WO2023134207A1 - 脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药治疗生殖泌尿系统肿瘤的应用 - Google Patents

脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药治疗生殖泌尿系统肿瘤的应用 Download PDF

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WO2023134207A1
WO2023134207A1 PCT/CN2022/120025 CN2022120025W WO2023134207A1 WO 2023134207 A1 WO2023134207 A1 WO 2023134207A1 CN 2022120025 W CN2022120025 W CN 2022120025W WO 2023134207 A1 WO2023134207 A1 WO 2023134207A1
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capsular polysaccharide
antibody
bacteroides fragilis
cancer
zwitterionic
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PCT/CN2022/120025
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French (fr)
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常秀娟
郭真
刘洋洋
王晔
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广州知易生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/09—Other cheese preparations; Mixtures of cheese with other foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
    • A23G9/34—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds characterised by carbohydrates used, e.g. polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
    • A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A23G9/363—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • 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
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • 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
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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

  • Bacteroides fragilis ZY-312 Bacteroides fragilis ZY-312
  • deposit number CGMCC No.10685 Bacteroides fragilis ZY-312 was isolated and obtained by the applicant unit of the present invention, and has been authorized for patent protection (patent number 201510459408.X). According to the provisions of the patent examination guidelines, the public can buy it from commercial channels or has been authorized without preservation, that is, No deposit certificate is required.
  • the invention relates to the field of biomedicine, in particular to the application of a zwitterionic capsular polysaccharide of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of genitourinary system tumor drugs.
  • Urogenital system tumors refer to tumors that occur in the urinary system and/or reproductive system. These include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ tumors such as kidney, bladder, urothelial, breast, ovarian, and prostate cancers.
  • Urogenital system tumors are major diseases that seriously endanger human health, with high morbidity and mortality. Statistics show that they account for 31% of all new cancer cases, posing a serious threat to human health and life. Its pathogenesis is heterogeneous and usually includes internal and external factors, such as physical factors, chemical factors, biological factors, genetic factors, endocrine factors, immune function and mental factors.
  • Surgical treatment is the primary treatment method for genitourinary system tumors. It is suitable for early and mid-stage solid tumors. It has a direct effect and can completely remove local tumors.
  • Renal cancer is one of the common tumors of the urinary system, accounting for 80% to 90% of malignant renal tumors. It originates from the urinary tubular epithelial system of the renal parenchyma, occurs in any part of the renal parenchyma, and is nodular or lobulated. Variation, hard, yellow cut surface, mostly solid, sometimes off-white similar to sarcoma, a few are multicystic, with transparent liquid in the cyst, necrosis and hemorrhage can be seen.
  • renal cancer is still unclear, and it is mainly believed to be related to genetics, smoking, hypertension, obesity and antihypertensive treatment.
  • smoking and obesity are recognized as important risk factors for renal cancer, and its pathological types are usually divided into clear cell Renal cell carcinoma, papillary renal cell carcinoma, chromophobe carcinoma, and collecting duct carcinoma are rare types of renal cell carcinoma.
  • the clinical manifestations of patients with RCC are complex and changeable, and early-stage patients usually lack clinical manifestations.
  • the main clinical manifestations include hematuria, low back pain, and mass.
  • the diagnostic methods of RCC include clinical manifestations, laboratory tests, and imaging examinations. Routine laboratory examinations mainly include urine routine, blood routine, erythrocyte sedimentation rate, blood sugar, blood calcium, kidney function, liver function, lactate dehydrogenase, alkaline phosphatase and other items;
  • the detection and staging, intraoperative positioning, and postoperative and non-surgical treatment follow-up are of great significance, including chest X-ray examination, ultrasonography, CT examination, MRI examination and PET examination.
  • kidney cancer patients usually refers to the results of imaging examinations to determine the clinical stage of the tumor, and uses auxiliary examinations to evaluate the patient's tolerance to treatment. According to the clinical stage and the patient's tolerance, the appropriate treatment is selected. Usually includes surgery, interventional therapy, radiotherapy and drug therapy. Among them, surgical treatment is the main treatment modality, usually suitable for patients with localized and locally advanced RCC
  • Bladder cancer is one of the three major tumors of the urinary system, and its incidence rate ranks 10th among malignant tumors in the world.
  • the incidence rate of bladder cancer in men rises to 6th, and its mortality rate ranks 9th among male malignant tumors , at present, its incidence rate is showing an increasing trend year by year, and bladder cancer has the characteristics of easy recurrence.
  • bladder tumors include bladder urothelial carcinoma, bladder squamous cell carcinoma, and bladder adenocarcinoma.
  • the most important pathological type is bladder urothelial carcinoma, accounting for more than 90% of all bladder cancers.
  • the etiology of bladder cancer is relatively complex, including genetic factors and environmental factors, such as smoking and the influence of external aromatic amines. Common symptoms include blood in the urine, frequent urination, painful urination, and low back pain.
  • the main detection methods are urinalysis, urine cytology examination, cystoscopy and so on.
  • the main treatments for bladder cancer include surgery, radiation therapy, chemotherapy and immunotherapy.
  • Common surgical treatment methods include transurethral resection combined with electrocautery, radical cystectomy, partial cystectomy, urinary diversion, etc.; radiation therapy uses high-energy X-rays or other types of radiation to kill cancer cells
  • a form of cancer treatment that prevents or prevents its growth, including external radiation therapy and internal radiation therapy, depending on the type and stage of the cancer being treated;
  • biological therapy which uses the patient's immune system to fight the cancer, uses Substances produced by the body or in a laboratory to boost, induce or restore the body's natural defense mechanisms against cancer.
  • breast cancer is a malignant tumor that occurs in the breast epithelium or ductal epithelium, mostly in women, and the incidence rate in men is about 1%. According to data released by the National Cancer Center, breast cancer is the malignant tumor with the highest incidence rate among women in my country, and the incidence rate is increasing year by year.
  • Breast cancer usually has no obvious symptoms in the early stage, and the main manifestations are: breast lumps, nipple changes, skin and appearance changes, breast pain, regional lymph node enlargement, and organ involvement after distant metastasis.
  • the etiology of breast cancer is still unclear, but it is considered to be closely related to age, hormones, genetic factors, menstrual marriage history, dietary structure, overdiagnosis, and environmental factors.
  • the main screening methods for breast cancer include imaging examination, tissue biopsy, breast cancer tumor marker examination, immunohistochemical examination, etc.
  • the main treatment methods for breast cancer include drug therapy, surgery and radiation therapy.
  • drug therapy can be divided into chemical drug therapy, endocrine therapy (hormone therapy) and targeted therapy according to different mechanisms of action; chemotherapy drugs generally interfere with the proliferation of cancer cells by changing or inhibiting the biochemical metabolic process of cancer cells.
  • Surgical treatment is the most commonly used method for breast cancer treatment. Surgical methods include radical mastectomy, modified radical mastectomy, extended radical mastectomy, breast-conserving radical mastectomy, sentinel lymph node biopsy, and breast reconstruction surgery.
  • Ovarian cancer refers to one of the most common malignant tumors growing on the ovary, of which 90% to 95% are primary cancers of the ovary, and the other 5% to 10% are primary cancers metastasized to other parts. Ovary, in recent years, its morbidity, recurrence rate and mortality rate are higher, and tend to be younger gradually. The early symptoms of ovarian cancer are not obvious and non-specific, so it is difficult for early screening and diagnosis. 60-70% of the patients are in the advanced stage, and the treatment effect is not good.
  • ovarian cancer The cause of ovarian cancer is still unclear, but it is believed to be closely related to genetic factors, continuous ovulation, eating habits and environmental factors through the investigation of its epidemiology.
  • Common diagnostic methods include ultrasonography, cytology, laparoscopy, tumor marker determination, and radiological diagnosis.
  • the treatment of ovarian cancer is usually based on surgery, supplemented by chemotherapy, radiotherapy, immunotherapy and so on.
  • the surgical method has a better curative effect and can be completely removed for early-stage patients, but this method has a high risk, the scope of treatment is relatively limited, and it is prone to side effects; chemotherapy can effectively control the progression of the disease and prolong the survival of patients, but the recurrence rate of this method is Higher, with more recurrences and shorter recurrence intervals; as a new treatment for ovarian cancer, immunotherapy has high specificity and few side effects, but at present, this method has not formed a complete system and diagnosis and treatment method.
  • prostate cancer in China has shown a continuous and rapid growth trend.
  • the overall incidence of prostate cancer in China was 10.23/100,000, and the mortality rate was 4.36/100,000. It is the tumor with the highest incidence rate in the reproductive system.
  • prostate cancer ranks sixth in the incidence of male malignancies and ninth in the mortality rate.
  • prostate cancer Early detection of prostate cancer relies on prostate cancer tumor markers, but currently used prostate-specific antigens have limited sensitivity and specificity. Patients with a large number of radically resected prostates (15% to 30%) were identified as overdiagnosed.
  • the immune checkpoint is a protective molecule in the human immune system, which acts like a brake to prevent inflammatory damage caused by excessive activation of T cells.
  • Tumor cells take advantage of the characteristics of the human immune system to overexpress immune checkpoint molecules, inhibit the response of the human immune system, and escape human immune surveillance and killing, thereby promoting the growth of tumor cells. Inhibiting the expression of immune checkpoint molecules and their ligands can enhance the killing effect of T cells on tumors and achieve the purpose of anti-tumor.
  • the published immune checkpoints include CTLA-4, PD-1/PD-L1, LAG-3, TIM-3, VISTA, A2aR, etc.
  • Programmed cell death protein 1 is expressed on a variety of lymphocytes, especially on tumor-specific T cells. In the tumor microenvironment, it leads to the expansion of malignant tumor cells by interfering with protective immune responses. It has two ligands, programmed cell death ligand 1 and 2 (PD-L1, PD-L2), among which PD-L1 is expressed by tumor cells to evade the anti-tumor response to it by the immune system. Blocking the interaction between PD-1 and PD-L1 can maintain the response of T cells after T cells enter the tumor microenvironment and ensure the anti-tumor effect of T cells.
  • PD-1 programmed cell death ligand 1 and 2
  • Antibodies against PD-1/PD-L1 have Nivolumab, Pembrolizumab, JQ1, Atezolizumab, Avelumab and Simi Cemiplimab. These mAbs are approved for the treatment of breast, lung, colorectal, cancer, bladder, pancreatic, prostate and diffuse large B-cell lymphoma (DLBCL).
  • DLBCL diffuse large B-cell lymphoma
  • the object of the present invention is to provide a combination of Bacteroides fragilis zwitterionic capsular polysaccharide and immune checkpoint inhibitors in the prevention and treatment of genitourinary system tumors.
  • the present invention unexpectedly finds that the combined administration of Bacteroides fragilis, especially the capsular polysaccharide A of Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685, and immune checkpoint inhibitors can regulate T cells and anti-tumor immune factor levels, enhance the body's anti-tumor immune response, and then achieve the goal of preventing and treating genitourinary system tumors.
  • Bacteroides fragilis zwitterionic capsular polysaccharide and an immune checkpoint inhibitor in the preparation of products for preventing and/or treating genitourinary cancer is provided.
  • the zwitterionic capsular polysaccharide is extracted from Bacteroides fragilis ZY-312 with the deposit number CGMCC No.10685.
  • the zwitterionic capsular polysaccharide comprises capsular polysaccharide A.
  • the structure of the capsular polysaccharide A is as follows:
  • the weight-average molecular weight of the capsular polysaccharide A is 80-90KD, the part with Mw distributed in 70-100KD accounts for 70-80% of the total, and the ratio of weight-average molecular weight/number-average molecular weight (Mw/Mn) 1.0-1.3.
  • the content of capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.
  • the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:
  • the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.
  • the acid solution in step (1) may be one or more of organic acids, inorganic acids and acidic buffers.
  • the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.
  • the organic acid can be acetic acid, citric acid, etc.
  • the molecular weight of the ultrafiltration membrane in step (2) may be 100, 50, 30, 10, 5, 3 KD or a range between any two molecular weight values.
  • the ion exchange column described in step (3) is preferably 16mm ⁇ 200mm of DEAE Sepharose Fast Flow, the flow rate during chromatography is 15-25mL/min, and pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, section collection, 100mL/bottle (component); The molecular weight of described ultrafiltration membrane is 10KD.
  • the genitourinary system tumor refers to a tumor occurring in the urinary system and/or reproductive system. These include tumors of the breast and genital organs in women, tumors of the genital organs in men, and tumors of the urinary organs. Exemplarily, one or more selected from breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.
  • the immune checkpoint inhibitors include PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies and other substances that can bind to immune checkpoints to activate or enhance endogenous anti-tumor immune responses .
  • the PD-1 antibodies include Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab ), Sindilimab (Cindilimab), Camrelizumab (Camrelizumab) and others can bind to PD-1, block PD-1/PD-L1 signaling pathway, up-regulate T cell activation, and activate endogenous Substances of the anti-tumor immune response.
  • the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and other antibodies that can bind to PD-L1 and block PD-L1.
  • the product is food or medicine.
  • the food product comprises milk powder, cheese, curd, yogurt, ice cream, or fermented cereal.
  • the food can also be animal food, such as feed and the like.
  • the dosage form of the medicine includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.
  • the medicine includes human medicine or animal medicine.
  • the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.
  • the Bacteroides fragilis zwitterionic capsular polysaccharide is administered simultaneously with the PD-1 antibody and/or the PD-L1 antibody.
  • Bacteroides fragilis zwitterionic capsular polysaccharide and the PD-1 antibody and/or PD-L1 antibody are administered separately.
  • the B. fragilis zwitterionic capsular polysaccharide is administered orally or enemaly.
  • compositions for preventing and treating genitourinary system tumors wherein the composition includes both B.
  • the polysaccharide is extracted from Bacteroides fragilis ZY-312 with the preservation number CGMCC No.10685.
  • the zwitterionic capsular polysaccharide comprises capsular polysaccharide A.
  • the structure of the capsular polysaccharide A is as follows:
  • the weight-average molecular weight of the capsular polysaccharide A is 80-90KD, the part with Mw distributed in 70-100KD accounts for 70-80% of the total, and the ratio of weight-average molecular weight/number-average molecular weight (Mw/Mn) 1.0-1.3.
  • the content of capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.
  • the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:
  • the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.
  • the acid solution in step (1) may be one or more of organic acids, inorganic acids and acidic buffers.
  • the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.
  • the organic acid can be acetic acid, citric acid, etc.
  • the molecular weight of the ultrafiltration membrane in step (2) may be 100, 50, 30, 10, 5, 3 KD or a range between any two molecular weight values.
  • the ion exchange column described in step (3) is preferably 16mm ⁇ 200mm of DEAE Sepharose Fast Flow, the flow rate during chromatography is 15-25mL/min, and pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, section collection, 100mL/bottle (component); The molecular weight of described ultrafiltration membrane is 10KD.
  • the immune checkpoint inhibitors include PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies and other substances that can bind to immune checkpoints to activate or enhance endogenous anti-tumor immune responses .
  • the PD-1 antibodies include Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab ), Sindilimab (Cindilimab), Camrelizumab (Camrelizumab) and others can bind to PD-1, block PD-1/PD-L1 signaling pathway, up-regulate T cell activation, and activate endogenous Substances of the anti-tumor immune response.
  • the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and other antibodies that can bind to PD-L1 and block PD-L1.
  • the composition is a medicament.
  • the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.
  • the medicine includes human medicine or animal medicine.
  • the Bacteroides fragilis zwitterionic capsular polysaccharide is administered simultaneously with the PD-1 antibody and/or the PD-L1 antibody.
  • Bacteroides fragilis zwitterionic capsular polysaccharide and the PD-1 antibody and/or PD-L1 antibody are administered separately.
  • the zwitterionic capsular polysaccharide is administered orally or enemaly.
  • the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.
  • the genitourinary system tumor refers to a tumor occurring in the urinary system and/or reproductive system. These include tumors of the breast and genital organs in women, tumors of the genital organs in men, and tumors of the urinary organs. Including one or more of breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.
  • the present invention also provides a method for treating tumors of the genitourinary system, comprising administering a therapeutically effective amount of the above product or composition to a patient.
  • Bacteroides fragilis especially Bacteroides fragilis ZY-312 with the deposit number CGMCC No. 10685, and its zwitterionic capsular polysaccharide are used in combination with immune checkpoint inhibitors, especially PD-1 antibody/
  • immune checkpoint inhibitors especially PD-1 antibody/
  • the combination of PD-L1 antibody can regulate the level of T cells and anti-tumor immune factors in mice, enhance the body's anti-tumor immune response, and effectively prevent and treat genitourinary system tumors.
  • Bacteroides fragilis ZY-312 that the present invention adopts does not contain BFT gene, is non-toxigenic bacterial strain, and acute toxicity proves, and this bacterial strain is all nonpathogenic to normal mouse and nude mouse (Wang Y, Deng H, Li Z, Tan Y , Han Y, Wang X, Du Z, Liu Y, Yang R, Bai Y, Bi Y, Zhi F. Safety Evaluation of a Novel Strain of Bacteroides fragilis. Front Microbiol. 2017 Mar 17; 8:435.).
  • 1A-1E are respectively the 1 H spectrum, 13 C spectrum, COZY spectrum, HSQC spectrum, and HMBC spectrum analyzed by the capsular polysaccharide A NMR spectrometer in Example 1 of the present invention;
  • Fig. 2 is the chemical structural formula of the structural unit of Bacteroides fragilis capsular polysaccharide A prepared in Example 1 of the present invention
  • Figures 3A-3B are respectively the expression levels of IFN- ⁇ and IL-2 in the serum of mice in each group in Example 2;
  • Figures 4A-4B are the expression levels of serum IFN- ⁇ and IL-2 in each group of mice in Example 3, respectively;
  • Figures 5A-5B are respectively the expression levels of serum IL-4 and IFN- ⁇ in each group of mice in Example 4;
  • Figures 6A-6C are respectively the levels of tumor infiltrating CD4 + T cells and CD8 + T cells and the ratio of CD4 + /CD8 + T cells in each group of mice in Example 5;
  • 7A-7B are respectively the levels of CD3 + T cells and CD4 + T cells in the peripheral blood of mice in each group in Example 6.
  • the raw materials and reagents used in the following examples are commercially available, all cells were purchased from ATCC; all cell culture materials were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; Or it can be prepared by known methods.
  • the experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.
  • step (2) Take 50g of the bacteria slime prepared in step (1), add 300g of purified water to resuspend the bacteria, adjust the pH to 3.5 with 1mol/L hydrochloric acid solution, extract at 100°C for 1.5h, cool to room temperature, and centrifuge at 12000g for 10min at room temperature , take the supernatant to obtain the crude sugar solution;
  • the prepared capsular polysaccharide A has a weight-average molecular weight of 80-90 kDa and a Mw/Mn of 1.0-1.3.
  • the chemical structure is shown in FIG. 2 .
  • Example 2 Drug efficacy test of Bacteroides fragilis zwitterionic capsular polysaccharides and immune checkpoint inhibitors in synergistic treatment of Renca renal carcinoma xenografts in mice
  • mice Male, SPF grade, body weight (20 ⁇ 2) g, and 80 of them were subcutaneously inoculated with 250 ⁇ L Renca cells (concentration: 1 ⁇ 107 cells/mL) on the outside of the right hind limb, daily Observe the growth of the tumor in the mice. After 7 days, a tumor nodule of about 5 mm can be seen at the injection site, which is regarded as a successful establishment of the renal cancer tumor-bearing mouse model.
  • the model group Bacteroides fragilis ZY-312 capsular polysaccharide A (PSA, 100 ⁇ g/mouse, prepared by Example 1, the same below ), PD-1 antibody (PD-1ab, product number BE0146, purchased from BioXcell, 200 ⁇ g/monkey), and low (50 ⁇ g/monkey), medium (100 ⁇ g/monkey), high (200 ⁇ g/monkey) doses of capsular polysaccharide A and In the PD-1 antibody combination group, 10 uncreated mice were used as the blank group.
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline daily, and intraperitoneally injected with 100 ⁇ L of PBS once a day; each administration group was given corresponding drugs at the same frequency, among which Bacteroides fragilis
  • the administration volume of the bacterial solution was 300 ⁇ L
  • the administration volume of the PD-1 antibody was 100 ⁇ L.
  • the nude mice in the model group formed tumors significantly, and the model was established successfully; compared with the model group, the body weight and tumor inhibition rate of the nude mice in each administration group increased, and the tumor weight decreased; the combination of PSA and PD-1 antibody The tumor weight of mice in the treatment group was significantly lower than that in the model group. This shows that the combination of Bacteroides fragilis ZY-312 capsular polysaccharide A and PD-1 antibody can effectively inhibit the growth of Renca renal carcinoma xenografts in mice. This inhibitory effect was dose-dependent with capsular polysaccharide A.
  • PSA combined with PD-1 antibody can up-regulate the levels of serum IFN- ⁇ and IL-2 in mice, enhance the body's anti-tumor immune response, and inhibit tumor growth, thereby achieving the prevention and treatment of renal cancer.
  • This preventive effect is dose-dependent with capsular polysaccharide A.
  • mice 100 male BALB/C-nude mice were taken, 80 of which were subcutaneously disinfected on the back, and each was inoculated with 0.2ml of T24 cell suspension (cell concentration: 2 ⁇ 10 7 /mL). After transplantation, the nude mice were kept in the SPF environment, and tumor nodules appeared at the inoculation site, and the texture was hard and other indicators were identified as tumor formation.
  • mice successfully modeled were randomly divided into 6 groups, 10 in each group, model group, Bacteroides fragilis ZY-312 capsular polysaccharide A (PSA, 100 ⁇ g/mouse) group, PD-1 antibody group (PD- 1ab, BE0146, BioXcell, 200 ⁇ g/body) and low (50 ⁇ g/body), medium (100 ⁇ g/body), high (200 ⁇ g/body) doses of Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody , and 10 uncreated mice were used as the blank group.
  • PSA Bacteroides fragilis ZY-312 capsular polysaccharide A
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline daily, and intraperitoneally injected with 100 ⁇ L of PBS every 2 days;
  • the administration volume of the Bacteroides bacteria solution was 300 ⁇ L, and the administration volume of the PD-1 antibody was 100 ⁇ L.
  • the model group significantly formed tumors, and the model was successfully established; compared with the model group, the body weight and tumor inhibition rate of nude mice in each administration group increased, and the tumor weight decreased.
  • the combination of PSA and PD-1 antibody group The tumor weight of the mice was significantly lower than that of the model group. This shows that the combination of Bacteroides fragilis ZY-312 capsular polysaccharide A and PD-1 antibody can effectively inhibit the growth of mouse T24 bladder cancer xenografts. This inhibitory effect was dose-dependent with capsular polysaccharide A.
  • PSA combined with PD-1 antibody can up-regulate the serum IFN- ⁇ and IL-2 levels in mice, enhance the body's anti-tumor immune response, and inhibit tumor growth, thereby achieving the prevention and treatment of bladder cancer.
  • This preventive effect is dose-dependent with capsular polysaccharide A.
  • penicillin (6 ⁇ 10 4 U/mL) was injected intramuscularly for anti-inflammation, 0.25 mL/day, and the incision was disinfected with povidone iodine for 3 consecutive days. Water and food were available ad libitum. With standard SPF feed, feeding for 30 days, the modeling was successful.
  • the castrated nude mice were disinfected with povidone iodine on the right chest wall and injected with 0.2 mL of 4T1 cell suspension. About 10 days later, tumor nodules (harder in texture, bigger and bigger, and growing rapidly) at the place where the breast pad was inoculated can be seen. The tumor grew to a size of 0.8 cm 3 . The tumor was removed by surgery and transplanted under the breast pads of the remaining 80 nude mice. After 4 to 5 days, solid tumor growth was seen on the second breast pad of the right chest wall, and tumors formed. Rate 100%.
  • mice successfully modeled were randomly divided into 6 groups, 10 in each group, model group, Bacteroides fragilis ZY-312 capsular polysaccharide A (PSA, 100 ⁇ g/mouse) group, PD-1 antibody group (PD -1ab, BE0146, BioXcell, 200 ⁇ g/body) and low (50 ⁇ g/body), medium (100 ⁇ g/body), high (200 ⁇ g/body) doses of Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody group, and 10 unmodeled nude mice were used as the blank group.
  • PSA Bacteroides fragilis ZY-312 capsular polysaccharide A
  • PD-1ab PD-1 antibody group
  • BioXcell 200 ⁇ g/body
  • medium 100 ⁇ g/body
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline daily, and intraperitoneally injected with 100 ⁇ L of PBS once a day; each administration group was given corresponding drugs at the same frequency, among which Bacteroides fragilis
  • the administration volume of the bacterial solution was 300 ⁇ L
  • the administration volume of the PD-1 antibody was 100 ⁇ L.
  • the nude mice in the model group had significant tumor mass, and the model was successfully established; compared with the model group, the body weight and tumor inhibition rate of the nude mice in each administration group increased, and the tumor weight decreased.
  • the tumor weight of mice in the Bacteroides ZY-312 capsular polysaccharide A combined with PD-1 antibody group was significantly lower than that in the model group. This shows that Bacteroides fragilis capsular polysaccharide A combined with PD-1 antibody can effectively inhibit the growth of 4T1 breast cancer in mice, and this inhibitory effect is dose-dependent with capsular polysaccharide A.
  • IL-4 can activate cytotoxic T cells, promote macrophages to promote antigens and kill tumor cells.
  • the levels of IL-4 and IFN- ⁇ in the serum of nude mice were measured by ELISA, and the experimental results are shown in FIG. 5 .
  • the serum IFN- ⁇ and IL-4 levels of the mice in the model group increased significantly (p ⁇ 0.05); compared with the model group, the serum IFN- ⁇ and IL-4 levels of the mice in each administration group increased, Among them, there was a significant difference between the high-dose PSA combined with PD-1 antibody group and the model group (p ⁇ 0.05). This shows that Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can regulate serum cytokines in mice and enhance the body's anti-tumor immune response.
  • PSA combined with PD-1 antibody can up-regulate serum IFN- ⁇ and IL-4 levels in mice, enhance the body's anti-tumor immune response, and inhibit tumor growth, thereby achieving the prevention and treatment of breast cancer.
  • This preventive effect is dose-dependent with capsular polysaccharide A.
  • mice 100 BALB/c-Nude nude mice were taken, 80 of which were inoculated with the human ovarian cancer cell line SKOV3 to construct the animal model.
  • the concentration was 5 ⁇ 10 7 cells/mL, mixed evenly, and 0.2 mL of SKOV3 cell suspension was subcutaneously inoculated on the back of each nude mouse for a total of 1 spot.
  • Two weeks after inoculation if the transplanted tumor grows to about 10 mm in diameter, it means that the nude mouse model bearing ovarian cancer has been successfully established.
  • mice successfully modeled were randomly divided into 6 groups, 10 in each group, model group, Bacteroides fragilis ZY-312 capsular polysaccharide A (PSA, 100 ⁇ g/mouse) group, PD-1 antibody group (PD -1ab, BE0146, BioXcell, 200 ⁇ g/body) and low (50 ⁇ g/body), medium (100 ⁇ g/body), high (200 ⁇ g/body) doses of capsular polysaccharide A combined with PD-1 antibody, and at the same time Ten nude mice were used as a blank group.
  • PSA Bacteroides fragilis ZY-312 capsular polysaccharide A
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline every day, and 100 ⁇ L of PBS was intraperitoneally injected once every 3 days; each administration group was given corresponding drugs at the same frequency, Among them, the administration volume of Bacteroides fragilis bacteria liquid is 300 ⁇ L, and the administration volume of PD-1 antibody is 100 ⁇ L.
  • mice 21 days after administration, the nude mice were sacrificed by cervical dislocation, and the complete transplanted tumor tissue was stripped and collected to measure the tumor weight and tumor inhibition rate, and CD4 + , CD8 + and CD4 + /CD8 + T lymphocytes were detected by flow cytometry Subgroup proportions.
  • capsular polysaccharide PSA group PD-1 antibody (PD-1ab) group
  • capsular polysaccharide PSA and PD-1 antibody (PD-1ab) combined group on tumor weight and tumor inhibition rate in nude mice are shown in Table 7 .
  • the model group significantly formed tumors, and the modeling was successful.
  • the tumor weight of nude mice in each administration group decreased, among which the PD-1 antibody group had a significant difference, and the Bacteroides fragilis ZY-312 capsular polysaccharide A combined with the PD-1 antibody group had a very significant difference. difference.
  • T cells are a kind of lymphocytes with immunosuppressive effect, and their subsets such as CD4 + , CD8 + etc. play an important regulatory role in the body's anti-tumor immune response.
  • the levels of CD4 + and CD8 + T cells and the ratio of CD4 + /CD8 + T cells in tumor tissues of different groups of nude mice were measured by flow cytometry, and the experimental results are shown in FIG. 6 . Compared with the blank group, the level of CD4 + T cells and the ratio of CD4 + /CD8 + T cells in the model group and the administration group decreased.
  • capsular polysaccharide PSA and PD-1 antibody Compared with the nude mice in the model group, capsular polysaccharide PSA and PD-1 antibody (PD-1ab) The level of CD4 + T cells and the ratio of CD4 + /CD8 + T cells in nude mice in the combined high-dose group were significantly increased (P ⁇ 0.05). This shows that Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can regulate the level of tumor-infiltrating CD4 + T cells and the ratio of CD4 + /CD8 + T cells in mice, thereby enhancing the body's anti-tumor immune response.
  • PSA combined with PD-1 antibody can up-regulate the infiltration levels of CD4 + and CD4 + /CD8 + T cells in mouse serum, enhance the body's anti-tumor immune response, and inhibit tumor growth, thereby achieving the prevention and treatment of ovarian cancer.
  • This preventive effect is dose-dependent with capsular polysaccharide A.
  • Prostate cancer cell PC-3 was cultured in RM1640 medium containing 10% FBS and 1% Penicium-Streptomycin at 37°C and 5% CO2 to the logarithmic growth phase, and the concentration was adjusted to 2 ⁇ 10 7 /mL.
  • mice Take 100 BALB/c-nude nude mice, aged 4 to 5 weeks, and 80 of them were subcutaneously inoculated with 0.1 mL of PC-3 cell suspension in the armpit of the forelimb, and raised under standard conditions.
  • the tumor volume was about 200 mm. For modeling success.
  • mice 60 successfully modeled mice were randomly divided into 6 groups, 10 in each group, respectively: model group, Bacteroides fragilis ZY-312 capsular polysaccharide A (PSA, 100 ⁇ g/mouse) group, PD-1 antibody group (PD-1ab, BE0146, BioXcell, 200 ⁇ g/monkey) and low (50 ⁇ g/big), medium (100 ⁇ g/big), high (200 ⁇ g/bill) doses of capsular polysaccharide A combined with PD-1 antibody, and at the same time 10 unmodeled nude mice were used as the blank group.
  • PSA Bacteroides fragilis ZY-312 capsular polysaccharide A
  • PD-1ab PD-1 antibody group
  • BioXcell 200 ⁇ g/monkey
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline every day, once every 2 days, and 100 ⁇ L of PBS was intraperitoneally injected; each administration group was given corresponding drugs at the same frequency, Among them, the administration volume of Bacteroides fragilis bacteria liquid is 300 ⁇ L, and the administration volume of PD-1 antibody is 100 ⁇ L.
  • the nude mice in the model group had significant tumor masses, and the model was successfully established; compared with the model group, the tumor weights of the nude mice in each administration group were reduced, and the capsular polysaccharide A of Bacteroides fragilis ZY-312 and The tumor weight of the mice in the PD-1 antibody combination group was significantly different from that in the model group.
  • Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can effectively inhibit the growth of mouse PC-3 prostate cancer xenografts, and this inhibitory effect is dose-dependent with capsular polysaccharide A.
  • T cells are a kind of lymphocytes with immunosuppressive effect, and their subsets such as CD3 + , CD4 + etc. play an important regulatory role in the body's anti-tumor immune response.
  • Flow cytometry was used to measure the ratio of CD3 + and CD4 + T cells in the peripheral blood of mice, and the experimental results are shown in FIG. 7 .
  • the levels of CD3 + , CD4 + T cells in the model group and each administration group were lower than those in the blank group.
  • the levels of CD3 + , CD4 + T cells in the peripheral blood of nude mice in each administration group increased.
  • the levels of CD3 + and CD4 + T cells in the high-dose PD-1 group were significantly higher than those in the model group (P ⁇ 0.05).
  • Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can regulate the levels of CD3 + and CD4 + T cells in the peripheral blood of mice, thereby enhancing the body's anti-tumor immune response.
  • PSA combined with PD-1 antibody can up-regulate the levels of CD3 + and CD4 + T cells in the peripheral blood of mice, enhance the body's anti-tumor immune response, and inhibit tumor growth, thereby achieving the prevention and treatment of prostate cancer.
  • This preventive effect is dose-dependent with capsular polysaccharide A.

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Abstract

本发明公开了脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药在防治生殖泌尿系统肿瘤中的应用。本发明通过大量实验证明，脆弱拟杆菌，尤其是保藏号为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312的荚膜多糖A与免疫检查点抑制剂联合用药，能够通过调控T细胞和抗肿瘤免疫因子水平，增强机体抗肿瘤免疫反应，进而达到防治生殖泌尿系统肿瘤的目标。

Description

脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药治疗生殖泌尿系统肿瘤的应用
本申请要求享有2022年1月12日向中国国家知识产权局提交的，专利申请号为202210034075.6，发明名称为“脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药治疗生殖泌尿系统肿瘤的应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
本发明在实施过程中所使用的微生物菌种已于2015年4月2日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院3号)保藏。分类命名：脆弱拟杆菌ZY-312(Bacteroides fragilis ZY-312)，保藏编号CGMCC No.10685。脆弱拟杆菌ZY-312由本发明申请单位自行分离获得，并且已经在授权专利保护(专利号201510459408.X)，按照专利审查指南的规定，公众能够从商业渠道买到或已经授权，不用保藏，即不用提供保藏证明。
技术领域
本发明涉及生物医药领域，具体涉及一种脆弱拟杆菌的两性离子荚膜多糖与免疫检查点抑制剂共同用药在制备生殖泌尿系统肿瘤的药物中的应用。
背景技术
泌尿生殖系统肿瘤是指病发于泌尿系统和(或)生殖系统的肿瘤。其中包括女性胸部和生殖器官肿瘤、男性生殖器官肿瘤以及泌尿器官肿瘤，如肾癌、膀胱癌、尿路上皮癌、乳腺癌、卵巢癌和前列腺癌等。
泌尿生殖系统肿瘤是严重危害人类健康的重大疾病，具有高发病率和死亡率，有数据表明，其占所有癌症新发病例的31％，对人类健康和生命造成严重威胁。其发病机制具有异质性，通常包含内因和外因，如物理因素、化学因素、生物学因素、遗传因素、内分泌因素、免疫功能和精神因素等。
目前对泌尿生殖系统(genitourinary，GU)肿瘤的诊断和监测方法多为侵袭性和/或缺乏敏感度和特异性。针对不同病症，治疗方式存在差异，主要包括手术治疗、放射治疗、化药治疗、靶向治疗和免疫治疗等。手术治疗是泌尿生殖系统肿瘤的首要治疗方法，适用于早、中期实体肿瘤，疗效直接，对于局部肿瘤可彻底清除，但该方法创伤性较大，部分部位不可切除，存在局部扩散风险；化疗方法对于不同肿瘤治愈效果不一，可作用于患者全身，但化疗药物的选择性差，且存在不同程度的毒副作用；放疗是一种局部治疗手段，该方法对于肿瘤的抑制效果及杀伤力较强，毒性相对较小，放疗后患者易存在后遗症，如肌肉、神经损伤等；生物治疗如免疫治疗、基因治疗、免疫治疗等疗效不一，价格昂贵且存在较强副作用。
肾癌是泌尿系统常见的肿瘤之一，占肾脏恶性肿瘤的80％～90％，其起源于肾实质泌尿小管上皮系统，发生于肾实质的任何部位，呈结节状或分叶状，大小不一，质硬，切面呈黄色，多为实性，有时灰白色类似肉瘤，少数为多囊性，囊内有透明液体，可见有坏死和出血。
目前，肾癌的病因尚不明晰，主要认为与遗传、吸烟、高血压、肥胖及抗高血压治疗有关，其中吸烟和肥胖是公认的重要致肾癌危险因素，其病理类型通常分为透明细胞癌、乳头状肾细胞癌、嫌色细胞癌，以及集合管癌等少见类型的肾细胞癌。
肾癌患者的临床表现复杂多变，且早期患者通常缺乏临床表现，主要临床表现包括血尿、腰痛和肿块，肾癌的诊断方式包括临床表现、实验室检查、影像学检查等。实验室常规检查的主要包括尿常规、血常规、红细胞沉降率、血糖、血钙、肾功能、肝功能、乳酸脱氢酶、碱性磷酸酶等项目；影像学检查对于肾癌的诊治、病灶的发现及分期、术中定位和术后及非手术治疗 随诊均具有重要意义，包括胸部X线检查、超声检查、CT检查、MRI检查及PET检查等。肾癌患者的治疗方式通常参考影像学检查的结果以确定肿瘤的临床分期，并利用辅助检查评估患者对治疗的耐受能力，根据临床分期并结合患者的耐受力，选择恰当的治疗方式，通常包括手术治疗、介入治疗、放疗和药物治疗等。其中，手术治疗是最主要的治疗方式，通常适用于局限性和局部进展性肾癌患者
膀胱癌是泌尿系统三大肿瘤之一，发病率在全世界范围内居恶性肿瘤的第10位，膀胱癌在男性中的发病率上升至第6位，死亡率在男性恶性肿瘤中居第9位，目前，其发病率呈现逐年上升的趋势，且膀胱癌具有易复发的特点。
常见膀胱肿瘤包括膀胱尿路上皮癌、膀胱鳞状细胞癌和膀胱腺癌，其中最主要的病理类型为膀胱尿路上皮癌，占膀胱癌总数的90％以上。
膀胱癌的病因较为复杂，包括遗传因素和环境因素，如吸烟和外界芳香胺类物质的影响等。常见症状包括血尿、尿频、排尿疼痛和下背痛等。主要检测方式有尿液分析、尿液细胞学检查、膀胱镜检查等。
膀胱癌的主要治疗手段包括手术治疗、放射治疗、化疗和免疫治疗。常用手术治疗方式有配合电灼术的经尿道切除术、根治性膀胱切除术、膀胱部分切除术、尿流改道术等；放射治疗是利用高能量X-射线或其他类型的辐射杀死癌细胞或阻止其增长的一种癌症治疗方式，包括外部放射疗法和内部放射疗法，具体方式取决于所治疗癌症的类型及所处阶段；生物治疗是利用患者的免疫系统对抗癌症的治疗方式，其使用身体或是在实验室生产的物质来促进、诱导或恢复身体对抗癌症的自然防御机制。
乳腺癌(breast cancer)是发生于乳腺上皮或导管上皮的恶性肿瘤，多发于女性，男性发病率约为1％。国家癌症中心发布的数据显示，乳腺癌是我国女性发病率最高的恶性肿瘤，且发病率逐年增高。
乳腺癌早期通常无明显症状，主要表现为：乳腺肿块、乳头改变、皮肤和外形改变、乳房疼痛、区域淋巴结肿大以及出现远处转移后的器官受累症状等。
乳腺癌病因尚不清楚，其被认为与年龄、激素、遗传因素、月经婚育史、膳食结构、过度诊断、环境因素等密切相关。乳腺癌的主要筛查手段包括影像学检查，组织活检，乳腺癌肿瘤标志物检查，免疫组化检查等。
乳腺癌主要治疗方式包括药物治疗、手术治疗和放射治疗等。其中，药物治疗根据作用机制不同，可分为化学药物治疗、内分泌治疗(激素治疗)以及靶向治疗；化疗药物一般通过改变或抑制癌细胞的生化代谢过程，从而干扰癌细胞的繁殖，主要成分以蒽环类和紫杉醇类为主；内分泌治疗是根据药物与细胞质内的雌激素受体结合从而使细胞发挥作用，主要包括三苯氧胺、雌激素拮抗剂和芳香化酶抑制剂等；药物治疗方法对于肿瘤细胞的转移和病症复发具有重要作用，但起效较慢，易对患者身体造成额外伤害，诱发消化系统等毒性作用和不良反应，并使患者产生耐药性。手术治疗方法是乳腺癌治疗的最常用方法，手术方式包括乳腺癌根治术、乳腺癌改良根治术、乳腺癌扩大根治术、保留乳房的乳腺癌根治术、前哨淋巴结活检和乳房重建手术等，其能够最大程度的局部控制病灶，有效提高患者生存率，但此方法恢复难度较大，较易复发，且一旦复发难以治愈。放射治疗通常用于局部治疗，适用于早期肿瘤患者，能够使患者获得较长生存期，以提高患者生存质量为前提缩小肿瘤扩散面积，但该方法费用昂贵、周期较长、并伴随一系列毒副作用和并发症。目前，临床尚缺乏更有效的乳腺癌治疗方法，虽然乳腺癌的诊疗技术不断提高，乳腺癌患者的死亡率仍无明显下降趋势。
卵巢癌(ovarian cancer)是指生长在卵巢上的最常见的恶性肿瘤之一，其中90％～95％为卵巢原发性的癌，另外5％～10％为其它部位原发的癌转移到卵巢，近年来，其发病率、复发率及病死率较高，且逐渐趋于年轻化。卵巢癌早期症状不明显且不特异，因此对于早期筛查及诊断较为困难，就诊患者60～70％为晚期，且治疗效果欠佳。
卵巢癌发病原因尚不明确，通过对其流行病学进行调查，认为与遗传因素、持续排卵、饮食习惯和环境因素等密切相关。常见的诊断方法包括超声波检查、细胞学检查、腹腔镜检查、肿瘤标记物测定和放射学诊断等。
卵巢癌的治疗方式通常以手术治疗为主，并辅以化学治疗、放射治疗、免疫治疗等。其中手术方法疗效较好，对于早期患者可彻底清除，但该方法风险较高，治疗范围较为局限，且易产生副作用；化疗方法能有效控制病情发展，延长患者的生存期，但该方法复发率较高，复发次数较多且复发间隔时间较短；免疫治疗方法作为治疗卵巢癌的新型疗法，特异性较高，副作用小，但目前该方法未形成完整体系及诊治方法。
近年来，中国前列腺癌(Prostate Cancer，PCa)的发病率呈现持续快速增长趋势，2015年中国前列腺癌的总体发病率为10.23/10万人，死亡率为4.36/10万人，成为中国男性泌尿生殖系统发病率最高的肿瘤，2020年世界癌症报告数据显示，前列腺癌位居男性恶性肿瘤发病率的第6位，死亡率的第9位。
早期发现前列腺癌依赖于前列腺癌肿瘤标志物，但目前应用的前列腺特异性抗原的敏感性和特异性有限。有大量根治性切除前列腺的患者(15％～30％)被确定为过度诊治。
目前，手术仍是恶性肿瘤的主要治疗手段。泌尿系统肿瘤手术治疗会严重影响患者的生活质量，临床使用的放化疗药物缺乏选择性和靶向性，会对癌症患者产生严重的不良反应。因此寻找高效低毒、具有靶向性和选择性抗癌作用的化疗药物是抗肿瘤药物研究的一个重要方向和迫切任务，对于不能耐受手术、传统化疗药物耐药、复发以及晚期的肿瘤患者，迫切需要一种新型抗肿瘤药物延长其生存期。
免疫检查点本是人体免疫系统中起保护作用的分子，起类似刹车的作用，防止T细胞过度激活导致的炎症损伤等。而肿瘤细胞利用人体免疫系统这一特性，通过过度表达免疫检查点分子，抑制人体免疫系统反应，逃脱人体免疫监视与杀伤，从而促进肿瘤细胞的生长。抑制免疫检查点分子及其配体的表达能够增强T细胞对肿瘤的杀伤效应，达到抗肿瘤的目的。已被公布的免疫检查点有CTLA-4、PD-1/PD-L1、LAG-3、TIM-3、VISTA、A2aR等。
程序性细胞死亡蛋白1(PD-1)在多种淋巴细胞上表达，尤其在肿瘤特异性T细胞上高表达。在肿瘤微环境中，它通过干扰保护性免疫应答而导致恶性肿瘤细胞的扩张。它具有两个配体，即程序性细胞死亡配体1和2(PD-L1、PD-L2)，其中，PD-L1被肿瘤细胞表达，以逃逸免疫系统对它进行的抗肿瘤反应。阻断PD-1和PD-L1间的作用可以在T细胞进入肿瘤微环境后保持T细胞的应答，保证T细胞的抗肿瘤作用。针对PD-1/PD-L1的抗体已有纳武单抗(Nivolumab)、派姆单抗(Pembrolizumab)、JQ1、阿特珠单抗(Atezolizumab)、阿维鲁单抗(Avelumab)和西米普利单抗(Cemiplimab)。这些单抗被批准用于治疗乳腺癌、肺癌、大肠癌、癌症、膀胱癌、胰腺癌、前列腺癌和弥漫性大B细胞淋巴瘤(DLBCL)。
尽管PD-1/PD-L1抗体抗癌疗效显著(总体无进展生存率达到80％)，但临床研究显示，仅有20-45％患者对其有所响应。
发明内容
为克服现有技术中所存在的上述缺陷，本发明的目的是提供一种脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药在防治生殖泌尿系统肿瘤中的应用。本发明出人预料地发现，脆弱拟杆菌，尤其是保藏号为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312的荚膜多糖A与免疫检查点抑制剂联合用药，能够通过调控T细胞和抗肿瘤免疫因子水平，增强机体抗肿瘤免疫反应，进而达到防治生殖泌尿系统肿瘤的目标。
为了实现上述目的，本发明采用如下技术方案：
第一方面，提供一种脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂在制备预防和/或治疗生殖泌尿系统癌症的产品中应用。
在其中一些实施例中，所述两性离子荚膜多糖提取自保藏编号为CGMCC No.10685的脆弱 拟杆菌ZY-312。
在其中一些实施例中，所述两性离子荚膜多糖含有荚膜多糖A。其中，所述荚膜多糖A的结构如下所示：
Figure PCTCN2022120025-appb-000001
根据本发明，所述荚膜多糖A的重均分子量为80-90KD，Mw分布于70-100KD的部分占总量的70-80％，重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
在其中一些实施例中，其中，所述两性离子荚膜多糖中荚膜多糖A的含量超过95wt％。
在其中一些实施例中，所述两性离子荚膜多糖的制备方法包括以下步骤：
(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物，即得脆弱拟杆菌菌泥；取菌泥，加入菌泥质量3～10倍的纯化水使菌体重悬，用酸溶液调节其pH至2.0～4.5，50～120℃提取0.5～3.0h，冷却至室温，常温离心，取上清，得到粗糖溶液；
(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
(3)回流液中加入等体积40mmol/L Tris-HCl转盐；离子交换柱层析，梯度洗脱，分段收集，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到脆弱拟杆菌提取物。
在其中一些实施例中，步骤(1)中所述离心为11000～13000g离心8～12min。
在其中一些实施例中，步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中，无机酸可以是盐酸、硫酸、磷酸等；有机酸可以是乙酸、柠檬酸等。
在其中一些实施例中，步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围。
在其中一些实施例中，步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm，层析时的流速15～25mL/min，pH5.0～9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)；所述超滤膜的分子量为10KD。
在其中一些实施例中，所述生殖泌尿系统肿瘤是指病发于泌尿系统和(或)生殖系统的肿瘤。其中包括女性胸部和生殖器官肿瘤、男性生殖器官肿瘤以及泌尿器官肿瘤。示例地，选自乳腺癌、宫颈癌、子宫体癌、卵巢癌、前列腺癌、肾癌、膀胱癌、睾丸癌中的一种或多种。
在其中一些实施例中，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体、CTLA-4抗体及其他能够与免疫检查点结合，激活或增强内源性抗肿瘤免疫反应的物质。
在其中一些实施例中，所述PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
在其中一些实施例中，所述PD-L1抗体包括阿特朱单抗(Atezolizumab)、阿维鲁单抗(Avelumab)、度伐鲁单抗(Durvalumab)及其他能够与PD-L1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
根据本发明的实施方案，所述产品为食品或药品。
在其中一些实施例中，所述食品包括奶粉、干酪、凝乳、酸奶酪、冰激凌或发酵谷类食品。所述食品还可以是动物食品，比如饲料等。
在其中一些实施例中，所述药品的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂。所述药品包括人用药或动物用药。
在其中一些实施例中，所述药品给药周期可为间歇给药、周期性给药、持续给药或长期给药。
在其中一些实施例中，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体同时给药。
在其中一些实施例中，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体分别给药。
在其中一些实施例中，脆弱拟杆菌两性离子荚膜多糖采用口服或灌肠方式给药。
第二方面，提供一种用于防治泌尿生殖系统肿瘤的组合物，其中，所述组合物同时包括脆弱拟杆菌两性离子荚膜多糖和免疫检查点抑制剂，所述脆弱拟杆菌两性离子荚膜多糖提取自保藏号为CGMCC No.10685的脆弱拟杆菌ZY-312。
在其中一些实施例中，所述两性离子荚膜多糖含有荚膜多糖A。其中，所述荚膜多糖A的结构如下所示：
Figure PCTCN2022120025-appb-000002
根据本发明，所述荚膜多糖A的重均分子量为80-90KD，Mw分布于70-100KD的部分占总量的70-80％，重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
在其中一些实施例中，其中，所述两性离子荚膜多糖中荚膜多糖A的含量超过95wt％。
在其中一些实施例中，所述两性离子荚膜多糖的制备方法包括以下步骤：
(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物，即得脆弱拟杆菌菌泥；取菌泥，加入菌泥质量3～10倍的纯化水使菌体重悬，用酸溶液调节其pH至2.0～4.5，50～120℃提取0.5～3.0h，冷却至室温，常温离心，取上清，得到粗糖溶液；
(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
(3)回流液中加入等体积40mmol/L Tris-HCl转盐；离子交换柱层析，梯度洗脱，分段收集，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到脆弱拟杆菌提取物。
在其中一些实施例中，步骤(1)中所述离心为11000～13000g离心8～12min。
在其中一些实施例中，步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中，无机酸可以是盐酸、硫酸、磷酸等；有机酸可以是乙酸、柠檬酸等。
在其中一些实施例中，步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围。
在其中一些实施例中，步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm，层析时的流速15～25mL/min，pH5.0～9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)；所述超滤膜的分子量为10KD。
在其中一些实施例中，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体、CTLA-4抗体及其他能够与免疫检查点结合，激活或增强内源性抗肿瘤免疫反应的物质。
在其中一些实施例中，所述PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
在其中一些实施例中，所述PD-L1抗体包括阿特朱单抗(Atezolizumab)、阿维鲁单抗(Avelumab)、度伐鲁单抗(Durvalumab)及其他能够与PD-L1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
根据本发明的实施方案，所述组合物为药物。
在其中一些实施例中，所述药物的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂。所述药品包括人用药或动物用药。
在其中一些实施例中，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体同时给药。
在其中一些实施例中，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体分别给药。
在其中一些实施例中，所述药物两性离子荚膜多糖采用口服或灌肠方式给药。
在其中一些实施例中，所述药物给药周期可为间歇给药、周期性给药、持续给药或长期给药。
根据本发明的实施方案，所述生殖泌尿系统肿瘤是指病发于泌尿系统和(或)生殖系统的肿瘤。其中包括女性胸部和生殖器官肿瘤、男性生殖器官肿瘤以及泌尿器官肿瘤。包括乳腺癌、宫颈癌、子宫体癌、卵巢癌、前列腺癌、肾癌、膀胱癌、睾丸癌中的一种或多种。
本发明还提供一种治疗生殖泌尿系统肿瘤的方法，包括向患者施用治疗有效量的上述产品或组合物。
本发明的有益效果：
本发明出人预料地发现，脆弱拟杆菌特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312及其两性离子荚膜多糖与免疫检查点抑制剂联用，尤其是PD-1抗体/PD-L1抗体联用，能够调节小鼠T细胞和抗肿瘤免疫因子水平，增强机体抗肿瘤免疫反应，有效防治生殖泌尿系统肿瘤。
本发明采用的脆弱拟杆菌ZY-312不含BFT基因，是非产毒菌株，急性毒性证实，该菌株对正常小鼠和裸鼠均无致病性(Wang Y，Deng H，Li Z，Tan Y，Han Y，Wang X，Du Z，Liu Y，Yang R，Bai Y，Bi Y，Zhi F.Safety Evaluation of a Novel Strain of Bacteroides fragilis.Front Microbiol.2017 Mar 17；8:435.)。根据专利ZL201510459408.X和科技文献Xu W，Su P，Zheng L，Fan H，Wang Y，Liu Y，Lin Y，Zhi F.In vivo Imaging of a Novel Strain of Bacteroides fragilis via Metabolic Labeling.Front Microbiol.2018 Oct 1；9:2298.的报道，该菌株对胃酸、胆盐有着较好的耐性，能够保证其在胃中的存活和有效定植。
附图说明
图1A-1E分别为本发明实施例1的荚膜多糖A核磁共振波谱仪分析的 1H谱、 13C谱、COSY谱、HSQC谱、HMBC谱图；
图2为本发明实施例1制备得到的脆弱拟杆菌荚膜多糖A的结构单元的化学结构式；
图3A-3B分别为实施例2各组小鼠血清IFN-γ、IL-2表达水平；
图4A-4B分别为实施例3各组小鼠血清IFN-γ、IL-2表达水平；
图5A-5B分别为实施例4各组小鼠血清IL-4、IFN-γ表达水平；
图6A-6C分别为实施例5各组小鼠肿瘤浸润CD4 +T细胞、CD8 +T细胞水平和CD4 +/CD8 +T细胞比值；
图7A-7B分别为实施例6各组小鼠外周血CD3 +T细胞、CD4 +T细胞水平。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，所有细胞购自ATCC；所有细胞培养材料购自Gibco；所有实验动物购自浙江维通利华实验动物技术有限公司；或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例1脆弱拟杆菌荚膜多糖A的制备
(1)将脆弱拟杆菌ZY-312菌种划线接种于血平皿，厌氧培养48h，选取单个菌落接种于植物源蛋白胨液体培养基中进行发酵培养8小时(温度为37℃)，所得菌液离心沉淀，转速3000r/min，离心15min，去上清，收集沉淀物，即得脆弱拟杆菌ZY-312菌泥。
(2)取步骤(1)中所制菌泥50g，加入300g纯化水使菌体重悬，用1mol/L盐酸溶液调节其pH至3.5，100℃提取1.5h，冷却至室温，12000g常温离心10min，取上清，得到粗糖溶液；
(3)粗糖溶液经10KD超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
(4)回流液中加入等体积40mmol/L Tris-HCl(pH8.5)转盐；DEAE Sepharose Fast Flow离子交换柱层析(16mm×200mm)，流速20mL/min，20mmol/L Tris-HCl(pH8.5，含0.2mol/L NaCl)梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，10KD超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到脆弱拟杆菌提取物；
(5)称量30mg步骤(4)所述的脆弱拟杆菌提取物，溶于0.5mL D 2O，加入1μl丙酮(1H，2.22；13C，30.89)定标。采用500MHz Bruker核磁共振波谱仪分析1H、13C、COSY、HSQC、HMBC谱(参见图1A-1E)，确证步骤(3)收集的脆弱拟杆菌提取物为荚膜多糖A，结合脂质含量低于0.02％，蛋白残留低于1％，核酸残留低于0.05％。通过GPC(凝胶渗透色谱)分析，制得的荚膜多糖A重均分子量为80-90kDa，Mw/Mn为1.0-1.3，化学结构参见图2。
实施例2脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂协同治疗小鼠Renca肾癌移植瘤的药效试验
1.实验设计和流程
(1)动物造模
取昆明种小鼠100只，雄性，SPF级，体质量(20±2)g，其中80只小鼠右后肢外侧皮下处接种250μL Renca细胞(浓度为1×10 7个/mL)，每日观察小鼠肿瘤生长情况，7d后注射处见 约5mm的肿瘤结节视为肾癌荷瘤小鼠模型建立成功。
(2)动物分组及给药
取造模成功的小鼠60只，随机分为6组，每组10只，模型组、脆弱拟杆菌ZY-312荚膜多糖A(PSA，100μg/只，由实施例一制得，下同)，PD-1抗体(PD-1ab，商品编号BE0146，购自BioXcell，200μg/只)以及低(50μg/只)、中(100μg/只)、高(200μg/只)剂量荚膜多糖A与PD-1抗体联用组，同时以未造模小鼠10只作为空白组。
建模成功后(D0)开始分组给药：从D0开始，空白组、模型组动物每日口服300μL生理盐水，每天一次腹腔注射100μL PBS；各给药组同频次给予对应药物，其中脆弱拟杆菌菌液给药体积为300μL，PD-1抗体给药体积为100μL。
给药14天后进行颈椎脱臼法处死小鼠，称量小鼠重量，剥离瘤体后，称取瘤体质量，计算抑瘤率，其中，抑瘤率＝(模型组肿瘤重量-给药组肿瘤重量)/模型组平均肿瘤重量×100％，并采用ELISA法测定小鼠血清中抗肿瘤免疫因子IFN-γ和IL-2水平。
2.实验结果
(1)小鼠重量、肿瘤重量及抑瘤率
表1荚膜多糖A与PD-1抗体对小鼠体重、肿瘤重量的影响
(均值±标准差，n＝8)
Figure PCTCN2022120025-appb-000003
注：“*”表示与模型组相比差异显著，其中*代表P＜0.05，**代表P＜0.01
与空白组相比，模型组裸鼠显著成瘤，造模成功；与模型组相比，各给药组裸鼠体重及抑瘤率升高，肿瘤重量降低；其中PSA与PD-1抗体联用组小鼠瘤重显著低于模型组。这说明脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用能够有效抑制小鼠Renca肾癌移植瘤的生长。这种抑制作用与荚膜多糖A具有一定的剂量依赖性。
(2)小鼠血清IFN-γ、IL-2水平
IFN-γ和IL-2均是已知的抗肿瘤免疫细胞因子。各给药组对小鼠血清IFN-γ、IL-2水平的影响如图3所示。与空白组相比，模型组小鼠血清IFN-γ、IL-2水平显著下降(p＜0.05)；与模型组相比，各给药组小鼠血清IFN-γ、IL-2水平上升，其中，高剂量PSA联用PD-1抗体组与模型组具有显著性差异(p＜0.05)。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节小鼠血清细胞因子，增强机体抗肿瘤免疫反应。这种调节作用与荚膜多糖A具有一定的剂量依赖性。
综上所述，PSA联用PD-1抗体能够上调小鼠血清IFN-γ、IL-2水平，增强机体抗肿瘤免疫反应，抑制肿瘤生长，从而达到对肾癌的防治作用。这种防治作用与荚膜多糖A具有一定的剂量依赖性。
实施例3脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂协同治疗小鼠膀胱癌(尿路上皮癌)移植瘤的应用
1.实验设计和流程
(1)动物造模
取雄性BALB/C-nude裸鼠100只，其中80只裸鼠于背部皮下消毒后，每只分别接种0.2ml T24细胞悬液(细胞浓度为2×10 7/mL)。移植后裸鼠继续饲养于SPF环境中，待在接种部位出现肿瘤结节，质地较硬等指标认定为成瘤。
(2)动物分组及给药
取造模成功的裸鼠60只，随机分成6组，每组10只，模型组、脆弱拟杆菌ZY-312荚膜多糖A(PSA，100μg/只)组，PD-1抗体组(PD-1ab，BE0146，BioXcell，200μg/只)以及低(50μg/只)、中(100μg/只)、高(200μg/只)剂量脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用组，同时以未造模小鼠10只作为空白组。
建模成功后(D0)开始分组给药：从D0开始，空白组、模型组动物每日口服300μL生理盐水，每2天一次腹腔注射100μL PBS；各给药组同频次给予对应药物，其中脆弱拟杆菌菌液给药体积为300μL，PD-1抗体给药体积为100μL。
给药14天后颈椎脱臼法处死小鼠，称量小鼠体重和肿瘤重量，计算抑瘤率，其中，抑瘤率＝(模型组肿瘤重量-给药组肿瘤重量)/模型组平均肿瘤重量×100％，收集小鼠血液，采用ELISA法测定血液中抗肿瘤免疫因子IFN-γ和IL-2水平。
2.实验结果
(1)小鼠体重、肿瘤重量和抑瘤率
表2荚膜多糖A与PD-1抗体对小鼠体重、肿瘤重量的影响
(均值±标准差，n＝8)
Figure PCTCN2022120025-appb-000004
注：“*”表示与模型组相比差异显著，其中*代表P＜0.05，**代表P＜0.01
与空白组相比，模型组显著成瘤，造模成功；与模型组相比，各给药组裸鼠体重及抑瘤率升高，肿瘤重量降低，其中PSA与PD-1抗体联用组小鼠瘤重显著低于模型组。这说明脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用能够有效抑制小鼠T24膀胱癌移植瘤的生长。这种抑制作用与荚膜多糖A具有一定的剂量依赖性。
(2)IFN-γ和IL-2水平
IFN-γ和IL-2均是已知的抗肿瘤免疫细胞因子。各给药组对小鼠血清IFN-γ、IL-2水平的影响如图4所示。与空白组相比，模型组小鼠血清IFN-γ、IL-2水平显著下降(p＜0.05)；与模型组相比，各给药组小鼠血清IFN-γ、IL-2水平上升，其中，高剂量PSA联用PD-1抗体组与模型组具有显著性差异(p＜0.05)。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节小鼠血清细胞因子，增强机体抗肿瘤免疫反应。这种调节作用与荚膜多糖A具有一定的剂量依 赖性。
综上所述，PSA联用PD-1抗体能够上调小鼠血清IFN-γ、IL-2水平，增强机体抗肿瘤免疫反应，抑制肿瘤生长，从而达到对膀胱癌的防治作用。这种防治作用与荚膜多糖A具有一定的剂量依赖性。
实施例4脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂协同治疗小鼠乳腺癌移植瘤的应用
1.实验设计和流程
(1)双侧卵巢切除(去势)模型的制备
取雌性BALB/C无胸腺裸鼠5只，以10％水合氯醛麻醉裸鼠后，将其俯卧并四肢固定于手术台中央，背部正中(肋弓下缘处)备皮并使用络合碘消毒，切开处位于脊柱旁0.5cm，距肋弓下缘0.5cm处皮肤，剪开腹膜。夹出卵巢并结扎，用缝合器分层缝合皮肤并消毒。术后肌肉注射青霉素(6×10 4U/mL)抗炎，0.25mL/日，并用碘伏消毒切口，连续3天。自由进食水。用标准SPF饲料，饲养30天，造模成功。
(2)动物造模
取去势后的裸鼠，在右侧胸壁利用碘伏进行消毒并注射0.2mL 4T1细胞悬液。10天左右，可见接种乳垫处的肿瘤结节(质地较硬，越来越大，长势迅速)。肿瘤长至0.8cm 3大小，利用手术的方式取下肿瘤，移植至其余80只裸鼠乳垫下，4～5d后右侧胸壁的第二乳垫处可见到有实体肿瘤的生长，成瘤率100％。
(3)动物分组及给药
取造模成功的小鼠60只，随机分为6组，每组10只，模型组、脆弱拟杆菌ZY-312荚膜多糖A(PSA，100μg/只)组，PD-1抗体组(PD-1ab，BE0146，BioXcell，200μg/只)以及低(50μg/只)、中(100μg/只)、高(200μg/只)剂量脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用组，同时以未造模裸鼠10只作为空白组。
建模成功后(D0)开始分组给药：从D0开始，空白组、模型组动物每日口服300μL生理盐水，每天一次腹腔注射100μL PBS；各给药组同频次给予对应药物，其中脆弱拟杆菌菌液给药体积为300μL，PD-1抗体给药体积为100μL。
给药4周后称量裸鼠体重，用脱颈法将裸鼠处死后，剥离裸鼠全部胸部肿瘤，计算裸鼠瘤重和抑瘤率，其中，抑瘤率＝(模型组肿瘤重量-给药组肿瘤重量)/模型组平均肿瘤重量×100％，并测定裸鼠血清中IL-4和IFN-γ水平。
2.实验结果
(1)体重、肿瘤重量和抑瘤率
表3荚膜多糖A与PD-1抗体(PD-1ab)对小鼠体重、肿瘤重量的影响及其抑瘤率
(均值±标准差，n＝8)
Figure PCTCN2022120025-appb-000005
Figure PCTCN2022120025-appb-000006
注：“*”表示与模型组相比差异显著，其中*代表P＜0.05，**代表P＜0.01
与空白组相比，模型组裸鼠出现显著瘤块，造模成功；与模型组相比，各给药组裸鼠体重及抑瘤率均有所升高，肿瘤重量有所降低，其中脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用组小鼠肿瘤重量显著低于模型组。这说明脆弱拟杆菌荚膜多糖A联用PD-1抗体能够有效抑制小鼠4T1乳腺癌生长，这种抑制作用与荚膜多糖A具有一定的剂量依赖性。
(2)IL-4和IFN-γ水平
IL-4能够活化细胞毒性T细胞，促进巨噬细胞提升抗原和杀伤肿瘤细胞。采用ELISA方法测定裸鼠血清中IL-4和IFN-γ水平，实验结果如图5所示。与空白组相比，模型组小鼠血清IFN-γ和IL-4水平显著上升(p＜0.05)；与模型组相比，各给药组小鼠血清IFN-γ、IL-4水平上升，其中，高剂量PSA联用PD-1抗体组与模型组相比具有显著性差异(p＜0.05)。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节小鼠血清细胞因子，增强机体抗肿瘤免疫反应。
综上所述，PSA联用PD-1抗体能够上调小鼠血清IFN-γ、IL-4水平，增强机体抗肿瘤免疫反应，抑制肿瘤生长，从而达到对乳腺癌的防治作用。这种防治作用与荚膜多糖A具有一定的剂量依赖性。
实施例5脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂协同治疗小鼠卵巢癌移植瘤的应用
1.实验设计和流程
(1)动物造模
取BALB/c-Nude裸鼠100只，其中80只采用接种人卵巢癌细胞株SKOV3的方法构建动物模型，具体实施方法为：将SKOV3细胞株传代培养至7～8代时消化细胞，调整细胞浓度为5×10 7个/mL，混匀，每只裸鼠背部皮下接种SKOV3细胞悬液0.2mL，共接种1点。接种2周后，若移植瘤直径长至约10mm，表示荷卵巢癌裸鼠模型建立成功。
(2)动物分组及给药
取造模成功的小鼠60只，随机分为6组，每组10只，模型组、脆弱拟杆菌ZY-312荚膜多糖A(PSA，100μg/只)组，PD-1抗体组(PD-1ab，BE0146，BioXcell，200μg/只)以及低(50μg/只)、中(100μg/只)、高(200μg/只)剂量荚膜多糖A与PD-1抗体联用组，同时以未造模裸鼠10只作为空白组。
建模成功后(D0)开始分组给药：从D0开始，空白组、模型组动物每日口服300μL生理盐水，每3天一次，于腹腔注射100μL PBS；各给药组同频次给予对应药物，其中脆弱拟杆菌菌液给药体积为300μL，PD-1抗体给药体积为100μL。
给药21天后采用颈椎脱臼法处死裸鼠，剥离、采集完整的移植瘤组织，测定肿瘤重量和抑瘤率，并采用流式细胞术检测CD4 +、CD8 +和CD4 +/CD8 +T淋巴细胞亚群比例。
2.实验结果
(1)肿瘤重量及抑瘤率
荚膜多糖PSA组、PD-1抗体(PD-1ab)组及荚膜多糖PSA和PD-1抗体(PD-1ab)联用组对裸鼠肿瘤重量及抑瘤率的影响如表7所示。与空白组相比，模型组显著成瘤，造模成功。
与模型组相比，各给药组裸鼠肿瘤重量均有所下降，其中PD-1抗体组具有显著差异，脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体各组具有极显著差异。
表4荚膜多糖A和PD-1抗体对卵巢癌细胞重量和抑瘤率的影响(％)
(均值±标准差，n＝6)
Figure PCTCN2022120025-appb-000007
Figure PCTCN2022120025-appb-000008
注：“*”表示与模型组相比差异显著，其中*代表P＜0.05，**代表P＜0.01
(2)CD4 +、CD8 +和CD4 +/CD8 +T淋巴细胞亚群
T细胞为一类具有免疫抑制作用的淋巴细胞，其亚群如CD4 +、CD8 +等在机体抗肿瘤免疫反应中起着重要的调控作用。采用流式细胞术测定不同组别裸鼠瘤组织中的CD4 +、CD8 +T细胞水平和CD4 +/CD8 +T细胞比值，实验结果如图6所示。与空白组相比，模型组和给药组CD4 +T细胞水平和CD4 +/CD8 +T细胞比值降低，与模型组裸鼠相比，荚膜多糖PSA和PD-1抗体(PD-1ab)联用高剂量组裸鼠CD4 +T细胞水平和CD4 +/CD8 +T细胞比值明显上升(P＜0.05)。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节小鼠肿瘤浸润CD4 +T细胞水平和CD4 +/CD8 +T细胞比值，进而增强机体抗肿瘤免疫反应。
综上所述，PSA联用PD-1抗体能够上调小鼠血清CD4 +、CD4 +/CD8 +T细胞浸润水平，增强机体抗肿瘤免疫反应，抑制肿瘤生长，从而达到对卵巢癌的防治作用。这种防治作用与荚膜多糖A具有一定的剂量依赖性。
实施例6脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂协同治疗小鼠前列腺癌移植瘤的应用
1.实验方法
(1)前列腺癌PC-3细胞培养及裸鼠人前列腺癌移植瘤模型的建立
将前列腺癌细胞PC-3在含有10％FBS和1％Penicium-Streptomycin的RM1640培养基中于37℃、5％CO 2条件下培养至对数生长期，用细胞培养液调整浓度为2×10 7/mL。
取BALB/c-nude裸鼠100只，鼠龄4～5周龄，其中80只小鼠前肢腋下皮下接种PC-3细胞悬液0.1mL，标准条件下饲养，肿瘤体积约200mm 3时视为造模成功。
(2)动物分组及给药
将60只造模成功的小鼠随机分为6组，每组10只，分别为：模型组、脆弱拟杆菌ZY-312荚膜多糖A(PSA，100μg/只)组，PD-1抗体组(PD-1ab，BE0146，BioXcell，200μg/只)以及低(50μg/只)、中(100μg/只)、高(200μg/只)剂量荚膜多糖A与PD-1抗体联用组，同时以未造模裸鼠10只作为空白组。
建模成功后(D0)开始分组给药：从D0开始，空白组、模型组动物每日口服300μL生理盐水，每2天一次，于腹腔注射100μL PBS；各给药组同频次给予对应药物，其中脆弱拟杆菌菌液给药体积为300μL，PD-1抗体给药体积为100μL。
给药4周后，用脱颈法将裸鼠处死后，称量裸鼠体重和肿瘤重量，计算抑瘤率，其中，抑瘤率＝(模型组肿瘤重量-给药组肿瘤重量)/模型组平均肿瘤重量×100％；处死前经眼眶采血，采用流式细胞术测小鼠外周血CD3 +、CD4 +T细胞水平。
2.实验结果
(1)小鼠体重、肿瘤重量和抑瘤率
表5荚膜多糖A与PD-1抗体(PD-1ab)对小鼠体重、肿瘤重量的影响及其抑瘤率
(均值±标准差，n＝8)
Figure PCTCN2022120025-appb-000009
Figure PCTCN2022120025-appb-000010
注：“*”表示与模型组相比差异显著，其中*代表P＜0.05，**代表P＜0.01
与空白组相比，模型组裸鼠出现显著瘤块，造模成功；与模型组相比，各给药组裸鼠肿瘤重量均有所降低，其中脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用组小鼠肿瘤重量与模型组具有极显著差异。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够有效抑制小鼠PC-3前列腺癌移植瘤的生长，这种抑制作用与荚膜多糖A具有一定的剂量依赖性。
(2)CD3 +、CD4 +T细胞
T细胞为一类具有免疫抑制作用的淋巴细胞，其亚群如CD3 +、CD4 +等在机体抗肿瘤免疫反应中起着重要的调控作用。采用流式细胞术测定小鼠外周血CD3 +、CD4 +T细胞比例，实验结果如图7所示。模型组及各给药组CD3 +、CD4 +T细胞水平低于空白组，与模型组相比，各给药组的裸鼠外周血中CD3 +、CD4 +T细胞水平增高，其中，PSA联用PD-1高剂量组的CD3 +、CD4 +T细胞水平显著高于模型组(P＜0.05)。这说明脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节小鼠外周血CD3 +、CD4 +T细胞水平，进而增强机体抗肿瘤免疫反应。
综上所述，PSA联用PD-1抗体能够上调小鼠外周血CD3 +、CD4 +T细胞水平，增强机体抗肿瘤免疫反应，抑制肿瘤生长，从而达到对前列腺癌的防治作用。这种防治作用与荚膜多糖A具有一定的剂量依赖性。
本发明还可有其它多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形，但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。

Claims (10)

  1. 一种脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂在制备预防和/或治疗生殖泌尿系统癌症的产品中应用。
  2. 根据权利要求1所述的应用，其特征在于，所述两性离子荚膜多糖提取自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
    优选，所述两性离子荚膜多糖含有荚膜多糖A。其中，所述荚膜多糖A的结构如下所示：
    Figure PCTCN2022120025-appb-100001
    优选地，所述荚膜多糖A的重均分子量为80-90KD，Mw分布于70-100KD的部分占总量的70-80％，重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
    更优选地，所述两性离子荚膜多糖中荚膜多糖A的含量超过95wt％。
    优选地，所述两性离子荚膜多糖的制备方法包括以下步骤：
    (1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物，即得脆弱拟杆菌菌泥；取菌泥，加入菌泥质量3～10倍的纯化水使菌体重悬，用酸溶液调节其pH至2.0～4.5，50～120℃提取0.5～3.0h，冷却至室温，常温离心，取上清，得到粗糖溶液；
    (2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
    (3)回流液中加入等体积40mmol/L Tris-HCl转盐；离子交换柱层析，梯度洗脱，分段收集，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到脆弱拟杆菌提取物。
    优选地，步骤(1)中所述离心为11000～13000g离心8～12min。
    优选地，步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中，无机酸可以是盐酸、硫酸、磷酸等；有机酸可以是乙酸、柠檬酸等。
    优选地，步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围。
    优选地，步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm，层析时的流速15～25mL/min，pH5.0～9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)；所述超滤膜的分子量为10KD。
  3. 根据权利要求1或2所述的应用，其特征在于，所述生殖泌尿系统肿瘤包括女性胸部和生殖器官肿瘤、男性生殖器官肿瘤以及泌尿器官肿瘤。优选地，选自乳腺癌、宫颈癌、子宫体癌、卵巢癌、前列腺癌、肾癌、膀胱癌、睾丸癌中的一种或多种。
  4. 根据权利要求1-3中任一所述的应用，其特征在于，所述免疫检查点抑制剂包括PD-1、PD-L1、PD-L2、CTLA-4、LAG-3、TIM-3、VISTA、A2aR抗体中的至少一种；优选地，免疫检查点抑制剂为PD-1抗体和/或PD-L1抗体。
    优选地，所述PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
    优选地，所述PD-L1抗体包括阿特朱单抗(Atezolizumab)、阿维鲁单抗(Avelumab)、度伐鲁单抗(Durvalumab)及其他能够与PD-L1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
  5. 根据权利要求1-4中任一所述的应用，其特征在于，所述产品为食品或药品。
    优选地，所述食品包括奶粉、干酪、凝乳、酸奶酪、冰激凌或发酵谷类食品。所述食品还可以是动物食品，比如饲料等。
    优选地，所述药品的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂。所述药品包括人用药或动物用药。
    优选地，脆弱拟杆菌与PD-1抗体和/或PD-L1抗体同时给药，或，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体分别给药。
    优选地，脆弱拟杆菌两性离子荚膜多糖采用口服或灌肠方式给药。
  6. 一种用于防治生殖泌尿系统肿瘤的组合物，其特征在于，所述组合物同时包括脆弱拟杆菌两性离子荚膜多糖和免疫检查点抑制剂。
  7. 根据权利要求6所述的组合物，其特征在于，所述脆弱拟杆菌两性离子荚膜多糖所述两性离子荚膜多糖提取自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
    优选，所述两性离子荚膜多糖含有荚膜多糖A。其中，所述荚膜多糖A的结构如下所示：
    Figure PCTCN2022120025-appb-100002
    优选地，所述荚膜多糖A的重均分子量为80-90KD，Mw分布于70-100KD的部分占总量的70-80％，重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
    更优选地，所述荚膜多糖A的含量超过95wt％。
    优选地，所述两性离子荚膜多糖的制备方法包括以下步骤：
    (1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物，即得脆弱拟杆菌菌泥；取菌泥，加入菌泥质量3～10倍的纯化水使菌体重悬，用酸溶液调节其pH至2.0～4.5，50～120℃提取0.5～3.0h， 冷却至室温，常温离心，取上清，得到粗糖溶液；
    (2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质，至电导率稳定，收集回流液；
    (3)回流液中加入等体积40mmol/L Tris-HCl转盐；离子交换柱层析，梯度洗脱，分段收集，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干，得到脆弱拟杆菌提取物。
    优选地，步骤(1)中所述离心为11000～13000g离心8～12min。
    优选地，步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中，无机酸可以是盐酸、硫酸、磷酸等；有机酸可以是乙酸、柠檬酸等。
    优选地，步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围。
    优选地，步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm，层析时的流速15～25mL/min，pH5.0～9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)；所述超滤膜的分子量为10KD。
  8. 根据权利要求6或7所述的组合物，其特征在于，所述生殖泌尿系统肿瘤包括女性胸部和生殖器官肿瘤、男性生殖器官肿瘤以及泌尿器官肿瘤。优选地，选自乳腺癌、宫颈癌、子宫体癌、卵巢癌、前列腺癌、肾癌、膀胱癌、睾丸癌中的一种或多种。
  9. 根据权利要求6-8中任一所述的组合物，其特征在于，所述免疫检查点抑制剂包括PD-1、PD-L1、PD-L2、CTLA-4、LAG-3、TIM-3、VISTA、A2aR抗体中的至少一种；优选的，免疫检查点抑制剂为PD-1抗体和/或PD-L1抗体。
    优选地，所述PD-1抗体包括纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)、卡瑞利珠单抗(Camrelizumab)及其他能够与PD-1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
    优选地，所述PD-L1抗体包括阿特朱单抗(Atezolizumab)、阿维鲁单抗(Avelumab)、度伐鲁单抗(Durvalumab)及其他能够与PD-L1结合，阻断PD-1/PD-L1信号通路，上调T细胞活化，激活内源性抗肿瘤免疫反应的物质。
  10. 根据权利要求6-9中任一所述的组合物，其特征在于，所述组合物为药物。
    优选地，所述药物的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂。所述药品包括人用药或动物用药。
    优选地，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体同时给药，或，脆弱拟杆菌两性离子荚膜多糖与PD-1抗体和/或PD-L1抗体分别给药。
    优选地，所述药物采用口服或灌肠方式给药。所述药物给药周期可为间歇给药、周期性给药、持续给药或长期给药。
PCT/CN2022/120025 2022-01-12 2022-09-20 脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂联合用药治疗生殖泌尿系统肿瘤的应用 WO2023134207A1 (zh)

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