WO2023134205A1 - 脆弱拟杆菌与pd-1及pd-l1抗体联合用药治疗呼吸系统肿瘤中的应用 - Google Patents

脆弱拟杆菌与pd-1及pd-l1抗体联合用药治疗呼吸系统肿瘤中的应用 Download PDF

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WO2023134205A1
WO2023134205A1 PCT/CN2022/120023 CN2022120023W WO2023134205A1 WO 2023134205 A1 WO2023134205 A1 WO 2023134205A1 CN 2022120023 W CN2022120023 W CN 2022120023W WO 2023134205 A1 WO2023134205 A1 WO 2023134205A1
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antibody
bacteroides fragilis
tumor
group
fragilis
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French (fr)
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郑丽君
易晓敏
常秀娟
林渝清
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广州知易生物科技有限公司
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    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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 present invention relates to an application technology of Bacteroides fragilis, in particular to the application of a combination of Bacteroides fragilis and PD-1 antibody or 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 among head and neck cancers, etc.
  • Lung cancer is one of the most dangerous and threatening malignant tumors in the world today. Globally, lung cancer is the second most common cancer with the highest mortality rate. In my country, the morbidity and mortality of lung cancer rank first, and among men, lung cancer is the number one cancer killer. Smoking is still the main risk factor for lung cancer, but factors such as air pollution, occupational exposure, and genetics also play a role.
  • Nasopharyngeal carcinoma is the most common malignant tumor of the head and neck. It refers to a malignant tumor arising from the epithelium covering the surface of the nasopharyngeal cavity or the nasopharyngeal crypt. Compared with other cancers, nasopharyngeal carcinoma is not common. However, in my country, the incidence of nasopharyngeal carcinoma ranks eighth among all malignant tumors. The occurrence of nasopharyngeal carcinoma is generally related to the combination of many factors, including heredity, environmental pollution, exposure to toxic chemicals and Epstein-Barr virus infection.
  • Laryngeal cancer refers to tumors whose primary site is in the larynx, and squamous cell carcinoma is the most common.
  • the incidence rate of laryngeal cancer is 6/100,000 people, which is lower than that of lung cancer and gastric cancer.
  • CA cardiovascular disease
  • the 5-year survival rate of laryngeal cancer has dropped from 66% to 63%, which is one of the few tumors with a decline in survival rate.
  • Multiple factors are thought to be involved in the pathogenesis of laryngeal cancer, including smoking, alcohol use, and exposure to certain toxic chemicals.
  • Human papillomavirus (HPV) infection is also considered a possible factor.
  • Most respiratory cancers are detected at an advanced stage. Most of the early lung cancers have no obvious symptoms, and most patients are already in the advanced stage when they present symptoms. Early diagnosis of nasopharyngeal carcinoma is extremely difficult, and patients are likely to miss the best opportunity for treatment due to missed diagnosis or misdiagnosis. Laryngeal cancer is also known as the "silent killer". For patients with respiratory system tumors who have no chance of radical surgery, comprehensive treatment mainly based on systemic drug therapy is currently adopted. Systemic drug therapy mainly includes chemotherapy drugs, molecular targeted drugs and immunotherapy drugs.
  • Immunotherapy drugs are currently the most popular cancer treatment.
  • Tumor immunotherapy mainly includes immune vaccines, immune checkpoint inhibitor therapy, adoptive immune cell therapy, cytokine therapy, etc.
  • immune checkpoint inhibitor therapy has attracted much attention for its remarkable clinical efficacy.
  • 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 and PD-1 antibody and/or PD-L1 antibody in the treatment of respiratory system tumors.
  • Bacteroides fragilis especially Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685, can correct the proportion of immune cells in the body, regulate the level of cancer-related cytokines in the body, improve the anti-tumor immune response of the body, and effectively Inhibits the growth of respiratory tumors.
  • a combination of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of drugs for preventing and/or treating respiratory system tumors is provided.
  • the immune checkpoint inhibitor includes one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, VISTA antibody, A2aR antibody or Multiple, preferably one or more of PD-1 antibodies and/or PD-L1 antibodies, preferably PD-1 and/or PD-L1 antibodies.
  • the Bacteroides fragilis is a live bacterium or an inactivated bacterium; preferably, the inactivated bacterium is an inactivated bacterium with a complete morphology or structure or an inactivated bacterium with an incomplete morphological structure.
  • the Bacteroides fragilis is live Bacteroides fragilis, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, Bacteroides fragilis Lysate, one or more of Bacteroides fragilis liquid culture supernatant.
  • Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.
  • the respiratory system tumor includes one or more of lung cancer and head and neck squamous cell carcinoma.
  • the tumor of the respiratory system includes one or more of small cell lung cancer, non-small cell lung cancer, laryngeal cancer and nasopharyngeal cancer.
  • 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.
  • Bacteroides fragilis is administered with PD-1 antibody and/or PD-L1 antibody at the same time.
  • Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.
  • the Bacteroides fragilis is administered orally or enemaly.
  • the present invention provides a drug, wherein the drug includes both a pharmaceutically effective dose of Bacteroides fragilis and an immune checkpoint inhibitor, for example, the drug simultaneously includes a pharmaceutically effective dose of Bacteroides fragilis and a PD-1 antibody and/or PD-L1 antibody.
  • the pharmaceutically effective dose is 10 6 -10 10 CFU.
  • the Bacteroides fragilis is one or more of live bacteria, inactivated bacteria with complete morphology and structure, or inactivated bacteria with incomplete morphology and structure.
  • the Bacteroides fragilis is live Bacteroides fragilis, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, Bacteroides fragilis Lysate, one or more of Bacteroides fragilis liquid culture supernatant.
  • Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.
  • the respiratory system tumor includes one or more of small cell lung cancer, non-small cell lung cancer, and head and neck squamous cell 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.
  • Bacteroides fragilis is administered with PD-1 antibody and/or PD-L1 antibody at the same time.
  • Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.
  • the Bacteroides fragilis 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, 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-mentioned drug to a patient.
  • the present invention unexpectedly finds that the combined use of Bacteroides fragilis, especially Bacteroides fragilis ZY-312 with the deposit number CGMCC No. Increase the body's cancer-related cytokine levels, improve the body's anti-tumor immune response, and effectively prevent and treat respiratory 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.).
  • Fig. 1 is the colony form diagram of embodiment one Bacteroides fragilis ZY-312 on the blood plate;
  • Fig. 2 is a Gram-stained microscope image of Bacteroides fragilis ZY-312 in Example 1.
  • the experimental methods used in the following examples, if no special instructions, are conventional methods; reagents, materials, etc. used in the following examples, if no special instructions, the raw materials and reagents used in the following examples are commercially available Commercial products, 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 Co., Ltd.; or 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.
  • Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.
  • a single colony was selected and inoculated in a plant-derived peptone liquid medium for fermentation and cultivation (at a temperature of 37° C.) for 8 hours to obtain a live bacterial liquid of Bacteroides fragilis ZY-312.
  • Bacteroides fragilis ZY-312 living bacteria liquid obtained by conventional heat inactivation to obtain Bacteroides fragilis inactivated bacteria liquid.
  • a total of 70 BALB/c female mice were selected and randomly divided into 7 groups according to their body weight intervals, namely blank group, model group, ZY-312 group (10 10 CFU/mouse), PD-1 antibody (PD-1ab) group (purchased From BioXcell, product number BE0146, the same below 200 ⁇ g/cell), ZY-312 live bacteria combined with PD-1 antibody group (10 10 CFU/cell), ZY-312 inactivated bacteria group (10 10 Cell/cell), ZY -312 inactivated bacteria combined with PD-1 antibody group (10 10 Cell/monkey), 10 rats in each group.
  • mice After the fourth administration of PD-1 antibody, 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.
  • TGI % or relative tumor proliferation rate T/C (%).
  • Intratumoral T cell subsets flow cytometry analysis of the proportion of CD4 + T cells and CD8 + T cells in CD3 + T cells in tumors.
  • Cytokine detection Luminex technology detects the content of IL-2 and IFN- ⁇ in mouse tumors.
  • SPSS statistical software 25.0 was used for statistical analysis.
  • Table 1 The antitumor efficacy calculated based on the tumor volume on the 15th day after group administration
  • Table 2 The antitumor efficacy calculated based on the tumor weight on the 15th day after group administration
  • the transplanted tumor mice produced obvious tumor masses, and the modeling was successful.
  • 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 by helper T (Th) cells, can bind to the non-polypeptide region of MHC class II molecules, and participate in the signal of T cell antigen receptor (TCR) recognizing antigens divert.
  • Th helper T
  • TCR T cell antigen receptor
  • CTL cytotoxic T lymphocytes
  • each administration group increased the proportion of CD3 + CD4 + T cells in the tumor to different degrees, the proportion of PD-1 antibody and Bacteroides fragilis ZY-312 or its inactivation
  • the proportion of CD3 + CD4 + /CD3 + T cells in the PD-1 antibody group was significantly greater than that in the model group.
  • the ratio of CD3 + CD4 + /CD3 + T cells in the ZY-312 combined with PD-1 antibody group was greater than that in the single use group.
  • each administration group up-regulated the proportion of CD3 + CD8 + T cells to the total cells in the tumor to varying degrees, and the CD3 + CD8 + /CD3
  • the proportion of + T cells was significantly greater than that of the model group.
  • the ratio of CD3 + CD8 + /CD3 + T cells in the ZY-312 combined with PD-1 antibody group was greater than that in the single use group.
  • B. fragilis ZY-312 combined with PD-1 antibody can up-regulate the proportion of CD3 + CD4 + and CD3 + CD8 + T cells in the tumor.
  • IFN- ⁇ and IL-2 are known anti-tumor immune cytokines. As shown in the table above, compared with the model group, each administration group up-regulated the level of IL-2 in the tumor to varying degrees, and there was a significant difference between the Bacteroides fragilis ZY-312 combined with PD-1 antibody group and the model group.
  • each administration group significantly up-regulated the level of intratumoral IFN- ⁇ .
  • the up-regulation range of Bacteroides fragilis ZY-312 combined with PD-1 antibody group was greater than that of the single administration group.
  • B. fragilis ZY-312 combined with PD-1 antibody can up-regulate the levels of cytokines IL-2 and IFN- ⁇ .
  • Bacteroides fragilis ZY-312 combined with PD-1 antibody can enhance the body's anti-tumor immune response by up-regulating the proportion of cytotoxic T cells and the level of anti-tumor cytokines, and effectively prevent and treat non-small cell lung cancer.
  • mice 70 BALB/c female mice were selected and randomly divided into 7 groups according to the weight range, namely blank group, model group, ZY-312 group (10 10 CFU/mouse), PD-L1 antibody (PD-L1ab) group (BE0101, BioXcell 200 ⁇ g/monkey), ZY-312 live bacteria combined with PD-L1 antibody group (10 10 CFU/monkey), ZY-312 inactivated bacteria group (10 10 Cell/monkey), ZY-312 inactivated Bacterial combination with PD-L1 antibody group (10 10 Cell/monkey), 10 rats in each group.
  • ZY-312 group 10 CFU/mouse
  • PD-L1ab PD-L1 antibody
  • BE0101 BioXcell 200 ⁇ g/monkey
  • ZY-312 live bacteria combined with PD-L1 antibody group (10 10 CFU/monkey
  • ZY-312 inactivated bacteria group 10 10 Cell/monkey
  • ZY-312 inactivated Bacterial combination with PD-L1 antibody group 10 Cell
  • mice After four doses of PD-L1 antibody administration, 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.
  • TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group)/(Average tumor volume at the end of treatment of the same type control group- The average tumor volume at the beginning of treatment in the same type control group)] ⁇ 100%.
  • Intratumoral T cell subsets Flow cytometry analysis of the ratio of CD4 + T cells and CD8 + T cells in tumors.
  • Cytokine detection luminex technology detects the content of IL-2 and IFN- ⁇ in mouse tumors.
  • SPSS statistical software 25.0 was used for statistical analysis.
  • Table 5 The antitumor efficacy calculated based on the tumor volume on the 15th day after group administration
  • Table 6 The antitumor efficacy calculated based on the tumor weight on the 15th day after group administration
  • the transplanted tumor mice had obvious tumor masses, and the modeling was successful.
  • 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. CD4 is mainly expressed in helper T (Th) cells, which can bind to the non-polypeptide region of MHC class II molecules and participate in the recognition of antigens by T cell antigen receptors (TCR). Signal Transduction. Studies have found that in tumor immunity, CD4 + T cells can activate CD8 + T cells through various mechanisms to differentiate into cytotoxic T lymphocytes (CTL), while maintaining and strengthening the anti-tumor response of CTL.
  • 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 Bacteroides fragilis ZY-312 combined with PD-L1 antibody group was greater than that of the single administration group, which was significantly different from the model group.
  • each administration group significantly up-regulated the proportion of CD3 + CD8 + T cells in the tumor to CD3 + T cells.
  • the up-regulation range of Bacteroides fragilis ZY-312 combined with PD-L1 antibody group was greater than that of the two single-use groups.
  • Bacteroides fragilis ZY-312 and PD-L1 antibody can up-regulate the proportion of CD3 + CD4 + and CD3 + CD8 + T cells in the tumor.
  • each administration group increased the level of IL-2 in the tumor to varying degrees, and the high-dose group of Bacteroides fragilis ZY-312 combined with PD-L1 antibody and the combination of inactivated bacteria There was a significant difference between the PD-L1 antibody group and the model group.
  • the up-regulation range of Bacteroides fragilis combined with PD-L1 antibody group was greater than that of the single administration group.
  • each administration group significantly up-regulated the level of intratumoral IFN- ⁇ .
  • the up-regulation range of Bacteroides fragilis ZY-312 combined with PD-L1 antibody group was greater than that of the single administration group, which was significantly different from the model group.
  • B. fragilis ZY-312 combined with PD-L1 antibody can up-regulate the levels of cytokines IL-2 and IFN- ⁇ .
  • Bacteroides fragilis ZY-312 combined with PD-L1 antibody can up-regulate the proportion of cytotoxic T cells and the level of anti-tumor cytokines, enhance the body's anti-tumor immune response, and effectively prevent and treat non-small cell lung cancer.
  • Example 4 The application of Bacteroides fragilis combined with PD-1 antibody in the prevention and treatment of head and neck squamous cell carcinoma
  • 70 C3H/HeOuJ female mice aged 6-8 weeks were randomly divided into 7 groups according to the weight range, namely blank group, model group, ZY-312 (10 10 CFU/mouse), PD-1 antibody (PD-1ab) group (BE0146, BioXcell 250 ⁇ g/monkey), ZY-312 live bacteria combined with PD-1 antibody group (10 10 CFU/monkey), ZY-312 inactivated bacteria group (10 10 Cell/monkey), ZY-312 inactivated bacteria group Bacteria combined with PD-1 antibody group (10 10 Cell/monkey), 10 mice in each group.
  • 1640 culture medium (purchased from BI, the same below) containing 10% calf serum (purchased from Gibco, the same below), penicillin (100 U/mL) and streptomycin (100 U/mL) under normal conditions (37 ° C, Culture SCC7 head and neck cancer cells in saturated humidity, 5% CO 2 ) to the logarithmic growth phase, adjust the cell concentration to 1 ⁇ 10 6 cells/mL, and inject 100 ⁇ L of cells subcutaneously into the right leg of each mouse in each group except the blank group Suspension, the right leg of the blank group was subcutaneously injected with 100 ⁇ L of normal saline.
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L of normal saline daily; starting from D3, 100 ⁇ L of PBS was injected intraperitoneally every three days; Corresponding drugs were administered to each administration group at the same frequency, in which the administration volume of Bacteroides fragilis was 300 ⁇ L, and the administration volume of PD-1 antibody was 100 ⁇ L, and the antibody was administered 3 times in total. Animals were observed daily for health and mortality, and tumor volume was measured every two days.
  • mice On D12, all mice were euthanized, and blood, tumor, spleen, feces, right cervical lymph and right axillary lymph were collected. Tumors were divided into two parts, one fixed in formalin and one sent in vitro for flow analysis.
  • Immune cell subsets flow cytometry analysis of the ratio of M1/M2 cells in the tumor.
  • Cytokine detection Luminex detects the content of IL-1 ⁇ and IP-10 in mouse serum.
  • SPSS statistical software 25.0 was used for statistical analysis.
  • the model group significantly formed tumors, and the modeling was successful.
  • each administration group significantly reduced the tumor volume, and there was a very significant difference between the Bacteroides fragilis ZY-312 combined with PD-1 antibody group and the model group. This shows that Bacteroides fragilis can inhibit the growth of head and neck cancer cells, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.
  • Tumor-associated macrophages are macrophages infiltrating in tumor tissue, and are the most abundant immune cells in the tumor microenvironment, which can secrete a variety of cytokines. Clear tumor cells, but with the occurrence and development of tumors, it also plays a key role in the growth, invasion and metastasis of tumors. Affected by cytokines in the tumor microenvironment, macrophages differentiate into different types of TAMs, which are mainly divided into M1 and M2 types. M1 type macrophages have the effect of killing tumor cells, while M2 type macrophages are shown to promote TAM. Tumor growth.
  • IL-1 is a cytokine released by many cell types that acts in an autocrine and/or paracrine manner, thereby stimulating multiple signaling pathways.
  • IL-1 ⁇ is an agonist in the IL-1 family, and it and its receptor have been shown to be important drivers of primary carcinogenesis and metastasis in mesenchymal and epithelial cells.
  • IP-10 is a chemokine induced by IFN- ⁇ , which can recruit neutrophils and inhibit tumor growth.
  • the IL-1 ⁇ in the model group was significantly increased; compared with the model group, each administration group significantly down-regulated the IL-1 ⁇ in the mouse serum, and the combination of Bacteroides fragilis ZY-312 and its inactivated bacteria
  • the PD-1 antibody group had extremely significant differences.
  • IP-10 in the model group increased, showing the body's spontaneous anti-tumor immune response; Bacillus ZY-312 combined with PD-1 antibody group has a very significant difference.
  • Bacteroides fragilis ZY-312 combined with PD-1 antibody can enhance the body's anti-tumor immune response and effectively prevent and treat head and neck cancer.
  • Example 5 The application of Bacteroides fragilis combined with PD-L1 antibody in the prevention and treatment of small cell lung cancer
  • mice When the mice showed obvious signs of weight loss, bowed back, and decreased activity, the mice were euthanized, and the tumor tissue was dissected. HE staining confirmed that it was small cell lung cancer. The tumor tissue was made into a cell suspension and frozen for transplantation. Tumor source.
  • group administration began: starting from D0, animals in the blank group and model group were orally administered 300 ⁇ L normal saline daily, and 100 ⁇ L PBS was intraperitoneally injected once every 7 days; each administration group was given corresponding drugs at the same frequency , where the administration volume of Bacteroides fragilis was 300 ⁇ L, the volume of PD-L1 antibody administration was 100 ⁇ L, and the antibody was administered 3 times in total. Animals were observed daily for health and mortality, and tumor volume was measured every two days. On D21, all mice were euthanized, and mouse blood, tumor, spleen, feces, right cervical lymph and right axillary lymph were collected. Tumors were divided into two parts, one fixed in formalin and one sent in vitro for flow analysis.
  • T cell subsets The proportion of CD3 + T cells and CD8 + T cells in the tumor was analyzed by flow cytometry.
  • SPSS statistical software 25.0 was used for statistical analysis.
  • the model group significantly formed tumors, and the modeling was successful.
  • each administration group can down-regulate the tumor volume of mice, and the Bacteroides fragilis ZY-312 combined with PD-L1 antibody group has a very significant difference. This shows that the combination of Bacteroides fragilis ZY-312 and PD-L1 antibody can effectively inhibit the growth of small cell lung cancer.
  • Bacteroides fragilis ZY-312 combined with PD-L1 antibody can effectively prevent small cell lung cancer in mice by enhancing the body's anti-tumor immune response, inhibiting the growth of tumor cells.

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Abstract

提供了一种包含脆弱拟杆菌，特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312，与PD-1抗体和/或PD-L1抗体的药物，及其用于修正机体免疫细胞比例，调节机体癌症相关细胞因子水平，提高机体抗肿瘤免疫反应，有效防治呼吸系统肿瘤中的应用。

Description

脆弱拟杆菌与PD-1及PD-L1抗体联合用药治疗呼吸系统肿瘤中的应用
本申请要求享有2021年10月12日向中国国家知识产权局提交的，专利申请号为202210034078.X，发明名称为“脆弱拟杆菌与PD-1及PD-L1抗体联合用药治疗呼吸系统肿瘤中的应用”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
本发明涉及一种脆弱拟杆菌的应用技术，特别是一种脆弱拟杆菌与PD-1抗体或PD-L1抗体联合用药在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、头颈癌中的鼻咽癌和喉癌等。
肺癌是当今世界上最危险、对人类威胁最大的恶性肿瘤之一。全球范围内，肺癌是发病率第二高，致死率最高的癌症。在我国，肺癌的发病率和死亡率都排名第一，在男性中，肺癌更是第一大癌症杀手。吸烟仍然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。
鼻咽癌是头颈部最常见的恶性肿瘤，指的是由披覆于鼻咽腔表面、或鼻咽隐窝的上皮发生的恶性肿瘤。鼻咽癌相对于其他癌症来说并不算常见，然而，在我国，鼻咽癌的发病率高居所有恶性肿瘤的第八位。鼻咽癌的发生一般来说与多种因素的综合作用有关，包括遗传、环境污染、接触有毒化学物和EB病毒感染等。
喉癌是指原发部位在喉部的肿瘤，以鳞状细胞癌最为常见。喉癌发病率在6/10万 人，相比于肺癌、胃癌等发病率较低。但在2016年《CA》的报道中，过去40年里，喉癌的5年生存率从66％下降到63％，是生存率下降的少数几种肿瘤之一。多种因素被视为与喉癌的发病机制有关，包括吸烟、喝酒以及暴露于某些有毒化学品。人乳头瘤病毒(HPV)感染也被视为可能因素之一。
多数呼吸系统癌症发现时已是晚期。早期肺癌多无明显症状，临床上多数患者出现症状就诊时已属晚期。鼻咽癌早期诊断难度极大，患者很容易因漏诊或误诊而错失最佳治疗时机。喉癌更是被称作“沉默杀手”。对于无手术根治机会的呼吸系统肿瘤患者，目前多采取以全身药物治疗为主的综合治疗。全身药物治疗主要包括化疗药物、分子靶向药物及免疫治疗药物。
免疫治疗药物是目前最热门的肿瘤治疗手段。肿瘤免疫治疗主要包括免疫疫苗、免疫检查点抑制剂治疗、过继性免疫细胞治疗、细胞因子治疗等，其中免疫检查点抑制剂治疗以其显著的临床疗效而备受瞩目。
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与PD-1抗体和/或PD-L1抗体联合用药在治疗呼吸系统肿瘤中的应用。本发明出人预料地发现，脆弱拟杆菌特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312能够修正机体免疫细胞比例，调节机体癌症相关细胞因子水平，提高机体抗肿瘤免疫反应，有效抑制呼吸系统肿瘤的生长。
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中的应用。
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、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抗体中的一种或多种，优选为PD-1和/或PD-L1抗体。
在其中一些实施例中，所述脆弱拟杆菌是活菌或灭活菌；优选地，所述灭活菌为形态结构完整的灭活菌或形态结构不完整的灭活菌。
在其中一些实施例中，所述脆弱拟杆菌是脆弱拟杆菌活菌体，经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液中的一种或多种。
在其中一些实施例中，所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
在其中一些实施例中，所述呼吸系统肿瘤包括肺癌、头颈部鳞癌中的一种或多种。
在其中一些实施例中，所述呼吸系统肿瘤包括小细胞肺癌、非小细胞肺癌、喉癌和鼻咽癌中的一种或多种。
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，所述药物同时包括药学有效剂量的脆弱拟杆菌和免疫检查点抑制剂，例如所述药物同时包括药学有效剂量的脆弱拟杆菌及PD-1抗体和/或PD-L1抗体。
在其中一些实施例中，所述药学有效剂量为10 6-10 10CFU。
在其中一些实施例中，所述脆弱拟杆菌是活菌、形态结构完整的灭活菌或形态结构不完整的灭活菌中的一种及以上。
在其中一些实施例中，所述脆弱拟杆菌是脆弱拟杆菌活菌体，经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液中的一种或多种。
在其中一些实施例中，所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
在其中一些实施例中，所述呼吸系统肿瘤包括小细胞肺癌、非小细胞肺癌、头颈部鳞癌的一种或多种。
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、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。
本发明还提供一种用于预防和/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。
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与PD-1抗体和/或PD-L1抗体联合应用，在修正机体免疫细胞比例，调节机体癌症相关细胞因子水平，提高机体抗肿瘤免疫反应，有效防治呼吸系统肿瘤。
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1是实施例一脆弱拟杆菌ZY-312在血平皿上的菌落形态图；
图2是实施例一脆弱拟杆菌ZY-312的革兰氏染色镜检图。
具体实施方式
下文将结合具体实施例对本发明的制备方法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。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，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。
实施例一 脆弱拟杆菌活菌液、灭活菌液的制备
将脆弱拟杆菌ZY-312菌种划线接种于血平皿，厌氧培养48h。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。
菌落特征：脆弱拟杆菌ZY-312在血平皿上37℃培养48h后，呈现圆形微凸、半透 明、白色、表面光滑、不溶血，菌落直径在1mm-3mm之间，参见图1。
显微镜下形态：脆弱拟杆菌ZY-312进行革兰氏染色镜检，为革兰阴性细菌，呈现典型的杆状，两端钝圆而浓染，菌体中间不着色部分形如空泡，参见图2。
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℃)，得脆弱拟杆菌ZY-312活菌菌液。
常规热灭活所得脆弱拟杆菌ZY-312活菌液，得脆弱拟杆菌灭活菌液。
实施例二 脆弱拟杆菌联合PD-1抗体在治疗非小细胞肺癌中的应用
1.材料与方法
(1)实验分组与给药方案
选用BALB/c雌性小鼠70只，按体重区间随机分为7组，即空白组、模型组、ZY-312组(10 10CFU/只)、PD-1抗体(PD-1ab)组(购自BioXcell，商品编号BE0146，下同200μg/只)、ZY-312活菌联用PD-1抗体组(10 10CFU/只)、ZY-312灭活菌组(10 10Cell/只)、ZY-312灭活菌联用PD-1抗体组(10 10Cell/只)，每组10只。除空白组外，其余各组动物背部注射1×10 6个LLC细胞，肿瘤体积达到50-100mm 3时(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，每周两次腹腔注射200μL 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200μL。每天观察动物的健康状况及死亡情况，每三天测量一次肿瘤体积。PD-1抗体第四次给药后，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。
检测项目与方法：
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(％)或相对肿瘤增殖率T/C(％)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％。
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：同型对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测 量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
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细胞在CD3 +T细胞中的比例。
细胞因子检测：Luminex技术检测小鼠肿瘤中IL-2和IFN-γ的含量。
数据统计与分析：使用SPSS统计软件25.0进行统计学分析。
2.试验结果
(1)肿瘤体积、肿瘤质量与肿瘤生长抑制率
表1基于分组给药后第15天肿瘤体积计算得出的抑瘤药效
Figure PCTCN2022120023-appb-000001
注：
a.平均值±SEM。
b.肿瘤生长抑制评价指标根据公式T/C％＝T RTV/C RTV×100％和TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％计算。
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。
表2基于分组给药后第15天肿瘤重量计算得出的抑瘤药效
Figure PCTCN2022120023-appb-000002
Figure PCTCN2022120023-appb-000003
注：
a.平均值±SEM。
b.肿瘤生长抑制评价指标根据公式T/C weight＝TW treatment/TW 同型对照计算。
c.根据肿瘤重量计算，两组间p值按照unpaired t-test(one-tailed)方法计算。
根据上表可知，与空白组相比，移植瘤小鼠产生明显肿瘤瘤块，造模成功。
与模型组相比，PD-1抗体、脆弱拟杆菌单独用药时，肿瘤生长速度有一定程度的减缓；但在联合用药时，肿瘤生长明显减缓。可见，脆弱拟杆菌ZY-312联用PD-1抗体能够有效抑制肿瘤生长。
(2)T细胞亚群
表3各组小鼠T细胞亚群比例(mean±SD)
Figure PCTCN2022120023-appb-000004
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，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细胞占总细胞的比例，PD-1抗体及脆弱拟杆菌ZY-312或其灭活菌联用PD-1抗体组的CD3 +CD4 +/CD3 +T细胞比例显著大于模型组。ZY-312联用PD-1抗体组的CD3 +CD4 +/CD3 +T细胞比例大于单用组。
与模型组相比，各给药组均不同程度地上调了肿瘤内CD3 +CD8 +T细胞占总细胞的比例，脆弱拟杆菌ZY-312联用PD-1抗体组的CD3 +CD8 +/CD3 +T细胞比例显著大于模型组。ZY-312联用PD-1抗体组的CD3 +CD8 +/CD3 +T细胞比例大于单用组。
可见，脆弱拟杆菌ZY-312联用PD-1抗体能够上调肿瘤内CD3 +CD4 +和CD3 +CD8 +T细胞的比例。
(3)细胞因子检测
表4各组小鼠肿瘤内细胞因子水平(mean±SD)
Figure PCTCN2022120023-appb-000005
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。
IFN-γ和IL-2均是已知的抗肿瘤免疫细胞因子。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IL-2的水平，脆弱拟杆菌ZY-312联用PD-1抗体组与模型组存在显著性差异。
与模型组相比，各给药组均显著上调了肿瘤内IFN-γ的水平。脆弱拟杆菌ZY-312联用PD-1抗体组的上调幅度大于单独给药组。
可见，脆弱拟杆菌ZY-312联用PD-1抗体能够上调细胞因子IL-2和IFN-γ的水平。
综上所述，脆弱拟杆菌ZY-312联用PD-1抗体能够通过上调细胞毒性T细胞的比例和抗肿瘤细胞因子的水平，增强机体抗肿瘤免疫反应，有效防治非小细胞肺癌。
实施例三 脆弱拟杆菌联合PD-L1抗体在治疗非小细胞肺癌中的应用
1.实验设计和流程
试验设计：选用BALB/c雌性小鼠70只，按体重区间随机分为7组，即空白组、模型组、ZY-312组(10 10CFU/只)、PD-L1抗体(PD-L1ab)组(BE0101，BioXcell 200μg/只)、ZY-312活菌联用PD-L1抗体组(10 10CFU/只)、ZY-312灭活菌组(10 10Cell/只)、ZY-312灭活菌联用PD-L1抗体组(10 10Cell/只)，每组10只。除空白组外，其余各组动物背部注射1×10 6个LLC细胞，肿瘤体积达到50-100mm 3时(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，每三天一次腹腔注射200μL 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-L1抗体给药体积为200μL。每天观察动物的健康状况及死亡情况，每三天测量一次肿瘤体积。PD-L1抗体给药四次后，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。
检测项目与方法：
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(％)或相对肿瘤增殖率T/C(％)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％。
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：同型对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
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。
细胞因子检测：luminex技术检测小鼠肿瘤中IL-2和IFN-γ的含量。
数据统计与分析：使用SPSS统计软件25.0进行统计学分析。
2.试验结果
(1)肿瘤体积、肿瘤重量与肿瘤生长抑制率
表5基于分组给药后第15天肿瘤体积计算得出的抑瘤药效
Figure PCTCN2022120023-appb-000006
注：
a.平均值±SEM。
b.肿瘤生长抑制评价指标根据公式T/C％＝T RTV/C RTV×100％和TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％计算。
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。
表6基于分组给药后第15天肿瘤重量计算得出的抑瘤药效
Figure PCTCN2022120023-appb-000007
注：
a.平均值±SEM。
b.肿瘤生长抑制评价指标根据公式T/C weight＝TW treatment/TW 同型对照计算。
c.根据肿瘤重量计算，两组间p值按照unpaired t-test(one-tailed)方法计算。
根据如上图表可知，与空白组相比，移植瘤小鼠产生明显肿瘤瘤块，造模成功。
与模型组相比，PD-L1抗体、脆弱拟杆菌单独用药时，肿瘤生长速度有一定程度的减缓；但在联合用药时，肿瘤生长明显减缓。可见，脆弱拟杆菌ZY-312联用PD-L1抗体能够有效抑制肿瘤生长。
(2)T细胞亚群
表7各组小鼠T细胞亚群比例(mean±SD)
Figure PCTCN2022120023-appb-000008
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，CD4主要表达于辅助T(Th)细胞，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7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联用PD-L1抗体组的上调幅度大于单独给药组，与模型组具有显著性差异。
与模型组相比，各给药组均显著上调了肿瘤内CD3 +CD8 +T细胞占CD3 +T细胞的比例。脆弱拟杆菌ZY-312联用PD-L1抗体组的上调幅度大于两者单用组。
可见，脆弱拟杆菌ZY-312联用PD-L1抗体能够上调肿瘤内CD3 +CD4 +和CD3 +CD8 +T细胞的比例。
(3)细胞因子检测
表8各组小鼠肿瘤内细胞因子水平(mean±SD)
Figure PCTCN2022120023-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，各给药组均不同程度地上调了肿瘤内IL-2的水平，脆弱拟杆菌ZY-312联用PD-L1抗体高剂量组、灭活菌联用PD-L1抗体组与模型组具有显著性差异。脆弱拟杆菌联用PD-L1抗体组的上调幅度大于单独给药组。
与模型组相比，各给药组均显著上调了肿瘤内IFN-γ的水平。脆弱拟杆菌ZY-312联用PD-L1抗体组的上调幅度大于单独给药组，与模型组具有极显著差异。
可见，脆弱拟杆菌ZY-312联用PD-L1抗体能够上调细胞因子IL-2和IFN-γ的水平。
综上所述，脆弱拟杆菌ZY-312联用PD-L1抗体能够上调细胞毒性T细胞比例和抗肿瘤细胞因子水平，增强机体抗肿瘤免疫反应，有效防治非小细胞肺癌。
实施例四 脆弱拟杆菌联用PD-1抗体防治头颈鳞癌的应用
1.试验设计和流程
6-8周龄C3H/HeOuJ雌性小鼠70只，按体重区间随机分为7组，即空白组、模型组、ZY-312(10 10CFU/只)、PD-1抗体(PD-1ab)组(BE0146，BioXcell 250μg/只)、ZY-312活菌联用PD-1抗体组(10 10CFU/只)、ZY-312灭活菌组(10 10Cell/只)、ZY-312灭活菌联用PD-1抗体组(10 10Cell/只)，每组10只。
采用含10％小牛血清(购自Gibco，下同)、青霉素(100U/mL)及链霉素(100U/mL)的1640培养液(购自BI，下同)在常规条件(37℃、饱和湿度、5％CO 2)下培养SCC7头颈癌细胞至对数生长期，调整细胞浓度为1×10 6个/mL，除空白组外，每组各小鼠右侧腿部皮下注射100μL细胞悬液，空白组右侧腿部皮下注射100μL生理盐水。
接种后8天(D0，此时肿瘤直径2-4mm)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；从D3开始，每三天一次腹腔注射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，抗体共给药3次。每天观察动物的健康状况及死亡情况，每两天测量一次肿瘤体积。D12，所有小鼠安乐死，采集小鼠血液、肿瘤、脾脏、粪便、右侧颈部淋巴以及右侧腋窝淋巴。肿瘤分为两份，一份于福尔马林中固定，一份送于体外用于流式分析。
检测项目与方法：
肿瘤体积与肿瘤生长抑制率：抑瘤率＝100％(模型组肿瘤平均体积-给药组肿瘤平均体积)/模型组肿瘤平均体积。
免疫细胞亚群：流式细胞术分析肿瘤内M1/M2细胞的比例。
细胞因子检测：Luminex检测小鼠血清中IL-1β、IP-10的含量。
数据统计与分析：使用SPSS统计软件25.0进行统计学分析。
2.试验结果
(1)肿瘤体积与肿瘤生长抑制率
表9各组小鼠肿瘤体积与肿瘤生长抑制率(mean±SD)
Figure PCTCN2022120023-appb-000010
Figure PCTCN2022120023-appb-000011
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。
与空白组相比，模型组显著成瘤，造模成功。
与模型组相比，各给药组均显著降低肿瘤体积，脆弱拟杆菌ZY-312联用PD-1抗体组与模型组具有极显著差异。这说明脆弱拟杆菌能够抑制头颈癌细胞的生长，脆弱拟杆菌ZY-312与PD-1抗体联用效果更佳。
(2)免疫细胞亚群
表10各组小鼠M1/M2细胞比(mean±SD)
Figure PCTCN2022120023-appb-000012
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。
肿瘤相关巨噬细胞(tumor-associated macrophages，TAM)是浸润在肿瘤组织中的巨噬细胞，是肿瘤微环境中最多的免疫细胞，可以分泌多种细胞因子，在肿瘤发生的初期，能够识别并清除肿瘤细胞，但随着肿瘤的发生发展，又对肿瘤的生长、侵袭、转移起着关键作用。受肿瘤微环境细胞因子的影响，巨噬细胞分化为不同类型的TAM，主要分为M1型和M2型，M1型巨噬细胞具有杀伤肿瘤细胞的作用，而M2型巨噬细胞则表现为促进肿瘤生长。与模型组相比，各给药组均上调了M1/M2的比值，脆弱拟杆菌ZY-312联用PD-1抗体组具有显著性差异。这说明脆弱拟杆菌能够调节TAM比例，促进机体的抗肿瘤免疫反应，脆弱拟杆菌ZY-312与PD-1抗体联用效果更佳。
(3)血清细胞因子
表11各组小鼠血清细胞因子(mean±SD)
Figure PCTCN2022120023-appb-000013
Figure PCTCN2022120023-appb-000014
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-1是一种由许多细胞类型释放的细胞因子，以自分泌和/或旁分泌的方式发挥作用，从而刺激多种信号通路。IL-1β是IL-1家族中的一种激动剂，它和它的受体已被证明是间充质和上皮细胞原发性癌变和转移的重要驱动因素。IP-10是IFN-γ诱导产生的趋化因子，能够募集中性粒细胞，抑制肿瘤生长。
与空白组相比，模型组IL-1β显著上升；与模型组相比，各给药组均显著下调了小鼠血清内的IL-1β，脆弱拟杆菌ZY-312及其灭活菌联用PD-1抗体组具有极显著差异。
与空白组相比，模型组IP-10有所上升，显示出机体自发的抗肿瘤免疫反应；与模型组相比，各给药组均显著上调了小鼠血清内的IP-10，脆弱拟杆菌ZY-312联用PD-1抗体组具有极显著差异。
上述结果说明，脆弱拟杆菌能够调节机体对肿瘤的免疫反应，脆弱拟杆菌ZY-312与PD-1抗体联用效果更佳。
综上，脆弱拟杆菌ZY-312联用PD-1抗体能够增强机体抗肿瘤免疫反应，有效防治头颈癌。
实施例五 脆弱拟杆菌联用PD-L1抗体防治小细胞肺癌的应用
1.试验设计和流程
(1)6-8周龄Trp53 loxP/loxP；Rb1 loxP/loxP；Rb12 loxP/loxP小鼠10只，腹腔注射麻醉剂麻醉，再以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)的共沉淀物插管吸入小鼠气管，每只小鼠吸入约100μL。待小鼠出现明显消瘦、弓背、活动度下降等表现，对小鼠进行安乐死，剖取肿瘤组织，HE染色验证为小细胞肺癌，将肿瘤组织制为细胞悬液，冻存，作为移植瘤瘤源。
(2)6-8周龄B6129F1雌性小鼠70只，按体重区间随机分为7组，即空白组、模型组、ZY-312(10 10CFU/只)、PD-L1抗体(PD-L1ab)组(9D2，BioXcell 300μg/只)、ZY-312活菌联用PD-L1抗体组(10 10CFU/只)、ZY-312灭活菌组(10 10Cell/只)、ZY-312灭活菌联用PD-L1抗体组(10 10Cell/只)，每组10只。
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，除空白组外，每组各小鼠右侧肋部皮下注射50μL细胞悬液，空白组右侧肋部皮下注射50μL Matrigel胶。
出现可触及肿瘤结节时(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7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-L1抗体给药体积为100μL，抗体共给药3次。每天观察动物的健康状况及死亡情况，每两天测量一次肿瘤体积。D21，所有小鼠安乐死，采集小鼠血液、肿瘤、脾脏、粪便、右侧颈部淋巴以及右侧腋窝淋巴。肿瘤分为两份，一份于福尔马林中固定，一份送于体外用于流式分析。
检测项目与方法：
肿瘤体积与肿瘤生长抑制率：抑瘤率＝100％(模型组肿瘤平均体积-给药组肿瘤平均体积)/模型组肿瘤平均体积。
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。
数据统计与分析：使用SPSS统计软件25.0进行统计学分析。
2.试验结果
(1)肿瘤体积与肿瘤生长抑制率
表12各组小鼠肿瘤体积与肿瘤生长抑制率(mean±SD)
Figure PCTCN2022120023-appb-000015
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。
与空白组相比，模型组显著成瘤，造模成功。
与模型组相比，各给药组均能够下调小鼠肿瘤体积，脆弱拟杆菌ZY-312联用PD-L1抗体组具有极显著差异。这说明脆弱拟杆菌ZY-312与PD-L1抗体联用能够有效抑制小细胞肺癌生长。
(2)T细胞亚群
表13各组小鼠CD3 +T细胞、CD8 +T细胞比例(mean±SD)
Figure PCTCN2022120023-appb-000016
Figure PCTCN2022120023-appb-000017
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。
与模型组相比，PD-L1抗体和ZY-312单用均未能明显提升小鼠肿瘤浸润CD3 +T细胞、CD8 +T细胞的比例；PD-L1抗体与脆弱拟杆菌ZY-312联用时，CD3 +T细胞、CD8 +T细胞比例显著上升。这说明脆弱拟杆菌ZY-312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。
综上，脆弱拟杆菌ZY-312联用PD-L1抗体能够通过增强机体抗肿瘤免疫反应，抑制肿瘤细胞生长，有效防治小鼠小细胞肺癌。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种脆弱拟杆菌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，所述脆弱拟杆菌是活菌、形态结构完整的灭活菌、形态结构不完整的灭活菌中的一种以上；和/或，所述脆弱拟杆菌是脆弱拟杆菌活菌体，经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液中的一种或多种。
  5. 根据权利要求1-4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。
  6. 一种药物，其中，所述药物同时包括脆弱拟杆菌与免疫检查点抑制剂，优选包括脆弱拟杆菌及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。
  7. 根据权利要求6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。
  8. 根据权利要求6或7所述的药物，其特征在于，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。
  9. 根据权利要求6-8中任一所述的应用，其特征在于，所述脆弱拟杆菌是活菌、形态结构完整的灭活菌、形态结构不完整的灭活菌中的一种以上；和/或，所述脆弱拟杆菌是脆弱拟杆菌活菌体，经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌，脆弱拟杆菌裂解物，脆弱拟杆菌液体培养上清液中的一种或多种。
  10. 根据权利要求6-9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。
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