Cytotoxic immunocompetent cell expressing Anti-CD38-car, method for producing same, and pharmaceutical composition containing said immunocompetent cell

Abstract

The present invention provides a cytotoxic immunocompetent cell that is derived from a pluripotent stem cell and that expresses a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain recognizes CD38 as an antigen. The present invention also provides a pharmaceutical composition or the like comprising the cytotoxic immunocompetent cell derived from a pluripotent stem cell.

Description

Cytotoxic immunocompetent cells expressing anti-CD38-CAR, method for producing the same, and pharmaceutical composition containing the immunocompetent cells

[0001] The present invention relates to cytotoxic immunocompetent cells that express anti-CD38-CAR, a method for producing the same, and a pharmaceutical composition containing the immunocompetent cells.

[0002] CD38 (Cluster of Differentiation 38) is a transmembrane glycoprotein with a molecular weight of 45 kDa, and is a surface marker for plasma cells, pre-B cells, etc. CD38 is involved in cell differentiation, activation, immune response control, apoptosis, etc., and is known to be expressed in the early stages of development in, for example, T cells, B cells, etc., disappear as they mature, but be expressed again upon activation.

[0003] As mentioned above, CD38 is also expressed on normal cells, but it is known that it is expressed at higher levels on the surface of hematologic malignant tumor cells such as myeloma (e.g., multiple myeloma) cells, malignant lymphoma cells, and leukemia cells than on normal cells (e.g., Non-Patent Document 1).

[0004] Furthermore, in recent years, as one type of cancer immunotherapy, research and application of CAR-T therapy using CAR-T cells obtained by introducing and expressing a gene encoding a chimeric antigen receptor (CAR) that recognizes a cancer cell surface antigen into T cells collected from a patient has been rapidly progressing (e.g., Patent Document 1).

[0005] Special Publication No. 2020-517244

[0006] Cytometry, 46:23-27, 2001

[0007] As described above, CD38 is often highly expressed on hematologic malignant tumor cells, and therefore can be a candidate cancer cell surface antigen for CAR gene therapy such as CAR-T therapy. On the other hand, cytotoxic immunocompetent cells (hereinafter sometimes referred to as "killer cells") generally express CD38 upon stimulation by gene transfer. Therefore, anti-CD38-CAR killer cells produced by transferring an anti-CD38-CAR gene into killer cells may undergo self-killing by fratricide in vitro or ex vivo, thereby impairing their anti-tumor activity. To address this issue, when transferring an anti-CD38-CAR gene into killer cells, CD38 expression can be suppressed by RNA interference using siRNA, DNA editing using Crispr-Cas9, or the like.

[0008] In view of the above circumstances, a main object of the present invention is to provide anti-CD38-CAR killer cells that can be easily prepared.

[0009] The present inventors attempted to produce anti-CD38-CAR killer cells using killer cells obtained by inducing the differentiation of pluripotent stem cells, and found that killer cells derived from pluripotent stem cells are CD38-negative even when gene transfer is performed, and that anti-CD38-CAR killer cells can be obtained while avoiding or suppressing fratricide, thereby completing the present invention.

[0010] [1] According to one aspect of the present invention, there is provided a cytotoxic immunocompetent cell derived from a pluripotent stem cell, in which a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain is expressed, and the extracellular antigen-binding domain recognizes CD38 as an antigen. [2] In the immunocompetent cell described in [1] above, the pluripotent stem cell may be a human induced pluripotent stem cell or a human embryonic pluripotent stem cell. [3] In the immunocompetent cell described in [1] or [2] above, the pluripotent stem cell may be a pluripotent stem cell partially or completely deficient in HLA molecules. [4] The immunocompetent cell described in any of [1] to [3] above may be a T cell. [5] The immunocompetent cell described in any of [1] to [4] above may be a CD8αβ heterodimer T cell. [6] In the immunocompetent cell described in any of [1] to [5] above, the pluripotent stem cell may be a pluripotent stem cell introduced with an exogenous TCR gene. [7] The immunocompetent cells according to any one of [1] to [6] above may be negative for CD38 expression. [8] In the immunocompetent cells according to any one of [1] to [7] above, the CD38 may be human CD38. [9] In the immunocompetent cells according to any one of [1] to [8] above, the chimeric antigen receptor may comprise the extracellular antigen-binding domain, hinge domain, transmembrane domain, costimulatory domain, and intracellular signaling domain, linked in this order.

[10] In the immunocompetent cells according to any one of [1] to [9] above, the extracellular antigen-binding domain may comprise the amino acid sequence represented by SEQ ID NO: 1 or a homologous sequence thereof and the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof.

[11] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease associated with CD38 expression, comprising the immunocompetent cells according to any one of [1] to

[10] above.

[12] The pharmaceutical composition according to

[11] above may be used in combination with an anti-CD38 antibody drug.

[13] Another aspect of the present invention provides a method for producing CAR-CD8 T cells derived from induced pluripotent stem cells, the method comprising: preparing CD8 T cells induced to differentiate from induced pluripotent stem cells; and introducing a gene encoding a chimeric antigen receptor into the CD8 T cells, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain recognizes CD38 as an antigen.

[0011] According to an embodiment of the present invention, anti-CD38-CAR killer cells with no or suppressed CD38 expression can be obtained by introducing an anti-CD38-CAR gene into killer cells derived from pluripotent stem cells.

[0012]

[0023] Figure 1 is a schematic diagram illustrating an example of a DNA construct comprising a gene encoding anti-CD38-CAR that can be used in the present invention. Figure 2 is a diagram showing a schematic configuration of a retroviral vector comprising a gene encoding anti-CD38-CAR. Figure 3 is a diagram showing the results of evaluating by flow cytometry the expression of the anti-CD38-CAR gene in cells used to produce anti-CD38-CAR-reCTL in Experimental Example 3. Figure 4 is a diagram showing the results of evaluating by flow cytometry the expression of CD38 in cells used to produce anti-CD38-CAR-reCTL in Experimental Example 3. Figure 5 is a diagram showing the results of evaluating in vitro the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells at various E:T ratios. Figure 6 is a diagram showing the results of evaluating in vitro the cytotoxic activity of anti-CD38-CAR-reCTL against KMM1 cells at various E:T ratios.

[0033] Figure 1 shows the results of in vitro evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells over time. Figure 2 shows the results of in vitro evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against KMM1 cells over time. Figure 3 shows the results of real-time analysis of the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells in vitro. Figure 4 shows the results of evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells in the presence of an anti-CD38 antibody drug. Figure 5 shows the results of in vitro evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against THP-1 cells. Figure 6 shows the results of in vitro evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against HT cells. 14 shows the results of evaluating the anti-tumor effect of anti-CD38-CAR-reCTL in vivo.

[0013] Preferred embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Furthermore, unless inappropriate in the context, the respective embodiments can be combined as appropriate. Furthermore, in this specification, the term "to" indicating a numerical range includes the upper and lower limits.

[0014] As used herein, "DP cells (double positive cells)" refer to CD4-positive CD8-positive T cells. "DN cells (double negative cells)" refer to CD4-negative CD8-negative T cells. "CD8 T cells" and "CD8 SP cells" both refer to CD4-negative CD8-positive T cells. Unless otherwise specified, "CD8" encompasses both CD8αα homodimers and CD8αβ heterodimers. "TCR" refers to T cell receptor. "CTL cells" refer to cytotoxic T cells. "T cells" refer to cells expressing an antigen receptor called a T cell receptor (TCR) on their surface.

[0015] A. Cytotoxic Immunocompetent Cells According to an embodiment of the present invention, there are provided pluripotent stem cell-derived cytotoxic immunocompetent cells (pluripotent stem cell-derived anti-CD38-CAR killer cells) that express a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and that the extracellular antigen-binding domain recognizes CD38 as an antigen. Generally, when CAR cells are produced using immunocompetent cells collected from a patient, a long production period (e.g., several weeks to several months) and high production costs (e.g., tens of millions of yen) are required. In contrast, by inducing differentiation of immunocompetent cells from pluripotent stem cells in advance and introducing an anti-CD38-CAR gene, the anti-CD38-CAR killer cells can be cryopreserved, enabling off-the-shelf storage.

[0016] The anti-CD38-CAR killer cells derived from pluripotent stem cells are preferably CD38-negative. Here, with regard to protein expression, "negative" means that the expression of the protein is completely or substantially not observed, and "positive" can mean that the expression of the protein is completely or substantially observed. In one embodiment, the expression of a protein in cells can be evaluated as positive in flow cytometry (FCM) analysis when the fluorescence intensity is clearly (in other words, significantly) distinguishable (e.g., clearly higher) when the cells are treated with a fluorescent-labeled antibody that specifically binds to the protein (e.g., a fluorescent-labeled anti-G4S linker antibody, a fluorescent-labeled anti-CD38 antibody) compared to when the cells are treated with a fluorescent-labeled antibody against an antigen that should not be present in the cells (a so-called isotype control antibody). Otherwise, the expression can be evaluated as negative. Comparison and analysis of the fluorescence intensities can be performed, for example, using flow cytometry analysis software.

[0017] Any immunocompetent cells having cytotoxic activity can be used as the cytotoxic immunocompetent cells (killer cells). Specific examples of killer cells include lymphocytes, monocytes, and polymorphonuclear leukocytes. Among these, lymphocytes are preferred, and killer T cells such as CD8 T cells (CTLs), natural killer (NK) T cells, and γδ T cells, and natural killer (NK) cells are more preferred.

[0018] The pluripotent stem cells are stem cells that have pluripotency, i.e., the ability to differentiate into many cells present in a living body, and also have the ability to self-renew. The pluripotent stem cells are preferably mammalian pluripotent stem cells, and more preferably human pluripotent stem cells.

[0019] Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells), etc. Among these, iPS cells or ES cells are preferred, and iPS cells are more preferred.

[0020] The pluripotent stem cells may be pluripotent stem cells that are partially or completely deficient in HLA molecules. Anti-CD38-CAR killer cells derived from such pluripotent stem cells can avoid being recognized as non-self by the patient's own killer T cells or helper T cells to which they are administered. HLA molecule deletion can be achieved, for example, using genome editing technology.

[0021] The pluripotent stem cells may be introduced with an exogenous TCR gene. For example, immunocompetent cells (typically, T cells) induced to differentiate from pluripotent stem cells (e.g., TCR-iPS cells or TCR-ES cells) into which an exogenous TCR gene has been introduced can express a desired TCR. By expressing a CAR in such immunocompetent cells, antigen recognition by the TCR (e.g., recognition of an antigen present within a cell) and antigen recognition by the CAR (e.g., recognition of an antigen present on the cell surface) can be used in combination.

[0022] The chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR preferably comprises an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, which are linked in tandem in this order.

[0023] The extracellular antigen-binding domain may be an anti-CD38 antibody or a functional portion thereof that recognizes CD38 as an antigen. Preferably, a fusion protein (so-called scFv) in which the light chain (VL) variable region and heavy chain (VH) variable region of anti-CD38 IgG are linked by a short linker peptide of, for example, about 10 to 25 amino acids is used as the extracellular antigen-binding domain. The extracellular antigen-binding domain may comprise the light chain (VL) variable region and heavy chain (VH) variable region in this order toward the transmembrane domain, or may comprise them in this order from the transmembrane domain. CD38 is preferably mammalian CD38, and more preferably human CD38.

[0024] As the anti-CD38 antibody from which the extracellular antigen-binding domain is derived, any suitable antibody capable of specifically binding to CD38, preferably an IgG antibody, can be used.

[0025] In one embodiment, the extracellular antigen-binding domain comprises a VL variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or a sequence homologous thereto.

[0026] In one embodiment, the extracellular antigen-binding domain comprises a VH variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 or a sequence homologous thereto.

[0027] In one embodiment, the scFv comprises a VL variable region comprising the amino acid sequence represented by SEQ ID NO: 1 or a homologous sequence thereof, and a VH variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof.

[0028] The homologous sequence may have, for example, 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with a reference amino acid sequence (specifically, the amino acid sequence represented by SEQ ID NO: 1 or 2). The homologous sequence may be a sequence in which 1 to 10, 8 or less, 5 or less, or 3 or less amino acids of the reference amino acid sequence have been substituted, deleted, or added with other amino acids. The light chain and heavy chain complementarity determining regions (CDRs) 1, 2, and 3 of the homologous sequence may have sufficiently high sequence identity (e.g., 3 or less, 2 or less, 1 or less, or 0 amino acid substitutions, deletions, or additions) with the light chain and heavy chain CDRs 1, 2, and 3 of the reference amino acid sequence, respectively.

[0029] The hinge domain, transmembrane domain, costimulatory domain, and intracellular signaling domain are not limited as long as the effects of the present invention are obtained, and any appropriate domains can be adopted as each of the domains constituting the CAR. The hinge domain is designed, for example, to provide space (length) and flexibility for the extracellular antigen-binding domain to access the target epitope. The transmembrane domain can include, for example, a transmembrane domain such as CD8 or CD28. The costimulatory domain can include, for example, either or both of a domain derived from the 4-1BB domain and a domain derived from the CD28 domain. The intracellular signaling domain can include, for example, a domain derived from the CD3ζ domain.

[0030] Hereinafter, a method for producing iPS cell-derived anti-CD38-CAR-CD8 T cells (hereinafter also referred to as "anti-CD38-CAR-iPS-CD8 T cells" or "anti-CD38-CAR-reCTL") will be specifically described as an example of pluripotent stem cell-derived anti-CD38-CAR killer cells according to an embodiment of the present invention. Anti-CD38-CAR-iPS-CD8 T cells are typically CD4-negative and CD8-positive.

[0031] Anti-CD38-CAR-iPS-CD8 T cells can be obtained, for example, by preparing CD8 T cells induced to differentiate from iPS cells (preferably human iPS cells), and introducing into the CD8 T cells a gene encoding a chimeric antigen receptor (anti-CD38-CAR) that recognizes CD38 as an antigen, and allowing the CD8 T cells to express the gene. As described above, CD8 T cells derived from pluripotent stem cells such as iPS cells are typically CD38-negative even after gene introduction. Therefore, by introducing a gene encoding anti-CD38-CAR, anti-CD38-CAR-iPS-CD8 T cells can be obtained simply and efficiently without causing the problem of fratricide.

[0032] Any appropriate method can be used as a method for inducing differentiation of iPS cells into CD8 T cells. In one embodiment, iPS cell-derived CD8 T cells can be obtained by a method comprising: step (1): inducing differentiation of iPS cells to obtain a cell culture containing DN cells and DP cells; step (2): removing DN cells from the cell culture obtained in step (1); and step (3): differentiating DP cells in the cell culture obtained in step (2) into CD8 T cells. This method can suitably obtain iPS cell-derived CD8 T cells that do not express CD38 even after stimulation. Killer cells that do not express CD38 even after stimulation can be used to easily and efficiently obtain anti-CD38-CAR killer cells without the need for suppressing CD38 expression by RNA interference, genome editing, or the like, or for masking CD38 on the killer cell surface by co-incubating with an anti-CD38 antibody.

[0033] In step (1), iPS cells are induced to differentiate to obtain a cell culture containing DN cells and DP cells.

[0034] The iPS cells used in step (1) may be iPS cells induced from T cells or somatic cells other than T cells. The iPS cells are preferably human-derived iPS cells.

[0035] T cells that are induced to become iPS cells are preferably T cells that express CD3 and at least one molecule selected from the group consisting of CD4 and CD8. Examples of such human T cells include helper / regulatory T cells, which are CD4-positive cells, cytotoxic T cells, which are CD8-positive cells, and naive T cells (CD45RA ＋ CD62L ＋ cells), central memory T cells (CD45RA － CD62L ＋ cells), effector memory T cells (CD45RA － CD62L － cells), and terminal effector T cells (CD45RA ＋ CD62L － cells).

[0036] Human T cells can be isolated from human tissues using known techniques. Examples of human tissues include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and diseased tissue. Peripheral blood and umbilical cord blood are particularly preferred. Known techniques for isolating human T cells include flow cytometry using a cell sorter and antibodies against cell surface markers such as CD3. Desired T cells can also be isolated using cytokine secretion or functional molecule expression as an indicator. In such cases, for example, T cells secrete different cytokines depending on whether they are Th1 or Th2 type, and T cells having the desired Th type can be isolated by sorting using such cytokines as an indicator. Cytotoxic (killer) T cells can also be isolated using secretion or production of granzymes, perforin, and the like as an indicator.

[0037] Somatic cells induced into iPS cells may be any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as sperm, spermatocytes, eggs, oocytes, and ES cells, or pluripotent cells. Somatic cells include somatic cells from fetuses (offspring), newborns (offspring), and somatic cells from mature, healthy individuals or individuals with diseases. They also include primary cultured cells, passaged cells, and established cell lines. Specifically, somatic cells include differentiated cells, such as tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, tissue progenitor cells, lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0038] iPS cells are induced from the T cells or somatic cells. Methods for obtaining iPS cells from T cells or somatic cells include, for example, the method described in Vizcardo et al., Cell Stem Cell 12, 31-36 (2013). For example, T-iPS cells can be obtained by obtaining antigen-specific T cells from a subject who has acquired immunity against a specific antigen and then introducing Yamanaka factors into these cells (Takahashi and Yamanaka, Cell 126, 663-673 (2006), Takahashi et al., Cell 131, 861-872 (2007), and Grskovic et al., Nat. Rev. Drug Dscov. 10, 915-929 (2011)).

[0039] iPS cells are artificial stem cells derived from somatic cells that have properties similar to those of ES cells and can be produced by applying specific reprogramming factors to somatic cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al.,Nat. Biotechnol. 26:101-106 (2008); WO2007 / 069666). The reprogramming factor may be composed of a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-coding RNA, or a low-molecular-weight compound. Examples of genes contained in the reprogramming factors include 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. 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, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO 2010 / 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, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, 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.

[0040] The reprogramming factors may be contacted with or introduced into somatic cells by any appropriate method depending on their form.

[0041] The iPS cells obtained as described above are induced to differentiate into a T cell population containing DP cells and DN cells. Examples of methods for inducing differentiation of iPS cells into T cells include the method described in Timmermans et al., Journal of Immunology, 2009, 182:6879-6888.

[0042] In one embodiment, iPS cells are co-cultured with OP9 stromal cells, e.g., a culture of mouse OP9 stromal cells, to obtain hematopoietic progenitor cells. The obtained hematopoietic progenitor cells are then co-cultured with OP9 / DLL1 cells. IL-7, FIT-3L, and stem cell factor (SCF) are added to the medium during the OP9 / DLL1 co-culture. When iPS cells are induced to differentiate by the above method, a cell culture containing DN cells and DP cells can be obtained.

[0043] In step (2), DN cells are removed from the cell culture obtained in step (1). When a cell culture containing DN cells and DP cells is stimulated with a CD3 antibody, the DP cells may be killed by the DN cells, but removing the DN cells can prevent this phenomenon.

[0044] Preferably, the DN cell-depleted cell culture is substantially free of DN cells, e.g., the DN cell content in the DN cell-depleted cell culture is 5% or less, preferably 3% or less, and more preferably less than 1%.

[0045] Hereinafter, the process of removing DN cells from a cell culture containing DN cells and DP cells may be referred to as "enrichment of DP cells," and the cell culture from which DN cells have been removed may be referred to as an "enriched DP cell culture." Similarly, the simultaneous or individual removal of cells other than DN cells from a cell culture may also be referred to as "enrichment of DP cells" or "enriched DP cell culture."

[0046] A cell culture containing DN cells and DP cells may contain CD8 SP cells. The CD8 SP cells may be CD8αα homodimeric T cells, and it is preferable to simultaneously remove CD8 SP cells, particularly CD8αα homodimeric T cells, when enriching DP cells (i.e., removing DN cells).

[0047] Any suitable method can be used to remove DN cells and / or CD8SP cells from cell culture. For example, DN cells and CD8SP cells can be removed by recovering CD4-positive cells using a carrier (e.g., MACS beads) bound to a CD4 antibody. Alternatively, CD8αα homodimer T cells can be removed by recovering CD8SPαβ heterodimer T cells using a carrier (e.g., MACS beads) bound to a CD8β antibody. Alternatively, a cell culture substantially free of DN cells can be obtained by removing the DN cell fraction using a cell sorter. Alternatively, a cell culture substantially free of DN cells and CD8SP cells can be obtained by collecting the DP cell fraction using a cell sorter. Alternatively, a cell culture substantially free of CD8SPαα homodimer cells can be obtained by collecting the CD8SPαβ heterodimer cell fraction using a cell sorter.

[0048] In the above step (3), DP cells in the cell culture obtained in step (2) are differentiated into CD8 SP cells. Differentiation of DP cells into CD8 SP cells can be induced by directly activating one of the activation pathways that occurs when the T cell receptor is stimulated. For example, T cells can be activated in a manner similar to TCR stimulation by adding PMA and ionomysin. An example of stimulation via TCR is culturing DP cells while stimulating them with a CD3 antibody. In addition to stimulation with a CD3 antibody, it is preferable to add IL-7 and IL-2 to the culture medium. The culture period in a culture medium containing a CD3 antibody may be 3 to 10 days, for example, 4 to 8 days, for example, about 6 days.

[0049] When a DP cell culture is a DP cell culture induced from iPS cells having a specific receptor (e.g., TCR) for a desired antigen, it is also possible to induce differentiation of the DP cells into CD8 SP cells by stimulating the DP cells with the antigen or with antigen-presenting cells that present the antigen.

[0050] CD8SP cells obtained by inducing differentiation of enriched DP cell cultures by directly activating one of the activation pathways that occurs when the T cell receptor is stimulated are mostly CD8αβ heterodimer-type T cells that express the CD8αβ heterodimer.

[0051] In the method for inducing differentiation of CD8 T cells from iPS cells, DP cells in the cell culture obtained in step (1) may be differentiated into CD8 SP cells in the presence of a substance that suppresses the cytotoxic activity of DN cells, without performing step (2). Examples of substances that suppress the cytotoxic activity of DN cells include perforin inhibitors, granzyme inhibitors, Fas pathway inhibitors, caspase inhibitors, and NK activating receptor inhibitor antibodies. Differentiation of DP cells into CD8 SP cells can be induced in the same manner as in step (3).

[0052] Details of the method for inducing differentiation of iPS cells into CD8 T cells are described in WO2016 / 010154, WO2017 / 179720, etc.

[0053] The CD8 T cells induced to differentiate from the iPS cells (iPS cell-derived CD8 T cells) can be cytotoxic T cells (CTLs) having cytotoxic activity, and CAR-T cells obtained by introducing and expressing a gene encoding an anti-CD38-CAR into the cytotoxic CD8 T cells can also have cytotoxicity.

[0054] 1 is a schematic diagram illustrating an example of a DNA construct comprising a gene encoding an anti-CD38-CAR that can be used in the present invention. DNA construct 1 encoding an anti-CD38-CAR has a configuration in which the following are sequentially linked: (a) a nucleotide sequence encoding a CD8α signal peptide; (b) a nucleotide sequence encoding an L-chain variable region of an anti-CD38 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 1); (c) a nucleotide sequence encoding a linker; (d) a nucleotide sequence encoding an H-chain variable region of an anti-CD38 antibody (e.g., the amino acid sequence represented by SEQ ID NO: 2); (e) a nucleotide sequence encoding a hinge domain and transmembrane domain of CD8α; (f) a nucleotide sequence encoding a 4-1BB intracellular domain; and (g) a nucleotide sequence encoding a CD3ζ chain. For details of anti-CD38-CAR and the DNA construct encoding it, see, for example, WO2007 / 142241.

[0055] Any appropriate method can be used as a method for introducing and expressing a gene encoding an anti-CD38-CAR into iPS cell-derived CD8 T cells. Examples include biological gene transfer methods using a viral vector, physical gene transfer methods such as electroporation, and chemical gene transfer methods such as lipofection and the calcium phosphate method. Among these, gene transfer methods using a viral vector can be preferably used.

[0056] The viral vector is not particularly limited, as long as the viral particles produced infect iPS cell-derived CD8 T cells and the cells express anti-CD38-CAR. From the viewpoint of enabling highly efficient integration of a transgene into the chromosome of a host cell and maintaining high expression of the gene, a retroviral vector can be preferably used. Here, the retroviral vector refers to a so-called oncoretroviral vector (hereinafter simply referred to as a retroviral vector), and includes a lentiviral vector and the like.

[0057] Gene transfer into iPS cell-derived CD8 T cells using a retroviral vector can be carried out, for example, by co-transfecting packaging cells (e.g., Cos cells, 293T cells) with a vector plasmid containing the gene sequence of interest, an LTR, and a packaging signal, and a packaging plasmid containing the gag sequence, pol sequence, and env sequence but no packaging signal, and then collecting the produced virus particles (virions) and infecting iPS cell-derived CD8 T cells. Alternatively, virus particles (virions) can be produced by transfecting a vector plasmid containing the gene sequence of interest into packaging cells that express gag, pol, and env and are producing empty virus particles.

[0058] Preferred examples of retroviral vector plasmids include MSCV (Murine Stem Cell Virus) vector plasmids and lentiviral vector plasmids. Various commercially available retroviral vector plasmids can be used, including the MSCV Retroviral Expression System (Clontech). A target gene sequence can be inserted into the multicloning site (MCS) of a retroviral vector plasmid using a standard cloning method.

[0059] In one embodiment, a retroviral vector plasmid having an IRES (internal ribosome entry site) sequence is used. The IRES sequence is a sequence present within an mRNA chain that encodes a structure to which a ribosome directly binds, and is used in a mechanism that initiates translation through direct ribosome binding, known as an internal initiation mechanism. The retroviral vector plasmid having an IRES sequence enables cap-independent translation of eukaryotic mRNA.

[0060] In one embodiment, a retroviral vector plasmid having a marker gene is used from the viewpoint of selecting, separating, etc., transfected cells. As the marker, for example, a fluorescent protein such as GFP or EGFP can be used.

[0061] As a method for transfecting the packaging cells and the retroviral vector plasmid into the packaging cells, methods that are commonly used for gene transfer using retroviral vectors can be applied.

[0062] The virus particles produced by the packaging cells can be obtained as a virus solution, for example, by filtering the culture supernatant.

[0063] Any appropriate method can be used to introduce the obtained virus particles into iPS cell-derived CD8 T cells and infect them. For example, a method (e.g., polybrene method, protamine method) can be used, in which the virus solution is mixed with a positively charged gene transfer adjuvant, and the resulting mixture is added to iPS cell-derived CD8 T cells and cultured. Alternatively, the retronectin method can be used.

[0064] After the infection, cells expressing fluorescence can be easily selected and isolated as cells expressing anti-CD38-CAR by using a fluorescent anti-G4S linker antibody or a fluorescent anti-mouse IgG antibody using, for example, a flow cytometer.

[0065] Although the method for producing anti-CD38-CAR-iPS-CD8 T cells has been described above, anti-CD38-CAR killer cells derived from pluripotent stem cells other than anti-CD38-CAR-iPS-CD8 T cells can also be produced in a similar manner to the above. For example, iPS cells may be induced to differentiate into other cytotoxic immunocompetent cells such as NK cells, or other pluripotent stem cells such as ES cells may be induced to differentiate into cytotoxic immunocompetent cells according to a previously reported method, and the anti-CD38-CAR gene may be introduced into and expressed in the resulting cytotoxic immunocompetent cells in a similar manner to the above.

[0066] B. Pharmaceutical Compositions According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease associated with CD38 expression, comprising the anti-CD38-CAR killer cells derived from the pluripotent stem cells described in Section A. The anti-CD38-CAR killer cells can be activated as needed to kill cells expressing CD38, preferably cells highly expressing CD38. Thus, pharmaceutical compositions according to embodiments of the present invention can be applied to, for example, CAR gene therapies such as CAR-T therapy (including CAR-γδT therapy) and CAR-NK therapy for diseases associated with CD38 expression.

[0067] Diseases associated with CD38 expression include malignancies such as cancer, including hematological malignancies such as leukemia, lymphoma, myeloma, and B-cell lymphoma. Examples of hematological malignancies include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) such as Burkitt's lymphoma (BL), chronic B-lymphocytic leukemia (B-CLL), acute B- and T-lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's macroglobulinemia, mantle cell lymphoma, Hodgkin's lymphoma (HL), plasma cell myeloma, precursor B-cell lymphoblastic leukemia / lymphoma, plasmacytoma, giant cell myeloma, plasma cell myeloma, heavy chain myeloma, light chain or Bence Jones myeloma, lymphomatoid granulomatosis, and the like.

[0068] The pharmaceutical composition can be used in combination with an anti-CD38 antibody drug. The additive or synergistic effect of the cytotoxic activity of the anti-CD38-CAR killer cells and the activity of the antibody drug (e.g., CDC activity, ADCC activity, ADCP activity, etc.) can more favorably achieve therapeutic or prophylactic effects against the diseases.

[0069] The pharmaceutical composition may be a combination containing the anti-CD38-CAR killer cells and an anti-CD38 antibody drug. Alternatively, the pharmaceutical composition does not contain the anti-CD38 antibody drug, and may be administered in combination with a separately prepared anti-CD38 antibody drug. In this case, the pharmaceutical composition and the anti-CD38 antibody drug may be administered simultaneously, or each may be administered separately at an appropriate timing. The dosage, administration rate, administration interval, etc. may be appropriately determined depending on the conditions of the subject to be treated (height, weight, age, sex, etc.), the condition, etc.

[0070] Currently, two types of anti-CD38 antibody drugs are clinically used, each recognizing a different epitope on CD38. Preferably, the anti-CD38-CAR killer cells bind to a site different from the epitopes recognized by these antibodies.

[0071] Examples of anti-CD38 antibody drugs include daratumumab and isatuximab.

[0072] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the description of the examples, cytotoxic iPS cell-derived CD8 T cells may be referred to as "reCTL."

[0073] [Cell preparation and culture] The retrovirus preparation cell line "G3T-hi cells" was purchased from TaKaRa. The retrovirus packaging cell line "PG13 cells," the myeloma-derived cell lines "RPMI8226 cells" and "KMM1 cells," and the acute monocytic leukemia-derived cell lines "THP-1 cells" and "HT cells" were purchased from ATCC. RPMI8226 cells, KMM1 cells, THP-1 cells, and HT cells were used as target cells. G3T-hi and PG13 cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) (Fujifilm Wako Pure Chemical Industries, Osaka) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA) and 100 U / mL penicillin and 100 μg / mL streptomycin (PS) (Fujifilm Wako Pure Chemical Industries, Osaka). RPMI8226, KMM1, THP-1, and HT cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS and 100 U / mL and 100 μg / mL PS. reCTLs were cultured in MEMα (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, 100 U / mL and 100 μg / mL PS, 10 ng / mL IL-2 (PeproTech, Cranbury, NJ), 5 ng / mL IL-7 (PeproTech, Cranbury, NJ), 10 ng / mL IL-21 (PeproTech, Cranbury, NJ), and 0.03 g / mL vitamin C (Nacalai Tesque, Kyoto). For reCTL expansion, anti-CD3 antibody (BioLegend, San Diego, CA) and anti-CD28 antibody (Thermo Fisher Scientific, Waltham, MA) were added to MEMα.

[0074] Experimental Example 1: Generation of iPS-derived CD8 T cells Human iPS cell-derived CD8 T cells (reCTLs) were obtained by the method described in WO 2017 / 179720. Specifically, the process is as follows. Human iPS cells were induced to differentiate into CD34CD43 double-positive hematopoietic progenitor cells by differentiation induction using the EB method, a mesoderm induction system. These cells were seeded on OP9DL1 feeder cells and cultured with the addition of a Notch signal, leading to induction into DP cells, which are T progenitor cells, to obtain a cell culture containing DN cells and DP cells. Next, the DP cells were isolated, and the isolated DP cells were differentiated into CD8 SP cells (reCTLs) by TCR stimulation using a CD3 antibody. The reCTLs generated in this manner could be expanded by repeated stimulation with a CD3 antibody and were used as the base cells for the present CAR-T cells.

[0075] [Experimental Example 2: Preparation of retroviral vector] A gene sequence encoding anti-CD38-CAR comprising a CD8α signal peptide, anti-CD38 scFV, hinge domain, CD8α transmembrane domain, 4-1BB intracellular domain, and CD3ζ domain was excised using a restriction enzyme from the lentiviral vector constructed in an Example of WO2007 / 142241 and inserted into the MCS of retroviral vector pMS3-1 (Takara Bio Inc.). The schematic configuration of the obtained retroviral vector is shown in Figure 2.

[0076] Experimental Example 3: Generation of anti-CD38-CAR-reCTL G3T-hi cells were seeded in a 6-well plate 24 hours before transduction. A retroviral vector containing the gene encoding the anti-CD38-CAR prepared in Experimental Example 2 was transfected together with a gag-pol expression vector and an env expression vector using TransIT-293 Reagent (Mirus, Madison, WI). After 48 hours, the supernatant containing the produced ecotropic retrovirus was collected, filtered through a 0.45 μm filter, and stored. PG13 cells were seeded in a 6-well plate, and after 24 hours, the ecotropic retroviral supernatant diluted 4-fold with D-MEM medium and polybrene adjusted to a final concentration of 8 μg / mL were added. After 4 hours or more, the supernatant was discarded, and fresh ecotropic retroviral supernatant and polybrene were added. The PG13 cells were infected five times in total. After the final infection, the PG13 cell culture supernatant was collected and filtered through a 0.45 μm filter to obtain a retrovirus solution. Immediately before transduction, the reCTLs were stimulated to proliferate with anti-CD3 and anti-CD28 antibodies. The activated reCTL suspension and retrovirus solution were added in a 1:1 ratio to a retronectin-coated polypropylene tube, and the tubes were centrifuged at 1,200 g for 1.5 hours to allow infection. After 24 hours, the cells were infected again with the retrovirus using the same procedure, to produce anti-CD38-CAR-reCTL.

[0077] [Experimental Example 4: Evaluation of anti-CD38-CAR expression in anti-CD38-CAR-reCTL] The expression of the anti-CD38-CAR gene in the cells used to generate the anti-CD38-CAR-reCTL in Experimental Example 3 was evaluated by flow cytometry. Specifically, an antibody against the linker sequence of a fluorescently labeled scFV (G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor （Ｒ）Infected cells were stained with 647 Conjugate #69782 (cell signaling) and analyzed using a BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using BD Accuri C6 Plus Software (FlowJo). A negative gate was set using the unstained sample, and a positive gate was set using the stained target cells. The dead cell population was excluded using propidium iodide (PI) staining. The results are shown in Figure 3. In the figure, "anti-Linker Ab" refers to G4S Linker (E7O2V) Rabbit mAb.

[0078] As shown in Figure 3, the transduction efficiency of the anti-CD38-CAR gene was 95% or more. Since the transduction efficiency of the CAR-T gene in the production of CAR-T cells used in CAR-T therapy currently being clinically deployed is approximately 25%, the transduction efficiency of the anti-CD38-CAR gene was significantly high.

[0079] Experimental Example 5: Evaluation of CD38 expression in anti-CD38-CAR-reCTL CD38 expression in cells used to generate anti-CD38-CAR-reCTL in Experimental Example 3 was evaluated by flow cytometry. Specifically, cells stained with a fluorescently labeled anti-CD38 antibody, CD38-APC (HIT2, BD Pharmingen), were measured using a BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using BD Accuri C6 Plus Software (FlowJo). A negative gate was set using unstained samples, and a positive gate was set using stained target cells. Cells stained with a fluorescently labeled antibody against the linker sequence of scFV were used as an isotype control. The dead cell population was excluded using propidium iodide (PI) staining. The results are shown in Figure 4.

[0080] As shown in Figure 4, the anti-CD38-CAR-reCTLs were negative for CD38 expression. The high transfer efficiency of the anti-CD38-CAR gene confirmed in Experimental Example 4 is presumably due to the fact that reCTLs did not express CD38 upon stimulation by gene transfer, thereby suppressing fratricide.

[0081] When anti-CD38-CAR-reCTLs were produced in the same manner as in Experimental Example 3 using reCTLs of different lots produced in the same manner as in Experimental Example 1, it was confirmed that all of the anti-CD38-CAR-reCTLs derived from three or more different lots were negative for CD38 expression. This demonstrates that reCTLs induced to differentiate from iPS cells do not express CD38 due to stimulation by gene transfer.

[0082] Experimental Example 6: Evaluation of in vitro cytotoxic activity 1 Target cells (RPMI8226 cells, KMM1 cells) and effector cells (anti-CD38-CAR-reCTL or reCTL) were co-cultured in a 96-well plate. To confirm the cytotoxic activity dependent on the number of effector cells, effector-to-target cells were seeded at an effector-to-target ratio (E:T) ranging from 1:20 to 1:2, and the number of surviving target cells after 48 hours was evaluated by flow cytometry. The results are shown in Figures 5 and 6.

[0083] As shown in Figures 5 and 6, anti-CD38-CAR-reCTLs exhibited cytotoxic activity against both RPMI8226 cells and KMM1 cells that was dependent on the number of effector cells.

[0084] [Experimental Example 7: Evaluation of in vitro cytotoxic activity 2] The cytotoxic activity dependent on the co-culture time was evaluated. Specifically, target cells (RPMI8226 cells, KMM1 cells) were cultured at 1 × 10 ５ cells / well, Effector cells 5 x 10 ４The cells were seeded at a density of 1000 cells / well, and the number of target cells was measured by flow cytometry between 0 and 48 hours. The results are shown in Figures 7 and 8. In the figures, the data shown as "CD38 CAR" and "Control" represent the measurement results using anti-CD38-CAR-reCTL and reCTL as effector cells, respectively.

[0085] As shown in Figures 7 and 8, anti-CD38-CAR-reCTLs exhibited cytotoxic activity against both RPMI8226 cells and KMM1 cells that depended on the co-culture time.

[0086] Experimental Example 8: Evaluation of in vitro cytotoxic activity 3 Real-time analysis of cytotoxic activity was performed using xCELLigence RTCA S16 (Agilent, Santa Clara, CA). Target cells (RPMI8226 cells) were cultured at 5 × 10 ４ Effector cells were seeded at a density of 1000 cells / well, with effector cells seeded at an E:T ratio of 1:5 to 1:1. RPMI8226 cells were adhered to the dedicated plates using the xCELLigence Immunotherapy Kit Liquid Tumor Killing Assay (anti-CD9) (Agilent, Santa Clara, CA). Cell seeding and time-course monitoring were performed according to the manufacturer's protocol. The results are shown in Figure 9.

[0087] As shown in Figure 9, anti-CD38-CAR-reCTLs exhibited effector cell number- and time-dependent cytotoxic activity against RPMI8226 cells.

[0088] Experimental Example 9: Evaluation of in vitro cytotoxic activity 4 A competitive test with an anti-CD38 antibody drug was carried out. Specifically, target cells (RPMI8226 cells) were cultured at 1 × 10 ５Then, daratumumab (Janssen Pharmaceutica, Beerse, Belgium) or isatuximab (Sanofi, Paris, France) was added to each well to a final concentration of 100 μg / mL or 1000 μg / mL, and after 2 hours, effector cells were added at a density of 5 × 10 ４ The effector cells were seeded at a density of 1000 cells / well. 48 hours after seeding, the number of surviving target cells was evaluated by flow cytometry. The results are shown in Figure 10.

[0089] As shown in Figure 10, it was confirmed that when anti-CD38-CAR-reCTL was coexisted with daratumumab or isatuximab, higher cytotoxic activity was obtained than when anti-CD38-CAR-reCTL was used alone.

[0090] Experimental Example 10: Evaluation of in vitro cytotoxic activity 5 Target cells (THP-1, HT) were cultured alone or co-cultured with reCTL or anti-CD38-CAR-reCTL in a 96-well plate. The ET ratio for co-culture was E:T = 1:5. The number of surviving target cells 48 hours after seeding was assessed by flow cytometry. The results are shown in Figures 11 and 12.

[0091] As shown in Figure 11, when THP-1 cells were cocultured with anti-CD38-CAR-reCTL, the number of recovered THP-1 cells was significantly smaller than when they were cultured alone or cocultured with reCTL. Furthermore, as shown in Figure 12, when HT cells were cocultured with anti-CD38-CAR-reCTL, the number of recovered HT cells was significantly smaller than when they were cocultured with reCTL. These results confirmed that anti-CD38-CAR-reCTL also exhibits cytotoxic activity against THP-1 cells and HT cells.

[0092] Experimental Example 11: Evaluation of antitumor effect in vivo RPMI8226 cells (RPMI8226-Luc) into which luciferase gene was introduced were injected into NOG mice at a concentration of 1 × 10 ６On days 4 and 11 after inoculation, 1 × 10 anti-CD38-CAR-reCTL (transduction efficiency: 70%) or 1 × 10 reCTL were injected intraperitoneally. ６ The mice were administered luciferin via the tail vein. On day 28 after inoculation, the luminescence intensity was measured by in vivo imaging. The results are shown in Figure 13.

[0093] As shown in Figure 13, tumor growth was suppressed in the anti-CD38-CAR-reCTL-administered group and the reCTL-administered group compared to the control group not administered with effector cells. In particular, most of the tumor disappeared in the anti-CD38-CAR-reCTL-administered group, confirming a remarkable antitumor effect.

[0094] The anti-CD38-CAR killer cells according to the embodiments of the present invention can be suitably used in the medical field, such as in CAR-T therapy.

Claims

1. A cytotoxic immunocompetent cell derived from a pluripotent stem cell, which expresses a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain recognizes CD38 as an antigen.

2. The immunocompetent cell according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells or human embryonic pluripotent stem cells.

3. The immunocompetent cell according to claim 1, wherein the pluripotent stem cell is a pluripotent stem cell partially or completely deficient in HLA molecules.

4. The immunocompetent cell described in claim 1, which is a T cell.

5. The immunocompetent cell described in claim 1, which is a CD8αβ heterodimer type T cell.

6. The immunocompetent cell according to claim 1, wherein the pluripotent stem cell is a pluripotent stem cell into which an exogenous TCR gene has been introduced.

7. The immunocompetent cell described in claim 1, wherein CD38 expression is negative.

8. The immunocompetent cell described in claim 1, wherein the CD38 is human CD38.

9. The immunocompetent cell of claim 1, wherein the chimeric antigen receptor comprises, linked in this order, the extracellular antigen-binding domain, the hinge domain, the transmembrane domain, the costimulatory domain, and the intracellular signaling domain.

10. The immunocompetent cell described in claim 1, wherein the extracellular antigen-binding domain comprises the amino acid sequence represented by SEQ ID NO: 1 or a homologous sequence thereof and the amino acid sequence represented by SEQ ID NO: 2 or a homologous sequence thereof.

11. A pharmaceutical composition for treating or preventing a disease associated with the expression of CD38, comprising the immunocompetent cells described in claim 1.

12. The pharmaceutical composition according to claim 11, which is used in combination with an anti-CD38 antibody drug.

13. A method for producing CAR-CD8 T cells derived from induced pluripotent stem cells, comprising: preparing CD8 T cells induced to differentiate from induced pluripotent stem cells; and introducing a gene encoding a chimeric antigen receptor into the CD8 T cells, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain recognizes CD38 as an antigen.

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