Method for producing CD3-positive cells

Abstract

The present invention relates to a method for producing CD3-positive cells. In order to be able to stably supply differentiated T cells, the present invention provides a method for producing CD3-positive cells, a method for enlarge culture, and a kit for enlarge culture, in which CD3, which is a T cell marker, is expressed on a cell membrane.

Description

[0001] This application is a divisional application of a Chinese invention patent application filed on August 8, 2019, with application number 201980052874.2 (international application number PCT / JP2019 / 031390), and invention name “Method for producing CD3-positive cells”. Technical Field

[0002] The present invention relates to a method for producing or expanding CD3-positive cells (or CD3-positive CD197-positive cells), and a kit for expanding CD3-positive cells, wherein the method comprises the step of culturing CD3-positive cells in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist. The present invention also relates to a method for maintaining CD3-positive cells as CD3-positive CD197-positive cells, and a kit for maintaining CD3-positive cells as CD3-positive CD197-positive cells, wherein the method comprises the step of stimulating CD30 signals in CD3-positive cells. Background Art

[0003] It is known that immunotherapy has shown a strong effect of prolonging life for cancers that have not been effectively treated with previous treatments, and it is believed that it will become the main force in cancer treatment. In CART therapy, autologous T cells with anti-CD19 chimeric antigen receptor (CAR) genes are implanted, which show a high complete remission rate for B-cell malignancies. In 2017, two CART therapies targeting B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma were approved by the FDA. In addition, TCR-T cell therapy, which introduces T cell receptor (TCR) genes that recognize cancer cell antigens, is also undergoing a large number of clinical trials. These gene-modified T cell therapies using the patient's own (autologous) T cells have shown very excellent clinical results, but on the other hand, the complicated processes that take more than several weeks, including the delivery and gene modification of T cells, cell culture, etc., make it difficult to reduce costs and quality management, and will cause cancer patients with rapid disease progression to lose treatment opportunities. Therefore, there is a strong demand for the development of off-the-shelf (finished product) allogeneic T cell therapies. As a method for expanding and culturing T cells recovered from patients, a method for making CD8-positive Tc1 lymphocytes or CD8-positive Tc2 lymphocytes by contacting an anti-CD3 agonist antibody and an anti-CD30 agonist antibody with a T cell group (Patent Document 1) and a method for obtaining a T cell group from a cancer patient and expanding and culturing tumor-specific T cells by contacting an anti-CD3 agonist antibody, an anti-CD28 antibody and a VEGF inhibitor with the T cell group (Patent Document 2) have been reported. However, the proliferation capacity of T cells from healthy people or patients is limited, and the number of genetically modified T cells that can be produced from T cells that can be recovered from a single blood component apheresis is limited. In addition, it has been reported that in the case of expanding and culturing T cells using TCR stimulation, the expression of CCR7 (CD197), a cell surface marker for immature cells and memory T cells, temporarily increases, but then gradually disappears within a few days (Non-Patent Document 1). In addition, it is reported that maintaining memory T cells ("T cell persistence") is important for in vivo anti-tumor activity, and by increasing the number of CART cells administered as memory T cells, a higher in vivo anti-tumor effect was observed (Non-patent Document 2). Therefore, when the CD197 expression of the cell population obtained by expanding the culture of T cells is low, it is not suitable for cancer treatment.

[0004] On the other hand, T cells (differentiated T cells) differentiated from pluripotent stem cells such as iPS cells and ES cells use pluripotent stem cells that can theoretically proliferate indefinitely as raw materials, so it is theoretically possible to manufacture genetically modified T cells without restriction. However, the method of expanding the culture of iPS cells while maintaining pluripotency requires high costs and is also accompanied by technical difficulties. Therefore, there is a limit to the method of proliferating iPS cells in large quantities and obtaining differentiated T cells, especially CD197-positive T cells, from them. As a result, other approaches for obtaining T cells, especially CD197-positive T cells, in large quantities are required.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2003 / 038062

[0008] Patent Document 2: U.S. Publication No. 2014 / 0255368

[0009] Non-patent literature

[0010] Non-patent document 1: Sallusto et al., Eur. J. Immunol. vol. 29, 2037-2045, 1999

[0011] Non-patent document 2: Kaartinen et al., Cytotherapy vol. 19, 689-702, 2017 Summary of the invention

[0012] Problems to be solved by the invention

[0013] The object of the present invention is to provide a method for producing or expanding CD3-positive cells expressing CD3 as a T cell marker on the cell membrane, and a kit for expanding the culture, in order to stably supply differentiated T cells, especially CD197-positive T cells. Another object of the present invention is to provide a method for maintaining CD197-positive T cells and a kit for maintaining CD197-positive T cells.

[0014] Means for solving problems

[0015] The inventors of the present application have repeatedly conducted intensive research to achieve the above-mentioned purpose, and found that, in a culture container obtained by solidifying anti-CD3 agonist antibodies and RetroNectin (registered trademark), using a culture medium containing anti-CD30 agonist antibodies to culture CD8-positive T cells (CD8-positive T cells derived from iPS cells) obtained from iPS cells into which TCR genes have been introduced, the T cells can be efficiently proliferated. Moreover, it has been confirmed that even if the aforementioned CD8-positive T cells derived from iPS cells are repeatedly proliferated using this culture method, the proliferation efficiency of T cells and the antigen-specific cell damage activity will not be affected. In addition, it was found that when the aforementioned CD8-positive T cells derived from iPS cells were cultured using anti-CD30 agonist antibodies, the expression ratio of CD197 was high compared to the case where the anti-CD30 agonist antibodies were not used, and the proliferated T cells existed as central memory T cells. In addition, similarly to CD8 positive T cells derived from iPS cells, CD8 positive T cells derived from human peripheral blood and CD8 positive T cells derived from iPS cells into which anti-CD19-CAR genes have been introduced (anti-CD19-CART cells derived from iPS cells) can also be cultured on a culture vessel obtained by solidifying anti-CD3 agonist antibodies / RetroNectin (registered trademark), and efficiently proliferate by adding anti-CD30 agonist antibodies to the culture medium. In addition, it was found that when the aforementioned CD8 positive T cells derived from human peripheral blood or the aforementioned anti-CD19-CART cells derived from iPS cells were cultured using anti-CD30 agonist antibodies, compared with the case where anti-CD30 agonist antibodies were not used, the expression of CD197 was maintained for a long time, and the aforementioned CD8 positive T cells derived from human peripheral blood were also able to survive for a long time in vivo. Based on the above findings, the present invention has been completed.

[0016] That is, the present invention provides the following solutions.

[0017] [1] A method for producing CD3-positive cells, comprising the following step (I), wherein the CD3-positive cells are cultured in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist.

[0018] [2] The method as described in [1] further comprises the following step (II), wherein the CD3-positive cells cultured in step (I) are cultured in the absence of a CD3 / TCR complex agonist and fibronectin or a modified form thereof, and in the presence of a CD30 agonist.

[0019] [3] The method according to [1] or [2], wherein the CD3-positive cells are derived from pluripotent stem cells.

[0020] [4] The method according to [3], wherein the pluripotent stem cells are iPS cells.

[0021] [5] The method according to any one of [1] to [4], wherein the CD3-positive cells are CD3-positive cells expressing a chimeric antigen receptor.

[0022] [6] The method according to any one of [1] to [5], wherein the CD3-positive cells are CD3-positive CD8-positive cells.

[0023] [7] The method as described in [6], wherein the CD3-positive CD8-positive cells are CD3-positive CD8-positive CD4-negative cells.

[0024] [8] The method according to any one of [1] to [7], wherein the CD3-positive cells are γTCR-positive and / or δTCR-positive cells.

[0025] [9] The method according to any one of [1] to [8], wherein the CD3 / TCR complex agonist is a CD3 agonist and / or a TCR agonist.

[0026]

[10] The method of [9], wherein the CD3 agonist is an anti-CD3 agonist antibody or a binding fragment thereof.

[0027]

[11] The method of

[10] , wherein the anti-CD3 agonist antibody or its binding fragment is an anti-CD3 agonist antibody or its binding fragment produced by UCHT1 clone.

[0028]

[12] The method of [9], wherein the TCR agonist is at least one selected from the group consisting of an anti-TCR antibody or a binding fragment thereof, an HLA / peptide complex or a multimer thereof, and an HLA / superantigen complex or a multimer thereof.

[0029]

[13] The method of

[10] or

[11] , wherein the anti-CD3 agonist antibody or binding fragment thereof is immobilized on a culture container.

[0030]

[14] The method according to

[13] , wherein the anti-CD3 agonist antibody or its binding fragment is immobilized on the solid phase by contacting 1 ng / ml to 50,000 ng / ml of the anti-CD3 agonist antibody or its binding fragment with a culture container.

[0031]

[15] The method according to any one of [1] to

[14] , wherein the fibronectin or a modified form thereof is RetroNectin (registered trademark).

[0032]

[16] The method according to

[15] , wherein the RetroNectin (registered trademark) is immobilized in a culture container.

[0033]

[17] The method according to

[16] , wherein the RetroNectin (registered trademark) is immobilized by bringing 1 to 150 μg / mL of the RetroNectin (registered trademark) into contact with a culture container.

[0034]

[18] The method according to any one of [1] to

[17] , wherein the CD30 agonist is at least one selected from the group consisting of an anti-CD30 agonist antibody or a binding fragment thereof and a CD30 ligand or a binding fragment thereof.

[0035]

[19] The method of

[18] , wherein the anti-CD30 agonist antibody or binding fragment thereof is contained in a culture medium.

[0036]

[20] The method of

[19] , wherein the concentration of the anti-CD30 agonist antibody or its binding fragment in the culture medium is 1 ng / ml to 1000 ng / ml.

[0037]

[21] The method according to any one of [1] to

[20] , wherein the culture medium contains at least one selected from the group consisting of IL-7, IL-15, IL-18, and IL-21.

[0038]

[22] The method according to

[21] , wherein the culture medium contains IL-7, IL-15, IL-18 and IL-21.

[0039]

[23] The method according to

[21] or

[22] , wherein the culture medium further contains TL1A and / or IL-12.

[0040]

[24] The method according to any one of [1] to

[23] , wherein the produced CD3-positive cells are also CD197-positive.

[0041]

[25] CD3-positive cells obtained by the method according to any one of [1] to

[24] .

[0042]

[26] A method for expanding CD3-positive cells, comprising the step of culturing CD3-positive cells in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist.

[0043]

[27] A kit for expanding and culturing CD3-positive cells, comprising the following (1) and (2):

[0044] (1) A culture container in which a CD3 / TCR complex agonist and fibronectin or a modified form thereof are solid-phased;

[0045] (2) A culture medium containing a CD30 agonist.

[0046]

[28] A method for maintaining CD3-positive cells as CD3-positive CD197-positive cells, comprising the step of stimulating CD30 signals in CD3-positive cells.

[0047]

[29] The method as described in

[28] , wherein the CD3-positive cells are CD3-positive CD8-positive cells.

[0048]

[30] The method as described in

[29] , wherein the CD3-positive CD8-positive cells are CD3-positive CD8-positive CD4-negative cells.

[0049]

[31] The method according to any one of

[28] to

[30] , wherein the culture medium used in the stimulation step contains at least one selected from the group consisting of IL-7, IL-15, IL-18 and IL-21.

[0050]

[32] The method according to

[31] , wherein the culture medium used in the stimulation step contains IL-7, IL-15, IL-18 and IL-21.

[0051]

[33] The method of

[31] or

[32] , wherein the culture medium used in the stimulation step further contains TL1A and / or IL-12.

[0052]

[34] A kit for maintaining CD3-positive cells as CD3-positive CD197-positive cells, comprising a CD30 agonist.

[0053]

[35] A chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises the intracellular signal transduction domain of CD30 or a modified form thereof.

[0054]

[36] A nucleic acid comprising a polynucleotide encoding the chimeric antigen receptor described in

[35] .

[0055]

[37] A chimeric antigen receptor expression vector comprising the nucleic acid described in

[36] .

[0056]

[38] A chimeric antigen receptor expressing cell comprising the chimeric antigen receptor expression vector described in

[37] .

[0057]

[39] The cell according to

[38] , wherein the chimeric antigen receptor-expressing cell is a CD3-positive cell.

[0058]

[40] A medicine comprising the cell described in

[38] or

[39] .

[0059]

[41] The drug according to

[40] , which is used for the prevention or treatment of a tumor expressing an antigen recognized by the chimeric antigen receptor according to

[35] .

[0060]

[42] An agent for killing cells expressing an antigen recognized by the chimeric antigen receptor described in

[35] , comprising the cells described in

[38] or

[39] .

[0061]

[43] The cell of

[38] or

[39] , which is used for the prevention or treatment of a tumor expressing an antigen recognized by the chimeric antigen receptor of

[35] .

[0062]

[44] Use of the cell described in

[38] or

[39] for producing a preventive or therapeutic agent for a tumor expressing an antigen recognized by the chimeric antigen receptor described in

[35] .

[0063]

[45] A method for preventing or treating a tumor expressing an antigen recognized by the chimeric antigen receptor of

[35] , comprising administering the cell of

[38] or

[39] .

[0064] Effects of the Invention

[0065] By culturing CD3 positive cells in the presence of CD3 / TCR complex agonists, fibronectin or its modified body, and CD30 agonists, T cells can be efficiently manufactured or expanded. In addition, the CD3 positive cells obtained by the above-mentioned culture manufacturing or expanded culture are CD197 positive cells, and therefore, when used for gene-modified T cell therapy, long-term drug efficacy can be expected. In addition, by stimulating the CD30 signal of CD3 positive cells, the cells can be maintained as CD197 positive cells for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] [ Figure 1 ] is a graph showing the concentrations of anti-CD3 agonist antibody and RetroNectin (registered trademark) suitable for solid-phase binding, measured using the ELISA method.

[0067] [ Figure 2 ] is a graph showing the number of iPSC-derived T cells (measured based on ATP amount) on day 12 after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0068] [ Figure 3] is a graph showing the proliferation curve of iPSC-derived T cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) or anti-CD3 / CD28 beads. The vertical axis represents the number of cells, and the horizontal axis represents the number of days from the start of the above-mentioned stimulation.

[0069] [ Figure 4 ] is a graph showing the proliferation curve of iPSC-derived T cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) in each stimulation.

[0070] [ Figure 5 ] is a graph showing the antigen-specific cell-damaging activity of iPSC-derived T cells that proliferated due to stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0071] [ Figure 6 ] is a graph showing the hyperproliferation of iPSC-derived T cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) due to the addition of anti-CD30 agonist antibody. The vertical axis represents the number of cells, and the horizontal axis represents the number of days from the start of the above-mentioned stimulation.

[0072] [ Figure 7 ] is a graph showing the proliferation curve of iPSC-derived T cells when stimulation (second stimulation) is performed again in the same manner after the proliferation test of iPSC-derived T cells using the first stimulation using solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) or solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody is completed. The vertical axis represents the number of cells, and the horizontal axis represents the number of days that have passed since the start of the second stimulation.

[0073] [ Figure 8 ] is a graph showing the expression of CD197 and CD45RA on the membrane surface of iPSC-derived T cells on the 7th day after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark), or solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody.

[0074] [ Fig. 9 ] is a graph showing the antigen-specific cell-damaging activity of iPSC-derived T cells that proliferated due to stimulation from solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody.

[0075] [ Fig.10] is a graph showing the proliferation curve of CD8-positive T cells derived from human peripheral blood stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark), or solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody. The vertical axis represents the number of cells, and the horizontal axis represents the number of days from the start of the above-mentioned stimulation.

[0076] [ Fig.11 ] is a graph showing the expression of CD197 on the membrane surface of CD8-positive T cells derived from human peripheral blood after stimulation with various ILs and anti-CD3 / CD28 beads containing anti-CD30 antibodies.

[0077] [ Fig.12 ] is a graph showing the number of human CD8-positive T cells surviving in the blood, spleen, and bone marrow of irradiated immunodeficient mice 4 weeks after intravenous transplantation of CD8-positive T cells derived from human peripheral blood stimulated with various ILs and anti-CD3 / CD28 beads containing anti-CD30 antibodies.

[0078] [ Fig.13 ] is a graph showing the proliferation curve of iPS cell-derived T cells (iPS-T) and iPS cell-derived anti-CD19-CART cells (iPS-CART anti-CD19) stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody. The vertical axis represents the number of cells, and the horizontal axis represents the number of days since the start of the above-mentioned stimulation.

[0079] [ Fig.14 ] is a graph showing the proliferation curve of anti-CD19-CART cells derived from iPS cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark), or solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody. The vertical axis represents the number of cells, and the horizontal axis represents the number of days since the start of the above-mentioned stimulation.

[0080] [ Fig.15 ] is a diagram showing the expression of CD197 on the surface of the anti-CD19-CART cell membrane derived from iPS cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark), or solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody.

[0081] [ Fig.16] is a graph showing the cell damage activity of anti-CD19-CART cells derived from iPS cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) against CD19-expressing Raji cells due to the addition of anti-CD30 agonist antibody.

[0082] [ Fig.17 ] is a graph showing the cell damaging activity of anti-CD19-CART cells (iCD19-CD30-CART) derived from iPS cells containing the intracellular domain from CD30 against CD19-expressing Raji cells.

[0083] [ Fig.18 ] is a graph showing the cell proliferation of γδT cells (iγδT cells) differentiated from iPS cells not derived from T cells (iγδT cells) stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) due to the addition of anti-CD30 agonist antibody. The vertical axis represents the cell proliferation multiple, and the horizontal axis represents the number of days from the start of the above-mentioned stimulation.

[0084] [ Fig.19 ] is a graph showing cell proliferation of anti-CD19-CARγδT cells (iCARγδT cells) stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) due to the addition of anti-CD30 agonist antibody. The vertical axis represents the number of cells, and the horizontal axis represents the number of days since the start of the above-mentioned stimulation.

[0085] [ Fig. 20 ] is a graph showing the antigen-specific cell-damaging activity of iCD19CAR / IL-15γδT cells that proliferated due to stimulation from solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0086] [ Fig.21 ] is a graph showing the effect of prolonging the survival days of tumor-bearing mice expressing human CD19, brought about by iCD19CAR / IL-15γδT cells proliferated due to stimulation from solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0087] [ Fig. 22 ] is a diagram showing the anti-tumor effect of iCD19CAR / IL-15αβT cells proliferated due to stimulation from solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0088] [ Fig.23] is a graph showing the concentrations suitable for solid-phase binding of anti-CD3 agonist antibody (UCHT1) and RetroNectin (registered trademark), measured using the ELISA method.

[0089] [ Fig.24 ] is a graph showing the proliferation rate of iPSC-derived T cells on day 13 after stimulation with solid-phase anti-CD3 agonist antibody (UCHT1) / RetroNectin (registered trademark) and anti-CD30 agonist antibody. DETAILED DESCRIPTION

[0090] In this specification, "gene expression" includes both the synthesis of mRNA from the specific nucleotide sequence of the gene (also called transcription or mRNA expression) and the synthesis of protein based on the information of the mRNA (also called translation or protein expression), but unless otherwise specified, "gene expression" or simply "expression" refers to protein expression.

[0091] In this specification, the so-called "positive" means that the protein or mRNA is expressed in an amount that can be detected using methods known in the art. The detection of protein can be performed using immunological analysis using antibodies, such as ELISA, immunostaining, Western blotting, and flow cytometry. In addition, a reporter protein can be expressed together with the protein, and the protein as the object can be detected by detecting the reporter protein. In addition, the presence of the protein can be detected by detecting the function of the target protein (for example, when the target protein is a transcription factor, the gene whose expression is controlled by the protein is detected; when the target protein is an enzyme, the substrate or product catalyzed by the protein is detected; etc.). The detection of mRNA can be implemented using nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0092] In this specification, "negative" means that the expression level of protein or mRNA is less than the detection limit of all or any of the known methods described above. The detection limit of protein or mRNA expression may vary depending on the method.

[0093] In this specification, "culture" means maintaining cells in an in vitro environment to proliferate (grow) and / or differentiate. "Cultivating" means maintaining cells outside a tissue or in vitro, such as in a cell culture dish or culture flask, to proliferate (grow) and / or differentiate.

[0094] In this specification, so-called " expanded culture ", expression is to cultivate for the purpose that desired cell group is propagated, cell quantity is increased.For the increase of cell quantity, as long as the quantity that increases due to cell proliferation exceeds the quantity that reduces due to death to reach, without the whole cell proliferation of cell group.The increase of cell quantity can be 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 300 times, 500 times, 1,000 times, 3,000 times, 5,000 times, 10,000 times, 100,000 times, 1,000,000 times or more relative to before starting expanded culture.

[0095] In this specification, the term "stimulation" means that a certain substance binds to various receptors and activates the downstream signal pathway.

[0096] In this specification, the so-called "enrichment" refers to increasing the amount of a specific constituent in a composition such as a composition of a cell, and the so-called "enriched" refers to a cell group in which the amount of a specific constituent is increased compared to the proportion of the constituent in the cell group before enrichment when used to illustrate a composition of a cell, such as a cell group. For example, a composition such as a cell group can be enriched according to a target cell type, so that the proportion of the target cell type is increased compared to the proportion of the target cells present in the cell group before enrichment. The cell group can also be enriched for the target cell type using cell screening and sorting methods known in the art. The cell group can also be enriched using a specific sorting or screening process described in this specification. In a specific embodiment of the present invention, by a method for enriching a target cell group, the cell group can be enriched in a ratio of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% according to the target cell group.

[0097] In the present specification, the term "cell group" refers to two or more cells of the same type or different types. The term "cell group" also refers to a mass of cells of the same type or different types.

[0098] The present invention provides a method for producing or expanding CD3-positive cells (hereinafter referred to as the producing method or expanding method of the present invention), comprising the step of culturing CD3-positive cells in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist.

[0099] CD3 / TCR complex and CD30 can be stimulated by using CD3 / TCR complex agonist and CD30 agonist, respectively.

[0100] In the manufacture method of the present invention or the method for expanding culture, the CD3 positive cells cultured are not particularly limited as long as they are cells expressing CD3 on the cell membrane. The aforementioned CD3 positive cells are preferably CD3 positive CD8 positive cells (CD3 positive CD8 positive CD4 positive cells or CD3 positive CD8 positive CD4 negative cells), more preferably CD3 positive CD8 positive CD4 negative cells. In addition, as other preferred aforementioned CD3 positive cells, CD3 positive CD4 positive cells (CD3 positive CD4 positive CD8 positive cells or CD3 positive CD4 positive CD8 negative cells) can be enumerated.

[0101] In addition, in the manufacturing method or the expanded culture method of the present invention, the cultured CD3 positive cells may be CD30 positive before culture, or may be differentiated into CD30 positive during culture. Therefore, in the case where the aforementioned CD3 positive cells are CD30 positive before culture, the aforementioned CD3 positive cells are preferably CD3 positive CD8 positive CD30 positive cells (CD3 positive CD8 positive CD4 positive CD30 positive cells or CD3 positive CD8 positive CD4 negative CD30 positive cells), more preferably CD3 positive CD8 positive CD4 negative CD30 positive cells. In addition, as other preferred aforementioned CD3 positive cells, CD3 positive CD4 positive CD30 positive cells (CD3 positive CD4 positive CD8 positive CD30 positive cells or CD3 positive CD4 positive CD8 negative CD30 positive cells) can be cited.

[0102] In the manufacture method of the present invention or the expanded culture method, as long as the CD3 positive cells obtained as the result of manufacture or expanded culture are cells expressing CD3 on cell membrane, there is no particular limitation, and the same cells as the CD3 positive cells cultivated in the manufacture method of the present invention or the expanded culture method can be enumerated. In addition, the CD3 positive cells obtained by the aforementioned manufacture or expanded culture are preferably still CD197 positive. In the case where the CD3 positive cells obtained by the aforementioned manufacture or expanded culture are still CD197 positive, the cell is specifically CD3 positive CD8 positive CD197 positive cells (CD3 positive CD8 positive CD4 positive CD197 positive cells or CD3 positive CD8 positive CD4 negative CD197 positive cells), more preferably CD3 positive CD8 positive CD4 negative CD197 positive cells. Or, the cell is CD3 positive CD4 positive CD30 positive CD197 positive cells (CD3 positive CD4 positive CD8 positive CD30 positive CD197 positive cells or CD3 positive CD4 positive CD8 negative CD30 positive CD197 positive cells). Among CD3-positive cells, CD197-positive cells are classified as stem cell-like memory T cells or central memory T cells according to the presence or absence of CD45RA expression. They are T cells with high autonomous proliferation ability that can supply effector T cells and are therefore suitable for anti-tumor immunity.

[0103] The CD3 positive cells obtained by the aforementioned manufacture or expanded culture are preferably CD197 positive CD45RA negative. In the case where the CD3 positive cells obtained by the aforementioned manufacture or expanded culture are still CD197 positive CD45RA negative, the cell is specifically CD3 positive CD8 positive CD197 positive CD45RA negative cell (CD3 positive CD8 positive CD4 positive CD197 positive CD45RA negative cell or CD3 positive CD8 positive CD4 negative CD197 positive CD45RA negative cell), more preferably CD3 positive CD8 positive CD4 negative CD197 positive CD45RA negative cell. Or, the cell is CD3 positive CD4 positive CD30 positive CD197 positive CD45RA negative cell (CD3 positive CD4 positive CD8 positive CD30 positive CD197 positive CD45RA negative cell or CD3 positive CD4 positive CD8 negative CD30 positive CD197 positive CD45RA negative cell). Among the CD3 positive cells, CD197 positive CD45RA negative cells are also particularly referred to as central memory T cells.

[0104] CD3 is a molecule that is expressed in the maturation process of T cells and forms a larger complex (CD3 / TCR complex) with T cell receptor (T cell receptor, TCR). It is also known as a T cell marker and is a complex of 4 polypeptides of γ, δ, ε, and ζ chains. In addition, the TCR that forms a complex with CD3 is a molecule that transmits signals into T cells, and a dimer composed of two of α chain (TCRα), β chain (TCRβ), γ chain (TCRγ) and δ chain (TCRδ) can be cited, and a heterodimer (αβTCR) composed of TCRα and TCRβ, a heterodimer (γδTCR) composed of TCRγ and TCRδ, and a homodimer composed of the same TCR chain can be cited. In vivo, CD8 is expressed in the maturation process of T cells in the same manner as CD3, and is co-expressed with CD4 in the early stages of maturation, but with differentiation to cytotoxic T cells, only the expression of CD4 is lost. CD8 functions as a co-receptor of the CD3 / TCR complex, is a molecule that recognizes MHC class I / antigen peptides, and is a homodimer composed of α chain polypeptides or a heterodimer of α and β chains. In vivo, CD4 is co-expressed with CD8 in the early stages of T cell maturation, but with differentiation into helper T cells, only CD8 expression is lost. Like CD8, CD4 also functions as a co-receptor of the CD3 / TCR complex, but unlike CD8, it is a molecule that recognizes MHC class II / antigen peptides. CD30 is known as a molecule expressed in activated lymphocytes (T cells and B cells), with 6 cysteine-rich pseudo-repeat motifs as extracellular domains, and as an intracellular domain, it has a TNF receptor-associated factor (TRAF) binding sequence that stimulates NFκB signals. CD197 and CD45RA are used as marker molecules to distinguish naive T cells or stem cell-like memory T cells (CD197 positive, CD45RA positive), central memory T cells (CD197 positive, CD45RA negative), effector memory T cells (CD197 negative, CD45RA negative), and effector T cells (CD197 negative, CD45RA positive) among T cells.

[0105] In the production method or expansion culture method of the present invention, the CD3-positive cells to be cultured may be cells collected from a mammal or cells differentiated from pluripotent stem cells, and are preferably cells differentiated from pluripotent stem cells.

[0106] When the CD3-positive cells are cells collected from a mammal, the cells can be separated and recovered, for example, by using an anti-CD3 antibody column, density gradient centrifugation, etc., after peripheral blood mononuclear cells (PBMC) are collected from the mammal by apheresis. Examples of mammals for recovering CD3-positive cells include humans, or animals other than humans (e.g., dogs, cats, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, monkeys, etc.), preferably humans.

[0107] When the CD3-positive cells are cells obtained by differentiating pluripotent stem cells, examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), and embryonic germ stem cells (EG cells), preferably ES cells or iPS cells (more preferably human iPS cells).

[0108] When the pluripotent stem cells are ES cells, they can be prepared by methods known per se. As methods for preparing ES cells, for example, methods for culturing the inner cell mass in the blastocyst stage of mammals (for example, see Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)), methods for culturing early embryos prepared by somatic cell nuclear transplantation (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Irigaya et al., Protein Nuclease, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Rideout III et al., Nature Genetics, 24, 109 (2000)), etc., but not limited to these. In addition, ES cells can be obtained from a predetermined institution or purchased commercially. For example, with respect to human ES cell lines, H1, H7, H9, H13 and H14 can be obtained from WiCell Research Institute in the United States, HES1 to 6 can be obtained from ES Cell International in Australia, SA002, SA181 and SA611 can be obtained from Cellartis AB in Sweden, HUES1 to 17 can be obtained from HUES Cell Facility in the United States, KhES-1 to KhES-5 can be obtained from the Institute of Regenerative Medicine, Kyoto University, and SEES1 to SEES7 can be obtained from the National Center for Developmental Medicine. When ES cells are produced by somatic cell nuclear transfer, the type of somatic cells and the source of the collected somatic cells are based on the case of producing iPS cells described below.

[0109] When the pluripotent stem cells are iPS cells, iPS cells can be produced by introducing a nuclear reprogramming substance into somatic cells. Somatic cells that can be used as starting materials for producing iPS cells may be any cells other than germ cells from mammals (eg, mice or humans). For example, keratinized epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the surface layer of the tongue), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), cells for metabolism / storage (e.g., hepatocytes), luminal epithelial cells constituting the boundary surface (e.g., type I alveolar cells), luminal epithelial cells of the intimal tube (e.g., vascular endothelial cells), cells having cilia with transport capabilities (e.g., tracheal epithelial cells), cells for extracellular matrix secretion (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), cells of the blood and immune system (e.g., T lymphocytes), cells related to sensation (e.g., rod photoreceptors), neurons of the autonomic nervous system (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., satellite cells), neurons and glial cells (e.g., astrocytes) of the central nervous system, pigment cells (e.g., retinal pigment epithelial cells), and their progenitor cells (tissue progenitor cells), etc. can be cited. The degree of cell differentiation is not particularly limited, and both undifferentiated progenitor cells (including adult stem cells) and terminally differentiated mature cells can be used as the source of somatic cells in the present invention. Here, as undifferentiated progenitor cells, for example, tissue stem cells (adult stem cells) such as adipose-derived stromal (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells can be cited.

[0110] Examples of nuclear reprogramming substances introduced into somatic cells for the purpose of producing iPS cells include various combinations of reprogramming genes reported so far (for example, see WO 2007 / 069666, Nature Biotechnology, 26, 101-106 (2008), Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007), Nat. Cell Biol., 11, 197-203 (2009), Nature, 451, 141-146 (2008), Science, 318, 1917-1920 (2007), Stem Cells, 26, 1998-2005 (2008), Cell Research (2008) 600-603, Nature 454: 646-650 (2008), Cell ... Stem Cells, 26, 1998-2005 (2008), Cell Stem Cells, 26, 1998-2005 (2008), Cell Stem Cells, 2 Cell, 2: 525-528 (2008), WO2008 / 118820, Nat. Cell Biol., 11, 197-203 (2009), Nat. Cell Biol., 11, 197-203 (2009), Science, 324: 797-801 (2009)). In addition, the protein encoded by the above-mentioned reprogramming gene can also be introduced into somatic cells as a nuclear reprogramming substance (Cell Stem Cell, 4: 381-384 (2009), Cell Stem Cell, doi: 10.1016 / j.stem.2009.05.005 (2009)). In particular, when considering the use of the obtained iPS cells for therapeutic purposes, the combination of the three factors Oct3 / 4, Sox2 and Klf4 is preferred.

[0111] Screening of iPS cell colonies can be performed by methods using drug resistance and reporter gene activity as indicators (Cell, 126, 663-676 (2006), Nature, 448, 313-317 (2007)) and by visually observing morphology (Cell, 131, 861-872 (2007)). Expression of various ES cell-specific genes and teratoma formation can be used as indicators to confirm that they are iPS cells.

[0112] At present, there are various iPS cells (iPSCs). In addition to iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPSCs derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131: 861-872.), Nanog-iPSCs established by sorting using Nanog expression as an index after the above four factors were introduced (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs produced by a method without c-Myc (Nakagawa M, Yamanaka S. et al., Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing 6 factors by a virus-free method (Okita K et al., Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al., Stem Cells. 31 (3): 458-66.), etc. In addition, iPSCs established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), iPSCs prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and iPSCs prepared by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007), etc. can also be used.

[0113] In addition, all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or any iPSC known in the art described in patents (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852).

[0114] As artificial pluripotent stem cell lines, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, in the case of human iPSC lines, HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, Nips-B2, etc. of RIKEN, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A31, FfI-01s04, etc. of Kyoto University can be cited, and 1231A3 is preferred.

[0115] In the case where CD3 positive cells are cells obtained by differentiating pluripotent stem cells, the TCR expressed in CD3 positive cells may include a dimer composed of two of TCRα, TCRβ, TCRγ and TCRδ, a heterodimer composed of TCRα and TCRβ (αβTCR), a heterodimer composed of TCRγ and TCRδ (γδTCR), and a homodimer composed of the same TCR chain. These TCRs may be endogenous dimers, exogenous dimers, and preferably dimers comprising exogenous TCRα and exogenous TCRβ.

[0116] In this specification, the so-called "exogenous TCR" refers to a heterodimer composed of exogenous TCRα and exogenous TCRβ, a heterodimer composed of exogenous TCRγ and exogenous TCRδ, and / or a homodimer composed of the same exogenous TCR chain.

[0117] In the case where TCR is a dimer comprising endogenous TCRα and endogenous TCRβ, pluripotent stem cells can be pluripotent stem cells obtained from cells containing nucleic acids containing base sequences encoding endogenous TCRα and nucleic acids containing base sequences encoding endogenous TCRβ. As such cells, for example, T cells (CD8 positive CD4 negative cells, CD8 negative CD4 positive cells, CD4 positive CD8 positive cells, etc.) can be cited. In addition, in the case where TCR is a dimer comprising exogenous TCRα and exogenous TCRβ, pluripotent stem cells can be cells containing nucleic acids containing base sequences encoding exogenous TCRα and nucleic acids containing base sequences encoding exogenous TCRβ.

[0118] In the present specification, the so-called "exogenous TCR nucleic acid" refers to a nucleic acid containing a base sequence encoding an exogenous TCRα, a nucleic acid containing a base sequence encoding an exogenous TCRβ, a nucleic acid containing a base sequence encoding an exogenous TCRγ, and / or a nucleic acid containing a base sequence encoding an exogenous TCRδ.

[0119] The method for introducing exogenous TCR nucleic acid (for example, nucleic acid containing a base sequence encoding exogenous TCRα and nucleic acid containing a base sequence encoding exogenous TCRβ) into pluripotent stem cells is not particularly limited, and for example, the following method can be used.

[0120] In the case where the exogenous TCR nucleic acid is in the form of DNA, for example, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into pluripotent stem cells using methods such as liposome transfection, liposomes, and microinjection. As viral vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai virus vectors, etc. can be exemplified. In addition, as artificial chromosome vectors, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. are included. As plasmids, mammalian cell plasmids can be used. The vector can contain control sequences such as promoters, enhancers, ribosome binding sequences, terminators, and polyadenylation sites in a manner that can express exogenous TCR. In addition, it can contain drug resistance genes (such as kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, diphtheria toxin gene screening marker sequences, fluorescent proteins, beta glucuronidase (GUS), reporter gene sequences such as FLAG, etc. as needed. As a promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-α promoter, CAG promoter and drug responsive promoter can be exemplified. The so-called drug responsive promoter can be exemplified by TRE promoter (a minimal CMV promoter with a Tet response sequence obtained by tetO sequence 7 times in a row) that makes gene expression in the presence of a corresponding drug. In the case of using a TRE promoter, it is preferably in the following form: by expressing the fusion protein formed by antisense tetR (rtetR) and VP16AD in the same cell simultaneously, gene expression is induced in the presence of a corresponding drug (for example, tetracycline or doxycycline can be exemplified). More preferably, the vector has both a TRE promoter and a fusion gene expressing rtetR and VP16AD in the same vector.

[0121] In the present invention, in order to express exogenous TCRα and exogenous TCRβ simultaneously, a polycistronic expression method may be adopted. For polycistronic expression, the coding gene sequences may be linked via IRES or foot-and-mouth disease virus (FMDV) 2A coding region.

[0122] As another embodiment, in the above-mentioned vector, in order to remove the sequence encoding exogenous TCR taken into the chromosome as needed, a transposon sequence may be provided before and after the expression cassette (a gene expression unit comprising a promoter, a gene sequence, and a terminator). As a transposon sequence, there is no particular limitation, and piggyBac can be exemplified. As another embodiment, based on the purpose of removing the expression cassette, a loxP sequence or a FRT sequence may be provided before and after the expression cassette.

[0123] When the exogenous TCR nucleic acid is in the form of RNA, it can be introduced into pluripotent stem cells by methods such as electroporation, liposome transfection, and microinjection.

[0124] In the production method or expansion culture method of the present invention, the cultured CD3-positive cells may also be cells expressing the chimeric antigen receptor.

[0125] In the present invention, "chimeric antigen receptor (CAR)" means a fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen binding domain of CAR includes a short-chain antibody (scFv) in which the light chain (VL) and the heavy chain (VH) of the variable region of the antibody are connected in series via a joint (eg, GS joint). After the CD3-positive cells expressing CAR recognize the antigen with the scFv region, the recognition signal is transmitted to the cell through the intracellular signal transduction domain. By introducing CAR into CD3-positive cells, specificity for the target antigen can be conferred. In addition, CAR can directly recognize antigen molecules without relying on HLA class I or class II, and therefore, even cells with reduced expression of HLA class I or class II genes can cause a high level of immune response.

[0126] As the antigen targeted by the above-mentioned CAR, for example, a tumor antigen can be mentioned, but it is not limited thereto. The tumor antigen can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). Specifically, the tumor antigen includes differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, tumor-specific multilineage antigens such as WT1, Glypican-3, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, embryonic antigens such as CEA, overexpressed tumor genes or mutated tumor suppressor genes such as p53, Ras, HER-2 / neu, intrinsic tumor antigens caused by chromosomal translocation such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. One or more antigens can be mentioned. Other tumor antigens include CD19, CD20, EGP2, erB2, 3, 4, GD2, GD3, Mesothelin, PSMA, 8H9, Lewis-Y, MUC1, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS, but are not limited to these.

[0127] As the transmembrane domain of CAR, for example, a transmembrane domain of a protein selected from the group consisting of α chain, β chain or ζ chain, CD28, CD3ε chain, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB (CD137) and CD154 of TCR can be cited, wherein the transmembrane domain from CD8 can be preferably cited. In addition, it is also preferred to use a transmembrane domain, for example, from the following molecules, which are the source molecules of the first intracellular signal transduction domain connected to the antigen binding domain. For example, when the source molecule of the first intracellular signal transduction domain connected to the antigen binding domain is CD28, the transmembrane domain may also be from CD28. Alternatively, an artificially designed transmembrane domain can be used.

[0128] As the intracellular signal transduction domain of CAR, for example, an intracellular domain of one or more proteins selected from the group consisting of FcRγ chain, FcRβ chain, CD3γ chain, CD3δ chain, CD3ε chain, CD3ζ chain, CD5, CD22, CD79a, CD79b and CD66d can be cited, but it is not limited to these. Among them, the intracellular domain from the CD3ζ chain is preferred. After the tyrosine phosphorylation of ITAM contained in the intracellular domain of the CD3ζ chain is triggered, intracellular Ca 2+ A series of responses such as the rise of concentration, cytokine secretion, and finally cell division occurs, so that CD3 positive cells show cell damage activity as CTL. In addition, the intracellular domain of the costimulatory molecule may also be included in the intracellular signal transduction domain, as the costimulatory molecule, for example, one or more intracellular domains of proteins selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83 can be cited, but not limited to these. Wherein, preferably from the intracellular domain of CD28, 4-1BB (CD137), CD30. CD28 can promote the proliferation of T cells by increasing the IL-2 produced. In addition, 4-1BB can promote differentiation to memory cells by suppressing apoptosis caused in the final stage of T cell activation. CD30 can further increase the proliferation of CD3 positive cells through CD3 antibodies, and can maintain them as CD197 positive cells for a long time. By selecting the type and quantity of costimulatory molecules, the intensity and duration of the activity of CAR can be controlled (for example, Mol Ther. 2009; 17: 1453-1464.). In the case where more than one intracellular domain is included in the intracellular signal transduction domain, their order is not limited.

[0129] A spacer can be incorporated between the antigen binding domain and the transmembrane domain of CAR, or between the intracellular signal transduction domain and the transmembrane domain of CAR. As the spacer, a peptide consisting of usually 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids can be used. Specifically, peptides including the hinge region from IgG1, the CH2CH3 region of immunoglobulin, and a portion of CD3 can be cited, but are not limited to these.

[0130] As specific CARs, there can be cited a first generation CAR in which scFv and CD3ζ chain are linked via a spacer sequence, a second generation CAR in which the transmembrane domain and intracellular domain from CD28 are incorporated between the scFv and CD3ζ chain of the first generation CAR in order to enhance the activation ability against T cells, and a third generation CAR in which the intracellular domain of a co-stimulatory molecule (4-1BB or OX40) different from CD28 is incorporated between the intracellular domain of CD28 and the CD3ζ chain of the second generation CAR, but the present invention is not limited to these.

[0131] In the manufacturing method or expansion culture method of the present invention, the cultured CD3 positive cells can be cells that also co-express CAR and a fusion protein (IL-15 / IL-15Rα) formed by IL-15 and IL-15Rα. The CAR co-expressed with IL-15 / IL-15Rα can be the same as the above-mentioned CAR.

[0132] In the present invention, the so-called "IL-15 / IL-15Rα" means a fusion protein comprising IL-15 and IL-15Rα. In the signal transduction system using IL-15, usually, IL-15Rα expressed on antigen presenting cells binds to IL-15 and presents IL-15 to the IL-15 receptor composed of Rβ and γc on CD8 positive CD4 negative cells (trans-presentation), thereby maintaining the cell damage activity of CD8 positive CD4 negative cells. Therefore, for CD3 positive cells expressing IL-15 / IL-15Rα, when the cells are CD8 positive CD4 negative, they can transmit IL-15 signals to their own cells via the IL-15 receptor. Alternatively, CD3 positive cells expressing IL-15 / IL-15Rα can transmit IL-15 signals to other CD8 positive CD4 negative cells via the IL-15 receptor. As described above, IL-15 / IL-15Rα can maintain the cell-damaging activity of CD8-positive CD4-negative cells, and therefore, a sustained cell-damaging effect on cells that are CAR targets can be expected.

[0133] IL-15 / IL-15Rα can be a transmembrane protein or a secretory protein. As for IL-15Rα, it is known that the IL-15 binding domain of the 1st to 65th amino acids from the N-terminus of the mature protein is the dominant region for binding to IL-15 (Wei X. et al., J. Immunol., 167: 277-282, 2001). Therefore, a transmembrane protein can be a protein that retains the IL-15 binding domain and the transmembrane domain of IL-15Rα. On the other hand, as a secretory protein, it can be a protein that retains the IL-15 binding domain and lacks the transmembrane domain of IL-15Rα.

[0134] For IL-15 / IL-15Rα, a spacer sequence may be incorporated between IL-15 and IL-15Rα, and as the spacer sequence, a peptide consisting of usually 300 amino acids or less, preferably 10 to 100 amino acids, and most preferably 20 to 50 amino acids may be used. Specifically, GS linkers may be mentioned, but are not limited to these.

[0135] As IL-15 / IL-15Rα, there is no particular limitation as long as it is a peptide in which IL-15 and IL-15Rα are bound via a spacer sequence. Specifically, a peptide comprising SEQ ID NO: 8 can be cited. Alternatively, as IL-15 / IL-15R, there is no limitation as long as it can bind to the IL-15 receptor and transmit the IL-15 signal into the cell. For example, a peptide comprising an amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more homology to the amino acid sequence shown in SEQ ID NO: 8 can be cited. The term "homology" as used herein means the ratio (%) of identical amino acids and similar amino acid residues to all overlapping amino acid residues in the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can consider introducing gaps into one or both sequences in order to achieve the optimal alignment). The term "similar amino acids" refers to amino acids that are similar in physicochemical properties, and examples thereof include aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr), amino acids with a small side chain (Gly, Ala, Ser, Thr, Met), etc., which are classified as the same group. It is expected that such substitutions with similar amino acids will not lead to phenotypic changes in the protein (i.e., conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various documents (e.g., see Bowie et al., Science, 247: 1306-1310 (1990)). The homology of the amino acid sequences in the present specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0136] When the CD3-positive cells cultured in the manufacturing method or expanded culture method of the present invention express a chimeric antigen receptor and / or IL-15 / IL-15Rα, the method for introducing the CAR nucleic acid and / or IL-15 / IL-15Rα nucleic acid into cells (e.g., pluripotent stem cells) is not particularly limited, and for example, the same method as the method for introducing the TCR nucleic acid can be used.

[0137] In the manufacturing method or the expanded culture method of the present invention, when the cultured CD3-positive cells are cells obtained by differentiating pluripotent stem cells, the pluripotent stem cells can be differentiated into CD3-positive cells according to a method known per se. Specific methods for differentiating pluripotent stem cells into CD3-positive cells are described in, for example, International Publication No. 2016 / 076415 and International Publication No. 2017 / 221975. As a specific method for differentiating pluripotent stem cells into CD3-positive cells, for example, it may include (1) a step of differentiating pluripotent stem cells into hematopoietic progenitor cells, and (2) a step of differentiating the hematopoietic progenitor cells into CD3-positive cells.

[0138] (1) Steps for differentiating pluripotent stem cells into hematopoietic progenitor cells

[0139] In step (1), hematopoietic progenitor cells (HPC) refer to CD34 positive cells, preferably CD34 positive CD43 positive cells. In this step (1), hematopoietic progenitor cells and hematopoietic stem cells are not distinguished, and unless otherwise specified, they represent the same cells.

[0140] There are no particular limitations on the method of differentiating pluripotent stem cells into hematopoietic progenitor cells as long as they can differentiate into hematopoietic progenitor cells. For example, there can be cited methods of culturing pluripotent stem cells in a culture medium that induces hematopoietic progenitor cells as described in International Publication No. 2013 / 075222, International Publication No. 2016 / 076415, and Liu S. et al., Cytotherapy, 17 (2015); 344-358, etc.

[0141] In step (1), the medium for inducing hematopoietic progenitor cells is not particularly limited, and a medium that can be used to culture animal cells can be prepared as a basal medium. Examples of the basal medium include Dulbecco's medium (e.g., IMDM), Eagle's medium (e.g., DMEM, EMEM, BME, MEM, and αMEM), Ham's medium (e.g., F10 medium and F12 medium), RPMI medium (e.g., RPMI-1640 medium and RPMI-1630 medium), MCDB medium (e.g., MCDB104, 107, 131, 151, and 153 medium), Fisher's medium, 199 medium, a medium for primate ES cells (a culture medium for primate ES / iPS cells, ReproCELL Inc.), a medium for mouse ES cells (TX-WES culture medium, Thrombo X), a serum-free medium (mTeSR, Stemcell Technology), ReproFF, StemSpan (registered trademark) SFEM, StemSpan (registered trademark) H3000, Stemline II, ESF-B medium, ESF-C medium, CSTI-7 medium, Neurobasal medium (Life Technologies Inc.), and the like. Technologies Co., Ltd.), StemPro-34 medium, StemFit (registered trademark) (e.g., StemFit AK03N, StemFit AK02N), etc., but are not limited to these. In addition, these media can also be mixed as needed, for example, DMEM / F12 medium can be mentioned. The basal medium may contain serum or may be used without serum. The basal culture medium may be appropriately supplemented with 10 to 20% serum (fetal bovine serum (FBS), human serum, horse serum) or serum substitutes (KSR, etc.), insulin, various vitamins, L-glutamine, various amino acids such as non-essential amino acids, 2-mercaptoethanol, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF (Stem cell factor)), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.), various extracellular matrices, various cell adhesion molecules, antibiotics such as penicillin / streptomycin and puromycin, pH indicators such as phenol red, etc., etc.In addition, as needed, the basal culture medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, L-alanyl-L-glutamine (e.g., Glutamax (registered trademark)), growth factors, small molecule compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0142] In step (1), the vitamin C refers to L-ascorbic acid and its derivatives, and the L-ascorbic acid derivatives refer to substances that become vitamin C through an enzyme reaction in a living body. Examples of the ascorbic acid derivatives used in step (1) include ascorbic acid phosphate, ascorbyl glucoside, ascorbic acid ethyl ether, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbyl 2-phosphate 6-palmitate. Preferred is ascorbic acid phosphate (e.g., ascorbic acid 2-phosphate), and examples thereof include L-ascorbate phosphates such as sodium phosphate-L-ascorbyl and magnesium phosphate-L-ascorbyl.

[0143] When vitamin C is used, it is preferred to add (supplement) vitamin C every 4 days, every 3 days, every 2 days, or every 1 day, and more preferably to add it every day. In one embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5ng / ml to 500ng / ml (for example, an amount equivalent to 5ng / ml, 10ng / ml, 25ng / ml, 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml). In another embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5μg / ml to 500μg / ml (for example, an amount equivalent to 5μg / ml, 10μg / ml, 25μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 300μg / ml, 400μg / ml, 500μg / ml).

[0144] The culture medium used in step (1) may be further supplemented with at least one cytokine selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), SCF (stem cell factor), TPO (thrombopoietin), FLT-3L (Flt3 ligand), and bFGF (basic fibroblast growth factor). More preferably, the culture medium is supplemented with BMP4, VEGF, and bFGF, and even more preferably, the culture medium is supplemented with BMP4, VEGF, SCF, and bFGF.

[0145] When cytokines are used, the concentration in the culture medium, for example, may be 5 ng / ml to 500 ng / ml for BMP4, 5 ng / ml to 500 ng / ml for VEGF, 5 ng / ml to 500 ng / ml for SCF, 3 ng / ml to 300 ng / ml for TPO, 1 ng / ml to 100 ng / ml for FLT-3L, and 5 ng / ml to 500 ng / ml for bFGF.

[0146] In addition, a TGFβ inhibitor may be added to the aforementioned culture medium. The so-called TGFβ inhibitor is a small molecule inhibitor that intervenes in the signal transduction of the TGFβ family, including, for example, SB431542, SB202190 (see RKLindemann et al., Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), etc. For example, when the TGFβ inhibitor is SB431542, the concentration in the culture medium is preferably 0.5 μM to 100 μM.

[0147] The culture of pluripotent stem cells can be adherent culture or suspension culture. In the case of adherent culture, a culture container coated with an extracellular matrix component can be used, or co-culture can be performed with feeder cells. As feeder cells, there is no particular limitation, for example, fibroblasts (mouse embryonic fibroblasts (MEF), mouse fibroblasts (STO), etc.) can be cited. Feeder cells are preferably inactivated using methods known per se, such as radiation (gamma rays, etc.), anticancer agents (mitomycin C, etc.) treatment, etc. As extracellular matrix components, fibrous proteins such as matrigel (Niwa A et al., PLoS One.6 (7): e22261, 2011), gelatin, collagen, elastin, glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, proteoglycans, fibronectin, vitronectin, cell adhesion proteins such as laminin, etc. can be cited.

[0148] The so-called suspension culture refers to culturing cells in a state where they are not adhered to a culture container, and is not particularly limited. It can be carried out using a culture container that has not been artificially treated for the purpose of improving adhesion to cells (e.g., coating treatment using an extracellular matrix, etc.), or a culture container that has been artificially treated to inhibit adhesion (e.g., coating treatment using polyhydroxyethyl methacrylate (poly-HEMA) or nonionic surfactant polyols (Pluronic F-127, etc.)). In the case of suspension culture, it is preferred to form embryoid bodies (EBs) and culture.

[0149] Hematopoietic progenitor cells can be prepared from a reticular structure (also called ES-sac or iPS-sac) obtained by culturing pluripotent stem cells. Here, the so-called "reticular structure" is a three-dimensional sac-like (with space inside) structure derived from pluripotent stem cells, and is formed by an endothelial cell group and contains hematopoietic progenitor cells inside.

[0150] The conditions of the culture temperature of step (1) are not particularly limited, for example, preferably about 37°C to 42°C, or about 37°C to 39°C. In addition, regarding the culture time, a person skilled in the art can appropriately determine it while monitoring the number of hematopoietic progenitor cells, etc. As long as hematopoietic progenitor cells can be obtained, the number of days is not particularly limited, for example, at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, preferably 14 days. In the manufacture of hematopoietic progenitor cells, a long culture time is generally not a problem, but it is preferably, for example, less than 35 days, and more preferably less than 21 days. In addition, culture can be carried out under hypoxic conditions. In this specification, the so-called hypoxic conditions can be exemplified by oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5% or less.

[0151] (2) Steps for differentiating hematopoietic progenitor cells into CD3-positive cells

[0152] There are no particular limitations on the method for differentiating hematopoietic progenitor cells into CD3-positive cells as long as they can differentiate hematopoietic progenitor cells into CD3-positive cells. For example, there can be mentioned a method of culturing hematopoietic progenitor cells under the same culture conditions as the method of inducing T cells from hematopoietic progenitor cells, as described in International Publication No. 2016 / 076415 or International Publication No. 2017 / 221975.

[0153] In step (2), the induction medium for differentiation into CD3 positive cells is not particularly limited, and a medium that can be used to culture animal cells can be prepared as a basal medium. The basal medium can include the same medium as the medium used in the above step (1). The medium can contain serum, or it can be used in a serum-free state. As needed, the basal medium can contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax (registered trademark)), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, etc.

[0154] When vitamin C is used in step (2), the vitamin C may be the same substance as the vitamin C described in step (1), and may be added in the same manner. In one embodiment, the concentration of the vitamin C in the culture medium or in the culture fluid is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added to the culture fluid in an amount equivalent to 5 μg / ml to 500 μg / ml (e.g., an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml).

[0155] In step (2), p38 inhibitors and / or SDF-1 (stromal cell derived factor 1) are preferably used. In this specification, the so-called "p38 inhibitor" refers to a substance that inhibits the function of p38 protein (p38 MAP kinase), for example, chemical inhibitors of p38, dominant negative mutants of p38 or nucleic acids encoding the same, but are not limited to these.

[0156] As chemical inhibitors of p38 in step (2), there can be exemplified SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives, SB239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol) and its derivatives, SB220025 and its derivatives, PD169316, RPR200765A, AMG-548, BIRB-796, SClO-469, SCIO-323, VX-702, and FR167653, but are not limited thereto. These compounds are commercially available, for example, SB203580, SB202190, SB239063, SB220025 and PD169316 can be obtained from Calbiochem, and SClO-469 and SCIO-323 can be obtained from Scios, etc. As chemical inhibitors of p38, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole) and its derivatives are preferred.

[0157] As dominant negative mutants of p38 in step (2), there can be mentioned p38T180A, which is obtained by point mutation of threonine at position 180 in the DNA binding region of p38 to alanine, and p38Y182F, which is obtained by point mutation of tyrosine at position 182 in p38 in humans and mice to phenylalanine. The p38 inhibitor can be contained in the culture medium in a range of, for example, about 1 μM to about 50 μM. When SB203580 is used as the p38 inhibitor, SB203580 can be contained in the culture medium in a range of 1 μM to 50 μM, 5 μM to 30 μM, or 10 μM to 20 μM.

[0158] The SDF-1 in step (2) may be SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε or SDF-1γ. Isoforms or mature forms thereof, or mixtures thereof in any ratios, etc. SDF-1α is preferably used. It should be noted that SDF-1 is sometimes also referred to as CXCL-12 or PBSF.

[0159] In step (2), as long as SDF-1 has its activity as a chemokine, one or more amino acids may be substituted, deleted, added and / or inserted in its amino acid sequence (such SDF-1 obtained by amino acid substitution, deletion, addition and / or insertion is also referred to as "SDF-1 mutant"). In addition, similarly, in SDF-1 or SDF-1 mutant, sugar chains may be substituted, deleted and / or added. As the mutant of the above-mentioned SDF-1, for example, there can be mentioned mutants that retain at least 4 cysteine ​​residues (in the case of human SDF-1α, Cys30, Cys32, Cys55 and Cys71) and have an identity of 90% or more with the amino acid sequence of the natural body, but it is not limited to this amino acid mutation. SDF-1 can be SDF-1 of mammals, such as humans, such as monkeys, sheep, cattle, horses, pigs, dogs, cats, rabbits, rats, mice and other non-human mammals. For example, as human SDF-1α, a protein registered with GenBank Accession No.: NP_954637 can be used, and as SDF-1β, a protein registered with GenBank Accession No.: NP_000600 can be used.

[0160] SDF-1 can be a commercial product, a substance purified from natural sources, or a substance produced by peptide synthesis or genetic engineering methods. SDF-1 can be contained in the culture medium in a range of, for example, about 10 ng / ml to about 100 ng / ml. In addition, an SDF-1 substitute having SDF-1-like activity can be used instead of SDF-1. As such an SDF-1 substitute, a CXCR4 agonist can be exemplified, and a small molecule compound having CXCR4 agonist activity can also be added to the culture medium instead of SDF-1.

[0161] In the culture medium used in step (2), at least one, preferably all, of the cytokines selected from the group consisting of SCF, TPO (thrombopoietin), FLT-3L and IL-7 may be further added to the culture medium. For example, their concentrations are 10 ng / ml to 100 ng / ml for SCF, 10 ng / ml to 200 ng / ml for TPO, 1 ng / ml to 100 ng / ml for IL-7, and 1 ng / ml to 100 ng / ml for FLT-3L.

[0162] In step (2), the hematopoietic progenitor cells can be cultured in an adherent culture or in a suspended culture. In the case of adherent culture, the culture container can be coated for use, or co-cultured with feeder cells, etc. As feeder cells for co-culture, OP9 cells of a bone marrow interstitial cell line (available from the RIKEN Bioresource Center) can be exemplified. The OP9 cells are preferably OP9-DL4 cells or OP9-DL1 cells that constantly express DLL4 or DLL1 (e.g., Holmes R1 and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009 (2)). In step (2), when OP9 cells are used as feeder cells, the culture can be performed by appropriately adding DLL4 or DLL1 prepared separately, or a fusion protein formed by DLL4 or DLL1 and Fc, etc., to the culture medium. When feeder cells are used, it is preferred to appropriately replace the feeder cells for culture. The feeder cells can be replaced by transferring the target cells in culture to feeder cells seeded in advance. The replacement can be performed every 5 days, every 4 days, every 3 days, or every 2 days. When hematopoietic progenitor cells are obtained by suspension culture of embryoid bodies, it is preferred to dissociate them into single cells and then culture them on a wall. Although co-culture with feeder cells is also possible, culture without feeder cells is preferred.

[0163] As coating agents for adherent culture and coating the culture container, for example, there can be mentioned matrigel (Niwa A et al., PLos One, 6(7):e22261, 2011)), collagen, gelatin, laminin, heparan sulfate proteoglycan, RetroNectin, DLL4 or DLL1, or a fusion protein formed by DLL4 or DLL1 and the Fc region of an antibody (hereinafter sometimes referred to as Fc) (e.g., DLL4 / Fc chimera), entactin, and / or a combination thereof, preferably a combination of RetroNectin and a fusion protein formed by DLL4 and Fc.

[0164] In step (2), the culture temperature conditions are not particularly limited, for example, about 37°C to about 42°C, preferably about 37 to about 39°C. In addition, regarding the culture time, those skilled in the art can appropriately determine while monitoring the number of CD3-positive cells, etc. As long as CD3-positive cells can be obtained, the number of days is not particularly limited, for example, at least 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 23 days, preferably 21 days. In addition, it is preferably 90 days or less, and more preferably 42 days or less.

[0165] The cells obtained by the above steps include CD3-positive cells, but the specific method for differentiating pluripotent stem cells into CD3-positive cells may further include the following step (3).

[0166] (3) A step of obtaining CD3-positive CD8-positive CD4-negative cells (or CD3-positive CD8-positive CD4-negative CD30-positive cells) or a step of enriching CD3-positive cells

[0167] As a method for obtaining CD3 positive CD8 positive CD4 negative cells (or CD3 positive CD8 positive CD4 negative CD30 positive cells), for example, there can be mentioned the methods described in International Publication No. 2016 / 076415 and International Publication No. 2017 / 221975, etc. In addition, CD3 positive cells can also be enriched using the same method.

[0168] In step (3), the medium for obtaining CD3-positive CD8-positive CD4-negative cells (or CD3-positive CD8-positive CD4-negative CD30-positive cells) or enriching CD3-positive cells is not particularly limited, and a medium that can be used to culture animal cells can be prepared as a basal medium. The basal medium can include the same medium as the medium used in the above step (1). The medium may contain serum, or it can be used in a serum-free state. As needed, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), fatty acids, trace elements, 2-mercaptoethanol, thioglycerol (e.g., α-monothioglycerol (MTG)), lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax (registered trademark)), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, hormones, etc.

[0169] When vitamin C is used in step (3), the vitamin C may be the same substance as the vitamin C described in step (1), and may be added in the same manner. In one embodiment, the concentration of the vitamin C in the culture medium or in the culture fluid is preferably 5 μg / ml to 200 μg / ml. In another embodiment, the vitamin C is added to the culture fluid in an amount equivalent to 5 μg / ml to 500 μg / ml (for example, an amount equivalent to 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml).

[0170] When a hormone is used in step (3), the hormone may be adrenocortical hormone. The adrenocortical hormone is a glucocorticoid or a derivative thereof, and examples thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone dipropionate. Dexamethasone is preferred. When the adrenocortical hormone is dexamethasone, its concentration in the culture medium is 1 nM to 100 nM.

[0171] In step (3), a CD3 / TCR complex agonist is contained in the culture medium. The CD3 / TCR complex agonist is not particularly limited as long as it can transmit the signal from the CD3 / TCR complex to the CD3 positive cell by specifically binding to at least a part of the CD3 / TCR complex. As a CD3 / TCR complex agonist, for example, a CD3 agonist and / or a TCR agonist can be cited. As a CD3 agonist, an anti-CD3 agonist antibody or its binding fragment can be cited, and as a TCR agonist, at least one selected from the group consisting of an anti-TCR antibody or its binding fragment, an MHC / antigen peptide complex or its multimer and an MHC / superantigen complex or its multimer can be cited. When an anti-CD3 agonist antibody is used, the anti-CD3 agonist antibody includes both a polyclonal antibody and a monoclonal antibody, preferably a monoclonal antibody. In addition, the antibody can be an antibody belonging to any immunoglobulin class among IgG, IgA, IgM, IgD or IgE, but preferably IgG. Examples of the anti-CD3 agonist antibody include antibodies produced by the OKT3 clone (OKT3) and antibodies produced by the UCHT1 clone (UCHT1), and UCHT1 is preferred. When the anti-CD3 agonist antibody is OKT3, OKT3 comprises RYTMH (SEQ ID NO: 9), YINPSRGYTNYNQKFKD (SEQ ID NO: 10), and YYDDHYCLDY (SEQ ID NO: 11) as complementarity determining regions 1 to 3 of the heavy chain variable region, and comprises SASSSVSYMN (SEQ ID NO: 12), DTSKLAS (SEQ ID NO: 13), and QQWSSNPFT (SEQ ID NO: 14) as complementarity determining regions 1 to 3 of the light chain variable region. In addition, OKT3 preferably comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22. When the anti-CD3 agonist antibody is UCHT1, UCHT1 comprises GYSFTGYTMN (SEQ ID NO: 15), LINPYKGVST (SEQ ID NO: 16), SGYYGDSDWYFDV (SEQ ID NO: 17) as complementarity determining regions 1 to 3 of the heavy chain variable region, and RASQDIRNYLN (SEQ ID NO: 18), YTSRLHS (SEQ ID NO: 19), QQGNTLPWT (SEQ ID NO: 20) as complementarity determining regions 1 to 3 of the light chain variable region. In addition, UCHT1 preferably comprises a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 24. The concentration of the anti-CD3 agonist antibody in the culture medium is, for example, 10 ng / ml to 1000 ng / ml, preferably 50 ng / ml to 800 ng / ml, and more preferably 250 ng / ml to 600 ng / ml.

[0172] When cytokines are used in step (3), examples of cytokines include IL-2 and IL-7. When the cytokine is IL-2, its concentration in the culture medium is 10 U / ml to 1000 U / ml, and when it is IL-7, its concentration in the culture medium is 1 ng / ml to 1000 ng / ml.

[0173] In step (3), the culture temperature conditions are not particularly limited, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. In addition, regarding the culture time, those skilled in the art can appropriately determine while monitoring the number of CD3-positive cells, etc. As long as CD3-positive cells can be obtained, the number of days is not particularly limited, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, preferably 6 days. In addition, it is preferably 28 days or less, and more preferably 14 days or less.

[0174] As described above, CD3-positive cells cultured in the production method or expansion culture method of the present invention can be obtained by differentiating pluripotent stem cells.

[0175] The production method or expansion culture method of the present invention comprises the step of culturing CD3-positive cells in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist (hereinafter referred to as step (I) of the present invention).

[0176] In step (I) of the present invention, the CD3 / TCR complex agonist may be the same CD3 / TCR complex agonist as that used in the aforementioned step (3).

[0177] In step (I) of the present invention (or the aforementioned step (3)), when the CD3 / TCR complex agonist used is an anti-CD3 agonist antibody or an anti-TCR antibody, polyclonal antibodies thereof can be prepared, for example, by the following method. For example, in the case of a polyclonal antibody against CD3, a mixture of CD3 or a partial peptide containing its epitope (e.g., γ, δ, ε, ζ or η chain) and Freund's complete or incomplete adjuvant (FCA or FIA) is used as a sensitizing antigen, and in the case of a polyclonal antibody against TCR, a mixture of TCR or a partial peptide containing its epitope (e.g., α, β, γ or δ chain) and Freund's complete or incomplete adjuvant (FCA or FIA) is used as a sensitizing antigen, and mammals such as rabbits, mice, rats, goats, guinea pigs or hamsters are immunized (one to multiple booster immunizations are performed approximately every 1 to 4 weeks from the initial immunization), and the antibody titer of the serum obtained by local blood sampling approximately 3 to 10 days after each booster immunization is measured using a conventionally known antigen-antibody reaction, and the increase is confirmed. Furthermore, whole blood is collected about 3 to 10 days after the final immunization, and the antiserum is purified. The polyclonal antibody can also be purified as a single immunoglobulin fraction using conventional separation techniques such as salting out by ammonium sulfate fractionation, centrifugation, dialysis, column chromatography, etc.

[0178] In addition, in step (I) of the present invention (or the aforementioned step (3)), when the anti-CD3 agonist antibody or anti-TCR antibody used is a monoclonal antibody, the monoclonal antibody can usually be obtained from a hybridoma (fusion cell) manufactured by cell fusion. That is, as in the case of the above-mentioned polyclonal antibody, cells producing antibodies are separated from mammals that are immunosensitized with CD3 or TCR or partial peptides containing their epitopes, and fused with myeloma cells to form hybridomas, which are cloned, and CD3 or TCR is used as a marker antigen to screen clones that produce antibodies that show specific affinity to them, thereby manufacturing. In addition, cells producing antibodies produced by causing CD3 to act on pre-separated spleen cells or lymphocytes in a culture medium can also be used. At this time, human cells producing antibodies can also be prepared.

[0179] The preparation of hybridomas secreting monoclonal antibodies can be carried out according to and Milstein's method (Nature, Vol. 256, pp. 495-497, 1975) and its improved method. That is, monoclonal antibodies can be prepared by culturing hybridomas, which are obtained by fusion of antibody-producing cells contained in spleen cells, thymocytes, lymph node cells, peripheral lymphocytes, myeloma cells or tonsil cells (preferably spleen cells) obtained from an animal sensitized as described above, and preferably myeloma cells (myeloma) of the same species of mice, rats, guinea pigs, hamsters, rabbits or humans, more preferably mice, rats or humans. Cultivation can be performed in vitro or in vivo (in the peritoneal cavity of mice, rats, guinea pigs, hamsters, rabbits and other mammals, preferably mice or rats, more preferably mice), and antibodies can be obtained from the culture supernatant or the ascites of mammals, respectively.

[0180] Myeloma cells that can be used for cell fusion include, for example, mouse myeloma (e.g., P3-NSI-1-Ag4-1, P3-X63-Ag8-U1, P3-X63-Ag8-653, SP2 / 0-Ag14, and BW5147, etc.), rat myeloma (e.g., 210RCY3-Ag1.2.3, etc.), and human myeloma (e.g., U-266AR1, GML500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, and CEM-T15, etc.).

[0181] Screening of hybridoma clones producing monoclonal antibodies can be performed by culturing hybridomas in, for example, a microtiter plate and measuring the reactivity against CD3 of the culture supernatant in the wells where proliferation was observed by radioimmunoassay, enzyme immunoassay, fluorescent immunoassay or the like.

[0182] The monoclonal antibody can be isolated and purified by subjecting the culture supernatant or ascites containing the antibody produced by the above method to ion exchange chromatography, affinity column chromatography such as an anti-immunoglobulin column or a protein G column.

[0183] The monoclonal antibody used in step (I) of the present invention (or the aforementioned step (3)) is not limited to the above-mentioned production method, and may be obtained by any method. In addition, monoclonal antibodies generally have sugar chains with different structures depending on the type of mammal to which they are immunized, but the monoclonal antibodies in the present invention are not limited by the structural differences of the sugar chains, and include monoclonal antibodies derived from any mammal.

[0184] In addition, anti-CD3 agonist antibodies or anti-TCR antibodies include natural antibodies such as the aforementioned polyclonal antibodies, monoclonal antibodies (mAb), chimeric antibodies (humanized antibodies) and single-chain antibodies produced using genetic recombination technology, as well as binding fragments of these antibodies. The so-called antibody binding fragment refers to a partial segment of the aforementioned antibody having specific binding activity, specifically, Fab, Fab', F(ab')2, scAb, scFv, or scFv-Fc, etc.

[0185] In addition, those skilled in the art can prepare fusion antibodies formed by anti-CD3 agonist antibodies or anti-TCR antibodies or their binding fragments with other peptides and proteins, and can also prepare modified antibodies formed by binding modifiers thereto. Other peptides and proteins used for fusion are not particularly limited as long as they do not reduce the binding activity of the antibody. For example, human serum albumin, various tag peptides, artificial helix motif peptides, maltose binding protein, glutathione S transferase, various toxins, and peptides or proteins that can promote polymerization can be cited. The modifier used for modification is not particularly limited as long as it does not reduce the binding activity of the antibody. For example, polyethylene glycol, sugar chains, phospholipids, liposomes, small molecule compounds, etc. can be cited.

[0186] In addition, commercially available reagents can be used as anti-CD3 agonist antibodies. Commercially available anti-CD3 agonist antibodies are not particularly limited, and for example, antibodies produced by the OKT3 clone (OKT3) (functional grade purified anti-human CD3 (clone: ​​OKT3) (eBioscience)) and antibodies produced by the UCHT1 clone (UCHT1) (CD3 antibody (clone: ​​UCHT1) (GeneTex)) can be used, and UCHT1 can be preferably used.

[0187] In addition, in step (I) of the present invention (or the aforementioned step (3)), when the CD3 / TCR complex agonist used is an MHC / antigen peptide complex, the MHC / antigen peptide complex is not particularly limited as long as it can be a molecule that can be specifically recognized by the CD3 / TCR complex of CD3-positive cells and can transmit signals to CD3-positive cells.

[0188] The MHC constituting the MHC / antigen peptide complex may be MHC class I or MHC class II, and is preferably MHC class I.

[0189] In step (I) of the present invention (or the aforementioned step (3)), MHC class I is not particularly limited as long as it forms a complex with an antigen peptide, is recognized by the CD3 / TCR complex and CD8, and transmits a signal to CD3-positive cells. MHC class I may include, for example, a dimer consisting of an α chain (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G in the case of humans; H-2K, H-2D or H-2L in the case of mice) and β2 microglobulin, preferably a dimer consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G and β2 microglobulin, and more preferably a dimer consisting of HLA-A, HLA-B or HLA-C and β2 microglobulin. In the manufacturing method or the expanded culture method of the present invention, as a specific MHC class I, for example, HLA-A*02:01, HLA-A*24:02 or HLA-A*01:01, etc. can be cited, but it is not limited to these. In addition, the antigenic peptide is not particularly limited as long as it can be a peptide presented by MHC class I. The antigenic peptide that can be presented by MHC class I can be cited as being derived from, for example, HER-2 / neu, MART-1, NY-ESO-1, Gp-100, MUC-1, p53, prostate-specific antigen (PSA), hTERT, WT1, survivin, CEA, MAGE-3, or peptides from proteins of virus groups highly correlated with the occurrence of malignant tumors, etc., and preferably a peptide derived from WT1 can be cited. Specific examples of peptides derived from WT1 include RMFPNAPYL (SEQ ID NO: 1), SLGEQQYSV (SEQ ID NO: 2), CMTWNQMNL (SEQ ID NO: 3) (its modified peptide CYTWNQMNL (SEQ ID NO: 4)), and the like.

[0190] In addition, in step (I) of the present invention (or the aforementioned step (3)), MHC class II is not particularly limited as long as it forms a complex with an antigenic peptide, is recognized by the CD3 / TCR complex and CD4, and transmits a signal to CD3-positive cells. For MHC class II, for example, HLA-DR, HLA-DQ and HLA-DP can be mentioned in the case of humans, and H-2A or H-2B can be mentioned in the case of mice, which are dimers composed of an α chain and a β chain (for example, HLA-DR has HLA-DRA as an α chain and HLA-DRB1 as a β chain), respectively, preferably HLA-DR, HLA-DQ or HLA-DP. In addition, the antigenic peptide is not particularly limited as long as it can be a peptide that can be presented by MHC class II. Antigenic peptides that can be presented by MHC II include, for example, peptides derived from WT1, PSA, MAGE-3, CEA, survivin, tyrosinase, or proteins from viral groups highly associated with the occurrence of malignant tumors, and preferably, peptides derived from WT1.

[0191] The MHC and / or antigenic peptide (hereinafter referred to as MHC etc.) may be a protein chemically synthesized or biochemically synthesized in a cell-free translation system, or may be a recombinant protein produced by a transformant into which a nucleic acid having a base sequence encoding MHC etc. has been introduced.

[0192] When manufacturing MHC etc. according to known peptide synthesis methods, for example, it can be either a solid-phase synthesis method or a liquid-phase synthesis method. The target protein can be manufactured by condensing amino acid derivatives obtained by imparting protecting groups to carboxyl groups and side-chain functional groups, and amino acid derivatives in which amino groups and side-chain functional groups have been protected, and then removing the protecting groups.

[0193] Here, condensation and removal of the protecting groups are carried out according to methods known per se, for example, the methods described in the following (1) to (5).

[0194] (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)

[0195] (2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

[0196] (3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)

[0197] (4) Haruaki Yajima and Shunpei Sakakibara, Biochemical Experiment Lectures 1, Chemistry of Proteins IV 205 (1977)

[0198] (5) Edited by Haruaki Yajima, Continued Development of Pharmaceuticals (続医薬品の開発), Volume 14, Peptide Synthesis, Hirokawa Shoten

[0199] The MHC etc. thus obtained can be purified and separated using known purification methods. Here, as purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, combinations thereof, etc. can be cited.

[0200] When the MHC etc. obtained by the above method is in the free form, the free form can be converted into a suitable salt by a known method or a method based thereon. Conversely, when the MHC etc. is obtained in the form of a salt, the salt can be converted into the free form or another salt by a known method or a method based thereon.

[0201] In addition, MHC and the like can also be produced by culturing a transformant containing a nucleic acid encoding the same, isolating and purifying MHC and the like from the obtained culture. The nucleic acid encoding MHC and the like can be DNA or RNA, or can be a DNA / RNA chimera. DNA can be preferably cited. In addition, the nucleic acid can be double-stranded or single-stranded. In the case of a double-stranded strand, it can be a double-stranded DNA, a double-stranded RNA, or a DNA:RNA hybrid. In the case of a single-stranded strand, it can be a sense strand (i.e., a coding strand) or an antisense strand (i.e., a non-coding strand).

[0202] Examples of DNA encoding MHC and the like include genomic DNA, cDNA derived from cells of warm-blooded animals (e.g., humans, cattle, monkeys, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits, hamsters, birds, etc.), and synthetic DNA. In the case of cDNA encoding MHC and the like, any cell from the aforementioned animals [e.g., hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, myocytes, adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes, or mesenchymal cells, or progenitor cells, stem cells, or cancer cells of these cells, etc.] or any group of these cells can also be used. Total RNA or mRNA fractions prepared from various tissues [e.g., brain, various parts of the brain (e.g., olfactory bulb, amygdala, basal ganglia of the brain, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid gland, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brown adipose tissue, white adipose tissue), skeletal muscle, etc.] are used as templates to directly amplify by polymerase chain reaction (hereinafter referred to as "PCR method") and reverse transcriptase PCR (hereinafter referred to as "RT-PCR method"). Alternatively, cDNA encoding MHC etc. can also be cloned from a cDNA library prepared by inserting the above-mentioned full-length RNA or mRNA fragments into an appropriate vector by colony hybridization method, plaque hybridization method, PCR method, etc. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

[0203] DNA encoding MHC etc. can be amplified by PCR method using synthetic DNA primers having a part of the base sequence encoding the MHC etc., or cloned by hybridizing DNA incorporated into a suitable expression vector with DNA obtained by labeling a part or full-length DNA fragment or synthetic DNA encoding MHC etc. Hybridization can be carried out according to a method known per se or a method based thereon, such as the method described in Molecular Cloning 2nd edition (J.Sambrook et al., Cold Spring Harbor Lab.Press, 1989). In addition, when using a commercially available library, hybridization can be carried out according to the method described in the accompanying instruction manual. Hybridization can preferably be carried out according to stringent conditions.

[0204] As highly stringent conditions, for example, hybridization reaction at 45°C in 6×SSC (sodium chloride / sodium citrate) followed by washing once or more at 65°C in 0.2×SSC / 0.1% SDS can be cited. A person skilled in the art can easily adjust to the desired stringency by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the temperature of the washing, etc. In addition, when using a commercially available library, hybridization can be performed according to the method described in the instruction manual attached to the library.

[0205] An expression vector containing a DNA encoding MHC or the like can be produced by, for example, cutting out a target DNA fragment from a DNA encoding MHC or the like and ligating the DNA fragment to the downstream of a promoter in an appropriate expression vector.

[0206] As expression vectors, plasmids derived from Escherichia coli (for example, pBR322, pBR325, pUC12, pUC13); animal cell expression plasmids (for example, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. can be used.

[0207] The promoter may be any promoter as long as it is an appropriate promoter corresponding to the host used for expressing the gene.

[0208] For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. can be used. Among them, CMV promoter, SRα promoter, etc. are preferred.

[0209] When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter and the like are preferred.

[0210] As an expression vector, in addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes referred to as SV40 ori), etc. can be used as desired. Examples of selection markers include dihydrofolate reductase gene (hereinafter, sometimes referred to as dhfr, methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter, sometimes referred to as amp r ), neomycin resistance gene (hereinafter, sometimes referred to as neo r , G418 resistance), etc. In particular, when using Chinese hamster cells with a dhfr gene deficiency and using the dhfr gene as a selection marker, a medium without thymidine can also be used to screen for the target gene.

[0211] In addition, a base sequence (signal codon) encoding a signal sequence that matches the host can be added to the 5' end of the DNA encoding MHC or the like (or replaced with the native signal codon) as needed. For example, when the host is an Escherichia bacterium, a PhoA signal sequence, an OmpA signal sequence, etc. are used; when the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. are used.

[0212] MHC and the like can be produced by transforming a host with an expression vector containing the DNA encoding MHC and the like described above and culturing the obtained transformant.

[0213] As the host, for example, bacteria of the genus Escherichia, animal cells, etc. can be used.

[0214] As Escherichia bacteria, for example, K12 DH1〔Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)〕, JM103〔Nucleic Acids Research, Vol. 9, 309 (1981)〕, JA221〔Journal of Molecular Biology, Vol. 120, 517 (1978)〕, HB101〔Journal of Molecular Biology, Vol. 41, 459 (1969)〕, C600〔Genetics, Vol. 39, 440 (1954)〕, etc. can be used.

[0215] As animal cells, for example, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter referred to as CHO cells), dhfr gene-deficient CHO cells (hereinafter referred to as CHO (dhfr - ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, etc.

[0216] Transformation can be carried out according to known methods depending on the type of host.

[0217] Escherichia bacteria can be transformed according to the methods described in, for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972), Genee, Vol. 17, 107 (1982), and the like.

[0218] Animal cells can be transformed according to the method described in, for example, Cell Engineering Special Issue 8 New Cell Engineering Experimental Plans, 263-267 (1995) (published by Shujunsha) or Virology, vol. 52, 456 (1973).

[0219] The transformant can be cultured according to a known method depending on the type of the host.

[0220] When the host is an Escherichia transformant, for example, an M9 medium containing glucose and casamino acids is preferably used [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 3β-indoleacrylic acid may be added to the medium for the purpose of making the promoter work efficiently.

[0221] The transformant whose host is bacteria of the genus Escherichia is usually cultured at about 15 to about 43° C. for about 3 to about 24 hours. Aeration and stirring may be performed as necessary.

[0222] As a medium for culturing a transformant whose host is an animal cell, for example, minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], Dulbecco's modified Eagle's medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI1640 medium [The Journal of the American Medical Association, Vol. 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)], etc. can be used. The pH of the medium is preferably about 6 to about 8. Cultivation is usually carried out at about 30° C. to about 40° C. for about 15 to about 60 hours. Aeration and stirring can be performed as needed.

[0223] By operating as described above, MHC and the like can be produced inside or outside the cells of the transformant.

[0224] MHC and the like can be isolated and purified from the culture obtained by culturing the above-mentioned transformant according to a method known per se.

[0225] For example, when extracting MHC from the cytoplasm of cultured bacteria or cells, the following method can be appropriately used: suspending bacteria or cells collected from the culture using a known method in a suitable buffer, destroying the bacteria or cells using ultrasound, lysozyme and / or freeze-thaw, and then obtaining a crude extract of soluble protein by centrifugation and filtration; etc. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, Triton X-100, TM When MHC and the like are secreted outside the bacteria (cells), a method of collecting the culture supernatant from the culture by centrifugation or filtration can be used.

[0226] The separation and purification of MHC and the like contained in the soluble fraction and culture supernatant thus obtained can be carried out according to a method known per se. As such methods, methods utilizing solubility such as salting out and solvent precipitation; methods mainly utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods utilizing charge differences such as ion exchange chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing hydrophobicity differences such as reversed-phase high performance liquid chromatography; methods utilizing isoelectric point differences such as isoelectric point electrophoresis; and the like can be used. These methods can also be appropriately combined.

[0227] The MHC / antigen peptide complex obtained as described above can be a polymer. By polymerizing the MHC / antigen peptide complex, a higher agonist effect for TCR can be expected. The method by which the MHC / antigen peptide complex of the present invention is polymerized is known, for example, the MHC / antigen peptide complex modified with biotin can be tetramerized via the tetramer of avidin or streptavidin. In addition, as other means, polymerization can also be carried out by connecting the MHC / antigen peptide complex to dextran.

[0228] In addition, in step (I) of the present invention (or the aforementioned step (3)), when the CD3 / TCR complex agonist used is an MHC / superantigen complex, the MHC / superantigen complex is a complex of MHC class II and a superantigen. As superantigens, Staphylococcus aureus enterotoxin, streptococcal febrile exotoxin, toxic shock syndrome toxin, etc. can be cited. MHC or superantigen can be manufactured using the same method as described in the aforementioned MHC, etc. In addition, the MHC / superantigen complex can be a polymer in the same manner as the MHC / antigen peptide complex. The method of polymerizing the MHC / superantigen complex of the present invention is the same as the method of polymerizing the MHC / antigen peptide complex.

[0229] The CD3 / TCR complex agonist used in step (I) of the present invention is not limited in form as long as it can be present in a manner that allows it to contact the CD3 / TCR complex on the surface of CD3-positive cells during culture. For example, it can be contained in the culture medium during culture, or it can be immobilized in the culture container, preferably immobilized in the culture container.

[0230] In the case where the medium contains a CD3 / TCR complex agonist, the medium is not particularly limited as long as it is a medium that can culture CD3-positive cells, and includes, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium (Life Technologies) and mixed mediums thereof. The medium may contain serum or may not contain serum. In the case of containing serum, the concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) may be generally 1% or more, preferably 5% or more as a lower limit, and may be generally 20% or less, preferably 15% or less as an upper limit. In addition, as needed, the culture medium may contain one or more serum substitutes such as knockout serum replacement (KSR) (a serum substitute for FBS when culturing ES cells), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), vitamin C (e.g., ascorbic acid), apotransferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, etc., and may also contain one or more substances such as lipids, amino acids, L-glutamine, L-alanyl-L-glutamine (e.g., Glutamax (registered trademark)), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics (e.g., penicillin, streptomycin), antioxidants, pyruvic acid, buffers, inorganic salts, selenic acid, progesterone and putrescine. In addition, when the culture medium contains a CD3 / TCR complex agonist, the concentration of the CD3 / TCR complex agonist may be 0.3 ng / ml or more, preferably 3 ng / ml or more, as a lower limit, and may be 10000 ng / ml or less, preferably 1000 ng / ml or less as an upper limit. In particular, when the CD3 / TCR complex agonist is an anti-CD3 agonist antibody, the concentration of the anti-CD3 agonist antibody in the culture medium is, for example, 10 ng / ml to 1000 ng / ml. Culture may be performed, for example, in a CO2 incubator, in an atmosphere of about 1 to about 10%, preferably about 2 to about 5%, at about 30 to about 40°C, preferably about 37°C.In addition, regarding the culture time in the medium containing the CD3 / TCR complex agonist, those skilled in the art can appropriately determine while monitoring the number of CD3 positive cells, etc. As long as CD3 positive cells can be obtained, the number of days is not particularly limited, for example, at least 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, preferably 16 to 72 hours. In addition, it is preferably 14 days or less, more preferably 7 days or less.

[0231] When vitamin C is used, it is preferred to add (supplement) vitamin C every 4 days, every 3 days, every 2 days, or every 1 day, and more preferably to add it every day. In one embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5ng / ml to 500ng / ml (for example, an amount equivalent to 5ng / ml, 10ng / ml, 25ng / ml, 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml). In another embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5μg / ml to 500μg / ml (for example, an amount equivalent to 5μg / ml, 10μg / ml, 25μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 300μg / ml, 400μg / ml, 500μg / ml).

[0232] The aforementioned culture medium used in step (I) of the present invention may also contain cytokines. As the cytokine contained in the culture medium, it is not particularly limited, and at least one selected from IL-7, IL-15, IL-18, and IL-21 can be cited, preferably containing all of IL-7, IL-15, IL-18, and IL-21. For the concentration of cytokines, as a lower limit, it can be more than 0.1ng / mL, preferably more than 10ng / mL, as an upper limit, it can be less than 1000ng / mL, preferably less than 300ng / mL. When using these cytokines, as the concentration in the culture medium, for example, IL-7 is 1 to 100ng / ml, IL-15 is 1 to 100ng / ml, IL-18 is 5 to 500ng / ml, and IL-21 is 2 to 200ng / ml.

[0233] The culture medium used in step (I) of the present invention may further contain TL1A and / or IL-12. In this case, the concentration of TL1A in the culture medium is 5 to 500 ng / ml, and the concentration of IL-12 in the culture medium is 5 to 500 ng / ml.

[0234] The culture medium used in step (I) of the present invention may contain TNF family cytokines as cytokines. Examples of TNF family cytokines include TNF-α, TNF-β, lymphotoxin α, Fas ligand, TRAIL, TWEAK, TL1A, RANK ligand, OX40 ligand, APRIL, AITRL, BAFF, 4-1BBL, and CD40 ligand, preferably TL1A. When TL1A is used, its concentration in the culture medium may be 5 to 500 ng / ml.

[0235] In addition, the apoptosis inhibitor used in step (I) of the present invention may include a protease inhibitor, for example, a caspase inhibitor. As a caspase inhibitor, Pan Caspase FMK inhibitor Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me)fluoromethylketone) is preferred, and its concentration in the culture medium may be 1 to 1000 μM.

[0236] In addition, when the CD3 / TCR complex agonist is immobilized on a culture container, the culture container is not particularly limited as long as it can culture CD3-positive cells, and a container, a slide, beads, or a combination thereof can be used. As the culture container, for example, a culture bottle, a culture bottle for tissue culture, a dish, a Petri dish, a dish for tissue culture, a multi-dish, a microplate, a microplate, a multi-plate, a multi-well plate, a microslide, a chamber slide, a culture dish, a tube, a tray, a culture bag, a roller bottle, and microbeads can be cited.

[0237] The solid phase of CD3 / TCR complex agonist to the culture container can be implemented based on known means. For example, the CD3 / TCR complex agonist is dissolved in a solvent (for example, PBS, etc.), added to the culture container, and left to stand overnight at 4°C, thereby the CD3 / TCR complex agonist can be solid phased in the culture container. The concentration of the CD3 / TCR complex agonist solution when the CD3 / TCR complex agonist is solid phased to the culture container can be appropriately determined by those skilled in the art according to the type of CD3 / TCR complex agonist. In one embodiment of the present invention, for example, when the CD3 / TCR complex agonist is an anti-CD3 agonist antibody or its binding fragment, usually, a solution of 0.3 to 10000 ng / ml of an anti-CD3 agonist antibody or its binding fragment can be contacted with the culture container, more preferably 3 to 5000 ng / ml, further preferably 3 to 3000 ng / ml, and particularly preferably 3 to 600 ng / ml. In another embodiment of the present invention, the concentration of the anti-CD3 agonist antibody or its binding fragment in the solution is, for example, 10 ng / ml to 10000 ng / ml, preferably 50 ng / ml to 5000 ng / ml, and more preferably 200 ng / ml to 4000 ng / ml. In another embodiment of the present invention, the concentration of the anti-CD3 agonist antibody or its binding fragment in the solution is, for example, 1 to 50000 ng / ml, preferably 3 to 30000 ng / ml, more preferably 30 to 30000 ng / ml, and even more preferably 300 to 30000 ng / ml. When the anti-CD3 agonist antibody or its binding fragment is an antibody (OKT3) produced by the OKT3 clone or its binding fragment, the concentration in the solution is, for example, 1 ng / ml to 50000 ng / ml, preferably 10 ng / ml to 10000 ng / ml, more preferably 50 ng / ml to 5000 ng / ml, and even more preferably 200 ng / ml to 4000 ng / ml. When the anti-CD3 agonist antibody or its binding fragment is an antibody produced by UCHT1 clone (UCHT1) or its binding fragment, its concentration in the solution is, for example, 1 ng / ml to 50,000 ng / ml, preferably 3 ng / ml to 30,000 ng / ml, more preferably 30 ng / ml to 30,000 ng / ml, and further preferably 300 ng / ml to 30,000 ng / ml.

[0238] In step (I) of the present invention, fibronectin is not particularly limited as long as it is a molecule that can bind to CD3-positive cells. Modified forms of fibronectin are not particularly limited as long as they are molecules that can bind to VLA-5 and VLA-4 on the surface of CD3-positive cells, and an example thereof is RetroNectin (registered trademark).

[0239] The fibronectin or its modified form used in step (I) of the present invention is not limited to the form in which it exists, as long as it can be present in contact with the surface of CD3-positive cells during culture, similarly to the CD3 / TCR complex agonist. For example, it can be contained in the culture medium during culture, or can be immobilized in the culture container, preferably immobilized in the culture container.

[0240] When the culture medium contains fibronectin or its modified form, the culture medium may be the same as the culture medium containing the CD3 / TCR complex agonist. In addition, the presence or absence of serum, additives, etc. may be the same as the culture medium containing the CD3 / TCR complex agonist. When the culture medium contains fibronectin or its modified form, the concentration of fibronectin or its modified form may be, as a lower limit, more than 10 ng / ml, preferably more than 100 ng / ml, and as an upper limit, may be less than 10000 μg / ml, preferably less than 1000 μg / ml.

[0241] In addition, when fibronectin or its modified body is solid-phased in a culture container, the culture container may be the same as the culture container for solid-phased CD3 / TCR complex agonist. In addition, the solid-phased fibronectin or its modified body to the culture container may be the same as the solid-phased CD3 / TCR complex agonist. The concentration of the solution of fibronectin or its modified body when solid-phased fibronectin or its modified body to the culture container can be appropriately determined by a person skilled in the art according to fibronectin or its modified body. For example, when fibronectin or its modified body is RetroNectin (registered trademark), it is preferred to contact a solution of 0.1 to 10000 μg / mL of RetroNectin (registered trademark) with the culture container. At this time, the concentration of the solution of RetroNectin is more preferably 0.1 to 1000 μg / mL, further preferably 1 to 300 μg / mL, and particularly preferably 1 to 150 μg / mL.

[0242] In step (I) of the present invention, the CD30 agonist is not particularly limited as long as it is a molecule that can transmit a signal from CD30 to the cell by specifically binding to CD30. Examples of the CD30 agonist include at least one selected from the group consisting of an anti-CD30 agonist antibody or a binding fragment thereof and a CD30 ligand or a binding fragment thereof.

[0243] The anti-CD30 agonist antibody and the binding fragment thereof used in step (I) of the present invention may be the same as the anti-CD3 agonist antibody and the anti-TCR antibody in terms of type and preparation method.

[0244] The CD30 ligand or its binding fragment used in step (I) of the present invention may be, for example, CD 153. The CD30 ligand or its binding fragment may be produced by the same method as the method for producing MHC and / or antigen peptide.

[0245] The CD30 agonist used in step (I) of the present invention is not limited in form as long as it can be in contact with CD30 during culture, similarly to the CD3 / TCR complex agonist. For example, it may be contained in the culture medium during culture or may be immobilized in the culture container, but is preferably contained in the culture medium.

[0246] When the culture medium contains a CD30 agonist, the culture medium may be the same as the culture medium containing a CD3 / TCR complex agonist. In addition, the presence or absence of serum, additives, etc. may be the same as the culture medium containing a CD3 / TCR complex agonist. When the culture medium contains a CD30 agonist, the concentration of the CD30 agonist in the culture medium may be appropriately determined by a person skilled in the art according to the type of CD30 agonist. For example, when the CD30 agonist is an anti-CD30 agonist antibody or a binding fragment thereof, the concentration of the anti-CD30 agonist antibody or a binding fragment thereof in the culture medium may generally be 1 ng / ml to 10000 ng / ml, preferably 1 ng / ml to 1000 ng / ml, more preferably 3 ng / ml to 300 ng / ml, and further preferably 30 ng / ml to 300 ng / ml.

[0247] In addition, when the CD30 agonist is solid-phased in the culture container, the culture container may be the same as the culture container in which the CD3 / TCR complex agonist is solid-phased. In addition, the method for solid-phased CD30 agonist in the culture container may be the same as the method for solid-phased CD3 / TCR complex agonist. The concentration of the CD30 agonist solution when the CD30 agonist is solid-phased in the culture container may be 0.1 ng / ml or more, preferably 1 ng / ml or more as a lower limit, and may be 10000 ng / ml or less, preferably 1000 ng / ml or less as an upper limit.

[0248] The production method or expansion culture method of the present invention may further include a step of culturing the CD3-positive cells cultured in step (I) of the present invention in the absence of a CD3 / TCR complex agonist and fibronectin or a modified form thereof, and in the presence of a CD30 agonist (hereinafter referred to as step (II) of the present invention).

[0249] The culture medium in step (II) of the present invention can use the culture medium used in step (I) above, and the culture medium may contain serum or may not contain serum. When serum is contained, the concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) may be generally 1% or more, preferably 5% or more as a lower limit, and may be generally 20% or less, preferably 15% or less as an upper limit. In addition, as required, the culture medium may contain, for example, one or more serum replacements selected from Knockout serum replacement (KSR) (a serum replacement for FBS when culturing ES cells), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, insulin, transferrin, selenium compounds (e.g., sodium selenite), vitamin C (e.g., ascorbic acid), apotransferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, etc., and may contain one or more substances selected from lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, selenic acid, progesterone, and putrescine, etc. The culture may be performed, for example, in a CO2 incubator in an atmosphere of about 1 to about 10%, preferably about 2 to about 5%, at about 30 to about 40°C, preferably about 37°C. In addition, regarding the culture time, those skilled in the art can appropriately determine it while monitoring the number of CD3-positive cells, etc. As long as CD3-positive cells can be obtained, the number of days is not particularly limited, for example, at least 3 days or more, 5 days or more, 7 days or more, 10 days or more, 14 days or more, 21 days or more, preferably 7 days or more and 15 days or less. In addition, preferably 30 days or less, more preferably 21 days or less.

[0250] When vitamin C is used, it is preferred to add (supplement) vitamin C every 4 days, every 3 days, every 2 days, or every 1 day, and more preferably to add it every day. In one embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5ng / ml to 500ng / ml (for example, an amount equivalent to 5ng / ml, 10ng / ml, 25ng / ml, 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 400ng / ml, 500ng / ml). In another embodiment, the vitamin C is added to the culture medium in an amount equivalent to 5μg / ml to 500μg / ml (for example, an amount equivalent to 5μg / ml, 10μg / ml, 25μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 300μg / ml, 400μg / ml, 500μg / ml).

[0251] The aforementioned culture medium used in step (II) of the present invention may also contain cytokines. As cytokines, there is no particular limitation, and at least one selected from IL-7, IL-15, IL-18, and IL-21 may be contained, preferably all of IL-7, IL-15, IL-18, and IL-21. For the concentration of cytokines, as a lower limit, it can be more than 0.1ng / mL, preferably more than 10ng / mL, and as an upper limit, it can be less than 1000ng / mL, preferably less than 300ng / mL. When using these cytokines, as the concentration in the culture medium, for example, IL-7 can be 1ng / ml~100ng / ml, IL-15 can be 1ng / ml~100ng / ml, IL-18 can be 5ng / ml~500ng / ml, and IL-21 can be 2ng / ml~200ng / ml.

[0252] The culture medium used in step (II) of the present invention may further contain TL1A and / or IL-12. In this case, the concentration of TL1A in the culture medium may be 5 ng / ml to 500 ng / ml, and the concentration of IL-12 in the culture medium may be 5 ng / ml to 500 ng / ml.

[0253] The aforementioned culture medium used in step (II) of the present invention may further contain an apoptosis inhibitor in the culture medium. Examples of apoptosis inhibitors include protease inhibitors, for example, caspase inhibitors. As a caspase inhibitor, Pan Caspase FMK inhibitor Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me)fluoromethylketone) (hereinafter sometimes referred to as "Z-VAD-FMK") is preferred, and its concentration in the culture medium may be 1 μM to 1000 μM, preferably 1 μM to 500 μM, more preferably 1 μM to 200 μM, and particularly preferably 1 μM to 50 μM.

[0254] In one embodiment of the present invention, the above steps (I) and (II) may be repeated in this order.

[0255] The present invention also provides CD3-positive cells obtained by the production method or expansion culture method of the present invention (hereinafter referred to as CD3-positive cells of the present invention). The CD3-positive cells of the present invention are the same as the CD3-positive cells obtained by the production method or expansion culture method of the present invention.

[0256] The present invention also provides a kit for expanding and culturing CD3-positive cells comprising the following (1) and (2) (hereinafter referred to as the kit of the present invention).

[0257] (1) a culture container in which a CD3 / TCR complex agonist and

[0258] A culture container in which fibronectin or a modified form thereof is immobilized; and

[0259] (2) a culture medium containing a CD30 agonist

[0260] The CD3 / TCR complex agonist, fibronectin or a modified form thereof, CD30 agonist, culture container and culture medium contained in the kit of the present invention may be the same as those described in the production method or expansion culture method of the present invention.

[0261] The present invention also provides a method for maintaining CD3-positive cells as CD3-positive CD197-positive cells (hereinafter referred to as the maintenance method of the present invention), the method comprising the step of stimulating CD30 signals in CD3-positive cells.

[0262] Usually, when naive T cells from peripheral blood are stimulated by antigens presented to dendritic cells, the intracellular signals of naive T cells are activated, and they proliferate and mature repeatedly, and differentiate into stem cell-like memory T cells, central memory T cells, effector memory T cells, and effector T cells in sequence. In the case of effector T cells, cytokines are produced in the case of CD3-positive CD4-positive CD8-negative cells (helper T cells), and CD3-positive CD4-negative CD8-positive cells (cytotoxic T cells) kill target cells through antigens, but cannot survive for a long time. However, by stimulating the CD30 signal in T cells, naive T cells that can differentiate into effector T cells and proliferate autonomously or stem cell-like memory T cells (CD197-positive, CD45RA-positive) or central memory T cells (CD197-positive, CD45RA-negative) can be maintained.

[0263] In the maintenance method of the present invention, the cultured CD3-positive cells may be the same as the CD3-positive cells cultured in the production method or the expansion culture method of the present invention. The CD3-positive cells are preferably CD3-positive CD197-positive cells.

[0264] The maintenance method of the present invention includes a step of stimulating CD30 signals in CD3-positive cells. The method of stimulating CD30 signals in CD3-positive cells is not particularly limited as long as it can transmit the signal to the downstream via the intracellular domain of CD30. As such a method, for example, a method including the step of culturing CD3-positive cells in the presence of a CD30 agonist can be cited (hereinafter, described as the maintenance method (1) of the present invention). By the maintenance method (1) of the present invention, CD3-positive cells can be maintained as CD3-positive CD197-positive cells.

[0265] In one embodiment of the maintenance method (1) of the present invention, the CD3-positive cells can be maintained as CD3-positive CD197-positive CD45RA-negative cells.

[0266] The CD30 agonist and culture conditions used in the maintenance method (1) of the present invention may be the same as those used in the production method or expansion culture method of the present invention.

[0267] The present invention also provides a kit for maintaining CD3-positive cells as CD3-positive CD197-positive cells (hereinafter referred to as the maintenance kit of the present invention), the kit comprising a CD30 agonist.

[0268] The CD30 agonist contained in the maintenance kit of the present invention may be the same as the CD30 agonist used in the maintenance method (1) of the present invention.

[0269] In addition, in the maintenance method of the present invention, as another method for stimulating the CD30 signal in CD3-positive cells, there can be mentioned a method comprising the step of culturing CD3-positive cells expressing a chimeric antigen receptor comprising the intracellular domain of CD30 in the presence of an antigen that stimulates the chimeric antigen receptor (hereinafter referred to as the maintenance method (2) of the present invention). By stimulating the chimeric antigen receptor comprising the intracellular domain of CD30, the signal can be transmitted downstream via the intracellular domain of CD30, so that the CD3-positive cells expressing the chimeric antigen receptor comprising the intracellular domain of CD30 can be maintained as CD3-positive CD197-positive cells.

[0270] In one embodiment of the maintenance method (2) of the present invention, CD3-positive cells expressing a chimeric antigen receptor comprising the intracellular domain of CD30 can be maintained as CD3-positive CD197-positive CD45RA-negative cells.

[0271] The culture conditions used in the maintenance method (2) of the present invention may be the same as the culture conditions used in the production method or expansion culture method of the present invention.

[0272] The antigen that stimulates the chimeric antigen receptor used in the maintenance method (2) of the present invention is not particularly limited as long as it can transmit the signal to the downstream via the intracellular domain of CD30 contained in the chimeric antigen receptor. The antigen that stimulates the chimeric antigen receptor may be, for example, the same antigen as that targeted by the chimeric antigen receptor of the CD3-positive cells expressing the chimeric antigen receptor cultured in the production method or expansion method of the present invention.

[0273] The present invention also provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain of the chimeric antigen receptor (hereinafter referred to as the chimeric antigen receptor of the present invention) comprises the intracellular domain of CD30 or a modified form thereof.

[0274] The antigen-binding domain contained in the chimeric antigen receptor of the present invention may be the same as the antigen-binding domain contained in the chimeric antigen receptor expressed in the CD3-positive cells cultured in the production method or expansion method of the present invention.

[0275] The transmembrane domain contained in the chimeric antigen receptor of the present invention may be the same as the transmembrane domain contained in the chimeric antigen receptor expressed in the CD3-positive cells cultured in the production method or expansion method of the present invention.

[0276] The intracellular signal transduction domain contained in the chimeric antigen receptor of the present invention comprises the intracellular domain of CD30 or a modified form thereof. The intracellular domain of CD30 is not particularly limited as long as the function of the antigen-binding domain contained in the chimeric antigen receptor of the present invention to recognize an antigen and transmit its recognition signal into the cell is maintained, and an example thereof includes a peptide comprising the amino acid sequence represented by SEQ ID NO: 6.

[0277] The modified form of the intracellular domain of CD30 is not limited as long as the above-mentioned function is maintained, and examples thereof include peptides comprising an amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more homology to the amino acid sequence shown in SEQ ID NO: 6. Here, the "homology" may be the same as the homology in the amino acid sequence of "IL-15 / IL-15Rα".

[0278] In addition, modified forms of the intracellular domain of CD30 include, for example, the following amino acid sequences: (1) an amino acid sequence in which one or two or more (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to more (2, 3, 4 or 5)) amino acids are deleted from the amino acid sequence represented by SEQ ID NO: 6; (2) an amino acid sequence in which one or two or more (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably one to more (2, 3, 4 or 5)) amino acids are added to the amino acid sequence represented by SEQ ID NO: 6; (5) an amino acid sequence obtained by inserting one or more (preferably about 1 to 50, preferably about 1 to 10, and more preferably one to more (2, 3, 4 or 5)) amino acids into the amino acid sequence represented by SEQ ID NO: 6; (4) an amino acid sequence obtained by replacing one or more (preferably about 1 to 50, preferably about 1 to 10, and more preferably one to more (2, 3, 4 or 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 6 with other amino acids; or (5) an amino acid sequence obtained by combining the above; etc. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the function of the intracellular domain of CD30 is maintained.

[0279] The intracellular signal transduction domain contained in the chimeric antigen receptor of the present invention may include an intracellular domain derived from another protein in addition to the intracellular domain of CD30 or its modified form. The further included intracellular domain may be the same as the intracellular domain contained in the intracellular signal transduction domain contained in the chimeric antigen receptor expressed in the CD3-positive cells cultured in the production method or expansion culture method of the present invention. The further included intracellular domain is not particularly limited, but is preferably an intracellular domain derived from CD28 or CD3 ζ chain.

[0280] The chimeric antigen receptor of the present invention may incorporate a spacer sequence between the antigen binding domain and the transmembrane domain, or between the intracellular signal transduction domain and the transmembrane domain. The spacer sequence may be the same as the spacer sequence contained in the chimeric antigen receptor expressed in the CD3-positive cells cultured in the production method or expansion culture method of the present invention.

[0281] As the chimeric antigen receptor of the present invention, specifically, there can be mentioned a chimeric antigen receptor comprising a scFv that recognizes CD19 as an antigen binding domain, a transmembrane domain of CD8 as a transmembrane domain, and an intracellular domain from CD28, an intracellular domain from CD30, and an intracellular domain from the CD3ζ chain as an intracellular signal transduction domain. The order of the above-mentioned intracellular domains contained in the intracellular signal transduction domain is not particularly limited, and for example, the intracellular domain from CD28, the intracellular domain from CD30, and the intracellular domain from the CD3ζ chain can be contained in this order. More specifically, the chimeric antigen receptor of the present invention comprises, for example, the amino acid sequence represented by SEQ ID NO: 7.

[0282] The chimeric antigen receptor of the present invention may be a protein synthesized chemically or biochemically in a cell-free translation system, or may be a recombinant protein produced by a transformant into which a nucleic acid having a base sequence encoding the chimeric antigen receptor of the present invention has been introduced. The chimeric antigen receptor of the present invention can be produced, isolated and purified in the same manner as the method for producing MHC and / or antigen peptides used in the production method or expansion culture method of the present invention.

[0283] The present invention also provides a nucleic acid comprising a polynucleotide encoding the chimeric antigen receptor of the present invention (hereinafter referred to as the nucleic acid of the present invention).

[0284] The nucleic acid of the present invention may be DNA, RNA, or a DNA / RNA chimera. Preferably, DNA is selected. In addition, the nucleic acid may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA, or a hybrid of DNA:RNA. In the case of single-stranded, it may be a sense strand (i.e., a coding strand) or an antisense strand (i.e., a non-coding strand).

[0285] The nucleic acid of the present invention can be obtained by polymerase chain reaction (hereinafter referred to as "PCR method"). First, genomic DNA or cDNA encoding each domain in the antigen binding domain, transmembrane domain and intracellular signal transduction domain contained in the chimeric antigen receptor of the present invention is obtained. In the case of cDNA, the total RNA or mRNA component prepared from cells can also be used as a template, respectively, and directly amplified by PCR method and reverse transcriptase PCR (hereinafter referred to as "RT-PCR method"). The obtained genomic DNA or cDNA can be used as a template, and a synthetic DNA primer composed of a part of the base sequence encoding each domain in the antigen binding domain, transmembrane domain and intracellular signal transduction domain, and a base sequence encoding a spacer sequence is used to amplify the nucleic acid encoding each domain by PCR method. The obtained domains can be used as templates for each other, and the aforementioned synthetic DNA primers are used to repeatedly implement the PCR method to obtain the nucleic acid of the present invention.

[0286] The present invention also provides a chimeric antigen receptor gene introduction vector containing the nucleic acid of the present invention (hereinafter referred to as the gene introduction vector of the present invention).

[0287] The gene introduction vector of the present invention can be prepared, for example, by connecting the nucleic acid of the present invention to the downstream of the promoter in the appropriate gene introduction vector. The gene introduction vector, promoter, and other elements can be the same as the vector, promoter, and other elements used when introducing exogenous TCR into pluripotent stem cells in the manufacturing method or expanded culture method of the present invention.

[0288] The present invention also provides a chimeric antigen receptor-expressing cell containing the gene-transfer vector of the present invention (hereinafter referred to as the chimeric antigen receptor-expressing cell of the present invention).

[0289] The chimeric antigen receptor expressing cell of the present invention can be produced by introducing the gene introduction vector of the present invention into cells and culturing them. The cells into which the gene introduction vector of the present invention is introduced can include CD3 positive cells or their progenitor cells (hematopoietic stem cells, common lymphoid progenitor cells, lymphoblasts, etc.) or pluripotent stem cells. As pluripotent stem cells, ES cells, iPS cells, EC cells, EG cells can be mentioned, and ES cells or iPS cells can be preferably mentioned.

[0290] As a method for introducing the gene transfer vector of the present invention into cells, introduction can be carried out by lipofection, liposome, microinjection, or the like.

[0291] The chimeric antigen receptor expressing cells of the present invention are preferably CD3 positive cells. The aforementioned CD3 positive cells are preferably CD3 positive CD8 positive cells (CD3 positive CD8 positive CD4 positive cells or CD3 positive CD8 positive CD4 negative cells), and more preferably CD3 positive CD8 positive CD4 negative cells. In addition, as other preferred aforementioned CD3 positive cells, CD3 positive CD4 positive cells (CD3 positive CD4 positive CD8 positive cells or CD3 positive CD4 positive CD8 negative cells) can be cited.

[0292] When the chimeric antigen receptor-expressing cells of the present invention are cells produced by introducing the gene introduction vector of the present invention into pluripotent stem cells, the pluripotent stem cells can be differentiated into CD3-positive cells according to a method known per se. The specific method for differentiating pluripotent stem cells into CD3-positive cells can be the same as the method for differentiating cultured pluripotent stem cells into CD3-positive cells in the production method or expansion culture method of the present invention.

[0293] The chimeric antigen receptor-expressing cells of the present invention obtained as described above are endowed with specificity for the target antigen via the chimeric antigen receptor, and therefore can exhibit cell-damaging activity against tumors expressing the target antigen. In addition, the chimeric antigen receptor can directly recognize antigen molecules independently of HLA class I or class II, and therefore, the chimeric antigen receptor-expressing cells of the present invention can induce a high level of immune response even against tumors in which the expression of HLA class I or class II genes is reduced.

[0294] Therefore, the present invention also provides a drug containing the chimeric antigen receptor expressing cell of the present invention as an active ingredient (hereinafter, recorded as the drug of the present invention). The drug containing the chimeric antigen receptor expressing cell of the present invention can be used for the prevention or treatment of tumors expressing an antigen recognized by the chimeric antigen receptor of the present invention, and can be administered to, for example, mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, humans), preferably to humans. Therefore, in one embodiment of the present invention, a drug of the present invention is provided, which is used for use in the prevention or treatment of tumors expressing an antigen recognized by the chimeric antigen receptor of the present invention. In addition, a method for preventing or treating tumors expressing an antigen recognized by the chimeric antigen receptor of the present invention is provided, which includes administering the chimeric antigen receptor expressing cell of the present invention, preferably in the form of a drug containing the cell.

[0295] The tumors to be prevented or treated using the drug of the present invention or the chimeric antigen receptor-expressing cells of the present invention are not particularly limited as long as they express an antigen recognized by the chimeric antigen receptor of the present invention. Here, the so-called tumors include benign tumors, malignant tumors (also called "cancer"), and tumors that may be diagnosed or determined to be benign or malignant, as described in, for example, "Daniel Baumhoer et al., Am J. Clin Pathol, 2008, 129, 899-906". Specific examples of tumors include liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., ovarian clear cell adenocarcinoma), childhood cancer, lung cancer (e.g., squamous cell carcinoma, small cell lung carcinoma), testicular cancer (e.g., non-seminomatous embryonal cell tumor), soft tissue tumors (e.g., liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g., cervical intraepithelial neoplasia, cervical squamous cell carcinoma), melanoma, adrenal tumors (e.g., adrenal adenoma), neurological tumors (e.g., schwannoma), gastric cancer (e.g., gastric adenocarcinoma), kidney cancer (e.g., Grawitz tumor), breast cancer (e.g., invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g., medullary carcinoma), laryngeal cancer (e.g., squamous cell carcinoma), bladder cancer (e.g., invasive transitional cell carcinoma), etc., but are not limited to these.

[0296] The chimeric antigen receptor expressing cells of the present invention can be cultured and / or stimulated using appropriate culture media and / or stimulatory molecules before administration to a subject. Examples of stimulatory molecules include cytokines, appropriate proteins, and other components, but are not limited to these. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, IFN-γ, and the like, and IL-2 can be preferably used. The concentration of IL-2 in the culture medium is not particularly limited, and is preferably, for example, 0.01 U / ml to 1×10 5 U / ml, more preferably 1U / ml to 1×10 4 U / ml. In addition, as suitable proteins, for example, antigens recognized by chimeric antigen receptors, CD3 ligands, CD28 ligands, and anti-IL-4 antibodies can be exemplified. In addition, lymphocyte stimulating factors such as lectins can also be added. In addition, serum and plasma can be added to the culture medium. The amount of addition to the culture medium is not particularly limited, and 0% to 20% by volume can be exemplified, and the amount of serum and plasma used can also be changed according to the culture stage. For example, the serum or plasma concentration can also be reduced step by step for use. As the source of serum or plasma, autologous or non-autologous is possible, but from the perspective of safety, serum or plasma of autologous origin is preferred.

[0297] The drug of the present invention is preferably administered to a subject non-orally. Examples of non-oral administration methods include intravenous, intra-arterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage can be appropriately selected according to the subject's condition, weight, age, etc., and is usually 1×10 per dose for a subject weighing 60 kg. 6 ~1×10 10 , preferably 1×10 7 ~1×10 9 , more preferably 5×10 7 ~5×10 8 The drug of the present invention can be administered in a single manner. In addition, it can be administered once or multiple times. The drug of the present invention can be prepared into a known form suitable for non-oral administration, such as an injection or an infusion. In addition, the drug of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc. in order to stably maintain cells. As a culture medium, there is no particular limitation, and culture media such as RPMI, AIM-V, and X-VIVO10 can be cited, but are not limited to these. In addition, a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. can be added to the drug for the purpose of stabilization.

[0298] Furthermore, the chimeric antigen receptor-expressing cells of the present invention can kill cells expressing an antigen recognized by the chimeric antigen receptor of the present invention, and therefore can be used as a killer of cells expressing the antigen. The killer can be prepared and used in the same manner as the above-mentioned drugs.

[0299] In addition, the present invention also includes the following embodiments: the use of the chimeric antigen receptor expressing cells of the present invention in the manufacture of a preventive or therapeutic agent for a tumor expressing an antigen recognized by the chimeric antigen receptor of the present invention, based on a drug containing the chimeric antigen receptor expressing cells of the present invention. The preventive or therapeutic agent for a tumor can be manufactured by a method known per se. For example, it can be manufactured in a known form suitable for parenteral administration, such as an injection or an infusion, in the same manner as the method for preparing the drug of the present invention described above.

[0300] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are merely illustrative and the present invention is not limited to these Examples.

[0301] Example

[0302] [Example 1]

[0303] 1. Preparation of iPS Cells

[0304] iPS cells used were the Ff-I01s04 strain provided by the iPS Cell Research Institute (CiRA) of Kyoto University. iPS cell culture was performed according to the protocol “Feeder-free culture of human iPS cells” published by CiRA.

[0305] 2. Introducing T cell receptor (TCR) genes into iPS cells

[0306] The TCR gene used was the HLA-A*24:02-restricted WT1-specific TCR (WT1-TCR) gene derived from TAK1 provided by Professor Masataka Yasukawa of the Graduate School of Medicine, Ehime University. Gene introduction into iPS cells was performed by incorporating the CS-UbC-RfA-IRES2-hKO1 lentiviral vector provided by the RIKEN Institute and infecting iPS cells with it.

[0307] 3. Differentiation of iPS cells into CD8-positive T cells (CTLs) after introduction of the WT1-TCR gene

[0308] The differentiation of iPS cells into CD8-positive T cells (CTLs) introduced with the WT1-TCR gene was carried out according to a known method (WO2017 / 221975). The cells were CD3-positive and CD8-positive. Hereinafter, the cells were used as T cells derived from iPS cells.

[0309] 4. Reagents and Antibodies

[0310] RetroNectin (registered trademark) was purchased from Takara Bio. As the anti-CD3 agonist antibody, functional grade purified anti-human CD3 (clone: ​​OKT3) purchased from eBioscience or CD3 antibody (clone: ​​UCHT1) purchased from GeneTex was used. Unless otherwise specified, OKT3 was used. As the anti-CD30 agonist antibody, CD30 agonist antibody purchased from R&D was used.

[0311] 5. Immobilize anti-CD3 agonist antibody and RetroNectin (registered trademark) on the culture plate

[0312] Anti-CD3 agonist antibody and RetroNectin (registered trademark) dissolved in PBS at required concentrations were added to a 96-well plate at 50 μL / well, and the plate was left to stand overnight at 4° C. After washing with PBS, the plate was used for the test.

[0313] 6. ELISA for detecting solid-phase anti-CD3 agonist antibodies and solid-phase RetroNectin (registered trademark)

[0314] The solid-phase anti-CD3 agonist antibody was detected using the HRP-conjugated goat anti-mouse IgG2a detection antibody included in the MouseIgG2a ELISA Quantitation Set purchased from Bethyl Laboratorie. The solid-phase RetroNectin (registered trademark) was detected using the peroxidase-labeled anti-RetroNectin antibody included in the RetroNectin EIA kit purchased from Takara Bio. After adding each detection antibody to the solid-phase plate, let it stand at room temperature for 1 hour. After washing with PBS containing 0.05% Tween 20, add 1-Step TM Ultra TMB-ELISA substrate solution (ThermoFisher) was allowed to stand at room temperature for 15 minutes, 1 M sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured using a microplate reader.

[0315] 7. Proliferation Assay

[0316] T cells derived from iPS cells whose cell number was adjusted to 100,000 cells / 200 μL using a culture medium (which was prepared by adding the cytokines listed in Table 1 to an α-MEM medium containing 15% FBS) were seeded on a culture plate on which anti-CD3 agonist antibodies (3, 30, 300, 3000 ng / ml) and RetroNectin (registered trademark) (0, 1.85, 2.25, 16.7, 50, 150 μg / ml) were solid-phased, and cultured at 5% CO2 / 37°C for 3 days.

[0317] On the third day of culture, cells were collected from the culture plate, the number of cells was counted using TC20 (Bio-rad), and an appropriate amount was suspended in a medium obtained by adding the cytokines and the like in Table 2 to an α-MEM medium containing 15% FBS, added to a 96-well plate without solid phase, and cultured at 5% CO2 / 37°C. Then, cells were collected from the culture plate once at any time on the 5th, 6th, 7th, 8th, 9th, 10th, 12th, 14th, and 16th days of culture, for a total of 4 to 7 times, the number of cells was counted, and an appropriate amount was suspended, added to a culture plate without solid phase, and cultured at 5% CO2 / 37°C.

[0318] [Table 1]

[0319]

[0320] [Table 2]

[0321]

[0322] 8. ATP test

[0323] ATP in the cells and culture supernatant on day 12 of the proliferation assay was measured using CellTiter-Glo(R) Luminescent Cell Viability Assay purchased from Promega according to a standard protocol.

[0324] 9. Determination of WT1 antigen-specific cell damage activity

[0325] HLA-A*24:02 positive LCL cells were purchased from the RIKEN Bio-Resource Center. They were cultured using RPMI1640 medium containing 10% FBS. WT1 antigen peptide (CMTWNQMNL: sequence number 3) was synthesized by SCRUM Inc. Cell damage test was performed using DELFIA Immunoassay purchased from PerkinElmer and evaluated according to the standard protocol.

[0326] 10. Proliferation assay with anti-CD30 agonist antibody

[0327] Anti-CD30 agonist antibodies diluted to a final concentration of 0, 30, 100, or 300 ng / mL were added to the culture medium in which the cells were suspended. Other than this, the assay was performed in the same manner as in "7. Proliferation assay".

[0328] In addition, as a proliferation test of iPS cell-derived T cells stimulated multiple times by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody, anti-CD30 agonist antibody diluted to a final concentration of 100 ng / mL was added to the culture medium when the cells were suspended. Other than this, the same method as in "7. Proliferation test" was used.

[0329] 11. Detection of CD197 and CD45RA on the cell membrane surface

[0330] The cells were collected on the 7th day of culture and stained with the antibodies listed in Table 3. TM The detection was performed using X-20 flow cytometer (BD Bioscience).

[0331] [Table 3]

[0332] Target Fluorescent Pigments clone Suppliers CD5 APC / Cy7 UCHT2 BioLegend CD8a PerCP / Cy5.5 SK1 BioLegend CD8b PE / Cy7 Sid18bee eBioscience CD197 AF647 G043H7 BioLegend CD45RA FITC HI100 BioLegend

[0333] [Test Example 1]

[0334] 1. Determination of the concentration of anti-CD3 agonist antibodies suitable for immobilization on culture plates and RetroNectin (registered trademark)

[0335] The concentrations of anti-CD3 agonist antibody and RetroNectin (registered trademark) suitable for immobilization on culture plates were determined by ELISA. Figure 1 ). The anti-CD3 agonist antibody was solid-phased in a concentration-dependent manner between 3 ng / ml and 3000 ng / mL. RetroNectin (registered trademark) was solid-phased in a concentration-dependent manner between 16.7 μg / ml and 150 μg / mL.

[0336] 2. Verification of the concentration range of the immobilized anti-CD3 agonist antibody and RetroNectin (registered trademark) required for the proliferation of iPS cell-derived T cells

[0337] After 3 days of stimulation on culture plates immobilized with anti-CD3 agonist antibody (0, 3, 30, 300, 3000 ng / mL) and RetroNectin (registered trademark) (0, 1.85, 5.56, 16.7, 50, 150 μg / mL), iPS cell-derived T cells were cultured on non-immobilized plates for 9 days, and the ATP level was measured ( Figure 2 ).

[0338] 3. Proliferation test of iPS cell-derived T cells stimulated by immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0339] T cells derived from iPS cells adjusted to 100,000 cells / 200 μL were stimulated for 3 days on a 96-well plate to which anti-CD3 agonist antibody (3000 ng / mL) and RetroNectin (registered trademark) (150 μg / mL) were immobilized, or with anti-CD3 / CD28 beads (Dynabeads) added in a ratio of 1:1 between the number of iPS cell-derived T cells and the number of beads, and the number of cells when cultured without stimulation was counted over time ( Figure 3 ). Compared with anti-CD3 / CD28 beads, iPS cell-derived T cells proliferated more when stimulated with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark).

[0340] 4. Proliferation test of iPS cell-derived T cells stimulated multiple times with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0341] The cell proliferation response of iPS cell-derived T cells to multiple stimulations with solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) was confirmed. A single proliferation test was performed by stimulating iPS cell-derived T cells on a solid-phased culture plate for 3 days and then culturing them on a non-solid-phased plate for 11 to 15 days. After the test, the cell number was adjusted and provided for the next test. iPS cell-derived T cells also responded to the fourth stimulation with solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) and proliferated ( Figure 4 ).

[0342] 5. Verification of WT1 antigen-specific cell-damaging activity of T cells derived from iPS cells introduced with WT1-TCR gene and proliferated by stimulation with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0343] The WT1 antigen-specific cell-damaging activity of the following cells was evaluated: the cells were T cells derived from iPS cells into which the WT1-TCR gene was introduced and proliferated by stimulation from a solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark). The WT1 antigen-specific cell-damaging activity was evaluated based on the difference between the damaging activity against LCL to which the WT1 antigen peptide was added and the damaging activity against LCL to which the WT1 antigen peptide was not added. T cells derived from iPS cells into which the WT1-TCR gene was introduced had almost no nonspecific cell-damaging activity and only showed WT1 antigen-specific cell-damaging activity ( Figure 5 ).

[0344] 6. Proliferation assay of iPS cell-derived T cells stimulated with anti-CD30 agonist antibody

[0345] We verified whether the addition of anti-CD30 agonist antibody (0, 30, 100, 300 ng / mL) has an effect on the proliferation of T cells derived from iPS cells during stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark). We used a solid-phase 96-well plate in which anti-CD3 agonist antibody (3000 ng / ml) and RetroNectin (registered trademark) (150 μg / ml) were solid-phased. T cells derived from iPS cells further proliferated due to the addition of anti-CD30 agonist antibody. Furthermore, they proliferated in a manner that depended on the concentration of the added anti-CD30 agonist antibody ( Figure 6 ).

[0346] 7. Proliferation test of iPS cell-derived T cells after multiple stimulations with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0347] We verified whether iPS cell-derived T cells showed proliferation responses to multiple stimulations from solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody. They were seeded on culture plates solid-phased with anti-CD3 agonist antibody (3000 ng / ml) and RetroNectin (registered trademark) (150 μg / ml) and cultured at 5% CO2 / 37°C for 3 days.

[0348] On the third day of culture, cells were collected from the culture plate, the number of cells was counted using TC20 (Bio-rad), and an appropriate amount was suspended in a medium obtained by adding the cytokines and the like in Table 2 to an α-MEM medium containing 15% FBS, and added to a culture plate without solid phase, and cultured at 5% CO2 / 37°C. Then, cells were collected from the culture plate once each on the 6th, 7th, 9th, 10th, 14th, and 16th days of culture, the number of cells was counted, and an appropriate amount was suspended, added to a culture plate without solid phase, and cultured at 5% CO2 / 37°C. A culture solution in which an anti-CD30 agonist antibody diluted to a final concentration of 100 ng / mL was added to the culture solution, and a culture solution to which an anti-CD30 agonist antibody was not added was used. The stimulation until the above-mentioned culture day 0 to 16 was repeated twice. Even in the second stimulation, iPS cell-derived T cells further proliferated due to stimulation with the solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody, compared with stimulation with the solid-phased anti-CD3 agonist antibody / RetroNectin (registered trademark) alone ( Figure 7 ).

[0349] 8. Detection of CD197 and CD45RA on the membrane surface of iPS cell-derived T cells after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0350] Using flow cytometry, we measured the expression of CD197 and CD45RA on the membrane surface of iPS cell-derived T cells 7 days after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) or with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody. In the cell population stimulated with anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody, CD197-positive CD45RA-negative central memory-like iPS cell-derived T cells accounted for more than half ( Figure 8 ).

[0351] 9. Verification of WT1 antigen-specific cell-damaging activity of T cells derived from iPS cells into which the WT1-TCR gene was introduced and proliferated by stimulation with immobilized anti-CD3 agonist antibody / RetroNectin and anti-CD30 agonist antibody

[0352] T cells derived from iPS cells into which the WT1-TCR gene had been introduced and proliferated by stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody were applied to HLA-A*24:02-positive LCL cells in the presence or absence of WT1 antigen peptides to evaluate WT1 antigen-specific cell-damaging activity. T cells derived from iPS cells into which the WT1-TCR gene had been introduced had almost no nonspecific cell-damaging activity and only showed WT1 antigen-specific cell-damaging activity ( Fig. 9 In addition, its WT1 antigen-specific cell-damaging activity was still maintained after proliferation caused by the second stimulation ( Fig. 9 ).

[0353] [Example 2]

[0354] 1. Preparation of CD8-positive T cells from human peripheral blood

[0355] Human peripheral blood was purchased as peripheral blood mononuclear cells from healthy subjects from Precision Bioservices, Inc. CD8+ T cells were isolated using a human CD8+ T cell isolation kit (CD8+ T cell isolation kit, human, Milteny).

[0356] 2. Proliferation test of CD8 positive T cells from human peripheral blood

[0357] The test was carried out in the same manner as in [Example 1] 7. Growth test.

[0358] 3. Cultivation of CD8-positive T cells from human peripheral blood for mouse implantation studies

[0359] To CD8-positive T cells derived from human peripheral blood suspended in 3 kinds of culture media obtained by adding the additives shown in Table 4 to α-MEM culture medium containing 15% FBS at a final concentration of 100,000 cells / mL, Dynabeads T-Activator CD3 / CD28 (gibco) adjusted in a manner that is 3 times the number of cells was added, and cultured at 5% CO2 / 37°C for 3 days. The cells were recovered from the culture plate on the third day of culture, suspended in a medium obtained by adding the cytokines in Table 5 to α-MEM culture medium containing 15% FBS, and cultured at 5% CO2 / 37°C. Then, cells were recovered from the culture plate on the 10th day of culture, the number of cells was counted, and the cells were suspended in an appropriate amount and administered to mice. In addition, cells were recovered from the culture plate on the 6th, 10th, 13th, and 17th days of culture, suspended in an appropriate amount, and provided for CD197 expression experiments.

[0360] [Table 4]

[0361]

[0362] w: Utilization (+ indicates the additives added to each group.)

[0363] [Table 5]

[0364]

[0365] w: Utilization (+ indicates the additives added to each group.)

[0366] 4. Administration of CD8-positive T cells derived from human peripheral blood to mice and recovery of transplanted cells

[0367] Eight-week-old male NSG mice (Charles River Japan) irradiated with 2 Gy of gamma rays on the day of cell administration were used. 5,000,000 cells prepared by culturing CD8-positive T cells derived from human peripheral blood for mouse implantation experiments in [Example 2] 3. were intravenously administered to the mice and then cultured for 4 weeks. The mice were then euthanized and bone marrow cells, spleen cells, and white blood cells in the blood were collected from the mice.

[0368] 5. Detection of CD8-positive T cells derived from human peripheral blood implanted in mouse tissues The recovered cells were stained with the antibodies listed in Table 3 and then analyzed using LSRFortessa TM X-20 (BD Bioscience) flow cytometer was used for detection.

[0369] [Test Example 2]

[0370] 1. Proliferation test of CD8-positive T cells from human peripheral blood stimulated by anti-CD30 agonist antibody

[0371] We examined whether the addition of an anti-CD30 agonist antibody affects the proliferation of CD8-positive T cells from human peripheral blood when stimulated by solid-phase anti-CD3 antibody / RetroNectin (registered trademark). The addition of an anti-CD30 agonist antibody increases the proliferation of CD8-positive T cells in human peripheral blood ( Fig.10 ).

[0372] 2. Detection of CD197 on the membrane surface of CD8-positive T cells from human peripheral blood after stimulation with anti-CD3 / CD28 beads and anti-CD30 agonist antibodies

[0373] Using flow cytometry, the expression of CD197 on the cell membrane surface of CD8-positive T cells derived from human peripheral blood, which were stimulated with anti-CD3 / CD28 beads in a medium containing IL-2, a medium containing IL-7 / IL-15, or a medium containing IL-7 / IL-15 / anti-CD30 antibodies, was measured on days 6, 10, 13, and 17 of culture. CD197 expression was confirmed in all groups on day 6, but CD197 expression in the group containing the medium containing IL-2 almost disappeared on day 13, and CD197 expression in the group containing the medium containing IL-7 / IL-15 almost disappeared on day 17. In contrast, CD197 expression was maintained even on day 17 in the group containing the medium containing IL-7 / IL-15 / anti-CD30 antibodies ( Fig.11 ).

[0374] 3. Evaluation of engraftment of CD8-positive T cells from human peripheral blood after stimulation with anti-CD3 / CD28 beads and anti-CD30 agonist antibodies

[0375] CD8 positive T cells derived from human peripheral blood on the 10th day of culture stimulated with anti-CD3 / CD28 beads in a medium containing IL-2 or a medium containing IL-7 / IL-15, or a medium containing IL-7 / IL-15 / anti-CD30 antibodies were administered to the veins of immunodeficient mice, and implantation in the blood and immune tissues was confirmed. Blood, spleen, and bone marrow were collected from mice on the 28th day after administration, and the human CD8 positive T cells contained were detected by flow cytometry. In any organ, human CD8 positive T cells were almost undetectable in the group containing the medium containing IL-2, while human CD8 positive T cells were detected in the group containing the medium containing IL-7 / IL-15 and the group containing the medium containing IL-7 / IL-15 / anti-CD30 antibodies. In addition, a larger number of human CD8 positive T cells were detected in the group containing the medium containing IL-7 / IL-15 / anti-CD30 antibodies. This indicates that CD8-positive T cells derived from human peripheral blood cultured in a medium containing IL-7 / IL-15 / anti-CD30 antibodies can not only maintain CD197 expression for a long time in culture, but also survive for a long time in vivo ( Fig.12 ).

[0376] [Example 3]

[0377] 1. Proliferation test of anti-CD19-CART cells derived from iPS cells

[0378] The anti-CD19-CART cells derived from iPS cells, which were adjusted to 100,000 cells / 200 μL with a culture medium (which was obtained by adding the cytokines in Table 1 to an α-MEM medium containing 15% FBS), were inoculated on a culture plate where anti-CD3 antibodies and RetroNectin (registered trademark) were solid-phased, and cultured at 5% CO2 / 37°C for 3 days. On the third day of culture, the cells were recovered from the culture plate, the number of cells was counted using TC20 (Bio-rad), and an appropriate amount was suspended in a culture medium obtained by adding the cytokines in Table 2 to an α-MEM medium containing 15% FBS, added to a non-solidified culture plate, and cultured at 5% CO2 / 37°C. Then, cells were recovered from the culture plate at any 4 to 7 times on the 5th, 6th, 7th, 8th, 9th, 10th, 12th, 14th, and 16th days of culture, the number of cells was counted, and an appropriate amount was suspended, added to a non-solidified culture plate, and cultured at 5% CO2 / 37°C.

[0379] 2. Anti-CD19-CAR gene

[0380] For the anti-CD19-CAR gene, an oligo DNA encoding a polypeptide (SEQ ID NO: 5) designed in the order of Table 6 from the N-terminus was artificially synthesized.

[0381] [Table 6]

[0382] Sequence from N-terminus Gene Number of amino acids 1 Immunoglobulin heavy chain leader sequence 22 2 Variable region of the light chain of anti-CD19 antibody (FMC60) 104 3 GGGGS connector 15 4 Variable region of the heavy chain of anti-CD19 antibody (FMC60) 120 5 Sequence from CD8 (including transmembrane region) 83 6 Intracellular domain of CD28 41 7 The intracellular domain of 4-1BB 47 8 The intracellular domain of CD3ζ 112

[0383] 3. Preparation of retroviral vector carrying anti-CD19-CAR gene

[0384] The artificial oligo DNA synthesized in [Example 3] 2. was inserted into the multiple cloning site of the pMEI-5 retroviral vector. The viral vector was produced by UNITECH.

[0385] 4. Production of anti-CD19-CART cells derived from iPS cells

[0386] The iPS cell-derived T cells prepared in [Example 1] 3. were infected with the retroviral vector carrying the anti-CD19-CAR gene prepared in [Example 3] 3. to prepare iPS cell-derived anti-CD19-CART cells.

[0387] [Test Example 3]

[0388] 1. Proliferation test of anti-CD19-CART cells derived from iPS cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0389] After 3 days of stimulation with solid-phase anti-CD3 antibody / RetroNectin (registered trademark) for iPS cell-derived T cells prepared in [Example 1] 3. and anti-CD19-CART cells derived from iPS cells prepared in [Example 3] 4., the number of cells when cultured without stimulation was counted over time. Anti-CD19-CART cells derived from iPS cells proliferated to the same extent as T cells derived from iPS cells due to stimulation with solid-phase anti-CD3 antibody / RetroNectin (registered trademark) ( Fig.13 ).

[0390] 2. Proliferation test of anti-CD19-CART cells derived from iPS cells stimulated by anti-CD30 agonist antibodies

[0391] We confirmed whether the addition of anti-CD30 agonist antibodies has an effect on the proliferation of anti-CD19-CART cells derived from iPS cells when stimulated by solid-phase anti-CD3 antibody / RetroNectin (registered trademark). The proliferation of anti-CD19-CART cells derived from iPS cells is enhanced by the addition of anti-CD30 agonist antibodies ( Fig.14 ).

[0392] 3. Detection of CD197 on the cell membrane surface of anti-CD19-CART cells derived from iPS cells after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0393] Using flow cytometry, the expression of CD197 on the surface of anti-CD19-CART cell membranes derived from iPS cells was measured on the 3rd and 7th days after stimulation with solid-phase anti-CD3 antibody / RetroNectin (registered trademark) or solid-phase anti-CD3 antibody / RetroNectin (registered trademark) / anti-CD30 agonist antibody. CD197 expression was confirmed in both groups on the 3rd day, but on the 7th day, it was confirmed that the expression of CD197 in the anti-CD3 antibody / RetroNectin (registered trademark) stimulation group almost disappeared. In contrast, the expression of CD197 was maintained in the anti-CD3 antibody / RetroNectin (registered trademark) + anti-CD30 agonist antibody stimulation group ( Fig.15 ).

[0394] 4. Verification of Raji cell-damaging activity of anti-CD19-CART cells derived from iPS cells after stimulation with immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0395] The cell-damaging activity of anti-CD19-CART cells derived from iPS cells obtained by proliferation of [Test Example 3] 1. against CD19-positive cancer cells was evaluated based on the damaging activity against CD19-positive Raji cells. Anti-CD19-CART cells derived from iPS cells showed cell-damaging activity against CD19-expressing Raji cells ( Fig.16 ).

[0396] [Example 4]

[0397] 1. Anti-CD19-CAR gene containing the intracellular domain from CD30

[0398] For the anti-CD19-CAR gene, an oligoDNA encoding a polypeptide (SEQ ID NO: 7) designed in the order of Table 7 from the N-terminus was artificially synthesized.

[0399] [Table 7]

[0400] Sequence from N-terminus Gene Number of amino acids 1 Immunoglobulin heavy chain leader sequence 22 2 Variable region of the light chain of anti-CD19 antibody (FMC60) 104 3 GGGGS connector 15 4 Variable region of the heavy chain of anti-CD19 antibody (FMC60) 120 5 Sequence from CD8 (including transmembrane region) 83 6 Intracellular domain of CD28 41 7 Intracellular domain of CD30 (SEQ ID NO: 6) 87 8 The intracellular domain of CD3ζ 112

[0401] 2. Preparation of a retroviral vector carrying an anti-CD19-CAR gene containing the intracellular domain from CD30

[0402] The artificial oligo DNA synthesized in [Example 4] 1. was incorporated into the multiple cloning site of the pMY retroviral vector, and a viral vector was prepared using FRY-RD18 cells for producing retroviral vectors.

[0403] 3. Production of anti-CD19-CART cells derived from iPS cells (iCD19-CD30-CART) containing the intracellular domain from CD30

[0404] The iPS cell-derived T cells prepared in [Example 1] 3. are infected with the retroviral vector carrying the anti-CD19-CAR gene prepared in [Example 4] 2. to prepare anti-CD19-CART cells (iCD19-CD30-CART) derived from iPS cells containing the intracellular domain from CD30.

[0405] [Test Example 4]

[0406] Based on the damaging activity against CD19-positive Raji cells, the cell damaging activity of iCD19-CD30-CART obtained in [Example 4] 3. against CD19-positive cancer cells was evaluated. iCD19-CD30-CART showed cell damaging activity against CD19-expressing Raji cells ( Fig.17 ).

[0407] [Example 5]

[0408] 1. Preparation of iPS Cells

[0409] iPS cells used were the Ff-I01s04 strain provided by the iPS Cell Research Institute (CiRA) of Kyoto University in the same manner as in Example 1. iPS cell culture was performed according to the protocol "Feeder-free Culture of Human iPS Cells" published by CiRA.

[0410] 2. Differentiation of iPS cells into γδTCR-positive T cells (γδT cells)

[0411] Differentiation of iPS cells into γδTCR-positive T cells (γδT cells) was carried out in the same manner as in [Example 1] according to a known method (WO2017 / 221975). As the anti-CD3 antibody used in the differentiation step, 3000 ng / mL UCHT1 (GeneTex) was used. The obtained CD3-positive cells were γδT cells (hereinafter referred to as "γδT cells derived from iPS cells (iγδT cells)").

[0412] 3. Expansion of γδT cells derived from iPS cells

[0413] The iγδT cells obtained in [Example 5] 2. were suspended at 2,000,000 cells / mL in a medium obtained by adding the cytokines listed in Table 8 to an α-MEM medium containing 15% FBS, seeded on a culture plate on which an anti-CD3 antibody (UCHT1) and RetroNectin (registered trademark) were immobilized, and cultured at 5% CO2 / 37°C for 3 days. On the third day of culture, the cells were collected from the culture plate, the number of cells was counted using NucleoCounter NC-200 (ChemoMetec), and an appropriate amount was suspended in a medium obtained by adding the cytokines listed in Table 9 to an α-MEM medium containing 15% FBS, added to a G-Rex 6-well plate (WILSONWOLF) that was not immobilized, and cultured at 5% CO2 / 37°C. Then, at any 4 to 6 times among the 5th, 6th, 7th, 8th, 9th, 10th, 11th, 14th, and 17th days of culture, a portion of the cells were collected from the culture plate and the number of cells was counted. Anti-CD3 antibody and RetroNectin (registered trademark) were immobilized on the culture plate by the following method. Anti-CD3 antibody (UCHT1, final concentration 3000 ng / mL) and RetroNectin (registered trademark) (final concentration 150 μg / mL) dissolved in PBS at the required concentration were added to the culture plate and left to stand overnight at 4°C. After washing with PBS, the plate was used for the experiment.

[0414] [Table 8]

[0415]

[0416] w / : used, w / 0: not used

[0417] Additives added to each group are shown with "+".

[0418] [Table 9]

[0419]

[0420] w / : added, w / o: not added

[0421] Additives added to each group are shown with "+".

[0422] [Test Example 5]

[0423] 1. Proliferation assay of γδT cells derived from iPS cells stimulated by anti-CD30 agonist antibody

[0424] We verified whether adding anti-CD30 agonist antibody (300 ng / ml) to the stimulation method using solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) in [Example 5] 3. would have an effect on the proliferation of γδT cells derived from iPS cells. T cells derived from iPS cells further proliferated due to the addition of anti-CD30 agonist antibody ( Fig.18 ).

[0425] [Example 6]

[0426] 1. IL-15Rα / IL-15 gene

[0427] Regarding the IL-15Rα / IL-15 gene, an oligo DNA encoding a polypeptide (SEQ ID NO: 8) designed in the order of Table 10 from the N-terminus was artificially synthesized.

[0428] [Table 10]

[0429] Sequence from N-terminus Gene Number of amino acids 1 Human IL-2 leader sequence 23 2 C-terminal sequence of human IL-15 114 3 GGGGS connector 24 4 C-terminal sequence of human IL-15RA 239

[0430] 2. Preparation of retroviral vector carrying IL-15Rα / IL-15 gene

[0431] The artificial oligo DNA synthesized in [Example 6] 1. was incorporated into the multiple cloning site of the pMY retroviral vector, and a viral vector was prepared using FRY-RD18 cells for producing retroviral vectors.

[0432] 3. Production of anti-CD19-CAR / IL-15γδT cells from iPS cells

[0433] The γδT cells (iγδT cells) derived from iPS cells prepared in [Example 5] 2. are infected with the retroviral vector carrying the anti-CD19-CAR gene produced in [Example 3] 3. and the retroviral vector carrying the IL-15Rα / IL-15 gene produced in [Example 6] 1. to prepare anti-CD19-CAR / IL-15γδT cells (iCD19CAR / IL-15γδT cells) derived from iPS cells.

[0434] 4. Expansion of anti-CD19-CAR / IL-15γδT cells derived from iPS cells

[0435] iCD19CAR / IL-15γδT cells were expanded and cultured in the same manner as in [Example 5] 3., except that IL-2 was not added.

[0436] [Test Example 6]

[0437] 1. Proliferation assay of anti-CD19-CAR / IL-15γδT cells derived from iPS cells stimulated by anti-CD30 agonist antibody

[0438] We verified whether the addition of anti-CD30 agonist antibody (300 ng / ml) during stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) in [Example 6] 4. has an effect on the proliferation of iCD19CAR / IL-15γδT cells. Further proliferation due to the addition of anti-CD30 agonist antibody ( Fig.19 ).

[0439] 2. Study on the cytotoxic activity of anti-CD19-CAR / IL-15γδT cells derived from iPS cells after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0440] The cell damage activity of the iCD19CAR / IL-15γδT cells obtained in [Example 6] 4. was evaluated. CD19-positive Raji cells and CD19-negative CCRF-CEN cells were used as target cells, and iCD19CAR / IL-15γδT cells were mixed at a ratio of 0.5, 1, 2, 4, 8, and 16 times relative to the target cells. Based on the death of target cells after 2 hours, the cell damage activity of iCD19CAR / IL-15γδT cells was evaluated. It was shown that the iCD19CAR / IL-15γδT cells obtained by expanding the culture medium containing anti-CD30 agonist antibodies had cell damage activity against CD19-positive Raji cells, but had no cell damage activity against CD19-negative CCRF-CEN cells ( Fig. 20 ).

[0441] 3. Effect of prolonging the survival days of anti-CD19-CAR / IL-15γδT cells derived from iPS cells after stimulation with solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark) and anti-CD30 agonist antibody

[0442] 5x10 IL-2RγKO (NOG) mice (Institute of Experimental Animals, female, 7-8 weeks old) were transplanted into the tail vein. 5 Nalm6 xenograft mice were prepared by using 5x10 Nalm6 cells (ATCC). On the 4th day after transplantation, iCD19CAR / IL-15γδT cells (5x10 6 After the cells were suspended in 0.1 mL of HBSS-buffer or an equal amount of HBSS-buffer, the survival days were confirmed. For mice transplanted with CD19-positive Nalm6 cancer cells via the tail vein, all test cases in the control administration group died within 3 weeks. In contrast, in the iCD19CAR / IL-15γδT cell administration group obtained by expanding culture in a medium containing anti-CD30 agonist antibodies, all test cases survived for at least 6 weeks ( Fig.21 ).

[0443] [Example 7]

[0444] 1. Production of anti-CD19-CAR / IL-15αβ T cells derived from iPS cells

[0445] The anti-CD19-CART cells derived from iPS cells produced using the method of [Example 3] 4. are infected with the retroviral vector carrying the IL15Rα / IL-15 gene produced in [Example 6] 2. to produce anti-CD19-CAR / IL-15αβT cells (iCD19CAR / IL-15αβT cells) derived from iPS cells.

[0446] 2. Expansion of iCD19CAR / IL-15αβT cells using immobilized anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0447] The iCD19CAR / IL-15αβT cells obtained in [Example 7] 1. were cultured until day 7 by the same method as [Example 6] 4. However, anti-human CD30 antibody was not added.

[0448] [Test Example 7]

[0449] 1. In vivo anti-tumor effect of iCD19CAR / IL-15αβT cells stimulated by solid-phase anti-CD3 agonist antibody / RetroNectin (registered trademark)

[0450] 5x10 IL-2RγKO (NOG) mice (Institute of Experimental Animals, female, 7-8 weeks old) were transplanted into the tail vein. 5 luciferase-expressing Nalm6 cells (ATCC) were used to prepare luciferase-expressing Nalm6 xenograft mice. On the 4th day after transplantation, iCD19CAR / IL-15αβT cells (5×10 6 A suspension obtained by suspending individual (cells) in 0.1 mL of HBSS-buffer or an equal amount of HBSS-buffer. Luciferin was administered through the tail vein every week after administration, and the activity of luciferase expressed by Nalm6 cells was measured using IVIS. In the control administration group, luminescence from Nalm6 cells was confirmed throughout the body 2 weeks after administration, and all test cases had died by the 3rd week after administration. In contrast, in the iCD19CAR / IL-15αβT cell administration group, no luminescence was detected until the 6th week after administration ( Fig. 22 ).

[0451] [Example 8]

[0452] 1. Production of γδT cells from iPS cells

[0453] iPS cell-derived γδT cells (iγδT cells) were produced in the same manner as in 1. and 2. of [Example 5].

[0454] [Test Example 8]

[0455] 1. Determination of the concentration of anti-CD3 agonist antibody (UCHT1) suitable for solid phase immobilization on a culture plate and the concentration of RetroNectin (registered trademark)

[0456] The anti-CD3 agonist antibody (UCHT1) and RetroNectin (registered trademark) were mixed and immobilized on the culture plate in the same manner as in [Example 1] 5. Then, the amount of immobilized antibody on the culture plate was measured using the ELISA method ( Fig.23). The anti-CD3 agonist antibody not mixed with RetroNectin (registered trademark) was confirmed to be solid-phased in a concentration-dependent manner between 3 ng / ml and 30000 ng / ml (0.003 μg / ml to 30 μg / ml), and the amount of solid-phased anti-CD3 agonist antibody decreased as the concentration of the mixed RetroNectin (registered trademark) increased. On the other hand, RetroNectin (registered trademark) was confirmed to be solid-phased in a concentration-dependent manner between 16.7 μg / ml and 150 μg / mL.

[0457] 2. Verification of the concentration range and mixing ratio of the anti-CD3 agonist antibody and RetroNectin (registered trademark) required for the immobilized anti-CD3 agonist antibody and the immobilized RetroNectin (registered trademark) suitable for the proliferation of iγδT cells

[0458] iγδT cells adjusted to 100,000 cells / 200 μL using a culture medium (which was prepared by adding the cytokines listed in Table 1 and the anti-CD30 agonist antibody diluted to a final concentration of 0, 3, 30, and 300 ng / ml to an IMDM medium containing 15% FBS) were seeded on a culture plate on which anti-CD3 agonist antibody (0, 300, 3000, 30000 ng / ml (0, 0.3, 3, 30 μg / ml)) and RetroNectin (registered trademark) (0, 2, 15, 150 μg / mL) were immobilized, and cultured at 5% CO2 / 37°C for 3 days.

[0459] On the third day of culture, cells were collected from the culture plate, suspended in an appropriate amount of culture medium obtained by adding the cytokines listed in Table 2 to an IMDM culture medium containing 15% FBS, inoculated in a non-solidified 96-well plate, and cultured at 5% CO2 / 37°C. Then, cells were collected from the culture plate once at any time on the fifth, sixth, seventh, eighth, ninth, tenth, and twelfth days of culture, for a total of 4 to 7 times, suspended in an appropriate amount of culture medium, inoculated in a non-solidified plate, and cultured at 5% CO2 / 37°C. On the thirteenth day of culture, the number of cells was counted using a hemocytometer, and the proliferation rate ( Fig.24 ). In the culture plates where the anti-CD3 agonist antibody and RetroNectin (registered trademark) were mixed and immobilized at 0.3 μg / mL and 2 μg / mL, 3 μg / mL and 15 μg / mL, and 30 μg / mL and 150 μg / mL, equivalent iγδT cell proliferation induction was observed. In addition, cell proliferation was confirmed to be concentration-dependent upon the addition of the anti-CD30 agonist antibody.

[0460] Industrial Applicability

[0461] By culturing CD3-positive cells in the presence of a CD3 / TCR complex agonist, fibronectin or a modified form thereof, and a CD30 agonist, T cells can be efficiently and repeatedly expanded. In addition, when the cultured CD3-positive cells are CD3-positive CD8-positive cells or CD3-positive CD8-positive CD30-positive cells, the cells produced or expanded can be easily transformed into effector T cells and exert cell-damaging activity, and therefore can be applied to T cell therapy.

[0462] This application is based on Japanese Patent Application No. 2018-151580 (filing date: August 10, 2018), Japanese Patent Application No. 2019-042666 (filing date: March 8, 2019), and Japanese Patent Application No. 2019-117878 (filing date: June 25, 2019) filed in Japan, and all the contents of the above applications are included in this specification.

Claims

1. A method for maintaining CD3-positive cells as CD3-positive CD197-positive cells, comprising the step of stimulating CD30 signals in CD3-positive cells.

2. The method of claim 1, wherein: The medium used in the stimulation step contains IL-21 and / or an apoptosis inhibitor.

3. The method of claim 1, wherein: The CD3-positive cells are derived from iPS cells.

4. The method of claim 1, wherein: The CD3 positive cells are CD3 positive CD8 positive cells.

5. The method of claim 4, wherein: The CD3-positive CD8-positive cells are CD3-positive CD8-positive CD4-negative cells.

6. The method according to any one of claims 1 to 5, wherein: The culture medium used in the stimulation step contains at least one selected from the group consisting of IL-7, IL-15, and IL-18.

7. The method of claim 6, wherein: The medium used in the stimulation step contains IL-7, IL-15, and IL-18.

8. The method of claim 6, wherein: The medium used in the stimulation step further contains TL1A and / or IL-12.

9. A kit for maintaining CD3-positive cells as CD3-positive CD197-positive cells, comprising a CD30 agonist.

10. The kit according to claim 9, further comprising IL-21 and / or an apoptosis inhibitor.

11. The kit according to claim 9, wherein The CD3-positive cells are derived from iPS cells.

12. The method according to any one of claims 9 to 11, wherein: The CD3 positive cells are CD3 positive CD8 positive cells.

Images