Application of TBK1 inhibitor and oncolytic virus in preparation of antitumor drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a TBK1 inhibitor and oncolytic virus in preparation of antitumor drugs. The research finds that the TBK1 inhibitor can improve the anti-tumor effect of the oncolytic virus so as to improve the treatment effectiveness of the oncolytic virus as an anti-tumor drug. Animal experiments prove that the combined application of the VSV delta 51 virus and the GSK8612 can obviously cause improvement of a tumor immune microenvironment, so that the inhibition effect on tumor cells is obviously enhanced. The invention provides a new treatment strategy for clinically treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a TBK1 inhibitor combined with an oncolytic virus in the preparation of an anti-tumor drug. Background Art

[0002] Oncolytic viruses are a class of replication-competent viruses that can selectively infect and kill tumor cells while leaving normal cells unharmed. Oncolytic virotherapy is an emerging tumor-targeted treatment strategy that uses natural or genetically engineered viruses to specifically infect tumor cells, causing them to replicate in the tumor microenvironment and cause cell lysis, with minimal impact on normal cells. Oncolytic viruses are mainly divided into natural viruses and genetically modified viruses. Natural oncolytic viruses such as reovirus, Newcastle disease virus, enterovirus and measles virus have natural tumor selectivity. Most modern oncolytic viruses have been genetically modified to enhance their ability to infect tumor cells, improve their selective replication and lysis potential, and enhance the host's anti-tumor immune response. Entering the 21st century, with the positive results of multiple clinical trials, oncolytic virotherapy has gained widespread attention. Researchers continue to explore its potential for application in tumor treatment, making it one of the important means of tumor treatment.

[0003] Vesicular stomatitis virus (VSV) is a non-pathogenic, enveloped, negative-strand RNA rhabdovirus and one of the oncolytic viruses. It can infect almost all cell types, but cannot induce productive infection in healthy cells due to the antiviral response mediated by type I interferon (IFN). However, IFN signaling defects often occur simultaneously with tumor development. Therefore, VSV can infect and selectively destroy tumor cells while causing minimal damage to normal cells. These characteristics make it an ideal oncolytic virus therapeutic agent. VSV has many advantages as an oncolytic virus, including a short replication cycle, the ability to target a variety of tumor cells, the ability to replicate normally under hypoxic conditions in the tumor microenvironment to exert tumor-killing effects, and a small viral genome that is easy to modify. However, wild-type VSV also has many defects, mainly manifested as potential neurotoxicity. In order to reduce neurotoxicity, researchers designed a VSV strain (VSVΔ51) by deleting the methionine at residue 51 of the matrix protein. Compared with the parent strain, VSVΔ51 acquired the ability to strongly induce IFN responses in cells.

[0004] TANK-binding kinase 1 (TBK1) is a multifunctional signaling protein kinase that is crucial in regulating immune responses, cell proliferation and apoptosis, and tumor progression. TBK1 mediates the induction of type I interferons (IFN-α / β) and antiviral innate immunity through Toll-like receptor (TLR)-dependent pathways in the innate immune system. When cells sense invading pathogens such as lipopolysaccharide, they can trigger the phosphorylation of interferon regulatory factor 3 (IRF3) through a TLR4-dependent pathway, releasing I-IFN. Increasing evidence indicates that abnormal activation of TBK1 kinase is closely associated with the development of cancer, including lung cancer, pancreatic cancer, and colorectal cancer. However, despite these findings, the relationship between TBK1 and reduced OV oncolytic sensitivity in chemotherapy-resistant tumors remains unclear, and the exact role and molecular mechanism of overcoming chemotherapy resistance require further investigation.

[0005] TBK1 inhibitors are gradually showing their potential in clinical research, especially in the treatment of cancer and other diseases. Currently, there are many TBK1 inhibitors and related drugs in clinical trials or research stages:

[0006] (1) BX-795, Mechanism of Action: BX-795 is an inhibitor with broad-spectrum anti-kinase activity that has been shown to inhibit the activity of TBK1 and IKKε. Clinical research: This is primarily intended to investigate its potential application in various types of cancer (e.g., combination therapy in tumor immunotherapy).

[0007] (2) M15, Mechanism of Action: M15 is a kinase inhibitor specifically targeting TBK1 and IKKε. It increases cell sensitivity to chemotherapy drugs by inhibiting the signaling pathways regulated by these kinases. Clinical Research: Its anti-tumor activity is currently being evaluated in early clinical trials.

[0008] (3) TIC10, Mechanism of Action: TIC10, as a small molecule immunomodulator, can activate interferon signaling and indirectly regulate TBK1. Clinical Research: TIC10 has previously demonstrated anti-tumor effects in multiple small animal models and has entered clinical trials.

[0009] (4) GSK2646264, Mechanism of Action: This is a selective inhibitor of TBK1 that has shown efficacy against certain cancer types. Clinical studies: This drug is currently under clinical investigation for related cancers.

[0010] (5) BMS-986016, Mechanism of Action: BMS-986016 is an inhibitor targeting TBK1, and studies have shown its potential in tumor treatment. Clinical research: This drug is currently in clinical trials. The use of small molecule inhibitors to selectively promote the growth and replication of OVs at tumor sites can be an effective strategy and shows great potential.

[0011] Although oncolytic virus therapy has shown good promise in a variety of tumor models, not all tumors are sensitive to this therapy. Some tumors may be resistant to the oncolytic effects of oncolytic viruses due to their unique biological characteristics or microenvironmental factors:

[0012] (1) Resistance of the tumor microenvironment: The microenvironment of some tumors may inhibit the replication and spread of oncolytic viruses, for example, by secreting antiviral proteins or immunosuppressive factors to limit the activity of the virus;

[0013] (2) Antiviral ability of tumor cells: Some tumor cells may effectively inhibit viral replication through powerful antiviral signaling pathways, thereby reducing the killing effect of oncolytic viruses;

[0014] (3) Tumor cell heterogeneity: The high heterogeneity of cells within the tumor may make some cell subpopulations insensitive to oncolytic viruses, which directly limits the overall effect of treatment;

[0015] (4) Immune escape mechanism of tumor cells: Some tumor cells evade the attack of the host immune system by regulating immune checkpoint molecules (such as PD-L1), which may weaken the ability of oncolytic viruses to activate anti-tumor immune responses;

[0016] (5) Genetic background of the tumor: Specific genetic variations or gene mutations may affect the sensitivity of the tumor to oncolytic viruses;

[0017] (6) Tumor differentiation status: The degree of differentiation of tumor cells may affect their sensitivity to oncolytic viruses. Generally, undifferentiated or poorly differentiated tumor cells are more resistant to viral infection and killing;

[0018] (7) Tumor angiogenesis: The angiogenesis status of the tumor directly affects the delivery efficiency of oncolytic viruses. Poor angiogenesis may limit the virus from reaching tumor cells, thereby affecting the therapeutic effect.

[0019] Therefore, screening small molecule compounds that have synergistic effects with oncolytic viruses may be an effective strategy to enhance their tumor-killing effect. Summary of the Invention

[0020] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide the use of TBK1 inhibitors combined with oncolytic viruses in the preparation of anti-tumor drugs.

[0021] To achieve the above object, the technical solution adopted by the present invention is:

[0022] In a first aspect, the present invention provides the use of a TBK1 inhibitor in combination with an oncolytic virus in the preparation of an anti-tumor drug.

[0023] TBK1 is an important serine / threonine kinase. TBK1 inhibitors are specific small molecules that target TBK1 function and are primarily used to treat TBK1-related diseases. Growing evidence indicates that abnormal activation of TBK1 kinase is closely associated with the development of tumors, including lung cancer, pancreatic cancer, and colorectal cancer. Studies have shown that TBK1 is an immune escape gene. Targeting TBK1 can enhance the response to PD-1 blockade by sensitizing tumor cells to cell death induced by effector cytokines. Targeting TBK1 can overcome resistance to cancer immunotherapy. The present invention has discovered that TBK1 inhibitors can enhance the anti-tumor effect of oncolytic viruses, thereby improving the therapeutic effectiveness of oncolytic viruses as anti-tumor drugs. Animal experiments have shown that the combined use of VSVΔ51 virus and GSK8612 can significantly improve the tumor immune microenvironment, thereby significantly enhancing the inhibitory effect on tumor cells. This invention provides a new therapeutic strategy for the clinical treatment of tumors.

[0024] In a second aspect, the present invention provides the use of a TBK1 inhibitor in the preparation of an oncolytic virus anti-tumor enhancer.

[0025] Preferably, the TBK1 inhibitor includes substances that reduce or inhibit the activity or expression level of TBK1.

[0026] The TBK1 inhibitor of the present invention is a substance that inhibits TBK1 activity, or a substance that degrades TBK1, or a genetic tool that reduces TBK1 levels.

[0027] Preferably, the substance comprises a compound or a salt form thereof, or a derivative thereof, or an isomer thereof, a nucleic acid molecule, an antibody, or an antibody fragment.

[0028] Preferably, the TBK1 inhibitor is selected from compounds, including but not limited to compounds or derivatives thereof having TBK1 inhibitory effects, or pharmaceutically acceptable salts, solvates, tautomers, isomers, and other compounds that inhibit TBK1 activity. Compounds may be obtained by, but are not limited to, in-house chemical isolation or synthesis or commercial purchase.

[0029] Preferably, the TBK1 inhibitor also includes tools for inhibiting TBK1 gene expression, including but not limited to gene, gene silencing, and gene editing or knockout tools.

[0030] Preferably, the TBK1 inhibitor also includes some small inhibitory nucleic acid molecules, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), ribozymes, and small hairpin RNA (shRNA), which can weaken or eliminate TBK1 expression.

[0031] Preferably, the TBK1 inhibitor further comprises one or more of an antibody, a functional fragment of an antibody, a peptide, and a peptidomimetic. The antibody comprises a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., a bispecific antibody), and / or an antibody fragment linked to PCSK9. The antibody may be a chimeric antibody, a humanized antibody, a CDR-grafted antibody, or a human antibody. The antibody fragment may be, for example, Fab, Fab', F(ab')2, Fv, Fd, a single-chain Fv (scFv), a disulfide-bonded FV (sdFv), or a VL or VH domain. The antibody may be in a conjugated form, for example, conjugated to a tag, a detectable marker, or a cytotoxic agent. The antibody may be of the IgG isotype (e.g., IgG1, IgG2, IgG3, IgG4), IgA, IgM, IgE, or IgD.

[0032] Preferably, the TBK1 inhibitor is a substance (e.g., a compound, amino acid sequence, nucleotide sequence, etc.) or tool that can knock down or affect TBK1 gene expression or reduce the amount or activity of TBK1. Those skilled in the art may modify, replace, or alter the inhibitory compound or genetic tool, but as long as it has the aforementioned TBK1 inhibitory effect, it is considered a TBK1 inhibitor of the present invention and is a homogeneous replacement for the aforementioned substance, compound, or tool.

[0033] Preferably, the oncolytic virus includes at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; the alphavirus includes M1 virus and Geta virus.

[0034] The oncolytic viruses (M1 virus, Geta virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus) mentioned in the present invention refer to currently available oncolytic viruses, but do not exclude some possible natural mutations or mutations (natural mutations, forced mutations, or selective mutations), genetic modifications, sequence additions or deletions, or partial replacements of viruses. The oncolytic viruses described herein include viruses that have undergone the above-mentioned changes, especially the above-mentioned changes do not affect the oncolytic viruses from playing the role described in the present invention.

[0035] Preferably, the alphavirus is the M1 virus with the deposit number CCTCC V201423 (deposited with the China Center for Type Culture Collection, deposit date July 17, 2014). As a virus that is likely derived from the same strain, Genbank Accession No. EF011023 records the sequence of an M1 strain. Getavirus is a virus with up to 97.8% homology to the M1 virus, and the two have a high degree of identity. The M1 virus is also classified as a Getavirus-like virus by some literature. It is expected that the two will have the same efficacy. A single alphavirus strain can also be administered. In other embodiments, multiple strains and / or types of alphavirus can also be used.

[0036] Preferably, the TBK1 inhibitor is at least one of compounds MRT67307 and GSK8612.

[0037] Preferably, the oncolytic virus is at least one of VSVΔ51 and M1 viruses.

[0038] Preferably, the tumor is a solid tumor or a hematological tumor; the solid tumor includes liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer.

[0039] In a third aspect, the present invention provides an anti-tumor pharmaceutical composition comprising a TBK1 inhibitor and an oncolytic virus.

[0040] Preferably, the present invention also provides a drug set for treating tumors, which comprises a TBK1 inhibitor or a derivative thereof or a combination thereof, and an oncolytic virus. The difference between the drug set and the composition is that the TBK1 inhibitor is different from the dosage form of the oncolytic virus, but is independently packaged (for example: a pill, or capsule, or tablet or ampoule containing a TBK1 inhibitor; another pill, or capsule, or tablet or ampoule containing an oncolytic virus).

[0041] In some embodiments, oncolytic viruses, TBK1 inhibitors, and combinations of oncolytic viruses and TBK1 inhibitors may also contain one or more adjuvants. The adjuvant refers to a component in the drug composition that can assist the efficacy of the drug. The drug set may also include independently packaged TBK1 inhibitors and independently packaged oncolytic viruses. The administration of the TBK1 inhibitor and the oncolytic virus in the drug set may be administered simultaneously or in any order, for example, administering the TBK1 inhibitor before the oncolytic virus, or administering the TBK1 inhibitor after the oncolytic virus, or administering both simultaneously.

[0042] In various embodiments, the patient can be a mammal. In some embodiments, the mammal can be a human. The TBK1 inhibitors include, but are not limited to, compounds that inhibit TBK1 activity, such as MRT67307 / GSK8612; or tools for inhibiting TBK1 gene expression, including, but not limited to, gene interference, gene silencing, and gene editing or knockout.

[0043] Preferably, the effective dose of the TBK1 inhibitor is 0.01-200 mg.

[0044] Preferably, the titer of the oncolytic virus is 10 3 -10 9 PFU.

[0045] In the composition or pharmaceutical set, the ratio of TBK1 inhibitor to oncolytic virus can be: 0.01-200 mg: 10 3 -10 9 PFU; preferably 0.1-200mg: 10 4 -10 9 PFU; further preferably 0.1-100mg: 10 5 -10 9 PFU. The preferred dosage is: anti-tumor drugs (such as Alirocumab) are used in the range of 0.01 mg / kg to 200 mg / kg, while the oncolytic virus is used at an MOI of 10 3 to 10 9 (PFU / kg); PCSK9 inhibitors (such as Alirocumab) are preferably used in the range of 0.1 mg / kg to 200 mg / kg, and oncolytic viruses are used at a titer of MOI from 10 4 to 10 9 (PFU / kg); more preferably, the PCSK9 inhibitor (e.g., Alirocumab) is used in the range of 0.1 mg / kg to 100 mg / kg, and the oncolytic virus is used at an MOI of 10 5 to 10 9 (PFU / kg).

[0046] Preferably, the dosage form of the drug includes injection, tablet, and capsule.

[0047] Preferably, the tumor is a tumor insensitive to oncolytic viruses.

[0048] More preferably, the tumor is a tumor insensitive to the VSVΔ51 oncolytic virus.

[0049] The beneficial effects of the present invention are:

[0050] The present invention has discovered that TBK1 inhibitors can enhance the anti-tumor effects of oncolytic viruses, thereby improving the therapeutic effectiveness of oncolytic viruses as anti-tumor drugs. Animal experiments have shown that the combined use of VSVΔ51 virus and GSK8612 can significantly improve the tumor immune microenvironment, thereby significantly enhancing the inhibitory effect on tumor cells.

[0051] The present invention combines GSK8612 and VSVΔ51 virus to act on a chemotherapy-resistant colorectal cancer subcutaneous transplanted tumor mouse model (immunodeficient / immunocompetent). It was found that when the TBK1 inhibitor GSK8612 and VSVΔ51 virus were used in combination, the tumor size and weight were significantly reduced and the survival rate of mice was improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The test results of cell survival after HCT116 / OXA, MC38 / OXA, SW620 / 5FU, LOVO / 5FU, HCT8 / PTX and their parental cells were treated with different concentrations of chemotherapy drugs (A) and the corresponding relative cell survival heat map (B).

[0053] Figure 2 These are the test results of the sensitivity of chemotherapy-sensitive and chemotherapy-resistant colorectal cancer cell lines to oncolytic viruses; Figure A is the VSVΔ51-treated group; Figure B is the HSV-1-treated group.

[0054] Figure 3 Figure 2 is the cell survival test result; Figure A shows the cell survival test results after HCT116 / OXA and SW620 / 5FU cells were treated with MRT67307 (MRT) in the presence or absence of VSVΔ51 (MOI = 0.001); Figure B shows the IC values of HCT116 / OXA and SW620 / 5FU cells after treatment with increasing titers of VSVΔ51 for 24 hours in the presence or absence of MRT. 50 and cell viability test results.

[0055] Figure 4 The dosing schedule for the HCT116 / OXA animal model.

[0056] Figure 5 These are the test results of the effects of drug treatment on the weight and volume of transplanted tumors of colorectal cancer strains in mice with HCT116 / OXA colorectal cancer model after grouping. Figure A shows the effect on the weight of transplanted tumors; Figure B shows the effect on the volume.

[0057] Figure 6 The dosing schedule for the MC38 / OXA animal model.

[0058] Figure 7The results show the effects of drug treatment on the weight and volume of transplanted tumors in MC38 / OXA colorectal cancer model mice after grouping. Figure A shows the effect on the weight of transplanted tumors; Figure B shows the effect on the volume.

[0059] Figure 8 These are the test results of the remission rate of mouse colorectal cancer transplanted tumors after grouping and drug treatment of MC38 / OXA colorectal cancer model mice; Figure A is the Control treatment group; Figure B is the GSK treatment group; Figure C is the VSVΔ51 treatment group; and Figure D is the combined treatment group (Comb). DETAILED DESCRIPTION

[0060] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Unless otherwise specified, the materials and reagents involved in the following experiments are all commercially available products.

[0061] The VSVΔ51 virus used in the experiment of the present invention is sourced from the Guangzhou National Laboratory; HCT116 / OXA is a human colorectal cancer resistant cell line, resistant to oxaliplatin; MC38 / OXA is a mouse colorectal cancer resistant cell line, resistant to oxaliplatin; the cell lines are sourced from Jinan University in Guangzhou.

[0062] Example 1: Sensitivity testing of chemotherapy-resistant colorectal cancer cell lines to chemotherapy drugs

[0063] HCT116 / OXA, MC38 / OXA, SW620 / 5FU, LOVO / 5FU, HCT8 / PTX and their parental cells were treated with different concentrations of chemotherapy drugs and IC 50 Quantify the sensitivity of cell lines to chemotherapeutic drugs.

[0064] Materials: High-glucose DMEM medium, RPMI-1640 medium, fetal bovine serum, chemotherapy-resistant colorectal cancer cell lines and their parental cells (HCT116 / HCT116-OXA (OXA indicates oxaliplatin resistance, and the same applies to the others), SW620 / SW620-5FU, LOVO / LOVO-5FU, MC38 / MC38-OXA, HCT / HCT-PTX), oxaliplatin (OXA), 5-fluorouracil (5FU), paclitaxel (PTX), and CCK-8 reagent.

[0065] method:

[0066] (1) HCT116, HCT116 / OXA, SW620, SW620 / 5FU, HCT8, HCT8 / PTX, LOVO, LOVO / 5FU, MC38, and MC38 / OXA in the logarithmic growth phase were selected. Chemotherapy-resistant cell lines were taken 1 week after being free from chemotherapy drugs. The cell density was adjusted to 1×10 5 cells / mL, inoculated into 96-well cell culture plates, 1×10 4 Pieces / hole.

[0067] (2) After 24 hours, the cells were allowed to adhere to the wall. An equal volume of culture medium without chemotherapy drugs was added to the control group, and an equal volume of PBS was added to the blank control group.

[0068] Experimental group:

[0069] HCT116 and HCT116 / OXA were added with full culture medium containing 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL of OXA, respectively, 100 μL / well;

[0070] SW620, SW620 / 5FU, LOVO, and LOVO / 5FU were added with full culture medium containing 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL of 5-FU, respectively, at 100 μL / well;

[0071] HCT8 and HCT8 / PTX were added with full culture medium containing 20, 10, 5, 2.5, 1.25, and 0.625 μg / mL of PTX, respectively, 100 μL / well;

[0072] MC38 and MC38 / OXA were added with 100 μL / well of OXA at 100, 50, 25, 12.5, 6.25, and 3.125 μg / mL, respectively. Cells were cultured for another 24 hours.

[0073] (3) Dilute the CCK-8 stock solution with complete culture medium (10:1) and add 20 μL to each well. When adding CCK-8, tilt the plate at a 45-degree angle and add the sample. Do not insert the sample directly below the culture medium surface, otherwise bubbles will easily form and affect the test results.

[0074] (4) After adding CCK-8, slightly tilt the culture plate to cover the remaining CCK-8 on the well wall. Gently tap the culture plate to mix the CCK-8 and culture medium. Continue incubation for 2-3 hours. After incubation, shake thoroughly and read the absorbance (OD) value of each group at a detection wavelength of 450 nm using a microplate reader.

[0075] (5) Calculate the cell survival rate of the experimental group according to the following formula: Cell survival rate = (average OD value of the experimental group - average OD value of the blank group) / (average OD value of the control group - average OD value of the blank group) × 100%. Set up four replicate wells for each concentration, and repeat the experiment three times.

[0076] The results are as follows Figure 1 As shown in Table 1, IC 50 The test results showed that the chemotherapy-resistant cell line had significantly reduced sensitivity to chemotherapy drugs compared with the parental colorectal cancer cell line.

[0077] Heatmap showing relative cell viability of the above colorectal cancer cell lines treated with various concentrations of chemotherapeutic drugs. Percent cell inhibition is color-coded by quartile.

[0078] Table 1 IC values of chemotherapy-sensitive and chemotherapy-resistant colorectal cancer cell lines to chemotherapy drugs 50

[0079] cell lines <![CDATA[IC 50 (μg / mL)]]> HCT116 57.23 HCT116 / OXA 215.80 MC38 19.01 MC38 / OXA 44.32 SW620 65.22 SW620 / 5FU 211.90 LOVO 71.48 LOVO / 5FU 131.40 HCT8 19.01 HCT8 / PTX 44.32

[0080] Example 2: Detection of sensitivity of chemotherapy-sensitive and chemotherapy-resistant colorectal cancer cell lines to oncolytic viruses

[0081] A and B: HCT116 / OXA, MC38 / OXA, SW620 / 5FU, LOVO / 5FU, HCT8 / PTX and their parental cells were treated with different MOI gradients of OVs (VSVΔ51, HSV-1). 50 Quantification of sensitivity to different OVs.

[0082] Materials: High-glucose DMEM, RPMI-1640 medium, fetal bovine serum, chemotherapy-resistant colorectal cancer cell lines and their parental cells (HCT116 / HCT116-OXA, SW620 / SW620-5FU, LOVO / LOVO-5FU, MC38 / MC38-OXA, HCT / HCT-PTX), VSVΔ51, HSV-1, and CCK-8 reagent.

[0083] method:

[0084] (1) HCT116, HCT116 / OXA, SW620, SW620 / 5FU, HCT8, HCT8 / PTX, LOVO, LOVO / 5FU, MC38, and MC38 / OXA in the logarithmic growth phase were selected. Chemotherapy-resistant cell lines were taken 1 week after being free from chemotherapy drugs. The cell density was adjusted to 1×10 5 cells / mL, inoculated into 96-well cell culture plates, 1×10 4 Pieces / hole.

[0085] (2) After 24 hours, cells were allowed to adhere to the wall. An equal volume of culture medium without chemotherapy drugs was added to the control group, and an equal volume of PBS was added to the blank control group. All cells were added with VSVΔ51 and HSV-1 at an MOI gradient of 10, 1, 0.1, 0.01, and 0.001, at 100 μL / well and 100 μL / well, respectively. Cells were cultured for another 24 hours.

[0086] (3) Dilute the CCK-8 stock solution with the whole culture medium (10:1) and add 20 μL to each well. When adding CCK-8, tilt the plate at a 45-degree angle and add the sample. Do not insert the sample directly under the culture medium surface, otherwise it will easily generate bubbles and affect the test results.

[0087] (4) After adding CCK-8, slightly tilt the culture plate to cover the remaining CCK-8 on the well wall. Gently tap the culture plate to mix the CCK-8 and culture medium. Continue incubation for 2-3 hours. After incubation, shake thoroughly and read the absorbance (OD) value of each group at a detection wavelength of 450 nm using a microplate reader.

[0088] (5) Calculate the cell survival rate of the experimental group according to the following formula: Cell survival rate = (average OD value of the experimental group - average OD value of the blank group) / (average OD value of the control group - average OD value of the blank group) × 100%. Set up four replicate wells for each concentration, and repeat the experiment three times.

[0089] The results are as follows Figure 2 As shown, IC 50 The test results showed that the chemotherapy-resistant cell lines were significantly less sensitive to OVs compared with the parental colorectal cancer cell lines. The chemotherapy-resistant colorectal cancer cell lines were significantly less sensitive to oncolytic viruses.

[0090] Example 3:

[0091] Materials: RPMI-1640 medium, fetal bovine serum, chemotherapy-resistant colorectal cancer cell lines (HCT116 / OXA and SW620 / 5FU), MRT67307, VSVΔ51, and CCK-8 reagent.

[0092] Experiment A: HCT116 / OXA and SW620 / 5FU cells were treated with increasing doses of the TBK1 inhibitor MRT67307 (MRT) in the presence or absence of VSVΔ51 (MOI = 0.001) for 24 hours, and then cell viability was assessed. 单 -AUC 联合 ) / AUC 联合 The difference in area under the curve (DAUC) was calculated, and the orange area represents DAUC. Statistical significance was determined using one-way ANOVA (n=3).

[0093] Experiment B: Treatment with increasing titers of VSVΔ51 for 24 h with or without MRT (5 μM) and determination of IC 50 The cell survival rate was assessed by one-way analysis of variance (ANOVA) to determine statistical significance (n=3).

[0094] Cell viability test method:

[0095] (1) Cell preparation: resuscitation and culture of HCT116 / OXA and SW620 / 5FU cells.

[0096] (2) Plating: After the cells reach the logarithmic phase, digest, centrifuge, and resuspend the cells to adjust the cell density to 1×10 5 cells / mL, inoculated into 96-well cell culture plates, 1×10 4 Pieces / hole.

[0097] (3) Experimental treatment: After 24 hours, the cells were allowed to adhere to the wall. The control group was treated with an equal volume of culture medium, and the blank control group was treated with an equal volume of PBS. The experimental groups were divided into MRT and MRT combined with VSVΔ51. MRT was diluted in a drug concentration gradient, and 100 μL / well of MRT containing 10, 3, 1, 0.3, and 0.1 μM was added to the whole culture medium. In the MRT combined with VSVΔ51 group, 50 μL / well of MRT containing 10, 3, 1, 0.3, and 0.1 μM was added to the whole culture medium. After 4 hours of continuous culture, VSVΔ51 (MOI = 0.001) was added to the combined group at 50 μL / well and cultured for 24 hours.

[0098] (4) After adding CCK-8, slightly tilt the culture plate to cover the remaining CCK-8 on the well wall. Gently tap the culture plate to mix the CCK-8 and culture medium. Continue incubation for 2-3 hours. After incubation, shake thoroughly and read the absorbance (OD) value of each group at a detection wavelength of 450 nm using a microplate reader.

[0099] (5) Calculate the survival rate of the cells in the experimental group according to the following formula: Cell survival rate = (average OD value of the experimental group - average OD value of the blank group) / (average OD value of the control group - average OD value of the blank group) × 100%. DAUC calculation method: (AUC 单 -AUC 联合 ) / AUC 联合 .

[0100] Shift in half-maximal inhibitory concentration (IC 50 shift) test method:

[0101] (1) Cell preparation: recovery, culture of HCT116 / OXA and SW620 / 5FU cells, and plating in 96-well plates.

[0102] (2) Experimental treatment: After 24 hours, the cells were allowed to adhere to the wall. The control group was added with an equal volume of culture medium, and the blank control group was added with an equal volume of PBS. The experimental components were VSVΔ51 and VSVΔ51 combined with MRT. VSVΔ51 was diluted in a gradient MOI titer, and 10, 1, 0.1, 0.01, and 0.001 of VSVΔ51 full culture were added at 100 μL / well. In the VSVΔ51 combined with MRT group, 10, 1, 0.1, 0.01, and 0.001 of VSVΔ51 full culture were added at 50 μL / well, and MRT (5 μM) was added at 50 μL / well. The cells were cultured for another 24 hours.

[0103] (3) Data processing: CCK-8 detection and calculation of cell viability were the same as above.

[0104] (4)IC 50 Shift calculation method: IC 50 (VSVΔ51 combined with MRT) / IC 50 (VSVΔ51).

[0105] The results are as follows Figure 3 As shown, the results showed that targeting TBK1 combined with oncolytic virus can enhance its oncolytic effect in chemotherapy-resistant colorectal cancer.

[0106] Example 4:

[0107] Materials: high-glucose DMEM medium, RPMI-1640 medium, VSVΔ51 virus, human colorectal cancer cell line HCT116 / OXA, mouse colorectal cancer cell line MC38 / OXA, 4-6 week-old female Nude mice (immunodeficient mice), 4-6 week-old female C57BL / 6J mice (immunocompetent mice).

[0108] method:

[0109] (1) This experiment adopted a randomized single-blind design. 6 HCT116 / OXA or MC38 / OXA cells were injected subcutaneously into the dorsal flanks of 4-6 week-old Nude or C57BL / 6J mice to generate colorectal cancer model mice.

[0110] (2) When the tumor size is ≤200 mm 3 The patients were divided into control group, GSK8612 group (intraperitoneal injection of GSK8612 10 mg / kg / day), VSVΔ51 group (intratumoral injection of 2×10 6PFU / time) and GSK8612 / VSVΔ51 combination group (the same dose of GSK8612 and VSVΔ51 were given in the same way), GSK8612 was injected continuously for 5 days, and then injected for another 5 days after a 2-day interval, for a total of two cycles, and VSVΔ51 was injected once in each cycle.

[0111] The dosing schedule for the HCT116 / OXA animal model is as follows: Figure 4 As shown; the administration schedule of MC38 / OXA animal model is as follows Figure 6 shown.

[0112] (3) The size of the tumor was measured every two days. The volume of the tumor was calculated based on the formula (length × width 2 ) / 2, and one-way ANOVA was performed after measuring the tumor volume. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0113] The results of group administration of HCT116 / OXA colorectal cancer cell model mice are as follows Figure 5 As shown in Figure 2, the results of group administration of MC38 / OXA colorectal cancer model mice are as follows: Figure 7 、 8 In two different tumor cell transplantation animal models, pathological anatomy of tumor volume showed that compared with the control group, the GSK8612 group and the VSVΔ51 group only caused a slight reduction in tumor volume, while the GSK8612 / VSVΔ51 combination group caused a significant reduction in tumor volume and weight.

[0114] In the chemotherapy-resistant colorectal cancer (MC38 / OXA) model mice (immune competent), the combination group (GSK8612 10 mg / kg / day, VSVΔ51 2×10 6 PFU / day) mice had significantly reduced tumor volume and weight. 6 The complete remission rate of mice treated with VSVΔ51 virus alone (VSVΔ51 2×10 6 The anti-tumor effect of GSK8612 (PFU / mouse) was significantly improved compared with the combination of GSK8612 and VSVΔ51.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of TBK1 inhibitors combined with oncolytic viruses in the preparation of anti-tumor drugs.

2. Application of TBK1 inhibitors in the preparation of oncolytic virus anti-tumor enhancers.

3. The use according to claim 1 or 2, characterized in that The TBK1 inhibitors include substances that reduce or inhibit the activity or expression level of TBK1.

4. The use according to claim 3, characterized in that The substances include compounds or salt forms thereof, or derivatives or isomers thereof, nucleic acid molecules, antibodies, and antibody fragments.

5. The use according to claim 1 or 2, characterized in that The oncolytic virus includes at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; the alphavirus includes M1 virus and Geta virus.

6. The use according to claim 1 or 2, characterized in that The TBK1 inhibitor is at least one of compounds MRT67307 and GSK8612; and / or the oncolytic virus is at least one of VSVΔ51 and M1 virus.

7. The use according to claim 1 or 2, characterized in that The tumor is a solid tumor or a hematological tumor; the solid tumor includes liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer.

8. An antitumor pharmaceutical composition, characterized in that: Including TBK1 inhibitors and oncolytic viruses.

9. The antitumor pharmaceutical composition according to claim 8, characterized in that The effective dose of the TBK1 inhibitor is 0.01-200 mg; and / or the titer of the oncolytic virus is 10 3 -10 9 PFU.

10. The antitumor pharmaceutical composition according to claim 8, characterized in that The dosage forms of the medicine include injection, tablet and capsule.

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