Composition for the prevention or treatment of cancer or inflammation containing a novel strain of Leuconostoc mesenteroides, and method for the prevention or treatment of cancer or inflammation using the same.

Abstract

The present invention provides compositions for the prevention, improvement, or treatment of cancer or inflammation. [Solution] A composition for the prevention, improvement, or treatment of cancer or inflammation is provided, comprising a novel Leuconostoc mesenteroides strain, its culture, or components derived therefrom.

Description

[Technical Field]

[0001] This application relates to a novel Leuconostoc mesenteroides strain, a culture thereof, or a composition for the prevention, improvement, or treatment of cancer or inflammation, an immunoenhancing composition containing the same, and a method for the prevention or treatment of cancer or inflammation using the same. [Background technology]

[0002] Cancer is the growth of abnormal cells caused by diverse changes in gene expression, which invades and destroys adjacent tissues, metastasizes to distant locations, and ultimately leads to death. Cancer has a high mortality rate globally and is the second most common cause of death in Western societies after cardiovascular disease. In particular, the Westernization of diets, the widespread consumption of high-fat foods, the rapid increase in environmental pollutants, and the increase in alcohol consumption have led to a sustained increase in colorectal cancer, lung cancer, breast cancer, and other cancers.

[0003] On the other hand, lactic acid bacteria are widely distributed in the oral cavity, intestines, vagina, and feces of humans and animals, as well as in fermented foods such as yogurt, cheonggukjang, and kimchi, and are closely related to human and animal health. Lactic acid bacteria have been reported to exhibit a variety of health-promoting effects, including regulating bowel function, suppressing harmful bacteria, regulating the immune system, lowering blood cholesterol, and anti-cancer effects (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2022 / 103138 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] This application provides a novel Leuconostoc mesenteroides strain, a composition for the prevention, improvement, or treatment of cancer or inflammation comprising its culture or components derived therefrom, an immunoenhancing composition comprising the same, and a method for the prevention or treatment of cancer or inflammation using the same. [Means for solving the problem]

[0006] One object of this application is to provide a pharmaceutical composition for the prevention or treatment of lung cancer or breast cancer, comprising the Leuconostoc mesenteroides strain, its culture, or components derived therefrom as an active ingredient.

[0007] Another object of this application is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising Leukonostok mecenteroides CJRS-10671 (accession number KCCM13059P) or Leukonostok mecenteroides CJRS-10672 (accession number KCCM13060P); its culture or components derived therefrom, as an active ingredient.

[0008] Another object of this application is to provide a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition of this application to an individual. Another object of this application is to provide a pharmaceutical composition for the prevention or treatment of colorectal cancer, comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as an active ingredient, to be administered in combination with an anticancer agent.

[0009] Another object of this application is to provide a method for preventing or treating lung cancer, breast cancer, or colorectal cancer, comprising administering the pharmaceutical composition of this application to an individual. Another object of the present application is to provide a food composition for preventing or improving lung cancer, breast cancer or colon cancer, which comprises Leuconostoc mesenteroides strain, its culture or a component derived therefrom as an active ingredient.

[0010] Another object of the present application is to provide a functional food for preventing or improving lung cancer, breast cancer or colon cancer, which comprises Leuconostoc mesenteroides strain, its culture or a component derived therefrom as an active ingredient.

[0011] Another object of the present application is to provide a feed composition for preventing or improving lung cancer, breast cancer or colon cancer, which comprises Leuconostoc mesenteroides strain, its culture or a component derived therefrom as an active ingredient.

[0012] Another object of the present application is to provide a food composition for preventing or improving cancer or inflammation, which comprises Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or a component derived therefrom as an active ingredient.

[0013] Another object of the present application is to provide a functional food for preventing or improving cancer or inflammation, which comprises Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or a component derived therefrom as an active ingredient.

[0014] Another object of the present application is to provide a feed composition for preventing or improving cancer or inflammation, which comprises Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or a component derived therefrom as an active ingredient.

[0015] Another object of this application is to provide an immunostimulatory pharmaceutical composition comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as active ingredients.

[0016] Another object of this application is to provide an immune-enhancing food composition containing Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom as active ingredients.

[0017] Another object of this application is to provide an immune-enhancing health functional food containing Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom as active ingredients.

[0018] Another object of this application is to provide an immune-enhancing feed composition containing Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom as active ingredients. [Effects of the Invention]

[0019] The novel Leuconostoc mesenteroides strain described in this application exhibits anti-cancer and anti-inflammatory effects, enhances CD8 T cell activity, and increases the secretion of immune-enhancing cytokines such as IL-6, IL-12p70, and IFNγ, thus demonstrating immune-enhancing effects. Therefore, it can be applied to the prevention or treatment of cancer or inflammation, and for immune enhancement purposes. [Brief explanation of the drawing]

[0020] [Figure 1] This figure shows the enzyme activity of Leuconostoc mesenteroides strains CJRS-10671 and CJRS-10672. [Figure 2] This figure shows the hemolytic activity of the CJRS-10671 and CJRS-10672 strains. [Figure 3] This figure shows the intestinal adhesion ability of the CJRS-10671 and CJRS-10672 bacterial strains. [Figure 4] This figure shows the confirmation of IL (interleukin)-6 secretion by the CJRS-10671 and CJRS-10672 bacterial strains in macrophagous cell lines. [Figure 5] This figure shows the confirmation of IL-12p70 secretion by the CJRS-10671 and CJRS-10672 bacterial strains in human peripheral blood mononuclear cells (PBMCs). [Figure 6] This figure shows the IFNγ (Interferon-gamma) secretion ability of CD8 T cells induced by the CJRS-10671 and CJRS-10672 strains in mouse spleen cells and human PBMCs. [Figure 7] This figure shows the cell cycle arrest effect of the CJRS-10671 and CJRS-10672 strains on cancer cells. [Figure 8] This figure shows the expression of cyclin B1 and CDK1 (Cyclin-Dependent Kinase 1), which are cell cycle arrest-related factors in cancer cells, by the CJRS-10671 and CJRS-10672 bacterial strains. [Figure 9] This figure shows the confirmation of cancer cell apoptosis induced by the CJRS-10671 and CJRS-10672 strains. [Figure 10] This figure shows the tumor-suppressing effects of CJRS-10671 and CJRS-10672 administration in a mouse lung cancer model (LLC1). [Figure 11] This figure shows the IFNγ secretion ability of CD8 T cells induced by CJRS-10671 administration in a mouse lung cancer model (LLC1). [Figure 12] This figure shows the IFNγ secretion ability of CD8 T cells induced by CJRS-10672 administration in a mouse lung cancer model (LLC1). [Figure 13] This figure shows the IFNγ secretion ability of CD8 T cells in tumor tissue in a mouse lung cancer model (LLC1) after administration of CJRS-10671 and CJRS-10672. [Figure 14] This figure shows the changes in myeloid-derived suppressor cells (MDSCs) in a mouse lung cancer model (LLC1) after administration of CJRS-10671 and CJRS-10672. [Figure 15] This figure shows the immune cell profiling (changes in immune cells) observed after administration of CJRS-10671 in a mouse lung cancer model (LLC1). [Figure 16] This figure shows the immune cell profiling (changes in immune cells) observed after administration of CJRS-10672 in a mouse lung cancer model (LLC1). [Figure 17] This figure shows the tumor-suppressing effect of CJRS-10672 administration in a mouse breast cancer model (4T1). [Figure 18] This figure shows the changes in MDSCs, which are immunosuppressive cells, in a mouse breast cancer model (4T1) after administration of CJRS-10672. [Modes for carrying out the invention]

[0021] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not to be limited by the specific descriptions described below. Moreover, a person with ordinary skill in the art can recognize and confirm many equivalents of the particular aspects of the invention described in this application using only ordinary experiments. Such equivalents are intended to be included in this application.

[0022] One aspect of this application provides a pharmaceutical composition for the prevention or treatment of lung cancer or breast cancer, comprising a Leuconostoc mesenteroides strain, its culture, or a component derived therefrom as an active ingredient.

[0023] In this application, the term "Leuconostoc mesenteroides" refers to a lactic acid bacterium that is round and spherical in shape, and is known as a probiotic or a lactic acid bacterium that ferments foods such as kimchi.

[0024] The Leuconostoc mesenteroides strain of this application may be one or more selected from CJRS-10671 deposited under accession number KCCM13059P or CJRS-10672 deposited under accession number KCCM13060P.

[0025] The CJRS-10671 strain of this application may have high enzymatic activity of β-galactosidase, α-glucosidase, and β-glucosidase, and the CJRS-10672 strain of this application may have high enzymatic activity of β-glucosidase.

[0026] Furthermore, the bacterial strain of this application has intestinal regulating and immune-enhancing effects. The bacterial strains of this application contained in the compositions thereof may exist as live or dead cells, and may also exist in a dried or freeze-dried form. The cultures of this application may include live bacterial cultures (i.e., the result of culturing live bacteria in a culture medium), cultures in which live bacteria have been killed in the live bacterial culture, or culture filtrates (i.e., the result of removing dead or live cells from the culture). Furthermore, components derived from the bacterial strains of this application may include, for example, cytoplasmic fractions obtained by crushing bacterial cells, such as those containing EVs (Extracellular Vesicles). In addition, suitable forms of lactic acid bacteria for inclusion in pharmaceutical compositions, formulation methods, and methods for separating components derived from bacterial strains are well known to those skilled in the art.

[0027] Specifically, the bacterial strains of this application may be strains obtained by inoculating 0.1-10% in MRS liquid medium and culturing at 25-40°C for 4-72 hours. Furthermore, the composition of this application may be administered in combination with an anticancer agent.

[0028] Another aspect of this application provides a pharmaceutical composition for the prevention or treatment of colorectal cancer, comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); a culture thereof; or a component derived therefrom, as an active ingredient, to be administered in combination with an anticancer agent.

[0029] The aforementioned bacterial strain, culture, and derived components are as described above. The bacterial strains of this application may also exhibit anti-cancer, anti-inflammatory, and immunostimulatory effects. In this application, cancer can be without limitation any cancer known to the art, such as lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, colorectal cancer (e.g., colon cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor), breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian carcinoma, extragonadal germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer), liver cancer (e.g., liver cancer). This may include, but is not limited to, cell carcinoma, primary liver cancer, extrahepatic cholangiocarcinoma, thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, endometrial cancer, uterine sarcoma), brain tumor (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinal nephroma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma), malignant bone tumor, bladder cancer, hematological cancer (e.g., multiple myeloma, leukemia, malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorder), cancer of unknown primary origin, etc., but specifically, it may include lung cancer, breast cancer, or colorectal cancer.

[0030] The pharmaceutical compositions of this application can be administered through any common route, as long as the compositions can reach the target tissue. A variety of administration methods may be considered, including intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration, but this application is not limited to such exemplary forms of administration. Preferably, the compositions can be administered in the form of injections. Alternatively, the pharmaceutical compositions can be administered by a specific device that allows the active ingredient to move into target cells.

[0031] The pharmaceutical compositions of this application may further include pharmaceutically acceptable carriers. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, and fragrances. For injectable preparations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers. For topical administration, pharmaceutically acceptable carriers may include bases, excipients, lubricants, and preservatives. The dosage forms of the pharmaceutical compositions of this application can be manufactured in a variety of dosage forms in combination with the pharmaceutically acceptable carriers described above. For example, for oral administration, they can be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers. For injectable preparations, they can be formulated as multi-dose dosage forms such as unit-dose ampoules or multi-dose containers. The composition can also be formulated into solutions, suspensions, tablets, pills, capsules, and sustained-release formulations.

[0032] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, fillers, anti-flocculants, lubricants, wetting agents, fragrances, and preservatives may be included.

[0033] The pharmaceutical composition of this application can be administered in a pharmaceutically effective amount. In this application, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective dose can be determined by factors including the individual's species and severity, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical compositions of this application can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or concurrently with conventional therapeutic agents. They can also be administered single or in multiple doses. It is important to administer an amount that takes all of the above factors into consideration and provides the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. The preferred dose of the pharmaceutical compositions of this application can be determined by various relevant factors, including not only the type of anticancer agent but also the type of cancer being treated, the route of administration, the patient's age, sex, weight, and disease severity. When the pharmaceutical compositions of this application are administered concurrently with anticancer agents, the dose of the concomitantly administered anticancer agent can be reduced, thereby reducing the side effects of the anticancer agent and increasing patient treatment compliance. The drug can be administered once a day or in several divided doses.

[0034] Furthermore, the pharmaceutical compositions of this application can be used not only as pharmaceuticals for human use but also in the form of veterinary pharmaceuticals. Here, "animal" includes livestock and pets. In this application, the pharmaceutical composition of this application may be administered in combination with an anticancer agent. The anticancer agent may be, but is not limited to, a taxane anticancer agent, a statin, an alkylating agent, a platinum-based drug, antimetabolites, an antibiotic, a vinca alkaloid anticancer agent, a targeted therapy agent, an immunosuppressant, a cancer vaccine, a cell therapy agent, an oncolytic virus, or a combination thereof, but more specifically, an immunosuppressant may be used.

[0035] Examples of the aforementioned taxane-based anticancer drugs include, but are not limited to, paclitaxel, docetaxel, larotaxel, and cabazitaxel.

[0036] The aforementioned statins may, but are not limited to, simvastatin, atorvastatin, lovastatin, fluvastatin, cerivastatin, and pitavastatin.

[0037] The alkylating drug may, but is not limited to, nitrogen mustard-based drugs, ethyleneimine and methylmelamine-based drugs, methylhydrazine derivatives, alkyl sulfonate-based drugs, nitrosourea-based drugs, and triazine-based drugs.

[0038] The platinum-based preparation may, but is not limited to, cisplatin, carboplatin, and oxaliplatin, for example.

[0039] The aforementioned antimetabolites may include, but are not limited to, folate antagonists, purine antagonists, and pyrimidine antagonists.

[0040] The aforementioned antibiotics may include, but are not limited to, etoposide, topotecan, irinotecan, idarubicin, epirubicin, dactinomycin, doxorubicin (adriamycin), daunorubicin, bleomycin, mitomycin C, and mitoxantrone.

[0041] The vinca alkaloid anticancer agent may, but is not limited to, vincristine, vinblastine, and vinorelbine.

[0042] Examples of the aforementioned targeted therapies include EGFR (Epidermal growth factor receptor) targeted therapies, HER2 (Human Epidermal growth factor Receptor 2) targeted therapies, CD20 (B cell marker) targeted therapies, CD33 (Myeloid Cell Surface Antigen) targeted therapies, CD52 (cluster of differentiation 52) targeted therapies, CD30 (Tumor Necrosis Factor Receptor Superfamily Member 8) targeted therapies, bcr-abl (breakpoint cluster region protein-Tyrosine-protein kinase) / c-Kit (tyrosine kinase receptor) targeted therapies, ALK (anaplastic lymphoma receptor tyrosine kinase) targeted therapies, anti-angiogenic (antiangiogenics) targeted therapies, mTOR (Mammalian target of rapamycin) targeted therapies, CDK4 / 6 (Cyclin-dependent kinase 4 / 6) targeted therapies, and PARP (Poly(ADP-ribose) This may include, but is not limited to, polymerase-targeted therapies, proteasome inhibitors, tyrosine kinase antagonists, protein kinase C inhibitors, and farnesyl transferase inhibitors.

[0043] The aforementioned immunosuppressant cancer agents may include, but are not limited to, anti-PD-1 (programmed cell death protein 1), anti-PD-L1 (programmed cell death-ligand 1), anti-CTLA4 (Cytotoxic T Lymphocyte associated Antigen 4), CTLA4 / B7-1 / B7-2 interaction inhibitors, CD47 / SIRP (Cluster of Differentiation 47 / Signal-regulatory protein) interaction inhibitors, anti-Tim3 (T-cell immunoglobulin and mucin domain 3), and anti-LAG3 (Lymphocyte Activating 3).

[0044] Specifically, the anticancer agents of this application may include, but are not limited to, anti-PD-1 immunoanticancer agents. In this application, concomitant administration may involve administering the pharmaceutical composition and the anticancer agent simultaneously, sequentially, or in reverse order. However, the order of administration is not limited, and as long as the administration interval is such that both pharmacological effects are exerted, it falls within the scope of this application. Furthermore, the pharmaceutical composition and the anticancer agent may be formulated individually or into a single formulation, but are not limited to this.

[0045] In this application, the administration of the pharmaceutical composition of this application may be used in combination with anticancer therapy. The anticancer therapy may be, but is not limited to, radiotherapy, neoadjuvant therapy, chemotherapy, targeted therapy, and photodynamic therapy, and may include all anticancer therapies known in the art.

[0046] In this application, the pharmaceutical composition of this application may exhibit an anticancer effect. The anticancer effect may include an effect of inhibiting cancer cell apoptosis or tumor growth (Examples 9 and 10-1).

[0047] For example, the pharmaceutical composition of this application may exhibit an anticancer effect by inhibiting the cell cycle of cancer cells, thereby inhibiting cancer cell apoptosis or tumor growth (Example 8).

[0048] As another example, the pharmaceutical composition of this application may exhibit anti-inflammatory or immune-enhancing effects by increasing the secretion of the immune-enhancing cytokines IL (interlukine)-6, IL-12p70, and IFNγ (interferon-gamma) (Example 7).

[0049] Another aspect of this application provides a method for preventing or treating lung cancer, breast cancer, or colorectal cancer, comprising administering the pharmaceutical composition of this application to an individual. The aforementioned pharmaceutical composition and cancer are as described above.

[0050] The therapeutic method of this application comprises the step of administering the pharmaceutical composition in a pharmaceutically effective amount into an individual suspected of having cancer. The individual means all mammals, including dogs, cattle, horses, rabbits, mice, rats, chickens, and humans, but the mammals of this application are not limited to the above examples. The pharmaceutical composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally. For local treatment, it may be administered by a suitable method, including intra-lesional administration, if necessary. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Preferred dosage forms are intravenous, subcutaneous, intradermal, intramuscular, or intravenous infusion. The preferred dosage of the pharmaceutical composition of this application may vary depending on the condition and weight of the individual, the severity of the disease, the drug form, the route of administration, and the duration, and can be appropriately determined by those skilled in the art.

[0051] Another aspect of this application is to provide a food composition for the prevention or improvement of lung cancer, breast cancer, or colorectal cancer, comprising the Leuconostoc mesenteroides strain, culture thereof, or components derived therefrom as an active ingredient.

[0052] Another aspect of this application is to provide a health functional food for the prevention or improvement of lung cancer, breast cancer, or colorectal cancer, comprising the Leuconostoc mesenteroides strain, culture thereof, or components derived therefrom as an active ingredient.

[0053] The aforementioned bacterial strains, cultures, derived components, and cancers are as described above. The aforementioned food composition may be in the form of a health functional food or a food additive. In this application, the term "health functional food" means a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body, as defined in the Act on Health Functional Foods (Article 3, Item 1), and "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological effects, on the structure and function of the human body (Article 3, Item 2).

[0054] The aforementioned food composition may further contain food additives. Unless otherwise specified, whether or not a food additive is suitable shall be determined according to the standards and criteria for the relevant item, based on the general provisions and general test methods of the Food Additives Code approved by the Food and Drug Administration.

[0055] Examples of items listed in the aforementioned "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar dye preparations.

[0056] Foods containing the active ingredient of this application include: confectionery such as bread, mochi, dried fruits, candies, chocolates, chewing gum, and jams; ice cream products such as ice cream, frozen fruit, and ice cream powder; dairy products such as milk, low-fat milk, lactose-free milk, processed milk, goat's milk, fermented milk, buttermilk, concentrated milk, fresh cream, buttermilk, natural cheese, processed cheese, milk powder, and whey; meat products such as processed meat products, egg products, and hamburgers; and Examples include, but are not limited to, fish products such as fish paste products like maboko, ham, sausage, and bacon; noodles such as ramen, dried noodles, fresh noodles, oil noodle soup, gelatinized dried noodles, improved aged noodles, frozen noodles, and pasta; beverages such as fruit drinks, vegetable drinks, carbonated drinks, soy milk, yogurt and other lactic acid bacteria drinks, and mixed drinks; seasonings such as soy sauce, miso, gochujang, Chinese miso, cheonggukjang, mixed miso, vinegar, sauces, tomato ketchup, curry, and dressings; and fermented foods.

[0057] In addition to the foregoing, the food compositions of this application may include various nutrients, vitamins, electrolytes, flavorings, colorings, pectin and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the food compositions of this application may include fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. Such components may be used individually or in combination.

[0058] Another aspect of this application is to provide a feed composition for the prevention or improvement of lung cancer, breast cancer, or colorectal cancer, comprising the Leuconostoc mesenteroides strain, culture thereof, or components derived therefrom as an active ingredient.

[0059] The aforementioned bacterial strains, cultures, derived components, and cancers are as described above. The aforementioned feed composition may be in the form of a feed additive composition in addition to a feed composition. When the composition is manufactured as a feed additive, it can be manufactured as a highly concentrated liquid of 20-90%, or in powder or granular form. The feed additive may further contain one or more of the following: organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenols, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When manufactured as feed, the composition may be formulated in the form of a normal feed and may also contain normal feed components.

[0060] The aforementioned feed and feed additives may further include grains, such as crushed or broken wheat, oats, barley, corn, and rice; plant-based protein feeds, such as feeds mainly composed of rapeseed, beans, and sunflowers; animal-based protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products, such as dried components consisting of various milk powders and whey powders, and may also further include nutritional supplements, digestive and absorption enhancers, growth promoters, and the like.

[0061] The feed additives may be administered to animals alone or in combination with other feed additives via an edible carrier. Furthermore, the feed additives can be readily administered to animals as top dressings, directly mixed into animal feed, or in oral dosage forms separate from the feed. When the feed additives are administered separately from animal feed, they can be prepared in combination with pharmaceutically acceptable edible carriers, as is well known in the art, to produce immediate-release or sustained-release dosage forms. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of tablets, capsules, powders, lozenges, sugar-containing tablets, or top dressings in a finely dispersed form. When a liquid carrier is used, the feed additive may be in the form of gelatin soft capsules, or syrups, suspensions, emulsions, or solutions.

[0062] Furthermore, the feed and feed additives may contain auxiliary agents, such as preservatives, stabilizers, wetting agents or emulsifiers, and solution enhancers. The feed additives may be used by injection, spraying, or mixing and adding them to animal feed.

[0063] The feed or feed additive of this application can be applied to a wide range of animal diets, including those of mammals, poultry, and fish. The aforementioned mammals include not only pigs, cattle, horses, sheep, rabbits, goats, rodents, and laboratory rodents such as rats, hamsters, and guinea pigs, but also pets (e.g., dogs, cats), and the aforementioned poultry include chickens, turkeys, ducks, geese, pheasants, and quail, and the aforementioned fish include carp, crucian carp, and trout, but are not limited to these.

[0064] Another aspect of this application is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising Leukonostok mecenteroides CJRS-10671 (accession number KCCM13059P) or Leukonostok mecenteroides CJRS-10672 (accession number KCCM13060P); its culture or a component derived therefrom, as an active ingredient.

[0065] Another aspect of this application provides a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition of this application to an individual. Another aspect of this application provides a food composition for the prevention or improvement of cancer or inflammation, comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as an active ingredient.

[0066] Another aspect of this application is to provide a health functional food for the prevention or improvement of cancer or inflammation, comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as an active ingredient.

[0067] Another aspect of this application is to provide a feed composition for the prevention or improvement of cancer or inflammation, comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as an active ingredient.

[0068] Another aspect of this application provides an immunostimulatory pharmaceutical composition comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as active ingredients.

[0069] Another aspect of this application is to provide an immune-enhancing food composition comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as active ingredients.

[0070] Another aspect of this application is to provide an immune-enhancing health functional food containing Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom as active ingredients.

[0071] Another aspect of this application is to provide an immune-enhancing feed composition comprising Leuconostoc mesenteroides CJRS-10671 (accession number KCCM13059P) or Leuconostoc mesenteroides CJRS-10672 (accession number KCCM13060P); its culture; or components derived therefrom, as active ingredients.

[0072] In the composition of the present application, the bacterial strain, culture, derived components, cancer, inflammation, and immune enhancement are as described in the other aspects described above. The present application will be described in more detail below through experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by an ordinary person skilled in the art of this application or a similar art. Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification as a whole by reference to more clearly explain the level of the art to which this application belongs and the content of this application.

[0073] Example 1: Confirmation of sugar fermentation characteristics of Leuconostoc mesenteroides strain Leuconostoc mesenteroides strains CJRS-10671 and CJRS-10672, isolated from kimchi, were cultured using Difco's MRS liquid medium and Agar medium. Each medium was prepared according to the protocol and sterilized at 121°C for 15 minutes before use. Two days prior to the experiment, each strain was inoculated at 1% in MRS liquid medium and incubated statically at 37°C for 20 hours, followed by a further 2% inoculation (activation) in MRS liquid medium and incubated statically at 37°C for 20 hours before use in the experiment.

[0074] The characteristics of sugar fermentation for 49 types of carbohydrates were confirmed using the API 50 CHL kit (Biomerieux, France) on the CJRS-10671 and CJRS-10672 strains cultured as described above. Specifically, after washing the strains that had undergone secondary subculturing with PBS (phosphate buffered saline), the strains were suspended in API 50 CHL medium using McFarland standard 2.0 as the reference, inoculated into strips, and cultured at 37°C for 24 hours. A yellow color when using carbohydrates was interpreted as a positive reaction, and a purple color when carbohydrates could not be utilized was interpreted as a negative reaction.

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] Example 2: Confirmation of enzyme activity of the bacterial strain To analyze the biochemical characteristics of the CJRS-10671 and CJRS-10672 strains of Example 1, the activity of 19 enzymes was investigated using the API zym kit (Biomerieux, France).

[0078] As a result, as shown in Table 2 and Figure 1 below, it was confirmed that CJRS-10671 showed high enzymatic activity of β-galactosidase, α-glucosidase, and β-glucosidase, while CJRS-10672 showed high enzymatic activity of β-glucosidase.

[0079] [Table 2]

[0080] Example 3: Confirmation of hemolytic properties of the bacterial strain To confirm the hemolytic properties of the CJRS-10671 and CJRS-10672 strains from Example 1, they were smeared onto blood agar containing 5% defibrinated sheep blood (MB cell, Korea) and incubated at 37°C for 24 hours. The presence or absence of a clear zone around the colonies was then checked.

[0081] As a result, as shown in Figure 2, the absence of a clear zone formed around the colonies due to the decomposition of red blood cells confirmed that the CJRS-10671 and CJRS-10672 strains are not hemolytic.

[0082] Example 4: Confirmation of antibiotic resistance in bacterial strains To confirm antibiotic resistance in the CJRS-10671 and CJRS-10672 strains of Example 1, antibiotic resistance evaluation was conducted to determine the MIC (Minimum Inhibitory Concentration) in accordance with the EFSA guidelines for antibiotic resistance assessment. In the case of Leuconostoc mesenteroides, the guidelines specify criteria for cut-off values ​​for eight antibiotics.

[0083] The experimental results of the MIC tests for strains CJRS-10671 and CJRS-10672 met the criteria for five antibiotics, but exceeded the MIC criteria for some antibiotics. Therefore, according to the EFSA guidelines, we conducted genetic analysis to determine whether the antibiotic resistance was intrinsic or acquired, and the results are shown in Table 3 below. According to the EFSA guidelines, if intrinsic resistance is present, it is usable because there is no possibility of external transfer of the antibiotic resistance gene to other bacteria. Leuconostoc strains have been reported to have resistance to chloramphenicol (MICs ≥ 4 μg / mL), but this is likely to be intrinsic resistance of the Leuconostoc genus itself, and it is known that horizontal gene transfer to other bacteria is unlikely (AB Flσrez, 2005).

[0084] [Table 3]

[0085] Example 5: Confirmation of acid and bile resistance of the bacterial strain Since it is important for oral formulations to stably reach the intestines and exert their effects, we evaluated acid resistance and bile resistance (bile resistance) to confirm their suitability as oral formulations. Specifically, the acid resistance test was conducted by inoculating 1% of a secondary passaged strain into a PBS solution corrected to pH 3.0 using 1N HCl, culturing at 37°C for 2 hours, and then comparing the initial viable cell count with the viable cell count after culturing.

[0086] Bile tolerance was measured by inoculating a 1% activated strain into a PBS solution containing 1% oxgall (Difco, USA), culturing at 37°C for 2 hours, and comparing the number of viable cells to the initial number of viable cells.

[0087] As a result, as shown in Table 4 below, strain CJRS-10671 showed 95.7% acid resistance and 136.6% gulp resistance based on an initial viable cell count of 100%, while strain CJRS-10672 showed 120.6% acid resistance and 67.9% gulp resistance based on an initial viable cell count of 100%.

[0088] [Table 4]

[0089] Example 6: Confirmation of the bacterial strain's ability to adhere to the intestines. To determine the stability and compatibility of the CJRS-10671 and CJRS-10672 strains of Example 1 with respect to the intestinal environment, intestinal adhesion ability was evaluated using the HT-29 cell line (Korean Cell Line Bank, KCLB; Seoul, Korea), which is an intestinal epithelial cell line. Specifically, the HT-29 cell line was cultured at 37°C and 5% CO2 in DMEM (Dulbecco's modified Eagle's minimal essential medium) medium supplemented with 10% FBS (fetal bovine serum) (Gibco, Carlsbad, CA, USA), 100 U / mL penicillin, and 0.1 mg / mL streptomycin. The HT-29 cell line was cultured in a 1.0X10 ratio to form a monolayer of approximately 80%. 5After seeding in 24-well cells, the cells were washed twice with PBS to remove antibiotics contained in the DMEM. After secondary passage, the strains were washed twice with PBS and then redissolved in antibiotic-free DMEM medium for 2x10⁶ cultures. 7 Cells were treated at a CFU / well ratio. After culturing at 37°C and 5% CO2 for 2 hours, non-adhering cells were removed by washing three times with PBS. Only the adhered HT-29 and bacterial strains were detached by adding trypsin-EDTA (ethylene-diamine-tetraacetic acid), and the number of viable cells was measured using MRS agar. The degree of intestinal adhesion was expressed as the number of attached bacterial strain CFUs per 100 HT-29 cell lines, and Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium lactis ATCC type strains were used as the control group.

[0090] As a result, the CJRS-10671 strain showed an intestinal adhesion capacity of approximately 217±21 bacteria / 100 cells, confirming that it exhibited an intestinal adhesion capacity equivalent to or greater than that of the control group strains (Figure 3).

[0091] Example 7: Confirmation of immunoenhancing cytokine secretion by bacterial strain 7-1. Confirmation of IL (interleukin)-6 secretion in macrophagous cell lines In this study, we measured whether CJRS-10671 and CJRS-10672 from Example 1 induced IL-6 secretion, a known immunoenhancing cytokine, in immune cells. Mouse macrophages (Raw 264.7) and human monocytes (THP-1) were used as immune cells. One week before the start of the experiment, the Raw 264.7 cell line was subcultured and maintained in DMEM medium containing 10% FBS and 1% Antibiotic-antimycotic, and 6x10 cells were cultured one day before the experiment. 4Cells were seeded in a flat 96-well plate at a rate of cells / well. The THP-1 cell line was subcultured and maintained in RPMI medium containing 10% FBS, 1% penicillin-streptomycin (PS), and 0.05 mM 2-mercaptoethanol (2-ME), and 8x10⁶ cells were cultured two days before the experiment. 4 After seeding cells / well in a flat 96-well plate, human monocytes were treated with 25 ng / ml of PMA (Phorbol 12-myristate 13-acetate) to differentiate them into human macrophages for 2 days. Subsequently, the culture medium for co-culturing with CJRS-10671 and CJRS-10672 was a 10% FBS-containing medium with antibiotics excluded. After culturing, the total bacterial count of CJRS-10671 and CJRS-10672 was measured and calculated using Novocyte equipment, and then treated with immune cells in a 1:10 ratio. After 24 hours of co-culturing, the cultured plates were centrifuged to settle the cells, and only the upper layer was obtained to check for cytokine secretion. The analytical samples were prepared using BD Biosciences' BD Cytometric Bead Array (CBA) Mouse / Human Flex Set, read using BD Lyric (flow cytometry), and the results were analyzed using the FCAP program.

[0092] As a result, we confirmed that CJRS-10671 and CJRS-10672 increase IL-6 secretion when co-cultured with mouse or human macrophages (Figure 4). 7-2. Confirmation of IL-12p70 secretion in human PBMCs (peripheral blood mononuclear cells) We measured whether CJRS-10671 and CJRS-10672 induce the secretion of IL-12p70, a known immunostimulatory cytokine, in human PBMCs. Human PBMCs were placed in 96-well plates in a 2x10⁶ format. 5Cells were seeded in a cell / well ratio. Subsequently, when co-culturing with CJRS-10671 and CJRS-10672, a medium containing 10% FBS with antibiotics excluded was used. After culturing, the total number of CJRS-10671 and CJRS-10672 was measured and calculated using Novocyte equipment, and then treated with immune cells in a 1:10 ratio. After 24 hours of culture, the supernatant was obtained and examined, confirming that CJRS-10671 and CJRS-10672 increased IL-12p70 secretion when co-culturing with human PBMCs (Figure 5).

[0093] 7-3. Confirmation of IFNγ (Interferon-gamma) secretion by CD8 T cells in mouse splenocytes and human PBMCs. The IFNγ secretion capacity of cytotoxic T cells, specifically CD8 T cells, is known to significantly influence their anticancer efficacy. To confirm the IFNγ secretion capacity of cytotoxic T cells from the CJRS-10671 and CJRS-10672 strains, mouse spleen cells and human PBMCs were used to study CD8 cells. + IFNγ + I confirmed it.

[0094] Specifically, to isolate mouse spleen cells, Hanks Balanced Salt Solution (HBSS) containing collagenase IV was injected into the inside of the mouse spleen tissue using a syringe to allow the cells to flow out. After repeating this several times, the tissue that retained its morphology was finely chopped using a syringe and incubated at 37°C for 25 minutes. It was then incubated again at 37°C for 5 minutes with the addition of 0.5M EDTA. After mixing the cells using a Pasteur pipette, the mixture was filtered through a cell strainer. After centrifugation at 1500 rpm for 3 minutes, red blood cells were removed using 1x RBC lysis buffer, and the spleen cells were suspended in fresh medium for experimental use.

[0095] Human PBMCs were purchased from stemcell technologies and stored in a nitrogen tank after receipt until use in experiments. On the day of the experiment, the stock was taken out of the nitrogen tank and placed in a water bath to thaw. After adding RPMI1640 medium supplemented with 10% FBS and centrifuging at 1500 rpm for 5 minutes, the PBMCs were resuspended in fresh medium and used in the experiment.

[0096] To confirm the IFNγ secretion ability in mouse spleen cells, since a T cell stimulator is required, one day before co-culture with the strain, anti-CD3 antibody was diluted with PBS at a concentration of 0.1 μg / ml and coated on a flat 96-well plate at a volume of 100 μl per well at a concentration of 0.1 μg / ml. On the day of co-culture, the coated plate was washed twice with PBS and prepared. Mouse spleen cells were separated according to the protocol and then seeded at 5x10 5 cells / well on the 96-well plate coated with purified anti-CD28 antibody at 1 μg / ml. Then, strains CJRS-10671 and CJRS-10672 were co-cultured with spleen cells at a ratio of 1:10 using antibiotic-free culture medium. Three days after co-culture, brefeldin A was added to advance golgi stop for 3 - 4 hours, and then the analysis samples were prepared by staining the markers to be confirmed using BD transcription factor buffer set and fluorescent antibodies. The analysis samples were analyzed using BD Lyric (flow cytometry) and the Flowjo program, and the results were confirmed.

[0097] Human PBMCs were seeded at 2x10 5 cells / well, and then the co-culture was performed in the same manner as in Example 7-2 above. As a result, it was confirmed that CJRS-10671 and CJRS-10672 increased the IFNγ secretion ability of CD8 T cells in mouse spleen cells and human PBMCs (Figure 6).

[0098] Example 8: Confirmation of the ability of the strain to regulate the cell cycle of cancer cells 8-1. Confirmation of the effect of cell cycle arrest on cancer cells The cell cycle regulatory ability of cancer cell lines by the CJRS-10671 and CJRS-10672 strains described in Example 1 was confirmed. Lung cancer cell line LLC1 (Lewis lung carcinoma, LL / 2, LLC1) and colorectal cancer cell line MC38 (Murine Carcinoma-38) cells were used as cancer cell lines. One week before the start of the experiment, LLC1 and MC38 cell lines were cultured and maintained in DMEM medium containing 10% FBS and 1% Anti-anti. LLC1 and MC38 cell lines were placed in 6-well plates, each containing 2.4 x 10⁶ cells. 5 cells / well, 1.2 x 10 5 Cells were seeded at a rate of cells / well. To synchronize the cell cycle to G0 / G1, after 24 hours, the cells were washed twice with 1xPBS and then replaced with culture medium containing 0.5% FBS and no antibiotics. CJRS-10671 and CJRS-10672 strains were added in cancer cell line:strain ratios of 1:10, 1:100, and 1:1000. After co-culturing for 24 hours, the cells were detached using trypsin-EDTA, washed with 1xPBS, and then stored overnight at -20°C in 5 ml of ice-cold 75% EtOH with only the cells remaining. Subsequently, the cells were stained with Ki67 and PI fluorescence, and the cell cycle results were analyzed using BD Lyric (flow cytometry) and the Flowjo program. The control group consisted of cells that were not treated with CJRS-10671 or CJRS-10672 strains.

[0099] As a result, the group treated with the bacterial strain showed a cell cycle arrest effect compared to the untreated group, and it was confirmed that the arrest effect mainly occurred in the G2 / M cycle due to CJRS-10671 (Figure 7).

[0100] 8-2. Confirmation of the expression of cyclin B1 and CDK1 (Cyclin-Dependent Kinase 1), which are cell cycle arrest-related factors in cancer cells. Changes in the expression of cyclin B1 and CDK1, G2 / M cell cycle regulation-related factors, in cancer cell lines induced by the CJRS-10671 and CJRS-10672 strains were confirmed. LLC1 cells were co-cultured with the CJRS-10671 and CJRS-10672 strains for 24 hours using the same method as in Example 8-1, and then RNA was extracted from the cells using trizol. cDNA was synthesized from this RNA, and relative expression levels were measured by PCR using QuatStudio 5 (real-time PCR machine). A control group consisted of cells that were not treated with the CJRS-10671 or CJRS-10672 strains (untreated group).

[0101] As a result, we confirmed a decrease in cyclin B1 and CDK1 associated with G2 / M arrest, and mainly in LLC1, we confirmed that the relative expression levels of cyclin B1 and CDK1 decreased in the treated group compared to the untreated group (Figure 8).

[0102] Example 9: Confirmation of cancer cell apoptosis by bacterial strain To confirm the change in the survival rate of cancer cell lines using the CJRS-10671 and CJRS-10672 strains of Example 1, LLC1 and MC38 cells were placed in 2.4 x 10⁶ well plates. 5 cells / well, 1.2 x 10 5 Cells were seeded at a rate of cells / well. After 24 hours, the medium was changed to one containing 0.5% FBS, followed by 24 hours of serum starvation. CJRS-10671 and CJRS-10672 were treated to achieve cancer cell line:strain ratios of 1:10, 1:100, and 1:1000, and co-cultured for 24 hours. After detaching the cells using trypsin-EDTA and washing with 1x PBS, the cells were fluorescently stained with Fixed Viability Stain 510 antibody to differentiate between live and dead cells. Cell viability was analyzed using BD Lyric (flow cytometry) and the Flowjo program. The ratio of live to dead cells, calculated with all cells as 100%, is shown in the figure.

[0103] As a result, it was confirmed that cell viability decreased in the group treated with the bacterial strain compared to the group without treatment (Figure 9). Example 10: Confirmation of anticancer effect of bacterial strain in mouse lung cancer (LLC1) model. 10-1. Tumor suppression effect To construct an animal model of lung cancer, we introduced 4x10 LLC1 cell lines into c57BL / 6 mice. 5 Cells / mouse were injected subcutaneously. After tumor cell injection, the tumor size was approximately 30-50 mm. 3 Upon reaching this stage, the mice were divided into the following groups: a negative control group (untreated after tumor cell injection), a group treated with CJRS-10671 or CJRS-10672 sample, a group treated with the immune checkpoint inhibitor anti-PD-1 (positive control group), and a group treated with both CJRS-10671 or CJRS-10672 sample and anti-PD-1. Each mouse received 10 units of CJRS-10671 or CJRS-10672 sample. 9 100 μl to obtain CFU The drug was diluted in PBS and administered a total of 10 times over 10 days, while anti-PD-1 (10 mg / kg, BioXCell) was administered intraperitoneally every other day. Tumor size was measured three times a week using calipers, and on the day after the end of administration, the tumor weight was measured and blood and organs were obtained and analyzed from sacrificial mice.

[0104] As a result, CJRS-10671, even when administered alone, reduced tumor size compared to the anti-PD-1 monotherapy group, confirming superior tumor suppression when administered in combination with anti-PD-1. CJRS-10672 showed minimal effect when administered alone, but demonstrated significantly superior tumor suppression when administered in combination with anti-PD-1 (Figure 10).

[0105] 10-2. Confirmation of IFNγ secretion capacity of CD8 T cells After completing the test in Example 10-1, spleen cells were isolated from the spleen tissue of each group, and then 10 6Cells were seeded in a round-bottom 96-well plate at a cell / well rate. After stimulating spleen cells with PMA / Ionomycin, which stimulates all immune cells, for 3-4 hours, surface marker staining and intracellular cytokine staining (ICS) were performed, and the amount of IFNγ secreted by CD8 T cells was confirmed via FACS. Furthermore, to confirm CD8 T cell activity, identically isolated spleen cells were stimulated for 1 day with CD3 / CD28 (anti-CD3 antibody 2 μg / ml, anti-CD28 antibody 2 μg / ml), which stimulates T cells, and then surface marker staining and ICS were performed, followed by the amount of IFNγ secreted by CD8 T cells via FACS.

[0106] As a result, we confirmed that in the group administered CJRS-10671 and anti-PD-1 in combination, the amount of IFNγ secreted by CD8 T cells increased significantly when all immune cells were stimulated, and we also confirmed a tendency for increased IFNγ secretion from CD8 T cells when T cells were stimulated (Figure 11). Furthermore, in the group administered CJRS-10672 and anti-PD-1 in combination, the amount of IFNγ secreted by CD8 T cells was similar to that of the group administered anti-PD-1 alone when all immune cells were stimulated, but we confirmed a significant increase in IFNγ secretion from CD8 T cells when T cells were stimulated (Figure 12).

[0107] 10-3. Confirmation of IFNγ secretion capacity of CD8 T cells in cancer tissue After completing the test in Example 10-1, tumor-infiltration lymphocytes (TILs) were isolated from the tumor tissue of each group. These T cells were then stimulated with CD3 / CD28 (anti-CD3 antibody 2 μg / ml, anti-CD28 antibody 2 μg / ml) for 1 day. After surface marker staining and ICS, the amount of IFNγ secreted by CD8 T cells was confirmed via FACS.

[0108] As a result, both the group receiving CJRS-10671 and anti-PD-1 combination therapy and the group receiving CJRS-10672 and anti-PD-1 combination therapy showed an increase in the amount of IFNγ secreted by CD8 T cells in response to T cell stimulation (Figure 13).

[0109] 10-4. Confirmation of changes in immunosuppressive cells After completing the test in Example 10-1, the changes in MDSCs (Myeloid-derived suppressor cells), known as immunosuppressive cells, in the spleen cells and tumor cells of each group were compared with CD11b + Gr-1 + The samples were fluorescently stained with a marker and confirmed via FACS.

[0110] As a result, MDSCs increased in spleen cells in the negative control group, but decreased in the CJRS-10671 and anti-PD-1 combination therapy group and the CJRS-10672 and anti-PD-1 combination therapy group. Furthermore, when examined with the same markers in tumors, a significant decrease in MDSCs was confirmed in the CJRS-10671 and anti-PD-1 combination therapy group and the CJRS-10672 and anti-PD-1 combination therapy group compared to the negative control group (Figure 14).

[0111] 10-5. Confirmation of changes in immune cells (profiling) After completing the test in Example 10-1, spleen cells were isolated from the spleen tissue of each group, and surface marker staining was performed using a fluorescent antibody to confirm changes in immune cells and immunosuppressive cells. The fluorescent marker used for each immune cell was as follows: M1(CD11b + F4 / 80 + CD86 + ), M2(CD11b + F4 / 80 + CD206 + ), DC (IA-IE + CD11b + CD11c + ), B cells (CD3 - B220 + ), NK cells (CD3 - NK1.1 + ), NKT cells (CD3 + NK1.1 + ), Treg(CD4 + CD25 + Foxp3 +The samples were analyzed using BD Lyric (flow cytometry) and the Flowjo program, and the results were confirmed using % and #.

[0112] As a result, in the groups receiving CJRS-10671, CJRS-10672 alone, or in combination with anti-PD-1, the percentage and number of M1, DC, and NKT cells increased compared to other cell types (Figures 15 and 16).

[0113] Example 11. Confirmation of anticancer effect of bacterial strain in a mouse mammary cancer (4T1) model. To construct an animal model of breast cancer, BALB / c mice were given 3x10 4T1 cell lines. 5 Cells / mouse were injected subcutaneously. After tumor cell injection, the tumor size was approximately 30-50 mm. 3 Upon reaching the target stage, the mice were divided into the following groups: a negative control group (untreated after tumor cell injection), a group treated with the CJRS-10672 sample, a group treated with the immune checkpoint inhibitor anti-PD-1 (positive control group), and a group treated with both the CJRS-10672 sample and anti-PD-1. The CJRS-10672 sample was administered at a rate of 10 per mouse. 9 The tumor was diluted in 100 μl of PBS to obtain a CFU and administered a total of 10 times over 10 days. Anti-PD-1 (10 mg / kg, BioXCell) was administered intraperitoneally every two days. Tumor size was measured three times a week using calipers, and on the day after the end of administration, the mice were sacrificed for tumor weight measurement and blood and organ analysis.

[0114] Furthermore, the number of MDSCs, which are immunosuppressive cells, among the obtained spleen-derived cells was confirmed using the same method as in Example 10-4. As a result, CJRS-10672 was found to reduce the size of breast cancers compared to the negative control group or the anti-PD-1 treated group, and in particular, individuals in the group treated with CJRS-10672 and anti-PD-1 showed reduced tumor size and weight (Figure 17).

[0115] Furthermore, the number of MDSCs, which are immunosuppressive cells in spleen cells, was significantly reduced in the group receiving CJRS-10672 and anti-PD-1 combination therapy (Figure 18). The aforementioned CJRS-10671 and CJRS-10672 strains were deposited with the Korean Culture Center of Microorganisms (KCCM), an international depositary under the Budapest Convention, on October 19, 2021, and were assigned deposit numbers KCCM13059P and KCCM13060P, respectively.

[0116] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0117] [Table 5]

[0118] [Table 6]

Claims

[Claim 1] A pharmaceutical composition for the prevention or treatment of lung cancer or breast cancer, comprising a strain of Leuconostoc mesenteroides, its culture, or components derived therefrom as an active ingredient.

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