WO2022218340A1 - 一种重组新城疫病毒rNDV-VEGF-Trap、其基因组、制备方法及其用途 - Google Patents

一种重组新城疫病毒rNDV-VEGF-Trap、其基因组、制备方法及其用途 Download PDF

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WO2022218340A1
WO2022218340A1 PCT/CN2022/086599 CN2022086599W WO2022218340A1 WO 2022218340 A1 WO2022218340 A1 WO 2022218340A1 CN 2022086599 W CN2022086599 W CN 2022086599W WO 2022218340 A1 WO2022218340 A1 WO 2022218340A1
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cancer
newcastle disease
disease virus
recombinant newcastle
vegf
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PCT/CN2022/086599
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English (en)
French (fr)
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肖伟
李德山
刘天艳
王振中
曹玉凯
刘芝航
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江苏康缘瑞翱生物医药科技有限公司
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Priority to JP2023562682A priority Critical patent/JP2024516370A/ja
Priority to US18/554,972 priority patent/US20240124852A1/en
Publication of WO2022218340A1 publication Critical patent/WO2022218340A1/zh

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Definitions

  • the present application belongs to the field of oncolytic viruses for cancer treatment, and in particular, relates to a recombinant Newcastle disease virus genome, a recombinant Newcastle disease virus comprising the genome and a preparation method thereof, and a DNA molecule encoding the above-mentioned recombinant Newcastle disease virus genome and their uses.
  • Cancer is a disease caused by the loss of normal regulation and excessive proliferation of body cells, and has become the number one killer that affects health.
  • my country is a high incidence area of cancer, especially lung cancer, stomach cancer, liver cancer and rectal cancer.
  • various new treatment methods, especially biological drug treatment have been continuously put into clinical use.
  • the needs of drug safety, efficacy and quality of life of patients are far from being met. It is imperative to develop new drugs or treatments.
  • Newcastle disease virus NDV
  • HSV-1 herpes simplex virus type 1
  • reovirus reovirus
  • oncolytic adenovirus oncolytic adenovirus
  • NDV an avian paramyxovirus with a negative-sense single-stranded RNA genome
  • NDV an avian paramyxovirus with a negative-sense single-stranded RNA genome
  • the effect of NDV as a single drug treatment is limited.
  • the body's antiviral immune response can clear the virus, and on the other hand, the body can produce neutralizing antibodies to resist the virus.
  • these viruses were subsequently used to deliver genes with antitumor activity to further enhance the activity.
  • genes include genes encoding cytokines or their receptors, immune checkpoint molecules, tumor suppressor proteins or immune stimulating proteins.
  • angiogenesis is an important target for tumor therapy.
  • Angiogenesis is the process of generating new blood vessels from existing endothelial cells to provide sufficient oxygen and nutrients to various organs. It is essential for tumor growth and metastasis.
  • Anti-angiogenic therapy is one of the important methods of cancer treatment. one.
  • the pro-angiogenic factors that support tumor growth mainly include: vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and angiogenin.
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • angiogenin angiogenin.
  • TGF- ⁇ transforming growth factor- ⁇
  • anti-angiogenic drugs target angiogenic growth factors and their receptors, or key molecules in downstream signaling pathways, thereby inhibiting tumor growth and metastasis by blocking tumor nutrient supply.
  • the current FDA-approved anti-angiogenic drugs mainly include macromolecular monoclonal antibodies and small-molecule targeted inhibitors.
  • anti-angiogenic factors mainly include platelet response protein-1 (TSP-1), angiostatin (angiostain), endostatin (endostain) and interferon- ⁇ (IFN- ⁇ ), etc., as well as VEGF blocking/antagonism Agents such as VEGF-Trap (obtained by fusing the Ig domain of VEGFR with the constant region of IgG molecules), these inhibitors can directly inhibit the proliferation and migration activities of vascular endothelial cells, thereby inhibiting angiogenesis, and blocking tumor growth and Metastasis, can exhibit beneficial effects in cancer therapy.
  • anti-angiogenic drugs have significant side effects.
  • the inventors of the present application provide a corresponding recombinant oncolytic virus by integrating the encoding genes of Angiostatin and VEGF-Trap into specific positions of the Newcastle disease virus genome respectively.
  • the anti-tumor effect of -Trap is significantly higher than that of rNDV group and rNDV-Angiostatin group, it can replicate in cancer cells with strong replication ability to kill host cancer cells, and at the same time, it has reliable safety for non-cancer cells. This solves the above technical problem.
  • the present application provides a recombinant Newcastle disease virus genome, wherein the genome includes a VEGF-Trap encoding gene, and the VEGF-Trap encoding gene is located between the P gene and the M gene of the Newcastle disease virus genome .
  • the present application provides a recombinant Newcastle disease virus, wherein the virus comprises the above-mentioned recombinant Newcastle disease virus genome.
  • the present application provides a DNA molecule encoding the above-mentioned recombinant Newcastle disease virus genome.
  • the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus and/or DNA molecules.
  • the application provides a method for preparing the above-mentioned recombinant Newcastle disease virus, wherein the method comprises:
  • the cloning vector containing the DNA sequence encoding the gene of VEGF-Trap and the NDV viral vector are respectively digested with enzymes, and the DNA sequence encoding the VEGF-Trap gene obtained by the enzyme digestion is digested with the NDV viral vector. connected to obtain a recombinant Newcastle disease virus plasmid;
  • the present application provides the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition in the preparation of a medicament for treating or ameliorating cancer.
  • the present application provides the above-described recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecules and/or pharmaceutical compositions for use in the treatment or amelioration of cancer.
  • the present application provides the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition for treating or improving cancer.
  • the present application provides a method of treating or ameliorating cancer, comprising administering the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition to a subject in need thereof.
  • the antitumor effect and oncolytic efficiency of the obtained recombinant oncolytic virus can be significantly improved.
  • Figure 1 shows the results of Western Blot detection of the allantoic fluid in Example 1, wherein the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in Example 1 can stably express the exogenous gene VEGF-Trap.
  • Figure 2 shows the proliferation curve of each recombinant Newcastle disease virus and parental virus inoculated in DF-1 cells.
  • Figure 3 shows tumor growth curves in mice of the negative control group and each recombinant Newcastle disease virus and parental virus treated group.
  • Figure 4 shows the tumor inhibition results of mice in the negative control group and each recombinant Newcastle disease virus and parental virus-treated group.
  • Figure 5 is a photograph showing tumors in mice of the negative control group and each of the recombinant Newcastle disease virus and parental virus treated groups.
  • Figure 6 shows the HE staining results of the negative control group and each recombinant Newcastle disease virus and parental virus treatment groups, wherein the tumor tissue structure of the mice in the negative control group is dense, the cell morphology is intact, and the growth is vigorous; the tumors of the mice in the rNDV group The lesions were disintegrated, and the tumor cell structure was relatively loose; the tumor structure of the mice in the rNDV-Angiostatin group was not significantly different from that in the rNDV group, while the tumor tissue lesions of the mice in the rNDV-VEGF-Trap group were largely disintegrated, the tumor cell structure was very loose, and there were many immune cells. Infiltrated, tumor cells were single scattered.
  • Figure 7 shows the results of immunohistochemical staining, in which the expression of CD34 in the negative control group was abundant, the rNDV group was similar to the negative control group, and the CD34 expression in the rNDV-VEGF-Trap group was significantly reduced.
  • Figure 8 shows the inhibition of tumor growth in mouse liver cancer model by rClone30-Anh-(F) treatment group, rClone30-Anh-(F)-Angiostatin treatment group and rClone30-Anh-(F)-VEGF-Trap treatment group .
  • FIG. 9 shows the genome sequence of the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in Example 1.
  • FIG. 9 shows the genome sequence of the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in Example 1.
  • Example 10 shows the genome sequence of the recombinant Newcastle disease virus rClone30-Anh-(F)-VEGF-Trap prepared in Example 5.
  • treating means curing, alleviating, alleviating, alleviating or ameliorating a disease or disease-related symptoms, or preventing, delaying, arresting, suspending, or halting a disease in a statistically significant manner, unless otherwise specified. or the onset or further development of related symptoms.
  • percent identity As used herein, the percent identity (degree of homology) between sequences can be compared between two sequences, for example, by using freely available computer programs commonly used for this purpose on the World Wide Web (such as BLASTp or BLASTn with default settings) to make sure.
  • Newcastle disease virus belongs to the family Paramyxoviridae, the order of single-molecule negative-stranded RNA viruses, and has an envelope; the nucleocapsid is located in the envelope and contains the RNA genome and nucleocapsid protein.
  • the full-length genome of the classic Newcastle disease virus is about 15-16kb, and it contains NP gene, P gene, M gene, F gene, HN gene and L gene from the 3' end to the 5' end, which are respectively used to encode the following 6 species Main proteins: Nucleocapsid Protein (NP), Phosphate Protein (P), Matrix Protein (M), Fusion Protein (F), Hemagglutinin-Neuraminidase Protein (Haemagglutinin Neuraminidase Protein, HN) and RNA-dependent RNA polymerase (Large Protein, L).
  • NP Nucleocapsid Protein
  • P Phosphate Protein
  • M Matrix Protein
  • F Fusion Protein
  • Haemagglutinin Neuraminidase Protein Haemagglutinin Neuraminidase Protein
  • L RNA-dependent RNA polymerase
  • the present application relates to a recombinant Newcastle disease virus genome, wherein the genome includes a VEGF-Trap encoding gene, and the VEGF-Trap encoding gene is located in the P gene and the M gene of the Newcastle disease virus genome between.
  • the gene encoding the VEGF-Trap may be in the form of DNA or RNA.
  • the gene encoding VEGF-Trap has the sequence shown in SEQ ID NO. 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical sequences.
  • the sequence of the recombinant Newcastle disease virus genome is shown as SEQ ID NO. 2 or SEQ ID NO. 5 (see Figures 9 and 10).
  • the present application relates to a recombinant Newcastle disease virus, wherein the virus comprises the above-mentioned recombinant Newcastle disease virus genome.
  • the starting strain of the Newcastle disease virus can be selected from but not limited to: attenuated strains LaSota, Hitchner B1, V4, virulent strains Mukteswar, Anhinga, virulent strains F48E9, JS/7/2017 Ch, juice, Herts/33, NDV-BJ; and any chimeric strain constructed by genetic engineering based on the originating strain, but not limited thereto.
  • the present application relates to a DNA molecule (eg, a recombinant Newcastle disease virus plasmid) encoding the recombinant Newcastle disease virus genome described above.
  • a DNA molecule eg, a recombinant Newcastle disease virus plasmid
  • the present application relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus and/or DNA molecules.
  • the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable pharmaceutical excipients can be selected from, for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc.
  • solvents for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc.
  • the present application relates to a method for preparing the above-mentioned recombinant Newcastle disease virus, wherein the method comprises:
  • the cloning vector containing the DNA sequence encoding the gene of VEGF-Trap and the NDV viral vector are respectively digested with enzymes, and the DNA sequence encoding the VEGF-Trap gene obtained by the enzyme digestion is digested with the NDV viral vector. connected to obtain a recombinant Newcastle disease virus plasmid;
  • the cloning vector can be constructed using a vector selected from the group consisting of: PUC57 vector, pMD18-T vector, pMD19-T vector, pBlueScript SK (+/-) vector, pBluescript II KS (+/-) ).
  • the NDV viral vector may be a full-length cDNA sequence of the genome of the NDV virus selected from the group consisting of: attenuated strains LaSota, Hitchner B1, V4, strains Mukteswar, Anhinga, virulent strains F48E9, JS /7/2017Ch, juice, Herts/33, NDV-BJ, but not limited to.
  • the NDV viral vector may be a pBluescript II KS(+/-)-NDV(pBrNDV), pCI-neo-NDV, pOLTV5-NDV vector.
  • the recombinant Newcastle disease virus plasmids were combined with helper plasmids NP, P and L capable of expressing nucleocapsid protein NP, phosphorylated protein P and RNA-dependent RNA polymerase L (they can be NP, P and L).
  • the gene is constructed to any NP, P, L recombinant plasmid obtained from any eukaryotic expression vector known in the art) and co-transfected into the cells.
  • the genes of the helper plasmids NP, P and L can be derived from any strain of NDV, such as LaSota, Anhinga, F48E9 and the like.
  • the recombinant Newcastle disease virus plasmid is co-transfected into cells with a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM-L; pCI-neo-NP, pCI- neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP(pBL-NP), pBluescript II KS(+/-)-P(pBL-P) and pBluescript II KS(+/- )-L(pBL-L), but not limited thereto.
  • a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM-L; pCI-neo-NP, pCI- neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP(pBL-NP), pBluescript II KS(+/
  • transfection is a technique for introducing exogenous nucleic acid substances (including DNA and RNA) into cells, mainly including physical mediation (electroporation, microinjection and gene gun), chemical mediation (calcium phosphate co- Precipitation method, liposome transfection, cationic substance-mediated) and biologically-mediated (protoplast transfection, virus-mediated transfection) three types of pathways.
  • specific operations can be selected by those skilled in the art based on common knowledge in the field (for example, please refer to "Molecular Cloning Experiment Guide” (4th Edition), edited by J. Sambrook et al., translated by He Fuchu, Science Press, 2017) Appropriate experimental conditions and procedures, or according to the instructions in commercially available kits.
  • the cells may be selected from, but not limited to, BHK-21 cells, BSR-T7/5 cells, VERO cells, DF-1 cells, 293 cells, MDCK cells.
  • the culture of the transfected cells can be carried out by those skilled in the art by selecting conventional culture medium and culture conditions according to the type of cells (Liu Bin, editor-in-chief, “Cell Culture (3rd Edition)", World Book Publishing Company, January 2018; Editor-in-chief of Lan Rong and Zhou Zhenhui, “Cell Culture Technology", Chemical Industry Press, August 2007; Editor-in-chief of Zhang Jingbo, “Tissue and Cell Culture Technology (3rd Edition)", People's Medical Publishing House, 2014 June, etc.).
  • the present application relates to the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition in the preparation of a medicament for the treatment or amelioration of cancer.
  • the present application provides the above-described recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecules and/or pharmaceutical compositions for use in the treatment or amelioration of cancer.
  • the present application provides the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition for treating or ameliorating cancer.
  • a method of treating or ameliorating cancer comprising administering the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition to a subject in need thereof.
  • the cancer may be selected from, but not limited to, colon cancer, liver cancer (eg, hepatocellular carcinoma), lung cancer (eg, non-small cell lung cancer, small cell lung cancer), gastric cancer, rectal cancer, leukemia, lymphatic Tumor, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell cancer, thyroid cancer, head and neck cancer cancer, testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, Sarcoma, Uterine Cancer, Glioblastoma, Medulloblastoma, Neuroblastoma, Kidney Cancer, Astrocytodecanceride,
  • a recombinant Newcastle disease virus genome wherein the genome comprises a gene encoding VEGF-Trap, and the gene encoding VEGF-Trap is located between the P gene and the M gene of the Newcastle disease virus genome.
  • a recombinant Newcastle disease virus wherein the virus comprises the recombinant Newcastle disease virus genome of any of paragraphs 1-4.
  • Newcastle disease virus as described in paragraph 5, wherein, the starting strain of described Newcastle disease virus is selected from: attenuated strain LaSota, Hitchner B1, V4, poisonous strain Mukteswar, Anhinga, virulent strain F48E9, JS/7 /5Ch, juice, Herts/33, NDV-BJ; and any chimeric strain constructed by genetic engineering based on the originating strain.
  • described pharmaceutical composition comprises the Newcastle disease virus genome of the reorganization described in any one paragraph in paragraph 1-4, the recombination Newcastle disease virus described in paragraph 5 or 6 and/or the recombination Newcastle disease virus described in paragraph 7. the DNA molecule described.
  • the pharmaceutically acceptable pharmaceutical adjuvant is selected from the group consisting of solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, disintegrant, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants or thinner.
  • the cloning vector containing the DNA sequence encoding the gene of VEGF-Trap and the NDV viral vector are respectively digested with enzymes, and the DNA sequence encoding the VEGF-Trap gene obtained by the enzyme digestion is digested with the NDV viral vector. connected to obtain a recombinant Newcastle disease virus plasmid;
  • the cloning vector is constructed using a vector selected from the group consisting of: PUC57 vector, pMD18-T vector, pMD19-T vector, pBlueScript SK (+/-) vector, pBluescript II KS ( +/-).
  • NDV viral vector is a full-length cDNA sequence selected from the genome of the following NDV viruses: attenuated strains LaSota, Hitchner B1, V4, strains Mukteswar, Anhinga, strong strains Strain F48E9, JS/7/2017Ch, juice, Herts/33, NDV-BJ.
  • NDV viral vector is a pBluescript II KS(+/-)-NDV(pBrNDV), pCI-neo-NDV, pOLTV5-NDV vector.
  • a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM -L; pCI-neo-NP, pCI-neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP, pBluescript II KS(+/-)-P and pBluescript II KS(+/ -)-L.
  • cells are selected from BHK-21 cells, BSR-T7/5 cells, VERO cells, DF-1 cells, 293 cells, or MDCK cells.
  • the recombinant Newcastle disease virus genome of any of paragraphs 1-4, the recombinant Newcastle disease virus of paragraphs 5 or 6, the DNA molecule of paragraph 7, and/or the paragraph of any of paragraphs 8-10 Use of the pharmaceutical composition in the preparation of a medicament for treating or improving cancer.
  • the cancer is selected from colon cancer, liver cancer, lung cancer, stomach cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urinary Epithelial cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell cancer, thyroid cancer, head and neck cancer, testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary gland cancer, Skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma , neuroblastoma, kidney cancer, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma,
  • the following examples relate to the following exogenous genes: the vascular inhibitor gene VEGF-Trap and the human angiostatin gene Angiostatin (Genbank Accession No. NG_016200.1).
  • pMD19-T was purchased from Bao Bioengineering (Dalian) Co., Ltd. (Dalian TaKaRa Company).
  • BHK-21 cells baby hamster kidney cells
  • human colon cancer cells HCT116 human colon cancer cells
  • mouse colon cancer cells CT26 mouse breast cancer cells 4T1
  • human umbilical vein endothelial cells EA.hy926 were purchased from ATCC.
  • DMEM high glucose
  • McCoy'5A medium fetal bovine serum
  • FBS fetal bovine serum
  • SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd.
  • Balb/c mice were purchased from Speifu (Beijing) Biotechnology Co., Ltd.
  • VEGF-Trap A VEGF blocker with potent antitumor effects, August 2002; https://doi.org/10.1073/pnas.172398299
  • SEQ ID NO. .1 The sequence of the VEGF-Trap gene (SEQ ID NO. .1), the sequence looks like this:
  • the recombinant plasmid pBrNDV-VEGF-Trap was constructed as follows:
  • VEGF-Trap gene containing the restriction sites of SacII enzyme (5') and PmeI enzyme (3') was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and ligated to the pUC57 vector to form pUC57-VEGF-Trap for use in follow-up experiments.
  • the plasmid pUC57-VEGF-Trap in step 1 was digested with restriction enzymes PmeI and SacII (purchased from NEB Company) according to the manufacturer's product instructions, and the digested product was identified by nucleic acid agarose gel electrophoresis. , after the identification is correct, the enzyme cleavage product is recovered by the gel recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: DP219) according to the manufacturer's product instructions.
  • PmeI and SacII purchased from NEB Company
  • step 4 Connect the enzyme-digested product of step 2 and the vector of step 3 with T4 DNA ligase (purchased from NEB) according to the manufacturer's product instructions to obtain a recombinant Newcastle disease virus plasmid pBrNDV-VEGF-Trap, wherein the VEGF-Trap gene Inserted between the P and M genes of the plasmid.
  • T4 DNA ligase purchased from NEB
  • the above-mentioned recombinant plasmids were subjected to PCR using Baobi taq enzyme (purchased from Baobi) according to the manufacturer's instructions (upstream primer: 5'TCAAGCGCCTTGCTCTAAATGGC 3' (SEQ ID NO.3); downstream primer: 5'GGGCAGAATCAAAGTACAGCCCAAT 3' (SEQ ID NO.4)) and PmeI and SacII double-enzyme digestion identification (37°C, 1h), the correct plasmid samples were packaged and sent to Shanghai Sangon Bioengineering Company for sequencing. The sequencing results were compared using the sequence analysis software DNAMAN. After sequencing, the sequenced sequence was consistent with the target sequence.
  • the recombinant Newcastle disease virus rNDV-VEGF-Trap was prepared by using the above-mentioned recombinant Newcastle disease virus plasmid by the following method:
  • step 2 Take 200 ⁇ L of the supernatant obtained in step 1, inoculate it into the allantoic cavity of 9-day-old SPF chicken embryos, and then incubate in an incubator at 37 °C and 5% CO 2 for 72 h to obtain allantoic fluid, and carry out Hemagglutination titer test (eg Zhou Ling, Li Y rotating, Ma Xiali, Isolation and identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary, 2015, 40(03): 8-10), positive Allantoic fluid was cryopreserved at -80°C, and the successfully rescued recombinant Newcastle disease virus was named rNDV-VEGF-Trap.
  • Hemagglutination titer test eg Zhou Ling, Li Y rotating, Ma Xiali, Isolation and identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary, 2015
  • step 4 Take the virus with the correct exogenous gene sequencing in step 3, inoculate it into the allantoic cavity of new 9-11-day-old SPF chicken embryos, place it at 37°C for 72 hours, and collect the chicken embryo allantoic fluid for HA detection (eg. See related methods recorded in Zhou Ling, Li Y rotating, Ma Xiali, Isolation and identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary Medicine, 2015, 40(03):8-10), select the HA titer Allantoic fluid greater than 29 was mixed and then dispensed for use.
  • HA detection eg. See related methods recorded in Zhou Ling, Li Y rotating, Ma Xiali, Isolation and identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary Medicine, 2015, 40(03):8-10
  • the recombinant Newcastle disease virus rNDV-Angiostatin was prepared according to the above method.
  • rNDV-VEGF-Trap and rNDV-Angiostatin refer to recombinant viruses prepared by the above methods using recombinant plasmids pBrNDV-VEGF-Trap and pBrNDV-Angiostatin, respectively.
  • the parental virus in the following embodiment 2-3 refers to starting from Newcastle disease virus LaSota (purchased from Harbin Veterinary Epidemic Prevention Station), according to Wang Yong et al. 2008, 48(5): 638-643), the engineering method of "gene replacement", using the F gene (GenBank accession number: AY508514.1) of the Newcastle disease virus virulent strain F48E9 to replace the LaSota strain.
  • the F gene is modified, and the parental strain is named rNDV herein.
  • Embodiment 2 Detection of recombinant virus proliferation stability
  • TCID50 The determination of TCID50 is carried out according to the following method:
  • rNDV-VEGF-Trap and rNDV-Angiostatin recombinant Newcastle disease virus
  • parental virus discard the original cell culture medium in the culture plate of step 1, and add 10% allantoic fluid, 180 ⁇ L of fresh DMEM medium with 1% antibiotics.
  • the mouse colon cancer cell CT26 was taken and stained with trypan blue to determine that the cell viability was over 95%.
  • the cell suspension was diluted with physiological saline to form a cell suspension of 1 ⁇ 10 6 /mL.
  • a dose of 0.1 mL was injected subcutaneously into the right abdomen of the mouse. After 8-12 days, the diameter of the solid tumor reaches 5-8mm, the modeling is successful, and subsequent experiments can be carried out. Individuals with large differences in tumor shape and size were removed, and mice with a tumor diameter of 5-8 mm were used as model mice.
  • mice were randomly divided into 4 groups with 10 mice in each group, and were treated as follows:
  • rNDV-VEGF-Trap group 0.2 mL of the PBS suspension of rNDV-VEGF-Trap virus prepared in Example 1 (prepared with 1 ⁇ PBS buffer; containing 10 7 pfu virus) was injected into the tumor of model mice every day for treatment 14 days;
  • rNDV-Angiostatin group 0.2 mL of the PBS suspension of rNDV-Angiostatin virus (prepared with 1 ⁇ PBS buffer; containing 10 7 pfu virus) prepared in Example 1 was injected into the tumor of model mice every day for 14 days;
  • rNDV group 0.2 mL of parental virus PBS suspension (prepared with 1 ⁇ PBS buffer; containing 10 7 pfu virus) was injected into the tumor of model mice every day for 14 days;
  • Negative control group (model group): 0.2 mL of SPF chicken embryo allantoic fluid was injected into the tumor of model mice every day for 14 days.
  • tumor volume was measured every other day, and a tumor growth curve was prepared based on the measurement results (Fig. 3).
  • the mice were euthanized, the tumors were exfoliated, and the tumor weight and size were measured (results are shown in Figures 4 and 5).
  • the average tumor volume in the negative control group was 1889.17 mm 3
  • the average tumor volume in the parental NDV (rNDV) treatment group was 728.49 mm 3
  • the average tumor volume in the rNDV-Angiostatin treatment group was 774.37 mm 3 ;
  • the mean tumor volume was 350.36 mm 3 .
  • the tumor tissue of each group of mice was taken, fixed with 4% paraformaldehyde, and the paraffin section of the tumor tissue with a thickness of 4 ⁇ m was prepared as follows: After HE staining, the tumor tissue morphology of each group was observed under a microscope, and the expression of CD34 protein in the tumor tissue of each group was detected by immunohistochemical staining. The specific operations are as follows:
  • the lung tissue of each group of mice was taken and placed in a pre-prepared 4% neutral formaldehyde fixative solution for 48 hours, the tissue was taken out, and the tissue block was trimmed to a size of about 1 cm with a razor blade.
  • Wax immersion The transparent tissue was transferred into the fully dissolved paraffin in (1), and placed in a 60°C incubator for 120 min.
  • Trimming the wax block After the paraffin in the carton has solidified, take out the wax block and trim it into a trapezoid shape with a blade.
  • the distance between the edge of the tissue and the edge of the wax block shall not be less than 2mm.
  • Dewaxing and hydration Immerse the prepared paraffin sections in xylene for dewaxing, immerse twice for 5 minutes each time, and then put in 100vol%, 95vol%, 90vol%, 80vol%, 70vol% of alcohol at all levels In the solution for 5min each, then rinsed twice in distilled water, 3min each time.
  • Blocking A blocking solution (purchased from Beyotime; Item No. P0260QuickBlock) was added dropwise to block the tissue section for 10 min.
  • Incubation of secondary antibody prepare a working solution of goat anti-rabbit secondary antibody (purchased from Abcam) with blocking solution according to the dilution ratio of the antibody specification, add the working solution of secondary antibody dropwise to each tissue section, and incubate at room temperature for 1 h. After secondary antibody incubation, tissue sections were washed three times with 1 ⁇ PBST buffer for 5 min each time.
  • a working solution of goat anti-rabbit secondary antibody purchased from Abcam
  • Color development 100 ⁇ l of DAB color development working solution (purchased from Beyotime) was added dropwise to fully cover the sample. Incubate in the dark at room temperature for 15 min. After color development, remove the DAB color development working solution and wash with distilled water for 1-2 times to stop the color reaction.
  • DAB color development working solution purchased from Beyotime
  • the tumor tissue structure of the mice in the negative control group was dense, the cell morphology was intact, and the growth was vigorous; the tumor lesions of the mice in the rNDV group were disintegrated, and the tumor cell structure was loose; the mice in the rNDV-Angiostatin group were The tumor structure of the rNDV-VEGF-Trap group was not significantly different from that of the rNDV group, while the tumor tissue lesions of the mice in the rNDV-VEGF-Trap group were largely disintegrated, the tumor cell structure was very loose, the immune cells were infiltrated in multiple places, and the tumor cells were single scattered (see Figure 6).
  • Embodiment 4 Safety detection of recombinant Newcastle disease virus
  • mice Healthy 6-week SPF Balb/c mice were selected and divided into groups, 10 mice in each group. The mice in the control group were fed normally. The mice in the experimental group were each intraperitoneally injected with 5 ⁇ 10 8 pfu (10 times the therapeutic dose) of recombinant Newcastle disease virus rNDV-VEGF-Trap and observed for 30 days. Mice with obvious adverse reactions such as lethargy, ruffled fur and death were positive.
  • mice in the experimental group stood up, and their diet and drinking were not affected.
  • the fur of the mice in the experimental group returned to normal, and the mice in the experimental group had no obvious adverse reactions (including lethargy, erect fur, etc.) ), and no mice died.
  • the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in this application has good safety.
  • the oncolytic virus rClone30-Anh-(F) as the parental strain provided in the following example is based on the LaSota strain of Newcastle disease virus (purchased from Harbin Veterinary Epidemic Prevention Station), according to Wang Yong et al. (Acta Microbiology, 2008, 48 (5). ): 638-643, the same as above)
  • the engineering method for "gene replacement" is obtained by replacing the F gene of the LaSota strain with the F gene of the Newcastle disease virus strain Anhinga (GenBank accession number: EF065682.1).
  • the genome of the recombinant Newcastle disease virus expressing VEGF-Trap of this example is shown in SEQ ID NO. 5 (see FIG. 10 ); the nucleic acid sequence of VEGF-Trap is shown in SEQ ID NO. 1.
  • VEGF-Trap gene SEQ ID NO.1
  • Angiostatin gene NG_016200.1
  • rClone30-Anh-(F)-VEGF-Trap rClone30-Anh-(F)-Angiostatin, respectively.
  • H22 cells purchased from Nanjing Kebai
  • H22 subcutaneous tumor-bearing model ie, a mouse liver cancer model.
  • the tumor grows to about 100 mm3
  • oncolytic viruses rClone30-Anh-(F)-Angiostatin and rClone30-Anh-(F)-VEGF-Trap
  • the parental strain rClone30-Anh into the tumor.
  • -(F) Suspension in PBS prepared with IX PBS buffer).
  • each oncolytic virus (rClone30-Anh-(F)-Angiostatin, rClone30-Anh-(F)-VEGF-Trap and rClone30-Anh-(F)) was injected intratumorally once a day, respectively, A total of 14 days of injection, each injection of 1 ⁇ 10 7 PFU; 1 ⁇ PBS buffer (without oncolytic virus) was injected into the tumor of the mouse liver cancer model as a negative control group (also called "PBS treatment group"); There were 6 animals in each group, and the therapeutic effect of each recombinant virus was observed by dissecting tumor tissue.
  • the average tumor volume of the negative control group was 1421.77 mm 3
  • the average tumor volume of the parental rClone30-Anh-(F) treatment group was 807.30 mm 3
  • the rClone30-Anh-(F)-Angiostatin The mean tumor volume in the treatment group was 668.60 mm3 ; the mean tumor volume in the rClone30-Anh-(F)-VEGF-Trap treatment group was 326.05 mm3 .
  • the results showed that, compared with the negative control group, the parental strain rClone30-Anh-(F) treatment group, rClone30-Anh-(F)-Angiostatin treatment group and rClone30-Anh-(F)-VEGF-Trap treatment group could inhibit the Tumor growth, especially in the rClone30-Anh-(F)-VEGF-Trap treated group, showed the smallest mean tumor volume.

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Abstract

本申请公开了一种重组的新城疫病毒基因组、包含该基因组的重组新城疫病毒rNDV-VEGF-Trap及其制备方法、编码上述的重组的新城疫病毒基因组的DNA分子以及它们在制备用于治疗癌症的药物中的用途。本申请所述的重组新城疫病毒涉及将VEGF-Trap的编码基因插入其基因组中，由此得到的重组新城疫病毒以较强的复制能力进行复制从而杀死宿主癌细胞，同时对非癌细胞具有可靠的安全性，并且表现出提高的抗肿瘤作用和溶瘤效率。

Description

一种重组新城疫病毒rNDV-VEGF-Trap、其基因组、制备方法及其用途 技术领域
本申请属于癌症治疗用溶瘤病毒领域，具体而言，涉及一种重组的新城疫病毒基因组、包含该基因组的重组新城疫病毒及其制备方法、编码上述的重组的新城疫病毒基因组的DNA分子以及它们的用途。
背景技术
癌症是由于机体细胞失去正常调控、过度增殖而引起的疾病，目前已成为影响健康的第一杀手。我国是癌症尤其是肺癌、胃癌、肝癌及直肠癌高发区。据统计，仅2016年一年全国新增各类癌症患者480万例，230万病人死于各种癌症。随着技术进步，各种新的治疗手段尤其是生物药物治疗不断投入临床使用。但药物的安全性、有效性及病人的生活质量等方面的需求远未得以满足。发展新药或治疗手段势在必行。
1991年，Martuza等人在《Science》杂志发表文章，证明转基因单纯疱疹病毒在恶性胶质瘤治疗中有一定的效果。至此以后，发展溶瘤病毒治疗癌症就日益受到关注。溶瘤病毒治疗癌症的原理是通过对自然界存在的一些致病力较弱的病毒进行基因改造，让其选择性地感染肿瘤细胞、在细胞内大量复制并最终摧毁肿瘤细胞。同时它还能激发免疫反应，吸引免疫细胞来继续杀死残余癌细胞或通过免疫反应杀灭已迁移的癌细胞。近几十年来，溶瘤病毒相关研究取得了巨大进展。人们先后用新城疫病毒(Newcastle disease virus，NDV)、1型单纯疱疹病毒(HSV-1)、呼肠孤病毒(reovirus)、溶瘤腺病毒(oncolytic adenovirus)来开发溶瘤病毒，但临床表现都远低于预期。例如，2005年CFDA批准了溶瘤腺病毒产品H101上市，然而其治疗效果并不理想。
NDV是一种具有负义单链RNA基因组的禽副粘病毒，它一直是癌症治疗的有希望的方法。但NDV作为单一药物治疗的效果有限，一方面，人体抗病毒免疫反应能够清除病毒，另一方面，人体能够产生中和抗体来抵抗病毒发挥作用。为了提高NDV在癌症中的治疗效率，这些病毒随后用来传递具有抗肿瘤活性的基因，以进一步增强活性。这样的基因包括编码细胞因子或其受体、免疫检查点分子、肿瘤抑制蛋白或免疫刺激蛋白的基因。
此前，本领域已对在其基因组中整合有编码细胞因子或其受体的基因的重组新城疫病毒进行了众多研究，但仍未取得临床上有益的进展。例如，Pascal Buijs等(Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment ofPancreatic Adenocarcinoma，Viruses 2015，7，2980-2998)研究了表达干扰素或干扰素拮抗蛋白的重组新城疫病毒。
研究表明血管生成是肿瘤治疗的重要靶标。血管生成是从现有的内皮细胞中生成新的血管，进而向各种器官提供充足氧气和营养的过程，其对于肿瘤的生长和转移至关重要，抗血管生成疗法是癌症治疗的重要方法之一。在肿瘤新生血管形成过程中，支持肿瘤生长的促血管生成因子主要有：血管内皮细胞生长因子(VEGF)、血小板源性生长因子(PDGF)、表皮生长因子(EGF)、血管生成蛋白(angiogenin)和转化生长因子-β(TGF-β)等，这些因子通过与相应的受体结合，激活下游信号通路，从而调控肿瘤新血管的生成。
大多数抗血管生成药物是靶向作用于促血管生长因子及其受体，或下游信号通路中的关键分子，通过阻断肿瘤的营养供应，从而抑制肿瘤的生长和转移。现有获FDA批准上市的抗血管生成药物主要包括大分子单抗药物和小分子靶向抑制剂。其中，抗血管生成因子主要有血小板应答蛋白-1(TSP-1)、血管抑素(angiostain)、内皮抑素(endostain)以及干扰素-α(IFN-α)等，以及VEGF阻断/拮抗剂如VEGF-Trap(通过将VEGFR的Ig结构域与IgG分子的恒定区融合得到)，这些抑制剂可直接抑制血管内皮细胞的增殖与迁移活性，从而抑制血管生成，并阻断肿瘤的生长与转移，能够在癌症治疗中表现出有益的效果。然而，抗血管生成药物的治疗副作用较大。
针对现有技术中存在的NDV作为单一药物的治疗效果有限、响应率以及肿瘤抑制率较小等限制溶瘤病毒的临床应用的问题，对于可以提高抗肿瘤效果、降低副作用的表达抗肿瘤活性的外源蛋白的重组NDV目前仍需要进行深入的研究。
发明内容
本申请的发明人通过将Angiostatin、VEGF-Trap的编码基因分别整合入新城疫病毒基因组的特定位置，提供一种相应的重组溶瘤病毒，经药效试验验证，发现重组溶瘤病毒rNDV-VEGF-Trap的抗肿瘤效果显著高于rNDV组和rNDV-Angiostatin组，其可以在癌细胞中以较强的复制能力进行复制从而杀死宿主癌细胞，同时对非癌细胞具有可靠的安全性，由此解决上述技术问题。
一方面，本申请提供了一种重组的新城疫病毒基因组，其中，所述基因组包括VEGF-Trap的编码基因，所述VEGF-Trap的编码基因位于新城疫病毒基因组的P基因与M基因之间。
另一方面，本申请提供了一种重组新城疫病毒，其中，所述病毒包含上述的重组的新城疫病毒基因组。
又一方面，本申请提供了一种编码上述的重组的新城疫病毒基因组的DNA分子。
又一方面，本申请提供了一种药物组合物，其中，所述药物组合物包含上述的重组的新城疫病毒基因组、重组新城疫病毒和/或DNA分子。
又一方面，本申请提供了一种制备上述重组新城疫病毒的方法，其中，所述方法包括：
(1)将含有VEGF-Trap的编码基因的DNA序列的克隆载体与NDV病毒载体分别进行酶切，并将酶切得到的所述VEGF-Trap的编码基因的DNA序列与所述NDV病毒载体进行连接，得到重组新城疫病毒质粒；
(2)将所述重组新城疫病毒质粒转染至细胞中并对经转染的细胞进行培养，获得所述重组新城疫病毒。
又一方面，本申请提供了上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物在制备用于治疗或改善癌症的药物中的用途。或者，本申请提供了用于治疗或改善癌症的上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物。或者本申请提供了上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物用于治疗或改善癌症的用途。或者本申请提供了一种治疗或改善癌症的方法，包括向有需要的受试者给予上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物。
通过将VEGF-Trap的编码基因整合入新城疫病毒基因组的特定位置，能够显著提高所得到的重组溶瘤病毒的抗肿瘤作用和溶瘤效率。
附图说明
为了更清楚地说明本申请示例性的技术方案，下面将对附图进行简单介绍，应当理解，以下附图仅示出了本申请的示例性方案，因此不应被看作是对保护范围的限定。
图1示出了实施例1中的尿囊液的Western Blot检测结果，其中，实施例1制备的重组新城疫病毒rNDV-VEGF-Trap能够稳定表达外源基因VEGF-Trap。
图2示出了接种于DF-1细胞中的各重组新城疫病毒和亲本病毒的增殖曲线。
图3示出了阴性对照组以及各重组新城疫病毒和亲本病毒处理组的小鼠中的肿瘤生长曲线。
图4示出了阴性对照组以及各重组新城疫病毒和亲本病毒处理组的小鼠的肿瘤抑制结果。
图5是示出了阴性对照组以及各重组新城疫病毒和亲本病毒处理组的小鼠中的肿瘤的照片。
图6示出了阴性对照组以及各重组新城疫病毒和亲本病毒处理组的HE染色结果，其中，阴性对照组小鼠的肿瘤组织结构致密，细胞形态完好，生长旺盛；rNDV组小鼠的肿瘤病灶解体，肿瘤细胞结构较为松散；rNDV-Angiostatin组小鼠的肿瘤结构与rNDV组无明显差异，而rNDV-VEGF-Trap组小鼠的肿瘤组织病灶大量解体，肿瘤细胞结构非常松散，免疫细胞多处浸润，肿瘤细胞呈单个分散。
图7示出了免疫组织化学染色结果，其中，阴性对照组小鼠的CD34表达量丰富，rNDV组与阴性对照组类似，而rNDV-VEGF-Trap组小鼠的CD34表达量明显减少。
图8示出了rClone30-Anh-(F)治疗组、rClone30-Anh-(F)-Angiostatin治疗组和rClone30-Anh-(F)-VEGF-Trap治疗组对小鼠肝癌模型肿瘤生长的抑制情况。
图9示出了实施例1制备的重组新城疫病毒rNDV-VEGF-Trap的基因组序列。
图10示出了实施例5制备的重组新城疫病毒rClone30-Anh-(F)-VEGF-Trap的基因组序列。
具体实施方式
在下文中对本发明的示例的实施方式进行描述，但是本发明的保护范围不限于此。除非另外定义，否则本文使用的技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。参见如Singleton等，Dictionary of Microbiology and Molecular Biology 2nd ed.，J.Wiley&Sons(New York，NY 1994)；Sambrook等，Molecular Cloning，A Laboratory Manual，Cold Springs Harbor Press(Cold Springs Harbor，NY 1989)。
在本文中，除非另有说明，术语“治疗”是指以统计学显著的方式治愈、减轻、缓解、减缓、缓和或改进疾病或疾病相关的症状，或者预防、延缓、阻止、中止或停止疾病或相关的症状的发作或进一步发展。
在本文中，除非另有说明，本文所使用的表示成分的量、测量值或反应条件的所有数字应理解为在所有情况下均由术语“约”修饰，以表示可能的测量误差。例如，当与百分比相连时，术语“约”可以表示在其所限定的对象值的±1％、例如±0.5％的范围内变化。
在本文中，除非另有说明，否则单数术语涵盖复数指代物，反之亦然。类似地，除非上下文另有明确指示，术语“或”意在包括“和”，反之亦然。
在本文中，除非另有说明，术语“包含、包括和含有(comprise、comprises和comprising)”或其等同物(例如contain、containing、include、including)为开放式表述，应理解为“包括但不限于”，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
在本文中，序列之间的同一性百分比(同源性程度)可以通过例如使用万维网上通常用于此目的的免费可用的计算机程序(例如具有默认设置的BLASTp或BLASTn)对两个序列进行比较来确定。
新城疫病毒(NDV)属于单分子负链RNA病毒目的副黏病毒科，具有包膜；核衣壳位于包膜内，含有RNA基因组和核衣壳蛋白。经典的新城疫病毒的基因组全长为约15～16kb，从3’端到5’端依次包含NP基因、P基因、M基因、F基因、HN基因和L基因，分别用于编码如下6种主要蛋白：核衣壳蛋白(Nucleocapsid Protein，NP)、磷酸化蛋白(Phosphate Protein，P)、基质蛋白(Matrix Protein，M)、融合蛋白(Fusion Protein，F)、血凝素-神经氨酸酶蛋白(Haemagglutinin Neuraminidase Protein，HN)和RNA依赖的RNA聚合酶(Large Protein，L)。
在一个实施方式中，本申请涉及一种重组的新城疫病毒基因组，其中，所述基因组包括VEGF-Trap的编码基因，所述VEGF-Trap的编码基因位于新城疫病毒基因组的P基因与M基因之间。
在一些优选的实施方式中，所述VEGF-Trap的编码基因可处于DNA或RNA的形式。
在一些优选的实施方式中，所述VEGF-Trap的编码基因具有如SEQ ID NO.1所示的序列或与其具有至少80％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的序列。
在一些优选的实施方式中，所述重组的新城疫病毒基因组的序列以SEQ ID NO.2或SEQ ID NO.5示出(见图9和图10)。
在一个实施方式中，本申请涉及一种重组新城疫病毒，其中，所述病毒包含上述的重组的新城疫病毒基因组。
在一些优选的实施方式中，所述新城疫病毒的出发毒株可选自但不限于：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ；以及基于所述出发毒株以基因工程手段构建的任意的嵌合毒株，但不限于此。
在一个实施方式中，本申请涉及一种编码上述的重组的新城疫病毒基因组的DNA分子(例如重组新城疫病毒质粒)。
在一个实施方式中，本申请涉及一种药物组合物，其中，所述药物组合物包含上述的重组的新城疫病毒基因组、重组新城疫病毒和/或DNA分子。
在一些优选的实施方式中，所述药物组合物进一步包含药学上可接受的辅料。所述药学上可接受的药用辅料可选自例如但不限于溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、抗氧剂、渗透促进剂、pH值调节剂、表面活性剂、稀释剂等。关于其它可用的药学上可接受的药用辅料，可参见例如《药用辅料手册》(第4版)，R.C.罗等著，郑泽民主译，2005年，化学工业出版社。
在一个实施方式中，本申请涉及一种制备上述重组新城疫病毒的方法，其中，所述方法包括：
(1)将含有VEGF-Trap的编码基因的DNA序列的克隆载体与NDV病毒载体分别进行酶切，并将酶切得到的所述VEGF-Trap的编码基因的DNA序列与所述NDV病毒载体进行连接，得到重组新城疫病毒质粒；
(2)将所述重组新城疫病毒质粒转染至细胞中并对经转染的细胞进行培养，获得所述重组新城疫病毒。
在一些优选的实施方式中，可采用选自如下的载体构建所述克隆载体：PUC57载体、pMD18-T载体、pMD19-T载体、pBlueScript SK(+/-)载体、pBluescript II KS(+/-)。
在一些优选的实施方式中，所述NDV病毒载体可为选自如下的NDV病毒的基因组的全长cDNA序列：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ，但不限于此。
优选地，所述NDV病毒载体可为pBluescript II KS(+/-)-NDV(pBrNDV)、pCI-neo-NDV、pOLTV5-NDV载体。
在本文中，将所述重组新城疫病毒质粒与能够表达核衣壳蛋白NP、磷酸化蛋白P和RNA依赖的 RNA聚合酶L的辅助质粒NP、P和L(它们可为NP、P和L的基因构建至本领域已知的任何真核表达载体上得到的任意的NP、P、L重组质粒)共转染至所述细胞中。在本文中，辅助质粒NP、P和L的基因可以来源于NDV任意毒株，如LaSota、Anhinga、F48E9等。在一些优选的实施方式中，将所述重组新城疫病毒质粒与选自如下的辅助质粒共转染至细胞中：pTM-NP、pTM-P和pTM-L；pCI-neo-NP、pCI-neo-P和pCI-neo-L；或者pBluescript II KS(+/-)-NP(pBL-NP)、pBluescript II KS(+/-)-P(pBL-P)和pBluescript II KS(+/-)-L(pBL-L)，但不限于此。
在本文中，转染是将外源性核酸物质(包括DNA和RNA)导入细胞内的技术，主要包括物理介导(电穿孔法、显微注射和基因枪)、化学介导(磷酸钙共沉淀法、脂质体转染、阳离子物质介导)和生物介导(原生质体转染、病毒介导的转染)三类途径。具体的操作可由本领域技术人员基于本领域的普通知识(例如可参见《分子克隆实验指南》(第4版)，J.萨姆布鲁克等编著，贺福初主译，科学出版社，2017年)选择合适的实验条件和步骤、或者根据可商购的试剂盒中的说明书进行。
在一些优选的实施方式中，所述细胞可选自但不限于BHK-21细胞、BSR-T7/5细胞、VERO细胞、DF-1细胞、293细胞、MDCK细胞。
在本文中，所述经转染的细胞的培养可由本领域技术人员根据细胞的种类选择常规的培养基和培养条件进行(刘斌主编，《细胞培养(第3版)》，世界图书出版公司，2018年01月；兰容、周珍辉主编，《细胞培养技术》，化学工业出版社，2007年8月；章静波主编，《组织和细胞培养技术(第3版)》，人民卫生出版社，2014年06月等)。
在一个实施方式中，本申请涉及上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物在制备用于治疗或改善癌症的药物中的用途。
或者，本申请提供了用于治疗或改善癌症的上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物。或者，本申请提供了上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物用于治疗或改善癌症的用途。或者一种治疗或改善癌症的方法，包括向有需要的受试者给予上述的重组的新城疫病毒基因组、重组新城疫病毒、DNA分子和/或药物组合物。
在一些优选的实施方式中，所述癌症可选自但不限于：结肠癌、肝癌(例如肝细胞癌)、肺癌(例如非小细胞肺癌、小细胞肺癌)、胃癌、直肠癌、白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、膀胱癌、尿路上皮癌、支气管癌、骨癌、前列腺癌、胰腺癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、肌肉瘤、子宫癌、成胶质细胞瘤、成神经管细胞瘤、神经母细胞瘤、肾癌、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征或肉瘤。
本发明的示例性的技术方案可通过如下编号段落的内容进行说明：
1.一种重组的新城疫病毒基因组，其中，所述基因组包括VEGF-Trap的编码基因，所述VEGF-Trap的编码基因位于新城疫病毒基因组的P基因与M基因之间。
2.如段落1所述的重组的新城疫病毒基因组，其中，所述VEGF-Trap的编码基因处于DNA或RNA的形式。
3.如段落1或2所述的重组的新城疫病毒基因组，其中，所述VEGF-Trap的编码基因具有如SEQ ID NO.1所示的序列或与其具有至少80％同一性的序列。
4.如段落1-3中任一段所述的重组的新城疫病毒基因组，其中，所述重组的新城疫病毒基因组的序列以SEQ ID NO.2或SEQ ID NO.5示出。
5.一种重组新城疫病毒，其中，所述病毒包含段落1-4中任一段所述的重组的新城疫病毒基因组。
6.如段落5所述的重组新城疫病毒，其中，所述新城疫病毒的出发毒株选自：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ；以及基于所述出发毒株以基因工程手段构建的任意的嵌合毒株。
7.一种编码段落1-4中任一段所述的重组的新城疫病毒基因组的DNA分子。
8.一种药物组合物，其中，所述药物组合物包含段落1-4中任一段所述的重组的新城疫病毒基因组、段落5或6所述的重组新城疫病毒和/或段落7所述的DNA分子。
9.如段落8所述的重组新城疫病毒，其中，所述药物组合物进一步包含药学上可接受的辅料。
10.如段落8或9所述的重组新城疫病毒，其中，所述药学上可接受的药用辅料选自溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、抗氧剂、渗透促进剂、pH值调节剂、表面活性剂或稀释剂。
11.一种制备段落5或6所述的重组新城疫病毒的方法，其中，所述方法包括：
(1)将含有VEGF-Trap的编码基因的DNA序列的克隆载体与NDV病毒载体分别进行酶切，并将酶 切得到的所述VEGF-Trap的编码基因的DNA序列与所述NDV病毒载体进行连接，得到重组新城疫病毒质粒；
(2)将所述重组新城疫病毒质粒转染至细胞中并对经转染的细胞进行培养，获得所述重组新城疫病毒。
12.如段落11所述的方法，其中，采用选自如下的载体构建所述克隆载体：PUC57载体、pMD18-T载体、pMD19-T载体、pBlueScript SK(+/-)载体、pBluescript II KS(+/-)。
13.如段落11或12所述的方法，其中，所述NDV病毒载体为选自如下的NDV病毒的基因组的全长cDNA序列：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ。
14.如段落13所述的方法，其中，所述NDV病毒载体为pBluescript II KS(+/-)-NDV(pBrNDV)、pCI-neo-NDV、pOLTV5-NDV载体。
15.如段落11-14中任一段所述的方法，其中，将所述重组新城疫病毒质粒与选自如下的辅助质粒共转染至所述细胞中：pTM-NP、pTM-P和pTM-L；pCI-neo-NP、pCI-neo-P和pCI-neo-L；或者pBluescript II KS(+/-)-NP、pBluescript II KS(+/-)-P和pBluescript II KS(+/-)-L。
16.如段落11-15中任一段所述的方法，其中，所述细胞选自BHK-21细胞、BSR-T7/5细胞、VERO细胞、DF-1细胞、293细胞或MDCK细胞。
17.段落1-4中任一段所述的重组的新城疫病毒基因组、段落5或6所述的重组新城疫病毒、段落7所述的DNA分子和/或段落8-10中任一段所述的药物组合物在制备用于治疗或改善癌症的药物中的用途。
18.如段落17所述的用途，其中，所述癌症选自：结肠癌、肝癌、肺癌、胃癌、直肠癌、白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、膀胱癌、尿路上皮癌、支气管癌、骨癌、前列腺癌、胰腺癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、肌肉瘤、子宫癌、成胶质细胞瘤、成神经管细胞瘤、神经母细胞瘤、肾癌、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征或肉瘤。
为使本申请实施例的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。除非另外说明，所用试剂、材料或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。以下结合实施例对本申请的特征和性能作进一步的详细描述。
实施例
除非另有说明，本申请中涉及的基因的设计、合成和克隆以及载体的构建和转染及电泳等操作，可按照本领域已知的技术进行(例如参见CURRENT PROTOCOLS IN MOLECULAR BIOLOGY的记载)。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段(例如参见：潘卫主编，《分子病毒学原理与实验技术》，上海第二军医大学出版社，2002年11月；黄祯祥主编，《医学病毒学基础及实验技术》，科学出版社，1990年2月等)。
下述的实施例涉及如下的外源基因：血管抑制剂基因VEGF-Trap和人源血管抑素基因Angiostatin(Genbank登录号NG_016200.1)。
pMD19-T购自宝生物工程(大连)有限公司(大连TaKaRa公司)。BHK-21细胞(幼仓鼠肾细胞)、人结肠癌细胞HCT116、小鼠结肠癌细胞CT26、小鼠乳腺癌细胞4T1、人脐静脉内皮细胞EA.hy926均购自ATCC。
DMEM(高糖)培养基、McCoy'5A培养基、RPMI 1640培养基、胰蛋白酶、新生牛血清(FCS)、胎牛血清(FBS)购自GIBCO公司。SPF鸡胚购自北京勃林格殷格翰维通生物技术有限公司。Balb/c小鼠(昆明小鼠)购自斯贝福(北京)生物技术有限公司。
实施例1重组新城疫病毒的制备
一、插入外源基因的重组新城疫病毒质粒的构建(以pBrNDV-VEGF-Trap为例)
根据文献Jocelyn Holash等(VEGF-Trap：A VEGF blocker with potent antitumor effects，2002年8月；https://doi.org/10.1073/pnas.172398299)的记载获得VEGF-Trap基因的序列(SEQ ID NO.1)，该序列如下所示：
Figure PCTCN2022086599-appb-000001
Figure PCTCN2022086599-appb-000002
按照如下方法构建重组质粒pBrNDV-VEGF-Trap：
1.通过上海生工生物工程公司合成含有SacII酶(5')和PmeI酶(3')的酶切位点的VEGF-Trap基因，并连接到pUC57载体上形成pUC57-VEGF-Trap，用于后续实验。
2.用限制性内切酶PmeI和SacII(购自NEB公司)根据制造商的产品说明书酶切步骤1中的质粒pUC57-VEGF-Trap，酶切产物通过核酸琼脂糖凝胶电泳进行酶切鉴定，鉴定正确后通过胶回收试剂盒(购自天根生化科技(北京)有限公司，货号：DP219)根据制造商的产品说明书胶回收酶切产物。
3.用限制性内切酶PmeI和SacII根据制造商的产品说明书酶切pBrNDV质粒(购自NEB)，通过胶回收试剂盒(购自天根生化科技(北京)有限公司，货号：DP219)根据制造商的产品说明书胶回收该质粒载体。
4.将步骤2的酶切产物和步骤3的载体用T4DNA连接酶(购自NEB)根据制造商的产品说明书进行连接，得到重组新城疫病毒质粒pBrNDV-VEGF-Trap，其中，VEGF-Trap基因插入质粒P基因和M基因之间。对上述重组质粒采用宝生物taq酶(购自宝生物)按照制造商的说明书进行PCR(上游引物：5’TCAAGCGCCTTGCTCTAAATGGC 3’(SEQ ID NO.3)；下游引物：5’GGGCAGAATCAAAGTACAGCCCAAT 3’(SEQ ID NO.4))以及PmeI和SacⅡ双酶切鉴定(37℃，1h)，鉴定正确的质粒样品分装并送至上海生工生物工程公司进行测序，测序结果使用序列分析软件DNAMAN进行比对，经测序，测序序列与目的序列一致。
采用与上述相同的方法构建和鉴定如下的插入外源Angiostatin基因(NG_016200.1)的重组质粒pBrNDV-Angiostatin(PCR扩增的引物同上)。
二、重组新城疫病毒的制备
通过如下方法利用上述的重组新城疫病毒质粒制备得到重组新城疫病毒rNDV-VEGF-Trap：
1.采用脂质体转染技术，将构建成功的重组新城疫病毒质粒pBrNDV-VEGF-Trap与三种辅助质粒pBL-NP、pBL-P和pBL-L(按照葛金英，新城疫病毒反向遗传操作基础与应用研究，2006年所述的方法构建)共转染至稳定表达T7RNA聚合酶的单层BHK-21细胞中，操作步骤参照Lipofectamine 3000转染试剂(购自Invitrogen)的说明书进行。孵育72h后，于-80℃下反复冻融3次，4℃下12000rpm离心收集上清液，加入0.001％胰酶。
2.取步骤1得到的上清液200μL，接种于9日龄SPF级鸡胚尿囊腔中，然后在孵化箱中于37℃、5％CO 2下培养72h后获取尿囊液，并进行血凝效价检测(例如周灵、李燕芳、马夏利，一株鸡源新城疫病毒的分离鉴定[J]，浙江畜牧兽医，2015，40(03):8-10中记载的方法)，阳性尿囊液冻存于-80℃冰箱保存，成功拯救的重组新城疫病毒命名为rNDV-VEGF-Trap。
3.将步骤2得到的重组新城疫病毒按照QIAamp Viral RNA Mini Kit(50)的说明书提取重组病毒RNA，利用随机引物获得cDNA样品，用P/M位点引物P/M-F(序列同SEQ ID NO.3)和P/M-R(序列同SEQ ID NO.4)对插入的VEGF-Trap基因进行RT-PCR扩增(Thermo Fisher RT-PCR试剂盒，按照试剂盒说明书合成cDNA第一链并进行PCR)，所扩增的PCR产物送至上海生工生物工程公司进行测序，测序结果使用序列分析软件DNAMAN进行比对，经测序，测序序列与目的序列一致。
4.取步骤3中的外源基因测序正确的病毒，接种于新的9-11日龄SPF鸡胚尿囊腔，置于37℃下培养72h，收集鸡胚尿囊液进行HA检测(例如参见记载于周灵、李燕芳、马夏利，一株鸡源新城疫病毒的分离鉴定[J]，浙江畜牧兽医，2015，40(03):8-10中的相关方法进行)，选取HA效价大于29的尿囊液进行混合后分装备用。
5.收取各组尿囊液进行Western Blot(例如，可参见记载于An Y,et al.Recombinant Newcastle disease virus expressing P53 demonstrates promising antitumor efficiency in hepatoma model[J].Journal of Biomedical Science,2016 23(1):55中的相关方法)检测各外源基因的表达，结果显示，重组新城疫病毒rNDV-VEGF-Trap能够稳定表达外源基因VEGF-Trap(参见图1)。
同样地，按照上述方法制备得到重组新城疫病毒rNDV-Angiostatin。
以下描述如无特别指明，rNDV-VEGF-Trap和rNDV-Angiostatin分别指使用重组质粒pBrNDV-VEGF-Trap和pBrNDV-Angiostatin通过上述方法制备的重组病毒。
以下的实施例2-3中的亲本病毒指从新城疫病毒LaSota(购自哈尔滨兽医防疫站)出发，按照王永等(置换HN基因对新城疫病毒LaSota株致病力的影响，微生物学报，2008，48(5)：638～643)中描述的关于“基因替换”的工程化方法，采用新城疫病毒强毒株F48E9的F基因(GenBank登录号:AY508514.1)替换该LaSota毒株的F基因改造得到，在本文中将该亲本毒株命名为rNDV。
实施例2重组病毒增殖稳定性的检测
按照如下的方法进行TCID50的测定：
1.用含有10％FBS、1％抗生素的DMEM培养基接种10000个DF-1细胞于96孔微量培养板内，37℃、5％CO 2培养箱中过夜培养。
2.接种各重组新城疫病毒(rNDV-VEGF-Trap和rNDV-Angiostatin)和亲本病毒前，将步骤1的培养板中的原有细胞培养基弃掉，向其中加入含有10％尿囊液、1％抗生素的180μL新鲜的DMEM培养基。
3.取20μL各重组新城疫病毒和亲本病毒分别接种于最上排细胞孔内，吹打混匀后，吸取20μL混合液向下方细胞孔做连续的10倍梯度稀释。每个病毒设置3个重复样品。
4.在37℃、5％CO 2细胞培养箱中，将接种的重组新城疫病毒和亲本病毒孵育1小时后，弃去培养液并用0.2ml的1×PBS缓冲液清洗一次，之后加入含10％尿囊液、1％抗生素的200μL新鲜的DMEM培养基。
5.在37℃、5％CO 2细胞培养箱中继续培养，分别于12h、24h、36h、48h、60h、72h后于光学倒置显微镜下观察细胞病变孔并记录病变孔的个数，根据Reed and Muench method方法计算该病毒的效价，并绘制增殖曲线。
结果如图2所示，重组新城疫病毒rNDV-VEGF-Trap和rNDV-Angiostatin与亲本病毒的增殖趋势无明显差异，说明外源基因的插入不影响病毒增殖。
实施例3重组新城疫病毒对肿瘤的治疗作用
1建立Balb/c小鼠结肠癌荷瘤动物模型
取小鼠结肠癌细胞CT26，台盼蓝染色确定细胞活度达95％以上，将其用生理盐水稀释成1×10 6/mL的细胞悬液，将该细胞悬液以每只Balb/c小鼠0.1mL的剂量注入右侧腹部皮下。8-12天后实体瘤的直径达到5-8mm，造模成功，可进行后续实验。去除肿瘤形态、大小差异大的个体，取肿瘤直径在5-8mm的小鼠作为模型小鼠。
2重组病毒对肿瘤的治疗作用
将模型小鼠随机分为4组，每组10只，分别处理如下：
rNDV-VEGF-Trap组：每天向模型小鼠的瘤内注射0.2mL实施例1制备的rNDV-VEGF-Trap病毒的PBS悬液(采用1×PBS缓冲液配制；含10 7pfu病毒)，治疗14天；
rNDV-Angiostatin组：每天向模型小鼠的瘤内注射0.2mL实施例1制备的rNDV-Angiostatin病毒的PBS悬液(采用1×PBS缓冲液配制；含10 7pfu病毒)，治疗14天；
rNDV组：每天向模型小鼠的瘤内注射0.2mL亲本病毒的PBS悬液(采用1×PBS缓冲液配制；含10 7pfu病毒)，治疗14天；
阴性对照组(模型组)：每天向模型小鼠的瘤内注射0.2mL SPF鸡胚尿囊液，治疗14天。
从治疗当天起，隔天测量肿瘤体积，并基于测量结果制作肿瘤生长曲线(图3)。治疗结束后，将小鼠安乐死，剥瘤，测量肿瘤重量和大小(结果在图4和图5中示出)。阴性对照组的肿瘤平均体积是1889.17 mm 3，亲本NDV(rNDV)治疗组的肿瘤平均体积是728.49mm 3，rNDV-Angiostatin治疗组的肿瘤平均体积是774.37mm 3；rNDV-VEGF-Trap治疗组的肿瘤平均体积是350.36mm 3。结果表明，与阴性对照组相比，亲本病毒和重组新城疫病毒均对肿瘤生长有显著的抑制效果，其中，rNDV-Angiostatin的抗肿瘤效果与rNDV组无明显差异性，而rNDV-VEGF-Trap的抗肿瘤效果显著高于rNDV组。
3肿瘤病理切片的观察
为观察重组新城疫病毒rNDV-VEGF-Trap对结肠癌及相关血管的抑制情况，取各组小鼠肿瘤组织，经4％多聚甲醛固定后，按如下方法制备4μm厚度的肿瘤组织石蜡切片，并经HE染色后显微镜下观察各组的肿瘤组织形态，通过免疫组织化学染色检测各组肿瘤组织中的CD34蛋白的表达。具体操作如下：
3.1石蜡切片的制备
(1)将石蜡放入1L大烧杯中，加入蜂蜡，放入60℃蜡箱，当蜡完全融化后，用滤纸过滤，取出后于室温冷却，然后重新放入蜡箱融化，反复2～3次。
(2)将载玻片逐个放入配好的洗液(重铬酸钾浓溶液：重铬酸钾25g，水75ml，浓硫酸400ml)中浸泡24h后，用自来水冲洗干净，再放入95vol％的酒精中浸泡24h，然后将载玻片用擦镜纸擦干于180℃干热灭菌6h。灭菌后，将载玻片逐个浸入到盛有APES处理剂(APES:丙酮＝1:50)的染缸中处理5min，取出后用蒸馏水冲洗两次，放入切片盒中，60℃烘干备用。
(3)在上述的治疗结束后，取各组小鼠的肺组织放入事先配制好的4％中性甲醛固定液中固定48h，取出组织，用刀片将组织块修成1cm左右大小。
(4)脱水与透明：30vol％乙醇-30min，50vol％乙醇-30min，70vol％乙醇4℃过夜；次日80vol％乙醇-30min，90vol％乙醇-30min，100vol％乙醇-30min(2次)。脱水后的组织于通风橱中浸入到二甲苯:无水乙醇＝1:1的染缸中漂洗20min后，放入纯二甲苯中漂洗20min，并重复2次。此时组织块颜色加深透明，二甲苯溶液澄清。
(5)浸蜡：透明后的组织转入到(1)中充分溶解的石蜡内，于60℃恒温箱放置120min。
(6)包埋：将蜡箱中的石蜡倒入纸盒内，用小镊子将各组织块按顺序排列，确保切面朝下。
(7)修蜡块：待纸盒内石蜡凝固后，将蜡块取出，用刀片将其修成梯形，组织边缘距蜡块边缘之间的距离不得小于2mm。
(8)切片：连续切片，厚度约4μm，将切片在40℃左右摊片机水浴中展开，用(2)中处理的载玻片从水浴中捞取切片，放在摊片机边框上，稍干后立即放入37℃烘箱中过夜，使切片紧贴于载玻片上并呈半透明状。过夜后，将载玻片置于玻片盒，备用。
3.2HE染色
(1)脱蜡：将蜡箱的温度预热到60℃时，将制备的肿瘤组织切片放入60℃烘箱中1h，切片上石蜡融化后浸入二甲苯中5min(3次)。无水乙醇-2min；90vol％乙醇-5min；80vol％乙醇-5min；70vol％乙醇-5min；50vol％乙醇-5min；水-5min(3次)。
(2)染色：切片用苏木精染色30sec后蒸馏水冲洗1min；插入伊红染缸中立即取出，并用蒸馏水浸洗1min(3次)。
(3)脱水：50vol％乙醇-1min；70vol％乙醇-1min；80vol％乙醇-1min；90vol％乙醇-1min；100vol％乙醇-5min(2次)；二甲苯1-5min(2次)。
(4)中性树胶封片：盖玻片干燥后放平，向中央滴加1～2滴中性树胶，载玻片带组织的一面轻轻盖在盖玻片上，让中性树胶沿着盖玻片充分漫延展开，之后倾斜载玻片，用滤纸将多余的二甲苯吸干，同时注意避免有气泡的产生。载玻片烘干后储存于切片盒中，显微镜下观察并比较各组小鼠瘤组织的病理变化。
3.3免疫组织化学染色
1、脱蜡和水化：将制备好的石蜡切片浸入二甲苯进行脱蜡，浸入两次，每次5min，然后放入100vol％、95vol％、90vol％、80vol％、70vol％的各级酒精溶液中各5min，再放入蒸馏水中漂洗两次，每次3min。
2、抗原修复：将0.01M柠檬酸钠缓冲液(pH＝6.0)水浴加热至95℃后，放入切片加热10min。1×PBS缓冲液漂洗3次，每次5min。
3、灭活内源性过氧化物酶：滴加适量的内源性过氧化物酶封闭液(购自Beyotime；货号P0100A)使其完全覆盖样品，室温孵育10min。1×PBS缓冲液液洗涤3次，每次3分钟。
4、封闭：滴加封闭液(购自Beyotime；货号P0260QuickBlock)封闭组织切片10min。
5、孵育一抗：用封闭液依照抗体说明书的稀释比配制CD34一抗工作液(购自Abcam)，向所述各组 织切片滴加一抗工作液，4℃孵育过夜。一抗(抗CD34抗体，购自Abcam)孵育后，1×PBST缓冲液冲洗组织切片3次，每次5min。
6、孵育二抗：用封闭液依照抗体说明书的稀释比配制羊抗兔二抗(购自Abcam)工作液，向所述各组织切片滴加二抗工作液，室温静置孵育1h。二抗孵育后，1×PBST缓冲液冲洗组织切片3次，每次5min。
7、显色：滴加100μl DAB显色工作液(购自Beyotime)，充分覆盖样品。室温避光孵育15min，显色后，去除DAB显色工作液，用蒸馏水洗涤1-2次以终止显色反应。
结果表明：治疗结束后，阴性对照组(模型组)小鼠的肿瘤组织结构致密，细胞形态完好，生长旺盛；rNDV组小鼠的肿瘤病灶解体，肿瘤细胞结构较为松散；rNDV-Angiostatin组小鼠的肿瘤结构与rNDV组无明显差异，而rNDV-VEGF-Trap组小鼠的肿瘤组织病灶大量解体，肿瘤细胞结构非常松散，免疫细胞多处浸润，肿瘤细胞呈单个分散(参见图6)。免疫组织化学染色结果显示出，模型组小鼠的CD34表达量丰富，rNDV组与模型组类似，而rNDV-VEGF-Trap组小鼠的CD34表达量明显减少。表明rNDV-VEGF-Trap具有抑制血管内皮细胞增生的疗效(参见图7)。
实施例4重组新城疫病毒的安全性检测
选用健康6周SPF级Balb/c小鼠分组，每组10只。对照组小鼠正常饲养。实验组小鼠每只腹腔注射5×10 8pfu(10倍治疗剂量)重组新城疫病毒rNDV-VEGF-Trap后观察30天。出现明显不良反应如精神萎靡、皮毛竖起及死亡的小鼠为阳性。
结果显示：注射第2天时，实验组中的3只小鼠皮毛竖起，饮食饮水未受到影响。连续注射重组新城疫病毒rNDV-VEGF-Trap一个星期后，实验组小鼠的皮毛都恢复正常，继续观察一个月，实验组中的各小鼠均未出现明显不良反应(包括精神萎靡、皮毛竖起)，且无小鼠死亡。
因此，本申请制备的重组新城疫病毒rNDV-VEGF-Trap具有良好的安全性。
实施例5
如下实施例提供的作为亲本毒株的溶瘤病毒rClone30-Anh-(F)从新城疫病毒LaSota毒株(购自哈尔滨兽医防疫站)出发，按照王永等(微生物学报，2008，48(5)：638～643，同上)中描述的关于“基因替换”的工程化方法，采用新城疫病毒中毒株Anhinga的F基因(GenBank登录号:EF065682.1)替换LaSota毒株的F基因改造得到。该实施例的表达VEGF-Trap的重组新城疫病毒的基因组以SEQ ID NO.5示出(参见图10)；VEGF-Trap的核酸序列以SEQ ID NO.1示出。
首先，按照实施例1中所述的方法采用VEGF-Trap基因(SEQ ID NO.1)和Angiostatin基因(NG_016200.1)分别构建得到相应的重组新城疫病毒质粒和重组新城疫病毒并成功拯救出重组病毒，将拯救成功并鉴定正确的重组新城疫病毒分别命名为rClone30-Anh-(F)-VEGF-Trap和rClone30-Anh-(F)-Angiostatin。
然后，按照实施例4中所述的方法采用H22细胞(购自南京科佰)建立H22皮下荷瘤模型(即，小鼠肝癌模型)。当肿瘤长到100mm 3左右，开始向瘤内注射100μL上述的各溶瘤病毒(rClone30-Anh-(F)-Angiostatin和rClone30-Anh-(F)-VEGF-Trap)以及亲本毒株rClone30-Anh-(F)的PBS悬液(利用1×PBS缓冲液配制)。在各治疗组中，将各溶瘤病毒(rClone30-Anh-(F)-Angiostatin、rClone30-Anh-(F)-VEGF-Trap和rClone30-Anh-(F))分别向瘤内每天注射一次，共注射14天，每次注射1×10 7PFU；向小鼠肝癌模型的瘤内注射1×PBS缓冲液(不含溶瘤病毒)作为阴性对照组(也称为“PBS治疗组”)；每组6只动物，通过解剖肿瘤组织，观察各重组病毒治疗效果。
如图8所示，治疗结束后，阴性对照组的肿瘤平均体积是1421.77mm 3，亲本rClone30-Anh-(F)治疗组的肿瘤平均体积是807.30mm 3，rClone30-Anh-(F)-Angiostatin治疗组的肿瘤平均体积是668.60mm 3；rClone30-Anh-(F)-VEGF-Trap治疗组的肿瘤平均体积是326.05mm 3。结果表明，比起阴性对照组，亲本毒株rClone30-Anh-(F)治疗组、rClone30-Anh-(F)-Angiostatin治疗组和rClone30-Anh-(F)-VEGF-Trap治疗组均能抑制肿瘤生长，尤其是rClone30-Anh-(F)-VEGF-Trap治疗组的肿瘤平均体积最小。
以上所述仅为本发明的优选实施例，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围内。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。

Claims (15)

  1. 一种重组的新城疫病毒基因组，其中，所述基因组包括VEGF-Trap的编码基因，所述VEGF-Trap的编码基因位于新城疫病毒基因组的P基因与M基因之间。
  2. 如权利要求1所述的重组的新城疫病毒基因组，其中，所述VEGF-Trap的编码基因处于DNA或RNA的形式。
  3. 如权利要求1或2所述的重组的新城疫病毒基因组，其中，所述VEGF-Trap的编码基因具有如SEQ ID NO.1所示的序列或与其具有至少80％同一性的序列。
  4. 如权利要求1-3中任一项所述的重组的新城疫病毒基因组，其中，所述重组的新城疫病毒基因组的序列以SEQ ID NO.2或SEQ ID NO.5示出。
  5. 一种重组新城疫病毒，其中，所述病毒包含权利要求1-4中任一项所述的重组的新城疫病毒基因组。
  6. 如权利要求5所述的重组新城疫病毒，其中，所述新城疫病毒的出发毒株选自：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ；以及基于所述出发毒株以基因工程手段构建的任意的嵌合毒株。
  7. 一种编码权利要求1-4中任一项所述的重组的新城疫病毒基因组的DNA分子。
  8. 一种药物组合物，其中，所述药物组合物包含权利要求1-4中任一项所述的重组的新城疫病毒基因组、权利要求5或6所述的重组新城疫病毒和/或权利要求7所述的DNA分子。
  9. 如权利要求8所述的药物组合物，其中，所述药物组合物进一步包含药学上可接受的辅料；
    优选地，所述药学上可接受的药用辅料选自溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、抗氧剂、渗透促进剂、pH值调节剂、表面活性剂或稀释剂。
  10. 一种制备权利要求5或6所述的重组新城疫病毒的方法，其中，所述方法包括：
    (1)将含有VEGF-Trap的编码基因的DNA序列的克隆载体与NDV病毒载体分别进行酶切，并将酶切得到的所述VEGF-Trap的编码基因的DNA序列与所述NDV病毒载体进行连接，得到重组新城疫病毒质粒；
    (2)将所述重组新城疫病毒质粒转染至细胞中并对经转染的细胞进行培养，获得所述重组新城疫病毒。
  11. 如权利要求10所述的方法，其中，采用选自如下的载体构建所述克隆载体：PUC57载体、pMD18-T载体、pMD19-T载体、pBlueScript SK(+/-)载体、pBluescript II KS(+/-)。
  12. 如权利要求10或11所述的方法，其中，所述NDV病毒载体为选自如下的NDV病毒的基因组的全长cDNA序列：弱毒株LaSota、Hitchner B1、V4，中毒株Mukteswar、Anhinga，强毒株F48E9、JS/7/05/Ch、Italien、Herts/33、NDV-BJ；
    优选地，所述NDV病毒载体为pBluescript II KS(+/-)-NDV(pBrNDV)、pCI-neo-NDV、pOLTV5-NDV载体。
  13. 如权利要求10-12中任一项所述的方法，其中，将所述重组新城疫病毒质粒与选自如下的辅助质粒共转染至所述细胞中：pTM-NP、pTM-P和pTM-L；pCI-neo-NP、pCI-neo-P和pCI-neo-L；或者pBluescript II KS(+/-)-NP、pBluescript II KS(+/-)-P和pBluescript II KS(+/-)-L。
  14. 如权利要求10-13中任一项所述的方法，其中，所述细胞选自BHK-21细胞、BSR-T7/5细胞、VERO细胞、DF-1细胞、293细胞或MDCK细胞。
  15. 用于治疗或改善癌症的权利要求1-4中任一项所述的重组的新城疫病毒基因组、权利要求5或6所述的重组新城疫病毒、权利要求7所述的DNA分子和/或权利要求8或9所述的药物组合物；
    优选地，所述癌症选自：结肠癌、肝癌、肺癌、胃癌、直肠癌、白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、膀胱癌、尿路上皮癌、支气管癌、骨癌、前列腺癌、胰腺癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、肌肉瘤、子宫癌、成胶质细胞瘤、成神经管细胞瘤、神经母细胞瘤、肾癌、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征或肉瘤。
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