Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition and application thereof. In a first aspect of the present application, a pharmaceutical composition is provided, the pharmaceutical composition comprising a microbial-derived ingredient. The pharmaceutical composition can be used for preventing and treating various different tumors or cancers such as solid tumors, soft tissue tumors, hematopoietic tumors, glandular tumors and metastatic tumors, and plays a good role in regulating an immune system or stimulating the immune system and regulating intestinal tracts.

Description

Technical Field

[0001] The present application relates to the technical field of microbiology, and in particular to a pharmaceutical composition and its application. Background Art

[0002] The intestine is the largest microecological environment in the human body. It participates in many important physiological processes such as nutrient absorption, energy metabolism, tissue and organ development, immune defense, and endocrine regulation, and is closely related to human health. Intestinal microecology has made great progress in the past decade, and human understanding of it has continued to deepen. There are a large number of symbiotic microorganisms in the human intestine, and the total amount of genetic information they carry is 50 to 100 times that of the human genome, namely the "gut microbiome", also known as the "second genome" of humans. The intestinal microbiome is the largest and most direct external environment of the human body, and plays an indispensable role in maintaining human health.

[0003] In recent years, with the rapid development of molecular biology, genomics, bioinformatics analysis technology, high-throughput sequencing technology and microbial culture technology, the impact and role of intestinal flora on intestinal and extra-intestinal diseases have become increasingly clear. The intestinal flora is more like an organ with metabolic, immune and endocrine functions, which can affect the human digestive system, circulatory system and nervous system, and is closely related to the occurrence of various chronic diseases (diabetes, hypertension, cardiovascular disease and brain disease, etc.) and tumors in humans. Studying the relationship between intestinal flora and human health and disease is not only an important scientific research, but also has important significance and value in clinical diagnosis, treatment, and even transformation.

[0004] PD-1 has good targeting function, and anti-PD-1 antibodies are commonly used drugs for treating tumors in clinical practice. However, anti-PD-1 antibodies may cause intolerance and non-response during tumor treatment. One of the main reasons is that the tumor microenvironment changes, which in turn affects the treatment effect. Therefore, it is necessary to provide a solution that can improve this situation.

[0005] Membrane vesicles are functional vesicles released by bacteria to the outside world. Their structure is similar to that of liposomes, and their diameter is usually 20 to 400 nm. In addition to the phospholipid bilayer, they also include the bacterial outer membrane and various proteins, peptidoglycan, lipopolysaccharide, DNA, RNA, etc. The release of membrane vesicles is an important secretion mechanism of bacteria. The released membrane vesicles play multiple roles in physiological activities such as bacterial growth, survival, information exchange, and virulence factor transmission. However, the membrane vesicles of intestinal bacteria need to be further understood and developed. Summary of the invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a pharmaceutical composition.

[0007] In a first aspect of the present application, a pharmaceutical composition is provided, which comprises a microbial-derived component.

[0008] In some embodiments, the microbial-derived component comprises membrane vesicles (MVs) isolated from a bacterial strain.

[0009] In some of these embodiments, the membrane vesicle comprises at least one of a lipid, a protein, and a nucleic acid fragment.

[0010] In some embodiments, the nucleic acid fragments include DNA fragments.

[0011] In some embodiments, the microbial-derived component comprises at least one of (1) to (5): (1) a bacterial strain having the function of stimulating the immune system or immunomodulating, (2) a culture of a bacterial strain having the function of stimulating the immune system or immunomodulating, (3) a fermentation supernatant of a bacterial strain having the function of stimulating the immune system or immunomodulating, (4) a metabolite of a bacterial strain having the function of stimulating the immune system or immunomodulating, and (5) a short-chain fatty acid of a bacterial strain having the function of stimulating the immune system or immunomodulating.

[0012] In some embodiments, the microbial-derived components include membrane vesicles (MVs) isolated from bacterial strains, and also include at least one of (1) to (5): (1) bacterial strains with immune system stimulating or immunomodulating functions, (2) cultures of bacterial strains with immune system stimulating or immunomodulating functions, (3) fermentation supernatants of bacterial strains with immune system stimulating or immunomodulating functions, (4) metabolites of bacterial strains with immune system stimulating or immunomodulating functions, and (5) short-chain fatty acids of bacterial strains with immune system stimulating or immunomodulating functions.

[0013] In some embodiments, the microbial-derived component includes at least 50wt%, 60wt%, 70wt%, 80wt%, 85wt%, 90wt%, 95wt%, 96wt%, 97wt%, 98wt%, or 99wt% of the membrane vesicles.

[0014] In some embodiments, the microbial-derived component includes at least 50 v / v%, 60 v / v%, 70 v / v%, 80 v / v%, 85 v / v%, 90 v / v%, 95 v / v%, 96 v / v%, 97 v / v%, 98 v / v%, or 99 v / v% of the membrane vesicles.

[0015] In some embodiments, the microbial-derived component includes at least 50wt%, 60wt%, 70wt%, 80wt%, 85wt%, 90wt%, 95wt%, 96wt%, 97wt%, 98wt%, 99wt%, and at least 50v / v%, 60v / v%, 70v / v%, 80v / v%, 85v / v%, 90v / v%, 95v / v%, 96v / v%, 97v / v%, 98v / v%, 99v / v% of the membrane vesicles.

[0016] In some embodiments, the membrane vesicles are derived from a microbial strain in the intestine of a mammal.

[0017] In some embodiments, the membrane vesicles are derived from genetically engineered microbial strains in the intestine of a mammal.

[0018] In some embodiments, the bacterial strain having the function of stimulating the immune system or immunomodulating is any one of the following A1) to A6):

[0019] A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1;

[0020] A2) bacteria with a deposit number of GDMCC No: 61121;

[0021] A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2);

[0022] A4) a bacterium whose genome has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% with the genome of any of the bacteria described in A1) to A3); and / or a bacterium whose genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% with the genome of any of the bacteria described in A1) to A3);

[0023] A5) Enterococcus bacteria;

[0024] A6) at least one bacterium selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ass, Enterococcus avium, Enterococcus canis, Enterococcus fusae, Enterococcus caecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus durgidis, Enterococcus yellow, Enterococcus gallus quail, Enterococcus light yellow, Enterococcus hemoperoxidis, Enterococcus spp., Enterococcus odoriferus, Enterococcus moravianus, Enterococcus monteronis, Enterococcus luminescentis, Enterococcus hoopoeus, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypolus, Enterococcus ratus, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharolyticus, Enterococcus marineus, and Enterococcus lactis.

[0025] In some embodiments, the membrane vesicles are isolated from any bacterial strain in A1) to A6) above.

[0026] In some embodiments, the mass percentage and / or volume percentage of the microbial-derived component in the pharmaceutical composition is 1-99%, 2-90%, 5-80%, 10-70%, 20-60%, or at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0027] In some embodiments, the pharmaceutical composition further comprises at least one of an excipient, a diluent, and a carrier.

[0028] In some embodiments, the pharmaceutical composition further comprises an excipient.

[0029] In some embodiments, the auxiliary material further includes at least one of a lubricant, a wetting agent, a binder, an emulsifier, a suspension stabilizer, a solubilizer, a preservative, a sweetener, and a flavoring.

[0030] In some embodiments, the pharmaceutical composition is a solid preparation or a liquid preparation.

[0031] In some embodiments, the dosage form of the pharmaceutical composition is any one of powder, granules, powder, tablet, capsule, gel, suspension, drops, pills, injection, suppository, aerosol, oral liquid, ointment, emulsion, and irrigation.

[0032] In some embodiments, the pharmaceutical composition further includes a product instruction sheet.

[0033] In some embodiments, the pharmaceutical composition further comprises a combined drug.

[0034] In some embodiments, the combination drug includes an anti-tumor drug.

[0035] In some embodiments, the anti-tumor drug includes at least one of a chemotherapeutic drug, a photosensitizer, a photothermal agent, and an immunotherapy drug.

[0036] In some embodiments, the chemotherapy drug includes at least one of paclitaxel, camptothecin, 5-fluorouracil, cisplatin, doxorubicin, mitomycin, and epirubicin.

[0037] In some embodiments, the photosensitizer includes at least one of boron dipyrrole, dihydrochlorin, and red bengal.

[0038] In some embodiments, the photothermal agent includes at least one of noble metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, and semiconductor nanomaterials.

[0039] In some embodiments, the immunotherapy drug includes at least one of an immune cell therapy drug and an immune checkpoint inhibitor.

[0040] In some embodiments, the immune cell therapy drug includes at least one of a T cell therapy drug, a tumor infiltrating lymphocyte therapy drug, and a NK cell therapy drug.

[0041] In some embodiments, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0042] In a second aspect of the present application, there is provided a use of the aforementioned pharmaceutical composition in the preparation of a drug having at least one of the following uses B1) to B4):

[0043] B1) Prevention or treatment of tumors or cancer;

[0044] B2) Modulate the immune system or stimulate the immune system;

[0045] B3) Regulate the intestines;

[0046] B4) Preventing and / or treating diseases mediated by the STING and / or NOD2 pathway.

[0047] In some embodiments, the tumor or cancer in B1) includes at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor.

[0048] In some embodiments, the solid tumor includes at least one of melanoma, colorectal cancer, liver cancer, lung cancer, fibrosarcoma, kidney cancer, head and neck cancer, upper gastrointestinal cancer, lower gastrointestinal cancer, hepatobiliary system cancer, neuroendocrine tumors, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer.

[0049] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0050] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of trunk and limbs, soft tissue sarcoma of abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of soft tissue sarcoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, and leukemia.

[0051] In some embodiments, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses.

[0052] In some embodiments, the bacteria or viruses include at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum.

[0053] In some embodiments, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) inhibiting the growth of tumor volume; (b) inhibiting the increase of tumor weight; (c) inhibiting the growth of tumor cells; (d) improving the response rate of tumor treatment; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways.

[0054] In some embodiments, the regulating or stimulating the immune system in B2) includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0055] In some embodiments, regulating the intestine in B3) includes at least one of regulating intestinal function and regulating intestinal flora.

[0056] In some embodiments, the regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0057] In some embodiments, the regulation of the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0058] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0059] In some embodiments, the disease in D1) is an intestinal disease.

[0060] In some embodiments, the drug of D2) is an anti-tumor drug.

[0061] In some embodiments, the anti-tumor drug of D2) includes an immunotherapy drug.

[0062] In some embodiments, the immunotherapy drug of D2) includes an immune checkpoint inhibitor.

[0063] In some embodiments, the immune checkpoint inhibitor of D2) includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0064] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammation, an allergy, and an autoimmune disease.

[0065] The third aspect of the present application provides the use of the aforementioned pharmaceutical composition in at least one of the following B1) to B4):

[0066] B1) Prevention or treatment of tumors or cancer;

[0067] B2) Modulate the immune system or stimulate the immune system;

[0068] B3) Regulate the intestines;

[0069] B4) Preventing and / or treating diseases mediated by the STING and / or NOD2 pathway.

[0070] In some embodiments, the tumor or cancer in B1) includes at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor.

[0071] In some embodiments, the solid tumor comprises at least one of head and neck cancer, upper gastrointestinal cancer, lower gastrointestinal cancer, hepatobiliary system cancer, neuroendocrine tumor, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer.

[0072] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0073] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of trunk and limbs, soft tissue sarcoma of abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of soft tissue sarcoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, and leukemia.

[0074] In some embodiments, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses.

[0075] In some embodiments, the bacteria or viruses include at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum.

[0076] In some embodiments, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) inhibiting the growth of tumor volume; (b) inhibiting the increase of tumor weight; (c) inhibiting the growth of tumor cells; (d) improving the response rate of tumor treatment; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways.

[0077] In some embodiments, the regulating or stimulating the immune system in B2) includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0078] In some embodiments, regulating the intestine in B3) includes at least one of regulating intestinal function and regulating intestinal flora.

[0079] In some embodiments, the regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0080] In some embodiments, the regulation of the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0081] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0082] In some embodiments, the disease in D1) is an intestinal disease.

[0083] In some embodiments, the drug of D2) is an anti-tumor drug.

[0084] In some embodiments, the anti-tumor drug of D2) includes an immunotherapy drug.

[0085] In some embodiments, the immunotherapy drug of D2) includes an immune checkpoint inhibitor.

[0086] In some embodiments, the immune checkpoint inhibitor of D2) includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0087] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammation, an allergy, and an autoimmune disease.

[0088] The fourth aspect of the present application provides a method for preventing or treating tumors or cancer, and / or regulating or activating the subject's immune system, and / or regulating the intestine, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathway, the method comprising administering an effective dose of the aforementioned composition to the subject.

[0089] In some embodiments, the tumor or cancer comprises at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor.

[0090] In some embodiments, the solid tumor comprises at least one of head and neck cancer, upper gastrointestinal cancer, lower gastrointestinal cancer, hepatobiliary system cancer, neuroendocrine tumor, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer.

[0091] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0092] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of trunk and limbs, soft tissue sarcoma of abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of soft tissue sarcoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, and leukemia.

[0093] In some embodiments, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses.

[0094] In some embodiments, the bacteria or viruses include at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum.

[0095] In some embodiments, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) inhibiting the growth of tumor volume; (b) inhibiting the increase of tumor weight; (c) inhibiting the growth of tumor cells; (d) improving the response rate of tumor treatment; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways.

[0096] In some embodiments, the regulating or stimulating the immune system includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0097] In some embodiments, the intestinal regulation includes at least one of regulating intestinal function and regulating intestinal flora.

[0098] In some embodiments, the regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0099] In some embodiments, the regulation of the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0100] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0101] In some embodiments, the disease in D1) is an intestinal disease.

[0102] In some embodiments, the drug of D2) is an anti-tumor drug.

[0103] In some embodiments, the anti-tumor drug of D2) includes an immunotherapy drug.

[0104] In some embodiments, the immunotherapy drug of D2) includes an immune checkpoint inhibitor.

[0105] In some embodiments, the immune checkpoint inhibitor of D2) includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0106] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammation, an allergy, and an autoimmune disease.

[0107] In some embodiments of the present application, the composition is administered at least once a day.

[0108] In some embodiments of the present application, when the administration frequency is twice or more per day, the same dose is administered each time.

[0109] In some embodiments of the present application, when the administration frequency is twice or more per day, each administration is at a different dose.

[0110] In some embodiments of the present application, the route of administration includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, oral administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

[0111] In a fifth aspect of the present application, there is provided the use of C1 and C2 in the preparation of an anti-tumor drug, wherein the anti-tumor drug includes the prevention or treatment of at least one of renal cancer, liver cancer, and fibrosarcoma;

[0112] Among them, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors.

[0113] In some embodiments of the present application, the Enterococcus bacteria include at least one of Enterococcus faecalis, Enterococcus faecium, Enterococcus donkey, Enterococcus avium, Enterococcus canis, Enterococcus lead yellow, Enterococcus cecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus quail and chicken, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestine, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus ratus, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0114] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0115] A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1;

[0116] A2) bacteria with a deposit number of GDMCC No: 61121;

[0117] A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2);

[0118] A4) has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% with the genome of the bacterium described in any one of A1) to A3); and / or, the alignment score of the genome with the genome of any one of A1) to A3) is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the bacteria described in any one of A1) to A3).

[0119] In some embodiments of the present application, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0120] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0121] In a sixth aspect of the present application, there is provided the use of C1 and C2 in preventing or treating at least one of renal cancer, liver cancer, and fibrosarcoma;

[0122] Among them, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors.

[0123] In some embodiments of the present application, the Enterococcus bacteria include at least one of Enterococcus faecalis, Enterococcus faecium, Enterococcus donkey, Enterococcus avium, Enterococcus canis, Enterococcus lead yellow, Enterococcus cecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus quail and chicken, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestine, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus ratus, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0124] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0125] A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1;

[0126] A2) bacteria with a deposit number of GDMCC No: 61121;

[0127] A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2);

[0128] A4) has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% with the genome of the bacterium described in any one of A1) to A3); and / or, the alignment score of the genome with the genome of any one of A1) to A3) is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the bacteria described in any one of A1) to A3).

[0129] In some embodiments of the present application, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0130] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0131] In a seventh aspect of the present application, a method for preventing or treating at least one of renal cancer, liver cancer, and fibrosarcoma is provided, comprising administering an effective dose of C1 and C2 to a subject;

[0132] Among them, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors.

[0133] In some embodiments of the present application, the Enterococcus bacteria include at least one of Enterococcus faecalis, Enterococcus faecium, Enterococcus donkey, Enterococcus avium, Enterococcus canis, Enterococcus lead yellow, Enterococcus cecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus quail and chicken, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestine, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus ratus, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0134] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0135] A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1;

[0136] A2) bacteria with a deposit number of GDMCC No: 61121;

[0137] A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2);

[0138] A4) has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% with the genome of the bacterium described in any one of A1) to A3); and / or, the alignment score of the genome with the genome of any one of A1) to A3) is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the bacteria described in any one of A1) to A3).

[0139] In some embodiments of the present application, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0140] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0141] In some embodiments of the present application, the C1 and the C2 are administered at least once a day.

[0142] In some embodiments of the present application, the C1 and the C2 are administered simultaneously, separately or sequentially.

[0143] In some embodiments of the present application, when the administration frequency is twice or more per day, the same dose is administered each time.

[0144] In some embodiments of the present application, when the administration frequency is twice or more per day, each administration is at a different dose.

[0145] In some embodiments of the present application, the route of administration includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, oral administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

[0146] In the embodiments of the present application, the membrane vesicles are secretions of intestinal bacteria, which contain a large amount of intestinal bacterial DNA and special proteins and other substances. Therefore, these specific components can activate the immune system of the whole body, thereby achieving the prevention and treatment of various tumors or cancers including solid tumors, soft tissue tumors, hematopoietic tumors, glandular tumors, metastatic tumors, etc., and play a good role in regulating or stimulating the immune system, and can even achieve better results than directly administering intestinal bacteria. In addition, regarding the activation of the STING and / or NOD2 pathways, the strain components including the supernatant of the strain and the membrane vesicles of the strain can play an activating role. Therefore, when preparing it into a drug, a variety of preparation forms can be adaptively provided according to different needs, including strains, strain metabolites, strain supernatants, and membrane vesicles secreted by strains. These components derived from strains can play a good anti-tumor effect.

[0147] In addition, for some clinically refractory tumors such as renal cancer, fibrosarcoma, and liver cancer, it is difficult to predict whether the drug will have an effective anti-tumor effect. In the embodiments of the present application, the combination of the relevant components of Enterococcus bacteria and immune checkpoint inhibitors can achieve good therapeutic effects. These tumor lesions originate from outside the gastrointestinal tract, and Enterococcus bacteria from the intestine, such as MNC-168, can still be used in combination with immune checkpoint inhibitors to achieve synergistic effects, so that the combination has a higher anti-tumor effect. It can be seen that the combination of the two significantly enhances the efficacy of immune checkpoint inhibitors such as anti-PD-1 antibodies, and experimental data show that the tumor disappearance rate is significantly improved through the intervention of the strains of the present application.

[0148] In addition, in the examples of the present application, the supernatant and membrane vesicle experiments of the strain showed that the STING activity was promoted to 3 times relative to the control, which may indicate that there are nucleic acid fragments (such as DNA fragments) that specifically bind to C-Gas, thereby effectively activating the STING and / or NOD2 pathways to exert anti-tumor activity.

[0149] On the other hand, it is noted that in diseases such as cancer, especially colorectal cancer, there will be an imbalance of intestinal flora. Even through immunotherapy such as anti-PD-1 antibodies, it is difficult to achieve a significant improvement. There is still a problem of decreased diversity of intestinal flora. This change in the tumor microenvironment may also be the cause of intolerance and non-response during the anti-PD-1 antibody treatment of tumors, thereby affecting the treatment effect. In this application, the use of the above-mentioned strain active ingredients has restored the intestinal flora to a greater extent, thereby improving this situation.

[0150] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] Figure 1 The experimental results of one embodiment of the present application that lactic acid enterococcus MNC-168 activates STING-dependent anti-tumor immunity through membrane vesicles. Among them, A is the particle size distribution of lactic acid enterococcus MNC-168 membrane vesicles separated by NanoSight technology; B is the morphology of lactic acid enterococcus MNC-168 membrane vesicles observed by transmission electron microscopy (TEM), scale = 100nm; C is the gel electrophoresis evaluation of the DNA components in lactic acid enterococcus MNC-168 vesicles, the marker is DL2000, and the three lanes are 10μL, 20μL and 30μL (vesicle concentration: 4.5×10 6 / μL) of isolated vesicles; D is the activation of STING activity by lactic acid Enterococcus MNC-168 membrane vesicles using THP-1-STING reporter cells, H-151 (10μM) is a specific inhibitor of STING; E and F are RT-PCR detection of IFN-β and TNF-α mRNA expression levels, THP-1 cells were treated with lactic acid Enterococcus MNC-168 vesicles for 24 hours to detect gene expression, H-151 (10μM) is a specific inhibitor of STING. One-Way ANOVA method was used for analysis with Tukey's multiple comparisons test.

[0152] Figure 2 The following is a curve showing the change in tumor volume of each group of mice during the intervention process in one embodiment of the present application. The mean value ± standard deviation of mouse tumor volume (mm 3 ) were used to present the data, and statistical analysis was performed using two-way ANOVA with Dunnett's multiple comparison analysis.

[0153] Figure 3This is a comparison of the tumor volumes of mice in each group at the intervention endpoint in one embodiment of the present application. The mean value ± standard deviation (mm) of the tumor volume at the intervention endpoint (D24) of mice 3 ) were used to present the data, and statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison analysis.

[0154] Figure 4 This is a comparison of the tumor weights of mice in each group at the intervention endpoint in one embodiment of the present application. The data are presented as the mean ± standard deviation (g) of the tumor weight at the intervention endpoint (D24) of the mice, and statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison analysis.

[0155] Figure 5 This is a comparison of the tumor inhibition rate of each group of mice at the intervention endpoint in one embodiment of the present application. The data are presented as the mean ± standard deviation (%) of the mouse tumor inhibition rate, and statistical analysis is performed using one-way ANOVA with Dunnett's multiple comparison analysis. The dotted line represents a 40% tumor inhibition rate, a tumor inhibition rate <40% represents an unresponsive anti-tumor drug efficacy, and a tumor inhibition rate ≥40% represents a responsive anti-tumor drug efficacy.

[0156] Figure 6 The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in each intervention mouse group in one embodiment of the present application. The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in the Control group, PD-1 group, MNC-168 group and MNC-168+PD-1 group. The average value of mouse tumor volume ± standard deviation (mm 3 ) presents data. The upper dotted line represents the average tumor volume of the Control group (2193.2 mm 3 ), the lower dotted line represents the tumor volume at 40% tumor inhibition rate relative to the Control group (1315.92mm 3 ), when the mouse volume>1315.92mm 3 When the volume of the mouse is ≤1315.92mm 3 When the tumor volume of mice is 0mm at the intervention endpoint, the tumor inhibition rate is ≥40%, which is a response to anti-tumor drug efficacy. 3 , then the tumor disappears (Cured), non-response rate = number of non-responding mice / total number of mice in the group × 100%, response rate = number of responding mice / total number of mice in the group × 100%, tumor disappearance rate = number of mice with endpoint tumor disappearance / total number of mice in the group × 100%.

[0157] Figure 7 The following is a curve showing the change in tumor volume of each group of mice during the intervention process in one embodiment of the present application. The mean value ± standard deviation of mouse tumor volume (mm 3 ) were used to present the data, and statistical analysis was performed using two-way ANOVA with Dunnett's multiple comparison analysis.

[0158] Figure 8 This is a comparison of the tumor volumes of mice in each group at the intervention endpoint in one embodiment of the present application. The mean ± standard deviation (mm) of the tumor volume at the intervention endpoint (D19) of mice 3 ) were used to present the data, and statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison analysis.

[0159] Fig. 9 This is a comparison of the tumor inhibition rate of each group of mice at the intervention endpoint in one embodiment of the present application. The data are presented as the mean ± standard deviation (%) of the mouse tumor inhibition rate, and statistical analysis is performed using one-way ANOVA with Dunnett's multiple comparison analysis. The dotted line represents a 40% tumor inhibition rate, a tumor inhibition rate <40% represents an unresponsive anti-tumor drug efficacy, and a tumor inhibition rate ≥40% represents a responsive anti-tumor drug efficacy.

[0160] Fig.10 The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in each intervention mouse group in one embodiment of the present application. The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in the control group, PD-1 group, MNC-168 group and MNC-168+PD-1 combination group. The average value of mouse tumor volume ± standard deviation (mm 3 ) presents data. The upper dotted line represents the average tumor volume of the Control group (2319.43 mm 3 ), the lower dotted line represents the tumor volume at 40% tumor inhibition rate relative to the Control group (1391.658mm 3 ), when the mouse volume>1391.658mm 3 When the volume of the mouse is ≤1391.658mm 3 When the tumor volume of mice is 0mm at the intervention endpoint, the tumor inhibition rate is ≥40%, which is a response to anti-tumor drug efficacy. 3 , then the tumor disappears (Cured), non-response rate = number of non-responding mice / total number of mice in the group × 100%, response rate = number of responding mice / total number of mice in the group × 100%, tumor disappearance rate = number of mice with endpoint tumor disappearance / total number of mice in the group × 100%.

[0161] Fig.11 The following is a curve showing the change in tumor volume of each group of mice during the intervention process in one embodiment of the present application. The mean value ± standard deviation of mouse tumor volume (mm 3 ) were used to present the data, and two-way ANOVA with Tukey's multiple comparison analysis was used for statistical analysis.

[0162] Fig.12 This is a comparison of the tumor volumes of mice in each group at the intervention endpoint in one embodiment of the present application. The mean ± standard deviation (mm) of the tumor volume at the intervention endpoint (D19) of mice 3 ) were used to present the data, and statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison analysis.

[0163] Fig.13 This is a comparison of the tumor weights of mice in each group at the intervention endpoint in one embodiment of the present application. The mean ± standard deviation (g) of the tumor weights at the intervention endpoint of mice was used for statistical analysis using one-way ANOVA with Tukey's multiple comparison analysis.

[0164] Fig.14 This is a comparison of the tumor inhibition rate of each group of mice at the intervention endpoint in one embodiment of the present application. The data are presented as the mean ± standard deviation (%) of the mouse tumor inhibition rate, and statistical analysis is performed using one-way ANOVA with Tukey's multiple comparison analysis. The dotted line represents a 40% tumor inhibition rate, a tumor inhibition rate <40% represents an unresponsive anti-tumor drug efficacy, and a tumor inhibition rate ≥40% represents a responsive anti-tumor drug efficacy.

[0165] Fig.15 The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in each intervention mouse group in one embodiment of the present application. The graph shows the change curve of tumor volume, the rate of anti-tumor drug effect and the rate of tumor disappearance of a single mouse in the control group, PD-1 group, MNC-168 group and MNC-168+PD-1 combination group. The average value of mouse tumor volume ± standard deviation (mm 3 ) presents data. The upper dotted line represents the average tumor volume of the Control group (1858.33 mm 3 ), the lower dotted line represents the tumor volume at 40% tumor inhibition rate relative to the Control group (1114.998mm 3 ), when the mouse volume>1114.998mm 3 When the volume of the mouse is ≤1114.998mm3 When the tumor volume of mice is 0mm at the intervention endpoint, the tumor inhibition rate is ≥40%, which is a response to anti-tumor drug efficacy. 3 , then the tumor disappears (Cured), non-response rate = number of non-responding mice / total number of mice in the group × 100%, response rate = number of responding mice / total number of mice in the group × 100%, tumor disappearance rate = number of mice with endpoint tumor disappearance / total number of mice in the group × 100%.

[0166] Fig.16 The experimental results of the activation of STING-dependent anti-tumor immunity by the supernatant of Enterococcus lactis MNC-168 in one embodiment of the present application. Among them, A is the use of THP-1-STING reporter cells to evaluate the activation of STING activity by the supernatant of Enterococcus lactis MNC-168, and H-151 (10μM) is a specific inhibitor of STING; B and C are RT-PCR detection of the mRNA expression levels of IFN-β and TNF-α, and THP-1 cells are treated with the supernatant of Enterococcus lactis MNC-168 for 24 hours to detect gene expression, and H-151 (10μM) is a specific inhibitor of STING. One-Way ANOVA method and Tukey's multiple comparisons test were used for analysis.

[0167] Fig.17 The experimental results of the activation of NOD2-dependent anti-tumor immunity by the supernatant of Enterococcus lactis MNC-168 in one embodiment of the present application. Among them, A and B are the mRNA expression levels of IL-1β and IL-6 detected by RT-PCR, and the gene expression is detected after THP-1 cells are treated with the supernatant of Enterococcus lactis MNC-168 for 24 hours, and GSK717 (10 μM) is a specific inhibitor of NOD2. One-Way ANOVA method and Tukey's multiple comparisons test were used for analysis.

[0168] Fig.18 The following are the curves of tumor volume changes in each group of mice during the intervention process and the detection results of corresponding cytokines and chemokines in mouse serum in one embodiment of the present application. A presents data as mean ± standard deviation of mouse tumor volume (mm3), using two-way ANOVA. B to E are the ELISA test results of CXCL1, CXCL9, CXCL10 and IFN-γ, respectively.

[0169] Fig.19This is an analysis of the diversity of fecal intestinal flora of each group of mice during the intervention process in one embodiment of the present application. Among them, A is a comparison of the types (left figure) and diversity (right figure) of intestinal flora in each group, and the one-way analysis of variance (One-Way ANOVA) method is used with Tukey's multiple comparisons test for analysis. All comparisons within or between pre and post groups, all comparisons with significant differences are shown in the figure as p values, and intra-group comparisons without marked p values ​​are all without significant differences; B is a PCA analysis of the diversity of intestinal flora in each group.

[0170] Fig. 20 The following are the tumor volume change curves of each group of mice during the intervention process and the tumor photography and weight results at the end of the experiment in Example 10. A is the tumor volume change curve, presenting the data as the mean ± standard deviation of the mouse tumor volume (mm3), and using two-way ANOVA with Tukey multiple comparison analysis for statistical analysis. B is the end of the experiment, the results of tumor photography and weighing comparison, presenting the data as the mean ± standard deviation of the mouse tumor weight at the end of the intervention (g), and using unpaired T-test comparative analysis for statistical analysis.

[0171] Fig.21 The results of flow cytometry analysis of immune cell populations in tumor tissues in Example 10. A is a comparison of the number of DCs in tumor tissues, B is an analysis of the CD206 / CD86 ratio of TAMs expressed in tumor tissues, and C is a comparison of the number of IFN-γ+CD8+T cells in tumor tissues. Data are presented as mean ± standard deviation, and statistical analysis was performed using unpaired T-test comparative analysis.

[0172] In the above figures, significant differences are indicated by *, no significant difference (ns), p value ≥ 0.05; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0173] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0174] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0175] In the description of this application, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, "above", "below", "within", etc. are understood to include the number itself, and "about" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0176] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0177] A first aspect of an embodiment of the present application relates to a pharmaceutical composition, which includes a microbial-derived component, and the microbial-derived component includes a bacterial membrane vesicle.

[0178] In some embodiments, the bacteria corresponding to the membrane vesicles in the above-mentioned pharmaceutical composition include a strain named Enterococcus lactis MNC-168, which is deposited in Guangdong Microbial Culture Collection Center (GDMCC), with a deposit number of GDMCC No: 61121, a deposit date of August 5, 2020, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, reference WO2022156119A1.

[0179] Among them, the term "strain" refers to a member of a bacterial species with genetic characteristics, so that it can be distinguished from closely related members of the same bacterial species. The genetic characteristic can be the total or partial absence of at least one gene, the total or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one non-natural plasmid, the presence of at least one antibiotic resistance box, or a combination thereof. The genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case where a strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic ability (e.g., auxotrophic strains), strains or nutrients / metabolites can be distinguished by selection or counter-selection using antibiotics.

[0180] It is known in the art that bacterial species can be classified and identified by traditional classification methods and molecular biological methods. Traditional classification methods include, for example, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biological methods include ribosomal RNA sequence determination methods, determination methods based on whole genome sequencing, etc.

[0181] Therefore, in some embodiments, the bacteria include, in addition to the above-mentioned strain MNC-168, also include variants based on strain MNC-168, for example, having at least one of the same morphological and / or physiological and biochemical identification characteristics as MNC-168.

[0182] In addition, the sequencing results of 16S rRNA of strain MNC-168 show that the sequence of its 16S rRNA is shown in SEQ ID No. 1 (the specific sequence is SEQ ID No. 1 in WO2022156119A1). 16S rRNA is a ribosomal RNA of prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is common to all bacteria, while the variable region has different degrees of difference between different bacteria. By comparing the gene sequences of bacterial 16S rRNA, according to the sequence difference base and its evolutionary distance, a biological evolutionary tree can be drawn.

[0183] When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, it can be judged that they belong to different species (see, Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequences similarity for species demarcation of prokaryotes. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 64 (Pt 2), 346-351, and Liu, C., Du, M.-X., Abuduaini, R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gut microbiomes with a culturedbiobank. Microbiome, 9(1), page P23; Pablo Yarza, Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNAgene sequences, Microbiology, volume 12, pages P635-P645, 2014).

[0184] When the identity between the 16S rRNA gene sequences of two strains is less than 95%, it can be judged that they belong to different genera (“Agenus definition for Bacteria and Archaea based on genome relatedness and taxonomic affiliation”, RA Barco, bioRxiv (doi: https: / / doi.org / 10.1101 / 392480); Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis, Jethro S. Johnson, Nature Communications 10, Article number: 5029 (2019)).

[0185] To this end, in some embodiments of the present application, the bacteria also include variants of strain MNC-168 based on the above 16S rRNA sequence, and these strain variants have at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity with strain MNC-168 in 16S rRNA sequence, and thus are strains of the same species or different species within the same genus as strain MNC-168. It can also be reasonably inferred that these strains of the same species or different species of the same genus have similar biological activities or uses.

[0186] The "identity" between the sequences of two nucleic acid molecules can be determined using a known computer algorithm, such as at least one of the GCG program packages, BLASTN or FASTA. Other commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

[0187] In addition, with the rapid development of second-generation and third-generation sequencing technologies, bacterial species identification based on whole genome sequencing has become possible, making the results of bacterial species identification more accurate. The average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. In the field of bacterial taxonomy, it is generally believed that the ANI value needs to reach 98.65% or above to be identified as belonging to the same species (Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARYMICROBIOLOGY, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini, R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gutmicrobiomes with a cultured biobank. Microbiome, 9(1), P23; Pablo Yarza, Uniting the classification of cultured and uncultured bacteria and archaea using16SrRNAgene sequences, Microbiology, volume 12, pages P635-P645, 2014).

[0188] When the identity between the 16S rRNA gene sequences of two strains is less than 95%, it can be judged that they belong to different genera (“Agenus definition for Bacteria and Archaea based on genome relatedness and taxonomic affiliation”, RA Barco, bioRxiv (doi: https: / / doi.org / 10.1101 / 392480); Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis, Jethro S. Johnson, Nature Communications 10, Article number: 5029 (2019)).

[0189] Using the above method, a person skilled in the art can determine whether a bacterium belongs to a new strain of the same genus but different species or the same species as strain MNC-168. For example, when the average nucleotide identity ANI value with the strain with the deposit number GDMCC No: 61121 is at least 87%, such as at least 87%, 90%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100%, it can be determined that the bacteria belong to the same genus or the same species.

[0190] At present, there are various mature ANI value calculation tools, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools. Therefore, the ANI value of the isolated strain and MNC-168 can be calculated by any of the above local calculation software or online calculation tools or other tools or methods known in the art.

[0191] To this end, in some embodiments of the present application, the bacteria also include variants of strain MNC-168 based on the above genome, and these strain variants have at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% average nucleotide identity compared to strain MNC-168 in the genome, and are therefore strains of the same species or different species within the same genus as strain MNC-168. It can also be reasonably inferred that these strains of the same species or different species of the same genus have similar biological activities or uses.

[0192] In some embodiments, based on the comparison results of 16S rRNA and ANI, the variant of the strain has at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity with the 16S rRNA of MNC-168, and at the same time, has at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% average nucleotide identity with the genome of MNC-168.

[0193] In some embodiments, the bacteria also include variants of the strain that combine the results of molecular biological identification and morphological and physiological and biochemical identification, for example, the variant of the strain has at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity with the 16S rRNA of MNC-168, and / or, has at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% average nucleotide identity with the genome of strain MNC-168; at the same time, the strain variant has at least one identical morphological or physiological and biochemical identification feature with MNC-168.

[0194] The term "variant" includes any bacteria generated from strain MNC-168, such as bacteria generated by natural mutation or recombination (such as mutation caused by cell proliferation and cell division) or resistance selection, as well as mutation or recombination caused by any other mechanism such as radiation, viruses, transposons or mutagenic chemicals. In addition, variants also include other strains isolated from the natural environment that have the same or similar taxonomic characteristics and properties as strain MNC-168, generally including other strains of the same genus but different species as MNC-168 under the genus Enterococcus, or other strains with different phenotypic variations within the same species.

[0195] In some other embodiments, the bacteria also include other existing species under the genus Enterococcus, such as Enterococcus faecalis, Enterococcus faecium, Enterococcus asini, Enterococcus avium, Enterococcus canis, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus malodoratus. , Moravian Enterococcus (E.moraviensis), Montenegrin Enterococcus (E.mundtii), Bright Yellow Enterococcus (E.pallens), Wood Hoopoe Enterococcus (E.phoeniculicola), Porcine Enterococcus (E.porcinus), False Bird Enterococcus (E.pseudoavium), Raffinosus Enterococcus (E.raffinosus), Mouse Enterococcus (E.ratti), Saccharolytic Enterococcus (E.saccharolyticus), Sulfur Enterococcus (E.sulfurous), Villorum Enterococcus (E.villorum), Davis Enterococcus (E.devriesei), Hermann Enterococcus (E.hermanniensis), Saccharominimus Enterococcus (E.saccharominimus) and Aquimarinus Enterococcus, etc.

[0196] The term "microorganism-derived components" refers to components derived from microorganisms, which may be intact microorganisms, microorganism secretions, microorganism metabolites, cell fragments, etc., such as small molecules (including amino acids, nucleosides, nucleotides, etc.), macromolecules (including polypeptides, carbohydrates, nucleic acids, proteoglycans, lipids, etc.), extracellular vesicles, membrane vesicles, etc. In addition, in some embodiments, microorganism-derived components also include microbial cultures, fermentation supernatants, etc.

[0197] In some embodiments, the microorganism of the microorganism-derived component is, for example, the aforementioned bacteria, such as the aforementioned bacterial strain having immune system stimulating or immunomodulating function.

[0198] In some embodiments, the membrane vesicles contain the above-mentioned small molecules or macromolecules, for example, the membrane vesicles contain at least one of lipids, proteins and nucleic acid fragments, wherein the nucleic acid fragments may be DNA fragments.

[0199] In addition, considering the source of the membrane vesicles, they can also be microbial strains from the intestines of mammals, or microbial strains that have been further genetically engineered.

[0200] In some embodiments, the mass percentage (wt %) of the microbial derived component in the pharmaceutical composition is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In some embodiments, the mass percentage content of the microbial-derived component in the pharmaceutical composition is 1-99%, 2-90%, 5-80%, 10-70%, 20-60%, etc. In some embodiments, the mass percentage content of the microbial-derived component in the pharmaceutical composition is at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%.

[0201] In some embodiments, the volume percentage (v / v%) of the microbial derived component in the pharmaceutical composition is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In some embodiments, the mass percentage content of the microbial-derived component in the pharmaceutical composition is 1-99%, 2-90%, 5-80%, 10-70%, 20-60%, etc. In some embodiments, the mass percentage content of the microbial-derived component in the pharmaceutical composition is at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%.

[0202] In some embodiments, the pharmaceutical composition can be a solid preparation or a liquid preparation. Considering the specific dosage form used, the pharmaceutical composition can be any one of a powder, a granule, a tablet, a capsule, a gel, a suspension, a drop, a pill, an injection, a suppository, an aerosol, an oral solution, an ointment, an emulsion, and an irrigant. In some embodiments, the pharmaceutical composition also includes at least one component in an excipient, a diluent, and a carrier to form the above-mentioned dosage form. Generally, acceptable carriers or diluents are well known to those skilled in the art. For example, lactose, starch, glucose, sucrose, methylcellulose, magnesium stearate, mannitol, sorbitol, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, syrup, and methylcellulose can be selected as a carrier or excipient. At least one of ethanol, glycerol, and water is used as a diluent. In addition, it is understood that the specific carrier, excipient or diluent can be selected according to the expected mode of administration, for example, different carriers, excipients or diluents can be selected for different dosage forms such as powders, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, lavage agents, etc. In addition, the pharmaceutical composition can also include at least one of any suitable lubricant, wetting agent, binder, emulsifier, suspension stabilizer, solubilizer, preservative, sweetener, spices, etc. as carrier, excipient or diluent. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, talcum, mineral oil, etc. Adhesives include starch, gelatin, natural sugars and natural or synthetic gums. Wherein natural sugars are such as glucose, anhydrous lactose, free-flowing lactose, β-lactose or corn sweeteners. Natural or synthetic gums such as gum arabic, tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Preservatives include sodium benzoate, sorbic acid or parabens. By preparing the strain or strain culture into any of the above compositions, the release of the effective active ingredients therein can be achieved in different requirements, such as rapid release, sustained release, etc.

[0203] In some embodiments, the pharmaceutical composition further comprises a product instruction sheet.

[0204] In some embodiments, the pharmaceutical composition further comprises a combined drug. By combining drugs, the efficacy of the drug is improved. In some embodiments, the combined drug comprises an oncology drug. In some embodiments, the oncology drug may include at least one of a chemotherapeutic drug, a photosensitizer, a photothermal agent, and an immunotherapy drug.

[0205] Among them, chemotherapy drugs include alkylating agents (such as cyclophosphamide, ifosfamide, thiotepa, melphalan, carmustine, lomustine, semustine, nimustine, fotemustine, estramustine, nitrogen methyl, nitrogen mustard, nitrosamic acid mustard, chlorambucil, hexamethylmelamine, busulfan, temozolomide), platinums (such as cisplatin, carboplatin, oxaliplatin, suplatin, lobaplatin, nedaplatin, dicycloplatin), podophyllotoxins (such as podophyllotoxin, etoposide, teniposide, etoposide phosphate), camptothecin At least one of alkaloids (such as camptothecin, hydroxycamptothecin, irinotecan, topotecan), taxanes (such as paclitaxel, docetaxel), anthracyclines (such as daunorubicin, doxorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, aclarubicin), fluoropyrimidines (such as gemcitabine, capecitabine, 5-fluorouracil, bifuroxidine, deoxyfluridine, tegafur, carmofur, trifluridine), antibiotics (such as mitomycin, bleomycin, bleomycin), etc.

[0206] The photosensitizer includes at least one of porphyrins (such as dihydrochlorins, phthalocyanines, bacteriochlorins), boron dipyrroles (such as fluoroboron dipyrroles, azafluoroboron dipyrroles), and rose bengal.

[0207] The photothermal agent includes at least one of precious metal nanoparticles (such as nanodots, nanorods, nanowires, nanosheets, etc. of precious metals such as gold, silver, platinum, palladium, etc.), organic polymers (such as polypyrrole, polyaniline, polyethylenedioxythiophene, polystyrene sulfonate, indocyanine green, porphyrin), carbon-based nanomaterials (such as graphene, carbon nanotubes, cellulose carbonization products), magnetic nanomaterials (such as Fe3O4), and semiconductor nanomaterials (such as copper sulfide, molybdenum sulfide, bismuth sulfide, antimony sulfide, gold sulfide, copper selenide, molybdenum selenide, bismuth selenide, antimony selenide, and gold selenide).

[0208] Immunotherapy drugs include at least one of immune cell therapy drugs and immune checkpoint inhibitors. Among them, immune cell therapy drugs include at least one of T cell therapy drugs (such as CAR-T, TCR-T), tumor infiltrating lymphocyte therapy drugs, and NK cell therapy drugs. Immune checkpoint inhibitors include inhibitors targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0209] The term "immune checkpoint inhibitor" is an antagonist that targets the immune checkpoint protein, which can enhance proteins that stimulate immune responses or block proteins that inhibit immune responses, thereby showing anti-cancer effects through immune responses. In some embodiments, the immune checkpoint inhibitor can be a protein or peptide, such as a soluble fusion protein; it can be an antibody or its antigen-binding fragment that binds to the immune checkpoint protein to be inhibited; it can be an inhibitory nucleic acid (such as a siRNA molecule, a shRNA molecule, an antisense RNA) that specifically binds to the mRNA encoding the immune checkpoint protein.

[0210] In some specific embodiments, the inhibitor is an antibody, such as anti-PD-1 antibodies (such as Camrelizumab, Cemiplimab (cemiplimab-rwlc), Dostarlimab, Enlonstobart, Nivolumab, Pembrolizumab, Penpulimab, Prolgolimab, Pucotenlimab, Retifanlimab, Serplulimab, Sintilimab, Tislelizumab, Toripalimab, Zimberelimab), anti-PD-L1 antibodies (such as Adebrelimab, Atezolizumab, Avelumab, Cosibelimab, Durvalumab, Envafolimab, Socazolimab, Sugemalimab, Tagitanlimab), anti-CTLA-4 antibodies (such as Ipilimumab, Tremelimumab), etc.

[0211] In some embodiments, the pharmaceutical composition is administered orally. Oral administration may involve swallowing, whereby the compound enters the gastrointestinal tract, and / or oral, lingual, or sublingual administration, whereby the compound enters the bloodstream directly from the oral cavity. Pharmaceutical dosage forms suitable for oral administration include solid suppositories, solid particulates, semisolids, and liquids (including multiphase or dispersed systems) such as tablets; soft or hard capsules containing multi-particles or nanoparticles, liquids (e.g., aqueous solutions), emulsions, or powders; lozenges (including liquid fillers); chewables, gels; rapidly dispersible dosage forms; ovules; sprays; and buccal / mucosal adhesive patches.

[0212] In some embodiments, the pharmaceutical composition is an enteric formulation, i.e., a gastro-resistant formulation (e.g., resistant to gastric pH) suitable for delivering the pharmaceutical composition to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g., susceptible to degradation under gastric conditions.

[0213] In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric coated dosage form. For example, the formulation can be an enteric tablet or an enteric capsule, etc. The enteric coating can be a conventional enteric coating, such as a conventional coating for tablets, capsules, etc. for oral delivery. The formulation can include a film coating, such as a film layer of an enteric polymer, such as an acid insoluble polymer. In some embodiments, the enteric formulation is enteric in nature, such as gastrointestinal and does not require an enteric coating. In some embodiments, the formulation is an inherently enteric capsule (e.g., (from Capsugel).

[0214] In some embodiments, the preparation is a soft capsule. A soft capsule is a capsule with certain elasticity and softness due to the addition of a softener such as glycerol, sorbitol, maltitol and polyethylene glycol present in the capsule shell. Soft capsules can be produced, for example, on the basis of gelatin or starch. Soft capsules based on gelatin are commercially available from various suppliers. According to the method of administration, for example, oral or rectal administration, soft capsules can have various shapes, and they can be, for example, circular, oval, elliptical or torpedo-shaped. Soft capsules can be produced by conventional processes, for example, by Scherer process, Accogel process or droplet or blow molding process.

[0215] In some embodiments, the formulation is a microencapsulated capsule that protects the composition from degradation by encapsulation until it is delivered to the target location, for example, by chemical or physical stimulation such as pressure, enzymatic activity or physical decomposition and rupture, which may be triggered by a change in pH. Any suitable encapsulation method can be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on a solid support surface, self-aggregation by flocculation or the use of a cross-linking agent, and mechanical containment behind a microporous membrane or microencapsulated capsule.

[0216] In some embodiments, the pharmaceutical combination product is a dosage form suitable for infants, children or adults, a dosage form for gastrointestinal administration or parenteral administration, an oral preparation or an injectable preparation, and the strain in the pharmaceutical combination product is at least partially capable of proliferating in the intestine of the subject, and the strain can be any of live bacteria (such as bacteria, or attenuated bacteria, or freeze-dried bacteria, or irradiated bacteria) and dead bacteria.

[0217] The second aspect of the embodiments of the present application relates to the use of the aforementioned pharmaceutical composition in the preparation of drugs for preventing or treating tumors or cancers, and / or regulating the immune system or stimulating the immune system, and / or regulating the intestines, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathways.

[0218] Wherein, the term "tumor" or "tumor" refers to a kind of abnormal mass or vegetation that grows out of cell division and proliferation exceeding normal levels in the body, and "cancer" generally refers to malignant tumors, which can be used universally in some cases in this application. Tumors or cancers generally include solid tumors, blood tumors, soft tissue tumors, glandular tumors, metastatic tumors, etc. The types of tumors or cancers include head and neck cancer, upper digestive tract cancer, lower digestive tract cancer, hepatobiliary system cancer, neuroendocrine tumors, chest cancer, osteoma, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, endocrine system cancer, and blood tumors include at least one tumor in blood, lymph and bone marrow. Among them, head and neck cancer includes oral cancer (such as lip cancer, tongue cancer, floor of mouth cancer, tonsil cancer, gingival cancer, etc.), salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, sinus cancer, laryngeal cancer, etc. Upper gastrointestinal tract cancer includes esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, etc. Lower gastrointestinal tract cancer includes appendix cancer, colon cancer, rectal cancer, anal canal cancer, etc. Hepatobiliary system cancer includes liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampulla cancer, pancreatic cancer, etc. Neuroendocrine tumors include gastric neuroendocrine tumors, duodenum and ampulla cancers. Neuroendocrine tumors, jejunal-ileal neuroendocrine tumors, appendiceal neuroendocrine tumors, colorectal neuroendocrine tumors, pancreatic neuroendocrine tumors, etc. Chest cancer includes thymic cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, etc.), malignant pleural mesothelioma, etc. Soft tissue sarcomas include head and neck sarcomas, trunk and limb soft tissue sarcomas, abdominal and thoracic organ soft tissue sarcomas, gastrointestinal stromal tumors, retroperitoneal soft tissue sarcomas, etc. Skin cancer includes Merkel cell carcinoma, skin malignant melanoma, etc. Female reproductive system cancer includes vulvar cancer, vaginal cancer, uterine cancer Cervical cancer, uterine cancer, uterine carcinosarcoma, uterine sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, etc. Male reproductive system cancer includes penile cancer, prostate cancer, testicular cancer, etc. Urinary system cancer includes kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethra cancer, etc. Eye cancer includes eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, ocular accessory organ lymphoma, etc. Central nervous system cancer includes brain cancer (such as glioma, meningioma, neuroblastoma, etc.) Endocrine system cancers include differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenocortical cancer, etc., as well as Hodgkin's lymphoma, non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, lymph node marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, etc.), cutaneous lymphoma, plasma cell myeloma, leukemia, etc.

[0219] In addition, tumors or cancers also include tumors or cancers caused by bacteria or viruses, for example, tumors caused by at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum.

[0220] In some embodiments, the aforementioned composition achieves the above-mentioned prevention or treatment of tumors or cancers by at least one of the following (a) to (j): (a) inhibiting tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) improving tumor treatment response rate; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting tumor cell spread or metastasis; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating STING and / or NOD2 pathway activity.

[0221] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0222] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0223] Among them, the intestine is mainly used to digest and absorb protein, fat and some carbohydrates in food, and to decompose cellulose and other substances through the water content and the fermentation of microorganisms to form feces. Therefore, the intestinal function mainly includes the following aspects: (1) movement, such as intestinal peristalsis; (2) secretion, such as intestinal fluid, including hormones, enzymes, etc.; (3) barrier, such as preventing the translocation of bacteria and toxins. In addition, as the largest immune organ in the human body, the intestine also has important immune functions. Therefore, in some embodiments, regulating intestinal function includes regulating at least one of the movement, secretion, barrier and immune functions of the intestine.

[0224] The intestinal flora refers to the microorganisms present in the intestine. Usually, the content of flora in the duodenum is relatively low, while the content of flora in the ileum and colon is relatively high. According to taxonomic characteristics, the intestinal flora mainly includes the Bacteroidetes and Metacobacteria, but also includes Proteus, Actinomycetes, Fusobacterium, Verrucomicrobia and Cyanobacteria; according to the spatial position of the intestinal flora in the intestine, it can also include membrane flora close to the intestinal mucosa and cavity flora swimming in the intestinal cavity; according to the function of the intestinal flora, it can include commensal bacteria, conditional pathogens and pathogens. These intestinal flora together construct the microecological environment of the intestine, so intestinal flora imbalance can also lead to problems in the digestive system, immune system, nervous system and other aspects.

[0225] To this end, in some embodiments, regulating the intestinal flora is to regulate at least one of the types, richness, uniformity, and diversity of the intestinal flora. Among them, the types of intestinal flora refer to the types of intestinal bacteria contained in the intestinal flora, such as the presence or absence, quantity, etc. of one or more intestinal bacteria; the richness of intestinal bacteria refers to the number of intestinal bacteria species, such as the number of intestinal bacteria at the species classification level, the number of intestinal bacteria at the genus classification level, the number of intestinal bacteria at the family classification level, the number of intestinal bacteria at the order classification level, etc.; the uniformity of intestinal bacteria refers to the difference in number between different intestinal bacteria, and high uniformity means that the number difference between specific or unspecific intestinal bacteria is small, and low uniformity means that the number difference between specific or unspecific intestinal bacteria is large; diversity is a comprehensive indicator of richness and uniformity, for example, it can be evaluated by at least one of the Simpson diversity index and the Shannon diversity index.

[0226] Normally, the intestinal flora can maintain a relatively stable distribution of its number, but due to diseases, medication, etc., the intestinal flora may be disturbed. Therefore, in some embodiments, regulating the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs. For example, a decrease in at least one of the richness, uniformity, and diversity, a decrease in the number of one or several symbiotic bacteria, an increase in the number of one or several conditional pathogens or pathogens, etc.

[0227] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0228] In some embodiments, the disease in D1) is an intestinal disease, such as at least one of intestinal cancer (including small intestine cancer, colon cancer, rectal cancer, etc.), intestinal infection, intestinal inflammation, intestinal allergy (such as allergic colitis), etc.

[0229] In some embodiments, the drug D2) is an anti-tumor drug, which can be an anti-colon cancer drug, such as an immunotherapy drug, such as an immune checkpoint inhibitor, specifically including an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA, such as anti-PD-1 antibodies (such as Camrelizumab, Cemiplimab (cemiplimab-rwlc), Dostarlimab, Enlonstobart, Ni Volumab, Pembrolizumab, Penpulimab, Prolgolimab, Pucotenlimab, Retifanlimab, Serplulimab, Sintilimab, Tislelizumab, Toripalimab, Zimberelimab), anti-PD-L1 antibodies (such as Adebrelimab, Atezolizumab, Avelumab, Cosibelimab, Durvalumab, Envafolimab, Socazolimab, Sugemalimab, Tagitanlimab), anti-CTLA-4 antibodies (such as Ipilimumab, Tremelimumab), etc.

[0230] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammatory disease, an allergic disease, and an autoimmune disease.

[0231] The third aspect of the embodiments of the present application relates to the use of the aforementioned composition in preventing or treating tumors or cancers, and / or regulating the immune system or stimulating the immune system, and / or regulating the intestine, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathways.

[0232] In some embodiments, the aforementioned composition achieves the above-mentioned prevention or treatment of tumors or cancers by at least one of the following (a) to (j): (a) inhibiting tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) improving tumor treatment response rate; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting tumor cell spread or metastasis; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating STING and / or NOD2 pathway activity.

[0233] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0234] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0235] In some embodiments, the regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0236] In some embodiments, the regulation of the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0237] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0238] In some embodiments, the disease in D1) is an intestinal disease.

[0239] In some embodiments, the drug of D2) is an anti-tumor drug.

[0240] In some embodiments, the anti-tumor drug of D2) includes an immunotherapy drug.

[0241] In some embodiments, the immunotherapy drug of D2) includes an immune checkpoint inhibitor.

[0242] In some embodiments, the immune checkpoint inhibitor of D2) includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0243] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammatory disease, an allergic disease, and an autoimmune disease.

[0244] The fourth aspect of the embodiments of the present application relates to a method for preventing or treating tumors or cancer, and / or regulating the immune system or stimulating the immune system, and / or regulating the intestine, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathways, comprising administering an effective dose of the aforementioned composition to a subject.

[0245] In some embodiments, the aforementioned composition achieves the above-mentioned prevention or treatment of tumors or cancers by at least one of the following (a) to (j): (a) inhibiting tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) improving tumor treatment response rate; (e) improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting tumor cell spread or metastasis; (g) reducing anti-tumor drug resistance; (h) promoting IFNβ transcriptional activity; (i) regulating or activating the subject's immune system; (j) increasing or activating STING and / or NOD2 pathway activity.

[0246] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune function in the gastrointestinal tract, regulating or stimulating the innate immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract.

[0247] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0248] In some embodiments, the regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0249] In some embodiments, the regulation of the intestinal flora is to regulate at least one change in the type, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0250] In some embodiments, the changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0251] In some embodiments, the disease in D1) is an intestinal disease.

[0252] In some embodiments, the drug of D2) is an anti-tumor drug.

[0253] In some embodiments, the anti-tumor drug of D2) includes an immunotherapy drug.

[0254] In some embodiments, the immunotherapy drug of D2) includes an immune checkpoint inhibitor.

[0255] In some embodiments, the immune checkpoint inhibitor of D2) includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0256] In some embodiments, the disease mediated by the STING and / or NOD2 pathway includes at least one of an infectious disease, a tumor or cancer, an inflammatory disease, an allergic disease, and an autoimmune disease.

[0257] In some embodiments, the composition is administered at least once a day. In some embodiments, when the administration frequency is more than twice a day, the same dose is administered each time. In some embodiments, when the administration frequency is more than twice a day, different doses are administered each time. In some embodiments, the route of administration includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, intragastric administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection. In some embodiments, when the composition includes a microbial-derived component and a combined drug, the two can be administered simultaneously, separately, or sequentially.

[0258] The term "subject", or patient, refers to any mammal. A subject or patient described as "in need" refers to a mammal that needs to be treated (or prevented) for a disease. For example, any one or more animals under the class Mammalia, such as monotremes, marsupials, insectivores, shrews, scaphoids, dermoptera, chiroptera, primates, edentata, phlonotida, lagomorpha, rodentia, carnivora, sirenians, hyraxes, tubulodonta, odd-toed ungulates, even-toed ungulates, cetaceans, and the like are included. Common mammals include rodents (such as mice, rats, hamsters, guinea pigs), lagomorphs (such as rabbits), odd-toed ungulates (such as horses, donkeys), even-toed ungulates (such as sheep, goats, camels, cattle, pigs), primates (such as monkeys, gorillas, chimpanzees, humans), carnivores (such as dogs, cats), and the like. It is understood that the subject may be healthy or may suffer from a disease at any stage of development.

[0259] The present application also relates to the combined use of C1 and C2, including the use of both in the preparation of anti-tumor drugs or for the prevention or treatment of tumors, wherein C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors. Among them, anti-tumor refers to the prevention or treatment of at least one tumor selected from renal cancer, liver cancer, and fibrosarcoma.

[0260] The term "treating" a disease in a subject or "treating" a subject having or suspected of having a disease refers to administering a drug therapy, such as administering one or more pharmaceutical agents, to a subject, thereby reducing or preventing at least one symptom of the disease from worsening. Therefore, in one embodiment, "treating" refers in particular to delaying progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, increasing the efficacy of alternative therapies, or reducing resistance to alternative therapies, or a combination thereof, for a tumor patient.

[0261] In some embodiments, the Enterococcus bacteria can be, for example, any of the aforementioned bacteria, and the immune checkpoint inhibitor can also be any of the aforementioned immune checkpoint inhibitors.

[0262] In some embodiments, the Enterococcus bacteria are live bacteria, attenuated bacteria, killed bacteria, freeze-dried bacteria, or irradiated bacteria.

[0263] In some embodiments, the Enterococcus bacteria is freeze-dried. In some embodiments, the Enterococcus bacteria is spray-dried. In some embodiments, the Enterococcus bacteria is freeze-dried or spray-dried and wherein it is alive. In some embodiments, the Enterococcus bacteria is reconstituted prior to administration. In some cases, reconstitution is performed by using a diluent as described herein.

[0264] The term "membrane vesicle" or "MV" refers to a composition derived from bacteria, which comprises bacterial lipids and bacterial proteins and / or bacterial nucleic acids and / or carbohydrate moieties contained in nanoparticles. These MVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different lipid species. The MVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different protein species. The MVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different nucleic acid species.

[0265] One skilled in the art can prepare membrane vesicles (MVs) from bacterial culture using methods known to one skilled in the art (S. Bin Park et al. PLoS ONE. 6(3):e17629 (2011)).

[0266] In addition, the present application also relates to a method for preventing or treating at least one tumor among renal cancer, liver cancer, and fibrosarcoma, comprising administering an effective dose of C1 and C2 to a subject. In some embodiments, C1 and C2 are administered at least once a day. In some embodiments, C1 and C2 are administered simultaneously, separately, or sequentially. In some embodiments, when the administration frequency is more than twice a day, the same dose is administered each time. In some embodiments, when the administration frequency is more than twice a day, different doses are administered each time. In some embodiments, the route of administration includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, oral gavage, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

[0267] The strain involved in the following examples is Enterococcus lactis MNC-168, which is deposited in Guangdong Microbial Culture Collection Center (GDMCC), with a deposit number of GDMCC No: 61121, deposited on August 5, 2020, and deposited at: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The source, screening method, physiological and biochemical characteristics, 16SrRNA sequence (SEQ ID No. 1), etc. of the strain are all recorded in WO2022156119A1, which is incorporated into this application in a combined manner.

[0268] The above implementation is described below in conjunction with specific examples.

[0269] Example 1: Preparation of Enterococcus lactis membrane vesicles

[0270] 1. Preparation of MNC-168 supernatant

[0271] After thawing the glycerol cryopreservation tube of Enterococcus lactis MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid culture medium, culture it anaerobically, obtain a sufficient number of live bacteria, centrifuge the cultured bacterial solution at 10000g for 10min to precipitate the bacteria, take the supernatant and filter it through a 0.22μm filter membrane, and use the filtered supernatant for subsequent experiments or freeze it at -80°C.

[0272] 2. Membrane Vesicle Isolation

[0273] The obtained MNC-168 supernatant was taken and bacterial membrane vesicles (MVs) were isolated using a bacterial membrane vesicle isolation kit (Runji Bio, #BacMV10-10).

[0274] 3. Identification of Membrane Vesicles

[0275] (1) The isolated bacterial membrane vesicle samples were diluted 200 times with PBS, and the size and distribution of membrane vesicles were detected using Nanosight NS300.

[0276] (2) 5 μL of the isolated bacterial membrane vesicle sample was dropped onto a copper grid and incubated for 1 min. The grid was then stained with 1% phosphotungstic acid for 2 min and quickly washed with 1 drop of pure water. The grid was washed twice and dried at room temperature for 10 min. The bacterial extracellular vesicles were observed using a transmission electron microscope (80 kV voltage).

[0277] (3) After adding DNA loading buffer to the isolated bacterial membrane vesicle sample, the reaction was carried out at 95°C for 5 min, and the DNA size was detected by 1% agarose gel electrophoresis.

[0278] The results are as follows Figure 1 As shown in A to C, the isolated membrane vesicles are round, with a diameter usually around 100 nm; at the same time, the electrophoresis results show that the membrane vesicles carry a large amount of DNA from bacteria, with the molecular weight mainly concentrated below 100 bp.

[0279] Example 2: Lactococcus lactis membrane vesicles activate STING pathway-dependent anti-tumor immunity

[0280] STING repoter cell evaluation: The evaluation was performed using THP-1 cells (a cell line built by Muen) that had been constructed to carry the IFNβ gene promoter reporter gene. The steps are as follows:

[0281] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1×10 cells per well. 5There were three groups, namely, the control group, the MNC-168MV group, and the MNC-168MV+H-151 group. The control group was a blank control, and the MNC-168MV group was a group in which 5uL membrane vesicles (concentration: 4.5×10 5 / μL), MNC-168MV+H-151 is 5uL membrane vesicles (concentration: 4.5×10 5 / μL) and H-151 (final concentration of 10μM), after continued culture for 24h, the cells were centrifuged at 300g for 5min, the culture supernatant was removed, 50μL 1×luciferase detection reagent was added, reacted for 1min, and the luminescence chemiluminescence value was detected by microplate reader. The relative fluorescence value (Relative Luminescence) was normalized to the control group to evaluate the effect of lactic acid Enterococcus MNC-168MVs on IFNβ transcription activity.

[0282] RT-PCR detection: THP-1 cells refer to the experimental steps of the above STING reporter cells, and finally collect the cells after different treatments, extract total RNA, reverse transcribe, and detect the expression of the target gene by qPCR.

[0283] The results are as follows Figure 1 D to F, the results showed that the vesicles of lactic acid Enterococcus MNC-168 can significantly promote the activity of STING, which can be increased by 3 times. At the same time, this activation can be inhibited by the STING specific inhibitor H-151. Furthermore, the detection results of STING downstream target genes IFN-β and TNF-α showed that lactic acid Enterococcus MNC-168 vesicles can also significantly activate the expression of the two genes, up-regulated by 5 times and 4 times, respectively, and this activation can also be inhibited by the STING specific inhibitor H-151.

[0284] These results indicate that lactic acid Enterococcus MNC-168 can induce STING pathway-dependent activation through membrane vesicles, thereby upregulating the expression of interferon and tumor necrosis factor that inhibit tumor cell proliferation, metastasis and angiogenesis, promote tumor cell apoptosis, and activate anti-tumor immune responses, thereby promoting anti-tumor immune activity.

[0285] Example 3: Combination of MNC-168 and anti-PD-1 antibody for the treatment of renal cancer

[0286] The therapeutic effect of Enterococcus lactis MNC-168 combined with anti-PD-1 antibody was verified in the mouse syngeneic tumor Renca (renal cancer) model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotechnology.

[0287] Test strains: After thawing the glycerol cryopreserved tube of lactic acid Enterococcus MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain is inoculated into MM01 liquid culture medium and cultured anaerobically to obtain a sufficient number of live bacteria. The cultured bacterial solution is concentrated by centrifugation and then resuspended in a solvent to obtain the purity and live bacterial count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0288] Tumor cells: Renca mouse renal carcinoma cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0289] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared into 2 mg / mL solution with normal saline, sourced from BioXcell.

[0290] Experimental animals: The experimental mice were BALB / c mice, 5 weeks old, a total of 55 mice, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0291] Animal experiment: BALB / c mice were divided into two groups according to their body weight (20.0-22.0 g), with 27-28 mice in each group, and divided into two groups, namely control group and lactic acid enterococcus MNC-168 group. On the day of tumor inoculation (D0), each group was gavaged with medium or 2×10 9 CFU / live suspension of Enterococcus lactis MNC-168, 5×10 4 The number of cells per mouse was subcutaneously inoculated with Renca renal cancer cells. Five days after tumor inoculation, the tumor grew to an average volume of 20-50 mm 3 At about 1:10 pm, the mice were further divided into four groups according to whether anti-mPD-1 (PD-1) was intraperitoneally injected or not. Each group finally enrolled 13 to 14 mice, including control, PD-1, MNC-168, and MNC-168+PD-1. The mice were given medium + 0.9% sodium chloride injection, medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. The medium or live bacterial drug suspension was administered by intragastric gavage once a day at 2×10 9CFU / 0.2ml / mouse, 0.9% sodium chloride injection or anti-mPD-1 (PD-1) was intraperitoneally injected at 200μg / 0.1ml / mouse on the 5th, 8th, 11th and 14th days. Animal characteristics, tumor changes, tumor size and mouse weight were monitored regularly at designated times during the experiment. After reaching the experimental endpoint, all surviving mice were dissected and sampled, tumor weight was measured and tumor volume was calculated, and finally statistical analysis and comparison of tumor volume curve change (Tumor volume), endpoint tumor volume (Tumor volume at endpoint), endpoint tumor weight (Tumor weight at endpoint) and endpoint tumor growth inhibition rate (Tumor Growth Inhibition at endpoint, TGI) were performed. Tumor growth inhibition rate (TGI) = 1-(individual tumor volume-individual tumor volume at grouping) / (Control group tumor average volume-Control group tumor average volume at grouping) × 100%, all data were expressed in mean±SD form, and GraphPad Prism8.0.2 software was used for plotting and statistical analysis. For comparisons between three or more groups, one-way ANOVA with Tukey's multiple comparisons test was used for analysis. For two-factor studies, two-way ANOVA with Sidak's multiple comparisons was used for analysis. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0292] The results are shown in the following table:

[0293] Table 1. Experimental results of MNC-168 combined with anti-PD-1 antibody for the treatment of renal cancer

[0294] Group <![CDATA[Volume (mm 3 )]]> Weight(g) TGI (%) Response rate (%) Disappearance rate (%) Control 2193 2.188 0 20.0 0 PD-1 1195 1.102 45.50 50.0 0 MNC-168 883 0.8880 59.57 85.7 14.3 MNC-168+PD-1 398 0.3786 81.88 87.5 37.5

[0295] The results are shown in Table 1 and Figures 2 to 6 The results showed that during the experiment, the statistical analysis results of the tumor volume curve of the MNC-168+PD-1 group were significantly smaller than those of the PD-1 group (e.g. Figure 2 At the end of the experiment, the average tumor volume of mice in the Control group was 2193mm 3 The average tumor weight was 2.188 g; the average tumor volume of mice in the PD-1 group was 1195 mm 3 The average tumor weight was 1.102 g; the average tumor volume of the MNC-168 group was 883 mm 3The average tumor weight was 0.8880 g; the average tumor volume of mice in the MNC-168+PD-1 group was 398 mm 3 The average tumor weight was 0.3786 g. Compared with the PD-1 alone group, the endpoint tumor volume and tumor weight of mice in the MNC-168+PD-1 antibody treatment group showed a significant downward trend ( Figure 3 , Figure 4 At the end of the experiment, the tumor growth inhibition rate (TGI) of PD-1 was 45.50%, and the tumor growth inhibition rate (TGI) of the MNC-168+PD-1 group was 81.88%, which was significantly higher than that of the PD-1 alone group ( Figure 5 At the end of the experiment, the response rate of PD-1 was 50.0%, and the response rate of MNC-168+PD-1 was 87.5%. The combination of MNC-168 and anti-PD-1 antibody significantly improved the tumor treatment response rate of anti-PD-1 antibody alone ( Figure 6 ). In addition, the corresponding tumor cure rate (disappearance rate) was 0% for PD-1, while it was 37.5% for MNC-168+PD-1. The combination of MNC-168 and anti-PD-1 antibody significantly improved the tumor cure rate of anti-PD-1 antibody alone. Therefore, these results show that MNC-168 can significantly improve the renal cancer treatment effect of anti-PD-1 antibody.

[0296] Example 4: Combination of MNC-168 and anti-PD-1 antibody for the treatment of fibrosarcoma

[0297] The therapeutic efficacy of MNC-168 combined with anti-PD-1 antibody was verified in the mouse syngeneic tumor WEHI 164 (fibrosarcoma) model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Munn Biotechnology.

[0298] Test strains: After thawing the glycerol cryopreserved tube of lactic acid Enterococcus MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain is inoculated into MM01 liquid culture medium and cultured anaerobically to obtain a sufficient number of live bacteria. The cultured bacterial solution is concentrated by centrifugation and then resuspended in a solvent to obtain the purity and live bacterial count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0299] Tumor cells: WEHI 164 mouse fibrosarcoma cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0300] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared into 2 mg / mL solution with normal saline, sourced from BioXcell.

[0301] Experimental animals: The experimental mice were BALB / c mice, 5 weeks old, a total of 55 mice, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0302] Animal experiment: BALB / c mice were divided into two groups according to their body weight (20.0-22.0 g), with 27-28 mice in each group, and divided into two groups, namely control group and lactic acid enterococcus MNC-168 group. On the day of tumor inoculation (D0), each group was gavaged with medium or 2×10 9 CFU / live suspension of Enterococcus lactis MNC-168, 5×10 4 WEHI 164 fibrosarcoma cells were subcutaneously inoculated into mice. Five days after inoculation, the tumors grew to an average volume of 40-100 mm. 3 At about 1:10 pm, the mice were further divided into four groups according to whether anti-mPD-1 (PD-1) was intraperitoneally injected or not. Each group finally enrolled 13 to 14 mice, including control, PD-1, MNC-168, and MNC-168+PD-1. The mice were given medium + 0.9% sodium chloride injection, medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. The medium or live bacterial drug suspension was administered by intragastric gavage once a day at 2×10 9CFU / 0.2ml / mouse, 0.9% sodium chloride injection or anti-mPD-1 (PD-1) was intraperitoneally injected at 200μg / 0.1ml / mouse on the 5th, 8th, 11th and 14th days. Animal characteristics, tumor changes, tumor size and mouse weight were monitored regularly at designated times during the experiment. After reaching the experimental endpoint, all surviving mice were dissected and sampled, tumor weight was measured and tumor volume was calculated, and finally statistical analysis and comparison of tumor volume curve change (Tumor volume), endpoint tumor volume (Tumor volume at endpoint) and endpoint tumor growth inhibition rate (Tumor Growth Inhibition at endpoint, TGI) were performed. Tumor growth inhibition rate (TGI) = 1-(individual tumor volume-individual tumor volume at grouping) / (Control group tumor average volume-Control group tumor average volume at grouping) × 100%, all data were expressed in mean±SD form, and GraphPad Prism 8.0.2 software was used for plotting and statistical analysis. For comparisons among more than three groups, one-way ANOVA with Tukey's multiple comparisons test was used for analysis. For two-factor studies, two-way ANOVA with Sidak's multiple comparisons test was used for analysis. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0303] The results are shown in the following table:

[0304] Table 2. Experimental results of MNC-168 combined with anti-PD-1 antibody in the treatment of fibrosarcoma

[0305] Group <![CDATA[Volume (mm 3 )]]> TGI (%) Response rate (%) Disappearance rate (%) Control 2320 0 00.0 0 PD-1 2061 11 40.0 0 MNC-168 1334 42 50.0 12.5 MNC-168+PD-1 771 67 57.1 14.3

[0306] The results are shown in Table 2 and Figures 7-10 The results showed that during the experiment, the statistical analysis results of the tumor volume curve of the MNC-168+PD-1 group were significantly smaller than those of the PD-1 group (see Figure 7 At the end of the experiment, the average tumor volume of mice in the Control group was 2320 mm 3 The average tumor volume of mice in the PD-1 group was 2061 mm 3 The average tumor volume of mice in the MNC-168+PD-1 group was 771 mm 3 Compared with the PD-1 alone group, the endpoint tumor volume of mice in the MNC-168+PD-1 antibody treatment group showed a significant downward trend ( Figure 8At the end of the experiment, the tumor growth inhibition rate (TGI) of PD-1 was 11%, and the tumor growth inhibition rate (TGI) of the MNC-168+PD-1 group was 67%, which was significantly higher than that of the PD-1 alone group ( Fig. 9 ). In terms of experimental endpoint response rate, the response rate of PD-1 was 40%, and the response rate of MNC-168+PD-1 was 57%. The combination of MNC-168 and anti-PD-1 antibody significantly improved the tumor treatment response rate of anti-PD-1 antibody alone ( Fig.10 ). In addition, the corresponding tumor cure rate (disappearance rate) of PD-1 is 0%, while that of MNC-168+PD-1 is 14.3%. The combination of MNC-168 and anti-PD-1 antibody significantly improves the tumor cure rate of anti-PD-1 antibody alone. Therefore, these results show that MNC-168 can significantly improve the therapeutic effect of anti-PD-1 antibody on fibrosarcoma.

[0307] Example 5: Combination of MNC-168 and anti-PD-1 antibody for the treatment of liver cancer

[0308] The therapeutic effect of MNC-168 combined with anti-PD-1 antibody was verified in the mouse syngeneic tumor H22 (liver cancer) model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotechnology.

[0309] Test strains: After thawing the glycerol cryopreserved tube of lactic acid Enterococcus MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain is inoculated into MM01 liquid culture medium and cultured anaerobically to obtain a sufficient number of live bacteria. The cultured bacterial solution is concentrated by centrifugation and then resuspended in a solvent to obtain the purity and live bacterial count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0310] Tumor cells: H22 mouse hepatoma cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0311] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared into 2 mg / mL solution with normal saline, sourced from BioXcell.

[0312] Experimental animals: The experimental mice were C57BL / 6J mice, 4 weeks old, a total of 45 mice, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0313] Animal experiment: C57BL / 6J mice were divided into three groups according to their body weight of 20.0-22.0 g: Control, PD-1, and MNC-168+PD-1. On the day of tumor inoculation (D0), each group was gavaged with medium or 2×10 9CFU / live suspension of Enterococcus lactis MNC-168, 5×10 5 The number of cells per mouse was subcutaneously inoculated with H22 liver cancer cells. Six days after tumor inoculation, the tumor grew to an average volume of 50 mm 3 At about 1:10 s, the following drugs were administered: culture medium + 0.9% sodium chloride injection, culture medium + PD-1, MNC-168 + PD-1, and culture medium or live bacterial drug suspension was administered by intragastric administration once a day at 2 × 10 9 CFU / 0.2ml / mouse, 0.9% sodium chloride injection or anti-mPD-1 (PD-1) was intraperitoneally injected at 200μg / 0.1ml / mouse on the 6th, 9th, 12th and 16th days. Animal characteristics, tumor changes, tumor size and mouse weight were monitored regularly at designated times during the experiment. After reaching the experimental endpoint, all surviving mice were dissected and sampled, tumor weight was measured and tumor volume was calculated, and finally statistical analysis and comparison of tumor volume curve change (Tumor volume), endpoint tumor volume (Tumor volume at endpoint), endpoint tumor weight (Tumor weight at endpoint) and endpoint tumor growth inhibition rate (Tumor Growth Inhibition at endpoint, TGI) were performed. Tumor growth inhibition rate (TGI) = 1-(individual tumor volume-individual tumor volume when grouped) / (Average tumor volume of group A-Average tumor volume of group A when grouped) × 100%, all data were expressed in the form of mean±SD, and GraphPad Prism 8.0.2 software was used for plotting and statistical analysis. For comparisons between three or more groups, one-way ANOVA with Tukey's multiple comparisons test was used for analysis. For two-factor studies, two-way ANOVA with Sidak's multiple comparisons test was used for analysis. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0314] Table 3. Experimental results of MNC-168 combined with anti-PD-1 antibody for the treatment of liver cancer

[0315] Group <![CDATA[Volume (mm 3 )]]> Weight(g) TGI (%) Response rate (%) Control 1858 2.065 -14.55 0 PD-1 1508 1.548 18.75 0 MNC-168+PD-1 748 0.5367 59.71 100

[0316] The results are shown in Table 3 and Figures 11 to 15 The results showed that during the experiment, the statistical analysis results of the tumor volume curve of the MNC-168+PD-1 group were significantly smaller than those of the PD-1 group (e.g. Fig.11At the end of the experiment, the average tumor volume of mice in the Control group was 1858 mm 3 The average tumor weight was 2.065 g; the average tumor volume of mice in the PD-1 group was 1508 mm 3 The average tumor weight was 1.548 g; the average tumor volume of mice in the MNC-168+PD-1 group was 748 mm 3 The average tumor weight was 0.5367 g. Compared with the PD-1 alone group, the endpoint tumor volume and tumor weight of mice in the MNC-168+PD-1 antibody treatment group were significantly reduced ( Fig.12 , Fig.13 At the end of the experiment, the tumor growth inhibition rate (TGI) of PD-1 was 18.75%, and the tumor growth inhibition rate (TGI) of the MNC-168+PD-1 group was 59.71%, which was significantly higher than that of the PD-1 group ( Fig.14 At the end of the experiment, the response rate of PD-1 was 0%, and the response rate of MNC-168+PD-1 was 100%. The combination of MNC-168 and anti-PD-1 antibody significantly improved the response rate of tumor treatment ( Fig.15 ). These results suggest that MNC-168 can significantly enhance the therapeutic efficacy of anti-PD-1 antibodies in liver cancer.

[0317] Example 6: Lactobacillus culture supernatant promotes STING pathway-dependent anti-tumor immunity

[0318] Preparation of MNC-168 supernatant: After thawing the glycerol cryopreservation tube of Enterococcus lactis MNC-168 strain at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid culture medium, culture it anaerobically, obtain a sufficient number of live bacteria, centrifuge the cultured bacterial solution at 10000g for 10min to precipitate the bacteria, take the supernatant and filter it through a 0.22μm filter membrane, and use the filtered supernatant for subsequent experiments or freeze it at -80°C.

[0319] STING repoter cell evaluation: The evaluation was performed using THP-1 cells (a cell line built by Muen) that had been constructed to carry the IFNβ gene promoter reporter gene. The steps are as follows:

[0320] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1×10 cells per well. 5cells. A total of three groups were set up, including the Control group, MNC-168SP group, and MNC-168SP+H-151 group. 10 μL of MM01 medium, 10 μL of MNC-168 culture supernatant, and 10 μL of MNC-168 culture supernatant (containing H-151 with a final concentration of 10 μM) were added, respectively. After culturing for 24 hours, the cells were centrifuged at 300 g for 5 minutes, the culture supernatant was removed, 50 μL of 1×luciferase detection reagent was added, the reaction was continued for 1 minute, and the luminescence chemiluminescence value was detected by an enzyme-labeled instrument. The relative fluorescence value (Relative Luminescence) was normalized with that of the Control group to evaluate the effect of the supernatant of Enterococcus lactis MNC-168 on the transcriptional activity of IFNβ.

[0321] RT-PCR detection: THP-1 cells refer to the experimental steps of the above STING reporter cells, and finally collect the cells after different treatments, extract total RNA, reverse transcribe, and detect the expression of the target gene by qPCR.

[0322] The results are as follows Fig.16 A to C, the results showed that the supernatant of lactic acid Enterococcus MNC-168 can significantly promote the activity of STING, which can be increased by 2 times. At the same time, this activation can be inhibited by the STING specific inhibitor H-151. Furthermore, the detection results of STING downstream target genes IFN-β and TNF-α showed that the supernatant of lactic acid Enterococcus MNC-168 can also significantly activate the expression of the two genes by about 4 times, and this activation can also be inhibited by the STING specific inhibitor H-151.

[0323] These results indicate that the supernatant of Enterococcus lactis MNC-168 can induce STING pathway-dependent activation, thereby upregulating the expression of interferon and tumor necrosis factor that inhibit tumor cell proliferation and metastasis, promote tumor cell apoptosis, and activate anti-tumor immune responses, thereby promoting anti-tumor immune activity.

[0324] Example 7: Lactobacillus culture supernatant promotes NOD2 pathway-dependent anti-tumor immunity

[0325] Preparation of MNC-168 supernatant: The MNC-168 supernatant prepared in Example 6 was used.

[0326] NOD2 activation evaluation experiment: THP-1 cells were seeded in 12-well plates, with 5×10 cells per well. 5cells. A total of three groups were set up, including the Control group, MNC-168SP group, and MNC-168SP+GSK717 group. 50 μL of MM01 medium, 50 μL of MNC-168 culture supernatant, and 50 μL of MNC-168 culture supernatant (containing GSK717 with a final concentration of 10 μM) were added, respectively. After culturing for 24 hours, the cells were centrifuged at 300 g for 5 minutes, the culture supernatant was removed, 50 μL of 1×luciferase detection reagent was added, the reaction was continued for 1 minute, and the luminescence chemiluminescence value was detected by an enzyme reader. The relative fluorescence value (Relative Luminescence) was normalized with that of the Control group to evaluate the effect of the supernatant of Enterococcus lactis MNC-168 on NOD2 activity.

[0327] RT-PCR detection: refer to the RT-PCR experimental steps in Example 6 to perform total RNA extraction, reverse transcription, and qPCR to detect the expression level of the target gene.

[0328] The results are as follows Fig.17 A and B showed that the supernatant of lactic enterococcus MNC-168 could significantly upregulate the expression of NOD2 downstream target genes IL-1β and IL-6, which were upregulated by more than 20 times and 10 times, respectively, and this activation could also be partially inhibited by the NOD2-specific inhibitor GSK717.

[0329] These results indicate that the supernatant of lactic enterococci MNC-168 can induce NOD2 pathway-dependent activation to a certain extent, thereby upregulating the expression of interferon and tumor necrosis factor that inhibit tumor cell proliferation and metastasis, promote tumor cell apoptosis, and activate anti-tumor immune response, thereby promoting anti-tumor immune activity.

[0330] Example 8: Lactococcus promotes activation of systemic immunity

[0331] The therapeutic effect of Enterococcus lactis MNC-168 combined with anti-PD-1 antibody and the effect on cytokines and chemokines in mouse fluid were verified in the mouse syngeneic tumor MC38 (colorectal cancer) model. This experimental protocol has been reviewed by the Animal Care and Use Committee of Muen Biotech.

[0332] Test strains: After thawing the glycerol cryopreserved tube of lactic acid Enterococcus MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain is inoculated into MM01 liquid culture medium and cultured anaerobically to obtain a sufficient number of live bacteria. The cultured bacterial solution is concentrated by centrifugation and then resuspended in a solvent to obtain the purity and live bacterial count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0333] Tumor cells: MC38 mouse colorectal cancer cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0334] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared into 2 mg / mL solution with normal saline, sourced from BioXcell.

[0335] Experimental animals: The experimental mice were C57BL / 6J mice, 5 weeks old, a total of 55 mice, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0336] Animal experiment: C57BL / 6J mice were divided into two groups according to their body weight (20.0-22.0 g), with 27-28 mice in each group, and divided into two groups, namely control group and lactic acid enterococcus MNC-168 group. On the day of tumor inoculation (D0), each group was intragastrically administered with medium or 2×10 9 CFU / live suspension of Enterococcus lactis MNC-168, 5×10 4 The number of cells per mouse was subcutaneously inoculated with Renca renal cancer cells. Five days after tumor inoculation, the tumor grew to an average volume of 20-50 mm 3 At about 1:10 pm, the mice were further divided into four groups according to whether anti-mPD-1 (PD-1) was intraperitoneally injected or not. Each group finally enrolled 13 to 14 mice, including control, PD-1, MNC-168, and MNC-168+PD-1. The mice were given medium + 0.9% sodium chloride injection, medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. The medium or live bacterial drug suspension was administered by intragastric gavage once a day at 2×10 9 CFU / 0.2ml / mouse, 0.9% sodium chloride injection or anti-mPD-1 (PD-1) antibody was administered by intraperitoneal injection at 200μg / 0.1ml / mouse on the 5th, 8th, 11th and 14th days. Animal characteristics, tumor changes, tumor size and mouse weight were regularly monitored at designated times during the experiment. After reaching the end of the experiment, the blood of the mice was collected, the serum was separated, and the concentration of the corresponding cytokines and chemokines in the serum was detected by ELISA kit.

[0337] The results are as follows Fig.18 As shown in Figure A, MNC-168 can significantly increase the therapeutic effect of anti-PD-1 antibody on MC38. ELISA was further used to detect the changes in cytokines and chemokines in serum. The results are as follows Fig.18 B~E, the combination of MNC-168 and anti-PD-1 antibody can further promote the increase of CXCL1, CXCL9, CXCL10 and IFN-γ concentrations in serum when anti-PD-1 antibody is used alone.

[0338] Example 9: Lactococcus lactis maintains intestinal flora diversity in tumor-bearing mice

[0339] Collection of mouse fecal samples: Fecal samples from the mouse homologous tumor MC38 (colorectal cancer) model in Example 8 were collected and stored at -80°C for subsequent experiments, including: fecal samples of four groups before (pre) and after (post) tumor inoculation and treatment, Control (CT), PD-1, MNC-168, and MNC-168+PD-1, namely, preCT, prePD-1, preMNC-168, preMNC-168+PD-1, postCT, postPD-1, postMNC-168, and postMNC-168-PD-1.

[0340] 16S RNA sequencing analysis: Mouse fecal samples were sent to a sequencing company for DNA extraction, 16S ribosomal RNA gene expansion, Illumina MiSeq sequencing, analysis and identification of bacteria and richness, and finally bacterial diversity analysis.

[0341] The results are as follows Fig.19 Before tumor inoculation and treatment, that is, between the treatment groups pre, there was no difference in the types and diversity of intestinal microorganisms in mice. However, after MC-38 colorectal cancer cells were inoculated, the preCT and postCT in the CT group were compared, and it was found that the types and diversity of intestinal microorganisms in the postCT group mice decreased significantly. After the implementation of MNC-168 after MC-38 inoculation, that is, postMNC-168, the types and diversity of intestinal microorganisms in mice did not change significantly compared with the preMNC-168 group before MC-38 inoculation. Compared with the changes in intestinal microorganisms of mice in the CT group before and after tumor inoculation, the implementation of MNC-168 treatment can restore the stability of the intestinal flora. The results of PD-1 or MNC-168+PD-1 implementation were similar to those of MNC-168 implementation ( Fig.19 A); similar results were found in the PCA analysis. In the CT group, the diversity of the intestinal flora of mice changed before and after cancer cell inoculation, but in the MNC-168 group, the diversity of the intestinal flora of mice was similar before and after cancer cell inoculation ( Fig.19 B) These results suggest that MNC-168 can restore the intestinal flora of mice.

[0342] These results show that MNC-168 can promote the therapeutic effect of anti-PD-1 antibodies. In addition, the combination of MNC-168 and anti-PD-1 antibodies can further promote the increase of host system immunity, thereby enhancing the immunity of the entire body. Therefore, it has therapeutic functions for soft tissue tumors, blood tumors, glandular tumors, metastatic tumors, and other tumors and cancers. MNC-168 can also improve the problem of decreased intestinal flora diversity caused by anti-PD-1 antibodies, thereby effectively maintaining the balance of intestinal flora.

[0343] Membrane vesicles are secretions of intestinal bacteria, which contain a large amount of intestinal bacterial DNA and special proteins and other substances. Therefore, these specific components can activate the immune system of the entire body, thereby achieving the prevention and treatment of various tumors or cancers including solid tumors, soft tissue tumors, hematopoietic tumors, adenoma, metastatic tumors, etc., and play a good role in regulating or stimulating the immune system, as well as regulating the intestines, and can even achieve better results than direct administration of intestinal bacteria. In addition, regarding the activation of the STING and / or NOD2 pathways, the strain components including the supernatant of the strain and the membrane vesicles of the strain can play an activating role. Therefore, when preparing it into a drug, a variety of preparation forms can be adaptively provided according to different needs, including strains, strain metabolites, strain supernatants, and membrane vesicles secreted by strains. These components derived from strains can play a good anti-tumor effect.

[0344] In addition, for some clinically refractory tumors such as renal cancer, fibrosarcoma, and liver cancer, it is difficult to predict whether the drug will have an effective anti-tumor effect. In the embodiments of the present application, the combination of the relevant components of Enterococcus bacteria and immune checkpoint inhibitors can achieve good therapeutic effects. These tumor lesions originate from outside the gastrointestinal tract, and Enterococcus bacteria from the intestine, such as MNC-168, can still be used in combination with immune checkpoint inhibitors to achieve synergistic effects, so that the combination has a higher anti-tumor effect. It can be seen that the combination of the two significantly enhances the efficacy of immune checkpoint inhibitors such as anti-PD-1 antibodies, and experimental data show that the tumor disappearance rate is significantly improved through the intervention of the strains of the present application.

[0345] In addition, in the examples of the present application, the supernatant and membrane vesicle experiments of the strain showed that the STING activity was promoted to 3 times relative to the control, which may indicate that there are nucleic acid fragments (such as DNA fragments) that specifically bind to C-Gas, thereby effectively activating the STING and / or NOD2 pathways to exert anti-tumor activity.

[0346] Example 10: Lactococcus lactis membrane vesicles for the treatment of colorectal cancer

[0347] The therapeutic efficacy of MNC-168 membrane vesicles was validated in the mouse syngeneic tumor CT-26 (colorectal cancer) model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotechnology.

[0348] Test strains: After thawing the glycerol cryopreserved tube of lactic acid Enterococcus MNC-168 at 37°C, inoculate it on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain is inoculated into MM01 liquid culture medium and cultured anaerobically to obtain a sufficient number of live bacteria. The cultured bacterial solution is concentrated by centrifugation and then resuspended in a solvent to obtain the purity and live bacterial count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0349] Preparation of MNC-168 membrane vesicles (MNC-168MV): refer to the preparation method in Example 1. The vesicles were diluted with PBS to adjust to the desired experimental concentration.

[0350] Tumor cells: CT-26 mouse colorectal cancer cells, from the American type culture collection (ATCC). The number of cells inoculated was 1×10 6 cells /

[0351] Experimental animals: The experimental mice were BALB / c mice, 8 weeks old, from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0352] Administration and dosage: MNC-168 membrane vesicles were injected into the tumor, 50 μL each time containing 10 μg of membrane vesicles, once every 2 days, for a total of 4 times. The group injected with PBS was the control group (Control).

[0353] Animal experiment: Several female BALB / c mice were ordered for this study. After the quarantine, tumor cells were inoculated to construct the CT26 colorectal cancer ectopic homologous tumor model. The day of tumor inoculation was Day 0, and tumor measurements were performed from Day 5. When the tumor grew to an average volume of 100 mm 3When the mice with abnormal tumor growth were eliminated, the number was at least 12, and the qualified mice were randomly stratified and grouped according to the tumor volume. MNC-168 membrane vesicles were injected intratumorally on the day of grouping. Animal characteristics, tumor changes, tumor size, and mouse weight were regularly monitored during the experiment. At the end of the experiment, tumor volume changes and weight were analyzed. The weight of all mice increased steadily throughout the experiment. All data were expressed in the form of mean±SD, and GraphPadPrism 8.0.2 software was used for drawing and statistical analysis. For comparisons between more than three groups, one-way analysis of variance (One-Way ANOVA) method with Tukey's multiple comparisons test was used for analysis. For two-factor investigations, two-way ANOVA with Sidak multiple comparisons was used for analysis. The significant difference was indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0354] Table 4. Results of MNC-168MV treatment of CT-26 mouse colorectal cancer

[0355] Group <![CDATA[Volume (mm 3 )]]> Weight(g) Control 1053 1.043 MNC-168MV 680.5 0.645

[0356] The test results show that according to Table 4 and Fig. 20 The results of A showed that the tumor growth curve of the MNC-168MV intratumoral injection group was significantly smaller than that of the Control group. At the end of the experiment, the tumor volume of the Control group was 1053 mm 3 The tumor volume of the MNC-168MV group was 680.5 mm 3 In addition, the end-point anatomical photography and weighing results showed that the weight of the tumor in the MNC-168MV group was significantly smaller than that in the Control group ( Fig. 20 B), the tumor in the Control group was 1.043 g, and that in the MNC-168MV group was 0.645 g. These experimental results show that MNC-168 membrane vesicles have the effect of inhibiting the growth of CT-26 tumors, suggesting that MNC-168 can achieve anti-tumor effects through secretion of membrane vesicles.

[0357] Example 11: Detection of biomarkers in the treatment of colorectal cancer using lactic acid Enterococcus membrane vesicles

[0358] In this study, flow cytometry was used to detect and analyze the changes in the immune cell population in Example 10 to clarify the anti-tumor mechanism of the membrane vesicles of Enterococcus lactis.

[0359] Preparation of single cell suspension of tumor tissue:

[0360] 1) Preparation of tumor tissue suspension: The tumor tissue is mechanically sheared, homogenized, consumed and ground to obtain a tumor grinding solution.

[0361] 2) The tumor grinding solution was centrifuged at 500g for 5 min at room temperature. The centrifuge tube was inverted on a paper towel for 5s-10s and the supernatant was discarded.

[0362] 2) Add 10 mL of red blood cell lysis solution and lyse the red blood cells for 10 minutes.

[0363] 3) Add PBS to the 30 ml mark of a 50 ml centrifuge tube.

[0364] 4) Centrifuge at 500g for 5 min at room temperature, invert the centrifuge tube on a paper towel and discard the supernatant.

[0365] 5) The cell pellet is resuspended in PBS and the cell suspension is passed through a 70 μm pore size membrane to obtain a single cell suspension of tumor tissue cells for subsequent cell staining and flow cytometry analysis.

[0366] Cell staining and flow cytometry indicators: refer to Table 5

[0367] Table 5. Flow cytometry indicators

[0368]

[0369] Flow cytometry results showed that compared with the Control group, the number of DC cells in the tumor of the MNC-168MV group increased significantly ( Fig.21 A). In addition, in the analysis of tumor-associated macrophages (TAM), the ratio of M2 TAM expressing CD206 to M1 TAM expressing CD86 was significantly decreased in the MNC-168MV group, indicating that MNC-168MV inhibits the effect of M2 TAM ( Fig.21 B); Finally, analysis of cytokines in T cells in the tumor also found that the number of cytotoxic T cells CD8 T cells expressing IFN-γ+ was significantly upregulated in the MNC-168MV group ( Fig.21 C). These results indicate that MNC-168 membrane vesicles can increase anti-tumor effects by promoting innate immunity and adaptive immunity. Combined with previous experimental results, it suggests that MNC-168 can promote innate immunity and adaptive immunity activation in vivo through secretory vesicles, thereby increasing anti-tumor effects.

[0370] The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A composition, characterized in that Includes ingredients of microbial origin; Preferably, the microbial-derived component comprises membrane vesicles (MVs) isolated from a bacterial strain; more preferably, the membrane vesicles comprise at least one of lipids, proteins and nucleic acid fragments; Preferably, the nucleic acid fragment comprises a DNA fragment; Preferably, the microbial-derived component comprises at least one of (1) to (5): (1) a bacterial strain having the function of stimulating the immune system or immunomodulating, (2) a culture of a bacterial strain having the function of stimulating the immune system or immunomodulating, (3) a fermentation supernatant of a bacterial strain having the function of stimulating the immune system or immunomodulating, (4) a metabolite of a bacterial strain having the function of stimulating the immune system or immunomodulating, (5) a short-chain fatty acid of a bacterial strain having the function of stimulating the immune system or immunomodulating; Preferably, the microbial-derived component comprises membrane vesicles (MVs) isolated from a bacterial strain, and further comprises at least one of (1) to (5): (1) a bacterial strain having a function of stimulating the immune system or immunomodulating, (2) a culture of a bacterial strain having a function of stimulating the immune system or immunomodulating, (3) a fermentation supernatant of a bacterial strain having a function of stimulating the immune system or immunomodulating, (4) a metabolite of a bacterial strain having a function of stimulating the immune system or immunomodulating, (5) a short-chain fatty acid of a bacterial strain having a function of stimulating the immune system or immunomodulating; Preferably, the microbial-derived component comprises at least 50 wt%, 60 wt%, 70 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, and / or, 50 v / v%, 60 v / v%, 70 v / v%, 80 v / v%, 85 v / v%, 90 v / v%, 95 v / v%, 96 v / v%, 97 v / v%, 98 v / v%, 99 v / v% of the membrane vesicles (MVs); Preferably, the membrane vesicles are derived from microbial strains in the intestine of a mammal; Preferably, the membrane vesicles are derived from genetically engineered microbial strains in the intestine of mammals; Preferably, the bacterial strain having the function of stimulating the immune system or immunomodulating is any one of the following A1) to A6): A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1; A2) bacteria with a deposit number of GDMCC No: 61121; A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2); A4) a bacterium whose genome has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% with the genome of any of the bacteria described in A1) to A3); and / or a bacterium whose genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% with the genome of any of the bacteria described in A1) to A3); A5) Enterococcus bacteria; A6) at least one bacterium selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus ass, Enterococcus avium, Enterococcus canis, Enterococcus fusae, Enterococcus caecum, Enterococcus pigeonus, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus gallus quailus, Enterococcus leucorus, Enterococcus hemoperoxidus, Enterococcus spp., Enterococcus malariae, Enterococcus malariae, Enterococcus moravianus, Enterococcus monterius, Enterococcus leucorus, Enterococcus wood hoopoeus, Enterococcus suis, Enterococcus pseudoavium, Enterococcus raffinose, Enterococcus ratus, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharolyticus, Enterococcus marineus, and Enterococcus lactis; Preferably, the membrane vesicles are isolated from any bacterial strain in A1) to A6) above; Preferably, the mass percentage and / or volume percentage of the microbial-derived component in the composition is 1-99%, 2-90%, 5-80%, 10-70%, 20-60%, or at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%; Preferably, the composition further comprises at least one of an excipient, a diluent, and a carrier; Preferably, the composition further comprises an auxiliary material; Preferably, the auxiliary material also includes at least one of a lubricant, a wetting agent, a binder, an emulsifier, a suspension stabilizer, a solubilizer, a preservative, a sweetener, and a flavoring agent; Preferably, the composition is a solid preparation or a liquid preparation; Preferably, the dosage form of the composition is any one of powder, granule, powder, tablet, capsule, gel, suspension, drop, pill, injection, suppository, aerosol, oral solution, ointment, emulsion, and lavage agent; Preferably, the composition further comprises a product instruction sheet.

2. The composition according to claim 1, characterized in that It also includes combination drugs; Preferably, the combination drug includes an anti-tumor drug; Preferably, the anti-tumor drug includes at least one of a chemotherapeutic drug, a photosensitizer, a photothermal agent, and an immunotherapy drug; Preferably, the chemotherapy drug includes at least one of paclitaxel, camptothecin, 5-fluorouracil, cisplatin, doxorubicin, mitomycin, and epirubicin; Preferably, the photosensitizer includes at least one of boron dipyrrole, dihydrochlorin, and red bengal; Preferably, the photothermal agent includes at least one of noble metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, and semiconductor nanomaterials; Preferably, the immunotherapy drug includes at least one of an immune cell therapy drug and an immune checkpoint inhibitor; Preferably, the immune cell therapy drug includes at least one of a T cell therapy drug, a tumor infiltrating lymphocyte therapy drug, and a NK cell therapy drug; Preferably, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

3. Use of the composition according to any one of claims 1 to 2 in the preparation of a medicament having at least one of the following uses B1) to B4): B1) Prevention or treatment of tumors or cancer; B2) Modulate the immune system or stimulate the immune system; B3) Regulate the intestines; B4) preventing and / or treating diseases mediated by the STING and / or NOD2 pathways; Preferably, the tumor or cancer in B1) includes at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor; Preferably, the solid tumor comprises at least one of head and neck cancer, upper digestive tract cancer, lower digestive tract cancer, hepatobiliary system cancer, neuroendocrine tumor, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer; Preferably, the blood tumor includes at least one tumor of blood, lymph and bone marrow; Preferably, the tumor or cancer includes oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, trunk and limb soft tissue sarcoma, abdominal and thoracic visceral soft tissue sarcoma, gastrointestinal stromal tumor, retroperitoneal soft tissue sarcoma at least one of the following: melanoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, leukemia; Preferably, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses; Preferably, the bacteria or virus includes at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum; Preferably, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) Inhibit tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) Improve the response rate of tumor treatment; (e) Improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reduce anti-tumor drug resistance; (h) Promote IFNβ transcription activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways; Preferably, the regulating or stimulating the immune system in B2) comprises: At least one of regulating or stimulating the inherent immune function in the gastrointestinal tract, regulating or stimulating the inherent immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract; Preferably, the intestinal regulation in B3) includes at least one of regulating intestinal function and regulating intestinal flora; Preferably, the regulating intestinal flora is regulating at least one of the types, richness, uniformity and diversity of intestinal flora; Preferably, the intestinal flora regulation is to regulate at least one of the changes in the type, richness, uniformity and diversity of the intestinal flora caused by diseases and / or drugs; Preferably, the change in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by the disease and / or drug includes at least one of the changes in the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) Infectious diseases, tumors or cancer, inflammation, allergic and autoimmune diseases; D2) Drugs for the prevention and / or treatment of D1); Preferably, the disease in D1) is an intestinal disease; Preferably, the drug D2) is an anti-tumor drug; Preferably, the anti-tumor drug of D2) includes an immunotherapy drug; Preferably, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitors of D2) include inhibitors targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the disease mediated by the STING and / or NOD2 pathway includes at least one of infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases.

4. Use of the composition according to any one of claims 1 to 2 in at least one of the following B1) to B4): B1) Prevention or treatment of tumors or cancer; B2) Modulate the immune system or stimulate the immune system; B3) Regulate the intestines; B4) preventing and / or treating diseases mediated by the STING and / or NOD2 pathways; Preferably, the tumor or cancer in B1) includes at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor; Preferably, the solid tumor comprises at least one of head and neck cancer, upper digestive tract cancer, lower digestive tract cancer, hepatobiliary system cancer, neuroendocrine tumor, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer; Preferably, the blood tumor includes at least one tumor of blood, lymph and bone marrow; Preferably, the tumor or cancer includes oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, trunk and limb soft tissue sarcoma, abdominal and thoracic visceral soft tissue sarcoma, gastrointestinal stromal tumor, retroperitoneal soft tissue sarcoma at least one of the following: melanoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, leukemia; Preferably, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses; Preferably, the bacteria or virus includes at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum; Preferably, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) Inhibit tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) Improve the response rate of tumor treatment; (e) Improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reduce anti-tumor drug resistance; (h) Promote IFNβ transcription activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways; Preferably, the regulating or stimulating the immune system in B2) comprises: At least one of regulating or stimulating the inherent immune function in the gastrointestinal tract, regulating or stimulating the inherent immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract; Preferably, the intestinal regulation in B3) includes at least one of regulating intestinal function and regulating intestinal flora; Preferably, the regulating intestinal flora is regulating at least one of the types, richness, uniformity and diversity of intestinal flora; Preferably, the intestinal flora regulation is to regulate at least one of the changes in the type, richness, uniformity and diversity of the intestinal flora caused by diseases and / or drugs; Preferably, the change in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by the disease and / or drug includes at least one of the changes in the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) Infectious diseases, tumors or cancer, inflammation, allergic and autoimmune diseases; D2) Drugs for the prevention and / or treatment of D1); Preferably, the disease in D1) is an intestinal disease; Preferably, the drug D2) is an anti-tumor drug; Preferably, the anti-tumor drug of D2) includes an immunotherapy drug; Preferably, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitors of D2) include inhibitors targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the disease mediated by the STING and / or NOD2 pathway includes at least one of infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases.

5. A method for preventing or treating tumors or cancers, and / or regulating or activating a subject's immune system, and / or regulating the intestinal tract, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathways, comprising administering to the subject an effective dose of a composition according to any one of claims 1 to 2: Preferably, the tumor or cancer comprises at least one of a solid tumor, a blood tumor, a soft tissue tumor, a glandular tumor, and a metastatic tumor; Preferably, the solid tumor comprises at least one of head and neck cancer, upper digestive tract cancer, lower digestive tract cancer, hepatobiliary system cancer, neuroendocrine tumor, chest cancer, bone tumor, soft tissue sarcoma, skin cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, urinary system cancer, eye cancer, central nervous system cancer, and endocrine system cancer; Preferably, the blood tumor includes at least one tumor of blood, lymph and bone marrow; Preferably, the tumor or cancer includes oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestine cancer, appendix cancer, colon cancer, rectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampullary cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampullary neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendix neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, trunk and limb soft tissue sarcoma, abdominal and thoracic visceral soft tissue sarcoma, gastrointestinal stromal tumor, retroperitoneal soft tissue sarcoma at least one of the following: melanoma, Merkel cell carcinoma, malignant melanoma of the skin, vulvar cancer, vaginal cancer, cervical cancer, uterine corpus cancer, uterine carcinosarcoma, uterine corpus sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, kidney cancer, renal pelvis cancer, ureter cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, leukemia; Preferably, the tumor or cancer includes a tumor or cancer caused by bacteria or viruses; Preferably, the bacteria or virus includes at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, Helicobacter pylori, and Fusobacterium nucleatum; Preferably, the prevention or treatment of tumors or cancer is achieved by at least one of the following (a) to (j): (a) Inhibit tumor volume growth; (b) inhibiting tumor weight increase; (c) inhibiting tumor cell growth; (d) Improve the response rate of tumor treatment; (e) Improving the efficacy of immunosuppressive drug treatment; for example, improving the efficacy of anti-PD-1 antibody treatment; (f) preventing and / or inhibiting the spread or metastasis of tumor cells; (g) reduce anti-tumor drug resistance; (h) Promote IFNβ transcription activity; (i) regulating or activating the subject's immune system; (j) increasing or activating the activity of STING and / or NOD2 pathways; Preferably, the regulating or stimulating the immune system in B2) comprises: At least one of regulating or stimulating the inherent immune function in the gastrointestinal tract, regulating or stimulating the inherent immune function outside the gastrointestinal tract, adding one or more beneficial immune functions in the gastrointestinal tract, and adding one or more beneficial immune functions outside the gastrointestinal tract; Preferably, the intestinal regulation includes at least one of regulating intestinal function and regulating intestinal flora; Preferably, the regulating intestinal flora is regulating at least one of the types, richness, uniformity and diversity of intestinal flora; Preferably, the intestinal flora regulation is to regulate at least one of the changes in the type, richness, uniformity and diversity of the intestinal flora caused by diseases and / or drugs; Preferably, the change in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by the disease and / or drug includes at least one of the changes in the types, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) Infectious diseases, tumors or cancer, inflammation, allergic and autoimmune diseases; D2) Drugs for the prevention and / or treatment of D1); Preferably, the disease in D1) is an intestinal disease; Preferably, the drug D2) is an anti-tumor drug; Preferably, the anti-tumor drug of D2) includes an immunotherapy drug; Preferably, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitors of D2) include inhibitors targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the disease mediated by the STING and / or NOD2 pathway includes at least one of infectious diseases, tumors or cancer, inflammation, allergies and autoimmune diseases.

6. The method according to claim 5, characterized in that The pharmaceutical composition is administered at least once a day; Preferably, when the administration frequency is twice or more per day, the same dose is administered each time; Preferably, when the administration frequency is twice or more per day, each administration is at a different dose; Preferably, the administration route includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

7. Use of C1 and C2 in the preparation of anti-tumor drugs, wherein the anti-tumor drugs include the prevention or treatment of at least one of renal cancer, liver cancer, and fibrosarcoma; in, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors; Preferably, the enterococcus bacteria include at least one of enterococci of the genus Enterococcus faecalis, Enterococcus faecium, Enterococcus ass, Enterococcus avium, Enterococcus canis, Enterococcus fusae, Enterococcus caecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus gallus quail, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestinalis, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus leucorhius, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharidus, Enterococcus marineus, and Enterococcus lactis; Preferably, the Enterococcus bacterium is any one of the following A1) to A4): A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1; A2) bacteria with a deposit number of GDMCC No: 61121; A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2); A4) a bacterium whose genome has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% with the genome of any of the bacteria described in A1) to A3); and / or a bacterium whose genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% with the genome of any of the bacteria described in A1) to A3); Preferably, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

8. Application of C1 and C2 in preventing or treating at least one of renal cancer, liver cancer and fibrosarcoma: in, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors; Preferably, the enterococcus bacteria include at least one of enterococci of the genus Enterococcus faecalis, Enterococcus faecium, Enterococcus ass, Enterococcus avium, Enterococcus canis, Enterococcus fusae, Enterococcus caecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus gallus quail, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestinalis, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus leucorhius, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharidus, Enterococcus marineus, and Enterococcus lactis; Preferably, the Enterococcus bacterium is any one of the following A1) to A4): A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1; A2) bacteria with a deposit number of GDMCC No: 61121; A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2); A4) a bacterium whose genome has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% with the genome of any of the bacteria described in A1) to A3); and / or a bacterium whose genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% with the genome of any of the bacteria described in A1) to A3); Preferably, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

9. A method for preventing or treating at least one of renal cancer, liver cancer, and fibrosarcoma, comprising administering to a subject an effective dose of C1 and C2: in, C1 includes at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, and supernatants of fermentation broths of Enterococcus bacteria, and C2 includes immune checkpoint inhibitors; Preferably, the enterococcus bacteria include at least one of enterococci of the genus Enterococcus faecalis, Enterococcus faecium, Enterococcus ass, Enterococcus avium, Enterococcus canis, Enterococcus fusae, Enterococcus caecum, Enterococcus pigeon, Enterococcus peculiaris, Enterococcus dulcis, Enterococcus yellow, Enterococcus gallus quail, Enterococcus light yellow, Enterococcus hemoperoxidus, Enterococcus small intestinalis, Enterococcus odoriferus, Enterococcus moravianus, Enterococcus montereyii, Enterococcus leucorhius, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharidus, Enterococcus marineus, and Enterococcus lactis; Preferably, the Enterococcus bacterium is any one of the following A1) to A4): A1) bacteria having 16S rRNA that is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identical to the sequence set forth in SEQ ID NO:1; A2) bacteria with a deposit number of GDMCC No: 61121; A3) bacteria having at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% identity to the 16S rRNA of the bacteria described in A2); A4) a bacterium whose genome has an average nucleotide identity of at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% with the genome of any of the bacteria described in A1) to A3); and / or a bacterium whose genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% with the genome of any of the bacteria described in A1) to A3); Preferably, the immune checkpoint inhibitor includes an inhibitor targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA; Preferably, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

10. The method according to claim 9, characterized in that The C1 and C2 are administered at least once a day; Preferably, the C1 and the C2 are administered simultaneously, separately or sequentially; Preferably, when the administration frequency is twice or more per day, the same dose is administered each time; Preferably, when the administration frequency is twice or more per day, each administration is at a different dose; Preferably, the administration route includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, oral administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

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