Antitumor bacterial strain, and composition and method using the same

Abstract

To provide Enterococcus faecium microorganisms or Enterococcus faecalis microorganisms having anti-cancer activity, or a composition containing the same, and a method of preventing or treating cancer by using the composition.SOLUTION: Provided is a bacterial strain that i) belongs to a species Enterococcus faecium; ii) has a lactate production ability compared to microbial growth (lactate / OD600) of 3 g / L or more, when cultured for 48 hours; iii) exhibits anti-tumor activity; and iv) has β-galactosidase activity, wherein the bacterial strain is at least one selected from specific groups.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an anti-tumor bacterial strain, a composition using the same, and a method thereof. More specifically, the present invention relates to a bacterial strain of the species Enterococcus faecium that exhibits anti-tumor activity, and a composition and a method for preventing or treating tumors using the same.

[0002] The present invention also relates to an anti-tumor bacterial strain, a composition using the same, and a method thereof. More specifically, the present invention relates to an Enterococcus faecalis microorganism having anti-cancer activity, a composition containing the same, and cancer prevention or cancer treatment using the same.

Background Art

[0003] It is known that gut bacteria can be used to prevent or treat various diseases and disorders.

[0004] International Publication No. WO2016 / 196605 discloses a method for treating or preventing cancer in a subject by modulating the levels of one or more commensal microbes, enhancing the immune response by the subject, and / or suppressing cancer growth or spread, and / or suppressing cancer immune evasion, and / or enhancing the efficacy of a therapeutic agent. The document has, as commensal microbes, examples such as Adlercreutzia, Oscillospira, Mollicutes, Butyrivibrio, Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Rikenella, Alistipes, Marinilabilia, Anaerostipes, Escherichia, Lactobacillus, and in particular discloses a method for treating cancer by combining Bifidobacterium bacteria and an immune checkpoint inhibitor.

[0005] International Publication No. WO2017 / 085520 discloses a composition for use in a method for treating or preventing cancer, comprising a bacterial strain of the Enterococcus gallinarum species.

[0006] International Publication No. WO2017 / 085518 discloses a composition for use in a method of treating or preventing a disease or disorder mediated by the IL-17 pathway or the Th17 pathway, comprising a bacterial strain of the species Enterococcus faecium.

[0007] To date, in the species Enterococcus faecium, there has been no known association between antitumor activity and the ability to produce specific metabolites in comparison to microbial growth.

[0008] Enterococcus faecalis is found in the gastrointestinal tracts of humans and other mammals. Enterococcus faecalis is a Gram-positive coccus bacterium. Enterococcus faecalis can grow at temperatures between 10°C and 45°C.

[0009] Korean Patent Publication 10-2020-0054589 discloses Enterococcus faecalis KACC92220P, which has the effect of being able to reduce lactose content. Korean Patent 10-2053730 discloses Enterococcus faecalis AMI-1001 strain, which has antioxidant, anti-salt or antibacterial activity.

[0010] However, even with the aforementioned prior art, there is a need to find Enterococcus faecalis with anti-cancer activity.

Summary of the Invention

Problems to be Solved by the Invention

[0011] A first object of the present disclosure is to provide a bacterial strain of the species Enterococcus faecium having antitumor activity characterized by the ability to produce lactate in comparison to microbial growth.

[0012] A second object of the present disclosure is to provide a composition for preventing or treating tumors, which contains, as an active ingredient, a bacterial strain of the Enterococcus faecium species.

[0013] A third object of the present disclosure is to provide a method for preventing or treating tumors in a subject, which includes the step of administering the bacterial strain of the Enterococcus faecium species or the composition to the subject.

[0014] A fourth object of the present disclosure is to provide a microorganism of Enterococcus faecalis LMT19-32 (accession number KCTC 14306BP) having antitumor activity, or a culture or an extract thereof.

[0015] A fifth object of the present disclosure is to provide a pharmaceutical composition for preventing or treating cancer, which contains, as an active ingredient, the microorganism of Enterococcus faecalis LMT19-32, or a culture or an extract thereof.

[0016] A sixth object of the present disclosure is to provide a food composition for preventing or improving cancer, which contains, as an active ingredient, the microorganism of Enterococcus faecalis LMT19-32, or a culture or an extract thereof.

[0017] A seventh object of the present disclosure is to provide a method for preventing or treating cancer in an individual, which includes the step of administering to the individual an effective amount of the microorganism of Enterococcus faecalis LMT19-32, or a culture or an extract thereof, effective for treating cancer.

Means for Solving the Problems

[0018] A first aspect of the present disclosure is i) belonging to the Enterococcus faecium species, ii) at the time of 48-hour culture, lactate production ability (lactate / OD in microbial growth comparison600 ) is 3 g / L or more, iii) It pertains to a bacterial strain showing antitumor activity.

[0019] The second aspect of the present disclosure pertains to a composition for preventing or treating tumors, containing, as an active ingredient, a bacterial strain of the Enterococcus faecium species.

[0020] The third aspect of the present disclosure pertains to a method for preventing or treating tumors in a subject, including the step of administering to the subject the bacterial strain of the Enterococcus faecium species, or the composition.

[0021] The fourth aspect of the present disclosure pertains to the microorganism Enterococcus faecalis LMT19 - 32 (Accession No. KCTC 14306BP) having antitumor activity, or its culture or its extract.

[0022] The fifth aspect of the present disclosure pertains to a pharmaceutical composition for preventing or treating cancer, containing, as an active ingredient, the microorganism Enterococcus faecalis LMT19 - 32, or its culture or its extract.

[0023] The sixth aspect of the present disclosure pertains to a food composition for preventing or improving cancer, containing, as an active ingredient, the microorganism Enterococcus faecalis LMT19 - 32, or its culture or its extract.

[0024] The seventh aspect of the present disclosure pertains to a method for preventing or treating cancer in an individual, including the step of administering to the individual an effective amount of the microorganism Enterococcus faecalis LMT19 - 32, or its culture or its extract for treating cancer.

Advantages of the Invention

[0025] The bacterial strain according to the present disclosure is tumor-infiltrating T cells, tumor-infiltrating CD8 T cells, and tumor-infiltrating IFNγ + increase the number of CD8 T cells, thereby exhibiting excellent tumor suppression activity and can be effectively used to prevent or treat tumors.

[0026] In another aspect, according to the Enterococcus faecalis LMT19-32 (accession number KCTC 14306BP) microorganism having antitumor activity according to the present disclosure, or its culture or its extract, it is also used to prevent or treat cancer.

[0027] In still another aspect, according to the pharmaceutical composition for preventing or treating cancer according to the present disclosure, it is also used to prevent or treat cancer.

[0028] In still another aspect, according to the food composition for preventing or improving cancer according to the present disclosure, cancer can be prevented or improved.

[0029] In still another aspect, according to the method for preventing or treating cancer of an individual according to the present disclosure, cancer can be efficiently prevented or treated.

Brief Description of the Drawings

[0030]

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Mode for Carrying Out the Invention

[0031] I. Enterococcus faecium This disclosure is based on the discovery that strains of Enterococcus faecium species, which are characterized by their lactate production ability in microbial growth comparison, exhibit excellent antitumor activity.

[0032] The terms "tumor" and "cancer" are used interchangeably and include, for example, solid and liquid tumors, such as diffuse or circulating tumors. It includes not only precancerous but also malignant cancers and tumors. It also includes primary malignant cells or tumors, and secondary malignant cells or tumors (e.g., metastatic tumors).

[0033] The terms "antitumor" and "anticancer" refer to biological effects that can be demonstrated by various means, including, but not limited to, for example, reduction of tumor size, decrease in the number of tumor cells, reduction of tumor cell proliferation, or reduction of tumor cell survival.

[0034] One aspect of the present disclosure is i) belonging to the species Enterococcus faecium, ii) having a lactate production ability (lactate / OD 600 ) of 3 g / L or more in microbial growth comparison during 48-hour culture, iii) related to a bacterial strain showing antitumor activity.

[0035] In one aspect, when cultured for 24 hours, the bacterial strain also has a lactate production ability (lactate / OD 600 ) exceeding 2.5 g / L in terms of microbial growth comparison.

[0036] In one aspect, the bacterial strain also shows a tumor growth inhibition rate of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more. In a specific example, the bacterial strain also shows a tumor growth inhibition rate of 10% to 40%, 15% to 40%, 20% to 40%, 25% to 40%, 30% to 40%, 35% to 40%.

[0037] In one aspect, the bacterial strain also has (iv) β-galactosidase activity.

[0038] In one aspect, the bacterial strain also has (v) the ability to decompose D-sorbitol.

[0039] In one aspect, the bacterial strain also has (vi) the ability to decompose D-tagatose.

[0040] In one aspect, the bacterial strain also has (vii) the ability to decompose methyl-aD-mannopyranoside.

[0041] In one aspect, the bacterial strain also satisfies three of the aforementioned characteristics (i) to (iii), four of the characteristics (i) to (iv), five of the characteristics (i) to (v), (i) to (iv), (i) to (vi), or (i) to (vii), six of the characteristics (i) to (vi), or (i) to (vii), or seven of the characteristics (i) to (vii).

[0042] In a specific example, the bacterial strain is also one or more of those shown in Table 1 below, but is not limited thereto.

[0043]

Table 1

[0044] The second aspect of the present disclosure relates to a composition for preventing or treating tumors, which contains the above-mentioned bacterial strain as an active ingredient. The terms "treat", "treating" or "treatment" mean, for example, healing of damaged or injured tissues, or applying changes, alterations, enhancements, improvements, refinements and / or embellishments to existing or recognized diseases, disorders or abnormalities to achieve a desired therapeutic result, including reduction or alleviation (including partial reduction, substantial reduction, almost complete reduction, and complete reduction), resolution or prevention (either temporary or permanent) of diseases, disorders or abnormalities.

[0045] The term "prevention" means delaying the onset of a disease, disorder or illness. If the onset of the disease, disorder or illness is delayed for a predetermined period, the prevention can be regarded as complete.

[0046] In one aspect, the bacterial strain contained in the composition can exist as viable cells or dead cells, and can also exist in a dried or lyophilized form.

[0047] In one aspect, the bacterial strain can be used in any form as long as it maintains antitumor activity, including cultures, isolated strains, etc., and they all belong to the scope of the present invention. The term "culture" means a composition containing cultured strains, their metabolites, residual nutrients, etc., obtained by culturing the above-mentioned strain in a medium capable of supplying nutrients for a certain period so that the bacterial strain of the present disclosure can grow and survive in a test tube.

[0048] In one aspect, the composition is also for oral or parenteral administration. The parenteral administration includes, but is not limited to, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intralung administration, rectal administration, intratumoral administration, etc. In a specific example, the composition is also for oral administration.

[0049] The composition is also a pharmaceutical composition, in which case, in addition to the active ingredient, it can be formulated by further including a pharmaceutically acceptable carrier or additive. The carrier or the additive includes excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricating agents, flavoring agents, coloring agents, diluents, dispersants, surfactants, antioxidants, buffer solutions, bacteriostatic agents, etc. The composition is also formulated into dosage forms for injection such as aqueous solutions, suspensions, emulsions, pills, powders, capsules, granules or tablets.

[0050] The composition is also a food or food additive composition, in which case, it also includes a foodologically acceptable diluent or carrier. The diluent is also water, a medium or a buffer solution such as PBS. The carrier is also a general excipient, disintegrant, binder, lubricant, thickening agent or filler. The food is also a health functional food. The food is also beverages, confectioneries, diet bars, chocolates, pizzas, ramen, other noodles, gums, ice creams, etc.

[0051] When the composition is for oral administration, it can be coated with a coating agent to enhance the acid resistance, heat resistance, bile resistance, survival rate, intestinal colonization ability, etc. of the bacterial strain. Coating agents that can be used include enteric coating agents; gelatin, polysaccharides, gums, etc.; water-soluble polymers, hyaluronic acid, porous particles and proteins; casein, coating agents, edible oils, extracellular polymeric substance of Lactobacillus plantarum, and alginic acid; silk fibroin, etc. However, they are not particularly limited thereto, and can be single-coated or multi-coated, for example, double-coated, triple-coated or quadruple-coated.

[0052] In one aspect, the tumor is also a solid tumor. Non-limiting examples of the solid tumor include breast cancer, lung cancer, head or neck cancer, colorectal cancer, esophageal cancer, laryngeal cancer, gastric cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, melanoma in the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer near the anus, rectal cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, lymphocyte lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but are not limited thereto.

[0053] In one aspect, the dosage varies depending on the patient's body weight, age, gender, health status, diet, administration time, administration method, administration period or interval, excretion rate, idiosyncrasy, nature of the formulation, severity of the disease, etc., and can be appropriately selected by those skilled in the art. For example, the bacterial strain as the active ingredient can be administered at 1×10 6 CFU or more, 1×10 7 CFU or more, 1×10 8 CFU or more, 1×10 9 CFU or more. For example, the bacterial strain can be administered at 1×10 15 CFU or less, 1×10 14 CFU or less, 1×1013 CFU or less, 1×10 12 CFU or less can be administered. For example, the bacterial strain can be administered at 1×10 6 ~1×10 15 CFU, 1×10 7 ~1×10 14 CFU, 1×10 8 ~1×10 13 CFU, 1×10 9 ~1×10 12 CFU, 1×10 10 ~1×10 12 CFU, 1×10 11 ~1×10 12 CFU can be administered. For example, the bacterial strain as the active ingredient can be administered once a day or divided into several times a day.

[0054] In one aspect, the composition is also for use in combination with one or more other therapeutic agents, such as anti-cancer agents, anti-viral agents, cytokines or immunomodulators.

[0055] The term "combination" refers to any form of two or more different therapeutic agents such that the second therapeutic agent is administered while the previously administered therapeutic agent is still effective in the body. For example, the two therapeutic agents are effective simultaneously in the subject, which also includes the synergistic effect of the two therapeutic agents. The different therapeutic agents can be administered simultaneously or sequentially, either in a single formulation or in separate formulations.

[0056] In a specific example, the anti-cancer agent is also a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, anti-cancer antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormonal drugs, hormone antagonists, drugs for treating leukopenia (neutropenia), drugs for treating thrombocytopenia, antiemetics, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, other immunotherapeutic drugs, and other anti-cancer drugs. Exemplary cytotoxic agents that can be co-administered include antimicrotubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents that can interfere with signal transduction pathways, agents that promote apoptosis, proteasome inhibitors, and radiation (local or systemic irradiation). Further non-limiting examples of therapeutic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.

[0057] In a specific example, the anti-cancer agent is also an immuno-oncology agent. The term "immuno-oncology agent" means a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and / or reduces the side effects of other anti-cancer therapies. Non-limiting examples of such immuno-oncology agents include cytokines, cancer vaccines, monoclonal antibodies, non-cytokine adjuvants, immune cells (such as T cells, NK cells, dendritic cells, B cells, etc.), immune checkpoint inhibitors, and the like. In a specific example, the immuno-oncology agent is an immune checkpoint inhibitor. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. For example, the immune checkpoint inhibitor is also administered to enhance the proliferative, migratory, persistent and / or cytotoxic activities of CD8+ T cells in a subject, and in particular, the tumor-infiltrating ability of the subject's CD8+ T cells. Typically, the immune checkpoint inhibitor is an antagonist that blocks immunosuppressive receptors expressed by NK cells, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 (PD-1), or various members of the killer cell immunoglobulin-like receptor (KIR) family, activated T lymphocytes, or an antagonist that blocks the major ligands of those receptors, such as the PD-1 ligand CD274 (most commonly known as PD-L1 or B7-H1). For example, the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof. Specifically, the immune checkpoint inhibitor is one or more selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDO1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, anti-B7H6 antibodies, and antigen-binding fragments thereof.More specifically, the immune checkpoint inhibitor is one or more selected from ipilimumab (Yervoy (registered trademark) (BMS / ONO)), tremelimumab (AstraZeneca), atezolizumab (Tecentriq (registered trademark) (Roche)), nivolumab (Opdivo (registered trademark) (BMS / ONO)), pembrolizumab (Keytruda (registered trademark) (MSD)), avelumab (Bavencio (registered trademark) (Firmenich / Merck (Germany)), durvalumab (Imfinzi (registered trademark) (AstraZeneca / MediMune), and antigen-binding fragments thereof, but is not limited thereto.

[0058] In one aspect, the immune anti-cancer agent is formulated into various forms such as a liquid, suspension, powder, granule, tablet, capsule, pill, extract, emulsion, syrup, aerosol, etc. by a conventional method so as to suit each purpose of use. The immune anti-cancer agent is administered orally or parenterally via various routes including local administration or injection into the vein, peritoneal cavity, subcutaneous, intradermal, intramuscular, spinal, intrathecal or rectal. The dosage varies depending on the patient's body weight, age, gender, health status, diet, administration time, administration method, administration period or interval, excretion rate, idiosyncrasy, nature of the formulation, severity of the disease, etc., but can be appropriately selected by those skilled in the art. For example, it is in the range of about 0.1 to about 10,000 mg / kg, but is not limited thereto, and is administered once or divided into several times a day.

[0059] The third aspect of the present disclosure relates to a method for preventing or treating a tumor in a subject, including the step of administering the bacterial strain or the composition to a subject in need of tumor prevention or treatment.

[0060] The term "administration" or "administering" means the stage of providing an active ingredient or a composition containing the same to a subject. The active ingredient or the composition containing the same is administered via various suitable routes.

[0061] The subjects to be administered include, without limitation, humans and animals such as humans, pigs, dogs, cats, cows, horses, mice, etc.

[0062] II. Enterococcus faecalis LMT19-32 The first aspect provides a microorganism, Enterococcus faecalis LMT19-32 (Accession No. KCTC 14306BP), having antitumor activity, or a culture thereof or an extract thereof.

[0063] The microorganism, or a culture thereof or an extract thereof, can suppress tumor growth. The microorganism, or a culture thereof or an extract thereof, can increase the level of immune cells in the tumor. The microorganism, or a culture thereof or an extract thereof, can increase the level of CD8 T cells in the tumor, for example, CD8 T cells expressing IFNγ. The microorganism, or a culture thereof or an extract thereof, can increase the infiltration of immune cells into the tumor. The immune cells are also CD8 T cells, CD4 T cells, NK cells, B cells, dendritic cells, macrophages and neutrophils. The microorganism, or a culture thereof or an extract thereof, can activate immune cells. The activation is also such that the microorganism, or a culture thereof or an extract thereof, promotes the production, secretion, or production and secretion of an antitumor-active cytokine or enzyme. The cytokine or enzyme is also one or more of interferon gamma and granzyme B. The microorganism is excellent in bile resistance and intestinal colonization. The microorganism was isolated from human feces.

[0064] The bile acid resistance is such that when cultured at 37°C for 2 hours in MRS medium containing 0.3% bile acid, the survival rate is 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 75 to 90%, 75 to 95%, 80 to 90%, 80% to 95%, 85% to 90%, or 90 to 95%.

[0065] The microorganism, or its culture or extract, can promote the infiltration of CD8 T cells into tumors. The promotion is such that the percentage of CD8 T cells to the number of T cells in the tumor is increased by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 5 to 100%, 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, or 90 to 100% compared to the case where the microorganism, or its culture or extract, is absent.

[0066] The microorganism, or its culture or extract, can promote the production, secretion, or both production and secretion of one or more of interferon gamma and granzyme B in tumor-infiltrating CD8 T cells. The promotion means that, compared with the case where the microorganism, or its culture or extract, is absent, the percentage of cells producing interferon gamma in CD8 T cells is increased by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%. Also, the promotion means that, compared with the case where the microorganism, or its culture or extract, is absent, the percentage of cells producing granzyme B in CD8 T cells is increased by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%.

[0067] The microorganism, or its culture or extract, can suppress the growth of tumors by CD8 T cells. The suppression reduces the tumor size by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100% compared to the case where the microorganism, or its culture or extract, is absent.

[0068] The second aspect provides a pharmaceutical composition for preventing or treating cancer, containing, as an active ingredient, the aforementioned microorganism, or its culture or extract.

[0069] In the pharmaceutical composition, the cancer is also a solid cancer. The cancer is also a solid cancer existing in other tissues rather than the tissues directly contacted when the microorganism is orally administered. The tissues directly contacted include the oral cavity, esophagus, stomach, duodenum, small intestine, large intestine, and rectum. Also, other tissues rather than the tissues directly contacted are the breast, lung, head, neck, liver, pancreas, bone, fallopian tube, visceral arch, vagina, vulva, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, bladder, kidney, ureter, or CNS (central nervous system). The cancer is also a metastatic cancer. The cancer is, for example, but not limited to, the cancers listed in "I. Enterococcus faecium".

[0070] In the pharmaceutical composition, the composition is also for suppressing the growth of cancer.

[0071] In the pharmaceutical composition, the composition is also for administration together with an immune checkpoint inhibitor. The immune checkpoint inhibitor is also for administration before, simultaneously with, or after administration of the aforementioned microorganism, or its culture or its extract.

[0072] The immune checkpoint inhibitor is, for example, one listed in "I. Enterococcus faecium", but is not limited thereto.

[0073] In the pharmaceutical composition, the composition is also for administration together with a chemotherapeutic agent. The chemotherapeutic agent is also for administration before, simultaneously with, or after administration of the aforementioned microorganism, or its culture or its extract.

[0074] The chemotherapeutic agent is, for example, one listed in "I. Enterococcus faecium", but is not limited thereto.

[0075] In the pharmaceutical composition, the composition also contains a pharmaceutically acceptable carrier. The carrier is also a stabilizer, excipient, diluent or adjuvant. The carrier is, for example, any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate buffered saline (PBS) solution), water, suspensions, emulsions (e.g., oil / water emulsion), various types of wetting agents, liposomes, dispersion media and coating agents suitable for pharmaceutical administration, particularly for oral administration. In pharmaceutical compositions, the use of such media and agents is well known in the art, and compositions containing such carriers are also formulated by well-known general methods.

[0076] The composition also contains the microorganism, or its culture or extract, in a "therapeutically effective amount". In the composition, the "therapeutically effective amount" means an amount sufficient to exhibit a therapeutic effect when administered to an individual in need of treatment, either once or multiple times. The term "treatment" means treating a cancer disease or medical symptoms of cancer in an individual, such as a mammal including a human, and includes the following: alleviating a cancer disease or medical symptoms of cancer; suppressing a cancer disease or medical symptoms of cancer, i.e., delaying or stopping the progression of the disease or medical symptoms of cancer in an individual; or reducing a cancer disease or medical symptoms of cancer in an individual. The "effective amount" can be appropriately selected by those skilled in the art. The "effective amount" is also 0.01 to 50% by weight, or 0.1 to 20% by weight, based on the weight of the composition. Further, the composition can be administered at 1×10 6 CFU / g or more, 1×10 7 CFU / g or more, 1×10 8 CFU / g or more, 1×10 9 CFU / g or more. For example, the bacterial strain can be administered at 1×10 15 CFU / g or less, 1×10 14 CFU / g or less, 1×10 13 CFU / g or less, 1×10 12 CFU / g or less. For example, it can be administered at 1×10 6 CFU / g to 1×10 15 CFU / g, 1×10 7 CFU / g to 1×10 14 CFU / g, 1×10 8 CFU / g to 1×10 13 CFU / g, 1×10 9 CFU / g to 1×10 12 CFU / g, 1×10 10 CFU / g to 1×10 12 CFU / g. For example, it can also contain a bacterial strain at 1×10

[0077] The composition can be administered orally. Accordingly, the composition can also be formulated into various forms such as tablets, capsules, and liquid preparations (e.g., aqueous solutions, dry syrups, or suspensions). In the case of oral tablets, excipients such as lactose and corn starch, and lubricants such as magnesium stearate can usually be added. In the case of oral capsules, lactose and / or dry corn starch are also used as diluents. When an oral aqueous suspension is required, the active ingredient can be combined with an emulsifier and / or a suspending agent. If necessary, specific sweeteners and / or flavoring agents can be added. In one specific example, the composition is also in a dosage form in which the microorganism, or its culture or its extract, can be stabilized in acidity such as gastric juice. For example, the composition is also a capsule containing the microorganism, or its culture or its extract inside, or a tablet in which the microorganism, or its culture or its extract is coated with a film.

[0078] A third aspect provides a food composition for preventing or ameliorating cancer, comprising, as an active ingredient, the aforementioned microorganism, or its culture or its extract.

[0079] The composition also contains a pharmaceutically acceptable carrier. The carrier is also a stabilizer, an excipient, a diluent, or an adjuvant. The carrier is, for example, any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate buffered saline (PBS) solution), water, suspensions, emulsions (e.g., oil / water emulsion), various types of wetting agents, liposomes, dispersion media, and coating agents suitable for pharmaceutical administration, particularly for oral administration. In pharmaceutical compositions, the use of such media and agents is well known in the art, and compositions containing such carriers are also formulated into dosage forms by well-known general methods.

[0080] The food is also a dairy product, a soy product, a vegetable product, a fruit product, or a food additive. The dairy product is also fermented milk, butter, cheese, or powdered milk. The food is also a health functional food. The health functional food is also a health functional food for cancer prevention or improvement. The food is also beverages, confectioneries, diet bars, chocolate, pizza, ramen, other noodles, gums, and ice creams.

[0081] The food also contains components generally added during food production, for example, protein, carbohydrate, fat, nutrients, seasonings, and flavoring agents.

[0082] The carbohydrates used in food production are monosaccharides (e.g., glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.); and polysaccharides (e.g., common sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol). Also, as flavoring agents, natural flavoring agents and synthetic flavoring agents such as saccharin and aspartame can be used. The natural flavoring agent is also a stevia extract such as somatin, rebaudioside A, and glycyrrhizin. The health functional food means that when ingested, it brings specific effects to health.

[0083] In the composition, the microorganism is also 0.01 to 50% by weight, or 0.1 to 20% by weight based on the weight of the composition. Also, the composition can be administered at 1x10 6 CFU / g or more, 1x10 7 CFU / g or more, 1x10 8 CFU / g or more, 1x10 9 CFU / g or more. For example, the bacterial strain can be administered at 1x10 15 CFU / g or less, 1x10 14 CFU / g or less, 1x10 13 CFU / g or less, 1x10 12 CFU / g or less. For example, it can be administered at 1x10 6 CFU / g to 1x10 15CFU / g, 1 x 10 7 CFU / g to 1 x 10 14 CFU / g, 1 x 10 8 CFU / g to 1 x 10 13 CFU / g, 1 x 10 9 CFU / g to 1 x 10 12 CFU / g, 1 x 10 10 CFU / g to 1 x 10 12 It also includes those containing bacterial strains of CFU / g.

[0084] The fourth aspect provides a method for preventing or treating cancer in an individual, which includes administering to the individual an effective amount of the aforementioned microorganism, or its culture or its extract for treating cancer.

[0085] The individual is also a mammal. The mammal is a human or a mammal other than human. The administration can also be oral administration.

[0086] The "effective amount for treating cancer" indicates an effective amount for preventing or treating cancer. The "effective amount for treating cancer" is 0.01 mg to 200 mg of the aforementioned microorganism, or its culture or its extract per kg of body weight, or 0.1 mg to 400 mg of the microorganism, or its culture or its extract per kg of body weight. Also, the "effective amount for treating cancer" can be administered at 1 x 10 6 CFU or more, 1 x 10 7 CFU or more, 1 x 10 8 CFU or more, 1 x 10 9 CFU or more. For example, the bacterial strain can be administered at 1 x 10 15 CFU or less, 1 x 10 14 CFU or less, 1 x 10 13 CFU or less, 1 x 10 12 CFU or less. For example, the bacterial strain can be administered at 1 x 10 6 CFU to 1 x 10 15 CFU, 1 x 10 7 CFU to 1 x 10 14 CFU, 1 x 10 8 CFU to 1 x 1013 CFU, 1 x 10 9 CFU or 1 x 10 12 CFU, 1 x 10 10 CFU or 1 x 10 12 It can be administered at CFU. For example, the bacterial strain as the active ingredient can be administered in two or several divided doses per day.

[0087] The method also includes a step of administering together with an immune checkpoint inhibitor. The immune checkpoint inhibitor can be administered before, simultaneously with, or after the administration of the aforementioned microorganism, or its culture or extract.

[0088] The immune checkpoint inhibitor is, for example, but not limited to, those listed in "I. Enterococcus faecium".

[0089] The method also includes a step of administering together with a chemotherapeutic agent. The chemotherapeutic agent can be administered before, simultaneously with, or after the administration of the aforementioned microorganism, or its culture or extract.

[0090] The chemotherapeutic agent is, for example, but not limited to, those listed in "I. Enterococcus faecium".

[0091] The following shows some desirable embodiments of Enterococcus faecalis LMT19 - 32 microorganism having antitumor activity, but are not limited thereto: 1. Enterococcus faecalis LMT19 - 32 (Accession No. KCTC 14306BP) microorganism having antitumor activity; 2. A pharmaceutical composition for preventing or treating cancer, comprising the microorganism of item 1, or its culture or extract as an active ingredient; 3. In item 2, the composition wherein the cancer is a solid cancer; 4. A composition for administration together with an immune checkpoint inhibitor in item 2; 5. The composition according to item 4, wherein the immune checkpoint inhibitor is a CTLA4 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor; 6. A food composition for preventing or improving cancer, comprising the microorganism of item 1, or a culture or extract thereof, as an active ingredient.

[0092] Hereinafter, the present disclosure will be specifically described with reference to examples, but they are only for helping the understanding of the present disclosure and do not limit the scope of the present disclosure in any way.

[0093] Examples I . Enterococcus faecium Example 1: Isolation and Identification of Strains 1. Isolation of Strains For the isolation of Enterococcus faecium strains, infant feces, adult feces, food samples, etc. were used. From this sample, for the isolation of Enterococcus faecium strains, it was spread on MRS medium (DeMan, Rogosa and Sharpe broth ((Difco Co. (USA))) and anaerobically cultured at 30°C. The sample was aseptically taken, diluted with 180 ml of 0.85% NaCl solution, homogenized with a stomacher for 5 minutes, and then a tube containing 9 ml of 0.85% NaCl solution was sterilized and prepared. The prepared sample was serially diluted into the prepared tube. Then, it was spread on MRS plate medium and cultured at 30°C for 2 - 3 days. The colonies that appeared were distinguished by morphology and color, and were also purely isolated. A total of 8 strains were selected. The selected strains were named LMT17-62, LMT17-74, LMT15-24, LMT17-25, LMT15-4, LMT17-43, LMT17-40, and LMT2-17, respectively.

[0094] 2. Identification of Selected Strains 1) Analysis of Morphological Characteristics The eight selected strains were cultured on MRS agar plates (Difco Co., USA), and the colony morphology was observed. The colony morphologies of the selected strains are shown in Table 2 below.

[0095]

Table 2

[0096] Figure 1 is an optical micrograph of Enterococcus faecium strain LMT17-62 and the standard strain (type strain) KCTC 13225 among the eight selected strains. As shown in Figure 1, the Enterococcus faecium strain LMT17-62 was confirmed to have a typical spherical appearance of the genus Enterococcus.

[0097] 2) 16S rDNA Analysis To analyze the 16S rDNA of the isolated strains, universal bacterial primer 27F (SEQ ID NO: 1) and universal bacterial primer 1492R (SEQ ID NO: 2) were used to amplify the 16S rRNA gene for sequence analysis ((Macrogen, Inc.)). The 16S rDNA sequences of the isolated strains are shown in SEQ ID NOs: 3 to 10 (SEQ ID NO: 3: 16S rDNA sequence of LMT17-62; SEQ ID NO: 4: 16S rDNA sequence of LMT17-74; SEQ ID NO: 5: 16S rDNA sequence of LMT15-24; SEQ ID NO: 6: 16S rDNA sequence of LMT17-25; SEQ ID NO: 7: 16S rDNA sequence of LMT15-4; SEQ ID NO: 8: 16S rDNA sequence of LMT17-40; SEQ ID NO: 9: 16S rDNA sequence of LMT2-17; and SEQ ID NO: 10: 16S rDNA sequence of LMT17-43). The results were analyzed using NCBI blast (http: / / www.ncbi.nlm.nih.gov / ), and as a result, the isolated strains were found to belong to the species Enterococcus faecium.

[0098] The isolated strains were deposited at the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, on August 26, 2020, and were finally named as Enterococcus faecium LMT17-62 (Accession No.: KCTC 14284BP), Enterococcus faecium LMT17-74 (Accession No.: KCTC 14285BP), Enterococcus faecium LMT15-24 (Accession No.: KCTC 14289BP), Enterococcus faecium LMT17-25 (Accession No.: KCTC 14288BP), Enterococcus faecium LMT15-4 (Accession No.: KCTC 14290BP), Enterococcus faecium LMT17-43 (Accession No.: KCTC 14286BP), Enterococcus faecium LMT17-40 (Accession No.: KCTC 14287BP), and Enterococcus faecium LMT2-17 (Accession No.: KCTC 14291BP), respectively.

[0099] Example 2: Evaluation of Antitumor Efficacy of Selected Enterococcus faecium Strains 1. Induction of Tumor Models in Mice and Administration of Enterococcus faecium Strains C57BL / 6 mice (male, 20 - 22 g) used in the mouse tumor induction model were purchased from Orient Bio Inc. and were acclimated to the environment for 1 week before the start of the experiment. 2.5×10 5 cells derived from the colon cancer of C57BL / 6 mice were injected into the subcutaneous tissue of the back of the mice. One week after the tumor injection, the mice were grouped based on the tumor size (50 - 70 mm 3 ). After the grouping, for 2 weeks, the Enterococcus faecium strains were administered once every 2 days using a probe at a dose of 1.0×10 9CFU was administered orally. As a positive control group, an anti-PD1 antibody (clone number: RMP1-14) was intraperitoneally administered to mice at a dose of 10 mg per kg of body weight twice a week. As a negative control group, PBS (phosphate buffered saline) was administered orally.

[0100] The tumor size was measured until the 21st day after inoculation of MC38 tumor cell line into mice. Thereafter, the mice were sacrificed using carbon dioxide, the tumors and immune organs were excised, and tumor-infiltrating immune cell analysis was performed.

[0101] 2. Evaluation of Antitumor Efficacy by Administration of Enterococcus faecium Strains To examine the tumor-suppressing efficacy of Enterococcus faecium strains, tumor suppression experiments were conducted on 8 selected microbial strains.

[0102] The tumor size was measured twice a week starting from 7 days after injection of the tumor cell line. To accurately measure the tumor size, Vernier calipers were used to measure the major axis and minor axis of the tumor, and the tumor size was calculated using the following formula.

[0103] Tumor size = (major axis × (minor axis) 2 ) / 2 The tumor growth inhibition rate (%) was calculated using the following formula. Tumor growth inhibition rate (%) = (1 - V t / V c ) × 100 V c : The average tumor size in the negative control group at the time of tumor size evaluation V t : The average tumor size in the experimental group at the time of tumor size evaluation.

[0104] The results are shown in Fig. 2A. As shown in Fig. 2A, the Enterococcus faecium strains LMT17-62, LMT17-74, LMT15-24, LMT17-25, and LMT15-4 showed tumor growth inhibition rates of 39%, 31%, 25%, 22%, and 15%, respectively. On the other hand, the Enterococcus faecium strains LMT17-40, LMT2-17, and LMT17-43 did not show antitumor efficacy, with tumor growth inhibition rates of 1%, -3%, and -5% or less, respectively. Therefore, it was confirmed that even within the same species, there are differences in antitumor efficacy depending on the strain.

[0105] Fig. 2B shows the tumor sizes in the Enterococcus faecium LMT17-62 strain-administered group until the 21st day when the experiment was terminated after the injection of the tumor cell line. As shown in Fig. 2B, it was confirmed that tumor growth was statistically significantly inhibited in the Enterococcus faecium LMT17-62-administered group and the positive control anti-PD1 antibody-administered group compared to the negative control group.

[0106] 3. Analysis of Tumor-Infiltrating Immune Cells and Evaluation of the Functionality of Immune Cells CD8 T cells, which are tumor-infiltrating immune cells, are important indicators of anti-tumor responses, and interferon gamma secreted by CD8 T cells is a functional cytokine indicating the activity of immune cells. To confirm whether the anti-tumor efficacy of Enterococcus faecium is due to the increase in the above elements, after the animal experiment, the tumors were excised and analyzed. The excised tumors were separated into single cells using RPMI1640 medium containing 50 μg / ml Liberase and 40 μg / ml DNaseI and a cell strainer. The separated cells were stimulated with 50 ng / ml PMA (phorbol 12-myristate 13-acetate) and 500 ng / ml ionomycin for 4 hours in RPMI1640 medium containing 10% fetal bovine serum (FBS). After stimulation, the cells were stained with the antibodies in Table 3 and analyzed using a CANTOII flow cytometer for immune cell analysis and confirmation of interferon gamma production.

[0107]

Table 3

[0108] Figure 2C shows the number of total tumor-infiltrating T cells, the number of CD8 T cells, and interferon gamma-producing CD8 T cells. Compared with the PBS-treated group, in the LMT17-62 strain-treated group, significantly, the tumor-infiltrating T cells and CD8 T cells increased. Also, it was confirmed that the production of interferon gamma, which is a CD8 T cell activator, also significantly increased in the LMT17-62 strain-treated group compared with the PBS control group.

[0109] Example 3: Analysis of Physiological Activity Characteristics of Antitumor-Positive Enterococcus faecium Strains 1. Evaluation of the Growth Curve and Metabolite Production Capacity of Antitumor-Positive Enterococcus faecium Strains To examine the growth pattern of the selected Enterococcus faecium strains, colonies of anti-tumor positive and anti-tumor negative strains were cultured in MRS liquid medium for 24 hours, and then for each strain, the initial OD 600(Optical density) values were inoculated into 50 ml or 250 ml Erlenmeyer flasks so that they matched. Subsequently, for the measurement of the growth curve of the strain, every hour, the OD 600 value of the strain was measured using a spectrophotometer Ultrospec 7000 (Biochrom (UK)). The results are shown in Figure 3A. As shown in Figure 3A, the LMT17-62 strain with antitumor efficacy stopped growing after 8 hours of culture, while the antitumor-negative LMT2-17 strain continued to grow. The amount of lactate, a metabolite of the strain present in the culture broth, was confirmed using a biochemical analyzer YSI2900 (Xylem (USA)).

[0110] The results are shown in Figure 3B. As shown in Figure 3B, in both the LMT17-62 strain with antitumor efficacy and the antitumor-negative LMT2-17 strain, the production of the metabolite lactate increased as the culture continued. Figure 3C shows the lactate production ability per OD 600 . As shown in Figure 3C, the group of Enterococcus faecium strains with antitumor efficacy (LMT17-62, LMT17-74, LMT15-24, LMT15-4, LMT17-25) showed a higher lactate production ability per OD 600 compared to the group of antitumor-negative Enterococcus faecium strains (LMT17-43, LMT17-40, LMT2-17). This became more prominent as the strain culture was continued from 24 hours to 48 hours. That is, at 48 hours of culture, the antitumor-negative strains showed lactate production per OD 600 not reaching 3 g / L, while the antitumor-positive strains consistently showed lactate production per OD 600 of 3 g / L or more, and the higher the lactate production ability per OD 600 , the more likely it was to have a higher antitumor activity.

[0111] 2. Evaluation of Enzyme Activity of Antitumor-Positive Enterococcus faecium Strains To investigate the biochemical properties of the selected Enterococcus faecium strains, the enzyme activity was evaluated using the Rapid ID 32A kit (BioMetrieux, France), and the results are shown in Table 4 below. The Enterococcus faecium strain groups (LMT17-62, LMT17-74, LMT15-24, LMT17-25) with antitumor efficacy different from the antitumor-negative strain group (LMT17-43, LMT17-40, LMT2-17) had β-galactosidase enzyme activity.

[0112]

Table 4

[0113] In Table 4, antitumor strains No.1, No.2, No.3, No.4, and No.5 represent LMT17-62, LMT17-74, LMT15-24, LMT17-25, and LMT15-4 respectively, and control strains No.1, No.2, and No.3 represent LMT17-43, LMT17-40, and LMT2-17 respectively.

[0114] 3. Sugar Fermentation Characteristics of Antitumor-Positive Enterococcus faecium Strains To analyze the sugar metabolism properties of the selected Enterococcus faecium strains, the API 50 CHL kit (BioMetrieux, France) was used, and the evaluation was carried out according to the experimental method of the supplier. The results are shown in Table 5. The Enterococcus faecium strain groups (LMT17-62, LMT17-74, LMT15-24) with antitumor efficacy had the sugar fermentation properties of D-sorbitol and D-tagatose. Also, in the case of the Enterococcus faecium strains (LMT17-62, LMT17-74) with antitumor efficacy, they had the sugar fermentation property of methyl-aD-mannopyranoside.

[0115]

Table 5

[0116] In Table 5, the antitumor strains No. 1, No. 2, No. 3, No. 4, and No. 5 respectively represent LMT17-62, LMT17-74, LMT15-24, LMT17-25, and LMT15-4, and the control strains No. 1, No. 2, and No. 3 respectively represent LMT17-43, LMT17-40, and LMT2-17.

[0117] 4. Evaluation of the Stability of Antitumor-Positive Enterococcus faecium Strains (1) Investigation of Acid Resistance To evaluate the acid resistance of Enterococcus faecium strains, experiments were conducted using the following method. The selected strains were inoculated into sterilized MRS liquid medium and then cultured at 37°C for 16 hours. Next, the pH was adjusted to 2.5 with HCl, inoculated into sterilized MRS liquid medium, cultured at 37°C for 2 hours, and then the viable cell count was confirmed. The viable cell counts before inoculation and 2 hours after inoculation were spread on MRS plate medium and then cultured, the number of colonies was compared and counted, and the results are shown in Figure 4.

[0118] As shown in Figure 4, the antitumor positive Enterococcus faecium strains were shown to have slightly lower acid resistance, but it is also desirable to use capsules, coating agents, etc. during formulation.

[0119] (2) Investigation of Bile Resistance To investigate the bile tolerance of Enterococcus faecium strains, experiments were conducted using the following method. The selected strains were inoculated into sterilized MRS liquid medium and then cultured at 37°C for 16 hours. Considering that the bile salt concentration in the intestinal tract is around 0.1%, only the bacterial cells were added to the MRS liquid medium containing 0.3% bile salts (Sigma (USA)) at a concentration of 10 8 ~10 9After inoculating to reach CFU / ml, it was cultured at 37°C for 2 hours, and then the viable cell count was confirmed. The viable cell counts before and 2 hours after inoculating the strain were spread on MRS plate medium and cultured, and the colony counts were measured. The results are shown in Fig. 5.

[0120] As shown in Fig. 5, since the anti-tumor positive Enterococcus faecium strain maintains an appropriate number of bacteria even at 0.3% which is higher than 0.1% similar to the actual intestinal concentration, it can be a basis for confirming that the selected anti-tumor positive Enterococcus faecium strain can survive sufficiently in the intestines of humans and animals.

[0121] (3) Investigation of Intestinal Colonization To evaluate the intestinal cell adhesion ability of the anti-tumor positive Enterococcus faecium strain, the adenocarcinoma cell Caco-2 cell line (KCLB 30037.1), which is a human rectal epithelial cell purchased from the Korean Cell Line Bank, was used. Caco-2 cells were dispensed to 7×10 4 cells / 100 μl in DMEM (Dulbecco's Modified Eagle's Medium) (Gibco, USA) culture medium containing 10% fetal bovine serum (FBS) (Gibco, USA) and cultured under the conditions of 5% CO2 and 37°C so that a single cell layer was formed on a 96-well plate (Corning, USA).

[0122] On the other hand, the selected anti-tumor positive Enterococcus faecium strain cultured in MRS liquid medium was washed with phosphate buffered saline (PBS), suspended in DMEM medium without added antibiotics, and the amount of Enterococcus faecium strain was 1×10 7Added to become CFU, cultured for 2 hours under the conditions of 5% CO2 and 37°C. To remove the cells not attached to Caco-2 cells, washed 5 times with PBS, detached the attached cells with 100 μl of 0.1% Triton x-100, and then smeared them on MRS solid medium. After culturing at 37°C for 24 hours, the number of colonies on the plate culture was counted to investigate the intestinal colonization of Enterococcus faecium strains.

[0123] Figure 6 shows the number of selected anti-tumor positive Enterococcus faecium strains attached to intestinal epithelial cells. As shown in Figure 6, for intestinal epithelial cells (Caco-2), it was confirmed that LMT17-62 had an intestinal colonization ability of 0.31%, LMT17-74 had 1.55%, LMT15-24 had 0.46%, and LMT17-25 had 0.48%.

[0124] II. Enterococcus faecalis LMT19-32 Example 1: Isolation and Identification of Enterococcus faecalis LMT19-32 Strains 1. Isolation of Enterococcus faecalis Strains Enterococcus faecalis strains were isolated from adult fecal samples. First, the samples were smeared on MRS medium (Difco Co., (USA)) and anaerobically cultured at 30°C. The sample pretreatment method was to take it aseptically, dilute it with 180 ml of 0.85% NaCl solution, and homogenize the fecal stock solution with a stomacher for 5 minutes. The homogenized sample was serially diluted into tubes containing 9 ml of sterilized 0.85% NaCl solution to prepare fecal samples. The samples were smeared on MRS plate medium (Difco Co., (USA)) and cultured at 37°C for 2 to 3 days, and the colonies that appeared were distinguished by morphology and color and further purified.

[0125] 2. Identification of Enterococcus faecalis Strains (1) Analysis of Morphological Characteristics The selected Enterococcus faecalis strains were cultured on MRS agar plates (Difco Co., USA), and colony morphology was observed. The colony morphology of the selected Enterococcus faecalis strains and KCTC3206, which is a standard Enterococcus faecalis strain, on MRS agar plates is shown in Table 6 below.

[0126]

Table 6

[0127] Figure 7 shows representative optical micrographs of the selected Enterococcus faecalis LMT19-32 strain and the standard strain KCTC3206. As shown in Figure 7, the LMT19-32 strain was cocci and was similar in appearance to the typical Enterococcus genus.

[0128] (2) 16S rDNA Analysis The 16S rRNA gene of the isolated LMT19-32 strain was amplified, and the nucleotide sequence of the amplified 16S rRNA gene was analyzed. The amplification was performed by PCR using the genomic DNA of the LMT19-32 strain as a template and a primer set consisting of the oligonucleotide of SEQ ID NO: 11 (Macrogen, Inc.) and the oligonucleotide of SEQ ID NO: 12 (Macrogen, Inc.). The nucleotide sequence of the 16S rDNA of the isolated LMT19-32 strain is shown in SEQ ID NO: 13. The confirmed nucleotide sequence of the 16S rDNA was compared with the known nucleotide sequences of 16S rDNA using NCBI blast (http: / / www.ncbi.nlm.nih.gov / ). As a result, the 16S rDNA of LMT19-32 had 100% sequence identity with the Enterococcus faecalis species. Also, as a result of phylogenetic tree analysis, LMT19-32 was the same as the Enterococcus faecalis species. As a result, it was confirmed that the LMT19-32 strain is a new strain belonging to a new Enterococcus faecalis species.

[0129] The inventors of the present invention named the Lactobacillus LMT19-32 as "Enterococcus faecalis LMT19-32" (Accession No.: KCTC 14306BP), and deposited it with the Korean Cell Line Bank (KCTC: Korean Collection for Type Cultures) located at the Korea Research Institute of Bioscience and Biotechnology on September 9, 2020.

[0130] Example 2: Analysis of Physiological Activity Characteristics of Enterococcus faecalis LMT19-32 Strains 1. Sugar Fermentation Characteristics of Enterococcus faecalis LMT19-32 Strains The sugar metabolism characteristics of the selected LMT19-32 strain were confirmed using an API 50 CHL kit (BioMetrieux, France) according to the experimental method provided by the supplier. Table 7 shows the sugar fermentation characteristics of the confirmed LMT19-32 strain.

[0131]

Table 7

[0132] 2. Evaluation of the Stability of Enterococcus faecalis LMT19-32 Strains In order to evaluate the acid resistance of the Enterococcus faecalis LMT19-32 strain, the following experiment was carried out. After inoculating the LMT19-32 strain into a sterilized MRS liquid medium, it was cultured at 37°C for 16 hours. Next, it was adjusted to pH 2.5 with HCl, 1% of the above strain was inoculated into the sterilized MRS liquid medium, and it was cultured at 37°C for 2 hours. Samples were collected immediately after inoculation of the strain and after 2 hours of culture, diluted in MRS liquid medium, spread on MRS plate medium, cultured at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of bacteria.

[0133] As shown in Table 8, the Enterococcus faecalis LMT19-32 strain is shown to have slightly low acid resistance, but it is also desirable to use capsule agents, coating agents, etc. during formulation.

[0134]

Table 8

[0135] (2) Investigation of Bile Resistance To confirm the effect of bile acids on the growth of Enterococcus faecalis strain LMT19-32, experiments were conducted in the following manner. The selected strain was inoculated into sterilized MRS liquid medium and then cultured at 37°C for 24 hours. Considering that the bile acid concentration in the intestinal tract is around 0.1%, the strain was inoculated at 1% into MRS liquid medium containing 0.3% bile salts (Sigma, USA) and cultured at 37°C for 2 hours. Samples were collected immediately after strain inoculation and after 2-hour culture, diluted in MRS liquid medium, spread on MRS plate medium, cultured at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the viable strain cell count. As a control group, in MRS liquid medium without 0.3% bile hydrochloride, the culture was carried out in the same way and the viable strain cell count was measured. Table 9 shows the results of measuring bile salt tolerance. As shown in Table 9, since the LMT19-32 strain maintained a survival rate of 93% at 0.3%, which is higher than 0.1% similar to the actual intestinal concentration, it provides a basis that the LMT19-32 strain can survive well in the intestines of humans and animals.

[0136]

Table 9

[0137] (3) Investigation of Intestinal Colonization To evaluate the degree of intestinal cell adhesion of Enterococcus faecalis strain LMT19-32, adenocarcinoma cells (human epithelial colorectal adenocarcinoma cell) Caco-2 cell line (KCLB 30037.1), which are human rectal epithelial cells purchased from the Korean Cell Line Bank, were used. Caco-2 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium (Gibco, USA)) containing 10% fetal bovine serum (FBS) (Gibco, USA) under the conditions of 5% CO2 and 37°C at 7x104 Dispensed to a concentration of cells of 100 μl per well, and cultured in a 96-well plate (Corning, USA) to form a monolayer of cells.

[0138] On the other hand, after washing the LMT19-32 strain cultured in MRS liquid medium with PBS, it was suspended in DMEM medium without added antibiotics, and the amount of the LMT19-32 strain was 1×10 7 CFU and added to the Caco-2 cells that constituted the monolayer of cells, and cultured under the conditions of 5% CO2 and 37 °C for 2 hours. In order to remove the cells not attached to the Caco-2 cells, it was washed 5 times with PBS, and the attached cells were detached with 100 μl of 0.1% Triton X-100, and then smeared on MRS solid medium. After culturing at 37 °C for 24 hours, the number of colonies on the plate culture was counted to investigate the intestinal colonization of the LMT19-32 strain.

[0139] Table 10 is a drawing showing the number of Enterococcus faecalis LMT19-32 strain attached to intestinal epithelial cells. As shown in Table 10, it was confirmed that both the novel Enterococcus faecalis LMT19-32 strain of the present invention and the comparative strain Enterococcus faecalis KCTC3206 have the ability to adhere to Caco-2, which is about 1% of intestinal epithelial cells.

[0140]

Table 10

[0141] Example 3: Evaluation of Antitumor Efficacy of Enterococcus faecalis LMT19-32 Strains 1. Induction of Tumor Models in Mice and Administration of Enterococcus faecalis LMT19-32 Strains C57BL / 6 mice (male, 20 - 22 g) used in the mouse tumor induction model were purchased from Orient Bio Inc. and adapted to the environment for 1 week before the start of the experiment. 2.5×10 5 cells derived from the colon cancer of C57BL / 6 mice were injected into the subcutaneous tissue of the back of the mice, and 1 week after the tumor injection, the tumor size (50 - 70 mm 3Based on , the groups were separated. Two weeks after group separation, the Enterococcus faecalis LMT19-32 strain was administered orally to each mouse at a dose of 1x10 9 CFU strain-containing PBS once every two days using a sonde. As a positive control group, an anti-PD1 antibody (clone number: RMP1-14 (manufacturer: Bioxcell (product name: Invivo MAb anti-mouse PD-1))) was intraperitoneally administered to the mice at a dose of 10 mg per kg of body weight twice a week. As a negative control group, PBS was administered orally. The experimental groups were composed of 10 mice each, with a total of 3 groups, and were configured as shown in Table 11.

[0142]

Table 11

[0143] After inoculating MC38 tumor cell line into the mice, the tumor size was measured until the 21st day. Then, using carbon dioxide, the mice were sacrificed, the tumors were excised, and tumor-infiltrating immune cell analysis was performed. The analysis results are shown in FIGS. 9A, 9B, and 9C. FIGS. 9A, 9B, and 9C show the results of analyzing T cells, CD8 T cells, and CD8 T cells expressing IFNγ, that is, IFNγ + CD8 T cells, after administering the Enterococcus faecalis LMT19-32 strain to mice with tumors. The MC38 tumor cell line is a mouse colon adenocarcinoma cell induced by subcutaneous injection of dimethylhydrazine into C57BL / 6 mice. The mouse tumor induction model used in this example shows that since the MC38 tumor cells are transplanted into the subcutaneous tissue, the effect of the orally administered strain can show cancer growth inhibition in other tissues that are not in direct contact with the strain, such as subcutaneous tissue, indicating that the influence of the bacteria can act not only on cancers related to the gastrointestinal tract or intestine, which are tissues in direct contact with the strain, but also on various solid carcinomas.

[0144] 2. Evaluation of Antitumor Efficacy by Administration of Enterococcus faecalis LMT19-32 Strains Figures 8A and 8B are drawings showing the results of measuring the tumor size after administering the Enterococcus faecalis LMT19-32 strain to mice with tumors. The administration was carried out according to Section 1. Specifically, the tumor size was measured twice a week from 7 days to 21 days after the injection of the tumor cell line. To measure the exact tumor size, Vernier calipers were used to measure the major axis and minor axis of the tumor, and the tumor size was calculated using the formula "major axis x (minor axis) 2 / 2". In Figures 8A and 8B, the control group was administered PBS as a negative control group, aPD1 was administered an anti-PD1 antibody as a positive control group, and LMT19-32 was administered LMT19-32 as an experimental group. The graph in Figure 8A shows the tumor size according to the number of days elapsed after cell injection, and Figure 8B shows the tumor size on the 21st day after cell injection. In Figure 8A, the horizontal axis measures the tumor size and indicates the number of days elapsed after tumor cell administration. In Figure 8B, the bars represent the tumor size for each individual on the 21st day after cell injection. As shown in Figures 8A and 8B, statistically significant tumor growth inhibition has been observed in the group administered the LMT19-32 strain alone.

[0145] 3. Tumor-infiltrating immune cell analysis and functional evaluation of immune cells CD8 T cells, which are tumor-infiltrating immune cells, are important indicators of anti-cancer responses. Interferon gamma and granzyme B secreted by CD8 T cells are functional cytokines that indicate the activation ability of immune cells. Figures 9A, 9B, and 9C show the results of analyzing CD8 T cells infiltrated into tumors and cytokine secretion therefrom after administering the Enterococcus faecalis LMT19-32 strain to mice with tumors. The administration was performed according to Section 1. Twenty-one days after administration, the tumors were excised. The excised tumors were separated into single cells using RPMI1640 medium containing 50 μg / ml Liberase and 40 μg / ml DNaseI and a cell strainer. To observe the interferon gamma production pattern of T cells, the separated cells were stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin for 4 hours in RPMI1640 medium containing 10% fetal bovine serum (FBS). The substances PMA and ionomycin are substances that provide signal stimulation so that T cells are activated, play a role like the actual antigen's T cell activation mechanism, and create an environment for an immune reaction to occur. Ionomycin, as an ionophore, increases protein kinase C (PKC), and in the case of PMA, phosphorylates PKC, thus playing a role of synergizing to target CD4 T cells and CD8 T cells. After stimulation, the cells were stained with the antibodies in Table 12 for immune cell analysis and confirmation of interferon gamma production, and analyzed using a CANTO II flow cytometry instrument. 2+ As an ionophore, it increases protein kinase C (PKC), and in the case of PMA, phosphorylates PKC, thus playing a role of synergizing to target CD4 T cells and CD8 T cells. After stimulation, the cells were stained with the antibodies in Table 12 for immune cell analysis and confirmation of interferon gamma production, and analyzed using a CANTO II flow cytometry instrument.

[0146]

Table 12

[0147] Figures 9A, 9B, and 9C show the number of total tumor-infiltrating T cells, the number of CD8 T cells in tumor-infiltrating T cells, and the number of IFNγ-expressing cells in CD8 T cells, respectively. As shown in Figure 9, compared with the PBS-treated group, the number of tumor-infiltrating T cells and the number of CD8 T cells significantly increased in the LMT19-32-administered group. Also, the number of CD8 T cells that produce the active factor interferon gamma significantly increased in the LMT19-32-treated group compared with the PBS control group.

[0148] TIFF2025094054000014.tif172170TIFF2025094054000015.tif172170TIFF2025094054000016.tif172170TIFF2025094054000017.tif172170TIFF2025094054000018.tif172170TIFF2025094054000019.tif172170TIFF2025094054000020.tif172170TIFF2025094054000021.tif172170TIFF2025094054000022.tif172170

Claims

1. i) belongs to the species Enterococcus faecium; ii) Lactate production capacity (lactate / OD) relative to microbial growth after 48 hours of culture ６００ ) is 3 g / L or more, iii) Bacterial strains exhibiting anti-tumor activity.

2. Lactate production capacity (lactate / OD) relative to microbial growth during 24-hour incubation ６００ 2. The bacterial strain of claim 1, wherein the total amount of ATP is greater than 2.5 g / L.

3. The bacterial strain of claim 1 or 2, which exhibits a tumor growth inhibition rate of 10% or more.

4. The bacterial strain according to any one of claims 1 to 3, which exhibits a tumor growth inhibition rate of 20% or more.

5. iv) A bacterial strain according to any one of claims 1 to 4, having β-galactosidase activity.

6. The bacterial strain according to any one of claims 1 to 5, which has a D-sorbitol decomposition ability.

7. vi) The bacterial strain according to any one of claims 1 to 6, having an ability to decompose D-tagatose.

8. vii) A bacterial strain according to any one of claims 1 to 7, having an ability to degrade methyl-aD-mannopyranoside.

9. 3. The bacterial strain according to claim 1 or 2, which is one or more selected from the following group: LMT17-62, deposited under accession number KCTC 14284BP; LMT17-74, deposited under accession number KCTC 14285BP; LMT15-24, deposited under accession number KCTC 14289BP; LMT17-25, deposited under accession number KCTC 14288BP; and LMT15-4, deposited under accession number KCTC 14290BP.

10. A composition for preventing or treating tumors, comprising as an active ingredient a bacterial strain according to any one of claims 1 to 9.

11. The composition of claim 10, which is for oral administration.

12. The composition of claim 10 or 11, wherein the bacterial strain is live or killed.

13. The composition of any one of claims 10 to 12, wherein the tumor is a solid tumor.

14. The bacterial strain was diluted to 1 × 10 ６ 14. The composition of any one of claims 10 to 13, comprising CFU or more.

15. 15. A composition according to any one of claims 10 to 14, for use in combination with one or more other therapeutic agents.

16. The composition of claim 15, wherein the therapeutic agent is an immunological anti-cancer agent.

17. The composition of claim 16, wherein the immune anti-cancer agent is an immune checkpoint inhibitor.

18. The composition of claim 17, wherein the immune checkpoint inhibitor is a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, or a combination thereof.

19. The composition of claim 18, wherein the immune checkpoint inhibitor is one or more selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

20. 20. A composition according to any one of claims 14 to 19, for administration simultaneously or sequentially with another therapeutic agent.

21. 21. A method for preventing or treating a tumor in a subject, comprising administering to a subject in need of such prevention or treatment a bacterial strain described in any one of claims 1 to 9 or a composition described in any one of claims 10 to 20.

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