Lactobacillus plantarum microorganism having anticancer activity, composition containing same, and method for preventing or treating cancer using same

Abstract

Provided are Lactobacillus plantarum microorganisms having anti-cancer activity, compositions containing the same, and methods for preventing or treating cancer using the same.

Description

[Technical field]

[0001] The present invention relates to a Lactobacillus plantarum microorganism having anticancer activity, a composition containing the same, and a method for preventing or treating cancer using the same. [Background technology]

[0002] Lactobacillus plantarum belongs to the genus Lactobacillus and is occasionally found in fermented food products and anaerobic plant materials. It is a Gram-positive, rod-shaped bacterium. It has a rounded end, is straight, and is generally rod-shaped, 0.9-1.2 μm wide and 3-8 μm long. It can grow at pH 3.4-8.8 and at temperatures between 12°C and 40°C.

[0003] Lactobacillus plantarum has antioxidant activity and helps maintain intestinal permeability. It is also effective in treating irritable bowel syndrome (IBS). For example, Korean Patent Publication 10-2011-0000871 discloses Lactobacillus plantarum HY7711 with antioxidant activity.

[0004] In cancer treatment, immunotherapy is a mechanism that attacks cancer cells through immune activation, but since only 20 to 30% of target patients respond, there is still a demand for efficient immunotherapy. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect provides a Lactobacillus plantarum LMT17-31 (Accession No. KCTC-14283BP) microorganism, or a culture thereof or an extract thereof, having anti-tumor activity.

[0006] Another embodiment provides a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, said microorganism, or a culture thereof or an extract thereof. Another embodiment provides a food composition for preventing or ameliorating cancer, comprising the microorganism, or a culture thereof or an extract thereof, as an active ingredient. Another aspect provides a method of preventing or treating cancer in an individual comprising administering to the individual an amount of said microorganism, or a culture or extract thereof, effective to treat the cancer. [Means for solving the problem]

[0007] A first aspect provides a Lactobacillus plantarum LMT17-31 (Accession No. KCTC-14283BP) microorganism, or a culture thereof or an extract thereof, having antitumor activity.

[0008] The microorganism, or its culture or extract thereof, can suppress tumor growth. In this specification, the terms "cancer" and "tumor" are used interchangeably. The microorganism, or its culture or extract thereof, can increase the level of intratumoral immune cells. The microorganism, or its culture or extract thereof, can increase the level of intratumoral CD8 T cells. The microorganism, or its culture or extract thereof, can increase the infiltration of immune cells into tumors. The immune cells can be CD8 T cells, CD4 T cells, NK cells, B cells, dendritic cells, macrophages, and neutrophils. The microorganism, or its culture or extract thereof, can activate immune cells. The activation can also promote the production, secretion, or production and secretion of cytokines or enzymes having antitumor activity by the microorganism, or its culture or extract thereof. The cytokines or enzymes can be one or more of interferon gamma and granzyme B. The microorganism has excellent acid resistance, bile resistance, and intestinal colonization. The microorganism was isolated from makgeolli.

[0009] The acid resistance is such that, when cultured in MRS medium at pH 2.5 and 37°C for 2 hours, the viability is 40% or more, 45% or more, 50% or more, 60% or more, 80 to 90%, 80% to 95%, 85% to 90%, or 90 to 95%.

[0010] The bile acid resistance is a viability of 65% or more, 70% or more, 75% or more, 80% or more, 90% or more, 95% or more, 75 to 90%, 75 to 95%, 80 to 90%, 80% to 95%, 85% to 90%, or 90 to 95% when cultured in MRS medium containing 0.3% bile acid at 37°C for 2 hours.

[0011] The microorganism, or a culture thereof, or an extract thereof, can promote the infiltration of CD8 T cells into tumors, and the promotion can also be an increase in the percentage of CD8 T cells relative to the number of T cells in the tumor 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 absence of the microorganism, or a culture thereof, or an extract thereof.

[0012] The microorganism, or a culture thereof or an extract thereof, may enhance the production, secretion, or production and secretion of one or more of interferon gamma and granzyme B by tumor-infiltrating CD8 T cells. The promotion may also be an increase in the percentage of cells producing interferon gamma in CD8 T cells 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 absence of the microorganism, or a culture thereof or an extract thereof. The promotion also refers to an increase in the percentage of cells producing granzyme B in CD8 T cells 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 absence of the microorganism, or a culture thereof or an extract thereof.

[0013] The microorganism, or a culture thereof or an extract thereof, is capable of suppressing tumor growth by CD8 T cells, the suppression also being such that the tumor size is reduced 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 absence of the microorganism, or a culture thereof or an extract thereof.

[0014] In a second aspect, there is provided a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the microorganism, or a culture thereof or an extract thereof. In the pharmaceutical composition, the cancer may be a solid cancer. The cancer may be a solid cancer not located in an internal organ. The cancer may be a metastatic cancer. Non-limiting examples of such cancers include, but are not limited to, breast cancer, lung cancer, head or neck cancer, colon cancer, esophageal cancer, laryngeal cancer, stomach cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, para-anal cancer, colon cancer, breast cancer, tracheal carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumors, primary CNS lymphomas, spinal cord tumors, brain stem gliomas, and pituitary adenomas.

[0015] In the pharmaceutical composition, the composition is also for suppressing the growth of cancer. In the pharmaceutical composition, the composition is also for administration together with an immune checkpoint inhibitor, which is for administration before, simultaneously with, or after administration of the microorganism, or a culture thereof or an extract thereof.

[0016] The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-IDO1 antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody, and antigen-binding fragments thereof. More specifically, the immune checkpoint inhibitor is ipilimumab (Yervoy). 登録商標 (BMS / ONO)), Tremelimumab (AstraZeneca), Atezolizumab (Tecentriq) 登録商標 (Roche), nivolumab (Opdivo 登録商標 (BMS / ONO)), pembrolizumab (Kitoiruda) 登録商標 (MSD)), Avelumab (Bavencio 登録商標 (Pfizer / Merck, Germany)), Durvalumab (Imfinzi) 登録商標 (AstraZeneca / MedImmune)), and antigen-binding fragments thereof, but are not limited thereto.

[0017] In said pharmaceutical composition, said composition is also for administration in conjunction with a chemotherapeutic agent, said chemotherapeutic agent being for administration before, simultaneously with, or after administration of said microorganism, or a culture thereof or an extract thereof.

[0018] Non-limiting examples of the chemotherapeutic agents include alkylating agents, nitrosoureases, antimetabolites, anti-cancer antibiotics, alkaloids of plant origin, topoisomerase inhibitors, hormonal drugs, hormone antagonists, drugs for treating leukopenia (e.g., 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 may be co-administered include anti-microtubule acting agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents that may interfere with signal transduction pathways, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or total body radiation). Non-limiting examples of additional 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.

[0019] In the pharmaceutical composition, the composition also includes a pharma- ceutically acceptable carrier. The carrier may be a stabilizer, excipient, diluent, or adjuvant. The carrier may be, for example, any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate-buffered saline (PBS) solutions), water, suspensions, emulsions (e.g., oil / water emulsions), various types of wetting agents, liposomes, dispersion media, and coating agents suitable for pharmaceutical administration, particularly oral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers may be formulated by well-known general methods.

[0020] The composition also includes the microorganism, or a culture thereof, or an extract thereof, in a "therapeutically effective amount". In the composition, the "therapeutically effective amount" means an amount sufficient to show a therapeutic effect when administered to an individual in need of treatment in one or more doses. The term "treatment" means treating a cancer disease or a medical symptom of cancer in an individual, for example, a mammal, including a human, including the following: alleviating a cancer disease or a medical symptom of cancer; inhibiting a cancer disease or a medical symptom of cancer, i.e., slowing or stopping the progression of a disease or medical symptom in an individual; or alleviating a cancer disease or a medical symptom in an individual. The "effective amount" can be appropriately selected by a person skilled in the art. The "effective amount" can be 0.01 to 50% by weight, or 0.1 to 20% by weight based on the weight of the composition. The composition can also be 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, a bacterial strain can be administered at a concentration of 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, 1x10 6 CFU / g or 1x10 15 CFU / g, 1x10 7 CFU / g or 1x10 14 CFU / g, 1x10 8 CFU / g or 1x10 13 CFU / g, 1x10 9 CFU / g or 1x10 12 CFU / g, 1x10 10 CFU / g or 1x10 12 It also contains CFU / g of bacterial strains.

[0021] The composition may be administered orally. Thus, the composition may be formulated in various forms such as tablets, capsules, aqueous liquids, dry syrups or suspensions. For oral tablets, excipients such as lactose, corn starch, and lubricants such as magnesium stearate may be added in general. For oral capsules, lactose and / or dry corn starch may be used as diluents. When an oral aqueous suspension is required, the active ingredient may be combined with an emulsifier and / or suspending agent. If necessary, specific sweeteners and / or flavoring agents may be added. In one embodiment, the composition may be in a dosage form that can stabilize the microorganism, or a culture thereof or an extract thereof, in acidic conditions such as gastric juice. For example, the composition may be a capsule in which the microorganism, or a culture thereof or an extract thereof is contained, or a tablet in which the microorganism, or a culture thereof or an extract thereof is coated with a coating.

[0022] A third aspect provides a food composition for preventing or ameliorating cancer, comprising as an active ingredient the above-mentioned microorganism, or a culture thereof, or an extract thereof.

[0023] The composition also includes a food-acceptable carrier. The carrier may be a stabilizer, excipient, diluent or adjuvant. The carrier may be, for example, any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate-buffered saline (PBS) solutions), water, suspensions, emulsions (e.g., oil / water emulsions), various types of wetting agents, liposomes, dispersion media and coating agents suitable for pharmaceutical administration, particularly oral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers may be formulated by well-known general methods.

[0024] The food may be a dairy product, a soy product, a vegetable or fruit product, or a food additive. The dairy product may be fermented milk, butter, cheese, or powdered milk. The food may be a functional health food. The functional health food may be a functional health food for cancer prevention or cancer improvement. The food may also be beverages, confectionery, diet bars, chocolates, pizza, ramen, other noodles, gums, or ice cream. The food products may also include ingredients that are commonly added during food production, including, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Carbohydrates used in food manufacturing include 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, and erythritol. In addition, natural flavors and synthetic flavors such as saccharin and aspartame can be used as flavorings. The natural flavors are also stevia extracts such as thaumatin, rebaudioside A, and glycylrhizin. Health functional foods refer to those that, when ingested, bring about a specific health effect.

[0025] In the composition, the microorganism is present in an amount of 0.01 to 50% by weight, or 0.1 to 20% by weight, based on the weight of the composition. 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, a bacterial strain can be administered at a concentration of 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, 1x10 6 CFU / g or 1x10 15 CFU / g, 1x10 7 CFU / g or 1x10 14CFU / g, 1x10 8 CFU / g or 1x10 13 CFU / g, 1x10 9 CFU / g or 1x10 12 CFU / g, 1x10 10 CFU / g or 1x10 12 It also contains CFU / g of bacterial strains.

[0026] A fourth aspect provides a method of preventing or treating cancer in an individual comprising administering to the individual an amount of said microorganism, or a culture thereof or an extract thereof, effective to treat the cancer. The individual may be a mammal. The mammal may be a human or a non-human mammal. The administration may be oral. The "effective amount for treating cancer" refers to an amount effective for preventing or treating cancer. The "effective amount for treating cancer" may be 0.01 mg to 200 mg of the microorganism, or a culture thereof, or an extract thereof, per kg of body weight, or 0.1 mg to 400 mg of the microorganism, or a culture thereof, or an extract thereof, per kg of body weight. The "effective amount for treating cancer" may be 1 x 10 6 CFU or more, 1x10 7 CFU or more, 1x10 8 CFU or more, 1x10 9 For example, a bacterial strain can be administered at a concentration of 1x10 15 CFU or less, 1x10 14 CFU or less, 1x10 13 CFU or less, 1x10 12 CFU or less. For example, a bacterial strain may be administered at a concentration of 1x10 6 CFU or 1x10 15 CFU, 1x10 7 CFU or 1x10 14 CFU, 1x10 8 CFU or 1x10 13 CFU, 1x10 9 CFU or 1x10 12 CFU, 1x10 10 CFU or 1x10 12For example, the active ingredient, a bacterial strain, can be administered twice or several times a day.

[0027] The method may also include co-administering an immune checkpoint inhibitor, either before, simultaneously with, or after administration of the microorganism, or a culture or extract thereof. The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-IDO1 antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody, and antigen-binding fragments thereof. More specifically, the immune checkpoint inhibitor is ipilimumab (Yervoy). 登録商標 (BMS / ONO)), Tremelimumab (AstraZeneca), Atezolizumab (Tecentriq) 登録商標 (Roche), nivolumab (Opdivo 登録商標 (BMS / ONO)), pembrolizumab (Kitoiruda) 登録商標 (MSD)), Avelumab (Bavencio 登録商標 (Pfizer / Merck, Germany)), Durvalumab (Imfinzi) 登録商標 (AstraZeneca / MedImmune)), and antigen-binding fragments thereof, but are not limited thereto.

[0028] The method may also include the step of co-administering a chemotherapeutic agent, which may be administered before, simultaneously with, or after administration of the microorganism, or a culture or extract thereof. Non-limiting examples of the chemotherapeutic agents include alkylating agents, nitrosoureases, antimetabolites, anti-cancer antibiotics, alkaloids of plant origin, topoisomerase inhibitors, hormonal drugs, hormone antagonists, drugs for treating leukopenia (e.g., 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 may be co-administered include anti-microtubule acting agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents that may interfere with signal transduction pathways, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or total body radiation). Non-limiting examples of additional 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. Effect of the Invention

[0029] According to one embodiment, the Lactobacillus plantarum LMT17-31 (Accession No. KCTC-14283BP) microorganism, or a culture thereof or an extract thereof, having antitumor activity may be used to prevent or treat cancer. According to another embodiment, the pharmaceutical composition for preventing or treating cancer can be used to prevent or treat cancer. According to another embodiment of the food composition for preventing or ameliorating cancer, cancer can be prevented or ameliorated. According to another embodiment of the method for preventing or treating cancer in an individual, cancer can be effectively prevented or treated. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows representative optical microscope photographs of the selected Lactobacillus plantarum LMT17-31 strain and the standard strain KCTC3108 strain. [Figure 2A] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice and then measuring tumor size. [Figure 2B] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice and then measuring tumor size. [Figure 3A] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. [Figure 3B] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. [Figure 3C] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. [Figure 3D] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. [Figure 3E] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. [Figure 3F] FIG. 1 shows the results of administering Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice, and then analyzing tumor-infiltrating CD8 T cells and their cytokine secretion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0032] Example 1: Isolation and identification of Lactobacillus plantarum LMT17-31 strain 1. Isolation of Lactobacillus plantarum strains A Lactobacillus plantarum strain was isolated from a makgeolli sample. First, the sample was smeared on MRS medium (Difco, USA) and cultured anaerobically at 30°C. The sample was pretreated by taking a sample under sterile conditions, diluting it with 180 ml of 0.85% NaCl solution, and homogenizing the original makgeolli solution for 5 minutes using a stomacher. The homogenized sample was serially diluted in a tube containing 9 ml of sterilized 0.85% NaCl solution to prepare a makgeolli sample. The sample was smeared on an MRS plate medium (Difco, USA) and cultured at 37°C for 2 to 3 days, and the colonies that appeared were classified by morphology and color, and further purified.

[0033] 2. Identification of Lactobacillus plantarum Strains (1) Morphological analysis The selected Lactobacillus plantarum strains were cultured on MRS plate medium (Difco, USA) and the colony morphology was observed. The colony morphology of the selected Lactobacillus plantarum strains and the standard strain KCTC3108 on MRS plate medium is shown in Table 1 below. [Table 1]

[0034] Representative optical micrographs of the selected Lactobacillus plantarum LMT17-31 strain and the standard strain KCTC3108 are shown in Figure 1. As shown in Figure 1, the LMT17-31 strain is a rod-shaped bacillus, which is similar to the typical characteristics of the genus Lactobacillus.

[0035] (2)16S rDNA analysis The 16S rRNA gene of the isolated LMT17-31 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 LMT17-31 strain as a template and the oligonucleotide of SEQ ID NO: 1 (Macrogen Co., Ltd.) and the oligonucleotide of SEQ ID NO: 2 (Macrogen Co., Ltd.) as a primer set. The nucleotide sequence of the 16S rDNA of the isolated LMT17-31 strain is shown in SEQ ID NO: 3. The confirmed nucleotide sequence of the 16S rDNA was compared with the nucleotide sequence of the publicly known 16S rDNA using NCBI blast (http: / / www.ncbi.nlm.nih.gov / ). As a result, the 16S rDNA of LMT17-31 had 100% sequence identity with the species Lactobacillus plantarum. In addition, the phylogenetic tree analysis showed that LMT17-31 was the same as the species Lactobacillus plantarum. As a result, the LMT17-31 strain was identified as a new strain belonging to the new Lactobacillus plantarum species.

[0036] The inventors named the LMT17-31 lactic acid bacterium "Lactobacillus plantarum LMT17-31" (deposit number: KCTC 14283BP) and deposited it at the Korean Collection for Type Cultures (KCTC), Korea Institute of Bioscience and Biotechnology, on August 26, 2020.

[0037] Example 2: Analysis of the bioactive properties of Lactobacillus plantarum LMT17-31 strain 1.Sugar fermentation characteristics of Lactobacillus plantarum LMT17-31 The sugar metabolism characteristics of the selected LMT17-31 strain were confirmed using API 50 CHL kit (bioMerieux BioMetrieux (France)) according to the supplier's experimental method. Table 2 shows the confirmed sugar fermentation characteristics of the LMT17-31 strain. [Table 2] TIFF0007599085000003.tif19170

[0038] 2. Stability evaluation of Lactobacillus plantarum LMT17-31 strain (1) Acid resistance test To evaluate the acid resistance of Lactobacillus plantarum LMT17-31 strain, an experiment was carried out as follows. The LMT17-31 strain was 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, and 1% of the strain was inoculated into sterilized MRS liquid medium and cultured at 37°C for 2 hours. Samples were collected immediately after inoculation and after 2 hours of culture, diluted in MRS liquid medium, smeared on MRS plate medium, and cultured at 37°C for 24 hours. The number of colonies on the plate medium was counted, and the number of bacteria was measured. As shown in Table 3, the Lactobacillus plantarum LMT17-31 strain exhibited 44.2% acid resistance at pH 2.5, and the standard strain KCTC3108 had 67.1% acid resistance. [Table 3]

[0039] (2) Bile tolerance test In order to confirm the effect of bile acid on the growth of Lactobacillus plantarum LMT17-31 strain, the following experiment was carried out. The selected strain was inoculated into sterilized MRS liquid medium and then cultured at 37℃ for 24 hours. Considering that the bile salt concentration in the intestinal tract is about 0.1%, the strain was inoculated at 1% into MRS liquid medium containing 0.3% bile salts (Sigma, USA) and cultured at 37℃ for 2 hours. Samples were collected immediately after inoculation and after 2 hours of culture, diluted in MRS liquid medium, smeared on MRS plate medium, and cultured at 37℃ for 24 hours, and the number of colonies on the plate medium was counted to measure the number of surviving strain cells. As a control group, the same culture was carried out in MRS liquid medium not containing 0.3% bile hydrochloric acid, and the number of surviving strain cells was measured. Table 4 shows the results of measuring bile salt resistance. As shown in Table 4, the LMT17-31 strain maintained a survival rate of 66.4% at 0.3%, which is higher than the actual concentration in the intestine of 0.1%, providing evidence that the LMT17-31 strain can survive sufficiently in the intestines of humans and animals. [Table 4]

[0040] (3) Intestinal adhesion To evaluate the colonization ability of Lactobacillus plantarum LMT17-31 strain in intestinal cells, human rectal epithelial colorectal adenocarcinoma cell line Caco-2 (KCLB 30037.1) was used. The Caco-2 cells were cultured at 7x10 in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) at 5% CO2 and 37℃. 4 The mixture was dispensed at a cell / 100 μl and cultured in a 96-well plate (Corning, USA) so that a cell monolayer was formed.

[0041] The LMT17-31 strain cultured in MRS liquid medium was washed with PBS and then suspended in antibiotic-free DMEM medium to form a monolayer of Caco-2 cells. The LMT17-31 strain was then added at a concentration of 1x10 7 The cells were added to the plate to obtain a total of 1000 CFU and incubated at 37°C for 2 hours in 5% CO2. To remove cells that could not adhere to the Caco-2 cells, the cells were washed five times with PBS, and the cells were detached from the plate with 100 μl of 0.1% Triton X-100 and then plated on MRS solid medium. After incubation at 37°C for 24 hours, the number of colonies on the plate was counted to investigate the intestinal colonization of the LMT17-31 strain.

[0042] Table 5 shows the number of Lactobacillus plantarum LMT17-31 strain attached to intestinal epithelial cells. As shown in Table 5, it was confirmed that the novel Lactobacillus plantarum LMT17-31 strain of the present invention has an adhesion ability to about 2% of intestinal epithelial cells, Caco-2, and the comparative strain Lactobacillus plantarum KCTC3108 has an adhesion ability to about 1% of intestinal epithelial cells. [Table 5]

[0043] Example 3: Evaluation of the antitumor efficacy of Lactobacillus plantarum LMT17-31 strain 1. Tumor model induction in mice and administration of Lactobacillus plantarum LMT17-31 C57BL / 6 mice (male, 20-22 g) used in the mouse tumor induction model were purchased from Orient Bio Co., Ltd. and allowed to adapt to the environment for one week before the start of the experiment. MC38 cells (derived from colon cancer in C57BL / 6 mice) (2.5 x 10 5 One week after tumor injection, the tumor size (50-70 mm) was measured. 3 For two weeks after group separation, Lactobacillus plantarum LMT17-31 strain was administered to each mouse at 1x10 9 PBS containing CFU strain was orally administered to the mice, and as a positive control group, anti-PD1 antibody (clone number: RMP1-14 (manufacturer: Bioxcell) (product name: InvivoMAb anti-mouse PD-1) was intraperitoneally administered at 10 mg per kg of mouse weight twice a week. As a negative control group, PBS was orally administered. There were a total of 4 experimental groups, each with 8 mice, as shown in Table 6. [Table 6]

[0044] The tumor size was measured up to 23 days after the injection of MC38 tumor cell line into the mice, and then the mice were sacrificed using carbon dioxide, the tumors were excised, and tumor-infiltrating immune cell analysis was performed. The analysis results are shown in Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, and Figure 3F. Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, and Figure 3F show the results of the analysis of tumor-infiltrating CD8 T cells and their cytokine secretion after the administration of Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice. 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 involved transplanting MC38 tumor cells into the subcutaneous tissue, demonstrating that the orally administered bacteria can suppress cancer growth in the subcutaneous tissue, indicating that the bacteria can have an effect not only on intestinal-related cancers but also on various solid cancers.

[0045] 2. Evaluation of antitumor efficacy by administration of Lactobacillus plantarum LMT17-31 strain Figures 2A and 2B show the results of measuring tumor size after administration of Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice. The administration was performed according to Section 1. Specifically, the tumor size was measured twice a week from 7 days after tumor cell line injection until the 23rd day. To measure the exact tumor size, a Vernier caliper was used to measure the long and short axes of the tumor, and the long axis x (short axis) was calculated. 2Tumor size was calculated by the formula: ρ / 2. In Figures 2A and 2B, the control group is a negative control group administered with PBS, aPD1 is a positive control group administered with anti-PD1 antibody, LMT17-31 is an experimental group administered with LMT17-31, and LMT17-31+aPD1 is an experimental group administered with LMT17-31 and anti-PD1 antibody. Figure 2A shows the tumor size according to the number of days after cell injection, and Figure 2B shows the tumor size on the 23rd day after cell injection. In Figure 2A, the horizontal axis indicates the day the tumor size was measured, and indicates the number of days after tumor cell injection. In Figure 2B, the bars represent the tumor size of each individual on the 23rd day after cell injection. As shown in Figures 2A and 2B, statistically significant tumor growth inhibition was observed in both the group administered with the LMT17-31 strain alone and the group administered with the LMT17-31 strain in combination with an anti-PD1 antibody.

[0046] 3. Analysis of tumor-infiltrating immune cells and evaluation of immune cell functionality CD8 T cells, which are tumor-infiltrating immune cells, are an important indicator of anti-cancer responses, and interferon gamma and granzyme B secreted by CD8 T cells are functional cytokines that indicate the activation of immune cells. Figure 3 shows the results of analyzing tumor-infiltrating CD8 T cells and their cytokine secretion after administration of Lactobacillus plantarum LMT17-31 strain to tumor-bearing mice. Administration was performed as described in Section 1. 23 days after administration, tumors were excised. The excised tumors were separated into single cells using RPMI1640 medium containing 50 μg / ml Liberase and 40 μg / ml DNase I and a cell strainer. To observe the interferon gamma production of T cells, the isolated cells were stimulated with 50ng / ml PMA (phorbol 12-myristate 13-acetate) and 500ng / ml ionomycin for 4 hours in RPMI1640 medium containing 10% FBS. PMA and ionomycin are substances that stimulate the activation of T cells and act as a mechanism for activating T cells by antigens, creating an environment in which an immune response can occur. Ionomycin inhibits Ca 2+ It is an ionophore that increases PKC (protein kinase C), and in the case of PMA, it plays a synergistic role in targeting CD4 T cells and CD8 T cells by phosphorylating PKC. After stimulation, cells were stained with the antibodies in Table 7 to analyze immune cells and confirm interferon gamma production, and analyzed using a CANTO II flow cytometer. [Table 7]

[0047] Figures 3A, 3B, and 3C show the percentage of CD8 T cells in tumor-infiltrating T cells, the percentage of IFNγ-expressing cells in CD8 T cells, and the percentage of granzyme B-expressing cells in CD8 T cells, respectively. Figures 3D, 3E, and 3F show the total number of tumor-infiltrating CD8 T cells, the number of IFNγ-expressing cells in CD8 T cells, and the number of granzyme B-expressing cells in CD8 T cells, respectively. As shown in Figure 3, the ratio and number of tumor-infiltrating CD8 T cells were significantly increased in the LMT17-31 treatment group and the anti-PD1 antibody and LMT17-31 combination treatment group compared to the PBS treatment group. In addition, the ratio and number of CD8 T cells that produce the activating factors interferon gamma and granzyme B were also significantly increased in the LMT17-31 treatment group and the anti-PD1 antibody and LMT17-31 combination treatment group compared to the PBS control group.

[0048] TIFF0007599085000009.tif180170

Claims

1. Lactobacillus plantarum LMT17-31 (Accession No. KCTC-14283BP) microorganism, having antitumor activity.

2. A pharmaceutical composition for preventing or treating cancer, comprising the microorganism according to claim 1 or a culture thereof as an active ingredient.

3. The composition of claim 2 , wherein the cancer is a solid tumor.

4. The composition of claim 2, for administration in combination with an immune checkpoint inhibitor.

5. The composition of claim 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 ameliorating cancer, comprising the microorganism or a culture thereof according to claim 1 as an active ingredient.

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