A combined preparation for regulating intestinal microecology for preparing a drug that enhances the effect of oncolytic virus immunotherapy

Abstract

The present invention relates to the field of preparations and methods for combined synergistic enhancement of oncolytic virus immunotherapy, and specifically to a preparation for regulating intestinal microecology and its use in the preparation of drugs that enhance the immunotherapy effect of oncolytic viruses and anti-tumor immune responses. The present invention discloses a mixed preparation that uses an antibiotic composition to remove intestinal sensitive bacteria and fungi. The preparation is used for oral administration to regulate the composition of intestinal flora, that is, to regulate the intestinal flora. Changes in intestinal microecology further affect the oncolytic virus-mediated anti-tumor immune response and tumor immunotherapy effect. Specifically, drinking deionized water containing mixed antibiotics can inhibit the growth and reproduction of sensitive microorganisms in the intestines of mice, accompanied by significant changes in intestinal flora, and further significantly improve the tumor immunotherapy effect of oncolytic viruses. Therefore, mixed antibiotics can be used to prepare drugs that enhance the immunotherapy effect of oncolytic viruses.

Description

Technical Field

[0001] The present invention relates to the field of oncolytic virus tumor immunotherapy, and in particular to the use of a preparation that regulates the composition of intestinal flora, i.e., regulates the intestinal microecology, to regulate the tumor immunotherapy effect of oncolytic viruses, and specifically to the use of a preparation that regulates the intestinal microecology in the preparation of a drug that enhances the immunotherapy effect of oncolytic viruses. Background Art

[0002] Tumor is one of the major diseases that threaten human health and life. In 2020, the number of newly diagnosed cancer cases worldwide reached 19.3 million, causing nearly 10 million deaths. It is expected that the incidence and mortality of patients with advanced cancer will increase significantly in the next few years. In addition, the trend of increasing cancer cases year by year will be inevitable in the next 20 years. It is estimated that by 2040, the number of confirmed cancer cases worldwide is expected to reach 28.4 million, an increase of 47% from 2020. Therefore, the prevention and treatment of tumors has become one of the most difficult and hot issues in the world. Tumor treatment includes chemotherapy, radiotherapy, surgical treatment and immunotherapy. With the development and progress of medicine, these methods have achieved remarkable achievements and progress.

[0003] 1. The application prospects of tumor immunotherapy are promising

[0004] Tumor immunotherapy is the fastest growing direction in the field of tumor treatment. The main tumor immunotherapy currently includes immune checkpoint inhibitor therapy, adoptive cell immunotherapy, neoantigen tumor vaccine therapy and oncolytic virus therapy. At present, the FDA alone has approved more than 50 immune checkpoint inhibitors for the treatment of human cancer, and their indications have been extended to many different tumor types. In terms of adoptive cell immunotherapy, especially CAR-T cell therapy, it has shown excellent therapeutic effects in patients with hematological malignancies and metastatic melanoma. In addition, neoantigen tumor vaccine therapy developed based on modern bioinformatics has demonstrated its clinical therapeutic value for patients with melanoma, glioma, etc. in strict phase I and II clinical trials. These research results have shown the world the great application value of tumor immunotherapy. However, a large number of studies have shown that tumor immunotherapy applied to the treatment of solid tumors in different patients will produce different treatment results. Some patients will achieve significant and lasting improvement in their condition, but some patients will have no effect at all. It can be seen that tumor immunotherapy has the problem of large individual response differences (low overall response rate) in solid tumors.

[0005] 2. The individual responses of tumor immune checkpoint inhibitors, adoptive cellular immunotherapy, and neoantigen tumor vaccine therapy on solid tumors vary greatly (the overall response rate is not high) and are associated with the patient's intestinal flora

[0006] There are many factors that affect the individual differences and overall response rate of immunotherapy for solid tumors. There is increasing evidence that host factors, including the host's intestinal flora, may affect the tumor's response to immunotherapy. Lee SHetal analyzed nearly 100 NSCLC patients who received different tumor treatments and classified them into responders and non-responders. After 16SrRNA sequencing of fecal samples from these two groups, it was found that Bifidobacterium bifidum in the feces of responding patients was significantly enriched compared with non-responding patients. In another group of metagenomic sequencing of feces from NSCLC and RCC patients, it was also found that the intestinal flora composition of patients with better and worse responses to immunotherapy targeting PD-1 was significantly different. In preclinical mouse models, multiple studies have found that the intestinal flora composition of different strains of mice is different, and there are also significant differences in the effects of tumor immunotherapy. Therefore, the individual response variability of tumor immunotherapy (the overall response rate is not high) is associated with the patient's intestinal flora. However, to date, the association between the individual differences of oncolytic virus-mediated tumor immunotherapy and the patient's intestinal flora has not been revealed.

[0007] 3. Oncolytic virus-mediated tumor immunotherapy

[0008] Oncolytic virus therapy, one of the important branches of tumor immunotherapy, has received increasing attention in recent years. Oncolytic viruses are viruses with anti-tumor effects developed by using the characteristics of certain viruses that can selectively infect and destroy cancerous tissues without damaging normal tissues. At present, the anti-tumor mechanism of oncolytic viruses includes two aspects. First, after the virus infects and enters the target cell, its genome "usurps" the biosynthetic pathway of the target cell to produce progeny viruses. After reaching a certain level, the infected cells are lysed, causing tumor cell death. In addition, oncolytic viruses can also inhibit tumor cells by destroying tumor blood vessels, expressing proteins encoded by modified genes, and inducing systemic anti-tumor immune responses. In 2015, the FDA approved the genetically modified oncolytic herpes simplex virus T-Vec for marketing, highlighting the value of oncolytic viruses in clinical applications. At present, many types of oncolytic viruses have entered the clinical research stage, including adenovirus, herpes virus, vaccinia virus and reovirus. At present, replicating oncolytic adenoviruses occupy the top two positions among the oncolytic viruses under development.

[0009] Similar to the performance of other immunotherapies on solid tumors, the therapeutic effects of oncolytic viruses also have strong individual response differences. Several clinical studies have shown that the activation state of individual patients' immune cells, the secretion of cytokines, and the expression of related proteins are correlated with the differences in the therapeutic effects of oncolytic viruses among different patients. There have been no public reports revealing a correlation between the differences in the intestinal flora of patients and the individual therapeutic effects in oncolytic virus-mediated tumor immunotherapy. There are also no reports of enhancing the efficacy of oncolytic virus immunotherapy by regulating the composition of the intestinal flora.

[0010] Therefore, the present invention hopes to achieve a breakthrough through our research, using a preparation that can intervene and regulate the intestinal flora to change the composition of the intestinal flora to see whether such a composition change will affect the immunotherapy effect of oncolytic viruses. So can the preparation of the present invention that regulates the intestinal flora enhance the immunotherapy effect of oncolytic viruses? Summary of the invention

[0011] In the present invention, we demonstrate the use of antibiotics to regulate the intestinal flora to significantly change the composition of the intestinal flora. After the composition of the intestinal flora, i.e., the intestinal microecology, is significantly changed, it will have a substantial impact on the oncolytic virus immunotherapy of tumors.

[0012] The preparation disclosed in the present invention for regulating intestinal flora to enhance the effect of oncolytic virus immunotherapy is an antibiotic or a combination thereof that is already on the market, so its safety is guaranteed.

[0013] In the colorectal cancer tumor model, the use of antibiotics or antibiotic combinations, administered orally to reach the digestive tract, significantly reduced the tumor size of the colorectal cancer-bearing mouse model and significantly prolonged the survival of colorectal cancer-bearing mice.

[0014] Therefore, the specific contents of the present invention are:

[0015] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic.

[0016] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is ampicillin.

[0017] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic and the antibiotic is streptomycin.

[0018] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is colistin.

[0019] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is a combination of ampicillin and streptomycin.

[0020] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is a combination of ampicillin and colistin.

[0021] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is a combination of streptomycin and colistin.

[0022] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the preparation is an antibiotic, and the antibiotic is a combination of ampicillin, streptomycin and colistin.

[0023] A preparation for regulating intestinal microecology is used in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, characterized in that the preparation is an antibiotic, the antibiotic is one or more of ampicillin, streptomycin or colistin, and the virus is an adenovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Experimental flow chart for establishing a CT26 / MC38 tumor model in immune-competent mice to detect the effect of ABX on oncolytic adenovirus anti-tumor immunotherapy

[0025] Figure 2 Effect of ABX treatment on the body weight of experimental mice before tumor loading

[0026] Figure 3 ABX treatment can significantly reduce the content of genomic DNA in mouse feces. On Day 0, the feces of mice in different treatment groups were randomly collected, and the genomic DNA in the feces was extracted and normalized for comparison.

[0027] Figure 4 ABX treatment can promote the inhibitory effect of oncolytic adenovirus on subcutaneous CT26 tumor growth. Balb / C mice were subcutaneously inoculated with 2×10 6 CT26 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, changes in tumor growth curve.

[0028] Figure 5ABX treatment can improve the inhibitory effect of oncolytic adenovirus on subcutaneous CT26 tumor growth. Balb / C mice were subcutaneously inoculated with 2×10 6 CT26 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, and the growth curve changes of the tumor in each mouse in each group.

[0029] Figure 6 ABX treatment can prolong the survival of mice inoculated with subcutaneous CT26 tumors treated with oncolytic adenovirus. Balb / C mice were subcutaneously inoculated with 2×10 6 CT26 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, changes in the survival curves of mice in each group.

[0030] Figure 7 ABX treatment can promote the inhibitory effect of oncolytic adenovirus on subcutaneous MC38 tumor growth. C57BL / 6 mice were subcutaneously inoculated with 2×10 6 MC38 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, changes in tumor growth curve.

[0031] Figure 8 ABX treatment can promote the inhibitory effect of oncolytic adenovirus on subcutaneous MC38 tumor growth. C57BL / 6 mice were subcutaneously inoculated with 2×10 6 MC38 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, and the growth curve changes of the tumor in each mouse in each group.

[0032] Fig. 9 ABX treatment can prolong the survival of mice inoculated with subcutaneous MC38 tumors treated with oncolytic adenovirus. C57BL / 6 mice were subcutaneously inoculated with 2×10 6 MC38 mouse colon cancer cells were injected at multiple sites within the tumor on days 7, 9, and 11. 8 pfu of oncolytic adenovirus, changes in the survival curves of mice in each group. DETAILED DESCRIPTION

[0033] All experimental materials and methods mentioned in the examples section

[0034] 1. In vivo study of the relationship between the anti-tumor effect of oncolytic viruses and the presence or absence of the mouse intestinal microbiome

[0035] All animal experiments were in accordance with the standards of the Animal Ethics Committee. Six-week-old C57BL / 6 male mice were selected and randomly divided into four groups: Control group (n=8), ADV group (n=20), ABX group (n=8) and ABX&ADV group (n=8). The ABX group and ABX&ADV group mice were fed with a broad-spectrum antibiotic mixture (ampicillin (1 mg / ml), streptomycin (5 mg / ml), and colistin (1 mg / ml) in drinking water) and continuously supplied throughout the experimental period to delete the background microorganisms in the mouse intestine. The other two groups of mice were fed with sterile water during the same period. Subsequently, a subcutaneous tumor model was established in the right axilla of each group of mice, and 1×10 CT-26 colon cancer cells were inoculated on one side of each mouse. 6 cells, and the length (a) and width (b) of the tumor were measured with a vernier caliper to calculate the tumor volume = ab 2 / 2, when the tumor size reaches 80mm 3 (about one week), ADV virus was injected into the right tumor according to the group, and the amount of virus injected each time was 5×10 8 pfu / 100 μl, three times, with one day between each injection, and follow-up measurement of tumor volume and body weight until the tumor volume is greater than 2500 mm 3 The mice were judged dead and their survival time was recorded. According to the therapeutic effect of oncolytic virus, the mice in the ADV group were divided into the response group (R, tumor volume ≤ 500 mm 3 ) and non-response group (NR, tumor volume ≥1500mm 3 ). Five days after virus injection, five mice were selected and killed to obtain tumor tissue, lung, liver, spleen, and intestine for histopathological analysis of inflammatory response in each tissue, immunohistochemistry analysis of T cell, NK cell, APC cell infiltration, virus infection, and PDL1 expression in tumor tissue; ELISA was used to detect IFN-γ, IL-6, IL-12, GZMB, TNF-α, and GM-CSF in serum and tumor tissue to analyze the immune status in the tumor microenvironment; tumor tissue and spleen lymphocytes were separated, and the proportion of tumor antigen-specific lymphocytes was detected using Elispot technology, and the proportion of infiltrating lymphocytes was detected by flow cytometry. Tumor tissues were used to detect ICD-related indicators such as CRT, HMGB1, and ATP. Similar experimental methods were used for other types of tumors.

[0036] 2. In vivo study of the effects of intestinal microecology on the specificity and memory of oncolytic virus-mediated immunotherapy and the distal anti-tumor effects

[0037] Similarly, 6-week-old C57BL / 6 male mice were selected and fed with broad-spectrum antibiotics (as described above) for 3 days in advance to delete the background microorganisms of the mice.

[0038] Regarding specificity, after the tumor was inoculated and the oncolytic virus treatment was completed, the cured mice in different groups were inoculated with other types of tumors at the same location, and the specific anti-tumor effects of oncolytic virus treatment in mice in different treatment groups were compared by differences in tumor volume and mouse survival.

[0039] Regarding memory, after the tumor was inoculated and the oncolytic virus treatment was completed, the cured mice in different groups were inoculated with the same tumor at the contralateral position, and the memory anti-tumor effect of mice in different treatment groups after oncolytic virus treatment was compared by differences in tumor volume and mouse survival.

[0040] Regarding the distal anti-tumor effect, tumors were inoculated in the left and right axillae of mice at the same time and oncolytic virus treatment was given only on one side. The distal anti-tumor effect of mice in different treatment groups after oncolytic virus treatment was compared by comparing the differences in contralateral tumor volume and mouse survival.

[0041] 3. Fecal microbiota transfer (FMT) to explore the impact of intestinal microbial composition on the anti-tumor effect of oncolytic viruses

[0042] Six-week-old C57BL / 6 male mice were also selected and fed with broad-spectrum antibiotics (as described above) for 7 days to delete the background microorganisms of the mice. The feces of the previously collected responding and non-responding mice were made into a suspension and gavaged to the ABX-treated mice once every three days for 14 days before inoculation of tumors. The subsequent treatment and detection analysis of tumor-bearing mice were the same as described above. The mice were maintained in a sterile isolator with irradiated food and autoclaved water.

[0043] 4. Extraction, quantification and 16S rRNA sequencing of fecal bacterial DNA.

[0044] 1) Sample collection

[0045] Feces of each mouse were collected at each stage of the experiment, including Day-14 before ABX treatment, Day 0 before tumor inoculation, Day 7 before oncolytic virus treatment, and Day 14 after oncolytic virus treatment. The collected feces were temporarily stored in a -80°C low-temperature refrigerator.

[0046] 2) DNA extraction and PCR amplification

[0047] according to The total DNA of the microbial community was extracted according to the instructions of soil DNA kit (Omega Bio-tek, Norcross, GA, US). The extraction quality of DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by NanoDrop2000. 338F (5'-ACTCCTACGGG AGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3-V4 variable region of the 16S rRNA gene by PCR. The amplification procedure was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI 9700 model). The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStart FastPfu DN polymerase, 10 ng of template DNA, and the total volume was 20 μL. Each sample was repeated 3 times.

[0048] 3) Illumina Miseq sequencing

[0049] The PCR products of the same sample were mixed and recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), detected by 2% agarose gel electrophoresis, and analyzed by Quantus TM Fluorometer (Promega, USA) was used to detect and quantify the recovered products. The NEXTflexTM Rapid DNA-Seq Kit (Bioo Scientific, USA) was used to construct the library: (1) linker connection; (2) use magnetic beads to screen and remove linker self-linked fragments; (3) use PCR amplification to enrich the library template; (4) use magnetic beads to recover the PCR product to obtain the final library. Sequencing was performed using Illumina's Miseq PE300 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The original data was uploaded to the NCBI SRA database.

[0050] 4) Data processing

[0051] The original sequencing sequence was quality controlled using fastp software [2] (https: / / github.com / OpenGene / fastp, version 0.20.0), and spliced ​​using FLASH software (http: / / www.cbcb.umd.edu / software / flash, version 1.2.7):

[0052] (1) Filter the bases with a quality value of less than 20 at the tail of the reads, set a 50 bp window, and if the average quality value in the window is less than 20, cut off the back end bases from the window, filter the reads below 50 bp after quality control, and remove the reads containing N bases;

[0053] (2) Based on the overlap relationship between PE reads, the paired reads were spliced ​​(merged) into a sequence with a minimum overlap length of 10 bp;

[0054] (3) The maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence is 0.2, and non-compliant sequences are screened out;

[0055] (4) Samples are distinguished based on the barcodes and primers at both ends of the sequence, and the sequence direction is adjusted. The allowed number of mismatches for the barcode is 0, and the maximum number of mismatches for the primers is 2.

[0056] Based on the default parameters, the optimized sequences after quality control splicing were denoised using the DADA2 plug-in in the Qiime2 process. The sequences after DADA2 denoising are usually called ASVs (i.e., amplicon sequence variants). In order to reduce the impact of sequencing depth on subsequent analysis results such as Alpha diversity and Beta diversity, the number of sequences after denoising for each sample was flattened to 4,000. After flattening, the average coverage (Good's coverage) of each sample can still reach 97.90%. Based on the Sliva16S rRNA database (v138), the Naive bayes (or Vsearch, or Blast) classifier in Qiime2 was used to perform species taxonomy analysis on ASVs. Subsequent data analysis was performed using the Diversity Cloud Analysis Platform (Qiime2 process) (cloud.majorbio.com) of Shanghai Major Biotechnology Co., Ltd.

[0057] 5. qPCR quantification of bacteria in fecal samples.

[0058] The QIAamp stool DNA mini kit (Qiagen) was used to extract genomic DNA from stool samples according to the instructions in the kit. The SYBR green qPCR system was used to detect the bacterial content in the genome of each sample using primers targeting the bacterial 16SV3-V4 region.

[0059] 6. Pathological examination of mouse tumor tissue and internal organs

[0060] Tumor tissues fixed in 10% formalin and tumor tissues, lung, liver, spleen, and intestine were embedded in paraffin and routinely stained with hematoxylin and eosin to quantify the level of cellular necrosis in each tissue. Necrotic areas and tumor margins were outlined using Adobe Photoshop Element. These outlined areas were used to calculate the percentage of necrosis in individual tumors. Gamma-H2AX immunohistochemistry was performed on formalin-fixed, paraffin-embedded subcutaneous tumor sections. Briefly, sections were routinely defatted and stained using an automated immunohistochemical stainer (Leica, IL). Slides were treated with endogenous peroxidase blocker and heated antigen retrieval in citrate buffer for 20 min. Biotinylated mouse anti-human phospho-histone H2AX antibody was used at a dilution of 1:100 for 60 min. Amplification was performed using streptavidin-horseradish peroxidase conjugate and visualization was performed with diaminobenzidine (Leica). Tissues were counterstained with hematoxylin. The center of the smallest necrotic area in each tumor tissue was imaged. Only nuclei that were morphologically consistent with tumor nuclei were quantified, and at least 1000 tumor nuclei were counted for each tumor tissue. Nuclei with morphology consistent with apoptosis (staining markers, chromatin condensation) were excluded. Nuclei were divided into three groups: nuclei with less than 4 foci were considered negative, and nuclei with more than 4 foci were considered positive. Within positive nuclei, nuclei with confluent foci were considered to have pan-nuclear labeling.

[0061] 7. Flow cytometry to detect immune cell activation levels in mouse tumors and tissues

[0062] Single cells were isolated from tumor tissue, spleen, small intestine, mesenteric lymph nodes (LN), colon draining lymph nodes, and inguinal lymph nodes. The spleen and lymph nodes were cleaned of fat and connective tissue, minced, and digested in RPMI-1640 supplemented with type IA collagenase and DNaseI in a 37°C incubator with shaking (220 rpm). The tissue was then filtered with a 40 μm cell strainer (ThermoFisher) and resuspended in PBS containing 2% heat-inactivated fetal bovine serum (FBS) and 2 mM EDTA. Fat, connective tissue, and Peyer's nodes were removed from the small intestine and then cut into small pieces of 0.5-1 cm. The tissue blocks were washed twice in pre-heated calcium- and magnesium-free HBSS (Sigma) containing 5 mM EDTA and shaken (220 rpm) in a 37°C incubator for 20 minutes. The supernatant containing intestinal epithelial cells and intraepithelial lymphocytes was discarded. The remaining tissue pieces were resuspended in pre-heated calcium- and magnesium-free HBSS containing type VIII collagenase (1 mg / ml; Sigma) and shaken (220 rpm) in a 37°C incubator for 20-25 minutes. The supernatant was filtered using a 100 μm and then a 40 μm cell strainer. Intracellular staining: The cells were placed in a 96-well U-bottom plate and 200 μl of IMDM supplemented with 10% fetal bovine serum, 50 μm 2-mercaptoethanol, 50 ng / ml phorbol (PMA), 750 ng / ml ionomycin and 10 μg / ml brefeldin A were added and incubated for 4 hours at 37°C, 5% CO2. The cells were then incubated with Fcγ receptor blocking antibodies (BD Biosciences) for 10 minutes at 4°C, followed by surface staining for 25 minutes at 4°C. For intracellular staining, eBioscience was used. TM The cells were fixed and permeabilized using the Foxp3 fixation and permeabilization kit. Intracellular markers were then stained overnight at 4°C. The cells were then washed and flow cytometry was performed on a FACSCanto (BD Biosciences). Data analysis was performed using Flowjo v10.5.3 (Treestar).

[0063] 8. Immunohistochemistry to detect the expression of cytokine levels in tumor tissue

[0064] Tumor tissues were fixed in formalin, embedded in paraffin, and sectioned at 3 μm thickness, placed on poly-lysine-coated slides, defatted and hydrated with graded alcohols and water. Antigens were retrieved by heating the sections in a buffer at 98°C for 30 min (0.01 M sodium citrate buffer at pH 6.0 for CD3 staining and 1 mM EDTA at pH 8.0 for FoxP3 staining). Endogenous peroxidase activity was first inhibited with 3% hydroperoxidase (DAKO) for 10 min, then permeabilized with PowerVision IHC / ISH blocking solution for 20 min, incubated with rabbit anti-human CD3 polyclonal antibody and rabbit anti-mouse FoxP3 polyclonal antibody for 1 h without washing, and then incubated with peroxidase-conjugated anti-rabbit secondary antibody. Peroxidase was detected with a diaminobenzidine (DAB)-peroxidase substrate kit (DAKO), and sections were counterstained with Mayer's hematoxylin stain. Immunohistochemical staining for CD4 was performed using an automated immunostainer. Heat-induced antigen retrieval was performed at 95°C for 32 minutes in EDTA buffer (pH 8.0). Anti-mouse CD4 primary antibody was diluted 1:500 with antibody diluent and incubated at 37°C for 1 hour. Biotin-free peroxidase detection technology system with DAB as the colorimetric agent was used.

[0065] 9. Antibody deletion experiments verify whether the mode of action of commensal microorganisms in affecting oncolytic virus anti-tumor immunity depends on NK cells and CD8 + T cells, CD4 + T cells or macrophages.

[0066] According to the previous method, mice with intestinal microbiota deletion were constructed and tumor-bearing (CT26 / MC38) were injected into the peritoneal cavity of mice once every 3 days, starting from the day before tumor bearing, 200 μg of NK cell-depleting and CD8 + T cells, CD4 + Antibodies to T cells or macrophages were used, and the injection of an equal amount of PBS was used as a control. The therapeutic effect of oncolytic virus in intestinal microbial deletion mice after the deletion of various types of immune cells was judged according to the tumor volume and survival of the mice.

[0067] Example 1 In vivo study of the relationship between the anti-tumor effect of oncolytic viruses and the presence or absence of intestinal microbiome in mice

[0068] 1) Effects of antibiotics (ABX) on the physiological state of mice

[0069] According to the experimental design ideas introduced in the "Research Plan" section ( Figure 1 ), we first pre-fed mice with a cocktail of antibiotics (ABX) in Balb / C strain mice to alter the composition of the mouse gut microbiota. ABX was continuously supplied throughout the experiment.

[0070] The weight changes of mice after ABX feeding for 14 days showed ( Figure 2 ), antibiotic ABX can significantly affect the weight of mice. However, after a significant decrease in weight in the first week, as the mice adapted to ABX, the weight gain rate of the mice in the second week was consistent with that of the normal drinking water group. This shows that ABX requires mice to have an adaptation process in the early stage of drinking, and has certain changes in physiological state, but no pathological damage occurs.

[0071] Monitoring of the water intake and food intake of normal mice and ABX-fed mice also revealed (Table 1) that in the first week of ABX feeding, the two groups were quite different, and ABX significantly suppressed the mice's food intake. However, in the second week, the effects of ABX on the mice's water intake and food intake significantly recovered, and there was basically no difference in feed intake, which again showed that antibiotics (ABX) had a weak effect on the physiological state of mice.

[0072] Table 1 Effects of ABX treatment on the diet and water intake of experimental mice before tumor bearing

[0073]

[0074] The results of this part show that drinking ABX can temporarily affect the eating habits of mice, but it is not enough to cause substantial damage to the mice. After two weeks of ABX pretreatment, the mice can recover to normal growth state. This lays the foundation for the subsequent evaluation criteria of tumor bearing and anti-tumor immune effects.

[0075] Example 2 Effect of antibiotics (ABX) on the total amount of intestinal microorganisms in mice

[0076] In order to preliminarily evaluate the effect of ABX on the deletion of intestinal microorganisms in mice before tumor bearing, the fecal pellets of mice in the Adv group and ABX&Adv group were randomly picked up on Day 0, and the total DNA content in the feces was extracted using a fecal genomic DNA extraction kit (TIANGEN DP328). After normalization, the DNA content was used to indicate the amount of microorganisms. The results showed that the DNA content in the feces of mice in the ABX&Adv group was significantly lower than that in the Adv group ( Figure 3 ), indicating that 14-day ABX treatment significantly reduced the microbial content in mouse feces.

[0077] This result indicates that drinking ABX may indeed inhibit the growth and reproduction of microorganisms in the mouse intestine, providing a basis for subsequent experimental studies.

[0078] Example 3 Effect of mixed antibiotics (ABX) on the therapeutic effect of oncolytic virus adv

[0079] According to the experimental design, Balb / C mice were inoculated with tumors 14 days after ABX treatment. First, CT26 colon tumors were inoculated in the right axilla of the mice. About one week after inoculation, when the tumor size was 80 mm 3 Around 2000, we started to use adv virus intratumoral injection to treat tumors, once every other day, with 1*10 virus injected each time 8 pfu / 100μl, three times in total. The volume change of the tumor showed that compared with the PBS control group, adv treatment could significantly delay tumor growth and had a therapeutic effect. ABX treatment alone had a certain ability to change tumor growth, but the improvement was not significant. The use of ABX combined with adv could significantly improve the effect of adv treatment alone ( Figure 4 ).

[0080] The tumor volume change curves of each mouse in each group of the experimental design showed that the tumor volume of mice in the PBS group and the ABX group increased rapidly, while the therapeutic effect of oncolytic adenovirus in the adv group was quite different, and the volume growth of most mice was significantly faster than that of a few mice with good treatment effects. After adv combined with ABX, the growth of tumor volume of mice in the group was generally inhibited ( Figure 5 ). This result indicates that ABX combined with adv can enhance the therapeutic effect of oncolytic adenovirus in inhibiting tumor growth.

[0081] The survival curve of mice was consistent with the results expressed by tumor volume. Compared with the PBS control group, adv could significantly prolong the survival of mice, while the survival of ABX&adv was significantly prolonged compared with the adv group ( Figure 6 ), indicating that ABX treatment can significantly improve the therapeutic effect of adv on the CT26 subcutaneous tumor model.

[0082] This result shows that ABX can promote the anti-tumor effect of oncolytic adenovirus adv on CT26 subcutaneous transplanted tumors, which gives us the confidence to continue exploring.

[0083] To further verify that oncolytic viruses under the action of ABX have better anti-tumor effects, we fed ABX to C57BL / 6 mice in the same treatment mode. About one week after inoculation, when the tumor size was 80 mm 3 Around 2000, we started to use adv virus intratumoral injection to treat tumors, once every other day, with 1*10 virus injected each time 8 pfu / 100μl, three times in total. The volume changes of the tumors showed that the growth of tumor volume in the ABX&adv group was significantly slower than that in the adv group. It is worth noting that the tumor progression in the ABX group that drank ABX alone was also delayed compared with the control group ( Figure 7 ).

[0084] The tumor volume change curves of each mouse in each group of the experimental design showed that the tumor volume of mice in the control group increased rapidly. Compared with it, the tumor volume growth rate of mice in the ABX group generally slowed down. The tumor volume of the adv group grew slowly in the early stage of treatment, but the tumor volume increased rapidly after the oncolytic adenovirus treatment was stopped. After adv combined with ABX, the tumor volume growth of mice in the group was generally significantly inhibited ( Figure 8 ). This result shows that in the MC38 tumor model, ABX combined with adv can significantly enhance the therapeutic effect of oncolytic adenovirus in inhibiting tumor growth.

[0085] Survival curve results of mice ( Fig. 9 ) is consistent with the results shown by tumor volume. It can be found that in the MC38 model, the therapeutic effect of oncolytic adenovirus is general, and compared with the Control group, it is not enough to significantly prolong the survival of mice. However, ABX showed a good therapeutic effect, and combined with adv, it can significantly promote the anti-tumor therapeutic effect of oncolytic adenovirus.

[0086] The above two mouse tumor models of colorectal cancer, CT26 and MC38, have proved that ABX treatment has a significant promoting effect on the anti-tumor immunotherapy of oncolytic adenovirus after affecting the intestinal flora of mice. The results show that antibiotic treatment has a clear and significant effect in promoting the anti-tumor immunotherapy effect of oncolytic adenovirus, and antibiotics can be used to prepare preparations or drugs that promote the anti-tumor immunotherapy effect of oncolytic adenovirus.

Claims

1. Use of a preparation for regulating the composition of intestinal flora in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, characterized by: The preparation is an antibiotic, which is a combination of ampicillin, streptomycin and colistin, and the oncolytic virus is an adenovirus.

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