WO2022033467A1 - 构建溶瘤病毒的方法 - Google Patents

构建溶瘤病毒的方法 Download PDF

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WO2022033467A1
WO2022033467A1 PCT/CN2021/111759 CN2021111759W WO2022033467A1 WO 2022033467 A1 WO2022033467 A1 WO 2022033467A1 CN 2021111759 W CN2021111759 W CN 2021111759W WO 2022033467 A1 WO2022033467 A1 WO 2022033467A1
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virus
tumor
oncolytic
cancer
ligand
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French (fr)
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吴可行
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上海行深生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

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  • the present invention belongs to the field of oncolytic viruses, and particularly relates to a method for constructing an oncolytic virus, an oncolytic virus, and the use of the oncolytic virus in the preparation of medicine, a composition and a kit.
  • oncolytic viruses In the past decade, the mechanism by which oncolytic viruses can kill tumors by inducing the body's anti-tumor immune response has gradually become clear. Since the German scientist Jean Rommelaere first called oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic virus has been accepted by the public as an important branch of tumor immunotherapy. Compared with other tumor immunotherapies, oncolytic viruses have the advantages of high killing efficiency, good targeting, less side effects, multiple tumor killing pathways to avoid drug resistance, and low cost.
  • viruses are more likely to replicate and spread in cancer cells.
  • PLR protein kinase R
  • scientists have made use of the differences in many signaling pathways and metabolisms between cancer cells and normal cells to continuously improve oncolytic virus resistance by screening specific virus species and modifying virus genomes.
  • the targeting of tumors can reduce the harm to normal cells and improve safety.
  • the approved T-vec knocks out the ⁇ 34.5 gene of HSV-1 (herpes simplex virus type 1).
  • the ⁇ 34.5 gene can inhibit the clearance mechanism of the virus in normal cells.
  • JX594 (Pexa-Vec), currently in phase III clinical trials, has knocked out the TK (thymidine kinase) gene of vaccinia viruses, and the replication of the virus is related to the level of TK in cells, so JX594 knocked out TK is only It can replicate in cancer cells with high TK activity, but cannot replicate in normal cells (normal cells with low TK activity).
  • CG0070 adds the E2F-1 promoter before the adenovirus replication-competent gene E1A.
  • E2F-1 is regulated by retinoblastoma inhibitory protein (Rb), and Rb is deleted in bladder cancer, and the loss of Rb activates E2F-1
  • Rb retinoblastoma inhibitory protein
  • the transcriptional activity of the E1A gene allows the virus to replicate specifically in bladder cancer.
  • Reolysin is an unmodified wild-type reovirus whose proliferation depends on the activation of the Ras signaling pathway, so it can only specifically proliferate in Ras-activated cancer cells.
  • the ideal approach is to develop a method for screening oncolytic viruses with high tumor targeting and affinity, with as few changes as possible to the sequence of the oncolytic virus itself.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the purpose of the present invention is to provide a general method for rapidly screening oncolytic viruses with high tumor cell specificity and broad spectrum of tumor therapy.
  • the present invention provides a method for constructing an oncolytic virus.
  • the method includes the following steps: a. determining a tumor cell-specific receptor; b. determining a target viral ligand based on the binding force of the viral ligand to the tumor cell-specific receptor; c. and constructing an oncolytic virus comprising the target viral ligand.
  • the method according to the embodiment of the present invention can rapidly screen oncolytic viruses with high tumor cell specificity and broad spectrum of tumor treatment.
  • the above method may further include at least one of the following additional technical features:
  • the tumor cell-specific receptor is determined by determining candidate cell membrane receptors whose expression in tumor cells is higher than that in normal cells Expression and/or differential expression; screening of said candidate cell membrane receptors based on binding to viral ligands to obtain said tumor-specific receptors, binding of the screened tumor-specific receptors to viral ligands
  • the capacity should be higher than the binding capacity of the viral ligand to cellular receptors on normal cells (non-cancerous/tumor cells).
  • the expression of the candidate cell membrane receptor in the tumor cell has a fold difference (log2FC) greater than or equal to 2.0 compared with the expression in the normal cell.
  • step b further includes: using the tumor cell-specific receptor obtained in step a, and determining a high affinity based on the binding ability of the tumor cell-specific receptor to the viral ligand viral ligand.
  • the binding ability of the receptor to the viral ligand is determined based on protein sequence analysis and/or protein co-precipitation method.
  • the protein sequence-based analysis is determined by determining the ZDOCK score of the receptor and the viral ligand.
  • the ZDOCK score not lower than a predetermined threshold is an indication that the viral ligand is a target viral ligand, wherein the predetermined threshold is based on the binding force of a predetermined oncolytic viral ligand and a receptor definite.
  • step c further includes: using the target viral ligand to obtain an oncolytic virus expressing the target viral ligand through virus assembly.
  • the method further comprises: verifying the oncolytic property of the oncolytic virus by the oncolytic ability.
  • the oncolytic properties include the ability of oncolytic virus to enter cells, the ability of intracellular proliferation, and the ability to lyse cells.
  • a method for constructing a recombinant oncolytic virus with improved oncolytic ability is also included by replacing other proteins other than the ligand protein of the oncolytic virus.
  • the oncolytic virus is selected from vaccinia virus, herpes virus, measles virus, Newcastle disease virus, adenovirus, alphavirus, parvovirus or rhabdovirus.
  • the virus is Coxsackie virus B (CVB virus), and the ligand is the VP1-VP4 protein of CVB virus.
  • CVB virus Coxsackie virus B
  • ligand is the VP1-VP4 protein of CVB virus.
  • the oncolytic virus is VSV virus.
  • the predetermined threshold is 1700
  • the ZDOCK score is not lower than 1700, preferably not lower than 1800, such as not lower than 1900, not lower than 2000, preferably not lower than 2100 , is an indication that the viral ligand is the target viral ligand.
  • the receptor on the surface of normal cells is LDLR.
  • the ligand is G protein of VSV virus, and the other protein is selected from M, P, N or L protein of VSV virus.
  • the present invention provides an oncolytic virus.
  • the oncolytic virus is constructed by the aforementioned method.
  • the oncolytic virus according to the embodiment of the present invention has stronger tumor targeting ability and stronger killing ability.
  • the present invention proposes the use of the oncolytic virus proposed in the second aspect in preparing a medicament for treating or preventing cancer or tumor.
  • the use may further include at least one of the following additional technical features:
  • the disease is cancer or tumor.
  • the oncolytic virus is formulated for intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous or intranasal administration.
  • the tumor is selected from lung cancer, melanoma, leukemia, ovarian cancer, central nervous system cancer, colon cancer, breast cancer, kidney cancer and prostate cancer.
  • the fourth aspect of the present invention provides a composition comprising the above-constructed oncolytic virus.
  • the administration mode of the composition is not particularly limited, and can be administered by intratumoral injection, intraperitoneal injection, intrapleural injection, brain injection administration or intravenous injection, etc.
  • the composition according to the embodiments of the present invention can effectively prevent or to treat cancer and/or tumors.
  • a fifth aspect of the present invention provides a kit, comprising: (a) a first container, wherein the first container contains a first pharmaceutical composition, and the first pharmaceutical composition contains the and (b) a second container containing a second pharmaceutical composition, the second pharmaceutical composition containing an additional tumor therapeutic agent and a pharmaceutically acceptable carrier.
  • the present invention provides a method for preventing or treating tumors. According to an embodiment of the present invention, it comprises administering to a subject at least one of: the oncolytic virus of the second aspect; the pharmaceutical composition of the fourth aspect; the kit of the fifth aspect.
  • the methods according to the embodiments of the present invention can provide a subject with the ability to effectively prevent tumors or inhibit the growth of tumor cells.
  • the above-mentioned method for preventing or treating tumors may further include at least one of the following additional technical features:
  • the tumor comprises at least one selected from the group consisting of lung cancer, melanoma, leukemia, ovarian cancer, central nervous system cancer, colon cancer, breast cancer, kidney cancer and prostate cancer.
  • Figure 1 shows the flow chart of the analysis of human membrane receptor genes based on a large sample of tumor tissue
  • Figure 2 shows a jittered scatterplot of the proportion of patients whose corresponding receptor genes were significantly up-regulated in each tumor
  • Figure 3 shows the results of ZDOCK scores reflecting the binding strength of candidate ligands to LRP2 respectively
  • Fig. 4 shows the results of the binding ability of CVB3-Nancy strain and CVB3-Woofruff strain to IL22RA2, GCGR receptor.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the present invention provides a method for constructing an oncolytic virus.
  • the method includes the following steps: a. determining a tumor cell-specific receptor; b. determining a target viral ligand based on the binding force of the viral ligand to the tumor cell-specific receptor; c. and constructing an oncolytic virus comprising the target viral ligand.
  • the method according to the embodiment of the present invention can rapidly screen oncolytic viruses with high tumor cell specificity and broad spectrum of tumor treatment.
  • the tumor cell-specific receptor is a cell membrane receptor
  • the expression of the cell membrane receptor in tumor cells is highly expressed and/or differentially expressed compared with the expression in normal cells.
  • the expression of the cell membrane receptor in tumor cells has a fold difference (log2FC) greater than or equal to 2.0 compared with the expression in normal cells.
  • tumor cell surface specific receptors in the present application refers to receptors with significantly up-regulated/down-regulated differential expression of tumor cell surface receptors relative to normal cell surface receptors, wherein the expression differential fold (log2FC) is greater than A difference equal to 2.0 was considered a significant up-regulation difference, and a p-value of t-test less than 0.01 was considered a statistically significant difference.
  • the binding ability of the VSV virus described in this application to specific receptors on the surface of tumor cells was evaluated by ZDOCK score function.
  • the present application uses computer simulation scoring and/or protein co-precipitation methods to determine the binding ability of VSV virus to specific receptors on the surface of tumor cells, and the binding ability ZDOCK score is higher than a predetermined threshold as a ligand with high binding ability.
  • a predetermined threshold as a ligand with high binding ability.
  • the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 ..
  • step b further includes: using the receptor obtained in step a to determine a high-affinity viral ligand based on the binding ability of the receptor to the viral ligand.
  • the binding ability of the receptor to the viral ligand is determined based on protein sequence analysis and/or protein co-precipitation methods.
  • step c further includes: using the target viral ligand to obtain an oncolytic virus expressing the target viral ligand through virus assembly.
  • the method further comprises: verifying the oncolytic property of the oncolytic virus by the oncolytic ability.
  • the oncolytic properties include the ability of the oncolytic virus to enter tumor cells, the ability to proliferate in cells, and the ability to lyse cells.
  • a method for constructing a recombinant oncolytic virus with improved oncolytic ability is also included by replacing other proteins other than the ligand protein of the oncolytic virus.
  • the oncolytic virus is selected from vaccinia virus, herpes virus, measles virus, Newcastle disease virus, adenovirus, alphavirus, parvovirus or rhabdovirus.
  • the virus is Coxsackie virus B (CVB virus), and the ligand is the VP1-VP4 protein of CVB virus.
  • Neonatal coxsackie virus belongs to the class of RNA viruses, Picornaviridae, enterovirus group. It is relatively stable at room temperature and only survives for 1 hour when the ambient temperature increases to 50°C.
  • the virus is highly tolerant to acidic environments, insensitive to antibiotics and chemical drugs, but sensitive to oxidants, and is easily killed by 1% potassium permanganate, 1% hydrogen peroxide or chlorine-containing oxidants. According to the characteristics of damage to organs, coxsackie virus is divided into two groups: A and B.
  • Group A mainly causes nerve, muscle and systemic infection, aseptic meningitis, paralytic disease, upper respiratory tract infection, and herpetic pharyngeal disease. Gorge, etc.
  • Group B mainly invades the heart, brain, liver and other organs, causing myocarditis, pericarditis, viral encephalitis and so on.
  • Coxsackie virus B group can be divided into 6 types (CVB1-6) by neutralization test and complement fixation test. According to recent reports, CVB3 and CVB5 infection are the most harmful to newborns and have the highest incidence.
  • the oncolytic virus is vesicular stomatitis virus (VSV virus).
  • VSV virus vesicular stomatitis virus
  • the predetermined threshold is 1700
  • the ZDOCK score is not less than 1700, for example, not less than 1800, not less than 1900, not less than 2000, preferably not less than 2100, which is the The viral ligand is an indication of the target viral ligand.
  • VSV vesicular stomatitis virus belongs to the Rhabdoviridae family of vesicular viruses and is divided into two serotypes: New Jersey (VSV-NJ) and Indiana (VSV-IND).
  • Virus particles are bullet-shaped or cylindrical, with a size of 150-180 nm ⁇ 50-70 nm.
  • the virus has an envelope, and the envelope is evenly covered with fibers about 10nm long.
  • Inside the virus is a tightly coiled helix-symmetric nucleocapsid.
  • the virus is named after the classic vesicular lesions in the oral mucosa, tooth pads, tongue, lips, nostrils, hoofs and nipples of sick animals.
  • Spread by insect vectors the disease is limited to its natural hosts such as horses, cattle and pigs. In humans, the infection is mild and asymptomatic.
  • the VSV genome is an unsegmented single-stranded negative-stranded RNA (ssRNA) virus with a length of approximately 11 KB. From the 3' end to the 5' end, there are five non-overlapping genes, N, NS, M, G, and L, which encode nuclear (N) protein, phosphate (P) protein, matrix (M) protein, sugar (G) protein, respectively. ) protein, and five different proteins including RNA-dependent RNA polymerase (L) protein.
  • the 3' end of the N gene is a leader sequence (Leader), the 5' end is a trailing sequence (Trailor), and there is a spacer sequence between each gene.
  • the 3'-end leader RNA is the earliest viral transcript in infected cells.
  • N protein is required to initiate the synthesis of the genome and can effectively protect the viral RNA from digestion by various nucleases. N protein has high immunogenicity, stimulates the body to produce cellular immunity, and plays an important role in transcriptional replication. It may be necessary to maintain the extended form of genomic RNA and is related to replication regulation.
  • the P protein, VSV-NJ is 41% homologous to the VSV-IND strain, and its role is to form a polymerase complex with polymerase L, nucleoprotein N and genomic RNA to maintain the transcriptional activity of the virus.
  • the M protein plays a key role in viral pathogenesis and viral replication, is rich in basic amino acids, and contains a highly basic amino-terminal domain that inhibits transcription by binding to the nucleocapsid while assisting virus budding from the host , is the only polypeptide involved in the budding process.
  • G protein is the main surface antigen of the virus, which determines the virulence of the virus and is also the protective antigen of the virus. It stimulates the body to produce neutralizing antibodies.
  • the L gene encodes the RNA poly E protein, which may determine the transcriptional activity of RNA, and binds to the P protein to catalyze the replication of mRNA.
  • This protein is a core component of the polymerase complex and replicase complex and is involved in initiation, elongation, methylation, capping, poly(A) tail formation, and more.
  • there is extensive homology in the spacer sequences between each gene and these sequences share a common structure, namely 3'-AUAC(U)7NAUUGUCNN-UAG-5'.
  • the conserved sequence between these genes is a key signal to affect the activity of the polymerase or the cleavage activity of the enzyme, and during replication, these signals are masked and not functional.
  • the ligand is G protein of VSV virus, and the other protein is selected from M, P, N or L protein of VSV virus.
  • the present invention provides an oncolytic virus.
  • the oncolytic virus is constructed by the aforementioned method.
  • the oncolytic virus according to the embodiment of the present invention has stronger tumor targeting ability, stronger affinity and stronger killing ability.
  • the present invention proposes the use of the oncolytic virus proposed in the second aspect in preparing a medicament for treating or preventing cancer or tumor.
  • the oncolytic virus is formulated for intraperitoneal, intravenous, intraarterial, intramuscular, intradermal, intratumoral, subcutaneous or intranasal administration.
  • the tumor is selected from lung cancer, melanoma, leukemia, ovarian cancer, central nervous system cancer, colon cancer, breast cancer, kidney cancer and prostate cancer.
  • the medicine provided by the present invention includes a pharmaceutically acceptable carrier and an effective amount of the following active ingredient: an oncolytic virus constructed according to the method of the present invention.
  • the term “effective amount” or “effective dose” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical compositions are preferably manufactured under sterile conditions.
  • additional tumor therapeutic agents may also be included in the medicament of the present invention.
  • the effective amount of the active ingredient of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or its combination.
  • the choice of carrier should match the mode of administration, as is well known to those of ordinary skill in the art.
  • the fourth aspect of the present invention provides a composition comprising the above-constructed oncolytic virus.
  • a fifth aspect of the present invention provides a kit, comprising: (a) a first container, wherein the first container contains a first pharmaceutical composition, and the first pharmaceutical composition contains the and (b) a second container containing a second pharmaceutical composition, the second pharmaceutical composition containing an additional tumor therapeutic agent and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is liquid or solid.
  • the pharmaceutical composition is a freeze-dried preparation.
  • freeze-dried preparation can be reconstituted into a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the content of the recombinant VSV virus is 1 ⁇ 10 3 to 1 ⁇ 10 10 /mL, preferably 1 ⁇ 10 4 to 1 ⁇ 10 9 /mL, more preferably 1 ⁇ 10 5 to 1 ⁇ 10 8 /mL.
  • the pharmaceutical composition contains buffer and auxiliary materials.
  • the buffer includes: phosphate buffer, acetate buffer, and amino acid salt buffer.
  • the pharmaceutical composition further contains an additional tumor therapeutic agent.
  • a method for treating tumors comprises the step of administering an oncolytic virus according to the second aspect of the present invention to a subject in need of treatment.
  • the subject is a human.
  • tumor-specific receptors with differential expression, it is possible to invent virus-binding receptors that are truly related to target cells, and the receptors can be different due to different tumor cell lines, which is the target of oncolytic viruses. Sex, specificity, and low binding to normal cells laid the foundation.
  • the present invention obtains receptors by screening and further selects corresponding high-binding viral ligands, which can further obtain oncolytic viruses with high affinity with tumor cells, which are highly specific to tumor cells and have little effect on normal tissues and cells. , so it has better safety performance and less side effects.
  • the present invention summarizes the receptor gene information expressed in human cells from existing research (reference: (Synchronous birth is a dominant pattern in receptor-ligand evolution, BMC Genomics. Grandchamp and Monget, 2018 Aug 14; 19) (1):611.).
  • the inventors downloaded the gene expression matrix (normalized value), gene mutation information and related clinical data of cancer patients from UCSC Xena ( http://xena.ucsc.edu/ ).
  • Carcinomas included are: adrenal cortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and cervical endometrial adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colon adenocarcinoma/Rectum adenocarcinoma, esophageal carcinoma, Lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, FFPE trial phase II, glioblastoma, glioma, head and neck squamous cell carcinoma, renal chromosome, pan-kidney cohort (KICH+KIRC+KIRP ), renal renal clear cell carcinoma, renal renal papillary cell carcinoma, acute myeloid leukemia, low-grade brain glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian se
  • the inventors first removed less than three sample tumor and normal tissue information from the downloaded data, and then performed differential expression analysis.
  • the inventors used limma software (version: 3.38.3) to perform differential expression analysis (reference: (Ritchie et al., 2015)).
  • the voom model of the limma R package was used in the analysis. Only when the gene satisfies the criterion
  • the fold difference (log2FC) and p-value of membrane receptor gene expression between groups were calculated using R language. Selecting
  • the inventors select genes that are significantly up-regulated (ie
  • genes that are significantly up-regulated ie
  • the inventors used ggplot2 and ggbeeswarm software to draw jitter scatter plots for the log2FC values of each gene in different tumor samples to show the proportion of patients whose genes were significantly up-regulated in each tumor.
  • the inventors docked the 13 receptors screened out with candidate ligands and selected them according to their binding strength.
  • the binding domain of the membrane receptor LRP2 was purified by in vitro protein expression, incubated with G protein with GST tag, and then purified by GST protein after incubation.
  • the purified protein was detected by SDS-PAGE, Coomassie brilliant blue The staining results showed two bands of SGT-G fusion protein and receptor binding domain, indicating that G protein can bind to receptors.
  • the inventors discovered and confirmed a novel cellular receptor that binds to oncolytic virus ligand proteins by using the specific expression of tumor cell membrane receptors.
  • the inventors expect that based on the newly discovered receptor, if a viral ligand with a high binding ability to the receptor can be obtained, it is possible to obtain a virus with a stronger ability to enter cells, and since the receptor is derived from a tumor-specific receptor. It is possible that it has stronger selectivity for tumors and thus achieves higher safety. In order to prove the above conjecture, the inventor further carried out the following verification.
  • LDLR is a vesicular stomatitis virus entry receptor known in the art
  • LDLR was used as a control in subsequent studies of the present invention.
  • the binding strength of different ligands to LDLR receptors was further analyzed.
  • 16 vesicular stomatitis virus homologous ligands were selected, modeled and docked with different tumor-specific receptors, respectively, and the binding strength was evaluated by the ZDOCK score function.
  • the inventors could screen out the tumor-specific receptor binding Powerful ligands. Taking the LRP2 receptor as an example (the results are shown in Figure 3, where the ligand number shown in Figure 3 and the corresponding ligand name and amino acid sequence capture number are shown in Table 2), its binding ability to G protein is relatively high. , and higher than the binding capacity of LDLR to G protein. The abscissa of the figure is different G proteins, and the ordinate is the binding ability.
  • LRP2 is one of the tumor-specific receptors associated with the VSV virus.
  • LRP2 is a member of the low-density lipoprotein superfamily, a class of receptor proteins on the cell surface. It is widely distributed in vivo and can bind to a variety of ligands, but it has not been reported to be related to VSV. By selecting differentially expressed tumor cell receptors and docking with a variety of ligand proteins, potential cell receptors with high binding ability to oncolytic viruses can be found.
  • the inventors further studied the relationship between the selected receptors and oncolytic virus ligands, and selected a vesicular stomatitis virus G protein ( VSV-G)-9_X03633.1-lig-FL ligand, the 9_X03633.1-lig-FL ligand has the strongest binding ability to the LRP2 receptor (as shown in Figure 3).
  • VSV-G vesicular stomatitis virus G protein
  • the following inventors constructed a recombinant oncolytic virus by using the screened LRP2 receptor high binding ligand 9_X03633.1-lig-FL (the amino acid sequence acquisition code is X03633.1) and the L, N, M, and P of the virus strain.
  • the plasmid used for the construction was the commercially available conventional vector PcDNA3.1. The specific construction process is as follows:
  • VSV In vitro recombination of VSV requires: the full-length plasmid (containing G protein) comprising the viral genome, and the helper plasmid (N, P, L, M) of the backbone protein required for virus packaging, and the plasmid is transferred into BHK21 by the method of in vitro transfection In cells, the virus is assembled and matured in the cell and then buds and released outside the cell (Reference: (Vesicular stomatitis virus-based vaccine protects hamsters again lethal challenge with Andes virus. Journal of virology 85, 12781-12791, doi: 10.1128/JVI. 00794-11 (2011), Brown, KS, Safronetz, D., Marzi, A., Ebihara, H. & Feldmann, H.).
  • Virus amplification uses Vero cells. A certain titer of virus is added to the cultured Vero cells. The virus can infect the cells and complete self-replication in the cells. The mature virus is released into the supernatant of the cell culture, and the cells are cultured. The supernatant was concentrated to obtain virus concentrate. This virus is an artificially synthesized gene recombinant vesicular stomatitis virus, marked as REV X03633.1.
  • the LRP2 receptor low-binding ligand 6_MK934319.1 (its amino acid sequence access code is MK934319.1)
  • the low-binding ligand 11_M35214.1 (its amino acid sequence access code is M35214.1)
  • the virus strain were constructed by the same method.
  • the lethality of the recombinant viruses REV X03633.1, REV MK934319.1 and REV M35214.1 constructed in Example 3 was detected by CCK.
  • CNK Cell Killing Assay
  • H460, HCT-8, HepG2, Su86.86, NCI-H358 and PANC-1 cells in good condition were made into a cell suspension of 5 ⁇ 10 4 cells/ml and added to a 96-well plate at 100 ⁇ L/well, and the edges were filled. The medium was reduced to evaporate and incubated overnight. Dilute the known titer virus (viruses are REV X03633.1, REV MK934319.1 and REV M35214.1) with Opti-MEM to a virus working solution of MOI: 0.01, and discard the culture solution in the 96-well plate.
  • viruses are REV X03633.1, REV MK934319.1 and REV M35214.1
  • virus diluent 50uL was added, and each dilution was repeated in 3 wells, and another 3 wells in Opti-MEM were taken as blank control.
  • the virus diluent was added for 2 hours and then the medium was changed, with 100 ⁇ L of 1% FBS medium per well. After 48/72h, add 10 ⁇ L of CCK8 detection solution to each well, incubate at 37°C for 2h and read on OD450 microplate reader.
  • CNK Cell Killing Assay
  • the normal lung normal cells BEAS-2B in good condition were made into a cell suspension of 5 ⁇ 10 4 cells/mL and added to a 96-well plate at 100 ⁇ L/well.
  • the medium was supplemented at the edge to reduce evaporation, and cultured overnight.
  • the virus diluent was added for 2 hours and then the medium was changed, with 100 ⁇ L of 1% FBS medium per well. After 72 hours, 10 ⁇ L of CCK8 detection solution was added to each well, and the samples were incubated at 37°C for 2 hours and read on an OD 450 microplate reader.
  • the experimental results are shown in Table 3.
  • the CCK test results showed that the virus working solution of REV X03633.1, REV MK934319.1 and REV M35214.1 recombinant virus strains at MOI: 0.01 had significantly lower killing effect on BEAS-2B cells than the three recombinant viruses on tumor cells.
  • the killing effect of REV X03633.1 on normal cells is smaller, showing its safety.
  • the tumor-specific receptors related to CVB were screened.
  • the inventors used the screened 13 receptors for molecular docking with CVB3-Nancy.
  • the obtained CVB tumor-specific receptors were: IL22RA2 (amino acid sequence capture code Gene ID: 116379), GCGR (amino acid sequence capture code Gene ID: 2642).
  • CVB3-Nancy surface ligand is CAR, amino acid sequence capture code JX312064.1
  • CVB3-Worduff surface ligand is CAR, amino acid sequence capture code U57056.1
  • HCC4006 and PC9 cells in good condition were made into a cell suspension of 5 ⁇ 10 4 cells/mL and added to a 96-well plate at 100 ⁇ L/well. The medium was supplemented at the edge to reduce evaporation, and cultured overnight. Dilute known titers of CVB3-Nancy virus and CVB3-Worduff with Opti-MEM to virus working solution with MOI: 0.1 respectively, aspirate the culture solution in 96-well plate, add 50 ⁇ L of virus dilution solution to each well, and each dilution solution Repeat 3 wells, and take Opti-MEM to repeat 3 wells as blank control.
  • the virus diluent was added for 2 hours and then the medium was changed, with 100 ⁇ L of 1% FBS medium per well. After 48/72h, add 10 ⁇ L of CCK8 detection solution to each well, incubate at 37°C for 2h and read on OD450 microplate reader.
  • the CCK detection results are shown in Table 4.
  • the CVB3-Nancy strain virus working solution with MOI: 0.1 has a significant killing effect on both PC9 and HCC4006 cells.
  • the virus working solution of CVB3-Woofruff strain had no obvious killing effect on PC9 and HCC4006 cells.
  • Table 4 % inhibition rate of virus on tumor cells at MOI: 0.1
  • CVB oncolytic viruses Through the screening of CVB oncolytic viruses, it was further confirmed that by screening virus-specific receptors and using the receptors to further screen high-affinity ligands to construct oncolytic viruses, high-affinity oncolytic viruses can be quickly obtained. virus, indicating the generalizability of the method.
  • the present invention verifies the selection of receptors specifically expressed by tumor cell membranes through VSV virus and CVB virus, and then selects a high-affinity ligand through the receptor, and constructs an oncolytic virus according to the ligand.
  • Oncoviruses have higher oncolytic capacity, and they have higher selectivity for tumor cells.
  • the feasibility of this method is proved.
  • the advantage of this method is that the selectivity of tumor cells is improved through the differential expression of cell membrane receptors, and on this basis, a high-affinity ligand is further selected through the receptor, which further improves the oncolytic ability and selectivity.
  • This screening idea has not been reported, and has high practicability, which greatly improves the screening speed and screening pertinence of dominant oncolytic viruses.

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Abstract

提供了一种构建溶瘤病毒的方法。该方法包括以下步骤：a、确定肿瘤细胞特异性受体；b、基于病毒配体与所述肿瘤细胞特异性受体的结合力，确定目标病毒配体；c、构建包含所述目标病毒配体的溶瘤病毒。

Description

构建溶瘤病毒的方法
优先权信息
本申请请求2020年08月14日向中国国家知识产权局提交的、专利申请号为202010816808.2的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于溶瘤病毒领域，具体涉及构建溶瘤病毒的方法、溶瘤病毒、溶瘤病毒在制备药物中的用途、组合物及试剂盒。
背景技术
近十几年，溶瘤病毒可以通过诱导机体的抗肿瘤免疫反应来杀伤肿瘤的机制逐渐明确。自2011年德国科学家Jean Rommelaere第一次将溶瘤病毒疗法称作肿瘤免疫治疗以来，溶瘤病毒目前已经被大众接受作为肿瘤免疫治疗的重要分支。而相较于其他肿瘤免疫疗法，溶瘤病毒具有杀伤效率高、靶向性好、副作用小、多种杀伤肿瘤途径避免耐药性和成本低廉等优势。
由于病毒基因组小，相对易于通过基因工程手段进行多种改造，且病毒的改造和包装是生物医学常规试验手段，技术相对成熟，价格也相对低廉。因此，溶瘤病毒更容易利用自身的特征和癌细胞与正常细胞的不同来做相应的改造，做到特异性靶向癌细胞。
由于大部分癌细胞自身清除病毒的机制受损(如正常细胞清除病毒的关键因子蛋白激酶R(PKR)在癌细胞中缺失)，所以病毒更容易在癌细胞中复制扩散。另外，近几十年，随着研究的不断深入，科学工作者们利用癌细胞和正常细胞中很多信号通路和代谢等的不同，通过筛选特异病毒品种和改造病毒基因组，不断提高溶瘤病毒对肿瘤的靶向性，降低其对正常细胞的危害，提高安全性。例如：已获批的T-vec敲除了HSV-1(单纯疱疹病毒1型)的γ34.5基因，γ34.5基因可以抑制正常细胞对病毒的清除机制，γ34.5基因敲除后，病毒不能在正常细胞中复制；而癌细胞中这一机制缺失，γ34.5基因敲除后不影响病毒在癌细胞中的复制。目前处于III期临床的JX594(Pexa-Vec)敲除了牛痘病毒(vaccinia viruses)的TK(胸苷激酶，thymidine kinase)基因，而病毒的复制与细胞中TK水平有关，所以敲除了TK的JX594仅能在TK活性高的癌细胞中进行复制，不能在正常细胞中复制(正常细胞TK活性低)。CG0070是在腺病毒主管复制的基因E1A前加了E2F-1启动子，E2F-1受视网膜母细胞瘤抑制蛋白(Rb)的调控，而Rb在膀胱癌中缺失，Rb的缺失激活E2F-1的转录活性，使得E1A基因表达，病毒可以特异性在膀胱癌中复制。Reolysin是未经改造的野生型呼肠孤病毒， 其增值依赖Ras信号通路的激活，所以仅能在Ras激活的癌细胞中特异性增值。
然而，目前的筛选和构建方法往往关注于病毒本身序列的改造，通过插入外源基因或者修改蛋白序列的方式，构建高亲和力的溶瘤病毒的效率较低，缺乏靶向性。导致目前研究进展缓慢，无法快速有效的获得对肿瘤细胞具有高亲和力的溶瘤病毒。而且通过外源序列的引入或基因突变等方法可能会影响病毒的靶向性、感染能力和溶瘤能力，并带来不可预知的副作用。
因此，理想的方法是开发能够具有高度肿瘤靶向性和亲和力的筛选溶瘤病毒的方法，并尽可能少的对溶瘤病毒本身的序列产生改变。
本领域迫切需要开发一种能够快速筛选具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的溶瘤病毒的通用方法。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
本发明的目的就是提供一种能够快速筛选具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的溶瘤病毒的通用方法。
在本发明的第一方面，本发明提供了一种构建溶瘤病毒的方法。根据本发明的实施例，所述方法包括以下步骤：a、确定肿瘤细胞特异性受体；b、基于病毒配体与所述肿瘤细胞特异性受体的结合力，确定目标病毒配体；c、构建包含所述目标病毒配体的溶瘤病毒。根据本发明实施例的方法能够快速筛选具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的溶瘤病毒。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述肿瘤细胞特异性受体是通过下列步骤确定的：确定候选细胞膜受体，所述候选细胞膜受体在肿瘤细胞的表达与在正常细胞中的表达相比具有高表达和/或差异表达；基于与病毒配体的结合力，对所述候选细胞膜受体进行筛选，以便获得所述肿瘤特异性受体，所筛选的肿瘤特异性受体与病毒配体的结合能力应高于病毒配体与正常细胞(非癌变/肿瘤细胞)上细胞受体的结合能力。
根据本发明的实施例，所述候选细胞膜受体在所述肿瘤细胞的表达与在所述正常细胞中的表达相比具有差异倍数(log2FC)大于等于2.0。
根据本发明的实施例，在步骤b中进一步包括：利用步骤a获得的所述肿瘤细胞特异性受体，基于所述肿瘤细胞特异性受体与所述病毒配体的结合能力，确定高亲和力病毒配体。
根据本发明的实施例，受体与病毒配体结合能力是基于蛋白质序列分析和/或蛋白共沉淀方法来确定。
根据本发明的实施例，所述基于蛋白质序列分析是通过确定所述受体与所述病毒配体的ZDOCK score确定的。
根据本发明的实施例，所述ZDOCK score不低于预定阈值为所述病毒配体是目标病毒配体的指示，其中，所述预定阈值是基于预定溶瘤病毒配体和受体的结合力确定的。
根据本发明的实施例，步骤c包括进一步包括：利用所述目标病毒配体，通过病毒组装，获得表达所述目标病毒配体的溶瘤病毒。
根据本发明的实施例，所述方法进一步包括：通过溶瘤能力验证所述溶瘤病毒的溶瘤性能。所述溶瘤性能包括溶瘤病毒的入胞能力、胞内增值能力以及裂解细胞的能力。
根据本发明的实施例，还包含通过替换溶瘤病毒的配体蛋白外的其他蛋白的方法，构建溶瘤能力提高的重组溶瘤病毒。
根据本发明的实施例，所述溶瘤病毒选自痘苗病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒或弹状病毒。
根据本发明的实施例，所述病毒为柯萨奇病毒B(CVB病毒)，配体为CVB病毒的VP1-VP4蛋白。
根据本发明的实施例，所述溶瘤病毒为VSV病毒。具体地，当所述溶瘤病毒为VSV病毒时，所述预定阈值为1700，所述ZDOCK score不低于1700，优选地不低于1800，例如不小于1900，不小于2000，优选不小于2100，为所述病毒配体是目标病毒配体的指示。
根据本发明的实施例，当选择的溶瘤病毒为VSV时，正常细胞表面的受体为LDLR。
根据本发明的实施例，所述配体为VSV病毒的G蛋白，所述其他蛋白选自VSV病毒的M、P、N或L蛋白。
在本发明的第二方面，本发明提出了一种溶瘤病毒。根据本发明的实施例，所述溶瘤病毒是通过前面所述的方法构建的。根据本发明实施例的溶瘤病毒具有更强的肿瘤靶向性和杀伤能力更强。
在本发明的第三方面，本发明提出了第二方面所提出的溶瘤病毒在制备药物中的用途，所述药物用于治疗或预防癌症或肿瘤。
根据本发明的实施例，所述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述疾病为癌症或肿瘤。
根据本发明的实施例，所述溶瘤病毒经配制用于腹膜内、静脉内、动脉内、肌内、皮内、瘤内、皮下或鼻内施用。
根据本发明的实施例，所述肿瘤选自肺癌、黑色素瘤、白血病、卵巢癌、中枢神经系统癌症、结肠癌、乳腺癌、肾癌和前列腺癌。
本发明的第四方面，提供了包含上述构建的溶瘤病毒的组合物。所述组合物的给药方 式不受特别限制，可通过瘤内注射、腹膜内注射、胸膜内注射、脑注射给药或静脉注射等方式给药，根据本发明实施例的组合物可有效预防或治疗癌症和/或肿瘤。
本发明的第五方面，提供了一种药盒，包括：(a)第一容器，所述的第一容器中含有第一药物组合物，所述的第一药物组合物含本发明所述的组合物；和(b)第二容器，所述的第二容器中含有第二药物组合物，所述的第二药物组合物含有额外的肿瘤治疗剂以及药学上可接受的载体。
在本发明的第六方面，本发明提出了一种预防或治疗肿瘤的方法。根据本发明的实施例，包括向受试者施用以下中的至少之一：第二方面所述的溶瘤病毒；第四方面所述的药物组合物；第五方面所述的药盒。根据本发明实施例的方法可使得受试者具有有效预防肿瘤或抑制肿瘤细胞生长的能力。
根据本发明的实施例，上述预防或治疗肿瘤的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述肿瘤包括选自肺癌、黑色素瘤、白血病、卵巢癌、中枢神经系统癌症、结肠癌、乳腺癌、肾癌和前列腺癌的至少之一。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了基于肿瘤组织大样本的人类膜受体基因的分析流程图；
图2显示了对应的受体基因在各肿瘤中显著上调的病人比例的抖动散点图；
图3显示了反映候选配体分别与LRP2结合强度的ZDOCK score结果图；
图4显示了CVB3-Nancy株、CVB3-Woofruff株与IL22RA2，GCGR受体的结合力的结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含 地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
构建溶瘤病毒的方法
在本发明的第一方面，本发明提供了一种构建溶瘤病毒的方法。根据本发明的实施例，所述方法包括以下步骤：a、确定肿瘤细胞特异性受体；b、基于病毒配体与所述肿瘤细胞特异性受体的结合力，确定目标病毒配体；c、构建包含所述目标病毒配体的溶瘤病毒。根据本发明实施例的方法能够快速筛选具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的溶瘤病毒。
根据本发明的实施例，所述肿瘤细胞特异性受体是细胞膜受体，所述细胞膜受体在肿瘤细胞的表达与在正常细胞的表达相比具有高表达和/或差异表达。根据本发明的实施例，所述细胞膜受体在肿瘤细胞的表达与在正常细胞的表达相比具有差异倍数(log2FC)大于等于2.0。本申请所述的“肿瘤细胞表面特异性受体”是指肿瘤细胞表面受体相对于正常细胞表面受体具有显著性上调/下调的差异表达的受体，其中，表达差异倍数(log2FC)大于等于2.0被视为有显著性上调差异，t检验的p值小于0.01被判定为差异有统计学意义。
本申请所述的VSV病毒与肿瘤细胞表面特异性受体的结合力通过ZDOCK score函数来评价。本申请通过计算机模拟评分和/或蛋白共沉淀方法来确定VSV病毒与肿瘤细胞表面特异性受体的结合力，结合力ZDOCK score高于预定阈值为高结合力的配体。本领域技术人员能够理解，通过输入G蛋白和细胞受体的序列，可以容易地获得G蛋白和细胞受体之间的结合力表征参数ZDOCK score。发明人发现，当ZDOCK score不低于预定阈值时，携带配体的病毒与携带对应受体的肿瘤细胞之间的结合力将显著提高。根据本发明的实施例，ZDOCK score是可以常规软件确定的，例如参见Pierce BG,Hourai Y,Weng Z.(2011)Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657.。
根据本发明的实施例，在步骤b中进一步包括：利用步骤a获得的受体，基于受体与病毒配体结合能力，确定高亲和力病毒配体。根据本发明的具体实施例，受体与病毒配体结合能力是基于蛋白质序列分析和/或蛋白共沉淀方法来确定。
根据本发明的实施例，步骤c包括进一步包括：利用所述目标病毒配体，通过病毒组装，获得表达所述目标病毒配体的溶瘤病毒。
根据本发明的实施例，所述方法进一步包括：通过溶瘤能力验证所述溶瘤病毒的溶瘤性能。所述溶瘤性能包括溶瘤病毒的入肿瘤细胞能力、胞内增殖能力以及裂解细胞的能力。
根据本发明的实施例，还包含通过替换溶瘤病毒的配体蛋白外的其他蛋白的方法，构建溶瘤能力提高的重组溶瘤病毒。
根据本发明的实施例，所述溶瘤病毒选自痘苗病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒或弹状病毒。
根据本发明的具体实施例，所述病毒为柯萨奇病毒B(CVB病毒)，配体为CVB病毒的VP1-VP4蛋白。新生儿柯萨奇病毒(CVB病毒)属于RNA病毒类，小RNA病毒科，肠道病毒群，在室温下相对稳定，当环境温度增高至50℃时仅存活1小时。该病毒对酸性环境耐受性较强，对抗生素、化学药物不敏感，但对氧化剂敏感，易于被1％高锰酸钾、1％过氧化氢或含氯氧化剂杀灭。依据对脏器的损害特点，柯萨奇病毒分为A与B两组，A组主要引起神经、肌肉及全身感染表现，发生无菌性脑膜炎、麻痹性疾病、上呼吸道感染、疱疹性咽峡炎等。B组主要侵犯心、脑、肝及其他脏器，引起心肌炎、心包炎、病毒性脑炎等。柯萨奇病毒B组用中和试验和补体结合试验测定抗原可分为6型(CVB1～6)，据近年报道以CVB3、CVB5型感染对新生儿危害最大，发生率也最高。
根据本发明的实施例，所述溶瘤病毒为水疱性口炎病毒(VSV病毒)。具体地，当所述溶瘤病毒为VSV病毒时，所述预定阈值为1700，所述ZDOCK score不低于1700，例如不小于1800，不小于1900，不小于2000，优选不小于2100，为所述病毒配体是目标病毒配体的指示。水疱性口炎病毒(Vesicular stomatitis Virus,VSV)属于弹状病毒科水疱病毒属的病毒，分为两个血清型：新泽西型(VSV-NJ)和印第安那型(VSV-IND)。病毒粒子为子弹状或圆柱状，大小为150～180nm×50～70nm。病毒有囊膜，囊膜上均匀密布有长约10nm的纤突。病毒内部为紧密盘旋的螺旋对称的核衣壳。该病毒根据患病动物的口腔粘膜，牙垫，舌头，嘴唇，鼻孔，蹄和乳头中的经典水疱性病变命名。通过昆虫媒介传播，疾病仅限于其自然宿主，例如马，牛和猪。在人类中，感染是轻度且无症状的。
VSV基因组为不分节段的单股负链RNA(ssRNA)病毒，长度约为11KB。从3’端→5’端依次排列着N、NS、M、G、L五个不重叠的基因，分别编码核(N)蛋白、磷酸(P)蛋白、基质(M)蛋白、糖(G)蛋白、及RNA依赖的RNA聚合酶(L)蛋白等5种不同的蛋白。N基因的3’端是先导序列(Leader)，5’端是拖尾序列(Trailor)，各基因间有间隔序列。3’端先导RNA在感染细胞中是最早的病毒转录物，长度为47个核苷酸，不戴帽也不翻译，其功能尚未完全清楚，可能是抑制宿主RNA的合成。N蛋白是启动基因组合成所必需的，可有效的保护病毒RNA免受各种核酸酶的消化。N蛋白有高的免疫原性，刺激机体产生细胞免疫，且在转录复制中担任了重要的角色，它对维持基因组RNA呈伸展形式可能是必要的，与复制调节有关。P蛋白，VSV-NJ与VSV-IND病毒株的同源性为41％，其作用是与聚合酶L，核蛋白N组成聚合酶复合物以及基因组RNA共同维持病毒的转录活性。M蛋白在病毒致病机制和病毒复制方面起关键作用，富含碱性氨基酸，并含有高度碱性的氨基末端结构域，可通过与核衣壳结合而抑制转录，同时辅助病毒从宿主中出芽，是涉及出芽过程的惟一多 肽。G蛋白是病毒的主要表面抗原，决定着病毒的毒力，也是病毒的保护性抗原。它可刺激机体产生中和抗体。L基因编码RNA poly E蛋白，它可能决定RNA的转录活性，与P蛋白结合以催化mRNA的复制。该蛋白是聚合酶复合物和复制酶复合物的核心成分，涉及起始、延伸、甲基化、戴帽、聚(A)尾形成等等。此外，在每个基因之间的间隔序列有广泛的同源性，这些序列有一个共同的结构，即3’-AUAC(U)7NAUUGUCNN-UAG-5’。这些基因之间的保守序列是一种关键信号，以影响多聚酶的活性或酶的切割活性，而在复制过程中，这些信号被掩盖，不起作用。
根据本发明的实施例，所述配体为VSV病毒的G蛋白，所述其他蛋白选自VSV病毒的M、P、N或L蛋白。
溶瘤病毒
在本发明的第二方面，本发明提出了一种溶瘤病毒。根据本发明的实施例，所述溶瘤病毒是通过前面所述的方法构建的。根据本发明实施例的溶瘤病毒具有更强的肿瘤靶向性、亲和力和杀伤能力更强。
溶瘤病毒在制备药物中的用途
在本发明的第三方面，本发明提出了第二方面所提出的溶瘤病毒在制备药物中的用途，所述药物用于治疗或预防癌症或肿瘤。
根据本发明的具体实施例，所述溶瘤病毒经配制用于腹膜内、静脉内、动脉内、肌内、皮内、瘤内、皮下或鼻内施用。根据本发明的实施例，所述肿瘤选自肺癌、黑色素瘤、白血病、卵巢癌、中枢神经系统癌症、结肠癌、乳腺癌、肾癌和前列腺癌。
本发明提供的药物，包括药学上可接受的载体和有效量的以下活性成分：根据本发明的方法所构建的溶瘤病毒。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
在一个实施方式中，本发明的药物中还可以包括额外的肿瘤治疗剂。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
组合物
本发明的第四方面，提供了包含上述构建的溶瘤病毒的组合物。
本发明的第五方面，提供了一种药盒，包括：(a)第一容器，所述的第一容器中含有第一药物组合物，所述的第一药物组合物含本发明所述的组合物；和(b)第二容器，所述的第二容器中含有第二药物组合物，所述的第二药物组合物含有额外的肿瘤治疗剂以及药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态或固态。
在另一优选例中，所述的药物组合物为冻干制剂。
在另一优选例中，所述的冻干制剂可重构为液体制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物(或重构的液体制剂)中，所述重组VSV病毒的含量为1×10 3～1×10 10个/mL，较佳地1×10 4～1×10 9个/mL，更佳地1×10 5～1×10 8个/mL。
在另一优选例中，所述的药物组合物中含有缓冲液和辅料。
在另一优选例中，所述的缓冲液包括：磷酸缓冲液、醋酸缓冲液、氨基酸盐缓冲液。
在另一优选例中，所述的药物组合物还含有额外的肿瘤治疗剂。
治疗肿瘤的方法
在本发明的第六方面，提供了一种治疗肿瘤的方法。根据本发明的实施例，所述方法包括步骤：给需要治疗的对象施用如本发明第二方面所述的溶瘤病毒。
在另一优选例中，所述的对象为人。
本发明的主要优点包括：
1)本发明中通过选择表达差异的肿瘤特异性受体，能够发明真正与目标细胞相关的病毒结合受体，而且该受体可因肿瘤细胞系的不同而不同，为溶瘤病毒的靶向性、特异性以及与正常细胞的低结合力奠定了基础。
2)本发明通过筛选获得受体进一步选择对应的高结合力病毒配体，可进一步获得与肿瘤细胞具有高亲和力的溶瘤病毒，对肿瘤细胞特异性高，而对正常组织和细胞几乎无影响，因此具有较好的安全性能，其副作用较小。
3)实验结果表明，利用本发明的方法，所获得的溶瘤病毒对多种肿瘤组织和细胞的均具有显著的溶瘤和杀伤效果，其杀伤效率高、靶向性好。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1基于肿瘤组织大样本的人类膜受体基因的分析
以下将参考图1详细描述基于肿瘤组织大样本的人类膜受体基因的分析的方法。
1.1人类膜受体基因及其表达数据预处理与分析
本发明从已有的研究中整理总结出在人类细胞中表达的受体基因信息(参考文献：(Synchronous birth is a dominant pattern in receptor-ligand evolution，BMC Genomics.Grandchamp and Monget,2018 Aug 14；19(1):611.)。发明人从UCSC Xena( http://xena.ucsc.edu/)下载了癌症病人的的基因表达矩阵(归一化值)，基因突变信息以及相关临床资料。数据中包含的癌种由：肾上腺皮质癌、膀胱尿路上皮癌、乳腺浸润癌、宫颈鳞状细胞癌和宫颈内膜腺癌、胆管癌、结肠腺癌、结肠腺癌/Rectum腺癌食管癌、淋巴样肿瘤弥漫性大B细胞淋巴瘤、食道癌、FFPE试验第二阶段、胶质母细胞瘤、胶质瘤、头颈部鳞状细胞癌、肾染色体、泛肾队列(KICH+KIRC+KIRP)、肾脏肾透明细胞癌、肾脏肾乳头状细胞癌、急性髓性白血病、脑低级脑胶质瘤、肝肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮瘤、卵巢浆液性囊腺癌、胰腺腺癌、嗜铬细胞瘤和副神经节瘤、前列腺腺癌、直肠腺癌、肉瘤、皮肤皮肤黑色素瘤、胃腺癌、胃和食道癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺瘤、子宫体子宫内膜癌、子宫癌肉瘤、葡萄膜黑色素瘤
发明人首先从下载的数据中首先剔除少于三个的样本肿瘤及正常组织信息，然后进行差异表达分析。发明人使用limma软件(版本：3.38.3)来执行差异表达分析(参考文献：(Ritchie et al.,2015))。分析中采用了limma R包的voom模型。只有当基因满足标准|log2FC|>1，P值<0.05才会被认定为是差异基因。
1.2数据分析
使用R语言计算各组间膜受体基因表达差异倍数(log2FC)和p值。选择|log2FC|大于等 于2.0被视为有显著性上调/下调差异表达基因。t检验的p值小于0.01被判定为差异有统计学意义。使用ComplexHeatmap R包生成每个比较组对的log2FC矩阵的热图。
然后，发明人根据一系列筛选条件，比如发明人选择在肠癌，肺癌，胰腺癌，胃癌和肝癌癌种中选择大于等于70％的癌症样本中显著上调的基因(即|log2FC|≥2.0)，结合本底表达量高等条件，选出备选受体；最后根据与备选配体的分子对接结果，最终选出目标受体。
具体地，发明人对每个基因在不同肿瘤样本中的log2FC值，使用ggplot2和ggbeeswarm软件绘制抖动散点图，用以展示该基因在各肿瘤中显著上调的病人比例。
另外，发明人将筛选出来的13个受体与备选配体做分子对接，按照结合力大小选择。
在本实施例中，所用到的各癌种简称、英文名称，及其中文名称总结于下表1。
表1：癌种简写和英文名称
简写 英文名称 中文名称
COAD Colon adenocarcinoma 结肠癌
LIHC Liver hepatocellular carcinoma 肝细胞肝癌
LUAD Lung adenocarcinoma 肺腺癌
LUSC Lung squamous cell carcinoma 肺鳞癌
PAAD Pancreatic adenocarcinoma 胰腺癌
STAD Stomach adenocarcinoma 胃癌
首先以VSV病毒为例，验证本申请所述方法的可行性。
结果如图2所示。结果表明，某些受体，如LRP2在5个癌种中的差异基因表达(即|log2FC|≥2.0)占比远大于LDLR。而LDLR是已发现的VSV入胞最为关键的膜受体，该研究结果提示，肿瘤细胞特异性表达的受体与正常细胞表面受体存在较大差异，因此要提高对肿瘤细胞的特异性，应当根据肿瘤细胞特异性表达受体进行研究，而不能局限于公认的入胞受体，这一发现与一般认识存在明显差异。
为了验证此发现，将膜受体LRP2的结合域进行体外蛋白表达纯化，与带有GST标签的G蛋白共同孵育，孵育之后进行GST蛋白纯化，将纯化的蛋白进行SDS-PAGE检测，考马斯亮蓝染色结果可以看到SGT-G融合蛋白及受体结合域两条带，表明G蛋白可以与受体结合。
因此，通过上述研究，发明人利用肿瘤细胞膜受体的特异表达，发现了并确认了一种新的与溶瘤病毒配体蛋白相结合的细胞受体。
发明人预期基于该新发现的受体，如果能够获得与该受体结合能力高的病毒配体，那么有可能获得入胞能力更强的病毒，而且由于该受体是从肿瘤特异性受体中选择获得的，有可能其对于肿瘤的选择性更强，从而获得更高的安全性。为了证明上述猜想，发明人进一 步进行了以下验证。
实施例2 受体与配体结合力分析
鉴于LDLR是本领域已知的水疱性口炎病毒入胞受体，因此，在本发明的后续研究中，将LDLR作为对照。在后续的实验中，进一步分析不同配体与LDLR受体的结合强弱。
选择16种水疱性口炎病毒同源配体，与不同肿瘤特异性受体分别进行建模和对接，通过ZDOCK score函数来评价结合的强弱，发明人可获得筛选出肿瘤特异性受体结合能力强的配体。以LRP2受体为例(结果如图3所示，其中，图3所示配体编号以及所对应的配体名称和氨基酸序列捕获号如表2所示)，其与G蛋白结合能力较高，且高于LDLR与G蛋白的结合能力。该图横坐标为不同的G蛋白，纵坐标为结合能力，可以看出大部分G蛋白与LRP2均有较强的结合能力，提示LRP2是与VSV病毒相关的肿瘤特异性的受体之一。LRP2是低密度脂蛋白超家族成员之一，是一类细胞表面的受体蛋白，体内分布范围广，可结合多种配体，但未见报道其与VSV相关。通过选择差异表达的肿瘤细胞受体，通过与多种配体蛋白的对接，能够发现潜在的与溶瘤病毒结合能力高的细胞受体。
表2：
Figure PCTCN2021111759-appb-000001
Figure PCTCN2021111759-appb-000002
实施例3 通过选择的受体进一步筛选溶瘤病毒配体
根据实施例2所描述的方法，发明人进一步研究所选择的受体与溶瘤病毒配体之间的关系，选择出一种和肿瘤特异性受体结合紧密的水疱性口炎病毒G蛋白(VSV-G)-9_X03633.1-lig-FL配体，该9_X03633.1-lig-FL配体与该LRP2受体结合能力最强(如图3所示)。
下面发明人利用所筛选出的LRP2受体高结合配体9_X03633.1-lig-FL(其氨基酸序列获取码为X03633.1)与病毒株的L、N、M、P构建重组溶瘤病毒。构建所使用的质粒为市售的常规的载体PcDNA3.1。具体构建过程如下所述：
体外重组VSV需要:包含病毒基因组的全长质粒(包含G蛋白)，以及病毒包装所需要的骨架蛋白的辅助质粒(N、P、L、M)，通过体外转染的方法将质粒转入BHK21细胞中，病毒在细胞内组装成熟后出芽释放到细胞外(参考文献：(Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus.Journal of virology 85,12781-12791,doi:10.1128/JVI.00794-11(2011)，Brown,K.S.,Safronetz,D.,Marzi,A.,Ebihara,H.&Feldmann,H.)。
病毒的扩增使用Vero细胞，将一定滴度的病毒加入到培养的Vero细胞中，病毒可以感染细胞，并且在细胞中完成自我复制，成熟的病毒释放到细胞培养的上清中，将细胞培养的上清浓缩即可得到病毒浓缩液。此病毒为人工合成的基因重组水疱性口炎病毒，标记为REV X03633.1。
用同样的方法构建了LRP2受体低结合配体6_MK934319.1(其氨基酸序列获取码为MK934319.1),低结合配体11_M35214.1(其氨基酸序列获取码为M35214.1)与病毒株的L、N、M、P构建重组溶瘤病毒，标记为REV MK934319.1与REV M35214.1。
实施例4 细胞杀伤结果
本实施例通过CCK检测实施例3所构建的重组病毒REV X03633.1，REV MK934319.1与REV M35214.1的杀伤力。
细胞杀伤实验(CCK)：
将状态良好的H460、HCT-8、HepG2、Su86.86、NCI-H358和PANC-1细胞制成5×10 4个/ml的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度病毒(病毒为REV X03633.1，REV MK934319.1与REV M35214.1)稀释为MOI：0.01的病毒工作液，将96孔板中培养液吸弃，每孔加入50uL病毒稀释液， 每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。48/72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
实验结果如表3所示。CCK检测结果显示，MOI：0.01的REV X03633.1病毒工作液对H460、HCT-8、HepG2、Su86.86、NCI-H358和PANC-1细胞(H460、HCT-8、HepG2、Su86.86、NCI-H358和PANC-1细胞表面LRP2表达相对较高)具有显著的杀伤效果。而REV MK934319.1与REV M35214.1在MOI：0.01的感染比下，对以上肿瘤细胞有杀伤效果，但杀伤效果明显低于REV X03633.1病毒工作液的杀伤效果。这与3种G蛋白配体和LRP2受体的结合力大小呈正相关关系。
表3：MOI:0.01时病毒对肿瘤细胞的抑制率％
Figure PCTCN2021111759-appb-000003
实施例5 重组病毒对正常细胞的杀伤作用
细胞杀伤实验(CCK)：
将状态良好的肺正常细胞BEAS-2B制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度病毒稀释为MOI：0.01的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD 450酶标仪读数。
实验结果如表3所示。CCK检测结果显示，REV X03633.1，REV MK934319.1与REV M35214.1重组病毒株在MOI：0.01时的病毒工作液均对BEAS-2B细胞杀伤效果要明显低于3株重组病毒对肿瘤细胞的杀伤效果，而且REV X03633.1对正常细胞的杀伤效果更小，显示了其安全性。
实施例6 构建CVB溶瘤病毒以及验证其杀伤效果
按照本申请实施例1的方法筛选与CVB相关的肿瘤特异性受体，发明人用筛选出的13个受体与CVB3-Nancy做分子对接，根据结合力大小，获得CVB肿瘤特异性受体为IL22RA2(氨基酸序列捕获码Gene ID:116379)，GCGR(氨基酸序列捕获码Gene ID:2642)。
用2种不同血清型CVB3病毒株CVB3-Nancy(表面配体为CAR，氨基酸序列捕获码JX312064.1)和CVB3-Worduff(表面配体为CAR，氨基酸序列捕获码U57056.1)，与筛选获得的CVB相关的肿瘤特异性受体IL22RA2、GCGR分别进行建模和对接，通过ZDOCK score函数来评价结合的强弱，方法如实施例2所述。
实验结果如图4所示，CVB3-Nancy株与IL22RA2、GCGR受体的结合力均强于CVB3-Woofruff株，因此CVB3-Nancy表面配体(氨基酸序列捕获码JX312064.1)为与肿瘤特异性受体结合能力高的配体，而CVB3-Worduff表面配体(氨基酸序列捕获码U57056.1)为与肿瘤特异性受体结合能力低的配体。
将状态良好的HCC4006和PC9细胞制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度CVB3-Nancy病毒和CVB3-Worduff分别稀释为MOI：0.1的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。48/72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
CCK检测结果如表4所示，MOI：0.1的CVB3-Nancy株病毒工作液均对PC9和HCC4006细胞均具有显著的杀伤效果。而CVB3-Woofruff株的病毒工作液对PC9和HCC4006细胞均无明显的杀伤效果。
表4：MOI:0.1时病毒对肿瘤细胞的抑制率％
  CVB Nancy CVB Woodruff
HCC4006 91.83 56.49
PC9 67.67 55.23
通过CVB溶瘤病毒的筛选，进一步证实，通过对病毒特异性受体的筛选，以及利用该受体进一步筛选高亲和力的配体，从而构建溶瘤病毒，能够很快的获得高亲和力的溶瘤病毒，表明该方法的普适性。
综上，本发明通过VSV病毒和CVB病毒，验证了通过肿瘤细胞细胞膜特异表达受体的选择，进而通过该受体选择高亲和力的配体，并根据该配体构建溶瘤病毒，得到的溶瘤病毒具有更高的溶瘤能力，其对于肿瘤细胞有更高的选择性。证明了该方法的可行性。该方法的优势在于通过细胞膜受体差异表达，提高了肿瘤细胞的选择性，在此基础上，进一步通过该受体选择高亲和力的配体，进一步提高了溶瘤能力和选择性。这种筛选思路未见报道，具有很高的实用性，大大提高了优势溶瘤病毒的筛选速度和筛选针对性。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (26)

  1. 一种构建溶瘤病毒的方法，其特征在于，包括以下步骤：
    a、确定肿瘤细胞特异性受体；
    b、基于病毒配体与所述肿瘤细胞特异性受体的结合力，确定目标病毒配体；
    c、构建包含所述目标病毒配体的溶瘤病毒。
  2. 根据权利要求1所述的方法，其特征在于，所述肿瘤细胞特异性受体是通过下列步骤确定的：
    (i)确定候选细胞膜受体，所述候选细胞膜受体在肿瘤细胞的表达与在正常细胞中的表达相比具有高表达和/或差异表达；
    (ii)基于与病毒配体的结合能力，对所述候选细胞膜受体进行筛选，以便获得所述肿瘤特异性受体，病毒配体与肿瘤特异性受体的结合能力高于病毒配体与正常细胞上的特异性受体的结合能力。
  3. 根据权利要求1-2所述的方法，其特征在于，所述候选细胞膜受体在所述肿瘤细胞的表达与在所述正常细胞中的表达相比具有差异倍数(log2FC)大于等于2.0。
  4. 根据权利要求1-3所述的方法，其特征在于，在步骤(b)中进一步包括：
    利用步骤a获得的所述肿瘤细胞特异性受体，基于所述肿瘤细胞特异性受体与所述病毒配体的结合能力，确定高亲和力病毒配体。
  5. 根据权利要求1-4所述的方法，其特征在于，受体与病毒配体结合能力是基于蛋白质序列分析和/或蛋白共沉淀方法来确定。
  6. 根据权利要求1-5所述的方法，其特征在于，所述基于蛋白质序列分析是通过确定所述受体与所述病毒配体的ZDOCK score确定的，
    可选的，所述ZDOCK score不低于预定阈值，为所述病毒配体是目标病毒配体的指示，其中，所述预定阈值是基于预定溶瘤病毒配体和受体的结合力确定的。
  7. 根据权利要求1-6所述的方法，其特征在于，步骤c包括进一步包括：
    利用所述目标病毒配体，通过病毒组装，获得表达所述目标病毒配体的溶瘤病毒。
  8. 根据权利要求1-7所述的方法，其特征在于，进一步包括：
    d、通过溶瘤能力验证实验判断所构建的溶瘤病毒的溶瘤性能。
  9. 根据权利要求1-8所述的方法，其特征在于，进一步包括：
    e、替换所构建的溶瘤病毒的目标病毒配体外的其他蛋白。
  10. 根据权利要求1-9所述的方法，其特征在于，所述溶瘤病毒为痘苗病毒、疱疹病毒、麻疹病毒、新城疫病毒、腺病毒、甲病毒、细小病毒、弹状病毒或肠道病毒。
  11. 根据权利要求1-10所述的方法，其特征在于，所述溶瘤病毒为柯萨奇病毒B。
  12. 根据权利要求1-10所述的方法，其特征在于，所述溶瘤病毒为VSV病毒。
  13. 根据权利要求6所述的方法，其特征在于，所述溶瘤病毒为VSV病毒，
    可选的，所述阈值为1700，所述ZDOCK score不小于1800，不小于1900，不小于2000或者不小于2100。
  14. 根据权利要求12所述的方法，其特征在于，所述病毒配体为VSV病毒的G蛋白。
  15. 根据权利要求14所述的方法，其特征在于，所述其他蛋白选自VSV病毒的M、P、N或L蛋白。
  16. 根据权利要求12所述的方法，其特征在于，正常细胞上的特异性受体为LDLR。
  17. 根据权利要求11所述的方法，其特征在于，所述病毒配体为柯萨奇病毒B的VP1-VP4蛋白。
  18. 一种溶瘤病毒，其特征在于，是通过权利要求1～17任一项所述的方法构建的。
  19. 根据权利要求18所述的溶瘤病毒，其特征在于，所述溶瘤病毒为REV X03633.1。
  20. 权利要求18所述的溶瘤病毒在制备药物中的用途，所述药物用于治疗或预防癌症或肿瘤。
  21. 根据权利要求20所述的用途，其特征在于，所述溶瘤病毒经配制用于腹膜内、静脉内、动脉内、肌内、皮内、瘤内、皮下或鼻内施用。
  22. 根据权利要求20所述的用途，其特征在于，所述癌症或肿瘤选自肺癌、黑色素瘤、白血病、卵巢癌、中枢神经系统癌症、结肠癌、乳腺癌、肾癌和前列腺癌。
  23. 一种组合物，其特征在于，包含权利要求18或19所述的溶瘤病毒。
  24. 一种药盒，其特征在于，包括：
    (a)第一容器，所述的第一容器中含有第一药物组合物，所述的第一药物组合物含有权利要求21所述的组合物；和
    (b)第二容器，所述的第二容器中含有第二药物组合物，所述的第二药物组合物含有额外的肿瘤治疗剂以及药学上可接受的载体。
  25. 一种预防或治疗肿瘤的方法，其特征在于，包括向受试者施用以下中的至少之一：
    权利要求18～19任一所述的溶瘤病毒；
    权利要求23所述的药物组合物；
    权利要求24所述的药盒。
  26. 根据权利要求25所述的方法，其特征在于，所述肿瘤包括选自肺癌、黑色素瘤、白血病、卵巢癌、中枢神经系统癌症、结肠癌、乳腺癌、肾癌和前列腺癌的至少之一。
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