Methods for producing immune cells that respond to cancer associated antigens
Patent Information
- Application Number
- JP2024077118
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-08-02
- Filing Date
- 2024-05-10
- Publication Date
- 2025-02-14
AI Technical Summary
【0062】 本発明によれば、がん関連抗原に反応する免疫細胞の製造方法を提供可能である。
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Abstract
Description
[Technical field]
[0001] The present invention relates to cell technology and to a method for producing immune cells that react to cancer-associated antigens. [Background technology]
[0002] Immunotherapies such as cancer vaccine therapy, dendritic cell therapy, and activated T cell therapy are used as cancer treatment methods. However, most of these therapies target a single tumor-associated antigen or even a specific antigen epitope. However, cancer cells are highly susceptible to genetic mutations, and gene expression patterns in cancer tissues are highly diverse. Therefore, immunotherapy targeting a single tumor antigen or antigen epitope is thought to be ineffective in cancer cells with antigen mutations or reduced expression, as it does not induce an immune response. [Prior art documents] [Non-patent literature]
[0003] [Non-Patent Document 1] Max Schnurr et al., "Tumor Cell Lysate-pulsed Human Dendritic Cells Induce a T-Cell Response against Pancreatic Carcinoma Cells: an in Vitro Model for the Assessment of Tumor Vaccines," CANCER RESEARCH 61, 6445-6450, September 1, 2001 Summary of the Invention [Problem to be solved by the invention]
[0004] An object of the present invention is to provide a method for producing immune cells that react to cancer-associated antigens. [Means for solving the problem]
[0005] [1] According to an embodiment, a method for producing immune cells responsive to a cancer-associated antigen includes stimulating immune cells with an antigen contained in pluripotent stem cells. The antigen contained in the pluripotent stem cells may be at least one of an undifferentiation-associated antigen, an immortalization-associated antigen, and a cell proliferation-associated antigen. The antigen may be at least one of a cancer-testis antigen and a carcinoembryonic antigen. The immune cells and the pluripotent stem cells may be derived from the same subject. The immune cells and the pluripotent stem cells may be derived from the same human. The method may be performed ex vivo or in vivo.
[0006] [2] In the method for producing immune cells reactive to a cancer-associated antigen according to the embodiment described in [1], the immune cells may be contained in monocytes. The monocytes and the pluripotent stem cells may be derived from the same subject. The monocytes and the pluripotent stem cells may be derived from the same human.
[0007] [3] In the method for producing immune cells that react to a cancer-associated antigen according to the embodiment described in [1] or [2], the immune cells may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0008] [4] In the method for producing immune cells that react to cancer-associated antigens according to the embodiment described in [3], the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0009] [5] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in [1] to [4], the immune cells may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0010] [6] In the method for producing immune cells reactive to a cancer-associated antigen according to any one of the embodiments described in [1] to [5], the pluripotent stem cells may be iPS cells.
[0011] [7] The method for producing immune cells reactive to a cancer-associated antigen according to any one of the embodiments described in [1] to [6] may further comprise lysing pluripotent stem cells to obtain the antigen.
[0012] [8] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in [1] to [7], the antigen may be a lysate of a pluripotent stem cell.
[0013] [9] In the method for producing immune cells that react to cancer-associated antigens according to any one of the embodiments described in [1] to [8], the stimulated immune cells may react to multiple types of cancer-associated antigens.
[0014]
[10] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in [1] to [9], the stimulated immune cells may be at least one of CD69-positive, CD107-positive, and IFN-γ-positive.
[0015]
[11] According to an embodiment, a method for producing immune cells responsive to a cancer-associated antigen comprises stimulating immune cells with at least a portion of pluripotent stem cells. The immune cells and the pluripotent stem cells may be from the same subject. The immune cells and the pluripotent stem cells may be from the same human. The method may be performed ex vivo or in vivo.
[0016]
[12] In the method for producing immune cells reactive to cancer-associated antigens according to the embodiment described in
[11] , the immune cells may be included in monocytes. The monocytes and the pluripotent stem cells may be derived from the same subject. The monocytes and the pluripotent stem cells may be derived from the same human.
[0017]
[13] In the method for producing immune cells that react to a cancer-associated antigen according to the embodiment described in
[11] or
[12] , the immune cells may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0018]
[14] In the method for producing immune cells that react to cancer-associated antigens according to the embodiment described in
[13] , the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0019]
[15] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in
[11] to
[14] , the immune cells may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0020]
[16] In the method for producing immune cells reactive to a cancer-associated antigen according to any one of the embodiments described in
[11] to
[15] , the pluripotent stem cells may be iPS cells.
[0021]
[17] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in
[11] to
[16] , at least a portion of the pluripotent stem cells may be a lysate of the pluripotent stem cells.
[0022]
[18] In the method for producing immune cells reactive to cancer-associated antigens according to any one of the embodiments described in
[11] to
[17] , the stimulated immune cells may react to multiple types of cancer-associated antigens.
[0023]
[19] In the method for producing immune cells that react to a cancer-associated antigen according to any one of the embodiments described in
[11] to
[18] , the stimulated immune cells may be at least one of CD69-positive, CD107-positive, and IFN-γ-positive.
[0024]
[20] According to an embodiment, a method for amplifying an immune cell response to a cancer-associated antigen includes stimulating the immune cell with an antigen contained in a pluripotent stem cell. The antigen contained in the pluripotent stem cell may be at least one of an undifferentiation-associated antigen, an immortalization-associated antigen, and a cell proliferation-associated antigen. The antigen may be at least one of a cancer-testis antigen and a carcinoembryonic antigen. The immune cell and the pluripotent stem cell may be derived from the same subject. The immune cell and the pluripotent stem cell may be derived from the same human. The method may be performed ex vivo or in vivo.
[0025]
[21] In the method for amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[20] , the immune cell may be comprised in a monocyte. The monocyte and the pluripotent stem cell may be derived from the same subject. The monocyte and the pluripotent stem cell may be derived from the same human.
[0026]
[22] In the method for amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[20] or
[21] , the immune cell may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0027]
[23] In the method for amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[22] , the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0028]
[24] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[23] , the immune cells may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0029]
[25] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[24] , the pluripotent stem cells may be iPS cells.
[0030]
[26] The method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[25] may further comprise lysing the pluripotent stem cells to obtain the antigen.
[0031]
[27] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[26] , the antigen may be a lysate of a pluripotent stem cell.
[0032]
[28] In the method for amplifying the response of immune cells to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[27] , the stimulated immune cells may respond to multiple types of cancer-associated antigens.
[0033]
[29] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[20] to
[28] , the stimulated immune cells may be at least one of CD69 positive, CD107 positive, and IFN-γ positive.
[0034]
[30] According to embodiments, a method of amplifying an immune cell response to a cancer associated antigen includes stimulating an immune cell with at least a portion of a pluripotent stem cell. The immune cell and the pluripotent stem cell may be from the same subject. The immune cell and the pluripotent stem cell may be from the same human. The method may be performed in vitro or in vivo.
[0035]
[31] In the method of amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[30] , the immune cells may be comprised in monocytes. The monocytes and the pluripotent stem cells may be derived from the same subject. The monocytes and the pluripotent stem cells may be derived from the same human.
[0036]
[32] In the method for amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[30] or
[31] , the immune cell may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0037]
[33] In the method for amplifying an immune cell response to a cancer-associated antigen according to the embodiment described in
[32] , the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0038]
[34] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[30] to
[33] , the immune cells may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0039]
[35] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[30] to
[34] , the pluripotent stem cells may be iPS cells.
[0040]
[36] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[30] to
[35] , at least a portion of the pluripotent stem cells may be a lysate of the pluripotent stem cells.
[0041]
[37] In the method for amplifying the response of immune cells to a cancer-associated antigen according to any one of the embodiments described in
[30] to
[36] , the stimulated immune cells may respond to multiple types of cancer-associated antigens.
[0042]
[38] In the method for amplifying an immune cell response to a cancer-associated antigen according to any one of the embodiments described in
[30] to
[37] , the stimulated immune cells may be at least one of CD69 positive, CD107 positive, and IFN-γ positive.
[0043]
[39] In accordance with an embodiment, the immune cells that respond to a cancer-associated antigen are immune cells stimulated with an antigen contained in the pluripotent stem cells. The antigen contained in the pluripotent stem cells may be at least one of an undifferentiation-associated antigen, an immortalization-associated antigen, and a cell proliferation-associated antigen. The antigen may be at least one of a cancer-testis antigen and a carcinoembryonic antigen. The immune cells and the pluripotent stem cells may be derived from the same subject. The immune cells and the pluripotent stem cells may be derived from the same human.
[0044]
[40] The immune cells according to the embodiment of
[39] may be included in the monocytes. The monocytes and the pluripotent stem cells may be derived from the same subject. The monocytes and the pluripotent stem cells may be derived from the same human.
[0045]
[41] The immune cells according to the embodiments described in
[39] or
[40] may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0046]
[42] In the immune cells according to the embodiment described in
[41] , the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0047]
[43] The immune cells according to any one of the embodiments described in
[39] to
[42] may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0048]
[44] In the immune cells according to any one of the embodiments described in
[39] to
[43] , the pluripotent stem cells may be iPS cells.
[0049]
[45] In the immune cells according to any one of the embodiments described in
[39] to
[44] , the antigen may be obtained by lysing pluripotent stem cells.
[0050]
[46] In the immune cell according to any one of the embodiments described in
[39] to
[45] , the antigen may be a lysate of a pluripotent stem cell.
[0051]
[47] The immune cells according to any one of the embodiments described in
[39] to
[46] may react to multiple types of cancer-associated antigens.
[0052]
[48] The immune cells according to any one of the embodiments described in
[39] to
[47] may be at least one of CD69 positive, CD107 positive, and IFN-γ positive.
[0053]
[49] In accordance with an embodiment, the immune cells that respond to a cancer-associated antigen are immune cells stimulated with at least a portion of a pluripotent stem cell. The immune cells and the pluripotent stem cells may be derived from the same subject. The immune cells and the pluripotent stem cells may be derived from the same human.
[0054]
[50] The immune cells according to the embodiment of
[49] may be included in the monocytes. The monocytes and the pluripotent stem cells may be derived from the same subject. The monocytes and the pluripotent stem cells may be derived from the same human.
[0055]
[51] The immune cells according to the embodiments described in
[49] or
[50] may be at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
[0056]
[52] In the immune cells according to the embodiment described in
[51] , the T cells may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0057]
[53] The immune cells according to any one of the embodiments described in
[49] to
[52] may be derived from at least one source selected from the group consisting of peripheral blood, umbilical cord blood, bone marrow, lymphatic fluid, and tissue fluid.
[0058]
[54] In the immune cells according to any one of the embodiments described in
[49] to
[53] , the pluripotent stem cells may be iPS cells.
[0059]
[55] In the immune cells according to any one of the embodiments described in
[49] to
[54] , at least a portion of the pluripotent stem cells may be a lysate of the pluripotent stem cells.
[0060]
[56] The immune cells according to any one of the embodiments described in
[49] to
[55] may react to multiple types of cancer-associated antigens.
[0061]
[57] The immune cells according to any one of the embodiments described in
[49] to
[56] may be at least one of CD69 positive, CD107 positive, and IFN-γ positive. Effect of the Invention
[0062] According to the present invention, it is possible to provide a method for producing immune cells that react to cancer-associated antigens. [Brief description of the drawings]
[0063] [Figure 1] 1 is a flow cytometer dot plot showing the results of Example 1. [Diagram 2] 1 is a flow cytometer dot plot showing the results of Example 1. [Diagram 3] 1 is a flow cytometer dot plot showing the results of Example 2. [Figure 4] 1 is a flow cytometer dot plot showing the results of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0064] Hereinafter, the embodiments of the present invention will be described in detail. Note that the following embodiments are merely examples for embodying the technical idea of the present invention, and the technical idea of the present invention is not limited to the combination of components described below. The technical idea of the present invention can be modified in various ways within the scope of the claims.
[0065] According to an embodiment, a method for producing immune cells reactive to cancer-associated antigens includes stimulating immune cells with an antigen contained in pluripotent stem cells or at least a portion of the pluripotent stem cells. The immune cells and the pluripotent stem cells are, for example, derived from the same subject. The immune cells and the pluripotent stem cells are, for example, derived from the same human individual. When the immune cells and the pluripotent stem cells are derived from different humans, the immune cells are stimulated mainly with non-self antigens and mainly acquire reactivity against non-self antigens. In contrast, when the immune cells and the pluripotent stem cells are derived from the same human, the immune cells mainly acquire reactivity to react with self cancer-associated antigens.
[0066] The immune cells may be derived from humans or non-human animals. The immune cells include, for example, mononuclear cells. Examples of immune cells include T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages. Examples of T cells include helper T cells, cytotoxic T cells, regulatory T cells, αβ T cells, and γδ T cells.
[0067] The immune cells are derived from, for example, blood, bone marrow, thymus, lymph, cavity, or tissue fluid. Examples of blood include, but are not limited to, peripheral blood and umbilical cord blood. Blood may be collected from adults or minors. Anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin, and biological product standard blood preservation solution A (ACD-A) may be used during blood collection. Examples of cavity fluid include ascites, pleural fluid, and pericardial fluid. Examples of tissue include tumor tissue.
[0068] Examples of pluripotent stem cells include iPS cells and ES cells. Pluripotent stem cells are undifferentiated cells that have the ability to differentiate into any cell, and express many types of undifferentiation-related antigens that are not expressed in terminally differentiated cells that constitute adults. Pluripotent stem cells also express immortalization-related antigens and cell proliferation-related antigens. In this disclosure, antigens include epitopes. Cancer cells and tumor cells express antigens that are different from normal cells, and antigens expressed in cancer cells and tumor cells are called tumor-associated antigens. Antigens contained in both undifferentiation-related antigens and tumor-associated antigens are called "cancer testis antigens" or "cancer fetal antigens". According to the findings of the present inventors, since pluripotent stem cells contain multiple types of cancer-associated antigens, it is possible to simultaneously generate reactivity against multiple types of cancer-associated antigens in immune cells by stimulating immune cells with at least a portion of the pluripotent stem cells or a lysate of the pluripotent stem cells. Cancer-associated antigens include, for example, cancer-specific antigens. Cancer-associated antigens include, for example, at least any of cancer testis antigens, cancer stem cell antigens, cancer undifferentiation antigens, cancer epithelial antigens, cancer proliferation-associated antigens, cancer immortalization antigens, cancer checkpoint inhibitor antigens, cancer invasion antigens, cancer surface antigens, cancer apoptosis inhibitor antigens, cancer epigenetic modeling antigens, cancer DNA repair antigens, cancer microRNA antigens, cancer nucleic acid antigens, cancer nuclear receptor antigens, cancer embryonic antigens, and cancer fetal antigens.
[0069] The pluripotent stem cells may be derived from humans. The pluripotent stem cells derived from humans may be at least one of TRA1-60 positive, TRA1-85 positive, NANOG positive, LIN28 positive, OCT positive, and Human Nuclear Antigen (HNA) positive. In addition, pluripotent stem cells include undifferentiated naive type and highly differentiated prime type. Mouse pluripotent stem cells are naive type, and human pluripotent stem cells are prime type. The pluripotent stem cells according to the present embodiment may be prime type. When culturing naive type pluripotent stem cells, leukemia inhibitory factor (LIF) is added to the medium. When culturing prime type pluripotent stem cells, basic fibroblast growth factor (bFGF) is added to the medium. The primed pluripotent stem cells may be at least one of CD24-positive, CD57-positive, CD90-positive, SSEA4-positive, HLA-A-positive, HLA-B-positive, and HLA-C-positive. The primed pluripotent stem cells may be at least one of SSEA1-negative, Stella-negative, and TFCP2L1-negative.
[0070] The antigen contained in the pluripotent stem cell or at least a part of the pluripotent stem cell may be, for example, a lysate of the pluripotent stem cell obtained by dissolving the pluripotent stem cell. The antigen contained in the pluripotent stem cell or at least a part of the pluripotent stem cell may be, for example, a lysate of the pluripotent stem cell obtained by disrupting the pluripotent stem cell. The lysate or lysate of the pluripotent stem cell may contain multiple types of undifferentiation-related antigens, immortalization-related antigens, and cell proliferation-related antigens. By stimulating immune cells with at least one of multiple undifferentiation-related antigens, immortalization-related antigens, and cell proliferation-related antigens contained in the pluripotent stem cell, immune cells that are reactive to multiple cancer antigens are activated. Examples of cancer antigens include cyclin B1, hyaluronan-mediated motility receptor, and heat shock protein 60.
[0071] The lysate of pluripotent stem cells can be obtained, for example, by repeatedly freezing and thawing the pluripotent stem cells. The density of the pluripotent stem cells in the solution containing the frozen pluripotent stem cells can be, for example, 0.5×10 4pcs / mL or more 5.0×10 6 pcs / mL or less, 1.0×10 4 pcs / mL or more 1×10 6 / mL or less, or 1.5 x 10 4 pieces / mL or more 5×10 5 The concentration is, but is not limited to, less than 1 / mL. Alternatively, the lysate of pluripotent stem cells can be obtained, for example, by dissolving the pluripotent stem cells with a chemical. The crushed product of pluripotent stem cells can be obtained, for example, by applying pressure to the pluripotent stem cells to physically crush the pluripotent stem cells, or by irradiating the pluripotent stem cells with ultrasound to crush the pluripotent stem cells.
[0072] The lysate of pluripotent stem cells may be, for example, an extract of pluripotent stem cells. The extract of pluripotent stem cells is prepared by the following method. Pluripotent stem cells are cultured on a matrigel coat or laminin coat using a culture medium. When the pluripotent stem cells become 80% or more confluent, the pluripotent stem cells are detached from the culture vessel using a release agent, and the solution containing the detached pluripotent stem cells is centrifuged to collect the pluripotent stem cells in a tube. Thereafter, the cell mass of the pluripotent stem cells is crushed or ground with, for example, a pestle such as a pellet pestle, an ultrasonic crusher, or a mortar, and the pluripotent stem cell extract containing the paste of the crushed or ground pluripotent stem cells is frozen with liquid nitrogen. The freezing temperature is, for example, -80°C, but is not particularly limited. The freezing may be instantaneous freezing. The pluripotent stem cells are further crushed by freezing, and the proteins contained in the pluripotent stem cells are released into the extract. The pluripotent stem cells may be disrupted by freezing without being crushed or mashed in advance. Alternatively, the pluripotent stem cells may be disrupted by repeatedly freezing and thawing without being crushed or mashed in advance. When using an extract of pluripotent stem cells, the extract of pluripotent stem cells is suspended in a culture medium and incubated overnight at 4°C, and the suspension is centrifuged the next day to remove cell debris from the solution. The solution is then filtered through a filter, and the solution that passes through the filter is used as an extract of pluripotent stem cells.
[0073] When stimulating immune cells, an antigen contained in the pluripotent stem cells or at least a part of the pluripotent stem cells may be added to a medium for culturing immune cells. The concentration of the pluripotent stem cell lysate in the solution containing the pluripotent stem cell lysate is, for example, 1% by mass to 50% by mass, 5% by mass to 40% by mass, or 10% by mass to 30% by mass, but is not particularly limited thereto. Immune cells may be cultured for 1 day or more, 3 days or more, 7 days or more, or 14 days or more in a medium to which an antigen contained in the pluripotent stem cells or at least a part of the pluripotent stem cells has been added. Examples of media for culturing immune cells include, but are not limited to, RPMI1640 medium, minimum essential medium (α-MEM), Dulbecco's modified Eagle medium (DMEM), and F12 medium.
[0074] When stimulating immune cells, a cytokine may be brought into contact with the immune cells to promote the proliferation of the immune cells. Examples of cytokines include interleukins. Examples of interleukins include IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, IL-21, and IL-33. The cytokine may be added to a medium in which the immune cells are cultured. The immune cells may be cultured in a medium containing an anti-cytokine for 1 day or more, 3 days or more, 7 days or more, or 14 days or more.
[0075] Immune cells stimulated with an antigen contained in pluripotent stem cells or at least a part of pluripotent stem cells can, for example, react to multiple types of cancer-associated antigens. Therefore, even if the cancer antigen mutates or the expression level changes in the cancer tissue, the immune effect is maintained better than that of immune cells reacting to a single cancer-associated antigen. In addition, the stimulated immune cells can be at least one of CD69 positive, CD107 positive, and IFN-γ positive.
[0076] Example 1 iPS cells were cultured, and when the colony size exceeded 2 mm or when the cells became 80% confluent, the culture medium was aspirated from the wells and the iPS cells were washed with PBS(-). The PBS(-) was removed from the wells, and TrypLE SELECT (ThermoFisher) was placed in the wells. The wells were incubated at 5% CO 2 The wells were then placed in a 37°C incubator for 5 minutes. TrypLE SELECT was then removed from the wells, and the iPS cells were washed with PBS(-). The PBS(-) was then removed from the wells, and a medium containing a ROCK inhibitor was placed in the wells, and the iPS cells were detached from the wells using a cell scraper.
[0077] The number of iPS cells was counted and found to be 2.5 x 10 5 The cell suspension containing iPS cells was placed in a 15 mL tube. The tube was centrifuged at 200 x g for 5 minutes, and the supernatant was aspirated. Culture medium was added to the tube, and the iPS cells were suspended by pipetting.
[0078] The iPS cells suspended in the medium without cell freezing solution were placed in a -80°C freezer for 1 hour to freeze the cell suspension and disrupt the cells. The cell viability was 1% or less. The cell suspension was then thawed at room temperature and mixed with a vortex mixer. The freezing and thawing process was repeated a total of four times to disrupt the iPS cells. The tube was then centrifuged at 300xg for 10 minutes, and the supernatant containing the iPS cell lysate was collected and placed in a 1.5mL tube. The supernatant was passed through a 0.22μm filter to remove cell debris from the supernatant, and placed in another 1.5mL tube as an iPS cell lysate solution.
[0079] RPMI1640 medium (ThermoFisher) containing 10% FBS, 1% GlutaMax, 1% non-essential amino acids (NEAA), 1% sodium pyruvate, 55 μmol / L mercaptoethanol, and 1% penicillin-streptomycin was prepared as the amplification medium.
[0080] Mononuclear cells derived from peripheral blood were placed in an expansion medium in a test tube. The iPS cells and peripheral blood were from the same human individual. Next, 20% iPS cell lysate solution, 1 μg / mL anti-CD28 antibody, and 100 U / mL IL-2 were placed in the expansion medium and incubated at 5% CO. 2 Mononuclear cell culture was started in a 37°C incubator, and immune cells that specifically react to antigens contained in iPS cells were activated, proliferated, and expanded in a test tube. On the third day after starting mononuclear cell culture, half of the medium was replaced with fresh expansion medium. On the seventh day after starting mononuclear cell culture, cells were harvested, placed in fresh expansion medium, and iPS cell lysate solution, anti-CD28 antibody, and IL-2 were placed in the expansion medium. On the tenth day after starting mononuclear cell culture, half of the medium was replaced with fresh expansion medium. On the 14th day after starting mononuclear cell culture, cells were harvested.
[0081] The mononuclear cells before being cultured in the expansion medium containing the iPS cell lysate solution were analyzed by flow cytometry. As shown in Figure 1, neither CD8-positive T cells nor CD4-positive T cells expressed the activation marker CD69. In contrast, as shown in Figure 2, the cells derived from the mononuclear cells cultured and expanded in the expansion medium containing the iPS cell lysate solution expressed the activation marker CD69 in both CD8-positive T cells and CD4-positive T cells, and included expanded and activated cells.
[0082] Example 2 The mononuclear cells cultured for 14 days in an expansion medium containing an iPS cell lysate solution were collected and washed with PBS in the same manner as in Example 1. As cancer antigen peptides, a peptide of cyclin B1, a peptide of hyaluronan-mediated motility receptor (HMMR), and a peptide of heat shock protein 60 (HSP60) were prepared, and each was added to the expansion medium at a concentration of 1 μmol / L. In addition, as a negative control, an expansion medium containing no cancer antigen peptide was prepared. The mononuclear cells were placed in each of the expansion media, and cultured in an incubator at 5% CO2 and 37°C.
[0083] After 5 hours, mononuclear cells were collected and stained using an IFN-γ secretion assay detection kit (Miltenyi Biotec, 130-054-202), anti-CD4 antibody, and anti-CD8 antibody, and analyzed by a flow cytometer. As shown in Figures 3 and 4, both CD8-positive T cells and CD4-positive T cells stimulated with cyclin B1 peptide, HMMR peptide, and HSP60 peptide were found to release IFN-γ, an activation marker. Both CD8-positive T cells and CD4-positive T cells in the negative control showed low release of IFN-γ, an activation marker.
Claims
1. A method for producing immune cells that respond to cancer-associated antigens, comprising stimulating immune cells with antigens contained in pluripotent stem cells.
2. The method of claim 1 , wherein the immune cells are mononuclear cells.
3. The method of claim 1, wherein the immune cell is at least one selected from the group consisting of T cells, B cells, memory B cells, plasma cells, NK cells, NKT cells, dendritic cells, monocytes, and macrophages.
4. The method of claim 1, further comprising lysing the pluripotent stem cells to obtain the antigen.
5. The method of claim 1 , wherein the antigen is a lysate of a pluripotent stem cell.
6. The method of claim 1 , wherein the stimulated immune cells respond to multiple cancer-associated antigens.
7. The method of claim 1, wherein the stimulated immune cells are at least one of CD69 positive, CD107 positive, and IFN-γ positive.
8. A method for producing immune cells that respond to cancer-associated antigens, comprising stimulating the immune cells with at least a portion of pluripotent stem cells.
9. The method of claim 8 , wherein the immune cells are mononuclear cells.
10. The method of claim 8 , wherein at least a portion of the pluripotent stem cells is a lysate of pluripotent stem cells.
11. The method of claim 8, wherein the stimulated immune cells respond to multiple cancer-associated antigens.
12. The method of claim 8, wherein the stimulated immune cells are at least one of CD69 positive, CD107 positive, and IFN-γ positive.
13. A method for amplifying an immune cell response to a cancer-associated antigen, comprising stimulating the immune cell with an antigen contained in pluripotent stem cells.
14. A method for amplifying an immune cell response to a cancer-associated antigen, comprising stimulating the immune cells with at least a portion of pluripotent stem cells.
15. A pharmaceutical composition for treating cancer, comprising immune cells stimulated with an antigen contained in pluripotent stem cells.
16. A pharmaceutical composition for treating cancer comprising immune cells stimulated with at least a portion of pluripotent stem cells.
17. Immune cells that react to cancer-associated antigens stimulated with antigens contained in pluripotent stem cells.
18. Immune cells stimulated with at least a portion of the pluripotent stem cells that respond to cancer-associated antigens.