Bispecific antibody fusion protein, recombinant oncolytic virus and application of bispecific antibody fusion protein and recombinant oncolytic virus in preparation of medicine for treating tumors

Abstract

The invention discloses a bispecific antibody fusion protein, a recombinant oncolytic virus and application of the bispecific antibody fusion protein and the recombinant oncolytic virus in preparation of drugs for treating tumors, the recombinant oncolytic virus can express and secrete a bispecific antibody HER2-BiTE, and the bispecific antibody HER2-BiTE can serve as an adapter to recruit T cell targeted HER2 positive tumor cells, so that the killing effect of the T cells on the tumor cells is improved; meanwhile, the oncolytic virus causes tumor cell lysis, tumor-associated antigen release and tumor microenvironment remodeling; the BiTE plays a role in tumor sites, so that the adverse reaction of systemic administration is relieved. The synergistic effect of the oncolytic virus and the bispecific antibody improves the tumor treatment effect.

Description

(I) Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bispecific antibody fusion protein, a recombinant oncolytic virus, and its application in the preparation of drugs for treating tumors. (II) Background Technology

[0002] Malignant tumors, especially breast cancer, remain a major challenge to global public health. While current mainstream treatments such as surgery, chemotherapy, radiotherapy, and targeted therapy have made some progress, they still face bottlenecks such as high toxicity, easy development of drug resistance, and limited effectiveness against metastatic lesions. Therefore, developing novel therapies that can precisely target tumors, stimulate sustained anti-tumor immunity, and have manageable side effects has become an urgent need in this field.

[0003] Oncolytic viruses (OVs) are genetically modified viruses used as a novel immunotherapy. They can specifically replicate and lyse tumor cells without harming normal cells. The mechanism of action of oncolytic viruses mainly includes two aspects: direct lysis of tumor cells and indirect immune activation. Immune activation is key to the anti-tumor effect of oncolytic viruses. After viral lysis of tumor cells, tumor-associated antigens are released, reshaping the tumor microenvironment and turning "cold" tumors into "hot" tumors, activating the body's systemic anti-tumor immune response. Furthermore, oncolytic viruses can be genetically engineered as vectors to enhance their functionality. However, single-agent oncolytic virus therapy faces challenges such as incomplete tumor clearance, weakened efficacy due to an immunosuppressive microenvironment, and the possibility of the virus being neutralized and cleared by the body's immune system during intravenous administration. Its efficacy still has significant room for improvement.

[0004] Bispecific antibodies (BsAbs) represent an important class of immune connectors. Among them, the bispecific T-cell connector (BiTE) is a typical design that involves binding to tumor cell surface antigens, bridging T cells and tumor cells, and killing tumor cells in a manner independent of MHC and TCR, thus bypassing tumor immune escape.

[0005] As a non-IgG bispecific antibody, BiTE does not have the Fc fragment of the antibody. Its small molecular weight and strong penetrability make it more suitable for short-acting and high tissue penetration treatment scenarios. However, its inherent short half-life means that current clinical protocols usually require continuous intravenous infusion to maintain an effective concentration in the blood. This brings inconvenience to treatment, increases medical costs, and systemic exposure may cause risks such as cytokine release syndrome.

[0006] Human epidermal growth factor receptor 2 (HER2 / ERBB2) is a member of the ErbB receptor tyrosine kinase family. HER2 uniquely functions as a ligandless receptor and is the preferred dimerization partner for other family members. Activation of the HER2 signaling pathway is a crucial mechanism for cell proliferation, survival, and differentiation. HER2 is expressed at low levels in normal human tissues, but high expression has been found in breast cancer, gastric cancer, esophageal cancer, lung cancer, and ovarian cancer, and is associated with poor tumor prognosis. Currently, targeted therapies against HER2 (trastuzumab, pertuzumab) are used clinically to treat cancer patients. However, chemotherapy drugs are administered systemically, involve high doses, and carry high risks; therefore, a more effective in-situ tumor-directed therapy is still needed.

[0007] Based on the above analysis, theoretically, combining the immune-activating ability of oncolytic viruses with the highly efficient T-cell recruitment ability of bispecific antibodies is a promising strategy. However, existing combined use of oncolytic viruses and bispecific antibodies is mostly limited to simple co-administration or sequential administration. This approach cannot guarantee the synergistic and targeted spatiotemporal distribution of the two therapeutic components at the tumor site, nor can it address the pharmacokinetic defects caused by systemic administration of bispecific antibodies.

[0008] Therefore, there is an urgent need in this field for an innovative technical solution that can deeply integrate the advantages of oncolytic viruses and bispecific antibodies, overcome the limitations of existing technologies, and thus develop more efficient, precise and safe anti-tumor therapies. (III) Summary of the Invention

[0009] The purpose of this invention is to provide a HER2-targeting bispecific antibody fusion protein, a recombinant oncolytic virus, and its application in the preparation of anti-tumor drugs. The bispecific antibody fusion protein (HER2-BiTE) is formed by the ligation of the gene sequences of HER2 on the surface of tumor cells and CD3 on the surface of T cells. The recombinant oncolytic virus prepared from the fusion protein can express HER2-BiTE in tumors and simultaneously target HER2 on the surface of tumor cells and CD3 on the surface of T cells, bridging tumor cells and T cells, mediating T cell killing of tumor cells, thereby enhancing the anti-tumor ability of the oncolytic virus. This invention solves the problems of systemic toxicity, short serum half-life, and the formation of an immunosuppressive tumor microenvironment associated with BiTE bispecific antibody therapy.

[0010] The technical solution adopted in this invention is:

[0011] In a first aspect, the present invention provides a bispecific antibody fusion protein targeting HER2, wherein the fusion protein is formed by linking and expressing the gene sequences of a HER2 antibody or a variant thereof on the surface of tumor cells and a CD3 antibody or a variant thereof on the surface of T cells, and the fusion protein can be used simultaneously for membrane localization of the cell surface HER2 antibody and CD3 antibody.

[0012] Furthermore, the nucleotide sequence of the light chain variable region of the CD3 antibody is shown in SEQ ID NO.4, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.5.

[0013] Furthermore, the nucleotide sequence of the light chain variable region of the HER2 antibody is shown in SEQ ID NO.6, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0014] Furthermore, the fusion protein structure is as follows: secretion signal peptide - CD3 antibody light chain variable region - linker 1 - CD3 antibody heavy chain variable region - linker 2 - HER2 antibody light chain variable region - linker 3 - HER2 antibody heavy chain variable region - FLAG tag protein; linker 1, linker 2 and linker 3 are each independently three groups (Gly-Gly-Gly-Gly-Ser, G4S).

[0015] Furthermore, the nucleotide sequence of the secretion signal peptide is shown in SEQ ID NO.3, and the nucleotide sequences of linker 1, linker 2, and linker 3 are all shown in SEQ ID NO.8. The encoding nucleotide sequence of the FLAG tag protein is shown in SEQ ID NO: 9.

[0016] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0017] Furthermore, the nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID NO.2.

[0018] Secondly, the present invention provides a recombinant oncolytic virus, wherein the recombinant oncolytic virus carries the encoding gene of the fusion protein.

[0019] Furthermore, the recombinant oncolytic virus is prepared by inserting the fusion protein gene into the thymidine kinase (TK) gene of the oncolytic virus genome, before the Pse / l promoter.

[0020] Furthermore, the recombinant oncolytic virus is obtained by loading the fusion protein gene into pRGB002 to obtain pRGB002-HER2-BiTE, and then homologously recombinating pRGB002-HER2-BiTE with wild-type oncolytic virus. The recombinant oncolytic virus can secrete and express HER2-BiTE, and HER2-BiTE has the function of activating T cell activity targeting HER2.

[0021] Thirdly, the present invention provides the application of the recombinant oncolytic virus in the preparation of a tumor treatment drug.

[0022] Furthermore, the tumor includes any one of HER2-positive breast cancer, gastric cancer, esophageal cancer, lung cancer, and ovarian cancer.

[0023] Furthermore, the drug is a drug that inhibits the proliferation of tumor cells; the tumor cells include human breast cancer cells Skbr3, human ovarian cancer cells Skov3, and human breast cancer cells Mdamb231.

[0024] Fourthly, the present invention provides a medicament for treating tumors, the medicament comprising the recombinant oncolytic virus described above.

[0025] Furthermore, the drug comprises at least one pharmaceutically acceptable carrier.

[0026] Furthermore, the pharmaceutically acceptable carrier is a T cell.

[0027] Furthermore, pharmaceutically acceptable carriers also include excipients or adjuvants that facilitate the processing of the recombinant oncolytic virus of the present invention into a formulation.

[0028] Furthermore, the drug includes solid or liquid formulations, and the liquid formulations can be prepared into formulations suitable for injection or infusion, including aqueous and non-aqueous sterile injections or aqueous and non-aqueous sterile suspensions.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] This invention provides a bispecific antibody fusion protein and a recombinant oncolytic virus. The recombinant oncolytic virus expresses and secretes the bispecific antibody HER2-BiTE, which acts as an adaptor to recruit T cells to target HER2-positive tumor cells, increasing the killing effect of T cells on tumor cells. Simultaneously, the oncolytic virus induces tumor cell lysis, releases tumor-associated antigens, and remodels the tumor microenvironment. BiTE functions at the tumor site, mitigating the adverse reactions of systemic administration. The synergistic effect of the oncolytic virus and the bispecific antibody improves the therapeutic effect on tumors. (iv) Description of the attached drawings

[0031] Figure 1 This is a schematic diagram of the pRGB002-HER2-BiTE structure.

[0032] Figure 2 Schematic diagrams of the structures of OVV, HER2-BiTE, and recombinant oncolytic virus OVV-HER2-BiTE.

[0033] Figure 3 The image shows an agarose gel electrophoresis diagram of the PCR amplification products; A represents the wild-type reserve strain; B represents OVV-HER2-BiTE.

[0034] Figure 4SDS-PAGE results for OVV-HER2-BiTE expression and secretion of HER2-BiTE.

[0035] Figure 5 Results of OVV-HER2-BiTE replication capability assay using the TCID50 method.

[0036] Figure 6 Results of flow cytometry analysis of the ability of OVV-HER2-BiTE to infect tumors.

[0037] Figure 7 Results of CCK8 assay for OVV-HER2-BiTE-mediated cytotoxicity.

[0038] Figure 8 The real-time RTCA curve for the T cell-tumor cell co-culture killing experiment. (V) Detailed Implementation Methods

[0039] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0040] Example 1: Preparation and identification of recombinant virus OVV-HER2-BiTE

[0041] (1) Synthesis of bispecific antibody fusion protein

[0042] Reference Figure 2 The HER2-BiTE fragment was synthesized by Nanjing Genscript Biotech Co., Ltd. by sequentially linking the genes of human CD3 antibody light chain variable region (anti-VLOKT3, SEQ ID NO.4), linker peptide (G4S)3 (SEQ ID NO.8), human CD3 antibody heavy chain variable region (anti-VHOKT3, SEQ ID NO.5), linker peptide (G4S)3, trastuzumab light chain variable region (anti-VLER2, SEQ ID NO.6), linker peptide (G4S)3, trastuzumab heavy chain variable region (anti-VHER2, SEQ ID NO.7), and FLAG tag protein (SEQ ID NO.9). The nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1.

[0043] SEQ ID NO.1

[0044] MDWVWTLLFLLSVTAGVHSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINGGGGSG GGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGG GSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSDYKDDDDK

[0045] (2) Construction of virus shuttle plasmid

[0046] The HER2-BiTE fragment was cloned into the EcoRI / XbaI restriction site of the vector pRGB002 (purchased from Nanjing GenScript Biotech Co., Ltd.) by Nanjing GenScript Biotech Co., Ltd., resulting in the viral shuttle plasmid pRGB002-HER2-BiTE. The sequence map after sequencing identification is shown below. Figure 1 As shown.

[0047] (3) Wild-type vaccinia virus reserve beads

[0048] One day in advance, add 2 mL of DMEM high-glucose complete medium (referred to as 10% DMEM medium) containing 10% FBS and 1% penicillin to each well of a 6-well plate, and inoculate 5 × 10⁶ cells / well. 5HEK293T cells were placed in a 37°C, 5% CO2 incubator for static culture. When the cell density reached 70%, the culture medium was replaced with antibiotic-free 10% DMEM medium, and 5 μL of wild-type vaccinia virus (WRVV, catalog number ATCC® VR-1354™) with an MOI of 0.05 was added. The cells were then placed in a 37°C, 5% CO2 incubator (hereinafter referred to as the incubator) for 3 h to allow the wild-type reserve beads to adsorb onto the surface of HEK293T cells.

[0049] (4) Plasmid transfection

[0050] Add 5 μL of LipoHigh liposome transfection reagent (Sangon Biotech Shanghai Co., Ltd., E607403) to 100 μL of DMEM high-glucose medium, mix gently, and incubate at room temperature for 5 min to obtain the transfection reagent solution. Add 2 μg of the target plasmid pRGB002-HER2-BiTE to 100 μL of DMEM high-glucose medium, mix gently, and incubate at room temperature for 5 min to obtain the plasmid mixture. Combine all the above plasmid mixtures with the transfection reagent solution and incubate at room temperature for 20 min to obtain the transfection mixture.

[0051] (5) Recombinant viral fluid

[0052] 200 μL of the transfection mixture from step (4) was evenly added to the culture wells of HEK293T cells that had been adsorbed with wild-type reserve beads and had been left to stand for 3 hours in step (3). After mixing, the cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. The cells were then collected and repeatedly frozen and thawed at -80°C and 37°C three times. After centrifugation at 2000 rpm for 5 minutes, the supernatant was collected, which was the recombinant virus solution.

[0053] (6) Preparation of screening media: Prepare 10 mg / mL mycophenolic acid (400×), xanthine (40×), and hypoxanthine (670×) using 0.1 M NaOH aqueous solution. Prepare screening media by adding 2.5 μL of mycophenolic acid (400×), 25 μL of xanthine (40×), and 15 μL of hypoxanthine (670×) to each mL of 10% DMEM medium.

[0054] (7) One day in advance, add 2 mL of 10% DMEM medium to each well of the 6-well plate, and inoculate 2 × 10⁶ cells per well. 5Vero cells were divided into experimental wells and control wells. On the second day, the medium was changed to 2 mL of antibiotic-free 10% DMEM medium. 0.5 mL of recombinant virus solution was added to the experimental wells and 0.5 mL of PBS was added to the control wells. The cells were placed in a constant temperature incubator and allowed to stand for 3 h. The medium was then changed to the screening medium from step (6) and cultured for about 96 h. The supernatant was collected and used to replace the recombinant virus solution for three rounds of drug screening.

[0055] (8) After serially diluting the supernatant from the last round of step (7) four times with PBS, take 0.2 mL of each dilution and add it to a 6-well plate with 2 mL of 10% DMEM medium in each well. Inoculate each well with 5 × 10⁶ ppm of the supernatant. 5 One Vero cell was prepared, and a control well was prepared (only Vero cells were inoculated). The cells were cultured at 37°C and 5% CO2 for 24 h. Then the culture medium was replaced with 10% DMEM medium containing 2% low-melting-point agarose and cultured continuously for 48 h. Viral plaques were picked under a microscope, which were then identified as monoclonal viruses.

[0056] (9) Add 0.5 mL of 10% DMEM medium to each well of a 12-well plate, then inoculate with monoclonal virus and 1×10 5 Vero cells were incubated at 37°C in a 5% CO2 incubator for 48 h. The culture medium was collected, and PCR was performed using virus identification primers. Under the same conditions, pRGB002-HER2-BiTE template was used as a positive control, and a template-free sample was used as a negative control. PCR products were detected by agarose gel electrophoresis. The electrophoresis image of the recombinant vaccinia virus target gene is shown below. Figure 3 As shown in lane B, 3-6, the target gene is 1575 bp in size. The culture medium of the correctly identified monoclonal virus, namely the recombinant oncolytic virus solution (denoted as OVV-HER2-BiTE), was selected for subsequent amplification and purification.

[0057] Using the same method, PCR was performed using primers for identifying wild-type reserve strains. Wild-type reserve strain DNA templates were used as positive controls, and template-free samples were used as negative controls. PCR products were detected by agarose gel electrophoresis. Results are shown below. Figure 3 A.

[0058] The primer nucleotide sequences for identifying the recombinant virus are: upstream primer-F: 5′-ATGGATTGGGTGTGGACCTT-3′, downstream primer-R: 5′-TTACTTGTCATCGTCGTCCT-3′; the primer nucleotide sequences for identifying the wild-type reserve strain are: TK-F: 5′-TGTGAAGAGAGATAAATTAATGATC-3′, TK-R: 5′-GTTTGCCATACGCTCACAG-3′.

[0059] Example 2: Amplification and purification of recombinant virus OVV-HER2-BiTE

[0060] (1) Add 8 mL of 10% DMEM medium to a 10 cm culture dish and inoculate with 2 × 10⁶ cells / mL. 5 One Vero cell was placed in a 37°C, 5% CO2 incubator and incubated statically until the density reached 80%.

[0061] (2) Take 200 μL of the recombinant oncolytic virus solution correctly identified in Example 1 and add it to a 10 cm culture dish with a Vero cell density of 80% in step (1). After static culture in a constant temperature incubator of 37℃ and 5% CO2 for 48 h, collect the cells and repeatedly freeze and thaw at -80℃ and 37℃ 3 times to obtain the virus seed solution.

[0062] (3) Take 200 μL of virus seed solution and add it to 30 15 cm culture dishes with a Vero cell density of 80%. Incubate at 37℃ and 5% CO2 for 72 h, then centrifuge at 300×g for 3 min to collect the cells. Finally, resuspend the cells in 500 μL of PBS in each dish.

[0063] (4) Repeat the freeze-thaw cycle at -80℃ and 37℃ three times with the resuspension in step (3), break the cells with an ultrasonic disruptor (rated power 750 W, 1 second interval, 2 min), centrifuge at 1800×g for 5 min, collect the supernatant, add a small amount of PBS to the cell pellet and sonicate again, and combine the two supernatants.

[0064] (5) The virus was purified by ultracentrifugation. 20 mL of 36% sucrose solution was added to the bottom of a 32 mL ultracentrifuge tube, followed by the slow addition of the supernatant collected in step (4), and finally the tube was filled with PBS. The tube was centrifuged at 78000×g for 1 h at 4℃. The precipitate at the bottom was resuspended in 2 mL of PBS to obtain the purified recombinant virus solution, denoted as OVV-HER2-BiTE (structure shown in [link to structure]). Figure 2 The viral titer (OVV-HER2-BiTE) was 1.66 × 10^9 PFU / mL.

[0065] The OVV virus solution was prepared using the same method, and the virus titer was 9.33 × 10^8 PFU / mL.

[0066] Example 3: Western Blot detection of expression and secretion of recombinant virus OVV-HER2-BiTE

[0067] (1) Add 2 mL of 10% DMEM medium to each well of a 6-well plate, and then inoculate each well with 2 × 10⁶ cells / well. 5Mdamb231 cells were incubated statically at 37°C with 5% CO2 for 12 h. The culture medium was discarded, and 2 mL of DMEM high-glucose medium was added. 5 μL of OVV or OVV-HER2-BiTE virus solution (MOI=10, Example 2) was added to each well. The cells were incubated statically at 37°C with 5% CO2 for 24 h to infect the cells. A PBS control group was also included. Cells were centrifuged at 800 rpm for 3 min at 4°C, and the cells and supernatant were collected separately.

[0068] (2) Step (1) The cells collected from each well were lysed on ice for 30 min with 70 μL of RIPA lysis buffer containing 10% 1× protease inhibitor, centrifuged at 12000 rpm for 15 min at 4℃, and the precipitate was discarded to obtain cell lysate.

[0069] (3) Step (1) Centrifuge the supernatant collected from each well at 2000 rpm for 5 min to remove cell debris, add 1× protease inhibitor, and obtain cell supernatant.

[0070] (4) The protein concentration of the cell lysate from step (2) was determined using the BCA quantitative method. The protein concentration of the cell lysate was quantified as 1 μg / μL, and the cell supernatant was not quantified. All samples were heated in a metal bath at 95℃ for 10 min to obtain the protein samples to be tested.

[0071] (5) The protein sample was subjected to SDS-PAGE with a 12% separating gel. For loading, 10 μL of cell lysis buffer and 30 μL of cell supernatant were taken from each lane. The stacking gel voltage was 80 V, and the separating gel voltage was 130 V. After removing excess gel residue, the membrane was transferred under ice-water bath conditions. A 0.22 μm PVDF membrane was selected, and the flow rate was constant at 260 mA for 70 min. The PVDF membrane was blocked at room temperature for 1 h with 5% skim milk dissolved in 1×TBST, followed by overnight incubation with rabbit anti-FLAG-TAG antibody at 4℃. After washing the membrane three times with 1×TBST, the PVDF membrane was incubated at room temperature for 1 h with goat anti-rabbit IgG secondary antibody, followed by three washes with 1×TBST. Developing solution was evenly added to the membrane, and fluorescence development was performed using a chemiluminescence analyzer. The image was formed and the results were recorded. The molecular weight of the target protein was approximately 60 KD. Figure 4 As shown, OVV-HER2-BiTE has the ability to express and secrete HER2-BiTE.

[0072] Example 4: TCID50 assay for the replication of recombinant virus OVV-HER2-BiTE in tumor cells

[0073] (1) Add 0.5 mL of 10% DMEM medium to each well of a 24-well plate, and seed three types of tumor cells, Skbr3, Skov3 and Mdamb231, into three plates respectively, with 100,000 cells per well. Each type of cell is seeded into 18 wells and placed in a 37°C, 5% CO2 constant temperature incubator for static culture. After the cells adhere to the plate, add 5 μL of OVV or OVV-HER2-BiTE virus solution with MOI=0.5 prepared in Example 2 to each well.

[0074] (2) The cells were continuously cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 h, 48 h and 72 h. Three replicates of each cell type were collected at each time point. The virus solution to be tested was obtained after repeated freeze-thaw cycles at -80℃ and 37℃.

[0075] (3) Add 100 μL of 10% DMEM medium to each well of a 96-well plate, and then inoculate with 1×10⁶ cells / well. 4 One Vero cell was cultured in a 37°C, 5% CO2 incubator until the cells adhered to the culture vessel.

[0076] (4) Take 100 μL of the virus solution to be tested from step (2) and dilute it serially with PBS to prepare 10 μL solutions. 1 -10 5 In step (3), 100 μL of diluent was added to each well of the plate, with 8 replicates for each dilution. 100 μL of PBS was added to the remaining two columns of wells as a control. After incubation at 37°C and 5% CO2 for 96 h, the percentage of wells with viral plaques at each dilution was recorded under a fluorescence microscope and denoted as S. T(TCID50) = 10 1 + d（S-0.5） Where d=1, the final virus titer is 0.7. T PFU / mL is expressed as T PFU / mL.

[0077] (5) Results are as follows Figure 5 As shown, compared with the control virus OVV, the replication ability of OVV-HER2-BiTE was not affected by the insertion of foreign genes.

[0078] Example 5: Infectivity of recombinant virus OVV-HER2-BiTE in tumor cells

[0079] (1) Skbr3, Skov3 and Mdamb231 tumor cells were seeded into 48-well plates, with 30,000 cells per well. The culture medium was 10% DMEM, 0.5 mL per well. The plates were placed in a 37°C, 5% CO2 incubator and allowed to stand for cell adhesion.

[0080] (2) Step (1) Add 10 μL of OVV or OVV-HER2-BiTE with MOI=0.1 prepared in Example 2 to each well of tumor cells. After static culture in a 37℃, 5% CO2 constant temperature incubator for 24 h, 48 h and 72 h respectively, collect the cells, wash once with PBS, and then analyze the proportion of GFP positive cells by flow cytometry. Each group has three replicates.

[0081] (3) The results are as follows Figure 6 As shown, the infectivity of the recombinant virus was not significantly different from that of the control virus.

[0082] Example 6: CCK8 assay for the cytotoxic effect of recombinant virus OVV-HER2-BiTE

[0083] (1) Skbr3, Skov3, and Mdamb231 were seeded in 96-well plates at a rate of 8000 cells per well. The medium was 10% DMEM, 200 μL per well. The plates were incubated at 37°C in a 5% CO2 incubator until adherence to the plate.

[0084] (2) Step (1) Add 10 μL of OVV or OVV-HER2-BiTE with different MOIs (0.1, 1, 10, 100) prepared in Example 2 to each well. After infecting cells for 72 h, discard the original culture medium and add 190 μL of 10% DMEM and 10 μL of CCK8 solution to each well. Under the same conditions, use no virus as the control and wells with 10% DMEM and CCK8 solution but no cells as blank control. After incubation at 37℃ and 5% CO2 for 1 h, measure the absorbance value at 450 nm with a microplate reader and record it as A value. Cell viability calculation: Cell viability (%) = [A (with virus) - A (blank)] / [A (without virus) - A (blank)] × 100.

[0085] (3) The results are as follows Figure 7 As shown, OVV and OVV-HER2-BiTE have comparable cytotoxic effects.

[0086] Example 7: T cell and tumor cell co-culture killing experiment

[0087] (1) Add 50 μL of 10% DMEM medium to each well of an E-Plate 16-well plate and place it in a real-time label-free analysis system (xCELLigence RTCA Dplus) for 1 min baseline detection. Then remove the plate and add 100 μL of 10% DMEM medium containing 10,000 tumor cells (Skbr3, Skov3, Mdamb231) to each well. Let it stand at room temperature for 30 min and continue to place it in the real-time label-free analysis system to continuously monitor the cell proliferation curve. After about 12 h, the cells are in the logarithmic growth phase.

[0088] (2) Preparation of OVV-HER2-BiTE supernatant: In a 6-well plate, add 2 mL of OVV-HER2-BiTE supernatant to each well. 5 Mdamb231 cells were cultured in 10% DMEM medium in a constant temperature incubator until the cells adhered. Each well was infected with 5 μL of OVV or OVV-HER2-BiTE purified from Example 2 (MOI=10) for 24 h. Cell pellet was removed by centrifugation at 400×g for 5 min, and the supernatant was filtered through a 0.22 μm PVDF membrane to obtain the supernatant of OVV and OVV-HER2-BiTE.

[0089] (3) Add 50,000 T lymphocytes to each E-Plate16 well in the logarithmic growth phase of step (1), add 10 μL of OVV supernatant and OVV-HER2-BiTE supernatant to each well, and add an equal amount of PBS as a control well.

[0090] (4) The cell proliferation curve was continuously monitored for 50 h, and the results were as follows: Figure 8 Compared with the HER2-negative Mdamb231 cell line, the HER2-BiTE secreted by the recombinant virus had a stronger killing effect on the HER2-positive tumor cell lines Skbr3 and Skov3.

[0091] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended 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 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 protection scope of the present invention.

Claims

1. A bispecific antibody fusion protein targeting HER2, characterized in that, The fusion protein is formed by the ligation of gene sequences of HER2 antibody or its variant on the surface of tumor cells and CD3 antibody or its variant on the surface of T cells.

2. The bispecific antibody fusion protein as described in claim 1, characterized in that, The nucleotide sequence of the light chain variable region of the CD3 antibody is shown in SEQ ID NO.4, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

5.

3. The bispecific antibody fusion protein as described in claim 1, characterized in that, The nucleotide sequence of the light chain variable region of the HER2 antibody is shown in SEQ ID NO.6, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

4. The bispecific antibody fusion protein as described in claim 1, characterized in that, The fusion protein structure is as follows: secretion signal peptide - CD3 antibody light chain variable region - linker 1 - CD3 antibody heavy chain variable region - linker 2 - HER2 antibody light chain variable region - linker 3 - HER2 antibody heavy chain variable region - FLAG tag protein; linker 1, linker 2 and linker 3 are each independently GGGGS.

5. The bispecific antibody fusion protein as described in claim 4, characterized in that, The nucleotide sequence of the secretion signal peptide is shown in SEQ ID NO.3; the nucleotide sequences of linker 1, linker 2 and linker 3 are all shown in SEQ ID NO.8; the encoding nucleotide sequence of the FLAG tag protein is shown in SEQ ID NO:

9.

6. The bispecific antibody fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

7. A recombinant oncolytic virus carrying the encoding gene of the fusion protein of claim 1.

8. The recombinant oncolytic virus as described in claim 7, characterized in that, The recombinant oncolytic virus is formed by inserting the fusion protein gene into the thymkinase TK gene of the oncolytic virus genome, before the Pse / l promoter.

9. The use of the recombinant oncolytic virus of claim 7 in the preparation of a tumor-treating drug.

10. A drug for treating tumors, characterized in that, The drug contains the recombinant oncolytic virus.

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