Cancer microenvironment improving agent, prophylactic or therapeutic agent for antitumor immune response, and method for improving cancer microenvironment

Abstract

Provided is a novel cancer microenvironment improving agent capable of improving a cancer microenvironment by microparticles derived from dental pulp-derived stem cells.SOLUTION: It is preferable that the agent is an agent for improving a cancer microenvironment containing microparticles derived from dental pulp-derived stem cells, an agent for improving an antitumor immune response, it is more preferable that the agent improves the cancer microenvironment in a direction in which proliferation of cancer cells can be suppressed by inhibiting the activity of CAF, and it is particularly preferable that the agent improves the cancer microenvironment in a direction in which proliferation of cancer cells can be suppressed by inducing M2-type macrophages into M1-type macrophages, and it is further preferable that the agent is a prophylactic or therapeutic agent for an antitumor immune response, or a method for improving a cancer microenvironment.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[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. [Background technology]

[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, which comprises 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 anti-cancer 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 reducing metastasis. That is, Patent Document 2 describes a method for treating patients with LCP-1-positive cancer. In particular, Example

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

[0055] of Patent Document 2 further describes the administration of these EVs to tumor-bearing mouse models, using a mouse model of prostate cancer, demonstrating that decreased LCP-1 expression inhibits metastasis, while increased LCP-1 expression and phosphorylation stimulate metastasis of primary tumors.

[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 efficacy in 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 efficacy in 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 in which four genes are introduced into deciduous dental pulp stem cells obtained from mammalian dental pulp to produce immortalized stem cells; and a conditioned medium preparation step in which the immortalized stem cells are cultured in serum-free medium for a predetermined time at 23-27°C under a hypoxic condition of 0.5% to less than 20% oxygen to prepare a conditioned medium. The conditioned medium contains 1.5 times more insulin-like growth factor (IGF-1) and 1.5 times more vascular endothelial growth factor (VEGF) than the conditioned medium prepared by culture 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 had accumulated, surrounding 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 enhanced 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 natural phagocytic ability while simultaneously controlling tumor growth via TGF-β. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2022-532396 [Patent Document 2] Special Publication No. 2021-533822 [Patent Document 3] Special Publication No. 2019-535691 [Patent Document 4] International Publication No. 2015 / 111712 Summary of the Invention [Problem to be solved by the invention]

[0009] Patent Documents 1 to 3 do not disclose or suggest microparticles derived from dental pulp-derived stem cells. In particular, the mechanisms of cancer metastasis inhibition, prevention, or treatment in patent documents 0024 and 0040 of Patent Document 1 require novel anticancer 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 the 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 experiments, 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. [Means for solving the problem]

[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 improvement agent containing microparticles derived from dental pulp-derived stem cells. [2] The agent for improving a cancer microenvironment according to [1], wherein the microparticles are exosomes. [3] A cancer microenvironment improving agent according to [1], which is an agent for improving anti-tumor immune response. [4] A cancer microenvironment improving agent described in [1], which improves the cancer microenvironment in a way 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 [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 microparticles are purified and isolated from the culture supernatant of dental pulp-derived stem cells; A cancer microenvironment improving agent described in [1] that does not contain components excluding exosomes from the culture supernatant of dental pulp-derived stem cells. [7] The agent for improving a cancer microenvironment according to [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 [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 the proliferation of cancer cells and is an agent for improving an anti-tumor immune response.

[10] The agent for improving a cancer microenvironment described in [1], which is 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.

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

[10] .

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

[10] .

[13] An immunostimulatory macrophage inducer comprising the agent for improving a cancer microenvironment according to any one 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] A method for improving a cancer microenvironment according to

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

[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. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing the administration of exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells to SCID-beige mice. [Figure 2] Figure 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. [Figure 3] Figure 3 is a graph showing the amount of luminescence in the whole body of a mouse after removal of the primary tumor 36 days after transplantation, when samples from Reference Example 1 (SGF EVs) and Reference Example 2 (PBS(-)) were added. [Figure 4] Figure 4 is a graph showing the number of metastatic foci 36 days after transplantation when the samples of Reference Example 1 (SGF EVs) and Reference Example 2 (PBS(-)) were added. [Figure 5] FIG. 5 is a schematic diagram showing 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] FIG. 6 is a graph showing the number of days since transplantation and the luminescence intensity on the back of the mice when the samples of Example 2 (SGF EVs) and Comparative Example 1 (saline) were added. [Figure 7] FIG. 7 shows images taken with an IVIS Imaging System of luminescence on the back of a mouse 21 days after transplantation when the samples of Example 2 (SGF EVs) and Comparative Example 1 (Saline) were added. [Figure 8] FIG. 8 is a schematic diagram showing an experimental system for the suppression of cancer-associated fibroblast (CAF) activation by exosomes (microparticles of Example 1) purified from dental pulp-derived stem cells. [Figure 9]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. [Figure 10] FIG. 10 is a schematic diagram showing an experimental system for the suppression of M2 macrophage activation by exosomes (microparticles of Example 1) purified from dental pulp-derived stem cells. [Figure 11] Figure 11 is a graph showing the expression levels of TNF-α and IL-10 when samples from Comparative Example 22 (untreated), Example 21 (SGF EVs10), Example 22 (SGF EVs100), and Comparative Example 21 (PBS(-)) were added. DETAILED DESCRIPTION OF THE INVENTION

[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] [Tumor microenvironment improver] The agent for improving a cancer microenvironment of the present invention contains microparticles derived from dental pulp-derived stem cells. More preferably, the agent for improving a cancer microenvironment of the present invention can prevent, treat, or improve an anti-tumor immune response by improving the cancer microenvironment. Preferred embodiments of the present invention will now be described.

[0020] <Cancer microenvironment> The tumor microenvironment is the environment surrounding cancer cells. It 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, immune 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 the cancer, forming a microenvironment unique to that cancer. The agent for improving a cancer microenvironment of the present invention preferably improves the cancer microenvironment, which is the environment surrounding cancer cells, in a direction that can suppress the proliferation of cancer cells.Furthermore, the agent for improving a cancer microenvironment of the present invention is preferably an agent for improving an anti-tumor immune response.More preferably, the agent for improving a cancer microenvironment of the present invention can reduce the volume of cancer tumors through the improvement of the anti-tumor immune response. On the other hand, the cancer microenvironment improver of the present invention preferably does not have the ability to suppress the proliferation of cancer cells in subjects without an immune system. More preferably, the cancer microenvironment improver of the present invention does not have the ability to suppress the proliferation of cancer cells in subjects without an immune system and is an improver of anti-tumor immune responses. That is, the cancer microenvironment improver of the present invention is preferably used for administering the cancer microenvironment improver to a subject with 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] Details on the cancer microenvironment are provided in "Challenging the Cancer Microenvironment at the Single Cell Level," in Experimental Medicine, Vol. 39, No. 12 (Special Edition), 2021, edited by Naoya Fujita. For example, immune checkpoint inhibitors are effective against many types of cancer, including malignant melanoma, lung cancer, and gastric cancer. However, while they produce long-term therapeutic effects in 20-30% of cases, they are ineffective in more than half of patients. Furthermore, immune checkpoint inhibitors are known to be ineffective against pancreatic cancer, a difficult-to-treat cancer, and these cases of non-response and acquired resistance 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, giving rise to 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, for which immune checkpoint inhibitors are ineffective, chemotherapy using anticancer drugs is the standard treatment, and among these, gemcitabine, oxaliplatin, and fluorouracil are considered preferable due to their ability to induce tumor immunity. However, in reality, tumor-bearing hosts contain molecules and cellular factors that suppress immune induction, resulting in resistance to treatment. Cancer cells are characterized by the ability to evade the host immune system by creating a diverse immunosuppressive environment. Among the immune cells infiltrating tumors, myeloid-derived suppressor cells (MDSCs), regulatory T cells, macrophages, and neutrophils have immunosuppressive properties, and these cells are important resistance factors to immunotherapy and chemotherapy. MDSCs inhibit both innate and adaptive immunity through immunosuppressive functions such as the production of immunosuppressive cytokines and the inhibition of 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 premetastatic 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 is significantly correlated with the stage and tumor burden, and the frequency of MDSCs in cancer tissue is significantly correlated with a poor prognosis.

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

[0023] (CAF activation inhibitor) The agent for improving a cancer microenvironment of the present invention is preferably an agent for suppressing the activation of cancer-associated fibroblasts (CAFs).More preferably, the agent for improving a cancer microenvironment of the present invention suppresses the activity of cancer-associated fibroblasts (CAFs) to improve the cancer microenvironment in a direction that suppresses the proliferation of cancer cells. It is known that fibroblasts, vascular endothelial cells, and immune cells in tumor tissue exhibit normal behaviors that differ from those found in normal tissues. Fibroblasts in tumor tissue are called cancer-associated fibroblasts (CAFs) and are involved in the progression of cancer. Fibroblasts present in tumor tissues are a mixture of CAFs that maintain activated myofibroblastic characteristics and normal fibroblast-like CAFs that lack these characteristics, surrounding the tumor. CAFs that maintain activated myofibroblastic characteristics produce extracellular matrix around the tumor, physically stiffening the tumor tissue and preventing the penetration of anticancer drugs and immune cells. CAFs, which are activated fibroblasts present in the tumor tissue of many solid tumors, 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 malignant factor. TGF-β induces the loss of characteristics of epithelial cancer cells, such as strong intercellular junctions, and conferring mesenchymal stem cell characteristics, such as high motility and invasiveness, to epithelial-mesenchymal transition (EMT), which induces the progression and metastasis of epithelial cancer. TGF-β also induces endothelial-mesenchymal transition in vascular endothelial cells, thereby reducing their barrier function and promoting the formation of cancer-associated fibroblasts (CAFs), contributing to cancer malignancy.

[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) strategies targeting CAFs themselves (treatments that eliminate CAFs); (2) strategies to reprogram CAFs into normal fibroblasts; (3) Strategies targeting the interaction between CAFs and cancer cells (treatments targeting TGF-β or HGF, a product secreted by CAFs). The agent for improving the 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. Inhibition of 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 measuring the relative expression levels 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 agent for improving a cancer microenvironment of the present invention is preferably an immunostimulatory macrophage inducer, and more preferably 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. It refers to a substance that induces (M2-type macrophages; tumor-supporting) to become immunostimulatory macrophages (M1-type 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, M1, and M2 macrophages. M0 macrophages are inactivated macrophages differentiated from human peripheral blood monocytes. M1 macrophages have strong antigen-presenting ability and are generally activated by interferon-γ, lipopolysaccharide (LPS), and tumor necrosis factor (TNF)-α, and have inflammatory and bactericidal functions. M2 macrophages are known to promote immunosuppression, tumorigenesis, and angiogenesis by releasing various extracellular matrix components, angiogenic factors, and chemotactic factors. Generally, M2 macrophages are induced by IL-4 and IL-13 and are distinct from M1 macrophages. M2 macrophages express specific M2 markers, such as arginase-1, mannose receptor (MMR, CD206), and scavenger receptor (SR-A, CD204). These macrophages are a type of white blood cell that play an important role in animals' defense against infection. Normally, macrophages function as immunostimulatory macrophages (M1 type), which have antitumor effects such as attacking cancer cells and inducing apoptosis. However, when cancer cells develop and proliferate, macrophages are attracted to the tumor site and differentiate into immunosuppressive macrophages (M2 type) due to immunosuppressive macrophage-inducing factors (e.g., IL-6 and IL-10) secreted by the cancer cells. These immunosuppressive macrophages (M2 type) promote angiogenesis through the expression of vascular endothelial growth factor (VEGF), creating an environment favorable for cancer cell proliferation. Therefore, in order to suppress cancer cell proliferation, it is important to induce immunostimulatory macrophages (M1 type) and suppress or reduce immunosuppressive macrophages (M2 type). The degree of induction of immunostimulatory macrophages (M1 type) and the degree of inhibition or 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 method) Known methods for treating cancer tumors include general anticancer drugs, immune checkpoint inhibitors, and agents that improve the cancer microenvironment. Common anticancer drugs target cancer cells, but the tumor microenvironment is involved in resistance to these drugs (drug resistance). Although the therapeutic effects of immune checkpoint inhibitors are cross-cancer, they are 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 the infiltration of regulatory T cells (Tregs) within the tumor 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 for improving the cancer 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 within tumor tissue. It is believed to have antitumor effects by suppressing the formation of new blood vessels within tumor tissue and by inducing the normalization of tumor blood vessels, thereby normalizing the cancer microenvironment. 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 bevacizumab 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 inside of dental pulp-derived stem cells by any method are the same as microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, they can be said 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 a state contained in the culture supernatant or in a state purified from the culture supernatant. The microparticles are preferably microparticles purified from the culture supernatant. The origin of the microparticles can be determined by known methods. For example, the method described in J Stem Cell Res Ther (2018) 8:2 can be used to determine whether the microparticles are derived from dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, or other stem cells. 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, and exovesicles, or microvesicles, and are 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 the surface of the microparticles contains tetraspanin molecules such as CD9, CD63, and CD81, and this may be CD9 alone, CD63 alone, or CD81 alone, or any combination of two or three of these. Hereinafter, a preferred embodiment in which exosomes are used as microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

[0029] Preferably, exosomes are extracellular vesicles that are released from cells upon fusion of multivesicular bodies with the plasma membrane. The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells. The exosomes preferably contain 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, and they are easily traceable and can be targeted to specific regions.

[0030] (Fine particle content) The content of the microparticles in the microparticle composition is not particularly limited. 8 It is preferable to contain more than 1.0 × 10 8 It is more preferable to include 2.0 × 10 8 It is particularly preferable that the number of atoms contained is 2.5 × 10 or more. 8 It is more particularly preferred that the number of atoms contained is 1.0 × 10 or more. 9 It is even more particularly preferred that the number of the hydroxyl groups contained is 1 or more. The concentration of the microparticles in the microparticle composition is not particularly limited. 8 It is preferable to contain more than 2.0 × 10 8 It is more preferable to have more than 4.0 × 10 8 It is particularly preferable that the content is 5.0 × 108 It is more particularly preferable that the content is 2.0 × 10 9 It is even more particularly preferred that the concentration is 1 / mL or more. A preferred embodiment of the microparticles used in the present invention is capable of sufficiently improving the cancer microenvironment by containing such a large amount or high concentration of microparticles.

[0031] <Other ingredients> In addition to the microparticles, the microparticle composition may contain other components depending on the type of animal to be administered and the purpose, 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, antiseptics, etc. Examples of nutritional components include fatty acids and vitamins. Examples of antibiotics include penicillin, streptomycin, and gentamicin. 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 may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, etc. The purpose of the pharmaceutical composition is to facilitate the administration of the microparticles to a subject.

[0032] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject to be administered and does not suppress the biological activity and properties of the administered compound. Examples of the carrier include propylene glycol; (physiological) saline; emulsion; buffer solution; culture medium, such as DMEM or RPMI; and cryopreservation medium 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 preferred that the microparticle composition does not contain any predetermined substances. For example, the microparticle composition preferably does not include dental pulp-derived stem cells. Furthermore, the microparticle composition preferably does not contain MCP-1. However, the microparticle composition may contain cytokines other than MCP-1. Examples of other cytokines include those described in paragraphs

[0014] to

[0020] of JP 2018-023343 A. Furthermore, the microparticle composition preferably does not contain Siglec 9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec 9. It is preferable that the microparticle composition is substantially free of serum (such as fetal bovine serum, human serum, or 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 contents (solid contents) of the other components described above are 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 of manufacturing microparticles> The method for producing the microparticles is not particularly limited. The 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 the 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 the 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 serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, 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 (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] There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, stem cells from deciduous teeth obtained by other methods, and stem cells from permanent teeth (DPSCs) can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, stem cells derived from dental pulp of animals other than humans, such as porcine deciduous tooth pulp stem cells, can also be used. 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, as well as 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 they can achieve the intended treatment. In particular, the present invention can use immortalized stem cells derived from dental pulp. By using immortalized stem cells that can proliferate virtually indefinitely, 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 stem cells derived from dental pulp. The immortalized stem cells are preferably non-cancerous immortalized stem cells. Immortalized stem cells derived from dental pulp can be prepared by adding the following low molecular weight compounds (inhibitors) 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, and the like. Examples of suitable pyrazoles include 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.). Y-27632 is preferred. 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). MEK inhibitors are not particularly limited as long as they have 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 from Selleck), PD98059, U0124, U0125 (all from Cosmo Bio Co., Ltd.), etc.

[0040] When the agent for improving a cancer microenvironment of the present invention is used in regenerative medicine, in accordance with the requirements of the Act on Safety of Regenerative Medicine, etc., the culture supernatant of dental pulp-derived stem cells or immortalized stem cells thereof, or a composition containing microparticles derived therefrom, does not contain somatic stem cells other than dental pulp-derived stem cells. The microparticle composition may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, but preferably does not contain them. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of somatic stem cells from the dermal system include epithelial stem cells, hair follicle stem cells, etc. Examples of somatic stem cells from the digestive system include pancreatic (general) stem cells, hepatic stem cells, etc. Examples of somatic stem cells from the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells, etc. Examples of somatic stem cells from the nervous system include neural stem cells, retinal stem cells, etc. The microparticle composition may contain, but preferably does not contain, stem cells other than somatic stem cells, including 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 treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may 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] It is preferable that the "dental pulp-derived stem cell culture supernatant" is a culture medium that does not contain the cells themselves 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 that contain dental pulp-derived stem cells, as well as 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 only of dental pulp-derived stem cell culture supernatant. The dental pulp-derived stem cell culture supernatant used in the present invention may contain the culture supernatant of 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 the deciduous dental pulp-derived stem cell culture supernatant as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the dental pulp-derived stem cell culture supernatant used in the present invention is a composition composed solely of the 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 ingredients 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 serum-free "dental pulp-derived stem cell culture supernatant," it is recommended to use 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 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 few subcultures in serum-free medium. Alternatively, serum-free dental pulp-derived stem cell culture supernatant can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or other methods.

[0045] The conditions commonly used for culturing dental pulp-derived stem cells to obtain a culture supernatant can be applied as is. 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 steps of isolating and selecting stem cells 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. In addition, special conditions may be applied to the culture of dental pulp-derived stem cells to produce large amounts of microparticles such as exosomes, such as low temperature, low oxygen, and microgravity conditions, or co-culture 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 it is preferable that it is substantially free of other components. However, each type of additive used in preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

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

[0048] Purification of microparticles is preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles. Microparticles can be isolated by separating them from non-associated components based on a property of the microparticle, for example, they can be isolated based on molecular weight, size, morphology, composition, or biological activity. In the present invention, microparticles can be purified by separating a specific fraction (e.g., precipitate) rich in microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the specified fraction may be removed. The removal of the solvent, dispersion medium, and unnecessary components from the microparticle composition does not have to be complete. Centrifugation conditions include 100 to 20,000 g for 1 to 30 minutes. In the present invention, microparticles can be purified by filtering the culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. 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 filtration membrane with an appropriate molecular weight or size cutoff using a known method. From the viewpoint of facilitating the separation of exosomes, the pore size of the filtration membrane is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. In the present invention, the culture supernatant of dental pulp-derived stem cells, its centrifuged product, or its filtered product can be further separated using a separation method 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. 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, etc. can be used to track activity in each fraction. As a method for purifying microparticles, the method described in

[0034] to

[0064] of JP-A No. 2019-524824 may be used, the contents of which are incorporated herein by reference.

[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 in a container together with a solvent or dispersion medium; microparticles gelled with a gel and packed in a container; or microparticles solidified by freezing and / or drying, formulated, or packed in 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 improver of the present invention has 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 improver of the present invention has the advantage of being highly safe from immunological and other standpoints and posing fewer ethical concerns when applied 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 improver of the present invention will be safer and pose fewer ethical concerns when applied to that patient. When the cancer microenvironment improving agent of the present invention is derived from the conditioned medium of dental pulp-derived stem cells, it can also be used in restorative medicine. In particular, compositions containing microparticles derived from the conditioned medium of dental pulp-derived stem cells are preferably used in restorative 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 solves the difficult issues associated with conventional stem cell transplantation, such as carcinogenesis, standardization, administration method, storage, and culture method, and makes restorative medicine possible using a composition using the conditioned medium of dental pulp-derived stem cells or microparticles derived therefrom. Compared to stem cell transplantation, the use of the cancer microenvironment improving agent of the present invention is safer, as it does not require cell transplantation, making it less likely to develop tumors. Furthermore, the cancer microenvironment improving agent of the present invention has the advantage of being of consistently standardized quality. It can be mass-produced and efficiently administered, allowing for low-cost use.

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

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

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

[0054] [Methods for improving the cancer microenvironment] The method of the present invention for improving a cancer microenvironment 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] There are no particular limitations on the step of administering the agent for improving a cancer microenvironment of the present invention to a subject who has developed a cancer microenvironment. Examples of administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, infusion, topical administration, and nasal drops, with minimal invasiveness being preferred. A preferred local administration method is injection. Electroporation is also preferred, which applies a voltage (electric pulse) to the skin surface to temporarily create minute holes in the cell membrane, allowing the active ingredient to penetrate into the dermis layer, which is difficult to reach with conventional care. Examples of local administration 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. The administration method in the present invention is particularly preferably intracavernous injection (ICI). In addition, various formulation techniques can be used to change the in vivo distribution of microparticles.Many methods for changing in vivo distribution are known to those skilled in the art.Examples of such methods include, for example, protecting exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates or synthetic polymers. The agent for improving a cancer microenvironment of the present invention administered to a subject who has developed cancer may circulate within the subject's body and reach a specific tissue. There are no particular limitations on the number of doses and the interval between doses. The number of administrations is preferably two or more times during the effective treatment period for the cancer microenvironment improving agent to be administered to a subject with a cancer tumor, 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 species and symptoms of the target. The agent for improving a cancer microenvironment of the present invention is preferably used to administer microparticles to a subject who has developed a cancer microenvironment at least once a week for an effective therapeutic period. When the subject 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 preferably set to 3 to 4 weeks initially, and if the improvement rate in the evaluation of the therapeutic effect is low, it is preferably extended to 6 to 8 weeks. 2.0×10 9 When using a culture supernatant of dental pulp-derived stem cells at a concentration of 1 / ml, 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) in a mouse model.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. 0.1×10 8 When microparticles at a concentration of exosomes / μg are used, in a mouse model, the amount 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 amount is preferably 1 to 100 μg per person, more preferably 5 to 50 μg, and even more preferably 10 to 30 μg. The agent for improving a cancer microenvironment of the present invention is preferably administered in an amount of 10 or more exosomes per cell, more preferably 50 or more exosomes per cell, and even more preferably 100 or more exosomes per cell. The preferred range of the dose per body weight for other animals can be calculated proportionally from the dose per body weight (approximately 25 g) for the model mouse, but can be adjusted appropriately depending on the symptoms of the subject.

[0056] There are no particular limitations on the animal (biological species) to which the cancer microenvironment improving agent of the present invention is administered. The 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 either 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] (Combined with anticancer drugs or immune checkpoint inhibitors) The agent for improving a cancer microenvironment of the present invention may be used in combination with a conventionally known therapeutic agent for cancer tumors. In the method for improving a cancer microenvironment of the present invention, it is preferable to administer the agent for improving a cancer microenvironment in combination with an anticancer agent or an immune checkpoint inhibitor to a subject. The agent for improving a cancer microenvironment of the present invention can enhance the therapeutic effect of a conventionally known therapeutic agent for cancer tumors, such as an anticancer agent or an immune checkpoint inhibitor, through improvement of the cancer microenvironment. For example, anticancer drugs such as alkylating agents (such as melphalan), anti-metabolites (such as methotrexate), anti-microtubule agents (such as paclitaxel and docetaxel), and platinum-based drugs (such as cisplatin) can be used in combination with the cancer microenvironment improving agent of the present invention. For example, immune checkpoint inhibitors such as PD-1 inhibitors (such as nivolumab), CTLA-4 inhibitors (such as ipilimumab), and PD-L1 inhibitors (such as durvalumab) can be used in combination with the cancer microenvironment improving agent of the present invention. On the other hand, the agent for improving a cancer microenvironment of the present invention may be used in combination with other agents for improving a cancer microenvironment, such as an angiogenesis inhibitor (bevacizumab, etc.). [Example]

[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 culture supernatant of dental pulp-derived stem cells> 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 DMEM / HamF12 mixed medium. Primary culture was performed with the addition of fetal bovine serum (FBS), and subculture was performed using primary culture medium. The supernatant of the subculture medium was separated so that it did not 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 culture supernatant of the obtained dental pulp-derived stem cells using the following method. The culture supernatant (100 mL) of primary dental pulp stem cells was filtered through a 0.22-micrometer pore size filter, and the 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 (approximately 100 μl) as the concentrated sample. Protein concentration was determined using a microBSA protein assay kit (Pierce, Rockford, IL). The exosome-containing composition (concentrated solution) was stored at -80°C. A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells 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 is 1.0 x 10 9 It is a highly concentrated exosome solution of 2.0 x 10 9 It was a highly concentrated exosome solution with 100 cells / 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 The ability of the microparticle composition of Example 1 to suppress tumor growth in SCID (Separate Combined ImmunoDeficiency)-beige mice, which are severely combined immunodeficient mice, was examined using an experimental system shown in the schematic diagram of FIG. MDA-MB-231-luc-D3H2LN cells, a highly metastatic breast cancer cell line, were injected into mice (SCID-beige mice, female, 6 weeks old, iv, n=10) at 2×10 6 The SCID mutation results in a severe combined immunodeficiency due to the lack of functional B and T lymphocytes. The beige gene results in a deficiency in NK cells. 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. Immediately after transplantation, and 7, 14, 21, 28, and 36 days after transplantation, the amount of luminescence from the mouse breast cancer cells was measured using an in vivo 2D / 3D luminescence, fluorescence, RI, X-ray, and CT imaging system, the IVIS Imaging System (Revvity). The results obtained are shown in Figure 2 (mean of n = 10) as the effect on the primary tumor. Furthermore, to examine the effect on metastasis, the luminescence intensity of the whole body of the mice was measured 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 tumors is also shown in Figure 4 (mean of n = 10).

[0063] In Reference Example 2, a 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 the experiment using severely combined immunodeficient mice with no immune system, no difference was observed between the group administered exosomes from dental pulp-derived stem cells 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 The experimental system shown in the schematic diagram of Figure 5 was used to examine whether the microparticle composition of Example 1 could suppress tumors in melanoma model mice equipped with a general immune system including T cells, B cells, and NK cells. 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 a dose of 2.5 × 10 4 cells / mouse transplanted. Two, five, eight and eleven days after transplantation, 5 μg (0.2 mL) of the microparticle composition of Example 1 (SGF EV) was administered to each mouse. Immediately after transplantation, and 7, 13, and 21 days after transplantation, the luminescence intensity on the back of the mice was measured using an in vivo 2D / 3D luminescence, fluorescence, RI, X-ray, and CT imaging system, the IVIS Imaging System (Revvity). The results are shown in Figure 6 (mean of n = 10). Figure 7 shows an image of luminescence on the back of the mouse taken with an IVIS Imaging System 21 days after transplantation. The IVIS Imaging System demonstrates a very good correlation between tumor volume and light intensity, making it possible to quantify tumor volume using relative values ​​converted into light intensity.

[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 an 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 that SGF EV administration has a significant inhibitory effect on melanoma cells. In other words, the cancer microenvironment improving agent of the present invention was found to be able to reduce cancer tumor volume.

[0068] The above Reference Examples 1 and 2, Example 2, and Comparative Example 1 suggest that the microparticle composition of Example 1 does not directly inhibit the proliferation of cancer cells (directly inhibit tumor proliferation), but exerts an anti-tumor suppression effect through the immune control mechanism of exosomes derived from dental pulp-derived stem cells (improvement of anti-tumor immunity). Therefore, next, we decided to confirm in vitro whether the microparticle composition of Example 1 has effects related to immune regulation, such as suppressing CAFs and converting M2 macrophages to M1 macrophages.

[0069] [Examples 11 and 12, Comparative Examples 11 and 12]: Inhibition of activation of cancer-associated fibroblasts Using the microparticle composition of Example 1, we investigated whether exosomes purified from dental pulp-derived stem cells could suppress the activation of cancer-associated fibroblasts (CAFs), which promote cancer progression, using the experimental system shown in the schematic diagram of Figure 8. Commercially available human fibroblast cells (normal human dermal fibroblasts (pediatric), manufactured by PromoCell) were seeded to 70% subconfluent and cultured for 12 hours. Stimulation with 10 ng / ml of TGF-β was then continued for 24 hours after seeding. Then, in Example 11, the microparticle composition of Example 1 was administered at a concentration such that the number of exosomes (number of EVs) per cell was 10, and the cells were collected 48 hours after seeding. RNAs such as αSMA and β-ACTIN were purified from the cells, and the relative expression levels of αSMA and β-ACTIN were quantified by qRT-PCR using the RNA obtained. αSMA (α-smooth muscle actin) is an indicator of CAFs, and expression levels were corrected or normalized with 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 (shown as TGFβ+EVs100 in Figure 9). In Comparative Example 11, the test was carried out in the same manner as in Example 11, except that phosphate buffered saline (PBS(-)) was used instead of the microparticle composition of Example 1 (shown as TGFβ+PBS(-) in Figure 9). In Comparative Example 12, the test was carried out in the same manner as in Example 11, except that no TGF-β stimulation was performed and the microparticle composition of Example 1 was not administered (shown as untreated control in FIG. 9).

[0071] From the above, it was found that the microparticle composition of Example 1 can suppress αSMA, 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, an experimental system shown in the schematic diagram of Figure 10 was used to examine whether exosomes purified from dental pulp-derived stem cells can suppress the activation of tumorigenic M2-type macrophages. Monocytes isolated from human whole blood were cultured for 4 days to generate human primary monocyte-derived macrophages (MDMs), which were then cultured for an additional 2 days with IL-4 (10 ng / ml) to induce M2 pro-tumorigenic macrophages. In Example 21, the microparticle composition of Example 1 was then administered at a concentration that resulted in 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 (mean of N=3). M2 macrophages produce little TNF-α and enhance IL-10. The ELISA test was performed using ELISA kits for TNF-α (ADI-900-099) and IL-10 (ADI-900-036) manufactured by Enzo Life Sciences, Inc. The results are shown in Figure 11 (denoted as SGF EVs10 in Figure 11).

[0073] In Example 12, 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 Figure 11). In Comparative Example 21, a test was carried out 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, the test was carried out 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.

Claims

1. An agent for improving the cancer microenvironment, comprising 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. 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 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 microparticles are purified and isolated from a culture supernatant of dental pulp-derived stem cells; The agent for improving a cancer microenvironment according to claim 1, which does not contain 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 according to 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.

Images