Use of bacteria in the preparation of potentiators of immune checkpoint inhibitors

Abstract

The present invention discloses the use of bacteria in the preparation of an enhancer for immune checkpoint inhibitors, which can be a live bacterial strain or an inactive whole-cell intestinal bacterium. The present invention utilizes a monomicrobial oral formulation using the human endogenous intestinal bacterium Lactobacillus alistipes in combination with an immune checkpoint inhibitor. By utilizing the antitumor immune protective response and intestinal microbiota reconstitution effect induced by oral administration of the active human commensal intestinal bacterium or the inactive whole-cell intestinal bacterium, the present invention significantly enhances the efficacy of immune checkpoint inhibitors against multiple tumors, enhances antitumor immune function, and helps improve the response rate of patients undergoing cancer immunotherapy. It also has superior safety and extends the overall survival of cancer patients, broadening the range of tumor patients eligible for cancer immunotherapy (immunotherapeutic checkpoint inhibitors). It also provides new combination therapy plans and therapeutic agents for the treatment of patients with tumors refractory to immune checkpoint inhibitors, thereby broadening the range of tumor patients eligible for cancer immunotherapy.

Description

[Technical field]

[0001] The present invention relates to the technical field of tumor therapy, and in particular to the use of bacteria in tumor therapy. [Background technology]

[0002] Malignant tumors are one of the biggest problems facing the world today, and among the various lethal factors that cause death every year, malignant tumors rank first. Conventional treatments such as surgery, radiotherapy and chemotherapy are difficult to completely remove tumor cells, and the recurrence rate is high, and because the therapeutic methods such as radiotherapy and chemotherapy have low targeting, they also have a killing effect on normal cells while killing tumor cells, which causes great harm to patients and seriously affects their quality of life.

[0003] Compared with conventional therapies, the target of tumor immunotherapy is not tumor cells or tissues, but the body's own immune system. Through the regulation and activation of the body's immune system, the autoimmune function is used to eliminate tumor cells, including those that have metastasized to distant sites, and the toxicity and side effects to normal cells are small, greatly improving the prognosis and quality of life of patients with advanced malignant tumors. Immune enhancers are a new drug category, also known as immunomodulators, and were previously called immune enhancers or immune stimulants. Many immune enhancers have been developed for the purpose of non-specific immunotherapy of tumors, and exert their therapeutic effects by improving the immune function of the body and promoting and inducing immune response reactions. In clinical practice, they are mainly used for immunodeficiency diseases, auxiliary treatment of malignant tumors, and refractory bacterial and viral infections. Immune enhancer drugs commonly used in clinical practice can be divided into five types according to their origin. 1. Bacterial immune enhancing drugs such as the BCG vaccine 2. Products of the human or animal immune system, such as thymosin, transfer factor, interferon, interleukin, etc. 3. Chemically synthesized immune enhancing drugs, such as levamisole, polyinosine-cytidine, etc. 4. Fungal polysaccharides, such as shiitake mushroom polysaccharides 5. Traditional Chinese medicines and others, such as the active ingredients of traditional Chinese medicines such as ginseng, astragalus root, etc.; phytohemagglutinin (PHA), concanavalin A, placental polysaccharides, etc.

[0004] The use of bacterial immune enhancers in cancer treatment has a long history. As early as 1893, William Coley, a pioneer in the field of cancer immunotherapy, repeatedly administered "Coley's toxin" prepared from Streptococcus pyogenes to terminal cancer patients, causing fever and stimulating the body's immune response, resulting in the regression of tumors in some patients. "Coley's toxin" became the catalyst for cancer immunotherapy and promoted the rise of modern cancer immunotherapy. In the past decade, cancer immunotherapy has achieved unprecedented development and success, completely changing the clinical management of various malignant tumors. Among them, immune checkpoint inhibitors (ICIs) are one of the most cutting-edge technologies in immunotherapy.

[0005] Currently, there are many types of tumor immunotherapy, among which immune checkpoint inhibitors (ICI) therapy reactivates T cells and reactivates the body's immune system's ability to identify and eliminate tumor cells by blocking immune suppressive pathways (e.g. PD-L1 / PD-1 and CTLA-4 / B7-1) hijacked by tumor cells, and has the advantages of good therapeutic effect and long duration of response, opening a new era of tumor treatment. ICI therapy has brought about a revolution in cancer treatment by its application to various types of tumors that lack treatment options, and has become a new milestone in cancer treatment.

[0006] Currently, patients treated with ICIs can achieve long-term remission even after treatment is discontinued, raising hopes for a cure for some patients. In particular, complete remission, i.e., the complete disappearance of all visible tumor metastases, is possible for melanoma patients. However, the low response rate of patients with ICIs alone is the biggest bottleneck in their development and use, and only 15% to 40% of patients achieve complete remission with ICIs alone. Therefore, there is an urgent need to develop combination strategies to improve the efficacy of immune checkpoint inhibitors.

[0007] The current mainstream combination strategy includes the combination of ICI with surgery, chemotherapy, radiation therapy, targeted therapy and other therapeutic methods, which can exert synergistic effects, enhance the durability and therapeutic effect of ICI, and solve the problem of low response rate of ICI. Although these combination therapies can improve the therapeutic effect and response rate of ICI to a certain extent, the toxicity is usually increased accordingly.

[0008] Research has found that factors related to the specificity of a patient's response rate to ICI treatment and drug resistance include internal factors of the tumor, such as the tumor microenvironment, as well as host-related factors, such as age, genetic diversity, diet, and intestinal microbiota. The influence of certain intestinal bacteria on the therapeutic effect of ICI through colonization, direct action of its own whole-cell components and the intestinal mucosal immune system, or indirect action of producing small molecule metabolites through its metabolic function has attracted the most attention. Therefore, among many combination therapies, many studies of manipulating endogenous intestinal microbes to support ICI treatment have become milestone events in the field of tumor treatment. The intestine is the largest lymphatic organ in the body, where more than 70% of T cells reside and where most memory T cells reside. The intestinal microbiota that resides in the intestine promotes the development of the human body's immune system through interaction with the intestinal immune organs, laying the foundation for the human body's immune strength and establishing its balance. For the human body's defense and immune systems, bacteria and tumors are both "foreign", so the human body uses the same defense mechanism to respond to bacteria and tumors, and the immune response stimulated by endogenous gut bacteria also has anti-tumor potential. Based on the anti-tumor immune cross-reaction theory of gut microbiota and tumor-lytic enzymes secreted by certain gut bacteria themselves, bacterial tumor treatment has a history of several hundred years. In addition, the anti-tumor effect induced by endogenous gut bacteria is derived from the body's own immune response, and is safer and less toxic and has less side effects than combined radiation therapy and chemotherapy.

[0009] An overview published in Cancer Discovery in August 2021 summarized 38 studies published between 2017 and 2020, and analyzed 11,959 tumor patients who received ICI treatment. The analysis showed that the use of antibiotics before or during immunotherapy significantly reduced the survival rate of patients (mortality risk ratio value HR = 1.81, p = 0.03), indicating that the immune response induced by gut bacteria in the body has an important effect on the therapeutic efficacy of ICI therapy (Derosa, Routy et al. 2021). Several clinical studies in China and abroad have shown that the combination of ICI therapy with measures to manipulate the gut microbiota, such as fecal bacteria transplantation and oral administration of pure strains and prebiotics, can reverse drug resistance to ICI in tumor patients, indicating that local treatment based on gut microbiota intervention can support intestinal immunity, stimulate effective antitumor immune surveillance, and trigger durable distal antitumor immunity. According to two recent clinical studies, fecal bacteria transplantation in patients who responded to ICI treatment (partial or complete remission) can induce partial or complete remission in patients with refractory metastatic melanoma who did not respond to ICI, without any toxicity or side effects, and has demonstrated the effect of endogenous gut microbes in enhancing the efficacy of ICI therapy (Baruch EN 2020, Zipkin 2021). However, fecal bacteria transplantation is complex in composition, and it is the entire gut microbes that are effective in fecal bacteria transplantation. Moreover, the gut microbiota is greatly influenced by the environment, diet, lifestyle, etc., and the lack of a sustainable and stable donor fecal bacteria limits the clinical application of fecal bacteria transplantation and ICI combined immunotherapy. Therefore, identifying and preparing a single-component antitumor gut bacteria that can be produced in a standardized quantitative manner is key to solving this problem. In the latest study, researchers isolated three types of bacteria -- Bifidobacterium pseudolongum, Lactobacillus johnsonii, and an Olsenella species -- from a mouse model of intestinal cancer treated with ICIs.These three special bacteria, when combined with immune checkpoint inhibitors, can enhance the therapeutic effect of ICI in four mouse cancer models. Further research has revealed that Bifidobacterium pseudolongum exerts an ICI enhancing effect via its metabolic product, inosine (Mager LF 2020).

[0010] With the integration of biology and chemistry with materials science, engineering, computer science and other fields, this study provides new avenues for the research and development of novel gut microbiota-derived immune enhancers. The powerful combination of gut microbiota-derived immune enhancers and immune checkpoint inhibitors is expected to be a breakthrough in next-generation tumor immunotherapy, providing an alternative therapeutic approach for the immunotherapy of patients with refractory tumors.

[0011] Alistipes is a genus of Bacteroidetes, a Gram-negative, obligately anaerobic, intestinal symbiotic bacterium. Existing research has shown that Alistipes is a new bacterial genus isolated from clinical samples, and that the species composition of Alistipes is diverse and complex, with different Alistipes species having unique functional properties and exerting health-promoting or pathogenic effects on the human body (Parker, Wearsch et al. 2020). According to the NCBI taxonomy database, the species of Alistipes identified and named so far are Alistipes communis, Alistipes dispar, Alistipes finegoldii, Alistipes ihumii, Alistipes indistinctus, Alistipes inops, Alistipes massiliensis, Alistipes megaguti, Alistipes okayasuensis, Alistipes onderdonkii, Alistipes provencensis, Alistipes It consists of 15 species: Alistipes provencensis, Alistipes putredinis, Alistipes senegalensis, Alistipes shahii, and Alistipes timonensis. In addition, it includes about 90 Alistipes species that are unnamed or predicted by macrogenome sequencing data. According to the Type Strain Genome database, there are 14 cultivable type strains of Alistipes species with accession numbers.Currently, only two Alistipes species, Alistipes shahii and Alistipes indistinctus, have been shown to have potential immunotherapy enhancing functions in animal studies (Iida N 2013, Routy B 2018). It is necessary to investigate whether other Alistipes species, or combinations of Alistipes species, could be used as gut bacteria-derived enhancers of immune checkpoint inhibitors.

[0012] From an ecological point of view, Alistipes species mainly reside in the intestines of healthy individuals and play an important role in maintaining a healthy intestinal immune status in the human body. For example, the reduction of Alistipes may promote the reduction of short-chain fatty acids and aggravate end-stage fibrosis in patients with non-alcoholic fatty liver disease. However, Alistipes species have also been isolated from other fluids in the human body, for example, Alistipes onderdonkii and Alistipes shahii have been isolated from abdominal abscesses, appendix tissue and urine in the human body, respectively, indicating the potential opportunistic pathogenicity of individual species of Alistipes.

[0013] In an example based on prior patent application CN110582291A, various single bacteria and species combinations, including Akkermansia municiphila, Enterococcus hirae 13144, Alistipes indistinctus, and a combination of Akkermansia municiphila + Enterococcus hirae 13144, were used as compensatory anti-cancer probiotics to compare their efficacy in reversing immune checkpoint inhibitor resistance in the mouse MCA205 murine fibrosarcoma model of tumor response. The results showed that all selected single bacteria or species combinations were able to significantly reduce the final tumor size in antibiotic-treated mice without FMT or with spontaneous gut microbiota reconstitution by FMT (derived from feces of patients not responding to immune checkpoint inhibitor treatment), among which the best responder group was Akkermansia municiphila + Enterococcus hirae 13144 administered by oral gavage. One of the best surrogate markers of activity of effective anticancer probiotic combinations is circulating blood (or splenic) CD4 + or CD8 + Upregulation of PD-L1 expression on T cells or upregulation of CCR9 on circulating T cells. Compared with the single-agent PD1 group, the combination of PD1 with Akkermansia municiphila + Enterococcus hirae 13144 significantly increased splenic tissue CD8 + PDL1 + and CD4 + PDL1 + The PD1 + Alistipes indistinctus group significantly increased the number of immune cells in spleen tissue, whereas the PD1 + Alistipes indistinctus group significantly increased the number of CD8 + PDL1 + and CD4 +PDL1 + No significant increase in immune cells was observed. CN110582291A further compared different compensatory anticancer probiotic combinations and the effect of each combination on tumor response to reverse immune checkpoint inhibitor resistance in the mouse MCA205 mouse fibrosarcoma model, including a combination of two Alistipes species (Alistipes onderdonkii + Alistipes finegoldii). The results showed that the more combinations, the better the therapeutic effect, and the combination of Akkermansia municiphila + Enterococcus limosum was the optimal response, while the combination of Alistipes onderdonkii + Alistipes finegoldii did not show any effect in improving the therapeutic effect of single-agent PD1 antibody. Summary of the Invention [Problem to be solved by the invention]

[0014] The Alistipes finegoldii of the present invention is abbreviated as Af in the prior priority applications CN2021108083661 and CN2021109396998, and as RX-af01 in the prior priority application CN2022101435293, and is abbreviated as Af in this application.

[0015] An object of the present invention is to provide the use of bacteria in the preparation of an enhancer of an immune checkpoint inhibitor.

[0016] The present invention provides combination therapies that enhance the therapeutic effect of immune checkpoint inhibitors that can be applied to one or more tumors.

[0017] The enhancer for enhancing the therapeutic effect of the immune checkpoint inhibitor of the present invention is a bacterium, preferably Alistipes finegoldii (Af), and the Af is one or more selected from live bacteria, inactive whole-cell bacteria, bacterial derivatives, and bacterial metabolites of Alistipes finegoldii.

[0018] The cancer immunotherapy of the present invention is selected from immune checkpoint inhibitor therapies, and the enhancement of the immunotherapy indicates an extension of the overall survival time of cancer patients, an improvement in the response rate of cancer immunotherapy patients, or an expansion of the range of tumor patients to which cancer immunotherapy can be applied.

[0019] The experimental data disclosed in the present invention shows that, when comparing the intestinal flora of cancer patients who responded to a combination of immune checkpoint inhibitor anti-PD1 antibody (αPD-1) and chemotherapy and those who did not respond to the combination, the genus Alistipes was significantly higher in the responding patients than in the non-responding patients, and such a difference did not exist in the chemotherapy alone group. In the intestines of colorectal cancer patients (CRC) who received αPD-1 combination chemotherapy, the progression-free survival (PFS) of Alistipes-positive patients was significantly longer than that of Alistipes-negative patients. In the intestines of esophageal cancer (ESCC) patients who received αPD-1 combination chemotherapy, the overall survival (OS) of patients with a high relative abundance of Alistipes was significantly longer than that of patients with a low relative abundance of Alistipes. Among the five Alistipes species detected, the relative abundance of intestinal Alistipes finegoldii was significantly higher in ESCC and CRC patients with a PFS of greater than 6 months after receiving αPD-1 combination chemotherapy than in patients with a PFS of less than 6 months.

[0020] We found that administration of Alistipes shahii alone or the combination of Alistipes shahii and Alistipes finegoldii did not enhance immune checkpoint inhibitors in multiple tumor models. However, administration of active Alistipes finegoldii alone or inactivated whole cell Alistipes finegoldii in combination with immune checkpoint inhibitors demonstrated that Alistipes finegoldii or its inactive bacterial components acted as enhancers of immune checkpoint inhibitors in cancer treatment, enhancing the tumor-killing immune function of the host.

[0021] The present invention provides a therapy that provides a clear improvement over the shortcomings of conventional therapies, such as high toxicity and side effects, a high likelihood of metastasis and recurrence, short duration of therapy, short survival time, and poor quality of life, and that provides a clear improvement over the shortcomings of therapy using an immune checkpoint inhibitor alone, such as a limited range of tumor activity and a small number of patients who respond to the drug, and that provides a clear improvement over the shortcomings of therapy using an immune checkpoint inhibitor in combination with radiation chemotherapy, such as high toxicity and side effects and a small number of patients who respond to the drug.

[0022] The treatment method provided by the present invention has excellent therapeutic effects on patients who are inoperable, for whom there are no available targeted drugs, and for whom radiation therapy, chemotherapy, etc. are ineffective; patients for whom immune checkpoint inhibitors alone are ineffective or who have drug resistance (primary, adaptive, and acquired drug resistance); and patients for whom the combined use of immune checkpoint inhibitors with radiation therapy, chemotherapy, or targeted therapy is ineffective or who have drug resistance (primary, adaptive, and acquired drug resistance). [Means for solving the problem]

[0023] The present invention achieves the above object through the following aspects. The use of bacteria in the treatment of tumors.

[0024] Preferably, the bacteria is one or more of live bacteria, inactive bacteria, bacterial derivatives and bacterial metabolic products.

[0025] Preferably, the inactivated bacteria is an inactivated whole cell bacteria.

[0026] Preferably, the live bacteria are whole bacteria and / or whole active bacteria.

[0027] Preferably, the use is the use of the bacterium as an active ingredient in the preparation of an enhancer of an immune checkpoint inhibitor.

[0028] Preferably, the use is an agent for enhancing an immune checkpoint inhibitor, thereby improving the effect of the immune checkpoint inhibitor in tumor treatment.

[0029] Preferably, the bacterium belongs to the genus Alistipes, i.e. the 16S rDNA sequence of the bacterial strain has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% homology with the 16S rDNA sequence of Alistipes.

[0030] Preferably, the bacteria belonging to the genus Alistipes is a combination of one or more species or strains of bacteria belonging to the genus Alistipes.

[0031] More preferably, the bacterium belonging to the genus Alistipes is Alistipes finegoldii.

[0032] More preferably, the 16S rDNA sequence contained in the Alistipes finegoldii has at least 99% homology with the 16S rDNA sequence of the Alistipes finegoldii species.

[0033] More preferably, the 16S rDNA sequence contained in the Alistipes finegoldii has at least 99.5% or 100% homology with the 16S rDNA sequence of Alistipes finegoldii strain DSM 17242.

[0034] Even more preferably, the Alistipes finegoldii strain is one or a combination of two or more Alistipes finegoldii strains.

[0035] More preferably, the strain of Alistipes finegoldii has the following strain names of subspecies classification in the National Center for Biotechnology Information (NCBI) genome database (https: / / www.ncbi.nlm.nih.gov / genome / browse / #! / prokaryotes / 11196 / ): Alistipes finegoldii DSM 17242, Alistipes finegoldii D53t1_180928_D3, Alistipes finegoldii 2789STDY5834947, Alistipes finegoldii 1001713B170207_170306_H2, Alistipes finegoldii DFI.2.31, Alistipes finegoldii BIOML-A1, Alistipes finegoldii DFI.2.16, Alistipes finegoldii DFI.2.10, Alistipes finegoldii aa_0143, Alistipes finegoldii 2789STDY5608890, Alistipes finegoldii MGBC116453, Alistipes finegoldii COPD076, or Alistipes finegoldii UBG195. Even more preferably, the Alistipes finegoldii strain is one or a combination of two or more of the following strains:

[0036] Alistipes feingoldii, deposited at the German Collection of Microorganisms and Species Depositary DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen = German collection of microorganisms and cell cultures) under the accession number DSM 17242 (NCBI: txid679935, https: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=679935); Alistipes finegoldii, deposited at the Japan Collection of Microorganisms (JCM Seed Depositary) under the accession number JCM16770; Alistipes finegoldii, deposited at the Korean Collection for Type Cultures (KCTC) under accession number KCTC 15236; Alistipes finegoldii, deposited at the Anaerobe Reference Laboratory, Helsinki Collection, National Public health Institute, Helsinki, Finland, accession number AHN2437; Alistipes finegoldii, deposited at the Swedish CCUG fungal species depository (Culture Collection University of Gothenburg) under accession number CCUG46020; Alistipes finegoldii, deposited at the French CIP Seed Depository Center (Collection de L'Institut Pasteur) under accession number CIP107999; Alistipes finegoldii , deposited at the Guangdong Microbial Species Depository Center, China, accession number GDMCC 1.2324. Preferably, the 16S rDNA sequence contained in the Alistipes finegoldii has at least 99% homology with the 16S rDNA sequence of the Alistipes finegoldii species shown in SEQ ID NO:1.

[0037] More preferably, the Alistipes finegoldii bacterium is the Alistipes finegoldii strain deposited at DSM with accession number 17242.

[0038] More preferably, the bacterium belonging to the genus Allosteris and the immune checkpoint inhibitor are administered simultaneously or separately.

[0039] Preferably, said bacterial metabolites include all molecules produced or modified by said bacteria as a result of the growth, survival, residence, transport or presence of the bacteria during preparation and storage of the bacteria, as well as during transit through the mammalian gastrointestinal tract.

[0040] More preferably, said bacterial metabolites include all organic acids, inorganic acids, bases, proteins and peptides, enzymes and coenzymes, amino acids and nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids, vitamins, all biologically active compounds, metabolites containing inorganic components, small molecules such as nitrogen- or sulfite-containing molecules.

[0041] Preferably, the bacterial derivative comprises bacterial components and genetic material and associated components, examples of which include bacterial derived components such as bacterial cell membranes, pili, flagella, LPS, nucleic acid material and the like.

[0042] Preferably, said inactive whole cell bacteria are obtained by amplifying the bacteria in culture and then inactivating them in various ways.

[0043] More preferably, the means for inactivating the bacteria is one or more selected from high temperature and / or high pressure inactivation, ultraviolet inactivation, radiation inactivation, and chemical inactivation using formaldehyde, acetone, phenols, etc.

[0044] More preferably, the chemical agent is one or more of formaldehyde, acetone, and phenols.

[0045] More preferably, the inactive whole cell bacteria comprises one or more of a bacterial whole cell component, a bacterial whole cell derivative, or a bacterial whole cell metabolic product.

[0046] More preferably, the bacterial whole cell components include bacterial constituents and genetic material and associated components, in particular one or more of bacterial derived components such as bacterial cell walls, cell membranes, pili, flagella, LPS, nucleic acid material, etc.

[0047] More preferably, the bacterial whole cell derivative comprises one or more of bacterial extracellular vesicles, bacterial associated exosomes, and prophages.

[0048] More preferably, the bacterial whole cell metabolites refer to metabolic products whose production is involved in all bacterial cell components, and include one or more of all organic acids, inorganic acids, bases, proteins and peptides, enzymes and coenzymes, amino acids and nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids, vitamins, all biologically active compounds, metabolites containing inorganic components, e.g. small molecules such as nitrogen-containing or sulfite-containing molecules.

[0049] Preferably, treatment of the tumor is manifested as tumor shrinkage or stabilization, increased overall survival, increased progression-free survival, and improved quality of life.

[0050] Preferably, the tumor is an adenoma, a malignant tumor, or an adenocarcinoma, and the tumor, when classified according to tissue origin or cell name, is one or more of adrenal cortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, glioblastoma multiforme, glioma, head and neck cancer, renal chromophobe cell carcinoma, mixed renal carcinoma, kidney cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, skin melanoma, gastric cancer, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, and soft tissue sarcoma.

[0051] In one particular embodiment of the present invention, the bacterium (Af) is a live bacterial strain of Alistipes finegoldii having accession number 17242 deposited at the DSM, the tumor is colon cancer or melanoma, and the immune checkpoint inhibitor is a PD-1 monoclonal antibody (αPD-1) having clone number G4C2, or a CTLA4 monoclonal antibody (αCTLA4) having clone number 9D9.

[0052] In one particular embodiment of the present invention, the bacterium (Af) is an inactivated bacterium of the Alistipes finegoldii strain deposited at the DSM with accession number 17242, the tumor is colon cancer or lung cancer, and the immune checkpoint inhibitor (αPD-1) is clone number G4C2.

[0053] Preferably, the tumor is a malignant tumor, a metastatic tumor or a non-metastatic tumor.

[0054] Preferably, herein, the tumor includes malignant, metastatic and non-metastatic types, including any stage of cancer (clinical stage: I, II, III, or IV; malignant tumor TNM classification: T1-4, N0-4, or M0-1; histological stage: G1, G2, G3, or G4, etc.).

[0055] More preferably, the immune checkpoint inhibitor is one or a combination of two or more blockers acting on T cell suppressive costimulatory (co-inhibitory) molecules and / or their respective ligands.

[0056] More preferably, the T cell inhibitory costimulatory (co-inhibitory) molecule and / or each ligand is one selected from CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and DcR3, or a combination of any two or more thereof.

[0057] Even more preferably, the blocker of the ligand of the T cell inhibitory costimulatory (co-inhibitory) molecule is selected from the group consisting of nivolumab (PD-1 monoclonal antibody), ipilimumab (CTLA-4 monoclonal antibody), pembrolizumab (PD-1 monoclonal antibody), azetolizumab (PD-L1 monoclonal antibody), atezolizumab (PD-L1 monoclonal antibody), camrelizumab (PD-L1 monoclonal antibody), tislelizumab (BGB-A317), durvalumab (PD-L1 monoclonal antibody), tramemumab (CTLA-4 monoclonal antibody), spartali Spartalizumab (PD-1 monoclonal antibody), avelumab (PD-L1 monoclonal antibody), sintilimab (PD-1 monoclonal antibody), toripalimab (PD-1 monoclonal antibody), cemiplimab (PD-1 monoclonal antibody), retifanlimab (PD-1 monoclonal antibody), MGD013 (tebotelimab, PD-1 / LAG-3 dual monoclonal antibody), MGD019 (PD-1 / CTLA-4 dual monoclonal antibody), enoblituzumab (B7-H3 monoclonal antibody), MGD009 (B7-H3 monoclonal antibody), MGC018 (B7-H3 monoclonal antibody), MEDI0680(PD-1 monoclonal antibody), PDR001 (PD-1 monoclonal antibody), FAZ053 (PD-L1 monoclonal antibody), PDR001FAZ053, TSR022 (TIM-3 monoclonal antibody), MBG453 (TIM-3 monoclonal antibody), relatlimab (BMS986016, LAG-3 monoclonal antibody), LAG525 (LAG-3 monoclonal antibody), IMP321 (LAG-3 monoclonal antibody), REGN3767 (LAG-3 monoclonal antibody), pexidartinib (CSF-1R monoclonal antibody), LY3022855 (CSF-1R monoclonal antibody), FPA008 (CSF-1R monoclonal antibody), BLZ945 (CSF-1R monoclonal antibody), GDC0919 (navoximod, The blocker is one selected from the group consisting of IDO monoclonal antibody, epacadostat (IDO monoclonal antibody), indoximid (IDO monoclonal antibody), BMS986205 (IDO monoclonal antibody), CPI-444 (A2AR monoclonal antibody), MEDI9447 (oleclumab, CD73 monoclonal antibody), PBF509 (A2AR monoclonal antibody), and lirilumab (KIR monoclonal antibody), or a combination of any two or more of these, and preferably, the blocker is one selected from the group consisting of nivolumab, pembrolizumab, toripalimab, cindirilumab, and cemiplimab, or a combination of any two or more of these.

[0058] Even more preferably, the immune checkpoint inhibitor is an inhibitor acting on the PD-1 / PD-L1 signal pathway and / or the PD-1 / PD-L2 signal pathway, where PD-1 stands for programmed cell death protein 1, also known as CD279, and PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273) are ligands of PD-1.

[0059] More preferably, the inhibitor of the PD-1 / PD-L1 signal pathway and / or the PD-1 / PD-L2 signal pathway is selected from the group consisting of nivolumab (PD-1 monoclonal antibody), pembrolizumab (PD-1 monoclonal antibody), azetolizumab (PD-L1 monoclonal antibody), atezolizumab (PD-L1 monoclonal antibody), camrelizumab (PD-L1 monoclonal antibody), and tissumab. Tislelizumab (BGB-A317), durvalumab (PD-L1 monoclonal antibody), spartalizumab (PD-1 monoclonal antibody), avelumab (PD-L1 monoclonal antibody), sintilimab (PD-1 monoclonal antibody), toripalimab (PD-1 monoclonal antibody), cemiplimab (PD-1 monoclonal antibody), MGA012 (retifanlimab, PD-1 monoclonal antibody), MGD013 (tebotelimab, PD-1 / LAG-3 dual monoclonal antibody), MGD019 (PD-1 / CTLA-4 dual monoclonal antibody), MEDI0680 (PD-1 monoclonal antibody), PDR001 (PD-1 monoclonal antibody), and FAZ053 (PD-L1 monoclonal antibody), or a combination of any two or more of these.

[0060] Even more preferably, the inhibitor of the PD-1 / PD-L1 signal pathway or the PD-1 / PD-L2 signal pathway is one selected from ipilimumab, tremelimumab, or MGD019, or a combination of any two or more thereof.

[0061] Even more preferably, the immune checkpoint inhibitor is an inhibitor acting on the CTLA-4 / B7-1 signal pathway and / or the CTLA-4 / B7-2 signal pathway, where CTLA-4 stands for cytotoxic T-lymphocyte protein 4, also known as CD152, and B7-1 (CD80) and B7-2 (CD86) are ligands for CTLA-4.

[0062] Even more preferably, it may be ipilimumab (CTLA-4 monoclonal antibody), tremelimumab (CTLA-4 monoclonal antibody), MGD019 (PD-1 and CTLA-4 dual monoclonal antibody), or a combination of any two or more of these.

[0063] In one particular embodiment of the present invention, the immune checkpoint inhibitor is an inhibitor acting on the PD-1 / PD-L1 signal pathway and / or the PD-1 / PD-L2 signal pathway, and / or an inhibitor acting on the CTLA-4 / B7-1 signal pathway and / or the CTLA-4 / B7-2 signal pathway. Specifically, the immune checkpoint inhibitor is a PD-1 monoclonal antibody or a CTLA-4 monoclonal antibody.

[0064] Preferably, chemotherapy, immunotherapy, radiotherapy is administered simultaneously, separately or sequentially with the administration of Af.

[0065] Preferably, the subject is a human, and the human is an infant, a child, an adolescent, an adult, or an elderly person.

[0066] Preferably, the subject of administration is a non-human primate, which is a mammal (e.g., dog, cat, ferret, horse, rabbit, guinea pig, gerbil, hamster, red squirrel, rat, mouse); bird; reptile; fish; amphibian; arthropod, or livestock animal (e.g., cow, pig, sheep, goat, alpaca, donkey, camel, buffalo, or mink).

[0067] Preferably, the bacterial CFU (colony forming units) is 10 5 ~10 12 Pieces or 10 7 ~10 11 , or 10 8 ~10 11 , or 10 9 ~10 11 , or 10 10 ~10 11 , more preferably 10 9 ~10 11 The bacteria (Af) is administered in a dose comprising:

[0068] The present invention further includes the following.

[0069] A tumor treatment kit comprising one or more of the immune checkpoint inhibitors and the bacterium, or consisting of one or more of the immune checkpoint inhibitors and the bacterium.

[0070] Preferably, the kit comprises a container.

[0071] A bacterium comprising a 16S rDNA sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% homology to the 16S rDNA sequence of Alistipes, and the bacterium is one or more of a live bacterium, an inactive bacterium, a bacterial derivative, or a bacterial metabolic product.

[0072] A bacterium comprising a 16S rDNA sequence having at least 99% homology with the 16S rDNA sequence of Alistipes finegoldii, and which is one or more of live bacteria, inactive bacteria, bacterial derivatives, and bacterial metabolic products.

[0073] A bacterium comprising a 16S rDNA sequence having at least 99% or 100% homology to the 16S rDNA sequence of any one of the following strains, which is one or more of live bacteria, inactive bacteria, bacterial derivatives, and bacterial metabolic products:

[0074] Alistipes feingoldii, deposited at the German Collection of Microorganisms and Species Depositary DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen = German collection of microorganisms and cell cultures) under the accession number DSM 17242 (NCBI: txid679935, https: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=679935); Alistipes finegoldii, deposited at the Japan Collection of Microorganisms (JCM Seed Depositary) under the accession number JCM16770; Alistipes finegoldii, deposited at the Korean Collection for Type Cultures (KCTC) under accession number KCTC 15236; Alistipes finegoldii, deposited at the Anaerobe Reference Laboratory, Helsinki Collection, National Public health Institute, Helsinki, Finland, accession number AHN 2437; Alistipes finegoldii, deposited at the Swedish CCUG fungal species depository (Culture Collection University of Gothenburg) under accession number CCUG46020; Alistipes finegoldii, deposited at the French CIP Seed Depository Center (Collection de L'Institut Pasteur) under accession number CIP107999; Alistipes finegoldii deposited at the Guangdong Microbial Species Depository Center, China, accession number GDMCC 1.2324.

[0075] Preferably, the inactivated bacteria is an inactivated whole cell bacteria.

[0076] Preferably, the bacteria are live whole bacteria and / or whole active bacteria.

[0077] Among them, the 16S rDNA sequence of Alistipes finegoldii DSM 17242 having the accession number DSM 17242 is shown in SEQ ID NO:1.

[0078] SEQ ID NO:1: agagtttgat cctggctcag gatgaacgct agcggcaggc ttaacacatg caagtcgagg ggcagcgggg agtagcaata ctccgccggc gaccggcgca cgggtgcgta acgcgtatgc aacctacctt taacaggggc ataacactga gaaattggta ctaattcccc ataacattcg agaaggcatc ttcttgggtt aaaaactccg gtggttaaag atgggcatgc gttgtattag ctagttggtg aggtaacggc tcaccaaggc aacgatacat agggggactg agaggttaac cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat tggtcaatgg acgcaagtct gaaccagcca tgccgcgtgc aggaagacgg ctctatgagt tgtaaactgc ttttgtacta gggtaaacgc ttttacgtgt aggagcctga aagtatagta cgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccaagcgtt atccggattt attgggttta aagggtgcgt aggcggtttg ataagttaga ggtgaaatac cggggctcaa ctccggaact gcctctaata ctgttgaact agagagtagt tgcggtaggc ggaatgtatg gtgtagcggt gaaatgctta gagatcatac agaacaccga ttgcgaaggc agcttaccaa actatatctg acgttgaggc acgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgcag taaacgatga taactcgttg tcggcgatac acagtcggtg actaagcgaa agcgataagt tatccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag gaaccttacc cgggcttgaa agttagtgac gattctggaa acaggatttc ccttcggggc acgaaactag gtgctgcatg gttgtcgtca gctcgtgccg tgaggtgtcg ggttaagtcc cataacgagc gcaaccccta ccgttagttg ccatcaggtc aagctgggca ctctggcggg actgccggtg taagccgaga ggaaggtggg gatgacgtca aatcagcacg gcccttacgt ccggggctac acacgtgtta caatggtagg tacagagggc cgctaccccg cgaggggatg ccaatctcga aagcctatct cagttcggat cggaggctga aacccgcctc cgtgaagttg gattcgctag taatcgcgca tcagccatgg cgcggtgaat acgttcccgg gccttgtaca caccgcccgt caagccatgg aagctggggg tgcctgaagt tcgtgaccgc aaggagcgac ctagggcaaa accggtgact ggggctaagt cgtaacaagg taaccaa

[0079] A formulation in which one or more of the above-mentioned bacterial compositions for tumor treatment are added in an effective amount to pharma- ceutically acceptable carriers and auxiliary materials, and prepared as a lyophilized powder, tablet, capsule, granule, or injection.

[0080] Here, the term "pharmaceutical acceptable carrier" refers to a carrier that does not cause obvious irritation to the living body and does not interfere with the biological activity and properties of the given compound. Here, the term "auxiliary material" refers to a solvent, diluent or other excipient, dispersant, and surfactant.

[0081] A composition comprising the bacteria and a pharma- ceutically acceptable carrier.

[0082] A composition comprising any of the above-described bacteria prepared for use as a pharmaceutical.

[0083] A composition comprising any of the above-described bacteria, prepared for use as a medical food.

[0084] A composition comprising a mixture of two or more of the bacterial strains described above, optionally further comprising a pharma- ceutically acceptable carrier.

[0085] A composition comprising an effective amount of two or more of the bacteria described above for treating tumors, optionally further comprising a pharma- ceutically acceptable carrier.

[0086] A composition comprising one or more live bacteria, inactive bacteria, bacterial derivatives, or bacterial metabolic products of any of the above-mentioned bacteria in an effective amount for tumor treatment, the composition optionally further comprising a pharma- ceutically acceptable carrier.

[0087] A food or drink, supplement, probiotic or health food formulated for enteral nutrition, comprising any of the bacteria described above.

[0088] A food, drink, supplement, probiotic or health food formulated for enteral nutrition comprising a mixture of two or more types of bacteria as described above.

[0089] The bacterium or composition described above, prepared as a freeze-dried powder, tablet, capsule, granule or injection.

[0090] Any of the above-mentioned bacteria, preparations, compositions, foods and beverages, supplements, probiotics or health foods, characterized in that the subject of administration is a human.

[0091] Preferably, the human is an infant, a child, an adolescent, an adult or an elderly person.

[0092] Any of the above-mentioned bacteria, preparations, compositions, foods and beverages, supplements, probiotics or health foods is administered to a non-human primate, wherein the non-human primate is a mammal (e.g., dog, cat, ferret, horse, rabbit, guinea pig, gerbil, hamster, red squirrel, rat, mouse); a bird; a reptile; a fish; an amphibian; an arthropod or a livestock animal (e.g., cow, pig, sheep, goat, alpaca, donkey, camel, buffalo or mink).

[0093] Use of any of the above-mentioned bacteria, preparations, compositions, foods and beverages, supplements, probiotics or health foods in tumor treatment.

[0094] Use of any of the above-described bacteria, or a formulation, composition, food or drink, supplement, probiotic or health food comprising an effective amount of the bacteria, in tumor treatment.

[0095] Use of any of the above-described bacteria, or a preparation, composition, food or drink, supplement, probiotic or health food containing an effective amount of the bacteria, in tumor treatment, characterized in that the tumor treatment comprises administering any of the above-described bacteria, preparation, composition, food or drink, supplement, probiotic or health food.

[0096] A composition comprising an effective amount of any of the bacteria or compositions described above for treating cancer, optionally further comprising a pharma- ceutically acceptable carrier.

[0097] A composition comprising an effective amount of two or more of the bacteria or compositions described above for the treatment of cancer, optionally further comprising a pharma- ceutically acceptable carrier.

[0098] A composition comprising live Alistipes finegoldii bacteria and an immune checkpoint inhibitor.

[0099] Preferably, the immune checkpoint inhibitor is a PD-1 monoclonal antibody (αPD-1) and / or a CTLA-4 monoclonal antibody (αCTLA4).

[0100] The present invention also includes the use of said composition in the preparation of a tumor medicament.

[0101] The present invention also includes a pharmaceutical product comprising the composition, specifically a pharmaceutical composition for tumor treatment comprising Alistipes finegoldii and an immune checkpoint inhibitor as active ingredients.

[0102] 2. Use of one or more live bacteria, inactive bacteria, bacterial derivatives, or bacterial metabolic products of bacteria belonging to the genus Alistipes in the preparation of an enhancer of an immune checkpoint inhibitor for tumor treatment.

[0103] Preferably, the use of Alistipes finegoldii as an active ingredient in the preparation of an enhancer of an immune checkpoint inhibitor, wherein the enhancer can improve the tumor therapeutic effect of the immune checkpoint inhibitor.

[0104] More preferably, the Alistipes finegoldii strain is one or a combination of two or more of the following strains:

[0105] Alistipes feingoldii, deposited at the German Collection of Microorganisms and Species Depositary DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen = German collection of microorganisms and cell cultures) under the accession number DSM 17242 (NCBI: txid679935, https: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=679935); Alistipes finegoldii, deposited at the Japan Collection of Microorganisms (JCM Seed Depositary) under the accession number JCM16770; Alistipes finegoldii, deposited at the Korean Collection for Type Cultures (KCTC) under accession number KCTC 15236; Alistipes finegoldii, deposited at the Anaerobe Reference Laboratory, Helsinki Collection, National Public health Institute, Helsinki, Finland, accession number AHN 2437; Alistipes finegoldii, deposited at the Swedish CCUG fungal species depository (Culture Collection University of Gothenburg) under accession number CCUG46020; Alistipes finegoldii, deposited at the French CIP Seed Depository Center (Collection de L'Institut Pasteur) under accession number CIP107999; Alistipes finegoldii deposited at the Guangdong Microbial Species Depository Center, China, accession number GDMCC 1.2324.

[0106] The present invention provides Alistipes finegoldii (Af), which is useful for treating tumor patients to enhance the therapeutic effect of immune checkpoint inhibitors. Specifically, the present invention proposes a combination therapy to enhance the therapeutic effect of immune checkpoint inhibition by administering immune checkpoint inhibition treatment simultaneously, separately or sequentially with active or inactive whole cell Af administration.

[0107] The administration of Alistipes finegoldii eliminates the indigenous intestinal flora.

[0108] A combination of antibiotics is used to eliminate the intestinal flora.

[0109] A combination of antibiotics is used to eliminate the indigenous gut flora and treatment is continued for 7 days.

[0110] Antibiotic combination: metronidazole 100 mg / kg, vancomycin 50 mg / kg, penicillin sodium 100 mg / kg, neomycin sulfate 100 mg / kg.

[0111] Af is administered orally.

[0112] The order of administration of the combination therapy is such that the Af is administered simultaneously with, before and / or after immune checkpoint inhibitor treatment.

[0113] The combination therapy may include dose delays and / or dose reductions and timing adjustments, if necessary, depending on the individual patient's tolerance to treatment.

[0114] The Af of the present invention may generally comprise an effective amount of Alistipes finegoldii dispersed in a pharma- ceutical or pharmacologically acceptable carrier.

[0115] The term "pharmacologically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to an animal (e.g., a human, as appropriate). Specific examples of pharmacologically acceptable carriers described herein are borate buffer or sterile saline solution.

[0116] The immune checkpoint inhibitor augmentation of the present invention is useful for immune checkpoint inhibitor refractory patients who exhibit congenital (primary) resistance to immune checkpoint inhibitor treatment, manifested as a lack of response or poor response to the checkpoint inhibitor treatment for at least about 8 or 12 weeks from the first agent.

[0117] The immune checkpoint inhibitor augmentation of the present invention is useful for immune checkpoint inhibitor refractory patients who exhibit acquired (secondary) resistance to treatment with the checkpoint inhibitor and who show an initial response to the checkpoint inhibitor, but subsequently relapse and progress with one or more tumors.

[0118] Evaluation of the therapeutic efficacy of the combination therapy of the present invention measures enhanced therapeutic efficacy by increased overall survival time.

[0119] Evaluation of the therapeutic efficacy of the combination therapy of the present invention measures the enhanced therapeutic efficacy by increased progression-free survival.

[0120] The therapeutic effect of the combination therapy of the present invention is evaluated by measuring the enhanced therapeutic effect by reducing or stabilizing tumor size of one or more tumors, including disease stabilization (SD), complete response (CR), or partial response (PR) of a target tumor, and / or disease stabilization (SD) or complete response (CR) of one or more non-target tumors, as defined by RECIST 1.1.

[0121] Evaluation of the therapeutic efficacy of the combination therapy of the present invention measures enhanced therapeutic efficacy by improved overall remission rate and / or improved quality of life.

[0122] The safety of the combination therapy of the present invention is evaluated by determining whether or not diarrhea or enteritis occurs. Effect of the Invention

[0123] Compared with the prior art, the present invention has the following beneficial effects:

[0124] By combining a monomicrobial oral preparation of a human commensal bacterium with an immune checkpoint inhibitor, the present invention significantly enhances the efficacy of immune checkpoint inhibitors against many types of tumors through the anti-tumor immune protective response induced by the bacteria, and also provides superior safety, extending the overall survival time of cancer patients and improving the response rate of people receiving cancer immunotherapy, thereby expanding the range of tumor patients to whom cancer immunotherapy (immunotherapy checkpoint inhibitors) can be applied. [Brief description of the drawings]

[0125] [Figure 1] Screening of gut bacteria that can enhance the effects of immune checkpoint inhibitors using the clinical cohort of Example 1. [Diagram 2] 1 shows a clinical cohort analysis of the relationship between each Alistipes species and the therapeutic effect of immune checkpoint inhibitors in Example 1. [Diagram 3] 1 is a flow chart showing the administration of each active Alistipes species to model mice with colon cancer and melanoma in Example 2. [Figure 4] 1 shows a tumor photograph of Example 2 (day 21). [Diagram 5] 1 shows a tumor volume change curve for Example 2. [Figure 6] FIG. 1 is a statistical diagram of tumor weight in Example 2. [Figure 7] 1 is a photograph of the mouse anus in Example 2 (day 21). [Figure 8] 1 shows HE staining of mouse intestinal tissue in Example 2 (day 21). [Figure 9] FIG. 1 shows an immunohistochemistry image of immune cells in tumor tissues in Example 2 (day 21). [Figure 10] Example 2: Detection of tumor-killing-associated immune-protective responses after administration of Alistipes finegoldii by flow cytometry. [Figure 11] FIG. 13 is a survival curve diagram when various doses of active Alistipes finegoldii were administered to model mice with colon cancer and melanoma in Example 3. [Figure 12]1 is a flow chart of administration of active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii to colon cancer model mice in Example 4. [Figure 13] FIG. 1 is a comparative electron micrograph of active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii of Example 4. [Figure 14] 13 shows a curve of change in tumor volume when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii were administered to colon cancer model mice in Example 4. [Figure 15] 13 shows PCoA analysis of β-diversity of intestinal microbiota when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii were administered to colon cancer model mice in Example 4. [Figure 16] FIG. 10 is a comparison of the intestinal flora reconstitution effect when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii are administered to colon cancer model mice in Example 4. [Figure 17] 13 is an analysis of the statistical difference of the top 20 bacterial genera in relative abundance in the intestinal microbiota of mice administered active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii to colon cancer model mice in Example 4. [Figure 18] 1 is a flow chart of administration of active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii to lung cancer model mice in Example 5. [Figure 19] 13 shows a curve of change in tumor volume when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii are administered to lung cancer model mice in Example 5. [Figure 20] FIG. 1 shows tumor weights at the experimental endpoints when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii were administered to lung cancer model mice in Example 5. [Figure 21] 13 shows tumor photographs at the experimental endpoint when active Alistipes fine-goldii and inactive whole-cell Alistipes fine-goldii were administered to lung cancer model mice in Example 5. [Figure 22] Example 6: Analysis of the relative abundance of Alistipes finegoldii in the intestine and other body sites of healthy individuals. [Diagram 23] Distribution and relative abundance in the intestines of healthy subjects and other subjects in Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0126] The present invention will be described in more detail below with reference to the drawings and specific examples in the specification, but the examples are used only to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the test methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0127] Example 1 Screening for gut bacteria capable of enhancing immune checkpoint inhibitors using clinical cohorts 1. Experimental Method 1. Dataset (1) Gut microbiota were obtained from 230 fecal samples from 108 patients with colorectal cancer (CRC) or esophageal cancer (ESCC) at different time points after treatment. Among them, 115 samples were obtained from patients in the immune checkpoint inhibitor group (ICI), and the treatment method was anti-PD1 antibody treatment combined with chemotherapy or targeted drugs, and 115 samples were obtained from patients in the chemotherapy group (Chemo) (Table 1). Evaluation of treatment efficacy mainly included progression-free survival (PFS), overall survival (OS), and optimal treatment effect. Patients with optimal treatment effect of complete remission (CR) and partial remission (PR) were defined as treatment-responsive patients (R), and patients with optimal treatment effect of stable disease (SD) and progressive disease (PD) were defined as treatment-non-responsive patients (NR). JPEG0007669065000001.jpg71168(2) After DNA extraction from 115 fecal samples, 16S rRNA gene V3-V4 region amplification and sequencing were performed (Illumina HiSeq platform). 16S rRNA sequencing data analysis used USEARCH software (version number 11.0.667) for quality control, filtering, and species identification. When there are fecal samples from multiple post-treatment time points for one patient, the abundance of gut microbiota species is taken as the average value of the different time points to account for subsequent analysis. Species identification was performed using the LTP (Living Tree Project) database so that the analysis results could be verified by subsequent strain-level pure culture experiments. The LTP database mainly collects sequences of typical strains and isolates classified based on observed traits. To maximize the accuracy of species-level bacterial identification by 16S rRNA sequencing, species annotation was performed using the SINTAX algorithm, which can score the confidence of the identified species, and enterobacteria annotated with a species confidence of more than 75% were screened for the following analysis. 2, Statistical analysis (1) Single-factor Cox regression analysis of PFS and OS: Using the R language pack survival (version number: 3.3-1), genus-level bacteria of all gut microbiota were subjected to single-factor Cox regression analysis. (2) Analysis of differences between groups: The Wilcoxon rank-sum test was used for statistical analysis of differences between groups. II. Experimental results Figure 1A shows the average relative abundance of intestinal Alistipes at different time points after treatment in treatment-responsive (R) and non-responsive (NR) patients in the Chemo and ICI groups. In the ICI group, the average relative abundance of intestinal Alistipes after treatment in R group patients was significantly higher than that in NR group patients (p<0.001), while in the Chemo group, the average relative abundance of intestinal Alistipes after treatment in R group patients was not significantly different from that in NR group patients (p=0.99). We showed that a high abundance of Alistipes after treatment correlates with a good therapeutic effect of immune checkpoint inhibitors. Figure 1B shows the average relative abundance of intestinal Alistipes at different time points in CRC and ESCC patients after receiving immune checkpoint inhibitors. As a result, after receiving immune checkpoint inhibitors, Alistipes showed zero values ​​in 50% of CRC patients, but Alistipes was detected in all ESCC patients. Therefore, CRC patients were divided into two groups: negative (relative abundance of Alistipes was zero) and positive (relative abundance of Alistipes was greater than zero) depending on whether the average relative abundance of Alistipes after receiving immune checkpoint inhibitor treatment was zero or not. ESCC patients were divided into two groups: high (above the average) and low (below the average) based on the average relative abundance of Alistipes after receiving immune checkpoint inhibitors. Figure 1C shows a comparison of the Kaplan-Meier (KM) curves of progression-free survival (PFS) between the negative and positive CRC patients. Figure 1D shows a comparison of the Kaplan-Meier (KM) curves of overall survival (OS) between the high and low ESCC patients. As a result, the PFS time of the CRC patients in the positive group was significantly longer than that of the negative group (log-rank p=0.02). The overall survival time of the ESCC patients with a high mean relative abundance of Alistipes was significantly longer than that of the patients with a low mean relative abundance of Alistipes (log-rank p=0.0071). These data suggest that after treatment with immune checkpoint inhibitors, a high abundance of Alistipes in the intestine may be beneficial for the therapeutic effects of immune checkpoint inhibitors. Figure 2 shows the relationship between each Alistipes species and the therapeutic effect time of immune checkpoint inhibitors. In this data set, we compared 11 Alistipes Operational Taxonomic Units (OTUs) with a confidence level of more than 0.75, including 5 Alistipes species, including 1 Alistipes finegoldii, 4 Alistipes indistinctus, 3 Alistipes onderdonkii, 2 Alistipes shahii, and 1 Alistipes timonensis, among which Otu1557 was identified as Alistipes finegoldii with a confidence level of 98% (Table 2). Among the five Alistipes species detected, the relative abundance of Alistipes finegoldii, Alistipes timonensis, and Alistipes shahii in the intestine was significantly higher in CRC patients with a PFS of more than 6 months after receiving αPD-1 combination chemotherapy than in patients with a PFS of less than 6 months. Meanwhile, the relative abundance of Alistipes finegoldii in the intestine was significantly higher in ESCC and CRC patients with a PFS of more than 6 months after receiving αPD-1 combination chemotherapy than in patients with a PFS of less than 6 months. The results suggest that Alistipes finegoldii may have a broader spectrum of enhancing effects against immune checkpoint inhibitors than other Alistipes species. JPEG0007669065000002.jpg120167

[0128] Example 2 Comparison of the enhancing effect of each active Alistipes species alone or in combination with other active Alistipes species on immune checkpoint inhibitors 1. Experimental Method 1. Experimental materials (1) Mouse strain: 6-week-old C57BL / 6J female mice (2) Tumor cell lines: rat melanoma cell line (B16-OVA, ATCC), rat colon carcinoma cell line (MC38, ATCC) (3) Alistipes sp. is selected from the following: Alistipes finegoldii (DSM No.: 17242, Type strain, its 16S rDNA sequence is shown in SEQ ID NO: 1), abbreviated as Af, was purchased from the German Depository Center for Microorganisms and Species, DSMZ (DSMZ official website: http: / / www.dsmz.de). Alistipes shahii (DSM No.:19121, Type strain), abbreviated as As, was purchased from the German Collection of Microorganisms (DSMZ). (4) Bacterial medium: the formulation mainly contained peptone, yeast extract, beef extract, and glucose, etc., and was liquid DSMZ104 medium purchased from the German Microbial Seed Collection Center DSMZ. (5) Immune checkpoint inhibitor: PD-1 monoclonal antibody (αPD-1), clone number G4C2, reagent provided by Shanghai Junshi Biopharmaceutical Technology Co., Ltd. (6) Antibiotic combination: metronidazole 100 mg / kg, vancomycin 50 mg / kg, penicillin sodium 100 mg / kg, neomycin sulfate 100 mg / kg. 2. Experimental group assignment The experimental groups are shown in Table 3. JPEG0007669065000003.jpg117170Aistipes.sp=1) Af: Alistipes finegoldii, 2) As: Alistipes shahii, 3) Af+As: Alistipes finegoldii+Alistipes shahii 3. Experimental steps (see Figure 3 for the flow) (1) Cultivation of active Alistipes species: Alistipes species (Af, As, or Af+As) were inoculated into DSMZ104 liquid medium and cultured in an anaerobic tank at 37°C for 18 hours, after which the concentration was 1×10 10 CFU / ml. (2) Tumor cells were 1 × 10 6 / mouse, B16-OVA cell line 5 × 10 5 The mice were inoculated subcutaneously at 1 / mouse. (3) Days 1-3: Antibiotics were administered intragastrically to mice in each group to eliminate the indigenous intestinal flora. (4) IgG or αPD-1 was administered intraperitoneally at 200 μg / mouse on days 5, 8, and 11, respectively. (5) Active Alistipes spp. treatment was administered intragastrically at 100 μl / animal on days 5, 7, 9, 11, 13, and 15, respectively. Single Af or As dose: 1 × 10 9 CFU / animal. Af+As combined dose: Af: 0.5×10 9 CFU+As: 0.5×10 9 CFU / animal. (6) Tumor size was measured on days 5, 8, 11, 14, 17, and 21, and tumor volume was calculated. Tumor volume = tumor width 2 ×Tumor length / 2 (7) On day 21, the mice were euthanized, and the tumor tissues were removed, photographed, and weighed. The intestinal tissues were stained with HE staining to reveal the state of enteritis. The tumor volume of mice was measured, tumor weight was measured at the endpoint, and the effect of immune cell infiltration in tumor tissue was evaluated by immunohistochemistry. We evaluated whether Alistipes finegoldii induces enteritis and its safety by HE staining of sections of mouse anal and intestinal tissues. Flow cytometry was used to detect tumor-killing-related immune cells in the blood of the mice to evaluate the system's anti-tumor immune response. II. Experimental results Figure 4 shows the tumor on day 21. Figure 5 shows the change curve of tumor volume, and Figure 6 shows the statistical diagram of tumor weight. In mouse models of MC38 colon cancer and B16-OVA melanoma, the monotherapy group (αPD-1) showed clear and significant (p<0.01) tumor shrinkage compared to the untreated group (IgG), while only the active Af combination therapy group (αPD-1+Af) showed clear and significant (p<0.01) tumor shrinkage compared to the monotherapy group (αPD-1), but there was no statistical difference between the active As combination therapy group (αPD-1+As) and the active Af+As combination therapy group (αPD-1+Af+As) and the monotherapy group (αPD-1). It was confirmed that A. faingoldii enhanced the antitumor effect of αPD-1. On the other hand, A. shahii (As) and the combination of A. shahii and A. faingoldii (Af+As) had no effect on enhancing the antitumor effect of αPD-1. In addition, there was no difference in tumors between mice treated with A. finegoldii alone (IgG+Af) and untreated (IgG), indicating that the antitumor effect of A. finegoldii is dependent on αPD-1. As is clear from the photographs of mouse anuses in Figure 7 and the HE staining of intestinal tissue sections in Figure 8, no enteritis was observed in either the active Af monotherapy group (IgG+Af) or the Af combination therapy group (αPD-1+Af), demonstrating the safety of enteral gastric administration of Alistipes finegoldii. As shown by the tumor tissue immunohistochemistry of the MC38 mouse model at the experimental endpoint in Figure 9, the active Af combination therapy group (IgG+Af) significantly increased the CD4 +It was shown that the infiltration of helper T cells could be significantly increased, and compared with the monotherapy group (αPD-1), the active Af combination therapy group (αPD-1+Af) significantly increased the CD4 + There was a trend towards increased helper T cell infiltration, but the difference was not statistically significant. Compared to the untreated group (IgG), mice in the monotherapy group (αPD-1) had significantly higher CD4 + Helper T cell infiltration also tended to increase, but was not statistically different. This result suggests that the effects of monotherapy (αPD-1) and active Af combination therapy (αPD-1+Af) on immune cell infiltration in mouse tumor tissue may be short-lived, and no significant differences were detected in tumor samples at the experimental endpoint. Af combination therapy (IgG+Af) had a longer-lasting effect on immune cell infiltration in mouse tumor tissue, with CD4 + Helper T cell infiltration was significantly increased, again demonstrating the modulatory effect of oral administration of active Alistipes finegoldii monobacteria on the tumor immune microenvironment. Figure 10 shows the tumor-killing-related immune cells in the MC38 mouse model, i.e., granulase-positive CD8 + T cells, granulase-positive NK cells and IL-6 + The detection of the amount of neutrophils. As a result, the active Af combination therapy group (αPD-1 + Af) had a higher number of granulase-positive CD8 + T cells, granulase-positive NK cells and IL-6 + It was shown to significantly increase the relative abundance of neutrophils. The combination of active Alistipes finegoldii and αPD-1 demonstrated an enhanced tumor-killing effect of the immune system.

[0129] Example 3: Prolongation of overall survival by combination of active Alistipes finegoldii and immune checkpoint inhibitors 1. Experimental Method 1. Experimental materials (1) Mouse strain: 6-week-old C57BL / 6J female mice (2) Tumor cell lines: rat melanoma cell line (B16-OVA, ATCC), rat colon carcinoma cell line (MC38, ATCC) (3) Alistipes finegoldii (DSM No: 17242, Type strain, the 16S rDNA sequence of which is shown in SEQ ID NO: 1), abbreviated as Af, was purchased from the DSMZ German National Culture Depository Center. (4) Bacterial medium: the formulation mainly contains peptone, yeast extract, beef extract and glucose, etc., and is liquid DSMZ104 medium purchased from DSMZ German National Culture Collection. (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number G4C2, reagent was a gift from Shanghai Junshi Biopharmaceutical Technology Co., Ltd. CTLA4 monoclonal antibody (αCTLA4), clone number 9D9, purchased from BioXcell, USA. (6) Antibiotic combination: metronidazole 100 mg / kg, vancomycin 50 mg / kg, penicillin sodium 100 mg / kg, neomycin sulfate 100 mg / kg. 2. Experimental group assignment The experimental groups are shown in Table 4. JPEG0007669065000004.jpg1751703, Experimental steps (1) Cultivation of active Alistipes finegoldii: Alistipes finegoldii was inoculated into DSMZ104 liquid medium and cultured in an anaerobic tank at 37°C for 18 hours, after which the concentration was 1×10 10 CFU / ml. (2) Tumor cells were 1 × 10 6 / mouse, B16-OVA cell line 5 × 10 5 The mice were inoculated subcutaneously at 1 / mouse. (3) Days 1-3: Antibiotics were administered intragastrically to mice in each group to eliminate the indigenous intestinal flora. (4) From the fifth day onwards, IgG, αPD-1 or αCTLA4 was administered intraperitoneally at 200 μg / mouse every three days. (5) From the fifth day onwards, the low-dose group was administered 100 μl / mouse, 1 × 109 CFU / animal, medium dose group (medium) 200 μl / animal, 2 × 10 9 CFU / animal, high dose group (high) 400 μl / animal, 4 × 10 9 Alistipes finegoldii treatments at each dose were administered by intragastric administration as CFU / animal. (6) From day 5 onwards, the tumor size was measured every 3 days, and the tumor volume was calculated. Tumor volume = tumor width 2 ×Tumor length / 2 (7) During the treatment cycle, the tumors in the mice were grown to a volume (2000 mm 3 Once tumors reached a critical mass, mice were either euthanized or died naturally without reaching a critical tumor mass. (8) The death status and time of each mouse were recorded, and a survival curve was created. II. Experimental results Figure 11 shows the survival curves of mice. In the MC38 colon cancer and B16-OVA melanoma mouse models, the combination therapy with high, medium and low doses of A. finegoldii (αPD-1+Af) significantly increased the overall survival time of tumor-bearing mice compared to the immune checkpoint inhibitor monotherapy group (αPD-1). In the B16-OVA melanoma mouse model, A. finegoldii showed a therapeutic effect enhancing effect on αCTLA4 immune checkpoint inhibitor, and the combination of αCTLA4 with high, medium and low doses of A. finegoldii significantly increased the overall survival time of tumor-bearing mice. On the other hand, in the MC38 colon cancer mouse model, the efficacy of the αCTLA4 immune checkpoint inhibitor was too strong, so the effect of A. finegoldii on αCTLA4 was not enhanced.

[0130] Example 4 Treatment of intestinal cancer with active and inactive whole-cell Alistipes finegoldii in combination with immune checkpoint inhibitors 1. Experimental Method 1. Experimental materials (1) Mouse strain: 6-week-old C57BL / 6J female mice (2) Tumor cell line: Mouse colon cancer cell line (MC38, ATCC) (3) Strain information: Alistipes finegoldii (DSM No.: 17242, type strain, its 16S rDNA sequence is shown in SEQ ID NO: 1), abbreviated as Af, was purchased from the German Microorganism Collection DSMZ (DSMZ official website: http: / / www.dsmz.de). (4) Medium components: The formula mainly includes peptone, yeast extract, beef extract and glucose, etc., and is liquid DSMZ104 medium purchased from DSMZ German National Culture Collection. (5) Immune checkpoint inhibitor: PD-1 monoclonal antibody (αPD-1), clone number G4C2, reagent provided by Shanghai Junshi Biopharmaceutical Technology Co., Ltd. (6) Antibiotic combination: metronidazole 100 mg / kg, vancomycin 50 mg / kg, penicillin sodium 100 mg / kg, neomycin sulfate 100 mg / kg. 2. Experimental group assignment The experimental groups are shown in Table 5. JPEG0007669065000005.jpg94163Note: Af is Alistipes finegoldii 3. Experimental steps (see Figure 12 for the flow) (1) Preparation of active Af (Af): Alistipes finegoldii was inoculated into DSMZ104 liquid medium and cultured in an anaerobic tank at 37°C for 18 h. After that, the concentration of Af was 1 × 10 10 The cells were centrifuged until the CFU / ml was reached. The cells were concentrated by washing three times with phosphate buffered saline (PBS) to wash away the residual medium. (2) Preparation of inactive whole-cell Af (heat inactivated, Af_heat killed): The cells prepared in (1) were washed and concentrated using phosphate buffered saline (PBS), and the cells were heated at a high temperature of 95°C for 5 minutes. (3) Subcutaneous inoculation of tumor cells: MC38 cell line 1 × 10 6 / animals. (4) Antibiotic treatment: Mice in each group were intragastrically administered antibiotics to eliminate the endemic intestinal flora, and then treated for 7 days. (5) Treatment by group: On days 6, 9, and 12, mice were intraperitoneally injected with phosphate buffered saline (PBS) or αPD-1 at 200 μg / mouse. On days 5, 7, 9, 11, and 13, mice were intraperitoneally injected with 100 μl / mouse or 1 × 10 9 Active Af treatment was performed by intragastric administration at CFU / mouse, or inactive whole-cell Af treatment was performed by oral administration. Tumor size was measured and tumor volume was calculated on days 0, 5, 7, 9, 11, 13, 16, 19, 22, and 25, respectively. (6) Formula for measuring tumor volume Tumor volume = tumor width 2 ×Tumor length / 2 (7) At the endpoint (day 25), intestinal contents of the mice were collected and the composition of the mouse intestinal microbiota was analyzed using 16S rRNA gene sequencing to compare the effects of intestinal cancer treatment with active Af and inactive whole-cell Af on the mouse intestinal microbiota. II. Experimental results Figure 13 shows a comparison of electron micrographs of active and inactive whole-cell Af, demonstrating that inactive whole-cell treatment preserved the whole-cell integrity of Af cells and that the components that mediate the antitumor effects of inactive whole-cell Af are derived from whole-cell components. Figure 14 shows the tumor volume change curves in colon cancer treatment with active Af and inactive whole cell Af. In comparison of tumor volume change, in MC38 colon cancer, the single-agent immune checkpoint inhibitor group (αPD-1) did not show significant (p=0.055) tumor shrinkage compared to the untreated group (PBS). In this example, colon cancer is resistant to single-agent αPD-1 treatment, while treatment with αPD-1 in combination with active Af or inactive whole cell Af showed significantly (***:p<0.001) superior tumor shrinkage to the single-agent αPD-1 group. Comparing the therapeutic effects of the two groups using active Af and inactive whole cell Af in combination with immune checkpoint inhibitors, αPD-1 combined with inactive whole cell Af showed significantly (*:p<0.05) superior tumor shrinkage to αPD-1 combined with active Af. These results demonstrate that both active Af and inactive whole-cell Af can enhance the antitumor effect of immune checkpoint inhibitors. When single-agent αPD-1 has poor therapeutic effect or drug resistance, αPD-1 can be combined with either active Af or inactive whole-cell Af to reverse the drug resistance of colon cancer to single-agent αPD-1 treatment, so that both active Af and inactive whole-cell Af are effective in treating, reducing, suppressing or controlling immune checkpoint inhibitor-refractory tumors, and the antitumor effect of inactive whole-cell Af is superior to that of active Af. Figure 15 shows the principal coordinate analysis of the β-diversity PCoA (principal co-ordinates analysis) of gut microbiota in colon cancer treatment with active Af and inactive whole-cell Af. Here, the β-diversity of gut microbiota refers to the difference in the composition of bacteria in the whole gut environment between different gut environments, and the statistical difference between the two groups was measured using the Anosim analysis (Analysis of Similarities), a non-parametric test method based on permutation tests and rank sum tests, to verify whether the difference between groups is significantly greater than the difference within groups and to determine whether the grouping is significant. This Anosim method has two main numerical results. One is R, which ranges from (-1, 1), and is used to determine whether there is a difference between different groups, where R>0 indicates that the difference between groups is greater than the difference within groups, and R<0 indicates that the difference between groups is smaller than the difference within groups, and the closer the value of R is to 1, the greater the difference between groups. The other is p, which indicates whether there is a significant difference between groups. As a result, a significant difference (R=0.6950, p=0.001) was observed in the beta diversity of gut microbiota in colon cancer treatment with active Af and inactive whole cell Af. It was demonstrated that there was a clear and significant difference in the composition of the whole gut microbiota by treating colon cancer with active Af and inactive whole cell Af, and furthermore, a significant difference in the intestinal mucosal immune status was generated. Therefore, in Example 5, the difference in the antitumor effect of inactive whole cell Af and active Af may lead to a difference in the remodeling effect on the gut microbiota structure, that is, when inactive whole cell Af and αPD-1 are combined to treat colon cancer, the remodeled gut microbiota structure is associated with enhanced antitumor immune function. Figure 16 compares the differences in genus-level composition of gut microbiota in colon cancer treatment with active and inactive whole-cell Af. Figure 17 shows a Wilcoxon rank-sum test statistical difference analysis comparing the top 20 bacterial genera by relative abundance in gut microbiota in colon cancer treatment with active and inactive whole-cell Af. As a result, significant statistical differences were observed in 8 of the top 20 bacterial genera in the relative abundance of the gut microbiota between colon cancer treatment with active Af and inactive whole-cell Af. Among them, the largest difference was in the genus Bacteroides, whose relative abundance was obviously higher in the active Af treatment group than in the inactive Af treatment group, and another striking difference was in the genus Desulfovibrio, which was only present in the inactive whole-cell combination therapy group (αPD-1+Af_heat killed). Meanwhile, the relative abundance of Aistipes, which belongs to Af, was significantly higher (p=0.0156) in the active combination therapy group than in the inactive whole-cell combination therapy group. It was demonstrated that the treatment of colon cancer with active Af and inactive whole-cell Af resulted in significant differences in the genus-level composition of the gut microbiota, and further differential gut mucosal immune status. Thus, in Example 4, the difference in antitumor efficacy between active and inactive whole-cell Af was associated with specific enrichment or attenuation of growth of certain bacterial genera in the intestine, further forming a differential intestinal mucosal immune status and stimulating a differential antitumor immune surveillance.

[0131] Example 5 Treatment of lung cancer by combined administration of active and inactive whole-cell Alistipes finegoldii with immune checkpoint inhibitors 1. Experimental Method 1. Experimental materials (1) Mouse strain: 6-week-old C57BL / 6J female mice (2) Tumor cell line: Mouse lung cancer cell line (LLC, ATCC) (3) Information on Af strain: Alistipes finegoldii (DSM No.: 17242, type strain, the 16S rDNA sequence of which is shown in SEQ ID NO: 1) was purchased from the German Strain Depository Center DSMZ (DSMZ official website: http: / / www.dsmz.de). (4) Medium components: The formula mainly includes peptone, yeast extract, beef extract and glucose, etc., and is liquid DSMZ104 medium purchased from DSMZ German National Culture Collection. (5) Immune checkpoint inhibitor: PD-1 monoclonal antibody (αPD-1), clone number G4C2, reagent provided by Shanghai Junshi Biopharmaceutical Technology Co., Ltd. (6) Antibiotic combination: metronidazole 100 mg / kg, vancomycin 50 mg / kg, penicillin sodium 100 mg / kg, neomycin sulfate 100 mg / kg. 2. Experimental group assignment The experimental groups are shown in Table 6. JPEG0007669065000006.jpg841633, Experimental steps (see Figure 18 for the flow) (1) Preparation of active Af (Af): Alistipes finegoldii was inoculated into DSMZ104 liquid medium and cultured in an anaerobic tank at 37°C for 18 h. After that, the concentration of Af was 1 × 10 10 The cells were centrifuged until the CFU / ml was reached. The cells were concentrated by washing three times with phosphate buffered saline (PBS) to wash away the residual medium. (2) Preparation of inactive whole-cell Af (heat inactivated, Af_heat killed): The cells prepared in (1) were washed and concentrated using phosphate buffered saline (PBS), and the cells were heated at a high temperature of 95°C for 5 minutes. (3) Subcutaneous inoculation of tumor cells: LLC cell line 1 × 10 6 / animals. (4) Antibiotic treatment: Mice in each group were intragastrically administered antibiotics to eliminate the endemic intestinal flora, and then treated for 7 days. (5) Treatment by group: PBS or αPD-1 was intraperitoneally injected at 150 μg / mouse on days 6, 9, 12, and 15, respectively. Active Af or inactive whole-cell Af was injected at 100 μl / mouse, 1 × 10 9 The tumors were intragastrically administered at CFU / mouse. The tumor sizes were measured on days 0, 5, 7, 10, 13, 16, 19, 22, 25, and 28, and the tumor volumes were calculated. (6) Formula for measuring tumor volume Tumor volume = tumor width 2 ×Tumor length / 2 (7) Tumor weight was measured at the endpoint (day 28), and statistics were performed by group. II. Experimental results FIG. 19. Tumor volume changes upon treatment of LLC mouse lung cancer model with active and inactive whole cell Af. Compared to the untreated group (PBS), the single-agent immune checkpoint inhibitor group (αPD-1) did not show significant (ns: p>0.05) tumor shrinkage. In this example, the single-agent immune checkpoint inhibitor group (αPD-1) had poor therapeutic effects in the LLC mouse lung cancer model, indicating resistance to single-agent αPD-1 treatment. When αPD-1 and active Af (αPD-1+Af) or αPD-1 and inactive whole-cell Af (αPD-1+Af_heat killed) were combined for treatment, both showed significantly (αPD-1+Af: **p<0.01; αPD-1+Af_heat killed: ****p<0.0001) superior tumor shrinkage compared to the untreated group (PBS). Compared with the single-agent immune checkpoint inhibitor group (αPD-1), αPD-1 and active Af (αPD-1+Af) or αPD-1 and inactive whole-cell Af (αPD-1+Af_heat killed) showed significantly superior tumor shrinkage compared to the untreated group (PBS). The groups in which αPD-1 was combined with active Af (αPD-1+Af) and inactive whole-cell Af (αPD-1+Af_heat killed) showed significantly (αPD-1+Af: *p<0.05; αPD-1+Af_heat killed: ****p<0.0001) superior tumor treatment effects to single-agent αPD-1. When comparing the therapeutic effects of αPD-1 combined with active Af (αPD-1+Af) and inactive whole-cell Af combined with αPD-1 (αPD-1+Af_heat killed), the therapeutic effect of inactive whole-cell Af tended to be superior to that of active Af, but there was no statistical difference (ns:p>0.05). FIG. 20 is a statistical diagram of lung cancer tumor weight at the endpoint (day 28) in the treatment of lung cancer in LLC mice with active Af and inactive whole cell Af. As can be seen from the tumor weight at the endpoint of the experiment, the single-agent immune checkpoint inhibitor group (αPD-1) did not show significant (ns: p>0.05) tumor shrinkage compared to the untreated group (PBS). The αPD-1 drug combined active Af did not show a significantly (ns: p>0.05) superior therapeutic effect than single-agent αPD-1, while the treatment with inactive whole cell Af combined αPD-1 drug showed a significantly (**p<0.01) superior therapeutic effect than single-agent αPD-1. When comparing the therapeutic effects of active Af combined αPD-1 and inactive whole cell Af combined αPD-1, in the inactive whole cell Af group, the average weight was lower than that of the active Af group. The results demonstrated that active Af and inactive whole cell Af can enhance the antitumor effect of immune checkpoint inhibitors. When the therapeutic effect of single-agent αPD-1 is poor or there is drug resistance, combining αPD-1 with active Af or inactive whole-cell Af can reverse the drug resistance to single-agent αPD-1 treatment of lung cancer. Therefore, both active Af and inactive whole-cell Af are effective in treating, reducing, suppressing or controlling immune checkpoint inhibitor-refractory tumors, and the antitumor effect of inactive whole-cell Af is superior to that of active Af. Figure 21 shows tumor images at the endpoint of an experiment using active Af and inactive whole-cell Af for lung cancer treatment. In the LLC mouse lung cancer model, one mouse in each of the two groups, active combination therapy (αPD-1+Af) and inactive whole-cell combination therapy (αPD-1+Af_heat killed), showed complete disappearance of tumors. Both active Af and inactive whole-cell Af were proven to enhance the antitumor effect of immune checkpoint inhibitors.

[0132] Example 6 Analysis of the relative abundance of Alistipes finegoldii in the intestine and other body sites of healthy individuals 1. Implementation method and steps (1) Original macrogenomic data source: Macrogenomic analysis data using public data resources from the Human Microbiome Project (HMP) of the National Institutes of Health (NIH) (https: / / www.hmpdacc.org / ). (2) Bacterial classification and strain-level identification analysis software: MetaPhlAn2 and StrainPhlAn (https: / / github.com / biobakery / metaphlan2,https: / / github.com / biobakery / metaphlan). By using MetaMetaPhlAn2 and StrainPhlAn together, macrogenomic data can be identified and analyzed at the strain level. When using both software, use the default settings. (3) After obtaining the relative abundance of the strains (the proportion of a given strain in the total microbial population) from (2), the abundance of Af in different parts of the body is visualized and analyzed. II. Results of the implementation Figure 22 shows the relative abundance of Af in the intestine and other body sites of healthy individuals. Analysis of 2335 samples from four body sites of healthy individuals (553 intestinal samples, 1259 oral samples, 309 skin samples, and 234 vaginal samples) showed that Af was mainly present in the intestine samples, with a positive detection rate of 73.6% (407 / 553) in the intestine. Meanwhile, the detection rates of Af were 1.4% (18 / 1259), 1.9% (6 / 309), and 1.7% (4 / 234) in the oral, skin, and vaginal samples, respectively. In the 407 Af-positive intestinal samples, the relative abundance of Af ranged from 0.00006% to 9.0%. According to the latest data on the estimation of the number of intestinal bacteria, the total number of bacteria in the intestine of a healthy adult male weighing 70 kg is approximately 3.8*10 13 According to the HMP database, the number of Af in the intestines of healthy individuals is approximately 10 6 ~10 13(Sender, Fuchs et al. 2016). Therefore, Af is a symbiotic intestinal bacterium specific to the human body, mainly colonizes the intestine and interacts with the human body, and the relative abundance of Af is in the position of a dominant species (defined as a species that accounts for more than 1% of the total intestinal flora) in some human groups. 5 ~10 12 It has been demonstrated that safety is ensured when

[0133] Example 7 Analysis of the relative abundance of Alistipes finegoldii in the intestines of healthy individuals and those with other diseases 1. Experimental Method Four human gut macrogenomic datasets were analyzed, including a total of 1396 human fecal samples, spanning nine different types of people. Macrogenomic sequencing technology can reach the accuracy of human gut bacterial species-level identification. The nine different types of populations included: 1) healthy adults, 2) colorectal adenoma patients, 3) colon cancer patients, 4) colon cancer post-surgery patients, 5) atherosclerotic disease patients, 6) non-small cell lung cancer patients before receiving immune checkpoint inhibitors (ICIs), 7) non-small cell lung cancer patients after receiving immune checkpoint inhibitors (ICIs), 8) renal cancer patients before receiving immune checkpoint inhibitors (ICIs), and 9) renal cancer patients after receiving immune checkpoint inhibitors (ICIs). Details of the dataset are shown in Table 7. JPEG0007669065000007.jpg91169 II. Experimental results According to the results of Figure 23 and Table 8, Alistipes finegoldii (Af) is present in each population, and its relative abundance is 0.001-0.07%. The relative abundance of Af varies among populations, and here, the relative abundance is the proportion of a specific species among all species of intestinal bacteria. The total number of bacterial species contained in the human intestine is about 10 14 CFU / ml, the Af in the intestines of humans in these groups was approximately 10 9 CFU / ml ~10 10CFU / ml. JPEG0007669065000008.jpg69169

[0134] It should be noted that the above examples are only used to describe the technical solutions of the present invention, and do not limit the scope of the present invention, and those skilled in the art can make various other changes or modifications based on the above description and ideas, and this specification does not need to cover all the embodiments, and cannot cover all the embodiments. All modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the patent scope of the claims of the present invention.

[0135] (References) 1. Baruch EN, YI, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L, Adler K, Dick-Necula D, Raskin S, Bloch N, Rotin D, Anafi L, Avivi C, Melnichenko J, Steinberg-Silman Y, Mamtani R, Harati H, Asher N, Shapira-Frommer R, Brosh-Nissimov T, Eshet Y, Ben-Simon S, Ziv O, Khan MAW, Amit M, Ajami NJ, Barshack I, Schachter J, Wargo JA, Koren O, Markel G, Boursi B. (2020). "Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients." Sciense 5(371(6529)): 8. 2. Derosa, L., B. Routy, A. Desilets, R. Daillere, S. Terrisse, G. Kroemer and L. Zitvogel (2021). "Microbiota-Centered Interventions: The Next Breakthrough in Immuno-Oncology?" Cancer Discov 11(10): 2396-2412. 3. Iida N, D. A., Stewart CA, Smith L, Bouladoux N, Weingarten RA, Molina DA, Salcedo R, Back T, Cramer S, Dai RM, Kiu H, Cardone M, Naik S, Patri AK, Wang E, Marincola FM, Frank KM, Belkaid Y, Trinchieri G, Goldszmid RS (2013). "Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment." Science 22(342(6161)): 4. 4. Mager LF, B. R., Pett N, Cooke NCA, Brown K, Ramay H, Paik S, Stagg J, Groves RA, Gallo M, Lewis IA, Geuking MB, McCoy KD. (2020). "Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy." Sciense 18(369(6510)): 10. 5. Parker, B.J., P.A. Wearsch, ACM Veloo and A. Rodriguez-Palacios (2020). “The Genus Alistipes: Gut Bacteria With Emerging Implications to Inflammation, Cancer, and Mental Health.” Frontiers in Immunology 11. 6. Routy B, LCE, Derosa L, Duong CPM, Alou MT, Daillere R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren K, Mondragon L, Jacquelot N, Qu B, Ferrere G, Clemenson C, Mezquita L, Masip JR, ​​Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria JC, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L (2018). "Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors." Sciense 5(359(6371)): 7. 7. Sender, R., S. Fuchs and R. Milo (2016). "Revised Estimates for the Number of Human and Bacteria Cells in the Body." PLoS Biol 14(8): e1002533. 8. Zipkin, M. (2021). "Fecal microbiota potentiate checkpoint inhibitors, unleash microbiome startups." Nat Biotechnol 39(5): 529-532.

Claims

1. A tumor treatment product comprising an immune checkpoint inhibitor and a bacterium, The immune checkpoint inhibitor is a PD-1 monoclonal antibody or a CTLA4 monoclonal antibody; A product for treating tumors, characterized in that the bacterium is only Alistipes finegoldii.

2. 2. The product according to claim 1, characterized in that the Alistipes finegoldii is Alistipes finegoldii deposited at the German Depository Center for Microorganisms and Species DSMZ, accession number DSM 17242.

3. 2. The product of claim 1, wherein the bacteria is an active bacteria.

4. 2. The product of claim 1, wherein the bacteria is an inactive whole cell bacteria.

5. A formulation for enhancing an immune checkpoint inhibitor for tumor treatment, comprising a bacterium, The preparation contains an effective amount of the bacteria as well as pharma- ceutically acceptable carriers and auxiliary materials, and is prepared as a lyophilized powder, tablet, capsule, granule, or injection; The pharma- ceutically acceptable carrier is a carrier that does not cause obvious irritation to a living body and does not interfere with the biological activity and properties of a given compound; the auxiliary material is a solvent, diluent, excipient, dispersant or surfactant; the bacterium is only Alistipes finegoldii, The formulation, wherein the immune checkpoint inhibitor is a PD-1 monoclonal antibody or a CTLA4 monoclonal antibody.

6. 6. The formulation of claim 5, wherein the bacterium is an active bacterium.

7. 6. The formulation of claim 5, wherein the bacteria is an inactive whole cell bacterium.

8. A composition for enhancing an immune checkpoint inhibitor for tumor treatment, comprising a bacterium, the bacterium is only Alistipes finegoldii, The composition, wherein the immune checkpoint inhibitor is a PD-1 monoclonal antibody or a CTLA4 monoclonal antibody.

9. 9. The composition of claim 8, wherein the bacteria is an active bacteria.

10. 9. The composition of claim 8, wherein the bacteria is an inactive whole cell bacterium.

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