WO2022156119A1 - 一种乳酸肠球菌、预防或治疗肿瘤的药物及应用 - Google Patents

一种乳酸肠球菌、预防或治疗肿瘤的药物及应用 Download PDF

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WO2022156119A1
WO2022156119A1 PCT/CN2021/098848 CN2021098848W WO2022156119A1 WO 2022156119 A1 WO2022156119 A1 WO 2022156119A1 CN 2021098848 W CN2021098848 W CN 2021098848W WO 2022156119 A1 WO2022156119 A1 WO 2022156119A1
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cancer
mnc
tumor
inhibitory
pharmaceutical composition
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PCT/CN2021/098848
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French (fr)
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李宝霞
林诠盛
贤一博
蒋先芝
梁婕凝
刘真真
陈�胜
苏雪
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慕恩（广州）生物科技有限公司
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Priority to US18/273,216 priority Critical patent/US20240082320A1/en
Priority to CN202180091324.9A priority patent/CN117480243A/zh
Priority to EP21920502.8A priority patent/EP4282950A1/en
Publication of WO2022156119A1 publication Critical patent/WO2022156119A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present disclosure relates to the field of medical biotechnology, in particular, to Enterococcus lactis, a medicine for preventing or treating tumors, and applications.
  • PD-1/PD-L1 immunotherapy is a new type of anti-tumor immunotherapy that has attracted worldwide attention and is widely researched. It aims to make full use of the body's own immune system to resist and fight tumors. By blocking PD The -1/PD-L1 signaling pathway enables tumor cell death, has the potential to treat multiple types of tumors, and substantially improve patient overall survival.
  • the existing immunotherapy has certain problems such as toxic side effects and treatment failure, and the introduction of intestinal microorganisms for immunotherapy treatment is a brand-new intervention method.
  • the present disclosure provides an application of Enterococcus lactis in the preparation of tumor suppressors.
  • the tumor optionally comprises at least one of the following solid tumors: colon cancer, rectal cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, kidney cancer, fibrosarcoma, lung cancer, and bile duct cancer .
  • the tumor further comprises the following solid tumors: ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, myeloma, lymphoma, brain tumor, spinal cord tumor, esophageal cancer, oropharyngeal cancer , laryngeal cancer, colorectal cancer, melanoma, neuroendocrine cancer, CNS cancer, non-Hodgkin's lymphoma, hematological malignancies, renal tumors, neuroblastoma, homeostasis sarcoma, Wing family sarcoma, retinal tumors, diffuse Large cell lymphoma, advanced CD70+ cancer.
  • solid tumors ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, myeloma, lymphoma, brain tumor, spinal cord tumor, esophageal cancer, oropharyngeal cancer , laryngeal cancer, colorectal cancer, melanoma, neuroendocrine cancer, CNS cancer, non-
  • the Enterococcus lactis is deposited in the Guangdong Provincial Microorganism Culture Collection Center, the deposit name is Enterococcus lactis (Enterococcus lactis) MNC-168, and the deposit number is GDMCC NO: 61121.
  • the characteristics of the colony culture of Enterococcus lactis are: on the bacterial medium, after 24 hours of culture, the colony is white, round, with a moist surface, opaque, and neat edges; Ellipsoid, 0.7-1.0 ⁇ m ⁇ 0.8-1.3 ⁇ m, arranged singly or in pairs, Gram-positive.
  • the bacterial culture medium is MRS medium.
  • the present disclosure provides a medicament for preventing or treating tumors, wherein the medicament comprises Enterococcus lactis.
  • the medicament for preventing or treating tumors further includes pharmaceutically acceptable additives or excipients.
  • the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections .
  • the pharmaceutically acceptable additives include one of fillers, diluents, wetting agents, disintegrants, glidants, lubricants, binders, colorants, or variety.
  • the pharmaceutically acceptable excipients include cyclodextrin, starch, sucrose, lactose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesium stearate, talc, oxidized One or more of iron and sodium hydroxymethyl starch.
  • the Enterococcus lactis is deposited in the Guangdong Provincial Microorganism Culture Collection Center, the deposit name is Enterococcus lactis (Enterococcus lactis) MNC-168, and the deposit number is GDMCC NO: 61121.
  • the present disclosure provides a pharmaceutical composition comprising the drug for preventing or treating tumors.
  • the pharmaceutical composition further includes a concomitant drug.
  • the combined drug is at least one of the following drugs:
  • Chemotherapy drugs photosensitizers, photothermal agents, antibodies to inhibitory second signal molecules, inhibitors of inhibitory second signal molecules, PD-L1 inhibitors, PD-1/PD-L1 monoclonal antibodies.
  • the combined drug is at least one of the following drugs:
  • Chemotherapy drugs photosensitizers, photothermal agents, antibodies to inhibitory signaling pathway molecules, inhibitors of inhibitory signaling pathway molecules, PD-L1 inhibitors, PD-1/PD-L1 monoclonal antibodies.
  • the inhibitory second signaling molecule includes at least one of the following signaling molecules: PD-1 and CTLA-4.
  • the inhibitory signaling pathway molecules include at least one of the following signaling molecules: PD-1 and CTLA-4.
  • the antibody to the inhibitory second signal molecule or the inhibitor of the inhibitory second signal molecule comprises at least one of the following antibodies: nivolumab, pembrolizumab, tisleliz Monoclonal antibody, nivolumab injection, pembrolizumab injection, toripalimab injection and sintilimab injection.
  • the antibody to the inhibitory signaling pathway molecule or the inhibitor of the inhibitory signaling pathway molecule comprises at least one of the following antibodies: nivolumab, pembrolizumab, tislelizumab , nivolumab injection, pembrolizumab injection, toripalizumab injection and sintilimab injection.
  • the PD-L1 inhibitor is selected from durvalumab, atezolizumab or avelumab.
  • the PD-1/PD-L1 monoclonal antibody drug is selected from pembrolizumab or nivolumab.
  • the present disclosure provides an Enterococcus lactis, which is preserved in the Guangdong Provincial Microbial Culture Collection Center, the preservation name is Enterococcus lactis MNC-168, and the preservation number is GDMCC NO: 61121.
  • the characteristics of the colony culture of Enterococcus lactis are: on the bacterial medium, after 24 hours of culture, the colony is white, round, with a moist surface, opaque, and neat edges; Ellipsoid, 0.7-1.0 ⁇ m ⁇ 0.8-1.3 ⁇ m, arranged singly or in pairs, Gram-positive.
  • the bacterial culture medium is MRS medium.
  • the present disclosure provides the use of the medicament and/or the pharmaceutical composition for the treatment of tumors.
  • the present disclosure provides a method of treating tumors, the method comprising:
  • a therapeutically effective amount of the drug or the pharmaceutical composition is administered to the subject in need thereof.
  • the administration comprises at least one of the following: oral, intravenous, subcutaneous, intraperitoneal, rectal, intramuscular, dermal, transdermal, topical, by any other parenteral route, pharmaceutically acceptable dosage form.
  • Fig. 1 is a graph of the growth status of MNC-168 on a plate
  • Figure 2A shows the changes of cytokine IL-1 ⁇ produced by macrophages and MNC-168 after co-culture at MOI (bacteria: cells) of 10, wherein the control group: PBS control group, LPS+INFr: M1 macrophage control group , MNC-168: test group;
  • Figure 2B shows the changes of cytokine IL-23 produced by macrophages and MNC-168 after co-culture at MOI (bacteria: cells) of 10, wherein the control group: PBS control group, LPS+INFr: M1 macrophage control group , MNC-168: test group;
  • Figure 2C shows the changes of cytokine TNF ⁇ produced by macrophages and MNC-168 after co-culture at MOI (bacteria: cells) of 10, in which, control group: PBS control group, LPS+INFr: M1 macrophage control group, MNC -168: test group;
  • Figure 3A shows the changes of cytokine IL-1 ⁇ produced by Primary PBMC and MNC-168 after co-culture at MOI (bacteria: cells) of 1, wherein control group: PBS control group, MNC-168: test group;
  • Figure 3B shows the changes of cytokine CXCL9 produced by Primary PBMC and MNC-168 after co-culture at MOI (bacteria: cells) of 1, wherein control group: PBS control group, MNC-168: test group;
  • Figure 4A shows the changes of cytokine CXCL9 produced by iDC cells and MNC-168 after co-culture at MOI (bacteria: cells) of 10, wherein, PBS: PBS control group, LPS: positive control group, MNC-168: test group;
  • Figure 4B shows the changes of cytokine IL-1 ⁇ produced by iDC cells and MNC-168 after co-culture at MOI (bacteria: cells) of 10, wherein, PBS: PBS control group, LPS: positive control group, MNC-168: test group ;
  • Figure 5 is the survival curve (absorbance value) of MNC-168 in simulated gastric juice
  • Figure 6 is the survival curve (absorbance value) of MNC-168 in simulated intestinal fluid
  • Figure 7 is a graph showing the results of the inhibitory effect of MNC-168 and PD-1 antibody on colorectal cancer (CT26);
  • Figure 8 is a graph showing the results of the inhibitory effect of MNC-168 and PD-1 antibody on colorectal cancer (MC38);
  • Figure 9 shows the non-response rate of MNC-168 reversing PD-1 antibody; (a) is the average tumor volume curve, (b) is the tumor volume curve of a single mouse;
  • Figure 10 shows that MNC-168 increases lymphocyte infiltration in tumors; (a) is the result of histochemical staining (CD4, CD8, INF- ⁇ , Foxp3), (b) is the result of flow staining (INF- ⁇ %/CD4) , (c) is the result of flow staining (INF- ⁇ %/CD8);
  • Figure 11 is a picture of MNC-168 after streaking on MRS medium for 24h;
  • Figure 12 is a morphological diagram of MNC-168 after staining under an optical microscope
  • Figure 13 is an electron microscope image of MNC-168
  • Figure 14 is a phylogenetic tree of 16S rDNA sequences of MNC-168 and related species
  • Fig. 15 is a graph showing the expression of CD14 in THP1 cells induced by PMA into M0 macrophages by flow cytometry, wherein the control group: PBS control group, and PMA: M0 macrophage group.
  • tumor suppressor refers to an agent that inhibits the division, growth of tumor cells.
  • combination drug refers to the simultaneous or sequential application of another additional drug or two or more drugs added for the purpose of treatment.
  • inhibitory signaling pathway molecule refers to a signaling molecule that suppresses an immune response.
  • antibody to inhibitory signaling pathway molecule refers to a protein produced by the body with a protective effect due to stimulation of an inhibitory signaling pathway molecule.
  • inhibitor of inhibitory signaling pathway molecule refers to an agent for the inhibition of inhibitory signaling pathway molecule production.
  • prevention refers to preventing or reducing the risk of developing a disorder in an individual.
  • treating can generally refer to treating, curing and/or ameliorating a disease, disorder, disorder or side effect, or reducing the rate of progression of a disease, disorder, disorder or side effect.
  • the term also includes within its scope enhancement of normal physiological function, palliative care and partial remedy for its disease, disorder, condition, side effect or symptom.
  • the term “dose” refers to the daily amount administered to an individual.
  • the daily amount or dose may be administered all at once, or may be administered in several divided doses throughout the day. Dosages can be administered by any known method, such as oral administration, injection, and the like.
  • the term “subject” refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, domestic animals, competitive animals, and pets. Tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro are also included.
  • the term "therapeutically effective amount” refers to an amount of an agent sufficient to produce a beneficial or desired result.
  • the therapeutically effective amount may vary depending on one or more of the subject being treated and the condition, the weight and age of the subject, the severity of the condition, the mode of administration, etc. It is readily determined by one of ordinary skill in the art.
  • M0 or "M0 type” refers to the unpolarized phase of macrophages, the M0 phase, eg, the terms “M0 type macrophages”, “M0 macrophages” refer to macrophages in the unpolarized phase
  • the polarized state period is the cell in the M0 phase.
  • the present disclosure provides an embodiment to provide Enterococcus lactis, a drug for preventing or treating tumors, and applications to solve the above-mentioned technical problems.
  • the present disclosure isolates and screened out a strain of Enterococcus lactis, which can inhibit the growth rate of tumors and can be used for the prevention and treatment of tumors.
  • the Enterococcus lactis can also be used in combination with other tumor suppressors.
  • the present disclosure provides an embodiment, providing an application of Enterococcus lactis in the preparation of a tumor suppressor; the tumor optionally includes at least one of the following solid tumors: colon cancer, rectal cancer, colorectal cancer, liver cancer, Pancreatic, breast, kidney, fibrosarcoma, lung and bile duct cancers.
  • the tumor further comprises the following solid tumors: ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, myeloma, lymphoma, brain tumor, spinal cord tumor, esophageal cancer, oropharyngeal cancer , laryngeal cancer, colorectal cancer, melanoma, neuroendocrine cancer, CNS cancer, non-Hodgkin's lymphoma, hematological malignancies, renal tumors, neuroblastoma, homeostasis sarcoma, Wing family sarcoma, retinal tumors, diffuse Large cell lymphoma, advanced CD70+ cancer.
  • solid tumors ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, myeloma, lymphoma, brain tumor, spinal cord tumor, esophageal cancer, oropharyngeal cancer , laryngeal cancer, colorectal cancer, melanoma, neuroendocrine cancer, CNS cancer, non-
  • Enterococcus lactis in the preparation of a colorectal cancer inhibitor.
  • the above-mentioned Enterococcus lactis is preserved in the Guangdong Provincial Microbial Culture Collection Center, the preservation name is Enterococcus lactis (Enterococcus lactis) MNC-168, and the preservation number is GDMCC NO: 61121.
  • the preservation time is August May 2020, and the preservation address is 5th Floor, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, and the classification name is Enterococcus lactis.
  • the characteristics of the colony culture of the above-mentioned Enterococcus lactis are: on the bacterial medium, after 24 hours of culture, the colony is white, round, with a moist surface, opaque, and neat edges;
  • the body is ellipsoid, 0.7-1.0 ⁇ m ⁇ 0.8-1.3 ⁇ m, arranged singly or in pairs, Gram-positive;
  • the bacterial culture medium is MRS medium.
  • the present disclosure also provides an embodiment to provide a medicament for preventing or treating tumors, which comprises Enterococcus lactis.
  • the above-mentioned drugs for preventing or treating tumors further include pharmaceutically acceptable additives or excipients.
  • pharmaceutically acceptable additives include one or more of fillers, diluents, wetting agents, disintegrants, glidants, lubricants, binders, colorants, .
  • the pharmaceutically acceptable adjuvants include cyclodextrin, starch, sucrose, lactose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesium stearate, talc, iron oxide, One or more of sodium hydroxymethyl starch.
  • the pharmaceutical composition dosage form is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.
  • the active ingredients of the above-mentioned drugs for preventing or treating tumors include but are not limited to Enterococcus lactis, and may also include other drugs or antibodies for preventing or treating tumors.
  • Enterococcus lactis was deposited in the Guangdong Provincial Microbial Culture Collection Center under the deposit name of Enterococcus lactis MNC-168, and the deposit number was GDMCC NO: 61121.
  • the present disclosure also provides a pharmaceutical composition comprising the above-mentioned drugs for preventing or treating tumors.
  • the above-mentioned pharmaceutical composition also includes a combined drug, and the combined drug is at least one of the following drugs:
  • Chemotherapy drugs photosensitizers, photothermal agents, antibodies to inhibitory second signal molecules, inhibitors of inhibitory second signal molecules, PD-L1 inhibitors, PD-1/PD-L1 monoclonal antibodies.
  • the combined drug is at least one of the following drugs:
  • Chemotherapy drugs photosensitizers, photothermal agents, antibodies to inhibitory signaling pathway molecules, inhibitors of inhibitory signaling pathway molecules, PD-L1 inhibitors, PD-1/PD-L1 monoclonal antibodies.
  • the inhibitory signaling pathway molecules include inhibitory second signaling pathway molecules.
  • the above-mentioned inhibitory second signal molecule includes at least one of the following signal molecules: PD-1 and CTLA-4.
  • the inhibitory second signaling molecule described above may be PD-1; CTLA-4; PD-1 and CTLA-4.
  • the above-mentioned inhibitory signaling pathway molecules include at least one of the following signaling molecules: PD-1 and CTLA-4.
  • the inhibitory signaling pathway molecules described above may be PD-1; CTLA-4; PD-1 and CTLA-4.
  • inhibitory second signaling molecule refers to a signaling molecule that suppresses an immune response. It is believed, without being bound by theory, that the aforementioned "inhibitory signaling pathway molecule” refers to a signaling molecule that suppresses an immune response.
  • the antibody to the second inhibitory signal molecule or the inhibitor of the second inhibitory signal molecule includes at least one of the following antibodies: nivolumab, pembrolizumab (pembrolizumab), tislelizumab (Tislelizumab), nivolumab injection (Opdivo), pembrolizumab injection (Keytruda), toripalizumab injection (Tuoyi JS001) and Xin Dilimumab injection (Daboshu IBI308).
  • the antibody to the inhibitory signaling pathway molecule or the inhibitor of the inhibitory signaling pathway molecule includes at least one of the following antibodies: nivolumab, pembrolizumab, tisleliz Fezizumab, Nivolumab Injection, Pembrolizumab Injection, Toripalizumab Injection, and Sintilimab Injection.
  • the aforementioned PD-L1 inhibitor is selected from durvalumab, atezolizumab, or avelumab.
  • the above-mentioned PD-1/PD-L1 monoclonal antibody drug is selected from pembrolizumab or nivolumab.
  • the Enterococcus lactis provided by the present disclosure for combined tumor therapy.
  • the present disclosure provides the use of the medicament and/or the pharmaceutical composition for the treatment of tumors.
  • the present disclosure provides a method of treating tumors, the method comprising:
  • a therapeutically effective amount of the drug or the pharmaceutical composition is administered to the subject in need thereof.
  • the tumor comprises at least one of the following solid tumors: colon cancer, rectal cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, kidney cancer, fibrosarcoma, lung cancer, and bile duct cancer.
  • the administration comprises at least one of the following: oral, intravenous, subcutaneous, intraperitoneal, rectal, intramuscular, dermal, transdermal, topical, by any other parenteral route, pharmaceutically acceptable dosage form.
  • a new Enterococcus lactis is isolated and screened, and the Enterococcus lactis has an important influence on the effect of tumor immunotherapy of cancer patients.
  • Gut microbes and immune cells can interact to jointly regulate the human immune system.
  • the Enterococcus lactis provided by the present disclosure can induce the differentiation and maturation of iDC (immature DC) cells, and the mature DCs can effectively activate T cells, thereby killing tumor cells; in addition, the Enterococcus lactis provided by the present disclosure can induce M0 macrophages Differentiation into M1 or M2 type cells significantly increased the secretion of IL-1 ⁇ , IL-23, and TNF ⁇ .
  • the Enterococcus lactis provided by the present disclosure can be used to prepare tumor suppressors and corresponding drugs, thereby realizing tumor prevention or treatment.
  • This example provides an Enterococcus lactis, the isolation, purification, and colony identification are as follows.
  • step (3) Pipet 100 ⁇ L of the 10 -6 dilution in step (2) into the anaerobic blood plate, and coat it with glass beads evenly. Oxygen culture for 3 to 5 days.
  • the isolated strain was named MNC-168, and its 16s RNA sequence is shown in SEQ ID NO.1.
  • Figure 11 shows the plate diagram of the isolated strains after streaking and culturing on MRS medium.
  • Figure 11 is a picture of MNC-168 cultured on MRS medium at 36°C for 24 hours. The colonies are white, round, moist and opaque with neat edges.
  • Figure 12 shows the morphology of MNC-168 under the light microscope after staining. Under the microscope, after culturing at 36°C for 24h in MRS medium, the cells are ellipsoid, 0.7-1.0 ⁇ m ⁇ 0.8-1.6 ⁇ m, single or Arranged in pairs, Gram-positive bacteria.
  • MNC-168 The morphology of MNC-168 under the electron microscope is shown in Figure 13. It can be seen from Figure 13 that the isolated MNC-168 is ellipsoid with a size of 0.7-1.0 ⁇ m ⁇ 0.8-1.6 ⁇ m, and is arranged singly or in pairs.
  • MNC-168 The physiological and biochemical characteristics of MNC-168 are shown in Table 1.
  • the biochemical identification reagent strip of API 20Strep BioMérieux, CN2060025 was used for the physiological and biochemical identification of microorganisms.
  • MNC-168 has certain resistance to erythromycin and rifampicin.
  • Antibacterial drugs MIC value ( ⁇ g/mL) drug susceptibility
  • Ampicillin (AM) 1.0 sensitive Vancomycin (VA) 0.38 sensitive Erythromycin (ERY) 8 drug resistance Tetracycline (TC) 0.38 sensitive Ciprofloxacin (CI) 0.75 sensitive
  • Nitrofurantoin (NI) 8 sensitive Rifampicin (RI) 4 drug resistance Chloramphenicol (CL) 3 sensitive Linezolid (LZ) 1.5 sensitive
  • Figure 14 shows the phylogenetic tree of the 16S rDNA sequence of MNC-168 and related species.
  • the 16S rDNA sequence phylogenetic tree of "MNC-168RCB” and related species was displayed by the adjacent ligation method, and the similarity was performed 1000 times. Repeat the calculation, the development tree node in the figure only shows that the Bootstrap value is greater than 50% of the value, and the superscript "T" represents the model strain.
  • Example 1 the genome of the original MNC-168 strain isolated and obtained in Example 1 was prepared, sequenced, assembled and analyzed.
  • the genome of the original strain of MNC-168 was fragmented by ultrasonic method (Covaris LE220R-plus, FastPreP-245G), and the fragmentation length ranged to ⁇ 350bp, and then a standard DNA library kit (NEB Ultra TM ) to construct Illumina sequencing libraries.
  • the constructed sequencing library was sequenced with 150 bp paired-end using NovaSeq (Illumina). Sequencing obtained 2.85Gbp data, of which Q20 accounted for 96.605%.
  • the genome raw sequencing data was filtered using fastp (version: 0.20.0), and the filtering parameter was "-q 15-l 50".
  • the filtered raw data was used for genome assembly using SPAdes (version: v3.13.1) with the assembly parameter "--isolate--cov-cutoff 10".
  • the total length of the genome was 2.84Mbp, the N50 length was 79.90kbp, and the GC content was 38.17%.
  • the model strain with the highest genome similarity is Enterococcus lactis, in which the average nucleotide similarity (ANI) is 98.36% and the gene coverage is 86.98%. Therefore, it can be identified as a strain of the same species as Enterococcus lactis. .
  • Genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation process prokka (version: 1.14.5), with the parameter "--gcode 11--evalue 1e-09--coverage 80". A total of 2769 CDS sequences were predicted, and the average CDS sequence length was 873 bp.
  • NCBI blastp version: 2.9.0
  • VFDB virulence factor database
  • MNC-168_00702 VFG002195 EF0818 62.413 MNC-168_00831 VFG002165 efaA 64.261 MNC-168_00926 VFG037100 msrA/B(pilB) 65.625 MNC-168_01264 VFG043518 acm 96.48
  • MNC-168_01317 VFG000964 hasC 75.427 MNC-168_01380 VFG002189 cpsB 74.34 MNC-168_01381 VFG002190 cpsA 78.652 MNC-168_01418 VFG002162 bsh 67.568 MNC-168_01432 VFG002162 bsh 78.086 MNC-168_01541 VFG006717 lap 69.525 MNC-168_01709 VFG048830 gnd 72.495 MNC-168_01754 VFG000080 clpE 61.821 MNC-168_
  • Macrophages are a heterogeneous population of myeloid cells in the innate immune system and participate in various physiological and pathological processes in the body. During inflammation or infection in the body, blood monocytes gather into tissues and differentiate into macrophages. Macrophages are highly plastic and have two main polarization states, classically activated type 1 (M1) and alternatively activated type 2 (M2). Mantovani et al. defined these two macrophage phenotypes as extremes of a continuum of functional states. M1 macrophages are macrophages that can produce pro-inflammatory cytokines, known as classic macrophages, which often occur after injury and infection.
  • M1 macrophages are macrophages that can produce pro-inflammatory cytokines, known as classic macrophages, which often occur after injury and infection.
  • M1 macrophages are characterized by the secretion of pro-inflammatory factors such as TNF ⁇ , IL -1 ⁇ (interleukin-1 ⁇ ), IL-6 (interleukin-6), IL-12 (interleukin-12), and IL-8 (interleukin-8), which play an important role in the early stage of inflammation.
  • M2 macrophage polarization can be controlled by Caused by different stimuli, mainly IL-4 (interleukin-4) and/or IL-13 (interleukin-13), etc.
  • cytokines such as IL-10 (Interleukin-10), CCL18 (chemokine 18) and CCL22 (chemokine 22)
  • M1 macrophages are cytotoxic to both pathogens and tumor cells. Their antitumor activity is related to their ability to secrete reactive nitro groups, reactive oxygen species, and pro-inflammatory cytokines. M2 macrophages promote tumor cell growth and survival by secreting many growth factors such as EGF, TGF- ⁇ or VEGF. Therefore, the detection of the expression of macrophage-related pro-inflammatory factors and anti-inflammatory factors induced by different strains can be used as a judgment index to determine whether the strain has possible anti-tumor characteristics.
  • THP-1 is a nucleocytic leukemia cell line
  • PBMC human peripheral blood mononuclear cell
  • CD14 lipopolysaccharide receptor
  • CD68 cytoplasmic glycoprotein
  • Collect the successfully induced M0 macrophages add a final concentration of 20ng/ml IFN ⁇ +10pg/ml LPS, and add M1 macrophages after 24 hours of LPS induction, add a final concentration of 20ng/ml IL-4+20ng/ml M2-type macrophages after IL-13 induction for 24 hr.
  • MNC-168 induced the differentiation of M0-type macrophages into M1 or M2-type cells.
  • the experimental scheme is as follows:
  • the THP-1 cell line was induced to become M0 macrophages after 48 hr treatment with PMA at a final concentration of 5 ng/ml.
  • the PMA-induced THP1 cell line is M0 macrophages as shown in Figure 15 for flow detection of CD14 expression.
  • M0 macrophages were collected, added with a final concentration of 20ng/ml IFN ⁇ +10pg/ml LPS, and M1 macrophages were induced after 24hr.
  • the THP-1 cell line (commercially purchased from Wuhan Proceeds Life Technology Co., Ltd.) was treated with PMA at a final concentration of 5 ng/ml for 48 hr to differentiate into M0 macrophages.
  • M0 macrophages were co-incubated with MNC-168 for 24 hr.
  • MOI number of viable cells:number of cells
  • M1 macrophages were set as a control group and M2 macrophages were set as a control group.
  • the combination antibiotics listed in the following Table 5 were added to kill the bacteria.
  • gut microbes has a profound impact on the surrounding immune system, and at the same time, gut microbes also have an important impact on the effect of immunotherapy in tumor patients, which is a key component of anti-tumor immunotherapy.
  • Gut microbes and immune cells can interact to jointly regulate the immune system. Intestinal strains can induce primary PBMC cells to differentiate into different cell types and secrete different inflammatory factors, so they can be used for the screening of immune-modulating strains.
  • PBMC Primary PBMC (purchased from TPCS, lot number A19Z289100) were used, thawed and cultured in PRMI1640 complete medium (10% heat-inactivated FBS, containing 1% L-glutamine, 0.1% ps (penicillin-streptomycin) Mixed solution), 10mg/ml DNase, the role of DNase is to avoid agglutination).
  • the combination of antibiotics listed in Table 6 killed the bacteria.
  • MNC-168 could induce a significant increase in the inflammatory factors IL1 ⁇ and CXCL9, and the secretion of IFN ⁇ and TNF ⁇ also increased to a certain extent, but the increase in the secretion of IFN ⁇ and TNF ⁇ was lower than that of IL1 ⁇ . , the increase in CXCL9 cytokines.
  • Gut microbes interact with immune cells to regulate the immune system.
  • Intestinal strains can induce iDC (immature DC, immature dendritic cells) cells to differentiate and mature, and mature DCs can effectively activate T cells, thereby killing tumor cells. Therefore, the induction and differentiation of iDC can be used for the screening of immune regulation strains.
  • iDC implant DC, immature dendritic cells
  • PBMC Primary PBMC (purchased from TPCS, lot number A19Z289100) were used, thawed and cultured in PRMI1640 complete medium (10% heat-inactivated FBS, containing 1% L-glutamine, 0.1% ps (penicillin-streptomycin) Mixed solution), 10mg/ml DNase, the role of DNase is to avoid agglutination). 5-6 hours later, GM-CSF with a final concentration of 50 ng/ml and IL-4 cytokines of 20 ng/ml were added to the culture to induce iDC, and the culture was continued for 5 days.
  • PRMI1640 complete medium 10% heat-inactivated FBS, containing 1% L-glutamine, 0.1% ps (penicillin-streptomycin) Mixed solution
  • DNase penicillin-streptomycin
  • the purpose of this experiment was to observe the differentiation of iDC cells into mature DC cells induced by MNC-168.
  • the experimental scheme is as follows:
  • MOI number of viable bacteria: number of cells
  • iDC cells clearly showed the characteristics of differentiation into mature DC cells.
  • IL-1 ⁇ and CXCL9 secreted by iDC cells all increased significantly, and the secretion of IFN ⁇ and TRAIL also increased to a certain extent, but the increase of IFN ⁇ and TRAIL secretion was not as obvious as that of the other two cytokines.
  • This experimental example provides the preparation method of MNC-168.
  • the bacterial liquid glycerol mixture was drawn into 0.1ml of bacterial strain cryopreserved solution and added to 0.9ml of normal saline for gradient dilution, diluted to a gradient of 10-8 , and 100 ⁇ l of dilutions with a concentration of 10-6 , 10-7 , and 10-8 gradient were taken, respectively.
  • Add to the anaerobic blood plate medium add glass beads and shake well, and measure the CFU by plate counting method.
  • the prepared strain cryopreservation solution was divided into sterile cryopreservation tubes, each tube was divided into 0.2ml bacterial solution, and each strain was divided into 10 tubes. Freeze at -80°C for cryopreservation. After being completely frozen (24h), a tube of frozen bacteria was taken out, thawed to room temperature, and the viable bacteria concentration (CFU) was measured by the dilution coating method.
  • CFU viable bacteria concentration
  • the tolerance of a bacterium to the digestive tract environment is related to whether it has the opportunity to be developed into a living biological drug. These indicators are also of great significance for the selection of dosage forms in the development of living biological drugs based on the bacterial strain. Therefore, it is necessary to test the acid resistance and bile salt tolerance of the strains.
  • SGF Fasting simulated gastric fluid
  • a solution with a pH of 1.0 (Chinese Pharmacopoeia) or 1.2 (US Pharmacopoeia) obtained by diluting with concentrated hydrochloric acid were prepared by adding pepsin.
  • pH 3 as the pH of gastric juice after meal, and this acidity is also similar to the normal pH of mouse gastric juice.
  • the simulated intestinal fluid (SIF) was prepared according to USP26 with potassium dihydrogen phosphate and sodium hydroxide in a certain proportion, and the pH was 6.8.
  • the duration of the test refers to the emptying time of the human gastrointestinal tract.
  • For the acid resistance test we select 2 hours as the test duration
  • for the bile salt tolerance test we select 6 hours as the test duration.
  • enzymes, inorganic salts, and bile salts were mainly considered. Among them, due to different sources and processes, the activities of pepsin and trypsin on the market are uneven.
  • MTT thiazole blue staining
  • Succinate dehydrogenase is one of the hubs connecting oxidative phosphorylation and electron transport, providing electrons for the respiratory chain of eukaryotic mitochondria and various prokaryotic cells for oxygen demand and energy production.
  • Succinate dehydrogenase can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan (Formazan) and deposit in cells, but dead cells do not have this function.
  • Form water-insoluble blue-violet crystalline formazan
  • the amount of formazan crystals formed is proportional to the number of living cells.
  • Dimethyl sulfoxide (DMSO) can dissolve the formazan precipitated in cells, and its light absorption value is measured at a wavelength of 570 nm, which can be indirectly Measure the number of viable cells. This method can avoid tedious concentration dilution and plating operations, and is very suitable for rapid determination of bacterial viability.
  • Simulated Gastric Fluid (SGF) experiment the experimental plan includes:
  • the preparation method of MTT (thiazole blue) dye includes:
  • sampling time point 0 this is the data at time 0
  • sample A, B, and C at 0.5h, 1h and 2h.
  • Table 8 Survival (%) of MNC-168 in simulated gastric juice.
  • time/h buffer set pH1.2 simulated gastric juice group pH3.0 simulated gastric juice group 0 100 100 100 0.5 100 -4 94 1 100 -6 100 2 100 -8 98
  • Figure 6 shows the survival curve of MNC-168 in simulated intestinal fluid with absorbance as the reference.
  • Table 9 Survival (%) of MNC-168 in simulated intestinal fluid.
  • MNC-168 can be used for tumor prevention and treatment.
  • mouse syngeneic tumor models to inhibit the growth of colorectal cancer. This experiment passed the ethical review of the Muen Bio-Animal Ethics Committee.
  • mice were purchased from Nanjing Model Animal Center, mouse colorectal cancer cells CT26 were purchased from ATCC, InVivoMAb anti-mouse PD-1 (BioXcell, BE0146, specifically anti-mPD-1 (CD279) Clone RMP1-14) .
  • mice were fed with MNC-168 bacteria by po method, MNC-168 was thawed and placed at room temperature until its temperature reached room temperature, and then fed at a dose of 2 ⁇ 10 9 CFU (colony formation unit)/day per mouse, continuously. 28 days.
  • the amount of cells inoculated with CT26 was 1 ⁇ 10 6 /cell.
  • the inoculation steps were as follows: CT26 cells were collected on the day of inoculation, the cells were washed once with PBS, resuspended in PBS, and the cell suspension was counted. The cell concentration was adjusted to 1 ⁇ 10 7 /mL, mixed 1:1 with pre-cooled Matrigel (basement membrane matrigel), and 0.1 mL/mouse was injected subcutaneously into mice. The day of tumor inoculation was set as D0 day.
  • mice were continuously fed with MNC-168 until the end of the experiment, and the feeding method was also p.o.
  • Anti-mouse PD-1 antibody was intraperitoneally injected on the 7th, 10th, 14th, and 17th days after tumor inoculation, and the injection dose was 100 ⁇ g per mouse each time.
  • the tumor size was measured every two days, and the tumor volume was calculated by the following formula:
  • Tumor volume 1/2 x tumor length and diameter 2 x tumor width.
  • Experimental group 1 (MNC-168+PD-1): feeding MNC-168 and injecting PD-1 antibody according to the method in Experimental Example 6;
  • Experimental group 2 MNC-168 treatment group: MNC-168 was fed according to the method in Experimental Example 6;
  • PD-1 treatment group (PD-1): only PD-1 antibody was injected according to the method in Experimental Example 6;
  • Control group no treatment.
  • the tumors of the non-treatment group ie the control group
  • the tumor growth rate of the PD-1 treatment group was slightly lower than that of the control group
  • the PD-1 treatment group PD-1
  • the tumor volume was about 1800mm 3 on the 19th day
  • the tumor volume of the MNC-168 treatment group ie the experimental group 2
  • the growth rate was significantly slower than that of the control group and the PD-1 treatment group (PD-1). 1), which shows that MNC-168 can significantly reduce the growth rate of colorectal cancer even when administered alone.
  • MNC-168 and PD-1 combined treatment group ie experimental group 1 (MNC-168+PD-1)
  • MNC-168+PD-1 The tumor volume of MNC-168 and PD-1 combined treatment group
  • PD-1 PD-1 treatment group
  • MNC-168 can be used for tumor prevention and treatment.
  • mouse syngeneic tumor models to inhibit the growth of colorectal cancer. This experiment passed the ethical review of the Muen Bio-Animal Ethics Committee.
  • mice were purchased from Nanjing Model Animal Center, and mouse colorectal cancer cells MC-38 were purchased from ATCC, InVivoMAb anti-mouse PD-1 (BioXcell, BE0146).
  • mice were fed with MNC-168 bacteria by po method, MNC-168 was thawed and placed at room temperature until its temperature reached room temperature, and then fed at a dose of 2 ⁇ 10 9 CFU (colony formation unit)/day per mouse, continuously. 28 days.
  • MC38 cells inoculated After feeding MNC-168 for seven days, the amount of MC38 cells inoculated was 2 ⁇ 10 6 /cell.
  • the inoculation steps were as follows: MC38 cells were collected on the day of inoculation, the cells were washed once with PBS, resuspended in PBS, and the cell suspension was counted. The cell concentration was adjusted to 2 ⁇ 10 7 /mL, mixed 1:1 with pre-cooled Matrigel, and then injected into mice subcutaneously with 0.1 mL/mouse. The day of tumor inoculation was set as D0 day.
  • mice inoculated with tumors were continuously fed with MNC-168 until the end of the experiment, and the feeding method was also p.o.
  • Anti-mouse PD-1 antibody was intraperitoneally injected on the 7th, 10th, 14th, and 17th days after tumor inoculation, and the injection dose was 200 ⁇ g per mouse each time.
  • the size of the seed tumor was measured every two days, and the tumor volume was calculated by the following formula:
  • Tumor volume 1/2 x tumor length and diameter 2 x tumor width.
  • Experimental group 1 (MNC-168+PD-1): feeding MNC-168 and injecting PD-1 antibody according to the method in Experimental Example 7;
  • Experimental group 2 (MNC-168 treatment group): only MNC-168 was fed according to the method in Experimental Example 7;
  • PD-1 treatment group (PD-1) only PD-1 antibody was injected according to the method in Experimental Example 7;
  • Blank control group no treatment.
  • the tumors of the non-treated group ie the control group
  • the tumor growth rate of the PD-1 treatment group (PD-1) was slightly lower than that of the control group
  • the PD-1 treatment group had a slightly lower tumor growth rate than the control group.
  • the tumor volume of the group was about 1200mm 3 on the 23rd day
  • the tumor volume of the MNC-168 treatment group (experimental group 2) was about 500mm 3 on the 23rd day, and the growth rate was faster than that of the control group and the PD-1 treatment group (PD-1). It is slow, thus demonstrating that MNC-168 alone can significantly reduce the growth rate of colorectal cancer.
  • the tumor volume of the MNC-168 combined with PD-1 treatment group was about 150 mm 3 on the 23rd day, and the growth rate was significantly slower than that of the control group and the PD-1 treatment group (PD-1).
  • This experiment demonstrated that MNC-168 can significantly reduce the growth rate of colorectal cancer and that MNC-168 can significantly reduce the growth rate of colorectal cancer in the presence of PD-1.
  • This experimental example explores that oral administration of MNC-168 can increase the response efficiency of anti-PD-1.
  • mice were purchased from Nanjing Model Animal Center, and mouse colorectal cancer cells CT26 were purchased from ATCC, InVivoMAb anti-mouse PD-1 (BioXcell, BE0146).
  • mice were fed with MNC-168 bacteria by po method, MNC-168 was thawed and placed at room temperature until its temperature reached room temperature, and then fed at a dose of 2 ⁇ 10 9 CFU (colony formation unit)/day per mouse, continuously. 28 days.
  • the amount of cells inoculated with CT26 was 1 ⁇ 10 6 /cell.
  • the inoculation procedure was as follows: cells were collected on the day of inoculation, washed once with PBS, resuspended in PBS, and the cell suspension was counted. The cell concentration was adjusted to 1 ⁇ 10 7 /mL, mixed 1:1 with pre-cooled Matrigel, and 0.1 mL/mouse was injected subcutaneously into mice. The day of tumor inoculation was set as D0 day.
  • mice were continuously fed with MNC-168 until the end of the experiment, and the feeding method was also p.o.
  • Anti-mouse PD-1 antibody was intraperitoneally injected on the 7th, 10th, 14th, and 17th days after tumor inoculation, and the injection dose was 100 ⁇ g per mouse each time.
  • the tumor size was measured every two days, and the tumor volume was calculated by the following formula:
  • Tumor volume 1/2 x tumor length and diameter 2 x tumor width.
  • Experimental group 1 (MNC-168+PD-1): feeding MNC-168 and injecting PD-1 antibody according to the method in Experimental Example 8;
  • Experimental group 2 MNC-168 treatment group: MNC-168 was fed according to the method of Experimental Example 8;
  • PD-1 treatment group (PD-1): only PD-1 antibody was injected according to the method in Experimental Example 8;
  • Control group no treatment.
  • the tumor in the non-treatment group ie, the control group
  • the tumor growth rate in the PD-1 treatment group was slightly lower than that in the control group, and the PD-1 treatment group ( The tumor volume of PD-1) was about 2500mm 3 on the 19th day, and the tumor volume of the MNC-168 treatment group (ie the experimental group 2) was about 1300mm 3 on the 19th day, and the growth rate was significantly slower than that of the control group and the PD-1 treatment group (PD-1), thus indicating that MNC-168 can significantly reduce the growth rate of colorectal cancer even when administered alone.
  • the tumor volume of the MNC-168 and PD-1 combined treatment group (ie the experimental group 1 (MNC-168+PD-1)) was about 600mm 3 on the 19th day, and the growth rate was significantly slower than that of the control group and the PD-1 treatment group ( PD-1).
  • MNC-168 can significantly reduce the growth rate of colorectal cancer and that MNC-168 can significantly reduce the growth rate of colorectal cancer in the presence of PD-1.
  • the experimental methods of this experimental example include:
  • CD4 leukocyte differentiation antigen 4
  • CD8 leukocyte differentiation antigen 8
  • FITC Rat Anti-Mouse CD4 fluorescein isothiocyanate rat anti-mouse CD4
  • PE anti-mouse CD8a Phycobilichrome protein anti-mouse CD8a
  • FITC Rat Anti-Mouse CD4 fluorescein isothiocyanate rat anti-mouse CD4
  • PE anti-mouse CD8a Phycobilichrome protein anti-mouse CD8a
  • 1X fixative was fixed at 4 degrees overnight
  • stained with cytokines PE-Cy7 anti-mouse IFN- ⁇ was diluted 1:100 in 1X permeabilization solution
  • Figure 10 shows flow cytograms of tumor-infiltrating immune cells CD4IFN ⁇ cells and CD8IFN ⁇ cells in tumor tissue activated by MNC-168.
  • PE-Cy7 anti-mouse IFN- ⁇ (Cat. No. 557649, BD), PMA (P1585, Sigma), ionomycin (Cat. No. 407952, Sigma), brefeldin A (Cat. No. 00-4506-51, Thermofisher), Transcription Factor Buffer Set (Cat. No. 00-4506-51, Thermofisher) 562574, BD), 1640 Medium (Cat. No. C11875500BT, Thermofisher), FBS (Cat. No. 10099-141, Gibico).
  • Enterococcus lactis of the present disclosure has an important influence on the effect of tumor immunotherapy in cancer patients.
  • Enterococcus lactis can induce the differentiation and maturation of iDC (immature DC) cells, and mature DCs can effectively activate T cells, thereby killing tumor cells;
  • Enterococcus lactis can induce the differentiation of M0-type macrophages into M1 or M2-type cells, resulting in a significant increase in the secretion of IL-1 ⁇ , IL-23 and TNF ⁇ .
  • the Enterococcus lactis of the present disclosure can be used to prepare tumor suppressors and corresponding drugs, so as to achieve tumor prevention or treatment.

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Abstract

提供了一种乳酸肠球菌、预防或治疗肿瘤的药物及应用，涉及生物医药技术领域。分离筛选出一种新的乳酸肠球菌，该乳酸肠球菌对癌症病人肿瘤免疫治疗的效果具有重要的影响。肠道微生物与免疫细胞之间能相互作用，共同调控人的免疫系统。提供的乳酸肠球菌可以诱导iDC(immature DC)细胞分化成熟，成熟DC能有效激活T细胞与增加具备抗肿瘤活性的肿瘤内浸润淋巴细胞的浸润，从而杀伤肿瘤细胞；此外，提供的乳酸肠球菌可以诱导M0型巨噬细胞向M1或M2型细胞的分化。

Description

一种乳酸肠球菌、预防或治疗肿瘤的药物及应用
相关申请的交叉引用
本公开要求于2021年01月19日提交中国专利局的申请号为CN202110072294.9、名称为“一种乳酸肠球菌、预防或治疗肿瘤的药物及应用”以及于2021年01月21日提交的申请号为CN202110085233.6、名称为“一种乳酸肠球菌、预防或治疗肿瘤的药物及应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及医药生物技术领域，具体而言，涉及一种乳酸肠球菌、预防或治疗肿瘤的药物及应用。
背景技术
肿瘤是免疫逃逸的结果。虽然肿瘤免疫治疗进展很快，各癌种标准疗法不断被改写。但单药有效率不高，存在原发和继发耐药，及获益人群少，提高疗效和使更多的肿瘤患者从中获益是亟需解决的临床问题。PD-1/PD-L1免疫疗法(immunotherapy)是当前全世界备受瞩目、广为研究的新一类抗肿瘤免疫疗法，旨在充分利用人体自身的免疫系统抵御、抗击肿瘤，通过阻断PD-1/PD-L1信号通路使肿瘤细胞死亡，具有治疗多种类型肿瘤的潜力，实质性改善患者总生存期。
目前，现有的免疫疗法存在一定的毒副作用及治疗失败等问题，而引入肠道微生物进行免疫疗法进行治疗，不失为一种全新的干预手段。
鉴于此，特提出本公开。
发明内容
本公开提供一种乳酸肠球菌在制备肿瘤抑制剂中的应用。在一些实施方式中，所述肿瘤可选地包括如下实体肿瘤中的至少一种：结肠癌、直肠癌、结肠直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌和胆管癌。
在可选的实施方式中，所述肿瘤还包括以下实体肿瘤：卵巢癌、宫颈癌、前列腺癌、膀胱癌、头颈癌、骨髓瘤、淋巴瘤、脑瘤、脊髓瘤、食管癌、口咽癌、喉癌、结直肠癌、黑色素瘤、神经内分泌癌、CNS癌、非霍奇金淋巴瘤、血液恶性肿瘤、肾脏肿瘤、神经母细胞瘤、恒温肌肉瘤、文氏家族肉瘤、视网膜瘤、弥漫性大细胞淋巴瘤、晚期CD70+癌症。
在可选的实施方式中，所述乳酸肠球菌保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。
在可选的实施方式中，所述乳酸肠球菌的菌落培养特征为：在细菌培养基上，培养24h后菌落呈白色，圆形，表面湿润，不透明，边缘整齐；在显微镜下，菌体呈椭球状，0.7-1.0μm×0.8-1.3μm，单个或成对排列，革兰氏阳性。
在可选的实施方式中，所述细菌培养基为MRS培养基。
本公开提供一种预防或治疗肿瘤的药物，其中，其包括乳酸肠球菌。
在可选的实施方式中，所述预防或治疗肿瘤的药物还包括药学上可接受的添加剂或辅料。在可选的实施方式中，所述药物组合物剂型选自片剂、丸剂、粉剂、混悬剂、凝胶、乳液、乳膏、颗粒剂、纳米颗粒、胶囊、栓剂、注射剂、喷雾和针剂。
在可选的实施方式中，所述药学上可接受的添加剂包括填充剂、稀释剂、润湿剂、崩解剂、助流剂、润滑剂、粘合剂、着色剂、中的一种或多种。
在可选的实施方式中，所述药学上可接受的辅料包括环糊精、淀粉、蔗糖、乳糖、羟丙基纤维素、羟丙基甲基纤维素、硬脂酸镁、滑石粉、氧化铁、羟甲淀粉钠中的一种或多种。
在可选的实施方式中，所述乳酸肠球菌保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。
本公开提供一种药物组合物，所述药物组合物包括所述的预防或治疗肿瘤的药物。
在可选的实施方式中，所述药物组合物还包括联用药物。在可选的实施方式中，所述联用药物为如下药物中的至少一种：
化疗药物，光敏剂，光热剂，抑制性第二信号分子的抗体、抑制性第二信号分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。
在可选的实施方式中，所述联用药物为如下药物中的至少一种：
化疗药物，光敏剂，光热剂，抑制性信号通路分子的抗体、抑制性信号通路分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。
在可选的实施方式中，所述抑制性第二信号分子包括如下信号分子中的至少一种：PD-1和CTLA-4。
在可选的实施方式中，所述抑制性信号通路分子包括如下信号分子中的至少一种：PD-1和CTLA-4。
在可选的实施方式中，所述抑制性第二信号分子的抗体或抑制性第二信号分子的抑制剂包括如下抗体中的至少一种：尼伏单抗、潘利珠单抗、替雷利珠单抗、纳武利尤单抗注射液、帕博利珠单抗注射液、特瑞普利单抗注射液和信迪利单抗注射液。
在可选的实施方式中，所述抑制性信号通路分子的抗体或抑制性信号通路分子的抑制剂包括如下抗体中的至少一种：尼伏单抗、潘利珠单抗、替雷利珠单抗、纳武利尤单抗注射液、帕博利珠单抗注射液、特瑞普利单抗注射液和信迪利单抗注射液。
在可选的实施方式中，所述PD-L1抑制剂选自度伐单抗、阿替珠单抗或阿维单抗。
在可选的实施方式中，所述PD-1/PD-L1单抗药物选自帕博利珠单抗或纳武利尤单抗。
本公开提供一种乳酸肠球菌，其中，保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。
在可选的实施方式中，所述乳酸肠球菌的菌落培养特征为：在细菌培养基上，培养24h后菌落呈白色，圆形，表面湿润，不透明，边缘整齐；在显微镜下，菌体呈椭球状，0.7-1.0μm×0.8-1.3μm，单个或成对排列，革兰氏阳性。
在可选的实施方式中，所述细菌培养基为MRS培养基。
本公开提供所述药物和/或药物组合物，用于治疗肿瘤的用途。
本公开提供一种治疗肿瘤的方法，所述方法包括：
向所述有此需要的受试者给药治疗有效量的所述药物或者所述药物组合物。
在可选的实施方式中，所述给药包括如下的至少一种：口服、静脉内、皮下、腹膜内、直肠、肌内、皮肤、经皮、局部、通过任何其他肠胃外途径、以药学上可接受的剂型。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为MNC-168在平板上的生长状况图；
图2A为巨噬细胞和MNC-168在MOI(细菌：细胞)为10共培养后产生细胞因子IL-1β的变化，其中，对照组：PBS对照组，LPS+INFr：M1巨噬细胞对照组，MNC-168：测试组；
图2B为巨噬细胞和MNC-168在MOI(细菌：细胞)为10共培养后产生细胞因子IL-23的变化，其中，对照组：PBS对照组，LPS+INFr：M1巨噬细胞对照组，MNC-168：测试组；
图2C为巨噬细胞和MNC-168在MOI(细菌：细胞)为10共培养后产生细胞因子TNFα的变化，其中，对照组：PBS对照组，LPS+INFr：M1巨噬细胞对照组，MNC-168：测试组；
图3A为Primary PBMC和MNC-168在MOI(细菌：细胞)为1共培养后产生细胞因子IL-1β的变化，其中，对照组：PBS对照组，MNC-168：测试组；
图3B为Primary PBMC和MNC-168在MOI(细菌：细胞)为1共培养后产生细胞因子CXCL9的变化，其中，对照组：PBS对照组，MNC-168：测试组；
图4A为iDC细胞和MNC-168在MOI(细菌：细胞)为10共培养后产生细胞因子CXCL9的变化，其中，PBS：PBS对照组，LPS：阳性对照组，MNC-168：测试组；
图4B为iDC细胞和MNC-168在MOI(细菌：细胞)为10共培养后产生细胞因子IL-1β的变化，其中，PBS：PBS对照组，LPS：阳性对照组，MNC-168：测试组；
图5为MNC-168在模拟胃液中的存活曲线(吸光度值)；
图6为MNC-168在模拟肠液中的存活曲线(吸光度值)；
图7为MNC-168和PD-1抗体联用对结直肠癌(CT26)的抑制作用结果图；
图8为MNC-168和PD-1抗体联用对结直肠癌(MC38)的抑制作用结果图；
图9为MNC-168逆转PD-1抗体未响应率；其中(a)为平均肿瘤体积曲线，(b)为单只小鼠肿瘤体积曲线；
图10为MNC-168增加肿瘤內淋巴細胞浸润；其中(a)为组织化学染色结果(CD4、CD8、INF-γ、Foxp3)，(b)为流式染色结果(INF-γ％/CD4)，(c)为流式染色结果(INF-γ％/CD8)；
图11为MNC-168在MRS培养基上划线培养24h后的图片；
图12为MNC-168经过染色后在光学显微镜下的形态图；
图13为MNC-168的电镜图；
图14为MNC-168与相关种的16S rDNA序列系统发育树；
图15为PMA诱导THP1细胞为M0型巨噬细胞流式检测CD14表达图，其中，对照组：PBS对照组，PMA：M0型巨噬细胞组。
实施方式
为使本公开实施例的技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。
术语定义
如本文所用，“肿瘤抑制剂”是指抑制肿瘤细胞的分裂、生长的制剂。
如本文所使用，“联用药物”是指为了达到治疗目的而额外添加的另外一种或两种及以上的药物同时或先后应用。如本文所用，术语“抑制性信号通路分子”是指抑制免疫反应的信号分子。
如本文所用，术语“抑制性信号通路分子的抗体”是指用于机体由于抑制性信号通路分子的刺激而产生的具有保护作用的蛋白质。
如本文所用，术语“抑制性信号通路分子的抑制剂”是指用于对抑制性信号通路分子产生的抑制的试剂。
如本文所用，术语“预防”是指预防个体发生病症或降低个体发生病症的风险。
如本文所用，“治疗”通常可以指治疗、治愈和/或改善疾病、疾患、病症或副作用，或降低疾病、疾患、病症或副作用的进展速度。该术语还包括在其范围内增强正常的生理功能、姑息治疗和对其疾病、疾患、病症、副作用或症状的部分补救。
如本文所用，术语“剂量”是指施用于个体的每日量。每日量或剂量可一次性全部施用，或者可在一天内分若干次施用。剂量可通过任何已知的方法施用，例如口服、注射等。如本文所使用，术语“受试者”是指脊椎动物，优选地是 哺乳动物，最优选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
如本文所用，术语“治疗有效量”是指足以产生有益的或期望的结果的药剂的量。治疗有效量可能会根据：接受治疗的受试者和疾病状况、受试者的体重和年龄、疾病状况的严重程度、给药的方式等中的一个或多个因素的不同而变化，其可由本领域普通技术人员容易地确定。
如本文所用，术语“M0”或“M0型”是指未极化的巨噬细胞时期即M0期，例如术语“M0型巨噬细胞”、“M0巨噬细胞”是指巨噬细胞在未极化状态时期即M0期的细胞。
本公开提供一实施方式，提供一种乳酸肠球菌、预防或治疗肿瘤的药物及应用以解决上述技术问题。
据信，不受理论的约束，基于现有的技术问题，本公开分离筛选出一株乳酸肠球菌，该乳酸肠球菌可以抑制肿瘤的生长速度，可以用于肿瘤的预防和治疗。此外，该乳酸肠球菌还可以与其他的肿瘤抑制剂联合使用。
本公开是这样实现的：
I.乳酸肠球菌在制备肿瘤抑制剂(预防或治疗肿瘤)中的应用
本公开提供一实施方式，提供一种乳酸肠球菌在制备肿瘤抑制剂中的应用；所述肿瘤可选地包括如下实体肿瘤中的至少一种：结肠癌、直肠癌、结肠直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌和胆管癌。
在可选的实施方式中，所述肿瘤还包括以下实体肿瘤：卵巢癌、宫颈癌、前列腺癌、膀胱癌、头颈癌、骨髓瘤、淋巴瘤、脑瘤、脊髓瘤、食管癌、口咽癌、喉癌、结直肠癌、黑色素瘤、神经内分泌癌、CNS癌、非霍奇金淋巴瘤、血液恶性肿瘤、肾脏肿瘤、神经母细胞瘤、恒温肌肉瘤、文氏家族肉瘤、视网膜瘤、弥漫性大细胞淋巴瘤、晚期CD70+癌症。
可选地，乳酸肠球菌在制备结肠直肠癌抑制剂中的应用。
可选地，上述的乳酸肠球菌，保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。保藏时间为2020年8月5月，保藏地址为广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，分类名称为Enterococcus lactis。
在本公开应用可选的实施方式中，上述乳酸肠球菌的菌落培养特征为：在细菌培养基上，培养24h后菌落呈白色，圆形，表面湿润，不透明，边缘整齐；在显微镜下，菌体呈椭球状，0.7-1.0μm×0.8-1.3μm，单个或成对排列，革兰氏阳性；
可选地，细菌培养基为MRS培养基。
II.预防或治疗肿瘤的药物
本公开还提供了一实施方式，提供一种预防或治疗肿瘤的药物，其包括乳酸肠球菌。
在本公开应用可选的实施方式中，上述预防或治疗肿瘤的药物还包括药学上可接受的添加剂或辅料。
在可选的实施方式中，药学上可接受的添加剂包括填充剂、稀释剂、润湿剂、崩解剂、助流剂、润滑剂、粘合剂、着色剂、中的一种或多种。
在可选的实施方式中，药学上可接受的辅料包括环糊精、淀粉、蔗糖、乳糖、羟丙基纤维素、羟丙基甲基纤维素、硬脂酸镁、滑石粉、氧化铁、羟甲淀粉钠中的一种或多种。
在可选的实施方式中，药物组合物剂型选自片剂、丸剂、粉剂、混悬剂、凝胶、乳液、乳膏、颗粒剂、纳米颗粒、胶囊、栓剂、注射剂、喷雾和针剂。
在其他可选的实施方式中，上述预防或治疗肿瘤的药物的有效成分包括并不限于乳酸肠球菌，还可以包括其他的预防或治疗肿瘤的药物或抗体。
乳酸肠球菌保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。
III.药物组合物
本公开还提供了一种药物组合物，其包括上述的预防或治疗肿瘤的药物。
可选的，上述药物组合物还包括联用药物，联用药物为如下药物中的至少一种：
化疗药物，光敏剂，光热剂，抑制性第二信号分子的抗体、抑制性第二信号分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。
在可选的实施方式中，所述联用药物为如下药物中的至少一种：
化疗药物，光敏剂，光热剂，抑制性信号通路分子的抗体、抑制性信号通路分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。其中，抑制性信号通路分子包括抑制性第二信号通路分子。
在本公开应用可选的实施方式中，上述抑制性第二信号分子包括如下信号分子中的至少一种：PD-1和CTLA-4。在其他实施方式中，上述抑制性第二信号分子可以是PD-1；CTLA-4；PD-1和CTLA-4。
在本公开应用可选的实施方式中，上述抑制性信号通路分子包括如下信号分子中的至少一种：PD-1和CTLA-4。在其他实施方式中，上述抑制性信号通路分子可以是PD-1；CTLA-4；PD-1和CTLA-4。
据信，不受理论的约束，上述抑制性第二信号分子是指抑制免疫反应的信号分子。据信，不受理论的约束，上述“抑制性信号通路分子”是指抑制免疫反应的信号分子。
在本公开应用可选的实施方式中，上述抑制性第二信号分子的抗体或抑制性第二信号分子的抑制剂包括如下抗体中的至少一种：尼伏单抗(nivolumab)、潘利珠单抗(pembrolizumab)、替雷利珠单抗(Tislelizumab)、纳武利尤单抗注射液(Opdivo)、帕博利珠单抗注射液(Keytruda)、特瑞普利单抗注射液(拓益JS001)和信迪利单抗注射液(达伯舒IBI308)。
在本公开应用可选的实施方式中，所述抑制性信号通路分子的抗体或抑制性信号通路分子的抑制剂包括如下抗体中的至少一种：尼伏单抗、潘利珠单抗、替雷利珠单抗、纳武利尤单抗注射液、帕博利珠单抗注射液、特瑞普利单抗注射液和信迪利单抗注射液。
在本公开应用可选的实施方式中，上述PD-L1抑制剂选自度伐单抗(durvalumab)、阿替珠单抗(atezolizumab)或阿维单抗(avelumab)。
在本公开应用可选的实施方式中，上述PD-1/PD-L1单抗药物选自帕博利珠单抗或纳武利尤单抗。
在其他可选的实施方式中，只要能抑制免疫反应的发生均可以与本公开提供的乳酸肠球菌配合进行联合肿瘤治疗。
本公开提供所述药物和/或药物组合物，用于治疗肿瘤的用途。
本公开提供一种治疗肿瘤的方法，所述方法包括：
向所述有此需要的受试者给药治疗有效量的所述药物或者所述药物组合物。
在可选的实施方式中，所述肿瘤包括如下实体肿瘤中的至少一种：结肠癌、直肠癌、结肠直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌和胆管癌。
在可选的实施方式中，所述给药包括如下的至少一种：口服、静脉内、皮下、腹膜内、直肠、肌内、皮肤、经皮、局部、通过任何其他肠胃外途径、以药学上可接受的剂型。
本公开具有以下有益效果：
本公开分离筛选出一种新的乳酸肠球菌，该乳酸肠球菌对癌症病人肿瘤免疫治疗的效果具有重要的影响。肠道微生物与免疫细胞之间能相互作用，共同调控人的免疫系统。研究表明，本公开提供的乳酸肠球菌可以诱导iDC(immature DC)细胞分化成熟，成熟DC能有效激活T细胞，从而杀伤肿瘤细胞；此外，本公开提供的乳酸肠球菌可以诱导M0型巨噬细胞向M1或M2型细胞的分化，使得IL-1β，IL-23，TNFα的分泌量显著的增加。本公开提供的乳酸肠球菌可以用于制备肿瘤抑制剂及相应的药物，从而实现肿瘤预防或治疗。
实施例
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种乳酸肠球菌，其分离、纯化，菌落鉴定如下所示。
菌株MNC-168的分离、纯化。
(1)在生物安全柜中分装生理盐水于无菌的10ml离心管中；提前24h将厌氧血平板和无菌生理盐水转移入厌氧工作台(H35 Hypoxystation,Whitley)中，并将5～7颗无菌的玻璃珠倒入已经凝固的厌氧血平板中备用。厌氧血平板购自江门凯林贸易有限公司，Bio-Caring。
(2)慕恩(广州)生物科技有限公司某员工(供体号403051)与公司签订粪便收集的知情同意书后，取其鲜粪便样本1g于厌氧操作台中使用漩涡振荡器震荡1min，混匀，吸取1mL样本到9mL生理盐水中，混匀为10 -1稀释液，依次梯度稀释至10 -6稀释液，用于涂平板用。
(3)吸取100μL步骤(2)中的10 -6稀释液于厌氧血平板中，并用玻璃珠涂布均匀，待平板表面干燥后，倒弃玻璃珠，在该厌氧工作台37℃厌氧培养3～5天。
(4)观察分离到的菌株生长状况，并用灭菌牙签挑取单菌落进行平板划线纯化、培养，对纯菌株进行16S rRNA基因分析，初步确定其分类地位。
分离到的菌株命名为MNC-168，其16s RNA序列见SEQ ID NO.1所示。
分离到的菌株在MRS培养基上划线培养后的平板图参照图11所示，图11为MNC-168在MRS培养基上以36℃，培养24h后的图片。菌落呈白色，圆形，表面湿润，不透明，边缘整齐。
MNC-168菌株在平板上的生长状况参照图1所示。
MNC-168经过染色后在光学显微镜下的形态参照图12所示，显微镜下，在MRS培养基中36℃培养24h后，菌体呈椭球状，0.7-1.0μm×0.8-1.6μm，单个或成对排列，为革兰氏阳性菌。
MNC-168在电镜下的形态参照图13所示，由图13可知，分离得到的MNC-168呈椭球形，大小为0.7-1.0μm×0.8-1.6μm，单个或成对排列。
MNC-168的生理生化特征参照表1所示，本实施例中采用API 20Strep(BioMérieux,CN2060025)生化鉴定试剂条进行微生物的生理生化鉴定。
表1 MNC-168的生理生化特征。
Figure PCTCN2021098848-appb-000001
Figure PCTCN2021098848-appb-000002
符号说明：“+”，阳性；“-”，阴性。
MNC-168的抗生素敏感性测试结果参照表2所示，由表2可知，MNC-168对红霉素和利福平具有一定的耐药性。
表2 MNC-168的抗生素敏感性测试表。
抗菌药物 MIC值(μg/mL) 药敏性
氨苄西林(AM) 1.0 敏感
万古霉素(VA) 0.38 敏感
红霉素(ERY) 8 耐药
四环素(TC) 0.38 敏感
环丙沙星(CI) 0.75 敏感
呋喃妥因(NI) 8 敏感
利福平(RI) 4 耐药
氯霉素(CL) 3 敏感
利奈唑胺(LZ) 1.5 敏感
图14为MNC-168与相关种的16S rDNA序列系统发育树，采用MEGA5.0软件，邻位连接法显示“MNC-168RCB”与相关种的16S rDNA序列系统发育树，进行1000次的相似度重复计算，图中发育树节点只显示Bootstrap值大于50％数值，上标的“T”表示模式菌株。
实施例2
本实施例对实施例1分离获得的MNC-168原始菌株进行基因组的制备、测序，组装和分析。
MNC-168原始菌株的基因组通过超声波法(Covaris LE220R-plus,FastPreP-245G)进行序列片段化，片段化长度范围～350bp，然后利用标准DNA建库试剂盒(NEB
Figure PCTCN2021098848-appb-000003
Ultra TM)构建Illumina测序文库。将构建好的测序文库利用NovaSeq(Illumina)进行双端150bp测序。测序得到2.85Gbp数据，其中Q20占比为：96.605％。
基因组原始测序数据使用fastp(版本：0.20.0)进行数据过滤，过滤参数：“-q 15-l 50”。过滤后的原始数据使用SPAdes(版本：v3.13.1)进行基因组组装，组装参数“—isolate--cov-cutoff 10”。基因组组装得到基因总长度2.84Mbp，N50长度为79.90kbp，GC含量为38.17％。基因组相似度最高的模式菌株为：乳酸肠球菌(Enterococcus lactis)，其中平均核苷酸相似度(ANI)为98.36％，基因覆盖度为86.98％，因此，可以认定为乳酸肠球菌同种的菌株。
基因组基因使用原核分析软件基因组注释流程prokka(版本：1.14.5)进行基因组基因预测分析，参数“—gcode 11--evalue 1e-09--coverage 80”。总共预测得到2769条CDS序列，其中平均CDS序列长度为873bp。
基因组中潜在的抗生素耐药基因使用RGI流程分析(版本：4.2.2)，其中抗生素耐药基因数据库为CARD(版本：3.0.0，https://card.mcmaster.ca/analyze/rgi)。详细对比信息参照表3所示。
表3耐药基因信息列表。
菌株基因 抗性基因 基因名称 比对一致性(％)
MNC-168_00879 ARO:3003954 efmA 100.00
MNC-168_01160 ARO:3002819 msrC 100.00
MNC-168_01954 ARO:3002556 AAC(6')-Ii 100.00
MNC-168_02155 ARO:3003761 eatAv 100.00
对基因组中潜在的毒力因子及相关基因的分析采用的是NCBI blastp(版本为：2.9.0)比对毒力因子数据库VFDB(virulence factor database，http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi，更新日期为2019年9月13日)。详细比对结果见表4。
表4 MNH-5168潜在毒性基因列表。
菌株基因 VFDB基因 基因名称 比对一致性(％)
MNC-168_00015 VFG043511 sgrA 95.918
MNC-168_00436 VFG045632 ecbA 92.093
MNC-168_00638 VFG002158 lplA1 62.09
MNC-168_00702 VFG002195 EF0818 62.413
MNC-168_00831 VFG002165 efaA 64.261
MNC-168_00926 VFG037100 msrA/B(pilB) 65.625
MNC-168_01264 VFG043518 acm 96.48
MNC-168_01317 VFG000964 hasC 75.427
MNC-168_01380 VFG002189 cpsB 74.34
MNC-168_01381 VFG002190 cpsA 78.652
MNC-168_01418 VFG002162 bsh 67.568
MNC-168_01432 VFG002162 bsh 78.086
MNC-168_01541 VFG006717 lap 69.525
MNC-168_01709 VFG048830 gnd 72.495
MNC-168_01754 VFG000080 clpE 61.821
MNC-168_01880 VFG000077 clpP 81.122
MNC-168_02111 VFG000964 hasC 70.068
MNC-168_02541 VFG002197 bopD 86.269
MNC-168_02708 VFG000079 clpC 62.162
MNC-168_02802 VFG048797 ugd 64.341
MNC-168_00015 VFG043511 sgrA 95.918
实验例1
本实验例研究实施例1分离获得的MNC-168对巨噬细胞的影响。
近年来的研究表明，癌症病人肠道微生物对肿瘤免疫治疗的效果有重要的影响，而调节和利用肠道微生物逐渐成为肿瘤免疫治疗的关键组成部分。已经证明：肠道微生物和免疫细胞之间存在直接相互作用的多种机制，而肠道淋巴组织中的树突状细胞和巨噬细胞是免疫调节微生物的关键靶细胞，特别是在小肠中，由于肠道表面积大，黏液层薄而弥散，使得微生物可以与免疫细胞紧密接触。微生物菌群中的不同的特异菌株能够抑制或激活全身免疫反应。
巨噬细胞是天然免疫系统中髓样细胞的异质性群体，参与机体多种生理和病理过程。在机体发生炎症或感染时，血液单核细胞聚集到组织中，分化成巨噬细胞。巨噬细胞具有很高的可塑性，其主要具有两种极化状态，经典活化的1型(M1)和替代活化的2型(M2)。Mantovani等人将这两种巨噬细胞表型定义为功能状态连续性的极端。M1巨噬细胞是可以产生促炎细胞因子的巨噬细胞，被称为经典型巨噬细胞，常发生在损伤及感染之后。在体外巨噬细胞的经典激活常通过细菌细胞壁(例如LPS(脂多糖)和TNFα(肿瘤坏死因子α)或IFNγ(干扰素γ)。M1巨噬细胞的特征在于分泌促炎因子例如TNFα、IL-1β(白介素-1β)、IL-6(白介素-6)、IL-12(白介素-12)和IL-8(白介素-8)，在炎症早期发挥重要的作用。M2巨噬细胞极化可由不同刺激引起，主要是IL-4(白介素-4)和/或IL-13(白介素-13)等。被IL-4和IL-13活化的巨噬细胞分泌抗炎细胞因子，如IL-10(白介素-10)、CCL18(趋化因子18)和CCL22(趋化因子22)，发挥抑制炎症反应及组织修复的作用。
M1巨噬细胞对病原体和肿瘤细胞都具有细胞毒性。它们的抗瘤活性与它们分泌的活性硝基、活性氧和促炎细胞因子的能力有关。M2巨噬细胞通过分泌许多生长因子例如EGF，TGF-β或VEGF等促进肿瘤细胞生长及存活。故通过检测不同菌株诱导巨噬细胞相关促炎因子及抑炎因子表达的检测可以作为判断该菌株是否具有可能的抗肿瘤特征的一个判断指标。
由于巨噬细胞分离过程比较麻烦，常用佛波醇12-十四酸酯13-乙酸酯(phorbol 12-myristate 13-acetate，PMA，Sigma，P8139)诱导THP-1(THP-1是人单核细胞白血病细胞系)，由于其与PBMC(人外周血单个核细胞)来源的单核细胞具有相似的反应，故被广泛用于研究单核细胞或单核来源的巨噬细胞免疫反应能力的模型。单核细胞系分化而成的M0巨噬细胞作为研究巨噬细胞功能的体外细胞模型。该巨噬细胞模型，具有以下优点：易于获取、分化及极化。
在培养的THP-1细胞系(武汉普诺赛生命科技有限公司)中加入PMA至终浓度为5ng/ml，37℃，5％CO 2继续培养48hr后，根据以下两个标准判断M0细胞诱导是否成功：
(1)细胞是否贴壁；
(2)流式检测CD14(脂多糖受体)，CD68(细胞浆糖蛋白)表达确认是否诱导成功(CD14表达下调)。
收集诱导成功的M0型巨噬细胞，加入终浓度为的20ng/ml IFNγ+10pg/ml LPS，LPS诱导24hr后M1类型巨噬细胞，加入终浓度为的20ng/ml IL-4+20ng/ml IL-13诱导24hr后M2型巨噬细胞。
首先，本实验例观察MNC-168诱导M0型巨噬细胞向M1或M2型细胞的分化。实验方案如下：
使用终浓度为5ng/ml的PMA处理THP-1细胞系48hr后诱导其为M0巨噬细胞。PMA诱导THP1细胞系为M0型巨噬细胞流式检测CD14表达图参照图15所示。
收集M0巨噬细胞，加入终浓度为的20ng/ml IFNγ+10pg/ml LPS，诱导24hr后为M1类型巨噬细胞。
其次，本实验例还观察了MNC-168诱导M0型巨噬细胞向M1型细胞的分化。
实验方案如下：
使用终浓度为5ng/ml的PMA处理THP-1细胞系(商购自武汉普诺赛生命科技有限公司)48hr使其分化为M0巨噬细胞。
M0巨噬细胞与MNC-168进行共孵育24hr。按照MOI(活菌数：细胞数)＝10：1的比例加入MNC-168。同时设置M1巨噬细胞作为对照组和M2巨噬细胞作为对照组，厌氧培养1hr后加入下述表5中所列的组合抗生素杀死细菌。
表5：使用抗生素列表。
抗生素类型 来源 终浓度
氨苄青霉素(Ampicillin) Sigma-Aldrich 1mg/ml
链霉素(Streptomycin) MCE 5mg/ml
粘菌素(Colistin) Sigma-Aldrich 1mg/ml
将上述培养物继续37℃，5％CO 2孵育24hr后，收集上清用ELISA方法检测其分泌的细胞因子浓度(结果参照图2A、图2B和图2C所示)。
结果显示，巨噬细胞与MNC-168共培养后，巨噬细胞明显表现出向M1型巨噬细胞分化的特征。其中，IL-1β，IL-23，TNFα的分泌都有了非常显著的增加，IL-27分泌有一定程度的增加，但IL-27的分泌水平的增加幅度小于其他三个细胞因子的分泌水平增加幅度。
实验例2
本实验例探究MNC-168对Primary PBMC(人原代外周血单个核细胞)的影响。
已有研究表明：肠道微生物的成分对周围的免疫系统有着深远的影响，同时肠道微生物对肿瘤病人免疫治疗的效果也有着重要的影响，是抗肿瘤免疫治疗的关键组成部分。肠道微生物与免疫细胞之间能相互作用，共同调控免疫系统。肠道菌株能够诱导primary PBMC细胞分化为不同的细胞类型，并分泌不同的炎症因子，故可用于免疫调控菌株的筛选。
本实验旨在观察MNC-168诱导Primary PBMC的分化。实验方案如下：
使用商业化的Primary PBMC(购买于TPCS，批号A19Z289100)，复苏后培养于PRMI1640完全培养基中(10％的热灭活FBS，含有1％L-谷氨酰胺，0.1％ps(青链霉素混合液)，10mg/ml DNase，DNase的作用为避免凝集)。
按照MOI(活菌数：细胞数)＝1：1的比例加入细菌，MNC-168与Primary PBMC共孵育，同时设置PBS对照组(PBS与Primary PBMC)共培养24hr，厌氧培养1hr后加入下述表6中所列抗生素组合杀死细菌。
表6：使用抗生素列表。
抗生素类型 来源 终浓度
氨苄青霉素 Sigma-Aldrich 1mg/ml
链霉素 MCE 5mg/ml
粘菌素 Sigma-Aldrich 1mg/ml
将上述培养物继续37℃，5％CO 2孵育24hr后，收集上清用ELISA方法检测其分泌的细胞因子浓度。实验结果参照图3A和图3B所示。
结果显示，Primary PBMC与MNC-168菌株共培养后，MNC-168能诱导炎症因子IL1β，CXCL9的显著增加，而IFNγ，TNFα分泌也有一定程度的增加，但IFNγ，TNFα分泌的增加幅度低于IL1β，CXCL9细胞因子的增加幅度。
实验例3
本实验例探究菌株MNC-168对DC细胞的影响。
肠道微生物与免疫细胞相互作用共同调控免疫系统。肠道菌株能诱导iDC(immature DC，未成熟的树突状细胞)细胞分化成熟，成熟的DC能有效激活T细胞，从而杀伤肿瘤细胞，故iDC的诱导分化可用于免疫调控菌株的筛选。
使用商业化的Primary PBMC(购买于TPCS，批号A19Z289100)，复苏后培养于PRMI1640完全培养基中(10％的热灭活FBS，含有1％L-谷氨酰胺，0.1％ps(青链霉素混合液)，10mg/ml DNase，DNase的作用为避免凝集)。5-6小时后培养物中加入终浓度为50ng/ml的GM-CSF及20ng/ml的IL-4细胞因子进行iDC的诱导，继续培养5天。
MNC-168对iDC细胞的影响。
本实验旨在观察MNC-168诱导iDC细胞向成熟DC细胞的分化。实验方案如下：
复苏Primary PBMC，37℃，5％CO 2培养5-6小时后，培养物中加入终浓度为50ng/ml的GM-CSF及20ng/ml的IL-4细胞因子进行iDC的诱导，继续培养5天。
诱导成功的iDC细胞与MNC-168进行共孵育24hr。按照MOI(活菌数：细胞数)＝10的比例加入细菌，同时设置空白对照组(PBS)和阳性对照组(LPS)，厌氧培养1hr后加入下述表7中所列抗生素组合杀死细菌。
表7：使用抗生素列表。
抗生素类型 来源 终浓度
氨苄青霉素 Sigma-Aldrich 1mg/ml
链霉素 MCE 5mg/ml
粘菌素 Sigma-Aldrich 1mg/ml
将上述培养物继续37℃，5％CO 2孵育24hr后，收集上清用ELISA方法检测其分泌的细胞因子浓度。实验结果参照图4A和图4B所示。
结果表明，iDC细胞和MNC-168共培养后，iDC细胞明显表现出向成熟DC细胞分化的特征。其中，iDC细胞分泌的IL-1β，CXCL9都有非常显著的增加，IFNγ和TRAIL分泌也有一定程度的增加，但IFNγ和TRAIL分泌的增加幅度没有其他两个细胞因子增加幅度明显。
实验例4
本实验例提供了MNC-168的制备方法。
用灭菌牙签挑取一个MNC-168单克隆到10ml的AC液体培养基(每升含：蛋白胨,20g；葡萄糖,5g；酵母提取物,3g；牛肉浸粉,3g；维生素C,0.2g；pH7.0)中培养1天，取1ml培养液进行质谱鉴定，鉴定结果正确后，吸取2ml菌悬液转接入80ml AC液体培养基中于厌氧操作台培养。
培养24小时后，吸取30ml菌悬液转接入400ml AC液体培养基中于厌氧操作台培养。培养24小时后，取1ml菌液进行质谱鉴定，鉴定结果正确后，将全部菌液转移到I L离心瓶中，6000rpm，4℃离心30min。取沉淀用AC液体培养基：无菌甘油(4：1)重悬。
将菌液甘油混合液吸取0.1ml菌株冻存液加入0.9ml生理盐水中进行梯度稀释，稀释至10 -8梯度，取浓度为10 -6，10 -7、10 -8梯度的稀释液100μl分别加入厌氧血平板培养基上，加入玻璃珠摇匀，采用平板计数法测定其CFU。
将制作好的菌株冻存液分装于无菌冻存管中，每管分装0.2ml菌液，每株菌分装10管。冷冻于-80℃冷冻保存。待完全冷冻后(24h)，取出一管冻存细菌，融化至室温，并采用稀释涂布法测量其活菌浓度(CFU)。
实验例5
本实验例探究MNC-168的抗逆性。
以活细菌为基础的活体生物药(Live Biotherapeutic Products)的开发在近年来获得了越来越多的关注。细菌在进入消化道后会面临相当恶劣的生存环境，一方面，胃部的低pH的胃酸以及肠道中一定浓度的胆盐对许多细菌有着很大的伤害，另一方面，不同的菌株对这些有害环境的抗性差异也很大。
一个细菌对消化道环境的耐受性关系到其是否有机会被开发成活体生物药，这些指标对以该细菌菌株为基础进行活体生物药开发中的剂型选择也具有重要的意义。因此，测试菌株的耐酸性以及胆盐耐受性是十分有必要的。
体外抗逆性测试所采取的方式是观察细菌在与体内环境相似的模拟液，即模拟胃液(SGF)和模拟肠液(SIF)，接入菌株，根据菌株在胃部及小肠的停留时间设定时间点取样，测定菌株的存活曲线。为排除菌株在自然情况下的生长/死亡带来的影响，我们以pH＝6.8的缓冲溶液作为对照。空腹模拟胃液(SGF)及以浓盐酸稀释法获得pH在1.0(中国药典)或1.2(美国药典)的溶液加入胃蛋白酶制成。考虑到餐前餐后胃液的变化，参考Margareth R.C.Marques等人所发表的文章，我们以pH＝3作为餐后的胃液pH，这个酸度也与小鼠的正常胃液pH相似。而模拟肠液(SIF)则根据USP26用磷酸二氢钾及氢氧化钠按一定比例配置，pH为6.8。
测试的时长参考人体胃肠道的排空时间，对于耐酸性试验，我们选取2小时作为测试时长，对于胆盐耐受性试验，我们选取6小时为测试时长。本实验中，主要考虑了酶、无机盐，以及胆盐。其中，由于不同的来源以及工艺，市面上胃蛋白酶、胰蛋白酶的活度参差不齐，我们参考M.Minekus等人，Leyuan Li等人和发表的文献，确定最终实验所用介质的配置及方案。
由于测试的取点时间相当密集，MRS涂板的方法操作时间长，受影响因素大，难以保证结果的准确性。因而在本实验中采用MTT(噻唑蓝)染色的方法进行活菌的鉴别。MTT作为一种染料常被当作底物通过检测细胞琥珀酸脱氢酶的活性来检测细胞的死活。琥珀酸脱氢酶是连接氧化磷酸化与电子传递的枢纽之一，可为真核细胞线粒体和多种原核细胞需氧和产能的呼吸链提供电子。
琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒(Formazan)并沉积在细胞中，而死细胞无此功能。在一定细胞数范围内，形成的甲瓒结晶的量与活细胞数成正比,二甲基亚砜(DMSO)能溶解细胞中沉淀的甲瓒，在570nm波长处测定其光吸收值，可间接测量活细胞的数量。该方法可以免去繁琐的浓度稀释和涂板操作，非常适用于快速进行细菌活率的测定。
一、模拟胃液(Simulated Gastric Fluid，SGF)实验，实验方案包括：
1.称取2.0g NaCl和2.67g胃蛋白酶(源叶生物胃蛋白酶，USP级，1:3000)；
2.加入7.0mL浓HCl(37％)，纯水定容至1000mL,混匀；
3.调pH分别至pH1.2(模拟人空腹胃液pH)和pH3.0(模拟人饭后胃液pH及小鼠空腹胃液pH)，过滤除菌，得到模拟胃液，应用于以下的耐酸性测试中。
二、模拟肠液(SIF)-Simulated Intestinal Fluid(SIF)实验，实验方案包括：
1、称取6.8g磷酸二氢钾以及0.9g氢氧化钠到1000mL纯水中；
2、加入0.1g胰蛋白酶(源叶生物胰蛋白酶，1:2500)，使溶液达到250U/mL的酶活力；
3、调节pH至6.8±0.1，过滤除菌，得到模拟肠液，应用于以下的胆盐耐受性测试中。
三、缓冲液的制备
将6.8g磷酸二氢钾及0.9g氢氧化钠溶于1000mL纯水中，调节pH至6.8±0.1，作为以下耐酸性测试和胆盐耐受性测试实验中使用的缓冲液(缓冲能力18.6mmol/L/pH)。
四、MTT(噻唑蓝)染色剂配制方法包括：
称量0.5g MTT(噻唑蓝Shanghai Acmec Biochemical Co.ltd,98％)至100mL无菌PBS(磷酸盐缓冲溶液，pH＝7.2)中，完全溶解后过滤除菌，储存于棕色试剂瓶，冷藏于4℃冰箱中。
五、耐酸性测试。
1、将待测菌粉0.7796g至10mL的缓冲液中，配置成浓度为2×10 9CFU/mL的菌液。
2、分别准备9mL缓冲液(对照组)；9mL pH1.2的模拟胃液(实验组1)；9mL pH3.0的模拟胃液(实验组2)至离心管中；
3、取本测试步骤1中的样品1mL加入本测试步骤2配制的模拟胃液及缓冲液中，摇匀获得样品A(缓冲液组)，B(pH1.2模拟胃液组)，和C(pH3.0模拟胃液组)。
4、在取样时间点取样：在0时时间点对样品A取样(以此为0时数据)；在0.5h，1h和2h时对样品A、B、C取样。
5、取1mL样品A，B和C加至4mL缓冲液中，混匀后取3mL混悬液加入1mL MTT(5mg/mL)染色液，黑暗环境下反应过夜。取3mL缓冲液加入1mL MTT作为空白对照。
6、过夜后，震荡摇匀，取1mL各反应的样品加入2mL二甲基亚砜(DMSO)用于溶解显色物质甲瓒)。
7、在570nM波长下测量样品的吸光值,并据此推算出活细菌的比例。
六、胆盐耐受性测试(在常温条件，超净工作台中进行)。
1、将待测菌液2mL加至含18mL的缓冲液的离心管中(缓冲液对照组)，将待测菌液2mL加至含18mL的模拟肠液的离心管中(模拟肠液组)。
2、取样时间点：0h(加入菌液混匀后马上取样)、0.5h、1h、2h、4h、6h。
3、在取样时间点分别取3mL对照组和模拟肠液组加入1mL MTT(5mg/mL)染色液，黑暗环境下反映过夜。
4、过夜后，震荡摇匀，取1mL各反应的样品加入5mL二甲基亚砜(DMSO)用于溶解显色物质甲瓒)，再次取1mL上述样品溶液到3mL二甲基亚砜中进行二次稀释。
5、在570nM波长下测量样品的吸光值,并据此推算出活细菌的比例。
七、耐酸性测试结果参照图5所示，图5中示出了MNC-168在模拟胃液中以吸光度值为参照的存活曲线。
以对照组的数值为基准，实验组相对于对照组的活菌存活率见表8，结果表明，2小时pH3.0的模拟胃液处理对MNC-168的存活几乎无影响，但该菌株无法抵抗pH1.2的模拟胃液(处理0.5小时后，几乎所有细菌均死亡)。
表8：MNC-168在模拟胃液中的存活率(％)。
时间/h 缓冲液组 pH1.2模拟胃液组 pH3.0模拟胃液组
0 100 100 100
0.5 100 -4 94
1 100 -6 100
2 100 -8 98
八、胆盐耐受性测试结果。
MNC-168在模拟肠液中以吸光度值为参照的存活曲线见图6所示。
以对照组的数值为基准，实验组相对于对照组的活菌存活率见表9。结果显示，MNC-168在肠液的环境中适应良好，并有所增殖，但随处理时间的增长，MNC-168存活率会有稍微下降。
表9：MNC-168在模拟肠液中的存活率(％)。
时间/h 缓冲液组 模拟肠液组
0 100 88
0.5 100 167
1 100 188
2 100 250
4 100 241
6 100 181
实验例6
本实验例探究了口服MNC-168可以用于肿瘤治疗结直肠癌。
为验证MNC-168是否可以用于肿瘤的预防和治疗，我们利用小鼠同源肿瘤模型进行了抑制结直肠癌的生长的实验。本实验通过了慕恩生物动物伦理委员会伦理审查。
C57B/6小鼠购自南京模式动物中心，小鼠结直肠癌细胞CT26购自ATCC，InVivoMAb anti-mouse PD-1(BioXcell,BE0146，具体为anti-mPD-1(CD279)Clone RMP1-14)。
小鼠被通过p.o.方式被喂食MNC-168细菌，MNC-168解冻后放置于室温至其温度达到室温，后按照每只小鼠2×10 9CFU(colony formation unit)/天的剂量喂食，连续28天。
在喂食MNC-168七天后，接种CT26的细胞量为1×10 6/只。接种的步骤如下：接种当天收集CT26细胞，细胞经PBS洗涤细胞一次后，用PBS重悬，并对细胞悬液进行计数。调整细胞浓度至1×10 7/mL，与预冷的Matrigel(基底膜基质胶)进行1：1混合均匀后注射0.1mL/只至小鼠皮下。接种肿瘤日设为D0天。
接种肿瘤后小鼠持续进行喂食MNC-168治疗直至实验结束，喂食方法同样采用p.o.方式。
在接种肿瘤后第7，10，14，17天腹腔注射抗鼠PD-1抗体，注射剂量为每次每只小鼠100μg。
每两天量取肿瘤大小，通过如下的公式计算肿瘤的体积：
肿瘤体积＝1/2×肿瘤长径 2×肿瘤宽径。
除实验组(MNC-168+PD-1)外，实验同时设置两组对照，分别为仅使用PD-1治疗组(PD-1)和不进行任何治疗的对照组(Control)。
本实验分为四组：
实验组1(MNC-168+PD-1)：按照实验例6方法中喂食MNC-168以及注射PD-1抗体；
实验组2(MNC-168治疗组)：按照实验例6方法中喂食MNC-168；
PD-1治疗组(PD-1)：按照实验例6方法中仅注射PD-1抗体；
对照组：不进行任何治疗。
结果参照图7所示，非治疗组(即对照组)肿瘤快速生长，在D19达到约2300mm 3，PD-1治疗组的肿瘤生长速度略低于对照组，PD-1治疗组(PD-1)在第19天肿瘤体积为约1800mm 3，MNC-168治疗组(即实验组2)在第19天肿瘤体积为约500mm 3，生长速度明显慢于对照组和PD-1治疗组(PD-1)，由此说明，MNC-168即使在单独给药的情况下，也可以明显降低结直肠癌的生长速度。
MNC-168与PD-1联合治疗组(即实验组1(MNC-168+PD-1))在第19天肿瘤体积为约200mm 3，生长速度明显慢于对照组和PD-1治疗组(PD-1)。该实验证明MNC-168可以明显降低结直肠癌的生长速度并且MNC-168可以在PD-1存在的情况下明显降低结直肠癌的生长速度。
实验例7
为验证MNC-168是否可以用于肿瘤的预防和治疗，我们利用小鼠同源肿瘤模型进行了抑制结直肠癌的生长的实验。本实验通过了慕恩生物动物伦理委员会伦理审查。
C57B/6小鼠购自南京模式动物中心，小鼠结直肠癌细胞MC-38购自ATCC，InVivoMAb anti-mouse PD-1(BioXcell,BE0146)。
小鼠被通过p.o.方式被喂食MNC-168细菌，MNC-168解冻后放置于室温至其温度达到室温，后按照每只小鼠2×10 9CFU(colony formation unit)/天的剂量喂食，连续28天。
在喂食MNC-168七天，接种MC38细胞量为2×10 6/只。接种的步骤如下：接种当天收集MC38细胞，细胞经PBS洗涤细胞一次后，用PBS重悬，并对细胞悬液进行计数。调整细胞浓度至2×10 7/mL，与预冷的Matrigel进行1：1混合均匀后注射0.1mL/只至小鼠皮下。接种肿瘤日设为D0天。
接种肿瘤后的小鼠持续进行喂食MNC-168治疗直至实验结束，喂食方法同样采用p.o.方式。
在接种肿瘤后的第7、10、14、17天腹腔注射抗鼠PD-1抗体，注射剂量为每次每只小鼠200μg。
每两天量取种瘤大小，通过如下的公式计算肿瘤的体积：
肿瘤体积＝1/2×肿瘤长径 2×肿瘤宽径。
除实验组(MNC-168+PD-1)外，实验同时设置两组对照，分别为仅使用PD-1治疗组(PD-1)和不进行任何治疗的对照组(Control)。
本实验分为四组：
实验组1(MNC-168+PD-1)：按照实验例7方法中喂食MNC-168以及注射PD-1抗体；
实验组2(MNC-168治疗组)：按照实验例7方法中仅喂食MNC-168；
PD-1治疗组(PD-1)：按照实验例7方法中仅注射PD-1抗体；
空白对照组：不进行任何治疗。
结果参照图8所示，非治疗组(即对照组)肿瘤快速生长，在D23达到约1500mm 3，PD-1治疗组(PD-1)的肿瘤生长速度略低于对照组，PD-1治疗组在第23天肿瘤体积为约1200mm 3，MNC-168治疗组(实验组2)在第23天肿瘤体积为约500mm 3，生长速度比对照组和PD-1治疗组(PD-1)更为缓慢，由此证明单独使用MNC-168可以明显降低结直肠癌的生长速度。
MNC-168与PD-1联合治疗组(实验组1)在第23天肿瘤体积为约150mm 3，生长速度明显慢于对照组和PD-1治疗组(PD-1)。该实验证明MNC-168可以明显降低结直肠癌的生长速度并且MNC-168可以在PD-1存在的情况下明显降低结直肠癌的生长速度。
实验例9
本实验例探究了口服MNC-168可以增加anti-PD-1的响应效率。
为验证MNC-168是否可以增加anti-PD-1的响应效率，我们利用小鼠抗生素处理过的同源肿瘤模型进行了抑制结直肠癌的生长的实验，抗生素列表参照表10所示。本实验通过了慕恩生物动物伦理委员会伦理审查。
表10：使用抗生素列表。
抗生素类型 来源 终浓度
氨苄青霉素 Sigma-Aldrich 1mg/ml
链霉素 MCE 5mg/ml
粘菌素 Sigma-Aldrich 1mg/ml
C57B/6小鼠购自南京模式动物中心，小鼠结直肠癌细胞CT26购自ATCC，InVivoMAb anti-mouse PD-1(BioXcell,BE0146)。
小鼠被通过p.o.方式被喂食MNC-168细菌，MNC-168解冻后放置于室温至其温度达到室温，后按照每只小鼠2×10 9CFU(colony formation unit)/天的剂量喂食，连续28天。
在喂食MNC-168七天后，接种CT26的细胞量为1×10 6/只。接种的步骤如下：接种当天收集细胞，细胞经PBS洗涤细胞一次后，用PBS重悬，并对细胞悬液进行计数。调整细胞浓度至1×10 7/mL，与预冷的Matrigel进行1：1混合均匀后注射0.1mL/只至小鼠皮下。接种肿瘤日设为D0天。
接种肿瘤后小鼠持续进行喂食MNC-168治疗直至实验结束，喂食方法同样采用p.o.方式。
在接种肿瘤后第7、10、14、17天腹腔注射抗鼠PD-1抗体，注射剂量为每次每只小鼠100μg。
每两天量取肿瘤大小，通过如下的公式计算肿瘤的体积：
肿瘤体积＝1/2×肿瘤长径 2×肿瘤宽径。
除实验组(MNC-168+PD-1)外，实验同时设置两组对照，分别为仅使用PD-1治疗组(PD-1)和不进行任何治疗的对照组(Control)。
本实验分为四组：
实验组1(MNC-168+PD-1)：按照实验例8方法中喂食MNC-168以及注射PD-1抗体；
实验组2(MNC-168治疗组)：按照实验例8方法中喂食MNC-168；
PD-1治疗组(PD-1)：按照实验例8方法中仅注射PD-1抗体；
对照组：不进行任何治疗。
结果参照图9(a)所示，非治疗组(即对照组)肿瘤快速生长，在D19达到约2300mm 3，PD-1治疗组的肿瘤生长速度略低于对照组，PD-1治疗组(PD-1)在第19天肿瘤体积为约2500mm 3，MNC-168治疗组(即实验组2)在第19天肿瘤体积为约1300mm 3，生长速度明显慢于对照组和PD-1治疗组(PD-1)，由此说明，MNC-168即使在单独给药的情况下，也可以明显降低结直肠癌的生长速度。
MNC-168与PD-1联合治疗组(即实验组1(MNC-168+PD-1))在第19天肿瘤体积为约600mm 3，生长速度明显慢于对照组和PD-1治疗组(PD-1)。该实验证明MNC-168可以明显降低结直肠癌的生长速度并且MNC-168可以在PD-1存在的情况下明显降低结直肠癌的生长速度。
实验例10
本实验例的实验方法包括：
取约0.3g肿瘤组织，用剪刀切成小块，在胶原酶IV消化液中(1mg/ml胶原酶IV，0.1mg/ml DNA酶，10％FBS)，37℃消化30分钟。组织消化后的混合液通过70um筛网，制成单细胞悬液。取一定量细胞悬液，加入刺激剂(1640培养基中加入100ng/ml PMA,1ug/ml离子霉素和6ug/ml BFA)，37℃刺激4小时。刺激后，进行表面分子CD4(白细胞分化抗原4)和CD8(白细胞分化抗原8)染色(FITC Rat Anti-Mouse CD4(异硫氰酸荧光素大鼠抗小鼠CD4)，PE anti-mouse CD8a(藻胆色素蛋白抗小鼠CD8a)按1：100稀释在1％FBS-PBS溶液)，4度避光染色30分钟。用Transcription Factor Buffer Set对细胞进行固定破膜，1X固定液在4度固定过夜，然后进行细胞因子染色(PE-Cy7 anti-mouse IFN-γ按1：100稀释在1x破膜液)，室温避光染色1小时。细胞重悬在PBS，上机检测。
图10中显示了MNC-168激活肿瘤组织中的肿瘤浸润免疫细胞CD4IFNγ细胞和CD8IFNγ细胞流式图。
实验材料：
FITC Rat Anti-Mouse CD4(货号553046,BD),PE Rat Anti-Mouse CD8a(货号553032,BD)。
PE-Cy7 anti-mouse IFN-γ(货号557649,BD),PMA(P1585,Sigma),ionomycin(货号407952,Sigma),brefeldin A(货号00-4506-51,Thermofisher),Transcription Factor Buffer Set(货号562574,BD),1640培养基(货号C11875500BT,Thermofisher),FBS(货号10099-141,Gibico)。
以上所述仅为本公开的可选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开的乳酸肠球菌对癌症病人肿瘤免疫治疗的效果具有重要的影响，乳酸肠球菌可以诱导iDC(immature DC)细胞分化成熟，成熟DC能有效激活T细胞，从而杀伤肿瘤细胞；同时本公开的乳酸肠球菌可以诱导M0型巨噬细胞向M1或M2型细胞的分化，使得IL-1β，IL-23，TNFα的分泌量显著的增加。本公开的乳酸肠球菌可以用于制备肿瘤抑制剂及相应的药物，从而实现肿瘤预防或治疗。

Claims (19)

  1. 一种乳酸肠球菌在制备肿瘤抑制剂中的应用；所述肿瘤包括如下实体肿瘤中的至少一种：结肠癌、直肠癌、结肠直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌和胆管癌。
  2. 根据权利要求1所述的应用，其特征在于，所述乳酸肠球菌保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121；
    优选地，所述乳酸肠球菌的菌落培养特征为：在细菌培养基上，培养24h后菌落呈白色，圆形，表面湿润，不透明，边缘整齐；在显微镜下，菌体呈椭球状，0.7-1.0μm×0.8-1.3μm，单个或成对排列，革兰氏阳性；
    优选地，所述细菌培养基为MRS培养基。
  3. 根据权利要求1或2所述的应用，其特征在于，
    所述肿瘤还包括以下实体肿瘤：卵巢癌、宫颈癌、前列腺癌、膀胱癌、头颈癌、骨髓瘤、淋巴瘤、脑瘤、脊髓瘤、食管癌、口咽癌、喉癌、结直肠癌、黑色素瘤、神经内分泌癌、CNS癌、非霍奇金淋巴瘤、血液恶性肿瘤、肾脏肿瘤、神经母细胞瘤、恒温肌肉瘤、文氏家族肉瘤、视网膜瘤、弥漫性大细胞淋巴瘤、晚期CD70+癌症。
  4. 一种预防或治疗肿瘤的药物，其特征在于，其包括乳酸肠球菌。
  5. 根据权利要求4所述的药物，其特征在于，所述预防或治疗肿瘤的药物还包括药学上可接受的添加剂或辅料，优选地，所述药物组合物剂型选自片剂、丸剂、粉剂、混悬剂、凝胶、乳液、乳膏、颗粒剂、纳米颗粒、胶囊、栓剂、注射剂、喷雾和针剂；
    优选地，所述乳酸肠球菌保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121。
  6. 根据权利要求5所述的药物，其特征在于，所述药学上可接受的添加剂包括填充剂、稀释剂、润湿剂、崩解剂、助流剂、润滑剂、粘合剂、着色剂、中的一种或多种；
    所述药学上可接受的辅料包括环糊精、淀粉、蔗糖、乳糖、羟丙基纤维素、羟丙基甲基纤维素、硬脂酸镁、滑石粉、氧化铁、羟甲淀粉钠中的一种或多种。
  7. 一种药物组合物，其特征在于，其包括权利要求4-5任一项所述的预防或治疗肿瘤的药物；
    优选地，所述药物组合物还包括联用药物，所述联用药物为如下药物中的至少一种：
    化疗药物，光敏剂，光热剂，抑制性第二信号分子的抗体、抑制性第二信号分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。
  8. 根据权利要求7所述的药物组合物，其特征在于，所述联用药物为如下药物中的至少一种：
    化疗药物，光敏剂，光热剂，抑制性信号通路分子的抗体、抑制性信号通路分子的抑制剂、抑制性第二信号通路分子的抗体、抑制性第二信号通路分子的抑制剂、PD-L1抑制剂、PD-1/PD-L1单抗药物。
  9. 根据权利要求7所述的药物组合物，其特征在于，所述抑制性第二信号分子包括如下信号分子中的至少一种：PD-1和CTLA-4。
  10. 根据权利要求8所述的药物组合物，其特征在于，所述抑制性信号通路分子包括如下信号分子中的至少一种：PD-1和CTLA-4。
  11. 根据权利要求9所述的药物组合物，其特征在于，所述抑制性第二信号分子的抗体或抑制性第二信号分子的抑制剂包括如下抗体中的至少一种：尼伏单抗、潘利珠单抗、替雷利珠单抗、纳武利尤单抗注射液、帕博利珠单抗注射液、特瑞普利单抗注射液和信迪利单抗注射液。
  12. 根据权利要求10所述的药物组合物，其特征在于，所述抑制性信号通路分子的抗体或抑制性信号通路分子的抑制剂包括如下抗体中的至少一种：尼伏单抗、潘利珠单抗、替雷利珠单抗、纳武利尤单抗注射液、帕博利珠单抗注射液、特瑞普利单抗注射液和信迪利单抗注射液。
  13. 根据权利要求7或8所述的药物组合物，其特征在于，所述PD-L1抑制剂选自度伐单抗、阿替珠单抗或阿维单抗。
  14. 根据权利要求7或8所述的药物组合物，其特征在于，所述PD-1/PD-L1单抗药物选自帕博利珠单抗或纳武利尤单抗。
  15. 一种乳酸肠球菌，其特征在于，保藏于广东省微生物菌种保藏中心，保藏名称为乳酸肠球菌(Enterococcus lactis)MNC-168，保藏编号为GDMCC NO:61121；
    优选地，所述乳酸肠球菌的菌落培养特征为：在细菌培养基上，培养24h后菌落呈白色，圆形，表面湿润，不透明，边缘整齐；在显微镜下，菌体呈椭球状，0.7-1.0μm×0.8-1.3μm，单个或成对排列，革兰氏阳性；
    优选地，所述细菌培养基为MRS培养基。
  16. 根据权利要求4-6中任一所述的药物，和/或根据权利要求7-14中任一所述的药物组合物，用于治疗肿瘤的用途。
  17. 一种治疗肿瘤的方法，其特征在于，所述方法包括：
    向所述有此需要的受试者给药治疗有效量的根据权利要求4-6中任一所述的药物，和/或根据权利要求7-14中任一所述的药物组合物。
  18. 根据权利要求4-6中任一所述的药物，和/或根据权利要求7-14中任一所述的药物组合物，和/或根据权利要求17所述的方法，其特征在于，所述肿瘤包括如下实体肿瘤中的至少一种：结肠癌、直肠癌、结肠直肠癌、肝癌、胰腺癌、乳腺癌、肾癌、纤维肉瘤、肺癌和胆管癌。
  19. 根据权利要求17所述的方法，其特征在于，所述给药包括如下的至少一种：口服、静脉内、皮下、腹膜内、直肠、肌内、皮肤、经皮、局部、通过任何其他肠胃外途径、以药学上可接受的剂型。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024037273A1 (en) * 2022-08-17 2024-02-22 Suzhou Precision Biotech Co., Ltd. Use of enterococcus lactis in preparation of drug for treating melanoma

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112618576B (zh) * 2021-01-19 2021-12-21 慕恩（广州）生物科技有限公司 一种乳酸肠球菌、预防或治疗肿瘤的药物及应用
CN114107133A (zh) * 2021-12-22 2022-03-01 苏州普瑞森生物科技有限公司 一种抗结直肠肿瘤的屎肠球菌及其应用
WO2024117703A1 (ko) * 2022-11-28 2024-06-06 주식회사 엠바이옴쎄라퓨틱스 신규 엔테로코커스 락티스 lcm001 균주 및 이의 용도

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2060025U (zh) 1989-04-14 1990-08-01 江苏省电力试验研究所 电子式可控(相)复励励磁调节器
CN101067120A (zh) * 2006-11-29 2007-11-07 康哲医药研究(深圳)有限公司 粪肠球菌cms－h001及其应用
CN104195075A (zh) * 2014-08-14 2014-12-10 生合生物科技股份有限公司 一种屎肠球菌ef08及包含它的饲料添加物和饲料
CN112618576A (zh) * 2021-01-19 2021-04-09 慕恩（广州）生物科技有限公司 一种乳酸肠球菌、预防或治疗肿瘤的药物及应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2060025U (zh) 1989-04-14 1990-08-01 江苏省电力试验研究所 电子式可控(相)复励励磁调节器
CN101067120A (zh) * 2006-11-29 2007-11-07 康哲医药研究(深圳)有限公司 粪肠球菌cms－h001及其应用
CN104195075A (zh) * 2014-08-14 2014-12-10 生合生物科技股份有限公司 一种屎肠球菌ef08及包含它的饲料添加物和饲料
CN112618576A (zh) * 2021-01-19 2021-04-09 慕恩（广州）生物科技有限公司 一种乳酸肠球菌、预防或治疗肿瘤的药物及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEROSA LISA, ROUTY BERTRAND, KROEMER GUIDO, ZITVOGEL LAURENCE: "The intestinal microbiota determines the clinical efficacy of immune checkpoint blockers targeting PD-1/PD-L1", ONCOIMMUNOLOGY, vol. 7, no. 6, 3 June 2018 (2018-06-03), pages e1434468, XP055954268, DOI: 10.1080/2162402X.2018.1434468 *
NAMI YOUSEF, HAGHSHENAS BABAK, HAGHSHENAS MINOO, ABDULLAH NORHAFIZAH, YARI KHOSROUSHAHI AHMAD: "The Prophylactic Effect of Probiotic Enterococcus lactis IW5 against Different Human Cancer Cells", FRONTIERS IN MICROBIOLOGY, vol. 6, XP055954267, DOI: 10.3389/fmicb.2015.01317 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024037273A1 (en) * 2022-08-17 2024-02-22 Suzhou Precision Biotech Co., Ltd. Use of enterococcus lactis in preparation of drug for treating melanoma

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