Cytotoxic immunocompetent cell expressing anti CD38-car, method for producing the same, and pharmaceutical composition including the immunocompetent cell

Abstract

To provide an anti CD38-CAR killer cell that can be simply fabricated.SOLUTION: Provided are a cytotoxic immunocompetent cell derived from a pluripotent stem cell in which a chimeric antigen receptor including an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain is expressed, where the extracellular antigen binding domain recognizes CD38 as an antigen, and a pharmaceutical composition or the like including the cytotoxic immunocompetent cell derived from the pluripotent stem cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to cytotoxic immunocompetent cells expressing an anti-CD38-CAR, a method for producing the same, and pharmaceutical compositions comprising the immunocompetent cells. [Background technology]

[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, control of immune responses, apoptosis, etc. For example, it is known that CD38 is expressed in the early stages of development in T cells, B cells, etc., disappears with maturation, but is expressed again upon activation.

[0003] As mentioned above, CD38 is also expressed on normal cells, and is known to be more highly expressed 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, research and application of CAR-T therapy, which uses CAR-T cells produced by introducing and expressing a gene encoding a chimeric antigen receptor (CAR) that recognizes cancer cell surface antigens into T cells collected from a patient, has been rapidly progressing as a cancer immunotherapy (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2020-517244 [Non-patent literature]

[0006] [Non-Patent Document 1] Cytometry, 46:23-27, 2001 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, CD38 is often highly expressed on hematologic malignant tumor cells and is therefore a potential candidate cancer cell surface antigen for CAR gene therapy, such as CAR-T therapy. On the other hand, since cytotoxic immunocompetent cells (hereinafter sometimes referred to as "killer cells") generally express CD38 upon stimulation by gene transfer, anti-CD38-CAR killer cells generated by introducing 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 introducing an anti-CD38-CAR gene into killer cells, the expression of CD38 can be suppressed by RNA interference using siRNA or DNA editing using Crispr-Cas9, etc.

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

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

[0010] [1] One aspect of the present invention provides a pluripotent stem cell-derived cytotoxic immunocompetent cell that expresses a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain recognizes CD38 as an antigen. [2] In the immunocompetent cells described in [1] above, the pluripotent stem cells may be human induced pluripotent stem cells or human embryonic pluripotent stem cells. [3] In the immunocompetent cells according to [1] or [2] above, the pluripotent stem cells may be pluripotent stem cells that are partially or completely deficient in HLA molecules. [4] The immunocompetent cells according to any one of [1] to [3] above may be T cells. [5] The immunocompetent cells according to any one of [1] to [4] above may be CD8αβ heterodimer T cells. [6] In the immunocompetent cell according to any one of [1] to [5] above, the pluripotent stem cell may be a pluripotent stem cell into which an exogenous TCR gene has been introduced. [7] The immunocompetent cells described in 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 cell according to any one of [1] to [8] above, the chimeric antigen receptor may comprise, linked in this order, the extracellular antigen-binding domain, the hinge domain, the transmembrane domain, the costimulatory domain, and the intracellular signaling domain.

[10] In the immunocompetent cell 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 the expression of CD38, comprising the immunocompetent cell described in any one of [1] to

[10] above.

[12] The pharmaceutical composition described in

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

[13] According to another aspect of the present invention, there is provided 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. Effect of the Invention

[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. [Brief description of the drawings]

[0012] [Figure 1] FIG. 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. [Diagram 2] FIG. 1 shows the schematic structure of a retroviral vector containing a gene encoding anti-CD38-CAR. [Diagram 3] This figure shows the results of evaluating the expression of the anti-CD38-CAR gene in cells after the production of anti-CD38-CAR-reCTL in Experimental Example 3 by flow cytometry. [Figure 4] This figure shows the results of flow cytometry evaluation of CD38 expression in cells after the production of anti-CD38-CAR-reCTL in Experimental Example 3. [Diagram 5] This figure shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells at various E:T ratios in vitro. [Figure 6] This figure shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against KMM1 cells at various E:T ratios in vitro. [Figure 7]This figure shows the results of time-course evaluation of the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells in vitro. [Figure 8] This figure shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against KMM1 cells over time in vitro. [Figure 9] This figure shows the results of real-time analysis of the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells in vitro. [Figure 10] FIG. 1 shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against RPMI8226 cells in the presence of an anti-CD38 antibody drug. [Figure 11] FIG. 1 shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against THP-1 cells in vitro. [Figure 12] FIG. 1 shows the results of evaluating the cytotoxic activity of anti-CD38-CAR-reCTL against HT cells in vitro. [Figure 13] FIG. 1 shows the results of in vivo evaluation of the anti-tumor effect of anti-CD38-CAR-reCTL. [Figure 14] FIG. 14 shows the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. In addition, each embodiment can be appropriately combined unless it is inappropriate in the context. Furthermore, in this specification, "~" representing a numerical range includes the upper and lower numerical limits.

[0014] As used herein, "DP cells (double positive cells)" refers to CD4 positive CD8 positive T cells. "DN cells (double negative cells)" refers to CD4 negative CD8 negative T cells. Both "CD8 T cells" and "CD8 SP cells" refer to CD4 negative CD8 positive T cells. Unless otherwise specified, "CD8" encompasses both the CD8αα homodimer and the CD8αβ heterodimer. "TCR" means T cell receptor. "CTL cells" refers to cytotoxic T cells. By "T cell" is meant a cell that expresses an antigen receptor called the T cell receptor (TCR) on its surface.

[0015] A. Cytotoxic immunocompetent cells According to an embodiment of the present invention, a chimeric antigen receptor including 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. A cytotoxic immunocompetent cell derived from a pluripotent stem cell (anti-CD38-CAR killer cell derived from a pluripotent stem cell) is provided. In general, 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 cells can be cryopreserved in the form of anti-CD38-CAR killer cells, making it possible to make them off-the-shelf.

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

[0017] As the cytotoxic immunocompetent cells (killer cells), any immunocompetent cells having cytotoxic activity can be used. Specific examples of killer cells include lymphocytes, monocytes, polymorphonuclear leukocytes, etc. Among them, lymphocytes are preferred, and killer T cells such as CD8 T cells (CTL), NKT cells, and γδ T cells, and NK cells are more preferred.

[0018] The pluripotent stem cells are stem cells that have pluripotency and can differentiate into many cells present in a living body, and also have the ability to self-proliferate. 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, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from 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. The deletion of HLA molecules can be performed, for example, using genome editing technology.

[0021] The above-mentioned 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 TCR (e.g., antigen recognition present inside cells) and antigen recognition by CAR (e.g., antigen recognition 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, and these regions are linked in tandem in this order.

[0023] As the extracellular antigen-binding domain, an anti-CD38 antibody or a functional portion thereof that recognizes CD38 as an antigen can be appropriately used. Preferably, a fusion protein (so-called scFv) in which the light chain (VL) variable region and the 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 contain the light chain (VL) variable region and the heavy chain (VH) variable region in this order toward the transmembrane domain, or may contain them in this order from the transmembrane domain. CD38 is preferably mammalian CD38, 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, may 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 homologous sequence thereof.

[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 homologous sequence thereof.

[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 above-mentioned 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 are 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 domain 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, the transmembrane domain of CD8, CD28, and the like. 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 CD3 zeta 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 and expressing a gene encoding a chimeric antigen receptor (anti-CD38-CAR) that recognizes CD38 as an antigen into the CD8 T cells. As described above, CD8 T cells derived from iPS cells are typically CD38 negative even after gene introduction, so that anti-CD38-CAR-iPS-CD8 T cells can be obtained simply and efficiently without causing the problem of fratricide by introducing a gene encoding an anti-CD38-CAR.

[0032] Any appropriate method can be used as a method for inducing differentiation of iPS cells into CD8 T cells. In one embodiment, the iPS cell-derived CD8 T cells are 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 the DP cells in the cell culture obtained in step (2) into CD8 T cells; The method may be obtained by a method comprising the steps of: According to the above method, it is possible to preferably obtain iPS cell-derived CD8 T cells that do not express CD38 even after stimulation. According to the killer cells that do not express CD38 even after stimulation, it is not necessary to suppress the expression of CD38 by RNA interference, genome editing, etc., or to mask CD38 on the surface of the killer cells by co-incubating with an anti-CD38 antibody, and it is possible to easily and efficiently obtain anti-CD38-CAR killer cells.

[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 by known methods. Examples of human tissues include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissues. Peripheral blood and umbilical cord blood are preferred. Known methods for isolating human T cells include, for example, flow cytometry using an antibody against a cell surface marker such as CD3 and a cell sorter. In addition, desired T cells can be isolated using the secretion of cytokines and the expression of functional molecules as indicators. In such cases, for example, T cells secrete different cytokines depending on whether they are Th1 type or Th2 type, so that T cells having a desired Th type can be selected using such cytokines as an indicator. In addition, cytotoxic (killer) T cells can be isolated using the secretion or production of granzymes, perforins, and the like as indicators.

[0037] The somatic cells induced to iPS cells may be any animal cell (preferably a mammalian cell, including a human cell) except for germline cells such as sperm, spermatocytes, eggs, oocytes, and ES cells, or pluripotent cells. Somatic cells include somatic cells of fetuses (offspring), somatic cells of newborns (offspring), and somatic cells of mature healthy individuals or somatic cells of individuals with diseases. They also include primary culture 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 (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0038] iPS cells are induced from the above T cells or somatic cells. As a method for obtaining iPS cells from T cells or somatic cells, for example, the method described in Vizcardo et al., Cell Stem Cell 12, 31-36 (2013) may be used. For example, desired antigen-specific T cells can be obtained from a subject who has acquired immunity against a disease to be treated, and Yamanaka factors can be introduced into these cells to obtain T-iPS 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 almost the same characteristics as ES cells and can be created 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 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 noncoding RNA, or a low molecular weight compound. Examples of genes contained in the reprogramming factor 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, or Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include 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, 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,RLJudson 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. are exemplified.

[0040] The reprogramming factor may be contacted with or introduced into a somatic cell by any appropriate method depending on its form.

[0041] The iPS cells obtained as described above are induced to differentiate into a T cell population containing DP cells and DN cells. A method for inducing differentiation from iPS cells into T cells includes, for example, 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, for example mouse OP9 stromal cell culture, to obtain blood cell progenitor cells. The obtained blood cell progenitor cells are then co-cultured with OP9 / DLL1 cells. During OP9 / DLL1 co-culture, IL-7, FlT-3L, and SCF (Stem Cell Factor) are added to the medium. 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 such a phenomenon can be prevented by removing the DN cells.

[0044] The cell culture from which DN cells have been depleted is preferably substantially free of DN cells, for example the content of DN cells in the cell culture from which DN cells have been depleted is 5% or less, preferably 3% or less, 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 "enriched DP cell culture." Similarly, when cells other than DN cells are simultaneously or individually removed from a cell culture, they may be referred to as "enrichment of DP cells" and "enriched DP cell culture."

[0046] The cell culture containing DN cells and DP cells may contain CD8SP cells. The CD8SP cells may be CD8αα homodimeric T cells, and it is preferable to simultaneously remove CD8SP cells, particularly CD8αα homodimeric T cells, when enriching DP cells (i.e., removing DN cells).

[0047] Any suitable method may be adopted as a method for removing DN cells and / or CD8SP cells from a 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. For example, 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. For example, a cell culture substantially free of DN cells can be obtained by removing a DN cell fraction using a cell sorter, or a cell culture substantially free of DN cells and CD8SP cells can be obtained by collecting a DP cell fraction using a cell sorter. For example, a cell culture substantially free of CD8SPαα homodimer cells can be collected by collecting a CD8SPαβ heterodimer cell fraction using a cell sorter.

[0048] In the above step (3), the DP cells in the cell culture obtained in step (2) are differentiated into CD8SP cells. Differentiation of DP cells into CD8SP cells can be induced by directly activating one of the activation pathways that occurs when a 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 the culture medium containing the CD3 antibody may be 3 to 10 days, for example 4 to 8 days, for example about 6 days.

[0049] When the 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 CD8SP cells by stimulating 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 an embodiment of the method for inducing differentiation of iPS cells into CD8 T cells, the DP cells in the cell culture obtained in step (1) may be differentiated into CD8 SP cells in the presence of a substance that inhibits the cytotoxic activity of DN cells without performing the above step (2). Examples of substances that inhibit 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 the above step (3).

[0052] Details of the method for inducing differentiation of iPS cells into CD8 T cells are described in WO / 2016 / 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, in which a gene encoding an anti-CD38-CAR has been introduced and expressed in the cytotoxic CD8 T cells, can also have cytotoxicity.

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

[0055] Any suitable method can be used to introduce and express the gene encoding anti-CD38-CAR into iPS cell-derived CD8 T cells. For example, biological gene transfer methods using viral vectors, physical gene transfer methods such as electroporation, and chemical gene transfer methods such as lipofection and calcium phosphate method can be used. Among them, gene transfer methods using viral vectors can be preferably used.

[0056] The viral vector is not particularly limited as long as the produced viral particles infect iPS cell-derived CD8T cells and the cells express anti-CD38-CAR. A retroviral vector can be preferably used because the introduced gene is incorporated into the chromosome of the host cell with high efficiency and the high expression of the gene can be maintained. Here, the retroviral vector means a so-called oncoretroviral vector (hereinafter, simply referred to as a retroviral vector), and includes lentiviral vectors and the like.

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

[0058] Preferred examples of retroviral vector plasmids include MSCV (Murine Stern Cell Virus) vector plasmids and lentiviral vector plasmids. Various commercially available products can be used as retroviral vector plasmids, and for example, MSCV Retroviral Expression System (manufactured by Clontech) can also be used. In the retroviral vector plasmid, a target gene sequence can be inserted into the multicloning site (MCS) using a standard cloning method.

[0059] In one embodiment, a retrovirus vector plasmid having an IRES (internal ribosome entry site) sequence is used. The IRES sequence is a sequence that exists inside an mRNA chain and codes for a structure to which a ribosome directly binds, and is used in a mechanism that initiates translation by direct binding of the ribosome, that is, a so-called internal initiation mechanism. The retrovirus vector plasmid having an IRES sequence can make the translation of eukaryotic cell mRNA cap structure-independent translation.

[0060] In one embodiment, a retroviral vector plasmid having a marker gene is used from the viewpoint of selection, sorting, etc. of 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, any method commonly used for gene transfer using a retroviral vector can be applied.

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

[0063] Any suitable 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 above-mentioned virus solution is mixed with a positively charged gene transfer aid, and the resulting mixture is added to iPS cell-derived CD8 T cells and cultured. The retronectin method can also be used.

[0064] After the above infection, cells expressing fluorescence can be easily selected and isolated as cells expressing anti-CD38-CAR, for example, by using a fluorescent anti-G4S linker antibody or a fluorescent anti-mouse IgG antibody using 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 the same manner as above. For example, differentiation of other cytotoxic immunocompetent cells such as NK cells from iPS cells or cytotoxic immunocompetent cells from other pluripotent stem cells such as ES cells can be induced according to previously reported methods, and the anti-CD38-CAR gene can be introduced into and expressed in the obtained immunocompetent cells in the same manner as 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 the expression of CD38, comprising 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 necessary to kill cells expressing CD38, preferably cells highly expressing CD38. Thus, the pharmaceutical composition according to the embodiment of the present invention can be applied to, for example, CAR gene therapy such as CAR-T therapy (including CAR-γδT therapy) and CAR-NK therapy for diseases associated with the expression of CD38.

[0067] Diseases associated with expression of CD38 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 cytotoxicity of the anti-CD38-CAR killer cells and the action of the antibody drug (e.g., CDC activity, ADCC activity, ADCP activity, etc.) can provide a more suitable therapeutic or preventive effect against the above 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 may not contain an 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 set depending on the conditions (height, weight, age, sex, etc.) and disease state of the subject to be treated.

[0070] Currently, two types of anti-CD38 antibody drugs that recognize different epitopes of CD38 are used clinically. It is preferable that 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. EXAMPLES

[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, the cytotoxic iPS cell-derived CD8 T cells may be referred to as "reCTLs".

[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. G3T-hi and PG13 cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA) and 100 U / mL penicillin·100 μg / mL streptomycin (PS) (Fujifilm Wako Pure Chemical Industries, Ltd., 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·100 μg / mL PS. reCTLs were cultured in MEMα (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, 100 U / mL·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 expansion of reCTLs, anti-CD3 antibody (BioLegend, San Diego, CA) and anti-CD28 antibody (Thermo Fisher Scientific, Waltham, MA) were added to MEMα.

[0074] [Experimental Example 1: Creation of iPS-derived CD8 T cells] Human iPS cell-derived CD8 T cells (reCTL) were obtained by the method described in WO2017 / 179720. Specifically, the method is as follows. iPS cells were induced to become CD34CD43 double positive blood cell precursor cells by differentiation induction using the EB method, which is a mesoderm induction system. These cells were seeded on OP9DL1 feeder cells and cultured while adding the Notch signal, and were induced to become DP cells, which are T precursor cells. Next, the DP cells were isolated, and the isolated DP cells were differentiated into CD8SP cells (reCTL) by adding TCR stimulation using a CD3 antibody. The reCTLs thus produced 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: Construction of retroviral vector] From the lentivirus vector constructed in the Examples of WO2007 / 142241, the gene sequence encoding the CD8α signal peptide, anti-CD38scFV, hinge domain, CD8α transmembrane domain, 4-1BB intracellular domain, and anti-CD38-CAR containing the CD3ζ domain was excised with a restriction enzyme and inserted into the MCS of the retrovirus vector pMS3-1 (Takara Bio Inc.). The schematic configuration of the obtained retrovirus vector is shown in FIG. 2.

[0076] [Experimental Example 3: Preparation of anti-CD38-CAR-reCTL] G3T-hi cells were seeded in a 6-well plate 24 hours before transduction. The retroviral vector containing the gene encoding the anti-CD38-CAR prepared in Experimental Example 2 was transfected together with the gag-pol expression vector and the 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 on a 6-well plate, and after 24 hours, ecotropic virus 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 new ecotropic virus supernatant and polybrene were added. The infection procedure for PG13 cells was performed 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, 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 polypropylene tube coated with Retronectin, and the tubes were centrifuged at 1200g for 1.5 hours to infect the cells. After 24 hours, the cells were infected again with the retrovirus using the same procedure to produce anti-CD38-CAR-reCTLs.

[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 anti-CD38-CAR-reCTL in Experimental Example 3 was evaluated by flow cytometry. Specifically, the antibody against the linker sequence of the fluorescently labeled scFV (G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor (R) After infection, 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" is 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 the CAR-T therapy currently being clinically deployed is approximately 25%, the transduction efficiency of the above-mentioned anti-CD38-CAR gene was remarkably 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 were stained with a fluorescently labeled anti-CD38 antibody: CD38-APC (HIT2, BD Pharmingen) and 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 the unstained sample, and a positive gate was set using stained target cells. Cells stained with an antibody against the linker sequence of fluorescently labeled 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, anti-CD38-CAR-reCTL was negative for CD38 expression. It is speculated that the high transfection efficiency of the anti-CD38-CAR gene confirmed in Experimental Example 4 is due to the fact that reCTL does not express CD38 upon stimulation of gene transfection, and fratricide is suppressed.

[0081] In addition, 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 CD38 expression was negative in all of the anti-CD38-CAR-reCTLs derived from three or more different lots. This shows that in reCTLs induced to differentiate from iPS cells, CD38 expression does not occur due to stimulation by gene transfer.

[0082] [Experimental Example 6: Evaluation of in vitro cytotoxic activity 1] Target cells (RPMI8226 cells, KMM1 cells) and 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 ratios (E:T) were seeded in the range of 1:20 to 1:2, and the number of surviving target cells was evaluated by flow cytometry after 48 hours. The results are shown in Figures 5 and 6.

[0083] As shown in Figures 5 and 6, anti-CD38-CAR-reCTL showed 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 was evaluated depending on the co-culture time. Specifically, target cells (RPMI8226 cells, KMM1 cells) were cultured at 1 × 10 5 cells / well, 5×10 effector cells 4 The number of target cells was measured by flow cytometry between 0 and 48 hours. The results are shown in Figures 7 and 8. The data shown as "CAR" and "Control" indicate the results of measurements using anti-CD38-CAR-reCTL and reCTL as effector cells, respectively.

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

[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 plated at 5 × 10 4 The cells were seeded at a density of 1000 cells / well, and effector cells were seeded at an E:T ratio of 1:5 to 1:1. RPMI8226 cells were attached to the dedicated plate using xCELLigence Immunotherapy Kit Liquid Tumor Killing Assay (anti-CD9) (Agilent, Santa Clara, CA). Cells were seeded and monitored over time according to the manufacturer's protocol. The results are shown in Figure 9.

[0087] As shown in Figure 9, anti-CD38-CAR-reCTL 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 anti-CD38 antibody was performed. Specifically, target cells (RPMI8226 cells) were cultured at 1 × 10 5 Then, 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 2 hours later, effector cells were added at a density of 5 × 10 4The effector cells were seeded at a density of 1000 cells / well. 48 hours after the seeding of the effector cells, 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 Daratumab 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 of the co-culture was E:T = 1:5. The number of surviving target cells was evaluated by flow cytometry 48 hours after seeding. The results are shown in Figure 11 and Figure 12.

[0091] As shown in Figure 11, when THP-1 cells were co-cultured with anti-CD38-CAR-reCTL, the number of THP-1 cells recovered was significantly smaller than when they were cultured alone or co-cultured with reCTL. Also, as shown in Figure 12, when HT cells were co-cultured with anti-CD38-CAR-reCTL, the number of HT cells recovered was significantly smaller than when they were co-cultured with reCTL. This 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 transfected with the luciferase gene (RPMI8226-Luc) were cultured in NOG mice at 1 × 10 6 After intraperitoneal inoculation, 1 × 10 anti-CD38-CAR-reCTL (transduction efficiency: 70%) or reCTL was administered on days 4 and 11. 6The mice were administered with Luciferin via the tail vein. On the 28th day after inoculation, the luminescence intensity was measured by in vivo imaging after Luciferin was administered to the mice. 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. In particular, most of the tumor disappeared in the anti-CD38-CAR-reCTL-administered group, confirming a remarkable antitumor effect. [Industrial Applicability]

[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 chimeric antigen receptor is expressed that contains an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, The extracellular antigen-binding domain recognizes CD38 as an antigen. Cytotoxic immunocompetent cells derived from pluripotent stem cells.

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

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 according to claim 1 , which is a T cell.

5. The immunocompetent cell according to claim 1 , which is a CD8αβ heterodimer 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 according to claim 1 , wherein expression of CD38 is negative.

8. The immunocompetent cell according to 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, a hinge domain, the transmembrane domain, a costimulatory domain, and the intracellular signaling domain.

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

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

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

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