WO2020258825A1 - 一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用 - Google Patents

一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用 Download PDF

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WO2020258825A1
WO2020258825A1 PCT/CN2020/070125 CN2020070125W WO2020258825A1 WO 2020258825 A1 WO2020258825 A1 WO 2020258825A1 CN 2020070125 W CN2020070125 W CN 2020070125W WO 2020258825 A1 WO2020258825 A1 WO 2020258825A1
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adenovirus
plasmid
sequence
promoter
tumor
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苏英晗
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江苏万戎生物医药科技有限公司
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Definitions

  • the present invention relates to the field of biomedicine technology, in particular to a set of fast and accurate three-plasmid oncolytic adenovirus recombination packaging system Ad5MixPlus and its application.
  • tumor cells infected by these oncolytic viruses can release a large amount of cytokines, and the lysed tumor cells release a large amount of tumor-associated antigens, and have immune activation effects. Therefore, combined with CAR-T cells and PD-1/PD-L1 antibodies, there are Conducive to the amplification of immunotherapy effects, the application range of oncolytic virus products can also be broadened.
  • Oncolytic virus is a type of virus that has been artificially genetically modified. It can specifically replicate in tumor cells in large numbers without affecting normal cells. The large number of replicated viruses dissolve and destroy tumor cells, releasing virus particles to continue The infection destroys more tumor cells.
  • the oncolytic virus is used as a carrier to carry anti-oncogenes. As the virus replicates and spreads, the number of copies of anti-oncogenes it carries increases, and the expression of gene protein products increases. The anti-cancer effect and oncolytic effect are synergistic and further enhanced Anti-cancer efficacy. Compared with CAR-T cell therapy, oncolytic viruses have broader indications; compared with monoclonal antibody drugs, oncolytic viruses are cheaper.
  • Oncolytic virus-mediated gene therapy has high killing efficiency, good targeting, high safety, low side effects and low cost, making it the next three conventional treatment methods (surgery, radiotherapy and chemotherapy) and immune cell therapy After that, another important emerging tumor treatment model is expected to become an important auxiliary means for comprehensive tumor treatment to synergistically improve the efficacy.
  • the top medical journal "Cell” reported a major breakthrough.
  • researchers from the United States, Switzerland, Spain, and Australia studied oncolytic virus therapy in a phase Ib clinical trial of 21 melanoma patients
  • the efficacy of combined therapy with PD-1 monoclonal antibody immunotherapy shows that oncolytic viruses can effectively improve the efficacy of immunotherapy.
  • the overall response rate is as high as 62%.
  • T-Vec oncolytic virus Talimogene laherparepvec
  • T-Vec expresses granulocyte-macrophage colony stimulating factor (GM-CSF). It is injected directly into the tumor and replicates in the tumor cells until it causes cell lysis (oncolysis). At the same time, GM-CSF is released locally in the tumor tissue to activate the systemic immune response.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • T-Vec works in two important and synergistic ways (Lancet Oncol. 2016 Nov; 17: 1485-1486) by inducing tumor cell lysis and stimulating systemic anti-tumor immune response.
  • T-Vec sales For the treatment market for melanin patients alone, Thomson Reuters expects T-Vec sales to be US$388 million per year by 2020.
  • OV has always been a hot spot for anti-tumor drug research and development, as well as a hot spot for capital markets and pharmaceutical groups.
  • Merck & Co. and Viralytics announced that Merck & Co. acquired Viralytics for US$394 million, which is equivalent to a 160% premium of the one-month volume-weighted average price of Viralytics stock.
  • Merck obtained the oncolytic virus immunotherapy developed by Viralytics. (CVA21) all rights. It is based on Viralytics' proprietary oncolytic virus (Coxsackievirus TypeA21) preparation, which 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 Merck’s ongoing PD-1 drug Trial, CAVATAK's next step or its PD-1 drug Used in combination for the treatment of melanoma, prostate cancer, lung cancer and bladder cancer.
  • the biotech giant Amgen acquired the OV drug T-Vec developed by BioVex for US$425 million.
  • the FDA approved the listing of T-Vec.
  • EU CHMP approval which became a milestone in OV treatment of tumors.
  • T-Vec has been widely used in the United States, Europe and Australia to treat relapsed melanoma at an average cost of US$65,000.
  • the OV products that have entered phase ⁇ clinical include Oncolytics' Reolysin for the treatment of head and neck tumors, ColdGenesys's CG0070 for the treatment of bladder cancer, Advantagene's Prost Atak TM for the treatment of prostate cancer, and Jennerex'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, and colon.
  • OV was developed to solve the shortcomings of low anti-cancer gene expression in gene therapy and the inability of the vector to target tumor cells.
  • oncolytic virus as a vector to mediate tumor gene therapy, it can target tumor cells, produce oncolytic effects, and produce synergistic anti-cancer effects with gene therapy, which has obvious application value.
  • many types of viruses can be transformed into oncolytic viruses.
  • T-Vec is transformed from herpes simplex virus
  • ProstAtak and G0070 are adenoviruses
  • Reolysin is reovirus
  • JX-594 is vaccinia virus.
  • Adenovirus is the most extensively studied type, and there are the most types of oncolytic viruses.
  • adenovirus There are many types of adenoviruses and their structures are complex. At present, more than 100 serotypes 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 effectively multiply in the host cell; the viral genome is not integrated into the host cell chromosome and has no insertional mutagenicity; high titer , Easy to prepare and purify, etc., the most suitable to develop safe OV products.
  • adenovirus also has some shortcomings: such as strong autoimmunity, poor specificity, and easy accumulation in the liver by intravenous injection, so it needs to be further modified. These complex features are just the best opportunity for us to carry out targeted and effective modification, and it is more conducive to the development of a multi-mechanism synergistic anti-cancer high-efficiency oncolytic adenovirus (OAV) product through comprehensive modification, and can be based on tumors. Type feature design to build a personalized OAV.
  • OAV oncolytic adenovirus
  • the modification of adenovirus mainly focuses on improving the specificity of targeted tumor cells, the transfection rate of infected cells, the expression of loaded anti-cancer genes, and avoiding the elimination of the virus by the body's immune system (Mol Cancer Ther. 2016 Jul; 15:1436-51).
  • OAV The construction of OAV is based on type 5 (Ad5) of the weakest human adenovirus C subgenus, which is mainly divided into the modification of the E1 region of the left arm of the genome and the modification of the coat protein, fibrin, and E3 region of the right arm.
  • Ad5 type 5 of the weakest human adenovirus C subgenus
  • E1a protein needs to be preserved and regulated by tumor-specific promoters to achieve tumor-specific replication.
  • E1a has 3 functional regions, namely CR1, CR2, CR3, CR1 region can inhibit the expression of Her-2/neu gene by combining with transcription regulator P300/CBP, CR2 region is combined with Rb protein family, CR3 region is transcription activation region . Therefore, E1a protein has an anti-tumor effect. It can inhibit the transcription of Her-2/neu gene, block the activity of NF- ⁇ B, increase the expression of p53, and inhibit protease genes such as type IV collagenase and plasminogen activator.
  • E1a can also cause non-specific immune response, improve the killing effect of CTL cells, NK cells, macrophages and other ways, 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 prevents it from binding to the Rb protein, ensuring that the dephosphorylated Rb protein forms a complex with the transcription factor E2F, blocking the transcription activity of E2F, and can enhance the anti-cancer activity.
  • the Elb transcription unit encodes Elb-55kDa and Elb-19kDa.
  • Elb-55kDa is a protein necessary for adenovirus proliferation and replication in normal cells but not necessary in tumor cells.
  • the selective deletion of the Elb-55kDa coding gene can enable adenovirus to maintain the ability to proliferate and replicate in tumor cells, while in normal cells Lost the ability to replicate.
  • Elb-55kDa protein can inactivate and degrade P53 protein.
  • Elb-55kDa deletion helps cells maintain the anti-tumor activity of P53, and at the same time improves the targeting of viral vectors.
  • the Elb-19kDa gene is homologous to the apoptosis-suppressing gene Bcl-2.
  • the Elb-19kDa protein can bind to Bax or/and Bak to initiate the downstream apoptosis-inhibiting program and protect infected cells from TNF- ⁇ -mediated killing.
  • Elb -19kDa deletion so the specificity of the virus mutant proliferation in tumor cells is improved, while the proliferation activity in normal cells is weakened.
  • the deletion of Elb-19kDa can promote the restoration of the apoptosis pathway of cancer cells, and facilitate the rapid clearance of the virus in normal cells and the rapid release and spread of the virus in tumor cells, making OAV more specific and more effective.
  • the double deletion of Elb-55kDa and Elb-19kDa affects the proliferation of OAV, and whether it can produce immune enhancement effect through modification of both needs further research.
  • 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’s immune response.
  • E3-gpl9k can attenuate the CTL-mediated killing effect of infected cells; RID can block the "dead"
  • the body includes TNF, Fas ligand and TRAIL, which mediate apoptosis; RID can also inhibit the activation of NF- ⁇ B necessary for cell survival mediated by IL-1 and TNF; E3-6.7k in addition to downregulate the TRAIL receptor In addition, it can also inhibit cell apoptosis induced by external and internal signaling pathways; E3-14.7k is a broad inhibitor of TNF-mediated cell apoptosis, and can also bind and inhibit Caspases-8 to prevent cell apoptosis initiated by Fas signaling pathway.
  • E3-14.7k interacts with the FIP protein (for 14.7K-interacting protein, FIP-1, -2, -3) in the infected cell, which can make E3-14.7k protein in the cell apoptosis and survival, inflammation response , Maintaining membrane stability, nucleoplasmic transport and other signal transduction pathways play an important role.
  • 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 deleting the E3 region during the OAV construction process can not only expand the vector capacity, but also promote the apoptosis of infected cancer cells.
  • E3 also relieves the immune resistance of the virus to the body, and the virus can be quickly eliminated. Therefore, it is necessary to strengthen the understanding of the protein encoded by the E3 region and avoid blindly deleting all the E3 regions in the construction of OAV, which 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 groups of adenoviruses use Coxsackievirus-adenovirus receptor (CAR) as their main adsorption receptor, which can effectively infect hematopoietic cells, hematopoietic stem cells, dendritic cells, and some tumor cells, especially tumor stem cells, lacking CAR. Very low. Group B adenovirus (Ad3, Ad11b, Ad14, Ad16, Ad21, Ad35, Ad50) mainly recognizes a widely expressed complement regulatory protein CD46.
  • CAR Coxsackievirus-adenovirus receptor
  • Using the fiber knob of group B adenovirus instead of Ad5 fiber protein to construct a chimeric virus will help improve the transduction efficiency of the virus to tumor cells, especially the ability to infect tumor stem cells, and it may more completely eradicate tumor recurrence The root cause.
  • Ad5 exists widely in nature, and most people have been infected and have produced neutralizing antibodies that can block the virus. Moreover, Ad5 is hepatotropic and can be adsorbed by hepatocytes. The hypervariable region (HVR) of the adenovirus coat protein Hexon is exposed to the surface of the adenovirus due to its position, which is a key part of the difference in liver infection ability and immunogenicity between different serotypes of adenovirus.
  • HVR hypervariable region
  • the 7 HVRs in the Hexon molecule of Ad5 are selectively chimerized with the Hexon corresponding regions of rare serotypes such as D subgroup (Ad37, Ad43) and B subgroup (Ad48) viruses, which helps Ad5 to avoid pre-existing immunity and liver uptake Effective method.
  • D subgroup Ad37, Ad43
  • B subgroup Ad48
  • the most used system for packaging adenovirus is Microbix Biosystems packaging technology, which uses the adenovirus left arm plasmid pDC series (such as pDC315, pDC316, pDC312, etc.) and the left arm skeleton plasmid (pBHGloxdelE13cre) that completely deletes the E1/E3 region for recombination ,
  • pDC315, pDC316, pDC312, etc. the left arm skeleton plasmid
  • pBHGloxdelE13cre left arm skeleton plasmid
  • the insert size of the left arm is limited, and 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, and the expression efficiency is reduced. We tried to separate them with insulator sequences in the early stage. Although there are improvements, the effect is not obvious.
  • the right arm skeleton is required
  • Large-scale transformation of plasmids requires not only screening of Fiber molecules that can enhance the efficiency of adenovirus infection among different serotypes of adenovirus, screening of Hexon molecules that can enable the virus to evade immune interception and liver uptake, and screening of different mechanisms and functions
  • a variety of foreign genes are inserted into the E3 region, and the screening and modification of each molecule requires a reconstruction of the large right arm skeleton vector.
  • a large-scale modification of adenovirus is required, almost involving the entire genome, mainly to improve the specificity of targeted tumor cells, The transfection rate of infected cells, the expression level of loaded anti-cancer genes, and the avoidance of virus clearance by the body's immune system.
  • OAV transformation and recombination were mostly concentrated in the E1 functional region of the left arm of the adenovirus genome.
  • the foreign anti-cancer genes were also inserted in the E1 region.
  • the tumor-specific promoters and anti-cancer gene promoters of E1a were both in the E1 region.
  • the present invention provides a fast and accurate three-plasmid oncolytic adenovirus recombination packaging system, which contains the following 3 adenovirus recombination plasmids:
  • Adenovirus right arm skeleton plasmid The adenovirus right arm skeleton plasmid is loaded with two sets of recombination sequences at different sites, one set of attL/attR in the Fiber/Hexon/E3 region, and a set of Cre/loxP in the E1 region; The E3 region also inserted the DB3.1 E. coli strain and the competent ccdB lethal gene;
  • Adenovirus right arm shuttle plasmid contains the reconstructed chimeric Hexon sequence and chimeric Fiber sequence.
  • the middle E3 region is preset with a foreign gene multi-cloning site, Hexon/E3 /Fiber sequence contains attL1/attL2 recombination sites at both ends;
  • Adenovirus left arm shuttle plasmid inserts an adenovirus early proliferation gene and loxP recombination site controlled by a tumor-specific promoter at its multiple cloning site;
  • the adenovirus right arm shuttle plasmid undergoes the first round of attL/attR site-specific recombination with the adenovirus right arm skeleton plasmid through the attL1/attL2 at both ends of the Hexon/E3/Fiber sequence, and the adenovirus right arm
  • the sequence between attL1/attL2 in the shuttle plasmid replaces the sequence between attR1/attR2 in the adenovirus right arm skeleton plasmid
  • the second round of Cre/loxP occurs between the adenovirus left arm shuttle plasmid and the adenovirus right arm skeleton plasmid
  • the E1a expression cassette controlled by the tumor-specific promoter in the adenovirus left arm shuttle plasmid is inserted into the E1 region deleted from the adenovirus right arm skeleton plasmid; after two rounds of site-specific recombination, Package the required on
  • the chimeric Hexon sequence is a chimera of Ad5, Ad48, Ad9, Ad37, Ad43 and any other serotype adenovirus Hexon and its mutant sequence.
  • the chimeric Hexon sequence is shown in SEQ ID NO: 5.
  • the chimeric Fiber sequence is a chimerization of Ad5 and Ad11b, Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55 and any other serotype adenovirus Fiber and its mutant sequence .
  • the chimeric Fiber sequence is shown in SEQ ID NO: 6.
  • the tumor-specific promoter is selected from: (a) carcinoembryonic antigen promoter, enhancer and mutant sequences thereof; (b) alpha-fetoprotein promoter, enhancer and mutations thereof (C) Human epidermal growth factor receptor family (EGFRs) receptor tyrosine kinase (including EGFR, Her-2, Her-3 and Her-4) promoters, enhancers and their mutant sequences; (d) Breast cancer related antigen DF3/MUC1 promoter, enhancer and its mutant sequence; (e) Vascular endothelial growth factor (VEGF) receptor KDR promoter, enhancer and its mutant sequence; (f)L -plastin promoter, enhancer and its mutant sequences; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) prostaglandin-specific antigen promoters, enhancers (I) Hypoxia inducible factor-1 (HIF-1) regulated hypo
  • the sequence of the tumor-specific promoter is shown in SEQ ID NO:7.
  • the adenovirus early proliferation genes include E1a and E1b, wherein E1a is selected from E1a wild sequence and its mutant sequence, and said E1b is selected from Elb-55kDa, Elb-19kDa and its mutant sequence.
  • sequence of the E1a expression cassette is shown in SEQ ID NO: 8.
  • sequence of the adenovirus right arm backbone plasmid is shown in SEQ ID NO: 1
  • sequence of the adenovirus right arm shuttle plasmid is shown in SEQ ID NO: 2.
  • sequence of the shuttle plasmid on the left arm of the virus is shown in SEQ ID NO: 3.
  • the sequence of the oncolytic adenovirus is shown in SEQ ID NO: 4.
  • the present invention also provides the application of the three-plasmid oncolytic adenovirus recombination packaging system in the preparation of oncolytic adenovirus or antitumor drugs.
  • the tumor is selected from the group consisting of digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer and pleuromas; blood system tumors such as leukemia , Lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast, ovarian, cervical, vulvar, testicular, prostate, penile cancer; nervous system tumors such as glioma, neuroblastoma, meninges Tumors; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, and thyroid cancer.
  • digestive system tumors such as esophageal cancer, gastric cancer, colorectal
  • the invention provides a set of fast and accurate three-plasmid oncolytic adenovirus recombination packaging system Ad5MixPlus and its application.
  • the system consists of 3 adenovirus recombinant plasmids, and its core technology is to cleverly load two sets of recombination sequences at different sites on the first type 5 adenovirus right arm skeleton plasmid pAd5MixPlus.
  • One set of attL/attR is in Fiber/Hexon. /E3 area, a set of Cre/loxP is in E1 area.
  • the second adenovirus right arm shuttle plasmid pAdH548F511LR contains the Fiber/Hexon/E3 modified sequence, and the E3 region is also preset with the foreign gene polyclonal insertion site.
  • This plasmid will pass through the attL1/attL2 at both ends of the Hexon/E3/Fiber sequence to connect with the gland
  • the first round of attL/attR site-specific recombination occurs in the virus right arm backbone plasmid pAd5MixPlus in bacteria.
  • the sequence between attL1/attL2 in pAdH548F511LR replaces the sequence between attR1/attR2 in pAd5MixPlus, and the ccdB gene in the E3 region of pAd5MixPlus is accurately used.
  • Screening; the third adenovirus left arm shuttle plasmid pAdSVPcreLoxP contains an E1a expression cassette controlled by a tumor-specific promoter and a loxP recombination site.
  • This plasmid will have a second round of Cre/loxP site specificity in eukaryotic cells with pAd5MixPlus Recombination, the E1a expression cassette controlled by the tumor-specific promoter in pAdSVPcreLoxP was inserted into the E1 region deleted by pAd5MixPlus. After two rounds of site-specific recombination, the ideal oncolytic adenovirus can be packaged accurately and quickly. All sequence modifications of the system are carried out on two small shuttle plasmids. The E1 region of the virus tumor-specific propagation is modified on the left arm shuttle plasmid. The protein structure of the virus Hexon, Fiber, and E3 region is modified, and the E3 region is inserted into foreign genes.
  • oncolytic adenovirus packaged in the present invention has strong specific proliferation activity in tumors, strong killing activity on tumor cells, and can obviously inhibit the increase of tumor volume.
  • Figure 1 Structure diagram of adenovirus right arm skeleton plasmid pAd5MixPlus.
  • Figure 2 The structure of the right arm shuttle plasmid pAdH548F511LR of adenovirus.
  • Figure 3 The structure diagram of the adenovirus left arm shuttle plasmid pAdSVPcreLoxP.
  • FIG. 1 Structure diagram of the oncolytic adenovirus AdSVPH548F511.
  • FIG. 1 Oncolytic adenovirus AdSVPH548F511 killing activity against tumor cells, (A) MTT method, (B) RTCA method.
  • Figure 8 The inhibitory effect of oncolytic adenovirus AdSVPH548F511 on transplanted renal carcinoma.
  • Example 1 A set of fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system Ad5MixPlus of the present invention
  • the set of fast and accurate three-plasmid oncolytic adenovirus recombination system Ad5MixPlus of the present invention is composed of three adenovirus recombination plasmids. These three adenovirus recombinant plasmids are Ad5MixPlus system right arm skeleton plasmid, Ad5MixPlus system right arm shuttle plasmid and Ad5MixPlus system left arm shuttle plasmid respectively. Now introduce these 3 adenovirus recombinant plasmids and the recombinant packaging of Ad5MixPlus, as follows:
  • the E3 region has also inserted the DB3.1 E. coli strain and the competent ccdB lethal gene, which is used to screen the successfully recombined virus clones.
  • the successfully recombined vector loses the ccdB gene between the attR1 and attR2 sites, and the competent bacteria survive. Clonal growth occurs; the unsuccessful recombination of the vector causes the bacteria to die under the action of its original ccdB gene product.
  • the full-length sequence of the adenovirus right arm skeleton plasmid pAd5MixPlus is: SEQ ID NO:1.
  • the second adenovirus right-arm shuttle plasmid pAdH548F511LR contains the modified Ad5H48 chimeric Hexon sequence and Ad5F11b chimeric Fiber sequence, the middle E3 region is preset with a foreign gene multiple cloning site, and the Hexon/E3/Fiber sequence ends Contains attL1/attL2 recombination sites ( Figure 2).
  • the full-length sequence of the adenovirus right arm shuttle plasmid pAdH548F511LR is: SEQ ID NO: 2.
  • the full-length sequence of the adenovirus left arm shuttle plasmid pAdSVPcreLoxP is: SEQ ID NO: 3.
  • the second adenovirus right arm shuttle plasmid pAdH548F511LR passes through the attL1/attL2 at both ends of the Hexon/E3/Fiber sequence and the adenovirus right arm skeleton plasmid pAd5MixPlus, the first round of attL/attR site specificity occurs in the DB3.1 E.
  • the oncolytic adenovirus AdSVPH548F511 of the present invention has a complete sequence of SEQ ID NO: 4.
  • the second adenovirus right arm shuttle plasmid pAdH548F511LR contains the modified Ad5H48 chimeric Hexon coding sequence.
  • the Hexon hypervariable region (HVR) is exposed to the surface of the adenovirus due to its location, which is a key part of the difference in liver infection ability and immunogenicity between different serotypes of adenovirus.
  • HVR Hexon hypervariable region
  • the Ad5H48 chimeric Hexon of the present invention replaces the corresponding sequence of Ad5 with the HVR of the D subgroup 48 adenovirus.
  • Ad48 generally lacks neutralizing antibodies in the population and has weak liver affinity. Therefore, replacing the corresponding part of Ad5 with the HVR of Ad48 to construct the adenovirus of Ad5 and Ad48 chimeric Hexon can avoid the interception of neutralizing antibodies and liver uptake, and improve the viability of the virus.
  • the chimeric Hexon of the present invention also includes the chimerism of Ad5 with Ad9, Ad37, Ad43 and any other serotype adenovirus Hexon and its mutant sequence.
  • Ad5H48 chimeric Hexon is as follows: SEQ ID NO: 5.
  • the second adenovirus right arm shuttle plasmid pAdH548F511LR contains the modified Ad5F11b chimeric Fiber coding sequence.
  • the human adenovirus family has 51 known serotypes, divided into 6 subgenres (A to F). Except for group B, all groups of adenoviruses use CAR as their main adsorption receptor (Ad5 belongs to group C). Group B adenoviruses 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.
  • group B adenovirus In recent years, derivatives of group B adenovirus have attracted attention as attractive gene therapy vectors because they can transduce target cells such as hematopoietic cells, hematopoietic stem cells, dendritic cells (DC cells), and malignant tumor cells. These cells are often not easily infected by commonly used adenovirus vectors (such as Ad5). Unlike many adenoviruses that infect cells through CAR receptors, group B adenoviruses use CD46 as an adsorption receptor. CD46 is a widely expressed complement regulatory protein, which exists on the surface of almost all human somatic cells.
  • Ad5F11b chimeric Fiber that is, the fiber knob of Ad11b is used to replace the corresponding sequence of the type 5 adenovirus fiber, so that the chimeric virus has high transduction characteristics to hematopoietic cells, stem cells, and tumor cells.
  • the chimeric Fiber of the present invention also includes the chimerization of Ad5 with Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55 and any other serotype adenovirus Fiber and its mutation sequence.
  • Ad5F11b chimeric Fiber The complete sequence of Ad5F11b chimeric Fiber is as follows: SEQ ID NO: 6.
  • the third adenovirus left arm shuttle plasmid pAdSVPcreLoxP inserts the E1a expression box controlled by the tumor-specific Survivin promoter at its multiple cloning site.
  • Survivin promoter has received more attention because of its high specificity and broad tumor spectrum. Survivin is almost not expressed in normal tissues, while its expression in malignant tumors is highly selective. It is highly expressed in most tumors such as lung cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer, and breast cancer. The recurrence and metastasis of patients are closely related to the poor prognosis of patients, making them a broad-spectrum molecular target for tumor gene therapy.
  • the oncolytic adenovirus regulated by the Survivin promoter can target cancer cells to proliferate, replicate and lyse cancer cells, and at the same time mediate the efficient expression of anti-tumor target genes. Therefore, the oncolytic adenovirus regulated by the Survivin promoter is expected to achieve a broad-spectrum and safe anti-cancer effect against most human tumors.
  • the tumor-specific promoter of the present invention in addition to the Survivin promoter, also includes: (a) carcinoembryonic antigen promoter, enhancer and its mutant sequence; (b) alpha-fetoprotein promoter, enhancer and its sequence Mutant sequence; (c) Human epidermal growth factor receptor family (EGFRs) receptor tyrosine kinase (including EGFR, Her-2, Her-3 and Her-4) promoters, enhancers and their mutant sequences (D) Breast cancer related antigen DF3/MUC1 promoter, enhancer and its mutant sequence; (e) Vascular endothelial growth factor (VEGF) receptor KDR promoter, enhancer and its mutant sequence; (f) L-plastin promoter, enhancer and its mutant sequences; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) prostaglandin-specific antigen promoters, Enhancer and its mutant sequence; (i) Hypoxia inducible factor
  • the coding sequence of the tumor-specific Survivin promoter of the present invention is as follows: SEQ ID NO: 7.
  • the third adenovirus left arm shuttle plasmid pAdSVPcreLoxP inserts the E1a expression box controlled by the tumor-specific Survivin promoter at its multiple cloning site. Place the adenovirus early proliferation gene E1a under the regulation of a tumor-specific promoter to achieve tumor-specific replication and oncolysis of the virus.
  • the adenovirus early proliferation gene of the present invention in addition to the wild E1a sequence, also includes the mutation modification of E1a, as well as Elb-55kDa, Elb-19kDa and their mutation sequences.
  • the E1a expression cassette controlled by the tumor-specific promoter of the present invention has the following E1a coding sequence: SEQ ID NO: 8.
  • HCCLM3, HepG2, Huh-7, MHCC97H, MHCC97L) and normal hepatocytes (WRL-68) and normal fibroblasts (BJ) in logarithmic growth phase, count them, and spread 96-well plates, 1 ⁇ 10 4 /Well, after the cells adhere to the wall, change to serum-free culture medium; add oncolytic adenovirus AdSVPH548F511 at MOI 1. After 2 hours of virus infection, adjust to 5% serum culture medium (at this time as infection start time 0h), continue to culture for 48h, 96h, collect cells in these three time periods respectively, and detect virus titer by TCID50 method.
  • AdSVPH548F511 has a very strong ability to specifically proliferate and replicate in liver cancer, and the proliferation folds are more than 10,000 times at 48h, and the highest is more than 50,000 times; after 96h, it can reach more than 100,000 to 800,000 times.
  • AdSVPH548F51 has mild proliferation in WRL-68 and BJ in normal cells, and the highest is only 200 times or less at 96h (Figure 6).
  • Example 3 The killing activity of oncolytic adenovirus AdSVPH548F511 on tumor cells
  • liver cancer cells HepG2, MHCC97H
  • L02 normal hepatocytes
  • BJ normal fibroblasts
  • MTT tetrazolium salt colorimetric test
  • AdSVPH548F511 had strong killing activity on HepG2 and MHCC97H, with IC50 values of 35.16 and 212.4, respectively; AdSVPH548F511 had no obvious effect on normal BJ cells, with IC50 value of 20035. It can be seen that the oncolytic adenovirus AdSVPH548F511 has the ability to specifically kill and destroy cancer cells (Figure 7 A).
  • RTCA Real Time Cellular Analysis
  • AdSVPH548F511 directly injected into the tumor at multiple points, once every other day, a total of 5 times.
  • the control group was injected with PBS instead of the virus, 100 ⁇ l ⁇ 5 times; the tumor size was measured regularly, with "a ⁇ b 2 ⁇ 0.5” formula to calculate the tumor volume (a: maximum diameter, b: minimum diameter), and draw the growth curve of transplanted tumor (Figure 8).
  • the results showed that AdSVPH548F511 can significantly inhibit the growth of OSRC-2 transplanted renal carcinoma.

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Abstract

一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用。该系统由3个腺病毒重组质粒组成，其核心技术就是巧妙地在第一个5型腺病毒右臂骨架质粒大载体上装载两套不同的位点重组序列，再由两个小的穿梭质粒分别提供右臂改造的Hexon/E3/Fiber序列和左臂肿瘤特异性启动子控制的E1a表达框，避开了对腺病毒骨架大载体改造的困难和障碍。经过两轮位点特异性重组，准确快速地包装出理想的溶瘤腺病毒。将病毒重组包装过程简单化、快速化，解决了腺病毒骨架大载体改造的挑战性难题，能快速重组并筛选到需要的溶瘤腺病毒，非常适合产业化需要，所包装的溶瘤腺病毒具备显著的抗肿瘤活性。

Description

一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用 技术领域
本发明涉及生物医学技术领域，具体地说，涉及一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用。
背景技术
恶性肿瘤已成为影响全人类生命的一大类疾病。2000年全球新发肿瘤病例约1000万人，死亡620万人，到2018年全球新发肿瘤病例已增至1810万，死亡达960万人(CA Cancer J Clin.2018 Nov；68:394-424)。我国2000年肿瘤发病人数约180-200万人，死亡140-150万人，而到2014年新增病例数占380.4万人，死亡占229.6万人，肿瘤已占死因的首位(Chin J Cancer Res.2018 Feb；30:1-12)。目前对恶性肿瘤的治疗除了常规的手术、放疗、化疗治疗手段以外，近几年发展起来的肿瘤免疫治疗有了突飞猛进的发展，特别是免疫检查点治疗如PD-1/PD-L1抗体和免疫细胞治疗如CAR-T，成为继放疗、化疗、手术治疗之后综合治疗中不可或缺的一部分。以CAR-T细胞、PD-1/PD-L1抗体为代表的免疫疗法已在临床应用，展示出强大疗效和巨大市场价值(Nat Biotechnol.2018 Oct；36:847-856；Cell.2018 Oct 4；175:313-326)。2017年8月，诺华公司治疗B细胞急性淋巴白血病的CAR-T产品Kymriah被FDA批准上市，定价47.5万美元。2个月后，FDA又批准Kite公司治疗大B细胞淋巴瘤的CAR-T药物Yescarta上市，定价37.3万美元。近来科学家又发现，多种能够作为CAR-T细胞、PD-1/PD-L1抗体免疫疗法增效剂的溶瘤病毒产品，不仅能增强放化疗作用，同样能提高抗体、免疫细胞治疗效果。同时，这些溶瘤病毒感染的肿瘤细胞能释放大量细胞因子，裂解的肿瘤细胞释放大量肿瘤相关抗原，兼有免疫激活作用，因此与CAR-T细胞、PD-1/PD-L1抗体联合，有利于免疫治疗效果的放大，溶瘤病毒产品应用范围也得以拓宽。
溶瘤病毒(oncolytic virus,OV)是经过人工基因改造的一类病毒，能够特异性地在肿瘤细胞中大量复制，而不影响正常细胞，大量复制的病毒溶解破坏肿瘤细胞，释放出病毒粒子继续感染破坏更多的肿瘤细胞。以溶瘤病毒作为载体携带抗癌基因，随着病毒的复制及扩散，其所携带的抗癌基因拷贝数随之增加，基因蛋白产物表达量增加，抗癌效果与溶瘤效应协同，进一步增强抗癌疗效。相比于CAR-T细胞治疗，溶瘤病毒适应症更广；相比于单抗药物，溶瘤病毒价格更低廉。溶瘤病毒介导的基因治疗对肿瘤细胞杀伤效率高、靶向性好、安全性高、副作用小和成本低廉，使其成为继三大常规治疗方法(手术、放疗和化疗)和免疫细胞治疗之后的又一重要的新兴肿瘤治疗模式，有望成为 肿瘤综合治疗协同提高疗效的重要辅助手段。2017年9月医学顶级期刊《Cell》报道一项重大突破，来自美国、瑞士、西班牙和澳大利亚的研究人员，在一项针对21名黑色素瘤患者的Ib期临床试验中，研究了溶瘤病毒疗法与PD-1单抗免疫治疗联合的疗效，结果提示溶瘤病毒能有效改善免疫疗法的疗效，总体反应率高达62％，联合治疗明显比单独治疗表现得更好，提示溶瘤病毒作为肿瘤综合治疗的方法之一，具有非常美好的前景(Cell.2017 Sep 7；170:1109-1119.e10)。而溶瘤病毒的里程碑事件是2015年Amgen公司的溶瘤病毒Talimogene laherparepvec(T-Vec)被美国和欧盟批准上市，T-Vec表达粒细胞-巨噬细胞集落刺激因子(GM-CSF)，通过直接注射入肿瘤，在肿瘤细胞中复制直至引起细胞裂解(溶瘤作用)，同时在肿瘤组织局部释放GM-CSF，激活系统性免疫反应。T-Vec通过引发肿瘤细胞溶解和激发全身抗肿瘤免疫反应两种重要且协同的方式发挥作用(Lancet Oncol.2016 Nov；17:1485-1486)。仅针对黑色素患者的治疗市场，汤森路透预计到2020年T-Vec销售额为3.88亿美元/年。
OV一直是抗肿瘤药研发热点，也是资本市场和制药集团关注热点。2018年初，默沙东公司和Viralytics公司宣布，默沙东以3.94亿美元价格收购Viralytics，相当于Viralytics股票一个月成交量加权平均价格的160％溢价，默沙东获得Viralytics研发的溶瘤病毒免疫疗法
Figure PCTCN2020070125-appb-000001
(CVA21)的全部权利。
Figure PCTCN2020070125-appb-000002
是基于Viralytics专有的溶瘤病毒(Coxsackievirus TypeA21)制剂，已被证明可优先感染并杀死癌细胞。CAVATAK目前正处于多个I期和II期临床试验，作为肿瘤内静脉注射剂，考虑到默沙东正在进行的PD-1药物
Figure PCTCN2020070125-appb-000003
的试验，CAVATAK下一步或与其PD-1药物
Figure PCTCN2020070125-appb-000004
联合使用，用于黑色素瘤、前列腺癌、肺癌和膀胱癌的治疗。2011年生物技术巨头安进公司以4.25亿美元收购BioVex公司研发的OV药物T-Vec，2015年FDA批准T-Vec上市，同年12月取得欧盟CHMP许可，成为OV治疗肿瘤里程碑。目前，T-Vec已在美国、欧洲和澳大利亚广泛用于复发黑色素瘤治疗，平均成本65000美元，汤森路透预计到2020年T-Vec销售额为3.88亿美元/年。进入ΙΙΙ期临床的OV产品还有Oncolytics公司治疗头颈部肿瘤的Reolysin、ColdGenesys公司治疗膀胱癌的CG0070、Advantagene公司治疗前列腺癌的Prost Atak TM、Jennerex公司治疗肝癌的PexaVec。OV是一个广谱抗肿瘤药，目前进入临床试验的OV治疗的肿瘤包括黑色素瘤、头颈部癌、前列腺癌、膀胱癌、胶质母细胞瘤、肝癌、乳腺癌、非小细胞肺癌、结肠癌、卵巢癌等，其中，前六种肿瘤将是最近几年的主要市场。据北京欧立信信息咨询有限公司预测，针对头颈部肿瘤、前列腺癌、膀胱癌、胶质细胞瘤和肝癌即将上市的OV产品，分别有11.7、71.5、29.25、22.75和48.64亿美元的年销售额。OV更大的应用价值是与放化疗联合，Oncolytics公司的Reolysin与紫杉 醇/卡铂联合治疗晚期头颈部癌，应答率为42％，而单用化疗仅为3-10％。因此，考虑OV可以和其他药物联合应用，其市场空间更加巨大。
我国在OV领域也取得不少成绩，国家科技部新药创制重大专项连续将OV研发立为支持项目，2005年CFDA抢先批准上海三维生物技术有限公司研发的溶瘤腺病毒“H101”(商品名：安柯瑞)一类新药许可，成为全球第一个上市的OV药物。2016年底，国内恒瑞医药与日本Oncolys BioPharma公司达成协议，以1.02亿美元获得Oncolys研发的溶瘤腺病毒产品Telomelysin TM(OBP-301)在中国大陆、香港和澳门的开发、生产及商业化的独家许可权。
OV是为解决基因治疗中抗癌基因表达量低及载体不能靶向肿瘤细胞等缺点而开始研发的。以溶瘤病毒为载体介导肿瘤的基因治疗，则能够靶向肿瘤细胞，产生溶瘤效应，并与基因治疗产生协同抗癌效应，具有明显的应用价值。理论上，很多种类的病毒都可以被改造成溶瘤病毒，如T-Vec由单纯疱疹病毒改造而来，ProstAtak和G0070是腺病毒，Reolysin是呼肠孤病毒，JX-594是牛痘病毒。腺病毒是研究最广泛深入的类型，形成溶瘤病毒的品种也最多。腺病毒种类多，结构复杂，目前已发现有100余个血清型，其基因组为线状双链DNA分子，大约35～36kb。与其他类型的病毒相比，腺病毒的优点有：几乎能感染所有类型的细胞，能在宿主细胞中有效进行增殖；病毒基因组不整合到宿主细胞染色体中，无插入致突变性；滴度高，易于制备和纯化等，最适宜开发安全的OV产品。但腺病毒作为溶瘤病毒，也存在一些缺点：如自身免疫原性强、特异性差和静脉注射易在肝脏富集等，因此需要对其进行进一步改造。这些复杂性特点正好成为我们进行靶向性和有效性改造的最佳机会，更有利于通过全方位的改造研发出多机制协同抗癌的高效溶瘤腺病毒(OAV)产品，并能根据肿瘤类型特征设计构建个性化的OAV。对腺病毒的改造主要集中于提高靶向肿瘤细胞的特异性、感染细胞的转染率、装载的抗癌基因的表达量、以及避免机体免疫系统对病毒的清除(Mol Cancer Ther.2016 Jul；15:1436-51)。
OAV构建以致病力最弱的人类腺病毒C亚属中的5型(Ad5)为基础，主要分为基因组左臂E1区的改造和右臂外壳蛋白、纤毛蛋白、E3区的改造。
1.腺病毒基因组左臂功能区改造
构建OAV，需要保留E1a蛋白表达并使其受肿瘤特异性启动子调控，实现肿瘤特异性复制。E1a有3个功能区，即CR1、CR2、CR3，CR1区通过与转录调节因子P300/CBP结合能抑制Her-2/neu基因的表达，CR2区与Rb蛋白家族结合，CR3区是转录活化区。因此E1a蛋白具有抗肿瘤作用，既可以抑制Her-2/neu基因的转录、阻断NF-κB的活性、 提高p53的表达，又可以抑制Ⅳ型胶原酶、纤溶酶原激活剂等蛋白酶基因表达；E1a还能引起非特异性免疫反应，提高CTL细胞、NK细胞、巨噬细胞的杀伤效应等多种途径，诱导肿瘤细胞凋亡，抑制肿瘤侵袭转移，提高肿瘤细胞对化疗、放疗的敏感性。对E1a的CR2区引入缺失突变，使其不能结合Rb蛋白，保证去磷酸化的Rb蛋白与转录因子E2F形成复合物，阻断E2F的转录活性，可以增强抗癌活性。改造E1a实现其抗癌活性、阐明其功能作用的分子机制和信号通路，还有很多问题需要深入研究，特别是OAV感染癌细胞释放大量细胞因子，裂解癌细胞释放大量肿瘤相关抗原，发挥进一步的免疫激活作用，因此OAV最大的应用价值是与免疫治疗的联合应用。
Elb转录单元编码Elb-55kDa和Elb-19kDa。Elb-55kDa是腺病毒在正常细胞增殖复制必需的而在肿瘤细胞中不必需的蛋白，Elb-55kDa编码基因的选择性缺失可以使腺病毒在肿瘤细胞内保持增殖复制的能力，而在正常细胞中失去复制能力。Elb-55kDa蛋白能够灭活并降解P53蛋白，Elb-55kDa缺失有利于细胞保持P53的抗肿瘤活性，同时提高病毒载体的靶向性。Elb-19kDa基因与凋亡抑制基因Bcl-2同源，Elb-19kDa蛋白能够结合Bax或/和Bak启动下游的凋亡抑制程序，保护感染的细胞免受TNF-α介导的杀伤作用，Elb-19kDa缺失，因而该病毒突变体在肿瘤细胞内增殖的特异性得以提高，而在正常细胞内的增殖活性被削弱。Elb-19kDa缺失能够促进癌细胞凋亡通路的恢复，且有利于病毒在正常细胞内的快速清除以及在肿瘤细胞内的快速释放和播散，使OAV特异性更好，效能更强。但Elb-55kDa和Elb-19kDa双缺失是否影响OAV增殖力、通过对两者的改造能否产生免疫增强作用，需要进一步研究。
2.腺病毒基因组右臂功能区改造
腺病毒基因组右臂E3转录单位有9个开放读框，编码蛋白保护感染细胞免受宿主免疫反应的杀伤，E3-gpl9k可减弱CTL介导的感染细胞杀伤效应；RID可阻断“死亡”配体包括TNF、Fas配体和TRAIL，介导的细胞凋亡；RID还可抑制IL-1和TNF介导的细胞存活所必须的NF-κB的激活；E3-6.7k除了使TRAIL受体下调之外还可单独抑制外部和内部信号通路诱导的细胞凋亡；E3-14.7k是TNF介导细胞凋亡的广泛抑制剂，也可结合并抑制Caspases-8从而阻止Fas信号通路启动的细胞凋亡；E3-14.7k与受感染的细胞内FIP蛋白(for 14.7K-interacting protein，FIP-1、-2、-3)作用，可使E3-14.7k蛋白在细胞凋亡和存活、炎症反应、维持膜稳定、核浆转运等信号转导通路中处于重要地位，这一多功能的E3蛋白的分子机制仍需深入研究。腺病毒死亡蛋白(ADP)可促进溶解细胞、病毒释放，然而分子机制不明。由此可见，在OAV构建过程中删除E3区，既可以扩大载体容量，又能够促进受感染的癌细胞的凋亡。但E3缺失也解除了病毒对机体的免疫抵 抗，病毒可被快速清除。因此有必要加强对E3区编码蛋白的认识，在OAV构建中避免盲目删除全部E3区，可能更有利于目的基因的长期表达。
人类腺病毒家族有52个血清型，分6个亚属(A至F)。除了B组以外，各组腺病毒都用Coxsackievirus-adenovirus receptor(CAR)作为其主要吸附受体，对缺乏CAR的造血细胞、造血干细胞、树突状细胞、部分肿瘤细胞尤其是肿瘤干细胞等感染效率很低。B组腺病毒(Ad3、Ad11b、Ad14、Ad16、Ad21、Ad35、Ad50)则主要识别一种广泛表达的补体调节蛋白CD46。采用B组腺病毒的纤毛蛋白(fiber knob)替代Ad5纤毛蛋白，构建嵌合病毒，则有利于提高病毒对肿瘤细胞转导效率，特别是感染肿瘤干细胞的能力，有可能更彻底地根除肿瘤复发的根源。
Ad5在自然界存在较广，大多数人已被感染并产生了中和抗体，能够拦截病毒。况且Ad5具有嗜肝细胞性，能够被肝细胞吸附。腺病毒外壳蛋白Hexon的高变区(HVR)因位置暴露于腺病毒表面，是导致不同血清型腺病毒之间对肝脏感染能力和免疫原性差异的关键部位。将Ad5的Hexon分子内7个HVR选择性地与稀有血清型如D亚群(Ad37、Ad43)、B亚群(Ad48)病毒的Hexon相应区域进行嵌合，是帮助Ad5逃避预存免疫和肝脏摄取的有效方法。然而，Hexon作为腺病毒的主要结构蛋白，对其进行改造往往会导致腺病毒载体结构不稳定，从而无法有效地包装病毒，因此这是一项很具有挑战性的研究。
3.腺病毒重组包装技术
目前包装腺病毒采用的最多的系统是Microbix Biosystems公司包装技术，以腺病毒左臂质粒pDC系列(如pDC315、pDC316、pDC312等)和完全删除E1/E3区的左臂骨架质粒(pBHGloxdelE13cre)进行重组，这个技术市场上用了近20年，而且现在一直还在用，产生的病毒都是缺少全部E3区的。实现E1a肿瘤特异性增殖以及外源抗癌基因表达都要靠pDC质粒携带，重组到腺病毒E1区。这样一来，左臂插入片段大小受限，而且E1a的启动子和抗癌基因启动子都在E1区，相距太近，互相干扰，表达效率都有下降。我们前期尝试用绝缘子序列隔开，虽有改善，但效果不明显。曾有人尝试将外源抗癌基因插入到E3区，外源基因表达效率较理想，但右臂质粒较大，以pBHGloxdelE13cre为例大小为34.707kb，做一次大载体的改造难度较大，成功率也不高，往往需要反复尝试很多次才能筛选到正确的载体。
对于构建新型的OAV来说，为了提高OAV靶向肿瘤细胞的特异性、感染细胞的转染率、装载的抗癌基因的表达量、以及避免机体免疫系统对病毒的清除，需要对右臂骨架质粒进行较大范围改造，不但要在不同的血清型腺病毒之间筛选能增强腺病毒感染效 率的Fiber分子、筛选能使病毒逃避机体免疫拦截和肝脏摄取的Hexon分子、以及筛选不同机制不同功能的多种外源基因插入E3区，每一个分子的筛选和改造都要重构一次右臂骨架大载体，且从Hexon、E3区到Fiber，基因组跨度达14kb，工作量大，难度高，费时耗力。如2013年博士论文“靶向胃癌相关成纤维细胞的纤毛蛋白修饰、六联体嵌合型溶瘤腺病毒载体的实验研究”，以嵌合型六联体基因替换原有的六联体基因，得到含有高变区的嵌合型六联体的腺病毒骨架质粒载体，即存在上述技术问题。为了解决OAV构建包装的技术难题，我们研发出一套快速准确的三质粒溶瘤腺病毒重组系统Ad5MixPlus，并在实际应用中得到证实，能够省时省力、准确快速地包装重组出理想的OAV新产品。
发明内容
实现腺病毒靶向肿瘤细胞增殖并产生溶瘤作用，以及介导抗癌基因高效表达，需要对腺病毒进行大范围的改造，几乎涉及到整个基因组，主要是提高靶向肿瘤细胞的特异性、感染细胞的转染率、装载的抗癌基因的表达量、以及避免机体免疫系统对病毒的清除。以往的OAV改造及重组，多集中于腺病毒基因组左臂E1功能区，外源抗癌基因也插入在E1区，E1a的肿瘤特异性启动子和抗癌基因启动子都在E1区，相距太近，互相干扰，表达效率都有下降。如果要想进一步优化并提高OAV的感染细胞的转染率、装载的抗癌基因的表达量、以及避免机体免疫系统对病毒的清除，则需要在不同血清型腺病毒之间筛选并建立Fiber嵌合和Hexon嵌合的杂合OAV，并根据肿瘤类型以及治疗需要优选多种抗癌基因装载到OAV的E3区，则要对腺病毒右臂骨架质粒的结构蛋白包括Hexon、Fiber、E3区等进行大范围多层次改造。但右臂质粒较大，以最常用的Microbix Biosystems公司的右臂骨架质粒pBHGloxdelE13cre为例大小为34.707kb，每一次改变每一个蛋白分子都要进行一次大载体构建。由于大载体改造难度较大，出错率极高，筛选难度大，因此费时费力费钱，往往需要反复尝试很多次才能筛选到正确的载体。
为解决上述技术问题，本发明提供了一种快速准确的三质粒溶瘤腺病毒重组包装系统，其包含以下3个腺病毒重组质粒：
a)腺病毒右臂骨架质粒：所述腺病毒右臂骨架质粒上装载两套不同的位点重组序列，一套attL/attR在Fiber/Hexon/E3区，一套Cre/loxP在E1区；E3区还插入了DB3.1大肠杆菌菌株及感受态ccdB致死基因；
b)腺病毒右臂穿梭质粒：所述腺病毒右臂穿梭质粒含有已改建好的嵌合型Hexon序列和嵌合型Fiber序列，中间E3区预置外源基因多克隆位点，Hexon/E3/Fiber序列两端含有attL1/attL2重组位点；
c)腺病毒左臂穿梭质粒：所述腺病毒左臂穿梭质粒在其多克隆位点插入肿瘤特异性 启动子控制的腺病毒早期增殖基因和loxP重组位点；
其中，所述腺病毒右臂穿梭质粒通过Hexon/E3/Fiber序列两端的attL1/attL2与所述腺病毒右臂骨架质粒发生第一轮attL/attR位点特异性重组，所述腺病毒右臂穿梭质粒中attL1/attL2间的序列置换所述腺病毒右臂骨架质粒中attR1/attR2间的序列；所述腺病毒左臂穿梭质粒与所述腺病毒右臂骨架质粒发生第二轮Cre/loxP位点特异性重组，所述腺病毒左臂穿梭质粒中的肿瘤特异性启动子控制的E1a表达框插入到所述腺病毒右臂骨架质粒缺失的E1区；经过两轮位点特异性重组，包装出所需要的溶瘤腺病毒。
作为本发明的一个优选例，所述嵌合型Hexon序列为Ad5与Ad48、Ad9、Ad37、Ad43及其它任一血清型腺病毒Hexon及其突变序列的嵌合。
作为本发明的另一优选例，所述嵌合型Hexon序列如SEQ ID NO:5所示。
作为本发明的另一优选例，所述嵌合型Fiber序列为Ad5与Ad11b、Ad3、Ad14、Ad16、Ad21、Ad35、Ad50、Ad55及其它任一血清型腺病毒Fiber及其突变序列的嵌合。
作为本发明的另一优选例，所述嵌合型Fiber序列如SEQ ID NO:6所示。
作为本发明的另一优选例，所述肿瘤特异性启动子选自：(a)癌胚抗原启动子、增强子及其突变体序列；(b)甲胎蛋白启动子、增强子及其突变体序列；(c)人表皮生长因子受体家族(EGFRs)的受体酪氨酸激酶(包括EGFR、Her-2、Her-3和Her-4)启动子、增强子及其突变体序列；(d)乳腺癌相关抗原DF3/MUC1启动子、增强子及其突变体序列；(e)血管内皮生长因子(VEGF)受体KDR的启动子、增强子及其突变体序列；(f)L-plastin启动子、增强子及其突变体序列；(g)凋亡抑制蛋白家族(IAP)成员的启动子、增强子及其突变体序列；(h)前列腺素特异性抗原的启动子、增强子及其突变体序列；(i)缺氧诱导因子-1(HIF-1)调控的缺氧反应元件保守序列；(j)转录因子E2F启动子、增强子及其突变体序列；(k)hTERT启动子、增强子及其突变体序列。
作为本发明的另一优选例，所述肿瘤特异性启动子的序列如SEQ ID NO:7所示。
作为本发明的另一优选例，所述腺病毒早期增殖基因包括E1a和E1b，其中E1a选自E1a野生序列及其突变序列，所述E1b选自Elb-55kDa、Elb-19kDa及其突变序列。
作为本发明的另一优选例，所述E1a表达框的序列如SEQ ID NO:8所示。
作为本发明的另一优选例，所述腺病毒右臂骨架质粒的序列如SEQ ID NO:1所示，所述腺病毒右臂穿梭质粒的序列如SEQ ID NO:2所示，所述腺病毒左臂穿梭质粒的序列如SEQ ID NO:3所示。
作为本发明的另一优选例，所述溶瘤腺病毒的序列如SEQ ID NO:4所示。
本发明还提供了所述的三质粒溶瘤腺病毒重组包装系统在制备溶瘤腺病毒或抗肿瘤 药物中的应用。
作为本发明的一个优选例，所述肿瘤选自消化系统肿瘤如食道癌、胃癌、结直肠癌、肝癌、胰腺癌、胆管及胆囊癌；呼吸系统肿瘤如肺癌、胸膜瘤；血液系统肿瘤如白血病、淋巴瘤、多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌、卵巢癌、宫颈癌、外阴癌、睾丸癌、前列腺癌、阴茎癌；神经系统肿瘤如胶质瘤、神经母细胞瘤、脑膜瘤；头颈部肿瘤如口腔癌、舌癌、喉癌、鼻咽癌；泌尿系统肿瘤如肾癌、膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤、脂肪肉瘤、甲状腺癌。
本发明优点在于：
本发明提供了一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus及其应用。该系统由3个腺病毒重组质粒组成，其核心技术就是巧妙地在第一个5型腺病毒右臂骨架质粒pAd5MixPlus上装载两套不同的位点重组序列，一套attL/attR在Fiber/Hexon/E3区，一套Cre/loxP在E1区。第二个腺病毒右臂穿梭质粒pAdH548F511LR含有Fiber/Hexon/E3改造序列，E3区还预置外源基因多克隆插入位点，该质粒将通过Hexon/E3/Fiber序列两端的attL1/attL2与腺病毒右臂骨架质粒pAd5MixPlus在细菌内发生第一轮attL/attR位点特异性重组，pAdH548F511LR中attL1/attL2间的序列置换pAd5MixPlus中attR1/attR2间的序列，由pAd5MixPlus中E3区的ccdB基因进行精准筛选；第三个腺病毒左臂穿梭质粒pAdSVPcreLoxP含有肿瘤特异性启动子控制的E1a表达框和loxP重组位点，该质粒将与pAd5MixPlus在真核细胞内发生第二轮Cre/loxP位点特异性重组，pAdSVPcreLoxP中肿瘤特异性启动子控制的E1a表达框插入到pAd5MixPlus缺失的E1区。经过两轮位点特异性重组，准确快速地包装出理想的溶瘤腺病毒。该系统所有序列改造均在两个小的穿梭质粒上进行，病毒肿瘤特异性增殖的E1区改造在左臂穿梭质粒上进行，病毒Hexon、Fiber、E3区蛋白结构改造以及E3区插入外源基因均在右臂穿梭质粒上进行，避开了对腺病毒骨架大载体改造的困难和障碍。根据实际需要，外源基因可以装在到任意一个穿梭质粒，分别插入到溶瘤腺病毒的E1区和E3区。因此，我们最新一代Ad5MixPlus重组系统，将病毒重组包装过程简单化、快速化、准确化，解决了腺病毒骨架大载体改造的挑战性难题，能快速重组并筛选到我们需要的溶瘤腺病毒，非常适合产业化需要。
进一步的实验表明，本发明所包装的溶瘤腺病毒在肿瘤中特异性增殖活性很强，对肿瘤细胞的杀伤活性很强，能明显抑制肿瘤体积的增加。
附图说明
图1.腺病毒右臂骨架质粒pAd5MixPlus结构图。
图2.腺病毒右臂穿梭质粒pAdH548F511LR结构图。
图3.腺病毒左臂穿梭质粒pAdSVPcreLoxP结构图。
图4.Ad5MixPlus系统重组包装溶瘤腺病毒AdSVPH548F511流程图。
图5.溶瘤腺病毒AdSVPH548F511结构图。
图6.TCID50法检测溶瘤腺病毒AdSVPH548F511的增殖活性。
图7.溶瘤腺病毒AdSVPH548F511对肿瘤细胞的杀伤活性，(A)MTT法，(B)RTCA法。
图8.溶瘤腺病毒AdSVPH548F511对肾癌移植瘤的抑制作用。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1 本发明的一套快速准确的三质粒溶瘤腺病毒重组包装系统Ad5MixPlus
本发明所述的一套快速准确的三质粒溶瘤腺病毒重组系统Ad5MixPlus，由3个腺病毒重组质粒组成。这3个腺病毒重组质粒分别是Ad5MixPlus系统右臂骨架质粒、Ad5MixPlus系统右臂穿梭质粒和Ad5MixPlus系统左臂穿梭质粒。现介绍这3个腺病毒重组质粒及Ad5MixPlus的重组包装，具体如下：
1.Ad5MixPlus系统右臂骨架质粒
在第一个5型腺病毒右臂骨架质粒pAd5MixPlus上装载两套不同的位点重组序列，一套attL/attR在Fiber/Hexon/E3区，一套Cre/loxP在E1区(图1)。E3区还插入了DB3.1大肠杆菌菌株及感受态ccdB致死基因，用于筛选重组成功的病毒克隆，重组成功的载体，失去了attR1和attR2位点间的ccdB基因，感受态细菌存活下来，出现克隆生长；重组不成功的载体，在其原有的ccdB基因产物作用下，细菌死亡。
腺病毒右臂骨架质粒pAd5MixPlus的全长序列为：SEQ ID NO:1。
2.Ad5MixPlus系统右臂穿梭质粒
第二个腺病毒右臂穿梭质粒pAdH548F511LR含有已改建好的Ad5H48嵌合型Hexon序列和Ad5F11b嵌合型Fiber序列，中间E3区预置外源基因多克隆位点，Hexon/E3/Fiber序列两端含有attL1/attL2重组位点(图2)。
腺病毒右臂穿梭质粒pAdH548F511LR的全长序列为：SEQ ID NO:2。
3.Ad5MixPlus系统左臂穿梭质粒
第三个腺病毒左臂穿梭质粒pAdSVPcreLoxP在其多克隆位点插入肿瘤特异性Survivin启动子控制的E1a表达框和loxP重组位点(图3)。
腺病毒左臂穿梭质粒pAdSVPcreLoxP的全长序列为：SEQ ID NO:3。
4.Ad5MixPlus系统重组包装程序
第二个腺病毒右臂穿梭质粒pAdH548F511LR通过Hexon/E3/Fiber序列两端的attL1/attL2与腺病毒右臂骨架质粒pAd5MixPlus在DB3.1大肠杆菌感受态细菌内发生第一轮attL/attR位点特异性重组，pAdH548F511LR中attL1/attL2间的序列置换pAd5MixPlus中attR1/attR2间的序列；第三个腺病毒左臂穿梭质粒pAdSVPcreLoxP与pAd5MixPlus在真核细胞内发生第二轮Cre/loxP位点特异性重组，pAdSVPcreLoxP中的肿瘤特异性启动子控制的E1a表达框插入到pAd5MixPlus缺失的E1区。经过两轮位点特异性重组，准确快速地包装出理想的溶瘤腺病毒AdSVPH548F511。Ad5MixPlus系统重组包装程序如图4所示，溶瘤腺病毒AdSVPH548F511如图5所示。
本发明所述的溶瘤腺病毒AdSVPH548F511，其完整序列为：SEQ ID NO:4。
需要说明的是：
1.嵌合型Hexon序列改造
第二个腺病毒右臂穿梭质粒pAdH548F511LR含有已改建好的Ad5H48嵌合型六邻体(Hexon)编码序列。Hexon高变区(HVR)因位置暴露于腺病毒表面，是导致不同血清型的腺病毒之间对肝脏感染能力和免疫原性差异的关键部位。利用基因工程改造腺病毒载体，将Ad5表面Hexon的7个HVR选择性地与稀有血清型病毒Hexon的相应区域嵌合，是帮助腺病毒逃避预存免疫、避免肝脏吸附的有效方法。本发明所述Ad5H48嵌合型Hexon，由D亚群48型腺病毒的HVR替换Ad5的相应序列，Ad48在人群中普遍缺乏中和抗体、且肝脏亲嗜性弱。因此，用Ad48的HVR替换Ad5的相应部分，构建Ad5和Ad48嵌合型Hexon的腺病毒，能够避免中和抗体拦截和肝脏摄取，提高病毒存活能力。本发明所述嵌合型Hexon，还包括Ad5与Ad9、Ad37、Ad43等其他任何血清型腺病毒Hexon及其突变序列的嵌合。
Ad5H48嵌合型Hexon完整序列如下：SEQ ID NO:5。
2.嵌合型Fiber序列改造
第二个腺病毒右臂穿梭质粒pAdH548F511LR含有已改建好的Ad5F11b嵌合型Fiber编码序列。人类腺病毒家族有51个已知血清型，分为6个亚属(A至F)。除了B组以外，各组腺病毒都用CAR作为其主要吸附受体(Ad5属于C组)。B组腺病毒又进一步分为B1和B2亚组，Ad11b、Ad14、Ad35为B2组腺病毒；Ad3、Ad16、Ad21、Ad50为B1组腺病毒。近年来，B组腺病毒的衍生体作为颇具吸引力的基因治疗载体而受到关注，因为它们可以转导造血细胞、造血干细胞、树突状细胞(DC细胞)和恶性肿瘤细胞等靶细胞，而这些细胞往往不易被常用的腺病毒载体(如Ad5)感染。与许多腺病毒是 通过CAR受体来感染细胞不同的是，B组腺病毒用CD46作为吸附受体。CD46是一种广泛表达的补体调节蛋白，这种蛋白几乎在所有的人类体细胞表面都存在。Ad5F11b嵌合型Fiber，即用Ad11b的纤突结(fiber knob)替代5型腺病毒的fiber相应序列，因而使该嵌合病毒对造血细胞、干细胞、肿瘤细胞具有高转导特点。本发明所述嵌合型Fiber，还包括Ad5与Ad3、Ad14、Ad16、Ad21、Ad35、Ad50、Ad55等其他任何血清型腺病毒Fiber及其突变序列的嵌合。
Ad5F11b嵌合型Fiber完整序列如下：SEQ ID NO:6。
3.肿瘤特异性启动子
第三个腺病毒左臂穿梭质粒pAdSVPcreLoxP在其多克隆位点插入肿瘤特异性Survivin启动子控制的E1a表达框。Survivin启动子因其特异性高、肿瘤谱广而受到更多关注。Survivin在正常组织中几乎不表达，而在恶性肿瘤中的表达具有高度选择性，在大多数肿瘤如肺癌、肝癌、结肠癌、胰腺癌、前列腺癌和乳腺癌中都呈高表达，并且与肿瘤的复发转移以及患者的预后不佳密切相关，使其成为肿瘤基因治疗广谱的分子靶标。Survivin启动子调控的溶瘤腺病毒能够靶向癌细胞增殖复制并裂解癌细胞，同时介导抗肿瘤目的基因高效表达。因此，以Survivin启动子调控的溶瘤腺病毒，有望实现针对大多数人体肿瘤的广谱而安全的抗癌效应。本发明所述的肿瘤特异性启动子，除Survivin启动子以外，还包括：(a)癌胚抗原启动子、增强子及其突变体序列；(b)甲胎蛋白启动子、增强子及其突变体序列；(c)人表皮生长因子受体家族(EGFRs)的受体酪氨酸激酶(包括EGFR、Her-2、Her-3和Her-4)启动子、增强子及其突变体序列；(d)乳腺癌相关抗原DF3/MUC1启动子、增强子及其突变体序列；(e)血管内皮生长因子(VEGF)受体KDR的启动子、增强子及其突变体序列；(f)L-plastin启动子、增强子及其突变体序列；(g)凋亡抑制蛋白家族(IAP)成员的启动子、增强子及其突变体序列；(h)前列腺素特异性抗原的启动子、增强子及其突变体序列；(i)缺氧诱导因子-1(HIF-1)调控的缺氧反应元件保守序列；(j)转录因子E2F启动子、增强子及其突变体序列；(k)hTERT启动子、增强子及其突变体序列。
本发明所述的肿瘤特异性Survivin启动子，其编码序列如下：SEQ ID NO:7。
4.腺病毒早期增殖基因E1a的调控
第三个腺病毒左臂穿梭质粒pAdSVPcreLoxP在其多克隆位点插入肿瘤特异性Survivin启动子控制的E1a表达框。将腺病毒早期增殖基因E1a置于肿瘤特异性启动子调控下，实现病毒的肿瘤特异性复制及溶瘤作用。本发明所述腺病毒早期增殖基因，除E1a野生序列以外，还包括对E1a进行的突变改造，以及Elb-55kDa、Elb-19kDa及其突 变序列。
本发明所述的肿瘤特异性启动子控制的E1a表达框，其E1a编码序列如下：SEQ ID NO:8。
实施例2 溶瘤腺病毒AdSVPH548F511的特异性增殖活性
收集对数生长期的肝癌细胞(HCCLM3、HepG2、Huh-7、MHCC97H、MHCC97L)和正常肝细胞(WRL-68)以及正常成纤维细胞(BJ)，计数，铺96孔板，1×10 4/孔，细胞贴壁后，换用无血清培养液；以MOI＝1加入溶瘤腺病毒AdSVPH548F511。病毒感染2h后调节至5％血清培养液(此时作为感染起始时间0h)，继续培养48h、96h，分别在这三个时间段收集细胞，TCID50方法检测病毒滴度。结果显示：AdSVPH548F511在肝癌中特异性增殖复制的能力非常强，48h时增殖倍数均在10000倍以上，最高达50000倍以上；96h后可达100000至800000倍以上。而AdSVPH548F51在正常细胞中WRL-68、BJ中有轻度增殖，96h时最高仅200倍以下(图6)。
实施例3 溶瘤腺病毒AdSVPH548F511对肿瘤细胞的杀伤活性
收集对数生长期的肝癌细胞(HepG2、MHCC97H)和正常肝细胞(L02)以及正常成纤维细胞(BJ)，计数，铺96孔板，1×10 4/孔，细胞贴壁后，换用无血清培养液。通过四唑盐比色实验(MTT)检测病毒AdSVPH548F511对细胞存活率的影响。Cell Proliferation Kit I(MTT)购于Roche Diagnostics GmbH。按梯度MOI加入病毒，对应于每个病毒设8个复孔，孵箱内培养2h；换血清培养液100μl/孔，培养48h后，弃培养液，加入0.1mol/L PBS溶液100μl/孔，再加MTT labeling reagent 10μl/孔至终浓度0.5mg/ml，置孵箱内4h；加Solubilization solution(10％SDS in 0.01mol/L HCl)100μl/孔，置孵箱内过夜；采用Model 550 Microplate Reader(BIO-RAD)测定570nm波长光吸收值，校正波长为655nm；绘制生存曲线，计算IC50值。结果显示：AdSVPH548F511对HepG2和MHCC97H杀伤活性很强，IC50值分别为35.16和212.4；AdSVPH548F511对正常BJ细胞无明显影响，IC50值为20035。可见溶瘤腺病毒AdSVPH548F511具有特异性杀伤破坏癌细胞的能力(图7中A)。
我们同时以实时无标记动态细胞分析技术(Real Time Cellular Analysis，RTCA)对AdSVPH548F511的细胞杀伤活性进行实时、动态、定量检测。向E-Plate检测板中加入培养基并测定背景阻抗值；收集对数生长期的肝癌细胞(HepG2)和正常肝细胞(L02)，计数，加入到E-Plate检测板中，室温超净台内放置30min；以无血清培养液稀释病毒并按MOI＝5、10、20的梯度加入病毒；将加入细胞的E-Plate检测板放入预先放入培养箱中的检测台上，进行实时动态的细胞增殖检测，绘制细胞实时动态生长曲线。结果发现， AdSVPH548F511对肝癌HepG2细胞的杀伤破坏活性，随病毒MOI的增加以及作用时间延长而增强，而AdSVPH548F511对正常肝细胞L02杀伤活性不明显，高强度感染病毒对L02细胞有一定抑制作用(图7中B)。
实施例4 溶瘤腺病毒AdSVPH548F511抗肿瘤活性的动物模型实验
健康纯种BALB/c裸鼠8只，6-8周龄，雄性，中科院上海实验动物中心提供，清洁级动物实验室饲养；取对数生长期的肾癌OSRC-2细胞，用PBS液调整细胞数至1×10 7/ml，消毒裸鼠侧腹部近腋下皮肤，取100μl细胞悬液注射于皮下，恒温、通风、无菌条件下饲养；每日定时观察瘤体生长情况，接种区皮下出现米粒大小肿瘤，即为种植成功。随机分为两组(病毒组AdSVP n＝5、对照组PBS n＝3)，编号、游标卡尺测量瘤体大小。开始治疗，以2×10 8pfu/100μl的病毒AdSVPH548F511直接瘤内多点注射，隔日一次，共5次，对照组以PBS代替病毒注射，100μl×5次；定时测量瘤体大小，以“a×b 2×0.5”公式计算瘤体体积(a:最大径，b:最小径)，绘制移植瘤生长曲线(图8)。结果显示，AdSVPH548F511能明显抑制肾癌OSRC-2移植瘤的生长。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (10)

  1. 一种快速准确的三质粒溶瘤腺病毒重组包装系统，其特征在于，其包含以下3个腺病毒重组质粒：
    a)腺病毒右臂骨架质粒：所述腺病毒右臂骨架质粒上装载两套不同的位点重组序列，一套attL/attR在Fiber/Hexon/E3区，一套Cre/loxP在E1区；E3区还插入了DB3.1大肠杆菌菌株及感受态ccdB致死基因；
    b)腺病毒右臂穿梭质粒：所述腺病毒右臂穿梭质粒含有已改建好的嵌合型Hexon序列和嵌合型Fiber序列，中间E3区预置外源基因多克隆位点，Hexon/E3/Fiber序列两端含有attL1/attL2重组位点；
    c)腺病毒左臂穿梭质粒：所述腺病毒左臂穿梭质粒在其多克隆位点插入肿瘤特异性启动子控制的腺病毒早期增殖基因和loxP重组位点；
    其中，所述腺病毒右臂穿梭质粒通过Hexon/E3/Fiber序列两端的attL1/attL2与所述腺病毒右臂骨架质粒发生第一轮attL/attR位点特异性重组，所述腺病毒右臂穿梭质粒中attL1/attL2间的序列置换所述腺病毒右臂骨架质粒中attR1/attR2间的序列；所述腺病毒左臂穿梭质粒与所述腺病毒右臂骨架质粒发生第二轮Cre/loxP位点特异性重组，所述腺病毒左臂穿梭质粒中的肿瘤特异性启动子控制的E1a表达框插入到所述腺病毒右臂骨架质粒缺失的E1区；经过两轮位点特异性重组，包装出所需要的溶瘤腺病毒。
  2. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述嵌合型Hexon序列为Ad5与Ad48、Ad9、Ad37、Ad43任一血清型腺病毒Hexon及其突变序列的嵌合。
  3. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述嵌合型Hexon序列如SEQ ID NO:5所示。
  4. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述嵌合型Fiber序列为Ad5与Ad11b、Ad3、Ad14、Ad16、Ad21、Ad35、Ad50、Ad55任一血清型腺病毒Fiber及其突变序列的嵌合。
  5. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述嵌合型Fiber序列如SEQ ID NO:6所示。
  6. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述肿瘤特异性启动子选自：(a)癌胚抗原启动子、增强子及其突变体序列；(b)甲胎蛋白启动子、增强子及其突变体序列；(c)人表皮生长因子受体家族(EGFRs)的受体酪氨酸激酶(包括EGFR、Her-2、Her-3和Her-4)启动子、增强子及其突变体序列；(d)乳腺癌相关抗原DF3 /MUC1启动子、增强子及其突变体序列；(e)血管内皮生长因子(VEGF)受体KDR的启动子、增强子及其突变体序列；(f)L-plastin启动子、增强子及其突变体序列；(g)凋亡抑制蛋白家族(IAP)成员的启动子、增强子及其突变体序列；(h)前列腺素特异性抗原的启动子、增强子及其突变体序列；(i)缺氧诱导因子-1(HIF-1)调控的缺氧反应元件保守序列；(j)转录因子E2F启动子、增强子及其突变体序列；(k)hTERT启动子、增强子及其突变体序列。
  7. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述肿瘤特异性启动子的序列如SEQ ID NO:7所示。
  8. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述腺病毒早期增殖基因包括E1a和E1b，所述E1a选自E1a野生序列及其突变序列，所述E1b选自Elb-55kDa、Elb-19kDa及其突变序列。
  9. 根据权利要求1所述的三质粒溶瘤腺病毒重组包装系统，其特征在于，所述E1a表达框的序列如SEQ ID NO:8所示。
  10. 权利要求1所述的三质粒溶瘤腺病毒重组包装系统在制备溶瘤腺病毒或抗肿瘤药物中的应用。
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