APPLICATION OF BACTERIA IN PREPARATION OF SYNERGIST FOR IMMUNE CHECKPOINT INHIBITOR

Abstract

The invention discloses an application of bacteria in preparation of a synergist for an immune checkpoint inhibitor. The bacteria are active bacteria or inactive whole-cell gut microbiota. By using a monobacterial oral preparation of human endogenous gut microbiota Alistipes combined with an immune checkpoint inhibitor, an anti-tumor immune protective response and an effect of remodeling gut microbiota is generated by stimulation of oral administration of active human commensal gut microbiota or inactive whole-cell human commensal gut microbiota, which significantly enhances an efficacy of the immune checkpoint inhibitor on multiple tumor species, enhances anti-tumor immune function, is conducive to improving the response rate of cancer immunotherapy populations, and has better safety, prolongs overall survival time of cancer patients, expands cancer patient population benefited from cancer immunotherapy (immunotherapy checkpoint inhibitors), provides new combination therapy regimens and therapeutic drugs to treat immune checkpoint inhibitor-refractory tumor patients, and expands the patient benefited from cancer immunotherapy.

Claims

1. A product for tumor treatment, the product comprising one or more of immune checkpoint inhibitors, and a bacterium; the immune checkpoint inhibitor is one of or a combination of a plurality of blockers acting on T cell negative costimulatory (coinhibitory) molecules and/or their respective ligands; the bacterium is Alistipes finegoldii; the bacterium is one or more of active bacteria, inactive whole-cell bacteria, bacterial derivatives and bacterial metabolites.

2. The product according to claim 1, wherein a 16S rDNA sequence of the Alistipes finegoldii has at least 99.5% consistency with a 16S rDNA sequence of a Alistipes finegoldii strain DSM17242.

3. The product according to claim 1, wherein the Alistipes finegoldii is one of or a combination of a plurality of Alistipes finegoldii strains.

4. The product according to claim 3, wherein the Alistipes finegoldii strain is one of or a combination of a plurality of the following strains: Alistipes finegoldii strain deposited at German collection of microorganisms and cell cultures DSMZ, under accession number DSM 17242; Alistipes finegoldii strain deposited at Japan JCM Culture Collection, under accession number JCM 16770; Alistipes finegoldii strain deposited at Korean KCTC Culture Collection, under accession number KCTC 15236; Alistipes finegoldii strain deposited at Finland Helsinki Anaerobe Reference Laboratory, under accession number AHN 2437; Alistipes finegoldii strain deposited at Sweden CCUG Culture Collection, under accession number CCUG 46020; Alistipes finegoldii strain deposited at French CIP Culture Collection, under accession number CIP 107999; and Alistipes finegoldii strain deposited at Guangdong Microorganism Culture Collection, under accession number GDMCC 1.2324.

5. The product according to claim 1, wherein the active bacteria are intact bacteria and/or intact viable bacteria.

6. The product according to claim 1, wherein the inactive whole-cell bacteria are obtained by first culturing and expanding the bacteria and then inactivating the bacteria by an inactivation method.

7. The product according to claim 6, wherein the inactivation method is selected from any one or more of high temperature inactivation, high pressure inactivation, ultraviolet inactivation, radiation inactivation and inactivation of at least one chemical agent, and the at least one chemical agent is selected from any one or more of formaldehyde, acetone, and phenol.

8. The product according to claim 1, wherein the bacterial derivatives comprise a bacterial constituent, a genetic material and any one or more of a bacterial cell membrane, a fimbriae, a flagella, LPS and a nucleic acid material.

9. The product according to claim 1, wherein the bacterial metabolite comprises molecules produced or modified by the bacterium as a result of bacterial growth, survival, retention, transport or existence during bacteria preparation and storage and during mammalian gastrointestinal transport.

10. The product according to claim 1, wherein the T cell negative costimulatory (coinhibitory) molecules and/or their respective ligands are selected from at least one of 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.

11. The product according to claim 10, wherein the blockers acting on ligands of T cell negative costimulatory (coinhibitory) molecules are selected from at least one of nivolumab, ipilimumab, pembrolizumab, azetolizumab, atezolizumab, camrelizumab, tislelizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, cemiplimab, MGA012, MGD013, MGD019, enoblituzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, PDR001FAZ053, TSR022, MBG453, relatlimab, LAG525, IMP321, REGN3767, pexidartinib, LY3022855, FPA008, BLZ945, GDC0919, epacadostat, indoximid, BMS986205, CPI-444, MEDI9447, PBF509, and lirilumab.

12. The product according to claim 1, wherein the immune checkpoint inhibitor is an inhibitor that acts on PD-1/PD-L1 signaling pathway and/or PD-1/PD-L2 signaling pathway, PD-1 refers to programmed cell death protein 1, PD-L1 refers to B7-H1 or CD274, and PD-L2 refers to B7-DC or CD273.

13. The product according to claim 12, wherein the inhibitor that acts on PD-1/PD-L1 signaling pathway and/or PD-1/PD-L2 signaling pathway is selected from one of or a combination of any of at least one of nivolumab, pembrolizumab, azetolizumab, atezolizumab, camrelizumab, tislelizumab, durvalumab, spartalizumab, avelumab, sintilimab, toripalimab, cemiplimab, MGA012, MGD013, MGD019 (PD-1/CTLA-4 double antibody), MEDI0680, PDR001, and FAZ053.

14. The product according to claim 1, wherein the immune checkpoint inhibitor is an inhibitor that acts on CTLA-4/B7-1 signaling pathway and/or CTLA-4/B7-2 signaling pathway, wherein CTLA-4 refers to cytotoxic T lymphocyte protein 4, B7-1 refers to CD80 and B7-2 refers to CD86.

15. The product according to claim 13, wherein the immune checkpoint inhibitor is selected from at least one of ipilimumab, tremelimumab, and MGD019.

16. A method for treating a tumor with a bacterium, wherein the immune checkpoint inhibitor and the bacterium in the product of claim 1 are administered simultaneously, separately or sequentially.

17. The method according to claim 16, wherein treating the tumor comprises one of or a combination of a plurality of shrinking or stabilizing the tumor, prolonging a total survival time, prolonging a progression-free survival, and improving a life quality.

18. The method according to claim 16, wherein the tumor is a adenomas, a malignant tumor, and a adenocarcinoma, wherein the tumor is one or more of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, glioblastoma multiforme, glioma, head and neck cancer, chromophobe renal cell carcinoma, mixed renal cancer, kidney cancer, leukemia, lymphadenoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, skin melanoma, stomach cancer, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, and soft tissue sarcoma.

19. The method according to claim 16, wherein the tumor is a malignant tumor, a metastatic tumor, or a non-metastatic tumor.

20. The method according to claim 16, wherein the bacterium is administered by oral administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1 shows use of clinical cohorts to screen gut microbiota that can promote immune checkpoint inhibitors in Embodiment 1.

[0153] FIG. 2 shows relationship between different Alistipes species and therapeutic efficacy of immune checkpoint inhibitors in the clinical cohorts of Embodiment 1.

[0154] FIG. 3 is a flowchart of administration of different active Alistipes species in mouse models of colon cancer and melanoma in Embodiment 2.

[0155] FIG. 4 is a tumor image (Day 21) in Embodiment 2.

[0156] FIG. 5 is a change curve in tumor volume in Embodiment 2.

[0157] FIG. 6 is a statistics graph of tumor weight in Embodiment 2.

[0158] FIG. 7 is an image of mouse anus (Day 21) in Embodiment 2.

[0159] FIG. 8 is a HE staining image of mouse intestinal tissues (Day 21) in Embodiment 2.

[0160] FIG. 9 is an immunohistochemical image of tumor tissue immune cells (Day 21) in Embodiment 2.

[0161] FIG. 10 shows tumor-killing related immune-protective responses after administration of Alistipes finegoldii by flow cytometry in Embodiment 2.

[0162] FIG. 11 is a survival curve of mouse models of colon cancer and melanoma administered with different doses of active Alistipes finegoldii in Embodiment 3,

[0163] FIG. 12 is a flowchart of administration of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in mouse model of colon cancer in Embodiment 4.

[0164] FIG. 13 is a comparison diagram of electron micrographs of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in Embodiment 4.

[0165] FIG. 14 is a graph showing changes in tumor volume in mouse model of colon cancer administered with active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in Embodiment 4.

[0166] FIG. 15 is β-diversity PCoA analysis of gut microbiome in mouse model of colon cancer administered with active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in Embodiment 4.

[0167] FIG. 16 is a comparison of remodeling effect on gut microbiome by administration of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in mouse model of colon cancer in Embodiment 4.

[0168] FIG. 17 shows a statistical difference analysis in relative abundance of main bacterial genera (top 20) in mouse model of colon cancer administered with active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in Embodiment 4.

[0169] FIG. 18 is a flowchart of administration of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in mouse model of lung cancer in Embodiment 5.

[0170] FIG. 19 is a graph showing changes in tumor volume in mouse model of lung cancer administered with active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in Embodiment 5.

[0171] FIG. 20 shows tumor weights at the experimental end point after administration of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in mouse model of lung cancer in Embodiment 5.

[0172] FIG. 21 is a tumor image at the experimental end point after administration of active Alistipes finegoldii and inactive whole-cell Alistipes finegoldii in mouse model of lung cancer in Embodiment 5.

[0173] FIG. 22 is relative abundance analysis of Alistipes finegoldii in gut and other body sites of healthy people in Embodiment 6.

[0174] FIG. 23 shows distribution and relative abundance of Alistipes finegoldii in gut of different healthy people and other populations in Embodiment 7.

DETAILED DESCRIPTION

[0175] The present invention will be further described in detail below in conjunction with the accompanying drawings of the description and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

EMBODIMENT 1 USE OF CLINICAL COHORT TO SCREEN GUT MICROBIOTA THAT CAN POTENTIATE IMMUNE CHECKPOINT INHIBITORS

I. Experimental Methods

1. Datasets

[0176] (1) Gut microbiota were obtained from 230 feces samples at different time points after treatment in 108 colorectal cancer (CRC) or esophageal cancer (ESCC) patients. Among them, 115 samples were from immune checkpoint inhibitor group (ICI) patients whose treatment method was anti-PD1 antibody therapy combined with chemotherapy or combined with targeted drugs, and 115 samples were from chemotherapy group (Chemo) patients (Table 1). Efficacy-related assessments mainly include progression-free survival (PFS), survival (OS), and optimal efficacy. We defined patients with the optimal efficacy of complete remission (CR) and partial response (PR) as treatment responder patients (R), and patients with the optimal efficacy of stable disease (SD) and progressive disease (PD) as treatment non-responder patients (NR).

TABLE-US-00002 TABLE 1 Baseline characteristics of patients in the clinical cohort name levels Chemo (N = 59) ICI (N = 49) p Tumor CRC 40 (67.8%) 15 (30.6%) <.001 ESCC 19 (32.2%) 34 (69.4%) Gender Female 15 (25.4%) 10 (20.4%) .699 Male 44 (74.6%) 39 (79.6%) Age Mean ± SD 57.7 ± 8.1 55.8 ± 9.9 272 BMI Mean ± SD 22.2 ± 3.1 22.3 ± 3.3 832 PFStime <6 m 19 (32.2%) 24 (57.1%) .022 >6 m 40 (67.8%) 18 (42.9%) Response NR 20 (33.9%) 19 (38.8%) .746 R 39 (66.1%) 30 (61.2%)

[0177] (2) DNA was extracted from 115 feces samples, and V3-V4 region of the 16S rRNA gene was amplified and sequenced (Illumina HiSeq platform). 16S rRNA sequencing data analysis was performed using USEARCH software (version 11.0.667) for quality control, filtering and species identification. If a patient included feces samples from multiple post-treatment time points, the species abundances of the gut microbiota were averaged at different time points for subsequent analysis. In order to enable the analysis results to be verified by pure culture experiments at a strain level, the LTP (Living Tree Project) database was used for species identification. The LTP database primarily collects sequences of typical strains and isolates classified according to observed traits. In order to improve the accuracy of 16S rRNA sequencing for species-level bacterial identification as much as possible, the SINTAX algorithm was used for species annotation, the algorithm can score and rank the identified species with confidence level, and screen the intestinal bacteria whose species annotation confidence level is greater than 75% for further analysis.

2. Statistical Analysis

[0178] (1) Univariate cox regression analysis of PFS and OS: All bacteria at a genus level of the gut microbiota were subjected to univariate cox regression analysis using the R language package survival (version number: 3.3-1).

[0179] (2) Difference analysis between groups: Wilcoxon rank-sum test was used to analyze statistical differences between groups.

II. Experimental Results

[0180] FIG. 1A shows mean relative abundances of Alistipes in the gut at different time points after treatment in Chemo and ICI responder patients (R) and non-responder patients (NR). In the ICI group, the mean relative abundance of intestinal Alistipes in the R group patients after treatment was significantly higher than that in the NR group patients (p<0.001), while in the Chemo group, the mean relative abundance of intestinal Alistipes in the R group patients after treatment was significantly higher than that in the NR group patients (p<0.001). There was no significant difference between patients in the NR group (p=0.99). This indicated that the higher abundance of Alistipes after treatment was associated with better efficacy of immune checkpoint inhibitors. FIG. 1B shows mean relative abundances of Alistipes in the gut of CRC and ESCC patients at different time points after receiving immune checkpoint inhibitors.

[0181] The results showed that Alistipes showed zero value in 50% of CRC patients after receiving immune checkpoint inhibitors, while Alistipes could be detected in all ESCC patients. Therefore, we divided CRC patients into two groups: Negative (relative abundance of Alistipes equal to zero) and Positive (relative abundance of Alistipes greater than zero) according to whether the mean relative abundance of Alistipes after receiving immune checkpoint inhibitor treatment was zero; ESCC patients were divided into two groups, High (greater than or equal to mean) and low (less than mean) according to the mean relative abundances of Alistipes after receiving immune checkpoint inhibitor treatment.

[0182] FIG. 1C shows a comparison of Kaplan-Meier (KM) curves of progression-free survival (PFS) of CRC patients in Negative group and Positive group. FIG. 1D shows a comparison of Kaplan-Meier (KM) curves of overall survival (OS) of ESCC patients in High group and low group. The results showed that the PFS of CRC patients in Positive group was significantly longer than that in Negative group (log-rank p=0.02); ESCC patients with high mean relative abundance of Alistipes had significantly longer overall survival than patients with low mean relative abundance of Alistipes (log-rank p=0.0071).

[0183] These data suggest that the higher abundance of Alistipes in the gut after receiving immune checkpoint inhibitors is beneficial for the efficacy of immune checkpoint inhibitors.

[0184] FIG. 2 is an analysis of the relationship between different Alistipes strains and immune checkpoint inhibitor treatment efficacy time. In this dataset, a total of 11 OTUs (Operational Taxonomic Units) belonging to Alistipes with a confidence level greater than 0.75 were compared, involving 5 Alistipes species, including 1 Alistipes finegoldii, 4 Alistipes indistinctus, 3 Alistipes onderdonkii, 2 Alistipes shahii and 1 Alistipes timonensis, of which Otu1557 was identified as Alistipes finegoldii with 98% confidence level (Table 2).

[0185] Among the 5 Alistipes species detected, the relative abundances of Alistipes finegoldii, Alistipes timonensis and Alistipes shahii in the gut of CRC patients with PFS greater than 6 months after receiving αPD-1 combined with chemotherapy were significantly higher than those of patients with PFS less than 6 months. The relative abundance of Alistipes finegoldii in the gut of ESCC and CRC patients with PFS greater than 6 months after receiving αPD-1 combined with chemotherapy was significantly higher than that of patients with PFS less than 6 months. The results show that Alistipes finegoldii may have a broader spectrum of potentiating immune checkpoint inhibitors than other Alistipes species.

TABLE-US-00003 TABLE 2 Alistipes species detected in the clinical cohort and their confidence levels OtuID SINTAX annotation (bootstrap confidence values) Otu1557 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—finegoldii(0.9800) Otu150 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—indistinctus(1.0000) Otu194 f: Rikenellaceae(0.9900), g: Alistipes(0.9801), s: Alistipes.sub.—indistinctus(0.8919) Otu379 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—indistinctus(0.9800) Otu942 f: Rikenellaceae(0.9700), g: Alistipes(0.9215), s: Alistipes.sub.—indistinctus(0.8201) Otu14 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—onderdonkii(1.0000) Otu1388 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—onderdonkii(0.8400) Otu2345 f: Rikenellaceae(0.9800), g: Alistipes(0.9604), s: Alistipes.sub.—onderdonkii(0.9028) Otu151 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—shahii(0.9100) Otu365 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—shahii(0.7800) Otu450 f: Rikenellaceae(1.0000), g: Alistipes(1.0000), s: Alistipes.sub.—timonensis(0.9000)

EMBODIMENT 2 COMPARISON OF EFFICACY OF DIFFERENT ACTIVE ALISTIPES STRAINS ALONE OR A COMBINATION OF ACTIVE ALISTIPES STRAINS TO POTENTIATE IMMUNE CHECKPOINT INHIBITORS

I. Experimental Methods

1. Experimental Materials

[0186] (1) Mouse: 6-week-old female C57BL/6J mice

[0187] (2) Tumor cell lines: murine melanoma cell line (B16-OVA, ATCC), murine colon cancer cell line (MC38, ATCC)

[0188] (3) Alistipes species (Alistipes.sp), selected from: [0189] Alistipes finegoldii (DSM No.: 17242, Type strain, which 16S rDNA sequence is shown in SEQ ID NO: 1), referred to as Af, commercially purchased from German collection of microorganisms and cell cultures DSMZ (official website of DSMZ: http://www.dsmz.de). [0190] Alistipes shahii (DSM No.: 19121, Type strain), referred to as As, commercially purchased from German collection of microorganisms and cell cultures DSMZ.

[0191] (4) Bacterial culture medium: liquid DSMZ104 culture medium, which formula mainly includes peptone, yeast extract, beef extract and glucose, etc., commercially purchased from German collection of microorganisms and cell cultures DSMZ.

[0192] (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number G4C2, the reagent was presented by Shanghai Junshi Biomedical Technology Co., Ltd.

[0193] (6) Antibiotic combination: metronidazole 100 mg/kg, vancomycin 50 mg/kg, penicillin sodium 100 mg/kg, and neomycin sulfate 100 mg/kg

2. Experimental Grouping

[0194] The experimental grouping is shown in Table 3.

TABLE-US-00004 TABLE 3 Experimental grouping Mouse Times of Frequency of Cell line Group quantity Dose (each) frequency treatment MC38 IgG 6 200 μg 3 times every two days IgG + 6 IgG: 200 μg, IgG for 3 times, IgG every two Alistipes. sp Alistipes. sp: Alistipes. sp for 6 days, Alistipes. sp 1 × 10.sup.9 CFU times every one day αPD-1 6 200 μg 3 times every two days αPD-1 + 6 αPD-1: 200 μg, αPD-1 for 3 times, αPD-1 every two Alistipes. sp Alistipes. sp: Alistipes. sp for 6 days, Alistipes. sp 1 × 10.sup.9 CFU times every one day B16-OVA IgG 6 200 μg 3 times every two days IgG + 6 IgG: 200 μg, IgG for 3 times, IgG every two Alistipes. sp Alistipes. sp: Alistipes. sp for 6 days, Alistipes. sp 1 × 10.sup.9 CFU times every one day αPD-1 6 200 μg 3 times every two days αPD-1 + 6 αPD-1: 200 μg, αPD-1 for 3 times, αPD-1 every two Alistipes. sp Alistipes. sp: Alistipes. sp for 6 days, Alistipes. sp 1 × 10.sup.9 CFU times every one day Alistipes. sp = 1) Af: Alistipes finegoldii; 2) As: Alistipes shahii; 3) Af + As: Alistipes finegoldii + Alistipes shahii

3. Experimental Steps (Process Shown in FIG. 3)

[0195] (1) Active Alistipes species cultivation: Alistipes species (Af, As or Af+As) was inoculated in DSMZ104 liquid medium, cultured in an anaerobic chamber at 37° C. for 18 hours, and then centrifuged to a concentration of 1×10.sup.10 CFU/ml.

[0196] (2) Tumor cells were inoculated subcutaneously, MC38 cells 1×10.sup.6/mouse, B16-OVA cells 5×10.sup.5/mouse.

[0197] (3) Day 1 to Day 3: the antibiotics combination was administered by gavage to each group of mice to eliminate gut inherent microbiota.

[0198] (4) On Day 5, Day 8 and Day 11 respectively, IgG or αPD-1 was injected intraperitoneally, 200 μg/mouse.

[0199] (5) On Day 5, Day 7, Day 9, Day 11, Day 13, and Day 15 respectively, active Alistipe species were administered by gavage for treatment, 100 μl/mouse, dosage of monobacterial Af or As is: 1×10.sup.9 CFU/mouse. A combined dosage of Af+As is: Af: 0.5×10.sup.9 CFU+As: 0.5×10.sup.9 CFU/mouse.

[0200] (6) On Day 5, Day 8, Day 11, Day 14, Day 17 and Day 21 respectively, a tumor size was measured and a tumor volume was calculated.

[00001]$\mathrm{Tumor}\mathrm{volume}=\frac{\mathrm{tumor}{\mathrm{width}}^2\times \mathrm{tumor}\mathrm{length}}{2}$

[0201] (7) The mice were euthanized on Day 21, tumor tissues were taken out, photographed and weighed, and intestinal tissues were taken for HE staining to confirm enteritis condition.

[0202] The mouse tumor volume was measured, the tumor weight at the endpoint was measured, an immunohistochemical evaluation was used to evaluate effect of immune cell infiltration in tumor tissues.

[0203] Mouse anal and intestinal tissue sections stained with HE were used to observe and evaluate whether Alistipes finegoldii would cause enteritis for safety evaluation.

[0204] Flow cytometry was used to detect tumor-killing-associated immune cells in mouse blood to assess systemic anti-tumor immune responses.

II. Experimental Results

[0205] FIG. 4 shows the tumors on Day 21, FIG. 5 is the change curve in tumor volume and FIG. 6 is the statistics graph of tumor weight. In a MC38 colon cancer and B16-OVA melanoma mouse models, compared with non-treatment group (IgG), monotherapy group (αPD-1) shows obvious and significant (p<0.01) tumor reduction. Compared with the monotherapy group (αPD-1), only active Af combination therapy group (αPD-1+Af) shows significant (p<0.01) tumor reduction, however there was no statistical difference between active As combination therapy group (αPD-1+As) and active Af+As combination therapy group (αPD-1+Af+As) and monotherapy group (αPD-1).

[0206] It proves that Alistipes finegoldii can enhance the anti-tumor effect of αPD-1. However, Alistipes shahii as well as combination of Alistipes shahii and Alistipes finegoldii (Af+As) could not enhance the anti-tumor effect of αPD-1.

[0207] In addition, there is no difference between the tumors of mice in the Alistipes finegoldii mono active bacterium treatment group (IgG+Af) and the non-treatment group (IgG), indicating that the anti-tumor effect of Alistipes finegoldii depends on αPD-1.

[0208] The images of mouse anus in FIG. 7 and the HE staining images of the intestinal tissue section in FIG. 8 show that the mice in the mono active Af therapy group (IgG+Af) and the Af combination therapy group (αPD-1+Af) are not found to have enteritis, which proves the safety of Alistipes finegoldii via gastrointestinal administration.

[0209] Immunohistochemistry of the tumor tissue of the MC38 mouse model at the experiment end in FIG. 9 shows that the active Af combination therapy group (IgG+Af) could significantly increase the infiltration of CD4+ T helper cell in the interstitial area of the tumor; compared with the monotherapy group (αPD-1), the active Af combination therapy group (αPD-1+Af) shows an increased trend of CD4+ T helper cell infiltration in the tumor interstitial area, but does not reach a statistical difference. Compared with the non-treatment group (IgG), the CD4+ T helper cell infiltration of the monotherapygroup (αPD-1) at the end point of mouse experiment only has an increased trend, but does not reach a statistical difference. This result shows that the effect of monotherapy (αPD-1) and active Af combination therapy (αPD-1+Af) on immune cell infiltration in mouse tumor tissue may be short-term, and no significant difference can be detected in the tumor samples at the end of the experiment. The effect of Alistipes finegoldii mono active bacterium treatment (IgG+Af) on immune cell infiltration in mouse tumor tissue lasts for a longer time, and there is still a significant increase in CD4+ T helper cell infiltration after the treatment is stopped for one week, which proves oral administration of Alistipes finegoldii monobacterium has a regulatory effect on tumor immune microenvironment.

[0210] FIG. 10 shows use of flow cytometry for detection of tumor-killing associated immune cells ie, abundancy of granzyme-positive CD8.sup.+ T cells, granzyme-positive NK cells and IL-6.sup.+ neutrophils, in MC38 mouse model. Results show that compared with the monotherapy group (αPD-1), active Af combination group (αPD-1+Af)significantly increases relative abundance of granzyme-positive CD8.sup.+ T cells, granzyme-positive NK cells and IL-6.sup.+ neutrophils, demonstrating that active Alistipes finegoldii combined with αPD-1 enhances the anti-tumor effect of the immune system.

EMBODIMENT 3 EFFECT OF ACTIVE ALISTIPES FINEGOLDII COMBINED WITH DIFFERENT IMMUNE CHECKPOINT INHIBITORS ON PROLONGATION OF OVERALL SURVIVAL

I. Experimental Methods

1. Experimental Materials

[0211] (1) Mice: 6-week-old female C57BL/6J mice

[0212] (2) Tumor cell lines: murine melanoma cell line (B16-OVA, ATCC), murine colon cancer cell line (MC38, ATCC)

[0213] (3) Alistipes finegoldii (DSM No.: 17242, Type strain, which 16S rDNA sequence is shown in SEQ ID NO: 1), referred to as Af, commercially purchased from DSMZ German National Culture Collection.

[0214] (4) Bacterial culture medium: liquid DSMZ104 culture medium, which formula mainly includes peptone, yeast extract, beef extract and glucose, etc., commercially purchased from DSMZ German National Culture Collection.

[0215] (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number G4C2, the reagent was presented by Shanghai Junshi Biomedical Technology Co., Ltd. CTLA4 monoclonal antibody (αCTLA4), clone number 9D9, was purchased from BioXcell, USA.

[0216] (6) Antibiotic combination: metronidazole 100 mg/kg, vancomycin 50 mg/kg, penicillin sodium 100 mg/kg, and neomycin sulfate 100 mg/kg

2. Experimental Grouping

[0217] The experimental grouping is shown in Table 4.

TABLE-US-00005 TABLE 4 Experimental grouping Mouse Frequency of Cell line Group quantity Dose (each) treatment MC38 IgG 6 200 μg every two days IgG + Af-low 6 IgG: 200 μg, Af: IgG every two days, 1 × 10.sup.9 CFU Af every one day IgG + Af -medium 6 IgG: 200 μg, Af: IgG every two days, 2 × 10.sup.9 CFU Af every one day IgG + Af-high 6 IgG: 200 μg, Af: IgG every two days, 4 × 10.sup.9 CFU Af every one day αPD-1/αCTLA4 6 200 μg every two days αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-low Af: 1 × 10.sup.9 CFU days, Af every one day αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-medium Af: 2 × 10.sup.9 CFU days, Af every one day αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-high Af: 4 × 10.sup.9 CFU days, Af every one day B16-OVA IgG 6 200 μg every two days IgG + Af-low 6 IgG: 200 μg, Af: IgG every two days, 1 × 10.sup.9 CFU Af every one day IgG + Af -medium 6 IgG: 200 μg, Af: IgG every two days, 2 × 10.sup.9 CFU Af every one day IgG + Af-high 6 IgG: 200 μg, Af: IgG every two days, 4 × 10.sup.9 CFU Af every one day αPD-1/αCTLA4 6 200 μg every two days αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-low Af: 1 × 10.sup.9 CFU days, Af every one day αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-medium Af: 2 × 10.sup.9 CFU days, Af every one day αPD-1/αCTLA4 + 6 αPD-1/αCTLA4: 200 μg, αPD-1/αCTLA4 every two Af-high Af: 4 × 10.sup.9 CFU days, Af every one day

3. Experimental Steps

[0218] (1) Active Alistipes finegoldii cultivation: Alistipes finegoldii was inoculated in DSMZ104 liquid medium, cultured in an anaerobic chamber at 37° C. for 18 hours, and then centrifuged to a concentration of 1×10.sup.10 CFU/ml.

[0219] (2) Tumor cells were inoculated subcutaneously, MC38 cells 1×10.sup.6/mouse, B16-OVA cells 5×10.sup.5/mouse.

[0220] (3) Day 1 to Day 3: the antibiotics combination was administered by gavage to each group of mice to eliminate intestinal inherent flora.

[0221] (4) Starting from Day 5, every 3 days, IgG or αPD-1 or αCTLA4 was intraperitoneally injected, 200 μg/mouse.

[0222] (5) Starting from Day 5, every 2 days, different doses of active Alistipes finegoldii were administered by gavage for treatment, wherein a low dose group (low) was 100 μl/mouse, 1×10.sup.9 CFU/mouse, and a medium dose group (medium) was 200 μl/mouse, 2×10.sup.9 CFU/mouse, and a high dose group (high) was 400 μl/mouse, 4×10.sup.9 CFU/mouse.

[0223] (6) Starting from Day 5, every 3 days, a tumor size was masured and a tumor volume was calculated.

[00002]$\mathrm{Tumor}\mathrm{volume}=\frac{\mathrm{tumor}{\mathrm{width}}^2\times \mathrm{tumor}\mathrm{length}}{2}$

[0224] (7) A treatment cycle was until the mouse tumor grew to a size (2000 mm.sup.3) and then the mouse was euthanized or the mouse did not reach the ethical size of the tumor and died spontaneously.

[0225] (8) A death status and time of each mouse was recorded, and a survival curve chart was drawn.

II. Experimental Results

[0226] FIG. 11 shows the survival curve of mice. In MC38 colon cancer and B16-OVA melanoma mouse models, compared with the single-agent immune checkpoint inhibitor group (αPD-1), the combination therapy groups (αPD-1+Af) wth high dose (high), medium dose (medium), and a low dose (low) of Alistipes finegoldii can all significantly increase survival time of tumor-bearing mice. In the B16-OVA melanoma mouse model, Alistipes finegoldii also shows the effect of promoting the efficacy of αCTLA4 immune checkpoint inhibitor: αCTLA4 combined with high, medium and low doses of Alistipes finegoldii can all significantly the increase survival time of the tumor-bearing mice. However, in the MC38 colon cancer mouse model, the effect of αCTLA4 immune checkpoint inhibitor is too strong, and a synergistic effect of Alistipes finegoldii on αCTLA4 is not observed.

EMBODIMENT 4 ADMINISTRATION OF ACTIVE AND INACTIVE WHOLE-CELL ALISTIPES FINEGOLDII COMBINED WITH IMMUNE CHECKPOINT INHIBITORS TO TREAT COLON CANCER

I. Experimental Methods

1. Experimental Materials

[0227] (1) Mice: 6-week-old female C57BL/6J mice

[0228] (2) Tumor cell lines: murine colon cancer cell line (MC38, ATCC)

[0229] (3) Strain information: Alistipes finegoldii (DSM No.: 17242, Type strain, which 16S rDNA sequence is shown in SEQ ID NO: 1), referred to as Af or Af, commercially purchased from German National Culture Collection DSMZ (official website of DSMZ: http://www.dsmz.de).

[0230] (4) Medium components: liquid DSMZ104 medium, the formula mainly includes peptone, yeast extract, beef extract and glucose, etc., commercially purchased from DSMZ German National Culture Collection.

[0231] (5) Immune checkpoint inhibitors: PD-1 monoclonal antibody (αPD-1), clone number G4C2, the reagent was presented by Shanghai Junshi Biomedical Technology Co., Ltd.

[0232] (6) Antibiotic combination: metronidazole 100 mg/kg, vancomycin 50 mg/kg, penicillin sodium 100 mg/kg, neomycin sulfate 100 mg/kg.

2. Experimental Grouping

[0233] The experimental grouping is shown in Table 5.

TABLE-US-00006 TABLE 5 Experimental grouping Mouse Frequency of Cell line Group quantity Dose (each) treatments MC38 colon Non-treatment group (PBS) 10 200 μl 3 times cancer Single-agent therapy group 10 200 μg 3 times (αPD-1) Active combination therapy 7 αPD-1: 200 μg, αPD-1 for 3 times, group (αPD-1 + Af) active Af: active Af for 5 times 1 × 10.sup.9 CFU Inactive whole-cell 7 αPD-1: 200 μg, αPD-1 for 3 times, combination therapy group inactive whole-cell inactive whole-cell (αPD-1 + Af_heat killed) Af: 1 × 10.sup.9 CFU Af for 5 times Note: Af means Alistipes finegoldii

3. Experimental Steps (Process Shown in FIG. 12)

[0234] (1) Preparation of active Af (Af): Alistipes finegoldii was inoculated in DSMZ104 liquid medium, cultured in an anaerobic chamber at 37° C. for 18 hours, and then centrifuged to a concentration of 1×10.sup.10 CFU/ml. Bacterial cells were washed and concentrated with phosphate buffered saline (PBS) for three times, till the residual medium was washed away.

[0235] (2) Preparation of inactive whole-cell Af (heat killed, Af_heat killed): Bacterial cells prepared in (1) were washed and concentrated with phosphate buffered saline (PBS), and heated at a high temperature of 95° C. for 5 minutes.

[0236] (3) Subcutaneous inoculation of tumor cells: MC38 cells 1×10.sup.6/mouse,

[0237] (4) Antibiotic treatment: the antibiotics combination was administered by gavage to each group of mice to eliminate intestinal inherent flora, the treatment was for 7 days.

[0238] (5) Group treatment: On Day 6, Day 9 and Day 12 respectively, phosphate buffered saline (PBS) or αPD-1 was injected intraperitoneally, 200 μg/mouse. On Day 5, Day 7, Day 9, Day 11, and Day 13 respectively, active Af was administered by gavage or inactive whole-cell Af was orally administered for treatment, 100 μl/mouse, 1×10.sup.9 CFU/mouse. On Day 0, Day 5, Day 7, Day 9, Day 11, Day 13, Day 16, Day 19, Day 22 and Day 25 respectively, a tumor size was measured and a tumor volume was calculated.

[0239] (6) Tumor volume measurement calculation formula:

[00003]$\mathrm{Tumor}\mathrm{volume}=\frac{\mathrm{tumor}{\mathrm{width}}^2\times \mathrm{tumor}\mathrm{length}}{2}$

[0240] (7) Intestinal contents of mice were collected at the endpoint (Day 25), and composition of the gut microbiota of mice was analyzed by 16S rRNA gene sequencing, and effects of treatment of colon cancer with active Af and inactive whole-cell Af on intestinal microbiota in mice were compared.

II. Experimental Results

[0241] FIG. 13 is a comparison diagram of electron micrographs of active Af and inactive whole-cell Af. It can be seen that inactive whole-cell treatment can maintain the whole cell integrity of Af cells, indicating that the component of inactive whole-cell Af that exerts an anti-tumor effect is derived from the whole-cell component.

[0242] FIG. 14 is the changes of tumor volume during the treatment of colon cancer with active Af and inactive whole-cell Af. From the comparison of tumor volume changes, in MC38 colon cancer model, compared with the non-treatment group (PBS), the single-agent immune checkpoint inhibitor treatment group (αPD-1) did not show significant (p=0.055) tumor reduction, indicating that colon cancer in this embodiment was resistant to single-agent αPD-1 therapy, while αPD-1 combined with active RX-af01 or inactive whole-cell Af shows significant (***: p<0.001) tumor reduction over the single-agent αPD-1 group after treatment. Comparing therapeutic effects of active Af and inactive whole-cell Af combined with immune checkpoint inhibitor, αPD-1 combined with inactive whole-cell Af shows significant (***: p<0.001) tumor reduction over αPD-1 combined with active Af.

[0243] The above results demonstrate that both active Af and inactive whole-cell Af can enhance the anti-tumor effect of immune checkpoint inhibitors. When single-agent αPD-1 is ineffective or resistant, simultaneous administration of αPD-1 combined with active Af or inactive whole-cell Af can reverse the resistance of colon cancer to single-agent αPD-1 treatment, therefore both active Af and inactive whole-cell Af have efficacy to treat, reduce, inhibit or control immune checkpoint inhibitors-refractory tumors, and inactive whole-cell Af may have better anti-tumor effect than active Af.

[0244] FIG. 15 is a principal coordinate analysis of β-diversity PCoA (principal co-ordinates analysis) of gut mcirobiome in mouse models of colon cancer treated with active Af and inactive whole-cell Af. The β-diversity of gut mcirobiome refers to composition differences in the overall gut environment bacteria between different intestinal environments; statistical differences between the two groups were measured using Anosim analysis method (Analysis of similarities), Anosim analysis is a nonparametric test method based on permutation test and rank sum test, which is used to test whether the difference between groups is significantly greater than the difference within the group, so as to judge whether the grouping is meaningful. The Anosim method mainly has two numerical results: one is R, its range is [−1, 1], which is used to judge whether there is a difference between different groups, R>0 means that the difference between groups is greater than the difference within the group, R<0 means that the difference between groups is less than the difference within the group, the closer the R value is to 1, the greater the difference between groups; the other is p, which is used to indicate whether there is a significant difference between groups.

[0245] Results show that there is a significant and large (R=0.6950, p=0.001) difference in the β-diversity of gut microbiome in mouse models of colon cancer between the treatment of active Af and inactive whole-cell Af. It is demonstrated that treatment of colon cancer with active Af and inactive whole-cell Af can lead to significant and large differences in the composition of the overall gut microbiota, which in turn leads to large differences in the immune status of the gut microbiome. Therefore, in Embodiment 5, the difference in the anti-tumor effects of inactive whole-cell Af and active Af may be related to their different roles of gut microbial remodeling, that is, the reconstruced gut microbiota induced by inactive whole-cell Af combined with αPD-1 correlates with enhanced anti-tumor immune function.

[0246] FIG. 16 is gut microbiome composition at genus level between the treatment of colon cancer with active Af and inactive whole-cell Af. FIG. 17 shows Wilcoxon rank-sum test statistical difference analysis of the top 20 bacterial genera in the relative abundance of gut microbiome in colon cancer model treated with active Af and inactive whole-cell Af.

[0247] Results show that among the top 20 bacterial genera of the gut microbiome in colon cancer model, 8 genera were showed significant statistical differences between the treatments of active Af and inactive whole-cell Af. Among them, the genus Bacteroides was showed the largest difference, and the relative abundance of Bacteroides in the active Af treatment group was significantly higher than that in the inactive Af treatment group; another prominent difference was for Desulfovibrio, which was only present in the inactive whole-cell combination therapy group (αPD-1+Af_heat killed). The relative abundance of Alistipes, to which Af belongs, was significantly (p=0.0156) higher in the active combination therapy group than that in the inactive whole-cell combination therapy group. These results demonstrated that treatment of colon cancer with active Af and inactive whole-cell Af lead to significant differences in the gut microbiome composition at the genus level, further resulted in differential intestinal mucosal immune status. Therefore, in Embodiment 4, differences in the anti-tumor effects of active whole-cell Af and inactive whole-cell Af correlates with specific enrichment or weakened of certain bacterial genera in the gut, thereby forming differential intestinal mucosal immune status and stimulating differential anti-tumor immune surveillance.

EMBODIMENT 5 ADMINISTRATION OF ACTIVE AND INACTIVE WHOLE-CELL ALISTIPES FINEGOLDII COMBINED WITH IMMUNE CHECKPOINT INHIBITORS TO TREAT LUNG CANCER

I. Experimental Methods

1. Experimental Materials

[0248] (1) Mice: 6-week-old female C57BL/6J mice

[0249] (2) Tumor cell line: mouse lung cancer cell line (LLC, ATCC)

[0250] (3) Af strain information: Alistipes finegoldii (DSM No.: 17242, Type strain, which 16S rDNA sequence is shown in SEQ ID NO: 1), commercially available from German National Culture Collection DSMZ (official website of DSMZ: http://www.dsmz.de).

[0251] (4) Medium components: liquid DSMZ104 medium, the formula mainly includes peptone, yeast extract, beef extract and glucose, etc., commercially purchased from DSMZ German National Culture Collection.

[0252] (5) Immune checkpoint inhibitor: PD-1 monoclonal antibody (αPD-1), clone number G4C2, the reagent was presented by Shanghai Junshi Biomedical Technology Co., Ltd.

[0253] (6) Antibiotic combination: metronidazole 100 mg/kg, vancomycin 50 mg/kg, penicillin sodium 100 mg/kg, neomycin sulfate 100 mg/kg

II. Experimental Grouping

[0254] The experimental grouping is shown in Table 6.

TABLE-US-00007 TABLE 6 Experimental grouping Mouse Frequency of Cell line Group quantity Dose (each) treatments LLC lung Non-treatment group (PBS) 6 150 μl 4 times cancer Single-agent therapy group 6 150 μg 4 times (αPD-1) Active combination therapy 6 αPD-1: 200 μg, αPD-1 for 4 times, group (αPD-1 + Af) active Af: active Af for 8 times 1 × 10.sup.9 CFU Inactive whole-cell 6 αPD-1: 150 μg αPD-1 for 4 times, combination therapy group inactive whole-cell inactive whole-cell (αPD-1 + Af_heat killed) Af: 1 × 10.sup.9 CFU Af for 8 times

3. Experimental Steps (Process Shown in FIG. 18)

[0255] (1) Preparation of active Af: Alistipes finegoldii was inoculated in DSMZ104 liquid medium, cultured in an anaerobic chamber at 37° C. for 18 hours, and then centrifuged to a concentration of 1×10.sup.10 CFU/ml. Bacterial cells were washed and concentrated with phosphate buffered saline (PBS) for three times, till the residual medium was washed away.

[0256] (2) Preparation of inactive whole-cell Af (heat killed, Af_heat killed): Bacterial cells prepared in (1) were washed and concentrated with phosphate buffered saline (PBS), and heated at a high temperature of 95° C. for 5 minutes.

[0257] (3) Subcutaneous inoculation of tumor cells: LLC cell line 1×10.sup.6/mouse,

[0258] (4) Antibiotic treatment: the antibiotics combination was administered by gavage to each group of mice to eliminate intestinal inherent flora, the treatment was for 7 days.

[0259] (5) Group treatment: On Day 6, Day 9 and Day 12 respectively, phosphate buffered saline (PBS) or αPD-1 was injected intraperitoneally, 150 μg/mouse. On Day 5, Day 7, Day 9, Day 11, Day 13, Day 15, Day 17 and Day 19 respectively, active Af or inactive whole-cell Af was orally administered by gavage for treatment, 100 μl/mouse, 1×10.sup.9 CFU/mouse. On Day 0, Day 5, Day 7, Day 10, Day 13, Day 16, Day 19, Day 22, Day 25 and Day 28 respectively, a tumor size was measured and a tumor volume was calculated.

[0260] (6) Tumor volume measurement calculation formula:

[00004]$\mathrm{Tumor}\mathrm{volume}=\frac{\mathrm{tumor}{\mathrm{width}}^2\times \mathrm{tumor}\mathrm{length}}{2}$

[0261] (7) A tumor weight was measured at the endpoint (Day 28), and group statistics were performed.

II. Experimental Results

[0262] FIG. 19 is the changes of tumor volume in lung cancer mouse models administered with active Af and inactive whole-cell Af. The single-agent immune checkpoint inhibitor group (αPD-1) did not show significant (ns: p>0.05) tumor reduction compared with the non-treatment group (PBS), indicating that the single-agent immune checkpoint inhibitor group (αPD-1) in this embodiment was less effective in this LLC mouse lung cancer models and resistant to single-agent αPD-1 therapy; treatment with αPD-1 combined with active Af (αPD-1+Af) or αPD-1 combined with inactive whole-cell Af (αPD-1+Af_heat killed) both showed significant (αPD-1+Af: **p<0.01; αPD-1+Af_heat killed: * ***p<0.0001) tumor reduction compared with non-treatment group (PBS); compared with the single-agent immune checkpoint inhibitor group (αPD-1), treatment with αPD-1 combined with active Af (αPD-1+RX -af01) or αPD-1 combined with inactive whole-cell Af (αPD-1+Af_heat killed) groups both showed significantly (αPD-1+Af: *p<0.05; αPD-1+RX-af01_heat killed: ****p<0.0001) better tumor treatment effect compared with the single-agent αPD-1; αPD-1 combined with active Af (αPD-1+Af) and αPD-1 combined with inactive whole-cell Af (αPD-1+Af_heat killed) show better tumor treatment effect compared with he single-agent αPD-1; comparing the treatment effects of αPD-1 combined with active Af (αPD-1+RX -af01) or αPD-1 combined with inactive whole-cell Af (αPD-1+Af_heat killed), inactive whole-cell Af shows a trend toward superior efficacy over active Af, but des not reach a statistical significance (ns: p>0.05). FIG. 20 is a tumor weight at the endpoint of treatment in LLC mouse model with active Af and inactive whole-cell Af. The tumor weight at the experimental endpoint shows that compared with non-treatment group (PBS), the single-agent immune checkpoint inhibitor group (αPD-1) does not show significant (ns: p>0.05) tumor reduction. αPD-1 combined with active Af treatment does not show a significantly (ns: p>0.05) superior therapeutic effect over single-agent αPD-1 treatment, while αPD-1 combined with inactive whole-cell Af treatment shows a significantly (**p<0.01) superior therapeutic effect over single-agent αPD-1 treatment. Comparing the therapeutic effects of αPD-1 combined with active Af and αPD-1 combined with inactive whole-cell Af, the average weight of inactive whole-cell Af group is lower than that of active Af group. The results demonstrate that both active Af and inactive whole-cell Af can enhance the anti-tumor effect of immune checkpoint inhibitors. When single-agent αPD-1 is ineffective or resistant, concurrent administration of αPD-1 in combination with active Af or inactive whole-cell Af can reverse lung cancer resistance to single-agent αPD-1 therapy, therefore both active Af and inactive whole-cell Af have potency to treat, reduce, inhibit or control immune checkpoint inhibitor-refractory tumors, and the anti-tumor effect of inactive whole-cell Af is superior to that of active Af.

[0263] FIG. 21 is the tumor images at the endpoint of the treatment with active Af and inactive whole-cell Af in LLC mouse model, there is one mouse with complete tumor disappearance in the active combination therapy group (αPD-1+Af) and the inactive whole-cell combination therapy group (αPD-1+Af_heat killed). It is demonstrated that both active Af and inactive whole-cell Af could enhance the anti-tumor effect of immune checkpoint inhibitors.

EMBODIMENT 6 ANALYSIS OF THE RELATIVE ABUNDANCE OF ALISTIPES FINEGOLDII IN THE GUT AND OTHER BODY SITES OF HEALTHY PEOPLE

I. Implementation Methods and Steps

[0264] (1) Source of original metagenomic data: metagenomic sequencing data of the public data resources of National Institutes of Health (NIH) Human Microbiome Project (HMP, https://www.hmpdacc.org/) were used.

[0265] (2) Bacterial classification and strain-level identification and analysis software: MetaPhlAn2 and StrainPhlAn (https://github.com/biobakery/metaphlan2, https://github.com/biobakery/metaphlan). Combining MetaPhlAn2 and StrainPhlAn can perform strain-level identification and analysis of metagenomic data. Default settings were used when using these two softwares.

[0266] (3) After obtaining the relative abundance of strains (the ratio of a certain strain to the total microbial population) from (2), the abundance of Af in different parts of the body was visualized.

II. Implementation Results

[0267] FIG. 22 shows the relative abundance of Af in the gut and other body sites of healthy people. Analysis of 2335 samples from 4 body sites of healthy people (553 fecal samples, 1259 oral samples, 309 skin samples, 234 vaginal samples) show that Af mainly exists in fecal samples, The positive detection rate in the feces is 73.6% ( 407/553). In oral, skin and vaginal samples, a detection rate of Af is 1.4% ( 18/1259), 1.9% ( 6/309) and 1.7% ( 4/234), respectively. The relative abundance of Af in 407 Af-positive fecal samples range from 0.00006% to 9.0%. According to the latest estimates of the number of gut microbiota, the total number of bacteria contained in the intestinal tract of a healthy adult male weighing 70 kg is about 3.8*1013, and the number of Af in the intestinal tract of healthy people in the HMP database is about 106-1013(Sender, Fuchs et al. 2016). Thus, Af interacts with human body as a symbiotic bacterium in intestine. The relative abundance of Af is the dominant species (defined as a bacterium with its relative abundance more than 1% in gut microbiome) in some individuals, which proves the safety of Af with the administration dose of 105-1012 in the present invention.

EMBODIMENT 7 ANALYSIS OF RELATIVE ABUNDANCE OF ALISTIPES FINEGOLDII IN THE GUT OF HEALTHY PEOPLE AND OTHER DISEASED POPULATIONS

I. Experimental Methods

[0268] Four gut metagenomic datasets from human were analyzed, containing 1,396 human feces samples, involving 9 cohorts with different health status. Metagenomic sequencing technology can reach an accuracy of bacterial species.

[0269] Nine different health status: 1) healthy adults; 2) patients with colorectal adenoma; 3) patients with colorectal cancer; 4) patients underwent resection for colorectal cancer ; 5) patients with atherosclerosis; 6) patients with non-small cell lung cancer before receiving immune checkpoint inhibitors (ICIs); 7) patients with non-small cell lung cancer after receiving immune checkpoint inhibitors (ICIs); 8) patients with renal cell carcinoma before receiving immune checkpoint inhibitors (ICIs); 9) patients with renal cell carcinoma after receiving immune checkpoint inhibitors (ICIs).

[0270] The specific situation of the data sets is shown in Table 7.

TABLE-US-00008 TABLE 7 Public data set of human intestinal metagenome Datasets Group Number Yachida_2019 (n = 347) Colorectal adenocarcinoma (Adenoma) 40 Colorectal cancer (CRC) 150 Healthy adults (Healthy) 127 Colorectal cancer after surgery (History_surgery) 30 PRJEB27928 (n = 575) Colorectal cancer (CRC) 285 Healthy adult (Healthy) 290 PRJEB22863 (n = 219) Non-small cell carcinoma before treatment 65 (Baseline_NSCLC) Renal cell carcinoma before treatment of 62 (Baseline_RCC) Non-small cell carcinoma after ICIs treatment of 53 (ICIs_treat_NSCLC) Renal cell carcinoma after ICIs treatment of 39 (ICIs_treat_RCC) VinodK.Gupta_2020 (n = 255) Atherosclerosis (ACVD) 152 Colorectal adenocarcinoma (Adenoma) 42 Colorectal cancer (CRC) 61

II. Experimental Results

[0271] Results in FIG. 23 and Table 8 show that Alistipes finegoldii (Af) exists in different populations with a relative abundance of 0.001 to 0.07%, and the relative abundances of Af in different populations have differences, where relative abundance refers to a proportion of a certain bacterial species to all bacterial species in the intestinal tract. According to an estimation that a total number of bacterial species contained in the human intestinal tract is about 10.sup.14 CFU/ml, the number of Alistipes finegoldii in the human intestinal tract in these groups is about 10.sup.9 CFU/ml to 10.sup.10 CFU/ml.

TABLE-US-00009 TABLE 8 Relative abundances of Af in different populations Group Relative abundance (%) Healthy 0.001361633 History_surgery 0.003098462 ICIs_treat_RCC 0.015572821 Baseline_RCC 0.022339194 Adenoma 0.024956603 Baseline_NSCLC 0.026339538 ICIs_treat_NSCLC 0.028744906 CRC 0.042939371 ACVD 0.078639934

[0272] Finally, it shall be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. For those of ordinary skill in the art, on the basis of the above description and ideas, other variations or changes can be further made in different forms and it is not necessary and impossible to enumerate all the implementations here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

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