Cell capable of differentiating into M2 macrophages in tumor tissue and expressing inflammatory cytokines when differentiated into M2 macrophages, and composition containing same

Abstract

Provided are: a cell capable of differentiating into an M2 macrophage in a tumor tissue and expressing an inflammatory cytokine when differentiated into an M2 macrophage; and a composition containing the cell.

Description

Technical Field

[0001] The present invention relates to cells that can differentiate into M2 macrophages in tumor tissues and express inflammatory cytokines when differentiated into M2 macrophages, and compositions containing the cells (eg, pharmaceutical compositions for treating cancer). Background Art

[0002] In current cancer treatments using anticancer drugs, drugs are typically administered multiple times (e.g., every two weeks) or at high doses to achieve a certain therapeutic effect [1-3]. This is due to drug metabolism in tumor tissue and a decrease in drug efficacy [4,5]. However, while increasing the drug dosage can achieve a higher effect, the risk of side effects also increases [6,7]. Due to such significant risks and burdens, there is a need for cancer treatments that do not rely on drugs. Against this backdrop, cancer immunotherapy is considered one of the promising solutions.

[0003] However, tumor tissue is generally immunosuppressive, and the tumor is not recognized as a foreign body by the immune system [8-13]. Therefore, cancer immunotherapy is not very effective. This is mainly due to the presence of intratumoral stromal cells such as M2 macrophages (anti-inflammatory) and regulatory T cells that secrete anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor-β (TGF-β) [8,14].

[0004] Tumor necrosis factor α (TNF-α) is one of the most important inflammatory cytokines and plays a crucial role in immune activation in peripheral tissues [15,16]. When TNF-α binds to TNF receptors 1 and 2, it activates signal transduction cascades such as the nuclear factor κB (NF-κB) pathway, leading to the expression of inflammatory cytokines such as IL-6 and IL-1 [16-19]. These inflammatory cytokines induce M1 macrophages (inflammatory) or activate other immune cells, transforming tissues into an inflammatory environment

[15] . This inflammatory induction function of TNF-α is attractive for cancer treatment based on the body's own foreign body clearance, but currently, even at doses far below the therapeutic range, systemic administration exhibits severe toxicity, hindering its clinical application

[20] . Furthermore, since the half-life of TNF-α administered in vivo is approximately 15 to 30 minutes, it is difficult to deliver sufficient TNF-α to tumor tissues [21,22].

[0005] To address this issue, recent reports have shown that TNF-α-antibody conjugates can effectively deliver TNF-α to tumor tissues.[23,24] Although these technologies can specifically deliver TNF-α to antigens expressed by cancer cells, their effectiveness is significantly reduced when the antigens are mutated or defective, so there is still room for technical improvement.[25,26]

[0006] Macrophages are one of the main immune cells and play an important role in tissue inflammation and immune response

[27] . In particular, the main characteristics of macrophages in tumor tissues can be listed as follows: (1) Macrophages migrate to tumor tissues through CC chemokine ligand 2 (CCL2) and granulocyte macrophage colony-stimulating factor (GM-CSF) secreted by cancer cells and stromal cells[28,29]; (2) Macrophages can polarize to the anti-inflammatory M2 phenotype in tumor tissues[18,30,31]. Macrophages transform into M1 or M2 phenotypes according to the inflammatory state of the tissue environment, thereby controlling the inflammatory environment. M2 macrophages participate in wound healing, tissue regeneration and immunosuppression[30,32]. In particular, in tumor tissues, macrophages polarize to M2 macrophages, playing an important role in the construction of an immunosuppressive environment

[30] .

[0007] Furthermore, it is known that arginase 1 (Arg1) activity is very high in mouse M2 macrophages

[33] . Arg1 catalyzes the hydrolysis of arginine into urea and ornithine. As a result, the amount of arginine decreases, promoting immunosuppression

[34] . Arg1 is not expressed much in M0 and M1 macrophages, so Arg1 activity is one of the important markers for identifying the M2 phenotype in mice

[35] . Summary of the Invention

[0008] The present invention provides cells that can differentiate into M2 macrophages in tumor tissues and express inflammatory cytokines when differentiated into M2 macrophages, and a composition containing the cells.

[0009] According to the present invention, for example, the following inventions can be provided.

[0010] (1) A cell having the ability to differentiate / polarize into an M2 macrophage, comprising a nucleic acid encoding an inflammatory cytokine operably linked to a regulatory sequence and capable of expressing the inflammatory cytokine after differentiation / polarization into an M2 macrophage.

[0011] (2) The cell according to (1) above, which is any cell selected from the group consisting of M0 macrophages, monocytes, common monocyte progenitor cells, monocyte dendritic cell progenitor cells, myeloid progenitor cells and hematopoietic stem cells, and any cell in the differentiation stage from hematopoietic stem cells to M0 macrophages.

[0012] (3) The cell according to (1) or (2) above, which is an iNOS, CD206, and Arg-1 negative macrophage.

[0013] (4) The cell according to any one of (1) to (3) above, which is a cell at any stage of differentiation from a monocyte to an M0 macrophage.

[0014] (5) The cell according to any one of (1) to (4) above, wherein the inflammatory cytokine is an inflammatory cytokine selected from the group consisting of TNF-α, IFN-α, IFN-γ, IL-1, IL-2, IL-6, IL-8, IL-12, IL-17, and IL-23.

[0015] (6) The cell according to any one of (1) to (5) above, wherein the inflammatory cytokine is TNF-α.

[0016] (7) The cell according to any one of (1) to (6) above, wherein the regulatory sequence is a regulatory sequence for specifically expressing the inflammatory cytokine in M2 macrophages.

[0017] (8) The cell according to any one of (1) to (7) above, wherein the regulatory sequence is a regulatory sequence of a gene selected from the group consisting of CD163, CD200R1, CD301, CXCR1, CXCR2, CD209, dendritic cell-associated C-type lectin-1 (dectin-1), FceRIa, IL-1RII, CD206, arginase 1, IFR4, PPARγ, STAT6, FIZZI, IL-1ra, IL-10, TGF-β, CCL1, CCL14, CCL18, CCL22, CCCL23, CCL24, CCL26, and YM1, or preferably a regulatory sequence of a gene selected from the group consisting of CCL-22, LOX, CISH, FCER15, ALOX15, F13A1, IDO1, MMP1, ALDH1A2, CD209, TGM2, VCAM1, MMP12, and SPINT2.

[0018] (9) A composition comprising the cell according to any one of (1) to (8) above.

[0019] (10) The composition according to (9) above, which is used for treating cancer.

[0020] (11) The composition according to (9) above, which is used for treating cancer in a subject having cancer.

[0021] (12) The composition according to (11) above, wherein the subject having cancer is a subject who has received cancer immunotherapy, a subject who is scheduled to receive cancer immunotherapy, or a subject who is currently receiving cancer immunotherapy.

[0022] (13) The composition according to (12) above, wherein the cancer immunotherapy is any one or more selected from the group consisting of chimeric antigen receptor-expressing cell therapy, T cell adoptive immunotherapy, and immune checkpoint inhibitor therapy.

[0023] (14) The composition according to (11) above, which is used in combination with cancer immunotherapy.

[0024] (15) The composition according to any one of (10) to (14) above, wherein the cancer has an immunosuppressive environment.

[0025] (16) The composition according to any one of (10) to (15) above, wherein the ratio of the number of M1 macrophages / the number of M2 macrophages in the cancer tissue is less than 1.

[0026] (17) The composition according to any one of (10) to (16) above, wherein the subject is resistant to immune checkpoint inhibitor therapy.

[0027] (18) The cell according to any one of (1) to (8) above, which is a human cell.

[0028] (19) The composition according to any one of (10) to (18) above, wherein the cells are human cells.

[0029] (20) A method for treating cancer in a subject having cancer, comprising: administering an effective amount of the cells described in any one of (1) to (8) and (18) to the subject, thereby exacerbating the inflammation of at least one immunosuppressive tumor possessed by the subject (for example, increasing the ratio of the number of M1 / M2 macrophages in the tumor); and administering an effective amount of an immune checkpoint inhibitor to the subject (preferably the subject having a tumor with exacerbated inflammation).

[0030] (21) A composition containing an effective amount of the cells described in any one of (1) to (8) and (18) above, for use in the method described in (20) above.

[0031] (22) A composition containing an effective amount of an immune checkpoint inhibitor, for use in the method described in (20) above.

[0032] (23) A kit comprising an effective amount of the cells described in any one of (1) to (8) and (18) above and an effective amount of an immune checkpoint inhibitor, for use in the method described in (20) above.

[0033] (24) A kit comprising a pharmaceutical composition containing an effective amount of cells according to any one of (1) to (8) and (18) above and a pharmaceutical composition containing an effective amount of an immune checkpoint inhibitor, for use in the method described in (20) above.

[0034] (25) A pharmaceutical composition comprising an effective amount of the cells described in any one of (1) to (8) and (18) above and an effective amount of an immune checkpoint inhibitor, for use in the method described in (20) above.

[0035] (26) Use of the cell according to any one of (1) to (8) and (18) above in the manufacture of the above-mentioned composition or kit.

[0036] (27) Use of cancer immunotherapy drugs (e.g., immune checkpoint inhibitors) in the manufacture of the above-mentioned compositions or kits.

[0037] (28) Use of the cells and cancer immunotherapy drugs (e.g., immune checkpoint inhibitors) described in any one of (1) to (8) and (18) above in the manufacture of the above-mentioned composition or kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results showed that the tumor tissue was an immunosuppressive environment with the presence of regulatory T cells and M2 macrophages, and minimal T cell infiltration.

[0039] Figure 2 This indicates that when tumor tissue is in an inflammatory environment, the immune system that an individual already possesses can shrink or eliminate the tumor tissue.

[0040] Figure 3 It shows that macrophages migrate into tumor tissues and polarize into M2 macrophages within the tumor tissues.

[0041] Figure 4 It is shown that macrophages that are M0 in normal tissues polarize to M2 in tumor tissues.

[0042] Figure 5A The present invention shows a scheme for introducing a nucleic acid containing a gene encoding an inflammatory cytokine (eg, TNF-α) under the control of an M2-specific promoter into M0 macrophages, inducing them into M2 macrophages, and evaluating the expression of the inflammatory cytokine (eg, TNF-α).

[0043] Figure 5B The results show that M0 macrophages treated with IL-4 or IL-13, which are M2-inducing cytokines, differentiate into M2 macrophages and M2-specifically produce TNF-α, and that M0 macrophages treated with cancer cell culture supernatant (TCM) differentiate into M2 macrophages and M2-specifically produce TNF-α.

[0044] Figure 5CIt is shown that M0 macrophages treated with IL-4 or IL-13 as M2-inducing cytokines differentiate into M2 macrophages and that M2 specifically produces cytokines (IL-2 and IL-12) derived from exogenous genes operably linked to an M2-specific promoter.

[0045] Figure 6A It was shown that the M1 marker was enhanced in the tumor tissue of the MacTrigger-administered group.

[0046] Figure 6B It was shown that the M1 / M2 ratio in the tumor tissue of the MacTrigger-administered group increased.

[0047] Figure 6C It was shown that MacTrigger polarized into M2 macrophages in tumor tissues and secreted TNF-α in tumor tissues.

[0048] Figure 7A Shown is a protocol for administering MacTrigger to tumor-bearing mice and measuring tumor size.

[0049] Figure 7B Shown are the changes in tumor size over time and the survival curve of the MacTrigger-administered group.

[0050] Figure 7C The figures are the results of an in vitro experiment showing the transition of the survival rate of MacTrigger cells cultured in the presence of M2-inducing cytokines or TCM.

[0051] Figure 7D The expression of Ki67, a cell proliferation marker, in tumor tissues of the MacTrigger-administered group is shown.

[0052] Figure 7E The graph shows the transition of body weight change in the MacTrigger-administered group.

[0053] Figure 8A This is a protocol to determine the ratio of NK cells to cytotoxic T cells in tumor tissues of the MacTrigger-administered group.

[0054] Figure 8B The ratio of NK cells to cytotoxic T cells in tumor tissues of the MacTrigger-administered group is shown.

[0055] Figure 9A The graph shows the dynamics of VT-680-labeled macrophages in mice after administration of VT-680-labeled macrophages.

[0056] Figure 9BThe figures show the amounts of VT-680-labeled macrophages in non-tumor tissues and tumor tissues of mice after administration of VT-680-labeled macrophages.

[0057] Figure 9C The figures show the amount of VT-680-labeled macrophages in various tissues of mice after administration of VT-680-labeled macrophages.

[0058] Figure 10A The analysis scheme of cells in tumor tissue and liver of the macrophage-administered group is shown.

[0059] Figure 10B The AST activity of macrophage-administered mice is shown.

[0060] Figure 10C The expression levels of CD206 (M2 marker) in tumor tissue and liver of the macrophage-administered group are shown.

[0061] Figure 10D The presence or absence of hepatomegaly and blood AST levels in the MacTrigger-administered group are shown.

[0062] Figure 10E The infiltration degree of immune cells in the liver of the MacTrigger-administered group is shown.

[0063] Figure 11A The effect of MacTrigger administration on larger tumors 10 days after tumor manipulation is shown. The left graph shows the change in tumor volume over time, and the right graph shows the ratio of M1 macrophages to M2 macrophages in the tumor.

[0064] Figure 11B The effect of MacTrigger administration on tumor-bearing mice transplanted with 4T1 cells or Colon-26 cells is shown. Figure 11B Shown are the changes in tumor volume over time after administration of MacTrigger.

[0065] Figure 12A The combined effect of MacTrigger and anti-PD-1 antibodies is shown in tumor-bearing mice transplanted with tumor cells resistant to immune checkpoint inhibitors. Figure 12A Shown are the changes in tumor volume over time after MacTrigger administration.

[0066] Figure 12B The graph shows the changes in the proportion of PD-1-positive cells in CD4 single-positive T cells after MacTrigger administration.

[0067] Figure 12CFigure 2 shows the body weight changes of tumor-bearing mice transplanted with tumor cells resistant to immune checkpoint inhibitors after combined administration of MacTrigger and anti-PD-1 antibodies.

[0068] Figure 12D Shown are the effects of MacTrigger combined with an anti-PD-1 antibody on liver weight (left) and spleen weight (right) in tumor-bearing mice transplanted with tumor cells resistant to immune checkpoint inhibitors.

[0069] Figure 13 The figures show the effects of MacTrigger administration and combined administration of MacTrigger and anti-PD-1 antibody on triple-negative breast cancer cell-bearing mice. Figure 13 Shown are the changes in tumor volume over time after administration.

[0070] Figure 14A The ratio of gene expression levels in human macrophages (expression level in M2 / expression level in M0) is shown. Indicates p<0.001.

[0071] Figure 14B The ratio of gene expression levels in human macrophages (expression level in M2 / expression level in M1) is shown. Indicates p<0.001.

[0072] Figure 14C The ratio of gene expression levels in human macrophages (expression level in M2 / expression level in M0) is shown. Indicates p<0.001.

[0073] Figure 15 The figure shows the production of M2 macrophage-specific cytokines induced by human MacTrigger.

[0074] Figure 16 A schematic diagram showing the cancer treatment strategy of the present invention. DETAILED DESCRIPTION

[0075] In the present invention, a "subject" is a vertebrate, and examples thereof include birds or mammals, for example, mammals such as mice, rats, hamsters, guinea pigs, horses, cows, pigs, goats, sheep, donkeys, dogs, and cats, as well as primates such as monkeys, chimpanzees, gorillas, orangutans, bonobos, and humans, particularly humans. As described above, in this specification, the term "subject" is used to include humans. When excluding humans, the term "non-human" is used.

[0076] In this specification, "treatment" can refer to both preventive treatment and therapeutic treatment. Preventive treatment is used to include preventing the occurrence of a disease, delaying the onset of the disease, and reducing the incidence of the disease. Therapeutic treatment is used to include reducing the rate of disease progression, delaying deterioration, preventing deterioration, alleviating disease symptoms, curing the disease, and alleviating the disease.

[0077] In this specification, "macrophage" is a type of white blood cell that migrates within a living organism through amebic movement. Macrophages have phagocytic properties and can ingest foreign matter such as bacteria, viruses, and dead cells. Macrophages are one of the main immune cells and play an important role in tissue inflammation and immune responses

[27] . In particular, the main characteristics of macrophages in tumor tissues include: (1) macrophages migrate to tumor tissues through CC chemokine ligand 2 (CCL2) and granulocyte macrophage colony-stimulating factor (GM-CSF) secreted by cancer cells and stromal cells [28,29]; (2) macrophages in tumor tissues can be polarized to the anti-inflammatory M2 phenotype [18,30,31]. Macrophages transform into M1 or M2 phenotypes depending on the inflammatory state of the tissue environment, thereby controlling the inflammatory environment. M2 macrophages participate in wound healing, tissue regeneration, and immunosuppression [30,32]. In particular, in tumor tissues, macrophages polarize into M2 macrophages, which play an important role in the construction of an immunosuppressive environment

[30] . In addition, it is known that the activity of arginase 1 (Arg1) in mouse M2 macrophages is very high

[33] . Arg1 catalyzes the hydrolysis of arginine into urea and ornithine. As a result, the amount of arginine decreases, and immunosuppression is promoted

[34] . Arg1 is not expressed much in M0 and M1 macrophages, so Arg1 activity is one of the important markers for identifying the mouse M2 phenotype

[35] . M1 macrophages express one or more or all molecules selected from the group consisting of CD80, CD86, TLR-2, TLR-4, iNOS and MHC-II, and preferably secrete one or more or all cytokines selected from the group consisting of interferon-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, IL-12, IL-23, CXCL9, CXCL16 and CCL5. M2 macrophages express one or more or all molecules selected from the group consisting of CD163, CD206, CD209, FIZZ1 and Ym1 / 2, and preferably secrete one or more or all cytokines selected from the group consisting of IL-10, TGF-β, CCL1, CCL17, CCL18, CCL22, CCL24, CXCL13 and VEGF. It is known that M1 macrophages are induced by monocytes using, for example, LPS, granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, and TNF-α, and M2 macrophages are induced by monocytes using, for example, IL-4, IL-13, IL-10 or IL-21. It is known that there are four subtypes of M2 macrophages (M2a, M2b, M2c, and M2d). Examples of human M2 macrophage markers include IDO, IL-10, TGF-β, CD115, CD204, CD163, CD206, CD209, FceR1, VSIG4, IRF4, and STAT6.Therefore, human M2 macrophages can be determined by using one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve species selected from the group consisting of IDO, IL-10, TGF-β, CD115, CD204, CD163, CD206, CD209, FceR1, VSIG4, IRF4, and STAT6. Untreated (naive) macrophages (macrophages that have not been stimulated) are referred to as M0 macrophages, and M0 macrophages are reversibly polarized into M1 macrophages and M2 macrophages. M0 macrophages are differentiated from monocytes. Monocytes are differentiated from hematopoietic stem cells via myeloid progenitor cells, monocyte dendritic cell progenitor cells, and common monocyte progenitor cells. M0 macrophages can be obtained by stimulating monocytes with phorbol 12-myristate 13-acetate (PMA) or macrophage colony-stimulating factor (M-CSF). M0 macrophages can be characterized by being negative for markers of M1 macrophages and markers of M2 macrophages.

[0078] In this specification, "regulatory sequence" refers to a sequence having the activity of driving a gene to which it is operably connected, or of transcribing RNA from the gene. A regulatory sequence is, for example, a promoter. Examples of promoters include class I promoters (which can be used for transcription of rRNA precursors), class II promoters (composed of a core promoter and an upstream promoter element, which can be used for transcription of mRNA), and class III promoters (which can be further subdivided into type I, type II, and type III). A regulatory sequence is also referred to as a regulatory sequence, as long as it is a promoter that can transcribe mRNA in cells such as animal cells and plant cells. For example, as the first regulatory sequence, various pol II promoters can be used. As pol II promoters, CMV promoter, EF1 promoter (EF1α promoter), SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, CBh promoter, etc. can be cited, but are not limited to these. In addition, promoters also include pol III promoters such as T7 promoter, T3 promoter, SP6 promoter, etc., which drive phage-derived RNA polymerases, and U6 promoters. The T7 promoter is preferably used for transcription from circular DNA, and the SP6 promoter is preferably used for transcription from linear DNA. In addition, the promoter can be an inducible promoter. An inducible promoter refers to a promoter that can induce the expression of a polynucleotide operably linked to the promoter only in the presence of an inducer that drives the promoter. Among inducible promoters, there are promoters that can induce the expression of a polynucleotide operably linked to the promoter only in the absence of an inhibitor that inhibits promoter activity. Inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters, but are not limited to these. In addition, inducible promoters also include promoters that can be driven by drugs. Examples of such drug-inducible promoters include the Cumate operator sequence, the λ operator sequence (e.g., 12×λOp), and tetracycline-inducible promoters. Examples of tetracycline-inducible promoters include promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or a reverse tetracycline-controlled transactivator (rtTA). An example of a tetracycline-inducible promoter is the TRE3G promoter. A regulatory sequence is capable of expressing a gene to which it is operably linked, at least in M2 macrophages. Operably linked means that a gene is linked to a regulatory sequence such that gene expression is driven by the regulatory sequence.

[0079] In this specification, "cell" refers to a vertebrate cell, preferably a mammal, more preferably a human cell. Cells with differentiation ability / polarization ability to M2 macrophages refer to hematopoietic stem cells, myeloid progenitor cells, monocyte dendritic cell progenitor cells, common monocyte progenitor cells, monocytes or M0 macrophages, or any cell in the differentiation process. These cells are all capable of differentiating into M2 macrophages in the body of an individual when administered to the individual. In particular, hematopoietic stem cells differentiate into monocytes in the bone marrow via myeloid progenitor cells, monocyte dendritic cell progenitor cells and common monocyte progenitor cells. The differentiated monocytes differentiate into macrophages in peripheral tissues. Macrophages migrate to tumor tissue and polarize into M2 macrophages.

[0080] In this specification, "differentiation / polarization capacity" refers to differentiation capacity or polarization capacity, or both differentiation capacity and polarization capacity. In this specification, "differentiation / polarization" refers to differentiation or polarization, or both differentiation and polarization. Polarization refers to the reversible change between M0, M1, and M2. Differentiation refers to the change in cell stage other than polarization.

[0081] According to the present invention, cells having the ability to differentiate / polarize into M2 macrophages can be provided. In a preferred embodiment, cells having the ability to differentiate / polarize into M2 macrophages contain a nucleic acid operably linked to a regulatory sequence, more preferably a nucleic acid encoding an inflammatory cytokine. According to the present invention, cells containing a nucleic acid encoding an inflammatory cytokine operably linked to a regulatory sequence can be provided. In a preferred embodiment, these cells are capable of expressing the aforementioned inflammatory cytokines after differentiation / polarization into M2 macrophages.

[0082] According to the present invention, there can be provided a cell having differentiation ability / polarization ability to M2 macrophages, which contains a nucleic acid encoding an inflammatory cytokine operably linked to a regulatory sequence and can express the above-mentioned inflammatory cytokine after differentiation / polarization into M2 macrophages. Such cells can differentiate / polarize into M2 macrophages in tumor tissue, thereby being able to express inflammatory cytokines in tumor tissue, alleviate the inflammatory suppression state of tumor tissue, and preferably convert the tumor tissue into an inflammatory environment (or an environment where the ratio of the number of M1 macrophages / the number of M2 macrophages (the ratio of the number of M1 macrophages / the number of M2 macrophages)>1). Tumor tissue is converted into an inflammatory environment, and NK cells or T cells are recruited into the tumor tissue, which can exert or enhance the anti-tumor effect on tumor tissue. The anti-tumor effect on tumor tissue of the object of the cell of the present invention is preferably obtained mainly by activation of the immune system of the above-mentioned object other than the administered cells (specifically, the recruitment and activation of immune cells such as NK cells and T cells possessed by the above-mentioned object).

[0083] In a preferred embodiment, the cell having the ability to differentiate / polarize to M2 macrophages is any cell selected from the group consisting of M0 macrophages, monocytes, common monocyte progenitor cells, monocyte dendritic cell progenitor cells, myeloid progenitor cells and hematopoietic stem cells, and any cell in the differentiation stage from hematopoietic stem cells to M0 macrophages. In a preferred embodiment, the cell having the ability to differentiate / polarize to M2 macrophages can be a cell in a state of differentiation between hematopoietic stem cells and myeloid progenitor cells. In a preferred embodiment, the cell having the ability to differentiate / polarize to M2 macrophages can be a cell in a state of differentiation between myeloid progenitor cells and monocyte dendritic cell progenitor cells. In a preferred embodiment, the cell having the ability to differentiate / polarize to M2 macrophages can be a cell in a state of differentiation between monocyte dendritic cell progenitor cells and common monocyte progenitor cells. In a preferred embodiment, the cells having the ability to differentiate / polarize to M2 macrophages can be cells having a state of differentiation between a common monocyte progenitor cell and a monocyte. In a preferred embodiment, the cells having the ability to differentiate / polarize to M2 macrophages can be cells having a state of differentiation between a monocyte and an M0 macrophage. In a more preferred embodiment, the cells having the ability to differentiate / polarize to M2 macrophages can be any cell in the differentiation stage from a monocyte to an M0 macrophage, more preferably an M0 macrophage. In a preferred embodiment, the cells having the ability to differentiate / polarize to M2 macrophages can be macrophages that are negative for iNOS, CD206, and Arg-1 (i.e., non-M1 macrophages and non-M2 macrophages).

[0084] In one embodiment, inflammatory cytokines can alleviate (reduce) the inflammatory suppression state of tumor tissue. In a preferred embodiment, inflammatory cytokines can convert tumor tissue from an immunosuppressive environment to an inflammatory environment. In a preferred embodiment, inflammatory cytokines are inflammatory cytokines selected from the group consisting of tumor necrosis factor-α (TNF-α), IFN-γ, IL-1 (such as IL-1β), IL-2, IL-6, IL-12, IL-17 and IL-23. In a preferred embodiment, inflammatory cytokines are inflammatory cytokines selected from the group consisting of tumor necrosis factor-α (TNF-α), IFN-α, IFN-γ, IL-1 (such as IL-1β), IL-2, IL-6, IL-8, IL-12, IL-17 and IL-23. In a preferred embodiment, the inflammatory cytokine can be TNF-α. Human TNF-α can, for example, have a sequence registered in GenBank: QCI55793.1 or a sequence of TNF-α corresponding to the sequence. Human TNF-α includes, for example, peptides that have a sequence identity of 90% or greater to the sequence registered in GenBank: QCI55793.1 and possess the function of human TNF-α. The function of human TNF-α may refer to the ability to induce inflammation in human subjects. When administered to humans, any inflammatory cytokine capable of inducing inflammation in humans can be used without particular limitation, but human cytokines are preferred.

[0085] In a preferred embodiment, the regulatory sequence is capable of expressing the above-mentioned genes of inflammatory cytokines, etc., which are operably linked to the regulatory sequence, in M2 macrophages. In a preferred embodiment, M2 macrophages can selectively or specifically express genes such as inflammatory cytokines, which are operably linked to the regulatory sequence. Here, M2 macrophage selectivity and specificity refer to driving stronger gene expression in M2 macrophages than at least in M0 macrophages and M1 macrophages (more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 20 times, more than 30 times, more than 40 times, more than 50 times, more than 100 times, more than 500 times or more than 1000 times stronger gene expression). In one embodiment, the regulatory sequence is a regulatory sequence of a gene selected from the group consisting of CD163, CD200 R1, CD301, CXCR1, CXCR2, CD209, dendritic cell-associated C-type lectin-1, Fc RIa, IL-1RII, CD206, arginase 1, IFR4, PPARγ, STAT6, FIZZI, IL-1ra, IL-10, TGF-β, CCL1, CCL14, CCL18, CCL22, CCCL23, CCL24, CCL26, and YM1. In one embodiment, the regulatory sequence may be a promoter of a gene selected from the group consisting of CCL-22, LOX, CISH, FCER15, ALOX15, F13A1, IDO1, MMP1, ALDH1A2, CD209, TGM2, VCAM1, MMP12, and SPINT2. In one preferred embodiment, the regulatory sequence may be a promoter of a gene selected from the group consisting of CCL-22, CISH, ALOX15, and TGM2. The regulatory sequence is preferably derived from the human genome. The regulatory sequence may preferably be a promoter of a human gene selected from the group consisting of CCL-22, CISH, ALOX15, and TGM2. In one embodiment, the regulatory sequence may be CCL-22. In another embodiment, the regulatory sequence may be CD209.

[0086] In a preferred embodiment, according to the present invention, cells at any stage of differentiation from monocytes to M0 macrophages can be provided, which contain nucleic acids encoding inflammatory cytokines operably linked to regulatory sequences selective or specific for M2 macrophages, and are capable of expressing the above-mentioned inflammatory cytokines after differentiation / polarization into M2 macrophages. In a preferred embodiment, according to the present invention, cells at any stage of differentiation from monocytes to M0 macrophages can be provided, which contain nucleic acids encoding TNF-α operably linked to regulatory sequences selective or specific for M2 macrophages, and are capable of expressing the above-mentioned TNF-α after differentiation / polarization into M2 macrophages. In a preferred embodiment, the cells are monocytes or M0 macrophages. In a preferred embodiment, the cells are macrophages that are neither M1 nor M2. In a preferred embodiment, the cells are, for example, macrophages that are negative for iNOS, CD206, and Arg-1.

[0087] In a preferred embodiment, the present invention provides M0 macrophages comprising a nucleic acid encoding an inflammatory cytokine operably linked to a regulatory sequence selective or specific for M2 macrophages, and capable of expressing the inflammatory cytokine after differentiation / polarization into M2 macrophages. In a preferred embodiment, the present invention provides M0 macrophages comprising a nucleic acid encoding TNF-α operably linked to a regulatory sequence selective or specific for M2 macrophages, and capable of expressing the TNF-α after differentiation / polarization into M2 macrophages.

[0088] In one embodiment, the cells of the present invention release inflammatory cytokines in tumor tissue. In one embodiment, the cells of the present invention increase the ratio of the number of M1 macrophages to the number of M2 macrophages (the ratio of the number of M1 macrophages to the number of M2 macrophages) in tumor tissue to 1 or more. In one embodiment, the cells of the present invention create an inflammatory environment in tumor tissue. In one embodiment, the cells of the present invention increase immune cells such as NK cells and / or T cells in tumor tissue.

[0089] According to the present invention, a composition containing the cells of the present invention can be provided. The composition of the present invention may contain, in addition to the cells, a pharmaceutically acceptable solution. The composition of the present invention may also contain, in addition to the cells, pharmaceutically acceptable additives (e.g., salts, buffers, thickeners, isotonic agents, dispersants, and other pharmaceutical additives). The composition of the present invention may also contain, in addition to the cells, a pharmaceutically acceptable solution and pharmaceutically acceptable additives (e.g., salts, buffers, thickeners, isotonic agents, dispersants, and the like).

[0090] According to the present invention, a composition for releasing inflammatory cytokines in tumor tissue is provided, comprising the cells of the present invention. According to the present invention, a composition for creating an inflammatory environment in tumor tissue is provided, comprising the cells of the present invention. According to the present invention, a composition for increasing the ratio of the number of M1 macrophages to the number of M2 macrophages in tumor tissue to 1 or greater is provided, comprising the cells of the present invention. According to the present invention, a composition for increasing immune cells such as NK cells and / or T cells in tumor tissue is provided, comprising the cells of the present invention.

[0091] In one embodiment, the composition of the present invention can be used to treat cancer. In one embodiment, the composition of the present invention can be used to treat cancer patients. In one embodiment, the composition of the present invention can be used to alleviate the inflammatory suppression state of cancer tissue in cancer patients. In one embodiment, the composition of the present invention can be used to convert the inflammatory suppression state of cancer tissue in cancer patients into an inflammatory state. In one embodiment, the composition of the present invention can be used to recruit immune cells to cancer tissue in cancer patients. In one embodiment, the composition of the present invention can be used to treat cancer in cancer patients (e.g., during treatment).

[0092] Examples of cancer include, but are not particularly limited to, hematological cancers such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, melanoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, breast cancer (including triple-negative breast cancer), prostate cancer, bladder cancer, vaginal cancer, neck cancer, head and neck cancer, uterine cancer, and cervical cancer. Solid cancers such as cancer of the lungs, liver, gallbladder, bile duct, kidney, pancreas, lung, colon, colorectal, rectal, small intestine, stomach, esophagus, testicular, ovarian, bladder, and brain; cancers of bone, cartilage, adipose, muscle, vascular, and hematopoietic tissues; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant neurilemmoma, osteosarcoma, and soft tissue sarcoma; and blastomas such as hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, and retinoblastoma.

[0093] According to the present invention, the composition or cell of the present invention can be administered parenterally to a subject. Examples of parenterally administered methods are not particularly limited, and include intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intrathoracic administration, intraarticular administration, intramyocardial administration, intraventricular administration, intracerebroventricular administration, intramedullary administration, and intracerebrospinal fluid administration. In a preferred embodiment, parenterally administered methods may be intravenous administration.

[0094] According to the present invention, a method for treating cancer in a subject in need thereof is provided, comprising administering a therapeutically effective amount of cells of the present invention to the subject. In one embodiment, the cancerous tissue of the subject is transformed into an inflammatory environment.

[0095] According to the present invention, a method for treating a subject having cancer is provided, comprising administering a therapeutically effective amount of cells of the present invention to the subject. In one embodiment, the cancerous tissue of the subject is transformed into an inflammatory environment.

[0096] According to the present invention, there can be provided a method for producing inflammatory cytokines in a tumor tissue of a subject having cancer, comprising administering an effective amount of cells of the present invention to the subject. According to the present invention, there can be provided a method for alleviating an inflammatory suppression state in a tumor tissue of a subject having cancer, comprising administering an effective amount of cells of the present invention to the subject. According to the present invention, there can be provided a method for converting a tumor tissue of a subject having cancer (e.g., from an inflammatory suppression state) into an inflammatory environment, comprising administering an effective amount of cells of the present invention to the subject. According to the present invention, there can be provided a method for causing the number of M1 macrophages / M2 macrophages in a tumor tissue of a subject having cancer to be greater than 1, comprising administering an effective amount of cells of the present invention to the subject.

[0097] According to the present invention, the subject with cancer can be a subject before, during or after immunotherapy. According to the present invention, the administration of the cells of the present invention can also be combined with cancer therapy to treat a subject with cancer. According to the present invention, a subject with cancer receives an effective amount of the cells of the present invention and cancer therapy. Administration of an effective amount of the cells of the present invention to a subject with cancer can ensure that the tumor becomes an inflammatory tumor, causing the immune system in the body to respond to the inflammatory tumor and begin to attack the tumor. Therefore, administration of an effective amount of the cells of the present invention to a subject with cancer can make all cancer immunotherapies and antibody therapies more effective. Cancer therapy can be, for example, antibody therapy. In antibody therapy, antibodies that bind to cancer antigens and have antibody-dependent cellular cytotoxicity activity (ADCC activity) or complement-dependent cytotoxicity activity (CDC activity) can be administered to cancer patients. Administration of the cells of the present invention can promote the recruitment of NK cells to tumor tissues by making non-inflammatory tumors inflammatory, thereby exerting the ADCC activity of the antibodies bound to cancer cells in the tumor tissue. Antibodies can be administered at, for example, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 Below M or 10 -11 M or less, or 10 -8 M~10 -12The binding dissociation constant (KD) of M is combined with cancer antigen. Ideally, the antibody preferably has high binding selectivity to cancer cells and does not show significant binding to normal cells. Cancer immunotherapy can be, for example, T cell adoptive immunotherapy, chimeric antigen receptor (CAR) expression immune cell therapy or immune checkpoint inhibitor therapy. T cell adoptive immunotherapy includes administering an effective amount of T cells combined with cancer antigens to an object with cancer. T cells can be T cells proliferated by cell culture, or T cells rejuvenated by initialization to iPS cells, for example, T cells combined with cancer antigens can be obtained from cancer patients and initialized to obtain iPS cells, then iPS cells are subjected to differentiation induction and obtained T cells (for example, with reference to US9,206,3934B). By cloning iPS cells and / or suppressing or stopping the expression of RAG1 and 2, T cells can become T cells with homogenized T cell receptors (TCR) (for example, with reference to US9,206,3934B). Chimeric antigen receptor (CAR) expression immune cell therapy includes administering an effective amount of CAR immune cells (such as CAR expressing T cells and CAR expressing NK cells, etc.) that bind to cancer antigens to a subject with cancer. Immune checkpoint inhibitor therapy includes administering an effective amount of immune checkpoint inhibitors to a subject with cancer. It is believed that immune checkpoint inhibitors can relieve immunosuppression, i.e., release the brakes, but the effectiveness is low when the immune system itself is not activated. Administration of an effective amount of cells of the present invention can increase the inflammatory nature of the tumor and activate immunity to the tumor, so it is expected to improve the effectiveness of immune checkpoint inhibitors.

[0098] According to the present invention, the cells or cancer immunotherapy agents of the present invention, or pharmaceutical compositions containing them, can be provided for use in a cancer treatment method comprising administering an effective amount of the cells of the present invention and cancer immunotherapy. According to the present invention, the use of the cells or cancer immunotherapy agents of the present invention, or both, in the manufacture of a medicament for use in a cancer treatment method comprising administering an effective amount of the cells of the present invention and cancer immunotherapy can be provided.

[0099] The above-mentioned antibodies are not particularly limited, and examples thereof include rituximab, trastuzumab, cetuximab, alemtuzumab, ofatumumab, obinutuzumab, daratumumab, elotuzumab, and avelumab. In antibody therapy, other humanized or human chimeric IgG1 antibodies that bind to cancer antigens can be administered.

[0100] As CAR expressing cells, CAR expressing immune cells can be enumerated. Immune cells are not particularly limited, and for example, T cells, NK cells, neutrophils, macrophages, etc. can be enumerated." Chimeric Antigen Receptor" (CAR) is a chimeric molecule having an antigen binding fragment (particularly scFv) of an antibody that binds to a cancer antigen and an activation domain of an immune cell. CAR is typically a molecule formed by connecting scFv, an extracellular hinge domain, a transmembrane domain (such as CD8α or CD28), and an activation signal transduction domain (such as CD3ζ). CAR can be introduced into cells and expressed on the cell surface. Cells expressing CAR can target specific antigens. For example, CAR is introduced into immune cells such as T cells or NK cells, and immune cells such as T cells or NK cells can be targeted to cancer. The first generation CAR is formed by connecting scFv, an extracellular hinge domain, a transmembrane domain (such as CD8α or CD28), and an activation signal transduction domain (such as CD3ζ). The second generation CAR also contains a costimulatory molecule signal transduction domain in order to activate the immune cells into which CAR is introduced. As the costimulatory molecule signal transduction domain, costimulatory factors such as CD28, 4-1BB, OX40, CD27 and ICOS can be used. Two or more costimulatory factors are integrated into the third generation CAR. In this way, improvements are introduced into CAR for the continued proliferation of immune cells introduced into CAR in vivo. Any domain other than the scFv portion is preferably derived from human protein.

[0101] The antigen of the antibody or antigen-binding fragment of the antibody (eg, scFv) may be an antigen expressed on the surface of cancer cells (eg, a cancer antigen). As a cancer antigen, for example, it can be one or more selected from the group consisting of CD16, CD19, CD20, CD22, CD123, CD171, epidermal growth factor receptor (EGFR), particularly EGFRvIII, type 3 EGFR, de2-7 EGFR and HER2, carcinoembryonic antigen (CEA), prostate stem cell antigen (PSCA), B cell maturation antigen (BCMA), CS1, NKG2D, NKp30, B7H6, MUC-16 (CA125), receptor tyrosine kinase-like orphan receptor 1 (ROR-1), GD3, GM2, glypican-3 (GPC3), mesothelin, IL13R, c-KIT, c-MET, NY-ESO-1, WT1, MAGE-A3, MAGE-A4, MAGE-A10, HPV E6, HPV E7, CMV, AFP, PRAME, SSX2, KRAS, HER2 and PD-L1. The antibody or antigen-binding fragment thereof (eg, scFv) may also be a protein tag, for example. The antibody or antigen-binding fragment thereof (eg, scFv) may be a fluorescent protein such as fluorescein isothiocyanate (FITC).

[0102] Examples of CAR-T therapy drugs approved by the U.S. Food and Drug Administration (FDA) include Abecma (generic name: Idecabtagene vicleucel), Breyanzi (generic name: Lisocabtagene maraleucel), Carvykti (generic name: Ciltacabtagene autoleucel), Kymriah (generic name: Tisagenlecleucel), Tecartus (generic name: Brexucabtagene autoleucel), and Yescarta (generic name: Axicabtagene ciloleucel). It is believed that CAR-T therapy drugs other than those listed above can also be advantageously combined with the administration of the cells of the present invention.

[0103] Immune checkpoint inhibitors (ICIs) can, for example, bind to immune checkpoint molecules or their ligands to inhibit the function of immune checkpoints. Examples of immune checkpoint molecules include programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), and dendritic cell-specific protein signal regulatory protein α (SIRPα). The immune checkpoints responsible for each are called PD-1 immune checkpoints, CTLA-4 immune checkpoints, TIM-3 immune checkpoints, LAG-3 immune checkpoints, VISTA immune checkpoints, and SIRPα immune checkpoints. For example, by inhibiting the binding of PD-1 to PD-L1 or PD-L2, the PD-1 immune checkpoint can be inhibited. Furthermore, by inhibiting the binding of CTLA-4 to CD80 or CD86, the CTLA-4 immune checkpoint can be inhibited. Furthermore, by inhibiting the binding of TIM-3 to galectin-9, the TIM-3 immune checkpoint can be inhibited. Furthermore, by inhibiting the binding of LAG-3 to MHC class II molecules, the LAG-3 immune checkpoint can be inhibited. Furthermore, by inhibiting the binding of VISTA to VSIG-3 / IGSF11, the VISTA immune checkpoint can be inhibited. Furthermore, by inhibiting the binding of SIRPα to CD47, the SIRPα immune checkpoint can be inhibited. In this way, it is possible to inhibit one or more immune checkpoints selected from the group consisting of PD-1 immune checkpoints, CTLA-4 immune checkpoints, TIM-3 immune checkpoints, Lag3 immune checkpoints, VISTA immune checkpoints, and SIRPα immune checkpoints. Antibodies that inhibit the binding of two proteins can bind to receptors or ligands. For example, antibodies that inhibit PD-1 immune checkpoints can be antibodies selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies (e.g., nivolumab, pembrolizumab, avelumab, atezolizumab, and durvalumab).In addition, the antibody that inhibits CTLA-4 immune checkpoints can be an antibody selected from the group consisting of anti-CDLA-4 antibodies, anti-CD80 antibodies and anti-CD86 antibodies (e.g., ipilimumab and tremelimumab). In addition, the antibody that inhibits TIM-3 immune checkpoints can be an antibody selected from the group consisting of anti-TIM-3 antibodies and anti-galectin-9 antibodies (e.g., MGB453). In addition, the antibody that inhibits VISTA immune checkpoints can be an antibody selected from the group consisting of anti-VISTA antibodies and anti-VSIG-3 / IGSF11 antibodies (e.g., JNJ-61610588). In addition, the antibody that inhibits SIRPα immune checkpoints can enumerate antibodies selected from the group consisting of anti-SIRPα antibodies and anti-CD47 antibodies (e.g., Magrolimab, CC-90002). Separately, molecules that inhibit SIRPα-based immune checkpoints have been developed, including proteins that bind the CD47-binding domain of human SIRPα to the Fc region of human IgG1 to inhibit the binding of SIRPα to CD47 (e.g., Optopercept, Maplirpacept, and Evorpacept).

[0104] The object preferably has cancer cells expressing immune checkpoint molecules. Even when the tumor of the object is in an immunosuppressive state (i.e., non-inflammatory tumor (cold tumor)), the tumor can be converted into an inflammatory tumor (hot tumor). Therefore, all objects including objects with non-inflammatory tumors or objects with immune checkpoint inhibitor-resistant tumors can become applicable objects of immune checkpoint inhibitors. Therefore, in one embodiment, the object is an object resistant to immune checkpoint inhibitors. Objects resistant to immune checkpoint inhibitors can be appropriately judged by medical practitioners such as doctors based on the treatment after administration. Alternatively, even when the object does not express immune checkpoint molecules or hardly expresses them (i.e., when immune checkpoint inhibitors are ineffective), administering the cells of the present invention is also effective and can induce the immune system in tumors. In addition, the administration of the cells of the present invention and the interaction of immune cells can express immune checkpoint molecules in cancer, and the combined administration of the cells of the present invention and immune checkpoint inhibitors may be effective. Cancer has a mechanism for expressing immune checkpoint molecules in order to suppress the immune cells it contacts. In one embodiment, a method for treating a cancer of an object having cancer cells that do not express or hardly express immune checkpoint molecules comprises: administering an effective amount of the cells of the present invention to the object; and administering an effective amount of an immune checkpoint inhibitor to the object. There is no restriction on the order of administration of cells and administration of immune checkpoint inhibitors, but it is preferred to administer cells first and then administer immune checkpoint inhibitors. The reason is that the administration of the cells of the present invention activates the immune of the cancer cells of the object receiving the cells and / or enhances the expression of immune checkpoint molecules in cancer cells, thereby improving the effectiveness of immune checkpoint inhibitors. In one embodiment, immune checkpoint inhibitors can be administered to an object in which cells are administered to an object and the expression of immune checkpoint molecules in cancer cells is confirmed to be increased. The expression of immune checkpoint molecules in cancer cells can be confirmed by conventional methods such as immunohistochemical staining, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the case where the object does not express or hardly expresses immune checkpoint molecules despite the administration of the cells of the present invention (i.e., when the immune checkpoint inhibitor is ineffective), the object may not be administered immune checkpoint inhibitors.

[0105] According to the present invention, there is provided a cell of the present invention for use in the above-mentioned method. According to the present invention, there is provided a composition of the present invention for use in the above-mentioned method.

[0106] According to the present invention, there is provided use of the cells of the present invention in producing a drug for use in the above-mentioned method. According to the present invention, there is provided use of the cells of the present invention in producing a drug for treating cancer.

[0107] Example

[0108] 2. Materials and Methods

[0109] Materials

[0110] Dulbecco's modified Eagle's medium (DMEM), Rexrothwell Parker Memorial Institute-1640 (RPMI-1640) medium, antibiotic-antimicrobial mix stock solution, 4% paraformaldehyde in phosphate-buffered saline (4% FA PBS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Nakalai Tesque Co., Ltd. (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from Gibco Inc. (Waltham, MA, USA). Lipopolysaccharide (LPS) from Escherichia coli serotype O55:B5 and Triton X-100 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Mouse interleukin-4, interleukin-13, TNF-α, and interferon-γ were purchased from Peprotech (Rocky Hill, NJ, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Research Institute (Kumamoto, Japan). Lipofectamine 3000 and SuperScript III First-Strand Synthesis System were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). NucleoBond Xtra Maxi Plus EF, In-Fusion Snap Assembly Master Mix, PrimeSTAR Max DNA Polymerase, DNase I, and RNAiso plus were purchased from Takara Bio Co., Ltd. (Shiga, Japan). G418 sulfate was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LightCycler FastStart DNA Master SYBR Green Kit and Liberase DH Research Grade were purchased from Roche Diagnostics (Basel, Switzerland). Vivo-Track 680 (VT-680) was purchased from PerkinElmer, Inc. Phycoerythrin (PE)-labeled anti-mouse CD206 antibody, Brilliant Violet (BV) 421-labeled anti-mouse F4 / 80 antibody, Alexa Fluor 700-labeled anti-mouse CD86 antibody, Alexa Fluor 488-labeled anti-mouse CD11b antibody, FITC (fluorescein isothiocyanate)-labeled anti-mouse NKp46 antibody, PE-labeled anti-mouse NK1.1 antibody, Alexa Fluor 700-labeled anti-mouse CD3 antibody, Alexa Fluor 488-labeled anti-mouse CD8 antibody, and enzyme-linked immunosorbent assay (ELISA) MAX TMDeluxe Set mouse TNF-α and TMB substrate reaction stop buffer were purchased from BioLegend, Inc. (San Diego, CA, USA). VersaLyse was purchased from Beckman Coulter Inc. (Brea, CA, USA). Aspartate aminotransferase (AST) colorimetric activity assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Small animal blood collection needles (Goldenrod Animal Lancet) were purchased from MEDIpoint, Inc. (Mineola, NY, USA). CD206 / MRC1 (E6T5J) XP (商标) Rabbit mAb, CD86 (E5W6H) Rabbit mAb, and Ki-67 (D3B5) Rabbit mAb were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). 3,3-Diaminobenzidine (DAB) and HRP-labeled rabbit antibodies were purchased from Dako, Inc. (Glostrup, Denmark). The pGL3-mArg1 promoter / enhancer (-31 / -3810) was a kind gift from Peter Murray (Addgene plasmid # 34571; http: / / n2t.net / addgene:34571; RRID: Addgene_34571)

[36] . All other chemicals were of the highest commercial grade available.

[0111] Cell culture

[0112] RAW264.7 mouse macrophages and CT-26 mouse colon carcinoma cells were purchased from the American Type Culture Collection (ATCC). RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM). CT-26 cells were cultured in RPMI-1640. All culture media were supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimicrobial stock solution. Cells were cultured at 37°C in an atmosphere containing 5% CO₂ and 95% air. The culture medium was changed every two days, and cells reaching 90% confluence were detached with a cell scraper and cultured again.

[0113] THP-1 human monocytes were purchased from JCRB. THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimicrobial mix stock solution. Cells were cultured at 37°C in an atmosphere containing 5% CO2 and 95% air. The medium was replaced every two days, and cells reaching 90% confluence were harvested and cultured again.

[0114] 2.3. Evaluation of macrophages as drug carriers

[0115] 2.3.1 In vitro expression assay

[0116] The expression of Arg1 mRNA in RAW264.7 cells was evaluated by quantitative real-time polymerase chain reaction (qPCR). 6 The cells were plated at a density of 10 cells / well in 2.5 mL of culture medium in a 6-well plate. After 24 hours, the culture medium was replaced with CT-26 (3×10 6 Tumor culture medium (TCM) was prepared by incubating cells (100 cells, 2.5 mL of culture medium) for 24 hours, and supplementing other wells with 25 ng / mL IL-4 or IL-13, 200 ng / mL LPS, and 100 ng / mL TNF-α. Cells were recovered after 3 hours, 6 hours, 12 hours, and 24 hours of culture, and total ribonucleic acid (RNA) was extracted using RNAiso Plus according to the manufacturer's protocol. Then, 5 μg of RNA was reverse transcribed using the SuperScript III First Strand Synthesis System. The LightCycler FastStart DNA Master SYBR Green Kit was used for PCR amplification. The following PCR conditions were used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene, and the relative expression of the target gene was determined by the comparative CT method

[37] . Primers for Arg1 and GAPDH were purchased from Fasmac. All primer sequences are shown in Table 1 [33,38].

[0117] [Table 1]

[0118]

[0119] Single-cell RNA-seq data from human THP-1-induced macrophages were selected and exported from the NCBI GEO Data Set (https: / / www.ncbi.nlm.nih.gov / ), converted into text files, and then analyzed using iDEP.96 (http: / / bioinformatics.sdstate.edu / idep96 / ) for heat map analysis.

[0120] Gene expression evaluation of human macrophages was performed as follows. THP-1 cells were differentiated into macrophages by adding 20 ng / mL of phorbol 12-myristate 13-acetate (PMA) to the culture medium. 5×10 6THP-1 cells were cultured at a density of 10 cells / well. After 16 hours, macrophages were stimulated with IL-4 and IL-13 (25 ng / mL, respectively) to polarize them toward M2 macrophages. For polarization toward M1 macrophages, LPS (200 ng / mL) and IFN-γ (100 ng / mL) were used for 4 hours. For CCL-22, LOX, CISH, FCER15, ALOX15, F13A1, IDO1, MMP1, ALDH1A2, CD209, TGM2, VCAM1, MMP12, and SPINT2, cells were harvested after 72 hours of culture and total RNA was extracted using RNAiso Plus according to the manufacturer's protocol. For CCL-22, CISH, ALOX15, and TGM2, cells were harvested after 24, 28, and 72 hours of culture and total RNA was extracted using RNAiso Plus according to the manufacturer's protocol. Then, 5µg of RNA was reverse transcribed using the SuperScript III First-Strand Synthesis System. The LightCycler FastStart SNA Master SYBR Green Kit was used for PCR amplification. The following PCR conditions were used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. By comparing C T The relative expression of target genes was determined by the primers shown below.

[0121] [Table 2]

[0122]

[0123] 2.3.2 In vivo evaluation of macrophage migration and liver injury

[0124] All animal experiments were performed with the approval of the Kyushu University Animal Care and Use Committee. Six- to eight-week-old female BALB / cAJcl mice were obtained from Kyudo Co., Ltd. (Saga Prefecture). After one week of feeding, CT-26 cells (1 × 10 6 Then, VT-680 labeled RAW264.7 macrophages (1×10 cells in 100 μL PBS) were intravenously (iv) administered 5 days after tumor inoculation. 6cells). In vivo fluorescence imaging was performed using the IVIS Spectrum (IVIS Lumina II, Zenogen Co., Ltd.) 1, 4, and 7 days after macrophage administration to visualize macrophage migration into tumor tissue. Blood was then collected using an animal blood collection needle 4 and 7 days after macrophage administration. Mice were sacrificed, and tumors, livers, spleens, kidneys, and lungs were removed and assessed for macrophage accumulation using the IVIS Spectrum. Blood AST activity was analyzed using an AST detection kit according to the manufacturer's protocol.

[0125] 2.3.3 Evaluation of macrophage polarization using flow cytometry

[0126] The tumor and liver were removed and washed with PBS. After washing, the tumor was cut into small pieces and digested with DMEM containing Liberase (0.15 mg / mL) and DNase I (1.4 units / mL) at 37°C for 60 minutes. After 60 minutes, the solution was filtered through a 100μm filter and centrifuged at 300×g for 5 minutes. After removing the supernatant, 4 mL of VersaLyse was added and incubated at room temperature for 5 minutes. After incubation, the solution was centrifuged at 300×g for 5 minutes and resuspended in FACS buffer (DPBS containing 2% FBS). The liver was homogenized using a plunger on a 100μm filter and suspended in cold DPBS. After centrifugation at 200×g for 5 minutes, 4 mL of VersaLyse was added and incubated at room temperature for 5 minutes. After incubation, the solution was centrifuged at 200×g for 5 minutes and resuspended in FACS buffer. After resuspension, fluorescently labeled antibodies (Alexa Fluor 488-labeled anti-mouse CD11b antibody, BV421-labeled anti-mouse F4 / 80 antibody, PE-labeled anti-mouse CD206 antibody) were added and incubated at 4°C for 60 minutes. After incubation, the cells were washed and resuspended in FACS buffer. The expression of CD206 (M2 macrophage marker) in the administered macrophages (CD11b+, F4 / 80+, VT-680+) was evaluated by flow cytometry (Beckman Coulter, CytoFLEX).

[0127] 2.4. MacTrigger Production and In Vitro Characterization

[0128] 2.4.1 Plasmid preparation and transfection into macrophages

[0129] The mArg1 promoter / enhancer (-31 / -3810) of pGL3-mArg1, an M2 macrophage-specific promoter, was amplified by PCR using PrimeSTAR Max DNA polymerase and inserted into the cytomegalovirus (CMV) promoter-target gene region of pcDNA3.1 using in-fusion snap assembly mastermix. The gene encoding mouse TNF-α, an inflammatory cytokine, was purchased from Integrated DNA Technology, Inc (Coralville, IA, USA) and cloned downstream of the M2 macrophage-specific Arg1 promoter to create pcDNA3.1-mArg1p-TNF-α. Furthermore, the mouse TNF-α gene and enhanced green fluorescent protein (EGFP) coding sequence were inserted downstream of the CMV promoter to generate pcDNA3.1-CMVp-TNF-α and pcDNA3.1-CMVp-EGFP, respectively. Plasmid DNA was extracted using NucleoBond Xtra MaxiPlus EF. The transfected RAW264.7 cells expressed TNF-α in response to the Arg1 promoter (Arg1p-TNFα), or in response to the CMV promoter (CMVp-TNFα), or in response to the CMV promoter (CMVp-EGFP). RAW264.7 cells were cultured at 2.0×10 5Cells were seeded at a density of 10 cells / well in a 24-well plate. Twenty-four hours later, RAW264.7 cells were transfected with Arg1p-TNF-α, CMVp-TNF-α, or CMVp-EGFP vectors using Lipofectamine 3000 reagent according to the manufacturer's protocol. Six hours after transfection, the medium was replaced with fresh medium. These cells were treated with 400 μg / mL G418 to generate stable cell lines. Stable cell lines expressing IL-1β (NCBI Reference Sequence: NP_032387.1), IL-2 (NCBI Reference Sequence: NP_032392.1), IL-6 (GenBank: ABG81953.1), IL-12 (IL-12α subunit: NCBI Reference Sequence: NP_001152896.2, IL-12β subunit: NCBI Reference Sequence: NP_001290173.1), and IFN-γ (NCBI Reference Sequence: NP_032363.1) were generated using the same procedures except that TNF-α was substituted. Untransfected macrophages, CMVp-TNF-α-transfected macrophages, CMVp-EGFP-transfected macrophages, and Arg1p-TNF-α-transfected macrophages were designated Mac(UTD), Mac(CMVp), Mac(EGFP), and MacTrigger, respectively.

[0130] In addition, a human MacTrigger was produced. The details are as follows. For regions other than the promoter-MCS (multiple cloning site) of pcDNA3.1, Prime STAR Max DNA polymerase was used to amplify by PCR to produce a linearized vector. After vector amplification, the amplified product was treated with DpnI restriction enzyme. A synthetic gene encoding human TNF-α (sequence number 32) (CCL22p-hTNF-α) operably linked to the CCL-22 promoter (sequence number 31) was purchased from IDT. The purchased synthetic DNA was amplified by PCR in the same manner as above. The linearized vector and synthetic DNA obtained by amplification were mixed with in fusion snapassembly master mix, incubated at 60°C for 15 minutes, and plasmid DNA was produced by in fusion method. The produced plasmid DNA was added to JM109 competent cells and cultured on LB agar medium containing ampicillin overnight. Single colonies were picked and cultured in 5 mL of LB medium for 6 hours. Plasmid DNA was extracted from the cultured Escherichia coli using QIAGEN's plasmid extraction reagent. After confirming the target sequence by sequence analysis, JM109 was transformed again and cultured. After culture, plasmid DNA was extracted using the NucleoBond (trademark) Xtra Maxi Plus EF kit under endotoxin-free conditions.

[0131] [Table 3]

[0132]

[0133] In order to transfect the prepared plasmid into THP-1 cells, THP-1 cells were plated at 5.0×10 6 Cells were seeded at a density of 10 cells / well in a 24-well plate. Twenty-four hours later, the CCL-22-TNF-α vector was transfected into THP-1 cells using TransIT-Jurkat reagent according to the manufacturer's protocol. Six hours after transfection, the culture medium was replaced with fresh medium. These cells were treated with G418 to generate a stable cell line. This cell line was stimulated with PMA (20 ng / mL) for 16 hours to generate the humanized MacTrigger.

[0134] 2.4.2 Evaluation of TNF-α production capacity in vitro

[0135] MacTrigger cells were plated with 500 μL of culture medium at a volume of 1 × 10 5 Cells were plated at a density of 10 cells / well in a 24-well plate. After 24 hours, the culture medium was supplemented with fresh culture medium, TCM, or culture medium containing 25 ng / mL IL-4 or IL-13. Supernatants were collected after 24, 48, and 72 hours and analyzed by ELISA MAX according to the manufacturer's protocol. TM The concentration of TNF-α was measured using Deluxe Set Mouse TNF-α. TNF-α production capacity was evaluated using human MacTrigger cells in the same manner.

[0136] 2.4.3 Evaluation of MacTrigger's Inflammation-Inducing Ability in Vitro

[0137] Mac(EGFP) cells were cultured with 500 μL of culture medium at a rate of 2 × 10 5 Cells were plated at a density of 1×10 cells / well in a 24-well plate. After 24 hours, 25 ng / mL IL-13 was added to the culture medium. After 24 hours, the cells were harvested and incubated with 500 μL of culture medium containing IL-13 at a density of 1×10 5 Then, Mac(UTD), Mac(CMVp) or MacTrigger cells were plated at a density of 1×10 5 cells / well, 2×10 5 cells / well or 4×10 5Cells were plated at a density of 10 cells / well (effector cell / target cell ratio of 1, 2, or 4) in a 24-well plate in DMEM supplemented with IL-13. After 72 hours of culture, cells were harvested and treated with PE-conjugated anti-mouse CD206 and Alexa Fluor 700-conjugated anti-mouse CD86 antibodies. After incubation at 4°C for 60 minutes, the ratios of CD206+ cells / EGFP+ cells and CD86+ cells / EGFP+ cells were analyzed using CytoFLEX.

[0138] 2.4.4 Evaluation of MacTrigger's in vitro survival time

[0139] MacTrigger cells were plated at 5 × 10 3 Cells were seeded at a density of 10 cells / well in a 96-well plate and 200 μL of culture medium was added. After 24 hours, fresh culture medium, TCM, or culture medium containing 25 ng / mL IL-4 or IL-13 was added and cultured for 1 to 6 days. After culture, cell viability was assessed using CCK-8 according to the manufacturer's protocol. (商标) The absorbance at 450 nm was measured using 200 PRO MPlex (manufactured by TECAN), and the cell survival rate was calculated using the following formula.

[0140]

[0141] Here, A1 is the absorbance in the presence of cytokines and TCM, Actrl is the absorbance in the absence of cytokines and TCM, and A0 is the absorbance in the absence of cytokines and cells.

[0142] 2.5. Antitumor Effect of Artificial Macrophages in Tumor-Bearing Mice

[0143] Tumor-bearing mice were used to evaluate the anti-tumor effect of artificial macrophages. CT-26 cells, 4T1 cells, or Colon-26 cells (1.0×10 cells in 100 μL PBS, respectively) were injected subcutaneously into the abdomen of 8-week-old male BALB / c mice. 6 PBS (100 μL), Mac(UTD), Mac(CMVp), MacTrigger (1.0×10 in 100 μL PBS) were intravenously administered 5 days (day 0) or 10 days after tumor inoculation. 6 cells). Tumor volume and body weight were measured every 2 days, and blood was collected every 4 days.

[0144]

[0145] The tumor volume on day 0 after administration was set as 1, and the relative tumor volume of each group was calculated.3 The experiment was terminated at 4 ℃ (humanitarian endpoint) and all mice were sacrificed. After sacrifice, the tumor tissue and liver were removed and the liver weight was measured. Cells were extracted from the tumor tissue according to the protocol described in 2.3.3. After resuspending in FACS buffer, fluorescently labeled antibodies (Alexa Fluor 488-labeled anti-mouse CD11b antibody, BV421-labeled anti-mouse F4 / 80 antibody, PE-labeled anti-mouse CD206 antibody and Alexa Fluor 700-labeled anti-mouse CD86 antibody, or Alexa Fluor 700-labeled anti-mouse CD3 antibody and Alexa Fluor 488-labeled anti-mouse CD8 antibody, or FITC-labeled anti-mouse NKp46 antibody and PE-labeled anti-mouse NK1.1 antibody) were added and incubated. 1 antibody was added) and incubated at 4 ℃ for 60 minutes. After incubation, the cells were washed twice and resuspended in FACS buffer. The expression of CD206, CD86, CD3, CD8, NK1.1 and NKp46 was evaluated by flow cytometry.

[0146] Histological analysis

[0147] Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) were performed according to conventional protocols. Tumor and liver tissues were fixed with 4% PFA-PBS. For IHC, sections embedded in paraffin blocks were stained for CD86, CD206, and Ki67. Tumor tissues were deparaffinized, hydrated, heated at 95°C for 15 minutes in citrate buffer (pH 6.0), and blocked with 3% skim milk for 10 minutes. Sections were incubated overnight with anti-CD86 antibody (1:100), anti-CD206 antibody (1:200), or anti-Ki67 antibody (1:200). After washing with PBS, sections were incubated in 0.3% hydrogen peroxide-methanol buffer for 30 minutes to remove endogenous peroxidase activity. After washing with PBS, HRP-labeled secondary antibody was added and incubated for 30 minutes. After washing with PBS, DAB as an enzyme substrate was added. For HE staining, tumor and liver tissues were deparaffinized and washed with water. Hematoxylin was added and incubated for 5 minutes. After incubation, the sections were washed and hydrated. Eosin was added and incubated for 5 minutes, followed by washing. HE and IHC images of the sections were observed using an integrated fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan).

[0148] Statistical analysis

[0149] Statistically significant differences were tested using a two-sided Student's t-test. 、 、 The p values ​​are smaller than 0.05, 0.01, and 0.005, respectively, and NS indicates no significant difference.

[0150] result

[0151] like Figure 5A As shown in the scheme, an inflammatory cytokine encoding gene driven by an M2 macrophage-specific promoter is introduced into macrophages to obtain M0 macrophages (MacTrigger). As inflammatory cytokines, TNF-α, IL-1β (NCBI reference sequence: NP_032387.1), IL-2 (NCBI reference sequence: NP_032392.1), IL-6 (GenBank: ABG81953.1), IL-12 (α subunit: NCBI reference sequence: NP_001152896.2 and β subunit: NCBI reference sequence: NP_001290173.1) and IFN-γ (NCBI reference sequence: NP_032363.1) are used as an example. Figure 5A As shown in Figure 2, MacTrigger was cultured in the presence of IL-4 or IL-13, which are M2 macrophage-inducing cytokines, or in the presence of cancer cell culture supernatant (TCM). The culture supernatant was recovered 24 hours, 48 ​​hours, and 72 hours after the start of culture, and the amount (concentration) of TNF-α, IL-2, and IL-12 produced by the cells was determined according to 2.4.2. The results are shown in Figure 2. Figure 5B As shown. Figure 5B As shown, in the presence of IL-4 or IL-13, which are M2 macrophage-inducing cytokines, MacTrigger produced TNF-α. In addition, in the presence of TCM, MacTrigger also produced TNF-α. This shows that MacTrigger differentiates into M2 macrophages in the presence of IL-4 or IL-13 or in the presence of TCM, and thereby produces TNF-α. In addition, the differentiation into M2 macrophages is promoted in the presence of TCM, indicating that tumor tissue has an environment that promotes polarization into M2 macrophages. In addition, as Figure 5C As shown, MacTriggers incorporating IL-2 and IL-12, respectively, also produce these cytokines specifically in M2 cells, suggesting that these MacTriggers also release cytokines specifically in tumor tissue. By driving cytokine production with an M2-specific promoter, MacTriggers accumulate in tumors, polarize to M2 macrophages within tumor tissue, and begin releasing cytokines. As long as the cytokines are inflammatory, MacTriggers are expected to transform tumors into ones with an inflammatory environment.

[0152] Mac(UTD), Mac(CMVp) or MacTrigger was administered to tumor-bearing mice according to the administration method described in 2.4.4. Tumor tissues were recovered 16 days after administration and the expression of CD86 and CD206 was confirmed by immunohistochemical staining. Figure 6A As shown. Figure 6A As shown in the figure, the expression of CD86, an M1 marker, was significantly increased in the MacTrigger-treated group. In addition, the ratio of the number of M1 macrophages to the number of M2 macrophages (M1 / M2) was determined based on the above staining results. Figure 6B As shown in the figure, in the groups administered with either Mac(UTD) or Mac(CMVp), M1 / M2 was less than 1, and M2 was dominant, while in the group administered with MacTrigger, M1 / M2 was greater than 1, and M1 was dominant. This indicates that the tumor tissue became inflammatory only in the group administered with MacTrigger. Figure 6C As shown, MacTrigger does not release inflammatory cytokines in normal tissues, but only differentiates into M2 macrophages in tumor tissues, secretes inflammatory cytokines in tumor tissues, causing the tumor tissue to become inflammatory, and induces the increase of inflammatory cells such as M1 macrophages in tumor tissues.

[0153] according to Figure 7A Mac(UTD), Mac(CMVp), or MacTrigger was administered to tumor-bearing mice using the protocol shown and the method described in 2.4.4. The tumor size of the mice was measured 16 days after administration. Figure 7B As shown in the left figure, the MacTrigger-administered group had the strongest suppression of tumor size increase. In addition, the survival rate of tumor-bearing mice was confirmed, and the results were as follows: Figure 7B As shown in the right figure, most mice died in the PBS-administered group, while no deaths were observed in the MacTrigger-administered group. Thus, the inflammatory transformation of tumor tissue by MacTrigger administration significantly suppressed subsequent tumor growth and mortality.

[0154] In the in vitro experiments, MacTrigger was cultured in the presence of IL-4 or IL-13 or TCM, and the survival rate of MacTrigger was confirmed by WST-8 test. Figure 7C As shown. Figure 7C As shown in Figure 2, most MacTrigger cells died after about 4 days of administration. It is believed that the death of MacTrigger cells is due to the effect of TNF-α secreted by the cells themselves. Considering the lifespan of MacTrigger cells, Figure 7BThe sustained inhibition of tumor size growth and the maintenance of survival rate shown above persisted even after the death of MacTrigger, suggesting the persistence of the inflammatory environment in tumor tissue induced by MacTrigger and its effects. Consistent with this, M1 macrophages were dominant in tumor tissue 16 days after MacTrigger administration (refer to Figure 6A and 6B ).

[0155] Tumor tissue was removed 16 days after MacTrigger administration, and the expression of Ki67, a proliferation marker, was confirmed by immunohistochemical staining. Figure 7D As shown. Figure 7D As shown in Figure 2, the expression of Ki67 in the MacTrigger-treated group was significantly decreased compared to the other groups, indicating that cell proliferation in the tumor tissue was inhibited. The body weight of the tumor-bearing mice was checked over time. Figure 7E As shown, no body weight loss was observed as a result of MacTrigger administration.

[0156] The number of immune cells in the tumor tissue of individuals administered with MacTrigger was evaluated. Specifically, tumor tissue was recovered from mice 16 days after administration of the above-mentioned MacTrigger. Figure 8A As described above, NK cells and cytotoxic T cells were gated by flow cytometry. Figure 8B As shown. Figure 8B As shown, the proportions of NK cells and cytotoxic T cells in tumor tissue significantly increased in the MacTrigger-treated group. This suggests that the inflammation of tumor tissue induced by MacTrigger administration not only increases the number of M1 macrophages in tumor tissue, but also increases the number of NK cells and cytotoxic T cells.

[0157] We investigated the dynamics of macrophages in mice after administration of VT-680-labeled macrophages. Specifically, we administered VT-680-labeled macrophages to tumor-bearing and non-tumor-bearing mice using the same method as described in 2.3.2. In vivo imaging was performed using IVIS spectrum 1, 4, and 7 days after administration. The results are shown in Figure 2. Figure 9A and 9B As shown, macrophages accumulate in tumor tissues.

[0158] Four and seven days after macrophage administration, various organs including tumor tissues were removed from mice, and the distribution of macrophages was analyzed based on the expression of VT-680. Figure 9C As shown in Figure 3, macrophages accumulate in large numbers in tumors and liver. These results suggest that macrophages may migrate to the liver.

[0159] To evaluate the hepatotoxicity caused by macrophage accumulation in the liver, macrophages were administered to tumor-bearing mice and non-tumor-bearing mice using the same method as described in 2.3.2, and the AST activity in the blood was measured 4 and 7 days after administration. Figure 10B As shown in the results, no significant difference in AST levels was observed between the macrophage-administered group and the non-administered group. This suggests that although macrophages accumulate in the liver, they do not cause damage to the liver. The levels of CD206, an M2 macrophage marker, in tumor tissue and liver were studied. Figure 10C As shown in Figure 2, a significant increase in M2 macrophages was observed in tumor tissue. In contrast, no significant increase in M2 macrophages was observed in the liver. This suggests that macrophages polarize into M2 macrophages only in tumor tissue.

[0160] In fact, when confirming the effect of MacTrigger administration on the liver, such as Figure 10D As shown in Figure 2, no significant differences were observed in the liver weight and AST values ​​of mice between the MacTrigger-treated group and the PBS-treated group 16 days after administration. Figure 10E As shown in the results, no immune cell infiltration was observed in the liver tissue sections of the MacTrigger-treated group. This indicates that MacTrigger did not significantly differentiate into M2 macrophages in the liver and did not cause liver damage. In contrast, in the Mac(CMVp)-treated group, the treated macrophages adopted a structure that stably secreted TNF-α, and TNF-α was also secreted in the liver, and hepatomegaly and an increase in blood AST values ​​were confirmed (refer to Figure 10D ) and triggered the infiltration of immune cells into the liver (refer to Figure 10E ).

[0161] Figure 7A and 7B In the case of the experiment, MacTrigger was administered 5 days after the tumor was implanted. To evaluate the effect of administering MacTrigger after further tumor enlargement, the same amount of MacTrigger was administered 10 days after the tumor was implanted. The tumor volume after 10 days was approximately twice that after 5 days. The results are shown in Figure 2. Figure 11A As shown. Figure 11A As shown, MacTrigger administration exerted a significant anti-tumor effect. The M1 / M2 ratio of tumor tissue in the MacTrigger administration group was greater than 1 (refer to Figure 11A ), it is believed that MacTrigger changes the tumor environment from an anti-inflammatory environment to an inflammatory environment, thereby exerting an anti-tumor effect.

[0162] MacTrigger was administered to mice transplanted with 4T1 cells, a triple-negative breast cancer cell line, or Colon-26 cells, a colon cancer cell line. As a result, the MacTrigger-administered group showed a significant anti-tumor effect compared to the negative control (PBS-administered group) (refer to Figure 11B ).

[0163] We tried to combine it with immune checkpoint inhibitors. As an immune checkpoint inhibitor, we used anti-PD-1 antibody (InVivoMAb anti-mouse PD-1, BioXcell, BE0146). 4T1 cells are PD-L1 low-expressing cells and have no response to treatment with anti-PD-1 antibodies. 2×10 5 4T1 cells were administered id to BALB / c mice (female, n=5) to establish a triple-negative breast cancer model. Five days after administration, (i) PBS, (ii) anti-PD-1 antibody (50 μg), or (iii) MacTrigger (10 6 cells), or (iv) anti-PD-1 antibody (50 μg) and MacTrigger (10 6 Tumor volume and body weight were measured every 2 days. Figure 12A As shown in the figure, the anti-PD-1 antibody administration group showed almost no effect as expected. In contrast, the MacTrigger administration group showed a significant anti-tumor effect (refer to Figure 12A In addition, compared with the MacTrigger-administered group, the anti-PD-1 antibody and MacTrigger-administered group showed a significant increase in anti-tumor effect (refer to Figure 12A ). The ratio of CD4 single positive / PD-1 positive T cells in T cells in cancer tissues was confirmed. As a result, the ratio of CD4 single positive / PD-1 positive T cells was confirmed to increase in the MacTrigger administration group (refer to Figure 12B ). In this way, although the recruitment of immune cells in tumors was promoted, the proportion of immune cells expressing immune checkpoint molecules was also increased. It should be noted that no significant weight loss was observed in these drug administration groups (refer to Figure 12C ), and no other toxicity was confirmed (refer to Figure 12DCompared to the ineffectiveness of the anti-PD-1 antibody alone, the combination of the anti-PD-1 antibody and MacTrigger demonstrated a significantly greater anti-tumor effect than the MacTrigger group, a surprising result. It is believed that MacTrigger specifically transforms tumors into immunologically active tumors and recruits and increases PD-1-positive T cells in anti-PD-1 antibody-unresponsive tumor tissues, thereby transforming the tumor environment into one in which the anti-PD-1 antibody can exert its effects.

[0164] Further experiments were conducted using a triple-negative breast cancer model. Five days after 4T1 cell administration, the negative control group was given PBS, the control group was given a combination of doxorubicin 40 μg and cyclophosphamide 2 mg, and the experimental group was given a combination of anti-PD-1 antibody (50 μg) and MacTrigger (10 6 cells), all of which were administered intravenously. Figure 13 As shown. Figure 13 As shown, the control group exhibited the strongest anti-tumor effect, but experienced severe side effects such as blood in stool, blood in urine, and weight loss, leading to the discontinuation of the control group trial. In contrast, the experimental group exhibited a significant anti-tumor effect without any identifiable side effects.

[0165] A human MacTrigger was prepared. Genes showing M2 macrophage-specific expression in M0, M1, and M2 macrophages were extracted from single-cell RNA-seq data. 61 genes showed M2-specific expression. Volcano plot analysis was performed to extract genes with small p-values ​​and large fold changes. The fold changes (log2(FC)) and p-values ​​of the extracted genes are shown in Table 3.

[0166] [Table 4]

[0167]

[0168] The expression of the underlined genes in Table 3 was further analyzed by RT-PCR. Figure 14A As shown in Figures 1 to 2C, nearly all genes identified showed M2-specific expression. Among these, CCL-22, CISH, ALOX15, and TGM2 were identified as M2 macrophage-specific, with a high ratio of expression in M2 to expression in M0, making them particularly preferred promoters. CCL-22 denotes chemokine C-C motif ligand 22, CISH denotes cytokine-inducible SH2-containing protein, ALOX15 denotes arachidonic acid 15-lipoxygenase, and TGM2 denotes transglutaminase 2.

[0169] The gene encoding human TNF-α was linked to the human CCL-22 promoter and introduced into THP-1 cells. The THP-1 cells into which the gene was introduced were induced into macrophages by PMA treatment (20 ng / mL for 16 hours). The M2 of human TNF-α was induced in a culture medium containing 25 ng / mL of IL-4 or IL-13. The expression level of human TNF-α in M2 and M0 macrophages was confirmed by ELISA, and the expression of M2-specific TNF-α was confirmed (refer to Figure 15 ).

[0170] MacTrigger is a novel cell-based drug that functions as a trigger to induce the immune system in tumor tissues to treat cancer. This example reports on a "MacTrigger" that functions as a trigger to manipulate macrophages to induce an inflammatory environment only in tumor tissues. This allows for a strong anti-tumor effect based on their ability to eliminate foreign matter. The advantage of this study lies in the utilization of two unique functions of macrophages: (1) the ability to migrate to tumor tissues with an immunosuppressive environment, and (2) polarization to the anti-inflammatory M2 phenotype in the presence of such tumor tissues. When MacTrigger polarizes to the M2 phenotype, it promotes the release of tumor necrosis factor-α (TNF-α), an inflammatory cytokine. Administration of this MacTrigger to tumor-bearing mice significantly inhibited tumor growth compared to the non-administered group, the group without macrophage introduction, and the group with artificial macrophages that randomly release TNF-α. Furthermore, the ratio of the M1 phenotype to the M2 phenotype in tumor tissue was greater than 1 only in the MacTrigger group. Furthermore, the ratio of natural killer cells to CD8+ T cells in tumor tissue in the MacTrigger-treated group increased compared to the other groups. These results suggest that MacTrigger induces inflammation in tumor tissue, resulting in effective anti-tumor effects. Furthermore, no significant side effects were observed in normal tissues, particularly the liver. This is believed to be because MacTrigger does not polarize to the M2 phenotype in the liver, thus failing to induce inflammation. These results demonstrate that MacTrigger is a "trigger" capable of inducing inflammation only in tumor tissue, allowing the innate immune system to attack tumor tissue.

[0171] The entire contents of the documents cited in this specification are incorporated herein by reference.

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Claims

A cell having differentiation / polarization ability into an M2 macrophage, comprising a nucleic acid encoding an inflammatory cytokine operably linked to a regulatory sequence and capable of expressing the inflammatory cytokine after differentiation / polarization into an M2 macrophage.

2. The cell according to claim 1, which is any cell selected from the group consisting of M0 macrophages, monocytes, common monocyte progenitor cells, monocyte dendritic cell progenitor cells, myeloid progenitor cells and hematopoietic stem cells, and any cell at the differentiation stage from hematopoietic stem cells to M0 macrophages. The cell according to claim 1 or 2, which is a macrophage negative for iNOS, CD206, and Arg-1. The cell according to any one of claims 1 to 3, which is a cell at any stage of differentiation from a monocyte to an M0 macrophage.

5. The cell according to any one of claims 1 to 4, wherein The inflammatory cytokine is an inflammatory cytokine selected from the group consisting of TNF-α, IFN-α, IFN-γ, IL-1, IL-2, IL-6, IL-8, IL-12, IL-17 and IL-23.

6. The cell according to any one of claims 1 to 5, wherein The inflammatory cytokine is TNF-α.

7. The cell according to any one of claims 1 to 6, wherein The regulatory sequence is a regulatory sequence for M2 macrophages to specifically express the inflammatory cytokine.

8. The cell according to any one of claims 1 to 7, wherein The regulatory sequences are those of genes selected from the group consisting of CD163, CD200 R1, CD301, CXCR1, CXCR2, CD209, dendritic cell-associated C-type lectin-1, Fce RIa, IL-1RII, CD206, arginase 1, IFR4, PPARγ, STAT6, FIZZI, IL-1ra, IL-10, TGF-β, CCL1, CCL14, CCL18, CCL22, CCCL23, CCL24, CCL26, and YM1. 9 . A composition comprising the cell according to claim 1 .

10. The composition according to claim 9, for use in treating cancer.

Images