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Definitions

• the present invention relates to proliferating macrophage-like cells (pMAC) derived from pluripotent stem cells, a method for producing the same, a method for producing CAR-pMAC, which includes introducing a chimeric antigen receptor (CAR) gene into the pMAC, and a method for producing the same. It relates to manufactured CAR-pMAC, etc.
• pMAC macrophage-like cells
• CAR chimeric antigen receptor
• Non-Patent Document 1 Cancer treatment using CAR-T cells kills cancer cells through a mechanism different from that of conventional anticancer drugs and radiation therapy, so it is effective even against intractable or treatment-resistant cancers. is expected.
• CAR-T cells have already been formulated for blood tumors such as some leukemias and malignant lymphomas. However, its effectiveness against solid cancers is limited.
• CCR2, CXCR2 cancer-directed receptors
• CAR cancer-directed receptors
• immune checkpoint control anti-PD-1 antibody, PD-1-CD28
• IL7 cytokine production ability
• iPS-T/NK cells SCM-T cells
• dual antigen recognition system there are currently no reports showing significant clinical effects on solid cancers.
• Non-Patent Document 2 a cell platform in which CAR is loaded on macrophages rather than T cells is effective in treating solid cancers.
• This is based on the following properties/functions inherent in macrophages. 1. 2. Expresses cancer-oriented receptors and accumulates and invades cancer. 3. Does not suffer from PD-1/PD-L1-mediated functional decline. Activation (type 1 macrophage (M1) conversion) induces a secondary immune response.
• Patent Documents 1 and 2 also describe CAR-equipped monocytes, macrophages, and dendritic cells, and pharmaceutical products containing these cells. A typical composition is described.
• Non-Patent Document 3 describes methods for differentiating myeloid cells from pluripotent stem cells. Furthermore, a technique for introducing CAR into iMAC based on iPS cell-derived macrophages has been developed (Non-Patent Document 3). However, with this method, the final product cannot be propagated, and the differentiation induction performed at any time is complicated, and there is a concern that the manufacturing cost will rise.
• the present invention aims to provide a novel CAR-equipped cell platform, particularly a CAR-equipped macrophage-like cell platform with excellent quality stability, stable supply, and economic efficiency.
• the present inventors focused on macrophage cells as immune cells that carry CAR.
• CAR-carrying macrophage cells derived from pluripotent stem cells it becomes possible to use allogeneic cells, and furthermore, by imparting cytokine-dependent proliferation activity to the macrophage cells, large amounts of They discovered that production is possible and completed the present invention. That is, the present invention is as follows.
• pMAC Proliferating macrophage - like cells
• GM-CSF granulocyte-macrophage colony-stimulating factor
• M-CSF macrophage colony-stimulating factor
• the pMAC according to [4], wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
• the pMAC according to [6], wherein the gene that inhibits macrophage-like function is the signal regulatory protein ⁇ (SIRP ⁇ ) gene.
• SIRP ⁇ signal regulatory protein ⁇
• CAR chimeric antigen receptor
• a method for producing proliferative macrophage-like cells comprising the steps of deleting or suppressing the expression of a gene that inhibits macrophage-like function, and expressing a gene that adds proliferative properties.
• the manufacturing method according to [9] comprising: [11] (A) Step A to obtain proliferative myeloid cells by expressing a gene that adds proliferative properties to myeloid cells differentiated from pluripotent stem cells (Step A), and (B) proliferative properties obtained in Step A.
• Step B The manufacturing method according to [9], comprising: [12] The production method according to any one of [9] to [11], wherein the gene that inhibits macrophage-like function is the signal regulatory protein ⁇ (SIRP ⁇ ) gene. [13] The production method according to any one of [9] to [12], wherein the gene that adds proliferation is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.
• a method for producing CAR-pMAC which comprises introducing a chimeric antigen receptor (CAR) into pMAC obtained by the production method according to any one of [9] to [13].
• CAR chimeric antigen receptor
• a method for producing CAR-pMAC comprising: [16] The production method according to [15], wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells. [17] The production method according to [15] or [16], wherein the induction of differentiation into myeloid cells in Step 1 is carried out by the method (i) or (ii) below. (i) Embryoid bodies (EBs) derived from pluripotent stem cells are cultured in a layered manner on feeder cells in a medium containing VEGF, and then further cultured in a medium containing VEGF, SCF and TPO.
• EBs Embryoid bodies
• embryoid bodies (EBs) derived from pluripotent stem cells are cultured in a monolayer without feeder cells in a medium containing BMP-4, VEGF and SCF, and then further cultured with VEGF, TPO and Further culturing in a medium containing GM-CSF.
• induction of pluripotent stem cells into embryoid bodies (EB) is performed by the following methods (a) and (b): (a) Pluripotent stem cells are seeded in a cell cluster-forming culture container to form embryoid bodies (EBs), and (b) the container is provided with a fine spheroid well at the bottom and adjacent wells. There should be no flat surface between them.
• step 1-1 Deleting or suppressing the expression of genes that inhibit macrophage-like functions in pluripotent stem cells (step 1-1),
• the pluripotent stem cells obtained in step 1-1, in which the gene that inhibits macrophage-like function has been deleted or whose expression has been suppressed, are placed in a cell with a fine spheroid well at the bottom, and a flat surface between adjacent wells.
• a method for producing CAR-pMAC comprising: [20] The production method according to any one of [15] to [19], wherein the gene that inhibits macrophage-like function is the signal regulatory protein ⁇ (SIRP ⁇ ) gene. [21] The production method according to any one of [15] to [20], wherein the gene that adds proliferation property is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.
• Cell groups along the macrophage cell lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, living body-derived monocytes, living body-derived macrophages M1, living body-derived macrophages M2, and living body-derived macrophages M0 were used as comparison targets.
• iPS cell-derived myeloid cells iPS cell-derived macrophages
• living body-derived monocytes living body-derived macrophages M1, living body-derived macrophages M2, and living body-derived macrophages M0
• proliferative macrophage-like cells that meet the following conditions are identified.
• Condition 1 Does not overlap with known macrophage cell lineage plots
• Condition 2 PC1 is 100 or more, and PC2 is plotted in the range of -80 to 0.
• a pharmaceutical composition comprising the proliferative macrophage-like cells described in [8] and a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier for the treatment of at least one selected from the group consisting of tumors or cancer-related diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis.
• Pharmaceutical compositions as described.
• At least one treatment method selected from the group consisting of vascular diseases, fibrotic diseases, and amyloidosis.
• CAR-equipped proliferative macrophage-like cells with excellent quality stability, stable supply, and economic efficiency can be produced.
• CAR-equipped proliferating macrophage-like cells are also effective in treating solid cancers.
• BM-derived monocytes bone marrow-derived monocytes
• BM-derived macrophages M0 bone marrow-derived macrophages M0
• mouse bone marrow-derived M1 type macrophages BM-derived macrophages M1
• mouse bone marrow-derived M2 type macrophages BM-de rived
• PC1 contribution rate 346%
• PC2 contribution rate 21%)
• Mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 type macrophages (BM-derived) macrophages M1), derived from mouse bone marrow Histogram showing the results of flow cytometer analysis of CD11b, F4/80, CD16/32, CD64, IA/E, and CD206 surface antigens of M2 type macrophages (BM-derived macrophages M2) and mouse pMAC.
• BM-derived monocytes mouse intraperitoneal macrophages
• BM-derived M1 type macrophages BM-derived M1 type macrophages
• the vertical axis represents the percentage of cells present (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 type macrophages (BM-derived) macrophages M1), derived from mouse bone marrow A histogram showing the results of flow cytometer analysis of CD163, Ly6C, CD80, CD86, SiglecF, and SIRP ⁇ surface antigens of M2 type macrophages (BM-derived macrophages M2) and mouse pMAC.
• the vertical axis represents the percentage of cells present (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 type macrophages (BM-derived) macrophages M1), derived from mouse bone marrow Histogram showing the results of TLR2, TLR4, CD124, CCR1, CCR2, CCR3, and CCR4 surface antigen analysis by flow cytometer of M2 type macrophages (BM-derived macrophages M2) and mouse pMAC.
• the vertical axis represents the percentage of cells present (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 type macrophages (BM-derived) macrophages M1), derived from mouse bone marrow Histogram showing the results of CCR5, CXCR2, and CXCR4 surface antigen analysis by flow cytometer of M2 type macrophages (BM-derived macrophages M2) and mouse pMAC.
• the vertical axis represents the percentage of cells present (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Results are shown for mouse pMAC (upper left), CAR-pMAC (upper right), pMAC-SIRP ⁇ -KO (lower left), and CAR-pMAC-SIRP ⁇ -KO (lower right).
• the vertical axis represents the concentration of formazan metabolized from the MTT reagent (evaluated as absorbance at 595 nm), and the horizontal axis represents the number of days of culture. There was no change in cytokine-dependent cell proliferation performance due to the deletion of the SIRP ⁇ gene and/or the introduction of CAR against the cancer antigen GPC3, which was carried out for the production of pMAC.
• Cytogram showing the results of hGPC3-FITC, SIRP ⁇ and tdTomato expression analysis of mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO using a flow cytometer.
• the vertical axis is the fluorescence intensity of hGPC3-FITC, and the horizontal axis is the fluorescence intensity of tdTomato (upper row).
• the vertical axis is the fluorescence intensity of SIRP ⁇ , and the horizontal axis is the fluorescence intensity of tdTomato (middle row).
• the vertical axis is the fluorescence intensity of SIRP ⁇
• the horizontal axis is the fluorescence intensity of hGPC3-FITC (lower row). Since pMAC expressing GPC3-CAR is obtained as a single cell population, it is found to be highly pure and contain almost no unintended cells.
• Mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, CAR-pMAC-SIRP ⁇ -KO, and mouse bone marrow-derived macrophages (BM-Macrophage) were subjected to M1 stimulation with medium without stimulating components, interferon ⁇ , and LPS.
• the vertical axis shows the measured cytokine concentration (pg/mL).
• the horizontal axis shows the culture time, and the vertical axis shows the Cell Index (CI), which indicates the degree of cell migration ability relative to the measurement start time (0 hr).
• Cytokines in the culture supernatant when mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO and mouse cancer cells (without GPC3 expression or with GPC3 expression) were co-cultured for 48 hours ELISA measurement results of IL-6 production.
• Cytokines in the culture supernatant when mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO and mouse cancer cells (without GPC3 expression or with GPC3 expression) were co-cultured for 48 hours ELISA measurement results of TNF ⁇ production.
• Cytokines in the culture supernatant when mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO and mouse cancer cells (without GPC3 expression or with GPC3 expression) were co-cultured for 48 hours ELISA measurement results of IL-10 production.
• Cytokines in the culture supernatant when mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO and mouse cancer cells (without GPC3 expression or with GPC3 expression) were co-cultured for 48 hours ELISA measurement results of IL-12 production.
• Lung cancer cell line LL/2 human GPC3 expression Absent or present; upper left
• colon cancer cell line CT26 no or present human GPC3 expression; upper right
• colon cancer cell line MC38 no or present human GPC3 expression; lower left
• breast cancer cell line 4T1 no human GPC3 expression
• Bar graph quantifying the luciferase activity of cancer cells after 48 hours of co-culture with or present (lower right).
• the vertical axis shows the reduction rate (%) of cancer cells.
• White bars represent cancer cells that do not express hGPC3
• black bars represent cancer cells that express hGPC3. It can be seen that CAR-introduced pMAC (D) eliminates cancer cells expressing the target cancer antigen human GPC (hGPC).
• Colon cancer cell line CT26 (with or without human GPC3 expression; upper left), colon cancer cell line MC38 (with or without human GPC3 expression; upper right), lung cancer cell line LL/2 (with or without human GPC3 expression; lower left) , and breast cancer cell line 4T1 (with or without human GPC3 expression; bottom right) were cultured for 24 hours, and then CAR-pMAC-SIRP ⁇ -KO was added at 10 times, 5 times, and 1 times the amount of the seeded cancer cells.
• a diagram plotting the cytotoxic activity (%Cytolysis) after 6 hours of co-culturing The vertical axis shows the cytotoxic activity (%Cytolysis) of cancer cells, and the horizontal axis shows the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRP ⁇ -KO) and target cells (cancer cells). .
• E:T ratio the mixing ratio of effector cells
• target mouse cancer cells with or without human GPC3 expression
• mouse CAR-pMAC-SIRP ⁇ -KO was used as an effector cell and co-cultured with target mouse cancer cells (with or without human GPC3 expression) at E:T ratios of 10:1, 5:1, and 1:1.
• CAR-pMAC-SIRP ⁇ -KO exhibits higher cytotoxic activity after 6 hours of co-culture with cancer cells compared to pMAC, CAR-pMAC, and pMAC-SIRP ⁇ -KO.
• Line graph showing phagocytes counted by time-lapse observation when mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, or CAR-pMAC-SIRP ⁇ -KO and mouse cancer cells MC38 (expressing GPC3) were co-cultured.
• the horizontal axis shows the co-culture time, and the vertical axis shows the number of phagocytic cells.
• CAR-pMAC-SIRP ⁇ -KO was administered to C57BL/6 line tumor-bearing mice that had been transplanted with MC38 (GPC3 expression) to form peritoneal dissemination, Day 0 before administration (upper row), 1 day later (middle row), Results of measuring the pharmacokinetics of CAR-pMAC-SIRP ⁇ -KO by quantitative PCR after 3 days (lower row).
• the horizontal axis shows each organ evaluated, and the vertical axis shows the common logarithm of the number of CAR-pMAC cells detected in 100 ⁇ g of genomic DNA of each organ.
• a mouse colon cancer cell line MC38 expressing the luciferase gene and the cancer antigen GPC3 was intraperitoneally administered to wild-type C57BL/6 mice, and the mice were untreated or the next day, 3rd day, 7th day, 10th day, and 14th day.
• the vertical axis of the lower left line graph represents the total flux of luciferase luminescence (unit: 10 10 photons/s), and the horizontal axis represents the number of days after cancer cell transplantation.
• the vertical axis of the lower right graph represents the mouse survival rate, and the horizontal axis represents the number of days after cancer cell transplantation.
• Human pMAC blood-derived CD14-positive cell-derived monocytes (CD14-derived monocytes), blood-derived CD14-positive cell-derived macrophages M0 (CD14-derived Macrophages M0), blood-derived CD14-positive cells-derived M1 type macrophages (CD14-derived Ma crophages M1) , blood-derived CD14-positive cell-derived M2 type macrophages (CD14-derived Macrophages M2), human iPS cell-derived myeloid cells (iPSC-derived Myeloid cells), and human iPS cell-derived macrophages (iPSC-derived macrophages) obtained by RNA sequence analysis of A diagram showing the principal component analysis of the gene expression profile of each cell and plotting the first principal component (PC1: contribution rate 40%) on the horizontal axis and the second principal component (PC2: contribution rate 18%) on the horizontal axis.
• PC1 contribution rate 40%
• PC2 contribution rate 18%
• Human blood collection-derived CD14-positive cell-derived monocytes (CD14-derived monocytes), pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC-SIRP ⁇ -KO, blood collection-derived CD14-positive cell-derived macrophages (CD14-derived macrophages M0), blood collection Origin CD14 M1-type macrophages derived from positive cells (CD14-derived macrophages M1), M2-type macrophages derived from CD14-positive cells derived from blood collection (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSCs) A histogram showing the results of HLA-ABC, HLA-DR, CD40, CD80, and CD83 surface antigen analysis using a flow cytometer.
• the vertical axis represents the abundance ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Human blood collection-derived CD14-positive cell-derived monocytes CD14-derived monocytes
• pMAC pMAC-SIRP ⁇ -KO
• CAR-pMAC-SIRP ⁇ -KO blood collection-derived CD14-positive cell-derived macrophages
• CD14-derived macrophages M0 blood collection Origin CD14 M1-type macrophages derived from positive cells (CD14-derived macrophages M1), M2-type macrophages derived from CD14-positive cells derived from blood collection (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSCs) -derived macrophages) using a flow cytometer to analyze CD86, CD68, CD163, CD206, and SIRPa surface antigens.
• the vertical axis represents the abundance ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Human blood collection-derived CD14-positive cell-derived monocytes CD14-derived monocytes
• pMAC pMAC-SIRP ⁇ -KO
• CAR-pMAC-SIRP ⁇ -KO blood collection-derived CD14-positive cell-derived macrophages
• CD14-derived macrophages M0 blood collection Origin CD14 M1-type macrophages derived from positive cells (CD14-derived macrophages M1), M2-type macrophages derived from CD14-positive cells derived from blood collection (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSCs)
• a histogram showing the results of CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CXCR2, CXCR4 and CD14 surface antigen
• the vertical axis represents the abundance ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.
• Human blood-derived CD14-positive cell-derived monocytes CD14-derived monocytes
• iPS cell-derived myeloid cells iPSC-derived myeloid cells
• pMAC blood-derived CD14-positive cell-derived macrophages
• CD14-derived macrophage CD14-derived macrophage
• CD14 positive from blood collection Phase-contrast microscope images of cell-derived M1 type macrophages (CD14-derived macrophages M1) and blood-derived CD14-positive cell-derived M2 type macrophages (CD14-derived macrophages M2).
• the scale bar indicates 40 ⁇ m.
• iPS cell-derived macrophages Human iPS cell-derived macrophages (iPSC-macrophages), pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO without stimulation (M0), interferon ⁇ and LPS stimulation (M1), IL-4 Phase contrast microscopy image under stimulation (M2) condition.
• MTT assay to examine the effect of medium containing GM-CSF and/or M-CSF ( ⁇ MEM containing 20% FBS) on the proliferation rate of human pMAC, pMAC-SIRP ⁇ -KO, and iPS cell-derived macrophages (iPSC-macrophages). Line graph showing measurement results.
• FIG. 1 Shows the results of human pMAC (left), pMAC-SIRP ⁇ -KO (center), and iPSC-macrophages (right).
• the vertical axis represents the concentration of formazan metabolized from the MTT reagent (evaluated as absorbance at 595 nm), and the horizontal axis represents the number of days of culture.
• Cytogram showing the results of hGPC3-FITC, SIRP ⁇ and tdTomato expression analysis using a flow cytometer of human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO.
• the vertical axis is the fluorescence intensity of hGPC3-FITC, and the horizontal axis is the fluorescence intensity of tdTomato (upper row).
• the vertical axis is the fluorescence intensity of SIRP ⁇ , and the horizontal axis is the fluorescence intensity of tdTomato (lower row). Since pMAC expressing GPC3-CAR is obtained as a single cell population, it is found to be highly pure and contain almost no unintended cells.
• the vertical axis shows the measured cytokine concentration (pg/mL).
• CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO Cell migration ability of CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO to culture supernatant derived from cancer cell line HepG2 (bottom row).
• the horizontal axis shows the culture time, and the vertical axis shows the Cell Index (CI), which indicates the degree of cell migration ability relative to the measurement start time (0 hr).
• Cytokines IL-6 left
• TNF ⁇ second from the left
• IL- in the culture supernatant when human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO were cultured for 48 hours. 10 (third from the left), ELISA measurement results of IL-12 (right) production.
• Cytokines in the culture supernatant when human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO was co-cultured with human cancer cell lines (without GPC3 expression or with GPC3 expression) for 48 hours ELISA measurement results of IL-6 production.
• the four types of pMAC described above liver cancer cell line SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top row), liver cancer cell line JHH7-GPC3-KO (no GPC3 expression) Or the result of co-culturing with JHH7 (expressing GPC3) (second row from the top).
• Cytokines in the culture supernatant when human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO was co-cultured with human cancer cell lines (without GPC3 expression or with GPC3 expression) for 48 hours ELISA measurement results of IL-10 production.
• the four types of pMAC described above the liver cancer cell line SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (third row from the top), the liver cancer cell line JHH7-GPC3-KO (GPC3 Results of co-culturing with JHH7 (no expression) or JHH7 (expressing GPC3) (bottom row).
• Cytokines in the culture supernatant when human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO was co-cultured with human cancer cell lines (without GPC3 expression or with GPC3 expression) for 48 hours ELISA measurement results of TNF ⁇ production.
• the four types of pMAC described above cancer cell line SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top row), cancer cell line JHH7-GPC3-KO (no GPC3 expression) or JHH7 (with GPC3 expression) (second row from top).
• Cytokines in the culture supernatant when human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC or CAR-pMAC-SIRP ⁇ -KO was co-cultured with human cancer cell lines (without GPC3 expression or with GPC3 expression) for 48 hours ELISA measurement results of IL-12 production.
• the four types of pMAC described above cancer cell line SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (third row from the top), cancer cell line JHH7-GPC3-KO (no GPC3 expression) ) or JHH7 (expressing GPC3) (bottom row).
• Human pMAC (A), CAR-pMAC (B), pMAC-SIRP ⁇ -KO (C) and CAR-pMAC-SIRP ⁇ -KO (D) and human liver cancer cell line JHH7-Luc into which luciferase (Luc) gene was introduced
• Human GPC3 expressed; left Human liver cancer cell line SK-Hep1-GPC3-Luc-Venus (human GPC3 expressed; second from left)
• human cancer cell line KOC7c-GPC3-Luc (human GPC3 expressed; Bar graph showing the results of quantifying the luciferase activity of cancer cells after 48 hours of co-culturing (third from the left).
• the vertical axis shows the cytotoxic activity (%Cytolysis) of cancer cells
• the horizontal axis shows the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRP ⁇ -KO) and target cells (cancer cells).
• E:T ratio the mixing ratio of effector cells
• target human cancer cells with or without human GPC3 expression
• Human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO were co-cultured with the target cell human SK-Hep1 (no GPC3 expression) at an E:T ratio of 10:1 as effector cells.
• a line graph (middle left) plotting the time course of Normalized Cell Index showing the cell survival of SK-Hep1 (no GPC3 expression) on the vertical axis and the co-culture time on the horizontal axis, human liver cancer cells SK- A line graph (bottom left) plotting the cytotoxic activity %Cytolysis of Hep1 (no GPC3 expression) on the vertical axis.
• Human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO were used as effector cells, and human cancer cells SK-Hep1 (with GPC3 expression) as target cells were used at an E:T ratio of 10:1.
• a line graph (middle right) plotting the time course of Normalized Cell Index indicating cell survival of SK-Hep1 (with GPC3 expression) when co-cultured on the vertical axis and the co-culture time on the horizontal axis.
• a line graph (bottom right) plotting the cytotoxic activity %Cytolysis of human liver cancer cells SK-Hep1 (with GPC3 expression) on the vertical axis. It can be seen that CAR-pMAC-SIRP ⁇ -KO and CAR-pMAC exhibit higher cytotoxic activity after 6 hours of co-culture with cancer cells compared to pMAC and pMAC-SIRP ⁇ -KO.
• the present invention provides macrophage cells characterized by their phagocytic ability and cytokine-dependent proliferation. Since the macrophage cells of the present invention have characteristics different from existing macrophages, they will be referred to as proliferating macrophage -like cells (pMAC) (hereinafter also referred to as pMAC of the present invention). ). Alternatively, the pMAC of the present invention can also be referred to as a cytokine-dependent proliferative macrophage. In one embodiment, the pMAC of the present invention has a gene that inhibits macrophage-like function deleted or whose expression is suppressed.
• pMAC proliferating macrophage -like cells
• cytokine stimulation specifically includes a step of culturing cells in a cytokine-containing medium.
• the cytokine may be added exogenously to the medium or may be produced intracellularly and secreted into the medium.
• GM-CSF granulocyte-macrophage colony-stimulating factor
• M-CSF macrophage colony-stimulating factor
• GM-CSF also known as CSF2
• CSF2 is produced when cells such as T cells, macrophages, endothelial cells, and fibroblasts receive immune stimulation, and functions as a hematopoietic growth factor and an immunomodulatory factor.
• GM-CSF induces bone marrow progenitor cells and promotes granulocyte and macrophage proliferation.
• M-CSF is a type of cytokine that induces proliferation and differentiation of the monocyte-macrophage system among bone marrow stem cell systems. Each cytokine is commercially available.
• sequence information of the protein, genomic DNA or cDNA of each cytokine for various biological species can be obtained from a database well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI). Since it can be obtained from information sources, it can be obtained by expressing the gene of the desired cytokine within the cell, producing the protein within the cell, and in some cases secreting it outside the cell, based on the information.
• NCBI National Center for Biotechnology Information
• the pMAC of the present invention proliferates in a cytokine-dependent manner, it is also characterized by being negative for CD83 and CD14.
• CD83 is known as a dendritic cell maturation marker expressed on the cell surface. It can be detected by performing immunostaining using an anti-CD83 antibody and analyzing it with a flow cytometer.
• CD14 is known as a marker for monocytes and macrophages expressed on the cell surface. It can be detected by performing immunostaining using an anti-CD14 antibody and analyzing it with a flow cytometer.
• CD83 and CD14 are negative means that CD83 and CD14 are not expressed or substantially not expressed. Specifically, this means that the cell surface markers CD83 antigen and CD14 antigen are not detected on the cell surface, are below the detection limit, or are detected in very small quantities. Compared to the expression levels of CD83 and CD14, it is 1/5 or less, preferably 1/10 or less, more preferably 1/20 or less, even more preferably 1/30 or less, and most preferably below the detection limit.
• the expression levels of CD83 and CD14 can be confirmed by known methods such as flow cytometry, quantitative PCR, microarray, Northern hybridization, immunohistochemical staining using antibodies against each antigen, and Western blotting.
• the pMAC of the present invention is characterized by a significantly altered gene expression profile compared to cells included in known macrophage cell lineages. do. Therefore, the pMAC of the present invention can also be defined using statistics based on gene expression profiles.
• the pMAC of the present invention can also be defined using statistics based on gene expression profiles.
• one example can be defined as follows, as demonstrated in detail in the Examples and Figures (particularly Figure 17): Whole transcripts using cell groups along the macrophage cell lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, living-derived monocytes, living-derived macrophages M1, living-derived macrophages M2, and living-derived macrophages M0 as comparison targets.
• the pMAC of the present invention has a PC1 of 100 or more, preferably 110 or more, 120 or more, 130 or more, or 140 or more, more preferably 150 or more, but is not limited thereto.
• the pMAC of the present invention may have a PC1 of 250 or less, preferably 220 or less, 210 or less, 200 or less, or 190 or less, more preferably 180 or less, but is not limited thereto.
• the pMAC of the present invention has a PC1 of 100 to 250 or less, preferably 110 to 220, 120 to 210, 130 to 200, or 140 to 190, more preferably 150 to 180, Not limited to these.
• the pMAC of the present invention is derived from pluripotent stem cells, more preferably from induced pluripotent stem cells or embryonic stem cells.
• Pluripotent stem cells are those that can be cultured in an undifferentiated state in vitro and are derived from all the cells that make up the body (excluding the placenta) (the three germ layers (ectoderm, mesoderm, endoderm)). stem cells that have the ability to differentiate into different tissues (pluripotency).
• "Pluripotent stem cells” also include embryonic stem cells (ES cells) isolated from embryos after fertilization before the formation of three germ layers, embryos and fetuses after the stage of organogenesis including primordial germ cells, or fetuses.
• ES cells embryonic stem cells isolated from embryos after fertilization before the formation of three germ layers, embryos and fetuses after the stage of organogenesis including primordial germ cells, or fetuses.
• induced pluripotent stem cells refer to somatic cells that have once lost their pluripotent ability to become pluripotent by directly reprogramming them by expressing several types of genes such as Oct3/4, Sox2, Klf4, and Myc. (hereinafter also referred to as iPSC or iPS cell). These "induced pluripotent stem cells” are also included in “pluripotent stem cells.” Pluripotent stem cells can be produced by known methods. As for the production method, for example, for human induced pluripotent stem cells, see Cell, 2007, 131 (5) pp. 861-872, Kitayama, S. (Stem Cell Reports.
• Pluripotent stem cells can also be obtained from animals subjected to embryo manipulation techniques, including cloned embryos, transgenic animals, and mutant animals produced by genome editing. Pluripotent stem cells are known to be capable of differentiating into various cell types, including myeloid cells (MC cells).
• MC cells myeloid cells
• iPSCs Human or mouse iPSCs can be maintained and expanded using the regenerative medicine medium StemFit (Ajinomoto). Laminin511 may be added at this time. Further, in order to reduce and remove damage to iPSCs caused by dissociating cells for cell subculture, a Rho kinase (ROCK) inhibitor, such as Y-27632, may be added to the medium at about 10 ⁇ M.
• ROCK Rho kinase
• Pluripotent stem cells can be obtained from designated institutions, or commercially available products can also be purchased.
• human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.
• EB5 cells which are mouse embryonic stem cells, are available from RIKEN, and the D3 strain is available from ATCC.
• Pluripotent stem cells can be maintained and cultured by known methods.
• human pluripotent stem cells can be maintained by culturing in a medium supplemented with Knockout TM Serum Replacement (Invitrogen).
• Mouse pluripotent stem cells can be maintained by adding fetal bovine serum (FBS) and Leukemia Inhibitory Factor (LIF) and culturing without feeders.
• FBS fetal bovine serum
• LIF Leukemia Inhibitory Factor
• pMAC can be produced by a method including a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function, and a step of expressing a gene that adds proliferative properties. Each step will be explained below.
• a "gene that inhibits macrophage-like function” refers to a gene that inhibits a function that can be provided by macrophages.
• “Functions that can be provided by macrophages” include phagocytosis, which contributes to the uptake and digestion of foreign substances, antigen presentation, which promotes the activation of other immune cells such as T cells, and secondary functions caused by activation. Examples include eliciting an immune response. Activation of macrophages is broadly classified into classical (M1) activation and selective (M2) activation.
• M1-activated macrophages are obtained by inducing differentiation of monocytes with inflammatory cytokines such as tumor necrosis factor (TNF)- ⁇ and interferon (IFN)- ⁇ , and are used to protect against pathogenic infections. and produce pro-inflammatory cytokines that reduce the infectivity of microorganisms.
• M2-activated macrophages are obtained by inducing differentiation with Th2-type cytokines such as IL-4 and IL-13, suppress host immune responses, promote wound healing and tissue remodeling, and promote intra-tissue activation.
• Th2-type cytokines such as IL-4 and IL-13
• the SIRP ⁇ gene is one of the membrane proteins present on the cell membrane that separates the inside and outside of a cell, and is abundantly present in macrophages. SIRP ⁇ weakens its phagocytosis by binding to CD47, another membrane protein on cells that are phagocytized by macrophages. That is, for example, cells lacking the SIRP ⁇ gene or whose expression is suppressed have lost the ability to bind to CD47 present on the cell membrane of cancer cells, so their phagocytosis is not weakened. As a method for deleting the gene or suppressing its expression, methods commonly practiced in the art can be used. One embodiment includes a nucleic acid that suppresses the expression of the SIRPa gene.
• the nucleic acid may act at any stage of the SIRPa gene, such as at the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. Therefore, examples of nucleic acids that suppress the expression of the SIRP ⁇ gene include nucleic acids that inhibit the transcription of the SIRP ⁇ gene (e.g., antigene), nucleic acids that inhibit the processing of early transcripts into mRNA, and nucleic acids that inhibit the translation of mRNA into protein. Nucleic acids that inhibit (eg, antisense nucleic acids, miRNA) or degrade (eg, siRNA, ribozymes, miRNA) mRNA, etc. are included.
• nucleic acids that inhibit eg, antisense nucleic acids, miRNA
• degrade eg, siRNA, ribozymes, miRNA
• nucleotide sequence of the SIRP ⁇ gene is known, and sequence information can be obtained from public databases (eg, NCBI, EMBL, DDBJ, etc.).
• nucleic acid that suppresses the expression of the SIRPa gene using genome editing includes a nucleic acid that suppresses the expression of the SIRP ⁇ gene using genome editing.
• the nucleic acid that suppresses the expression of the SIRP ⁇ gene may be a nucleic acid that can inactivate (knock out) the SIRP ⁇ gene.
• a nucleic acid includes a nucleic acid sequence recognition module (e.g., CRISPR/Cas9, ZF motif, TAL effector, etc.) that can specifically recognize a partial nucleotide sequence in the gene as a target, and a nucleic acid sequence within or near the target sequence. and a nuclease that introduces a double-strand break (DSB) into the gene.
• a nucleic acid sequence recognition module e.g., CRISPR/Cas9, ZF motif, TAL effector, etc.
• the gene can be knocked out by insertion or deletion mutations due to non-homologous end joining (NHEJ) repair errors.
• gene knockout by homologous recombination (HR) repair can be achieved by combining with a targeting vector in which a marker gene (e.g., a reporter gene such as a fluorescent protein gene, a selection marker gene such as a drug resistance gene) is inserted into the gene sequence. You can also do it.
• a marker gene e.g., a reporter gene such as a fluorescent protein gene, a selection marker gene such as a drug resistance gene
• macrophage-like cells can be prepared by deleting or suppressing the expression of genes that inhibit macrophage-like functions.
• a "gene that adds proliferation ability” is a gene that has the function of promoting cell proliferation by being introduced into a cell and expressed within the cell, and is a gene for various factors. can be mentioned. In this specification, it is also referred to as a proliferation gene. Specific examples include, but are not limited to, genes such as c-MYC, BMI1, and MDM2 of any biological species, or homologs or orthologs thereof.
• the biological species is selected to be compatible with the animal species from which the cells to be introduced are derived.
• at least one, preferably two, and more preferably all three genes selected from the group consisting of c-MYC, BMI1, and MDM2 are introduced and expressed.
• expressing means producing a desired gene or protein within a cell, and secreting it outside the cell as the case may be, so that the function of the gene or protein is exerted.
• gene introduced means that a desired gene is introduced into a specific cell, and the gene or the protein encoded by the gene is produced within the cell and, in some cases, secreted outside the cell. , refers to the ability of a protein to perform its functions.
• One embodiment of the method for producing pMAC of the present invention includes the following method (Aspect 1): (1) After deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells, myeloid cells are a step of inducing differentiation into macrophage-like cells (MAC) (step 1), and (2) expressing a gene that adds proliferative properties to the MAC obtained in step 1 to form proliferative macrophage-like cells (pMAC). Step of obtaining (Step 2) method including.
• step 1 of aspect 1 the above-mentioned "1. step of deleting or suppressing the expression of a gene that inhibits macrophage-like function" is performed on pluripotent stem cells.
• the pluripotent stem cells are preferably induced pluripotent stem cells or embryonic stem cells, more preferably induced pluripotent stem cells.
• pluripotent stem cells lacking or suppressing the expression of a gene that inhibits macrophage-like functions for example, pluripotent stem cells (especially iPS cells) lacking or suppressing the expression of the SIRP ⁇ gene, are induced to differentiate into myeloid cells. (hereinafter also referred to as "myeloid differentiation"), macrophage-like cells (MAC) can be obtained.
• Myeloid cells are a general term for monocytes, macrophages, dendritic cells, and progenitor cells that belong to cell lineages that differentiate into these cells, among leukocytes differentiated from stem cells.
• myeloid differentiation of pluripotent stem cells in which genes that inhibit macrophage-like functions are deleted or whose expression is suppressed, the differentiated myeloid cells become macrophage-like cells.
• Specific methods for obtaining macrophage-like cells (MAC) by myeloid differentiation of pluripotent stem cells in which genes that inhibit macrophage-like functions are deleted or whose expression is suppressed include the following methods.
• Embryoid bodies (EBs) derived from pluripotent stem cells are cultured in a layered manner on feeder cells in a medium containing VEGF, and then further cultured in a medium containing VEGF, SCF and TPO. that, or, (ii) Embryoid bodies (EBs) induced from the pluripotent stem cells are cultured in a monolayer without feeder cells in a medium containing BMP-4, VEGF and SCF, and then further cultured in a medium containing VEGF, TPO and GM. - Cultivation in medium containing CSF.
• the pluripotent stem cells are pluripotent stem cells in which a gene that inhibits macrophage-like function is deleted or whose expression is suppressed.
• Step 2 of Embodiment 1 the MAC obtained in Step 1 is subjected to the above-mentioned "2. Step of expressing a gene that adds proliferation ability".
• Another embodiment of the method for producing pMAC of the present invention includes the following method (Aspect 2): (A) Step A to obtain proliferative myeloid cells by expressing a gene that adds proliferative properties to myeloid cells differentiated from pluripotent stem cells (Step A); and (B) In the proliferative myeloid cells obtained in Step A. , a step of deleting or suppressing the expression of a gene that inhibits macrophage-like functions and then inducing differentiation to obtain proliferative macrophage-like cells (pMAC) (step B).
• Step A of Embodiment 2 the above-mentioned "2.
• Step of expressing a gene that adds proliferative ability is performed on myeloid cells differentiated from pluripotent stem cells (myeloid cells derived from pluripotent stem cells).
• the pluripotent stem cells are preferably induced pluripotent stem cells or embryonic stem cells, more preferably induced pluripotent stem cells.
• myeloid cells endowed with proliferative properties can be obtained.
• Specific methods for inducing differentiation of pluripotent stem cells into myeloid cells include the following methods.
• Embryoid bodies (EBs) derived from pluripotent stem cells are cultured in a layered manner on feeder cells in a medium containing VEGF, and then further cultured in a medium containing VEGF, SCF and TPO. that, or, (ii) Embryoid bodies (EBs) induced from the pluripotent stem cells are cultured in a monolayer without feeder cells in a medium containing BMP-4, VEGF and SCF, and then further cultured in a medium containing VEGF, TPO and GM. - Cultivation in medium containing CSF.
• Step B of Embodiment 2 the proliferative myeloid cells obtained in Step A are subjected to the above-mentioned "1.
• Step of deleting or suppressing the expression of a gene that inhibits macrophage-like function By deleting or suppressing the expression of a gene that inhibits macrophage-like functions in proliferative myeloid cells, such as the SIRP ⁇ gene, pMAC can be obtained by being induced to become macrophage-like cells.
• embryoid body in the present invention refers to a cell mass formed by adhesion of undifferentiated pluripotent stem cells. Due to the close cell-to-cell interaction within the cell cluster, the pluripotent stem cells that make up the cell cluster have a limited repertoire of cell differentiation, which is similar to the developmental stage in which the germ layer is formed after implantation. similar to the embryo of
• the size of the embryoid bodies in the present invention is not particularly limited, but the diameter is preferably 50 to 1000 ⁇ m, more preferably 50 to 500 ⁇ m, and particularly preferably 50 to 200 ⁇ m. If the size of the embryoid body is too large than the above range, oxygen supply to the inside of the embryoid body may become insufficient and cell necrosis may occur. If the size of the embryoid body is smaller than the above range, no embryoid body will be formed and the single cell may undergo cell death due to 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 feeder cells.
• VEGF vascular endothelial growth factor
• SCF serotonin
• TPO myeloid cells
• BMP-4 myeloid cells
• VEGF, SCF, TPO, BMP-4" and/or "GM-CSF” of the present invention are derived from “VEGF”, “SCF”, “ VEGF, SCF, TPO of biological species other than the embryoid body species to be cultured, provided that they have the same degree of differentiation-inducing activity as “TPO”, “BMP-4" and/or “GM-CSF", Using BMP-4 and/or GM-CSF protein, or a variant of VEGF, SCF, TPO, BMP-4 and/or GM-CSF protein of the same biological species as the embryoid body to be cultured. Can be done.
• Flt-3L, IL-3 and/or bFGF are also used in culturing the cells of the present invention together with the above-mentioned VEGF, SCF, TPO, BMP-4 and/or GM-CSF, etc.
• VEGF vascular endothelial growth factor
• SCF vascular endothelial growth factor
• TPO vascular endothelial growth factor
• BMP-4 vascular endothelial growth factor-4
• GM-CSF etc.
• recombinant protein reagents for example, Recombinant Human Flt-3 Ligand/FLT3L Protein (308-FKN) and Recombinan from R&D Systems (https://www.rndsystems.com/) are available.
• t Human IL-3 Protein (203-IL ), Recombinant Human FGF basic/FGF2/bFGF (145 aa) Protein (3718-FB), Recombinant Human VEGF 165 Protein (29 3-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.
• VEGF vascular endothelial growth factor
• SCF vascular endothelial growth factor
• TPO vascular endothelial growth factor
• BMP-4 GM-CSF
• pluripotent stem cells used as the above-mentioned gene or protein.
• the pluripotent stem cells are human-derived, it is preferable to use human-derived VEGF, SCF, TPO, BMP-4, GM-CSF, Flt-3L, IL-3 and/or bFGF;
• the pluripotent stem cells are mouse-derived, it is also preferable to use the mouse-derived gene or protein.
• medium containing substance X means a medium to which exogenous substance X is added or a medium containing exogenous substance X
• medium containing no substance means a medium to which no exogenous substance X is added or a medium containing no exogenous substance X
• exogenous substance X means substance X that is foreign to cells or tissues cultured in the medium, and includes endogenous substance Not possible.
• a medium containing VEGF is a medium to which exogenous VEGF is added or a medium containing exogenous VEGF.
• the dose of VEGF may be influenced by other growth factors, it is preferably 0.2 ng/mL to 200 ng/mL, more preferably 2 ng/mL to 100 ng/mL, and 5 ng/mL to 50 ng/mL is particularly preferred.
• VEGF-free medium is a medium to which exogenous VEGF is not added or a medium that does not contain exogenous VEGF.
• a medium containing SCF, TPO, BMP-4, and/or GM-CSF refers to a medium containing exogenous SCF, TPO, BMP-4, and/or GM.
• the dose of SCF, TPO, BMP-4 and/or GM-CSF may be influenced by other growth factors, etc., but is preferably 0.2 ng/mL to 200 ng/mL, and 2 ng/mL to 200 ng/mL. /mL to 100ng/mL is more preferable.
• TPO it is more preferably 5 ng/mL to 15 ng/mL.
• SCF, BMP-4 and GM-CSF it is more preferably 5 ng/mL to 70 ng/mL.
• SCF, TPO, BMP-4 and/or GM-CSF-free medium means a medium to which exogenous VEGF is not added or exogenous SCF, TPO, BMP-4 and/or GM-CSF is not added. This is a medium that does not contain
• ⁇ MEM As the culture medium for mouse embryoid bodies in the present invention, ⁇ MEM supplemented with 20% fetal bovine serum (FBS) can be used.
• FBS fetal bovine serum
• feeder cells mouse mesenchymal C3H10T1/2 cells or mouse bone marrow-derived stromal OP9 cells can be used.
• a medium for human embryoid bodies StemFit (Ajinomoto), a completely synthetic medium, can be used.
• ⁇ MEM supplemented with 20% FBS can be used as the medium.
• feeder cells are other cell types used to adjust the culture conditions for undifferentiated maintenance culture or differentiation induction culture of iPS cells. Feeder cells are used after their proliferation is stopped by mitomycin C treatment or radiation irradiation.
• Mouse bone marrow-derived stromal cells OP9, mouse-derived mesenchymal cells C3H10T1/2, and the like can be used.
• multilayer culture refers to co-culturing by seeding desired iPS cells or embryoid bodies into a feeder cell layer culture system that has been cultured in a predetermined culture container.
• monolayer culture refers to culturing a single cell type by adhering it to a tissue culture container.
• monolayer culture without feeder cells refers to seeding iPS cells or embryoid bodies of interest in a culture container and culturing them statically.
• the present invention includes forming embryoid bodies from pluripotent stem cells by the following method. (i) Seeding a single-cell dispersion of pluripotent stem cells in a cell cluster forming culture container to form embryoid bodies (EBs), and (ii) The container is provided with fine spheroid wells at the bottom and there is no flat surface between adjacent spheroid wells.
• EBs embryoid bodies
• Form an embryoid body means to dissociate pluripotent stem cells that have been proliferating in an undifferentiated state to obtain a single-cell dispersion of pluripotent stem cells, and to combine the pluripotent stem cells with each other. This refers to forming a qualitatively uniform cell mass by culturing in suspension or statically under conditions that allow cells to adhere and aggregate. The formation of embryoid bodies is confirmed by visual observation using a microscope to confirm the presence of viable cells.
• suspension culture refers to culturing under conditions in which cells or cell aggregates are maintained in a suspended state so that they do not adhere to a substrate such as the bottom or wall of a culture container.
• the incubator used when performing suspension culture is not particularly limited as long as it is capable of "sustaining culture", and can be appropriately determined by those skilled in the art.
• Such culture vessels include, for example, flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multiplates, multiwell plates, chamber slides, Examples include petri dishes, tubes, trays, culture bags, or roller bottles.
• These culture vessels are preferably non-cell-adhesive in order to enable floating culture.
• a culture vessel whose surface has not been artificially treated (for example, coated with an extracellular matrix or the like) for the purpose of improving adhesion with cells can be used.
• pluripotent stem cells it is preferable to rapidly aggregate pluripotent stem cells to form embryoid bodies derived from pluripotent stem cells.
• a cell mass of pluripotent stem cells is formed in this way and then transferred to a medium supplemented with VEGF at a concentration of 50 ng/mL and cultured, the cells in the mass are transformed into myeloid cells, similar to blood islands in the yolk sac. Can be differentiated into cell lineages.
• a culture vessel such as a plate (for example, 96-well plate) equipped with many small-diameter holes (wells) or a vessel for cell cluster formation culture may be used in a small space.
• These methods include trapping the cells in a small centrifuge tube, and clumping the cells together by centrifuging them for a short time in a small centrifuge tube.
• Culture conditions for cells or aggregates of pluripotent stem cells include swirl culture, shaking culture, and other suspension cultures, as well as pluripotent stem cell culture, in which the surface of the substrate in contact with the cells in the culture container is treated in advance to make it hydrophobic. Stationary culture can also be used, provided that the adhesion between sexual stem cells and the substrate surface of the culture container is reduced or suppressed.
• embryoid bodies as a cell mass of pluripotent stem cells and the differentiation of embryoid body cells into myeloid cells can be confirmed by confirming the size and cell number of embryoid bodies using a microscope or FACS. , confirmation of microscopic morphology of embryoid bodies in culture, confirmation of histological or cytochemical findings, and/or expression of differentiation and undifferentiation markers, their uniformity, regulation of expression of differentiation markers, and synchronization Alternatively, judgment can be made based on characteristics including, but not limited to, confirmation of reproducibility of differentiation efficiency between embryoid bodies.
• a certain cell is "positive" for a certain marker when the cell is measured by flow cytometry, and an average fluorescence intensity ratio of positive cells relative to a negative control of 10 times or more is defined as +, and 2 times or more or more Less than 10 times is low, and less than 2 times is -.
• the expression level of a certain gene in a certain cell is measured by real-time PCR method, and housekeeping genes ⁇ -actin, GAPDH (glyceraldehyde-3-phosphate)
• HPRT 1 hyperxanthine phosphoribosyltransferase 1
• ⁇ 2-microglobulin is considered high, and less than that is considered low.
• the single cell dispersion refers to cultured cells that are washed with PBS or the like, treated with cell detachment solution Trypsin-EDTA (Life Technologies) or TrypLE Select (Life Technologies) for 4 to 5 minutes, and then treated with PBS, etc. It is prepared by resuspending a pellet of target cells obtained by centrifugation after washing with a desired culture medium by pipetting.
• a cell cluster-forming culture container is one in which cells seeded during culture can form cell aggregates without adhering to the container.
• the container is provided with fine spheroid wells on the bottom, which is the culture surface, and there is no flat surface between adjacent spheroid wells, and the adjacent spheroid wells They are separated by a curved surface that slopes toward the spheroid, and because the curved surface is surface-treated to suppress cell adhesion, most of the cells seeded in the culture container are either spheroids or spheroids. Fall into the well.
• a container is a container in which a spheroid well with a diameter of approximately 400 to 800 ⁇ m and a depth of 100 to 800 ⁇ m is formed uniformly on the culture surface (bottom) without any gaps and down to the wall surface.
• the spheroid well has a diameter of 400-500 ⁇ m and a depth of 50-200 ⁇ m. More preferably, the spheroid well is a container with a diameter of about 500 ⁇ m and a depth of about 400 ⁇ m, a container with a diameter of about 800 ⁇ m and a depth of about 400 ⁇ m, or a container with a diameter of about 800 ⁇ m and a depth of about 300 ⁇ m. Most preferably, the spheroid well is a container with a diameter of about 400 ⁇ m and a depth of 100-200 ⁇ m.
• the container is one in which the spheroid well is processed uniformly down to the wall surface without any gaps on the culture surface (bottom).
• the spheroid well is processed uniformly down to the wall surface without any gaps on the culture surface (bottom).
• there are no flat surfaces between adjacent spheroid wells in the container and the seeded single cell suspension will fall into one of the spheroid wells, forming uniform aggregates. This is because it can be done.
• EZSPHERE® microfabricated cell culture vessel manufactured by AGC.
• pMAC can be produced by either the method of Aspect 1 or Aspect 2, it is preferably produced by the method of Aspect 1.
• Aspect 1 By performing the step of expressing genes that add proliferative properties after the step of deleting or suppressing the expression of genes that inhibit macrophage-like functions, a cell population containing highly pure pMAC with few non-target cells can be stabilized. It can be manufactured using More specific procedures of aspect 1 are as follows.
• step 1-1 A step of deleting or suppressing the expression of genes that inhibit macrophage-like functions in pluripotent stem cells (step 1-1),
• the pluripotent stem cells obtained in step 1-1, in which the gene that inhibits macrophage-like function has been deleted or whose expression has been suppressed, are placed in a cell with a fine spheroid well at the bottom, and a flat surface between adjacent wells.
• step 2 A step (step 2) of obtaining proliferative macrophage-like cells (pMAC) by expressing a gene that adds proliferative properties to the MAC obtained in step 1-3.
• the gene that inhibits macrophage-like functions is the SIRP ⁇ gene.
• the thus obtained pMAC has phagocytic ability similar to macrophages, and in addition, has characteristics different from existing macrophages, such as proliferating in a cytokine-dependent manner.
• the present invention provides a pMAC (hereinafter also referred to as CAR-pMAC) containing a chimeric antigen receptor (CAR) and a method for producing the same.
• CAR-pMAC a pMAC
• CAR-pMAC chimeric antigen receptor
• the CAR gene is introduced into pMAC that has been endowed with cytokine-dependent proliferative properties as described in the above section "Method for producing pMAC of the present invention”.
• the method for producing CAR-pMAC of the present invention includes the step of expanding and culturing the obtained CAR-pMAC.
• An embodiment of the method for producing CAR-pMAC of the present invention includes the following method (Aspect I).
• a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells, and then inducing differentiation into myeloid cells to obtain macrophage-like cells (step 1); (2) a step (step 2) of expressing a gene that adds proliferative properties to the macrophage-like cells obtained in step 1 to obtain proliferative macrophage-like cells (pMAC); and (3) a step of obtaining a chimeric antigen receptor (CAR).
• Process to be introduced (process 3)
• Another embodiment of the method for producing CAR-pMAC of the present invention includes the following method (Aspect II).
• A a step of expressing a gene that adds proliferation to myeloid cells differentiated from pluripotent stem cells to obtain proliferative myeloid cells (step A);
• B A step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in the proliferative myeloid cells obtained in step A, and then inducing differentiation to obtain pMAC (step B), and
• C chimeric antigen Step of introducing receptor (CAR) (Step C).
• Step A and Step B in this embodiment can be carried out in the same manner as Step A and Step B in Aspect 2 of the above-described "method for producing pMAC of the present invention", respectively.
• Another embodiment of the method for producing CAR-pMAC of the present invention includes the following method (Aspect III) (1') A step of introducing a chimeric antigen receptor (CAR) into pluripotent stem cells (step 1'), (2') After the pluripotent stem cells into which the CAR gene obtained in step 1' has been introduced, the gene that inhibits macrophage-like function is deleted or suppressed, and the CAR gene is induced to differentiate into myeloid cells. (Step 2') of obtaining macrophage-like cells into which the CAR gene obtained in Step 2' has been introduced, and (3') expressing a gene that adds proliferative properties to the macrophage-like cells into which the CAR gene obtained in Step 2' has been introduced.
• a step of introducing a chimeric antigen receptor (CAR) into pluripotent stem cells step 1'
• step 2' After the pluripotent stem cells into which the CAR gene obtained in step 1' has been introduced, the gene that inhibits macrophage-like
• Step of obtaining introduced proliferative macrophage-like cells (CAR-pMAC) (step 3')
• a method for producing CAR-pMAC comprising: Step 2' and Step 3' in this embodiment are respectively Step 1 and Step 2 of Aspect 1 of the above-described "method for producing pMAC of the present invention" except that the CAR gene is introduced into the target cells in advance. It can be implemented in the same manner as .
• Another embodiment of the method for producing CAR-pMAC of the present invention includes the following method (Aspect IV) (A') Step of introducing chimeric antigen receptor (CAR) into pluripotent stem cells (Step A'), (B') A step of expressing a gene that adds proliferative properties to myeloid cells differentiated from the CAR gene-introduced pluripotent stem cells obtained in step A' to obtain proliferative myeloid cells into which the CAR gene has been introduced. (Step B') and (C') In the proliferative myeloid cells into which the CAR gene obtained in Step B' has been introduced, genes that inhibit macrophage-like functions are deleted or expression suppressed, and then differentiation is induced.
• Step C' Step of obtaining proliferative macrophage-like cells (CAR-pMAC) into which the CAR gene has been introduced (step C').
• Step A' and Step B' in this embodiment are respectively Step A and Step B of Aspect 2 of the above-mentioned "method for producing pMAC of the present invention” except that the CAR gene is introduced into the target cells in advance. It can be implemented in the same manner as .
• Chimeric antigen receptor (also referred to herein as CAR) CAR is a structure that contains, from the N-terminal side to the C-terminal side of the protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for effector functions of immune cells.
• the gene is the one that codes for this receptor.
• the extracellular domain contains an antigen recognition site that exhibits target-specific binding.
• a transmembrane domain exists between the extracellular domain and the intracellular signaling domain.
• Intracellular signaling domains transmit signals necessary for immune cells to exert their effector functions. That is, when the extracellular domain binds to a target antigen, an intracellular signal domain is used that is capable of transmitting a signal necessary for activating immune cells.
• CAR gene into pMAC introduction of the CAR gene is carried out by introducing the CAR gene into desired cells using a CAR expression vector.
• the cells to which the CAR gene is introduced include, for example, the pMAC provided by the present invention described above, as well as pluripotent stem cells before or after deletion or expression suppression of genes that inhibit macrophage-like functions. It will be done.
• CAR expression vector means a nucleic acid molecule that can transport a nucleic acid molecule encoding a CAR gene into a target cell, regardless of whether it is DNA or RNA, as well as its form, origin, etc. There are also no particular limitations, and various types of vectors can be used. Vectors can be viral or non-viral vectors.
• viral vectors examples include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors, vaccinia virus vectors, and the like.
• retrovirus vectors, lentivirus vectors, and adeno-associated virus vectors the target gene inserted into the vector is integrated into the host chromosome, and stable and long-term expression can be expected.
• Each viral vector can be produced according to a conventional method or using a commercially available dedicated kit.
• Non-viral vectors include plasmid vectors, liposome vectors, and positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987 ), YAC vectors, BAC vectors, artificial chromosome vectors, etc.
• a CAR expression vector contains an expression unit for expressing a CAR gene, and the expression unit usually contains a promoter, a CAR gene, and a polyA addition signal.
• the expression unit is derived from various organisms or viruses or is composed of an arbitrary sequence, and includes similar sequences and modified units thereof. Promoters that can be used in the CAR expression cassette include CAG promoter, CMV-IE (cytomegalovirus early gene-derived promoter), SV40ori, retrovirus LTRSR ⁇ , EF1 ⁇ , ⁇ -actin promoter, and the like.
• Examples of the poly A addition signal sequence include the poly A addition sequence of SV40, the poly A addition sequence of the bovine growth hormone gene, and the globulin poly A addition sequence.
• the CAR gene In order to control the expression of the CAR gene by the promoter, the CAR gene is usually linked directly to the 3' end of the promoter or via another sequence, and a polyA addition signal sequence is placed downstream of the CAR gene. be done.
• the CAR gene is transcribed into messenger RNA (mRNA) by such an expression unit, and CAR is translated from the mRNA and presented on the cell surface.
• the expression unit contains detection genes (reporter genes, cell- or tissue-specific genes, selection marker genes, etc.) to enable detection of gene expression, enhancer sequences, WRPE sequences, etc. to improve expression efficiency. may be included.
• the detection gene is used for determining the success or failure and efficiency of introduction of the CAR expression vector, detecting the expression of the CAR gene or determining the expression efficiency, and selecting and sorting cells in which the CAR gene is expressed.
• Detection genes include the neo gene that confers resistance to neomycin, the kmr gene and nptII gene that confer resistance to kanamycin, etc. (Bernd Reiss et al. EMBO J. 3 (1984), 3317-3322), and the resistance to hygromycin.
• hph gene that confers resistance to methotrexate (Blochlinger & Diggelmann, Mol Cell Bio 4:2929-2931), DHFR gene that confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7)), etc. (marker genes); Luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), ⁇ -glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett.
• reporter genes genes for fluorescent proteins such as tdTomato or its variants (pTdTomato, etc.) (hereinafter referred to as reporter genes); epidermal growth factor receptor lacking an intracellular domain ( A gene such as the EGFR) gene can be used.
• the detection gene may be linked to the CAR gene via, for example, a bicistronic control sequence (eg, internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleavable peptide.
• Self-cleavable peptides include the 2A peptide (T2A) derived from Thosea asigna virus.
• Other self-cleavable peptides include 2A peptide (F2A) derived from picornavirus, 2A peptide (F2A) derived from foot disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), and porcine teschovirus.
• F2A 2A peptide
• F2A foot disease virus
• E2A 2A peptide
• E2A derived from equine rhinitis A virus
• porcine teschovirus examples include, but are not limited to, 2A peptide (P2A) derived from (PTV-1), and 2A peptide derived from rotavirus, insect virus, aphthovirus, or trypanosome virus. Examples include similar sequences and partially modified sequences.
• the CAR gene expression vector prepared for gene introduction is introduced into pMAC by a conventional method.
• viral vectors they are introduced into cells by infection with a virus.
• non-viral vectors such as plasmids
• conventional methods such as electroporation, liposome, calcium phosphate, and nucleofection can be used for introduction into cells, and it is preferable that the vector be introduced by electroporation. Ru.
• the transposon method is a non-viral gene introduction method that uses a pair of gene enzyme (transposase) and its specific recognition sequence to cause gene transposition, and can integrate any gene into the host chromosome.
• transposase a pair of gene enzyme
• the transposon method for example, the piggyBac transposon method can be used.
• the piggyBac transposon method uses transposons isolated from insects (Fraser MJ et al., Insect Mol Biol.
• the transposon method is not limited to the one using piggyBac, for example, Sleeping Beauty (Ivics Z, hackett PB, Plasterk RH, Izsvak Z (1997) Cell 91: 501-510.), Frog Prince (Miskey C, Izsvak Z , Plasterk RH, Ivics Z (2003) Nucleic Acids Res 31: 6873-6881.), Tol1 (Koga A, Inagaki H, Bessho Y, Hori H. Mol Gen Genet. 1995 Dec 10;249(4):400-5 ; Koga A, Shimada A, Kuroki T, Hori H, Kusumi J, Kyono-Hamaguchi Y, Hamaguchi S. J Hum Genet.
• transposon method can be performed using conventional methods.
• a vector carrying a gene encoding piggyBac transposase (transposase plasmid) and a vector (transposon plasmid) having a structure in which a CAR gene expression unit is sandwiched between piggyBac inverted repeat sequences are prepared.
• These vectors can be introduced into target cells by various techniques such as electroporation, nucleofection, lipofection, and calcium phosphate method.
• CAR-pMAC As a treatment after introducing the CAR gene, CAR-pMAC is expanded and cultured to the required scale.
• CAR-pMAC of the present invention can be proliferated in a cytokine-dependent manner. Therefore, in the method for producing CAR-pMAC of the present invention, the aforementioned expansion culture may be carried out in the presence of at least one, preferably two cytokines selected from the group consisting of GM-CSF and M-CSF. preferable.
• the medium for preparing CAR-pMAC is not particularly limited, and a medium used for normal cell culture, such as RPMI1640, MEM, X-VIVO, IMDM, DMEM, DC medium, OptiMEM, etc., can be used.
• the medium may be a medium to which serum (human serum, fetal bovine serum, etc.) is added according to a conventional method, or a serum-free medium. It is preferable to use a serum-free medium because it is highly safe in clinical application and the culture efficiency is less likely to differ due to differences between serum lots.
• serum-free medium examples include TexMACS TM (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific), ALyS culture medium (Cell Science Institute, Inc.), and the like.
• autologous serum that is, serum collected from the individual from whom the CAR-expressing immune cells are derived (more specifically, a patient receiving the cell population obtained by the production method of the present disclosure) may be used. It may also be an artificial serum. In the present invention, it is preferable to use autologous serum.
• TexMACSTM Miltenyi Biotec
• AIM V registered trademark
• ALyS culture medium Cell Science Institute, Inc.
• Other culture conditions may be those suitable for cell survival and proliferation, and general conditions can be adopted. For example, culturing in a CO 2 incubator (CO 2 concentration 5%) set at 37°C, hypoxic culture (oxygen concentration 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%), etc. can be mentioned.
• the recovery operation may be performed in a conventional manner. For example, it is collected by pipetting, centrifugation, etc.
• a step of culturing the expanded cells in the presence of a stimulating substance is performed before the recovery operation. This step enables efficient expansion culture and also has the advantage of increasing cell survival rate.
• the CAR-pMAC population after expansion culture may be subjected to a cell separation step such as bead separation. By performing the cell separation step, the purity of CAR-pMAC can be increased with higher efficacy.
• the cell population containing CAR-pMAC produced by the method of the present invention can be used for the treatment of diseases associated with tumors or cancer, particularly for the treatment of cancers that express the target antigen of the CAR-expressing immune cells.
• a disease associated with a tumor or cancer may be a solid tumor or a hematological tumor.
• Specific cancers include various B-cell lymphomas (follicular malignant lymphoma, diffuse large B-cell malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), bone marrow Proliferative tumors, myelodysplastic/myeloproliferative tumors (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinal buds Cytoma, small cell lung cancer, non-small cell lung cancer, melanoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer , colon cancer, etc., but are not limited to these.
• the cancer is a solid tumor.
• solid tumors include neuroblastoma, brain tumor, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcoma, and kidney cancer.
• pancreatic cancer malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, and colorectal cancer.
• the cell population containing CAR-pMAC produced by the method of the present invention can be used for the treatment of neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis.
• Neurodegenerative diseases include tauopathy, ⁇ -synucleopathy, presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, Lewy body dementia, Down syndrome, multiple system atrophy, amyotrophic side Examples include ALS, Hallerforden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, and prion disease.
• Inflammatory diseases include systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infectious diseases, hereditary periodic fever, malignant tumors, Systemic vasculitis, cystic fibrosis, bronchiectasis, epidermolysis bullosa, periodic neutropenia, acquired or inherited immunodeficiency, injection-drug use, and acne clumps. , Muckle-Wells disease (MWS), familial Mediterranean fever (FMF), etc.
• MWS Muckle-Wells disease
• FMF familial Mediterranean fever
• cardiovascular disease examples include atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, heart failure, and the like.
• Fibrosis diseases include pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, and interstitial fibrosis. Examples include lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, bone marrow fibrosis, and skin fibrosis.
• Amyloidosis includes primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), other familial amyloidosis, ⁇ -2 microglobulin amyloidosis, localized amyloidosis, and heavy chain amyloidosis (AH).
• AL light chain amyloidosis
• primary systemic amyloidosis ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, apolipoprotein C2 amyloidosis, apolipoprotein C3 amyloidosis, corneal lactoferrin amyloidosis, transthyretin-related amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Examples include Lect2 amyloidosis (ALECT2) and lysozyme amyloidosis.
• the cell population containing CAR-pMAC produced by the method of the present invention is administered in a therapeutically effective amount appropriately determined depending on the age, weight, body surface area, symptoms, etc. of the subject.
• the subject in this disclosure is usually a human, preferably a cancer patient.
• the cell population containing CAR-pMAC produced by the method of the present invention can be administered at, for example, 1 ⁇ 10 4 to 1 ⁇ 10 10 cells at a time.
• the route of administration is not particularly limited, and administration can be intratumoral, peritumoral, intraventricular, intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular, or intraperitoneal.
• the cell populations of the present disclosure may be administered systemically or locally, and local administration includes direct injection into the target tissue, organ, or organ.
• the administration schedule is appropriately determined depending on the subject's age, body weight, body surface area, symptoms, etc., and may be a single administration or continuous or periodic multiple administrations.
• a cell population containing CAR-pMAC produced by the method of the present invention can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
• pharmaceutically acceptable carrier include components commonly used in pharmaceuticals, such as conventional excipients, binders, buffers, water for injection, isotonizing agents, preservatives, and soothing agents.
• drugs such as dimethyl sulfoxide (DMSO) and serum albumin for the purpose of cell protection, antibiotics, etc. for the purpose of preventing bacterial contamination, cell activation, proliferation or differentiation induction, etc. It may also contain various components (vitamins, cytokines, growth factors, steroids, etc.) aimed at.
• Pharmaceutical compositions can be prepared by conventional methods.
• Example 1 Production of human iPSCs Kitayama, S.; Human iPSCs were produced by the method described in (Stem Cell Reports. 6: 213-227, (2016)). Maintenance culture of human iPSCs was performed using a regenerative medicine medium StemFit AK02N (Ajinomoto Healthy Supply) on a polystyrene tissue culture plate coated with iMatrix511 (Nippi). Replace the medium every 1 to 2 days, and once a week depending on cell proliferation, treat the cells with the cell detachment solution TrypLE Select (Life Technologies) for 4 to 5 minutes to recover a single cell dispersion. The seeds were seeded in a culture container of the same size and culture was continued. The seeded iPSCs were cultured overnight in the presence of Rock inhibitor 10 ⁇ M Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), and the medium was replaced the next day to remove Y-27632.
• Example 2 Production of mouse iPSCs Araki, R.
• Mouse iPSCs were produced by the method described in (Nature, 494:100-104. (2013)).
• ESM DMEM (Fujifilm Wako Pure Chemicals) containing 15% KSR (Life Technologies)
• cESM complete ESM
• cESM complete medium prepared by adding Factor (LIF) (Merck, trade name: ESGROmLIF), on feeder cells of mouse embryonic fibroblasts MEF (Reprocell) seeded on a 6-well tissue culture plate. I went there.
• Feeder cells were prepared by seeding MEFs (Reprocell) whose cell proliferation had been stopped by Mitomycin C treatment on a gelatin-coated dish, and used for maintenance culture of mouse iPSCs from the day after the MEFs adhered.
• Example 3 Deletion of SIRP ⁇ gene by genome editing Single guide RNA of human SIRP ⁇ and mouse SIRP ⁇ was synthesized by nucleic acid synthesis using sequence information from the National Center for Biotechnology Information (NCBI) database. .
• a Cas9 protein guide RNA complex (RNP) was prepared by mixing the single guide RNA of each gene with Guide-it TM Recombinant Cas9 protein (Takara Bio), added to the iPSC single cell dispersion, and electroporation method. RNA was introduced into the cells.
• An iPSC clone lacking the SIRPa gene was obtained by sequence analysis of the genomic DNA sequence targeted by the guide RNA and expression analysis by flow cytometry using the differentiated cells obtained by differentiation induction in Example 5.
• Example 4 Production of embryoid bodies derived from iPSCs Embryoid bodies (hereinafter referred to as human EBs) derived from human iPSCs (wild type or SIRP ⁇ -deficient type) were produced using a 6-well plate type cell cluster formation culture. This was carried out by seeding human iPSCs at a predetermined density prepared with StemFit containing 10 ⁇ M Y-27632 into a container EZ SPHERE SP (AGC Techno Glass). One to four days after the formation of embryoid bodies with a diameter of 50 to 200 ⁇ m, differentiation induction culture was performed as described in Example 5 below.
• mouse EBs embryoid bodies derived from mouse iPSCs (wild type or SIRP ⁇ -deficient type) derived from mouse iPSCs (wild type or SIRP ⁇ -deficient type)
• mouse iPSCs at a predetermined density prepared in a complete medium cESM are seeded in a 6-hole plate type EZ SPHERE. So I went there.
• Example 5 Induction of differentiation of embryoid bodies into myeloid cells (1) Preparation of feeder cells Mouse-derived cultured cell line C3H10T1/2 or OP9 was seeded on a gelatin-coated dish, and the cell line was incubated the next day. used. The feeder cells used for inducing differentiation of embryoid bodies derived from human iPSCs were irradiated with 60 Gy of radiation to stop proliferation before use. BME medium containing 10% FBS (Life Technologies) was used for culturing C3H10T1/2 cells, and ⁇ MEM medium containing 20% FBS (Life Technologies) was used for culturing OP9 cells.
• Mouse EBs prepared from mouse iPSCs were cultured on feeder cells OP9 for 7 to 10 days using differentiation induction medium 20% FBS-containing ⁇ MEM. Afterwards, the collected cells were replated onto freshly prepared OP9 feeders. Differentiated cells including myeloid cells were obtained by culturing in the presence of 55 ⁇ M 2-mercaptoethanol and 50 ng/mL GM-CSF for 7 to 10 days.
• the cells were cultured for 7 to 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.
• the upper and lower limits can be any two concentrations within the range of 0.2 ng/mL to 200 ng/mL, for example, 0.2 ng/mL.
• the upper and lower limits can be any two concentrations.
• Example 6 Production of proliferating macrophage-like cells pMAC by introducing proliferative genes
• Preparation of lentiviral vector Gene synthesis was performed using sequence information from the database of the National Center for Biotechnology Information (NCBI) in the United States. Human c-MYC, BMI1, and MDM2 cDNAs were synthesized by. The cDNA fragment of each gene was inserted into the lentiviral vector pSL or CSII-EF-MSC-IRES2-Veneus.
• NCBI National Center for Biotechnology Information
• -Rev was introduced into 293T cells, which are packaging cells (virus-producing cells).
• the cell culture solution was collected, filtered through a filter with a pore size of 0.45 ⁇ m, and then the virus particles were concentrated and collected using a Lenti-X concentrator (Clontech).
• the recovered recombinant virus particles were suspended in a DMEM solution, then dispensed into cryovials and stored in a -150°C environment.
• the post-infected cells were cultured in the presence of 50 ng/mL GM-CSF and 50 ng/mL M-CSF using ⁇ MEM containing 20% FBS as a culture medium for myeloid cells after virus infection.
• the cells into which the gene had been introduced continued to proliferate for over three months.
• cells derived from wild-type iPSCs are referred to as pMACs
• cells derived from pluripotent stem cells lacking the SIRP ⁇ gene that inhibits macrophage-like functions are referred to as SIRP ⁇ -deficient proliferating macrophage-like cells (pMACs).
• pMACs cells derived from pluripotent stem cells lacking the SIRP ⁇ gene that inhibits macrophage-like functions
• SIRP ⁇ -deficient proliferating macrophage-like cells pMACs
• -SIRP ⁇ -KO macrophage-like cells
• BM-derived monocytes bone marrow-derived monocytes
• peritoneal macrophages bone marrow-derived monocytes
• Bone marrow-derived monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum in the presence of 50 ng/mL M-CSF on a 6-well tissue culture plate for 4 days to obtain bone marrow-derived macrophages M0 (BM-derived macrophages M0) were induced.
• bone marrow-derived macrophages M1 bone marrow-derived macrophages M1
• BM-derived macrophages M2 bone marrow-derived macrophages M2
• thioglycollate medium Merck
• RNAseq analysis (species Mus musculus , reference genome mm10), which is a whole transcriptome analysis, on pMAC prepared from iPS cells and biologically derived macrophages, and quantified the expression of transcripts. I did it.
• Total RNA was extracted from each cell using QIAGEN RNeasy mini kit, and NEBNext (R) Poly (A) mRNA Magnetic Isolation Module (model number E7490), N EBNext(R ) Using Ultra TM II Directional RNA Library Prep Kit (model number E7760), PCR amplification was performed using primers containing the Index sequence to prepare a sequence library.
• the base sequence of the library prepared sample was obtained using the next generation sequencer NovaSeq 6000 (Illumina), and the read length was PE150 (150bp x 2 paired-end), the data amount was 4G bases per sample, and the number of reads was 26.7M reads per sample ( 13.3M pairs) of data were acquired.
• informatics analysis was performed to perform principal component analysis (PCA).
• PCA principal component analysis
• the quality scores Scores indicating sequence information error rate
• R1 (Read 1) and R2 (Read 2) were calculated using the software FastQC (version 0.11.7), and abnormalities were detected. None (Entirely Normal, quality score >28) was confirmed.
• LEADING 20
• TRAILING 20
• SLIDINGWINDW 4:15
• adapter sequences, short reads, etc. were removed based on the results of FastQC quality confirmation.
• the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0), the read mapping rate was calculated, and it was confirmed that all samples were over 98%. Subsequently, raw read counts mapped to known exon regions for each gene were calculated using the software featureCounts (version 1.6.3).
• BM-derived monocytes Mouse bone marrow-derived monocytes (BM-derived monocytes) and pMAC were collected by pipetting.
• es M1 was treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes and collected by pipetting.
• the cells include anti-CD11b antibody, anti-F4/80 antibody, anti-CD16/32 antibody, anti-CD64 antibody, anti-IA/E antibody, anti-CD206 antibody, anti-CD163 antibody, anti-Ly6C antibody, anti-CD80 antibody, anti-CD86 antibody, Anti-SiglecF antibody, anti-SIRP ⁇ antibody, anti-TLR2 antibody, anti-TLR4 antibody, anti-CD124 antibody, anti-CCR1 antibody, anti-CCR2 antibody, anti-CCR3 antibody, anti-CCR4 antibody, anti-CCR5 antibody, anti-CXCR2 antibody and anti-CXCR4 antibody or isotype Staining was performed using a matched control antibody.
• Example 7 Production of iPSC-derived chimeric antigen receptor-introduced macrophage-like cells (CAR-pMAC) (1) Preparation of lentiviral vector A chimeric antigen receptor (CAR-pMAC) having the tdTomato gene and/or the GC33scFv domain and the CD3 ⁇ domain was generated by gene synthesis. ) was synthesized and inserted into the lentiviral vector pSL in the same manner as in Example 6 (1), and then virus particles were prepared.
• Figure 4 shows the plasmid map and CAR gene expression construct used to prepare the lentiviral vector. The recovered recombinant virus particles were suspended in a DMEM solution, then dispensed into cryovials and stored in a -150°C environment.
• iPSC-derived pMAC human/mouse
• pMAC-SIRP ⁇ -KO human or mouse proliferated macrophage-like cells prepared in Example 6 (2)
• a lentivirus suspension expressing GC33 CAR is added thereto. This caused the infection.
• the culture scale was expanded by adding culture medium according to cell proliferation. Culture was performed in the presence of 50 ng/mL GM-CSF and 50 ng/mL human M-CSF using ⁇ MEM containing 20% FBS as the culture medium. Note that human GM-CSF was used for human pMAC, and mouse GM-CSF was used for mouse pMAC.
• FIG. 5 shows the measurement results of MTT assay for various mouse pMACs. Approximately 50 ng/mL of GM-CSF or M-CSF was required in the culture medium for the proliferation of pMAC. Furthermore, the combined use of GM-CSF and M-CSF had the effect of promoting proliferation. Deletion of the SIRP ⁇ gene and introduction of the CAR gene performed for the production of pMAC had no effect on cell proliferation, indicating that CAR-pMAC has the same cell proliferation ability as pMAC.
• FIG. 6 shows the results of examining the expression of GC33 ⁇ CAR in pMAC, CAR-pMAC, pMAC-SIRP ⁇ -KO, and CAR-pMAC-SIRP ⁇ -KO using a flow cytometer. It can be seen that CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO bind hGPC antigen-specifically compared to pMAC and pMAC-SIRP ⁇ -KO.
• Cytokine production by mouse pMAC Macrophages existing in the body are divided into functional phenotypes classified as M1 type and M2 type depending on cell-cell interactions in the microenvironment of cancer tissues.
• cytokines produced from mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO were evaluated.
• Mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, CAR-pMAC-SIRP ⁇ -KO, and mouse bone marrow-derived macrophages (2 ⁇ 10 5 cells) were seeded in 48-well tissue culture plates, and 20 ng/mL interferon ⁇ (BioLegend ), M1 type was induced by culturing for 48 hours in the presence of 50 ng/mL LPS (Fujifilm Wako Pure Chemical Industries, Ltd.).
• mouse bone marrow-derived macrophage mouse bone marrow-derived macrophage M0 prepared in Example 6 (3) was used.
• M2 type was induced by culturing in the presence of 20 ng/mL IL-4 (BioLegend) for 48 hours.
• IL-6, TNF ⁇ , IL-12, and IL-10 which are known as M1 or M2 type markers, was quantified (FIG. 7). It was found that pMAC induced the production of IL-6, TNF ⁇ , and IL-10 by M1 stimulation, regardless of the presence or absence of SIRP ⁇ or CAR expression, whereas IL-12 was not produced.
• Mouse CAR-pMAC-SIRP ⁇ -KO (3 ⁇ 10 5 cells) were seeded in 20% FCS-containing ⁇ MEM containing CSF and M-CSF and cultured at 37° C. and 5% CO 2 . Due to the attracting factor in the lower chamber, the CAR-pMAC in the upper chamber passes through the pores of the transwell and is adsorbed to the electrode sensor on the back surface of the membrane, thereby increasing the electrical resistance value. Cell migration of mouse CAR-pMAC can be evaluated by measuring this electrical resistance value using the xCELLigence RTCA DP system (Agilent).
• mouse cancer cell culture supernatant mouse MC38, LL/2, 4T1, and CT26 cells (1 ⁇ 10 6 cells each) were seeded in a 6-well tissue culture plate. The next day, the medium was replaced with 2 mL of fresh 10% FCS-containing RPMI-1640 medium and cultured for an additional 24 hours to prepare a culture supernatant. A culture supernatant was prepared using T cells and B cells (1 ⁇ 10 6 cells) isolated from the spleen of a c57BL/6 strain mouse as normal cells.
• cytochalasin D cytochalasin D
• PTX Pertussis Toxin
• mouse recombinant chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CX3CL1, CXCL1, CXCL2, CXCL5, CXCL12SDF-1 ⁇ and CXCL12SDF-1 ⁇ (both manufactured by BioLegend) were used at a concentration of 100 ng/mL.
• CCL3, CCL4, and CCL7 both manufactured by BioLegend
• Cytokine production of mouse pMAC by co-culture with cancer cells Co-culture of mouse pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, CAR-pMAC-SIRP ⁇ -KO with cancer cells expressing or not expressing the cancer antigen hGPC3. The cells were cultured and cytokine production was evaluated.
• FIG. 10 shows the results of examining the survival of target cells by quantifying the bioluminescence obtained by adding Bright-Glo Luciferase Assay (Promega), a luciferase substrate, to a 96-well tissue culture plate after co-culture. .
• Bright-Glo Luciferase Assay Promega
• the cell-killing effect on target cells was about 20%, but when co-cultivated with CAR-pMAC-SIRP ⁇ -KO, it improved to about 60%. I know that there is.
• Cancer cells are detected by green fluorescence (excitation wavelength 470 nm, absorption wavelength 525 nm) derived from the fluorescent protein Venus, and pMAC or CAR-pMAC-SIRP ⁇ -KO is detected by red fluorescence (excitation wavelength 545 nm, absorption wavelength) derived from the fluorescent protein tdTomato. 605 nm).
• Cancer cells co-cultured with pMAC proliferate, but cancer cells co-cultured with CAR-pMAC-SIRP ⁇ -KO are surrounded by CAR-pMAC-SIRP ⁇ -KO and are eliminated by being phagocytosed. I understand.
• Cancer cytotoxicity of mouse CAR-pMAC To examine the cancer cytotoxicity of mouse CAR-pMAC, cancer cells and CAR-pMAC-SIRP ⁇ -KO were cultured in a 16-well plate E-Plate 16 (Agilent). were co-cultured at 37°C and 5% CO2 . Cancer cells that adhered to the electrode sensor on the plate during pre-culture are phagocytosed by co-culture with CAR-pMAC-SIRP ⁇ -KO, thereby weakening cell adhesion and decreasing electrical resistance. By measuring this electrical resistance value using the xCELLigence RTCA DP system (Agilent), the cancer cytotoxic activity of CAR-pMAC-SIRP ⁇ -KO can be evaluated.
• Target mouse cancer cells hGPC3-introduced or non-expressing CT26 (5 x 10 3 cells), hGPC3-introduced or non-expressing MC38 (1 x 10 4 cells), hGPC3-introduced or non-expressing LL/2 (1 x 10 4 cells) , hGPC3-introduced or non-expressing 4T1 (5 ⁇ 10 3 cells) were seeded on E-Plate 16 coated with iMatrix-511 and cultured for 24 hours at 37°C and 5% CO 2 , and the effector cells, mouse CAR-pMAC- Co-culture was performed by adding 1 times, 5 times, or 10 times the amount of cancer cells targeting SIRP ⁇ -KO.
• Mouse cancer cells hGPC-introduced or non-expressing CT26 (5 ⁇ 10 3 cells) were seeded on E-Plate 16 coated with iMatrix-511, and after culturing at 37°C for 24 hours, effector cells mouse pMAC, pMAC-SIRP ⁇ - KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO (5 ⁇ 10 4 cells) were added and co-cultured. It can be seen that antigen-specific cytotoxic activity (%Cytolysis) is improved by SIRP ⁇ deletion and GC33- ⁇ -CAR introduction against hGPC3-introduced CT26 (FIG. 13).
• FIG. 16 shows images captured by in vivo imaging and changes over time in the total flux of luciferase luminescence.
• CAR-pMAC-SIRP ⁇ -KO significantly suppressed tumor growth and significantly prolonged survival.
• Example 8 Functional analysis of human CAR-pMAC (1) Preparation of human organism-derived macrophages and iPS cell-derived macrophages CD14 monocytes (Lonza) derived from blood collected from healthy human donors were incubated with ImmunoCult-SF Macrophage Differentiation Medium (Stemcell technology). s), 50 ng /mL of human M-CSF in a 6-well tissue culture plate (2 x 10 6 cells/well) and cultured for 4 days to induce CD14-derived macrophages M0 (Human CD14-derived macrophages M0). did.
• CD14-derived macrophages M1 Human CD14-derived macrophages M1
• CD14 monocyte-derived macrophages M0 Human CD14-derived macrophages M0
• IL-4 Human interferon ⁇
• CD14 monocyte-derived macrophages M2 Human CD14-derived macrophages M2
• Human iPS cell-derived macrophages were induced by culturing the differentiated cells including myeloid cells obtained in Example 5 (3) in the presence of 50 ng/mL M-CSF and 20 ng/mL IL-3 for 7 to 14 days.
• RNAseq analysis (species Human , reference genome hg38), which is a whole transcriptome analysis, was performed on pMAC prepared from iPS cells, biologically derived macrophages, and iPSC-derived macrophages, and the transcripts were analyzed. Expression quantification was performed. Total RNA was extracted from each cell using the QIAGEN RNeasy mini kit, and NEBNext® Poly (A) mRNA Magnetic Isolation Module (model number E7490) was extracted using the strand-specific library preparation method (dUTP method).
• NEBNext R Using Ultra TM II Directional RNA Library Prep Kit (model number E7760), PCR amplification was performed using primers containing the Index sequence to prepare a sequence library.
• the base sequence of the library prepared sample was obtained using the next generation sequencer NovaSeq 6000 (Illumina), and the read length was PE150 (150bp x 2 paired-end), the data amount was 4G bases per sample, and the number of reads was 26.7M reads per sample ( 13.3M pairs) of data were acquired. Using the acquired data, informatics analysis was performed to perform principal component analysis (PCA).
• PCA principal component analysis
• the quality scores (scores indicating sequence information error rate) of R1 (Read 1) and R2 (Read 2) were calculated using the software FastQC (version 0.11.7), and abnormalities were detected. None (Entirely Normal, quality score >28) was confirmed.
• the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0), the read mapping rate was calculated, and it was confirmed that all samples were over 98%. Subsequently, raw read counts mapped to known exon regions for each gene were calculated using the software featureCounts (version 1.6.3). Additionally, we perform a count of mapped fragments and calculate the Fragments Per Kilobase of exon per Million mapped fragments (FPKM) value, Fragments Per Kilobase of Transcript per Million mapped reads upper quartile (FPKM-UQ) value, and Transcripts Per Million (TPM) value. The value was calculated.
• FPKM Fragments Per Kilobase of exon per Million mapped fragments
• FPKM-UQ Fragments Per Kilobase of Transcript per Million mapped reads upper quartile
• TPM Transcripts Per Million
• Human pMAC is plotted in the range of PC1 (150 to 200) and PC2 (-20 to -40), and is compared to other biological CD14-positive cell-derived monocytes, CD14-positive cell-derived macrophages (M0, M1, M2 types), Furthermore, it was found that myeloid cells derived from iPS cells and macrophages derived from iPS cells exist in a different region.
• CD14-derived macrophages M1 was treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes and recovered by pipetting.
• the cells include anti-HLA-ABC antibody, anti-HLA-DR antibody, anti-CD40 antibody, anti-CD80 antibody, anti-CD83 antibody, anti-CD86 antibody, anti-CD68 antibody, anti-CD163 antibody, anti-CD206 antibody, anti-SIRP ⁇ antibody, anti-CCR1 antibody, Staining was performed using anti-CCR2 antibody, anti-CCR3 antibody, anti-CCR4 antibody, anti-CCR5 antibody, anti-CCR7 antibody, anti-CXCR2 antibody, anti-CXCR4 antibody and anti-CD14 antibody, or isotype-matched control antibody.
• FIG. 18 shows the results of surface antigen analysis using a flow cytometer after antibody staining. From the results of this analysis, it was found that human pMAC (pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC-SIRP ⁇ -KO) has CD83, CD14, which is expressed in CD14-positive cell-derived macrophages (M0, M1, and M2) and iPSC-derived macrophages. It turns out that the test results are negative.
• FIG. 19-1 shows phase-contrast microscope images of human CD14-positive monocytes, iPSC-derived myeloid cells, pMAC, and CD14-positive cell-derived macrophages (unstimulated M0, M1, and M2 stimulation conditions).
• Phase-contrast microscopic images of cell-derived macrophages, pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO are shown in FIG. 19-2.
• GM-CSF or M-CSF was required in the culture medium for the proliferation of human pMAC and pMAC-SIRP ⁇ -KO. Furthermore, the combined use of GM-CSF and M-CSF had the effect of promoting proliferation. On the other hand, no cytokine-dependent cell proliferation was observed in iPS cell-derived macrophages, indicating that pMAC has high cell proliferation ability.
• FIG. 21 shows the results of examining the expression of GC33 ⁇ CAR in human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO using a flow cytometer. It can be seen that CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO bind hGPC antigen-specifically compared to pMAC and pMAC-SIRP ⁇ -KO.
• M2 type was induced by culturing in the presence of 50 ng/mL IL-4 (BioLegend) for 48 hours.
• IL-6, TNF ⁇ , IL-12, and IL-10 which are known as M1 or M2 type markers, was quantified (FIG. 22). It can be seen that pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO are induced to produce IL-6, TNF ⁇ , and IL-10 by interferon ⁇ and LPS stimulation. On the other hand, no production of IL-12 was detected.
• human cancer cell culture supernatant human HepG2, JHH-7, KOC7c cells (2 ⁇ 10 cells each), and SK-Hep1 ( 5 ⁇ 10 cells) were seeded in a 6-well tissue culture plate. . The next day, the medium was replaced with 2 mL of fresh 10% FCS-containing RPMI-1640 medium and cultured for an additional 24 hours to prepare a culture supernatant. A culture supernatant was prepared using HEK293 (5 ⁇ 10 5 cells) as normal cells. The results of measuring the cell migration ability of human CAR-pMAC-SIRP ⁇ -KO in the culture supernatant of cancer cells are shown in FIG.
• the Cell Index value increases with respect to the culture supernatant of human cancer cells HepG2, SK-Hep1, and JHH-7, indicating that human CAR-pMAC-SIRP ⁇ -KO has migration ability.
• the migration ability toward cancer cell KOC7c was low, and the Cell Index value was comparable to that of normal cell HEK293 (FIG. 23, upper left).
• cancer cells HepG2, SK-Hep1, and KOC7c all had CI values similar to those of the negative control Medium and normal cell HEK293. ( Figure 23, upper right).
• the above results show that CAR-pMAC-SIRP ⁇ -KO has higher cell migration ability than iPS-macrophage against trigger factors produced by multiple cancer cells.
• human recombinant chemokines CCL2 (BioLegend), CCL3 (BioLegend), CCL3LI (BioLegend), CCL4 (BioLegend), CCL5 (BioLegend), CCL7 (R&D Systems), CCL8 (BioLegend), CCL13 (R&D Systems), CCL14 (Novus Biologicals), CCL15 (R&D Systems), CCL23 (BioL egend), CX3CL1 (R&D Systems), CXCL1 (BioLegend), CXCL2 (BioLegend), CXCL5 (BioLegend), CXCL12SDF-1 ⁇ (BioLegend), and CXCL12SDF-1 ⁇ (BioLegend) were all used at a concentration of 100 ng/mL.
• CAR-pMAC-SIRP ⁇ -KO As a result of evaluating the cell migration ability of human CAR-pMAC-SIRP ⁇ -KO against human recombinant chemokines, it was found that it responds to CCL2, CCL3LI, CCL5, CCL7, CCL23, and SDF-1 ⁇ (CXCL12). (Second and third rows from the top in Figure 23). Furthermore, cell migration of CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO to the culture supernatant of cancer cell HepG2 was investigated (bottom row of FIG. 23). It can be seen that CAR-pMAC-SIRP ⁇ -KO has improved cell migration ability compared to CAR-pMAC derived from wild-type iPS cells due to SIRP ⁇ deficiency.
• cytokine production was evaluated by co-culturing cultured cancer cells expressing or not expressing the cancer antigen hGPC3 with human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, and CAR-pMAC-SIRP ⁇ -KO.
• human CAR-pMAC and CAR-pMAC-SIRP ⁇ -KO produce IL-6, TNF ⁇ , and IL-10 by introducing CAR. It can be seen that no induction of antigen-specific cytokine production is observed by co-culture with human cancer cells, hGPC3-introduced or non-expressing SK-Hep1, and hGPC3-expressing or hGPC3-deficient JHH-7 cells. IL-12 was not detected.
• Figure 25 shows the results of examining the survival of target cells by quantifying the bioluminescence obtained by adding Bright-Glo Luciferase Assay (Promega), a luciferase substrate, to a 96-well tissue culture plate after co-culture. .
• Bright-Glo Luciferase Assay Promega
• the killing effect on target cells was about 20-40%, but in co-culture with CAR-pMAC-SIRP ⁇ -KO, it was about 60-90%. You can see that it improves.
• the cell-killing effect of CAR-pMAC-SIRP ⁇ -KO was not observed in target cells SK-Hep1-mock-Luc-V, which do not express hGPC3. It can be seen that it has the ability to phagocytose cancer cells.
• Human cancer cells with or without hGPC3 expression SK-Hep1 (5 x 10 4 cells) were seeded on E-Plate 16 and cultured at 37°C for 24 hours, followed by effector cells human pMAC, pMAC-SIRP ⁇ -KO, CAR- pMAC and CAR-pMAC-SIRP ⁇ -KO (5 ⁇ 10 5 cells) were added and co-cultured. It can be seen that the cytotoxic activity (%Cytolysis) of hGPC3-introduced SK-Hep1 is improved by the introduction of GC33- ⁇ -CAR (FIG. 27, upper left).
• cytotoxic activity is significantly higher than the cytotoxic activity against SK-Hep1, which does not express the cancer antigen hPGC3 (Figure 27, upper right).
• An adhesion signal (Normalized Cell Index value; CI value) that indicates survival of cancer cells by co-culturing cancer cells with human pMAC, pMAC-SIRP ⁇ -KO, CAR-pMAC, CAR-pMAC-SIRP ⁇ -KO,
• the time course of the cytotoxic activity (%Cytolysis) of cancer cells normalized to the case where cancer cells were cultured alone is shown in the middle and lower rows of FIG. 27.
• GC33- ⁇ -CAR introduction and SIRP ⁇ deficiency caused the most remarkable decrease in CI value and increase in %Cytolysis of cancer cells over time.
• a novel CAR-loaded platform uses proliferating macrophage-like cells (pMAC).
• pMAC proliferating macrophage-like cells
• CAR-equipped pMAC is also effective in treating solid cancers.

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