Use of sutterella wadsworthensis in preparation of synergist of immune checkpoint inhibitor

Abstract

According to the present invention, Sutterella wadsworthensis and an immune checkpoint inhibitor are used in combination for treating tumors. An enhanced treatment effect is measured on the basis of an increased overall survival time and an increased progression-free survival time, and results show that Sutterella wadsworthensis can significantly improve the tumor treatment effect of the immune checkpoint inhibitor. The present invention has important significance for improving the overall efficacy of immunotherapy for tumor patients and promoting translational applications of pharmacomicrobiomics in personalized precision medicine.

Description

Application of S. waldensis in the preparation of potentiators of immune checkpoint inhibitors Technical Field

[0001] The present invention relates to the field of cancer treatment, and in particular to the use of S. waldensis in the preparation of enhancers of immune checkpoint inhibitors. Background Art

[0002] Malignant tumors are a serious disease that is fatal and difficult to treat. Recent studies have found that treatment with immune checkpoint inhibitors (ICIs) can significantly prolong the survival of patients with various solid tumors, including non-small cell lung cancer, melanoma, and renal cell carcinoma. However, only about 30% of patients show a definite clinical efficacy in response to ICIs, and about 60-70% of patients develop primary or secondary drug resistance. Therefore, it is of great clinical significance to identify biomarkers of ICI response and elucidate their biological mechanisms, and to develop treatment options that can significantly enhance the sensitivity of ICIs.

[0003] The use of multi-omics (genome, transcriptome, epigenome, metabolome, and microbiome) to explore biomarkers that determine the clinical efficacy of ICIs and design reasonable ICI combination therapy regimens is the key to improving the overall therapeutic effect of ICIs and is also one of the key research directions in the current field of pharmacogenomics and personalized precision therapy. PD-L1 expression is currently the most commonly used biomarker to predict the efficacy of ICIs. High PD-L1 expression has been found to be associated with a higher objective response rate in patients with non-small cell lung cancer, melanoma, and renal cell carcinoma who receive PD-1 monoclonal antibodies. However, PD-L1 is not applicable to all cancer types, and its use as a predictive marker for ICI efficacy is only applicable to some cancer patients. The spatial heterogeneity of PD-L1 expression and its dynamic evolution with the immune microenvironment reduce its reliability as a biomarker for ICI treatment.

[0004] The gut microbiome has become one of the potential markers for predicting the response to ICIs in tumors. In preclinical models and clinical tumor patients, the composition of gut microbes is significantly correlated with the efficacy of ICIs. Studies have found that broad-spectrum antibiotics can cause intestinal flora disorders in patients with non-small cell lung cancer, melanoma, etc. treated with ICIs, leading to a significant shortening of overall survival (2 months vs. 26 months, HR=7.4), and a significant increase in the non-response rate; higher α diversity of the gut microbiota has been shown to be associated with prolonged progression-free survival in melanoma patients receiving ICIs. When fecal microbiota from patients who had a complete response (R) after PD-1 monoclonal antibody treatment was transplanted to terminal metastatic melanoma patients who had no effect (NR), approximately 30% of the recipient patients (NR) had new significant clinical responses, of which 10% even had complete remission. A high abundance of Fusobacterium nucleatum (F. nucleatum) in the intestine is associated with a good response to PD-1 monoclonal antibody treatment in colorectal cancer patients. Bifidobacterium pseudolongum, isolated from mice with orthotopic colon cancer that responds well to ICIs, can enhance the anti-colorectal cancer efficacy of PD-1 monoclonal antibodies through its metabolite creatinine. This suggests that the gut microbiota has a significant impact on the efficacy of ICIs in treating malignancies such as colorectal cancer, melanoma, and non-small cell lung cancer, and that specific gut microbiota may become a new class of biomarkers for predicting responsiveness to ICIs.

[0005] Intestinal flora and its metabolites can improve the responsiveness of ICIs treatment by regulating the body's innate immunity and adaptive immunity. In terms of innate immune regulation, intestinal flora can affect the functions of dendritic cells (DCs), monocytes and macrophages, and natural killer cells (NK). For example, A. muciniphila can activate the TLR2 / NF-κB and NLRP3 pathways and induce macrophages to polarize to the M1 type, thereby inhibiting the progression of colon cancer. Lactobacillus rhamnousus GG (LGG) activates the cGAS / STING / TBK1 / IRF1 signaling pathway in DCs to induce them to secrete IFN-β, thereby improving the anti-tumor effect of PD-1 monoclonal antibodies. In terms of adaptive immune regulation, intestinal flora can affect CD8 + T and CD4 + The intestinal microbiota metabolite butyrate directly enhances CD8 T cell activity through DNA binding inhibitor 2 (ID2)-IL-12 +The tumor-killing effect of T cells; the secondary metabolite inosine of Bifidobacterium pseudolongum (B. pseudolongum) activates the phosphorylation of cAMP response element binding protein (pCREB) through the T cell-specific A2AR-cAMP-PK4 signaling pathway, upregulating IL12Rβ2 and IFNγ transcription, activating Th1 cell immune responses, and thus enhancing the efficacy of ICIs. Therefore, in-depth exploration of the functional properties of different types of intestinal flora and their metabolites is crucial for identifying the response mechanism of anti-tumor immunotherapy and improving the efficacy of ICIs. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of S. waldensis in the preparation of a synergist for immune checkpoint inhibitors to solve the problems existing in the above-mentioned prior art. By combining S. waldensis with immune checkpoint inhibitors, the effect of immune checkpoint inhibitors in treating tumors can be significantly improved.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides use of Sutterella wadsworthensis in preparing a synergist of immune checkpoint inhibitors for tumor treatment.

[0009] The present invention also provides the use of S. waldensis combined with immune checkpoint inhibitors in the preparation of drugs for treating tumors.

[0010] The tumor treatment includes reduction or stabilization of tumor volume, extension of overall survival time of tumor patients, extension of progression-free survival time, and improvement of quality of life.

[0011] Preferably, the Sutterella wadsworthensis comprises a 16S rDNA sequence, and the Sutterella wadsworthensis of the present invention refers to any strain having at least 99% identity with the 16S rDNA sequence of Sutterella wadsworthensis. More preferably, the Sutterella wadsworthensis is a combination of one or more Sutterella wadsworthensis strains. More preferably, the strain of Sutterella wadsworthensis is a strain with a subspecies classification in the National Center for Biotechnology Information (NCBI) genome database (https: / / www.ncbi.nlm.nih.gov / genome / browse / #! / prokaryotes / Sutterella%20wadsworthensis): Sutterella wadsworthensis DSM 14016, Sutterella wadsworthensis FDAARGOS_1159, Sutterella wadsworthensis 351h, Sutterella wadsworthensis 809h, Sutterella wadsworthensis 351h, Sutterella wadsworthensis DFI.4.78, Sutterella wadsworthensis 934h, Sutterella wadsworthensis MCC752, Sutterella wadsworthensis 239h, Sutterella wadsworthensis 333h, Sutterella wadsworthensis 228h, Sutterella wadsworthensis 1122h, Sutterella wadsworthensis HGA0223, Sutterella wadsworthensis 3_1_45B, Sutterella wadsworthensis 2_1_59BFAA, Sutterella wadsworthensis min17_bin46, Sutterella wadsworthensis CD33_MAG38, Sutterella wadsworthensis UBA11514, Sutterella wadsworthensisUBA11458, Sutterella wadsworthensis UBG025, Sutterella wadsworthensis SUG770, Sutterella wadsworthensis 1017h, Sutterella wadsworthensis 1045h, Sutterella wadsworthensis 2733h, Sutterella wadsworthensis 3358, Sutterella wadsworthensis 876h, Sutterella wadsworthensis 831h, Sutterella wadsworthensis 856h, Sutterella wadsworthensis 830h, Sutterella wadsworthensis 877h, Sutterella wadsworthensis 910h, Sutterella wadsworthensis 627h, Sutterella wadsworthensis UBA10685 and Sutterella wadsworthensis 823h or a combination of one or more thereof. More preferably, the waldesartensis strain is one or a combination of the following strains: waldesartensis, deposited in the German DSM collection of microorganisms and cell cultures, with a preservation number of DSM14016 (NCBI: taxid40545, https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / tree / ?taxon=40545); deposited in the American ATCC type culture collection, with a preservation number of ATCC51579; deposited in the Japan Collection of Microorganisms, with a preservation number of JCM32440; deposited in the Korean Collection for Type Cultures, with a preservation number of JCM32440; deposited in the Korean Collection for Type Cultures, with a preservation number of JCM32440; deposited in the Korean Collection for Type Cultures, with a preservation number of JCM32440; deposited in the Korean Collection for Type Cultures, with a preservation number of JCM32440; deposited in the Korean Collection for Type Cultures, with a preservation number of JCM32440. Culturers), with the accession number KCTC15691; deposited in the CCUG Culture Collection University of Gothenburg, Sweden, with the accession number CCUG69352; deposited at the Culture Collection University of Gothenburg (CIP) in France with accession number CIP 104799; deposited at the National Collection of Type Cultures (NCTC) in the United Kingdom with accession number NCTC 12926; and deposited at the Guangdong Provincial Center for Microbiological Cultures (GDMCC) in China with accession number GDMCC 1.2562. As an embodiment of the present invention, the present invention is described using the Wardesartella with accession number DSM 14016 as an example.

[0012] The immune checkpoint inhibitor is one or more combinations of blockers acting on T cell negative costimulation (co-inhibition) molecules and / or their respective ligands. It is more preferred that T cell negative costimulation (co-inhibition) molecules and / or their respective ligands are 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, LAG3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, DcR3.More preferably, the blocker of the ligand of the negative co-stimulatory (co-inhibitory) molecule of T cell is selected from 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 (durvalumab , PD-L1 monoclonal antibody), tremelimuab (CTLA-4 monoclonal antibody), spartalizumab (PD-L1 monoclonal antibody), avelumab (PD-L1 monoclonal antibody), sintilimab (PD-1 monoclonal antibody), toripalimab (PD-L1 monoclonal antibody), cemiplimab (PD-1 monoclonal antibody), MGA012 (retifanlimab, PD-1 monoclonal antibody), MGD013 (tebotelimab, PD-1 / LAG-3 dual antibody), MGD019 (PD-1 / CTLA-4 dual 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), 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), pexidatinib (pexidatinib) rtinib (GSF-1R monoclonal antibody), LY3022855 (CSF-1R monoclonal antibody), FPA008 (CSF-1R monoclonal antibody), BLZ945 (CSF-1R monoclonal antibody), GDC0919 (navoximod (IDO monoclonal antibody), epacadostat (IDO monoclonal antibody), indoximid (IDO monoclonal antibody), BMS986205 (IDO monoclonal antibody), CPT-444 (A2AR monoclonal antibody), MEDI9447 (oleclumab (CD73 monoclonal antibody), PBF509 (A2AR monoclonal antibody), lirilumab (KIR monoclonal antibody) or any combination thereof.More preferably, the blocker is selected from nivolumab, pembrolizumab, toripalimab, sintilimab, cemiplimab, or a combination of any of them. More preferably, the immune checkpoint inhibitor is an inhibitor that acts on the PD-1 / PD-L1 signaling pathway and / or the PD-1 / PD-L2 signaling pathway, wherein PD-1 refers to 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. More preferably, the inhibitor of the PD-1 / PD-L1 signaling pathway or the PD-1 / PD-L2 signaling pathway is selected from nivolumab (PD-1 monoclonal antibody), perbrolizumab (PD-1 monoclonal antibody), azetolizumab (PD-L1 monoclonal antibody), atezolizumab (PD-L1 monoclonal antibody), camrelizuman (PD-L1 monoclonal antibody), tislelizumab (BGB-A317), durvalumab (PD-L1 monoclonal antibody), spartalizumab (PD-L1 monoclonal antibody), and spartalizumab. umab (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 antibody), MGD019 (PD-1 / CTLA-4 dual antibody), MEDI0680 (PD-1 monoclonal antibody), PDR001 (PD-1 monoclonal antibody), FAZ053 (PD-L1 monoclonal antibody), or any combination thereof. More preferably, the immune checkpoint inhibitor is an inhibitor that acts on the CTLA-4 / B7-1 signaling pathway and / or the CTLA-4 / B7-2 signaling pathway, wherein CTLA-4 refers to cytotoxic T lymphocyte protein 4, also known as CD152, B7-1 (CD80) and B7-2 (CD86) are ligands of CTLA-4. More preferably, it can be selected from ipilimumab (CTLA-4 monoclonal antibody), tremelimumab (CTLA-4 monoclonal antibody), MGD019 (PD-1 and CTLA-4 dual antibody) or any combination thereof.As a specific embodiment of the present invention, the immune checkpoint inhibitor is an inhibitor that acts on the PD-1 / PD-L1 signaling pathway and / or the PD-1 / PD-L2 signaling pathway and / or an inhibitor that acts on the CTLA-4 / B7-1 signaling pathway and / or the CTLA-4 / B7-2 signaling pathway. Specifically, the immune checkpoint inhibitor is a PD-1 monoclonal antibody or a CTLA-4 monoclonal antibody.

[0013] Preferably, the tumor includes a reduction or stabilization of tumor volume, a prolonged overall survival time of tumor patients, a prolonged progression-free survival time, and an improved quality of life.

[0014] Preferably, the tumor is an adenoma, a malignant tumor, and adenocarcinoma, wherein the tumor is classified according to tissue origin or cell name, including: bladder urothelial carcinoma, adrenocortical carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck cancer, chromophobe renal carcinoma, mixed nerve carcinoma, renal cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, 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. As a specific embodiment of the present invention, the tumor is colorectal cancer. Preferably, the tumor is a malignant tumor, a metastatic tumor, or a non-metastatic tumor. More preferably, any stage of cancer is included (clinical stage I, II, III, or IV, TNM classification of malignant tumors T1-4, N0-4, or M0-1, histological grade G1, G2, G3, or G4, etc.).

[0015] The present invention also provides use of a pharmaceutical composition or preparation containing S. waldensis in the preparation of a synergist of immune checkpoint inhibitors for tumor treatment.

[0016] Preferably, the Wardsutteria is a combination of one or more Wardsutteria strains (as described above). As an embodiment of the present invention, the deposit number of Wardsutteria is DSM NO: 14016, and the immune checkpoint inhibitor includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody or CTLA-4 monoclonal antibody.

[0017] Preferably, the pharmaceutical composition or the preparation uses the Wardsutterella as an active ingredient, and the Wardsutterella is a live bacterium;

[0018] The pharmaceutical composition or formulation further comprises a pharmaceutically acceptable carrier and excipient. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals (e.g., humans, if appropriate). Specific examples of pharmaceutically acceptable carriers as described herein are borate buffer or sterile saline solution.

[0019] Preferably, the pharmaceutical composition is prepared into tablets, capsules, granules, suspensions or injections.

[0020] Preferably, the tumor includes one or more of bladder urothelial carcinoma, adrenocortical carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck cancer, chromophobe renal cell carcinoma, mixed renal carcinoma, kidney cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, 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.

[0021] The present invention also provides a pharmaceutical composition or preparation for treating tumors, comprising an effective dose of S. waldenii combined with an immune checkpoint inhibitor, wherein the deposit number of S. waldenii is DSM NO: 14016, and the immune checkpoint inhibitor comprises PD-1 monoclonal antibody, PD-L1 monoclonal antibody or CTLA-4 monoclonal antibody.

[0022] The present invention proposes a combination therapy: immune checkpoint inhibition therapy is performed simultaneously, separately or sequentially with the use of S. waldensis, thereby increasing the therapeutic effect of immune checkpoint inhibition.

[0023] The route of administration for the administration of S. waldensis is oral.

[0024] The order of using the combination therapy is: using the said Wardsatella simultaneously with, before and / or after the immune checkpoint inhibition therapy.

[0025] In the combination therapy, dose delays and / or dose reductions and time adjustments are performed as needed based on the individual patient's tolerance to treatment.

[0026] The Waldersartella described in the present invention may comprise an effective amount of Waldersartella generally dispersed in a pharmaceutically or pharmacologically acceptable carrier.

[0027] The term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals (e.g., humans, if appropriate). Specific examples of pharmacologically acceptable carriers as described herein are borate buffer or sterile saline solution.

[0028] The enhanced immune checkpoint inhibitors of the present invention can be used for patients who are refractory to immune checkpoint inhibitors, wherein the patients who are refractory to immune checkpoint inhibitors exhibit innate (primary) resistance to treatment with the immune checkpoint inhibitors, which is manifested as a lack of response or insufficient response to treatment with the checkpoint inhibitors that lasts for at least about 8 weeks or 12 weeks from the first dose.

[0029] The enhanced immune checkpoint inhibitors described in the present invention can be used for patients who are refractory to immune checkpoint inhibitors, wherein the patients who are refractory to immune checkpoint inhibitors show acquired (secondary) resistance to treatment with multiple checkpoint inhibitors, which is manifested as an initial response to the checkpoint treatment, but one or more tumors subsequently relapse and develop.

[0030] The present invention discloses the following technical effects:

[0031] The present invention relates to a combination therapy that enhances the efficacy of immune checkpoint inhibitors, suitable for colorectal cancer and various other tumors. This invention combines the bacterium Waldensella as a novel immune adjuvant with ICIs for tumor treatment, achieving remarkable technical results. This approach provides a scientific basis for combined bacterial and ICI therapies and offers a new solution for improving the clinical efficacy of ICIs targeting intestinal microbes. This approach is of great significance for improving the overall efficacy of immunotherapy for cancer patients and promoting the translational application of pharmacomicrobiome in personalized precision medicine.

[0032] The present invention uses an oral preparation of a human symbiotic bacterium (Wardiella) in combination with immune checkpoint inhibitors. The anti-tumor immune protection response stimulated by Wardiella can significantly enhance the efficacy of immune checkpoint inhibitors against various tumors, with higher safety, prolong the overall survival time of tumor patients, improve the response rate of cancer immunotherapy population, and expand the population of tumor patients who benefit from cancer immunotherapy (immunotherapy checkpoint inhibitors). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] FIG1 is an experimental flow chart of Example 1;

[0035] Figure 2 shows the curve of tumor volume change; MC38 tumor model (left), CT26 tumor model (right);

[0036] Figure 3 shows the statistical graph of tumor weight; MC38 tumor model (left), CT26 tumor model (right);

[0037] Figure 4 is a HE staining image of the intestinal tissue of MC38 tumor model mice (day 19);

[0038] Figure 5 shows the results of multicolor flow cytometry staining of mouse tumor tissue at the end of the experiment; CD4 + ICOS+T(A), CD8 + IFN-γ+TNF-α+T(B), CD4 + The proportion of IFN-γ+TNF-α+T(C) cells; CD8 + IFN-γ+TNF-α+T(D), CD4 + IFN-γ+TNF-α+T(E) cell ratio;

[0039] Figure 6 shows the RNA-seq results of tumor tissues (day 19) in MC38 tumor model mice;

[0040] Figure 7 shows the distribution and relative abundance of S. waldensis in different populations;

[0041] Figure 8 shows the relationship between the abundance of S. waldensis and the efficacy of immune checkpoint inhibitors in patients with renal cell carcinoma; A is progression-free survival; B is treatment effect, where SD is stable disease, CR is complete remission, PD is progressive disease, and PR is stable disease. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The present invention significantly improves the conventional therapies, which have serious toxic and side effects, are prone to recurrence and metastasis, have short duration of therapeutic effects, short patient survival, heavy economic burden and poor quality of life. It also significantly improves the shortcomings of immune checkpoint monotherapy, such as a small number of drug-responsive populations and a small number of effective tumor types. It also significantly improves the shortcomings of immune checkpoint combined chemoradiotherapy, such as severe adverse reactions and a limited number of drug-responsive populations.

[0048] The treatment regimen provided by the present invention has a good therapeutic effect on the following patients: patients who are not suitable for surgery, have no effectively available targeted drugs, and are ineffective with radiotherapy and chemotherapy; patients with tumors for whom immune checkpoint inhibitors alone are ineffective or have developed drug resistance (primary, adaptive, and acquired); patients with tumors for whom immune checkpoint inhibitors combined with radiotherapy, chemotherapy, and targeted therapy are ineffective or have developed drug resistance (primary, adaptive, and acquired).

[0049] The treatment scheme provided by the present invention is further illustrated below with reference to specific examples.

[0050] Example 1 The therapeutic effect of S. waldensis combined with immune checkpoint inhibitors on tumors

[0051] 1. Experimental methods

[0052] 1.1 Experimental Materials

[0053] (1) Mouse strains: 6-week-old female C57BL / 6J mice and Balb / c mice.

[0054] (2) Tumor cell lines: mouse intestinal cancer cell line (MC38, ATCC), mouse intestinal cancer cell line (CT26, ATCC).

[0055] (3) Bacterial preparation: Sutterella wadsworthensis (DSM NO: 14016, Type strain, whose 16S rDNA sequence is shown in SEQ ID NO: 1), referred to as SW, was purchased from DSMZ, German National Culture Collection (official website of DSMZ: http: / / www.dsmz.de).

[0056] (4) Bacterial culture medium: Liquid mGAM medium, the formula mainly includes peptone, yeast extract, soluble starch, beef extract and glucose, etc., purchased from Nissui Pharmaceutical Co., Ltd., Japan. Fumaric acid (2.86 g / L) and formate (2.86 g / L) were additionally added to this medium.

[0057] (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number RPM1-14, the reagent was purchased from BioXCell, USA.

[0058] (6) Antibiotic combination: ampicillin (50 mg / kg), neomycin sulfate (50 mg / kg), vancomycin (25 mg / kg), metronidazole (50 mg / kg).

[0059] 1.2 Experimental Grouping

[0060] The experimental groups are shown in Table 1 below.

[0061] Table 1

[0062] 1.3 Experimental methods

[0063] The experimental flow chart is shown in Figure 1. The specific operations are as follows:

[0064] (1) Bacterial culture: Inoculate S. waldensis in mGAM liquid medium (containing formate and fumaric acid), culture in an anaerobic chamber at 37°C for 48 hours, and then centrifuge to a concentration of 1×10 9 CFU / mL.

[0065] (2) Subcutaneous inoculation of tumor cells: MC38 cell line 5×10 5 / mouse, CT26 cell line 5×10 5 / Only.

[0066] (3) 14 to 8 days before tumor cell inoculation: Each group of mice was gavaged with a combination of antibiotics to eliminate the intestinal flora.

[0067] (4) 7 to 16 days before tumor cell inoculation: SW live bacterial liquid preparation treatment was given by gavage, 100 μL / mouse, 1×10 8 CFU / piece.

[0068] (5) IgG or PD-1 mAb were injected intraperitoneally on days 7, 10, 13, and 16, respectively, at 200 μg / mouse.

[0069] (6) Measure the tumor size on days 7, 10, 13, 16, and 18, and calculate the tumor volume. Tumor volume = (tumor width 2 × tumor long diameter) × 1 / 2

[0070] (7) On the 18th day, the mice were euthanized, the tumor tissues were removed, photographed and weighed, and the intestinal tissues were taken for HE staining to determine the intestinal inflammation.

[0071] (8) Measure the volume of mouse tumors, measure the weight of mouse tumors at the end time point, and evaluate the infiltration of immune cells in tumor tissues by multicolor flow cytometry to evaluate the efficacy.

[0072] (9) HE staining of mouse intestinal tissue sections was used to observe and evaluate whether Waldesworm can cause enteritis for safety assessment.

[0073] 2. Experimental results

[0074] As shown in Figures 2 and 3, which are the tumor volume change curves and tumor weight statistics, respectively, the results show that in the MC38 and CT26 colorectal cancer mouse models, compared with the placebo treatment group (IgG) and the single-drug immune checkpoint inhibitor group (PD-1 mAb), the combination therapy group (PD-1 mAb+SW) showed obvious and significant tumor reduction (p<0.01), proving that S. waldensis can enhance the anti-tumor effect of PD-1 mAb.

[0075] As shown in Figure 4, it is the HE staining result of the intestinal tissue section of the MC38 mouse model at the end point of the experiment. The results show that no enteritis was found in the mice in the Waldenström single bacteria group (IgG+SW) and the combination therapy group (PD-1 mAb+SW), proving the safety of oral gastrointestinal administration of Waldenström.

[0076] As shown in Figure 5, it is the result of multicolor flow cytometry staining of tumor tissues of MC38 (AC in Figure 5) and CT26 (DE in Figure 5) mice at the experimental endpoint. The results show that the single-bacterial group of S. waldensis (IgG+SW) can significantly increase the infiltration of CD4+ICOS+T cells in tumor tissues. Compared with the single-drug immune checkpoint inhibitor group (PD-1 mAb), the combination therapy group (PD-1 mAb+SW) also has significantly increased CD4 + ICOS+T cell infiltration; at the same time, it was observed in both MC38 and CT26 mouse tumor models that compared with the single-agent immune checkpoint inhibitor group (PD-1mAb), the combination therapy group (PD-1 mAb+SW) had increased CD8 + IFN-γ+TNF-α+and CD4 + IFN-γ+TNF-α+T cells confirmed that S. waldensis could enhance the anti-tumor immune response of PD-1 mAb. Compared with the placebo treatment group (IgG), the S. waldensis single bacteria group (IgG+SW) showed an increase in tumor CD8 + IFN-γ+TNF-α+T cells and CD4 + The trend of IFN-γ+TNF-α+T cell infiltration confirmed the regulatory effect of oral administration of S. waldensis on systemic immunity.

[0077] As shown in Figure 6, it is the RNA-seq result of mouse tumor tissue. The results show that compared with the single-agent immune checkpoint inhibitor group (PD-1 mAb), the combination therapy group (PD-1 mAb+SW) showed activated anti-tumor immune response signals, indicating that oral administration of S. waldensis significantly enhanced the anti-tumor immune response during immune checkpoint inhibitor treatment.

[0078] Example 2: Waldesartella is an endogenous intestinal commensal bacterium in humans

[0079] 1. Experimental methods

[0080] The GMrepo public database was used to analyze a human gut metagenomic dataset, comprising 2,676 human fecal samples from 14 different populations. Metagenomic sequencing technology can accurately identify human gut bacteria at the species level.

[0081] The gut microbiota metagenomics data of 14 different types of populations include: 1) healthy people; 2) diarrhea; 3) inflammatory bowel disease; 4) colorectal cancer; 5) autism spectrum disorder; 6) cirrhosis; 7) cardiovascular disease; 8) type 1 diabetes; 9) rheumatoid arthritis; 10) type 2 diabetes; 11) non-alcoholic fatty liver disease; 12) Parkinson's disease; 13) hypertension; 14) renal cell carcinoma patients before treatment with immune checkpoint inhibitors (ICIs).

[0082] The number of samples of relevant types of population included is shown in Table 2.

[0083] Table 2 Public human intestinal metagenomic datasets

[0084] 2. Experimental results

[0085] As shown in Figure 7 and Table 3, the results show that Sutterella wadsworthensis exists in different populations, with a relative abundance ranging from 0.47% to 12.5%. The relative abundance of Sutterella wadsworthensis varies among different populations. Relative abundance refers to the proportion of a certain species to all bacterial species in the intestine. 14 According to the estimation of CFU / mL, the number of Waldesertella in the human intestine is about 10 11 CFU / mL~10 13 CFU / mL.

[0086] Table 3 Relative abundance of S. waldensis in different populations

[0087] Example 3: Intestinal abundance of S. waldenii is associated with responsiveness to ICIs in patients with renal cell carcinoma

[0088] 1. Experimental methods

[0089] We analyzed the public gut metagenomic dataset PRJEB22863 from renal cell carcinoma (RCC) patients before immune checkpoint inhibitor (ICI) treatment, and established a relationship between the abundance of Sutterella wadsworthensis in the stool of RCC patients at baseline and their responsiveness to ICIs treatment.

[0090] 2. Experimental results

[0091] As shown in FIG8 , the results showed that renal cell carcinoma patients with high abundance of Sutterella wadsworthensis in the intestine had a higher proportion of progression-free survival (PFS) greater than or equal to 6 months, better disease control, and higher response rate.

[0092] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of Sutterella wadsworthensis in the preparation of potentiators of immune checkpoint inhibitors for tumor treatment.

2. Application of S. waldensis combined with immune checkpoint inhibitors in the preparation of drugs for treating tumors.

3. The use according to claim 1 or 2, characterized in that The deposit number of the bacterium Waldersartella is DSM NO: 14016, and the immune checkpoint inhibitor includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody or CTLA-4 monoclonal antibody.

4. The use according to claim 1 or 2, characterized in that The tumor includes one or more of bladder urothelial carcinoma, adrenocortical carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck cancer, renal chromophobe cell carcinoma, mixed nerve carcinoma, kidney cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, 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.

5. Use of a pharmaceutical composition or preparation comprising S. waldersartella in the preparation of a synergist for immune checkpoint inhibitors for tumor treatment.

6. The use according to claim 5, characterized in that The deposit number of the bacterium Waldersartella is DSM NO: 14016, and the immune checkpoint inhibitor includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody or CTLA-4 monoclonal antibody.

7. The use according to claim 5, characterized in that The pharmaceutical composition or the preparation uses the Wardsutterella as an active ingredient, and the Wardsutterella is a live bacterium; The pharmaceutical composition or the preparation further includes pharmaceutically acceptable carriers and excipients.

8. The use according to claim 5, characterized in that The pharmaceutical composition is prepared into tablets, capsules, granules, suspensions or injections.

9. The use according to any one of claims 5 to 8, characterized in that The tumor includes one or more of bladder urothelial carcinoma, adrenocortical carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck cancer, renal chromophobe cell carcinoma, mixed nerve carcinoma, kidney cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, 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.

10. A pharmaceutical composition or preparation for treating tumors, characterized in that: It includes an effective dose of Waldenström combined with an immune checkpoint inhibitor, the deposit number of the Waldenström is DSM NO: 14016, and the immune checkpoint inhibitor includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody or CTLA-4 monoclonal antibody.

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