WO2023068855A1

WO2023068855A1 - Composition for alleviation, prevention or treatment of cancer using veillonella parvula strain having anti-cancer activity

Info

Publication number: WO2023068855A1
Application number: PCT/KR2022/016090
Authority: WO
Inventors: 고광표; 윤성현; 유현주; 최성훈; 조선아; 김현정
Original assignee: 주식회사 고바이오랩; 서울대학교산학협력단
Priority date: 2021-10-20
Filing date: 2022-10-20
Publication date: 2023-04-27
Also published as: KR20230057980A; KR20230056619A; WO2023068857A1

Abstract

The present invention relates to use of Veillonella parvula strain for the prevention, treatment or alleviation of cancer.

Description

Composition for alleviating, preventing or treating cancer using a strain of Baylonella parbula having anticancer activity

The present invention relates to the use of Veillonella parvula strains for preventing, treating or improving cancer having anticancer effects.

Probiotics refer to microorganisms and products produced by the microorganisms having antibacterial activity and enzymatic activity that help the intestinal microbial balance. In addition, probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dry cells or fermentation products to improve the intestinal flora. Probiotics have the characteristics of non-pathogenicity and non-toxicity, inhabit the human intestine, and must survive while going to the intestine, so they must have resistance to acids, enzymes, and bile in the intestinal environment. Furthermore, probiotics must retain viability and activity prior to consumption in delivery foods, must be sensitive to antibiotics used to prevent infection, and must not contain antibiotic-resistant plasmids. Probiotics refer to microorganisms and products produced by the microorganisms having antibacterial activity and enzymatic activity that help the intestinal microbial balance. In addition, probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dry cells or fermentation products to improve the intestinal flora. Probiotics have the characteristics of non-pathogenicity and non-toxicity, inhabit the human intestine, and must survive while going to the intestine, so they must have resistance to acids, enzymes, and bile in the intestinal environment. Furthermore, probiotics must retain viability and activity prior to consumption in delivery foods, must be sensitive to antibiotics used to prevent infection, and must not contain antibiotic-resistant plasmids.

These probiotics include Bacillus sp. , which has an excellent ability to produce digestive enzymes (amylase, protease, lipase, cellulase, and phosphatase), Lactobacillus sp. Examples include photosynthetic bacteria that use substances (ammonia, hydrogen sulfide, amines, etc.) in metabolic processes to prevent odors.

Veillonella parvula , which is treated in the present invention, is a microorganism found in the human intestine, and is one of probiotics that have a beneficial effect on the intestinal environment while living in the human intestine.

On the other hand, cancer refers to a group of abnormal cells generated by continuous division and proliferation when the balance between cell division and death is destroyed for various reasons, and is also referred to as a tumor or a neoplasm. In general, it develops in more than 100 different body parts, including organs, white blood cells, bones, and lymph nodes, and develops into serious symptoms through infiltration into surrounding tissues and metastasis to other organs (WHO, 2006). Causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital genetic mutations (Klauunig & Kamendulis, Annu Rev Pharmacol Toxicol 2004, 44:239-267 ).

Although remarkable progress has been made in finding new targets, including regulation of cell cycle or apoptosis and oncogenes or cancer suppressor genes, the incidence of cancer continues to increase. . Currently, the treatment of cancer patients relies on surgical operation, radiation therapy, and chemotherapy by administration of anticancer substances showing strong cytotoxicity. Today, about 60 kinds of various anticancer drugs are used, and as knowledge about the occurrence of cancer and the characteristics of cancer cells has recently become known, research on the development of new anticancer drugs is being actively conducted. In anticancer chemotherapy, many patients suffer from side effects of anticancer drugs, and in particular, due to the toxicity of anticancer drugs, limited administration is being performed. Anticancer substances used clinically affect not only cancer cells but also normal cells, and have problems such as side effects and resistance to anticancer drugs, such as failure of treatment as the number of times of administration is repeated.

Accordingly, the inventors of the present invention have devoted themselves to probiotics research as a method that can replace drug-based treatment of cancer for which there is no conventional satisfactory treatment, and as a result, the cancer growth inhibitory effect of Veillonella parvula strains A new discovery of excellence led to the completion of the present invention.

An object of the present invention is to provide a probiotic strain showing excellent effects in the alleviation, prevention or treatment of cancer, a composition containing the same, and a composition for co-administration thereof with an anti-cancer therapeutic agent.

In order to solve the above problems, the present invention provides a Veillonella parvula KBL1041 strain deposited with accession number KCTC 5019.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising at least one strain selected from the group consisting of at least one strain, a culture of the strain, a lysate of the strain, and an extract of the strain. .

In addition, the present invention provides a food composition for preventing or improving cancer, comprising at least one selected from the group consisting of the strain, culture of the strain, lysate of the strain, and extract of the strain. In order to solve this, the present invention is Veillonella parvula ( Veillonella parvula ) strain, a culture of the strain, a lysate of the strain, and a drug for preventing or treating cancer, including at least one selected from the group consisting of an extract of the strain A scientific composition is provided.

In addition, the present invention is a Veillonella parvula strain, a culture of the strain, a lysate of the strain, and a food composition for preventing or improving cancer, including at least one selected from the group consisting of an extract of the strain to provide.

In addition, the present invention provides a feed composition comprising at least one selected from the group consisting of a Veillonella parvula strain, a culture of the strain, a lysate of the strain, and an extract of the strain.

The Bailonella parbula strain according to the present invention, specifically the KCTC 5019 strain (KBL1041 strain), and its culture, lysate and extract show the effect of reducing the size of tumors when administered to animals with cancer, Increases immune cells related to improvement or treatment of In addition, since it exhibits an improved tumor suppression effect even when administered in combination with an anti-cancer treatment, the composition comprising at least one selected from the group consisting of the strain of the present invention, its culture, lysate and extract exhibits cancer alleviation, prevention or treatment effects As a result, it can be used industrially very usefully.

Figure 1 shows the secretion of IFN-γ, IL-10 and IL-12p70, which are anticancer-related cytokines, by treating splenocytes and BMDC with the KBL1041 strain (KCTC 5019 strain) of the present invention compared with other strains through ELISA analysis is a result

Figure 2 is a diagram showing the results of analyzing the death rate (%) of cancer cells after co-cultivating the KBL1041 strain of the present invention with splenocytes and MC38 colorectal cancer cells.

Figure 3 is a graph showing the tumor volume increase inhibitory effect when the KBL1041 strain of the present invention was administered to mice induced with colorectal cancer and melanoma.

Figure 4 is a result of analyzing immune cells through flow cytometry after administering the KBL1041 strain of the present invention to mice induced with colon cancer.

Figure 5 is a graph showing changes in tumor volume and weight measured in the KBL1041 strain of the present invention in a neutrophil-free model using an anti-Ly6G antibody.

Figure 6 is a graph showing changes in tumor volume and weight measured after administration of KBL1041 of the present invention to NSGA mice in which only neutrophils among immune cells function normally.

7 is a result of analyzing the ratio of tumor-infiltrating neutrophils by performing flow cytometry on tumor tissues obtained from the NSGA mice.

8 is a result of RT-qPCR analysis of the relative expression level of neutrophil phenotype-related genes after administration of the KBL1041 strain of the present invention to mice induced with colon cancer.

9 is a view showing the results of analyzing the death rate (%) of MC38 colon cancer cells co-cultured with neutrophils isolated from colon cancer-induced mice administered with the KBL1041 strain of the present invention.

10 is a result of analyzing the ratio of neutrophils having anticancer efficacy by performing flow cytometry on tumor tissues obtained after administration of the KBL1041 strain of the present invention to mice induced with colon cancer.

11 shows the mean fluorescence intensity (MFI) of reactive oxygen species (ROS) and the secretion of nitric oxide in tissues obtained after administration of the KBL1041 strain of the present invention to mice in which colon cancer was induced. This is the result of analyzing the ratio of neutrophils to

12 is a result of analyzing the relative expression level of the pattern recognition receptor gene by RT-qPCR after administering the KBL1041 strain of the present invention to mice induced with colon cancer.

13 is a flow cytometry analysis result of measuring the mean fluorescence intensity (MFI) of TLR2 and TLR4 after administering the KBL1041 strain of the present invention to mice induced with colon cancer.

14 is a graph showing changes in tumor volume and weight measured after single or combined administration of the KBL1041 strain of the present invention and an anti-TLR2 antibody to mice induced with colon cancer.

15 is a view showing the results of analyzing the death rate (%) of cancer cells after co-cultivating MC38 cells co-cultured with neutrophils treated with an anti-TLR2 antibody with KBL1041 strain.

16 is a graph showing changes in tumor volume and weight measured after single or combined administration of the KBL1041 strain of the present invention and an anti-PD1 antibody, an immuno-anticancer agent, to mice induced with colon cancer.

Figure 17 analyzes the survival rate of cancer cells after treatment with KBL1041 strain in lung cancer cell line A549, breast cancer cell line MDA-MB-231, liver cancer cell line HepG2, melanoma cell line B16F10, pancreatic cancer cell line PANC1, renal cancer cell line RENCA, and colon cancer cell line MC38 it is a graph

FIG. 18 is a view showing analysis of non-settled formation ability of cancer cells after treatment of strain KBL1041 with lung cancer cell line A549.

FIG. 19 is a view showing analysis of non-settled formation ability of cancer cells after KBL1041 strain was treated with renal cancer cell line RENCA.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

The present invention is a Veillonella parvula strain, a culture of the strain, a lysate of the strain, and a pharmaceutical composition for preventing or treating cancer, comprising at least one member selected from the group consisting of an extract of the strain to provide.

The Bailonella parbula strain may be a KCTC 5019 strain, wherein the KCTC 5019 strain is a known strain classified as a Parbula species of the genus Veilonella, and can be easily purchased by a person skilled in the art or can be obtained In the present specification, the KCTC 5019 strain is also referred to as “KBL1041 strain”.

Since the strain is excellent in the effect of preventing, treating or improving cancer, it can be used for this purpose.

Specifically, the strain may have an activity of increasing the number and activity of immune cells in cancer cells.

In this specification, the term “and/or” is used to refer to any combination of components derived from a plurality of components.

Of course, the composition of the present invention may further include other strains in addition to the above strains, and may further include other Bailonella parbula strains.

In the composition of the present invention, strains may be included in the form of live cell cells, dead cell cells and dried strains, cultures thereof, lysates thereof, or extracts thereof.

As used herein, the term "culture" refers to a product obtained by culturing a strain in a known medium, and the product may include the strain itself. The medium may be selected from known liquid medium or solid medium, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium, but is not limited thereto.

As used herein, the term “lysate” refers to any product obtained by disrupting a strain by enzyme treatment, homogenization, or ultrasonic treatment.

In addition, as used herein, the term “extract” refers to a product obtained by extracting a strain with a known extraction solvent. In the present invention, "extract" includes a water extract and/or an organic solvent extract of the strains according to the present invention.

The organic solvent extract of the strain according to the present invention may be an organic solvent extract having 1 or more and 10 or less carbon atoms. For example, a substituted or unsubstituted alcohol extract having 1 to 10 carbon atoms, 1 to 5 carbon atoms, or 1 to 3 carbon atoms may be used as the extraction solvent. alcohol extracts such as, for example, methanol extracts, ethanol extracts, iso-propanol extracts, n-propanol extracts, n-butanol extracts, iso-butanol extracts, tert-butanol extracts and/or phenol extracts; ether extracts such as dialkyl ethers such as dimethyl ether, diethyl ether, and methyl ethyl ether; n-hexane, ethyl acetate, dichloromethane, chloroform and/or acetone extracts.

Also, in the present invention, the term “live cell” refers to the strain itself of the present invention, and “dead cell” refers to a strain that has been sterilized by heating, pressurization, or drug treatment.

The composition of the present invention may be provided as a composition capable of being further combined with pharmaceutically acceptable additives such as carriers or media. The additives used in the present invention are solvents, dispersants, coatings, absorption accelerators, controlled release agents (i.e. sustained release agents), and one or more inactive excipients (starch, polyols, granules, microfine cellulose, microcrystalline cellulose). (including, for example, cellphere, cellphere beads, diluents, lubricants, binders, disintegrants, etc.), etc. If necessary, tablet formulations of the disclosed compositions may be standard aqueous or Non-limiting examples of excipients for use as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers and the additional ingredients include binders, fillers, disintegrants, lubricants. , antimicrobial agents and coating agents.

In one embodiment of the present invention, the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.

As used herein, the term “cancer” medically refers to a problem in the normal division, differentiation, or death control function of cells, resulting in abnormal overproliferation, infiltration of surrounding tissues or organs, formation of lumps, and destruction or transformation of existing structures. means any condition. Due to such uncontrolled and abnormal cell growth, a cell mass called a tumor is formed, infiltrating surrounding normal tissues or organs, destroying the normal tissues or organs, and taking the life of the subject.

As used herein, the term "alleviation" means that the disease is not cured, but the severity of symptoms is reduced.

As used herein, the term “prevention” means delaying the onset of a disease, disorder or condition.

As used herein, the term “treatment” means, unless otherwise stated, to reverse, alleviate, inhibit the progress of, or reverse the disease or condition to which the term applies, or one or more symptoms of the disease or condition, or It means to prevent, and the term "treatment" used in the present invention refers to the act of treating when "treating" is defined as above. Thus, treatment or therapy for a disease in a mammal may include one or more of the following:

(1) inhibit the growth of the disease, i.e., arrest its development;

(2) prevent the spread of disease;

(3) alleviate disease;

(4) preventing disease recurrence; and

(5) Alleviate the symptoms of the disease

The composition of the present invention according to the conventional method according to each purpose of use, oral formulations such as solutions, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, injections of sterile injection solutions It can be formulated and used in various forms such as oral administration or parenteral administration through various routes including intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, spinal, intrathecal, or rectal topical administration or injection. The dosage may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, administration period or interval, excretion rate, constitutional specificity, nature of the preparation, severity of the disease, etc. there is.

In this specification, the term “liquid formulation” refers to a medicine to be taken in the form of a liquid medicine dissolved in water or an organic solvent. Compared to suspensions or solid formulations, liquid formulations have the advantage of more effective drug absorption from the intestinal tract to the systemic circulation, and the liquid formulations may contain additional solutes in addition to pharmaceuticals, and additives providing color, odor, sweetness, or stability. may also be included.

As used herein, the term “suspending agent” refers to any agent capable of providing the desired solubility and/or dispersibility of an alginate-containing composition, i.e., an aqueous formulation that is substantially clear and free of sedimentation and lumps. means

As used herein, the term "powder" refers to finely divided drugs, chemicals, or a dry mixture of both. “Powder” may include a mixture in a lyophilized state. For example, powders may contain conventional additives used in lyophilized formulations such as lyophilized strains and lyophilized preservatives.

In this specification, the term "granule" refers to a drug or a mixture of drugs in the form of granules, which usually passes through a sieve of 4.76 to 20 mm. Granules are generally produced by wetting the powder or mixture of powders and passing the mass through a sieve or granulator of appropriate mesh size depending on the size of the granules required. Since granules, like powders, are granular, the degree of contact of the drug to the tongue is high, so when a drug having a bitter taste is used in the form of granules, it may cause discomfort to patients, especially children or the elderly.

In this specification, the term "tablet" means a powdered medicine made easy to take by compressing it into a small disc shape. Tablets may include uncoated tablets, film-coated tablets, coated tablets, multilayer tablets, press-coated tablets, inner core tablets, orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets.

As used herein, the term “capsule” refers to a product made by filling a drug into a capsule in the form of a liquid, suspension, water, powder, granule, mini-tablet, or pellet, or encapsulated with a capsule base. For example, a pharmaceutical composition according to one embodiment may include a lyophilized strain. For example, the pharmaceutical composition of one embodiment may be formulated in the form of a capsule formulation of the lyophilized strain. When the composition of one embodiment includes the strain in a lyophilized form, the storage period of the strain can be improved by minimizing the loss of the strain that may occur due to heat. In addition, the biological activity of the strain can be inactivated to improve formulation stability, and the strain can be more easily induced to rehydrate than other formulation methods, so that the original activity and growth ability can be quickly restored after supplying water to the dry strain. In addition, the freeze-dried preparation is easy to formulate, such as tableting and encapsulation, and can be prepared in various dosage forms. In addition, regeneration, which is the most important feature of LBP (Live Biotherapeutic Products) such as strain preparations, can proceed very stably in freeze-dried strains.

In one embodiment, when the strain is formulated by lyophilization, skim milk, sugars (eg, trehalose, sucrose, maltose or glucose, etc.), sugar alcohols (eg, to prevent a rapid decrease in activity during freeze-drying of the strain) For example, mannitol, inositol, or sorbitol, etc.) may be mixed with conventional freeze-drying preservatives (or protecting agents) used in the art and then freeze-dried. However, it goes without saying that any lyophilization method commonly used in the art can be used without particular limitation.

As used herein, the term "pill" is meant to encompass small, round solid dosage forms containing multiparticulates mixed with binders and other excipients.

In this specification, the term "syrup" means a thick homemade sugar or sugar substitute. In the present invention, the syrup is an easy-to-take medicine with an unpleasant taste, such as a bitter taste, in a liquid form, and is particularly suitable for use by children. In the present invention, the syrup may include, in addition to purified water and extract, sugar or a substitute for sugar used to give sweetness and viscosity, an antibacterial preservative, a flavoring agent, or a coloring agent, but is not limited thereto. Examples of sweeteners that may be included in such syrups include, but are not limited to, white sugar, mannitol, sorbitol, xylitol, aspartame, stevioside, fructose, lactose, sucralose, saccharin, or menthol.

As used herein, the term "injection" refers to an aseptic preparation applied to the body through the skin or mucous membrane, and in particular, any form of administration such as subcutaneous injection, intramuscular injection, intradermal injection, and intraperitoneal injection may be used as an injection route. It is possible, and the dosage form is selected according to the characteristics of each pharmacologically active substance.

In one embodiment, the composition of the present invention may be a composition for oral administration. As used herein, the term "oral administration" means that the active substance is administered to the gastrointestinal tract through the oral route for absorption. Non-limiting examples of the formulation for oral administration include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, and the like. can be heard In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol may be used, and sweeteners, aromatics, syrups, and the like may also be used. Furthermore, in the case of capsules, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

The pharmaceutical composition of the present invention may be provided as an enteric-coated enteric preparation, particularly as a unit dosage form for oral use. "Enteric coating" in the present specification includes all types of pharmaceutically acceptable known coatings that are not decomposed by gastric acid and the coating is maintained, but sufficiently decomposed in the small intestine so that the active ingredient can be released into the small intestine. do. The "enteric coating" of the present invention is maintained for at least 2 hours when artificial gastric juice such as an HCl solution of pH 1 is brought into contact at 36°C to 38°C, preferably after that, a KH ₂ PO ₄ buffer solution of pH 6.8. Refers to coatings that disintegrate within 30 minutes in artificial intestinal juices such as

The enteric coating of the present invention is coated in an amount of about 16 to 30, preferably 16 to 20 or 25 mg or less per core. When the thickness of the enteric coating of the present invention is 5 to 100 μm, preferably 20 to 80 μm, satisfactory results are obtained as an enteric coating. The material of the enteric coating is appropriately selected from known high molecular materials. Suitable polymeric materials are described in a number of well-known literature (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373 H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359; Hagers Handbuchder pharmazeutischen Praxis, 4th edition, Vol. 7, pp. 739-742, and 766-778, (Springer Verlag, 1971); 1970), cellulose ester derivatives, cellulose ethers, methyl acrylate copolymers of acrylic resins, and copolymers of maleic acid and phthalic acid derivatives may be included therein.

The enteric coating of the present invention can be prepared using a conventional enteric coating method in which an enteric coating solution is sprayed onto a core. Suitable solvents used in the enteric coating process include alcohols such as ethanol, ketones such as acetone, and halogenated hydrocarbon solvents such as dichloromethane (CH ₂ Cl ₂ ), and a mixed solvent of these solvents may be used. An emollient such as di(di)-n-butylphthalate or triacetin is added to the coating solution in a ratio of 1 to about 0.05 to about 0.3 (coating material to softener). It is appropriate to carry out the spraying process continuously and it is possible to adjust the amount of spraying taking into account the conditions of the coating. The spray pressure can be varied, and satisfactory results are generally obtained with spray pressures of about 1 to about 1.5 bar.

The composition of the present invention may be a composition for parenteral administration. As used herein, the term "parenteral administration" means intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, spinal, intrathecal or rectal topical administration or injection. Parenteral administration is by injecting a suppository preparation, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. At this time, in order to formulate a formulation for parenteral administration, the composition may be mixed in water with a stabilizer or a buffer to prepare a solution or suspension, which may be prepared in a unit dosage form in an ampoule or vial.

The preferred dosage of the composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, the excretion rate, And the range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.

The present invention provides a food composition for preventing or alleviating cancer or a food additive composition comprising at least one selected from the group consisting of a Bailonella parbula strain, a culture of the strain, a lysate of the strain, and an extract of the strain to provide.

The Bailonella parbula strain may be a KCTC 5019 strain.

The food may be a health functional food.

The food composition or composition for food additives may be included in foods effective in preventing or alleviating cancer. For example, the food composition of the present invention can be easily utilized as a main ingredient, supplementary ingredient, food additive, health functional food or functional beverage of food.

The food composition means a natural product or processed product containing one or more nutrients, and preferably means a product that can be directly eaten through a certain degree of processing, and in a conventional sense, food, It refers to food additives, health functional foods, and functional beverages.

Foods to which the food or food additive composition according to the present invention can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and functional foods. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), bread, health supplement food, seasoning Foods (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

In this specification, the term “health functional food” refers to a food group or food composition that has added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It refers to food designed and processed to fully express the body's regulatory functions related to disease prevention and recovery. The functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

Furthermore, in addition to the above, the food containing the food composition of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectins acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. and can be used.

In the food or food containing the food additive composition of the present invention, the amount of the composition according to the present invention may be 0.001% to 100% by weight of the total weight of the food, preferably 1% to 99% by weight. %, and in the case of beverages, it may be included in a ratio of 0.001 g to 10 g, preferably 0.01 g to 1 g based on 100 mL, but for health and hygiene purposes or health control purposes In the case of long-term ingestion, it may be less than the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The food or food additive composition of the present invention may be prepared by adding a Veilonella parbula strain, such as a Veilonella parbula KCTC 5019 strain, independently or to an acceptable carrier, or in the form of a composition suitable for consumption by humans or animals. can That is, it can be added to and used in foods that do not contain other probiotic bacteria and foods that already contain some probiotic bacteria.

The present invention provides an animal feed or composition for addition to animal feed comprising at least one selected from the group consisting of a Bailonella parbula strain, a culture of the strain, a lysate of the strain, and an extract of the strain.

The Bailonella parbula strain may be a KCTC 5019 strain.

The additive for animal feed of the present invention may be in the form of a dry or liquid preparation, and may further contain other non-pathogenic microorganisms in addition to the Bailonella parbula strain, for example, the Bailonella parbula KCTC 5019 strain.

In using the Bailonella Parbula strain of the present invention, such as the Bailonella Parbula KCTC 5019 strain, as an additive for animal feed, the feed raw materials include various grains and soybean proteins, peanuts, peas, sugar beets, pulp, and grain by-products , animal intestine powder, fish meal powder, etc. may be used, and these may be used unprocessed or processed without limitation.

The present invention provides a therapeutically effective amount of a pharmaceutical composition comprising at least one member selected from the group consisting of a Baylonella parbula strain, a culture of the strain, a lysate of the strain, and an extract of the strain administered to a subject in need of treatment It provides a cancer prevention or treatment method comprising the step of doing.

The Bailonella parbula strain may be a KCTC 5019 strain.

The present invention is a use of a composition comprising at least one selected from the group consisting of a Baylonella parbula strain, a culture of the strain, a lysate of the strain, and an extract of the strain in the manufacture of a drug for preventing or treating cancer provides

The Bailonella parbula strain may be a KCTC 5019 strain.

The present invention provides a use for preventing or treating cancer of a pharmaceutical composition comprising at least one member selected from the group consisting of a Bailonella parbula strain, a culture of the strain, a lysate of the strain, and an extract of the strain.

The Bailonella parbula strain may be a KCTC 5019 strain.

The pharmaceutical composition of the present invention may be used alone or in other therapies, such as surgery, radiation therapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, hormone therapy, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy. etc. can be performed.

The pharmaceutical composition of the present invention may be administered in combination with an anti-cancer therapeutic agent. That is, the composition of the present invention may be a composition for concomitant administration of anticancer drugs.

The composition of the present invention may further contain an anti-cancer agent. The composition of the present invention may include the strain and the anticancer agent as a single agent, or may be included as separate agents.

The composition of the present invention may include a first agent containing a strain and a second agent including an anti-cancer agent. The composition of the present invention may include the first agent and the second agent as separate and separate agents. The first agent and the second agent may be administered through the same route of administration or different routes of administration. The first agent and the second agent may be administered simultaneously or sequentially via the same route of administration or separate routes of administration.

The anticancer drug may be an anti-PD1 antibody.

In a specific embodiment of the present invention, the anticancer effect was confirmed when the Baylonella parbula KCTC 5019 (KBL1041) strain and the anti-PD1 antibody were treated in vitro , and the anti-PD1 antibody was the veil of the present invention. It can be included in the composition as an anti-cancer treatment that can be used in combination with Ronella parbula.

In one embodiment, examples of anticancer therapeutics include chemotherapeutic agents, targeted anticancer therapeutics, and immunotherapeutic agents.

In one embodiment, the immunotherapeutic agent is an immune checkpoint inhibitor, an immune checkpoint protein activity inhibitor, an immune checkpoint protein expression inhibitor, an immune cell therapy (eg, a tumor infiltrating lymphocyte, a T cell receptor or a chimeric antigen receptor cell therapy, etc.) and an anticancer agent. It may be at least one selected from vaccines. For example, an immunotherapeutic agent may be an immune checkpoint inhibitor.

In the present invention, the term “immune checkpoint inhibitor” refers to MHC class presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, and cytokine, chemokine or immune cell proliferation and/or differentiation. It refers to any substance that prevents suppression of any mechanism in the immune system, such as signal transduction for cancer, and can treat cancer by suppressing immune evasion of cancer by blocking immune checkpoints that prevent the progression of immune responses in cancer with high immunosuppression. .

In the present invention, an anti-CTLA4 antibody as a non-limiting example of an immune checkpoint inhibitor In one embodiment, examples of anticancer therapeutics include chemotherapeutic agents, targeted anticancer therapeutics, and immunotherapeutic agents.

In the present invention, non-limiting examples of immune checkpoint inhibitors include anti-CTLA4 antibodies or antigen-binding fragments thereof; anti-PD-L1 antibody or antigen-binding fragment thereof; and one or more immune checkpoint inhibitors selected from the group consisting of anti-PD-1 antibodies or antigen-binding fragments thereof. For example, non-limiting examples of immune checkpoint inhibitors in the present invention include anti-PD-1 antibodies or antigen-binding fragments thereof.

Non-limiting examples of immune checkpoint inhibitors in the present invention include pembrolizumab, nivolumab, semiflamab, atezolizumab, avelumab, durvalumab, and/or ipilimumab and antigen-binding fragments thereof. there is.

The composition of the present invention may exhibit a synergistic or additive effect on cancer alleviation, prevention, or treatment by being administered simultaneously with an immune checkpoint inhibitor or sequentially administered in combination with an immune checkpoint inhibitor. For example, synergistic or additive effects on tumor size reduction can be achieved by administering the composition of the present invention in combination with the aforementioned immune checkpoint inhibitor.

In the present invention, the cancer is lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer (eg, melanoma), uterine cancer, ovarian cancer, colon cancer, colorectal cancer, breast cancer, uterine sarcoma , fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia , may include at least one selected from the group consisting of childhood solid tumor, lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, axial contraction tumor, brain stem glioma, Merkel cell tumor, urinary tract tumor, and pituitary adenoma. .

For example, in the present invention, cancer may include at least one selected from the group consisting of non-small cell lung cancer, melanoma, colorectal cancer, colorectal cancer, renal cancer, and liver cancer.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예 1. 베일로넬라 파르불라 KBL1041(KCTC 5019) 균주의 항암 효과 관련 사이토카인 분비 증가 효과 확인Example 1. Confirmation of cytokine secretion increase effect related to anti-cancer effect of Baylonella parbula KBL1041 (KCTC 5019) strain

By treating splenocytes and bone marrow-derived dendritic cells (BMDC) with the Bailonella parbula KBL1041 strain of the present invention, anticancer-related cytokines IFN-γ, IL-10 and IL-12p70 secreted from them The amount of secretion was measured and confirmed by ELISA.

Specifically, splenocytes were isolated through magnetic-activated cell sorting (MACS). For the production of BMDC, the isolated bone marrow cells were cultured for 7 days and then in RPMI medium supplemented with 40 ng/ml GM-CSF, 10% PBS, 100 IU/ml penicillin/streptomycin and 50 μM 2-mercaptoethanol. It was cultured for 7 days in a 37 ℃, 5% CO ₂ humidity incubator, and divided and cultured every 3-4 days. In addition, splenocytes and BDMC were treated with dead cells of various strains including the Bailonella parbula KBL1041 strain of the present invention at a ratio of 1:100 for 16 hours, and then the supernatant was obtained and ELISA was performed.

As a result of ELISA analysis, the Veillonella parbula KBL1041 strain of the present invention increased the secretion of the anti-cancer cytokines IFN-γ and IL-12p70 compared to other strains, whereas the secretion of IL-10, one of the carcinogenic cytokines, decreased. It did not increase significantly compared to other strains (Fig. 1). Therefore, the group treated with the Baylonella parbula KBL1041 strain showed an excellent anti-cancer cytokine secretion effect by immune cells.

실시예 2. 베일로넬라 파르불라 KBL1041 균주의 암 세포의 사멸능 확인Example 2. Confirmation of cancer cell killing ability of Baylonella parbula KBL1041 strain

MC38 cells, a colorectal cancer cell line isolated from mice with tumors, were co-cultured with the Bailonella parbula KBL1041 strain of the present invention or Akkermansia muciniphila , a control strain, and the control strain was tested by LDH-mediated cytotoxicity assay. Compared to the present invention, KBL1041 was analyzed to determine whether or not the ability to kill cancer cells was superior.

Specifically, the isolated MC38 colorectal cancer cells were cultured in a 96-well culture plate one day prior to analysis of cell killing ability (5x10 ³ cells/well). MC38 cells were co-cultured with the KBL1041 strain of the present invention and the pasteurized Akkermansia muciniphila, a control strain, at a ratio of 1:10000, and when analyzed by co-culture with splenocytes, the ratio of MC38 cells to splenocytes was 1:1. 100 was co-cultured. PBS was treated as a negative control group. Cytotoxicity was analyzed using CyQUANT LDH Cytotoxicity Assay (Invitrogen).

As a result, splenocytes co-cultured with KBL1041 showed better cancer cell killing ability (Cytotoxicity %) than splenocytes co-cultured with the control strain, but cancer cell killing ability was significant when only tumor cells were cultured without splenocytes. There was no difference (Fig. 2). This indicates that the Veillonella parbula KBL1041 strain of the present invention induces cell death by activating immune cells.

실시예 3. 암 유도 마우스에서 베일로넬라 파르불라 KBL1041 균주 투여 시의 종양 증가 억제 효과 확인Example 3. Confirmation of tumor growth inhibitory effect upon administration of Baylonella parbula KBL1041 strain in cancer-induced mice

In order to confirm the effect of intraperitoneal or oral administration of the Bailonella parbula KBL1041 strain of the present invention to mice induced with various types of cancer, the change in tumor volume after administration was measured.

To induce tumors, colon cancer cells MC38 and melanoma cells B16F10 were subcutaneously injected into 8-week-old C57BL/6 mice, and colon cancer cells CT26 cells were subcutaneously injected into BALB/c mice at 2.5x10 ⁵ cells/100 μl, respectively. induced. From the 7th day after induction, 5x10 ⁹ cells/200 μl/mouse of the prepared Bailonella parbula KBL1041 strain was intraperitoneally administered once every 3 days for a total of 3 times. In the case of oral administration, 1x10 ¹² cells/200 μl/mouse was administered daily from the 7th day after induction. PBS was administered as a negative control group.

From 7 days after induction, changes in tumor volume were measured using a caliper every 2-4 days.

As a result of the measurement, when the Bailonella parbula KBL1041 strain of the present invention was administered, the increase in tumor size was significantly reduced compared to PBS administration in all mice whose cancer was induced by MC38, CT26, and B16F10, and KBL1041 strain was intraperitoneally administered. Tumor growth was also suppressed not only in the case of oral administration but also in the case of oral administration (FIG. 3).

As can be seen from the above experimental results, it was confirmed that the KBL1041 strain of the present invention had an effect of inhibiting tumor growth in mice induced with colorectal cancer or melanoma, and both intraperitoneal and oral administration were effective was able to confirm

실시예 4. 암 유도 마우스에서 베일로넬라 파르불라 KBL1041 균주 투여 시의 면역세포 증가 효과 확인Example 4. Confirmation of immune cell increasing effect upon administration of Baylonella parbula KBL1041 strain in cancer-induced mice

In addition, immune cells were analyzed in the tumor tissues of the mice to which KBL1041 was administered through flow cytometry, and the ratios of NK cells, macrophages, monocytes, and neutrophils expressing specific components were confirmed.

For tumor induction, the pasteurized KBL1041 strain of the present invention was intraperitoneally administered 4 times every 3 days to a mouse whose colon cancer was induced with MC38, a colon cancer cell, and then tissue, spleen and bone marrow tissue were collected from the mouse. Immune cells were analyzed by flow cytometry.

As a result, when compared to the analysis results in the PBS-administered group treated with the negative control group, when the KBL1041 strain was treated, CD3-NK1.1+ NK cells, F4/80+CD11b+ macrophages, and Ly6ChiCD11b monocytes were significantly reduced, and Ly6G +CD11b+ neutrophils increased (FIG. 4). As can be seen from the above experimental results, it was confirmed that the Baylonella parbula KBL1041 strain caused anticancer efficacy by specifically increasing neutrophils among immune cells.

실시예 5. 항-Ly6G 항체를 이용한 호중구 부재 모델에서 베일로넬라 파르불라 KBL1041 균주의 항암 효능 소멸 확인Example 5. Confirmation of extinction of anti-cancer efficacy of Baylonella parbula KBL1041 strain in neutrophil absence model using anti-Ly6G antibody

In order to confirm that the Baylonella parbula KBL1041 strain of the present invention exhibits anticancer efficacy through neutrophils, it is confirmed whether the tumor increase inhibitory effect disappears in a neutrophil-free tumor model using an anti-Ly6G antibody that neutralizes neutrophils did

For neutrophil depletion studies, mice were injected with MC38, a colorectal cancer cell, to induce cancer, and from day 6 after induction, control IgG antibody and anti-Ly6G antibody, an anti-cancer drug, were intratumorally administered at 50 μg per mouse per day. did The next day after antibody administration, the Baylonella parbula KBL1041 strain of the present invention was intraperitoneally administered every 3 days. PBS was administered as a negative control group.

As a result of measuring the change in tumor volume and tumor weight, when the control antibody, IgG, was administered, the tumor growth inhibitory effect was significantly shown in the mice administered with the KBL1041 strain of the present invention compared to the mice administered with PBS, whereas the anti-antibody When neutrophils were absent by administration of the -Ly6G antibody, there was no difference in tumor growth between the PBS-administered group and the KBL1041-administered group of the present invention (FIG. 5). Through the above experimental results, it was verified that the strain of Baylonella parbula KBL1041 of the present invention exerts anticancer efficacy through neutrophils.

실시예 6. 면역세포 중 호중구만 정상적으로 기능하는 NSGA 마우스에서 베일로넬라 파르불라 KBL1041 균주의 호중구 조절을 통한 종양 억제 효과 확인 Example 6. Confirmation of tumor suppression effect through neutrophil control of Baylonella parbula KBL1041 strain in NSGA mice in which only neutrophils among immune cells function normally

MC38, a colorectal cancer cell, was inoculated into an NSGA mouse, which is a mouse with normal functioning of only neutrophils, no T cells and natural killer cells, and functional disorders of dendritic cells and macrophages, and then the dead cells of the Baylonella parbula KBL1041 strain of the present invention were injected into the tumor. It was verified that the KBL1041 strain of the present invention exhibits anticancer efficacy by neutrophils, not other immune cells, by intraperitoneal administration at 5x10 ⁹ cells/200 μl/mouse from day 7 after transplantation.

As a result, when the KBL1041 strain of the present invention was administered, the increase in both the volume and weight of the tumor was significantly reduced when compared to the PBS-treated mice (FIG. 6). In addition, flow cytometry was performed on the tumor tissues obtained from the NSGA mice to analyze the ratio of neutrophils. Significantly increased when compared to (FIG. 7).

As can be seen from the above experimental results, it was verified that the Bailonella parbula KBL1041 of the present invention increases the ratio of neutrophils and exhibits anticancer efficacy through neutrophils, regardless of the presence or function of other immune cells.

실시예 7. 베일로넬라 파르불라 KBL1041 균주 투여에 따른 호중구 표현형 관련 유전자의 상대적 발현량 확인Example 7. Verification of relative expression level of genes related to neutrophil phenotype according to administration of Baylonella parbula KBL1041 strain

In the same manner as in the above example, after administering the Bailonella parbula KBL1041 strain of the present invention to mice whose colon cancer was induced by MC38, splenic neutrophils isolated from the mice were analyzed. Relative expression levels of ccl2, icam1, il1b, tnfa, cxcl10 and cd206 genes associated with the neutrophil phenotype in isolated neutrophils were measured by real-time PCR. Total RNA expressed from the splenic neutrophils was extracted, and then complementary cDNA was synthesized to confirm the expression level of the genes through RT-qPCR.

As a result, compared to PBS-administered mice treated as a negative control group, expression levels of ccl2, icam1, il1b, tnfa, and cxcl10 genes, which are markers of neutrophils having anticancer functions, were found in mice administered with the Bailonella parbula KBL1041 strain of the present invention. Significantly increased (FIG. 8).

As can be seen from the above experimental results, it was confirmed at the gene expression level that the Baylonella parbula KBL1041 of the present invention induces a neutrophil phenotype to a neutrophil phenotype having anticancer efficacy.

실시예 8. 베일로넬라 파르불라 KBL1041 균주를 투여한 암 유도 마우스에서 분리한 호중구의 암 세포 사멸 능력 확인 Example 8. Confirmation of cancer cell killing ability of neutrophils isolated from cancer-induced mice administered with Baylonella parbula KBL1041 strain

After administering Bailonella parbula KBL1041 strain or control PBS to MC38 colorectal cancer-induced mice, respectively, neutrophils were isolated from the spleen and co-cultured with MC38 cells at ratios of 1:10, 1:50, and 1:100 for 16 hours did.

As a result, neutrophils isolated from mice administered with KBL1041 showed a significantly increased cancer cell death rate (%) compared to neutrophils isolated from mice administered with PBS. was further increased (FIG. 9). As can be seen from the above experimental results, it was confirmed that the Baylonella parbula KBL1041 of the present invention not only increased the simple anti-cancer phenotype of neutrophils but also increased the ability of neutrophils to kill cancer cells.

실시예 9. 암 유도 마우스에서 베일로넬라 파르불라 KBL1041 균주 투여 시 항암 표현형을 갖는 호중구의 증가 효과 확인Example 9. Confirmation of the effect of increasing neutrophils having an anti-cancer phenotype when Baylonella parbula KBL1041 strain was administered to cancer-induced mice

Immune cells were analyzed in the tumor tissues of mice administered with KBL1041 through flow cytometry. After intraperitoneal administration of killed bacteria of the KBL1041 strain of the present invention to mice induced with colon cancer by MC38, flow cytometry analysis of tissue collected from the mice was compared with the results of analysis in the PBS-administered group treated as a negative control group, KBL1041 strain When treated, CD16+CD54+ neutrophils, which are neutrophils with an anticancer phenotype, increased (FIG. 10).

As can be seen from the above experimental results, it was confirmed not only at the gene level confirmed in Example 7 that the Baylonella parbula KBL1041 of the present invention induces the phenotype of neutrophils to the phenotype of neutrophils having anticancer efficacy, but also at the actual cell level did.

실시예 10. 베일로넬라 파르불라 KBL1041 균주 투여 시의 종양 침윤 호중구의 ROS 및 iNOS+ 호중구 분석Example 10. ROS and iNOS+ neutrophil analysis of tumor-infiltrating neutrophils when Baylonella parbula KBL1041 strain was administered

After intraperitoneal administration of dead bacteria of the KBL1041 strain of the present invention to mice induced with colon cancer by MC38, mean fluorescence intensity (MFI) of reactive oxygen species (ROS) of infiltrating neutrophils in tumor tissue collected from mice through flow cytometry was measured. The ratio of iNOS+ neutrophils, which are neutrophils that secrete nitric oxide, was analyzed.

As a result of the measurement, when the KBL1041 strain of the present invention was treated, the ROS MFI value of neutrophils and the ratio of iNOS+ neutrophil cells significantly increased compared to the negative control, PBS-treated group (FIG. 11).

Through the above experimental results, it was confirmed that the Veillonella parbula KBL1041 strain of the present invention induces cancer cell death by increasing the secretion of ROS or NO from neutrophils.

실시예 11. 베일로넬라 파르불라 KBL1041 균주 투여에 따른 패턴 인식 수용체(PRR) 유전자의 발현 증가 확인Example 11 Confirmation of increase in pattern recognition receptor (PRR) gene expression following administration of Baylonella parbula KBL1041 strain

In the same manner as in the above example, after administering the Bailonella parbula KBL1041 strain of the present invention to mice whose colon cancer was induced by MC38, splenic neutrophils isolated from the mice were analyzed. Relative expression levels of pattern recognition receptor (PRR) genes tlr1, tlr2, tlr4, tlr5, tlr6, tlr9, nod1 and nod2 in isolated neutrophils were measured by real-time PCR. Total RNA expressed from the splenic neutrophils was extracted, and then complementary cDNA was synthesized to confirm the expression level of the genes through RT-qPCR.

As a result, compared to PBS-administered mice treated as a negative control group, the expression level of only the tlr2 gene significantly increased in mice administered with the Bailonella parbula KBL1041 strain of the present invention (FIG. 12). In addition, the mean fluorescence intensity (MFI) of TLR2 and TLR4 in tumor-infiltrating neutrophils was measured. As a result, when the KBL1041 strain of the present invention was treated, only the MFI value of TLR2 significantly increased compared to the PBS-treated group, which is a negative control group (FIG. 13).

As can be seen from the above experimental results, it was confirmed that the Bailonella parbula KBL1041 strain of the present invention exhibits anticancer efficacy by specifically activating TLR2 of neutrophils.

실시예 12. TLR2가 중립화된 대장암 유도 마우스에서 베일로넬라 파르불라 KBL1041 균주의 종양 억제 효과 소멸 확인Example 12. Confirmation of disappearance of tumor suppression effect of Baylonella parbula KBL1041 strain in TLR2-neutralized colon cancer-induced mice

In order to determine whether the Baylonella parbula KBL1041 strain of the present invention induces anticancer efficacy of immune cells through TLR2, whether the tumor growth inhibitory effect disappears in a TLR2 neutralizing tumor model using an anti-TLR2 antibody that neutralizes TLR2 It was confirmed whether

C57BL/6 mice were injected with MC38, a colorectal cancer cell, to induce cancer, and from day 6 after induction, a control IgG antibody and an anti-TLR2 antibody, an anti-cancer drug, were intratumorally administered at 50 μg per mouse per day. The next day after antibody administration, the Baylonella parbula KBL1041 strain of the present invention was intraperitoneally administered every 3 days. PBS was administered as a negative control group.

As a result of measuring the change in tumor volume and weight, when TLR2 was not neutralized by administering IgG as a control antibody, as can be seen in the above examples, the KBL1041 strain of the present invention was administered as compared to the control PBS-administered mice. In one mouse, the effect of inhibiting tumor growth was significant. However, when anti-TLR2 antibody was administered to neutralize TLR2, the tumor growth inhibitory effect of the KBL1041 strain of the present invention disappeared (FIG. 14).

In addition, in order to confirm in more detail that KBL1041 of the present invention activates TLR2 of neutrophils to induce anticancer function of neutrophils, it is verified whether the ability to kill cancer cells disappears when the KBL1041 strain of the present invention is treated with an anti-TLR2 antibody. did Spleen neutrophils were treated with 20 μg/ml anti-TLR2 antibody 4 hours before KBL1041 strain treatment, and MC38 cells were co-cultured with the neutrophils at a ratio of 1:100. Neutrophils and killed KBL1041 strain were co-cultured at a ratio of 1:100.

As a result of LDH-mediated cytotoxicity analysis, cancer cell death rate (%) was significantly increased when treated with KBL1041 alone, but cancer cell death ability was rather decreased when treated in combination with an anti-TLR2 antibody (FIG. 15) .

Through the above experimental results, it was verified that the strain of Baylonella parbula KBL1041 of the present invention specifically activates TLR2 of neutrophils to increase the cancer cell killing function of neutrophils.

실시예 13. 베일로넬라 파르불라 KBL1041 균주와 항암제 병용 투여 시의 종양 억제 효과 확인 Example 13. Confirmation of tumor suppression effect when co-administered with Baylonella parbula KBL1041 strain and anticancer agent

Whether the effect of inhibiting tumor growth when the Baylonella parbula KBL1041 strain of the present invention is administered in combination with an anti-PD1 antibody, a type of immuno-anticancer agent, is increased compared to the case of administering the KBL1041 strain or the anti-PD1 antibody alone confirmed.

C57BL/6 mice were injected with MC38, a colorectal cancer cell, to induce cancer, and from the 6th day after the induction, a control IgG antibody and an immuno-anticancer anti-PD1 antibody were intraperitoneally administered at 200 μg per mouse every 3 days. From the day following the start of antibody administration, on the 7th day after cancer induction, the Baylonella parbula KBL1041 strain of the present invention was intraperitoneally administered every 3 days. PBS was administered as a negative control group.

As a result of measuring the change in tumor volume and weight, in the case of mice administered with PBS, the tumor volume of mice administered with anti-PD1 antibody, an immuno-anticancer agent, was about 52.2%, compared to mice administered with control IgG antibody, and tumor volume was approximately 52.2%. Weight decreased by about 42.6% respectively. In addition, in mice administered with the KBL1041 strain of the present invention, tumor volume and tumor weight were reduced by about 65.1% and 63%, compared to mice administered with PBS, when control IgG antibody was administered without immunotherapy. Even when the anti-PD1 antibody, an immuno-anticancer drug, was administered, the tumor volume and tumor weight were reduced by 76.5% and 79.6% in mice administered with the KBL1041 strain of the present invention, compared to control mice administered with PBS. Above all, when the KBL1041 strain and the anti-PD1 antibody of the present invention were administered in combination, the tumor weight was reduced by about 68.4% and 79.6%, respectively, compared to the case of administering the KBL1041 strain or the anti-PD1 antibody alone (FIG. 16).

As can be seen from the above experimental results, it was confirmed that the Baylonella parbula KBL1041 strain of the present invention exhibits a combined effect with an immuno-anticancer agent, thereby confirming that the KBL1041 strain of the present invention can be used as an adjuvant to increase the reactivity of an immuno-anticancer agent did.

실시예 14. 베일로넬라 파르불라 KBL1041 균주의 다양한 암종의 암 세포주 생존율 저감 효과 확인Example 14. Confirmation of the effect of reducing the viability of cancer cell lines of various carcinomas of the strain Baylonella parbula KBL1041

In order to confirm the anticancer effect of the Baylonella parbula KBL1041 strain of the present invention, lung cancer cell line A549, breast cancer cell line MDA-MB-231, liver cancer cell line HepG2, melanoma cell line B16F10, pancreatic cancer cell line PANC1, renal cancer cell line RENCA and colorectal cancer cell line After treatment with MC38, the survival rate of cancer cells was measured.

Specifically, each cancer cell was cultured in a 96-well culture plate, and the dead cells of the Bailonella parbula KBL1041 strain (pasteurization, 70 ℃, 30 minutes) were 1:10 ¹ , 1:10 ² compared to the number of cancer cells. , 1:10 ³ , 1:10 ⁴ , 1:10 ⁵ , and reacted for 24 to 72 hours. After 72 hours of strain treatment, cell viability was measured by dissolving formazan reduced by reductase in mitochondria in DMSO using an MTT assay kit (Promega, #G4000) and measuring absorbance at 570 nm.

As a result of the measurement, the survival rate was significantly decreased in all types of cancer cell lines in a concentration-dependent manner after 72 hours of treatment with the KBL1041 strain (FIG. 17).

As can be seen from the above experimental results, it was confirmed that the KBL1041 strain of the present invention exhibits a growth inhibitory effect in various cancers such as lung cancer, breast cancer, liver cancer, melanoma, pancreatic cancer, kidney cancer, and colorectal cancer.

실시예 15. 베일로넬라 파르불라 KBL1041 균주의 폐암 및 신장암 세포주의 비정착 형성 저해 효과 확인Example 15. Confirmation of the effect of inhibiting non-establishment formation of lung cancer and renal cancer cell lines of Baylonella parbula KBL1041 strain

For the lung cancer cell line A549, after treatment with KBL1041 strain, it was confirmed whether the non-settled growth ability of A549 was reduced.

Specifically, the lung cancer cell line A549 was cultured in a 6-well culture plate (A549: 1x10 ⁴ cells/well, bottom agar: 0.6%, top agar: 0.3%), and the dead cells of the Baylonella parbula KBL1041 strain (pasteurization, 70 ° C.) , 30-minute method) was treated at a ratio of 1:10 ² , 1:10 ³ to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.

As a result of the measurement, there was no ^significant difference in the number of colonies of the lung cancer cell line A549 when the KBL1041 strain was treated compared to the control group. The size of the cluster was also significantly reduced (FIG. 18). Therefore, it was confirmed that the KBL1041 strain of the present invention has an effect of inhibiting the ability of lung cancer cell lines to form non-settled cells.

Similarly, the same experiment was performed with RENCA, a renal cancer cell line. Specifically, the renal cancer cell line RENCA was cultured in a 6-well culture plate (RENCA: 5x10 ³ cells/well, bottom agar: 0.6%, top agar: 0.3%), and the dead cells of the Baylonella parbula KBL1041 strain (pasteurization, 70 ℃, 30 minutes) was treated at a ratio of 1:10 ² , 1:10 ³ to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.

As a result of the measurement, there was no significant difference in the number of colonies of the pancreatic cancer cell line PANC1 compared to the control group when KBL1041 strain was treated at a ratio of 1:10 ³ , but when KBL1041 strain was treated at a ratio of 1:10 ² The number of was significantly decreased, and the size of the cluster was also significantly decreased (FIG. 19). Therefore, it was confirmed that the KBL1041 strain of the present invention exhibited an inhibitory effect on non-settled formation ability in renal cancer cell lines.

Claims

Veillonella parvula strain, a culture of the strain, a lysate of the strain, and a pharmaceutical composition for preventing or treating cancer, comprising at least one member selected from the group consisting of an extract of the strain.
The method of claim 1,

The Bailonella Parbula strain is a KCTC 5019 strain, a pharmaceutical composition for preventing or treating cancer.
The method of claim 1,

The cancers include lung cancer, non-small cell lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, colon cancer, colorectal cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, uterus Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, sarcoma of the soft tissue, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, solid tumor of childhood, lymphoma, bladder cancer , A pharmaceutical composition comprising at least one selected from the group consisting of renal cancer, renal cell carcinoma, renal pelvic carcinoma, axial contraction tumor, brain stem glioma, Merkel cell tumor, urinary tract tumor and pituitary adenoma.
The method of claim 1,

The composition further comprises an anti-cancer therapeutic agent, a pharmaceutical composition.
The method of claim 4,

Wherein the anti-cancer treatment is an immune checkpoint inhibitor, a pharmaceutical composition
The method of claim 5,

Wherein the anti-cancer drug is an anti-PD1 antibody, a pharmaceutical composition
Veillonella parvula ( Veillonella parvula ) strain, a culture of the strain, a lysate of the strain, and at least one selected from the group consisting of an extract of the strain, a food composition for preventing or improving cancer.
The method of claim 7,

The Bailonella Parbula strain is a KCTC 5019 strain, a food composition for preventing or improving cancer.
Veillonella parvula strain, a culture of the strain, a lysate of the strain, and an animal feed composition comprising at least one selected from the group consisting of an extract of the strain.
The method of claim 9,

The Bailonella Parbula strain is KCTC 5019 strain, animal feed composition.