Recombinant construct, recombinant oncolytic virus as well as construction and application of recombinant oncolytic virus

Abstract

The invention relates to the field of biotechnology and targeted therapy, in particular to a recombinant construct, a recombinant oncolytic virus and construction and application of the recombinant oncolytic virus. The virus takes a wild poxvirus genome as a skeleton, thymokinase TK genes are deleted through homologous recombination, and a poxvirus P-se / l promoter is used for promoting expression of a CD19 membrane localization antibody. The constructed recombinant oncopoxvirus aCD19-TM-OVV can improve the infection efficiency and gene delivery efficiency on B lymphoma in a CD19 antigen target-dependent manner, promote virus replication of the oncopoxvirus in CD19 positive B lymphoma cells, and significantly enhance the tumor growth inhibition effect on the CD19 positive B lymphoma cells in vitro and in mouse B lymphoma models.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and targeted therapy, and specifically to a recombinant construct, a recombinant oncolytic virus, and their construction and applications. Background Art

[0002] Human CD19 belongs to the immunoglobulin (Ig) superfamily and is encoded by the cd19 gene located on the short arm of human chromosome 16 at 16p11.2. It is a 95kd transmembrane glycoprotein composed of 556 amino acids and shows extensive conservation between humans and mice. CD19 is highly conserved and expressed in most acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma, making it an ideal therapeutic target for lymphoma.

[0003] In recent years, chimeric antigen receptor T (CAR-T) cell therapy has shown good efficacy and potential in the treatment of refractory and relapsed hematological malignancies. Cappell et al. analyzed the long-term follow-up data of 10 international clinical studies. The complete remission (CR) rate of autologous CD19-CAR-T in the treatment of refractory and relapsed B-cell non-Hodgkin lymphoma (B-NHL) was 28-68%. Durable remissions have been observed in patients with aggressive lymphoma, follicular lymphoma (FL), mantle cell lymphoma (MCL), and chronic lymphocytic leukemia (CLL). Literature reports that the CR rate of dual antigen-targeted CAR-T therapy for CD19 and CD20 in relapsed / refractory non-Hodgkin lymphoma is 71%, and the progression-free survival rate at 12 months is 64%. However, the problem of drug resistance in CAR-T cell therapy remains an urgent problem to be solved, and its efficacy is mainly limited by the following factors: (1) abnormal differentiation and function of CAR-T cells, insufficient persistence in vivo; (2) inhibition by the tumor microenvironment (TME), abnormal vascular structures and extracellular matrix, etc. hinder the infiltration of CAR-T cells, and tumor-associated macrophages (TAM) and regulatory T (Treg) cells lead to cell exhaustion, and metabolites such as lactic acid and immune checkpoints such as the PD-1 / PD-L1 pathway negatively regulate the function of CAR-T cells; (3) antigen escape, including the loss of CD19, CD20, and BCMA antigens, and its mechanisms are caused by coding gene mutations, lineage conversion, splicing, abnormal antigen processing and presentation, CAR masking, and trogocytosis. Therefore, new strategies that weaken or are not negatively regulated by the tumor microenvironment are needed to solve the problem of CAR-T drug resistance.

[0004] Oncolytic virus drugs are one of the potential candidate drugs. Oncolytic viruses are a class of viruses that can selectively replicate in tumor cells, produce progeny viruses, and lyse and kill tumor cells either naturally or through genetic engineering modification. They may be one of the promising drugs to overcome the tumor immunosuppressive microenvironment. In addition to killing tumors through oncolysis, oncolytic viruses (OVs) also have the following biological characteristics: (1) Genetically modified OVs can selectively infect tumor cells, causing the release of tumor-associated antigens (TAAs), cytokines, and pathogen-associated molecular patterns (PAMPs), triggering a local immune response in the tumor; the release of tumor-associated molecular patterns (TAMPs) can stimulate a systemic anti-tumor immune response; (2) They can disrupt the vascular endothelial cells and tumor vascular beds in the tumor microenvironment (TME); recruit immune cells. (3) Further genetic engineering modification to make OVs express anti-tumor genes can significantly enhance their anti-tumor effects.

[0005] Currently, several oncolytic virus drugs have been approved for clinical treatment: In 2005, oncolytic adenovirus H101 was approved for the combined treatment of nasopharyngeal carcinoma with chemotherapy drugs. The objective remission rate of single chemotherapy in clinical trials was 40.3%, and the objective remission rate of chemotherapy combined with H101 was 72.7%. In 2015, oncolytic herpes simplex virus T-VEC was approved by the US Food and Drug Administration for the treatment of unresectable stage IIIB-IV melanoma. The durable response rate of patients receiving granulocyte-macrophage colony-stimulating factor therapy was 2.1%, and the median overall survival was 18.9 months. While the durable response rate of patients receiving T-VEC treatment was 16.3%, and the median overall survival was 23.3 months. In 2021, oncolytic herpes simplex virus Teserpaturev was approved by the Japanese Ministry of Health, Labour and Welfare for follow-up administration after radiotherapy and temozolomide treatment for glioblastoma. The progression-free survival was 4.7 months, and the median overall survival was 20.2 months. Oncolytic virus drugs have relatively high safety, and there are no lethal toxic and side effects in current clinical trials, but the efficacy of single administration needs to be further improved. In addition, in recent decades, the research on oncolytic viruses in cancer treatment has mainly focused on using oncolytic virus vectors to express different therapeutic genes in solid tumors, with insufficient research on the targeting of oncolytic viruses at the level of target cell infection and the treatment of hematological tumors with low virus infection efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant construct, a recombinant oncolytic virus, and their construction and applications to solve the problems raised in the above background technology.

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] A recombinant construct encoding a membrane-localized antibody, its fragment or variant that can be modified on the surface of an oncolytic virus.

[0009] Preferably, the nucleic acid sequence of the recombinant construct is as shown in SEQ ID NO.1, its fragment or variant.

[0010] Preferably, the amino acid sequence encoded by the recombinant construct is as shown in SEQ ID NO.2, its fragment or variant.

[0011] Preferably, the membrane-localized antibody is a CD19 membrane-localized antibody.

[0012] A recombinant oncolytic virus, comprising a recombinant construct as described in any one of the foregoing.

[0013] Preferably, the TK gene of the recombinant oncolytic virus is replaced or inserted with the recombinant construct.

[0014] Preferably, the recombinant oncolytic virus is an oncolytic poxvirus.

[0015] A pharmaceutical composition, comprising a recombinant construct as described in any one of the foregoing or a recombinant oncolytic virus as described in any one of the foregoing and at least one pharmaceutically acceptable carrier.

[0016] Use of a recombinant construct as described in any one of the foregoing or a recombinant oncolytic virus as described in any one of the foregoing or the pharmaceutical composition as described above in the preparation of a kit for diagnosing / treating malignant tumors or autoimmune diseases.

[0017] Use of a recombinant construct as described in any one of the foregoing or a recombinant oncolytic virus as described in any one of the foregoing or the pharmaceutical composition as described above in the preparation of a medicament for diagnosing / treating malignant tumors or autoimmune diseases.

[0018] Preferably, the malignant tumor is any one of acute lymphoblastic leukemia, chronic lymphocytic leukemia or B-cell lymphoma.

[0019] A method for constructing a recombinant oncolytic virus, wherein the TK gene of the recombinant oncolytic virus is replaced or inserted with a recombinant construct as described in any one of the foregoing.

[0020] The present invention belongs to the field of tumor targeted therapy, and relates to a recombinant construct, a recombinant oncolytic virus and their construction and application. The CD19 membrane localization antibody sequence is inserted into the oncolytic poxvirus genome through genetic engineering, and the recombinant oncolytic poxvirus aCD19-TM-OVV is obtained through poxvirus packaging, purification and identification. The surface of target cells infected with aCD19-TM-OVV can express the CD19 membrane localization antibody, and the surface of the generated progeny virus structure is also modified with the CD19 membrane localization antibody. aCD19-TM-OVV can improve the infectivity and killing ability against CD19 antigen-positive B lymphoma cells in a CD19 antigen target-dependent manner. In a mouse B lymphoma model, compared with the PBS treatment group and the control oncolytic poxvirus OVV treatment group, aCD19-TM-OVV has a stronger inhibitory effect on B lymphoma growth. These research results reveal the application prospect of the recombinant oncolytic poxvirus aCD19-TM-OVV targeted and modified with the CD19 membrane localization antibody in the development of drugs for the treatment of CD19 antigen-positive B lymphoma.

[0021] The present invention provides the construction and application of an oncolytic poxvirus targeted and modified with the CD19 membrane localization antibody. The virus uses the wild-type poxvirus genome as the backbone, homologous recombination deletes the thymidine kinase TK gene, and the CD19 membrane localization antibody is expressed under the initiation of the poxvirus P-se / l promoter. By inserting the aCD19-TM gene into the oncolytic poxvirus in the present invention, the recombinant oncolytic poxvirus can effectively express the CD19 membrane localization antibody aCD19-TM after infecting target cells, and can modify aCD19-TM on the surface of the oncolytic poxvirus structure, without affecting its biological characteristics and having good safety. The recombinant oncolytic poxvirus aCD19-TM-OVV constructed in the present invention can improve the infection efficiency and gene delivery efficiency against B lymphoma in a CD19 antigen target-dependent manner, promote virus replication of the oncolytic poxvirus in CD19-positive B lymphoma cells, and significantly enhance the inhibitory effect on the tumor growth of CD19-positive B lymphoma cells in vitro and in a mouse B lymphoma model, laying the foundation for the research and development of this oncolytic poxvirus as a tumor targeted therapeutic drug, and the application of the recombinant oncolytic poxvirus aCD19-TM-OVV in the preparation of drugs for the treatment of CD19-positive B lymphoma.

[0022] Oncolytic poxvirus is a large double-stranded DNA virus in the poxvirus family. It has the following advantages compared to other oncolytic viruses: (1) It replicates in the cytoplasm, which can reduce the risk of integration into the host genome; (2) It has a large cloning capacity and can allow the insertion of gene fragments of relatively large lengths; (3) It has high safety and was early made into a vaccine to combat the smallpox virus. By deleting the thymidine kinase gene (TK) of the poxvirus, the virus can be made to preferentially replicate in cancer cells rich in nucleotides, which can reduce the toxic and side effects on normal tissues. In the previous research of the team led by Qian Wenbin at Zhejiang University, it was found that the recombinant oncolytic poxvirus (OVV-CD19BiTE) expressing CD19 bispecific T cell engager (CD19BiTE) had similar virus infection, replication, and oncolytic abilities compared to the control oncolytic poxvirus (OVV) in an in vitro B lymphoma model. However, the supernatant after its infection of cells could induce the activation and proliferation of human T cells, as well as the bystander effect; OVV-CD19BiTE could selectively replicate in mouse B lymphoma tumor tissues. Compared with OVV and the clinical first-line blinatumomab, it had a higher percentage of CD3+CD8+ T cells and a therapeutic effect of long-term tumor remission.

[0023] The insufficient targeting of oncolytic poxvirus to B lymphoma cells is an important factor restricting its efficacy. The infection of cells by oncolytic poxvirus depends on its envelope protein D8L binding to chondroitin sulfate on the cell surface. However, chondroitin sulfate is widely expressed in most cells and is not a B lymphoma cell-specific antigen. Although deleting the thymidine kinase gene (TK) of the poxvirus can make the virus preferentially replicate in cancer cells rich in nucleotides and reduce the toxic and side effects on normal tissues, the virus amount reaching the tumor tissue will be reduced due to the infection of non-tumor cells, thus reducing its efficacy. Improving the antigen-specific targeting ability of oncolytic poxvirus to B lymphoma may be an ideal strategy to enhance the efficacy of oncolytic poxvirus drugs.

[0024] The antigen chimeric receptor technology has been maturely applied in immunocyte therapy. The tumor cell antigen-specific antibody is designed as a membrane-localized protein, enabling it to be anchored on the envelopes of cells such as T cells, NK cells, and macrophages, endowing the cells with the ability to specifically recognize and bind the antibody. Oncolytic poxvirus is a virus with an envelope structure. In theory, the antigen chimeric receptor technology can also be applied to the modification of oncolytic poxvirus to endow it with the ability to target and infect tumor cells.

[0025] In summary, oncolytic vaccinia virus has the advantages of high safety, large gene modification capacity, not being negatively regulated by the tumor microenvironment and immune checkpoints, and being able to activate the body's immunity and improve the tumor immunosuppressive microenvironment. It is an ideal anti-tumor drug and a new strategy that may overcome CAR-T drug resistance. Considering the insufficient specific infectivity of oncolytic vaccinia virus to B lymphoma cells, can the use of chimeric antigen receptor technology modify the CD19 membrane-localized antibody on the surface of oncolytic vaccinia virus to improve the infectivity and killing ability of oncolytic vaccinia virus to CD19 antigen-positive B lymphoma cells? To address this issue, the present invention constructs a recombinant oncolytic vaccinia virus aCD19-TM-OVV expressing the CD19 membrane-localized antibody and explores the inhibitory effect of the recombinant oncolytic vaccinia virus aCD19-TM-OVV on the tumor growth of CD19-positive B lymphoma in vitro and in a mouse tumor model.

[0026] One of the objectives of the present invention is to provide a recombinant oncolytic vaccinia virus aCD19-TM-OVV that can infect and kill B lymphoma cells in a CD19 antigen-specific-dependent manner. This virus uses the oncolytic vaccinia virus genome as a backbone and homologously recombines and replaces the insertion of the sequence expressing the membrane-localized antibody against CD19 (anti-CD19 scFv-TM, aCD19-TM) in the TK gene region.

[0027] The DNA sequence of the anti-CD19 membrane-localized protein (aCD19-TM) is shown in SEQ ID No.1.

[0028] The amino acid sequence of the anti-CD19 membrane-localized protein (aCD19-TM) is shown in SEQ ID No.2.

[0029] The preparation method of the recombinant oncolytic vaccinia virus aCD19-TM-OVV of the present invention is achieved through the following steps:

[0030] The sequence of the anti-CD19 membrane-localized antibody aCD19-TM was synthesized on behalf of Nanjing Genscript Biotech Co., Ltd. This sequence connects the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-CD19 antibody (FMC63) with a (GGGGS)3 linker, adds a CD8 membrane localization signal peptide at the N-terminus and a CD8 transmembrane region (the extracellular and transmembrane region design structure of the chimeric antigen receptor in classical CD19 CAR-T), and inserts it between the two restriction endonucleases EcoR I and Xba I of the vaccinia virus shuttle plasmid pRGB (the DNA sequence is shown in SEQ ID NO.3, and the plasmid map is as Figure 1 shown, constructed and obtained by the team of Wenbin Qian from Zhejiang University), and the plasmid is named pRGB-aCD19-TM.

[0031] Vero cells of simian embryo kidney in good growth state were seeded at 5×10 per well5 Cells were inoculated in a six-well plate culture dish. After the cells adhered to the wall, Vero cells were infected with wild-type vaccinia virus (WTVV) at 1 MOI, and 2 μg of pRGB-aCD19-TM plasmid was transfected 3 h later. Three days later, the virus mixture was collected, frozen and thawed three times at -80 °C and 37 °C, centrifuged at 2000 rpm for 5 min to discard cell debris. 200 μL of the virus supernatant was added to a six-well plate seeded with Vero cells. After 4 h of virus infection, the drug screening medium (containing 1× mycophenolic acid, xanthine and hypoxanthine) was replaced, and the target screening was carried out using the gpt gene drug screening principle (wild-type vaccinia virus without the gpt gene cannot be amplified, while recombinant virus containing the target gene and the gpt gene can continue to be amplified). After at least 3 rounds of drug screening experiments, low melting point agarose was poured, and single clone viruses were obtained by picking plaques. Then, the virus genome was identified by PCR. The recombinant oncolytic vaccinia virus seed we needed was the one without the TK gene and containing the aCD19-TM gene, named aCD19-TM-OVV. Subsequently, virus amplification, sucrose density gradient centrifugation purification and TCID50 virus titer determination were carried out in Vero cells to obtain the recombinant oncolytic vaccinia virus aCD19-TM-OVV.

[0032] Another object of the present invention is the use of the recombinant oncolytic vaccinia virus aCD19-TM-OVV in the preparation of a tumor drug for targeted treatment of CD19-positive B lymphoma. The application is that aCD19-TM-OVV can express a membrane-localized CD19 antibody, which is armed on the virus surface, and can promote the infectivity of B lymphocytes and the inhibitory effect on tumor growth in a CD19 antigen-dependent manner. The tumor is CD19 antigen-positive B lymphoma.

[0033] The present invention successfully constructed the recombinant oncolytic vaccinia virus aCD19-TM-OVV by molecular cloning. Flow cytometry was used to verify that cells infected with aCD19-TM-OVV could effectively express the aCD19-TM protein, and this antibody protein was also armed on the surface of the oncolytic vaccinia virus envelope structure. In vitro flow cytometry experiments for detecting virus infection rate showed that aCD19-TM-OVV could promote the infection efficiency of CD19-positive B lymphoma cells and the inhibitory effect on tumor growth in a CD19 antigen-dependent manner. TCID50 experiments for measuring virus replication ability showed that aCD19-TM-OVV could replicate to produce more progeny oncolytic vaccinia viruses. Experiments on NSG mice bearing B lymphoma tumors showed that aCD19-TM-OVV had a stronger inhibitory effect on tumor growth in vivo and less toxic and side effects. The recombinant oncolytic vaccinia virus aCD19-TM-OVV provided by the present invention has the prospect of translational application, and can be used to develop therapeutic drugs for CD19-positive B lymphoma or combined with immunocyte therapies such as CAR-T to overcome the drug resistance problem of B lymphoma.

[0034] The beneficial effects of the present invention are as follows: (1) By inserting an anti-CD19 membrane-localized antibody into the poxvirus genome, the present invention enables the antibody to be modified on the surface of the poxvirus envelope structure, enhancing the infectivity and gene delivery efficiency of B lymphoma cells positive for the CD19 antigen; (2) The recombinant oncolytic poxvirus aCD19-TM-OVV constructed in the present invention has been verified to have a stronger tumor growth inhibitory effect than the control virus treatment group in in vitro and mouse tumor model experiments, laying the foundation for the research and development of this oncolytic poxvirus as a tumor-targeted therapeutic drug; (3) This application is a creative exploration of the targeting research of oncolytic viruses at the level of target cell infection. After demonstration, it has good targeting efficiency compared with that before modification. In addition, the exploration of the treatment of hematological tumors with low virus infection efficiency in this application has not been studied by any team or research institution in the prior art. It is generally recognized in the industry that the virus has low infection efficiency for hematological tumors and high infection efficiency for solid tumors, so the research on treating hematological tumors with viruses has been abandoned. This application overcomes the industry prejudice, enabling the modified virus to greatly improve the infection efficiency for hematological tumors and laying a solid foundation for the subsequent further research on treating hematological tumors with viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. In the accompanying drawings:

[0036] Figure 1 is the map of the poxvirus shuttle plasmid;

[0037] Figure 2 is the identification of whether the genome of the aCD19-TM-OVV recombinant oncolytic poxvirus contains the wild-type TK gene;

[0038] Figure 3 is the identification of whether the genome of the aCD19-TM-OVV recombinant oncolytic poxvirus contains the aCD19-TM gene;

[0039] Figure 4 is the schematic diagram of the genome structure of the aCD19-TM-OVV recombinant oncolytic poxvirus;

[0040] Figure 5 is the result diagram of verifying the expression of the aCD19-TM protein on the surface of Vero cells infected with PBS;

[0041] Figure 6 is the result diagram of verifying the expression of the aCD19-TM protein on the surface of Vero cells infected with OVV;

[0042] Figure 7It is a result graph verifying the expression of aCD19-TM protein on the surface of Vero cells infected with aCD19-TM-OVV;

[0043] Figure 8 It is a result graph verifying the expression of aCD19-TM protein on the surface of Vero cells after incubating Vero cells with PBS on ice for one hour;

[0044] Figure 9 It is a result graph verifying the expression of aCD19-TM protein on the surface of Vero cells after incubating OVV with Vero cells on ice for one hour;

[0045] Figure 10 It is a result graph verifying the expression of aCD19-TM protein on the surface of Vero cells after incubating aCD19-TM-OVV with Vero cells on ice for 30 min;

[0046] Figure 11 It is a result graph showing that aCD19-TM-OVV can promote the infectivity of CD19 antigen-positive B lymphoma Jeko-1 cells in vitro;

[0047] Figure 12 It is a result graph showing that aCD19-TM-OVV can promote the infectivity of CD19 antigen-positive B lymphoma Raji cells in vitro;

[0048] Figure 13 It is a result graph showing that aCD19-TM-OVV does not affect the infectivity of triple-negative breast cancer MDA-MB-231 cells negative for CD19 antigen in vitro;

[0049] Figure 14 It is a result graph showing that the infectivity of aCD19-TM-OVV on CD19 antigen-knockout cells Jeko-1-CD19KO-GFP is significantly reduced in vitro;

[0050] Figure 15 It is a result graph showing that the infectivity of aCD19-TM-OVV on CD19 antigen-knockout cells Raji-CD19KO-GFP is significantly reduced in vitro;

[0051] Figure 16 It is a result graph showing that the viral replication ability of aCD19-TM-OVV is enhanced in B lymphoma Raji cells;

[0052] Figure 17 It is a result graph showing that the viral replication ability of aCD19-TM-OVV is not affected in triple-negative breast cancer MDA-MB-231 cells;

[0053] Figure 18It is the result graph of the killing effect of aCD19-TM-OVV on Raji cells positive for CD19 antigen in vitro;

[0054] Figure 19 It is the result graph of the disappearance of the killing promotion effect of aCD19-TM-OVV on Raji-CD19KO cells with CD19 antigen knockout in vitro;

[0055] Figure 20 It is the result graph of the killing effect of aCD19-TM-OVV on Jeko-1 cells positive for CD19 antigen in vitro;

[0056] Figure 21 It is the result graph of the disappearance of the killing promotion effect of aCD19-TM-OVV on Jeko-1-CD19KO cells with CD19 antigen knockout in vitro;

[0057] Figure 22 It is the schematic diagram of the operation of establishing a B lymphoma model with Raji cells and the oncolytic virus treatment method in NSG mice;

[0058] Figure 23 It is the result graph of the body weight change of the significant inhibition of tumor growth by aCD19-TM-OVV in the tumor model of NSG mice bearing B lymphoma Raji;

[0059] Figure 24 It is the result graph of the tumor volume of the significant inhibition of tumor growth by aCD19-TM-OVV in the tumor model of NSG mice bearing B lymphoma Raji. Detailed implementation manners

[0060] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0061] Example 1: Packaging, identification, amplification and purification of recombinant oncolytic vaccinia virus aCD19-TM-OVV

[0062] Experimental method: Vero cells of rhesus monkey kidney (ATCC, HZ-0242) in good growth state were seeded at 5×10 per well 5The cells were seeded in a six-well plate culture dish (using DMEM medium containing 10% fetal bovine serum, DMEM: Cat#11965092, Thermo Fisher; fetal bovine serum: Cat#16000044, Gibco). After the cells adhered, Vero cells were infected with wild-type vaccinia virus WTVV (ATCC, VR-1354) at 1 MOI. After 3 h, 2 μg of pRGB-aCD19-TM plasmid was transfected. The cell growth was observed under a microscope. When there were many plaques with cytopathic effects (CPE) such as cell rounding and detachment in the cells (usually taking 2 - 4 days), the virus mixture was collected. The collected cells were frozen at -80 °C, then dissolved in a 37 °C water bath and vortexed at high speed. This step was repeated 3 - 4 times to fully lyse the cells (releasing the virus). After centrifuging at 2000 rpm for 5 min to discard the cell debris, 200 μL of the virus supernatant was added to a six-well plate seeded with Vero cells. After 4 h of virus infection, the drug screening medium (containing 1× mycophenolic acid, xanthine, and hypoxanthine) was replaced. The target virus was screened using the gpt gene drug screening principle (wild-type vaccinia virus without the gpt gene cannot be amplified, while recombinant virus containing the target gene and the gpt gene can continue to be amplified). When there were many virus plaques after 2 - 4 days, the cell and virus mixture was collected, and this drug screening step was repeated. At least 3 rounds or more of drug screening experiments were required. The virus solution after more than three rounds of drug screening was diluted in a 10-fold serial dilution and added to a six-well plate seeded with Vero cells. After 4 h, the old medium was aspirated, and 3 mL of low melting point agar (a 1:1 mixture of 5% low melting point agar and DMEM medium containing 10% serum) was added. After 2 - 4 days, the plaques were picked from the well plate with the highest dilution to obtain monoclonal virus, and the monoclonal virus was added to a 24-well plate culture dish seeded with Vero cells for small-scale virus amplification. After 2 - 3 days, the virus solution was collected. Half of the virus solution was used to extract the virus genome, and PCR amplification was performed using primer sequence 4 (SEQ ID No.4) and primer sequence 5 (SEQ ID No.5) to identify whether it contained wild-type virus, and PCR amplification was performed using primer sequence 6 (SEQ ID No.6) and primer sequence 7 (SEQ ID No.7) to identify whether it contained the target gene. The target recombinant oncolytic vaccinia virus seed that does not contain the TK gene and contains the aCD19-TM gene is named aCD19-TM-OVV. Subsequently, large-scale virus amplification was carried out in Vero cells, and purification was performed by sucrose density gradient centrifugation. Then, the virus titer was determined by the TCID50 method.

[0063] Experimental results: PCR of the wild-type vaccinia virus genome in lane 1 could amplify a TK gene band of approximately 600 bp in size, while there was no band in the genomes of the 8 monoclonal viruses in lanes 2 - 9 ( Figure 2); PCR of 8 monoclonal virus genomes with 2-9 lanes can amplify a target gene band of aCD19-TM about 1000bp in size, while the wild-type poxvirus genome in lane 1 does not( Figure 3 ), and the recombinant oncolytic poxvirus aCD19-TM-OVV was successfully constructed. Figure 4 It is a schematic diagram of the recombinant virus genome.

[0064] Example 2: Expression identification of aCD19-TM

[0065] Experimental method: Vero cells were infected with PBS, 10 MOI OVV (control oncolytic poxvirus with TK gene deletion, constructed and obtained by the team of Wenbin Qian from Zhejiang University), and aCD19-TM-OVV respectively. After 24 hours, the expression of aCD19-TM was detected by flow cytometry; 1×10 7 PFU of OVV and aCD19-TM-OVV were incubated with 5×10 4 Vero cells on ice for 30 minutes, and then incubated with an anti-aCD19-TM antibody (Shanghai Xingwan Biotechnology Co., Ltd., product number: 300402) for 30 minutes (without membrane permeabilization), and the expression of aCD19-TM protein was detected by flow cytometry.

[0066] Experimental results: The surface of Vero cells infected with aCD19-TM-OVV had the expression of aCD19-TM protein, while the PBS group and the control virus OVV group did not( Figures 5 to 7 ). Vero cells themselves do not express aCD19-TM. After incubating with aCD19-TM-OVV virus for 30 minutes, aCD19-TM positive and GFP protein negative can be detected, while the expression of aCD19-TM and GFP cannot be detected after incubating with the control virus OVV( Figures 8 to 10 ). GFP negative indicates that the virus has not infected the cells, while aCD19-TM positive indicates that aCD19-TM is on the surface of the envelope structure of aCD19-TM-OVV virus. The expression of aCD19-TM on the oncolytic poxvirus envelope has the ability to improve the recognition and infection of B lymphoma cells positive for CD19 antigen, which is similar to the function of CD19CAR in CAR-T drugs; after aCD19-TM-OVV infects target cells, aCD19-TM protein will also be expressed on the surface of the target cells, enabling B lymphoma cells infected with aCD19-TM-OVV to bind adjacent B lymphoma cells not infected by the oncolytic virus, having the potential to promote the reinfection of B lymphoma cells by progeny viruses of oncolytic poxvirus and reduce the dispersion of B lymphoma cells.

[0067] Example 3: aCD19-TM-OVV can enhance the infectivity of B lymphocytes through the CD19 target

[0068] Experimental methods: B lymphoma cells Jeko-1 (Chinese Academy of Sciences, TCHu194), Raji (Chinese Academy of Sciences, TCHu44) and triple-negative breast cancer MDA-MB-231 (ATCC, YS4076C) were infected with 1 MOI and 10 MOI OVV and aCD19-TM-OVV respectively. After 24 h, flow cytometry was used to detect the percentage of GFP-positive cells to represent the virus infection rate. B lymphoma cells Jeko-1-GFP (GFP gene inserted into Jeko-1, constructed and preserved by the team of Wenbin Qian at Zhejiang University), Jeko-1-CD19KO-GFP (GFP gene inserted into Jeko-1 and CD19 expression gene knocked out, constructed and preserved by the team of Wenbin Qian at Zhejiang University), Raji-GFP (GFP gene inserted into Raji, constructed and preserved by the team of Wenbin Qian at Zhejiang University), Raji-CD19KO-GFP (GFP gene inserted into Raji and CD19 expression gene knocked out, constructed and preserved by the team of Wenbin Qian at Zhejiang University) were infected with 1 MOI and 10 MOI OVV and aCD19-TM-OVV respectively. After 24 h, flow cytometry was used to detect the positive rate of aCD19-TM-expressing cells to represent the virus infection rate and gene delivery efficiency.

[0069] Experimental result 3: aCD19-TM-OVV significantly enhanced the infection rate of poxvirus-resistant CD19-positive B lymphoma Jeko-1 and Raji cells, and there was no significant change in the infection rate of CD19-negative triple-negative breast cancer MDA-MB-231 ( Figures 11 to 13 ). The virus infection rate of aCD19-TM-OVV in CD19KO cell lines decreased significantly ( Figures 14 to 15 ), similar to the infection rate of OVV ( Figures 11 to 15 ). These results indicate that aCD19-TM-OVV can enhance the virus infection rate and gene delivery efficiency of poxvirus to CD19-positive B lymphoma through the CD19 antigen target.

[0070] Example 4: Detection of the virus replication ability of aCD19-TM-OVV

[0071] Experimental methods: Raji and MDA-MB-231 were infected with 1 MOI OVV and aCD19-TM-OVV respectively. Virus replication samples were collected at 24 h and 48 h, and the virus titer of each sample was determined by the TCID50 method. Based on the dose of virus added at 0 h, the virus proliferation multiple at different times was calculated.

[0072] Experimental results: In MDA-MB-231 cells, there was no significant difference in the virus replication ability of aCD19-TM-OVV compared with OVV. In Raji cells, the virus replication ability of aCD19-TM-OVV was significantly enhanced ( Figures 16 to 17) This may be the result of the increased infection rate of aCD19-TM-OVV virus on B lymphoma cells.

[0073] Example 5: Enhanced killing effect of aCD19-TM-OVV on CD19-positive B lymphoma cells in vitro

[0074] Experimental method: Raji, Jeko-1, Raji-CD19KO and Jeko-1-CD19KO cells in good growth state were seeded in 24-well plates at a density of 4×10 5 per well, and PBS, 1MOI and 10MOI of OVV and aCD19-TM-OVV were added respectively. After incubation for 72 h, the absolute number of viable cells in each sample was counted by flow cytometry. Taking the number of viable cells in the PBS group as 100%, the cell survival rate was statistically analyzed.

[0075] Experimental results: aCD19-TM-OVV significantly enhanced the killing effect on CD19-positive Raji and Jeko-1 cells; there was no significant difference in the killing effect on CD19-knockout Raji-CD19KO and Jeko-1-CD19KO cells ( Figures 18 to 21 ). It shows that aCD19-TM-OVV can promote the killing effect of oncolytic poxvirus on tumor cells in a CD19 antigen target-dependent manner.

[0076] Example 6: Enhanced anti-B lymphoma effect of aCD19-TM-OVV in a severe immunodeficient mouse tumor model with less toxic and side effects

[0077] Experimental method: NOD-SCID mice with severe immunodeficiency were subcutaneously inoculated with 5×10 6 Raji cells mixed with Matrigel at a ratio of 1:1 on the right wing, and tumor growth was monitored. When the tumor grew to an average volume of 100 mm 3 , PBS, 2×10 7 PFU OVV and aCD19-TM-OVV were injected intratumorally respectively, once every other day for a total of three times, and the tumor volume growth of mice and the body weight change of mice were monitored.

[0078] Experimental results: aCD19-TM-OVV had a stronger anti-B lymphoma effect in the NSG mouse tumor model; in the OVV treatment group, a significant decrease in mouse body weight was observed from the start of drug administration to one week after the third drug administration, indicating certain toxic and side effects, while in the aCD19-TM-OVV treatment group, no significant decrease in mouse body weight was observed, indicating less toxic and side effects ( Figures 22 to 24 ).

[0079] DNA sequence of anti-CD19 transmembrane protein (aCD19-TM) (SEQ ID NO.1): ATGGCACTGCCTGTGACCGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCAAGGCCAGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGATAGGGTGACCATCTCCTGCCGCGCCTCTCAGGACATCAGCAAGTATCTGAACTGGTACCAGCAGAAGCCTGACGGCACAGTGAAGCTGCTGATCTATCACACCTCCAGGCTGCACTCTGGCGTGCCAAGCCGCTTTTCCGGCTCTGGCAGCGGCACAGATTACTCCCTGACCATCTCTAACCTGGAGCAGGAGGACATCGCCACCTATTTTTGCCAGCAGGGCAATACACTGCCATACACCTTCGGCGGCGGCACAAAGCTGGAGATCACCGGAGGAGGAGGATCCGGCGGAGGAGGCTCTGGCGGCGGCGGCAGCGAGGTGAAGCTGCAGGAGTCCGGACCAGGACTGGTGGCACCTTCCCAGTCTCTGAGCGTGACATGTACCGTGTCTGGCGTGAGCCTGCCCGACTACGGCGTGTCTTGGATCCGGCAGCCCCCTAGAAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACATACTATAATAGCGCCCTGAAGTCCCGGCTGACAATCATCAAGGATAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACAGACGATACCGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGACTACTGGGGCCAGGGCACATCTGTGACCGTGTCTAGCACCACAACCCCAGCACCAAGGCCACCAACACCTGCACCAACCATCGCCTCTCAGCCACTGAGCCTGAGGCCAGAGGCATGTAGGCCTGCAGCAGGAGGCGCCGTGCACACCAGAGGCCTGGATTTCGCCTGCGACATCTATATCTGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGTCCCTGGTCATCACCCTGTATTGC.

[0080] Amino acid sequence of anti-CD19 membrane-localized protein (aCD19-TM) (SEQ ID No. 2):

[0081] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0082] DNA sequence of poxvirus shuttle plasmid pRGB (SEQ ID NO. 3):

[0083] CCTATCACGGAGAAATCTGTAATTGATTCCAAGACATCACATAGTTTAGTTGCTTCCAAT

[0084] GCTTCAAAATTATTCTTATCATGCGTCCATAGTCCCGTTCCGTATCTATTATCGTTAGAATA

[0085] TTTTATAGTCACGCATTTATATTGAGCTATTTGATAACGTCTAACTCGTCTAATTAATTCTG

[0086] TACTTTTACCTGAAAACATGGGGCCGATTATCAACTGAATATGTCCGCCGTTCATGATGA

[0087] CAATAAAGAATTAATTATTGTTCACTTTATTCGACTTTAATATATCCATCACGTTAGAAAAT

[0088] GCGATATTGCGACGAGGATCTATGTATCTAACAGGATCTATTGCGGTGGTAGCTAGAGAG

[0089] GATTCTTTTTTGAATCGCATCAAACTAATCACAAAGTCGAACAAATATCCTTTATTAAGTT

[0090] TGACCCTTCCATCTGTAACAATAGGGACCTTGTTAAACAGTTTTTTAAAATCTTGAAAGT

[0091] CTGTGAATTTTGTCAATTGTCTGTATTCCTCTGAAAGAGATTCATAACAATGACCCACGG

[0092] CTTCTAATTTATTTTTTGATTGGATCAATAATAATAACAGAAAGTCTAGCTAGATATTGAG

[0093] TGATTTGCAATATATCAGATAATGAAGATTCATCATCTTGACTAGCCAAATACTTAAAAAA

[0094] TGAATCATCATCTGCGAAGAACATCGTTAAGAGATACTGGTTGTGATCCATTTATTGATC

[0095] CCAAAAGCTCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAA

[0096] AATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTC

[0097] CATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGA

[0098] TCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGC

[0099] CAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGC

[0100] GAAGTGATCTTCCGTCACAGGTATTTATTCGAAGACGAAAGGGCCTCGTGATACGCCTA

[0101] TTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG

[0102] GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT

[0103] CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTAT

[0104] TCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCT

[0105] CACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG

[0106] GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAA

[0107] CGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTT

[0108] GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGA

[0109] GTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA

[0110] GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA

[0111] GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGA

[0112] TCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG

[0113] CCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC

[0114] TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC

[0115] GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGG

[0116] TCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC

[0117] TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG

[0118] GTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT

[0119] TGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTC

[0120] ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA

[0121] GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA

[0122] AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC

[0123] CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

[0124] TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC

[0125] CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG

[0126] ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG

[0127] CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAG

[0128] AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG

[0129] TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG

[0130] TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG

[0131] GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCCGAGTCAGTCTCATGTTCTCACC

[0132] GGTATAAATACTTAATAATCTCATTTCAGCTGAATATGAAGGAGCAAAAGGTTGTAACAT

[0133] TTTATTACCGTGTGGGATATAAAAGTCCTTGATCCATTGATCTGGAAACGGGCATCTCCA

[0134] TTTAAGACTAGACGCCACGGGGTTTAAAATACTAATCATGACATTTTGTAGAGCGTAATT

[0135] ACTTAGTAAATCCGCCGTACTAGGTTCATTTCCTCCTCGTTTGGATCTCACATCAGAAATT

[0136] AAAATAATCTTAGAAGGATGCAGTTGTTTTTTGATGGATCGTAGATATTCCTCATCAACG

[0137] AACCGAGTCACTAGAGTCACATCACGCAATCCATTTAAAATAGGATCATGATGGCGGCC

[0138] GTCAATTAGCATCCATTTGATGATCACTCCTAAATTATAGAAATGATCTCTCAAATAACGT

[0139] ATATGTGTACCGGGAGCAGATCCTATATACACTACGGTGGCACCATCTAATATACCGTGTC

[0140] GCTGTAACTTACTAAGAAAAAATAATTCTCCTAGTAATAGTTTTAACTGTCCTTGATACG

[0141] GCAGTTTTTTTGCGACCTCATTTGCACTTTCTGGTTCGTAATCTAACTCATTATCAATTTC

[0142] CTCAAAATACATAAACGGTTTATCTAACGACACAACATCCATTTTTAAGTATTATATTAAA

[0143] ATTTAATCAATGTTTATTTTTAGTTTTTTAGATAAAAAATATAATATTATGAGTCGATGTAA

[0144] CACTTTCTACACACCGATTGATACATATCATTACCTCCTATTATTTCTATCTCGGTTTCCTC

[0145] ACCCAATCGTTTAGAAAAGGAAGCCTCCTTAAAGCATTTCATACACACAGCAGTTAGTT

[0146] TTACCACCATTTCAGATAATGGAATAAGATTCAAAATATTATTAAACGGTTTACGTTGAAA

[0147] TGTCCCATCGAGTGCGGCTACTATAACTGCGCGCAATTAACCCTCACTAAAGGGAACAA

[0148] AAGCTGGGATCCGCAGGTTTGCCTGTGTCATGGATGCAGCCTCCAGAATACTTACTGGA

[0149] AACTATTGTAACCCGCCTGAAGTTAAAAAGAACAACGCCCGGCAGTGCCAGGCGTTGA

[0150] AAAGATTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTC

[0151] CAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGC

[0152] TCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTG

[0153] GTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCA

[0154] CCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGT

[0155] ACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCG

[0156] ATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCC

[0157] GTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTG

[0158] AAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGG

[0159] TCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGA

[0160] ACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAA

[0161] CTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAAC

[0162] AGCTCCTCGCCCTTGCTCACCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCC

[0163] TCCACCGCGACCGGAGATTGGCGGGACGAATACGACGCCCATATCCCACGGCTGTTCAA

[0164] TCCAGGTATCTTGCGGGATATCAACAACATAGTCATCAACCAGCGGACGACCAGCCGGT

[0165] TTTGCGAAGATGGTGACAAAGTGCGCTTTTGGATACATTTCACGAATCGCAACCGCAGT

[0166] ACCACCGGTATCCACCAGGTCATCAATAACGATGAAGCCTTCGCCATCGCCTTCTGCGC

[0167] GTTTCAGCACTTTAAGCTCGCGCTGGTTGTCGTGATCGTAGCTGGAAATACAAACGGTA

[0168] TCGACATGACGAATACCCAGTTCACGCGCCAGTAACGCACCCGGTACCAGACCGCCAC

[0169] GGCTTACGGCAATAATGCCTTTCCATTGTTCAGAAGGCATCAGTCGGCTTGCGAGTTTAC

[0170] GTGCATGGATCTGCAACATGTCCCAGGTGACGATGTATTTTTCGCTCATGTGAAGTGTCC

[0171] CAGCCTGTTTATCTACGGCTTAAAAAGTGTTCGAGGGGAAAATAGGTTGCGCGAGATTA

[0172] TAGAGATCCCCAATTCCTCGAGTTATGATCTACTTCCTTACCGTGCAATAAATTAGAATAT

[0173] ATTTTCTACTTTTACGAGAAATTAATTATTGTATTTATTATTTATGGGTGAAAAACTTACTA

[0174] TAAAAAGCGGGTGGGTTTGGAATTAGTGAAAGCTTAAAAATTGAAATTTTATTTTTTTTT

[0175] TTTGGAATATAAATAAGCTCGAAGTCGACAGATCTAGGCCTGAGCTTGATATCGAATTCC

[0176] TGCAGCCCGGGGGATCCACTAGTTCTAGACTCCACAATAAAAACAGATCACCTGATGGA

[0177] TAAAAAGGCGGTTAACCGCGCAGATAAAAAGAGCTCCAATTCGCCCTATAGTGAGTCGT

[0178] ATTACGCGCG

[0179] Primer sequence 4 (SEQ ID No.4):

[0180] ATGGCAAACGAAGGAAAAAT

[0181] Primer sequence 5 (SEQ ID No.5):

[0182] TTATGAGTCGATGTAACACT

[0183] Primer sequence 6 (SEQ ID No.6):

[0184] ATGGCACTGCCTGTGACCGC

[0185] Primer sequence 7 (SEQ ID No.7):

[0186] TTAGCAATACAGGGTGATGAC

[0187] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Finally, it should be stated that the above are only preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant construct, characterized in that, The coding can modify a membrane-localized antibody, its fragment or variant on the surface of the oncolytic virus.

2. A recombinant construct according to claim 1, wherein The nucleic acid sequence of the recombinant construct is as shown in SEQ ID NO.1, its fragment or variant.

3. A recombinant construct according to claim 1, wherein The amino acid sequence encoded by the recombinant construct is as shown in SEQ ID NO.2, its fragment or variant.

4. A recombinant construct according to claim 1, wherein The membrane-localized antibody is a CD19 membrane-localized antibody.

5. A recombinant oncolytic virus, characterized in that, It includes a recombinant construct as described in any one of claims 1 to 4.

6. The recombinant oncolytic virus according to claim 5, characterized in that, The TK gene of the recombinant oncolytic virus is replaced or inserted with the recombinant construct.

7. A recombinant oncolytic virus according to claim 5, characterized in that, The recombinant oncolytic virus is an oncolytic poxvirus.

8. A pharmaceutical composition, characterized in that: It includes a recombinant construct as described in any one of claims 1 to 4 or a recombinant oncolytic virus as described in any one of claims 5 to 7 and at least one pharmaceutically acceptable carrier.

9. Use of a recombinant construct as described in any one of claims 1 to 4 or a recombinant oncolytic virus as described in any one of claims 5 to 7 or the pharmaceutical composition as claimed in claim 8 in the preparation of a kit for diagnosing / treating malignant tumors or autoimmune diseases.

10. Use of a recombinant construct as described in any one of claims 1 to 4 or a recombinant oncolytic virus as described in any one of claims 5 to 7 or the pharmaceutical composition as claimed in claim 8 in the preparation of a drug for diagnosing / treating malignant tumors or autoimmune diseases.

11. Use according to claim 10 in the preparation of a medicament for diagnosing / treating malignant tumors or autoimmune diseases, characterized in that, The malignant tumor is any one of acute lymphoblastic leukemia, chronic lymphocytic leukemia or B-cell lymphoma.

12. A method for constructing a recombinant oncolytic virus, characterized in that, Replace or insert the TK gene of the recombinant oncolytic virus with a recombinant construct as described in any one of claims 1 to 4.

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