Recombinant oncolytic adenovirus and application thereof

Abstract

The invention provides a recombinant oncolytic adenovirus and application thereof. A recombinant oncolytic adenovirus, the genome of which comprises a nucleotide sequence encoding a bispecific T cell adaptor (BiTE) targeting B7H3. The B7H3BiTE encoded by the gene has the binding capacity to B7H3 on the surface of a tumor cell and CD3 on the surface of a T cell. The oncolytic viruses of the present invention mediate in vitro an effective cytotoxic effect against a B7-H3 positive cancer cell line. The oncolytic virus also shows effective anti-tumor activity in adoptive transfer xenograft mouse models. Results show that the oncolytic virus disclosed by the invention has the potential of being used for treating B7-H3 positive solid tumors.

Description

Technical Field

[0001] The present invention relates to the fields of medical biotechnology and gene therapy, and in particular to a drug for treating malignant tumor-related diseases and its application. Background Art

[0002] Traditional tumor treatments include surgical treatment, radiotherapy and chemotherapy, which are the main means of treating malignant tumors. Surgical treatment has a certain effect on the long-term control of benign or non-metastatic tumors, but the long-term effect on many advanced metastatic tumors is not ideal. Although the tumor can be removed, it may not be completely removed or cause tumor metastasis during the operation, which is very harmful to the patient's body and may cause organ damage and reduced immunity. Radiotherapy can achieve the effect of surgical resection in some tumors, but radiotherapy is costly, has a long cycle, and has many complications. Chemotherapy, as a systemic treatment, has obvious and rapid effects, but it may kill normal cells while killing tumor cells, resulting in systemic side effects. Current conventional cancer treatments have little effect on reducing the annual mortality rate of cancer. In particular, conventional treatments for advanced patients have almost no effect in prolonging survival or improving the quality of life of patients. Not only is the cure rate of most malignant metastatic cancers very low, but these conventional treatments often make the condition worse.

[0003] Oncolytic viruses (ONCs) are a new type of cancer immunotherapy drug. Oncolytic viruses are genetically modified viruses that can selectively replicate in and destroy tumor cells without causing harm to normal cells. The bio-amplification of ONCs is its main advantage over other cancer treatments. Oncolytic viruses work in two ways: 1. Oncolytic viruses directly infect tumor cells and endothelial cells, causing their lysis. 2. Recruit immune cells into the tumor microenvironment. The bio-amplification of ONCs in tumor cells is one of the main advantages of ONCs therapy over other cancer treatments. Compared with traditional immunotherapy, ONCs have many advantages, including accurate targeting, efficient killing, and few adverse reactions.

[0004] BiTE is a bispecific antibody composed of two single-chain antibody fragments (scFv), one end of which targets tumor-associated antigens and the other end targets CD3, an invariant component of the T cell receptor complex. Targeting these two targets allows BiTE to bind to T cells and tumor cells at the same time, while mediating T cell killing of recognized tumor cells. The structure of BiTE is entirely derived from the variable region of the parent antibody. Compared with the complete antibody, it is small in size (about 55kDa) and its various regions are very flexible in spatial position. Therefore, it can bring T cells into close contact with tumor cells and bind to its two targets at the same time. BiTE does not have the constant region of the antibody, especially the Fc segment, which not only has pharmacokinetic significance, but also avoids the occurrence of related drug toxicity caused by binding to Fc receptors. However, BiTE has a short biological half-life in the tumor microenvironment, a short residence time in the tumor, and requires frequent administration, which limits its application in clinical practice.

[0005] B7H3 is a type I transmembrane protein and a member of the B7 family with immunomodulatory functions. B7H3 is absent or lowly expressed in normal human tissues, but highly expressed in human malignant tumors. The expression of B7H3 promotes tumorigenesis, metastasis, and malignant behavior through multiple mechanisms and is associated with poor prognosis. In recent years, B7H3 has been considered a promising target for future immunotherapy. Several therapeutic antibodies targeting B7H3 have been developed, but they still require systemic administration, with large dosages, multiple dosing times, and high risks. A method of effectively delivering antibodies in situ in tumors is still needed. Summary of the invention

[0006] In view of the above-mentioned deficiencies of the prior art, in order to enhance the anti-tumor ability of oncolytic viruses, we invented a B7H3 BiTE and its encoding gene fragment.

[0007] Another object of the present invention is to provide an oncolytic virus encoding B7H3 BiTE. The oncolytic virus can express B7H3 BiTE in tumors. B7H3 BiTE simultaneously targets B7H3 on the surface of tumor cells and CD3 on the surface of T cells, connects tumor cells and T cells, and mediates T cells to kill tumor cells, thereby enhancing the anti-tumor ability of oncolytic viruses.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A bispecific T cell engager B7H3-BiTE targeting B7H3, wherein the B7H3-BiTE has the following structure:

[0010] Secretion signal peptide-VH(CD3)-Linker 1-VL(CD3)-Linker 2-VH(B7H3)-Linker 3-VL(B7H3); the amino acid sequence of the B7H3-BiTE is shown in SEQ ID NO:3.

[0011] As a preferred embodiment of the present invention, the encoding nucleotide sequence of the B7H3-BiTE is shown in SEQ ID NO:2.

[0012] In some embodiments of the present invention, the BiTE simultaneously targets the T cell CD3 antigen and the tumor-associated antigen B7H3.

[0013] The B7H3-BiTE comprises a human B7H3 antibody sequence fragment and two scFv sequences of CD3.

[0014] In some embodiments of the present invention, the two scfv sequences of B7H3 and CD3 are connected with a DNA sequence encoding a G4S linker.

[0015] The gene fragment encoding the bispecific T cell engager B7H3-BiTE, in the B7H3-BiTE structure, the encoding nucleotide sequence of the secretory signal peptide is shown in SEQ ID NO: 1, the encoding nucleotide sequence of VH (CD3) is shown in SEQ ID NO: 4, the encoding nucleotide sequence of VL (CD3) is shown in SEQ ID NO: 5, the encoding nucleotide sequence of VH (B7H3) is shown in SEQ ID NO: 6, and the encoding nucleotide sequence of VL (B7H3) is shown in SEQ ID NO: 7; linker 1, linker 2 or linker 3 is a group of G4S linkers or a plurality of groups of G4S linkers connected in series; the nucleotide sequence of linker 1 is shown in SEQ ID NO: 8, the nucleotide sequence of linker 2 is shown in SEQ ID NO: 9, and the nucleotide sequence of linker 3 is shown in SEQ ID NO: 10.

[0016] As a preferred embodiment of the present invention, the encoding nucleotide sequence of the B7H3-BiTE is shown in SEQ ID NO:2.

[0017] The use of the gene fragment of the present invention in the preparation of a drug for treating tumors is preferably used in the preparation of an oncolytic virus for treating tumors, and further preferably used in the preparation of an oncolytic virus for treating B7H3-positive solid tumors.

[0018] The B7H3-positive solid tumor is preferably synovial sarcoma, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, neuroblastoma, neurofibrosarcoma, prostate cancer, breast cancer, ovarian cancer, bladder cancer, mesothelial tumor, Wilms' cell carcinoma, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and rhabdomyosarcoma.

[0019] A recombinant oncolytic adenovirus expressing the bispecific T cell engager B7H3-BiTE.

[0020] As a preferred embodiment of the present invention, the genome thereof comprises the gene fragment described in any one of claims 3 to 4.

[0021] The present invention uses hTERT promoter to start the expression of E1A, a gene essential for viral replication, so that the virus selectively replicates in tumor cells but not in normal cells. The B7H3 BiTE sequence is placed after the mCMV promoter to start the expression of B7H3 BiTE. A secretion signal peptide is added before B7H3 BiTE to enhance its secretion.

[0022] In a first aspect of the present invention, the present invention provides a recombinant oncolytic adenovirus, which is constructed based on adenovirus type 5, and whose genome contains a nucleotide sequence encoding a BiTE antibody or antibody fragment. In some embodiments of the present invention, the structure of the recombinant oncolytic adenovirus of the present invention is as follows Figure 4 shown.

[0023] In some embodiments of the present invention, the virus used in the present invention is adenovirus type 5, and the virus is packaged using the Ad-Max system.

[0024] The use of the recombinant oncolytic adenovirus of the present invention in the preparation of a drug for treating tumors is preferably used in the preparation of a drug for treating B7H3-positive solid tumors.

[0025] The B7H3-positive solid tumor is preferably synovial sarcoma, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, neuroblastoma, neurofibrosarcoma, prostate cancer, breast cancer, ovarian cancer, bladder cancer, mesothelial tumor, Wilms' cell carcinoma, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and rhabdomyosarcoma.

[0026] In an embodiment of the present invention, the recombinant oncolytic adenovirus RCAd-B7H3-BiTE of the present invention has a good killing effect on B7H3-positive cancer cells, for example, it has a good oncolytic effect on various tumors including U87 and MGC-803, and the killing ability can be significantly improved in the presence of PBMCs.

[0027] A pharmaceutical composition for treating B7H3-positive solid tumors, comprising the recombinant oncolytic adenovirus as an effective ingredient.

[0028] The present invention provides a method for treating solid tumors, comprising administering to a subject a therapeutically effective amount of the recombinant oncolytic adenovirus described in the first aspect above, or a composition or pharmaceutical preparation comprising the recombinant oncolytic adenovirus of the present invention.

[0029] The "subject" refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

[0030] The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals. The optimal dosage should be determined according to the specific treatment. Usually, it is started with a small dose and gradually increased until the most suitable dose is found.

[0031] Compared with the prior art, the advantages of the present invention are: the present invention provides a recombinant oncolytic adenovirus, which can express and secrete a bispecific antibody, especially B7H3 BiTE, which can bridge tumor cells and T cells, induce immune synapse formation, and mediate T cell killing of tumor cells. In particular, the RCAd-B7H3-BiTE obtained after modification can simultaneously target T cell CD3 antigen and tumor cell B7H3 antigen, which not only enhances the targeting of oncolytic adenovirus, but also avoids the adverse reactions caused by systemic administration of BiTE, and increases the killing effect of oncolytic adenovirus on solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the oncolytic virus shuttle plasmid RCAD vector structure

[0033] Figure 2 The result of restriction digestion of RCAD plasmid

[0034] Figure 3 Enzyme digestion results after RCAd-B7H3-BiTE construction

[0035] Figure 4 Schematic diagram of the structure of RCAd-B7H3-BiTE

[0036] Figure 5 The results of adenovirus cesium chloride density gradient centrifugation and virus purity detection in the embodiment of the present invention are shown in FIG.

[0037] Figure 6 Results for RCAd-B7H3-BiTE expression of B7H3 BiTE

[0038] Figure 7 Detection of B7H3 expression in cell lines

[0039] Figure 8 Cytotoxicity assay for RCAd-B7H3-BiTE

[0040] Fig. 9 Replication of RCAd-B7H3-BiTE in tumor cells

[0041] Fig.10 The killing effect of RCAd-B7H3-BiTE on B7H3-positive tumors

[0042] Fig.11 The inhibition of tumor growth by RCAd-B7H3-BiTE in tumor-bearing mice DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with embodiments:

[0044] Example 1 Construction of viral shuttle plasmid:

[0045] The oncolytic virus shuttle plasmid RCAd vector (Yuan Meng, Haotian Liu, Haoran Zhu et al. RCAd-LTH-shPD-L1, a double-gene recombinant oncolytic adenovirus with enhanced antitumor immunity, increases lymphocyte infiltration and reshapes the tumor microenvironment [J]. Journal for ImmunoTherapy of Cancer 2024; 12: e007171. doi: 10.1136 / jitc-2023-007171) was transformed from the plasmid pDC316-ZsGreen1-shRNA (purchased from Miaoling Bio), deleting the mCMV sequence and ZsGreen1 sequence and replacing them with the E1A sequence with the hTERT promoter. The nucleotide sequence of E1A with the hTERT promoter is shown in SEQ ID NO: 11. The structure of the oncolytic virus shuttle plasmid RCAd vector is as shown in Figure 1 In the present invention, the U6 promoter is deleted in the oncolytic virus shuttle plasmid RCAd vector and replaced with a B7H3 BiTE sequence with an mCMV promoter and a secretion signal peptide.

[0046] (1) Gene synthesis The gene sequence of B7H3 BiTE with mCMV promoter and secretion signal peptide is shown in SEQ ID NO: 12.

[0047] (2) Use restriction nucleases MluⅠ and BamHI to digest the RCAd vector overnight in a 37℃ water bath, digest the RCAd plasmid, and remove the U6 promoter. Weigh 0.3g agarose, dissolve it in 30ml TAE solution, add nucleic acid dye at a ratio of 1:10000, mix well, pour into the agarose gel preparation tank, and cool at room temperature for 30min. Add the RCAd vector after digestion to the sample tank and perform electrophoresis at 120V for 30min using a gel analyzer. The fragment size is 4514bp, such as Figure 2 As shown, the fragments shown are consistent with the corresponding enzyme cutting lengths, indicating that the plasmid enzyme cutting identification was successful.

[0048] (3) The vector and the insert fragment were homologously recombined at a molar ratio of 1:2. The vector and the insert fragment were ligated using the homologous recombinase 2×ClonExpress Mix, and the reaction system was mixed and placed in a 50°C water bath for 15 min, and then placed on ice.

[0049] (4) Resuscitate competent cells E. coli DH5α on ice, add homologous recombination products to the ice-water mixture, mix well on ice, and let stand on ice for 30 minutes. Heat shock in a 42°C water bath for 90 seconds, cool on ice for 2 minutes, transfer all bacterial liquid to a 50mL centrifuge tube, then add 1mL LB liquid medium (without antibiotics), and shake at 200rpm for 1 hour on a 37°C shaker. Centrifuge at 4000rpm for 5 minutes, discard 1mL of supernatant, and resuspend in LB liquid medium. Use a sterile spreader to evenly spread on LB solid medium containing 100μg / mL ampicillin, and place in an incubator at 37°C for inversion culture for 12 to 16 hours.

[0050] (5) Pick a single clone and extract the plasmid. After digestion with BamHI and Nhe, the lengths of the two fragments are 5723 bp and 1037 bp, respectively. After digestion with BamHI and PacⅠ, the lengths of the two fragments are 4928 bp and 1832 bp, respectively. Figure 3 As shown, the fragments shown are consistent with the corresponding enzyme cutting lengths, indicating that the plasmid enzyme cutting identification was successful.

[0051] (6) The successfully identified plasmid was sequenced and the sequence was consistent, indicating that the plasmid was successfully constructed.

[0052] Example 2

[0053] Virus packaging:

[0054] The virus was packaged using the Ad-Max system.

[0055] (1) HEK293 cells were co-transfected with the shuttle plasmid and the backbone plasmid pBHGlox (delta) E13Cre (purchased from Miaoling Biotechnology). The density of HEK293 cells was adjusted to 30%-40% in DMEM medium, inoculated into 6-well plates, and cultured in a 37°C, 5% CO2 incubator. Transfection was started when the cell density reached 70%.

[0056] Dissolve 8 μg of transfection reagent PEI in 500 ml opt-MEM and mix well. Add 4 μg of backbone plasmid: shuttle plasmid in a ratio of 1:1 into 500 ml opt-MEM and mix well.

[0057] (2) Add the PEI dilution solution dropwise to the plasmid dilution solution, mix gently, and let stand at room temperature for 20 minutes to allow the DNA and transfection reagent to fully combine to form a stable transfection complex.

[0058] (3) Take out the 6-well plate, slowly and evenly add 1 ml of the mixed reagent to the wells, and place it in the incubator. After 6 hours of culture, replace the culture medium and add 1.5 ml of fresh complete culture medium. When the culture medium turns yellow during the culture process, replace half of the culture medium, aspirate 500 μl of the culture medium, and add 500 μl of the culture medium.

[0059] (4) Virus plaques appear in about 10 days. After the cells are completely diseased, collect all the cells and supernatant into a centrifuge tube.

[0060] (5) Place the centrifuge tube in liquid nitrogen for quick freezing for 10 minutes, then place it in 37°C until completely thawed, vortex for 2 minutes, place it in liquid nitrogen again, and repeat this process three times. Centrifuge at 5000g, 4°C for 30 minutes, take the supernatant as the virus stock solution, and store it at -80°C.

[0061] Example 3

[0062] Virus Amplification:

[0063] (1) Adjust the density of HEK293 cells to 30%-40%, inoculate them in a 6 cm dish, and place them in a 37°C, 5% CO2 incubator for culture. When the cell density reaches 70%, start restaining. When most cells show CPE lesions, collect the cells and supernatant. Repeat freezing and thawing in liquid nitrogen and 37°C water bath for 3 times, freeze-thaw and shake at 70°C / 37°C for 3 times, centrifuge at 4°C, 5000g, 4°C for 30 minutes, and store the supernatant at -80°C.

[0064] (2) Adjust the density of HEK293 cells to 30%-40%, inoculate them in a 10 cm dish, and place them in a 37°C, 5% CO2 incubator for culture. When the cell density reaches 70%, start restaining. When most cells show CPE lesions, collect the cells and supernatant. Repeat freezing and thawing in liquid nitrogen and 37°C water bath for 3 times, freeze-thaw and shake at 70°C / 37°C for 3 times, centrifuge at 4°C, 5000g, 4°C for 30 minutes, and store the supernatant at -80°C.

[0065] (3) Adjust the density of HEK293 cells to 30%-40%, inoculate them into 20 15 cm dishes, and place them in a 37°C, 5% CO2 incubator for culture. When the cell density reaches 70%, start restaining. When most cells show CPE lesions, collect the cells and supernatant. Repeat freezing and thawing in liquid nitrogen and 37°C water bath for 3 times, freeze-thaw and shake at 70°C / 37°C for 3 times, centrifuge at 4°C, 5000g, and store the supernatant at -80°C.

[0066] Example 4

[0067] Virus purification:

[0068] (1) Add 15 mL of virus precipitation solution (20% PEG8000, 2.5 M NaCl) to every 30 mL of supernatant of the virus stock solution, mix by inversion, and incubate at 4°C overnight to precipitate the virus.

[0069] (2) Centrifuge the mixture at 7000 g and 4°C for 20 min, discard the supernatant, and resuspend the precipitate in 10 mL of CsCl with a density of 1.10 g / mL (solvent: 20 mM Tris-HCl, pH 8.0).

[0070] (3) Add 5 mL of 1.40 g / mL CsCl solution (solvent is the same as above) to the ultracentrifuge tube. Then add 7.5 mL of 1.30 g / mL CsCl solution. Finally, add 13.5 mL of the above virus suspension. Centrifuge at 26000 rpm and 4°C for 2 h. Extract the virus when the virus band is between 1.30-1.40 g / mL.

[0071] (4) Perform a second centrifugation on the virus. Add 9 mL of 1.34 g / mL CsCl solution to an ultracentrifuge tube, then add 3.5 mL of the above virus supernatant, and centrifuge at 155,000 g and 18 degrees for 22 hours. Extract the virus and dialyze it.

[0072] Example 5

[0073] Viral dialysis:

[0074] Take a 5ml syringe and inject a small amount of air into the dialysis membrane, absorb the virus, inject it into the dialysis membrane, and extract the air in the membrane so that the virus is completely in contact with the dialysis membrane. Draw out the syringe, place the dialysis membrane in the dialysis solution, put in the rotor, and place it on a magnetic stirrer at 4℃ overnight. Draw out the dialyzed virus with a syringe and store it at -80℃.

[0075] Example 6

[0076] Virus silver staining:

[0077] Take 10μl of dialyzed virus and add 6× protein loading buffer, mix well and cook at 95℃ for 10min. Add the sample to the loading well of 10% SDS-PAGE gel. Perform electrophoresis at a voltage of 90V-120V. Fixation: Take out the gel after electrophoresis, fix it with fixative for 30min on a shaker, then perform silver staining identification, and discard the fixative. Discard the fixative and wash with 30% ethanol for 10min. Then wash with 200mL ddH2O for 10min. Put the washed SDS-PAGE gel in sensitizing solution for 2min, and wash again for 1min, for a total of two washes. Put the washed SDS-PAGE gel in silver staining solution for silver staining for 10min, then wash briefly for color development. The silver staining results are as follows: Figure 5 As shown on the right, RCAd-B7H3-BiTE corresponds to RCAd in each viral capsid band.

[0078] Example 7

[0079] Western blotting to detect B7H3 BiTE expression:

[0080] HEK293 cells were seeded into 12-well plates at 3 × 10 cells per well. 5 cells, infected with the virus RCAd or RCAd-B7H3-BiTE at an MOI of 5. After 48 hours of infection, the cell culture supernatant was collected, and 10 μl was taken for SDS agarose gel electrophoresis. The PVDF membrane was blocked with 5% skim milk at room temperature for 45 minutes, and then incubated with anti-CD3 scFv antibody at 4°C overnight. The membrane was then washed 3 times with TBST. Then incubated with anti-rabbit secondary antibody (1:5000 dilution) at room temperature for 1 hour. Wash the membrane 3 times with TBST, spread the membrane flat in a chemiluminometer, evenly add the developer on the membrane, perform fluorescent imaging and record the results. The molecular weight of the target protein is approximately 57 kDa. Figure 6 As shown, RCAd-B7H3-BiTE has the ability to express and secrete B7H3 BiTE.

[0081] Example 8

[0082] B7H3 antigen expression detection:

[0083] To detect antigen expression, MGC-803, U87, and Raji were stained with PE B7H3 or isotype control antibodies at 4°C for 30 min and then subjected to flow cytometry. Figure 7 As shown, MGC-803 and U87 expressed B7H3, while Raji did not express B7H3.

[0084] Example 9

[0085] Virus cytotoxicity assay:

[0086] MGC-803 or U87 cells were seeded into 96-well plates at 2 × 10 cells per well. 4 The tumor cells were infected with RCAd or RCAd-B7H3-BiTE at different concentration gradients. Four days after infection, the cell activity was detected using CCK-8 reagent. Figure 8 As shown, the cytotoxic effects of RCAd and RCAd-B7H3-BiTE were comparable.

[0087] Example 10

[0088] Viral amplification test:

[0089] MGC-803 or U87 cells were seeded into 96-well plates at 2 × 10 cells per well. 4 Cells were infected with the virus at an MOI of 0.5. Tumor cells were collected at different time points and cell DNA was extracted using a blood / cell / tissue genomic DNA extraction kit. 20 ng of total DNA was taken to detect the content of the viral E1A gene using qPCR, and its expression relative to that of uninfected cells was calculated. Fig. 9 As shown, viral amplification of RCAd and RCAd-B7H3-BiTE was comparable.

[0090] Embodiment 11

[0091] PBMCs and tumor cells co-culture killing experiment:

[0092] HEK293 cells were infected with RCAd or RCAd-B7H3-BiTE at an MOI of 0.5, and the culture supernatant was collected 48 hours later. Target cells were prestained with CFSE and seeded into 96-well plates at 2×10 4 After 24 hours, 2×10 5PBMCs were co-cultured with tumor cells. RCAd supernatant was then added to the wells as the RCAd group; or RCAd-B7H3-BiTE supernatant was added as the RCAd-B7H3-BiTE group; no supernatant was added as the Mock group, and the supernatant was added to the co-culture system at a dilution ratio of 1:3000. After 48 hours of co-culture, cells were collected for live-death staining and detected by flow cytometry. The proportion of target cell death was calculated as: CFSE + Zombie-Dye + / CFSE + .like Fig.10 As shown, RCAd-B7H3-BiTE had the strongest tumor killing effect.

[0093] Example 12

[0094] Mouse experiments

[0095] Six-week-old female M-NSG mice were purchased from Shanghai Model Organisms Technology Co., Ltd. and housed in an SPF environment. The mice were intraperitoneally injected with 1×10 7 Freshly extracted PBMCs were injected subcutaneously into mice 3 days later at a concentration of 2 × 10 6 MGC-803 tumor cells. When the tumor volume reaches 50mm 3 The mice were randomly divided into three groups, with 5 mice in each group. PBS, RCAd or RCAd-B7H3-BiTE were injected into the tumors of the three groups of mice, respectively. The virus injection dose was 1×10 8 pfu, treated three times, with an interval of 3 days between each treatment. The tumor size of the mice was measured every two days. Fig.11 As shown, RCAd-B7H3-BiTE had the best tumor growth inhibition effect.

[0096] Finally, it should be noted that the above 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 aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bispecific T cell engager B7H3-BiTE targeting B7H3, characterized in that: The B7H3-BiTE has the following structure: Secretion signal peptide-VH(CD3)-Linker 1-VL(CD3)-Linker 2-VH(B7H3)-Linker 3-VL(B7H3); the amino acid sequence of the B7H3-BiTE is shown in SEQ ID NO:

3.

2. The bispecific T cell engager B7H3-BiTE according to claim 1, characterized in that The coding nucleotide sequence of the B7H3-BiTE is shown in SEQ ID NO:

2.

3. The gene fragment encoding the bispecific T cell engager B7H3-BiTE according to claim 1, characterized in that: In the B7H3-BiTE structure, the encoding nucleotide sequence of the secretory signal peptide is shown in SEQ ID NO: 1, the encoding nucleotide sequence of VH (CD3) is shown in SEQ ID NO: 4, the encoding nucleotide sequence of VL (CD3) is shown in SEQ ID NO: 5, the encoding nucleotide sequence of VH (B7H3) is shown in SEQ ID NO: 6, and the encoding nucleotide sequence of VL (B7H3) is shown in SEQ ID NO: 7; linker 1, linker 2 or linker 3 is a group of G4S linkers or a plurality of groups of G4S linkers connected in series; the nucleotide sequence of linker 1 is shown in SEQ ID NO: 8, the nucleotide sequence of linker 2 is shown in SEQ ID NO: 9, and the nucleotide sequence of linker 3 is shown in SEQ ID NO:

10.

4. The gene fragment according to claim 3, characterized in that The coding nucleotide sequence of the B7H3-BiTE is shown in SEQ ID NO:

2.

5. Use of the gene fragment according to any one of claims 3 to 4 in the preparation of a drug for treating tumors, preferably in the preparation of an oncolytic virus for treating tumors, and further preferably in the preparation of an oncolytic virus for treating B7H3-positive solid tumors; the B7H3-positive solid tumors are preferably synovial sarcoma, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, neuroblastoma, neurofibrosarcoma, prostate cancer, breast cancer, ovarian cancer, bladder cancer, mesothelial tumors, Wilms' carcinoma, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and rhabdomyosarcoma.

6. A recombinant oncolytic adenovirus, characterized in that: Expressing the bispecific T cell engager B7H3-BiTE described in claim 1.

7. The recombinant oncolytic adenovirus according to claim 6, characterized in that Its genome contains the gene fragment according to any one of claims 3 to 4.

8. Use of the recombinant oncolytic adenovirus according to claim 6 or 7 in the preparation of a drug for treating tumors.

9. The use according to claim 8, characterized in that The use of the recombinant oncolytic adenovirus in the preparation of a drug for treating B7H3-positive solid tumors; the B7H3-positive solid tumors are preferably synovial sarcoma, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, neuroblastoma, neurofibrosarcoma, prostate cancer, breast cancer, ovarian cancer, bladder cancer, mesothelial tumors, Wilms' carcinoma, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and rhabdomyosarcoma.

10. A pharmaceutical composition for treating B7H3-positive solid tumors, characterized in that: The recombinant oncolytic adenovirus according to claim 6 or 7 is used as an effective ingredient.

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