WO2023134204A1 - 脆弱拟杆菌荚膜多糖a与pd-1及pd-l1抗体联合治疗呼吸系统肿瘤的应用 - Google Patents

脆弱拟杆菌荚膜多糖a与pd-1及pd-l1抗体联合治疗呼吸系统肿瘤的应用 Download PDF

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WO2023134204A1
WO2023134204A1 PCT/CN2022/120022 CN2022120022W WO2023134204A1 WO 2023134204 A1 WO2023134204 A1 WO 2023134204A1 CN 2022120022 W CN2022120022 W CN 2022120022W WO 2023134204 A1 WO2023134204 A1 WO 2023134204A1
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capsular polysaccharide
antibody
zwitterionic
bacteroides fragilis
acid
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PCT/CN2022/120022
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French (fr)
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邝高波
李平
刘洋洋
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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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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 invention relates to the field of biomedicine, in particular, the present invention relates to the application of a combination of Bacteroides fragilis zwitterionic capsular polysaccharide and PD-1 antibody/PD-L1 antibody in the treatment of respiratory system tumors.
  • Respiratory system neoplasm refers to any disease characterized by anatomically located malignant cells in the respiratory system, including lung cancer, nasopharyngeal cancer, and laryngeal cancer.
  • Clinically common types include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and head and neck squamous cell carcinoma (HNSCC). Pharyngeal cancer and laryngeal cancer, etc.
  • HNSCC Head and neck squamous cell carcinoma
  • HNSCC is the main pathological type of malignant tumors of the head and neck, and its incidence ranks sixth among common malignant tumors, accounting for about 90% of all head and neck tumors. cells, including the oral cavity, oropharynx, hypopharynx, and larynx.
  • HNSCC is a highly immunocompromised tumor with an annual death toll of approximately 315,000 worldwide and a 5-year survival rate of less than 40%.
  • the lesion site of HNSCC is relatively hidden and lacks specific clinical manifestations. It is often difficult to find in the early stage and prone to cervical lymph node metastasis. More than 70% of the patients are already in the middle and late stage when they are diagnosed. For patients with late metastasis or recurrence, most of them are currently Comprehensive treatment including surgery, chemotherapy, and radiation is used.
  • Lung cancer is the malignant tumor with the highest morbidity and mortality among all cancers, accounting for about 18.4% of total cancer deaths.
  • the occurrence of lung cancer is related to environmental factors and genetic factors. Smoking is the main risk factor for lung cancer, but air pollution Factors such as occupational exposure and genetics also have an impact.
  • IARC International Agency for Research on Cancer
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • the 5-year overall survival rate is about 16%.
  • NSCLC is mainly treated with chemotherapy, immunotherapy, and anti-angiogenic therapy. Whether it is first-line therapy or maintenance therapy, clinical trials have confirmed that combined immunotherapy on the basis of chemotherapy and anti-angiogenic therapy can benefit patients.
  • Immune checkpoint inhibitors are monoclonal antibodies that mainly target immune checkpoints in the body. Eliminating tumor cells to prolong the survival of patients is a popular treatment direction for SCLC and NSCLC.
  • PD-1/PD-L1 antibodies PD-1, Programmed Death 1, programmed death receptor; PD-L1, Programmed Cell Death-Ligand 1, programmed death receptor-ligand 1
  • CTLA-4 Cytotoxic T-Lymphocyte-Associated Antigen 4, cytotoxic T-lymphocyte-associated antigen 4
  • 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
  • immune checkpoint blockade therapy brings durable tumor suppression in clinical application, it is only effective for some patients. How to improve the response rate of immune checkpoint antibody drugs is the main problem currently facing. Current medical research believes that continuous treatment of immune checkpoint blockade can bring better therapeutic effect. However, in patients with limited immune checkpoint response, strong side effects (such as severe gastrointestinal adverse reactions, severe skin itching, etc.) , liver injury, pneumonia, renal impairment, etc.) make these patients have to suspend immunotherapy, so that they cannot obtain the expected clinical benefit.
  • the object of the present invention is to provide a combination of Bacteroides fragilis zwitterionic capsular polysaccharide and PD-1 antibody and/or PD-L1 antibody in the prevention and treatment of respiratory system tumors .
  • the present invention unexpectedly finds that the zwitterionic capsular polysaccharide of Bacteroides fragilis, especially the zwitterionic capsular polysaccharide extracted from Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685, and PD-1 antibody/PD-L1
  • the combination of antibodies can regulate the proportion of immune cells and the level of immune factors, enhance the body's anti-tumor immune response, and effectively prevent and treat respiratory system tumors.
  • the present invention provides a combination of Bacteroides fragilis zwitterionic capsular polysaccharide and an immune checkpoint inhibitor for the preparation of a drug for preventing and/or treating respiratory system tumors.
  • the B. fragilis zwitterionic capsular polysaccharide is extracted from B. fragilis ZY-312 with deposit number CGMCC No. 10685.
  • the immune checkpoint inhibitors include PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody, A2aR antibody One or more of them, preferably PD-1 antibody and/or PD-L1 antibody.
  • 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 respiratory system tumor includes non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma.
  • the head and neck squamous cell carcinoma includes nasopharyngeal carcinoma and laryngeal carcinoma.
  • 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 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.
  • the present invention provides a drug, wherein the drug simultaneously includes Bacteroides fragilis zwitterionic capsular polysaccharide and immune checkpoint inhibitors, for example, simultaneously includes Bacteroides fragilis zwitterionic capsular polysaccharide and PD-1 and/or or PD-L1 antibody.
  • the B. fragilis zwitterionic capsular polysaccharide is extracted from B. fragilis ZY-312 with the deposit number of 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, and the part of Mw distributed in 70-100KD accounts for 70-80% of the total amount, and the weight average molecular weight/number average molecular weight (Mw/Mn ) ratio is 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 velocity 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 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.
  • the medicament is used for preventing and/or treating tumors of the respiratory system.
  • the respiratory system tumors include non-small cell lung cancer, small cell lung cancer and head and neck squamous cell carcinoma.
  • the head and neck squamous cell carcinoma includes nasopharyngeal carcinoma and laryngeal carcinoma.
  • the present invention also provides a method for preventing and/or treating respiratory system tumors, comprising administering a therapeutically effective amount of the above drug to a patient.
  • prevention includes prevention and/or treatment.
  • the present invention unexpectedly finds that Bacteroides fragilis, especially Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685 and its zwitterionic capsular polysaccharide, especially capsular polysaccharide A (PSA), and PD-1 antibody
  • PSA capsular polysaccharide A
  • the combination of /PD-L1 antibody can regulate the proportion of immune cells and the level of immune factors, enhance the body's anti-tumor immune response, effectively prevent the occurrence, development, recurrence and metastasis of respiratory tumors, and improve the quality of life of patients.
  • 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.
  • the raw materials and reagents used in the following examples are commercially available, all cells were purchased from ATCC; all cell culture materials and trypsin were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Ltd; or may 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;
  • step (4) Weigh 30 mg of the Bacteroides fragilis extract described in step (4), dissolve it in 0.5 mL of D 2 O, and add 1 ⁇ L of acetone ( 1 H, 2.22; 13 C, 30.89) for calibration. Using a 500MHz Bruker NMR spectrometer to analyze 1H, 13C, COZY, HSQC, and HMBC spectra (see Figures 1A-1E), it was confirmed that the Bacteroides fragilis extract collected in step (3) was capsular polysaccharide A (PSA), which binds lipids The content is lower than 0.02%, the protein residue is lower than 1%, and the nucleic acid residue is lower than 0.05%.
  • PSA capsular polysaccharide A
  • 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 .
  • the prepared PSA was named PSA-ZY-312.
  • C57BL/6 mice were selected for experiments in this example, and the method of transplanted tumor model was used to establish small Mouse non-small cell lung cancer model.
  • the tumor cells of the passaged Lewis lung cancer tumor-bearing mice dilute the cell suspension with a concentration of 1 ⁇ 107 cells/mL with normal saline, and inoculate 0.2 mL subcutaneously in the right axilla of the mice to establish Lewis lung cancer tumor-bearing mice. mouse model.
  • the model mice were randomly divided into model group, PD-1 antibody group, PSA-ZY-312 group, low-dose PSA-ZY-312+PD-1 antibody group, medium-dose PSA-ZY-312+PD -1 antibody group and high-dose PSA-ZY-312+PD-1 antibody group, 10 rats in each group.
  • 10 uninfected mice of the same species were used as the blank group.
  • the day of grouping was taken as Day0 (D0), and the administration was carried out according to Table 1, for a total of 21 days. On D21, all mice were euthanized, and mouse serum, tumor, feces, right cervical lymph and right axillary lymph were collected. All tumors were weighed and photographed. Tumors were divided into three parts, one part was frozen for cytokine detection, one part was fixed in formalin, and one part was sent in vitro for flow cytometry analysis.
  • Tumor volume and tumor growth inhibition rate Tumor diameter was measured twice a week with a vernier caliper.
  • Intratumoral T cell subsets Flow cytometry analysis of the ratio of CD4 + T cells and CD8 + T cells in tumors.
  • the tumor mass volume of tumor-bearing mice in each group is shown in Table 2.
  • the model group had obvious tumor mass, and the model was successfully established; compared with the model group, each administration group could reduce the tumor volume.
  • the combined administration of PD-1 antibody and PSA-ZY-312 can significantly reduce the volume of tumor mass (p ⁇ 0.05 or 0.01).
  • Within the PSA group there was a certain dose dependence. This shows that the combination of Bacteroides fragilis capsular polysaccharide A and PD-1 antibody can effectively inhibit the growth of Lewis lung cancer in mice.
  • CD8 + T cells can kill target cells expressing antigens, and they are important effector cells in antiviral infection, acute allograft rejection and tumor cell killing.
  • CD4 + T cells are an important immune cell in the human immune system, mainly differentiated from helper T (Th) cells, can bind to the non-polypeptide region of MHC class II molecules, and participate in T cell antigen receptor (TCR) recognition of antigens signal transduction.
  • Th helper T
  • TCR T cell antigen receptor
  • CTL cytotoxic T lymphocytes
  • each administration group up-regulated the proportion of CD3 + CD4 + T cells in the tumor to CD3 + T cells to varying degrees.
  • the up-regulation range of the Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody group was greater than that of the single administration group; there was a certain dose difference within the PSA group, and the medium and high doses of PSA combined with PD-1 antibody group and the model groups have significant differences.
  • each administration group up-regulated the proportion of CD3 + CD8 + T cells in the tumor to CD3 + T cells.
  • the up-regulation range of the Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody group was greater than that of the single administration group; there was a certain dose-dependence in the PSA group, and the high-dose PSA combined with PD-1 antibody group had a significant difference with the model group. Significant difference.
  • capsular polysaccharide A of Bacteroides fragilis ZY-312 combined with PD-1 antibody can up-regulate the proportion of CD3 + CD4 + and CD3 + CD8 + T cells in the tumor.
  • Bacteroides fragilis ZY-312 capsular polysaccharide A combined with PD-1 antibody can regulate the proportion of tumor-infiltrating T cells, enhance the body's anti-tumor immune response, and effectively prevent and treat Lewis lung cancer in mice.
  • This preventive effect has a certain dose-dependent relationship with capsular polysaccharide A.
  • this example selects Trp53 loxP/loxP ; Rb1 loxP/loxP ; Rb12 loxP/loxP mice for experiments , and established a small cell lung cancer model in mice using the method of xenograft tumor model.
  • the model mice were randomly divided into model group, PD-L1 antibody group, PSA-ZY-312 group, low-dose PSA-ZY-312+PD-L1 antibody group, medium-dose PSA-ZY-312+PD group -L1 antibody group and high-dose PSA-ZY-312+PD-L1 antibody group, 10 rats in each group.
  • 10 uninfected mice of the same species were used as the blank group.
  • the day of grouping was taken as Day0 (D0), and the administration was carried out according to Table 4, for a total of 21 days. On D21, all mice were euthanized, and mouse serum, tumor, feces, right cervical lymph and right axillary lymph were collected. All tumors were weighed and photographed. Tumors were divided into three parts, one part was frozen for cytokine detection, one part was fixed in formalin, and one part was sent in vitro for flow cytometry analysis.
  • Tumor volume and tumor growth inhibition rate Tumor diameter was measured twice a week with a vernier caliper.
  • T cell subsets The proportion of CD3 + T cells and CD8 + T cells in the tumor was analyzed by flow cytometry.
  • the p value between the two groups is calculated according to the unpaired t-test (one-tailed) method
  • the tumor volumes of the tumor-bearing mice in each group during the administration period are shown in Table 5.
  • the model group formed a significant tumor, and the model was successfully established.
  • each administration group could reduce the tumor volume, and the combined administration of PD-L1 antibody and PSA-ZY-312 could significantly reduce the tumor volume (p ⁇ 0.05 or 0.01).
  • Within the PSA group there was a certain dose dependence. This shows that the combination of Bacteroides fragilis capsular polysaccharide A and PD-L1 antibody can effectively inhibit the growth of SCLC lung cancer in mice. This inhibitory effect has a certain dose-dependent relationship with capsular polysaccharide A.
  • Example 4 Application of Bacteroides fragilis zwitterionic capsular polysaccharide combined with PD-1 antibody in the treatment of Hep-2 laryngeal carcinoma xenografts in mice
  • this example selected 6-week-old male BALB/c pure nude mice for the experiment, and used Hep-2
  • the method of xenotransplantation nude mouse model established a mouse laryngeal cancer xenograft tumor model.
  • the day of grouping was taken as Day0 (D0), and the administration was carried out according to Table 7 for a total of 27 days.
  • D0 Day0
  • mice On D27, all mice were euthanized, and mouse serum, tumor, feces, right cervical lymph and right axillary lymph were collected. All tumors were weighed and photographed. Tumors were divided into three parts, one part was frozen for cytokine detection, one part was fixed in formalin, and one part was sent in vitro for flow cytometry analysis.
  • Tumor volume and tumor growth inhibition rate Tumor diameter was measured twice a week with a vernier caliper.
  • Cytokine detection Luminex technology detects the content of IL-2 and IL-10 in mouse tumors.
  • the tumor volumes of tumor-bearing mice in each group are shown in Table 8.
  • the model group formed a significant tumor, and the model was successfully established; compared with the model group, the tumor volume of each administration group was down-regulated , PSA-ZY-312 combined with PD-1 antibody had significant difference among groups (p ⁇ 0.05 or 0.01). It can be seen that the combination of Bacteroides fragilis ZY-312 capsular polysaccharide A and PD-1 antibody can inhibit the growth of Hep-2 laryngeal cancer cells in mice, and this inhibitory effect has a certain dose-dependent relationship with capsular polysaccharide A.
  • IL-2 is a known anti-tumor immune cytokine.
  • IL-10 is widely accepted as an immunosuppressive cytokine and is thought to promote tumor immune evasion by reducing antitumor immune responses in the tumor microenvironment.
  • each administration group significantly down-regulated the level of intratumoral IL-10, and the combination of medium and high doses of Bacteroides fragilis ZY-312 capsular polysaccharide A and PD-1 antibody had Very significant difference.
  • each administration group significantly up-regulated the level of intratumoral IL-2, and there was a very significant difference when the high dose of Bacteroides fragilis ZY-312 capsular polysaccharide A was combined with PD-1 antibody.

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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PD-1抗体和/或PD-L1抗体联合用药在防治呼吸系统肿瘤中的应用。本发明出人预料地发现，脆弱拟杆菌两性离子荚膜多糖，特别是提取自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312的两性离子荚膜多糖与PD-1抗体/PD-L1抗体联用能够调节免疫细胞比例和免疫因子水平，增强机体抗肿瘤免疫反应，有效防治呼吸系统肿瘤。

Description

脆弱拟杆菌荚膜多糖A与PD-1及PD-L1抗体联合治疗呼吸系统肿瘤的应用
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202210034086.4，发明名称为“脆弱拟杆菌荚膜多糖A与PD-1及PD-L1抗体联合治疗呼吸系统肿瘤的应用”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。
技术领域
本发明涉及生物医药领域，具体而言，本发明涉及一种脆弱拟杆菌两性离子荚膜多糖与PD-1抗体/PD-L1抗体联合用药治疗呼吸系统肿瘤中的应用。
背景技术
呼吸系统肿瘤是指任何特征为在呼吸系统中解剖学定位为恶性细胞的疾病，包括肺癌、鼻咽癌、喉癌等。临床上常见类型包括非小细胞肺癌(non-small cell lung cancer,NSCLC)、小细胞肺癌(small cell lung cancer,SCLC)、头颈部鳞癌(head and neck squamous cell carcinoma,HNSCC)中的鼻咽癌与喉癌等。
头颈部鳞癌(HNSCC)是头颈部恶性肿瘤的主要病理类型，发病率居于常见恶性肿瘤的第6位，约占所有头颈部肿瘤的90％，起源于上呼吸消化道的黏膜上皮细胞，包括口腔、口咽部、喉咽部和喉部。HNSCC是一种具有高度免疫缺陷的肿瘤，全球每年死亡人数约31.5万，5年生存率不足40％。HNSCC发生病变的部位较为隐蔽，且缺乏特异性临床表现，早期常难以发现，并易发生颈部淋巴结转移，七成以上患者确诊时已经处于中晚期阶段，对于晚期转移或复发的患者，目前多采用包括手术、化疗和放疗在内的综合性治疗。
肺癌是所有癌症中发病率与病死率最高的恶性肿瘤，约占总癌症死亡的18.4％，肺癌的发生与环境因素和遗传因素都有关系，吸烟是导致肺癌发生的主要危险因素，但空气污染、职业原因造成的暴露和遗传等因素也有影响。2020年世界卫生组织国际癌症研究机构(IARC)发布的最新癌症负担数据显示全球新发癌症病例约2000万例。
肺癌分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)。其中，SCLC约占支气管源性肺癌的15％-20％，其特点为确诊时多已转移，治疗后易复发，预后差，约67％的小细胞肺癌患者确诊时有明显的肺外转移病灶，而仅有33％的局限期患者病变局限于胸腔 的单一放射野内。而NSCLC是世界上死亡率最高的恶性肿瘤，约占总体肺癌的85％。NSCLC发现时多为中晚期，传统治疗手段效果不佳，5年总体生存率约为16％。NSCLC以化疗、免疫治疗、抗血管生成治疗为主要治疗手段，无论是一线治疗还是维持治疗，临床试验均证实在化疗、抗血管生成治疗的基础上联合免疫治疗可使患者受益。免疫检查点抑制剂(Immune checkpoint inhibitor,ICI)是主要针对机体免疫检查点的单抗，其能够有效阻断T淋巴细胞负性共刺激信号途径，进而恢复机体的抗肿瘤作用，促使T淋巴细胞清除肿瘤细胞，以延长患者的生存期，是SCLC和NSCLC的热门治疗方向。
近年来，肿瘤免疫治疗特别是免疫检查点抑制剂在临床试验中取得巨大成功，尤其是PD-1/PD-L1抗体(PD-1，Programmed Death 1,程序性死亡受体；PD-L1，Programmed Cell Death-Ligand 1,程序性死亡受体-配体1)和CTLA-4(Cytotoxic T-Lymphocyte-Associated Antigen 4，细胞毒性T淋巴细胞相关抗原4)抗体在黑色素瘤、肺癌、膀胱癌、CRC、霍奇金淋巴瘤等的治疗中取得革命性突破，被FDA(Food and Drug Administration，美国食品药品监督管理局)批准用于临床治疗，如Opdivo、Keytruda和Tecentriq通过阻断细胞表面的PD-L1发挥作用。
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)。
虽然免疫检查点阻断治疗在临床应用中带来持久的肿瘤抑制作用，但仅对部分患者有效，如何提高免疫检查点抗体药物应答率是目前面临的主要难题。目前医学研究认为，免疫检查点阻断治疗的持续治疗能带来更好的治疗效果，然而在有限的免疫检查点应答患者中，强烈的副反应(如严重的消化道不良反应、皮肤剧烈瘙痒、肝损伤、肺炎、肾功能损害等)使得这些患者不得不中止免疫治疗，从而无法获得预期的临床受益。
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PD-1抗体和/或PD-L1抗体联合用药在防治呼吸系统肿瘤中的应用。本发明出人预料地发现，脆弱拟杆菌两性离子荚膜多糖，特别是提取自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312的两性离子荚膜多糖与PD-1抗体/PD-L1抗体联用能够调节免疫细胞比例和免疫因子水平，增强机体抗肿瘤免疫反应，有效防治呼吸系统肿瘤。
为此，本发明采用的技术方案如下：
第一方面，本发明提供了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制备预防和/或治疗呼吸系统肿瘤药物的应用。
在其中一些实施例中，所述脆弱拟杆菌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，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体、A2aR抗体中的一种或多种，优选为PD-1抗体和/或PD-L1抗体。
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 PCTCN2022120022-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，得到脆弱拟杆菌两性离子荚膜多糖。
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，所述呼吸系统肿瘤包括非小细胞肺癌、小细胞肺癌、头颈部鳞癌。
根据本发明的实施方案，所述头颈部鳞癌包括鼻咽癌和喉癌。
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。
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，本发明提供一种药物，其中，所述药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，例如同时包括脆弱拟杆菌两性离子荚膜多糖及PD-1和/或PD-L1抗体。
在其中一些实施例中，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 PCTCN2022120022-appb-000002
根据本发明的实施方案，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，得到脆弱拟杆菌两性离 子荚膜多糖。
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。
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。
根据本发明的实施方案，所述药物用于预防和/或治疗呼吸系统肿瘤。其中，所述呼吸系统肿瘤包括非小细胞肺癌、小细胞肺癌和头颈部鳞癌。
根据本发明的实施方案，所述头颈部鳞癌包括鼻咽癌和喉癌。
本发明还提供一种预防和/或治疗呼吸系统肿瘤的方法，包括向患者施用治疗有效量的上述药物。
其中，所述“防治”包括预防和/或治疗。
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，尤其是荚膜多糖A(PSA)，与PD-1抗体/PD-L1抗体联用能够调节免疫细胞比例和免疫因子水平，增强机体抗肿瘤免疫反应，有效预防呼吸系统肿瘤的发生发展及其复发转移，提高患者的生活质量。
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。
具体实施方式
为了便于理解本发明，下面将对本发明进行更详细的描述。但是，应当理解，本发明可以许多不同的形式来实现，并不限于本文所描述的实施方式或实施例。相反地，提供这些实施方式或实施例的目的是使对本发明的公开内容的理解更加透彻全面。下文中，仅仅是为说明，只在实施例中描述了其中一少部分，然而不应将其理解为对本发明的限制。
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；所有细胞培养材料及胰酶购自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ZY-312两性离子荚膜多糖的制备
(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(PSA)，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。
制得的PSA命名为PSA-ZY-312。
实施例2脆弱拟杆菌两性离子荚膜多糖联合PD-1抗体在治疗小鼠Lewis肺癌中的应用
一.实验设计及流程
为了验证实施例1提供的脆弱拟杆菌两性离子荚膜多糖联合PD-1抗体对非小细胞肺癌的治疗作用，本实施例选用C57BL/6小鼠进行实验，并采用移植瘤模型的方法建立小鼠非小细胞肺癌模型。取传代Lewis肺癌荷瘤小鼠的肿瘤细胞，以生理盐水稀释成瘤细胞浓度1×10 7个/mL的细胞悬液，取0.2mL接种于小鼠右侧腋窝皮下，建立Lewis肺癌荷瘤小鼠模型。建模成功后将模型小鼠随机分为模型组、PD-1抗体组、PSA-ZY-312 组、低剂量PSA-ZY-312+PD-1抗体组、中剂量PSA-ZY-312+PD-1抗体组和高剂量PSA-ZY-312+PD-1抗体组，每组10只。另以10只未接瘤同种小鼠作为空白组。以分组当日作为Day0(D0)，按表1进行给药，共给药21天。D21，所有小鼠安乐死，采集小鼠血清、肿瘤、粪便、右侧颈部淋巴以及右侧腋窝淋巴。所有肿瘤称重和拍照。肿瘤分为三份，一份冻存用于细胞因子检测，一份于福尔马林中固定，一份送于体外用于流式分析。
表1实验动物分组及给药方案
Figure PCTCN2022120022-appb-000003
二.肿瘤测量和实验指标
肿瘤体积与肿瘤生长抑制率：每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100％。
肿瘤内T细胞亚群：流式细胞术分析肿瘤内CD4 +T细胞和CD8 +T细胞的比例。
三.统计分析
基于实验结束时获得的数据进行统计学分析评估组间差异。两组间比较用T-test(one tailed)进行统计分析。使用SPSS统计软件25.0进行统计学分析，p<0.05认为有显著性 差异。
四.实验结果
1.肿瘤体积与肿瘤生长抑制率
表2.受试物对小鼠Lewis肺癌模型的抑瘤药效评价
(基于分组给药后第21天肿瘤体积计算得出)
Figure PCTCN2022120022-appb-000004
注：
a.平均值±SEM；
b.TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％；
c.根据肿瘤体积计算，两组间p值按照unpaired t-test(one-tailed)方法计算。
给药期间各组荷瘤小鼠的瘤块体积如表2所示，与空白组相比，模型组出现明显瘤块，造模成功；与模型组相比，各给药组均可降低肿瘤体积，而PD-1抗体与PSA-ZY-312联合给药可显著降低瘤块体积(p＜0.05或0.01)。在PSA组内，存在一定的剂量依赖性。这说明脆弱拟杆菌荚膜多糖A与PD-1抗体联用能够有效抑制小鼠Lewis肺癌的生长。
2.T细胞亚群
表3.各组小鼠T细胞亚群比例(mean±SD)
Figure PCTCN2022120022-appb-000005
Figure PCTCN2022120022-appb-000006
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
CD8 +T细胞(TC或CTL细胞)能杀伤表达抗原的靶细胞，它在抗毒感染、急性同种异型移植物排斥和对肿瘤细胞的杀伤作用是重要的效应细胞。CD4 +T细胞是人体免疫系统中的一种重要免疫细胞，主要由辅助T(Th)细胞分化而来，可与MHCⅡ类分子的非多肽区结合，参与T细胞抗原受体(TCR)识别抗原的信号转导。研究发现，在肿瘤免疫中，CD4 +T细胞启动后可以通过多种机制激活CD8 +T细胞，使其分化为细胞毒性T淋巴细胞(CTL)，同时维持并加强CTL的抗肿瘤反应。
如上表所示，与模型组相比，各给药组均不同程度地上调了肿瘤内CD3 +CD4 +T细胞占CD3 +T细胞的比例。脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体组的上调幅度大于单独给药组；PSA组内具有一定的剂量差异性，中、高剂量PSA联用PD-1抗体组与模型组具有显著性差异。
与模型组相比，各给药组均上调了肿瘤内CD3 +CD8 +T细胞占CD3 +T细胞的比例。脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体组的上调幅度大于单独给药组；PSA组内具有一定的剂量依赖性，高剂量PSA联用PD-1抗体组与模型组具有显著性差异。
可见，脆弱拟杆菌ZY-312的荚膜多糖A联用PD-1抗体能够上调肿瘤内CD3 +CD4 +和CD3 +CD8 +T细胞的比例。
由以上实施例的结果可见，脆弱拟杆菌ZY-312荚膜多糖A联用PD-1抗体能够调节肿瘤浸润T细胞的比例，增强机体抗肿瘤免疫反应，有效防治小鼠Lewis肺癌。这种防治作用与荚膜多糖A具有一定的剂量依赖关系。
实施例3荚膜多糖A联合PD-L1抗体在治疗小细胞肺癌中的应用
一.实验设计
为了验证实施例1提供的脆弱拟杆菌两性离子荚膜多糖联合PD-1抗体对小细胞肺癌的治疗作用，本实施例选用Trp53 loxP/loxP；Rb1 loxP/loxP；Rb12 loxP/loxP小鼠进行实验，并采用移植瘤模型的方法建立小鼠小细胞肺癌模型。以Ad-CreCaPi(Ad-CreCaPi：滴度为10 7PfU的Ad-Cre 2μL加入到246.8μL的MEM中，再加入1.2μL的2mol/L CaCl 2，购自 VECTOR BIOLABS)的共沉淀物诱导Trp53 loxP/loxP；Rb1 loxP/loxP；Rb12 loxP/loxP小鼠得到的小细胞肺癌细胞(SCLC细胞)，以Matrigel胶(基质胶：无血清培养基＝1:1，基质胶购自Solarbio)调整SCLC细胞浓度为1×10 7个/mL，取50μL接种于Trp53 loxP/loxP；Rb1 loxP/loxP；Rb12 loxP/loxP小鼠右侧肋部皮下，建立小细胞肺癌荷瘤小鼠模型。建模成功后将模型小鼠随机分为模型组、PD-L1抗体组、PSA-ZY-312组、低剂量PSA-ZY-312+PD-L1抗体组、中剂量PSA-ZY-312+PD-L1抗体组和高剂量PSA-ZY-312+PD-L1抗体组，每组10只。另以10只未接瘤同种小鼠作为空白组。以分组当日作为Day0(D0)，按表4进行给药，共给药21天。D21，所有小鼠安乐死，采集小鼠血清、肿瘤、粪便、右侧颈部淋巴以及右侧腋窝淋巴。所有肿瘤称重和拍照。肿瘤分为三份，一份冻存用于细胞因子检测，一份于福尔马林中固定，一份送于体外用于流式分析。
表4实验动物分组及给药方案
Figure PCTCN2022120022-appb-000007
二.肿瘤测量和实验指标
肿瘤体积与肿瘤生长抑制率：每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100％。
T细胞亚群：流式细胞术分析肿瘤内CD3 +T细胞、CD8 +T细胞的比例。
三.统计分析
基于实验结束时获得的数据进行统计学分析评估组间差异。两组间比较用T-test(two-tailed)进行统计分析。使用SPSS 25.0软件进行所有数据分析，p<0.05认为有显著性差异。
四.实验结果
1.肿瘤体积与肿瘤生长抑制率
表5.受试物对小鼠SCLC模型的抑瘤药效评价
(基于分组给药后第21天肿瘤体积计算得出)
Figure PCTCN2022120022-appb-000008
注：
a.平均值±SEM；
b.TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％；
c.根据肿瘤体积计算，两组间p值按照unpaired t-test(one-tailed)方法计算；
组别中的“ab”为抗体的简写。
给药期间各组荷瘤小鼠的瘤块体积如表5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，各给药组均可降低肿瘤体积，而PD-L1抗体与PSA-ZY-312联合给药可显著降低瘤块体积(p＜0.05或0.01)。在PSA组内，存在一定的剂量依赖性。这说明脆弱拟杆菌荚膜多糖A与PD-L1抗体联用能够有效抑制小鼠SCLC肺癌的生长。这种抑制作用与荚膜多糖A具有一定的剂量依赖关系。
2.T细胞亚群
表6各组小鼠CD3 +T细胞、CD8 +T细胞比例(mean±SD)
Figure PCTCN2022120022-appb-000009
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
如上表所示，与模型组相比，PD-L1抗体、脆弱拟杆菌ZY-312荚膜多糖A单用均未能明显提升小鼠肿瘤内浸润CD3 +T细胞、CD8 +T细胞的比例；PD-L1抗体与脆弱拟杆菌ZY-312荚膜多糖A联用时，CD3 +T细胞、CD8 +T细胞比例显著上升。PSA组内具有一定剂量依赖性。这说明脆弱拟杆菌ZY-312荚膜多糖A与PD-L1抗体联用能够调节T细胞水平，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关系。
由以上实施例的结果可见，脆弱拟杆菌ZY-312荚膜多糖A与PD-L1抗体联用能够调节肿瘤内T细胞水平，增强机体抗肿瘤免疫反应，有效防治小鼠SCLC肺癌。这种防治作用与荚膜多糖A具有一定的剂量依赖关系。
实施例4脆弱拟杆菌两性离子荚膜多糖联合PD-1抗体在治疗小鼠Hep-2喉癌移植瘤中的应用
一.实验设计
为了验证实施例1提供的脆弱拟杆菌两性离子荚膜多糖联合PD-1抗体对喉癌的治疗作用，本实施例选用6周龄雄性BALB/c纯系裸鼠进行实验，并采用Hep-2异种移植裸鼠模型的方法建立小鼠喉癌移植瘤模型。将对数生长期Hep-2喉癌细胞制成无血清细胞悬液，接种于裸鼠背部，每只接种100μL，含有2×10 6个细胞，待瘤体直径约50mm 3时，选择肿瘤生长大小均匀的动物，随机分为空白组、模型组、PD-1抗体组、PSA-ZY-312组、低剂量PSA-ZY-312+PD-1抗体组、中剂量PSA-ZY-312+PD-1抗体组和高剂量PSA-ZY-312+PD-1抗体组，每组10只。另设置10只未接瘤同种小鼠作为空白组。以分组当日作为Day0(D0)，按表7进行给药，共给药27天。D27，所有小鼠安乐死，采集小鼠血清、肿瘤、粪便、右侧颈部淋巴以及右侧腋窝淋巴。所有肿瘤称重和拍照。肿瘤分为三份，一份冻存用于细胞因子检测，一份于福尔马林中固定，一份送于体外用于流式分析。
表7实验动物分组及给药方案
Figure PCTCN2022120022-appb-000010
二.肿瘤测量和实验指标
肿瘤体积与肿瘤生长抑制率：每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(同型对照组治疗结束时平均瘤体积－同型对照组开始治疗时平均瘤体积)]×100。
细胞因子检测：Luminex技术检测小鼠肿瘤中IL-2和IL-10的含量。
三.统计分析
基于实验结束时获得的数据进行统计学分析评估组间差异。两组间比较用T-test(two-tailed)进行统计分析。使用SPSS 25.0软件进行所有数据分析，p<0.05认为有显著性差异。
四.实验结果
1.肿瘤体积与肿瘤生长抑制率
表8.受试物对小鼠喉癌移植瘤模型的抑瘤药效评价
(基于分组给药后第27天肿瘤体积计算得出)
Figure PCTCN2022120022-appb-000011
Figure PCTCN2022120022-appb-000012
注：
a.平均值±SEM；
b.TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％；
c.根据肿瘤体积计算，两组间p值按照unpaired t-test(one-tailed)方法计算。
给药期间各组荷瘤小鼠的瘤块体积如表8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，各给药组均下调了肿瘤体积，PSA-ZY-312联用PD-1抗体各组具有显著性差异(p＜0.05或0.01)。可见脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用可抑制Hep-2喉癌细胞在小鼠体内的生长，这种抑制作用与荚膜多糖A有一定的剂量依赖关系。
2.细胞因子水平
表9.各组小鼠肿瘤内细胞因子水平(mean±SD)
Figure PCTCN2022120022-appb-000013
注：与模型组比较，*表示差异显著p<0.05；**表示差异极显著p<0.01。
IL-2是已知的抗肿瘤免疫细胞因子。IL-10作为一种免疫抑制细胞因子被广泛接受，被认为通过降低肿瘤微环境中的抗肿瘤免疫反应来促进肿瘤免疫逃逸。
如表9所示，与模型组相比，各给药组均显著下调了肿瘤内IL-10的水平，中、高剂量脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用时具有极显著差异。
与模型组相比，各给药组均显著上调了肿瘤内IL-2的水平，脆弱拟杆菌ZY-312荚膜多糖A高剂量与PD-1抗体联用时具有极显著差异。
上述结果说明脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用能够调节肿瘤内免疫因子，增强机体抗肿瘤免疫反应。这种调节作用与荚膜多糖A有一定的剂量依赖关系。
由以上实施例的结果可见，脆弱拟杆菌ZY-312荚膜多糖A与PD-1抗体联用能够调节小鼠喉癌Hep-2移植瘤内的免疫因子水平，抑制肿瘤生长，有效防治小鼠喉癌。这种防治作用与荚膜多糖A具有一定的剂量依赖关系。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。

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协同制备预防和/或治疗呼吸系统肿瘤药物的应用，其特征在于，所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
  2. 根据权利要求1所述的应用，其特征在于，所述免疫检查点抑制剂包括PD-1抗体、PD-L1抗体、PD-L2抗体、CTLA-4抗体、LAG-3抗体、TIM-3抗体、VISTA抗体、A2aR抗体中的一种或多种，优选为PD-1抗体和/或PD-L1抗体。
  3. 根据权利要求1或2所述的应用，其特征在于，所述呼吸系统肿瘤包括头颈部癌、非小细胞肺癌、小细胞肺癌。优选地，所述头颈部癌还包括鼻咽癌、喉癌。
  4. 根据权利要求1-3中任一所述的应用，其特征在于，所述两性离子荚膜多糖含夹膜多糖A。优选地，所述荚膜多糖A的结构如下所示：
    Figure PCTCN2022120022-appb-100001
    更优选地，所述夹膜多糖A的重均分子量为80-90KD；所述两性离子荚膜多糖中夹膜多糖A的含量超过95wt％。
  5. 根据权利要求1-4任一项所述的应用，其特征在于，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，得到脆弱拟杆菌两性离子荚膜多糖。
    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。
  6. 根据权利要求1-5任一项所述的应用，其特征在于，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。
  7. 一种药物，其特征在于，所述药物同时包括脆弱拟杆菌两性离子荚膜多糖与免疫检查点抑制剂；优选地，所述药物同时包括脆弱拟杆菌两性离子荚膜多糖及PD-1和/或PD-L1抗体。优选地，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。
  8. 根据权利要求7所述的药物，其特征在于，所述两性离子荚膜多糖含夹膜多糖A。优选地，所述荚膜多糖A的结构如下所示：
    Figure PCTCN2022120022-appb-100002
    更优选地，所述夹膜多糖A的重均分子量为80-90kD；所述两性离子荚膜多糖中夹膜多糖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，得到脆弱拟杆菌两性离子荚膜多糖。
    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。
  9. 根据权利要求7或8所述的药物，其特征在于，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7-9中任一所述的药物，其特征在于，所述呼吸系统肿瘤包括非小细胞肺癌、小细胞肺癌、头颈部鳞癌。优选地，所述头颈部鳞癌选自鼻咽癌、喉癌。
PCT/CN2022/120022 2022-01-12 2022-09-20 脆弱拟杆菌荚膜多糖a与pd-1及pd-l1抗体联合治疗呼吸系统肿瘤的应用 WO2023134204A1 (zh)

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