A recombinant construct, a recombinant oncolytic virus and methods of construction and use thereof

Abstract

This invention relates to the fields of biotechnology and targeted therapy, specifically to a recombinant construct, a recombinant oncolytic virus, its construction method, and its applications. The recombinant oncolytic virus CD19 BIKE is prepared by inserting exogenous genes for CD16 and CD19 into the genome of an oncolytic vaccinia virus. The antibodies secreted by this recombinant virus can not only bridge NK cells and CD19-positive B-cell lymphoma cells, increasing the contact between NK cells and tumor cells and their accumulation within tumor tissue, but also synergize with the tumor-lysing effect of the oncolytic virus, breaking down the stromal barrier, reshaping the tumor microenvironment, and enhancing therapeutic efficacy.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and targeted therapy, and particularly to a recombinant construct, a recombinant oncolytic virus, its construction method, and its application. Background Technology

[0002] Oncolytic viruses are tumor-killing viruses with self-replicating and amplifying capabilities, representing an important branch of tumor immunotherapy. By modifying the genome of oncolytic viruses, recombinant oncolytic viruses with low pathogenicity and high safety can be obtained. Specifically, on the one hand, recombinant oncolytic viruses can selectively infect tumor cells, occupying the intracellular protein translation mechanism of tumor cells, inhibiting protein synthesis in tumor cells, and then infecting neighboring tumor cells with progeny viruses after self-replication within tumor cells, continuously lysing the tumor. On the other hand, the intracellular replication of recombinant oncolytic viruses leads to endoplasmic reticulum stress, mitochondrial apoptosis, or necrotizing apoptosis. The resulting damage-associated pattern molecules (DAMPs) and pathogen-associated pattern molecules (PAMPs) recruit effector T cells and related macrophages, thereby reshaping the tumor microenvironment and inducing immunogenic death (ICD). Therefore, oncolytic viruses possess the dual advantages of direct oncolysis and immune activation, demonstrating unique advantages in the treatment of malignant tumors.

[0003] The core of bispecific or trispecific antibodies is the integration of two or three different antigen-binding domains into a single molecule. This serves as a "bridge" connecting single or multiple immune cells with tumor cells or other targets, guiding immune cells to precisely attack target cells, thereby breaking immune tolerance and achieving targeted immune redirection. Currently, the U.S. Food and Drug Administration (FDA) has approved 12 bispecific antibodies, most of which target endogenous CD3 cells. Examples include the BCMA / CD3 series (Teclistamab, Elrexfio, Linvoseltamab) and the CD20 / CD3 series (Glofitamab and Epcoritamab), all of which have achieved breakthroughs in the treatment of hematological malignancies. Simultaneously, bridging natural killer (NK) cells and tumor cells with bispecific antibodies is also a NK cell redirection technique. Its main characteristic is that bispecific antibodies bind to the CD16a receptor (FcγRIIIa) on the surface of NK cells, mediating antibody-dependent cytotoxicity (ADCC) and cytokines to directly activate NK cells, forming immune synapses and releasing cytotoxic molecules to induce apoptosis and eliminate target cells. Among the existing bispecific antibodies that bridge NK cells, CD33 / CD16 and B7-H3 / CD16 have achieved certain results in clinical trials for the treatment of hematologic malignancies and solid tumors. They have also been used in combination with other immunotherapies to produce synergistic effects. Their unique mechanism of action and significant effects provide new directions for tumor immunotherapy and show broad application prospects. Summary of the Invention

[0004] The present invention aims to provide a recombinant construct, a recombinant oncolytic virus, its preparation method, and its applications. A CD19 BIKE recombinant oncolytic virus is prepared by inserting exogenous genes encoding CD16 and CD19 into an oncolytic virus, enabling the recombinant oncolytic virus to secrete and express the bispecific antibody CD19 BIKE. Mechanistically, the oncolytic virus produces oncolysis and immune activation. The secreted CD19 BIKE spatially pulls CD16-positive NK cells closer to CD19-positive tumor cells, redirecting NK cells and increasing NK cell infiltration in tumor tissue. Under this triple anti-tumor effect, the killing efficacy against CD19-positive tumor cells is significantly enhanced.

[0005] Specifically, Cluster of Differentiation 19 (CD19) is a transmembrane glycoprotein unique to the surface of B lymphocytes, belonging to the immunoglobulin superfamily, and is a core molecule for B cell activation, development, and functional regulation. Firstly, CD19 is distributed only on the surface of B lymphocytes and their derived tumor cells, exhibiting high specificity. Secondly, CD19 mediates the survival and proliferation of B lymphocyte-related tumors, demonstrating its functional role. Thirdly, CD19 expression is stable in B lymphocyte-related tumors and is not easily lost during treatment. Based on these characteristics, CD19 has become a core target for B lymphocyte-related tumors.

[0006] CD16a (Cluster of Differentiation 16, FcγRIIIa) is a key member of the immunoglobulin G (IgG) Fc receptor family and is expressed on NK cells, macrophages, monocytes, and dendritic cells. CD16a can not only mediate antibody-dependent cytotoxicity (ADCC) to eliminate tumor cells, but also act as a "co-stimulatory receptor" to activate NK cells and amplify effector functions.

[0007] Based on the above description, the present invention provides the following technical solution:

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

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

[0010] Preferably, the recombinant construct encodes an amino acid sequence as shown in SEQ ID NO.2, or a fragment thereof or a variant thereof.

[0011] Preferably, the membrane-localizing antibody is a bispecific antibody that co-localizes CD19 and CD16 membranes.

[0012] A recombinant oncolytic virus comprising a recombinant construct as described in any of the preceding claims.

[0013] Preferably, the recombinant construct replaces or inserts the TK gene of the recombinant oncolytic virus.

[0014] The CD16 and CD19 sequences are inserted into the recombinant construct or into the TK gene of the recombinant oncolytic virus.

[0015] The CD16 and CD19 sequences are loaded onto pCB to obtain pCB-CD19 BIKE, and pCB-CD19 BIKE and oncolytic virus homologous recombination.

[0016] The exogenous gene of CD19 BIKE can secrete and express CD19 BIKE, and CD19 BIKE has the function of activating NK cell activity.

[0017] The coding sequence of the CD19 BIKE exogenous gene is any DNA sequence capable of encoding CD19 BIKE. Preferably, the sequence is SEQ ID NO.1 or its complementary sequence. In this case, the coding sequence of the CD19 BIKE exogenous gene of the present invention can be a polynucleotide or its complementary sequence that hybridizes with the nucleotide sequence of SEQ ID NO.1 under strict conditions and encodes CD19 BIKE with NK cell activation activity.

[0018] Preferably, the recombinant oncolytic virus is an oncolytic vaccinia virus.

[0019] A pharmaceutical composition comprising a recombinant construct or a recombinant oncolytic virus as described in any of the preceding claims, and at least one pharmaceutically acceptable vector.

[0020] Preferably, the pharmaceutically acceptable carrier is an NK cell.

[0021] The method for preparing NK cells includes: collecting peripheral blood from healthy individuals and obtaining mononuclear cells by Ficoll density gradient centrifugation; obtaining autologous plasma by centrifugation; sorting NK cells using magnetic beads; the basal culture medium (X-VIVO 15, LONZA, 04-418Q) can be 50 mL with a final concentration of 20 μg / mL IFN-γ, 10 μg / mL IL-2, 10 μg / mL IL-21, 50 μg / mL IL-15, and 2.5 mL autologous plasma; culturing in a constant temperature incubator at 37℃ and 5.0% CO2, with subsequent supplementation of culture medium according to cell density to maintain a cell density of 1.0~2.5×10⁻⁶ cells / mL. 6 / mL.

[0022] The pharmaceutically acceptable carriers include excipients and adjuvants that facilitate the processing of the recombinant vaccinia virus of the present invention into formulations, and the pharmaceutical composition can be prepared into formulations suitable for injection or infusion, including aqueous and non-aqueous sterile injections or aqueous and non-aqueous sterile suspensions.

[0023] The pharmaceutical composition is in solid or injectable form. Preferably, the pharmaceutical composition is packaged in a medicine box; more preferably, the medicine box also contains instructions for use of the pharmaceutical composition.

[0024] The use of any of the recombinant constructs described in any one of the foregoing claims, or any of the recombinant oncolytic viruses described in any one of the foregoing claims, or any of the pharmaceutical compositions described in the foregoing claims in the preparation of drugs for treating tumors or cancer. Wherein, tumor or cancer refers to CD19-positive B-cell tumors, which may be any one of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or B-cell lymphoma.

[0025] A method for constructing a recombinant oncolytic virus, wherein the method involves inserting CD16 and CD19 nucleotide sequences into an oncolytic virus to obtain a recombinant oncolytic virus. Specifically, the nucleotide sequences of CD16 and CD19 are loaded into pCB particles to obtain pCB-CD19 BIKE; the plasmid pCB-CD19 BIKE undergoes homologous recombination with a wild-type oncolytic virus, and the CD16 and CD19 sequences are inserted into the TK gene of the wild-type oncolytic virus.

[0026] This invention has the following characteristics compared to the prior art:

[0027] 1) Using a self-replicating oncolytic virus as a vector, it continuously secretes a functional bispecific antibody, CD19 BIKE, as a bridge to connect NK cells and CD19-positive B-cell lymphoma cells, increasing the contact between NK cells and tumor cells or their accumulation within tumor tissue, thus breaking down the stromal barrier; 2) The oncolytic virus exerts an infectious lysis effect on tumor cells, and various cellular stress responses during progeny virus production lead to the remodeling of the tumor microenvironment; 3) The oncolytic virus and its secreted CD19 BIKE antibody form a synergistic anti-tumor system, specifically addressing or alleviating the stromal barrier and suppressive immune microenvironment in B-cell lymphomas, thereby improving treatment efficacy. Attached Figure Description

[0028] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0029] Figure 1 This is the plasmid map of the pCB shuttle plasmid in Example 1 of the present invention;

[0030] Figure 2 This is a schematic diagram of the genome structure of the recombinant oncolytic vaccinia virus (OVV-CD19 BIKE) in Example 1 of the present invention;

[0031] Figure 3 This is a schematic diagram illustrating the detection of CD19 BIKE expression levels in the cytoplasm and cell supernatant after Vero cells were infected with recombinant oncolytic vaccinia virus CD19 BIKE (OVV-CD19 BIKE) in Example 2 of the present invention.

[0032] Figure 4This is a graph showing the results of detecting the binding of cell supernatant carrying CD19 BIKE to CD19 or CD16 positive cells in Example 3 of the present invention;

[0033] Figure 5 This is a graph showing the results of detecting the activation effect of cell supernatant carrying CD19 BIKE on NK cells in Example 4 of the present invention;

[0034] Figure 6 This is a graph showing the results of detecting the effect of cell supernatant carrying CD19 BIKE on NK cell killing of tumor cells in Example 5 of the present invention;

[0035] Figure 7 This is a schematic diagram illustrating the operation of the NSG mouse B-lymphoma model created using Raji cells and the oncolytic virus treatment method in Example 6 of this invention;

[0036] Figure 8 These are the results of the changes in tumor volume and body weight of mice, as well as the circulation of NK cells in vivo, in a mouse lymphoma model, as shown in Example 6 of this invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1: Preparation of recombinant oncolytic virus OVV-CD19 BIKE.

[0039] Experimental methods:

[0040] 1) A foreign gene sequence is synthesized by gene synthesis method, wherein the foreign gene can express CD16 and CD19 antibodies; the nucleotide sequence of the foreign gene is shown in SEQ ID NO.1; the amino acid sequence of the foreign gene is shown in SEQ ID NO.2; and the amino acid sequence of the foreign gene is supplemented with a Flag tag protein sequence as shown in SEQ ID NO.3.

[0041] 2) The exogenous gene sequence is loaded into the pCB shuttle plasmid (plasmid map as shown). Figure 1 The pCB-CD19 BIKE plasmid was obtained by interfering with two restriction endonucleases, EcoRI and XbaI (as shown).

[0042] 3) pCB-CD19 BIKE plasmid and wild-type vaccinia virus (ATCC, VR-1354) were transformed into HEK293 cells (Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, SCSP-5500). In HEK293 cells, the TK-L and TK-R sequences on the pCB-CD19 BIKE plasmid underwent homologous recombination with the TK sequence of the wild-type vaccinia virus, and finally the foreign gene sequence was inserted into the TK gene of the wild-type vaccinia virus to obtain recombinant oncolytic vaccinia virus (OVV-CD19 BIKE); the wild-type vaccinia virus was selected after being attenuated.

[0043] 4) The vaccinia virus was screened sequentially using xanthine, hypoxanthine and mycophenolic acid to obtain the purified recombinant oncolytic vaccinia virus.

[0044] Experimental results: The structural diagram of recombinant oncolytic vaccinia virus (OVV-CD19 BIKE) is shown below. Figure 2 As shown.

[0045] Example 2: Detection of cell supernatant and expression level of CD19 BIKE in cells after infection with recombinant oncolytic vaccinia virus (OVV-CD19 BIKE).

[0046] Experimental method: 5×10⁶ cells were seeded into each well of a 6-well plate. 5 Monkey embryonic kidney Vero cells (Cell Bank of the Chinese Academy of Sciences / Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used. The experimental group received 0.5 MOI of recombinant oncolytic vaccinia virus (OVV-CD19 BIKE), while the control group received an equal amount of oncolytic vaccinia virus (OVV). Normal culture medium was used as the basal control (NC). Cells were cultured at 37℃ and 5% CO2. Cells and cell supernatants were collected after 48 h. After cell lysis, Western blotting was used to detect the expression of CD19 BIKE in the cell supernatant and intracellularly.

[0047] Experimental results: such as Figure 3 As shown, no expression of Flag (CD19 BIKE) was detected in the cell supernatant in the NC and OVV groups, while it was detected in the OVV-CD19 BIKE group, indicating that Vero cells secreted CD19 BIKE antibody after OVV-CD19 BIKE infection.

[0048] CD19 BIKE antibody was also detected in the cell lysate in the OVV-CD19 BIKE group, and the concentration was much higher than that in the cell supernatant.

[0049] Example 3: Detection of the binding of cell supernatant carrying CD19 BIKE to CD19 or CD16 positive cells.

[0050] Experimental methods:

[0051] Experimental group: NK cells (CD16) in good condition were collected. + / - CD19 - Raji (CD19) + CD16 - Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, TCHU44) and Jeko-1 (CD19) + CD16 - Cell Bank of the Type Culture Collection Committee, Chinese Academy of Sciences / Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, TCHU194) 1×10 5 Cells (1 mL each) were incubated at 4°C for 60 minutes with different supernatant concentrations containing CD19 BIKE (0.3%, 3%, 30%, 60%, V / V; for example, 60% could be prepared by adding 0.6 mL of CD19 BIKE supernatant from Example 2 to 0.4 mL of phosphate buffer). After washing once with phosphate buffer, Flag-PE antibody (Biolegend, 637309, 1:200, V / V) was added and incubated at 4°C for 20 minutes. Cells were then washed once with phosphate buffer and resuspended in 300 μL of phosphate buffer. The Flag-PE status was detected by flow cytometry and statistically analyzed.

[0052] Control group: Incubated with supernatant after infection with oncolytic vaccinia virus, with other treatments the same as the experimental group;

[0053] Experimental results: The results are as follows Figure 4 As shown, Raji, Jeko-1, and NK cells can all bind to the supernatant of cells carrying CD19 BIKE, and the proportion of Flag-positive cells increases with the increase of supernatant volume, showing a certain concentration dependence; while no binding was detected in the control group.

[0054] Example 4: Flow cytometry detection of the activation effect of cell supernatant carrying CD19 BIKE on NK cells.

[0055] Experimental methods:

[0056] Experimental group: 1×10⁶ healthy NK cells were collected. 5Cells were seeded in 48-well plates with either 30% or 60% CD19BIKE supernatant (to a final volume of 500 μL) and incubated at 37°C and 5% CO2 for 24 hours (with a protein transport inhibitor, Thermo (004980-93), added after approximately 6-8 hours of incubation). After incubation, cells were removed, washed once with phosphate-buffered saline (PFS), and incubated with CD107a (Biolegend, 328631, 1:150, V / V) and NKp46 (Biolegend, 331931, 1:150, V / V) at 4°C for 20 minutes. Cells were washed once with PFS and resuspended in 300 μL of PFS. The proportions of CD107a and NKp46 in NK cells were analyzed by flow cytometry and statistically analyzed (CD107a and NKp46 can both serve as indicators of NK cell activation).

[0057] Control group: Cultured using supernatant after infection with oncolytic vaccinia virus, with other treatments consistent with the experimental group;

[0058] Experimental results: such as Figure 5 As shown, after stimulation, both the control group and the experimental group showed a certain degree of increase in CD107a and NKp46, as well as the unstimulated group, and there were significant differences between the control group and the experimental group at different concentrations; with the increase of CD19 BIKE concentration, the induction of NK cells also increased.

[0059] Example 5: Results of detecting the effect of cell supernatant carrying CD19 BIKE on NK cell killing of tumor cells.

[0060] Experimental methods: Healthy tumor cells Raji-CBRluc-GFP, Jeko-1-CBG99-BFP, and Raji-luc-GFP / CD19 were collected. - Press 2×10 5 Tumor cells were uniformly suspended at a density of / mL in cell supernatant containing 60% (V / V) CD19-bike-carrying cells. NK cells were also uniformly suspended in the same supernatant. 50μL of tumor cells were seeded into each well of an opaque 96-well plate, with varying densities of NK cells added to establish an effector / target cell ratio. After culturing at 37℃ and 5% CO2 for 5 hours, luciferase substrate was added, and the microplate reader was set to detect the cells. Cell viability was calculated using the values ​​obtained from tumor cells without NK cells and cell supernatant as 100%.

[0061] Raji-CBRluc-GFP and Jeko-1-CBG99-BFP cells are obtained by transfecting Raji or Jeko-1 cells with lentivirus to express fluorescent proteins, followed by flow cytometry sorting. Raji-CBRluc-GFP / CD19 - The cells were first constructed using CRISPR-Cas9 technology to select the sgRNA corresponding to CD19 from the database and construct a CD19 knockout Raji cell line (constructed by Genewiz). The cells were then transfected with lentivirus to express fluorescent protein and obtained by flow cytometry sorting.

[0062] Experimental results: such as Figure 6 As shown, CD19 BIKE can significantly enhance the killing effect on CD19-positive Raji and Jeko-1 cells; and on CD19-knockout Raji-luc-GFP / CD19 cells. - There was no significant difference in the cytotoxic effects.

[0063] Example 6: Antitumor effect of OVV-CD19 BIKE in a severely immunodeficient mouse B lymphoma model and in vivo circulation of NK cells.

[0064] Experimental method: The operation diagram is shown below. Figure 7 As shown, NOD-SCID mice with severe immunodeficiency were subcutaneously inoculated with 5×10⁶ styrene mixed with a 1:1 matrix gel in the right flank. 5 Raji-luc-GFP cells were used to detect tumor growth. Tumors grew to an average volume of approximately 80 mm². 3 At that time, PBS and 2×10 were injected intratumorally respectively. 7 PFU-conjugated OVV and OVV-CD19 BIKE; the next day, NK cells labeled with DiR (Thermo Fisher Scientific, D12731) were injected via tail vein; the drug was administered every 2 days and NK cells were injected every 2 days, for a total of three times; changes in tumor volume and NK cell circulation in mice were detected using a small animal imaging system, while tumor diameter was measured using calipers and mouse weight was measured using a scale. (The tumor fluorescence value was detected by a small animal imaging system after mice were injected intraperitoneally with fluorescein substrate (Lambolid, D-fluorescein sodium salt, weighed 150 mg, dissolved in 10 mL phosphate buffer, filtered through a 0.22 μM filter membrane, 150 μL / mouse).

[0065] Experimental results: such as Figure 8 As shown,

[0066] 1) By Figure 8 a (changes in tumor fluorescence values ​​monitored by small animal imaging) and Figure 8b (tumor volume measured by vernier calipers) shows that the OVV-CD19 BIKE+NK cell group has a stronger tumor-suppressing effect compared with the OVV+NK cell group and the OVV-CD19 BIKE group; there is no significant difference between the OVV+NK cell group and the PBS group in terms of tumor volume or total fluorescence value, while there is a significant difference between the OVV-CD19 BIKE+NK cell group and the PBS group.

[0067] 2) By Figure 8 As shown in c, the mice's weight did not change much throughout the monitoring process, indicating that there were no toxic side effects.

[0068] 3) By Figure 8 As can be seen from day 12, the fluorescence value of NK cells indicates that the NK cells in mice in the OVV+NK cell group and PBS group showed a rapid decline, while the decline in the OVV-CD19 BIKE group was not significant, and the cells circulated to the tumor site, indicating that the CD16 terminus of CD19 BIKE is playing a role in recruiting NK cells.

[0069] Nucleotide sequence of anti-CD19 BIKE antigen (SEQ ID NO.1):

[0070]

[0071] Amino acid sequence of anti-CD19 BIKE antigen (SEQ ID NO.2):

[0072] MGWSCIILFLVATATGVHSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSQVQLQESGGGSVQTGGSLRLSCAASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLYLQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSDYKDDDDK

[0073] The amino acid sequence of the flag tag (SEQ ID NO.3): DYKDDDDK

[0074] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant construct, characterized in that, The recombinant construct encodes a membrane-localizing antibody that can be modified on the cell surface, and the amino acid sequence encoded by the recombinant construct is shown in SEQ ID NO.

2.

2. The recombinant construct according to claim 1, characterized in that, The nucleic acid sequence of the recombinant construct is shown in SEQ ID NO.

1.

3. The recombinant construct according to claim 1, characterized in that, The membrane-localizing antibody refers to a bispecific antibody that co-localizes CD19 and CD16 membranes.

4. A recombinant oncolytic virus, characterized in that, Includes a recombinant construct as described in any one of claims 1 to 3.

5. A recombinant oncolytic virus according to claim 4, characterized in that, The recombinant construct replaces or inserts the TK gene of the recombinant oncolytic virus.

6. The recombinant oncolytic virus according to claim 4, characterized in that, The recombinant oncolytic virus mentioned is an oncolytic vaccinia virus.

7. A pharmaceutical composition, characterized in that: Includes a recombinant construct according to any one of claims 1 to 3 or a recombinant oncolytic virus according to any one of claims 4 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that, Including at least one pharmaceutically acceptable carrier.

9. A pharmaceutical composition according to claim 8, characterized in that, The pharmaceutically acceptable carrier is NK cells.

10. The pharmaceutical composition according to claim 9, characterized in that, The method for preparing NK cells includes: collecting peripheral blood from healthy individuals and obtaining mononuclear cells by Ficoll density gradient centrifugation; obtaining autologous plasma by centrifugation; obtaining NK cells by magnetic bead sorting; adding IFN-γ, IL-2, IL-21, IL-15, and autologous plasma to the basal culture medium at final concentrations of 20 μg / mL, 10 μg / mL, 10 μg / mL, and 50 μg / mL; culturing in a constant temperature incubator at 37℃ and 5.0% CO2, and replacing the culture medium with fresh medium according to the cell growth status.

11. The use of a recombinant construct according to any one of claims 1 to 3, or a recombinant oncolytic virus according to any one of claims 4 to 6, or a pharmaceutical composition according to any one of claims 7 to 10, in the preparation of a medicament for treating tumors or cancer; wherein the tumor or cancer is a CD19-positive B-system tumor.

12. The application according to claim 11, characterized in that, The CD19-positive B-cell lymphoma is selected from any one of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or B-cell lymphoma.

13. A method for constructing a recombinant oncolytic virus, characterized in that, The recombinant construct according to any one of claims 1 to 3 replaces or inserts the TK gene of the recombinant oncolytic virus.

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