JP7535285B2 - Tumor-associated macrophage activating agent - Google Patents

Description

Application of Article 30, paragraph 2 of the Patent Act ▲1▼ Publication date: February 28, 2019 Publication address: http://pharmacology.main.jp/92nenkai/pdf/pdf/02.pdf ▲2▼ Publication date: February 28, 2019 Publication address: http://pharmacology.main.jp/92nenkai/pdf/pdf/04.pdf ▲3▼ Publication date: February 28, 2019 Publication address: http://pharmacology.main.jp/92nenkai/pdf/pdf/09.pdf pdf ▲4▼ Date: March 14, 2019 Meeting name: 92nd Annual Meeting of the Japanese Pharmacological Society ▲5▼ Date: March 15, 2019 Meeting name: 92nd Annual Meeting of the Japanese Pharmacological Society ▲6▼ Date: March 15, 2019 Meeting name: 92nd Annual Meeting of the Japanese Pharmacological Society

The present invention relates to a tumor-associated macrophage activator that can suppress tumor growth by itself.

In tumor tissue, blood and vascular permeability changes, reducing the supply of oxygen and nutrients, as well as the delivery of drugs to the tumor tissue. This is one of the reasons for the resistance of tumors to radiation therapy or drug therapy. In particular, highly malignant cancer types have even lower sensitivity to anticancer drugs and a low response rate. Furthermore, treatment with cytotoxic anticancer drugs, which are standard therapeutic drugs, often results in significant side effects, due to the low specificity of the cytotoxic effect on tumors.

The most abundant immune cells in tumor tissue are macrophages. Tumor-associated macrophages (TAMs) are classified into inflammatory (M1) and anti-inflammatory (M2) phenotypes. M1 macrophages are known to have high phagocytosis and are 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 exclude cytotoxic lymphocytes from tumors such as lung cancer and melanoma, which are classified as T cell-poor and TAM-rich tumors. Thus, strong polarization into M2 macrophages suppresses tumor immune response function and causes treatment resistance.

Therefore, development of tumor immunotherapy that reactivates host immunity in tumor tissue is underway. Examples of tumor immunotherapy include immune cell therapy, antibody therapy, and cytokine therapy. Another example of tumor immunotherapy is immune checkpoint inhibitors, which reactivate cytotoxic T cells by deactivating signals that suppress the activity of cytotoxic T cells. Immune checkpoint inhibitors are thought to be effective against a wide range of cancers, and have attracted attention around the world, with various clinical trials being conducted with the aim of expanding the range of diseases for which they can be used. As a result, ipilimumab (trade name: Yervoy), pembrolizumab (trade name: Keytruda), nivolumab (trade name: Opdivo), and other drugs have been approved in succession in the United States, Japan, and other countries.

On the other hand, hypoxia-inducible factor (HIF) is a major regulator of cellular responses to hypoxia. HIF stability is post-transcriptionally regulated by oxygen availability via prolyl hydroxylase (PHD). It has been reported that administration of prolyl hydroxylase inhibitors to tumors normalizes vascular function in tumors, and that while prolyl hydroxylase inhibitors alone do not exert an antitumor effect, their combined use with anticancer drugs can enhance the antitumor effect of the anticancer drug (Patent Document 1). In addition, several reports have disclosed that normalization of vascular function leads to accelerated tumor progression (Non-Patent Document 3, Non-Patent Document 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.

Patent Application No. 2018-35112

Conventional tumor immunotherapy has focused on controlling the adaptive immune system to induce specific responses through immune memory. However, according to conventional tumor immunotherapy, the drug delivery efficiency is low in tumor tissues with incomplete blood flow and low tissue osmotic pressure, and drug therapy via the blood flow is not efficient. In particular, it is difficult to target the deep part of a tumor. Even immune checkpoint inhibitors, which have attracted attention worldwide, show a certain response rate, but their effects are still not sufficient. Since immune checkpoint inhibitors exert their efficacy through a mechanism of action that reactivates T cells, they are particularly ineffective against tumors such as lung cancer and melanoma, which are classified as tumors with few T cells and rich in TAMs. In other words, there is a limit to the antitumor effects that can be obtained with the mechanism of action of drugs used in conventional tumor immunotherapy.

The present invention therefore aims to provide a drug that activates tumor immunity through a mechanism of action different from that of drugs used in previous tumor immunotherapy.

After extensive research, the present inventors have newly discovered that prolyl hydroxylase inhibitors, known as drugs that normalize vascular function in tumors, exert a tumor growth-inhibiting effect on their own. In light of the fact that it has been known that prolyl hydroxylase inhibitors do not exert an antitumor effect on their own, and that the normalization of vascular function, which is known as the mechanism of action of prolyl hydroxylase inhibitors, has generally been reported to be capable of accelerating tumor progression, the newly discovered tumor growth-inhibiting effect of prolyl hydroxylase inhibitors was unexpected.

After further research, the inventors found that prolyl hydroxylase inhibitors significantly increase the levels of tumor phagocytic macrophages at a time when tumor blood vessel normalization does not occur. Since tumor blood vessel normalization does not have an effect of suppressing tumor growth, but an increase in the level of tumor phagocytic macrophages does, this result demonstrated that prolyl hydroxylase inhibitors activate tumor-associated macrophages through a mechanism of action different from that of tumor blood vessel normalization. In addition, while the tumor vascular normalization ability reported for prolyl hydroxylase inhibitors is associated with mouse Ly6C-negative monocytes, the tumor-associated macrophage activation ability found enhances tumor phagocytosis by mouse Ly6C-positive monocytes, and it was found that while prolyl hydroxylase inhibitors enhance tumor phagocytosis by mouse Ly6C-positive monocytes, the level of Ly6C-negative monocytes does not increase. This further supports the idea that tumor vascular normalization and tumor-associated macrophage activation by prolyl hydroxylase inhibitors are due to different mechanisms of action.

In addition, because increased levels of tumor-phagocytic macrophages were also observed in macrophages isolated from bone marrow, it was found that prolyl hydroxylase inhibitors are drugs with a mechanism of action that directly affects macrophages, that is, drugs that activate tumor immunity through a mechanism of action different from that of drugs used in previous tumor immunotherapy. Furthermore, the tumor growth inhibitory effect of prolyl hydroxylase inhibitors alone was found in tumor tissues with few T cells, further supporting the idea that prolyl hydroxylase inhibitors and immune checkpoint inhibitors have different mechanisms of action.

The present invention was completed based on these findings and through further investigation.

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

The tumor-associated macrophage activating agent of the present invention activates tumor immunity through a new mechanism of action (natural immune system) that differs from drugs used in previous tumor immunotherapy, thereby making it possible to effectively suppress tumor growth. In addition, the new mechanism of action of the tumor-associated macrophage activating agent of the present invention makes it possible to suppress tumor growth even in tumor tissues with few T cells, for which immune checkpoint inhibitors have shown little response. In addition, because the tumor-associated macrophages are directly activated by the tumor-associated macrophages activating agent of the present invention, a pharmaceutical for cancer treatment can be prepared by treating the tumor-associated macrophages in vitro.

(i) LLC tumor growth curves showing the results of treating mice with vehicle (Veh) or 100 mg/kg PHD inhibitor (FG) on day 10 after transplantation of lung cancer LLC tumor cells into mice (n=15). (ii) LLC tumor weights on day 16 (n=15). The results are based on two-way ANOVA, with * indicating p<0.05 and **** indicating p<0.0001. (i) LLC tumor growth curves showing the results of treating mice with vehicle (Veh) or 100 mg/kg PHD inhibitor (FG) on day 10 after implantation of melanoma B16F10 tumor cells (n=8). (ii) B16F10 tumor weights on day 16 (n=8). The results are from a Mann-Whitney test, with * indicating p<0.05 and **** indicating p<0.0001. (i) Quantitative results of the ratio of CC3-positive cells among all cells constituting the tumor tissue (n=3), and (ii) results of tumor cell proliferation assay at 72 hours after FG treatment (n=3). The results are from one-way analysis of variance, and ns indicates no significant difference. Quantification of tumor infiltration: (i) CD45 ⁺ cell ratio (n=12-13) and (ii) CD11b ⁺ F4/80 ⁺ cell ratio (n=8) in LLC tumors by flow cytometry analysis. The results are based on the Mann-Whitney test, and * indicates p<0.05. Quantification of tumor infiltration: CD4 ⁺ and CD8 ⁺ cell ratios (n=5) in lung cancer LLC tumors analyzed by flow cytometry. The results are based on the Mann-Whitney test, and ns indicates no significant difference. The results of quantification of the number of F4/80 ⁺ cells per 1 mm ² of tumor (n=5) are shown. The results are based on the Mann-Whitney test, and ** indicates p<0.01. Quantification of tumor infiltration: CD4 ⁺ and CD8 ⁺ cell ratios (n=4) in melanoma B16F10 tumors by flow cytometry analysis. 1 shows the gating strategy for flow cytometry of macrophages. The figures show the quantification results (n=6) of the ratios of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in lung cancer LLC tumors by flow cytometry analysis. The results are the results of a Mann-Whitney test, with ** indicating p<0.01 and ns indicating no significant difference. Quantification of the ratios of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in melanoma B16F10 tumors by flow cytometry analysis (n=4) is shown. This shows tumor growth curves (n=4) of LLC tumor mouse models administered clodronate liposomes (Clo) or plain control liposomes (Lip) and then treated with vehicle (Veh) or 100 mg/kg PHD inhibitor (FG). The results are from a two-way analysis of variance, with *** indicating p<0.001 and ns indicating no significant difference. The results of quantification of the number of F4/80 ⁺ cells per mm ² of the tumor in Fig. 11 (n = 3) are shown. The results are those of a Mann-Whitney test, and *** indicates p < 0.001. Quantification results (n=3) of tumor vascular density and vascular lumen area 48 hours after administration of vehicle (Veh) or PHD inhibitor (FG) in a GFP-labeled LLC tumor model. Note that the results are from a Mann-Whitney test, and ns indicates no significant difference. The results of quantification (n=5) of the number of phagocytes (GFP ⁺ F4/80 ⁺ ) in the tumor 48 hours after administration of a GFP-labeled LLC tumor model treated with a vehicle (Veh) or a PHD inhibitor (FG) are shown. The results are those of a Mann-Whitney test, and ** indicates p<0.01. (i) Schematic diagram of the in vitro bead phagocytosis assay and (ii) quantification results of the proportion of phagocytic BMDMs (bone marrow derived macrophages) (n=3). The gating strategy for flow cytometry of GFP ⁺ macrophages is shown. The results of quantification of the ratio of Ly6C-fractionated GFP ⁺ macrophages (Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages) to total GFP ⁺ macrophages by flow cytometry analysis (n=5-6) are shown. (i) Schematic diagram of ex vivo bead phagocytosis assay for Ly6C positive macrophages, (ii) quantification of the proportion of phagocytic Ly6C ^lo macrophages (n=5), and (iii) quantification of the proportion of phagocytic Ly6C ^hi macrophages (n=4-5). The results are from the Mann-Whitney test, and * indicates p<0.05. (i) Schematic diagram of ex vivo bead phagocytosis assay for Ly6C-negative macrophages and quantification results of the ratio of phagocytic Ly6-negative macrophages (n=3-4). Note that the results are from the Mann-Whitney test, and ns indicates no significant difference. (i) Tumor growth curves of LLC tumor models of Vhl ^fl/fl LysM-Cre ^-/- (control mice (Ctrl); n=9) and Vhl ^fl/fl LysM-Cre ^+/- (macrophage-specific VHL knockout mice (VHL KO); n=7), and (ii) tumor weights on day 16 (n=7 to 9). (i) is the result of two-way analysis of variance, (ii) is the result of Mann-Whitney test, and ** indicates p<0.01. The figures show the results of flow cytometry analysis (n=6) of the proportions of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in a VHL KO LLC tumor model, where the results were obtained by Mann-Whitney test and * indicates p<0.05. qPCR analysis of bone marrow-derived macrophages (BMDM) isolated from mice treated with vehicle (Veh) or PHD inhibitor (FG) showing quantitative analysis of HIF downstream genes. (i) Schematic diagram of the procedure for transplanting Ly6C ^lo macrophages selected from LLC tumor mouse model into the tumor site of another LLC tumor mouse model; (ii) Tumor growth curves (n=6) in LLC tumor mouse models transplanted with Ly6C ^lo macrophages from PBS-injected or vehicle (Veh)- or FG-treated mouse tumors; and (iii) tumor weights (n=6) on day 16. (ii) The results of two-way ANOVA analysis, ** indicates p<0.01. (iii) The results of Mann-Whitney test. (i) Tumor weights at day 16 in LLC tumor mouse models (n=5-6) injected with PBS or implanted with Ly6C ^lo or Ly6C ^neg macrophages from vehicle (Veh) or FG-treated mouse tumors; (ii) quantification of tumor vascular density (n=3); and (iii) quantification of tumor vascular lumen area (n=3). (i) is the result of Mann-Whitney test; (ii) and (iii) are the results of unpaired Student's t-test, * indicates p<0.05, ** indicates p<0.01. (i) Schematic diagram of the procedure for treating Ly6C ^lo macrophages selected from tumors in LLC tumor mouse model with vehicle (Veh) or FG and transplanting them into another LLC tumor mouse model; (ii) Tumor growth curves (n=5) in LLC tumor mouse model injected with PBS or transplanted with vehicle (Veh) or FG-treated Ly6C ^lo macrophages; and (iii) tumor weights (n=5) on day 16. (ii) Results of two-way ANOVA analysis, * indicates <0.05, *** indicates p<0.001. (iii) Results of unpaired Student's t-test, * indicates p<0.05. (i) Schematic diagram of the procedure in which bone marrow derived macrophage fraction (BMDM) of LLC tumor mouse model was treated with vehicle (Veh) or FG and transplanted into another LLC tumor mouse model; (ii) Tumor growth curves (n=5) in LLC tumor mouse model injected with PBS or transplanted with vehicle (Veh) or FG-treated BMDM.

1. Tumor-associated macrophage activating agent (TAM activating agent)
(Active ingredient)
The tumor-associated macrophage activator of the present invention (hereinafter also referred to as "TAM activator" in the present specification) contains a prolyl hydroxylase inhibitor (hereinafter also referred to as "PHD inhibitor" in the present specification) as an active ingredient. Prolyl hydroxylase (PHD) is an enzyme that, under normal oxygen concentration conditions, hydroxylates specific proline residues in the α-subunit (HIFα) of hypoxia-inducible factor (HIF), thereby leading to the decomposition of HIF and thereby suppressing the hypoxic response, while its enzymatic 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 the activation of tumor-associated macrophages.

PHD inhibitors are not particularly limited as long as they are compounds that have the effect of inhibiting the activity of prolyl hydroxylase (PHD) and upregulating the HIF signaling pathway, or are generally known PHD inhibitors, and examples thereof include dimethyloxalylglycine (DMOG), pyrazolopyrimidine compounds (such as the pyrazolopyrimidine compounds described in International Publication No. 2014-030716), FG-4592 (roxadustat), FG-2216, FG-4497, BAY-85-3934 (molidustat), AKB-6548 (Vadadustat), GSK127886 (daprodustat), JTZ-951, TM-6089, and DS-1093. Note that 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 also known as Roxadustat and is a compound also known as N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine.

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

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

AKB-6548 (development number) is also known as Vadadustat, a compound also known as (5-(3-chlorophenyl)-3-hydroxypicolinoyl)glycine.

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

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

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

The PHD inhibitor may be a synthetic one or a commercially available one. The method for synthesizing the PHD inhibitor is not particularly limited, and may be any known synthesis method.

In the present invention, commercially available products can also be used as PHD inhibitors. Commercially available PHD inhibitors that can be used in the present invention include, for example, FG4592 (Roxadustat), FG2216, and FG4497 from FiveGen, BAY-85-3934 (Molidustat) from Bayer Yakuhin, AKB-6548 (Vadadustat) from Akebia, GSK127886 (daprodustat) from GlaxoSmithKline, JTZ-961 from Japan Tobacco Inc., TM-6089 from Professor Miyata Toshio of Tohoku University, and DS-1093 from Daiichi Sankyo Co., Ltd.

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

The TAM activator of the present invention may contain pharma- ceutically acceptable carriers and additives as necessary to prepare the desired dosage form and formulation. Examples of such carriers and additives include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizing agents, stabilizers, sweeteners, colorants, flavoring agents, odorants, surfactants, moisturizers, preservatives, pH adjusters, buffers, thickeners, etc.

The dosage form of the TAM activator of the present invention is not particularly limited and may be appropriately set depending on the mode of administration, etc. Specific dosage forms of the TAM activator of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; solid preparations such as tablets, hard capsules, soft capsules, granules, powders, and pills; topical preparations such as creams, gels, ointments, sprays, and patches, inhalants, etc., and may be any dosage form for internal or external use. In addition, when made into an injection, it may be in the form of a powder (e.g., a freeze-dried powder) to be dissolved in physiological saline or the like before use.

The TAM activating agent of the present invention is used for the purpose of activating tumor-associated macrophages. When tumor-associated macrophages are activated, the tumor phagocytosis of the tumor-associated macrophages is enhanced. Therefore, the TAM activating agent of the present invention can be used as an agent for enhancing the tumor phagocytosis of tumor-associated macrophages, and as an anticancer agent that suppresses tumor growth by activating tumor-associated macrophages.

Tumor-associated macrophages that enhance tumor phagocytosis in the TAM activating agent of the present invention include Ly6C positive macrophages in mice, more specifically Ly6C ^low macrophages (also referred to as Ly6C ^lo macrophages in the present specification) and Ly6C ^high macrophages (also referred to as Ly6C ^hi macrophages in the present specification). Similarly, in the case of other mammals such as humans, hamsters, guinea pigs, cows, sheep, pigs, goats, monkeys, and rabbits, counterparts of mouse Ly6C ^low macrophages and Ly6C ^high macrophages in the other mammals can be included. For example, in the case of humans, human counterparts of Ly6C ^low macrophages and Ly6C ^high macrophages, respectively, include CD14 ⁺ CD16 ^- macrophages and CD14 ^lo/- CD16 ⁺ macrophages. The TAM activating agent of the present invention can be used as an agent for enhancing tumor phagocytosis of these specific tumor-associated macrophages.

From the viewpoint of obtaining a higher tumor phagocytosis, preferred tumor-associated macrophages that enhance tumor phagocytosis in the TAM activator of the present invention include Ly6C ^low macrophages in mice and CD14 ⁺ CD16 ^- macrophages in humans. Furthermore, in the case of mammals other than mice and humans, preferred tumor-associated macrophages that enhance tumor phagocytosis in the TAM activator of the present invention from the viewpoint of further enhancing tumor phagocytosis include counterparts in other mammals of mouse Ly6C ^low macrophages and human CD14 ⁺ CD16 ^- macrophages.

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

Other anticancer agents that may be combined with the TAM activator of the present invention are not particularly limited, but include cytocidal anticancer agents (specifically, alkylating agents, metabolic antagonists, platinum preparations, topoisomerase inhibitors, microtubule-active anticancer agents, anticancer antibiotics, etc.), cancer vaccines used in immune cell therapy, molecular targeted drugs used in antibody therapy, drugs used in cytokine therapy, immunoactivators used in BRM therapy, hormones, immune checkpoint inhibitors (specifically, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, etc.), etc.

Examples of alkylating agents include ifosfamide, carboquone, cyclophosphamide, dacarbazine, thiotepa, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine.

Examples of metabolic antagonists include enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocfosfate, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, doxifluridine, nelarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, and methotrexate.

Examples of platinum preparations include oxaliplatin, carboplatin, cisplatin, and nedaplatin.

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

Examples of microtubule-active anticancer drugs include eribulin, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, etc.

Examples of anticancer antibiotics include actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone, and liposomal doxorubicin.

Cancer vaccines include, for example, peptide vaccines and dendritic cell vaccines.

Examples of molecular targeted drugs include ibritumomab tiuxetan, nivolumab, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, trastuzumab emtansine, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib, rituximab, vemurafenib, and alectinib.

Drugs used in cytokine therapy include, for example, interleukin-2 (Aldesleukin), interferon-α, interferon-β, and interferon-γ.

Examples of immunostimulants include Maruyama vaccine, OK-432 (picibanil), ubenimex, lentinan, krestin, BCG (Immucyst, Immunobladder), etc.

Examples of hormonal agents include anastrozole, exemestane, estramustine, ethinyl estradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin, and letrozole.

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

These other anticancer agents may be used alone or in combination of two or more. Among these other anticancer agents, those that can be taken up into tumor cells and exert an antitumor effect are preferred, with cytocidal anticancer agents (alkylating agents, metabolic antagonists, platinum preparations, topoisomerase inhibitors, microtubule-acting anticancer agents, and anticancer antibiotics) being more preferred, platinum preparations being even more preferred, and cisplatin being particularly preferred.

The TAM activator of the present invention can significantly improve the antitumor effect by combining with other anticancer drugs, and therefore the dosage of the other anticancer drugs can be reduced compared to conventional methods, thereby reducing side effects in anticancer treatment. For example, by combining with the TAM activator of the present invention, the dosage of the other anticancer drugs 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 dosage is usually about 6 to 30 mg/kg (body weight) per dose, either once or twice a day, in mice, and usually about 0.5 to 2.5 mg/kg (body weight) per dose, either once or twice a day, in humans. However, when used in combination with the TAM activator of the present invention, the dosage can be reduced to 2 to 20 mg/kg, preferably 2 to 15 mg/kg (body weight), and more preferably 2 to 10 mg/kg (body weight), in mice, and to 0.16 to 1.6 mg/kg, preferably 0.16 to 1.2 mg/kg (body weight), and more preferably 0.16 to 0.8 mg/kg (body weight), per dose, in humans.

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 macrophages accumulate in large numbers. Cancers in which macrophages accumulate in large numbers are known, and specific examples include lung cancer, melanoma, glioma, breast cancer, esophageal cancer, intraductal bile duct cancer, gastric cancer, gallbladder cancer, pancreatic cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and leiomyosarcoma. Other specific examples of cancers in which macrophages accumulate in large numbers include advanced cancers and treatment-resistant cancers.

Furthermore, from the viewpoint of efficiently activating tumor-associated macrophages and achieving even greater tumor phagocytosis, the cancer types to which the TAM activating agent of the present invention is administered are more preferably cancers in which specific macrophages, such as the above-mentioned Ly6C ^low macrophages and Ly6C ^high macrophages, preferably Ly6C ^low macrophages (in the case of mice); CD14 ⁺ CD16 ^- macrophages and CD14 ^lo/- CD16 ⁺ macrophages, preferably CD14 ⁺ CD16 ^- macrophages (in the case of humans); and their counterparts (in the case of mice and mammals other than humans), have accumulated in large amounts.

Examples of cancers in which the specific macrophages accumulate in large amounts include cancers in which the average ratio (the average for n≧3, preferably n≧4, more preferably ≧6) of Ly6C ^low macrophages (in the case of mice) or their counterparts (in the case of other mammals) among all macrophages in the tumor is 35% or more, preferably 40% or more, and the average ratio (the average for n≧3, preferably n≧4, more preferably ≧6) of Ly6C ^high macrophages (in the case of mice) or their counterparts (in the case of other mammals) among all macrophages in the tumor is 6% or more, preferably 15% or more, more preferably 20% or more.

From the above viewpoint, preferred examples of cancer types to which the TAM activating agent of the present invention is administered include lung cancer, melanoma, breast cancer, glioma, esophageal cancer, and intrahepatic bile duct cancer, more preferably lung cancer and melanoma, and even more preferably lung cancer. From the same viewpoint, preferred examples of cancer types to which the TAM activating agent 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 for which an antitumor effect is desired, and examples of such organisms include, in addition to humans, mammals such as mice, rats, hamsters, guinea pigs, cows, sheep, pigs, goats, monkeys, and rabbits.

The method of administration of the TAM activator of the present invention is not particularly limited and may be appropriately selected depending on the type of disease to which it is applied, and may be systemic or local administration. Specific examples include oral, intravascular (intraarterial or intravenous), transdermal, enteral, and pulmonary administration. Intravascular administration also includes intravascular injection and continuous infusion. The method of administration of the TAM activator of the present invention may be the same as or different from the method of administration of the anticancer agent to be targeted for enhancing the antitumor effect. When the method of administration of the TAM activator of the present invention is the same as the other anticancer agent to be targeted for enhancing the antitumor effect, the TAM activator of the present invention and the other anticancer agent may be administered in a mixed state, or each may be administered separately.

The dosage of the TAM activator of the present invention may be appropriately set depending on the type of PHD inhibitor, the type of other anticancer agent when combined with the other anticancer agent, the age, sex, weight, severity of symptoms, administration method, etc. of the subject. Specifically, the dosage of the PHD inhibitor in the TAM activator of the present invention is, for example, at least twice, preferably at least 2.5, and more preferably at least 3 times the dosage when the PHD inhibitor is a tumor vascular function normalizer that does not exert an antitumor effect by itself. In addition, the upper limit of the dosage of the PHD inhibitor in the TAM activator of the present invention is not particularly limited, but may be up to 8 times, preferably up to 6 times, and more preferably up to 4 times the dosage when the PHD inhibitor is a tumor vascular function normalizer.

Specifically, when the active ingredient, a PHD inhibitor, is FG4592, the dose in terms of PHD inhibitor per administration for mice is, for example, 55 to 310 mg/kg (body weight), preferably 67 to 185 mg/kg (body weight), more preferably 80 to 145 mg/kg (body weight), and even more preferably 85 to 120 mg/kg (body weight), and for humans is, for example, 4.5 to 25 mg/kg (body weight), preferably 5.5 to 15 mg/kg (body weight), more preferably 6.5 to 12 mg/kg (body weight), and even more preferably 7 to 10 mg/kg (body weight). Furthermore, when the active ingredient PHD inhibitor is DMOG, the amount of PHD inhibitor per administration is about 800 to 2000 mg/kg (body weight) for mice, preferably 1000 to 1500 mg/kg (body weight), and more preferably 1100 to 1300 mg/kg (body weight), and about 64 to 400 mg/kg (body weight) for humans, preferably 80 to 200 mg/kg (body weight), and more preferably 90 to 100 mg/kg (body weight).

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 may be before administration of the other anticancer agent or simultaneously with administration of the other anticancer agent, but is preferably before administration of the anticancer agent. When the TAM activator of the present invention is administered before administration of the other anticancer agent, the administration timing can be, for example, about 48 hours to 12 days, preferably 3 to 10 days, more preferably 5 to 8 days before administration of the other anticancer agent.

2. Method for Producing a Drug for Cancer Treatment As described above, a tumor-associated macrophage (TAM) activating agent can directly activate tumor-associated macrophages. That is, the tumor-associated macrophage activating agent of the present invention can convert tumor-associated macrophages with poor phagocytic activity into an enhanced phagocytic phenotype. Therefore, a drug for cancer treatment can be prepared by treating tumor-associated macrophages in vitro. That is, the present invention also provides a method for producing a drug for cancer treatment, which comprises acting a prolyl hydroxylase inhibitor on a macrophage fraction containing tumor-associated macrophages in vitro.

In the method for producing a pharmaceutical for cancer treatment of the present invention, the tumor-associated macrophages on which a prolyl hydroxylase inhibitor (PHD inhibitor) is applied are tumor-associated macrophages activated by the above-mentioned "1. Tumor-associated macrophage activator (TAM activator)". In other words, examples of the tumor-associated macrophages on which a PHD inhibitor is applied include Ly6C ^low monocytes and Ly6C ^high monocytes, preferably Ly6C ^low monocytes (in the case of mice); CD14 ⁺ CD16 ^- monocytes and CD14 ^lo/- CD16 ⁺ monocytes, preferably CD14 ⁺ CD16 ^- monocytes (in the case of humans); and their counterparts (in the case of mice and mammals other than humans).

Examples of macrophage fractions containing tumor-associated macrophages include macrophage fractions obtained from the bone marrow of cancer patients and macrophage fractions obtained from tumor tissues. In addition, the macrophage fraction may contain tumor-associated macrophages, and the macrophages may be substantially only tumor-associated macrophages (i.e., the macrophage fraction itself is an isolated fraction of tumor-associated macrophages), or may contain other macrophages in addition to tumor-associated macrophages. A method for obtaining a macrophage fraction can be appropriately determined by a person skilled in the art based on known methods.

Regarding PHD inhibitors, please refer to the above section "1. Tumor-associated macrophage activators (TAM activators)."

To treat isolated tumor-associated macrophages, a PHD inhibitor may be contacted with the isolated tumor-associated macrophages and incubated. The amount of PHD inhibitor used in the culture medium may be 50 to 150 μM, preferably 80 to 120 μM, and more preferably 90 to 110 μM. The incubation temperature may be, for example, 10 to 35°C, preferably 20 to 25°C, and the incubation time may be, for example, 8 to 20 hours, preferably 10 to 15 hours.

Tumor-associated macrophages treated with a PHD inhibitor are activated and acquire an enhanced phagocytic phenotype. The tumor-associated macrophages thus prepared with enhanced tumor phagocytosis are themselves useful as a cancer treatment drug in tumor immunotherapy. This cancer treatment drug can be returned to the tumor tissue of the subject from which the tumor-associated macrophages were collected, thereby suppressing the proliferation of the tumor tissue of the subject.

3. Drugs for Cancer Treatment and Pharmaceutical Compositions for Cancer Treatment As described above, the antitumor effect of the tumor-associated macrophage (TAM) activator can be significantly improved by combining it with other anticancer drugs. Therefore, the present invention also provides drugs for cancer treatment and pharmaceutical compositions for cancer treatment in which the above-mentioned TAM activator is combined with other anticancer drugs.

The pharmaceutical for cancer treatment of the present invention is used when treating cancer by administering the above-mentioned TAM activator by the same or different administration method as another anticancer drug, and is characterized by comprising a first formulation containing the other anticancer drug and a second formulation containing the above-mentioned TAM activator.

The pharmaceutical composition for cancer treatment of the present invention is used when treating cancer by administering the above-mentioned TAM activator in the same manner as other anticancer drugs, and is characterized in that the other anticancer drugs and the above-mentioned TAM activator are contained in the same formulation.

The pharmaceutical composition for cancer treatment of the present invention and the TAM activator and other anticancer agents which are components of the pharmaceutical composition for cancer treatment, their dosages, and modes of use, etc. are as described above in the section "1. Tumor-associated macrophage activator (TAM activator)."

The present invention will be explained below using test examples, but the present invention is not limited to these test examples.

(Animal Subjects)
All animal experiments were conducted with the approval of the Osaka City University Animal Experiment Management Regulations and the Japan Laboratory Animal Association. Furthermore, animal research and handling were conducted in strict compliance with the guidelines of the Animal Experiment Management Regulations. Subject animals were C57BL/6 male mice obtained from SLC Japan, Inc. LysM Cre, VHL floxed mice obtained from Jackson Laboratories and bred in the Osaka City University facility. Mice were used in cages with a 12-hour light/dark cycle and an environment of 22±1°C, with food and water provided ad libitum.

(Statistical Analysis)
Statistical analysis was performed using unpaired t-tests followed by Bonferroni post-hoc tests. Details are provided in the figure briefs. All statistical analyses were performed using GraphPad Prism (version 7.02) software. Statistical significance was defined as p<0.05. Additionally, p<0.01, p<0.001, and p<0.0001 are also presented to indicate the level of confidence. All analyses were performed using two-tailed tests.

Test Example 1: Tumor proliferation suppression effect by PHD inhibitors Tumor-implanted model mice were treated with PHD inhibitors, and the cell proliferation suppression effect was examined.

Cell Culture, Tumor Xenograft Models, and PHD Inhibitor Treatment
Lung cancer LLC cells and melanoma B16F10 cells (RIKEN BRC) were maintained in Dulbecco's modified 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% ^CO2 atmosphere. Cultured cells were harvested and resuspended in phosphate-buffered saline (PBS) at 1 x ¹⁰⁷ cells/mL. A portion of the cells (1 x ¹⁰⁶ cells) was subcutaneously implanted into the right flank of 8-12 week-old mice. Ten days after tumor implantation, mice were intraperitoneally administered 3 mg (100 mg/kg; in 5 v/v % DMSO aqueous solution; 600 μl; the dose to mice was the same in the following test examples) of PHD inhibitor FG4592 (Selleck Chemicals, TX, USA) or 600 μl of 5 v/v % DMSO aqueous solution as vehicle (Veh) (eligible tumor size for this test was ^100-400 mm3). Tumors were measured in two dimensions using calipers once every two days. Tumor tissue volume was calculated using the following formula 1. Mice were sacrificed at 0°C or when the tumor volume exceeded ⁴⁵⁰⁰ mm3 or when the tumor ruptured, and tumor tissues were excised and weighed.

(result)
The tumor volume transition and tumor weight for the mice transplanted with lung cancer LLC cells, and the tumor volume transition and tumor weight for the mice transplanted with melanoma B16F10 cells are shown in Figure 1(i) and Figure 1(ii) and Figure 2(i) and Figure 2(ii), respectively. As is clear from these figures, tumor growth was significantly inhibited in FG-treated mice for both lung cancer LLC cells and melanoma B16F10 cells. In addition, although not shown, it was confirmed that there was a dose-dependent inhibition of tumor growth by FG, and that repeated FG treatment further suppressed tumor growth and extended survival time.

Test Example 2: Tumor-associated macrophage activation effect by PHD inhibitors
Test Example 2-1
To determine whether FG induced apoptosis or proliferation inhibition in tumor cells, quantification of caspase-3 positive cells by cleaved caspase-3 (CC3) immunostaining and in vitro cell proliferation assays were performed on tumor tissues treated with the PHD inhibitor FG4592 (FG) or vehicle (Veh).

(Immunostaining)
Tumor tissues treated with the PHD inhibitor FG4592 (FG) or vehicle (Veh) 2 or 6 days after treatment were sliced into 8 μm-thick frozen sections at −20°C and air-dried. Sectioned tissue samples were rehydrated with PBS for 10 min and fixed with 4 (w/v)% cold paraformaldehyde for 10 min. Sections were washed with PBS and permeabilized with 0.5% (v/v) Triton™ X-100 in PBS for 10 min. Sections were blocked in 5% normal goat serum for 30 min at room temperature (20-25°C). Sections were incubated with the primary antibody anti-CC3 (1:400; Cell Signaling Technologies, MA, USA) overnight at 4°C. Sections were washed in 0.1% Tween 20 in PBS and incubated with the appropriate fluorophore secondary antibody (AlexaFluor 488 or Cy3, goat anti-rat or goat anti-rabbit IgG; Biolegend, CA, USA) for 1 h at room temperature. Sections were washed in 0.1% Tween 20 in PBS, dehydrated in ethanol and air-dried. Subsequently, they were mounted with Vectashield mounting medium (Vector Laboratories, CA, USA) with 4',6-diamidino-2-phenylindole stain (1:5000) and a coverslip.

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

Cell proliferation assay
Lung cancer LLC cells were seeded in 96-well plates at 2 × ¹⁰⁴ cells per well. Then, FG was added to the medium at a given concentration (0, 50, 100, 200, and 400 μM). Six hours after FG treatment, the medium was replaced, and after another 72 hours, AlamarBlue reagent (Bio-Rad) was added, and the plate was incubated at 37°C in a 5% _CO2 incubator for 6 hours. Finally, the optical density (OD) was measured at 570 and 600 nm, and the cell viability was calculated. Each experiment was performed in triplicate.

(result)
The quantitative results of the ratio of CC3-positive cells among all cells constituting the tumor tissue are shown in FIG. 3(i), and the results of the tumor cell proliferation assay at 72 hours after FG treatment are shown in FIG. 3(ii). As is clear from these figures, there was no significant difference in the CC3 expression level and cell proliferation with or without FG treatment. In other words, the inhibition of tumor proliferation observed by FG treatment in Test Example 1 was not due to apoptosis or proliferation inhibition in tumor cells. This result suggests that FG does not directly affect tumor cells.

Test Example 2-2
To examine whether the PHD inhibitor FG4592 (FG) altered immune cell responses, tumor infiltration was quantified by measuring the ratios of CD45 ⁺ cells, CD45 ⁺ CD11b ⁺ F4/80 ⁺ cells, and T cell populations, and the number of macrophages in tumor tissues was also quantified based on immunofluorescence staining.

(Tumor Dissociation)
Tumors treated with PHD inhibitor FG4592 (FG) or vehicle (Veh) 2 or 6 days after treatment were minced and digested with 1 mg/mL collagenase IV containing 50 μg/mL DNase I for 45 min at 37°C with shaking. Cells were filtered through a 100 μm nylon mesh and washed in isolation buffer (PBS containing 2% FBS and 2 mM EDTA). Red blood cells were lysed using RBC lysis buffer (BioLegend). Cells were resuspended in isolation buffer and Fc receptors were blocked with CD16/32 blocking antibody (Biolegend) for 15 min on ice.

(Flow cytometry and cell sorting)
Dissociated tumor single cells were diluted and stained for 30 min on ice in the dark with the following fluorochrome-conjugated antibodies: for macrophages, anti-mouse F4/80-PE (clone BM8, BioLegend), anti-mouse CD11b-APC-Cy7 (clone M1/70, BioLegend), anti-mouse CD45PE-y7 (clone 30-F11, BioLegend), and for T cells, anti-mouse CD4-APC (clone GK1.5, BioLegend), and anti-mouse CD8-APC-Cy7 (clone 53-6.7, BioLegend). DAPI was added immediately before analysis to exclude dead cells. Flow cytometric analysis and cell sorting were performed on a BD LSR or BD FACSAria. 100,000 cells were analyzed per sample per mouse using BD FACSDiva software Ver. 8.0 (BD Bioscience).

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

(result)
The CD45 ⁺ cell ratios (n=12-13) in lung cancer LLC tumors are shown in Figure 4(i), and the CD11b ⁺ F4/80 ⁺ cell ratios (n=8) are shown in Figure 4(ii). The CD4 ⁺ and CD8 ⁺ cell ratios (n=5) in LLC tumors are shown in Figure 5. 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 no significant change, i.e., the tumor-infiltrating T cell population was very low. The quantification results of the number of F4/80 ⁺ cells per ^mm2 of tumor (n=5) are shown in Figure 6. As is clear from this figure, an increase in the number of macrophages was observed in the tumor tissues by immunofluorescence staining as well. The above data indicate that LLC tumors are rich in tumor-associated macrophages (TAM) and contain almost no T cells. The CD4 ⁺ and CD8 ⁺ cell ratios (n=5) in melanoma B16F10 tumors are shown in Figure 7. As is clear from this figure, the T cell population did not show any significant change in melanoma B16F10 tumors, and the population of tumor-infiltrating T cells was very low, showing the same tendency as in lung cancer LLC tumors (Figure 5).

Test Example 2-3
We focused on the M1 marker (CD80) and M2 marker (CD206) that have been reported to date as cell surface markers reflecting the differentiation state of tumor-associated macrophages (TAMs), as well as other markers, and investigated their functions when TAMs were subdivided by Ly6C.

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

(Flow cytometry and cell sorting)
Further, 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) were used as fluorescent dye-conjugated antibodies, and flow cytometry and cell selection were performed in the same manner as in Test Example 2-2 above. The gating strategy in flow cytometry of macrophages is shown in Figure 8.

(result)
As is clear from Figure 8, analysis of tumor-infiltrating macrophages using CD80 as an M1 marker and CD206 as an M2 marker showed that these markers were expressed in macrophages from both vehicle-treated and FG-treated tumors, and no differences in macrophages based on these markers were observed in either treatment group.

On the other hand, the quantification results (n=6) of the ratios of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in lung cancer LLC tumors by flow cytometry analysis are shown in Figure 9. As is clear from this figure, the ratio of Ly6C ^lo macrophages in total macrophages was increased by FG treatment. Furthermore, although not shown, when the selected macrophages were stained with a Diffquick staining kit and then observed under a microscope, it was confirmed that the morphology of the Ly6C ^lo macrophages selected from the FG-treated tumor was such that they had formed phagosomes, which was different from the Ly6C ^lo macrophages selected from the Veh-treated tumor.

Furthermore, the quantification results (n=4) of the ratios of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in melanoma B16F10 tumors by flow cytometry analysis are shown in Figure 10. As is clear from this figure, the ratio of Ly6C ^lo macrophages among total macrophages was increased by FG treatment, similar to the lung cancer LLC tumor (Figure 9).

As is clear from Figures 9 and 10, the ratios of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in mice were common between lung cancer LLC tumors and melanoma B16F10 tumors. For example, the average ratio of Ly6C ^lo to all macrophages in the tumor was 35% or more, specifically 40% or more, on average, for lung cancer LLC tumors (n=6) and melanoma B16F10 tumors (n=4), and the average ratio of Ly6C ^{hi to all macrophages in the tumor was 6% or more, specifically 40% or more, for lung cancer LLC tumors (n=6) and melanoma B16F10 tumors (n=4), and the average ratio of Ly6C hi} to all macrophages in the tumor was 6% or more, on average, for lung cancer LLC tumors (n=6) and melanoma B16F10 tumors (n=4).

Test Example 2-4
Tumor-infiltrating macrophages were depleted with clodronate liposomal Clo prior to administration of the PHD inhibitor FG4592 (FG) to examine whether FG affects the ability of tumor-infiltrating macrophages to suppress tumor growth.

In vivo macrophage depletion
Mice were intraperitoneally administered 100 μL of clodronate-loaded liposomes Clo (F70101C-A, FormuMax Scientific Inc.) or 100 μL of plain control liposomes Lip (F70101C-A, FormuMax Scientific Inc.) 24 h prior to injection of the PHD inhibitor FG4592 (FG) or vehicle (Veh). In addition, 72 h after injection of FG or Veh, mice were intraperitoneally administered 70 μL of the same liposomes as a second dose.

(Quantification of Immunostaining and Immunofluorescence Images)
The macrophage depletion efficiency was evaluated by immunostaining. Immunostaining was performed in the same manner as in Test Example 2-1, and the immunofluorescence images were quantified.

(result)
Figure 11 shows tumor growth curves (n=4) of LLC tumor mouse models administered with clodronate liposomes (Clo) or plain control liposomes (Lip) and treated with vehicle (Veh) or 100 mg/kg PHD inhibitor (FG). As is clear from this figure, in the control liposome (Lip) group, tumors treated with FG showed suppression of tumor growth compared to tumors treated with vehicle (Veh). However, in the clodronate liposome (Clo) treatment group, there was no significant difference in tumor growth between tumors treated with FG and tumors treated with vehicle (Veh).

The quantification results (n=3) of the number of F4/80 ⁺ cells per mm ² of tumor are shown in Figure 12. As is clear from this figure, the efficiency of macrophage depletion by clodronate liposomes (Clo) was 80% of that observed when the plain control liposomes (Lip) were administered in both tumors treated with the vehicle (Veh) and FG.

These 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 directly inhibiting tumor growth through phagocytosis. We therefore investigated the activation of macrophage phagocytosis by the PHD inhibitor FG4592 (FG) using a GFP-labeled LLC tumor model. We also investigated the effect of the PHD inhibitor FG4592 (FG) on tumor vascular normalization. Furthermore, we investigated the activation of macrophage phagocytosis by the PHD inhibitor FG4592 (FG) using macrophages isolated from bone marrow.

(Preparation of GFP-labeled LLC tumor model)
The cDNA encoding GFP1 was double-digested with SalI and NotI from pAcGFP1 (Clontech, No. 632468) and inserted into the SalI and NotI sites of pEBMulti-Hyg vector (WAKO, 050-08121). This expression vector was transfected into LLC using Lipofectamine LTX reagent (Invitrogen), and GFP-stably expressing 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%). Cells were cultured for 7 days and medium was changed every 2 days. Fractionated macrophages were identified by F4/80 antibody staining and flow cytometry.

Phagocytosis Assay
Figure 15(i) shows a schematic diagram of the in vitro bead phagocytosis assay (for BMDMs). Macrophages harvested and sorted 48 hours after GFP-labeled LLC tumor models administered vehicle (Veh) or PHD inhibitor (FG) before seeding in 96-well plates, or isolated BMDMs, were stained with CellVue Claret (Sigma-Aldrich). Sorted cells were incubated at 37°C for 2 hours to allow cells to become quiescent after sorting. Subsequently, 100 μM FG or an equal volume of DMSO was added to the medium after 24 hours. After 12 hours, latex bead-rabbit IgG-FITC conjugates (Cayman) were 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 the equipped software. More than 800 cells were analyzed for each group.

(Tumor vascular staining)
Tumor tissues harvested from GFP-labeled LLC tumor models administered vehicle (Veh) or PHD inhibitor (FG) 48 hours after administration were 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)
Quantification results of tumor vascular density and vascular lumen area (n=3) at 48 hours after administration of vehicle (Veh) or PHD inhibitor (FG) in GFP-labeled LLC tumor models are shown in FIG. 13, and quantification results of the number of phagocytes (GFP ⁺ F4/80 ⁺ ) in the tumor (n=5) are shown in FIG. 14. As can be seen from these figures, tumor tissues at 48 hours after FG administration did not show normalized tumor vasculature at that time point, but there was a significant increase in tumor phagocytic macrophages (GFP ⁺ macrophages). Quantification results of the proportion of BMDM (bone marrow-derived macrophages) isolated from bone marrow (n=3) are shown in FIG. 15(ii). As can be seen from this figure, in vitro BMDM phagocytosis assay showed that the level of phagocytic macrophages was increased in BMDM treated with FG. These results indicate that FG directly affected macrophages.

Test Example 2-6
We investigated which Ly6C macrophage populations were activated by the PHD inhibitor FG4592 (FG).

(Flow cytometry and cell sorting)
Flow cytometry and cell selection were performed on GFP ⁺ macrophages in the same manner as in Test Example 2-3. The gating strategy in flow cytometry of GFP ⁺ macrophages is shown in Figure 16.

Phagocytosis Assay
Schematic diagrams of the ex vivo bead phagocytosis assay are shown in Figure 18(i) and Figure 19(i). GFP ⁺ macrophages were collected from the GFP-labeled LLC tumor model administered with a vehicle (Veh) or a PHD inhibitor (FG) 6 days after administration (day 16), and each fraction selected by flow cytometry was subjected to a phagocytosis assay in the same manner as in Test Example 2-5.

(result)
Quantification results (n=5-6) of the ratio of Ly6C-fractionated GFP ⁺ macrophages (Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages) to total GFP ⁺ macrophages by flow cytometry analysis are shown in Figure 17. As is clear from this figure, FG treatment increased the ratio of the Ly6C-positive population (Ly6C ^hi and Ly6C ^lo ), but did not increase the ratio of the Ly6C-negative population.

In addition, the quantification results of the ratio of phagocytic Ly6C ^lo macrophages (n=5) by the ex vivo bead phagocytosis assay are shown in FIG. 18(ii), and the quantification results of the ratio of phagocytic Ly6C ^hi macrophages (n=4-5) are shown in FIG. 18(iii). As is clear from these figures, the ratio of phagocytic macrophages was significantly higher for Ly6C ^lo macrophages selected from tumors treated with FG than for Ly6C ^lo macrophages selected from tumors treated with vehicle, and the ratio of phagocytic macrophages was higher for Ly6C ^hi macrophages selected from tumors treated with FG than for Ly6C ^hi macrophages selected from tumors treated with vehicle. Meanwhile, the quantification results of the ratio of phagocytic Ly6C negative macrophages (n=3-4) are shown in FIG. 19(ii). As is clear from this figure, the Ly6C-negative macrophages selected from tumors treated with FG were almost the same as those selected from tumors treated with vehicle, and even showed a slight tendency to decrease. The above data, that is, these results show that FG treatment specifically enhances the phagocytosis of Ly6C-positive macrophages among macrophages subdivided by Ly6C, and in particular, the phagocytosis of Ly6C ^lo macrophages is further enhanced.

Test Example 3: Inhibitory effect of PHD inhibitors on macrophage-mediated tumor growth via HIF PHD inhibitors inhibit prolyl hydroxylation of HIF and proteasomal degradation of HIF, thereby upregulating the HIF signaling pathway. Therefore, we investigated whether the macrophage-mediated tumor growth inhibitory effect of PHD inhibitors is due to HIF inhibition.

(Tumor growth inhibition in macrophage-specific VHL knockout mice)
To genetically mimic FG drug response, macrophage-specific von Hippel-Lindau (VHL) knockout mice (VHLfl/flLys-MCre ^+/- ; VHL KO mice) were used. VHL KO mice show HIF upregulation. Tumor volume transition was examined for VHL KO mice in the same manner as in Test Example 1.

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

(Quantitative analysis of HIF downstream genes in bone marrow-derived macrophages)
Mice from which bone marrow-derived macrophages (BMDM) were harvested were administered 100 μM FG-4592 or vehicle (Veh) 12 hours prior to harvest. BMDM were isolated 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 Prime Script RT Reagent Kit (Takara). Transcripts were analyzed with the 7500 Fast Real-Time PCR System using Thunderbird SYBR qPCR Mix. qRT-PCR was performed for each sample pair. Measured transcript data were normalized to 18S ribosomal RNA content and analyzed using 7500 Software v2.3 (Applied Biosystems).

(result)
Tumor growth curves of LLC tumor models in Vhl ^fl/fl LysM-Cre ^-/- (control (Ctrl); n=9) and Vhl ^fl/fl LysM-Cre ^+/- (VHL KO; n=7) mice are shown in Figure 20(i), and tumor weights on day 16 (n=7-9) are shown in Figure 20(ii). As is clear from these figures, the LLC tumor xenograft model in VHL KO mice, which genetically mimics the FG drug response, showed similar tumor growth inhibition as the LLC tumor xenograft mouse model treated with FG (Figure 1). In addition, flow cytometry analysis results (n=6) of the proportions of Ly6C ^hi , Ly6C ^lo , and Ly6C ^neg macrophages in the VHL KO LLC tumor model are shown in Figure 21. As is clear from this figure, the ratio of Ly6C ^lo macrophages to total tumor-infiltrating macrophages was also increased in VHL KO mice, similar to the LLC tumor-implanted mouse model treated with FG (FIG. 9). These results indicate that FG exerted a macrophage-mediated tumor growth inhibitory effect via the PHD-HIF axis.

Quantitative analysis of HIF downstream genes from bone marrow-derived macrophages (BMDMs) isolated from mice administered vehicle (Veh) or PHD inhibitor (FG) is shown in Figure 22. As can be seen from this figure, HIF downstream gene expression was upregulated in BMDMs from mice treated with FG. These results indicate that PHD inhibitors affect the HIF signaling pathway in 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) was examined in a mouse model.

(Transplantation of macrophages collected and selected from LLC tumor model mice into tumor tissue)
A schematic diagram of the procedure for transplanting Ly6C ^lo macrophages selected from an LLC tumor mouse model into the tumor site of another LLC tumor mouse model is shown in Figure 23(i). 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, and Ly6C ^lo macrophages were selected in the same manner as in Test Example 2-3. Using a 26-gauge needle, 1 x ¹⁰⁵ Ly6C ^lo macrophages 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 the mice were sacrificed on the 16th day, after which the tumor tissue was excised and weighed.

A similar transplantation experiment was also carried out using Ly6C ^neg macrophages, and the weight of the tumor excised from the transplanted mice was measured.

Furthermore, in the same manner as in Test Example 2-5, the vascular density and vascular lumen area in the tumor tissues excised from the transplanted mice were quantified.

(result)
The tumor volume transition and tumor weight for mice transplanted with Ly6C ^lo macrophages or injected with PBS are shown in Figure 23 (ii) and Figure 23 (iii), respectively. As is clear from these figures, tumor growth suppression effect was observed in mice transplanted with Ly6C ^lo macrophages selected from tumors of mice treated with FG. In addition, the comparison of tumor weight for mice transplanted with Ly6C ^lo macrophages or Ly6C ^neg macrophages is shown in Figure 24 (i); the comparison of the quantification results of tumor vascular density is shown in Figure 24 (ii); and the comparison of the quantification results of tumor vascular lumen area is shown in Figure 24 (iii). As is clear from these figures, in mice transplanted with Ly6C ^neg macrophages selected from tumors of mice treated with FG, the tumor weight was almost the same as that of the vehicle (Veh), while a significant decrease in tumor vascular density and a significant increase in vascular lumen area were observed. These data indicate that Ly6C ^lo macrophages suppress tumor growth by enhancing phagocytosis, whereas Ly6C ^neg macrophages normalize tumor vasculature.

Test Example 5: Production of a drug for cancer treatment by in vitro treatment of PHD inhibitors (1)
The tumor growth-suppressing effect of macrophages prepared by exposing Ly6C ^lo macrophages isolated from tumors in vehicle (Veh)-administered LLC tumor model mice to the PHD inhibitor FG4592 (FG) in vitro was examined in a tumor mouse model.

(Transplantation of macrophages collected and selected from LLC tumor model mice into tumor tissue)
A schematic diagram of the procedure for transplanting Ly6C ^lo macrophages selected from an LLC tumor mouse model into the tumor site of another LLC tumor mouse model is shown in FIG. 25(i). As shown in this figure, macrophages were collected from LLC tumor model mice administered with a 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×g for 3 minutes, and resuspended in 20 μL of PBS. The selected macrophages were seeded on a 10 cm dish and incubated for 12 hours using a medium containing 100 μM FG or Veh. The macrophages were then collected and resuspended in 20 μL of PBS. Using a 26-gauge needle, 1 x ¹⁰⁵ prepared macrophages (medicinal drug for cancer treatment) or PBS was injected into the tumor of LLC tumor-bearing mice on day 10 after tumor implantation. After intratumoral injection, the tumors were measured twice every two days in the same manner as in Test Example 1, and the mice were sacrificed on day 16, after which the tumor tissues were excised and weighed.

(result)
The changes in tumor volume and tumor weight for mice implanted with the prepared macrophages (a drug for cancer treatment) or injected with PBS are shown in Figure 25(ii) and Figure 25(iii), respectively. As is clear from these figures, tumor growth was significantly suppressed in tumor tissues implanted with Ly6C ^lo macrophages that had been treated with FG in vitro.

The above results demonstrate that FG directly affects Ly6C-positive macrophages (especially Ly6C ^lo macrophages) and induces their activation, thereby inhibiting tumor growth, and therefore Ly6C-positive macrophages (especially Ly6C ^lo macrophages) treated in vitro with FG are useful as a pharmaceutical for cancer treatment.

Test Example 6: Production of a drug for cancer treatment by in vitro treatment of PHD inhibitors (2)
Macrophages obtained from the bone marrow of LLC tumor model mice were exposed to the PHD inhibitor FG4592 (FG) in vitro to prepare macrophages, and the tumor growth suppression effect in tumor mouse models was examined. A schematic diagram of the procedure in this test example is shown in Figure 26(i).

Isolation of bone marrow derived macrophages (BMDM)
Bone marrow was harvested 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 replaced every 2 days. The resulting macrophages were washed in PBS, centrifuged at 200×g for 3 minutes, and resuspended in 20 μL of PBS. This resulted in the bone marrow-derived macrophage fraction.

(Transplantation of BMDM into tumor tissue)
The obtained bone marrow-derived macrophage fraction was seeded on a 10 cm dish and incubated with a medium containing 100 μM FG or Veh for 12 hours. The macrophages were then harvested and resuspended in 20 μL of PBS. Using a 26-gauge needle, 1× ¹⁰⁵ prepared macrophages (cancer treatment drug) or PBS was injected into the tumors of LLC tumor-bearing mice 10 days after tumor implantation. After intratumoral injection, the tumors were measured twice every 2 days in the same manner as in Test Example 1.

(result)
The tumor volume transition for mice transplanted with the prepared macrophages (cancer therapeutic drug) or injected with PBS is shown in Fig. 26 (ii). As is clear from this figure, tumor growth was suppressed in tumor tissue transplanted with bone marrow-derived macrophages treated in vitro with FG (FG+BMDM) compared with tumor tissue injected with PBS (PBS) and tumor tissue transplanted with bone marrow-derived macrophages treated with Veh (Ctrl+BMDM).

These results demonstrate that FG directly affects tumor-associated macrophages in bone marrow-derived macrophages from cancer-bearing mice, inducing their activation and thereby inhibiting tumor growth, demonstrating that bone marrow-derived macrophages from cancer patients treated with FG in vitro are useful as a pharmaceutical for cancer treatment.

Claims (3)

A method for producing a pharmaceutical for treating cancer, comprising in vitro administering to a macrophage fraction containing tumor-associated macrophages at least one prolyl hydroxylase inhibitor selected from the group consisting of FG-4592 (Roxadustat), FG-2216, FG-4497, BAY-85-3934 (Molidustat), AKB-6548 (Vadadustat), GSK1278863 (Daprodustat), JTZ-951, TM-6089, and DS-1093. The method according to claim 1 , wherein the macrophage fraction is obtained from the bone marrow of a cancer patient. The method of claim 1 or 2 , wherein the tumor-associated macrophages comprise Ly6C-positive macrophages or their human counterparts.