Gene-modified pluripotent stem cell, immunocompetent cell derived therefrom, method for producing said cells, and use thereof

Abstract

The present invention provides: pluripotent stem cells expressing (a) an exogenous gene encoding CC chemokine receptor type 2B (CCR2B) and (b) an exogenous gene encoding CC chemokine ligand 19 (CCL19); NK cells derived from said pluripotent stem cells; or precursor cells thereof.

Description

Genetically modified pluripotent stem cells, immunocompetent cells derived therefrom, methods for producing them, and uses thereof

[0001] The present invention relates to genetically modified pluripotent stem cells, immunocompetent cells derived from the pluripotent stem cells, methods for producing them, and uses thereof. More specifically, the present invention relates to genetically modified pluripotent stem cells with enhanced function for cellular immunotherapy, natural killer (NK) cells or their precursor cells derived from the pluripotent stem cells, methods for producing them which include introducing an exogenous gene into the pluripotent stem cells to enhance their function, and uses of them as cell medicines.

[0002] Cells involved in cancer immunity include lymphocytes such as T cells, NK cells, and natural killer T (NKT) cells, and research and development of cell therapy using these immune cells has a history of over half a century. In recent years, technological advances such as the development of iPS cells and differentiation induction methods, as well as genetic modification methods for 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. This has made it possible to develop a variety of treatment strategies using immune cells.

[0003] Currently, only two gene-modified immune cell-based cell therapies have been approved: CAR-T cells targeting CD19 (Kymriah and Yescarta). NK cells, on the other hand, do not require presensitization to cancer cells and can respond without being bound by antigen recognition. NK cells directly recognize and kill target cells, and also activate T cells and macrophages through the production of various cytokines, improving immune function. Furthermore, unlike T cells, NK cells are less likely to develop problems such as GvHD, even when using donor cells with a completely mismatched HLA type, and are less likely to cause cytokine release syndrome (CRS), a side effect associated with T cells. Given the recent positive clinical results of T cell therapy, expectations are high for immune cell therapy using NK cells, which are also anti-tumor effector cells.

[0004] In fact, various clinical trials are currently underway targeting cell-based medicines using modified NK cells (Non-Patent Document 1). For example, Fate Therapeutics is developing modified NK cells derived from iPS cells that have been knocked out (KO) of CD38 and express an IL-15 / IL-15 receptor fusion protein and CD16 for the treatment of solid tumors (e.g., Patent Document 1). Takeda Pharmaceutical Company Limited, in collaboration with The University of Texas MD Anderson Cancer Center, is developing CD19-CAR NK cells that express IL-15 (e.g., Non-Patent Document 2). However, none of these have yet been approved by authorities.

[0005] Known functional enhancing factors used in NK cells include IL-15 and CD16, which have been introduced into products developed by Fate Therapeutics and Takeda Pharmaceutical Co., Ltd. (see above). IL-15 is known to proliferate and activate NK cells themselves, as well as activate surrounding T cells (Non-Patent Documents 3-5). Furthermore, CD16, when expressed on the surface of NK cells, is known to contribute to NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) (Non-Patent Document 6). Therefore, attempts to express these factors, which are already well-known to function in NK cells, in NK cells to further enhance their function have already been adopted in various modified NK cells, including those developed by Fate Therapeutics and Takeda Pharmaceutical Co., Ltd. (see above).

[0006] Meanwhile, attempts have been made to enhance immune cells' ability to attack cancer cells by expressing various chemokines and chemokine receptors to enhance their migratory ability. For example, Patent Document 2 discloses cancer immunotherapy using T cells expressing various chemokines and chemokine receptors, such as CCR2. Patent Document 3 discloses that expressing CCL19 together with various interleukins enhances the antitumor activity of T cells and improves their proliferation. Patent Document 4 also discloses CAR immune cells expressing immunostimulatory factors, and states that NK cells are also included as target CAR immune cells. IL-15 and CCR2B are also listed as immunostimulatory factors.

[0007] However, these disclosures only concern methods for enhancing the function of immune cells such as T cells by expressing the factor, and do not involve the use of genetically modified iPS cells. Furthermore, none of the documents suggests the combination of CCR2B and CCL19 being introduced into NK cells.

[0008] WO 2019 / 126748WO 2018 / 152572WO 2020 / 045610WO 2017 / 133633

[0009] Nat Rev Drug Discov. 2020 Mar;19(3):200-218N Engl J Med. 2020 Feb 6; 382(6): 545-553J Exp Med. 1994 Oct 1; 180(4): 1395-1403Cancer Immunnol Immunother. 2012 Sep; 61(9): 1451-1461Nat Rev Immunnol. 2003 Apr; 3(4): 269-279Front Immunnol. 2015 Jul 27; 6: 368

[0010] There is a need for genetically modified immune cells that offer additional therapeutic options. Therefore, an object of the present invention is to provide genetically modified immunocompetent cells with high functionality, particularly genetically modified NK cells derived from pluripotent stem cells such as iPS cells.

[0011] To achieve the above-mentioned objectives, the present inventors conceived the idea of ​​overexpressing chemokines and chemokine receptors in NK cells. From among the many chemokines and chemokine receptors available, they selected CCR2B as the chemokine receptor and CCL19 as the chemokine, and exogenously introduced these into iPS cells by genetic modification. The iPS cells were then induced to differentiate into NK cells, and their functions were examined. The modified NK cells exhibited higher levels of function than expected. It is known that cytokines, including chemokines, in immune cells can have dual roles in cancer immunity. For example, CCL2 can promote the recruitment of immunosuppressive cells and suppress T cell function (e.g., Cancer Lett 2007 Jul 8;252(1):86-92). Therefore, even if multiple chemokines and chemokine receptors are combined, it is completely unknown whether the desired bonus effect can be achieved. Therefore, the present inventors' findings are surprising. Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0012] That is, the present invention is as follows. Item [1] Pluripotent stem cells expressing the following (a) and (b): (a) an exogenous gene encoding CC chemokine receptor type 2B (CCR2B), (b) an exogenous gene encoding CC chemokine ligand 19 (CCL19), or NK cells or their precursor cells derived from the pluripotent stem cells. Item [2] The cell according to Item [1], further expressing the following (c) and / or (d): (c) an exogenous gene encoding interleukin-15 (IL-15), or (d) an exogenous gene encoding CD16. Item [3] The cell according to Item [1] or [2], further expressing the following (e): (e) an exogenous gene encoding NKG2D or NKG2D-CAR. Item [4] The cell according to Item [3], wherein (e) is an exogenous gene encoding NKG2D-CAR. Item [5] The cell according to Item [3], wherein (e) is an exogenous gene encoding NKG2D and further co-expresses (f) an exogenous gene encoding DAP10. Item [6] The cell according to any one of Item [2] to [5], wherein the cell expresses an exogenous gene encoding IL-15, wherein the IL-15 is secreted IL-15. Item [7] The cell according to any one of Item [2] to [5], wherein the cell expresses an exogenous gene encoding IL-15 and co-expresses an exogenous gene encoding IL-15 receptor α (IL-15Rα). Item [8] The cell according to any one of Item [2] to [7], wherein the cell expresses an exogenous gene encoding CD16, wherein the CD16 has a high-affinity mutation or a non-cleavable mutation. Item [9] The cell according to Item [8], wherein the CD16 has a mutation selected from F176V (F158V) and S197P. Item

[10] A pharmaceutical comprising the cell according to any one of items [1] to [9]. Item

[11] The pharmaceutical according to item

[10] , which is a therapeutic drug for cancer. Item

[12] A method for producing genetically modified cells with enhanced homing function to cancer tissues and enhanced function to recruit immunocompetent cells, the method comprising introducing the following (a) and (b): (a) an exogenous gene encoding CCR2B, and (b) an exogenous gene encoding CCL19 into pluripotent stem cells.Item

[13] The method of Item

[12] , further comprising inducing differentiation of the pluripotent stem cells into which the exogenous gene has been introduced into NK cells or their precursor cells. Item

[14] A construct for producing genetically modified cells, comprising the following (a) and (b): (a) an exogenous gene encoding CCR2B, and (b) an exogenous gene encoding CCL19. Item

[15] The construct of Item

[14] , in which the exogenous genes are contained in separate constructs. Item

[16] An expression cassette comprising a gene expression control region and the construct of Item

[14] or

[15] under the control of the control region. Item

[17] A vector comprising the expression cassette of Item

[16] . Item

[18] The vector of Item

[17] , in which the expression cassette is flanked by a pair of transposon inverted repeat sequences. Item

[19] A kit for producing genetically modified cells comprising the vector of Item

[18] and a transposase expression vector, and expressing the following (a) and (b): (a) an exogenous gene encoding CCR2B, and (b) an exogenous gene encoding CCL19. Item

[20] The kit of Item

[19] , wherein the transposase is PiggyBac transposase. Item

[21] A method for treating cancer, comprising administering the cell of any one of Item [1] to [9] to a cancer patient. Item

[22] The cell of any one of Item [1] to [9] for use in a method for treating cancer. Item

[23] Use of the cell of any one of Item [1] to [9] in the manufacture of a medicament for cancer treatment.

[0013] The modified NK cells or their precursor cells of the present invention are derived from pluripotent stem cells transfected with genes specifically designed to enhance cancer immunity. By expressing CCR2B and CCL19, they possess superior cancer tissue homing and recruitment capabilities to immunocompetent cells such as T cells compared to conventional cells, resulting in high antitumor activity. Furthermore, high expression of CD16 enhances antibody-dependent cellular cytotoxicity, while expression of IL-15 and IL-15 receptor α improves persistence and proliferation. Furthermore, high expression of NKG2D(-CAR) and DAP10 enhances activation signals stimulated by NKG2D ligands. Therefore, the use of modified NK cells or their precursor cells of the present invention can improve the therapeutic efficacy of immunotherapy. Furthermore, the modified NK cells or their precursor cells of the present invention may be involved in the activation of not only NK cells themselves but also surrounding immune cells such as T cells, potentially making them useful for treating diseases for which existing therapies have limited efficacy. Furthermore, since the modified NK cells or their precursor cells of the present invention can be obtained by inducing differentiation of genetically modified pluripotent stem cells, it is possible to produce larger quantities of modified NK cells or their precursor cells more easily than cells obtained by genetically modifying NK cells.

[0014] By using the combination of predetermined factors of the present invention, various modified immune cells with high anti-tumor activity can be obtained, not only in immune cells in general, but also in T cells, monocytes / macrophages, dendritic cells, etc., when genes are introduced and expressed in these cells.

[0015] 1 is a diagram showing that CCR2B-expressing iNK cells have high migratory activity toward CCL2. FIG. 2 is a diagram showing that CCL19-expressing iNK cells have the ability to enhance the migratory activity toward PBMCs. FIG. 3 is a diagram showing that CD16-expressing iNK cells have enhanced cytotoxic activity in the presence of an anti-EGFR antibody (Cetuximab). FIG. 4 is a diagram showing that the number of IL-15-expressing iNK cells does not decrease even when cultured in the absence of IL-15. FIG. 5 is a diagram showing that the IFN-γ secretion activity of NKG2D-expressing iNK cells is enhanced by stimulation in coculture with A549 cells. FIG. 6 is a diagram showing that CCR2B / CCL19-coexpressing iNK cells have high migratory activity toward CCL2. FIG. 7 is a diagram showing that CCR2B / CCL19-coexpressing iNK cells have high attracting activity toward DCs. FIG. 8 is a diagram showing that IL2sp / IL-15 expression enhances the proliferation and survival of iNK cells, and that this effect is further enhanced by coexpression of full-length or soluble IL-15Rα.

[0016] (I) Genetically modified pluripotent stem cells of the present invention The present invention provides genetically modified pluripotent stem cells (hereinafter also referred to as "modified pluripotent stem cells of the present invention") suitable for differentiation into genetically modified NK cells or their precursor cells with enhanced immunocompetent cell function. The pluripotent stem cells 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).

[0017] Target cells such as cancer cells express CC chemokine ligand 2 (CCL2), and 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 target tissues such as cancer tissues.

[0018] 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.

[0019] As used herein, "pluripotent stem cells" refer to cells with 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.

[0020] 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); Iritani A. 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 to 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 of the somatic cell are the same as those for iPS cell production, described below.

[0021] 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).

[0022] As used herein, the term "somatic cells" refers to any human cell except for 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, somatic cells include, for example, (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, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0023] 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 small molecules. 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.

[0024] 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 by the expression of various ES cell-specific genes and teratoma formation.

[0025] In addition, various human iPS cell lines established by designated institutions, 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.

[0026] As used herein, "introducing an exogenous gene" refers to introducing an exogenous gene into a target site in the genome, thereby enabling the exogenous gene to be expressed in the target cell. A specific method for introducing an exogenous gene is to isolate the DNA of the exogenous gene according to conventional methods, and then insert a DNA fragment of the exogenous gene into the target site of the target cell, thereby constructing a DNA strand (hereinafter referred to as a gene introduction targeting vector) having a DNA sequence that results in the exogenous gene being expressed in the target cell. A preferred method is to integrate the DNA strand into the target site of the target cell by homologous recombination. Since the gene insertion site is fixed using homologous recombination, the absence of random integration is expected to result in minimal differences in expression levels between clones and minimal impact on other genes.

[0027] The homologously recombinant cells can be obtained, for example, by introducing the above-mentioned targeting vector into a subject cell.

[0028] 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:

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 genetic 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 medium at a concentration of cells / ml and transferred to a cuvette, to which 10-100 μg of a targeting vector for gene deletion is added, followed by application of an electric pulse of 200-600 V / cm.

[0035] 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.

[0036] 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.

[0037] 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 outside 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 to be differentiated 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 little effect on other genes.

[0038] 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:

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The modified pluripotent stem cells of the present invention may further express the following (c) and / or (d): (c) an exogenous gene encoding interleukin-15 (IL-15); and (d) an exogenous gene encoding CD16.

[0048] 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 themselves, which are induced to differentiate from the pluripotent stem cells, are activated, and surrounding T cells are also activated, thereby further enhancing the immune function of NK cells.

[0049] 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.

[0050] In another preferred embodiment of the modified pluripotent stem cells 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 the 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.

[0051] 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.

[0052] 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.

[0053] The modified pluripotent stem cells of the present invention may further express the following (e): (e) an exogenous gene encoding NKG2D or NKG2D-CAR. NKG2D is an activating receptor expressed on NK cells, and upon stimulation by an NKG2D ligand on a target cell, it transmits an activation signal, 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 the pluripotent stem cells can be further enhanced.

[0054] 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.

[0055] 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 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.

[0056] 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.

[0057] (II) Modified NK Cells of the Present Invention The modified pluripotent stem cells thus obtained can be induced to differentiate into NK cells by a method known per se, for example, the method described in Matsubara et al. Biochem Biophys Res Commun. 2019 Jul 12;515(1):1-8. Therefore, the present invention also provides NK cells or their precursor cells (hereinafter, precursor cells are also collectively referred to as "modified NK cells of the present invention") induced to differentiate from the modified pluripotent stem cells of the present invention.

[0058] The modified NK cells of the present invention highly express at least exogenous CCR2B and CCL19, and thus can efficiently home to and kill target tissues (e.g., cancer tissues) expressing CCL2. They can also induce the migration of surrounding T cells and dendritic cells expressing CCR7, thereby exerting synergistic antitumor activity. Furthermore, in one embodiment, the modified NK 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 by these function-enhancing factors. Therefore, the modified NK cells of the present invention can be used, for example, as a pharmaceutical for any disease for which killing CCL2-expressing cells can produce a therapeutic effect.

[0059] In pharmaceuticals containing the modified NK cells of the present invention as active ingredients, the NK cells may be cultured in 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 NK cells. Furthermore, serum or plasma may be added to the medium. The amount of these substances 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 a safety perspective.

[0060] Pharmaceuticals containing the modified NK cells of the present invention as active ingredients are preferably administered parenterally to a subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but typically, a single dose of 1 x 10 cells is administered to a subject weighing 60 kg. 6 ~1×10 10 Preferably 1 x 10 7 ~1×10 9 5×10 7 ~5×10 8 The pharmaceutical composition is administered so that the total number of cells reaches 100. The pharmaceutical composition may be administered once or multiple times. The pharmaceutical composition may be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition may contain a pharmacologically acceptable excipient as appropriate. The pharmaceutical composition 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, and X-VIVO10. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc. for stabilization purposes.

[0061] A medicament containing the modified NK cells of the present invention as an active ingredient can be a therapeutic agent for cancer. The cancer to which the medicament is applied is not particularly limited, and examples thereof include acute lymphocytic cancer, 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, cervical, 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 cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia), and the like. , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), 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 tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.

[0062] (III) CAR-NK Cells Furthermore, by introducing an antigen-specific CAR into the modified NK cells of the present invention (CAR-NK cells), they can be used as a cellular immunotherapeutic agent specific to cells expressing the antigen.

[0063] To produce CAR-NK cells from the modified NK cells of the present invention, a method known per se can be appropriately selected and used.

[0064] 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 selected targets 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.

[0065] The CAR introduced into the modified NK cells of the present invention comprises an antigen-binding domain of an antibody that can specifically recognize the surface antigen to be recognized by the modified NK cells (e.g., a cancer antigen peptide, a surface receptor whose expression is increased in cancer cells, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0066] Examples of surface antigens specifically recognized by the antigen-binding domain include various cancers (e.g., acute lymphocytic carcinoma, 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, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, 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 cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute surface receptors whose expression is upregulated in tumors such as myeloid leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), 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.), e.g., CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, 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.).

[0067] 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, they can be designed in the order light chain variable region-linker peptide-heavy chain variable region from the N-terminus. Known linker peptides 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.

[0068] 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.

[0069] 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.

[0070] 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. DNA encoding a CAR can be obtained by ligating the thus obtained DNA fragments encoding the antigen-binding domain, extracellular hinge domain, transmembrane domain, and intracellular signaling domain using standard methods.

[0071] 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.

[0072] (IV) Methods for producing modified pluripotent stem cells and modified NK cells, and reagents and kits therefor The present invention also provides a method for producing genetically modified cells with enhanced homing function to cancer tissues and enhanced function to recruit immunocompetent cells, the method comprising introducing the following (a) and (b): (a) an exogenous gene encoding CCR2B; and (b) an exogenous gene encoding CCL19 into pluripotent stem cells.

[0073] In the above production method, preparation of an exogenous gene, gene transfer, selection of genetically modified pluripotent stem cells, etc. can be performed in the same manner as described above. In addition, one or more exogenous genes selected from IL-15 (and IL-15Rα), CD16, and NKG2D (including the NKG2D / DAP10 complex and NKG2D-CAR) may be further transferred.

[0074] The resulting modified pluripotent stem cells can be induced to differentiate into NK cells or their precursor cells by a method known per se.

[0075] The present invention also provides a construct for producing a genetically modified cell, comprising the following (a) and (b): (a) an exogenous gene encoding CCR2B, and (b) an exogenous gene encoding CCL19.

[0076] Here, the two exogenous genes may be constructed to enable polycistron expression via the 2A peptide or IRES, but are preferably designed to be contained in separate constructs.

[0077] These constructs are placed under the control of functional gene expression control regions (e.g., promoters, enhancers, polyadenylation signals, etc.) to form an expression cassette. When two exogenous genes are contained in separate constructs, the expression cassettes containing these constructs may be carried on a single vector or on separate vectors.

[0078] In a preferred embodiment, the expression cassette is carried on a vector flanked by a pair of transposon inverted repeats. This transposon vector can be combined with a transposase expression vector, preferably a PiggyBac transposase (PBase) expression vector, to integrate an exogenous gene of interest into a cellular chromosome. Therefore, the present invention also provides a kit for generating genetically modified cells that include a transposon vector containing the expression cassette and a transposase expression vector, and that express the following (a) and (b): (a) an exogenous gene encoding CCR2B; and (b) an exogenous gene encoding CCL19.

[0079] 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.

[0080] Production Example 1: Production of Genetically Modified iPS Cells iPS cell clone 06E (TC-1133HKK_06E_MCB) was used as the parent line of iPS cells. This parent line will be referred to as "unedited iPS cells" hereafter. The following target genes were forced to express in these unedited iPS cells. The PiggyBac method was used for gene transfer. Furthermore, the constitutively active EF1 alpha (EF1A) promoter region was used to force the cDNA expression of each factor.

[0081] The following plasmid DNAs were prepared to express each gene of interest: #CCR2B-expressing PiggyBac plasmid #CCL19-expressing PiggyBac plasmid #IL-15-expressing PiggyBac plasmid #IL-15 receptor α-expressing PiggyBac plasmid #Secreted IL-15 receptor α-expressing PiggyBac plasmid #Signal peptide-modified IL-15-expressing PiggyBac plasmid #CD16a-expressing PiggyBac plasmid #NKG2D-expressing PiggyBac plasmid #NKG2D-CAR-expressing PiggyBac plasmid #DAP10-expressing PiggyBac plasmid

[0082] In addition, the following plasmid DNA was prepared to express PiggyBac transposase: #hPBase expression plasmid

[0083] Plasmid DNA used for gene transfer using the PiggyBac method was constructed as follows. A construct consisting of the PiggyBac 3' ITR sequence (SEQ ID NO: 1), a restriction enzyme multiple cloning site (MCS), and the PiggyBac 5' ITR sequence (SEQ ID NO: 2) was artificially synthesized and inserted into the restriction enzyme site of the pHSG298 plasmid (Takara Bio). Plasmid DNA expressing each gene of interest was constructed by inserting the EF1A promoter (PCR amplified), each gene of interest (sequences described below), an IRES (synthetic: SEQ ID NO: 3), a drug resistance gene (sequences described below), and human growth hormone polyA (synthetic: SEQ ID NO: 4) into the MCS of the PiggyBac ITR plasmid. The pBApo-EF1a Pur DNA plasmid (Takara Bio) was used as the PCR template for EF1A promoter PCR amplification.

[0084] The target genes (artificially synthesized) used were any of CCR2B (artificially synthesized: SEQ ID NO: 5), CCL19 (artificially synthesized: SEQ ID NO: 6), IL-15 (artificially synthesized: SEQ ID NO: 7), full-length IL-15 receptor α (IL-15Rα) (artificially synthesized: SEQ ID NO: 8), soluble IL-15 receptor α (hereinafter, may be referred to as "sIL-15Rα") (artificially synthesized: SEQ ID NO: 9), signal peptide-modified IL-15 (hereinafter, may be referred to as "IL-2sp / IL-15") (artificially synthesized: SEQ ID NO: 10), CD16a (artificially synthesized: SEQ ID NO: 11), NKG2D (artificially synthesized: SEQ ID NO: 12), NKG2D-CAR (artificially synthesized: SEQ ID NO: 13), and DAP10 (artificially synthesized: SEQ ID NO: 14), or a combination thereof. The drug resistance gene used was any one of puromycin (artificially synthesized: SEQ ID NO: 15), hygromycin (artificially synthesized: SEQ ID NO: 16), zeocin (artificially synthesized: SEQ ID NO: 17), and neomycin (artificially synthesized: SEQ ID NO: 18).

[0085] The hPBase expression plasmid DNA was constructed as follows: the EF1A promoter (amplified by PCR), human codon-optimized PBase (artificially synthesized, SEQ ID NO: 19), and human growth hormone poly A (artificially synthesized, SEQ ID NO: 4) were inserted into the restriction enzyme site within the MCS of the pHSG298 plasmid (Takara Bio Inc.).

[0086] The unedited iPS cells 06E were transfected with the above transfectant expression plasmid DNA (one or more, depending on the cell type) and hPBase expression plasmid DNA using electroporation (Neon Transfection System, Thermo Fisher Scientific). The day after transfection, the cells were cultured for 3 to 7 days in liquid medium containing one to four drugs: puromycin, hygromycin, zeocin, and neomycin, and drug-resistant cells were selected. In this way, a transfectant-expressing iPS cell pool was generated (Table 1).

[0087]

[0088] If necessary, single cell cloning was performed from the iPS cell pool to isolate iPS cell clones expressing the introduced factors.

[0089] Production Example 2: Induction of Differentiation into NK Cells. The iPS cell pool or clones expressing the target gene prepared in Production Example 1 were expanded and then induced to differentiate into NK cells. The differentiation of iPS cells into NK cells was performed according to the literature (Matsubara et al. Biochem Biophys Res Commun. 2019 Jul 12;515(1):1-8.). To evaluate the differentiation potential into NK cells, the cell surface expression of NK cell-specific proteins was measured by flow cytometry. NK cell markers were anti-CD56 antibody (clone B159, BD Biosciences, Catalogue No. 555518) and anti-CD16 antibody (clone 3G8, BD Biosciences, Catalogue No. 555407). Both target gene-expressing iPS cells were successfully differentiated into NK cells.

[0090] Test Example 1: Cell migration activity of CCR2B-expressing iNK cells The cell migration activity of iNK cells (Example 4) induced to differentiate from the CCR2B-expressing iPS cells obtained in Production Example 2 in a medium containing CCL2 recombinant protein was examined using a Boyden chamber. Each iNK cell was labeled with CFSE (5- or 6-(N-Succinimidyloxycarbonyl)fluorescein 3',6'-diacetate) and then cultured at a concentration of 5 x 10 5 The upper chamber was immersed in a medium containing 100 ng / mL of recombinant CCL2, and the upper chamber was cultured at 37°C under 5% CO2 for 4 hours. The number of iNK cells that migrated from the upper chamber to the lower chamber was then counted using a flow cytometer, using CFSE fluorescence as an indicator. The results are shown in Figure 1.

[0091] The number of cells that migrated into the lower chamber was significantly higher in CCR2B-expressing iNK cells than in control iNK cells differentiated from iPSCs transfected with an empty vector, demonstrating that CCR2B-expressing iNK cells have a high migratory activity toward CCL2.

[0092] Test Example 2: Cell migration activity of CCL19-expressing cells The cell migration activity of peripheral blood mononuclear cells (PBMCs) toward the culture supernatant of iNK cells (Example 5) induced to differentiate from CCL19-expressing iPS cells in Production Example 2 was examined using a Boyden chamber. PBMCs that had been thawed and recovered were labeled with CFSE, and then placed in the upper chamber at 4 × 10 5 The cells were seeded at 500 μL / well. The cells were immersed in the lower chamber containing the culture supernatant of CCL19-expressing iNK cells or control iNK cells differentiated from iPSCs transfected with an empty vector, and cultured at 37°C and 5% CO2 for 1.5 hours. PBMCs that had migrated into the lower chamber were then photographed using a fluorescence microscope using CFSE fluorescence as an indicator, and the number of cells was counted using a flow cytometer. The results are shown in Figure 2.

[0093] The culture supernatant of CCL19-expressing iNK cells showed higher chemotactic activity toward PBMCs than the culture supernatant of control iNK cells differentiated from iPS cells transfected with an empty vector. These results demonstrate that CCL19-expressing iNK cells have the ability to enhance the chemotactic activity toward PBMCs.

[0094] Test Example 3: Cytotoxic Activity of CD16-Expressing iNK Cells iNK cells (Example 6) induced to differentiate from the CD16-expressing iPS cells obtained in Production Example 2 were confirmed by flow cytometry to confirm the correct expression of CD16 on the cell surface (Figure 3, left). Next, the cytotoxic activity of these CD16-expressing iNK cells against EGFR-expressing A549 cells was examined using an LDH assay (Takara Bio LDH Cytotoxicity Detection Kit) in the presence of an anti-EGFR antibody (Cetuximab). Specifically, adherent A549 cells were co-cultured with iNK cells in the presence of an anti-EGFR antibody (Cetuximab) for 4 hours, and the amount of LDH released from A549 cells due to iNK cell-mediated cytotoxicity was measured using its enzyme activity as an indicator. The results are shown in Figure 3, right. Compared to control iNK cells induced to differentiate from empty vector-transfected iPS cells, the CD16-expressing iNK cells exhibited approximately two-fold higher cytotoxic activity (ADCC activity).

[0095] Test Example 4: Cytokine secretion activity of IL-15-expressing iNK cells iNK cells (Example 7) were differentiated from the IL-15-expressing iPS cells obtained in Production Example 2. 6 After 4 days of culture in the absence of IL-15, the number of viable cells was counted using a NucleoCounter NC-200 cell counter. iNK cells induced to differentiate from iPS cells transfected with an empty vector were used as a negative control. The results are shown in Figure 4.

[0096] Compared to control iNK cells, the number of IL-15-expressing iNK cells was more than twice as high, indicating that IL-15 expression enhances the proliferation and survival of iNK cells.

[0097] Test Example 5: IFNγ secretion activity of NKG2D-expressing iNK cells IFNγ production was investigated in various forms of NKG2D-expressing iNK cells obtained in Production Example 2. Each NKG2D-iNK cell (NKG2D-CAR iNK cells (Example 8), NKG2D / DAP10-coexpressing iNK cells (Example 9)) was co-cultured with A549 cells expressing an NKG2D ligand such as ULBP2 for 24 hours, and the culture supernatant was then collected, and the amount of IFNγ expression was confirmed by ELISA.

[0098] When co-cultured with A549 cells, all NKG2D-iNK cells showed significantly increased IFNγ production compared to control iNK cells differentiated from iPS cells transfected with an empty vector (Figure 5). These results demonstrate that all NKG2D-expressing iNK cells exhibit enhanced NKG2D-specific IFNγ production.

[0099] Test Example 6: Cell migration activity of CCR2B / CCL19 co-expressing iNK cells The cell migration activity of iNK cells (Example 12), which were induced to differentiate from the CCR2B / CCL19 co-expressing iPS cells obtained in Production Example 2, in a medium containing CCL2 recombinant protein was examined using an Incucyte Clearview 96-well plate for chemotaxis (Sartorius). This plate is also divided into an upper chamber and a lower chamber, and cell migration can be evaluated using the same principle as in the Boyden chamber.

[0100] Each iNK cell was labeled with DiO (3,3'-Dioctadecyloxacarbocyanine perchlorate) and then cultured at 1 × 10 4 Cells were seeded in the upper chamber and immersed in the lower chamber containing medium with or without 1 μg / mL recombinant CCL2. They were then cultured at 37°C under 5% CO2. The number of cells remaining in the upper chamber was quantified using DiO fluorescence every 2 hours for 30 hours. The results are shown in Figure 6.

[0101] In CCR2B / CCL19 co-expressing iNK cells, the addition of CCL2 to the lower chamber significantly reduced fluorescence in the upper chamber compared to when CCL2 was not added. This result indicates that CCR2B / CCL19 co-expressing iNK cells migrated from the upper chamber to the lower chamber in a CCL2-dependent manner. In contrast, control iNK cells differentiated from iPSCs transfected with an empty vector did not exhibit a CCL2-dependent reduction in fluorescence in the upper chamber. These results demonstrate that CCR2B / CCL19 co-expressing iNK cells exhibit a high migration ability in response to CCL2.

[0102] Test Example 7: Cell migration activity of CCR2B / CCL19 co-expressing cells The cell migration activity of dendritic cells (DCs) toward the culture supernatant of iNK cells (Example 12) that had been induced to differentiate from the CCR2B / CCL19 co-expressing iPS cells obtained in Production Example 2 was examined using an Incucyte Clearview 96-well plate for chemotaxis (Sartorius).

[0103] DCs were differentiated from CD14-positive cells (monocytes) isolated from peripheral blood mononuclear cells and labeled with DiO. 8.4 × 10 3 DCs were seeded onto the nuclei of the nuclei. The nuclei were then immersed in the lower chamber containing the culture supernatant of CCR2B / CCL19 co-expressing iNK cells or control iNK cells differentiated from iPSCs transfected with an empty vector, in the presence or absence of 10 μg / mL of an anti-CCL19 antibody (clone A15093C, BioLegend, Catalogue No. 612803). The cells were then cultured at 37°C and 5% CO2. DCs remaining in the upper chamber were quantified using DiO fluorescence at 2-hour intervals for 72 hours. The results are shown in Figure 7.

[0104] When the conditioned medium of CCR2B / CCL19 co-expressing iNK cells was placed in the lower chamber, a significant decrease in fluorescence was observed in the upper chamber compared to when the conditioned medium of control iNK cells was placed in the lower chamber. This decrease in fluorescence was also suppressed by the addition of anti-CCL19 antibody. This indicates that DC migrated to the conditioned medium of CCR2B / CCL19 co-expressing iNK cells in a CCL19-dependent manner. These results demonstrate that CCR2B / CCL19 co-expressing iNK cells have a high ability to attract DCs.

[0105] Test Example 8: Effects of IL2sp / IL-15, full-length IL-15Rα, and soluble IL-15Rα expression iNK cells induced to differentiate from IL2sp / IL-15-expressing iPS cells obtained in Production Example 2 (Example 13), iNK cells co-expressing IL2sp / IL-15 and full-length IL-15Rα (Example 10), iNK cells co-expressing IL2sp / IL-15 and soluble IL-15Rα (Example 11), and iNK cells induced to differentiate from iPSCs transfected with an empty vector (control) were cultured for 7 days in the absence of IL-15. On days 4 and 7 of culture, the number of viable cells was counted using a NucleoCounter NC-200 cell counter. The results are shown in Figure 8. The measured values ​​are expressed as a percentage of the number of cells at the time of seeding.

[0106] Compared with control iNK cells, significantly higher numbers of viable cells were observed in IL2sp / IL-15-expressing iNK cells, IL2sp / IL-15-coexpressing iNK cells with full-length IL-15Rα, and IL2sp / IL-15-coexpressing soluble IL-15Rα. Furthermore, more viable cells were measured in iNK cells coexpressing full-length IL-15Rα and soluble IL-15Rα than in iNK cells expressing IL2sp / IL15 alone. These results demonstrate that IL2sp / IL-15 expression enhances the proliferation and survival of iNK cells, and that this effect is further enhanced by coexpression of full-length or soluble IL-15Rα.

[0107] The modified pluripotent stem cells and modified NK cells of the present invention are useful as active ingredients and supply materials thereof for cellular immunotherapy, particularly cancer immunotherapy.

[0108] This application is based on patent application No. 2021-184197 filed in Japan (filing date: November 11, 2021), the contents of which are incorporated in their entirety herein.

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); Pluripotent stem cells expressing the above-mentioned NK cells, or NK cells or their precursor cells derived from the pluripotent stem cells.

2. (c) and / or (d) below: (c) an exogenous gene encoding interleukin 15 (IL-15) (d) an exogenous gene encoding CD16 The cell of claim 1 further expressing:

3. (e) below: (e) an exogenous gene encoding NKG2D or NKG2D-CAR The cell of claim 1 further expressing:

4. The cell of claim 3, wherein (e) is an NKG2D-CAR.

5. (e) is NKG2D, and (f) an exogenous gene encoding DAP10 The cell of claim 3, wherein the cell co-expresses

6. The cell according to claim 2, which expresses an exogenous gene encoding IL-15, and the IL-15 is secreted IL-15.

7. The cell according to claim 2, which expresses an exogenous gene encoding IL-15 and co-expresses an exogenous gene encoding IL-15 receptor alpha (IL-15Rα).

8. The cell according to claim 2, which expresses an exogenous gene encoding CD16, wherein the CD16 is a CD16 having a high affinity mutation or a non-cleavable mutation.

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

10. A pharmaceutical comprising the cells according to any one of claims 1 to 9.

11. The pharmaceutical composition according to claim 10, which is a therapeutic agent for cancer.

12. A method for producing genetically modified cells having enhanced homing function to cancer tissues and enhanced immunocompetent cell recruitment function, comprising the steps of: (a) and (b) (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 into pluripotent stem cells.

13. The method according to claim 12, further comprising inducing differentiation of the pluripotent stem cells into which the exogenous gene has been introduced into NK cells or their precursor cells.

14. (a) and (b) below: (a) an exogenous gene encoding CCR2B (b) containing an exogenous gene encoding CCL19; Constructs for manufacturing genetically modified cells.

15. The construct of claim 14 , wherein the exogenous gene is contained in a separate construct.

16. An expression cassette comprising a gene expression control region and the construct according to claim 14 or 15 under the control of said control region.

17. A vector comprising the expression cassette of claim 16.

18. The vector according to claim 17, wherein the expression cassette is flanked by a pair of transposon inverted repeat sequences.

19. The vector of claim 18 and a transposase expression vector, comprising the following (a) and (b): (a) an exogenous gene encoding CCR2B (b) an exogenous gene encoding CCL19 A kit for producing genetically modified cells that express the gene.

20. 20. The kit of claim 19, wherein the transposase is PiggyBac transposase.