WO2024022036A1 - Produit d'akkermansia muciniphila pour prévention et traitement de tumeurs et son utilisation - Google Patents

Produit d'akkermansia muciniphila pour prévention et traitement de tumeurs et son utilisation Download PDF

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WO2024022036A1
WO2024022036A1 PCT/CN2023/105255 CN2023105255W WO2024022036A1 WO 2024022036 A1 WO2024022036 A1 WO 2024022036A1 CN 2023105255 W CN2023105255 W CN 2023105255W WO 2024022036 A1 WO2024022036 A1 WO 2024022036A1
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tumor
cancer
tumors
antibody
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PCT/CN2023/105255
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English (en)
Chinese (zh)
Inventor
梁德宝
叶思璇
郭真
蔡琰
黄铄雅
赵泽普
侯凤仪
郑丽君
常秀娟
刘洋洋
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广州知易生物科技有限公司
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Publication of WO2024022036A1 publication Critical patent/WO2024022036A1/fr

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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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the field of pharmaceutical technology, and particularly relates to an Akkermansia muciniphila product for preventing and treating tumors and its application.
  • stage IV colorectal cancer The 5-year relative survival rate of stage IV colorectal cancer is only 14%. 70% to 80% of patients with hepatocellular carcinoma are already in the advanced stage when diagnosed and can only receive palliative treatment methods such as systemic therapy, and the prognosis is extremely poor.
  • Systemic drug treatment mainly includes chemotherapy drugs, molecular targeted drugs and immunotherapy drugs.
  • Molecular targeted drugs and immunotherapy-related drugs targeting corresponding gene mutations are constantly being developed, especially immunotherapy drugs such as anti-PD-1/L1 monoclonal antibodies that have been approved by the U.S. Food and Drug Administration (FDA) It is used in the first-line treatment of patients with a variety of advanced solid tumors.
  • FDA U.S. Food and Drug Administration
  • immune checkpoint blockade therapy brings lasting tumor suppression in clinical applications, it is only effective for some patients. How to improve the response rate of immune checkpoint antibody drugs is the main problem currently faced. Current medical research believes that continued treatment with immune checkpoint blockade therapy can bring better therapeutic effects. However, in patients with limited immune checkpoint response, strong side effects such as severe gastrointestinal adverse reactions and severe skin itching occur. , liver damage, pneumonia, renal damage, etc., forcing these patients to discontinue immunotherapy and thus unable to obtain expected clinical benefits.
  • the present disclosure provides an Akkermansia muciniphila product for preventing and treating tumors, which at least includes Akkermansia muciniphila for preventing and treating tumors;
  • the Akkermansia muciniphila includes: one or both of AM06 and AM02;
  • the AM06 is Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793; the AM02 is Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794.
  • the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 were both deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021.
  • nucleic acid sequence of AM06 is shown in SEQ ID NO.1; the nucleic acid sequence of AM02 is shown in SEQ ID NO.2.
  • the Akkermansia muciniphila product for preventing and treating tumors also includes a monoclonal antibody
  • the monoclonal antibody includes one or more of PD-1 antibody, CD20 antibody and CTLA-4 antibody.
  • the Akkermansia muciniphila is one or more of live bacteria, inactivated bacteria with complete morphological structure, and inactivated bacteria with incomplete morphological structure.
  • the Akkermansia muciniphila is a viable cell of Akkermansia, Akkermansia that has undergone inactivation treatment, genetic recombination, transformation or modification, attenuation, chemical treatment or physical treatment. bacteria, Akkermansia lysate, and Akkermansia liquid culture supernatant.
  • the product includes a composition
  • the composition includes the Akkermansia muciniphila and an anti-tumor drug.
  • composition is a pharmaceutical composition
  • the dosage forms of the pharmaceutical composition include enema, injection, external pharmaceutical dosage and oral dosage.
  • the administration mode of the pharmaceutical composition includes oral administration, enema, subcutaneous injection, intravenous injection and intramuscular injection.
  • the oral dosage forms include powders, suspensions, tablets, capsules, films and granules.
  • the release mode of the oral dosage form includes sustained release and non-sustained release.
  • the composition is a probiotic composition.
  • the anti-tumor drug is an immune checkpoint inhibitor.
  • the Akkermansia muciniphila and the immune checkpoint inhibitor are administered simultaneously or separately.
  • the immune checkpoint inhibitor is selected from one or two combinations of antibodies and compounds.
  • the immune checkpoint of the immune checkpoint inhibitor is selected from the group consisting of PD-1/PD-L1, CTLA-4, LAG-3, TIM-3, VISTA, A2aR, B7/H3, 4-1BB, OX One or more of -40, TIGHT, CD-28, TNFR2, NKG2A, CD86, CD80, ICOS, CD40 and GITR.
  • the immune checkpoint of the immune checkpoint inhibitor is PD-1/L1.
  • the monoclonal antibody agent whose immune checkpoint is PD-1/L1 is selected from the group consisting of nivolumab, pembrolizumab, cimepilimab, toripalimab, sintilimab, One or more of reslizumab, atezolizumab, avelumab, and durvalumab.
  • the composition also includes auxiliary materials;
  • the auxiliary materials include one or more of diluents, excipients, binders, lubricants, suspending agents, flavoring agents, coating agents and solubilizers.
  • the present disclosure also provides the use of any of the above Akkermansia muciniphila products for preventing and treating tumors in the preparation of products for preventing and treating tumors, including digestive system tumors, lymphatic system tumors, genitourinary system tumors, One or more of respiratory system tumors and skin tumors.
  • the tumor is a digestive system tumor.
  • the digestive system tumors include one or more of rectal cancer, gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, and liver cancer.
  • the tumor is a lymphoid system tumor.
  • the lymphatic system tumor includes one or more of non-Hodgkin lymphoma and Hodgkin lymphoma.
  • the tumor is a genitourinary system tumor.
  • the genitourinary system tumors include one or more of kidney cancer, bladder cancer, urothelial cancer, ovarian cancer, breast cancer, cervical cancer and prostate cancer.
  • the tumor is a respiratory system tumor.
  • the respiratory system tumors include one or more of head and neck cancer, non-small cell lung cancer, and small cell lung cancer.
  • the head and neck cancer includes nasopharyngeal cancer and laryngeal cancer.
  • the tumor is a skin tumor.
  • the skin tumor includes one or more of melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • the present disclosure also provides the use of the Akkermansia muciniphila product for preventing and treating tumors in the preparation of products for preventing and treating tumors, and the products for preventing and treating tumors include food, health food, medicines, detection reagents and detection kits.
  • the dosage form of the medicine includes powder, suspension, pill, tablet, granule, capsule, oral liquid or tube feeding preparation, and enema.
  • the medicines include medicines for humans or animals;
  • the dosage forms of the health food include powders, suspensions, oral liquids, tablets, capsules and granules.
  • the present disclosure also provides Akkermansia muciniphila
  • the Akkermansia muciniphila includes: one or both of AM06 and AM02;
  • the AM06 is Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793; the AM02 is Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794.
  • the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 were both deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021.
  • the present disclosure also provides any of the above Akkermansia muciniphila products for preventing and treating tumors or the use of Akkermansia muciniphila for preventing and treating tumors, including digestive system tumors, lymphoid One or more of systemic tumors, genitourinary system tumors, respiratory system tumors, and skin tumors.
  • the tumor is a digestive system tumor.
  • the digestive system tumors include one or more of rectal cancer, gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, and liver cancer.
  • the tumor is a lymphoid system tumor.
  • the lymphatic system tumor includes one or more of non-Hodgkin lymphoma and Hodgkin lymphoma.
  • the tumor is a genitourinary system tumor.
  • the genitourinary system tumors include one or more of kidney cancer, bladder cancer, urothelial cancer, ovarian cancer, breast cancer, cervical cancer and prostate cancer.
  • the tumor is a respiratory system tumor.
  • the respiratory system tumors include one or more of head and neck cancer, non-small cell lung cancer, and small cell lung cancer.
  • the head and neck cancer includes nasopharyngeal cancer and laryngeal cancer.
  • the tumor is a skin tumor.
  • the skin tumor includes one or more of melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • the present disclosure also provides a method for treating tumor-related diseases, the method comprising
  • the tumors include digestive system tumors, One or more of lymphatic system tumors, genitourinary system tumors, respiratory system tumors, and skin tumors.
  • the tumor is a digestive system tumor.
  • the digestive system tumors include one or more of rectal cancer, gastric cancer, esophageal cancer, bile duct cancer, pancreatic cancer, and liver cancer.
  • the tumor is a lymphoid system tumor.
  • the lymphatic system tumor includes one or more of non-Hodgkin lymphoma and Hodgkin lymphoma.
  • the tumor is a genitourinary system tumor.
  • the genitourinary system tumors include one or more of kidney cancer, bladder cancer, urothelial cancer, ovarian cancer, breast cancer, cervical cancer and prostate cancer.
  • the tumor is a respiratory system tumor.
  • the respiratory system tumors include one or more of head and neck cancer, non-small cell lung cancer, and small cell lung cancer.
  • the head and neck cancer includes nasopharyngeal cancer and laryngeal cancer.
  • the tumor is a skin tumor.
  • the skin tumor includes one or more of melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • Figure 1 shows the colony characteristics of AM02
  • Figure 2 shows the colony characteristics of AM06
  • Figure 3 is a microscopic observation of AM02 after Gram staining
  • Figure 4 is a microscopic observation of AM06 after Gram staining
  • Figure 5 is a graph showing the ratio of CD8+ T cells to Treg cells in tumor cells of NCG mouse tumor-bearing mice administered SUDHL-6 cells subcutaneously in the method of Example 3 of the present disclosure;
  • Figure 6 shows the effects of each group on the number of CD8+ effector T cells in mouse tumors in Example 6 of the present disclosure
  • Figure 7 shows the effect of each group on the number of CD4+CD25+Treg cells in mouse tumors in Example 6 of the present disclosure
  • Figure 8 shows the effects of each group on IL-1 in mouse tumors in Example 6 of the present disclosure
  • Figure 9 shows the effects of each group on IL-6 in mouse tumors in Example 6 of the present disclosure.
  • Figure 10 shows the effects of each group on IL-8 in mouse tumors in Example 6 of the present disclosure
  • Figure 11 shows the effects of each group on VEGF in mouse tumors in Example 6 of the present disclosure
  • Figure 12 shows the effect of Akkermansia muciniphila on body weight changes of liver cancer mice in the method of Example 8.
  • the terms “comprises,” “comprising,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unrecited elements or method steps. .
  • the term “consisting of” is considered to be a preferred embodiment of the term “comprising”. If in the following a certain group is defined as containing at least a certain number of embodiments, this is also to be understood as revealing a group that optionally consists only of these embodiments.
  • a “therapeutically effective amount” refers to an amount effective in dosage and for the specified period of time necessary to achieve the desired therapeutic result.
  • the therapeutically effective amount of an agent can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the agent to elicit the desired response in the individual.
  • a therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the agent.
  • the terms "subject” and “patient” are used interchangeably herein and refer to a vertebrate animal, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, domestic animals, competitive animals, and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also included.
  • the term "monoclonal antibody” refers to a highly uniform antibody produced by a single B cell clone and directed only against a specific antigenic epitope.
  • “synergistic effect,” “synergy,” or “synergy” refers to an effect produced between two or more molecules, compounds, substances, factors, or compositions that is greater than or different from the sum of their individual effects.
  • an Akkermansia muciniphila product for preventing and treating tumors wherein the Akkermansia muciniphila product for preventing and treating tumors at least includes Akkermansia muciniphila;
  • the Akkermansia muciniphila includes: one or both of AM06 and AM02.
  • the Akkermansia muciniphila product for preventing and treating tumors may be a single strain of Akkermansia muciniphila or a combination of the strain and other substances.
  • Akkermansia muciniphila is also provided.
  • Akkermansia muciniphila including: one or both of AM06 and AM02;
  • the AM06 is Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793; the AM02 is Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794.
  • AM02 Classification name: Akkermansiamuciniphila, depository unit: General Microbiology Center of China Microbial Culture Collection Committee, depository address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Institute of Scientific Microbiology, Postcode 100101, Beijing ( CN), preservation date: June 28, 2021, preservation number: CGMCC No.22794
  • the present disclosure provides an application of Akkermansia muciniphila in the preparation of products for preventing and treating tumors, wherein the provided Akkermansia muciniphila includes: one or both of AM06 and AM02; wherein , the AM06 is Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793; the AM02 is Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794.
  • nucleic acid sequence of AM06 is shown in SEQ ID NO.1; the nucleic acid sequence of AM02 is shown in SEQ ID NO.2.
  • Akkermansiamuciniphila (also known as Akkermansia muciniphila, Akkermansia, Akkella) is a type of normal bacteria in the human intestinal tract and belongs to mucin-decomposing bacteria. It has been One of the disclosed strains of Akkermansia muciniphila is the ATCC BAA-835 strain, or BAA-835. In this disclosure, AM06 and AM02 are provided, two strains belonging to Akkermansia muciniphila and different from BAA-835.
  • Akkermansia muciniphila (AM06 and AM02) in the Akkermansia muciniphila products for preventing and treating tumors provided in the present disclosure have significant advantages compared with the existing ATCC BAA-835 strain. The role of preventing and treating tumors.
  • the Akkermansia muciniphila product for preventing and treating tumors further includes a monoclonal antibody.
  • the monoclonal antibody includes one or more of PD-1 antibody, CD20 antibody and CTLA-4 antibody.
  • monoclonal antibodies are secreted by plasma cells transformed from B lymphocytes. Each B lymphocyte strain can only produce its own proprietary antibody against a specific antigenic determinant. This kind of antibody produced from a single cell line is called a monoclonal antibody (McAb), or monoclonal antibody for short.
  • the monoclonal antibody can be a monoclonal antibody drug.
  • Monoclonal antibody drugs are generally divided into: monoclonal antibody agents for treating diseases (especially tumors), anti-tumor monoclonal antibody conjugates, and monoclonal antibodies for treating other diseases.
  • the targets of monoclonal antibodies are usually disease-related antigens or specific receptors on the cell surface.
  • Akkermansia muciniphila products (AM06 and AM02) in the tumor prevention and treatment have significant tumor prevention and treatment effects relative to the ATCC BAA-835 strain.
  • the role of immune checkpoint inhibitors, and when combined with immune checkpoint inhibitors, can significantly enhance the anti-tumor effect of immune checkpoint inhibitors.
  • PD-1 antibodies are monoclonal antibodies.
  • PD-1 programmed death receptor 1
  • CD279 cluster of differentiation 279
  • CD279 cluster of differentiation 279
  • Commercially available PD-1 antibody products include sintilimab (trade name Tyvyt), slulimab (trade name Hanselimab), and so on.
  • CD20 monoclonal antibody is a monoclonal antibody preparation used to treat B lymphoma.
  • CD20 refers to a molecular marker present on the surface of B lymphocytes. There is no CD20 molecular marker in other types of lymphocytes. Since this molecular marker only exists in B lymphocytes, monoclonal antibodies to CD20 were invented clinically. The antibody currently developed against CD20 is CD20 monoclonal antibody.
  • Commercially available CD20 monoclonal antibody products such as rituximab (trade name Rituximab), Ripertuzumab (Anpingxi), etc.
  • CTLA-4 antibody is cytotoxic T lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152), which is a protein receptor that functions as an immune checkpoint and downregulates immune responses.
  • CD152 cluster of differentiation 152
  • CTLA4 is constitutively expressed in regulatory T cells but is only upregulated in conventional T cells upon activation - a phenomenon that is particularly pronounced in cancer.
  • CD80 or CD86 When bound to CD80 or CD86 on the surface of antigen-presenting cells, it acts as an "off" switch.
  • Commercially available products include ipilimumab (Evo).
  • Akkermansia muciniphila products for preventing and treating tumors can include Akkermansia muciniphila and monoclonal antibodies, among which Akkermansia muciniphila includes AM06 and AM02, and The monoclonal antibody pool can include one or more of PD-1 antibodies, CD20 antibodies and CTLA-4 antibodies, so different combinations of the above components can be used in Akkermansia muciniphila products for the prevention and treatment of tumors. Different Akkermansia muciniphila products are available for the prevention and treatment of tumors.
  • the Akkermansia muciniphila is one or more of live bacteria, inactivated bacteria with complete morphological structure, and inactivated bacteria with incomplete morphological structure.
  • inactivated bacteria are beneficial bacteria that retain the original structure and characteristics of beneficial bacteria through special inactivation technology, but no longer have the ability to grow and reproduce. Inactivation is to maintain the activity of probiotics and resist the killing effect of environmental factors. Engineering and technical personnel often use wrapping technology, which can be supplemented by a full cold chain. Inactivated bacteria are here divided into those with complete morphological structure and those with incomplete morphological structure according to their morphological structure.
  • the Akkermansia muciniphila is one or more of live bacteria, inactivated bacteria with complete morphological structure, and inactivated bacteria with incomplete morphological structure.
  • the Akkermansia muciniphila is a living organism of Akkermansia, an Akkermansia strain that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, physically treated or inactivated.
  • intestinal microorganisms can affect the host's immune system and even affect immune tests through their surface molecules (such as capsular polysaccharides, flagella and surface proteins) and metabolites (such as short-chain fats, indole and inosine) Efficacy of point inhibitors. Studies have shown that immune checkpoint inhibitors can weaken the intestinal barrier function, allowing inosine, a key metabolite produced by B. pseudolongum, to enter the blood and promote anti-tumor T cell immunity.
  • Inosine acts on the adenosine 2A receptor expressed by T cells, and under the co-stimulation of conventional dendritic cells, it can promote the differentiation and activation of Th1 cells, thereby enhancing the therapeutic effect of immune checkpoint inhibitors; in intestinal cancer, bladder Inosine supplementation enhances the efficacy of immune checkpoint inhibitors in mouse models of cancer and melanoma.
  • Akkermansia muciniphila also known as Akkermansia muciniphila.
  • Akkermansia muciniphila In 2004, Dutch scholar Derrien et al. isolated and identified a new mucus-degrading bacterium, Akkermansia muciniphila, from human feces.
  • Everard et al. found that the abundance of Akkermansia muciniphila in the intestinal tract of obese and type 2 diabetic mice was significantly reduced; treatment with Akkermansia muciniphila could reverse the metabolic disorder caused by a high-fat diet. Including increased fat mass, metabolic endotoxemia, adipose tissue inflammation and insulin resistance; it also reduced intestinal inflammation and improved intestinal barrier function.
  • AKK is the first member and only representative of the Verrucomicrobiain phylum in the human intestine. Its 16S rRNA gene sequence is relatively easy to detect. AKK is one of the three major enterotypes of the human intestinal microbiome (Ruminococcus-rich type III). Major member, accounting for 1% to 5% of our gut microbes. Research to develop AKK in tumor treatment is warranted.
  • the Akkermansia muciniphila products for preventing and treating tumors provided in this embodiment include AM06 and AM02.
  • a large number of experiments have proven that the live and inactivated bacteria of Akkermansia muciniphila AM06 and AM02 .
  • Bacterial lysate has the effect of preventing and treating tumors, such as colorectal cancer and gastric cancer, and when combined with immune checkpoint inhibitors such as PD-1 antibodies or CTLA-4 antibodies, it can significantly enhance immune checkpoint inhibition. anti-tumor effect of the agent.
  • the product includes a composition
  • composition includes the Akkermansia muciniphila and an anti-tumor drug
  • the composition can be a composition used to prevent and treat tumors, a tumor suppressor drug for anti-tumor, preventing and treating cancer, or a tumor detection drug.
  • composition is a pharmaceutical composition
  • the dosage forms of the pharmaceutical composition include enema, injection, external pharmaceutical dosage and oral dosage.
  • the administration mode of the pharmaceutical composition includes oral administration, enema, subcutaneous injection, intravenous injection and intramuscular injection.
  • the oral dosage forms include powders, suspensions, tablets, capsules, films and granules.
  • the release mode of the oral dosage form includes sustained release and non-sustained release.
  • the composition is a probiotic composition.
  • the anti-tumor drug is an immune checkpoint inhibitor.
  • the Akkermansia muciniphila and the immune checkpoint inhibitor are administered simultaneously or separately.
  • the immune checkpoint inhibitor is selected from one or two combinations of antibodies and compounds.
  • the compound may be one or more combinations of macromolecular compounds and small molecule compounds.
  • antibodies may include, but are not limited to, monoclonal antibodies and polyclonal antibodies.
  • the immune checkpoint of the immune checkpoint inhibitor is selected from the group consisting of PD-1/PD-L1, CTLA-4, LAG-3, TIM-3, VISTA, A2aR, B7/H3, 4-1BB, OX One or more of -40, TIGHT, CD-28, TNFR2, NKG2A, CD86, CD80, ICOS, CD40 and GITR.
  • the immune checkpoint of the immune checkpoint inhibitor is PD-1/L1;
  • the monoclonal antibody agent whose immune checkpoint is PD-1/L1 is selected from the group consisting of nivolumab, pembrolizumab, cimepilimab, toripalimab, sintilimab, One or more of reslizumab, atezolizumab, avelumab, and durvalumab.
  • the composition also includes auxiliary materials;
  • the auxiliary materials include one or more of diluents, excipients, binders, lubricants, suspending agents, flavoring agents, coating agents and solubilizers.
  • one embodiment of the present disclosure also provides an application of an Akkermansia muciniphila product for preventing and treating tumors in the preparation of a product for preventing and treating tumors.
  • the tumors include one or more of digestive system tumors, lymphatic system tumors, genitourinary system tumors, respiratory system tumors, and skin tumors.
  • One embodiment of the present disclosure also provides the above-mentioned Akkermansia muciniphila product for preventing and treating tumors or the use of Akkermansia muciniphila for preventing and treating tumors, wherein the tumors include digestive system tumors, lymphatic system tumors, reproductive and urinary tract tumors, and digestive system tumors.
  • the tumors include digestive system tumors, lymphatic system tumors, reproductive and urinary tract tumors, and digestive system tumors.
  • One embodiment of the present disclosure also provides a method for treating tumor-related diseases, the method comprising
  • Akkermansia muciniphila product or Akkermansia muciniphila for preventing and treating tumors is administered to the subject in need thereof.
  • the tumor is a digestive system tumor.
  • the digestive system tumors include rectal cancer, gastric cancer, esophageal cancer, One or more of cholangiocarcinoma, pancreatic cancer, and liver cancer.
  • the tumor is a lymphatic system tumor
  • the lymphatic system tumors include one or more of non-Hodgkin lymphoma and Hodgkin lymphoma.
  • the tumor is a genitourinary system tumor.
  • the genitourinary system tumors include one or more of kidney cancer, bladder cancer, urothelial cancer, ovarian cancer, breast cancer, cervical cancer and prostate cancer.
  • the tumor is a respiratory system tumor.
  • the respiratory system tumors include one or more of head and neck cancer, non-small cell lung cancer, and small cell lung cancer.
  • the head and neck cancer includes nasopharyngeal cancer and laryngeal cancer.
  • the tumor is a skin tumor.
  • the skin tumor includes one or more of melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • the present disclosure provides an Akkermansia muciniphila product for preventing and treating tumors and an application thereof, wherein the Akkermansia muciniphila product for preventing and treating tumors at least includes Akkermansia muciniphila; Akkermansia muciniphila includes: one or both of AM06 and AM02; the AM06 is Akkermansia muciniphila AM06 with the deposit number CGMCC No. 22793; the AM02 is the deposit number It is Akkermansia muciniphila AM02 of CGMCC No. 22794.
  • Akkermansia muciniphila in the Akkermansia muciniphila product for preventing and treating tumors provided by the present disclosure has been proven through a large number of experiments that its live bacteria, inactivated bacteria and cell lysates have the effect of preventing and treating tumors, and When other immune checkpoint inhibitors such as PD-1 antibodies or CTLA-4 antibodies are used in combination, the anti-tumor effect of immune checkpoint inhibitors can be significantly enhanced.
  • Example 2 Preparation of inactivated bacteria and lysate of Akkermansia muciniphila
  • AKK inactivated bacteria preparation method :
  • Preparation method of lysate take an appropriate amount of resuspended Akkermansia muciniphila bacterial liquid, ultrasonicate at 50W for 10 seconds in an ice bath, then stop for 10 seconds, repeat ultrasonic until the bacteria rupture, and prepare Akkermansia mucinophila. Mann's inactivated bacterial lysate.
  • step 3 Perform vacuum freeze-drying of the original solution obtained in step 2, pre-freeze at -40 ⁇ 2°C for 1.5h, then pre-freeze at -20 ⁇ 2°C for 1h, and finally pre-freeze at -40 ⁇ 2°C for 1.5h under 0.25mbar vacuum. After one drying (-5 ⁇ 2°C and 0 ⁇ 2°C) and 0.1mbar analytical drying (35 ⁇ 2°C), the bacterial powder is prepared. The bacterial count of the bacterial powder reaches more than 1 ⁇ 10 11 cells/g. . used in the following examples.
  • Example 4 Efficacy experiment of Akkermansia muciniphila in treating diffuse large B-cell lymphoma (DLBCL) mice
  • NCG immunodeficient NCG (NOD Prkdc em26Cd52 Il2rg em26Cd22 /NjuCrl) mice were used for experiments, and a mouse DLBCL model was established using a transplanted tumor model method.
  • model group CD20 antibody (rituximab, rituximab) group, ATCC BAA-835 group, medium-dose AM06 group, medium-dose AM02 group, high-dose AM06, high-dose AM02, AM06 inactivated bacteria group, AM02 Inactivated bacteria group, ATCC BAA-835 inactivated bacteria group, medium-dose AM06+CD20 antibody group, medium-dose AM02+CD20 antibody group, high-dose AM06+CD20 antibody group and high-dose AM02+CD20 antibody group, 10 animals in each group mice.
  • the day of grouping was used as day 0, and administration was carried out as shown in Table 1 for a total of 22 days.
  • Akkermansia muciniphila and the inactivated bacteria were prepared by using the methods of Example 1 and Example 2 respectively.
  • tumor diameter was measured with a vernier caliper every 2 days.
  • TGI (%) [1-(Average tumor volume at the end of treatment in a certain treatment group/Average tumor volume at the beginning of treatment in the treatment group)/(Average tumor volume at the end of treatment in the same control group/ The average tumor volume of the isotype control group at the beginning of treatment)] ⁇ 100%.
  • mice were euthanized. Tumors from mice in each group were collected, and all tumors were weighed. The tumors were divided into three parts, one part was quick-frozen, one part was fixed in formalin, and one part was used for pathological sectioning.
  • Flow cytometry detection Collect freshly collected tumor samples and cut them into small pieces, prepare a single cell suspension of tumor tissue, and then extract 1 ⁇ 10 6 viable cells for antibody staining. After antibody staining and fixation, the cells are resuspended and transferred to a flow tube for on-machine detection. At least 10,000 viable cells are collected from each sample.
  • Table 3a Evaluation of the anti-tumor efficacy of the test substance on the A431 model (calculated based on the tumor volume on the 22nd day after group administration)
  • the tumor volume of each group of tumor-bearing mice during the administration period is shown in Table 2a and Table 2b.
  • CD20 antibody rituximab rituximab
  • Akkermansia muciniphila BAA-835, AM06, and AM02 can reduce the tumor volume.
  • the volume of small tumors (p ⁇ 0.0001), live Akkermansia muciniphila and inactivated bacteria are equally effective; Akkermansia muciniphila AM06 is more effective in reducing tumor volume than the CD20 antibody rituximab (rituximab) (p ⁇ 0.05); Akkermansia muciniphila AM06 and AM02 are both more effective in reducing tumor volume than Akkermansia muciniphila ATCC BAA-835 (p ⁇ 0.05); The combined administration of tuximab and Akkermansia muciniphila AM06/AM02 can significantly reduce the tumor volume (p ⁇ 0.0001).
  • the tumor inhibition rates of the 835 inactivated bacteria group, medium-dose AM06+CD20 antibody group, medium-dose AM02+CD20 antibody group, high-dose AM06+CD20 antibody group, and high-dose AM02+CD20 antibody group were 29.96%, 26.95%, and 37.25 respectively. %, 34.23%, 36.35%, 33.26%, 36.59%, 32.32%, 25.37%, 87.45%, 84.92%, 87.55% and 84.34% (Table 3a).
  • CD8+ T cells play an important role in tumor immunity and are effector cells that directly kill tumor cells.
  • Treg regulatory T cells
  • the ratio of CD8+T/Treg is an important indicator that reflects the effect of immunotherapy and changes in the tumor microenvironment.
  • the CD20 antibody group, the medium-dose AM06 group, the medium-dose AM02 group, and the high-dose AM06 CD8 of the group high-dose AM02 group, AM06 inactivated group, AM02 inactivated group, medium-dose AM06+CD20 antibody group, medium-dose AM02+CD20 antibody group, high-dose AM06+CD20 antibody group, and high-dose AM02+CD20 antibody group
  • the +T/Treg ratio increased (p ⁇ 0.05); in addition, compared with the CD20 antibody group, the CD8+T/Treg ratio was lower in the medium-dose AM06+CD20 antibody group, the medium-dose AM02+CD20 antibody group, and the high-dose AM06+ There was a significant increase in the CD20 antibody group and the high-dose AM02+rituximab combined administration group (p ⁇ 0.0001).
  • Example 5 Efficacy experiment of Akkermansia muciniphila in treating classical Hodgkin lymphoma (HL) mice
  • mice 4 to 5-week-old (20-22 grams body weight) BNX (NIH-Beige-NudeXID, NIH-III) triple combination immunized mice were used for experiments, and a mouse HL model was established using a transplanted tumor model.
  • mice weighing 20-22g 5 ⁇ 10 6 human H-RS cell lines (L1236 or HD-MyZ) in the logarithmic growth phase were subcutaneously injected into the abdominal surface. Palpable tumors will be formed within 5 days. When the tumor volume reaches After 60mm 3 (L1236)/160mm 3 (HD-MyZ), mice with uniform tumor sizes were selected and randomly grouped.
  • mice in each group.
  • the day of grouping was regarded as day 0, and administration was carried out as shown in Table 4 for a total of 22 days.
  • Akkermansia muciniphila and the inactivated bacteria were prepared by using the methods of Example 1 and Example 2 respectively.
  • TGI (%) [1-(Average tumor volume at the end of treatment in a certain treatment group/Average tumor volume at the beginning of treatment in the treatment group)/(Average tumor volume at the end of treatment in the same control group/ The average tumor volume of the isotype control group at the beginning of treatment)] ⁇ 100%.
  • mice were euthanized. Tumors from mice in each group were collected, and all tumors were weighed. The tumors were divided into three parts, one part was quick-frozen, one part was fixed in formalin, and one part was used for pathological sectioning.
  • the tumor volumes of tumor-bearing mice in each group during the administration period are shown in Table 5 and Table 6.
  • PD-1 antibodies sintilimab, Tyvyt
  • Akkermansia muciniphila BAA-835, AM06, AM02 and their inactivated bacteria can all reduce the size of tumors (p ⁇ 0.0001).
  • the live Akkermansia muciniphila bacteria are equivalent to the inactivated bacteria.
  • AM06 and AM02 are both more effective in inhibiting tumor growth than the inactivated bacteria.
  • the combined administration of BAA-835; PD-1 antibody and Akkermansia muciniphila AM06/AM02 can significantly reduce the tumor volume (p ⁇ 0.0001).
  • the combination of PD-1 antibody and Akkermansia muciniphila AM06 can completely eliminate mouse tumors established by L1236 cells after 18 days of administration (Table 5); HD-MyZ can be completely eliminated after 22 days of administration. cells established mouse tumors (Table 6).
  • the combination of PD-1 antibody and Akkermansia muciniphila AM02 can reduce the tumor volume to about 3mm3 .
  • the tumor inhibition rates (Table 7) were 47.46%, 58.62%, 45.00%, 51.67%, 61.67%, 51.72%, 58.33%, 55.00%, 49.15%, 100.00%, 95.00%, 100.00% and 96.67% respectively;
  • the tumor inhibition rates (Table 8) of mouse tumors induced by HD-MyZ cells were 56.84%, 58.97, 53.18%, 56.64%, 59.64%, 57.20%, 58.36%, 55.62%, 52.13%, 100.00%
  • AM02 and AM06 can significantly inhibit the growth of tumors.
  • AM02 and AM06 can enhance the therapeutic effect of the PD-1 antibody, showing a significant synergistic effect.
  • Example 6 Efficacy experiment of Akkermansia muciniphila in treating mouse B16F10 skin melanoma model
  • this example uses B16F10 melanoma cells to establish a tumor model in C57BL/6 mice and conduct experiments. Observed the tumor volume, relative tumor proliferation rate, tumor weight, tumor inhibition rate, CD8+ effector T cells, regulatory Changes in T cells (Treg cells) and related cytokines.
  • Mouse skin melanoma cell culture Take B16F10 cells and place them in RPMI 1640 complete medium (containing 10% FBS and 1% double antibody) and culture them in an incubator at 37°C and 5% CO2 . Observe under the microscope. When the cell density reaches about 80%, add 2-3 mL of trypsin for digestion. Transfer the cell suspension to a 50 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 5 mL of antibiotic-free solution containing 10% fetal bovine. Resuspend cells in serum-based RPMI 1640 medium.
  • mice 7-week-old male C57BL/6 mice were used in the experiment. After the mice grew naturally for 1 week, tumor inoculation was performed. All mice except the blank group were inoculated with tumor cells, and mouse-derived B16F10 cells were used for subcutaneous injection in the lower abdomen to create a model. The injection cell volume was 5 ⁇ 10 5 cells/mL ⁇ 0.2mL/mouse. Observe tumor growth in mice.
  • Signs of successful modeling The mice showed signs of exhaustion such as weight loss, hunched back, and lethargy, and palpable masses were visible at the inoculated site of the mice.
  • the administration time point is 10 a.m. on the administration day.
  • the combined medication group is administered PD-1 antibody first, and then live bacteria/inactivated bacteria.
  • Test items tumor volume and relative tumor proliferation rate: measure the long diameter and short diameter length after 0 days of administration and 14 days to calculate and record the tumor volume.
  • Relative tumor proliferation rate T/C (%) TRTV / CRTV ⁇ 100% ( TRTV : RTV of treatment group, CRTV : RTV of control group).
  • RTV V t /V 0 (the mean value of tumor volume at each measurement of V t and the mean value of tumor volume measured when V 0 is administered).
  • Tumor volume The tumor volume on day 0 and 14 after administration is as shown in Table 10 below.
  • T/C % is the relative tumor proliferation rate.
  • the AM06 group has a significantly better tumor inhibition effect than the PD-1 antibody group (P ⁇ 0.05), and is extremely significantly better than BAA-835.
  • group (P ⁇ 0.01) which was equivalent to the AM02 group (P>0.05).
  • the AM02 group's tumor inhibition efficacy was equivalent to that of the PD-1 antibody group (P>0.05), and significantly better than the BAA-835 group (P ⁇ 0.05).
  • the anti-tumor efficacy of the AM06 inactivated bacteria group is equivalent to that of the PD-1 antibody group (P>0.05), significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.05), and equivalent to the AM02 group (P>0.05).
  • the AM02 group The tumor inhibitory effect is equivalent to that of the PD-1 antibody group and BAA-835 inactivated bacteria (P>0.05); compared with the use of Akkermansia or PD-1 antibody alone, Akkermansia or inactivated Akkermansia The combination of Mannheimia AM06, AM02 and PD-1 antibodies inhibited tumors more significantly.
  • the p value between the two groups is calculated according to the unpaired t test (two-tailed) method.
  • PD-1 antibodies and Akkermansia can inhibit the growth of tumors, among which the AM06 group has extremely significant tumor inhibition effect Better than the PD-1 antibody group and BAA-835 group (P ⁇ 0.01), and equivalent to the AM02 group (P>0.05).
  • the AM02 group's tumor inhibitory effect was significantly better than the PD-1 antibody group and BAA-835 group (P ⁇ 0.05).
  • the anti-tumor efficacy of the AM06 inactivated bacteria group is equivalent to that of the PD-1 antibody group (P>0.05), significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.05), and equivalent to the AM02 group (P>0.05).
  • the AM02 group The tumor inhibitory effect was significantly better than that of the PD-1 antibody group (P ⁇ 0.05), and extremely significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.01); compared with Akkermansia or PD-1 antibody alone , Akkermansia or inactivated Akkermansia AM06 and AM02 combined with PD-1 antibodies were more effective in inhibiting tumors, and the anti-tumor effect was better than the sum of single strains and single antibodies (p ⁇ 0.01 ), showing that strains and antibodies have a strong synergistic effect.
  • Flow cytometry was used to analyze the proportion of CD8+ effector T cells in tumors in each group after treatment and administration.
  • the number of CD8+ effector T cells in tumors was significantly higher in the model group than in the blank group, and in the experimental group was significantly higher than that in the model group.
  • the Akkermansia AM06 combined with PD-1 inhibitor group was also significantly higher than the Akkermansia or PD-1 antibody alone group, as shown in Figure 6.
  • Flow cytometry was used to detect the proportion of Treg cells in the spleen and tumor.
  • the number of tumor Treg cells in the model group was significantly higher than that in the blank group, while the experimental group was lower than the model group. There was no significant difference in each group in the Akkermansia AM06 combined with PD-1 inhibitor group, and they were all higher than those in the blank group, as shown in Figure 7 .
  • Cytokines Use luminex technology to detect the levels of IL-1, IL-6, IL-8, VEGF and other cytokines in skin melanoma mouse models.
  • Figures 8, 9, 10 and 11 show the changes in IL-1, IL-6, IL-8 and VEGF respectively.
  • pro-inflammatory factors such as IL-1, IL-6, IL-8, and VEGF were significantly increased in the model group.
  • PD-1 inhibitors can effectively regulate the above-mentioned cytokines and down-regulate the levels of pro-inflammatory factors such as IL-1, IL-6, IL-8, VEGF, etc., while Akkermansia AM06 and inactivated bacteria can strengthen PD-1 inhibitors. Effect.
  • Example 7 Efficacy experiment of Akkermansia muciniphila in treating A431 skin basal cell tumor model
  • this example uses A431 epidermal cancer cells to establish a tumor model in C57BL/6 mice and conduct experiments.
  • the changes in tumor volume, relative tumor proliferation rate, tumor weight, and tumor inhibition rate in mouse tumor models treated with Akkermansia combined with PD-1 inhibitor (product number BE0146, purchased from BioXcell) were observed.
  • A431 epidermal cancer cells Place A431 cells in DMEM high-glucose complete medium and culture them in an incubator at 37°C and 5% CO2 . Observe under the microscope. When the cell density reaches about 80%, add 2-3 mL of trypsin for digestion. Transfer the cell suspension to a 50 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 5 mL of antibiotic-free solution containing 10% fetal bovine. Resuspend cells in serum-rich DMEM high-glucose complete medium. Pipette 10 ⁇ L of cell suspension into a 1.5 mL centrifuge tube, add 10 ⁇ L of Tripan blue dye solution, and mix well. Add 10 ⁇ L of the mixed liquid to the cell counting plate, and put it on the machine for counting. According to the counting results, adjust the cell concentration to 5 ⁇ 10 5 cells/mL. Place on ice and set aside.
  • mice 7-week-old male C57BL/6 mice were used in the experiment. After the mice grew naturally for 1 week, tumor inoculation was performed. All mice except the blank group were inoculated with tumor cells, and A431 cells were used for subcutaneous injection on the back to create a model. The injection cell volume was 5 ⁇ 10 5 cells/mL ⁇ 0.2mL/mouse. Observe tumor growth in mice.
  • Signs of successful modeling The mice showed signs of exhaustion such as weight loss, hunched back, and lethargy, and palpable masses were visible at the inoculated site of the mice.
  • the administration time point is 10 a.m. on the administration day.
  • the combined medication group is administered PD-1 antibody first, and then live bacteria/inactivated bacteria.
  • Relative tumor proliferation rate T/C (%) TRTV / CRTV ⁇ 100% ( TRTV : RTV of treatment group, CRTV : RTV of control group).
  • RTV V t /V 0 (the mean value of tumor volume at each measurement of V t and the mean value of tumor volume measured when V 0 is administered).
  • Tumor volume The tumor volume on day 0 and 14 after administration is as shown in Table 13 below.
  • the p value between the two groups is calculated according to the unpaired t test (two-tailed) method.
  • PD-1 antibodies and Akkermansia can inhibit the growth of tumors.
  • the AM06 group has a significantly better tumor inhibition effect than the PD-1 antibody group (P ⁇ 0.05), and is extremely significantly better than BAA-835.
  • the AM02 group's tumor inhibition efficacy is significantly better than the PD-1 antibody group (P ⁇ 0.05), and extremely significantly better than the BAA-835 group (P ⁇ 0.01) ;
  • the anti-tumor efficacy of the AM06 inactivated bacteria group was significantly better than that of the PD-1 antibody group (P ⁇ 0.05), extremely significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.01), and equivalent to the AM02 group (P>0.05) ;
  • the anti-tumor efficacy of the AM02 group was equivalent to that of the PD-1 antibody group (P>0.05), and extremely significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.01); compared with Akkermansia alone or PD- 1 antibody, the combination of Akkermansia or inactivated Akkermansia AM06, AM02 and PD-1 antibodies inhibited tumors more significantly, reflecting the significant synergistic effect of the combination of strains and antibodies.
  • the p value between the two groups is calculated according to the unpaired t test (two-tailed) method.
  • the AM06 group has a significantly better tumor inhibition effect than the PD-1 antibody group and BAA-835 group (P ⁇ 0.01). Compared with the AM02 group The efficacy is equivalent (P>0.05).
  • the AM02 group has a significantly better tumor inhibition effect than the PD-1 antibody group and the BAA-835 group (P ⁇ 0.01).
  • the AM06 inactivated bacteria group has a significantly better tumor inhibition effect than the PD-1 antibody group (P ⁇ 0.01). P ⁇ 0.05), significantly better than the BAA-835 inactivated bacteria group (P ⁇ 0.05), and equivalent to the AM02 group (P>0.05).
  • the AM02 group’s tumor inhibitory effect is the same as that of the PD-1 antibody group and BAA-835 inactivated bacteria.
  • the groups were equivalent (P>0.05); compared with Akkermansia or PD-1 antibody alone, Akkermansia AM06, AM02 or inactivated Akkermansia AM06, AM02 and PD-1 antibody combination
  • the tumor inhibition was more significant, and the tumor inhibition effect was better than the sum of the strain and the antibody alone (p ⁇ 0.01), showing that the strain and the antibody have a strong synergistic effect.
  • the above results illustrate that Akkermansia AM06 or AM02 and its inactivated bacteria combined with PD-1 inhibitors can significantly improve the tumor microenvironment in mice.
  • Example 8 Efficacy test of Akkermansia muciniphila in the treatment of esophageal cancer
  • mice 130 wistar male rats aged 6 to 8 weeks were randomly divided into a modeling group (120 rats, subcutaneously injected with 0.15% MBNA solution) and a normal group (10 rats, after adaptive feeding for 2 weeks). Subcutaneous injection of 0.9% normal saline) twice a week for 10 consecutive weeks. After 6 weeks of mutagenesis, 3 rats were randomly selected for esophageal mucosal hyperplasia.
  • Model group positive drug group (PD-1 antibody, BE0146, BioXcell 200 ⁇ g/animal), AM06 live bacteria group, AM02 live bacteria group, AM06 inactivated bacteria group, AM02 inactivated bacteria group, AM06 supernatant group, AM02 upper Serum group and ATCC BAA-835 live bacteria group, 10 in each group.
  • Administration began after 7 weeks of mutagenesis.
  • the blank group and model group were given physiological saline.
  • the administration group was administered morning and evening according to the corresponding dosage of drugs, and was administered continuously for 13 Week; among them, the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 was 10 10 CFU/mL/animal, and the concentration of PD-1 antibody (BE0146, BioXcell) was 200 ⁇ g/animal.
  • Akkermansia muciniphila was prepared by the method described in Example 1, and the supernatant of Akkermansia muciniphila was prepared by the method described in Example 4 (1).
  • the rats were weighed at the 0, 7, 9, 11, 13, 15, 17, and 20 weeks of modeling.
  • the rats were fasted and water-free the day before the end of the administration (i.e., the 20th week), and the rats were weighed.
  • blood was taken from the abdominal aorta, the esophagus was removed and weighed, the number of esophageal epithelial tumors larger than 1mm3 was counted, the long and short diameters of the esophagus were measured, the volume of esophageal tumors was calculated, and each tumor was detected by ELISA.
  • the expression levels of COX-2 and PTX3 in the serum of rats in the group were weighed at the 0, 7, 9, 11, 13, 15, 17, and 20 weeks of modeling. The rats were fasted and water-free the day before the end of the administration (i.e., the 20th week), and the rats were weighed.
  • blood was taken from the abdominal aorta, the esophagus was removed and weighed,
  • the body weight of the rats in the model group was significantly lower than that of the blank control group from 7 to 20 weeks; from 7 to 9 weeks, there was no significant change in the body weight of the rats in each administration group and the model group, and from 11 to 20 weeks week, the body weight of rats in each administration group was significantly higher than that of the model group.
  • the degree of improvement in the body weight of rats with different administrations was: positive drug group ⁇ AM06 ⁇ AM02 > BAA-835.
  • the expression of COX-2 was down-regulated and the expression of PTX3 was up-regulated in each administration group and the blank group.
  • the positive drug group, AM06 and AM02 groups could It significantly affects the expression of COX-2 and PTX3, and the improvement effect is significantly better than that of the BAA-835 group.
  • the degree of influence of each administration group is: positive drug group ⁇ AM06 ⁇ AM02 > BAA-835 group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with BAA-835 group.
  • Akkermansia muciniphila can effectively improve esophageal cancer in rats, and its positive drug ⁇ AM06 ⁇ AM02>BAA-835 group.
  • Example 9 Efficacy test of combining Akkermansia muciniphila with PD-1 antibody for the prevention and treatment of esophageal cancer
  • Eca-109 cell culture The esophageal cancer cell line Eca-109 is cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (containing 100 U/mL penicillin and 0.1 mg/mL streptomycin, and 7.4% NaHCO3 Adjust the pH value to 7.2 ⁇ 7.4), culture it in a constant temperature incubator at 37°C, 5% CO2 and 95% air saturated humidity. Take the cells in the logarithmic growth phase, digest the cells with 0.25% trypsin and 0.02% EDTA, and add Complete culture medium RPMI 1640 was used to terminate the digestion.
  • FBS fetal bovine serum
  • the cells were washed twice with serum-free RPMI 1640 culture medium, centrifuged at 1500 rpm for 5 min, resuspended in physiological saline, and the cell concentration was adjusted to 1 ⁇ 10 7 /mL.
  • mice in the modeling group were inoculated with esophageal cancer Eca-109 cells to establish the esophageal cancer model (the cells were inoculated subcutaneously in the left axilla and right buttock of nude mice, 0.2mL/site).
  • the mice in each administration group were inoculated on the 3rd day Carry out gastric administration twice a day, in which the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 is 10 10 CFU/mL/animal, and the concentration of PD-1 antibody (BE0146, BioXcell) is 200 ⁇ g/animal. Administration was given for a total of 4 weeks.
  • tumor inhibition rate (%) (tumor weight in the control group - tumor weight in the experimental group)/tumor weight in the control group ⁇ 100 %, RT-PCR method to detect COX-2mRNA expression.
  • the tumor weight of mice in the model group was significantly higher than that of each administration group.
  • Different administration groups can inhibit mouse tumor growth.
  • AM06 and PD-1 antibody combined group, AM02 and PD-1 antibody The tumor inhibitory effect of the combination group was significantly better than that of each AKK strain alone group and the positive drug alone group, and the tumor inhibition rate was significantly better than the sum of the AKK strain alone group and the positive drug alone group, reflecting The combination of the strains and PD-1 antibody has obvious synergistic effect; and the single use effect of AM06 and AM02 is significantly better than that of the BAA-835 group, that is, the degree of tumor inhibition in different administration groups is: AM06 combined with PD-1 antibody Use group ⁇ AM02 combined with PD-1 antibody group > AM06 group ⁇ positive drug group ⁇ AM02 group > BAA-835 group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with BAA-835 group.
  • COX-2mRNA expression Take mouse tumor tissue and use RT-PCR to detect COX-2mRNA expression (amplification fragment 255bp, upstream primer: 5'-AGAATGCATTGGAAACATCGACAG-3' (SEQ ID NO.3); downstream Primer: 5'-CCTGTTGCGGAGAAAGGAGTC-3' (SEQ ID NO.4)), the measurement results are shown in Table 18 below.
  • the expression of COX-2mRNA in the tumor tissue of the mice in the blank group and each administration group was down-regulated, among which AM06 or AM02 and The expression of COX-2 in the PD-1 antibody combined group and blank group was significantly lower than the model group, AKK alone group and positive drug group.
  • AM06 and AM02 alone were significantly better than those in the BAA-835 group.
  • Different administration groups had different effects on The degree of influence is: AM06 combined with PD-1 antibody group ⁇ AM02 combined with PD-1 antibody group > AM06 group ⁇ AM02 group ⁇ positive drug group > BAA-835 group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with the BAA-835 group.
  • the combination of Akkermansia muciniphila and PD-1 antibody can effectively improve esophageal cancer in rats.
  • Example 10 Efficacy test of Akkermansia muciniphila for preventing and treating liver cancer
  • the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 is 10 10 CFU/mL/bird, PD-1
  • the antibody (BE0146, BioXcell) concentration was 200 ⁇ g/animal.
  • Each administration group was administered the drug 24 hours after inoculation of the tumor strain, once a day, for 10 consecutive days.
  • Akkermansia muciniphila was prepared by the method described in Example 1, and the supernatant of Akkermansia muciniphila was prepared by the method described in Example 4 (1).
  • Tumor body weight and tumor inhibition rate As shown in Table 19 below, compared with the model group, the solid tumor tumors of mice in each drug group were significantly reduced, and the positive drug group, AM06 and AM02 live bacteria and inactivated bacteria were all There was a significant difference. Among them, the positive drug group, AM06 and AM02 groups were significantly better than the BAA-835 group in inhibiting tumor weight.
  • the inhibitory rate of liver cancer tumors by different administration methods was mainly as follows: positive drug group ⁇ AM06 ⁇ AM02>BAA-835.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group.
  • NK cell activity and T lymphocyte proliferation activity The occurrence and development of tumors are closely related to the body's immune function. The proliferation of tumor cells is usually accompanied by a weakening of the immune response. NK cells, as a spectrum killer cell, play a role in immune surveillance. Important role; lymphocyte proliferation activity can reflect the level of immune response mediated by T cells in the body.
  • the MTT method was used to measure the NK cell activity and T lymphocyte proliferation activity of mice.
  • the experimental results are shown in Table 20 below.
  • the NK cell activity and T lymphocyte proliferation activity of mice in the administration group were significantly increased, and AM06,
  • the degree of improvement in the AM02 and positive drug groups was significantly better than that in the BAA-835 group, that is, the immune mediating ability of the mice under different administrations was: positive drug group ⁇ AM06 ⁇ AM02 > BAA-835.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with BAA-835 group.
  • Akkermansia muciniphila can effectively prevent and treat liver cancer in mice.
  • Example 11 Efficacy test of the combination of Akkermansia muciniphila and immunosuppressants for the prevention and treatment of liver cancer
  • the rats were randomly divided into 11 groups: blank control group, model group, positive drug group (PD-1 antibody, BE0146, BioXcell 200 ⁇ g/rat), AM06 live bacteria group, AM02 live bacteria group, and AM06 inactivated Bacteria group, AM02 inactivated bacteria group, AM06 live bacteria combined with PD-1 antibody, AM02 live bacteria combined with PD-1 antibody, AM06 inactivated bacteria combined with PD-1 and AM02 inactivated bacteria combined with PD-1 antibody, 10 per group.
  • PD-1 antibody positive drug group
  • BE0146 BioXcell 200 ⁇ g/rat
  • AM06 live bacteria group AM02 live bacteria group
  • AM06 inactivated Bacteria group AM02 inactivated bacteria group
  • AM06 live bacteria combined with PD-1 antibody AM02 live bacteria combined with PD-1 antibody
  • AM06 inactivated bacteria combined with PD-1 and AM02 inactivated bacteria combined with PD-1 antibody 10 per group.
  • the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 was 10 10 CFU/mL/animal
  • the concentration of PD-1 antibody (BE0146, BioXcell) was 200 ⁇ g/animal, once a day for a total of 4 weeks.
  • the tumor weight of rats in the model group was significantly higher than that of each administration group.
  • Different administration groups can inhibit the growth of rat tumors.
  • AKK live bacteria, inactivated bacteria
  • PD-1 antibody are used in combination.
  • the degree of tumor inhibition was significantly better than that of the AKK alone group and the positive drug group, and the tumor inhibition rate was significantly better than the sum of the AKK strain alone group and the positive drug alone group, reflecting the AKK (live bacteria) , inactivated bacteria) and PD-1 antibody have obvious synergistic effects; and the tumor size of mice in the AM06 and AM02 groups is significantly smaller than that of the BAA-835 group, that is: AM06 and PD-1 antibody combination group ⁇ AM02 and PD -1 Antibody combination group>AM06 group ⁇ AM02 group ⁇ Positive drug group>BAA-835 group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with BAA-835 group.
  • Programmed necrosis is a type of inflammation-related programmed death, mediated by signaling pathways downstream of TNF- ⁇ , which intensifies inflammation and causes cell death, thereby promoting cancer cell metastasis.
  • IL-1 ⁇ plays an important role in the late stages of cancer. It can be produced in the tumor microenvironment and drive angiogenesis through cell signaling interactions with angiogenic factors to achieve tumor metastasis and spread.
  • the TNF- ⁇ and IL-1 ⁇ contents in the serum of rats in the blank group and each administration group were significantly lower than those in the model group. That is, the combination of AKK bacteria and PD-1 antibodies can effectively inhibit tumor cell metastasis, among which The improvement effect of the AKK combination group was significantly better than that of the AKK alone group and the positive drug group, reflecting the obvious synergistic effect of the combination of the strain and the PD-1 antibody; the improvement effect of the positive drug group, AM06 and AM02 groups was significantly better than that of the BAA- 835 group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with the BAA-835 group
  • bbb means P ⁇ 0.001 compared with the BAA-835 group.
  • the combination of Akkermansia muciniphila and PD-1 antibody can effectively improve liver cancer in rats.
  • Example 12 Efficacy test of Akkermansia muciniphila in preventing and treating pancreatic cancer
  • pancreatic cancer nude mouse tumor-bearing model and experimental grouping 130 4-6 week old healthy BALB/c female nude mice (weight 18-20g) were divided into blank group (10 mice) and model group ( 120 mice), among which the mice in the modeling group were inoculated dorsoventrally with the SW1990 cell suspension (1 mL/mouse) prepared in (1). The tumor status was checked one week after inoculation. If there was tumor growth, it indicated that the modeling was successful.
  • the tumor-bearing mice were randomly divided into 9 groups: model group, positive drug group (PD-1 antibody, BE0146, BioXcell 200 ⁇ g/mouse), AM06 live bacteria group, AM02 live bacteria group, AM06 Inactivated bacteria group, AM02 inactivated bacteria group, AM06 supernatant group, AM02 supernatant group, ATCC BAA-835 live bacteria group, 10 in each group, the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 is the same
  • the concentration of PD-1 antibody (BE0146, BioXcell) was 10 10 CFU/mL/bird, and the concentration of PD-1 antibody (BE0146, BioXcell) was 200 ⁇ g/bird.
  • Each administration group was administered once a day for 2 consecutive weeks.
  • Akkermansia muciniphila was prepared by the method described in Example 1, and the supernatant of Akkermansia muciniphila was prepared by the method described in Example 9(1).
  • the weight of solid tumor tumors in mice in each administration group was significantly reduced.
  • the positive drug group, AM06 and AM02 live bacteria and inactivated bacteria all had significant differences, and the above-mentioned administration groups
  • the tumor weight of the mice in the medium was significantly lower than that of the BAA-835 group, that is, the inhibitory rate of liver cancer tumors by different administration methods was mainly expressed as: positive drug group ⁇ AM06 > AM02 > BAA-835.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group.
  • the Caspase family plays a very important role in mediating cell apoptosis.
  • Caspase-3 as a key executive molecule, plays a role in many pathways of apoptosis signaling. Its level indirectly reflects cell apoptosis. level; in the mitochondrial apoptosis pathway, Bcl-2 family proteins regulate the release of pro-apoptotic factors in mitochondria and play an important role in cell apoptosis and survival.
  • Bax is a water-soluble related protein homologous to Bcl-2 , is a gene that promotes cell apoptosis in the Bcl-2 gene family. Its overexpression can antagonize the protective effect of Bcl-2 and cause cells to die. It is used together with Bcl-2 in tumor research.
  • the removed tumor tissue was ground and RNA was extracted.
  • the CT values of Bcl-2, Bax and Caspase-3 mRNA were detected by Real-time PCR, and the expression levels of Bcl-2, Bax and Caspase-3 mRNA were calculated using the 2- ⁇ CT method (Livak). , the results obtained are shown in Table 24 below.
  • the expression of Bal-2 in each drug group and the blank group was significantly down-regulated, and the expression of Bax and Caspase-3 mRNA was significantly up-regulated.
  • the positive drug group, AM06 and AM02 groups had a significant ability to regulate apoptosis-related genes.
  • BAA-835 group that is, AKK bacteria can effectively regulate cell apoptosis, and the ability of different drug groups to regulate cell apoptosis is: positive drug group ⁇ AM06 > AM02 > BAA-835.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • b means P ⁇ 0.05 compared with the BAA-835 group
  • bb means P ⁇ 0.01 compared with the BAA-835 group.
  • Akkermansia muciniphila can effectively prevent and treat pancreatic cancer in mice by regulating cell apoptosis.
  • Example 13 Efficacy test of the combination of Akkermansia muciniphila and immunosuppressants for the prevention and treatment of pancreatic cancer
  • Cell culture Cell line BxPC-3 was cultured in RPMI1640 culture medium (containing 10% fetal bovine serum, 2.0mmol/L glutamine, 100U/mL penicillin and 100mg/L streptomycin) at 37°C and 5% CO Culture under 2 conditions to prepare a single cell suspension with a concentration of 1.0 ⁇ 10 6 cells/mL.
  • RPMI1640 culture medium containing 10% fetal bovine serum, 2.0mmol/L glutamine, 100U/mL penicillin and 100mg/L streptomycin
  • pancreatic cancer mouse model 130 Balb/cnu/nu nude mice (mouse age 4-6 weeks, male:female ratio 3:1, body weight 18-22g), raised under SPF conditions, randomly divided into : Blank control group (10 animals) and modeling group (120 animals). Under sterile conditions, take 0.5mL of BxPC-3 cell suspension and inoculate it subcutaneously on the back of the scapula of the nude mice in the modeling group. When the tumor grows to a diameter of about 0.8cm (2 to 3 months), the transplanted tumor model will be considered successful. Nude mice in the normal group were injected with physiological saline.
  • nude mice were randomly divided into 11 groups: blank control group, model group, positive drug group (PD-1 antibody, BE0146, BioXcell 200 ⁇ g/mouse), AM06 live bacteria group, AM02 live bacteria group, and AM06 inactivated bacteria Group, AM02 inactivated bacteria group, AM06 live bacteria combined with PD-1 antibody, AM02 live bacteria combined with PD-1 antibody, AM06 inactivated bacteria combined with PD-1 and AM02 inactivated bacteria combined with PD-1 antibody, each Group of 10.
  • the concentration of bacterial liquid in each group of AM06, AM02 and ATCC BAA-835 was 10 10 CFU/mL/animal
  • the concentration of PD-1 antibody (BE0146, BioXcell) was 200 ⁇ g/animal. It was administered once every other day for a total of 8 weeks.
  • Tumor weight and tumor inhibition rate As shown in Table 25 below, compared with the model group, the tumor weight of solid tumors in mice in each administration group was significantly reduced, that is, each AKK strain and combination with PD-1 antibody can be effective Inhibit tumor growth, and the inhibitory effect of the combination group is significantly better than that of each AKK bacteria single-use group and the positive drug group; the combined use of AKK (live bacteria, inactivated bacteria) and PD-1 antibodies has a significantly better inhibitory effect on tumors than The tumor inhibition rate of each AKK single-use group and the positive drug group was significantly better than the sum of the two groups of each AKK strain single-use group and the positive drug single-use group, reflecting the obvious synergy of the combination of strains and PD-1 antibodies. Effect; The degree of inhibition of liver cancer tumors by different administration methods is mainly as follows: AM06 combined with PD-1 antibody group ⁇ AM02 combined with PD-1 antibody group > AM06 group ⁇ AM02 group > positive drug group.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the positive drug group
  • bb means compared with the positive drug group P ⁇ 0.01 compared with the positive drug group.
  • NF- ⁇ B directly acts on the cell cycle or modulates cell proliferation, apoptosis, inflammatory response, blood vessel growth and related MDR genes to cause cancer;
  • the proto-oncogene cyclin D 1 as the NF- ⁇ B transcription target gene, is a restricted regulatory G1
  • the overexpression and misregulation of the most important positive regulatory genes in /S phase transition can lead to abnormal cell cycle regulation, thereby promoting tumorigenesis.
  • a means P ⁇ 0.05 compared with the model group
  • aa means P ⁇ 0.01 compared with the model group
  • aaa means P ⁇ 0.001 compared with the model group
  • b means P ⁇ 0.05 compared with the positive drug group
  • bb means compared with the positive drug group P ⁇ 0.01 compared with the positive drug group
  • bbb means P ⁇ 0.001 compared with the positive drug group.
  • the combination of Akkermansia muciniphila and PD-1 antibodies can effectively inhibit pancreatic cancer and have a synergistic effect.
  • Example 14 Efficacy test of the application of Akkermansia muciniphila in the prevention and treatment of colorectal cancer
  • mice were randomly divided into two groups, namely: blank control group (10 mice) and modeling group (80 mice).
  • AOM azoxymethane
  • mice in the modeling group were given free drinking 3% dextran sodium sodium (DSS) for 6 consecutive days. sky.
  • DSS dextran sodium sodium
  • mice in the model group were randomly divided into 7 groups according to their body weight, namely: model group (normal saline, 1mL/mouse, intragastric administration), oxaliplatin group (10 mg/kg/time, intraperitoneal injection), BAA-835 live bacteria group (10 9 CFU/animal, intragastric injection), AM02 live bacteria group (10 9 CFU/animal, intragastric injection), AM06 live bacteria group (10 9 CFU/animal, gavage), AM06 inactivated bacteria group (10 9 /animal, gavage), AM06 lysate group (0.5mL/animal, gavage), 10 animals in each group.
  • the corresponding test substances were administered on the day of grouping. Except for the oxaliplatin group, which was administered twice a week, the other groups were administered the corresponding test substances once a day for 6 weeks.
  • mice were anesthetized with ether. The abdominal cavity of the mouse was cut open, and 8 cm of the colon was cut from the anus upward in an ice bath. After flushing with physiological saline, the colon was blotted dry with filter paper, and the body weight was weighed. The number of colorectal tumors was recorded, and the total volume of all tumors was measured and calculated.
  • a represents P ⁇ 0.05
  • b represents P ⁇ 0.05
  • Example 15 Efficacy test of Akkermansia muciniphila combined with CTLA-4 antibody in the prevention and treatment of colorectal cancer
  • tumor inhibition rate [1-(tumor volume on day 1 of the drug treatment group - tumor volume on day 25 of the drug treatment group)/(tumor volume on day 1 of the model group - day 25 of the model group) Tumor volume)]*100%.
  • the tumor volume of each group of tumor-bearing nude mice is shown in Table 29 below. It can be seen that the CTLA-4 antibody was administered alone or any Akkermansia muciniphila was administered alone or the CTLA-4 antibody was combined with any The combined administration of Akkermansia muciniphila can significantly reduce the tumor volume (P ⁇ 0.05 or P ⁇ 0.01).
  • the combined administration of CTLA-4 antibody and any kind of Akkermansia muciniphila was significantly better than the administration of CTLA-4 antibody alone or the same kind of Akkermansia muciniphila alone (P ⁇ 0.01) , and at the same dose level, the tumor volume after AM02 live bacteria or AM06 live bacteria combined with CTLA-4 antibody was significantly smaller than that of BAA-835 live bacteria combined with CTLA-4 antibody (P ⁇ 0.05 or P ⁇ 0.01); and The tumor inhibition rate of the combined administration was significantly better than the sum of the corresponding strain alone group and CTLA-4 antibody alone group, reflecting the obvious synergistic effect of the combination of the strain and CTLA-4 antibody.
  • this example can prove through the vascular calcification intervention experiment that Akkermansia muciniphila can significantly reduce the symptoms of vascular calcification, and the combination of Akkermansia muciniphila and propionate can significantly reduce the symptoms of vascular calcification.
  • Symptoms of vascular calcification, the combination of compound drugs and strain drugs has obvious effects on preventing and treating symptoms of vascular calcification, indicating that the two have significant synergistic effects, providing a new source of drugs for the prevention and treatment of vascular calcification in current clinical applications.
  • New ideas and new methods have important clinical significance in preventing and treating vascular calcification.
  • Example 16 Efficacy test of Akkermansia muciniphila combined with PD-1 antibody in the prevention and treatment of gastric cancer
  • BALB/c nude mice were used for experiments, and a mouse gastric cancer model was established using a transplanted tumor model.
  • tumor inhibition rate [1-(tumor volume on day 1 of the drug treatment group - tumor volume on day 25 of the drug treatment group)/(tumor volume on day 1 of the model group - day 25 of the model group) Tumor volume)]*100%.
  • the tumor volume after the combination of AM02 live bacteria or AM06 live bacteria and PD-1 antibody was significantly smaller than that of BAA-835 live bacteria and PD-1 antibody (P ⁇ 0.05 or P ⁇ 0.01); and the combination
  • the tumor inhibition rate of the administration group was significantly better than the sum of the corresponding strain alone group and PD-1 antibody alone group, reflecting the obvious synergistic effect of the combination of the strain and PD-1 antibody.
  • a represents P ⁇ 0.05
  • aa represents P ⁇ 0.01
  • b represents P ⁇ 0.05
  • bb represents P ⁇ 0.01
  • d represents P ⁇ 0.05
  • dd represents P ⁇ 0.01.
  • Example 17 Efficacy experiment of Akkermansia muciniphila promoting apoptosis of mouse lung cancer cell line Lewis cell line through macrophages in vitro
  • Mouse lung cancer cells Lewis cells and RAW264.7 cells were grown in DMEM medium containing 10% FBS (complete medium), adding 1% penicillin/streptomycin, at 37°C, 5% CO 2 and saturated Culture in a humidified incubator and replace the culture medium every two days. a) Take out the cell line from liquid nitrogen and quickly melt it in a constant temperature water bath at 37°C. Open the cryovial under sterile conditions, transfer the liquid to a 15 mL centrifuge tube, resuspend the cells in 2-3 mL of complete culture medium, and centrifuge at 1000 rpm for 5 min.
  • Cell staining Add 5 ⁇ L Annexin V-FITC and 5 ⁇ L PI Staining Solution, and gently blow evenly; incubate for 10 minutes at room temperature (20-25°C) in the dark; add 400 ⁇ L 1 ⁇ Binding Buffer, and mix gently. After staining, the samples were detected by flow cytometry within 1 hour.
  • Cell apoptosis Calculate the early apoptotic cells that are Annexin V-FITC single positive (Annexin V-FITC+/PI-) and the late apoptotic cells that are double positive for Annexin V-FITC and PI (Annexin V-FITC+/PI+) The sum of the proportion of apoptotic cells.
  • aa means compared with negative control, P ⁇ 0.01.
  • bb means P ⁇ 0.01 compared with ATCC BAA-835.
  • Akkermansia can promote the apoptosis of lung cancer cells through RAW264.7 cells, suggesting that it has the effect of treating lung cancer, and the effect is significantly better than the standard strain ATCC BAA-835.
  • Example 18 Efficacy test of Akkermansia muciniphila in treating non-small cell lung cancer in mice
  • the Lewis cells of the mouse lung cancer cell line in the logarithmic growth phase were adjusted to a single cell suspension with a cell concentration of 2 ⁇ 10 4 cells/mL, and the Lewis cells were inoculated into SPF using a syringe at an amount of 0.2 mL/animal under sterile conditions.
  • Lewis lung cancer tumor-derived mice were prepared subcutaneously in the right axilla of C57BL/6 mice. Re-inoculation was performed once on the 11th day.
  • Lewis lung cancer tissue with good growth was selected and sacrificed by cervical vertebra dislocation under sterile conditions. The tumor tissue was peeled off from the armpit, cut into pieces, homogenized, and ground, and the tissue was analyzed according to the tumor quality.
  • mice When the experiment is over or the tumor-bearing mice show obvious signs of exhaustion such as weight loss, hunched back, lethargy, etc., they will be sacrificed by dislocation of the cervical vertebrae. The weight of the transplanted tumor will be measured and the tumor inhibition rate will be calculated. The serum will be taken and frozen in a -80°C refrigerator. For detecting cytokines, etc.
  • Lewis cell culture Lewis cells are cultured in DMEM medium containing 10% fetal bovine serum. The culture temperature is 37°C, the gas environment is 5% CO 2 , and the humidity is saturated humidity; according to the cell growth rate and the culture medium When the color changes, the culture medium is replaced and digested with 0.25% trypsin for passage. According to the cell growth conditions, cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell concentration was adjusted to 2 ⁇ 10 4 cells/mL.
  • Lewis lung cancer tumor-derived mice Take 10 SPF grade C57BL/6 mice that are 6 to 8 weeks old and weigh about (20 ⁇ 2) g. Use the mouse lung cancer cell line in the logarithmic growth phase. Adjust the cell concentration of Lewis cells to a single cell suspension of 2 ⁇ 10 4 /mL. Use a syringe to inoculate the Lewis cells subcutaneously into the right axilla of the mouse at an amount of 0.2mL/mouse under sterile conditions. When the subcutaneous transplanted tumor grows to a diameter The transplanted tumor was peeled off when it was about 1cm.
  • mice were randomly divided into 12 groups, with 10 mice in each group: normal saline group, DDP group, AM02 low-dose group (10 7 CFU/mouse), AM02 medium dose group (10 8 CFU/only), AM02 high dose group (10 9 CFU/only), AM02 inactivated bacteria group (10 9 /only), AM06 low dose group (10 7 CFU/only), AM06 Medium dose group (10 8 CFU/bird), AM06 high dose group (10 9 CFU/bird), AM06 inactivated bacteria group (10 9 /bird), ATCC BAA-835 live bacteria group (10 9 CFU/bird) , ATCC BAA-835 inactivated bacteria group (10 9 / only).
  • Normal saline group 0.2 mL/animal, once a day, continuous intragastric injection for 3 weeks
  • DDP group intraperitoneal injection at 0.3 mL/time, 3 mg/kg, once a week, continuous injection for 3 weeks
  • AM02, AM06 And ATCC BAA-835 related group dosage group 0.2mL/animal, once a day, continuously for 3 weeks.
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of normal saline group - tumor weight of administration group)/average tumor weight of normal saline group.
  • c The p value between the two groups is calculated according to the t test (two-tailed) method.
  • d Comparison between AM02 and AM06 live bacteria and ATCC BAA-835 live bacteria group, and comparison between AM02 and AM06 inactivated bacteria and ATCC BAA-835 inactivated bacteria group.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 33 above.
  • Table 33 The tumor weight and tumor inhibition rate of each group are shown in Table 33 above.
  • the AM02 low-dose group, ATCC BAA-835 live bacteria group and inactivated bacteria group have statistical significance, while the other groups of AM02 and AM06 have no statistical significance, indicating that AM02 and AM06 can inhibit the growth of tumors and inhibit tumor growth.
  • the effect is similar to that of positive drugs.
  • the AM02 and AM06 live bacteria groups with the same dose have statistical differences; compared with the ATCC BAA-835 inactivated bacteria group, the AM02 and AM06 inactivated bacteria groups with the same dose have statistical differences. difference. The results showed that AM02 and AM06 could inhibit the growth of tumors, and the inhibitory effect was similar to that of the positive drugs, and significantly better than the standard strain ATCC BAA-835.
  • aa means P ⁇ 0.01 compared with the normal saline group
  • aaa means P ⁇ 0.001 compared with the normal saline group
  • bbb means P ⁇ 0.001 compared with the ATCC BAA-835 live bacteria group
  • ccc means P ⁇ 0.001 compared with the ATCC BAA-835 inactivated bacteria group Comparison P ⁇ 0.001.
  • IL-2 is mainly produced by T cells and has the functions of activating T cells, stimulating NK cell proliferation, promoting B cell proliferation and antibody secretion.
  • IL-6 is a cytokine closely related to tumor treatment. Studies have shown that IL-6 can weaken the differentiation of tumor-specific T cells and promote the survival of tumor cells, and inhibiting IL-6 can block the growth of tumors.
  • IFN- ⁇ is mainly produced by macrophages and has the function of enhancing the anti-tumor activity of macrophages.
  • each group of AM02 and AM06 live bacteria and inactivated bacteria
  • the ATCC BAA-835 live bacteria group can promote the content of IL-2 in the serum of tumor mice, which is better than the normal saline group. significant difference.
  • each group of AM02 and AM06 live bacteria and inactivated bacteria
  • the ATCC BAA-835 live bacteria group can reduce the content of IL-6 in the serum of tumor mice, and there is a significant difference compared with the normal saline group. .
  • each group of AM02 and AM06 (live bacteria and inactivated bacteria) and ATCC BAA-835 (live bacteria and inactivated bacteria) can promote the content of IFN- ⁇ in the serum of tumor mice, which is the same as the normal saline group. There is a significant difference in comparison. This shows that Akkermansia upregulates the levels of tumor suppressor factors IL-2 and IFN- ⁇ and downregulates the level of IL-6 in the serum of tumor mice.
  • the same dose of AM02 and AM06 live bacteria can significantly increase the expression of IL-2 and INF- ⁇ , and downregulate the expression of IL-6; compared with the ATCC BAA-835 inactivated bacteria group, The same dose of AM02 and AM06 inactivated bacteria can significantly up-regulate the expression of IL-2 and INF- ⁇ , and down-regulate the expression of IL-6.
  • Akkermansia AM02 and AM06 can regulate the levels of tumor-related immune factors and effectively treat non-small cell lung cancer transplanted tumors in mice, and the effect is significantly better than the standard strain ATCC BAA-835.
  • Example 19 Efficacy test of Akkermansia muciniphila in treating small cell lung cancer in mice
  • the model is established and drug administration is started in groups: AM02 low-dose group (10 7 CFU/animal), AM02 high-dose group (10 9 CFU/animal), and AM02 inactivated bacteria low-dose group (10 7 /bird), AM02 inactivated bacteria high-dose group (10 9 /bird), AM06 low-dose group (10 7 CFU/bird), AM06 high-dose group (10 9 CFU/bird), AM06 inactivated bacteria high Dosage group (10 7 /bird), AM06 inactivated bacteria high-dose group (10 9 /bird), ATCC BAA-835 live bacteria group (10 9 CFU/bird), ATCC BAA-835 inactivated bacteria group (10 9 /animal) were orally administered continuously for 3 weeks; the DDP (cisplatin, Shandong Qilu Pharmaceutical Co., Ltd.) group was intraperitoneally injected once a week for 3 consecutive weeks.
  • mice When the experiment is over or the tumor is When the mice showed obvious signs of exhaustion such as weight loss, hunched back, and lethargy, they were killed by dislocation of the cervical spine. The weight of the transplanted tumors was measured and the tumor inhibition rate was calculated. The serum was taken and frozen in a -80°C refrigerator for detection of cytokines, etc.
  • NCI-H446 cells were cultured in RPMI-1640 medium containing 10% calf serum, and cultured in a 37°C, 5% CO 2 cell incubator. NCI-H446 cells in the logarithmic growth phase were digested and dispersed with trypsin, washed twice with PBS, centrifuged at 2000 rpm for 5 min, and then prepared a suspension with PBS. Adjust the cell concentration to 3 ⁇ 10 7 /mL, and take 0.2 mL (6 ⁇ l0 6 /mouse) were inoculated subcutaneously in the right ribs of 110 nude mice and raised in an SPF (Specific pathogen Free) environment. Tumors appeared in situ after about 1 week of inoculation. When the tumor diameter was about 5 ⁇ Treatment experiments were conducted at 8 mm.
  • mice that have been successfully modeled will be randomly divided into 12 groups, with 10 mice in each group: normal saline group, DDP group (3mg/kg) , AM02 low-dose group (10 7 CFU/bird), AM02 high-dose group (10 9 CFU/bird), AM02 inactivated bacteria low-dose group (10 7 /bird), AM02 inactivated bacteria high-dose group (10 9 pcs/bird), AM06 low-dose group (10 7 CFU/bird), AM06 high-dose group (10 9 CFU/bird), AM06 inactivated bacteria high-dose group (10 7 pcs/bird), AM06 inactivated bacteria high-dose group (10 9 /bird), ATCC BAA-835 live bacteria group (10 9 CFU/bird), ATCC BAA-835 inactivated bacteria group (10 9 /bird).
  • Normal saline group 0.2mL/animal, once a day, continuous gavage for 3 weeks;
  • DDP group Intraperitoneal injection at 0.3 mL/time, 3 mg/kg, once a week, for 3 consecutive weeks;
  • AM02, AM06 and ATCC BAA-835 related group dosage groups 0.2mL/animal, once a day, continuously for 3 weeks.
  • Elisa was used to detect IL-2 (R&D Systems, M2000, the same below), IL-6 (R&D Systems, M6000B, the same below) and IFN- ⁇ ( R&D Systems, MIF00, the same below) and other cytokine levels.
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of normal saline group - tumor weight of administration group)/average tumor weight of normal saline group.
  • c The p value between the two groups is calculated according to the t test (two-tailed) method.
  • d Comparison between AM02 and AM06 live bacteria and ATCC BAA-835 live bacteria group, and comparison between AM02 and AM06 inactivated bacteria and ATCC BAA-835 inactivated bacteria group.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 35 above.
  • the DDP, AM02 and AM06 groups, and the ATCC BAA-835 live bacteria and inactivated bacteria groups which inhibit the growth of tumors.
  • the DDP group there is no statistical significance in the AM02 high-dose group, AM02 inactivated bacteria high-dose group, AM06 high-dose group, and AM06 inactivated bacteria high-dose group.
  • AM02 and AM06 low-dose groups and ATCC BAA-835 live bacteria group There was statistical significance with the inactivated bacteria group.
  • the AM02 and AM06 live bacteria groups with the same dose have statistical differences; compared with the ATCC BAA-835 inactivated bacteria group, the AM02 and AM06 inactivated bacteria groups with the same dose have statistical differences. difference.
  • the results show that AM02 and AM06 can inhibit the growth of tumors.
  • the high-dose inhibitory effect is similar to that of positive drugs and significantly better than the standard strain ATCC BAA-835.
  • aa means P ⁇ 0.01 compared with the normal saline group
  • aaa means P ⁇ 0.001 compared with the normal saline group
  • bb means P ⁇ 0.01 compared with the ATCC BAA-835 live bacteria group
  • bbb means compared with the ATCC BAA-835 live bacteria group P ⁇ 0.001
  • c means P ⁇ 0.05 compared with ATCC BAA-835 inactivated bacteria group
  • ccc means P ⁇ 0.001 compared with ATCC BAA-835 inactivated bacteria group.
  • IL-2 is mainly produced by T cells and has the functions of activating T cells, stimulating NK cell proliferation, promoting B cell proliferation and antibody secretion.
  • IL-6 is a cytokine closely related to tumor treatment. Studies have shown that IL-6 can weaken the differentiation of tumor-specific T cells and promote the survival of tumor cells, and inhibiting IL-6 can block the growth of tumors.
  • IFN- ⁇ is mainly produced by macrophages and has the function of enhancing the anti-tumor activity of macrophages.
  • each group of AM02 and AM06 live bacteria and inactivated bacteria
  • ATCC BAA-835 live bacteria group and inactivated bacteria group can promote the content of IFN- ⁇ in the serum of tumor mice, which is the same as the normal saline group.
  • Akkermansia upregulates the levels of tumor suppressor factors IL-2 and IFN- ⁇ and downregulates the level of IL-6 in the serum of tumor mice.
  • the same dose of AM02 and AM06 live bacteria groups significantly increased the expression of IL-2 and INF- ⁇ , and downregulated the expression of IL-6; compared with the ATCC BAA-835 inactivated bacteria group , the same dose of AM02 and AM06 inactivated bacteria groups significantly up-regulated the expression of IL-2 and INF- ⁇ , and down-regulated the expression of IL-6.
  • Akkermansia AM02 and AM06 can regulate the levels of tumor-related immune factors and effectively treat transplanted tumors of small cell lung cancer in mice, and the effect is significantly better than the standard strain ATCC BAA-835.
  • Example 20 Efficacy test of Akkermansia muciniphila in treating nasopharyngeal carcinoma in mice
  • RPMI-1640 medium containing fetal bovine serum to culture the human nasopharyngeal carcinoma cell line HONE1.
  • HONE1 cells under sterile conditions, adjust the cell concentration of the HONE1 cells in the logarithmic growth phase to a single cell suspension of 1 ⁇ 10 7 cells/mL.
  • the cell suspension was inoculated into female nude mice 5-7 weeks old and weighing 20-24g at about 0.2mL each.
  • mice When the experiment is over or the tumor-bearing mice show obvious signs of exhaustion such as weight loss, hunched back, lethargy, etc., they will be sacrificed by dislocation of their cervical vertebrae. The weight of the transplanted tumor will be measured and the tumor inhibition rate will be calculated. The tumor tissue will be frozen and stored in a -80°C refrigerator. , used to detect the expression of apoptotic proteins, etc.
  • HONE1 cells Human nasopharyngeal carcinoma HONE1 cells were cultured in RPMI-1640 medium containing 10% calf serum at 37°C and 5% CO 2 in a cell incubator. HONE1 cells in the logarithmic growth phase were digested and dispersed with trypsin, washed three times with PBS, centrifuged at 1000 rpm for 5 min, and then prepared a suspension with PBS. Adjust the cell concentration to 1 ⁇ l0 7 /mL, and take 0.2 mL (2 ⁇ l0 6 /mouse) were inoculated subcutaneously in the right ribs of 110 nude mice and raised in an SPF (specific pathogen-free) environment. Tumors appeared in situ about one week after inoculation. Tumors were considered to be hard when the tumor mass was larger than 5 mm in length and diameter and hard to touch. Criteria for successful modeling.
  • mice One week after the establishment of the transplanted tumor model, 120 successfully modeled mice were randomly divided into 12 groups, 10 mice in each group: normal saline group, DDP group, AM02 low-dose group (10 7 CFU/ only), AM02 medium dose group (10 8 CFU/only), AM02 high dose group (10 9 CFU/only), AM02 inactivated bacteria group (10 9 /only), AM06 low dose group (10 7 CFU/only) ), AM06 medium-dose group (10 8 CFU/bird), AM06 high-dose group (10 9 CFU/bird), AM06 inactivated bacteria group (10 9 /bird), ATCC BAA-835 live bacteria group (10 9 CFU / only), ATCC BAA-835 inactivated bacteria group (10 9 / only).
  • Normal saline group 0.2mL/animal, once a day, continuous gavage for 3 weeks;
  • DDP group Intraperitoneal injection at 0.3 mL/time, 3 mg/kg, once a week, for 3 consecutive weeks;
  • Dosage group of AM02 and AM06 related groups 0.2mL/animal, once a day, continuously for 3 weeks.
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of normal saline group - tumor weight of administration group)/average tumor weight of normal saline group.
  • c The p value between the two groups is calculated according to the t test (two-tailed) method.
  • d Comparison between AM02 and AM06 live bacteria and ATCC BAA-835 live bacteria group, and comparison between AM02 and AM06 inactivated bacteria and ATCC BAA-835 inactivated bacteria group.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 37 above.
  • the positive drug, AM02 and AM06 groups and the ATCC BAA-835 live bacteria group all had statistical differences in inhibiting tumor growth.
  • AM02 and AM06 low-dose group, medium-dose group, and ATCCATCC BAA-835 live bacteria group and inactivated bacteria group have statistical significance.
  • Compared with the live bacteria group there were statistical differences between the AM02 and AM06 live bacteria groups at the same dose; compared with the ATCC BAA-835 inactivated bacteria group, there were statistical differences between the AM02 and AM06 inactivated bacteria groups at the same dose.
  • the results show that AM02 and AM06 can inhibit the growth of tumors.
  • the high-dose inhibitory effect is similar to that of positive drugs, and is significantly better than the standard strain ATCC BAA-835.
  • b means P ⁇ 0.05 compared with ATCC BAA-835 viable bacteria group, bb means P ⁇ 0.01 compared with ATCC BAA-835 viable bacteria group, bbb means P ⁇ 0.001 compared with ATCC BAA-835 viable bacteria group;
  • c means compared with ATCC BAA -835 inactivated bacteria group P ⁇ 0.05, cc means P ⁇ 0.01 compared with ATCC BAA-835 inactivated bacteria group ccc means P ⁇ 0.001 compared with ATCC BAA-835 inactivated bacteria group.
  • the Bax gene has the effect of promoting cell apoptosis.
  • the results are shown in Table 38 above.
  • the positive drug, each AM02 and AM06 group, and the ATCC BAA-835 live bacteria group can significantly promote the expression of Bax mRNA after administration.
  • the Bcl-2 gene i.e., B-cell lymphoma/leukemia-2 gene
  • the results are shown in Table 38.
  • each group of positive drug, AM02 and AM06 could significantly inhibit the expression of Bcl-2 mRNA after administration (except the AM02 low-dose group).
  • Bax/Bcl-2 is a related protein that regulates cell apoptosis.
  • the expression of Bax is enhanced and the expression of Bcl-2 is weakened. Cells tend to undergo apoptosis.
  • the results are shown in the table above. Compared with the normal saline group, the ratio of Bax/Bcl-2 in each group of positive drug, AM02 and AM06 showed statistical significance after administration.
  • the same dose of AM02 and AM06 live bacteria groups significantly up-regulated the expression of BaxmRNA and down-regulated the expression of Bcl-2 mRNA; compared with the ATCC BAA-835 inactivated bacteria group, the same dose of AM02, AM06 The AM06 inactivated bacteria group significantly upregulated the expression of BaxmRNA and downregulated the expression of Bcl-2mRNA.
  • Akkermansia AM02 and AM06 can effectively treat mouse nasopharyngeal carcinoma transplanted tumors by promoting tumor cell apoptosis, and the effect is significantly better than the standard strain ATCC BAA-835.
  • Example 21 Efficacy test of Akkermansia muciniphila combined with PD-1 antibody in the treatment of non-small cell lung cancer in mice
  • the Lewis cells of the mouse lung cancer cell line in the logarithmic growth phase were adjusted to a single cell suspension with a cell concentration of 2 ⁇ 10 4 cells/mL, and the Lewis cells were inoculated into SPF using a syringe at an amount of 0.2 mL/animal under sterile conditions.
  • Lewis lung cancer tumor-derived mice were prepared subcutaneously in the right axilla of C57BL/6 mice. Re-inoculation was performed once on the 11th day.
  • Lewis lung cancer tissue with good growth was selected and sacrificed by cervical vertebra dislocation under sterile conditions. The tumor tissue was peeled off from the armpit, cut into pieces, homogenized, and ground, and the tissue was analyzed according to the tumor quality.
  • Lewis cell culture Lewis cells are cultured in DMEM medium containing 10% fetal bovine serum. The culture temperature is 37°C, the gas environment is 5% CO2, and the humidity is saturated humidity; according to the cell growth rate and the color of the culture medium Change the culture medium and digest with 0.25% trypsin for passage. According to the cell growth conditions, cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell concentration was adjusted to 2 ⁇ 10 4 cells/mL.
  • Lewis lung cancer tumor-derived mice Take 10 SPF grade C57BL/6 mice that are 6 to 8 weeks old and weigh about (20 ⁇ 2) g. Use the mouse lung cancer cell line in the logarithmic growth phase. Adjust the cell concentration of Lewis cells to a single cell suspension of 2 ⁇ 10 4 /mL. Use a syringe to inoculate the Lewis cells subcutaneously into the right axilla of the mouse at an amount of 0.2mL/mouse under sterile conditions. When the subcutaneous transplanted tumor grows to a diameter The transplanted tumor was peeled off when it was about 1cm.
  • a N Number of mice in each group; b Dosing volume: adjusted according to mouse body weight (10 ⁇ L/g). AM02 and AM06 are administered in a fixed volume: 200 ⁇ L/animal.
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of model group - tumor weight of administration group)/average tumor weight of model group.
  • c The p value between the two groups is calculated according to the t test (two-tailed) method.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 40 above.
  • the PD-1 antibody group, AM02 alone group and combined with PD-1 antibody group, AM06 alone group and combined with PD-1 antibody group all inhibited Tumor growth was statistically different from the model group, while the standard strain ATCC BAA-835 did not inhibit tumor growth.
  • CD8+ T cells can kill target cells expressing antigens. They are important effector cells in resisting viral infections, acute allograft rejection, and killing tumor cells.
  • CD4+ T cells are an important immune cell in the human immune system. They are mainly differentiated from helper T (Th) cells. They can bind to the non-peptide region of MHC class II molecules and participate in T cell antigen receptor (TCR) recognition of antigens. of signal transduction.
  • Th helper T
  • TCR T cell antigen receptor
  • Akkermansia AM02 and AM06 combined with PD-1 antibodies can regulate the proportion of infiltrating T cells in the tumor microenvironment, enhance the body's anti-tumor activity, and effectively treat non-small cell lung cancer transplanted tumors in mice.
  • Example 22 Efficacy test of Akkermansia muciniphila combined with PD-1 antibody in the treatment of small cell lung cancer in mice
  • model group normal saline
  • PD-1 antibody Keytruda, pembrolizumab, 10 mg/kg
  • AM02 group (10 8 CFU /bird)
  • AM02+PD-1 antibody group (10 8 CFU/bird + 10mg/kg
  • AM06 group (10 8 CFU/bird)
  • AM06+PD-1 antibody group (10 8 CFU/bird)
  • the serum will be taken and frozen in a -80°C refrigerator. For detecting cytokines, etc.
  • NCI-H446 cells were cultured in RPMI-1640 medium containing 10% calf serum, and cultured in a 37°C, 5% CO2 cell incubator. NCI-H446 cells in the logarithmic growth phase were digested and dispersed with trypsin, washed twice with PBS, centrifuged at 2000 rpm for 5 min, and then prepared a suspension with PBS. Adjust the cell concentration to 3 ⁇ 10 7 /mL, and take 0.2 mL (6 ⁇ l0 6 /mouse) were inoculated subcutaneously in the right ribs of 110 nude mice and raised in an SPF (specific pathogen-free) environment. Tumors appeared in situ after about 1 week of inoculation. Treatment was performed when the tumor diameter was approximately 5 to 8 mm. experiment.
  • a.N Number of mice in each group; b. Administration volume: adjusted according to mouse body weight (10 ⁇ L/g). AM02 and AM06 are administered in a fixed volume: 200 ⁇ L/animal.
  • Elisa was used to detect the levels of cytokines such as IL-2 (R&D Systems, M2000, the same below) and IFN- ⁇ (R&D Systems, MIF00, the same below) in the serum of the lung cancer xenograft mouse model. .
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of model group - tumor weight of administration group)/average tumor weight of model group.
  • c The p value between the two groups is calculated according to the t test (two-tailed) method.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 43 above.
  • the PD-1 antibody group, AM02 alone group and combined with PD-1 antibody group, AM06 alone group and combined with PD-1 antibody group all inhibited Tumor growth was statistically different from the model group; while the standard strain ATCC BAA did not inhibit tumor growth.
  • the tumor weight of the combination group of AM02 and PD-1 antibody, and the combination group of AM06 and PD-1 antibody was lower, and the tumor inhibition rate was significantly larger. ;
  • the tumor inhibition rate of combined administration was significantly better than the sum of the corresponding strain alone group and PD-1 antibody alone group, reflecting the obvious synergistic effect of the combination of the strain and PD-1 antibody. It shows that AM02 and AM06 can inhibit the growth of tumors. When combined with the immunosuppressant PD-1 antibody, AM02 and AM06 can enhance the therapeutic effect of the PD-1 antibody, showing a significant synergistic effect.
  • aaa means P ⁇ 0.001 compared with the model group water group.
  • IL-2 is mainly produced by T cells and has the functions of activating T cells, stimulating NK cell proliferation, promoting B cell proliferation and antibody secretion.
  • IFN- ⁇ is mainly produced by macrophages and has the function of enhancing the anti-tumor activity of macrophages. It can be seen from Table 44 above that the PD-1 antibody group, AM02 and AM06 alone or in combination can promote the content of IL-2 in the serum of tumor mice, and there is a significant difference compared with the model group. AM02 and AM06 and PD- 1 Antibody combination has a greater increase than IL-2 alone. The PD-1 antibody group, AM02 and AM06 alone or in combination can all promote the IFN- ⁇ content in the serum of tumor mice, with significant differences compared with the model group.
  • Akkermansia AM02 and AM06 combined with PD-1 antibodies can regulate the levels of tumor-related immune factors and effectively treat transplanted small cell lung cancer tumors in mice.
  • Example 23 Efficacy test of Akkermansia muciniphila using PD-1 antibody to treat nasopharyngeal carcinoma in mice
  • RPMI-1640 medium containing fetal bovine serum to culture the human nasopharyngeal carcinoma cell line HONE1.
  • HONE1 cells under sterile conditions, adjust the cell concentration of the HONE1 cells in the logarithmic growth phase to a single cell suspension of 1 ⁇ 10 7 cells/mL.
  • the cell suspension was inoculated into female nude mice 5-7 weeks old and weighing 20-24g at about 0.2mL each.
  • HONE1 cells Human nasopharyngeal carcinoma HONE1 cells were cultured in RPMI-1640 medium containing 10% calf serum at 37°C and 5% CO2 in a cell incubator. HONE1 cells in the logarithmic growth phase were digested and dispersed with trypsin, washed three times with PBS, centrifuged at 1000 rpm for 5 min, and then prepared a suspension with PBS. Adjust the cell concentration to 1 ⁇ l0 7 /mL, and take 0.2 mL (2 ⁇ l0 6 /mouse) were inoculated subcutaneously in the right ribs of 110 nude mice and raised in an SPF (specific pathogen-free) environment. Tumors appeared in situ about one week after inoculation. Tumors were considered to be hard when the tumor mass was larger than 5 mm in length and diameter and hard to touch. Criteria for successful modeling.
  • a.N Number of mice in each group; b. Administration volume: adjusted according to mouse body weight (10 ⁇ L/g). AM02 and AM06 are administered in a fixed volume: 200 ⁇ L/animal.
  • Transplanted tumor weight and tumor inhibition rate After three weeks of continuous administration, take out the transplanted tumor and weigh it and calculate the tumor inhibition rate.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of the model group - average tumor weight of the administration group )/average tumor weight of the model group.
  • cytokines in tumor tissues Collect freshly collected tumor samples and cut them into small pieces, and use the qPCR method to detect the levels of cytokines such as IL-2 and IFN- ⁇ in the tumors.
  • a Mean ⁇ SD.
  • Tumor inhibition rate % 100% ⁇ (average tumor weight of model group - tumor weight of administration group)/average tumor weight of model group.
  • c p value between two groups according to Calculated using t-test (two-tailed) method.
  • the tumor weight and tumor inhibition rate of each group are shown in Table 46 above.
  • the PD-1 antibody group, AM02 alone group and combined with PD-1 antibody group, AM06 alone group and combined with PD-1 antibody group all inhibited Tumor growth was statistically different from the model group, while the standard strain ATCC BAA-835 did not inhibit tumor growth.
  • the AM02 and AM06 groups there was no statistical significance in the AM02 and AM06 groups; the AM02 and PD-1 antibody combination group, and the AM06 and PD-1 antibody combination group had lower tumor weights and greater tumor inhibition rates;
  • the tumor inhibition rate of the combination group was significantly better than the sum of the corresponding strain alone group and PD-1 antibody alone group, reflecting the obvious synergistic effect of the combination of the strain and PD-1 antibody. It shows that AM02 and AM06 can inhibit the growth of tumors. When combined with the immunosuppressant PD-1 antibody, AM02 and AM06 can enhance the therapeutic effect of the PD-1 antibody, showing a significant synergistic effect.
  • aaa means P ⁇ 0.001 compared with the model group.
  • IL-2 is mainly produced by T cells and has the functions of activating T cells, stimulating NK cell proliferation, promoting B cell proliferation and antibody secretion.
  • IFN- ⁇ is mainly produced by macrophages and has the function of enhancing the anti-tumor activity of macrophages. It can be seen from Table 47 above that the PD-1 antibody group, AM02 and AM06 alone or in combination can promote the content of tumor factor IL-2, and there is a significant difference compared with the model group. AM02 or AM06 combined with PD-1 antibody The increase was greater with IL-2 than with IL-2 alone.
  • the PD-1 antibody group, the individual groups of AM02 and AM06, and the combination group with PD-1 antibody can all promote the content of IFN- ⁇ in tumor factors, and there is a significant difference compared with the model group.
  • AM02 or AM06 and The combination of PD-1 antibody and IFN- ⁇ increased more than that of IFN- ⁇ alone. This shows that the combination of Akkermansia and PD-1 antibodies can increase the levels of tumor factors IL-2 and IFN- ⁇ .
  • Akkermansia AM02 or AM06 alone or in combination with PD-1 antibody can regulate the levels of tumor-related immune factors and effectively treat small cell nasopharyngeal carcinoma xenografts in mice.
  • Example 24 Experiment on the efficacy of Akkermansia muciniphila in treating mouse Caki2 renal cancer cell transplantation
  • Caki2 renal cancer cell culture Caki2 renal cancer cells were grown in McCoy'5A medium with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were cultured in a humidified cell culture incubator at 37°C and 5% CO2. Cells were passaged when the cells covered more than 80% of the area in the field of view under the microscope. First, pour away the waste liquid in the fine culture bottle, rinse it twice with PBS, then add 2-3mL trypsin, put it into the cell culture incubator for digestion for 1-2 minutes, then use 10% serum-containing medium to neutralize the trypsin, and use Tap the cells with a dropper and place into a 15 mL centrifuge tube.
  • mice 6-week-old male nude mice were used in the experiment. After the mice grew naturally for 1 week, tumor inoculation was performed. All mice except the normal control group were inoculated with tumor cells, and Caki2 renal cancer cells were planted on the backs of mice. The injection cell volume was 5 ⁇ 10 6 cells/mL ⁇ 0.2mL/mouse. Observe tumor growth in mice.
  • Signs of successful modeling The mice showed signs of exhaustion such as weight loss, hunched back, and lethargy, and palpable masses were visible at the inoculated site of the mice. Mice were successfully inoculated with tumor cells and grouped into groups 7 days later.
  • Administration method will begin 7 days after tumor inoculation. The administration time point is 10 a.m. on the administration day. The combined medication group will be administered PD-1 antibody first, and then live bacteria/inactivated bacteria. Administration was given for a total of 3 weeks.
  • tumor inhibition rate (%) 100% (average tumor weight of model group - average tumor weight of drug administration group)/average tumor weight of model group.
  • T cell subsets in tumor tissue Flow cytometry analysis of CD80+ and CD86+ T cells in tumor tissue.
  • CD80 and CD86 molecules participate in the antigen presentation process and T cell-mediated cellular immunity, and play an important role in the body's anti-tumor effect.
  • Recent studies have shown that the expression of CD80 and CD86 in renal cell carcinoma tissue is significantly lower than that in peritumoral tissue and normal renal tissue, which weakens the antigen presentation effect and prevents tumor antigens from eliciting an effective immune response, thus inducing T cells.
  • the incompetent state ultimately leads to the body's immune system being unable to eliminate tumor cells, which is an important reason for the unrestricted growth of tumors.
  • Cytokine detection detects the levels of IL-2, IL-4, and IFN- ⁇ in mouse serum. IL-2, IL-4, and IFN- ⁇ can induce the body to produce immune effects and have anti-tumor effects.
  • SPSS statistical software 25.0 was used for statistical analysis.
  • the model group Compared with the blank group, the model group obviously formed tumors, and the model was successfully constructed. Compared with the model group, the tumor weight of the PD-1 group, AM02 live bacteria group, AM06 live bacteria group, AM02 inactivated bacteria group, and AM06 inactivated bacteria group was significantly reduced (p ⁇ 0.05); compared with the model group, AM02 The tumor weight of the live bacteria + PD-1 antibody group, AM06 live bacteria + PD-1 antibody group, AM02 inactivated bacteria + PD-1 antibody group, and AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.01) . AM02 live/killed bacteria and AM06 live/killed bacteria have the effect of inhibiting tumors and are better than BAA-835 standard strain live/killed bacteria.
  • the tumor weight of the AM02 live bacteria group and AM06 live bacteria group was significantly reduced (p ⁇ 0.05).
  • the tumor weight of the AM02 live bacteria + PD-1 antibody group and AM06 live bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.01).
  • the tumor weight of the AM02 inactivated bacteria + PD-1 antibody group and the AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.01).
  • AM02 live/inactivated bacteria and AM06 live/inactivated bacteria can achieve better anti-tumor effects when combined with PD-1 antibodies, which are better than BAA-835 standard strain live/inactivated bacteria.
  • the content of CD80+ and CD86+ cells in the normal control group was lower (p ⁇ 0.05). The reason was that no tumor cells were inoculated and there was no tumor-related immune response.
  • the percentages of CD80+ and CD86+ T cells in the PD-1 antibody group, AM02 live bacteria/killed bacteria, and AM06 live bacteria/killed bacteria increased (p ⁇ 0.05).
  • AM02 live bacteria/inactivated bacteria + PD-1 antibody group (combination)
  • AM06 live bacteria/inactivated bacteria + PD-1 The antibody group (combination) can increase the percentage of CD80+ and CD86+ T cells in tumor tissue more significantly (p ⁇ 0.001). This shows that Akkermansia AM02 and AM06 not only have anti-tumor effects, but can also effectively enhance the anti-tumor effect of PD-1 antibodies.
  • a means compared with the model group, the difference is significant p ⁇ 0.05
  • aaa means compared with the model group, the difference is significant p ⁇ 0.001
  • b means compared with the BAA-835 standard strain live bacteria group, the difference is significant p ⁇ 0.05
  • c means compared with the BAA-835 standard strain live bacteria group, the difference is significant p ⁇ 0.05
  • dd means compared with the BAA-835 standard strain live bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01
  • ee means compared with the BAA-835 standard strain live bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01 Compared with the inactivated bacteria group + PD-1 antibody group, the difference was significant p ⁇ 0.01.
  • the normal control group had lower levels of IL-2, IL-4, and IFN- ⁇ (p ⁇ 0.01). The reason was that no tumor cells were inoculated and there was no tumor-related immune response.
  • IL-2, IFN- ⁇ , and IL-4 are immune activating factors that can stimulate the body's immunity to produce anti-tumor effects.
  • the -1 antibody group significantly increased the levels of the above-mentioned cytokines.
  • the combination of AM02 live/inactivated bacteria group and AM06 live/inactivated bacteria group with PD-1 antibody can enhance the effect of PD-1 antibody in up-regulating cytokine levels.
  • AM02 live bacteria/inactivated bacteria and AM06 live bacteria/inactivated bacteria have better up-regulated cytokine levels than BAA-835 live bacteria/inactivated bacteria.
  • Akkermansia muciniphila AM02 live/inactivated bacteria and AM06 live/inactivated bacteria not only have anti-tumor effects, but also can increase the efficacy of PD-1 antibodies when combined with PD-1 antibodies. , enhance the body's anti-tumor immunity and inhibit the growth of renal cell carcinoma transplanted tumors.
  • Example 25 Efficacy experiment of Akkermansia muciniphila in treating T24 cell bladder cancer transplanted tumors in mice
  • T24 bladder cancer cell culture (1) Preheat the culture reagents in a constant-temperature water bath in advance, irradiate the clean bench with ultraviolet light for 30 minutes, and wipe all items brought into the clean bench with 75% alcohol cotton; (2) Add 3 mL of serum-free RPMI 1640 medium to a 15 mL centrifuge tube, take out the cells to be revived, and quickly place them in a 37°C water bath to melt them quickly; (3) Quickly move the cells to a 15 mL centrifuge tube, and gently pipet evenly.
  • mice 6-week-old male nude mice were used in the experiment. After the mice grew naturally for 1 week, tumor inoculation was performed. All mice except the normal control group were inoculated with tumor cells, and T24 cells were planted on the backs of mice. The injection cell volume was 5 ⁇ 10 6 cells/mL ⁇ 0.2mL/mouse. Observe tumor growth in mice.
  • Signs of successful modeling The mice showed signs of exhaustion such as weight loss, hunched back, and lethargy, and palpable masses were visible at the inoculated site of the mice. Mice were successfully inoculated with tumor cells and grouped into groups 7 days later.
  • Administration method will begin 7 days after tumor inoculation. The administration time point is 10 a.m. on the administration day. The combined medication group will be administered PD-1 antibody first, and then live bacteria/inactivated bacteria. Administration was given for a total of 3 weeks.
  • T cell subsets in tumor tissue Flow cytometry analysis of CD4+ and CD8+ T cells in tumor tissue. Cytokine detection: ELISA detects the levels of IL-2 and IFN- ⁇ in mouse serum. Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.
  • the model group Compared with the blank group, the model group obviously formed tumors and the modeling was successful; compared with the model group, the PD-1 group, AM02 live bacteria group, AM06 live bacteria group, AM02 inactivated bacteria group, AM06 inactivated bacteria group, the tumor The weight decreased significantly (p ⁇ 0.05); compared with the model group, AM02 live bacteria + PD-1 antibody group, AM06 live bacteria + PD-1 antibody group, AM02 inactivated bacteria + PD-1 antibody group, AM06 inactivated bacteria The tumor weight in the +PD-1 antibody group decreased significantly (p ⁇ 0.001). AM02 live/killed bacteria and AM06 live/killed bacteria have the effect of inhibiting tumors and are better than BAA-835 standard strain live/killed bacteria.
  • the tumor weight of the AM02 live bacteria group and AM06 live bacteria group was significantly reduced (p ⁇ 0.05).
  • the tumor weight of the AM02 live bacteria + PD-1 antibody group and AM06 live bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.001).
  • the tumor weight of the AM02 inactivated bacteria + PD-1 antibody group and the AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.001).
  • AM02 live/inactivated bacteria and AM06 live/inactivated bacteria can achieve better anti-tumor effects when combined with PD-1 antibodies, which are better than BAA-835 standard strain live/inactivated bacteria.
  • the normal control group had lower levels of CD4+ and CD8+ cells (p ⁇ 0.05). The reason was that no tumor cells were inoculated and there was no tumor-related immune response. Compared with the model group, the percentages of CD4+ and CD8+ T cells in the PD-1 antibody group, AM02 live/inactivated bacteria, and AM06 live/inactivated bacteria increased significantly (p ⁇ 0.05).
  • the AM02 live bacteria/killed bacteria + PD-1 antibody group and the AM06 live bacteria/killed bacteria + PD-1 antibody group were administered group can increase the percentage of CD4+ and CD8+ T cells in tumor tissue more significantly (p ⁇ 0.001). This shows that Akkermansia AM02 and AM06 can effectively enhance the anti-tumor effect of PD-1 antibodies, and the effect is significantly better than the BAA-835 standard strain live/inactivated bacteria.
  • the normal control group had lower IL-2 and IL-4 contents (p ⁇ 0.01). The reason was that no tumor cells were inoculated and there was no tumor-related immune response. IL-2 and IFN- ⁇ are immune activating factors that can stimulate the body's immunity to produce anti-tumor effects.
  • AM02 live/inactivated bacteria, AM06 live/inactivated bacteria, and PD-1 antibodies were significantly increased.
  • the combination of AM02 live/inactivated bacteria, AM06 live/inactivated bacteria and PD-1 antibody can enhance the effect of PD-1 antibody on up-regulating the above-mentioned cytokine levels, which is better than BAA-835 live bacteria. /kill bacteria.
  • the combination of Akkermansia muciniphila AM02 live/killed bacteria and AM06 live/killed bacteria with PD-1 antibodies can increase the efficacy of PD-1 antibodies and enhance the body's anti-tumor immunity. Inhibits the growth of bladder tumors.
  • Example 26 Efficacy experiment of Akkermansia muciniphila in treating upper urinary tract cell carcinoma UMUC3 cell metastases in mice
  • Upper urinary tract cell carcinoma UMUC3 cell culture (1) Preheat the culture reagents in a constant temperature water bath in advance, irradiate the ultra-clean bench with ultraviolet light for 30 minutes, and wipe all items brought into the ultra-clean bench with 75% alcohol cotton; ( 2) Add 3 mL of serum-free culture medium to a 15 mL centrifuge tube, take out the cells to be revived, and quickly place them in a 37°C water bath to melt them quickly; (3) Quickly move the cells to a 15 mL centrifuge tube, and gently pipet evenly.
  • Tumor inoculation 6-week-old female severe combined immunodeficiency (SCID) mice were used in the experiment. After the mice grew naturally for 1 week, all mice except the normal control group were inoculated with tumor cells. , inject 1.0 ⁇ 10 6 cells/mL ⁇ 150 ⁇ L into the mouse bladder through the urethra, ligate the urethra to prevent the mouse from urinating, and loosen the ligation suture 4 hours later. About 10-20% of UMUC3-luc cells reflux through the vesicoureter. urethra.
  • Administration method will begin 7 days after tumor inoculation. The administration time point is 10 a.m. on the administration day. The combined medication group will be administered PD-1 antibody first, and then live bacteria/inactivated bacteria. Administration was given for a total of 3 weeks.
  • T cell subsets in peripheral blood Flow cytometry analysis of CD4+ and CD8+ T cells in peripheral blood. Cytokine detection: ELISA detects the levels of IL-2 and IFN- ⁇ in mouse serum. Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.
  • the model group Compared with the blank group, the model group obviously formed tumors, and the model was successfully constructed. Compared with the model group, the tumor weight of the PD-1 group, AM02 live bacteria group, AM06 live bacteria group, AM02 inactivated bacteria group, AM06 inactivated bacteria group, and BAA-835 standard strain live bacteria group was significantly reduced (p ⁇ 0.05) ; Compared with the model group, AM02 live bacteria + PD-1 antibody group, AM06 live bacteria + PD-1 antibody group, AM02 inactivated bacteria + PD-1 antibody group, AM06 inactivated bacteria + PD-1 antibody group, BAA The tumor weight of the -835 standard strain live bacteria + PD-1 antibody group decreased significantly (p ⁇ 0.01; p ⁇ 0.001). AM02 live/killed bacteria and AM06 live/killed bacteria have the effect of inhibiting tumors and are better than BAA-835 standard strain live/killed bacteria.
  • the tumor weight of the AM02 group and AM06 group was significantly reduced (p ⁇ 0.05).
  • the tumor weight of the AM02 live bacteria + PD-1 antibody group and AM06 live bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.001).
  • the tumor weight of the AM02 inactivated bacteria + PD-1 antibody group and the AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.01).
  • the combination of AM02 live/inactivated bacteria, AM06 live/inactivated bacteria and PD-1 antibody can exert better anti-tumor effect, which is better than BAA-835 standard strain live/inactivated bacteria.
  • a means compared with the model group, the difference is significant p ⁇ 0.05
  • aaa means compared with the model group, the difference is significant p ⁇ 0.01
  • b means compared with the BAA-835 standard strain inactivated bacteria group, the difference is significant p ⁇ 0.05
  • cc means Compared with the BAA-835 standard strain live bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01
  • dd means that compared with the BAA-835 standard strain inactivated bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01.
  • the body's immunity does not respond to the tumor. There is no statistical difference between the CD4+ and CD8+ in the model group and the normal control group. Compared with the model group, the percentages of CD4+ and CD8+ T cells in the PD-1 antibody group, AM02 live/inactivated bacteria, AM06 live/inactivated bacteria, and BAA-835 standard strain live bacteria groups increased (p ⁇ 0.05) .
  • the AM02 live bacteria/killed bacteria + PD-1 antibody group and the AM06 live bacteria/killed bacteria + PD-1 antibody group were administered group can increase the percentage of CD4+ and CD8+ T cells in tumor tissue more significantly (p ⁇ 0.01). This shows that Akkermansia AM02 and AM06 can effectively enhance the anti-tumor effect of PD-1 antibodies.
  • a means compared with the model group, the difference is significant p ⁇ 0.05
  • aaa means compared with the model group, the difference is significant p ⁇ 0.01
  • b means compared with the BAA-835 standard strain inactivated bacteria group, the difference is significant p ⁇ 0.05
  • cc means Compared with the BAA-835 standard strain live bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01
  • dd means that compared with the BAA-835 standard strain inactivated bacteria group + PD-1 antibody group, the difference is significant p ⁇ 0.01.
  • IL-2 and IFN- ⁇ are immune activating factors that can stimulate the body's immunity to produce anti-tumor effects.
  • AM02 live/inactivated bacteria, AM06 live/inactivated bacteria, and PD-1 antibodies were significantly increased.
  • the above-mentioned cytokine levels; AM02 live bacteria/inactivated bacteria, AM06 live bacteria/inactivated bacteria combined with PD-1 antibodies can enhance the effect of PD-1 antibodies on up-regulating the above-mentioned cytokine levels, and are better than BAA-835 live bacteria. bacteria/killed bacteria.
  • Akkermansia muciniphila AM02 live/killed bacteria and AM06 live/killed bacteria are used alone or in combination with PD-1 antibodies to increase the number of effector T cells and upregulate the level of immune activating factors. , enhance the body's anti-tumor immunity and inhibit the growth of upper urinary tract cell transplanted tumors.
  • Example 27 Efficacy experiment of Akkermansia muciniphila in treating Tramp-C1 prostate cancer xenografts in mice
  • TRAMP-C1 cells are routinely cultured in fresh DMEM culture medium containing 10% inactivated FBS and 0.01 nmol/L dehydroepiandrosterone at 37°C and 5% CO2 in a sterile incubator. When the cells reached 80% to 90% confluence, they were digested and subcultured with 0.25% trypsin. Select cells in good condition and in the logarithmic growth phase. After digestion, centrifuge at 1500 rpm to collect the cell pellet. Resuspend the cells in PBS for 3 times. After the last resuspension, count the cells and dilute to 1.0 ⁇ 10 8 cells/mL. suspension and keep on ice until use.
  • mice 6-week-old male C57BL/6 mice were used in the experiment. After the mice grew naturally for 1 week, tumor inoculation was performed. Before modeling, all mice were anesthetized with 10% chloral hydrate at 3.5 ⁇ L/g body weight. The mice were fixed on the sterile operating table with their abdomen facing up. The lower abdomen was cut open to reveal the abdominal cavity, and the operation was performed under a 10x microscope. After careful operation, find the mouse bladder and gonads and push them to both sides to expose the prostate. All mice, except the control group, use microsyringes to inject Tramp-C11 cell suspension 1.0 ⁇ 10 8 cells/mL into the lateral lobes of the prostate on both sides.
  • mice show signs of exhaustion such as emaciation, curling up, and listlessness. Mice were successfully inoculated with tumor cells and grouped into groups 7 days later.
  • the administration time point is 10 a.m. on the administration day.
  • the combined medication group is administered PD-1 antibody first, and then live/inactivated bacteria.
  • the PD-1 antibody is administered for a total of 4 weeks. .
  • T cell subsets in tumor tissue Flow cytometry analysis of CD3+ and CD8+ T cells in tumor tissue. Cytokine detection: ELISA detects the levels of IL-2 and IFN- ⁇ in mouse serum. Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.
  • the model group Compared with the blank group, the model group obviously formed tumors, and the model was successfully constructed. Compared with the model group, the tumor weight of the PD-1 group, AM02 live bacteria group, AM06 live bacteria group, AM02 inactivated bacteria group, and AM06 inactivated bacteria group was significantly reduced (p ⁇ 0.05); compared with the model group, AM02 The tumor weight of the live bacteria + PD-1 antibody group, AM06 live bacteria + PD-1 antibody group, AM02 inactivated bacteria + PD-1 antibody group, and AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.001) .
  • AM02 live/killed bacteria and AM06 live/killed bacteria have the effect of inhibiting tumors and are better than BAA-835 standard strain live/killed bacteria.
  • the tumor weight of the AM02 live bacteria group and AM06 live bacteria group was significantly reduced (p ⁇ 0.05).
  • the tumor weight of the AM02 live bacteria + PD-1 antibody group and AM06 live bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.001).
  • the tumor weight of the AM02 inactivated bacteria + PD-1 antibody group and AM06 inactivated bacteria + PD-1 antibody group was significantly reduced (p ⁇ 0.01).
  • the combination of AM02 live/inactivated bacteria, AM06 live/inactivated bacteria and PD-1 antibody can exert better anti-tumor effect, which is better than BAA-835 standard strain live/inactivated bacteria.
  • the normal control group had lower levels of CD3+ and CD8+ cells (p ⁇ 0.05). The reason was that no tumor cells were inoculated and there was no tumor-related immune response. Compared with the model group, the percentages of CD3+ and CD8+ T cells in the PD-1 antibody group, AM02 live/inactivated bacteria, and AM06 live/inactivated bacteria increased (p ⁇ 0.05).
  • the AM02 live bacteria + PD-1 antibody group and AM06 live bacteria + PD-1 antibody group can increase the percentage of CD3+ and CD8+ T cells in tumor tissue. The increase is more significant (p ⁇ 0.01).
  • the AM02 inactivated bacteria + PD-1 antibody group and AM06 inactivated bacteria + PD-1 antibody group can make tumor tissue CD3+, CD8+ The percentage of T cells increased more significantly (p ⁇ 0.01). It shows that Akkermansia AM02 live/inactivated bacteria and AM06 live/inactivated bacteria can effectively enhance the anti-tumor effect of PD-1 antibodies.
  • the normal control group had lower levels of IL-2 and IFN- ⁇ (p ⁇ 0.01). The reason was that no tumor cells were inoculated and there was no tumor-related immune response. IL-2 and IFN- ⁇ are immune activating factors that can stimulate the body's immunity to produce anti-tumor effects.
  • AM02 live/inactivated bacteria, AM06 live/inactivated bacteria, and PD-1 antibodies were significantly increased.
  • the combination of AM02 live/inactivated bacteria, AM06 live/inactivated bacteria and PD-1 antibody can enhance the effect of PD-1 antibody on up-regulating the above-mentioned cytokine levels.
  • AM02, AM06 live bacteria/ Killed bacteria are better than BAA-835 live bacteria/killed bacteria.
  • This disclosure further verifies the effect of Akkermansia muciniphila AM06 and AM02 in preventing and treating other tumors, including other types of tumors of the digestive system (such as liver cancer, esophageal cancer, Pancreatic cancer, etc.), genitourinary system tumors (such as kidney cancer, bladder cancer, urothelial cancer, ovarian cancer, prostate cancer, etc.), skin tumors (such as melanoma, basal cell carcinoma, squamous cell carcinoma, etc.), respiratory system Tumors (such as head and neck cancer, non-small cell lung cancer, small cell lung cancer, etc.), etc.
  • Akkermansia muciniphila AM06 and AM02 provided by the present disclosure have good potential and prospects for application in anti-tumor drugs.
  • Akkermansia AM02 and AM06 live/inactivated bacteria alone or in combination with PD-1 antibodies can increase the number of effector T cells, upregulate the level of immune activating factors, enhance the body's anti-tumor immunity, and inhibit the prostate. Cancer xenografts grow.
  • the present disclosure provides Akkermansia muciniphila and its products and applications for preventing and treating tumors, wherein the live bacteria, inactivated bacteria and cell lysates of Akkermansia muciniphila of the present disclosure have the ability to prevent and treat tumors.

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Abstract

L'invention a trait à l'Akkermansia muciniphila qui comprend AM06 et/ou AM02. AM06 est l'Akkermansia muciniphila dont le numéro d'accession CGMCC est 22793, et AM02 est l'Akkermansia muciniphila dont le numéro d'accession CGMCC est 22794. Il a été prouvé par des expériences que les bactéries viables, les bactéries inactivées et les lysats bactériens de l'Akkermansia muciniphila ont un effet préventif et curatif sur les tumeurs. L'invention concerne un produit d'Akkermansia muciniphila destiné à la prévention et au traitement des tumeurs et son utilisation. Le produit comprend au moins l'Akkermansia muciniphila et peut en outre comprendre un inhibiteur de point de contrôle immunitaire, tel qu'un anticorps PD-1, un anticorps CTLA-4, etc. Lorsque l'Akkermansia muciniphila est utilisée en association avec l'inhibiteur de point de contrôle immunitaire, l'Akkermansia muciniphila peut renforcer de manière considérable l'effet antitumoral de l'inhibiteur de point de contrôle immunitaire.
PCT/CN2023/105255 2022-07-29 2023-06-30 Produit d'akkermansia muciniphila pour prévention et traitement de tumeurs et son utilisation WO2024022036A1 (fr)

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