Pharmaceutical composition and use thereof

Abstract

Disclosed are a pharmaceutical composition and a use thereof. Provided is a pharmaceutical composition, wherein the pharmaceutical composition comprises a microorganism-derived component. The pharmaceutical composition can achieve prevention and treatment of various tumors or cancers comprising solid tumors, soft tissue tumors, hematopoietic tumors, adenoma, metastatic tumors and the like, and play a good role in regulating or stimulating the immune system, and in regulating the intestinal tract.

Description

Pharmaceutical composition and its application Technical Field

[0001] The present application relates to the technical field of microbiology, and in particular to pharmaceutical compositions and applications thereof. 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. In the past decade or so, intestinal microecology has made great strides, 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. This is 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, high-throughput sequencing, and microbial culture technologies, the impact and role of the gut microbiome on intestinal and extraintestinal diseases has become increasingly clear. The gut microbiome is more like an organ with metabolic, immune, and endocrine functions, influencing the human digestive, circulatory, and nervous systems. It is closely associated with the development of various chronic diseases (diabetes, hypertension, cardiovascular disease, brain diseases, etc.) and tumors. Studying the interrelationship between the gut microbiome and human health and disease is not only important scientific research, but also has important significance and value in clinical diagnosis, treatment, and even translational research.

[0004] PD-1 has excellent targeting capabilities, and anti-PD-1 antibodies are commonly used in clinical tumor treatment. However, intolerance and non-response can occur during anti-PD-1 antibody treatment, primarily due to alterations in the tumor microenvironment, which in turn affect treatment efficacy. Therefore, it is necessary to provide solutions that can improve this situation.

[0005] Membrane vesicles are functional vesicles released by bacteria. Their structure is similar to liposomes, with diameters typically ranging from 20 to 400 nm. In addition to a phospholipid bilayer, they also include the bacterial outer membrane and various proteins, peptidoglycan, lipopolysaccharide, DNA, and RNA. The release of membrane vesicles is a crucial bacterial secretion mechanism, playing multiple roles in bacterial growth, survival, information exchange, and virulence factor transmission, among other physiological activities. However, further research is needed on the membrane vesicles of intestinal bacteria.

[0006] Summary of the Invention

[0007] 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.

[0008] In a first aspect of the present application, a pharmaceutical composition is provided, comprising a microbial-derived component.

[0009] In some embodiments, the microbial-derived component comprises membrane vesicles (MVs) isolated from a bacterial strain.

[0010] In some embodiments, the membrane vesicle comprises at least one of a lipid, a protein, and a nucleic acid fragment.

[0011] In some embodiments, the nucleic acid fragment comprises a DNA fragment.

[0012] 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.

[0013] In some embodiments, the microbial-derived component includes membrane vesicles (MVs) isolated from a bacterial strain, and further includes 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.

[0014] In some embodiments, 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%, or 99 wt% of the membrane vesicles.

[0015] In some embodiments, the microbial-derived component comprises 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.

[0016] In some embodiments, the microbial-derived component includes 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 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%, 99 v / v% of the membrane vesicles.

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

[0018] In some embodiments, the membrane vesicles are derived from genetically engineered microbial strains in the mammalian intestine.

[0019] In some embodiments, the bacterial strain having immune system stimulating or immunomodulating function is any one of the following A1) to A6):

[0020] A1) bacteria whose 16S rRNA is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the sequence set forth in SEQ ID NO: 1;

[0021] A2) bacteria deposited with GDMCC No: 61121;

[0022] 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%, or 100% identity to the 16S rRNA of the bacterium described in A2);

[0023] 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%, or 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%, or 99% with the genome of any of the bacteria described in A1) to A3);

[0024] A5) Enterococcus bacteria;

[0025] A6) at least one bacterium selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, Enterococcus donkey, Enterococcus avium, Enterococcus canis, Enterococcus leucoflavus, Enterococcus caecum, Enterococcus pigeonis, Enterococcus peculiaris, Enterococcus durgidus, Enterococcus yellow, Enterococcus gallinarum, Enterococcus paleum, Enterococcus hemoperoxidase, Enterococcus spp., Enterococcus malariae, Enterococcus moravianus, Enterococcus montereyii, Enterococcus leucoflavus, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus raffinose, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marinum, and Enterococcus lactis.

[0026] In some embodiments, the membrane vesicles are isolated from any one of the bacterial strains A1) to A6) above.

[0027] 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%.

[0028] In some embodiments, the pharmaceutical composition further comprises at least one of an excipient, a diluent, and a carrier.

[0029] In some embodiments, the pharmaceutical composition further comprises an excipient.

[0030] 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.

[0031] In some embodiments, the pharmaceutical composition is a solid preparation or a liquid preparation.

[0032] In some embodiments, the pharmaceutical composition is in the form of any one of powders, granules, powders, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and lavage solutions.

[0033] In some embodiments, the pharmaceutical composition further includes product instructions.

[0034] In some embodiments, the pharmaceutical composition further comprises a combined drug.

[0035] In some embodiments, the combination drug includes an anti-tumor drug.

[0036] In some embodiments, the anti-tumor drug includes at least one of a chemotherapy drug, a photosensitizer, a photothermal agent, and an immunotherapy drug.

[0037] In some embodiments, the chemotherapy drug comprises at least one of paclitaxel, camptothecin, 5-fluorouracil, cisplatin, doxorubicin, mitomycin, and epirubicin.

[0038] In some embodiments, the photosensitizer includes at least one of boron dipyrrole, dihydrochlorin, and rose bengal.

[0039] In some embodiments, the photothermal agent includes at least one of noble metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, and semiconductor nanomaterials.

[0040] In some embodiments, the immunotherapy drug includes at least one of an immune cell therapy drug and an immune checkpoint inhibitor.

[0041] 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.

[0042] 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.

[0043] In a second aspect of the present application, there is provided use of the aforementioned pharmaceutical composition in the preparation of a medicament having at least one of the following uses B1) to B4):

[0044] B1) Prevention or treatment of tumors or cancer;

[0045] B2) Modulate the immune system or stimulate the immune system;

[0046] B3) Regulate the intestines;

[0047] B4) Preventing and / or treating diseases mediated by the STING and / or NOD2 pathway.

[0048] 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.

[0049] In some embodiments, the solid tumor comprises 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 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.

[0050] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0051] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity 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, appendiceal neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of the trunk and limbs, soft tissue sarcoma of the abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of the following: 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 neoplasia, 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, ocular adnexal lymphoma, 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.

[0052] In some embodiments, the tumor or cancer comprises a tumor or cancer caused by bacteria or a virus.

[0053] In some embodiments, 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.

[0054] 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 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.

[0055] In some embodiments, the regulating or stimulating the immune system in B2) includes at least one of regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0056] In some embodiments, regulating the intestine in B3) includes at least one of regulating intestinal function and regulating intestinal flora.

[0057] In some embodiments, regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0058] In some embodiments, regulating the intestinal flora is regulating changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0059] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0060] In some embodiments, the disease in D1) is an intestinal disease.

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

[0062] In some embodiments, the anti-tumor drug of D2) comprises an immunotherapy drug.

[0063] In some embodiments, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor.

[0064] In some embodiments, 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.

[0065] 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.

[0066] 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):

[0067] B1) Prevention or treatment of tumors or cancer;

[0068] B2) Modulate the immune system or stimulate the immune system;

[0069] B3) Regulate the intestines;

[0070] B4) Preventing and / or treating diseases mediated by the STING and / or NOD2 pathway.

[0071] 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.

[0072] 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.

[0073] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0074] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity 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, appendiceal neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of the trunk and limbs, soft tissue sarcoma of the abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of the following: 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 neoplasia, 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, ocular adnexal lymphoma, 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.

[0075] In some embodiments, the tumor or cancer comprises a tumor or cancer caused by bacteria or a virus.

[0076] In some embodiments, 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.

[0077] 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 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.

[0078] In some embodiments, the regulating or stimulating the immune system in B2) includes at least one of regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0079] In some embodiments, regulating the intestine in B3) includes at least one of regulating intestinal function and regulating intestinal flora.

[0080] In some embodiments, regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0081] In some embodiments, regulating the intestinal flora is regulating changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0082] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0083] In some embodiments, the disease in D1) is an intestinal disease.

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

[0085] In some embodiments, the anti-tumor drug of D2) comprises an immunotherapy drug.

[0086] In some embodiments, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor.

[0087] In some embodiments, 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the hematological tumor comprises a tumor of at least one of blood, lymph and bone marrow.

[0093] In some embodiments, the tumor or cancer comprises oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity 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, appendiceal neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, head and neck sarcoma, soft tissue sarcoma of the trunk and limbs, soft tissue sarcoma of the abdominal and thoracic organs, gastrointestinal stromal tumor, retroperitoneal At least one of the following: 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 neoplasia, 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, ocular adnexal lymphoma, 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.

[0094] In some embodiments, the tumor or cancer comprises a tumor or cancer caused by bacteria or a virus.

[0095] In some embodiments, 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.

[0096] 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 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.

[0097] In some embodiments, the regulating or stimulating the immune system includes at least one of regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0098] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0099] In some embodiments, regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0100] In some embodiments, regulating the intestinal flora is regulating changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0101] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0102] In some embodiments, the disease in D1) is an intestinal disease.

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

[0104] In some embodiments, the anti-tumor drug of D2) comprises an immunotherapy drug.

[0105] In some embodiments, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor.

[0106] In some embodiments, 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.

[0107] 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.

[0108] In some embodiments of the present application, the composition is administered at least once a day.

[0109] In some embodiments of the present application, when the administration frequency is twice a day or more, the same dose is administered each time.

[0110] In some embodiments of the present application, when the administration frequency is twice or more per day, each administration is at a different dose.

[0111] 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.

[0112] 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;

[0113] 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.

[0114] 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 gallinarum, Enterococcus light yellow, Enterococcus hemoperoxidase, Enterococcus small intestine, Enterococcus odoriferous, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudobirdii, Enterococcus raffinose, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0115] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0116] A1) bacteria whose 16S rRNA is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the sequence set forth in SEQ ID NO: 1;

[0117] A2) bacteria deposited with GDMCC No: 61121;

[0118] 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%, or 100% identity to the 16S rRNA of the bacterium described in A2);

[0119] 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 genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the genome of any one of A1) to A3).

[0120] 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.

[0121] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0122] 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;

[0123] 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.

[0124] 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 gallinarum, Enterococcus light yellow, Enterococcus hemoperoxidase, Enterococcus small intestine, Enterococcus odoriferous, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudobirdii, Enterococcus raffinose, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0125] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0126] A1) bacteria whose 16S rRNA is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the sequence set forth in SEQ ID NO: 1;

[0127] A2) bacteria deposited with GDMCC No: 61121;

[0128] 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%, or 100% identity to the 16S rRNA of the bacterium described in A2);

[0129] 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 genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the genome of any one of A1) to A3).

[0130] 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.

[0131] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0132] 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;

[0133] 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.

[0134] 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 gallinarum, Enterococcus light yellow, Enterococcus hemoperoxidase, Enterococcus small intestine, Enterococcus odoriferous, Enterococcus moravianus, Enterococcus montereyii, Enterococcus bright yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudobirdii, Enterococcus raffinose, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulfur, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisacchari, Enterococcus marineus, and Enterococcus lactis.

[0135] In some embodiments of the present application, the Enterococcus bacterium is any one of the following A1) to A4):

[0136] A1) bacteria whose 16S rRNA is at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to the sequence set forth in SEQ ID NO: 1;

[0137] A2) bacteria deposited with GDMCC No: 61121;

[0138] 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%, or 100% identity to the 16S rRNA of the bacterium described in A2);

[0139] 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 genome has an alignment score of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% with the genome of any one of A1) to A3).

[0140] 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.

[0141] In some embodiments of the present application, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0142] In some embodiments of the present application, the C1 and the C2 are administered at least once a day.

[0143] In some embodiments of the present application, the C1 and the C2 are administered simultaneously, separately or sequentially.

[0144] In some embodiments of the present application, when the administration frequency is twice a day or more, the same dose is administered each time.

[0145] In some embodiments of the present application, when the administration frequency is twice or more per day, each administration is at a different dose.

[0146] 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.

[0147] In the embodiments of the present application, membrane vesicles, as secretions of intestinal bacteria, contain a large amount of intestinal bacterial DNA and special proteins and other substances. Therefore, these specific components can activate the entire body's immune system, thereby achieving the prevention and treatment of a variety of different 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, 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 it is prepared into a drug, a variety of preparation forms can be provided according to different needs. These components derived from the strain, including the strain, its metabolites, the supernatant of the strain, and the membrane vesicles secreted by the strain, can all exert good anti-tumor effects.

[0148] In addition, for some clinically refractory tumors such as renal cancer, fibrosarcoma, and liver cancer, it is difficult to expect that drugs will have an effective anti-tumor effect. However, in the embodiments of the present application, a combination of 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, thereby achieving a higher anti-tumor effect after combination. 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 by the intervention of the strain of the present application.

[0149] 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 pathway to exert anti-tumor activity.

[0150] On the other hand, it is noted that in diseases such as cancer, especially colorectal cancer, an imbalance of intestinal flora occurs. Even with immunotherapy such as anti-PD-1 antibodies, it is difficult to achieve significant improvement. There is still a problem of decreased intestinal flora diversity. 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 active ingredients of the above-mentioned strains has greatly restored the intestinal flora, thereby improving this situation.

[0151] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 shows the experimental results of an embodiment of the present application in which MNC-168 of lactic acid bacteria activates STING-dependent anti-tumor immunity through membrane vesicles. A shows the particle size distribution of MNC-168 membrane vesicles isolated by NanoSight technology; B shows the morphology of MNC-168 membrane vesicles observed by transmission electron microscopy (TEM), with a scale of 100 nm; C shows the evaluation of DNA content in MNC-168 vesicles by gel electrophoresis, with a DL2000 marker and 3 lanes of 10 μL, 20 μL, and 30 μL samples (vesicle concentration: 4.5×10 6 Figure 4 shows the volume of isolated vesicles (μL / μL). D shows the activation of STING activity by E. lactis MNC-168 membrane vesicles using THP-1-STING reporter cells, with H-151 (10 μM) a specific STING inhibitor. E and F show the mRNA expression levels of IFN-β and TNF-α detected by RT-PCR. Gene expression was measured in THP-1 cells treated with E. lactis MNC-168 vesicles for 24 hours, with H-151 (10 μM) a specific STING inhibitor. Analyses were performed using one-way ANOVA with Tukey's multiple comparisons test.

[0153] Figure 2 is a curve showing the changes 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 the tumor volume of mice (mm 3 ) were used to present the data, and two-way ANOVA with Dunnett's multiple comparison analysis was used for statistical analysis.

[0154] Figure 3 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 of the tumor volume at the intervention endpoint (D24) of mice (mm 3 ) Data were presented and statistically analyzed using one-way ANOVA with Tukey's multiple comparison analysis.

[0155] Figure 4 compares tumor weights of mice in each group at the end of treatment, according to one embodiment of the present invention. Data are presented as mean ± SD tumor weights (g) at the end of treatment (D24). Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons.

[0156] Figure 5 compares the tumor inhibition rates of mice in each group at the endpoint of the intervention, according to one example of the present application. Data are presented as mean ± standard deviation (%) of tumor inhibition rates in mice. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons. The dotted line represents a 40% tumor inhibition rate. A tumor inhibition rate <40% indicates no anti-tumor response, and a tumor inhibition rate ≥40% indicates a response.

[0157] Figure 6 shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each intervention mouse group in one embodiment of the present application. The graph shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each mouse in the Control group, PD-1 group, MNC-168 group and MNC-168+PD-1 group. The mean ± standard deviation of the tumor volume (mm) of each drug intervention group is shown in Figure 6. 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 (1315.92 mm 3 ), when the mouse volume > 1315.92 mm 3 When the tumor inhibition rate is less than 40%, it means no response to anti-tumor drug efficacy. When the volume of mice is ≤1315.92mm 3 When the tumor volume of the mouse is 0 mm 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 tumor disappearance at the endpoint / total number of mice in the group × 100%.

[0158] Figure 7 is a curve showing the changes 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 the tumor volume of mice (mm 3 ) were used to present the data, and two-way ANOVA with Dunnett's multiple comparison analysis was used for statistical analysis.

[0159] Figure 8 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 of the tumor volume at the intervention endpoint (D19) of mice (mm 3 ) Data were presented and statistically analyzed using one-way ANOVA with Dunnett's multiple comparison analysis.

[0160] Figure 9 compares the tumor inhibition rates of mice in each group at the endpoint of the intervention, according to one embodiment of the present application. Data are presented as mean ± standard deviation (%) of tumor inhibition rates in mice. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons. The dotted line represents a 40% tumor inhibition rate. A tumor inhibition rate <40% indicates no anti-tumor response, and a tumor inhibition rate ≥40% indicates a response.

[0161] Figure 10 shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each intervention mouse group in one embodiment of the present application. The graph shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each mouse in the control group, PD-1 group, MNC-168 group and MNC-168 + PD-1 combination group. The mean ± standard deviation of the tumor volume (mm) of each drug intervention group is shown in Figure 10. 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 (1391.658 mm 3 ), when the mouse volume > 1391.658 mm 3 When the tumor inhibition rate is less than 40%, it means no response to anti-tumor drug efficacy. When the volume of mice is ≤1391.658mm 3 When the tumor volume of the mouse is 0 mm 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 tumor disappearance at the endpoint / total number of mice in the group × 100%.

[0162] Figure 11 is a curve showing the changes 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 the tumor volume of mice (mm 3 ) were used to present the data, and two-way ANOVA with Tukey's multiple comparison analysis was used for statistical analysis.

[0163] Figure 12 is a comparison of the tumor volumes of mice in each group at the end of intervention in one embodiment of the present application. The mean ± standard deviation of the tumor volume at the end of intervention (D19) of mice (mm 3 ) Data were presented and statistically analyzed using one-way ANOVA with Tukey's multiple comparison analysis.

[0164] Figure 13 shows a comparison of tumor weights of mice in each group at the intervention endpoint in one embodiment of the present application. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison analysis, using the mean ± standard deviation (g) of tumor weight at the intervention endpoint.

[0165] Figure 14 compares the tumor inhibition rates of mice in each group at the endpoint of the intervention, according to one embodiment of the present application. Data are presented as mean ± standard deviation (%) of tumor inhibition rates. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons. The dotted line represents a 40% tumor inhibition rate. A tumor inhibition rate <40% indicates no anti-tumor response, and a tumor inhibition rate ≥40% indicates a response.

[0166] Figure 15 shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each intervention mouse group in one embodiment of the present application. The graph shows the curve of tumor volume change, anti-tumor drug effect and tumor disappearance rate of each mouse in the control group, PD-1 group, MNC-168 group and MNC-168 + PD-1 combination group. The mean ± standard deviation of the tumor volume (mm) of each drug intervention group is shown in Figure 15. 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 (1114.998 mm 3 ), when the mouse volume > 1114.998 mm 3 When the tumor inhibition rate is less than 40%, it means no response to anti-tumor drug efficacy. When the volume of mice is ≤1114.998mm 3 When the tumor volume of the mouse is 0 mm 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 tumor disappearance at the endpoint / total number of mice in the group × 100%.

[0167] Figure 16 shows the experimental results of an embodiment of the present application showing that the supernatant of Enterococcus lactis MNC-168 activates STING-dependent anti-tumor immunity. Figure A shows the activation of STING activity by the supernatant of Enterococcus lactis MNC-168 using THP-1-STING reporter cells, with H-151 (10 μM) being a specific STING inhibitor. Figures B and C show the mRNA expression levels of IFN-β and TNF-α detected by RT-PCR. THP-1 cells were treated with the supernatant of Enterococcus lactis MNC-168 for 24 hours and gene expression was measured using H-151 (10 μM), a specific STING inhibitor. One-way ANOVA was used for analysis with Tukey's multiple comparisons test.

[0168] Figure 17 shows the experimental results of an embodiment of the present application showing that the supernatant of Enterococcus lactis MNC-168 activates NOD2-dependent anti-tumor immunity. Figures A and B show RT-PCR detection of IL-1β and IL-6 mRNA expression levels. Gene expression was measured in THP-1 cells treated with the supernatant of Enterococcus lactis MNC-168 for 24 hours. GSK717 (10 μM) is a specific inhibitor of NOD2. One-way ANOVA with Tukey's multiple comparisons test was used for analysis.

[0169] Figure 18 shows the changes in tumor volume during the intervention period for each group of mice in one example of the present application, along with the results of corresponding cytokine and chemokine assays in mouse serum. Data (A) are presented as mean ± standard deviation (SD) tumor volume (mm³), using a two-way ANOVA. Data (B-E) are the ELISA results for CXCL1, CXCL9, CXCL10, and IFN-γ, respectively.

[0170] Figure 19 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. Wherein, A is a comparison of the species (left figure) and diversity (right figure) of the intestinal flora of each group, which is analyzed by one-way analysis of variance (One-Way ANOVA) method and Tukey's multiple comparisons test. All comparisons within or between pre and post groups, all comparisons with significant differences are shown in the figure as p values, and the intra-group comparisons without marked p values ​​are all no significant differences; B is a PCA analysis of the diversity of intestinal flora in each group.

[0171] Figure 20 shows the tumor volume changes during the intervention period for each group of mice in Example 10, as well as the tumor images and weights at the end of the study. A shows the tumor volume change curves, with data presented as mean ± standard deviation (mm³) of mouse tumor volume. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons. B shows the results of tumor images and weight comparisons at the end of the study. Data are presented as mean ± standard deviation (g) of mouse tumor weight at the end of the intervention period. Statistical analysis was performed using unpaired T-tests.

[0172] Figure 21 shows the results of flow cytometric analysis of immune cell populations in tumor tissues from Example 10. A shows a comparison of DC numbers in tumor tissues, B shows the CD206 / CD86 ratio of TAMs in tumor tissues, and C shows a comparison of IFN-γ+CD8+ T cell numbers in tumor tissues. Data are presented as mean ± SD, and statistical analysis was performed using unpaired T-tests.

[0173] 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

[0174] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0175] 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.

[0176] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. "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. The terms "first" and "second" are used solely to distinguish technical features and are not to be construed as indicating or implying relative importance, or as implicitly indicating the quantity of the indicated technical features, or as implicitly indicating the order of the indicated technical features.

[0177] In the description of this application, 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 this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0178] A first aspect of the embodiments of the present application relates to a pharmaceutical composition comprising a microbial-derived component, wherein the microbial-derived component comprises bacterial membrane vesicles.

[0179] 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 the 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 Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, reference WO2022156119A1.

[0180] Among them, the term "strain" refers to a member of a bacterial species with genetic characteristics that make it distinguishable 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) gains or loses antibiotic resistance or gains or loses biosynthetic ability (e.g., auxotrophic strains), strains or nutrients / metabolites can be distinguished by selection or counter-selection using antibiotics.

[0181] It is known in the art that bacterial species can be classified and identified using traditional classification methods and molecular biology methods. Traditional classification methods include, for example, cell morphology observation, Gram staining, flagellar staining, and various metabolic experiments. Molecular biology methods include ribosomal RNA sequencing and whole genome sequencing-based methods.

[0182] Therefore, in some embodiments, the bacteria include not only the aforementioned strain MNC-168 but also variants based on the strain MNC-168, for example, having at least one identical morphological and / or physiological and biochemical identification feature as MNC-168.

[0183] In addition, the sequencing results of the 16S rRNA of strain MNC-168 showed that its 16S rRNA sequence 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 shared by all bacteria, while the variable region varies to varying degrees between different bacteria. By comparing the gene sequences of bacterial 16S rRNA, according to the sequence difference base and their evolutionary distance, an evolutionary tree can be drawn.

[0184] When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, they can be judged to 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 sequence 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 cultured biobank. Microbiome, 9(1), page P23; Pablo Yarza, Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences, Microbiology, volume 12, pages P635-P645, 2014).

[0185] When the identity between the 16S rRNA gene sequences of two strains is less than 95%, they can be judged to 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)).

[0186] To this end, in some embodiments of the present application, the bacteria further include variants of strain MNC-168 based on the aforementioned 16S rRNA sequence. 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%, or 100% identity with strain MNC-168 in the 16S rRNA sequence, and are therefore strains of the same species or different species within the same genus as strain MNC-168. Based on this, it is reasonable to infer that these strains of the same species or different species within the same genus have similar biological activities or uses.

[0187] 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 commercially or publicly available programs may include the DNAStar "MegAlign" program.

[0188] In addition, with the rapid development of second- and third-generation sequencing technologies, bacterial species identification based on whole-genome sequencing has become possible, making the identification results 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 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 cultured biobank. Microbiome, 9(1), P23; Pablo Yarza, Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences, Microbiology, volume 12, pages P635-P645, 2014).

[0189] When the identity between the 16S rRNA gene sequences of two strains is less than 95%, they can be judged to belong to different genera (“A genus 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)).

[0190] Using the above method, one 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 deposited with 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%, or 100%, it can be determined to belong to the same genus or species.

[0191] Currently, various mature tools for calculating ANI values ​​are available, such as the local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), and online calculation tools ANI Calculator (http: / / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani), and ANItools. Therefore, the ANI values ​​of isolates and MNC-168 can be calculated using any of these local calculation software or online calculation tools, or other tools or methods known in the art.

[0192] To this end, in some embodiments of the present application, the bacteria further include variants of strain MNC-168 based on the aforementioned genome, wherein 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%, or 100% average nucleotide identity with strain MNC-168, and thus are strains of the same species or different species within the same genus as strain MNC-168. Based on this, it is reasonable to infer that these strains of the same species or different species within the same genus have similar biological activities or uses.

[0193] 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%, or 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%, or 100% average nucleotide identity with the genome of MNC-168.

[0194] In some embodiments, the bacteria also include variants of the strain that combine molecular biological identification results with morphological and physiological and biochemical identification results, for example, the strain variant has at least 95%, 96%, 97%, 98%, 98.5%, 98.65%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 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%, or 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.

[0195] The term "variant" includes any bacteria derived from strain MNC-168, such as those derived through natural mutation or recombination (e.g., mutations resulting from cell proliferation or cell division), resistance selection, or mutation or recombination caused by any other mechanism, such as radiation, viruses, transposons, or mutagenic chemicals. Furthermore, 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 strains of the same genus but different species as MNC-168, or strains within the same species with different phenotypic variations.

[0196] In some other embodiments, the bacteria further include other existing species under the genus Enterococcus, such as Enterococcus faecalis, Enterococcus faecium, Enterococcus assini, 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), Raffinosaccharide 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.

[0197] The term "microbial-derived components" refers to components derived from microorganisms, which may be intact microorganisms, microbial secretions, microbial 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, microbial-derived components also include microbial cultures, fermentation supernatants, etc.

[0198] In some embodiments, the microorganism of the microbial-derived component is, for example, the aforementioned bacteria, such as the aforementioned bacterial strain having immune system stimulating or immunomodulating functions.

[0199] In some embodiments, the membrane vesicles contain the aforementioned 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, for example, DNA fragments.

[0200] 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.

[0201] 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 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 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 of microbial-derived ingredients 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%, or at least 99%.

[0202] 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 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%, or at least 99%.

[0203] In some embodiments, the pharmaceutical composition can be a solid preparation or a liquid preparation. Taking into account the specific dosage form used, the pharmaceutical composition can be any one of a powder, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and lavage agents. In some embodiments, the pharmaceutical composition further comprises at least one component of an excipient, a diluent, or 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, at least one of 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 used as a carrier or excipient. At least one of ethanol, glycerol, and water can be 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, and lavages. In addition, the pharmaceutical composition can also contain at least one of any suitable lubricant, wetting agent, binder, emulsifier, suspension stabilizer, solubilizer, preservative, sweetener, and flavoring agent in addition to the carrier, excipient or diluent. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, talc, mineral oil, etc. Binders include starch, gelatin, natural sugars, and natural or synthetic gums. Natural sugars include glucose, anhydrous lactose, free-flowing lactose, β-lactose, or corn sweeteners. Natural or synthetic gums such as gum arabic, gum tragacanth, sodium alginate, carboxymethyl cellulose or polyethylene glycol. Preservatives include sodium benzoate, sorbic acid or parabens. By preparing the strain or strain culture into any of the above compositions, different release requirements of the effective active ingredient therein can be achieved, such as rapid release, sustained release, etc.

[0204] In some embodiments, the pharmaceutical composition further includes product instructions.

[0205] In some embodiments, the pharmaceutical composition further comprises a combination drug. By combining the drugs, the efficacy of the drug is enhanced. In some embodiments, the combination drug comprises an oncology drug. In some embodiments, the oncology drug may comprise at least one of a chemotherapeutic drug, a photosensitizer, a photothermal agent, and an immunotherapy drug.

[0206] Among them, chemotherapy drugs include alkylating agents (such as cyclophosphamide, ifosfamide, thiotepa, melphalan, carmustine, lomustine, semustine, nimustine, fotemustine, estramustine, nitrogen methyl, nitrogen mustard, nitrocarb, phthalate nitrogen mustard, chlorambucil, hexamethylmelamine, busulfan, temozolomide), platinums (such as cisplatin, carboplatin, oxaliplatin, suplatin, lobaplatin, nedaplatin, dicycloplatin), podophyllums (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, fufurantoin, deoxyfluridine, tegafur, carmofur, trifluridine), antibiotics (such as mitomycin, bleomycin, bleomycin), etc.

[0207] The photosensitizer includes at least one of porphyrins (such as dihydrochlorin, phthalocyanine, bacteriochlorin), boron dipyrroles (such as fluoroboron dipyrrole, azafluoroboron dipyrrole), and rose bengal.

[0208] 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, and palladium), organic polymers (such as polypyrrole, polyaniline, polyethylenedioxythiophene, polystyrene sulfonate, indocyanine green, porphyrin), carbon-based nanomaterials (such as graphene, carbon nanotubes, and 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).

[0209] 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.

[0210] The term "immune checkpoint inhibitor" refers to an antagonist that targets an immune checkpoint protein. The immune checkpoint inhibitor can enhance proteins that stimulate the immune response or block proteins that inhibit the immune response, thereby exhibiting an anti-cancer effect through the immune response. 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 antigen-binding fragment thereof that binds to the immune checkpoint protein to be inhibited; it can be an inhibitory nucleic acid (such as an siRNA molecule, shRNA molecule, antisense RNA) that specifically binds to the mRNA encoding the immune checkpoint protein.

[0211] 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.

[0212] 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- or nanoparticles, liquids (e.g., aqueous solutions), emulsions, or powders; lozenges (including liquid fillers); chewables, gels; rapidly dispersing dosage forms; ovules; sprays; and buccal / mucosal adhesive patches.

[0213] In some embodiments, the pharmaceutical composition is an enteric formulation, i.e., a gastro-resistant formulation (e.g., resistant to gastric pH) suitable for delivery of 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.

[0214] 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-coated tablet or an enteric-coated 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 comprise 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-coated capsule (e.g., from Capsugel).

[0215] In some embodiments, the preparation is a soft capsule. A soft capsule is a capsule having certain elasticity and pliability due to the addition of a softening agent 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.

[0216] In some embodiments, the formulation is a microencapsulated capsule that protects the composition from degradation by encapsulation until delivery to the target site, for example, by rupture by chemical or physical stimuli such as pressure, enzymatic activity, or physical decomposition, 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.

[0217] 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 dosage form or an injectable dosage form, and the strain in the pharmaceutical combination product is capable of at least partially 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) or dead bacteria.

[0218] The second aspect of the embodiments of the present application relates to the use of the aforementioned pharmaceutical composition in the preparation of a drug for preventing or treating tumors or cancer, and / or regulating or stimulating the immune system, and / or regulating the intestine, and / or preventing and / or treating diseases mediated by the STING and / or NOD2 pathway.

[0219] Wherein, the term "tumor" or "tumor" refers to an abnormal mass or growth that grows when cells in the body divide and proliferate beyond normal levels, and "cancer" generally refers to a malignant tumor, which can be used interchangeably 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 hematological 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, gum cancer, etc.), salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, laryngeal cancer, etc. Upper gastrointestinal cancer includes esophageal cancer, gastroesophageal junction cancer, stomach cancer, small intestine cancer, etc. Lower gastrointestinal 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, duodenal and ampulla cancers. Neuroendocrine tumors, jejunal-ileal neuroendocrine tumors, appendiceal neuroendocrine tumors, colorectal neuroendocrine tumors, pancreatic neuroendocrine tumors, etc.; chest cancers include 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, soft tissue sarcomas of the trunk and limbs, soft tissue sarcomas of the abdominal and thoracic organs, gastrointestinal stromal tumors, retroperitoneal soft tissue sarcomas, etc.; skin cancers include Merkel cell carcinoma, malignant melanoma of the skin, etc.; female reproductive system cancers include 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 adnexa lymphoma, etc. Central nervous system cancer includes brain cancer (such as glioma, meningioma, neuroblastoma, etc.) The scope of the disease includes: endocrine system cancers such as thyroid carcinoma, medullary pituitary adenoma, primary CNS lymphoma and central nervous system germ cell tumor, etc.), spinal cord tumors, etc.; endocrine system cancers including differentiated thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, parathyroid cancer, adrenal cortical cancer, etc.; Hodgkin lymphoma, non-Hodgkin lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, lymph node marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, etc.), cutaneous lymphoma, plasma cell myeloma, leukemia, etc.

[0220] 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.

[0221] 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.

[0222] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0223] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0224] The intestine is primarily used to digest and absorb protein, fat, and some carbohydrates in food, as well as to decompose cellulose and other substances into feces through the fermentation of moisture and microorganisms. Therefore, intestinal function mainly includes the following aspects: (1) motility, such as intestinal peristalsis; (2) secretion, such as intestinal fluid, which contains hormones and enzymes; (3) barrier function, 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 motility, secretion, barrier function, and immune function of the intestine.

[0225] The intestinal flora refers to the microorganisms present in the intestines. Typically, the duodenum contains fewer microorganisms, while the ileum and colon contain more. Based on taxonomic characteristics, the intestinal flora primarily includes the Bacteroidetes and Posterior Wall bacteria, but also includes Proteobacteria, Actinomycetes, Fusobacteria, Verrucomicrobia, and Cyanobacteria. Based on their spatial location in the intestine, they can also include membrane flora that adhere closely to the intestinal mucosa and luminal flora that roam in the intestinal lumen. Based on their function, they can include commensal bacteria, opportunistic pathogens, and pathogens. These intestinal flora collectively create the intestinal microecological environment, and therefore, intestinal flora imbalance can lead to problems with the digestive system, immune system, nervous system, and other aspects.

[0226] To this end, in some embodiments, regulating the intestinal flora is regulating at least one of the species, richness, uniformity, and diversity of the intestinal flora. The species of the intestinal flora refers to the species of intestinal bacteria contained in the intestinal flora, such as the presence or absence, abundance, etc. of one or more intestinal bacteria; the richness of the intestinal flora 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 the intestinal flora refers to the difference in number between different intestinal bacteria, with high uniformity indicating a small difference in number between specific or unspecific intestinal bacteria, and low uniformity indicating a large difference in number between specific or unspecific intestinal bacteria; and diversity is a comprehensive indicator of richness and uniformity, which can be evaluated, for example, by at least one of the Simpson diversity index and the Shannon diversity index.

[0227] Under normal circumstances, the intestinal flora can maintain a relatively stable distribution of its population, but due to diseases, medication, and other factors, the intestinal flora may be disturbed. Therefore, in some embodiments, regulating the intestinal flora is to regulate changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or medications. For example, at least one of the following may be reduced in richness, uniformity, and diversity; a decrease in the number of one or more commensal bacteria; an increase in the number of one or more opportunistic pathogens or pathogens; and the like.

[0228] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0229] 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.

[0230] In some embodiments, the drug of 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 (c emiplimab-rwlc), Dostarlimab, Enlonstobart, Nivolumab, Pembrolizumab, Penpulimab, Prolgolimab, Pucotenlimab, Retifanlimab, Serplul imab, 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0235] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0236] In some embodiments, regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0237] In some embodiments, regulating the intestinal flora is regulating changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0238] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0239] In some embodiments, the disease in D1) is an intestinal disease.

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

[0241] In some embodiments, the anti-tumor drug of D2) comprises an immunotherapy drug.

[0242] In some embodiments, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor.

[0243] In some embodiments, 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.

[0244] 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.

[0245] 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 pathway, comprising administering an effective dose of the aforementioned composition to a subject.

[0246] 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.

[0247] In some embodiments, regulating the immune system or stimulating the immune system includes: regulating or stimulating the innate immune effects in the gastrointestinal tract, regulating or stimulating the innate immune effects outside the gastrointestinal tract, adding one or more beneficial immune effects in the gastrointestinal tract, and adding one or more beneficial immune effects outside the gastrointestinal tract.

[0248] In some embodiments, regulating the intestine includes at least one of regulating intestinal function and regulating intestinal flora.

[0249] In some embodiments, regulating the intestinal flora is regulating at least one of the types, richness, uniformity, and diversity of the intestinal flora.

[0250] In some embodiments, regulating the intestinal flora is regulating changes in at least one of the types, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs.

[0251] In some embodiments, the changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by diseases and / or drugs include changes in at least one of the species, richness, uniformity, and diversity of the intestinal flora caused by any one of the following D1) to D2): D1) infectious diseases, tumors or cancers, inflammation, allergies, and autoimmune diseases; D2) drugs for preventing and / or treating D1).

[0252] In some embodiments, the disease in D1) is an intestinal disease.

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

[0254] In some embodiments, the anti-tumor drug of D2) comprises an immunotherapy drug.

[0255] In some embodiments, the immunotherapy drug of D2) comprises an immune checkpoint inhibitor.

[0256] In some embodiments, 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.

[0257] 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.

[0258] 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, a different dose is 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 administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection. In some embodiments, when the composition includes a microbial-derived component and a combination drug, the two can be administered simultaneously, separately, or sequentially.

[0259] The term "subject" or "patient" refers to any mammal. A subject or patient described as "in need" refers to a mammal in need of treatment (or prevention) of a disease. For example, it includes any one or more animals under the class Mammalia, such as Monotremes, Marsupials, Insectivora, Shrews, Climbing Order, Dermoptera, Chiroptera, Primates, Edentata, Pholidota, Lagomorpha, Rodentia, Carnivora, Sirenia, Hyrax, Tubulodonta, Perissodactyla, Artiodactyla, Cetacea and the like. Common mammals include Rodentia (such as mice, rats, hamsters, guinea pigs), Lagomorpha (such as rabbits), Perissodactyla (such as horses, donkeys), Artiodactyla (such as sheep, goats, camels, cattle, pigs), Primates (such as monkeys, gorillas, chimpanzees, humans), Carnivora (such as dogs, cats), etc. It is understood that the subject may be healthy or may have a disease at any stage of development.

[0260] The present application also relates to the combined use of C1 and C2, including their use in the preparation of anti-tumor drugs or for the prevention or treatment of tumors, wherein C1 comprises at least one of Enterococcus bacteria, metabolites of Enterococcus bacteria, cultures of Enterococcus bacteria strains, or supernatants of Enterococcus bacteria fermentation broths, and C2 comprises an immune checkpoint inhibitor. The anti-tumor drug refers to the prevention or treatment of at least one of renal cancer, liver cancer, and fibrosarcoma.

[0261] 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 one or more pharmaceutical agents, to a subject to reduce or prevent worsening of at least one symptom of the disease. Thus, in one embodiment, "treating" refers, inter alia, 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, in a tumor patient.

[0262] In some embodiments, the Enterococcus bacterium can be, for example, any of the aforementioned bacteria, and the immune checkpoint inhibitor can also be any of the aforementioned immune checkpoint inhibitors.

[0263] In some embodiments, the Enterococcus bacteria are live bacteria, attenuated bacteria, killed bacteria, freeze-dried bacteria, or irradiated bacteria.

[0264] 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 is alive. In some embodiments, the Enterococcus bacteria is reconstituted prior to administration. In some cases, reconstitution is performed using a diluent as described herein.

[0265] The term "membrane vesicle" or "MV" refers to a composition derived from bacteria that comprises bacterial lipids and bacterial proteins and / or bacterial nucleic acids and / or carbohydrate moieties contained within nanoparticles. These MVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different lipid species. MVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different protein species. MVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different nucleic acid species.

[0266] Those skilled in the art can prepare membrane vesicles (MVs) from bacterial culture using methods known to those skilled in the art (S. Bin Park et al. PLoS ONE. 6(3):e17629 (2011)).

[0267] In addition, the embodiments of the present application also relate 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 twice or more per day, the same dose is administered each time. In some embodiments, when the administration frequency is twice or more per day, a different dose is 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 administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

[0268] The strain involved in the following examples is Enterococcus lactis MNC-168, which was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) under the deposit number GDMCC No: 61121 on August 5, 2020, at the Guangdong Institute of Microbiology, Building 59, 5th Floor, 100 Xianlie Middle Road, Guangzhou. The source, screening method, physiological and biochemical properties, and 16S rRNA sequence (SEQ ID No. 1) of this strain are described in WO2022156119A1, which is incorporated into this application by reference.

[0269] The above implementation manner is described below with reference to specific examples.

[0270] Example 1: Preparation of Enterococcus lactis membrane vesicles

[0271] 1. Preparation of MNC-168 supernatant

[0272] Thaw a cryopreserved glycerol tube of Enterococcus lactis MNC-168 at 37°C and activate it by inoculating it onto an anaerobic blood plate in an anaerobic workstation. Inoculate the activated strain into MM01 liquid medium and culture anaerobically to obtain a sufficient number of viable bacteria. Centrifuge the culture at 10,000 g for 10 minutes to precipitate the bacteria. Pass the supernatant through a 0.22 μm filter membrane and use it for subsequent experiments or freeze it at -80°C.

[0273] 2. Membrane Vesicle Isolation

[0274] 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).

[0275] 3. Identification of Membrane Vesicles

[0276] (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.

[0277] (2) 5 μL of the isolated bacterial membrane vesicle sample was dropped onto a copper grid, incubated for 1 min, stained with 1% phosphotungstic acid for 2 min, quickly washed with 1 drop of pure water, washed twice, dried at room temperature for 10 min, and observed the bacterial extracellular vesicles under a transmission electron microscope (80 kV voltage).

[0278] (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.

[0279] The results are shown in Figure 1A to C. The isolated membrane vesicles are round, with a diameter of usually around 100 nm. At the same time, electrophoresis results show that the membrane vesicles carry a large amount of DNA from bacteria, with the molecular weight mainly concentrated below 100 bp.

[0280] Example 2: Lactococcus lactis membrane vesicles activate STING pathway-dependent anti-tumor immunity

[0281] STING repoter cell evaluation: The evaluation was performed using THP-1 cells (a cell line developed by Muen) that have been constructed and carry the IFNβ gene promoter reporter gene. The steps are as follows:

[0282] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1×10 cells per well. 5There were three groups: Control group, MNC-168MV group, and 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 After 24 hours of culture, cells were centrifuged at 300g for 5 minutes, the supernatant was removed, and 50μL of 1× luciferase detection reagent was added. The reaction was allowed to proceed for 1 minute, and luminescence was measured using a microplate reader. Relative luminescence values ​​were normalized to those of the control group to evaluate the effect of Enterococcus lactis MNC-168MVs on IFNβ transcriptional activity.

[0283] RT-PCR detection: THP-1 cells were treated with the same experimental steps as those for STING reporter cells. The cells after different treatments were collected and total RNA was extracted, reverse transcribed, and the expression of the target gene was detected by qPCR.

[0284] The results, shown in Figures 1D-F, show that vesicles from Enterococcus lactis MNC-168 significantly boosted STING activity by up to 3-fold, a phenomenon that was inhibited by the STING-specific inhibitor H-151. Furthermore, testing of STING's downstream target genes, IFN-β and TNF-α, revealed that vesicles from Enterococcus lactis MNC-168 also significantly activated the expression of these two genes, increasing their expression by 5-fold and 4-fold, respectively, and this activation was also inhibited by the STING-specific inhibitor H-151.

[0285] 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, which inhibit tumor cell proliferation, metastasis and angiogenesis, promote tumor cell apoptosis, and activate anti-tumor immune responses, thereby promoting anti-tumor immune activity.

[0286] Example 3: Combination of MNC-168 and anti-PD-1 antibody for the treatment of renal cancer

[0287] The therapeutic efficacy of Enterococcus lactis MNC-168 combined with an anti-PD-1 antibody was validated in a mouse syngeneic tumor model of Renca (renal carcinoma). This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotech.

[0288] Test strains: After thawing the glycerol cryopreserved tube of Enterococcus lactis MNC-168 at 37°C, the strain was inoculated on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain was inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient number of viable bacteria. The cultured liquid was centrifuged and concentrated, and the bacteria were resuspended in a solvent to obtain the purity and viable count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0289] Tumor cells: Renca mouse renal carcinoma cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0290] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared in normal saline at 2 mg / mL, sourced from BioXcell.

[0291] Experimental animals: The experimental mice were 55 BALB / c mice, 5 weeks old, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0292] Animal experiment: BALB / c mice were divided into two groups with a body weight of 20.0-22.0 g, with 27-28 mice in each group. They were divided into two groups, control and Enterococcus lactis MNC-168. On the day of tumor inoculation (D0), each group was gavaged with culture medium or 2×10 9 CFU / mouse live bacterial suspension of Enterococcus lactis MNC-168, on the day of tumor inoculation (D0), 5×10 4 The number of cells per mouse was subcutaneously inoculated with Renca renal carcinoma cells. Five days after tumor inoculation, the tumor grew to an average volume of 20-50 mm. 3 Around 30 days, 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 the control, PD-1, MNC-168, and MNC-168 + PD-1 groups. The mice were given the following drugs: culture medium + 0.9% sodium chloride injection, culture medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. Culture medium or live bacterial drug suspension was administered by oral gavage once a day at 2×10 9CFU / 0.2ml / mouse. 0.9% sodium chloride injection or anti-mPD-1 (PD-1) at 200μg / 0.1ml / mouse was administered via intraperitoneal injection on days 5, 8, 11, and 14. Animal health, tumor progression, tumor size, and mouse body weight were monitored regularly at designated times throughout the study. At the endpoint, all surviving mice were dissected, and tumor weight and volume were measured. Statistical analysis and comparison were performed using tumor volume curves, endpoint tumor volume, endpoint tumor weight, and tumor growth inhibition (TGI). TGI = 1 - (individual tumor volume - individual tumor volume at grouping) / (mean tumor volume of the control group - mean tumor volume of the control group at grouping) × 100%. All data are presented as mean ± SD and analyzed using GraphPad Prism 8.0.2 software. For comparisons between three or more groups, one-way ANOVA with Tukey's multiple comparisons test was used. For two-way comparisons, two-way ANOVA with Sidak's multiple comparisons test was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0293] The results are shown in the following table:

[0294] Table 1. Experimental results of MNC-168 combined with anti-PD-1 antibody for the treatment of renal cancer

[0295] The results are shown in Table 1 and Figures 2 to 6. The results show 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 (as shown in Figure 2). At the end of the experiment, the average tumor volume of the mice in the control group was 2193 mm 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 mice in the MNC-168 group was 883 mm 3 The average tumor weight was 0.8880 g; the average tumor volume of mice in the MNC-168+PD-1 group was 398 mm 3The average tumor weight was 0.3786g. Compared to the PD-1 alone group, the endpoint tumor volume and weight of mice treated with MNC-168 plus PD-1 antibody showed a significant decrease (Figures 3 and 4). At the endpoint, the tumor growth inhibition rate (TGI) for PD-1 was 45.50%, while that for the MNC-168 plus PD-1 group was 81.88%, significantly higher than that for the PD-1 alone group (Figure 5). At the endpoint, the response rate for PD-1 was 50.0%, while that for MNC-168 plus PD-1 was 87.5%. The combination of MNC-168 and anti-PD-1 antibody significantly improved the response rate of tumors treated with anti-PD-1 antibody alone (Figure 6). Furthermore, the corresponding tumor cure rate (disappearance rate) was 0% for PD-1 and 37.5% for MNC-168 plus PD-1. The combination of MNC-168 and anti-PD-1 antibody significantly improved the cure rate of tumors treated with anti-PD-1 antibody alone. Therefore, these results indicate that MNC-168 can significantly enhance the therapeutic efficacy of anti-PD-1 antibodies in renal cancer.

[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 an anti-PD-1 antibody was validated in the WEHI 164 mouse syngeneic tumor model (fibrosarcoma). This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Mun Biopharmaceuticals.

[0298] Test strains: After thawing the glycerol cryopreserved tube of Enterococcus lactis MNC-168 at 37°C, the strain was inoculated on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain was inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient number of viable bacteria. The cultured liquid was centrifuged and concentrated, and the bacteria were resuspended in a solvent to obtain the purity and viable 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 in normal saline at 2 mg / mL, sourced from BioXcell.

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

[0302] Animal experiment: BALB / c mice were divided into two groups with a body weight of 20.0-22.0 g, with 27-28 mice in each group. They were divided into two groups, control and Enterococcus lactis MNC-168. On the day of tumor inoculation (D0), each group was gavaged with culture medium or 2×10 9 CFU / mouse live bacterial suspension of Enterococcus lactis MNC-168, on the day of tumor inoculation (D0), 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 Around 30 days, 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 the control, PD-1, MNC-168, and MNC-168 + PD-1 groups. The mice were given the following drugs: culture medium + 0.9% sodium chloride injection, culture medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. Culture medium or live bacterial drug suspension was administered by oral gavage once a day at 2×10 9 CFU / 0.2ml / mouse. 0.9% sodium chloride injection or anti-mPD-1 (PD-1) at 200μg / 0.1ml / mouse was administered via intraperitoneal injection on days 5, 8, 11, and 14. Animal health, tumor progression, tumor size, and mouse body weight were monitored regularly at designated intervals throughout the study. At the endpoint, all surviving mice were dissected, and tumor weight and volume were measured. Statistical analysis and comparison were performed using tumor volume curves, endpoint tumor volume, and tumor growth inhibition at endpoint (TGI). TGI = 1 - (individual tumor volume - individual tumor volume at grouping) / (mean tumor volume of the control group - mean tumor volume of the control group at grouping) × 100%. All data are presented as mean ± SD and analyzed using GraphPad Prism 8.0.2 software. Comparisons between three or more groups were performed using one-way ANOVA with Tukey's multiple comparisons test. Two-way ANOVA with Sidak's multiple comparisons test was used for analysis of variance. 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] The results are shown in Table 2 and Figures 7 to 10. The results show 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 the 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 8). At the experimental endpoint, 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 (Figure 9). In terms of the 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 (Figure 10). In addition, the corresponding tumor cure rate (disappearance rate) was 0% for PD-1, while it was 14.3% for MNC-168 + PD-1. The combination of MNC-168 and anti-PD-1 antibody significantly improved the tumor treatment cure rate of anti-PD-1 antibody alone. Therefore, these results suggest that MNC-168 can significantly enhance the therapeutic efficacy of anti-PD-1 antibodies in fibrosarcoma.

[0306] Example 5: Combination of MNC-168 and anti-PD-1 antibody for the treatment of liver cancer

[0307] The therapeutic efficacy of MNC-168 combined with an anti-PD-1 antibody was validated in the H22 mouse syngeneic tumor model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Mun Biopharmaceuticals.

[0308] Test strains: After thawing the glycerol cryopreserved tube of Enterococcus lactis MNC-168 at 37°C, the strain was inoculated on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain was inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient number of viable bacteria. The cultured liquid was centrifuged and concentrated, and the bacteria were resuspended in a solvent to obtain the purity and viable count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0309] Tumor cells: H22 mouse hepatocellular carcinoma cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0310] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared in normal saline at 2 mg / mL, sourced from BioXcell.

[0311] Experimental animals: The experimental mice were C57BL / 6J mice, 4 weeks old, a total of 45, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0312] Animal experiment: C57BL / 6J mice were divided into three groups based on body weight (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 9 CFU / mouse live bacterial suspension of Enterococcus lactis MNC-168, on the day of tumor inoculation (D0), 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 14 h, the following drugs were administered: culture medium + 0.9% sodium chloride injection, culture medium + PD-1, MNC-168 + PD-1, culture medium or live bacterial drug suspension was administered by oral gavage once a day at 2 × 10 9CFU / 0.2 ml / mouse. 0.9% sodium chloride injection or anti-mPD-1 (PD-1) at 200 μg / 0.1 ml / mouse was administered via intraperitoneal injection on days 6, 9, 12, and 16. Animal health, tumor progression, tumor size, and mouse body weight were monitored regularly at designated times throughout the study. At the endpoint, all surviving mice were dissected, and tumor weight and volume were measured. Statistical analysis and comparison were performed using tumor volume curves, endpoint tumor volume, endpoint tumor weight, and tumor growth inhibition (TGI). TGI = 1 - (individual tumor volume - individual tumor volume at grouping) / (mean tumor volume of group A - mean tumor volume of group A at grouping) × 100%. All data are presented as mean ± SD and analyzed using GraphPad Prism 8.0.2 software. For comparisons between three or more groups, one-way ANOVA with Tukey's multiple comparisons test was used. For two-way comparisons, two-way ANOVA with Sidak's multiple comparisons test was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0313] Table 3. Experimental results of MNC-168 combined with anti-PD-1 antibody for the treatment of liver cancer

[0314] The results are shown in Table 3 and Figures 11 to 15. The results show 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 (as shown in Figure 11). At the end of the experiment, the average tumor volume of the 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 3The average tumor weight was 0.5367g. Compared to 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 (Figures 12 and 13). At the experimental endpoint, 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%, significantly higher than that of the PD-1 group (Figure 14). At the experimental endpoint, 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 antibodies significantly improved the response rate of tumor treatment (Figure 15). These results indicate that MNC-168 can significantly enhance the therapeutic effect of anti-PD-1 antibodies in liver cancer.

[0315] Example 6: Lactobacillus lactis culture supernatant promotes STING pathway-dependent anti-tumor immunity

[0316] Preparation of MNC-168 supernatant: Thaw a cryovial of Enterococcus lactis MNC-168 at 37°C and activate it by inoculating it onto an anaerobic blood plate in an anaerobic workstation. Inoculate the activated strain into MM01 liquid medium and culture anaerobically to obtain a sufficient number of viable bacteria. Centrifuge the culture at 10,000 g for 10 minutes to pellet the bacteria. Pass the supernatant through a 0.22 μm filter membrane and use it for subsequent experiments or freeze it at -80°C.

[0317] STING repoter cell evaluation: The evaluation was performed using THP-1 cells (a cell line developed by Muen) that have been constructed and carry the IFNβ gene promoter reporter gene. The steps are as follows:

[0318] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1×10 cells per well. 5 Three groups (control, MNC-168SP, and MNC-168SP+H-151) were set up. 10 μL of MM01 medium, 10 μL of MNC-168 culture supernatant, and 10 μL of MNC-168 culture supernatant (containing H-151 at a final concentration of 10 μM) were added, respectively. After 24 hours of culture, the cells were centrifuged at 300 g for 5 minutes, the supernatant removed, and 50 μL of 1× luciferase detection reagent added. The reaction was allowed to proceed for 1 minute, and luminescence was measured using a microplate reader. Relative fluorescence values ​​(Relative Luminescence) were normalized to those of the control group to evaluate the effect of the MNC-168 supernatant on IFNβ transcriptional activity.

[0319] RT-PCR detection: THP-1 cells were treated with the same experimental steps as those for STING reporter cells. The cells after different treatments were collected and total RNA was extracted, reverse transcribed, and the expression of the target gene was detected by qPCR.

[0320] The results, shown in Figures 16A to 16C, show that the supernatant of Enterococcus lactis MNC-168 significantly boosted STING activity by 2-fold, and this activation was inhibited by the STING-specific inhibitor H-151. Furthermore, testing of the STING downstream target genes IFN-β and TNF-α showed that the supernatant of Enterococcus lactis MNC-168 also significantly activated the expression of these two genes, increasing their expression by approximately 4-fold, and this activation was also inhibited by the STING-specific inhibitor H-151.

[0321] 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, which inhibit tumor cell proliferation and metastasis, promote tumor cell apoptosis, and activate anti-tumor immune responses, thereby promoting anti-tumor immune activity.

[0322] Example 7: Lactobacillus lactis culture supernatant promotes NOD2 pathway-dependent anti-tumor immunity

[0323] Preparation of MNC-168 supernatant: The MNC-168 supernatant prepared in Example 6 was used.

[0324] NOD2 activation evaluation experiment: THP-1 cells were seeded in 12-well plates, with 5×10 cells per well. 5 Three groups (control, MNC-168SP, and MNC-168SP+GSK717) were set up. 50 μL of MM01 medium, 50 μL of MNC-168 culture supernatant, and 50 μL of MNC-168 culture supernatant (containing a final concentration of 10 μM GSK717) were added, respectively. After 24 hours of culture, the cells were centrifuged at 300 g for 5 minutes, the supernatant removed, and 50 μL of 1× luciferase detection reagent added. The reaction was allowed to proceed for 1 minute, and luminescence was measured using a microplate reader. Relative fluorescence values ​​(Relative Luminescence) were normalized to those of the control group to evaluate the effect of the supernatant of Enterococcus lactis MNC-168 on NOD2 activity.

[0325] 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.

[0326] The results are shown in Figure 17A and B, which show that the supernatant of Enterococcus lactis MNC-168 can significantly promote the upregulation of the expression of NOD2 downstream target genes IL-1β and IL-6, which are increased by more than 20 times and 10 times, respectively. This activation can also be partially inhibited by the NOD2-specific inhibitor GSK717.

[0327] These results indicate that the supernatant of Enterococcus lactis MNC-168 can induce NOD2 pathway-dependent activation to a certain extent, thereby upregulating the expression of interferon and tumor necrosis factor, which have the effects of inhibiting tumor cell proliferation and metastasis, promoting tumor cell apoptosis, and activating anti-tumor immune responses, thereby promoting anti-tumor immune activity.

[0328] Example 8: Lactobacillus promotes activation of systemic immunity

[0329] The therapeutic efficacy of Enterococcus lactis MNC-168 combined with anti-PD-1 antibodies and their effects on cytokines and chemokines in mouse fluid were evaluated in the MC38 mouse syngeneic tumor model. This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotech.

[0330] Test strains: After thawing the glycerol cryopreserved tube of Enterococcus lactis MNC-168 at 37°C, the strain was inoculated on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain was inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient number of viable bacteria. The cultured liquid was centrifuged and concentrated, and the bacteria were resuspended in a solvent to obtain the purity and viable count (1×10 10 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0331] Tumor cells: MC38 mouse colorectal cancer cells, sourced from Nanjing Kebai Biotechnology Co., Ltd.

[0332] Anti-PD-1 antibody: anti-mPD-1 antibody (RMP1-14), prepared in normal saline at 2 mg / mL, sourced from BioXcell.

[0333] Experimental animals: The experimental mice were C57BL / 6J mice, 5 weeks old, a total of 55, from Nanjing Jicui Yaokang Biotechnology Co., Ltd.

[0334] Animal experiment: C57BL / 6J mice were divided into two groups based on body weight (20.0-22.0 g), with 27-28 mice in each group. They were divided into two groups: 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 / mouse live bacterial suspension of Enterococcus lactis MNC-168, on the day of tumor inoculation (D0), 5×104 The number of cells per mouse was subcutaneously inoculated with Renca renal carcinoma cells. Five days after tumor inoculation, the tumor grew to an average volume of 20-50 mm. 3 Around 30 days, 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 the control, PD-1, MNC-168, and MNC-168 + PD-1 groups. The mice were given the following drugs: culture medium + 0.9% sodium chloride injection, culture medium + PD-1, MNC-168 + 0.9% sodium chloride injection, and MNC-168 + PD-1, respectively. Culture medium or live bacterial drug suspension was administered by oral gavage once a day at 2×10 9 CFU / 0.2ml / mouse. 0.9% sodium chloride injection or anti-mPD-1 (PD-1) antibody (200μg / 0.1ml / mouse) was administered via intraperitoneal injection on days 5, 8, 11, and 14. Animal symptoms, tumor progression, tumor size, and mouse body weight were monitored regularly during the study. At the end of the study, blood was collected from the mice, serum was separated, and serum concentrations of corresponding cytokines and chemokines were measured using ELISA kits.

[0335] The results, as shown in Figure 18A, show that MNC-168 significantly enhanced the therapeutic effect of anti-PD-1 antibodies on MC38 cells. Further ELISA analysis of serum cytokine and chemokine levels, as shown in Figures 18B-E, showed that the combination of MNC-168 and anti-PD-1 antibodies further enhanced the increases in serum CXCL1, CXCL9, CXCL10, and IFN-γ concentrations compared to anti-PD-1 antibody alone.

[0336] Example 9: Enterococcus lactis maintains intestinal flora diversity in tumor-bearing mice

[0337] Mouse fecal sample collection: 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 the four groups of Control (CT), PD-1, MNC-168, and MNC-168+PD-1 before (pre) and after (post) tumor inoculation and treatment, namely preCT, prePD-1, preMNC-168, preMNC-168+PD-1, postCT, postPD-1, postMNC-168, and postMNC-168-PD-1.

[0338] 16S RNA sequencing analysis: Mouse fecal samples were sent to a sequencing company for DNA extraction, 16S ribosomal RNA gene amplification, Illumina MiSeq sequencing, bacterial identification and richness analysis, and finally bacterial diversity analysis.

[0339] The results, as shown in Figure 19, showed no differences in gut microbial species and diversity between the treatment groups before and after tumor inoculation (pre-CT). However, after MC-38 colorectal cancer cell inoculation, a comparison of the pre-CT and post-CT groups revealed a significant decrease in gut microbial species and diversity in the post-CT group. However, after MNC-168 administration following MC-38 inoculation (post-MNC-168), there was no significant change in gut microbial species and diversity compared to the pre-MNC-168 group before MC-38 inoculation. Compared to the changes in gut microbiota before and after tumor inoculation in the CT group, MNC-168 treatment restored gut microbial stability. The results of PD-1 or MNC-168+PD-1 administration were similar to those of MNC-168 administration (Figure 19A). PCA analysis also revealed similar results: in the CT group, gut microbial diversity changed before and after cancer cell inoculation, but after MNC-168 administration, gut microbial diversity remained similar before and after cancer cell inoculation (Figure 19B). These results suggest that MNC-168 can restore the intestinal flora of mice.

[0340] These results demonstrate that MNC-168 can enhance the therapeutic effects of anti-PD-1 antibodies. Furthermore, the combination of MNC-168 and anti-PD-1 antibodies further boosts the host immune system, thereby enhancing the overall immune capacity of the body. Therefore, it has therapeutic potential against a variety of tumors and cancers, including soft tissue tumors, hematologic tumors, adenoma, and metastatic tumors. Furthermore, MNC-168 can also mitigate the decrease in gut microbial diversity caused by anti-PD-1 antibodies, thereby effectively maintaining a balanced gut microbiome.

[0341] Membrane vesicles, secretions of intestinal bacteria, contain a large amount of intestinal bacterial DNA and special proteins and other substances. Therefore, these specific components can activate the entire body's immune system, thereby achieving the prevention and treatment of a variety of different 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, as well as regulating the intestine, 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 provided according to different needs. These components derived from the strain, including the strain, its metabolites, the supernatant of the strain, and the membrane vesicles secreted by the strain, can all exert good anti-tumor effects.

[0342] In addition, for some clinically refractory tumors such as renal cancer, fibrosarcoma, and liver cancer, it is difficult to expect that drugs will have an effective anti-tumor effect. However, in the embodiments of the present application, a combination of 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, thereby achieving a higher anti-tumor effect after combination. 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 by the intervention of the strain of the present application.

[0343] 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 pathway to exert anti-tumor activity.

[0344] Example 10: Lactococcus lactis membrane vesicles for the treatment of colorectal cancer

[0345] The therapeutic efficacy of MNC-168 membrane vesicles was validated in a mouse syngeneic tumor model of CT-26 (colorectal cancer). This experimental protocol has been reviewed by the Institutional Animal Care and Use Committee of Muen Biotechnology.

[0346] Test strains: After thawing the glycerol cryopreserved tube of Enterococcus lactis MNC-168 at 37°C, the strain was inoculated on an anaerobic blood plate in an anaerobic workstation for activation. The activated strain was inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient number of viable bacteria. The cultured liquid was centrifuged and concentrated, and the bacteria were resuspended in a solvent to obtain the purity and viable count (1×1010 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0347] 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.

[0348] Tumor cells: CT-26 mouse colorectal cancer cells, from the American type culture collection (ATCC). The inoculated cell number was 1×10 6 cells /

[0349] Experimental animals: BALB / c mice, 8 weeks old, were purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0350] Administration and Dosage: MNC-168 membrane vesicles were injected intratumorally, with 50 μL containing 10 μg of membrane vesicles per dose, once every two days for a total of four doses. The control group was injected with PBS.

[0351] Animal experiment: Several female BALB / c mice were ordered for this study. After quarantine, tumor cells were inoculated to establish a CT26 colorectal cancer ectopic syngeneic tumor model. The day of tumor inoculation was designated as Day 0. Tumor measurements were taken starting from Day 5. When the tumor grew to an average volume of 100 mm 3 After excluding mice with abnormal tumor growth, at least 12 mice were randomly stratified into groups based on tumor volume. MNC-168 membrane vesicles were injected intratumorally on the day of grouping. Animal characteristics, tumor progression, tumor size, and mouse body weight were regularly monitored throughout the study. Tumor volume and weight changes were analyzed at the endpoint. Body weights of all mice increased steadily throughout the study. All data are presented as mean ± SD and analyzed using GraphPad Prism 8.0.2 software. Comparisons between groups of three or more were analyzed using one-way ANOVA with Tukey's multiple comparisons test. Two-way ANOVA with Sidak's multiple comparisons test were used for analysis of differences. Significant differences are indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0352] Table 4. Results of MNC-168MV treatment of CT-26 mice with colorectal cancer

[0353] The experimental results showed that, according to the results in Table 4 and Figure 20A, 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, endpoint autopsy, photography, and weighing revealed that the tumor weight in the MNC-168MV group was significantly smaller than that in the control group (Figure 20B). The control group tumor weighed 1.043 g, while the MNC-168MV group weighed 0.645 g. These experimental results demonstrate that MNC-168 membrane vesicles inhibit CT-26 tumor growth, suggesting that MNC-168 can achieve its anti-tumor effects through secretion of membrane vesicles.

[0354] Example 11: Detection of biomarkers in the treatment of colorectal cancer using Enterococcus lactis membrane vesicles

[0355] This study used flow cytometry to detect and analyze the changes in the immune cell population in Example 10 to elucidate the anti-tumor mechanism of action of lactic acid Enterococcus membrane vesicles.

[0356] Preparation of single cell suspension of tumor tissue:

[0357] 1) Preparation of tumor tissue suspension: The tumor tissue is mechanically minced, homogenized, consumed, and ground to obtain a tumor grinding solution.

[0358] 2) Centrifuge the tumor grinding solution at 500g for 5 minutes at room temperature. Invert the centrifuge tube on a paper towel for 5-10 seconds and discard the supernatant.

[0359] 2) Add 10 mL of red blood cell lysis solution and lyse the red blood cells for 10 minutes.

[0360] 3) Add PBS to the 30ml mark of a 50ml centrifuge tube.

[0361] 4) Centrifuge at 500g for 5 minutes at room temperature, invert the tube on a paper towel, and discard the supernatant.

[0362] 5) Resuspend the cell pellet in PBS and pass the cell suspension 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.

[0363] Cell staining and flow cytometry indicators: refer to Table 5

[0364] Table 5. Flow cytometry indicators

[0365] 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 (Figure 21A). In addition, in the analysis of tumor-associated macrophages (TAM), the ratio of M2 TAM expressing CD206 to M1 TAM expressing CD86 decreased significantly in the MNC-168MV group, indicating that MNC-168MV inhibited the effect of M2 TAM (Figure 21B); 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 (Figure 21C). These results indicate that MNC-168 membrane vesicles can increase anti-tumor effects by promoting innate immunity and adaptive immunity. Combined with the results of previous experiments, it is suggested that MNC-168 can promote the activation of innate immunity and adaptive immunity in the body by secreting vesicles, thereby increasing the anti-tumor effect.

[0366] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a 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); The average nucleotide identity of the genome of A4) to the genome of any of the bacteria described in A1) to A3) is at least 87%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100% of the bacteria; and / or, the alignment score of the genome with the genome of any of the bacteria described in A1) to A3) is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the bacteria; 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 viruses include 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 viruses include 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 viruses include 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 luminescent yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharides, 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 luminescent yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharides, 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 luminescent yellow, Enterococcus wood hoopoe, Enterococcus suis, Enterococcus pseudoavium, Enterococcus gossypii, Enterococcus muris, Enterococcus saccharolyticus, Enterococcus sulphureus, Enterococcus villus, Enterococcus davis, Enterococcus hermannii, Enterococcus paucisaccharides, 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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