Method for producing proliferative macrophage-like cells (pMAC)

Abstract

The present invention provides: a proliferative macrophage-like cell (pMAC) characterized by cytokine-dependent proliferation; a method for producing the same; and a method for producing CAR-pMAC comprising the following step, (1) A step for obtaining macrophage-like cells (MAC) by inducing differentiation into myeloid cells after deletion or inhibition of expression of a gene that inhibits macrophage-like function in pluripotent stem cells (step 1), (2) a step for obtaining proliferative macrophage-like cells (pMAC) by expressing a proliferative gene in the MAC obtained in step 1 (step 2), and (3) and a step (step 3) for introducing a chimeric antigen receptor (CAR). Further, the present invention provides a medical use of CAR-pMAC. According to the present invention, it is possible to provide a novel CAR-mounted cell platform, in particular, a CAR-mounted cell platform having excellent economical efficiency and stable quality and stable supply, and more particularly, a CAR-mounted cell platform excellent as a pharmaceutical product.

Description

Technical Field

[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 comprising the step of introducing a chimeric antigen receptor (CAR) gene into the pMAC, and CAR-pMAC produced by the method. Background Art

[0002] As one of the immunotherapies for cancer patients, there is a treatment method based on chimeric antigen receptor (CAR) T cells (hereinafter referred to as CAR-T cells) (non-patent literature 1), which refers to the genetic modification of the T cell receptor (TCR) possessed by cytotoxic T cells (CTL), so that CTL can directly and selectively recognize tumor cells and exert anti-tumor effects. Cancer therapy based on CAR-T cells kills cancer cells through a mechanism different from existing anticancer drugs and radiotherapy, so it is expected to be effective for refractory or treatment-resistant cancers. CAR-T cells for some blood tumors such as leukemia and malignant lymphoma have been formulated. However, the effect on solid cancers is limited.

[0003] CAR-T cell therapy inherently has the following problems.

[0004] 1. No cancer tissue targeting (no receptors for approaching cancer tissue)

[0005] 2. No infiltration into cancerous tissue

[0006] 3. Functional decline and dysfunction (fatigue) due to interaction with the tumor microenvironment (TME)

[0007] 4. Loss of target antigen due to cancer gene mutation = cancer escape (treatment resistance)

[0008] 5. Concerns about cytokine release syndrome (CRS)

[0009] In view of these problems, both domestic and foreign attempts to improve them are underway. For example, the introduction of cancer-directed receptors (CCR2, CXCR2); the improvement of the intracellular signal transduction region of CAR; immune checkpoint control (anti-PD-1 antibody, PD-1-CD28); cytokine production ability (IL7, 12, 15, 18); regeneration of T / NK cells (iPS-T / NK cells, SCM-T cells); dual antigen recognition system; etc.

[0010] However, there are currently no reports showing significant clinical effects on solid cancers.

[0011] In recent years, preclinical trial results have been reported that a cell platform in which CAR is carried on macrophages rather than T cells is effective in treating solid cancers (Non-Patent Document 2).

[0012] This is based on the following properties / functions inherent to macrophages.

[0013] 1. Express cancer-directed receptors, aggregate and infiltrate cancer

[0014] 2. No PD-1 / PD-L1-mediated functional decline

[0015] 3. Cause secondary immune response through activation (type 1 macrophage (M1) transformation)

[0016] In addition, Patent Documents 1 and 2 describe monocytes, macrophages, and dendritic cells carrying CAR, and pharmaceutical compositions containing these cells.

[0017] In this way, CAR-macrophage therapy can become a very promising cell immunotherapy, but in order to realize cell preparation, quality stability, standardization (stable supply), and mass production (economic efficiency) are required. From the perspective of quality stability and stable supply, it is preferred that the cells carrying CAR are allogeneic. Patent documents 2 and 3 describe a method for differentiating myeloid cells from pluripotent stem cells.

[0018] In addition, a technology for introducing CAR into iMAC 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 performed at any time is cumbersome, which may lead to high manufacturing costs.

[0019] Prior art literature

[0020] Patent Literature

[0021] Patent Document 1: Japanese Patent Application No. 2021-511809

[0022] Patent Document 2: Japanese Patent Application Publication No. 2022-28831

[0023] Patent Document 3: International Publication No. 2021 / 230304

[0024] Non-patent literature

[0025] Non-patent literature 1: Eshhar Z. et al., Proc Natl Acad Sci USA, 1993, 90: 720-724.

[0026] Non-patent document 2: Klichinsky M. et al., Nat Biotechnol. 38(8): 947-953, 2020 Non-patent document 3: Su S. et al., Cells. 2022, 11: 1652 Summary of the invention

[0027] The object of the present invention is to provide a novel CAR-carrying cell platform, in particular, to provide a CAR-carrying macrophage-like cell platform with excellent quality stability, stable supply, and economic efficiency.

[0028] In view of the above problems, the present inventors focused on macrophages as immune cells carrying CARs. They found that allogeneic cells can be utilized by using macrophages induced by pluripotent stem cells as macrophages carrying CARs, and mass production can be achieved by giving cytokine-dependent proliferation activity to the macrophages, thereby completing the present invention.

[0029] That is, the present invention is as follows.

[0030] [1] A proliferating macrophage-like cell (pMAC) characterized by proliferating in a cytokine-dependent manner.

[0031] [2] The pMAC according to [1], wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) and / or macrophage colony-stimulating factor (M-CSF).

[0032] [3] The pMAC according to [1] or [2], wherein CD14 and CD83 are negative.

[0033] [4] The pMAC according to any one of [1] to [3], which is derived from pluripotent stem cells.

[0034] [5] The pMAC according to [4], wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0035] [6] The pMAC according to any one of [1] to [5], wherein a gene that inhibits macrophage-like function is deleted or its expression is suppressed.

[0036] [7] The pMAC according to [6], wherein the gene that inhibits macrophage-like function is the signal regulatory protein α (SIRPα) gene.

[0037] [8] The pMAC according to any one of [1] to [7], wherein the pMAC further comprises a chimeric antigen receptor (CAR).

[0038] [9] A method for producing proliferative macrophage-like cells (pMAC), comprising: a step of deleting or inhibiting the expression of a gene that inhibits macrophage-like function; and a step of expressing a gene that imparts proliferative properties.

[0039]

[10] The manufacturing method according to [9], comprising:

[0040] (1) Step 1, a step of obtaining macrophage-like cells (MACs) by inducing differentiation into myeloid cells after deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells, and

[0041] (2) Step 2 is a step of causing the MAC obtained in step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC).

[0042]

[11] The manufacturing method according to [9], comprising:

[0043] (A) Step A, a step of causing myeloid cells differentiated from pluripotent stem cells to express a gene that imparts proliferation to obtain proliferative myeloid cells, and

[0044] (B) Step B is a step of obtaining proliferative macrophage-like cells (pMAC) by 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.

[0045]

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

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

[0046]

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

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

[0047]

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

[13] .

[0048]

[15] A method for producing a CAR-pMAC, comprising:

[0049] (1) Step 1, a step of obtaining macrophage-like cells (MACs) by deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells and then inducing differentiation into myeloid cells,

[0050] (2) Step 2, a step of causing the MAC obtained in step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC), and

[0051] (3) Step 3: a step of introducing a chimeric antigen receptor (CAR).

[0052]

[16] The production method according to

[15] , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0053]

[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).

[0054] (i) culturing embryoid bodies (EBs) induced from pluripotent stem cells on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF and TPO, or

[0055] (ii) Embryoid bodies (EBs) induced from pluripotent stem cells are cultured as a feeder-free monolayer in a medium containing BMP-4, VEGF and SCF, and then further cultured in a medium containing VEGF, TPO and GM-CSF.

[0056]

[18] The method according to

[17] , wherein the induction of pluripotent stem cells into embryoid bodies (EBs) is carried out by the following methods (a) and (b):

[0057] (a) seeding pluripotent stem cells in a culture vessel for cell cluster formation to form embryoid bodies (EBs); and

[0058] (b) The container has fine spherical holes on the bottom, and there is no flat surface between adjacent holes.

[0059]

[19] A method for producing a CAR-pMAC, comprising:

[0060] (1) Step 1-1, a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells,

[0061] Step 1-2, a step of forming embryoid bodies (EBs) by inoculating the pluripotent stem cells obtained in step 1-1 in which the gene that inhibits macrophage-like function is deleted or the expression is inhibited, into a cell cluster formation culture container having fine spheroid holes in the bottom and no flat surface between adjacent holes,

[0062] Step 1-3 is a step of obtaining macrophage-like cells (MAC) from the EBs obtained in step 1-2 by the following method (i) or (ii),

[0063] (i) culturing the EBs in a medium containing VEGF on a feeder layer of cells, and then further culturing them in a medium containing VEGF, SCF and TPO, or

[0064] (ii) culturing the EBs in a culture medium containing BMP-4, VEGF and SCF without feeder cells as a monolayer, and then further culturing them in a culture medium containing VEGF, TPO and GM-CSF;

[0065] (2) Step 2, a step of causing the MAC obtained in Steps 1-3 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC); and

[0066] (3) Step 3: a step of introducing a chimeric antigen receptor (CAR) into the pMAC obtained in step 2 to obtain CAR-pMAC.

[0067]

[20] The production method according to any one of

[15] to

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

[0068]

[21] The production method according to any one of

[15] to

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

[0069]

[22] A proliferative macrophage-like cell, wherein a gene expression profile is obtained by performing whole transcriptome analysis using a cell group along the macrophage lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, organism-derived monocytes, organism-derived macrophages M1, organism-derived macrophages M2, and organism-derived macrophages M0 as a comparison object, and principal component analysis is performed based on the gene expression profile. When the index of the top two components with the highest contribution rate is used as the analysis object, the following conditions are satisfied:

[0070] Condition 1: No overlap with known macrophage lineage maps, and,

[0071] Condition 2: Plot when PC1 is greater than 100 and PC2 is between -80 and 0.

[0072]

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

[0073]

[24] The pharmaceutical composition according to

[23] , which is used to treat at least one selected from the group consisting of tumor- or cancer-related diseases, neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases, and amyloidosis.

[0074]

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

[0075]

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

[0076] According to the method of the present invention, it is possible to produce CAR-carrying proliferative macrophage-like cells (CAR-pMAC) with stable quality, stable supply, and excellent economic efficiency. CAR-carrying proliferative macrophage-like cells are also effective in the treatment of solid cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is a graph obtained by performing principal component analysis on the gene expression profiles of mouse pMAC, BM-derived monocytes, BM-derived macrophages M0, mouse BM-derived macrophages M1, mouse BM-derived macrophages M2, and intraperitoneal macrophages obtained by RNA sequencing analysis, and plotting the first principal component (PC1: contribution rate 34%) and the second principal component (PC2: contribution rate 21%) on the horizontal axis. The closer the plot distance between cells, the more similar the gene expression profiles are, and conversely, the farther the plot distance between cells, the more different the gene expression profiles are.

[0078] Figure 2-1This is a histogram showing the results of flow cytometric analysis of CD11b, F4 / 80, CD16 / 32, CD64, I-A / E, and CD206 surface antigens of mouse bone marrow-derived monocytes, mouse intraperitoneal macrophages, mouse bone marrow-derived macrophages M0, mouse bone marrow-derived M1 macrophages M1, mouse bone marrow-derived M2 macrophages M2, and mouse pMAC. The vertical axis represents the cell presence ratio (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0079] Figure 2-2 This is a histogram showing the results of flow cytometric analysis of CD163, Ly6C, CD80, CD86, SiglecF, and SIRPα surface antigens of mouse bone marrow-derived monocytes, mouse intraperitoneal macrophages, mouse bone marrow-derived macrophages M0, mouse bone marrow-derived M1 macrophages M1, mouse bone marrow-derived M2 macrophages M2, and mouse pMAC. The vertical axis represents the cell presence ratio (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0080] Figure 2-3 This is a histogram showing the results of TLR2, TLR4, CD124, CCR1, CCR2, CCR3, and CCR4 surface antigen analysis of mouse bone marrow-derived monocytes, mouse intraperitoneal macrophages, mouse bone marrow-derived macrophages (BM-derivedmacrophages M0), mouse bone marrow-derived M1 macrophages (BM-derived macrophages M1), mouse bone marrow-derived M2 macrophages (BM-derived macrophages M2), and mouse pMAC by flow cytometry. The vertical axis represents the cell presence ratio (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0081] Figure 2-4This is a histogram showing the results of CCR5, CXCR2, and CXCR4 surface antigen analysis of mouse bone marrow-derived monocytes, mouse 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 by flow cytometry. The vertical axis represents the cell presence ratio (%), and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0082] Figure 3 The images are microscopic observation images of mouse pMAC and pMAC-SIRPα-KO lacking the SIRPα gene, using May-Giemsa staining. The scale bar indicates 20 μm. Compared with pMAC, no characteristic morphological changes due to the lack of the SIRPα gene were observed.

[0083] Figure 4 The vector map (upper layer) and CAR expression construct (lower layer) of the lentiviral vector used to introduce the GPC3-specific chimeric antigen receptor (GPC3-CAR) gene into pMAC.

[0084] Figure 5 This is a line graph showing the results of the MTT analysis to investigate the effect of a culture medium (αMEM containing 20% ​​FBS) containing GM-CSF and / or M-CSF that affects the proliferation rate of pMAC. The results of mouse pMAC (upper left), CAR-pMAC (upper right), pMAC-SIRPα-KO (lower left), and CAR-pMAC-SIRPα-KO (lower right) are shown. The vertical axis represents the concentration of formazan metabolized by the MTT reagent (evaluated by absorbance at 595nm), and the horizontal axis represents the number of days in culture. The deletion of the SIRPα gene and / or the introduction of CAR against the cancer antigen GPC3 implemented to produce pMAC did not change the cytokine-dependent cell proliferation performance.

[0085] Figure 6It is a scatter plot (cytogram) showing the results of hGPC3-FITC, SIRPα and tdTomato expression analysis based on flow cytometry for mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC and CAR-pMAC-SIRPα-KO. The vertical axis is the fluorescence intensity of hGPC3-FITC, and the horizontal axis is the fluorescence intensity of tdTomato (upper layer). The vertical axis is the fluorescence intensity of SIRPα, and the horizontal axis is the fluorescence intensity of tdTomato (middle layer). The vertical axis is the fluorescence intensity of SIRPα, and the horizontal axis is the fluorescence intensity of hGPC3-FITC (lower layer). Since pMAC expressing GPC3-CAR is obtained as a single cell population, it can be seen that the purity is high and almost no cells other than the target are contained.

[0086] Figure 7 The results are measured by ELISA for the production of cytokines IL-6, TNFα, IL-12, or IL-10 when medium without stimulating components was added to mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO, and mouse bone marrow-derived macrophages (BM-Macrophage), interferon γ and LPS were used to stimulate M1, and IL-4 was used to stimulate M2. The vertical axis represents the concentration of the measured cytokine (pg / mL).

[0087] Figure 8The results of measuring the cell migration ability of mouse CAR-pMAC-SIRPα-KO using the xCELLigence DP system using the Boyden chamber method. The cell migration ability in the culture supernatant and fresh medium derived from mouse cancer cells MC38, LL / 2, 4T1, and CT26 (upper left); the cell migration ability in the culture supernatant and fresh medium derived from mouse cancer cells MC38, mouse embryonic fibroblasts MEF, T cells, and B cells (upper center); the cell migration ability of mouse CAR-pMAC-SIRPα-KO in the culture supernatant of mouse cancer cells MC38 or fresh medium after untreated, cytochalasin D (+CytD) treatment, or pertussis toxin (+PTX) treatment (upper right). Cell migration ability in recombinant CCL2, CCL3, CCL4, CCL5 and fresh medium (Medium) (lower left); Cell migration ability in recombinant CCL7, CCL8, CXCL1, CXCL2 and fresh medium (Medium) (lower center); Cell migration ability in recombinant CXCL5, SDF-1α (CXCL12), SDF-1β (CXCL12), CX3CL1 and fresh medium (Medium) (lower right). The horizontal axis represents the culture time, and the vertical axis represents the cell index (Cell Index, CI) showing the degree of cell migration ability relative to the start time of the measurement (0 hr).

[0088] Figure 9-1 The results of ELISA measurement of cytokine IL-6 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with mouse cancer cells (without or with GPC3 expression) for 48 hours. The above four types of pMAC were co-cultured with breast cancer cell line 4T1-mock (without GPC3 expression) or 4T1-GPC3 (with GPC3 expression) (top), colorectal cancer cell line CT26-mock (without GPC3 expression) or CT26-GPC3 (with GPC3 expression) (second layer from the top), lung cancer cell line LL / 2-mock (without GPC3 expression) or LL / 2-GPC3 (with GPC3 expression) (third layer from the top), and colorectal cancer cell line MC38-mock (without GPC3 expression) or MC38-GPC3 (with GPC3 expression) (bottom).

[0089] Figure 9-2The results are ELISA measurements of TNFα production in the culture supernatant of mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO and mouse cancer cells (with or without GPC3 expression) when co-cultured for 48 hours. The above four pMACs were co-cultured with breast cancer cell line 4T1-mock (without GPC3 expression) or 4T1-GPC3 (with GPC3 expression) (top), colorectal cancer cell line CT26-mock (without GPC3 expression) or CT26-GPC3 (with GPC3 expression) (second from the top), lung cancer cell line LL / 2-mock (without GPC3 expression) or LL / 2-GPC3 (with GPC3 expression) (third from the top), and colorectal cancer cell line MC38-mock (without GPC3 expression) or MC38-GPC3 (with GPC3 expression) (bottom).

[0090] Figure 9-3 The results of ELISA measurement of cytokine IL-10 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with mouse cancer cells (without or with GPC3 expression) for 48 hours. The above four types of pMAC were co-cultured with breast cancer cell line 4T1-mock (without GPC3 expression) or 4T1-GPC3 (with GPC3 expression) (top), colorectal cancer cell line CT26-mock (without GPC3 expression) or CT26-GPC3 (with GPC3 expression) (second layer from the top), lung cancer cell line LL / 2-mock (without GPC3 expression) or LL / 2-GPC3 (with GPC3 expression) (third layer from the top), and colorectal cancer cell line MC38-mock (without GPC3 expression) or MC38-GPC3 (with GPC3 expression) (bottom).

[0091] Figure 9-4The results of ELISA measurement of cytokine IL-12 production in the culture supernatant when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were co-cultured with mouse cancer cells (without or with GPC3 expression) for 48 hours. The above four types of pMAC were co-cultured with breast cancer cell line 4T1-mock (without GPC3 expression) or 4T1-GPC3 (with GPC3 expression) (top), colorectal cancer cell line CT26-mock (without GPC3 expression) or CT26-GPC3 (with GPC3 expression) (second layer from the top), lung cancer cell line LL / 2-mock (without GPC3 expression) or LL / 2-GPC3 (with GPC3 expression) (third layer from the top), and colorectal cancer cell line MC38-mock (without GPC3 expression) or MC38-GPC3 (with GPC3 expression) (bottom).

[0092] Fig.10 This is a bar graph showing the quantitative luciferase activity of cancer cells after co-culture of mouse pMAC (A), CAR-pMAC (B), pMAC-SIRPα-KO (C), and CAR-pMAC-SIRPα-KO (D) with the luciferase (Luc) gene-introduced lung cancer cell line LL / 2 (no or human GPC3 expression; upper left), colorectal cancer cell line CT26 (no or human GPC3 expression; upper right), colorectal cancer cell line MC38 (no or human GPC3 expression; lower left), and breast cancer cell line 4T1 (no or human GPC3 expression; lower right) for 48 hours. The vertical axis indicates the reduction ratio (%) of cancer cells. White bars indicate cancer cells that do not express hGPC3, and black bars indicate cancer cells that express hGPC3. It can be seen that pMAC (D) into which CAR has been introduced eliminates cancer cells that express the target cancer antigen human GPC (hGPC).

[0093] Fig.11Fluorescence microscopic images of pMAC and CAR-pMAC-SIRPα-KO mice expressing tdTomato and cancer cell lines expressing Venus (expressing human GPC3) after co-culture for 2 and 4 days (upper layer). Cancer cells are detected by green fluorescence (excitation wavelength 470nm, absorption wavelength 525nm) from the fluorescent protein Venus, and pMAC or CAR-pMAC-SIRPα-KO are detected by red fluorescence (excitation wavelength 545nm, absorption wavelength 605nm) from the fluorescent protein tdTomato. The bar graph is plotted by plotting the area where the green fluorescence signal of cancer cells and the red fluorescence signal of pMAC or CAR-pMAC-SIRPα-KO are repeatedly displayed (lower layer, left side). The bar graph is plotted by plotting the area where the green fluorescence signal of cancer cells is detected (lower layer, right side). It can be seen that although cancer cells co-cultured with pMAC proliferate, cancer cells co-cultured with CAR-pMAC-SIRPα-KO are eliminated by being surrounded and engulfed by CAR-pMAC-SIRPα-KO.

[0094] Figure 12-1 This is a 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. After culturing the colon cancer cell line CT26 (no or human GPC3 expression; upper left), colon cancer cell line MC38 (no or human GPC3 expression; upper right), lung cancer cell line LL / 2 (no or human GPC3 expression; lower left), and breast cancer cell line 4T1 (no or human GPC3 expression; lower right) for 24 hours, 10-fold, 5-fold, and 1-fold the amount of CAR-pMAC-SIRPα-KO as much as the inoculated cancer cells were added for co-culture, and the cytotoxic activity (% cytolysis) after 6 hours was plotted. The vertical axis represents the cytotoxic activity (% cytolysis) of cancer cells, and the horizontal axis represents the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRPα-KO) and target cells (cancer cells).

[0095] Figure 12-2The results of the measurement of the normalized cell index (Normalized Cell Index) of the cell survival signal of the cancer cell CT26 caused by mouse CAR-pMAC-SIRPα-KO (top layer) and the cytotoxic activity % cytolysis of mouse CT26 cells (second layer from the top) using the xCELLigence RTCADP system when mouse CAR-pMAC-SIRPα-KO was used as effector cells and co-cultured with mouse cancer cells (no or human GPC3 expression) as target cells at an E:T ratio of 10:1, 5:1, and 1:1. The results of the measurement of the normalized cell index of the cell survival signal of the cancer cell MC38 caused by mouse CAR-pMAC-SIRPα-KO (third layer from the top) and the cytotoxic activity % cytolysis of mouse MC38 cells (bottom layer) using the xCELLigence RTCA DP system.

[0096] Figure 12-3 The results of the xCELLigence RTCA DP system measurement of the normalized cell index (top) of the cell survival signal of the cancer cell LL / 2 caused by mouse CAR-pMAC-SIRPα-KO and the cytotoxic activity % cytolysis of the mouse LL / 2 cells (second layer from the top) when mouse CAR-pMAC-SIRPα-KO was used as effector cells and co-cultured with mouse cancer cells (no or human GPC3 expression) as target cells at an E:T ratio of 10:1, 5:1, and 1:1. The results of the xCELLigence RTCA DP system measurement of the normalized cell index (third layer from the top) of the cell survival signal of the cancer cell 4T1 caused by mouse CAR-pMAC-SIRPα-KO and the cytotoxic activity % cytolysis of the mouse 4T1 cells (bottom layer) were used.

[0097] Fig.13The bar graph is a plot of the cytotoxic activity of mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO as effector cells and the cytotoxic activity of CT26 cells when they were co-cultured with the target cell mouse CT26 (expressing GPC3) at an E:T ratio of 10:1 (top left). The bar graph is a plot of the cytotoxic activity of mouse CAR-pMAC-SIRPα-KO as effector cells and the cytotoxic activity of CT26 cells when they were co-cultured with the target cell mouse cancer cell CT26 (without or with GPC3 expression) at an E:T ratio of 10:1 (top right). A line graph (middle left) showing the change over time of the Normalized Cell Index representing cell survival of CT26 (without GPC3 expression) when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with mouse CT26 (without GPC3 expression) as target cells at an E:T ratio of 10:1, with the co-culture time as the horizontal axis; and a line graph (bottom left) showing the cytotoxic activity % cell lysis of mouse CT26 cells (without GPC3 expression) as the vertical axis. A line graph (middle right) with the time-dependent change of the normalized cell index indicating cell survival of CT26 (with GPC3 expression) as the vertical axis and the co-culture time as the horizontal axis when mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with mouse CT26 (with GPC3 expression) as the target cell at an E:T ratio of 10:1, and a line graph (bottom right) with the cytotoxic activity % cell lysis of mouse CT26 cells (with GPC3 expression) as the vertical axis. It can be seen that CAR-pMAC-SIRPα-KO showed higher cytotoxic activity than pMAC, CAR-pMAC, and pMAC-SIRPα-KO when co-cultured with cancer cells for 6 hours.

[0098] Fig.14 This is a line graph obtained by co-culturing mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC or CAR-pMAC-SIRPα-KO with mouse cancer cell MC38 (GPC3 expression) and counting phagocytes by time lapse observation. The horizontal axis represents the co-culture time, and the vertical axis represents the number of phagocytes.

[0099] Fig.15The results are as follows: CAR-pMAC-SIRPα-KO was administered to C57BL / 6 tumor-bearing mice that were intraperitoneally inoculated with MC38 (expressing GPC3) and the in vivo dynamics of CAR-pMAC-SIRPα-KO were measured by quantitative PCR on Day 0 (upper layer), 1 day (middle layer), and 3 days (lower layer) before administration. The horizontal axis represents the organs 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.

[0100] Fig.16 The images of all individuals in vivo imaging were taken when the mouse colorectal cancer cell line MC38 expressing the luciferase gene and the cancer antigen GPC3 was intraperitoneally administered to wild-type C57BL / 6 mice, and the surviving cancer cells in the CAR-pMAC-SIRPα-KO colorectal cancer peritoneal inoculation model mice that were not treated or were irradiated with 10 Gy on the next day, day 3, day 7, day 10, day 14, and day 17 were measured by biochemiluminescence (top), a line graph showing the temporal changes in the total luciferase luminescence flux (bottom left), and the survival curve of each tumor-bearing mouse individual (bottom right). The in vivo imaging was taken at the time of intraperitoneal administration of MC38 (Day 0), day 3 (Day 3), day 7 (Day 7), day 10 (Day 10), and day 14 (Day 14). On the right side of the image above is a scale that represents the luminescence intensity of luciferase in color, with the minimum luminescence intensity being 2.00×e 8 , the maximum luminous intensity is 1.00×e 10 (Unit: p / sec / cm 2 / sr). The vertical axis of the lower left line graph represents the total luminescence flux of luciferase (unit: 10 10 photons / s), and the horizontal axis represents the number of days after cancer cell transplantation. The vertical axis of the graph on the lower right represents the mouse survival rate, and the horizontal axis represents the number of days after cancer cell transplantation. It can be seen that although cancer cell growth was observed in untreated mice, the growth of cancer cells was inhibited in mice treated with CAR-pMAC-SIRPα-KO even after radiation exposure, and the development of peritoneal inoculation of colorectal cancer was inhibited, thereby prolonging the survival of individual mice.

[0101] Fig.17This is a graph obtained by performing principal component analysis on the gene expression profiles of human pMAC, blood-derived CD14-positive cell-derived monocytes, blood-derived CD14-positive cell-derived macrophages M0, blood-derived CD14-positive cell-derived M1 macrophages, blood-derived CD14-positive cell-derived M2 macrophages, human iPS cell-derived myeloid cells, and human iPS cell-derived macrophages, and plotting the first principal component (PC1: contribution rate 40%) on the horizontal axis and the second principal component (PC2: contribution rate 18%) on the horizontal axis. The closer the plotted distances between cells, the more similar the gene expression profiles are. Conversely, the farther the plotted distances between cells, the more different the gene expression profiles are. The gene expression profile of pMAC is very different from that of known macrophages derived from blood or iPS cells.

[0102] Figure 18-1 This is a histogram showing the results of HLA-ABC, HLA-DR, CD40, CD80 and CD83 surface antigen analysis of human blood-derived CD14-positive cell-derived monocytes (CD14-derivedMonocyte), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive cell-derived macrophages (CD14-derived macrophages M0), blood-derived CD14-positive cell-derived M1 macrophages (CD14-derived macrophages M1), blood-derived CD14-positive cell-derived M2 macrophages (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derivedMyeloid cells), and iPS cell-derived macrophages (iPSC-derived macrophages) obtained by flow cytometry. The vertical axis represents the presence ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0103] Figure 18-2This is a histogram showing the results of CD86, CD68, CD163, CD206 and SIRPα surface antigen analysis of human blood-derived CD14-positive cell-derived monocytes (CD14-derivedMonocyte), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive cell-derived macrophages (CD14-derived macrophages M0), blood-derived CD14-positive cell-derived M1 macrophages (CD14-derived macrophages M1), blood-derived CD14-positive cell-derived M2 macrophages (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derivedMyeloid cells), and iPS cell-derived macrophages (iPSC-derived macrophages) obtained by flow cytometry. The vertical axis represents the presence ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0104] Figure 18-3 The figure is a histogram showing the results of CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CXCR2, CXCR4 and CD14 surface antigen analysis of human blood-derived CD14-positive cell-derived monocytes (CD14-derived monocytes), pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive cell-derived macrophages (CD14-derived macrophages M0), blood-derived CD14-positive cell-derived M1 macrophages (CD14-derived macrophages M1), blood-derived CD14-positive cell-derived M2 macrophages (CD14-derived macrophages M2), iPS cell-derived myeloid cells (iPSC-derived myeloid cells), and iPS cell-derived macrophages (iPSC-derived macrophages) obtained by flow cytometry. The vertical axis represents the existence ratio (%) of each cell, and the horizontal axis represents the fluorescence intensity of each measurement marker.

[0105] Figure 19-1Phase contrast microscopic images of CD14-derived monocytes, iPSC-derived myeloid cells, pMAC, CD14-derived macrophages M0, M1, and M2. The scale bar indicates 40 μm.

[0106] Figure 19-2 Phase contrast microscopy images of human iPS cell-derived macrophages (iPSC-macrophages), pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO under no stimulation (M0), interferon γ and LPS stimulation (M1), and IL-4 stimulation (M2).

[0107] Fig. 20 This is a line graph showing the results of the MTT assay to investigate the effects of a culture 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). Results for human pMAC (left), pMAC-SIRPα-KO (center), and iPSC-macrophages (right) are shown. The vertical axis represents the concentration of formazan metabolized by the MTT reagent (evaluated by absorbance at 595 nm), and the horizontal axis represents the number of days of culture.

[0108] Fig.21 This is a scatter plot showing the results of hGPC3-FITC, SIRPα, and tdTomato expression analysis using flow cytometry for human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO. The vertical axis is the fluorescence intensity of hGPC3-FITC, and the horizontal axis is the fluorescence intensity of tdTomato (upper layer). The vertical axis is the fluorescence intensity of SIRPα, and the horizontal axis is the fluorescence intensity of tdTomato (lower layer). The pMAC expressing GPC3-CAR was obtained as a single cell population, so it can be seen that the purity is high and almost no cells other than the target are contained.

[0109] Fig. 22The results of ELISA measurement of the production of cytokines IL-6, TNFα, IL-12, or IL-10 when adding medium without stimulating components to human pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive cell-derived macrophages (CD14-Macrophage), and iPS cell-derived macrophages (iPSC-Macrophage), interferon γ and LPS were used to apply M1 stimulation, and IL-4 was used to apply M2 stimulation. The vertical axis represents the concentration of the measured cytokine (pg / mL).

[0110] Fig.23The results of real-time measurement of cell migration ability of human CAR-pMAC-SIRPα-KO using the xCELLigenceDP system in Boyden chamber format. Cell migration ability in culture supernatants from human cancer cell lines HepG2, SK-Hep1, JHH7, KOC7c, and human embryonic kidney cell line HEK293, and in fresh culture medium (upper left), and cell migration ability of human CAR-pMAC-SIRPα-KO in culture supernatants from human cancer cell line HepG2, human fetal kidney cell line HEK293, or in fresh culture medium (upper center) after untreated, cytochalasin D (+CytD)-treated, or pertussis toxin (+PTX)-treated cells. Cell migration ability of human iPSC-macrophages in culture supernatants from cancer cell lines HepG2, SK-Hep1, JHH7, KOC7c, and human embryonic kidney cell line HEK293, and in fresh culture medium (upper right). The cell migration ability of human CAR-pMAC-SIRPα-KO in recombinant CCL2, CCL3, CCL3LI, CCL4 and fresh medium (left, second layer from the top). The cell migration ability in recombinant CCL5, CCL7, CCL8, CCL14 and fresh medium (center, second layer from the top), the cell migration ability in recombinant CCL15, CCL17, CCL23, CX3CL1 and fresh medium (right, second layer from the top). The cell migration ability in recombinant CXCL1, CXCL2, CXCL5 and fresh medium (left, third layer from the top). The cell migration ability in recombinant CCL13, SDF-1α (CXCL12), SDF-1β (CXCL12) and fresh medium (right, third layer from the top). Cell migration ability of CAR-pMAC and CAR-pMAC-SIRPα-KO in the culture supernatant derived from the cancer cell line HepG2 (bottom). The horizontal axis represents the culture time, and the vertical axis represents the cell index (CI) showing the degree of cell migration ability relative to the measurement start time (0 hr).

[0111] Figure 24-1 These are the results of ELISA measurement of the production of cytokines IL-6 (left), TNFα (second from the left), IL-10 (third from the left), and IL-12 (right) in the culture supernatant when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO were cultured for 48 hours.

[0112] Figure 24-2The results of ELISA measurement of 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 (without or with GPC3 expression) for 48 hours. The results of co-culture of the above four pMACs with liver cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top layer), liver cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (second layer from the top). The results of ELISA measurement of 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 (without or with GPC3 expression) for 48 hours. Results of co-culturing the above four types of pMAC with liver cancer cell lines SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (GPC3 expression) (third layer from the top), and liver cancer cell lines JHH7-GPC3-KO (no GPC3 expression) or JHH7 (GPC3 expression) (bottom layer).

[0113] Figure 24-3 The results of ELISA measurement of 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 (without or with GPC3 expression) for 48 hours. The results of co-culture of the above four pMACs with cancer cell lines SK-Hep1 (without GPC3 expression) or SK-Hep1-GPC3 (with GPC3 expression) (top layer), cancer cell lines JHH7-GPC3-KO (without GPC3 expression) or JHH7 (with GPC3 expression) (second layer from the top). The results of ELISA measurement of 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 (without or with GPC3 expression) for 48 hours. Results of co-culturing the above four types of pMAC with cancer cell lines SK-Hep1 (no GPC3 expression) or SK-Hep1-GPC3 (GPC3 expression) (third layer from the top), and cancer cell lines JHH7-GPC3-KO (no GPC3 expression) or JHH7 (GPC3 expression) (bottom layer).

[0114] Fig.25The bar graph shows the results of quantitative analysis of luciferase activity of cancer cells after 48 hours of co-culture of human pMAC (A), CAR-pMAC (B), pMAC-SIRPα-KO (C), and CAR-pMAC-SIRPα-KO (D) with 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 the left), and human cancer cell line KOC7c-GPC3-Luc (expressing human GPC3; third from the left). The bar graph shows the results of quantitative analysis of luciferase activity of 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 indicates the reduction ratio (%) of cancer cells. White bars indicate cancer cells that do not express hGPC3, and black bars indicate cancer cells that express hGPC3. It is known that pMAC (D) into which CAR is introduced eliminates cancer cells that express the target cancer antigen human GPC (hGPC).

[0115] Figure 26-1 This is the cytotoxic activity of cancer cells caused by human CAR-pMAC-SIRPα-KO when two types of human cancer cells are co-cultured with human CAR-pMAC-SIRPα-KO as effector cells and target cells. After culturing the human liver cancer cell line SK-Hep1 (without or with human GPC3 expression) for 24 hours, 10-fold, 5-fold, and 1-fold the amount of CAR-pMAC-SIRPα-KO as the inoculated cancer cells were added for co-culture, and the cytotoxic activity (% cell lysis) after 6 hours was plotted. The vertical axis represents the cytotoxic activity (% cell lysis) of cancer cells, and the horizontal axis represents the mixing ratio (E:T ratio) of effector cells (CAR-pMAC-SIRPα-KO) and target cells (cancer cells).

[0116] Figure 26-2 The normalized cell index (upper layer) of the cell survival signal of the hepatoma cell line SK-Hep1 caused by human CAR-pMAC-SIRPα-KO and the cytotoxic activity % cell lysis (lower layer) of the human hepatoma cell line SK-Hep1 were measured using the xCELLigence RTCA DP system when human CAR-pMAC-SIRPα-KO was used as effector cells and human cancer cells (without or with human GPC3 expression) as target cells at an E:T ratio of 10:1, 5:1, and 1:1.

[0117] Fig. 27The bar graph is a plot of cytotoxic activity % cytolysis of SK-Hep1 when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells and co-cultured with human hepatoma cell SK-Hep1 (expressing GPC3) at an E:T ratio of 10:1 (top left). The bar graph is a plot of cytotoxic activity % cytolysis of SK-Hep1 when human CAR-pMAC-SIRPα-KO was used as effector cells and co-cultured with human hepatoma cell SK-Hep1 (without or with GPC3 expression) at an E:T ratio of 10:1 (top right). A line graph showing the change over time of the normalized cell index of cell survival of SK-Hep1 (without GPC3 expression) when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were co-cultured as effector cells with human SK-Hep1 (without GPC3 expression) as target cells at an E:T ratio of 10:1 (middle left), and a line graph showing the cytotoxic activity % cell lysis of human liver cancer cells SK-Hep1 (without GPC3 expression) was plotted on the vertical axis (bottom left). A line graph showing the time-dependent changes in the normalized cell index of SK-Hep1 (GPC3-expressing) cell survival when human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were co-cultured as effector cells with human cancer cells SK-Hep1 (GPC3-expressing) at an E:T ratio of 10:1 (middle right). A line graph showing the cytotoxic activity % cell lysis of human liver cancer cells SK-Hep1 (GPC3-expressing) was plotted on the vertical axis (bottom right). It can be seen that CAR-pMAC-SIRPα-KO and CAR-pMAC showed higher cytotoxic activity than pMAC and pMAC-SIRPα-KO after 6 hours of co-culture with cancer cells. DETAILED DESCRIPTION

[0118] The present invention will be described below. Unless otherwise specified, the terms used in this specification have the meanings generally used in the field.

[0119] <Proliferating macrophage-like cells>

[0120] The present invention provides a macrophage, characterized in that it proliferates in a cytokine-dependent manner in addition to its phagocytic ability. The macrophage of the present invention is called a proliferating macrophage-like cell (pMAC) (hereinafter also referred to as the pMAC of the present invention) because it has characteristics different from those of existing macrophages. Alternatively, the pMAC of the present invention can also be referred to as a cytokine-dependent proliferating macrophage. In one embodiment, the gene that inhibits the macrophage-like function of the pMAC of the present invention is deleted or its expression is inhibited.

[0121] In this specification, "proliferation in a cytokine-dependent manner" means that the proliferation of cells is promoted by stimulation with cytokines, and the proliferation can be cultured for more than 3 months and is significantly better than that of cells not stimulated by cytokines. As cytokine stimulation, specifically, a process of culturing cells in a culture medium containing cytokines can be cited. Cytokines can be added to the culture medium exogenously, or they can be produced intracellularly and secreted into the culture medium. As a cytokine, one is selected from granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF), and preferably two are used in combination. It is preferred to use GM-CSF and M-CSF in combination.

[0122] GM-CSF, also known as CSF2, is produced by immune stimulation of cells such as T cells, macrophages, endothelial cells, and fibroblasts, and functions as a hematopoietic growth factor and immunomodulatory factor. GM-CSF induces bone marrow progenitor cells and promotes the proliferation of granulocytes and macrophages.

[0123] M-CSF is a cytokine that induces proliferation and differentiation of bone marrow stem cells, especially monocyte-macrophage cells.

[0124] Each cytokine can be obtained commercially. Alternatively, the sequence information of each cytokine protein, genomic DNA or cDNA of various biological species can be obtained from information sources known to those skilled in the art, such as the gene in the database of the National Center for Biotechnology Information (NCBI), so based on this information, the desired cytokine gene can be expressed in cells, the protein can be produced in cells, and it can be secreted outside the cells as appropriate.

[0125] The pMAC of the present invention has the characteristic of being CD83 and CD14 negative in addition to the characteristic of proliferating in a cytokine-dependent manner.

[0126] CD83 is known as a maturation marker of dendritic cells expressed on the cell surface and can be detected by immunostaining with an anti-CD83 antibody and analyzing using a flow cytometer.

[0127] CD14 is known as a marker for monocytes and macrophages expressed on the cell surface and can be detected by immunostaining with an anti-CD14 antibody and analyzing with a flow cytometer.

[0128] Here, "CD83 and CD14 are negative" means that CD83 and CD14 are not expressed or are substantially not expressed. Specifically, it means that the CD83 antigen and CD14 antigen as cell surface markers are not detected on the cell surface, or are less than the detection limit, and even if detected, they are extremely trace. Specifically, it means that compared with the expression of CD83 and CD14 in existing macrophages, it is less than 1 / 5, preferably less than 1 / 10, more preferably less than 1 / 20, further preferably less than 1 / 30, and most preferably less than the detection limit. The expression 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 protein blotting.

[0129] In addition, as will be demonstrated in detail in the following examples and drawings, the pMAC of the present invention is characterized in that its gene expression profile is greatly changed compared to cells included in the known macrophage lineage. Therefore, the pMAC of the present invention can also be defined using statistics based on the gene expression profile. For example, in the case of human pMAC, as an example, according to the examples and drawings (particularly Fig.17 ) and are defined as follows:

[0130] A proliferative macrophage-like cell, wherein a gene expression profile is obtained by performing whole transcriptome analysis using a cell group along the macrophage lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, organism-derived monocytes, organism-derived macrophages M1, organism-derived macrophages M2, and organism-derived macrophages M0 as a comparison object, and principal component analysis is performed based on the gene expression profile. When the indexes of the components ranked first in contribution rate are used as the analysis object, the following conditions are satisfied:

[0131] Condition 1: No overlap with known macrophage lineage maps, and

[0132] Condition 2: Plot when PC1 is greater than 100 and PC2 is between -80 and 0.

[0133] In one embodiment, the PC1 of the pMAC of the present invention may be 100 or more, preferably 110 or more, 120 or more, 130 or more, or 140 or more, and more preferably 150 or more, but the present invention is not limited thereto. In addition, the PC1 of the pMAC of the present invention may be 250 or less, preferably 220 or less, 210 or less, 200 or less, or 190 or less, and more preferably 180 or less, but the present invention is not limited thereto. In one embodiment, the PC1 of the pMAC of the present invention is 100-250, preferably 110-220, 120-210, 130-200, or 140-190, and more preferably 150-180, but the present invention is not limited thereto.

[0134] The pMAC of the present invention is derived from pluripotent stem cells, more preferably induced pluripotent stem cells or embryonic stem cells. Pluripotent stem cells refer to stem cells that can be cultured in an undifferentiated state in vitro and have the ability (pluripotency) to differentiate into all cells (tissues from three germ layers (ectoderm, mesoderm, endoderm)) that constitute an organism, excluding the placenta. Embryonic stem cells (ES cells) isolated from embryos before the formation of the three germ layers after fertilization, embryos after the organ formation stage containing primordial germ cells, fetuses or fetuses are also included in "pluripotent stem cells".

[0135] In the present invention, "induced pluripotent stem cells" refer to cells (hereinafter also referred to as iPSCs or iPS cells) in which somatic cells that have temporarily lost their pluripotency are directly initialized by expressing several genes such as Oct3 / 4, Sox2, Klf4, and Myc to induce pluripotency. Such "induced pluripotent stem cells" are also included in "pluripotent stem cells". Pluripotent stem cells can be prepared using known methods. As preparation methods, for example, for human induced pluripotent stem cells, the methods described in Cell, 2007, 131 (5) pp. 861-872, Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)) can be cited, and for mouse induced pluripotent stem cells, the methods described in Cell, 2006, 126 (4) pp. 663-676 can be cited. "Pluripotent stem cells" can also be obtained from animals subjected to embryo manipulation techniques represented by cloned embryos, transgenic animals, and mutant animals based on genome editing. It is known that pluripotent stem cells can differentiate into various cell types including myeloid cells (MC cells).

[0136] Human or mouse iPSCs can be maintained and expanded using the regenerative medicine medium StemFit (Ajinomoto). At this time, laminin 511 can be added. In addition, about 10 μM of Rho kinase (ROCK) inhibitors such as Y-27632 can be added to the culture medium to reduce the damage to iPSCs caused by the dissociation of cells for cell subculture.

[0137] Pluripotent stem cells can be obtained from a designated institution or purchased commercially. For example, KhES-1, KhES-2, and KhES-3, which are human embryonic stem cells, can be obtained from the Institute of Regenerative Medicine, Kyoto University. EB5 cells, which are mouse embryonic stem cells, can be obtained from the RIKEN Institute of Physical and Chemical Research, and the D3 cell line, which is mouse embryonic stem cells, can be obtained from ATCC.

[0138] Pluripotent stem cells can be maintained and cultured using known methods. For example, human pluripotent stem cells can be maintained and cultured using Knockout TM Mouse pluripotent stem cells can be maintained by culturing in a medium containing serum replacement (Invitrogen). Mouse pluripotent stem cells can be maintained by culturing in a feeder-free condition supplemented with fetal bovine serum (FBS) and leukemia inhibitory factor (LIF).

[0139] <Method for producing proliferative macrophage-like cells>

[0140] In the present invention, pMAC can be produced by a method including a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function and a step of expressing a gene that enhances proliferation. Each step is described below.

[0141] 1. Step of deleting or inhibiting the expression of a gene that inhibits macrophage-like function

[0142] "Genes that inhibit macrophage-like functions" refer to genes that inhibit functions that can be provided by macrophages. Examples of "functions that can be provided by macrophages" include phagocytosis, which helps in the ingestion and digestion of foreign bodies, antigen presentation, which promotes the activation of other immune cells such as T cells, and induction of secondary immune responses through activation. Macrophage activation can be roughly divided into classical (M1) activation and selective (M2) activation. M1-activated macrophages (M1 macrophages) are obtained by differentiation and induction of monocytes by inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and play a role in defending against pathogenic infections and producing pro-inflammatory cytokines that reduce the infectivity of microorganisms. M2-activated macrophages (M2 macrophages) are obtained by differentiation and induction by Th2-type cytokines such as IL-4 and IL-13, and play a role in suppressing the host's immune response, promoting wound healing and tissue remodeling, and producing growth factors and anti-inflammatory cytokines that improve metabolism and endocrine signaling in tissues. The "gene that inhibits macrophage-like function" refers to a gene whose expression can inhibit such a function that can be provided by macrophages. Examples of such a gene include the signal regulatory protein α (SIRPα) gene, but the invention is not limited thereto.

[0143] The SIRPα gene is one of the membrane proteins present on the cell membrane that separates the inside and outside of the cell, and is present in large quantities in macrophages. SIRPα weakens the phagocytic function of macrophages by binding to another membrane protein CD47 on cells that become phagocytic targets. That is, for example, cells that lack the SIRPα gene or whose expression is suppressed lose the ability to bind to CD47 present on the cell membrane of cancer cells, so their phagocytic function is not weakened.

[0144] The method for deleting the gene or inhibiting its expression can be a method commonly used in the art.

[0145] As one embodiment, nucleic acids that inhibit the expression of SIRPα gene can be cited. The nucleic acid can play a role at any stage such as the transcription level, post-transcriptional regulation level, protein translation level, and post-translational modification level of the SIRPα gene. Therefore, as a nucleic acid that inhibits the expression of the SIRPα gene, for example, nucleic acids that inhibit the transcription of the SIRPα gene (for example, antigene), nucleic acids that inhibit the processing of primary transcription products into mRNA, nucleic acids that inhibit the translation of mRNA into protein (for example, antisense nucleic acids, miRNA) or degrade mRNA (for example, siRNA, ribozymes, miRNA), etc. Substances that play a role at any stage can be used, but preferably substances that complementarily bind to mRNA and inhibit translation into protein or degrade mRNA.

[0146] The nucleotide sequence of the SIRPα gene is known, and sequence information can be obtained from, for example, public databases (eg, NCBI, EMBL, DDBJ, etc.).

[0147] As another preferred embodiment, nucleic acids that use genome editing to inhibit the expression of SIRPα gene can be cited. Specifically, the nucleic acid that inhibits the expression of SIRPα gene can be a nucleic acid that can inactivate (knock out) SIRPα gene. As such nucleic acids, nucleic acid sequences that encode nucleic acid sequence recognition modules (e.g., CRISPR / Cas9, ZF motifs, TAL effectors, etc.) that can specifically recognize a partial nucleotide sequence in the gene as a target and a nucleic acid that introduces a double-strand break (DSB) into the gene inside or near the target sequence can be cited. Nucleic acids. After DSB is introduced, the gene can be knocked out by repairing erroneous insertion or deletion mutations based on non-homologous end joining (NHEJ). Alternatively, by combining with a targeting vector that inserts a marker gene (e.g., a reporter gene such as a fluorescent protein gene, a drug resistance gene, etc.) into the gene sequence, homologous recombination (HR) repair can also be used to perform gene knockout.

[0148] Therefore, macrophage-like cells can be prepared by deleting or inhibiting the expression of genes that inhibit macrophage-like functions.

[0149] 2. Step of expressing a gene that increases proliferation

[0150] "Additional proliferative gene" refers to a gene that has the function of promoting cell proliferation by being introduced into cells and expressed in cells, and genes of various factors can be cited. In this specification, it is also called a proliferation gene. Specifically, genes such as c-MYC, BMI1, MDM2, or their homologs or orthologs of any biological species can be cited, but it is not limited to this. The biological species is selected to be a biological species that is suitable for the animal species from which the cells to be introduced are derived. It is preferred to introduce and express at least one, preferably two, and more preferably all three genes selected from c-MYC, BMI1 and MDM2.

[0151] Sequence information of proteins, genomic DNA or cDNA of various biological species such as "c-MYC", "BMI1" and "MDM2" can be obtained from information sources known to those skilled in the art, such as the Gene database of the National Center for Biotechnology Information (NCBI).

[0152] In the present invention, expression means that a desired gene or protein is produced in a cell and, in some cases, is secreted outside the cell to exert the function of the gene or protein.

[0153] In the present invention, "introducing a gene" means introducing a desired gene into a specific cell, producing the gene or a protein encoded by the gene in the cell, and optionally secreting the gene outside the cell to exert the function of the protein.

[0154] One embodiment of the method for producing pMAC of the present invention is the following method (embodiment 1), which comprises: (1) 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

[0155] (2) A step of causing the MAC obtained in step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC) (step 2).

[0156] In step 1 of method 1, the above-mentioned "1. step of deleting or inhibiting the expression of a gene that inhibits macrophage-like function" is performed 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, macrophage-like cells (MAC) can be obtained by inducing differentiation into myeloid cells (hereinafter also referred to as "myeloid differentiation") of pluripotent stem cells in which a gene that inhibits macrophage-like function is deleted or its expression is inhibited, such as pluripotent stem cells in which the SIRPα gene is deleted or its expression is inhibited (particularly iPS cells).

[0157] Myeloid cells refer to the general term for monocytes, macrophages, dendritic cells and progenitor cells belonging to the cell lineages that differentiate into these cells in the white blood cells differentiated from stem cells. Myeloid differentiation is carried out by causing a gene that inhibits macrophage-like function to be deleted or whose expression is suppressed to cause a pluripotent stem cell to undergo myeloid differentiation, so that the differentiated myeloid cells become macrophage-like cells. As a method for obtaining macrophage-like cells (MAC) by causing a gene that inhibits macrophage-like function to be deleted or whose expression is suppressed to cause a pluripotent stem cell to undergo myeloid differentiation, specifically, the following method can be cited.

[0158] (i) culturing embryoid bodies (EBs) induced from pluripotent stem cells on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF and TPO, or

[0159] (ii) Embryoid bodies (EBs) induced from the above pluripotent stem cells are cultured as a feeder-free monolayer in a medium containing BMP-4, VEGF and SCF, and then further cultured in a medium containing VEGF, TPO and GM-CSF.

[0160] In the above (i) and (ii), the pluripotent stem cells are pluripotent stem cells in which a gene that inhibits macrophage-like function is deleted or the expression of the gene is suppressed.

[0161] In step 2 of embodiment 1, the MAC obtained in step 1 is subjected to the above-mentioned "2. step of expressing a gene imparting proliferation property".

[0162] As another embodiment of the method for producing pMAC of the present invention, the following method (method 2) can be cited:

[0163] (A) a step of causing myeloid cells differentiated from pluripotent stem cells to express a gene that imparts proliferation properties to obtain proliferative myeloid cells (step A); and

[0164] (B) A step of obtaining proliferative macrophage-like cells (pMAC) by 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 (step B).

[0165] In step A of embodiment 2, the myeloid cells differentiated from pluripotent stem cells (myeloid cells derived from pluripotent stem cells) are subjected to the above-mentioned "2. step of expressing a gene imparting proliferative properties". Here, the pluripotent stem cells are preferably induced pluripotent stem cells or embryonic stem cells, and more preferably induced pluripotent stem cells. Myeloid cells endowed with proliferative properties can be obtained by the above-mentioned steps.

[0166] Specifically, the following methods can be used to induce differentiation from pluripotent stem cells into myeloid cells.

[0167] (i) culturing embryoid bodies (EBs) induced from pluripotent stem cells on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF and TPO, or

[0168] (ii) Embryoid bodies (EBs) induced from the above pluripotent stem cells are cultured as a feeder-free monolayer in a medium containing BMP-4, VEGF and SCF, and then further cultured in a medium containing VEGF, TPO and GM-CSF.

[0169] In step B of embodiment 2, the proliferative myeloid cells obtained in step A are subjected to the above-mentioned "1. step of deleting or inhibiting the expression of a gene that inhibits macrophage-like function". pMAC can be obtained by inducing proliferative myeloid cells to become macrophage-like cells by deleting or inhibiting the expression of a gene that inhibits macrophage-like function, such as SIRPα gene.

[0170] The "embryoid body" in the present invention refers to a cell mass formed by the adhesion of undifferentiated pluripotent stem cells. The pluripotent stem cells that form the cell mass by utilizing the close cell-cell interactions in the cell mass are similar to the embryo in the developmental stage of forming germ layers after implantation in terms of the restricted differentiation spectrum of cells.

[0171] The size of the embryoid body in the present invention is not particularly limited, and the diameter is preferably 50 to 1000 μm, more preferably 50 to 500 μm, and particularly preferably 50 to 200 μm. If the size of the embryoid body is too large than the above range, the oxygen supply to the inside of the embryoid body becomes insufficient, and cell necrosis may occur. If the size of the embryoid body is too small than the above range, the embryoid body is not formed and a single cell may cause cell death due to apoptosis. Mouse embryoid bodies are sometimes cultured using mouse mesenchymal stem cell line C3H10T1 / 2 cells or mouse bone marrow-derived matrix OP9 cells as feeder layer cells. Human embryoid bodies can be cultured without using feeder layer cells.

[0172] The "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 and their homologs or orthologs of any biological species that have the activity of inducing differentiation from human or non-human mammalian pluripotent stem cells into myeloid cells (MCs).

[0173] For the "VEGF", "SCF", "TPO", "BMP-4" and / or "GM-CSF" of the present invention, VEGF, SCF, TPO, BMP-4 and / or GM-CSF proteins of a biological species other than the biological species of the embryoid body cultured under conditions that have the same degree of differentiation-inducing activity as the "VEGF", "SCF", "TPO", "BMP-4" and / or "GM-CSF" of the biological species of the cultured embryoid body, or mutants of VEGF, SCF, TPO, BMP-4 and / or GM-CSF proteins of a biological species the same as the biological species of the cultured embryoid body can be used.

[0174] In the present specification, Flt-3L, IL-3 and / or bFGF may also be used for 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 information sources known to those skilled in the art, such as the gene database of the National Center for Biotechnology Information (NCBI). In addition, as genetic recombinant protein reagents, for example, recombinant human Flt-3 ligand / FLT3L protein (308-FKN), recombinant human IL-3 protein (203-IL), recombinant human FGF basic / FGF2 / bFGF (145aa) protein (3718-FB), recombinant human VEGF 165 protein (293-VE), recombinant human SCF protein (255-SC), recombinant human Thrombopoietin protein (288-TP), recombinant human BMP-4 protein (314-BP), and recombinant human GM-CSF protein (215-GM) can also be purchased from R&D Systems (https: / / www.rndsystems.com / ).

[0175] The sequence information of various biological species of the "VEGF", "SCF", "TPO", "BMP-4" and / or "GM-CSF" proteins, genomic DNA or cDNA of the present invention can be obtained from information sources known to those skilled in the art, such as the gene database of the National Center for Biotechnology Information (NCBI).

[0176] It should be noted that the above-mentioned genes or proteins are preferably homologous to the pluripotent stem cells used. For example, when the pluripotent stem cells are of human origin, VEGF, SCF, TPO, BMP-4, GM-CSF, Flt-3L, IL-3 and / or bFGF of human origin are preferably used, and when the pluripotent stem cells are of mouse origin, the same genes or proteins of mouse origin are preferably used.

[0177] In the present invention, "a medium containing substance X" refers to a medium to which an exogenous substance X is added or a medium containing exogenous substance X, and "a medium not containing substance X" refers to a medium to which no exogenous substance X is added or a medium not containing exogenous substance X. Here, "exogenous substance X" refers to a substance X that is exogenous to cells or tissues cultured in the medium, and does not include endogenous substance X produced by the cells or tissues.

[0178] For example, "a medium containing VEGF" refers to a medium to which exogenous VEGF is added or a medium containing exogenous VEGF. The dosage of VEGF is sometimes affected by other growth factors, etc., and is preferably 0.2ng / mL to 200ng / mL, more preferably 2ng / mL to 100ng / mL, and particularly preferably 5ng / mL to 50ng / mL. "A medium not containing VEGF" refers to a medium to which exogenous VEGF is not added or a medium not containing exogenous VEGF.

[0179] Similarly, for SCF, TPO, BMP-4, and GM-CSF, a culture medium containing SCF, TPO, BMP-4, and / or GM-CSF also refers to a culture medium to which exogenous SCF, TPO, BMP-4, and / or GM-CSF is added or a culture medium containing exogenous SCF, TPO, BMP-4, and / or GM-CSF. Therefore, the dosage of SCF, TPO, BMP-4, and / or GM-CSF is sometimes affected by other growth factors, etc., and 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, it is more preferably 5 ng / mL to 15 ng / mL. In the case of SCF, BMP-4 and GM-CSF, it is more preferably 5 ng / mL to 70 ng / mL.

[0180] The “medium not containing SCF, TPO, BMP-4 and / or GM-CSF” refers to a medium to which exogenous VEGF is not added or a medium not containing exogenous SCF, TPO, BMP-4 and / or GM-CSF.

[0181] As a culture medium for mouse embryoid bodies in the present invention, αMEM supplemented with 20% fetal bovine serum (FBS) can be used. As feeder cells, mouse mesenchymal stem cell line C3H10T1 / 2 cells or mouse bone marrow-derived matrix OP9 cells can be used. As a culture medium for human embryoid bodies, a complete synthetic culture medium StemFit (Ajinomoto) can be used.

[0182] As the culture medium, αMEM supplemented with 20% FBS or the like can be used.

[0183] The "feeder layer cells" in the present invention refer to other cell types used to adjust the culture conditions for the undifferentiated maintenance culture and differentiation induction culture of iPS cells. Feeder layer cells are used by stopping proliferation by treatment with mitomycin C or irradiation. Mouse bone marrow-derived stromal cells OP9, mouse-derived mesenchymal stem cell line cells C3H10T1 / 2, etc. can be used.

[0184] In the present invention, "stacking culture" refers to inoculating target iPS cells and embryoid bodies into a culture system of a feeder cell layer that has been cultured in a specified culture container for co-culture. In the present invention, "monolayer culture" refers to culturing a single cell type by adhering to a tissue culture container.

[0185] The feeder-free monolayer culture in the present invention refers to inoculating the target iPS cells or embryoid bodies into a culture container for static culture.

[0186] In addition, as one embodiment of the present invention, the present invention includes a step of forming an embryoid body from pluripotent stem cells using the following method. That is, it includes:

[0187] (i) inoculating a single cell dispersion of pluripotent stem cells into a cell cluster formation culture vessel to form an embryoid body (EB); and

[0188] (ii) The container has fine spherical holes at the bottom, and there is no flat surface between adjacent spherical holes.

[0189] "Embryoid formation" refers to the formation of uniform cell clusters by dissociating pluripotent stem cells that proliferate in an undifferentiated state to obtain a single cell dispersion of pluripotent stem cells, and subjecting the dispersion to suspension culture or static culture under conditions where pluripotent stem cells adhere to each other. Confirmation of embryoid formation is performed by visually observing the presence of living cells using a microscope.

[0190] In this specification, "suspension culture" means culturing cells or cell clusters under conditions where they are kept in a suspended state and do not adhere to a base material such as a bottom surface or a wall surface of a culture container.

[0191] The incubator used during suspension culture is not particularly limited as long as it can carry out "suspension culture", and can be appropriately determined for those skilled in the art. As such incubator, for example, flask, flask for tissue culture, culture dish, petri dish, culture dish for tissue culture, porous culture dish, microporous culture plate, microwell plate, micropore, porous culture plate, multi-well plate, chamber slide, watch glass, tube, square dish, culture bag or roller bottle can be enumerated. In order to be able to carry out suspension culture, these incubators are preferably cell non-adhesive. Crop cell non-adhesive incubator, the surface of the incubator can be used without artificial treatment (for example, coating treatment based on extracellular matrix etc.) for the purpose of improving the adhesion of the cell as a culture device etc.

[0192] In the present invention, it is preferred to rapidly aggregate pluripotent stem cells to form embryoid bodies derived from pluripotent stem cells. If a cell mass of pluripotent stem cells is formed in this way and then transferred to a medium supplemented with VEGF at a concentration of 50 ng / mL for culture, the cells in the cell mass can be differentiated into a cell lineage that becomes a myeloid cell, similar to the blood islands of the yolk sac.

[0193] As experimental operations for forming embryoid bodies, for example, there can be mentioned methods of enclosing cells in a small space using culture plates (e.g., 96-well plates) with multiple small-caliber wells, cell cluster formation culture containers, and other culture containers, and methods of aggregating cells by short-term centrifugation using microcentrifuge tubes. In addition to rotary culture, shaking culture, and suspension culture, the culture conditions for aggregates of cells or pluripotent stem cells can also be such that the surface of the substrate in contact with the cells of the culture container is treated to be hydrophobic in advance to reduce or inhibit the adhesion of pluripotent stem cells to the substrate surface of the culture container, and static culture is performed under this condition.

[0194] The formation of embryoid bodies as cell clusters that serve as pluripotent stem cells and the differentiation of cells of the embryoid bodies into myeloid cells can be judged based on the following characteristics, including: 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 under culture, confirmation by tissue or cytochemical observation, and / or confirmation of the expression of differentiation and undifferentiation markers, their uniformity, control of the expression of differentiation markers, their synchronization, or the reproducibility of differentiation efficiency among embryoid bodies, but is not limited to these.

[0195] In the present invention, a marker possessed by a certain cell is "positive" when the cell is measured by flow cytometry, and the average fluorescence intensity ratio of the positive cells to the negative control is 10 times or more, which is recorded as +, 2 times or more and less than 10 times, which is recorded as low, and less than 2 times, which is recorded as -.

[0196] In addition, in the present invention, whether the expression of a certain gene in a certain cell is high or low means that the expression level of the gene is measured by the real-time PCR method, and a value of 1 or more relative to the expression level of β-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HPRT 1 (hypoxanthinephosphoribosyl transferase 1) or β2-microglobulin as a housekeeping gene is considered high, and a value of less than this value is considered low.

[0197] The single cell dispersion in the present invention refers to a dispersion 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 then centrifuging to obtain a target cell mass, and resuspending the target cell mass with a desired culture medium or the like by pipetting.

[0198] The cell cluster formation culture container in the present invention refers to a container that can form cell aggregates without the seeded cells adhering to the container during culture. Preferably, the container has fine spheroid holes on its bottom as a culture surface, and there is no flat surface between adjacent spheroid holes, and the adjacent spheroid holes are separated by a curved surface inclined toward any spheroid hole, and since the curved surface is subjected to a surface treatment for inhibiting cell adhesion, the cells seeded in the culture container almost all fall into any spheroid hole.

[0199] As an example of a container, there can be mentioned a container in which spheroid holes with a diameter of about 400 to 800 μm and a depth of 100 to 800 μm are evenly arranged on the culture surface (bottom) without gaps to the wall surface. Preferably, the spheroid holes are 400 to 500 μm in diameter and 50 to 200 μm in depth. More preferably, the spheroid holes are containers with a diameter of about 500 μm and a depth of about 400 μm, containers with a diameter of about 800 μm and a depth of about 400 μm, or containers with a diameter of about 800 μm and a depth of about 300 μm. Most preferably, the spheroid holes are containers with a diameter of about 400 μm and a depth of 100 to 200 μm.

[0200] As a container, it is preferred that the spheroid holes are processed uniformly from the culture surface (bottom) to the wall surface without gaps. The reason is that in the case of such a container, there is no flat surface between adjacent spheroid holes in the container, and the inoculated single cell suspension falls into any spheroid hole, so that a uniform aggregate can be formed. More preferably, it is a microfabricated cell culture container (EZSPHERE (registered trademark)) manufactured by AGC.

[0201] pMAC can be produced by any of the methods of Mode 1 and Mode 2, and is preferably produced by the method of Mode 1. By performing a step of expressing a gene that adds proliferation after a step of deleting or inhibiting the expression of a gene that inhibits macrophage-like function, a cell population containing pMAC with high purity and few cells other than the target can be stably produced. The more specific steps of Mode 1 are as follows.

[0202] (1) a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells (step 1-1),

[0203] a step of forming embryoid bodies (EBs) by seeding the pluripotent stem cells obtained in step 1-1 in which the gene suppressing macrophage-like function is deleted or the expression is suppressed in a cell cluster formation culture container having fine spheroid holes in the bottom and no flat surface between adjacent holes (step 1-2);

[0204] a step of obtaining macrophage-like cells (MAC) from the EBs obtained in step 1-2 by the following method (i) or (ii) (step 1-3),

[0205] (i) culturing the EBs in a medium containing VEGF on a feeder layer of cells, and then further culturing them in a medium containing VEGF, SCF and TPO, or

[0206] (ii) culturing the EBs in a culture medium containing BMP-4, VEGF and SCF without feeder cells as a monolayer, and then further culturing them in a culture medium containing VEGF, TPO and GM-CSF,

[0207] (2) A step of causing the MAC obtained in step 1-3 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC) (step 2).

[0208] Here, the gene that inhibits macrophage-like function is the SIRPα gene.

[0209] The pMAC thus obtained has phagocytic ability similar to macrophages, but also has characteristics different from existing macrophages, such as cytokine-dependent proliferation.

[0210] <pMAC containing a chimeric antigen receptor and method for producing the same>

[0211] The present invention provides a pMAC (hereinafter also referred to as CAR-pMAC) comprising a chimeric antigen receptor (CAR) and a method for producing the same. In the method for producing CAR-pMAC of the present invention, the introduction of the CAR gene can be carried out, for example, before the gene that inhibits macrophage-like function is deleted or the expression is inhibited in pluripotent stem cells, or after the gene that inhibits macrophage-like function is deleted or the expression is inhibited in pluripotent stem cells, in addition to the introduction into the pMAC that is endowed with cytokine-dependent proliferation as described in the above-mentioned "method for producing pMAC of the present invention". The method for producing CAR-pMAC of the present invention includes a step of expanding and culturing the obtained CAR-pMAC.

[0212] As one embodiment of the method for producing CAR-pMAC of the present invention, the following method (embodiment I) can be cited. The method for producing CAR-pMAC comprises:

[0213] (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 (step 1);

[0214] (2) a step of causing the macrophage-like cells obtained in step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells (pMAC) (step 2); and

[0215] (3) Step of introducing a chimeric antigen receptor (CAR) (step 3).

[0216] Step 1 and Step 2 in this embodiment can be carried out in the same manner as Step 1 and Step 2 in Embodiment 1 of the above-mentioned "Method for producing pMAC of the present invention", respectively.

[0217] Another embodiment of the method for producing CAR-pMAC of the present invention is the following method (Embodiment II).

[0218] (A) a step of causing myeloid cells differentiated from pluripotent stem cells to express a gene that imparts proliferation to obtain proliferative myeloid cells (step A),

[0219] (B) a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in the proliferating myeloid cells obtained in step A, and then inducing differentiation to obtain pMAC (step B); and

[0220] (C) Step of introducing a chimeric antigen receptor (CAR) (step C).

[0221] Step A and step B in this embodiment can be carried out in the same manner as step A and step B in embodiment 2 of the above-mentioned "method for producing pMAC of the present invention", respectively.

[0222] As another embodiment of the method for producing CAR-pMAC of the present invention, the following method (embodiment III) can be cited. The method for producing CAR-pMAC comprises:

[0223] (1') a step of introducing a chimeric antigen receptor (CAR) into pluripotent stem cells (step 1'),

[0224] (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

[0225] (3′) A step of causing the CAR gene-transferred macrophage-like cells obtained in step 2′ to express a gene that enhances proliferation to obtain CAR gene-transferred proliferative macrophage-like cells (CAR-pMAC) (step 3′).

[0226] Step 2' and step 3' in this embodiment can be carried out in the same manner as step 1 and step 2 in embodiment 1 of the above-mentioned "method for producing pMAC of the present invention", except that the CAR gene is introduced into the target cells in advance.

[0227] As another embodiment of the method for producing CAR-pMAC of the present invention, the following method (Embodiment IV) can be mentioned:

[0228] (A') a step of introducing a chimeric antigen receptor (CAR) into pluripotent stem cells (step A'),

[0229] (B') a step of causing myeloid cells differentiated from the pluripotent stem cells into which the CAR gene has been introduced obtained in step A' to express a gene that adds proliferation properties, thereby obtaining proliferative myeloid cells into which the CAR gene has been introduced (step B'); and

[0230] (C') A step of obtaining CAR gene-transferred proliferative macrophage-like cells (CAR-pMAC) by deleting or suppressing the expression of a gene that inhibits macrophage-like function in the proliferative myeloid cells obtained in step B' (step C').

[0231] Step A' and step B' in this embodiment can be carried out in the same manner as step A and step B in embodiment 2 of the above-mentioned "method for producing pMAC of the present invention", except that the CAR gene is introduced into the target cells in advance.

[0232] Hereinafter, the introduction of the CAR gene into cells in step 3 of embodiment I and embodiment II, step 1′ of embodiment III, and step A′ of embodiment IV will be described in detail.

[0233] Chimeric Antigen Receptor (also referred to as CAR in this specification)

[0234] CAR is a structure that includes a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for the effector function of immune cells from the N-terminal side of the protein toward the C-terminal side. The CAR gene is a gene encoding the receptor. The extracellular domain contains an antigen recognition site that shows target-specific binding. The transmembrane domain is present between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain conducts the signal required for the effector function of immune cells. That is, when the extracellular domain binds to the target antigen, an intracellular signaling domain that can conduct the signal required for the activation of immune cells is used.

[0235] It should be noted that to date, there have been some reports on 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 in the present invention can be constructed with reference to these reports.

[0236] Introduction of CAR gene into pMAC

[0237] In the present invention, the introduction of the CAR gene is implemented by introducing the CAR gene into the desired cell using a CAR expression vector. Here, for the cells into which the CAR gene is introduced, for example, in addition to the pMAC provided by the present invention, pluripotent stem cells before or after the gene that inhibits macrophage-like function is deleted or the expression is inhibited can also be cited. The CAR expression vector refers to a nucleic acid molecule that can deliver a nucleic acid molecule encoding a CAR gene to a cell as a target, whether it is DNA or RNA. In addition, there is no particular limitation on the form, source, etc., and various types of vectors can be used. The vector can be a viral vector or a non-viral vector. As a viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes virus vector, a Sendai virus vector, a vaccinia virus vector, etc. can be cited. Among them, in retroviral vectors, lentiviral vectors, and adeno-associated viral vectors, the target gene integrated into the vector is integrated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to conventional methods or using a commercially available dedicated kit. Examples of 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, and artificial chromosome vectors.

[0238] The CAR expression vector contains an expression unit for expressing the CAR gene, which usually contains a promoter, a CAR gene, and a polyA tailing signal. The expression unit is composed of various biological, viral or arbitrary sequences, and also contains similar sequences and their modified units. As promoters that can be used in the CAR expression cassette, CAG promoter, CMV-IE (cytomegalovirus early gene source promoter), SV40ori, retrovirus LTRSRα, EF1α, β-actin promoter, etc. can be cited. As polyA tailing signal sequences, polyA tailing sequences of SV40, polyA tailing sequences of bovine growth hormone genes, and globulin polyA tailing sequences can be cited. In order to control the expression of the CAR gene by the promoter, the CAR gene is usually connected directly or via other sequences to the 3' end of the promoter, and a polyA tailing signal sequence is configured downstream of the CAR gene. The CAR gene is transcribed into messenger RNA (mRNA) by such an expression unit, and CAR is translated by the mRNA and presented to the cell surface.

[0239] The expression unit may also contain a detection gene (reporter gene, cell- or tissue-specific gene, selection marker gene, etc.) that can detect gene expression, an enhancer sequence for improving expression efficiency, a WRPE sequence, etc.

[0240] The detection gene is used to determine the success or failure of the introduction of the CAR expression vector, the efficiency, the detection of CAR gene expression or the determination of expression efficiency, the selection and sorting of cells expressing the CAR gene, etc. As detection genes, the neo gene that confers neomycin resistance, the kmr gene or nptII gene that confers kanamycin resistance (Bernd Reiss et al. EMBO J. 3 (1984), 3317-3322), the hph gene that confers hygromycin resistance (Blochlinger & Diggelmann, Mol Cell Bio 4: 2929-2931), the DHFR gene that confers methotrexate resistance (Bourouis et al., EMBO J. 2 (7)), etc. (these are marker genes); luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β-glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett.389 (1996), 44-47) or its mutants (EGFP, d2EGFP, etc.), tdTomato or its mutants (pTdTomato, etc.) and other fluorescent protein genes (the above are reporter genes); genes such as the epithelial growth factor receptor (EGFR) gene lacking the intracellular domain. The detection gene can be connected to the CAR gene via, for example, a bicistronic control sequence (e.g., an internal ribosomal recognition sequence (IRES)) and a sequence encoding a self-cleaving peptide. As a self-cleaving peptide, a 2A peptide (T2A) derived from Thosea asignavirus can be cited. Other self-cleaving peptides include, but are not limited to, 2A peptides derived from picornavirus (F2A), 2A peptides derived from foot-and-mouth disease virus (FMDV), 2A peptides derived from equine rhinitis virus A (ERAV), 2A peptides derived from porcine teschovirus (PTV-1), 2A peptides derived from rotavirus, insect virus, foot-and-mouth disease virus (Aphthovirus), or trypanosome virus, etc. Similar sequences, partially mutated sequences, etc. can also be mentioned.

[0241] The CAR gene expression vector prepared for gene introduction purposes is introduced into pMAC by conventional methods. In the case of a viral vector, it is introduced into cells by infecting the virus. In the case of a non-viral vector such as a plasmid, in order to introduce into cells, conventional methods such as electroporation, liposome method, calcium phosphate method, nuclear transfection method, etc. can be used, preferably using electroporation to introduce.

[0242] In order to improve the integration efficiency into the host chromosome, it is preferred to use the transposon method for gene introduction. The transposon method is one of the non-viral gene introduction methods. It can use the pairing of gene enzymes (transposases) and their specific recognition sequences to cause gene transposition and integrate any gene into the host chromosome. As a transposon method, for example, the piggyBac transposon method can be used. The piggyBac transposon method uses transposons isolated from insects (Fraser MJ et al., Insect Mol Biol. 1996 May; 5 (2): 141-51.; Wilson MH et al., Mol THER 2007 Jan; 15 (1): 139-45.), which can achieve high-efficiency integration into mammalian chromosomes. The piggyBac transposon method is actually used for gene introduction (for example, see Nakazawa Y, et al., J Immunother 32: 826-836, 2009; Nakazawa Y et al., J Immunother 6: 3-10, 2013, etc.).

[0243] The transposon method is not limited to the use of piggyBac. For example, it can also be the use of 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 2007Jun 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.) and other transposon methods.

[0244] The operation of gene introduction using the transposon method can be performed by conventional methods. For example, in order to use the piggyBac transposon method, a vector (transposase plasmid) carrying a gene encoding the piggyBac transposase and a vector (transposon plasmid) having a structure in which the CAR gene expression unit is sandwiched in the piggyBac inverted repeat sequence can be prepared, and these vectors are introduced into the target cells using various methods such as electroporation, nuclear transfection, liposome transfection, and calcium phosphate method.

[0245] As a treatment after CAR gene introduction, CAR-pMAC is expanded and cultured to a desired scale. The CAR-pMAC of the present invention can proliferate in a cytokine-dependent manner. Therefore, the method for producing CAR-pMAC of the present invention is preferably carried out in the presence of at least one, preferably two, cytokines selected from GM-CSF and M-CSF.

[0246] The culture medium used in the preparation of CAR-pMAC is not particularly limited, and the culture medium used in conventional cell culture, such as RPMI1640, MEM, X-VIVO, IMDM, DMEM, DC medium, OptiMEM, etc., can be used. The culture medium can be a culture medium to which serum (human serum, fetal bovine serum, etc.) is added according to conventional methods, or it can be a serum-free culture medium. Because of its high safety during clinical application and the fact that differences in culture efficiency are not easily caused by differences between serum batches, it is preferred to use a serum-free culture medium. As serum-free culture media, TexMACS TM (Miltenyi Biotec), AIMV (registered trademark) (Thermo Fisher Scientific), ALyS culture medium (Cell Science Research Institute Co., Ltd.), etc. can be cited. When using serum, autologous serum, that is, serum taken from an individual from whom CAR-expressing immune cells are derived (more specifically, a patient who receives a cell population obtained by the manufacturing method disclosed in the present invention), can be used, or it can be artificial serum. In the present invention, autologous serum is preferably used. As the basal medium, any basal medium suitable for cell culture may be used, and the above-mentioned TexMACS TM (Miltenyi Biotec), AIMV (registered trademark), ALyS culture medium (Cell Science Institute Co., Ltd.) may be used. Other culture conditions may be general conditions as long as they are suitable for cell survival and proliferation. For example, culture in a CO2 incubator (CO2 concentration 5%) set at 37°C, hypoxic culture (oxygen concentration 0 to 20%, preferably 0 to 10%, more preferably 1 to 5%), etc. may be mentioned.

[0247] By expanding the culture of CAR-pMAC, a cell population containing CAR-pMAC of sufficient quantity and quality for clinical use can be obtained.

[0248] Recover CAR-pMAC after expansion culture. The recovery operation can be performed using conventional methods. For example, it can be recovered by pipetting, centrifugation, etc. In a preferred embodiment, before the recovery operation, a step of culturing the cells after expansion culture in the presence of a stimulating substance is performed. According to this step, expansion culture can be effectively performed, and there is also the advantage of improving cell survival rate. If necessary, the CAR-pMAC population after expansion culture can be subjected to a cell separation process such as magnetic bead separation. By implementing the cell separation process, the purity of CAR-pMAC with a higher effect can be improved.

[0249] The cell population containing CAR-pMAC produced by the method of the present invention can be used to treat diseases related to tumors or cancers, especially cancers expressing the target antigen of the CAR-expressing immune cells. Diseases related to tumors or cancers can be solid tumors or blood tumors. Specific cancers include various B-cell lymphomas (follicular malignant lymphoma, diffuse large B-cell malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), myelodysplastic tumors, myelodysplastic / marrow hyperplastic tumors (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, neuroblastoma, brain tumor, Ewing sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, bone and soft tissue sarcoma, renal cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, colorectal cancer, etc., but are not limited thereto. In a preferred embodiment, the cancer is a solid tumor. Examples of solid tumors include neuroblastoma, brain tumors, Ewing sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcoma, renal cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, and colorectal cancer.

[0250] In addition, the cell population containing CAR-pMAC produced by the method of the present invention can be used for the treatment of neurodegenerative diseases, inflammatory diseases, cardiovascular diseases, fibrotic diseases and amyloidosis. As "neurodegenerative diseases", Tau disease, α-synuclein disease, presenile dementia, senile dementia, Alzheimer's disease, chromosome 17-related Parkinson's disease (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, Lewy body dementia, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallewarden-Spatz disease, polyglutamine disease, trinucleotide repeat disease, prion disease, etc. can be cited. Examples of "inflammatory diseases" include systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infection, hereditary periodic fever, malignant tumor, systemic vasculitis, cystic fibrosis, bronchiectasis, bullous dermatosis, cyclical neutropenia, acquired or inherited immunodeficiency, injection-drug use and acne vulgaris, Muckwell syndrome (MWS), familial Mediterranean fever (FMF), etc. Examples of "cardiovascular diseases" include atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, heart failure, etc. Examples of the “fibrotic disease” include pulmonary fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis, and skin fibrosis. Examples of “amyloidosis” include primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), other familial amyloidosis, β-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 amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and lysozyme amyloidosis.

[0251] The cell population containing CAR-pMAC produced by the method of the present invention is administered in a therapeutically effective amount appropriately determined according to the age, 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 can be administered at 1×10 4 ~1×10 10 The administration route is not particularly limited, and the administration may be into the tumor, around the tumor, into the ventricle, into the vein, into the artery, into the portal vein, into the skin, subcutaneously, into the muscle, or into the peritoneal cavity. The cell population disclosed herein may be administered systemically or topically, and as a topical administration, direct injection into the target tissue, organ, or organ may be cited. The dosage regimen is appropriately determined based on the age, weight, body surface area, symptoms, etc. of the subject, and may be administered as a single dose or may be administered multiple times continuously or periodically.

[0252] 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. As a "pharmaceutically acceptable carrier", conventional excipients, adhesives, buffers, water for injection, isotonic agents, preservatives, pain-relieving agents and other ingredients commonly used in medicine can be exemplified. In addition to the cell population to be administered to the subject, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained for the purpose of protecting the cells, antibiotics may be contained for the purpose of preventing bacteria from mixing in, and various ingredients (vitamins, cytokines, growth factors, steroids, etc.) may be contained for the purpose of activating, proliferating or inducing differentiation of cells. Pharmaceutical compositions can be prepared by conventional methods.

[0253] Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited in any way. The reagents and materials used, as long as they are not particularly limited, can be obtained commercially, or can be prepared according to known literature etc. In addition, those skilled in the art will appreciate that as long as they have the same effect and function, they can be substituted.

[0254] Example

[0255] Example 1: Preparation of human iPSCs

[0256] Human iPSCs were prepared according to the method described in Kitayama, S. et al. (Stem Cell Reports. 6: 213-227, (2016)). The maintenance culture of human iPSCs was carried out using a polystyrene tissue culture plate coated with iMatrix511 (NIPPO) using the regenerative medicine culture medium StemFit AK02N (AJINOMOTO HEALTHY SUPPLY). The culture medium was replaced every 1 to 2 days, and depending on the proliferation of the cells, the cells were treated with the cell detachment solution TrypLESelect (Life Technologies) for 4 to 5 minutes once a week, recovered as a single cell dispersion, and inoculated in a culture vessel of an appropriate size for continued culture. The inoculated iPSCs were cultured overnight in the presence of 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), a Rock inhibitor, and the culture medium was replaced the next day to remove Y-27632.

[0257] Example 2: Preparation of mouse iPSCs

[0258] Mouse iPSCs were prepared according to the method described in Araki, R. et al. (Nature, 494: 100-104. (2013)). Mouse iPSCs were maintained in ES cell medium ESM (DMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 15% KSR (Life Technologies)) supplemented with 55 μM 2-mercaptoethanol (Life Technologies) and 10 4 The cells were cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) (ReproCELL) seeded on 6-well tissue culture plates using complete ESM (cESM) medium prepared with 100 U / mL leukemia inhibitory factor (LIF) (Merck, trade name ESGRO mLIF).

[0259] Feeder cells were prepared by seeding MEFs (ReproCELL) whose cell proliferation was stopped by mitomycin C treatment on a gelatin-coated culture dish, and used for maintenance culture of mouse iPSCs from the day after MEF attachment.

[0260] Example 3: Deletion of SIRPα gene by genome editing

[0261] Using the sequence information from the database of the National Center for Biotechnology Information (NCBI), single-stranded guide RNAs for human SIRPα and mouse SIRPα were synthesized by nucleic acid synthesis.TM Recombinant Cas9 protein (Takara Bio) was mixed to prepare Cas9 protein-guide RNA complex (RNP), added to iPSC single cell dispersion, and RNA was introduced into cells using electroporation. Based on sequence analysis of the target genomic DNA sequence of the guide RNA and expression analysis based on flow cytometry using differentiated cells obtained by differentiation induction in Example 5, iPSC clones with SIRPα gene deletion were obtained.

[0262] Example 4: Preparation of iPSC-derived embryoid bodies

[0263] The embryoid bodies (hereinafter referred to as human EBs) derived from human iPSCs (wild type or SIRPα-deficient) were prepared by inoculating human iPSCs of a predetermined density prepared with StemFit containing 10 μM Y-27632 in a 6-well plate-type cell cluster formation culture vessel EZ SPHERE SP (AGC TECHNO GLASS). One to four days after the formation of embryoid bodies with a diameter of 50 to 200 μm, differentiation induction culture was performed as described in Example 5 below.

[0264] The preparation of embryoid bodies (hereinafter referred to as mouse EBs) derived from mouse iPSCs (wild type or SIRPα-deficient) was carried out by inoculating mouse iPSCs of a predetermined density prepared with complete medium cESM into a 6-well plate type EZ SPHERE. One to four days after the formation of embryoid bodies with a diameter of 50 to 200 μm, differentiation induction culture described in Example 5 was performed.

[0265] Example 5: Differentiation induction of embryoid bodies into myeloid cells

[0266] (1) Preparation of feeder cells

[0267] Mouse-derived culture cell lines C3H10T1 / 2 or OP9 were inoculated in a culture dish coated with gelatin and used the next day. Feeder cells used in the differentiation induction of human iPSC-derived embryoids were used after irradiation with 60 Gy to stop proliferation. C3H10T1 / 2 cells were cultured with BME medium containing 10% FBS (Life Technologies), and OP9 cells were cultured with αMEM medium containing 20% ​​FBS (Life Technologies).

[0268] (2) Differentiation induction culture of mouse iPSCs on feeder cells

[0269] Mouse EBs prepared from mouse iPSCs (wild type or SIRPα-deficient) were cultured on feeder cells OP9 for 7 to 10 days using differentiation induction medium αMEM containing 20% ​​FBS, and then recovered and inoculated on freshly prepared OP9 feeder cells. Differentiated cells including myeloid cells were obtained by culturing for 7 to 10 days in the presence of 55 μM 2-mercaptoethanol and 50 ng / mL GM-CSF.

[0270] (3) Feeder-free cell differentiation induction culture of human iPSCs

[0271] Human EBs prepared from human iPSCs (wild type or SIRPα-deficient) were cultured on polystyrene tissue culture plates coated with iMatrix511 and Retronectin (Takara Bio), and then cultured for 5 to 7 days in the presence of 50 ng / mL bFGF, 50 ng / mL BMP-4, 50 ng / mL VEGF, and 50 ng / mL SCF using differentiation induction medium X-VIVO15 (Lonza). Then, cultured for 7 to 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. 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 it is sometimes affected by other growth factors, any two concentrations in the range of 0.2ng / mL to 200ng / mL can be used as the upper and lower limits. For example, any two concentrations of 0.2ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL, and 100ng / mL to 200ng / mL can be used as the upper and lower limits.

[0272] Example 6: Production of proliferating macrophage-like cells pMAC based on introduction of proliferation genes

[0273] (1) Preparation of lentiviral vector

[0274] The cDNAs of human c-MYC, BMI1, and MDM2 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. The genes introduced into the lentiviral vector pSL or CSII-EF-MSC-IRES2-Veneus prepared above were introduced into 293T cells as packaging cells (virus-producing cells) using the liposome transfection method, respectively, with the packaging construct pCAG-HIVgp and the envelope and Rev construct pCMV-VSV-G-RSV-Rev.

[0275] Three days after the gene was introduced into 293T cells, the cell culture medium was recovered, filtered through a filter with a pore diameter of 0.45 μm, and concentrated and recovered using Lenti-X lentivirus concentration reagent (Clontech). The recovered recombinant virus particles were suspended in DMEM solution, dispensed into cryopreservation tubes, and stored at -150°C.

[0276] (2) Imparting proliferation ability by introducing proliferation genes into iPSC-derived myeloid cells

[0277] The myeloid cells prepared in the previous item were cultured in a 48-well tissue culture plate, and in the case of mouse myeloid cells, a lentiviral suspension expressing c-MYC was added thereto at the same time for infection. In addition, in the case of human myeloid cells, a lentiviral suspension expressing c-MYC, BMI1 and MDM2 was added at the same time for infection. From the second day after the gene was introduced, the culture medium was added according to the proliferation of the cells, and the culture scale was expanded. αMEM containing 20% ​​FBS was used as the culture medium for the myeloid cells after virus infection, and the infected cells were cultured in the presence of 50ng / mL GM-CSF and 50ng / mL M-CSF. The cells into which the gene was introduced continued to proliferate for more than 3 months. In each embodiment of the present specification, the proliferative macrophage-like cells derived from wild-type iPSCs are referred to as pMAC, and the cells derived from pluripotent stem cells in which the SIRPα gene that inhibits macrophage-like function is deleted 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 in which a gene suppressing macrophage-like function was deleted.

[0278] (3) Preparation of mouse macrophages

[0279] In order to prepare organism-derived macrophages from C57BL / 6 mice, bone marrow-derived monocytes and peritoneal macrophages were collected. Bone marrow-derived monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum in the presence of 50 ng / mL M-CSF for 4 days on a 6-well tissue culture plate to induce bone marrow-derived macrophages M0. Then, bone marrow-derived macrophages M1 were induced by culturing in the presence of 20 ng / mL interferon-γ (FUNAKOSHI) and 50 ng / mL lipopolysaccharide (Fujifilm Wako Pure Chemical Industries, Ltd.) for 24 hours. 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. In addition, 2 mL of thioglycollate medium (Merck) was intraperitoneally administered to mice, and macrophages in the peritoneal cavity were collected 3 days after the administration.

[0280] (4) Gene expression profile of mouse pMAC

[0281] RNAseq analysis (biological species: mouse (Mus musculus), reference genome mm10) was performed on pMACs prepared from iPS cells and macrophages derived from organisms as a whole transcriptome analysis to quantify the expression of transcripts. Total RNA was extracted from each cell using the QIAGEN RNeasymini kit, and the strand-specific library preparation method (dUTP method) was used with the NEBNext(R) Poly(A) mRNA Magnetic Separation Module (Model E7490), NEBNext(R) Ultra TM IIDirectionalRNA Library Prep Kit (model E7760) was used to prepare the sequence library by PCR amplification using primers containing the Index sequence. The base sequence of the sample for library preparation was obtained using the next-generation sequencer NovaSeq 6000 (illumina), with a read length of PE150 (150bp×2 double-end sequencing), a data volume of 4G bases per sample, and a read count of 26.7Mreads (13.3M pairs) per sample.

[0282] Using the acquired data, informatics analysis was performed and principal component analysis (PCA) was performed. In order 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 error rate of sequence information) of R1 (Read1) and R2 (Read2), and it was confirmed that there were no abnormalities (completely normal, quality score>28). Next, the software Trimmomatic (version 0.38) was used to trim the sequence reads according to the set values ​​(ILLUMINACLIP 2:30:10, LEADING=20, TRAILING=20, SLIDINGWINDW=4:15, MINLEN=36). In order to obtain data of good quality, in view of the results of FastQC quality confirmation, the ends of the reads with low quality, the adapter sequences, short reads, etc. were trimmed. Next, the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0), and the mapping rate of the reads was calculated, confirming that all samples exceeded 98%. Next, the raw read counts mapped to the known exon regions of each gene were calculated using the software featureCounts (version 1.6.3). Furthermore, the mapped fragments were counted, and the fragments per kilobase of exon per million mapped reads (Fragments Per Kilobase of exon per Million mapped fragments, FPKM) value, the upper quartile of FPKM (Fragments Per Kilobase of Transcript per Million mapped reads upper quartile, FPKM-UQ) value, and the transcripts per kilobase of transcript per million mapped reads (Transcripts Per Million, TPM) value were calculated. The software stats (version 3.6.1) was used to perform principal component analysis of the samples for all genes tested based on the raw reads, FPKM values, and TPM values. The index reflecting the most data by the component with the largest deviation of each data is set as principal component (PC) 1 on the horizontal axis, and the index reflecting the second most data by the component with the second largest deviation is set as PC2 on the vertical axis to plot each sample to create a plot data ( Figure 1 ). It was found that mouse pMAC was plotted in the range of PC1 (-100 to -75) and PC2 (-75 to -50), and existed in a region different from monocytes and macrophages derived from other organisms.

[0283] (5) Surface antigen analysis of mouse pMAC

[0284] Mouse bone marrow-derived monocytes and 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) at 37°C for 15 minutes and collected by pipetting, and bone marrow-derived macrophages M1 were treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes and collected by pipetting. The cells were stained with anti-CD11b antibody, anti-F4 / 80 antibody, anti-CD16 / 32 antibody, anti-CD64 antibody, anti-I-A / E antibody, anti-CD206 antibody, anti-CD163 antibody, anti-Ly6C antibody, anti-CD80 antibody, anti-CD86 antibody, anti-SiglecF antibody, anti-SIRPα antibody, anti-TLR2 antibody, anti-TLR4 antibody, anti-CD124 antibody, anti-CCR1 antibody, anti-CCR2 antibody, anti-CCR3 antibody, anti-CCR4 antibody, anti-CCR5 antibody, anti-CXCR2 antibody and anti-CXCR4 antibody or isotype control antibody. Then, the cells were washed twice with PBS containing 2% FBS or PBS. The washed cells were analyzed using a flow cytometer analyzer (trade name: BD Accuri C6 FlowCutometer, manufactured by Beckton Dickinson). The results of surface antigen analysis based on flow cytometry after antibody staining are shown in FIG2. The results of this analysis revealed that mouse pMAC has properties of both bone marrow-derived macrophages M1 and M2 used as a comparative control.

[0285] (6) Morphology of mouse pMAC

[0286] The pMAC and pMAC-SIRPα-KO mice did not show changes in cell morphology caused by SIRPα deficiency, and maintained Figure 3 The myeloid cells shown exhibit an irregular morphology with processes.

[0287] Example 7: Preparation of iPSC-derived macrophage-like cells into which chimeric antigen receptors are introduced (CAR-pMAC)

[0288] (1) Preparation of lentiviral vector

[0289] The tdTomato gene and / or cDNA of a chimeric antigen receptor (CAR) having a GC33scFv domain and a CD3ζ domain were synthesized by gene synthesis, and inserted into the lentiviral vector pSL in the same manner as in Example 6 (1), and viral particles were prepared. Figure 4 The plasmid map and CAR gene expression construct used to prepare the lentiviral vector are shown in . The recovered recombinant virus particles were suspended in DMEM solution, dispensed into cryovials, and stored at -150°C.

[0290] (2) Introduction of CAR gene into iPSC-derived pMAC (human and mouse)

[0291] The pMAC or pMAC-SIRPα-KO cells, which are human or mouse proliferating macrophage-like cells prepared in Example 6 (2), were cultured in a 48-well tissue culture plate, and a lentivirus suspension expressing GC33 CAR was added thereto for infection.

[0292] From the second day after gene introduction, culture medium was added according to cell proliferation, and the culture scale was expanded. The culture medium used was αMEM containing 20% ​​FBS, and culture was performed in the presence of 50ng / mL GM-CSF and 50ng / mL human M-CSF. It should be noted that human GM-CSF was used for human pMAC and mouse GM-CSF was used for mouse pMAC.

[0293] (4) Effects of genetic manipulation of mouse pMAC on cell proliferation

[0294] After gene introduction, the cells were cultured for more than 2 weeks, and then 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 the presence of GM-CSF and M-CSF was investigated by MTT analysis. pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were recovered and seeded in 96-well tissue culture plates (3 × 10 3Cells were cultured at 100 μg / mL (100 μg / mL) per well (cells / well). The proliferation rates were then compared between the culture medium containing GM-CSF and M-CSF and the culture medium without GM-CSF and M-CSF. 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 from the start of culture in a 96-well tissue culture plate, and the metabolized formazan was measured using the absorbance at a wavelength of 595 nm 4 hours later. The amount of metabolized formazan is proportional to the number of cells at the time of MTT reagent addition, i.e., the rate of cell proliferation.

[0295] Figure 5 The results of the MTT assay of various mouse pMACs are shown. The proliferation of pMAC requires about 50 ng / mL of GM-CSF or M-CSF in the culture medium. In addition, the combined use of GM-CSF and M-CSF has the effect of promoting proliferation. The deletion of the SIRPα gene and the introduction of the CAR gene for the production of pMAC did not affect cell proliferation, indicating that CAR-pMAC has the same cell proliferation ability as pMAC.

[0296] (5) Analysis of cell surface antigens of mouse CAR-pMAC

[0297] The expression of tdTomato, GC33ζCAR, and SIRPα in mouse pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO was investigated by flow cytometry. The nucleic acid sequence at the 3' end of the GC33ζCAR cDNA integrated into the lentiviral vector was expressed as a fluorescent protein, tdTomato, via the T2A sequence. In addition, FITC-labeled human glypican 3 (hGPC3), which is the target antigen of CAR, was added to the cell sample. Thus, the expression of CAR can be analyzed by flow cytometry using the fluorescence of tdTomato and hGPC3-FITC as indicators. Figure 6 The results of investigating the expression of GC33ζCAR using flow cytometry in pMAC, CAR-pMAC, pMAC-SIRPα-KO, and CAR-pMAC-SIRPα-KO are shown in FIG. Compared with pMAC and pMAC-SIRPα-KO, it can be seen that CAR-pMAC and CAR-pMAC-SIRPα-KO bind antigen-specifically to hGPC. In addition, compared with pMAC and CAR-pMAC, it can be seen that the expression of SIRPα in pMAC-SIRPα-KO and CAR-pMAC-SIRPα-KO is reduced. According to each scatter plot, it can be seen that the various cells produced are a single cell population, and almost no cells as impurities are contained, which can be said to be cells of high purity.

[0298] (6) Cytokine production by mouse pMAC

[0299] Macrophages present in vivo are classified into functional phenotypes of M1 and M2 based on the cell-cell interactions in the microenvironment of cancer tissues, etc. In order to compare the phenotypes of macrophages derived from organisms, the cytokines produced by mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were evaluated.

[0300] Mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO and mouse bone marrow-derived macrophages (2×10 5 Cells) were inoculated in a 48-well tissue culture plate and cultured for 48 hours in the presence of 20 ng / mL interferon γ (BioLegend) and 50 ng / mL LPS (Fujifilm Wako Pure Chemical Industries, Ltd.) to induce the M1 type. As mouse bone marrow-derived macrophages, mouse bone marrow-derived macrophages M0 prepared in Example 6 (3) were used. Similarly, the M2 type was induced by culturing for 48 hours in the presence of 20 ng / mL IL-4 (BioLegend). Using the obtained culture supernatant, the production of IL-6, TNFα, IL-12, and IL-10, which are known to be M1 or M2 type markers, was quantified ( Figure 7 ). It was found that pMAC induced the production of IL-6, TNFα, and IL-10 by M1 stimulation regardless of whether SIRPα or CAR was expressed, but did not produce IL-12.

[0301] (7) Cell migration ability of CAR-pMAC-SIRPα-KO mice

[0302] To investigate the cell migration ability of mouse CAR-pMAC-SIRPα-KO, 160 μL of culture supernatant of cancer cells or RPMI-1640 medium containing 10% FCS supplemented with recombinant chemokines was added to the lower chamber of a 16-well Boyden transwell plate CIM-Plate 16 (Agilent) coated with bovine fibronectin (Fujifilm Wako Pure Chemical Industries, Ltd.). Mouse CAR-pMAC-SIRPα-KO (3×10 5Cells) were inoculated in the upper chamber and cultured at 37°C and 5% CO2. Due to the induction factor in the lower chamber, the CAR-pMAC in the upper chamber passed through the pores of the transwell and adsorbed to the electrode sensor on the back of the membrane, increasing the resistance value. The resistance value was measured using the xCELLigence RTCA DP system (Agilent), thereby evaluating the cell migration of mouse CAR-pMAC.

[0303] To prepare the culture supernatant of mouse cancer cells, mouse MC38, LL / 2, 4T1, and CT26 cells (1×10 6 The next day, the culture medium was replaced with 2 mL of fresh RPMI-1640 medium containing 10% FCS, and the culture supernatant was prepared by further culturing for 24 hours. As normal cells, T cells and B cells (1×10 6 cells) to prepare the culture supernatant.

[0304] The results of measuring the cell migration ability of CAR-pMAC-SIRPα-KO to the culture supernatant of cancer cells are shown in Figure 8 Upper layer. It can be seen that the resistance value, i.e., the cell index value, increased compared to the culture supernatant of mouse cancer cells MC38, LL / 2, CT26, and 4T1, and that mouse CAR-pMAC-SIRPα-KO has migration ability ( Figure 8 When the culture supernatant of normal mouse spleen-derived B cells and T cells was used as a negative control, the cell index value was as low as that of RPMI1640 (Medium) containing 10% FCS without induction factors, and CAR-pMAC did not show cell migration activity ( Figure 8 In addition, the increase in the cell index value of the culture supernatant of CAR-pMAC-SIRPα-KO against cancer cell MC38 cultured in the presence of cytochalasin D (Fujifilm Wako Pure Chemical Industries, Ltd.) or pertussis toxin (Fujifilm Wako Pure Chemical Industries, Ltd.) 200 ng / mL, which are known inhibitors of cell migration, was partially reduced by cytochalasin D (CytD) and completely suppressed by pertussis toxin (PTX) ( Figure 8 (Upper right) Based on the above results, it can be seen that CAR-pMAC-SIRPα-KO has the ability to induce cell migration against the induction factors produced by various cancer cells.

[0305] Next, in order to investigate the cell migration ability of CAR-pMAC-SIRPα-KO against recombinant chemokines, mouse recombinant chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CX3CL1, CXCL1, CXCL2, CXCL5, CXCL12SDF-1α, and CXCL12SDF-1β (all manufactured by BioLegend) as candidate induction factors were used at a concentration of 100 ng / mL. The results are shown in Figure 8 Lower level.

[0306] The cell migration ability of mouse CAR-pMAC-SIRPα-KO against mouse recombinant chemokines was evaluated, and the results showed that it responded to CCL3, CCL4, and CCL7 ( Figure 8 lower left and center).

[0307] (8) Producing cytokines in mouse pMAC by co-culturing with cancer cells

[0308] Cancer cells expressing or not expressing cancer antigen hGPC3 were co-cultured with mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO to evaluate cytokine production. Mouse cancer cells hGPC3-introduced or not expressing 4T1, hGPC3-introduced or not expressing CT26, hGPC3-introduced or not expressing LL / 2, and hGPC3-introduced or not expressing MC38 (all 5×10 4 cells) and mice pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO (all 2×10 5 The cells were inoculated into 48-well tissue culture plates and cultured at 37°C and 5% CO2 for 48 hours. The obtained culture supernatant was 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 the co-culture of 4T1-GPC3, CT26-GPC3, LL / 2-GPC3, MC38-GPC3 and CAR-pMAC-SIRPα-KO induced the production of IL-6 and TNFα. There was no difference in the production of IL-10 under the experimental conditions, and IL-12 was not detected.

[0309] (9) Antigen-specific cancer cell phagocytosis based on mouse CAR-pMAC

[0310] Mouse pMAC, CAR-pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO (3×10 4Cells / well) were seeded in 96-well tissue culture plates, and RPMI-1640 containing 10% FBS and 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 3 The cells were co-cultured for 2 days (cells / well). To evaluate the phagocytosis of CAR target antigen-specific cancer cells, cancer cells into which the cancer antigen hGPC3 was introduced were used. The survival of the target cells was investigated by quantifying the bioluminescence obtained by adding Bright-GlO Luciferase Assay (Promega), a substrate for luciferase, to the 96-well tissue culture plate after co-culture, and the results are shown in Fig.10 . It can be seen that when co-cultured with pMAC, CAR-pMAC, and pMAC-SIRPα-KO, the killing effect of target cells was about 20%, and when co-cultured with CAR-pMAC-SIRPα-KO, it increased to about 60%. In addition, since the cell killing effect based on CAR-pMAC-SIRPα-KO did not appear in target cells that did not express hGPC3, it can be seen that CAR-pMAC-SIRPα-KO has the ability to phagocytose cancer cells that are specific to the target antigen.

[0311] Mice expressing tdTomato, pMAC or CAR-pMAC-SIRPα-KO (5×10 4 cells / well) and colorectal cancer cells MC38 cells expressing Venus and introduced with hGPC3 (5×10 3 The cells / well were co-cultured in 48-well tissue culture plates using RPMI-1640 medium containing 10% FBS. After 2 days and 4 days, fluorescence observation was performed using a fluorescence microscope (KEYENCE BZ-X810). The results are shown in Fig.11 Cancer cells were detected by green fluorescence from the fluorescent protein Venus (excitation wavelength 470 nm, absorption wavelength 525 nm), and pMAC or CAR-pMAC-SIRPα-KO was detected by red fluorescence from the fluorescent protein tdTomato (excitation wavelength 545 nm, absorption wavelength 605 nm). The bar graph was plotted with the area where the green fluorescence signal of cancer cells and the red fluorescence signal of pMAC or CAR-pMAC-SIRPα-KO overlapped. Fig.11 The bar graph is a plot of the area where the green fluorescence signal of cancer cells was detected ( Fig.11(right side of the lower layer). It can be seen that cancer cells co-cultured with pMAC proliferated, but cancer cells co-cultured with CAR-pMAC-SIRPα-KO were surrounded by CAR-pMAC-SIRPα-KO and phagocytosed and eliminated.

[0312] (10) Cancer cell cytotoxicity of mouse CAR-pMAC

[0313] In order to investigate the cancer cell toxicity of mouse CAR-pMAC, cancer cells and CAR-pMAC-SIRPα-KO were co-cultured in a 16-well plate E-Plate 16 (Agilent) and cultured at 37°C and 5% CO2. Cancer cells attached to the electrode sensor on the plate after pre-culture were engulfed by co-culture with CAR-pMAC-SIRPα-KO, cell adhesion weakened, and resistance decreased. By measuring this resistance value using the xCELLigenceRTCA DP system (Agilent), the cancer cell toxicity of CAR-pMAC-SIRPα-KO can be evaluated.

[0314] The target mouse cancer cells hGPC3 were introduced into or non-expressing CT26 (5×10 3 cells), hGPC3-introduced or non-expressing MC38 (1×10 4 cells), hGPC3-introduced or non-expressing LL / 2 (1×10 4 cells), hGPC3-introduced or non-expressing 4T1 (5×10 3 10 cells) were inoculated on E-Plate 16 coated with iMatrix-511, and cultured at 37°C and 5% CO2 for 24 hours. Then, mouse CAR-pMAC-SIRPα-KO effector cells were added at 1, 5, or 10 times the amount of target cancer cells for co-culture. The cancer cell cytotoxicity (% cell lysis, cell lysis rate) of CAR-pMAC-SIRPα-KO at effector cell: target cell ratio (E:T ratio) = 1:1, 5:1, and 10:1 at 6 hours after the start of co-culture (T = 30 hours) showed superior activity with higher expression of cancer antigen hGPC3 and E:T ratio regardless of the cancer cell target (Figure 12-1). The time-dependent changes in adhesion signals (normalized cell index value, Normalized Cell Index value; CI value) indicating cancer cell survival when cancer cells were co-cultured with CAR-pMAC-SIRPα-KO, and normalized cancer cell cytotoxic activity (% cell lysis) when cancer cells were cultured alone are shown in Figures 12-2 and 12-3. It was found that the CI value of cancer cells decreased over time and the % cell lysis increased over time, depending on the expression of cancer antigen hGPC3 and the number of CAR-pMAC-SIRPα-KO cells (E:T ratio).

[0315] (11) Study on the importance of SIRPα gene deletion or GC33-ζ-CAR introduction in mouse CAR-pMAC-induced cytotoxicity

[0316] To investigate the importance of SIRPα gene deletion or GC33-ζ-CAR introduction in the cancer cell toxicity caused by CAR-pMAC, human or mouse pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells to evaluate cancer cell toxicity at an E:T ratio of 10:1 using the xCELLigence RTCA DP system.

[0317] Mouse cancer cells hGPC were introduced into or not expressing CT26 (5×10 3 Cells) were inoculated on E-Plate 16 coated with iMatrix-511 and cultured at 37°C for 24 hours. Effector cells of mice pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (5×10 4 It was found that the antigen-specific cytotoxic activity (% cell lysis) of CT26 cells into which hGPC3 was introduced was improved by SIRPα deletion and GC33-ζ-CAR introduction ( Fig.13 ).

[0318] (12) Time-lapse observation of cancer cell phagocytosis based on mouse CAR-pMAC

[0319] To evaluate the phagocytic ability of cancer cells based on CAR-pMAC, MC38-GPC3-Luc-Venus cancer cells (5×10 5 cells / mL) and pMAC, pMAC-SIRPα-KO, CAR-pMAC, or CAR-pMAC-SIRPα-KO (5×10 6 Cells / mL) were co-cultured in RPMI-1640 medium containing 10% FCS and 12-well tissue culture plates. Four specific fields of view were photographed every 10 minutes using a fluorescence microscope (KEYENCE BZ-X810), and each field of view was photographed 50 times for 8 hours of time-lapse observation. The results are shown in Fig.14. If pHrodo-labeled cancer cells are engulfed and taken up into pMAC cells, a captured image is obtained in which red fluorescence (excitation wavelength 620nm, absorption wavelength 700nm) from pHrodo is detected in response to a decrease in pH. The time-dependent changes in the number of phagocytic cells were measured by image analysis (time-series cell counting) using the BZ-X810 analyzer analysis application. It was found that the phagocytic ability of CAR-pMAC-SIRPα-KO was improved compared to pMAC, pMAC-SIRPα-KO, and CAR-pMAC.

[0320] (13) In vivo dynamics of mouse CAR-pMAC in tumor-bearing mice

[0321] MC38 cells (5×10 5 The colorectal cancer peritoneal inoculation model (n=6) was established by intraperitoneal administration of CAR-pMAC-SIRPα-KO (1×10 7 Cells) were collected before treatment and 1 and 3 days after treatment. 100 μg of genomic DNA extracted from each organ was used as a template. A customized PrimeTime real-time PCR probe (Integrated DNA Technologies) that specifically recognizes CAR (GC33-ζ) was used to measure the amount of CAR-pMAC in the lung, liver, kidney, heart, spleen, bone marrow, tumor, and peripheral blood using a real-time PCR device 7500Fast (Thermo Fisher Scientific). Fig.15 ). It can be seen that: CAR signals were not detected in mice before treatment. On the first day after intraperitoneal administration, CAR-pMAC-SIRPα-KO was detected in the lungs, liver, kidneys, heart, spleen, and tumors, but not in bone marrow and peripheral blood. On the third day after intraperitoneal administration, the number of mice in which CAR-pMAC-SIRPα-KO was detected in normal tissues of the lungs, kidneys, heart, and spleen decreased, but CAR-pMAC was detected in tumor tissues in 5 out of 6 mice, and the amount was also high.

[0322] (14) Anti-tumor effect of CAR-pMAC in vivo

[0323] MC38 cells (5×10 5 The colorectal cancer peritoneal inoculation model was established by intraperitoneal administration of CAR-pMAC-SIRPα-KO (3×10 6The therapeutic effect was evaluated by measuring the survival curve of mice with a body weight loss of more than 20% or a body temperature below 30°C. The survival of MC38 cells introduced with hGPC3 was measured every 3 to 4 days using an IVIS imaging system (PerkinElmer) based on biochemiluminescence. Fig.16 The images captured by in vivo imaging and the temporal changes in the total flux of luciferase luminescence are shown in the figure. The untreated group that did not receive CAR-pMAC-SIRPα-KO saw significant tumor growth after cancer cell transplantation. In contrast, CAR-pMAC-SIRPα-KO significantly suppressed tumor growth and significantly prolonged survival.

[0324] Example 8 Functional Analysis of Human CAR-pMAC

[0325] (1) Preparation of macrophages from human organisms and iPS cells

[0326] CD14 monocytes (Lonza) derived from blood samples from healthy human donors were seeded in 6-well tissue culture plates (2×10 6 Cells / well) were cultured for 4 days to induce CD14 monocyte-derived macrophages M0. Then, CD14 monocyte-derived macrophages M1 were induced by culturing for 2 days in the presence of 50 ng / mL lipopolysaccharide and 50 ng / mL human interferon-γ. In addition, CD14 monocyte-derived macrophages M0 were cultured for 2 days in the presence of 50 ng / mL IL-4 (BioLegend) to induce CD14 monocyte-derived macrophages M2.

[0327] The differentiated cells including myeloid cells obtained in Example 5(3) were cultured for 7 to 14 days in the presence of 50 ng / mL M-CSF and 20 ng / mL IL-3 to induce human iPS cell-derived macrophages.

[0328] (2) Gene expression profile of human pMAC

[0329] RNAseq analysis (biological species: human, reference genome hg38) was performed on pMACs prepared from iPS cells, as well as macrophages derived from organisms and iPSCs, as a whole transcriptome analysis, to quantify the expression of transcripts. Total RNA was extracted from each cell using the QIAGEN RNeasymini kit, and the strand-specific library preparation method (dUTP method) was used with the NEBNext(R) Poly(A) mRNA Magnetic Separation Module (Model E7490), NEBNext(R) Ultra TM IIDirectional RNA Library Prep Kit (model E7760) was used to prepare the sequence library by PCR amplification with primers containing the Index sequence. The base sequence of the sample prepared by the library was obtained using the next-generation sequencer NovaSeq6000 (illumina), and the data of the read length PE150 (150bp×2 double-end sequencing), the data volume of each sample was 4G bases, and the reading of each sample was 26.7Mreads (13.3M pairs).

[0330] The acquired data were used to implement informatics analysis and principal component analysis (PCA). In order to confirm the quality of the sequence reads, the quality scores (scores indicating the error rate of sequence information) of R1 (Read1) and R2 (Read2) were calculated using the software FastQC (version 0.11.7), and it was confirmed that there were no abnormalities (completely normal, quality score>28). Next, the software Trimmomatic (version 0.38) was used to trim the sequence reads according to the set values ​​(ILLUMINACLIP2:30:10, LEADING=20, TRAILING=20, SLIDINGWINDW=4:15, MINLEN=36). In order to obtain data of good quality, in view of the results of FastQC quality confirmation, the ends of the reads with low quality, the adapter sequences, short reads, etc. were trimmed. Next, the trimmed sequence reads were mapped to the reference genome using the software HISAT2 (version 2.1.0) to calculate the mapping rate of the reads, and it was confirmed that all samples exceeded 98%. Next, the software featureCounts (version 1.6.3) was used to calculate the original read counts mapped to the known exon regions of each gene. In addition, the mapped fragments were counted to calculate the Fragments Per Kilobase of exon per Million mapped fragments (FPKM) value, Fragments Per Kilobase of Transcript per Million mapped reads upper quartile (FPKM-UQ) value, and Transcripts Per Million (TPM) value. Using the software stats (version 3.6.1), principal component analysis of the samples was performed based on the original reads, FPKM values, and TPM values ​​for all genes detected. The index reflecting the most data from the component with the largest deviation in each data was used as principal component (PC) 1 on the horizontal axis, and the index reflecting the second most data from the component with the second largest deviation was used as PC2 on the vertical axis to plot each sample to create a plot data ( Fig.17 ). It was found that human pMAC was plotted in the range of PC1 (150 to 200) and PC2 (-20 to -40), and existed in a region different from other organism-derived CD14-positive cell-derived monocytes, CD14-positive cell-derived macrophages (M0, M1, M2 types), iPS cell-derived myeloid cells, and iPS cell-derived macrophages.

[0331] (3) Analysis of surface antigens of human iPSC-pMAC

[0332] Human pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO, blood-derived CD14-positive monocytes (CD14-derived monocytes), iPSC-derived myeloid cells (iPSC-derivedMyeloid cells), and iPSC-derived macrophages (iPSC-macrophages) were recovered by pipetting. CD14 monocyte-derived macrophages M0 (CD14-derived macrophages M0) and CD14 monocyte-derived macrophages M2 (CD14-derived macrophages M2) were treated with D-PBS (Nacalai Tesque) containing 2 mM EDTA (Nacalai Tesque) at 37°C for 15 minutes and recovered by pipetting, and CD14 monocyte-derived macrophages M1 (CD14-derived macrophages M1) were treated with Accutase (Nacalai Tesque) at 37°C for 15 minutes and recovered by pipetting.

[0333] 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 control antibodies. Then, the cells were washed twice with PBS or PBS containing 2% FBS. The washed cells were analyzed using a flow cytometer analyzer (trade name: BD Accuri C6 Flow Cutometer, manufactured by Beckton Dickinson). The results of the surface antigen analysis based on flow cytometry after antibody staining are shown in Figure 18. The results of this analysis revealed that human pMAC (pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO) was negative for CD83 and CD14 expressed in CD14-positive cell-derived macrophages (M0, M1, and M2) and iPSC-derived macrophages.

[0334] (4) Morphology of human pMAC

[0335] Phase contrast microscopic observation images of human CD14-positive monocytes, iPSC-derived myeloid cells, pMAC, and CD14-positive cell-derived macrophages (unstimulated, M0, M1, and M2 stimulation conditions) are shown in Figure 19-1, and phase contrast microscopic observation images of iPS cell-derived macrophages, pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (unstimulated, M0, M1, and M2 stimulation conditions) are shown in Figure 19-2. It can be seen that pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO do not show changes in cell morphology due to the loss of SIRPα and the introduction of CAR, and have the irregular morphology with protrusions exhibited by the blood-collected CD14-positive cell-derived macrophages shown in Figure 19-1.

[0336] (5) Human pMAC cell proliferation

[0337] The cell proliferation of human pMAC, pMAC-SIRPα-KO, and iPS cell-derived macrophages as a control in response to GM-CSF and M-CSF was investigated by MTT analysis after continued culture for more than 2 weeks after gene introduction. pMAC and pMAC-SIRPα-KO were collected and inoculated in 96-well tissue culture plates (3×10 3 Then, the proliferation rates of the cells were compared between the culture medium containing GM-CSF and M-CSF and the culture medium not containing GM-CSF and M-CSF.

[0338] Fig. 20 The results of the MTT assay are shown in Figure 2. The proliferation of human pMAC and pMAC-SIRPα-KO requires about 50 ng / mL of GM-CSF or M-CSF in the culture medium. In addition, the combined use of GM-CSF and M-CSF has the effect of promoting proliferation. On the other hand, there is no cytokine-dependent cell proliferation in iPS cell-derived macrophages, indicating that pMAC has a high cell proliferation ability.

[0339] (6) Analysis of cell surface antigens of human CAR-pMAC

[0340] The expression of tdTomato, GC33ζCAR, and SIRPα in human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO was investigated using flow cytometry. The nucleic acid sequence on the 3' end side of the GC33ζCAR cDNA integrated into the lentiviral vector was used to express tdTomato as a fluorescent protein via the T2A sequence. In addition, FITC-labeled human glypican 3 (hGPC3), which is the target antigen of CAR, was added to the cell sample. In this way, the fluorescence of tdTomato and hGPC3-FITC can be used as indicators to analyze the expression of CAR using flow cytometry. Fig.21 The results of investigating the expression of GC33ζCAR in human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO using flow cytometry are shown in Figure 2. It can be seen that CAR-pMAC and CAR-pMAC-SIRPα-KO bind to hGPCs with antigen-specific binding compared to pMAC and pMAC-SIRPα-KO. In addition, it can be seen that the expression of SIRPα in pMAC-SIRPα-KO and CAR-pMAC-SIRPα-KO is reduced compared to pMAC and CAR-pMAC. According to each scatter plot, it can be seen that the various cells produced are single cell populations, and almost no cells as impurities are contained, which can be said to be cells of high purity.

[0341] (7) Cytokine production based on human CAR-pMAC

[0342] The human CD14 monocyte-derived macrophages M0 type and human iPS cell-derived macrophages prepared in Example 8 (1), the human pMAC and pMAC-SIRPα-KO prepared in Example 6 (2), and the CAR-pMAC and CAR-pMAC-SIRPα-KO prepared in Example 7 (2) (2×10 5 Cells were seeded in 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, Ltd.) to induce the M1 type. Similarly, the M2 type was induced by culturing for 48 hours in the presence of 50 ng / mL IL-4 (BioLegend). The obtained culture supernatant was used to quantify the production of IL-6, TNFα, IL-12, and IL-10, which are known markers of the M1 or M2 type ( Fig. 22). It was found that pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were induced to produce IL-6, TNFα, and IL-10 by stimulation with interferon γ and LPS. On the other hand, the production of IL-12 was not detected.

[0343] (8) Cell migration ability of human CAR-pMAC

[0344] To investigate the cell migration ability of human CAR-pMAC-SIRPα-KO, 160 μL of culture supernatant of cancer cells or RPMI-1640 medium containing 10% FCS supplemented with recombinant chemokines was added to the lower chamber of a 16-well Boyden chamber (transwell) plate CIM-Plate 16 (Agilent) coated with bovine fibronectin (Fujifilm Wako Pure Chemical Industries, Ltd.), and human CAR-pMAC-SIRPα-KO (3×10 5 Cells) are inoculated and cultured at 37°C and 5% CO2. Due to the induction factor in the lower chamber, the CAR-pMAC in the upper chamber passes through the pores of the transfer well and adsorbs to the electrode sensor on the back of the membrane, increasing the resistance value. By measuring this resistance value using the xCELLigenceRTCA DP system (Agilent), the cell migration of CAR-pMAC can be evaluated.

[0345] In order to prepare the culture supernatant of human cancer cells, human HepG2, JHH-7, and KOC7c cells (2×10 6 cells), SK-Hep1 (5×10 5 HEK293 cells (5×10 5 cells) to prepare the culture supernatant.

[0346] The results of measuring the cell migration ability of human CAR-pMAC-SIRPα-KO into the culture supernatant of cancer cells are shown in Fig.23 The cell index value of the culture supernatant of human cancer cells HepG2, SK-Hep1, and JHH-7 increased, indicating that human CAR-pMAC-SIRPα-KO has migration ability. The migration ability of cancer cell KOC7c is low, and the cell index value is the same as that of normal cell HEK293 ( Fig.23In addition, when CAR-pMAC-SIRPα-KO was cultured in the presence of 200 ng / mL of cytochalasin D (Fujifilm Wako Pure Chemical Industries, Ltd.) or pertussis toxin (Fujifilm Wako Pure Chemical Industries, Ltd.), which are known inhibitors of cell migration, the increase in the cell index value of the culture supernatant against the cancer cell MC38 was partially reduced by cytochalasin D (CytD) and completely suppressed by pertussis toxin (PTX) ( Fig.23 The cell migration ability of human iPS-derived macrophages (iPS-macrophage) into the culture supernatant of cancer cells was measured. As a result, the cancer cells HepG2, SK-Hep1, and KOC7c all showed CI values ​​similar to those of the negative control Medium and normal cells HEK293 ( Fig.23 The above results indicate that the induction factor produced by CAR-pMAC-SIRPα-KO against various cancer cells has a higher cell migration ability than that of iPS-derived macrophages (iPS-macrophage).

[0347] Next, in order to investigate the cell migration ability of CAR-pMAC-SIRPα-KO against 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), CX3CL1 (R&D Systems), CXCL1 (BioLegend), CXCL2 (BioLegend), CXCL5 (BioLegend), CXCL12SDF-1α (BioLegend), and CXCL12SDF-1β (BioLegend) as candidates for inducing factors were used at a concentration of 100 ng / mL.

[0348] The cell migration ability of human CAR-pMAC-SIRPα-KO cells against human recombinant chemokines was evaluated, and the results showed that they responded to CCL2, CCL3LI, CCL5, CCL7, CCL23, and SDF-1α (CXCL12). Fig.23The second and third layers from the top). Furthermore, the cell migration of CAR-pMAC and CAR-pMAC-SIRPα-KO against the culture supernatant of HepG2 cancer cells was investigated ( Fig.23 (bottom row) It was found that the cell migration ability of CAR-pMAC-SIRPα-KO cells was improved by the loss of SIRPα compared with that of CAR-pMAC derived from wild-type iPS cells.

[0349] (9) Cytokine production by human CAR-pMAC (co-culture with cancer cells)

[0350] Human pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO (all 2×10 5 Cells) were inoculated in 48-well tissue culture plates using RPMI-1640 containing 10% FCS and cultured at 37°C and 5% CO2 for 48 hours. The culture supernatant obtained was used to quantify the production of IL-6, TNFα, IL-10, and IL-12 (Figure 24-1). In addition, cancer cells expressing or not expressing cancer antigen hGPC3 were co-cultured with human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO to evaluate cytokine production. Human cancer cells hGPC3 were introduced with or without SK-Hep1 expression, hGPC3 expression or lack of JHH-7 (all 5×10 4 cells) and human pMAC, pMAC-SIRPα-KO, CAR-pMAC, CAR-pMAC-SIRPα-KO (all 2×10 5 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 in the culture supernatant obtained by co-culturing with WT cells.

[0351] It was found that human CAR-pMAC and CAR-pMAC-SIRPα-KO produced IL-6, TNFα, and IL-10 by the introduction of CAR. It was found that the production of antigen-specific cytokines was not induced by the introduction of human cancer cells hGPC3 or co-culture with non-expressing SK-Hep1 and hGPC3 expressing or lacking JHH-7. IL-12 was not detected.

[0352] (10) Harmfulness of cancer cells induced by human CAR-pMAC

[0353] Human pMAC, CAR-pMAC, pMAC-SIRPα-KO, CAR-pMAC-SIRPα-KO (3×10 4Cells / well) were seeded in 96-well tissue culture plates, and RPMI-1640 containing 10% FBS and target cells of cancer cells JHH7-Luc, SK-Hep1-GPC3-Luc-Venus, and KOC7c-GPC3-Luc (3×10 3 In order to evaluate the phagocytosis of CAR target antigen-specific cancer cells, cancer cells that endogenously express the cancer antigen hGPC3 or have been introduced with the hGPC3 gene were used. The survival of target cells was investigated by quantifying the bioluminescence obtained by adding the Bright-Glo Luciferase Assay (Promega), a substrate for luciferase, to the 96-well tissue culture plate after co-culture, and the results are shown in Fig.25 . It can be seen that the killing effect of target cells when co-cultured with pMAC, CAR-pMAC, and pMAC-SIRPα-KO was about 20-40%, and it increased to about 60-90% when co-cultured with CAR-pMAC-SIRPα-KO. In addition, since the cell killing effect caused by CAR-pMAC-SIRPα-KO did not appear in the target cells SK-Hep1-mock-Luc-V that did not express hGPC3, it can be seen that CAR-pMAC-SIRPα-KO has the ability to phagocytose target antigen-specific cancer cells.

[0354] (11) Human CAR-pMAC-SIRPα-KO cancer cell toxicity (real-time measurement)

[0355] Human CAR-pMAC-SIRPα-KO was used as effector cells, and the cytotoxicity to human cancer cells was evaluated using the xCELLigence RTCA DP system (Figures 26-1 and 26-2). hGPC3 was introduced into SK-Hep1 cells (5×10 4 Cells) were inoculated on E-Plate 16, cultured at 37°C and 5% CO2 for 24 hours, and then human CAR-pMAC-SIRPα-KO was added to make E:T ratio = 1:1, 5:1, and 10:1 for co-culture. It can be seen that human CAR-pMAC-SIRPα-KO showed excellent cytotoxic activity as hGPC3 expression and E:T ratio increased. The time-dependent changes in the adhesion signal (cell index value; CI value) indicating cancer cell survival when cancer cells were co-cultured with CAR-pMAC-SIRPα-KO and the normalized cytotoxic activity of cancer cells (% cell lysis) when cancer cells were cultured alone are shown in Figure 26-2. It can be seen that the CI value of cancer cells decreased over time and the % cell lysis increased depending on the expression of cancer antigen hGPC3 and the number of CAR-pMAC-SIRPα-KO cells (E:T ratio).

[0356] (12) Study on the importance of SIRPα gene deletion or GC33-ζ-CAR introduction in CAR-pMAC-induced cytotoxicity

[0357] To investigate the importance of SIRPα gene deletion or GC33-ζ-CAR introduction in the cancer cell toxicity caused by CAR-pMAC, human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO were used as effector cells to evaluate cancer cell toxicity at an E:T ratio of 10:1 using the xCELLigence RTCA DP system.

[0358] Human cancer cells hGPC3 were transfected with or without SK-Hep1 expression (5×10 4 Cells) were inoculated on E-Plate 16 and cultured at 37°C for 24 hours. Then, effector cells human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO (5×10 5 It was found that the cytotoxic activity (% cell lysis) against SK-Hep1 cells introduced with hGPC3 was improved by the introduction of GC33-ζ-CAR ( Fig. 27 Upper left). It can be seen that the cytotoxic activity is significantly higher than that against SK-Hep1 which does not express the cancer antigen hPGC3 ( Fig. 27 The adhesion signal (normalized cell index value; CI value) indicating cancer cell survival when cancer cells were co-cultured with human pMAC, pMAC-SIRPα-KO, CAR-pMAC, and CAR-pMAC-SIRPα-KO, and the cytotoxic activity (% cell lysis) of normalized cancer cells when cancer cells were cultured alone are shown over time. Fig. 27 Middle and lower layers: The most significant decrease in CI value and increase in % cell lysis over time of cancer cells was observed by GC33-ζ-CAR introduction and SIRPα deficiency.

[0359] Industrial Applicability

[0360] According to the method of the present invention, a novel CAR-carrying platform with stable quality, stable supply and excellent economic efficiency can be provided. The novel CAR-carrying platform uses proliferative macrophage-like cells (pMAC). CAR-carrying pMAC is also effective in the treatment of solid cancers.

[0361] This application is based on Japanese Patent Application No. 2022-127477 (filing date: August 9, 2022) filed in Japan, and all the contents are incorporated herein by reference.

Claims

1. A proliferative macrophage-like cell, i.e., pMAC, characterized in that: Proliferation is cytokine dependent.

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

3. The pMAC according to claim 1, wherein: CD14 and CD83 were negative.

4. The pMAC according to claim 1, wherein: Genes that inhibit macrophage-like function are deleted or their expression is suppressed.

5. The pMAC according to claim 4, wherein: The gene that inhibits macrophage-like function is the signal regulatory protein α, i.e., SIRPα gene.

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

7. A method for producing proliferative macrophage-like cells (pMAC), comprising: a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function, and a step of expressing a gene that increases proliferation.

8. The manufacturing method according to claim 7, wherein: include: (1) Step 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 (MACs); and (2) Step 2 is a step of causing the MAC obtained in step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells, namely, pMAC.

9. The manufacturing method according to claim 7, wherein: include: (A) Step A, a step of causing myeloid cells differentiated from pluripotent stem cells to express a gene that imparts proliferation to obtain proliferative myeloid cells, and (B) Step B is 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, namely, pMAC.

10. The manufacturing method according to claim 7, wherein: The gene that inhibits macrophage-like function is the signal regulatory protein α, or SIRPα gene.

11. The manufacturing method according to claim 7, wherein: The gene that increases proliferation is at least one selected from the group consisting of c-MYC, BMI1, and MDM2. 12 . A method for producing CAR-pMAC, comprising introducing a chimeric antigen receptor (CAR) into pMAC obtained by the production method according to claim 7 .

13. A method for producing CAR-pMAC, comprising: (1) Step 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 (MACs), (2) Step 2, a step of causing the MAC obtained in Step 1 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells, i.e., pMAC; and (3) Step 3: the step of introducing a chimeric antigen receptor, namely, CAR.

14. The manufacturing method according to claim 13, wherein: The differentiation induction into myeloid cells in step 1 is carried out by the following method (i) or (ii), (i) EBs induced from pluripotent stem cells are stacked and cultured on feeder cells in a medium containing VEGF, and then further cultured in a medium containing VEGF, SCF and TPO, or (ii) Embryoid bodies (EBs) induced from pluripotent stem cells are cultured as a monolayer in a medium containing BMP-4, VEGF and SCF without feeder cells, and then further cultured in a medium containing VEGF, TPO and GM-CSF.

15. The method according to claim 14, wherein: Induction of pluripotent stem cells into embryoid bodies (EBs) is carried out by the following methods (a) and (b): (a) seeding pluripotent stem cells into a culture vessel for cell cluster formation to form embryoid bodies (EBs); and (b) The container has fine spherical holes at the bottom, and there is no flat surface between adjacent holes.

16. A method for producing CAR-pMAC, comprising: (1) Step 1-1, a step of deleting or suppressing the expression of a gene that inhibits macrophage-like function in pluripotent stem cells, Step 1-2 is a step of seeding the pluripotent stem cells obtained in step 1-1 in which the gene that inhibits macrophage-like function is deleted or the expression is inhibited, into a cell cluster formation culture container having fine spheroid holes in the bottom and no flat surface between adjacent holes to form embryoid bodies (EBs), Step 1-3 is a step of obtaining macrophage-like cells (MAC) from the EBs obtained in step 1-2 by the following method (i) or (ii), (i) culturing the EBs on feeder cells in a medium containing VEGF, and then further culturing them in a medium containing VEGF, SCF and TPO, or (ii) culturing the EBs in a culture medium containing BMP-4, VEGF and SCF in a monolayer culture without feeder cells, and then further culturing them in a culture medium containing VEGF, TPO and GM-CSF; (2) Step 2, a step of causing the MAC obtained in Steps 1-3 to express a gene that imparts proliferation to obtain proliferative macrophage-like cells, namely, pMAC; as well as (3) Step 3 is a step of introducing CAR, a chimeric antigen receptor, into pMAC obtained in step 2 to obtain CAR-pMAC.

17. The production method according to any one of claims 13 to 16, wherein The gene that inhibits macrophage-like function is the signal regulatory protein α, or SIRPα gene.

18. The production method according to any one of claims 13 to 16, wherein The gene that increases proliferation is at least one selected from the group consisting of c-MYC, BMI1, and MDM2.

19. A proliferative macrophage-like cell, wherein a gene expression profile is obtained by performing a whole transcriptome analysis using a cell group along the macrophage lineage consisting of iPS cell-derived myeloid cells, iPS cell-derived macrophages, organism-derived monocytes, organism-derived macrophages M1, organism-derived macrophages M2, and organism-derived macrophages M0 as a comparison object, and principal component analysis is performed based on the gene expression profile, and when the indexes of the components ranked first in contribution rate are used as the analysis object, the following conditions are satisfied: Condition 1: No overlap with known macrophage lineage maps, and Condition 2: Plot when PC1 is greater than 100 and PC2 is between -80 and 0.

20. A pharmaceutical composition comprising the proliferative macrophage-like cells according to claim 6 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, which is used for treating at least one selected from the group consisting of a disease associated with a tumor or cancer, a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, and amyloidosis.

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