JP2021024797A - Tumor-associated macrophage activator - Google Patents

Abstract

To provide an agent to activate tumor immunity by an action mechanism different from those of conventional agents used in tumor immunity therapy.SOLUTION: A tumor-associated macrophage activator having a prolyl hydroxylase inhibitor as an active ingredient, activates tumor immunity by a new action mechanism (natural immunity system) different from those of conventional agents used in tumor immunity therapy, thereby effectively inhibiting tumor proliferation. According to the tumor-associated macrophage activator herein, a tumor-associated macrophage is directly activated, so that a tumor-associated macrophage is treated in vitro, enabling the preparation of a pharmaceutical for cancer treatment.SELECTED DRAWING: None

Description

The present invention relates to a tumor-related macrophage activator that can independently suppress tumor growth.

In tumor tissue, blood and vascular permeability is altered, reducing oxygen and nutrient supply and delivery of the drug to the tumor tissue. This contributes to the tumor's resistance to radiation or drug therapy. In particular, highly malignant cancer types are less sensitive to anticancer drugs and have lower response rates. In addition, treatment with a cell-killing anticancer drug, which is a standard therapeutic agent, has a low specificity in a tumor having a cell-killing effect, so that side effects are often increased.

The most abundant immune cells in tumor tissue are macrophages. Macrophages in tumors, called tumor-associated macrophages (TAMs), are classified into inflammatory (M1) and anti-inflammatory (M2) phenotypes. M1 macrophages have a high phagocytosis and are known to be inhibitory to tumor growth (Non-Patent Document 1). On the other hand, M2 macrophages promote tumor progression and metastasis and are strongly polarized in the tumor microenvironment (Non-Patent Document 2). M2 macrophages eliminate cytotoxic lymphocytes from tumors such as lung cancer and melanoma, which are classified as TAM-rich tumors with few T cells. Therefore, strong polarization to M2 macrophages suppresses tumor immune response function and causes treatment resistance.

Therefore, the development of tumor immunotherapy that reactivates host immunity in tumor tissues is underway. Examples of tumor immunotherapy include immunocell therapy, antibody therapy, cytokine therapy and the like. Other examples of tumor immunotherapy include immune checkpoint inhibitors that reactivate cytotoxic T cells by releasing signals that suppress the action of cytotoxic T cells. It is thought that there are a wide variety of cancers for which immune checkpoint inhibitors are effective, and it is attracting attention all over the world, and various clinical trials are being conducted with the aim of expanding the indications for the disease. As a result, ipilimumab (trade name: Yervoy), pembrolizumab (trade name: Keytruda), nivolumab (trade name: Opdivo), etc. have been approved one after another in the United States and Japan.

On the other hand, hypoxia-inducing factor (HIF) is a major regulator of cellular response to hypoxia. HIF stability is post-transcriptionally regulated by oxygen availability via prolyl isomerase (PHD). By administering a prolyl hydroxylase inhibitor to a tumor, the function of blood vessels in the tumor is normalized, and while the prolyl hydroxylase inhibitor does not exert an antitumor effect alone, it can be used in combination with an anticancer drug. It has been reported that the antitumor effect of the anticancer agent can be enhanced (Patent Document 1). In addition, it is disclosed in a plurality of reports that normalization of vascular function causes acceleration of tumor progression (Non-Patent Documents 3 and 4).

Nature. 2017 Mar 16; 543 (7645): 428-432. J Clin Invest. 2016; 126 (10): 3672-3679. Cancer Cell. 2008 Mar; 13 (3): 206-20. Nature. 2008 Dec 11; 456 (7223): 814-8.

Japanese Patent Application No. 2018-35112

Conventional tumor immunotherapy has focused on the control of the adaptive immune system, which induces a specific response by immune memory. However, according to conventional tumor immunotherapy, drug delivery efficiency is low in tumor tissues with incomplete blood flow and low tissue osmolality, and drug treatment via blood flow is not efficient. In particular, it is difficult to target deep tumors. Even immune checkpoint inhibitors, which are attracting worldwide attention, show a certain response rate, but their effects are still not sufficient. Immune checkpoint inhibitors exert their efficacy by a mechanism of action that reactivates T cells, and therefore, especially for tumors such as lung cancer and melanoma, which are classified as TAM-rich tumors with few T cells. Is poorly successful. In other words, there is a limit to the antitumor effect that can be obtained by the mechanism of action of the drugs used in conventional tumor immunotherapy.

Therefore, an object of the present invention is to provide a drug that activates tumor immunity by a mechanism of action different from that of the drugs used in conventional tumor immunotherapy.

As a result of diligent research by the present inventors, it has been newly found that a prolyl isomerase inhibitor, which is known as a drug for normalizing the function of blood vessels in a tumor, exerts a tumor growth inhibitory effect alone. Until now, it has been known that prolyl hydroxylase inhibitors do not exert antitumor effects by themselves, and normalization of vascular function, which is known as the mechanism of action of prolyl hydroxylase inhibitors, In general, the tumor growth inhibitory effect of the newly discovered prolyl hydroxylase inhibitor was unexpected in view of the fact that it has been reported that it can accelerate tumor progression.

As a result of further studies by the present inventors, it has been found that a prolyl isomerase inhibitor significantly improves the level of tumor-phagocytic macrophages when tumor blood vessels do not normalize. Since normalization of tumor blood vessels does not have a tumor growth inhibitory effect, and improvement of the level of tumor-eating macrophages has a tumor growth inhibitory effect, this result indicates that the prolyl hydroxylase inhibitor is the normalization of tumor blood vessels. It was found to activate tumor-related macrophages by a different mechanism of action. In addition, the tumor vascular normalization ability reported for prolyl hydroxylase inhibitors is associated with mouse Ly6C-negative monocytes, while the tumor-related macrophage activation ability found is associated with mouse Ly6C-positive monocytes. It was found that the tumor phagocytosis was enhanced and that prolyl hydroxylase inhibitors enhanced the tumor phagocytosis of mouse Ly6C-positive monocytes but did not increase the levels of Ly6C-negative monocytes. This further supported the different phagocytic mechanisms of tumor vascular normalization and tumor-related macrophage activation by prolyl hydroxylase inhibitors.

And since the level of tumor-poor macrophages was improved in macrophages isolated from bone marrow, the prolyl hydroxylase inhibitor is a drug having a mechanism of action that directly affects macrophages, that is, , It has been found that it is a drug that activates tumor immunity by a mechanism of action different from that of the drugs used in conventional tumor immunotherapy. In addition, the tumor growth inhibitory effect of the prolyl isomerase inhibitor alone was found in the tumor tissue lacking T cells because of the different mechanism of action between the prolyl isomerase inhibitor and the immune checkpoint inhibitor. Was further supported.

The present invention has been completed by further studies based on these findings.

That is, the present invention provides the inventions of the following aspects.
Item 1. A tumor-related macrophage activator containing a prolyl isomerase inhibitor as an active ingredient.
Item 2. Item 2. The tumor-related macrophage activator according to Item 1, which is a tumor phagocytosis enhancer for Ly6C-positive macrophages or their human counterparts.
Item 3. Item 2. The tumor-related macrophage activator according to Item 2, wherein the human counterpart is CD14 ⁺ CD16 ^- macrophages and / or CD14 ^{lo /} -CD16 ^{+ macrophages.}
Item 4. Item 2. The tumor-related macrophage activator according to Item 1 or 2, which is a tumor phagocytosis enhancer for ^{Ly6C low macrophages or their human counterparts.}
Item 5. Item 4. The tumor-related macrophage activator according to Item 4, wherein the human counterpart is CD14 ⁺ CD16 ^{-macrophages.}
Item 6. The prolyl isomerase inhibitors are FG-4592 (Roxadustat), FG-2216, FG-4497, dimethyloxalylglycine, BAY-85-3934 (Molidustat), AKB-6548 (Vadadustat), GSK1278863 (Daprodustat), JTZ. Item 3. The tumor-related macrophage activator according to any one of Items 1 to 3, which is at least one selected from the group consisting of -951, TM-6089, and DS-1093.
Item 7. Item 4. The tumor-related macrophage activator according to any one of Items 1 to 6, which is an anticancer agent.
Item 8. Lung cancer, melanoma, glioma, breast cancer, esophageal cancer, intraductal bile duct cancer, gastric cancer, bile sac cancer, pancreatic cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, Item 4. The tumor-related macrophage activator according to any one of Items 1 to 7, which is administered for rhabdomidal myoma, smooth myoma, or advanced cancer or treatment-resistant cancer.
Item 9. A method for producing a drug for treating cancer, which comprises reacting a macrophage fraction containing a tumor-related macrophage with a prolyl isomerase inhibitor in vitro.
Item 10. Item 9. The production method according to Item 9, wherein the macrophage fraction is a macrophage fraction obtained from the bone marrow of a cancer patient.
Item 11. Item 9. The production method according to Item 9 or 10, wherein the tumor-related macrophage comprises a Ly6C-positive macrophage or a human counterpart thereof.
Item 12. A drug for treating cancer, which comprises a first preparation containing the tumor-related macrophage activator according to any one of Items 1 to 8 and a second preparation containing an anticancer agent other than the tumor-related macrophage activator.
Item 13. A pharmaceutical composition for treating cancer, which comprises the tumor-related macrophage activator according to any one of Items 1 to 8 and an anticancer agent other than the tumor-related macrophage activator.

According to the tumor-related macrophage activator of the present invention, tumor immunity is activated by a new mechanism of action (innate immune system) different from the agents used in conventional tumor immunotherapy, thereby improving tumor growth. Can be suppressed. In addition, by the new mechanism of action of the tumor-related macrophage activator of the present invention, it is possible to suppress tumor growth even in a tumor tissue lacking T cells, which was poorly responded to by an immune checkpoint inhibitor. Further, according to the tumor-related macrophage activator of the present invention, the tumor-related macrophage is directly activated. Therefore, by treating the tumor-related macrophage in vitro, a drug for treating cancer can be prepared.

(I) It is an LLC tumor growth curve showing the result (n = 15) of mouse treatment with vehicle (Veh) or 100 mg / kg PHD inhibitor (FG) 10 days after mouse transplantation of lung cancer LLC tumor cells. (Ii) Shows the LLC tumor weight (n = 15) on day 16. It should be noted that it is the result of using the two-way ANOVA, where * indicates p <0.05 and *** indicates p <0.0001. (I) An LLC tumor growth curve showing the result (n = 8) of mouse treatment with a vehicle (Veh) or a 100 mg / kg PHD inhibitor (FG) 10 days after mouse transplantation of melanoma B16F10 tumor cells. (Ii) The B16F10 tumor weight (n = 8) on the 16th day is shown. It should be noted that it is the result of performing the Mann-Whitney test, where * indicates p <0.05 and *** indicates p <0.0001. (I) Quantitative results of the ratio of CC3-positive cells in all cells constituting the tumor tissue (n = 3), and (ii) Results of tumor cell proliferation assay at 72 hours after FG treatment (n = 3). Is shown. It should be noted that it is the result of one-way ANOVA, and ns indicates that there is no significant difference. It is a quantification result of tumor infiltration, and (i) CD45 ⁺ cell ratio (n = 12 to 13) and (ii) CD11b ⁺ F4 / 80 ⁺ cell ratio (n = 8) in the LLC tumor by flow cytometric analysis. Shown. It should be noted that it is the result of performing the Mann-Whitney test, and * indicates p <0.05. It is a quantification result of tumor infiltration, and shows the CD4 ⁺ and CD8 ⁺ cell ratio (n = 5) in lung cancer LLC tumor by flow cytometric analysis. It is the result of the Mann-Whitney test, and ns indicates that there is no significant difference. The quantification result (n = 5) of F4 / 80 ⁺ cell number per 1 mm ^{2 of the tumor is shown.} It should be noted that it is the result of performing the Mann-Whitney test, and ** indicates p <0.01. It is a quantification result of tumor infiltration, and shows the CD4 ⁺ and CD8 ⁺ cell ratio (n = 4) in the melanoma B16F10 tumor by flow cytometry analysis. The gating strategy in the flow cytometry of macrophages is shown. The quantification results (n = 6) ^{of Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophage ratios in lung cancer LLC tumors by flow cytometric analysis are shown. It should be noted that it is the result of performing the Mann-Whitney test, ** indicates p <0.01, and ns indicates that there is no significant difference. The quantification results (n = 4) ^{of Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophage ratios in melanoma B16F10 tumor by flow cytometric analysis are shown. Tumor growth curve (n = 4) by treating LLC tumor mouse models treated with clodronate liposomes (Clo) or plain control liposomes (Lip) with vehicle (Veh) or 100 mg / kg PHD inhibitor (FG). Shown. It should be noted that it is the result of two-way ANOVA, where *** indicates p <0.001 and ns indicates that there is no significant difference. The quantification result (n = 3) of F4 / 80 ^{+ cell number per 1 mm 2} of the tumor in FIG. 11 is shown. It should be noted that it is the result of performing the Mann-Whitney test, and *** indicates p <0.001. It is a quantification result (n = 3) of the vascular density and the vascular lumen area of a tumor 48 hours after administration of a GFP-labeled LLC tumor model to which a vehicle (Veh) or a PHD inhibitor (FG) was administered. It is the result of the Mann-Whitney test, and ns indicates that there is no significant difference. Quantification results (n = 5) of the number of ^{phagocytic cells (GFP +} F4 / 80 ⁺ ) in the tumor 48 hours after administration of a GFP-labeled LLC tumor model administered with a vehicle (Veh) or PHD inhibitor (FG). Shown. It should be noted that it is the result of performing the Mann-Whitney test, and ** indicates p <0.01. (I) Schematic diagram of in vitro bead phagocytosis assay and (ii) quantification result (n = 3) of phagocytotic BMDM (bone marrow-derived macrophage) ratio are shown. The gating strategy in the flow cytometry of GFP ^{+ macrophages is shown.} The quantification result (n = 5-6) of the ratio of ^{GFP +} macrophages (Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages) subdivided by Ly6C to ^{total GFP + macrophages by flow cytometric analysis is shown.} (I) Schematic of the exvibobead phagocytosis assay for Ly6C-positive macrophages; (ii) Phagocytotic Ly6C ^lo macrophage ratio quantification results (n = 5); and (iii) Phagocytotic Ly6C ^hi macrophage ratio quantification The conversion result (n = 4 to 5) is shown. It should be noted that it is the result of performing the Mann-Whitney test, and * indicates p <0.05. (I) Schematic diagram of the exvibobead phagocytosis assay for Ly6C-negative macrophages; and quantification results of the phagocytotic Ly6-negative macrophage ratio (n = 3-4). It is the result of the Mann-Whitney test, and ns indicates that there is no significant difference. (I) Vhl ^{fl / fl} LysM-Cre ^-/- (Control mouse (Ctrl); n = 9), and Vhl ^{fl / fl} LysM-Cre ^+/- (Macrophage-specific VHL knockout mouse (VHL KO); n = The tumor growth curve of the LLC tumor model of 7) and (ii) the tumor weight on day 16 (n = 7-9) are shown. Note that (i) is the result of two-way ANOVA, (ii) is the result of Mann-Whitney test, and ** indicates p <0.01. The flow cytometric analysis results (n = 6) of the ratios of ^{Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophages in the LLC tumor model of VHL KO are shown. It should be noted that it is the result of performing the Mann-Whitney test, and * indicates p <0.05. It is a qPCR analysis of bone marrow-derived macrophages (BMDM) isolated from mice administered with a vehicle (Veh) or a PHD inhibitor (FG), and shows the results of quantitative analysis of HIF downstream genes. (I) ^{Schematic of the procedure for transplanting Ly6C lo} macrophages selected from an LLC tumor mouse model into a tumor site in another LLC tumor mouse model; (ii) PBS-injected or vehicle (Veh) or FG-treated mouse tumor. Tumor growth curve (n = 6); and (iii) day 16 tumor weight (n = 6) in an LLC tumor mouse model transplanted with Ly6C ^{lo macrophages from.} (Ii) is the result of two-way ANOVA analysis, and ** indicates p <0.01. (Iii) is the result of the Mann-Whitney test. (I) Tumor weight at day 16 (n = 5-6) in an LLC tumor mouse model transplanted with ^{PBS-injected or transplanted Ly6C lo} macrophages or Ly6C ^neg macrophages from vehicle (Veh) or FG-treated mouse tumors; ( ii) Results of quantification of tumor vascular density (n = 3); and (iii) Results of quantification of tumor vascular lumen area (n = 3). Note that (i) is the result of the Mann-Whitney test; (ii) and (iii) are the results of the unpaired Student's t-test, where * is p <0.05 and ** is. It shows p <0.01. ^{(I) Schematic of the procedure of treating Ly6C lo} macrophages selected from tumors of an LLC tumor mouse model with a vehicle (Veh) or FG and transplanting them into another LLC tumor mouse model; (ii) PBS injection or Tumor growth curves (n = 5); and (iii) day 16 tumor weight (n = 5) in an LLC tumor mouse model transplanted with vehicle (Veh) or FG-treated Ly6C ^{lo macrophages.} (Ii) is the result of two-way ANOVA analysis, where * indicates <0.05 and *** indicates p <0.001. (Iii) is the result of unpaired Student's t-test, and * indicates p <0.05. (I) Schematic of the procedure of treating a macrophage fraction (BMDM) obtained from the bone marrow of an LLC tumor mouse model with a vehicle (Veh) or FG and transplanting it into another LLC tumor mouse model; (ii) PBS injection. Or, the tumor growth curve (n = 5) in the LLC tumor mouse model transplanted with vehicle (Veh) or FG-treated BMDM is shown.

1. 1. Tumor-related macrophage activator (TAM activator)
(Active ingredient)
The tumor-related macrophage activator of the present invention (hereinafter, also referred to as “TAM activator” in the present specification) is also referred to as a prolyl hydroxylase inhibitor (hereinafter, also referred to as “PHD inhibitor” in the present specification). ) Is the active ingredient. Prolyl hydroxylase (PHD) leads HIF to degradation by hydroxylating specific proline residues of the α-subunit (HIFα) of hypoxia-inducible factor (HIF) under normal oxygen concentration environments. It is an enzyme that suppresses the hypoxia response, while the enzyme activity can be inactivated when the oxygen concentration decreases. By inhibiting the enzymatic activity of this PHD with a PHD inhibitor, the HIF signaling pathway is upregulated and tumor growth is suppressed through activation of tumor-related macrophages.

The PHD inhibitor is not particularly limited as long as it is a compound having an action of inhibiting the activity of prolyl isomerase (PHD) to upwardly control the HIF signal transduction pathway, or a compound generally known as a PHD inhibitor. , For example, dimethyloxalylglycine (DMOG), pyrazolopyrimidine compounds (pyrazolopyrimidine compounds described in International Publication No. 2014-030716, etc.), FG-4592 (roxadustat), FG-2216, FG-4497, BAY-85. Examples thereof include -3934 (molidustat), AKB-6548 (Vadadustat), GSK127886 (daprodustat), JTZ-951, TM-6089, and DS-1093. In addition, FG4592, FG2216, FG4497, BAY-85-3934, AKB-6548, GSK127886, JTZ-951, TM-6089, and DS-1093 are all development numbers.

FG-4592 (development number) is a compound also referred to as Roxadustat and also N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl) carbonyl] -glycine.

FG-2216 (development number) is a compound also referred to as N-[(1-chloro-4-hydroxyisoquinoline-3-yl) carbonyl] -glycine.

BAY-85-3934 (development number), also known as Moldustat, 1,2-dihydro-2- [6- (4-morpholinyl) -4-pyrimidinyl] -4- (1H-1,2,3-triazole) -1-yl) -3H-pyrazole-3-one is also a compound.

AKB-6548 (development number) is a compound also referred to as Vadadustat and (5- (3-chlorophenyl) -3-hydroxypicolinoyl) glycine.

GSK1278863 (development number) is a compound also referred to as Daprodustat and N-[(1,3-dicyclohexylhexahydro-2,4,6-trioxopyrimidine-5-yl) carbonyl] -glycine.

TM-6089 (development number) is a compound also referred to as 6-amino-1,3-dimethyl-5-[2- (pyridin-2-ylsulfanyl) -acetyl] -1H-pyrimidine-2,4-dione. Is.

These PHD inhibitors may be used alone or in combination of two or more. Among them, those having an action mechanism of an enzyme-inhibiting analog (2-OG) of PHD are preferable, and specifically, FG4592 (Roxadustat), FG2216, FG4497, DMOG, BAY-85-3934 (Molidustat), or AKB. -6548 (Vadadustat) is preferable, and FG4592 (Roxadustat) is more preferable.

The PHD inhibitor may be synthetic or commercially available. The method for synthesizing the PHD inhibitor is not particularly limited, and examples thereof include known synthetic methods.

In the present invention, a commercially available product can also be used as the PHD inhibitor. Commercially available PHD inhibitors that can be used in the present invention include, for example, Fivegen's FG4592 (Roxadustat), FG2216, FG4497, Bayer Yakuhin's BAY-85-3934 (Molidustat), and Akevia's AKB-6548. (Vadadustat), GSK127886 (daprodustat) of GlaxoSmithKline, JTZ-961 of Japan Tobacco Inc., TM-6089 of Professor Toshio Miyata of Tohoku University, DS-1093 of Daiichi Sankyo Corporation, etc. ..

The TAM activator of the present invention preferably contains 25 to 100% by mass of a PHD inhibitor as an active ingredient, and more preferably 30 to 80% by mass.

The TAM activator of the present invention may optionally contain a pharmaceutically acceptable carrier or additive to prepare for the desired dosage and formulation form. Such carriers and additives include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizers, stabilizers, sweeteners, colorants, flavoring agents, and odorants. Examples thereof include agents, surfactants, moisturizers, preservatives, pH adjusters, buffers, thickeners and the like.

The dosage form of the TAM activator of the present invention is not particularly limited, and may be appropriately set according to the administration form and the like. Specific examples of the dosage form of the TAM activator of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; tablets, hard capsules, soft capsules, granules, and powders. , Solid preparations such as pills; external preparations such as creams, gels, ointments, sprays, patches, etc., inhalants, etc., and may be of any dosage form for internal or external use. Further, when it is used as an injection, it may be in the form of a powder for preparation at the time of use (for example, lyophilized powder) which is dissolved in physiological saline or the like before use.

The TAM activator of the present invention is used for the purpose of activating tumor-related macrophages. In the activation of tumor-related macrophages, the tumor phagocytosis of tumor-related macrophages is enhanced. Therefore, the TAM activator of the present invention can be used as a tumor phagocytosis enhancer for tumor-related macrophages and as an anticancer agent that suppresses ulcer growth by activating tumor-related macrophages.

Examples of tumor-related macrophages that enhance tumor phagocytosis in the TAM activator of the present invention include Ly6C-positive macrophages in the case of mice, and more specifically, Ly6C ^low macrophages (in the present specification). , Ly6C ^lo macrophages) and Ly6C ^high macrophages (also referred to herein as Ly6C ^hi macrophages). Similarly, in the case of other mammals such as humans, hamsters, guinea pigs, cows, sheep, pigs, goats, monkeys, rabbits, the counterparts of mouse Ly6C ^low macrophages and Ly6C ^high macrophages in the other mammals are mentioned. Be done. For example, in the case of humans, the human counterparts of ^{Ly6C low} macrophages and Ly6C ^{high macrophages include CD14 +} CD16 ^- macrophages and CD14 ^{lo /} -CD16 ⁺ macrophages. The TAM activator of the present invention of the present invention can be used as a tumor phagocytosis enhancer for these specific tumor-related macrophages.

From the viewpoint of obtaining even higher tumor phagocytosis, preferred tumor-related macrophages that enhance tumor phagocytosis in the TAM activator of the present invention include Ly6C ^{low macrophages in the case of mice and CD14 +} CD16 ^- macrophages in the case of humans. Can be mentioned. In the case of mice and mammals other than humans, mice are preferred tumor-related macrophages that enhance tumor phagocytosis in the TAM activator of the present invention from the viewpoint of further enhancing tumor phagocytosis. ^{Ly6C low} macrophages, the counterparts of ^{human CD14 +} CD16 ^- macrophages in the other mammal.

The TAM activator of the present invention acts alone as an anticancer agent, but when used in combination with another anticancer agent (other than the TAM activator of the present invention), the antitumor effect can be significantly improved.

Other anti-cancer agents that may be combined with the TAM activator of the present invention are not particularly limited, but specifically, cell-killing anti-cancer agents (specifically, alkylating agents, antimetabolites, platinum preparations, topoisomerase inhibitors). Drugs, microtubule anticancer agents, anticancer antibiotics, etc.), cancer vaccines used in immune cell therapy, molecular target drugs used in antibody therapy, drugs used in cytokine therapy, immunostimulators used in BRM therapy, Examples thereof include hormonal agents, immune checkpoint inhibitors (specifically, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, etc.).

Examples of the alkylating agent include ifosfamide, carbocon, cyclophosphamide, dacarbazine, thiotepa, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine.

Metabolic antagonists include, for example, enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocphosfert, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxiflulysin, neralabine, hydroxycarbamide, fluorouracil. Examples thereof include pemetrexed, pentostatin, mercaptopurine, methotrexate and the like.

Examples of the platinum preparation include oxaliplatin, carboplatin, cisplatin, nedaplatin and the like.

Examples of topoisomerase inhibitors include irinotecan, etoposide, nogitecan and the like.

Examples of the microtubule-acting anticancer agent include eribulin, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine and the like.

Examples of the anticancer antibiotics include actinomycin D, acralubicin, amrubicin, idarubicin, epirubicin, diostatinstimalamar, daunorubicin, doxorubicin, pirarubicin, bleomycin, pepromycin, mitomycin C, mitoxantrone, liposomaldoxorubicin and the like. Can be mentioned.

Examples of cancer vaccines include peptide vaccines and dendritic cell vaccines.

Molecular-targeted drugs include, for example, ibritsumomabutiuxetane, nibolumab, imatinib, everolimus, elrotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, trastuzumab emtanib, trastuzumab emtanib. , Vemurafenib, Alexinib, etc.

Examples of the drug used in cytokine therapy include interleukin-2 (Aldesleukin), interferon-α, interferon-β, interferon-γ and the like.

Examples of the immunostimulatory agent include Maruyama vaccine, OK-432 (Picibanil), Ubenimex, Lentinan, Krestin, BCG (Imcyst, Immunoblader) and the like.

Examples of hormonal agents include anastrozole, exemestane, estramustine, ethinyl estradiol, chlormaginone, goserelin, tamoxifen, flutamide, prednisolone, phosfestol, mitotan, methyltestosterone, medroxyprogesterone, and mepitithiostan. , Leuprorelin, letrozole and the like.

Examples of the PD-1 inhibitor include nivolumab (trade name: Opdivo) and pembrolizumab (trade name: Keytruda). Examples of the PD-L1 inhibitor include durvalumab (trade name: Imfinzi), atezolizumab (trade name: Tecentriq), avelumab (trade name: Babencio) and the like. Examples of the CTLA-4 inhibitor include ipilimumab (trade name: Yervoy) and the like.

These other anticancer agents may be used alone or in combination of two or more. Among these other anticancer agents, those capable of being taken up into tumor cells and exerting an antitumor effect are preferable, and cell-killing anticancer agents (alkylating agents, antimetabolites, platinum preparations, topoisomerase inhibitors, microtubule-acting anticancer agents, etc. And anti-cancer antibiotics) are more preferred, platinum preparations are even more preferred, and cisplatin is particularly preferred.

Since the TAM activator of the present invention can significantly improve the antitumor effect by being combined with other anticancer agents, the dose of the other anticancer agent is reduced as compared with the conventional case, thereby treating the anticancer drug. Side effects can be reduced. For example, by combining with the TAM activator of the present invention, the dose of the other anticancer agent can be reduced to about 1/4 to 1/2 of the conventional dosage. For example, when cisplatin is used as another anticancer agent, the conventional dose thereof is usually about 6 to 30 mg / kg (body weight) per dose in mice or daily administration, and usually 0. When used in combination with the TAM activator of the present invention, which is a single dose or daily dose of about 5 to 2.5 mg / kg (body weight), the dosage is 2 to 20 mg / kg, preferably 2 to 20 mg / kg in mice. 2 to 15 mg / kg (body weight), more preferably 2 to 10 mg / kg (body weight), usually 0.16 to 1.6 mg / kg, preferably 0.16 to 1.2 mg / kg (body weight) per dose in humans ), More preferably 0.16 to 0.8 mg / kg (body weight).

The type of cancer to which the TAM activator of the present invention is administered is not particularly limited, but is preferably a cancer in which a large amount of macrophages are accumulated. Cancers in which a large amount of macrophages are accumulated are known, and specific examples thereof include lung cancer, melanoma, glioma, breast cancer, esophageal cancer, intraductal bile duct cancer, gastric cancer, bile sac cancer, pancreatic cancer, colon cancer, tongue cancer, and thyroid cancer. Examples thereof include kidney cancer, prostate cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, horizontal print myoma, and smooth myoma. In addition, other specific examples of cancers in which a large amount of macrophages are accumulated include advanced cancers and treatment-resistant cancers.

In addition, as the cancer type to which the TAM activator of the present invention is administered, the above-mentioned Ly6C ^low macrophages and Ly6C ^high macrophages are used from the viewpoint of efficiently obtaining activation of tumor-related macrophages to obtain even higher tumor phagocytosis. , Preferably Ly6C ^low macrophages (for mice); CD14 ⁺ CD16 ^- macrophages and CD14 ^{lo /} -CD16 ⁺ macrophages, preferably CD14 ⁺ CD16 ^- macrophages (for humans); and their counterparts (non-mouse and non-human). It is more preferable that the cancer has a large accumulation of specific macrophages (in the case of mammals).

Examples of cancers in which a large amount of the specific macrophages are accumulated include ^{the average value (n) of the abundance ratio of Ly6C low} macrophages (in the case of mice) or their counterparts (in the case of other mammals) in all macrophages in the tumor. ≧ 3, preferably n ≧ 4, more preferably ≧ 6) is 35% or more, preferably 40% or more, and Ly6C ^high macrophages (in the case of mice) or the same among all macrophages in the tumor. The average value (mean value at n ≧ 3, preferably n ≧ 4, more preferably ≧ 6) of the abundance ratio of the counterpart (in the case of other mammals) is 6% or more, preferably 15% or more, more preferably. Cancers that account for 20% or more can be mentioned.

From the above viewpoint, preferable examples of the cancer type to which the TAM activator of the present invention is administered include lung cancer, melanoma, breast cancer, glioma, esophageal cancer, intrahepatic bile duct cancer and the like, and more preferably lung cancer. , Melanoma, and more preferably lung cancer. From the same viewpoint, preferable examples of the cancer type to which the TAM activator of the present invention is administered include advanced cancer and treatment-resistant cancer.

The organism to which the TAM activator of the present invention is administered may be any organism that is required to have an antitumor effect, and in addition to humans, mice, rats, hamsters, guinea pigs, cows, sheep, pigs, goats, monkeys, and rabbits. Such as mammals and the like.

The method for administering the TAM activator of the present invention is not particularly limited, and may be appropriately selected depending on the type of disease to be applied, and may be systemic administration or topical administration. Specific examples thereof include oral, transvascular (intravenous or intravenous), transdermal, transintestinal, and transpulmonary administration. Intravenous administration also includes intravascular injection and continuous infusion. The method of administering the TAM activator of the present invention may be the same as or different from the method of administering the anticancer agent whose antitumor effect is to be enhanced. When the administration method of the TAM activator of the present invention is the same as that of other anticancer agents whose antitumor effect is to be enhanced, the TAM activator of the present invention and the anticancer agent are administered in a mixed state. It may be administered separately, or each may be administered separately.

Regarding the dose of the TAM activator of the present invention, the type of PHD inhibitor, the type of the other anticancer agent when combined with other anticancer agents, the age, sex, body weight, degree of symptoms, administration method of the subject to be administered. It may be set appropriately according to the above. Specifically, the dose of the PHD inhibitor in the TAM activator of the present invention is, for example, twice the dose when the PHD inhibitor is a tumor vascular function normalizing agent that does not exert an antitumor effect by itself. As mentioned above, preferably 2.5 or more, more preferably 3 or more. The upper limit of the dose of the PHD inhibitor in the TAM activator of the present invention is not particularly limited, but is 8 times or less, preferably 6 times or less the dose when the PHD inhibitor is a tumor vascular function normalizing agent. , More preferably 4 times or less.

Specifically, when the active ingredient PHD inhibitor is FG4592, for example, 55 to 310 mg / kg (body weight), preferably 67 to 185 mg / kg (body weight) per PHD inhibitor conversion. , More preferably 80 to 145 mg / kg (body weight), still more preferably 85 to 120 mg / kg (body weight), for example 4.5 to 25 mg / kg (body weight), preferably 5.5 to 5 to humans. 15 mg / kg (body weight), more preferably 6.5-12 mg / kg (body weight), still more preferably 7-10 mg / kg (body weight). When the PHD inhibitor which is the active ingredient is DMOG, the amount of PHD inhibitor is about 800 to 2000 mg / kg (body weight), preferably 1000 to 1500 mg / kg (body weight), more per dose. It is preferably 1100 to 1300 mg / kg (body weight), and is about 64 to 400 mg / kg (body weight), preferably 80 to 200 mg / kg (body weight), and more preferably 90 to 100 mg / kg (body weight) with respect to humans. Can be mentioned.

When the TAM activator of the present invention is used in combination with another anticancer agent, the administration timing of the TAM activator of the present invention is not particularly limited, and is before the administration of the other anticancer agent or the administration of the other anticancer agent. However, it is preferable before administration of the anticancer drug. When the TAM activator of the present invention is administered before administration of another anticancer agent, for example, 48 hours to 12 days before administration of the other anticancer agent, preferably 3 days to 10 days before administration, more preferably 5 About 1 to 8 days ago can be mentioned.

2. 2. Method for Producing Pharmaceuticals for Cancer Treatment As described above, the tumor-related macrophage (TAM) activator can directly activate the tumor-related macrophage. That is, the tumor-related macrophage activator of the present invention can change tumor-related macrophages having poor phagocytosis into an enhanced phagocytotic phenotype. Therefore, by treating tumor-related macrophages in vitro, a drug for treating cancer can be prepared. That is, the present invention also provides a method for producing a drug for treating cancer, which comprises allowing a prolyl isomerase inhibitor to act on a macrophage fraction containing a tumor-related macrophage in vitro.

The tumor-related macrophages on which a prolyl hydroxylase inhibitor (PHD inhibitor) acts in the method for producing a drug for treating cancer of the present invention are activated by the above-mentioned "1. Tumor-related macrophage activator (TAM activator)". It is a tumor-related macrophage that is transformed. That is, as the tumor-associated macrophages exerting PHD inhibitors, Ly6C ^low monocytes and Ly6C ^high monocytes, preferably (for mice) Ly6C ^low monocytes; CD14 ⁺ CD16 ^- monocytes and CD14 ^lo / - CD16 ⁺ single spheres, preferably CD14 ⁺ CD16 ^- monocytes (in humans); (in the case of mice and non-human mammals) and their counterparts and the like.

Examples of the macrophage fraction containing tumor-related macrophages include a macrophage fraction obtained from the bone marrow of a cancer patient, a macrophage fraction obtained from a tumor tissue, and the like. Further, the macrophage fraction may contain tumor-related macrophages, and the macrophages may be substantially only tumor-related macrophages (that is, the macrophage fraction itself is an isolated fraction of tumor-related macrophages). Macrophages may include other macrophages in addition to tumor-related macrophages. A method for obtaining a macrophage fraction can be appropriately determined by those skilled in the art based on a known method.

The PHD inhibitor is as described in the column of "1. Tumor-related macrophage activator (TAM activator)" above.

Treatment of isolated tumor-related macrophages may be performed by contacting the isolated tumor-related macrophages with a PHD inhibitor and incubating them. The amount of the PHD inhibitor used is 50 to 150 μM, preferably 80 to 120 μM, and more preferably 90 to 110 μM in the culture medium. The incubation temperature is, for example, 10 to 35 ° C., preferably 20 to 25 ° C., and the incubation time is, for example, 8 to 20 hours, preferably 10 to 15 hours.

Tumor-related macrophages treated with PHD inhibitors are activated to acquire an enhanced phagocytic phenotype. Tumor-related macrophages with enhanced tumor phagocytosis thus prepared are themselves useful as cancer therapeutic agents in tumor immunotherapy. By returning the tumor-related macrophage to the target tumor tissue from which the tumor-related macrophages were collected, the cancer therapeutic drug can suppress the growth of the target tumor tissue.

3. 3. Drugs for treating cancer and pharmaceutical compositions for treating cancer As described above, tumor-related macrophages (TAM) activators can significantly improve antitumor effects when combined with other anticancer agents. Therefore, the present invention also provides a cancer therapeutic drug and a cancer therapeutic pharmaceutical composition in which the above-mentioned TAM activator is combined with another anticancer agent.

The drug for treating cancer of the present invention is used when the above-mentioned TAM activator is administered by the same or different administration method as other anticancer agents to treat cancer, and the first preparation containing other anticancer agents. And a second preparation containing the above-mentioned TAM activator.

Further, the pharmaceutical composition for treating cancer of the present invention is used when treating cancer with the above-mentioned TAM activator by the same administration method as other anticancer agents, and is used with other anticancer agents and the above-mentioned TAM activation. The agent is contained in the same preparation.

The TAM activator and other anticancer agents which are the constituent elements of the pharmaceutical composition for cancer treatment and the pharmaceutical composition for cancer treatment of the present invention, their doses, usage modes and the like are described in the above-mentioned "1. Tumor-related macrophage activation". It is as described in the column of "agent (TAM activator)".

Hereinafter, the present invention will be described with reference to test examples, but the present invention is not limited to these test examples.

(Test animal)
All animal experiments were conducted with the approval of the Osaka City University Animal Experiment Management Regulations and the Japan Laboratory Animal Association. In addition, animal research and handling was carried out in strict compliance with the guidelines of the Animal Care and Use Regulations. The test animals were C57BL / 6 male mice, and SLC Japan, Inc. Obtained from. LysM Cre and VHL frozen mice were obtained from Jackson Laboratories and bred at a facility at Osaka City University. Mice were fed freely in cages with food and water under a 12-hour light-dark cycle and an environment of 22 ± 1 ° C.

(Statistical analysis)
Statistical analysis was performed using an unpaired t-test followed by Bonferroni's post-test. Details are given in the brief description of the drawings. All statistical analyzes were performed using GraphPad Prism (version 7.02) software. The statistically significant difference was defined as p <0.05. In addition, p <0.01, p <0.001, and p <0.0001 are also shown to indicate the level of reliability. All analyzes were performed using a two-sided test.

Test Example 1: Tumor growth inhibitory effect by PHD inhibitor Tumor transplant model mice were treated with PHD inhibitor and the cell growth inhibitory effect was examined.

(Cell culture, tumor transplant model, and PHD inhibitor treatment)
Lung cancer LLC cells and melanoma B16F10 cells (RIKEN BRC) were maintained in Dulbecco's Improved Eagle's Medium (DMEM) and Roswell Park Memorial Institute 1640 Medium (RPMI1640). These media were supplemented with 10% fetal bovine serum and penicillin / streptomycin and cultured at 37 ° C. ^{in a 5% CO 2 atmosphere.} The cultured cells were harvested and resuspended at 1 × 10 ⁷ cells / mL in phosphate buffered saline (PBS). Were subcutaneously transplanted part of cells (1 × 10 ⁶ cells) into the right flank of 8-12 week old mice. 10 days after tumor transplantation, 3 mg (100 mg / kg; in 5 v / v% DMSO aqueous solution; 600 μl; the dose to mice is the same in the following test examples) PHD inhibitor FG4592 (Selleck Chemicals, TX, USA). ^{) Or 600 μl of 5} v / v% DMSO aqueous solution as a vehicle (Veh) was administered intraperitoneally (tumor size eligible for this study was 100-400 mm 3). Tumors were measured two-dimensionally using calipers once every two days. The tumor tissue volume was calculated using Equation 1 below. Mice were ^{sacrificed when the tumor volume exceeded 4500 mm 3} or when the tumor ruptured, and the tumor tissue was removed and weighed.

(result)
Tumor volume transition and tumor weight for lung cancer LLC cell transplanted mice, and tumor volume transition and tumor weight for melanoma B16F10 cell transplanted mice are shown in FIGS. 1 (i), 1 (ii) and 2 (i), respectively. And FIG. 2 (ii). As is clear from these figures, tumor growth was significantly inhibited in FG-treated mice in both lung cancer LLC cells and melanoma B16F10 cells. Although not shown, it was also confirmed that there was a dose-dependent inhibition of tumor growth by FG, and that repeated FG treatment further suppressed tumor growth and prolonged survival.

Test Example 2: Tumor-related macrophage activation effect by PHD inhibitor
Test Example 2-1
Cleaved caspase 3 (CC3) immunostaining for tumor tissue treated with the PHD inhibitor FG4592 (FG) or vehicle (Veh) to determine whether FG induced apoptosis or growth inhibition in tumor cells. A quantification of caspase 3 positive cells was performed and an in vitro cell proliferation assay was performed.

(Immunostaining)
Tumor tissue treated with the PHD inhibitor FG4592 (FG) or vehicle (Veh) 2 or 6 days after treatment was sliced into frozen sections 8 μm thick at −20 ° C. and air dried. The sectioned tissue sample was rehydrated with PBS for 10 minutes and fixed with 4 (w / v)% cold paraformaldehyde for 10 minutes. Sections were washed with PBS and permeabilized with PBS containing 0.5% (v / v) Triton TM X-100 for 10 minutes. Sections were blocked in 5% normal goat serum at room temperature (20-25 ° C.) for 30 minutes. Sections were incubated overnight at 4 ° C. with the primary antibody anti-CC3 (1: 400; Cell Signaling Technologies, Massachusetts, USA). Sections were washed with 0.1% Tween 20 in PBS and incubated with a suitable fluorophore secondary antibody (AlexaFluor 488 or Cy3, goat anti-rat or goat anti-rabbit IgG; BioLegend, CA, USA) for 1 hour at room temperature. Sections were washed with 0.1% Tween 20 in PBS, dehydrated with ethanol and air dried. They were subsequently encapsulated with a Vector Shield encapsulant (Vector Laboratories, CA, USA) containing a 4', 6-diamidino-2-phenylindole stain (1: 5000) and a cover glass.

(Quantification of immunofluorescence images)
Immunofluorescent images taken with a microscope (BZX-710, KEYENCE, Osaka, Japan) were quantified using the equipped software. For each sample, at least 15 fields of view were analyzed at 100x, 200x, or 400x magnification. Each experiment was performed with at least 3 animals from each group.

(Cell proliferation assay)
Lung cancer LLC cells were seeded on a 96-well plate at 2 × 10 ^{4 cells per well.} Subsequently, FG was added to the medium to a predetermined concentration (0, 50, 100, 200, and 400 μM). The medium was changed 6 hours after the FG treatment, an additional 72 hours later, the AramarBlue reagent (Bio-Rad) was added, and the plates were _{incubated in a 5% CO 2} incubator at 37 ° C. for 6 hours. Finally, optical densities (OD) were measured at 570 and 600 nm to calculate cell viability. The experiments were performed 3 times per experiment.

(result)
The quantification result of the ratio of CC3-positive cells in all the cells constituting the tumor tissue is shown in FIG. 3 (i), and the result of the tumor cell proliferation assay at 72 hours after the FG treatment is shown in FIG. 3 (ii). As is clear from these figures, there was no significant difference in CC3 expression level and cell proliferation depending on the presence or absence of FG treatment. That is, the inhibition of tumor growth observed by FG treatment in Test Example 1 was neither due to apoptosis in tumor cells nor due to inhibition of growth. This result suggests that FG does not directly affect tumor cells.

Test Example 2-2
To determine if the PHD inhibitor FG4592 (FG) results in altered immune cell response, ^{by measuring the proportions of CD45 +} cell population, CD45 ⁺ CD11b ⁺ F4 / 80 ⁺ cell population, and T cell population, respectively. Tumor infiltration was quantified. We also quantified the number of macrophages in tumor tissue based on immunofluorescence staining.

(Tumor dissociation)
Treat with the PHD inhibitor FG4592 (FG) or vehicle (Veh), finely chop the tumor 2 or 6 days after treatment, and use 1 mg / mL collagenase IV containing 50 μg / mL DNase I for 45 minutes at 37 ° C. Digested with shaking. Cells were filtered through a 100 μm nylon mesh and washed in isolation buffer (PBS containing 2% FBS and 2 mM EDTA). Erythrocytes were lysed using RBC lysis buffer (BioLegend). Cells were resuspended in isolation buffer and Fc receptors were blocked on ice with CD16 / 32 blocking antibody (Biolegend) for 15 minutes.

(Flow cytometry and cell selection)
The dissociated tumor single cells were diluted on ice in the dark for 30 minutes and stained with the following fluorescent dye-binding antibody. For macrophages, anti-mouse F4 / 80-PE (clone BM8, BioLegend), anti-mouse CD11b-APC-Cy7 (clone M1 / 70, BioLegend), and anti-mouse CD45PE-y7 (clone 30-F11, BioLegend) were used. As for T cells, anti-mouse CD4-APC (clone GK1.5, BioLegend) and anti-mouse CD8-APC-Cy7 (clone 53-6.7, BioLegend) were used. DAPI was added just prior to analysis to eliminate dead cells. Flow cytometric analysis and cell selection were performed on BD LSR, or BD FACSAria. 100,000 cells per mouse sample were analyzed using BD FACSDiva software Ver. 8.0 (BD Bioscience).

(Immunostaining)
Using anti-mouse F4 / 80-PE (clone BM8, BioLegend), immunostaining and immunofluorescence image quantification were performed in the same manner as in Test Example 2-1.

(result)
^{The CD45 +} cell ratio (n = 12-13) in a lung cancer LLC tumor is shown in FIG. 4 (i), and the CD11b ⁺ F4 / 80 ⁺ cell ratio (n = 8) is shown in FIG. 4 (ii). In addition, the CD4 ⁺ and CD8 ⁺ cell ratios (n = 5) in LLC tumors are shown in FIG. As is clear from these figures, in tumors treated with FG, the CD45 ⁺ leukocyte cell population ratio and the CD45 ⁺ CD11b ⁺ F4 / 80 ⁺ MI cell population ratio were significantly increased, while the T cell population showed significant changes. Not shown, that is, the population of tumor-invasive T cells was very low. In addition, the quantification result (n = 5) of F4 / 80 ^{+ cell number per 1 mm 2 of the tumor is shown in FIG.} As is clear from this figure, an increase in the number of macrophages was observed in the tumor tissue by immunofluorescence staining. These data indicate that LLC tumors are rich in tumor-related macrophages (TAMs) and contain very few T cells. Note that FIG. 7 shows the CD4 ⁺ and CD8 ⁺ cell ratios (n = 5) in melanoma B16F10 tumors. As is clear from this figure, the T cell population does not show a significant change in the melanoma B16F10 tumor, and the tumor-invasive T cell population is very low, which is similar to that of the lung cancer LLC tumor (Fig. 5). Was done.

Test Example 2-3
Focusing on the previously reported M1 marker (CD80), M2 marker (CD206) and other markers as cell surface markers reflecting the differentiation state of tumor-related macrophages (TAM), TAM was measured by Ly6C. The function when subdivided was investigated.

(Tumor dissociation)
Tumors treated with the PHD inhibitor FG4592 (FG) or vehicle (Veh) (7 days later) were dissociated in the same manner as in Test Example 2-2 above.

(Flow cytometry and cell selection)
Further, as fluorescent dye-binding antibodies, anti-mouse Ly6C-PerCP-Cy5.5 (clone HK1.4, BioLegend), anti-mouse CD206-APC (clone C068C2, BioLegend), and anti-mouse CD80 FITC (clone 16-10A1, BioLegend) Flow cytometry and cell selection were performed in the same manner as in Test Example 2-2. The gating strategy in the flow cytometry of macrophages is shown in FIG.

(result)
As is clear from FIG. 8, from the analysis results of tumor-infiltrating macrophages using CD80 as an M1 marker and CD206 as an M2 marker, these markers were found in both vehicle-treated and FG-treated tumors. It was expressed, and no difference in macrophages based on these markers was observed in any of the treatment groups.

On the other hand, FIG. 9 shows the quantification results (n = 6) ^{of Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophage ratios in lung cancer LLC tumors by flow cytometric analysis. As is clear from this figure, the Ly6C ^lo macrophage ratio in all macrophages was increased by FG treatment. Furthermore, although not shown, when the selected macrophages were stained with the Diffquick staining kit and then observed under a microscope ^{, the morphology of the Ly6C lo} macrophages selected from the FG-treated tumor was as if they formed phagosomes, and they were treated with Veh. It was also confirmed that it was different from the ^{Ly6C lo} macrophages selected from the tumors.

Furthermore, the quantification results (n = 4) ^{of Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophage ratios in melanoma B16F10 tumors by flow cytometric analysis are shown in FIG. ^{As is clear from this figure, the Ly6C lo} macrophage ratio in all macrophages was increased by FG treatment as in the case of lung cancer LLC tumor (FIG. 9).

As is clear from FIGS. 9 and 10, lung cancer LLC tumor and melanoma B16F10 tumor had a common tendency of ^{Ly6C hi} , Ly6C ^lo , and Ly6C ^{neg macrophage ratio in mice. For example, the average value of the abundance ratio of Ly6C lo in} all macrophages in the tumor is n = 6 for lung cancer LLC tumor and n = 4 for melanoma B16F10 tumor, both of which average 35% or more, specifically about 40. ^{The average value of the abundance ratio of Ly6C hi in} all macrophages in the tumor was n = 6 in the lung cancer LLC tumor and n = 4 in the melanoma B16F10 tumor, and both were 6% or more.

Test Example 2-4
It was investigated whether FG affects the tumor growth inhibitory ability of tumor-invasive macrophages by depleting the tumor-invasive macrophages with the clodronate liposome Clo before administration of the PHD inhibitor FG4592 (FG).

(In vivo macrophage depletion)
Twenty-four hours prior to administration (injection) of the PHD inhibitor FG4592 (FG) or vehicle (Veh), mice were treated with 100 μL clodronate-loaded liposome Clo (F70101C-A, FormuMax Scientific Inc.) or 100 μL plain control liposome Lip (F70101C-). A, FormuMax Scientific Inc.) was administered intraperitoneally. Furthermore, 72 hours after the administration of FG or Veh, 70 μL of the same liposome was intraperitoneally administered as the second administration.

(Immunostaining and quantification of immunofluorescence images)
Macrophage depletion efficiency was evaluated by immunostaining. Immunostaining was performed in the same manner as in Test Example 2-1 to quantify immunofluorescent images.

(result)
Tumor growth curve (n = 4) by treating LLC tumor mouse models treated with clodronate liposomes (Clo) or plain control liposomes (Lip) with vehicle (Veh) or 100 mg / kg PHD inhibitor (FG). It is shown in FIG. As is clear from this figure, in the control liposome (Lip) group, the tumor treated with FG showed suppression of tumor growth as compared with the tumor treated with vehicle (Veh). However, in the clodronate liposome (Clo) treated group, there was no significant difference in tumor growth between the tumor treated with FG and the tumor treated with vehicle (Veh).

In addition, the quantification result (n = 3) of F4 / 80 ^{+ cell number per 1 mm 2 of the tumor is shown in FIG.} As is clear from this figure, the macrophage depletion efficiency by clodronate liposomes (Clo) is 80% depletion when plain control liposomes (Lip) are administered in both vehicle (Veh) and FG-treated tumors. showed that.

The above results indicate that FG affects the ability of tumor infiltrating macrophages to inhibit tumor growth.

Test Example 2-5
Macrophages play an important role in the innate immune system in that they directly inhibit tumor growth through phagocytosis. Therefore, using a GFP-labeled LLC tumor model, the activation of phagocytosis of macrophages by the PHD inhibitor FG4592 (FG) was investigated. At the same time, the normalization of tumor blood vessels by the PHD inhibitor FG4592 (FG) was also investigated. Furthermore, using macrophages isolated from bone marrow, the activation of phagocytosis of macrophages by the PHD inhibitor FG4592 (FG) was investigated.

(Preparation of GFP-labeled LLC tumor model)
The cDNA encoding GFP1 was double digested with SalI and NotI (Clontech, No. 632468) from pAcGFP1 and inserted into the SalI and NotI sites of the pEBMulti-Hyg vector (WAKO, 050-08121). This expression vector was transfected into LLC using Lipofectamine LTX reagent (Invitrogen), and GFP stable expression LLC (GFP-labeled LLC tumor model) was selected by hygromycin B treatment (250 μg / mL) for 7 days.

(Isolation of bone marrow-derived macrophages (BMDM))
Bone marrow was harvested from the tibia and femur of 8-12 week old C57BL / 6 mice and incubated in DMEM supplemented with L929 cell conditioned medium (20%). The cells were cultured for 7 days and the medium was changed every 2 days. Fractionated macrophages were identified by F4 / 80 antibody staining and flow cytometry.

(Phagocytosis assay)
FIG. 15 (i) shows a schematic diagram of an in vitro bead phagocytosis assay (in the case of BMDM). Macrophages collected and sorted 48 hours after a GFP-labeled LLC tumor model administered with a vehicle (Veh) or PHD inhibitor (FG) prior to seeding in a 96-well plate, or isolated BMDMs were used in CellVue Claret (Sigma). -Stained with Aldrich). The sorted cells were incubated at 37 ° C. for 2 hours and the cells were allowed to stand after sorting. Subsequently, after 24 hours, 100 μMFG or equal volume DMSO was added to the medium. After 12 hours, latex beads-rabbit IgG-FITC complex (Cayman) was added to the medium (1: 400). After 45 minutes, images of phagocytic macrophages were taken, counted using a microscope (BZX-710, KEYENCE, Osaka, Japan) and analyzed using equipped software. Over 800 cells were analyzed for each group.

(Staining of tumor blood vessels)
Tumor tissue collected 48 hours after administration from a GFP-labeled LLC tumor model administered with a vehicle (Veh) or PHD inhibitor (FG) was subjected to CD31 staining to stain vascular endothelial cells. Based on the staining, the vascular density and vascular lumen area of the tumor tissue were quantified.

(result)
FIG. 13 shows the quantification results (n = 3) of the vascular density and the vascular lumen area of the tumor 48 hours after the administration of the GFP-labeled LLC tumor model to which the vehicle (Veh) or PHD inhibitor (FG) was administered. The quantification result (n = 5) of the number of phagocytic cells (GFP ⁺ F4 / 80 ^{+) in the tumor is shown in FIG.} As is clear from these figures, the tumor tissue 48 hours after FG administration did not show normalized tumor blood vessels at that time, but there was a significant increase ^{in tumor phagocytic macrophages (GFP + macrophages).} It was. In addition, the quantification result (n = 3) of the ratio of BMDM (bone marrow-derived macrophage) isolated from bone marrow is shown in FIG. 15 (ii). As is clear from this figure, in vitro phagocytotic assays of BMDM showed that levels of phagocytic macrophages were increased in FG-treated BMDMs. The above results indicate that FG directly affected macrophages.

Test Example 2-6
It was investigated which Ly6C macrophage population was activated by the PHD inhibitor FG4592 (FG).

(Flow cytometry and cell selection)
For GFP ⁺ macrophages, flow cytometry and cell selection were performed in the same manner as in Test Example 2-3. The gating strategy in flow cytometry of GFP ^{+ macrophages is shown in FIG.}

(Phagocytosis assay)
18 (i) and 19 (i) show a schematic diagram of the Exvivobead phagocytosis assay. ^{GFP +} macrophages were collected 6 days (16th day) after administration from a GFP-labeled LLC tumor model administered with a vehicle (Veh) or PHD inhibitor (FG), and each fraction selected by flow cytometry was used as a test example. A phagocytosis assay was performed in the same manner as in 2-5.

(result)
Quantification results (n = 5-6) of the ratio of ^{GFP +} macrophages (Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages) subdivided by Ly6C to ^{total GFP + macrophages by flow cytometric analysis are shown in FIG.} Shown in. As is clear from this figure, the FG treatment ^{increased the proportion of the Ly6C positive population (Ly6C hi} and Ly6C ^lo ), but did not increase the proportion of the Ly6C negative population.

Further, the quantification result (n = 5) of the phagocytotic ^{Ly6C lo} macrophage ratio by the phagocytotic Ly6C ^{lo macrophage assay is shown in FIG. 18 (ii), and the quantification result of the phagocytotic Ly6C hi} macrophage ratio (n = 4 to 4 to 5). 5) is shown in FIG. 18 (iii). As is clear from these figures, the Ly6C ^lo macrophages selected from FG-treated tumors had a significantly higher proportion of phagocytic macrophages than ^{the Ly6C lo} macrophages selected from vehicle-treated tumors, and FG. ^{The Ly6C hi} macrophages selected from the tumors treated with the above had a higher ratio of phagocytic macrophages than ^{the Ly6C hi} macrophages selected from the tumors treated with the vehicle. On the other hand, the quantification results (n = 3 to 4) of the phagocytic Ly6C negative macrophage ratio are shown in FIG. 19 (ii). As is clear from this figure, the Ly6C-negative macrophages selected from the FG-treated tumors were almost the same as the Ly6C-negative macrophages selected from the vehicle-treated tumors, and even a slight downward trend was observed. The above data, that is, from these results, the phagocytosis of Ly6C-positive macrophages among the macrophages subdivided by FG treatment was specifically enhanced, and the ^{phagocytosis of Ly6C lo} macrophages was further enhanced. It shows that it is enhanced.

Test Example 3: HIF-mediated macrophage-mediated tumor growth inhibitory effect of PHD inhibitors PHD inhibitors inhibit HIF prolyl hydroxylation and HIF proteasome degradation, thereby upregulating the HIF signaling pathway. .. Therefore, it was investigated that the macrophage-mediated tumor growth inhibitory effect of the PHD inhibitor is caused by HIF inhibition.

(Inhibition of tumor growth in macrophage-specific VHL knockout mice)
Macrophage-specific von Hippel-Lindau (VHL) knockout mice (VHLfl / flLys-MCre ^+/- ; VHL KO mice) were used to genetically mimic the FG drug response. VHL KO mice exhibit HIF upregulation. For VHL KO mice, the tumor volume transition was examined in the same manner as in Test Example 1.

(Flow cytometry and cell selection)
^{Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophages were selected for macrophage-specific VHL knockout mice in the same manner as in Test Example 2-3.

(Quantitative analysis of HIF downstream genes of bone marrow-derived macrophages)
Mice collecting bone marrow-derived macrophages (BMDM) were administered 100 μM FG-4592 or vehicle (Veh) 12 hours prior to collection. BMDM was isolated in the same manner as in Test Example 2-5, and RNA was extracted from BMDM using ISOGEN II (NIPPON GENE). Complementary DNA was reverse transcribed from 1 μg of total RNA using the Prime Script RT Reagent Kit (Takara). Transcripts were analyzed on a 7500 Fast Real-Time PCR system using the Thunderbird SYBR qPCR Mix. QRT-PCR was performed on each sample pair. The measured transcript data was standardized for 18S ribosomal RNA content and analyzed using 7500 software v2.3 (Applied Biosystems).

(result)
Vhl ^{fl / fl} LysM-Cre ^-/- (Control (Ctrl); n = 9) and Vhl ^{fl / fl} LysM-Cre ^+/- (VHL KO; n = 7) Tumor growth curves of the mouse LLC tumor model. The tumor weight (n = 7-9) on the 16th day is shown in FIG. 20 (i) and shown in FIG. 20 (ii). As is clear from these figures, the LLC tumor transplantation model of VHL KO mice genetically mimicking the FG drug response showed the same tumor growth inhibition as the FG-treated LLC tumor transplantation mouse model (FIG. 1). In addition, the flow cytometric analysis results (n = 6) of the ratios of ^{Ly6C hi} , Ly6C ^lo , and Ly6C ^neg macrophages in the LLC tumor model of VHL KO are shown in FIG. As is clear from this figure, in VHL KO mice, the ^{ratio of Ly6C lo} macrophages to total tumor infiltrating macrophages was also increased as in the FG-treated LLC tumor transplanted mouse model (FIG. 9). The above results indicate that FG exerted a macrophage-mediated tumor growth inhibitory effect via the PHD-HIF axis.

In addition, qPCR analysis of bone marrow-derived macrophages (BMDM) isolated from mice administered with vehicle (Veh) or PHD inhibitor (FG), and the quantitative analysis results of HIF downstream genes are shown in FIG. As is clear from this figure, HIF downstream gene expression was upregulated in BMDM of FG-treated mice. These results indicate that PHD inhibitors affect the HIF signaling pathway of macrophages.

Test Example 4: Tumor Growth Inhibition by Macrophages Activated by PHD Inhibitor The tumor growth inhibitory effect of macrophages activated by the PHD inhibitor FG4592 (FG) in a mouse model was investigated.

(Transplantation of macrophages collected and selected from LLC tumor model mice into tumor tissue)
^{FIG. 23 (i) shows a schematic diagram of a procedure for transplanting Ly6C lo} macrophages selected from an LLC tumor mouse model into a tumor site of another LLC tumor mouse model. As shown in this figure, macrophages were collected from LLC tumor model mice administered with FG-4592 (FG) or vehicle (Veh) 7 days (16 days) after administration, in the same manner as in Test Example 2-3. , Ly6C ^lo macrophages were selected. Using a 26-gauge needle, Ly6C ^lo macrophages 1 × 10 ⁵ cells or PBS were injected into the tumor of LLC tumor mice 10 days after tumor transplantation. After intratumoral injection, the tumor was measured twice every 2 days in the same manner as in Test Example 1, and after sacrifice on the 16th day, the tumor tissue was removed and the weight was measured.

In addition, a ^{similar transplantation experiment was performed on Ly6C neg} macrophages, and the weight of the tumor removed from the transplanted mice was measured.

Furthermore, in the same manner as in Test Example 2-5, the blood vessel density and the blood vessel lumen area in the tumor tissue removed from the transplanted mouse were quantified.

(result)
Tumor volume transitions and tumor weights of mice that received Ly6C ^lo macrophage transplantation or PBS injection are shown in FIGS. 23 (ii) and 23 (iii), respectively. As is clear from these figures, a tumor growth inhibitory effect was observed in the mice transplanted with ^{Ly6C lo} macrophages selected from the tumors of the mice treated with FG. In addition, ^{the comparison of tumor weights for mice transplanted with Ly6C lo} macrophages or Ly6C ^neg macrophages is shown in FIG. 24 (i); the comparison of the results of quantification of the vascular density of tumors is shown in FIG. 24 (ii); The comparison of the quantification results of the vascular lumen area is shown in FIG. 24 (iii). As is clear from these figures, in the mice ^{transplanted with Ly6C neg} macrophages selected from the tumors of the mice treated with FG, the weight of the tumor was almost the same as that of the vehicle (Veh), while the tumor blood vessels A significant decrease in density and a significant increase in vascular lumen area were observed. The above data indicate that Ly6C ^lo macrophages suppress tumor growth by enhancing phagocytosis, while Ly6C ^neg macrophages normalize tumor blood vessels.

Test Example 5: Production of a drug for treating cancer by in vitro treatment of a PHD inhibitor (1)
^{Tumor growth inhibitory effect of macrophages prepared by exposing Ly6C lo} macrophages isolated from tumors of vehicle-administered LLC tumor model mice to the PHD inhibitor FG4592 (FG) in vitro in tumor mouse models. Examined.

(Transplantation of macrophages collected and selected from LLC tumor model mice into tumor tissue)
^{FIG. 25 (i) shows a schematic diagram of the procedure for transplanting Ly6C lo} macrophages selected from an LLC tumor mouse model into a tumor site of another LLC tumor mouse model. As shown in this figure, macrophages were collected from LLC tumor model mice administered with vehicle (Veh) 7 days (16 days) after administration, and Ly6C ^lo macrophages were selected in the same manner as in Test Example 2-3. .. The selected Ly6C ^lo macrophages were washed in PBS, centrifuged at 200 xg for 3 minutes and resuspended in 20 μL PBS. The selected macrophages were seeded in a 10 cm dish and incubated for 12 hours in medium containing 100 μM FG or Veh. Macrophages were then collected and resuspended in 20 μL PBS. Using a 26-gauge needle, prepared macrophages (cancer therapeutic drug) 1 × 10 ⁵ cells or PBS were injected into the tumor of LLC tumor mice 10 days after tumor transplantation. After intratumoral injection, the tumor was measured twice every 2 days in the same manner as in Test Example 1, and after sacrifice on the 16th day, the tumor tissue was removed and the weight was measured.

(result)
The tumor volume transition and tumor weight of the prepared macrophage (cancer therapeutic drug) transplanted or PBS injection are shown in FIGS. 25 (ii) and 25 (iii), respectively. As is clear from these figures, tumor growth was significantly suppressed in the tumor tissue transplanted with ^{Ly6C lo macrophages treated with FG in vitro.}

The above results show that FG directly affects Ly6C-positive macrophages (particularly Ly6C ^lo macrophages), induces their activation, and thereby inhibits tumor growth. Therefore, in vitro FG-treated Ly6C-positive macrophages (particularly) Ly6C ^lo macrophages) have been demonstrated to be useful as therapeutic agents for cancer.

Test Example 6: Production of drug for cancer treatment by in vitro treatment of PHD inhibitor (2)
The tumor growth inhibitory effect of macrophages prepared by exposing macrophages obtained from the bone marrow of LLC tumor model mice to the PHD inhibitor FG4592 (FG) in vitro in a tumor mouse model was investigated. A schematic diagram of the procedure in this test example is shown in FIG. 26 (i).

(Isolation of bone marrow-derived macrophages (BMDM))
Bone marrow was collected from the tibia and femur of LLC tumor model mice and incubated in DMEM supplemented with L929 cell conditioned medium (20%). The cells were cultured for 7 days and the medium was changed every 2 days. The resulting macrophages were washed in PBS, centrifuged at 200 xg for 3 minutes and resuspended in 20 μL PBS. As a result, a macrophage fraction derived from bone marrow was obtained.

(Transplantation of BMDM into tumor tissue)
The resulting bone marrow-derived macrophage fraction was seeded in a 10 cm dish and incubated for 12 hours in medium containing 100 μM FG or Veh. Macrophages were then harvested and resuspended in 20 μL PBS. Using a 26-gauge needle, prepared macrophages (cancer therapeutic drug) 1 × 10 ⁵ cells or PBS were injected into the tumor of LLC tumor mice 10 days after tumor transplantation. After intratumoral injection, the tumor was measured twice every two days in the same manner as in Test Example 1.

(result)
The tumor volume transition of the prepared macrophage (medicine for treating cancer) transplantation or PBS injection is shown in FIG. 26 (ii). As is clear from this figure, in the tumor tissue (FG + BMDM) transplanted with FG-treated bone marrow-derived macrophages in vitro, the tumor tissue (PBS) injected with PBS and the Veh-treated bone marrow-derived macrophages were transplanted. Tumor growth was suppressed compared to tumor tissue (Ctrl + BMDM).

The above results show that FG directly affects tumor-related macrophages contained in bone marrow-derived macrophages of cancer-bearing mice, induces their activation, and thereby inhibits tumor growth. Therefore, in vitro FG-treated cancers We have demonstrated that the patient's bone marrow-derived macrophages are useful as drugs for treating cancer.

Claims (13)

A tumor-related macrophage activator containing a prolyl isomerase inhibitor as an active ingredient. The tumor-related macrophage activator according to claim 1, which is a tumor phagocytosis enhancer for Ly6C-positive macrophages or their human counterparts. The tumor-related macrophage activator according to claim 2, wherein the human counterpart is CD14 ⁺ CD16 ^- macrophages and / or CD14 ^{lo /} -CD16 ^{+ macrophages.} The tumor-related macrophage activator according to claim 1 or 2, which is a tumor phagocytosis enhancer for Ly6C ^{low macrophages or their human counterparts.} The tumor-related macrophage activator according to claim 4, wherein the human counterpart is CD14 ⁺ CD16 ^{-macrophages.} The prolyl isomerase inhibitors are FG-4592 (Roxadustat), FG-2216, FG-4497, dimethyloxalylglycine, BAY-85-3934 (Molidustat), AKB-6548 (Vadadustat), GSK1278863 (Daprodustat), JTZ. The tumor-related macrophage activator according to any one of claims 1 to 3, which is at least one selected from the group consisting of −951, TM-6089, and DS-1093. The tumor-related macrophage activator according to any one of claims 1 to 6, which is an anticancer agent. Lung cancer, melanoma, glioma, breast cancer, esophageal cancer, intraductal bile duct cancer, gastric cancer, bile sac cancer, pancreatic cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer, osteosarcoma, cartiloma The tumor-related macrophage activator according to any one of claims 1 to 7, which is administered for rhombic myoma, smooth myoma, or advanced cancer or treatment-resistant cancer. A method for producing a drug for treating cancer, which comprises reacting a macrophage fraction containing a tumor-related macrophage with a prolyl isomerase inhibitor in vitro. The production method according to claim 9, wherein the macrophage fraction is a macrophage fraction obtained from the bone marrow of a cancer patient. The production method according to claim 9 or 10, wherein the tumor-related macrophage comprises a Ly6C-positive macrophage or a human counterpart thereof. A drug for treating cancer, which comprises a first preparation containing the tumor-related macrophage activator according to any one of claims 1 to 8 and a second preparation containing an anticancer agent other than the tumor-related macrophage activator. A pharmaceutical composition for treating cancer, which comprises the tumor-related macrophage activator according to any one of claims 1 to 8 and an anticancer agent other than the tumor-related macrophage activator.