Novel Prevotella merde immunoactis strain and its uses

Abstract

This invention relates to a novel strain of Prevotella merde immunoactis and its uses. The inventors have confirmed that the novel strain of Prevotella merde immunoactis (deposit number: KCTC 14922BP) not only increases T cell proliferation, increases T cell IFN-γ secretion, increases the expression or activity level of IFN-γ or Cxcl9, and decreases the expression or activity level of Ccl22, but also enhances the anticancer effect of immune checkpoint inhibitors. Therefore, the novel strain of Prevotella merde immunoactis or its culture medium is expected to be useful for immune enhancement and enhancing the anticancer effect of immune checkpoint inhibitors.

Description

[Technical Field]

[0001] This invention relates to a novel strain of Prevotella merde immunoactis and its uses.

[0002] This invention claims priority under Republic of Korea Patent Application No. 10-2023-0036857, filed on 21 March 2023, and all contents disclosed in the specification and drawings of said application are incorporated herein by reference.

[0003] (blank) [Background technology]

[0004] Cancer is one of the intractable diseases that humanity must overcome, and enormous amounts of capital are being invested worldwide in the development of cures for it. In South Korea, since the 1990s, it has been the leading cause of death, with over 100,000 people diagnosed with cancer annually and over 60,000 deaths. Factors that trigger cancer include smoking, ultraviolet radiation, chemicals, food, stress, and other environmental factors. Because these triggers are so diverse, developing treatments is difficult, and the effectiveness of treatments varies depending on the site of the cancer.

[0005] Cancer treatments include surgical removal of tumor tissue, radiation therapy, chemotherapy, and immunotherapy. The anticancer drugs used in cancer treatment are applied differently depending on the type of cancer. Generally, chemotherapy drugs prescribed to cancer patients affect the survival mechanisms of normal cells, including cancer cells, leading to side effects such as hair loss, fever, diarrhea, rash, and weakened immunity. Subsequently, based on genetic research into cancer, targeted anticancer drugs that suppress specific gene mutations in cancer have been developed, significantly improving the side effects of conventional chemotherapy. However, the rapid mutation rate of cancer cells can induce resistance to targeted anticancer drugs, making it difficult to expect 100% sustained anticancer effects from targeted anticancer drugs. In recent years, research on the tumor microenvironment has been active, leading to the development and use of immunotherapy drugs targeting various immune checkpoints that regulate the patient's immune system. Among these, immunosuppressants that inhibit PD-1 / PD-L1 are known to show a high therapeutic response in patients with melanoma, gastric cancer, and lung cancer. Such immunosuppressants work by activating the function of immune cells in the tumor microenvironment, thereby suppressing the proliferation of cancer cells. However, immunosuppressants do not show the same anticancer effect in all patients.

[0006] (blank) [Overview of the project] [Problems that the invention aims to solve]

[0007] The technical problem that the present invention aims to solve is to provide a novel strain of Prevotella merde immunoactis, an immunoenhancing composition containing the strain or culture medium as an active ingredient, and a composition for enhancing anticancer effects. The inventors have confirmed that the novel strain of Prevotella merde immunoactis increases T cell proliferation, increases IFN-γ secretion by T cells, and enhances the anticancer effect of immune checkpoint inhibitors.

[0008] Therefore, an object of the present invention is to provide a Prevotella merdae Immunoactis strain deposited under the accession number KCTC 14922BP.

[0009] Another object of the present invention is to provide an immunopotentiating composition comprising, as an active ingredient, a Prevotella merdae Immunoactis strain according to the present invention or a culture broth thereof.

[0010] Still another object of the present invention is to provide a pharmaceutical composition for enhancing an anti-cancer effect, comprising, as an active ingredient, a Prevotella merdae Immunoactis strain according to the present invention or a culture broth thereof.

[0011] Still another object of the present invention is to provide a food composition for enhancing an anti-cancer effect, comprising, as an active ingredient, a Prevotella merdae Immunoactis strain according to the present invention or a culture broth thereof.

[0012] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising, as active ingredients, a Prevotella merdae Immunoactis strain according to the present invention or a culture broth thereof, and an immune checkpoint inhibitor.

[0013] However, the technical problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the technical field from the following description.

[0014] (Blank)

Means for Solving the Problems

[0015] To achieve the above object of the present invention, the present invention provides a Prevotella merdae Immunoactis strain deposited under the accession number KCTC 14922BP.

[0016] Furthermore, the present invention provides an immunoenhancing composition comprising the Prevotella merde immunoactis strain or its culture solution according to the present invention as an active ingredient.

[0017] Furthermore, the present invention provides a pharmaceutical composition for enhancing anticancer effects, comprising the Prevotella merde immunoactis strain or its culture solution according to the present invention as an active ingredient.

[0018] Furthermore, the present invention provides a food composition for enhancing anticancer effects, comprising the Prevotella merde immunoactis strain or its culture solution according to the present invention as an active ingredient.

[0019] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the Prevotella merde-immunoactis strain or its culture medium according to the present invention, and an immune checkpoint inhibitor as active ingredients.

[0020] In one embodiment of the present invention, the bacterial strain may, but is not limited to, include a DNA sequence encoding the 16S rRNA sequence of Sequence ID No. 1.

[0021] In other embodiments of the present invention, the bacterial strain may enhance immunity or increase the anticancer effect of anticancer drugs, but is not limited thereto.

[0022] In yet another embodiment of the present invention, the pharmaceutical composition for enhancing anticancer effects may be used in combination with an anticancer agent, but is not limited thereto.

[0023] In yet another embodiment of the present invention, the immune checkpoint inhibitor may be, but is not limited to, a PD-L1 inhibitor or a PD-1 inhibitor.

[0024] In yet another embodiment of the present invention, the PD-L1 inhibitor may be, but is not limited to, at least one selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, and BMS-986189.

[0025] In yet another embodiment of the present invention, the PD-1 inhibitor may be, but is not limited to, at least one selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, and dostarlimab, INCMGA00012, AMP-224, and AMP-514.

[0026] In yet another embodiment of the present invention, the Prevotella merde-immunoactis strain or its culture medium and the immune checkpoint inhibitor may be administered simultaneously or sequentially, but are not limited thereto.

[0027] In yet another embodiment of the present invention, the Prevotella merde immunoactis strain or its culture medium may satisfy one or more of the following characteristics, but is not limited thereto.

[0028] (a) CD4 + T cells and CD8 + It increases the proliferation of T cells.

[0029] (b) Increases the secretion of interferon-γ (IFN-γ) from T cells.

[0030] (c) Increase the expression or activity level of Inf-γ or Cxcl9.

[0031] (b) Reduce the expression or activity level of Ccl22.

[0032] In yet another embodiment of the present invention, the cancer may be at least one selected from the group consisting of colorectal cancer, breast cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, hematological cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, trumpet duct cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, hydroureteral cancer, renal cell carcinoma, renal pelvic cancer, CNS tumors, primary CNS lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma.

[0033] Furthermore, the present invention provides an immunoenhancing method comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture medium according to the present invention to an individual in need.

[0034] Furthermore, the present invention provides an immunoenhancing application for a composition containing the Prevotella merde immunoactis strain or its culture medium according to the present invention.

[0035] Furthermore, the present invention provides a use for producing an immunostimulant using the Prevotella merde immunoactis strain or its culture medium according to the present invention.

[0036] Furthermore, the present invention provides a method for enhancing the anticancer effect of an anticancer agent, comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture solution according to the present invention to an individual in need.

[0037] Furthermore, the present invention provides a composition containing the Prevotella merde immunoactis strain or its culture solution according to the present invention for enhancing the anticancer effect of anticancer agents.

[0038] Furthermore, the present invention provides the use of the Prevotella merde immunoactis strain or its culture medium according to the present invention for producing an anticancer effect enhancer for anticancer agents.

[0039] Furthermore, the present invention provides a method for preventing or treating cancer, comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, to an individual in need.

[0040] Furthermore, the present invention provides for the use of a composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, for the prevention or treatment of cancer.

[0041] Furthermore, the present invention provides the use of a composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, for the production of cancer therapeutic agents.

[0042] (blank) [Effects of the Invention]

[0043] The inventors have confirmed that the novel Prevotella merde immunoactis strain (deposit number: KCTC 14922BP) increases T cell proliferation, increases T cell IFN-γ secretion, enhances T cell cancer cell killing ability, increases the expression or activity level of IFN-γ or Cxcl9 and decreases the expression or activity level of Ccl22 in the tumor microenvironment, and enhances the anticancer effect of immune checkpoint inhibitors. Therefore, the novel Prevotella merde immunoactis strain or its culture medium is expected to be useful for immune enhancement and enhancing the anticancer effect of immune checkpoint inhibitors.

[0044] (blank) [Brief explanation of the drawing]

[0045] [Figure 1] Figure 1 shows the proliferation effect of CD4+ T cells when treated with the culture supernatant of the Prevotella merde immunoactis strain.

[0046] [Figure 2] Figure 2 shows the proliferation effect of CD8+ T cells when treated with the culture supernatant of the Prevotella merde immunoactis strain.

[0047] [Figure 3] Figure 3 shows IFN-γ secreted from CD4+ T cells and CD8+ T cells treated with the culture supernatant of Prevotella merde immunoactis strain.

[0048] [Figure 4] Figure 4 shows that the combination of Prevotella merde immunoactis strain and α-PD-1 antibody reduces the growth of colon cancer tumors.

[0049] [Figure 5] Figure 5 shows the effects of combining the Prevotella merde immunoactis strain with an α-PD-1 antibody on the expression of Inf-γ, Cxcl9, and Ccl22.

[0050] [Figure 6] Figure 6 shows that the combination of Prevotella merde immunoactis strain and α-PD-1 antibody increases the populations of CD4+ T cells, CD4+CD44+CD62L-T cells (CD4+ effector T cells), and TNF-α+ cells.

[0051] [Figure 7]Figure 7 shows that the combination of Prevotella merde immunoactis strain and α-PD-1 antibody increases the populations of CD8+ T cells, CD8+CD44+CD62L-T cells, and PD-1+CD8+ T cells.

[0052] [Figure 8] Figure 8 shows the results of the cytotoxicity assessment of the Prevotella merde immunoactis strain. [Modes for carrying out the invention]

[0053] (blank)

[0054] In one embodiment of the present invention, a bacterial strain isolated from a patient who responded to anti-PD-1 treatment was identified and confirmed to be the same species as the publicly disclosed strain Prevotella merdae sp. Marseille-P4119, but belonging to a different strain. This novel strain was named Prevotella merdae Immunoactis (see Example 1).

[0055] In another embodiment of the present invention, when the culture supernatant of the Prevotella merde immunoactis strain is treated, the strain culture solution contains CD4 + T cells and CD8 + We confirmed that T cell proliferation was significantly promoted and IFN-γ secretion from T cells was significantly increased (see Example 3).

[0056] In yet another embodiment of the present invention, it was confirmed that co-administration of Prevotella merde immunoactis strain and a PD-1 inhibitor resulted in remarkably superior tumor growth inhibition (see Example 4).

[0057] In still other embodiments of the present invention, when Prevotella mellea immunostimulating strain and a PD-1 inhibitor are administered in combination, it was confirmed that the expressions of Inf-γ and Cxcl9 increased and the expression of Ccl22 decreased in the tumor tissues of mice. Further, by the combined administration, + CD4 + CD44 + CD62L - T cells (CD4 + effector T cells), TNF-α + cells, CD8 + T cells, CD8 + CD44 + CD62L - T cells, and PD-1 + CD8 + T cell populations were confirmed to increase (see Example 5).

[0058] In still other embodiments of the present invention, when the culture supernatant of Prevotella mellea immunostimulating strain is treated with OT-1 T cells, it was confirmed that Ovalbumin-pulsed T cells treated with the culture supernatant of Prevotella mellea immunostimulating strain more effectively killed MC38 colon cancer cells.

[0059] Therefore, the Prevotella mellea immunostimulating strain or culture solution of the present invention can be usefully used not only for the use of enhancing immunity and promoting anti-cancer effects, but also is expected to be effectively used as an anti-cancer composition by combination with an immune checkpoint inhibitor based on the synergistic effect with the immune checkpoint inhibitor.

[0060] (blank)

[0061] Hereinafter, the present invention will be described in detail.

[0062] (blank)

[0063] The present invention provides a strain of Prevotella merdae Immunoactis deposited under deposit number KCTC 14922BP. According to one embodiment of the present invention, analysis of the full-length dielectric and 16S rRNA sequence to identify the strain confirmed that the strain's 16S rRNA is encoded by the sequence of Sequence ID No. 1.

[0064] According to one embodiment of the present invention, the strain was found to have a similarity of 97.82% to the publicly disclosed strain Prevotella merdae sp. Marseille-P4119, and was found to be a novel strain that is the same species as Prevotella merdae sp. Marseille-P4119 but belongs to a different lineage.

[0065] Therefore, the inventors named the strain Prevotella merdae Immunoactis and deposited it with the Korea Center for Bioresources (KCTC) of the Korea Institute of Biotechnology on March 24, 2022, where it was assigned accession number KCTC 14922BP.

[0066] According to one embodiment of the present invention, the bacterial strain not only increases the proliferation of T cells, increases the secretion of IFN-γ by T cells, increases the expression or activity level of IFN-γ or Cxcl9, and decreases the expression or activity level of Ccl22, but also enhances the anticancer effect of immune checkpoint inhibitors, thus enhancing immunity or enhancing the anticancer effect.

[0067] Accordingly, the present invention provides an immunostimulatory composition, a pharmaceutical composition for enhancing anticancer effects, or a pharmaceutical composition for the prevention or treatment of cancer, comprising the Prevotella merde immunoactis strain or its culture solution according to the present invention as an active ingredient.

[0068] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the Prevotella merde-immunoactis strain or its culture medium according to the present invention, and an immune checkpoint inhibitor as active ingredients.

[0069] In this invention, "immune enhancement" means increasing the immune response or activity in the immune system within a living organism. Immunity is broadly divided into innate immunity and acquired immunity. Innate immunity reacts nonspecifically to antigens without special memory mechanisms and has the function of protecting against antigen invasion. Innate immunity includes many anatomical and biological protective mechanisms, such as the skin, mucous tissue, highly acidic gastric acid, and complement present in the blood. Cells responsible for innate immunity include macrophages and polymorphonuclear leukocytes, which perform phagocytic activity, and natural killer cells (NK cells) that kill infected cells and cancer cells. Acquired immunity refers to a stronger immune response, including immunological memory, which means the function of remembering the label of an antigen. It is antigen-specific and requires the recognition of non-self antigens through the antigen presentation process.

[0070] The Prevotella merde immunoactis strain of the present invention promotes T cell proliferation and has excellent IFN-γ secretion-promoting effects on T cells, thus enhancing innate and adaptive immune function.

[0071] In the present invention, "enhancement of anticancer effect" refers to one example of the anticancer effect of an anticancer agent when used in combination with an anticancer agent, and may also include, but is not limited to, enhancing the tumor growth inhibitory effect of the anticancer agent. In one embodiment of the present invention, when an anti-PD-1 antibody, which is an immune checkpoint inhibitor, and the culture supernatant of the Prevotella merde-immunoactis strain were administered in combination, it was confirmed that not only was the growth of colon cancer suppressed, but the proportion of T cells also increased significantly (see Example 3).

[0072] Therefore, in the present invention, the pharmaceutical composition for enhancing anticancer effects may be used in combination with an anticancer agent, but is not limited thereto.

[0073] In the present invention, the anticancer agent may be one or more selected from the group consisting of cisplatin, 5-fluorouracil, paclitaxel, doxorubicin, daunorubicin, vinblastine, vincristine, actinomycin D, teniposide, etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin, immune checkpoint inhibitors, and mitomycin-C.

[0074] In this invention, the term "cultivation" refers to all actions taken to grow microorganisms under appropriately artificially controlled environmental conditions, and in this invention, it is a concept that includes "fermentation."

[0075] In the present invention, the bacterial cells of Prevotella merde immunoactis may include not only the live bacteria themselves obtained from the culture medium, but also any processing form of lactic acid bacteria known to those skilled in the art, such as cell crushed products, dried products, frozen products, etc.

[0076] In the terminology used in this invention, "culture medium" includes "fermentation liquid," and may include, but is not limited to, the culture medium itself obtained by culturing in a liquid medium, or processed products derived from the culture medium itself, such as the filtrate (centrifugally separated supernatant) obtained by filtering or centrifuging the culture medium to remove bacterial strains.

[0077] In the present invention, the immune checkpoint inhibitor may be a PD-L1 inhibitor or a PD-1 inhibitor, but is not limited thereto.

[0078] In the present invention, the PD-L1 inhibitor may be an anti-PD-L1 antibody, but is not limited thereto. The PD-L1 inhibitor may be at least one selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, and BMS-986189, but is not limited thereto.

[0079] In the present invention, the PD-1 inhibitor may be an anti-PD-1 antibody, but is not limited thereto. The PD-1 inhibitor may be at least one selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514, but is not limited thereto.

[0080] In the present invention, the Prevotella merde immunoactis strain or its culture medium, and the immune checkpoint inhibitor are available in the following ratios: 0.5-10:1, 0.5-9:1, 0.5-8:1, 0.5-7:1, 0.5-6:1, 0.5-5:1, 0.5-4.5:1, 1-10:1, 1-9:1, 1-8:1, 1-7:1, 1-6:1, 1 The mixture may be mixed in volume ratios of ~5:1, 1~4.5:1, 2~10:1, 2~9:1, 2~8:1, 2~7:1, 2~6:1, 2~5:1, 2~4.5:1, 3~10:1, 3~9:1, 3~8:1, 3~7:1, 3~6:1, 3~5:1, 3~4.5:1, 3.5~4.5:1, or 4:1, but is not limited thereto. In addition, the Prevotella merde immunoactis strain or its culture medium and the immune checkpoint inhibitor may be administered separately in the aforementioned volume ratios.

[0081] Furthermore, the Prevotella merde immunoactis strain or its culture medium, and the PD-1 inhibitor may be mixed in volume ratios of 0.1-10:1, 0.1-9:1, 0.1-8:1, 0.1-7:1, 0.1-6:1, or 0.1-5:1, but are not limited thereto.

[0082] In the present invention, the Prevotella merde-immunoactis strain or its culture medium may be administered simultaneously with or sequentially with the immune checkpoint inhibitor, but is not limited thereto. In the present invention, the composition containing the Prevotella merde-immunoactis strain or its culture medium as an active ingredient may be formulated to exist in a single container as a mixture with the immune checkpoint inhibitor, or it may be formulated to exist in separate containers and be administered simultaneously or sequentially. Furthermore, the composition containing the Prevotella merde-immunoactis strain or its culture medium as an active ingredient may be administered before treatment with the immune checkpoint inhibitor, or it may be administered as a second-line therapy after treatment with the immune checkpoint inhibitor, but is not limited thereto.

[0083] In the present invention, the Prevotella merde immunoactis strain or its culture medium may satisfy one or more of the following characteristics, but is not limited thereto.

[0084] (a) CD4 + T cells and CD8 + It increases the proliferation of T cells.

[0085] (b) Increases the secretion of interferon-γ (IFN-γ) from T cells.

[0086] (c) Increase the expression or activity level of Inf-γ or Cxcl9.

[0087] (b) Reduce the expression or activity level of Ccl22.

[0088] In this invention, the term "cancer" refers to a disease related to the control of cell death, specifically a disease that occurs when cells excessively proliferate due to an imbalance in normal apoptosis. These abnormally proliferating cells may, in some cases, invade surrounding tissues or organs and form lumps, potentially causing the destruction or deformation of normal structures within the body. This condition is collectively referred to as a tumor. Generally, tumors can be divided into benign tumors and malignant tumors. Malignant tumors grow much faster than benign tumors, invade surrounding tissues, cause metastasis, and can be life-threatening. Such malignant tumors are usually referred to as "cancer."

[0089] In the present invention, the cancer may be at least one selected from the group consisting of breast cancer, colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, hematological cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, trumpet duct cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, hydroureteral cancer, renal cell carcinoma, renal pelvic cancer, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. In one embodiment of the present invention, the cancer may be colon cancer, but is not limited thereto.

[0090] The content of the Prevotella merde immunoactis strain or its culture solution in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progression of the symptoms, the patient's condition, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry weight after removing the solvent.

[0091] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be at least one selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0092] The pharmaceutical compositions according to the present invention may be used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elsilics, emulsions, suspensions, alcoholic preparations, lozenges, aromatic preparations, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, softened extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injections, or aerosols, respectively, by conventional methods. The external preparations may also have dosage forms such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasms.

[0093] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0094] When formulating, the product is typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients.

[0095] Excipients for tablets, powders, granules, capsules, pills, and lozenges according to the present invention include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC1928, HPMC2208, HPMC2906, HPMC2910, propylene glycol, casein, calcium lactate, primozel, gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, and casein. Thrium, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, and other binders may be used. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum (GuarDisintegrants such as gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and hard anhydrous silicic acid may be used. Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, PEG6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silicic acid may also be used.

[0096] Additives to the liquid formulation according to the present invention may include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

[0097] The syrup according to the present invention may contain a solution of sucrose, other sugars, or sweeteners, and may also contain, if necessary, fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc.

[0098] Purified water may be used in the emulsion of the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0099] The suspending agent according to the present invention may contain suspending agents such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, HPMC1828, HPMC2906, and HPMC2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0100] The injectable preparation according to the present invention includes distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG (PEG), lactating Ringer's injection solution, solvents such as ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate, solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidinedione, propylene glycol, twins, dijontinamide, hexamine, and dimethylacetamide, weak acids and their salts (acetic acid and sodium acetate), and weak bases and their salts (ammonia and ammonium acetate). It may also contain buffering agents such as organic compounds, proteins, albumin, peptone, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfurizing agents such as 0.1% sodium bisulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Twin 80, and aluminum monostearate.

[0101] The suppositories according to the present invention include cocoa butter, lanolin, vitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocoat 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-en, Paramount-B, Sposyl (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecobi (W, R, S, M, Fs), and Tezestr triglyceride base (TG-95, MA, 57) may be used.

[0102] Orally administered solid dosage forms include tablets, pills, powders, granules, and capsules. Such solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stylate talc are also used.

[0103] Oral liquid formulations include suspensions, liquid preparations, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0104] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including the type and severity of the patient’s disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0105] The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple agent. Taking all of the above factors into consideration, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects, which may be readily determined by a person of the skill in the art to which the present invention belongs.

[0106] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All methods of administration are predictable, but may include, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, perispinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0107] The pharmaceutical composition of the present invention is determined by the type of drug that is the active ingredient, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

[0108] Furthermore, the present invention provides an immunoenhancing method comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture medium according to the present invention to an individual in need.

[0109] Furthermore, the present invention provides an immunoenhancing application for a composition containing the Prevotella merde immunoactis strain or its culture medium according to the present invention.

[0110] Furthermore, the present invention provides a use for producing an immunostimulant of the Prevotella merde immunoactis strain or its culture medium according to the present invention.

[0111] Furthermore, the present invention provides a method for enhancing anticancer effects, comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture medium according to the present invention to an individual in need.

[0112] Furthermore, the present invention provides a use for enhancing the anticancer effect of compositions containing the Prevotella merde immunoactis strain or its culture solution according to the present invention.

[0113] Furthermore, the present invention provides a use for producing an anti-cancer effect enhancer of the Prevotella merde immunoactis strain or its culture medium according to the present invention.

[0114] Furthermore, the present invention provides a method for preventing or treating cancer, comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, to an individual in need.

[0115] Furthermore, the present invention provides for the use of a composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, for the prevention or treatment of cancer.

[0116] Furthermore, the present invention provides the use of a composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, for the production of cancer therapeutic agents.

[0117] In the present invention, "individual" means an animal that requires treatment for a disease, and more specifically, may be, but is not limited to, a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0118] In the present invention, "administration" means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0119] In the present invention, "prevention" means all actions that suppress or delay the onset of a desired disease; "treatment" means all actions that improve or beneficially alter the symptoms of a desired disease and associated metabolic disorders by administering a pharmaceutical composition according to the present invention; and "improvement" means all actions that reduce parameters related to a desired disease, such as the severity of symptoms, by administering a composition according to the present invention.

[0120] (blank)

[0121] Furthermore, the present invention provides a food composition for enhancing immunity or promoting anticancer effects, which contains Prevotella merde immunoactis strain (deposit number: KCTC 14922BP) or its culture solution as an active ingredient. The food composition may, but is not limited to, a health functional food composition.

[0122] When using the Prevotella merde immunoactis strain or its culture solution of the present invention as a food additive, the Prevotella merde immunoactis strain or its culture solution can be added as is, or used in combination with other foods or food ingredients, and can be used as appropriate according to the usual methods. The amount of active ingredient mixed may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the Prevotella merde immunoactis strain or its culture solution of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, relative to the raw materials. However, in the case of long-term intake for health and hygiene purposes, or for health regulation purposes, the amount may be less than the above range, and the active ingredient may be used in an amount greater than the above range, as there are no safety issues.

[0123] There are no particular restrictions on the types of food products to which the substance may be added. Examples of foods to which the substance may be added include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the usual sense.

[0124] The health beverage composition according to the present invention may contain various flavorings or natural carbohydrates as additional ingredients, as in ordinary beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates is generally about 0.01 to 0.20 g, or about 0.04 to 0.10 g, per 100 mL of the composition of the present invention.

[0125] In addition to the foregoing, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. Furthermore, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not particularly important, but it is generally selected from a range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0126] In this specification, "health functional foods" is the same term as "foods for special health use (FoSHU)," and refers to foods with high medical and therapeutic effects that are processed to efficiently exhibit biological regulatory functions in addition to providing nutrients. The said foods may be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain effects that are useful for preventing or improving obesity.

[0127] The health functional food of the present invention can be manufactured by methods commonly used in the industry, and during such manufacturing, raw materials and ingredients commonly added in the industry may be added. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food as a raw material and not having the side effects that may occur when pharmaceuticals are taken long-term, and it is highly portable.

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[0129] Because the present invention can be subjected to various transformations and has various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather should be understood to encompass all transformations, equivalents, or substitutes that fall within the spirit and technical scope of the present invention. In the description of the present invention, if it is determined that a specific description of the relevant prior art may obscure the gist of the invention, such detailed description will be omitted.

[0130] [Modes for carrying out the invention]

[0131] The following examples are provided to aid in understanding the present invention. However, these examples are provided to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0132] (blank) [Examples]

[0133] [Examples]

[0134] Example 1. Isolation and identification of Prevotella merdae Immunoactis strain.

[0135] Prevotella merdae Immunoactis (P. merdae Immunoactis) was isolated from one patient who responded to anti-PD-1 treatment. Feces from the patient who responded to anti-PD-1 treatment were diluted with PBS and then streaked onto BHI (brain-heart infusion) agar medium (#237500, BD Bioscience) supplemented with 0.5% yeast extract (#212750, BD Bioscience), 0.05% L-cysteine-HCl (#C7880, Sigma-Aldrich), 0.0005% hemin (#51280, Sigma-Aldrich), 0.0001% vitamin K1 (#95271, Sigma-Aldrich), 0.01% kanamycin (#HKA01, Rd-tech), and 5% defibrinated sheep blood (#S1876, KisanBio). The colonies were incubated in an anaerobic incubator (GasPak 100 system, #260626, BD) at 37°C for 48 hours. After randomly selecting colonies, P. merdae Immunoactis was isolated using species-specific PCR.

[0136] For full-length dielectric analysis of isolated P. merdae Immunoactis, genomic DNA was extracted from P. merdae Immunoactis pellets and sequenced. Specifically, a library was prepared from the genomic DNA using the TruSeq Nano DNA sample prep kit (Illumina), and sequencing was performed. The sequenced files were quality checked using FastQC (version 0.11.8), and low-quality sequencing reads were removed using Bbduk (version 38.34). A full-length dielectric assembly was prepared using Unicycler (version 0.4.9b), and the similarity of the full-length dielectrics with Prevotella stercorea DSM 18206 and Prevotella merdae sp. Marseille-P4119 was confirmed and compared using OrthoANI (version 0.93.1) to identify the strain. As a result, P.merdae Immunoactis was found to have a similarity of 78.40% to Prevotella stercorea DSM 18206, and the highest similarity of 97.41% to Prevotella merdae sp.Marseille-P4119. It was found to be the same species as Prevotella merdae sp.Marseille-P4119, but a different strain.

[0137] Furthermore, to confirm the 16S rRNA sequence of the isolated P. merdae Immunoactis, genome annotation was performed using Prokka on contigs obtained by full-length dielectric analysis. By identifying the contigs corresponding to the 16S rRNA region (partial), it was confirmed that the 16S rRNA of P. merdae Immunoactis was encoded by the nucleotide sequence of Sequence ID No. 1.

[0138] [Table 1]

[0139] Example 2. Preparation of analytical samples of Prevotella merdae Immunoactis strain.

[0140] The Prevotella merdae Immunoactis isolated in Example 1 was grown under anaerobic conditions at 37°C. To maintain anaerobic conditions, anaeropack (#A-06, MGC) was added to the anaerobic incubator (Oxoid). P. merdae Immunoactis was grown in sterilized BHI (brain-heart infusion) medium (#237500, BD Bioscience) supplemented with 0.5% yeast extract (#212750, BD Bioscience), 0.05% L-cysteine ​​HCl (#C7880, Merck), 0.005% hemin (#H9039, Sigma-Aldrich), and 0.0001% vitamin K1 (#95271, Sigma-Aldrich). Two mL of a bacterial culture culture incubated overnight was inoculated into eight mL of RPMI 1640 medium (#10-040-CV, Corning), and the bacteria were cultured for two days. The RPMI culture was centrifuged at 6,000 rpm for five minutes, and the supernatant was filtered through a 0.2 μm syringe filter (#17823-K, Sartorius). A 1 mL aliquot of the supernatant was stored at -80°C until use. Subsequently, the P. merdae Immunoactis culture was centrifuged at 6,000 rpm for five minutes, the supernatant was discarded, and the pellet was resuspended in PBS. The resuspended culture was filtered at 600 nm (OD 600 The optical density at ) is OD 600 I adjusted it to =6.

[0141] (blank)

[0142] Example 3. Analysis of T cell proliferation and activation

[0143] To confirm the immune-related effects of P. merdae Immunoactis, human T cells were treated with the culture supernatant of P. merdae Immunoactis. Specifically, human T cells were isolated from human blood samples. First, with prior consent obtained from the Gwangju Advanced Institute of Science and Technology (GIST) in Korea, 40-50 mL of blood samples were collected from healthy donors in their 20s. Then, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using a Ficoll Paque Plus (#GE17-1440-02, Merck). Next, CD4 + T cells and CD8 + T cells are each CD4 + T cell isolation kit (#130-096-533, Miltenyi biotec) and CD8 +T cells were isolated using a T cell isolation kit (#130-096-495, Miltenyi biotec). The isolated T cells were resuspended in RPMI1640 medium (#10-040-CV, Corning) supplemented with 10% FBS (#92916754, MP biomedicals), 1% penicillin-streptomycin (#15140-122, Thermo Fisher Scientific), 1×NEAA (#11140-050, Thermo Fisher Scientific), 200mM HEPES (#15630-080, Thermo Fisher Scientific), 2mM sodium pyruvate, 2mM L-glutamine (#25030-081, Thermo Fisher Scientific), and 1×2-mercaptoethanol (#21985-023, Thermo Fisher Scientific). Next, the isolated T cells were stained with CFSE (#C34554, Thermo Fisher Scientific) at 37°C under a 5% CO2 atmosphere for 30 minutes. 96-well untreated plates (#32096, SPL) were pre-coated with 0.2 mg of α-CD3e antibody (#100340, BioLegend) per well and cultured overnight at 4°C. CFSE-stained T cells were then placed in 96-well pre-coated plates (#32096, SPL) at a rate of 2 × 10⁶ cells per well. 5 Cellular cells were dispensed and cultured individually or with 10% P. merdae Immunoactis culture supernatant at 37°C under a 5% CO2 atmosphere for 72 hours. After 72 hours of culture, T cells were harvested and washed with FACS buffer (PBS + 10% FBS + 2 mM EDTA). Stained cells were analyzed using CANTO II (BD Bioscience) and BDFACS Diva software v.8.0.2 (BD Bioscience), and data analysis was performed using FlowJo software (v.10, TreeStar).

[0144] As a result, as shown in Figures 1 and 2, combined treatment with the culture supernatant of P. merdae Immunoactis and α-CD3e antibody showed a higher CD4 count compared to treatment with α-CD3e antibody alone. + T cells and CD8 + It significantly promoted the proliferation of T cells.

[0145] Furthermore, the supernatant of the cultured T cells was collected and stored at -80°C until use in ELISA. Subsequently, IFN-γ levels were measured using a human IFN-γ ELISA kit according to the manufacturer's protocol (#88-7316-88, Thermo Fisher Scientific).

[0146] As a result, as shown in Figure 3, it was confirmed that IFN-γ secretion increased when α-CD3e antibody was used in combination with P. merdae Immunoactis culture supernatant compared to when α-CD3e antibody was used alone.

[0147] (blank)

[0148] Example 4. Synergistic effect of P. merdae Immunoactis administration on α-PD-1 tumor suppression.

[0149] To confirm the anticancer effect of P. merdae Immunoactis, mice were inoculated with MC38 colon cancer cells and administered α-PD-1 in syngeneic mouse experiments. Specifically, all animal experiments were conducted according to protocols approved by the GIST Institutional Animal Care and Use Committee (GIST-2021-096). All animals used in this study were kept and handled in accordance with the approved policies of the research protocol. MC38 colon cancer cells (#EZH204, Kerafast) were maintained under conditions of 5% CO2 and 37°C in DMEM (#11965-118, Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (#92916754, MP biomedicals) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Subsequently, MC38 cells were subcultured at 70-80% confluence. In the syngeneic tumor model, 2 × 10⁶ female C57B6 / N mice (Orientbio) were given 7 weeks old. 5 MC38 colon cancer cells were prepared by subcutaneous injection (Day 0). The size of the tumor was measured three times a week until the end of the experiment, and the volume of the tumor was calculated as "length × width". 2 The calculation was performed using "×0.5". PBS (Phosphate Buffered saline, #10010-049, Thermo Fisher Scientific) and isolated P. merdae Immunoactis (OD 600 PBS (6, 200 μl) was administered orally 6 days a week starting 14 days before tumor inoculation (-14 days) and continued for 3 weeks after tumor inoculation (23 days). To investigate combination therapy, 2 mg / kg of IgG isotype (clone 2A3, #BE0089, BioXCell) and 2 mg / kg of α-PD-1 mAb (clone RMP1-14, #BE0146, BioXCell) were administered intraperitoneally on 7, 9, 11, 14, 16, and 18 days after tumor inoculation. PBS and IgG isotype were used as control groups for bacterial administration and α-PD-1 mAb injection, respectively.

[0150] As shown in Figure 4, the experimental results demonstrated that the combined administration of P. merdae Immunoactis and α-PD-1 significantly suppressed tumor growth compared to α-PD-1 monotherapy.

[0151] (blank)

[0152] Example 5. Improved immune activity and penetration effect of α-PD-1 into the tumor environment by administration of P. merdae Immunoactis.

[0153] To demonstrate the role of P. merdae Immunoactis in the immune system, 2 mg / kg of α-PD-1 and OD were administered. 600 In tumors of a mouse model of tumors treated with 6,200 μl of P. merdae Immunoactis, we performed profiling of immune-related gene expression and immune cell populations.

[0154] Specifically, lymph nodes and tumor tissue were isolated from mice 21 days after inoculation with MC38 cells. Subsequently, five types of immune cells were analyzed: T cells, NK cells, dendritic cells (DCs), and regulatory T cells (T). regRegulatory T cells and macrophages were analyzed. Lymph nodes were pulverized with 1 mL of PBS (#10010-049, Thermo Fisher Scientific) using a 70 μm cell strainer (#352350, Falcon), and lymphocytes were collected. After washing with PBS, the cell count was calculated. Tumor tissue was cut into small pieces, transferred to 5 mL of dissociation medium, and cultured at 37°C for 40 minutes. Tumor dissociation media was prepared using T cell medium supplemented with 2.5 mg / ml collagenase type 1 (#17100-017, Thermo Fisher Scientific), 1.5 mg / ml collagenase type 2 (#17101-015, Thermo Fisher Scientific), 1 mg / ml collagenase type 4 (#17104-019, Thermo Fisher Scientific), 50 μg / ml DNase type 1 (#10104159001, Merck), and 0.25 mg / ml hyaluronidase type IV-S (#H3884, Merck). After dissociation, the samples were centrifuged, the supernatant was discarded, and the cells were resuspended in 5 mL of 1 RBC lysis buffer (#420301, Biolegend) and cultured at room temperature for 10 minutes. The sample was filtered through a 70 μm cell strainer and the cell number was counted. To block the Fc receptor, this sample was cultured with anti-mouse CD16 / CD32 (BioLegend, #101330) at room temperature for 10 minutes. After Fc blockade, the antibody mixture was added to the sample for surface marker staining and cultured at 4°C for 1 hour. reg For macrophages, samples were fixed and permeabilized using a fixation / permeabilization buffer set (BioLegend, #424401), and an Ab mixture was added for marker staining. The cells were then cultured at 4°C for 1 hour. Stained cells were analyzed using CANTO II (BD Bioscience) and BDFACS Diva software v.8.0.2 (BD Bioscience), and data analysis was performed using FlowJo software (v.10, TreeStar). The staining markers used for each cell type are as follows:

[0155] T cells: CD45 (#103116, Biolegend), CD3 (#100218, Biolegend), CD4 (#100422, Biolegend), CD8a (#563068, BD Bioscience), CD44 (#563970, BD Bioscience), CD62L (#104412, Biolegend), and PD-1 (#109104, Biolegend);

[0156] NK cells: CD45 (#103116, Biolegend), CD3 (#100218, Biolegend), NK1.1 (#550627, BD Bioscience), and NKG2A (#11-5896-82, eBioscience);

[0157] DC: CD11c (#562782, BD Bioscience), CD11b (#566416, BD Bioscience), and B220 (#103224, Biolegend);

[0158] T reg :CD45(#103116, Biolegend), CD3(#100218, Biolegend), CD4(#100422, Biolegend), CD8a(#563068, BD Bioscience), CD25(#101910, Biolegend), Foxp3(#562996, BD Bioscience), and IFN-g (#163503, Biolegend);

[0159] Macrophages: CD45 (#103116, Biolegend), CD11b (#566416, BD Bioscience), F4 / 80 (#565411, Biolegend), and TNF-α (#506308, Biolegend).

[0160] The experimental results, as shown in Figure 5, showed that compared to tumor tissue from mice administered α-PD-1 alone, tumor tissue from mice administered P. merdae Immunoactis and α-PD-1 showed increased expression of Ifn-γ and Cxcl9, but decreased expression of Ccl22. Ifn-γ is an important cytokine in innate and acquired immunity, and Cxcl9 is a chemokine that plays a role in promoting the differentiation and proliferation of leukocytes. Furthermore, the suppression of Ccl22 expression leads to T reg The accumulation of this substance may be suppressed, resulting in an antitumor effect.

[0161] Furthermore, as shown in Figure 6, compared to tumor tissue from mice administered α-PD-1 alone, tumor tissue from mice administered P. merdae Immunoactis and α-PD-1 showed a higher CD4 level. + T cells, CD4 + CD44 + CD62L - T cells (CD4 + Effector T cells, TNF-α + We were able to confirm that the cell population increased.

[0162] Furthermore, as shown in Figure 7, compared to tumor tissue from mice administered α-PD-1 alone, tumor tissue from mice administered P. merdae Immunoactis and α-PD-1 showed a higher CD8 level. + T cells, CD8 + CD44 + CD62L - T cells, PD-1 + An increase in the population of CD8+ T cells was confirmed.

[0163] From these results, it was found that when α-PD-1 and P. merdae Immunoactis are administered in combination, immune activity is improved and the activity of penetrating the tumor environment is enhanced compared to when PD-1 is administered alone.

[0164] (blank)

[0165] Example 6. Analysis of cytotoxicity of P. merdae Immunoactis

[0166] To analyze cytotoxicity, OT-1 T cells (ovalbumin-specific CD8 + T cells were used. Briefly, the spleen was isolated from OT-1 mice (#003831, C57BL / 6-Tg[TcraTcrb]1100Mjb / J, The Jackson Laboratory) and pulverized with 3 mL PBS in a 70 μm cell strainer (#352350, Falcon). The cells were resuspended in 10 mL of 1 RBC lysis buffer (#420301, BioLegend) and cultured at room temperature for 10 minutes. OT-1 T cells were CD8 + T cells were isolated using the T Cell Isolation Kit (#130-096-495, Miltenyi Biotec). The isolated OT-1 T cells were resuspended in T cell medium. 60 mm untreated plates (#32096, SPL) were pre-coated with 5 mg of anti-CD3e antibody (#100340, BioLegend) and 0.5 mg of anti-CD28 antibody (#102116, BioLegend), and cultured overnight at 4°C. In addition, 1-2 × 10⁶ cells were cultured in 60 mm plates pre-coated with OT-1 T cells along with 5% bacterial culture supernatant. 6 Cells were distributed in cell / well units and cultured at 37°C under a 5% CO2 atmosphere for 48 hours. After 48 hours of culture, OT-1 T cells were co-cultured with Ova-treated MC38 colon cancer cells in T cell medium in a 5:1 ratio for 6 hours. Next, the cells were harvested and washed with FACS buffer. Finally, the cells were stained using the Annexin V-PI staining kit (#ALX-850-020-K101, Enzo Life Science) and analyzed by flow cytometry. Prior to co-culture with OT-1 T cells, MC38 colon cancer cells were treated with ovalbumin (323-339) (#O1641, Merck) and cultured overnight at 37°C under a 5% CO2 atmosphere.

[0167] As a result, as shown in Figure 8, it was confirmed that ovalbumin-pulsed T cells treated with P. merdae Immunoactis culture supernatant more effectively killed MC38 colon cancer cells. Based on these results, it was concluded that the P. merdae Immunoactis strain can increase the cancer cell killing ability of T cells.

[0168] (blank)

[0169] The above description of the present invention is illustrative, and a person with ordinary skill in the art will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting.

[0170] (blank) [Industrial applicability]

[0171] The novel Prevotella merde-immunoactis strain of the present invention (deposit number: KCTC 14922BP) increases T cell proliferation, increases T cell IFN-γ secretion, enhances T cell cancer cell killing ability, increases the expression or activity level of IFN-γ or Cxcl9 and decreases the expression or activity level of Ccl22 in the tumor microenvironment, and enhances the anticancer effect of immune checkpoint inhibitors. Therefore, the novel Prevotella merde-immunoactis strain or its culture medium can be usefully used for immune enhancement and enhancing the anticancer effect of immune checkpoint inhibitors, and has industrial applicability.

[0172] (blank)

[0173] <Accession Number>

[0174] Depository name: Korea Institute of Biotechnology

[0175] Accession number: KCTC 14922BP

[0176] Date of acceptance: 20220324

[0177] [Table 2]

Claims

1. A strain of Prevotella merdae Immunoactis deposited under deposit number KCTC 14922BP.

2. The strain according to claim 1, characterized in that the strain contains a DNA sequence encoding the 16S rRNA sequence of Sequence ID No.

1.

3. The strain according to claim 1, characterized in that the strain enhances immunity or increases anti-cancer effects.

4. An immunoenhancing composition comprising the Prevotella merde immunoactis strain or its culture solution as described in claim 1 as an active ingredient.

5. A pharmaceutical composition for enhancing anticancer effects, comprising the Prevotella merde immunoactis strain or its culture solution as described in claim 1, as an active ingredient.

6. The pharmaceutical composition for enhancing anticancer effects according to claim 5, characterized in that the aforementioned pharmaceutical composition for enhancing anticancer effects is used in combination with an anticancer agent.

7. A food composition for enhancing anticancer effects, comprising the Prevotella merde immunoactis strain or its culture solution as described in claim 1, as an active ingredient.

8. A pharmaceutical composition for the prevention or treatment of cancer, comprising the Prevotella merde immunoactis strain or its culture medium as described in claim 1, and an immune checkpoint inhibitor as an active ingredient.

9. The pharmaceutical composition according to claim 8, characterized in that the immune checkpoint inhibitor is a PD-L1 inhibitor or a PD-1 inhibitor.

10. The pharmaceutical composition according to claim 9, characterized in that the PD-L1 inhibitor is at least one selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, AUNP12, CA-170, and BMS-986189.

11. The pharmaceutical composition according to claim 9, characterized in that the PD-1 inhibitor is at least one selected from the group consisting of nivolumab, pembrolizumab, cemiprimab, spartalizumab, camrelizumab, sintilimab, tislerizumab, tripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

12. The pharmaceutical composition according to claim 8, characterized in that the Prevotella merde immunoactis strain or its culture medium and the immune checkpoint inhibitor are administered simultaneously or sequentially.

13. The pharmaceutical composition according to claim 8, wherein the Prevotella merde immunoactis strain or its culture solution satisfies one or more of the following characteristics. (a) CD4 ＋ T cells and CD8 ＋ It increases the proliferation of T cells. (b) Increase the secretion of interferon-γ (IFN-γ) from T cells. (c) Increase the expression or activity level of Inf-γ or Cxcl9. (b) Reduce the expression or activity level of Ccl22.

14. The pharmaceutical composition according to claim 8, characterized in that the cancer is at least one selected from the group consisting of colorectal cancer, breast cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, hematological cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, trumpet duct cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, hydroureteral cancer, renal cell carcinoma, renal pelvic cancer, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

15. An immunoenhancing method comprising the step of administering a composition containing the Prevotella merde immunoactis strain or culture medium described in claim 1 to an individual in need thereof.

16. An immunoenhancing application of a composition comprising the Prevotella merde immunoactis strain or its culture solution as described in claim 1.

17. Use of the Prevotella merde immunoactis strain or culture medium described in claim 1 for the production of an immunostimulant.

18. A method for enhancing the anticancer effect of an anticancer agent, comprising the step of administering a composition containing the Prevotella merde immunoactis strain or its culture solution as described in claim 1 to an individual in need thereof.

19. The use of a composition containing the Prevotella merde immunoactis strain or its culture solution as described in claim 1 for enhancing the anticancer effect of an anticancer agent.

20. Use of the Prevotella merde immunoactis strain or culture medium described in claim 1 for the production of an anticancer effect enhancer for anticancer drugs.

21. A method for preventing or treating cancer, comprising the step of administering a composition comprising the strain of Prevotella merde immunoactis described in claim 1 or its culture medium, and an immune checkpoint inhibitor, to an individual in need.

22. A composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, for use in the prevention or treatment of cancer.

23. Use of a composition comprising the Prevotella merde immunoactis strain or its culture medium, and an immune checkpoint inhibitor, as described in claim 1, for the production of a cancer treatment agent.

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