KR20230123429A - Pharmaceutical composition for preventing or treating cancere - Google Patents

Abstract

The present invention relates to an extracellular vesicle (EV) derived from a Faecalibacterium sp. strain; And it relates to a pharmaceutical composition for preventing or treating cancer and a health functional food for preventing or improving cancer, including a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of the present invention exhibits excellent effects in preventing or treating cancer.

Description

Pharmaceutical composition for preventing or treating cancer {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CANCERE}

The present invention relates to a pharmaceutical composition for preventing or treating cancer, and more particularly, to a pharmaceutical composition for preventing or treating cancer containing an extracellular vesicle (EV) derived from a strain of the genus Picalibacterium .

Cancer is a product of uncontrolled and disorderly cell proliferation caused by an excess of abnormal cells, and a malignant tumor leaves its primary site and invades other tissues and grows rapidly, threatening life.

In order to treat cancer, various therapeutic approaches such as chemotherapy using various anticancer agents, radiotherapy, and antibody therapy targeting specific molecules related to cancer have been attempted. However, in the case of chemotherapy or radiation therapy, since it affects normal cells, side effects are serious, and cancer cells acquire resistance to anticancer drugs, and treatment fails or recurs frequently.

According to recent research reports, extracellular vesicles have been reported to play an important role in processes such as intercellular signal transduction and disposal management, and interest in clinical applications has recently increased. Using the characteristics of specific extracellular vesicles and cell membranes of target cells, it is expected that it will be possible to develop a therapeutic agent that can prevent side effects on other normal cells and specifically treat only diseased cells, including cancer cells.

On the other hand, the importance of studies on the effect of combination therapy of probiotics and immune checkpoint inhibitors, which have beneficial effects in the body such as strengthening immunity, is emphasized at the level of pharmabiotics. It is still in a state of obscurity.

KR 10-2019-0129739 A KR 10-2307603 B

The present invention is to solve the above-described technical problem, one object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing extracellular vesicles derived from pharmabiotics.

Another object of the present invention is to provide a health functional food for preventing or improving cancer.

Another object of the present invention is to provide a veterinary composition or feed additive for preventing or treating cancer.

Another object of the present invention is to provide a method for treating cancer of a patient by activating the patient's immune system using pharmabiotics-derived extracellular vesicles.

One aspect of the present invention for achieving the above object is,

Extracellular vesicles (EV) derived from Faecalibacterium sp. strains; And it relates to a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient.

The strain of the genus Picalibacterium is a Faecalibacterium prausnitzii strain, preferably a strain of Picalibacterium prosnitz EB-FPDK3 (KCCM12619P ) , F. prausnitzii EB-FPDK9 strain (KCCM12620P), F. prausnitzii EB-FPDK11 strain (KCCM12621P), and F. prausnitzii EB-FPYYK1 strain (KCCM12622P).

The pharmaceutical composition for preventing or treating cancer of the present invention may further include a cancer treatment agent such as a chemo-anticancer agent or an immuno-anticancer agent. The immuno-anticancer agent may be selected from the group consisting of anti-PD1, anti-PDL1, anti-CTLA, anti-Tim3 and anti-LAG3. Extracellular vesicles (EVs) derived from strains of the genus Picalibacterium and chemotherapeutic agents or immuno-anticancer agents may be administered simultaneously in one formulation, or simultaneously or sequentially in separate formulations.

Another aspect of the present invention is a strain of the genus Picylobacterium ; And it relates to a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient.

Another aspect of the present invention, extracellular vesicles (EV) derived from strains of the genus Picalibacterium ; And it relates to a health functional food for preventing or improving cancer containing a physiologically acceptable carrier or excipient.

Another aspect of the present invention is a strain of the Picalibacterium genus in a therapeutically effective amount to a subject or It relates to a cancer treatment method characterized by administering an extracellular vesicle (EV) derived from a strain of the genus Picalibacterium .

Another aspect of the present invention is a strain of the genus Picalibacterium or an extracellular vesicle (EV) derived from a strain of the genus Picalibacterium; And it relates to a veterinary composition for preventing or treating cancer comprising an acceptable carrier or excipient.

The present invention is a novel Picylobacterium prosnich EB-FPDK3 strain (KCCM12619P), F. prausnitzii EB-FPDK9 strain ( KCCM12620P ), F. prausnitzii EB-FPDK11 strain ( KCCM12621P ), and F. prausnitzii EB-FPYYK1 strain (KCCM12622P).

The pharmaceutical composition for preventing or treating cancer comprising the strain of the genus Picalibacterium or the extracellular vesicles (EV) derived from the strain of the genus Picalibacterium of the present invention reduces tumor size, reduces tumor growth, By preventing metastasis or preventing angiogenesis, it will be possible to develop an effective anticancer agent.

The pharmaceutical composition for preventing or treating cancer comprising the strain of the genus Picalibacterium or the extracellular vesicles (EV) derived from the strain of the genus Picalibacterium of the present invention not only exhibits excellent anticancer effects when administered alone, When administered in combination with a chemo-anticancer agent or an immuno-anticancer agent, the side effects of the anti-cancer agent are reduced and the efficacy is more activated, resulting in a superior anti-cancer effect compared to administering them alone.

Figure 1 shows the Picalibacterium prosniche EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention and the type strain Picalibacter This is the result of microscopic observation of the standard strain of Leeum Prosnich A2-165.
Figure 2 is a Picylobacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention and Picylobacterium prosnich A2-165 standard This is the result of PCR analysis of the strain.
Figure 3 is a Picylobacterium prosniche EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention and Picalibacterium prosnich A2-165 standard This is the result of RAPD (Random Amplified Polymorphic DNA) analysis of the strain's genomic DNA.
Figure 4 is a phylogenetic diagram of F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains based on the 16S rRNA sequence.
Figure 5 is a Picylobacterium prosniche EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains and Picalibacterium prosnich A2-165 standard of the present invention The result of confirming whether the strain has hemolytic activity is shown.
Fig. 6 is an electron micrograph of extracellular vesicles derived from the strains of F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 according to the present invention.
Figure 7 (a) and (b) is a Picylobacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strain alone or derived from strains of the present invention It is a schematic diagram for explaining the animal experiment procedure of Example 4 and Example 5 for confirming the anticancer activity of extracellular vesicles (EV).
8 and 9 show the results of the F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains and aPD in mice transplanted with tumors of the present invention. It is a figure showing the tumor formation inhibitory effect by the combined administration of -1.
Figure 10 shows the use of Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains and aPD-1 antibody of the present invention in Example 4 It is a photograph comparing the tumor size of mice of each experimental group for 20 days in an allograft anti-cancer animal model to measure the effect of suppressing cancer development by administration.
Figure 11 shows the cancer of extracellular vesicles derived from the Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention in Example 5. It is a graph showing the change in tumor size over time in an allograft anti-cancer animal model to measure the effect of inhibiting development.
Figure 12 is an allograft anti-cancer animal model of Ficcolibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains derived from extracellular vesicles and anti- It is a graph showing the change in tumor weight for 25 days when PD1 was administered together and when only anti-PD1 was administered.
Figure 13 shows the cancer development inhibitory effect of extracellular vesicles derived from the Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention. This is a picture comparing the tumor size of mice in each experimental group for 25 days in an allograft anti-cancer animal model to measure.
Figures 14a and 14b show the F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention using a wound healing assay in HT29 cells. The results of evaluation of the anticancer activity of the derived extracellular vesicles are shown.
Figure 15 shows the scheme of animal experiments to confirm the anticancer effect by administration of Picylobacterium prosniche EB-FPDK9 or EB-FPDK9-derived extracellular vesicles (EB-FPDK9EVs) in an allograft mouse animal model using melanoma. It is also a model.
Figure 16 shows tumor growth curves upon administration of Ficcalibacterium prosnich EB-FPDK9 strain or EB-FPDK9 EVs.
17 is a photograph comparing the size of tumors at the end of administration of the Ficcolibacterium prosnich EB -FPDK9 strain or EB-FPDK9 EVs.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

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 the present specification, when a certain part 'includes' a certain component, this means that it may further include other components without excluding other components unless otherwise stated.

As used herein, the terms 'treat', 'treatment' and the like mean temporarily or permanently alleviating symptoms, removing the cause of symptoms, or preventing or delaying the onset of symptoms of a disease or condition. .

As used herein, the term 'prevention' refers to any activity that suppresses or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term 'improvement' refers to any action that reduces a parameter associated with an abnormal state, for example, the severity of a symptom.

As used herein, the term 'pharmaceutically acceptable' means that the benefit / risk ratio is reasonable without excessive toxicity, irritation, allergic reaction or other problems or complications, and is suitable for use in contact with the tissue of a subject (eg, human) means a composition that is within the scope of sound medical judgment.

As used herein, the term 'immune checkpoint inhibitor' refers to a type of drug that blocks certain proteins produced by certain types of immune system cells, such as T lymphocytes, and some cancer cells, which are immune responses. and prevents T lymphocytes from killing cancer cells. PD-1/PD-L1 and CTLA-4/B7-1/B7-2 are well known as "immune checkpoint inhibitors".

One aspect of the present invention, the genus Picalibacterium ( Faecalibacterium sp.) Extracellular vesicles (EV) derived from the strain; And it relates to a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient.

Pharmabiotics are defined as human-derived bacteria or their products that have a proven pharmacological role for health or disease (“Probiotics and pharmabiotics,” Bioeng Bugs. 2010 Mar-Apr; 1 (2):79-84) The pharmaceutical composition of the present invention contains pharmabiotics as a main component and can be safely used without side effects.

The strain of the genus Picalibacterium may be a strain of Faecalibacterium prausnitzii . Specifically, the strains of the Ficcolibacterium genus are F. prausnitzii EB-FPDK3 strain (KCCM12619P), F. prausnitzii EB-FPDK9 strain (KCCM12620P), F. prausnitzii EB-FPDK11 strain (KCCM12621P), or F. prausnitzii. EB-FPYYK1 strain (KCCM12622P).

Extracellular vesicles (EVs) are nano-vesicles secreted from cells that contain powerful anti-inflammatory proteins including the immunologically important proteins main histocompatibility complex (MHC) and heat shock proteins. It induces a tumor immune response and contains anti-inflammatory microRNAs and microRNAs that regulate the accumulation of collagen.

Extracellular vesicles (EVs) derived from the Faecalibacterium sp. strain of the present invention can be used as an excellent anticancer agent by simultaneously inhibiting cancer cell proliferation, reducing cancer cell mobility, and inhibiting angiogenesis. It may be administered in combination with chemotherapy or immunotherapy.

Another aspect of the present invention, the genus Picalibacterium ( Faecalibacterium sp.) strain; And it relates to a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient. The strain may be live or dead.

The strain of the genus Picalibacterium or the extracellular vesicles (EV) derived from the strain of the genus Picalibacterium of the present invention are administered simultaneously with the chemotherapeutic agent or immuno-anticancer agent as one agent, or simultaneously or sequentially as separate agents. can be administered.

In the present invention, the method for separating extracellular vesicles is not limited, and, for example, in culture medium, centrifugation, ultra-high-speed centrifugation, filter filtration, gel filtration chromatography, free-flow electrophoresis, capillary electrophoresis, polymer It can be separated using a method such as separation using a method and a combination thereof, preferably by centrifugation/ultracentrifugation. In this case, centrifugation/ultracentrifugation may be performed sequentially at 100 to 300,000 g, preferably 150 to 150,000 g to remove cell debris, non-extracellular vesicle fractions, dead cells, and the like.

Fractional Centrifugation : The most preferred method for extracellular vesicles is fractional centrifugation. This method consists of several steps, preferably carried out at about 4 ° C, and includes at least the following three steps 1) to 3):

Step 1) low-speed centrifugation to remove cells and cell debris,

Step 2) high-speed spin to remove large vesicles >100 nm, and

Step 3) High-speed centrifugation to pellet extracellular vesicles.

Density gradient centrifugation : This approach combines ultracentrifugation with a sucrose density gradient. More specifically, density gradient centrifugation is used to separate extracellular vesicles from non-vesicular particles, such as proteins and protein/RNA aggregates. Thus, this method separates vesicles from particles of different densities.

Size Exclusion Chromatography : Size exclusion chromatography is used to separate macromolecules based on size, not molecular weight. This technology applies a column packed with porous polymer beads containing a plurality of pores and tunnels. Molecules pass through beads according to their diameter. These macromolecules, which take longer to migrate through the pores of the column, elute faster from the column. Size exclusion chromatography allows precise separation of large and small molecules.

Filtration : Ultrafiltration membranes can also be used for isolation of extracellular vesicles. Depending on the size of the microvesicles, this method allows the separation of extracellular vesicles from proteins and other macromolecules.

Polymer-based sedimentation : Polymer-based sedimentation methods generally include mixing biological fluids with polymer-containing precipitation liquids, incubation at 4°C, and centrifugation at low speeds. One of the most common polymers used for polymer-based precipitation is polyethylene glycol (PEG). Precipitation with this polymer has many advantages, including mild effects on isolated extracellular vesicles and use of neutral pH.

Isolation by sieving : This technique isolates extracellular vesicles by sieving them in a biological fluid through a membrane and filtering them by pressure or electrophoresis.

The pharmaceutical composition of the present invention has an excellent effect in preventing or treating cancer.

Specifically, the strains belonging to the genus Picalibacterium or the extracellular vesicles derived from the strains belonging to the genus Picalibacterium are absorbed into cancer cells, suppress the EMT action and activate the immune system, thereby inhibiting cancer cell invasion and metastasis.

Preferably, the strains of the genus Ficcolibacterium are Ficcolibacterium prosnich EB-FPDK3 strain, Ficcallybacterium prosnich EB-FPDK9 strain, Ficcallybacterium prosnich EB-FPDK11 strain, or Picalibacterium prosnich. It may be the EB-FPYYK1 strain. Preferably, the extracellular vesicles derived from strains of the genus Ficcolibacterium are Ficcolibacterium prosnich EB-FPDK3 strain, Ficcalibacterium prosnich EB-FPDK9 strain, Ficcalibacterium prosnich EB-FPDK11 strain, p It may be an extracellular vesicle derived from the Caulibacterium prosnich EB-FPYYK1 strain. Hereinafter, these extracellular vesicles are abbreviated as EB-FPDK3 EV, EB-FPDK9 EV, EB-FPDK11 EV, and EB-FPYYK1 EV, respectively. These EB-FPDK3 EV, EB-FPDK9 EV, EB-FPDK11 EV, and EB-FPYYK1 EV play an important role in regulating T cells to activate innate and adaptive immune systems. In addition, regulatory T cells (Treg), characterized by the expression of Foxp3, are known to suppress anticancer immunity, hinder protective immune surveillance of tumors, and oppose effective antitumor immune responses. EB-FPDK11 EV and EB-FPYYK1 EV are presumed to exhibit anticancer effects by activating T helper cells, activating cytotoxic T cells, and suppressing Treg cells. Accordingly, the pharmaceutical composition of the present invention has excellent effects in preventing, treating, and inhibiting metastasis of cancer.

When used in the present invention, the term 'cancer' is meant to include tumors, neoplasias, and malignant tissues or cells. The cancer is colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, proximal anal cancer, colon cancer, breast cancer, Fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute including cancers such as leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, or a combination of one or more of these cancers do.

In the present invention, the extracellular vesicles (EV) derived from the strain of the genus Picalibacterium according to the present invention included as an active ingredient in the pharmaceutical composition have a size of 20 to 300 nm.

The effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, and body weight, and is generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, administered daily or every other day, or daily. It can be administered in 1 to 3 divided doses.

The pharmaceutical composition according to the present invention can be used as a single anti-cancer therapy. In addition, the pharmaceutical composition according to the present invention can be used simultaneously, separately or sequentially with radiation or chemotherapy or immunotherapy, if necessary according to circumstances. The combination therapy of the present invention is for use in at least one of reducing tumor size, preventing tumor growth, preventing metastasis, or preventing angiogenesis.

Specifically, the pharmaceutical composition of the present invention may be administered sequentially or simultaneously with conventional radiation therapy or anticancer therapy. In addition, it can be single or multiple administration, and it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

In some embodiments, an immunotherapeutic agent is an immune checkpoint inhibitor. Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate the immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. An immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof that binds to and inhibits an immune checkpoint protein. Examples of immune checkpoint inhibitors are nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR -012 and STI-A1010, but is not necessarily limited thereto.

Unlike conventional chemotherapy that directly kills cells, immune checkpoint inhibitors are next-generation anticancer drugs with fewer side effects caused by chemotherapy, such as hair loss, anemia, and suppression of bone marrow function, which reduce the quality of life of cancer patients. is being popularized as However, immune checkpoint inhibitors have very low response rates for some cancers (e.g., gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, etc.) and cause severe immune-related adverse reactions such as enteritis, hepatitis, pneumonia, hypothyroidism, and pituitary glanditis. It is also known to do Most of the side effects of using immune checkpoint inhibitors appear as minor side effects, but in rare cases in the nervous system or heart system, they are reported to be serious and fatal. The pharmaceutical composition comprising the extracellular vesicles derived from the strain of the genus Picalibacterium of the present invention can overcome the reaction rate limitations of immune checkpoint inhibitors, minimize side effects, and enhance anticancer efficacy.

The Ficalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains used in the present invention were isolated from the feces of healthy Koreans, and had a concentration of 0.5-1 μm. It is a monococcal or diplococcal, anaerobic, non-motile, Gram-negative, non-endospore forming, mucin-degrading bacterium. F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains produce several mucolytic enzymes that can use mucus as a carbon and nitrogen source. It can metabolize various carbon sources, including glucose, galactose, N-acetylglucosamine and lactose, and produces short-chain fatty acids such as propionic acid and acetic acid as major metabolites.

The pharmaceutical composition for preventing or treating cancer of the present invention includes strains of the genus Picalibacterium, including strain cells, cell lysates, cultures of the strains, culture solutions obtained by removing cells from the cultures of the strains, cell extracts of the strains, and strains of the strains. It may be selected from extracts of cultures, or extracts of cultures obtained by removing cells from cultures of strains.

In the present invention, strains of the genus Picalibacterium may be live or dead bacteria. The F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention are cultured, recovered by a separation process such as centrifugation, and dried , For example, it may be prepared and used in the form of a probiotic by lyophilization. The dead cells are also cells killed by heat treatment or pasteurized inactivated strains. Pasteurization of the Picylobacterium prosnich strain means heating at a temperature of 50 ° C or more and less than 100 ° C for 10 minutes or more. For example, it can be pasteurized at 70° C. for 30 minutes.

In the present invention, the term 'dead cells' refers to cells sterilized by heating, pressurization, drug treatment, etc. The method for producing killed cells is not particularly limited as long as it is performed by a method for killing lactic acid bacteria known in the art. As an example, the dead cells of the present invention may be prepared by a killing method including heat treatment (by heat treatment). The heat treatment may be performed only on live cells separated from the culture medium, or may be performed on the culture medium containing the live cells. The heat treatment temperature is not particularly limited as long as the properties of the cells are maintained and other general bacteria are sterilized, but the temperature condition may be 80 ° C to 150 ° C, preferably 80 ° C to 110 ° C. can

In one embodiment of the present invention, strains of the genus Ficcolibacterium or F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains The pharmaceutical composition containing the extracellular vesicles of is, according to a conventional method, in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions, respectively. It may be formulated and used, but is not necessarily limited thereto.

In some embodiments, the pharmaceutical composition of the present invention is an aqueous liquid dispersion, self-emulsifying dispersion, solid solution, liposomal dispersion, aerosol, solid body form, powder, immediate release formulation, controlled release formulation, fast melt formulation, tablet, capsule, pill , delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

The pharmaceutical composition of the present invention may be used as an aqueous suspension, isotonic saline solution, or sterile, injectable solution containing a pharmacologically compatible dispersing agent and/or wetting agent for intravenous, intratumoral or intranasal administration. Water, alcohol, polyol, glycerol, vegetable oil and the like can be used as excipients.

The pharmaceutical composition of the present invention may be formulated as a product for enteral or oral administration. In addition, the pharmaceutical composition of the present invention can be enterically coated and commercialized so that the active ingredient, extracellular vesicles (EV), can be rapidly released into the intestine by passing through the stomach and reaching the small intestine using a known method.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers and/or excipients in addition to the above active ingredients, and in addition, various additives commonly used pharmaceutically, such as binders, disintegrants, coating agents, lubricants, and the like. It can be formulated with

A pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, various drug delivery materials used for oral administration may be included. In addition, carriers for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like. The pharmaceutical composition of the present invention may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Pharmaceutically acceptable carriers and agents that may be included in the pharmaceutical composition of the present invention may be referenced as described in the following literature (Remington's Pharmaceutical Sciences, ^19th ed., Mack Publishing Company, Easton, PA, 1995).

Excipients usable in the present invention include sugars such as sucrose, lactose, mannitol, glucose and the like, and starches such as corn starch, potato starch, rice starch, and partially pregelantinated starch. Binders include polysaccharides such as dextrin, sodium alginate, carrageenan, guar gum, acacia, agar, tragacanth, gelatin, naturally-occurring macromolecular substances such as gluten, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl Cellulose derivatives such as cellulose, ethyl cellulose, hydroxypropylethyl cellulose, and carboxymethyl cellulose sodium, and polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resins. contains polymers;

Decomposers usable in the present invention include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose, and sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially starch. Starches such as gelatinized starch may be used.

Examples of the lubricant usable in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrosilicone dioxide, various types of waxes and hydrogenated oils.

As the coating agent, dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-chlorotrimethylammonium ethyl methacrylate Copolymers, water insoluble polymers such as ethyl cellulose, methacrylic acid-ethyl acrylate copolymers, enteric polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, and methylcellulose, hydroxypropylmethylcellulose , polyvinylpyrrolidone, and water-soluble polymers such as polyethylene glycol, but are not necessarily limited thereto.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the dosage of the active ingredient, the extracellular vesicles derived from the strain of the genus Picalibacterium, can vary according to the type of various diseases, the age, weight, sex of the patient, the medical condition of the patient, and the condition. It can be determined according to factors including severity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. Therefore, the dosage regimen can vary widely, but it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, taking into account all the above factors, which can be easily determined by those skilled in the art using standard methods. .

Another aspect of the present invention relates to a health functional food comprising a strain of the genus Picalibacterium or an extracellular vesicle (EV) derived from a strain of the genus Picalibacterium . A strain of the genus Picalibacterium may be live or dead. The extracellular endoplasmic reticulum is preferably an extracellular vesicle derived from the strains F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains. The health functional food of the present invention can be used to prevent or improve cancer.

The health functional food of the present invention includes all forms such as nutraceutical food, nutritional supplement, health food, food additives and feed.

The health functional food of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods, fish, meat and their processed foods, bread and noodles, fruit juice, various drinks, cookies, taffy, dairy products, edible vegetable oils, margarine , vegetable protein, retort food, frozen food, various sauces, etc. derived from F. prausnitzii EB-FPDK3 strain, F. prausnitzii EB-FPDK9 strain, F. prausnitzii EB-FPDK11 strain and F. prausnitzii EB-FPYYK1 strain of the genus Ficcolibacterium It can be prepared by adding the extracellular vesicles of

In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH regulators, stabilizers, and preservatives. , glycerin, alcohol or carbonation agent, and the like.

Another aspect of the present invention is a strain of the genus Picalibacterium Or extracellular vesicles (EV) derived from strains of the genus Picalibacterium ; And it relates to a veterinary composition for preventing or treating cancer comprising a physiologically acceptable carrier or excipient. Here, the animal is not particularly limited, and may refer to pets such as dogs, cats, guinea pigs, hamsters, rats, mice, ferrets, rabbits, and the like. The veterinary composition may be a feed additive.

Another aspect of the invention relates to a method of treating cancer in a subject. In the method of the present invention, a subject is treated with a therapeutically effective amount of a strain of the genus Picalibacterium described herein or and administering extracellular vesicles derived from a strain of the genus Picalibacterium .

Another aspect of the present invention provides novel EB-FPDK3 strains (KCCM12619P), EB-FPDK9 strains (KCCM12620P), EB-FPDK11 strains (KCCM12621P), and EB-FPYYK1 strains (KCCM12622P) of the genus Picalibacterium . The above strains were each deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on November 1, 2019.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example

Example 1: Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9; F. prausnitzii EB-FPDK11, and F. prausnitzii Isolation and identification of EB-FPYYK1 strains

1.1. Isolation and identification of strains

In order to isolate the strains of the genus Picalibacterium from feces of a healthy Korean (female, 7 years old, BMI 19.9), an anaerobic chamber was used according to Martin's method under strict anoxic conditions (5% H ₂ , 5% CO ₂ and 90 % N ₂ ), YBHI medium supplemented with 0.5% yeast extract, 0.1% D-cellobiose, and 0.1% D-maltose (brain heart infusion medium supplemented with 0.5% yeast extract) (Difco, Detroit, USA). After that, EOS (Extremely Oxygen Sensitivity) strains were selected and isolated (Martin et al., 2017). The standard strain , Picalibacterium prosnich A2-165, was distributed from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and used in the test.

1.2. microscope observation

In order to confirm whether the isolated strain is a strain of the genus Picalibacterium , the isolated strain was observed under a microscope, and the results are shown in FIG. 1. As shown in FIG. 1, the standard strain, the Picylobacterium prosnich A2-165 standard strain, and the Ficcybacterium prosnich EB-FPDK3, EB-FPDK9, EB-FPDK11, and EB-FPYYK1 isolated strains of the present invention As a result of observation at a magnification of 1,000 times, the shapes of the strains were all observed as straight or curved rod cells.

1.3. PCR analysis

In order to confirm whether it is a strain of the genus Picylobacterium , the isolated strain was subjected to PCR analysis using FP-specific primers (SEQ ID NO: 1 and SEQ ID NO: 2) in Table 1, and the results are shown in FIG. 2 did In Fig. 2, lane M is a DNA size marker, lane 1 is a positive control (A2-165), lanes 2 to 5 are isolates, and lane 6 is the result of a negative control (distilled water).

As shown in FIG. 2, the isolated strains of the present invention, that is, Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains, are It was confirmed that the result value came out with the same band as the Prosnich A2-165 standard strain.

Designation Direction Sequence (5’→3’) Amplicon size sequence number FP1 Forward ACT CAA CAA GGA AGT GA 192 bp SEQ ID NO: 1 FP2 Reverse AAT TCC GCC TAC CTC TG SEQ ID NO: 2

1.4. Random Amplified Polymorphic DNA (RAPD) analysis

Ficaulibacterium prosnich isolated as described above, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains and previously reported strains of Picalibacterium prosnich A2-165 In order to verify the similarity of the standard strain, RAPD, a type of molecular typing, was performed. To this end, the genomic DNA extracted from the cells was amplified using the universal primers in Table 2 below, electrophoresed on a 1% agarose gel for 1 hour and 30 minutes, and the DNA fragmentation pattern was compared on a UV perforator. Results are shown in FIG. 3 .

Designation direction Sequence (5' → 3') sequence number ERIC-1 Forward ATG TAA GCT CCT GGG GAT TCA C SEQ ID NO: 3 ERIC-2 Reverse AAG TAA GTG ACT GGG GTG AGC G SEQ ID NO: 4 (GTG) ₅ Forward/Reverse GTG GTG GTG GTG GTG SEQ ID NO: 5

As confirmed through FIG. 3, the F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention are Compared to Nichi A2-165 standard strain, they showed different RAPD band patterns. It is known that the RAPD band patterns of Picalibacterium prosnich are different when the species are different, and therefore, the RAPD band patterns of Picalibacterium prosnitch EB-FPDK3, F. prausnitzii EB-FPDK9, F of the present invention prausnitzii EB-FPDK11 and F. prausnitzii EB-FPYYK1 strains belong to the same species as the standard strain of Ficcolibacterium prosnichi A2-165, but it was confirmed that they are different strains.

1.5. Phylogenetic tree analysis using full-length 16S rRNA gene sequences

Full-length 16S rRNA gene sequencing analysis of Ficcolibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains isolated as described above For this purpose, the 16S rRNA gene was amplified using the 27F and 1492R primers in Table 3 below, and then the nucleotide sequence was determined using a 3730xl DNA analyzer.

The thus obtained Picylobacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, EB-FPD 11 and the previously published Ficca bacterium prosnich strains (A2-165, ATCC2766, ATCC27768) additionally belong to the Ruminococcaceae family ( family), a complete rRNA sequence database was created by collecting the 16S rRNA gene sequences of a total of eight strains of the Ruminococcus albus DSM20455 strain. In addition, homology and query coverage were calculated using the Nucleotide-Nucleotide BLAST 2.9.0+ program and are shown in Table 4. In addition, the phylogeny was analyzed based on the 16S rRNA gene sequences of a total of 8 strains. Phylogenetic analysis was performed using MEGA-X, and a phylogenetic tree was constructed through a neighbor-joining method using 1000 bootstraps, and is shown in FIG. 4(A). Average nucleotide identity (ANI) values were evaluated for evolutionary distances by applying the pyani v0.2.7 program with -m ANIb settings. Ficcallybacterium prosnitch A2-165 strain (RefSeq assembly accession: GCF_000162015.1), Ficcallybacterium prosnich ATCC27766 strain (RefSeq assembly accession: GCF_003324115.1), Ficcallybacterium prosnich ATCC27768 strain (RefSeq assembly accession . downloaded and used. A phylogenetic tree was constructed using the 16S rRNA gene sequences of other strains of the same species and is shown in FIG. 4(B).

Designation Direction Sequence (5’ → 3’) sequence number Amplicon size 27F Forward AGA GTT TGA TCM TGG CTC AG SEQ ID NO: 6 1,472 bp 1492R Reverse GGT TAC CTT GTT ACG ACT T SEQ ID NO: 7

strain F. prausnitzii
A2-165 F. prausnitzii
ATCC27768 Ruminococcus albus
DSM20455 sameness
(identity%) Queries
coverage
(Query coverage %) sameness
(identity%) Queries
coverage
(Query coverage %) sameness
(identity%) Queries
coverage
(Query coverage %) EB-FPYYK1 99.735 100 97.95 100 86.868 100 EB-FPDK3 98.676 100 98.082 100 86.623 100 EB-FPDK9 97.95 100 99.603 100 86.588 100 EB-FPDK11 98.613 100 97.82 100 86.82 100

As shown in Figures 4 (A) and 4 (B) and Table 4, in the case of the Picylobacterium prosnich EB-FPYYK1 strain, the nucleotide sequence of the 16S rRNA gene was previously published. -99.735% identity and 100% query coverage with strain ATCC27768, 97.95% identity with strain ATCC27768, and 100% query coverage with 86.868% identity and 100% query coverage with strain Ruminococcus albus DSM20455 belonging to the Ruminococcaceae family confirmed to have coverage. In the case of the Picalibacterium prosnich EB-FPDK3 strain, it was confirmed that it had 98.676% identity with the A2-165 strain, 98.082% identity with the F. prausnitzii ATCC27768 strain, and 86.623% identity with the R. albus DSM20455 strain. In the case of the EB-FPDK9 strain, it was confirmed to have 97.95% identity with the A2-165 strain, 99.603% identity with the ATCC27768 strain, and 86.623% identity with the R. albus DSM20455 strain. In the case of the EB-FPDK11 strain, it was confirmed that it had 98.613% identity with the F. prausnitzii A2-165 standard strain, 97.82% identity with the F. prausnitzii ATCC27768 strain, and 86.82% identity with the DSM20455 strain. That is, as a result of analyzing the phylogenetic tree of evolutionary relatedness through 16S rRNA gene sequencing , F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. It was confirmed that the prausnitzii EB-FPYYK1 strains are genetically belonging to the Picylobacterium prosnichi .

Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention isolated from human feces were isolated from Picalibacterium prosnich (A2 -165) was identified through biochemical methods (API) and molecular biological methods (16S rRNA sequencing, 16S rRNA BLAST analysis, RAPD) as a control, and through the antibiotic resistance test described later, safe probiotics that can have the function of probiotics It was confirmed that it was a strain. Based on these results, the isolated Ficcolibacterium prosnich strains were named Ficcolibacterium prosnich EB-FPDK3 strains, F. prausnitzii EB-FPDK9 strains, F. prausnitzii EB-FPDK11 strains, and F. prausnitzii EB-FPYYK1 strains. and deposited with the Korea Research Institute of Bioscience and Biotechnology, and were assigned accession numbers KCCM12619P, KCCM12620P, KCCM12621P, and KCCM12622P, respectively.

Example 2: Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9; F. prausnitzii EB-FPDK11, and F. prausnitzii Mycological characteristics and safety analysis of EB-FPYYK1 strains

2.1. Confirmation of antimicrobial susceptibility of the isolated strain

In order to determine the antimicrobial susceptibility of the Ficalibacterium prosnich strains isolated as described above, an antimicrobial agent for anaerobic bacteria (piperacillin-tazobactam (PTZ), ceftidoxime (CTZ), chloramphenicol (CHL), clindamycin (CLI), meropenem (MEM), moxifloxacin (MXF), metronidazole (MTZ), ciprofloxacin (CIP) MIC) was determined (CLSI, 2017), and the results are shown in Table 5 below.

Antibiotic MICa Breakpoints
(μg/mL) QC test strain S I R ATCC ^29741a A2-165 EB-FPDK3 EB-FPDK9 EB-FPDK11 EB-FPYYK1 PTZ ≤32/4 64/4 ≥128/4 8/4 >256/4 (R) 32/4 (S) 32/4 (S) >256/4 (R) >256/4 (R) CTZ ≤32 64 ≥128 16 64 (I) 16(S) 128 (R) 128 (R) 128 (R) CHL ≤8 16 ≥32 8 64 (R) 8(S) 32 (R) 8(S) 256 (R) CLI ≤2 4 ≥8 4 ≤0.125 (S) ≤0.125 (S) ≤0.125 (S) ≤0.125 (S) ≤0.125 (S) MEM ≤4 8 ≥16 0.5 >64 (R) >64 (R) >64 (R) >64 (R) >64 (R) MXF ≤2 4 ≥8 8 16 (R) >32 (R) 32 (R) 32 (R) >32 (R) MTZ ≤8 16 ≥32 2 4(S) 1(S) <0.25 (S) 0.5 (S) 2(S) CIP ≤1 2 ≥4 >32 32 (R) 32 (R) >32 (R) 16 (R) 32 (R) PTZ: Piperacillin-tazobactam, CTZ: ceftizoxime ( ^3rd gen), CHL: chloramphenicol,
CLI: clindamycin, MEM: meropenem, MXF: moxifloxacin ( ^4th gen),
MTZ: metronidazole, CIP: ciprofloxacin ( ^2nd gen),
^a MIC : minimal inhibitory concentration, ^b Bacteroides thetiotaomicron ATCC 29741

As can be seen in Table 5, the Picylobacterium prosnichi strains of the present invention showed different resistance patterns, all strains were sensitive to metronidazole, and all strains were sensitive to fluoroquinolone antibiotics, moxifloxacin and ciprofloxacin. The strains were showing resistance. Resistance to fluoroquinolone-based antibiotics is the same resistance that exists in the same Picylobacterium prosnich and is judged as intrinsic resistance. In addition, antibiotic resistance gene database MEGARes ( https://megares. meglab.org/ ), genes encoding antibiotic resistance genes were investigated on the whole genome of the strains of Picalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9 and EB-FPDK11 of the present invention, The results are shown in Table 6 below.

Genome MEGAResID Ident Align Coverage Class Mechanism Group A2-165 MEG_988 99.755 816 100 Aminoglycosides Aminoglycoside O-nucleotidyltransferases ANT6 EB-FPYYK1 MEG_988 99.885 867 100 Aminoglycosides Aminoglycoside O-nucleotidyltransferases ANT6 EB-FPDK3 MEG_2793 100 738 100 MLS 23S rRNA methyltransferases ERMB EB-FPDK9 MEG_2793 100 738 100 MLS 23S rRNA methyltransferases ERMB EB-FPDK11 MEG_7216 99.844 1920 100 Tetracyclines Tetracycline resistance ribosomal protection proteins TETW

As can be seen in Table 6, Aminoglycoside O-nucleotidyltransferases genes were detected in the case of the Picylobacterium prosnich A2-165 standard strain. In addition, in the case of the Picylobacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11 strains of the present invention, Aminoglycoside O-nucleotidyltransferases, 23S rRNA methyltransferases, 23S rRNA methyltransferases, Tetracycline resistance ribosomal protection proteins genes, respectively has been detected It was confirmed that the antibiotic resistance genes detected through the MEGARes database also appear through Resfinder (https://cge.cbs.dtu.dk/services/ResFinder/), a bioinformatics-based antibiotic resistance gene detection program. Through these results, it was confirmed that the antibiotic resistance gene did not appear identically in the Picylobacterium prosnich strains, but appeared differently for each strain.

PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) and Mobile Element Finder (cge.cbs.dtu.dk/services/MobileElementFinder ) program was applied to conduct additional investigations. PlasmidFinder program was applied to examine the presence of plasmids on the entire genomes of the strains of Picalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11 of the present invention, and it was confirmed that they were not detected. In addition, as a result of confirming mobile genetic elements on the whole genome of the Picalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11 strains of the present invention through the Mobile Element Finder program, transposons were not detected. confirmed that it is not. Through the results, it was confirmed that the antibiotic resistance gene detected was not transmitted to other strains. Therefore, it can be confirmed that the F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the genus Picalibacterium according to the present invention are safe strains.

2.3. Analysis of hemolytic activity and virulence factors of isolated strains

To verify the safety of the Ficalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains isolated as described above, whether or not they had hemolytic activity was evaluated. For this, tryptic soy agar (17.0 g/L pancreatic digest of casein, 3.0 g/L pancreatic digest of soybean, 2.5 g/L dextrose, 5.0 g/L sodium chloride, 2.5 g/L potassium phosphate, 15 g/L The strain was cultured using a blood agar medium prepared by adding 5% w/v defibrinated sheep blood to L agar), and the results are shown in FIG. 5 . As can be seen from Figure 5, the Picalibacterium prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains of the present invention have β related to pathogenicity. -hemolysis (completely transparent area around the colony) was not seen.

Based on the reference database for bacterial virulence factors (VFDB, http://www.mgc.ac.cn/VFs), which is a pathogenic bacterial database, the gene encoding the virulence factor is the Picalibacterium prosnich EB-FPYYK1 of the present invention , EB-FPDK3, EB-FPDK9 and EB-FPDK11 strains were investigated on the whole genome. The Virulence Factor Database (VFDB) is a comprehensive online resource for screening information on the virulence factors of bacterial pathogens, providing an in-depth look at the major virulence factors of the most well-characterized bacterial pathogens. The VFDB database used for analysis included 3,688 and 32,772 sequence data involved in toxicity. In the case of the analysis, the protein identity was at least 80%, the coverage was at least 80%, and the alignment length was at least 50 bp. As a result of the analysis, virulence factors were not detected in the strains of the present invention , Ficalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11. Additionally, virulence factor genes based on VirulenceFinder (https://cge.cbs.dtu.dk/services/VirulenceFinder/) were investigated. VirulenceFinder is a database of genome sequences of four well-known pathogens ( E. coli , Enterococcus , Listeria and Staphylococcus aureus ). As a result of the analysis, E. coli shiga toxin gene, S. aureus exoenzyme genes, and host immune alteration or evasion on the whole genome of the strains of Picalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11 of the present invention Genes related to genes and toxin genes were not detected. Overall, it can be seen that the strains of Picalibacterium prosnich EB-FPYYK1, EB-FPDK3, EB-FPDK9, and EB-FPDK11 of the present invention are harmless in the human body.

2.4. Confirmation of short-chain fatty acids (SCFAs) production ability of Picalibacterium prosnich strains

Short chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, are metabolites produced by intestinal bacteria and play an important role in host energy metabolism. ) and is involved in energy balance as a signaling mediator acting on

Short-chain fatty acids (SCFAs) decrease intestinal motility and increase intestinal transit speed through GPR41 in enteroendocrine cells. Through this, it induces PYY (peptide YY) secretion to reduce energy intake and prevent obesity. In addition, GPR43 by short-chain fatty acids induces GPL-1 (Glucagon-like peptide 1) to increase satiety through increased insulin sensitivity, and the activity of GPR43 inhibits insulin signaling in adipose tissue to prevent fat accumulation. Short-chain fatty acids (SCFAs) can enhance glucose metabolism and activate intestinal gluconeogenesis (IGN), which can reduce food intake through the gut-brain neural circuit.

In order to confirm changes in the functional metabolome of the strains of Picalibacterium prosnich, after culturing in vitro, changes in the content of major short-chain fatty acids (butyric acid and acetic acid) contained in the culture medium were analyzed by Gas Chromatography (GC). To this end, the culture solution was centrifuged at 12,000 xg for 5 minutes to recover the supernatant, and the supernatant was filtered using a 0.2 μm syringe filter and used for analysis. Gas chromatography (Agilent 7890N) equipped with a FFAP column (30 m × 0.320 mm, 0.25 μm phase) was used, the conditions were set as in Table 7, and the analysis results are shown in Table 8 below.

Flow _H2 : 40 ml/min, Air: 350 ml/min Injector temp. 240℃ Detector temp. 250℃ Oven temp. 40℃ (hold 2 min)→ 65℃/10 min (hold 2 min)→ 240℃/10 min (hold 5 min) Injection vol. 2 μL Split ratio 20:1

strain Amount of short-chain fatty acids produced/consumed (μg/ml) Butyrate production P value ^* Acetate consumption P value ^* A2-165 508.75 ± 64.90 - 198.25 ± 13.47 - EB-FPDK3 326.44 ± 13.84 <0.0001 246.10 ± 17.80 <0.0001 EB-FPDK9 358.83 ± 24.28 <0.0001 160.89 ± 14.84 <0.0001 EB-FPDK11 354.22 ± 8.83 <0.0001 154.42 ± 21.75 <0.0001 EB-FPYYK1 587.74 ± 17.46 0.0003 197.67 ± 7.25 >0.9999 * P < 0.05 significantly different compared to A2-165 (type strain).

As can be seen from the results of Table 8, the Picylobacterium prosnich EB-FPDK strains of the present invention showed different short-chain fatty acid production/consumption abilities. In detail, it was confirmed that the standard strain of Picalibacterium prosnich A2-165 and the strains EB-FPYYK1, EB-FPDK9 and EB-FPDK11 had similar consumption of acetic acid and production of butyrate, respectively. -FPDK3 strain produced the lowest butyric acid among the five strains, but consumed the highest acetic acid. Picalibacterium prosnich is known as a representative butyric acid-producing bacterium, and since butyric acid is synthesized from acetic acid in their metabolic pathway, acetic acid is consumed.

Example 3: Separation of extracellular vesicles from Picalibacterium prosnich strains

To obtain extracellular vesicles (EVs) of the strains of F. prausnitzii EB - FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains, Cultures of Prosnich EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains were subjected to high-speed centrifugation at 10,000 xg and 4°C for 20 minutes, and the supernatant was After recovery, the supernatant was filtered with a 0.45 μm filter and a 0.22 μm filter. The filtered supernatant was subjected to high-speed centrifugation for 2 hours at 150,000xg and 4° C. to obtain a pellet, which was then dissolved in sterile physiological saline (PBS) and used for protein quantification and efficacy testing.

The separated extracellular vesicles were observed under an electron microscope (Zeiss, Germany) (150,000X), and the results are shown in FIG. 6 . As shown in Figure 6 , the extracellular vesicles (Extracellular vesicles , EVs) are spherical, and it can be confirmed that the approximate size is in the range of 20 to 300 nm (scale bar 500 nM).

Example 4: Anticancer effect of combined administration of aPD-1 immunotherapeutic agent and Ficcallybacterium prosnich EB-FPDK3, EB-FPDK9, EB-FPDK11, EB-FPYYK1 live bacteria in an allograft mouse animal model using melanoma

4.1. strain sample

The Picylobacterium prosniche A2-165 standard strain (control group) and the Piculybacterium prosnich EB-FPDK3 strain (KCCM12619P), EB-FPDK9 strain (KCCM12620P), EB-FPDK11 strain (KCCM12621P), and EB-FPYYK1 strain (KCCM12622P) viable cells were prepared at a concentration of 1x10 ⁸ CFU/150 μl PBS (25% glycerol, 0.05% cysteine/PBS).

4.2. animal testing

Animal experiments were conducted in compliance with the Animal use and Care Protocol of the Institutional Animal Care and Use Committee (IACUC). For cancer induction, 8-week-old female C57BL/6 mice were purchased, and after a week-long adaptation period, they were bred for 5 weeks. The breeding environment maintained a constant temperature (22 ℃) and relative humidity (40 ~ 60%), and was bred while adjusting the light and shade in a 12-hour cycle.

To prepare a syngeneic mouse anti-cancer animal model, mouse-derived melanoma cells (B16-F10) were used. The syngeneic model is a technique in which a cell line of a mouse grown in vitro is transplanted into a real mouse and grown, and tumor rejection does not occur in the same host and cell line strain.

After one week of acclimatization, mice were pretreated with antibiotics for one week using the antibiotics shown in Table 9 below.

Antibiotic density Ampicillin (Sigma-A0166) 1g/L Vancomycin (Sigma SBR00001) 0.5g/L Metronidazole (Sigma M1547) 1g/L Neomycin (Sigma N6386) 1g/L Amphotericini B (Sigma PHR1662) 0.1g/L

Subsequently, B16-F10 cells, 2×10 ⁴ cells, along with 100 μl Matrigel were injected subcutaneously into the thighs of mice (SC, subcutaneous injection). The animal experiment scheme in this embodiment is shown in FIG. 7a.

4.3. Sample administration and experimental group setup

Each drug shown in Table 10 was orally administered to mice every day for 2 weeks. As a positive control group, anti-PD1 antibody was orally administered at 250 μg/100 μl600/head.

From the 6th day, cancer cells rose, and from this point on, F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPYYK1 strains were injected at 10 ⁸ CFU was administered orally daily. From the 6th day, 250 μg of aPD-1 antibody was intraperitoneally injected every 4 days. At this time, as the aPD-1 antibody, BioXCell's InVivoMab anti-mouse PD-1 (RMP1-14), catalog #BE0146 was used, and InVivoPure pH 7.0 Dilution Buffer, catalog #IP0070 was used at a concentration of 250 μg/100 μl. diluted and used.

During tumor growth, mice were weighed twice a week and monitored daily, and from the 6th day when cancer cells appeared, tumor size was monitored every other day using a computerized caliper (see FIG. 8).

Tumor volume was calculated according to the following equation by measuring the two diameters of the major and minor diameters of each tumor.

[Formula 1]

Tumor volume (㎣) = [long diameter × short diameter ² ]/2

group experimental group administered drug I normal control PBS administration II Positive control group (Anti-PD1 administration group) 250 μg/100 μl/head III Anti-PD1+ F. prausnitzii EB-FPDK3 strain administration group EB-FPDK3 Live Cell, 1x10 ⁸ CFU +
anti-PD1 (250 μg/100 μl/head) IV Anti-PD1+ F. prausnitzii EB-FPDK9 strain administration group EB-FPDK9 Live Cell, 1x10 ⁸ CFU +
anti-PD1 (250 μg/100 μl/head) V Anti-PD1+ F. prausnitzii EB-FPDK11 strain administration group EB-FPDK11 Live Cell, 1x10 ⁸ CFU +
anti-PD1 (250 μg/100 μl/head) VI Anti-PD1+ F. prausnitzii EB-FPYYK1 strain administration group EB-FPYYK1 Live Cell, 1x10 ⁸ CFU +
anti-PD1 (250 μg/100 μl/head)

Referring to FIGS. 8 to 10, compared to the control group in which B16-F10 cells were allotransplanted, in the group orally administered with F. prosniche EB-FPDK3, EB-FPDK9, EB-FPDK11, EB-FPYYK1 live cells It was confirmed that the tumor size was remarkably reduced. Compared to the normal control group's tumor size of 726.8 ± 66.7 mm 3 , the tumor size of the aPD-1 administered group was 419.5 ± 80.43 mm 3 ^, (p<0.05), a 42% decrease. 400.6±67.45 ㎣, (P<0.01), 455.7±67.67 ㎣, (P<0.05), 190.7±42.5 ㎣, respectively in the aPD-1 and EB-FPDK3, EB-FPDK9, EB-FPDK11, EB-FPYYK1 co-administration group (P<0.001), 355.6±50.47 ㎣, (P<0.01) showed a significant decrease in size. In particular, the aPD-1 and EB-FPDK11 combined administration group showed the best anticancer effect with a reduction of about 74% compared to the normal control group.

Example 5: Combined administration of aPD-1 immunotherapeutic agent and Picalibacterium prosnich EB-FPDK3, EB-FPDK9, EB-FPDK11, EB-FPYYK1-derived extracellular vesicles (EV) in an allograft mouse animal model using melanoma Anticancer effect by

Referring to FIG. 7B, from the 6th day when cancer cells were raised, 250 μg of aPD-1 antibody and F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii EB-FPDK11, and F. 100 μg of extracellular vesicles (EV) derived from prausnitzii EB-FPYYK1 strains were intraperitoneally injected every 4 days. At this time, as the aPD-1 antibody, BioXCell's InVivoMab anti-mouse PD-1 (RMP1-14), catalog #BE0146 was used, and InVivoPure pH 7.0 Dilution Buffer, catalog #IP0070 was used at a concentration of 250 μg/100 μl. diluted and used.

The tumor size was monitored once every two days from the time the tumor was measured, and the tumor size was calculated as in Equation 1 above, and the results are shown graphically in FIG. 11 below. Extracellular vesicles were tested for their efficacy in a mouse tumor model alone or in the presence or absence of anti-PD1.

11 and 13, compared to the control group in which B16-F10 cells were allotransplanted, F. prausnitzii EB-FPDK3, F. prausnitzii EB-FPDK9, F. prausnitzii EB-FPDK11, and F. prausnitzii In the group to which extracellular vesicles (EV) derived from EB-FPYYK1 strains were orally administered, it was confirmed that the size of the tumor was remarkably reduced. Compared to the normal control group's tumor size of 570.4 ± 160.7 mm 3 , the tumor size of the aPD-1 administered group was 378.1 ± 128 mm 3 , which was confirmed to be reduced by 34%. In the aPD-1 and EB-FPDK3 EV, EB-FPDK9 EV, EB-FPDK11 EV, and EB-FPYYK1EV co-administration groups, the size of 165.6±57.54 ㎣, 152.7±40.44 ㎣, 230.2±83.91 ㎣, and 237.1±73.95 ㎣, respectively, were significantly showed a decrease. In particular, the aPD-1 and EB-FPDK9 combined administration group showed the best anticancer effect with a reduction of about 73% compared to the normal control group.

Referring to FIG. 12 , tumors were removed and weighed after sacrifice of the mice. As a result, the average tumor weight of the normal control group was 2.8±0.6622 g and that of the aPD-1 group was 1.89±0.5806 g, showing no significant difference. However, in the EB-FPDK3 EV, EB-FPDK9 EV, and EB-FPDK11 EV groups administered with PD-1 and EB-FP EV, a significant decrease was confirmed compared to the normal control group. Compared to the tumor weight of the normal control group, 2.8±0.6622 g, each tumor weight was EB-FPDK3 EV; 0.7696±0.2281 g (P<0.01), EB-FPDK9 EV; 0.8688±0.2224 g (P<0.05), EB-FPDK11; It was confirmed that it decreased significantly to 0.7409 ± 0.2423 g (P <0.05).

As described above, the inventors confirmed the inhibitory effects of cancer cell growth and metastasis of extracellular vesicles (EV) derived from Faecalibacterium sp. strains and anti-PD1 immune checkpoint inhibitors in vivo. , It was confirmed that tumor size and weight were effectively reduced when tumor-transplanted mice were injected with extracellular vesicles (EV) derived from a strain of the genus Picalibacterium and an anti-PD1 immune checkpoint inhibitor (Fig. 11 and see Figure 12). Therefore, the pharmaceutical composition of the present invention can be usefully used as a pharmaceutical composition for preventing or treating cancer because it has significantly superior tumor growth inhibitory effect compared to the administration of anti-PD1 alone.

Example 6: Wound healing assay using HT29 cells and B16-F10

In order to examine the anticancer activity of the extracellular vesicles derived from the Picalibacterium prosnich strain of the present invention, a wound healing activity test was performed.

HT29 human colorectal cancer cells were cultured in McCoy's medium containing 10% FBS and 1% gentamicin in 5% CO ₂ , at 37°C. HT29 colorectal cancer cells are dispensed into a 6-well plate for cell culture and cultured confluently. Thereafter, the 6-well plate is regularly scratched using a pipette tip. Thereafter, EB-FPDK3, EB-FPDK9, EB-FPDK11, and EB-FPYYK1EV were treated with 1 or 10 μg/ml for 24 hours and observed under a microscope. The cell area was calculated using the Image J program.

Referring to FIGS. 14A and 14B , as a result of wound healing experiments using HT29 cells, metastatic reduction of 25.97% and 55.25% were observed at 1 μg/ml and 10 μg/ml of EB-FPDK3EV, respectively, compared to the normal control group. In addition, in the group treated with 10 μg/ml, a significant decrease was confirmed with P<0.05. In the experimental groups treated with 1 μg/ml and 10 μg/ml of EB-FPDK9EV, 66.91% and 58.95% reductions in metastasis were respectively observed compared to the normal control group, and a significant decrease was confirmed at P<0.05 at both concentrations. In the experimental group treated with EB-FPDK11EV 1 μg/ml and 10 μg/ml concentrations, 54.48% and 49.45% reduction in metastatic effects were observed compared to the normal control group, respectively, and significant decreases were observed at both concentrations (P<0.05, respectively). , P<0.1). Finally, in the experimental group treated with 1 μg / ml and 10 μg / ml of EB-FPYYK1EV, 49.45% and 59.4% reductions in metastatic properties were observed compared to the normal control group, respectively, and a significant decrease was observed at both concentrations ( P<0.1, P<0.05, respectively).

Example 7: Anticancer effect by administration of F. prosniche EB-FPDK9 strain or EB-FPDK9-derived extracellular vesicles (EB-FPDK9EVs) in an allograft mouse animal model using melanoma

7.1 Experimental method

A 5-week-old C57BL/6 female mouse was purchased, and after a one-week acclimatization period, the melanoma cell line B16F10 was subcutaneously injected into the mouse as 5X10 ⁵ cells to construct a syngenic mouse. A mixture of 15 mg/ml of antibiotics ampicillin, 15 mg/ml of neomycin, 10 mg/ml of metronidazole, and 7.5 mg/ml of vancomycin was administered orally to all mice in an amount of 250 ul for 2 days.

As a positive control, 200 ug/200 ul of anti-PD-1 immuno-anticancer drug (αPD-1) was intraperitoneally administered once every 3 days from the 7th day after tumor transplantation, and Ficcolibacterium prosnich EB-FPDK9 strain was 1X10 ⁸ CFU was administered orally twice every 3 days, and 50 μg of Picalibacterium prosniche EB-FPDK9 EVs was administered intravenously (see FIG. 15).

The size of the tumor was measured using a ruler once every 3 days, and the size of the tumor was calculated as the square of the major axis X the minor axis X 0.5 (mm ³ ).

7.2 Experimental results

The tumor size was measured and tracked on the 7th day after transplantation of the B16F10 cell line, and the mice were sacrificed on the 19th day to measure the tumor weight. From the 13th day, it was confirmed that tumor growth was inhibited in the experimental group administered with the αPD-1 experimental group, which was a positive control group, and the Ficcolibacterium prosnich EB-FPDK9 EVs, compared to the normal control group.

Referring to FIG. 16, compared to the normal control group tumor size of 260.4±64.33 mm ³ on the 19th day, the day of sacrifice of the mice, the EB-FPDK9 administration group was 135.6±56.46 mm ³ , about 48%, and the EB-FPDK9 EVs administration group was 53.41± The tumor size was statistically significantly reduced by about 80% to 26.51 mm ³ .

Referring to FIG. 17, it was confirmed that the tumor weight of the normal control group was significantly reduced in the group administered with the positive control αPD-1 , Ficcolibacterium prosniche EB-FPDK9, and Ficcolibacterium prosnich EB-FPDK9EVs. , especially in the group administered with Picalibacterium prosnich EB-FPDK9 EVs, there was a statistically significant decrease.

When compared with the results of the tumor size and weight of the αPD-1 administration group, similar levels were observed in both the Ficcolibacterium prosnich EB-FPDK9 strain or the Ficcolibacterium prosnich EB-FPDK9 EVs administration group (FIG. 16 and FIG. 17). reference). This proves that the single administration of the Ficcolibacterium prosnich EB-FPDK9 strain and the Ficcalibacterium prosnich EB-FPDK9EVs has anticancer efficacy as much as that of αPD-1.

The specific examples described in this specification are only for describing preferred embodiments of the present invention, and should not be construed as limiting the present invention. The present invention can be practiced with various modifications and changes without departing from the spirit and scope of the present invention, and these facts will be apparent to those skilled in the art. The protection scope of the present invention should be defined by the appended claims, and various modifications and changes of the above are intended to be included in the protection scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology KCCM12619P (EB-FPDK3) 20191101 Korea Research Institute of Bioscience and Biotechnology KCCM12620P (EB-FPDK9) 20191101 Korea Research Institute of Bioscience and Biotechnology KCCM12621P (EB-FPDK11) 20191101 Korea Research Institute of Bioscience and Biotechnology KCCM12622P (EB-FPYYK1) 20191101

Claims (14)

Extracellular vesicles (EV) derived from strains of the genus Picalibacterium ; And a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the strain of the genus Picalibacterium is a strain of Faecalibacterium prausnitzii . The method of claim 1, wherein the strain of the genus Picalibacterium is Picalibacterium prosnich EB-FPDK3 strain (KCCM12619P), F. prausnitzii EB-FPDK9 strain (KCCM12620P), F. prausnitzii EB-FPDK11 strain (KCCM12621P), or F. prausnitzii EB-FPYYK1 strain (KCCM12622P). pharmaceutical composition for use. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the extracellular vesicles (EV) derived from the Faecalibacterium sp. strain have an average diameter of 20 to 300 nm. The method of claim 1, wherein the cancer is colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, cervical cancer, ovarian cancer, rectal cancer, or near anus. cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Select from the group consisting of prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition for preventing or treating cancer, characterized in that any one of which is. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the composition further comprises a chemo-anticancer agent or an immuno-anticancer agent. The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the immuno-anticancer agent is at least one selected from the group consisting of anti-PD1, anti-PDL1, anti-CTLA, anti-Tim3 and anti-LAG3. . The method of claim 6, wherein the extracellular vesicles (EV) derived from Faecalibacterium sp. strain and the chemotherapeutic agent or immunoanticancer agent are administered simultaneously in one formulation, or simultaneously or sequentially in separate formulations. A pharmaceutical composition for preventing or treating cancer, characterized in that it is administered. strains of the genus Picalibacterium ; And a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient. [Claim 10] The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the strain of the genus Picalibacterium is live or dead. [ Claim 12] The pharmaceutical composition for preventing or treating cancer according to claim 11, wherein the strain of the genus Picalibacterium is a strain of Faecalibacterium prausnitzii . The method of claim 11, wherein the strain of the genus Picalibacterium is Picalibacterium prosnich EB-FPDK3 strain (KCCM12619P), F. prausnitzii EB-FPDK9 strain (KCCM12620P), F. prausnitzii EB-FPDK11 strain (KCCM12621P), or F. prausnitzii EB-FPYYK1 strain (KCCM12622P). pharmaceutical composition for use. Extracellular endoplasmic reticulum (EV) derived from strains of the genus Picalibacterium or strains of the genus Picalibacterium ; and a physiologically acceptable carrier or excipient for preventing or improving cancer health functional food. Extracellular endoplasmic reticulum (EV) derived from strains of the genus Picalibacterium or strains of the genus Picalibacterium ; And a veterinary composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier or excipient.