Application of Megasphaera bacteria in the preparation of drugs for treating cancer

Abstract

The present invention relates to the use of Megasphaera bacteria in the preparation of drugs for treating cancer, belonging to the field of biomedicine technology. The present invention provides the use of Megasphaera bacteria XA-511 in the preparation of drugs for preventing and / or treating cancer. XA-511 can cooperate with anti-PD-1 antibodies to reduce the number and volume of tumors in mice with orthotopic colorectal cancer models, inhibit the formation of tumors in mice with orthotopic colorectal cancer models, and can cooperate with anti-PD-1 antibodies to enhance the expression of CD8 + T and CD4 + XA-511 can synergize with anti-PD-1 antibodies to reduce tumor weight and volume in pancreatic cancer model mice, and can synergize with anti-PD-1 antibodies to reduce CD4 + T infiltration, and can cooperate with anti-PD-1 antibodies to increase CD8 + Infiltration of T.

Description

Technical Field

[0001] The invention relates to application of Megasphaera bacteria in preparing medicine for treating cancer, and belongs to the technical field of biomedicine. Background Art

[0002] Cancer generally refers to malignant tumors, which are caused by malignant cell proliferation. They are invasive and can metastasize. They manifest as a localized mass that continues to grow, destroying normal tissue structures and potentially metastasizes to other parts of the body. Currently, cancers with high mortality rates include colorectal cancer, pancreatic cancer, breast cancer, lung cancer, stomach cancer, liver cancer, and esophageal cancer.

[0003] Surgical resection is a basic means of treating cancer. In clinical practice, it is generally combined with radiotherapy, chemotherapy, targeted therapy and immunotherapy for cancer. However, existing radiotherapy, chemotherapy, targeted therapy and immunotherapy methods all have many problems in clinical application. For example, PD-1 / PD-L1 immunotherapy is a type of anti-cancer immunotherapy that blocks the PD1 / PD-L1 signaling pathway to cause cancer cell death, thereby treating various types of cancer and improving patients' overall survival. However, since the start of clinical trials, PD-1 inhibitors, which PD-1 / PD-L1 immunotherapy relies on, have shown resistance in 15-20% of patients. In addition, PD-1 inhibitors have relatively strong side effects. In addition, some patients do not respond to PD-1 / PD-L1 immunotherapy. This secondary resistance caused by low response efficiency also limits the clinical application of PD-1 / PD-L1 immunotherapy (see reference: Jin-YuSun et al. 2005).

[0004] Therefore, there is an urgent need to find drugs that can effectively treat cancer with less drug resistance and side effects, or to find methods that can reduce the drug resistance and side effects of PD-1 inhibitors and improve the response efficiency of PD-1 / PD-L1 immunotherapy. Summary of the Invention

[0005] To solve the above problems, the present invention provides a use of Megasphaera indica XA-511 in the preparation of a drug. The Megasphaera indica XA-511 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 23400 and a deposit date of September 13, 2021. The drug has at least one of the following uses:

[0006] (a) preventing and / or treating cancer; and / or,

[0007] (b) Promote CD8 +T cells produce tumor suppressor factors.

[0008] The Megasphaera bacteria XA-511 was derived from frozen fecal samples of healthy people in the Shenzhen area. The strain was sequenced and analyzed, and its 16SrDNA sequence was shown as SEQ ID NO.1. The sequence obtained by sequencing was compared with the nucleic acid sequence in NCBI, and the results showed that the strain belonged to the Megasphaera bacteria and was named Megasphaera bacteria XA-511.

[0009] In one embodiment of the present invention, the tumor suppressor factors include IFN-γ, Granzyme B and / or TNF-α.

[0010] In one embodiment of the present invention, the cancer includes pancreatic cancer and / or colorectal cancer; the colorectal cancer includes colon cancer and / or rectal cancer.

[0011] In one embodiment of the present invention, the active ingredient of the drug comprises live bacteria, inactivated bacteria and / or metabolites of Megasphaera bacteria XA-511.

[0012] In one embodiment of the present invention, the method for preparing the active ingredient comprises: culturing Megasphaera bacteria XA-511 in a culture medium to obtain a culture fluid; and treating the culture fluid to obtain live bacteria, inactivated bacteria, and / or culture supernatant of Megasphaera bacteria XA-511 to obtain the active ingredient. The culture supernatant contains metabolites of Megasphaera bacteria XA-511.

[0013] In one embodiment of the present invention, the treatment includes: centrifuging the culture solution to obtain live bacteria and / or culture supernatant of Megasphaera bacteria XA-511; and / or sterilizing the culture solution and then centrifuging it to obtain inactivated bacteria and / or culture supernatant of Megasphaera bacteria XA-511.

[0014] In one embodiment of the present invention, the components of the culture medium include a carbon source; the carbon source includes fructose, glucose, maltose, mannitol, sucrose and / or lactose.

[0015] In one embodiment of the present invention, the culture medium is RCM medium, RCM medium with additional fructose as a carbon source, acetic acid medium with additional glucose as a carbon source, acetic acid medium with additional maltose as a carbon source, acetic acid medium with additional mannitol as a carbon source, acetic acid medium with additional sucrose as a carbon source, or acetic acid medium with additional lactose as a carbon source.

[0016] In one embodiment of the present invention, the active ingredient of the drug further comprises a cancer immunotherapeutic agent.

[0017] In one embodiment of the present invention, the cancer immunotherapeutic agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

[0018] In one embodiment of the present invention, the composition of the drug further comprises a pharmaceutically acceptable carrier.

[0019] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes a surfactant, an excipient, a stabilizer, a suspending agent, an isotonic agent, a preservative, a filler, a disintegrant, an antioxidant, an emulsifier, a coating agent, an adhesive, a lubricant and / or a flavoring agent.

[0020] In one embodiment of the present invention, the dosage form of the drug is ointment, suppository, aerosol, oral liquid, injection, tablet, capsule, granule or powder; the oral liquid is solution, syrup, emulsion or suspension.

[0021] In one embodiment of the present invention, the drug is administered into the digestive system or injected into the tumor.

[0022] In one embodiment of the present invention, the administration into the digestive system is oral or enema.

[0023] The present invention also provides a pharmaceutical composition, wherein the composition comprises a pharmaceutically active ingredient; the pharmaceutically active ingredient comprises Megasphaera bacteria XA-511; the Megasphaera bacteria XA-511 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 23400 and a deposit date of September 13, 2021, and the pharmaceutical composition has at least one of the following uses:

[0024] (a) preventing and / or treating cancer; and / or,

[0025] (b) Promote CD8 + T cells produce tumor suppressor factors.

[0026] In one embodiment of the present invention, the tumor suppressor factors include IFN-γ, Granzyme B and / or TNF-α.

[0027] In one embodiment of the present invention, the cancer includes pancreatic cancer and / or colorectal cancer; the colorectal cancer includes colon cancer and / or rectal cancer.

[0028] In one embodiment of the present invention, the pharmaceutical active ingredient comprises live bacteria, inactivated bacteria and / or metabolites of Megasphaera bacteria XA-511.

[0029] In one embodiment of the present invention, a method for preparing the active pharmaceutical ingredient comprises: culturing Megasphaera bacteria XA-511 in a culture medium to obtain a culture fluid; and treating the culture fluid to obtain live bacteria, inactivated bacteria, and / or culture supernatant of Megasphaera bacteria XA-511 to obtain the active ingredient. The culture supernatant contains metabolites of Megasphaera bacteria XA-511.

[0030] In one embodiment of the present invention, the treatment includes: centrifuging the culture solution to obtain live bacteria and / or culture supernatant of Megasphaera bacteria XA-511; and / or sterilizing the culture solution and then centrifuging it to obtain inactivated bacteria and / or culture supernatant of Megasphaera bacteria XA-511.

[0031] In one embodiment of the present invention, the components of the culture medium include a carbon source; the carbon source includes fructose, glucose, maltose, mannitol, sucrose and / or lactose.

[0032] In one embodiment of the present invention, the culture medium is RCM medium, RCM medium with additional fructose as a carbon source, acetic acid medium with additional glucose as a carbon source, acetic acid medium with additional maltose as a carbon source, acetic acid medium with additional mannitol as a carbon source, acetic acid medium with additional sucrose as a carbon source, or acetic acid medium with additional lactose as a carbon source.

[0033] In one embodiment of the present invention, the pharmaceutical active ingredient further comprises a cancer immunotherapeutic agent.

[0034] In one embodiment of the present invention, the cancer immunotherapeutic agent comprises an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

[0035] In one embodiment of the present invention, the ingredients of the pharmaceutical composition further include a pharmaceutically acceptable carrier.

[0036] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes a surfactant, an excipient, a stabilizer, a suspending agent, an isotonic agent, a preservative, a filler, a disintegrant, an antioxidant, an emulsifier, a coating agent, an adhesive, a lubricant and / or a flavoring agent.

[0037] In one embodiment of the present invention, the dosage form of the pharmaceutical composition is ointment, suppository, aerosol, oral liquid, injection, tablet, capsule, granule or powder; the oral liquid is solution, syrup, emulsion or suspension.

[0038] In one embodiment of the present invention, the pharmaceutical composition is administered by injection into the digestive system or into a tumor.

[0039] In one embodiment of the present invention, the administration into the digestive system is oral or enema.

[0040] The technical solution of the present invention has the following advantages:

[0041] The present invention provides the use of Megasphaeraindica XA-511 in the preparation of drugs for preventing and / or treating cancer. Studies have shown that

[0042] For CD8 + T cells: The culture supernatant of Megasphaera bacteria XA-511 can significantly promote CD8 + T cells produce IFN-γ, Granzyme B, and TNF-α (IFN-γ, Granzyme B, and TNF-α are all tumor suppressor factors);

[0043] For colorectal cancer: Live and inactivated bacteria of the genus Megasphaera XA-511 can synergize with anti-PD-1 antibodies to significantly reduce the number and volume of tumors in mice with orthotopic colorectal cancer models, and inhibit the formation of tumors in mice with orthotopic colorectal cancer models; Live bacteria of the genus Megasphaera XA-511 can synergize with anti-PD-1 antibodies to significantly enhance the expression of CD8 + T cells and CD4 + T cell infiltration and stimulation of the production of the tumor suppressor cytokine IFN-γ by these two types of cells;

[0044] For pancreatic cancer: Live bacteria of the genus Megasphaera XA-511 can significantly reduce the tumor weight and volume in pancreatic cancer model mice, and can also reduce the number of Treg cells and CD4 in the tumor tissue of pancreatic cancer model mice. + The live bacteria of Megasphaera XA-511 can synergize with anti-PD-1 antibodies to significantly reduce the tumor weight and volume in pancreatic cancer model mice, and can synergize with anti-PD-1 antibodies to significantly reduce the number of CD4 T cells in the tumor tissue of pancreatic cancer model mice. + Furthermore, it can synergize with anti-PD-1 antibodies to significantly increase the number of CD8 T cells in tumor tissues of pancreatic cancer model mice. + T cell infiltration,

[0045] Therefore, the Megasphaera bacterium XA-511 has great application prospects in the preparation of drugs for preventing and / or treating cancer.

[0046] Biomaterial Deposit

[0047] A strain of Megasphaeraindica XA-511, taxonomically named Megasphaera asp., was deposited in the General Microbiology Center of the China Culture Collection Administration on September 13, 2021, with the deposit number CGMCC No. 23400, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Statistics of the total number of tumors and the number of tumors of different sizes in each group at the end point of the in situ colorectal cancer model mouse experiment with Megasphaera bacteria XA-511. Figure 1 In the table, “*” indicates that the P value of the difference between the two groups is less than 0.05, indicating that there is a significant difference.

[0049] Figure 2 : Distribution of total tumor volume in each group at the end point of the in situ colorectal cancer model mouse experiment with Megasphaera bacteria XA-511. Figure 2 In the table, “*” indicates that the P value of the difference between the two groups is less than 0.05, indicating that there is a significant difference.

[0050] Figure 3 : Photos of tumors in different groups of mice at the end point of the orthotopic colorectal cancer model mouse model induced by Megasphaera bacteria XA-511.

[0051] Figure 4 :The experimental endpoint of the in situ colorectal cancer model mouse model of Megasphaera XA-511 was the expression of CD8 + T cells and CD8 IFN-γ-producing cytokines + The relative number of T cells. Figure 4 In the table, “*” indicates that the P value of the difference between the two groups is less than 0.05, indicating that there is a significant difference.

[0052] Figure 5 :The experimental endpoint of the in situ colorectal cancer model mouse model of Megasphaera XA-511 was the number of CD4 + T cells and CD4 T cells that produce IFN-γ cytokines + The relative number of T cells. Figure 5 In the table, “*” indicates that the P value of the difference between the two groups is less than 0.05, indicating that there is a significant difference.

[0053] Figure 6 : Line graph showing the relative volume changes of tumors in different groups of mice in the pancreatic cancer model mouse experiment with Megasphaera bacteria XA-511. Figure 6In the figure, the dotted line represents the control group, the square dotted line represents the anti-PD-1 antibody monotherapy group, the upper triangle line represents the XA-511 monotherapy group, and the lower triangle line represents the XA-511 and anti-PD-1 antibody combination group. "*" indicates that the difference between the two groups is significantly different with a P value less than 0.0001.

[0054] Figure 7 : Distribution of tumor weights in each group of mice at the end of the experiment in a pancreatic cancer model mouse model induced by Megasphaera bacteria XA-511. Figure 7 In the table, “***” indicates that the P value of the difference between the two groups is less than 0.001, indicating a significant difference.

[0055] Figure 8 :The experimental endpoint of pancreatic cancer model mice induced by Megasphaera bacteria XA-511 was the expression of Treg cells and CD8 + T cells, CD4 + The distribution ratio of T cells and MDSC cells. Figure 8 In the table, “*” indicates that the P value of the difference between the two groups is less than 0.05, indicating that there is a significant difference. DETAILED DESCRIPTION

[0056] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0057] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0058] The culture media involved in the following examples are shown in Table 1.

[0059] Table 1 Culture medium

[0060]

[0061] Experimental Example 1: Acquisition of Megasphaera bacteria XA-511

[0062] This experimental example provides a process for obtaining Megasphaera bacteria XA-511. The specific process is as follows:

[0063] Selection of mycobacterial samples:

[0064] Shenzhen Weizhijun Biotechnology Co., Ltd. recruited a large number of healthy donors, obtained fresh stool samples, extracted metagenomic DNA, and analyzed the bacterial flora and abundance within the stool samples. Based on the sequencing results, a donor sample with a high abundance of the genus Megasphaera was selected, designated F14 (Megasphaera abundance: 2.10%), for targeted screening of Megasphaera strains.

[0065] Strain isolation:

[0066] In the anaerobic operating table, 1 g of fecal sample from the donor was evenly suspended in 10 mL of PBS buffer (pH 7.4 ± 0.1, 1×, purchased from Corning Cellgro, product number 21-040-CV), and gradient diluted to 10 -6 Dilution. Pipette 100mL of 10 -6 The dilution was placed on a reinforced Clostridium medium (ATCC 2107) agar plate and evenly spread using a disposable sterile spreading stick, and anaerobically cultured at 37°C for 3 days.

[0067] After 3 days, colonies with a diameter of 2.0 to 5.0 mm, slightly raised, round, shiny, and light yellowish surface were selected. Single colonies were picked with a sterile loop and streaked onto plates for purification and culture to obtain a pure strain. This strain was designated as XA-511.

[0068] Strain sequencing and identification:

[0069] A single colony was picked from a single colony of XA-511 bacteria by self-division and propagation using a sterile inoculating loop and transferred to 5 mL of fortified Clostridium liquid medium (ATCC 2107) and cultured in an anaerobic operating chamber for 20 to 24 hours. 1 mL of bacterial suspension was taken and strain DNA was extracted from the bacterial suspension using a DNA extraction kit. The strain gene fragment was PCR amplified using universal primers 27F (SEQ ID NO: 2) / 1492R (SEQ ID NO: 3). The amplified product was gel-cleaved and purified, and the fragment was extracted and recovered using a SanPrep column-based DNA gel recovery kit (REF: 518131-0100). The target fragment was ligated to a T vector using a pGM-T cloning kit (Cat# VT302-02) at 16°C overnight. The ligation product was transformed into TOP10 competent cells and screened for blue-white spots. Positive recombinant clones were selected and cloned using universal primers T7 (SEQ ID NO: 4) / SP6 (SEQ ID NO: 5). NO:5) was sequenced; the 16S ribosomal RNA gene sequence of the isolated bacterium XA-511 is shown in SEQ ID NO:1;

[0070] The components and contents of the 50 μL PCR amplification reaction system are shown in Table 2, and the reaction conditions are shown in Table 3.

[0071] Table 2 PCR amplification reaction system

[0072] Volume / μl Reagents 25 Taq PCR Mix (2X, containing blue dye) 2 27F 2 1492R 3 XA-511 DNA (concentration: 38.1 ng / μL) 18 <![CDATA[ddH2O]]>

[0073] Table 3 PCR amplification reaction conditions

[0074]

[0075] Strain preservation:

[0076] XA-511 (Megasphaera sp.) was deposited with the China General Microbiology Center under the China Culture Collection Administration on September 13, 2021, under the accession number CGMCC No. 23400. The address of the deposit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Further identification of XA-511 confirmed it to be Megasphaera indica.

[0077] Experimental Example 2: Effect of Megasphaera XA-511 on CD8 + Effects of cytokine production by T cells

[0078] CD8 + T cells, also called cytotoxic T lymphocytes, are a type of specific T cells that can secrete various cytokines to participate in immune responses. + T cells, under the stimulation of anti-CD3 / CD28, can produce functional cytokines such as IFN-γ, Granzyme B and TNF-α. Adding different strains of heat-killed bacteria or culture supernatants to the culture medium while stimulating with anti-CD3 / CD28 can detect whether different strains of heat-killed bacteria or culture supernatants can directly promote or inhibit CD8 + T cells produce cytokines, which reflects the effects of heat-killed bacteria of different strains and culture supernatants on CD8 + Therefore, this experimental example uses this reaction system to explore the effects of heat-killed Megasphaera XA-511 bacteria and culture supernatant on CD8 + The experimental process is as follows:

[0079] Preparation of culture supernatant:

[0080] Single colonies of the Megasphaera bacteria XA-511 obtained in Experimental Example 1 were picked and inoculated into fructose medium, RCM medium, glucose medium, maltose medium, mannitol medium, sucrose medium and lactose medium, and cultured anaerobically at 37°C until the end of the logarithmic growth phase to obtain the corresponding culture fluids; the OD values ​​of the XA-511 culture fluids were respectively adjusted using fructose medium, RCM medium, glucose medium, maltose medium, mannitol medium, sucrose medium and lactose medium. 600 The concentration was adjusted to 1 to obtain the corresponding bacterial solution; the bacterial solution was centrifuged at 4°C and 5000g for 5 min, and then filtered using a 0.2 μm filter (Millipore) to obtain the XA-511 culture supernatant; the culture supernatant was aliquoted into sterile tubes according to 1 mL and stored at -80°C for later use.

[0081] OT-1 mouse lymph node CD8 + Isolation and culture of T cells:

[0082] CD8 T cells were extracted from OT-1 mouse lymph nodes of C57BL / 6 background OT-1 mice (6-8 weeks old, weighing 20-24 g, SPF grade, obtained from the Institute of Immunology, Tsinghua University). + T cells. Animals were housed in the SPF IVC animal room at the Tsinghua University Experimental Animal Center under a 12:12 light cycle. A sterile PBS solution containing 0.5 μg / mL anti-CD3 (purchased from Thermo Fisher) and 1 μg / mL anti-CD28 (purchased from Thermo Fisher) was added to a 24-well plate at a volume of 500 μL per well. The plates were incubated at 4°C for 16 hours to obtain 24-well plates coated with anti-CD3 / 28. Mice were killed, and inguinal, axillary, and mesenteric lymph nodes were obtained. Lymph nodes were minced and ground using a syringe plunger. The cells were filtered through a 70 μm yellow mesh into a flow cytometer. The cells in the flow cytometer were centrifuged at 1700 rpm for 5 minutes, the supernatant discarded, and the cells counted. Biotin-labeled antibody staining solution (Biotin anti-mouse / human CD11b, model 101204; Biotin anti-mouse / human CD45R / B220, model 103204; Biotin anti-mouse Ly-6G / Ly-6C (GR-1), model 108403; Biotin anti-mouse CD4, model 100404; all the above antibody reagents were purchased from Biolegend) was prepared and mixed according to the kit. 6Add 100 cells to the flow tube to resuspend the cells and stain at 4°C for 30 minutes. After staining for 30 minutes, first add 4mL PBS buffer to the flow tube to wash the cells, then centrifuge the cells in the flow tube at 1700rpm for 5 minutes, discard the supernatant, and end the staining. After the staining is completed, add 1mL Isolation buffer (PBS buffer with 1% fetal bovine serum and 2mM EDTA, where % refers to volume percentage) to the flow tube to resuspend the cells. The resuspended cells were added with 50μg Dynabeads per 10 6 Add the amount of cells to the EP tube containing the cleaned Dynabeads Biotin Binder kit (purchased from Thermo Fisher) and incubate at 4°C for 30 minutes. After the incubation, place the EP tube on a magnet for 2 minutes, take the supernatant and add it to a new EP tube, centrifuge at 1700 rpm for 5 minutes to obtain the cell pellet. Use fresh CD8 + Resuspend the cells in the EP tube in T cell culture medium (i.e., 1640 medium supplemented with 10% fetal bovine serum, 50 mM β-mercaptoethanol, 300 IU / mL interleukin-2, and 0.5 μg / L phosphatidylserine, where % refers to volume percentage, and 1640 medium was purchased from Gibco) to a concentration of 5 × 10 5 The resuspended cells were added to a 24-well plate coated with anti-CD3 / 28 and cultured in a constant temperature incubator at 37°C and 5% (v / v) CO2. The cells were passaged at a ratio of 1:3 every day to obtain cells containing OT-1 mouse lymph node CD8 + T cell culture medium.

[0083] The supernatants of different culture media of XA-511 stimulated CD8 + T cells:

[0084] After adding 0.5 μg / mL anti-CD3 (purchased from ThermoFisher) and 1 μg / mL anti-CD28 (purchased from Thermo Fisher) anti-CD3 / 28 antibody solution (solvent is sterile PBS solution) to a 96-well plate at a volume of 100 μL per well, the plate was incubated at 4°C for 16 hours to obtain a 96-well plate coated with anti-CD3 / 28. + T cells were cultured at 1 × 10 5 OT-1 mouse lymph node CD8 +T cells were seeded into 96-well plates coated with anti-CD3 / 28. Then, blank culture medium (fructose, RCM, glucose, maltose, mannitol, sucrose, and lactose) and XA-511 culture supernatants from the corresponding culture medium were added to the anti-CD3 / 28-coated 96-well plates at 1% of the cell culture volume. The plates were incubated in a 37°C, 5% (v / v) CO2 incubator for 21 hours. After the incubation period, brefeldin A was added to the anti-CD3 / 28-coated 96-well plates at a volume of 1 μL per well and incubated in a 37°C, 5% (v / v) CO2 incubator for 3 hours to block cytokine secretion into the culture medium. After the incubation, the 96-well plate was centrifuged at 1700 rpm for 5 min, and the supernatant was used to detect the secretion of cytokines (Granzyme B, IFN-γ and TNF-α) by ELISA. The cell pellet was washed with FACS buffer and then analyzed by flow cytometry to detect the secretion of cytokines (Granzyme B, IFN-γ and TNF-α) (ELISA kit purchased from Biolegend: ELISA MAX TM Standard Set Mouse Granzyme B, Cat. No. 439207; ELISAMAX TM Standard Set Mouse IFN-γ, Catalog No. 430801; ELISA MAX TM Standard Set Mouse TNF-α, Catalog No. 430901). Detection and analysis results are shown in Tables 4 and 5.

[0085] As shown in Tables 4 and 5, compared with the control, the culture supernatants obtained by culturing Megasphaera XA-511 using fructose medium, RCM medium, glucose medium, maltose medium, mannitol medium, sucrose medium and lactose medium can increase the production of IFN-γ, Granzyme B and TNF-α in CD8 + The percentage of T cells, among which, the culture supernatant obtained by culturing Megasphaera XA-511 with fructose medium, RCM medium, glucose medium, maltose medium and lactose medium increased the CD8 T cells that produce IFN-γ, Granzyme B and TNF-α. + The effect of T cells was particularly significant.

[0086] Table 4 XA-511 culture supernatants obtained from different culture media stimulated CD8 + The result of cytokine production by T cells

[0087]

[0088]

[0089] Table 5 XA-511 culture supernatants obtained from different culture media stimulated CD8 + The result of cytokine production by T cells

[0090]

[0091] Experimental Example 3: Effects of Megasphaera XA-511 on Colorectal Cancer Model Mice

[0092] This experiment provides an experimental study on the effects of Megasphaera bacteria XA-511 on a colorectal cancer model mouse model. This experiment was reviewed by the Experimental Animal Care and Use Committee of Shenzhen Top Biotechnology Co., Ltd. The experimental procedures are as follows:

[0093] Preparation of live bacteria and inactivated bacteria:

[0094] A single colony of the Megasphaera bacteria XA-511 obtained in Experimental Example 1 was selected and inoculated into a fortified Clostridium liquid medium (ATCC 2107), and cultured anaerobically at 37°C for 24 hours to obtain a culture medium; the culture medium was divided into two portions, one portion was centrifuged at 1700 rpm for 5 minutes, and then the cell pellet was obtained to obtain live bacteria of the Megasphaera bacteria XA-511, and the other portion was first heated at 110°C for 15 minutes, and then centrifuged at 1700 rpm for 5 minutes, and then the cell pellet was obtained to obtain inactivated bacteria of the Megasphaera bacteria XA-511.

[0095] Model establishment and drug administration:

[0096] Fifty-five SPF-grade Balb / c mice (male, 6 weeks old, purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were randomly divided into 6 groups: blank control group, experimental control group, experimental observation group, Anti-PD-1 antibody monotherapy group, XA-511 + anti-PD-1 antibody combination therapy group, and XA-511 inactivated bacteria + anti-PD-1 antibody combination therapy group.

[0097] All mice were individually caged and housed in an SPF animal room for one week at a room temperature of (24±2)°C, with a 12-hour day-night cycle and free access to water. After acclimation, mice other than the blank control group were injected with azoxymethane (AOM) to induce orthotopic colorectal cancer. Model establishment and drug administration began after grouping. The following procedures were performed: On day 1 of the experiment, mice other than the blank control group were intraperitoneally injected with 12.5 mg / kg of AOM (the day of AOM injection was designated Day 0). One week later, mice were given 3% (w / v, g / 100 mL) DSS solution for 7 days, followed by normal drinking water for 2 weeks. On week 4, mice were given 2.5% (w / v, g / 100 mL) DSS solution for 7 days, followed by normal drinking water for 2 weeks. Each cycle consisted of one week of DSS followed by two weeks of normal drinking water, with a repeat cycle starting on week 7. On day 37 of the experiment, mice in each group began drug administration according to Table 6.

[0098] The experiment ended on day 79. After the experiment, the mice were dissected, the colorectal length of each mouse was recorded, and photos were taken. The colon was cut open longitudinally, the number of tumors was counted, the tumor weight was weighed, the long / short diameter of each tumor was measured, and the tumor volume was calculated (tumor volume = 1 / 2 × tumor long diameter). 2 × tumor short diameter). The spleen of the mouse was taken (after processing the fat), the spleen weight was measured, and the spleen was stored in RPMI medium. The proportion of different immune cells in the spleen was detected by flow cytometry. The detection and analysis results are shown in Figures 1 to 5 .

[0099] The flow cytometry analysis process is as follows:

[0100] Place the tumor in a 100mm 2Rinse the culture dish with PBS to remove fat, lesions, and necrotic areas from the tumor tissue. After thoroughly mincing the tumor tissue with forceps and scissors, transfer it to a Miltenyi Gentle MACS tube C and add 5 mL of Miltenyi Tumor Dissociation Kit digestion buffer. Tighten the tube C and insert it upside down into the Gentle MACS sleeve. Select the appropriate Gentle MACS program and run it. After the program terminates, remove the tube C and place it in a constant temperature shaking water bath at 37°C and 220 rpm for 30 minutes. After digestion, insert the tube C upside down into the Gentle MACS sleeve again and run the appropriate program. Add 5 mL of PBS buffer to the tube C, filter through a 70 μm filter into a 50 mL centrifuge tube, and collect the filtrate. Centrifuge at 400 g for 5 minutes and discard the supernatant (retain the filtered tissue clumps when discarding the supernatant). Lyse the red blood cells: Add 5 mL of Miltenyi Red Blood Cell Lysis Buffer and incubate at room temperature (25°C) in the dark for 5 minutes. Add 5 mL of PBS buffer and shake to mix thoroughly. Centrifuge at 400 g for 5 min and discard the supernatant. Add 5 mL of PBS buffer and mix thoroughly for subsequent staining steps.

[0101] 1×10 6 Resuspend the cells in 100 μL of live / dead solution (1 / 1000 live / dead in PBS) and incubate at 4°C for 10 min. Wash once with 1 mL of stain buffer, centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend the cells in 50 μL of Fcblock (1 / 100 Fcblock in stain buffer) and incubate at room temperature (25°C) in the dark for 5 min. Without washing the cells, add the surface antibody mixture prepared according to the manufacturer's instructions (see Table 7 for dye and antibody details) to the corresponding tubes and incubate at room temperature (25°C) in the dark for 15 min. Wash once with 1 mL of PBS, centrifuge at 400 g for 5 min, and discard the supernatant. Add 1 mL of FoxP3 fixative, mix on a shaker, and incubate at room temperature (25°C) in the dark for 40 min. Centrifuge at 400 g for 5 min, and discard the supernatant. Add 1 mL of 1X permeabilization buffer, wash once with 1 mL of PBS, and centrifuge at 400 g for 5 min, and discard the supernatant. Add 200 μL 1X stain buffer and load the sample (for details of other flow cytometry reagents, see Table 8). Use flow cytometer to collect data and use Kaluza 2.1 software to analyze the CD45 of different immune cell subsets. + (immune cells), CD4 + / CD45 + (CD4 T cells) and CD8 + / CD45 +Results for CD8 T cells were obtained. Dunnett's multi-comparison test was used to compare the two groups. A statistically significant difference was considered if p < 0.05.

[0102] Depend on Figure 1 Compared with the control group, the number of tumors in the anti-PD-1 antibody monotherapy group showed a nonsignificant downward trend, while the number of tumors in the XA-511 + anti-PD-1 antibody combination therapy group and the XA-511 inactivated bacteria + anti-PD-1 antibody combination therapy group was significantly reduced (p < 0.05). Furthermore, compared with the control group and the anti-PD-1 antibody monotherapy group, the total number of tumors in the XA-511 + anti-PD-1 antibody combination therapy group and the XA-511 inactivated bacteria + anti-PD-1 antibody combination therapy group was lower, with a higher proportion of tumors with a diameter of 1-2 mm and a lower proportion of tumors with a diameter of 3-4 mm. There were no tumors with a diameter greater than 4 mm, indicating that the number of large tumors in the XA-511 + anti-PD-1 antibody combination therapy group and the XA-511 inactivated bacteria + anti-PD-1 antibody combination therapy group was significantly reduced. Therefore, gavage of live and killed XA-511 bacteria synergistically with anti-PD-1 antibodies can significantly reduce the number of tumors, especially large tumors, in in situ colorectal cancer.

[0103] Depend on Figure 2 It can be seen that compared with the experimental control group, the total tumor volume of the anti-PD-1 antibody monotherapy group showed a trend of decrease, while the XA-511 + anti-PD-1 antibody combination therapy group and the XA-511 inactivated bacteria group

[0104] The total tumor volume in the combined treatment group with anti-PD-1 antibodies was significantly reduced (p<0.05), indicating that gavage with live and inactivated XA-511 bacteria can synergize with anti-PD-1 antibodies to significantly reduce tumor volume in in situ colorectal cancer.

[0105] Depend on Figure 3 The results showed that tumor formation was most severe in the control group, while the anti-PD-1 antibody monotherapy group showed some tumor suppression. The XA-511 + anti-PD-1 antibody combination therapy group and the XA-511 inactivated bacteria + anti-PD-1 antibody combination therapy group showed significant combined anti-tumor efficacy. Therefore, gavage with live and inactivated XA-511 bacteria can synergize with anti-PD-1 antibodies to inhibit the progression of in situ colorectal cancer.

[0106] Depend on Figure 4 It can be seen that compared with the experimental control group, the CD8 +The number of T cells showed an increasing trend, and the CD8 + The production of IFN-γ, a cytokine produced by T cells, was significantly enhanced (p<0.05), indicating that XA-511 live bacteria can synergize with anti-PD-1 antibodies to significantly enhance the expression of CD8 + T cell infiltration and production of tumor suppressor factors.

[0107] Depend on Figure 5 It can be seen that in the spleen of the XA-511+anti-PD-1 antibody combined treatment group, compared with the experimental control group, CD4 + The amount of T cell infiltration also tended to increase, and CD4 + The cytokine IFN-γ produced by T cells also increased significantly (p<0.05). + The main lineage in T cells mainly produces IFN-γ, so it is speculated that XA-511 live bacteria can synergize with anti-PD-1 antibodies to significantly increase the infiltration of Th1 cells and the production of tumor suppressor factors.

[0108] In summary, feeding XA-511 live bacteria can increase CD8 + T and CD4 + The infiltration of T cells stimulates the production of the tumor suppressor factor IFN-γ, thereby promoting the anti-PD-1 antibody to inhibit the development of in situ colorectal cancer.

[0109] Currently, immune checkpoint inhibitor (ICI) therapy is the mainstay of clinically used immunotherapy for colorectal cancer. However, this type of therapy demonstrates promising results only for certain CRC subtypes, with suboptimal results for others, such as pMMR / MSS and dMMR / MSI-L. Furthermore, due to the heterogeneity of solid tumors and the external microenvironment, ICI immunotherapy for solid tumors has been less effective than anticipated. In this experimental case, XA-511 achieved a synergistic anti-PD-1 antibody therapeutic effect against in situ colorectal cancer by enhancing the infiltration of anti-tumor immune cells and the production of tumor suppressor factors in tumor tissue.

[0110] Table 6 Dosage

[0111]

[0112] Table 7 Antibody information

[0113] Fluorescent dyes index Species supplier Item No. L / D AF700 / BD 564997 CD45 PC5.5 anti-mouse Biolegend 103132 CD3e APC-CY7 anti-mouse BD 557596 CD4 APC anti-mouse BD 553051 CD8a PE-CF594 anti-mouse Biolegend 100762 CD25 BV421 anti-mouse BD 562606 FOXP3 PE-CY7 anti-mouse / rat invitrogen 25-5573-82 IFN-γ PE anti-mouse Biolegend 505808 TNF-a BV605 anti-mouse Biolegend 506329 Granzyme B FITC anti-huamn / mouse Biolegend 372206

[0114] Table 8 Other reagent information for flow cytometry experiment

[0115] Reagents factory Item No. AOPI Count Star C0211 <![CDATA[BD Pharmingen TM Stain Buffer(BSA)]]> BD 554657 CytoFLEX Daily QC Fluorospheres BECKMAN B53230 mouse Fc Block BD 553142 PMA InvivoGen tlrl-pma Calcium ionophore-NFAT Activator InvivoGen inh-ion Brefeldin A Solution (1,000X) Biolegend 420601 Fix / Perm buffer <![CDATA[eBioscience TM ]]> 00-5523-00

[0116] Experimental Example 4: Effects of Megasphaera XA-511 on Pancreatic Cancer Model Mice

[0117] This experiment describes the effects of Megasphaera bacteria XA-511 on pancreatic cancer mice. This study was reviewed by the Experimental Animal Care and Use Committee of Pengli Biopharmaceutical Technology (Shanghai) Co., Ltd. The experimental procedures are as follows:

[0118] Preparation of live bacteria:

[0119] A single colony of the Megasphaera bacteria XA-511 obtained in Experimental Example 1 was selected and inoculated into a fortified Clostridium liquid medium (ATCC 2107), and cultured anaerobically at 37°C for 24 hours to obtain a culture solution; the culture solution was centrifuged at 1700 rpm for 5 minutes, and the cell pellet was obtained to obtain live bacteria of the Megasphaera bacteria XA-511.

[0120] Model establishment and drug administration:

[0121] Forty SPF-grade C57BL / 6 mice (female, 6-7 weeks old, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were selected. All mice were housed in separate cages in an SPF-grade animal room for one week at room temperature of (24±2)°C, with a 12-hour day and night cycle and free access to water.

[0122] After adaptive feeding, all mice were inoculated with Pan02 tumor cells (purchased from Beina Biotechnology) to induce pancreatic cancer. The specific operation was as follows: before inoculation, the mice were anesthetized with 3% (w / v, g / 100 mL) isoflurane; after anesthesia, 5×10 6 Pan02 tumor cells were resuspended in 100 μL PBS buffer and inoculated subcutaneously into the left armpit of each group of mice with an inoculation volume of 100 μL. 3 40 mice were randomly divided into 4 groups, namely: experimental control group, Anti-PD-1 antibody monotherapy group, XA-511 bacteria treatment group and XA-511 + anti-PD-1 treatment group. The drugs were administered according to Table 9, and the day of grouping was defined as Day 0.

[0123] One week after cell inoculation, the long / short diameter of the tumor of each mouse was measured weekly and the tumor volume was calculated (tumor volume = 1 / 2 × tumor long diameter 2 × tumor short diameter), and the measurements were continued for 4 weeks. The experiment ended on the 27th day of administration. After the experiment, the mice were dissected, the tumor tissues were taken, the weight of the tumor tissues was measured, and the tumor tissues were stored in RPMI medium. The proportion of different immune cells in the spleen was detected by flow cytometry. The detection and analysis results are shown in Figures 6-8 .

[0124] The flow cytometry analysis process is as follows:

[0125] Place the tumor in a 100mm 2 Rinse the culture dish with PBS to remove fat, lesions, and necrotic areas from the tumor tissue. After thoroughly mincing the tumor tissue with forceps and scissors, transfer it to a Miltenyi GentleMACS tube C and add 5 mL of Miltenyi Tumor Dissociation Kit digestion buffer. Tighten the tube C and insert it upside down into the GentleMACS sleeve. Select the appropriate GentleMACS program and run it. After the program terminates, remove the tube C and place it in a constant temperature shaking water bath at 37°C and 220 rpm for 30 minutes. After digestion, insert the tube C upside down into the GentleMACS sleeve again and run the appropriate program. Add 5 mL of PBS buffer to the tube C, filter through a 70 μm filter into a 50 mL centrifuge tube, and collect the filtrate. Centrifuge at 400 g for 5 minutes and discard the supernatant (retain the filtered tissue clumps when discarding the supernatant). Lyse the red blood cells: Add 5 mL of Miltenyi Red Blood Cell Lysis Buffer and incubate at room temperature (25°C) in the dark for 5 minutes. Add 5 mL of PBS buffer and shake to mix thoroughly. Centrifuge at 400 g for 5 min and discard the supernatant. Add 5 mL of PBS buffer and mix thoroughly for subsequent staining steps.

[0126] 1×10 6Resuspend the cells in 100 μL of live / dead solution (1 / 1000 live / dead in PBS) and incubate at 4°C for 10 min. Wash once with 1 mL of stain buffer, centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend the cells in 50 μL of Fcblock (1 / 100 Fcblock in stain buffer) and incubate at room temperature (25°C) in the dark for 5 min. Without washing the cells, add the surface antibody mixture prepared according to the manufacturer's instructions (see Table 10, Panel 1 for specific dye and antibody information) to the corresponding tubes and incubate at room temperature (25°C) in the dark for 15 min. Wash once with 1 mL of PBS, centrifuge at 400 g for 5 min, and discard the supernatant. Add 1 mL of FoxP3 fixative, mix well on a shaker, and incubate at room temperature (25°C) in the dark for 40 min. Centrifuge at 400 g for 5 min, and discard the supernatant. Add 100 μL 1X permeabilization buffer to resuspend the cells, add the mixed intracellular antibody (see Table 10 Panel 2 for specific information on dyes and antibodies) according to the instructions, and incubate at 4°C for 30 minutes in the dark. Add 1 mL 1X permeabilization buffer, wash once with 1 mL PBS buffer, centrifuge at 400 g for 5 minutes and discard the supernatant. Add 200 μL 1X stain buffer and load the sample (see Table 11 for specific information on other flow cytometry-related reagents). Use flow cytometry for data acquisition and use Kaluza 2.1 software to analyze different immune cell subsets CD45 + (immune cells), CD4 + / CD45 + (CD4 T cells) and CD8 + / CD45 + Results for CD8 T cells were obtained. Dunnett's multi-comparison test was used to compare the two groups. A statistically significant difference was considered if p < 0.05.

[0127] Depend on Figure 6 It can be seen that the tumor volume of the experimental control group continued to grow. 14 days after grouping, the average tumor volume in the group was 80.02 mm 3 43 days after grouping, the average tumor volume in each group was 807.02 mm 3 , indicating that the subcutaneous transplant tumor model of pancreatic cancer Pan02 cell line C57BL / 6 mice was successfully established in this experiment. The average tumor volume of the anti-PD-1 antibody monotherapy group was 80.42 mm 14 days after grouping. 3 43 days after grouping, the average tumor volume in each group was 592.33 mm 3Compared with the experimental control group during the same period, the tumor volume and relative tumor volume were significantly reduced from 18 to 28 days after grouping (p < 0.01). The average tumor volume in the XA-511 treatment group was 79.81 mm on day 14 after grouping. 3 43 days after grouping, the average tumor volume in each group was 405.14 mm 3 Fourteen days after grouping, the average tumor volume in the XA-511+anti-PD-1 treatment group was 79.68 mm 3 43 days after grouping, the average tumor volume in each group was 379.86 mm 3 Compared with the control group and the Anti-PD-1 antibody monotherapy group, tumor volumes in the XA-511 bacteria treatment group and the XA-511 + anti-PD-1 treatment group decreased significantly from 18 to 43 days after grouping (p < 0.01), indicating that both XA-511 bacteria treatment and XA-511 + anti-PD-1 combination therapy had significantly better anti-tumor effects than anti-PD-1 antibody monotherapy.

[0128] Depend on Figure 7 It can be seen that compared with the experimental control group, the tumor weight of the XA-511 bacteria treatment group and the XA-511+anti-PD-1 treatment group were significantly reduced (p<0.001), indicating that XA-511 single bacteria treatment and XA-511+anti-PD-1 combination treatment have significant anti-tumor effects.

[0129] Depend on Figure 8 It can be seen that according to the flow cytometry results of tumor tissues, compared with the Anti-PD-1 antibody monotherapy group, the XA-511 bacteria treatment group and the XA-511 + anti-PD-1 treatment group downregulated Treg cells and CD4 + In addition, compared with the XA-511 bacteria treatment group, the XA-511+anti-PD-1 treatment group also increased CD8 + The proportion of T cells and the reduction of CD4 + The ratio of T cells and immune suppressive cells MDSC. The experimental results suggest that XA-511 can inhibit tumors by reducing the infiltration of Treg cells. And when XA-511 is used in combination with anti-PD-1 antibodies, it can also increase the CD8 + T cell infiltration, reduced CD4 + T cells and immunosuppressive cells MDSC infiltrate, thereby promoting the effect of anti-PD-1 antibody in treating tumors.

[0130] Pancreatic cancer immunotherapy has been a research hotspot in recent years. In this example, XA-511 can not only synergize with anti-PD-1 antibodies to achieve a good therapeutic effect on pancreatic cancer by reducing the infiltration of immune cells with immunosuppressive activity in tumor tissues, but XA-511 alone can also achieve a good therapeutic effect on pancreatic cancer.

[0131] Table 9 Dosage

[0132]

[0133] Table 10 Antibody information

[0134]

[0135]

[0136] Table 11 Other reagents information for flow cytometry experiment

[0137] Reagents factory Item No. AOPI Count Star C0211 <![CDATA[BD Pharmingen TM Stain Buffer(BSA)]]> BD 554657 CytoFLEX Daily QC Fluorospheres BECKMAN B53230 mouse Fc Block BD 553142 PMA InvivoGen tlrl-pma Calcium ionophore-NFAT Activator InvivoGen inh-ion Brefeldin A Solution (1,000X) Biolegend 420601 Fix / Perm buffer <![CDATA[eBioscience TM ]]> 00-5523-00

[0138] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in preparing a drug for treating cancer, characterized in that: The cancer is in situ colorectal cancer or pancreatic cancer; the components of the pharmaceutical composition include active pharmaceutical ingredients; When the cancer is in situ colorectal cancer, the active pharmaceutical ingredient is composed of Megasphaera bacteria and anti-PD-1 antibodies; when the cancer is pancreatic cancer, the active pharmaceutical ingredient is Megasphaera bacteria, or the active pharmaceutical ingredient is composed of Megasphaera bacteria and anti-PD-1 antibodies; The Megasphaera bacteria ( Megasphaera indica ) is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.23400.

2. The use according to claim 1, characterized in that The in situ colorectal cancer is in situ colon cancer and / or in situ rectal cancer.

3. The use according to claim 1 or 2, characterized in that When the cancer is in situ colorectal cancer, the drug comprises live bacteria of the genus Megasphaera and an anti-PD-1 antibody, or the drug comprises inactivated bacteria of the genus Megasphaera and an anti-PD-1 antibody; When the cancer is pancreatic cancer, the component of the drug is live bacteria of the genus Megasphaera, or the component of the drug consists of live bacteria of the genus Megasphaera and anti-PD-1 antibodies.

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