Application of lactobacillus muricatum in developing medicine for enhancing curative effect of immunodetection point inhibitor

Abstract

The invention discloses an application of lactobacillus muricatum in developing a medicine for enhancing the curative effect of an immunodetection point inhibitor. The invention finds that the treatment effect of the alphaPD-1 monoclonal antibody is enhanced by the lactobacillus murinus, the infiltration of T cells in a tumor microenvironment is reduced, and the infiltration of CD8 + T cells is enhanced; targeted metabonomics analysis finds that the content of butyric acid in tumor tissue colonized with L.murinus is remarkably increased, and the butyric acid is co-cultured with CD8 + T cells in vitro and can inhibit the growth of mouse tumor cells MFC. Therefore, a scientific basis is provided for using the L.murinus as an auxiliary means for enhancing the therapeutic effect of the alphaPD-1 antibody, and valuable information is provided for developing a new tumor immunotherapy strategy.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of Lactobacillus murinus in developing drugs that enhance the efficacy of immune checkpoint inhibitors. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors. With the further study of microbial detection technology and bacteria resident in tumors, more and more evidence shows that microorganisms in tumors are crucial to the tumor microenvironment (TME). These microorganisms are usually in a dynamic equilibrium with the host and affect a variety of physiological processes. The disruption of this balance is associated with the occurrence of a series of diseases, and its impact on tumor progression, drug resistance and treatment response has attracted the close attention of more and more researchers. Some studies have shown that intratumoral microorganisms or their metabolites can promote cancer progression and metastasis by changing the proportion and function of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. For example, the colonization of gastric cancer tumors by Streptococcus anginosus can accelerate the progression and metastasis of gastric cancer through the interaction between TMPC and ANXA2. The metabolite methylamine-N-oxide associated with intratumoral symbiotic microorganisms can promote CD8 + T cell-mediated anti-tumor immunity. However, the impact of other potential microorganisms in tumors on tumors and their mechanisms of action remain to be further explored. With increasing evidence showing the presence of microorganisms in tumors, intratumoral microorganisms are gradually considered to be part of the tumor microenvironment. The impact of intratumoral microorganisms on tumor progression and their potential mechanisms remain to be fully elucidated.

[0003] Through in-depth studies of immune checkpoint molecules such as programmed cell death receptor 1 (PD-1), its ligand (PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), people have found that immune checkpoints have several pathways that regulate immunosuppression. Immune checkpoint inhibitors (ICBs) targeting PD-1, PD-L1 and CTLA-4 can block these inhibitory pathways, thereby enhancing the killing effect on tumor cells and delaying tumor progression. PD-1, also known as CD279, is an important immunosuppressive molecule in the immunoglobulin superfamily. The PD-1 protein on the surface of the T cell membrane specifically recognizes its ligand PD-L1 expressed by tumor cells, and regulates the immune response to prevent tumor cells from being damaged or destroyed. In the tumor microenvironment, after PD-L1 interacts with PD-1, it recruits protein tyrosine phosphatase SHP-2 to weaken the activation signal of T cells and mediate immune escape, ultimately inducing T cell apoptosis, anergy and functional failure, allowing tumor cells to escape the surveillance of the immune system. Blocking the combination of PD-1 and PD-L1 can reverse the immunosuppressive tumor microenvironment and enhance the body's immune cells' ability to kill tumor cells. In this regard, immunotherapy represented by ICB provides a reliable treatment strategy for patients with different refractory tumors. It has been widely used in the clinic to treat tumors such as lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, and Hodgkin's lymphoma. However, more than half of the patients treated with ICB showed drug resistance, which limits the widespread use of ICB. Elucidating the relevant mechanisms that affect ICB treatment of tumors may be a potential strategy to overcome ICB treatment resistance. At present, the mechanisms of resistance to PD-1 inhibitors are mainly the following: insufficient immunogenicity of tumor antigens, major histocompatibility complex (MHC) dysfunction, CD8 + T cells are severely exhausted and the tumor microenvironment is suppressed. More and more studies have found that the microbiome can affect the response of various tumor cells to immunotherapy, including gastric cancer, colorectal cancer, and melanoma. The immune-related toxicity of ICB is also related to the composition of the intestinal microbiome. A clinical trial showed that transplanting fecal microbiota from immunotherapy responders can overcome the resistance of melanoma patients to immunotherapy. It is reported that Akkermansia muciniphila, Bifidobacterium longum, and Faecalibacterium prausnitzii show good efficacy in ICB treatment. Summary of the invention

[0004] The present invention aims to explore the potential link between microorganisms in tumors and the development of gastric cancer. The results show that Lactobacillus murinus enhances the therapeutic effect of αPD-1 monoclonal antibody, reduces the infiltration of Treg cells in the tumor microenvironment, and enhances the CD8 +Through targeted metabolomics analysis, it was found that the butyrate content in tumor tissues colonized with Lactobacillus murinus was significantly increased, and butyrate was related to CD8 + T cell co-cultivation can inhibit the growth of mouse tumor cells MFC.

[0005] Therefore, the first object of the present invention is to provide a use of Lactobacillus murinus in the preparation of a drug for enhancing the efficacy of immune checkpoint inhibitors.

[0006] Preferably, the immune checkpoint inhibitor is a monoclonal antibody against the PD-1 / PD-L1 immune checkpoint.

[0007] Preferably, the immune checkpoint inhibitor is a PD-1 monoclonal antibody.

[0008] The second object of the present invention is to provide the use of Lactobacillus murinus in the preparation of anti-tumor drugs. Preferably, the anti-tumor drugs include but are not limited to drugs for ovarian cancer, breast cancer cells, cervical cancer, lung cancer, liver cancer, colorectal cancer or gastric cancer.

[0009] The third object of the present invention is to provide an anti-tumor composition comprising Lactobacillus murinus and an immune checkpoint inhibitor.

[0010] Preferably, the immune checkpoint inhibitor is a monoclonal antibody against the PD-1 / PD-L1 immune checkpoint.

[0011] Preferably, the immune checkpoint inhibitor is a PD-1 monoclonal antibody.

[0012] The fourth object of the present invention is to provide the use of the above antitumor composition in the preparation of antitumor drugs. Preferably, the antitumor drugs include but are not limited to drugs for ovarian cancer, breast cancer cells, cervical cancer, lung cancer, liver cancer, colorectal cancer or gastric cancer.

[0013] The fifth object of the present invention is to provide an anti-tumor drug, which comprises an effective amount of Lactobacillus murinus or the above anti-tumor composition as an active ingredient, and a pharmaceutically acceptable carrier.

[0014] The present invention uses an MFC cell (mouse gastric cancer cell) tumor-bearing mouse model to evaluate the effect of Lactobacillus murinus colonization on tumor growth and the efficacy of αPD-1 monoclonal antibody. Immunohistochemistry and flow cytometry are used to evaluate the expression of regulatory T (Treg) cells and CD8+ A targeted metabolomics approach was used to compare the differential metabolites in mouse tumor tissues, and cell proliferation experiments and flow cytometry were used to verify the effects of differential metabolites on mouse CD8 + The results showed that Lactobacillus murinus enhanced the therapeutic effect of αPD-1 monoclonal antibody, reduced the infiltration of T cells in the tumor microenvironment, and enhanced the CD8 + Targeted metabolomics analysis revealed that the butyrate content was significantly increased in tumor tissues colonized with Lactobacillus murinus, and butyrate was involved in the infiltration of CD8 + T cell co-cultivation can inhibit the growth of mouse tumor cells MFC. Lactobacillus murine has inhibitory activity against a variety of tumor cells, including ovarian cancer cells, breast cancer cells, cervical cancer cells, lung cancer cells, liver cancer cells, colorectal cancer cells, and gastric cancer cells, and is expected to be used in the development of anti-tumor drugs.

[0015] The findings of the present invention provide a scientific basis for using L.murinus as an auxiliary means to enhance the therapeutic effect of αPD-1 antibodies. L.murinus enhances the anti-tumor immune response by regulating immune cells and metabolites in the tumor microenvironment, thereby improving the therapeutic effect of αPD-1 antibodies. These findings provide valuable information for the development of new tumor immunotherapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Enhancement of anti-PD-1 efficacy in gastric cancer by L. murinus. (A) Representative tumor images. (B) Changes in subcutaneous MFC tumor volume in mice treated with αPD-1 antibody or αPD-1 antibody combined with L. murinus. n = 6 mice per group. (C) Tumor weights of mice. (D) Representative scanning electron microscopy (SEM) images of MFC cells after co-culture with L. murinus. High-magnification images show attachment between bacteria and cells, scale bar 1:200.

[0017] Figure 2 Immunohistochemical staining was used to evaluate the effect of αPD-1 antibody combined with L. murinus on immune cells in the tumor immune microenvironment. (A) Effect of αPD-1 antibody combined with L. murinus on Foxp 3+ (B) Effects of αPD-1 antibody combined with L. murinus on mouse CD8 + (C) Effect of αPD-1 antibody combined with L. murinus on CD4 T cells in mice 4+(D) Effect of αPD-1 antibody combined with L. murinus on GranzymeB expression in mouse tumor tissues. Scale bar = 50 μm.

[0018] Figure 3 L. murinus produces butyrate and its derivative metabolites in vitro and in vivo. (A) Targeted metabolomics showing heat map analysis of saline and L. murinus in mouse tumors (n=6) or culture supernatants (D) (n=6). (B) Pathway enrichment analysis of differential metabolites of L. murinus in mouse tumors and culture supernatants using pathway-associated metabolite sets (SMPDB) (E). (C) Volcano plot showing differential metabolites based on one-dimensional statistical screening of saline and L. murinus in mouse tumors or culture supernatants (F). (G) Venn diagram showing overlapping differential metabolites of L. murinus supernatants (red) and mouse tumors (blue). (H) Contents of 2-hydroxybutyrate, 2-hydroxy-3-methylbutyrate, and butyrate in mouse tumors or culture supernatants (n=6).

[0019] Figure 4 Butyrate directly promotes CD8 + T cells inhibit MFC tumor growth. (A) Butyrate content in tumor tissues of mice treated with different treatment groups. (B) Butyrate content in serum samples of mice treated with different treatment groups. (C) Effect of butyrate on cell viability of MFC and AGS cell lines in vitro. (D) CD3 / CD28 and CD8 + T cells inhibited the colony formation of MFC cells. (E) Butyrate can inhibit the volume and reduce the weight of subcutaneous MFC tumors in mice. DETAILED DESCRIPTION

[0020] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0021] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0022] Example 1: PD-1 antibody treatment experiment

[0023] MFC cells (8×10 5 The tumor volume reaches 50-100mm 3 After that, mice treated with or without αPD-1 antibody were injected intratumorally with 2×10 7CFU Lactobacillusmurinus or an equal amount of saline was administered twice a week. IgG2a isotype control or αPD-1 antibody was injected intraperitoneally twice a week. 3 When , the treatment was terminated and the mice were euthanized.

[0024] The results of the combined treatment of L. murinus and αPD-1 monoclonal antibody in the 615 mouse gastric cancer subcutaneous transplant tumor model are shown in Figure 1 .Depend on Figure 1 It can be seen that L. murinus can enhance the anti-gastric cancer effect of αPD-1 monoclonal antibody, and can significantly reduce the volume of subcutaneous MFC tumors in mice and reduce tumor weight.

[0025] Example 2: Immunohistochemical staining

[0026] (1) Dewaxing of paraffin sections: Soak the sections in dewaxing solution I, II, and III for 15 min, then transfer to anhydrous ethanol I, anhydrous ethanol II, 85% alcohol for 5 min, 75% alcohol, and distilled water for 5 min each.

[0027] (2) Antigen retrieval: Place tissue sections in a citric acid antigen retrieval buffer (pH 6.0) in a microwave oven on medium heat for 8 min until boiling, then turn off the heat for 8 min to keep warm and then turn to medium-low heat for 6 min. After natural cooling, place the sections in PBS solution and wash on a shaker three times, 5 min each time.

[0028] (3) Blocking endogenous peroxidase: Add 3% hydrogen peroxide solution to the tissue surface and incubate at room temperature in the dark for 10 min. Place the slide in PBS solution and wash on a shaker three times, 5 min each time.

[0029] (4) Serum blocking: Add 3% BSA solution into the tissue culture circle and block at room temperature for 30 minutes.

[0030] (5) Add primary antibody: Add primary antibody prepared in PBS solution and incubate overnight in a humidified chamber at 4°C.

[0031] (6) Adding secondary antibody: Wash the sections in PBS solution three times, 5 min each time. After drying, add secondary antibody working solution at a ratio of 1:200 and incubate at room temperature for 50 min.

[0032] (7) DAB color development and counterstaining of cell nuclei: Wash the sections in PBS three times, 5 min each time. After drying, add DAB color development solution for staining. After staining, rinse with tap water. Counterstain the sections with hematoxylin for 3 min, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, blue with hematoxylin blueing solution, and rinse with running water.

[0033] (8) Dehydration and sealing: Immerse the sections in 75% alcohol, 85% alcohol, anhydrous ethanol I, anhydrous ethanol II, and xylene I in sequence for 5 min each, then allow to dry slightly and seal.

[0034] (9) Examination under a microscope and counting the number of positive cells.

[0035] Immunohistochemical staining was used to detect the changes in immune cells in the tumor immune microenvironment during L.murinus combined with αPD-1 antibody treatment. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that L.murinus combined with αPD-1 antibody treatment can significantly reduce the number of regulatory T cells (Foxp 3+ ) ratio, increasing the MFC tumor infiltration CD 8+ The results showed that L.murinus combined with αPD-1 antibody treatment may enhance anti-tumor immune response through multiple mechanisms, including reducing immunosuppressive cells (such as regulatory T cells), increasing effector T cells (such as CD4 T cells), and increasing the expression of Granzyme B in tumor tissues. 8+ T cells) infiltration, and increased expression of cytotoxic proteins (such as Granzyme B), thereby improving the therapeutic effect.

[0036] Example 3: Targeted metabolomics analysis

[0037] Targeted metabolomics analysis was performed by Metabo-Profile (Shanghai, China). Tumor tissues of mice in the model group and L. murinus group were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Tissue samples were taken and mixed with zirconium oxide beads and deionized water, and then methanol containing internal standards was added to extract metabolites. After the samples were stirred for several minutes, the supernatant was transferred to a 96-well plate and centrifuged. Finally, the plate was sealed and subjected to liquid chromatography-mass spectrometry analysis. Based on the results of the OPLS-DA model, differential metabolites were screened out using the volcano plot. Those variables with a projected effect (VIP) greater than 1.0 were considered to be significantly different between different categories. Univariate statistical analysis (T test or Mann-Whitney U test) was used to obtain the differential metabolites between the two groups. The thresholds in the volcano plot were set at p < 0.05 and |log2FC| ≥ 0. Pathway enrichment analysis of differential metabolites was performed using the Pathway-Associated Metabolite Set (SMPDB) library.

[0038] A non-targeted metabolomics approach was used to compare differential metabolites in mouse tumor tissues. Figure 3 .Depend on Figure 3 It can be seen that compared with the model group, the relative abundance of 10 differential metabolites in the Lactobacillus murinus treatment group increased significantly, and 1 differential metabolite was downregulated ( Figure 3A to Figure 3 C). We also analyzed the metabolites of Lactobacillus murinus itself and found that compared with the blank culture medium, 30 differential metabolites were up-regulated and 52 differential metabolites were down-regulated in the culture supernatant of Lactobacillus murinus ( Figure 3 D to Figure 3 F). Butyric acid derivatives 2-hydroxybutyric acid and 2-hydroxy-3-methylbutyric acid were increased both in tumor tissue and in the metabolites of Lactobacillus murinus itself. In tumor tissue, butyric acid content was significantly increased ( Figure 3 G to Figure 3 H).

[0039] Example 4: Mouse CD8 + T cell extraction and activation

[0040] The mice were euthanized and the spleen was quickly removed. The spleen was placed on a 200-mesh nylon mesh in a culture dish and ground with a syringe, and PBS solution was continuously added during the grinding process. The spleen cell suspension was placed in a centrifuge tube and centrifuged at 350g for 5 minutes, and the supernatant was discarded. 1 mL of 1×ACK red blood cell lysis buffer was added, and the red blood cells were gently pipetted to mix. The cells were lysed at room temperature for 2 minutes, centrifuged at 350g for 5 minutes, and resuspended in RPMI 1640 complete medium and counted. Mouse CD8 + T cells were purified using an isolation kit and then cultured in RPMI-1640 medium supplemented with mouse CD3 / CD28 magnetic beads and 30 U / mL recombinant mouse IL-2.

[0041] Example 5: Flow cytometry analysis

[0042] (1) Sample preparation: CD8 + T cells were counted and added to 96-well plates at 3 × 10 5 Cells / well were added, butyrate was diluted to 500 μM in RPMI 1640 (containing 10% FBS, 1% PS) cell culture medium, and then diluted to 250 μM in multiple ratios, and cultured for 48 hours. Cells in the 96-well plate were aspirated and washed 3 times with PBS solution.

[0043] (2) Staining: Add CD3 and CD8 flow cytometry antibodies respectively and incubate at 4°C in the dark for 30 min.

[0044] (3) Fixation and permeabilization: Add 200 μL 1% BSA / PBS solution to resuspend the cells, then add 200 μL Fixation Buffer, mix gently, incubate at room temperature in the dark for 30 min, add 1 mL 1× Permeabilization working solution, centrifuge at 600×g for 5 min, discard the supernatant. Add 100 μL 1× Permeabilization Working Solution to resuspend the cells.

[0045] (4) Add antibodies: Add the corresponding intracellular antibodies Granzyme B and IFN-γ to the flow tube, mix well and incubate at room temperature in the dark for 30 minutes. Add 2 mL of 1% BSA / PBS solution to each tube, centrifuge at 600×g for 5 minutes, and discard the supernatant. Add 200 μL of 1% BSA / PBS solution to resuspend.

[0046] (5) The data were analyzed using FlowJo 7.6 software.

[0047] Example 6: Detection of anti-tumor activity of butyric acid

[0048] The changes in the content of butyric acid in the tumor tissue and serum of mice in each treatment group of Example 1 were detected respectively. The results are shown in Figure 4 A and 4B. Figure 4 A and 4B show that compared with the model group, the butyric acid content in the tumor tissue and serum of the mice treated with Lactobacillus murinus increased significantly, especially the butyric acid content in the tumor tissue increased more significantly. This result reveals that the effect of Lactobacillus murinus on enhancing the anti-gastric cancer effect of αPD-1 monoclonal antibody is related to the increase in the butyric acid content in the tumor tissue, and butyric acid may have anti-tumor activity. To this end, the present invention further carried out the anti-tumor activity detection of butyric acid, specifically:

[0049] 1. Cell proliferation assay CCK8 method

[0050] Human gastric cancer cells AGS and mouse gastric cancer cells MFC were inoculated in 96-well plates, 3000 cells per well, 3 replicates per group, and PBS was added to the edge wells of the 96-well plate. After culturing overnight in a 37°C, 5% CO2 incubator, 200mM sodium butyrate was added, and then diluted with culture medium in turn to form a concentration gradient, cultured at 37°C for 48h, the cell supernatant was discarded and washed 3 times with PBS, 10μL of CCK8 detection solution was added to each well, gently shaken and tapped to mix, and continued to incubate in the dark for 4 hours, and then the absorbance value was measured at a wavelength of 450nm using an enzyme marker.

[0051] Results Figure 4 C. By Figure 4 C shows that the IC of AGS cells treated with sodium butyrate 50=3.130±0.462mM, IC of MFC cells 50 =17.43±4.66mM, indicating that sodium butyrate can effectively inhibit the activity of the above two gastric cancer cells.

[0052] 2. Colony formation

[0053] The cells were inoculated on a 6-well plate and cultured overnight (5% CO2, 37°C); different proportions of lymphocytes and tumor cells were added to the 6-well plate and cultured for 48 hours; the drug-containing culture medium was discarded and rinsed twice with 1×PBS, and then a culture medium containing 10% serum was added, and the culture was continued for about 5 days until visible and easy-to-count colonies were obtained and the culture was stopped; the cells in the six-well plate were fixed with 4% paraformaldehyde for 15-20 minutes, washed with 1×PBS after fixation, and then stained with 0.1% crystal violet for 30 minutes. After staining, the residual crystal violet was fully washed away, and it was allowed to air dry naturally. The colony size was observed under a digital biological microscope (Sony, Japan), and the colony image was taken.

[0054] Results Figure 4 D. By Figure 4 D shows that CD3 / CD28 and CD8 provided by IL-2 in mice treated with sodium butyrate + T cells inhibited the colony formation of MFC cells.

[0055] 3. Butyrate inhibits tumor growth

[0056] Experimental animals: 36 5-6 week-old male 615 mice (18-20 g) were purchased from Guangdong Zhiyuan Biopharmaceutical Technology Co., Ltd. (license number SYXK (Yue) 2021-0251). The experimental mice were raised under SPF conditions, the bedding was changed every three days, the light pattern of alternating light and dark was implemented, the temperature was maintained at 20-24 ° C, and the humidity was maintained at 35-50%. MCF tumor cells (8×10 4 100 μL of cell suspension resuspended in RPMI 1640 medium was subcutaneously injected into the skin of the inner thigh of the mouse until the tumor volume reached 50-100 mm 3 The mice were randomly divided into 2 groups (n=6 in each group) according to their tumor volumes. The tumor volume was calculated according to the following formula: Volume (mm 3 )=0.5×long diameter(mm)×short diameter 2 (mm). Use a 0.3mL×12.7mm disposable syringe to inject 100μL of 200mM butyric acid into the tumor. The control group mice were injected with 100μL PBS solution twice a week. The tumor volume and body weight of the mice were measured twice a week. When the tumor volume reached 1200-1500mm 3 The experimental mice were killed by cervical dislocation, and the tumors were removed and the tumor weights were recorded.

[0057] Results Figure 4 E. By Figure 4 E It can be seen that butyrate can inhibit the volume of subcutaneous MFC tumors in mice and reduce tumor weight.

[0058] Example 7: Anti-tumor activity detection of Lactobacillus murinus

[0059] The inhibition rate (%) of Lactobacillus murine (multiplicity of infection MOI = 20) on tumor cell growth was calculated by cell viability experiment. The specific steps were as follows: tumor cell lines were cultured with RPMI 1640 cell culture medium (containing 10% FBS, 1% PS). When the cell lines reached half the growth period, Lactobacillus murine (multiplicity of infection MOI = 20) was cultured in cell culture medium without penicillin and streptomycin at 37°C for 6 hours, the cell supernatant was discarded and washed 3 times with PBS, 10 μL of CCK8 detection solution was added to each well, and the mixture was gently shaken and tapped to mix, and the cells were incubated in the dark for 4 hours. The absorbance was then measured at a wavelength of 450 nm using an enzyme reader, and the inhibition rate was calculated. The results are shown in Table 1.

[0060] Table 1 Inhibition rate of tumor cell growth by Lactobacillus murinus (MOI=20) (%)

[0061] Cell Type Inhibition rate (%) A2780 (human ovarian cancer) 43.34±0.48％ OV2008 (human ovarian cancer) 23.84±1.39％ MCF-7 (human breast cancer) 47.7±1.58％ MB-231 (human breast cancer) 65.58±3.13％ Siha (human cervical cancer) 54.81±6.72％ HeLa (human cervical cancer) 27.6±4.19％ A549 (human lung cancer) 53.91±1.21％ HepG2 (human liver cancer) 24.6±5.41％ MC38 (human colorectal cancer) 30.31±1.24％ MCF (mouse gastric cancer cell) 40.26±3.37％ AGS (human gastric cancer cells) 45.39±6.32％

[0062] As shown in Table 1, Lactobacillus murine has inhibitory activity against a variety of tumor cells, including ovarian cancer cells, breast cancer cells, cervical cancer cells, lung cancer cells, liver cancer cells, colorectal cancer cells, and gastric cancer cells, and is expected to be used in the development of anti-tumor drugs.

[0063] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of Lactobacillus murinus in the preparation of drugs that enhance the efficacy of immune checkpoint inhibitors.

2. The use according to claim 1, characterized in that: The immune checkpoint inhibitor is a monoclonal antibody against the PD-1 / PD-L1 immune checkpoint.

3. The use according to claim 2, characterized in that: The immune checkpoint inhibitor is a PD-1 monoclonal antibody.

4. Application of Lactobacillus murinus in the preparation of anti-tumor drugs.

5. An antitumor composition, characterized in that: Contains Lactobacillus murinus and an immune checkpoint inhibitor.

6. The antitumor composition according to claim 5, characterized in that: The immune checkpoint inhibitor is Monoclonal antibodies targeting the PD-1 / PD-L1 immune checkpoints.

7. The antitumor composition according to claim 6, characterized in that: The immune checkpoint inhibitor is a PD-1 monoclonal antibody.

8. Use of the antitumor composition according to any one of claims 5 to 7 in the preparation of antitumor drugs.

9. The use according to claim 4 or 8, characterized in that: The anti-tumor drug is a drug for ovarian cancer, breast cancer cells, cervical cancer, lung cancer, liver cancer, colorectal cancer or gastric cancer.

10. An anti-tumor drug, characterized in that: The invention comprises an effective amount of Lactobacillus murinus or the anti-tumor composition according to any one of claims 5 to 7 as an active ingredient, and a pharmaceutically acceptable carrier.

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