Application of combination of recombinant oncolytic virus and monoclonal antibody in preparation of medicine for treating HBV (Hepatitis B Virus) positive liver cancer

Abstract

The invention provides an application of combination of a recombinant oncolytic virus and a monoclonal antibody in preparation of a medicine for treating HBV positive liver cancer. The recombinant oncolytic virus is a recombinant attenuated vesicular stomatitis virus carrying an HBV HBsAg gene and a 4-1BBL gene (wherein the 51st amino acid M in the M gene of the virus is mutated into R). The curative effect of the recombinant oncolytic virus is proved in a tumor model, and the recombinant oncolytic virus can be used for preparing therapeutic vaccines for HBV positive tumors such as HBV positive liver cancer and HBV positive diffuse large B-cell lymphoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to application of a combined drug composition in preparation of a drug for treating HBV-positive liver cancer. BACKGROUND

[0002] Liver cancer is a major malignant tumor, which seriously threatens human health, increases the economic burden of patients, and the global incidence is rising. Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for about 90% of all cases, and 84% of primary hepatocellular carcinoma in China is caused by HBV infection. The development of liver cancer usually goes through the process of chronic hepatitis, cirrhosis to liver cancer. At present, liver cancer is mainly treated by surgical resection and liver transplantation, supplemented by radiotherapy, chemotherapy, targeted therapy and immunotherapy. Even after strict monitoring, many liver cancer patients are in the middle and late stages of tumor, missing the best opportunity for surgery and liver transplantation, which often leads to ineffective treatment. In addition, liver resection and transplantation still face serious damage to liver function and shortage of liver donors. Radiotherapy and chemotherapy lack specificity for tumor cells and have extensive side effects.

[0003] In recent years, significant progress has been made in the research of HCC immunotherapy, which has changed the treatment mode of cancer. In particular, the immunotherapy represented by immune checkpoint inhibitors (ICIs) has broken the single treatment pattern of molecular targeted drugs. Tumor immunotherapy is a treatment method that actively or passively induces the body to produce tumor-specific immune response and plays its role in inhibiting and killing tumor cells. It has the advantages of high specificity and efficiency, and makes the body immune to harmful treatment. However, in clinical practice, immunotherapy only brings lasting survival benefit to a small number of patients, and most patients still face the problem of immunotherapy resistance. Once first-line treatment fails, there is usually no effective treatment, so suitable treatment strategies still need to be developed for HBV-positive liver cancer patients.

[0004] Gene therapy is a biological therapy in which a vector introduces an exogenous gene into target cells to change gene expression. Many studies on liver cancer gene therapy have confirmed that the effect of combined gene therapy is better than that of single gene therapy. Selecting oncolytic viruses (OVs) with oncolytic effects as delivery vectors can achieve higher expression of tumor-specific antigens in the tumor microenvironment, stimulate stronger local tumor immunity. In the past decade, the progress and improvement of immunotherapy combined with improved viral engineering have brought new treatment ideas for OV therapy. Compared with single therapy, the combination of OV therapy and immunotherapy can improve the outcome of cancer.

[0005] HBV belongs to the hepadnavirus family, and has a partially double-stranded, circular DNA genome of 3.2 kb. The HBV genome has four open reading frames, which encode the envelope protein (S), core protein (C), polymerase (P), and X protein (HBX). More than 90% of HBV-related HCC genomes contain integrated HBV-DNA, i.e., most HBV-related HCC cells contain HBV-DNA fragments that do not encode complete HBV antigens. Studies have shown that HCC cells that do not express complete HBV antigens contain short HBV mRNA, which encodes epitopes that are recognized and activated by HBV-specific T cells. Some secondary HCC patients can express complete HBV surface antigen epitopes. In these specific patients, the above phenomena provide guidance for the feasibility of recombinant oncolytic virus treatment. Moreover, the "hot" tumor microenvironment produced by inflammatory cells recruited by oncolytic viruses can also ensure the optimal anti-tumor activity of tumor-specific cytotoxic T cells, which is lacking in current therapeutic HBV tumor vaccine methods.

[0006] OV is a virus that can specifically infect and kill tumor cells. After genetic engineering modification, the specificity of OV infection to tumor cells is increased, and the effect on normal cells is reduced. Oncolytic viruses mediate anti-tumor activity mainly through two ways: one is to replicate in tumor cells, and eventually cause tumor cell death through lysis; the other is to activate the body's systemic anti-tumor immune response by releasing intracellular immunologically related molecules such as damage-associated molecular patterns, tumor-associated antigens, and pathogen-associated molecular patterns. Oncolytic viruses lyse tumors locally to release tumor-associated antigens (TAAs), which is similar to the principle of tumor vaccines, but may not be sufficient to induce tumor-specific T cell responses. More TAAs can be expressed by integrating TAA genes into the viral genome to enhance specific T cell immune responses. Many studies have shown that tumor-specific antigens have high immunogenicity, and spontaneous neoantigen-specific T cells are detected in tumor patients, so in the design of tumor therapeutic vaccines, tumor-specific antigens are preferentially selected as targets, which have higher immunogenicity than self-antigens and reduce the risk of autoimmune. Chronic viral infection is prone to induce cancer by causing repeated tissue damage, inflammation, and integration of viral genes with the host genome. Integrated viral genes can be expressed by host mechanisms to produce viral-derived protein fragments, which are then degraded into viral peptides and assembled with MHC class I to be presented on the cell surface. Therefore, these cancer cells are specifically labeled by viral peptides as tumor antigens. At the same time, these cancer proteins are also tumor-specific, which can cause T cell recognition and killing of the antigen.

[0007] Therefore, it is necessary to develop an effective therapeutic drug for HBV-positive liver cancer. SUMMARY

[0008] The application aims to overcome the defects of the prior art, and provides application of a combined drug composition in preparation of a medicine for treating HBV-positive liver cancer.

[0009] The application is achieved in the following manner:

[0010] In a first aspect of the application, application of a combined drug composition in preparation of a medicine for treating HBV-positive liver cancer is provided, wherein the combined drug composition contains a recombinant oncolytic virus and a monoclonal antibody, the recombinant oncolytic virus is an attenuated vesicular stomatitis virus carrying an HBV HBsAg gene, and the monoclonal antibody is a 4-1BB monoclonal antibody.

[0011] In the embodiments of the application, the recombinant oncolytic virus is referred to as rVSV M51R -S (see patent application CN115806944A).

[0012] Further, the attenuated vesicular stomatitis virus is a vesicular stomatitis virus in which the 51st amino acid of the M gene is mutated from M to R.

[0013] Further, the HBV HBsAg gene is from D-type HBV, and the nucleotide sequence is shown in SEQ ID NO. 1, and the HBV HBsAg gene is located between the G gene and the L gene of the attenuated vesicular stomatitis virus.

[0014] In some embodiments, the 4-1BB monoclonal antibody is a commercial InVivoMAb anti-mouse 4-1BB (CD137) (Bio x cell; item # BE0239).

[0015] In a second aspect of the application, a medicine for treating HBV-positive liver cancer is provided, which contains a recombinant oncolytic virus and a monoclonal antibody, the recombinant oncolytic virus is an attenuated vesicular stomatitis virus carrying an HBV HBsAg gene, and the monoclonal antibody is a 4-1BB monoclonal antibody.

[0016] Further, the medicine for treating HBV-positive liver cancer contains a pharmaceutically acceptable carrier and / or an excipient. The excipient is selected from one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a colorant.

[0017] Further, the dosage form of the drug for treating HBV-positive liver cancer is selected from one of granules, tablets, pills, capsules, injections and dispersions.

[0018] In a third aspect of the present application, a multi-gene fusion recombinant oncolytic virus is provided, which is an attenuated vesicular stomatitis virus carrying an HBV HBsAg gene and a 4-1BBL gene, and the attenuated vesicular stomatitis virus is a vesicular stomatitis virus in which the 51st amino acid of the M gene is mutated from M to R.

[0019] Further, the HBsAg gene and the 4-1BBL gene are located between the G gene and the L gene of the vesicular stomatitis virus, and the HBsAg gene and the 4-1BBL gene are linked by a P2A cleavage peptide.

[0020] Further, the nucleotide sequence of the HBV HBsAg gene is shown in SEQ ID NO. 1, the nucleotide sequence of the P2A is shown in SEQ ID NO. 2, and the nucleotide sequence of the 4-1BBL gene is shown in SEQ ID NO. 3.

[0021] In a fourth aspect of the present application, a construction method of the multi-gene fusion recombinant oncolytic virus is provided, and the method comprises:

[0022] PCR amplification is performed on the primer pair shown in SEQ ID NO. 4-SEQ ID NO. 5 and the pAAV-HBV1.3 as a template to obtain an HBsAg-P2A nucleic acid fragment;

[0023] PCR amplification is performed on the primer pair shown in SEQ ID NO. 6-SEQ ID NO. 7 and the mouse lung tissue cDNA genome as a template to obtain a 4-1BBL-P2A nucleic acid fragment;

[0024] The HBsAg-P2A nucleic acid fragment and the 4-1BBL-P2A nucleic acid fragment are amplified by the primer pair shown in SEQ ID NO. 4, SEQ ID NO. 7 to link the two target genes to obtain a linking fragment;

[0025] The linking fragment is inserted into the Xho I and Nhe I enzyme cutting sites of the pXN2-M51R-GFP vector by enzyme cutting and enzyme ligation to obtain a pXN2-M51R-HBsAg-P2A-4-1BBL plasmid;

[0026] The pXN2-M51R-HBsAg-P2A-4-1BBL plasmid is co-transfected with a packaging plasmid of the vesicular stomatitis virus into cells for packaging to obtain a multi-gene fusion recombinant oncolytic virus.

[0027] The multi-gene fusion recombinant oncolytic virus of the application is also called recombinant oncolytic virus rVSV M51R -S-4-1BBL.

[0028] In the fifth aspect of the application, the use of the multi-gene fusion recombinant oncolytic virus in the preparation of a medicament for treating HBV-positive liver cancer is provided.

[0029] In the sixth aspect of the application, a tumor therapeutic vaccine is provided, and the effective component of the tumor therapeutic vaccine is the multi-gene fusion recombinant oncolytic virus.

[0030] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0031] In the application, the gene encoding the surface antigen of hepatitis B virus is inserted into the attenuated oncolytic virus vector, and the surface antigen of hepatitis B virus is expressed in a large amount by infecting the HBV-positive liver cancer cells, and the T cell agonist 4-1BB monoclonal antibody is used in combination, so as to enhance the tumor-specific cellular immune response and improve the clearance of tumor cells by the killer T cells. Meanwhile, the gene encoding the surface antigen of hepatitis B virus and the gene of the ligand 4-1BBL of the T cell agonist receptor 4-1BB are inserted into the attenuated oncolytic virus vector, the antigen delivery to DCs is strengthened, the MHC I processing and presentation are promoted, the activation of the functionally exhausted T cells is promoted, and the therapeutic effect of the oncolytic virus expressing a single tumor antigen gene is further enhanced.

[0032] The recombinant oncolytic virus rVSV of the application M51R The -S and 4-1BB monoclonal antibody combination system, the recombinant oncolytic virus rVSV M51R The -S-4-1BBL therapeutic vaccine can obviously inhibit the tumor growth of the HBV-positive liver cancer model mice and activate a stronger immune response. The recombinant oncolytic virus rVSV M51R The survival rate of the HBV-positive liver cancer mice using the -S and 4-1BB monoclonal antibody in combination reaches 44%, which is much higher than that of the control group; after the treatment of rVSV M51R The tumor control effect of the HBV-positive liver cancer mice after the treatment of -S-4-1BBL is obviously much higher than that of the control rVSV M51R The -S group. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The recombinant oncolytic virus rVSV M51R The modification diagram of -S-4-1BBL.

[0034] Figure 2 The construction of the virus packaging plasmid pXN2-M51R-HBs-4-1BBL is shown. In the construction, Figure 2 The plasmid pXN2-M51R-HBs-4-1BBL map is shown.

[0035] Figure 3 is a recombinant oncolytic virus rVSV M51R Construction and identification of -S-4-1BBL. Figure 3 (A) shows the recombinant oncolytic virus rVSV M51R Infection of VERO-E6 cells with -S-4-1BBL, and verification of expression of hepatitis B virus surface protein in cell lysate by ELISA. Figure 3 (B) shows the recombinant oncolytic virus rVSV M51R Infection of VERO-E6 cells with -S, and verification of expression of 4-1BBL (ligand of 4-1BB) protein by Western Blot;

[0036] Figure 4 is a tumor therapeutic vaccine recombinant oncolytic virus rVSV M51R Evaluation of therapeutic effect of combination of -S and 4-1BB monoclonal antibody on HBV-positive hepatocarcinoma. Figure 4 (A) shows tumor volume in different treatment groups in a subcutaneous tumor-bearing model of HBV-positive hepatocarcinoma. Figure 4 (B) shows survival rate comparison in a subcutaneous tumor-bearing model of HBV-positive hepatocarcinoma.

[0037] Figure 5 is a tumor therapeutic vaccine recombinant oncolytic virus rVSV M51R Construction and identification of -S-4-1BBL and recombinant oncolytic virus rVSV M51R Construction and identification of -S, recombinant oncolytic virus rVSV M51R Evaluation and comparison of therapeutic effect of combination of -S and 4-1BB monoclonal antibody on HBV-positive hepatocarcinoma. Figure 5 (A) shows tumor volume in different treatment groups in a subcutaneous tumor-bearing model of HBV-positive hepatocarcinoma. Figure 5 (B) shows body weight change of mice in different treatment groups in a subcutaneous tumor-bearing model of HBV-positive hepatocarcinoma. DETAILED DESCRIPTION

[0038] The advantages and various effects of the present application will be more clearly presented hereinafter in combination with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, rather than limit the present application.

[0039] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood in the manner as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. If there is a conflict, the present specification takes precedence.

[0040] Unless otherwise specifically explained, various materials, reagents, instruments and equipment that are used in the present application are commercially available or are prepared by conventional methods.

[0041] The application of the combined pharmaceutical composition of the present application in the preparation of a medicament for treating HBV-positive liver cancer will be described in detail below in conjunction with examples and experimental data.

[0042] As used herein, "combination" or "combination therapy" refers to the administration of two or more active agents, where the agents can be administered simultaneously, or sequentially in any order.

[0043] As used herein, "effective amount" refers to an amount that is sufficient to prevent or treat the symptoms or disease of a medical condition. After being used in a particular patient or medical subject, the following changes can be produced: the condition to be treated is improved, the overall health of the patient / subject is improved. The effective amount can also be a dosage regimen below the maximum dosage that avoids significant side effects or toxic effects.

[0044] Example 1, Multi-gene fusion recombinant oncolytic virus and its preparation method

[0045] This example further describes the recombinant oncolytic virus using the vesicular stomatitis virus system as an example:

[0046] rVSV M51R -S-4-1BBL (see the modification schematic diagram Figure 1 ) construction process, specifically comprising the following steps: through molecular biology experimental means, the 51st amino acid of the M gene of the vesicular stomatitis virus is mutated from M to R, and the mutated M gene is cloned into the plasmid pXN2-GFP containing the full-length genome of VSV through XbaI and MIuI enzyme cutting sites (the plasmid pXN2-GFP is a gift from Professor John K Rose of Yale University, and the construction of the plasmid is described in the literature 1. Dalton KP, Rose JK. Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology. 2001 Jan

[0047] 20; 279(2): 414-21.) and plasmid pXN2-M51R-GFP was prepared (the construction of the plasmid is described in 2. Gaddy DF, Lyles DS. Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways. J Virol. 2005

[0048] Apr; 79(7): 4170-9. doi: 10.1128 / JVI.79.7.4170-4179.2005.).

[0049] Then we obtained the HBsAg sequence to clone the plasmid pAAV-HBV1.3 (the construction of the plasmid pAAV-HBV1.3 is described in 3. Xu Z, Zhao L, Zhong Y, Zhu C, Zhao K, Teng Y, Cheng X, Chen Q, Xia Y. A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA. Cell Mol Gastroenterol Hepatol. 2022; 13(4): 1001-1017.).

[0050] In addition, we obtained the 4-1BBL sequence to design primers with the original mouse lung tissue cDNA genome in the laboratory as a template.

[0051] According to the pXN2-M51R-GFP, HBsAg sequence, 4-1BBL sequence, design homologous recombination upstream and downstream primers, add restriction enzyme cutting sites Xho I and Nhe I to the upstream and downstream respectively, and add P2A cleavage peptide sequence in the middle of HBsAg sequence and 4-1BBL sequence, add Flag tag to the downstream of 4-1BBL sequence, and the sequence primers are shown in the following table.

[0052] Table 1

[0053]

[0054] (1) Amplification of HBsAg-P2A sequence: pAAV-HBV1.3 was used as a template, and PCR amplification was performed under the following conditions: the PCR reaction system included:

[0055] pAAV-HBV1.3: 1 μL;

[0056] pXN2-HBsAg-F1: 1 μL;

[0057] HBsAg-P2A-R1: 1 μL;

[0058] 2x Primer Star Mix: 25 μL;

[0059] ddH2O: 22 μL.

[0060] PCR reaction conditions: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 35 cycles of PCR, 72°C sufficient extension for 10 min, and 4°C preservation. After 1% agarose gel electrophoresis of the PCR product, the target band was cut and DNA gel recovery was performed according to the instructions of the gel recovery kit of Axygen Company.

[0061] (2) Amplification of 4-1BBL-P2A sequence: mouse lung tissue cDNA genome as a template, PCR amplification was performed as follows: the PCR reaction system included:

[0062] Human PBMC cDNA: 1 μL;

[0063] P2A-4-1BBL-F2: 1 μL;

[0064] pXN2-4-1BBL-R2: 1 μL;

[0065] 2x Primer Star Mix: 25 μL;

[0066] ddH2O: 22 μL.

[0067] PCR reaction conditions: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 35 cycles of PCR, 72°C sufficient extension for 10 min, and 4°C preservation. After 1% agarose gel electrophoresis of the PCR product, the target band was cut and DNA gel recovery was performed according to the instructions of the gel recovery kit of Axygen Company.

[0068] (3) Enzymatic digestion of pXN2-M51R-GFP vector: pXN2-M51R-GFP vector was double-digested with Xho I and Nhe I restriction endonucleases, and the reaction system was as follows:

[0069] pXN2-M51R-GFP: 2 μg;

[0070] Xho I: 1 μL;

[0071] Nhe I: 1 μL;

[0072] 10X M Buffer: 5 μL.

[0073] 37℃ enzyme cutting for 50 min, and the enzyme cutting product was detected by 1% DNA agarose gel electrophoresis and recovered by cutting the gel.

[0074] (4) Two target genes were connected, and the connection system was as follows: HBsAg-P2A sequence: 1 μL; P2A-4-1BBL sequence: 1 μL; pXN2-HBsAg-F1: 1 μL; pXN2-4-1BBL-R2: 1 μL; 2X Primer Star Mix: 25 μL; ddH2O: 21 μL. PCR reaction condition: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 PCR cycles, 72℃ sufficient extension for 10 min, 4℃ preservation. After 1% agarose gel electrophoresis of the PCR product, the target band was cut and recovered by DNA gel recovery, and the operation was performed according to the instructions of the gel recovery kit of Axygen company.

[0075] (5) Connection of target gene and vector, and the connection system was as follows:

[0076] HBsAg-P2A-4-1BBL target gene fragment: 3.5 μL;

[0077] pXN2-M51R-GFP enzyme cutting product: 2.5 μL;

[0078] 2X Basic assembly mix: 6 μL.

[0079] 50℃ reaction for 45 min.

[0080] (6) Transformation of the connection system: 50 μL stable competent cells were added to 12 μL connection system, and the mixture was placed on ice for 30 min, 42℃ heat shock for 90 s, and placed on ice for 2 min. 1 mL of LB medium without antibiotics was added, and the mixture was cultured at 37℃ and 250 rpm for 1 h. The mixture was centrifuged at 4000 rpm for 30 s, and the supernatant was discarded. 100 μL of LB medium was added to resuspend the bacteria, and the bacteria were uniformly coated on LB solid plate containing Kana antibiotic. After 8-12 h of inverted culture at 37℃, single colonies were picked and amplified in liquid LB for 8-12 h. Referring to the instructions of AXY GEX plasmid small amount extraction kit, the pXN2-M51R-HBsAg-P2A-4-1BBL plasmid was extracted in small amount.

[0081] (7) Sequencing of the target fragment, and the reaction system was as follows:

[0082] pXN2-M51R-HBsAg-P2A-4-1BBL plasmid: 1 μL;

[0083] pXN2-HBsAg-F1: 1 μL;

[0084] pXN2-4-1BBL-R2: 1 μL;

[0085] 2x Primer Star Mix: 25 μL;

[0086] ddH2O: 21 μL.

[0087] PCR reaction conditions: 94°C pre-denaturation 2 min, 94°C denaturation 30 s, 60°C annealing 30 s, 72°C extension 30 s, 35 PCR cycles, 72°C sufficient extension 10 min, 4°C storage. The PCR product was subjected to 1% agarose gel electrophoresis and photographed by gel imaging system.

[0088] In this embodiment, we find the DNA sequence of the surface antigen of D-type hepatitis B virus, and design primers with plasmid AAV-HBV1.3 and mouse lung tissue cDNA genome as templates, PCR to obtain S-HBs gene fragment and 4-1BBL gene fragment, and clone the target gene between G and L genes of pXN2-M51R vector with M51R mutation by homologous recombination, to obtain recombinant plasmid pXN2-M51R-HBsAg-P2A-4-1BBL, and the recombinant plasmid map is as Figure 2 , the inserted target gene is 1686 bp, and the company sequencing comparison is correct.

[0089] Four plasmids required for packaging recombinant vesicular stomatitis virus: pXN2-M51R-GFP / pXN2-M51R-HBsAg-P2A-4-1BBL, pP, pN, pL (plasmids pP, pN, pL were kindly gifted by Professor John K Rose of Yale University, and their construction was described in the literature 1. Dalton KP, Rose JK. Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology. 2001 Jan 20;279(2):414-21.). When the growth density of BSR cells (a clonal cell line of the commercial cell line BHK-21 cells, with a T7 promoter inserted into its genome) reached 80% in a 10 cm cell culture dish, the vesicular stomatitis virus packaging plasmids were added to the culture medium by liposome transfection method (Lipo3000 transfection reagent) according to the following ratio:

[0090] (a) pXN2-M51R-GFP / pXN2-M51R-HBsAg: pP: pN: pL = 2.5: 1.25: 1: 0.5,

[0091] (b) PEI volume: total mass of four plasmids = 3: 1

[0092] After transfecting BSR cells for 4 h, vaccinia virus containing T7 RNA polymerase (its construction was described in the literature 4. Yang F, Tan J, Fang Y, Chen G, Zhang Y, Hu Q, Han W, Liu Y, Fu B, Jing Z, Li W. The Multiplicity of Infection of Recombinant Vaccinia Virus Expressing the T7 RNA Polymerase Determines the Rescue Efficiency of Vesicular Stomatitis Virus. Front Microbiol. 2022 Apr 4;13:846426.) was added to the culture medium at a MOI of 1, and the BSR cells were infected for 2 h.

[0093] The supernatant was collected 48h after infecting BSR cells, and filtered with a 0.22 μm filter to remove the poxvirus. The collected virus supernatant was used to infect Vero cells again, and 24h later, the cytopathic effect was observed. If the cells were rounded, it indicated that the VSV virus was successfully packaged in vitro.

[0094] The mutant attenuated strain rVSV was rescued in BSR cells by reverse genetics M51R -GFP, rVSV M51R -S-4-1BBL. Then the rVSV M51R -S-4-1BBL was used to infect Vero-E6 cells, and ELISA was used to verify that the S-HBsAg protein of HBV was efficiently expressed in the lysate of the infected tumor cells. The specific results are shown in Figure 3 A. Western blotting was used to verify that 4-1BBL was efficiently expressed in the lysate of the infected Vero-E6 cells. The specific results are shown in Figure 3 B.

[0095] The cultured Vero cells were discarded from the original culture medium, and 10 mL of DMEM medium containing 2% serum was added to each plate of cells. The recombinant VSV virus (rVSV M51R -GFP, rVSV M51R -S-4-1BBL) was used to infect Vero cells at a dose of MOI = 0.01. Since VSV virus can be transmitted by aerosol, the VSV-infected cells were cultured separately to prevent cross-contamination. The cell morphology was observed 24h after infection. After the recombinant VSV virus successfully infected the Vero cells, the cells were rounded. When most of the cells were rounded and more than 50% of the cells were floating, the amplified VSV virus was collected and centrifuged at 3500 rpm for 20 min to discard the cell debris.

[0096] A solution of 40% PEG8000 / 2.5M NaCl and 1x TNF buffer were prepared and sterilized by passing through a 0.22 μm filter. For every 40 mL of VSV virus-infected supernatant, 10 mL of 40% PEG8000 / 2.5M NaCl solution was added to form a mixed solution with a final concentration of 8% PEG8000 / 0.5M NaCl, and thoroughly mixed and placed at 4°C overnight. The virus solution that had been standing overnight was centrifuged at 12000 rpm for 1h, and at this time the precipitated virus appeared on the wall of the centrifuge tube, and the virus was resuspended with 2 mL of 1x TNE buffer.

[0097] Next, the virus is subjected to angle ultra-centrifugation. 2 mL of the virus suspension and 30 mL of 1xTNF buffer solution are mixed into a sterile ultracentrifuge tube, and the ultracentrifuge is used to centrifuge the tube at the lowest acceleration, for 2.5 h. After the ultracentrifugation is completed, the supernatant is discarded, and the virus precipitate is resuspended with 200 μL of 1xTNF buffer solution, aliquoted into 20 μL / tube, and stored at -80°C.

[0098] Example 2, recombinant oncolytic virus rVSV M51R The use of -S and 4-1BB monoclonal antibodies in combination in the preparation of a medicament for treating HBV-positive liver cancer

[0099] This example describes the construction of recombinant oncolytic virus rVSV M51R The use of -S and 4-1BB monoclonal antibodies in combination in the treatment of HBV-positive liver cancer is described in detail.

[0100] 1, rVSV M51R The construction process of -S (see the modification schematic diagram Figure 1 ) has been disclosed in patent application CN 115806944A, which specifically includes the following steps:

[0101] Through molecular biology experimental means, the 51st amino acid of the M gene of the vesicular stomatitis virus is mutated from M to R, and the mutated M gene is cloned into the plasmid pXN2-GFP containing the full-length genome of VSV through XbaI and MIuI enzyme cutting sites (the plasmid pXN2-GFP was donated by Professor John K Rose of Yale University, and the construction of the plasmid is described in the literature 1. Dalton KP, Rose JK. Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology. 2001 Jan

[0102] 20; 279(2): 414-21.) and plasmid pXN2-M51R-GFP was prepared (the construction of the plasmid is described in 2. Gaddy DF, Lyles DS. Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways. J Virol. 2005

[0103] Apr; 79(7): 4170-9. doi: 10.1128 / JVI.79.7.4170-4179.2005.).

[0104] Then we obtained the HBsAg sequence to clone the plasmid pAAV-HBV1.3 (the construction of the plasmid pAAV-HBV1.3 is described in 3. Xu Z, Zhao L, Zhong Y, Zhu C, Zhao K, Teng Y, Cheng X, Chen Q, Xia Y. A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA. Cell Mol Gastroenterol Hepatol. 2022; 13(4): 1001-1017.) as a template. And add the Flag tag sequence upstream, the primers are as shown in the following table:

[0105]

[0106] (1) Amplification of HBsAg: pAAV-HBV1.3 as a template, PCR amplification was performed as follows: the PCR reaction system included:

[0107] pAAV-HBV1.3: 1 μL;

[0108] pXN2-HBsAg-F: 1 μL;

[0109] pXN2-HBsAg-R: 1 μL;

[0110] 2x Primer Star Mix: 25 μL;

[0111] ddH2O: 22 μL

[0112] PCR reaction conditions: 94℃ pre-denaturation 2 min, 94℃ denaturation 30 s, 60℃ annealing 30 s, 72℃ extension 1 min, 35 PCR cycles, 72℃ full extension 10 min, 4℃ preservation. After 1% agarose gel electrophoresis of the PCR product, the target band was cut and the DNA gel was recovered according to the instructions of the Axygen gel recovery kit.

[0113] (2) Enzymatic digestion of pXN2-M51R-GFP vector: the pXN2-M51R-GFP vector was double-digested with Xho I and Nhe I restriction endonucleases, and the reaction system was as follows:

[0114] pXN2-M51R-GFP: 2 μg;

[0115] Xho I: 1 μL;

[0116] Nhe I: 1 μL;

[0117] 10×M Buffer: 5 μL 37℃ enzyme digestion for 2 h, and the enzyme digestion product was detected by 1% DNA agarose gel electrophoresis and gel recovery.

[0118] (2) Ligation of the target gene and the vector, and the ligation system was as follows:

[0119] HBsAg target gene fragment: 3.5 μL;

[0120] pXN2-M51R-GFP enzyme digestion product: 2.5 μL;

[0121] 2×Basic assembly mix: 6 μL,

[0122] 50℃ reaction for 30 min.

[0123] Ligation system transformation: 50 μL stable competent cells were added to 12 μL ligation system, and the mixture was placed on ice for 30 min, 42℃ heat shock for 90 s, and placed on ice for 2 min. 1 mL of LB medium without antibiotics was added, and the mixture was cultured at 37℃ and 250 rpm for 1 h. After centrifugation at 12000 rpm for 1 min, the supernatant was discarded, 100 μL of LB medium was left to resuspend the bacteria, and the bacteria were uniformly coated on an LB solid plate containing Kana antibiotic. After 8-12 h of inverted culture at 37℃, single colonies were picked and expanded in liquid LB for 8-12 h. Referring to the AXY GEX Plasmid Miniprep Kit Instructions, the pXN2-M51R-HBsAg plasmid was extracted in small amounts.

[0124] Plasmid enzyme digestion verification and sequencing, 37℃ enzyme digestion for 2 h, and the enzyme digestion product was detected by 1% DNA agarose gel electrophoresis, and the plasmid with correct enzyme digestion results was sent to the company for sequencing.

[0125] 2、This embodiment is to use the rVSV M51R -S combined with 4-1BB monoclonal antibody for the treatment of HBV-positive liver cancer, using a viral vector system to express hepatitis B virus antigens to activate specific anti-tumor immune responses. The specific embodiment involves the treatment of the virus expressed by the specific purified recombinant non-segmented, negative-strand RNA virus vector.

[0126] (1) H22-DE-HBV liver cancer subcutaneous tumor model establishment and immunotherapy

[0127] H22-DE-HBV cells were cultured in 1640 complete medium, and when the cells grew to 70-80% density, tumor cells were inoculated. Before inoculating the tumor cells, their state was observed, and the cell growth rate was maintained in the exponential growth phase. One day before cell inoculation, the right thigh lateral skin of the mice was shaved with an electric shaver. The type of tumor-bearing mice was selected as 6-8 week-old C57BL / 6 male mice, and the H22-DE-HBV tumor cells were resuspended with PBS, and the cell density was 1×10 6 / 200μL / mouse (i.e. 5×10 6 / mL), and each mouse was subcutaneously inoculated with tumor cells 200μL on the lateral groin of the thigh. Generally, tumors were formed in about 6 days, the tumor size was measured and the mice were grouped after the results were counted, and the average volume of the mouse tumor was generally maintained at 50mm 3 around. The tumor size was counted every other day, and the tumor volume was calculated, and the tumor volume calculation formula was V=0.52×L×W 2 (L=length, W=width), and when the tumor volume reached 1500mm 3 , the mice were sacrificed by cervical dislocation according to the experimental humane endpoint. On the 6th day, the mice with appropriate tumor size in the HBV-positive liver cancer model were divided into 3 groups:

[0128] (a) PBS group;

[0129] (b) Oncolytic virus rVSV M51R -S group

[0130] (c) Oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group;

[0131] Oncolytic virus administration: 5×10 7 PFU (50μL) was injected intratumorally with a 1mL insulin syringe on the 6th, 8th, and 10th days.

[0132] Monoclonal antibody administration: 5mg / kg / time was injected intraperitoneally with a 1mL insulin syringe on the 6th, 9th, 12th, and 15th days.

[0133] The tumor size was counted every other day and the tumor volume was calculated. The formula for tumor volume calculation is: V = 0.52 × L × W 2 (L = length, W = width), wait until the tumor volume reaches 1500mm 3 According to the humane endpoint of the experiment, mice were killed by cervical dislocation.

[0134] Oncolytic virus rVSV M51R -S group, oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group, such as Figure 4 As shown in A, on day 16 after viral treatment, the average volume of the PBS group was 1045 mm 3 , rVSV M51R The average tumor volume in the S group was 317.8 mm 3 , oncolytic virus rVSV M51R The average tumor volume in the S group and the 4-1BB monoclonal antibody combination group was approximately 28.4 mm 3 about.

[0135] Compared with the control group PBS group, the other three groups had significant tumor inhibition effects; M51R Compared with the -S group, oncolytic virus rVSV M51R The -S group and the 4-1BB monoclonal antibody combination group further improved the tumor inhibition rate, and the treatment effect was the best among the three groups.

[0136] (2) Establishment of H22-DE-HBV liver cancer subcutaneous tumor model and observation of survival rate

[0137] H22-DE-HBV cells were cultured with 1640 complete medium. When the cells grew to 70-80% density, tumor cells were inoculated. Observe the status of the tumor cells before inoculation. The cell growth rate should be maintained in the exponential growth phase. One day before cell inoculation, shave the epidermal hair on the outer side of the right thigh of the mouse with an electric shaver. The tumor-bearing mice were 6-8 weeks old C57BL / 6 male mice. H22-DE-HBV tumor cells were resuspended in PBS and the inoculation cell density was 1×10 6 / 200μL / mouse (i.e. 5×10 6 Each mouse was inoculated with 200 μL of tumor cells subcutaneously in the groin of the thigh. Tumors usually formed in about 6 days. The tumor size was measured and the mice were grouped after statistical analysis. The average tumor volume of mice was generally maintained at 50 mm. 3 Every other day, the tumor size was counted and the tumor volume was calculated. The formula for tumor volume calculation is: V = 0.52 × L × W 2 (L = length, W = width), wait until the tumor volume reaches 1500mm 3According to the humane endpoint of the experiment, mice were killed by cervical dislocation. On the 6th day, mice with appropriate tumor size in the mouse HBV-positive liver cancer model were divided into 4 groups:

[0138] (a) PBS group;

[0139] (b) Oncolytic virus rVSV M51R -S group (recombinant oncolytic virus previously constructed in the laboratory)

[0140] (c) 4-1BB mAb alone / combination group;

[0141] (d) Oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group;

[0142] Oncolytic virus administration: On days 6, 8, and 10, the virus was injected intratumorally using a 1 mL insulin syringe at a dose of 5 × 10 7 PFU (50 μL) was administered intratumorally.

[0143] Monoclonal antibody administration: On days 6, 9, 12, and 15, the monoclonal antibody was intraperitoneally injected using a 1 mL insulin syringe at a dose of 5 mg / kg / time.

[0144] PBS group, oncolytic virus rVSV M51R -S group, 4-1BB monoclonal antibody group, oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group, such as Figure 4 As shown in B, the survival rate of the PBS group was 0%, and the rVSV group was 0%. M51R The survival rate of the -S group was 15.4%, the survival rate of the 4-1BB monoclonal antibody group was 11.1%, and the survival rate of the oncolytic virus rVSV group was 15.4%. M51R The survival rate of the -S group and the 4-1BB monoclonal antibody combination group was about 44%, which was the best among the four groups.

[0145] Compared with the control group PBS group, the other three groups had significant tumor inhibition effects; M51R Compared with the group using 4-1BB monoclonal antibody alone, the oncolytic virus rVSV M51R The combination of the -S group and the 4-1BB monoclonal antibody group further improved the tumor inhibition rate, with the best therapeutic effect among the four groups, and also increased the survival rate of mice.

[0146] Example 3, oncolytic virus rVSV M51R -Application of S-4-1BBL in the preparation of drugs for treating HBV-positive liver cancer

[0147] Oncolytic virus rVSV M51R The treatment of HBV-positive liver cancer in mice using S-4-1BBL is described in detail as an example.

[0148] This example is the rVSV constructed using Example 1 M51R -S-4-1BBL evaluates the efficacy of HBV-positive liver cancer treatment, using a viral vector system to express hepatitis B virus antigens and activate specific anti-tumor immune responses. Specific implementation cases involve the use of specific purified recombinant non-segmented, negative-strand RNA viral vectors to express viral antigens for treatment.

[0149] (1) Establishment of H22-DE-HBV liver cancer subcutaneous tumor model and immunotherapy

[0150] H22-DE-HBV cells were cultured with 1640 complete medium. When the cells grew to 70-80% density, tumor cells were inoculated. Observe the status of the tumor cells before inoculation. The cell growth rate should be maintained in the exponential growth phase. One day before cell inoculation, shave the epidermal hair on the outer side of the right thigh of the mouse with an electric shaver. The tumor-bearing mice were 6-8 weeks old C57BL / 6 male mice. H22-DE-HBV tumor cells were resuspended in PBS and the inoculation cell density was 1×10 6 / 200μL / mouse (i.e. 5×10 6 Each mouse was inoculated with 200 μL of tumor cells subcutaneously in the groin of the thigh. Tumors usually formed in about 6 days. The tumor size was measured and the mice were grouped after statistical analysis. The average tumor volume of mice was generally maintained at 50 mm. 3 Every other day, the tumor size was counted and the tumor volume was calculated. The formula for tumor volume calculation is: V = 0.52 × L × W 2 (L = length, W = width), wait until the tumor volume reaches 1500mm 3 According to the humane endpoint of the experiment, mice were killed by cervical dislocation. On day 6, mice with appropriate tumor size in the mouse HBV-positive liver cancer model were divided into 3 groups:

[0151] (a) Oncolytic virus rVSV M51R -S group (recombinant oncolytic virus previously constructed in the laboratory)

[0152] (b) Oncolytic virus rVSV M51R -S-4-1BBL group;

[0153] (c) Oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group;

[0154] Oncolytic virus administration: On days 6, 8, and 10, the virus was injected intratumorally using a 1 mL insulin syringe at a dose of 5 × 10 7 PFU (50 μL) was administered intratumorally.

[0155] Oncolytic virus rVSV M51RPharmacodynamic performance of -S-4-1BBL, such as Figure 5 As shown, three consecutive days of injection treatment, rVSV M51R -S-4-1BBL and control virus rVSV M51R -S can significantly inhibit tumor growth and improve the survival of tumor-bearing mice. By analysis, it can be seen that on the 18th day after virus treatment, rVSV M51R -S group, the average tumor volume was about 441mm 3 ,rVSV M51R -S-4-1BBL treatment group, the average tumor volume was about 303mm 3 , oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group was about 91.1mm 3 . From the results, rVSV M51R -S-4-1BBL treatment group has a stronger tumor inhibition effect than rVSV M51R -S group, but the effect is not as good as rVSV M51R -S group and 4-1BB monoclonal antibody combination group.

[0156] Comparing the body weight of mice in different treatment groups, rVSV M51R -S treatment group, the average weight was 25.2g, rVSV M51R -S-4-1BBL treatment group, the average weight was 25.1g, oncolytic virus rVSV M51R -S group and 4-1BB monoclonal antibody combination group, the average weight was 27g, and there was no significant difference between the three groups, indicating that different treatment groups had no obvious toxic side effects on mice.

[0157] The above results suggest that the insertion of hepatitis B surface antigen and 4-1BBL in the attenuated vesicular stomatitis virus plays an important role in anti-mouse tumor, and has a stronger therapeutic effect than the original single expression of hepatitis B surface antigen attenuated vesicular stomatitis virus.

[0158] The present application designs and develops a treatment method of chimeric expression of hepatitis B surface antigen attenuated vector system and monoclonal antibody 4-1BB combination, which greatly improves the treatment effect of liver cancer model, and fills the long-term unsolved problem at home and abroad in this demand.

[0159] The present application inserts the nucleotide sequence encoding hepatitis B surface antigen and 4-1BBL into the modified virus expression vector by gene editing means, and recombines in a specific eukaryotic cell to obtain a stable expression of chimeric hepatitis B surface antigen and 4-1BBL attenuated virus system, and screens rVSV M51RThe S-4-1BBL oncolytic virus is used to further evaluate the therapeutic effect of the oncolytic virus on HBV-positive hepatoma tumors in a HBV-positive hepatoma model, thereby providing a new technical solution and option for developing a therapeutic product for solid tumors.

[0160] Finally, it is also necessary to note that the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article or device that includes a series of elements not only includes those elements, but also includes other elements not explicitly listed, or inherent to such a process, method, article or device.

[0161] Although preferred embodiments of the present application have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once they have been given the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present application.

[0162] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, the present application is also intended to include these modifications and variations, provided that they fall within the scope of the claims of the present application and their equivalents.

Claims

1. Use of a combined pharmaceutical composition in the preparation of a drug for treating HBV-positive liver cancer, characterized in that: The combination pharmaceutical composition contains a recombinant oncolytic virus and a monoclonal antibody, wherein the recombinant oncolytic virus is an attenuated vesicular stomatitis virus carrying the HBV HBsAg gene, and the monoclonal antibody is a 4-1BB monoclonal antibody.

2. The use according to claim 1, characterized in that The attenuated vesicular stomatitis virus is a vesicular stomatitis virus in which the 51st amino acid of the M gene is mutated from M to R.

3. The use according to claim 1, characterized in that The HBV HBsAg gene is derived from type D HBV, and the nucleotide sequence is shown in SEQ ID NO.

1. The HBV HBsAg gene is located between the G gene and the L gene of the attenuated vesicular stomatitis virus.

4. A drug for treating HBV-positive liver cancer, characterized in that: The medicine for treating HBV-positive liver cancer contains a recombinant oncolytic virus and a monoclonal antibody.

5. A multi-gene fusion recombinant oncolytic virus, characterized in that: The multi-gene fusion recombinant oncolytic virus is an attenuated vesicular stomatitis virus carrying the HBV HBsAg gene and the 4-1BBL gene, and the attenuated vesicular stomatitis virus is a vesicular stomatitis virus in which the 51st amino acid of the M gene is mutated from M to R.

6. The multi-gene fusion recombinant oncolytic virus according to claim 5, characterized in that The HBsAg gene and the 4-1BBL gene are located between the G gene and the L gene of the vesicular stomatitis virus, wherein the HBsAg gene and the 4-1BBL gene are linked by a P2A cleavage peptide.

7. The multi-gene fusion recombinant oncolytic virus according to claim 6, characterized in that The nucleotide sequence of the HBV HBsAg gene is shown in SEQ ID NO.1, the nucleotide sequence of the P2A is shown in SEQ ID NO.2, and the nucleotide sequence of the 4-1BBL gene is shown in SEQ ID NO.

3.

8. A method for constructing a multi-gene fusion recombinant oncolytic virus according to any one of claims 5 to 7, characterized in that: The method comprises: PCR amplification was performed using the primer pair shown in SEQ ID NO.4-SEQ ID NO.5 and pAAV-HBV1.3 as a template to obtain the HBsAg-P2A nucleic acid fragment; PCR amplification was performed using the primer pair shown in SEQ ID NO.6-SEQ ID NO.7 and the mouse lung tissue cDNA genome as a template to obtain the 4-1BBL-P2A nucleic acid fragment; Amplifying the HBsAg-P2A nucleic acid fragment and the 4-1BBL-P2A nucleic acid fragment using primers shown in SEQ ID NO. 4 and SEQ ID NO. 7 to connect the two target genes and obtain a connection fragment; The ligated fragments were inserted into the Xho I and Nhe I restriction sites of the pXN2-M51R-GFP vector by enzyme digestion to obtain the pXN2-M51R-HBsAg-P2A-4-1BBL plasmid; The pXN2-M51R-HBsAg-P2A-4-1BBL plasmid and the packaging plasmid of vesicular stomatitis virus are co-transfected into cells for packaging to obtain a multi-gene fusion recombinant oncolytic virus.

9. Use of the multi-gene fusion recombinant oncolytic virus according to any one of claims 5 to 7 in the preparation of a medicament for treating HBV-positive liver cancer.

10. A tumor therapeutic vaccine, characterized in that: The active ingredient of the tumor therapeutic vaccine is the multi-gene fusion recombinant oncolytic virus according to any one of claims 5 to 7.

Images