Membrane protein replacement type oncolytic virus vector and application thereof

Abstract

The invention discloses a cell membrane protein replacement type oncolytic virus vector and application thereof. The cell membrane protein replacement type oncolytic virus vector is rhabdoviridae virus, and a nucleotide sequence for coding G protein in a genome of the rhabdoviridae virus is replaced by a nucleotide sequence for coding an antibody and a nucleotide sequence for coding a spike protein truncation of coronavirus. The invention also discloses a construction method of the cell membrane protein replacement type oncolytic virus vector for expressing the antibody, the non-replicated virus vector is used for expressing the antibody sequence for the first time, and meanwhile, the novel coronavirus cell membrane protein is embedded into the virus surface, so that the cell membrane protein replacement type oncolytic virus vector can be rapidly produced in a suspension cell in a large scale; through removal of virus cell membrane protein genes, the virus cell membrane protein genes cannot be continuously replicated in vivo, so that the safety of the virus cell membrane protein genes is ensured, the tumor immunosuppression condition is improved, and an organism can be stimulated to generate a neutralizing antibody for resisting new coronavirus while tumor cells are killed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a cell membrane protein replacement type oncolytic virus vector and application thereof. BACKGROUND

[0002] Immunotherapy is an effective treatment method for cancer, and oncolytic virus therapy is one of the important methods of tumor immunotherapy. In the field of cancer treatment, recombinant virus vectors are considered as an ideal oncolytic platform because they can target tumor cells, activate cellular immunity, promote the release of tumor-associated antigens (TAA), and activate and regulate anti-tumor immune responses. Natural or genetically engineered oncolytic viruses (OV) can selectively replicate and kill tumor cells in tumor cells. At present, a variety of oncolytic viruses are in the clinical development stage worldwide.

[0003] Hepatocellular carcinoma (HCC) has developed into a global health problem and is one of the research hotspots of solid tumors. At present, the main treatment methods are still surgical operation and local regional treatment, and the strategies for treating hepatocellular carcinoma (HCC) are limited.

[0004] Oncolytic Vesicular Stomatitis Virus (VSV), a single-stranded negative-sense RNA virus. Since the establishment of reverse genetics to recover infectious VSV from cDNA, VSV has become an important research tool and paved the way for the application of recombinant VSV as a platform for the development of biological drugs. Currently, there are two types of recombinant VSV (rVSV) widely used as a tool for virology research: replication-defective rVSV and replication-competent rVSV. Replication-defective rVSV usually contains a genome in which VSV G is deleted (VSVΔG) and replaced by a gene encoding a fluorescent reporter gene. At the same time, during the virus rescue process, the transient expression of heterologous glycoproteins from different viruses leads to the production of "pseudotyped" virions inlaid with non-native glycoproteins. Since the replication-defective rVSV genome does not contain a glycoprotein gene, the virus can only replicate once unless the glycoprotein is provided in trans. In contrast, the replication-competent rVSV genome has the VSV G gene replaced by a gene for a heterologous glycoprotein, thus producing a recombinant virus capable of multiple rounds of replication. In addition, these viruses can be engineered to encode reporter genes to facilitate quantitative applications. Both types of rVSV have been widely used to study various applications of different viruses, such as in the field of cancer treatment. Several VSV-derived vectors, such as VSV-G / GFP, VSV-dG-GFP, VSV-CT9, VSV-M51, etc. have been widely evaluated as viral therapy and vaccine vectors.

[0005] However, there are still many problems to be overcome in the clinical development and preclinical research of VSV. The main obstacles for clinical translation include safety issues such as neurotoxicity, hepatotoxicity, and the low sensitivity of malignant tumors to VSV treatment. SUMMARY

[0006] The technical problem to be solved by the present application is the lack of VSV oncolytic vectors that can be safely and effectively used for tumor treatment in the prior art. The present application provides a cell membrane protein replacement type oncolytic virus vector and its application. The cell membrane protein replacement type oncolytic virus vector of the present application can simultaneously display the S protein of SARS-CoV-2 virus on the surface of the virion to stimulate the production of neutralizing antibodies, and can target liver cancer and effectively inhibit the growth of liver cancer cells, with considerable safety.

[0007] The skilled person knows that SARS-CoV-2 virus is the pathogen that causes novel coronavirus infection, and its spike protein S protein plays an important role in the process of virus and cell infection. It is a trimeric structure, and the S protein is the main antigen that stimulates the body's immune response, which can produce high levels of specific neutralizing antibodies in the body.

[0008] The inventors found in previous studies that the vesicular stomatitis virus (VSV) as a cell membrane protein replacement oncolytic viral vector has the following advantages compared with other viral vectors: VSV can quickly transcribe and translate the inserted target gene antibody, and will not integrate into the host genome, while the expressed S protein of the novel coronavirus is completely displayed on the surface of the virion, can induce a comparable level of neutralizing antibody, and can significantly enhance the strength of the immune response of the body, improve the local immunosuppression of the tumor by inducing T cell and B cell immune responses.

[0009] Therefore, the inventors take advantage of the advantages of the replication-defective VSV viral vector to propose a cell membrane protein replacement oncolytic viral vector and a construction method thereof. The cell membrane protein replacement oncolytic viral vector expressing the antibody can be treated by various methods such as intramuscular injection, intravenous injection and intraperitoneal injection. Different treatment approaches can affect the antibody immune level induced and the expression of the antibody after infection. The cell membrane protein replacement oncolytic viral vector expressing the antibody designed herein can embed the S protein on the surface of the cell line into the surface of the virion during the budding process of the virus, and display the trimeric structure to induce the immune response of the body. Meanwhile, the cell membrane protein replacement oncolytic viral vector is a replication-defective vector, which will not replicate in vivo, further improving the safety of use, and different immune approaches can be suitable for different types of tumor models to achieve more effective killing of tumors. The cell membrane protein replacement oncolytic viral vector can kill tumor cells and prevent the novel coronavirus at the same time, thereby completing the present application.

[0010] The present application solves the above technical problems through the following technical solutions.

[0011] The first aspect of the present application provides a cell membrane protein replacement oncolytic viral vector, which is a rhabdovirus. The nucleotide sequence encoding the G protein in the genome of the rhabdovirus is replaced by a nucleotide sequence encoding an antibody and a nucleotide sequence encoding a spike protein truncation of a coronavirus.

[0012] In some embodiments, the rhabdovirus is a vesicular stomatitis virus.

[0013] In some embodiments, the vesicular stomatitis virus is an Indiana strain, for example, a wild-type vesicular stomatitis virus Indiana strain.

[0014] In some embodiments, the coronavirus is selected from SARS-CoV-2 and variants thereof.

[0015] In some embodiments, the spike protein truncation of the coronavirus lacks 19-26 amino acids at the C-terminus of the spike protein.

[0016] In some embodiments, the S protein truncation lacks 19, 20, 21, 22, 23, 24, 25, or 26 amino acids at the C-terminus of the S protein.

[0017] In some embodiments, the S protein truncation lacks 21 amino acids at the C-terminus of the S protein.

[0018] In some embodiments, the nucleotide sequence encoding the S protein truncation is set forth in SEQ ID NO: 31.

[0019] In some embodiments, the S protein truncation comprises an amino acid sequence set forth in SEQ ID NO: 26.

[0020] In some embodiments, the N-terminus of the S protein truncation further comprises a signal peptide.

[0021] In some embodiments, the signal peptide has an amino acid sequence set forth in SEQ ID NO: 25.

[0022] In some embodiments, the nucleotide sequence encoding the signal peptide is set forth in SEQ ID NO: 32.

[0023] In some embodiments, the nucleotide sequence encoding the antibody is located between the M gene and the L gene in the genome of the Rhabdoviridae virus, and the nucleotide sequence encoding the antibody is upstream or downstream of the nucleotide sequence encoding the S protein truncation.

[0024] In some embodiments, the antibody targets one or more antigens; preferably, the antibody targets GPC3 and / or CD3. For example, the antibody targets GPC3. For example, the antibody targets GPC3 and CD3.

[0025] In some preferred embodiments, the antibody targeting GPC3 is an IgG1 antibody.

[0026] In some preferred embodiments, when the antibody targets GPC3, the antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region, for example, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, respectively, and the light chain variable region, for example, comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, 6, respectively.

[0027] In some embodiments, the N-terminus of the antibody is linked to an antibody signal peptide.

[0028] In some embodiments, the antibody signal peptide has an amino acid sequence as set forth in SEQ ID NO: 29.

[0029] In some embodiments, the nucleotide sequence encoding the signal peptide is as set forth in SEQ ID NO: 30.

[0030] In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region.

[0031] In some embodiments, when the antibody targets GPC3 and CD3, the antibody targeting GPC3 is an IgGl antibody, and the antibody targeting CD3 is a single chain antibody.

[0032] In some embodiments, the single chain antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as set forth in SEQ ID NOs: 13, 14, 15, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as set forth in SEQ ID NOs: 16, 17, 18, respectively.

[0033] In some embodiments, the single chain antibody further comprises a linker connecting the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region.

[0034] In some embodiments, the linker has an amino acid sequence as set forth in SEQ ID NO: 21.

[0035] In some embodiments, the single chain antibody targeting CD3 is connected at the C-terminus of the light chain of the antibody targeting GPC3.

[0036] In some embodiments, the heavy chain variable region of the antibody targeting GPC3 has an amino acid sequence as set forth in SEQ ID NO: 7 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 7, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 8 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 8.

[0037] In some embodiments, the heavy chain variable region of the antibody targeting CD3 has an amino acid sequence as set forth in SEQ ID NO: 19 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 19, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 20 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 20.

[0038] In some embodiments, the heavy chain of the antibody targeting GPC3 has an amino acid sequence as set forth in SEQ ID NO: 9 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 9, and the light chain has an amino acid sequence as set forth in SEQ ID NO: 11 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 11.

[0039] In some embodiments, when the antibody targets GPC3 and CD3, the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID NO: 9 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 9, and the light chain has an amino acid sequence as set forth in SEQ ID NO: 22 or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 22.

[0040] In the present application, "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. In general, the comparison is made at the amino acid or nucleotide level, when the two sequences are aligned for maximum correspondence.

[0041] In some specific embodiments, when the antibody targets GPC3, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID NO:10, and the nucleotide sequence encoding the light chain of the antibody is shown in SEQ ID NO:12.

[0042] In some specific embodiments, when the antibody targets GPC3 and CD3, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID NO:10, and the nucleotide sequence encoding the light chain of the antibody is shown in SEQ ID NO:23.

[0043] In some embodiments, the vesicular stomatitis virus is a recombinant virus with three mutations in the M protein, wherein the three mutations are the deletion of methionine at position 51, and the mutation of valine at position 221 and glycine at position 226 in the M protein into phenylalanine and arginine, respectively.

[0044] In this invention, the vesicular stomatitis virus can be a vesicular stomatitis virus that is routinely obtained by those skilled in the art, such as the one described in the inventor's prior patent application CN113817753A.

[0045] In some specific implementations, the oncolytic virus vector with membrane protein replacement is a VSV virus whose membrane protein has been removed from its genome. The recombinant virus is harvested by infecting CHO cells and suspension cells such as Expi293F with the rescued G-VSV-mAb that express the extracellular segment of the viral spike protein S gene. The G-VSV-mAb is defined as a recombinant VSV virus in which the G protein gene is replaced by the antibody heavy light chain gene in the genome.

[0046] Preferably, the cell lines CHO and Expi293F are transfected with pIRES-SΔ19-26 plasmid, and the method of cell line construction does not affect cell viability.

[0047] Preferably, the cell membrane protein replacement oncolytic virus vector harvested by G-VSV infection has the highest efficiency when the number of C-terminal amino acid deletions is preferably 21.

[0048] Preferably, the oncolytic virus vector with cell membrane protein replacement is a replication-deficient vector, which fully ensures its safety during use.

[0049] Preferably, the oncolytic virus vector with cell membrane protein replacement can simultaneously kill tumor cells and prevent the novel coronavirus.

[0050] A second aspect of the present invention provides a method for constructing an antibody-expressing oncolytic virus vector with membrane protein substitution, the method comprising the following steps:

[0051] (1) Construct expression plasmids for truncated spike S protein of SARS-CoV-2 and antibody expression plasmids, respectively;

[0052] (2) Transfect cells with the antibody expression plasmid in (1) and rescue vesicular stomatitis virus with replication deficiency to obtain vesicular stomatitis virus with replication deficiency carrying nucleotide sequences encoding antibodies; transfect cells with the spike protein truncated expression plasmid in (1) to obtain cells expressing spike protein truncated forms.

[0053] (3) Transfect the cells in (2) with the virus obtained in (2) to obtain cells that express and secrete a truncated spike protein of SARS-CoV-2 and the antibody.

[0054] In some specific implementations, the method is as follows:

[0055] S1. The S-truncated gene of the coronavirus was obtained using synthetic biology and constructed into the multiple cloning site region corresponding to the pIRES plasmid to obtain the pIRES-SΔ19-26 plasmid, wherein SΔ19-26 is located as the gene sequence abbreviation corresponding to the deletion of 19-26 amino acids at the C-terminus of the coronavirus spike protein S gene.

[0056] S2. Obtain the gene of any of the antibody sequences described above using synthetic biology, clone the gene fragment into the multiple cloning site region of the pVSV plasmid, and obtain the pVSV-mAb plasmids respectively.

[0057] S3. Transfect the obtained pIRES-SΔ19-26 plasmid into suspension cell lines CHO and Expi293F. The transfection step involves mixing pIRES-SΔ19-26 with cationic polymer PEI transfection reagent (25kDa linear polyetherimide (PEI) Polysciences, Inc.) at a ratio of 1:3 and incubating for 25 minutes. Then, add cells at densities of 6e6 / ml for CHO and 1e6 / ml for Expi293F, ensuring that the cell viability is higher than 90%.

[0058] S4. Infect BHK21 cells with VT7 poxvirus; plasmids for which endotoxin was removed 1 hour later included: pBS-G, pBS-N, pBS-P, pBS-L and pVSV-mAb co-transfection, where pBS-G, N, P and L are expression plasmids that clone the VSV G, N, P and L protein genes respectively. The plasmids were used to prepare a transfection mixture to express the G, N, P and L proteins required for virus rescue.

[0059] S5. During the observation period of 48-72 hours, when cytopathic effects appear, collect the cells and supernatant separately. Filter the virus solution through a 0.22μm filter membrane to amplify it in BHK21 cells expressing G protein. The virus described above is G-VSV-mAb.

[0060] S6. The amplified G-VSV-mAb was then used to infect and transfect pIRES-SΔ19-26 SΔ19-26-CHO and SΔ19-26-Expi293F suspension cell lines at an MOI of 1. These cells are cell lines expressing the truncated form of the novel coronavirus S protein. The supernatant viral fluid was harvested at an appropriate time. The virus was SΔ19-26-VSV-mAb.

[0061] S7. After collecting the above-mentioned viral fluid, Western blotting was used to identify whether the novel coronavirus S protein was embedded in the non-replicating oncolytic virus vector SΔ19-26-VSV-mAb. At the same time, reverse transcription PCR was used to identify the gene copy number of SΔ19-26-VSV-mAb.

[0062] A third aspect of the present invention provides a cell membrane protein-replacement oncolytic virus vector prepared according to the method described in the second aspect.

[0063] A third aspect of the present invention provides a composition comprising a cell membrane protein-replacement oncolytic virus vector as described in the first aspect.

[0064] In some embodiments, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipients.

[0065] In other embodiments, the composition is a vaccine.

[0066] The fourth aspect of the present invention provides the use of a cell membrane protein-replacement oncolytic virus vector as described in the first aspect, a cell membrane protein-replacement oncolytic virus vector as described in the third aspect, or a composition as described in the third aspect in the preparation of a medicament for the prevention and / or treatment of a disease;

[0067] The disease in question is either a tumor or a coronavirus infection.

[0068] In some specific implementations, the tumor is liver cancer.

[0069] In some specific implementations, the coronavirus infection is SARS-CoV-2 infection.

[0070] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0071] The reagents and raw materials used in this invention are all commercially available.

[0072] The positive and progressive effects of this invention are as follows:

[0073] Compared to oncolytic virus vectors, this invention, for the first time, utilizes a replication-deficient VSV virus packaging system. By constructing cell lines and replacing the virus's own cell membrane proteins with exogenous ones, the virus can be retargeted, simultaneously expressing antibodies. This yields a recombinant replication-deficient virus that displays the SARS-CoV-2 virus S protein on its surface and expresses specific antibodies in its genome. This virus can be used for treatment through various pathways, carrying antibody genes to specific tumor tissues in vivo to express specific antibodies and kill tumor cells. Simultaneously, it can induce a specific humoral immune response in ACE2 mice. Compared to traditional replication-deficient viral vectors (adenovirus vectors), the antibody-expressing, cell membrane protein-replaced oncolytic virus vector involved in this invention can be rapidly and massively produced using stable cell lines and suspension cell lines, laying the foundation for its industrial application. Furthermore, it can activate the body's immune system, improving immunosuppression in cancer patients. Because it is a replication-deficient viral vector, this ensures its safety for use in cancer patients. Attached Figure Description

[0074] Figure 1 In the diagram, A is a schematic diagram of the gene structure of the replication-deficient VSV vector expressing antibodies, B is a schematic diagram of its killing effect, and C is a diagram of GPC3 and GPC3×CD3 antibody expression after Huh7 cells are infected by Western blotting.

[0075] Figure 2 Electrophoresis image of the PCR amplification results of the truncated S protein variant SΔ19-26.

[0076] Figure 3 In the table, A shows the weight change of antibody-expressing replication-deficient VSV in Nod-SCID mice after treatment with Huh7, and B shows the tumor volume monitoring results of antibody-expressing membrane replacement replication-deficient VSV in Nod-SCID mice after treatment with Huh7.

[0077] Figure 4 Neutralizing antibody titers in hACE2 mice of VSV with membrane replacement replication defects expressing antibodies. Detailed Implementation

[0078] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0079] The reagents and consumables used in the examples are as follows:

[0080] Rapid PCR polymerase PrimeSTAR Max DNA Polymerase (Baori Biotechnology (Beijing) Co., Ltd.), Homologous recombination kit (Novazan Biotechnology Co., Ltd.), Endotoxin-free plasmid mass production kit (Omega Beijing Biotechnology Co., Ltd.), Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), DMEM basal medium (Gibco Thermo Fisher Scientific), PBS (Hyclone Thermo Fisher Scientific), 96-well cell culture plate (Corning Corporation), 6-well cell culture plate (Corning Corporation), 100mm cell culture dish (Corning Corporation), 0.22μm filter (Millipore Merck Co., Ltd.), 250ml Erlenmeyer Flask (Corning Corporation), 1L Erlenmeyer Flask (Corning Corporation). Cell lines: BHK21, CHO, Expi293F, ACE2-293T, and Huh7. SMMC7721 and QGY7701 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0081] ACE2-293T, HepG2, Huh7 and Hep3B cells were cultured in DMEM medium at 37℃ with 5% CO2 in an adherent state. CHO and Expi293F cells were cultured in serum-free medium at 37℃ with 8% CO2 in a shaker at 120 rpm.

[0082] The mice used in the examples were 6 weeks old.

[0083] Example 1: Construction of S protein truncated expression plasmid pIRES-SΔ19-26

[0084] To determine the optimal intracellular tail length for constructing a replication-deficient chimeric VSV virus from the SARS-CoV-2 spike protein, the inventors first constructed a plasmid encoding the spike S protein gene and its mutants with deleted C-terminal amino acid sequences. SFL, SΔ19, SΔ20, SΔ21, SΔ22, SΔ23, SΔ24, SΔ25, and SΔ26 represent the full-length S protein (amino acid sequence as shown in SEQ ID NO:24, and the signal peptide amino acid sequence as shown in SEQ ID NO:25) or truncated mutants of the S protein, respectively, with 19-26 amino acids truncated from the intracellular C-terminus. The mutant genes were inserted into the eukaryotic expression plasmid pIRES. These plasmids were named pIRES-SΔ19-26 or -SFL. The gene sequences synthesized after codon optimization were evaluated for annealing temperature using Premier, and appropriate amplification primers were selected. At the same time, when designing primers, the C-terminus 19-26 amino acids of the intracellular region of the S protein were truncated. The Kozak sequence, restriction enzyme site and protective base were also introduced when designing primers. Figure 2 Electrophoresis diagram showing the PCR amplification results of the S protein truncated variant SΔ19-26.

[0085] The obtained pIRES-SΔ19-26 plasmid was transfected into suspension cell lines CHO and Expi293F, respectively. The transfection step involved mixing pIRES-SΔ19-26 with cationic polymer PEI transfection reagent (25kDa linear polyetherimide (PEI) Polysciences, Inc.) at a ratio of 1:3 and incubating for 25 minutes. Then, CHO cells with a cell density of 6e6 / ml and Expi293F cells with a cell density of 1e6 / ml were added to ensure that the cell viability was higher than 90%.

[0086] Example 2: Design and packaging of a membrane-replacement vector for expressing GPC3 monoclonal antibody using a replication-defective VSV viral vector.

[0087] like Figure 1As shown, antibody sequences targeting GPC3 were selected based on clinical trials and constructed into the pVSV vector genome (the G protein in the pVSV genome itself was deleted through genetic engineering). The codons of the antibody sequence were optimized for eukaryotic expression (codon optimization can increase mRNA expression, which is beneficial for the secretion of higher levels of antibodies after viral infection). In this embodiment, the GPC3 sequence (the sequence encoding the heavy chain is shown in SEQ ID NO:10, the sequence encoding the heavy chain is shown in SEQ ID NO:12, and the sequence encoding the signal peptide is shown in SEQ ID NO:29) was synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pIRES eukaryotic expression vector and pVSV viral vector, respectively. After PCR amplification of the target gene, the fragment and the vector were ligated using a homologous recombination kit and transformed into DH5α competent cells. Positive clones were selected for verification by bacterial PCR. Positive clones were screened and the constructed plasmids were confirmed and named pIRES-GPC3 Hc, pIRES-GPC3 Lc, and pVSV-GPC3, respectively (the monoclonal antibody Hc and Lc were ligated by the IRES sequence). After transfecting pIRES-GPC3 Hc and pIRES-GPC3 Lc into 293F cells, the antibodies were harvested.

[0088] Replication-deficient G-VSV-GPC3 cells were rescued in BHK21 cells using transgenesis. Specifically, BHK-21 cells in logarithmic growth phase were seeded into 100 mm cell culture dishes and cultured in a cell culture incubator for 12-16 hours until cell coverage reached 80-90%. One hour before plasmid transfection, BHK21 cells were infected with poxvirus vTF7-3 at an MOI of 3-10, followed by washing the cells once with 2 mL of DPBS, discarding the washed cells, and then adding 8 mL of DMEM medium. The recombinant plasmid pVSV-GPC3 was co-transfected into BHK21 cells with helper plasmids pBS-P, pBS-N, and pBS-L, respectively. After mixing the helper plasmid and recombinant plasmid, 250 μL of serum-free Opti-MEM medium and the mixed plasmid were added to EP tube A. Simultaneously, 30 μL of Lipo2000 was added to 250 μL of serum-free Opti-MEM medium in EP tube B. A and B were incubated separately at room temperature for 5 min. Then, A and B were mixed and incubated for 25 min. Finally, the mixture was added to a cell culture dish, added evenly, and cultured at 37℃ for 4-6 h. After 48-72 h, the supernatant was collected to obtain G-VSV-GPC3 virus, which was then quantified. The G-VSV-GPC3 virus was used to infect a 293T cell line expressing the SARS-CoV-2 virus S protein, and a replication-defective virus, S-VSV-GPC3, with SARS-CoV-2 virus S protein embedded on its surface and GPC3 antibody expressed in its genome, was harvested.

[0089] Example 3: Design and packaging of a membrane-replacement vector for expressing GPC3 bispecific antibodies using a replication-defective VSV viral vector.

[0090] like Figure 1 As shown, antibody sequences targeting GPC3 and CD3 were selected based on clinical trials (the sequence encoding the heavy chain is shown in SEQ ID NO:10, the sequence encoding the heavy chain is shown in SEQ ID NO:23, and the sequence encoding the signal peptide is shown in SEQ ID NO:29). These sequences were constructed into the pVSV vector genome. The codons of the antibody sequences were optimized for eukaryotic expression. (In this embodiment, the sequences of GPC3 and CD3 antibodies were synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pIRES eukaryotic expression vector and pVSV viral vector, respectively. After PCR amplification of the target genes, the fragments and vectors were ligated using a homologous recombination kit and transformed into DH5α competent cells. Positive clones were selected for verification by bacterial culture. Positive clones were screened to confirm the constructed plasmids (named pIRES-BsAb-GPC3 Hc, pIRES-BsAb-GPC3 Lc, and pVSV-GPC3×CD3, respectively). pIRES-BsAb-GPC3 Hc and pIRES-BsAb-GPC3...) Lc transfection into 293F cells yielded the bispecific antibody GPC3×CD3.

[0091] Replication-deficient G-VSV-GPC3×CD3 was rescued in BHK21 cells using transgenetic technology. G-VSV-GPC3×CD3 virus was obtained by treating pVSV-GPC3×CD3 in the same manner as in Example 2. The G-VSV-GPC3×CD3 virus was used to infect 293T cell lines expressing SARS-CoV-2 virus S protein, and replication-deficient virus S-VSV-GPC3×CD3 with SARS-CoV-2 virus S protein embedded on its surface and bispecific antibody GPC3×CD3 expressed in its genome was harvested.

[0092] Example 4: Preparation of VSV membrane-replacement vector expressing antibodies by large-scale expansion of Expi293F suspension cells

[0093] The obtained pIRES-SΔ19-26 plasmid was transfected into the suspension cell line Expi293F. After incubating pIRES-SΔ19-26 with cationic liposome transfection reagent at a 1:3 ratio for 25 minutes, the mixture was added at a cell density of 1e6 / mL, ensuring cell viability was above 90%. Amplified G-VSV-GPC3 and G-VSV-GPC3×CD3 were then transfected into the pIRES-SΔ19-26 SΔ19-26-CHO and SΔ19-26-Expi293 cells at an MOI of 1. F suspension cell lines, the above cells are cell lines expressing truncated versions of the SARS-CoV-2 virus S protein. The supernatant viral fluid was harvested at an appropriate time. The above viruses were S-VSV-GPC3 and S-VSV-GPC3×CD3. Western blotting was used to identify whether the novel coronavirus S protein was embedded in the oncolytic virus vector with a cell membrane protein replacement and to identify the expression of the recombinant oncolytic virus bispecific antibody protein. Reverse transcription PCR was used to identify the viral gene copy number. The fluorescent probe primers used were Forward 5'-TGATCGACTTTGGATTGTCTTCTAA-3' (SEQ ID NO:27) and Reverse: 5'-TCTGGTGGATCTGAGCAGAAGAG-3' (SEQ ID NO:28).

[0094] The above method can be used to obtain high-titer non-replicating recombinant viruses, which can be amplified in large quantities for use in engineered production.

[0095] Example 5: Oncolytic therapeutic effect in Nod-Scid mice using a VSV viral vector-based oncolytic virus vector with membrane protein substitution.

[0096] Based on the in vitro cellular activity of GPC3 antibodies and bispecific antibodies, the antitumor effects of GPC3 monoclonal antibodies, GPC3×CD3 BsAb, and recombinant VSV expressed in GPC3 were evaluated in vivo in Nod-Scid mice inoculated with Huh7 hepatocellular carcinoma. Female Nod-Scid mice were purchased from Charles River. A tumor model was first established by inoculating Nod-Scid mice with Huh7 hepatocytes, with tumors reaching a size of 150 mm. 3 At that time, 10 were injected in situ. 8 TCID 50 The recombinant S-VSV-GPC3 and S-VSV-GPC3×CD3 viruses were used to assess the in vivo killing effect on tumor cells by measuring mouse body weight and tumor size daily. Figure 3As shown in the figure. The results indicate that the S-VSV-GPC3 and S-VSV-GPC3×CD3 recombinant viruses can achieve highly efficient killing of tumor cells, especially S-VSV-GPC3×CD3, which can completely inhibit tumor growth. The recombinant virus can combine antibody therapy and oncolytic virus therapy to achieve a synergistic effect, resulting in complete tumor regression while ensuring safety.

[0097] Example 6: Immunogenicity study of VSV-based oncolytic virus vector with membrane protein substitution in hACE2 mice

[0098] Since the surface of the replication-defective virus is replaced with the spike S protein of the SARS-CoV-2 virus and is displayed on the surface of the virus in the form of a trimer, it indicates that it can also play the role of a vaccine in stimulating specific neutralizing antibodies against the novel coronavirus. Therefore, the inventors chose to evaluate its ability to stimulate the body's immunity by intramuscular and intraperitoneal injection in hACE2 male mice (constructed by the method described in Ke Y, Zhang E, Guo J, Zhang X, Wang L, Chen D, Fang X, Zhu J, Li F, Sun T, Zhang B. Immunogenicity of mucosal COVID-19vaccinecandidates based on the highly attenuated vesicular stomatitis virus vector (VSVMT) in golden syrian hamster. Acta Pharm Sin B. 2023 Dec; 13(12):4856-4874). After immunization with S-VSV-GPC3 and S-VSV-GPC3×CD3 via the two immunization routes, serum samples were collected at 3, 7, 14, and 21 days, respectively. The neutralizing antibody titers were then serially diluted to determine their neutralization levels. Figure 4 As shown in the figure. The results indicate that both recombinant viruses can elicit an immune response in the body through intramuscular and intraperitoneal injection, and high levels of neutralizing antibodies were detected in serum, thus blocking SARS-CoV-2 infection. S-VSV-GPC3 and S-VSV-GPC3×CD3 can also elicit specific neutralizing antibodies against the novel coronavirus through intramuscular and intraperitoneal injection, indicating that they also have a vaccine effect.

[0099] The sequences involved in the embodiments of the present invention are shown below.

[0100] GPC3 HCDR1:

[0101] DYEMH (SEQ ID NO:1)

[0102] GPC3 HCDR2:

[0103] ALDPKTGDTAYSQKFKG(SEQ ID NO:2)

[0104] GPC3 HCDR3:

[0105] FYSYTY(SEQ ID NO:3)

[0106] GPC3 LCDR1:

[0107] RSSQSLVHSNRNTYLH(SEQ ID NO:4)

[0108] GPC3 LCDR2:

[0109] KVSNRFS(SEQ ID NO:5)

[0110] GPC3 LCDR3:

[0111] SQNTHVPPT(SEQ ID NO:6)

[0112] GPC3 VH:

[0113] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALD PKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTL VTVSS(SEQ ID NO:7)

[0114] GPC3 VL:

[0115] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK(SEQ ID NO:8)

[0116] GPC3 H

[0117] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:9)

[0118] GPC3 H

[0119]

[0120] GPC3 L

[0121] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:11)

[0122] GPC3 L

[0123] GACGTGGTCATGACCCAGAGCCCTCTGAGCCTGCCCGTGACCCCCGGAGAGCCTGCTTCCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAGCAATAGGAACACCTACCTGCACTGGTACTTGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAACAGGTTCTCCGGCGTGCCTGATAGGTTCTCCGGATCTGGCAGCGGCACCGATTTCACCCTGAAGATCAGCAGGGTGGAGGCTGAGGATGTGGGCGTGTACTACTGTAGCCAGAACACCCACGTGCCTCCTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCTGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGA(SEQ ID NO:12)CD3HCDR1:

[0124] RYTMH(SEQ ID NO:13)

[0125] CD3 HCDR2:

[0126] YINPSRGYTNYNQKFKD(SEQ ID NO:14)

[0127] CD3 HCDR3:

[0128] YYDDHYCLDY(SEQ ID NO:15)

[0129] CD3 LCDR1:

[0130] SASSSVSYMN(SEQ ID NO:16)

[0131] CD3 LCDR2:

[0132] DTSKLAS (SEQ ID NO:17)

[0133] CD3 LCDR3:

[0134] QQWSSNPFT (SEQ ID NO:18)

[0135] CD3 VH:

[0136] QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSS (SEQ ID NO:19)

[0137] CD3 VL:

[0138] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLAS GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR (SEQ ID NO:20)

[0139] Linker

[0140] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:21)

[0141] GPC3×CD3 bispecific antibody light chain full length:

[0142] MGWSLILLFLVAVATSSRADVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKCLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR(SEQ ID NO:22)

[0143] Full length of GPC3×CD3 bispecific antibody light chain:

[0144] ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGAGCAGCAGGGCCGACGTGGTCATGACCCAGAGCCCTCTGAGCCTGCCCGTGACCCCCGGAGAGCCTGCTTCCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAGCAATAGGAACACCTACCTGCACTGGTACTTGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAACAGGTTCTCCGGCGTGCCTGATAGGTTCTCCGGATCTGGCAGCGGCACCGATTTCACCCTGAAGATCAGCAGGGTGGAGGCTGAGGATGTGGGCGTGTACTACTGTAGCCAGAACACCCACGTGCCTCCTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCTGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGGAGGAGGAGGCAGCGGAGGAGGCGGAAGCGGAGGAGGAGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTAAGGCTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTCCGCCAGGCTCCAGGCAAGTGCCTGGAGTGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACCGATTCACCATCTCCAGAGACAACTCCAAGAACACGGCGTTTCTGCAAATGGACAGCCTGAGACCCGAGGACACGGGTGTGTATTTCTGTGCGAGATATTATGATGATCATTACTGTCTTGACTACTGGGGCCAAGGCACCCCTGTCACAGTCTCCTCAGGCGGAGGCGGATCTGGCGGAGGAGGATCTGGCGGCGGCGGATCTGGAGGCGGAGGATCTGGAGGAGGCGGCAGCGGAGGCGGAGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGTGCCAGCTCAAGTGTAAGTTACATGAACTGGTATCAGCAGACCCCAGGGAAAGCCCCTAAGCGCTGGATCTACGACACATCCAAACTGGCTTCTGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTATACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAGCAGTGGAGTAGTAACCCATTCACGTTTGGCTGTGGTACCAAACTGCAGATTACCCGCTGA(SEQ ID NO:23)

[0145] Full length of S protein:

[0146] MSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGS(SEQ ID NO:24)

[0147] S protein signal peptide:

[0148] MFVFLVLLPLVS(SEQ ID NO:25)

[0149] S protein truncation (21):

[0150]

[0151] Forward:

[0152] TGATCGACTTTGGATTGTCTTCTAA(SEQ ID NO:27)

[0153] Reverse:

[0154] TGGTGGATCTGAGCAGAAGAG(SEQ ID NO:28)

[0155] Antibody signal peptide:

[0156] MGWSLILLFLVAVATSSRA(SEQ ID NO:29)

[0157] Antibody signal peptide encoding sequence:

[0158] ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGAGCAGCAG GGCC(SEQ ID NO:30)

[0159] Full-length coding sequence of S protein:

[0160]

[0161] S protein signal peptide coding sequence:

[0162] ATGTTCGTTTTCCTTGTTCTGTTGCCTCTCGTTAGT(SEQ ID NO:32)

[0163] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A cell membrane protein-substituted oncolytic virus vector, characterized in that, The oncolytic virus vector with membrane protein replacement is a Rhabdoviridae virus, in which the nucleotide sequence encoding the G protein in the genome of the Rhabdoviridae virus is replaced with a nucleotide sequence encoding an antibody and a nucleotide sequence encoding a truncated spike protein of a coronavirus.

2. The oncolytic virus vector with cell membrane protein replacement according to claim 1, characterized in that, The rhabdoviridae virus is a vesicular stomatitis virus, such as the Indiana strain of vesicular stomatitis virus; and / or, the coronavirus is selected from SARS-CoV-2 and its variants; and / or, the spike protein truncated form of the coronavirus has 19-26 amino acids missing from the C-terminus of the spike protein.

3. The oncolytic virus vector with cell membrane protein replacement according to claim 1 or 2, characterized in that, The spike protein truncated form has 21 amino acids missing from the C-terminus of the spike protein; and / or, the N-terminus of the spike protein truncated form also includes a signal peptide. Preferably, the spike protein truncated form comprises the amino acid sequence shown in SEQ ID NO:26; and / or, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:25; More preferably, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO:32; and / or, the nucleotide sequence encoding the spike protein truncated form is shown in SEQ ID NO:

31.

4. The oncolytic virus vector with cell membrane protein replacement according to any one of claims 1-3, characterized in that, The nucleotide sequence encoding the antibody is located between the M and L genes in the genome of the Rhabdoviridae virus, and the nucleotide sequence encoding the antibody is located upstream or downstream of the nucleotide sequence encoding the spike protein truncated form. And / or, the antibody targets one or more antigens; And / or, the N-terminus of the antibody is attached to an antibody signal peptide; Preferably, the antibody targets GPC3 and / or CD3; the antibody targeting GPC3 is preferably an IgG1 antibody, including a heavy chain variable region and a light chain variable region; the heavy chain variable region includes, for example, the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, 2, and 3, respectively, and the light chain variable region includes, for example, the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, 5, and 6, respectively; the antibody targeting CD3 is preferably a single-chain antibody, including a heavy chain variable region and a light chain variable region; the heavy chain variable region includes, for example, the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, 14, and 15, respectively, and the light chain variable region includes, for example, the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, 17, and 18, respectively; and / or, the antibody signal peptide has the amino acid sequence shown in SEQ ID NO:29; More preferably, the antibody targeting GPC3 further includes a heavy chain constant region and a light chain constant region; the antibody targeting CD3 further includes a linker connecting the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region of the antibody targeting CD3, the linker preferably having an amino acid sequence as shown in SEQ ID NO:21; the antibody targeting CD3 is preferably linked to the C-terminus of the light chain of the antibody targeting GPC3.

5. The oncolytic virus vector with cell membrane protein replacement according to claim 4, characterized in that, The heavy chain variable region of the antibody targeting GPC3 has an amino acid sequence as shown in SEQ ID NO:7 or having at least 85% sequence identity with SEQ ID NO:7, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:8 or having at least 85% sequence identity with SEQ ID NO:8; and / or, The heavy chain variable region of the antibody targeting CD3 has an amino acid sequence as shown in SEQ ID NO:19 or having at least 85% sequence identity with SEQ ID NO:19, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:20 or having at least 85% sequence identity with SEQ ID NO:

20. Preferably, the heavy chain of the antibody targeting GPC3 has an amino acid sequence as shown in SEQ ID NO:9 or having at least 85% sequence identity with SEQ ID NO:9, and the light chain has an amino acid sequence as shown in SEQ ID NO:11 or having at least 85% sequence identity with SEQ ID NO:

11. More preferably, when the antibody targets GPC3 and CD3, the heavy chain of the antibody has an amino acid sequence as shown in SEQ ID NO:9 or having at least 85% sequence identity with SEQ ID NO:9, and the light chain has an amino acid sequence as shown in SEQ ID NO:22 or having at least 85% sequence identity with SEQ ID NO:22; For example, when the antibody targets GPC3, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID NO:10, and the nucleotide sequence encoding the light chain of the antibody is shown in SEQ ID NO:12; when the antibody targets both GPC3 and CD3, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID NO:10, and the nucleotide sequence encoding the light chain of the antibody is shown in SEQ ID NO:23; the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO:

30.

6. The oncolytic virus vector with cell membrane protein replacement according to any one of claims 2-5, characterized in that, The vesicular stomatitis virus is a recombinant virus resulting from three mutations in the M protein. The three mutations are the deletion of methionine at position 51, and the mutation of valine at position 221 and glycine at position 226 in the M protein into phenylalanine and arginine, respectively.

7. A method for constructing an antibody-expressing oncolytic virus vector with membrane protein substitution, characterized in that, The method includes the following steps: (1) Construct expression plasmids for truncated spike proteins and antibody expression plasmids for SARS-CoV-2, respectively; (2) Transfect cells with the antibody expression plasmid in (1) and rescue vesicular stomatitis virus with replication deficiency to obtain vesicular stomatitis virus with replication deficiency carrying nucleotide sequences encoding antibodies; transfect cells with the spike protein truncated expression plasmid in (1) to obtain cells expressing spike protein truncated forms. (3) Transfect the cells in (2) with the virus obtained in (2) to obtain cells that express and secrete a truncated spike protein of SARS-CoV-2 and the antibody.

8. A cell membrane protein-replacement oncolytic virus vector prepared according to the method of claim 7.

9. A composition, characterized in that, The composition comprises the oncolytic virus vector with membrane protein replacement as described in any one of claims 1-6; Preferably, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier and / or excipients; or, the composition is a vaccine.

10. The use of the oncolytic virus vector with cell membrane protein replacement as described in any one of claims 1-6, the oncolytic virus vector with cell membrane protein replacement as described in claim 8, or the composition as described in claim 9 in the preparation of a medicament for the prevention and / or treatment of a disease; The disease is either a tumor or a coronavirus infection; Preferably, the tumor is liver cancer; the coronavirus infection is SARS-CoV-2 infection.

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