WO2023068857A1 - Novel bacterial strain having anticancer activity and composition for relieving, preventing, or treating cancer using same - Google Patents

Abstract

The present invention relates to an Erysipelatoclostridium ramosum strain and/or a Clostridium scindens strain, and a use thereof for preventing, treating, or relieving cancer.

Description

Novel bacterial strains having anticancer activity and compositions for relieving, preventing or treating cancer using the same

The present invention relates to Erysipelatoclostridium ramosum strains and Clostridium scindens strains having anticancer effects, and their cancer prevention, treatment, or improvement uses.

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 species ( Bac probiotics), which have an excellent ability to produce digestive enzymes (amylase, protease, lipase, cellulase, phosphatase), are microorganisms with antibacterial activity and enzyme activity that help balance intestinal microorganisms, and the microorganisms are produced 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. Because they have the intestinal tract as their habitat and must survive on their way to the intestine, they must be resistant to acids, enzymes, and bile in the intestinal environment.Moreover, probiotics maintain their viability and activity before being consumed in delivered food, and as a preventive measure against infection. It should be sensitive to the antibiotics used and should not carry 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.

Treated in the present invention, Erysipelatoclostridium ramosum ( Erysipelatoclostridium ramosum ) and Clostridium sindens ( Clostridium scindens ) are microorganisms found in the human intestine, and while living in the human intestine, they have a beneficial effect on the intestinal environment It is one of the probiotics.

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 devoted themselves to probiotics research as a method that can replace drug-based treatment of cancer for which there is no conventional satisfactory treatment. ( Erysipelatoclostridium ramosum ) Strain and Clostridium syndenseu ( Clostridium scindens ) The strain was newly developed to complete 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 an Erysiphellatoclostridium ramosum strain and a Clostridium syndens strain having anticancer activity.

In addition, the present invention is an Erysipella toclostridium lamosum KBL1038 strain deposited with accession number KCTC14615BP, an Erysipella toclostridium lamosum KBL1039 strain deposited with accession number KCTC14609BP, and a Clostridium deposited with accession number KCTC13277BP Provided is a Clostridium sindense KBL987 strain and a Clostridium sindense KBL1037 strain deposited under accession number KCTC14608BP.

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, including at least one selected from the group consisting of one or more strains of the strains, cultures of the strains, lysates of the strains, and extracts of the strains.

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

Erysipella toclostridium lamosum KBL1038 strain and KBL1039 strain and Clostridium syndens KBL987 strain and KBL1037 strain, and extracts thereof according to the present invention reduce the size of tumors when administered to animals having cancer The effect appears, and it increases immune cells related to improvement or treatment of cancer. 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. Erysipelato Clostridium lamosum strain and Clostridium syndens strain, and their cultures, lysates and extracts according to the present invention are administered to animals with cancer It has the effect of reducing the size of the tumor when it becomes, and increases the immune cells related to the improvement or treatment of cancer. 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.

1 is a view showing the experimental process of animal experiments to confirm the tumor size after administering the extracts of KBL1038, KBL1039, KBL987 and KBL1037 of the present invention to melanoma-induced mice.

Figure 2 is a graph showing the tumor volume increase inhibitory effect when Clostridium syndens KBL987 strain extract and Erysipelato Clostridium lamosum KBL1039 strain extract were administered to melanoma mice.

Figure 3 is a graph showing the tumor volume increase inhibitory effect when Clostridium syndens KBL1037 strain extract and Erysipelato Clostridium lamosum KBL1038 strain extract were administered to melanoma mice.

Figure 4 shows Clostridium syndens KBL1037 strain extract, Clostridium syndens KBL987 strain extract, Erysipella toclostridium lamosum KBL1038 strain extract and Erysipella toclostridium lamosum KBL 1039 strain extract melanoma It is a graph showing the tumor volume increase inhibitory effect when administered to mice.

Figure 5 is a view showing the experimental process of animal experiments to determine the tumor size after administering Erysipelato Clostridium lamosum KBL1038 strain to melanoma-induced mice.

Figure 6 is a graph showing the tumor volume increase inhibitory effect when Erysipella toclostridium lamosum KBL1038 strain was administered to melanoma mice compared to other strains.

Figure 7 is a graph showing the change in tumor volume increase over three times after administration of Erysipelato Clostridium lamosum KBL1038 strain to melanoma mice (upper figure), and immune cells in tumor tissue using flow cytometry It is a diagram showing the analysis result (bottom figure).

8 is a diagram illustrating an experimental process of an animal experiment for confirming the size of a tumor after administering an Erysiphelatoclostridium lamosum KBL1038 strain to a mouse in which colon cancer was induced.

9 is a graph showing the change in tumor volume increase after administration of Erysipelatoclostridium lamosum KBL1038 strain to colorectal cancer mice (left figure), and the results of analyzing immune cells in tumor tissue using flow cytometry ( It is a drawing showing the figure on the right).

Figure 10 shows the experimental process of an animal experiment to confirm the tumor size by orally administering Erysipelato Clostridium ramosum KBL1038 strain after administering antibiotics to melanoma-induced mice for 0, 7, or 14 days. it is a drawing

11 is a graph showing changes in tumor volume increase after oral administration of Erysiphelatoclostridium lamosum KBL1038 strain to melanoma mice in an antibiotic and oral model.

Figure 12 shows the experimental process of animal experiments to determine the size of tumors after administering the anti-PD-1 antibody, an anti-cancer treatment, alone or in combination with the Erysipelatoclostridium lamosum KBL1038 strain to melanoma-induced mice. it is a drawing

13 is a graph showing changes in tumor volume increase when Erysiphelatoclostridium lamosum KBL1038 strain and anti-PD-1 antibody were administered alone or in combination to melanoma mice.

14 is a graph (left figure) showing changes in tumor volume increase when Erysiphelatoclostridium lamosum KBL1038 strain and anti-PD-1 antibody are administered alone or in combination to colorectal cancer mice (left figure) and tumor It is a diagram showing the result of analyzing immune cells in tissues (right figure).

Figure 15 shows the experimental process of animal experiments to confirm the size of tumors by administering Erysipella toclostridium ramosum KBL1038 strain to melanoma and colorectal cancer induced mice in a T cell deficient mouse model (athymic nude mouse). It is an illustrated drawing.

16 is a graph showing changes in tumor volume increase of Erysiphelatoclostridium ramosum KBL1038 strain for T cell deficient mice.

17 is a graph illustrating the survival rate of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in colorectal cancer cell lines HCT116 and DLD-1.

18 is a diagram showing the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in HCT116, a colorectal cancer cell line.

19 is a diagram illustrating the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in colorectal cancer cell line DLD-1.

FIG. 20 is a view showing analysis of non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in lung cancer cell line A549.

FIG. 21 is a diagram illustrating the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in pancreatic cancer cell line PANC1.

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 an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Provides Clostridium scindens KBL987 strain and/or Clostridium scindens KBL1037 strain deposited with accession number KCTC14608BP.

The KBL1038 strain may have a 16s rDNA sequence of SEQ ID NO: 1 below.

<SEQ ID NO: 1> 16s rDNA sequence of strain Erysipelato Clostridium lamosum KBL1038

The present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Clostridium sindens deposited as ( Clostridium scindens ) KBL987 strain and / or the basic invention with accession number KCTC14608BP Erysipelato Clostridium ramosum having anticancer activity ( Erysipelatoclostridium ramosum ) and Clostridium sindens ( Clostridium scindens ) strains provides

The present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Provides Clostridium scindens KBL987 strain and/or Clostridium scindens KBL1037 strain deposited with accession number KCTC14608BP.

The KBL1038 strain may have a 16s rDNA sequence of SEQ ID NO: 1 below.

<SEQ ID NO: 1> 16s rDNA sequence of strain Erysipelato Clostridium lamosum KBL1038

CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCGTTAGTTACCATCATTAAGTTGGGGACTCTAGCGAGACTGCCAGTGACAAGCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTGCAGAGGGAAGCGAAGCCGCGAGGTGAAGCAAAACCCATAAAACCATTCAGTTGGATCACTTCACATTCAGTTGAGTCACTCAACTTA GAATCAGCATGTCGCGGTGATACGTTCTCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTGATAACACCCGAAGCCGGTGGCCTAACCGCAAGGAAGGAGCTGTCTAAGGTGGATGAAGGG

For example, the KBL1038 strain has a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 1 It may be an Erysipella toclostridium ramosum strain.

The KBL1039 strain may have a 16s rDNA sequence of SEQ ID NO: 2 below.

<SEQ ID NO: 2> 16s rDNA sequence of strain Erysipelato Clostridium lamosum KBL1039

AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCGTTAGTTACCATCATTAAGTTGGGGACTCTAGCGAGACTGCCAGTGACAAGCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTGCAGAGGGAAGCGAAGCCGCGAGGTGAAGCAAAACCCTGGAAAACCATTCAGTTCGGATTGTGAGTCAATTCAATTACACT TCGCGAATCAGCATGTCGCGGTGATACGTTCTCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTGATAACACCCGAAGCCGGTGGCCTAACCGCAAGGAAGGAGCTTTCTAATGGTGATTAGT

For example, the KBL1039 strain has a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 2 It may be an Erysipella toclostridium ramosum strain.

The KBL987 strain may have a 16s rDNA sequence of SEQ ID NO: 3 below.

<SEQ ID NO: 3> 16s rDNA sequence of Clostridium syndens KBL987

ATCCTTAGTAGCCAGCACATGATGGTGGGCACTCTAGGGAGACTGCCGGGGATAACCCGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGACAGCGATGTTGAGCGAATCCCAAAAATAACGTCCCAGTTCGCGGACTGCAGTCTGCAGCTCGACTGCACGAAGGGCTGGGTCTGCTAGTCAGAATGATA CCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACCGAAAGG

For example, Clostridium syndense is a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 3 It may be a Clostridium syndens strain having.

The KBL1037 strain may have a 16s rDNA sequence of SEQ ID NO: 4 below.

<SEQ ID NO: 4> 16s rDNA sequence of Clostridium syndens KBL1037

GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCAGTAGCCAGCATTATGGATGGGCACTCTGGAGAGACTGCCAGGGAGAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCAGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAGGCGAACCCGCGAGGGTGGGCAAATCGCGTCTGAGTTCCATTCGGAACT TAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGCCGGTGACCCAACCCGTAAGGGAGGGAGCCGTCGAAGTGACCCGTTTGC

For example, Clostridium syndense is a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 4. It may be a Clostridium syndens strain having.

Since the above strains are excellent in preventing, treating or improving cancer, they can be used for this purpose.

Specifically, the strains may have an activity of increasing the number and activity of immune cells in cancer cells, and may suppress tumors through T cells.

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

The present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited with the accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited with accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number Clostridium sindens deposited as KCTC13277BP ( Clostridium scindens ) KBL987 strain and / or deposited with accession number KCTC14608BP Clostridium scindens ( Clostridium scindens ) At least one or more strains of the KBL1037 strain, a culture of the strain, a lysate of the strain, and It provides a pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of extracts of the strain.

The composition of the present invention may further include other strains in addition to the above strains, and may include both other strains of Erysipelato Clostridium ramosum and Clostridium syndens, of course.

In the composition of the present invention, the strains are in the form of live cell cells, dead cell cells and dried strains, and the present invention is Erysipelatoclostridium ramosum ( Erysipelatoclostridium ramosum ) At least one strain and Clostridium syndens ( Clostridium scindens ) strain It provides a pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of the above strains, cultures of the strains of the strains, lysates of the strains, and extracts of the strains.

The strain is Erysipelatoclostridium ramosum KBL1038 strain, deposited with accession number KCTC14615BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Clostridium sindens deposited as ( Clostridium scindens ) KBL987 strain and Clostridium sindens deposited as accession number KCTC14608BP ( Clostridium scindens ) It may be at least one strain of KBL1037 strain.

The composition of the present invention may further include other strains in addition to the above strains, and may include both other strains of Erysipelato Clostridium ramosum and Clostridium syndens, of course.

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 a pH 1 HCl solution, is brought into contact at 36°C to 38°C, and is preferably followed by a pH 6.8 KH ₂ PO ₄ buffer solution. 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, Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a food composition or food additive composition for preventing or alleviating cancer, including at least one selected from the group consisting of an extract of.

The strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 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 composition for food or food additives of the present invention is at least one of Erysipella toclostridium lamosum strains (eg KBL1038 strain and / or KBL1039 strain) and Clostridium syndens strain (eg KBL987 strain and / or KBL1037 strain) The above strains may be added independently or to an acceptable carrier, or prepared in the form of a composition suitable for consumption by humans or animals. 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, Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a composition for adding animal feed or animal feed, including at least one selected from the group consisting of extracts of.

The strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.

The additive for animal feed of the present invention may be in the form of a dry or liquid formulation, and is an Erysipherato Clostridium ramosum strain (eg, KBL1038 strain and / or KBL1039 strain) and a Clostridium syndens strain (eg, KBL987 strain and / or KBL1037 strain) may further include other non-pathogenic microorganisms in addition to at least one or more strains.

At least one strain of the Erysipelato Clostridium ramosum strain (eg KBL1038 strain and / or KBL1039 strain) and Clostridium syndens strain (eg KBL987 strain and / or KBL1037 strain) of the present invention as an additive for animal feed In use, various grains and soybean proteins, including peanuts, peas, sugar beets, pulp, grain by-products, animal intestine powder and fish meal powder, etc. may be used as raw materials for feed, and these may be used unprocessed or processed without limitation. can

The present invention, Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a cancer prevention or treatment method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one selected from the group consisting of an extract of to a subject in need of treatment.

The strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.

The present invention, in the manufacture of a drug for preventing or treating cancer, Erysipelato Clostridium ramosum ( Erysipelatoclostridium ramosum ) At least one strain of the strain and Clostridium sindens ( Clostridium scindens ) strain, of the strain of the strain It provides the use of a composition comprising at least one selected from the group consisting of a culture, a lysate of the strain, and an extract of the strain.

The strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.

The present invention, Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a use for preventing or treating cancer of a pharmaceutical composition comprising at least one selected from the group consisting of extracts of.

The strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 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.

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, 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.

Example 1. Confirmation of anti-tumor effect of strain extract

In order to confirm the antitumor effect of the Erysiphellatoclostridium lamosum strain and Clostridium syndens strain of the present invention, the cancer alleviating, preventive and therapeutic effects of the extracts of the strains were confirmed. Specifically, after culturing Erysipella toclostridium lamosum KBL1038 strain, Erysipella toclostridium lamosum KBL1039 strain, Clostridium syndens KBL987 strain and Clostridium syndens KBL1037 strain, their extracts were black By treating species-induced mice, the tumor volume increase inhibitory effect was confirmed.

1-1. Cultivation and preparation of strains

Erysipella toclostridium lamosum KBL1038, KB Example 1. Confirmation of anti-tumor effect of strain extract

In order to confirm the antitumor effect of the Erysiphellatoclostridium lamosum strain and Clostridium syndens strain of the present invention, the cancer alleviating, preventive and therapeutic effects of the extracts of the strains were confirmed. Specifically, after culturing Erysipella toclostridium lamosum KBL1038 strain, Erysipella toclostridium lamosum KBL1039 strain, Clostridium syndens KBL987 strain and Clostridium syndens KBL1037 strain, their extracts were black By treating species-induced mice, the tumor volume increase inhibitory effect was confirmed.

1-1. Cultivation and preparation of strains

In order to confirm the effect of the extracts of Erysipelato Clostridium ramosum KBL1038 and KBL1039 strains and Clostridium syndens KBL987 and KBL1037 strains of the present invention, the strains were first cultured. Each of the strains was cultured in YBHI medium supplemented with 0.5% cysteine, activated through a total of two subcultures at 9 and 12 hour intervals, and then used in the experiment.

1-2. Preparation of strain extract

In order to obtain extracts of Erysipelato Clostridium lamosum KBL1038 and KBL1039 strains and Clostridium syndens KBL987 and KBL1037 strains cultured in Example 1-1, the following extraction process was performed.

The above strains cultured in 1.8 liter YBHI medium were centrifuged at 6000 g, 20 minutes, 4 ° C, the culture medium was removed, and the pellet was reproduced with 40 mL of phosphate-buffered saline (PBS). Resuspension was made. After washing with PBS to completely remove the culture medium, it was centrifuged under the same conditions. After centrifugation, the supernatant was discarded, and the remaining pellet was again dissolved in 20 mL of PBS, and then ultrasonicated using a sonicator for 60 minutes or treated three times for 30 seconds using an ultra-high speed blender. Samples treated with ultrasound or ultra-high-speed blender were phase separated by centrifugation at 10,000 g for 20 minutes at 4 °C. The obtained supernatant was transferred to a dedicated ultracentrifuge tube and then ultracentrifuged using an ultracentrifuge at 100,000 g, 1 hour, and 4 °C. After ultracentrifugation, the supernatant was discarded and the remaining pellet was resuspended in 200uL of PBS. After protein quantification using the BCA assay, the effect was confirmed using the extract of the strain of the present invention.

1-3. Confirmation of the inhibitory effect of the strain extract on the increase in tumor volume

In order to confirm the tumor-inhibiting effect of the extracts of the strains of the present invention obtained through Examples 1-1 and 1-2 in melanoma mice, after inducing melanoma in mice, the strain extracts were administered for a certain period of time, Changes in tumor volume of mice were measured (FIG. 1).

For tumor induction, 5-week-old C57BL/6 male mice were used. Mice introduced into the animal testing facility were subjected to a one-week acclimatization and stabilization period, and their weights were measured to match the mean and standard deviation of the weights among the animals.

After preparing C57BL/6 mice, each mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 1x10 ⁶ cells/100 μl to induce cancer. On the 6th day after induction, 6 animals per group were randomized, 3 animals per cage, and the extracts prepared from the 7th day after induction (Erysipherato Clostridium lamosum KBL1038, KBL1039 strains and Clostridium syndens KBL987 , KBL1037 strain extract) was intraperitoneally administered daily at 5 μg/100 μl/mouse. For the negative control group, 100 μl of PBS was administered, Roseburia intestinalis, Escherichia coli, Clostridium clostridioforme , and Lactobacillus luminis. luminis ) was administered to compare the effect with the strain extract of the present invention.

From day 6 to day 22 after administration of the extract, changes in tumor volume were measured using a caliper twice or three times a week, and are shown in FIGS. 2 to 4 .

As a result of the measurement, the mice administered with the Clostridium syndens KBL987 strain extract and the Erysiphelatoclostridium ramosum KBL1038 strain extract of the present invention were mice administered with PBS or Roseburia intranasal strain extract and S. The size of the tumor was significantly reduced compared to the mice administered with Kerichia coli strain extract (FIG. 2).

In addition, mice administered with the Clostridium syndens KBL1037 strain extract and the Erysiphelatoclostridium ramosum KBL1038 strain extract of the present invention, mice administered with PBS, Roseburia internalis, Escherichia coli And the size of the tumor was reduced compared to the mice administered with Clostridium clostrdioform extract (FIG. 3).

Clostridium sindens KBL987 strain extract, Clostridium sindens KBL1037 strain extract, Erysipelato Clostridium ramosum KBL1038 strain extract and Erysipelato Clostridium ramosum KBL1039 strain extract of the present invention were administered , The size of tumors was significantly reduced compared to mice administered with PBS, and the size of tumors was reduced compared to mice administered with extracts of Roseburia intranas and Lactobacillus luminis strains (FIG. 4).

As can be seen from the results of Figures 2 to 4, Clostridium sindens KBL987 strain extract, Clostridium sindens KBL1037 strain extract, Erysiphera toclostridium ramosum KBL1038 strain extract and Erysipellato of the present invention It can be seen that the Clostridium ramosum KBL1039 strain extract has the effect of significantly suppressing the increase in tumor size compared to PBS or other strain extracts. In particular, since tumors derived from B16F10 cells are less sensitive to anticancer drugs than tumors derived from other tumor cells, the tumor size inhibitory effect observed after administration of the strain extract of the present invention was more unpredictable. From this, it was confirmed that the significantly superior cancer alleviation, prevention or treatment effect of the present invention was obtained.

Example 2. Confirmation of tumor suppression effect in various tumor mouse models of Erysipelato Clostridium ramosum KBL1038 strain

2-1. Confirmation of effect in melanoma-induced mice

In addition to confirming the anticancer effect of the strain extract of the present invention through Example 1, dead cells of the Erysiphelatoclostridium lamosum KBL1038 strain were directly administered to melanoma-induced mice, and then the volume change of the tumor was measured. The tumor growth inhibitory effect was confirmed (FIG. 5).

For tumor induction, each C57BL/6 mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 5x10 ⁵ cells/100 μl to induce cancer. On the 6th day after induction, 7 animals per group were randomly assigned to 3 or 4 animals per cage, and from the 7th day after induction, the pasteurized bacteria of the Erysiphelatoclostridium ramosum KBL1038 strain were 1x10 ⁹ cells/200 μl/mouse was intraperitoneally administered once every 3 days for a total of 5 times. For the negative control group, 200 μl of PBS was administered, Enterococcus faecalis SNUV414 strain, Clostridium syndense SNUG40297 and Veillonella parvula strain were administered to compare the effects with the strains of the present invention did

From day 6 to day 21 after induction, the change in tumor volume was measured using a caliper twice or three times a week and is shown in FIG. 6 .

As a result of the measurement, mice administered with the Erysipelato Clostridium lamosum KBL1038 strain of the present invention were mice administered with PBS, or Enterococcus faecalis SNUV414 strain, Clostridium syndens SNUG40297 and Baylonella Parbula strain administration. Compared to mice, the tumor size was significantly reduced (FIG. 6).

As can be seen from the above experimental results, when the KBL1038 strain of the present invention is administered, there is an effect of suppressing tumor growth in melanoma-induced mice, and even when compared to the case of administering other types of strains showing related effects, tumor It was confirmed that the inhibitory effect was better.

In addition, using the melanoma-induced mouse, the KBL1038 strain was intraperitoneally administered once every 3 days for a total of 4 times in the same way, and then calipers twice or 3 times a week from the 6th day to 20-21 after induction The volume change of the tumor was measured using and shown in FIG. 7 , and then immune cells were analyzed in the tumor tissue using a flow cytometer, and the results were shown in FIG. 7 .

As a result of the measurement, mice administered with the strain of Erysiphelatoclostridium lamosum KBL1038 of the present invention had a significantly reduced tumor size compared to mice administered with PBS (upper part of FIG. 7). In addition, as a result of flow cytometry analysis, CD8 T cells secreting the inflammatory factor IFNγ and the cell killing factor Granzyme B increased in the tumor tissue of mice administered with the KBL1038 strain (bottom of FIG. 7).

As can be seen from the above experimental results, it was confirmed once again that the KBL1038 strain of the present invention had an effect of inhibiting tumor growth in melanoma-induced mice, and immune cells that play an important role in anti-cancer mechanisms It was confirmed that there is an effect of increasing the distribution of .

2-2. Confirmation of effect in colorectal cancer-induced mice

After directly administering dead cells of the Erysiphelatoclostridium ramosum KBL1038 strain to mice induced with colon cancer, the tumor growth inhibitory effect was confirmed by measuring the change in tumor volume (FIG. 8).

To induce tumors, colon cancer cells, MC38, were subcutaneously injected into each C57BL/6 mouse at 5×10 ⁵ cells/100 μl to induce cancer. On the 6th day after induction, 6 animals per group were randomized, 3 animals per cage, and from the 7th day after the induction, the prepared bacteria of the Erysiphelatoclostridium lamosum KBL1038 strain were 1x10 ⁹ cells/ 200 μl/mouse was intraperitoneally administered once every 3 days for a total of 4 times. For the negative control group, 200 μl of PBS was administered.

From day 6 to day 16 after induction, the change in tumor volume was measured using a caliper two or three times a week, and then immune cells were analyzed in the tumor tissue using flow cytometry, and the results are shown in FIG. 9 shown in

As a result of the measurement, mice administered with the strain of Erysiphelatoclostridium lamosum KBL1038 of the present invention had a significantly reduced tumor size compared to mice administered with PBS (left side of FIG. 9). In addition, as a result of flow cytometry analysis, CD8 and CD4 T cells secreting the inflammatory factor IFNγ and the cell killing factor Granzyme B were increased in the tumor tissues of mice administered with the KBL1038 strain (right side of FIG. 9).

As can be seen from the above experimental results, when the KBL1038 strain of the present invention is administered, there is an effect of suppressing tumor growth in mice induced with colon cancer, and an effect of increasing the distribution of immune cells that play an important role in anticancer mechanisms. It could be confirmed that there is

Example 3. Confirmation of antitumor inhibitory effect of Erysiphelatoclostridium ramosum KBL1038 strain in antibiotics and oral administration model

It was confirmed whether the anticancer effect was exhibited even when the Erysiphelatoclostridium lamosum KBL1038 strain of the present invention was administered orally to melanoma mice administered with antibiotics. To this end, the KBL1038 strain of the present invention was orally administered to melanoma mice not administered with antibiotics or melanoma mice administered with antibiotics for 7 days or 14 days for 7 days to confirm the effect (FIG. 10).

To induce tumors, cancer was induced by subcutaneous injection of 5x10 ⁵ cells/100 μl of B16F10 suspension, which is a melanoma cell, into each C57BL/6 mouse, and antibiotics (5 g/L Streptomycin, 1 g/L Colistin sulfate, 1 g/L Ampicillin ) was supplied for 0, 7 or 14 days, after which the KBL1038 strain was supplied at various doses (5x10 ⁸ cells/200 μl/mouse, 1x10 ⁹ cells/200 μl/mouse, 5x10 ⁹ cells/200 μl/mouse). It was orally administered for 7 days. For the negative control group, 200 μl of PBS was administered.

After administration of the strain, the volume change of the tumor was measured using a caliper 2 or 3 times and shown in FIG. 11 .

As a result of the measurement, when the KBL1038 strain was orally administered to melanoma mice not separately administered with antibiotics, the tumor size was reduced compared to the PBS-administered mice, especially at the doses of 1x10 ⁹ cells/200 μl/mouse and 5x10 ⁹ cells/200 μl/mouse. showed a significant reduction in In addition, when the KBL1038 strain was administered at a dose of 5x10 ⁹ cells/200 μl/mouse in mice after administration of antibiotics, the tumor size was significantly reduced (FIG. 11). That is, in the case of mice orally administered with the KBL1038 strain at a concentration of 5x10 ⁹ cells / 200 μl / mouse, the size of the tumor is significantly reduced compared to mice administered with PBS, which is a negative control, regardless of the antibiotic supply period.

As can be seen from the above experimental results, the KBL1038 strain of the present invention shows excellent tumor suppression effect even when administered by oral administration as well as intraperitoneal administration, and tumor suppression of the orally administered KBL1038 strain even when antibiotics are administered It was confirmed that the effect appeared.

Example 4. Erysipelato Clostridium Ramosum KBL1038 Confirmation of tumor suppression effect when administered in combination with anticancer drugs

When the Erysiphelatoclostridium lamosum KBL1038 strain of the present invention was administered in combination with an anti-PD-1 antibody, which is a kind of anticancer drug, it was confirmed how much the tumor growth inhibitory effect was exhibited.

4-1. Confirmation of effect in melanoma-induced mice

For tumor induction, each C57BL/6 mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 5x10 ⁵ cells/100 μl to induce cancer. On the 6th day after induction, 6 animals per group were randomized, 3 animals per cage, and the dead cells of the Erysipella toclostridium lamosum KBL1038 strain prepared from the 10th day after the induction (1x10 ⁹ cells / 200 μl / mouse) ) and immune checkpoint inhibitor anti-PD-1 antibody (BioxCell, Cat# BE0146, 150 μg/mouse/time) were intraperitoneally administered once every 3 days for a total of 3 times. For the negative control group, 200 μl of PBS was administered (FIG. 12).

From day 7 to day 21 after induction, the change in tumor volume was measured using a caliper twice or three times a week and is shown in FIG. 13 .

As a result of the measurement, in the case of mice administered with the Erysiphelatoclostridium lamosum KBL1038 strain alone and in combination with the anti-PD-1 antibody of the present invention, mice administered with PBS or anti-PD-1 antibody The size of the tumor was significantly reduced compared to the mice administered with only the mouse alone (FIG. 13).

As can be seen from the above experimental results, the KBL1038 strain of the present invention not only has a superior tumor suppression effect compared to the anti-PD-1 antibody, which is an existing anti-cancer drug, but also uses the KBL1038 strain in combination with the anti-PD-1 antibody. Even when administered, it was confirmed that the tumor inhibitory effect was very excellent.

4-2. Confirmation of effect in colorectal cancer-induced mice

For tumor induction, colon cancer cell MC38 was subcutaneously injected into each C57BL/6 mouse at 1x10 ⁵ cells/100 μl to induce cancer. On the 7th day after induction, 6 animals per group were randomized, 3 animals per cage, and the dead cells of the Erysiphelatoclostridium lamosum KBL1038 strain prepared from the 10th day after the induction (1x10 ⁹ cells / 200 μl / mouse) ) and immune checkpoint inhibitor anti-PD-1 antibody (BioxCell, Cat# BE0146, 150 μg/mouse/time) were intraperitoneally administered once every 3 days for a total of 3 times. For the negative control, IgG was administered.

From day 10 to day 21 after induction, the change in tumor volume was measured using a caliper twice or three times a week, and then immune cells were analyzed in the tumor tissue using flow cytometry, and the results are shown in FIG. 14 shown in

As a result of the measurement, in the case of mice administered in combination with the anti-PD-1 antibody of the Erysiphelatoclostridium lamosum KBL1038 strain of the present invention, the tumor size was significantly reduced compared to the mice administered with IgG (Fig. left of 14). In addition, as a result of flow cytometry analysis, CD4 T cells expressing the immunosuppressive factors FoxP3 and PD-1 decreased, and CD8 T cells secreting the cell killing factor Granzyme B increased in the tumor tissue of mice administered with the combination. (Right in Fig. 14).

As can be seen from the above experimental results, when the KBL1038 strain of the present invention is administered in combination with an anti-PD-1 antibody, the tumor suppression effect is very excellent and the immunological factor representing cytotoxic T cells is increased. was able to confirm

Example 5. Confirmation of anticancer action mechanism of Erysipelato Clostridium ramosum KBL1038 strain

In order to confirm the mechanism of action of the Erysipelato Clostridium ramosum KBL1038 strain on tumors of the present invention, the change in tumor size after administration of the KBL1038 strain using a T cell deficient mouse model (athymic nude mouse) It was confirmed (FIG. 15).

To induce tumors, melanoma cells B16F10 and colon cancer cells MC38 were subcutaneously injected at 2x10 ⁵ cells/100 μl into each athymic nude mouse to induce melanoma and colon cancer, respectively. On day 6 after induction, 7 animals per group were randomized, and dead cells (1x10 ⁹ cells/200 μl/mouse) of the Erysiphelatoclostridium lamosum KBL1038 strain prepared from day 7 after induction were added for 3 days. was administered intraperitoneally once for a total of 4 times. For the negative control group, 200 μl of PBS was administered.

From day 7 to day 19 after induction, the change in tumor volume was measured using a caliper twice or three times a week and is shown in FIG. 16 .

As a result of the measurement, even when the Erysiphelatoclostridium ramosum KBL1038 strain of the present invention was administered to immunodeficient mice lacking T cells, there was no significant change in tumor size compared to mice administered with PBS (Fig. 16).

As can be seen from the above experimental results, when the T cells are deficient, the anticancer effect of the KBL1038 strain of the present invention does not appear, and some of the mechanisms that allow the anticancer effect of the KBL1038 strain to appear are tumor mediated by T cells. It was found to be suppressed.

Example 6. Confirmation of colorectal cancer cell line survival rate reduction effect of Erysipelato Clostridium lamosum KBL1038 strain

In order to confirm the anticancer effect of the Erysiphelatoclostridium ramosum KBL1038 strain of the present invention, colorectal cancer cell lines HCT116 and DLD-1 were treated and then the survival rate of cancer cells was measured.

Specifically, a colon cancer cell line (HCT116, DLD-1) was cultured in a 96-well culture plate (HCT116: 1x10 ⁴ cells/well, DLD-1: 2x10 ³ cells/well), and Erysipella toclostridium lamosum Killed cells of strain KBL1038 (killed by pasteurization, 70 ℃, 30 minutes) are treated at a ratio of 1:10 ¹ , 1:10 ² , 1:10 ³ , 1:10 ⁴ to the number of cancer cells, and then treated for 24 to 72 hours reacted 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 of the colorectal cancer cell line was significantly decreased in a concentration-dependent manner after 72 hours of treatment with the KBL1038 strain (FIG. 17).

As can be seen from the above experimental results, when the colorectal cancer cell line was treated with the KBL1038 strain of the present invention, it was confirmed that the growth inhibitory effect of the colorectal cancer cell line was dependent on the concentration of the treated KBL1038 strain.

Example 7. Confirmation of non-settled formation inhibitory effect of colorectal cancer cell line of Erysipelato Clostridium ramosum KBL1038 strain

HCT116 is a human-derived colorectal cancer cell line having a KRAS mutation. After treatment with KBL1038 strain, it was confirmed whether the non-establishment growth ability of HCT116 was reduced.

Specifically, the colorectal cancer cell line HCT116 was cultured in a 6-well culture plate (HCT116: 5x10 ³ cells/well, bottom agar: 0.6%, top agar: 0.3%), and the dead cells of the Erysipella toclostridium lamosum KBL1038 strain (pasteurization, 70 ℃, dead cells by way of 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 colon cancer cell line HCT116 colonies compared to the control group when KBL1038 strain was treated, but the number of clusters with a size of 20% or more decreased significantly depending on the concentration of the treated strain, and the size of the clusters also decreased. (FIG. 18). In particular, when the KBL1038 strain is treated at a ratio of 1:10 ³ , the number of colon cancer cell line clusters is reduced by less than about 30%, and the KBL1038 strain of the present invention has the effect of significantly inhibiting the non-establishment formation ability of the colon cancer cell line was able to confirm

Similarly, the same experiment was performed with another colorectal cancer cell line, DLD-1. DLD-1 is a human p53-defective colorectal cancer cell line.

Specifically, the colon cancer cell line DLD-1 was cultured in a 6-well culture plate (DLD-1: 1x10 ⁴ cells/well, bottom agar: 0.6%, top agar: 0.3%), and Erysipella toclostridium lamosum Killed cells of the KBL1038 strain (killed by pasteurization, 70 ° C., 30 minutes) were treated at a ratio of 1:10 ² and 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, when the KBL1038 strain was treated, the number of colon cancer cell line DLD-1 colonies was significantly reduced compared to the control group in proportion to the treated strain, and the size of clusters and clusters of 20% or more were also reduced (FIG. 19). Accordingly, it was confirmed that the KBL1038 strain of the present invention showed an inhibitory effect on non-settled formation ability not only in HCT116 but also in the DLD-1 colorectal cancer cell line.

Example 8. Confirmation of non-establishment formation inhibitory effect of lung cancer and pancreatic cancer cell lines of Erysipelato Clostridium ramosum KBL1038 strain

For the lung cancer cell line A549, after treatment with KBL1038 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 dead cells of the Erysipella toclostridium lamosum KBL1038 strain ( pasteurization, 70 ℃, 30 minutes) were 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 KBL1038 strain was treated compared to the control group. The size of the cluster was also significantly reduced (FIG. 20). Therefore, it was confirmed that the KBL1038 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 pancreatic cancer cell line PANC1. Specifically, the pancreatic cancer cell line PANC1 was cultured in a 6-well culture plate (PANC1: 5x10 ³ cells/well, bottom agar: 0.6%, top agar: 0.3%), and dead cells of the Erysipella toclostridium lamosum KBL1038 strain ( pasteurization, 70 ℃, 30 minutes) were 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, when the KBL1038 strain was treated at a ratio of 1:10 ³ , the number of colonies of pancreatic cancer cell line PANC1 was reduced by more than 30% in size (FIG. 21). Therefore, it was confirmed that the KBL1038 strain of the present invention exhibits an effect of inhibiting non-settled formation to some extent even in pancreatic cancer cell lines.

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14615BP

Entrusted date: 2021616

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14609BP

Entrusted date: 2021611

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13277BP

Entrusted date: 2017529

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14068BP

Entrusted date: 2021611

Claims (13)

Erysipelatoclostridium ramosum having anticancer activity ( Erysipelatoclostridium ramosum ) strain. The method of claim 1, The strain is an Erysipella toclostridium ramosum KBL1038 strain deposited with accession number KCTC14615BP. The method of claim 1, The strain is an Erysipella toclostridium ramosum KBL1039 strain deposited with accession number KCTC14609BP. Clostridium scindens strain having anticancer activity. The method of claim 4, The strain is Clostridium syndens KBL987 strain deposited with accession number KCTC13277BP. The method of claim 4, The strain is Clostridium syndens KBL1037 strain deposited with accession number KCTC14608BP. A pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of at least one or more strains of claims 1 to 6, a culture of the strain, a lysate of the strain, and an extract of the strain. The method of claim 7, 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 Carcinoma of the neck, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the larynx, cancer of the small intestine, 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 member selected from the group consisting of renal cancer, renal cell carcinoma, renal pelvic carcinoma, axial contraction tumor, brainstem glioma, Merkel cell tumor, urinary tract tumor and pituitary adenoma. The method of claim 7, The composition further comprises an anti-cancer therapeutic agent, a pharmaceutical composition. The method of claim 9, The anti-cancer treatment is an immune checkpoint inhibitor, a pharmaceutical composition. The method of claim 10, The pharmaceutical composition of claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. A food composition for preventing or improving cancer, comprising at least one strain selected from the group consisting of at least one or more strains of claims 1 to 6, a culture of the strain, a lysate of the strain, and an extract of the strain. An animal feed composition comprising at least one strain selected from the group consisting of at least one or more strains of claims 1 to 6, a culture of the strain, a lysate of the strain, and an extract of the strain.

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