Antitumor immune response enhancer

Abstract

To provide means for maintaining and enhancing the anti-tumor activity of TADC in a tumor microenvironment.SOLUTION: It was found that when a composition, "cPLs adjuvant," containing three types of phospholipids is administered, the degree of growth of a tumor transplanted into a mouse is remarkably reduced. It was also confirmed that immature dendritic cells are cultured in vitro in the presence of the cPLs adjuvant, and then become mature myeloid dendritic cells and induce the production of cytokines.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antitumor immune response enhancer, and more particularly to an antitumor immune response enhancer comprising phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. [Background technology]

[0002] Dendritic cells (DCs) are known to be the most potent antigen-presenting cells that can prime both naive T cells and memory T cells and induce antigen-specific antitumor immunity (see, for example, Non-Patent Document 1). However, DCs in the tumor microenvironment, i.e., tumor-associated dendritic cells (TADCs), are known to exhibit low expression of costimulatory molecules and immature phenotypes (see, for example, Non-Patent Document 2), or to exhibit suppressive and dysfunctional phenotypes, which can contribute to cancer cells escaping the host's immune surveillance network (see, for example, Non-Patent Document 3), thus potentially causing immunosuppression or immune tolerance. Furthermore, there are reports that TADCs secrete various types of cytokines that suppress the activation of antitumor-type T cells and promote tumor cell proliferation (see, for example, Non-Patent Document 4). Therefore, it is known that in the tumor microenvironment, DCs can be a double-edged sword, having both positive and negative effects on the antitumor response (see, for example, Non-Patent Document 5), and it is said that conventional cancer immunotherapy may result in reduced antitumor activity.

[0003] On the other hand, a method for activating tumor-infiltrating lymphocytes (TILs) has been proposed, which involves administering a fermentation composition produced through fermentation in a culture medium of symbiotic microorganisms to subjects requiring TIL activation (see, for example, Patent Document 1). Furthermore, compositions for ex vivo dendritic cell activation containing one or more lipids having at least one cationic lipid and at least one antigen (see, for example, Patent Document 2), and the use of all-trans retinoic acid injections characterized by enabling the reduction of activity of abnormal myeloid-derived immunosuppressive cells, induction of differentiation of myeloid-derived immunosuppressive cells, and suppression of tumor growth and recurrence in tumor patients (see, for example, Patent Document 3). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2021-517587 [Patent Document 2] Japanese Patent Publication No. 2022-36961 [Patent Document 3] Special Publication No. 2019-528315 [Non-patent literature]

[0005] [Non-Patent Document 1] Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature. 2007;449(7161):419-26 [Non-Patent Document 2] Oncotarget. 2016;7(39):63204-14 [Non-Patent Document 3] J Cancer. 2013;4(1):36-44 [Non-Patent Document 4] J Leukoc Biol. 2017;102(2):317-324 [Non-Patent Document 5] Frontiers in Oncology 2013 Volume 3 Article 90:1-12

Summary of the Invention

Problems to be Solved by the Invention

[0006] The problem of the present invention is to provide a means for maintaining and enhancing the antitumor activity of TADC in the tumor microenvironment.

Means for Solving the Problems

[0007] The present inventors focused on the microenvironment in mouse tumors and continued to examine various components in order to maintain and enhance the antitumor activity of TADC, which is considered to be immature DC. However, after the tumor transplanted into the mouse reached a predetermined size, a composition containing three types of phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) (Combined PhosphoLipids adjuvant: cPLs adjuvant) (hereinafter also referred to as "cPLs adjuvant") was administered, and it was found that the degree of growth of the tumor transplanted into the mouse was significantly suppressed. In addition, when immature DC was cultured in vitro in the presence of cPLs adjuvant, it was confirmed that such cultured DC became mature bone marrow-derived dendritic cells (BMDCs) and induced the production of inflammatory cytokines. Furthermore, tumor tissue was excised from cancer cell transplanted mice administered with cPLs adjuvant, and the characteristics of tumor infiltrating leukocytes in the tumor tissue were analyzed. As a result, it was confirmed that markers expressed by mature DC were expressed, the production of T cells was promoted, and the production of inflammatory cytokines was promoted, leading to the completion of the present invention.

Effects of the Invention

[0008] By administering the antitumor immune response enhancer of the present invention, it is possible to suppress the degree of tumor growth, mature immature bone marrow-derived dendritic cells and TADCs, and also to mature immature dendritic cells in vitro by culturing them in the presence of the antitumor immune response enhancer.

[0009] That is, the present invention is as follows. [1] An antitumor immune response enhancer containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. [2] The antitumor immune response enhancer according to [1] above, for use in suppressing tumor growth in a subject. [3] The antitumor immune response enhancer according to [1] above, for use in converting immature dendritic cells into mature dendritic cells. [4] Immature dendritic cells into CD11b + CD11c + The antitumor immune response enhancer according to [3] above, for use in making viable cells. [5] The antitumor immune response enhancer according to [4] above, wherein the immature dendritic cells are immature standard dendritic cells or tumor-associated dendritic cells. [6] The antitumor immune response enhancer according to [1] or [2] above, characterized in that phosphatidylcholine is formulated at 50 to 90%, phosphatidylethanolamine at 5 to 25%, and phosphatidylserine at 5 to 25%. [7] The antitumor immune response enhancer according to [1] or [2] above, further comprising one or more anticancer agents. [8] A method for preparing mature dendritic cells by culturing immature dendritic cells in vitro in the presence of the antitumor immune response enhancer according to [1] or [2] above.

[0010] Furthermore, the present invention relates to an agent comprising phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine for use in antitumor immune response enhancement therapy; a cytokine production promoter comprising phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; a combination of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine used to enhance the antitumor immune response; a method for enhancing the antitumor immune response in a subject, comprising administering an antitumor immune response enhancer comprising phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to the subject; and a method for enhancing the antitumor immune response using phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine as required to enhance the antitumor immune response. A method for enhancing an antitumor immune response, comprising the step of administering to a subject; a method for activating tumor-associated dendritic cells in a subject, comprising the step of administering an antitumor immune response enhancer containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to a subject requiring enhancement of the antitumor immune response in the tumor microenvironment; a method for administering an agent that reduces the immunosuppressiveness of the tumor microenvironment in a subject and enhances clinical utility; and cancer immunotherapy using mature dendritic cells activated by an antitumor immune response enhancer, tumor-associated dendritic cells activated by an antitumor immune response enhancer, mature dendritic cells activated by an antitumor immune response enhancer, or cancer immunotherapy using tumor-associated dendritic cells activated by an antitumor immune response enhancer. [Brief explanation of the drawing]

[0011] [Figure 1](a) is a graph showing the changes in tumor volume up to 16 days post-transplantation in mice that received a subcutaneous injection of 4 mg / kg mouse body weight or 12 mg / kg mouse body weight of cPLs adjuvant in the flank after transplantation of MO4-Luc cells, and in control mice that did not receive cPLs adjuvant. The horizontal axis represents the number of days after transplantation, and the vertical axis represents the tumor volume in MO4-Luc cell transplanted mice. (b) is a histogram of tumor volume at 16 days post-transplantation in mice that received a subcutaneous injection of 4 mg / kg mouse body weight or 12 mg / kg mouse body weight of cPLs adjuvant, and in control mice that did not receive cPLs adjuvant. (c) is a graph showing the changes in tumor volume up to 16 days post-transplantation in mice that received a subcutaneous injection of 12 mg / kg mouse body weight of cPLs adjuvant in the flank after transplantation of C26 cells, and in control mice that did not receive cPLs adjuvant. (d) is a histogram of tumor volume at 16 days post-transplant in mice administered cPLs adjuvant at 12 mg / kg mouse body weight and in control mice not administered cPLs adjuvant. (e) is a graph showing the changes in tumor volume up to 16 days post-transplant in MO4-Luc cell transplanted mice administered cPLs adjuvant, PC, PE, and PS, respectively. [Figure 2] This shows flow cytometry analysis of standard mouse immature bone marrow dendritic cells cultured in the presence of cPLs adjuvants. [Figure 3] These graphs show the expression of (a) IA / IE, (b) CD80, and (c) CD40 surface markers after culturing standard mouse bone marrow dendritic cells in the presence of cPLs adjuvants, expressed as the mean fluorescence intensity (MFI) delta (Δ) value. [Figure 4] The graphs below show the expression of (a) IL-1β, (b) IL-12, and (c) IL-6 in imDCs cultured for 2 days in the presence of cPL adjuvants, expressed as MFI delta values. [Figure 5]Both (a) and (b) show flow cytometry analysis of TIL in the cPLs adjuvant-treated group. [Figure 6] The graphs below show (a) the expression of CD86 and (b) the expression of IA / IE in the cPL adjuvant-treated group of TILs, expressed as the delta value of MFI. [Figure 7] This graph shows the expression of (a) IL-1β, (b) IL-12, and (c) IFN-γ in the cPL adjuvant-treated group, expressed as the delta value of MFI. [Figure 8] This graph shows the production of (a) IFN-γ, (c) TNF-α, and (e) IL-2 in CD4+ T cells, and the production of (b) IFN-γ, (d) TNF-α, and (f) IL-2 in CD8+ T cells, expressed as the delta value of MFI. [Modes for carrying out the invention]

[0012] The antitumor immune response enhancer of the present invention is not particularly limited as long as it is an agent capable of enhancing the antitumor immune response and contains phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Examples include agents that do not contain phospholipids other than PC, PE, and PS, or agents that do not contain phospholipids other than PC, PE, and PS as essential components. Examples of animals to which the above antitumor immune response enhancer can be administered include mammals such as mice, rats, sheep, pigs, cattle, cattle, dogs, monkeys, and humans.

[0013] As an example of a method for producing the antitumor immune response enhancer of the present invention, a method can be described in which PC, PE, and PS are dissolved and mixed in a solvent sequentially or as a mixture to produce the antitumor immune response enhancer. As the solvent, examples include solvents that can achieve the effects of the present invention and are not invasive to the target, but ethanol is a suitable example. The concentration of the solution can be 0.5 to 2 mg / mL, preferably 0.8 to 1.5 mg / mL, for PC, PE, and PS. The mixing ratio of PC, PE, and PS contained in such a solution can be 50 to 90% PC, 5 to 25% PE, and 5 to 25% PS, with 60 to 80% PC, 10 to 20% PE, and 10 to 20% PS being preferred, and 65 to 75% PC, 12.5 to 17.5% PE, and 12.5 to 17.5% PS being more preferred.

[0014] Examples of the above-mentioned polysaccharides (PCs) include naturally derived PCs such as soybean-derived PCs and egg yolk PCs, as well as synthetic PCs containing saturated or unsaturated carboxylic acids with 7 to 22 carbon atoms. Specific examples of synthetic PCs include dilauroyl PC, dimyristoyl PC, dioleoyl PC, dipalmitoyl PC, palmitooleoyl PC, and distearoyl PC. Furthermore, the fatty acid residues attached to positions 1 and 2 of glycerol may be the same or different.

[0015] Examples of the above-mentioned PE include naturally derived PE such as soybean-derived PE and soybean-derived hydrogenated PE, as well as synthetic PE such as PE containing saturated or unsaturated carboxylic acids with 7 to 22 carbon atoms. Specifically, examples include dilauroyl PE, dimyristoyl PE, dipalmitoyl PE, dioleoyl PE, palmitooleoyl PE, and distearoyl PE. Furthermore, the fatty acid residues attached to the 1st and 2nd positions of glycerol may be the same or different.

[0016] Examples of the above-mentioned PS include naturally derived PS such as soybean-derived PS and soybean-derived hydrogenated PS, as well as synthetic PS such as polyphosphate (PS) containing saturated or unsaturated carboxylic acids with 7 to 22 carbon atoms. Specifically, examples include dilauroyl PS, dimyristoyl PS, dipalmitoyl PS, dioleoyl PS, palmitooleoyl PS, and distearoyl PS. Furthermore, the fatty acid residues attached to positions 1 and 2 of glycerol may be the same or different.

[0017] The tumors used in the present invention are not particularly limited as long as their growth is suppressed by administering the antitumor immune response enhancer of the present invention, but malignant tumors are preferred. Examples of malignant tumors include hematopoietic cell malignancies, head and neck cancers, brain tumors, breast cancers, endometrial cancers, cervical cancers, ovarian cancers, esophageal cancers, gastric cancers, appendiceal cancers, colorectal cancers, liver cancers, gallbladder cancers, bile duct cancers, pancreatic cancers, kidney cancers, adrenal cancers, gastrointestinal stromal tumors, mesotheliomas, thyroid cancers, lung cancers, osteosarcomas, bone cancers, prostate cancers, testicular tumors, bladder cancers, skin cancers, and anal cancers.

[0018] The antitumor immune response enhancer of the present invention can be used to suppress tumor growth in a target. Suppression of tumor growth can include reducing the degree of increase in tumor volume, decreasing the tumor volume, or eliminating the tumor when the antitumor immune response enhancer of the present invention is administered, compared to when the antitumor immune response enhancer of the present invention is not administered. There are no particular limitations on cases in which the degree of increase in tumor volume is reduced, but for example, tumor volume (mm) 3 ) = 1 / 2 (tumor length × (tumor width) 2 When calculating as follows, one example is when the tumor volume after a predetermined time has elapsed since administration of the antitumor immune response enhancer of the present invention is smaller than the tumor volume when not administered. Specifically, one example is when the tumor volume when administered is 4 / 5 or less, preferably 3 / 5 or less, and more preferably 1 / 2 or less, of the tumor volume when not administered.

[0019] The antitumor immune response enhancer of the present invention can be used to convert immature dendritic cells into mature dendritic cells, preferably by converting immature dendritic cells to CD11b + CD11c + These cells can be used to produce living cells. Examples of immature dendritic cells include immature standard bone marrow dendritic cells and TADCs that exist in vivo, but these cells can also be prepared in vitro.

[0020] The method for preparing the above-mentioned immature standard dendritic cells in vitro is not particularly limited as long as it is a known method, but involves collecting bone marrow cells from the femur and / or tibia of mammals to be administered, such as mice, rats, sheep, pigs, cattle, cats, dogs, monkeys, and humans, and culturing the collected bone marrow cells in RPMI-1640 medium containing FBS, penicillin streptomycin, sodium pyruvate, and MEM non-essential amino acid solution, GlutaMAX-I, 2-mercaptoethanol, IL-4, and GM-CSF, thereby producing bone A method for preparing immature standard dendritic cells derived from bone marrow can be exemplified. In the case of mouse cells, a suitable method is to prepare immature standard dendritic cells derived from bone marrow by culturing them 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% 50 μM 2-mercaptoethanol, 10 ng / mL mouse IL-4, and 10 ng / mL GM-CSF.

[0021] As a method for preparing mature dendritic cells in vitro by culturing immature dendritic cells, an example can be given of culturing immature dendritic cells collected from a subject in vitro in the presence of the antitumor immune response enhancer of the present invention. For example, this can be done by culturing the cells in a culture medium containing 0.1 to 10 mg / mL, preferably 0.5 to 5 mg / mL, more preferably 0.8 to 1.6 mg / mL, and even more preferably 1.0 to 1.4 mg / mL of the antitumor immune response enhancer for 12 to 72 hours, preferably 24 to 72 hours, and more preferably 36 to 60 hours.

[0022] The above-mentioned antitumor immune response enhancer can be used to prepare mature dendritic cells by culturing immature dendritic cells collected from a subject in vitro, and the mature dendritic cells prepared in vitro can also be administered into the subject's body.

[0023] 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, a tumor excised from a mammal's body is washed with PBS, finely cut with a scalpel, the cut tumor tissue is further dispersed to prepare a single-cell suspension, the single-cell suspension is filtered, washed with PBS, and mononuclear cells are isolated by centrifugation using a Percoll gradient. The TADC thus prepared can be evaluated in vitro for the degree of tumor growth inhibition, etc.

[0024] The above-mentioned tumor microenvironment refers to an in vivo environment in which cells other than malignant tumor (cancer) cells are contained within a solid tumor or coexist around a solid tumor, and in which 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, and in which immunity against the tumor is suppressed.

[0025] As a method for confirming that the antitumor immune response enhancer of the present invention transforms immature dendritic cells such as immature standard dendritic cells and TADCs into mature dendritic cells, an example of such a method is to determine that the test cells have become mature dendritic cells by detecting markers expressed by mature dendritic cells or activated cytokines using means that can perform multiple parameter analyses of the physical and chemical characteristics of individual cells, such as flow cytometry.

[0026] Methods for confirming that immature standard dendritic cells have matured with the antitumor immune response enhancer of the present invention include determining, through the above-mentioned parameter analysis, that the expression of cell surface markers such as CD (Cluster of Differentiation) 11b, CD11c, IA / IE, CD80, and CD40 is significantly increased compared to when the antitumor immune response enhancer of the present invention is not administered (added), and / or determining that the production of cytokines such as interleukin (IL)-1β, IL-12, and IL-6 is significantly increased compared to when the antitumor immune response enhancer of the present invention is not administered (added) due to the maturation and activation of standard dendritic cells.

[0027] Methods for confirming that TADCs have matured with the antitumor immune response enhancer of the present invention include determining, through the above-mentioned parameter analysis, that the expression levels of cell surface markers such as CD11b, CD11c, CD86, and IA / IE in CD45 living cells are significantly increased compared to cases where the antitumor immune response enhancer of the present invention is not administered (added). Alternatively, confirmation can be obtained by determining that the production levels of cytokines such as IL-1β, IL-12, and IFN-γ are significantly increased compared to cases where the antitumor immune response enhancer of the present invention is not administered (added) due to the maturation and activation of standard dendritic cells.

[0028] A method for confirming that TADCs have matured with the antitumor immune response enhancer of the present invention may further include confirming that the production of IFN-γ and IL-2 in CD4-positive TADCs is significantly increased compared to when the antitumor immune response enhancer of the present invention is not administered (added), and that the production of TNF-α is significantly decreased compared to when the antitumor immune response enhancer of the present invention is not administered.

[0029] Furthermore, a method for confirming that TADCs have matured with the antitumor immune response enhancer of the present invention may further include confirming that the production of IFN-γ, IL-2, and TNF-α in CD8-positive TADCs is significantly increased compared to when the antitumor immune response enhancer of the present invention is not administered (added).

[0030] The target recipients of the antitumor immune response enhancer of the present invention are preferably those carrying tumors, but it can also be administered to non-tumor-carrying subjects as a preventive measure to prevent the development of (malignant) tumors.

[0031] The manner in which the antitumor immune response enhancer of the present invention is administered is not particularly limited as long as it achieves the effects of the present invention, but parenteral administration by subcutaneous injection, intramuscular injection, intravenous injection, etc. is preferred, and if the location of the solid tumor can be identified, administration to the solid tumor, the area around the solid tumor, or the vicinity of the solid tumor is preferred.

[0032] The dosage of the antitumor immune response enhancer of the present invention is not particularly limited, as long as it produces the effects of the present invention and does not cause serious side effects. For example, when determining the dosage of the antitumor immune response enhancer of the present invention for humans, the dosage for humans can be determined after accumulating experimental data using known methods, such as the HED conversion method which estimates the dose that produces an equivalent effect in humans from the body surface area of ​​test animals such as mice, or by examining data on peak blood concentration (Cmax) and area under the time curve (AUC), as is commonly done in the art.

[0033] The antitumor immune response enhancer of the present invention can enhance the anticancer effect of other anticancer agents when used in combination with them. Other anticancer drugs include alkylating agents such as cyclophosphamide, bendamustine, eosfamide, and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 6-mercaptopurine, and enocitabine; molecular targeted agents such as rituximab, cetuximab, and trastuzumab; kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubidine, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum-based drugs such as cisplatin, oxaliplatin, and carboplatin; and immunosuppressants such as interferon, nivolumab, and pembrolizumab.

[0034] In this specification, the terms “increased,” “enhance,” “enhance,” or “activate” are all used to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “enhance,” “enhance,” or “(an action by increasing) is activated” may mean an increase of at least 10% compared to the control level, e.g., 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 than an increase of 100%, or any increase between 10% and 100% compared to the reference (control) level, or an increase of 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.

[0035] (Statistical analysis method) In this specification, the terms "statistically significant" or "significantly significant" refer to statistical significance, which can also be defined as p<0.05.

[0036] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Examples]

[0037] [Example 1] (Preparation of lipid composition) Solutions were prepared by dissolving three types of 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 Corporation), and soybean-derived phosphatidylserine (PS) (Lipamine® PS 90 PN, manufactured by Nagase ChemteX Corporation)—in ethanol in 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.

[0038] (mouse) Eight-week-old female C57BL / 6NCrSlc mice (hereinafter referred to as "B6 mice") and eight-week-old female Balb / c mice were purchased from SLC Japan Co., Ltd. Each mouse was fed filtered water and food for one week prior to the experiment. The mouse rearing method followed the guidelines for laboratory animal welfare set forth by the National Center for Child Health and Development (NCCHD).

[0039] (Preparation of cancer cell lines) OVA-expressing mouse melanoma cells expressing firefly luciferase (hereinafter referred to as "MO4-Luc cells"), provided by Dr. Jun Fang of the Faculty of Pharmaceutical Sciences, Sojo University, and C26 cells, a mouse colon cancer cell line, were cultured at 37°C under a 5% CO2 concentration in RPMI1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific).

[0040] (Statistical analysis method) Each result shown in the examples was analyzed as the mean ± standard deviation (SD). The data were analyzed using GraphPad Prism software (version 7.0, manufactured by GraphPad Software). For the comparison of two groups (normal distribution), one-sided unpaired (in independent samples) or paired (in three independent experiments) Student's t-test was used. For the comparison of two groups, one-sided Wilcoxon matched-pairs signed-rank test was used (abnormal distribution). For the comparison of multiple groups, one-way analysis of variance (One-way ANOVA) with Tukey's multiple comparison test was used. The term "significantly" refers to statistical significance. The term "remarkably" means that the significant difference is very large. Statistical significance was set at p < 0.05.

[0041] (Transplantation of MO4-Luc cells into mice) The cultured MO4-Luc cells were washed twice with PBS and resuspended in PBS at a concentration of 3×10 6 cells / mL. The resuspended MO4-Luc cells were subcutaneously injected (3×10 5 / 100 μL s.c.) into the shaved right flank of the above B6 mice to obtain MO4-Luc cell transplanted mice (day 0 after transplantation).

[0042] For the above MO4-Luc cell transplanted mice, the tumor volume was calculated by measuring the tumor length and width using calipers every two days from 6 days after MO4-Luc cell transplantation. The formula for calculating the tumor volume was 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 > 10 mm 3We selected MO4-Luc cell transplanted mice and administered cPLs adjuvant. Specifically, 4 mg / kg mouse body weight or 12 mg / kg mouse body weight of cPLs adjuvant was injected intraperitoneally on days 8, 11, and 14 after MO4-Luc cell transplantation. Mice that did not receive cPLs adjuvant were used as control mice, and tumor volume was measured in the same manner. Figure 1(a) shows a graph (**p<0.01, ****p<0.0001) showing the change in tumor volume up to 16 days after transplantation, and Figure 1(b) shows a histogram (**p<0.01, ****p<0.0001) of the tumor volume at 16 days after MO4-Luc melanoma transplantation, expressed as mean ± SD using one-way ANOVA with Tukey's multiple comparison test.

[0043] (result) As is clear from Figure 1(a), in MO4-Luc cell transplanted mice, the increase in tumor volume in mice administered 12 mg / kg mouse body weight of cPLs adjuvant and mice administered 4 mg / kg mouse body weight of cPLs adjuvant was significantly smaller than the increase in tumor volume in untreated control mice. In Figure 1(a), the tumor volume of mice administered 12 mg / kg mouse body weight of cPLs adjuvant 16 days after MO4-Luc cell transplantation was approximately half the tumor volume of untreated control mice, which was remarkably small. The tumor volume of mice administered 12 mg / kg mouse body weight of cPLs adjuvant was approximately 3 / 5 the tumor volume of mice administered 4 mg / kg mouse body weight of cPLs adjuvant, which was significantly smaller.

[0044] As is clear from Figure 1(b), a significant difference in tumor volume was observed between mice treated with 12 mg / kg of cPLs adjuvant and untreated control mice 16 days after MO4-Luc cell transplantation. A significant difference in tumor volume was also observed between mice treated with 12 mg / kg of cPLs adjuvant and mice treated with 4 mg / kg of cPLs adjuvant.

[0045] These results confirm that administration of cPLs adjuvants in MO4-Luc cell-transplanted mice results in excellent tumor growth inhibition.

[0046] [Example 2] (Transplantation of C26 cells into mice) The cultured C26 cells described above were washed twice with PBS and then 3 × 10⁶ cells were removed. 6 The cells were resuspended in PBS to a concentration of cells / mL. The resuspended C26 cells were subcutaneously injected (3 × 10) into the shaved right flank of the Balb / c mice. 5 (100 μL s.c.) was used to transplant C26 cells into mice (0 days post-transplant).

[0047] For the C26 cell-transplanted mice described above, the tumor length and width were measured every two days starting from day 6 after C26 cell transplantation using calipers, and the tumor volume was calculated in the same manner as for the MO4-Luc cell-transplanted mice described above. On day 8 after cell transplantation, the tumor was 100 mm 3 > Tumor volume > 10 mm 3 C26 cell-transplanted mice were selected, and 4 mg / kg mouse body weight or 12 mg / kg mouse body weight of cPLs adjuvant was injected intraperitoneally on days 8, 11, and 14 post-transplant. Mice that did not receive cPLs adjuvant were used as control mice, and tumor volume was measured in the same manner. Similar to Example 1, a graph showing the change in tumor volume over time (****p<0.0001) is shown in Figure 1(c), and a histogram (****p<0.0001) is shown in Figure 1(d).

[0048] (result) As is clear from Figure 1(c), in C26 cell-transplanted mice, the increase in tumor volume in mice administered 12 mg / kg of cPLs adjuvant was significantly smaller than the increase in tumor volume in untreated control mice. In Figure 1(c), the tumor volume of mice administered 12 mg / kg of mouse body weight of cPLs adjuvant 16 days after C26 cell transplantation was approximately 1 / 4 of the tumor volume of untreated control mice, which was remarkably smaller. Furthermore, as is clear from Figure 1(d), a comparison of tumor volumes between mice administered 12 mg / kg of cPLs adjuvant and untreated control mice 16 days after C26 cell transplantation showed that the tumor size was significantly smaller in mice administered with cPLs adjuvant.

[0049] These results confirm that, in C26 cell-transplanted mice, administration of cPLs adjuvants also resulted in excellent tumor growth inhibition.

[0050] [Example 3] (Synergistic effect of cPL adjuvants) A PC solution was prepared by dissolving 0.84 mg of PC in 1 mL of ethanol, a PE solution was prepared by dissolving 0.18 mg of PE in 1 mL of ethanol, and a PS solution was prepared by dissolving 0.18 mg of PS in 1 mL of ethanol. Instead of the 12 mg / kg mouse body weight cPLs adjuvant mentioned above, 8.4 mg / kg of PC, 1.8 mg / kg mouse body weight of PE, or 1.8 mg / kg mouse body weight of PS was administered to MO4-Luc cell-transplanted mice, respectively, following the procedure of Example 1. The changes in tumor volume up to 16 days post-transplant in each mouse are shown in the graph in Figure 1(e) (*p<0.05, **p<0.01).

[0051] As is clear from Figure 1(e), which shows the changes in tumor volume in MO4-Luc transplanted mice, the increase in tumor volume in MO4-Luc cell transplanted mice administered 12 mg / kg mouse body weight of cPLs adjuvant was smaller than that in untreated control mice or in mice administered PC, PE, or PS individually. Furthermore, in Figure 1(e), the tumor size 16 days post-transplant in MO4-Luc mice administered 12 mg / kg mouse body weight of cPLs adjuvant was significantly smaller, less than half the tumor volume of untreated control mice, and significantly smaller than that of mice administered PE, demonstrating the excellent antitumor effect of cPLs adjuvant.

[0052] [Example 4] [Phenomena analysis of standard dendritic cells] To investigate the effects of cPLs adjuvants on DCs in more detail, we first examined the effects of cPLs adjuvants on immature, conventional dendritic cells (DCs) derived from bone marrow.

[0053] (Preparation of immature mouse bone marrow-derived dendritic cells) Bone marrow cells were collected from the femur and tibia of 8-12 week old B6 mice, and immature, standard-type mouse bone marrow dendritic cells (imDCs) were prepared from the collected bone marrow cells. Specifically, imDCs were prepared by culturing B6 mouse-derived bone marrow cells 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).

[0054] (Addition of cPLs adjuvant to immature dendritic cells derived from mouse bone marrow) The above imDCs were cultured for an additional 2 days on a 24-well plate to which 1.2 mg / mL of cPLs adjuvant was further added. Cells were then collected and the number of cells was counted. The cell viability was over 90%.

[0055] (Flow cytometry analysis of dendritic cells after addition of cPLs adjuvant) Cells cultured for 2 days in the presence of the above cPLs adjuvant were analyzed by flow cytometry. After washing, the cells cultured for 2 days in the presence of the above cPLs adjuvant were suspended in PBS at the optimal concentration. For intracellular cytokine staining, the cells were stimulated at 37°C for 4 hours with 50 ng / mL of PMA (Phorbor 12-myristate 13-acetate) and 500 ng / mL of ionomycin (Sigma-Aldrich) in the presence of Brefeldin A solution (Thermo Fisher Scientific). Subsequently, to facilitate intracellular staining, the cells were permeabilized using intracellular immobilization and permeabilization buffer (cat#00-5523-00, Thermo Fisher Scientific). The results of the flow cytometry analysis are shown in Figure 2.

[0056] From this point forward, flow cytometry analysis was performed using either the LE-SP6800 cell analyzer (manufactured by Sony Corporation), BD FACSymphony™ (manufactured by BD Biosciences), or FlowJo software (version 10.5.0; manufactured by BD Biosciences).

[0057] (Staining reagents) From this point onward, the following reagents were used for intracellular staining. LIVE / DEAD TM The following anti-mouse monoclonal antibodies (all from BioLegend) that bind to one of the following antibodies were used for staining: Fixable Aqua Dead Cell Stain Kit, for 405nm excitation (L34957, Thermo Fisher Scientific), mouse Fc blocking reagent (130-092-575, Milteny), and Brilliant Violet605, Alexa Fluor 700, APC / Cyanine7, PE / Cyanine7, PE / Dazzle594, PerCP / Cy5.5, APC, PE, or FITC. CD11b (M1 / 70), CD11c(N418), CD40 (3 / 23), CD80 (16-10A1), CD86 (GL-1), IA / IE (M5 / 114.15.2), CD8 (53-6.7), CD4 (RM4-5), CD45 (30-F11), IL-10 (JES5-16E3), IL-12 (C15.6), IL-6 (MP5-20F3), IL-1β(NJTEN3), TNF-α (MP6-XT22), IFN-γ(XMG1.2).

[0058] (result) As is clear from Figure 2, the above imDCs were cultured for 2 days in the presence of cPLs adjuvants, and then CD11b expressed in standard dendritic cells (cDCs) was observed. + CD11c + It was confirmed that it was expressed. Therefore, immature mouse DCs, when cultured in the presence of cPLs adjuvants, become mature mouse bone marrow dendritic cells (mature BMDCs), and CD11b + CD11c + It was confirmed that it expresses [the condition].

[0059] (Marker expression) Figure 3 shows graphs representing the expression of (a) IA / IE, (b) CD80, and (c) CD40 surface markers of the mature BMDC described above, expressed as MFI delta values.

[0060] (result) 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 compared to imDCs without cPLs adjuvant treatment (horizontal axis: imDC). Furthermore, CD86 expression also showed an increasing trend when imDCs were cultured for 2 days in the presence of cPLs adjuvant.

[0061] These results demonstrate that in the presence of cPLs adjuvants, imDCs mature sufficiently into mature BMDCs, increasing the expression of IA / IE antigens and CD40, and thus exhibiting an antitumor immune-enhancing effect.

[0062] (Cytokine production) Figure 4 shows graphs representing the production of (a) IL-1β, (b) IL-12, and (c) IL-6 in dendritic cells (DCs) that matured into BMDCs after being cultured for 2 days in the presence of the cPL adjuvant described above, expressed as the delta value of MFI.

[0063] (result) imDCs cultured for 2 days in the presence of cPLs adjuvants (horizontal axis: cPLs adjuvant) showed a significant increase in the production of IL-1β, IL-12, and IL-6 compared to imDCs without cPLs adjuvant treatment (horizontal axis: imDC).

[0064] The results above indicate that the secretion of IL-1β, a cytokine that activates T cells and promotes antigen recognition, increased, as did the secretion of IL-12, a cytokine that induces Th1 cells and modulates inflammatory responses. Furthermore, the secretion of IL-6, an inflammatory cytokine, also increased. These results demonstrate that in the presence of cPL adjuvants, imDCs mature sufficiently into mature BMDCs, increasing the expression of IL-1β, IL-12, and IL-6, and thus exhibiting an antitumor immune-enhancing effect.

[0065] [Example 5] [Phenomorphic analysis of tumor-associated dendritic cells (TADCs)] To evaluate the effect of cPLs adjuvants on TADCs (transcatheter arterial ducts) commensal to tumor tissue in TILs, TILs were isolated from tumor tissue, and markers expressed on TADCs were analyzed.

[0066] (Preparation of tumor-infiltrating leukocytes) In Example 1, tumor-infiltrating leukocytes were prepared from tumors excised 16 days after transplantation in MO4-Luc cell-transplanted mice that had been administered cPLs adjuvant (12 mg / kg mouse body weight) or untreated mice that had not been administered cPLs adjuvant. The excised tumors were washed with PBS, finely dissected with a scalpel, and the dissected mouse tumor tissue was further dispersed using the Miltenyi Tumor Dissociation Kit (Cat No. 130-096-730, Miltenyi Biotech) and GentleMACS Octo dissociator (Cat No. 130-093-235, Miltenyi Biotech) to prepare single-cell suspensions. The tumor, now a single-cell suspension, was filtered through a 70 μM pre-separation filter (Thermo Fisher Scientific), washed with PBS, and isolated mononuclear cells by centrifugation (600 × g) using a Percoll gradient (55% and 70%, GE Healthcare) at 20°C for 20 minutes, after which each TIL was prepared.

[0067] (Analysis of surface markers of TADC contained in TILs) Markers expressed in TILs of MO4-Luc cell-transplanted mice administered with cPLs adjuvant (12 mg / kg) were analyzed by flow cytometry using the procedure described in Example 4 above. The results are shown in Figure 5.

[0068] (result) As is evident from the flow cytometry analysis in Figure 5, in TILs prepared from tumors excised from mice administered with cPLs adjuvants, TADC was found to be CD45 + CD11b + CD11c +The cells were shown as living cells. Therefore, it was confirmed that they expressed markers for mature dendritic cells, similar to the markers for mature standard dendritic cells confirmed in Example 4 above.

[0069] Figure 6 shows graphs representing (a) CD86 expression and (b) IA / IE expression in the above TILs, expressed as MFI delta values.

[0070] In TILs prepared from tumors excised from mice administered the above-mentioned cPLs adjuvant, a significant increase in the expression of CD86 and IA / IE, which are dendritic cell maturation markers, was confirmed. Therefore, this indicates that the cPLs adjuvant promotes the maturation of TADCs in TILs and can exert an antitumor immune-enhancing effect, suggesting that the cPLs adjuvant enables dendritic cells to mature sufficiently in the tumor microenvironment and exert an antitumor immune-enhancing effect.

[0071] (Cytokine production) The functionality of T cells included in TILs was evaluated from the perspective of cytokine production. Figure 7 shows graphs representing the expression of the cytokines (a) IL-1β, (b) IL-12, and (c) IFN-γ as MFI delta values.

[0072] (result) In the TILs of the cPLs adjuvant-treated group (horizontal axis: cPLs adjuvant), it was confirmed that the production of IL-1β, IL-12, and IFN-γ was significantly increased in TILs prepared from tumors excised from mice administered the cPLs adjuvant compared to TILs of the control group without cPLs adjuvant treatment (horizontal axis: control).

[0073] In TADCs prepared from tumors excised from mice administered the above-mentioned cPLs adjuvant, the secretion of cytokines that promote antigen recognition and T cell activity, namely IL-1β, IL-12, and IFN-γ, was increased. This confirmed that cPLs adjuvant promoted the maturation of TADCs in TILs. Therefore, it was suggested that cPLs adjuvant enabled dendritic cells to mature sufficiently even in the tumor microenvironment, thereby achieving an antitumor immune-enhancing effect.

[0074] CD4 included in TILs + In T cells, (a) production of IFN-γ, (c) TNF-α, and (e) IL-2, and CD8 + The delta values ​​of MFI were calculated for the production of (b) IFN-γ, (d) TNF-α, and (f) IL-2 in T cells. The results are shown in Figure 9.

[0075] (result) As is clear from Figure 9, the CD4 of the cPLs adjuvant treatment group + In T cells (horizontal axis: cPLs adjuvant), CD4 cells are shown in the same size as those untreated with cPLs adjuvant. + Compared to T cells (horizontal axis: control), the production of IFN-γ and IL-2 was significantly higher in the cPLs adjuvant-treated group (CD4). On the other hand, for TNF-α, the CD4 cells in the cPLs adjuvant-treated group were significantly higher. + The amount produced in T cells was compared to CD4 in the cPLs adjuvant-free control group. + The number of T cells was significantly lower compared to other cells.

[0076] The results above indicate that activated CD4+ helper T cells (Th) increase the production of cytokines such as IL-2 and IFN-γ, and that administration of cPLs adjuvants enhances CD4 production in the tumor microenvironment. + The study showed that T cells were activated.

[0077] CD8 of the cPLs adjuvant treatment group + In T cells, CD8 cells were not treated with cPLs adjuvant. +Compared to T cells, the production of IFN-γ, IL-2, and TNF-α was significantly higher.

[0078] CD8 + In T cells, cPLs adjuvants were confirmed to enhance the expression of IFN-γ, IL-2, and TNF-α. This suggests that administration of cPLs adjuvants enhances the antitumor immune effect in the tumor microenvironment.

[0079] (summary) One mechanism for the release of immune tolerance in the tumor microenvironment was suggested to be T cell activation induced by cPL adjuvant administration.

Claims

1. An antitumor immune response enhancer comprising phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.

2. An antitumor immune response enhancer according to claim 1, for use in suppressing tumor growth in a target.

3. An antitumor immune response enhancer according to claim 1, for use in converting immature dendritic cells into mature dendritic cells.

4. CD11b in immature dendritic cells ＋ CD11c ＋ An antitumor immune response enhancer according to claim 3, for use in producing viable cells.

5. The antitumor 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. An antitumor immune response enhancer according to claim 1 or 2, characterized in that it contains 50-90% phosphatidylcholine, 5-25% phosphatidylethanolamine, and 5-25% phosphatidylserine.

7. The antitumor immune response enhancer according to claim 1 or 2, further comprising one or more anticancer agents.

8. A method for preparing mature dendritic cells by culturing immature dendritic cells in vitro in the presence of an antitumor immune response enhancer according to claim 1 or 2.

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