METHOD FOR PRODUCING PROLIFERATIVE MACROPHAGE-LIKE CELLS (pMAC)

Abstract

The present invention provides: proliferative macrophage-like cells (pMAC), characterized by proliferating in a cytokine-dependent manner; a method for producing the same; and a method for producing chimeric antigen receptor (CAR)-proliferative macrophage-like cells (pMAC) that includes (1) a step (step 1) that deletes or suppresses the expression of a gene that inhibits macrophage-like function in pluripotent stem cells, then induces differentiation into myeloid cells to obtain macrophage-like cells (MAC), (2) a step (step 2) that brings about expression of a gene that adds a proliferative property to the MAC obtained in step 1 and obtains proliferative macrophage-like cells (pMAC), and (3) a step (step 3) that introduces a CAR. The present invention also provides the pharmaceutical use of CAR-pMAC. The present invention makes it possible to provide a novel CAR-loaded cell platform, especially a CAR-loaded cell platform having excellent quality stability, stable supply, and economy, and especially to provide a CAR-loaded cell platform that is excellent as a pharmaceutical.

Description

Method for producing proliferative macrophage-like cells (pMACs)

[0001] The present invention relates to proliferative macrophage-like cells (pMAC) derived from pluripotent stem cells and a method for producing the same, a method for producing CAR-pMAC, which comprises introducing a chimeric antigen receptor (CAR) gene into the pMAC, and CAR-pMAC produced by the method, etc.

[0002] One immunotherapy for cancer patients is a treatment using chimeric antigen receptor (CAR) T cells (hereinafter simply referred to as CAR-T cells), which involves genetically modifying the T cell receptor (TCR) of cytotoxic T cells (CTLs) to allow the CTLs to directly and selectively recognize tumor cells and exert an antitumor effect (Non-Patent Document 1). Because cancer treatment using CAR-T cells kills cancer cells using a mechanism different from that of conventional anticancer drugs or radiation therapy, it is expected to be effective against intractable or treatment-resistant cancers. CAR-T cells have already been formulated for some hematological tumors, such as leukemia and malignant lymphoma. However, their effectiveness against solid cancers is limited.

[0003] CAR-T cell therapy faces the following fundamental problems: 1. Lack of cancer tissue tropism (lack of receptors to approach cancer tissue) 2. Lack of infiltration into cancer tissue 3. Functional decline and dysfunction (exhaustion) due to interactions with the tumor microenvironment (TME) 4. Loss of target antigen due to cancer genetic mutations = cancer escape (treatment resistance) 5. Concerns about cytokine release syndrome (CRS) In light of these issues, attempts to improve the therapy have been made both in Japan and overseas. Examples include the introduction of cancer-targeting receptors (CCR2, CXCR2); improvement of the intracellular signaling domain of CAR; immune checkpoint control (anti-PD-1 antibody, PD-1-CD28); conferring cytokine production capabilities (IL7, IL12, IL15, IL18); rejuvenated T / NK cells (iPSC-T / NK cells, SCM-T cells); and dual antigen recognition systems. However, there are currently no reports demonstrating significant clinical efficacy in solid cancers.

[0004] Recently, preclinical results have been reported showing that a cell platform in which CARs are loaded onto macrophages, rather than T cells, is effective in treating solid tumors (Non-Patent Document 2). This is based on the following inherent properties / functions of macrophages: 1. Expressing cancer-targeting receptors to accumulate and infiltrate tumors; 2. Avoiding PD-1 / PD-L1-mediated functional decline; and 3. Inducing a secondary immune response through activation (type 1 macrophage (M1) transformation). Furthermore, Patent Documents 1 and 2 describe CAR-loaded monocytes, macrophages, and dendritic cells, as well as pharmaceutical compositions containing these cells. Thus, CAR-macrophage therapy is a promising cellular immunotherapy, but cell formulation requires stable quality, standardization (stable supply), and mass production (economic efficiency). From the perspectives of stable quality and stable supply, it is preferable that the CAR-loaded cells be allogeneic. Patent Documents 2 and 3 describe methods for differentiating myeloid cells from pluripotent stem cells. Furthermore, a technique for introducing a CAR into iMACs based on iPS cell-derived macrophages has been developed (Non-Patent Document 3). However, this method does not allow the final product to proliferate, and the differentiation induction required at each stage is cumbersome, raising concerns about rising production costs.

[0005] JP 2021-511809 A JP 2022-28831 A International Publication No. 2021 / 230304

[0006] Eshhar Z. et al., Proc Natl Acad Sci USA, 1993, 90: 720-724.Klichinsky M. et al., Nat Biotechnol. 38(8):947-953, 2020Su S. et al., Cells. 2022, 11: 1652

[0007] An objective of the present invention is to provide a novel CAR-equipped cell platform, in particular, to provide a CAR-equipped macrophage-like cell platform that is stable in quality, stable in supply, and economical.

[0008] In view of the above problems, the present inventors focused on macrophage cells as immune cells carrying a CAR. They found that using macrophage cells induced from pluripotent stem cells as CAR-carrying macrophage cells makes it possible to use allogeneic cells, and that imparting cytokine-dependent proliferation activity to the macrophage cells enables mass production, and thus completed the present invention. Specifically, the present invention is as follows.

[0009] [1] Proliferating macrophage-like cells (pMACs), characterized by cytokine-dependent proliferation. [2] The pMAC of [1], wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or macrophage colony-stimulating factor (M-CSF). [3] The pMAC of [1] or [2], which is CD14 and CD83 negative. [4] The pMAC of any of [1] to [3], which is derived from pluripotent stem cells. [5] The pMAC of [4], wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells. [6] The pMAC of any of [1] to [5], characterized in that a gene inhibiting macrophage-like function is deleted or its expression is suppressed. [7] The pMAC of [6], wherein the gene inhibiting macrophage-like function is the signal regulatory protein α (SIRPα) gene. [8] The pMAC according to any one of [1] to [7], further comprising a chimeric antigen receptor (CAR).

[0010] [9] A method for producing proliferative macrophage-like cells (pMACs), comprising the steps of deleting or suppressing the expression of a gene that inhibits macrophage-like function and expressing a gene that confers proliferative properties.

[10] The method according to [9], comprising: (1) a step (step 1) 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 (MACs), and (2) a step (step 2) of expressing a gene that confers proliferative properties in the MACs obtained in step 1 to obtain proliferative macrophage-like cells (pMACs).

[11] The method according to [9], comprising: (A) a step (step A) of expressing a gene that confers proliferative properties in myeloid cells differentiated from pluripotent stem cells to obtain proliferative myeloid cells, and (B) a step (step B) 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 proliferative macrophage-like cells (pMACs).

[12] The method according to any one of [9] to

[11] , wherein the gene that inhibits macrophage-like function is the signal regulatory protein α (SIRPα) gene.

[13] The method according to any one of [9] to

[12] , wherein the gene that imparts proliferation ability is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.

[0011]

[14] A method for producing CAR-pMAC, comprising introducing a chimeric antigen receptor (CAR) into pMAC obtained by the production method according to any one of [9] to

[13] .

[15] A method for producing CAR-pMAC, comprising: (1) a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in a pluripotent stem cell, and then inducing differentiation into myeloid cells to obtain macrophage-like cells (MAC) (Step 1); (2) a step of expressing a gene that confers proliferation ability to the MAC obtained in Step 1 to obtain proliferative macrophage-like cells (pMAC) (Step 2); and (3) a step of introducing a chimeric antigen receptor (CAR) (Step 3).

[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 following method (i) or (ii): (i) culture embryoid bodies (EBs) induced from pluripotent stem cells in a medium containing VEGF in a multilayer on feeder cells, followed by further culturing in a medium containing VEGF, SCF, and TPO, or (ii) culture embryoid bodies (EBs) induced from pluripotent stem cells in a monolayer without feeder cells in a medium containing BMP-4, VEGF, and SCF, followed by further culturing in a medium containing VEGF, TPO, and GM-CSF.

[18] The method according to

[17] , wherein the induction of embryoid bodies (EBs) from pluripotent stem cells is carried out by the following methods (a) and (b): (a) seeding pluripotent stem cells into a cell cluster formation culture vessel to form embryoid bodies (EBs), and (b) the vessel has fine spheroid wells at the bottom, with no flat surface between adjacent wells.

[19] (1) a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells (step 1-1); a step of seeding 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, into a cell cluster formation culture vessel having fine spheroid wells at the bottom and no flat surface between adjacent wells, to form embryoid bodies (EBs) (step 1-2); a step of obtaining macrophage-like cells (MACs) from the EBs obtained in step 1-2 by the following method (i) or (ii) (step 1-3); (i) performing a multilayer culture of the EBs on feeder cells in a medium containing VEGF, followed by further culturing in a medium containing VEGF, SCF, and TPO, or (ii) performing a monolayer culture of the EBs without feeder cells in a medium containing BMP-4, VEGF, and SCF, followed by further culturing in a medium containing VEGF, TPO, and GM-CSF, (2) A method for producing CAR-pMAC, comprising: (Step 2) expressing a gene that confers proliferation to the MAC obtained in Steps 1-3 to obtain proliferative macrophage-like cells (pMAC); and (3) introducing a chimeric antigen receptor (CAR) into the pMAC obtained in Step 2 to obtain CAR-pMAC (Step 3).

[20] The production method according to any of

[15] to

[19] , wherein the gene that inhibits macrophage-like function is signal regulatory protein α (SIRPα) gene.

[21] The production method according to any of

[15] to

[20] , wherein the gene that confers proliferation is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.

[0012]

[22] Proliferative macrophage-like cells that satisfy the following conditions when principal component analysis is performed based on gene expression profiles obtained by performing whole transcriptome analysis using as comparison subjects cell groups along the macrophage cell lineage, consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, biologically-derived monocytes, biologically-derived macrophage M1, biologically-derived macrophage M2, and biologically-derived macrophage M0, and the indicators of the top two components with the highest contribution rates are used as the analysis subjects: Condition 1: the cells do not overlap with plots of known macrophage cell lineages, and Condition 2: PC1 is 100 or greater, and PC2 is plotted in the range of -80 to 0.

[0013]

[23] A pharmaceutical composition comprising the proliferative macrophage-like cells of [8] and a pharmaceutically acceptable carrier.

[24] The pharmaceutical composition of

[23] for treating at least one disease selected from the group consisting of tumor- or cancer-associated diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis.

[25] A method for treating at least one disease selected from the group consisting of tumor- or cancer-associated diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis, comprising administering an effective amount of the proliferative macrophage-like cells of [8] to a subject in need thereof.

[26] The proliferative macrophage-like cells of [8] for use in treating at least one disease selected from the group consisting of tumor- or cancer-associated diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis.

[0014] According to the method of the present invention, it is possible to produce CAR-loaded proliferative macrophage-like cells (CAR-pMAC) that are stable in quality, stable in supply, and economical. The CAR-loaded proliferative macrophage-like cells are also effective in treating solid cancers.

[0015] This figure shows the gene expression profiles of mouse pMAC, bone marrow-derived monocytes (BM-derived monocytes), bone marrow-derived macrophages M0 (BM-derived macrophages M0), mouse bone marrow-derived M1 macrophages (BM-derived macrophages M1), mouse bone marrow-derived M2 macrophages (BM-derived macrophages M2), and peritoneal macrophages (intraperitoneal macrophages) obtained by RNA sequencing analysis. Principal component analysis was performed on the gene expression profiles of each cell, and the first principal component (PC1: contribution rate 34%) was plotted on the horizontal axis, while the second principal component (PC2: contribution rate 21%) was plotted on the horizontal axis. The closer the distance between the plots of each cell, the more similar the gene expression profiles. Conversely, the greater the distance between the plots of each cell, the more dissimilar the gene expression profiles. Histogram showing the results of flow cytometric analysis of CD11b, F4 / 80, CD16 / 32, CD64, I-A / E, and CD206 surface antigens in mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (intraperitoneal macrophages), mouse bone marrow-derived macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 macrophages (BM-derived macrophages M1), mouse bone marrow-derived M2 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 measured marker. Histogram showing the results of flow cytometric analysis of CD163, Ly6C, CD80, CD86, SiglecF, and SIRPα surface antigens in mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages, mouse bone marrow-derived macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1-type macrophages (BM-derived macrophages M1), mouse bone marrow-derived 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. Histograms showing the results of flow cytometric analysis of TLR2, TLR4, CD124, CCR1, CCR2, CCR3, and CCR4 surface antigens in mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (intraperitoneal macrophages), mouse bone marrow-derived macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1-type macrophages (BM-derived macrophages M1), mouse bone marrow-derived 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. Histogram showing the results of flow cytometric analysis of CCR5, CXCR2, and CXCR4 surface antigens in mouse bone marrow-derived monocytes (BM-derived monocytes), mouse intraperitoneal macrophages (intraperitoneal macrophages), mouse bone marrow-derived macrophages (BM-derived macrophages M0), mouse bone marrow-derived M1 macrophages (BM-derived macrophages M1), mouse bone marrow-derived M2 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 measured marker. Microscopic images of May-Giemsa stained mouse pMAC and pMAC-SIRPα-KO lacking the SIRPα gene. The scale bar indicates 20 μm. Compared to pMAC, no characteristic morphological changes due to SIRPα gene deletion were observed. The vector map (top) and CAR expression construct (bottom) of the lentiviral vector used to introduce the GPC3-specific chimeric antigen receptor (GPC3-CAR) gene into pMAC are shown. A line graph shows the results of an MTT assay that examined the effect of a medium (αMEM containing 20% ​​FBS) containing GM-CSF and / or M-CSF on the growth rate of pMAC. The results are shown for mouse pMAC (upper left), CAR-pMAC (upper right), pMAC-SIRPα-KO (lower left), and CAR-pMAC-SIRPα-KO (lower right).The vertical axis represents the concentration of formazan metabolized from the MTT reagent (assessed as absorbance at 595 nm), and the horizontal axis represents the number of days of culture. The deletion of the SIRPα gene and / or the introduction of CAR against the cancer antigen GPC3, which were performed to produce pMAC, did not affect cytokine-dependent cell proliferation. Cytogram showing the results of flow cytometric analysis of hGPC3-FITC, SIRPα, and tdTomato expression in mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO. The vertical axis represents the fluorescence intensity of hGPC3-FITC, and the horizontal axis represents the fluorescence intensity of tdTomato (top). The vertical axis represents the fluorescence intensity of SIRPα, and the horizontal axis represents the fluorescence intensity of tdTomato (middle). The vertical axis shows the fluorescence intensity of SIRPα, and the horizontal axis shows the fluorescence intensity of hGPC3-FITC (lower panel). Because pMAC expressing GPC3-CAR is obtained as a single cell population, it is clear that it is highly pure and contains almost no non-target cells. Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO, and mouse bone marrow-derived macrophages (BM-Macrophages) were cultured in a medium containing no stimulating components, or stimulated M1 with interferon-γ and LPS, or stimulated M2 with IL-4. The production of cytokines IL-6, TNFα, IL-12, and IL-10 was measured by ELISA. The vertical axis shows the concentration (pg / mL) of the measured cytokines. The results of real-time measurement of cell migration ability of mouse CAR-pMAC-SIRPα-KO using the Boyden chamber-type xCELLigence DP system. Cell migration ability toward culture supernatants derived from mouse cancer cells MC38, LL / 2, 4T1, and CT26, and fresh medium (top left); cell migration ability toward culture supernatants derived from mouse cancer cells MC38, normal mouse embryonic fibroblasts (MEF), T cells, and B cells, and fresh medium (top center); and cell migration ability of untreated, cytochalasin D (+CytD), or Bordetella pertussis toxin (+PTX)-treated mouse CAR-pMAC-SIRPα-KO toward culture supernatants from mouse cancer cells MC38, or fresh medium (top right).Cell migration toward recombinant CCL2, CCL3, CCL4, CCL5, and fresh medium (lower left); cell migration toward recombinant CCL7, CCL8, CXCL1, CXCL2, and fresh medium (lower center); cell migration toward recombinant CXCL5, SDF-1α (CXCL12), SDF-1β (CXCL12), CX3CL1, and fresh medium (lower right). The horizontal axis represents the incubation time, and the vertical axis represents the Cell Index (CI), which indicates the degree of cell migration relative to the start of measurement (0 hr). ELISA measurement results of cytokine IL-6 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO was co-cultured with mouse cancer cells (with or without GPC3 expression) for 48 hours. The results are from co-culturing the four types of pMAC mentioned above with breast cancer cell lines 4T1-mock (no GPC3 expression) or 4T1-GPC3 (GPC3 expression) (top row), colon cancer cell lines CT26-mock (no GPC3 expression) or CT26-GPC3 (GPC3 expression) (second row from the top), lung cancer cell lines LL / 2-mock (no GPC3 expression) or LL / 2-GPC3 (GPC3 expression) (third row from the top), and colon cancer cell lines MC38-mock (no GPC3 expression) or MC38-GPC3 (GPC3 expression) (bottom row). ELISA measurement results of cytokine TNFα production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO was co-cultured with mouse cancer cells (with or without GPC3 expression) for 48 hours. The results are from co-culturing the four types of pMAC mentioned above with breast cancer cell lines 4T1-mock (no GPC3 expression) or 4T1-GPC3 (GPC3 expression) (top row), colon cancer cell lines CT26-mock (no GPC3 expression) or CT26-GPC3 (GPC3 expression) (second row from the top), lung cancer cell lines LL / 2-mock (no GPC3 expression) or LL / 2-GPC3 (GPC3 expression) (third row from the top), and colon cancer cell lines MC38-mock (no GPC3 expression) or MC38-GPC3 (GPC3 expression) (bottom row).ELISA measurement results of cytokine IL-10 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO was co-cultured with mouse cancer cells (with or without GPC3 expression) for 48 hours. The results are from co-culturing the four types of pMAC mentioned above with breast cancer cell lines 4T1-mock (no GPC3 expression) or 4T1-GPC3 (GPC3 expression) (top row), colon cancer cell lines CT26-mock (no GPC3 expression) or CT26-GPC3 (GPC3 expression) (second row from the top), lung cancer cell lines LL / 2-mock (no GPC3 expression) or LL / 2-GPC3 (GPC3 expression) (third row from the top), and colon cancer cell lines MC38-mock (no GPC3 expression) or MC38-GPC3 (GPC3 expression) (bottom row). ELISA measurement results of cytokine IL-12 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO was co-cultured with mouse cancer cells (with or without GPC3 expression) for 48 hours. The results are from co-culturing the four types of pMAC mentioned above with breast cancer cell lines 4T1-mock (no GPC3 expression) or 4T1-GPC3 (GPC3 expression) (top row), colon cancer cell lines CT26-mock (no GPC3 expression) or CT26-GPC3 (GPC3 expression) (second row from the top), lung cancer cell lines LL / 2-mock (no GPC3 expression) or LL / 2-GPC3 (GPC3 expression) (third row from the top), and colon cancer cell lines MC38-mock (no GPC3 expression) or MC38-GPC3 (GPC3 expression) (bottom row). Bar graph showing the quantification of luciferase activity in the luciferase (Luc)-transfected lung cancer cell line LL / 2 (with or without human GPC3 expression; upper left), colon cancer cell line CT26 (with or without human GPC3 expression; upper right), colon cancer cell line MC38 (with or without human GPC3 expression; lower left), and breast cancer cell line 4T1 (with or without human GPC3 expression; lower right) after 48 hours of co-culture with mouse pMAC (A), CAR-pMAC (B), pMAC-SIRPα-KO (C), and CAR-pMAC-SIRPα-KO (D). The vertical axis shows the percentage reduction in cancer cells.White bars represent cancer cells that do not express hGPC3, and 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). Mouse pMAC and CAR-pMAC-SIRPα-KO expressing tdTomato were co-cultured with a cancer cell line expressing Venus (human GPC3 expression), and the images (top row) were observed under a fluorescence microscope 2 and 4 days later. Cancer cells were detected by green fluorescence (excitation wavelength 470 nm, absorption wavelength 525 nm) derived from the fluorescent protein Venus, while pMAC or CAR-pMAC-SIRPα-KO were detected by red fluorescence (excitation wavelength 545 nm, absorption wavelength 605 nm) derived from the fluorescent protein tdTomato. Bar graph (lower left) plotting the area of ​​the region where the green fluorescent signal of cancer cells and the red fluorescent signal of pMAC or CAR-pMAC-SIRPα-KO overlap. Bar graph (lower right) plotting the area of ​​the region where the green fluorescent signal of cancer cells was detected. It can be seen that cancer cells co-cultured with pMAC proliferate, but cancer cells co-cultured with CAR-pMAC-SIRPα-KO are surrounded by CAR-pMAC-SIRPα-KO and eliminated by phagocytosis. Graph showing the cytotoxic activity of mouse CAR-pMAC-SIRPα-KO against cancer cells when mouse CAR-pMAC-SIRPα-KO was used as effector cells and co-cultured with four types of mouse cancer cells as target cells. The 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; lower right) were cultured for 24 hours, and then CAR-pMAC-SIRPα-KO was added in amounts 10-fold, 5-fold, or 1-fold the amount of the seeded cancer cells for co-culture. The cytotoxic activity (% Cytolysis) after 6 hours is plotted. The vertical axis represents the cytotoxic activity (% Cytolysis) of the cancer cells, and the horizontal axis represents the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRPα-KO) to target cells (cancer cells).Mouse CAR-pMAC-SIRPα-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. The cell survival signal of the cancer CT26 cells induced by mouse CAR-pMAC-SIRPα-KO was measured using the xCELLigence RTCA DP system. The normalized cell index (top row) and the cytotoxic activity (% cytolysis) of mouse CT26 cells were measured using the xCELLigence RTCA DP system. Cell survival signal of MC38 cancer cells induced by mouse CAR-pMAC-SIRPα-KO. Normalized Cell Index (third row from the top) and cytotoxic activity %Cytolysis (bottom row) of mouse MC38 cells measured using the xCELLigence RTCA DP system. Mouse CAR-pMAC-SIRPα-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. The cell survival signal of LL / 2 cancer cells induced by mouse CAR-pMAC-SIRPα-KO was measured using the xCELLigence RTCA DP system. Normalized Cell Index (top row) and cytotoxic activity (%Cytolysis) of mouse LL / 2 cells were measured using the xCELLigence RTCA DP system. Mouse CAR-pMAC-SIRPα-KO cell survival signal in 4T1 cancer cells (Normalized Cell Index, third row from the top) and mouse 4T1 cell cytotoxicity %Cytolysis (bottom row) measured using the xCELLigence RTCA DP system. Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were co-cultured as effector cells with mouse CT26 (GPC3-expressing) target cells at an E:T ratio of 10:1. The bar graph (top left) plots the cytotoxicity %Cytolysis of CT26 cells on the vertical axis. Mouse CAR-pMAC-SIRPα-KO was used as an effector cell and co-cultured with mouse cancer cell CT26 (with or without GPC3 expression) as target cells at an E:T ratio of 10:1. The bar graph (top right) plots the cytotoxic activity (% Cytolysis) of CT26 cells on the vertical axis.Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with mouse CT26 (no GPC3 expression) target cells at an E:T ratio of 10:1. The graph plots the time course of the normalized cell index (which indicates cell viability of CT26 cells) on the vertical axis and the time of co-culture on the horizontal axis (middle left). The graph also plots the cytotoxic activity (% cytolysis) of mouse CT26 cells (no GPC3 expression) on the vertical axis (bottom left). Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO effector cells were co-cultured with mouse CT26 (GPC3-expressing) target cells at an E:T ratio of 10:1. The graph (middle right) plots the time course of the normalized cell index (indicating cell viability) on the vertical axis and the time of co-culture on the horizontal axis, and the graph (bottom right) plots the cytotoxic activity (% cytolysis) of mouse CT26 cells (GPC3-expressing) on ​​the vertical axis. It can be seen that CAR-pMAC-SIRPα-KO exhibits higher cytotoxic activity after 6 hours of co-culture with cancer cells than pMAC, CAR-pMAC, and pMAC-SIRPα-KO. This is a line graph showing the count of phagocytic cells by time-lapse observation in which mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with mouse cancer cells MC38 (GPC3 expression). The horizontal axis represents the time of co-culture, and the vertical axis represents the number of phagocytic cells. CAR-pMAC-SIRPα-KO was administered to C57BL / 6 cancer-bearing mice that had been transplanted with MC38 (GPC3 expression) to form peritoneal dissemination. The in vivo kinetics of CAR-pMAC-SIRPα-KO were measured by quantitative PCR on Day 0 (top row), 1 day (middle row), and 3 days (bottom row) before administration. The horizontal axis represents each organ evaluated, and the vertical axis represents the common logarithm of the number of CAR-pMAC cells detected in 100 μg of genomic DNA from each organ.Wild-type C57BL / 6 mice were intraperitoneally administered the mouse colon cancer cell line MC38, which expresses the luciferase gene and the cancer antigen GPC3. Surviving cancer cells in these peritoneally disseminated colon cancer model mice were either untreated or administered CAR-pMAC-SIRPα-KO, which was then irradiated with 10 Gy on the following day, day 3, day 7, day 10, day 14, and day 17. Images of all individual mice were measured for viable cancer cells by biochemiluminescence (top), along with a line graph (bottom left) showing the time course of the total flux of luciferase luminescence and a survival curve (bottom right) for each individual tumor-bearing mouse. In vivo imaging was performed on day 0, day 3 (Day 3), day 7 (Day 7), day 10 (Day 10), and day 14 (Day 14) of intraperitoneal administration of MC38. On the right of the image above is a scale showing the luciferase luminescence intensity in chromatic colors, with the minimum luminescence intensity being 2.00 x e. ８ The maximum emission intensity is 1.00 x e １０ (unit: p / sec / cm ２ / sr). The vertical axis of the line graph at the bottom left is the total flux of luciferase luminescence (unit: 10 １０The vertical axis of the graph on the bottom right shows the mouse survival rate, and the horizontal axis shows the number of days after cancer cell transplantation. While cancer cell growth is observed in untreated mice, CAR-pMAC-SIRPα-KO-treated mice suppressed cancer cell growth even after radiation exposure, suppressing the progression of peritoneal dissemination of colon cancer and extending the survival of individual mice. Human pMAC, monocytes derived from blood-derived CD14-positive cells (CD14-derived monocytes), macrophages M0 derived from blood-derived CD14-positive cells (CD14-derived macrophages M0), M1-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M1), M2-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M2), human iPS cell-derived myeloid cells (iPSC-derived myeloid cells), and human iPS cell-derived macrophages (iPSC-derived This figure shows the gene expression profiles of each cell obtained by RNA sequencing analysis of pMAC macrophages, with the first principal component (PC1: contribution rate 40%) plotted on the horizontal axis and the second principal component (PC2: contribution rate 18%) plotted on the horizontal axis. The closer the plots between cells, the more similar the gene expression profiles are; conversely, the farther the plots between cells, the more different the gene expression profiles are. It can be seen that the gene expression profile of pMAC is significantly different from that of known blood-derived or iPS cell-derived macrophages.Monocytes derived from human blood-derived CD14-positive cells (CD14-derived monocytes), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M0), M1-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M1), M2-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSC-derived Histogram showing the results of HLA-ABC, HLA-DR, CD40, CD80, and CD83 surface antigen analysis using a flow cytometer on a 100-well platelet macrophage. The vertical axis represents the percentage of each cell type present, and the horizontal axis represents the fluorescence intensity of each measured marker. Monocytes derived from human blood-derived CD14-positive cells (CD14-derived monocytes), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M0), M1-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M1), M2-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSC-derived 1 is a histogram showing the results of CD86, CD68, CD163, CD206, and SIRPα surface antigen analysis using a flow cytometer on a 10-cell chromatin immunoglobulin macrophage. The vertical axis represents the percentage of each cell type present, and the horizontal axis represents the fluorescence intensity of each measured marker.Monocytes derived from human blood-derived CD14-positive cells (CD14-derived monocytes), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M0), M1-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M1), M2-type macrophages derived from blood-derived CD14-positive cells (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), iPS cell-derived macrophages (iPSC-derived 1 is a histogram showing the results of CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CXCR2, CXCR4, and CD14 surface antigen analysis using a flow cytometer (Cell Phosphage). The vertical axis represents the percentage of each cell type present, and the horizontal axis represents the fluorescence intensity of each measured marker. Phase-contrast microscopic images of human blood-derived CD14-positive cell-derived monocytes (CD14-derived monocytes), iPSC-derived myeloid cells (iPSC-derived myeloid cells), pMAC, blood-derived CD14-positive cell-derived macrophages (CD14-derived macrophages M0), blood-derived CD14-positive cell-derived M1-type macrophages (CD14-derived macrophages M1), and blood-derived CD14-positive cell-derived M2-type macrophages (CD14-derived macrophages M2). Scale bar indicates 40 μm. Phase-contrast microscopic images of human iPS cell-derived macrophages (iPSC-macrophages), pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO under unstimulated conditions (M0), stimulated with interferon-γ and LPS (M1), and stimulated with IL-4 (M2). Line graph showing the results of an MTT assay investigating the effect of a medium (αMEM containing 20% ​​FBS) containing GM-CSF and / or M-CSF on the proliferation rate of human pMAC, pMAC-SIRPα-KO, and iPS cell-derived macrophages (iPSC-macrophages).The graph shows the results for human pMAC (left), pMAC-SIRPα-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. Cytograms showing the results of analysis of hGPC3-FITC, SIRPα, and tdTomato expression by flow cytometry for human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO. The vertical axis represents the fluorescence intensity of hGPC3-FITC, and the horizontal axis represents the fluorescence intensity of tdTomato (top). The vertical axis represents the fluorescence intensity of SIRPα, and the horizontal axis represents the fluorescence intensity of tdTomato (bottom). Since pMAC expressing GPC3-CAR is obtained as a single cell population, it is clear that it is highly pure and contains almost no non-target cells. The production of cytokines IL-6, TNFα, IL-12, or IL-10 was measured by ELISA for human pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO, macrophages derived from CD14-positive cells (CD14-Macrophage) derived from blood collection, and iPS cell-derived macrophages (iPSC-Macrophage) in a medium without stimulating components, M1 stimulation with interferon-γ and LPS, or M2 stimulation with IL-4. The vertical axis indicates the concentration of the measured cytokines (pg / mL). The results of real-time measurement of cell migration ability of human CAR-pMAC-SIRPα-KO using the Boyden chamber-type xCELLigence DP system. Cell migration ability toward culture supernatants from human cancer cell lines HepG2, SK-Hep1, JHH7, and KOC7c, and the human embryonic kidney cell line HEK293, and toward fresh medium (upper left). Cell migration ability of untreated, cytochalasin D (+CytD)-treated, or Bordetella pertussis toxin (+PTX)-treated human CAR-pMAC-SIRPα-KO toward culture supernatants from the human cancer cell line HepG2, the human embryonic kidney cell line HEK293, or toward fresh medium (upper center).The cell migration ability of human iPSC-macrophages toward culture supernatants from cancer cell lines HepG2, SK-Hep1, JHH7, and KOC7c, and the human embryonic kidney cell line HEK293, and toward fresh medium (top right). The cell migration ability of human CAR-pMAC-SIRPα-KO toward recombinant CCL2, CCL3, CCL3LI, and CCL4, and toward fresh medium (second row from the top, left). Cell migration toward recombinant CCL5, CCL7, CCL8, CCL14, and fresh medium (middle, second row from the top), recombinant CCL15, CCL17, CCL23, CX3CL1, and fresh medium (middle, second row from the top), recombinant CXCL1, CXCL2, CXCL5, and fresh medium (middle, third row from the top), and recombinant CCL13, SDF-1α (CXCL12), SDF-1β (CXCL12), and fresh medium (middle, third row from the top). Cell migration ability of CAR-pMAC and CAR-pMAC-SIRPα-KO toward culture supernatant derived from the cancer cell line HepG2 (bottom panel). The horizontal axis represents culture time, and the vertical axis represents Cell Index (CI), which indicates the degree of cell migration ability relative to the start of measurement (0 hr). ELISA measurement results for the production of cytokines IL-6 (left), TNFα (second from left), IL-10 (third from left), and IL-12 (right) in the culture supernatant after 48 hours of culture of human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO. These are ELISA measurement results of cytokine IL-6 production in the culture supernatant when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with human cancer cell lines (with or without GPC3 expression) for 48 hours. The results are from co-culture of the four types of pMAC mentioned above with the liver cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top row), and the liver cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (second row from the top).These are ELISA measurement results of cytokine IL-10 production in the culture supernatant when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with human cancer cell lines (with or without GPC3 expression) for 48 hours. These are results of co-culture of the four types of pMAC described above with the liver cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (third row from the top), and the liver cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (bottom row). These are ELISA measurement results of cytokine TNFα production in the culture supernatant when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with human cancer cell lines (with or without GPC3 expression) for 48 hours. The results are from co-culture of the four types of pMAC mentioned above with the cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top row), and the cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (second row from the top). These are ELISA measurement results of cytokine IL-12 production in the culture supernatant when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with human cancer cell lines (with or without GPC3 expression) for 48 hours. The results are from co-culture of the four types of pMAC described above with the cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (third row from the top), and the cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (bottom row). Bar graph showing the results of quantification of luciferase activity in the human liver cancer cell line JHH7-Luc (expressing human GPC3; left), human liver cancer cell line SK-Hep1-GPC3-Luc-Venus (expressing human GPC3; second from left), and human cancer cell line KOC7c-GPC3-Luc (expressing human GPC3; third from left) transfected with luciferase (Luc) gene after 48 hours of co-culture with human pMAC (A), CAR-pMAC (B), pMAC-SIRPα-KO (C), and CAR-pMAC-SIRPα-KO (D).Bar graph showing the results of quantifying luciferase activity in cancer cells after 48 hours of co-culture of CAR-pMAC-SIRPα-KO (D) with the human liver cancer cell line SK-Hep1-GPC3-Luc-Venus (with or without human GPC3; right). The vertical axis represents the percentage reduction in cancer cells. White bars represent cancer cells that do not express hGPC3, and black bars represent cancer cells that express hGPC3. It can be seen that CAR-introduced pMAC (D) eliminates cancer cells that express the target cancer antigen, human GPC (hGPC). Cytotoxic activity of human CAR-pMAC-SIRPα-KO on cancer cells when human CAR-pMAC-SIRPα-KO was used as effector cells and co-cultured with two types of human cancer cells as target cells. The human liver cancer cell line SK-Hep1 (with or without human GPC3 expression) was cultured for 24 hours, and then CAR-pMAC-SIRPα-KO was added in amounts 10, 5, or 1 times the amount of the seeded cancer cells for co-culture, and the cytotoxic activity (% Cytolysis) after 6 hours was plotted. The vertical axis represents the cytotoxic activity (% Cytolysis) of the cancer cells, and the horizontal axis represents the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRPα-KO) to target cells (cancer cells). Human CAR-pMAC-SIRPα-KO was used as an effector cell and co-cultured with target human cancer cells (with or without human GPC3 expression) at E:T ratios of 10:1, 5:1, and 1:1. The cell survival signal of human CAR-pMAC-SIRPα-KO in SK-Hep1 hepatoma cells was measured using the xCELLigence RTCA DP system. The normalized cell index (top) and cytotoxic activity (% cytolysis) of SK-Hep1 human hepatoma cells were measured using the xCELLigence RTCA DP system. Human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with human hepatoma cells SK-Hep1 (expressing GPC3) as target cells at an E:T ratio of 10:1. The bar graph plots the cytotoxic activity (%Cytolysis) of SK-Hep1 on the vertical axis (top left).Human CAR-pMAC-SIRPα-KO was used as an effector cell and co-cultured with human hepatoma SK-Hep1 cells (with or without GPC3 expression) as target cells at an E:T ratio of 10:1. The bar graph plots the cytotoxic activity (% Cytolysis) of the hepatoma SK-Hep1 cells on the vertical axis (top right). Human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with human SK-Hep1 (without GPC3 expression) as target cells at an E:T ratio of 10:1. The graph plots the time course of the normalized cell index (indicating cell viability) on the vertical axis and the time of co-culture on the horizontal axis (middle left). The graph also plots the cytotoxic activity (% cytolysis) of human hepatoma SK-Hep1 (without GPC3 expression) on the vertical axis (bottom left). Human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with human cancer cells SK-Hep1 (with GPC3 expression) at an E:T ratio of 10:1. The normalized cell index (normalized cell index) indicates cell viability of SK-Hep1 (with GPC3 expression) over time, and the horizontal axis represents the time of co-culture (middle right). The vertical axis represents the cytotoxic activity (% cytolysis) of human hepatoma cells SK-Hep1 (with GPC3 expression) (bottom right). It can be seen that CAR-pMAC-SIRPα-KO and CAR-pMAC exhibit higher cytotoxic activity after 6 hours of co-culture with cancer cells compared to pMAC and pMAC-SIRPα-KO.

[0016] The present invention will now be described. Terms used in this specification have the meanings commonly used in the art unless otherwise specified.

[0017] <Proliferating Macrophage-Like Cells> The present invention provides macrophage cells characterized by cytokine-dependent proliferation in addition to phagocytic activity. The macrophage cells of the present invention have characteristics different from existing macrophages, and are therefore referred to as proliferating macrophage-like cells (pMACs) (hereinafter also referred to as pMACs of the present invention). Alternatively, the pMACs of the present invention can be referred to as cytokine-dependent proliferating macrophages. In one aspect, the pMACs of the present invention are deleted or have their expression suppressed for a gene that inhibits macrophage-like function.

[0018] As used herein, "cytokine-dependent proliferation" means that cell proliferation is promoted by cytokine stimulation, allowing for culturing for three months or more, and showing significantly superior proliferation compared to cells without cytokine stimulation. Cytokine stimulation specifically includes culturing cells in a cytokine-containing medium. Cytokines may be exogenously added to the medium, or may be produced intracellularly and secreted into the medium. Cytokines used include one or, preferably, two cytokines selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). A combination of GM-CSF and M-CSF is preferred. GM-CSF, also known as CSF2, is produced by cells such as T cells, macrophages, endothelial cells, and fibroblasts in response to immune stimulation, and functions as a hematopoietic growth factor and immunoregulatory factor. GM-CSF induces bone marrow progenitor cells and promotes proliferation of granulocytes and macrophages. M-CSF is a type of cytokine that induces proliferation and differentiation of bone marrow stem cells, particularly the monocyte-macrophage system. Each cytokine is commercially available. Alternatively, sequence information for the protein, genomic DNA, or cDNA of each cytokine from various organisms can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI). Based on this information, M-CSF can be obtained by expressing the gene for the desired cytokine in cells, producing the protein within the cells, and, in some cases, secreting it extracellularly.

[0019] The pMAC of the present invention is characterized by its cytokine-dependent proliferation and also by its CD83 and CD14 negativity.

[0020] CD83 is known as a dendritic cell lineage maturation marker expressed on the cell surface, and can be detected by immunostaining with an anti-CD83 antibody and analyzing with a flow cytometer.

[0021] CD14 is known as a marker expressed on the cell surface of monocytes and macrophages, and can be detected by immunostaining with an anti-CD14 antibody and analyzing with a flow cytometer.

[0022] Here, "negative for CD83 and CD14" means that CD83 and CD14 are not expressed or are 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 only in very small amounts. Specifically, this means that the expression levels of CD83 and CD14 in existing macrophages are 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.

[0023] Furthermore, as demonstrated in detail in the following Examples and Figures, the pMACs of the present invention are characterized by significantly altered gene expression profiles compared to cells belonging to known macrophage cell lineages. Therefore, the pMACs of the present invention can also be defined using statistics based on the gene expression profiles. For example, in the case of human pMACs, as demonstrated in detail in the Examples and Figures (particularly Figure 17), they can be defined as follows: A whole-transcriptome analysis is performed using a group of cells along the macrophage cell lineage, consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, biologically-derived monocytes, biologically-derived macrophage M1, biologically-derived macrophage M2, and biologically-derived macrophage M0, to compare the gene expression profiles obtained. When the indicators of the top two components with the highest contribution rates are used as the analysis targets, the proliferative macrophage-like cells satisfy the following conditions: Condition 1: The plot does not overlap with the plot of known macrophage cell lineages, and Condition 2: PC1 is 100 or greater, and PC2 is plotted in the range of -80 to 0. In one aspect, 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, and more preferably, but is not limited to, 150 or more. Furthermore, the pMAC of the present invention has a PC1 of 250 or less, preferably 220 or less, 210 or less, 200 or less, or 190 or less, and more preferably, but is not limited to, 180 or less. In one aspect, 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, and more preferably, but is not limited to, 150 to 180.

[0024] 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 refer to stem cells that can be cultured in vitro while maintaining an undifferentiated state and have the ability (pluripotency) to differentiate into all cells that constitute a living body except for the placenta (tissues derived from the three germ layers (ectoderm, mesoderm, and endoderm)). Embryonic stem cells (ES cells) isolated from embryos before the formation of the three germ layers after fertilization, and embryos and fetuses from the organogenesis stage or later including primordial germ cells, are also included in the term "pluripotent stem cells."

[0025] In the present invention, "induced pluripotent stem cells" refer to cells in which pluripotency has been induced by directly reprogramming somatic cells that have once lost pluripotency through the expression of several genes such as Oct3 / 4, Sox2, Klf4, and Myc (hereinafter also referred to as iPSCs or iPS cells). These "induced pluripotent stem cells" are also included in "pluripotent stem cells." Pluripotent stem cells can be produced by known methods. Examples of production methods include those described in Cell, 2007, 131(5) pp. 861-872 and Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)) for human induced pluripotent stem cells, and those described in Cell, 2006, 126(4) pp. 663-676 for mouse induced pluripotent stem cells. Pluripotent stem cells can also be obtained from animals that have undergone embryonic manipulation techniques, including cloned embryos, transgenic animals, and genome-edited mutant animals. Pluripotent stem cells are known to be capable of differentiating into a variety of cell types, including myeloid cells (MC cells).

[0026] Human or mouse iPSCs can be maintained and expanded using the regenerative medicine medium StemFit (Ajinomoto). Laminin 511 may be added at this time. Furthermore, to reduce or eliminate damage to iPSCs caused by cell dissociation for subculture, a Rho kinase (ROCK) inhibitor, such as Y-27632, may be added to the medium at approximately 10 μM.

[0027] Pluripotent stem cells are available from designated institutions, and also commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Mouse embryonic stem cells, EB5 cells, are available from RIKEN, a national research and development agency, and the D3 line is available from ATCC.

[0028] Pluripotent stem cells can be maintained and cultured by known methods. For example, human pluripotent stem cells can be maintained and cultured by the method described in Knockout et al. ＴＭ Mouse pluripotent stem cells can be maintained by culturing them in a medium supplemented with fetal bovine serum (FBS) and leukemia inhibitory factor (LIF) in a feeder-free environment.

[0029] <Method for producing proliferative macrophage-like cells> In the present invention, pMAC can be produced by a method comprising the steps of deleting or suppressing the expression of a gene that inhibits macrophage-like function and expressing a gene that confers proliferative properties. Each step will be described below.

[0030] 1. A process of deleting or suppressing the expression of a gene that inhibits macrophage-like function. A "gene that inhibits macrophage-like function" refers to a gene that inhibits a function that can be provided by macrophages. Examples of "functions that can be provided by macrophages" include phagocytosis (engulfment), 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 the induction of secondary immune responses through activation. Macrophage activation is broadly divided into classical (M1) activation and alternative (M2) activation. M1-activated macrophages (M1 macrophages) are obtained by inducing the differentiation of monocytes with inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and produce pro-inflammatory cytokines that act to defend against pathogenic infections and reduce the infectivity of microorganisms. M2-activated macrophages (M2 macrophages) are obtained by inducing differentiation with Th2 cytokines such as IL-4 and IL-13, and are responsible for producing growth factors and anti-inflammatory cytokines that suppress host immune responses, promote wound healing and tissue remodeling, and improve intra-tissue metabolism and endocrine signaling. A "gene that inhibits macrophage-like function" is a gene whose expression can inhibit the functions that can be provided by such macrophages. Examples of such genes include, but are not limited to, the signal regulatory protein α (SIRPα) gene.

[0031] The SIRPα gene is a membrane protein present on the cell membrane that separates the inside and outside of a cell, and is abundant in macrophages. SIRPα binds to another membrane protein, CD47, present on cells that are phagocytic targets of macrophages, thereby weakening their phagocytic activity. That is, for example, cells in which the SIRPα gene is deleted or whose expression is suppressed lose the ability to bind to CD47 present on the cell membrane of cancer cells, and therefore their phagocytic activity is not weakened. Methods commonly used in the art can be used to delete or suppress the expression of the gene. One embodiment includes a nucleic acid that suppresses the expression of the SIRPα gene. The nucleic acid may act at any stage of the SIRPα gene, such as 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 SIRPα gene include nucleic acids that inhibit the transcription of the SIRPα gene (e.g., antigene), nucleic acids that inhibit the processing of initial transcription products into mRNA, and nucleic acids that inhibit the translation of mRNA into protein (e.g., antisense nucleic acids, miRNA) or degrade mRNA (e.g., siRNA, ribozymes, miRNA). While substances that act at any stage can be used, substances that bind complementarily to mRNA to inhibit translation into protein or degrade mRNA are preferred. The nucleotide sequence of the SIRPα gene is known, and sequence information can be obtained, for example, from public databases (e.g., NCBI, EMBL, DDBJ, etc.).

[0032] Another preferred embodiment includes a nucleic acid that suppresses the expression of the SIRPα gene using genome editing. Specifically, the nucleic acid that suppresses the expression of the SIRPα gene can be a nucleic acid that can inactivate (knock out) the SIRPα gene. Examples of such nucleic acids include nucleic acids encoding artificial nucleases consisting of 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 nuclease that introduces a double-strand break (DSB) into the gene within or near the target sequence. After introducing the DSB, the gene can be knocked out by an insertion or deletion mutation caused by a non-homologous end joining (NHEJ) repair error. Alternatively, gene knockout can be achieved by homologous recombination (HR) repair by combining the gene with a targeting vector in which a marker gene (e.g., a reporter gene such as a fluorescent protein gene, or a selectable marker gene such as a drug resistance gene) has been inserted into the gene sequence. Thus, macrophage-like cells can be prepared by deleting or suppressing the expression of genes that inhibit macrophage-like functions.

[0033] 2. Step of expressing a gene that confers proliferation properties A "gene that confers proliferation properties" is a gene that has the function of promoting cell proliferation when introduced into a cell and expressed within the cell, and examples of such genes include genes for various factors. Herein, this 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 their homologs or orthologs. The biological species selected is one that is compatible with the animal species from which the cells to be introduced are derived. Preferably, at least one gene, preferably two genes, and more preferably all three genes selected from the group consisting of c-MYC, BMI1, and MDM2 are introduced and expressed.

[0034] Sequence information for "c-MYC," "BMI1," and "MDM2" proteins, genomic DNA, or cDNA of various biological species can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI). In the present invention, "expression" refers to producing a desired gene or protein intracellularly and, in some cases, secreting it extracellularly to allow the gene or protein to exert its function.

[0035] In the present invention, "gene-introduced" means that a desired gene is introduced into a specific cell, and the gene or a protein encoded by the gene is produced within the cell, and in some cases, secreted outside the cell, thereby allowing the protein to exert its function.

[0036] One embodiment of the method for producing pMAC of the present invention is the following method (Aspect 1): (1) 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 (MAC) (Step 1), and (2) a step of expressing a gene that confers proliferative properties to the MAC obtained in Step 1 to obtain proliferative macrophage-like cells (pMAC) (Step 2). In 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 carried out on pluripotent stem cells. Here, the pluripotent stem cells are preferably induced pluripotent stem cells or embryonic stem cells, and more preferably induced pluripotent stem cells. Next, pluripotent stem cells lacking or suppressed in expression of a gene that inhibits macrophage-like function, for example, pluripotent stem cells (particularly iPS cells) lacking or suppressed in expression of the SIRPα gene, are induced to differentiate into myeloid cells (hereinafter also referred to as "myeloid differentiation"), thereby obtaining macrophage-like cells (MAC).

[0037] Myeloid cells are a collective term for monocytes, macrophages, dendritic cells, and progenitor cells that belong to the cell lineages that differentiate into these cells, among the white blood cells differentiated from stem cells. By myeloid differentiation of pluripotent stem cells lacking or suppressed in the expression of a gene that inhibits macrophage-like function, the differentiation-induced myeloid cells become macrophage-like cells. Specific methods for obtaining macrophage-like cells (MACs) by myeloid differentiation of pluripotent stem cells lacking or suppressed in the expression of a gene that inhibits macrophage-like function include the following methods. (i) culturing embryoid bodies (EBs) induced from pluripotent stem cells in a medium containing VEGF in a layer on feeder cells, followed by further culturing in a medium containing VEGF, SCF, and TPO, or (ii) culturing embryoid bodies (EBs) induced from the pluripotent stem cells in a monolayer without feeder cells in a medium containing BMP-4, VEGF, and SCF, followed by further culturing in a medium containing VEGF, TPO, and GM-CSF. In the above (i) and (ii), the pluripotent stem cells are pluripotent stem cells in which a gene that inhibits macrophage-like function has been deleted or expression of the gene has been suppressed.

[0038] In step 2 of embodiment 1, the MAC obtained in step 1 is subjected to the above-mentioned "2. Step of expressing a gene that confers proliferation ability."

[0039] Another embodiment of the method for producing pMAC of the present invention is the following method (Aspect 2): (A) a step of expressing a gene that confers proliferation capability to myeloid cells differentiated from pluripotent stem cells to obtain proliferative myeloid cells (Step A), and (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 proliferative macrophage-like cells (pMAC) (Step B). In Step A of Aspect 2, the above-mentioned "2. Step of expressing a gene that confers proliferation capability" is carried out on myeloid cells differentiated from pluripotent stem cells (myeloid cells derived from pluripotent stem cells). Here, the pluripotent stem cells are preferably induced pluripotent stem cells or embryonic stem cells, and more preferably induced pluripotent stem cells. Myeloid cells conferred with proliferation capability can be obtained by these steps. Specific methods for inducing differentiation of pluripotent stem cells into myeloid cells include the following. (i) culture embryoid bodies (EBs) induced from pluripotent stem cells in a medium containing VEGF in a layer on feeder cells, followed by further culturing in a medium containing VEGF, SCF, and TPO; or (ii) culture embryoid bodies (EBs) induced from the pluripotent stem cells in a monolayer without feeder cells in a medium containing BMP-4, VEGF, and SCF, followed by further culturing in a medium containing VEGF, TPO, and GM-CSF.

[0040] In step B of embodiment 2, the above-mentioned "1. step of deleting or suppressing the expression of a gene that inhibits macrophage-like function" is carried out on the proliferative myeloid cells obtained in step A. By deleting or suppressing the expression of a gene that inhibits macrophage-like function, such as the SIRPα gene, in proliferative myeloid cells, the cells can be induced to become macrophage-like cells, and pMAC can be obtained.

[0041] In the present invention, the term "embryoid body" refers to a cell mass formed by the adhesion of undifferentiated pluripotent stem cells. Due to the close intercellular interactions within the cell mass, the pluripotent stem cells that make up the cell mass have a limited repertoire of cell differentiation, similar to an embryo at the developmental stage in which germ layers are formed after implantation.

[0042] The size of the embryoid bodies of the present invention is not particularly limited, but a diameter of 50 to 1000 μm is preferred, 50 to 500 μm is more preferred, and 50 to 200 μm is particularly preferred. If the size of the embryoid bodies is larger than the above range, there is a risk of insufficient oxygen supply to the interior of the embryoid bodies, resulting in cell necrosis. If the size of the embryoid bodies is smaller than the above range, there is a risk of single cells not forming embryoid bodies and undergoing cell death by apoptosis. Mouse embryoid bodies may be cultured using mouse mesenchymal C3H10T1 / 2 cells or mouse bone marrow-derived stromal OP9 cells as feeder cells. Human embryoid bodies can be cultured without the use of feeder cells.

[0043] "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the present invention refer to VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins of any biological species, and homologs or orthologs thereof, which have the activity of inducing differentiation of human or non-human mammalian pluripotent stem cells into myeloid cells (MCs).

[0044] The "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the present invention may be VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins from a biological species other than the biological species of the embryoid bodies being cultured, or mutants of VEGF, SCF, TPO, BMP-4, and / or GM-CSF proteins from the same biological species as the biological species of the embryoid bodies being cultured, provided that they have the same level of differentiation-inducing activity as the "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the biological species of the embryoid bodies being cultured.

[0045] Herein, Flt-3L, IL-3, and / or bFGF may also be used in culturing the cells of the present invention, together with the above-mentioned VEGF, SCF, TPO, BMP-4, and / or GM-CSF, etc. These can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI). Furthermore, recombinant protein reagents are available from R&D Systems (https: / / www.rndsystems.com / ), including Recombinant Human Flt-3 Ligand / FLT3L Protein (308-FKN), Recombinant Human IL-3 Protein (203-IL), Recombinant Human FGF basic / FGF2 / bFGF (145 aa) Protein (3718-FB), Recombinant Human VEGF 165 Protein (293-VE), and Recombinant Human SCF (3718-FB). It is also possible to purchase Recombinant Human GM-CSF Protein (215-GM), Recombinant Human Thrombopoietin Protein (255-SC), Recombinant Human BMP-4 Protein (314-BP), and Recombinant Human GM-CSF Protein (215-GM).

[0046] Sequence information of proteins, genomic DNAs, or cDNAs of various biological species of "VEGF," "SCF," "TPO," "BMP-4," and / or "GM-CSF" of the present invention can be obtained from sources well known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI).

[0047] The above-mentioned genes or proteins are preferably genes or proteins of the same species as those of the pluripotent stem cells used. For example, when the pluripotent stem cells are derived from humans, it is preferable to use human-derived VEGF, SCF, TPO, BMP-4, GM-CSF, Flt-3L, IL-3, and / or bFGF, and when the pluripotent stem cells are derived from mice, it is similarly preferable to use mouse-derived genes or proteins.

[0048] In the present invention, "medium containing substance X" means a medium to which exogenous substance X has been added or a medium containing exogenous substance X, and "medium not containing substance X" means a medium to which exogenous substance X has not been added or a medium not containing exogenous substance X. Here, "exogenous substance X" means substance X that is foreign to cells or tissues cultured in the medium, and does not include endogenous substance X produced by the cells or tissues.

[0049] For example, a "VEGF-containing medium" refers to a medium to which exogenous VEGF has been added or a medium containing exogenous VEGF. The dose of VEGF may be affected by other growth factors, but is preferably 0.2 ng / mL to 200 ng / mL, more preferably 2 ng / mL to 100 ng / mL, and particularly preferably 5 ng / mL to 50 ng / mL. A "VEGF-free medium" refers to a medium to which exogenous VEGF has not been added or a medium containing no exogenous VEGF.

[0050] Similarly, for SCF, TPO, BMP-4, and GM-CSF, a medium containing SCF, TPO, BMP-4, and / or GM-CSF refers to a medium supplemented with exogenous SCF, TPO, BMP-4, and / or GM-CSF, or a medium containing exogenous SCF, TPO, BMP-4, and / or GM-CSF. Therefore, although the dose of SCF, TPO, BMP-4, and / or GM-CSF may be affected by other growth factors, it is preferably 0.2 ng / mL to 200 ng / mL, and more preferably 2 ng / mL to 100 ng / mL. In the case of TPO, the dose is more preferably 5 ng / mL to 15 ng / mL. In the case of SCF, BMP-4, and GM-CSF, the dose is more preferably 5 ng / mL to 70 ng / mL.

[0051] "Culture medium free of SCF, TPO, BMP-4 and / or GM-CSF" refers to a culture medium to which exogenous VEGF has not been added or a culture medium free of exogenous SCF, TPO, BMP-4 and / or GM-CSF.

[0052] In the present invention, αMEM supplemented with 20% fetal bovine serum (FBS) can be used as a medium for mouse embryoid bodies. Mouse mesenchymal C3H10T1 / 2 cells or mouse bone marrow-derived stromal OP9 cells can be used as feeder cells. StemFit (Ajinomoto), a fully defined medium, can be used as a medium for human embryoid bodies.

[0053] The medium may be αMEM supplemented with 20% FBS.

[0054] In the present invention, "feeder cells" refer to other cell types used to prepare culture conditions for maintaining the undifferentiated state of iPS cells or for inducing differentiation. Feeder cells are used after stopping their growth by treatment with mitomycin C or irradiation. Mouse bone marrow-derived stromal cells OP9 and mouse-derived mesenchymal cells C3H10T1 / 2, etc., can be used.

[0055] In the present invention, "multilayer culture" refers to the co-culturing of iPS cells or embryoid bodies of interest seeded on a culture system of a feeder cell layer cultured in a predetermined culture vessel. In the present invention, "monolayer culture" refers to the culturing of a single cell type adhered to a vessel for tissue culture.

[0056] In the present invention, feeder cell-free monolayer culture refers to static culture of iPS cells or embryoid bodies of interest seeded in a culture vessel.

[0057] Furthermore, one embodiment of 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 into a vessel for cell cluster formation culture to form embryoid bodies (EBs), and (ii) the vessel has fine spheroid wells at the bottom, with no flat surface between adjacent spheroid wells.

[0058] "Forming embryoid bodies" refers to dissociating pluripotent stem cells that have been growing in an undifferentiated state to obtain a single-cell dispersion of pluripotent stem cells, and then subjecting the dispersion to suspension culture or static culture under conditions that allow the pluripotent stem cells to adhere to each other and aggregate, thereby forming qualitatively uniform cell masses. Embryoid body formation is confirmed by visually confirming the presence of viable cells through microscopic observation.

[0059] As used herein, "suspension culture" refers to culturing under conditions in which cells or cell aggregates are kept in a suspended state and do not adhere to a substrate such as the bottom or wall of a culture vessel or the like.

[0060] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and those skilled in the art can appropriately determine the appropriate vessel. Examples of such vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surface has not been artificially treated (e.g., coated with an extracellular matrix) to improve cell adhesion.

[0061] In the present invention, it is preferable to rapidly aggregate pluripotent stem cells to form embryoid bodies derived from the pluripotent stem cells. When cell clusters of pluripotent stem cells are formed in this way and then transferred to a medium containing VEGF at a concentration of 50 ng / mL for culture, the cells of the cell clusters can be differentiated into myeloid cell lineages, similar to the blood islands in the yolk sac.

[0062] Experimental procedures for forming embryoid bodies include, for example, confining cells in a small space using a culture vessel such as a plate with many small-diameter holes (wells) (e.g., a 96-well plate) or a vessel for cell aggregate formation culture, and a method of aggregating cells by short-term centrifugation using a small centrifuge tube. Culture conditions for cells or pluripotent stem cell aggregates include rotational culture, shaking culture, and other suspension cultures, as well as static culture, provided that the substrate surface of the culture vessel that comes into contact with the cells is previously treated to make it hydrophobic, thereby reducing or suppressing adhesion between the pluripotent stem cells and the substrate surface of the culture vessel.

[0063] The formation of embryoid bodies as cell masses of pluripotent stem cells and the differentiation of cells from embryoid bodies into myeloid cells can be determined based on characteristics including, but not limited to, confirmation of the size and cell number of the embryoid bodies using a microscope or FACS, confirmation of the microscopic morphology of the embryoid bodies in culture, confirmation of histological or cytochemical findings, and / or confirmation of the expression of differentiated and undifferentiated markers, their uniformity, control of the expression of differentiation markers, and confirmation of the synchrony or reproducibility of differentiation efficiency between embryoid bodies.

[0064] In the present invention, a cell is "positive" for a certain marker when the cell is measured by flow cytometry, and a ratio of the mean fluorescence intensity of the positive cells to the negative control is 10 times or more, defined as "+", 2 times to less than 10 times, defined as "low", and less than 2 times, defined as "-". Furthermore, in the present invention, the expression of a certain gene in a certain cell is high or low when the expression level of the gene is measured by real-time PCR, and an expression level of the housekeeping gene β-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HPRT 1 (hypoxanthine phosphoribosyltransferase 1), or β2-microglobulin is 1 time or more, defined as "high", and an expression level less than that is defined as "low".

[0065] In the present invention, the single cell dispersion is prepared by washing cultured cells with PBS or the like, treating them with a cell detachment solution such as trypsin-EDTA (Life Technologies) or TrypLE Select (Life Technologies) for 4 to 5 minutes, further washing them with PBS or the like, and centrifuging them to obtain a pellet of target cells, and then resuspending the pellet in a desired culture medium or the like by pipetting.

[0066] In the present invention, a vessel for cell aggregate formation culture refers to a vessel in which seeded cells can form cell aggregates without adhering to the vessel during culture. Preferably, the vessel has minute spheroid wells on its bottom, which is the culture surface, and adjacent spheroid wells are separated by a curved surface that slopes toward one of the spheroid wells, with no flat surface between them, and the curved surface is surface-treated to suppress cell adhesion, so that most of the cells seeded in the culture vessel fall into one of the spheroid wells.

[0067] An example of a vessel is one in which a spheroid well with a diameter of approximately 400 to 800 μm and a depth of 100 to 800 μm is uniformly distributed along the wall surface without gaps on the culture surface (bottom). Preferably, the spheroid well has a diameter of 400 to 500 μm and a depth of 50 to 200 μm. More preferably, the spheroid well is a vessel with a diameter of approximately 500 μm and a depth of approximately 400 μm, a vessel with a diameter of approximately 800 μm and a depth of approximately 400 μm, or a vessel with a diameter of approximately 800 μm and a depth of approximately 300 μm. Most preferably, the spheroid well is a vessel with a diameter of approximately 400 μm and a depth of 100 to 200 μm.

[0068] Preferably, the vessel has spheroid wells that are uniformly processed all the way to the wall surface, with no gaps on the culture surface (bottom). In such a vessel, there are no flat surfaces between adjacent spheroid wells, and the seeded single-cell suspension falls into one of the spheroid wells, allowing the formation of uniform aggregates. More preferably, the vessel is a microfabricated cell culture vessel (EZSPHERE®) manufactured by AGC.

[0069] pMAC can be produced by either the method of Embodiment 1 or Embodiment 2, but is preferably produced by the method of Embodiment 1. By carrying out a step of expressing a gene that confers proliferation ability after a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function, a cell population containing highly pure pMAC with few undesired cells can be stably produced. A more specific procedure of Embodiment 1 is as follows. (1) a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells (step 1-1); a step of seeding 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, into a cell cluster formation culture vessel having fine spheroid wells at the bottom and no flat surface between adjacent wells, to form embryoid bodies (EBs) (step 1-2); a step of obtaining macrophage-like cells (MACs) from the EBs obtained in step 1-2 by the following method (i) or (ii) (step 1-3); (i) performing a multilayer culture of the EBs on feeder cells in a medium containing VEGF, followed by further culturing in a medium containing VEGF, SCF, and TPO, or (ii) performing a monolayer culture of the EBs without feeder cells in a medium containing BMP-4, VEGF, and SCF, followed by further culturing in a medium containing VEGF, TPO, and GM-CSF, (2) A step (step 2) of expressing a gene that imparts proliferation to the MAC obtained in steps 1-3 to obtain proliferative macrophage-like cells (pMAC). Here, the gene that inhibits macrophage-like function is the SIRPα gene. The pMAC thus obtained has phagocytic ability similar to macrophages, and in addition, has characteristics that differ from existing macrophages, such as cytokine-dependent proliferation.

[0070] <pMAC Comprising a Chimeric Antigen Receptor and a Method for Producing the Same> The present invention provides a pMAC comprising a chimeric antigen receptor (CAR) (hereinafter also referred to as CAR-pMAC) and a method for producing the same. In the method for producing CAR-pMAC of the present invention, the CAR gene can be introduced into pMAC to which cytokine-dependent growth ability has been imparted as described above in the section "Method for Producing pMAC of the Present Invention." In addition, the CAR gene can also be introduced before or after deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells. The method for producing CAR-pMAC of the present invention includes a step of expanding the obtained CAR-pMAC. One embodiment of the method for producing CAR-pMAC of the present invention is as follows (Aspect I). A method for producing CAR-pMAC, comprising: (1) a step (step 1) 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, (2) a step (step 2) of expressing a gene that confers proliferative ability to the macrophage-like cells obtained in step 1 to obtain proliferative macrophage-like cells (pMAC), and (3) a step (step 3) of introducing a chimeric antigen receptor (CAR). Steps 1 and 2 in this embodiment can be performed in the same manner as steps 1 and 2 of embodiment 1 of the above-mentioned "method for producing pMAC of the present invention," respectively.

[0071] Another embodiment of the method for producing CAR-pMAC of the present invention is the following method (Aspect II): (A) a step of expressing a gene that confers proliferation ability 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) a step of introducing a chimeric antigen receptor (CAR) (Step C). Steps A and B in this aspect can be performed in the same manner as steps A and B in Aspect 2 of the above-mentioned "Method for producing pMAC of the present invention," respectively.

[0072] 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') a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in the pluripotent stem cells into which the CAR gene has been introduced obtained in Step 1', and then inducing differentiation into myeloid cells to obtain macrophage-like cells into which the CAR gene has been introduced (Step 2'), and (3') a step of expressing a gene that confers proliferative ability in the macrophage-like cells into which the CAR gene has been introduced obtained in Step 2', to obtain proliferative macrophage-like cells into which the CAR gene has been introduced (CAR-pMAC) (Step 3'). Steps 2' and 3' in this aspect can be performed in the same manner as steps 1 and 2, respectively, of Aspect 1 of the above-mentioned "Method for producing pMAC of the present invention," except that the CAR gene has been introduced into the target cells in advance.

[0073] Another embodiment of the method for producing CAR-pMAC of the present invention includes the following method (Aspect IV): (A') a step of introducing a chimeric antigen receptor (CAR) into pluripotent stem cells (Step A'), (B') a step of expressing a gene that confers proliferation ability in myeloid cells differentiated from the pluripotent stem cells into which the CAR gene has been introduced obtained in Step A', thereby obtaining CAR gene-introduced proliferative myeloid cells (Step B'), and (C') a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in the CAR gene-introduced proliferative myeloid cells obtained in Step B', and then inducing differentiation to obtain CAR gene-introduced proliferative macrophage-like cells (CAR-pMAC) (Step C'). Steps A' and B' in this aspect can be performed in the same manner as steps A and B of Aspect 2 of the above-mentioned "Method for producing pMAC of the present invention," respectively, except that the CAR gene has been introduced into the target cells in advance.

[0074] The introduction of a CAR gene into cells in Step 3 of Aspect I and Aspect II, Step 1' of Aspect III, and Step A' of Aspect IV is described in detail below. Chimeric Antigen Receptor (hereinafter also referred to as CAR) CAR is a structure comprising, from the N-terminus to the C-terminus of a protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for the effector function of immune cells, and the CAR gene is a gene encoding this receptor. The extracellular domain contains an antigen recognition site that exhibits target-specific binding affinity. The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain transmits a signal required for immune cell effector function. That is, an intracellular signaling domain is used that can transmit a signal required for immune cell activation when the extracellular domain binds to a target antigen.

[0075] There have been several reports of experiments and clinical studies using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum Gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20 (R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pule MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720, 2012; Porter DL, et al. N Engl J Med 365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73,2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5:177ra38, 2013), the CAR of the present invention can be constructed with reference to these reports.

[0076] Introduction of a CAR Gene into pMAC In the present invention, introduction of a CAR gene is carried out by introducing the CAR gene into a desired cell using a CAR expression vector. Examples of cells into which the CAR gene is introduced include the pMAC provided by the present invention as described above, as well as pluripotent stem cells before or after deletion or suppression of a gene that inhibits macrophage-like function. A CAR expression vector refers to a nucleic acid molecule capable of delivering a nucleic acid molecule encoding a CAR gene into a target cell. It may be DNA or RNA, and there are no particular limitations on its form or origin; various types of vectors can be used. The vector may be a viral or non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, retroviral vectors, lentiviral vectors, and adeno-associated viral vectors are expected to integrate a target gene incorporated into the vector into the host chromosome, resulting in stable and long-term expression. Each viral vector can be prepared according to a conventional method or by using a commercially available dedicated kit. Non-viral vectors include plasmid vectors, liposome vectors, 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.

[0077] A CAR expression vector contains an expression unit for expressing the CAR gene, and the expression unit typically includes a promoter, a CAR gene, and a poly(A) addition signal. The expression unit may be derived from various organisms or viruses or may consist of any sequence, including sequences similar to or modified from these. Examples of promoters that can be used for the CAR expression cassette include the CAG promoter, CMV-IE (cytomegalovirus early gene-derived promoter), SV40 ori, retrovirus LTRSRα, EF1α, and β-actin promoters. Examples of poly(A) addition signal sequences include the SV40 poly(A) addition sequence, the bovine growth hormone gene poly(A) addition sequence, and the globulin poly(A) addition sequence. To control the expression of the CAR gene by the promoter, the CAR gene is typically linked to the 3'-end of the promoter directly or via another sequence, and a poly(A) addition signal sequence is located downstream of the CAR gene. Such an expression unit transcribes the CAR gene into messenger RNA (mRNA), which is then translated into CAR and presented on the cell surface. The expression unit may contain a detection gene (e.g., a reporter gene, a cell- or tissue-specific gene, a selection marker gene) for detecting gene expression, an enhancer sequence for improving expression efficiency, a WRPE sequence, etc. The detection gene is used for determining the success or efficiency of introduction of the CAR expression vector, detecting CAR gene expression or determining expression efficiency, selecting or sorting cells in which the CAR gene is expressed, etc.Examples of detection genes include the neo gene that confers resistance to neomycin, the kmr gene and nptII gene that confers resistance to kanamycin and the like (Bernd Reiss et al. EMBO J. 3 (1984), 3317-3322), the hph gene that confers resistance to hygromycin (Blochlinger & Diggelmann, Mol Cell Bio 4:2929-2931), and the DHFR gene that confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7)) (all of these are marker genes); the luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), the β-glucuronidase (GUS) gene, and GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) or its variants (e.g., EGFP and d2EGFP), tdTomato or its variants (e.g., pTdTomato) (all of these are reporter genes); or genes such as the epidermal growth factor receptor (EGFR) gene lacking an intracellular domain can be used. The detection gene may be linked to the CAR gene via, for example, a bicistronic regulatory sequence (e.g., an internal ribosomal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is the 2A peptide (T2A) derived from Thosea asigna virus. Other self-cleaving peptides include, but are not limited to, 2A peptides derived from picornaviruses (F2A), 2A peptides derived from foot disease virus (FMDV), 2A peptides derived from equine rhinitis A virus (ERAV), 2A peptides derived from porcine teschovirus (PTV-1), and 2A peptides derived from rotaviruses, insect viruses, aphthoviruses, or trypanosoma viruses. Examples of similar sequences and partially modified sequences are also included.

[0078] A CAR gene expression vector prepared for gene transfer is introduced into pMAC by a conventional method. In the case of a viral vector, the vector is introduced into cells by viral infection. In the case of a non-viral vector such as a plasmid, the vector can be introduced into cells by conventional methods such as electroporation, liposome, calcium phosphate, and nucleofection, and is preferably introduced by electroporation.

[0079] To improve the efficiency of integration into the host chromosome, gene introduction using the transposon method is preferred. The transposon method is a non-viral gene introduction method that utilizes a pair of a gene enzyme (transposase) and its specific recognition sequence to induce gene transposition, allowing any gene to be integrated into the host chromosome. For example, the piggyBac transposon method can be used as the transposon method. The piggyBac transposon method utilizes a transposon isolated from an insect (Fraser MJ et al., Insect Mol Biol. 1996 May;5(2):141-51; Wilson MH et al., Mol THER 2007 Jan;15(1):139-45), enabling highly efficient integration into mammalian chromosomes. The piggyBac transposon method has actually been used to introduce genes (see, for example, Nakazawa Y, et al., J Immunother 32:826-836, 2009; Nakazawa Y et al., J Immunother 6:3-10, 2013).

[0080] Transposon methods are not limited to those 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. 2007;52(7):628-35. Epub 2007 Jun 7), Tol2 (Koga A, Hori H, Sakaizumi M (2002) Mar Biotechnol 4: 6-11; Johnson Hamlet MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K, Mead PE (2006) Genesis 44: 438-445; Choo BG, Kondrichin I, Parinov S, Emelyanov A, Go W, Toh WC, Korzh V (2006) BMC Dev Biol 6: 5), or the like may also be used.

[0081] Gene introduction using the transposon method can be performed by a conventional method. For example, for the piggyBac transposon method, a vector carrying a gene encoding the piggyBac transposase (transposase plasmid) and a vector having a structure in which a CAR gene expression unit is sandwiched between piggyBac inverted repeat sequences (transposon plasmid) are prepared, and these vectors can be introduced into target cells by various methods such as electroporation, nucleofection, lipofection, and the calcium phosphate method.

[0082] As a treatment after CAR gene introduction, CAR-pMAC is expanded to a required scale. The CAR-pMAC of the present invention can be grown in a cytokine-dependent manner. Therefore, in the method for producing CAR-pMAC of the present invention, the expansion culture is preferably carried out in the presence of at least one, and preferably two, cytokines selected from the group consisting of GM-CSF and M-CSF.

[0083] The medium used for preparing CAR-pMAC is not particularly limited, and media used in ordinary cell culture, such as RPMI1640, MEM, X-VIVO, IMDM, DMEM, DC medium, and OptiMEM, can be used. The medium may be a medium supplemented with serum (human serum, fetal bovine serum, etc.) in accordance with conventional methods, or a serum-free medium. It is preferable to use a serum-free medium because it is highly safe for clinical application and is less likely to produce differences in culture efficiency due to differences between serum lots. Examples of serum-free media include TexMACS™ (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific), and ALyS culture medium (Cell Science Institute, Inc.). When serum is used, autologous serum, i.e., serum collected from an individual from which 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, or artificial serum may be used. In the present invention, autologous serum is preferably used. The basal medium used may be one suitable for cell culture, and the above-mentioned TexMACS™ (Miltenyi Biotec), AIM V (registered trademark), or ALyS culture medium (Cell Science Institute, Inc.) may be used. Other culture conditions may be any suitable for cell survival and proliferation, and general conditions may be adopted. For example, the culture may be performed under CO 2 set at 37°C. ２ Incubator (CO ２ Examples of suitable oxygen-rich culture include culturing in an oxygen-rich environment (oxygen concentration of 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%), and low-oxygen culture (oxygen concentration of 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%).

[0084] By expanding CAR-pMAC, a cell population containing CAR-pMAC of sufficient quantity and quality for clinical use can be obtained.

[0085] After expansion, CAR-pMAC is recovered. The recovery procedure may be performed by a conventional method. For example, recovery is performed by pipetting, centrifugation, or the like. In a preferred embodiment, a step of culturing the cells after expansion in the presence of a stimulating substance is performed before the recovery procedure. This step enables efficient expansion and also has the advantage of increasing the cell survival rate. If desired, the CAR-pMAC population after expansion may be subjected to a cell separation step such as bead separation. By performing the cell separation step, the purity of CAR-pMAC, which has a higher effect, can be increased.

[0086] The cell population containing CAR-pMAC produced by the method of the present invention can be used to treat diseases associated with tumors or cancers, particularly cancers that express the target antigen of the CAR-expressing immune cells. The diseases associated with tumors or cancers may be solid tumors or hematological tumors. Specific cancers include, but are not limited to, various B-cell lymphomas (follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative neoplasms, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinoblastoma, 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, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer. In a preferred embodiment, the cancer is a solid tumor. Examples of 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, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer.

[0087] Furthermore, cell populations containing CAR-pMAC produced by the methods of the present invention can be used to treat neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis. Examples of "neurodegenerative diseases" include tauopathies, α-synucleopathies, 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, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, trinucleotide repeat diseases, and prion diseases. "Inflammatory diseases" include systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infections, hereditary periodic fevers, malignant tumors, systemic vasculitis, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, acquired or hereditary immunodeficiency, injection drug use, and acne conglobata, Muckle-Wells (MWS) disease, familial Mediterranean fever (FMF), etc. "Cardiovascular diseases" include atherosclerosis, coronary artery disease, peripheral arterial disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, heart failure, etc. "Fibrotic diseases" include pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, hepatic fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, and dermal fibrosis.Examples of "amyloidosis" include primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), other familial amyloidoses, beta-2 microglobulin amyloidosis, localized amyloidosis, heavy chain amyloidosis (AH), light chain amyloidosis (AL), 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, Lect2 amyloidosis (ALECT2), lysozyme amyloidosis, and the like.

[0088] The cell population containing CAR-pMAC produced by the method of the present invention is administered at a therapeutically effective dose that is determined appropriately depending on the age, body weight, body surface area, symptoms, etc. of the subject. The subject in the present disclosure is usually a human, preferably a cancer patient. The cell population containing CAR-pMAC produced by the method of the present invention is administered at a therapeutically effective dose of, for example, 1 x 10 ４ pieces ~ 1x10 １０ The cell population of the present disclosure may be administered in individual doses. The route of administration is not particularly limited, and may be administered intratumorally, peritumorally, intraventricularly, intravenously, intraarterially, intraportally, intradermally, subcutaneously, intramuscularly, or intraperitoneally. The cell population of the present disclosure may be administered systemically or locally, and local administration may include direct injection into the target tissue, organ, or tissue. The administration schedule is determined appropriately depending on the subject's age, weight, body surface area, symptoms, etc., and may be a single administration or multiple continuous or regular administrations.

[0089] The 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. Examples of "pharmaceutically acceptable carriers" include conventional excipients, binders, buffers, water for injection, isotonicity agents, preservatives, soothing agents, and other components typically used in pharmaceuticals. In addition to the cell population to be administered to a subject, the composition may contain other components, such as dimethyl sulfoxide (DMSO) or serum albumin for cell protection, antibiotics for preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for cell activation, proliferation, or differentiation induction. Pharmaceutical compositions can be prepared by conventional methods. The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature. Those skilled in the art will understand that alternatives with similar effects and actions are also possible.

[0090] Example 1: Preparation of human iPSCs Human iPSCs were prepared using the method described in Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)). Human iPSCs were maintained in a polystyrene tissue culture plate coated with iMatrix511 (Nippi) using the regenerative medicine medium StemFit AK02N (Ajinomoto Healthy Supply). The medium was changed every 1-2 days, and once a week, depending on cell growth, cells were treated with the cell detachment solution TrypLE Select (Life Technologies) for 4-5 minutes to recover a single-cell suspension. The cells were then seeded into an appropriately sized culture vessel and cultured. The seeded iPSCs were cultured overnight in the presence of 10 μM Rock inhibitor Y-27632 (Fujifilm Wako Pure Chemical Industries), and the medium was replaced the next day to remove Y-27632.

[0091] Example 2: Preparation of mouse iPSCs Mouse iPSCs were prepared by the method described in Araki, R. et al. (Nature, 494:100-104. (2013)). Mouse iPSCs were maintained in an ES cell medium (ESM) (DMEM (Fujifilm Wako Pure Chemical Industries) containing 15% KSR (Life Technologies)) supplemented with 2-mercaptoethanol (Life Technologies) at a final concentration of 55 μM and 10 ４ The culture was carried out on mouse embryonic fibroblast (MEF) (ReproCell) feeder cells seeded on a 6-well tissue culture plate using complete ESM (cESM) medium prepared by adding 100 U / mL Leukemia Inhibitory Factor (LIF) (Merck, trade name ESGROM LIF).

[0092] Feeder cells were prepared by seeding MEFs (Reprocells), whose cell growth had been stopped by mitomycin C treatment, onto a gelatin-coated dish, and were used for the maintenance culture of mouse iPSCs from the day after the MEFs attached.

[0093] Example 3: Deletion of SIRPα gene by genome editing Single guide RNAs for human SIRPα and mouse SIRPα were synthesized by nucleic acid synthesis using sequence information from the database of the National Center for Biotechnology Information (NCBI). ＴＭ The Cas9 protein-guide RNA complex (RNP) was prepared by mixing with Recombinant Cas9 protein (Takara Bio), and added to a single iPSC cell suspension. The RNA was then introduced into the cells using electroporation. Sequence analysis of the genomic DNA sequence targeted by the guide RNA and expression analysis by flow cytometry using differentiated cells obtained by the differentiation induction described in Example 5 yielded iPSC clones lacking the SIRPα gene.

[0094] Example 4: Preparation of embryoid bodies derived from iPSCs Embryoid bodies (hereinafter referred to as human EBs) derived from human iPSCs (wild-type or SIRPα-deficient) were prepared by seeding human iPSCs at a predetermined density prepared with StemFit containing 10 μM Y-27632 into a 6-well plate-type cell cluster formation culture vessel EZ SPHERE SP (AGC Technoglass). 1 to 4 days after the formation of embryoid bodies with a diameter of 50 to 200 μm, differentiation-inducing culture was performed as described in Example 5 below.

[0095] Embryoid bodies (hereinafter referred to as mouse EBs) derived from mouse iPSCs (wild-type or SIRPα-deficient) were prepared by seeding mouse iPSCs prepared in complete medium cESM at a predetermined density onto a 6-well EZ SPHERE plate. One to four days after the formation of embryoid bodies with a diameter of 50 to 200 μm, differentiation-inducing culture was performed as described in Example 5 below.

[0096] Example 5: Induction of differentiation of embryoid bodies into myeloid cells (1) Preparation of feeder cells Mouse-derived cultured cell lines C3H10T1 / 2 or OP9 were seeded onto gelatin-coated dishes, and the cell lines were used the following day. Feeder cells used to induce differentiation of human iPSC-derived embryoid bodies were irradiated with 60 Gy to stop proliferation before use. C3H10T1 / 2 cells were cultured in BME medium (Life Technologies) containing 10% FBS, and OP9 cells were cultured in αMEM medium (Life Technologies) containing 20% ​​FBS.

[0097] (2) On-feeder differentiation-inducing culture of mouse iPSCs Mouse EBs generated from mouse iPSCs (wild-type or SIRPα-deficient) were cultured on OP9 feeder cells for 7 to 10 days using differentiation-inducing medium αMEM containing 20% ​​FBS, and the collected cells were then replated on freshly prepared OP9 feeders. They were cultured for 7 to 10 days in the presence of 55 μM 2-mercaptoethanol and 50 ng / mL GM-CSF to obtain differentiated cells, including myeloid cells.

[0098] (3) Feeder-free differentiation of human iPSCs. Human EBs generated from human iPSCs (wild-type or SIRPα-deficient) were cultured on polystyrene tissue culture plates coated with iMatrix511 and Retronectin (Takara Bio). They were then cultured for 5-7 days in differentiation medium X-VIVO15 (Lonza) in the presence of 50 ng / mL bFGF, 50 ng / mL BMP-4, 50 ng / mL VEGF, and 50 ng / mL SCF. They were then cultured for 7-10 days in the presence of 50 ng / mL bFGF, 50 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL Flt-3L, 10 ng / mL TPO, 20 ng / mL IL-3, and 50 ng / mL GM-CSF. The cells were then 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. Although this may be affected by other growth factors, any two concentrations within the range of 0.2 ng / mL to 200 ng / mL can be used as the upper and lower limits. For example, any two concentrations from 0.2 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL to 200 ng / mL can be used as the upper and lower limits.

[0099] Example 6: Generation of proliferating macrophage-like cells, pMAC, by introduction of proliferation genes (1) Preparation of lentiviral vectors Human c-MYC, BMI1, and MDM2 cDNAs were synthesized by gene synthesis using sequence information from the database of the National Center for Biotechnology Information (NCBI). The cDNA fragments of each gene were inserted into the lentiviral vector pSL or CSII-EF-MSC-IRES2-Veneus. Using lipofection, each of the genes introduced into the lentiviral vectors pSL or CSII-EF-MSC-IRES2-Veneus prepared above, along with the packaging construct pCAG-HIVgp and the envelope and Rev construct pCMV-VSV-G-RSV-Rev, were introduced into 293T cells, which are packaging cells (virus-producing cells). Three days after gene introduction into 293T cells, the cell culture medium was collected and filtered through a 0.45 μm pore size filter. The viral particles were then concentrated and collected using a Lenti-X concentrator (Clontech). The collected recombinant viral particles were suspended in DMEM solution, dispensed into cryovials, and stored at -150°C.

[0100] (2) Imparting proliferation potential to iPSC-derived myeloid cells by transfection with proliferation-inducing genes. The myeloid cells prepared in the previous section were cultured in 48-well tissue culture plates. Mouse myeloid cells were infected by simultaneously adding a lentivirus suspension expressing c-MYC. Human myeloid cells were infected by simultaneously adding a lentivirus suspension expressing c-MYC, BMI1, and MDM2. The culture scale was expanded by adding additional medium depending on cell growth from the day after transfection. After virus infection, myeloid cells were cultured in αMEM containing 20% ​​FBS in the presence of 50 ng / mL GM-CSF and 50 ng / mL M-CSF. Transfected cells continued to proliferate for more than three months. In each example herein, cells derived from wild-type iPSCs are referred to as pMAC, and cells derived from pluripotent stem cells deficient in the SIRPα gene that inhibits macrophage-like function are referred to as SIRPα-deficient proliferative macrophage-like cells (pMAC-SIRPα-KO). In this example, SIRPα-deficient iPSCs were used as pluripotent stem cells deficient in the gene that inhibits macrophage-like function.

[0101] (3) Preparation of Mouse Macrophages To prepare macrophages from C57BL / 6 mice, bone marrow-derived monocytes (BM-derived monocytes) and peritoneal macrophages were collected. The bone marrow-derived monocytes were cultured for 4 days in 6-well tissue culture plates in RPMI-1640 medium containing 10% fetal bovine serum in the presence of 50 ng / mL M-CSF to induce bone marrow-derived macrophages (M0). Subsequently, bone marrow-derived macrophages (M1) were induced by culturing for 24 hours in the presence of 20 ng / mL interferon-γ (Funakoshi) and 50 ng / mL lipopolysaccharide (Fujifilm Wako Pure Chemical Industries). Bone marrow-derived macrophages M2 were induced by culturing bone marrow-derived macrophages M0 in the presence of 20 ng / mL IL-4 for 24 hours. Mice were intraperitoneally administered 2 mL of thioglycollate medium (Merck), and peritoneal macrophages were collected 3 days after administration.

[0102] (4) Gene Expression Profiling of Mouse pMAC pMACs prepared from iPS cells and macrophages derived from living organisms were subjected to RNA-seq analysis (Mus musculus, reference genome mm10), which is a whole transcriptome analysis, to quantify the expression of transcripts. Total RNA was extracted from each cell using the QIAGEN RNeasy mini kit, and then purified using a strand-specific library preparation method (dUTP method) using the NEBNext® Poly (A) mRNA Magnetic Isolation Module (product number E7490) and NEBNext® Ultra. ＴＭUsing the 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 sample prepared from the library was obtained using the next-generation sequencer NovaSeq 6000 (Illumina), and data of read length PE150 (150 bp x 2 paired-end), data volume 4G bases per sample, and number of reads 26.7M reads per sample (13.3M pairs) was obtained. Using the acquired data, informatics analysis was performed, and principal component analysis (Principal Component Analysis; PCA) was performed. To confirm the quality of the sequence reads, the software FastQC (version 0.11.7) was used to calculate the quality scores (scores indicating the sequence information error rate) of R1 (Read1) and R2 (Read2), and no abnormalities were confirmed (Entirely Normal, quality score > 28). Next, using the software Trimmomatic (version 0.38), the sequence reads were trimmed based on the settings (ILLUMINACLIP 2:30:10, LEADING = 20, TRAILING = 20, SLIDINGWINDW = 4:15, MINLEN = 36), and in order to obtain high-quality data, low-quality read ends, adapter sequences, short reads, etc. were removed based on the results of the FastQC quality check. Next, the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0) to calculate the read mapping rate, which was confirmed to be over 98% for all samples. Subsequently, the raw read counts mapped to known exon regions for each gene were calculated using the software featureCounts (version 1.6.3).Additionally, mapped fragments were counted and Fragments Per Kilobase of Exon Per Million Mapped Fragments (FPKM), Fragments Per Kilobase of Transcript Per Million Mapped Reads Upper Quartile (FPKM-UQ), and Transcripts Per Million (TPM) were calculated. Principal component analysis (PCA) was performed on all detected genes using the software STATS (version 3.6.1) based on the number of raw reads, FPKM, and TPM values. Each sample was plotted on a graph, with principal component (PC) 1 representing the component with the greatest variability and the index most representative of the data on the horizontal axis, and PC2 representing the component with the second greatest variability and the index most representative of the data on the vertical axis (Figure 1). Mouse pMACs were plotted within the range of PC1 (-100 to -75) and PC2 (-75 to -50), indicating that they exist in a region distinct from other biologically derived monocytes and macrophages.

[0103] (5) Surface antigen analysis of mouse pMAC Mouse bone marrow-derived monocytes (pMAC) were collected by pipetting. Bone marrow-derived macrophages M0 and M2 were treated with D-PBS (Nacalai Tesque) containing 2 mM EDTA (Nacalai Tesque), and bone marrow-derived macrophages M1 were treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes, and then collected by pipetting. Cells were stained with anti-CD11b, anti-F4 / 80, anti-CD16 / 32, anti-CD64, anti-IA / E, anti-CD206, anti-CD163, anti-Ly6C, anti-CD80, anti-CD86, anti-SiglecF, anti-SIRPα, anti-TLR2, anti-TLR4, anti-CD124, anti-CCR1, anti-CCR2, anti-CCR3, anti-CCR4, anti-CCR5, anti-CXCR2, and anti-CXCR4 antibodies or isotype-matched control antibodies. Then, cells were washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer (product name: BD Accuri C6 Flow Cutometer, manufactured by Beckton Dickinson). Figure 2 shows the results of surface antigen analysis using a flow cytometer after antibody staining. The results of this analysis showed that mouse pMACs possess the properties of both bone marrow-derived M1 and M2 macrophages, which were used as controls.

[0104] (6) Morphology of Mouse pMAC Mouse pMAC and pMAC-SIRPα-KO showed no change in cell morphology due to SIRPα deficiency, and maintained the distorted morphology with processes characteristic of myeloid cells shown in Figure 3.

[0105] Example 7: Preparation of iPSC-derived chimeric antigen receptor-transduced macrophage-like cells (CAR-pMAC) (1) Preparation of lentiviral vector cDNA for a chimeric antigen receptor (CAR) having the tdTomato gene and / or the GC33scFv domain and CD3ζ domain was synthesized by gene synthesis and inserted into the lentiviral vector pSL in the same manner as in Example 6(1), followed by preparation of viral particles. Figure 4 shows the plasmid map and CAR gene expression construct used in preparation of the lentiviral vector. The recovered recombinant viral particles were suspended in DMEM solution, dispensed into cryovials, and stored at -150°C.

[0106] (2) Introduction of CAR gene into iPSC-derived pMAC (human / mouse) pMAC or pMAC-SIRPα-KO, which are human or mouse expanded macrophage-like cells prepared in Example 6(2), were cultured in a 48-well tissue culture plate and infected by adding a lentivirus suspension expressing GC33 CAR. From the day after gene introduction, the culture scale was expanded while adding culture medium according to cell growth. 20% FBS-containing αMEM was used as the culture medium, and the cells were cultured in the presence of 50 ng / mL GM-CSF and 50 ng / mL human M-CSF. Human GM-CSF was used for human pMAC, and mouse GM-CSF was used for mouse pMAC.

[0107] (4) Effect of Genetic Manipulation of Mouse pMAC on Cell Proliferation After gene transfer and continued culture for 2 weeks or more, the cell proliferation of human or mouse pMAC, SIRPα-deficient pMAC-SIRPα-KO, CAR-introduced pMAC (CAR-pMAC), and SIRPα-deficient CAR-introduced pMAC (CAR-pMAC-SIRPα-KO) in response to GM-CSF and M-CSF was examined by MTT assay. pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were harvested and seeded (3 x 10 cells) onto 96-well tissue culture plates. ３Cells were cultured in 96-well tissue culture plates. The proliferation rates were compared between cultures containing and excluding GM-CSF and M-CSF. Immediately after the initiation of culture in 96-well tissue culture plates, 0.5 mg / mL of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Merck) was added every 24 hours. After 4 hours, the amount of metabolized formazan was measured by absorbance at 595 nm. The amount of metabolized formazan is proportional to the cell number at the time of addition of the MTT reagent, i.e., the rate of cell proliferation. Figure 5 shows the results of the MTT assay for various mouse pMACs. Approximately 50 ng / mL of GM-CSF or M-CSF was required in the culture medium for pMAC proliferation. Furthermore, the combined use of GM-CSF and M-CSF promoted proliferation. The deletion of the SIRPα gene and the introduction of the CAR gene, which were performed to prepare pMAC, did not affect cell proliferation, indicating that CAR-pMAC has the same cell proliferation ability as pMAC.

[0108] (5) Cell Surface Antigen Analysis of Mouse CAR-pMAC The expression of tdTomato, GC33ζCAR, and SIRPα in mouse pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO was examined using a flow cytometer. The fluorescent protein tdTomato was expressed via a T2A sequence in the nucleic acid sequence at the 3' end of the GC33ζCAR cDNA incorporated into a lentiviral vector. Additionally, FITC-labeled human glypican 3 (hGPC3), the target antigen of CAR, was added to the cell sample. This enabled analysis of CAR expression using the fluorescence of tdTomato and hGPC3-FITC as indicators using a flow cytometer. Figure 6 shows the results of flow cytometry analysis of GC33ζCAR expression in pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO. Compared to pMAC and pMAC-SIRPα-KO, CAR-pMAC and CAR-pMAC-SIRPα-KO show antigen-specific binding to hGPC. Furthermore, compared to pMAC and CAR-pMAC, pMAC-SIRPα-KO and CAR-pMAC-SIRPα-KO show reduced SIRPα expression. The various cells prepared from each cytogram are seen to be single cell populations, containing almost no impurity cells and indicating high purity.

[0109] (6) Cytokine production by mouse pMAC Macrophages present in vivo are divided into functional phenotypes, M1 and M2, depending on cell-cell interactions in the microenvironment of cancer tissue. To compare with the phenotype of macrophages derived from living organisms, cytokines produced by mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were evaluated. Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO, and mouse bone marrow-derived macrophages (2 × 10 ５M1 type was induced by culturing the mouse bone marrow-derived macrophages (M0) prepared in Example 6(3) for 48 hours in the presence of 20 ng / mL IL-4 (BioLegend). The M2 type was induced by culturing the mouse bone marrow-derived macrophages for 48 hours in the presence of 20 ng / mL IL-4 (BioLegend). The resulting culture supernatant was used to quantify the production of IL-6, TNFα, IL-12, and IL-10, which are known to be markers of M1 or M2 type ( Figure 7 ). It was found that M1 stimulation of pMAC induced the production of IL-6, TNFα, and IL-10, regardless of the presence or absence of SIRPα or CAR expression, whereas IL-12 was not produced.

[0110] (7) Cell migration ability of mouse CAR-pMAC-SIRPα-KO To examine the cell migration ability of mouse CAR-pMAC-SIRPα-KO, 160 μL of 10% FCS-containing RPMI-1640 medium supplemented with cancer cell culture supernatant or recombinant chemokine was added to the lower chamber of a 16-well Boyden chamber (transwell type) CIM-Plate 16 (Agilent) coated with bovine fibronectin (Fujifilm Wako Pure Chemical Industries, Ltd.), and mouse CAR-pMAC-SIRPα-KO (3 × 10 cells) was cultured in 20% FCS-containing αMEM containing GM-CSF and M-CSF in the upper chamber. ５ The cells were seeded and incubated at 37°C, 5% CO ２ The cells were cultured at 1×10 s. The CAR-pMAC in the upper chamber was attracted to the electrode sensor on the backside of the membrane by the attractant in the lower chamber, passing through the pores of the transwell, and increased in electrical resistance. The electrical resistance was measured using an xCELLigence RTCA DP system (Agilent) to evaluate the cell migration of mouse CAR-pMAC. Mouse MC38, LL / 2, 4T1, and CT26 cells (1×10 s each) were cultured in the mouse cancer cell culture supernatant. ６The normal cells were T cells and B cells (1 × 10 cells) isolated from the spleen of a c57BL / 6 mouse strain. ６ Culture supernatant was prepared using the culture medium.

[0111] The results of measuring the cell migration ability of CAR-pMAC-SIRPα-KO in the culture supernatant of cancer cells are shown in the upper panel of Figure 8. The electrical resistance (Cell Index) value increased when used in the culture supernatant of mouse cancer cells MC38, LL / 2, CT26, and 4T1, demonstrating that mouse CAR-pMAC-SIRPα-KO has migration ability (Figure 8, upper left). When culture supernatant of normal mouse spleen-derived B cells and T cells was used as a negative control, the Cell Index value was low, similar to that of RPMI 1640 (medium) containing 10% FCS and no inducer, demonstrating that CAR-pMAC did not exhibit cell migration activity (Figure 8, upper center). Furthermore, when CAR-pMAC-SIRPα-KO was cultured in the presence of 200 ng / mL of cytochalasin D (Fujifilm Wako Pure Chemical Industries) or Pertussis Toxin (Fujifilm Wako Pure Chemical Industries), which are known inhibitors of cell migration, the increase in cell index value in the culture supernatant of MC38 cancer cells was partially reduced by cytochalasin D (CytD) and completely suppressed by Pertussis Toxin (PTX) (Figure 8, top right). These results demonstrate that CAR-pMAC-SIRPα-KO has the ability to induce cell migration in response to attracting factors produced by multiple cancer cells. Next, to examine the cell migration ability of CAR-pMAC-SIRPα-KO in response to recombinant chemokines, mouse recombinant chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CX3CL1, CXCL1, CXCL2, CXCL5, CXCL12SDF-1α, and CXCL12SDF-1β (all manufactured by BioLegend) were used as candidate chemotactic factors, each at a concentration of 100 ng / mL. The results are shown in the lower panel of Figure 8. Evaluation of the cell migration ability of mouse CAR-pMAC-SIRPα-KO in response to mouse recombinant chemokines revealed that it responded to CCL3, CCL4, and CCL7 (lower left and center panel of Figure 8).

[0112] (8) Cytokine production by mouse pMAC in co-culture with cancer cells. Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were co-cultured with cancer antigen hGPC3-expressing or non-expressing cancer cells to evaluate cytokine production. Mouse cancer cells: 4T1 with or without hGPC3 expression, CT26 with or without hGPC3 expression, LL / 2 with or without hGPC3 expression, and MC38 with or without hGPC3 expression (all 5 × 10 ４ cells) and mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (all 2 × 10 ５ Cells) were seeded in a 48-well tissue culture plate using RPMI-1640 containing 10% FCS and incubated at 37°C, 5% CO ２ The cells were cultured at 4°C for 48 hours. The resulting culture supernatants were used to quantify the production of IL-6 (Figure 9-1), TNFα (Figure 9-2), IL-10 (Figure 9-3), and IL-12 (Figure 9-4). It was found that co-culture of 4T1-GPC3, CT26-GPC3, LL / 2-GPC3, and MC38-GPC3 with CAR-pMAC-SIRPα-KO induced the production of IL-6 and TNFα. No difference in the amount of IL-10 produced was observed under the test conditions, and IL-12 was not detected.

[0113] (9) Antigen-specific phagocytosis of cancer cells by mouse CAR-pMAC. Mouse pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO (3 × 10) were cultured in a 96-well tissue culture plate as effector cells. ４ The target cells, luciferase gene-transfected mouse cancer cells hGPC3-introduced or non-expressing 4T1, hGPC3-introduced or non-expressing CT26, hGPC3-introduced or non-expressing LL / 2, and hGPC3-introduced or non-expressing MC38 (all 3 × 10 ３The CAR-pMAC-SIRPα-KO cells were co-cultured with CAR-pMAC-SIRPα-KO (cells / well) for two days. To evaluate the phagocytosis of cancer cells specific to the CAR target antigen, cancer cells transfected with the cancer antigen hGPC3 were used. After co-culture, Bright-Glo Luciferase Assay (Promega), a luciferase substrate, was added to the 96-well tissue culture plate, and the survival of the target cells was examined by quantifying the bioluminescence obtained. The results are shown in Figure 10. It can be seen that the cytotoxicity of target cells was approximately 20% in co-culture with pMAC, CAR-pMAC, and pMAC-SIRPα-KO, but increased to approximately 60% in co-culture with CAR-pMAC-SIRPα-KO. Furthermore, the cell-killing effect of CAR-pMAC-SIRPα-KO was not observed in target cells that did not express hGPC3, indicating that CAR-pMAC-SIRPα-KO has the ability to phagocytose cancer cells specific to the target antigen. Mouse pMAC or CAR-pMAC-SIRPα-KO (5 × 10) expressing tdTomato were cultured in a 48-well tissue culture plate. ４ cells / well) and hGPC3-transfected colon cancer cells MC38 (5 × 10 ３ The cancer cells (100 cells / well) were co-cultured in RPMI-1640 medium containing 10% FBS, and fluorescence observation was performed using a fluorescence microscope (Keyence BZ-X810) after 2 and 4 days. The results are shown in Figure 11 . Cancer cells were detected by green fluorescence (excitation wavelength 470 nm, absorption wavelength 525 nm) derived from the fluorescent protein Venus, and pMAC or CAR-pMAC-SIRPα-KO was detected by red fluorescence (excitation wavelength 545 nm, absorption wavelength 605 nm) derived from the fluorescent protein tdTomato. The bar graph plots the area of ​​the region where the green fluorescence signal of the cancer cells and the red fluorescence signal of pMAC or CAR-pMAC-SIRPα-KO overlap (lower left of Figure 11 ). The bar graph plots the area of ​​the region where the green fluorescence signal of the cancer cells was detected (lower right of Figure 11 ). It was found that cancer cells co-cultured with pMAC proliferated, whereas cancer cells co-cultured with CAR-pMAC-SIRPα-KO were surrounded by CAR-pMAC-SIRPα-KO and eliminated by phagocytosis.

[0114] (10) Cancer Cell Toxicity of Mouse CAR-pMAC To examine the cancer cell cytotoxicity of mouse CAR-pMAC, cancer cells and CAR-pMAC-SIRPα-KO were co-cultured in a 16-well plate E-Plate 16 (Agilent) and incubated at 37°C and 5% CO. ２ The cancer cells that had adhered to the electrode sensor on the plate during pre-culture were phagocytosed by co-culture with CAR-pMAC-SIRPα-KO, weakening cell adhesion and reducing the electrical resistance value. The electrical resistance value was measured using an xCELLigence RTCA DP system (Agilent) to evaluate the cytotoxic activity of CAR-pMAC-SIRPα-KO on cancer cells. The target mouse cancer cells, CT26 (5 x 10 ３ cells), hGPC3-transfected or non-expressing MC38 (1 × 10 ４ cells), hGPC3-transfected or non-expressing LL / 2 (1 × 10 ４ cells), hGPC3-transfected or non-expressing 4T1 cells (5 × 10 ３ Cells) were seeded onto an E-Plate 16 coated with iMatrix-511 and incubated at 37°C, 5% CO ２ After 24 hours of culture, mouse CAR-pMAC-SIRPα-KO effector cells were added in an amount 1x, 5x, or 10x the amount of target cancer cells, and co-cultured. Six hours after the start of co-culture (T = 30 hours), the cancer cell cytotoxicity (% Cytolysis) of CAR-pMAC-SIRPα-KO at effector:target ratios (E:T ratios) of 1:1, 5:1, and 10:1 showed superior activity with higher expression of the cancer antigen hGPC3 and higher E:T ratios, regardless of the cancer cell type targeted ( Figure 12-1 ). Figures 12-2 and 12-3 show the time-dependent changes in adhesion signals (Normalized Cell Index values; CI values) indicating cancer cell survival when cancer cells are co-cultured with CAR-pMAC-SIRPα-KO, and the cytotoxic activity (%Cytolysis) of cancer cells normalized to that of cancer cells cultured alone. It can be seen that the CI value of cancer cells decreases over time, and %Cytolysis increases, depending on the expression of the cancer antigen hGPC3 and the number of CAR-pMAC-SIRPα-KO cells (E:T ratio).

[0115] (11) Investigation of the Importance of SIRPα Gene Deficiency or GC33-ζ-CAR Transduction in the Cytotoxicity of Mouse CAR-pMAC To investigate the importance of SIRPα gene deficiency or GC33-ζ-CAR transduction in the cytotoxicity of CAR-pMAC to cancer cells, human or mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were used as effector cells, and cancer cytotoxicity was evaluated at an E:T ratio of 10:1 using an xCELLigence RTCA DP system. Mouse cancer cells, hGPC-transfected or non-expressing CT26 (5 × 10 ３ cells) were seeded on an E-Plate 16 coated with iMatrix-511 and cultured at 37°C for 24 hours, after which effector cells mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO (5 × 10 ４ Cells) were added and co-cultured. It can be seen that antigen-specific cytotoxic activity (% Cytolysis) is improved by SIRPα deficiency and GC33-ζ-CAR introduction compared to hGPC3-introduced CT26 ( FIG. 13 ).

[0116] (12) Time-lapse observation of cancer cell phagocytosis by mouse CAR-pMAC To evaluate the phagocytosis of cancer cells by CAR-pMAC, MC38-GPC3-Luc-Venus cancer cells (5 × 10 cells) were labeled in the presence of the pH-sensitive red fluorescent dye pHrodo Deep Red (Thermo Fisher Scientific) at 20°C for 2 hours. ５ cells / mL) and pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO (5 × 10 ６1 mL of pHrodo-labeled cancer cells (1000 cells / mL) were co-cultured in 10% FCS-containing RPMI-1640 medium in a 12-well tissue culture plate. Four specific fields of view were photographed 50 times every 10 minutes using a fluorescence microscope (Keyence BZ-X810) for 8 hours. The results are shown in Figure 14. When pHrodo-labeled cancer cells were phagocytosed and taken into pMAC cells, images were obtained in which red fluorescence (excitation wavelength 620 nm, absorption wavelength 700 nm) derived from pHrodo was detected in response to a decrease in pH. Time-dependent changes in the number of phagocytic cells were measured using the BZ-X810 analyzer analysis application (time-series cell counting). Compared to pMAC, pMAC-SIRPα-KO, and CAR-pMAC, CAR-pMAC-SIRPα-KO exhibited improved phagocytic activity.

[0117] (13) Pharmacokinetics of mouse CAR-pMAC in tumor-bearing mice. C57BL6 / N mice were intraperitoneally injected with hGPC3-introduced MC38 (5 × 10 ５ A colon cancer peritoneal dissemination model (n=6) was prepared by transplanting CAR-pMAC-SIRPα-KO (1×10 ７ CAR-pMAC-SIRPα-KO was intraperitoneally administered to mice. 100 μg of genomic DNA was extracted from each organ removed before treatment and 1 and 3 days after treatment. Using 100 μg of genomic DNA as a template, a custom-made PrimeTime real-time PCR probe (Integrated DNA Technologies) that specifically recognizes CAR (GC33-ζ) was used with a real-time PCR system 7500Fast (Thermo Fisher Scientific) to measure the abundance of CAR-pMAC in the lungs, liver, kidneys, heart, spleen, bone marrow, tumor, and peripheral blood ( FIG. 15 ). No CAR signal was detected in mice before treatment. 1 day after intraperitoneal administration, CAR-pMAC-SIRPα-KO was detected in the lungs, liver, kidneys, heart, spleen, and tumor, but not in the bone marrow or peripheral blood. Three days after intraperitoneal administration, the number of mice in which CAR-pMAC-SIRPα-KO was detected in normal tissues of the lung, kidney, heart, and spleen decreased, but CAR-pMAC was detected in tumor tissues in 5 out of 6 mice, indicating that it was present in high amounts.

[0118] (14) In vivo antitumor effect of CAR-pMAC. hGPC3-introduced MC38 (5 × 10 ５ A colon cancer peritoneal dissemination model was created by transplanting CAR-pMAC-SIRPα-KO (3 × 10 ６ The therapeutic effect was evaluated by intraperitoneal administration of CAR-pMAC-SIRPα-KO cells a total of six times. Mice were judged as dead when they lost 20% or more of their body weight or their body temperature dropped below 30°C, and a survival curve was created. Cell survival of hGPC3-introduced MC38 cells in vivo was measured by biochemiluminescence every 3 to 4 days using an IVIS imaging system (PerkinElmer). Figure 16 shows in vivo imaging images and the time course of total luciferase luminescence flux. The no-treatment group, which did not receive CAR-pMAC-SIRPα-KO, showed significant tumor growth after cancer cell transplantation. In contrast, CAR-pMAC-SIRPα-KO significantly suppressed tumor growth and significantly extended survival.

[0119] Example 8 Functional Analysis of Human CAR-pMAC (1) Preparation of Human Biologically Derived Macrophages and iPS Cell-Derived Macrophages CD14 monocytes (Lonza) derived from blood collected from healthy human donors were seeded (2 × 10) onto a 6-well tissue culture plate in the presence of 50 ng / mL human M-CSF using ImmunoCult-SF Macrophage Differentiation Medium (Stemcell Technologies). ６The cells were cultured for 4 days to induce CD14 monocyte-derived macrophages M0 (human CD14-derived macrophages M0). Subsequently, the cells were cultured for 2 days in the presence of 50 ng / mL lipopolysaccharide and 50 ng / mL human interferon-γ to induce CD14 monocyte-derived macrophages M1 (human CD14-derived macrophages M1). Furthermore, CD14 monocyte-derived macrophages M2 were induced by culturing CD14 monocyte-derived macrophages M0 (human CD14-derived macrophages M0) for 2 days in the presence of 50 ng / mL IL-4 (BioLegend). Human iPS cell-derived macrophages were induced by culturing differentiated cells containing myeloid cells obtained in Example 5(3) for 7 to 14 days in the presence of 50 ng / mL M-CSF and 20 ng / mL IL-3.

[0120] (2) Gene Expression Profiling of Human pMAC pMACs generated from iPS cells, biologically derived macrophages, and iPSC-derived macrophages were subjected to RNA-seq analysis (human species, reference genome hg38), which is a whole transcriptome analysis, to quantify the expression of transcripts. Total RNA was extracted from each cell using the QIAGEN RNeasy mini kit, and then subjected to strand-specific library preparation (dUTP method) using the NEBNext® Poly (A) mRNA Magnetic Isolation Module (product number E7490), NEBNext® Ultra ＴＭUsing the 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 sample prepared from the library was obtained using the next-generation sequencer NovaSeq 6000 (Illumina), and data of read length PE150 (150 bp x 2 paired-end), data volume 4G bases per sample, and number of reads 26.7M reads per sample (13.3M pairs) was obtained. Using the acquired data, informatics analysis was performed, and principal component analysis (Principal Component Analysis; PCA) was performed. To confirm the quality of the sequence reads, the software FastQC (version 0.11.7) was used to calculate the quality scores (scores indicating the sequence information error rate) of R1 (Read1) and R2 (Read2), and no abnormalities were confirmed (Entirely Normal, quality score > 28). Next, using the software Trimmomatic (version 0.38), the sequence reads were trimmed based on the settings (ILLUMINACLIP 2:30:10, LEADING = 20, TRAILING = 20, SLIDINGWINDW = 4:15, MINLEN = 36), and in order to obtain high-quality data, low-quality read ends, adapter sequences, short reads, etc. were removed based on the results of the FastQC quality check. Next, the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0) to calculate the read mapping rate, which was confirmed to be over 98% for all samples. Subsequently, the raw read counts mapped to known exon regions for each gene were calculated using the software featureCounts (version 1.6.3).Additionally, mapped fragments were counted and Fragments Per Kilobase of Exon Per Million Mapped Fragments (FPKM), Fragments Per Kilobase of Transcript Per Million Mapped Reads Upper Quartile (FPKM-UQ), and Transcripts Per Million (TPM) were calculated. Principal component analysis (PCA) of samples was performed using the software STATS (version 3.6.1) for all detected genes based on the number of raw reads, FPKM, and TPM values. Each sample was plotted with the component with the greatest variability representing the most significant data as principal component (PC) 1 on the horizontal axis and the component with the second greatest variability representing the second most significant data as PC2 on the vertical axis to create a plot (Figure 17). Human pMAC was plotted in the range of PC1 (150-200) and PC2 (-20 to -40), and was found to exist in a different region from other biologically derived CD14-positive cell-derived monocytes and CD14-positive cell-derived macrophages (M0, M1, M2 types), as well as iPS cell-derived myeloid cells and iPS cell-derived macrophages.

[0121] (3) Surface antigen analysis of human iPSC-pMAC Human pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive monocytes (CD14-derived monocytes), iPSC-derived myeloid cells, and iPSC-macrophages (iPSC-macrophages) were collected by pipetting. CD14 monocyte-derived macrophages M0 and CD14 monocyte-derived macrophages M2 were treated with D-PBS (Nacalai Tesque) containing 2 mM EDTA (Nacalai Tesque), and CD14 monocyte-derived macrophages M1 were treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes, and then collected by pipetting. The cells were stained with anti-HLA-ABC antibodies, anti-HLA-DR antibodies, anti-CD40 antibodies, anti-CD80 antibodies, anti-CD83 antibodies, anti-CD86 antibodies, anti-CD68 antibodies, anti-CD163 antibodies, anti-CD206 antibodies, anti-SIRPα antibodies, anti-CCR1 antibodies, anti-CCR2 antibodies, anti-CCR3 antibodies, anti-CCR4 antibodies, anti-CCR5 antibodies, anti-CCR7 antibodies, anti-CXCR2 antibodies, anti-CXCR4 antibodies, and anti-CD14 antibodies, or isotype-matched control antibodies. The cells were then washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer (product name: BD Accuri C6 Flow Cutometer, manufactured by Beckton Dickinson). Figure 18 shows the results of surface antigen analysis using a flow cytometer after antibody staining. The results of this analysis show that human pMAC (pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO) are negative for CD83 and CD14, which are expressed on CD14-positive cell-derived macrophages (M0, M1, and M2) and iPSC-derived macrophages.

[0122] (4) Morphology of human pMAC Figure 19-1 shows phase-contrast microscopic images of human CD14-positive monocytes, iPSC-derived myeloid cells, pMAC, and CD14-positive cell-derived macrophages (unstimulated M0, M1, and M2 stimulated conditions), and Figure 19-2 shows phase-contrast microscopic images of iPS cell-derived macrophages, pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (unstimulated M0, M1, and M2 stimulated conditions). pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO showed no changes in cell morphology due to SIRPα deficiency or CAR introduction, and were found to have the distorted morphology with processes exhibited by the blood-derived CD14-positive cell-derived macrophages shown in Figure 19-1.

[0123] (5) Cell proliferation of human pMAC After gene transfer, human pMAC, pMAC-SIRPα-KO, and iPS cell-derived macrophages (controls) were cultured for more than two weeks. The cell proliferation of these cells was examined by MTT assay. pMAC and pMAC-SIRPα-KO were harvested and seeded (3 × 10 cells) on a 96-well tissue culture plate. ３ Cells were cultured (1000 x 1000 cells / well). The proliferation rates were compared between cultures containing GM-CSF and M-CSF and those not containing GM-CSF. Figure 20 shows the results of the MTT assay. Human pMAC and pMAC-SIRPα-KO required approximately 50 ng / mL of GM-CSF or M-CSF in the culture medium for proliferation. Furthermore, the combined use of GM-CSF and M-CSF was effective in promoting proliferation. On the other hand, cytokine-dependent cell proliferation was not observed in iPS cell-derived macrophages, indicating that pMAC has high cell proliferation potential.

[0124] (6) Analysis of Cell Surface Antigens in Human CAR-pMAC The expression of tdTomato, GC33ζCAR, and SIRPα in human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO was examined using a flow cytometer. The fluorescent protein tdTomato was expressed via a T2A sequence in the nucleic acid sequence at the 3' end of the GC33ζCAR cDNA incorporated into a lentiviral vector. In addition, FITC-labeled human glypican 3 (hGPC3), the target antigen of CAR, was added to the cell sample. This enabled analysis of CAR expression using the fluorescence of tdTomato and hGPC3-FITC as indicators using a flow cytometer. Figure 21 shows the results of flow cytometry analysis of GC33ζCAR expression in human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO. Compared to pMAC and pMAC-SIRPα-KO, CAR-pMAC and CAR-pMAC-SIRPα-KO show antigen-specific binding to hGPC. Furthermore, compared to pMAC and CAR-pMAC, pMAC-SIRPα-KO and CAR-pMAC-SIRPα-KO show reduced SIRPα expression. The various cells prepared from each cytogram are seen to be single cell populations, containing almost no impurity cells and indicating high purity.

[0125] (7) Cytokine production by human CAR-pMAC Human CD14 monocyte-derived macrophages M0 type and human iPS cell-derived macrophages prepared in Example 8(1), human pMAC and pMAC-SIRPα-KO prepared in Example 6(2), and CAR-pMAC and CAR-pMAC-SIRPα-KO prepared in Example 7(2) were cultured at 2 × 10 ５Cells) were seeded into 48-well tissue culture plates and cultured for 48 hours in the presence of 50 ng / mL interferon-γ (BioLegend) and 50 ng / mL LPS (Fujifilm Wako Pure Chemical Industries) to induce M1 type. Similarly, M2 type was induced by culturing for 48 hours in the presence of 50 ng / mL IL-4 (BioLegend). Using the resulting culture supernatant, production of IL-6, TNFα, IL-12, and IL-10, known as markers of M1 or M2 type, was quantified ( Figure 22 ). It was found that interferon-γ and LPS stimulation induced the production of IL-6, TNFα, and IL-10 in pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO. However, IL-12 production was not detected.

[0126] (8) Cell migration ability of human CAR-pMAC To examine the cell migration ability of human CAR-pMAC-SIRPα-KO, 160 μL of 10% FCS-containing RPMI-1640 medium supplemented with cancer cell culture supernatant or recombinant chemokine was added to the lower chamber of a 16-well Boyden chamber (transwell type) CIM-Plate 16 (Agilent) plate coated with bovine fibronectin (Fujifilm Wako Pure Chemical Industries, Ltd.), and human CAR-pMAC-SIRPα-KO (3 × 10 cells) was cultured in 20% FCS-containing αMEM containing GM-CSF and M-CSF in the upper chamber. ５ The cells were seeded and incubated at 37°C, 5% CO ２ The cells were cultured at 100°C for 1 hour. The CAR-pMAC in the upper chamber was attracted to the electrode sensor on the backside of the membrane by the inducer in the lower chamber, passing through the pores of the transwell and increasing the electrical resistance. The electrical resistance was measured using an xCELLigence RTCA DP system (Agilent) to evaluate the migration of CAR-pMAC cells. Human HepG2, JHH-7, and KOC7c cells (2 x 10 cells each) were cultured to prepare the human cancer cell culture supernatant. ６ cells), SK-Hep1 (5 × 10 ５The following day, the medium was replaced with 2 mL of fresh RPMI-1640 medium containing 10% FCS, and the medium was further cultured for 24 hours to prepare the culture supernatant. HEK293 (5 × 10 ５Culture supernatants were prepared using human CAR-pMAC-SIRPα-KO cells. The results of measuring the cell migration ability of human CAR-pMAC-SIRPα-KO against the culture supernatants of cancer cells are shown in Figure 23. The Cell Index value increased against the culture supernatants of human cancer cells HepG2, SK-Hep1, and JHH-7, demonstrating that human CAR-pMAC-SIRPα-KO has migration ability. The migration ability against cancer cells KOC7c was low, and the Cell Index value was similar to that of normal cells HEK293 (top left of Figure 23). Furthermore, when CAR-pMAC-SIRPα-KO cells were cultured in the presence of 200 ng / mL of cytochalasin D (Fujifilm Wako Pure Chemical Industries) or Pertussis Toxin (Fujifilm Wako Pure Chemical Industries), known inhibitors of cell migration, the increase in the cell index value of the MC38 cancer cell culture supernatant was partially reduced by cytochalasin D (CytD) and completely suppressed by Pertussis Toxin (PTX) (Figure 23, upper center). When the cell migration ability of human iPS-macrophage cells to the cancer cell culture supernatant was measured, the HepG2, SK-Hep1, and KOC7c cancer cells all showed CI values ​​comparable to those of the negative control medium and normal HEK293 cells (Figure 23, upper right). The above results demonstrate that CAR-pMAC-SIRPα-KO has a higher cell migration ability than iPS-macrophage in response to inducers produced by multiple cancer cells.Next, to examine the cell migration ability of CAR-pMAC-SIRPα-KO in response to recombinant chemokines, 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 (BioLegend), and CX3CL1 (R&D Systems) were used as candidate chemotactic factors. The following chemotactic markers were used: CXCL1 (BioLegend), CXCL2 (BioLegend), CXCL5 (BioLegend), CXCL12SDF-1α (BioLegend), and CXCL12SDF-1β (BioLegend), all at a concentration of 100 ng / mL. The cell migration ability of human CAR-pMAC-SIRPα-KO in response to human recombinant chemokines was evaluated, revealing that it responds to CCL2, CCL3LI, CCL5, CCL7, CCL23, and SDF-1α (CXCL12) (Figure 23, second and third rows from the top). Furthermore, the cell migration of CAR-pMAC and CAR-pMAC-SIRPα-KO toward the culture supernatant of HepG2 cancer cells was examined (bottom panel of Figure 23). It was found that the cell migration ability of CAR-pMAC-SIRPα-KO is improved due to the SIRPα deficiency, compared to CAR-pMAC derived from wild-type iPS cells.

[0127] (9) Cytokine production by human CAR-pMAC (co-culture with cancer cells) Human pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO (all 2 × 10 ５ Cells) were seeded in a 48-well tissue culture plate using RPMI-1640 containing 10% FCS and incubated at 37°C, 5% CO ２The cells were cultured for 48 hours at 5×10. The resulting culture supernatant was used to quantify the production of IL-6, TNFα, IL-10, and IL-12 (Figure 24-1). Furthermore, human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were co-cultured with cultured cancer antigen hGPC3-expressing or non-expressing cancer cells to evaluate cytokine production. Human cancer cells, SK-Hep1 with or without hGPC3 expression, and JHH-7 with or without hGPC3 expression (all at 5×10 ４ cells) and human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (2 × 10 ５ Using the culture supernatant obtained by co-culturing human CAR-pMAC and CAR-pMAC-SIRPα-KO cells, the production of IL-6 (Figure 24-2), TNFα (Figure 24-3), IL-10 (Figure 24-2), and IL-12 (Figure 24-3) was quantified. It was found that human CAR-pMAC and CAR-pMAC-SIRPα-KO cells produced IL-6, TNFα, and IL-10 upon introduction of CAR. It was also found that co-culturing with human cancer cells (hGPC3-introduced or non-expressing SK-Hep1, and hGPC3-expressing or -deficient JHH-7) did not induce antigen-specific cytokine production. IL-12 was not detected.

[0128] (10) Cancer cell toxicity by human CAR-pMAC. Human pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO (3 × 10) cells were cultured in a 96-well tissue culture plate as effector cells. ４ The luciferase gene-transfected target cancer cells JHH7-Luc, SK-Hep1-GPC3-Luc-Venus, and KOC7c-GPC3-Luc (3 × 10 cells / well) were seeded in RPMI-1640 containing 10% FBS. ３The CAR-pMAC-SIRPα-KO cells were co-cultured with CAR-pMAC-SIRPα-KO cells (cells / well) for two days. To evaluate the phagocytosis of cancer cells specific to the CAR target antigen, cancer cells that endogenously express or have been transfected with the cancer antigen hGPC3 were used. After co-culture, Bright-Glo Luciferase Assay (Promega), a luciferase substrate, was added to the 96-well tissue culture plate, and the survival of the target cells was examined by quantifying the bioluminescence obtained. The results are shown in Figure 25. It can be seen that the cytotoxicity of target cells was approximately 20-40% in co-culture with pMAC, CAR-pMAC, and pMAC-SIRPα-KO, but increased to approximately 60-90% in co-culture with CAR-pMAC-SIRPα-KO. Furthermore, the cell-killing effect of CAR-pMAC-SIRPα-KO was not observed in the target cells SK-Hep1-mock-Luc-V, which do not express hGPC3, indicating that CAR-pMAC-SIRPα-KO has the ability to phagocytose cancer cells specific to the target antigen.

[0129] (11) Cancer cell cytotoxicity of human CAR-pMAC-SIRPα-KO (real-time measurement) Using human CAR-pMAC-SIRPα-KO as effector cells, the cytotoxicity against human cancer cells was evaluated using the xCELLigence RTCA DP system (Figures 26-1 and 26-2). hGPC3-introduced or non-expressing SK-Hep1 (5 x 10 ４ The cells were seeded on E-Plate 16 and incubated at 37°C, 5% CO ２After 24 hours of culture, human CAR-pMAC-SIRPα-KO was added at E:T ratios of 1:1, 5:1, and 10:1 for co-culture. It can be seen that human CAR-pMAC-SIRPα-KO exhibits superior cytotoxic activity as hGPC3 expression and the E:T ratio increase. Figure 26-2 shows the time course of the adhesion signal (Cell Index; CI) indicating cancer cell survival when cancer cells are co-cultured with CAR-pMAC-SIRPα-KO, as well as the cytotoxic activity (%Cytolysis) of cancer cells normalized to that of cancer cells cultured alone. It can be seen that the CI value of cancer cells decreases and %Cytolysis increases over time, depending on the expression of the cancer antigen hGPC3 and the number of CAR-pMAC-SIRPα-KO cells (E:T ratio).

[0130] (12) Investigation of the Importance of SIRPα Gene Deficiency or GC33-ζ-CAR Transduction in the Cytotoxicity of CAR-pMAC To investigate the importance of SIRPα gene deficiency or GC33-ζ-CAR transduction in the cytotoxicity of CAR-pMAC to cancer cells, human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells, and the cytotoxicity was evaluated at an E:T ratio of 10:1 using the xCELLigence RTCA DP system. Human cancer cells, hGPC3-transfected or non-expressing SK-Hep1 (5 × 10 ４ cells) were seeded on E-Plate 16 and cultured at 37°C for 24 hours, after which effector cells human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (5 × 10 ５Cells) were added and co-cultured. It can be seen that the cytotoxic activity (% Cytolysis) of SK-Hep1 cells introduced with GC33-ζ-CAR is improved by the introduction of GC33-ζ-CAR against hGPC3-introduced SK-Hep1 (top left of Figure 27). It can be seen that the cytotoxic activity is significantly higher than that against SK-Hep1 cells that do not express the cancer antigen hPGC3 (top right of Figure 27). The middle and bottom panels of Figure 27 show the time course of adhesion signals (Normalized Cell Index values; CI values) indicating cancer cell survival when cancer cells are co-cultured with human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO, as well as the cytotoxic activity (% Cytolysis) of cancer cells normalized to that of cancer cells cultured alone. GC33-ζ-CAR introduction and SIRPα deficiency most significantly reduced the CI value of cancer cells over time and increased % cytolysis.

[0131] The method of the present invention can provide a novel CAR-equipped platform that is stable in quality, stable in supply, and economical. The novel CAR-equipped platform uses proliferative macrophage-like cells (pMAC). CAR-equipped pMAC is also effective in the treatment of solid cancers. This application is based on Japanese Patent Application No. 2022-127477 (filing date: August 9, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. Proliferating macrophage-like cells (pMACs) are characterized by cytokine-dependent proliferation and are negative for CD14 and CD83.

2. The pMAC according to claim 1, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or macrophage colony-stimulating factor (M-CSF).

3. The pMAC according to claim 1, characterized in that a gene that inhibits macrophage-like function is deleted or its expression is suppressed.

4. The pMAC according to claim 3, wherein the gene that inhibits the macrophage-like function is the signal regulatory protein α (SIRPα) gene.

5. The pMAC according to any one of claims 1 to 4, wherein the pMAC further comprises a chimeric antigen receptor (CAR).

6. A method for producing proliferative macrophage-like cells (pMACs), 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.

7. (1) A step (step 1) in which a gene that inhibits macrophage-like function is deleted or its expression is suppressed in pluripotent stem cells, and then differentiation into myeloid cells is induced to obtain macrophage-like cells (MACs), and (2) Step 2: Expressing a gene that adds proliferative properties to the MAC obtained in Step 1 to obtain proliferative macrophage-like cells (pMACs). The manufacturing method according to claim 6, including

8. (A) A step (Step A) of expressing a gene that adds proliferative properties to myeloid cells differentiated from pluripotent stem cells in order to obtain proliferative myeloid cells, and (B) Step (Step B) involves deleting or suppressing the expression of genes that inhibit macrophage-like function in the proliferative myeloid cells obtained in Step A, and then inducing differentiation to obtain proliferative macrophage-like cells (pMACs). The manufacturing method according to claim 6, including

9. The method for producing a product according to claim 6, wherein the gene that inhibits macrophage-like function is the signal regulatory protein α (SIRPα) gene.

10. The manufacturing method according to claim 6, wherein the gene that adds proliferative properties is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.

11. A method for producing CAR-pMAC, comprising introducing a chimeric antigen receptor (CAR) into a pMAC obtained by the production method described in any one of claims 6 to 10.

12. (1) Step 1: In pluripotent stem cells, a gene that inhibits macrophage-like function is deleted or its expression is suppressed, and then differentiation into myeloid cells is induced to obtain macrophage-like cells (MACs). (2) A step (step 2) to obtain proliferative macrophage-like cells (pMACs) by expressing a gene that adds proliferative properties to the MACs obtained in step 1, and (3) Step 3: Introducing a chimeric antigen receptor (CAR) A method for manufacturing CAR-pMAC, including [the specified element].

13. The manufacturing method according to claim 12, wherein the induction of differentiation into myeloid cells in step 1 is carried out by the following method (i) or (ii). (i) Embryoid bodies (EBs) derived from pluripotent stem cells are cultured in a medium containing VEGF on feeder cells, and then further cultured in a medium containing VEGF, SCF, and TPO, or (ii) 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 in a medium containing VEGF, TPO, and GM-CSF.

14. The method according to claim 13, wherein the induction of pluripotent stem cells into germ bodies (EBs) is carried out by the following methods (a) and (b): (a) Pluripotent stem cells are seeded in a cell aggregate culture vessel to form embryoid bodies (EBs), and (b) the vessel has fine spheroid wells at the bottom and no flat surfaces between adjacent wells.

15. (1) A step of deleting or suppressing the expression of genes that inhibit macrophage-like function in pluripotent stem cells (step 1-1), Step 1-2 involves seeding pluripotent stem cells obtained in Step 1-1, which have a gene that inhibits macrophage-like function deleted or whose expression is suppressed, into a cell aggregate culture vessel having fine spheroid wells at the bottom and no flat surfaces between adjacent wells, in order to form embryoid bodies (EBs). Step 1-3: Obtaining macrophage-like cells (MACs) from the EB obtained in Step 1-2 by the following method (i) or (ii), (i) The EB is cultured in a medium containing VEGF on feeder cells, and then further cultured in a medium containing VEGF, SCF and TPO, or (ii) The EB is 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-CSF. (2) A step (step 2) to obtain proliferative macrophage-like cells (pMACs) by expressing a gene that adds proliferative properties to the MACs obtained in steps 1-3, and (3) Step 3: Introducing a chimeric antigen receptor (CAR) into the pMAC obtained in step 2 to obtain CAR-pMAC. A method for manufacturing CAR-pMAC containing [the specified substance].

16. The method for producing a macrophage-like function, wherein the gene that inhibits macrophage-like function is the signal regulatory protein α (SIRPα) gene, according to any one of claims 12 to 15.

17. The manufacturing method according to any one of claims 12 to 15, wherein the gene that adds proliferative capacity is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.

18. Principal component analysis based on gene expression profiles obtained by performing whole transcriptome analysis using a cell population following a macrophage cell lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, in vivo-derived monocytes, in vivo-derived macrophages M1, in vivo-derived macrophages M2, and in vivo-derived macrophages M0 as a comparison group, and analyzing the top two components with the largest contribution rates, proliferative macrophage-like cells that satisfy the following conditions: Condition 1: Does not overlap with known macrophage cell lineage plots, Condition 2: PC1 is 100 or greater, and PC2 is plotted in the range of -80 to 0.

19. A pharmaceutical composition comprising proliferative macrophage-like cells according to claim 5 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition according to claim 19, for the treatment of at least one disease selected from the group consisting of tumors or cancer-related diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrous diseases, and amyloidosis.