Methods for producing CD8α+β+ cell-damaging T cells

Abstract

To provide a method of efficiently producing cytotoxic T cells having intrinsic properties of lymphocytes of the acquired immune system suitable for cellular immunotherapy.SOLUTION: A method of producing CD8α+β+ cytotoxic T cells comprises: the step (A) for culturing CD4 / CD8 double-positive T cells in a medium containing IL-7 and T-cell receptor activators to induce them into CD8α+β+ cytotoxic T cells; and the step (B) for culturing the CD8α+β+ cytotoxic T cells resulting from the step (A) in a medium containing IL-7 and IL-15 and also containing at least one selected from a group consisting of IL-21, IL-18, IL-12 and TL1A.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to CD8α + β + A method for producing cytotoxic T cells, preferably pluripotent stem cells CD8α from cells + β + The present invention relates to a method for producing cytotoxic T cells. [Background technology]

[0002] Cellular immunotherapy using cytotoxic T lymphocytes (CTLs) specific to disease-related antigens may be extremely effective in controlling malignant tumors and chronic intractable infectious diseases. Attempts have been made to produce antigen-specific CTLs in vitro, but Due to the inevitable cell exhaustion, it is difficult to prepare sufficient numbers of antigen-specific CTLs. However, the recent emergence of induced pluripotent stem cell (iPSC) technology is showing a way to fundamentally solve the cell source problem. In other words, it is becoming possible to generate iPSCs that can proliferate indefinitely from a small number of exhausted disease antigen-specific CTLs, thereby enabling a strategy to infinitely regenerate CTLs in vitro.

[0003] For example, Patent Document 1 discloses a method for producing T cells, which includes the steps of inducing hematopoietic progenitor cells from pluripotent stem cells, inducing CD4CD8 dual-positive cells from the hematopoietic progenitor cells, and inducing CD8 dual-positive T cells from the CD4CD8 dual-positive cells. Furthermore, Patent Document 2 discloses a method for producing CD8-positive T cells by inducing CD4CD8-positive T cells from pluripotent stem cells using a medium supplemented with vitamin C and culturing the cells in a medium containing a corticosteroid. However, these methods are not sufficient to generate cytotoxic T cells from pluripotent stem cells. I can't say.

[0004] Furthermore, Patent Document 3 and Non-Patent Document 1 disclose that iPSCs were induced from human peripheral blood CTLs, hematopoietic progenitor cells were produced from the iPSCs, and the iPSCs were co-cultured with OP9 / DLL1 cells that express the Notch ligand DLL-1 in a medium containing IL (interleukin)-2, IL-7, and IL-15, thereby successfully leading to terminal differentiation into CTLs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO 2011 / 096482 [Patent Document 2] WO / 2016 / 076415 [Patent Document 3] WO / 2013 / 176197 [Non-patent literature]

[0006] [Non-Patent Document 1] Cell Stem Cell. 2013 Jan 3;12(1):114-26. Summary of the Invention [Problem to be solved by the invention]

[0007] The redifferentiated CTLs disclosed in Patent Document 3 and Non-Patent Document 1 retain the antigen specificity of the original CTLs while possessing all of the CTL functions, such as high cytotoxic activity, cytokine production, and proliferation ability. However, it was also revealed that it also possessed NK cell-like properties not observed in normal CTLs. In other words, while normal CTLs, including the original CTLs, belong to the adaptive immune system lymphocytes, the redifferentiated CTLs possessed the properties of the innate immune system lymphocytes to which NK cells belong. Among the characteristics of innate immune lymphocytes observed in these redifferentiated CTLs, there are some that have particular implications for clinical application. The factors that are thought to have the greatest impact are: (1) natural killer (NK) activity, which damages targets non-specifically to antigens; (2) the constitutive expression of various activating molecules (NKp46, etc.) that induce NK activity; and (3) ) Relatively low proliferation and survival ability compared to normal adaptive immune system CTLs, (4) complementation of antigen receptors The accessory molecule, CD8, is a CD8αα homodimer, not the usual CD8αβ heterodimer. These are the reduced antigen recognition ability caused by the lack of CD8αβ expression. While (1) and (2) are expected to be effective against escape variants, they are associated with the risk of unpredictable graft-versus-host disease (GVHD). Regarding (3), low proliferation and viability, previous reports have shown that insufficient in vivo persistence worsens the outcome of cellular immunotherapy. Furthermore, (4) lack of CD8αβ expression must be corrected because it directly leads to a reduced antigen recognition ability of CTLs. Since such characteristics of innate immune lymphocytes are not necessarily suitable for cellular immunotherapy, the present invention provides a method for efficiently producing CTLs that have the characteristics of original adaptive immune lymphocytes that are suitable for cellular immunotherapy. The objective is to provide a method for manufacturing the same. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. When culturing the cells, a step of culturing them in a medium containing IL-7 and a T cell receptor activator is carried out. As a result, we succeeded in producing CTLs that do not exhibit NK activity and maintain CD8αβ expression for a long period of time, i.e., CTLs that are closer to CTLs of the normal adaptive immune system. Based on these findings, we have completed the present invention.

[0009] That is, the present invention provides the following inventions. [1] CD8α + β + A method for producing cytotoxic T cells, comprising: CD4CD8 positive T cells were cultured in a medium containing IL (Interleukin)-7 and T cell receptor activators. Cultivate CD8α + β + A method comprising a step of inducing cytotoxic T cells. [2] The method described in [1], wherein the T cell receptor activator is an anti-CD3 antibody. [3] The medium further contains IL-21 and Flt3L (Flt3 Ligand), [1] or [2]. The method described. [4] The method according to any one of [1] to [3], comprising the following steps (a) and (b): (a) culturing CD4CD8 bi-positive T cells in a medium containing IL-7 and a T cell receptor activator; and (b) The cells obtained in step (a) are cultured in a medium containing IL-7 and not containing a T cell receptor activator. The process of culturing the cells. [5] The culture (b) contains a fibronectin fragment and / or a Notch ligand. The method described in [4] is carried out using an incubator. [6] The method according to [5], wherein the fibronectin fragment is retronectin and the Notch ligand is Delta-like 4 (DLL4). [7] The method according to [5] or [6], further comprising the following step (c): (c) The cells obtained in step (b) are treated with a fibronectin fragment and a Notch ligand. and culturing the cells in a medium containing IL-7, IL-21, and Flt3L using an incubator that does not contain any of the above. [8] The method according to [7], comprising the following steps (a1), (b1) and (c1): (a1) culturing CD4CD8 dual-positive T cells in a medium containing IL-7, Flt3L, IL-21, and an anti-CD3 antibody; (b1) Incubating the cells obtained in step (a1) with IL-7, Flt3L, and IL-21, and with an anti-CD3 antibody. Culturing the cells in a medium containing no fibronectin fragments using an incubator containing the fibronectin fragments; (c1) Culturing the cells obtained in step (b1) in a medium containing IL-7, IL-21, and Flt3L using an incubator that does not contain any of fibronectin fragments or Notch ligands. Process. [9] The method according to [7], comprising the following steps (a2), (b2) and (c2): (a2) culturing CD4CD8 dual-positive T cells in a medium containing IL-7, Flt3L, and an anti-CD3 antibody; , (b2) The cells obtained in step (a2) are cultured in a medium containing IL-7 and Flt3L but not containing anti-CD3 antibody, using an incubator containing a fibronectin fragment and a Notch ligand. The process of nourishing (c2) Culturing the cells obtained in step (b2) in a medium containing IL-7, IL-21, and Flt3L using an incubator that does not contain any of the fibronectin fragments and the Notch ligand. Process.

[10] The method according to any one of [1] to [9], wherein the culture is carried out without using feeder cells.

[11] Furthermore, the obtained CD8α + β + The method according to any one of [1] to

[10] , which comprises a step of selecting cytotoxic T cells.

[12] The method according to

[11] , wherein the selection step is carried out using one or more of CD8β positivity, CD5 positivity, CD336 negativity, and CD1a negativity as indicators.

[13] Furthermore, CD8α + β + The method according to any one of [1] to

[12] , which comprises a step of expanding cytotoxic T cells in a medium containing IL-7, IL-15 and IL-21.

[14] Furthermore, CD8α + β + Cytotoxic T cells are stimulated by IL-7 and IL-15, as well as IL-21 and IL-18. [1] to

[12] , which include a step of expanding the cells in a medium containing one or more of IL-12 and TL1A. A method according to any one of the preceding claims.

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

[0014] , wherein the CD4CD8 bi-positive T cells are induced from pluripotent stem cells.

[16] The method according to

[15] , wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

[17] Induction of CD4CD8 dual-positive T cells from pluripotent stem cells by the following steps (a) and (b): The method according to

[15] or

[16] , comprising: (a) Pluripotent stem cells are cultured in a medium supplemented with vitamin C to induce hematopoietic progenitor cells. Process, and (b) A step of culturing the hematopoietic progenitor cells obtained in step (a) in a medium containing vitamin C, FLT3L, and IL-7 to induce CD4CD8 co-positive T cells.

[18] CD8α from pluripotent stem cells + β + A method for producing cytotoxic T cells, comprising: (a) Pluripotent stem cells are cultured in a medium supplemented with vitamin C to induce hematopoietic progenitor cells. The course, (b) culturing the hematopoietic progenitor cells obtained in step (a) in a medium containing vitamin C, FLT3L, and IL-7 to induce CD4CD8 co-positive T cells; and (c) The CD4CD8 bi-positive T cells obtained in step (b) are cultured in a medium containing IL-7 and a T cell receptor activator to induce CD8α + β + A method comprising a step of inducing cytotoxic T cells.

[19] The method according to

[18] , wherein the concentration of IL-7 in the medium in step (c) is higher than the concentration of IL-7 in the medium in step (b).

[20] Produced CD8α + β + The method according to any one of [1] to

[19] , wherein the cytotoxic T cells do not exhibit natural killer (NK) activity.

[21] CD8α obtained by the method described in any of [1] to

[20] + β + Cytotoxic T cell cultures.

[22] CD8α obtained by the method described in any of [1] to

[20] + β + A pharmaceutical composition comprising cytotoxic T cells. [Effects of the Invention]

[0010] According to the present invention, normal cells that do not exhibit NK activity and maintain CD8αβ expression even after long-term culture are It is possible to produce CTLs that are closer to CTLs of the adaptive immune system. Furthermore, the CTLs obtained by the method of the present invention not only undergo a differentiation shift from innate immune system to adaptive immune system lymphocytes, but also show higher antigen-specific cytotoxic activity, cytokine production, and proliferation ability than conventional CTLs. The excellent properties of maturation into naive / memory cells without cell exhaustion and maintaining their phenotypes It has a remarkable proliferation ability, capable of expanding in vitro by more than 100 trillion times. Therefore, the present invention will make a significant contribution to the current cellular immunotherapy, which has difficulties in obtaining cells. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the results of flow cytometry of modified iPSC-CTLs produced by the method of the present invention, the T cell clone from which the iPS cells were derived (original CTL clone), and iPSC-CTLs produced by conventional methods. The upper panel shows plots based on CD8α and CD8β, and the lower panel shows plots based on TCR and HLA-tetramer staining intensities. [Figure 2]Figure 2 shows the results of examining the proliferation ability (upper panel: vertical axis indicates cell number, horizontal axis indicates CFSE fluorescence intensity) and the production of two cytokines, IFNγ and IL-2, of improved iPSC-CTLs (modified CD8b+ CTLs) and conventional iPSC-CTLs (conventional CD8b- CTLs) produced by the method of the present invention when they were stained with CFSE and stimulated with K562 / HLA A-24 cells presenting a specific antigen peptide (lower panel). [Figure 3] FIG. 3 shows the relationship between the amount of antigen peptide and cytotoxic activity when improved iPSC-CTLs (modified CD8b+ CTLs) produced by the method of the present invention and conventional iPSC-CTLs (conventional CD8b- CTLs) were co-cultured with K562 / HLA A-24 cells pulsed with a specific antigen peptide. [Figure 4] Figure 4 shows the results of FACS analysis of improved iPSC-CTLs (modified CD8b+ CTLs) and conventional iPSC-CTLs (conventional CD8b- CTLs) produced by the method of the present invention, as well as the original T cell clones, using CD28, CD27, and CCR7 (left) and the combination of CD45RA and CD45RO (right). [Figure 5] Figure 5 shows the results of analyzing the proliferation of improved iPSC-CTLs (modified CD8b+ CTLs) produced by the method of the present invention and conventional iPSC-CTLs (conventional CD8b- CTLs) (the start of expansion culture is shown as Day 0). [Figure 6] Figure 6 shows the results of quantification of cytokine (IFNγ) production and cytotoxic molecule (Granzyme B) expression levels after stimulation of improved iPSC-CTLs (modified CD8b+ CTLs) produced by the method of the present invention with PHA + PBMCs or in a feeder-free environment (with or without IL-21). [Figure 7] FIG. 7 shows the results of measuring cytokine (IFNγ) production and cytotoxic activity of improved iPSC-CTLs (modified CD8b+ CTLs) produced by the method of the present invention. [Figure 8]FIG. 8 shows the results of quantifying cytokine production and the expression levels of cytotoxic molecules in improved iPSC-CTLs (modified CD8b+ CTLs) prepared by the method of the present invention and the original CTLs. [Figure 9] FIG. 9 shows the results of analyzing the effects of adding IL-7 and IL-21 on the amount of CD8α+β+ CTL produced and on surface markers. [Figure 10] FIG. 10 shows the results of an analysis of the cytokine production profiles of the generated iPSC-CTLs and primary CTLs. [Figure 11] FIG. 11 shows the results of FACS analysis of CD8β / CD5 positive cells sorted from iPSC-CTLs matured by the conventional method and subjected to PHA on-feeder expansion culture for 2 weeks. [Figure 12] FIG. 12 shows an analysis of proliferation ability after a second expansion culture under feeder-free conditions in which conventional iPSC-CTLs were divided into CD8β + CD5 bright cells and CD8β + CD5 − cells after expansion culture. [Figure 13] FIG. 13 shows the results of FACS analysis of CD8β / CD5 positive cells sorted from improved iPSC-CTLs and subjected to four rounds of PHA on-feeder expansion culture. [Figure 14] FIG. 14 shows the results of analyzing the proliferation of improved iPSC-CTLs and naive CTLs expanded with or without feeders. [Figure 15] Figure 15 shows the results of an analysis of the proliferation of improved iPSC-CTLs (modified CD8b+ CTLs) and the parental CTL clone (H25-4) after expansion culture in media containing each cytokine for 2 weeks (shown as the growth rate relative to the start of expansion culture). [Figure 16] FIG. 16 shows the results of measuring the cytotoxic activity, cytokine production, and proliferation rate of improved iPSC-CTLs (modified CD8b+ CTLs) after expansion culture in media containing each cytokine for 2 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to CD8α + β + A method for producing cytotoxic T cells, comprising: CD4CD8 co-positive T cells were cultured in a medium containing IL-7 and T cell receptor activators to induce CD8α + β + The present invention provides a method for inducing cytotoxic T cells.

[0013] In the present invention, CD8α + β + Cytotoxic T cells are T cells that are positive for both the surface antigens CD8α and CD8β (CD4 is negative) and have cytotoxic activity. means. Cytotoxic T cells (CTLs) recognize antigenic peptides derived from viruses, tumors, etc., that are presented together with class 1 major histocompatibility complexes (MHC class 1, HLA class 1) on antigen-presenting cells via T cell receptors (TCRs) present on their cell surfaces, and exert cytotoxic activity specifically against cells presenting the foreign antigenic peptides. Cytotoxic activity can be confirmed, for example, using the secretion or production of granzymes, perforin, etc. as indicators.

[0014] On the other hand, CD4CD8 dual positive T cells are T cells that are positive for both the surface antigens CD4 and CD8 (CD8 + CD4 + ) means that CD4CD8 dual positive T cells can be induced to differentiate into CD4 positive cells (CD8 - CD4 + ) or CD8-positive cells (CD8 + CD4 - In the present invention, CD4CD8 dual positive T cells can be differentiated into CD8α + β + Induce cytotoxic T cells.

[0015] The origin of CD4CD8 dual positive T cells is not particularly limited, but they can be obtained by inducing differentiation from pluripotent stem cells. Preferably, the T cells are CD4CD8 bi-positive T cells.

[0016] pluripotent stem cells In the present invention, pluripotent stem cells are stem cells that have pluripotency and can differentiate into many cells present in the body, and also have proliferation ability, and can be induced into any CD4CD8 positive T cells. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts, and bone marrow cells. These include pluripotent cells derived from stem cells (Muse cells). The preferred pluripotent stem cells are iPS cells, as they can be obtained without destroying embryos, eggs, etc. during the manufacturing process. More preferably, they are human iPS cells.

[0017] Methods for producing iPS cells are known in the art, and involve introducing reprogramming factors into somatic cells. Here, examples of reprogramming factors include genetic factors such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. Examples of reprogramming factors include molecules or gene products, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, W O2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotechnol., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. 46 1:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0018] Somatic cells include fetal (baby) somatic cells, neonatal (baby) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. In the present invention, for the purpose of producing CTLs, it is preferable to produce iPS cells using lymphocytes (T cells) in which T cell receptor (TCR) gene rearrangement has been performed as somatic cells. When lymphocytes are used as somatic cells, it is preferable to activate the lymphocytes by stimulating them with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-2 prior to the reprogramming step. Such stimulation can be carried out, for example, by adding IL-2, anti-CD3 antibody, and anti-CD28 antibody to a culture medium to activate the lymphocytes. This can be achieved by culturing the lymphocytes for a certain period of time. Alternatively, instead of adding these antibodies to the medium, the lymphocytes can be cultured in a culture dish with anti-CD3 and anti-CD28 antibodies bound to the surface. The T cells may be stimulated by culturing them on a medium containing feeder cells for a certain period of time. Furthermore, the T cells may be stimulated by adding an antigen peptide recognized by the human T cells to the medium together with the feeder cells.

[0019] CD8α produced in the present invention + β + Cytotoxic T cells preferably have a desired antigen specificity. Therefore, lymphocytes from which iPS cells are derived should have the desired antigen specificity. The lymphocytes may be specifically isolated by purification using an affinity column or the like to which a desired antigen is immobilized. For example, the lymphocytes may be specifically isolated by purification using an MHC (major histocompatibility complex) to which a desired antigen is bound. The MHC tetramer is a tetramer of the MHC-compatible complex (MHC-compatibility complex). Alternatively, a method can be employed in which lymphocytes having the desired antigen specificity are purified from animal tissue.

[0020] The mammalian individual from which the somatic cells are collected is not particularly limited, but is preferably a human. + β + When using cytotoxic T cells in cell immunotherapy, the somatic cells from which iPS cells are derived are CD8α, which is an antigen that can be easily matched to the patient's human leukocyte antigen (HLA) type. + β +Preferably, the cytotoxic T cells are isolated from the recipient.

[0021] The method for obtaining CD4CD8 dual-positive T cells from pluripotent stem cells is not particularly limited, and known methods can be used. For example, a method including the following steps (a) and (b) can be used. (a) Pluripotent stem cells are cultured in a medium supplemented with vitamin C to induce hematopoietic progenitor cells. and (b) A step of culturing the hematopoietic progenitor cells obtained in step (a) in a medium containing vitamin C, FLT3L, and IL-7 to induce CD4CD8 co-positive T cells. These steps are explained in detail below, although the method for inducing CD4CD8 bi-positive T cells from pluripotent stem cells in the present invention is not limited to the following.

[0022] A step of inducing hematopoietic progenitor cells from pluripotent stem cells As used herein, hematopoietic progenitor cells (HPCs) refer to: Hematopoietic progenitor cells are cells that can differentiate into blood cells such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. Hematopoietic progenitor cells are, for example, positive for the surface antigens CD34 and / or CD43. It can be recognized by

[0023] Hematopoietic progenitor cells can be obtained by, for example, culturing pluripotent stem cells in a medium supplemented with vitamin C. It can be produced by a method including the steps of:

[0024] In the present invention, vitamin C means L-ascorbic acid and its derivatives, Ascorbic acid derivatives are substances that become vitamin C through an enzymatic reaction in the body. Derivatives of L-ascorbic acid include vitamin C phosphate, ascorbic acid glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, and stearic acid. Examples include ascorbyl and ascorbyl-2-phosphate-6-palmitate. Vitamin C phosphate is preferred, including L-ascorbate phosphate salts such as sodium L-ascorbate phosphate or magnesium L-ascorbate phosphate.

[0025] The medium used in the hematopoietic progenitor cell induction step is not particularly limited, but can be prepared by adding vitamin C to a basal medium used for culturing animal cells. Examples of suitable basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixtures thereof. Serum may be added to the medium, or serum-free media may be used. If necessary, the basal medium may also contain one or more substances, such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. The basal medium is, for example, IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, L-glutamine, and ascorbic acid.

[0026] The medium used in the hematopoietic progenitor cell induction step may be supplemented with a cytokine selected from the group consisting of BMP4 (Bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), SCF (Stem cell factor), and FLT3L (Flt3 Ligand). In this case, a medium supplemented with VEGF, SCF, and FLT3L is used.

[0027] Vitamin C is added to the medium in an amount corresponding to, for example, 5 ng / ml to 100 μg / ml. VEGF is added to the medium in an amount corresponding to, for example, 10 ng / ml to 100 ng / ml. SCF is added to the medium in an amount corresponding to, for example, 10 ng / ml to 100 ng / ml. FLT3L is added to the medium in an amount corresponding to, for example, 1 ng / ml to 100 ng / ml.

[0028] In the hematopoietic progenitor cell induction step, pluripotent stem cells are cultured in adherent culture or suspension culture. In the case of adherent culture, the culture may be performed using a culture vessel coated with a coating agent, or the stem cells may be co-cultured with other cells. Examples of other cells to be co-cultured include C3H10T1 / 2 (Takayama N., et al. J Exp Med. 2817-2830, 2010), heterologous stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77.) An example of a coating agent is Matrigel (Niwa A, et al. PLoS One.6(7):e22261, 2011). , Chadwick et al. Blood 2003, 102: 906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4: 248-62, and Saeki et al. Stem Cells 2009, 27: 59-67. will be done.

[0029] Hematopoietic progenitor cells are derived from net-like structures (ES-sacs or ESSCs) obtained by culturing pluripotent stem cells. These can also be prepared from iPS cells (also called iPS-sacs). Here, the term "net-like structure" refers to a three-dimensional sac-like structure (with an internal space) derived from pluripotent stem cells, formed from endothelial cell populations and containing hematopoietic progenitor cells.

[0030] The temperature conditions for the hematopoietic progenitor cell induction step are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. The culture period is, for example, 6 days or more. The cells may be cultured under hypoxic conditions, and hypoxic conditions include 15%, 10%, 9%, 8%, 7%, 6%, 5% or Examples of oxygen concentrations below these levels are given below.

[0031] The hematopoietic progenitor cell induction step can be performed by appropriately combining the above conditions. The combinations are as follows: (i) pluripotent stem cells are cultured in a basal medium supplemented with vitamin C, and C3H10T1 / 2 and (ii) culturing VEGF, SCF, and FLT3L under hypoxic conditions in (i). and culturing the medium under normal oxygen conditions. The period for carrying out step (a) is 6 days or more, and the period for carrying out step (ii) is 6 days or more.

[0032] A process for inducing CD4CD8 dual-positive T cells from hematopoietic progenitor cells CD4CD8 bi-positive T cells can be induced, for example, by culturing hematopoietic progenitor cells in a medium supplemented with vitamin C.

[0033] The medium used for inducing CD4CD8 dual positive T cells is not particularly limited, but may be a medium used for culturing animal cells. The medium can be prepared by adding vitamin C to the basal medium. , such as Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Examples of basal media include Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's, Neurobasal Medium (Life Technologies), and mixtures thereof. The media may contain serum or may be serum-free. If necessary, the basal medium may also contain one or more substances, such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. The preferred basal medium for the induction of CD4CD8 dual positive T cells is serum-, transferrin-, and cerebrospinal fluid. The basal medium is an αMEM medium containing phosphorus and L-glutamine. The type and concentration of vitamin C added to the basal medium are the same as those used for the induction of hematopoietic progenitor cells described above.

[0034] The medium used for inducing CD4CD8 dual positive T cells of the present invention preferably further contains a cytokine selected from the group consisting of FLT3L and IL-7. The concentration of IL-7 in the medium used for inducing CD4CD8 positive T cells is, for example, 0.01 ng / ml. to 100 ng / ml, preferably 0.1 ng / ml to 10 ng / ml. The concentration of FLT3L in the medium used to induce CD4CD8 dual positive T cells is, for example, 1 ng / ml to 200 ng / ml. The average concentration is 100 ng / ml.

[0035] In the production of CD4CD8 dual-positive T cells, hematopoietic progenitor cells may be cultured in adherent or suspension culture. In the case of adherent culture, the culture vessel may be coated, or co-culture with feeder cells or the like may be performed. An example of the feeder cells to be co-cultured is the bone marrow stromal cell line OP9 cells (available from the RIKEN BioResource Center). The OP9 cells may preferably be OP-DL1 cells that constitutively express Dll1 (Holmes R1 and Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009(2)). When using OP9 cells as feeder cells, they should be prepared separately. Alternatively, this can be achieved by adding fused Dll1 or a fusion protein of Dll1 and Fc, etc., to the culture medium as appropriate. Dll1 has the NCBI accession number NM_005618 for humans and NM_005619 for mice. In this case, the protein encoded by the gene having the nucleotide sequence set forth in NM_007865 These include naturally occurring variants that have high sequence identity (e.g., 90% or more) and equivalent functions. When using feeder cells, it is preferable to appropriately replace the feeder cells during culture. Feeder cells can be replaced by transferring the target cells being cultured onto feeder cells that have been seeded in advance.

[0036] The culture temperature conditions for culturing hematopoietic progenitor cells to induce CD4CD8 dual-positive T cells are particularly important. Although the temperature is not limited to, for example, about 37°C to about 42°C, and about 37°C to about 39°C are preferred. Regarding the culture period, those skilled in the art can determine the temperature while monitoring the number of CD4CD8 positive T cells, etc. The number of days is not particularly limited as long as hematopoietic progenitor cells can be obtained, but is, for example, 10 days or more.

[0037] CD4CD8 co-positive T cells to CD8α + β + Inducing cytotoxic T cells In the present invention, CD8α + β+ Cytotoxic T cells can be produced by culturing CD4CD8 bi-positive T cells in a medium containing IL-7 and a T cell receptor activator. This step is preferably performed by adherent culture, preferably without the use of feeder cells, and preferably by culturing CD4CD8 bi-positive T cells by direct adhesion to a culture vessel coated with a fibronectin fragment and / or a Notch ligand. CD8α + β + The concentration of IL-7 in the medium used for inducing cytotoxic T cells was determined according to the above-mentioned " The concentration of IL-7 in the medium used in the "step of inducing CD4CD8 dual-positive T cells from blood precursor cells" It is preferable to use a higher concentration of IL-7 than the normal range, for example, 0.05 ng / ml to 500 ng / ml, preferably 0.1 ng / ml to 100 ng / ml, more preferably 0.5 ng / ml to 50 ng / ml. be.

[0038] Examples of T cell receptor activators include PHA (phytohemagglutinin), anti-CD3 antibody, anti-CD28 antibody, PMA, and ionomycin. For example, the T cell receptor (TCR) can be stimulated by adding an anti-CD3 antibody or the like to the medium and culturing CD4CD8 positive T cells for a certain period of time. Alternatively, the T cells may be bound to an anti-CD3 antibody or other suitable antibody. Furthermore, instead of adding an antibody to the medium, the T cells may be stimulated by culturing them for a certain period of time on a culture dish with an anti-CD3 antibody bound to its surface. This also corresponds to culturing in a medium containing a TCR activator. The concentration of the anti-CD3 antibody bound to the surface of the culture dish to stimulate the TCR of CD4CD8 co-positive T cells is not particularly limited, but is, for example, 0.1 to 100 μg / ml. T cell receptor activators are used to stimulate the activation of CD4CD8-positive T cells to CD8α + β +At the start of the "step of inducing cytotoxic T cells," the medium is added together with IL-7, and after culturing for preferably 1 to 2 days, the medium is preferably changed to one containing IL-7 but not containing a T cell receptor activator (for example, a T cell receptor activator concentration of less than 1 ng / mL to the detection limit or below), and the culture is continued. In this case, it is preferable to increase the fibronectin fraction at the timing of changing to a medium not containing a T cell receptor activator. It is preferable to culture the cells using a culture vessel coated with a fragment and / or a Notch ligand. It's nice. Then, a mixture containing neither a fibronectin fragment nor a Notch ligand was prepared. It is more preferable to further culture the cells using a culture vessel that is not coated with these materials, preferably in a medium containing IL-7, IL-21, and Flt3L.

[0039] CD8α + β + The medium used in the step of inducing cytotoxic T cells is not particularly limited, but may be any medium suitable for use in animal The medium used for cell culture can be prepared by adding IL-7 and TCR activators to the basal medium. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, and Fischer's medium. , Neurobasal Medium (Life Technologies), and mixed media thereof. The medium may contain serum or may be serum-free. Optionally, the basal medium may also contain one or more substances, such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. A preferred basal medium contains serum, transferrin, serine, L-glutamine, and ascorbic acid. The αMEM medium contains:

[0040] CD8α + β + The medium used in the cytotoxic T cell induction step further contains cytotoxic T cells other than IL-7. It is preferable that the medium contains cytokines such as FLT3L and IL-21. The medium does not contain IL-15 (for example, the IL-15 concentration is less than 1 ng / mL to 1000 ng / mL). By using a medium that does not contain IL-15, the maturation of innate immune lymphocytes, which have a higher requirement for IL-15, is suppressed, and CTLs with the characteristics of adaptive immune lymphocytes are developed. In addition, CD8α + β + Used in the induction process of cytotoxic T cells The medium may contain a caspase inhibitor, such as Pan Caspase fmk Inhibitor Z-VAD.

[0041] CD8α + β + The type and concentration of vitamin C used to induce cytotoxic T cells are determined based on the above-mentioned structure. This is similar to the induction of hematopoietic progenitor cells. CD8α + β + The concentration of FLT3L in the medium used for inducing cytotoxic T cells is, for example, 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of IL-21 in the medium used for inducing cytotoxic T cells is, for example, 1 ng / ml to 100 ng / ml.

[0042] In the present invention, a Notch ligand is a molecule that binds to a Notch signal receptor and transmits a Notch signal. Notch signal receptors are single-pass transmembrane proteins. The Notch receptor, which consists of an extracellular domain (NECD), a transmembrane domain (TM), and an intracellular domain (NICD), is processed into TM-NICD, which is then synthesized from NECD and TM-NICD. Ligands for this Notch signal receptor include members of the Delta-like family (DLL1, DLL3, DLL4) and the Jagged family (JAG1, JAG2). The ligand for the Notch signal receptor may be a recombinant substance, and may be linked to Fc or the like. The ligand of the Notch signal receptor used in the present invention may be a fusion protein, which is commercially available from, for example, Adipogen and is readily available. , DLL4 or JAG1.

[0043] In the present invention, the fibronectin (FN) fragment is selected from fragments contained in the FN-binding domain, cell adhesion domain, or heparin-binding domain, for example, III1, III2, III3, III7, III8, III9, III 11 , III 12 , III 13The fragment may contain at least one fragment selected from the group consisting of CS-1 and III1, and may further comprise multiple domains linked together repeatedly. For example, fragments containing a cell adhesion domain containing a VLA-5 ligand, a heparin-binding domain, a CS-1 domain that is a VLA-4 ligand, III1, etc., can be used in the present invention. Examples of such fragments include CH-271, CH-296, H-271, and H-296, as well as derivatives and modifications thereof, which are described in J. Biochem., Vol. 110, pp. 284-291 (1991). CH-296 is commercially available under the name RetroNectin (registered trademark). The C-terminal two-thirds of the polypeptide of III1 is commercially available under the name Fibronectin Fragment III1-C.

[0044] The FN fragment may be used by immobilizing it on an appropriate solid phase, such as a cell culture carrier such as cell cultureware, beads, membranes, or glass slides. Immobilization of the FN fragment on a solid phase can be carried out, for example, according to the method described in International Publication No. 00 / 09168. The concentration of the FN fragment used in the present invention is not particularly limited; for example, it may be added to the medium so that the final concentration is 0.001 to 500 μg / mL, preferably 0.01 to 500 μg / mL. When the FN fragment is used in an immobilized form, it may be immobilized on a solid phase using an FN fragment solution at the aforementioned concentration. The FN fragment may be added to the medium at a final concentration of 0.001 to 500 μg / mL, for example, 0.01 to 500 μg / mL. The culture of a cell population in the presence of a fragment is described in detail in International Publication No. 03 / 080817, and can be carried out by referring to this document. Notch ligands can also be immobilized on cell cultureware at the same concentration and by the same method. This can be done.

[0045] In the present invention, CD8α + β + CD4CD8 positive T cells were used to induce cytotoxic T cells. The temperature conditions for culturing are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, those skilled in the art can appropriately determine the culture period while monitoring the number of CD8-positive T cells, etc. The number of days is not particularly limited as long as hematopoietic progenitor cells are obtained, but is, for example, one day or more.

[0046] CD8α + β + Expansion culture process of cytotoxic T cells CD8α obtained as described above + β + By expanding cytotoxic T cells, a large amount of CD8α + β + Cytotoxic T cells can be obtained. Expansion culture can be performed using a medium containing, for example, IL-7, IL-15, and IL-21. Alternatively, in addition to IL-7 and IL-15, IL-21, IL-18, IL-12, and TL1A (TNF-like ligand 1A, also known as vascular endothelial growth factor 1A) can be used. Inhibitor (VEGI) or TNF superfamily member 15 (TNFSF15) This can be done using the earth. In addition, CD8α was isolated from the cell population obtained from CD4CD8 positive T cells by FACS or affinity column. + β + It is preferable to further select (sort) the cytotoxic T cells before subjecting them to the above-mentioned expansion culture. The selection can be carried out, for example, using one or more of the following as indicators: CD8β positive, CD5 positive, CD336 negative, and CD1a negative. Since CD8β is a TCR co-receptor, CD8β positive sorters are preferred. By this method, it is possible to select CTLs with high antigen recognition ability, and also to distinguish abnormal NK-like cells. By selecting CD5 positive cells, it is possible to select cells that stably express CD8β and have high proliferation potential. In addition, CD1a is an immature T cell marker. Therefore, immature cells can be removed by selecting CD1a-negative cells. Furthermore, CD336 is not expressed by normal CTLs, but is a marker expressed only by innate immune cells. Abnormal NK-like cells can be removed by selection based on sex.

[0047] In the present invention, CD8α + β + The medium used in the expansion of cytotoxic T cells is not particularly limited, and can be prepared by adding cytokines such as IL-7, IL-15, and IL-21 to a basal medium such as a medium used for culturing animal cells. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixtures thereof. The medium may contain serum or may be serum-free. Optionally, the basal medium may also contain one or more substances, such as, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, etc. Preferred basal media contain serum, transferrin, This is αMEM medium containing serine, L-glutamine, and ascorbic acid.

[0048] CD8α + β + The medium used in the expansion step of cytotoxic T cells preferably further contains an anti-CD3 antibody and vitamin C. In the present invention, CD8α + β+ The type and concentration of vitamin C used in the expansion and culture process of cytotoxic T cells are the same as those described above. CD8α + β + The concentration of IL-7 in the medium used in the expansion culture step of cytotoxic T cells is For example, from 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of IL-15 in the medium used in the expansion culture step of cytotoxic T cells is For example, from 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of IL-21 in the medium used in the expansion step of cytotoxic T cells is, for example, 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of IL-12 in the medium used in the expansion step of cytotoxic T cells is, for example, 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of IL-18 in the medium used in the expansion step of cytotoxic T cells is, for example, 1 ng / ml to 100 ng / ml. CD8α + β + The concentration of TL1A in the medium used in the expansion culture process of cytotoxic T cells is For example, from 1 ng / ml to 100 ng / ml.

[0049] The anti-CD3 antibody is not particularly limited as long as it specifically recognizes CD3, and examples thereof include antibodies produced from the OKT3 clone. The concentration of the anti-CD3 antibody in the medium is, for example, , 10ng / ml to 10μg / ml. Instead of adding anti-CD3 antibody to the culture medium, The anti-CD3 antibody may be removed during the expansion process.

[0050] CD8α +β + The temperature conditions for the expansion culture step of cytotoxic T cells are not particularly limited, but for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. + β + It is possible to determine the dose appropriately while monitoring the number of cytotoxic T cells. + β + The number of days is not particularly limited as long as cytotoxic T cells can be obtained, but is, for example, 5 days or more. In the expansion culture step, it is preferable that IL-7 and IL-15 are present in the medium throughout the expansion culture step, but IL-21, IL-18, IL-12, and and TL1A were measured at the beginning of the expansion culture process (e.g., 12, 16, and 24 hours after the start of expansion culture). If the protein is present in the medium for 72 hours or 30 minutes, it does not need to be present in the medium thereafter.

[0051] In such cultures, CD8α + β + Cytotoxic T cells were co-cultured with feeder cells There are no particular limitations on the feeder cells, but CD8α + β + From the viewpoint of further promoting the maturation and proliferation of cytotoxic T cells, peripheral blood mononuclear cells (PBMCC) are preferred.

[0052] The expansion culture step can also be applied to T cells other than cells derived from pluripotent stem cells, such as T cell clones and T cells isolated from living organisms. In the method, T cells are cultured in a culture medium containing IL-7 and IL-15, and one or more of IL-21, IL-18, IL-12, and TL1A. The present invention also provides a medium for culturing T cells, which comprises IL-7 and IL-15, and one or more of IL-21, IL-18, IL-12, and TL1A. The T cells referred to herein are preferably cytotoxic T cells, more preferably CD8α + β + Cell damage However, as mentioned above, it is not limited to T cells other than those derived from pluripotent stem cells, and T cells The T cells may be cloned or isolated from an organism.

[0053] <CD8α + β + Pharmaceutical composition containing cytotoxic T cells, cellular immunotherapy CD8α produced by the method of the present invention + β + As shown in the Examples below, the cytotoxic T cells (culture) have antigen-specific cytotoxic activity. + β + Cytotoxic T cells, preferably human CD8α + β + cytotoxic T cells are useful in the treatment or prevention of diseases such as, for example, tumors, infectious diseases (eg, chronic infections), and autoimmune disorders.

[0054] Therefore, the present invention provides a CD8α antibody produced by the method of the present invention. + β + Pharmaceutical composition containing cytotoxic T cells and said CD8α + β + We provide cellular immunotherapy using cytotoxic T cells.

[0055] In the cellular immunotherapy of the present invention, T cells (CD8α + β + CTL) administration, especially Although not limited thereto, parenteral administration is preferred, for example, intravenous, intraperitoneal, subcutaneous or intramuscular administration. The drug may be administered intravenously, more preferably intravenously, or may be administered locally to the affected area.

[0056] The pharmaceutical composition of the present invention comprises CD8α produced by the method of the present invention. + β + cytotoxic T cells These can be prepared by formulating the above-mentioned compounds by known pharmaceutical methods, and can be administered primarily parenterally, for example, as capsules, liquids, film-coated formulations, suspensions, emulsions, injections (intravenous injections, drip infusions, etc.), etc. In preparing these formulations, the composition may be appropriately combined with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, solubilizing agent, or other additives, etc. Furthermore, the composition may be used in combination with known pharmaceutical compositions or immunostimulants used in the treatment or prevention of the above-mentioned diseases.

[0057] When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected depending on the age, weight, symptoms, health condition of the subject, type of composition (drug, food, drink, etc.), etc.

[0058] The CD8α of the present invention + β + Cellular immunotherapy using cytotoxic T cells involves the steps of isolating T cells with desired antigen specificity from a human, and then generating iPS cells from the T cells with the desired antigen specificity. a step of inducing iPS cells into CD4CD8 bi-positive T cells; and a step of differentiating the iPS cells into CD4CD8 bi-positive T cells. CD8α cells + β + a step of inducing differentiation into cytotoxic T cells, and + β + cell and administering the cytotoxic T cells into the body of a mammal such as a human.

[0059] When performing cell immunotherapy, from the perspective of avoiding rejection reactions, the subject from whom T cells can be isolated is preferably one whose HLA type matches the subject to whom the CD8α + β + cytotoxic T cells obtained by the present invention are administered, and more preferably is the same subject as the subject to whom the CD8α + β + cytotoxic T cells obtained by the present invention are administered. The cells to be administered may be the CD8α + β + cytotoxic T cells produced by the method of the present invention administered as they are, or, as described above, may be administered in the form of a formulated pharmaceutical composition.

Examples

[0060] The present invention will be further specifically described with the following examples, but the scope of the present invention is not limited to those examples.

[0061] <Preparation of CD8αβ+ CTL> cell iPS cells (TKT3v 1-7 strain) were established from human CD3-positive T cells isolated after obtaining consent after notification using the method described in Nishimura T, et al., Cell Stem Cell. 12(1):114-126, 2013. C3H10T1 / 2 cells and OP9 / DLLl cells were obtained from the RIKEN BioResource Center and used. K562 / HLA A-24 was provided by Dr. Tachikawa of the University of Tokyo (currently the National Institute of Infectious Diseases).

[0062] Induction of CD4CD8 positive cells (DP cell induction process) On confluent C3H10T1 / 2 cells in a 10 cm dish, small pieces of the TKT3v 1-7 strain were seeded (Day0), and EB medium (15% fetal bovine serum (FBS), 10 μg / mL human insulin, 5.5 μg / mL h The cells were cultured in IMDM (supplemented with 50 μg / mL phosphoascorbic acid, 5% O, 5% CO ...

[0063] Subsequently, 20 ng / mL VEGF, 30 ng / mL SCF, and 10 ng / mL FLT3L (Peprotech) were added. The cells were cultured under normal oxygen pressure for 7 days (Day 14).

[0064] The resulting net-like structures (also called iPS-SACs) contained hematopoietic cells (CD34 + Hematopoietic progenitor cells (HPCs) were harvested and seeded on OP9 / DLL1 cells. They were cultured for 23 days under normoxic conditions in OP9 medium (αMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 50 μg / ml phosphoascorbic acid, 100 ng / ml streptomycin, 5.5 μg / ml human transferrin, and 5 ng / ml sodium selenite) supplemented with 10 ng / ml FLT3L and 1 ng / ml IL-7. (Day 37). The cells were seeded onto fresh OP9 / DLL1 cells every 3 to 4 days.

[0065] Differentiation induction from CD4 / CD8 positive cells (maturation process) (Protocol 1) On day 37, while maintaining the co-culture of OP9 / DLL1 and differentiated cells, the cells were resuspended in 20% FBS, PSG (penicillin-streptomycin-L-glutamine), ITS (insulin-transferrin-selenite), and NADH. Thorium), 50μg / ml phospho ascorbic acid, 10μM Pan Caspase fmk Inhibitor Z-VAD αMEM medium containing (FMK001, R&D), 10 ng / ml IL-7, 20 ng / ml IL-21, 10 ng / ml Flt3L, and 2 μg / ml anti-human CD3 antibody (OKT3) was added.

[0066] On Day 38, the medium was thoroughly washed and transferred to a plate coated with 5 μg / ml Retronectin (Takara Bio Inc.). The medium was changed from Day 37 to Day 38, containing only anti-CD3 antibody. The Retronectin-coated culture vessel was used for culturing these solutions. The cells were placed in a dish and left to stand overnight at 4°C, and then washed with PBS.

[0067] On day 41, the medium was replaced with the same medium composition as day 38, but with an IL-21 concentration of 10 ng / ml, and the cells were transferred to a plate not coated with Fc-DLL4 or retronectin, and the culture was continued. On day 43, cells were collected and CD8β was detected by FACS. + CD336 - CD5 + CD1a - The cells were sorted.

[0068] Feeder-free expansion culture (Expansion culture - Part 1) 1 μg / ml of anti-CD3 antibody (OKT3) diluted in PBS was added to a 96-well flat bottom plate and incubated for 4 hours. The OKT3-coated plate was then left to stand overnight at ℃. + CD5 + CD1a - cell The cells were suspended in αMEM medium containing 20% ​​FBS, PSG, ITS, 50 μg / ml phosphoascorbic acid, 5 ng / ml IL-7, 5 ng / ml IL-15, and 10 ng / ml IL-21, transferred to an OKT3-coated plate, and cultured for 16 hours. The cells were then transferred to wells not coated with OKT3, and the culture was continued with a half-medium change every three days. An equal amount was always added during medium changes, and 5 ng / ml of IL-21 was added only during the first medium change. CD8α cells were cultured from 14 to 21 days after the start of expansion. + β + These were used as CTLs in the following experiments ( improved iPSC-CTL).

[0069] As a control, CTLs differentiated by a conventional protocol (conventional CTLs) were used. As a conventional protocol, a method was adopted in which expansion culture was directly carried out after the above-mentioned DP cell induction step without going through the maturation step.

[0070] Feeder-free expansion culture (Expansion culture - Part 2) 1 μg / ml of anti-CD3 antibody (OKT3) diluted in PBS was added to a 96-well flat bottom plate and incubated for 4 hours. The OKT3-coated plate was then left to stand overnight at ℃. + CD5 + CD1a - cell The cells were suspended in αMEM medium containing 20% ​​FBS, PSG, ITS, 50μg / ml phospho ascorbic acid, 10μM Pan Caspase fmk Inhibitor Z-VAD (FMK001, R&D), 5ng / ml IL-7, 5ng / ml IL-15, 20ng / ml IL-21, 50ng / ml IL-12, 50ng / ml IL-18, and 50ng / ml TL1A (2x10^4 cells / 200ul / well), transferred to OKT3-coated plates, and cultured for 16 hours. The cells were then transferred to wells not coated with OKT3, and culture was continued with half-medium changes every three days. The medium changes were performed using αMEM containing 20% ​​FBS, PSG, ITS, 50 μg / ml phosphoascorbic acid, 5 ng / ml IL-7, and 5 ng / ml IL-15. On the 14th day after the start of expansion culture, the cells were transfected with CD8α + β + These were used as CTLs in the following experiments (improved iPSC-CTLs).

[0071] <CD8α + β + CTL evaluation 1. CD8αβ expression and antigen binding analysis The expression of CD8α and CD8β was confirmed using a flow cytometer for the improved iPSC-CTLs produced by the method of the present invention, conventional CTLs produced by the conventional method, and the original CTLs from which the iPS cells were derived. As shown in Figure 1, the improved iPSC-CTLs maintained the expression of CD8α even after repeated expansion culture. Furthermore, when the antigen binding activity of the TCR of each of the above cells was quantified by the tetramer method, the improved TCR produced by the method of the present invention was found to be stable. The binding of the iPSC-CTL to the HLA-tetramer was as high as that of the original CTL. Although conventional CTLs obtained from this study expressed TCR to the same extent, they did not express CD8β and therefore had significantly weaker binding to HLA-tetramers.

[0072] 2. Analysis of cell proliferation and cytokine production Next, we compared the improved iPSC-CTLs (modified CD8b+ CTLs) with conventional iPSC-CTLs (conventional CD8b - CTLs were stained with CFSE and treated with 0 (unpulsed), 10 nM, or 100 nM of a specific antigen peptide (Nef138-8). The mice were stimulated with K562 / HLA A-24, which presented the IL-11 IgG1-associated HLA-binding domain. The decrease in CFSE levels and the production of two cytokines, IFNγ and IL-2, were investigated during proliferation. is a reagent used in proliferation assays because its fluorescence intensity decreases with each proliferation (J Vis Exp. 2010; (44): 2259) (The more proliferated the cells, the lower the CFSE fluorescence). The results are shown in Figure 2. In this CFSE assay, the improved iPSC-CTLs demonstrated higher proliferation capacity than conventional iPSC-CTLs. Furthermore, the improved iPSC-CTLs also demonstrated significantly higher cytokine production capacity.

[0073] 3. Analysis of cytotoxic activity Modified iPSC-CTL(Modified CD8b + We co-cultured improved iPSC-CTLs (CD8b- CTLs) and conventional iPSC-CTLs (conventional CD8b- CTLs) with K562 / HLA A-24 cells pulsed with various concentrations of a specific antigen peptide (Nef138), and examined their cytotoxic activity using LDH activity as an indicator. The results are shown in Figure 3. While the improved iPSC-CTLs lost NK activity, their antigen-specific cytotoxic activity was superior to that of the conventional type, and both types exhibited equivalent cytotoxic activity against target cells presenting sufficient amounts of antigen.

[0074] 4. Analysis of Cellular Traits Modified iPSC-CTL(Modified CD8b + CTL) and conventional iPSC-CTL (conventional CD8b - The original T cell clones were analyzed by FACS. CD28 and CD27 are costimulatory molecules expressed only on naive or central memory cells, and CCR7 is a homing molecule essential for immune surveillance. As shown in Figure 4, these molecules were expressed during the ex vivo culture process. Expression was lost in the original CTLs, but expression was restored only in the improved iPSC-CTLs. In addition, in the combination of CD45RA and CD45RO, which is most commonly used to distinguish between naive and memory cells, both improved and conventional iPSC-CTLs express CD45RA + CD45RO - showed the characteristics of naive cells.

[0075] 5. Analysis of proliferation ability in expansion culture Improved iPSC-CTL (Modified CD8b + CTL) and conventional iPSCs - CTL (conventional CD8b - As shown in Figure 5, αMEM was used as the basal medium, and ITS and phosphoascorbic acid were added as supplements. In the improved on-feeder expansion culture system, improved iPSC-CTLs were obtained at 10 20 showed extremely high proliferation ability did.

[0076] 6. Analysis of IFNγ production and expression of cytotoxic molecules during expansion culture Improved iPSC-CTL (Modified CD8b + CTLs) were stimulated with PHA + PBMC or in a feeder-free environment (with or without IL-21), and the production of the cytokine IFNγ and the expression of the cytotoxic molecule Granzyme B were quantified. As shown in Figure 6, the addition of IL-21 to feeder-free expansion cultures enhanced not only cytokine production but also Granzyme B expression, maintaining functionality comparable to that of the on-feeder method.

[0077] 7. Analysis of cytokine production and cytotoxic activity under antigen stimulation The improved iPSC-CTLs (modified CD8b+ CTLs) produced by the method of the present invention (expansion culture - part 1) were stimulated with PHA + PBMCs and examined for cytokine production and cytotoxic activity. As shown in Figure 7, the improved iPSC-CTLs acquired cytotoxic activity with repeated stimulation, perhaps reflecting their naive phenotype. However, at the same time, they lost the ability to produce IFNγ.

[0078] 8. Cytokine production and expression analysis of cytotoxic molecules The improved iPSC-CTLs (modified CD8b+ CTLs) prepared by the method of the present invention (expansion culture - part 1) and the original CTLs were stimulated with PHA + PBMC, and the production of cytokine IFNγ and the expression level of the cytotoxic molecule Granzyme B were quantified after 5 expansion times (Figure 7). When iPSC-CTLs prepared using a combination of on-feeder and feeder-free expansion culture, which is expected for clinical application, were compared in function with the original CTLs, it was found that their cytokine production ability was slightly inferior. It was revealed that the cytotoxic activity of both was equivalent.

[0079] 9. CD8α with IL-7 and IL-21 + β + Analysis of CTL production and effects on surface markers The effects of adding IL-15 and IL-21 on the iPSC-CTL maturation process were examined. The results are shown in Figure 9. The upper graph in Figure 9 shows the effect of CD8α on DP cells. + β + The efficiency of CTL production was shown by adding IL-15. CD8α + β + It is clear that the efficiency of CTL production has increased dramatically. However, in the lower panel of Figure 9, the representative markers of naive CTLs and important chemokines The expression of CCR7, which is also a kinase receptor, and CD5, which is thought to be related to proliferation ability, was significantly reduced by the addition of IL-15. Furthermore, NK markers, which are not expressed in normal CTLs, CD56 and CD336, which are CD8α receptors, are induced by the addition of IL-15. + β + Generation of CTLs Although this improves efficiency, it has been shown that the cells obtained do not have the inherent properties of desirable CTLs. was suggested. On the other hand, the addition of IL-21 inhibited CD8α + β + Although no contribution to CTL production efficiency was observed, NK-related markers In addition to not having any side effects such as increasing CCR7, the drug was found to have the effect of increasing the expression of CCR7 and CD28, an important co-stimulatory molecule.

[0080] 10. Analysis of Cytokine Production Profiles of Generated iPSC-CTLs Previous reports have shown that mature CTLs can be classified into naive, central memory (CM), effector memory (EM), The IL-2 and IFNγ production profiles were changed during the differentiation process to the most differentiated EMRA cells. It is known that naive cells are more inclined to produce IL-2, and as differentiation progresses, cells that simultaneously produce IFNγ and cells that only produce IFNγ appear. The important point here is that naive cells, which are less differentiated, have stronger antitumor effects in vivo than more differentiated EM and EMRA. As shown in Figure 10, the IL-2 / IFNγ profile of the improved iPSC-CTLs prepared by the method of the present invention is intermediate between naive and CM, and shows a shallow differentiation. These results suggest that iPSC-CTLs have naive / CM-like properties and high antitumor activity. Furthermore, the results of Figure 9 above suggest that the addition of IL-15 inhibits the production of naive CTLs, and in agreement with this, it was confirmed that the addition of IL-15 reduces the ability to produce IL-2. On the other hand, the addition of IL-21 was found to have the effect of boosting IL-2 production. Taking the results of Figures 9 and 10 together, the addition of IL-21 is particularly useful in the maturation of iPSC-CTLs. It was suggested that this was the case.

[0081] 11. Other Aspects of the Maturation Process Differentiation induction from CD4 / CD8 positive cells (maturation process) (Protocol 2) After the above-mentioned DP cell induction process, on day 37, while maintaining the co-culture of OP9 / DLL1 and differentiated cells, the cells were cultured in 20% FBS, PSG (penicillin-streptomycin-L-glutamine), ITS (insulin-transcription steroids), and ATP. The cells were cultured in αMEM medium containing spherin-sodium selenite, 50 μg / ml phosphoascorbic acid, 10 μM Pan Caspase fmk Inhibitor Z-VAD (FMK001, R&D), 10 ng / ml IL-7, 10 ng / ml Flt3L, and 2 μg / ml anti-human CD3 antibody (OKT3).

[0082] On Day 38, the medium was thoroughly washed and then transferred to a plate coated with 5 μg / ml Retronectin (Takara Bio Inc.) and 1 μg / ml Fc-DLL4 (Sino Biological Inc.). The medium used was the same as that used on Day 37, except that the anti-CD3 antibody had been removed. For the Fc-DLL4 and Retronectin-coated incubators, these solutions were placed in the incubator and left to stand overnight at 4°C. and then washed with PBS.

[0083] On Day 44, the medium was replaced with the medium from Day 38 supplemented with 10 ng / ml of IL-21, and Fc-DLL4 and The cells were transferred to a plate coated with Retronectin and not coated with Retronectin, and the culture was continued. On day 58, cells were collected and analyzed by FACS to identify CD8β+ CD336 - CD5 + CD1a - The cells were sorted.

[0084] 12. Comparison of marker expression and proliferation ability between conventional and improved iPSC-CTLs Figure 11 shows the FACS results of sorting CD8β / CD5 positive cells from iPSC-CTLs matured by the conventional method, followed by two weeks of PHA on-feeder expansion culture. Even if we start with β / CD5 positive cells, the expression stability of both molecules (especially CD5) is low. After one expansion, the expression of CD8β was significantly reduced. + CD5 bright cells and CD8β + CD5 - The cells were then divided into two groups and expanded for a second time under feeder-free conditions. + CD5 bright Cells are CD8b + CD5 - It has been revealed that the cells have a much higher proliferation ability than This is consistent with previous reports showing that CD5 is an indicator of highly proliferative CTLs (Fig. 12). (Nature Immunology, 16.1 (2015), 107-117.) Based on these results, it is clear that the conventional method can also detect CD5 bright Highly proliferative iPSC-CTLs are generated, but their efficiency and stability are very low, and in many cases, they are CD4+ with low proliferation capacity. 5- It was suggested that cells were primarily produced.

[0085] On the other hand, the improved iPSC-CTLs induced by Protocol 2 showed high CD5 expression immediately after maturation. The expression was maintained at a high level comparable to that of primary CTLs even after four rounds of expansion (Fig. 13). 20 , feeder-free also 10 15 all of which are inferior to primary naive CTLs The cells showed extremely high proliferation potential suitable for clinical application (Figure 14).

[0086] 13. Analysis of proliferation ability in expansion culture Improved iPSC-CTL (Modified CD8b + The proliferation of the parent CTL clone (H25-4) was analyzed. As shown in Figure 15, the addition of one or more of IL-21, IL-12, IL-18, and TL1A in addition to the basal cytokines IL-7 and IL-15 enhanced proliferation when the cells were expanded. In addition to the basic cytokines IL-7+IL-15, IL-21, IL-12, IL-18, and TL1A were added. When expanded, the effect of enhancing the proliferation of the parental CTL clone was also observed.

[0087] 14. Analysis of cytokine production and cytotoxic activity under antigen stimulation Improved iPSC-CTL (Modified CD8b + CTLs) were stimulated with PHA + PBMCs to examine cytokine production and cytotoxic activity. As a result, as shown in Figure 16, in addition to the basic cytokines IL-7 and IL-15, IL-21, IL-12, IL-18, and a type of TL1A were produced. It was found that when the cells were expanded with the addition of these ingredients, not only the proliferation rate but also the cytokine production ability and cytotoxic activity improved.

Claims

1. CD4CD8 co-positive T cells were cultured in a medium containing IL-7 and T cell receptor activators to induce CD8α ＋ β ＋ CD8α including the step (A) of inducing it into cytotoxic T cells ＋ β ＋ A method for producing cytotoxic T cells, comprising: CD8α obtained by the above step (A) ＋ β ＋ (B) culturing cytotoxic T cells in a medium containing IL-7 and IL-15, and also containing IL-18 and IL-12; ＋ β ＋ Method for producing cytotoxic T cells.

2. The step (B) comprises IL-7, IL-15, IL-18, IL-12, and IL-21 and / or TL1A. The method according to claim 1, which comprises culturing the bacteria in a medium.

3. The method of claim 1, wherein the T cell receptor activator is an anti-CD3 antibody.

4. The method according to claim 2, wherein the culture in the step (B) is carried out without using feeder cells. Law.

5. The CD4CD8 bi-positive T cells according to claim 1, wherein the CD4CD8 bi-positive T cells are induced from pluripotent stem cells. Manufacturing method.

6. The method of claim 5, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

7. The induction of CD4CD8 bi-positive T cells from the pluripotent stem cells comprises the following steps (a) and (b): The method of claim 5, (a) Pluripotent stem cells are cultured in a medium supplemented with vitamin C, and hematopoietic progenitor cells are induced. conducting the (b) A step of culturing the hematopoietic progenitor cells obtained in step (a) in a medium containing vitamin C, FLT3L, and IL-7 to induce CD4CD8 co-positive T cells.

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