Patent Publication Number: US-2022235332-A1

Title: Fast and Accurate Three-Plasmid Oncolytic Adenovirus Recombinant Packaging System AD5MIXPLUS and Application Thereof

Description:
TECHNICAL FIELD OF THE INVENTION 
     The present invention refers to the field of biomedical technology, particularly to a fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system Ad5MixPlus and application thereof. 
     BACKGROUND OF THE INVENTION 
     Malignant tumor has become a major disease affecting all human life. In 2000, there were about 10 million new tumor cases and 6.2 million deaths worldwide. By 2018, the number of new cancer cases worldwide had increased to 18.1 million, and the number of deaths had increased to 9.6 million (CA cancer J Clin. 2018 November; 68:394-424). In 2000, there were about 1.8-2 million tumor cases and 1.4-1.5 million tumor deaths in China, but by 2014, the number of new cases was 3.804 million and the number of deaths was 2.296 million, making tumor the leading cause of death (Chin J Cancer Res. 2018 February; 30:1-12). At present, in addition to conventional surgery, radiotherapy, and chemotherapy for malignant tumors, the tumor immunotherapy developed in recent years has made rapid progress, especially immune checkpoint therapy such as PD-1/PD-L1 antibody and immunocyte therapy, such as CAR-T, and has become an indispensable part of comprehensive treatment following radiotherapy, chemotherapy, and surgery. Immunotherapy represented by CAR-T cells and PD-1/PD-L1 antibodies has been used in clinical practice, showing strong curative effect and great market value (Nat Biotechnol. 2018 October; 36:847-856; Cell. 2018 October 4; 175:313-326). In August 2017, Novartis&#39;s CAR-T product Kymriah for the treatment of B-cell acute lymphoblastic leukemia was approved by FDA for marketing, with a price of US$475,000. Two months later, Kite&#39;s CAR-T drug Yescarta for the treatment of large B-cell lymphoma was approved by FDA for marketing, with a price of US$373,000. Recently, scientists have discovered that a variety of oncolytic virus products that can be used as CAR-T cells and PD-1/PD-L1 antibody immunotherapy synergists can not only enhance the effect of radiotherapy and chemotherapy, but also improve the therapeutic effect of antibodies and immune cells. At the same time, tumor cells infected by these oncolytic viruses release a large amount of cytokines, and the lysed tumor cells release a large amount of tumor-associated antigens which have immune activation effects. Therefore, the combined use of oncolytic virus products with CAR-T cells or PD-1/PD-L1 antibodies is conducive to the amplification of immunotherapy effects, and the application scope of oncolytic virus products can be widened. 
     Oncolytic virus (OV) is a type of virus that has been artificially genetically modified, which can specifically replicate in tumor cells in large quantities without affecting normal cells. Tumor cells containing a large amount of virus are lysed and destroyed, and virus particles are released to continue to infect and destroy more tumor cells. The oncolytic virus is used as a vector to carry antitumor genes. As the virus replicates and spreads, the number of copies of the antitumor genes it carries increases, which further increases the expression amount of protein products. Therefore, the anti-cancer effect is enhanced under the synergistic effects by the anti-cancer effect of the antitumor genes and the oncolytic effect of oncolytic virus. Oncolytic virus therapy has broader indications than CAR-T and has a lower cost than monoclonal antibody drugs. Due to the high efficiency of killing tumor cells, good targeting, high safety, low side effects and low cost, oncolytic virus-mediated gene therapy for tumors has become an important new tumor treatment method following the three conventional treatment methods (surgery, radiotherapy and chemotherapy) and immunotherapy, and may become an important auxiliary method to assist comprehensive treatment of tumors to improve curative effect. In September 2017, the top medical journal “Cell” reported a breakthrough study. In this study, the efficacy of oncolytic virus therapy combined with PD-1 monoclonal antibody immunotherapy was studied in a phase Ib clinical trial of 21 melanoma patients by researchers from the U.S., Switzerland, Spain, and Australia. The results suggest that the efficacy of immunotherapy is effectively improved by oncolytic viruses with an overall response rate of up to 62%, and combination therapy is significantly better than single treatment, which proved that oncolytic virus has a very bright future as one of the methods of comprehensive tumor treatment (Cell. 2017 Sep. 7; 170:1109-1119.e10). The milestone of the oncolytic virus is that Amgen&#39;s oncolytic virus Talimogene laherparepvec (T-Vec) was approved for marketing in the United States and the European Union in 2015. T-Vec, expressing granulocyte-macrophage colony stimulating factor (GM-CSF), can be directly injected into tumors, and replicate in tumor cells until cell lysis (oncolysis) occurs. Then, GM-CSF is released to the tumor tissue to activate the systemic immune response. In general, T-Vec works in two important and synergistic ways by inducing tumor cell lysis and stimulating the systemic anti-tumor immune response (Lancet Oncol. 2016 November; 17: 1485-1486). Thomson Reuters estimates that T-Vec sales for melanoma treatment will be $388 million/year by 2020. 
     OV has always been a hot spot in the research and development of antitumor drugs, as well as the capital market and pharmaceutical groups. At the beginning of 2018, MSD and Viralytics announced that MSD acquired Viralytics at a price of $394 million which is equivalent to a 160% premium of the weighted average price of Viralytics stock in one month. MSD acquired all rights about the oncolytic virus immunotherapy product CAVATAK® (CVA21) developed by Viralytics through the acquisition. CAVATAK®, a preparation based on Viralytics&#39; proprietary oncolytic virus Coxsackievirus Type A21, has been proven to infect and kill cancer cells preferentially. CAVATAK® is currently in multiple phase I and phase II clinical trials as an intra-tumor intravenous injection. Taking into account MSD&#39;s ongoing PD-1 drug KEYTRUDA® trial, CAVATAK® may be used in the next step with PD-1 drug KEYTRUDA® for the treatment of melanoma, prostate cancer, lung cancer and bladder cancer. The OV drug T-Vec, developed by BioVex and acquired by biotech giant Amgen at a price of $425 million in 2011, was approved by the FDA for marketing in 2015 and obtained EU CHMP approval in December of the same year, becoming a milestone in OV treatment for tumors. At present, T-Vec has been widely used in the United States, Europe and Australia to treat relapsed melanoma at an average cost of $65,000. Thomson Reuters estimates that T-Vec&#39;s sales will be $388 million per year by 2020. The OV products that have entered phase III clinical include Oncolytics&#39; Reolysin for the treatment of head and neck tumors, ColdGenesys&#39;s CG0070 for the treatment of bladder cancer, Advantagene&#39;s Prost Atak™ for the treatment of prostate cancer, and Jennerex&#39;s PexaVec for the treatment of liver cancer. OV is a broad-spectrum anti-tumor drug. The tumors treated by OV currently entering clinical trials include melanoma, head and neck cancer, prostate cancer, bladder cancer, glioblastoma, liver cancer, breast cancer, non-small cell lung cancer, colon cancer, ovarian cancer, etc., among them, the first six types of tumors will be the most important on the market in recent years. According to Beijing OLXOZ Information Consulting Co., Ltd., the upcoming OV products for head and neck cancer, prostate cancer, bladder cancer, glioma and liver cancer will have annual sales of 1.17, 7.15, 2.925, 2.275, and 4.864 billion U.S. dollars, respectively. The greater value of OV is to be used in combination with radiotherapy or chemotherapy. Oncolytics&#39; Reolysin combined with paclitaxel or carboplatin in the treatment of advanced head and neck cancer has a response rate of 42%, while chemotherapy alone is only 3%-10%. Therefore, the OV market space is even greater if it is combined with other drugs. 
     China has also made a lot of achievements in the field of OV drugs. OV research and development has been supported by the major special fund for new drug creation of the Ministry of Science and Technology for many consecutive years. In 2005, the oncolytic adenovirus “H101” (trade name: Oncorine) developed by Shanghai Sunway Biotechnology Co., Ltd. was first approved by China Food and Drug Administration (CFDA) as a first-class new drug and became the world&#39;s first OV drug to be marketed. At the end of 2016, reaching an agreement with Japan&#39;s Oncolys BioPharma, China&#39;s Hengrui pharmaceutical company obtained an exclusive license for $102 million to develop, produce and commercialize Oncolys&#39; oncolytic adenovirus product Telomelysin™ (OBP-301) in mainland China, Hong Kong and Macao. 
     OV was developed to solve the shortcomings of low expression of anti-cancer genes and the inability of the vector to target tumor cells in gene therapy. Cancer gene therapy mediated by oncolytic viruses can target tumor cells, produce oncolytic effects, and produce synergistic anti-cancer effects with gene therapy, which has obvious application value. In theory, many types of viruses can be transformed into oncolytic viruses. For example, T-Vec is transformed from herpes simplex virus, ProstAtak and G0070 are transformed from adenovirus, Reolysin is transformed from reovirus, and JX-594 is transformed from the vaccinia virus. Among them, adenovirus is the most extensively studied type, and the types of oncolytic viruses transformed from adenoviruses are also the most. There are many types of adenoviruses and their structures are complex. At present, more than 100 serotypes of adenoviruses have been found. Their genomes are linear double-stranded DNA molecules, about 35-36 kb. Compared with other types of viruses, adenovirus has the following advantages: it can infect almost all types of cells and can replicate in large numbers in host cells; the viral genome does not integrate into the host cell chromosome, and there is no risk of insertional mutagenicity; the titer is high so it is easy to prepare and purify, etc. Adenovirus is most suitable for developing safe OV products. However, the use of adenovirus as an oncolytic virus also has some disadvantages: such as strong autoimmunity, poor specificity, and easy accumulation in the liver by intravenous injection, so it needs to be further modified. These complex characteristics of adenovirus provide us the best opportunity for targeted and effective modification, which is more conducive to the development of a multi-mechanism synergistic anti-cancer efficient oncolytic adenovirus (OAV) product through a comprehensive modification, and the design and construction of personalized OAV suitable for different tumor types. The main purpose of the modification of adenovirus is to improve the specificity of targeting, the efficiency of infection, the expression of loaded anti-cancer genes, and to avoid the elimination of the virus by the body&#39;s immune system (Mol Cancer Ther. 2016 July; 15:1436-51). 
     The construction of OAV based on the least virulent human adenovirus C subgenus type 5 (Ad5) mainly includes the modification of the E1 region of the left arm of the genome and the modification of the capsid protein, fiber protein, and E3 region of the right arm of the genome. 
     1. Modification of Left Arm Functional Region of Adenovirus Genome 
     To construct OAV, it is necessary to retain the expression of E1a protein and make it regulated by tumor-specific promoters, so that the virus can specifically replicate in the tumor. E1a has 3 functional areas, CR1, CR2, and CR3. The CR1 region inhibits the expression of Her-2/neu gene by binding to the transcription regulator P300/CBP, the CR2 region binds to the Rb protein family, and the CR3 region is the transcription activation region. Therefore, E1a protein can not only inhibit the transcription of Her-2/neu gene, block the activity of NF-κB, increase the expression of p53, but also inhibit the expression of protease genes such as type IV collagenase and plasminogen activator, thereby exerting resistance tumor effect. E1a can also cause non-specific immune responses, improve the killing effect of CTL cells, NK cells, and macrophages, induce tumor cell apoptosis, inhibit tumor invasion and metastasis, and improve tumor cell sensitivity to chemotherapy and radiotherapy. The introduction of deletion mutations in the CR2 region of E1a can prevent E1a from binding to the Rb protein, ensuring that the dephosphorylated Rb protein forms a complex with the transcription factor E2F to block the transcriptional activity of E2F and ultimately enhances the anti-cancer activity. There are still many questions that need to be studied in depth, such as how to modify E1a to make it have anti-cancer activity, and what are the molecular mechanisms and signaling pathways involved in E1a. In addition, a large number of cytokines released when OAV infects cancer cells and a large number of tumor-associated antigens released when OAV lyses cancer cells can play an immune activation effect. Therefore, the greatest application value of OAV is its combined application with immunotherapy. 
     The Elb transcription unit encodes Elb-55 kDa and Elb-19 kDa. Elb-55 kDa is a protein necessary for adenovirus replication in normal cells but not necessary in tumor cells. The selective deletion of the Elb-55 kDa coding gene can enable adenovirus to maintain the ability of replication in tumor cells, and lost the ability to replicate in normal cells. Because the Elb-55 kDa protein can inactivate and degrade the P53 protein, the Elb-55 kDa deletion helps cells maintain the anti-tumor activity of P53. The Elb-19 kDa gene is homologous to the apoptosis-inhibiting gene Bcl-2. The Elb-19 kDa protein can bind to Bax or/and Bak to initiate the downstream apoptosis-inhibiting program and protect infected cells from TNF-α-mediated killing. The deletion of Elb-19 kDa increases the specificity of the virus mutant&#39;s replication in tumor cells, while the replication activity in normal cells is weakened. The deletion of Elb-19 kDa can promote the recovery of apoptosis pathway in cancer cells, and is conducive to the rapid elimination of viruses in normal cells and the rapid release and dissemination of viruses in tumor cells, so that OAV has better specificity and stronger efficacy. However, whether the double deletion of Elb-55 kDa and Elb-19 kDa affects the replication of OAV, and whether the modification of the two can produce immune enhancement effect needs further study. 
     2. Modification of the Right Arm Functional Region of Adenovirus Genome 
     The E3 transcription unit on the right arm of the adenovirus genome has 9 open reading frames, which encode proteins that protect infected cells from the host&#39;s immune response. E3-gp19k can attenuate the killing effect of CTL-mediated infected cells. RID can block apoptosis mediated by “death” ligands including TNF, Fas ligand and TRAIL. RID can also inhibit the activation of NF-κB necessary for cell survival mediated by IL-1 and TNF. In addition to down-regulating the TRAIL receptor, E3-6.7k can individually inhibit cell apoptosis induced by external and internal signaling pathways. E3-14.7k is a broad inhibitor of TNF-mediated apoptosis. It can also bind to and inhibit Caspases-8 to prevent apoptosis initiated by the Fas signaling pathway. E3-14.7k interacts with FIP protein (for 14.7K-interacting protein, FIP-1, -2,-3) in infected cells, making E3-14.7k protein play an important role in signal transduction pathways such as apoptosis and survival, inflammatory response, maintaining membrane stability, and nuclear and plasma transport. The molecular mechanism of this multifunctional E3 protein still needs to be studied in depth. Adenovirus death protein (ADP) can promote cell lysis and virus release, but the molecular mechanism is unknown. It can be seen that the deletion of the E3 region during the OAV construction process can not only expand the vector capacity, but also promote the apoptosis of infected cancer cells. The immune resistance of the virus to the body is also relieved by the loss of E3, and the virus can be quickly eliminated. Therefore, increasing the understanding of the function of the protein encoded by the E3 region and avoiding deleting all the E3 regions blindly in the construction of OAV may be more conducive to the long-term expression of the target gene. 
     The human adenovirus family has 52 serotypes, divided into 6 subgenres (A to F). Except for group B, all subgenres of adenoviruses use coxsackievirus-adenovirus receptor (CAR) as their main recognition receptor, and the infection efficiency for CAR-deficient cells such as hematopoietic cells, hematopoietic stem cells, dendritic cells, some tumor cells, especially tumor stem cells is very low. Adenoviruses subgenres B (Ad3, Ad11b, Ad14, Ad16, Ad21, Ad35, Ad50) mainly recognize CD46, a widely expressed complement regulatory protein. Using fiber knob of adenoviruses subgenres B instead of fiber knob of Ad5 to construct a chimeric virus will help improve the infection efficiency of the virus to tumor cells, especially the ability to infect tumor stem cells, and may more completely prevent tumor recurrence. 
     Ad5 exists widely in nature, and most people have been infected and have produced neutralizing antibodies that can block the virus. Ad5 is hepatotropic and can be adsorbed by hepatocytes. The hypervariable region (HVR) of the adenovirus capsid protein Hexon is exposed to the surface of the adenovirus, which is the key part that causes the difference in liver infection ability and immunogenicity between different serotypes of adenovirus. An effective way to help Ad5 avoid pre-existing immunity and liver uptake is to selectively chimerize the 7 HVRs in the Hexon molecule of Ad5 with the corresponding regions of Hexon of rare serotypes such as subgenres D (Ad37, Ad43) and subgenres B (Ad48). However, modification of Hexon, the main structural protein of adenovirus, often results in structural instability of the adenovirus vector, which makes it impossible to effectively package the virus. Therefore, this is a very challenging study. 
     3. Technology for Recombination and Packaging of Adenovirus 
     At present, the most commonly used system for packaging adenoviruses is from Microbix Biosystems. The adenovirus left arm plasmid pDC series (such as pDC315, pDC316, pDC312, etc.) and the left arm backbone plasmid (pBHGloxdelEl3cre) with complete deletion of E1/E3 region are recombined. This technology has been used on the market for nearly 20 years, and is still in use now. The produced virus lacks all the E3 area. Both E1a gene for specific replication in tumor and exogenous antitumor genes must be carried by pDC plasmids before they are further recombined into the adenovirus E1 region. As a result, the size of the inserted fragment in the left arm is limited, and since the E1a promoter and the anti-cancer gene promoter are both in the E1 region, they are too close to each other and interfere with each other, the expression efficiency of them are all reduced. We tried to separate them with insulator sequences in the early stage. Although there are improvements, the effect is not obvious. There have been attempts to insert exogenous anti-cancer genes into the E3 region. The expression efficiency of exogenous genes is ideal, but the right arm plasmid is large, taking pBHGloxdelE13cre as an example, the size is 34.707 kb. The transformation of a large vector is difficult, and the success rate is not high. It often requires repeated attempts to screen the correct vector. 
     When a new type of OAV is constructed, in order to improve the targeting efficiency of tumor cells, the transfection rate, the expression of the loaded anti-cancer genes, and to avoid the elimination of the virus by the body&#39;s immune system, the right arm backbone plasmid needs to be greatly modified, including screening Fiber molecules that can enhance the efficiency of adenovirus infection, Hexon molecules that can make the virus evade the body&#39;s immune interception and liver uptake, and a variety of exogenous genes with different mechanisms and functions, and inserting the expression sequence of the above molecules into the E3 region. For the screening and modification of each molecule, the large right arm backbone vector must be reconstructed, and the genome spanning from Hexon and E3 region to Fiber is up to 14 kb, therefore, the modification is a lot of work, difficult, time-consuming and labor-intensive. For example, replacing the original Hexon gene with a chimeric Hexon gene to construct an adenovirus backbone plasmid vector containing chimeric Hexon with hypervariable region, reported by a doctoral dissertation titled “Fiber-modified Hexon-chimeric Oncolytic Adenoviral Vectors Targeting Gastric Cancer-Associated Fibroblasts” published in 2013, has the disadvantage of large workload mentioned above. In order to solve the technical difficulties of OAV construction and packaging, we have developed a fast and accurate three-plasmid oncolytic adenovirus recombinant system Ad5MixPlus, which has been confirmed in practical applications. The ideal new OAV product can be packaged accurately and quickly, saving time and effort. 
     SUMMARY OF THE INVENTION 
     Adenovirus has to be modified on a large scale, involving almost the whole genome, mainly to improve the specificity of targeting tumor cells, and the transfection rate, increase the expression level of loaded anti-cancer genes, and avoid the elimination of the virus by the body&#39;s immune system. In the past, the modification and recombination of OAV were mostly concentrated in the E1 region of the left arm of the adenovirus genome. Exogenous anti-cancer genes were also inserted in the E1 region. Since the E1a promoter and the anti-cancer gene promoter are both in the E1 region, they are too close to each other and interfere with each other, the expression efficiency of them are all reduced. If you want to further optimize and increase the transfection rate of OAV, the expression level of loaded anti-cancer genes, and avoid the elimination of the virus by the body&#39;s immune system, it is necessary to screen useful molecules from different serotypes of adenovirus to construct Fiber chimeric and Hexon chimeric hybrid OAV, and variety of anti-cancer genes to load them into the E3 region of OAV according to tumor type and treatment needs. Therefore, the structural proteins of the adenovirus right arm backbone plasmid, including Hexon, Fiber, E3 region, etc., need to be modified in a wide range. But the right arm plasmid is very large. Take the most commonly used Microbix Biosystems right arm backbone plasmid pBHGloxdelE13cre as an example, the size is 34.707 kb. Thus, a large vector construction is required for each modification of each protein molecule. Because the modification of large vectors is difficult, the error rate is high, and the selection is difficult, the modification of OVA is time-consuming, laborious and costly, and screening the correct vector often requires repeated attempts. 
     In order to solve the above technical problems, the present invention provides a fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system, which involving the following 3 adenovirus recombinant plasmids: 
     a) adenovirus right arm backbone plasmid: the said adenovirus right arm backbone plasmid is loaded with two sets of recombinant sequences at different sites, one set of attL/attR in the Fiber/Hexon/E3 region, and the other set of Cre/loxP in the E1 region; the E3 region is also inserted the ccdB lethal gene of DB3.1  E. coli  strain and competent cells; 
     b) adenovirus right arm shuttle plasmid: the said adenovirus right arm shuttle plasmid contains the reconstructed chimeric Hexon sequence and chimeric Fiber sequence; the E3 region is preset with multiple cloning sites for exogenous gene insertion; Hexon/E3/Fiber sequence contains attL1/attL2 recombination sites at both ends; 
     c) adenovirus left arm shuttle plasmid: the said adenovirus left arm shuttle plasmid is inserted tumor-specific promoter-controlled adenovirus early replication gene and loxP recombination site at its multiple cloning sites; 
     wherein, the first round of attL/attR site-specific recombination is performed between the adenovirus right arm shuttle plasmid and the adenovirus right arm backbone plasmid based on attL1/attL2 at both ends of the Hexon/E3/Fiber sequence, resulting that the sequence between attL1/attL2 in the adenovirus right arm shuttle plasmid replaces the sequence between attR1/attR2 in the adenovirus right arm backbone plasmid; the second round of Cre/loxP site-specific recombination is performed between the adenovirus left arm shuttle plasmid and the adenovirus right arm backbone plasmid, resulting that the E1a expression cassette controlled by the tumor-specific promoter in the adenovirus left arm shuttle plasmid is inserted into the E1 region of the adenovirus right arm backbone plasmid; the required oncolytic adenovirus is packaged after the above two rounds of site-specific recombination. 
     As a preferred example of the present invention, the chimeric Hexon sequence is a chimeric sequence formed by Hexon of Ad5 and Hexon or its mutant sequence of Ad48, Ad9, Ad37, Ad43 or any other serotype adenovirus. 
     As another preferred example of the present invention, the sequence of the said chimeric Hexon is SEQ ID NO: 5. 
     As another preferred example of the present invention, the chimeric Fiber sequence is a chimeric sequence formed by Fiber of Ad5 and Fiber or its mutant sequence of Ad11b, Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55 or any other serotype adenovirus. 
     As another preferred example of the present invention, the sequence of the said chimeric Fiber is SEQ ID NO: 6. 
     As another preferred example of the present invention, the tumor-specific promoter is selected from: (a) promoter, enhancer and mutant sequence of carcinoembryonic antigen; (b) promoter, enhancer and mutant sequence of alpha fetoprotein; (c) promoters, enhancers and mutant sequences of receptor tyrosine kinases (including EGFR, Her-2, Her-3 and Her-4) of the human epidermal growth factor receptor family (EGFRs); (d) promoter, enhancer and mutant sequence of breast cancer related antigen DF3/MUC1; (e) promoter, enhancer and mutant sequence of vascular endothelial growth factor (VEGF) receptor KDR; (f) promoter, enhancer and mutant sequence of L-plastin; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) promoters, enhancers and mutant sequences of prostaglandin-specific antigens; (i) conserved sequences of hypoxia response elements regulated by hypoxia inducible factor-1 (HIF-1); (j) promoter, enhancer and mutant sequence of transcription factor E2F; (k) promoter, enhancer and mutant sequence of hTERT. 
     As another preferred example of the present invention, the sequence of the said tumor-specific promoter is SEQ ID NO:7. 
     As another preferred example of the present invention, the said adenovirus early replication gene is E1a or E1b, wherein E1a is wild or mutant, and the E1b is E1b-55 kDa, E1b-19 kDa or mutants thereof. 
     As another preferred example of the present invention, the sequence of the said E1a expression cassette is SEQ ID NO:8. 
     As another preferred example of the present invention, the sequence of the said adenovirus right arm backbone plasmid is SEQ ID NO: 1, the sequence of the said adenovirus right arm shuttle plasmid is SEQ ID NO: 2, and the sequence of the said adenovirus left arm shuttle plasmid is SEQ ID NO:3. 
     As another preferred example of the present invention, the sequence of the oncolytic adenovirus is SEQ ID NO: 4. 
     The present invention also provides the application of the three-plasmid oncolytic adenovirus recombinant packaging system in the preparation of oncolytic adenovirus or antitumor drugs. 
     As a preferred example of the present invention, the tumor is selected from a digestive system tumor such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumor such as lung cancer and pleuromas; blood system tumor such as leukemia, lymphoma and multiple myeloma; gynecological and reproductive system tumor such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer and penile cancer; nervous system tumor such as glioma, neuroblastoma and meningioma; head and neck tumor such as oral cancer, tongue cancer, laryngeal cancer and nasopharyngeal cancer; urinary system tumor such as kidney cancer and bladder cancer, and skin and other system tumor such as skin cancer, melanoma, osteosarcoma, liposarcoma and thyroid cancer. 
     The advantages of the present invention are listed as follows: 
     This invention provides a fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system Ad5MixPlus consisting of 3 adenovirus recombinant plasmids and the application thereof. The core technology of the present invention is to cleverly load two sets of different sites for recombination on the first adenovirus recombinant plasmid, type 5 adenovirus right arm backbone plasmid pAd5MixPlus, one set of attL/attR in the Fiber/Hexon/E3 region, the other set of Cre/loxP in the E1 area. The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Fiber/Hexon/E3 sequence, and the E3 region is preset with multiple cloning sites for exogenous gene insertion. Basing on attL1/attL2 at both ends of the Hexon/E3/Fiber sequence, the adenovirus right arm shuttle plasmid undergoes the first round of specific recombination at attL/attR site with adenovirus right arm backbone plasmid pAd5MixPlus in bacteria, and the sequence between attR1/attR2 in pAd5MixPlus is replaced by the sequence between attL1/attL2 in pAdH548F511LR. Then the bacteria carrying the recombinant plasmid are accurately screened with the help of ccdB gene in the E3 region of pAd5MixPlus. The third adenovirus recombinant plasmid, the adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by a tumor-specific promoter and a loxP recombination site. The adenovirus left arm shuttle plasmid undergoes the second round of specific recombination at Cre/loxP site with pAd5MixPlus in eukaryotic cells, and the E1a expression cassette controlled by the tumor-specific promoter in pAdSVPcreLoxP is inserted into the E1 region of pAd5MixPlus. Finally, the ideal oncolytic adenovirus is packaged accurately and quickly after the above two rounds of site-specific recombination. All sequence modifications are carried out on two small shuttle plasmids. The modification of the E1 region for tumor-specific replication of virus is carried out on the said adenovirus left arm shuttle plasmid, while the modification of the protein structure of the virus Hexon, Fiber, E3 region and the insertion of exogenous genes in the E3 region are all carried out on the said adenovirus right arm shuttle plasmid. Therefore, the difficulty of modification of the large adenovirus backbone plasmid vector is solved. The exogenous gene can be loaded into any shuttle plasmid to be inserted into the E1 or E3 region of the oncolytic adenovirus genome according to actual needs. Basing on our Ad5MixPlus recombination system, the viral recombination and packaging process is simplified, fast and accurate, which solves the challenging problem of the large adenovirus backbone plasmid vector modification. The required oncolytic adenovirus can be quickly recombined and screened, which is very suitable for industrialization needs. 
     Further experiments showed that the oncolytic adenovirus packaged in the present invention had strong specific replication activity in tumor cells, strong killing activity on tumor cells, and significantly inhibited the increase of tumor volume. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the structure of the adenovirus right arm backbone plasmid pAd5MixPlus. 
         FIG. 2  shows the structure of the adenovirus right arm shuttle plasmid pAdH548F511LR. 
         FIG. 3  shows the structure of the adenovirus left arm shuttle plasmid pAdSVPcreLoxP. 
         FIG. 4  shows the recombinant packaging process of oncolytic adenovirus AdSVPH548F511 based on the Ad5MixPlus system. 
         FIG. 5  shows the structure of the oncolytic adenovirus AdSVPH548F511. 
         FIG. 6  shows the replication activity of the oncolytic adenovirus AdSVPH548F511 detected by the TCID50 method. 
         FIG. 7  shows the killing activity of the oncolytic adenovirus AdSVPH548F511 on tumor cells. (A) MTT method, (B) RTCA method. 
         FIG. 8  shows the inhibitory effect of the oncolytic adenovirus AdSVPH548F511 on renal cell carcinoma xenograft. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The specific embodiments of the present invention are described in detail with reference to drawings. 
     Example 1 Fast and Accurate Three-Plasmid Oncolytic Adenovirus Recombinant Packaging System Ad5MixPlus of the Present Invention 
     The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of the present invention involves 3 adenovirus recombinant plasmids, adenovirus right arm backbone plasmid of Ad5MixPlus system, adenovirus right arm shuttle plasmid of Ad5MixPlus system, and adenovirus left arm shuttle plasmid of Ad5MixPlus system. The recombination and packaging process of these 3 adenovirus recombinant plasmids and Ad5MixPlus is as follows. 
     1. Adenovirus Right Arm Backbone Plasmid of Ad5MixPlus System 
     The first adenovirus recombinant plasmid, adenovirus right arm backbone plasmid pAd5MixPlus, was constructed based on adenovirus type 5 and was loaded with two sets of recombinant sites, one attL/attR in Fiber/Hexon/E3 region and the other Cre/loxP in E1 region ( FIG. 1 ). The ccdB lethal gene that causes the death of the DB3.1  E. coli  strain and competent cells was inserted in the E3 region to screen successful recombinant clones. The principle is that the successful recombinant vector lost the ccdB lethal gene between attR1 and attR2 sites, and there was no ccdB lethal gene expression, so the competent bacteria survived and formed clones, while the unsuccessful recombinant vector expressed the original ccdB gene and resulted in bacterial death. 
     The full-length sequence of adenovirus right arm backbone plasmid pAd5MixPlus is SEQ ID NO: 1. 
     2. Adenovirus Right Arm Shuttle Plasmid of Ad5MixPlus System 
     The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5H48 chimeric Hexon sequence and Ad5F11b chimeric Fiber sequence. Multiple cloning sites for exogenous genes insertion were preset in the E3 region. Hexon/E3/Fiber sequence contains attL1/attL2 recombination sites at both ends ( FIG. 2 ). 
     The full-length sequence of adenovirus right arm shuttle plasmid pAdH548F511LR is SEQ ID NO: 2. 
     3. Adenovirus Left Arm Shuttle Plasmid of Ad5MixPlus System 
     The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin and a loxP recombination site ( FIG. 3 ). 
     4. Recombinant Packaging Procedure for Ad5MixPlus System 
     Adenovirus right arm shuttle plasmid pAdH548F511LR was recombined with adenovirus right arm backbone plasmid pAd5MixPlus at attL1/attL2 located at both ends of the Hexon/E3/Fiber sequence in DB3.1  E. coli  competent cells. After the first round of recombination mentioned above, the sequence between attR1/attR2 in pAd5MixPlus was replaced by the sequence between attL1/attL2 in pAdH548F511LR. Then, the adenovirus left arm shuttle plasmid pAdSVPcreLoxP was recombined with pAd5MixPlus in eukaryotic cells, which is the second round of recombination, leading to that the E1a expression cassette controlled by the tumor-specific promoter in pAdSVPcreLoxP was inserted into the E1 region of pAd5MixPlus. The ideal oncolytic adenovirus was packaged accurately and quickly after the above two rounds of site-specific recombination. The recombinant packaging procedure for Ad5MixPlus system was shown in  FIG. 4 . The structure of the oncolytic adenovirus AdSVPH548F511 was shown in  FIG. 5 . 
     The complete genome sequence of the oncolytic adenovirus AdSVPH548F511 of the present invention is: SEQ ID NO: 4. 
     The contents that need special explanation are as follows. 
     1. Modification of Chimeric Hexon Sequence 
     The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5H48 chimeric Hexon sequence. Due to exposure to the surface of adenovirus, the hypervariable region (HVR) of Hexon is the key site for the difference of liver infection ability and immunogenicity between different serotypes of adenovirus. Using genetic engineering to modify the adenovirus vector, seven HVRs of Hexon on the surface of Ad5 are selectively chimeric with the corresponding regions of Hexon of rare serotype viruses, which is an effective method to help adenovirus evade pre-existing immunity and avoid liver adsorption. 
     The modified Ad5H48 chimeric Hexon of the present invention was prepared by replacing the corresponding sequence of Ad5 with the HVR of type 48 adenovirus of subgroup D. The population generally lacks neutralizing antibodies against Ad48, and Ad48 has weak liver affinity. Therefore, interception from neutralizing antibodies and uptake by the liver can be avoided and virus survivability is improved by replacing the corresponding part of Ad5 with the HVR of Ad48 to construct Ad5 and Ad48 chimeric Hexon adenovirus. The chimeric Hexon sequence described in this present invention also can be a chimeric sequence constructed by Hexon of Ad5 and Hexon of any other serotype adenovirus, such as Ad9, Ad37, Ad43, or a mutant sequence thereof. 
     The complete sequence of Ad5H48 chimeric Hexon is SEQ ID No: 5. 
     2. Modification of Chimeric Fiber Sequence 
     The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5F11b chimeric Fiber sequence. The human adenovirus family has 51 known serotypes and is divided into 6 subgenus (A to F). CAR is the main recognition receptor of each subgenus adenovirus except B (Ad5 belongs to Subgenus C). Adenoviruses of subgenus B are further divided into subgroups B1 and B2. Ad11b, Ad14, and Ad35 are group B2 adenoviruses; Ad3, Ad16, Ad21, and Ad50 are group B1 adenoviruses. In recent years, subgenus B adenovirus derivatives have attracted much attention as attractive gene therapy vectors because they can infect target cells such as hematopoietic cells, hematopoietic stem cells, dendritic cells (DCs) and malignant tumor cells, which are often not easily infected by commonly used adenovirus vectors such as Ad5. Different from many adenoviruses that infect cells through CAR receptors, subgenus B adenoviruses use CD46 as recognition receptor. CD46 is a widely expressed complement regulatory protein that is present on the surface of almost all human cells. The chimeric Fiber of Ad5F11b is prepared by replacing the corresponding sequence of adenovirus type 5 Fiber with the Fiber knob of Ad11b, so that the chimeric virus has high infection characteristics to hematopoietic cells, stem cells and tumor cells. The chimeric Fiber sequence described in the invention also can be a chimeric sequence constructed by the Fiber of Ad5 and Fiber of any other serotype adenovirus, such as Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55, or a mutant sequence thereof. 
     The complete sequence of Ad5F11b chimeric Fiber is SEQ ID No: 6. 
     3. Tumor Specific Promoter 
     The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin inserted at multiple cloning sites of pAdSVPcreLoxP. Survivin promoter has attracted much attention due to its high specificity and wide tumor spectrum. Survivin is rarely expressed in normal tissues, but highly selectively expressed in malignant tumors. It is highly expressed in most tumors such as lung cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer and breast cancer, and is closely related to tumor recurrence and metastasis and poor prognosis of patients, making it a broad-spectrum molecular target for tumor gene therapy. The oncolytic adenovirus regulated by the Survivin promoter can target cancer cells, replicate in cancer cells, and lyse cancer cells, and at the same time mediate the high-efficiency expression of anti-tumor target genes. Therefore, oncolytic adenovirus regulated by Survivin promoter is expected to be used to obtain broad-spectrum and safe anti-cancer effect against most human tumors. In addition to being a Survivin promoter, the tumor specific promoter of the invention can also be any of the following: (a) promoter, enhancer and mutant sequence of carcinoembryonic antigen; (b) promoter, enhancer and mutant sequence of alpha fetoprotein; (c) promoters, enhancers and mutant sequences of receptor tyrosine kinases (including EGFR, Her-2, Her-3 and Her-4) of the human epidermal growth factor receptor family (EGFRs); (d) promoter, enhancer and mutant sequence of breast cancer related antigen DF3/MUC1; (e) promoter, enhancer and mutant sequence of vascular endothelial growth factor (VEGF) receptor KDR; (f) promoter, enhancer and mutant sequence of L-plastin; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) promoters, enhancers and mutant sequences of prostaglandin-specific antigens; (i) conserved sequences of hypoxia response elements regulated by hypoxia inducible factor-1 (HIF-1); (j) promoter, enhancer and mutant sequence of transcription factor E2F; (k) promoter, enhancer and mutant sequence of hTERT. 
     The nucleotide sequence of the tumor-specific promoter Survivin in this invention is SEQID NO: 7. 
     4. Adenovirus Early Replication Gene E1a 
     The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin. Adenovirus early replication gene E1a is placed under the regulation of tumor-specific promoter to achieve the purpose of tumor-specific replication and oncolysis of the virus. The sequence of the early replication gene of adenovirus described in this invention can be a wild sequence of E1a, a mutant sequence of E1a, or E1b-55 kDa, E1b-19 kDa or their mutant sequences. 
     The nucleotide sequence of E1a in the E1a expression cassette controlled by the tumor-specific promoter in this invention is SEQ ID NO: 8. 
     Example 2 Specific Replication Activity of Oncolytic Adenovirus AdSVPH548F511 
     Hepatocellular carcinoma cells (HCCLM3, HepG2, Huh-7, MHCC97H, MHCC97L), normal hepatocytes (WRL-68) or normal fibroblasts (BJ) in logarithmic phase were collected and counted. The cells were seeded on 96-well plates at 1×10 4  cells/well, changing to serum-free culture medium after cell adherence. The oncolytic adenovirus AdSVPH548F511 was added to the culture medium at MOI=1. After 2 h of viral infection, the medium was changed to 5% serum medium (This is the starting time of infection 0 h), and the cells were further cultured for 48 h and 96 h. The cells were collected at these three time points, and the virus titer was detected by TCID50 method. The results showed that AdSVPH548F511 had a very strong ability of specific replication in liver cancer cells, and the replication multiples were all above 10,000 times at 48 h, with the maximum was above 50,000 times. After 96 h, it reached 100,000 to 800,000 times. The replication ability of AdSVPH548F51 in normal cells WRL-68 or BJ was very low, and the highest replication multiple at 96 h was below 200 times ( FIG. 6 ). 
     Example 3 Killing Activity of Oncolytic Adenovirus AdSVPH548F511 on Tumor Cells 
     Hepatocellular carcinoma cells (HepG2, MHCC97H), normal hepatocytes (L02) and normal fibroblasts (BJ) in logarithmic phase were collected and counted. The cells were seeded on 96-well plates at 1×10 4  cells/well, changing to serum-free culture medium after cell adherence. The effect of AdSVPH548F511 on cell viability was detected by MTT assay. Cell Proliferation Kit I (MTT) was purchased from Roche Diagnostics GmbH. The viruses were added into culture wells with a gradient of MOIs, with 8 replicates per treatment. Then the cells were cultured in the incubator. After 2 h, the medium was replaced with serum medium for further culture, with the addition amount of 100 μl/well. After 48 hours, the culture medium was discarded, 0.1 mol/L PBS solution was added to the culture well with the dosage 100 μl/hole. Then MTT labeling reagent was added to the culture well to the final concentration of 0.5 mg/ml, and the culture plates were placed in incubator. After 4 h, 100 μl/well of Solubilization solution (10% SDS in 0.01 mol/L HCl) was added to the culture well, and the cells were cultured overnight in an incubator. The Model 550 Microplate Reader (BIO-RAD) was used to measure the light absorption value at 570 nm wavelength, and the corrected wavelength was 655 nm. The survival curve was drawn, and the IC50 value was calculated. The results showed that AdSVPH548F511 had strong killing activity against HepG2 and MHCC97H with IC50 values of 35.16 and 212.4, respectively, while AdSVPH548F511 had no significant effect on normal BJ cells with IC50 value of 20035. It can be seen that oncolytic adenovirus AdSVPH548F511 has the ability to specifically kill and destroy cancer cells ( FIG. 7A ). 
     At the same time, the killing activity of AdSVPH548F511 on cells was detected in real time, dynamically and quantitatively by Real Time Cellular Analysis (RTCA). E-Plate plate was added with culture medium and background impedance was measured. Hepatocellular carcinoma cells (HepG2) or normal liver cells (L02) in logarithmic growth phase were collected, counted and added to the E-Plate detection plate, and placed in the ultra-clean worktable at room temperature for 30 min. The virus was diluted in serum-free medium and added to E-Plate at MOI=5, 10 or 20. The E-Plate detection plate was placed on the detection platform pre-placed in the incubator for real-time dynamic cell proliferation detection, and the real-time dynamic cell growth curves were plotted. The results showed that the killing activity of AdSVPH548F511 on HepG2 cells increased with the increase of MOIs and time. However, the killing activity of AdSVPH548F511 on normal liver cell L02 was not high. L02 cells were inhibited to some extent only when given high-MOI virus infection ( FIG. 7  B). 
     Example 4 Antitumor Animal Experiment of Oncolytic Adenovirus AdS VPH548F511 
     Eight healthy purebred male BALB/c nude mice aged 6-8 weeks were provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and kept in a clean animal laboratory. Renal carcinoma OSRC-2 cells in logarithmic growth phase were adjusted to 1×10 7  cells/ml with PBS. Near-axillary skin on the abdominal side of nude mice was disinfected and subcutaneously injected with 100 μL cell suspension. Then the nude mice were raised under the condition of constant temperature, ventilation and sterility. Tumor growth was regularly observed daily and the presence of a rice-size tumor underneath the inoculation site was considered a success. Model animals were randomly divided into two groups, virus group (AdSVP) n=5, control group (PBS) n=3. Animals were numbered and vernier caliper was used to measure the tumor size. Then treatment was started. The virus AdSVPH548F511 with a concentration of 2×10 8  pfu/100 μl was injected directly into the tumor at multiple sites, once every other day, 5 times in total. Control animals were injected with PBS instead of virus, 100 μl×5 times. The tumor size was measured regularly, and the tumor volume was calculated with the formula of ‘a×b 2 ×0.5’ (a: maximum diameter, b: minimum diameter). The growth curve of transplanted tumor was plotted ( FIG. 8 ). The results showed that AdSVPH548F511 could significantly inhibit the growth of renal cell carcinoma OSRC-2 xenograft. 
     The above are only the preferred embodiments of the present invention. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. Such changes and modifications are intended to be encompassed by the scope of the following claims.