALB / c mice using a Pan T Cell Isolation Kit II (Miltenyi Biotec) and a MidiMACS Separator (Miltenyi Biotec) (Day 0). The process for producing CAR-T cells from the isolated T cells is described below (Figure 5). The isolated T cells were seeded in CAR-T medium supplemented with 0.32% DMSO (Day 0). The T cells were activated by culturing this medium on plates coated with anti-CD3 antibody (Biolegend) and anti-CD28 antibody (Biolegend) for 48 hours. Separately, Platinum-E cells (Cosmo Bio) were transfected with a CD19 chimeric antigen receptor (CAR) plasmid (1D3-28Z.1-3; Addgene) to prepare a "retroviral vector." This retroviral vector was seeded onto a non-tissue culture plate coated with RetroNectin (Takara), centrifuged, and allowed to adhere to create a "retrovirus-treated non-tissue culture plate."

[0046] Next, 1.0 × 10 ５ The T cells activated for 48 hours and adjusted to 1000 mg / mL were seeded onto a separately prepared "retrovirus-treated non-tissue culture plate," followed by centrifugation, and the CD19CAR gene was introduced into the T cells (Day 2). After another 24 hours, the retrovirus supernatant was seeded again, followed by centrifugation, and the CAR gene was introduced into the T cells (Day 3). After another 24 hours, control cells into which the CD19CAR gene had been introduced (control CAR-T cells) were collected (Day 4).

[0047] (2) Production of Ferrostatin-1-Treated CAR-T Cells The isolated T cells were seeded in CAR-T medium supplemented with a 0.32% DMSO solution of ferrostatin-1 (Fer-1 / DMSO; Fer-1 concentration: 32 μM) (Day 0) (Fer-1 concentration in the medium: 32 μM), and the medium was cultured on an anti-CD3 antibody (biolegend) and anti-CD28 antibody (biolegend) adherent plate for 48 hours to activate the T cells. Then, on Day 4, T cells transfected with CD19CAR under Fer-1 treatment (Fer-1-treated CAR-T cells) were recovered in the same manner as above.

[0048] 2. Verification of Gene Transfer Efficiency Control CAR-T cells and Fer-1-treated CAR-T cells collected on Day 4 were stained with anti-TCR-β antibody, a T cell population marker, and biotin anti-protein L antibody, a CAR-expressing population marker, and the gene transfer efficiency was verified by flow cytometry. The gene transfer efficiency was calculated from the ratio of the protein L-positive cell population within the TCR-β-positive cell population, and was 44.3% for control CAR-T cells. The efficiency was 46.6% for Fer-1-treated CAR-T cells, which was equivalent to that of the control CAR-T cells ( FIG. 6 ).

[0049] 3. Evaluation of intracellular reactive oxygen species levels and lipid peroxidation Using control CAR-T cells as a reference, the accumulation of ROS and the progression of lipid peroxidation in Fer-1-treated CAR-T cells collected on Day 4 were evaluated. A suspension of control CAR-T cells or Fer-1-treated CAR-T cells was incubated at 37°C and 5% CO in the presence of CellROX® Green (Invitrogen) to evaluate the amount of ROS accumulation, or in the presence of the Lipid Peroxidation Sensor included in the Lipid Peroxidation Assay Kit (cell based) (ab243377) (Abcam) to evaluate lipid peroxidation. ２CAR-T cells were fluorescently stained by culturing them for 30 minutes in an incubator under the specified conditions. The fluorescently stained control CAR-T cells or Fer-1-treated CAR-T cells were then washed and analyzed by flow cytometry within 2 hours of staining. Regarding lipid peroxidation, the fluorescence of the Lipid Peroxidation Sensor changes from PE (non-peroxidized lipids) to FITC (peroxidized lipids) as lipid peroxidation progresses, so the PE / FITC ratio was used for evaluation. The results are shown in Figure 7.

[0050] CD4 ＋ T cells and CD8 ＋ In all cases, the fluorescence intensity of the Fer-1-treated CAR-T cells was lower than that of the control CAR-T cells (indicated as DMSO in Figure 7). This indicates that the method for producing CAR-T cells of the present invention, which involves treatment with ferrostatin-1, reduces the amount of ROS accumulation (Figure 7a). ＋ T cells and CD8 ＋ In both cases, the PE / FITC ratio of the Fer-1-treated CAR-T cells was higher than that of the control CAR-T cells, demonstrating that lipid peroxides are reduced by the method for producing CAR-T cells of the present invention using ferrostatin-1 treatment ( FIG. 7 b).

[0051] 4. Evaluation of Cytokine Production Ability. Cytokine production ability was evaluated by activating control CAR-T cell and Fer-1-treated CAR-T cell suspensions collected on Day 4 with 50 ng / mL PMA (phorbol 12-myristate 13-acetate) and 1000 ng / mL ionomycin for 1 hour, followed by the addition of Goldistop (BD) and further activation for 4 hours. For intracellular cytokine staining, cells were fixed using a Cytofix / Cytoperm kit (BD) and washed with 1x Perm / wash buffer. For transcription factor staining, cells were fixed using Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent (Invitrogen) and washed with Permeabilization buffer (Invitrogen). Flow cytometry was performed using a Canto II (BD) or Aria III (BD), and analysis was performed using FlowJo. The parameters used were the combination of transcription factors T cell factor 1 (TCF1) and Tim3, IFNγ and TNFα, CD8 and granzyme B. The results obtained are shown in Figures 8 to 10.

[0052] Fer-1-treated CAR-T cells showed significantly higher TCF1 expression than control CAR-T cells. ＋ Time 3 －The proportion of CD8-positive CAR-T cells that simultaneously produced IFNγ and TNFα was increased (Figure 8). This indicates an increase in the stem cell-like cell population. It has been reported that the higher the stem cell-like proportion of CAR-T cells, the more effective the antitumor effect (CIENCE TRANSLATIONAL MEDICINE, 5 Apr 2023, Vol. 15, Issue 690 DOI: 10.1126 / scitranslmed.abk1900). Figure 8 suggests that the Fer-1-treated CAR-T cell production method of the present invention may increase the proportion of stem cell-like cells, thereby improving antitumor effect. Compared to control CAR-T cells, Fer-1-treated CAR-T cells showed a decreased proportion of CD8-positive CAR-T cells that simultaneously produced IFNγ and TNFα (Figure 9). IFNγ and TNFα were selected as representative cytokines, and Figure 9 shows that the method for producing CAR-T cells of the present invention using Fer-1 treatment increases cell populations with low cytokine production capacity, i.e., cell populations with a low degree of differentiation. The results in Figure 9 are considered to be consistent with the characteristics of the stem cell-like population described above in Figure 8. Compared to control CAR-T, the proportion of CD8-positive CAR-T expressing granzyme B was reduced in Fer-1-treated CAR-T (Figure 10). Because granzyme B is the most common granule secreted by cytotoxic CD8 T cells, Figure 10 shows that the method for producing CAR-T cells of the present invention using Fer-1 treatment increases cell populations with low granzyme B expression and a low degree of differentiation. The results in Figure 10 are considered to be consistent with the characteristics of the stem cell-like population described above in Figure 8.

[0053] 5. Assessment of CAR-T cell proliferation To assess cell proliferation, control CAR-T cells or Fer-1-treated CAR-T cells collected on Day 4 were cultured in CAR-T medium supplemented with 5 μM CellTrace® Violet (CTV; ThermoFisher) at 37°C and 5% CO ２The CAR-T cells were then cultured in a 1:1 incubator at room temperature for 20 minutes. The CAR-T cells were then washed and cultured in CAR-T medium at a 1:1 ratio of CAR-T cells (effector) to A20 cells (target) for 72 hours (Figure 11). The A20 cell line (ATCC TIB-208) was purchased from ATCC. A20 cells are a murine B-cell lymphoma cell line. The A20 cell line was cultured in CAR-T medium. After 72 hours (3 days) of culture, the expanded cells were harvested, and the degree of decay of CellTrace® Violet fluorescent staining (CTV) from Day 0 for control CAR-T cells and Fer-1-treated CAR-T cells was analyzed by flow cytometry (Figure 12a). CellTrace® Violet fluorescent staining allows for tracking cell generations or cell division without affecting cell morphology or physiological function. That is, the mean fluorescence intensity (MFI) decreases as the cells divide, and the number of generations after fluorescent labeling can be determined by the attenuation of the mean fluorescence intensity in the cell population.

[0054] CD4 ＋ T cells and CD8 ＋ In both cases, the mean fluorescence intensity (CTVMFI) of Fer-1-treated CAR-T cells was reduced to about half that of control CAR-T cells, demonstrating that the method for producing CAR-T cells of the present invention promotes cell proliferation ( FIG. 12 a).

[0055] 6. Measurement of CAR-T Cytotoxic Activity For the measurement of the in vitro cytotoxic activity of CAR-T cells, A20 cells were cultured in CAR-T medium supplemented with 5 μM CellTrace® Violet (CTV; ThermoFisher) at 37°C and 5% CO ２The cells were then cultured for 20 minutes in a constant temperature incubator. Then, on Day 4, control CAR-T cells or Fer-1-treated CAR-T cells (effector) were harvested and co-cultured with A20 cells (target) in CAR-T medium in a 96-well plate at a ratio of 0.25:1, 0.5:1, 1:1, or 2:1. Each concentration was cultured in triplicate wells for 10 hours. A20 cells were cultured alone to assess the baseline apoptosis rate. After incubation, the cells were harvested and stained with Fixable ViAblity Stain 780 (BD biosciences) for 30 minutes before analysis by flow cytometry. The cell killing rate (% cytolysis) by Protein L-positive T cells was calculated as [(number of dead A20 cells) / (number of dead A20 cells + number of viable A20 cells)] × 100 - baseline apoptosis. The results are shown in Figure 12b.

[0056] At all effector:target ratios, the cell-killing rate of Fer-1-treated CAR-T cells was higher than that of control CAR-T cells, demonstrating that the method for producing CAR-T cells of the present invention improves in vitro cytotoxic activity ( FIG. 12 b).

[0057] 7. Creation of a B-cell acute leukemia model The process for creating a mouse model of B-cell acute leukemia is described below (Figure 13). Female BALB / c mice aged 7 to 12 weeks were subjected to total body irradiation (TBI) at 3.0 Gy and intraperitoneally administered 200 mg / kg of cyclophosphamide. The following day (Day 0), 1.0 x 10 ６ The mice were then given 1.0 × 10 luciferase-expressing A20 cells by intravenous injection into the retroorbital venous plexus and reared for 7 days (Day 7), at which point they were designated as B-All mice. ５Control CAR-T cells or Fer-1-treated CAR-T cells were administered intravenously via the retroorbital venous plexus. Bioluminescence images were taken using an in vivo imaging system on days 7, 10, and 14. Tumor growth was evaluated by measuring the leukemic tumor volume over time. Specifically, the leukemic tumor volume was evaluated using the luminescence intensity on day 7 [(total luminous flux (p / s)] as the reference value and the rate of change in luminescence intensity on day X [(Day X, total luminous flux) / (Day 7, total luminous flux)]. The results are shown in Figure 14a. To evaluate the number of CAR-T cells in the peripheral blood, peripheral blood was collected from the tail of B-All mice on day 16, and the number of CAR-T cells in the peripheral blood was counted by cytometry. The results are shown in Figure 14b.

[0058] The leukemic tumor burden increased from Day 7 to Day 10 in both the control CAR-T cells and the Fer-1-treated CAR-T cells. This indicates that the CAR-T cells did not proliferate sufficiently in vivo. From Day 10 onwards, the rate of increase in the leukemic tumor burden slowed in the control CAR-T cells, while the leukemic tumor burden significantly decreased in the Fer-1-treated CAR-T cells ( Figure 14a ). This demonstrates that the method for producing CAR-T cells of the present invention improves the in vivo antitumor effect. The number of Fer-1-treated CAR-T cells in the peripheral blood counted on Day 16, a long time after inoculation into B-All mice, was greater than that of the control CAR-T cells ( Figure 14b ). This demonstrates that the method for producing CAR-T cells of the present invention achieves a long-lasting in vivo antitumor effect.

[0059] Example 2: The antitumor effect of mouse CAR-T cells treated with deferoxamine, an iron chelator, during the manufacturing process was evaluated. In this example, deferoxamine (DFO; Funakoshi) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 μM DMSO solution, and DMSO was used as a control.

[0060] 1. T Cell Isolation and Production of Deferoxamine-Treated CAR-T Cells (1) Production of Control CAR-T Cells. T cells were isolated from the lymph nodes of 7- to 12-week-old female BALB / c mice using the Pan T Cell Isolation Kit II (Miltenyi Biotec) and a MidiMACS Separator (Miltenyi Biotec) (Day 0). The process for producing CAR-T cells from the isolated T cells is described below (Figure 4). The isolated T cells were seeded in CAR-T medium supplemented with 0.32% DMSO (Day 0). This medium was then cultured on an anti-CD3 antibody (biolegend) and anti-CD28 antibody (biolegend) adherent plate for 48 hours to activate the T cells. Separately, a "retroviral vector" was prepared by transfecting Platinum-E cells (Cosmo Bio) with a CD19 chimeric antigen receptor (CAR) plasmid. This retroviral vector was inoculated onto a non-tissue culture plate coated with RetroNectin (Takara), centrifuged, and allowed to adhere to create a "retrovirus-treated non-tissue culture plate."

[0061] Next, 1.0 × 10 ５ The T cells after 48 hours of activation, adjusted to 1 mL / mL, were plated onto a separately prepared "retrovirus-treated non-tissue culture plate," and centrifuged to transduce the CD19CAR gene into the T cells (Day 2). After another 24 hours, the retrovirus supernatant was plated again, and the plate was centrifuged for gene transduction (Day 3). After another 24 hours, control cells transfected with the CD19CAR gene (control CAR-T cells) were collected (Day 4).

[0062] (2) Preparation of deferoxamine-treated CAR-T cells. The isolated T cells were activated by culturing them for 48 hours on anti-CD3 antibody (Biolegend) and anti-CD28 antibody (Biolegend) adherent plates in CAR-T medium supplemented with 0.32% DMSO. Separately, Platinum-E cells (Cosmo Bio) were transfected with a CD19 chimeric antigen receptor (CAR) plasmid (1D3-28Z.1-3; Addgene) to prepare a retroviral supernatant. This retroviral supernatant was seeded on a non-tissue culture plate coated with RetroNectin (Takara), centrifuged, and allowed to adhere to create a "retrovirus-treated non-tissue culture plate." Next, 1.0 x 10 T cells were cultured in CAR-T medium supplemented with a 0.32% deferoxamine DMSO solution (DFO / DMSO; DFO concentration 20 μM) (the DFO concentration in the medium was 10 μM). ５ The T cells after 48 hours of activation, adjusted to a concentration of 1 mL / mL, were seeded onto a separately prepared "retrovirus-treated non-tissue culture plate" and centrifuged to transduce the CD19CAR gene into the T cells. Thereafter, on Day 4, T cells transduced with the CD19CAR gene under DFO treatment (DFO-treated CAR-T cells) were collected in the same manner as above.

[0063] 2. Verification of gene transfer efficiency The resulting CAR-T cells were stained with an anti-TCR-β antibody, a T cell population marker, and a biotin anti-protein L antibody, a CAR-expressing population marker, and the gene transfer efficiency was verified by flow cytometry. The gene transfer efficiency was calculated from the ratio of the protein L-positive cell population within the TCR-β-positive cell population, and was 63.1% for the control CAR-T cells. The DFO-treated CAR-T cells had a gene transfer efficiency of 63.0%, which was equivalent to that of the control CAR-T cells ( FIG. 15 ).

[0064] 3. Assessment of intracellular reactive oxygen species levels and lipid peroxidation ROS accumulation and progression of lipid peroxidation in DFO-treated CAR-T cells were assessed using control CAR-T cells as a reference. A suspension of control CAR-T cells or DFO-treated CAR-T cells was incubated at 37°C and 5% CO in the presence of CellROX® Green (Invitrogen) to assess ROS accumulation, or in the presence of the Lipid Peroxidation Sensor included in the Lipid Peroxidation Assay Kit (cell-based) (ab243377) (Abcam) to assess lipid peroxidation. ２ CAR-T cells were fluorescently stained by culturing them for 30 minutes in an incubator under the specified conditions. The fluorescently stained control CAR-T cells or DFO-treated CAR-T cells were then washed and analyzed by flow cytometry within 2 hours after staining. Regarding lipid peroxidation, the fluorescence of the lipid peroxidation sensor changes from PE (non-peroxidized lipids) to FITC (peroxidized lipids) as lipid peroxidation progresses, so the PE / FITC ratio was used for evaluation. The results are shown in Figure 16.

[0065] CD4 ＋ T cells and CD8 ＋ There was no significant difference in fluorescence intensity between the control CAR-T cells (indicated as DMSO in Figure 16) and the DFO-treated CAR-T cells. This suggests that the method for producing CAR-T cells of the present invention using DFO treatment does not affect the amount of ROS accumulation (Figure 16a). On the other hand, CD4 ＋ T cells and CD8 ＋ In both cases, the PE / FITC ratio of DFO-treated CAR-T cells was higher than that of control CAR-T cells. This demonstrated that lipid peroxidation was reduced by the method for producing CAR-T cells of the present invention using DFO treatment ( Figure 16b ). Lipid peroxidation was suppressed by the method for producing CAR-T cells of the present invention using DFO treatment, even when ROS accumulation occurred.

[0066] 3. Evaluation of Cytokine Production Ability Cytokine production ability was evaluated by activating control CAR-T cell and DFO-treated CAR-T cell suspensions with 50 ng / mL PMA and 1000 ng / mL ionomycin for 1 hour, followed by the addition of Goldistop (BD), followed by an additional 4 hours of activation. For intracellular cytokine staining, cells were fixed using a Cytofix / Cytoperm kit (BD) and washed with 1x Perm / wash buffer. For transcription factor staining, cells were fixed using Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent (Invitrogen) and washed with Permeabilization buffer (Invitrogen). Flow cytometry was performed using a Canto II (BD) or Aria III (BD), and analysis was performed using FlowJo. The parameters used were the combination of transcription factors T cell factor 1 (TCF1) and Tim3, IFNγ and TNFα, CD8 and granzyme B. The results obtained are shown in Figures 17 to 19.

[0067] DFO-treated CAR-T cells showed significantly higher TCF1 expression than control CAR-T cells. ＋ Time 3 －The proportion of CD8-positive CAR-T cells increased ( Figure 17 ). This indicates an increase in the stem cell-like cell population. It has been reported that the higher the stem cell-like proportion of CAR-T cells, the better the antitumor effect. Figure 17 suggests that the DFO treatment method for producing CAR-T cells of the present invention may increase the proportion of stem cell-like cells, thereby improving the antitumor effect. Compared to control CAR-T cells, DFO-treated CAR-T cells increased the proportion of CD8-positive CAR-T cells that simultaneously produced IFNγ and TNFα ( Figure 18 ). Figure 18 shows that the DFO treatment method for producing CAR-T cells of the present invention increases cell populations with low cytokine production capacity, i.e., cell populations with low differentiation levels. The results in Figure 18 are considered consistent with the characteristics of the stem cell-like population described above in Figure 17 . Compared to control CAR-T cells, DFO-treated CAR-T cells increased the proportion of CD8-positive CAR-T cells that expressed granzyme B ( Figure 19 ). Figure 19 shows that the method for producing CAR-T cells of the present invention using Fer-1 treatment increases a population of cells with low levels of granzyme B expression and a low degree of differentiation. The results in Figure 19 are considered to be consistent with the characteristics of the stem cell-like population described above in Figure 17.

[0068] 5. Measurement of CAR-T Cytotoxic Activity For the measurement of the in vitro cytotoxic activity of CAR-T cells, A20 cells were cultured in CAR-T medium supplemented with 5 μM CellTrace® Violet (CTV; ThermoFisher) at 37°C and 5% CO ２The cells were then cultured for 20 minutes in a constant temperature incubator. Then, on Day 4, control CAR-T cells or DFO-treated CAR-T cells (effector) were harvested and co-cultured with A20 cells (target) in CAR-T medium in a 96-well U-bottom plate at a ratio of 0.25:1, 0.5:1, 1:1, or 2:1. Each concentration was cultured in triplicate in three wells for 10 hours. The baseline apoptosis rate was assessed by culturing A20 cells alone. After incubation, the cells were harvested and stained with Fixable Viability Stain 780 (BD biosciences) for 30 minutes before analysis by flow cytometry. The cell killing rate (% cytolysis) by Protein L-positive T cells was calculated as [(number of dead A20 cells) / (number of dead A20 cells + number of viable A20 cells)] × 100 - baseline apoptosis. The results are shown in Figure 20.

[0069] At all effector:target ratios, the cell killing rate of DFO-treated CAR-T cells was higher than that of control CAR-T cells, demonstrating that the method for producing CAR-T cells of the present invention improves in vitro cytotoxic activity ( FIG. 20 ).

[0070] 7. Creation of a B-cell acute leukemia model The process for creating a mouse model of B-cell acute leukemia is described below (Figure 13). Female BALB / c mice aged 7 to 12 weeks were subjected to total body irradiation (TBI) at 3.0 Gy and intraperitoneally administered 200 mg / kg of cyclophosphamide. The following day (Day 0), 1.0 x 10 6 The mice were then given 1.0 × 10 luciferase-expressing A20 cells via intravenous injection into the retroorbital plexus and reared for 7 days (Day 7), at which point they were designated B-All mice. ５Control CAR-T cells or DFO-treated CAR-T cells were administered intravenously via the retro-orbital plexus. Bioluminescence images were taken using an in vivo imaging system on Days 7, 10, and 14. Tumor growth was evaluated by measuring the leukemic tumor mass over time. Specifically, the leukemic tumor mass was evaluated using the luminescence intensity on Day 7 [(total luminous flux (p / s)] as the reference, and the rate of change in luminescence intensity on Day X [(Day X, total luminous flux) / (Day 7, total luminous flux)]. The results are shown in Figure 21.

[0071] Example 3: Evaluating the antitumor effect of human CAR-T cells treated with ferrostatin-1, a ferroptosis inhibitor, during the manufacturing process. In this example, ferrostatin-1 (Fer-1) was prepared in the same manner as in Example 1.

[0072] 1. Human T Cell Isolation and Production of Ferrostatin-1-Treated Human CAR-T Cells (1) Production of Control Human CAR-T Cells T cells were isolated from human peripheral blood using the Pan T Cell Isolation Kit II (Miltenyi Biotec) and a MidiMACS Separator (Miltenyi Biotec) (Day 0). The process for producing CAR-T cells from the isolated T cells is described below ( Figure 22 ). As in Example 1, the isolated T cells were seeded in CAR-T medium supplemented with a 0.32% DMSO solution of ferrostatin-1 (Fer-1 / DMSO; Fer-1 concentration: 32 μM) (Day 0). Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher) was mixed with the T cells at a 1:1 ratio to activate the T cells. Next, the CD19CAR gene was introduced on Day 1, and the dynabeads were removed on Day 2. Thereafter, T cells into which the CD19CAR gene had been introduced under Fer-1 treatment (Fer-1-treated human CAR-T cells) were recovered on Day 4 in the same manner as described above.

[0073] 2. Verification of Gene Transfer Efficiency Untransduced human T cells, and control and Fer-1-treated human CAR-T cells collected on Day 4 were stained with anti-CD3 antibody, a T cell population marker, and CD19 CAR Detection Reagent (Miltenyi Biotec), a CAR-expressing population marker, to verify gene transfer efficiency by flow cytometry. Gene transfer efficiency was calculated from the proportion of CD19 CAR Detection Reagent-positive cells (denoted as CAR) within the CD3-positive cell population. The gene transfer efficiency was 0.04% for untransduced human T cells, 45.9% for control CAR-T cells, and 48.4% for Fer-1-treated CAR-T cells, indicating that the Fer-1-treated CAR-T cells exhibited a similar level of CAR transfer efficiency to that of the control human CAR-T cells ( Figure 23 ).

[0074] 3. Evaluation of intracellular reactive oxygen species levels and lipid peroxidation The progression of lipid peroxidation in Fer-1-treated human CAR-T cells collected on Day 4 was evaluated using control human CAR-T cells as a reference in the same manner as in Example 1. The results obtained are shown in Figure 24.

[0075] CD4 ＋ T cells and CD8 ＋ In all cases, the PE / FITC ratio of the Fer-1-treated human CAR-T cells was higher than that of the control CAR-T cells (indicated as DMSO in Figure 24). This demonstrates that the method for producing CAR-T cells of the present invention, which involves treatment with ferrostatin-1, also reduces lipid peroxides in human CAR-T cells.

[0076] 4. Evaluation of Cytokine Production Ability Cytokine production ability was evaluated using the parameters of CD8 and granzyme B, and the parameters of the transcription factors T cell factor 1 (TCF1) and Tim3, as in Example 1. The results obtained are shown in Figures 25 and 26, respectively.

[0077] In Fer-1-treated CAR-Ts, the ratio of CD4-positive CAR-T cells and CD8-positive CAR-T cells expressing granzyme B was reduced compared to control CAR-Ts (Figure 25). Figure 25 shows that the method for producing CAR-T cells of the present invention using Fer-1 treatment increases the population of less differentiated cells with low granzyme B expression. A less differentiated cell population is considered to be a stem cell-like population. Therefore, an increase in the population with low granzyme B expression means an increase in the stem cell-like population (Figure 25). In Fer-1-treated CAR-T cells, the TCF1 expression was significantly higher compared to control CAR-T cells. ＋ Time 3 － CD4-positive CAR-T cells and TCF1 ＋ Time 3 － The ratio of CD8-positive CAR-T cells increased ( FIG. 26 ). This indicates an increase in the stem cell-like cell population. It has been reported that the higher the stem cell-like ratio of CAR-T cells, the more effective the antitumor effect. FIG. 26 suggests that the method for producing CAR-T cells of the present invention using Fer-1 treatment improves the antitumor effect.

[0078] 6. Creation of a Xenograft Model The process of creating a mouse model of B-cell acute leukemia is described ( FIG. 27 ). Nalm6 cells (CRL-3273 (registered trademark)) (1.0 × 10 cells), a B-cell leukemia cell line obtained from ATCC, were transfected into NOG mice purchased from the Central Institute for Experimental Animals. ６ On Day 5, 0.4 × 10 cells were inoculated into B-All mice. ６ Control CAR-T cells or Fer-1-treated CAR-T cells were administered intravenously via the retroorbital venous plexus. After tumor inoculation, blood samples were taken twice a week for 30 days, and the time course of CAR-T cell counts in 10 μL of peripheral blood was measured. The results are shown in Figure 28. The number of Fer-1-treated CAR-T cells in the peripheral blood of B-All mice was counted over approximately 30 days after inoculation, and the number increased compared to the control CAR-T cells from 11 days after inoculation onward. This demonstrates that the in vivo antitumor effect of the CAR-T cell production method of the present invention is sustained for a long period of time.

[0079] According to the present invention, the use of a ferroptosis inhibitor in the production of CAR-T cells prevents the progression of lipid peroxides that occur during the production process, thereby improving the antitumor effect of the resulting CAR-T cells. The present invention can be applied to the treatment of intractable malignant tumors that are difficult to treat using normal immune function alone.

Claims

1. A method for producing modified white blood cells in the presence of a ferroptosis inhibitor.

2. The method of claim 1, wherein the method of producing modified leukocytes comprises the step of: (b) modifying the leukocytes to express at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR) by transfecting the leukocytes with a nucleic acid or vector encoding at least one xenogeneic T cell receptor (TCR) or chimeric antigen receptor (CAR).

3. The method of claim 2, further comprising at least one of the following steps: (a) activating the leukocytes; (c) expanding and / or proliferating the activated or modified leukocytes; (d) culturing the activated or modified leukocytes or a population thereof; or (e) recovering and / or cryopreserving the activated or modified leukocytes or a population thereof.

4. The method of claim 3, wherein in at least one step, the modified leukocytes are produced in the presence of a ferroptosis inhibitor.

5. The method according to claim 4, wherein a ferroptosis inhibitor is present in the steps (a) of activating leukocytes and / or (b) of modifying leukocytes.

6. The method of claim 1, wherein the ferroptosis inhibitor is a lipid peroxidation inhibitor and / or an iron chelator.

7. The method of claim 1, wherein the ferroptosis inhibitor is ferrostatin-1 and / or deferoxamine.

8. The method of claim 1, wherein the leukocytes are T cells or NK cells.

9. The method of claim 8, wherein the white blood cells are T cells.

10. The method of claim 1, wherein the leukocytes are monocytes or granulocytes.

11. A modified leukocyte produced by the method of any one of claims 1 to 9.

12. A kit for producing modified white blood cells according to claim 11, comprising at least a ferroptosis inhibitor.

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