Composition containing oncolytic virus and application of composition in field of preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition containing oncolytic viruses and application of the composition in the field of preparation of antitumor drugs. In order to solve the problem that the anti-tumor effect is poor when an oncolytic virus is independently used, the invention provides an anti-tumor interferon inhibitor and oncolytic virus composition, the active ingredients of the composition comprise an interferon inhibitor (a gemarane type sesquiterpene lactone compound EM-2) and an oncolytic virus VSV delta51 which are active ingredients with a synergistic effect, in the preparation of anti-tumor drugs, a remarkable anti-tumor effect can be achieved through combined use. It is found for the first time that the gemmarane type sesquiterpene lactone compound can be used as an interferon inhibitor and can be used as an anti-tumor synergist or a drug resistance reversal agent of oncolytic viruses, the anti-tumor effectiveness of the oncolytic viruses is remarkably improved by inhibiting interferon signal channels, and the gemmarane type sesquiterpene lactone compound has important application value and wide clinical transformation prospects and is worthy of popularization and application. And a new direction is expected to be provided for clinical treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a composition containing an oncolytic virus and application thereof in the field of preparation of anti-tumor drugs. BACKGROUND

[0002] Oncolytic viruses are key tools in the field of tumor immunotherapy, and the core mechanism of action is to directly lyse tumor cells and activate the body's anti-tumor immune response to achieve tumor inhibition. However, in the prior art, the application of oncolytic viruses alone has obvious technical pain points: the anti-tumor effect is poor in clinical treatment, and it is difficult to meet the treatment needs of advanced solid tumors. In-depth research shows that the core cause of the above technical pain points is the abnormal activation of the interferon signaling pathway in the tumor microenvironment. After the pathway is activated, it significantly enhances the resistance of tumor cells to oncolytic viruses, which can be manifested in two key aspects: one is to directly inhibit the replication efficiency of oncolytic viruses in tumor cells, and the other is to weaken the anti-tumor activity mediated by the virus. The two effects work together to ultimately cause a significant decline in the clinical treatment effect of oncolytic viruses.

[0003] At present, there is still a lack of compositions that can specifically inhibit the interferon signaling pathway in tumors and have a synergistic effect with oncolytic viruses. In view of the urgent need for efficient and safe anti-tumor drugs in clinical practice, it is of great clinical value and broad application prospect to develop a composition that can target the regulation of the interferon pathway and synergistically exert anti-tumor effects with oncolytic viruses. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the present application first found that gimalin sesquiterpene lactone compounds can be used as an anti-tumor synergist or a drug resistance reversing agent for oncolytic viruses. The specific technical solution is to construct an anti-tumor composition composed of sesquiterpene lactone compound EM-2 (which acts as an interferon inhibitor) and oncolytic virus VSVΔ51 (Vesicular Stomatitis Virus Δ51), which are active components with synergistic effects. In the preparation of anti-tumor drugs, the combination of the two can achieve a significant anti-tumor effect.

[0005] In order to achieve the above purpose, the technical solution adopted by the present application is: The first aspect of the present application provides a composition containing an oncolytic virus, which comprises oncolytic virus VSVΔ51 and interferon inhibitor gimalin sesquiterpene lactone compound EM-2. The structural formula of EM-2 can be found in Chinese invention patent CN114129558A, and is specifically as follows: .

[0006] The second aspect of the present application also provides the use of the composition comprising the oncolytic virus of the first aspect in the preparation of an antitumor drug.

[0007] The gimmeran-type sesquiterpene lactone EM-2 is used as a synergist of oncolytic virus in the preparation of an antitumor drug, which significantly improves the effectiveness of oncolytic virus against tumors by inhibiting the interferon signaling pathway (acting as an interferon inhibitor).

[0008] Preferably, the tumor is a solid tumor with high expression level of ISG15 (Interferon-Stimulated Gene 15). The tumor tissue has a higher mRNA or protein normalized expression level of ISG15 than the corresponding non-tumor tissue (the ratio of the normalized expression level of ISG15 in the tumor tissue to that in the non-tumor tissue is >1), which is considered as high expression of ISG15. For the following treatment objects, it is more necessary to use the combination therapy of EM-2 and oncolytic virus: tumors with a normalized expression level of ISG15 in the tumor tissue to that in the non-tumor tissue of >1.5, especially >2.

[0009] More preferably, the tumor includes liver cancer, colorectal cancer, breast cancer, melanoma, pancreatic cancer, or lung cancer.

[0010] Preferably, the effective dose of oncolytic virus VSVΔ51 is 1×10 7 -10 8 PFU (Plaque-Forming Unit) / mL, and the effective dose of gimmeran-type sesquiterpene lactone EM-2 is 1-100 mg / kg.

[0011] More preferably, the effective dose of oncolytic virus VSVΔ51 is 1×10 7 PFU / mL, and the effective dose of gimmeran-type sesquiterpene lactone EM-2 is 4 mg / kg.

[0012] Preferably, gimmeran-type sesquiterpene lactone EM-2 competes with p-IRF3 for binding to ISG15, thereby blocking the ISGylation modification of p-IRF3, promoting the degradation of IRF3 protein, and further inhibiting the interferon signaling pathway, which significantly improves the effectiveness of oncolytic virus against tumors.

[0013] The present application first discovers that the gimmer alkane type sesquiterpene lactone compound (EM-2) can be used as an interferon inhibitor, the target point of which is ISG15 protein, and the EM-2 can block the ISGylation of p-IRF3 (Phosphorylated Interferon Regulatory Factor 3) by competitively binding ISG15 with p-IRF3, promote the degradation of IRF3 (Interferon Regulatory Factor 3) protein, and then inhibit the interferon signal pathway, and finally promote the antitumor effect of the oncolytic virus VSVΔ51.

[0014] Preferably, the antitumor drug further comprises a pharmaceutically acceptable excipient.

[0015] More preferably, the excipient comprises at least one of excipient, solubilizer, emulsifier, coloring agent, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, coating material, pH regulator, absorbent, diluent, antioxidant.

[0016] More preferably, the administration mode of the antitumor drug comprises oral administration, injection administration, subcutaneous administration, inhalation administration, rectal administration.

[0017] More preferably, the dosage form of the antitumor drug comprises injection, tablet, capsule, granule, powder, syrup, or oral liquid.

[0018] Compared with the prior art, the present application has the following beneficial effects: The present application discloses an interferon inhibitor and oncolytic virus composition for anti-tumor, which active ingredients include an interferon inhibitor (gymnamide sesquiterpene lactone compound EM-2) and an oncolytic virus VSVΔ51, both of which are active components of synergistic action, and can achieve significant anti-tumor effect by combined use in the preparation of anti-tumor drugs. The present application first discovers that the gymnamide sesquiterpene lactone compound can be used as an interferon inhibitor, and can be used as an anti-tumor synergist or drug resistance reversing agent for oncolytic virus, the target of which is ISG15 protein, which competes with p-IRF3 to bind ISG15, blocks the ISGylation modification of p-IRF3, promotes the degradation of IRF3 protein, and then inhibits the interferon signaling pathway, and significantly improves the effectiveness of oncolytic virus in anti-tumor, which has important application value and broad clinical transformation prospect, and is expected to provide a new direction for clinical treatment of tumors. The interferon inhibitor and oncolytic virus composition of the present application is especially suitable for the treatment of solid tumors with high expression level of ISG15 (ubiquitin-like protein 15, Interferon-Stimulated Gene 15), such as colorectal cancer, liver cancer and other solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 2 is a graph showing the effect of combined treatment of compound EM-2 and oncolytic virus VSVΔ51 on the survival rate of human colorectal cancer and liver cancer cell lines; wherein the left graph shows the survival rate detection results of human colorectal cancer cell line HT29 treated with EM-2 and VSVΔ51, and the right graph shows the survival rate detection results of human liver cancer cell line SNU387 treated with EM-2 and VSVΔ51.

[0020] Figure 2 Figure 3 is a graph showing the effect of combined treatment of compound EM-2 and oncolytic virus VSVΔ51 on the apoptosis rate of patient-derived colorectal cancer and liver cancer organoids; wherein a is the cleaved Caspase-3 (activated cysteine-containing aspartate-specific protease 3) fluorescence staining result of colorectal cancer organoids (CRCO, Colorectal Cancer Organoid), and b is the cleaved Caspase-3 fluorescence staining result of hepatocellular carcinoma organoids (HCCO, Hepatocellular Carcinoma Organoid).

[0021] Figure 3Figure of the inhibitory effect of the combination of compound EM-2 and oncolytic virus VSVΔ51 on the interferon pathway in human colorectal cancer and liver cancer cell lines; wherein a is the effect of the combination of EM-2 and VSVΔ51 on the expression of interferon pathway-related proteins in HT29 cells; b is the effect of the combination of EM-2 and VSVΔ51 on the expression of interferon pathway-related proteins in SNU387 cells; c is the Co-IP (Co-Immunoprecipitation) binding verification result of EM-2 and ISG15; d is the reverse effect of ISG15 supplement experiment on the expression of interferon pathway-related proteins; e is the expression level of ISG15 protein between normal cells and tumor cells; f is the effect of the combination of EM-2 and VSVΔ51 on the interferon pathway in normal human intestinal epithelial cells NCM460; g is the effect of the combination of EM-2 and VSVΔ51 on the interferon pathway in normal human liver cells MIHA.

[0022] Figure 4 Figure of the inhibitory effect of the combination of compound EM-2 and oncolytic virus VSVΔ51 on the subcutaneous transplanted tumor of murine colorectal cancer cell line MC38; wherein a is the subcutaneous transplanted tumor growth curve, b is the mouse weight change curve, c is the H&E (Hematoxylin-Eosin) staining result of the main organs of mice, and d is the IHC (Immunohistochemistry) staining result of the subcutaneous tumor tissue.

[0023] Figure 5 Figure of the inhibitory effect of the combination of compound EM-2 and oncolytic virus VSVΔ51 on the orthotopic transplanted tumor of murine liver cancer cell line Hepa1-6; wherein a is the orthotopic transplanted tumor live imaging fluorescence signal result, b is the mouse weight change curve, c is the H&E staining result of the main organs of mice, and d is the IHC staining result of the orthotopic tumor tissue. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are further described below. It should be noted that the description of these embodiments is intended for purposes of illustration, of understanding, and not for limiting the present application. Moreover, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the experimental materials used in the following examples are all commercially available unless otherwise specified.

[0026] In the following examples, the sesquiterpene lactone compound EM-2 is prepared by the following method: (1) White flower dry grass 5 kg of whole plant into coarse powder, 80 mesh sieve. With 95% ethanol for cold immersion extraction, each immersion 24 hours, a total of three times, combined extract and reduced pressure concentration to no ethanol taste, get ethanol extract about 504 g.

[0027] (2) The ethanol extract with the right amount of water suspension, and then with petroleum ether and ethyl acetate extraction. After concentration under reduced pressure, respectively, get petroleum ether extract 315 g and ethyl acetate extract 120 g.

[0028] (3) The petroleum ether extract was dissolved in petroleum ether and mixed with 200 g of silica gel (80-100 mesh) evenly, and dried under reduced pressure to obtain the sample. Then take 2.5 kg of silica gel (200-300 mesh) wet column (inner diameter x length = 10 x 75 cm), the sample is loaded on the column and covered with a layer of silica gel, first eluted with petroleum ether, then eluted with petroleum ether-ethyl acetate gradient (100:0→0:100). Collection of petroleum ether-ethyl acetate (volume ratio 2:1) elution site, concentrated and dried to get mixed solvent eluate about 45 g.

[0029] (4) The mixed solvent eluate was dissolved in 30 mL of methanol, and mixed with 50 g of silica gel (80-100 mesh) to obtain the sample. Then take 200 g of ODS column chromatography material dry column (inner diameter x length = 5.5 x 50 cm), the sample is loaded on the column, eluted with methanol-water gradient. Collection of methanol-water (volume ratio 6:4) elution site, concentrated and dried to get white flower grass extract about 6.7 g.

[0030] (5) The white flower grass extract was dissolved in 3 mL of methanol, and loaded on a Sephadex LH-20 gel column with a volume of 50 mL. The fractions with retention time of 90-100 min were collected by LC-MS tracking analysis and detection, and concentrated under reduced pressure to obtain about 2.6 g of sesquiterpene lactone crude extract.

[0031] (6) The sesquiterpene lactone crude extract was further purified by preparative HPLC to obtain compound EM-2.

[0032] Example 1: Gemma-type sesquiterpene lactone compound EM-2 combined with oncolytic virus VSVΔ51 treatment reduces the survival rate of human colorectal cancer and liver cancer cell lines 1. Experimental materials: Human colorectal cancer cell line HT29, human hepatocellular carcinoma cell line SNU387 (both purchased from ATCC), VSVΔ51 oncolytic virus (donated by Professor Xing Fan of Guangdong General Hospital), RPMI 1640 medium (Gibco, USA), fetal bovine serum (Gibco, USA), penicillin-streptomycin (Thermo Fisher, USA), CCK-8 kit (Yikesheng, China), 37℃ / 5% CO2 incubator (Thermo Scientific, USA), microplate reader (Bioteck, USA), 96-well cell culture plate (Thermo Fisher, USA).

[0033] 2. Experimental method: (1) Cell culture: HT29 and SNU387 cells were inoculated in culture bottles containing RPMI 1640 medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin) and cultured in a 37℃, 5% CO2 incubator. When the cells grew to the logarithmic phase (confluence 70%-80%), they were passaged for future use.

[0034] (2) Grouping and treatment: two kinds of cells were inoculated in 96-well plates at a density of 5×10 3 cells / well, and cultured for 24 h. Then they were divided into 4 groups, with 3 replicate wells for each treatment condition: a) Control group: only RPMI 1640 medium was added.

[0035] b) EM-2 single treatment group: EM-2 solution was added, with a final concentration of 8 μM for HT29 and 4 μM for SNU387; c) VSVΔ51 single treatment group: VSVΔ51 virus solution was added, with a final concentration of 0.01 MOI (multiplicity of infection) for HT29 and 1 MOI for SNU387; d) EM-2+VSVΔ51 combined treatment group: the final concentration of EM-2 was the same as b, and the final concentration of VSVΔ51 virus solution was the same as c; After 24 h of treatment, the cell survival rate was detected (CCK-8 detection): the culture medium was aspirated, 100 μL of fresh culture medium containing 10% CCK-8 reagent was added, and the cells were incubated at 37℃ for 2 h. Then the OD value (optical density value) at 450 nm was detected by a microplate reader, and the cell survival rate was calculated according to the formula: cell survival rate (%) = (experimental group OD value / control group OD value) x 100%.

[0036] 3. Experimental results: AsFigure 1 As shown in the left graph in FIG. 11B, the survival rate of human colorectal cancer cell line HT29 cells treated with VSVΔ51 alone was 80%. After EM-2 and VSVΔ51 were combined, the survival rate of the cells was significantly reduced, only 21%. As shown in the right graph in FIG. 11B, after human liver cancer cell line SNU387 cells were treated with EM-2 and VSVΔ51, the survival rate of the cells was also significantly reduced compared with the single treatment group. The above results show that EM-2 and VSVΔ51 combined treatment can significantly reduce the survival rate of human colorectal cancer and liver cancer cell lines. Figure 1 As shown in the left graph in FIG. 11B, the survival rate of human colorectal cancer cell line HT29 cells treated with VSVΔ51 alone was 80%. After EM-2 and VSVΔ51 were combined, the survival rate of the cells was significantly reduced, only 21%. As shown in the right graph in FIG. 11B, after human liver cancer cell line SNU387 cells were treated with EM-2 and VSVΔ51, the survival rate of the cells was also significantly reduced compared with the single treatment group. The above results show that EM-2 and VSVΔ51 combined treatment can significantly reduce the survival rate of human colorectal cancer and liver cancer cell lines.

[0037] Example 2 Gemma-type sesquiterpene lactone compound EM-2 combined with oncolytic virus VSVΔ51 to improve the apoptosis rate of patient-derived colorectal cancer and liver cancer organoids 1. Experimental materials: Colorectal cancer organoids (CRCO; long-term passage stage) derived from patients with advanced colorectal cancer or liver cancer, liver cancer organoids (HCCO; long-term passage stage) derived from patients, VSVΔ51 oncolytic virus, EM-2, human colorectal cancer organoid medium (Merck, DEU), human liver cancer organoid medium (Yixing Biological, China), DAPI (4', 6-diamidino-2-phenylindole; Merck, DEU), cleaved Caspase-3 antibody (Abcam, UK), goat anti-rabbit IgG-Alexa Fluor 488 anti (Thermo Fisher, USA), fluorescence microscope (Nikon, Japan).

[0038] 2. Experimental method: (1) Organoid culture: The conventional organoid culture method in the art (referring to “Organoids: opportunities and challenges of cancer therapy. Volume 11-2023 | https: / / doi.org / 10.3389 / fcell.2023.1232528”) was used. CRCO and HCCO were inoculated into sterile culture dishes containing organoid special medium, and cultured in a 37°C, 5% CO2 (carbon dioxide) incubator to maintain the growth activity of the organoids to the logarithmic phase.

[0039] (2) Grouping and treatment: Each of the two types of organoids was divided into 4 groups, with 3 replicates in each group: a) Control group: only containing organoid special medium; b) EM-2 single treatment group: EM-2 was added to adjust the final concentration to 0.28 μM (CRCO), 0.26 μM (HCCO); c) VSVΔ51 single treatment group: VSVΔ51 was added to adjust the final concentration to 0.01 MOI (CRCO), 1 MOI (HCCO); d) EM-2 + VSVΔ51 combined treatment group: EM-2 and VSVΔ51 were added at the above concentrations at the same time; After 3 days of treatment in each group, subsequent apoptosis detection (immunofluorescence staining) was performed: a) Fixing and permeabilization: the culture medium was aspirated, 4% paraformaldehyde was added to fix the organoids for 30 min, and PBS (Phosphate-Buffered Saline) was washed 3 times; 0.1% Triton X-100 was added for permeabilization for 15 min, and PBS was washed 3 times; b) Antibody incubation: cleaved Caspase-3 primary antibody (1:500 dilution) was added, and incubated at 4°C overnight; after PBS washing 3 times, fluorescent secondary antibody (1:1000 dilution) was added, and incubated at room temperature for 1 h; c) Nucleus staining: DAPI staining solution (1:1000 dilution) was added, and incubated at room temperature for 5 min, and PBS was washed 3 times; d) Observation: the green fluorescence intensity of cleaved Caspase-3 in each group was observed by fluorescence microscopy, and the fluorescence signal value was quantitatively analyzed by ImageJ software.

[0040] 3、Experimental results: As Figure 2 shown, in CRCO ( Figure 2 a) and HCCO ( Figure 2 b), the green fluorescence intensity of cleaved Caspase-3 in the Control group, the EM-2 single treatment group, and the VSVΔ51 single treatment group was weak, and the quantitative signal value was low; while the green fluorescence of the combined treatment group was significantly enhanced. The above results show that the combined treatment of EM-2 and VSVΔ51 can significantly increase the expression level of cleaved Caspase-3 in patient-derived colorectal cancer and liver cancer organoids, and significantly increase the apoptosis rate of organoids.

[0041] Example 3 Combined treatment of gimalin sesquiterpene lactone compound EM-2 and oncolytic virus VSVΔ51 inhibits the interferon pathway in human colorectal cancer and liver cancer cell lines 1、Experimental materials: Human colorectal cancer cell line HT29, human hepatocellular carcinoma cell line SNU387, VSVΔ51 oncolytic virus, EM-2, RIG-I (Cell Signaling Technology, USA), MDA5 (Cell Signaling Technology, USA), MAVS (Cell Signaling Technology, USA), TBK1 (Cell Signaling Technology, USA), IRF7 (Cell Signaling Technology, USA), IRF3 (Cell Signaling Technology, USA), a-Tubulin (Cell Signaling Technology, USA), ISG15 overexpression plasmid (Yikang, China), BCA protein quantification kit (Biyuntian, China), color pre-dye protein marker (Biosharp, China), cell transfection reagent (Thermo Fisher, USA), protein lysis buffer (Thermo Scientific, USA), chemiluminescence developing solution (Advansta, USA), RNA extraction kit (Yikang, China), reverse transcription kit (containing Oligo (dT) primer; Thermo Fisher, USA), qPCR primer (Ribo, China), PCR thermal cycler (Thermo Scientific, USA), real-time fluorescent quantitative PCR kit (Yikang, China), enzyme-free water (Yikang, China), 96-well PCR plate (Beyotime, China), RNase-free centrifuge tube (Thermo Fisher, USA), 37℃ / 5% CO2 incubator, ultracentrifuge (Beckman, USA), Western Blot (protein blotting method, Western Blot Electrophoresis) electrophoresis tank (Bai Sha, China), chemiluminescence imaging system (Bio-Rad, USA), Nanodrop detector (Thermo Fisher, USA), real-time fluorescent quantitative PCR instrument (Bio-Rad, USA).

[0042] 2. Experimental method: (1) Cell culture and grouping treatment: HT29 and SNU387 cells were inoculated in 6-well plates, and after culture to the logarithmic phase, they were divided into 4 groups (3 replicates for each group): a) Control group: only containing RPMI 1640 medium; b) EM-2 single treatment group: EM-2 was added to adjust the final concentration to 8 μM (HT29), 4 μM (SNU387); c) VSVΔ51 single treatment group: VSVΔ51 was added to adjust the final concentration to 0.01 MOI (HT29), 1 MOI (SNU387); d) EM-2+VSVΔ51 combined treatment group: EM-2 and VSVΔ51 were added at the above concentrations at the same time; After 24 h of treatment, the cells were collected for subsequent detection.

[0043] (2) Co-IP detection (EM-2 and p-IRF3 competitively bind ISG15): a) Take the HT29 cell lysate (lysed by protein lysate and add protease inhibitor), add ISG15 antibody (1:100), 4°C incubate overnight; b) Add Protein A / G (Protein A / G Agarose Beads) agarose beads, incubate at room temperature for 2 h, centrifuge at 4°C, 12000 rpm (revolutions per minute) for 5 min, collect the precipitate; c) The precipitate was washed with PBS for 3 times, added with loading buffer, boiled for 10 min, and Western Blot detection was performed, primary antibody (ISG15, IRF3, p-IRF3; 1:1000), to observe the protein expression of ISG15, IRF3 and p-IRF3.

[0044] (3) Western Blot detection (interferon pathway proteins): a) Take the cell lysate of each group, measure the protein concentration, and take 30 μg of protein for SDS-PAGE electrophoresis (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis); b) Transfer to PVDF membrane (Polyvinylidene Fluoride Membrane), block with 5% skim milk for 1 h; add primary antibody (p-IRF3 1:1000, IRF3 1:1000, α-Tubulin 1:5000), incubate at 4°C overnight; c) Add HRP (Horseradish Peroxidase-Labeled Secondary Antibody) labeled secondary antibody (1:5000), incubate at room temperature for 1 h, chemiluminescence development, ImageJ quantitative analysis of protein gray value.

[0045] (4) ISG15 backfill experiment: a) Take HT29 cells, inoculate in 6-well plates, culture for 24 h, then use cell transfection reagent to transfer ISG15 expression plasmid into cells (empty plasmid as control); b) After 48 h of transfection, perform 4 groups of treatment (synchronous step (1)), collect cells after 24 h, and detect the expression level of interferon pathway related proteins by Western Blot.

[0046] (5) PCR detection (gene expression level): a) Total RNA extraction: Take cells of each group (including Control group, EM-2 single treatment group, VSVΔ51 single treatment group, EM-2+VSVΔ51 combined treatment group), extract total RNA according to the operation of Trizol reagent instruction book. Use Nanodrop detector to detect the concentration and purity of RNA (20-200 ng / μL).

[0047] b) RNA reverse transcription: Take 1 μg of total RNA, use RNA reverse transcription kit, and follow the kit operation steps to reverse transcribe RNA into cDNA (complementary DNA). The reaction system is usually 20 μL, containing RNA template, reverse transcriptase, dNTPs (deoxynucleotide triphosphates), primers and other components. Under specific temperature conditions (such as 42℃ incubation for 30 minutes, then 95℃ denaturation for 5 minutes), reverse transcription is completed, and the obtained cDNA can be used for subsequent PCR reaction or temporarily stored in -20℃ refrigerator.

[0048] c) Real-time quantitative PCR (qPCR): Using cDNA as a template, a 20 μL reaction system is prepared using a real-time quantitative PCR kit. This typically includes TB Green™ Premix Ex Taq™ II (or other quantitative PCR premix), upstream and downstream primers (Table 1; the final primer concentration is generally 0.2 μM), cDNA template (2 μL, adjusted according to concentration), and enzyme-free water. The reaction program on the real-time quantitative PCR instrument is typically set as follows: pre-denaturation: 95℃ for 30 seconds; then 40 cycles, each cycle including denaturation (95℃ for 5 seconds), annealing (adjusted according to primer annealing temperature, such as 60℃ for 30 seconds), and extension (72℃ for 30 seconds); finally, melting curve analysis is performed to verify the specificity of the PCR product. After the reaction, the 2^(-ΔΔCt) method (where ΔCt = Ct) is used with the software provided with the PCR instrument. 目标基因 -Ct 内参基因 , ΔΔCt = ΔCt 实验组 -ΔCt 对照组 The relative expression levels of each gene were calculated and analyzed to assess the changes in the expression of interferon pathway-related genes in different treatment groups and different cells.

[0049] Table 1 qPCR primers

[0050] 3. Experimental Results: like Figure 3 As shown, Figure 3 a(HT29) and Figure 3 In group b (SNU387), the gray values ​​of interferon pathway-related proteins in the combined treatment group were significantly lower than those in the other three groups; for example... Figure 3 As shown in c, the Co-IP experiment showed that the p-IRF3 protein content decreased in the presence of EM-2, proving that EM-2 competitively binds to ISG15 with p-IRF3; Figure 3 As shown in d, after HT29 cells were reintroduced with ISG15, the expression levels of p-IRF3 and IRF3 proteins in the combined treatment group were significantly higher than those in the non-introduced group, and the inhibitory effect of the interferon pathway was reversed; Figure 3 As shown in e, compared with normal human intestinal epithelial cells NCM460 and normal human hepatocytes MIHA, the expression levels of ISG15 protein were higher in human colorectal cancer cells HT29 and human hepatocyte cells SNU387; Figure 3 As shown in f, combined treatment with EM-2 and VSVΔ51 does not inhibit the interferon pathway in normal human intestinal epithelial cells NCM460; Figure 3 As shown in g, combined treatment with EM-2 and VSVΔ51 does not inhibit the interferon pathway of MIHA in normal human hepatocytes.

[0051] The above results show that EM-2 significantly inhibits the interferon pathway in human colorectal cancer (HT29), liver cancer (SNU387) cell lines by specifically binding ISG15, but has no inhibitory effect on the interferon pathway of normal cells (NCM460, MIHA); combined with the characteristics of high expression of ISG15 in tumor cells, it is suggested that EM-2 combined with VSVΔ51 therapy is suitable for tumors with high expression levels of ISG15.

[0052] Example 4 Gemma-type sesquiterpene lactone compound EM-2 combined with oncolytic virus VSVΔ51 treatment inhibits the growth of subcutaneous transplanted tumors of mouse colorectal cancer cell line MC38 1. Experimental materials: Mouse colorectal cancer cell line MC38 (purchased from ATCC), SPF (Specific Pathogen Free) C57BL / 6J mice (6-8 weeks old, body weight 18-22 g; purchased from Jisui Yaoke), VSVΔ51 oncolytic virus (titer 1×10 7 PFU / mL), general tissue fixative (BaiSha, China), H&E staining kit (Solebao, China), cleaved Caspase-3 antibody (Abcam, UK), Ki-67 antibody (Abcam, UK), VSV-G (Kerafast, USA), IRF3 (Abcam, UK), IHC detection kit (Abcam, UK), electronic balance (Liangping, China), vernier caliper (Mitutoyo, Japan), pathological section machine (Leica, DEU), inverted phase contrast microscope (Olympus, Japan), small animal live imaging instrument (PerkinElmer, USA).

[0053] 2. Experimental method: (1) Construction of subcutaneous transplanted tumor model: a) Take the logarithmic phase MC38 cells, resuspend with sterile PBS, adjust the concentration to 1×10 6 cells / mL; b) Subcutaneously inject 100 μL of cell suspension (containing 1×10 5 cells) into the right back of each mouse, and raise normally (SPF environment, free feeding and drinking); c) When the transplanted tumor volume reaches 50-100 mm 3 , the mice are randomly divided into 4 groups (3 mice in each group): Control group, EM-2 single treatment group, VSVΔ51 single treatment group, and combined treatment group.

[0054] (2) Grouping treatment: a) Control group: Intraperitoneal injection of sterile saline once a day for a total of 5 times; b) EM-2 monotherapy group: Intraperitoneal injection of EM-2 solution (4 mg / kg) once daily for a total of 5 times; c) VSVΔ51 monotherapy group: Intratumoral injection of VSVΔ51 virus solution (1×10⁻⁶) 7 (PFU / mL), once daily for a total of 3 times; d) Combined treatment group: Simultaneous intraperitoneal injection of EM-2 and intratumoral injection of VSVΔ51, with the same dosage and frequency as the single treatment group.

[0055] (3) Indicator monitoring: a) Tumor volume monitoring: The long diameter (L) and short diameter (W) of the transplanted tumor are measured every 2 days using calipers, and the result is calculated using the formula V = 0.5 × L × W. 2 Calculate the volume and plot the growth curve; b) Mouse weight monitoring: The weight of mice was measured every 2 days using an electronic balance to assess the impact of the treatment on the overall health of the mice.

[0056] (4) Tissue sample testing: a) Sample collection: Five days after treatment, mice were euthanized by cervical dislocation, and the transplanted tumor and major organs (heart, liver, spleen, lungs, and kidneys) were removed. After rinsing with physiological saline, the mice were fixed with 4% paraformaldehyde for 24 hours. b) H&E staining: Paraffin sections (5 μm thick) were prepared from the fixed organs and stained according to the H&E kit instructions (dewaxing → hydration → hematoxylin staining → differentiation → eosin staining → dehydration → clearing → mounting). The tissue morphology was then observed under a microscope to assess toxicity. c) IHC staining: Tumor tissue sections were stained according to the IHC kit. The primary antibodies were cleaved Caspase-3 antibody (1:500) and IRF3 antibody (1:500). After DAB staining (3,3'-Diaminobenzidine Developing Solution) and hematoxylin counterstaining, the positive cell rate was observed.

[0057] 3. Experimental Results: like Figure 4 As shown in a, the tumor volume in the combined treatment group was significantly smaller than that in the other three groups, and the growth rate was significantly slowed down; as shown in a... Figure 4 As shown in b, there was no significant difference in body weight among the groups of mice, and no weight loss was observed, indicating that the treatment had no obvious toxicity; Figure 4 As shown in c, H&E staining of the major organs of mice in the combined treatment group showed normal tissue morphology, with no inflammatory infiltration, cell necrosis, or other damage, demonstrating good in vivo safety;Figure 4 As shown in Figure 2D, the tumor tissue cleaved Caspase-3 positive rate of the combination treatment group was significantly higher than that of the Control group, and the IRF3 positive rate was significantly lower than that of the Control group.

[0058] The above results show that EM-2 combined with VSVΔ51 can significantly inhibit the growth of MC38 subcutaneous transplanted tumors, and has good safety, and the mechanism is related to the inhibition of the interferon pathway of colorectal cancer cells.

[0059] Example 5 Gemma-type sesquiterpene lactone compound EM-2 combined with oncolytic virus VSVΔ51 to inhibit the growth of murine hepatoma cell line Hepa1-6 orthotopic transplanted tumor 1. Experimental materials: Murine hepatoma cell line Hepa1-6 (purchased from ATCC), SPF C57BL / 6J mice (6-8 weeks old, body weight 18-22 g; purchased from Jisui Yaoke), VSVΔ51 oncolytic virus (titer 1×10 7 PFU / mL), general tissue fixative (BaiSha, China), H&E staining kit (Solebao, China), cleaved Caspase-3 antibody (Abcam, UK), Ki-67 antibody (Abcam, UK), VSV-G (Kerafast, USA), IRF3 (Abcam, UK), p-IRF3 antibody (Abcam, UK), IHC detection kit (Abcam, UK), electronic balance (Liangping, China), fluorescent substrate for in vivo imaging (D-luciferin, Yixing, China), small animal in vivo imaging instrument (PerkinElmer, USA), pathological sectioning machine (Leica, DEU), inverted phase contrast microscope (Olympus, Japan).

[0060] 2. Experimental methods: (1) Orthotopic transplanted tumor model construction: a) Take the logarithmic phase Hepa1-6 cells, resuspend with sterile PBS, adjust the concentration to 1×10 7 cells / mL; b) After abdominal anesthesia of the mice, the liver was exposed by laparotomy, and 100 μL of cell suspension (containing 1×10 6 cells) was injected into the liver of each mouse, the wound was sutured, and the mice were routinely fed; c) After 2 weeks of feeding, the small animal in vivo imaging instrument (D-luciferin was injected to detect the fluorescence signal) was used to confirm that the transplanted tumor was successfully established, and then the mice were randomly divided into 4 groups (3 mice in each group): Control group, EM-2 single treatment group, VSVΔ51 single treatment group, and combination treatment group.

[0061] (2) Group processing: a) Control group: intraperitoneal injection of sterile saline, once a day, a total of 5 times; b) EM-2 single treatment group: intraperitoneal injection of EM-2 solution (4 mg / kg), once a day, a total of 5 times; c) VSVΔ51 single treatment group: tail vein injection of VSVΔ51 virus solution (1×10 7 PFU / mL), once a day, a total of 3 times; d) Combination treatment group: intraperitoneal injection of EM-2 and tail vein injection of VSVΔ51 were performed simultaneously, with the same dose and frequency as the single treatment group.

[0062] (3) Index monitoring: a) Tumor growth monitoring: one day before and after administration, the fluorescence signal intensity of the tumor was detected by a small animal live imaging instrument, and the change in tumor volume was quantitatively analyzed; b) Body weight monitoring: body weight was measured every 2 days, and the changes were recorded.

[0063] (4) Tissue sample detection: a) Sample collection: after 5 days of treatment, the mice were sacrificed, and the liver tumor and main organs (heart, spleen, lung, kidney, skeletal muscle, brain) were stripped and fixed with 4% paraformaldehyde for 24 h; b) H&E staining: as in Example 4, organ toxicity was observed; c) IHC staining: tumor sections were stained to detect the positive expression rates of cleaved Caspase-3 (apoptosis), Ki-67 (proliferation), IRF3, VSV-G, and p-IRF3.

[0064] 3. Experimental results: As shown in Figure 5 a, the fluorescence signal intensity of the tumor in the combination treatment group was significantly lower than that in the other three groups, indicating that the tumor growth was significantly inhibited; as shown in Figure 5 b, there was no significant difference in body weight among the mice in each group, and no obvious weight loss was observed, indicating good safety of the treatment; as shown in Figure 5 c, the H&E staining of the main organs of the mice in the combination treatment group was normal, and there was no toxic injury; as shown in Figure 5 d, the positive rate of Cleaved Caspase-3 in the tumor tissue of the mice in the combination treatment group was significantly higher than that in the Control group, the positive rate of Ki-67 was significantly lower than that in the Control group, and the expression level of p-IRF3 was significantly reduced.

[0065] The above results show that EM-2 combined with VSVΔ51 can significantly inhibit the growth of Hepa1-6 orthotopic transplanted tumor, inhibit tumor cell proliferation and promote apoptosis, the mechanism is related to the inhibition of interferon pathway, and the in vivo safety is good.

[0066] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A composition comprising an oncolytic virus, characterized in that, The composition comprises oncolytic virus VSVΔ51 and interferon inhibitor gemmaline-type sesquiterpene lactone compound EM-2.

2. The use of the composition comprising oncolytic virus according to claim 1 in the preparation of an antitumor drug.

3. The application according to claim 2, characterized in that, The tumor is a solid tumor with high ISG15 expression levels.

4. The application according to claim 3, characterized in that, The tumors include liver cancer, colorectal cancer, breast cancer, melanoma, pancreatic cancer, or lung cancer.

5. The application according to claim 2, characterized in that, The effective dose of oncolytic virus VSVΔ51 is 1×10⁻⁶. 7 -10 8 The effective dose of EM-2, a gemmae-type sesquiterpene lactone compound, is 1-100 mg / kg (PFU / mL).

6. The application according to claim 2, characterized in that, Gemmaline-type sesquiterpene lactone compound EM-2 competitively binds to ISG15, thereby blocking the ISGylation modification of p-IRF3, promoting IRF3 protein degradation, and thus inhibiting the interferon signaling pathway, significantly enhancing the antitumor efficacy of oncolytic viruses.

7. The application according to claim 2, characterized in that, The antitumor drugs also include pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The administration methods of the antitumor drugs include oral administration, injection administration, subcutaneous administration, inhalation administration, and rectal administration.

9. The application according to claim 7, characterized in that, The dosage forms of the antitumor drugs include injections, tablets, capsules, granules, powders, syrups, or oral liquids.

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