Anti-tumor immune response enhancer

Abstract

The present invention addresses the problem of providing a method for maintaining / enhancing the antitumor activity of TADC in a tumor microenvironment. The inventors have found that if administered a composition "cPLs adjuvant" containing three phospholipids, the degree of growth of a tumor transplanted into a mouse is very significantly inhibited, and furthermore, it has been confirmed that immature dendritic cells become mature bone marrow dendritic cells, inducing the production of cytokines, if cultured in vitro in the presence of the cPLs adjuvant.

Description

Technical Field

[0001] The present invention relates to an anti-tumor immune response enhancer, and more particularly to an anti-tumor immune response enhancer containing phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Background Art

[0002] Dendritic cells (DC) are known to be the most potent antigen presenting cells that can activate both naive T cells and memory T cells and induce antigen-specific anti-tumor immunity (see, for example, non-patent document 1, etc.). However, it is known that DCs in the tumor microenvironment, namely tumor-associated dendritic cells (hereinafter also referred to as [tumor associated dendritic cells: (TADC)], have low expression of co-stimulatory molecules and present an immature phenotype (see, for example, non-patent document 2, etc.), or present an inhibitory and dysfunctional phenotype that helps cancer cells escape host immune surveillance (see, for example, non-patent document 3, etc.), etc., and may cause immunosuppression and immune tolerance. There are also reports that TADC secretes various cytokines that inhibit the activation of anti-tumor T cells and promote the proliferation of tumor cells (see, for example, non-patent document 4, etc.). Therefore, it is known that in the tumor microenvironment, DCs may become a double-edged sword that acts both in the positive and negative directions for anti-tumor responses (see, for example, non-patent document 5, etc.), and in previous cancer immunotherapy, there are cases where anti-tumor activity is reduced.

[0003] On the other hand, a method for activating tumor-infiltrating lymphocytes (TIL) has been proposed, which comprises administering a fermentation composition produced by fermentation of a symbiotic microbiota in a culture medium to a subject requiring activation of TIL (see, for example, Patent Document 1, etc.). In addition, a composition for in vitro dendritic cell activation comprising one or more liposomes having at least one cationic liposome and at least one antigen has been proposed (see, for example, Patent Document 2, etc.), and a method for using an all-trans retinoic acid injection, etc., characterized in that the activity of abnormal bone marrow-derived immunosuppressive cells in tumor patients can be reduced, differentiation of bone marrow-derived immunosuppressive cells can be induced, and proliferation and recurrence of tumors can be inhibited (see, for example, Patent Document 3, etc.).

[0004] [Prior art literature]

[0005] [Patent Document]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2021-517587

[0007] [Patent Document 2] Japanese Patent Application Publication No. 2022-36961

[0008] [Patent Document 3] Japanese Patent Publication No. 2019-528315

[0009] [Non-patent literature]

[0010] [Non-patent document 1] Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature. 2007; 449(7161): 419-26

[0011] [Non-patent document 2] Oncotarget. 2016; 7(39): 63204-14

[0012] [Non-patent document 3] J Cancer. 2013; 4(1): 36-44

[0013] [Non-patent document 4] J Leukoc Biol. 2017; 102(2): 317-324

[0014] [Non-patent document 5] Frontiers in Oncology 2013 Volume 3 Article 90: 1-12 Summary of the invention

[0015] [Problems to be solved by the invention]

[0016] The present invention aims to provide a method for maintaining / enhancing the anti-tumor activity of TADC in the tumor microenvironment.

[0017] [Solution to the problem]

[0018] The present inventors focused on the microenvironment in mouse tumors and continued to study various components in order to maintain / enhance the antitumor activity of TADCs, which are considered to be immature DCs. As a result, they found that if a combination of three phospholipids, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), is administered after the tumor transplanted into the mouse reaches a certain size (Combined PhosphoLipids adjuvant: cPLs adjuvant) (hereinafter also referred to as "cPLs adjuvant"), the growth of the tumor transplanted into the mouse is significantly inhibited. In addition, it was confirmed that if immature DCs are cultured in vitro in the presence of cPLs adjuvant, the cultured DCs become mature bone marrow-derived dendritic cells (BMDCs) and induce the production of inflammatory cytokines. Furthermore, tumor tissues were removed from cancer cell-transplanted mice administered with cPLs adjuvant, and the phenotype of tumor-infiltrating leukocytes in the tumor tissues was analyzed. As a result, markers expressed by mature DCs were expressed, and it was confirmed that the production of T cells was promoted and the production of inflammatory cytokines was enhanced, thereby completing the present invention.

[0019] [Effects of the invention]

[0020] Administration of the anti-tumor immune response enhancer of the present invention can inhibit tumor growth and mature immature bone marrow-derived dendritic cells and TADCs. Furthermore, immature dendritic cells can be matured by culturing them in vitro in the presence of the anti-tumor immune response enhancer.

[0021] Specifically, the present invention is as follows.

[0022] [1] An anti-tumor immune response enhancer comprising phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

[0023] [2] The anti-tumor immune response enhancer according to [1] above, for use in inhibiting tumor growth in a subject.

[0024] [3] The anti-tumor immune response enhancer according to [1] above, which is used to convert immature dendritic cells into mature dendritic cells.

[0025] [4] The anti-tumor immune response enhancer as described in [3] above, for use in converting immature dendritic cells into CD11b + CD11c + Living cells.

[0026] [5] The anti-tumor immune response enhancer according to [4] above, wherein the immature dendritic cells are immature standard dendritic cells or tumor-associated dendritic cells.

[0027] [6] The anti-tumor immune response enhancer according to [1] or [2] above, characterized in that it contains 50% to 90% phosphatidylcholine, 5% to 25% phosphatidylethanolamine and 5% to 25% phosphatidylserine.

[0028] [7] The anti-tumor immune response enhancer according to [1] or [2] above, further comprising one or more anticancer drugs.

[0029] [8] A method for preparing mature dendritic cells, comprising culturing immature dendritic cells in vitro in the presence of the anti-tumor immune response enhancer described in [1] or [2] above, thereby preparing mature dendritic cells.

[0030] In addition, the present invention may include: a preparation containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine for use in anti-tumor immune response enhancement therapy; a cytokine production promoter containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; a combination of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine for enhancing anti-tumor immune response; a method for enhancing the anti-tumor immune response of a subject, comprising administering an anti-tumor immune response enhancer containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to a subject; a method for enhancing the anti-tumor immune response of a subject, comprising administering phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to a subject in need of enhancing the anti-tumor immune response. Response method; comprising the step of administering an anti-tumor immune response enhancer containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to a subject in need of enhancing the anti-tumor immune response in the tumor microenvironment, a method for activating the subject's tumor-associated dendritic cells, or a method for reducing the immunosuppressive nature of the subject's tumor microenvironment to enhance clinical practicality, a cancer immunotherapy using mature dendritic cells activated by an anti-tumor immune response enhancer, tumor-associated dendritic cells activated by an anti-tumor immune response enhancer, mature dendritic cells activated by an anti-tumor immune response enhancer, or a cancer immunotherapy using tumor-associated dendritic cells activated by an anti-tumor immune response enhancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1(a) is a graph showing changes in tumor volume until 16 days after MO4-Luc cell transplantation in mice to which 4 mg / kg or 12 mg / kg of cPLs adjuvant was subcutaneously injected into the flank and in control mice to which cPLs adjuvant was not administered. The horizontal axis represents the number of days after mouse transplantation, and the vertical axis represents the tumor volume of MO4-Luc cell transplanted mice. Figure 1 (b) is a histogram of tumor volumes on day 16 after transplantation in mice subcutaneously injected with cPLs adjuvant at 4 mg / kg or 12 mg / kg of mouse body weight and control mice not given cPLs adjuvant. Figure 1 (c) is a graph showing changes in tumor volume until 16 days after C26 cell transplantation in mice to which 12 mg / kg of cPLs adjuvant was subcutaneously injected into the flank and in control mice to which no cPLs adjuvant was administered. Figure 1 (d) is a histogram of tumor volumes on day 16 after transplantation in mice to which 12 mg / kg of mouse body weight of cPLs adjuvant was administered and control mice to which cPLs adjuvant was not administered. Figure 1 (e) is a graph showing changes in tumor volume until 16 days after transplantation in cases where cPLs adjuvant, PC, PE, and PS were administered to MO4-Luc cell-transplanted mice, respectively.

[0032] Figure 2 Shown is the flow cytometric analysis of mouse standard immature bone marrow dendritic cells cultured in the presence of cPLs adjuvant.

[0033] Figure 3 The graphs show the delta (Δ) values ​​of mean fluorescence intensity (MFI) for the expression of cell surface markers (a) IA / IE, (b) CD80, and (c) CD40 after culturing mouse standard immature bone marrow dendritic cells in the presence of cPLs adjuvant.

[0034] Figure 4 Graphs showing the expression of (a) IL-1β, (b) IL-12, and (c) IL-6 in imDC cultured for 2 days in the presence of cPLs adjuvant, respectively, expressed as delta values ​​of MFI.

[0035] Figure 5 (a) Figure 5 (b) Both show flow cytometric analysis of TILs of the cPLs adjuvant-treated group.

[0036] Figure 6Graphs showing the expression of (a) CD86 and (b) IA / IE in TILs of the cPLs adjuvant-treated group expressed as delta values ​​of MFI.

[0037] Figure 7 The graphs show the expression of (a) IL-1β, (b) IL-12, and (c) IFN-γ in TILs of the cPLs adjuvant-treated group as delta values ​​of MFI.

[0038] Figure 8 CD4 + Production of (a) IFN-γ, (c) TNF-α, and (e) IL-2 in T cells, and CD8 + Graph showing the production of (b) IFN-γ, (d) TNF-α, and (f) IL-2 in T cells using delta values ​​of MFI. DETAILED DESCRIPTION

[0039] The anti-tumor immune response enhancer of the present invention is not particularly limited as long as it is a preparation that contains phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) and can enhance the anti-tumor immune response. Examples include preparations that do not contain phospholipids other than PC, PE and PS, or preparations that do not contain phospholipids other than PC, PE and PS as essential ingredients. Examples of the subjects to whom the anti-tumor immune response enhancer is administered include mammals such as mice, rats, sheep, pigs, cattle, cats, dogs, monkeys, and humans.

[0040] As a method for preparing the anti-tumor immune response enhancer of the present invention, a method for preparing the anti-tumor immune response enhancer by dissolving / mixing PC, PE and PS in a solvent in sequence or as a mixture can be exemplified; as the above-mentioned solvent, a solvent that can exert the effect of the present invention and is non-invasive to the object can be cited, and ethanol can be preferably cited. As the concentration of the solution, PC, PE and PS can be cited as 0.52 mg / mL, preferably 0.8-1.5 mg / mL; as the ratio of PC, PE and PS contained in the solution, PC can be 50%-90%, PE can be 5%-25%, PS can be 5%-25%, preferably PC is 60%-80%, PE is 10%-20%, PS is 10%-20%, and more preferably PC is 65%-75%, PE is 12.5%-17.5%, and PS is 12.5%-17.5%.

[0041] Examples of the PC include PC derived from soybeans, PC derived from egg yolks, and other PC derived from natural sources, or synthetic PC containing saturated or unsaturated carboxylic acids having 7 to 22 carbon atoms. Specific examples of synthetic PC include dilauroyl PC, dimyristoyl PC, dioleoyl PC, dipalmitoyl PC, palmitoleoyl PC, and distearoyl PC. In addition, the fatty acid residues bonded to the 1- and 2-positions of glycerol may be the same or different.

[0042] Examples of the PE include naturally derived PE such as soybean-derived PE and hydrogenated PE derived from soybean, and synthetic PE such as PE containing a saturated or unsaturated carboxylic acid having 7 to 22 carbon atoms. Specifically, examples include dilauroyl PE, dimyristoyl PE, dipalmitoyl PE, dioleoyl PE, palmitoleoyl PE, and distearoyl PE. In addition, the fatty acid residues bonded to the 1st and 2nd positions of glycerol may be the same or different.

[0043] Examples of the PS include naturally derived PS such as soybean-derived PS and hydrogenated PS derived from soybean, and synthetic PS such as PS containing a saturated or unsaturated carboxylic acid having 7 to 22 carbon atoms. Specifically, examples include dilauroyl PS, dimyristoyl PS, dipalmitoyl PS, dioleoyl PS, palmitoleoyl PS, and distearoyl PS. The fatty acid residues bonded to the 1- and 2-positions of glycerol may be the same or different.

[0044] The tumor in the present invention is not particularly limited as long as it is a tumor whose growth is suppressed by administering the anti-tumor immune response enhancer of the present invention, but is preferably a malignant tumor; examples of malignant tumors include hematopoietic cell malignancies, head and neck cancer, brain tumors, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastrointestinal stromal tumors, mesothelioma, thyroid cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular tumors, bladder cancer, skin cancer, and anal cancer.

[0045] The anti-tumor immune response enhancer of the present invention can be used to inhibit the growth of tumors in a subject. Examples of the inhibition of tumor growth include: when the anti-tumor immune response enhancer of the present invention is administered, the degree of increase in tumor volume is reduced, the volume of the tumor is reduced, or the tumor disappears, compared to when the anti-tumor immune response enhancer of the present invention is not administered; the case where the degree of increase in tumor volume is reduced is not particularly limited, and examples include the following: when the tumor volume (mm 3)=1 / 2(tumor length×(tumor width)2), the tumor volume after a certain period of time after administration in the case where the anti-tumor immune response enhancer of the present invention is administered becomes smaller than that in the case where it is not administered; specifically, the following cases can be cited: the tumor volume in the case of administration is less than 4 / 5 of the tumor volume in the case of non-administration, preferably less than 3 / 5, and more preferably less than 1 / 2.

[0046] The anti-tumor immune response enhancer of the present invention can be used to convert immature dendritic cells into mature dendritic cells, preferably to convert immature dendritic cells into CD11b + CD11c + Living cells. Examples of immature dendritic cells include immature standard bone marrow dendritic cells and TADCs existing in vivo, but these cells can also be prepared in vitro.

[0047] The method for preparing the immature standard dendritic cells in vitro is not particularly limited as long as it is a known method, but the following method can be exemplified: collecting bone marrow cells from the femur and / or tibia of a mammal to be administered, such as mice, rats, sheep, pigs, cattle, cats, dogs, monkeys, and humans, and incubating the collected bone marrow cells in RPMI-1640 medium containing FBS, penicillin-streptomycin, sodium pyruvate, MEM non-essential amino acid solution, GlutaMAX-I, 2-mercaptoethanol, IL-4, and GM-CSF. In the case of mouse cells, the following method can be preferably used: immature standard dendritic cells derived from bone marrow are prepared by culturing in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin, 1% sodium pyruvate, 1% MEM non-essential amino acid solution, 1% GlutaMAX-I, 0.4% 50μM 2-mercaptoethanol, 10ng / mL mouse IL-4, and 10ng / mL GM-CSF to prepare immature standard dendritic cells derived from bone marrow.

[0048] As a method for preparing mature dendritic cells in vitro by culturing immature dendritic cells, the following method can be exemplified: immature dendritic cells collected from a subject are cultured in vitro in the presence of the anti-tumor immune response enhancer of the present invention; for example, the following method can be cited: culturing for 12 hours to 72 hours, preferably 24 hours to 72 hours, and more preferably 36 hours to 60 hours in a culture medium to which 0.1 to 10 mg / mL, preferably 0.5 to 5 mg / mL, more preferably 0.8 to 1.6 mg / mL, and particularly preferably 1.0 to 1.4 mg / mL of the anti-tumor immune response enhancer is added.

[0049] The anti-tumor immune response enhancer can be used to prepare mature dendritic cells by culturing immature dendritic cells collected from a subject in vitro, and can also be used to administer the mature dendritic cells prepared in vitro into the subject's body.

[0050] The method for preparing the above-mentioned TADC in vitro is not particularly limited as long as it is a known method for isolating TADC present in the tumor microenvironment. For example, the following method can be mentioned: a tumor removed from a mammal is washed with PBS, finely cut with a scalpel, the cut tumor tissue is further dispersed, a single-cell suspension is prepared, the tumor to become a single-cell suspension is filtered, washed with PBS, and mononuclear cells are isolated by centrifugation based on a Percoll gradient; the TADC prepared in this way can be evaluated in vitro for the degree of tumor growth inhibition, etc.

[0051] The above-mentioned tumor microenvironment includes the following environments: an in vivo environment in which cells other than malignant tumor (cancer) cells are contained in a solid tumor or coexist around a solid tumor, and various cells such as dendritic cells exist that have characteristics such as promoting tumor cell growth, promoting tumor metastasis, and / or being able to evade host immunity, thereby suppressing immunity to the tumor.

[0052] As a method for confirming that the anti-tumor immune response enhancer of the present invention has caused immature standard dendritic cells, TADC and other immature dendritic cells to become mature dendritic cells, the following method can be exemplified: when markers expressed by mature dendritic cells and / or activated cytokines are detected by a method such as flow cytometry that can perform multi-parameter analysis of the physical and chemical properties of each cell, it is determined that the detected cells have become mature dendritic cells, thereby confirming.

[0053] As a method for confirming that immature standard dendritic cells have matured by the anti-tumor immune response enhancer of the present invention, the following method can be cited: through the above-mentioned parameter analysis, it is determined that the expression of cell surface markers such as CD (Cluster of Differentiation) 11b, CD11c, IA / IE, CD80, CD40, etc. has increased significantly compared with the case where the anti-tumor immune response enhancer of the present invention is not administered (added), and / or it is determined that the production of cytokines such as interleukin (IL)-1β, IL-12, IL-6, etc. has increased significantly due to the maturation and activation of standard dendritic cells compared with the case where the anti-tumor immune response enhancer of the present invention is not administered (added), and the like, thereby confirming.

[0054] As a method for confirming that the TADC has been matured by the anti-tumor immune response enhancer of the present invention, the following method can be cited: by the above-mentioned parameter analysis, it is determined that the expression of cell surface markers such as CD11b, CD11c, CD86, IA / IE in CD45-positive living cells is significantly increased compared to the case where the anti-tumor immune response enhancer of the present invention is not given (added). In addition, the following method can be cited: by maturation and activation of standard dendritic cells, the production of cytokines such as IL-1β, IL-12, IFN-γ is significantly increased compared to the case where the anti-tumor immune response enhancer of the present invention is not given (added), etc., thereby confirming.

[0055] As a method for confirming that TADC has been matured by the anti-tumor immune response enhancer of the present invention, the following method may also be included: in TADC that is CD4-positive, the production of IFN-γ and IL-2 is significantly increased compared to the case where the anti-tumor immune response enhancer of the present invention is not administered (added), or the production of TNF-α is significantly reduced compared to the case where the anti-tumor immune response enhancer of the present invention is not administered (added).

[0056] In addition, as a method for confirming that TADC has been matured by the anti-tumor immune response enhancer of the present invention, the following method may also be included: in TADC that is CD8 positive, the production of IFN-γ, IL-2 and TNF-α is confirmed to be significantly increased compared to the case where the anti-tumor immune response enhancer of the present invention is not administered (added).

[0057] The subject to which the anti-tumor immune response enhancer of the present invention is administered is preferably a subject carrying a tumor, but even a subject not carrying a tumor can be administered as a preventive measure to prevent the occurrence of a (malignant) tumor.

[0058] The administration method of the anti-tumor immune response enhancer of the present invention is not particularly limited as long as the effect of the present invention can be exerted, but parenteral administration by subcutaneous injection, intramuscular injection, intravenous injection, etc. is preferred. In addition, when the location of a solid tumor can be determined, administration to the solid tumor, around the solid tumor, or near the solid tumor is preferred.

[0059] The dosage of the anti-tumor immune response enhancer of the present invention is not particularly limited as long as the effects of the present invention can be exerted and no serious side effects are caused; for example, when determining the dosage of the anti-tumor immune response enhancer of the present invention to humans, the dosage to humans can be determined by accumulating experimental data using a known method such as a HED conversion method that estimates a dose that exhibits an equivalent effect in humans based on the body surface area of ​​a test animal such as a mouse, or studying data such as the maximum blood concentration (Cmax) and the area under the time curve (AUC), as is commonly done in the art.

[0060] The anti-tumor immune response enhancer of the present invention can improve the anti-cancer effect of anti-cancer drugs by being used in combination with other anti-cancer drugs. As other anti-cancer drugs, alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine can be cited; metabolic antagonists such as pentostatin, fludarabine, cladribine, methotrexate, 6-mercaptopurine, and enocitabine; molecular targeted agents such as rituximab, cetuximab, and trastuzumab; kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; calcineurin inhibitors such as cyclosporine and tacrolimus; anti-cancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; and immunomodulators such as interferon, nivolumab, and pembrolizumab.

[0061] In the present specification, the terms "increased", "increased", "enhanced" or "activated" are used here to indicate a statistically significant increase in amount. In some embodiments, the terms "increased", "increased", "enhanced" or "activated (by increase)" can mean: an increase of at least 10% compared to the control level, for example, including an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or more, which is a 100% increase, or any increase between 10% and 100% compared to the reference (control) level, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more.

[0062] (Statistical Analysis Method)

[0063] The terms "statistically significant" or "significant" in this specification refer to statistical significance, and statistical significance can also be set as p<0.05.

[0064] The present invention will be described in more detail below by way of examples, but the technical scope of the present invention is not limited to these examples.

[0065] [Example]

[0066] [Example 1]

[0067] (Preparation of Lipid Composition)

[0068] The following solution was prepared: three phospholipids, phosphatidylcholine (PC) (Soy PC (95%), manufactured by Avanti Polar Lipids), phosphatidylethanolamine (PE) (Lipoid S PE, (purified phosphatidylethanolamine derived from soybeans, 98% or more) manufactured by Lipoid Co., Ltd.) and phosphatidylserine (PS) derived from soybeans (Lipamine (registered trademark) PS 90PN, manufactured by Nagase Chemical Technology Co., Ltd.) were dissolved in EtOH at a mass ratio of PC:PE:PS of 14:3:3. Specifically, 0.84 mg of PC, 0.18 mg of PE and 0.18 mg of PS were dissolved in 1 mL of ethanol to prepare a 1.2 mg / mL lipid composition cPLs adjuvant.

[0069] (Mouse)

[0070] 8-week-old female C57BL / 6NCrSlc mice (hereinafter referred to as "B6 mice") and 8-week-old female Balb / c mice were purchased from Japan SLC Co., Ltd. Each mouse was fed with filtered water and feed from 1 week before the experiment. The feeding method of mice followed the guidelines for experimental animal welfare of the National Center for Child Health and Research (NCCHD) of Japan.

[0071] (Preparation of cancer cell lines)

[0072] OVA-expressing mouse melanoma cells expressing firefly luciferase (hereinafter referred to as “MO4-Luc cells”) provided by Dr. Jun Fang of the Department of Pharmacy of Sojo University and C26 cells as a mouse colon cancer cell line were cultured in RPMI1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 5% CO. 2 The concentrations were cultured at 37°C.

[0073] (Statistical Analysis Method)

[0074] Each result shown in the embodiment is analyzed with mean ± standard deviation (SD). GraphPadPrism software (version 7.0, GraphPad Software) is used to analyze the data. In the comparison of two groups (normal distribution), a one-sided unpaired (in independent samples) or paired (in three independent experiments) Student's t test is used. In the comparison of two groups, Wilcoxon matched-pairs signed-rank test (abnormal distribution) is used. In the comparison of multiple groups, one-way ANOVA with Tukey's multiple comparison test is used. The term "significant" refers to statistical significance. The term "very significant" means that the significant difference is very large. Statistical significance is set to p < 0.05.

[0075] (Transplantation of MO4-Luc cells into mice)

[0076] The MO4-Luc cells cultured above were washed twice with PBS and resuspended in PBS to a volume of 3×10 6 The resuspended MO4-Luc cells were injected subcutaneously (3×10 5 / 100 μL sc) was injected into the shaved right abdomen of the above B6 mice to make them MO4-Luc cell-transplanted mice (day 0 after transplantation).

[0077] For the MO4-Luc cell transplanted mice, the length and width of the tumor were measured with a vernier caliper every 2 days starting from the 6th day after MO4-Luc cell transplantation, and the tumor volume was calculated. The calculation formula of tumor volume is: Tumor volume (mm 3 ) = 1 / 2 (tumor length × (tumor width) 2). On the 8th day after MO4-Luc cell transplantation, 100 mm 3 >Tumor volume >10mm 3 MO4-Luc cells were transplanted into mice and cPLs adjuvant was administered. That is, 4 mg / kg or 12 mg / kg of mouse body weight of cPLs adjuvant was injected into the peritoneal cavity on days 8, 11, and 14 after transplantation. In addition, mice not given cPLs adjuvant were used as control mice and tumor volume was measured in the same manner. Figure 1 (a) shows the change in tumor volume until 16 days after transplantation (**p<0.01, ****p<0.0001). Figure 1(b) shows a histogram of the tumor volume on day 16 after MO4-Luc melanoma transplantation expressed as mean ± SD by one-way ANOVA using Tukey's multiple comparison test (**p<0.01, ****p<0.0001).

[0078] (result)

[0079] from Figure 1 (a) It can be clearly seen that in MO4-Luc cell transplanted mice, the increase in tumor volume in mice given 12 mg / kg of cPLs adjuvant and 4 mg / kg of cPLs adjuvant was significantly smaller than that in untreated control mice. Figure 1 In (a), the tumor volume of mice given 12 mg / kg of cPLs adjuvant on day 16 after MO4-Luc cell transplantation was about 1 / 2 of the tumor volume of untreated control mice, which was significantly smaller. The tumor volume of mice given 12 mg / kg of cPLs adjuvant was about 3 / 5 of the tumor volume of mice given 4 mg / kg of cPLs adjuvant, which was significantly smaller.

[0080] from Figure 1 (b) It can be clearly seen that the tumor volume of mice given 12 mg / kg of cPLs adjuvant and untreated control mice on day 16 after MO4-Luc cell transplantation showed a very significant difference. In addition, a significant difference was also confirmed between the tumor volume of mice given 12 mg / kg of cPLs adjuvant and mice given 4 mg / kg of cPLs adjuvant.

[0081] The above results confirmed that an excellent tumor growth inhibitory effect was obtained by administration of cPLs adjuvant in MO4-Luc cell-transplanted mice.

[0082] [Example 2]

[0083] (Transplantation of C26 cells into mice)

[0084] The cultured C26 cells were washed twice with PBS and resuspended with PBS to a concentration of 3×106 cells / mL. The resuspended C26 cells were subcutaneously injected (3×105 / 100 μL subcutaneously (sc)) into the shaved right flank of the Balb / c mice to make C26 cell transplanted mice (day 0 after transplantation).

[0085] For the C26 cell-transplanted mice, the length and width of the tumor were measured using a vernier caliper every 2 days starting from the 6th day after transplantation, and the tumor volume was calculated in the same way as for the MO4-Luc cell-transplanted mice. 3 >Tumor volume >100mm 3 C26 cells were transplanted into mice, and 4 mg / kg or 12 mg / kg of cPLs adjuvant was injected into the peritoneal cavity on days 8, 11, and 14 after transplantation. Mice not given cPLs adjuvant were used as control mice, and tumor volume was measured in the same manner. Figure 1 (c) shows a graph of tumor volume changes (****p<0.0001). Figure 1 Histogram is shown in (d) (****p<0.0001).

[0086] from Figure 1 (c) It can be clearly seen that in C26 cell transplanted mice, the increase in tumor volume in mice given 12 mg / kg of cPLs adjuvant was significantly smaller than that in untreated control mice. Figure 1 In (c), the tumor volume of mice given 12 mg / kg of cPLs adjuvant on day 16 after C26 cell transplantation was approximately 1 / 4 of the tumor volume of untreated control mice, which was significantly smaller. Figure 1 (d) It is clear that when the tumor volumes of mice administered with 12 mg / kg of cPLs adjuvant were compared with those of untreated control mice on day 16 after C26 cell transplantation, the tumor volume of mice administered with cPLs adjuvant was significantly smaller.

[0087] The above results confirmed that an excellent tumor growth inhibitory effect was also obtained by administering cPLs as an adjuvant in C26 cell-transplanted mice.

[0088] [Example 3]

[0089] (Additive effect of cPLs adjuvant)

[0090] A PC solution prepared by dissolving 0.84 mg of PC in 1 mL of ethanol, a PE solution prepared by dissolving 0.18 mg of PE in 1 mL of ethanol, and a PS solution prepared by dissolving 0.18 mg of PS in 1 mL of ethanol were prepared respectively, and instead of the above-mentioned 12 mg / kg of cPLs adjuvant, 8.4 mg / kg of PC, 1.8 mg / kg of PE or 1.8 mg / kg of PS were administered to MO4-Luc cell-transplanted mice in the order of Example 1. Figure 1The graph in (e) shows the changes in tumor volume until day 16 after transplantation in each mouse (*p<0.05, **p<0.01).

[0091] Figure 2 shows the changes in tumor volume in MO4-Luc transplanted mice. Figure 1 (e) It is clear that in MO4-Luc-transplanted mice, the increase in tumor volume in mice given 12 mg / kg of cPLs adjuvant was smaller than that in untreated control mice and mice given PC, PE or PS alone. Figure 1 In (e), the tumor size of MO4-Luc mice given 12 mg / kg of cPLs adjuvant on the 16th day after transplantation was less than 1 / 2 of the tumor volume of the untreated control mice, which was significantly smaller. In addition, it was significantly smaller than the tumor volume of mice given PE, indicating the excellent anti-tumor effect of the cPLs adjuvant.

[0092] [Example 4]

[0093] [Phenotypic analysis of standard dendritic cells]

[0094] In order to study the effect of cPLs adjuvant on DC in more detail, the effect of cPLs adjuvant on bone marrow-derived immature conventional dendritic cells (DC) was first studied.

[0095] (Preparation of Immature Mouse Bone Marrow-Derived Dendritic Cells)

[0096] Bone marrow cells of femur and tibia were collected from 8-12 week old B6 mice, and immature standard mouse bone marrow dendritic cells (hereinafter also referred to as "imDC") were prepared from the collected bone marrow cells. That is, bone marrow cells derived from B6 mice were cultured for 5 days in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin (Gibco Thermo Fisher Scientific), 1% sodium pyruvate (Gibco Thermo Fisher Scientific), 1% MEM non-essential amino acid solution (Gibco Thermo Fisher Scientific), 1% GlutaMAX-I (Gibco Thermo Fisher Scientific), 0.4% 50 μM 2-mercaptoethanol (Wako Pure Chemical Industries, Ltd.), 10 ng / mL mouse IL-4 (PeproTech), and 10 ng / mL GM-CSF (BioLegend), thereby preparing imDC.

[0097] (Addition of cPLs adjuvant to mouse bone marrow-derived immature dendritic cells)

[0098] The above imDCs were cultured for another 2 days on a 24-well plate to which 1.2 mg / mL cPLs adjuvant was added, and the cells were harvested and counted. The cell survival rate was above 90%.

[0099] (Flow cytometry analysis of dendritic cells after cPLs adjuvant addition)

[0100] Cells cultured for 2 days in the presence of the above-mentioned cPLs adjuvant were subjected to flow cytometry analysis. The cells cultured for 2 days in the presence of the above-mentioned cPLs adjuvant were washed and suspended in PBS at an optimal concentration. For intracellular cytokine staining, 50 ng / mL of PMA (Phorbol 12-myristate 13-acetate) and 500 ng / mL of ionomycin (Sigma-Aldrich) were used to stimulate the cells at 37°C for 4 hours in the presence of Brefeldin A solution (manufactured by Thermo Fisher Scientific). Thereafter, in order to facilitate intracellular staining, the cells were permeabilized using an intracellular fixation / permeabilization buffer (cat#00-5523-00, manufactured by Thermo Fisher Scientific). The results of the analysis based on flow cytometry are shown in Figure 2 .

[0101] In the subsequent flow cytometry analysis, the cell analyzer LE-SP6800 (manufactured by Sony Corporation) or BD FACSymphony TM (manufactured by BD Biosciences) and FlowJo software (version 10.5.0; manufactured by BD Biosciences) were used for analysis.

[0102] (Staining Reagents)

[0103] Thereafter, the following reagents were used for intracellular staining.

[0104] In staining, LIVE / DEAD for 405 nm excitation was used. TM Fixable Aqua Dead Cell Stain Kit (L34957, manufactured by Thermo Fisher Scientific), mouse Fc blocking reagent (130-092-575, manufactured by Miltenyi), and the following anti-mouse monoclonal antibodies (all manufactured by BioLegend) that bind to Brilliant Violet 605, AlexaFluor 700, APC / Cyanine7, PE / Cyanine7, PE / Dazzle594, PerCP / Cy5.5, APC, PE, and FITC.

[0105] CD11b(M1 / 70),

[0106] CD11c(N418),

[0107] CD40 (3 / 23),

[0108] CD80 (16-10A1),

[0109] CD86(GL-1),

[0110] IA / IE(M5 / 114.15.2),

[0111] CD8(53-6.7),

[0112] CD4(RM4-5),

[0113] CD45(30-F11),

[0114] IL-10 (JES5-16E3),

[0115] IL-12 (C15.6),

[0116] IL-6 (MP5-20F3),

[0117] IL-1β(NJTEN3),

[0118] TNF-α(MP6-XT22),

[0119] IFN-γ(XMG1.2).

[0120] (result)

[0121] from Figure 2 It was clearly confirmed that after culturing the above imDCs in the presence of cPLs adjuvant for 2 days, they expressed CD11b expressed in standard dendritic cells (cDCs). + and CD11c + Thus, it was confirmed that immature mouse DCs became mature mouse bone marrow dendritic cells (mature BMDCs) by culturing in the presence of cPLs adjuvant, expressing CD11b + and CD11c + .

[0122] (Expression of markers)

[0123] exist Figure 3 In the figure, regarding the surface markers of the mature BMDCs, graphs are shown showing the expression of (a) IA / IE, (b) CD80, and (c) CD40 as delta values ​​of MFI.

[0124] (result)

[0125] Compared with imDCs not treated with cPLs adjuvant (horizontal axis: imDCs), mature BMDCs cultured for 2 days in the presence of cPLs adjuvant (horizontal axis: cPLs adjuvant) showed significantly increased expression of IA / IE, CD80, and CD40. In addition, CD86 was also found to have an increasing expression trend when imDCs were cultured for 2 days in the presence of cPLs adjuvant.

[0126] The above results show that imDCs fully matured into mature BMDCs in the presence of cPLs adjuvant, with increased expression of IA / IE antigens and CD40, and were able to exert an anti-tumor immune enhancement effect.

[0127] (Cytokine Production)

[0128] exist Figure 4 In the figure, graphs are shown showing the production of (a) IL-1β, (b) IL-12, and (c) IL-6 as delta values ​​of MFI for DCs that have become mature BMDCs after being cultured for 2 days in the presence of cPLs adjuvant.

[0129] (result)

[0130] It was confirmed that the production of IL-1β, IL-12, and IL-6 by imDCs cultured for 2 days in the presence of cPLs adjuvant (horizontal axis: cPLs adjuvant) was significantly increased compared with imDCs not treated with cPLs adjuvant (horizontal axis: imDC).

[0131] According to the above results, the secretion of IL-1β, a cytokine that activates T cells and promotes antigen recognition, increased, and the secretion of IL-12, a cytokine that induces Th1 cells and regulates inflammatory responses, increased. In addition, the secretion of IL-6, an inflammatory cytokine, increased. These results show that imDCs fully matured in the presence of cPLs adjuvant to become mature BMDCs, and the expression of IL-1β, IL-12, and IL-6 increased, which can exert anti-tumor immune enhancement effects.

[0132] [Example 5]

[0133] [Phenotypic analysis of tumor-associated dendritic cells (TADC)]

[0134] In order to evaluate the effect of cPLs adjuvant on TADCs resident in tumors in TILs, TILs were isolated from tumor tissues and markers expressed in TADCs were analyzed.

[0135] (Preparation of Tumor Infiltrating Leukocytes)

[0136] In Example 1, tumor-infiltrating leukocytes were prepared from tumors removed from MO4-Luc cell-transplanted mice that had been treated with cPLs adjuvant (12 mg / kg mouse body weight) or untreated mice 16 days after transplantation. The removed tumors were washed with PBS, finely cut with a scalpel, and the cut mouse tumor tissues were further dispersed using a Miltenyi Tumor Dissociation Kit (Tumor Dissociation Kit) (Cat No. 130-096-730, manufactured by Miltenyi Biotec) and a GentleMACS Octo dissociator (Cat No. 130-093-235, manufactured by Miltenyi Biotec) to prepare single cell suspensions. The tumor to be a single cell suspension was filtered using a 70 μM pre-separation filter (manufactured by ThermoFisher Scientific), washed with PBS, and centrifuged (600×g) based on a Percoll gradient (55% and 70%, GE Healthcare) at 20°C for 20 minutes to isolate mononuclear cells and prepare individual TILs.

[0137] (Analysis of surface markers of TADC contained in TILs)

[0138] According to the procedure described in Example 4 above, the markers expressed in TILs of MO4-Luc cell-transplanted mice given cPLs adjuvant (12 mg / kg) were analyzed by flow cytometry. The results are shown in Figure 5 .

[0139] (result)

[0140] according to Figure 5 Flow cytometry-based analysis clearly showed that TADC was presented as CD45 in TILs prepared from tumors removed from mice given cPLs adjuvant. + CD11b + CD11c + Therefore, it was confirmed that the mature dendritic cell markers similar to the mature standard dendritic cell markers confirmed in Example 4 above were expressed.

[0141] exist Figure 6 , graphs showing the expression of (a) CD86 and (b) IA / IE in terms of MFI delta values ​​are shown for TADC in the above TILs.

[0142] In TILs prepared from tumors removed from mice given cPLs adjuvant, it was confirmed that the expression of CD86 and IA / IE, which are maturation markers of dendritic cells, increased significantly. Therefore, this indicates that cPLs adjuvant promotes the maturation of TADC in TILs and can exert an anti-tumor immunity enhancement effect, indicating that cPLs adjuvant can fully mature dendritic cells even in the tumor microenvironment and can exert an anti-tumor immunity enhancement effect.

[0143] (Cytokine Production)

[0144] The functionality of T cells contained in TILs was evaluated from the perspective of cytokine production. Figure 7 Graphs showing the expression of cytokines (a) IL-1β, (b) IL-12, and (c) IFN-γ in terms of MFI delta values ​​are shown in FIG.

[0145] (result)

[0146] In the TILs of the above-mentioned cPLs adjuvant-treated group (horizontal axis: cPLs adjuvant), compared with the TILs of the cPLs adjuvant-untreated control group (horizontal axis: control), it was confirmed that the production of IL-1β, IL-12 and IFN-γ was significantly increased in TADC prepared from tumors removed from mice administered with cPLs adjuvant.

[0147] In TADCs prepared from tumors removed from mice given cPLs adjuvant, the secretion of IL-1β, IL-12, and IFN-γ, which are cytokines that recognize antigens and promote T cell activity, increased, confirming that the maturation of TADCs in TILs is promoted by cPLs adjuvant. Therefore, this suggests that cPLs adjuvants can fully mature dendritic cells even in the tumor microenvironment and can exert anti-tumor immunity enhancement effects.

[0148] The delta values ​​of MFI were calculated for the production of (a) IFN-γ, (c) TNF-α, and (e) IL-2 by CD49+ T cells contained in TILs, and (b) IFN-γ, (d) TNF-α, and (f) IL-2 by CD8+ T cells. The results are shown in FIG9 .

[0149] (result)

[0150] As can be clearly seen from Figure 9, in the CD4+ T cells of the cPLs adjuvant-treated group (horizontal axis: cPLs adjuvant), there was a significant difference in CD4+ T cells compared with those without cPLs adjuvant treatment. + Compared with T cells (horizontal axis: control), the production of IFN-γ and IL-2 was significantly higher. On the other hand, regarding TNF-α, the CD4 + The production of CD4 T cells was compared with the control group without cPLs adjuvant treatment. + T cells were significantly less.

[0151] The above results show that activated CD4 + Helper T cells (Th) increase the production of cytokines such as IL-2 and IFN-γ. Through the administration of cPLs adjuvant, CD4 + T cells are activated.

[0152] CD8 + T cells, compared with CD8 + Compared with T cells, the production of IFN-γ, IL-2 and TNF-α was significantly higher.

[0153] Confirmed in CD8 + In T cells, cPLs adjuvant enhanced the expression of IFN-γ, IL-2, and TNF-α, indicating that the anti-tumor immune effect in the tumor microenvironment was enhanced by the administration of cPLs adjuvant.

[0154] (Summarize)

[0155] This suggests that one of the mechanisms of immune tolerance release in the tumor microenvironment is the activation of T cells caused by the administration of cPLs adjuvant.

Claims

1. An anti-tumor immune response enhancer, characterized in that: Contains phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

2. The anti-tumor immune response enhancer according to claim 1, characterized in that For inhibiting tumor growth in a subject.

3. The anti-tumor immune response enhancer according to claim 1, characterized in that Used to convert immature dendritic cells into mature dendritic cells.

4. The anti-tumor immune response enhancer according to claim 3, characterized in that Used to convert immature dendritic cells into CD11b+CD11c+ living cells.

5. The anti-tumor immune response enhancer according to claim 4, characterized in that The immature dendritic cells are immature standard dendritic cells or tumor-associated dendritic cells.

6. The anti-tumor immune response enhancer according to claim 1 or 2, characterized in that: Contains 50% to 90% phosphatidylcholine, 5% to 25% phosphatidylethanolamine and 5% to 25% phosphatidylserine.

7. The anti-tumor immune response enhancer according to claim 1 or 2, characterized in that: It also contains one or more anticancer drugs.

8. A method for preparing mature dendritic cells, characterized in that: Mature dendritic cells are prepared by culturing immature dendritic cells in vitro in the presence of the anti-tumor immune response enhancer according to claim 1 or 2.

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