Application of virococcus giganteus in preparation of synergist of immune checkpoint inhibitor

Abstract

The invention discloses application of levirococcus in preparation of a synergist of an immune checkpoint inhibitor, and belongs to the technical field of tumor treatment. According to the present invention, the small virococcus and the immune checkpoint inhibitor are combined to treat tumors, the enhanced treatment effect is measured by reducing the tumor volume, and the result shows that the small virococcus can significantly improve the tumor treatment effect of the immune checkpoint inhibitor. The invention has important significance for improving the overall curative effect of tumor patient immunotherapy and promoting the transformation application of drug microbiomics in individualized precise medication.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and in particular to the application of *Veillonella spp.* in the preparation of potentiators for immune checkpoint inhibitors. Background Technology

[0002] Malignant tumors are a class of deadly and difficult-to-treat diseases. Recent studies have found that immune checkpoint inhibitors (ICIs) can significantly prolong the survival of patients with various solid tumors, including advanced non-small cell lung cancer and melanoma. However, only about 30% of patients show a definitive clinical response to ICIs, and approximately 60-70% develop primary or secondary resistance. Therefore, identifying biomarkers of ICI response and elucidating their biological mechanisms, as well as developing treatment regimens that can significantly improve ICI sensitivity, is of significant clinical importance.

[0003] Utilizing multi-omics approaches (genomics, transcriptomics, epigenetics, metabolomics, and microbiome) to explore biomarkers determining the clinical efficacy of ICIs and design rational ICI combination therapy regimens is key to enhancing the overall therapeutic benefit of ICIs and is also one of the key research directions in the fields of pharmacogenomics and personalized precision medicine. PD-L1 expression is currently the most commonly used biomarker for predicting the efficacy of ICIs. High PD-L1 expression has been found to be associated with higher objective response rates 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 biomarker for ICI efficacy is only suitable for certain 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 a potential biomarker for predicting response to tumor immunotherapy (ICIs). In preclinical studies and cancer patients, the composition of the gut microbiota is significantly correlated with the efficacy of ICI treatment. Exploring specific microbial characteristics and modulating the gut microbiota through methods such as microbiota transplantation holds promise as an adjunct therapy for tumor immunotherapy. Studies have found that broad-spectrum antibiotics can cause gut microbiota dysbiosis in patients with non-small cell lung cancer and melanoma treated with ICIs, leading to a significant reduction in overall survival (2 months vs. 26 months, HR = 7.4) and a significant increase in non-response rate. High alpha diversity in the gut microbiota has been shown to be associated with prolonged progression-free survival in melanoma patients receiving ICI treatment. Transplanting fecal microbiota from patients who achieved complete remission (R) after PD-1 monoclonal antibody therapy to patients with unresponsive (NR) end-stage metastatic melanoma resulted in approximately 30% of recipient patients (NR) showing significant new clinical responses, with 10% even achieving complete remission. High abundance of *Fusobacterium nucleatum* in the gut...F.nucleatum ) was associated with a good response to PD-1 monoclonal antibody therapy in colorectal cancer patients. *Bifidobacterium pseudolongum* was isolated from mice with orthotopic colon cancer that had a good response to ICIs therapy. B.pseudolongum It can enhance the anti-colorectal cancer effect of PD-1 monoclonal antibodies through its metabolite creatinine. Therefore, the gut microbiota has a significant impact on the efficacy of ICIs in treating malignant tumors such as colorectal cancer, melanoma, and non-small cell lung cancer, and specific gut microbiota may become a new class of biomarkers for predicting the responsiveness to ICIs treatment.

[0005] Gut microbiota and its metabolites can improve the responsiveness to ICIs (intrinsic immunizations) by modulating innate and adaptive immunity. Regarding innate immune regulation, gut microbiota can influence the function of dendritic cells (DCs), monocytes / macrophages, and natural killer (NK) cells. For example, *Ekkermansia myxophilus* (… A. muciniphila It can activate the TLR2 / NF-κB and NLRP3 pathways and induce macrophage polarization towards the M1 type, thereby inhibiting the progression of colon cancer. *Lactobacillus rhamnosus* ( L.rhamnosus GG (Gastrointestinal G ... + T and CD4 + T cell activity. The gut microbiota metabolite butyrate directly enhances CD8+ by binding to the DNA-binding inhibitory factor 2 (ID2)-IL-12. + The tumor-killing effect of T cells; Bifidobacterium pseudolongum ( B.pseudolongum Creatinine, a secondary metabolite of creatinine, activates cAMP response element-binding protein (pCREB) phosphorylation via 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 characteristics of different types of gut microbiota and their metabolites is crucial for elucidating the response mechanisms of antitumor immunotherapy and improving the efficacy of ICIs. Summary of the Invention

[0006] The purpose of this invention is to provide the application of *Veillonella spp.* in the preparation of potentiators for immune checkpoint inhibitors, in order to solve the problems existing in the prior art. By combining *Veillonella spp.* with immune checkpoint inhibitors, the efficacy of immune checkpoint inhibitors in treating tumors can be significantly improved.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides *Veillonella spp.* (…) Veillonella parvulaThe above-mentioned *Veillonella* strain is used in the preparation of potentiators for immune checkpoint inhibitors in tumor therapy. Its preservation number is ATCC 17745. The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer. The *Veillonella* strain is a live bacterium.

[0008] This invention also provides the use of *Veillonella spp.* in combination with immune checkpoint inhibitors in the preparation of drugs for treating tumors. The *Veillonella spp.* strain has the accession number ATCC 17745. The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer. The *Veillonella spp.* strain is a live bacterium.

[0009] In this invention, *Veillonella spp.* includes, but is not limited to, the strain with accession number ATCC 17745; *Veillonella spp.* contains a 16S rDNA sequence, and the *Veillonella spp.* strain of this invention refers to any strain with at least 99% identity to the 16S rDNA sequence of *Veillonella spp.* More preferably, the aforementioned *Veillonella spp.* strain is a combination of one or more *Veillonella spp.* strains. More preferably, the strain of *Veillonella spp.* is the strain name of subspecies classification in the National Center for Biotechnology Information (NCBI) genome database (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ?taxon=29466) as follows: Veillonella parvula NCTC11810 Veillonella parvula DSM 2008 Veillonella parvula PK1910 Veillonella parvula DSM2007 Veillonella parvula UTDB1-3 Veillonella parvula SKV38 Veillonella little girl FDAARGOS_1046 Veillonella parvula f34 Veillonella parvula ATCC 17745 Veillonella parvula CM18GR_62_ID1101 Veillonella parvula DFI.7.67 Veillonella little girl CM85Y_47 Veillonella parvula K67 Veillonella parvula DFI.7.66 Veillonella parvula CM711B_41 Veillonella parvulaAM72-29pH10A, Veillonella little girl CLA-JM-H54, Veillonella parvula 1001295B_180824_H5, Veillonella little girl CLA-AA-H2, Veillonella parvula UMB0371, Veillonella parvula MCC755, Veillonella parvula DFI.7.83, Veillonella parvula DFI.7.93, Veillonella little girl DFI.7.72, Veillonella parvula BIOML-A1, Veillonella parvula BIOML-A2, Veillonella parvula KHUD_VP2, Veillonella parvula UMB0138, Veillonella little girl DFI.7.86, Veillonella parvula AF04-47, Veillonella parvula 1001216B_150713_F11, Veillonella parvula DNF00876, Veillonella parvula 2218st1_D6_2218SCRN_220325, Veillonella parvula ACS-068-V-Sch12, Veillonella parvula HSIVP1, Veillonella parvula SHI-1, Veillonella parvula DFI.1.137 and Veillonella little girlOne or more of AM48-14BH. More preferably, the above-mentioned *Veillonella* strain is one or more of the following strains: *Veillonella* deposited at the German Collection of Microorganisms and Cell Collection, accession number DSM 2008 (NCBI: taxid 479436, https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / 479436 / ); *Veillonella* deposited at the American Type Culture Collection, accession number ATCC 17745 (NCBI: taxid 686660, https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / 686660 / ); and *Veillonella* deposited at the National Collection of Type Cultures, accession number NCTC 11810. As an embodiment of the present invention, *Veillarios valerianii* with accession number ATCC 17745 is used as an example for illustration.

[0010] In this invention, the aforementioned immune checkpoint inhibitors include, but are not limited to, PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies, which are one or more combinations of blockers acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands. More preferably, the T cell negative co-stimulatory (co-inhibitory) 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, and DcR3.More preferably, the blocking agents of the ligands of the aforementioned T-cell negative co-stimulatory (co-inhibitory) molecules are derived 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), and 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 bispecific antibody), MGD019 (PD-1 / CTLA-4 bispecific 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 ( 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 aforementioned blocking agent is selected from nivolumab, pembrolizumab, toripalimab, sintilimab, cimiprimab, or any combination thereof. More preferably, the aforementioned immune checkpoint inhibitor is an inhibitor acting on the PD-1 / PD-L1 signaling pathway and / or the PD-1 / PD-L2 signaling pathway, wherein PD-1 refers to programmed 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 inhibitors of the aforementioned PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathways are 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), and spartalizumab (PD-L2 monoclonal antibody). The following are drug names: 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 bispecific antibody), MGD019 (PD-1 / CTLA-4 bispecific antibody), MEDI0680 (PD-1 monoclonal antibody), PDR001 (PD-1 monoclonal antibody), FAZ053 (PD-L1 monoclonal antibody), or any combination thereof. More preferably, the aforementioned immune checkpoint inhibitor is an inhibitor acting 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, and 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 bispecific antibody), or any combination thereof.As a specific embodiment of the present invention, the aforementioned immune checkpoint inhibitor is an inhibitor acting on the PD-1 / PD-L1 signaling pathway and / or the PD-1 / PD-L2 signaling pathway and / or the CTLA-4 / B7-1 signaling pathway and / or the CTLA-4 / B7-2 signaling pathway. Specifically, in this embodiment of the present invention, the aforementioned immune checkpoint inhibitor is illustrated using PD-1 monoclonal antibody or CTLA-4 monoclonal antibody as an example.

[0011] In this invention, tumor treatment or treatment of tumors refers to tumor volume reduction or stabilization, extension of overall survival time of tumor patients, extension of progression-free survival, and improvement of quality of life.

[0012] In this invention, the aforementioned tumors include, but are not limited to, melanoma and non-small cell lung cancer. The tumors can be adenomas, malignant tumors, and adenocarcinomas, and are 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, multifocal glioma, glioma, head and neck cancer, chromophobe renal carcinoma, mixed renal 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, cutaneous melanoma, gastric cancer, esophageal cancer, testicular cancer, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, and soft tissue sarcoma, one or more of these. Preferably, the tumors are malignant tumors, metastatic tumors, or non-metastatic tumors. More preferably, it includes any stage of cancer (clinical stage I, II, III, or IV; TNM classification of malignant tumors as T1-4, N0-4, or M0-1; histological grade G1, G2, G3, or G4, etc.). In this embodiment of the invention, melanoma and non-small cell lung cancer are used as examples.

[0013] This invention also provides the use of a pharmaceutical composition or formulation containing *Veillonella spp.* in the preparation of a potentiator for immune checkpoint inhibitors used in tumor therapy, wherein the *Veillonella spp.* accession number is ATCC 17745. The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer. The pharmaceutical composition or formulation uses *Veillonella spp.* as the active ingredient, wherein *Veillonella spp.* is a live bacterium, and the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0014] This invention also provides a pharmaceutical composition or formulation for treating tumors, wherein the pharmaceutical composition or formulation comprises *Veillonella spp.* and an immune checkpoint inhibitor, wherein the *Veillonella spp.* accession number is ATCC 17745. The immune checkpoint inhibitor includes PD-1 monoclonal antibody, PD-L1 monoclonal antibody, or CTLA-4 monoclonal antibody; the tumor includes melanoma and non-small cell lung cancer. The *Veillonella spp.* is a live bacterium.

[0015] In this invention, a combination therapy is proposed: immune checkpoint inhibition therapy is performed simultaneously with, separately or sequentially using *Virginia valerate*, thereby increasing the therapeutic effect of immune checkpoint inhibition.

[0016] The administration route of Veillonella is oral.

[0017] The order of use for combination therapy is as follows: use the aforementioned Veillonella microphylla before and / or after immune checkpoint inhibitory therapy.

[0018] In combination therapy, dose delays and / or dose reductions, as well as time adjustments, may be performed as needed, depending on the individual patient's tolerance to the treatment.

[0019] In this invention, "pharmaceutically acceptable" means a molecular entity and composition that does not produce adverse reactions, allergic reactions, or other adverse effects when used in animals (e.g., humans, if appropriate). Specific examples of pharmaceutically acceptable excipients as described herein are borate buffer solutions or sterile saline solutions.

[0020] In this invention, the potentiator can be used in patients refractory to immune checkpoint inhibitors, wherein the patients exhibit innate (primary) resistance to the treatment of the immune checkpoint inhibitors, manifested as a lack of or insufficient response to the treatment of the checkpoint inhibitors for at least about 8 or 12 weeks from the start of the first dose.

[0021] In this invention, the potentiator can be used in patients refractory to immune checkpoint inhibitors, wherein the patients exhibit acquired (secondary) resistance to the treatment of the checkpoint inhibitors, characterized by an initial response to the checkpoint treatment, but subsequent recurrence and progression of one or more tumors.

[0022] The present invention discloses the following technical effects: This invention relates to a combination therapy that enhances the efficacy of immune checkpoint inhibitors, applicable to melanoma, non-small cell lung cancer, and various other tumors. This invention utilizes *Veillonella spp.* as a novel immune adjuvant in combination with immune checkpoint inhibitors (ICIs) for tumor treatment, achieving significant technical effects. It provides a scientific basis for bacterial and ICI combination therapy, offers a new solution for improving the clinical efficacy of ICIs by targeting gut microbiota, and is of great significance for improving the overall efficacy of immunotherapy in cancer patients and promoting the translational application of pharmacomicrobiome in personalized precision medicine.

[0023] This invention utilizes an oral formulation of a single symbiotic bacterium (Virronella spp.) in combination with an immune checkpoint inhibitor. The anti-tumor immune protective response stimulated by Virronella spp. significantly enhances the efficacy of immune checkpoint inhibitors against various tumors, while also improving safety, prolonging the overall survival time of cancer patients, increasing the response rate in cancer immunotherapy patients, and expanding the beneficiary population of cancer immunotherapy (immunotherapy checkpoint inhibitors). Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Flowchart of an experimental study on the combined use of Veillonella microphylla and immune checkpoint inhibitors for tumor treatment; Figure 2 The tumor volume change curve (A) and tumor photograph at the experimental endpoint (B) are shown after treatment with Vibrio vulnificus combined with PD-1 mAb. Figure 3 The tumor volume change curve (A) and tumor photograph at the experimental endpoint (B) are shown after treatment with Vibrio vulnificus combined with CTLA-4 mAb. Figure 4 Abundance of Veillonella spp. in melanoma patients treated with PD-1 mAb monotherapy; Figure 5 Abundance of *Veillonella spp.* in melanoma patients treated with combination therapy of PD-1 mAb and CTLA-4 mAb; Figure 6 The abundance of Veillonella spp. in NSCLC patients treated with PD-1 mAb monotherapy. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] This invention significantly improves upon the drawbacks of conventional therapies, such as severe toxic side effects, high recurrence and metastasis rates, short duration of treatment effects, short patient survival, heavy economic burden, and poor quality of life. It also significantly improves upon the shortcomings of immune checkpoint monotherapy, such as a small number of patients responding to the drug and a limited range of tumor types it targets. Furthermore, it significantly improves upon the drawbacks of immune checkpoint combined radiotherapy and chemotherapy, such as severe adverse reactions and a limited number of patients responding to the drug.

[0032] The treatment regimens proposed in this invention have good therapeutic effects on the following patients: patients for whom surgery is not an option, where no effective targeted drugs are available, and where radiotherapy and chemotherapy are ineffective; patients with tumors for whom immune checkpoint inhibitors are ineffective or have developed resistance (primary, adaptive, and acquired); and patients with tumors for whom immune checkpoint inhibitors combined with radiotherapy, chemotherapy, and targeted therapy are ineffective or have developed resistance (primary, adaptive, and acquired).

[0033] The treatment plan provided by the present invention will be further illustrated below with specific embodiments.

[0034] Example 1: The therapeutic effect of *Veillonella spp.* combined with immune checkpoint inhibitors on tumors. 1. Experimental Methods 1.1 Experimental Materials (1) Mouse strain: 6-week-old female C57BL / 6J mice.

[0035] (2) Tumor cell line: mouse melanoma cell line (B16F10, ATCC).

[0036] (3) Bacterial preparations: Villous cocci ( Veillonella parvula ATCC NO: 17745, Type strain (its 16S rDNA sequence is shown in SEQ ID NO: 1), abbreviated as VP, was commercially purchased from the ATCC Type Culture Collection (ATCC's official website is: https: / / www.atcc.org / ). SEQ ID NO: 1 is:

[0037] (4) Bacterial culture medium: liquid RCM medium, the main ingredients of which are peptone, yeast extract, soluble starch, cysteine ​​hydrochloride, agar, sodium chloride, sodium acetate and glucose, etc., purchased from Thermo Fisher Scientific. Sodium lactate (3.0 g / L) was added to this medium.

[0038] (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number RPM1-14, CTLA-4 monoclonal antibody, clone number 9H10, reagents were purchased from BioXCell, USA.

[0039] (6) Antibiotic combination: ampicillin (50 mg / kg), neomycin sulfate (50 mg / kg), vancomycin (25 mg / kg), metronidazole (50 mg / kg).

[0040] 1.2 Experimental Grouping The experimental groups are shown in Table 1 below.

[0041] Table 1 1.3 Experimental Methods See the experimental flowchart. Figure 1 The specific steps are as follows: (1) Bacterial culture: Inoculate *Veillonella spp.* into RCM liquid medium (containing lactic acid), incubate at 37°C in an anaerobic incubator for 48 hours, centrifuge, and adjust the concentration to 1×10⁻⁶. 10 CFU / mL.

[0042] (2) Subcutaneous inoculation of tumor cells: 5 × 10⁶ B16f10 cell line 5 / Only.

[0043] (3) One week before tumor cell inoculation: mice in each group were given a combination of antibiotics by gavage to clear the intestinal flora.

[0044] (4) After tumor cell inoculation: VP live bacterial solution was administered via gavage, 200 μL / animal, 2 × 10 9 CFU / each.

[0045] (5) IgG2a / PD-1 mAb / CTLA-4 mAb, 200 μg / animal, were injected intraperitoneally on days 7, 10, 13 and 16 respectively.

[0046] (6) Measure the tumor size and calculate the tumor volume on days 7, 10, 13, 16 and 19 respectively.

[0047] (7) The mice were euthanized on day 19 and the tumor tissue was removed and photographed.

[0048] (8) Measurement of tumor volume in mice.

[0049] 2. Experimental Results like Figure 2 and Figure 3 As shown, the results are the tumor volume change curves. The results show that in the B16f10 melanoma mouse model, compared with the placebo treatment group (IgG2a) and the single-drug immune checkpoint inhibitor group (PD-1 mAb or CTLA-4 mAb), the combination therapy group (PD-1 mAb / CTLA-4 mAb+VP) showed significant tumor reduction (p<0.01), demonstrating that the live bacteria preparation of *Vibrio vulnificus* can enhance the anti-tumor effect of PD-1 mAb or CTLA-4 mAb.

[0050] Example 2: *Veillonella spp.* is an endogenous gut commensal bacterium for humans. 1. Experimental Methods The human gut metagenomic dataset was analyzed using the GMrepo public database (https: / / gmrepo.humangut.info / home), which contains 29,529 independent healthy human fecal samples.

[0051] 2. Experimental Results *Veillarum valgum* was detected in only 3,920 samples, representing approximately 13.275%, indicating that this bacterium is a low-prevalence species in the gut of healthy individuals. The relative abundance exhibited an extremely right-skewed distribution: the median (0.0239%) differed significantly from the mean (75.079%). The standard deviation (385.263%) was extremely high, with a coefficient of variation (CV) > 5, demonstrating its ability to become the absolutely dominant bacterium in a small number of individuals, indicating its strong niche-occupancy ability in specific gut microenvironments.

[0052] Example 3: Abundance of *Veillonella spp.* in the gut is associated with ICI treatment responsiveness in patients with melanoma and non-small cell lung cancer. 1. Experimental Methods We analyzed the gut metagenomic dataset (PRJEB22863) of melanoma (PRJNA541981, PRJNA399742, PRJNA397906, PRJNA762360, PRJNA678737) and non-small cell lung cancer (NSCLC) patients. The dataset included metagenomic stool samples from 154 patients – 115 patients treated with PD-1 mAb monotherapy and 39 patients treated with PD-1 mAb and CTLA-4 mAb combination therapy before treatment – ​​and 65 patients treated with PD-1 mAb monotherapy before treatment with NSCLC. This established the baseline presence of *Veillariosporinus* in the stool of melanoma and NSCLC patients. Veillonella parvula The relationship between abundance and patient responsiveness to ICIs treatment.

[0053] 2. Experimental Results like Figure 4-Figure 6 As shown, the results indicate that the gut microbiota has a high abundance of *Veillonella* (Veillonella). Veillonella parvula Patients with melanoma and NSCLC have a higher response rate to ICIs.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Varionella microphylla ( Veillonella parvula Its application in the preparation of potentiators for immune checkpoint inhibitors used in tumor therapy is characterized by, The preservation number of the *Veillonella* species is ATCC 17745.

2. The application of *Veilonella spp.* in combination with immune checkpoint inhibitors in the preparation of drugs for treating tumors, characterized in that... The preservation number of the *Veillonella* species is ATCC 17745.

3. The application as described in claim 1 or 2, characterized in that, The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer.

4. The application as described in claim 1 or 2, characterized in that, The *Veillonella* species mentioned are live bacteria.

5. The use of a pharmaceutical composition or preparation containing *Veillarum valgum* in the preparation of a potentiator for immune checkpoint inhibitors used in tumor therapy, characterized in that... The preservation number of the *Veillonella* species is ATCC 17745.

6. The application as described in claim 5, characterized in that, The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer.

7. The application as described in claim 5, characterized in that, The pharmaceutical composition or the formulation uses *Veillonella spp.* as the active ingredient, *Veillonella spp.* being a live bacterium, and the pharmaceutical composition further includes pharmaceutically acceptable excipients.

8. A pharmaceutical composition or formulation for treating tumors, characterized in that, The pharmaceutical composition or formulation comprises *Veillonella spp.* and an immune checkpoint inhibitor, wherein the *Veillonella spp.* accession number is ATCC 17745.

9. The pharmaceutical composition or formulation for treating tumors as described in claim 8, characterized in that, The immune checkpoint inhibitors include PD-1 monoclonal antibodies, PD-L1 monoclonal antibodies, or CTLA-4 monoclonal antibodies; the tumors include melanoma and non-small cell lung cancer.

10. The pharmaceutical composition or formulation for treating tumors as described in claim 8, characterized in that, The *Veillonella* species mentioned are live bacteria.

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