Recombinant adenovirus and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a recombinant adenovirus and application thereof. The technical problem to be solved by the invention is to provide the recombinant adenovirus with good tumor inhibition effect and high efficiency. The technical scheme of the invention is a recombinant adenovirus. The recombinant adenovirus is a replication-defective adenovirus, and an exogenous nucleic acid fragment is operably inserted into a viral genome; the exogenous nucleic acid fragment is a coding gene of human p53 protein, a coding gene of GM-CSF and a coding gene of RGD. The coding gene of the human p53 protein, the coding gene of the GM-CSF and the coding gene of the RGD are jointly constructed into the replication-deficient adenovirus, so that the adenovirus with multiple advantages is obtained, the tumor cell killing efficiency is improved by 2-4 times compared with that before recombination, and a new effective choice is provided for tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a recombinant adenovirus and an application thereof. Background Art

[0002] In recent years, with the continuous development of molecular biology, research into the causes, occurrence, and progression of diseases has deepened to the molecular level. Gene therapy offers advantages such as high selectivity, minimal side effects, and effectiveness against advanced tumors and metastases, making it a promising treatment option following surgery, radiotherapy, chemotherapy, and biological therapies.

[0003] The human tumor suppressor gene TP53 encodes the p53 protein, which has a wide range of biological functions. Normally expressed wild-type p53 protein in cells can regulate the cell cycle, induce apoptosis, regulate DNA replication and transcription, and even participate in the regulation of the body's immune system. However, the p53 gene is one of the most frequently mutated genes in human cancers, with p53 reportedly inactivated in approximately half of all cancers. This makes wild-type p53 (WT-p53) a promising candidate for gene therapy. Several reports have demonstrated that introducing wild-type p53 into tumor cells can restore its tumor suppressor function, achieving the goal of tumor treatment.

[0004] As a new treatment approach, tumor immunotherapy is being increasingly researched and applied. Tumor immunotherapy primarily activates the body's immune system and stimulates tumor-specific immune responses, thereby controlling or killing tumor cells. GM-CSF is a cytokine that has attracted much attention in tumor immunotherapy. It is the most promising inducer of long-term activation of the body's specific anti-tumor immunity, promoting the maturation of dendritic cells and enhancing the function of antigen-presenting cells and natural killer cells. Clinically, GM-CSF-based tumor immunotherapy includes: GM-CSF monotherapy, GM-CSF-secreting cancer cell vaccines, GM-CSF-fused tumor-associated antigen protein vaccines, GM-CSF-based DNA vaccines, and GM-CSF combination therapy.

[0005] Adenovirus vectors are one of the most promising viral vectors for gene therapy. Their advantages include: ① They target a wide range of cell types, are highly efficient infecting both dividing and non-dividing cells, and offer high transfer efficiency. ② They are easy to prepare and purify, have high titers, and possess a large transfection capacity. ③ The exogenous gene they carry does not integrate into the host chromosome, thus avoiding the risk of insertional mutagenesis. ④ They can carry large exogenous fragments (up to 36 kb). ⑤ Furthermore, adenoviruses are highly efficient in both ex vivo and in vivo gene transfer, and their operation is simple. Consequently, they have been widely used for in vivo gene transfer in clinical gene therapy.

[0006] Adenoviruses primarily enter cells by binding to the coxsackievirus receptor (CAR) on the cell membrane via the knob domain of the fiber protein on their outer coat. However, most tumor cells express little or no CAR receptor, which greatly limits the application of recombinant adenoviruses. The current tumor suppression effects of adenoviruses are not ideal, and there is considerable room for improvement. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant adenovirus with good tumor inhibition effect and high efficiency.

[0008] The technical solution of the present invention is a recombinant adenovirus.

[0009] The recombinant adenovirus is a replication-deficient adenovirus, and can be operable to insert exogenous nucleic acid fragments into the viral genome; the exogenous nucleic acid fragments are the coding genes of human p53 protein, GM-CSF and RGD.

[0010] Specifically, the amino acid sequence of the human p53 protein is shown in SEQ ID No. 2.

[0011] The amino acid sequence of GM-CSF is shown in SEQ ID No. 4.

[0012] Furthermore, the amino acid sequence of the RGD is shown in SEQ ID No.6.

[0013] Specifically, the nucleotide sequence of the gene encoding the human p53 protein is shown in SEQ ID No. 1.

[0014] Wherein, the nucleotide sequence of the GM-CSF encoding gene is shown as SEQ ID No.3.

[0015] Furthermore, the nucleotide sequence of the RGD encoding gene is shown in SEQ ID No.5.

[0016] Particularly, the gene encoding the human p53 protein is inserted into the ΔE1 region of the recombinant adenovirus.

[0017] The GM-CSF coding gene is inserted into the ΔE3 region of the recombinant adenovirus.

[0018] Specifically, the RGD coding gene is inserted into the knob domain of the fiber region of the recombinant adenovirus.

[0019] Preferably, in the above-mentioned recombinant adenovirus, the adenovirus is an adenovirus of serotype belonging to subgenus A, subgenus B, subgenus C, subgenus D, subgenus E, subgenus F or subgenus G.

[0020] Furthermore, in the above-mentioned recombinant adenovirus, the adenovirus serotype includes but is not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48 and 91.

[0021] The present invention also provides a host cell containing the recombinant adenovirus.

[0022] Furthermore, the above-mentioned host cell is a eukaryotic cell.

[0023] The present invention also provides the use of the recombinant adenovirus or the host cell in preparing anti-tumor drugs.

[0024] Specifically, the above-mentioned tumor is lung cancer or intestinal cancer.

[0025] The present invention further provides a drug for treating tumors, which is prepared by adding pharmaceutically acceptable auxiliary components to the above-mentioned recombinant adenovirus or the above-mentioned host cell.

[0026] The present invention also provides a method for preparing the above-mentioned recombinant adenovirus:

[0027] a) The gene encoding human p53 protein was constructed into the shuttle plasmid, and the gene encoding GM-CSF was connected to the backbone plasmid;

[0028] Alternatively, the gene encoding human p53 protein is constructed into a backbone plasmid, and the gene encoding GM-CSF is connected into a shuttle plasmid;

[0029] b) ligating the RGD coding gene into the backbone plasmid;

[0030] c) The shuttle plasmid obtained in step a) and the backbone plasmid obtained in step b) are transferred into packaging cells to package and obtain recombinant adenovirus.

[0031] Furthermore, the shuttle plasmid is pDC516.

[0032] Specifically, the backbone plasmid described in the above method is pBHGfrtdelE13FLP.

[0033] Particularly, the packaging cells are HEK293 or 293A cells.

[0034] Specifically, the gene encoding human p53 protein was introduced into the pDC516 shuttle plasmid by seamless cloning.

[0035] Among them, the GM-CSF coding gene and the RGD coding gene were concatenated into a cDNA and constructed into a backbone plasmid.

[0036] Furthermore, the cDNA was constructed between the PacI and SwaI restriction sites of pBHGfrtdelE13FLP.

[0037] The beneficial effects of the present invention are as follows: RGD is modified in the knob region of the fiber protein of the replication-deficient adenovirus, thereby enhancing the specific infection efficiency of tumor cells and preventing large-scale replication in the body, thereby increasing both effectiveness and ensuring safety. The gene encoding the human p53 protein is inserted into the ΔE1 region of the recombinant adenovirus, and the gene encoding GM-CSF is inserted into the ΔE3 region of the recombinant adenovirus, so that the recombinant replication-deficient adenovirus specifically infects tumor cells and expresses the p53 protein in the tumor cells, causing tumor cell apoptosis and releasing GM-CSF into the tumor microenvironment, promoting the infiltration of Th, Tc, and NK cells at the tumor site, thereby further killing the tumor. The present invention co-constructs the gene encoding the human p53 protein, the gene encoding GM-CSF, and the gene encoding RGD into the replication-deficient adenovirus, resulting in an adenovirus with multiple advantages. The tumor cell killing efficiency is increased by 2 to 4 times compared to the original recombination, providing a new and effective option for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 , schematic diagram of the construction of Ad-RGD-p53-GM CSF recombinant adenovirus.

[0039] Figure 2 , Ad-RGD-p53-GM CSF recombinant adenovirus construction route.

[0040] Figure 3 , in vitro tumor cell (Caco-2) inhibition curve.

[0041] Figure 4 , in vitro tumor cell (NCI-H1299) inhibition curve.

[0042] Figure 5 , in vitro tumor cell (A549) inhibition curve.

[0043] Figure 6 , the changing trend of mouse tumor volume after administration of different samples.

[0044] Figure 7 , schematic diagram of the pDC516-TP53 plasmid map.

[0045] Figure 8 , schematic diagram of the pBHGFF-RGD-GM-CSF plasmid map. DETAILED DESCRIPTION

[0046] In order to provide a new option for tumor treatment, the applicant considered using recombinant adenovirus to express substances that have an inhibitory effect on tumors.

[0047] The human p53 gene is a tumor suppressor gene. Mutations in this gene occur in over 50% of all malignant tumors. The protein encoded by this gene is a transcription factor that controls the initiation of the cell cycle. The p53 protein inhibits tumor cell growth by inhibiting the G1 phase, inducing tumor cell suicide or apoptosis, and inhibiting tumor angiogenesis. Therefore, the present invention first constructed a recombinant adenovirus expressing the human p53 gene product. Both in vitro and in vivo experiments verified that this recombinant adenovirus has an inhibitory effect on tumors.

[0048] The p53 protein controls cell cycle initiation, inducing tumor cell suicide or apoptosis. To achieve even better results, the present invention considered further enhancing the immune system's response. Therefore, the inventors added an element expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) to a recombinant adenovirus expressing the human p53 gene product. GM-CSF is a cytokine produced by activated T cells, macrophages, vascular endothelial cells, and fibroblasts. It promotes the differentiation, maturation, and activation of antigen-presenting cells (APCs) such as dendritic cells (DCs) and upregulates CD86 expression to stimulate an immune response against tumors. In vitro, it has been shown to promote the proliferation of myeloid progenitor cells, enhance the phagocytosis and ADCC of tumor cells by neutrophils, monocytes, macrophages, and eosinophils, and promote the infiltration of Th, T cells, and NK cells into tumor sites, thereby killing the tumor. Subsequent in vitro and in vivo experiments demonstrated that combining these two approaches can enhance tumor suppression.

[0049] Considering that recombinant adenoviruses need to enter cells to express and secrete proteins to target cells, further improving the recognition between target proteins and tumor cells may further enhance the efficiency of tumor suppression. To this end, the inventors further integrated RGD peptides into the expression system, primarily used to modify the knob region of the recombinant adenovirus fiber protein. RGD peptides are short peptides containing arginine, glycine, and aspartic acid, and serve as recognition sites for interactions between integrins and their ligands, mediating interactions between cells and the extracellular matrix, as well as between cells themselves.

[0050] Based on the above research, the present invention ultimately developed a technical solution for a recombinant adenovirus expressing p53 protein, GM-CSF, and RGD peptide. Tumor inhibition experiments using this recombinant adenovirus demonstrated that the resulting recombinant adenovirus, through the integration of three distinct aspects, exhibited the best tumor suppression effect and multiple advantages, particularly a 2- to 4-fold increase in tumor cell killing efficiency compared to the original recombinant adenovirus.

[0051] In the process of constructing recombinant adenovirus, a variety of shuttle plasmids and backbone vectors can be used. In the examples, the AdMax system (pDC516 shuttle plasmid + pBHGfrtdelE13FLP backbone plasmid) was selected to construct recombinant adenovirus.

[0052] Example 1 Construction and preparation of recombinant adenovirus

[0053] 1. Construction of recombinant adenovirus

[0054] The AdMax system (pDC516 shuttle plasmid + pBHGfrtdelE13FLP backbone plasmid) was used to construct the recombinant Ad-RGD-p53-GM-CSF recombinant adenovirus, codenamed WTS102. The specific experimental plan is as follows (see Figure 2 ):

[0055] The human TP53 gene (encoding human p53 protein) was introduced into the pDC516 shuttle plasmid by seamless cloning to construct pDC516-TP53. The schematic diagram of the pDC516-TP53 plasmid is shown in Figure 2. Figure 7 .

[0056] Find the appropriate PacI and SwaI restriction sites on the pBHGfrtdelE13FLP backbone vector, and concatenate the sequence between the PacI and SwaI restriction sites of the backbone vector and the GM-CSF and RGD-4C sequences into a full-length cDNA in vitro (see Figure 2 ), and then the cDNA was constructed between the PacI and SwaI restriction sites of pBHGfrtdelE13FLP to finally construct pBHGFF-RGD-GM-CSF. The plasmid map is shown in FIG. Figure 8 .

[0057] pDC516-TP53 and pBHGFF-RGD-GM-CSF were co-transfected into 293A cells for virus packaging.

[0058] Human wild-type TP53 cDNA (NM_000546.6) was purchased from Beijing Sino-Bio Technology Co., Ltd.; human GM-CSF cDNA (NM_000758.4) was fully synthesized by Suzhou Genewise Biotechnology Co., Ltd.; and RGD peptide was synthesized by Chengdu Kangjianxing Biotechnology Co., Ltd.

[0059] SEQ ID No. 1 human TP53 cDNA (1182 bp):

[0060]

[0061] SEQ ID No. 2 Human TP53 Amino Acid Sequence (393 aa):

[0062] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD。

[0063] SEQ ID No. 3 Human GM-CSF cDNA Nucleotide Sequence (435 bp):

[0064] ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA。

[0065] SEQ ID No. 4 human GM-CSF amino acid sequence (144aa):

[0066] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVE VISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE.

[0067] SEQ ID No. 5: Nucleotide sequence of RGD peptide (27 bp): TGTGACTGCCGCGGAGACTGTTTCTGC. SEQ ID No. 6: Amino acid sequence of RGD peptide (9 aa): CDCRGDCFC.

[0068] 2. Preparation of recombinant adenovirus

[0069] Cell culture: 293A cells were cultured in square flasks, digested with trypsin (Gibco / 25200072), and counted. 1.0×10 8 Cells were plated onto a 10-layer cell factory (Corning / 3320) in a medium of high-glucose DMEM (Gibco / 12100061) + 10% FBS (Gibco / 10099) with a total culture volume of 1000 mL at 37° C. and 5% CO 2 .

[0070] Infection: Expand 293A cells into 10 cell factories as described above. When cells reach 90% confluency, dilute the viral seed in high-glucose DMEM + 2% FBS medium and infect the cells at an MOI of 15. Incubate at 37°C, 5% CO2 for 72 hours.

[0071] Harvest: Collect cells and culture supernatant, centrifuge at 4500 rpm for 20 min, discard the supernatant, and collect the cell pellet.

[0072] Cell lysis: Resuspend the cell pellet in 800 mL of DMEM + 2% FBS medium, freeze and thaw repeatedly at -80°C and 37°C for 3 times, centrifuge the freeze-thawed sample at 4500 rpm for 20 min, and collect the supernatant.

[0073] Filtration and Concentration: The collected supernatant was filtered through a 0.8+0.45μm PES / CA membrane. The collected filtrate was concentrated by ultrafiltration using a 300kD molecular weight cutoff (MWCO) membrane, resulting in an 8- to 10-fold sample concentration. The concentrated sample was further diafiltered using a buffer solution of 20mol / L Tris-HCl, 2mmol / L magnesium chloride, and 2% sucrose, pH 8.0, for a 6- to 8-fold diafiltration coefficient. The transmembrane pressure was maintained at 10-20psi throughout the ultrafiltration process. The diafiltered sample was incubated at 37°C in a water bath and 10-20U / mL of Benzonase nuclease was added for 1 hour at 37°C to degrade free nucleic acids.

[0074] Chromatography: Recombinant adenovirus was isolated and prepared using anion exchange chromatography at room temperature. Cytiva Q Sepharose XL was used for anion chromatography. The column was disinfected with 0.5 mol / L NaOH before use. The column was equilibrated with 20 mol / L Tris-HCl, 2 mmol / L MgCl2, and 2% sucrose, pH 8.0. The nuclease-digested sample was loaded with a retention time of 5 min. After loading, the equilibration solution was flushed for three column volumes. Contaminants were washed with 20 mol / L Tris-HCl, 2 mmol / L MgCl2, 2% sucrose, and 0.45 mol / L NaCl, pH 8.0. The virus sample was then eluted and collected with 20 mol / L Tris-HCl, 2 mmol / L MgCl2, 2% sucrose, and 0.6 mol / L NaCl. The column was regenerated with 1 mol / L NaCl.

[0075] Ultrafiltration / diafiltration: The collected chromatography samples were ultrafiltration and concentrated. The ultrafiltration membrane had a molecular weight cutoff of 300 kD and the target concentration of concentrated viruses was 1 to 1.2 × 10 12 VP / mL, and after concentration, diafiltration was performed to change the buffer system to 20mol / L Tris-HCl, 2mmol / L magnesium chloride, 2% sucrose pH8.0, with a diafiltration coefficient of 6-8 times. After the diafiltration, the virus concentration was adjusted to 0.8-1×10 12 VP / mL. The transmembrane pressure during the entire ultrafiltration process is controlled at 10-20 psi.

[0076] The diafiltration sample was sterilized and filtered to obtain a sterile sample, which was stored in a refrigerator at -80°C for a long term. The recombinant adenovirus sample of the present invention was named WTS102.

[0077] Using a similar method, empty virus (Ad-Null), recombinant adenovirus containing only RGD (Ad-RGD), recombinant adenovirus containing only human p53 and GM-CSF (Ad-p53-GM), and recombinant adenovirus containing only human p53 (Ad-p53) were prepared for use in subsequent experiments.

[0078] Example 2 Identification of the Expression Activity of Exogenous Genes from Recombinant Adenovirus

[0079] 1. P53 expression

[0080] The NCI-H1299 cells were digested with 0.25% trypsin-EDTA solution, collected and resuspended in 5% DMEM culture medium at a cell concentration of 2.5×10 5 cells / mL, seeded in 6-well plates, 5×10 5 Cells / 2mL / well, incubate at 37℃, 5% CO2 overnight. Dilute the sample with DMEM 2% as sample diluent, take 100μL of the sample of the present invention and dilute 10 times to a final virus concentration of 1×10 6 IU / mL. Before virus infection, remove the culture medium and add 1 mL of diluted virus sample (MOI = 1) to each well. Incubate at 37°C, 5% CO2 for 2 hours. Perform three replicate wells for each dilution, and also set up three negative control wells.

[0081] After 2 hours, 1 mL of DMEM2% was added to each well and cultured for a further 72 hours. The infected cells served as the test samples. P53 protein expression was assayed using a Human p53 ELISA Kit (Abcam, Catalog No. ab171571). The protocols for Ad-p53-GM, Ad-p53, Ad-RGD, and Ad-Null were the same as for the samples of the present invention.

[0082] The results showed that NCI-H1299 cells infected with the recombinant adenovirus of the present invention expressed p53 protein at a level of 599.9±37.7 ng / mL. Both Ad-p53-GM and Ad-p53 expressed p53 protein at levels of 554.2±42.3 ng / mL and 676±44.2 ng / mL, respectively. No p53 protein expression was detected in Ad-RGD or Ad-Null.

[0083] 2. GM-CSF expression

[0084] HeLa cells were digested with 0.25% trypsin-EDTA solution, collected and resuspended in 5% DMEM culture medium at a cell concentration of 2.5×10 5 cells / mL, seeded in 6-well plates, 5×10 5 Cells / 2ml / well, incubate at 37℃, 5% CO2 overnight. Dilute the sample with DMEM 2% as sample diluent, take 100 μL of the sample of the present invention and dilute 10 times to a final virus concentration of 1×10 6IU / mL. Before virus infection, remove the culture medium and add 1 mL of diluted virus sample (MOI = 1) to each well. Incubate at 37°C, 5% CO2 for 2 hours. Perform three replicate wells for each dilution, and also set up three negative control wells.

[0085] After 2 hours, 1 mL of DMEM 2% was added to each well and culture was continued for 72 hours. The supernatant of the infected cells was used as the sample to be tested.

[0086] The level of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant was determined using a double antibody sandwich method. Anti-GM-CSF antibody (2 μg / mL, 100 μL / well) was coated on an ELISA plate and incubated at 4°C overnight. The plate was washed five times with PBST (20 μmol / L phosphate buffer, pH 7.2, containing 0.05% Tween 20); 200 μL of blocking solution (0.02% BSA, 20 μmol / L phosphate buffer, pH 7.2, containing 0.05% Tween 20) was added and incubated at 37°C for 1 h; the plate was washed five times with PBST (20 μmol / L phosphate buffer, pH 7.2, containing 0.05% Tween 20); the cell culture supernatant to be tested and GM-CSF standard were added to set up blank wells and negative controls (blank cell culture supernatant), respectively, and incubated at 37°C for 1 h; the plate was washed five times with PBST; 100 μL of HRP-labeled anti-GM-CSF antibody (1:1000) was added and incubated at 37°C for 1 h; the plate was washed five times with PBST; 200 μL of chromogenic substrate (o-phenylenediamine) was added to each well, and after standing at room temperature for 30 min, 50 μL of 1 mol / L The reaction was terminated with H2SO4.

[0087] The operation mode of Ad-p53-GM, Ad-p53, Ad-Null and Ad-RGD is the same as that of the samples of the present invention.

[0088] The results showed that the supernatant of Hela cells infected with the recombinant adenovirus of the present invention expressed GM-CSF at a level of 7.1±0.2 ng / mL. Ad-p53-GM also expressed GM-CSF at a level of 9.2±0.9 ng / mL. No GM-CSF expression was detected with Ad-p53, Ad-RGD, or Ad-Null.

[0089] Example 3 Infectivity test of recombinant adenovirus to tumor cells

[0090] 293A cells were taken and digested with 0.25% trypsin-EDTA solution, the cells were collected and resuspended in DMEM 2% culture medium to prepare 1×10 5 cells / mL, a total of 30 mL.

[0091] Add 100 μL of cell suspension to each well of a 96-well plate, add three 96-well plates in total, and culture in a 37°C, 5% CO2 incubator for 2 hours.

[0092] Dilute the sample with DMEM 2% as sample diluent, make a 10-fold dilution gradient, and dilute to 10 -13 . Make 3 dilutions respectively.

[0093] According to the dilution from high to low (from 10 -13 to 10 -6 ) Add 100 μL of the same dilution of sample to each row of a 96-well plate. Repeat 10 wells for each dilution (i.e., add 100 μL of DMEM 2% to wells 11-12 of each row as a blank control. Prepare three replicate plates. Incubate the plates at 37°C in a 5% CO2 incubator for 10 days.

[0094] The negative control in the 96-well plate has normal cell morphology and no cytopathic effect. The lowest dilution concentration in the 96-well plate should show 100% cytopathic effect, while the highest dilution concentration should show 0% cytopathic effect. The results of this test are valid.

[0095] The cytopathic effect (CPE) of each well of the 96-well plate was observed, and the ratio of CPE effect at each dilution was calculated (100% CPE effect was 1, and so on).

[0096] The test data were used The result is calculated according to the following formula:

[0097] Virus titer of 100 μL sample = 10 1+(s-0.5)

[0098] S is the sum of the ratios of CPE effects at each dilution (from 10 -1 start)

[0099] Then, the virus titer of 1 mL sample = 10 × 10 1+(s-0.5) =10 2+(s-0.5) (IU / mL)

[0100] The operation mode of Ad-p53-GM, Ad-p53 and Ad-RGD is the same as that of the samples of the present invention.

[0101] The results showed that the recombinant adenovirus of the present invention had a cytopathic effect on 293A cells, with a titer of 4.37×10 10 IU / mL. The titers of Ad-p53-GM, Ad-p53, Ad-RGD, and Ad-Null were 4.12×10 10 IU / mL, 5.38×1010 IU / mL, 3.76×10 10 IU / mL, 3.92×10 10 IU / mL.

[0102] Example 4 Determination of the anti-tumor activity of recombinant adenovirus in vitro

[0103] Caco-2 cells (human colon cancer cells), NCI-H1299 cells (human non-small cell lung cancer cell line), and A549 cells (human non-small cell lung cancer cell line) were used at 1×10 4 cells / well in a 96-well plate, and the sample of the present invention (WTS102), Ad-p53-GM (a recombinant adenovirus control containing only human p53 and GM-CSF but no RGD peptide sequence), and Ad-Null (an empty recombinant adenovirus control) were diluted to 1×10 6 After Vp / mL, 8 to 10 gradients of 4-fold dilution were added, and 100 μL of the sample of the same dilution was added in sequence. Three replicates were made for each dilution. After 72 hours of culture, CellTiter Cell viability was determined using the AQueous non-radioactive cell proliferation assay (MTS) kit.

[0104] The results are shown in Table 1. Figures 3-5 As shown, WTS102 and Ad-p53-GM all had significant inhibitory effects on A549 cells, NCI-H1299 cells, and Caco-2 tumor cells, while Ad-Null and the negative control had no inhibitory effect on tumor cells. Compared to Ad-p53-GM (a recombinant adenovirus control containing only human p53 and GM-CSF, without the RGD peptide sequence), the samples of the present invention had a higher killing activity against Caco-2 and A549 tumor cells, which was 2-4 times that of the latter.

[0105] Table 1 In vitro tumor cell inhibition effect

[0106]

[0107]

[0108] Example 5 Tumor Inhibitory Effect of Recombinant Adenovirus Based on Animal Models

[0109] 1. Experimental Animals

[0110] Species and strain: BALB / c; Age: 6-8 weeks; Gender: female; Animal level: SPF.

[0111] 2. Tumor Cell Inoculation and Grouping

[0112] CT26 cells (mouse colon cancer cell line) were digested with 0.25% trypsin-EDTA solution, collected and resuspended in 1640 culture medium, and washed twice with 1640 culture medium. The final cell concentration was 7.5×10 6 cells / mL, inoculated subcutaneously on the right side of the abdomen of the experimental mice, 100 μL / mouse, i.e. 0.75×10 6 / Only.

[0113] Six days after tumor inoculation, the average size of the tumor in mice was 75 mm 3 The mice were randomly divided into 6 groups with 6 animals in each group.

[0114] 3. Dosage regimen

[0115] Administration was performed according to Table 2, where Ad-Null is an empty recombinant adenovirus; WTS102 is a sample of the present invention; Ad-p53-GM is a recombinant adenovirus containing only human p53 and GM-CSF but not the RGD peptide sequence; Ad-p53 is a recombinant adenovirus containing only human p53; and Ad-RGD is a recombinant adenovirus containing only the RGD sequence.

[0116] Table 2 Dosage regimen

[0117] Group sample Number of animals Dosage (PFU / mouse) Route of administration Dosage cycle A Ad-Null 6 <![CDATA[2×10 7 ]]> ip Q3D×5 B WTS102 6 <![CDATA[2×10 7 ]]> ip Q3D×5 C Ad-p53-GM 6 <![CDATA[2×10 7 ]]> ip Q3D×5 D Ad-p53 6 <![CDATA[2×10 7 ]]> ip Q3D×5 E NC (sample buffer) 6 50 μL ip Q3D×5 F Ad-RGD 6 <![CDATA[2×10 7 ]]> ip Q3D×5

[0118] Note: The administration volume is 50 μL / mouse; ip: intratumoral injection, Q3D×5: administration once every 3 days, for a total of 5 administrations.

[0119] 4. Detection indicators

[0120] a. Animal Post-Dosing Reactions: After all animals were grouped, their body weight and tumor size were measured before each dosing, i.e., every three days. Changes in body weight and tumor size were recorded in relation to dosing time. Following the completion of the five dosing cycles, body weight and tumor size were measured and recorded three times every three days. After all measurements were completed, the mice were sacrificed, the tumors were excised from their subcutaneous tissue, and the tumors were weighed and photographed.

[0121] b. Measure tumor size: Use a vernier caliper to measure the length and width of the tumor. The same person should use the same vernier caliper to measure the tumors of all mice during the entire experimental period. Calculate tumor volume: Tumor size = tumor length * tumor width 2 Calculate the mean and SD values of the tumors in each group of mice and draw a linear graph of the relationship between administration time and tumor size.

[0122] 5. Experimental Results

[0123] After 5 doses, the mice were observed for 9 days. The trend of tumor volume changes after administration of different samples was shown in Figure 2. Figure 6 As shown in Table 3, tumors in the negative control (NC), empty virus (Ad-Null), and RGD-only virus (Ad-RGD) groups all grew rapidly. Ad-p53 exhibited a significant tumor-suppressing effect, with a significant difference compared to the negative control (P < 0.05). Ad-p53-GM and the samples of the present invention both exhibited very significant tumor-suppressing effects, with extremely significant differences compared to the negative control (P < 0.01). The samples of the present invention exhibited the most pronounced tumor-suppressing effect, exhibiting a higher tumor-suppressing ability than the recombinant adenovirus control containing only p53 (Ad-p53), with an extremely significant difference compared to Ad-p53 (P < 0.01). The average tumor volume was 24.5% of that of Ad-p53. The samples of the present invention exhibited a significant difference (P < 0.05) compared to the recombinant adenovirus control containing only p53 and GM-CSF, without the RGD peptide sequence (Ad-p53-GM), with an average tumor volume of 53.6% of that of Ad-p53-GM.

[0124] Table 3 Tumor volume of mice after administration of different samples ( n=6)

[0125]

[0126] Note: Compared with the negative control group, **: P < 0.01, *P < 0.05.

Claims

1. A recombinant adenovirus, characterized in that The recombinant adenovirus is a replication-deficient adenovirus, and can be operable to insert exogenous nucleic acid fragments into the viral genome; the exogenous nucleic acid fragments are the coding genes of human p53 protein, GM-CSF and RGD.

2. The recombinant adenovirus according to claim 1, characterized in that The amino acid sequence of the human p53 protein is shown as SEQ ID No. 2; or, the amino acid sequence of GM-CSF is shown as SEQ ID No. 4; or, the amino acid sequence of RGD is shown as SEQ ID No.

6.

3. The recombinant adenovirus according to claim 2, characterized in that The nucleotide sequence of the gene encoding human p53 protein is shown in SEQ ID No. 1; or, the nucleotide sequence of the gene encoding GM-CSF is shown in SEQ ID No. 3; or, the nucleotide sequence of the gene encoding RGD is shown in SEQ ID No.

5.

4. The recombinant adenovirus according to claim 1, characterized in that Meet at least one of the following: 1) The gene encoding the human p53 protein is inserted into the ΔE1 region or ΔE3 region of the recombinant adenovirus; 2) The GM-CSF encoding gene is inserted into the ΔE3 region or ΔE1 region of the recombinant adenovirus; 3) The RGD coding gene is inserted into the knob domain of the fiber region of the recombinant adenovirus; 4) The recombinant adenovirus is an adenovirus of serotype A, B, C, D, E, F or G.

5. A host cell containing the recombinant adenovirus according to any one of claims 1 to 4; further, the host cell is a eukaryotic cell.

6. Use of the recombinant adenovirus according to any one of claims 1 to 4 or the host cell according to claim 5 in the preparation of an anti-tumor drug, wherein the tumor is lung cancer, liver cancer, head and neck cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, prostate cancer, various sarcomas or malignant body cavity effusion.

7. A drug for treating tumors, characterized in that: The invention is prepared by adding pharmaceutically acceptable auxiliary components to the recombinant adenovirus according to any one of claims 1 to 4 or the host cell according to claim 5.

8. A method for preparing the recombinant adenovirus according to any one of claims 1 to 4, characterized in that The steps include: a) The gene encoding human p53 protein was constructed into the shuttle plasmid, and the gene encoding GM-CSF was connected to the backbone plasmid; Alternatively, the gene encoding human p53 protein is constructed into a backbone plasmid, and the gene encoding GM-CSF is connected into a shuttle plasmid; b) ligating the RGD coding gene into the backbone plasmid; c) The shuttle plasmid obtained in step a) and the backbone plasmid obtained in step b) are transferred into packaging cells to package and obtain recombinant adenovirus.

9. The method according to claim 8, characterized in that Meet at least one of the following: 1) The shuttle plasmid is pDC516; 2) The backbone plasmid is pBHGfrtdelE13FLP; 3) The packaging cells are HEK293 or 293A cells.

10. The method according to claim 9, characterized in that Meet at least one of the following: a) introducing the gene encoding human p53 protein into the pDC516 shuttle plasmid by seamless cloning; b) The GM-CSF and RGD coding genes were concatenated into a single cDNA and constructed into a backbone plasmid; c) constructing the cDNA between the PacI and SwaI restriction sites of pBHGfrtdelE13FLP.

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