WO2023134208A1 - Application of bacteroides fragilis and pd-1 or pd-l1 antibody drug combination in treating genitourinary system cancer - Google Patents

Abstract

Provided is an application of bacteroides fragilis and a PD-1 antibody and/or PD-L1 antibody drug combination in preventing and treating a genitourinary system tumor. A large number of experiments haven proven that bacteroides fragilis, and in particular bacteroides fragilis ZY-312 having preservation number CGMCC No. 10685, can improve a state of an immune cell, enhance the anti-tumor immune response of an organism, and effectively prevent and treat a genitourinary system tumor by means of regulating an immune factor.

Description

Combination of Bacteroides fragilis and PD-1 or PD-L1 antibody in the treatment of genitourinary cancer

This application claims the patent application submitted to the State Intellectual Property Office of China on January 12, 2022. The patent application number is 202210034082.6, and the title of the invention is "Application of Bacteroides fragilis and PD-1 or PD-L1 Antibody Combination Medication in the Treatment of Genitourinary Cancer" the priority interest of the earlier application. The entirety of said prior application is incorporated by reference into this application.

The microbial strains used in the implementation of the present invention have been preserved on April 2, 2015 at the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing). Classification name: 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.

technical field

The present invention relates to the field of application of Bacteroides fragilis, in particular to the application of co-medication of Bacteroides fragilis and PD-1 antibody or PD-L1 antibody in the prevention and treatment of genitourinary system cancer.

Background technique

According to the World Health Organization (WHO) statistics in 2019, cancer is currently the first or second leading cause of death in 112 countries, and the third or fourth leading cause of death in 23 countries. Cancer causes a huge disease burden.

Cancers of the female reproductive system include cancers of the breast, cervix, ovary, and uterus. Among women, gynecological cancers (including reproductive organs and breasts) are the tumors with the highest morbidity and mortality and the second leading cause of death worldwide. Surgical treatment of breast cancer can achieve good results, but the loss of breasts and femininity often brings heavy psychological pressure to patients and affects family life. 13% of cervical cancer patients are at an advanced stage when they are diagnosed, while the 5-year survival rate of metastatic cervical cancer is only 16.5%, with a median survival time of 8-13 months. Due to the heterogeneous nature of metastatic cervical cancer, there is still no standard treatment. Ovarian cancer is the deadliest gynecological malignancy. Surgery and chemotherapy are the mainstays of treatment for ovarian cancer; however, patients often relapse due to chemotherapy resistance within years of initial treatment.

Cancers of the urinary and male reproductive system mainly include prostate cancer, bladder cancer, kidney cancer and testicular cancer. According to the latest cancer statistics, in 2020, prostate cancer, bladder cancer, and kidney cancer rank second, sixth, and ninth in the global cancer spectrum, respectively. Despite improvements in primary prevention, early detection, and treatment, urological cancers remain characterized by increasing morbidity and mortality worldwide and a poor prognosis. Prostate cancer (PCa) is a major health problem among older male populations worldwide and is the fifth most common cause of cancer-related death in the world. The androgen receptor plays a crucial role in the development of PCa, and androgen deprivation therapy is the first-line treatment for newly diagnosed PCa patients. However, most patients progress to castration-resistant PCa, often associated with metastasis. Bladder cancer (BCa) is three times more common in men than in women. The most common surgical method for bladder tumors is transurethral resection of bladder tumors, after which patients need to be routinely treated with chemical drug infusion. The MVAC (methotrexate, vincristine, pirarubicin and cisplatin) regimen is currently the first-line regimen of chemotherapy for bladder cancer (hereinafter referred to as chemotherapy), but the curative effect of intravesical infusion chemotherapy is not very satisfactory. Although the patient's condition can be effectively relieved or even cured after routine postoperative intravesical infusion chemotherapy, the recurrence rate is still as high as 60%. The malignancy of recurrent bladder tumors will gradually increase, and eventually develop into muscle-invasive bladder tumors. The incidence of renal cell carcinoma (RCC) is steadily increasing in most parts of the world. Total or partial nephrectomy is the best primary treatment. However, RCC recurs in 20–40% of patients after resection, which is associated with worse tumor stage and grade. Some urinary tract malignancies are age-related and, therefore, their incidence is likely to continue to rise due to an aging global population.

For patients with reproductive and urinary system tumors who have no chance of radical surgery, or breast cancer patients who are willing to preserve their breasts and their physical condition permits, 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. Among them, 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 (PD-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. 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).

Although the anti-cancer effect of PD-1/PD-L1 antibody is significant (the overall progression-free survival rate reaches 80%), clinical studies show that only 20-45% of patients respond to it.

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the object of the present invention is to provide an application of Bacteroides fragilis in combination with PD-1 antibody and/or PD-L1 antibody in the prevention and treatment of genitourinary system tumors. The present invention proves through a large number of experiments that Bacteroides fragilis, especially Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685, can improve the state of immune cells, enhance the body's anti-tumor immune response, and effectively prevent and treat genitourinary system tumors by regulating immune factors .

In order to achieve the above object, the present invention adopts the following technical solutions:

The first aspect provides an application of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of products for preventing and/or treating genitourinary system tumors.

In some of these embodiments, 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.

In some of these embodiments, 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.

In some of these embodiments, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.

In some of these embodiments, 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. Preferably, it is selected from one or more of breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.

In some of these embodiments, the immune checkpoint inhibitor includes at least one of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, VISTA, and A2aR antibodies; preferably Preferably, the immune checkpoint inhibitor is PD-1 antibody and/or PD-L1 antibody.

In some of these embodiments, 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.

In some of these embodiments, the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and other antibodies that can bind to PD-L1 and block PD-L1. -1/PD-L1 signaling pathway, upregulation of T cell activation, substances that activate endogenous anti-tumor immune responses.

According to an embodiment of the invention, the product is food or medicine.

In some of these embodiments, 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.

In some of these embodiments, 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. In some of these embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

In some of these embodiments, Bacteroides fragilis is administered with PD-1 antibody and/or PD-L1 antibody at the same time.

In some of these embodiments, Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.

In some of these embodiments, the Bacteroides fragilis is administered orally or enemaly.

In the second aspect, a pharmaceutical composition for preventing and treating tumors of the genitourinary system is provided, wherein the pharmaceutical composition simultaneously includes Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody.

In some of these embodiments, 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.

In some of these embodiments, 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.

In some of these embodiments, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.

In some of these embodiments, the genitourinary system tumors include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ tumors. Preferably, it is selected from one or more of breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.

In some of these embodiments, 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.

In some of these embodiments, the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and other antibodies that can bind to PD-L1 and block PD-L1. -1/PD-L1 signaling pathway, upregulation of T cell activation, substances that activate endogenous anti-tumor immune responses.

According to an embodiment of the invention, the composition is a medicament.

In some of these embodiments, 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.

In some of these embodiments, Bacteroides fragilis is administered with PD-1 antibody and/or PD-L1 antibody at the same time.

In some of these embodiments, Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.

In some of these embodiments, the drug is administered orally or enemaly.

In some of these embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

The present invention also provides a method for preventing and treating genitourinary system cancer, comprising administering a therapeutically effective dose of the above-mentioned product or pharmaceutical composition to a patient.

Wherein, the "prevention" includes prevention and/or treatment.

Beneficial effects of the present invention:

The present invention proves through a large number of experiments that B. fragilis, especially B. fragilis ZY-312 with the deposit number CGMCC No. 10685, combined with PD-1 antibody and/or PD-L1 antibody can improve immune factors by regulating immune factors in vivo. Immune cell status, enhance the body's anti-tumor immune response, and effectively prevent and treat reproductive and urinary 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.). According to patent ZL201510459408.X and scientific literature 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. reported that the strain has good tolerance to gastric acid and bile salts, which can ensure its survival and effective colonization in the stomach.

Description of drawings

Fig. 1 is the colony morphology diagram of Bacteroides fragilis in embodiment 1;

Fig. 2 is a Gram microscope image of Bacteroides fragilis in Example 1.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

Unless otherwise specified, 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.

Unless otherwise defined or clearly indicated by background, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Embodiment 1 Bacteroides fragilis living bacteria liquid, the preparation of inactivated bacteria liquid

Streak inoculation of Bacteroides fragilis ZY-312 strain on blood plate, anaerobic culture for 48h. Observe the colony morphological characteristics, staining characteristics, size, club shape and distribution, etc.

Colony characteristics: After Bacteroides fragilis ZY-312 was cultured on a blood plate at 37°C for 48 hours, it was slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1mm and 3mm, see Figure 1.

Morphology under the microscope: 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.

Example 2 Bacteroides fragilis combined with PD-1 antibody in the treatment of 4T1 breast cancer xenografts in mice

Experimental design: 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 (10 ¹⁰ CFU/mouse), PD-1 antibody (PD-1ab) group (product number BE0146, purchased from BioXcell, 200μg/cell), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ cells/cell), ZY-312 inactivated bacteria combined PD-1 antibody group, 10 rats in each group. Except for the blank group, animals in the other groups were inoculated with 1×10 ⁶ 4T1 cells under the fourth pair of mammary fat pads, and when the tumor volume reached 100-150mm ³ (D0), the animals were divided into groups: starting from D0, blank group, model group Animals in the group were orally administered 300 μL of normal saline daily, and 200 μL of PBS was injected intraperitoneally twice a week; each administration group was given corresponding drugs at the same frequency, in which the Bacteroides fragilis bacterial liquid was administered in a volume of 300 μL, and the PD-1 antibody was administered in a volume of 200 μL. Animals were observed daily for health and mortality, and tumor volume was measured every two days. On the 14th day after administration (D14), all mice were euthanized, and mouse serum, tumor, spleen, 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.

Test items and methods:

Tumor volume and tumor growth inhibition rate: Tumor diameter was measured twice a week with a vernier caliper. The formula for calculating the tumor volume is: V=0.5a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively.

The antitumor efficacy of compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects tumor growth inhibition rate. Calculation of TGI (%): 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.

Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C%=T _RTV /C _RTV ×100% (T _RTV : RTV of the treatment group; C _RTV : RTV of the isotype control group). The relative tumor volume (RTV) was calculated according to the results of tumor measurement, and the calculation formula was RTV=V _t /V ₀ , where V ₀ was the average tumor volume measured during group administration (i.e. d0), and V _t was The average tumor volume at a certain measurement, T _RTV and C _RTV take the data of the same day.

T cell subsets in the spleen: flow cytometry analysis of the ratio of CD4 ⁺ T cells and CD8 ⁺ T cells in the tumor.

Cytokine detection: ELISA was used to detect the contents of IL-2 and IFN-γ in mouse serum.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

1. Test results

(1) Tumor volume, tumor weight and tumor growth inhibition rate

Table 1 The antitumor efficacy calculated based on the tumor volume on the 14th day after group administration

Note:

a. Mean ± SEM.

b. Tumor growth inhibition evaluation index was calculated according to the formula T/C%=T _RTV /C _RTV ×100% and TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100.

c. Calculated according to the tumor volume, the p value between the two groups was calculated according to the unpaired t-test (one-tailed) method.

Table 2 The antitumor efficacy calculated based on the tumor weight on the 14th day after group administration

Note:

a. Mean ± SEM.

b. The tumor growth inhibition evaluation index is calculated according to the formula T/C _weight = TW _treatment / TW _{isotype control} .

c. Calculated according to the tumor weight, the p value between the two groups was calculated according to the unpaired t-test (one-tailed) method.

According to the above table, compared with the blank group, the transplanted tumor mice produced obvious tumor masses, and the modeling was successful. Compared with the model group, when the PD-1 antibody and Bacteroides fragilis ZY-312 were administered alone, the tumor growth rate was slowed down to a certain extent; but when ZY-312 was administered in combination with the PD-1 antibody, the tumor growth was significantly slowed down. It can be seen that Bacteroides fragilis ZY-312 combined with PD-1 antibody can effectively inhibit tumor growth.

(2) T cell subsets

Table 3 Ratio of spleen T cell subsets in each group of mice (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

As shown in the table above, compared with the model group, each administration group up-regulated the proportion of CD4 ⁺ T cells in the spleen to the total cells to varying degrees. The up-regulation range of Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significantly different from that of the model group, which was greater than that of the single administration group.

Compared with the model group, each administration group up-regulated the ratio of CD8 ⁺ T cells to the total cells in the spleen to varying degrees. The up-regulation range of Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significantly different from that of the model group, which was greater than that of the single administration group.

It can be seen that B. fragilis ZY-312 combined with PD-1 antibody can up-regulate the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen.

(3) Cytokine detection

Table 4 Serum cytokine levels of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

As shown in the table above, compared with the model group, each administration group increased the level of serum IL-2 to varying degrees. The up-regulation range of Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significantly different from that of the model group, which was greater than that of the single administration group.

Compared with the model group, each administration group increased the level of serum IFN-γ to varying degrees. The up-regulation range of Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significantly different from that of the model group, which was greater than that of the single administration group.

It can be seen that B. fragilis ZY-312 combined with PD-1 antibody can up-regulate the levels of cytokines IL-2 and IFN-γ.

In summary, Bacteroides fragilis ZY-312 combined with PD-1 antibody can effectively prevent and treat breast cancer by enhancing the anti-tumor immune function of mice.

Example 3 Bacteroides fragilis combined with PD-1 antibody in the treatment of mouse ID8 ovarian cancer ascites tumor

1. Experimental design and process

70 C57BL/6 female mice aged 4-6 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 (BE0273, BioXcell, 200μg/monkey), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ CFU/monkey), ZY-312 inactivated bacteria combined with PD-1 Antibody group (10 ¹⁰ CFU/rat), 10 rats in each group.

ID8 ovarian cancer cells were cultured in DMEM medium containing 10% calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) under conventional conditions (37°C, saturated humidity, 5% CO ₂ ) until In the growth phase, the cell concentration was adjusted to 2×10 ⁷ cells/mL. Except for the blank group, each mouse in each group was intraperitoneally injected with 0.2 mL of cell suspension, and the blank group was intraperitoneally injected with 0.2 mL of normal saline.

Three weeks after inoculation (D0), group administration began: starting from D0, animals in the blank group and model group were orally administered 300 μL of normal saline daily, and intraperitoneally injected with 100 μL of PBS once every two days; The administration volume of Bacteroides bacteria liquid was 300 μL, the volume of PD-1 antibody administration was 100 μL, and the antibody was administered 9 times in total. Animals were observed daily for health and mortality, and tumor volume was measured every two days. Two weeks after the last administration, all mice were euthanized, and mouse serum, ascites, 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.

Test items and methods:

Ascites volume: 5mL syringe, 18-gauge needle to extract ascites.

Intratumoral T cell subsets: Flow cytometry analysis of the proportion of CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells in tumors.

Cytokine detection: ELISA was used to detect the content of IL-2 in ascites of mice.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

2. Test results

(1) Volume of ascites

Table 5 Mouse ascites volume in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Ascites tumor was modeled by intraperitoneal injection of ID8 ovarian cancer cells in mice. Compared with the blank group, the model group showed significant ascites, and the modeling was successful. Compared with the model group, each administration group reduced the volume of ascites, and the volume of ascites in the Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significantly smaller than that in the model group.

(2) T cell subsets

Table 6 T cell subsets in tumors of mice in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Intraperitoneal tumors dispersed as small, friable intraperitoneal micronodules attached to the mesentery, 2–7 cm in diameter. The tumor micronodules present in each mouse were gently dissected from the mesentery and collected to make a cell suspension for flow cytometry detection.

Compared with the model group, each administration group increased the ratio of CD3 ⁺ CD8 ⁺ T cells in living cells, except for the ZY-312 group, the other groups had significant differences with the model group; Bacteroides fragilis ZY-312 The up-regulation range was higher in the PD-1 antibody group.

Compared with the model group, each administration group significantly increased the proportion of CD3 ⁺ CD4 ⁺ T cells in living cells, and the up-regulation range was higher in the Bacteroides fragilis ZY-312 combined with PD-1 antibody group. This shows that both PD-1 antibody and Bacteroides fragilis ZY-312 can increase the proportion of infiltrating T cells in the tumor.

(3) Cytokines

Table 7 IL-2 content in ascites of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

IL-2 is an immune activator, and its level reflects the function of effector T cells. Compared with the model group, each administration group significantly increased the level of IL-2 in ascites, and the increase in the Bacteroides fragilis ZY-312 combined with PD-1 antibody group was higher. This shows that both PD-1 antibody and Bacteroides fragilis ZY-312 can enhance the function of effector T cells.

In summary, Bacteroides fragilis can increase the proportion of infiltrating T cells in tumors, enhance the function of effector T cells, and effectively prevent and treat ovarian cancer.

Example 4 Bacteroides fragilis combined with PD-1 antibody in the treatment of cervical cancer xenografts in mice

1. Experimental design and process

70 C57BL/6 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 (BE0273, BioXcell, 200μg/monkey), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ CFU/monkey), ZY-312 inactivated bacteria combined with PD-1 Antibody group (10 ¹⁰ CFU/rat), 10 rats in each group.

TC-1 cells were cultured in DMEM medium containing 10% calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) under conventional conditions (37°C, saturated humidity, 5% CO ₂ ) until In the growth phase, the cell concentration was adjusted to 6×10 ⁶ cells/mL. Except for the blank group, 0.1 mL of cell suspension was subcutaneously injected into the right armpit of each mouse in each group, and 0.1 mL of normal saline was subcutaneously injected into the right armpit of the blank group. .

7 days after the inoculation (D0), group administration began: starting from D0, animals in the blank group and the model group were orally administered 300 μL of normal saline daily, and intraperitoneally injected with 100 μL of PBS once every seven days; The administration volume of bacterial solution was 300 μL, the volume of PD-1 antibody administration was 100 μL, 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. One week after the last administration, all mice were euthanized, and mouse serum, tumor, spleen, feces, right cervical lymph and right axillary lymph were collected.

Test items and methods:

Tumor weight and tumor growth inhibition rate: tumor inhibition rate=100% (average tumor weight in model group-average tumor weight in administration group)/average tumor weight in model group.

Cytotoxicity Assay: Cytotoxicity of splenocytes was measured using a non-radioactive cytotoxicity assay.

Cytokine detection: ELISA was used to detect the expressions of vascular endothelial growth factor (VEGF) and IL-10 in mouse tumors, and the levels of IFN-γ and IL-4 in the culture supernatant of splenic lymphocytes.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

2. Test results

(1) Tumor weight and tumor growth inhibition rate

Table 8 Tumor weight and tumor inhibition rate of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the blank group, the model group obviously formed tumors, and the modeling was successful.

Compared with the model group, the tumor weight of each administration group decreased, and the Bacteroides fragilis ZY-312 combined with PD-1 antibody group had a significant difference. This shows that Bacteroides fragilis can effectively inhibit the growth of cervical cancer, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

(2) Cytotoxicity Assay

Table 9 Cytotoxicity of mouse spleen cells in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Cytotoxicity reflects the ability of immune cells to kill target cells. Compared with the model group, each administration group up-regulated the cytotoxicity of splenocytes to tumor cells, and there was a significant difference in the Bacteroides fragilis ZY-312 combined with PD-1 antibody group.

(3) Cytokines

Table 10 Mouse cytokines in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

CD4+ T cells are an important component of effector T cells. According to the cytokines produced and the biological function characteristics of effector cells, they can be divided into Th1, Th2, Treg and Th17. Th1 mainly secretes IFN-γ and IL-2 to promote cellular immunity, while Th2 mainly secretes IL-4, IL-5, IL-10 and IL-13 to promote humoral immunity. Tumor cells can induce the Th1/Th2 polarization balance to shift to Th2, thereby up-regulating the level of immunosuppressive cells regulating T cells. VEGF is the key mechanism of tumor angiogenesis and the main target of anti-angiogenic therapy for various malignant tumors.

In the tumor microenvironment, compared with the model group, each administration group down-regulated the levels of VEGF and IL-10 in different ranges, and the combination group was significant.

In the spleen, compared with the model group, each administration group down-regulated the ratio of IL-4/IFN-γ in different ranges, and the combination group was significant. This shows that the combination of Bacteroides fragilis and PD-1 antibody can regulate the balance of Th1/Th2 and inhibit Th2 polarization.

The above results show that the combination of Bacteroides fragilis and PD-1 antibody can inhibit tumor angiogenesis and promote anti-tumor immune response.

In summary, Bacteroides fragilis can enhance the function of CTLs, promote anti-tumor immune response, inhibit tumor angiogenesis, and effectively prevent and treat cervical cancer.

Example 5 Bacteroides fragilis combined with PD-1 antibody in the treatment of Tramp-C1 prostate cancer xenografts in mice

1. Experimental design and process

Prostate xenograft model preparation

(1) Tramp-C1 prostate cancer cells were cultured under conventional conditions (37°C, saturated humidity, 5% CO ₂ ) in 1640 culture medium containing 10% calf serum, 1% mixed solution of penicillin and streptomycin until During several growth phases, adjust the cell concentration to 1×10 ⁸ cells/mL. C57BL/6 male mice were subcutaneously injected with 0.1 mL of cell suspension in the left groin under aseptic conditions, and the tumor-bearing mice were killed when the tumor grew to a diameter of 2-3 cm, and used as a tumor source for transplantation.

(2) 70 C57BL/6 male mice were randomly divided into 7 groups according to body weight range, namely blank group, model group, ZY-312 (10 ¹⁰ CFU/mouse), PD-1 antibody (PD-1ab) group ( BE0273, BioXcell, 200μg/cell), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ CFU/cell), ZY-312 inactivated bacteria combined with PD-1 antibody group (10 ¹⁰ CFU/only), 10 in each group. The tumor source was divided into small pieces with a diameter of 0.4 cm, and implanted subcutaneously in the left groin of each mouse except the blank group. Tumors formed in the mice one week later. The blank group underwent the same operation, but no tumor mass was implanted.

After tumor formation (D0), group administration began: starting from D0, animals in the blank group and model group were orally administered 300 μL normal saline daily, and 100 μL PBS was intraperitoneally injected once a week; The administration volume of bacterial solution was 300 μL, the volume of PD-1 antibody administration was 100 μL, and the antibody was administered 7 times in total. Animals were observed daily for health and mortality, and tumor volume was measured every two days. Two weeks after the last administration, all mice were euthanized, and mouse serum, tumor, spleen, feces, right cervical lymph and right axillary lymph were collected. Spleen and tumor were divided into two parts, one was fixed in formalin, and the other was sent in vitro for flow analysis.

Test items and methods:

Tumor weight and tumor growth inhibition rate: tumor inhibition rate=100% (average tumor weight in model group-average tumor weight in administration group)/average tumor weight in model group.

T cell subsets: the ratio of CD3 ⁺ CD8 ⁺ T cells in spleen and tumor was analyzed by flow cytometry.

Cytokine detection: ELISA was used to detect the contents of IL-2, IFN-γ and TNF-α in mouse serum.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

2. Test results

(1) Tumor weight and tumor growth inhibition rate

Table 11 Tumor weight and tumor growth inhibition rate of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the blank group, the model group significantly formed tumors, and the modeling was successful.

Compared with the model group, the tumor weights of each administration group decreased, and the tumor weight of the combination group decreased significantly. It shows that Bacteroides fragilis ZY-312 can inhibit the growth of prostate tumors, and ZY-312 combined with PD-1 antibody has a better tumor inhibitory effect.

(2) T cell subsets

Table 12 T cell subsets in the spleen and tumor of mice in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the model group, each administration group up-regulated the ratio of spleen CD8 ⁺ T cells to CD3 ⁺ T cells, and the group with B. fragilis ZY-312 combined with PD-1 antibody had a significant effect.

Compared with the model group, each administration group up-regulated the ratio of intratumoral infiltrating CD8 ⁺ T cells to CD3 ⁺ T cells, and the Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significant. The above results indicate that Bacteroides fragilis can increase the proportion of infiltrating effector T cells in the spleen and tumor, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

(3) Cytokines

Table 13 Serum cytokines of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

IL-2, IFN-γ and TNF-α are immune stimulators. Compared with the model group, the levels of serum cytokines were up-regulated in each administration group, and the Bacteroides fragilis ZY-312 combined with PD-1 antibody group was significant. This shows that Bacteroides fragilis can up-regulate the level of immune activation factors, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

In conclusion, Bacteroides fragilis combined with PD-1 antibody can increase the number of effector T cells, up-regulate the level of immune activation factors, enhance the body's anti-tumor immune response, and effectively prevent and treat prostate cancer.

Example 6 Bacteroides fragilis combined with PD-1 antibody in the treatment of MB49 bladder cancer xenografts in mice

1. Experimental design and process

Seventy C57BL/6 mice aged 6-8 weeks, half male and half male, were randomly divided into 7 groups according to body weight, namely blank group, model group, ZY-312 (10 ¹⁰ CFU/mouse), PD-1 antibody (PD -1ab) group (BE0273, BioXcell, 100μg/monkey), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ CFU/monkey), ZY-312 inactivated bacteria combined PD-1 antibody group (10 ¹⁰ CFU/rat), 10 rats in each group.

Using 1640 culture medium containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) to culture MB49 bladder cancer cells under normal conditions (37°C, saturated humidity, 5% CO ₂ ) to the right In the growing period, the cell concentration was adjusted to 1×10 ⁶ cells/mL. Except for the blank group, 0.1 mL of cell suspension was subcutaneously injected into the back of the hind legs of each mouse, and 0.1 mL of normal saline was subcutaneously injected into the back of the hind legs of the mice in the blank group.

On the day of inoculation (D0), group administration began: starting from D0, animals in the blank group and model group were orally administered 300 μL of normal saline daily, and intraperitoneally injected with 100 μL of PBS every four days; each administration group was given corresponding drugs at the same frequency, among which Bacteroides fragilis The volume of bacterial liquid administration was 300 μL, the volume of PD-1 antibody administration was 100 μL, and the antibody was administered 4 times in total. Animals were observed daily for health and mortality, and tumor volume was measured every two days. One week after the last administration, 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.

Test items and methods:

Tumor weight and tumor growth inhibition rate: tumor inhibition rate=100% (average tumor weight in model group-average tumor weight in administration group)/average tumor weight in model group.

T cell subsets: flow cytometry analysis of the ratio of CD4 ⁺ , CD8 ⁺ T cells in peripheral blood.

Cytokine detection: ELISA was used to detect the contents of IL-10, IL-12 and IFN-γ in mouse serum.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

2. Test results

(1) Tumor weight and tumor growth inhibition rate

Table 14 Tumor weight and tumor growth inhibition rate (mean ± SD) of mice in each group

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the blank group, the model group obviously formed tumors, and the modeling was successful.

Compared with the model group, the tumor weights of each administration group decreased, and the tumor weight of the combination group decreased significantly. It shows that Bacteroides fragilis ZY-312 can inhibit the growth of bladder tumors, and ZY-312 combined with PD-1 antibody has a better tumor inhibitory effect.

(2) T cell subsets

Table 15 Peripheral blood T cell subsets of mice in each group (mean±SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the model group, the proportion of peripheral blood CD4 ⁺ and CD8 ⁺ T cells in each administration group increased, and the group with Bacteroides fragilis ZY-312 combined with PD-1 antibody was significant. It shows that Bacteroides fragilis can up-regulate the proportion of effector T cells in peripheral blood, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

(3) Cytokines

Table 16 Serum cytokines of mice in each group

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the blank group, all cytokines in the model group increased, showing the body's immune response against the tumor.

IL-10 is an immunosuppressive factor. Compared with the model group, the level of IL-10 in the PD-1 antibody group increased; ZY-312 and the combination group down-regulated the level of IL-10. The combined group has a larger reduction.

Both IL-12 and IFN-γ are immune activation factors. Compared with the model group, the levels of IL-12 were up-regulated in each administration group. The combination group is significant.

Compared with the model group, each administration group significantly up-regulated the level of IFN-γ (p<0.05), and the combination group had a larger up-regulation range (p<0.01).

The above results indicate that Bacteroides fragilis can down-regulate the level of immunosuppressive factors and up-regulate the level of immune activating factors, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

In conclusion, Bacteroides fragilis combined with PD-1 antibody can increase the number of effector T cells, down-regulate the level of immunosuppressive factors, up-regulate the level of immune activating factors, enhance the body's anti-tumor immunity, and effectively prevent and treat bladder cancer.

Example 7 Bacteroides fragilis combined with PD-1 antibody in the treatment of Renca renal cell carcinoma xenografts in mice

1. Experimental design and process

70 BALB/c male mice aged 6-8 weeks were randomly divided into 7 groups according to body weight range, namely blank group, model group, ZY-312 (10 ¹⁰ CFU/mouse), PD-1 antibody (PD-1ab) group (BE0146, BioXcell, 200μg/monkey), ZY-312 live bacteria combined with PD-1 antibody group, ZY-312 inactivated bacteria group (10 ¹⁰ CFU/monkey), ZY-312 inactivated bacteria combined with PD-1 Antibody group (10 ¹⁰ CFU/rat), 10 rats in each group.

Using 1640 culture medium containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) to culture Renca renal cell carcinoma cells under normal conditions (37°C, saturated humidity, 5% CO ₂ ) to the right In the growing period, the cell concentration was adjusted to 2×10 ⁷ cells/mL. Except for the blank group, 50 μL of cell suspension was subcutaneously injected into the left flank of each mouse in each group, and 50 μL of normal saline was subcutaneously injected into the left flank of the blank group.

When the tumor volume reached 500 mm3 (D0), group administration began: starting from D0, animals in the blank group and model group were orally administered 300 μL of normal saline daily, and intraperitoneally injected with 100 μL of PBS once every three days; The administration volume of Bacteroides bacteria liquid was 300 μL, the volume of PD-1 antibody administration was 100 μL, 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 D24, 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.

Test items and methods:

Tumor weight and tumor growth inhibition rate: tumor inhibition rate=100% (average tumor weight in model group-average tumor weight in administration group)/average tumor weight in model group.

T cell subsets: The ratio of CD80 ⁺ and CD86 ⁺ T cells in tumor-draining lymph nodes was analyzed by flow cytometry.

Cytokine detection: ELISA was used to detect the contents of IL-2, IFN-γ, IL-4 and IL-10 in mouse serum.

Data statistics and analysis: SPSS statistical software 25.0 was used for statistical analysis.

2. Test results

(1) Tumor weight and tumor growth inhibition rate

Table 17 Tumor weight and tumor growth inhibition rate of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Compared with the blank group, the model group significantly formed tumors, and the modeling was successful.

Compared with the model group, each administration group reduced the tumor weight, and the Bacteroides fragilis ZY-312 combined with PD-1 antibody group had a significant difference. It shows that Bacteroides fragilis can inhibit the growth of kidney cancer, and the effect of Bacteroides fragilis ZY-312 combined with PD-1 antibody is better.

(2) T cell subsets

Table 18 T cell subsets in tumor draining lymph nodes of mice in each group (mean±SEM)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

Dendritic cells express PD-L1 ligand, which can immature dendritic cells and promote immune escape of tumor cells. Systematic application of PD-1 antibody can theoretically promote the maturation of dendritic cells and enhance the body's anti-tumor immune response. Compared with the model group, each administration group increased the proportion of mature dendritic cells in the tumor draining lymph nodes, and there was a significant difference in the PD-1 antibody group combined with Bacteroides fragilis ZY-312. It shows that Bacteroides fragilis can promote the maturation of dendritic cells and enhance the body's anti-tumor immune response, and the combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

(3) Cytokines

Table 19 Serum cytokines of mice in each group (mean ± SD)

Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

IL-2 and IFN-γ are Th1-type cytokines, and IL-4 and IL-10 are Th2-type cytokines. Compared with the model group, each administration group had little effect on Th2 cytokines, but up-regulated Th1 cytokines, and the up-regulation range of the Bacteroides fragilis ZY-312 combined with PD-1 antibody group was greater than that of the two drugs alone group . This shows that Bacteroides fragilis can regulate Th1/Th2 balance, enhance the body's Th1 immune response, and then enhance the anti-tumor immune response. The combination of Bacteroides fragilis ZY-312 and PD-1 antibody has a better effect.

In summary, Bacteroides fragilis ZY-312 combined with PD-1 antibody can promote the maturation of dendritic cells, enhance the body's Th1 immune response, and effectively prevent and treat renal cancer.

The present invention can also have other various embodiments, without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes and All deformations should belong to the protection scope of the appended claims of the present invention.

Claims (10)

1. An application of Bacteroides fragilis and immune checkpoint inhibitors in the preparation of products for preventing and/or treating genitourinary system tumors.
2. The application according to claim 1, wherein 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.

   Preferably, the Bacteroides fragilis is a live Bacteroides fragilis cell, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, a lysate of Bacteroides fragilis, a lysate of Bacteroides fragilis, One or more of Bacteroides liquid culture supernatant.

   Preferably, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No.10685.
3. The application according to claim 1 or 2, characterized in that the genitourinary system tumor refers to a tumor occurring in the urinary system and/or reproductive system.

   Preferably, female thoracic and reproductive organ tumors, male reproductive organ tumors, and urinary organ tumors are included. Preferably, it is selected from one or more of breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.
4. The application according to any one of claims 1-3, wherein the immune checkpoint inhibitors include PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, At least one of VISTA and A2aR antibodies; preferably, the immune checkpoint inhibitor is PD-1 antibody and/or PD-L1 antibody.

   Preferably, the PD-1 antibody includes Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Sindhi Cindilimab, Camrelizumab and others can bind to PD-1, block PD-1/PD-L1 signaling pathway, up-regulate T cell activation, and activate endogenous anti-tumor immune response substance.

   Preferably, the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and others that can bind to PD-L1 and block PD-1/PD -L1 signaling pathway, upregulation of T cell activation, substances that activate endogenous anti-tumor immune responses.
5. The use according to any one of claims 1-4, characterized in that the product is food or medicine.

   Preferably, the food product comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereals. The food can also be animal food, such as feed and the like.

   Preferably, 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.

   Preferably, Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered simultaneously, or Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.

   Preferably, the Bacteroides fragilis is administered orally or by enema.
6. A pharmaceutical composition for preventing and treating genitourinary system tumors, wherein the pharmaceutical composition simultaneously includes Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody.
7. The composition according to claim 6, wherein 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.

   Preferably, the Bacteroides fragilis is a live Bacteroides fragilis cell, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, a lysate of Bacteroides fragilis, a lysate of Bacteroides fragilis, One or more of Bacteroides liquid culture supernatant.

   Preferably, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No.10685.
8. The composition according to claim 6 or 7, characterized in that the genitourinary system tumors include female breast and reproductive organ tumors, male reproductive organ tumors and urinary organ tumors. Preferably, it is selected from one or more of breast cancer, cervical cancer, uterine body cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, and testicular cancer.
9. The composition according to any one of claims 6-8, wherein the PD-1 antibody comprises Nivolumab, Pembrolizumab, Cimiprizumab (Cemiplimab), Toripalimab, Cindilimab, Camrelizumab and others that can bind to PD-1 and block PD-1/PD-L1 Signaling pathways, upregulation of T cell activation, substances that activate endogenous anti-tumor immune responses.

   Preferably, the PD-L1 antibody includes Atezolizumab, Avelumab, Durvalumab and others that can bind to PD-L1 and block PD-1/PD -L1 signaling pathway, upregulation of T cell activation, substances that activate endogenous anti-tumor immune responses.
10. The composition according to any one of claims 6-9, wherein the composition is a medicine.

    Preferably, 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.

    Preferably, Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered simultaneously, or Bacteroides fragilis and PD-1 antibody and/or PD-L1 antibody are administered separately.

    Preferably, the drug is administered orally or enemaly. Preferably, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

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