Method for producing human professional antigen-presenting cells

Abstract

A method for producing professional antigen-presenting cells and professional antigen-presenting cells are provided. [Solution] We provide a method for producing professional antigen-presenting cells, which includes obtaining proliferative myeloid cells (pMCs) by expressing c-MYC or the like in myeloid cells (MCs) differentiated from pluripotent stem cells, and obtaining professional antigen-presenting cells (pAPCs) by expressing GM-CSF and / or M-CSF in the pMCs, as well as human professional antigen-presenting cells produced by this method.

Description

[Technical Field]

[0001] The present invention relates to a method for producing human professional antigen-presenting cells derived from pluripotent stem cells, human professional antigen-presenting cells derived from pluripotent stem cells produced by the method, and a method for inducing T cell proliferation using the human professional antigen-presenting cells. [Background technology]

[0002] Professional antigen-presenting cells are cells that present fragments of bacteria, virus-infected cells, cancer cells, and other substances that have invaded the body as antigens on their cell surface, activating T cells. Professional antigen-presenting cells are specialized cells that express not only human leukocyte antigen (HLA) class I molecules, which are present in normal nucleated cells, but also HLA class II molecules, and include macrophages, dendritic cells, B cells, and the like. Dendritic cells (hereinafter also referred to as DCs) are antigen-presenting cells that have potent T cell-stimulating activity and play a role in eliciting immune responses to foreign antigens. DC vaccine therapy, in which cancer-reactive T cells in the body are activated by loading DCs with "cancer antigens" and administering them to the body, is considered a promising "cancer immunotherapy" (Non-Patent Documents 1 and 2).

[0003] Current DC therapy has many problems, including: (1) the need for apheresis from patients to obtain DC precursor cells; (2) the efficiency of DC induction varies between patients, making it difficult to obtain sufficient DC function and cell numbers for treatment; and (3) the high cost of personalized medicine using autologous cells.

[0004] To achieve clinical efficacy using DCs as a cell preparation, it is necessary to obtain the necessary cells, which cannot be obtained from blood collection10. 8 ~10 9 Because the number of DCs present in in vivo tissues is finite and their expansion by ex vivo culture is difficult, it is difficult to extract and prepare large quantities of DCs from in vivo tissues.

[0005] Techniques for inducing DC differentiation from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) (Non-Patent Documents 3-7) enable the preparation of DCs from pluripotent stem cells, which are an unlimited cell source. However, the process of differentiating ESCs and iPSCs into DCs via hematopoietic differentiation is complicated and requires more than a month of production time and expense, preventing the practical application of DC vaccine methods using this method. Human iPSC-derived myeloid blood cell lines (iPS-MLs), reported as prior art, are produced through two main steps: differentiation induction and gene transfer (Patent Document 1). Previously reported methods for producing iPS-MLs have low induction efficiency and reproducibility, making it impossible to achieve a stable supply as a cell preparation. iPS-MLs cannot function as DCs, and some form of differentiation induction, such as culturing in the presence of cytokines, is required to enable them to function as DCs, posing a challenge to their practical application.

[0006] There have also been reports on the production of human myeloid blood cells capable of proliferation in vitro (Patent Document 1) and a method for producing human myeloid blood cells with suppressed tumorigenicity (Patent Document 2). However, none of these prior art documents discloses or suggests a method for producing human myeloid blood cells with antigen-presenting ability, or the existence or characteristics of cells with antigen-presenting ability. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5861191 [Patent Document 2] Japanese Patent Application Publication No. 2018-171005 [Non-patent literature]

[0008] [Non-Patent Document 1] Immunity 39: 38-48, 2013. [Non-patent document 2] Nat Rev Cancer 17, 209-222, 2017. [Non-patent document 3] Blood 101: 3501-8, 2003. [Non-patent document 4] J Immunol 172: 776-86, 2004. [Non-Patent Document 5] J Immunol 174: 1888-97, 2005. [Non-patent document 6] J Immunol 178: 918-25, 2007. [Non-Patent Document 7] Stem Cells 25: 2720-29, 2007. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for producing human professional antigen-presenting cells derived from pluripotent stem cells that have the ability to proliferate in vitro, allowing for a stable supply as a cell preparation, and that have antigen-presenting ability comparable to that of living body-derived DCs, and the human professional antigen-presenting cells produced by this method.A further object of the present invention is to provide a method for promoting the proliferation of antigen-specific or non-specific T cells using the human professional antigen-presenting cells of the present invention, and the T cells whose proliferation is promoted and / or produced by this method.Further object of the present invention is to provide a pharmaceutical composition for promoting T cell proliferation, which comprises the professional antigen-presenting cells derived from pluripotent stem cells of the present invention, and an anticancer agent comprising antigen-specific or non-specific T cells whose proliferation is promoted and / or produced using the human professional antigen-presenting cells of the present invention. [Means for solving the problem]

[0010] The present invention provides a method for producing professional antigen-presenting cells derived from pluripotent stem cells, and the like.

[0011] That is, the present invention provides: expressing c-MYC, BMI1, and MDM2 in myeloid cells (MCs) to obtain proliferative myeloid cells (pMCs); and expressing GM-CSF and / or M-CSF in the pMCs to obtain professional antigen-presenting cells (pAPCs), A method for producing professional antigen-presenting cells is provided.

[0012] In the method for producing professional antigen-presenting cells of the present invention (hereinafter referred to as "the production method of the present invention"), the myeloid cells may be myeloid cells differentiated from pluripotent stem cells.

[0013] In the production method of the present invention, the pluripotent stem cells may be induced pluripotent stem cells or embryonic stem cells.

[0014] In the production method of the present invention, the c-MYC, BMI1, and MDM2 may be genes introduced into myeloid cells.

[0015] In the production method of the present invention, the GM-CSF and / or M-CSF may be gene-transfected into pMC.

[0016] In the production method of the present invention, when the myeloid cells are myeloid cells (MCs) differentiated from pluripotent stem cells, (i) layering embryoid bodies (EBs) induced from the pluripotent stem cells on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF, and TPO; or (ii) The method may comprise the steps of: culturing embryoid bodies (EBs) induced from the pluripotent stem cells in a monolayer culture without feeder cells in a medium containing BMP-4, VEGF, and SCF; and then further culturing the EBs in a medium containing VEGF, TPO, and GM-CSF, thereby differentiating the pluripotent stem cells into myeloid cells (MCs).

[0017] In the production method of the present invention, when the embryoid bodies (EBs) are formed from pluripotent stem cells, the production method further comprises: (i) seeding pluripotent stem cells into a cell cluster culture vessel to form embryoid bodies (EBs); and (ii) The method may include forming embryoid bodies (EBs) from pluripotent stem cells by a process in which the container has fine spheroid wells at the bottom and there is no flat surface between adjacent wells.

[0018] The manufacturing method of the present invention comprises: (i) seeding pluripotent stem cells into a cell cluster formation culture vessel having fine spheroid wells at the bottom and no flat surface between adjacent spheroid wells to form embryoid bodies (EBs); (ii) The following step (ii)-1 or (ii)-2: (ii)-1. Layering the embryoid bodies on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF, and TPO, or (ii)-2 culturing the embryoid bodies in a monolayer culture without feeder cells in a medium containing BMP-4, VEGF, and SCF, and then culturing them in a medium containing VEGF, TPO, and GM-CSF; Obtaining myeloid cells (MCs) from the embryoid bodies (EBs) by (iii) expressing c-MYC, BMI1, and MDM2 in the myeloid cells (MCs) obtained in (ii) to obtain proliferative myeloid cells (pMCs); and (iv) expressing GM-CSF and / or M-CSF in the proliferative myeloid cells (pMCs) obtained in (iii) to obtain professional antigen-presenting cells (pAPCs).

[0019] In the production method of the present invention including the steps (i) to (iv), c-MYC, BMI1 and MDM2 may be transfected into myeloid cells (MCs), and GM-CSF and / or M-CSF may be transfected into proliferating myeloid cells (pMCs).

[0020] In the production method of the present invention, which includes forming embryoid bodies (EBs) from pluripotent stem cells, the size of the embryoid bodies (EBs) may be 50 to 200 μm.

[0021] The production method of the present invention may further comprise irradiating the professional antigen-presenting cells (pAPCs) with radiation.

[0022] The present invention provides professional antigen-presenting cells (pAPCs) produced by any of the production methods of the present invention.

[0023] The professional antigen-presenting cells (pAPCs) of the present invention may be professional antigen-presenting cells (pAPCs) that, when administered to a living body after radiation irradiation, disappear from the living body at least 3 to 4 days after administration.

[0024] The professional antigen-presenting cells (pAPCs) of the present invention may be professional antigen-presenting cells (pAPCs) that have low expression of CD11b / c and express CD74.

[0025] The present invention provides professional antigen-presenting cells (pAPCs) derived from pluripotent stem cells that have low expression of CD11b / c and express CD74.

[0026] Professional antigen-presenting cells (pAPCs) produced by any of the production methods of the present invention, or professional antigen-presenting cells (pAPCs) derived from pluripotent stem cells of the present invention, may further express CD33.

[0027] In the professional antigen-presenting cells (pAPCs) derived from pluripotent stem cells of the present invention, the pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells.

[0028] The pluripotent stem cell-derived professional antigen-presenting cells (pAPCs) of the present invention may express exogenous GM-CSF and / or M-CSF.

[0029] In the pluripotent stem cell-derived professional antigen-presenting cells (pAPCs) of the present invention that express exogenous GM-CSF and / or M-CSF, the GM-CSF and / or M-CSF may be genetically introduced.

[0030] Professional antigen-presenting cells (pAPCs) produced by the production method of the present invention, or professional antigen-presenting cells (pAPCs) derived from the pluripotent stem cells of the present invention that have low expression of CD11b / c and express CD74, may have the ability to self-proliferate and exhibit apoptosis resistance.

[0031] The present invention provides a pharmaceutical composition for inducing and / or promoting the proliferation of CD8-expressing T cells, comprising any of the professional antigen-presenting cells of the present invention.

[0032] The present invention provides methods for promoting the proliferation of antigen-nonspecific or antigen-specific CD8-expressing T cells in vivo and in vitro by co-culturing CD8-expressing naive T cells in vitro with any of the professional antigen-presenting cells of the present invention or GM-CSF.

[0033] In the method of the present invention for promoting the proliferation of CD8-expressing T cells, the professional antigen-presenting cells may be professional antigen-presenting cells loaded with a desired antigen.

[0034] The method of the present invention for promoting proliferation of CD8-expressing T cells may further comprise ex vivo irradiation of the antigen-loaded professional antigen-presenting cells.

[0035] In the method of the present invention for promoting the proliferation of CD8-expressing T cells, when the irradiated professional antigen-presenting cells are administered to a living body after ex vivo irradiation, they may disappear in the living body within at least 3 to 4 days.

[0036] In the method of the present invention for promoting the proliferation of CD8-expressing T cells, the CD8-expressing T cells may be CD8-expressing T cells specific to an antigen loaded onto professional antigen-presenting cells.

[0037] The present invention provides CD8-expressing T cells whose proliferation is promoted by the method of promoting the proliferation of CD8-expressing T cells of the present invention.

[0038] The present invention provides a method for producing antigen-nonspecific or -specific CD8-expressing T cells using the method of the present invention for promoting the proliferation of CD8-expressing T cells.

[0039] The present invention provides antigen-nonspecific or -specific CD8-expressing T cells produced by the method of the present invention for producing antigen-nonspecific or -specific CD8-expressing T cells.

[0040] The present invention provides a method for producing antigen-specific CD8-expressing T cells for administration to the body. The method for producing antigen-specific CD8-expressing T cells for administration to the body of the present invention includes loading professional antigen-presenting cells (pAPCs) of the present invention with an antigen, irradiating the antigen-loaded professional antigen-presenting cells, and co-culturing the irradiated professional antigen-presenting cells with naive CD8-expressing T cells ex vivo to promote proliferation of the antigen-specific CD8-expressing T cells. The professional antigen-presenting cells (pAPCs) may be capable of self-proliferation and may be resistant to apoptosis.

[0041] The present invention provides antigen-specific CD8-expressing T cells for administration to the body, which are produced by the method of the present invention for producing antigen-specific CD8-expressing T cells for administration to the body.

[0042] In the case of antigen-specific CD8-expressing T cells for administration to the body, which are produced by the method of the present invention for producing antigen-specific CD8-expressing T cells for administration to the body, when the T cells are administered to a living body after irradiation, the professional antigen-presenting cells derived from the irradiated pluripotent stem cells may disappear at least 3 to 4 days after administration to the living body.

[0043] The present invention provides an anticancer agent. The anticancer agent of the present invention comprises CD8-expressing T cells whose proliferation is promoted by the method of the present invention for promoting the proliferation of CD8-expressing T cells, or antigen-specific CD8-expressing T cells for administration to the body that are produced by the method of the present invention for producing antigen-specific CD8-expressing T cells for administration to the body, and the antigen in the method of the present invention for promoting the proliferation of CD8-expressing T cells and the method of the present invention for producing antigen-specific CD8-expressing T cells for administration to the body is a cancer-specific antigen. [Effects of the Invention]

[0044] The pluripotent stem cell-derived professional antigen-presenting cells provided by the present invention are cells that have acquired the ability to self-proliferate. Once constructed, the pluripotent stem cell-derived professional antigen-presenting cells of the present invention do not require redifferentiation or regeneration from pluripotent stem cells into myeloid cells, and have an excellent antigen-presenting ability comparable to that of DCs, which has not been reported until now. [Brief explanation of the drawings]

[0045] [Figure 1]This line graph shows the results of an MTT assay that examined the effect of medium containing GM-CSF and / or M-CSF (αMEM containing 20% ​​FBS) on the proliferation rate of iPSC-derived proliferative myeloid cells (pMCs) cultured with or without feeder cells. The vertical axis represents the concentration of formazan metabolized from the MTT reagent (assessed as absorbance at 595 nm), and the horizontal axis represents the number of days in culture. [Figure 2] Histogram showing the results of flow cytometric analysis of CD11b, CD11c, and CD33 surface antigens in iPSC-derived pMCs cultured with or without feeder cells. [Figure 3] Histograms showing the results of flow cytometric analysis of GM-CSF-Venus surface antigens in mouse pMCs and ipAPC-GM (upper left), M-CSF-dCD19 surface antigens in human pMCs and ipAPC-M (lower left), GM-CSF-dCD19 surface antigens in human pMCs and ipAPC-GM (upper right), and GM-CSF-M-CSF-CD19 surface antigens in human pMCs and ipAPC-GMM (lower right). [Figure 4] Bar graphs showing the results of quantification by ELISA of GM-CSF production in the culture supernatant of mouse pMCs and ipAPC-GM (left), and M-CSF production (center) and GM-CSF production (right) in the culture supernatant of human pMCs, ipAPC-M, ipAPC-GM, and ipAPC-GMM. [Figure 5] Line graph showing the results of an MTT assay examining the effect of medium (αMEM containing 20% ​​FBS) containing GM-CSF and / or M-CSF on the proliferation rate of mouse ipAPC-GM (top) and human ipAPC-GMM (bottom). Measurement results are the mean of n=3 replicates, and error bars indicate the standard deviation. [Figure 6] Histogram showing the results of flow cytometric analysis of human ipAPC-GM, ipAPC-M, and ipAPC-GMM for HLA-ABC, HLA-DR, CD40, CD80, CD83, CD86, CCR7, CD74, CD11b, CD11c, and CD33 surface antigens. [Figure 7] Histogram showing the results of flow cytometric analysis of DCs for HLA-ABC, HLA-DR, CD40, CD80, CD83, CD86, CCR7, CD74, CD11b, CD11c, and CD33 surface antigens. [Figure 8] Plots of the profiles of mouse pMCs, mouse ipAPC-GM, and GM-CSF-supplemented mouse pMCs (C57BL / 6 (left) and 129 / Sv (right)) against the two most highly contributing axes (PC1 and PC2) obtained by multivariate principal component analysis (PCA) after whole-transcriptome analysis (RNA-seq analysis) of GM-CSF-supplemented mouse pMCs. [Figure 9] FIG. 1 shows the results of gene set enrichment analysis (GSEA) of mouse ipAPC-GM relative to mouse pMC and GM-CSF-added mouse pMC. [Figure 10A] Cytograms showing the results of Annexin V and 7-AAD analysis by flow cytometer of mouse ipAPC-GM (lower panel) and mouse pMC (upper panel) cultured in 20% FBS-containing αMEM without exogenous cytokines on the first day of culture (Day 0, left) and on Day 4 of culture (Day 4, right). In Figure 10A, the vertical axis represents the fluorescence intensity of 7-AAD, and the horizontal axis represents the fluorescence intensity of Annexin V. [Figure 10B]The line graphs in Figure 10B (lower left, lower right, and upper right) show the results of flow cytometric analysis of the percentage of cells gated on each fraction out of the total cells analyzed by flow cytometry for Annexin V-negative and 7-AAD-negative, Annexin V-positive and 7-AAD-negative, and Annexin V-positive and 7-AAD-positive fractions, respectively, as shown in Figure 10A. The percentages were determined by sampling daily from the initiation of culture through day 4 of culture, and the percentages were determined by flow cytometry for the Annexin V-negative and 7-AAD-negative fractions, Annexin V-positive and 7-AAD-negative fractions, Annexin V-positive and 7-AAD-positive fractions, as shown in Figure 10A. The vertical axis of the line graphs in Figure 10B (lower left, lower right, and upper right) represents the percentage of cells gated on each fraction out of the total cells analyzed by flow cytometry for mouse ipAPC-GM or mouse pMC on each sampling day. The horizontal axis represents the sampling days from the initiation of culture through day 4 of culture. [Figure 10C] Similar cytograms (Day 0 and Day 3) of human ipAPC-GMM (1st row), ipAPC-M (2nd row), ipAPC-GM (3rd row), and pMC (4th row). [Figure 10D] Line graphs showing the percentages of live cells (lower left), early apoptotic cells (lower right), and late apoptotic cells (upper right). [Figure 11] Left: Plot of the results of quantification of the mixed allo-lymphocyte reaction (allo-MLR) by scintillation counting of [3H]-thymidine incorporation when C57BL / 6 mouse ipAPC-GM and pMC were co-cultured at various ratios with unstimulated T cells prepared from BALB / c mice. Right: Plot of the results of quantification of the allo-MLR when human ipAPC-GMM, ipAPC-GM, ipAPC-M, and pMC were co-cultured in the same manner as alloreactive T cells. The vertical axis represents the scintillation counting results of [3H]-thymidine incorporation (unit: cpm), and the horizontal axis represents the ratio of ipAPC-GMM, ipAPC-GM, ipAPC-M, or pMC cell counts to unstimulated T cells. [Figure 12A]Cytogram (left) showing flow cytometry evaluation of human pMC, ipAPC-GM, ip-APC-M, ip-APC-GM, and DC loaded with DQ-OVA protein, and bar graph (right) showing the percentage of cells that took up and intracellularly digested DQ-OVA. [Figure 12B] Mouse ipAPC-GM were dose-dependently loaded with either the OVA peptide OVA257-264 or OVA protein, and then irradiated to stop proliferation of the ipAPC-GM. The ipAPC-GM were then co-cultured with naive CD8+ T cells, OT-1, that possess the OVA-specific T cell receptor (TCR). The in vitro proliferation of antigen-specific CD8+ T cells was quantified by scintillation counting of [3H]-thymidine incorporation. The vertical axis represents the scintillation counting results of [3H]-thymidine incorporation (unit: 104 cpm), and the horizontal axis represents the concentration of the OVA peptide OVA257-264 or OVA protein. [Figure 13] Bar graph comparing the OT-1 proliferation-inducing activity of mouse ipAPC-GM and mouse pMC cells loaded with 10 μM OVA peptide (left) or 100 μg / mL OVA protein (right). The vertical axis represents the scintillation count results of [H]-thymidine incorporation (units: 104 cpm (left) and 103 cpm (right)), and the horizontal axis represents cells co-cultured with OT-1. An asterisk (*) indicates p<0.05 by two-tailed Student's t test. [Figure 14]This line graph compares the inhibitory effect of mouse ipAPC-GM and mouse pMC loaded with OVA peptide with that of mouse ipAPC-GM and mouse pMC not loaded with peptide on subcutaneously implanted tumors containing OVA-expressing cancer cells. The vertical axis represents the volume of the implanted tumor (unit: cm), and the horizontal axis represents the number of days after tumor implantation. Asterisks (*) indicate p-values ​​from one-way ANOVA with Tukey's test: *p<0.05, **p<0.01, ***p<0.001. Animals not administered mouse ipAPC-GM or mouse pMC showed the weakest inhibitory effect, while mouse ipAPC-GM and mouse pMC not loaded with peptide showed similar inhibitory effects. Mouse ipAPC-GM loaded with OVA peptide showed the strongest inhibitory effect, followed by mouse pMC loaded with OVA peptide. [Figure 15] Survival curves of tumor-bearing mice treated with each cell type in Figure 15. Asterisks (*) indicate p-values ​​based on log-rank tests: *p<0.05, **p<0.01, ***p<0.001. [Figure 16] Photographs of stained culture dishes show the results of an ELISPOT assay to examine the frequency of OVA peptide antigen-specific T cells in peripheral blood mononuclear cells collected from mice 56 days after tumor inoculation, along with a bar graph summarizing the results. The upper composite photograph shows the frequency of interferon-γ-producing antigen-specific T cells in ELISPOT assays using mouse ipAPC-GM (right) stimulated with OVA peptide (bottom) or control SIY peptide (top) and naive T cells (left). The lower bar graph quantitatively illustrates the results of the upper composite photograph, showing the frequency of interferon-γ-producing antigen-specific T cells induced by mouse ipAPC-GM or naive T cells stimulated with OVA peptide (gray) or control SIY peptide (white). The vertical axis of the graph represents the number of spots reactive with interferon-γ (unit: 1 x 102). [Figure 17]Bar graph showing proliferation of naive OT-1 CD8+ T cells in medium containing ipAPC-GM or pMC culture supernatant. The vertical axis represents scintillation counting of [H]-thymidine incorporation into T cells (unit: 103 cpm). Asterisks (**) indicate p<0.01 by two-tailed Student's t test. [Figure 18] Bar graph showing the effect of adding anti-GM-CSF neutralizing antibody (αGM-CSF Ab) or control antibody (Control Ab) to medium containing ipAPC-GM culture supernatant on the proliferation of naive OT-1 CD8+ T cells. The vertical axis represents the scintillation count results of [3H]-thymidine incorporation into T cells (unit: 103 cpm). [Figure 19] Bar graph showing the effect of co-culture with ipAPC-GM or pMC on T cell proliferation. The vertical axis represents scintillation counting of [H]-thymidine incorporation into T cells (unit: 10 cpm). Asterisks (**) indicate p<0.01 by two-tailed Student's t test. [Figure 20] Histograms showing the results of flow cytometric analysis of T cell receptor (TCR) Vβ frequencies in naive CD8+ T cells (left), after co-culture of ipAPC-GM with naive CD8+ T cells (center), and after co-culture of splenocytes with naive CD8+ T cells (right). The horizontal axis represents each TCR Vβ repertoire, and the vertical axis represents the frequency of use of each repertoire. [Figure 21] Phase-contrast micrographs comparing the effects of different intensities of radiation on the cell morphology of ipAPC-GM (right) or pMC (left). The absorbed doses of the cells in the top to bottom rows are 0 Gy, 65 Gy, 85 Gy, and 100 Gy, respectively. The magnification of the photographs is 400x. The scale bar indicates 50 μm. [Figure 22]Bar graph comparing the effects of different intensities of radiation on cell proliferation of mouse ipAPC-GM (center) or mouse pMC (left), human ipAPC-GMM, ipAPC-GM, ipAPC-M, and pMC (right). The horizontal axis represents the absorbed dose of radiation (unit: Gy), and the vertical axis represents the scintillation count results of [3H]-thymidine incorporation into ipAPC-GM (right) or pMC (left) after radiation exposure under each condition (unit: 102 cpm (left), 104 cpm (center), cpm (right)). [Figure 23] The top panel of Figure 23 shows cytograms of Annexin V and 7-AAD analysis by flow cytometry of mouse ipAPC-GM (bottom panel) and mouse pMC (top panel) before and 24 hours after 0 or 85 Gy irradiation. The bottom left and bottom right panels of Figure 23 show bar graphs similar to those in the top panel. The Annexin V-positive and 7-AAD-negative fractions and the Annexin V-positive and 7-AAD-positive fractions were sampled before and 24 hours after 0 or 85 Gy irradiation, respectively. These fractions were designated as early apoptotic cells (bottom left) and late apoptotic cells (bottom right). The bar graphs show the percentages of cells gated on each fraction compared to the total cells. The vertical axis of the cytogram in Figure 23 represents the fluorescence intensity of 7-AAD, and the horizontal axis represents the fluorescence intensity of Annexin V. The vertical axes of the bar graphs at the bottom left and bottom right of Figure 23 represent the percentage of cells gated in each fraction out of the total cells analyzed by flow cytometry on each sampling day for mouse ipAPC-GM or mouse pMC, and the horizontal axes represent samples taken before irradiation and 24 hours after irradiation with 0 Gy or 85 Gy. [Figure 24] This is a line graph showing the results of in vitro cell proliferation of mouse ipAPC-GM or mouse pMC irradiated with 0 or 85 Gy, assessed by MTT assay for 3 consecutive days after irradiation. The vertical axis represents the concentration of formazan metabolized from the MTT reagent (measured by absorbance at 595 nm), and the horizontal axis represents the number of days of culture. [Figure 25]Luciferase-expressing ipAPC-GM (ipAPC-GM-Luc) or pMC (pMC-Luc) irradiated with 85 Gy was subcutaneously administered to mice of the same strain. Viable cells were measured daily from the day of administration (Day 0) through Day 4 (Day 4) by bioluminescence. Images (top) of all mice were in vivo imaged, and a line graph (bottom) shows the time course of the total luciferase luminescence flux. To the right of the top image is a scale showing luciferase luminescence intensity in chromatic colors, with the minimum luminescence intensity at 5.46 x e5 and the maximum at 4.31 x e6 (units: p / sec / cm2 / sr). The vertical axis of the line graph (bottom) represents the total luciferase luminescence flux (units: 107 p / s), and the horizontal axis represents the number of days after administration. [Figure 26] ipAPC-GM, whose proliferation was stopped by irradiation, were loaded with OVA peptide and administered intraperitoneally. MO4 cancer cells were then implanted subcutaneously, and tumor diameter was evaluated every 3 to 4 days. This line graph shows the progression of tumor volume over time. The vertical axis represents tumor volume (unit: 103 mm3), and the horizontal axis represents the number of days after tumor implantation. The tumor-suppressing effect was weakest when no antigen-presenting cells were administered, the second weakest in the group administered pMCs after 85 Gy irradiation, and the tumor-suppressing effect of ipAPC-GM after 85 Gy irradiation was the highest, the same as unirradiated bone marrow-derived dendritic cells (BM-DCs). [Figure 27] Figure 26 shows the survival curves of tumor-bearing mice treated with each cell type. [Figure 28]Photographs of stained culture dishes show the results of an ELISPOT assay to examine the frequency of OVA peptide antigen-specific T cells in peripheral blood mononuclear cells collected from mice on day 56 after tumor inoculation, along with a bar graph summarizing the results. The upper composite photograph shows the frequency of interferon-γ-producing antigen-specific T cells measured by ELISPOT assay in mouse ipAPC-GM cells (ipAPC-GM 85 Gy) loaded with OVA peptide (lower panel) or control SIY peptide (upper panel) and then irradiated with 85 Gy, mouse pMC cells (pMC 85 Gy) loaded with OVA peptide (lower panel) or control SIY peptide (upper panel) and then irradiated with 85 Gy, bone marrow dendritic cells (BM-DCs, far right) loaded with OVA peptide (lower panel) or control SIY peptide (upper panel), and naive T cells (far left). The bar graph below quantitatively shows the results of the combined photographs above, showing the frequency of interferon-γ-producing antigen-specific T cells induced by irradiated mouse ipAPC-GM and pMC loaded with OVA peptide (gray) or control SIY peptide (white), bone marrow dendritic cells loaded with OVA peptide (gray) or control SIY peptide (white), and naive T cells. The vertical axis of the graph represents the number of spots (unit: 1 × 102) that react with interferon-γ. [Figure 29] A line graph shows the time course of total luciferase luminescence flux obtained by in vivo imaging of all mice from day 0 to day 7 after subcutaneous injection of luciferase-expressing ipAPC-GM (ipAPC-GM-HSV-TK-Luc or ipAPC-GM-iCasp9-Luc) or luciferase-expressing ipAPC-GM (ipAPC-GM-Luc) transfected with the suicide gene HSV-TK or iCasp9 into the same strain of mice. Viable cells were measured by biochemiluminescence from day 0 to day 7. The graph also shows the results of intraperitoneal injection of the suicide gene inducers GCV, AP1903, or saline (medium) without inducer for five consecutive days from day 0 to day 4. * indicates p<0.05 by two-tailed Student's t-test, and ** indicates p<0.01. [Figure 30]Luciferase-expressing ipAPC-GM (ipAPC-GM-iCasp9-Luc) transfected with the suicide gene iCasp9 was subcutaneously administered to mice of the same strain. Viable cells were measured by biochemiluminescence from the day of administration (Day 0) to Day 7 (Day 7). The line graph (left) shows the time course of the total luciferase luminescence flux obtained by in vivo imaging of all mice. The signal intensity on Day 0 was set as 100%, and the bar graph (right) shows the signal reduction rate on each measurement day. For five consecutive days, from Day 0 to Day 4 (Day 4), the mice were intraperitoneally administered with the iCasp9 inducer AP1903, the Survivin inhibitor YM-155, or saline (medium) without any inducer. * indicates p<0.05 by two-tailed Student's t-test. [Figure 31] Mice were implanted subcutaneously with tumor-forming MO4 cancer cells, which are insensitive to immune checkpoint inhibitors (ICIs). Tumors were formed in the mice, and OVA peptide-loaded ipAPC-GM, ICI (anti-CTLA-4 antibody and anti-PD-L1 antibody), or a combination of ipAPC-GM and ICI were administered intraperitoneally. Tumor size was assessed every 3–4 days. The left graph shows the progression of tumor volume over time, and the right graph shows the survival curves of individual tumor-bearing mice. The vertical axis of the left graph represents tumor volume (unit: 103 mm3), and the horizontal axis represents the number of days after tumor implantation. The vertical axis of the survival curve (right) represents the mouse survival rate, and the horizontal axis represents the number of days after tumor implantation. One-way ANOVA with Tukey's test indicates *p<0.05, **p<0.01, and ***p<0.001. The tumor-suppressing effect was weakest when no ICI or antigen-presenting cells were administered, the group administered ICI had the second weakest tumor-suppressing effect, and the combination of ipAPC-GM and ipAPC-GM with ICI after 85 Gy irradiation had the highest tumor-suppressing effect. [Figure 32]Mice were subcutaneously implanted with ICI-sensitive cancer cell lines MC38 to form tumors. ipAPC-GM loaded with mAdpgk or wtAdpgk peptide as a cancer antigen, or ICI (anti-CTLA-4 antibody and anti-PD-L1 antibody), or a combination of ipAPC-GM and ICI were intraperitoneally administered. Tumor size was assessed every 3–4 days. The graph (left) shows the progression of tumor volume over time, and the graph (right) shows the survival curves of individual tumor-bearing mice. The vertical axis of the graph (left) represents tumor volume (unit: 103 mm3), and the horizontal axis represents the number of days after tumor implantation. The vertical axis of the survival curve (right) represents the mouse survival rate, and the horizontal axis represents the number of days after tumor implantation. One-way ANOVA with Tukey's test indicates *p<0.05, **p<0.01, and ***p<0.001. Administration of no ICI or antigen-presenting cells showed the weakest tumor-suppressing effect, while administration of wtAdpgk peptide-loaded ipAPC-GM showed the second weakest tumor-suppressing effect. Compared with wtAdpgk peptide-loaded ipAPC-GM, the mAdpgk peptide-loaded ipAPC-GM treatment group showed a tendency toward suppression of tumor size and significantly prolonged mouse survival, although the difference was not significant. Compared with ICI alone, there was no significant difference in the time course of tumor size or mouse survival with mAdpgk peptide-loaded ipAPC-GM. The combination of ICI and mAdpgk peptide-loaded ipAPC-GM significantly suppressed tumor size and prolonged mouse survival compared with ICI alone, although there was no significant difference. [Figure 33] Cytogram (left) shows the results of flow cytometry analysis of Gr-1 and CD11b surface antigens on CD45-positive immune cells in tumors after treatment of tumor-bearing mice implanted with MO4 cancer cells with ipAPC-GM alone or in combination with an immune checkpoint inhibitor (ICI). The gate numbers indicate the percentage of myeloid-derived immunosuppressive cells (MDSCs). The right panel shows a bar graph showing the percentage of MDSCs calculated from the results of flow cytometry analysis. ** indicates p<0.01 by two-tailed Student's t-test, and *** indicates p<0.001. [Figure 34]Cytogram (left) shows the results of flow cytometry analysis of GzmB and Perforin surface antigens in CD8+ T cells within tumors after treatment of tumor-bearing mice implanted with MO4 cancer cells with ipAPC-GM alone or in combination with an immune checkpoint inhibitor (ICI). The gate numbers indicate the percentage of measured CD8+ T cells. The right is a bar graph showing the percentage of GzmB- and Perforin-positive CD8+ T cells calculated from the results of flow cytometry analysis. ** indicates p<0.01 by two-tailed Student's t-test, and *** indicates p<0.001. [Figure 35] Cytogram (top) showing the results of CD34 and CD43 or CD11b and CD11c surface antigen analysis of differentiated cells derived from human iPS cells prepared by the feeder-free differentiation induction method (Comparative Example 1) described in Example 3(4) and a non-patent document (Gene Therapy 2011 18, 874-883). Table (bottom) showing the frequency of CD34-positive cells on day 18 of culture and the frequency of CD11b-positive cells on day 25 of culture. ND stands for Not Detected. DETAILED DESCRIPTION OF THE INVENTION

[0046] Hereinafter, embodiments of the present invention will be described in detail.

[0047] In one embodiment, the present invention provides a method for producing a myeloid cell (pMC) by expressing c-MYC, BMI1, and MDM2 in a myeloid cell (MC), and a method for producing a professional antigen-presenting cell (pAPC) by expressing GM-CSF and / or M-CSF in the pMC. The present invention relates to a method for producing professional antigen-presenting cells.

[0048] This method enabled the first ex vivo production of professional antigen-presenting cells (pAPCs) with antigen-presenting ability from myeloid cells.

[0049] In the present invention, myeloid cells are a collective term for leukocytes differentiated from stem cells, including monocytes, macrophages, and dendritic cells, as well as progenitor cells that differentiate into these cells. Preferably, myeloid cells are differentiated from pluripotent stem cells. Hereinafter, myeloid cells are also referred to as MCs.

[0050] In the present invention, the term "stem cell" refers to a cell that maintains the ability to differentiate into cells having a specific function even after undergoing cell division at the same level as before cell division.

[0051] In the present invention, pluripotent stem cells refer to stem cells that can be cultured in vitro while maintaining an undifferentiated state and that have the ability (pluripotency) to differentiate into all cells that constitute the living body except for the placenta (tissues derived from the three germ layers (ectoderm, mesoderm, and endoderm)). Embryonic stem cells (ES cells) isolated from embryos before the formation of the three germ layers after fertilization, and embryos and fetuses from the organogenesis stage or later, including primordial germ cells, are also included in the term "pluripotent stem cells." Preferably, the pluripotent stem cells in the present invention are induced pluripotent stem cells.

[0052] In the present invention, "induced pluripotent stem cells" refer to cells in which pluripotency has been induced by directly reprogramming somatic cells that have once lost pluripotency through the expression of several genes, such as Oct3 / 4, Sox2, Klf4, and Myc (hereinafter also referred to as iPSCs or iPS cells). These "induced pluripotent stem cells" are also included in the "pluripotent stem cells." Pluripotent stem cells can be produced by known methods. Examples of production methods include those described in Cell, 2007, 131(5), pp. 861-872 and Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)) for human induced pluripotent stem cells, and in Cell, 2006, 126(4), pp. 663-676 for mouse induced pluripotent stem cells. Pluripotent stem cells can also be obtained from animals that have undergone embryonic manipulation techniques, including cloned embryos, transgenic animals, and genome-edited mutant animals. Pluripotent stem cells are known to be capable of differentiating into various cell types, including myeloid cells. Professional antigen-presenting cells (pAPCs) created from induced pluripotent stem cells are called iPSC-derived professional antigen-presenting cells (ipAPCs).

[0053] Human or mouse iPSCs can be maintained and expanded using the regenerative medicine medium StemFit (Ajinomoto). Laminin 511 may be added at this time. Furthermore, to reduce damage to iPSCs caused by cell dissociation during subculture, a lock inhibitor, such as Y-27632, may be added at approximately 10 μM.

[0054] Pluripotent stem cells are available from designated institutions or commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Mouse embryonic stem cells, EB5 cells, are available from RIKEN, and the D3 line is available from ATCC.

[0055] Pluripotent stem cells can be maintained in culture by known methods. For example, human pluripotent stem cells can be maintained by culturing them in a medium supplemented with KnockOut™ Serum Replacement (Invitrogen). Mouse pluripotent stem cells can be maintained by culturing them in a feeder-free medium supplemented with fetal bovine serum (FBS) and leukemia inhibitory factor (LIF).

[0056] In the present invention, proliferative myeloid cells refer to myeloid cells that can be cultured for a long period of time (3 months or more) and have superior proliferation properties compared to myeloid cells normally present in embryos or adults. Hereinafter, proliferative myeloid cells are also referred to as pMCs.

[0057] In the present invention, professional antigen-presenting cells are cells specialized for antigen presentation that express not only human leukocyte antigen (HLA) class I molecules, which are present in ordinary nucleated cells, but also HLA class II molecules, and include macrophages, dendritic cells, B cells, etc. In this specification, professional antigen-presenting cells are also referred to as pAPCs.

[0058] In the present invention, "c-MYC," "BMI1," "MDM2," "GM-CSF," and "M-CSF" refer to the c-MYC, BMI1, MDM2, GM-CSF, and M-CSF genes, or their homologs or orthologs, of any biological species that have the activity of inducing differentiation of myeloid cells derived from human or non-human mammalian pluripotent stem cells into professional antigen-presenting cells.

[0059] For example, when inducing differentiation of human pluripotent stem cells into professional antigen-presenting cells, genomic DNA or cDNA of human c-MYC, BMI1, MDM2, GM-CSF, and M-CSF, as well as their mutants, are used.

[0060] Furthermore, genomic DNA or cDNA of c-MYC, BMI1, MDM2, GM-CSF, and / or M-CSF from other biological species can also be used. Similarly, for professional antigen-presenting cells derived from pluripotent stem cells of biological species other than humans, genomic DNA or cDNA of c-MYC, BMI1, MDM2, GM-CSF, and / or M-CSF from the relevant biological species can also be used. Furthermore, genomic DNA or cDNA of c-MYC, BMI1, MDM2, GM-CSF, and / or M-CSF from other biological species can also be used if the desired cells can be obtained.

[0061] Sequence information for the proteins, genomic DNA or cDNA of the "c-MYC", "BMI1", "MDM2", "GM-CSF" and "M-CSF" of the present invention from various biological species can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI).

[0062] In the present invention, "expression" refers to producing a desired gene or protein within a cell, and in some cases secreting it outside the cell, thereby allowing the function of the gene or protein to be exerted.

[0063] In the present invention, "gene introduced" means that a desired gene is introduced into a specific cell, and the gene or a protein encoded by the gene is produced within the cell, and in some cases, secreted outside the cell, thereby allowing the protein to exert its function.

[0064] In the present invention, the desired gene or protein refers to "c-MYC," "BMI1," "MDM2," "GM-CSF," "M-CSF," and the like.

[0065] In the present invention, preferably, c-MYC, BMI1, and MDM2 are transfected into myeloid cells, and GM-CSF and / or M-CSF are transfected into proliferating myeloid cells.

[0066] In addition, as one embodiment of the present invention, the present invention further relates to a method for producing professional antigen-presenting cells, which comprises differentiating pluripotent stem cells into myeloid cells (MCs) by the following method (i) or (ii): (i) culturing embryoid bodies (EBs) induced from the pluripotent stem cells in a medium containing VEGF on feeder cells in a layered manner, and then further culturing the EBs in a medium containing VEGF, SCF, and TPO; or (ii) culturing the embryoid bodies induced from the pluripotent stem cells in a monolayer culture without feeder cells in a medium containing BMP-4, VEGF, and SCF, and then culturing them in a medium containing VEGF, TPO, and GM-CSF.

[0067] In the present invention, the term "embryoid body" refers to a cell mass formed by the adhesion of undifferentiated pluripotent stem cells. Due to the close intercellular interactions within the cell mass, the pluripotent stem cells that make up the cell mass have a limited repertoire of cell differentiation, similar to an embryo at the developmental stage in which germ layers are formed after implantation.

[0068] The size of the embryoid bodies of the present invention is not particularly limited, but a diameter of 50 to 1000 μm is preferred, 50 to 500 μm is more preferred, and 50 to 200 μm is particularly preferred. If the size of the embryoid bodies is larger than the above range, there is a risk of insufficient oxygen supply to the interior of the embryoid bodies, resulting in cell necrosis. If the size of the embryoid bodies is smaller than the above range, there is a risk of single cells not forming embryoid bodies and undergoing cell death by apoptosis. Mouse embryoid bodies may be cultured using mouse mesenchymal C3H10T1 / 2 cells or mouse bone marrow-derived stromal OP9 cells as feeder cells. Human embryoid bodies can be cultured without the use of feeder cells.

[0069] "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the present invention refer to VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins of any biological species, and their homologs or orthologs, which have the activity of inducing differentiation of human or non-human mammalian pluripotent stem cells into myeloid cells (MCs).

[0070] The "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the present invention may be VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins from a biological species other than that of the embryoid bodies being cultured, or mutants of VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins from the same biological species as the embryoid bodies being cultured, provided that they have the same level of differentiation-inducing activity as that of the "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the biological species of the embryoid bodies being cultured.

[0071] Herein, Flt-3L, IL-3, and / or bFGF may also be used in culturing the cells of the present invention together with the above-mentioned VEGF, SCF, TPO, BMP-4, and / or GM-CSF, etc. These can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI). Additionally, recombinant protein reagents such as Recombinant Human Flt-3 Ligand / FLT3L Protein (308-FKN), Recombinant Human IL-3 Protein (203-IL), Recombinant Human FGF basic / FGF2 / bFGF (145 aa) Protein (3718-FB), Recombinant Human VEGF 165 Protein (293-VE), Recombinant Human SCF Protein (255-SC), Recombinant Human Thrombopoietin Protein (288-TP), Recombinant Human BMP-4 Protein (314-BP), and Recombinant Human GM-CSF Protein (215-GM) can also be purchased from R&D Systems (https: / / www.rndsystems.com / ).

[0072] Sequence information of proteins, genomic DNA or cDNA of "VEGF", "SCF", "TPO", "BMP-4" and / or "GM-CSF" of the present invention from various biological species can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI).

[0073] The above-mentioned genes or proteins are preferably the same as those of the pluripotent stem cells used. For example, if the pluripotent stem cells are of human origin, it is preferable to use human-derived VEGF, SCF, TPO, BMP-4, GM-CSF, Flt-3L, IL-3, and / or bFGF, and if the pluripotent stem cells are of mouse origin, it is similarly preferable to use mouse-derived genes or proteins.

[0074] In the present invention, "medium containing substance X" means a medium to which exogenous substance X has been added or a medium containing exogenous substance X, and "medium not containing substance X" means a medium to which exogenous substance X has not been added or a medium not containing exogenous substance X. Here, "exogenous substance X" means substance X that is foreign to the cells or tissues cultured in that medium, and does not include endogenous substance X produced by those cells or tissues.

[0075] For example, a "VEGF-containing medium" refers to a medium supplemented with exogenous VEGF or a medium containing exogenous VEGF. The dose of VEGF may be affected by other growth factors, but is preferably 0.2 ng / mL to 200 ng / mL, more preferably 2 ng / mL to 100 ng / mL, and particularly preferably 5 ng / mL to 50 ng / mL. A "VEGF-free medium" refers to a medium not supplemented with exogenous VEGF or a medium containing no exogenous VEGF.

[0076] Similarly, for SCF, TPO, BMP-4, and GM-CSF, a medium containing SCF, TPO, BMP-4, and / or GM-CSF refers to a medium supplemented with exogenous SCF, TPO, BMP-4, and / or GM-CSF or a medium containing exogenous SCF, TPO, BMP-4, and / or GM-CSF. Therefore, although the dose of SCF, TPO, BMP-4, and / or GM-CSF may be affected by other growth factors, it is preferably 0.2 ng / mL to 200 ng / mL, more preferably 2 ng / mL to 100 ng / mL, even more preferably 5 ng / mL to 50 ng / mL, particularly preferably 5 ng / mL to 15 ng / mL, and most preferably 10 ng / mL.

[0077] "Culture medium free of SCF, TPO, BMP-4 and / or GM-CSF" refers to a culture medium to which exogenous VEGF has not been added or a culture medium free of exogenous SCF, TPO, BMP-4 and / or GM-CSF.

[0078] In the present invention, αMEM supplemented with 20% fetal bovine serum (FBS) can be used as the medium for mouse embryoid bodies. Mouse mesenchymal C3H10T1 / 2 cells or mouse bone marrow-derived stromal OP9 cells can be used as feeder cells. The fully defined medium X-VIVO15 (Lonza) can be used as the medium for human embryoid bodies. For differentiation from hematopoietic progenitor cells (HPCs), the CD11b-positive fraction can be enriched with magnetic beads.

[0079] The medium may be αMEM supplemented with 20% FBS.

[0080] The proliferative myeloid cells (pMCs) of the present invention can be transfected with GM-CSF and / or M-CSF genes.

[0081] In the present invention, "feeder cells" refer to other cell types used to prepare culture conditions for maintaining iPS cells in an undifferentiated state or for differentiation-inducing culture. Feeder cells are used after their proliferation is arrested by treatment with mitomycin C or irradiation. Mouse-derived fibroblasts can be used for maintaining undifferentiated states, while mouse bone marrow-derived stromal cells OP-9 and mouse-derived mesenchymal cells C3H10T1 / 2 can be used for differentiation-inducing culture.

[0082] In the present invention, "multilayer culture" refers to the co-culturing of iPS cells or embryoid bodies of interest seeded on a culture system of a feeder cell layer cultured in a specified culture vessel. In the present invention, "monolayer culture" refers to the culturing of a single cell type adhered to a vessel for tissue culture.

[0083] In the present invention, feeder cell-free monolayer culture refers to static culture of iPS cells or embryoid bodies of interest seeded in a culture vessel.

[0084] Furthermore, as one embodiment of the present invention, the present invention further relates to a method for producing professional antigen-presenting cells, which comprises forming embryoid bodies from pluripotent stem cells by the following method. (i) seeding a single-cell dispersion of pluripotent stem cells into a vessel for cell cluster formation culture to form embryoid bodies (EBs); and (ii) The container has minute spheroid wells at the bottom, and there is no flat surface between adjacent spheroid wells.

[0085] "Forming embryoid bodies" refers to dissociating pluripotent stem cells that have been growing in an undifferentiated state to obtain a single-cell dispersion of pluripotent stem cells, and then subjecting the dispersion to suspension culture or static culture under conditions that allow the pluripotent stem cells to adhere to each other and aggregate, thereby forming qualitatively uniform cell masses. Embryoid body formation is confirmed by visually confirming the presence of viable cells through microscopic observation.

[0086] As used herein, "suspension culture" refers to culturing under conditions in which cells or cell aggregates are kept in a suspended state and do not adhere to a substrate such as the bottom or wall of a culture vessel or the like.

[0087] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and can be appropriately determined by one skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surface has not been artificially treated (e.g., coated with an extracellular matrix) to improve cell adhesion.

[0088] In the present invention, it is preferable to rapidly aggregate pluripotent stem cells to form embryoid bodies derived from pluripotent stem cells. When cell clusters of pluripotent stem cells are formed in this way and then transferred to a medium containing VEGF at a concentration of 50 ng / mL for culture, the cells of the cell clusters can be differentiated into the cell lineage from hematopoietic stem cells to myeloid cells, similar to the blood islands in the yolk sac.

[0089] Experimental procedures for forming embryoid bodies include, for example, confining cells in a small space using a culture vessel such as a plate with many small-diameter holes (e.g., a 96-well plate) or a vessel for cell aggregate formation culture, and a method of aggregating cells by short-term centrifugation using a small centrifuge tube. Culture conditions for cells or pluripotent stem cell aggregates include rotational culture, shaking culture, and other suspension cultures, as well as static culture, provided that the substrate surface in contact with the cells of the culture vessel is treated in advance to be hydrophobic or otherwise reduced or inhibited from adhering to the substrate surface of the culture vessel.

[0090] The formation of embryoid bodies as cell masses of pluripotent stem cells and the differentiation of cells from embryoid bodies into myeloid cells can be determined based on characteristics including, but not limited to, confirmation of the size and cell number of the embryoid bodies using a microscope or FACS, confirmation of the microscopic morphology of the embryoid bodies in culture, confirmation of histological or cytochemical findings, and / or confirmation of the expression of differentiated and undifferentiated markers, their uniformity, control of the expression of differentiation markers, and confirmation of the synchrony or reproducibility of differentiation efficiency between embryoid bodies.

[0091] In the present invention, a cell is "positive" for a certain marker when the cell is measured by flow cytometry, and the ratio of the mean fluorescence intensity of the positive cells to the negative control is 10 times or more, defined as "+", 2 times or more but less than 10 times, defined as "low", and defined as "less than 2 times". In addition, in the present invention, high or low expression of a gene in a certain cell is determined by measuring the expression level of the gene by real-time PCR, and an expression level of the gene that is 1-fold or more relative to the housekeeping genes β-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HPRT 1 (hypoxanthine phosphoribosyltransferase 1), or β2-microglobulin is considered high, and an expression level less than that is considered low.

[0092] In the present invention, a single-cell dispersion is prepared by washing cultured cells with PBS or the like, treating them with a cell detachment solution such as trypsin-EDTA (Life Technologies) or TrypLE Select (Life Technologies) for 4 to 5 minutes, washing them further with PBS or the like, and centrifuging them to obtain a pellet of the target cells, and then resuspending the pellet in a desired culture medium or the like by pipetting.

[0093] In the present invention, a vessel for cell aggregate formation culture refers to a vessel in which seeded cells can form cell aggregates without adhering to the vessel during culture. Preferably, the vessel has minute spheroid wells on its bottom, which is the culture surface, and adjacent spheroid wells are separated by a curved surface that slopes toward one of the spheroid wells, with no flat surface between them, and the curved surface is surface-treated to suppress cell adhesion, so that most of the cells seeded in the culture vessel fall into one of the spheroid wells.

[0094] An example of a vessel is one in which a spheroid well with a diameter of approximately 400 to 800 μm and a depth of 100 to 800 μm is uniformly distributed across the culture surface (bottom) with no gaps. Preferably, the spheroid well has a diameter of 400 to 500 μm and a depth of 50 to 200 μm. More preferably, the spheroid well has a diameter of approximately 500 μm and a depth of approximately 400 μm, a diameter of approximately 800 μm and a depth of approximately 400 μm, or a diameter of approximately 800 μm and a depth of approximately 300 μm. Most preferably, the spheroid well has a diameter of approximately 400 μm and a depth of 100 to 200 μm.

[0095] A preferred vessel is one in which the spheroid wells are uniformly processed all the way to the wall surface, with no gaps on the culture surface (bottom). In such a vessel, there are no flat surfaces between adjacent spheroid wells, and the seeded single-cell suspension falls into one of the spheroid wells, allowing the formation of uniform aggregates. A more preferred vessel is the microfabricated cell culture vessel (EZSPHERE®) manufactured by AGC.

[0096] The present invention further comprises: (i) seeding pluripotent stem cells into a cell cluster formation culture vessel having fine spheroid wells at the bottom and no flat surface between adjacent spheroid wells to form embryoid bodies (EBs); (ii) obtaining myeloid cells (MCs) from the embryoid bodies by the following method (ii)-1 or (ii)-2; (ii)-1. Layering the embryoid bodies on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF, and TPO, or (ii)-2 culturing the embryoid bodies in a monolayer culture without feeder cells in a medium containing BMP-4, VEGF, and SCF, and then culturing them in a medium containing VEGF, TPO, and GM-CSF; (iii) expressing c-MYC, BMI1, and MDM2 in the myeloid cells (MCs) obtained in (ii) to obtain proliferative myeloid cells (pMCs); and (iv) expressing GM-CSF and / or M-CSF in the proliferative myeloid cells (pMCs) obtained in (iii) to obtain professional antigen-presenting cells (pAPCs), The present invention relates to a method for producing professional antigen-presenting cells.

[0097] Furthermore, as one embodiment of the present invention, the present invention relates to a method for producing professional antigen-presenting cells, in which c-MYC, BMI1 and MDM2 are genetically introduced into myeloid cells, and GM-CSF and / or M-CSF are genetically introduced into proliferative myeloid cells (pMCs).

[0098] In addition, as one embodiment of the present invention, the present invention further relates to a method for producing professional antigen-presenting cells, wherein the size of the embryoid bodies (EBs) is 50 to 200 μm.

[0099] When the size of the embryoid bodies is 50 to 200 μm, pluripotent stem cells can be efficiently differentiated into myeloid cells in both cases of on-feeder and feeder-free differentiation induction.

[0100] Furthermore, as one embodiment of the present invention, the present invention relates to a method for producing professional antigen-presenting cells (pAPCs), further comprising irradiating the professional antigen-presenting cells (pAPCs) with radiation (R / pAPC step) and / or introducing a suicide gene into the pAPCs.

[0101] In the present invention, radiation may be any radiation, including gamma rays, X-rays, ultraviolet rays, visible light, infrared rays, and microwave radiation, preferably X-rays.

[0102] In the present invention, "irradiating" refers to exposing cells to radiation. When professional antigen-presenting cells are irradiated with radiation in the present invention, any absorbed dose may be used as long as the irradiated professional antigen-presenting cells lose their ability to self-proliferate but maintain their ability to promote T cell proliferation.

[0103] In the present invention, introducing a suicide gene into pAPC means that the suicide gene is retained within the pAPC and is in a functional state. A suicide gene is a gene that can cause the death of a cell into which it is introduced. Cell elimination using a suicide gene involves first introducing a suicide gene into a cell, encoding a protein that causes the cell to die only when the suicide gene is activated by a specific compound. The suicide gene may also encode an enzyme that selectively converts non-toxic compounds into highly toxic metabolites, resulting in the specific elimination of cells expressing the enzyme. In some embodiments, the suicide gene is the herpes virus thymidine kinase (HSV-tk) gene, and the trigger is ganciclovir, AP1903 (available from MedChemExpress), or YM-155 (available from Cayman Chemical). In another embodiment, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene, and its trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10):1932-1943 (2012); Xu et al., Cell Res. 8:73-8 (1998), both of which are incorporated by reference in their entireties).

[0104] In another embodiment, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In a preferred embodiment, the inducible caspase protein is iCasp9, which comprises the sequence of human FK506-binding protein (FKBP12) with the F36V mutation linked via a series of amino acids to a gene encoding human caspase 9. FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903. Therefore, the suicide function of iCasp9 in the present invention is triggered by administering a chemical inducer of dimerization (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization rapidly induces apoptosis (see WO2011146862; Stasi et al, N. Engl. J. Med 365;18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is incorporated herein by reference in its entirety).

[0105] A suicide gene can be introduced into cells using a gene transfer vector containing the desired suicide gene. Gene transfer vectors include viral vectors and non-viral vectors. Examples of viral vectors include lentiviral vectors and retroviral vectors, with lentiviral vectors being preferred. Non-viral vectors include transposon vectors and plasmid DNA. Alternatively, the suicide gene can be introduced into the desired cells by integrating it into genomic DNA using genome editing.

[0106] Surprisingly, the present inventors found that when professional antigen-presenting cells are irradiated, they lose their ability to self-proliferate; however, when irradiated to an extent that allows them to maintain their ability to promote T cell proliferation, the professional antigen-presenting cells disappear from the body within at least 3 to 4 days after administration in vivo.

[0107] The radiation dose of the present invention may be any within a range in which the irradiated professional antigen-presenting cells disappear from the body within 3 to 4 days after in vivo administration. Examples include 5 to 100 Gy, 10 to 85 Gy, 10 to 75 Gy, 10 to 65 Gy, and 10 to 55 Gy. Preferably, the dose is 10 Gy to 85 Gy, more preferably 10 Gy to 75 Gy, even more preferably 10 Gy to 65 Gy, particularly preferably 10 Gy to 55 Gy, and most preferably 10 Gy to 40 Gy. When mouse pAPCs are irradiated with radiation, the radiation dose of the present invention is preferably 5 to 85 Gy, more preferably 10 to 85 Gy, even more preferably 20 to 85 Gy, and particularly preferably 25 to 85 Gy. Within the above ranges, the irradiated mouse pAPCs will disappear from the body within 3 to 4 days after in vivo administration. When irradiating human pAPCs, the radiation dose of the present invention is preferably 5 to 80 Gy, more preferably 10 to 80 Gy, even more preferably 20 to 80 Gy, and particularly preferably 20 to 40 Gy. Within the above ranges, irradiated human pAPCs will disappear from the body within 3 to 4 days after in vivo administration.

[0108] T cells are lymphocytes that recognize antigens presented by pAPCs via HLA / MHC and initiate a cellular immune response, damaging and killing cells they perceive as foreign bodies. Naive T cells are a population of cells that are CD3-positive on the surface marker differentiated from hematopoietic stem cells, and further exist as subtypes, such as CD4-positive helper T cells, CD8-positive killer T cells, and Foxp3-positive regulatory T cells, depending on the co-expressed surface markers. In the present invention, CD8-positive killer T cells specific to the antigen loaded on the pAPC are particularly desirable as cytotoxic T cells induced by pAPCs.

[0109] Furthermore, as one embodiment of the present invention, the present invention relates to professional antigen-presenting cells (pAPCs) produced by the production method described herein. Furthermore, as one embodiment of the present invention, the present invention relates to professional antigen-presenting cells (pAPCs) produced by the production method described herein, which, when administered to a living body after radiation exposure, disappear from the living body within at least 3 to 4 days after administration.

[0110] Furthermore, one embodiment of the present invention relates to a professional antigen-presenting cell (pAPC) that has low expression of CD11b / c and expresses CD74.

[0111] Furthermore, one embodiment of the present invention relates to a professional antigen-presenting cell (pAPC) that has low expression of CD11b / c and expresses CD74 and CD33.

[0112] The professional antigen-presenting cells can be produced by the methods described herein.

[0113] CD11b is a myeloid cell differentiation marker expressed on the cell surface. It can be detected by immunostaining with anti-CD11b antibody and analyzing with a flow cytometer.

[0114] CD11c is a differentiation marker for dendritic cells that is expressed on the cell surface. It can be detected by immunostaining with anti-CD11c antibody and analyzing with a flow cytometer.

[0115] CD33 is a myeloid cell differentiation marker expressed on the cell surface and can be detected by immunostaining with anti-CD33 antibody and analyzing with a flow cytometer.

[0116] CD74 is a cell surface marker involved in cross-presentation. It can be detected by immunostaining with anti-CD74 antibody and analyzing with a flow cytometer.

[0117] Expression is defined as a ratio of the mean fluorescence intensity of positive cells to that of the negative control of 2-fold or more. Low expression refers to a ratio of the average fluorescence intensity of positive cells to that of the negative control that is 2-fold or more but less than 10-fold.

[0118] The present inventors have surprisingly found for the first time that professional antigen-presenting cells that express CD74 or CD74 and CD33 with low expression of CD11b and c promote the antigen-nonspecific proliferation of T cells, particularly naive T cells expressing CD8. Furthermore, the present inventors have surprisingly found that irradiation of these professional antigen-presenting cells promotes the proliferation of CD8-expressing T cells in vivo, but that these professional antigen-presenting cells disappear from the body within 3 to 4 days after administration.

[0119] "Disappearance" refers to the fact that the cells administered to the living body cannot be detected or are immunologically eliminated. Specifically, for example, by the method described in Example 8 below, the time-dependent changes in in vivo imaging images and total flux of luciferase luminescence can be confirmed by checking whether they are below the detection limit. Furthermore, confirmation can also be performed using equivalent methods using fluorescent dyes, radioactive compounds, etc., in addition to luciferase.

[0120] Furthermore, after administering the irradiated professional antigen-presenting cells of the present invention to a living body, it can also be confirmed by examining blood samples from the living body and finding that T cells expressing CD11b / c are low and CD33 and CD74 are below the detection limit.

[0121] Any method can be used to confirm the professional antigen-presenting cells of the present invention or T cells that have low expression of CD11b / c and express CD33 and CD74. If the presence of the antigen-presenting cells or T cells cannot be detected by these methods, it can be determined that the professional antigen-presenting cells of the present invention have disappeared.

[0122] In one embodiment of the present invention, the present invention further provides a cell line having a self-renewal ability, and The present invention relates to the above-mentioned professional antigen-presenting cells (pAPCs) that exhibit apoptosis resistance.

[0123] The professional antigen-presenting cells can be produced by the methods described herein.

[0124] Self-proliferation ability refers to the ability of cells to proliferate by activating cell proliferation signals through cytokines that are produced and secreted by the cells themselves. Self-proliferation ability can be confirmed by the proliferation of cells seeded in a cytokine-free culture medium, such as RPMI-1640 medium containing 10% fetal bovine serum (FBS). Methods for confirming cell proliferation include counting the number of cells before and after culture, MTT assay, and 3 This can be evaluated by measuring the incorporation activity of [H]-thymidine.

[0125] Apoptosis resistance is the ability to avoid apoptotic cell death. It can be assessed by staining cells with fluorescent dye-labeled reagents, FITC-labeled Annexin-V, 7-AAD, and PI, which can detect cell death, followed by analysis using a flow cytometer.

[0126] Furthermore, as one embodiment of the present invention, the present invention relates to a method for promoting the proliferation of antigen-nonspecific or antigen-specific CD8-expressing T cells in vivo and in vitro, by co-culturing the above-mentioned professional antigen-presenting cells or GM-CSF with naive T cells expressing CD8 in vitro.

[0127] Furthermore, as one embodiment, the present invention relates to a method for producing T cells expressing antigen-nonspecific or antigen-specific CD8, using the above-mentioned method for promoting the proliferation of T cells expressing antigen-nonspecific or antigen-specific CD8.

[0128] The professional antigen-presenting cells can be produced by the methods described herein.

[0129] Furthermore, the present inventors have surprisingly found that GM-CSF alone can promote the proliferation of T cells, particularly CD8-expressing T cells, in an antigen-nonspecific manner.

[0130] Naive T cells expressing CD8 are a population of cells expressing the T cell surface marker CD3 that have not been stimulated by antigen. Non-specific T cells are a population of T cells that maintain a state of T cell receptor diversity, without being biased toward a population of T cells with T cell receptors that recognize specific antigens.

[0131] Furthermore, the professional antigen-presenting cells may be professional antigen-presenting cells loaded with a desired antigen.

[0132] Antigen-specific T cells are T cells that express a T cell receptor that specifically recognizes a particular antigen upon stimulation by antigen-presenting cells loaded with the desired antigen.

[0133] In the present invention, the term "antigen" refers to any protein, sugar chain, or other molecule capable of eliciting an immune response specific to the antigen, particularly a cellular immune response. The antigen of the present invention is preferably an antigen that elicits cellular immunity specific to cells harmful to the body, such as cancer cells, cells that have undergone neurodegeneration or other tissue degeneration, or cells that have undergone physiological or pathological processes and have begun to elicit harmful responses to the body, in order to eliminate these cells. In the present invention, cancer-specific antigens are particularly preferred.

[0134] In the present invention, the term "cancer-specific antigen" refers to a protein, sugar chain, or other molecule that is expressed only when a specific cell type undergoes malignant transformation and proliferation, and is not present in a living body in which such malignant cells do not exist. A typical cancer-specific antigen is the so-called carcinoembryonic antigen, which is expressed only up to the embryonic or fetal stage during normal development but is expressed in cancer cells.

[0135] Furthermore, the professional antigen-presenting cells may be irradiated ex vivo.

[0136] By irradiating the professional antigen-presenting cells of the present invention using the above-described method, the cells can be administered to a living body and then promote the proliferation of antigen-specific or non-specific CD8-expressing T cells in the living body. However, these professional antigen-presenting cells can be eliminated from the living body within 3 to 4 days after administration.

[0137] Furthermore, in one embodiment, the present invention relates to a pharmaceutical composition for inducing the proliferation of CD8-expressing T cells, which further comprises the above-mentioned professional antigen-presenting cells.

[0138] In the present invention, the term "pharmaceutical composition for inducing T cell proliferation" refers to a pharmaceutical composition for inducing T cell proliferation. In the present invention, this refers to a pharmaceutical composition that acts on naive T cells, which are in a dormant state or a state close to dormant with respect to cell proliferation, to induce proliferation. This pharmaceutical composition includes cytokines themselves that promote T cell proliferation, cells that produce and secrete such cytokines, and pharmaceutical compositions that not only induce proliferation but also induce T cells that proliferate with cytotoxicity as specific cytotoxic T cells. This pharmaceutical composition can activate the immune system in the body.

[0139] Furthermore, as one embodiment of the present invention, the present invention provides a method for producing CD8-expressing T cells for administration into the body, the method comprising: loading the above-mentioned professional antigen-presenting cells with an antigen; irradiating the antigen-loaded professional antigen-presenting cells; and co-culturing the irradiated professional antigen-presenting cells in vitro with CD8-expressing naive T cells to promote the proliferation of the antigen-specific CD8-expressing T cells; and the CD8-expressing T cells produced by this method.

[0140] The CD8-expressing T cells produced by this method can be administered to the body. The irradiated professional antigen-presenting cells of this method can disappear from the body 3 to 4 days after administration. The CD8-expressing T cells produced by this method can be antigen-specific CD8-expressing T cells loaded onto the professional antigen-presenting cells, and the antigen can be a cancer-specific antigen.

[0141] The professional antigen-presenting cells or CD8-expressing T cells of the present invention can be used in combination with anti-cancer agents and / or other cancer treatment methods. Additionally, the professional antigen-presenting cells or CD8-expressing T cells of the present invention can be used in combination with immune checkpoint inhibitors.

[0142] As used herein, anticancer agents and / or other cancer treatment methods include, but are not limited to, surgical therapy to physically remove and excise cancer cells; chemotherapy and chemotherapeutic agents to administer chemicals, including but not limited to alkylating agents, platinum compounds, metabolic antagonists, topoisomerase inhibitors, microtubule inhibitors, and antibiotics; immunotherapy and immunotherapeutic agents to attack cancer cells as non-self cells by immune cells; and radiation therapy to inhibit the growth of and kill cancer cells by irradiating them with radiation, including but not limited to X-rays, electron beams, gamma rays, and neutron beams. Chemotherapeutic drugs for treating cancer include 6-O-(N-chloroacetylcarbamoyl) fumagillol, bleomycin, methotrexate, actinomycin D, mitomycin C, daunorubicin, adriamycin, neocarzinostatin, cytosine arabinoside, fluorouracil, tetrahydrofuryl-5-fluorouracil, picibanil, lentinan, levamisole, bestatin, azimexon, glycyrrhizin, doxorubicin hydrochloride, aclarubicin hydrochloride, bleomycin hydrochloride, levothyroxine ... These include omycin, hepatomycin sulfate, vincristine sulfate, vinblastine sulfate, irinotecan hydrochloride, cyclophosphamide, melphalan, busulfan, thiotepa, procarbazine hydrochloride, cisplatin, azathioprine, mercaptopurine, tegafur, carmofur, cytarabine, methyltestosterone, testosterone propionate, testosterone enanthate, mepitiostane, fosfestrol, chlormadinone acetate, leuprorelin acetate, buserelin acetate, etc. The other treatment methods may also include surgical removal of cancer tissue and / or irradiation to kill cancer cells.

[0143] As used herein, immune checkpoint inhibitors are anticancer drugs that suppress cancer growth by binding to immune checkpoint molecules that suppress T cell activity through antigen presentation and inhibiting their signal transduction. Immune checkpoint molecules can include both receptors and ligands that function as immune checkpoints.

[0144] In one embodiment of the present invention, the term "immune checkpoint inhibitor" includes, but is not limited to, any antibody or compound that can inhibit, for example, the binding or interaction between PD-L1 and PD-1, the binding or interaction between CD80 / CD86 and CTLA4, the binding or interaction between CD137L and CD137, the binding or interaction between MHC and LAG-3 / KIR, the binding or interaction between CD48 and CD244, the binding or interaction between GAL9 and TIM3, the binding or interaction between HVEM and BTLA / CD160, the binding or interaction between CD40L and CD40, the binding or interaction between OX40L and OX40, and the binding or interaction between GITRL and GITR.

[0145] In another embodiment of the present invention, the "immune checkpoint inhibitor" is selected from the group consisting of, but not limited to, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-B7 antibody, an anti-C27 antibody, an anti-KIR antibody, an IDO inhibitor, an anti-CD137 antibody, and an anti-TIM3 antibody.

[0146] In yet another embodiment of the present invention, the "immune checkpoint inhibitor" is selected from, but not limited to, Atezolizumab, Durvalumab, Avelumab, Nivolumab, Pembrolizumab, Pidilizumab, BMS-936559, Ipilimumab, Tremelimumab, Enoblituzumab, Varlilumab, Lirilumab, Epacadostat, Utomilumab, Urelumab, and TSR-022.

[0147] In yet another embodiment of the present invention, the "immune checkpoint inhibitor" is preferably an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-CTLA4 antibody, more preferably an anti-PD-L1 antibody or an anti-CTLA4 antibody.

[0148] All documents mentioned in this specification, as well as the specifications and drawings of Japanese Patent Application Nos. 2020-084821 and 2020-149486, which are the basic applications of this application, are incorporated herein by reference in their entirety.

[0149] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention. [Example]

[0150] Example 1: Generation of embryoid bodies derived from human iPSCs Human iPSCs were generated using the method described by Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)). Human iPSCs were maintained in polystyrene tissue culture plates coated with iMatrix511 (Nippi) using the regenerative medicine medium StemFit AK02N (Ajinomoto Healthy Supply). Medium was changed every 1–2 days. Depending on cell growth, cells were treated with the cell detachment solution TrypLE Select (Life Technologies) for 4–5 minutes once a week to recover single-cell suspensions. The cells were then seeded into appropriate culture vessels for further culture. The seeded iPSCs were cultured overnight in the presence of 10 μM Rock inhibitor Y-27632 (Wako Pure Chemical Industries), and the medium was changed the following day to remove Y-27632.

[0151] Human iPSC-derived embryoid bodies (hereafter referred to as human EBs) were prepared by seeding human iPSCs at a predetermined density in a 6-well plate-type cell mass formation culture vessel, EZ SPHERE SP (AGC Technoglass), prepared with StemFit containing 10 μM Y-27632. One to four days after the formation of embryoid bodies with diameters of 50 to 200 μm, differentiation-inducing culture was performed as described in Example 3 below.

[0152] Example 2: Generation of embryoid bodies derived from mouse iPSCs Mouse iPSCs were generated using the method described in Araki, R. et al. (Nature, 494:100-104. (2013)). Mouse iPSCs were maintained in ES cell medium ESM (DMEM (Wako Pure Chemical Industries) containing 15% KSR (Life Technologies)) supplemented with a final concentration of 55 μM 2-mercaptoethanol (Life Technologies) and 10 4 The culture was performed on mouse embryonic fibroblast (MEF) (ReproCell) feeder cells seeded on a 6-well tissue culture plate using complete ESM (cESM) medium prepared with the addition of 100 U / mL Leukemia Inhibitory Factor (LIF) (Merck, trade name ESGROmLIF).

[0153] Feeder cells were prepared by seeding MEFs (Reprocells) whose cell growth had been stopped by Mitomycin C treatment onto gelatin-coated dishes, and were used for the maintenance culture of mouse iPSCs from the day after the MEFs attached.

[0154] Mouse iPSC-derived embryoid bodies (hereafter referred to as mouse EBs) were prepared by seeding mouse iPSCs prepared in complete medium cESM at a predetermined density onto a 6-well EZ SPHERE plate. One to four days after the formation of embryoid bodies with diameters of 50 to 200 μm, differentiation-inducing culture was performed as described in Example 3 below.

[0155] Example 3: Induction of differentiation of embryoid bodies into myeloid cells (1) Preparation of feeder cells Mouse-derived cultured cell lines C3H10T1 / 2 or OP9 were seeded onto gelatin-coated dishes and used the following day. Feeder cells used to induce differentiation of human iPSC-derived embryoid bodies were irradiated with 60 Gy to stop proliferation before use. C3H10T1 / 2 cells were cultured in BME medium (Life Technologies) containing 10% FBS, and OP9 cells were cultured in αMEM medium (Life Technologies) containing 20% ​​FBS.

[0156] (2) On-feeder differentiation induction culture of mouse iPSCs Mouse EBs generated from mouse iPSCs were cultured on OP9 feeder cells in αMEM containing 20% ​​FBS for 7-10 days. The recovered cells were then replated on freshly prepared OP9 feeder cells. They were cultured for 7-10 days in the presence of 55 μM 2-mercaptoethanol and 50 ng / mL GM-CSF to obtain differentiated cells, including myeloid cells.

[0157] (3) On-feeder differentiation induction culture of human iPSCs Human EBs generated from human iPSCs were cultured on C3H10T1 / 2 feeder cells in 20% FBS-containing αMEM with 20 ng / mL VEGF for 7–10 days. Subsequently, the cells were cultured for 7–10 days in the presence of 20 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL Flt-3L, 10 ng / mL TPO, and 20 ng / mL IL-3. The recovered cells were then replated on freshly prepared OP9 feeder cells. The replated cells were then cultured for 7–10 days in the presence of 50 ng / mL SCF, 50 ng / mL Flt-3L, and 50 ng / mL GM-CSF to obtain differentiated cells, including myeloid cells.

[0158] (4) Feeder-free differentiation-inducing culture of human iPSCs Human EBs generated from human iPSCs were cultured in differentiation medium X-VIVO15 (Lonza) for 5–7 days in the presence of 50 ng / mL bFGF, 50 ng / mL BMP-4, 50 ng / mL VEGF, and 50 ng / mL SCF. Subsequently, they were cultured for 7–10 days in the presence of 50 ng / mL bFGF, 50 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL Flt-3L, 10 ng / mL TPO, 20 ng / mL IL-3, and 50 ng / mL GM-CSF. These were then cultured for 7–10 days in the presence of 50 ng / mL GM-CSF, 50 ng / mL SCF, and 50 ng / mL Flt-3L to obtain differentiated cells, including myeloid cells. Although it may be affected by other growth factors, any two concentrations within the range of 0.2 ng / mL to 200 ng / mL can be used as the upper and lower limits, for example, any two concentrations from 0.2 ng / mL, 2 ng / mL, 2 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL to 200 ng / mL can be used as the upper and lower limits.

[0159] Example 4: Generation of proliferative myeloid cells (pMC) by introducing proliferation genes (1) Preparation of lentiviral vector Human c-MYC, BMI1, and MDM2 cDNAs were synthesized by gene synthesis using sequence information from the National Center for Biotechnology Information (NCBI) database. The cDNA fragments of each gene were inserted into the lentiviral vector pSL or CDII-EF-MSC-IRES2-Veneus. Using lipofection, the genes introduced into the lentiviral vector pSL or CDII-EF-MSC-IRES2-Veneus, along with the packaging construct pCAG-HIVgp and the envelope and Rev construct pCMV-VSV-G-RSV-Rev, were transfected into 293T cells, which are used as packaging (virus-producing) cells.

[0160] Three days after gene transfer into 293T cells, the cell culture medium was collected and filtered through a 0.45 μm pore size filter. The viral particles were concentrated and collected using a Lenti-X concentrator (Clontech). The collected recombinant viral particles were suspended in DMEM solution, dispensed into cryovials, and stored at -150°C.

[0161] (2) Improving proliferation ability by introducing proliferation genes into iPSC-derived myeloid cells The myeloid cells prepared in the previous section were cultured in 48-well tissue culture plates and infected with a lentivirus suspension expressing c-MYC (mouse myeloid cells) simultaneously. Human myeloid cells were infected with a lentivirus suspension expressing c-MYC, BMI1, and MDM2 simultaneously. The day after transfection, the culture was expanded by adding additional medium depending on cell growth. The infected myeloid cells were cultured in αMEM containing 20% ​​FBS in the presence of 50 ng / mL GM-CSF and 50 ng / mL M-CSF. Transfected cells continued to proliferate for more than three months. The proliferative myeloid cells thus prepared were named proliferating myeloid cells (pMCs).

[0162] (3) Examination of the necessity of GM-CSF and M-CSF for the proliferation of pMCs After gene transfection, pMCs were collected after culturing for more than 2 weeks and seeded (2 × 10 3 The proliferation rate was compared between cultures containing GM-CSF and M-CSF and those without.

[0163] Immediately after the start of culture in a 96-well tissue culture plate, 0.5 mg / mL of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Merck) was added every 24 hours, and the amount of metabolized formazan was measured 4 hours later by absorbance at 595 nm. The amount of metabolized formazan is proportional to the cell number at the time of addition of the MTT reagent, i.e., the rate of cell proliferation.

[0164] The results of the MTT assay are shown in Figure 1. These results indicated that approximately 50 ng / mL of GM-CSF or M-CSF was required in the culture medium for pMC proliferation. Furthermore, the combined use of GM-CSF and M-CSF had the effect of promoting proliferation.

[0165] (4) Morphological and surface antigen analysis of pMCs The resulting pMCs showed no change in cell morphology due to the introduction of c-MYC, BMI1, or MDM2, and retained the distorted morphology with processes characteristic of myeloid cells.

[0166] pMCs were collected by pipetting and stained with anti-CD11b, anti-CD11c, and anti-CD33 antibodies, or an isotype-matched control antibody. The cells were then washed twice with PBS containing 2% FBS or PBS. After washing, the cells were analyzed using a flow cytometer (BD Accuri C6 Flow Cutometer, Beckton Dickinson). Figure 2 shows the results of surface antigen analysis using a flow cytometer after antibody staining. This analysis revealed that pMCs exhibited reduced expression of CD11b and CD11c on the cell surface, which were previously observed in differentiated cells. Furthermore, pMCs expressed CD33, a physiological myeloid cell marker, on their cell surface.

[0167] Example 5: Generation of iPSC-derived professional antigen-presenting cells (ipAPCs) (1) Preparation of lentiviral vector (human) Human GM-CSF and M-CSF cDNAs were synthesized by gene synthesis, and the cDNA fragments of each gene were inserted into the lentiviral vector pSL, followed by preparation of viral particles, as described in Example 4(1). The collected recombinant viral particles were suspended in DMEM solution, dispensed into cryovials, and stored at -150°C.

[0168] (2) Introduction of GM-CSF and M-CSF genes into iPSC-derived pMCs (human and mouse) Human proliferative myeloid cells (pMC) prepared in Example 4(2) were cultured in a 48-well tissue culture plate and infected with a lentivirus suspension expressing human GM-CSF only, human M-CSF only, or human GM-CSF and human M-CSF. Similarly, mouse proliferative myeloid cells (pMC) prepared in Example 4(2) were cultured in a 48-well tissue culture plate and infected with a lentivirus suspension expressing mouse GM-CSF only.

[0169] The culture scale was expanded from the day after gene transfection, with additional medium being added depending on cell proliferation. The culture medium was αMEM containing 20% ​​FBS, and the cells were cultured in the presence of 50 ng / mL GM-CSF and 50 ng / mL human M-CSF. Human GM-CSF was used for human proliferated myeloid cells (pMC), and mouse GM-CSF was used for mouse proliferated myeloid cells (pMC).

[0170] The transfected cells self-proliferated due to their own production of GM-CSF and M-CSF, even without the addition of exogenous cytokines. The self-proliferating pMCs generated in this way were named iPSC-derived professional antigen-presenting cells-GMM (ipAPC-GMM). Similarly, pMCs capable of self-proliferation due to their own production of GM-CSF were named ipAPC-GM, and pMCs capable of self-proliferation due to their own production of GM-CSF were named ipAPC-M.

[0171] (3) Examination of the production of cytokines GM-CSF or M-CSF (in mice and humans) introduced into ipAPC-GM, ipAPC-M, and ipAPC-GMM. The expression of GM-CSF in the mouse ipAPC-GM, GM-CSF in the human ipAPC-GM, M-CSF in the ipAPC-M, and GM-CSF and M-CSF in the ipAPC-GMM was examined using a flow cytometer. The 3'-terminal nucleic acid sequence of the mouse GM-CSF cDNA incorporated into the lentiviral vector was engineered to express the fluorescent protein Venus via an IRES sequence. This enabled analysis of GM-CSF expression in mouse ipAPC-GM using Venus fluorescence as an indicator. The 3'-terminal nucleic acid sequence of the human GM-CSF or M-CSF cDNA was engineered to express a truncated dCD19 protein lacking the activation domain. This enabled analysis of dCD19 expression using a fluorescent dye-conjugated anti-CD19 antibody as an indicator of GM-CSF expression in human ipAPC-GM, M-CSF in ipAPC-M, and GM-CSF and M-CSF in ipAPC-GMM. Figure 3 shows the results of flow cytometry analysis of GM-CSF-Venus expression in mouse pMCs and ipAPC-GM, GM-CSF-dCD19 expression in human pMCs and ipAPC-GM, M-CSF-dCD19 expression in ipAPC-M, and GM-CSF-M-CSF-dCD19 expression in ipAPC-GMM. Compared to pMCs before cytokine introduction, mouse ipAPC-GM emits Venus fluorescence, and human ipAPC-GM, ipAPC-M, and ipAPC-GMM express CD19.

[0172] Mouse ipAPC-GM, or human ipAPC-GM, ipAPC-M, or ipAPC-GMM were cultured at a constant cell density (1 × 10) in αMEM containing 20% ​​FBS without the addition of exogenous cytokines. 5 ~1×10 6The cultures were maintained at a constant concentration of 100 ng / mL. The GM-CSF levels in the culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) (BioLegend) as shown in Figure 4. Control pMCs did not produce GM-CSF or M-CSF, whereas mouse ipAPC-GM produced approximately 150 ng / mL of GM-CSF, human ipAPC-GM and ipAPC-GMM produced approximately 40 ng / mL of GM-CSF, and human ipAPC-M and ipAPC-GMM produced approximately 15 ng / mL of M-CSF. Mouse pMCs exhibited GM-CSF-dependent growth, while human pMCs exhibited GM-CSF- and M-CSF-dependent growth, demonstrating that mouse ipAPC-GM and human ipAPC-GMM produced sufficient GM-CSF or M-CSF for their own proliferation.

[0173] (4) Examination of the self-proliferation ability of ipAPC-GM and the requirement of GM-CSF and M-CSF for proliferation (human and mouse) The mouse ipAPC-GM or on-feeder differentiation-induced human ipAPC-GMM obtained as described above was collected and seeded (2 × 10 3 The proliferation rates were compared by MTT assay between the culture medium containing 50 ng / mL GM-CSF only, 50 ng / mL M-CSF only, or 50 ng / mL GM-CSF and 50 ng / mL M-CSF, and the culture medium containing none of these.

[0174] Figure 5 shows the results of the MTT assay for mouse ipAPC-GM and human ipAPC-GMM. These results demonstrate that ipAPC-GM and ipAPC-GMM proliferated even in the absence of GM-CSF or M-CSF in the culture medium. Therefore, ipAPC-GM and ipAPC-GMM self-proliferated without the addition of cytokines. Furthermore, the addition of exogenous GM-CSF to the culture medium did not promote proliferation. However, the addition of 50 ng / mL of M-CSF to the culture medium was found to promote proliferation of ipAPC-GM and ipAPC-GMM.

[0175] (5) Morphology of ipAPC-GM (mouse) The mouse ipAPC-GM prepared in Example 5(2) showed no change in cell morphology due to the introduction of GM-CSF, and had the morphology of myeloid-like cells with processes.

[0176] (6) Morphology of ipAPC-GMM and surface antigen analysis of dendritic cells (human) Similar to the mouse ipAPC-GM, the human ipAPC-GMM prepared in Example 5(2) did not show any change in cell morphology due to the introduction of GM-CSF and M-CSF.

[0177] The ipAPC-GMM, ipAPC-GM, and ipAPC-M prepared in Example 5(2) were seeded onto 12-well tissue culture plates and cultured in αMEM medium containing 20% ​​FBS and 50 ng / mL GM-CSF and 50 ng / mL M-CSF. Dendritic cells (DCs) were seeded onto 12-well tissue culture plates in cytokine-free RPMI-1640 medium containing 10% FBS and cultured for 48 hours. DCs were induced by culturing CD14-positive monocytic cells derived from healthy donor blood in the presence of 50 ng / mL GM-CSF and 50 ng / mL IL-4 for 5 days. The collected cells were stained with anti-HLA-ABC, anti-HLA-DR, anti-CD40, anti-CD80, anti-CD83, anti-CD86, anti-CCR7, anti-CD74, anti-CD11b, anti-CD11c, and anti-CD33 antibodies or isotype-matched antibodies and analyzed by flow cytometry.

[0178] Figure 6 shows the results of surface antigen analysis of human ipAPC-GM, ipAPC-M, and ipAPC-GMM. ipAPC-GMM were positive for CD86, HLA-ABC, HLA-DR, CD74, and CD33, with low expression of CD40 and CD80, low to positive expression of CD11b and CD11c, and negative for CD83 and CCR7. Positive expression indicates a surface marker intensity ratio (RFI) of 10 or greater relative to the mean fluorescence intensity of an isotype-matched antibody. Low expression indicates an RFI of 2 or greater but less than 10. Negative expression indicates an RFI of less than 2.

[0179] Figure 7 shows the results of DC surface antigen analysis. DCs were found to be positive for all markers except for CD83 and CCR7, which were expressed at low levels. These results indicate that ipAPC-GM, ipAPC-M, and ipAPC-GMM express the antigen-presenting molecules HLA-ABC and HLA-DR, as well as the costimulatory molecule CD86, and also express CD74, which is important for cross-presentation, similar to DCs.

[0180] (7) Comprehensive gene expression analysis of mouse ipAPC-GM The mouse ipAPC-GM prepared in Example 5(2) was subjected to RNA-seq analysis, a whole-transcriptome analysis, to quantify transcript expression. Total RNA was extracted from the three types of cells: mouse ipAPC-GM, mouse pMC, and mouse pMC supplemented with exogenous GM-CSF, using the QIAGEN RNeasy Mini Kit. The total RNA was sequenced using a BGISEQ (BGI) next-generation sequencer, and principal component analysis (PCA) and gene set enrichment analysis (GSEA) were performed.

[0181] The results of PCA are shown in Figure 8. This figure plots the profiles of mouse pMC, mouse ipAPC-GM, and GM-CSF-supplemented mouse pMC against the two axes (PC1 and PC2) with the highest contribution rates obtained by multivariate analysis. The gene expression profile of mouse ipAPC-GM was found to be significantly separated from that of mouse pMC and GM-CSF-supplemented mouse pMC.

[0182] Figure 9 shows the results of GSEA of mouse ipAPC-GM compared with mouse pMCs and GM-CSF-treated mouse pMCs. The gene sets that showed significantly increased expression in mouse ipAPC-GM included transcripts involved in the cell cycle, its processes, cell division, and its regulation. Specifically, genes involved in the cell cycle included XRCC2, KNTC1, TTK, PKMYT1, AURKA, HMGN5, ANKLE1, KIF2C, MCM8, CDC45, MCM7, CDCA5, CABLES1, CDC6, CDK1, SGOL1, TPX2, NUSAP1, TACC3, UBE2C, MCM5, OSM, UHRF1, CCND1, ABCB1A, APITD1, NSL1, DSCC1, HAUS5, FOXM1, CHEK1, KIF4, BUB1, and SKA. 1, ERCC6L, RECQL4, GINS1, EXO1, CENPN, MKI67, DLGAP5, KIF18A, SYCE2, BIRC5, CDC20, CDC25C, CDKN3, GSG2, CENPH, FAM64A, EPS8, NUP62, RASSF1, KIF20B, CENPT, UTP14B, and genes involved in cell cycle processes such as HAUS5, XRCC2, FOXM1, KNTC1, TTK, AURKA, CHEK1, HMGN5, ANKLE1, KIF2C, and ERCC6L. CDC45, KIF4, BUB1, SKA1, CDCA5, CABLES1, ERCC6L, RECQL4, GINS1, CDC6, CENPN, CDK1, MKI67, SGOL1, TPX2, KIF18A, NUSAP1, SYCE2, B IRC5, CDC20, CDKN3, UBE2C, TACC3, CDC25C, GSG2, CENPH, OSM, FAM64A, CCND1, ABCB1A, EPS8, NUP62, APITD1, NSL1, RASSF1, KIF20B, C Genes involved in ENPT, DSCC1, and cell mitosis include XRCC2, HAUS5, FOXM1, KNTC1, TTK, AURKA, CHEK1, KIF2C, CDC45, KIF4, BUB1, SKA1, CDCA5, CABLES1, ERCC6L, GINS1, CDC6, CDK1, CENPN, MKI67, SGOL1, TPX2, KIF18A, NUSAP1, BIRC5, CDC20, CDKN3, UBE2C, TACC3, CDC25C, GSG2, CENPH, andExpression of genes involved in mitotic regulation, such as FAM64A, CCND1, ABCB1A, EPS8, NUP62, APITD1, NSL1, KIF20B, CENPT, and DSCC1, was found to be elevated in ipAPC-GM. These genes include HAUS5, FOXM1, KNTC1, TTK, AURKA, CHEK1, KIF2C, CDC45, KIF4, BUB1, SKA1, CDCA5, CABLES1, ERCC6L, GINS1, CDC6, CDK1, CENPN, SGOL1, TPX2, KIF18A, NUSAP1, BIRC5, CDC20, CDKN3, UBE2C, TACC3, CDC25C, GSG2, CENPH, FAM64A, CCND1, ABCB1A, EPS8, NUP62, APITD1, NSL1, KIF20B, CENPT, and DSCC1.

[0183] (8) Apoptosis analysis of ipAPC-GM Mouse ipAPC-GM and human ipAPC-GM, ipAPC-M, and ipAPC-GMM cells prepared in Example 5(2) were analyzed for cell death using fluorescently labeled Annexin V and 7-Amino-Actinomycin D (7-AAD). Annexin-V is a protein that specifically binds to phosphatidylserine (PS), a phospholipid component of cell membranes. Viable cells do not expose PS on the cell surface, but Annexin V binds to it upon inversion of the cell membrane during apoptosis. 7-AAD penetrates dead cells whose cell membranes have collapsed due to late apoptosis or necrosis, intercalates into DNA strands, and emits red fluorescence. This allows for the analysis of apoptosis in cultured cells.

[0184] Mouse ipAPC-GM and mouse pMC, or human ipAPC-GM, ipAPC-M, ipAPC-GMM, and human pMC, were cultured in α-MEM containing 20% ​​FBS without the addition of exogenous cytokines. Mouse-derived cells were cultured for 4 days, and human-derived cells were cultured for 3 days. Cells cultured under these conditions were sampled daily and stained with Annexin V and 7-AAD before analysis by flow cytometry. Figure 10A shows the results of flow cytometry analysis of mouse-derived cells. On day 4 of culture, mouse ipAPC-GM maintained a low level of Annexin V and 7-AAD positivity, which indicates cell death, compared to control mouse pMC. Figure 10C shows the results of flow cytometry analysis of human-derived cells. On day 3 of culture, human ipAPC-GMM and ipAPC-M, like mouse ipAPC-GM, maintained lower Annexin V and 7-AAD positivity rates, which indicate cell death, compared to human pMC. On the other hand, human ipAPC-GM, like human pMC, showed a higher frequency of cell death. Figure 10B (mouse-derived cells) and Figure 10D (human-derived cells) show line graphs of the time course of viable cells, early apoptotic cells, and late apoptotic cells, separated by four quadrants of the cytogram. Mouse ipAPC-GM, human ipAPC-GMM, and human ipAPC-M maintained high viable cell rates and low apoptotic cell rates throughout the culture period. However, mouse pMC, human pMC, and human ipAPC-GMM began to show a decrease in viable cell rates and an increase in the apoptotic cell rate from day 2 of culture. These findings indicate that mouse ipAPC-GM, and human ipAPC-GMM and human ipAPC-M, are more resistant to apoptosis than pMC when GM-CSF is introduced, and when M-CSF alone or in combination with GM-CSF is introduced, respectively.

[0185] Example 6: Examination of antigen-presenting function of ipAPC-GM (1) In vitro proliferation of alloreactive T cells by mouse ipAPC-GM, and human ipAPC-GM, ipAPC-M, and ipAPC-GMM The mixed allo-lymphocyte reaction (allo-MLR) induction activity was evaluated using mouse ipAPC-GM prepared from C57BL / 6 mouse iPSCs in the same manner as described above, and human ipAPC-GM, human ipAPC-M, and human ipAPC-GMM prepared in Example 5(2). Naive T cells (1.5 × 10 ) that had not received antigen stimulation and were prepared from BALB / c mice were used. 5 Cells / well were co-cultured with mouse ipAPC-GM or mouse pMC derived from different strains, C57BL / 6, for 6 days, and then [ 3 [H]-thymidine (PerkinElmer, 1 μCi / well) was added, and the next day, high-molecular-weight DNA in the cells was captured on a glass filter using a cell harvester (PerkinElmer). The allo-MLR induction activity of human ipAPC-GM, human ipAPC-M, and human ipAPC-GMM was assessed by co-culturing them with CD3-positive naive T cells derived from peripheral blood mononuclear cells of healthy human donors. The [H]-thymidine binding to high-molecular-weight DNA was then assessed. 3 Incorporation of [H]-thymidine into high molecular weight DNA was measured by scintillation counting. 3 Incorporation of [H]-thymidine is proportional to the rate of DNA synthesis and thus the rate of cell proliferation.

[0186] Figure 11 shows the results of scintillation counting of naive T cell stimulatory activity. As the ratio of mouse ipAPC-GM to T cells increased, the proliferation of alloreactive naive CD8+ T cells was induced. On the other hand, the proliferation of alloreactive naive T cells in response to mouse pMC was weak. Similar to mouse ipAPC-GM, the proliferation of alloreactive naive CD8+ T cells was induced as the ratio of human ipAPC-GM and ipAPC-GMM increased. On the other hand, the proliferation of alloreactive naive T cells in response to human pMC and ipAPC-M was weak. These results demonstrate that the immune response induced by pMC is enhanced by the effect of the cytokine GM-CSF introduced into ipAPC-GM and ipAPC-GMM.

[0187] (2) Examination of the proliferation of antigen-specific CD8+ T cells in vitro using peptide- and protein-loaded ipAPC-GM Typically, antigen-presenting cells (APCs) internalize extracellular antigens, process them into peptides, and then present the peptide antigens to CD4+ T cells via major histocompatibility complex (MHC) class II antigen-presenting molecules. Specialized APCs, such as dendritic cells (DCs), present internalized extracellular antigens to CD8+ T cells via MHC class I molecules. This mechanism is called cross-presentation. The antigen internalization ability of human ipAPC-GM, ipAPC-M, and ipAPC-GMM, prepared in Example 5(2), was examined using ovalbumin (DQ-OVA), which is labeled with a self-quenching fluorescent dye and emits green fluorescence upon internalization and digestion into peptides. Cross-presentation by mouse ipAPC-GM was examined using ovalbumin (OVA) and OVA-specific T cells.

[0188] The human ipAPC-GM, ipAPC-M, and ipAPC-GMM prepared in Example 5(2) were plated in a 24-well tissue culture plate at 5 × 10 4 The ipAPC-GM, ipAPC-M, and ipAPC-GMM were seeded at 1000 cells / well, and the ipAPC-GM, ipAPC-M, and ipAPC-GMM were loaded with 10 μg / mL DQ-OVA and then incubated at 37°C for 4 hours. As a negative control without antigen uptake, a sample incubated at 4°C was also prepared. The frequency of antigen uptake was compared with that of the DCs derived from living bodies induced in Example 5(6). Next, mouse ipAPC-GM prepared from C57BL / 6 strain mouse iPSCs described in Example 6(1) was plated at 5 × 10 4 Cells were seeded at 1000 cells / well, and the ipAPC-GM cells were transfected with OVA peptide OVA. 257-264 Alternatively, ipAPC-GM cells were dose-dependently loaded with OVA protein and then irradiated (85 Gy) to stop proliferation. Then, naive CD8+ T cells OT-1 (1 × 10 ) bearing OVA-specific T cell receptors (TCRs) were transfected. 4The cells were co-cultured with 100% BASF / well for 4 days.

[0189] The results of measuring the intracellular uptake of DQ-OVA by flow cytometry are shown in Figure 12A (left). 3 The results of measurement by [H]-thymidine incorporation are shown in Figure 12A (right). Human ipAPC-GM, ipAPC-M, and ipAPC-GMM were found to take up antigen at a high frequency of approximately 95%, similar to pMC before cytokine introduction. In contrast, the frequency of antigen uptake by DCs derived from living organisms was approximately 75%. Mouse ipAPC-GM was treated with OVA peptide OVA. 257-264 Alternatively, after loading OVA protein in a concentration-dependent manner, the proliferation of ipAPC-GM was stopped by irradiation, and the proliferation of antigen-specific CD8+ T cells in vitro when co-cultured with naive CD8+ T cells OT-1 that have OVA-specific T cell receptors (TCR) was measured. 3 Figure 12B shows a plot of the results quantified by scintillation counting of [H]-thymidine incorporation. Mouse ipAPC-GM induced OT-1 proliferation in a concentration-dependent manner depending on the OVA peptide and protein. Figure 13 shows the results of comparing the OT-1 proliferation-inducing activity of mouse ipAPC-GM and mouse pMC when loaded with 10 μM OVA peptide or 100 μg / mL OVA protein. These results demonstrate that human ipAPC-GM, ipAPC-M, and ipAPC-GMM had a higher frequency of antigen uptake than DCs, and mouse ipAPC-GM was more effective at stimulating antigen-specific T cell proliferation than mouse pMCs.

[0190] (3) Induction of antigen-specific cancer-reactive T cells in vivo The in vivo antigen-specific T cell stimulatory activity of ipAPC-GM was examined using a mouse prevention model. C57BL / 6 mice were treated with mouse ipAPC-GM or mouse pMC alone prepared from C57BL / 6 mouse iPSCs as described in Example 6(1), or 1×10 mouse ipAPC-GM or mouse pMC alone loaded with OVA peptide or OVA protein. 5The cells were intraperitoneally administered twice at 7-day intervals, and then 2 × 10 5 Cells were implanted into mice by subcutaneous injection. Starting on day 5 after implantation, when tumor diameters reached 2–3 mm, tumor diameters were measured twice weekly in mice treated with mouse ipAPC-GM or mouse pMC, as well as in the control group. This measurement continued until the mice died or the tumor diameter reached 20 mm.

[0191] Figure 14 shows the time course of the volume of the transplanted tumor. ipAPC-GM significantly suppressed the increase in tumor volume. The suppressive effect of ipAPC-GM was greater than that of pMC, and the effect was more pronounced when both ipAPC-GM and pMC were loaded with OVA peptide or OVA protein.

[0192] The survival curves of mice transplanted with cancer cells are shown in Figure 15. Consistent with the tumor volume measurements mentioned above, the group administered ipAPC-GM loaded with OVA peptide or OVA protein showed a significantly longer survival than the group administered pMC loaded with OVA peptide or OVA protein.

[0193] Figure 16 shows the results of in vitro detection of OVA peptide antigen-specific T cells in the spleen 56 days after transplantation. CD8+ T cells were isolated from the collected peripheral blood mononuclear cells using beads coated with anti-CD8 antibody. The resulting cells were stimulated with OVA peptide or control SIY peptide, and 36 hours later, the frequency of antigen-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assay to detect interferon-γ. OVA-specific T cells were detected at a high frequency in the ipAPC-GM-treated mice. These findings demonstrate that the ipAPC-GM of the present invention has the function of presenting desired antigens, such as cancer antigens, to CD8+ T cells and further promotes the proliferation of CD8+ T cells bearing the antigen-specific T cell receptor.

[0194] Example 7: Induction of homeostatic expansion of CD8+ T cells by ipAPC-GM (1) Promotion of naive T cell proliferation by GM-CSF and ipAPC-GM culture supernatant The effect of GM-CSF produced by the mouse ipAPC-GM prepared in Example 5 on the proliferation of naive OT-1 CD8-positive T cells was examined. Naive OT-1 CD8-positive T cells (1 × 10 5 T cells (1000 cells / well) were cultured for 6 days in the presence of various concentrations of recombinant GM-CSF protein or ipAPC-GM culture supernatant. The contribution of GM-CSF was also verified by adding an anti-GM-CSF neutralizing antibody to the ipAPC-GM culture supernatant. T cell proliferation was [ 3 H]-thymidine incorporation was assessed.

[0195] Naive OT-1 CD8+ T cells were cultured in the presence of various concentrations of recombinant GM-CSF. T cell proliferation depended on the GM-CSF concentration, with GM-CSF concentrations of at least 1.5 ng / mL required. Figure 17 shows the results of T cell proliferation in the presence of ipAPC-GM or pMC culture supernatant. ipAPC-GM culture supernatant promoted T cell proliferation, while pMC culture supernatant did not. Figure 18 shows the results of T cell proliferation assessed in the presence of ipAPC-GM culture supernatant with the addition of an anti-GM-CSF neutralizing antibody. The anti-GM-CSF antibody inhibited the T cell proliferation activity of ipAPC-GM culture supernatant. These results demonstrate that ipAPC-GM not only induces antigen-specific T cells, but also promotes the proliferation of naive CD8+ T cells through the GM-CSF secreted by the cells. In particular, we found that GM-CSF at 1.5 ng / mL or higher is required for the homeostatic expansion of naive CD8+ T cells.

[0196] (2) Enhanced proliferation of naive T cells co-cultured with ipAPC-GM The mouse ipAPC-GM prepared in Example 5 was subjected to 85 Gy of irradiation to stop proliferation of the mouse ipAPC-GM or mouse pMC (5×10 4 The cells were co-cultured with naive CD8+ T cells prepared from C57BL / 6 mice for 4 days to investigate the proliferation of T cells.3 The proliferation activity of T cells cocultured with ipAPC-GM or pMC was evaluated by [H]-thymidine incorporation. Figure 19 shows the proliferation activity of T cells cocultured with ipAPC-GM or pMC. It can be seen that coculture with ipAPC-GM significantly proliferated T cells. It can be seen that the T cell proliferation-promoting effect of ipAPC-GM was significantly higher than that of pMC. Figure 20 shows the results of flow cytometry analysis of T cell receptor (TCR) Vβ frequency in naive CD8+ T cells before and after coculture with ipAPC-GM. It can be seen that coculture with ipAPC-GM promotes the homeostatic expansion of polyclonal T cells without affecting specific TCR clones.

[0197] Example 8: Irradiation of ipAPC-GM controls in vivo dynamics while retaining antigen-presenting ability (disappears from the body within 3-4 days) (1) Apoptosis analysis of ipAPC-GM irradiated with 85 Gy As described above in Examples 5(7) and (8), we found that ipAPC-GM undergo active cell division due to the GM-CSF they produce, resulting in improved viability compared to pMC. While the high self-proliferation capacity of ipAPC-GM is useful for producing the number of cells required for treatment, concerns remain regarding its tumorigenicity after administration in vivo. As described in Science 311, 1160-1164, ipAPC-GM must survive in vivo for several days after transplantation in order for antigen-presenting cells to activate CD8+ T cells, but it is desirable for them to be rapidly cleared thereafter. To address this issue, we irradiated ipAPC-GM and examined the effect on their self-proliferation capacity.

[0198] Mouse ipAPC-GM and mouse pMC prepared in Example 5 were irradiated with 65, 85, and 100 Gy of radiation and cultured for 4 days at 37°C and 5% CO2 in αMEM containing 20% ​​FBS in the absence of cytokines. Human pMC, ipAPC-GM, ipAPC-M, and ipAPC-GMM were irradiated with 2.5, 5, 10, 20, 40, and 80 Gy of radiation and cultured for 6 days in the presence of cytokines 50 ng / mL GM-CSF and 50 ng / mL M-CSF. The cell morphology is shown in Figure 21. ipAPC-GM maintained its morphology even after irradiation. In contrast, control pMC were largely destroyed and damaged by irradiation. Figure 22 shows the cell proliferation of mouse ipAPC-GM, human pMC, ipAPC-GM, ipAPC-M, and ipAPC-GMM after irradiation. 3 The results of evaluation using [H]-thymidine incorporation activity are shown. Cell proliferation was significantly suppressed for both mouse ipAPC-GM and pMC at all irradiation doses. Cell proliferation of human pMC, ipAPC-GM, ipAPC-M, and ipAPC-GMM was arrested at 20 Gy or higher, whereas cell proliferation was observed at low irradiation doses of 10 Gy or lower. These results demonstrate that cell proliferation of human ipAPC-GM, ipAPC-M, and ipAPC-GMM can be controlled by irradiation of 20 Gy or higher.

[0199] The mouse ipAPC-GM prepared in Example 5 was seeded in a 96-well plate and irradiated with 85 Gy. The mouse ipAPC-GM and mouse pMC (1 × 10 5 The ipAPC-GM cells were stained with Annexin V and 7-AAD and analyzed for in vitro viability using a flow cytometer. Figure 23 shows the results. 24 hours after 85 Gy irradiation, the majority of ipAPC-GM cells were negative for both Annexin V and 7-AAD, indicating viability. In contrast, the majority of pMC cells were positive for both Annexin V and 7-AAD, indicating apoptosis.

[0200] (2) Examination of cell proliferation ability of irradiated ipAPC-GM FIG. 24 shows the results of the mouse ipAPC-GM prepared in Example 5, seeded in a 96-well plate, and irradiated with 85 Gy, and the mouse pMCs (2×10 4 The results show that in vitro cell proliferation of ipAPC-GM (77 cells / well) was assessed by MTT assay for three consecutive days after irradiation. Irradiation with 85 Gy suppressed the proliferation of ipAPC-GM, which was observed in non-irradiated cells (0 Gy). In pMC, radiation irradiation caused a significant decrease in MTT activity. This result is consistent with the results in Figure 21, in which pMC were destroyed due to radiation-induced cell damage.

[0201] (3) Pharmacokinetics of irradiated ipAPC-GM A lentiviral vector expressing luciferase as a reporter gene was introduced into the mouse ipAPC-GM prepared in Example 5. Mouse pMCs transfected with the luciferase gene were also prepared in the same manner. Cells expressing the luciferase gene emit bioluminescence upon addition of the luminescent substrate luciferin. This allows optical investigation of the pharmacokinetics of ipAPC-GM administered in vivo.

[0202] Luciferase-expressing ipAPC-GM or pMC (1 × 10 6 Cells) were subcutaneously administered to C57BL / 6 mice. In vivo cell survival of ipAPC-GM and pMC was measured daily by biochemiluminescence using an IVIS imaging system (PerkinElmer). Figure 25 shows in vivo imaging images and the time course of total luciferase luminescence flux. Immediately after administration, ipAPC-GM or pMC were detected as a clear luminescence signal locally at the transplant site, which disappeared over the course of days. ipAPC-GM was retained in vivo for 3 days after subcutaneous administration and disappeared by the fourth day. In contrast, pMC almost disappeared from the body the day after administration.

[0203] From the above, it was found that the ipAPC-GM of the present invention persists in vivo for 3 days after administration, even after radiation exposure, and disappears after 4 days. This indicates that when ipAPC-GM is administered to the body after radiation exposure, it disappears 3 to 4 days after administration.

[0204] (4) In vivo cross-presentation ability of irradiated ipAPC-GM The murine ipAPC-GM and pMC prepared in Example 5 were irradiated to stop their proliferation, then loaded with OVA peptide and administered intraperitoneally to C57BL / 6 mice. MO4 cancer cells were then implanted subcutaneously, and tumor size and mouse survival were assessed every 3 to 4 days. Non-irradiated bone marrow-derived dendritic cells (BM-DCs) served as a positive control, and an untreated group served as a negative control. ELISPOT assays for interferon-γ were performed 54 days after MO4 implantation.

[0205] Figure 26 shows the progression of tumor volume. The ipAPC-GM-administered group significantly suppressed tumor growth compared to the untreated and pMC-administered groups. The tumor-suppressing effect of ipAPC-GM was comparable to that of the positive control, bone marrow-derived dendritic cells (BM-DCs).

[0206] The survival curves of the mice are shown in Figure 27. The ipAPC-GM-administered group showed a significant prolongation of survival compared to the untreated group. It was found that the survival-prolonging effect of ipAPC-GM administration was not significantly different from that of BM-DCs or pMCs.

[0207] The results of the ELISPOT assay are shown in Figure 28. The ipAPC-GM-administered group significantly induced CD8+ T cells specific for the cancer antigen OVA compared to the untreated and pMC-administered groups. The induction of cancer-specific CD8+ T cells by ipAPC-GM was found to be equivalent to that by BM-DCs.

[0208] From the above, it was found that the ipAPC-GM of the present invention has the function of presenting desired antigens, such as cancer antigens, to CD8-positive T cells even after radiation exposure, and furthermore, it proliferates CD8-positive T cells that have T cell receptors specific to the desired antigen.

[0209] Example 9: Control of in vivo dynamics by introducing a suicide gene into ipAPC-GM Lentiviral vectors expressing HSV-TK or iCasp9 as suicide genes were transduced into mouse ipAPC-GM prepared in Example 5. The ipAPC-GM transduced with HSV-TK is referred to as ipAPC-GM-HSV-TK, and the ipAPC-GM transduced with iCasp9 is referred to as ipAPC-GM-iCasp9. A lentiviral vector expressing luciferase as a reporter gene was further transduced into ipAPC-GM-HSV-TK or ipAPC-GM-iCasp9, and the in vivo kinetics of ipAPC-GM-HSV-TK or ipAPC-GM-iCasp9 administered in vivo were optically analyzed. The luciferase-transduced cells are referred to as ipAPC-GM-Luc, ipAPC-GM-HSV-TK-Luc, and ipAPC-GM-iCasp9-Luc. ipAPC-GM expressing luciferase and a suicide gene or ipAPC-GM expressing only the luciferase gene (1 × 10 6 C57BL / 6 mice were subcutaneously injected with ipAPC-GM-HSV-TK cells. Subsequently, for five consecutive days, from Day 0 to Day 4, mice were intraperitoneally injected with 100 mg / kg GCV, an inducer of the suicide gene HSV-TK, or 2.5 mg / kg AP1903, an inducer of iCasp9. In vivo cell survival of ipAPC-GM-HSV-TK, ipAPC-iCasp9, and ipAPC-GM cells was measured daily by bioluminescence using an IVIS imaging system (PerkinElmer).

[0210] Figure 29 shows the time course of luciferase luminescence obtained from in vivo imaging images. Immediately after injection, HSV-TK-transfected, iCasp9-transfected, and suicide gene-free ipAPC-GM showed clear luminescence signals at the transplantation site, which decreased over time. In mice treated with ipAPC-GM-Luc, no significant signal reduction was observed after GCV or AP1903 administration. In mice treated with ipAPC-HSV-TK-Luc or ipAPC-GM-iCasp9-Luc, the signal from cells retained in vivo after GCV or AP1903 administration significantly decreased and completely disappeared by day 7 after transplantation.

[0211] Figure 30 shows the combined effect of the suicide gene iCasp9 and 5 mg / kg YM-155, an inhibitor of the anti-apoptotic factor Survivin. In mice treated with ipAPC-GM-iCasp9-Luc, treatment with 5 mg / kg YM-155 alone showed a similar signal decay profile to that observed with saline (medium), whereas treatment with AP1903 resulted in a significantly greater signal reduction than AP1903 alone on the day after cell transplantation. These results suggest that the combination of YM-155 and AP1903 induces early apoptosis of the suicide gene iCasp9 in transplanted cells, resulting in rapid cell elimination. Furthermore, the combination of a suicide gene and an anti-apoptotic factor enabled rapid elimination of ipAPCs.

[0212] Example 10: Examination of the safety of in vivo administration of ipAPC-GM (1) Measurement of blood cytokines in mice administered ipAPC-GM The mouse ipAPC-GM prepared in Example 5 was administered ipAPC-GM alone twice, seven days apart, and blood cytokines were measured the day after the second administration. A group administered 4.0 mg / kg of lipopolysaccharide (LPS) was used as a positive control for comparison. No increases in blood levels of IL-6, TNFα, or GM-CSF were observed in mice administered ipAPC-GM, indicating that inflammation was not induced in the body.

[0213] (2) Changes in blood cell fractions in mice administered ipAPC-GM The mouse ipAPC-GM prepared in Example 4(2) was examined by flow cytometry for changes in leukocyte fractions the day after administration of ipAPC-GM. Administration of ipAPC-GM did not significantly affect the frequencies of cell populations such as T cells (B220- / CD3+), B cells (B220+ / CD3-), myeloid cells (CD11b+ / Gr-1-), or bone marrow-derived immunosuppressive cells (CD11b+ / Gr-1+), indicating no effect on leukocyte fractions.

[0214] Example 11: Comparison of the effects of ipAPC-GM with immune checkpoint inhibitors The antitumor effects of ipAPC-GM were compared with those of immune checkpoint inhibitors (ICIs), a known existing cancer immunotherapy, and the combined effects of ICIs and ipAPC-GM were also examined. Mice were implanted with tumor cells by subcutaneous injection and intraperitoneally administered ipAPC-GM, ICIs (anti-CTLA-4 antibody and anti-PD-L1 antibody), or a combination of ipAPC-GM and ICIs. Tumor size and mouse survival were assessed every 3–4 days. Figure 31 shows the antitumor effects against MO4 tumors, a cancer cell line that is insensitive to immune checkpoint inhibitors (ICIs). OVA was used as the cancer antigen for MO4 tumors. 257-264 Peptides were loaded onto ipAPC-GM. Compared with ICI treatment alone, ipAPC-GM significantly suppressed tumor growth and prolonged mouse survival. Furthermore, the combination of ICI and ipAPC-GM tended to suppress tumor growth and prolong mouse survival more than ipAPC-GM treatment alone.

[0215] Next, the antitumor effect on ICI-sensitive MC38 tumor cells is shown in Figure 32. ipAPC-GM was loaded with mutant Adpgk (mAdpgk) peptide as a cancer antigen for MC38 tumors, and wild-type Adpgk (wtAdpgk) peptide as a negative control for the cancer antigen. Compared with wtAdpgk peptide-loaded ipAPC-GM, the mAdpgk peptide-loaded ipAPC-GM treatment group showed a tendency toward suppression of tumor size growth, although no significant difference was observed, and significantly prolonged mouse survival. Compared with ICI treatment alone, no significant differences were observed in the time course of tumor size or mouse survival for mAdpgk peptide-loaded ipAPC-GM. The combined use of ICI and mAdpgk peptide-loaded ipAPC-GM significantly suppressed tumor size growth and prolonged mouse survival compared with ICI treatment alone, although no significant differences were observed.

[0216] Example 12: Effect of ipAPC-GM on intratumoral myeloid-derived immunosuppressive cells (MDSCs) Figure 33 shows the effect of combining ipAPC-GM with immune checkpoint inhibitors (ICIs), a known cancer immunotherapy, on intratumoral myeloid-derived suppressor cells (MDSCs). Mice bearing subcutaneous tumors of the ICI-resistant cancer cell line MO4 were treated with ipAPC-GM or with a combination of ICIs (anti-CTLA-4 antibody and anti-PD-L1 antibody) and ipAPC-GM. After treatment, intratumoral MDSCs were assessed by flow cytometry. MDSCs suppress immune responses and are thought to contribute to resistance to cancer immunotherapy. Compared to 15.5% in untreated tumor tissue, treatment with ipAPC-GM alone significantly reduced the percentage of intratumoral MDSCs to 12.1%, and treatment with ipAPC-GM in combination with an ICI significantly reduced the percentage to 4.33%.

[0217] Example 13: Effect of ipAPC-GM on intratumoral CD8-positive T cells Figure 34 shows the results of flow cytometry analysis of intratumoral CD8+ T cells after treatment, as obtained in Example 11. Perforin and granzyme (GzmB) are cytotoxic granules secreted by CD8+ T cells that recognize antigenic peptides. Perforin polymerizes on the membrane of target cancer cells, creating pores through which GzmB invades the target cancer cells, inducing cell death. Administration of ipAPC-GM increased the percentage of intratumoral CD8+ T cells expressing Perforin and GzmB from 0.52% to 6.19% compared with untreated control. Treatment with ipAPC-GM in combination with an ICI (anti-CTLA-4 antibody + anti-PD-L1 antibody) further increased this percentage to 12.3%. The reduction in intratumoral MDSCs and the increase in Perforin- and GzmB-positive CD8+ T cells are thought to contribute to the antitumor effect of ipAPC-GM. These results also revealed that the combined use of ipAPC-GM with various ICIs could produce a stronger antitumor effect.

[0218] Comparative Example 1: Feeder-free differentiation-inducing culture of human iPSCs The differentiation induction in Example 3(4) was compared with the differentiation induction method described in a non-patent document (Gene Therapy 2011 18, 874-883). Human EBs generated from human iPSCs were seeded onto 12-well tissue culture plates coated with recombinant human fibronectin and cultured overnight in StemFit medium at 37°C in a 5% CO2 incubator. iPSC colonies attached to the plates were cultured for 10 days in a 1:1 mixture of AIM-V (Life Technologies) and X-VIVO15 (Lonza) in the presence of 50 ng / mL BMP-4. Further culture was performed for 14 days using X-VIVO15 in the presence of 50 ng / mL BMP-4. The resulting cells were detached and collected using TripLE (Life Technologies), seeded onto low-adhesion plates (Corning), and cultured for 7 days in the presence of 50 ng / mL GM-CSF and 50 ng / mL M-CSF using X-VIVO15 to obtain differentiated cells. Differentiated cells on days 18 and 25 of culture were examined by flow cytometry for the expression of hematopoietic differentiation markers CD34 and CD43, or myeloid differentiation markers CD11b and CD11c.

[0219] The differentiated cells were collected by pipetting and stained with anti-CD11b, anti-CD11c, anti-CD34, anti-CD43, or an isotype-matched control antibody. The cells were then washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer. Figure 35 shows the results of surface antigen analysis using a flow cytometer after antibody staining. The results of this analysis showed that the differentiated cells prepared in Example 3(4) had a frequency of hematopoietic differentiation marker CD34-positive cells of 19.4% on day 18 of differentiation induction. On day 25, the frequency of myeloid differentiation marker CD11b-positive cells was 48.3%.

[0220] Differentiated cells (Comparative Example 1) prepared by the differentiation induction method described in a non-patent document (Gene Therapy 2011 18, 874-883) were negative for the hematopoietic differentiation marker CD34 on day 18 of differentiation induction. Subsequently, the cells died, and significantly fewer cells were recovered. On day 25, the cells were negative for the myeloid differentiation marker CD11b. These results indicated that the differentiation induction protocol used was not reproducible and had poor differentiation induction efficiency. [Industrial Applicability]

[0221] The human professional antigen-presenting cells derived from pluripotent stem cells of the present invention have the ability to proliferate in vitro and antigen-presenting ability comparable to that of DCs derived from living organisms, making it possible to ensure a stable supply as a cell preparation.

Claims

1. Professional antigen-presenting cells (pAPCs) derived from embryonic stem cells or induced pluripotent stem cells, which have low CD11b / c expression, express CD74 and CD33, have been transfected with GM-CSF and / or M-CSF genes, and express exogenous GM-CSF and / or M-CSF.

2. The professional antigen-presenting cell (pAPC) according to claim 1, which has the ability to self-replicate and exhibits resistance to apoptosis.

3. A pharmaceutical composition for inducing and / or promoting the proliferation of CD8-expressing T cells, comprising the professional antigen-presenting cells described in Claim 1 or 2.

4. A method for promoting the proliferation of T cells expressing antigen-nonspecific or antigen-specific CD8, comprising co-culturing professional antigen-presenting cells or GM-CSF according to claim 1 or 2 with naive T cells expressing CD8 in vitro.

5. The method according to claim 4, wherein the professional antigen-presenting cells are professional antigen-presenting cells loaded with a desired antigen.

6. The method according to claim 4 or 5, further comprising irradiating the professional antigen-presenting cells loaded with the antigen in vitro.

7. The method according to any one of claims 4 to 6, wherein the professional antigen-presenting cells irradiated with radiation disappear within at least 3 to 4 days when administered to a living organism after in vitro radiation irradiation.

8. The method according to any one of claims 4 to 7, wherein the T cells expressing CD8 are T cells that express CD8 specific to the antigen loaded onto the professional antigen-presenting cells.

9. A method for producing T cells expressing antigen-nonspecific or specific CD8, comprising producing T cells expressing antigen-nonspecific or specific CD8 using the method described in any one of claims 4 to 8.

10. (i) Loading the professional antigen-presenting cells described in Claim 2 with an antigen, (ii) Irradiating professional antigen-presenting cells loaded with the antigen, and (iii) Co-culturing professional antigen-presenting cells irradiated with the aforementioned radiation and naive T cells expressing CD8 in vitro, and promoting the proliferation of T cells expressing antigen-specific CD8. A method for producing T cells expressing antigen-specific CD8 for administration into the body, including [the specified component].

Images