Application of bud plant bacteria in preparation of anti-tumor drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a germicide bacterium in preparation of an anti-tumor drug. The invention provides an application of a germaria bacterium in preparation of a medicine for preventing and / or treating cancers. Specifically, a strain of bud plant bacteria XA-2595 is separated, the strain can activate an immune system and promote immune cells to secrete various proinflammatory factors and chemotactic factors, the proportion of CD4 + positive cells and CD8 + positive cells in intestinal tracts and tumors is increased by adjusting immune-related signal channels in a tumor microenvironment, and cold tumors are converted into hot tumors. The XA-2595 single drug has an anti-tumor effect, and can also be combined with a PD-1 antibody to play a synergistic effect, so that the anti-tumor effect is further improved. The bud plant bacteria found by the invention have good safety, and the bud plant bacteria have good application prospects in preparation of medicines for preventing and / or treating tumors by single use or combined immunotherapy.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of bacteria of the genus Phytophthora in the preparation of anti-tumor drugs. Background Art

[0002] The human intestinal microbiome, or what we usually call the intestinal flora, plays an indispensable role in maintaining health. Numerous studies have revealed that the intestinal flora not only affects the body's nutritional metabolism, drug absorption, intestinal development and immune system maturation in infancy, but also effectively resists the invasion of pathogens. These microorganisms regulate local and distal organs through specific structures or metabolites on their surfaces. They can also maintain microecological balance by regulating the diversity and composition of the intestinal flora, thereby promoting the absorption of nutrients, regulating immune function, building body barriers, and exerting antibacterial effects. The huge gene pool of the intestinal flora, with an estimated number of functional genes that is 100 times that of the human genome, shows great potential for functional regulation. Therefore, the intestinal flora is also called the "second genome" of the human body. With the maturity and popularization of gene sequencing technology, the connection between human microecological imbalance and various diseases has gradually been revealed, and the relationship between intestinal flora and human diseases has also been widely confirmed. Studies have shown that cancer, inflammatory bowel disease, obesity, diabetes, and even neurological diseases and cardiovascular diseases are closely related to the intestinal flora. These research advances indicate that the use of intestinal flora for disease prevention and treatment has great potential and important significance.

[0003] Cancer is a group of diseases caused by gene mutations that cause cells to lose normal growth and differentiation control and form tumors. Cancer can affect almost all tissues and organs in the body and has the ability to invade locally and metastasize to distant sites, seriously threatening human health. Cancer treatment includes a variety of methods such as surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy. As a revolutionary tumor treatment, immunotherapy has shown great potential. It uses the patient's own immune system to attack and destroy tumor cells, and has higher intelligence and specificity than traditional treatments. At present, immunotherapy, including checkpoint inhibitors, CAR-T cell therapy, tumor vaccines and other forms, has achieved remarkable results in the treatment of various malignant tumors. Although immunotherapy has shown significant efficacy in some patients, it still faces challenges, as there are primary and secondary drug resistance problems due to low tumor immune infiltration.

[0004] The clinical treatment dilemma of tumors in terms of immune infiltration is that the immunosuppressive mechanism in the tumor microenvironment may hinder the effective attack of immune cells on tumor cells, thereby weakening the efficacy of immunotherapy. The interactions between tumor cells and immune cells are complex and diverse, which may lead to immune escape and the development of drug resistance, making it difficult for immunotherapy to achieve the desired therapeutic effect. In the clinical treatment dilemma of tumor immune infiltration, bacteria may have potential efficacy. At present, there are no reports on the therapeutic strategy of using Gemmiger qucibialis to regulate the tumor microenvironment, convert cold tumors into hot tumors, and promote the treatment of tumors with immune checkpoint inhibitors. Summary of the invention

[0005] To solve the above problems, the present invention provides a new application of a Gemmiger qucibialis bacterium and a specific Gemmiger qucibialis bacterium (Gemmiger qucibialis XA-2595).

[0006] The first aspect of the present invention aims to provide a new application of bacteria of the genus Phytophthora.

[0007] The second aspect of the present invention aims to provide a strain of Phytophaga bacteria. The third aspect of the present invention aims to provide a product.

[0008] The fourth aspect of the present invention aims at the use of the Phytophthora bacteria according to the second aspect of the present invention and the product according to the third aspect of the present invention.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides the use of bacteria of the genus Phytophthora in the preparation of drugs for preventing and / or treating cancer.

[0011] In some embodiments of the present invention, the Phytophthora bacterium is the Phytophthora bacterium Gemmigerqucibialis XA-2595.

[0012] The second aspect of the present invention provides a strain of Gemmiger qucibialis XA-2595. The Gemmiger qucibialis XA-2595 is deposited in the General Microbiological Center of the China Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a taxonomic name of Gemmiger qucibialis, a deposit number of CGMCC: 27813, and a deposit date of July 5, 2023. Or, a Bacterium bacteria having a 16S rRNA gene sequence as shown in SEQ ID NO: 1; or, a Bacterium bacteria comprising a 16S rDNA sequence having at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 1; or, a Bacterium bacteria comprising a genome sequence having at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98% or at least 99% identity with the Bacterium bacteria deposited with CGMCC No. 27813.

[0013] Preferably, the colonies of the Bacillus bacteria XA-2595 are round, with a smooth, protruding, moist surface, a diameter of about 1 to 2 mm, and are milky white and shiny.

[0014] The third aspect of the present invention provides a product, comprising at least one of a1) to a8):

[0015] a1) Bacteria belonging to the genus Phytophaga according to the second aspect of the present invention;

[0016] a2) a bacterial agent containing the Phytophthora bacteria XA-2595 according to the second aspect of the present invention;

[0017] a3) containing the live bacterial solution of the Phytophthora bacteria XA-2595 according to the second aspect of the present invention;

[0018] a4) a dead bacteria solution containing the Phytophthora bacteria XA-2595 according to the second aspect of the present invention;

[0019] a5) a culture product containing the Bacillus bacteria XA-2595 according to the second aspect of the present invention;

[0020] a6) containing the metabolites of the Bacillus bacteria XA-2595 according to the second aspect of the present invention;

[0021] a7) an extract containing the Phytophaga bacteria XA-2595 according to the second aspect of the present invention;

[0022] a8) A culture supernatant containing the Bacillus bacteria XA-2595 according to the second aspect of the present invention.

[0023] Preferably, the product includes medicines, health products, foods, functional foods, skin care products or medical devices.

[0024] In one embodiment of the present invention, the preparation method of the product comprises: inoculating the above-mentioned Phytophthora bacteria into a liquid culture medium for culturing to obtain a culture solution; treating the culture solution to obtain live bacteria, inactivated bacteria and / or culture supernatant of the above-mentioned Phytophthora bacteria to obtain the active ingredient. The culture supernatant contains the metabolites of the above-mentioned Phytophthora bacteria.

[0025] In one embodiment of the present invention, the treatment includes: centrifuging the culture solution to obtain the live bacteria and / or culture supernatant of the above-mentioned Phytophthora bacteria; and / or sterilizing the culture solution and then centrifuging it to obtain the inactivated bacteria and / or culture supernatant of the above-mentioned Phytophthora bacteria.

[0026] In one embodiment of the present invention, the culture medium is YCFA medium.

[0027] The fourth aspect of the present invention provides the use of the Phytophthora bacteria of the second aspect of the present invention and the product of the third aspect of the present invention in any one of b1) to b5):

[0028] b1) Preparation of products for the treatment and / or prevention of cancer;

[0029] b2) preparing products for regulating the intestinal microenvironment;

[0030] b3) preparing products for regulating tumor microenvironment;

[0031] b4) Preparation and activation of intratumoral CD4 + Cell products;

[0032] b5) Preparation and activation of intratumoral CD8 + Cell products.

[0033] In some embodiments of the present invention, the product includes pharmaceutical excipients;

[0034] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0035] Furthermore, for the convenience of medication, the Phytophthora bacteria XA-2595 can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder, etc.), and disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.). silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0036] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable excipients can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994); Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients.

[0037] In some embodiments of the present invention, the dosage form of the product includes powders, powders, drops, gels, lozenges, tablets, pills, soft / hard gelatin capsules, patches, lyophilized powders, sprays, granules, suspensions, syrups, suppositories, granules, emulsions, creams, ointments, injections, sterile powders, gels, oral solutions and nanoparticles.

[0038] In some embodiments of the present invention, the product includes other anti-tumor drugs.

[0039] In some embodiments of the present invention, the other anti-tumor drugs include at least one of immune checkpoint inhibitors, anti-tumor factors, tumor antibodies, cancer vaccines, cell therapy and small molecule drugs.

[0040] Further preferably, the immune checkpoint inhibitors include PD-1 antibody, PD-L1 antibody, and CTLA-4 antibody.

[0041] More preferably, the anti-tumor factors include tumor necrosis factor and interferon.

[0042] In some embodiments of the present invention, the tumor microenvironment includes but is not limited to immune cells, inflammatory cells, metabolites, and inflammatory factors.

[0043] Preferably, regulating the tumor microenvironment includes upregulating the amount of immune activation factors and / or downregulating the amount of anti-inflammatory factors.

[0044] Preferably, the immune activation factors include but are not limited to TNF-α, IFN-γ, IL-6; the anti-inflammatory factors include but are not limited to IL-10.

[0045] In some embodiments of the invention, the cancer includes solid tumors and hematological tumors.

[0046] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial embryonic cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal cancer, Langerhans cell Histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumors, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer and single myeloma.

[0047] Preferably, the hematological tumor is selected from B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia and preleukemia.

[0048] In some embodiments of the invention, the tumor comprises colorectal cancer.

[0049] In one embodiment of the present invention, the prevention and / or treatment of cancer includes preventing and / or treating cancer by regulating the tumor microenvironment.

[0050] Preferably, the regulation of the tumor microenvironment includes regulating the metabolite components in the tumor, regulating the infiltration of immune cells in the tumor, and regulating the infiltration of inflammatory cells in the tumor.

[0051] The method of regulating the infiltration of immune cells in tumors includes activating CD4 + cells and / or activated intratumoral CD8 + cell.

[0052] In one embodiment of the present invention, the prevention and / or treatment of cancer includes converting a cold tumor into a hot tumor by regulating the tumor microenvironment, thereby promoting the prevention and / or treatment of cancer by cancer immunotherapeutics.

[0053] The beneficial effects of the present invention are:

[0054] The present invention provides the use of Gemmiger qucibialis XA-2595 in the preparation of a drug for preventing and / or treating cancer. The results show that:

[0055] (1) Activation of immune cells and immune responses: XA-2595 from the genus Phytophthora can activate the immune system and promote immune cells to secrete a variety of pro-inflammatory factors and chemokines.

[0056] (2) Regulating the intestinal and tumor immune microenvironment: XA-2595, a bacterium of the genus Phytophthora, regulates immune-related signaling pathways in the tumor microenvironment, increasing the proportion of CD4+ positive cells and CD8+ positive cells in the intestine and tumors, and transforming cold tumors into hot tumors. This not only helps XA-2595 alone to exert its anti-tumor effect, but also helps to enhance the efficacy of immunotherapy.

[0057] (3) It can be used alone or in combination with PD-1 antibodies to exert anti-tumor effects: XA-2595, a bacterium of the genus Phytophthora, can effectively inhibit tumor growth when used alone; it can also be used in combination with PD-1 antibodies to exert a synergistic anti-tumor effect.

[0058] (4) Good safety: The Bacillus bacteria XA-2595 has good safety while exerting anti-tumor effects.

[0059] Therefore, the bacteria XA-2595 of the genus Phytophthora alone or in combination with immunotherapy has great application prospects in the preparation of drugs for preventing and / or treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0061] Figure 1 : Plate growth status of Bacillus bacteria XA-2595;

[0062] Figure 2 : Transmission electron micrographs of the bacterium XA-2595 of the genus Phytophthora; wherein A: negative staining transmission electron micrograph of the bacterium XA-2595 of the genus Phytophthora; B: positive staining transmission electron micrograph of the bacterium XA-2595 of the genus Phytophthora.

[0063] Figure 3 : CIRCOS map of Bacillus sp. XA-2595.

[0064] Figure 4 : Phylogenetic tree comparison result of Bacillus bacteria XA-2595.

[0065] Figure 5 :Comparison of IFN-γ and TNF-α levels induced by live and killed bacteria in human PBMCs.

[0066] Figure 6: The level of cytokine production induced by Phytochrome Bacteria XA-2595 in human PBMCs; A: The effect of Phytochrome Bacteria XA-2595 and its metabolites on the production of IFN-γ by human PBMCs; B: The effect of Phytochrome Bacteria XA-2595 and its metabolites on the production of TNF-α by human PBMCs; C: The effect of Phytochrome Bacteria XA-2595 and its metabolites on the production of IL-6 by human PBMCs; D: The effect of Phytochrome Bacteria XA-2595 and its metabolites on the production of IL-10 by human PBMCs.

[0067] Figure 7 : The levels of cytokine production by macrophages induced by human monocytes THP-1 induced by XA-2595 of the genus Phytophthora; A: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of TNF-α by THP-1; B: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of IL-6 by THP-1; C: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of IL-1β by THP-1; D: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of IL-1RA by THP-1; E: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of CXCL-10 by THP-1; F: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of TARC by THP-1; G: The effect of XA-2595 of the genus Phytophthora and its metabolites on the production of IL-10 by THP-1.

[0068] Figure 8 : Preventive administration of Bacillus bacteria XA-2595 inhibits tumor growth in MC38 colorectal cancer model mice.

[0069] Fig. 9 : Effects of preventive administration of Bacillus bacteria XA-2595 combined with PD-1 antibody on tumor weight and tumor pictures of MC38 colorectal cancer model mice; A: Effects of Bacillus bacteria XA-2595 on tumor weight of MC38 colorectal cancer model mice; B: Pictures of tumors in MC38 colorectal cancer model mice after administration of Bacillus bacteria XA-2595.

[0070] Fig.10 : Effects of preventive administration of Bacillus bacteria XA-2595 combined with PD-1 antibody on the levels of multiple cytokines in the plasma of MC38 colorectal cancer model mice.

[0071] Fig.11: Effect of XA-2595 of the genus Phytophthora on the infiltration of CD4+T cells and CD8+T cells in tumors; A: Immunohistochemical staining images of CD4 and CD8 antibodies in tumors of MC38 colorectal cancer model mice; B: Effect of XA-2595 of the genus Phytophthora on the area of ​​CD8-positive cells in tumors; C: Effect of XA-2595 of the genus Phytophthora on the area of ​​CD4-positive cells in tumors.

[0072] Fig.12 :Therapeutic administration of Bacillus bacteria XA-2595 combined with PD-1 antibody inhibits tumor growth in MC38 colorectal cancer model mice.

[0073] Fig.13 : Effects of therapeutic administration of Phytophthora bacteria XA-2595 combined with PD-1 antibody on tumor weight and tumor pictures of MC38 colorectal cancer model mice; A: Effects of Phytophthora bacteria XA-2595 on tumor weight of MC38 colorectal cancer model mice; B: Pictures of tumors in MC38 colorectal cancer model mice after administration of Phytophthora bacteria XA-2595.

[0074] Fig.14 : Expression levels of differentially expressed genes in tumors of MC38 colorectal cancer model mice after treatment with Phylogenes XA-2595; A: Effect of Phylogenes XA-2595 on Ikbkb gene expression in tumors of MC38 colorectal cancer model mice; B: Effect of Phylogenes XA-2595 on Ccnd3 gene expression in tumors of MC38 colorectal cancer model mice; C: Effect of Phylogenes XA-2595 on Cxcl10 gene expression in tumors of MC38 colorectal cancer model mice; D: Effect of Phylogenes XA-2595 on Lifr gene expression in tumors of MC38 colorectal cancer model mice; E: Effect of Phylogenes XA-2595 on Irf3 gene expression in tumors of MC38 colorectal cancer model mice.

[0075] Fig.15 :Effects of therapeutic administration of Bacillus bacteria XA-2595 combined with PD-1 antibody on the number of colorectal tumors in AOM / DSS-induced colorectal cancer model mice.

[0076] Fig.16 : Images of colorectal tumors in mice with AOM / DSS-induced colorectal cancer model treated with Bacillus bacteria XA-2595 combined with PD-1 antibody.

[0077] Fig.17: Effect of XA-2595 of the genus Phytophthora on the infiltration of CD4+T cells and CD8+T cells in colorectal in situ tumors; A: Effect of XA-2595 of the genus Phytophthora on the area of ​​CD4-positive cells in the tumor; B: Effect of XA-2595 of the genus Phytophthora on the area of ​​CD8-positive cells in the tumor.

[0078] Fig.18 : Changes in body weight of mice after repeated oral administration of the highest administrable dose of XA-2595 from the genus Phytophthora.

[0079] Fig.19 : Changes in the weights of major organs after repeated oral administration of the highest administrable dose of XA-2595 from the genus Phytophthora; A: Effects of repeated administration of XA-2595 from the genus Phytophthora on the heart weight of mice; B: Effects of repeated administration of XA-2595 from the genus Phytophthora on the liver weight of mice; C: Effects of repeated administration of XA-2595 from the genus Phytophthora on the spleen weight of mice; D: Effects of repeated administration of XA-2595 from the genus Phytophthora on the lung weight of mice; E: Effects of repeated administration of XA-2595 from the genus Phytophthora on the kidney weight of mice; F: Effects of repeated administration of XA-2595 from the genus Phytophthora on the thymus weight of mice; G: Effects of repeated administration of XA-2595 from the genus Phytophthora on the colon length of mice.

[0080] Figures 1 to 19 In the table, “*” means p<0.05, “**” means p<0.01, and “***” means p<0.001. DETAILED DESCRIPTION

[0081] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0082] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0083] The culture media involved in the following examples are shown in Table 1.

[0084] Table 1 Culture medium

[0085]

[0086]

[0087]

[0088] Example 1 Isolation and identification of strain XA-2595

[0089] 1. Isolation of XA-2595 strain of Bacillus

[0090] XA-2595 strain was isolated from healthy human fecal samples. The specific isolation method is as follows:

[0091] (1) Preparation: In a biosafety cabinet, dispense 20 mL of sterile, deoxygenated PBS (1×) containing 2 g / L L-cysteine ​​hydrochloride into a sterile 50 mL centrifuge tube. Transfer the anaerobic blood plate (YCFA agar medium) and sterile PBS solution to the anaerobic workbench 24 h in advance. Pour 5 to 7 sterile glass beads into the solidified anaerobic blood plate.

[0092] (2) Collecting stool samples: Use a disposable sterile stool collector to collect fresh stool samples from the donor in a clean environment and immediately transfer them to an anaerobic operating table. Take about 1 g of stool sample and add it to sterile PBS. Vortex the sample thoroughly and mix until no obvious particles are visible. Let it stand for 5 minutes.

[0093] (3) Full nutrition pre-culture: Prepare 30 mL of PBS (1×) containing 1 g / L L-cysteine, put it in a 100 mL anaerobic bottle, sterilize it at 121°C for 20 min, and then put it in an anaerobic box for use. In the anaerobic incubator, add 30 mL of PBS, 4 mL of blood culture medium (Meike Biotech), 2 mL of sterile defibrinated sheep blood (final concentration 5%) and 4 mL of sterile clarified rumen fluid (final concentration 10%) to the anaerobic blood culture bottle, and then use a syringe to extract 0.5 mL of the supernatant of the fecal sample after mixing and standing, add it to the blood culture bottle, mix well, and obtain the pre-culture mixed bacterial solution. The pre-culture mixed bacterial solution is cultured in a 37°C constant temperature incubator for 3 days to obtain the pre-culture mixed bacterial solution after 3 days of culture.

[0094] (4) Mixed bacterial solution dilution and coating: 100 μL to 900 μL of the pre-cultured mixed bacterial solution and the pre-cultured mixed bacterial solution after 3 days of culture were extracted and added to PBS and mixed. The mixture was diluted to 10-10-6 with a total of 6 gradients using a 10-fold gradient dilution method. The pre-cultured mixed bacterial solution after 3 days of culture (i.e., the sample without pre-culture) and the sample after 3 days of pre-culture were plated with 10-4, 10-5, and 10-6 gradients. 100 μL of the bacterial solution of the corresponding dilution gradient was plated on each plate. The same gradient dilution bacterial solution was plated on 3 YCFA solid plates in parallel, and evenly spread with dry sterile glass beads. The plates were incubated in a 37°C constant temperature incubator for 3 days.

[0095] (5) Monoclonal selection: After the solid plates of various conditions are cultured, solid plates with 100 to 500 monoclonal numbers are selected for monoclonal selection. 4 to 6 monoclonals of each type are selected based on their size, color, and morphology.

[0096] (6) Monoclonal liquid enrichment: Liquid culture medium was added to a 96-well deep-well plate in advance, with 1 mL of YCFA liquid culture medium placed in each well. Four to six clones from the same monoclone were selected for culture. One clone from each 96-well deep-well plate was selected from the plates corresponding to the samples pre-cultured for 0 days and the samples pre-cultured for 3 days. The plates were covered with sterile plastic film and incubated in a 37°C constant temperature incubator for 3 days.

[0097] (7) Bacterial plate streaking and purification: Prepare sufficient BHI plates and BHI liquid culture medium according to experimental requirements. Divide each BHI culture plate into three equal parts. Streak a single colony enriched in the above liquid on each part and culture it in a 37°C constant temperature incubator for 3 days.

[0098] (8) Liquid amplification: After the single colony on the BHI culture plate is cultured, observe whether the color and morphology of the single colony are uniform. Take one of the single colonies that are determined to be pure and inoculate it into a 1.5 mL sterile EP tube containing 800 μL BHI liquid culture medium. Incubate it in a 37°C incubator for 2-3 days. It was identified as Gemmiger qucibialis by sequencing and named XA-2595. At the same time, preserve the bacterial liquid, mark the corresponding strain number, and freeze it in a -80°C refrigerator.

[0099] 2. Identification of strains

[0100] (1) Culture characteristics, microscopic examination and morphological features

[0101] The isolated XA-2595 strain was streaked on YCFA agar medium and cultured anaerobically for 24 to 48 hours. Figure 1 It can be seen that the colonies of the XA-2595 strain are round, with a smooth, protruding and moist surface, a diameter of about 1 to 2 mm, milky white, shiny, and easily picked up by an inoculation loop. Figure 2 This is an individual morphological picture of the strain observed under a transmission electron microscope. The bacteria have a cell wall and cell membrane structure unique to Gram-negative bacteria, no flagella, and are mostly dumbbell-shaped.

[0102] (2) Biochemical identification of strain XA-2595

[0103] 2ANC ID card and 2 identification system, following internal routine procedures and the manufacturer's instructions. All culture treatments were carried out in an anaerobic operating chamber at 37°C. The XA-4490 strain prepared above was used 2ANC ID card identification was performed by incubation on Brucella blood agar for 48-72 hours to obtain a single clone. The inoculum suspension was prepared with 0.45% NaCl aqueous solution and a calibrated Vitek 2Densichek instrument (bioMérieux, Marcy l'etoile, France) was used to achieve a McFarland standard turbidity of 2.70-3.30. 2ANC ID card detects 36 biochemical tests for carbon source utilization and enzyme activity. 2Compact instrument detects positive and negative results of biochemical tests for carbon source utilization and enzyme activity. The identification results are shown in Table 2.

[0104] Table 2

[0105]

[0106]

[0107] (3) Whole genome sequencing and identification

[0108] ① The DNA of the Bacillus XA-2595 strain was extracted using a bacterial genomic DNA rapid extraction kit. After the genomic DNA was extracted, the purity and integrity of the DNA was detected by agarose gel electrophoresis and quantified using Qubit.

[0109] ②Library construction: The Nanopore platform was used to build the library. The BluePippin fully automatic nucleic acid fragment recovery system was first used to recover large DNA fragments, and then the ends were repaired. The EXP-NBD104 kit from Oxford Nanopore Technologies was used to add Barcodes using the PCR-free method. The fragment size was then detected using the AATI fully automatic capillary electrophoresis instrument, and the samples were mixed in equal moles. Finally, the SQK-LSK109 ligation kit from Oxford Nanopore Technologies was used for linker connection to construct a 10K library, which was then sequenced using the Nanopore platform. The Illumina platform was used for library construction and library testing. DNA samples that passed the electrophoresis test were randomly broken into fragments of approximately 350 bp in length using a Covaris ultrasonic disruptor. After the processing, the DNA fragments were used The UltraTM DNA Library Prep Kit for Illumina (NEB, USA) kit completes the entire library preparation through steps such as end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification.

[0110] ③After the library is constructed, Qubit 2.0 is used for preliminary quantification, and the library is diluted to 2ng / ul. Then, the insert fragment of the library is detected using Agilent2100. After the insert size meets the expectations, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library. After the library is qualified, different libraries are sequenced by Nanopore PromethION and Illumina NovaSeq PE150 according to the effective concentration and target data volume.

[0111] ④ After obtaining the offline data, use cutadapt (version 2.5) software to remove adapters and low-quality sequences; after obtaining the filtered reads sequence, use fastqc (version 0.11.9) software to statistically analyze the data profile of reads before and after filtering; then use SPAdes to assemble all bacterial sequences (including plasmid sequences) and only assemble plasmid sequences; from all bacterial sequences, use blast software to distinguish plasmid and chromosome sequences; display the basic situation of the contigs, GC content, gene information, tRNA, rRNA position information, and VFDB, CARD and other annotation information of the genome in the CIRCOS diagram (such as Figure 3 shown).

[0112] ⑤Species identification

[0113] The species were classified and annotated based on the Genome Taxonomy Database (GTDB), see Table 3.

[0114] Table 3 GTDB-Tk species classification

[0115]

[0116] ⑥The sequence of 16s rDNA of Bacillus subtilis XA-2595 is shown in SEQ ID NO:1.

[0117] ⑦Evolutionary tree analysis: The 16srDNA sequence of the obtained Bacillus XA-2595 was compared with the bacterial sequence obtained from the NCBI database to construct an evolutionary tree. The analysis diagram of the evolutionary tree is shown in the figure below. Figure 4 As shown, XA-2595 was identified as Gemmiger qucibialis.

[0118] The bacterium of the genus Gemmigerqucibialis XA-2595 was deposited in the General Microbiology Center of the China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the taxonomic name Gemmigerqucibialis, the deposit number CGMCC: 27813, and the deposit date of July 5, 2023.

[0119] Example 2 Screening of high potential strains with the potential to activate immune response

[0120] It is known that IFN-γ and TNF-α play a key role in regulating inflammatory responses and immune cell activation, and can activate immune cells and enhance their ability to recognize and kill tumor cells. Therefore, the upregulation of these two pro-inflammatory factors may help enhance anti-tumor immune responses. Based on this, this study used the experiment of cytokine secretion by human PBMCs as a strategy for screening anti-tumor bacteria, aiming to select bacteria that can activate human PBMCs and promote their secretion of high levels of IFN-γ and TNF-α.

[0121] The experimental steps are as follows: resuspend human PBMCs cells (purchased from Maishun Biotechnology Co., Ltd.) in RPMI-1640 complete medium to 1×10 6 The cell suspension was inoculated into a 96-well plate at a volume of 100 μL per well, and PBS buffer (blank control) and MOI = 1 (1×10 6 CFU) of multiple strains of live and inactivated bacteria (including XA-2595, and multiple strains from Gemmiger sp., Phocaeicola sp., Bifidobaterium sp., Enterocloster sp., all of which are other candidate strains obtained by the applicant in the initial screening of Example 1), live bacteria interacted on a 37°C anaerobic workbench for 2 hours, and then transferred to a 5% (v / v) CO2, 37°C cell culture incubator for continued culture for 22 hours; inactivated bacteria were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 24 hours. After the culture was completed, PBMCs cells were collected and the cell viability was detected using a CCK-8 kit (purchased from Tongren Chemical), and the cell supernatant was collected and the cytokine concentration was detected using a multi-factor kit (purchased from Biolegend).

[0122] Figure 5The ratio of the levels of IFN-γ and TNF-α secreted by human PBMCs stimulated by live and inactivated experimental strains relative to the levels of IFN-γ and TNF-α secreted by the control group was shown. The results showed that live and inactivated bacteria of the two strains of Phytophthora bacteria significantly enhanced the secretion of IFN-γ and TNF-α by human PBMCs, indicating that Phytophthora bacteria are significantly superior to other genera in activating immune cells, among which inactivated XA-2595 bacteria have the most significant effect in promoting IFN-γ secretion, and its effect is twice that of other experimental Phytophthora bacteria. Therefore, XA-2595 was selected as a candidate strain for further immune activation testing and anti-tumor effectiveness studies.

[0123] Example 3: Phytophthora bacteria XA-2595 activates immune response of human PBMCs

[0124] Resuspend human PBMCs cells (purchased from Maishun Biotechnology Co., Ltd.) in RPMI-1640 complete medium to 1×10 6 / mL, and obtain a cell suspension; the cell suspension was inoculated into a 96-well plate at an inoculum of 100 μL per well, and PBS buffer (blank control), 1 μg / mL commercially available LPS solution (with PBS buffer as solvent) (positive control) and MOI = 1 (1×10 6 CFU) of XA-2595 live bacteria were incubated at 37°C anaerobic bench for 2 hours and then transferred to a 5% (v / v) CO2, 37°C cell culture incubator for further culture for 22 hours. After the culture, PBMCs were collected and the cell viability was detected using a CCK-8 kit (purchased from Tongren Chemical), and the cell supernatant was collected and the cytokine concentration was detected using a multifactor kit (purchased from Biolegend).

[0125] The experimental results are as follows Figure 6 As shown, XA-2595 of the genus Phytophthora can promote the release of multiple cytokines from human PBMCs, including IFN-γ, TNF-α, IL-6, and IL-10, and the levels of TNF-α, IL-6, and IL-10 are higher than those of the positive control LPS. This indicates that XA-2595 of the genus Phytophthora can significantly activate the immune system and promote the release of cytokines from immune cells.

[0126] Example 4: Bacteria XA-2595 of the genus Phytophthora promotes the release of multiple cytokines and chemokines from macrophages derived from human THP-1

[0127] This example provides an experiment on the effect of the Bacillus bacteria XA-2595 on the release of cytokines and chemokines from human THP-1 differentiated M0 macrophages. The experimental process is as follows:

[0128] (1) THP-1 polarization: Resuspend human THP-1 cells to 1×10 in RPMI-1640 complete medium containing PMA (phorbol ester) at a final concentration of 50 ng / mL. 6 / mL to obtain a cell suspension; the cell suspension was added to each well at 200 μL (i.e., 2×10 5 / well) were inoculated into a 96-well plate and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 hours. After 48 hours of culture, the supernatant was discarded, washed once with PBS, and replaced with RPMI-1640 complete medium for a further 72 hours of static culture.

[0129] (2) Interaction between XA-2595 live bacteria and cells: After resting THP-1 cells for 72 h, the supernatant was discarded and the cells were washed once with PBS. PBS buffer (blank control), 1 μg / mL commercially available LPS solution (with PBS buffer as solvent) (positive control), MOI = 10 (2×10 6 CFU) of XA-2595 live bacteria and MOI = 1 (2×10 5 CFU) of XA-2595 live bacteria were incubated at 37°C anaerobic bench for 2 hours and then transferred to a 5% (v / v) CO2, 37°C cell culture incubator for further culturing for 22 hours. After the culture was completed, the cells were collected and the cell viability was detected using a CCK-8 kit (purchased from Tongren Chemical), and the cell supernatant was collected and the cytokine concentration was detected using a multifactor kit (purchased from Biolegend).

[0130] (3) Interaction between XA-2595 inactivated bacteria, culture supernatant and cells: After resting THP-1 cells for 72 h, the supernatant was discarded and the cells were washed once with PBS. PBS buffer (blank control), 1 μg / mL commercially available LPS solution (with PBS buffer as solvent) (positive control), MOI = 10 (2×10 6 CFU) of XA-2595 inactivated bacteria and MOI = 1 (2×10 5 CFU) of XA-2595 inactivated bacteria, 10% YCFA, and the supernatant of 10% YCFA cultured XA-2595 to the stationary phase were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 24 h. After the culture, the cells were collected and the cell viability was detected using a CCK-8 kit (purchased from Tongren Chemical), and the cell supernatant was collected and the cytokine concentration was detected using a multi-factor kit (purchased from Biolegend).

[0131] The experimental results are as follows Figure 7As shown in the results, live bacteria, inactivated bacteria and culture supernatant of XA-2595 of the genus Phytophthora can significantly promote the release of multiple cytokines (TNF-α, IL-6, IL-1β, IL-1RA and IL-10) and chemokines (CXCL-10 and TARC) by M0 macrophages, and the levels of each factor are higher than those of the positive control LPS. This indicates that various forms of XA-2595 of the genus Phytophthora can significantly activate mononuclear macrophages, promote the release of cytokines and chemokines, and help attract related immune cells into target organs.

[0132] Example 5 Effect of preventive administration of Bacillus bacteria XA-2595 combined with PD-1 antibody on MC38 colorectal cancer model mice

[0133] This example provides an experiment on the effect of preventive administration of Bacillus bacteria XA-2595 combined with immune checkpoint inhibitor PD-1 antibody on MC38 colorectal cancer model mice. The experimental process is as follows:

[0134] Preparation of live bacteria: Pick a single colony of the bacteria XA-2595 of the genus Phytophthora and inoculate it into YCFA medium, and culture it anaerobically at 37° C. for 24 hours to obtain a culture solution; after centrifuging the culture solution, take out the cell precipitate to obtain live bacteria of the bacteria XA-2595 of the genus Phytophthora.

[0135] Model establishment and drug administration: 45 SPF-grade C57BL / 6 mice (male, 6 weeks old, purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were selected. All mice were adaptively raised in SPF-grade animal rooms for 1 week, with room temperature of (24±2)℃, day and night alternation every 12h, and free access to water. After adaptive feeding, the animals were randomly divided into 3 groups according to body weight: G1 Vehicle group, G2 PD-1 antibody group and G3 XA-2595+PD-1 antibody combination group, with 15 mice in each group. Preventive administration began on the day of grouping (D-14), and mice in the G3 group were given 2×10 9 CFU / mouse intragastrically with XA-2595 (in 100 μL PBS buffer) for intervention, once a day. The mice in group G1 were intragastrically administered with an equal volume of PBS buffer as a control. After 4 days of preventive administration, MC38 cells (purchased from Kyowa Cell Bank) were inoculated at 1×10 6 The cells were inoculated into the subcutaneous tissue of the left upper limb of the mouse (with 100 μL PBS buffer as solvent) to establish the model (the day of cell inoculation was defined as D0). On D11 of cell inoculation, 10 mice were selected from each group to make the average tumor volume 200 mm 3, PD-1 antibody intraperitoneal injection treatment was started. Mice in groups G2 and G3 were intraperitoneally injected with PD-1 antibody (purchased from BioXcell, with 100 μL PBS buffer as solvent) at a dose of 5 mg / kg / time, and mice in group G1 were intraperitoneally injected with an equal dose of IgG2a (purchased from BioXcell, with 100 μL PBS buffer as solvent) as a control. The mice were intraperitoneally injected once every 4 days, for a total of 4 times. During this period, XA-2595 bacterial solution was continued to be gavaged every day until D30, for a total of 45 days. The experiment ended on D31.

[0136] Body weight monitoring: Body weight was monitored twice a week starting from the day of grouping, and was measured 3 times a week together with tumor measurement during the efficacy observation period.

[0137] Tumor volume measurement: After tumor formation, the tumor growth of mice was observed three times a week and the tumor volume was monitored. The longest diameter (length) and shortest diameter (width) of the tumor were measured with a vernier caliper on the edge of the tumor. The measurement data were recorded and the tumor volume was calculated. The calculation results are shown in Figure 8 Tumor volume calculation formula: Tumor volume = 1 / 2 × tumor long diameter × tumor short diameter 2 .

[0138] Status observation: During the administration period, observe the mice's mental state, whether their activity is reduced, whether their hair is rough, whether they have paralysis, breathing difficulties, etc., as well as their digestion and defecation conditions.

[0139] Sample collection: At the end of the experiment, blood was collected from the mouse orbits, plasma was prepared, and stored at -80°C; the tumors were weighed and photographed, and divided into 2 parts, 1 part was fixed with formalin solution, and the other part was stored at -80°C.

[0140] The experimental results are as follows Figure 8 As shown, the combined administration group of Bacillus bacteria XA-2595 + PD-1 antibody gradually showed an anti-tumor effect superior to PD-1 antibody starting from D17 (p<0.05). Fig. 9 The tumor weight and tumor images at the end of the experiment showed that the tumors of some animals in the XA-2595+PD-1 antibody combined administration group showed regression. This result shows that XA-2595 has a significant synergistic effect on the anti-tumor function of PD-1 antibody.

[0141] Fig.10 For plasma cytokine levels, the levels of pro-inflammatory factors TNF-α, IFN-α, IFN-β, IFN-γ, IL-1β and IL-12 in the plasma of mice treated with XA-2595+PD-1 antibody were higher than those in the control group and the PD-1 antibody monotherapy group, and the anti-inflammatory factor IL-10 also increased significantly. This result shows that the combined synergistic anti-tumor effect of XA-2595 may be related to its significant improvement of the immune response level in the body's circulation.

[0142] Immunohistochemical analysis Fig.11 It can be seen that the area of ​​CD8+ and CD4+ positive cells in the tumors of mice in the XA-2595+PD-1 antibody combined treatment group was significantly increased compared with the control group and the PD-1 antibody monotherapy group. This result shows that XA-2595 can increase the proportion of cytotoxic immune cells in the tumor, transform cold tumors into hot tumors, and play a role in killing tumor cells. Example 6 Effect of XA-2595 therapy combined with PD-1 antibody on MC38 colorectal cancer model mice

[0143] This example provides an experiment on the effect of the combined administration of XA-2595, a bacterium of the genus Phytophthora, and the immune checkpoint inhibitor PD-1 antibody on MC38 colorectal cancer model mice. The experimental process is as follows:

[0144] Preparation of live bacteria: Pick a single colony of the bacteria XA-2595 of the genus Phytophthora and inoculate it into YCFA medium, and culture it anaerobically at 37° C. for 24 hours to obtain a culture solution; after centrifuging the culture solution, take out the cell precipitate to obtain live bacteria of the bacteria XA-2595 of the genus Phytophthora.

[0145] Model establishment and drug administration: 30 SPF-grade C57BL / 6 mice (male, 6 weeks old, purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were selected. All mice were adaptively raised in SPF-grade animal rooms for 1 week at room temperature (24±2)℃, with day and night alternation every 12h, and free access to water. After adaptive feeding, MC38 cells (purchased from Concord Cell Bank) were cultured at 1×10 6 The inoculation amount of 100 μL PBS buffer was inoculated into the subcutaneous tissue of the left upper limb of the mouse to establish the model (the day of cell inoculation was defined as D0). 3 At 14:00 pm, the mice were divided into three groups according to their body weight: G1 Vehicle group, G2 PD-1 antibody group, and G3 XA-2595 + PD-1 antibody combination group, with 10 mice in each group. On the day of grouping (D12), intragastric administration began. The mice in the G3 group were given 5×10 8 CFU / mouse intragastrically administered with XA-2595 (in 100 μL PBS buffer) once a day. The mice in the G1 and G2 groups were intragastrically administered with an equal volume of PBS buffer as a control. 3At D2, PD-1 antibody was injected intraperitoneally. Mice in the G2 and G3 groups were injected intraperitoneally with PD-1 antibody (purchased from BioXcell, with 100 μL PBS buffer as solvent) at a dose of 5 mg / kg / time. Mice in the G1 group were injected intraperitoneally with an equal dose of IgG2a (purchased from BioXcell, with 100 μL PBS buffer as solvent) as a control. The mice were injected intraperitoneally once every 4 days, for a total of 4 times. During this period, XA-2595 bacterial solution was continued to be gavaged every day until D24, for a total of 13 days. The experiment ended on D25.

[0146] Body weight monitoring: Starting from the day of grouping, body weight was monitored three times a week along with tumor measurement.

[0147] Tumor volume measurement: Observe the tumor growth of mice and monitor the tumor volume three times a week. Use a vernier caliper to clamp the edge of the tumor and measure the longest diameter (length) and shortest diameter (width) of the tumor. Record the measurement data and calculate the tumor volume. The calculation results are shown in Fig.12 Tumor volume calculation formula: Tumor volume = 1 / 2 × tumor long diameter × tumor short diameter 2 .

[0148] Status observation: During the administration period, observe the mice's mental state, whether their activity is reduced, whether their hair is rough, whether they have paralysis, breathing difficulties, etc., as well as their digestion and defecation conditions.

[0149] Sample collection: At the end of the experiment, blood was collected from the mouse orbits, and plasma was prepared and stored at -80°C. The tumors were weighed and photographed, and divided into two parts, one of which was fixed with formalin solution for pathological examination, and the other was stored at -80°C for transcriptome sequencing.

[0150] The experimental results are as follows Fig.12 As shown, the combined administration group of Bacillus bacteria XA-2595 + PD-1 antibody gradually showed an anti-tumor effect superior to PD-1 antibody starting from D18 (p<0.05). Fig.13 The tumor weight and tumor images were the endpoints of the experiment. More than half of the animals in the XA-2595+PD-1 antibody combined administration group had tumor regression. This result showed that XA-2595 treatment was effective at a lower dose of 5×10 8 CFU / unit can significantly enhance the anti-tumor function of PD-1 antibody. It is speculated that the effective dose of XA-2595 is lower than 5×10 8 CFU / piece.

[0151] Fig.14The differentially expressed genes mined from each group of tumors and their expression levels are shown in Figure 2. The differentially expressed genes in the tumors of mice treated with XA-2595+PD-1 antibody combination therapy include ccnd related to the JAK-STAT signaling pathway, lifr related to the JAK-STAT, TNF and p53 signaling pathways, ikbkb related to the cGAS-STING, TNF and IL-17 signaling pathways, irf related to the cGAS-STING and NF-kappaB signaling pathways, and cxcl10 related to the IL-17 signaling pathway. Among them, the gene expression of ikbkb, ccnd, lifr and cxcl10 in the tumors of mice treated with XA-2595+PD-1 antibody combination therapy group was lower than that in the PD-1 antibody monotherapy group, while the expression of irf was higher than that in the PD-1 antibody monotherapy group. This result indicates that the combined synergistic anti-tumor effect of XA-2595 may be related to its regulation of immune-related signaling pathways in the tumor microenvironment.

[0152] Example 7 Effects of Bacillus bacteria XA-2595 combined with PD-1 antibody on AOM / DSS-induced colorectal cancer model mice

[0153] This example provides an experiment on the effect of XA-2595 of the genus Phytophthora combined with the immune checkpoint inhibitor PD-1 antibody on AOM / DSS-induced colorectal cancer model mice. The experimental process is as follows:

[0154] (1) Preparation of live bacteria: A single colony of the bacteria XA-2595 of the genus Phytophthora was selected and inoculated into a YCFA medium, and cultured anaerobically at 37° C. for 24 h to obtain a culture solution; the culture solution was centrifuged and the cell pellet was collected to obtain live bacteria of the bacteria XA-2595 of the genus Phytophthora.

[0155] (2) Establishment of CRC model: 41 SPF C57BL / 6 mice (male, 6 weeks old, purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were selected. All mice were adaptively raised in SPF animal rooms for 1 week, with room temperature (24±2)℃, day and night alternation every 12 hours, and free access to water. After adaptive feeding, 36 mice were intraperitoneally injected with the gene mutation inducer azoxymethane (AOM) on D0 (the day of AOM administration was D0), and were given normal drinking water for 1 week. Then, three rounds of inflammatory induction cycles were performed: first, drinking water containing the inflammatory agent DSS (dextran sulfate sodium) was maintained for 1 week, and then normal drinking water was changed for 2 weeks as one cycle; the DSS concentration in the first and second times was 3%, and the DSS concentration in the third time was 2.5%). The remaining 5 mice were given normal drinking water.

[0156] (3) XA-2595 bacterial liquid treatment:

[0157] According to the DAI (Disease Activity Index) score and animal weight of the model mice, the AOM / DSS model mice were divided into 4 groups on D64: G2 Vehicle (model group), G3 PD-1 antibody monotherapy group, G4 XA-2595 monotherapy group, G5 XA2595+PD-1 antibody combination group, with 10 animals in each group. Five normal mice were set up as G1 Normal (normal group). XA-2595 was administered orally on the day of grouping. Mice in the G4 and G5 groups were given 2×10 9 CFU / mouse gavage of XA-2595 bacteria of the genus Phytophthora (with 100 μL PBS buffer as solvent) was used for intervention, and the drug was administered once a day. The mice in the G1, G2 and G3 groups were gavaged with an equal volume of PBS buffer every day as a control. The PD-1 antibody was intraperitoneally injected at D71. The mice in the G3 and G5 groups were intraperitoneally injected with PD-1 antibodies (purchased from BioXcell, with 100 μL PBS buffer as solvent) at a dose of 10 mg / kg / time. The mice in the G1, G2 and G4 groups were intraperitoneally injected with an equal dose of IgG2a (purchased from BioXcell, with 100 μL PBS buffer as solvent) as a control, and the drugs were administered intraperitoneally once every 4 days for a total of 6 times. During this period, XA-2595 bacterial solution was continued to be gavaged every day until D96, for a total of 33 days. The experiment ended at D97.

[0158] Weight monitoring: Starting from the day of grouping, weight monitoring should be performed 2-3 times a week.

[0159] Status observation: During the administration period, observe the mice's mental state, whether their activity is reduced, whether their hair is rough, whether they have paralysis, breathing difficulties, etc., as well as their digestion and defecation conditions.

[0160] Sample collection: At the end of the experiment, blood was collected from the mouse orbits, and plasma was prepared and stored at -80°C; the colorectum was photographed and the number and volume of tumors were calculated, and the samples were fixed with formalin solution for pathological examination.

[0161] Intestinal tumor volume measurement: At the end of the experiment, the colon and rectum were dissected and removed, the number of tumors was counted, and the longest diameter (length) and shortest diameter (width) of a single tumor were measured with a vernier caliper to calculate the tumor volume. Tumor volume calculation formula: Tumor volume = 1 / 2 × tumor long diameter × tumor short diameter 2 All tumor volumes of a single animal were summed to give the final tumor volume for that animal.

[0162] Tumor volume results and intestinal tumor images are as follows Fig.15 and Fig.16As shown, tumor growth was observed in the colorectal region of each mouse in the Vehicle control group. The tumor volume in the PD-1 antibody monotherapy group was slightly lower than that in the control group. The XA-2595 monotherapy group and the XA-2595+PD-1 antibody combination group significantly inhibited tumors, with tumor inhibition rates of 39% and 43%, respectively. Compared with the control group and the PD-1 antibody group, the XA-2595+PD-1 antibody combination group did not reach a statistical difference of p<0.05 due to large individual differences in animals and large data dispersion, but it had significant biological significance.

[0163] Immunohistochemical analysis of colorectal tissue Fig.17 It can be seen that the area of ​​CD4+ and CD8+ positive cells in the colorectal tissue of mice in the XA-2595 group was increased compared with that in the control group. This result shows that XA-2595 can activate intestinal immunity and promote the infiltration of immune cells in the intestine, thereby exerting an anti-tumor effect on colorectal tumors.

[0164] Example 8 Safety evaluation of repeated administration of Bacillus bacteria XA-2595

[0165] This example provides a safety evaluation experiment of the Bacillus bacteria XA-2595 administered to mice for two consecutive weeks at the highest administrable dose. The experimental process is as follows:

[0166] (1) Preparation of live bacteria: A single colony of the bacteria XA-2595 of the genus Phytophthora was selected and inoculated into a YCFA medium, and cultured anaerobically at 37° C. for 24 h to obtain a culture solution; the culture solution was centrifuged and the cell pellet was collected to obtain live bacteria of the bacteria XA-2595 of the genus Phytophthora.

[0167] (2) Repeated administration of XA-2595 bacterial solution: 18 SPF-grade Balb / c mice (male, 6 weeks old, purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were selected. All mice were adaptively raised in cages in an SPF-grade animal room for 1 week, with a room temperature of (24±2)℃, alternating day and night every 12 hours, and free access to water. After adaptive feeding, the mice were divided into 3 groups according to their body weight on D0: G1 normal control group, G2 XA-2595-low-dose group, and G3 XA-2595 high-dose group, with 6 mice in each group. On the day of grouping, mice in the G2 XA-2595-low-dose group were fed 5×10 9 CFU / mouse gavage (with 400 μL PBS buffer as solvent), and mice in the G3 XA-2595 high-dose group were gavaged with 1×10 10 CFU / mouse (the highest administrable dose) was administered intragastrically (with 400 μL PBS buffer as the solvent), and the G1 normal control group mice were administered intragastrically with an equal amount of PBS buffer as a control. The drug was administered once a day until D16, for a total of 17 days, and the experiment ended on D17.

[0168] Weight monitoring: Starting from the day of grouping, weight measurement is performed 3 times a week. The weight monitoring results are shown in Fig.17 .

[0169] Status observation: During the experiment, the mice’s mental state, reduced activity, rough fur, paralysis, breathing difficulties, and death were observed.

[0170] Sample collection: At the end of the experiment, blood was collected from the mouse orbits, and plasma was prepared and stored at -80°C; the heart, liver, spleen, lung, kidney and thymus were weighed and photographed, and the colon was measured in length.

[0171] Weight results are as follows Fig.18 As shown in the figure, during the administration of the low- and high-dose groups of the Bacillus bacteria XA-2595, the body weights were comparable to those of the control group, and no drug-related changes in body weight were observed. Fig.19 As shown in the figure, except that the kidney weight was slightly lower than that of the control group, no difference was found in other organs. The low and high doses of XA-2595 bacterial solution tested in this experiment were effective doses (5×10 8 The results showed that XA-2595 had good safety and a large safety window, and kidneys were the main target organs, which may be related to the fact that the strain was mainly metabolized by the kidneys after oral administration.

Claims

1. Use of Gemmiger qucibialis in the preparation of drugs for preventing and / or treating cancer.

2. A strain of Phytophaga, characterized in that: The Gemmiger bacterium is Gemmiger XA-2595, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a taxonomic name of Gemmiger qucibialis, a deposit number of CGMCC: 27813, and a deposit date of July 5, 2023; Or, a Bacillus bacterium having a 16SrRNA gene sequence as shown in SEQ ID NO: 1; or, a Phytophthora bacterium comprising a 16S rDNA sequence having at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 1; Or, a Bacillus bacterium comprising a genome sequence that is at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98% or at least 99% identical to the Bacillus bacterium deposited with CGMCC No. 27813.

3. A product, characterized in that: Including at least one of a1) to a8): a1) The bacterium of the genus Phytophaga according to claim 1 or 2; a2) a bacterial agent containing the bacteria of the genus Phytophorum according to claim 2; a3) containing a live bacterial solution of the Phytophaga bacterium according to claim 2; a4) containing the dead bacteria solution of the Phytophaga bacteria according to claim 2; a5) containing the culture product of the Phytophaga bacterium according to claim 2; a6) containing the metabolites of the bacteria of the genus Phytophorum according to claim 2; a7) containing an extract of the Phytophaga bacterium according to claim 2; a8) A culture supernatant containing the Phytosporum bacterium according to claim 2.

4. The product according to claim 3, characterized in that: The products include medicines, health products, foods, functional foods, skin care products or medical devices.

5. Use of the Phytophaga bacterium according to claim 1 or 2, or the product according to claim 3, in any one of b1) to b5): b1) Preparation of products for the treatment and / or prevention of cancer; b2) preparing products for regulating the intestinal microenvironment; b3) preparing products for regulating tumor microenvironment; b4) Preparation and activation of intratumoral CD4 + Cell products; b5) Preparation and activation of intratumoral CD8 + Cell products.

6. The use according to claim 5, characterized in that: The product includes pharmaceutical excipients; Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

7. The use according to claim 5, characterized in that: The products mentioned include other anti-tumor drugs; Preferably, the other anti-tumor drugs include at least one of immune checkpoint inhibitors, anti-tumor factors, tumor antibodies, cancer vaccines, cell therapy and small molecule drugs; Preferably, the immune checkpoint inhibitors include but are not limited to PD-1 antibodies, PD-L1 antibodies, and CTLA-4 antibodies. Further preferably, the anti-tumor factors include but are not limited to at least one of tumor necrosis factor and interferon; Preferably, the small molecule drug includes but is not limited to at least one of doxorubicin, paclitaxel, docetaxel, cisplatin, mitoxantrone, daunorubicin, vincristine, all-trans retinoic acid, farubicin, rutotecan, irinotecan, 2-methoxyestradiol, gemcitabine, vinorelbine, 5-fluorouracil, methotrexate, capecitabine, lomustine, and etoposide.

8. The use according to claim 5, characterized in that: The dosage forms of the product include powders, drops, gels, lozenges, tablets, pills, soft / hard gelatin capsules, patches, lyophilized powders, sprays, granules, suspensions, syrups, suppositories, granules, emulsions, creams, ointments, injections, sterile powders, gels, oral solutions and nanoparticles.

9. The use according to claim 5, characterized in that: The tumor microenvironment includes but is not limited to immune cells, inflammatory cells, metabolites, and inflammatory factors; Preferably, regulating the tumor microenvironment includes upregulating the level of immune activation factors and / or downregulating the level of anti-inflammatory factors; Preferably, the immune activation factors include but are not limited to TNF-α, IFN-γ, IL-6; the anti-inflammatory factors include but are not limited to IL-10.

10. The use according to claim 5, characterized in that: The cancers include solid tumors and hematologic tumors; solid tumors include, but are not limited to, colon, rectal and prostate tumors; esophageal, stomach, pancreatic and liver tumors; bladder and gallbladder tumors; breast, lung and chest solid tumors; ovarian and uterine tumors; nasopharyngeal tumors, laryngeal tumors, brain tumors and head and neck tumors; skin tumors such as melanoma; hematologic tumors include, but are not limited to, leukemias, acute myeloid leukemia, chronic myeloid leukemia, blast crisis of chronic myeloid leukemia, myelodysplasia and myeloproliferative syndrome; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma.

Images