Therapeutic agent for solid cancer

Abstract

To provide a therapeutic agent for solid cancer employing genetically engineered NK cells derived from pluripotent stem cells in a novel dosage form, thereby providing therapeutic means for solid cancer that combines therapeutic effect, ease of use, and safety.SOLUTION: A therapeutic agent for solid cancer comprising NK cells or precursor cells thereof derived from pluripotent stem cells that express the following (a) and (b): (a) an exogenous gene encoding CC chemokine receptor type 2B (CCR2B); and (b) an exogenous gene encoding CC chemokine ligand 19 (CCL19), the agent being characterized by being administered in the vicinity of a tumor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for solid cancer, which comprises natural killer (NK) cells or their precursor cells derived from genetically modified pluripotent stem cells. More specifically, the present invention relates to a therapeutic agent for solid cancer, which comprises administering the genetically modified pluripotent stem cell-derived NK cells or their precursor cells to the vicinity of the tumor. [Background technology]

[0002] Cancer immunotherapy using immune cells, including CAR-T cells, has been shown to be highly effective against blood cancers, but is less effective against solid cancers, and research to improve its effectiveness is being actively conducted.Recent advances in iPS cells and differentiation induction methods, as well as technological advances in methods for genetically modifying iPS cells, have made it possible to produce immune cells with high functionality by genetically modifying iPS cells and inducing their differentiation into target immune cells.

[0003] NK cells do not require presensitization to cancer cells and can respond without being bound by antigen recognition. NK cells not only directly recognize and kill target cells, but also activate T cells, macrophages, and other cells through the production of various cytokines, improving immune function. NK cells, unlike T cells, are less likely to cause problems such as GvHD even when using donor cells with completely mismatched HLA types, and are also less likely to cause cytokine release syndrome, a side effect of T cells. These advantageous properties have raised expectations for immune cell therapy using NK cells.

[0004] Healios has discovered that NK cells induced to differentiate from genetically modified pluripotent stem cells that co-express exogenous CC chemokine receptor type 2B (CCR2B) and CC chemokine ligand 19 (CCL19), and optionally express one or more other exogenous genes such as cytokines, have superior properties as anti-tumor effector cells for immune cell therapy compared to NK cells derived from unmodified pluripotent stem cells (Patent Document 1), and is currently conducting research and development into these cells.

[0005] Among solid cancers, mesothelioma is a rare disease with few treatment options and a poor prognosis. Mesothelioma is very hard, making it difficult to administer drugs directly into the tumor with a needle. Therefore, conventional drug therapies have attempted to use intravenous administration of anticancer drugs and immune checkpoint inhibitors. However, systemic administration poses many challenges in terms of both efficacy and side effects, and a breakthrough is desired.

[0006] In a Phase I / II trial of CAR-T cell therapy for malignant pleural mesothelioma, there have been reported clinical cases in which CAR-T cells were injected into the pleural cavity (pleural effusion) or into the tumor (Non-Patent Document 1). However, intrapleural administration may result in the cells spreading throughout the pleural cavity, potentially reducing efficacy. Furthermore, the procedure requires the interventional radiology (IVR) specialist to perform the procedure under image guidance using CT, PET-CT, or ultrasound, making it less versatile. Furthermore, no treatment using NK cells (including genetically modified NK cells) has yet been reported. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 2023 / 085356 [Non-patent literature]

[0008] [Non-Patent Document 1] Lung Cancer. 2022, 165: 1-9. Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for an immune cell therapy agent that has enhanced therapeutic efficacy against solid cancers and that can be administered easily and safely. Therefore, an object of the present invention is to provide a solid cancer therapy agent with a novel administration form that uses genetically modified NK cells derived from pluripotent stem cells such as iPS cells, thereby providing a means of treating solid cancer that combines therapeutic efficacy with ease and safety. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above-mentioned objectives, the present inventors found that when NK cells derived from pluripotent stem cells transduced with the CCR2B gene and CCL19 gene were administered near the tumor in mice implanted with mesothelioma cells, the NK cells infiltrated into the tumor and exhibited an antitumor effect equivalent to that achieved when administered directly into the tumor. Surprisingly, such a high antitumor effect was observed, even though the number of NK cells infiltrating into the tumor was less than 10% of that observed when administered intratumorally. Furthermore, no tumor infiltration was observed when non-genetically modified pluripotent stem cell-derived NK cells were administered near the tumor. Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0011] That is, the present invention is as follows. [Section 1] (a) and (b) below: (a) A foreign gene encoding CC chemokine receptor type 2B (CCR2B) (b) an exogenous gene encoding CC chemokine ligand 19 (CCL19); A therapeutic agent for solid cancer, comprising natural killer (NK) cells or their precursor cells derived from pluripotent stem cells expressing the gene encoding the NK-1 gene, characterized in that the agent is administered in the vicinity of the tumor. [Section 2] NK cells or their precursor cells are selected from the group consisting of (c) and / or (d) below: (c) an exogenous gene encoding interleukin 15 (IL-15) (d) an exogenous gene encoding CD16 Item 1. The agent according to Item 1, further expressing: [Section 3] NK cells or their precursor cells are selected from the group consisting of the following (e): (e) an exogenous gene encoding NKG2D or NKG2D-CAR Item 3. The agent according to Item 1 or 2, further expressing: [Section 4] Item 4. The agent according to Item 3, wherein (e) is NKG2D-CAR. [Section 5] (e) is NKG2D, and the NK cells or their precursor cells are further (f) an exogenous gene encoding DAP10 Item 4. The agent according to Item 3, wherein the agent co-expresses [Section 6] Item 6. The agent according to any one of Items 2 to 5, wherein the NK cells or their precursor cells express an exogenous gene encoding IL-15, and the IL-15 is secreted IL-15. [Section 7] Item 6. The agent according to any one of Items 2 to 5, wherein the NK cells or their precursor cells express an exogenous gene encoding IL-15 and co-express an exogenous gene encoding IL-15 receptor α (IL-15Rα). [Section 8] Item 8. The agent according to any one of Items 2 to 7, wherein the NK cells or their precursor cells express an exogenous gene encoding CD16, and the CD16 is CD16 having a high-affinity mutation or a non-cleavable mutation. [Section 9] Item 9. The agent according to Item 8, wherein CD16 has a mutation selected from F176V (F158V) and S197P. [Section 10] Item 10. The agent according to any one of Items 1 to 9, wherein the solid cancer is mesothelioma. [Section 11] A method for treating solid cancer, comprising the steps of: (a) administering to a subject a therapeutic agent selected from the group consisting of: (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 A method comprising administering an effective amount of NK cells or their precursor cells derived from pluripotent stem cells expressing the gene to the vicinity of the tumor of a subject with solid cancer. [Section 12] (a) and (b) of the following for use in the treatment of solid tumors: (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 NK cells or their precursor cells derived from pluripotent stem cells that express the NK cell gene, which are administered near a tumor. [Section 13] In the manufacture of a solid cancer therapeutic agent, the following (a) and (b): (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 Use of NK cells or their precursor cells derived from pluripotent stem cells expressing the above-mentioned compound, wherein the therapeutic agent is administered in the vicinity of a tumor. [Effects of the Invention]

[0012] According to the present invention, by administering genetically modified NK cells or their precursor cells near a tumor, the cells infiltrate into the tumor and achieve antitumor effects equivalent to those achieved by local administration into the tumor. Therefore, even for solid cancers for which intratumoral administration (including administration to the peritumor area, which allows unobstructed access to the tumor) is difficult or requires specialized equipment or skilled techniques, this method enables effective, simple, and minimally invasive treatment. Furthermore, since genetically modified NK cells or their precursor cells can be obtained by inducing differentiation of genetically modified pluripotent stem cells, it is easier to provide larger amounts of modified NK cells or their precursor cells than cells obtained by genetically modifying NK cells. [Brief explanation of the drawings]

[0013] [Figure 1] This figure shows the infiltration of eNK cells into tumors when eNK cells were administered near the tumor in mice implanted with MSTO-211H mesothelioma cells (left panel). The upper panel shows the results of HE staining, and the lower panel shows the results of CD45 immunostaining. The right panel shows the results of intratumoral administration. [Figure 2]Comparison of eNK cell and iNK cell infiltration into tumors in mice implanted with MESO-4 mesothelioma cells. Right panel: eNK cells, left panel: iNK cells. The upper panel shows the results of HE staining, and the lower panel shows the results of CD45 immunostaining. [Figure 3] This figure shows a comparison of the intratumoral infiltration of eNK cells and iNK cells in mice implanted with MESO-4 mesothelioma cells. This is an enlarged view of the boxed area in Figure 2. The arrow indicates CD45-positive cells (eNK cells). [Figure 4] This figure shows intratumoral infiltration of eNK cells and tumor cell necrosis in mice implanted with MESO-4 mesothelioma cells. The left image is an enlarged portion of the photograph of eNK cell administration in Figure 3, and the right image shows the results of TUNEL staining. The arrows in the left image indicate CD45-positive cells (eNK cells), and the arrows in the right image indicate TUNEL-positive cells (tumor cells undergoing apoptosis). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides (a) an exogenous gene encoding CCR2B, and (b) an exogenous gene encoding CCL19 The present invention provides a therapeutic agent for solid cancers (hereinafter also referred to as "therapeutic agent of the present invention"), which comprises, as an active ingredient, NK cells derived from pluripotent stem cells that express the gene, or their precursor cells. In this specification, NK cells derived from pluripotent stem cells that contain at least the above-mentioned genetic modification, or their precursor cells, are collectively referred to as "eNK cells."

[0015] Since cancer cells of solid cancers express CC chemokine ligand 2 (CCL2), by introducing and expressing its receptor, CCR2B, into pluripotent stem cells, NK cells induced to differentiate from the pluripotent stem cells can be made to home to cancer tissues.

[0016] On the other hand, CCL19 is a ligand for CC chemokine receptor 7 (CCR7), which is expressed in T cells and dendritic cells. Therefore, by introducing and expressing CCL19 into pluripotent stem cells, NK cells induced to differentiate from the pluripotent stem cells can stimulate the migration of immune cells such as T cells and dendritic cells around the cells, thereby enhancing the effects of NK cells.

[0017] As used herein, "pluripotent stem cells" refer to cells that have pluripotency, and examples thereof include ES cells and iPS cells, with iPS cells being preferred. NK cells induced to differentiate from iPS cells are referred to as "iNK cells" herein.

[0018] ES cells can be prepared by methods known per se. Methods for producing ES cells include, for example, a method for culturing the inner cell mass of a human blastocyst stage embryo (see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)), a method for culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Akira Iritani et al., Proteins, Nucleic Acids, and Enzymes, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Rideout III et al., Nature Genetics, 24,109 (2000)). ES cells can be obtained from designated institutions or commercially available. For example, human ES cell lines H1, H7, H9, H13, and H14 are available from the WiCell Research Institute in the United States; HES1-6 are available from ES Cell International in Australia; SA002, SA181, and SA611 are available from Cellartis AB in Sweden; HUES1-17 are available from the HUES Cell Facility in the United States; KhES-1 to KhES-5 are available from the Institute for Frontier Medical Sciences, Kyoto University; and SEES1-SEES7 are available from the National Center for Child Health and Development. When ES cells are produced by somatic cell nuclear transfer, the type of somatic cell and the source from which the somatic cells are collected are the same as those for producing iPS cells, described below.

[0019] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of nucleic acids (DNA or RNA) or proteins. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (Takahashi, K. and S. Yamanaka (2006) Cell, 126: 663-676; Takahashi, K. et al. (2007) Cell, 131: 861-872; Yu, J. et al. (2007) Science, 318: 1917-1920; Nakagawa, M. et al. (2008) Nat. Biotechnol. 26: 101-106; WO2007 / 069666).

[0020] As used herein, the term "somatic cells" refers to any human cell excluding germline cells such as eggs, oocytes, and ES cells, and totipotent cells. Somatic cells include, but are not limited to, fetal somatic cells, neonatal somatic cells, and mature, healthy or diseased somatic cells, as well as primary cultured cells, passaged cells, and established cell lines. Specifically, examples of somatic cells include: (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0021] The reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products, or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds. Examples of genes included 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 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, WO2 009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 06895 5, 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, Judson, RL et al. (2009) Nat. Biotechnol., 27: 459-461, Lyssiotis, CA et al. (2009) Proc. Natl. Acad. Sci. USA, 106: 8912-8917, Kim, Examples of such combinations include those described in 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, and Maekawa, M. et al. (2011) Nature, 474: 225-9.

[0022] iPS cell colonies can be selected using drug resistance and reporter activity as indicators (Cell, 126, 663-676 (2006), Nature, 448, 313-317 (2007)) or by visual morphological observation (Cell, 131, 861-872 (2007)). Identification of iPS cells can be confirmed using the expression of various ES cell-specific genes and teratoma formation as indicators.

[0023] In addition, various human iPS cell lines established by designated organizations, such as the National Institutes of Health (NIH), RIKEN, Kyoto University, etc., can be used as iPS cells. Examples include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain, and Kyoto University's 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A31 strain, and FfI-01s04 strain.

[0024] As used herein, "introducing an exogenous gene" refers to introducing the exogenous gene into a target site in the genome, thereby rendering the exogenous gene expressible in the target cell. A preferred method for introducing an exogenous gene involves isolating the DNA of the exogenous gene using conventional methods, inserting a DNA fragment of the exogenous gene into the target site in the target cell, and then integrating a DNA strand (hereinafter referred to as a gene introduction targeting vector) having a DNA sequence constructed so that the exogenous gene is expressed in the target cell by homologous recombination into the target site in the target cell. Since homologous recombination fixes the gene insertion site, it is expected that, in the absence of random integration, there will be little difference in expression levels between clones and little effect on other genes.

[0025] The homologously recombinant cells can be obtained, for example, by introducing the above-mentioned targeting vector into a subject cell.

[0026] For example, when a targeting vector for gene introduction is designed to insert a DNA fragment of an exogenous gene (which can be cloned by conventional methods based on the sequence information of the cDNA of the human CCR2B gene (see, for example, Refseq NM_001123396) and the cDNA of the human CCL19 gene (see, for example, Refseq NM_006274)) into a target site so that the exogenous gene is expressed in a target cell, the vector can be configured, for example, as follows:

[0027] First, in order for the DNA fragment of the exogenous gene to be inserted into the target site by homologous recombination, the targeting vector for gene introduction must contain sequences (5' arm and 3' arm) that are homologous to the target site 5' upstream and 3' downstream of the DNA fragment of the exogenous gene, respectively.

[0028] To select target cells in which the targeting vector for gene transfer has been integrated into the chromosome, it is preferable that the targeting vector for gene transfer contain a drug resistance gene and a reporter gene in addition to the exogenous gene to be inserted. Examples of drug resistance genes include, but are not limited to, the neomycin phosphotransferase II (nptII) gene and the hygromycin B phosphotransferase (hph) gene, and examples of reporter genes include, but are not limited to, the β-galactosidase (lacZ) gene and the chloramphenicol acetyltransferase (cat) gene.

[0029] The drug resistance or reporter gene is preferably under the control of any gene expression control region that can function in the target cell, including, but not limited to, viral promoters such as the SV40-derived early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR, and adenovirus (AdV)-derived early promoter, as well as the β-actin gene promoter, PGK gene promoter, and transferrin gene promoter.

[0030] Furthermore, the targeting vector for gene transfer preferably has a polyA signal downstream of the drug resistance or reporter gene, and for example, a terminator sequence derived from a viral gene or from various mammalian or avian genes can be used, preferably an SV40-derived terminator sequence.

[0031] Typically, genetic recombination in cells is largely non-homologous, with introduced DNA randomly integrated at any chromosomal location. Therefore, selection by detecting drug resistance or reporter gene expression (positive selection) is not sufficient to efficiently select clones in which homologous recombination has occurred at the target site; instead, Southern hybridization or PCR analysis is required to confirm the integration site for all selected clones. Therefore, if, for example, the herpes simplex virus-derived thymidine kinase (HSV-tk) gene, which confers ganciclovir sensitivity, is ligated to the outside of the sequence homologous to the target site of the gene deletion targeting vector, cells into which the vector has been randomly integrated will contain the HSV-tk gene and therefore will be unable to grow in ganciclovir-containing media. However, cells into which homologous recombination has occurred at the endogenous locus will lack the HSV-tk gene and will therefore be resistant to ganciclovir and will be selected (negative selection). Alternatively, if the HSV-tk gene is replaced with, for example, the diphtheria toxin gene, cells into which the vector has been randomly inserted will be killed by the toxin they themselves produce (positive selection), and homologous recombinants can be selected in the absence of drugs.

[0032] Any of the calcium phosphate coprecipitation, electroporation, lipofection, retroviral infection, aggregation, microinjection, gene gun (particle gun), and DEAE-dextran methods can be used to introduce a targeting vector for gene deletion into target cells. However, as mentioned above, most gene recombination in cells is non-homologous, and the frequency of obtaining homologous recombinants is low. Therefore, electroporation is generally chosen because it allows for easy processing of a large number of cells. For electroporation, the same conditions as those used for gene transfer into normal animal cells can be used. For example, target cells in the logarithmic growth phase are treated with trypsin to disperse them into single cells, and then 10 6 ~10 8The cells are suspended in a medium at a concentration of cells / ml and transferred to a cuvette, to which 10 to 100 μg of a targeting vector for gene deletion is added, followed by application of an electric pulse of 200 to 600 V / cm.

[0033] Target cells incorporating a gene-deficient targeting vector can be identified by Southern hybridization or PCR screening of chromosomal DNA isolated from colonies obtained by culturing single cells. However, if a drug resistance gene or reporter gene is used as another DNA fragment, transformants can be selected at the cell stage using their expression as an indicator. For example, if a vector containing the nptII gene is used as a positive selection marker, the target cells after gene transfection are cultured in a medium containing a neomycin-based antibiotic such as G418, and the resulting resistant colonies are selected as candidate transformants. Alternatively, if a vector containing the HSV-tk gene is used as a negative selection marker, the cells are cultured in a medium containing ganciclovir, and the resulting resistant colonies are selected as candidate homologously recombinant cells. The resulting colonies are transferred to culture plates and repeatedly treated with trypsin and exchanged with medium. Some are kept for culture, while the remaining colonies are subjected to PCR or Southern hybridization to confirm the presence of the introduced DNA.

[0034] Furthermore, when a virus is used as a targeting vector for gene introduction, an example is a method in which target cells are infected with a virus containing DNA in which an exogenous gene and a positive selection marker gene are inserted between the 5' and 3' arms and a negative selection marker gene is inserted outside the arms. The virus, the method for infecting cells, and the method for selecting cells into which the vector has been incorporated may be the same as those used for the targeting vector for gene deletion.

[0035] The target site of a gene transfer targeting vector is not particularly limited as long as it can render the exogenous gene expressible in the target cell. Examples of such sites include safe harbor regions within the genome. A safe harbor region is a region where the integration of an exogenous gene does not result in phenotypic changes, and where the locus is open in many differentiated cells, resulting in relatively stable expression of the introduced factor. This region is selected as a target site for the integration of an exogenous gene into cells to be used as a pharmaceutical. Examples of such safe harbor regions include the AAVS1 (Adeno-associated virus integration site 1) region, the CCR5 (CC chemokine receptor 5) region, and the ROSA26 region. Introducing an exogenous gene into a site other than a safe harbor region can result in the disruption of the gene at the introduced site, resulting in an unexpected phenotype or suppression of the expression of the introduced exogenous gene. Therefore, the region must be carefully selected depending on the type of cell the cell is to be differentiated into and used as a pharmaceutical. When an exogenous gene is introduced into a safe harbor region, the integration site of the exogenous gene is fixed, so it is expected that there will be little difference in the expression level of the exogenous gene between the obtained homologous recombinants and that the gene will have little effect on other genes.

[0036] Another embodiment for introducing an exogenous gene is the PiggyBac method. The PiggyBac method uses a transposon vector incorporating a DNA fragment containing the exogenous gene and a transposase expression vector that expresses a transposase. The genes and other components contained in the transposon vector and transposase expression vector may be contained in the above-mentioned separate vectors or in a single vector. The transposon vector and transposase expression vector can have, for example, the following configurations:

[0037] To enable transposase to excise a DNA fragment containing a foreign gene from a transposon-based vector, the transposon-based vector contains inverted terminal repeats 5' upstream and 3' downstream of the DNA fragment containing the foreign gene. Transposase recognizes the inverted terminal repeats contained in the transposon-based vector and excises the DNA fragment containing the foreign gene flanked by the inverted terminal repeats from the transposon-based vector.

[0038] To select target cells in which an exogenous gene has been integrated into the target site, it is preferable that the DNA fragment containing the exogenous gene also contains a drug resistance gene or a reporter gene in the transposon vector. Here, the drug resistance gene and reporter gene may be the same as those used in the targeting vector for gene transfer.

[0039] The drug resistance and reporter genes are preferably under the control of any gene expression control region that can function in the target cells, which may be the same as that used in the targeting vector for gene transfer.

[0040] Furthermore, the transposon vector preferably has a polyA signal downstream of the drug resistance or reporter gene, and may be the same as that used in the targeting vector for gene transfer.

[0041] Furthermore, in addition to the gene encoding the transposase, the transposase expression vector may contain a drug resistance gene, a reporter gene, a gene expression control region, a polyA signal, etc. The drug resistance gene, reporter gene, gene expression control region, and polyA signal may be the same as those contained in the transposon-based vector.

[0042] The transposon vector and transposase expression vector may be introduced into target cells using the same methods as those used for targeting vectors for gene introduction.

[0043] Cells into which an exogenous gene has been integrated into the target site may be selected by the same method as that for selecting homologously recombinant cells into which a targeting vector for gene transfer has been integrated.

[0044] As described above, the transposon vector incorporating the DNA fragment of a foreign gene and the transposase expression vector can be used to integrate the DNA fragment of the foreign gene into the transposase target sequence TTAA in the genome of the target cell. Unlike the homologous recombination method using the above-mentioned targeting vector for gene introduction, this method does not allow for the site of integration of the foreign gene to be limited because the target sequence is TTAA. However, it is possible to subsequently remove the foreign gene integrated into the genome without leaving any trace by expressing the transposase.

[0045] The eNK cells used in the therapeutic agent of the present invention are selected from the group consisting of the following (c) and / or (d): (c) an exogenous gene encoding IL-15 (d) an exogenous gene encoding CD16 It may further express the following.

[0046] IL-15 is produced by monocytes, macrophages, dendritic cells, etc., and induces the proliferation and activation of tumoricidal cells such as CTLs and NK cells. Therefore, by introducing and expressing the IL-15 gene into pluripotent stem cells, the NK cells differentiated from the pluripotent stem cells are activated, and surrounding T cells are also activated, further enhancing the immune function of NK cells.

[0047] The exogenous IL-15 gene to be introduced is not particularly limited and includes, for example, the various forms described in WO2020 / 045610, but is preferably one that encodes secreted IL-15. The secreted IL-15 may be natural secreted IL-15 containing a native signal peptide, but modified IL-15 in which a heterologous signal peptide such as the IL-2 signal peptide has been substituted may also be preferably used.

[0048] In another preferred embodiment of the present invention, an exogenous gene encoding IL-15 receptor α (hereinafter sometimes referred to as "IL-15Rα") is introduced and coexpressed with the IL-15 gene. The gene may encode full-length (membrane-bound) IL-15Rα or soluble (secreted) IL-15Rα. Here, the IL-15Rα gene may be introduced into pluripotent stem cells as an expression construct separate from the IL-15 gene, or may be introduced as an expression construct constructed to express a fusion protein with IL-15. Examples of such IL-15 can be found in, for example, WO2020 / 045610.

[0049] CD16 is a low-affinity receptor for the Fc portion of aggregated IgG, and can bind to the Fc portion of target cell-specific antibodies to induce antibody-dependent cellular cytotoxicity. Therefore, by introducing and expressing the CD16 gene into pluripotent stem cells, NK cells induced to differentiate from the pluripotent stem cells have enhanced antibody-dependent cellular cytotoxicity and can exhibit more potent tumoricidal activity.

[0050] CD16 exists in two isoforms: transmembrane CD16a, which is expressed on most NK cells and a portion of monocytes, and GPI-anchored CD16b, which is expressed on neutrophils. In the present invention, the CD16 used as an exogenous gene may encode either isoform as long as it is capable of inducing antibody-dependent cytotoxicity, but CD16a is preferred. Furthermore, the CD16 gene may be a wild-type gene. In a preferred embodiment, the CD16 gene may be a high-affinity mutant with improved affinity for the Fc portion of IgG, or a non-cleavable mutant that is resistant to degradation by ADAM17. For example, a high-affinity mutant may be F176V, in which phenylalanine at position 176 is replaced with valine (F158V, in which phenylalanine at position 158 of the mature form is replaced with valine), and a non-cleavable mutant may be S197P, in which serine at position 197 is replaced with proline.

[0051] The eNK cells used in the therapeutic agent of the present invention are selected from the group consisting of the following (e): (e) an exogenous gene encoding NKG2D or NKG2D-CAR may be further expressed. NKG2D is an activating receptor expressed on NK cells that transmits an activation signal upon stimulation by an NKG2D ligand on target cells, enhancing the cytotoxic activity of NK cells and enhancing the production of cytokines such as IFN-γ and TNFα in NK cells. Therefore, by introducing and expressing the NKG2D gene in pluripotent stem cells, the immunocompetent function of NK cells induced to differentiate from these pluripotent stem cells can be further enhanced.

[0052] The NKG2D gene to be introduced can be the endogenous NKG2D gene of NK cells or a modified gene with enhanced function. The effect of NKG2D can be further enhanced by co-expressing the NKG2D gene with an exogenous gene encoding the coactivator DAP10. Alternatively, a gene encoding a fusion protein in which DAP10 is linked to the intracellular domain of NKG2D can be introduced.

[0053] Alternatively, a gene encoding an NKG2D-CAR, which fuses the extracellular domain of NKG2D with the transmembrane domain, costimulatory domain, and signaling domain of a chimeric antigen receptor (CAR), can be introduced. The CAR used here can have the same combination of transmembrane domain, costimulatory domain, and signaling domain as those used in conventional CAR-T cells, but preferably has a transmembrane domain of CD8a, a costimulatory domain of 2B4, and a signaling domain of CD3z.

[0054] The exogenous genes encoding the above-mentioned IL-15 (and IL-15Rα), CD16, and NKG2D can be introduced into pluripotent stem cells using similar methods by constructing expression constructs, in the same way as the exogenous genes encoding CCR2B and CCL19.

[0055] The genetically modified pluripotent stem cells obtained as described above can be induced to differentiate into NK cells or their precursor cells by methods known per se, such as the method described in Matsubara et al. Biochem Biophys Res Commun. 2019 Jul 12;515(1):1-8.

[0056] The eNK cells used in the therapeutic agents of the present invention highly express at least exogenous CCR2B and CCL19, and therefore can efficiently home to solid cancer tissues expressing CCL2 and kill the cancer cells, and can induce the migration of surrounding T cells and dendritic cells expressing CCR7, thereby exerting synergistic antitumor activity. Furthermore, in one embodiment, the eNK cells further express one or more exogenous genes selected from IL-15 (and IL-15Rα), CD16, and NKG2D (including the NKG2D / DAP10 complex and NKG2D-CAR), thereby achieving further functional enhancement brought about by these function-enhancing factors.

[0057] The solid cancers to which the therapeutic agent of the present invention is applicable are not particularly limited, and examples thereof include alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder, or pleural cancer, nose, nasal cavity, or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoma, and the like. Examples of solid cancers include, but are not limited to, id tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc. Preferably, the solid cancer is malignant mesothelioma (e.g., malignant pleural mesothelioma, malignant peritoneal mesothelioma, malignant pericardial mesothelioma, malignant tunica vaginalis testis mesothelioma), more preferably malignant pleural mesothelioma.

[0058] For the therapeutic agent of the present invention, eNK cells may be cultured using an appropriate medium before administration to a subject. Stimulatory molecules may also be added to the medium to maintain and / or amplify the activation and / or proliferation of eNK cells. Furthermore, serum or plasma may be added to the medium. The amount of these to be added to the medium is not particularly limited, but examples include 0% to 20% by volume. The amount of serum or plasma used can be varied depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources, but autologous sources are preferred from the viewpoint of safety.

[0059] The therapeutic agents of the present invention can be administered intratumorally or peritumorally to subjects with solid cancer. Considering the number of eNK cells delivered into the tumor, intratumoral administration (including the peritumoral area, which can reach the tumor without any barriers) is preferred. However, although the number of eNK cells infiltrating into the tumor is reduced when administered peritumorally, the therapeutic agents of the present invention exert an antitumor effect equivalent to that of intratumoral administration. Therefore, even when intratumoral administration is difficult (e.g., in the case of solid cancers such as mesothelioma, where the tumor is very hard and injection is difficult) or when such administration is not very versatile (e.g., when specialized equipment or skilled techniques are required for administration), sufficient therapeutic effects can be obtained by administering the therapeutic agents peritumorally. Therefore, in a preferred embodiment, the therapeutic agents of the present invention are administered peritumorally.

[0060] As used herein, "vicinity of tumor" refers to the area around the tumor that requires infiltration of eNK cells to reach the tumor.

[0061] Administration near the tumor can be performed using, for example, a catheter or injection needle. The dosage is selected appropriately depending on the condition, weight, age, type and progression of cancer of the subject. Usually, the number of eNK cells is 1 × 10 per administration for a subject weighing 60 kg. 6 ~1×10 10 Preferably 1 x 10 7 ~1×10 9 5×10 7 ~5×10 8 The therapeutic agent is administered so that the cells reach a total volume of 1000 cells / ml. The therapeutic agent may be administered once or multiple times. The therapeutic agent may be in a known form suitable for parenteral administration, such as an injection or infusion. The therapeutic agent may contain a pharmacologically acceptable excipient as appropriate. The therapeutic agent may contain saline, phosphate-buffered saline (PBS), a culture medium, etc. to stably maintain the cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, X-VIVO10, etc. Furthermore, the therapeutic agent may contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. for the purpose of stabilization.

[0062] The therapeutic agent of the present invention can be used in combination with other cancer treatments, such as surgery, chemotherapy, radiation therapy, and other cancer immunotherapies (e.g., immune checkpoint inhibitors).

[0063] Furthermore, by introducing an antigen-specific chimeric antigen receptor (CAR) into eNK cells (CAR-NK cells), they can be made into cellular immunotherapeutic agents specific to cells expressing that antigen.

[0064] To produce CAR-NK cells from eNK cells, a method known per se can be appropriately selected and used.

[0065] CARs are artificially engineered hybrid proteins containing an antibody antigen-binding domain (e.g., scFv) linked to a T cell signaling domain. CARs feature the ability to utilize the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing NK cells the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor escape.

[0066] The CAR introduced into eNK cells contains an antigen-binding domain of an antibody that can specifically recognize the surface antigen that eNK cells should recognize (e.g., a cancer antigen peptide, a surface receptor whose expression is increased on cancer cells, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0067] Examples of surface antigens specifically recognized by the antigen-binding domain include various solid cancers (e.g., alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder, or pleural cancer, nose, nasal cavity, or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney ... Surface receptors with elevated expression in cancers such as pancreatic cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer, e.g., CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, and B-cell Examples of antigens include, but are not limited to, mutation antigens, CD123, CD3, prostate specific membrane antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2, PD-L1, CEA, chondroitin sulfate proteoglycan-4, IL-13 receptor α chain, IgGκ light chain, and cancer antigen peptides (e.g., peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.).

[0068] The antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment capable of specifically recognizing a target antigen. However, considering the ease of CAR production, a single-chain antibody (scFv) in which a light chain variable region and a heavy chain variable region are linked via a linker peptide is desirable. The arrangement of the light chain variable region and heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstitute a functional antigen-binding domain. Typically, the arrangement is designed in the following order from the N-terminus: light chain variable region-linker peptide-heavy chain variable region. As the linker peptide, a publicly known linker peptide commonly used in the production of single-chain antibodies can be used. DNA encoding the light chain variable region and DNA encoding the heavy chain variable region can be prepared, for example, by cloning the light chain gene and heavy chain gene, respectively, from antibody-producing cells and performing PCR using them as templates, or by chemical synthesis using the sequence information of an existing antibody. DNA encoding a single-chain antibody can be obtained by ligating the resulting DNA fragments with DNA encoding the linker peptide using an appropriate method. It is preferable that a leader sequence be further added to the N-terminus of the antigen-binding domain in order to present the CAR on the cell surface of the modified NK cell.

[0069] As the extracellular hinge domain and transmembrane domain, domains derived from T cell surface molecules commonly used in the art can be used as appropriate, including, but not limited to, domains derived from CD8α and CD28.

[0070] Examples of intracellular signaling domains include those having a CD3ζ chain, those having an additional costimulatory motif such as CD28, CD134, CD137, LCK, DAP10, ICOS, or 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more costimulatory motifs, but are not limited to these, and any combination of domains commonly used in the art can be used.

[0071] Nucleic acid sequence information encoding the extracellular hinge domain, transmembrane domain, and intracellular signaling domain is well known in the art, and a person skilled in the art can easily obtain DNA fragments encoding each domain from T cells based on this information. The DNA fragments thus obtained, each encoding the antigen-binding domain, extracellular hinge domain, transmembrane domain, and intracellular signaling domain, can be ligated using standard methods to obtain DNA encoding a CAR.

[0072] The resulting DNA encoding the CAR can be inserted directly or after adding an appropriate linker and / or nuclear localization signal, etc., into an expression vector, preferably a plasmid vector, containing a gene expression control region functional in NK cells. Examples of gene expression control regions functional in NK cells include, but are not limited to, the constitutive SRα promoter in mammalian cells, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney murine leukemia virus) LTR, and the HSV-TK (herpes simplex virus thymidine kinase) promoter. Gene promoters specifically expressed in NK cells, such as CD16, CD56, and NKG2D, can also be used.

[0073] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and do not limit the present invention in any way. [Example]

[0074] (Production of iNK cells and eNK cells) Unmodified control iNK cells and eNK cells transfected with the CCR2B gene and CCL19 gene were generated according to the method described in WO 2023 / 085356.

[0075] Example 1 Antitumor effects of intratumoral and peritumoral administration of eNK cells MSTO-211H human mesothelioma cells (obtained from ATCC) 3 x 10 6 The cells / mouse were subcutaneously transplanted into immunodeficient mice (obtained from Invivo Science Co., Ltd.) (day 0), and the tumor volume (mm 3 ) = Long diameter (mm) x Short diameter (mm) x Height (mm) x π / 6 = 45 mm 3 1x10 eNK cells were added 24 days after tumor inoculation, when the tumor size reached approximately 1x10 7 Cells were administered intratumorally or near the tumor at 0.05 mL / mouse, three times a week for 3 weeks (days 24, 26, 28, 31, 33, 35, 38, 40, and 42). IL-2 was also administered intraperitoneally at 5 μg / 0.1 mL / mouse every day from day 24 to day 42.

[0076] On day 44 after tumor inoculation, the mice were euthanized, and tumor tissue was collected and fixed in 4% paraformaldehyde. Paraffin sections were prepared and histological examination was performed. The sections were stained with hematoxylin and eosin (HE) and with anti-human CD45 antibody to label eNK cells. Histological evaluation was performed on three mice per group, and a representative sample is shown in Figure 1.

[0077] Following intratumoral administration, HE staining revealed tumor necrosis, and anti-human CD45 antibody staining demonstrated the presence of numerous eNK cells within the tumor. In the photograph of administration near the tumor, the upper right shows the skin covering the tumor, and the lower left shows the center of the tumor. Anti-human CD45 antibody staining demonstrated eNK cell infiltration into the tumor, and HE staining clearly demonstrated tumor cell necrosis. From the above, it is clear that eNK cells that infiltrated into the tumor demonstrated an anti-tumor effect.

[0078] The results of histological examination using HE staining and anti-human CD45 antibody staining were analyzed by image analysis. The results are shown in Table 1.

[0079] [Table 1]

[0080] The tumor area in the sections examined histologically showed no significant difference between the intratumoral and peritumoral administration groups, and there was also no significant difference in the area ratio of necrotic and degenerative areas within the tumor. This demonstrates that the antitumor effect is due to the cytotoxic activity of eNK cells, and that this effect is equivalent for both administration methods. Meanwhile, although the number of human CD45-positive cells (eNK cells) in the peritumoral administration group was less than 10% of that in the intratumoral administration group in this study, the above results demonstrate that peritumoral administration also results in the infiltration of eNK cells sufficient to induce tumor necrosis and degeneration, i.e., the antitumor effect.

[0081] Example 2 Comparison of infiltration into tumors of eNK cells and iNK cells administered near tumors MESO-4 human mesothelioma cells (obtained from RIKEN) 5x10 6 The cells / mouse were subcutaneously transplanted into immunodeficient mice (obtained from Invivo Science Co., Ltd.) (day 0), and the tumor volume (mm 3 ) = long diameter (mm) x short diameter (mm) x height (mm) x π / 6 = 35 mm 3 On day 66 after tumor inoculation, when the tumor size reached approximately 100%, the eNK and iNK administration groups were administered 1x10 eNK cells or 1x10 iNK cells, respectively. 7 Cells were administered in 0.05 mL / mouse into the tumor area in the control group, and 0.05 mL / mouse of Hank's balanced salt solution (HBSS) was administered once near the tumor. IL-2 (5 μg / 0.1 mL / mouse) was also administered intraperitoneally on days 66, 68, 70, and 72.

[0082] Mice were euthanized on days 67, 70, and 73. Tumor tissues were collected and fixed in 4% paraformaldehyde. Paraffin sections were prepared and histologically evaluated (n = 3). Histological evaluation was performed using HE staining and anti-human CD45 antibody to label iNK cells and eNK cells. TUNEL staining was also performed to assess tumor cell apoptosis. The results are shown in Figures 2–4.

[0083] In the eNK-administered group, eNK cells were not observed inside the tumor on days 67 and 70, i.e., 1 and 4 days after eNK cell administration. However, on day 73, i.e., 7 days after eNK administration, the presence of eNK cells inside the tumor was clear in 2 out of 3 cases, demonstrating their infiltration into the tumor. Figure 3, an enlarged view of the boxed area in Figure 2, shows infiltration from the edge of the tumor (right side of the photograph), with some of the cells reaching the tumor interior (arrow).

[0084] Figure 4 is a further enlarged view of a portion of Figure 3. The arrows indicate CD45-positive eNK cells (left photo) and apoptotic TUNEL-positive tumor cells (right photo). These results demonstrate that apoptosis of tumor cells was observed in areas where eNK cells had infiltrated, and that the infiltrated eNK cells exerted an antitumor effect through their cytotoxic activity.

[0085] On the other hand, in the iNK-treated group, iNK cells were not observed in the tumor tissue on days 67, 70, and 73, i.e., 1, 4, and 7 days after iNK cell administration. The iNK cells at the injection site were CD45 negative, indicating that they had been necrotic for a long time and had a shorter survival time than eNK cells.

[0086] These findings demonstrate that eNK cells migrate into tumors after administration near the tumor and exert antitumor effects.

[0087] The results of histological examination of anti-human CD45 antibody staining were analyzed using images. The results are shown in Table 2.

[0088] [Table 2]

[0089] The number of CD45-positive cells observed per unit area in the iNK-administered group was similar to that in the HBSS-administered group, whereas the eNK-administered group showed a significantly higher number of CD45-positive cells. The results of image analysis also revealed that eNK cells infiltrated into the tumor more than iNK cells. [Industrial Applicability]

[0090] The therapeutic agent of the present invention is useful as a cellular immunotherapeutic agent for solid cancers including mesothelioma.

Claims

1. (a) and (b) below: (a) an exogenous gene encoding CC chemokine receptor type 2B (CCR2B) (b) an exogenous gene encoding CC chemokine ligand 19 (CCL19); A therapeutic agent for solid cancer, comprising natural killer (NK) cells or their precursor cells derived from pluripotent stem cells expressing the gene encoding the NK-1 gene, characterized in that the agent is administered near the tumor.

2. NK cells or their precursor cells are selected from the following (c) and / or (d): (c) an exogenous gene encoding interleukin 15 (IL-15) (d) an exogenous gene encoding CD16 The agent according to claim 1, further expressing:

3. NK cells or their precursor cells are selected from the group consisting of the following (e): (e) an exogenous gene encoding NKG2D or NKG2D-CAR The agent according to claim 1 or 2, further expressing:

4. The agent according to claim 3, wherein (e) is NKG2D-CAR.

5. (e) is NKG2D, and the NK cells or their precursor cells further (f) an exogenous gene encoding DAP10 The agent according to claim 3, which co-expresses

6. The agent according to any one of claims 2 to 5, wherein the NK cells or their precursor cells express an exogenous gene encoding IL-15, and the IL-15 is secreted IL-15.

7. The agent according to any one of claims 2 to 5, wherein the NK cells or their precursor cells express an exogenous gene encoding IL-15 and co-express an exogenous gene encoding IL-15 receptor α (IL-15Rα).

8. The agent according to any one of claims 2 to 7, wherein the NK cells or their precursor cells express an exogenous gene encoding CD16, and the CD16 is CD16 having a high-affinity mutation or a non-cleavable mutation.

9. The agent according to claim 8, wherein CD16 has a mutation selected from F176V (F158V) and S197P.

10. The agent according to any one of claims 1 to 9, wherein the solid cancer is mesothelioma.

11. A method for treating solid cancer, comprising the following (a) and (b): (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 A method comprising administering an effective amount of NK cells or their precursor cells derived from pluripotent stem cells expressing the gene to the vicinity of the tumor of a subject with solid cancer.

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