Composition for promoting immune checkpoint inhibition therapy

Abstract

The present invention addresses the problem of providing a composition for promoting immune checkpoint suppression therapy. Provided is a composition for promoting immune checkpoint inhibition therapy comprising an exopolysaccharide of lactic acid bacteria. A preferred example of the lactic acid bacteria is a bacterium of the genus Lactobacillus (Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1).

Description

[0001] This application is a divisional application of a Chinese patent application filed on June 13, 2019, with application number 201980039616.0 and invention name “Composition for Promoting Immune Checkpoint Inhibition Therapy”. Technical Field

[0002] The present invention relates to a composition containing a culture of lactic acid bacteria for promoting immune checkpoint inhibition therapy. Background Art

[0003] It is known that most tumors use the immunosuppressive function based on immune checkpoint molecules to avoid being attacked by the immune system. Immune checkpoint inhibition therapy is a therapy that relieves the activation inhibition of immune cells including T cells by inhibiting the transmission of immunosuppressive signals based on immune checkpoint molecules, thereby improving the patient's immune activity and enhancing the anti-tumor effect. So far, as immune checkpoint inhibitors, PD-1 (Programmed Death-1) inhibitory antibody drugs, CTLA-4 (Cytotoxic T-lymphocyte-associatedantigen 4) inhibitory antibody drugs, etc. have been approved for specific tumors.

[0004] The use of immune checkpoint inhibitors in combination with other active ingredients has also been studied. For example, Patent Document 1 discloses a combination of more than one anti-cancer antibody for treating cancer and a specific immune adjuvant compound, but records that the combined composition can be administered in combination with an antibody that blocks immunosuppressive function. In addition, Patent Document 2 discloses a peptide whose linear sequence consists of 9 amino acids as a compound for treating tumors by combining, sequentially administering, or separately administering with immune checkpoint inhibitors. Furthermore, Patent Document 3 discloses that a compound (e.g., an anti-SIRPa antibody) is used in combination with a second therapeutic agent, the compound being used to treat any condition that can be improved or prevented by differentiation of myeloid-derived suppressor cells (MDSC) into non-suppressor cells, and blocking the interaction between signal regulatory protein α (SIRPa) and at least one of its ligands. In addition, as a second therapeutic agent, it is recorded that a group consisting of chemotherapeutic agents, radiotherapy, surgery, immunotherapeutic agents, antibiotics, and probiotics can be selected.

[0005] On the other hand, lactic acid bacteria produce various substances during their fermentation process, one of which is exopolysaccharides (EPS). EPS of lactic acid bacteria are known to have some physiological activities, such as the prevention of autoimmune diseases (Patent Document 4), NK cell activation (Patent Document 5), the prevention of pneumococcal infection (Patent Document 6), the improvement of fatigue (Patent Document 7), and the suppression of the decline of acquired immune function based on anti-influenza drugs (Patent Document 8).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2012-211196

[0009] Patent Document 2: Japanese Patent Application Publication No. 2016-128399

[0010] Patent Document 3: Japanese Patent Application No. 2017-538669

[0011] Patent Document 4: Japanese Patent Application Publication No. 2000-247895

[0012] Patent Document 5: Japanese Patent Application Publication No. 2005-194259

[0013] Patent Document 6: International Publication No. WO2011 / 065300

[0014] Patent Document 7: International Publication No. WO2015 / 133638

[0015] Patent Document 8: International Publication No. WO2017 / 183595 Summary of the invention

[0016] Problem that the invention aims to solve

[0017] Immune checkpoint inhibition therapy activates the patient's original immunity to cause tumor regression, so it is less burdensome to the patient than conventional surgical resection and radiotherapy. It is further desired to have a composition that can promote the effect of this therapy.

[0018] Solutions for solving problems

[0019] The present inventors orally administered yogurt made by fermentation of Lactobacillus bacteria (Lactobacillus delbrueckii subspecies bulgaricus OLL1073 R-1) or exopolysaccharides obtained from its lactic acid bacteria to colorectal cancer model mice, and the results showed that the effects of CTLA-4 inhibition therapy and PD-1 inhibition therapy were enhanced, and tumor proliferation was observed to be inhibited. In addition, more CCR6-positive CD8+T cells were observed in the subjects. Based on such insights, the present invention was completed.

[0020] The present invention provides the following solutions.

[0021] [1] A composition for promoting immune checkpoint inhibition therapy, comprising exopolysaccharide of lactic acid bacteria.

[0022] [2] The composition according to 1, wherein the immune checkpoint inhibition therapy is CTLA-4 (cytotoxic T lymphocyte-associated antigen) inhibition therapy or PD-1 (programmed death-1) inhibition therapy.

[0023] [3] The composition according to 1 or 2, wherein the lactic acid bacteria are classified into the genus Lactobacillus.

[0024] [4] The composition according to any one of 1 to 3, wherein the lactic acid bacteria is classified as Lactobacillus delbrueckii subspecies bulgaricus.

[0025] [5] The composition according to any one of 1 to 4, which is fermented milk.

[0026] [6] A composition for increasing CCR6-positive CD8+T cells, comprising exopolysaccharide of lactic acid bacteria.

[0027] [7] A non-medical method for promoting immune checkpoint inhibition therapy, comprising the step of allowing a subject to ingest an effective amount of a composition containing exopolysaccharides of lactic acid bacteria.

[0028] [8] A non-medical method for increasing CCR6-positive CD8+ T cells in a subject, comprising the step of allowing the subject to ingest an effective amount of a composition containing exopolysaccharides of lactic acid bacteria.

[0029] [9] A method for increasing CCR6-positive CD8+ T cells, using exopolysaccharide of lactic acid bacteria (excluding medical treatment for humans).

[0030] In addition, the present invention also provides the following solutions.

[0031]

[10] A method for promoting immune checkpoint inhibition therapy in a subject, comprising the step of (orally) administering to the subject (causing the subject to ingest) an exopolysaccharide of lactic acid bacteria or a composition containing the same.

[0032]

[11] Use of an exopolysaccharide of lactic acid bacteria in the manufacture of a composition for promoting immune checkpoint inhibition therapy.

[0033]

[12] An exopolysaccharide of lactic acid bacteria or a composition comprising the same, for use in a method for promoting immune checkpoint inhibition therapy.

[0034]

[13] A non-therapeutic method for promoting immune checkpoint inhibition therapy in a subject, comprising the step of (orally) administering to the subject (causing the subject to ingest) an exopolysaccharide of lactic acid bacteria or a composition containing the same.

[0035]

[14] Application of an exopolysaccharide of lactic acid bacteria for promoting immune checkpoint inhibition therapy.

[0036]

[15] A method for increasing CCR6-positive CD8+ T cells in a subject, comprising the step of (orally) administering (causing the subject to ingest) an exopolysaccharide of lactic acid bacteria or a composition containing the same.

[0037]

[16] Use of an exopolysaccharide of lactic acid bacteria in the manufacture of a composition for increasing CCR6-positive CD8+T cells.

[0038]

[17] An exopolysaccharide of lactic acid bacteria or a composition containing the same, for use in a method for increasing CCR6-positive CD8+ T cells.

[0039]

[18] A non-therapeutic method for increasing CCR6-positive CD8+ T cells in a subject, comprising the step of (orally) administering (causing the subject to ingest) an exopolysaccharide of lactic acid bacteria or a composition containing the same.

[0040]

[19] A use of an exopolysaccharide of lactic acid bacteria for increasing CCR6-positive CD8+T cells.

[0041]

[20] A method for producing a composition for promoting immune checkpoint inhibition therapy or increasing CCR6-positive CD8+ T cells, comprising the step of mixing an exopolysaccharide of lactic acid bacteria and an additive acceptable as a drug.

[0042] Effects of the Invention

[0043] According to the present invention, immune checkpoint inhibition therapy can be effectively promoted. In addition, according to the present invention, CCR6-positive CD8+T cells can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A The tumor volume of the CTLA-4 inhibition therapy group (data for all individuals). The black solid line: yogurt-administered mice, the gray dotted line: control mice. The Student-t test was performed at the time of the final measurement of tumor volume, and the p value is shown.

[0045] Figure 1B The tumor volume of the CTLA-4 inhibition therapy group (both the yogurt administration group and the control group are the data of the lower half of the tumor volume). The black solid line: yogurt administration mice, the gray dotted line: control mice. The Student-t test was performed at the time of the final measurement of tumor volume to show the p value.

[0046] Figure 1C It is the CCR6 positive rate of CD8+T cells. The results are shown as mean±SE.

[0047] Figure 1D is the tumor volume of the PD-1 inhibition therapy group. The black solid line: yogurt-administered mice, the gray dotted line: control mice.

[0048] Figure 2A The tumor volume of mice (data for all individuals). Black solid line: EPS-administered mice, gray dotted line: control mice. Student-t test was performed at the time of the final measurement of tumor volume, and p values ​​are shown.

[0049] Figure 2B The tumor volume of mice (EPS-administered group and control group are both data of the lower half of the tumor volume). Black solid line: EPS-administered mice, gray dotted line: control mice. Student-t test was performed at the time of the final measurement of tumor volume, and p value is shown.

[0050] Figure 3 TG40 cells CCR6 positive rate (%) In the EPS-added system, the CCR6 positive rate increased significantly (P<0.001) compared with the control without EPS addition (Student-t test). DETAILED DESCRIPTION

[0051] Hereinafter, the present invention will be described in detail.

[0052] The present invention relates to a composition for promoting immune checkpoint inhibition therapy, which contains exopolysaccharide (EPS) produced by lactic acid bacteria as an effective ingredient.

[0053] [Active ingredients]

[0054] The composition of the present invention contains EPS of lactic acid bacteria as an effective ingredient.

[0055] Lactic acid bacteria is a general term for microorganisms that assimilate glucose and produce more than 50% lactic acid based on the yield of sugar. As a physiological property, it is a coccus or bacillus of Gram-positive bacteria, and has characteristics such as non-motility, no spore-forming ability, and catalase negativity. Lactic acid bacteria have been consumed worldwide through fermented milk and the like since ancient times, and can be said to be extremely safe microorganisms. Lactic acid bacteria are divided into multiple genera. The EPS of the lactic acid bacteria contained in the composition of the present invention is preferably produced by Lactobacillus lactic acid bacteria classified as the genus Lactobacillus.

[0056] As Lactobacillus lactic acid bacteria, for example, Bulgaria species, cheese species, acidophilus species, plant species, etc. can be listed. Among these Lactobacillus lactic acid bacteria, in the present invention, it is preferably separated into the lactic acid bacteria of Bulgaria species (also referred to as Lactobacillus bulgaricus). Furthermore, among the Lactobacillus lactic acid bacteria, it is more preferably classified as Lactobacillus delbrueckii subsp.bulgaricus. In a particularly preferred embodiment, the lactic acid bacteria is Lactobacillus delbrueckii subsp.bulgaricusOLL1073R-1 bacteria (Lactobacillus delbrueckii subsp.bulgaricusOLL1073 R-1) (preservation number: FERM BP-10741) (sometimes referred to as "Lactobacillus bulgaricus R-1 strain".).

[0057] The Lactobacillus bulgaricus R-1 strain was internationally deposited at the National Institute of Advanced Industrial Science and Technology, International Patent Organisms Depository (IPOD, NITE) (Room 120, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) based on the Budapest Treaty (depositor: Meiji Co., Ltd., deposit date: November 29, 2006, deposit number: FERM BP-10741).

[0058] The EPS used as an effective ingredient in the composition of the present invention is not particularly limited as long as it is produced by lactic acid bacteria (preferably lactic acid bacteria of the genus Lactobacillus classified as Lactobacillus) and has the target effect. The EPS produced by lactic acid bacteria is structurally classified as homopolysaccharides and heteropolysaccharides (for example, composed of galactose and glucose), and is sometimes modified by phosphorylation, sulfation, etc., but can all be used as an effective ingredient of the composition of the present invention. A preferred example is an acidic EPS containing a large number of phosphate groups. An example of such EPS is the EPS produced by Lactobacillus bulgaricus R-1 strain (see the above-mentioned patent document 6).

[0059] The EPS of lactic acid bacteria contained in the composition of the present invention can be contained in the form of lactic acid bacteria fermentation products. Lactic acid bacteria fermentation products include not only the fermentation products obtained from lactic acid bacteria themselves, but also their processed products. The lactic acid bacteria fermentation products themselves include, for example, fermented milk (specifically, yogurt, etc.). The processed products include, for example, crude purified products, culture filtrate obtained by filtering, centrifuging or sterilizing the fermentation products by membrane separation, culture supernatant, concentrate obtained by concentrating the culture filtrate / culture supernatant, and dried products of the concentrate.

[0060] The method for preparing EPS from lactic acid bacteria can refer to the above-mentioned Patent Document 6, etc. In addition, when the EPS from lactic acid bacteria is made into a lactic acid bacteria fermentation product, the lactic acid bacteria that produce EPS can be added as a starter to raw milk to ferment it, and EPS is produced in the fermentation product, thereby producing fermented milk containing EPS. The conditions during the fermentation, such as raw milk, fermentation temperature, and fermentation time, are not particularly limited as long as the lactic acid bacteria used can produce EPS, and those skilled in the art can set them appropriately.

[0061] [use]

[0062] The composition of the present invention can be used to promote immune checkpoint inhibition therapy. Promoting immune checkpoint inhibition therapy means: when used in combination with immune checkpoint inhibition therapy, obtaining the effect brought by immune checkpoint inhibition therapy earlier, improving the effect brought by immune checkpoint inhibition therapy, improving the response rate of immune checkpoint inhibition therapy, and obtaining the effect by administering a lower dose of immune checkpoint inhibitors. The effects brought by immune checkpoint inhibition therapy include, for example: killing cancer cells to shrink cancer (reducing tumor volume), slowing the growth of cancer (inhibiting the increase of tumor volume), curing cancer, preventing cancer metastasis / recurrence, and killing cancer cells that may have metastasized.

[0063] Immune checkpoint molecules are a group of molecules that suppress immune responses to the self in order to maintain immune homeostasis and to suppress excessive immune responses. Originally, they existed to suppress the overactivation of immune cells including T cells without attacking the self, but in the process of carcinogenesis, cancer cells use them to avoid attacks from the immune system and proliferate. Currently, various immune checkpoint molecules and their ligands have been identified.

[0064] As an example of immune checkpoint molecules, PD-1, CTLA-4, TIM-3, BTLA, LAG-3 / TCR can be listed, but the composition of the present invention can be used in any therapy for the inhibition of related immune checkpoint molecules. As an example of particularly preferred therapy, the therapy using CTLA-4 inhibitors and the therapy using PD-1 inhibitors can be listed. The example of CTLA-4 inhibitors is Ipilimumab (Yervoy), Avelumab (Avelumab), Atezolizumab (Atezolizumab), and the example of PD-1 inhibitors is Nivolumab (Opdivo), Keytruda (Keytruda), Pembrolizumab (Pembrolizumab). The composition of the present invention can be used for any immune checkpoint inhibition therapy using any of these.

[0065] It should be noted that low response rate is also a major issue of immune checkpoint inhibition therapy. The main reason is that there are some checkpoints (nodes, brake) from the time the patient's immune system functions to the actual damage to cancer cells. That is, even if one node can be released with one immune checkpoint inhibitor, due to the presence of other nodes, sufficient therapeutic effects cannot be obtained in most cases. The composition of the present invention has the effect of increasing certain cytotoxic T cells (CCR6 positive CD8+T cells). Therefore, it can be considered that among the checkpoints possessed by various immune cells, only by releasing a node possessed by cytotoxic T cells such as CTLA-4, PD-1, etc., the effect is greatly increased, and the therapeutic effect is clearly observed. Therefore, the composition of the present invention can also be expected to make up for the low response rate of immune checkpoint inhibition therapy.

[0066] Examples of cancers for which immune checkpoint inhibition therapy is used include malignant melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck tumors, gastric cancer, urothelial carcinoma, Merkel cell carcinoma, etc. The composition of the present invention can also be used for immune checkpoint inhibition therapy for any of these.

[0067] According to the present inventors, by giving the composition of the present invention, the increase of CCR6 positive CD8+T cells was observed in the tumor of the object, and the proliferation of the tumor was suppressed. In addition, when EPS was added to culture cell strain TG40 derived from T cells for cultivation, the CCR6 positive rate increased compared with the control without EPS. Thus, it is revealed that EPS has the possibility of directly increasing the CCR6 positive rate of T cells. That is, it is known that the composition of the present invention has the function of assisting in identifying and killing the CCR6 positive CD8+T cells of the cells (cancer cells, virus-infected cells, etc.) that become foreign bodies in the object (for example, the increase of the T cells, the induction promotion to foreign bodies, etc.). Therefore, the composition of the present invention can be used to increase CCR6 positive CD8+T cells in the body of the object, and can be used to increase the CCR6 positive CD8+T cells in the tumor in the object in addition. It should be noted that when it is called CCR6 positive rate, unless otherwise stated, it refers to the proportion of CCR6 positive cells in T cells. The CCR6 positivity rate can be calculated as (number of CD3+, CD8+, and CCR6-positive cells) / (number of CD3+ and CD8+ cells), and in an experimental system using TG40, it can be calculated as (number of CCR6-positive cells) / (total TG40 cells).

[0068] Even if immune checkpoint inhibition therapy is used together with other therapies for treating cancer, the composition of the present invention can also be used. Such other therapies include: surgery (surgical treatment), therapy based on drugs other than immune checkpoint inhibitors (e.g., metabolic antagonists, alkylating agents, anticancer antibiotics, microtubule inhibitors, etc.), including radiotherapy, a large amount of chemotherapy, hematopoietic stem cell transplantation after pre-transplantation treatment of radiotherapy for the whole body, immunotherapy other than immune checkpoint inhibition therapy (e.g., cytokine therapy, administration of immune activators, immune cell transplantation, etc.), alternative cancer therapies based on health foods and nutritional enhancers, etc.

[0069] [Composition]

[0070] (Food composition, etc.)

[0071] The composition of the present invention can be made into a food composition or a pharmaceutical composition. Unless otherwise stated, food and medicine are not only for humans, but also for animals other than humans. Unless otherwise stated, food includes general food, functional food, nutritional composition, and therapeutic food (for therapeutic purposes. Food cooked based on a menu prepared by a doctor's dietician, etc.), diet therapy food, formula food, nursing food, and treatment support food. Unless otherwise stated, food includes not only solid food, but also liquid food, such as beverages, beverages, liquid food, and soup. Functional food refers to food that can give a specified functionality to an organism, such as specific health food (including conditional special protection [specific health food]), functional labeled food, health functional food including nutritional functional food, special purpose food, nutritional supplement food, health supplement food, nutritional enhancer (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquid preparations and other various dosage forms of food), beauty food (for example, weight loss food), etc., all health foods. In addition, the "functional food" in the present invention includes health foods with health claims based on applicable food standards of the Codex (FAO / WHO Joint Food Standards Commission).

[0072] (Target)

[0073] The composition of the present invention is suitable for ingestion / administration to a subject who has received immune checkpoint inhibition therapy or a subject who is scheduled to receive immune checkpoint inhibition therapy.

[0074] (Route of administration)

[0075] The composition of the present invention can be administered parenterally, for example, via a tube (gastrostomy, enterostomy), or nasally or orally, but is preferably administered orally.

[0076] (Content / dosage of active ingredient)

[0077] The content of EPS of lactic acid bacteria in the composition of the present invention is an amount that can exert the target effect. For the composition, taking into account various factors such as the age, weight, symptoms of the subject, the dosage or intake can be appropriately set, and the amount of EPS of lactic acid bacteria in the daily dose can be set to 0.1 mg or more, preferably 0.6 mg or more, and more preferably 3 mg or more. The upper limit of the EPS amount in the daily dose can be set to 480 mg or less, preferably 360 mg or less, more preferably 180 mg or less, and particularly preferably 90 mg or less in any case.

[0078] The amount of EPS per dose or per meal, i.e., per dose of lactic acid bacteria, can be set, for example, to 0.03 mg or more, preferably to 0.2 mg or more, and more preferably to 1 mg or more. The upper limit of the amount of EPS per dose can be set to 160 mg or less, preferably to 120 mg or less, more preferably to 60 mg or less, and particularly preferably to 30 mg or less, when the lower limit is any.

[0079] When the EPS of lactic acid bacteria in the composition of the present invention is used as fermented milk, the daily dose of the fermented milk can be, for example, 10 g or more, preferably 30 g or more, and more preferably 60 g or more. The upper limit of the daily dose of the fermented milk can be, for example, 1500 g or less, preferably 1200 g or less, and more preferably 600 g or less, when the lower limit is any.

[0080] The dosage of fermented milk per dose or per meal, i.e., one dose can be, for example, 3 g or more, preferably 10 g or more, and more preferably 20 g or more. The upper limit of the dosage of fermented milk can be, for example, 800 g or less, preferably 400 g or less, and more preferably 200 g or less, while the lower limit is arbitrary.

[0081] The composition can be administered / ingested once a day, or multiple times a day, for example, three times per meal. The composition contains EPS of lactic acid bacteria with rich dietary experience as an active ingredient. Therefore, the composition of the present invention can be repeatedly or long-term ingested, and can be continuously administered / ingested, for example, for more than 1 week, preferably more than 4 weeks.

[0082] (Other ingredients, additives)

[0083] The composition of the present invention may contain other effective ingredients and nutrients that are permitted as food or medicine. Examples of such ingredients are amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid and niacin), minerals (e.g., copper, zinc, iron, cobalt, manganese), antibiotics, dietary fiber, protein, lipids, etc.

[0084] In addition, the composition may further contain food or additives permitted as medicines. Examples of such additives are inactive carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH regulators, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, water, other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevioside, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

[0085] (Dosage form / form)

[0086] The pharmaceutical composition of the present invention can be prepared into solid preparations such as tablets, granules, powders, pills, capsules, etc. suitable for oral administration; liquid preparations such as solutions, suspensions, slurries, etc.; and any dosage forms such as gels and aerosols.

[0087] The food composition of the present invention can be made into any form such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule, etc. In addition, the food composition of the present invention can be made into any form such as dairy products, nutritional fortifiers, candies, beverages, beverages, flavoring agents, processed foods, side dishes, soups, etc. More specifically, the composition of the present invention can be made into forms such as milk beverages, refreshing drinks, fermented milk, yogurt, ice cream, tablets, cheese, bread, biscuits, crackers, pizza, formula milk powder, liquid food, patient food, nutritious food, frozen food, processed food, etc., and can also be made into forms such as granules, powders, pastes, concentrates, etc., which are mixed in beverages and foods for ingestion.

[0088] (other)

[0089] In the manufacture of the composition of the present invention, the compounding stage of the EPS of lactic acid bacteria can be appropriately selected. As long as the characteristics of the EPS of lactic acid bacteria are not significantly damaged, the compounding stage is not particularly limited. For example, it can be mixed into the raw materials for compounding in the initial stage of manufacture. Or when the composition of the present invention is implemented in the form of fermented milk, the lactic acid bacteria that produce EPS are added as a starter to the raw milk to ferment it to produce EPS, thereby being able to manufacture fermented milk containing EPS.

[0090] The composition of the present invention may be labeled with content that can be used to promote immune checkpoint inhibition therapy, and may also be labeled with content that is recommended for ingestion for specific subjects. Labeling may be direct or indirect, and examples of direct labeling include records on tangible objects such as the product itself, packaging, containers, labels, and logos, and examples of indirect labeling include advertising / promotional activities based on places or means such as web pages, stores, brochures, exhibitions, books, newspapers, magazines, television, radio, mail, email, and audio.

[0091] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples.

[0092] Example

[0093] <Preparation of Yogurt and EPS (Exopolysaccharide)>

[0094] In Example 1, Lactobacillus delbrueckii subsp.bulgaricus OLL1073R-1 and Streptococcus thermophilus were added as lactic acid bacteria starters to a skim milk powder medium, and the skim milk powder medium was fermented for 2.5 hours at a temperature of 43°C to prepare yogurt, which was used. In Examples 2 and 3, EPS was purified from a culture obtained by culturing Lactobacillus delbrueckii subsp.bulgaricus OLL1073R-1 in a 10% by mass skim milk powder medium. That is, trichloroacetic acid was added to the culture obtained by culturing at 37°C for 18 hours to remove denatured proteins in a final concentration of 10% by mass, and cold ethanol was added and allowed to stand at 4°C for 2 hours to obtain a precipitate containing EPS. The solution was dialyzed with MilliQ water using a dialysis membrane (molecular weight cutoff 6-8 kDa) to decompose nucleic acid and protein by enzymatic decomposition, and then ethanol precipitation was performed again to obtain a precipitate, which was dissolved in MilliQ water, dialyzed again, and freeze-dried to purify EPS.

[0095] <Example 1>

[0096] A total of 32 BALB / c mice, male, 7 weeks old (CHARLES RIVER LABORATORIES JAPAN, INC.) were divided into control mice (n=16) and yogurt-administered mice (n=16). Control mice were given 10% (w / v) skim milk powder, and yogurt-administered mice were given yogurt, and 400 μL / mouse were forced to be orally administered. Oral administration was implemented once a day until the day before the autopsy. It should be noted that when quantified by HPLC using a corona charged particle detector, the amount of EPS in 400 μL of yogurt was 28 μg.

[0097] On the day of oral administration, a single cell suspension of colon26, a mouse colon cancer-derived cell line, was prepared (suspended in PBS), and 6.0×10 5 Cells / 200 μL were transplanted subcutaneously into the left abdomen of all mice. Furthermore, the mice in each group (n=16) were further divided into two groups according to the treatment method, one group was the CTLA-4 inhibition therapy group (n=8), and the other group was the PD-1 inhibition therapy group (n=8). In the CTLA-4 inhibition therapy group, each therapeutic drug (anti-mouse CTLA-4 antibody or anti-mouse PD-1 antibody) was intraperitoneally administered 3 times (150 μg / mouse / time) within 10 days after tumor transplantation, and in the PD-1 inhibition therapy group, each therapeutic drug (anti-mouse CTLA-4 antibody or anti-mouse PD-1 antibody) was intraperitoneally administered 5 times (240 μg / mouse / time) within 18 days after tumor transplantation.

[0098] The course of the disease was monitored by measuring tumor volume with a ruler three times a week. In the CTLA-4 inhibition therapy group, tumors were dissected 15 days after transplantation, and tumor-infiltrating T cells were analyzed and compared. In the PD-1 inhibition therapy group, tumor volume exceeded 2000mm 3 Euthanasia was carried out one by one at the time due to humanitarian checkpoints.

[0099] In the CTLA-4 inhibition therapy group, no significant difference was observed in the tumor volume of the two mice on the closest dissection day ( Figure 1A ), but when the tumor volume was half of the low value, which is considered to have a therapeutic effect of the drug, the tumor volume of the yogurt-treated mice was significantly smaller than that of the control mice ( Figure 1B ).

[0100] In addition, the collected tumor mass was cut into small pieces with a scalpel and cultured in RPMI1640 medium containing collagenase (Roche) and DNase (Roche) (37°C, 1 hour) to prepare a single cell suspension. The cell group was stained with fluorescently labeled antibody MIX containing anti-mouse CD3 antibody, anti-mouse CD8 antibody, and anti-mouse CCR6 antibody (all from BD, Japan), and tumor-infiltrating T cells were analyzed using a flow cytometer (FACS Verse, BD, Japan).

[0101] As a result, CCR6-positive CD8+ T cells increased significantly in yogurt-administered mice ( Figure 1C ). Thus, it was confirmed that in the yogurt-administered mice, cellular immunity in cancer tissues was activated and tumor growth was suppressed.

[0102] In the PD-1 inhibition therapy group, the course of tumor growth in two mice was observed. Only in the yogurt-treated mice group were tumor growth significantly suppressed during the drug administration period (~18 days after tumor transplantation) ( Figure 1D ).

[0103] The above results show that, in CTLA-4 inhibition therapy or PD-1 inhibition therapy, oral ingestion of the yogurt of the present invention or EPS contained therein can enhance the therapeutic effect.

[0104] <Example 2>

[0105] A total of 24 7-week-old male BALB / c mice (CHARLES RIVER LABORATORIES JAPAN, INC.) were divided into control mice (n=12) and EPS-administered mice (n=12). The control mice were allowed to drink distilled water freely until the day of autopsy, and the EPS-administered mice were allowed to drink EPS aqueous solution (25 μg / ml) freely (intake of about 4-8 mL / day was confirmed).

[0106] On the day of free drinking, a colon26 single cell suspension (suspended in PBS) was prepared, and 6.0×10 5 Cells / 200 μL were transplanted subcutaneously into the left abdomen of all mice. Furthermore, anti-mouse CTLA-4 antibody was intraperitoneally administered (150 μg / mouse / time) three times within 10 days after tumor transplantation to all mice. Tumor volume was measured three times a week and the course of the disease was observed. All mice were dissected 15 days after tumor transplantation.

[0107] Comparison of tumor volumes of the two mice on the day of autopsy showed that the EPS-administered group had a significantly smaller tumor volume ( Figure 2A), and when the tumor volume was half of the low value, more significant tumor growth inhibition was observed ( Figure 2B ).

[0108] The above results show that, in CTLA-4 inhibition therapy or PD-1 inhibition therapy, oral ingestion of the yogurt of the present invention or EPS contained therein can enhance the therapeutic effect.

[0109] <Example 3>

[0110] EPS (150 ug / ml) was added to the cultured cell line TG40 (mouse T cell origin) and co-cultured for 6 hours. A culture medium was prepared by adding 10% by mass fetal bovine serum (FBS) to RPMI. 6 The cells were seeded in a well plate at 37°C and 5% CO2 concentration.

[0111] The cells were stained with a fluorescently labeled anti-mouse CCR6 antibody and analyzed using a flow cytometer (FACS Verse, BD Japan).

[0112] As a result, the CCR6 positive rate in the EPS-added system was significantly (P<0.001) increased compared with the control without EPS (Student-t test) ( Figure 3 ). This result suggests that EPS also directly increases CCR6-positive CD8+ T cells in addition to those in tumors.

Claims

1. Use of an exopolysaccharide of lactic acid bacteria in the manufacture of a composition for increasing CCR6-positive CD8+T cells.

2. The use according to claim 1, wherein: The exopolysaccharide is a processed product of a lactic acid bacteria fermentation product, and the processed product is one or more selected from a crude purified product, a culture filtrate, a culture supernatant, a culture filtrate concentrate, a culture supernatant concentrate, and a dried product of a concentrate.

3. The use according to claim 1, wherein: Lactic acid bacteria are classified into the genus Lactobacillus.

4. The use according to claim 1, wherein: The lactic acid bacteria are classified as Lactobacillus delbrueckii subsp. bulgaricus.

5. The use according to claim 1, wherein: The composition is a fermentation product obtained by using Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus.

6. The use according to claim 1, which is for ingestion by a subject who has received immune checkpoint inhibition therapy and / or a subject who is scheduled to receive immune checkpoint inhibition therapy.

7. A non-therapeutic method for promoting immune checkpoint inhibition therapy, which uses exopolysaccharides of lactic acid bacteria.

8. The method according to claim 7, wherein: Lactic acid bacteria are classified into the genus Lactobacillus.

9. The method according to claim 7, wherein: The lactic acid bacteria are classified as Lactobacillus delbrueckii subsp. bulgaricus.

10. Use of an exopolysaccharide of lactic acid bacteria in the manufacture of a composition for promoting immune checkpoint inhibition therapy.

11. Use of an exopolysaccharide of lactic acid bacteria for preparing a composition for ingestion by a subject who has received immune checkpoint inhibition therapy and / or a subject who is scheduled to receive immune checkpoint inhibition therapy.

12. The use according to claim 10 or 11, wherein: The exopolysaccharide is a processed product of a lactic acid bacteria fermentation product, and the processed product is one or more selected from a crude purified product, a culture filtrate, a culture supernatant, a culture filtrate concentrate, a culture supernatant concentrate, and a dried product of a concentrate.

13. The use according to claim 10 or 11, wherein: Lactic acid bacteria are classified into the genus Lactobacillus.

14. The use according to claim 10 or 11, wherein: The lactic acid bacteria are classified as Lactobacillus delbrueckii subsp. bulgaricus.

15. The use according to claim 10 or 11, wherein: The composition is for ingestion by a subject who has received immune checkpoint inhibition therapy and / or a subject who is scheduled to receive immune checkpoint inhibition therapy.

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