Cancer microenvironment-improving agent, prophylactic or therapeutic agent for Anti-tumor immune response, and method for improving cancer microenvironment

Abstract

The present invention relates to a novel cancer microenvironment-improving agent which can improve a cancer microenvironment by the action of microparticles derived from dental pulp-derived stem cells. More specifically, it is preferable that there are provided a cancer microenvironment-improving agent and an anti-tumor immune response-ameliorating agent, each containing microparticles derived from dental pulp-derived stem cells; it is more preferable that the cancer microenvironment can be improved by suppressing the activity of CAFs so that the proliferation of cancer cells can be suppressed; and it is especially preferable that the cancer microenvironment can be improved by inducing M1-type macrophages from M2-type macrophages so that the proliferation of cancer cells can be suppressed. The present invention also relates to: a prophylactic or therapeutic agent for an anti-tumor immune response; and a method for improving a cancer microenvironment.

Description

Agent for improving cancer microenvironment, preventive or therapeutic agent for anti-tumor immune response, and method for improving cancer microenvironment

[0001] The present invention relates to an agent for improving a cancer microenvironment, a preventive or therapeutic agent for an anti-tumor immune response, and a method for improving a cancer microenvironment. The present invention also relates to an agent for suppressing the activation of cancer-associated fibroblasts and an agent for inducing immunostimulatory macrophages.

[0002] By improving the tumor microenvironment in a way that suppresses cancer cell proliferation, it is possible to improve the anti-tumor immune response, which in turn improves cancer resistance to treatment. As a result, it is expected that this will lead to the treatment of cancers that cannot be treated with conventional anti-cancer drugs.

[0003] On the other hand, exosomes derived from mesenchymal stem cells are not known as agents for improving the cancer microenvironment, and are only known to be able to suppress cancer growth and metastasis.

[0004] For example, Patent Document 1 describes a pharmaceutical composition for suppressing, preventing, or treating cancer metastasis, comprising extracellular vesicles containing (i) lactate dehydrogenase B (LDHB), (ii) peroxisome proliferator-activated receptor gamma activator 1-α (PGC-1α), and (iii) calcium calmodulin-activated kinase 1B (CaMK1B), calcium calmodulin-activated kinase 2B (CaMK2B), calcium calmodulin-activated kinase 5 (CaMK5), myocyte-specific enhancer factor 2B (MEF2B), myocyte-specific enhancer factor 2C (MEF2C), and cyclic adenosine monophosphate (cAMP). It also describes that the extracellular vesicles further contain (iv) HLA-G1 and HLA-G5 proteins, and (v) human chorionic gonadotropin (hCG) and placenta growth factor (PlGF). Example 3 of Patent Document 1 describes that the anticancer extracellular vesicles obtained by culturing, passage, and centrifuging the following: (1) a matrix gel for in vitro culture (polylactic acid was added instead of hyaluronic acid in Example 1) in which polylactic acid was added to extracellular vesicles containing hcG and PIGF obtained by co-culturing human amniotic membrane-derived stem cells, human amniotic fluid-derived stem cells, and human mesenchymal stem cells of unknown origin that are an alternative to FBS; (2) extracellular vesicles derived from trophoblast (human placenta) cells that secrete and express HLA-G1 and HLA-G5 (Example 2); and (3) extracellular vesicles secreted during hepatocyte differentiation from one type of mesenchymal stem cell selected from mesenchymal stem cells derived from human bone marrow, adipose tissue, umbilical cord blood, placenta, amniotic epithelium, chorion, umbilical cord, and amniotic fluid (Example 3), contain the proteins (i) to (v) listed in Table 3. Example 4 describes the effects of absorption, lactic acid, and pH change of the anticancer extracellular vesicles prepared in Example 3 on breast cancer cells in vitro. Example 5,

[0018] describes data on the anticancer efficacy in an in vivo mouse cancer model.

[0005] Patent Document 2 describes extracellular vesicles (EVs) loaded with a panel of specific miRNAs. Patent Document 2 describes a mechanism by which reducing the expression and / or phosphorylation of LCP-1 in tumor cells may interfere with the aggressiveness, migration, and invasion of tumor cells, thereby potentially reducing metastasis. That is, Patent Document 2 describes a method for treating LCP-1-positive cancer patients. In particular, Example

[0053] of Patent Document 2 describes EVs containing the endogenous miRNAs listed in Table 2, further loaded with miR-885-5p as an additional miRNA. Furthermore, it describes that administration of these EVs to breast cancer cells or the like induced apoptosis, inhibited cell proliferation, and attenuated tumor growth and metastasis in a dose- and time-dependent manner (FIGS. 5 and 6). In Example

[0055] of Patent Document 2, it is described that such EVs were administered to cancer-bearing model mice, and using a mouse model of prostate cancer, it was further demonstrated that a decrease in LCP-1 expression inhibits metastasis, while an increase in LCP-1 expression and phosphorylation stimulates metastasis of the primary tumor.

[0006] Patent Document 3 describes a method for treating a disease or condition, comprising: (a) providing an enriched population of placental chorion-derived mesenchymal stem cells (CH-MSCs); and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one of: i. the CH-MSC population; ii. extracellular vesicles derived from the CH-MSC population; iii. conditioned medium from the CH-MSC population; and iv. extracellular matrix secreted by the CH-MSC population. Furthermore, Example 6 of Patent Document 3 describes that CH (placental chorion)-MSCs have excellent effects on colon cancer, prostate cancer, lung cancer metastasis, breast cancer metastasis, glioma, meningioma, neuroblastoma, medulloblastoma, and head and neck cancer cells (Table 12). Furthermore, Example 7 of Patent Document 3 also describes their effects on brain metastasis.

[0007] Patent Document 4 describes a pharmaceutical composition for cancer treatment, which is prepared by a method comprising: a stem cell preparation step of introducing four types of genes into deciduous dental pulp stem cells obtained from mammalian dental pulp to produce immortalized stem cells; and a conditioned medium preparation step of culturing the immortalized stem cells in a serum-free medium for a predetermined period of time at 23-27°C under a low oxygen concentration of 0.5% or more but less than 20% to prepare a conditioned medium, the conditioned medium containing 1.5 times or more insulin-like growth factor (IGF-1) and 1.5 times or more vascular endothelial growth factor (VEGF) compared to a conditioned medium prepared by culturing under the same conditions except for an oxygen concentration of 20%. Furthermore, Example 5 of Patent Document 4 describes that when breast cancer-prone mice (C3H / He) were injected with the mouse squamous cell carcinoma line SCCVII and further administered the culture supernatant of deciduous dental pulp stem cells as a pharmaceutical composition for cancer treatment, the increase in tumor diameter was significantly slowed or the tumor was treated, and survival time was extended. Example 6 and Figure 9 of Patent Document 4 describe that in the treatment group GIV, macrophages began to migrate within 1 hour of administration of the culture supernatant of deciduous dental pulp stem cells, and after 24 hours, they had accumulated to surround the entire tumor, and that in the early stages of tumor development, there was a high proportion of M2-type macrophages, which express a large amount of the TGF-β superfamily. Example 7 of Patent Document 4 describes that the culture supernatant of deciduous dental pulp stem cells administered as a therapeutic pharmaceutical composition increased the migration ability of macrophages, leading to their high accumulation in tumor tissue, and that the macrophages that accumulated in the tumor destroyed tumor tissue using their inherent phagocytic ability while simultaneously controlling tumor growth via TGF-β.

[0008] Patent Publication No. 2022-532396 Patent Publication No. 2021-533822 Patent Publication No. 2019-535691 International Publication No. 2015 / 111712

[0009] Patent Documents 1 to 3 do not disclose or suggest microparticles derived from dental pulp-derived stem cells. In particular, the mechanism of cancer metastasis inhibition, prevention, or treatment in paragraphs

[0024] and

[0040] of Patent Document 1 requires novel anti-cancer extracellular vesicles obtained by culturing extracellular vesicles from human amniotic membrane-derived stem cells, extracellular vesicles from human amniotic fluid-derived stem cells, extracellular vesicles derived from trophoblasts (human placenta), and extracellular vesicles secreted by stem cells during hepatocyte differentiation, and transferring their protein secretion function. Furthermore, Patent Document 1 only discloses data on the rate of tumor volume increase of cancer cells.

[0010] Patent Document 2 merely exemplifies that EVs are derived from stem cells derived from umbilical cord, Wharton's gel, blood, umbilical cord blood, or bone marrow. However, Figure 6 of Patent Document 2 does not suggest that cancer metastasis in cancer model mice is suppressed.

[0011] Although the examples in Patent Document 3 also examine the characteristics of MSCs derived from bone marrow (BM), placental amniotic membrane (AM), adipose tissue (AD), umbilical cord (UC), dental pulp (DP), exfoliated teeth (DD), and dental follicles (DF), the effect of suppressing cancer metastasis other than that of CH (placental chorion)-MSCs was not clearly stated.

[0012] On the other hand, the examples in Patent Document 4 state that when culture supernatant of dental pulp-derived stem cells is administered to cancer-implanted mice, the cancer is treated and survival time is extended, but when the inventor conducted further tests, no such effect was observed.

[0013] The problem to be solved by the present invention is to provide a novel agent for improving the cancer microenvironment, which can improve the cancer microenvironment using microparticles derived from dental pulp-derived stem cells.

[0014] The present inventors have found that microparticles derived from dental pulp-derived stem cells can improve the cancer microenvironment.

[0015] Specifically, the present invention and preferred configurations thereof are as follows: [1] A cancer microenvironment improver comprising microparticles derived from dental pulp-derived stem cells. [2] The cancer microenvironment improver according to [1], wherein the microparticles are exosomes. [3] The cancer microenvironment improver according to [1], which is an agent for improving anti-tumor immune responses. [4] The cancer microenvironment improver according to [1], which improves the cancer microenvironment in a direction that allows cancer cell proliferation to be suppressed by suppressing the activity of cancer-associated fibroblasts (CAFs). [5] The cancer microenvironment improver according to [1], which improves the cancer microenvironment in a direction that allows cancer cell proliferation to be suppressed by inducing immunosuppressive macrophages (M2-type macrophages) to immunostimulatory macrophages (M1-type macrophages). [6] The cancer microenvironment improver according to [1], wherein the microparticles are purified and isolated from the culture supernatant of dental pulp-derived stem cells, and does not contain components obtained by excluding exosomes from the culture supernatant of dental pulp-derived stem cells. [7] The agent for improving a cancer microenvironment according to [1], which is used to administer the agent for improving a cancer microenvironment in an amount of 10 or more exosomes per cell. [8] The agent for improving a cancer microenvironment according to [1], which is used to administer the agent for improving a cancer microenvironment to a subject having a cancer tumor at least twice during the effective treatment period. [9] The agent for improving a cancer microenvironment according to [1], which does not have the ability to inhibit cancer cell proliferation and is an agent for improving an anti-tumor immune response.

[10] The agent for improving a cancer microenvironment according to [1], which is used to administer the agent for improving a cancer microenvironment to a subject having an immune system having all of T cells, B cells, and NK cells.

[11] A preventive or therapeutic agent for anti-tumor immune response, comprising the agent for improving a cancer microenvironment according to any of [1] to

[10] .

[12] An agent for suppressing the activation of cancer-associated fibroblasts (CAFs), comprising the agent for improving a cancer microenvironment according to any of [1] to

[10] .

[13] An immunostimulatory macrophage inducer, which comprises the agent for improving a cancer microenvironment according to any of [1] to

[10] .

[14] A method for improving a cancer microenvironment, comprising administering an effective amount of the agent for improving a cancer microenvironment according to any one of [1] to

[10] to a subject having a cancer tumor.

[15] The method for improving a cancer microenvironment according to

[14] , wherein the agent for improving a cancer microenvironment is administered to a subject in combination with an anticancer drug or an immune checkpoint inhibitor.

[16] A cancer microenvironment improver comprising extracellular vesicles derived from dental pulp-derived stem cells.

[17] The cancer microenvironment improver according to

[16] , wherein the extracellular vesicles are exosomes.

[18] The cancer microenvironment improver according to

[16] , which is an agent for improving anti-tumor immune responses.

[19] The cancer microenvironment improver according to

[16] , which improves the cancer microenvironment in a direction that allows cancer cell proliferation to be suppressed by suppressing the activity of cancer-associated fibroblasts (CAFs).

[20] The cancer microenvironment improver according to

[16] , which improves the cancer microenvironment in a direction that allows cancer cell proliferation to be suppressed by inducing immunosuppressive macrophages (M2 macrophages) to immunostimulatory macrophages (M1 macrophages).

[21] The cancer microenvironment improver according to

[16] , wherein the extracellular vesicles are exosomes purified and isolated from the culture supernatant of dental pulp-derived stem cells, and which does not contain components remaining from the culture supernatant of the dental pulp-derived stem cells excluding the exosomes.

[22] The agent for improving a cancer microenvironment according to

[16] , wherein the agent for improving a cancer microenvironment is administered in an amount of 10 or more exosomes per cell.

[23] The agent for improving a cancer microenvironment according to

[16] , wherein the agent for improving a cancer microenvironment is administered to a subject having a cancer tumor at least twice during the effective treatment period.

[24] The agent for improving a cancer microenvironment according to

[16] , wherein the agent for improving a cancer microenvironment does not have the ability to suppress the proliferation of cancer cells in a subject without an immune system and is an agent for improving an anti-tumor immune response.

[25] The agent for improving a cancer microenvironment according to

[16] , wherein the agent for improving a cancer microenvironment is administered to a subject having an immune system having all of T cells, B cells, and NK cells.

[26] A preventive or therapeutic agent for an anti-tumor immune response, comprising the agent for improving a cancer microenvironment according to any of

[16] to

[25] .

[27] An inhibitor of cancer-associated fibroblast (CAF) activation, comprising the agent for improving a cancer microenvironment according to any of

[16] to

[25] .

[28] An immunostimulatory macrophage inducer comprising the agent for improving a cancer microenvironment according to any one of

[16] to

[25] .

[29] A method for improving a cancer microenvironment, comprising administering an effective amount of the agent for improving a cancer microenvironment according to any one of

[16] to

[25] to a subject having a cancer tumor.

[30] The method for improving a cancer microenvironment according to

[29] , wherein the agent for improving a cancer microenvironment is administered to the subject in combination with an anticancer drug or an immune checkpoint inhibitor.

[31] The agent for improving a cancer microenvironment according to [1] or

[16] , wherein the dental pulp-derived stem cells are deciduous dental pulp stem cells.

[0016] According to the present invention, a novel agent for improving a cancer microenvironment can be provided, which can improve the cancer microenvironment using microparticles derived from dental pulp-derived stem cells.

[0017] FIG. 1 is a schematic diagram of the administration of exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells to SCID-beige mice. FIG. 2 is a graph showing the number of days since transplantation and the luminescence intensity of mouse breast cancer-derived cells when samples from Reference Example 1 (SGF EVs) and Reference Example 2 (PBS(-)) were added. FIG. 3 is a graph showing the luminescence intensity of the whole body of a mouse after removal of the primary lesion 36 days after transplantation when samples from Reference Example 1 (SGF EVs) and Reference Example 2 (PBS(-)) were added. FIG. 4 is a graph showing the number of metastatic lesions 36 days after transplantation when samples from Reference Example 1 (SGF EVs) and Reference Example 2 (PBS(-)) were added. FIG. 5 is a schematic diagram of the administration of exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells to melanoma model mice. Figure 6 is a graph showing the number of days since transplantation and the luminescence intensity on the back of mice when samples from Example 2 (SGF EVs) and Comparative Example 1 (saline) were added. Figure 7 is an IVIS Imaging System image of the luminescence on the back of mice 21 days after transplantation when samples from Example 2 (SGF EVs) and Comparative Example 1 (saline) were added. Figure 8 is a schematic diagram showing an experimental system for the inhibition of cancer-associated fibroblast (CAF) activation by exosomes (microparticles from Example 1) purified from dental pulp-derived stem cells. Figure 9 is a graph showing the relative expression levels of αSMA / β-ACTIN mRNA by qRT-PCR when samples from Comparative Example 12 (untreated control), Example 11 (TGFβ + EVs10), Example 12 (TGFβ + EVs100), and Comparative Example 11 (TGFβ + PBS(-)) were added. Fig. 10 is a schematic diagram showing an experimental system for the suppression of M2 macrophage activation by exosomes purified from dental pulp-derived stem cells (microparticles of Example 1). Fig. 11 is a graph showing the expression levels of TNF-α and IL-10 when the samples of Comparative Example 22 (untreated), Example 21 (SGF EVs10), Example 22 (SGF EVs100), and Comparative Example 21 (PBS(-)) were added.

[0018] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0019] [Cancer microenvironment improving agent] The cancer microenvironment improving agent of the present invention comprises microparticles derived from dental pulp-derived stem cells. More preferably, the cancer microenvironment improving agent of the present invention can prevent, treat, or improve anti-tumor immune responses by improving the cancer microenvironment. Preferred embodiments of the present invention are described below.

[0020] <Cancer Microenvironment> The cancer microenvironment is the environment surrounding cancer cells. The cancer microenvironment plays an important role in the malignant progression of cancer and resistance to cancer treatment. Cancers are composed of cancer cells, stromal cells (mesenchymal cells, vascular endothelial cells, immunocompetent cells, etc.), and the extracellular matrix produced by these cells, forming a structure characteristic of each cancer. Various levels of intercellular interactions occur between the cells that make up cancer, forming a microenvironment unique to each cancer. The cancer microenvironment improver of the present invention preferably improves the cancer microenvironment, the environment surrounding cancer cells, in a manner that suppresses cancer cell proliferation. Furthermore, the cancer microenvironment improver of the present invention is preferably an agent for improving anti-tumor immune responses. More preferably, the cancer microenvironment improver of the present invention can reduce cancer tumor volume through improved anti-tumor immune responses. On the other hand, the cancer microenvironment improver of the present invention preferably does not have the ability to suppress cancer cell proliferation in subjects without an immune system. More preferably, the cancer microenvironment improver of the present invention does not have the ability to suppress cancer cell proliferation in subjects without an immune system and is an agent for improving anti-tumor immune responses. That is, the agent for improving a cancer microenvironment of the present invention is preferably used for administering the agent for improving a cancer microenvironment to a subject having an immune system that has all of T cells, B cells, and NK cells. The agent for improving a cancer microenvironment of the present invention may also be capable of suppressing cancer metastasis.

[0021] The cancer microenvironment is described in detail in "Challenging the Cancer Microenvironment at the Single-Cell Level," edited by Naoya Fujita, Experimental Medicine, Vol. 39, No. 12 (Special Edition), 2021. For example, immune checkpoint inhibitors are effective against many cancer types, including malignant melanoma, lung cancer, and gastric cancer. However, while they provide long-term therapeutic benefit in 20-30% of cases, they are ineffective in more than half of patients. Immune checkpoint inhibitors are also known to be ineffective against pancreatic cancer, a difficult-to-treat cancer. These non-response and acquired resistance cases are important issues in clinical oncology. In recent years, it has become clear that anticancer drugs not only directly damage cancer cells but also induce tumor immunity, leading to the emergence of the therapeutic concept of chemoimmunotherapy. Their mechanism of action is to suppress the tumor immune escape mechanisms of cancer cells and to suppress the number and function of immunosuppressive cells (such as regulatory T cells). For pancreatic cancer, where immune checkpoint inhibitors are ineffective, chemotherapy using anticancer drugs is the standard treatment. Among these, gemcitabine, oxaliplatin, and fluorouracil are considered preferable due to their ability to induce tumor immunity. However, tumor-bearing hosts contain molecules and cellular factors that suppress immune induction, making them resistant to treatment. Cancer cells evade the host's immune system by creating a diverse immunosuppressive environment, a hallmark of cancer. Among immune cells infiltrating tumors, myeloid-derived suppressor cells (MDSCs), regulatory T cells, macrophages, and neutrophils possess immunosuppressive properties, and these cells are important resistance factors in immunotherapy and chemotherapy. MDSCs inhibit both innate and adaptive immunity through their immunosuppressive functions, such as producing immunosuppressive cytokines and suppressing T cell infiltration into tumors. MDSCs are involved in the process of tumor progression and metastasis through mechanisms such as the construction of an immunosuppressive microenvironment, the formation of a pre-metastatic niche, the induction of stem cell-like phenotypes through the production of TGF-β, EGF, and HGF, and the promotion of epithelial-mesenchymal transition. Furthermore, the number of MDSCs in the blood shows a significant correlation with the stage and tumor volume, and the frequency of MDSCs in cancer tissue correlates significantly with poor prognosis.

[0022] In the present invention, examples of improving the cancer microenvironment include a method of suppressing the activity of cancer-associated fibroblasts (CAFs) and a method of inducing immunostimulatory macrophages.

[0023] (CAF Activation Inhibitor) The cancer microenvironment improving agent of the present invention is preferably an activation inhibitor of cancer-associated fibroblasts (CAFs). More preferably, the cancer microenvironment improving agent of the present invention improves the cancer microenvironment in a direction that inhibits cancer cell proliferation by inhibiting the activity of cancer-associated fibroblasts (CAFs). Here, it is known that fibroblasts, vascular endothelial cells, immune cells, etc. in tumor tissue exhibit normality that differs from cells found in normal tissue. Fibroblasts in tumor tissue are called cancer-associated fibroblasts (CAFs) and are involved in the malignant progression of cancer. Fibroblasts present in cancer tissue are a mixture of CAFs that maintain the characteristics of activated myofibroblasts and normal fibroblast-like CAFs that do not have these characteristics, surrounding the cancer. CAFs that maintain the characteristics of activated myofibroblasts produce extracellular matrix around the cancer, physically hardening the cancer tissue and preventing the penetration of anticancer drugs and immune cells. CAFs, activated fibroblasts present in the tumor tissues of many solid cancers, contribute to the progression and malignancy of cancer by producing various growth factors that promote cancer cell proliferation and angiogenesis. For example, CAFs are known to induce myeloid-derived suppressor cells (MDSCs), recruit MDSCs into tumors, and activate MDSC proliferation. One cytokine abundant in the tumor microenvironment is transforming growth factor-β (TGF-β), a cancer malignancy factor. TGF-β induces the loss of traits possessed by epithelial cancer cells, such as high intercellular junctions, and endows them with mesenchymal stem cell traits, such as high motility and invasiveness, thereby inducing a phenomenon known as epithelial-mesenchymal transition (EMT), which induces the progression and metastasis of epithelial cancers. Furthermore, TGF-β induces endothelial-mesenchymal transition in vascular endothelial cells, thereby reducing their barrier function and contributing to cancer malignancy through the formation of cancer-associated fibroblasts (CAFs).

[0024] To date, therapies targeting the interaction between CAFs and cancer cells have been investigated. These therapies can be divided into the following strategies. However, the mechanism of the present invention is not limited to any one of the following strategies: (1) a strategy targeting CAFs themselves (treatment that eliminates CAFs); (2) a strategy that reprograms CAFs into normal fibroblasts; and (3) a strategy that targets the interaction between CAFs and cancer cells (treatment targeting TGF-β or treatment targeting HGF, a product secreted by CAFs). The agent for improving a cancer microenvironment of the present invention preferably improves the cancer microenvironment by suppressing the activation of cancer-associated fibroblasts (CAFs) formed under the influence of TGF-β. Inhibiting CAF activation reduces the production of cancer growth factors, suppressing cancer cell proliferation and angiogenesis, and improving immunosuppression caused by the interaction between CAFs and MDSCs. The degree of inhibition of CAF activation by the cancer microenvironment improving agent of the present invention can be determined by the relative expression level of αSMA / β-ACTIN by qRT-PCR. αSMA (α-smooth muscle actin) is an indicator of CAF and a representative marker for identifying CAF. The expression level of αSMA can be corrected or normalized with β-ACTIN. The relative expression level of αSMA / β-ACTIN is preferably 6 or less, more preferably 5 or less, particularly preferably 4 or less, and even more particularly preferably 3 or less.

[0025] (Immunostimulatory Macrophage Inducer) The cancer microenvironment improving agent of the present invention is preferably an immunostimulatory macrophage inducer. More preferably, the cancer microenvironment improving agent of the present invention exerts a cancer microenvironment improving effect through the induction of immunostimulatory macrophages and the suppression of immunosuppressive macrophages. As used herein, the term "immunostimulatory macrophage inducer" refers to an agent that induces immunosuppressive macrophages (M2 macrophages; tumor-supporting) into immunostimulatory macrophages (M1 macrophages; tumor-suppressing). Macrophages are important innate immune cells found in almost all tissues. They originate from the bone marrow, circulate in the blood, and differentiate in tissues via extravasation. These macrophages are classified into three phenotypes: M0 macrophages, M1 macrophages, and M2 macrophages. M0 macrophages are inactivated macrophages differentiated from human peripheral blood monocytes. M1 macrophages have strong antigen-presenting capabilities and are generally activated by interferon-γ, lipopolysaccharide (LPS), and tumor necrosis factor (TNF)-α, exerting inflammatory and bactericidal functions. M2 macrophages are known to promote immunosuppression, tumor development, and angiogenesis by releasing various extracellular matrix components, angiogenic factors, and chemotactic factors. Generally, M2 macrophages are induced by IL-4 and IL-13, distinguishing them from M1 macrophages. M2 macrophages express unique M2 markers, such as arginase-1, mannose receptor (MMR, CD206), and scavenger receptor (SR-A, CD204). These macrophages are a type of leukocyte and play an important role in animal defense mechanisms against infection. Normally, macrophages function as immunostimulatory macrophages (M1 type) with antitumor effects, such as attacking cancer cells and inducing apoptosis. On the other hand, when cancer cells develop and increase in number, macrophages are attracted to the cancer site and differentiate into immunosuppressive macrophages (M2 type) due to immunosuppressive macrophage-inducing factors (IL-6, IL-10, etc.) secreted by the cancer cells.Immunosuppressive macrophages (M2 type) create an environment that further favors cancer cell proliferation by promoting angiogenesis through the expression of vascular endothelial growth factor (VEGF). Therefore, in order to inhibit cancer cell proliferation, it is important to induce immunostimulatory macrophages (M1 type) and suppress and reduce immunosuppressive macrophages (M2 type). The degree of induction of immunostimulatory macrophages (M1 type) and the degree of suppression and reduction of immunosuppressive macrophages (M2 type) by the cancer microenvironment improving agent of the present invention can be determined by the amount of TNF-α or IL-10. The amount of TNF-α is preferably 5 pg / ml or more, more preferably 10 pg / ml or more, and particularly preferably 12 pg / ml or more. The amount of IL-10 is preferably 20 pg / ml or less, more preferably 15 pg / ml or less, and particularly preferably 12 pg / ml or less.

[0026] (Cancer Tumor Treatment Methods) Known cancer tumor treatment methods include conventional anticancer drugs, immune checkpoint inhibitors, and agents that improve the tumor microenvironment. Conventional anticancer drugs target cancer cells, but the tumor microenvironment is involved in resistance to these drugs (therapeutic drug resistance). Although the therapeutic effect of immune checkpoint inhibitors is cross-cancer, it is ineffective in more than half of cases, making it necessary to improve the immune status within the tumor microenvironment. In the tumor microenvironment, exhaustion of tumor-infiltrating lymphocytes (TILs) and intratumoral infiltration of regulatory T cells (Tregs) are known to contribute to TIL exhaustion. It is known that some Tregs are activated by the administration of immune checkpoint inhibitors (PD-1 inhibitors), which strongly suppress anti-tumor immunity. Known agents that improve the tumor microenvironment include cancer therapeutic drugs that target non-cancerous cells within tumor tissue. For example, bevacizimab, a VEGF-neutralizing antibody, is known as a cancer therapeutic drug that targets non-cancerous cells in tumor tissue, and is believed to exhibit antitumor effects by suppressing the formation of new blood vessels in tumor tissue and normalizing the cancer microenvironment by inducing the normalization of tumor blood vessels. The agent for improving a cancer microenvironment of the present invention preferably contains microparticles derived from dental pulp-derived stem cells as an active ingredient, and is a novel agent different from bevacizimab and the like.

[0027] <Details of Microparticles> The microparticles used in the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion, and are exuded, released, or shed into the cell culture medium. The microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably, are microparticles derived from the culture supernatant of dental pulp-derived stem cells. However, microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily have to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if microparticles isolated from the interior of dental pulp-derived stem cells by any method are identical to microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, they can still be considered to be microparticles derived from the culture supernatant of dental pulp-derived stem cells. The microparticles derived from the culture supernatant of dental pulp-derived stem cells may be used in the state contained in the culture supernatant or purified from the culture supernatant. Preferably, the microparticles are microparticles purified from the culture supernatant. The origin of the microparticles can be determined by known methods. For example, the microparticles can be determined to be derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, and umbilical cord-derived stem cells, using the method described in J Stem Cell Res Ther (2018) 8:2. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticles.

[0028] (Types of Microparticles) The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles, and more preferably exosomes. The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. Furthermore, it is desirable that tetraspanin molecules such as CD9, CD63, and CD81 are present on the surface of the microparticles, and this may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three of these. Below, preferred embodiments using exosomes as microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

[0029] Exosomes are preferably extracellular vesicles (EVs) released from cells upon fusion of multivesicular bodies with the plasma membrane. The surface of the exosomes preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells. The interior of the exosomes preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins. Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.

[0030] (Content of Microparticles) There is no particular limitation on the content of microparticles in the microparticle composition. ８ It is preferable that the number of atoms contained is 1.0 × 10 or more. ８ It is more preferable that the number of atoms contained is 2.0 × 10 or more. ８ It is particularly preferable that the number of atoms contained is 2.5 × 10 or more. ８ It is more particularly preferable that the number of atoms contained is 1.0 × 10 or more. ９It is even more particularly preferable that the microparticle composition contains 1.0 × 10 or more microparticles. In addition, there is no particular limitation on the concentration of the microparticles contained in the microparticle composition. ８ It is preferable that the concentration is 2.0 × 10 ８ It is more preferable that the concentration is 4.0 × 10 ８ It is particularly preferable that the concentration is 5.0 × 10 ８ It is more particularly preferable that the concentration is 2.0 × 10 ９ It is even more particularly preferable that the microparticles used in the present invention contain such a large amount or high concentration of microparticles, which can sufficiently improve the cancer microenvironment.

[0031] <Other Components> In addition to microparticles, the microparticle composition may contain other components depending on the type and purpose of administration to the animal to which it is administered, as long as the effects of the present invention are not impaired. Examples of other components include nutritional components, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, and antiseptics. Examples of nutritional components include fatty acids and vitamins. Examples of antibiotics include penicillin, streptomycin, and gentamicin. Examples of carriers include materials known as pharmaceutically acceptable carriers. The microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or it may be a pharmaceutical composition further containing pharmaceutically acceptable carriers, excipients, and the like. The purpose of the pharmaceutical composition is to facilitate the administration of the microparticles to the recipient.

[0032] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject to administration and does not abrogate the biological activity and properties of the administered compound. Examples of carriers include propylene glycol, (physiological) saline, emulsions, buffer solutions, culture media such as DMEM or RPMI, and cryopreservation media containing components that scavenge free radicals.

[0033] The microparticle composition may contain an active ingredient of a conventionally known therapeutic agent for the cancer microenvironment, and those skilled in the art can appropriately modify the composition depending on the intended use, the subject of administration, etc.

[0034] On the other hand, it is preferable that the microparticle composition does not contain a specific substance. For example, it is preferable that the microparticle composition does not contain dental pulp-derived stem cells. It is also preferable that the microparticle composition does not contain MCP-1. However, it may contain cytokines other than MCP-1. Examples of other cytokines include those described in paragraphs

[0014] to

[0020] of JP 2018-023343 A. It is also preferable that the microparticle composition does not contain Siglec-9. However, it may contain sialic acid-binding immunoglobulin-like lectins other than Siglec-9. It is also preferable that the microparticle composition is substantially free of serum (e.g., fetal bovine serum, human serum, sheep serum). It is also preferable that the microparticle composition is substantially free of conventional serum substitutes such as Knockout serum replacement (KSR). In the microparticle composition, the content (solid content) of each of the above-mentioned other components is preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0035] <Method for Producing Microparticles> There are no particular limitations on the method for producing microparticles. Microparticles may be prepared by preparing a culture supernatant of dental pulp-derived stem cells and subsequently purifying the microparticles from the culture supernatant of dental pulp-derived stem cells. Alternatively, the agent for improving a cancer microenvironment of the present invention may be prepared by purifying microparticles from a culture supernatant of commercially purchased dental pulp-derived stem cells. Furthermore, the agent for improving a cancer microenvironment of the present invention may be prepared by obtaining a composition containing a culture supernatant of dental pulp-derived stem cells that had been discarded (or by appropriately purifying the composition), and purifying microparticles from the composition.

[0036] (Method for preparing culture supernatant of dental pulp-derived stem cells) The culture supernatant of dental pulp-derived stem cells is not particularly limited. The culture supernatant of dental pulp-derived stem cells is preferably substantially free of serum. For example, the culture supernatant of dental pulp-derived stem cells preferably contains 1% by mass or less of serum, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0037] Dental pulp-derived stem cells may be derived from humans or non-human animals, including the same animals (biological species) as those to which the agent for improving a cancer microenvironment of the present invention described below is administered, and mammals are preferred.

[0038] The dental pulp-derived stem cells used in the culture supernatant are not particularly limited. Stem cells from exfoliated deciduous teeth, stem cells from exfoliated deciduous teeth obtained by other methods, and stem cells from permanent teeth (dental pulp stem cells; DPSCs) can be used. Stem cells from human exfoliated deciduous teeth (SHED) and stem cells from human permanent teeth can also be used, as well as stem cells derived from dental pulp of animals other than humans, such as porcine deciduous teeth. In addition to exosomes, dental pulp-derived stem cells can produce various cytokines such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines. They can also produce many other physiologically active substances. In the present invention, it is preferable to use exosomes derived from the culture supernatant of deciduous dental pulp stem cells as exosomes derived from dental pulp-derived stem cells.

[0039] The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved. In particular, the present invention can use immortalized dental pulp-derived stem cells. By using immortalized stem cells that are capable of virtually unlimited proliferation, the amount and composition of biological factors contained in the stem cell culture supernatant can be stabilized over a long period of time. There are no particular limitations on the immortalized dental pulp-derived stem cells. It is preferable that the immortalized stem cells are non-cancerous immortalized stem cells. Immortalized dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors), either alone or in combination, to dental pulp-derived stem cells and culturing them. The TGFβ receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of transforming growth factor (TGF) β receptor, and examples thereof include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyridine, Examples of suitable ROCK inhibitors include pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all manufactured by Merck), and SB431542 (Sigma-Aldrich). A-83-01 is preferred. The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-associated kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Fujifilm Wako Pure Chemical Industries, Ltd.). Preferred is Y-27632.The GSK3 inhibitor is not particularly limited as long as it inhibits GSK-3 (glycogen synthase kinase 3), and examples include A 1070722, BIO, and BIO-acetoxime (all manufactured by TOCRIS). The MEK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and examples include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all manufactured by Selleck), PD98059, U0124, U0125 (all manufactured by Cosmo Bio Co., Ltd.), and the like.

[0040] When the cancer microenvironment improving agent of the present invention is used in regenerative medicine, due to the requirements of the Act on Safety of Regenerative Medicine, the culture supernatant of dental pulp-derived stem cells or these immortalized stem cells, and compositions containing microparticles derived therefrom, are embodied as not containing somatic stem cells other than dental pulp-derived stem cells. The microparticle composition may contain, but preferably does not contain, mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermis, digestive system, bone marrow, nervous system, etc. Examples of somatic stem cells of the dermis include epithelial stem cells and hair follicle stem cells. Examples of somatic stem cells of the digestive system include pancreatic (general) stem cells and hepatic stem cells. Examples of somatic stem cells of the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells. Examples of somatic stem cells of the nervous system include neural stem cells and retinal stem cells. The microparticle composition may contain, but preferably does not contain, stem cells other than somatic stem cells. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

[0041] There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used. The culture supernatant of dental pulp-derived stem cells is a culture medium obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant usable in the present invention can be obtained by separating and removing cellular components after culturing dental pulp-derived stem cells. Culture supernatants that have been appropriately subjected to various processes (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) can also be used.

[0042] Dental pulp-derived stem cells for obtaining the conditioned medium can be selected by conventional methods based on cell size or morphology, or as adhesive cells. Adhesive cells or their subcultured cells can be selected from dental pulp cells collected from shed deciduous or permanent teeth. The conditioned medium for dental pulp-derived stem cells can be obtained by culturing selected stem cells.

[0043] The "dental pulp-derived stem cell culture supernatant" is preferably a culture medium that does not contain cells obtained by culturing dental pulp-derived stem cells. In one embodiment, the dental pulp-derived stem cell culture supernatant used in the present invention preferably does not contain cells (regardless of cell type) as a whole. This characteristic clearly distinguishes the composition of this embodiment from various compositions containing dental pulp-derived stem cells, as well as from dental pulp-derived stem cells themselves. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed solely of dental pulp-derived stem cell culture supernatant. The dental pulp-derived stem cell culture supernatant used in the present invention may contain both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention preferably contains deciduous dental pulp-derived stem cell culture supernatant as an active ingredient, more preferably at least 50% by weight, and preferably at least 90% by weight. It is particularly preferable that the dental pulp-derived stem cell culture supernatant used in the present invention be a composition composed solely of deciduous dental pulp-derived stem cell culture supernatant.

[0044] The culture medium for dental pulp-derived stem cells to obtain the culture supernatant can be a basal medium or a basal medium supplemented with serum, etc. Examples of basal media that can be used include Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Examples of components that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals. However, to prepare a serum-free "dental pulp-derived stem cell culture supernatant," it is recommended to use a serum-free medium throughout the entire process or for the final or penultimate few subcultures. For example, serum-free dental pulp-derived stem cell culture supernatant can be prepared by culturing dental pulp-derived stem cells in a serum-free medium. Serum-free dental pulp-derived stem cell culture supernatant can also be obtained by performing one or more subcultures and culturing the final or penultimate subculture in a serum-free medium. Serum-free dental pulp-derived stem cell culture supernatant can also be obtained by removing serum from the recovered culture supernatant using dialysis, solvent replacement using a column, or the like.

[0045] Conventional conditions can be applied to the cultivation of dental pulp-derived stem cells to obtain the culture supernatant. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells depending on the type of stem cells. Furthermore, special conditions may be applied to the cultivation of dental pulp-derived stem cells in order to produce large amounts of microparticles such as exosomes. Examples of special conditions include low-temperature conditions, low-oxygen conditions, microgravity conditions, and conditions for co-culturing with some kind of stimuli.

[0046] The culture supernatant of dental pulp-derived stem cells used in the present invention for preparing microparticles such as exosomes may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but is preferably substantially free of other components. However, various additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

[0047] (Preparation of Microparticles) Microparticles can be prepared by purifying them from the culture supernatant of dental pulp-derived stem cells.

[0048] Purification of microparticles is preferably the separation of a microparticle-containing fraction from the dental pulp-derived stem cell culture supernatant, and more preferably the isolation of microparticles. Microparticles can be isolated by separating them from non-associated components based on their properties. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity. In the present invention, microparticles can be purified by centrifuging the dental pulp-derived stem cell culture supernatant and isolating a specific fraction (e.g., precipitate) rich in microparticles. Unnecessary components (insoluble components) from fractions other than the specified fraction may be removed. Complete removal of the solvent, dispersion medium, and unnecessary components from the microparticle composition is not required. Centrifugation conditions include 100 to 20,000 g for 1 to 30 minutes. In the present invention, microparticles can be purified by filtering the dental pulp-derived stem cell culture supernatant or its centrifuged product. Unnecessary components can be removed by filtration. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed simultaneously. The material and pore size of the filtration membrane used for filtration are not particularly limited. Filtration can be performed using a known method using a membrane with an appropriate molecular weight or size cutoff. From the perspective of facilitating exosome isolation, the pore size of the membrane is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. Tangential flow filtration (TFF) is also preferred for filtration. TFF is a type of membrane filtration technology in which a liquid flows parallel to the membrane surface to separate and purify components based on molecular size. TFF can be widely used for the concentration, desalting, buffer exchange, and size fractionation of biomolecules such as extracellular vesicles (exosomes). Ultrafiltration membranes such as hollow fiber membranes can be used for TFF. In the present invention, the culture supernatant of dental pulp-derived stem cells, its centrifuged product, or its filtrate can be separated using additional separation methods such as column chromatography. For example, high-performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column.An example of column chromatography is a fully automated exosome isolation system that combines chromatography and hollow fiber membrane separation technology. Such a system enables energy-saving, highly efficient exosome isolation. Furthermore, when combining TFF filtration with column chromatography, TFF filtration and size exclusion column chromatography are preferred. One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing stage. For example, light scattering, refractive index, dynamic light scattering, or UV-visible light detectors can be used to track the microparticles. Alternatively, specific enzyme activity can be used to track activity in each fraction. Microparticle purification methods may also be used, such as those described in paragraphs

[0034] to

[0064] of JP2019-524824A, the contents of which are incorporated herein by reference. Preferred purification methods include a combination of centrifugation and filtration of the dental pulp-derived stem cell culture supernatant, TFF of the dental pulp-derived stem cell culture supernatant (more preferably a combination of TFF and column chromatography), and column chromatographic separation of the dental pulp-derived stem cell culture supernatant (more preferably a combination of column chromatography and hollow fiber membrane separation). All of these methods can produce the cancer microenvironment improver of the present invention. That is, it is possible to obtain a cancer microenvironment improver that improves the cancer microenvironment in a way that suppresses cancer cell proliferation by suppressing the activity of cancer-associated fibroblasts (CAFs), or a cancer microenvironment improver that improves the cancer microenvironment in a way that suppresses cancer cell proliferation by inducing immunosuppressive macrophages (M2 macrophages) to immunostimulatory macrophages (M1 macrophages).

[0049] The final form of the microparticle composition is not particularly limited. For example, the microparticle composition may be in the form of microparticles packed together with a solvent or dispersion medium in a container; microparticles gelled together with a gel and packed into a container; or microparticles solidified by freezing and / or drying, formulated, or packed into a container. Examples of the container include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

[0050] Compared to conventional compositions that can be used as therapeutic or preventive agents for cancer microenvironments, the cancer microenvironment improvers of the present invention have advantages such as ease of mass production, the ability to utilize stem cell culture medium that was previously discarded as industrial waste, and reduced disposal costs for stem cell culture medium. In particular, when the dental pulp-derived stem cell culture supernatant is a culture supernatant from human dental pulp-derived stem cells, the cancer microenvironment improvers of the present invention are highly safe from immunological and other standpoints and pose fewer ethical concerns when administered to humans. When the dental pulp-derived stem cell culture supernatant is a culture supernatant from dental pulp-derived stem cells from a cancer patient, the cancer microenvironment improvers of the present invention are safer and pose fewer ethical concerns when administered to the patient. When the cancer microenvironment improvers of the present invention are derived from a dental pulp-derived stem cell culture supernatant, they can also be used in reparative medicine. In particular, compositions containing microparticles derived from a dental pulp-derived stem cell culture supernatant are preferably used in reparative medicine. It is known that in regenerative medicine based on stem cell transplantation, stem cells are not the main players in regeneration, but rather the liquid components produced by stem cells, together with the patient's own stem cells, repair organs. This overcomes the difficult issues associated with conventional stem cell transplantation, such as carcinogenesis, standardization, administration methods, storage, and culture methods, making regenerative medicine possible using dental pulp-derived stem cell culture supernatant or compositions containing microparticles derived therefrom. Compared to stem cell transplantation, the use of the cancer microenvironment improver of the present invention is safer, as it is less likely to cause tumorigenesis because cells are not transplanted. Furthermore, the cancer microenvironment improver of the present invention has the advantage of being of consistently standardized quality. It can be mass-produced and efficiently administered, allowing for cost-effective use.

[0051] [Preventive or therapeutic agent for anti-tumor immune response] The preventive or therapeutic agent for anti-tumor immune response of the present invention includes the agent for improving a cancer microenvironment of the present invention. As used herein, "prevention" refers to preventing the onset of a disease (herein, cancer microenvironment). Furthermore, as used herein, "treatment" refers to alleviating, suppressing, or preventing the progression of symptoms of an onset disease, and ameliorating the symptoms. The preventive or therapeutic agent for anti-tumor immune response of the present invention is preferably a therapeutic agent for an anti-tumor immune response.

[0052] [Cancer-associated fibroblast (CAF) activation inhibitor] The cancer-associated fibroblast (CAF) activation inhibitor of the present invention includes the cancer microenvironment improving agent of the present invention.

[0053] [Immunostimulatory Macrophage Inducer] The immunostimulatory macrophage inducer of the present invention includes the agent for improving a cancer microenvironment of the present invention.

[0054] [Method for Improving Cancer Microenvironment] The method for improving a cancer microenvironment of the present invention comprises administering an effective amount of the agent for improving a cancer microenvironment of the present invention to a subject having a cancer tumor.

[0055] The process of administering the agent for improving a cancer microenvironment of the present invention to a subject with a cancer microenvironment is not particularly limited. Administration methods include spraying or inhaling into the oral cavity, nasal cavity, or airway, infusion, topical administration, nasal drops, etc., with minimal invasiveness being preferred. A preferred local administration method is injection. Electroporation, which temporarily creates tiny holes in the cell membrane by applying a voltage (electrical pulse) to the skin surface, allowing the active ingredient to penetrate into the dermis layer, which is difficult to reach with conventional care, is also preferred. Local administration can include intracavernous administration, intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, and intraperitoneal administration, with intracavernous administration being more preferred. Intracavernous injection (ICI) is particularly preferred as the administration method in the present invention. Furthermore, various formulation techniques can be used to alter the in vivo distribution of microparticles. Numerous methods for altering in vivo distribution are known to those skilled in the art. Examples of such methods include the protection of exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers. When administered to a subject with cancer, the cancer microenvironment improving agent of the present invention may circulate throughout the subject's body and reach the desired tissue. The number of administrations and the administration interval are not particularly limited. The number of administrations is preferably two or more times during the effective therapeutic period, more preferably three or more times, and particularly preferably four or more times. The number of administrations can be one or more times per month, preferably one to ten times per month, more preferably two to six times per month, and particularly preferably four times per month (once per week). The administration interval is preferably one hour to two weeks, more preferably one to ten days, and particularly preferably two to seven days. However, this can be adjusted appropriately depending on the target organism and the target's symptoms. The agent for improving a cancer microenvironment of the present invention is preferably used in such a manner that the microparticles are administered to a subject who has developed a cancer microenvironment at least once a week for the effective therapeutic period.When the subject of administration is a human, the effective therapeutic period is preferably 1 to 10 weeks, more preferably 2 to 8 weeks, and particularly preferably 3 to 6 weeks. The effective therapeutic period is initially set at 3 to 4 weeks, and if the improvement rate in the evaluation of the therapeutic effect is low, it is preferable to extend it to 6 to 8 weeks. 2.0 x 10 ９ When using a culture supernatant of dental pulp-derived stem cells at a concentration of 0.1 x 10 cells / ml, in a mouse model, the amount is preferably 0.1 to 5 ml, more preferably 0.3 to 3 ml, and even more preferably 0.5 to 1 ml per mouse (approximately 25 g). When the subject of administration is a human, the amount is preferably 0.1 to 10 ml, more preferably 0.5 to 5 ml, and even more preferably 1 to 3 ml per person. ８ When using microparticles at a concentration of exosomes / μg, in a mouse model, the dose is preferably 1 to 50 μg per mouse (approximately 25 g), more preferably 3 to 30 μg, and even more preferably 5 to 25 μg. When the subject of administration is a human, the dose is preferably 1 to 100 μg per person, more preferably 5 to 50 μg, and even more preferably 10 to 30 μg. The cancer microenvironment improving agent of the present invention is preferably administered at an amount of 10 or more exosomes per cell, more preferably at an amount of 50 or more exosomes per cell, and even more preferably at an amount of 100 or more exosomes per cell. The preferred range of dose per body weight for other animals can be calculated proportionally from the dose per body weight (approximately 25 g) for a mouse model. However, this can be adjusted appropriately depending on the symptoms of the subject.

[0056] There are no particular limitations on the target animal (biological species) to which the cancer microenvironment improving agent of the present invention is administered. The target animal to which the cancer microenvironment improving agent of the present invention is administered is preferably a mammal, bird (chicken, quail, duck, etc.), or fish (salmon, trout, tuna, bonito, etc.). The mammal may be a human or a non-human mammal, with humans being particularly preferred. The non-human mammal is more preferably a cow, pig, horse, goat, sheep, monkey, dog, cat, mouse, rat, guinea pig, or hamster.

[0057] (Combination with Anticancer Agents or Immune Checkpoint Inhibitors, etc.) The cancer microenvironment improver of the present invention may be used in combination with a conventionally known cancer tumor therapeutic agent. In the cancer microenvironment improving method of the present invention, the cancer microenvironment improver is preferably administered to a subject in combination with an anticancer agent or an immune checkpoint inhibitor. The cancer microenvironment improver of the present invention can enhance the therapeutic effect of a cancer tumor therapeutic agent, such as a conventionally known anticancer agent or immune checkpoint inhibitor, through improvement of the cancer microenvironment. For example, as anticancer agents, alkylating agents (e.g., melphalan), antimetabolites (e.g., methotrexate), antimicrotubule agents (e.g., paclitaxel, docetaxel), and platinum-based agents (e.g., cisplatin) can be used in combination with the cancer microenvironment improver of the present invention. For example, as immune checkpoint inhibitors, PD-1 inhibitors (e.g., nivolumab), CTLA-4 inhibitors (e.g., ipilimumab), and PD-L1 inhibitors (e.g., durvalumab) can be used in combination with the cancer microenvironment improver of the present invention. Meanwhile, the cancer microenvironment improver of the present invention may be used in combination with other cancer microenvironment improvers. As other agents for improving the cancer microenvironment, angiogenesis inhibitors (such as bevacizumab) can be used in combination with the agent for improving the cancer microenvironment of the present invention.

[0058] The features of the present invention will be explained in more detail below with reference to examples, comparative examples, and reference examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0059] Example 1 Preparation of Dental Pulp-Derived Stem Cell Culture Supernatant A culture supernatant of human deciduous dental pulp stem cells was prepared and separated according to the method described in Example 6 of Japanese Patent No. 6296622, except that DMEM medium was used instead of the DMEM / HamF12 mixed medium. Primary culture was performed with the addition of fetal bovine serum (FBS), and subculture was performed using the primary culture medium. The supernatant of the subculture medium was separated so as not to contain FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. Note that DMEM is Dulbecco's Modified Eagle's Medium, and F12 is Ham's F12 medium.

[0060] <Exosome Preparation> Dental pulp-derived stem cell exosomes were purified from the resulting dental pulp-derived stem cell culture supernatant using the following method. The culture supernatant (100 mL) of deciduous dental pulp stem cells was filtered through a 0.22-micrometer pore filter, and the resulting solution was centrifuged at 100,000 × g for 60 minutes at 4°C. The supernatant was decanted, and the exosome-enriched pellet was resuspended in phosphate-buffered saline (PBS). The resuspended sample was centrifuged at 100,000 × g for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 μl). Protein concentration was determined using a microBSA protein assay kit (Pierce, Rockford, IL). The exosome-containing composition (concentrated solution) was stored at -80°C. The composition containing exosomes purified from the dental pulp-derived stem cell culture supernatant was used as the microparticle composition sample of Example 1.

[0061] The average particle size and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated. The average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm. The microparticle composition of Example 1 had a particle size of 1.0 × 10 ９ It is a highly concentrated exosome solution of 2.0 × 10 ９It was a highly concentrated exosome solution with exosome concentrations of 1000 / ml. Furthermore, the components of the obtained microparticle composition of Example 1 were analyzed by known methods. As a result, it was found that the microparticle composition of Example 1 did not contain stem cells derived from dental pulp, MCP-1, or Siglec-9. Therefore, it was found that the active ingredient of the microparticle composition of Example 1 was an active ingredient different from MCP-1 and Siglec-9, which are active ingredients in the culture supernatant of mesenchymal stem cells, and their analogs.

[0062] [Reference Examples 1 and 2]: Administration to SCID-beige model mice Using the microparticle composition of Example 1, whether tumors in severely combined immunodeficient SCID (Severe Combined ImmunoDeficiency)-beige mice could be suppressed was examined using the experimental system shown in the schematic diagram of Figure 1. MDA-MB-231-luc-D3H2LN cells, which are highly metastatic breast cancer cells, were administered to mice (SCID-beige mice, female, 6 weeks old, iv, n=10) at 2 x 10 ６ The SCID mutation results in a lack of functional B and T lymphocytes, resulting in severe combined immunodeficiency. The beige gene results in NK cell deficiency. In Reference Example 1, 10 μg (0.2 mL) of the microparticle composition (SGF EV) of Example 1 was administered per mouse 5, 8, 11, 14, 17, 20, 23, and 26 days after transplantation. The amount of luminescence from breast cancer-derived cells in the mice was measured immediately after transplantation and 7, 14, 21, 28, and 36 days after transplantation using an in vivo 2D / 3D luminescence, fluorescence, RI, X-ray, and CT imaging system, IVIS Imaging System (Revvity). The results obtained are shown in Figure 2 (mean of n = 10) as the effect on primary tumors. Furthermore, to examine the effect on metastasis, the luminescence intensity of the whole body of the mice was measured in the same manner after removal of the primary tumor 36 days after transplantation, and the results are shown in Figure 3 (mean of n = 10). The number of metastatic lesions is also shown in Figure 4 (mean of n = 10).

[0063] In Reference Example 2, the test was carried out in the same manner as in Reference Example 1, except that phosphate buffered saline (PBS(-)) was used instead of the microparticle composition of Example 1 (shown as PBS(-) in Figures 2 to 4).

[0064] As shown in Figure 2, there was no significant difference in the effect on primary tumors between the dental pulp-derived stem cell exosome administration group and the control group (PBS(-) administration group). As shown in Figures 3 and 4, there was no significant difference in the effect on cancer metastasis between the dental pulp-derived stem cell exosome administration group and the control group (PBS(-) administration group). From the above, in experiments using severe combined immunodeficient mice with no immunity, no difference was observed between the dental pulp-derived stem cell exosome administration group and the control group. In other words, it was found that the microparticle composition of Example 1 cannot directly inhibit the proliferation of cancer cells.

[0065] [Example 2 and Comparative Example 1]: Antitumor suppression in melanoma model mice Using the microparticle composition of Example 1, whether tumor suppression in melanoma model mice equipped with a general immune system including T cells, B cells, and NK cells was investigated using the experimental system shown in the schematic diagram of Figure 5. Mouse (C57BL / 6)-derived melanoma cells (B16F10-luc-G5) were inoculated into mice (C57BL / 6 Albino mice, female, 6 weeks old, iv, n=10) at 2.5 x 10 ４ The tumors were transplanted at a dose of 10 ... Therefore, tumor volume can be converted into a relative value of light intensity and quantified.

[0066] In Comparative Example 1, a test was carried out in the same manner as in Example 2, except that physiological saline was used instead of the microparticle composition of Example 1 (shown as Saline in FIGS. 6 and 7).

[0067] As shown in Figures 6 and 7, the luminescence intensity on the back of the mice measured using the IVIS Imaging System was significantly lower in the SGF EV-administered group than in the control group (saline-administered group) 21 days after transplantation. This suggests a significant melanoma cell-suppressing effect of SGF EV administration. In other words, it was found that the cancer microenvironment improving agent of the present invention can reduce cancer tumor volume in immunocompetent individuals.

[0068] The above-mentioned Reference Examples 1 and 2, Example 2, and Comparative Example 1, particularly the results of Figures 2 and 6, suggest that the microparticle composition of Example 1 does not directly inhibit cancer cell proliferation (direct tumor growth inhibition), but rather exerts an anti-tumor suppression effect through the immunoregulatory mechanism of action (improvement of anti-tumor immunity) possessed by exosomes derived from dental pulp-derived stem cells. Therefore, next, we decided to confirm in vitro whether the microparticle composition of Example 1 exerts immunoregulatory-related effects, such as suppressing CAFs and converting M2 macrophages to M1 macrophages.

[0069] [Examples 11-12, Comparative Examples 11-12]: Inhibition of Cancer-Associated Fibroblast Activation Using the microparticle composition of Example 1, the experimental system shown in the schematic diagram of Figure 8 was used to examine whether exosomes purified from dental pulp-derived stem cells could inhibit the activation of cancer-associated fibroblasts (CAFs), which promote cancer progression. Commercially available human fibroblasts (human fibroblast cells) (normal human dermal fibroblasts (pediatric), manufactured by PromoCell) were seeded to 70% subconfluent and cultured for 12 hours. Cells were stimulated with 10 ng / ml of TGF-β and cultured for 24 hours after seeding. In Example 11, the microparticle composition of Example 1 was then administered at a concentration that resulted in 10 exosomes (EVs) per cell, and the cells were harvested 48 hours after seeding. RNAs such as αSMA and β-ACTIN were purified from the resulting cells, and the relative expression levels of αSMA / β-ACTIN were quantified by qRT-PCR using the resulting RNA. αSMA (α-smooth muscle actin) is an indicator of CAF, and expression levels were corrected or normalized by β-ACTIN (mean of N = 2). The results are shown in Figure 9 (denoted as TGFβ + EVs10 in Figure 9).

[0070] In Example 12, a test was performed in the same manner as in Example 11, except that the microparticle composition of Example 1 was administered at a concentration such that the number of exosomes (number of EVs) per cell was 100 (referred to as TGFβ+EVs100 in FIG. 9). In Comparative Example 11, a test was performed in the same manner as in Example 11, except that phosphate-buffered saline PBS(-) was used instead of the microparticle composition of Example 1 (referred to as TGFβ+PBS(-) in FIG. 9). In Comparative Example 12, a test was performed in the same manner as in Example 11, except that no TGF-β stimulation was performed and no microparticle composition of Example 1 was administered (referred to as untreated control in FIG. 9).

[0071] From the above, it was found that the microparticle composition of Example 1 can suppress α-SMA, which is an indicator of CAF. Therefore, it was suggested that the microparticle composition of Example 1 can suppress the activation of cancer-associated fibroblasts (CAF). In other words, it was suggested that the microparticle composition of Example 1 can improve the cancer microenvironment.

[0072] [Examples 21-22, Comparative Examples 21-22]: Inhibition of M2 Macrophage Activation Using the microparticle composition of Example 1, the experimental system shown in the schematic diagram of Figure 10 was used to investigate whether exosomes purified from dental pulp-derived stem cells could inhibit the activation of tumorigenic M2 macrophages. Monocytes isolated from human whole blood were cultured for four days to produce human primary monocyte-derived macrophages (MDM). IL-4 (10 ng / ml) was then added, and the culture was continued for an additional two days to induce tumorigenic M2 macrophages (M2 pro-tumorigenic macrophages). In Example 21, the microparticle composition of Example 1 was then administered at a concentration sufficient to achieve 10 exosomes (EVs) per cell. 24 hours later, the degree of inhibition of M2 macrophage activation was measured using TNF-α and IL-10 ELISA kits (average of N=3). M2 macrophages produce little TNF-α and increased IL-10. The ELISA test was performed using TNF-α (ADI-900-099) and IL-10 (ADI-900-036) ELISA kits manufactured by Enzo Life Sciences, Inc. The results are shown in Figure 11 (labeled SGF EVs10 in Figure 11).

[0073] In Example 22, a test was performed in the same manner as in Example 21, except that the microparticle composition of Example 1 was administered at a concentration such that the number of exosomes (number of EVs) per cell was 100 (shown as "SGF EVs100" in FIG. 11 ). In Comparative Example 21, a test was performed in the same manner as in Example 11, except that PBS(-) was used instead of the microparticle composition of Example 1 (shown as "PBS(-)" in FIG. 11 ). In Comparative Example 22, a test was performed in the same manner as in Example 21, except that IL-4 was added but the microparticle composition of Example 1 was not administered (shown as "untreated" in FIG. 11 ).

[0074] From the above, it was found that the microparticle composition of Example 1 can suppress the degree of activation of M2-type macrophages, suggesting that the microparticle composition of Example 1 can improve the cancer microenvironment.

[0075] [Examples 31 and 32]: Modification of Purification Method In Example 31, instead of purifying the culture supernatant of the obtained deciduous dental pulp stem cells by a combination of filter filtration and centrifugation as in Example 1, the culture supernatant was purified by TFF separation to obtain a composition containing exosomes (concentrated solution). The obtained composition containing exosomes was designated the microparticle composition sample of Example 31. In Example 32, instead of purifying the culture supernatant of the obtained deciduous dental pulp stem cells by a combination of filter filtration and centrifugation as in Example 1, the culture supernatant was purified by a combination of column chromatography and hollow fiber membrane separation using Daicel's DiagExo® Human Body Fluid Exosome Isolation kit (trade name), to obtain a composition containing exosomes (concentrated solution). The obtained composition containing exosomes was designated the microparticle composition sample of Example 32. When the performance of the microparticle composition samples of Examples 31 and 32 was evaluated, they were found to be equivalent to the microparticle composition sample of Example 1, with equivalent average particle size, concentration, etc. When the microparticle composition samples of Examples 31 and 32 were used to evaluate antitumor inhibition in melanoma model mice, they exhibited effects comparable to those of the microparticle composition of Example 1. When the microparticle composition samples of Examples 31 and 32 were used to evaluate inhibition of cancer-associated fibroblast activation, they exhibited effects comparable to those of the microparticle composition of Example 1. When the microparticle composition samples of Examples 31 and 32 were used to evaluate inhibition of M2 macrophage activation, they exhibited effects comparable to those of the microparticle composition of Example 1.

Claims

1. A cancer microenvironment improvement agent containing microparticles derived from dental pulp-derived stem cells.

2. The agent for improving a cancer microenvironment according to claim 1, wherein the microparticles are exosomes.

3. The agent for improving a cancer microenvironment according to claim 1, which is an agent for improving an anti-tumor immune response.

4. A cancer microenvironment improving agent according to claim 1, which improves the cancer microenvironment in a direction that suppresses the proliferation of cancer cells by suppressing the activity of cancer-associated fibroblasts (CAFs).

5. The agent for improving a cancer microenvironment according to claim 1, which improves the cancer microenvironment in a direction that suppresses the proliferation of cancer cells by inducing immunosuppressive macrophages (M2 type macrophages) to immunostimulatory macrophages (M1 type macrophages).

6. The agent for improving a cancer microenvironment according to claim 1, wherein the microparticles are purified and isolated from the culture supernatant of dental pulp-derived stem cells, and do not contain any components other than the exosomes from the culture supernatant of the dental pulp-derived stem cells.

7. The agent for improving a cancer microenvironment according to claim 1, wherein the agent for improving a cancer microenvironment is administered in an amount of 10 or more exosomes per cell.

8. The agent for improving a cancer microenvironment according to claim 1, wherein the agent for improving a cancer microenvironment is administered to a subject having a cancer tumor at least twice during the effective treatment period.

9. The agent for improving a cancer microenvironment according to claim 1, which does not have the ability to suppress the proliferation of cancer cells in subjects without an immune system and is an agent for improving anti-tumor immune responses.

10. The agent for improving a cancer microenvironment according to claim 1, wherein the agent for improving a cancer microenvironment is administered to a subject having an immune system that has all of T cells, B cells, and NK cells.

11. A preventive or therapeutic agent for anti-tumor immune response, comprising the agent for improving a cancer microenvironment according to any one of claims 1 to 10.

12. An agent for inhibiting the activation of cancer-associated fibroblasts (CAFs), comprising the agent for improving a cancer microenvironment according to any one of claims 1 to 10.

13. An immunostimulatory macrophage inducer comprising the agent for improving a cancer microenvironment according to any one of claims 1 to 10.

14. A method for improving a cancer microenvironment, comprising administering an effective amount of the agent for improving a cancer microenvironment according to any one of claims 1 to 10 to a subject having a cancer tumor.

15. The method for improving a cancer microenvironment described in claim 14, wherein the agent for improving a cancer microenvironment is administered to the subject in combination with an anticancer drug or an immune checkpoint inhibitor.

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