Recombinant Newcastle Disease Virus rNDV-VEGF-Trap, Its Genome, Preparation Method Therefor and Its Use

Abstract

The present application relates to a recombinant Newcastle disease virus genome, a recombinant Newcastle disease virus rNDV-VEGF-Trap containing the genome and a preparation method therefor, a DNA molecule encoding the recombinant Newcastle disease virus genome, and the use of the genome and the recombinant Newcastle disease virus in the preparation of a drug for the treatment of cancer. The recombinant Newcastle disease virus provided by the present application is characterized in that the recombinant Newcastle disease virus obtained by inserting a coding gene for VEGF-Trap into the genome of the recombinant Newcastle disease virus replicates with a strong replicative ability, thereby killing host cancer cells, and further, that the recombinant Newcastle disease virus has a reliable safety against non-cancer cells and shows improved antitumor effect and tumor lysis efficiency.

Description

[Technical field]

[0001] The present application is in the field of oncolytic viruses for cancer treatment, and in particular relates to a recombinant Newcastle Disease Virus genome, a recombinant Newcastle Disease Virus containing the genome and a preparation method therefor, a DNA molecule encoding the recombinant Newcastle Disease Virus genome, and uses thereof. [Background technology]

[0002] Cancer is a disease caused by the loss of normal regulation of the body's cells and their excessive proliferation. At present, cancer has become the number one cause of death affecting health. China is an area with high incidence of cancer, especially lung, stomach, liver and rectal cancer. According to statistics, in 2016 alone, 4.8 million new cases of various cancer patients occurred in China, and 2.3 million patients died from various cancers. With the development of technology, various new treatment methods, especially biologic therapy, are continuously entering clinical use. However, the needs for drug safety, efficacy and quality of life of patients are far from being met. The development of new drugs or treatment methods is essential.

[0003] In 1991, Martuza et al. published a paper in Science demonstrating that transgenic herpes simplex virus has certain efficacy in treating glioblastoma. Since then, there has been increasing attention on the development of oncolytic viruses to treat cancer. The principle of oncolytic virus treatment of cancer is to genetically modify some naturally occurring viruses with weak pathogenicity, selectively infect them into tumor cells, replicate widely in the cells, and finally destroy the tumor cells. At the same time, it also stimulates the immune response and attracts immune cells to continue killing the remaining cancer cells, or kill the cancer cells that have migrated through the immune response. In recent decades, research on oncolytic viruses has progressed tremendously. Newcastle disease virus (NDV), herpes simplex virus 1 (HSV-1), reovirus, and oncolytic adenovirus have been successfully used to develop oncolytic viruses, but their clinical findings have been far below expectations. For example, in 2005, the CFDA approved the oncolytic adenovirus product H101 for marketing, but its therapeutic effect was not ideal.

[0004] NDV is an avian paramyxovirus with a negative-sense single-stranded RNA genome and has always been a promising method for cancer treatment. However, the effectiveness of NDV as a single drug treatment is limited. In one aspect, the antiviral immune response in the human body can eliminate the virus. In another aspect, the human body can produce neutralizing antibodies to resist the virus and affect its effectiveness. To improve the therapeutic efficacy of NDV in cancer, these viruses have then been used to deliver genes with antitumor activity to further enhance their activity. Such genes include genes encoding cytokines or their receptors, immune checkpoint molecules, tumor suppressor proteins, or immune stimulatory proteins.

[0005] Many studies on recombinant Newcastle disease viruses, whose genomes incorporate genes encoding cytokines or their receptors, have been carried out in the field before, but no clinically useful progress has been made.For example, Pascal Buijs et al. (Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma, Viruses 2015, 7, pp. 2980-2998) studied recombinant Newcastle disease viruses expressing interferon or interferon antagonist proteins.

[0006] Studies show that angiogenesis is an important target for tumor treatment. Angiogenesis is the process by which new blood vessels develop from existing endothelial cells to provide various organs with sufficient oxygen and nutrients, which is important for tumor growth and metastasis. Antiangiogenic therapy is one of the important methods for cancer treatment. During tumor neovascularization, the pro-angiogenic factors that support tumor growth mainly include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), angiogenin, and transforming growth factor beta (TGF-β). These factors activate downstream signaling pathways by binding to the corresponding receptors, thereby regulating the formation of new blood vessels in tumors.

[0007] Most antiangiogenic drugs target proangiogenic factors and their receptors, or key molecules in downstream signaling pathways, thereby inhibiting tumor growth and metastasis through blocking the supply of nutrients to tumors. Antiangiogenic drugs currently approved by the FDA mainly include macromolecular monoclonal antibodies and small molecule targeted inhibitors. Among them, antiangiogenic factors mainly include thrombospondin 1 (TSP-1), angiostatin, endostatin, and interferon-α (IFN-α), etc., as well as VEGF blockers / antagonists, 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 activity of vascular endothelial cells, thereby inhibiting angiogenesis, blocking tumor growth and metastasis, and showing beneficial effects in cancer treatment. However, antiangiogenic drugs have significant therapeutic side effects. Summary of the Invention [Problem to be solved by the invention]

[0008] Considering the challenges in the prior art that limit the clinical application of oncolytic viruses, such as the limited therapeutic efficacy, low response rate and low tumor inhibition rate of NDV as a single drug, further research into recombinant NDV expressing exogenous proteins with antitumor activity, which can improve antitumor efficacy and reduce side effects, is still needed. [Means for solving the problem]

[0009] The inventors of the present application provide the corresponding recombinant oncolytic virus by integrating the coding genes of angiostatin and VEGF-Trap into the specific position of Newcastle disease virus genome. After being verified through pharmacodynamic testing, it was found that the antitumor effect of the recombinant oncolytic virus rNDV-VEGF-Trap is significantly higher than that of the rNDV group and the rNDV-angiostatin group. This can be reproduced in cancer cells with strong replicative ability, and can kill host cancer cells, while having reliable safety to non-cancer cells, thus solving the above technical problems.

[0010] In one aspect, the present application provides a recombinant Newcastle Disease Virus genome, wherein the genome comprises a gene encoding VEGF-Trap located between the P gene and the M gene of the Newcastle Disease Virus genome.

[0011] In another aspect, the present application provides a recombinant Newcastle Disease Virus, wherein the virus comprises a recombinant Newcastle Disease Virus genome as described above.

[0012] In yet another aspect, the present application provides a DNA molecule encoding a recombinant Newcastle Disease Virus genome as described above.

[0013] In yet another aspect, the present application provides a pharmaceutical composition comprising the recombinant Newcastle Disease Virus genome, recombinant Newcastle Disease Virus and / or DNA molecule as described above.

[0014] In yet another aspect, the present application provides a method for preparing the above-mentioned recombinant Newcastle disease virus, comprising: (1) Enzymatically cleaving a cloning vector containing a DNA sequence of a VEGF-Trap coding gene and an NDV viral vector, respectively, and ligating the resulting DNA sequence of the VEGF-Trap coding gene with the NDV viral vector to obtain a recombinant Newcastle disease virus plasmid; (2) transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain a recombinant Newcastle disease virus. The present invention provides a method comprising:

[0015] In yet another aspect, the present application provides a 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. Alternatively, the present application provides a use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and / or pharmaceutical composition for use in treating or ameliorating cancer. Alternatively, the present application provides a 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. Alternatively, the present application provides a method of treating or ameliorating cancer, comprising administering to a subject in need thereof the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and / or pharmaceutical composition.

[0016] By integrating the VEGF-Trap coding gene into a specific location in the Newcastle disease virus genome, the antitumor effect and oncolytic efficiency of the resulting recombinant oncolytic virus can be significantly improved.

[0017] In order to more clearly illustrate the exemplary technical solutions of the present application, the accompanying drawings are briefly introduced below. It should be understood that the following drawings only illustrate the exemplary technical solutions of the present application, and therefore should not be regarded as limiting the protection scope. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the Western blot detection results of allantoic fluid in Example 1, in which the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in Example 1 can stably express exogenous gene VEGF-Trap. [Diagram 2] 1 is a graph showing the growth curves of each recombinant Newcastle disease virus and the parental virus inoculated into DF-1 cells. [Diagram 3] 1 is a graph showing tumor growth curves in mice from the negative control group and each of the recombinant Newcastle Disease virus-treated and parental virus-treated groups. [Figure 4] 1 is a graph showing tumor inhibition results in mice from the negative control group and each of the recombinant Newcastle Disease virus-treated and parental virus-treated groups. [Diagram 5] Photographs showing tumors in mice from the negative control group and each of the recombinant Newcastle disease virus-treated and parental virus-treated groups. [Figure 6] FIG. 1 shows the HE staining results of the negative control group and each recombinant Newcastle disease virus-treated group and parent virus-treated group, in which the tumor tissue structure of the mice in the negative control group is dense, with intact cell morphology and vigorous growth; the tumor lesions of the mice in the rNDV group are collapsed, and the tumor cell structure is relatively loose; the tumor structure of the mice in the rNDV-angiostatin group is not significantly different from that of the rNDV group, but the tumor tissue lesions of the mice in the rNDV-VEGF-Trap group are widely collapsed, the tumor cell structure is very loose, immune cells are infiltrated in multiple places, and tumor cells are scattered individually. [Figure 7] FIG. 1 shows the results of immunohistochemical staining, in which the expression of CD34 in mice of the negative control group is abundant and that in the rNDV group is similar to that of the negative control group, while the expression of CD34 in mice of the rNDV-VEGF-Trap group is significantly reduced. [Figure 8]FIG. 13 shows inhibition of tumor growth in a mouse liver cancer model by rClone30-Anh-(F)-treated group, rClone30-Anh-(F)-angiostatin-treated group, and rClone30-Anh-(F)-VEGF-Trap-treated group. [Figure 9-1] FIG. 2 shows the genomic sequence of the recombinant Newcastle Disease virus rNDV-VEGF-Trap prepared in Example 1. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 10-1] FIG. 1 shows the genomic sequence of the recombinant Newcastle disease virus rClone30-Anh-(F)-VEGF-Trap prepared in Example 5. [Figure 10-2] Continued from Figure 10-1. [Figure 10-3] Continued from Figure 10-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The exemplary embodiments of the present application are described below, but the scope of protection of the present application is not limited thereto.Unless otherwise defined, technical and scientific terms used herein have the same meaning as that commonly understood by those skilled in the art to which this disclosure belongs, as can be found in, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0020] Unless otherwise specified, the term "treatment" herein means to cure, reduce, alleviate, slow, palliate or ameliorate a disease or related symptoms in a statistically significant manner, or to prevent, delay, halt, interrupt or halt the onset or further development of a disease or related symptoms.

[0021] Unless otherwise specified, all numbers used herein to express amounts of ingredients, measurements, or reaction conditions should be understood to be modified in all instances by the term "about" to indicate possible measurement error. For example, when connected to percentages, the term "about" can represent a variation within ±1%, e.g., ±0.5%, of the value intended to qualify it.

[0022] Unless otherwise specified, singular terms herein include plural referents and vice versa. Similarly, unless the context clearly dictates otherwise, the word "or" is intended to include "and" and vice versa.

[0023] Unless otherwise specified, the terms "comprise," "comprises," and "comprising" or their equivalents (e.g., contain, containing, include, including) in this specification are open-ended and should be understood as "including, but not limited to," meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may be included.

[0024] The percentage of identity (degree of homology) between sequences herein can be determined, for example, by aligning two sequences using freely available computer programs commonly used for this purpose on the World Wide Web, such as BLASTp or BLASTn, with default settings.

[0025] Newcastle disease virus (NDV) belongs to the order Mononegavirales, family Paramyxoviridae, and has an envelope, and the nucleocapsid is located within the envelope and contains an RNA genome and nucleocapsid protein. The genome of classical Newcastle disease virus is about 15-16 kb long, and contains NP gene, P gene, M gene, F gene, HN gene and L gene from the 3' end to the 5' end direction, which are used to code for the following six main proteins: nucleocapsid protein (NP), phosphate protein (P), matrix protein (M), fusion protein (F), hemagglutinin neuraminidase protein (HN) and large protein (L).

[0026] In one embodiment, the present application relates to a recombinant Newcastle Disease Virus genome, wherein the genome comprises a VEGF-Trap coding gene, and the VEGF-Trap coding gene is located between the P gene and the M gene of the Newcastle Disease Virus genome.

[0027] In some preferred embodiments, the VEGF-Trap encoding gene may be in the form of DNA or RNA.

[0028] In some preferred embodiments, the VEGF-Trap encoding gene has a sequence as set forth in SEQ ID NO:1 or a sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity thereto.

[0029] In some preferred embodiments, the sequence of the recombinant Newcastle Disease Virus genome is shown in SEQ ID NO:2 or SEQ ID NO:5 (see Figures 9 and 10).

[0030] In one embodiment, the present application relates to a recombinant Newcastle Disease Virus, wherein the virus comprises a recombinant Newcastle Disease Virus genome as described above.

[0031] In some preferred embodiments, the starting strain of Newcastle Disease Virus can be selected from, but is not limited to, low pathogenic strains LaSota, Hitchner B1 or V4; moderate pathogenic strains Mukteswar or Anhinga; high pathogenic strains F48E9, JS / 7 / 05 / Ch, Italien, Herts / 33 or NDV-BJ; and any chimeric strain constructed by genetic engineering means based on the starting strain, but is not limited to them.

[0032] In one embodiment, the present application relates to a DNA molecule (eg, a recombinant Newcastle Disease Virus plasmid) encoding a recombinant Newcastle Disease Virus genome as described above.

[0033] In one aspect, the present application relates to a pharmaceutical composition comprising a recombinant Newcastle Disease Virus genome, a recombinant Newcastle Disease Virus and / or a DNA molecule as described above.

[0034] In some preferred embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient. The pharma-ceutically acceptable excipient 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, permeation enhancers, pH adjusters, surfactants, diluents, etc. For other available pharma-ceutically acceptable pharmaceutical excipients, they can be found, for example, in "Handbook of Pharmaceutical Excipients" (4th ed.), edited by RC Rowe et al., translated by Junmin ZHENG, 2005, Chemical Industry Press.

[0035] In one embodiment, the present application provides a method for preparing the above-mentioned recombinant Newcastle Disease Virus, comprising: (1) Enzymatically cleaving a cloning vector containing a DNA sequence of a VEGF-Trap coding gene and an NDV viral vector, respectively, and ligating the resulting DNA sequence of the VEGF-Trap coding gene with the NDV viral vector to obtain a recombinant Newcastle disease virus plasmid; (2) transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain a recombinant Newcastle disease virus. The present invention relates to a method comprising the steps of:

[0036] In some preferred embodiments, the cloning vector can be constructed using a vector selected from a PUC57 vector, a pMD18-T vector, a pMD19-T vector, a pBlueScript SK(+ / -) vector, a pBluescript II KS(+ / -).

[0037] In some preferred embodiments, the NDV viral vector may be a full-length cDNA sequence of the genome of an NDV virus selected from, but not limited to, low pathogenic strains LaSota, Hitchner B1 or V4; moderate pathogenic strains Mukteswar or Anhinga; highly pathogenic strains F48E9, JS / 7 / 05 / Ch, Italien, Herts / 33 or NDV-BJ.

[0038] Preferably, the NDV viral vector can be pBluescript II KS(+ / -)-NDV (pBrNDV), pCI-neo-NDV or pOLTV5-NDV vector.

[0039] In the present disclosure, the recombinant Newcastle Disease Virus plasmid is co-transfected into the cell with helper plasmids NP, P and L (which can be any NP, P and L recombinant plasmids obtained by constructing NP, P and L genes in any eukaryotic expression vector known in the art) capable of expressing nucleocapsid protein NP, phosphoprotein P and RNA-dependent RNA polymerase L. In the present disclosure, the genes of the helper plasmids NP, P and L can be derived from any strain of NDV, e.g., LaSota, Anhinga, F48E9, etc. In some preferred embodiments, the recombinant Newcastle Disease Virus plasmid is co-transfected into cells with a helper plasmid selected from, but not limited to, 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).

[0040] Transfection in this specification refers to a technique for introducing exogenous nucleic acid substrates (including DNA and RNA) into cells, and mainly includes three routes: physical mediation (electroporation, microinjection and gene gun), chemical mediation (calcium phosphate co-precipitation, liposome transfection, cationic substrate-mediated) and biological mediation (protoplast transfection, virus-mediated transfection). Specific operations can be performed by those skilled in the art based on general knowledge in the art (for example, can be found in "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by J. Sambrook et al., translated by Fuchu HE, Science Press, 2017) by selecting appropriate experimental conditions and steps, or can be performed according to the instructions in a commercially available kit.

[0041] In some preferred embodiments, the cells may be selected from, but are not limited to, BHK-21 cells, BSR-T7 / 5 cells, VERO cells, DF-1 cells, 293 cells, and MDCK cells.

[0042] The culture of the transfected cells herein can be performed by those skilled in the art by selecting conventional culture media and culture conditions according to the cell type (e.g., "Cell Culture (3rd Edition)", Editor in Chief Bin LIU, World Publishing Corporation, January 2018; "Cell Culture Technology", Editor in Chief Rong LAN and Zhenhui ZHOU, Chemical Industry Press, August 2007; "Tissue and Cell Culture Technology (3rd Edition)", Editor in Chief Jingbo ZHANG, People's Medical Publishing House, June 2014, etc.).

[0043] In one embodiment, 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 treating or ameliorating cancer.

[0044] Alternatively, the present application provides the above-mentioned recombinant Newcastle Disease Virus genome, recombinant Newcastle Disease Virus, DNA molecule and / or pharmaceutical composition for use in treating or ameliorating cancer. Alternatively, 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. Alternatively, 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.

[0045] In some preferred embodiments, the cancer may be selected from, but is not limited to, colon cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), gastric cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial carcinoma, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine cancer, adrenal gland cancer, pituitary cancer, skin cancer, soft tissue cancer, hemangioma, brain cancer, nerve cancer, eye cancer, meningioma, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma, neuroblastoma, kidney cancer, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

[0046] Exemplary technical solutions of the present invention can be described by the contents in the following numbered items.

[0047] 1. A recombinant Newcastle Disease Virus genome, wherein the genome comprises a VEGF-Trap coding gene, and the VEGF-Trap coding gene is located between the P gene and the M gene of the Newcastle Disease Virus genome. 2. The recombinant Newcastle Disease Virus genome described in item 1, wherein the VEGF-Trap-encoding gene is in the form of DNA or RNA. 3. The recombinant Newcastle Disease Virus genome according to item 1 or 2, wherein the VEGF-Trap-encoding gene has the sequence shown in SEQ ID NO: 1 or a sequence having at least 80% identity thereto. 4. A recombinant Newcastle Disease Virus genome described in any one of paragraphs 1 to 3, wherein the sequence of the recombinant Newcastle Disease Virus genome is set forth in SEQ ID NO: 2 or SEQ ID NO: 5. 5. A recombinant Newcastle Disease Virus, wherein the virus comprises a recombinant Newcastle Disease Virus genome according to any one of paragraphs 1 to 4. 6. The recombinant Newcastle disease virus according to paragraph 5, wherein the starting strain of the Newcastle disease virus is selected from the group consisting of low pathogenic strains LaSota, Hitchner B1 or V4; moderate pathogenic strains Mukteswar or Anhinga; high pathogenic strains F48E9, JS / 7 / 05 / Ch, Italian, Herts / 33 or NDV-BJ; and any chimeric strain constructed by genetic engineering means based on the starting strain. 7. A DNA molecule encoding a recombinant Newcastle Disease Virus genome according to any one of paragraphs 1 to 4. 8. A pharmaceutical composition comprising a recombinant Newcastle disease virus genome according to any one of paragraphs 1 to 4, a recombinant Newcastle disease virus according to paragraph 5 or 6, and / or a DNA molecule according to paragraph 7. 9. The recombinant Newcastle Disease Virus described in item 8, wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient. 10. The recombinant Newcastle disease virus according to item 8 or 9, wherein the pharma- ceutically acceptable excipient is selected from a solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, disintegrant, filler, lubricant, wetting agent, osmotic regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, antioxidant, permeation enhancer, pH adjuster, surfactant or diluent, etc. 11. A method for preparing the recombinant Newcastle disease virus according to item 5 or 6, comprising: (1) Enzymatically cleaving a cloning vector containing a DNA sequence of a VEGF-Trap coding gene and an NDV viral vector, respectively, and ligating the resulting DNA sequence of the VEGF-Trap coding gene with the NDV viral vector to obtain a recombinant Newcastle disease virus plasmid; (2) transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain a recombinant Newcastle disease virus. A method comprising: 12. The method according to item 11, wherein 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, and pBluescript II KS(+ / -). 13. The method according to item 11 or 12, wherein the NDV viral vector is a full-length cDNA sequence of the genome of an NDV virus selected from the group consisting of low pathogenic strains LaSota, Hitchner B1 or V4; medium pathogenic strains Mukteswar or Anhinga; and high pathogenic strains F48E9, JS / 7 / 05 / Ch, Italien, Herts / 33 or NDV-BJ. 14. The method according to item 13, wherein the NDV viral vector is pBluescript II KS(+ / -)-NDV (pBrNDV), pCI-neo-NDV or pOLTV5-NDV vector. 15. The method of any one of paragraphs 11 to 14, wherein the recombinant Newcastle Disease Virus plasmid is co-transfected into the cell with a helper plasmid selected from 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. 16. The method of any one of paragraphs 11 to 15, wherein the cell is selected from a BHK-21 cell, a BSR-T7 / 5 cell, a VERO cell, a DF-1 cell, a 293 cell, or an MDCK cell. 17. Use of a recombinant Newcastle disease virus genome described in any one of clauses 1 to 4, a recombinant Newcastle disease virus described in clause 5 or 6, a DNA molecule described in clause 7, and / or a pharmaceutical composition described in any one of clauses 8 to 10 in the preparation of a medicament for treating or ameliorating cancer. 18. The use according to item 17, wherein the cancer is selected from colon cancer, liver cancer, lung cancer, gastric cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial carcinoma, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine cancer, adrenal cancer, pituitary cancer, skin cancer, soft tissue cancer, hemangioma, brain cancer, nerve cancer, eye cancer, meningioma, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma, neuroblastoma, kidney cancer, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

[0048] In order to clarify the purpose, technical problem and advantages of the examples of the present application, the technical problem in the examples of the present application is clearly and completely described below. If no specific conditions are given in the examples, the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, the reagents, materials or equipment used are conventional products that can be purchased commercially unless otherwise indicated by the manufacturer. Below, a more detailed description of the features and performance of the present application is provided in conjunction with the examples.

[0049] [Example] Unless otherwise stated, the design, synthesis and cloning of genes, as well as the construction and transfection of vectors, and electrophoresis, etc., contained in this application can be carried out according to techniques known in the art (see, for example, records in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY). If not specifically specified, the technical means used in the examples are conventional means well known to those skilled in the art (see, for example, "Principle and Experimental Technology of Molecular Virology", Editor in Chief Wei PAN, Shanghai Second Military Medical University Press, November 2002; "Fundamentals and Experimental Technologies of Medical Virology", Editor in Chief Zhenxiang HUANG, Science Press, February 1990, etc.).

[0050] The following examples include the following exogenous genes: the vascular inhibitor gene VEGF-Trap and the human angiostatin gene angiostatin (Genbank accession number NG_016200.1).

[0051] pMD19-T was purchased from TaKaRa Bioengineering (Dalian) Co., Ltd. (Dalian TaKaRa Company). BHK-21 cells (baby hamster kidney cells), human colon cancer cells HCT116, mouse colon cancer cells CT26, mouse breast cancer cells 4T1, and human umbilical vein endothelial cells EA.hy926 were all purchased from ATCC.

[0052] DMEM (high glucose) medium, McCoy's 5A medium, RPMI 1640 medium, trypsin, calf serum (FCS) and fetal bovine serum (FBS) were purchased from GIBCO company. SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd. Balb / c mice (Kunming mice) were purchased from Sipeifu (Beijing) Biotechnology Co., Ltd.

[0053] [Example 1] Preparation of recombinant Newcastle disease viruses I. Construction of recombinant Newcastle Disease Virus plasmids with exogenous genes inserted (e.g., to obtain pBrNDV-VEGF-Trap) According to the literature records of Jocelyn Holash et al. (VEGF-Trap: A VEGF blocker with potent antitumor effects, August 2002; https: / / doi.org / 10.1073 / pnas.172398299), the sequence of the VEGF-Trap gene (SEQ ID NO: 1) shown as follows was obtained: TIFF0007680564000001.tif15170TIFF0007680564000002.tif78170

[0054] The recombinant plasmid pBrNDV-VEGF-Trap was constructed according to the following method. 1. The VEGF-Trap gene containing the enzyme cleavage sites for SacII enzyme (5') and PmeI enzyme (3') was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into pUC57 vector to form pUC57-VEGF-Trap, which was used for subsequent experiments. 2. The plasmid pUC57-VEGF-Trap from step 1 was cleaved with restriction endonucleases PmeI and SacII (purchased from NEB Company) according to the manufacturer's instructions. The enzyme cleavage product was identified by nucleic acid agarose gel electrophoresis. After the enzyme cleavage product was identified as correct, it was recovered by gel purification kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP219) according to the manufacturer's instructions. 3. Plasmid pBrNDV (purchased from NEB) was cleaved with restriction endonucleases PmeI and SacII according to the manufacturer's instructions, and the plasmid vector was recovered by gel purification kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP219) according to the manufacturer's instructions. 4. The enzymatic cleavage product of step 2 was ligated into the vector of step 3 by using T4 DNA ligase (purchased from NEB) according to the manufacturer's instructions to obtain the recombinant Newcastle Disease Virus plasmid pBrNDV-VEGF-Trap, in which the VEGF-Trap gene was inserted between the P and M genes of the plasmid. PCR was performed according to the manufacturer's instructions to ligate the above recombinant plasmid (upstream primer: 5' TIFF0007680564000003.tif41613' (SEQ ID NO: 3); downstream primer: 5' TIFF0007680564000004.tif41613' (SEQ ID NO: 4)) was used with Takara Bio taq enzyme (purchased from Takara Bio), and identification of double enzyme digestion was performed with PmeI and SacII (37°C, 1 hour), and the correct plasmid sample was packaged and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were aligned using sequence analysis software DNAMAN. After sequencing, the sequenced sequence was consistent with the target sequence.

[0055] The following recombinant plasmid pBrNDV-angiostatin, into which the exogenous angiostatin gene (NG_016200.1) was inserted, was constructed and identified using the same method as above (the primers for PCR amplification were the same as above).

[0056] II. Preparation of recombinant Newcastle disease virus The recombinant Newcastle disease virus rNDV-VEGF-Trap was prepared using the recombinant Newcastle disease virus plasmid described above by the following method.

[0057] 1. Using liposome transfection technology, the successfully constructed recombinant Newcastle disease virus plasmid pBrNDV-VEGF-Trap was co-transfected with three helper plasmids pBL-NP, pBL-P and pBL-L (constructed according to the method described in Jinying GE, Basic and applied research on reverse genetic operation of Newcastle disease virus, 2006) into a monolayer of BHK-21 cells stably expressing T7 RNA polymerase. The operation steps were performed according to the instructions of Lipofectamine 3000 transfection reagent (purchased from Invitrogen). After 72 hours of incubation, the cells were frozen and thawed three times at -80°C, centrifuged at 12000 rpm at 4°C, and the supernatant was collected, to which 0.001% trypsin was added.

[0058] 2. Take 200 μL of the supernatant obtained in step 1 and inoculate it into the allantoic cavity of 9-day-old SPF-grade chicken embryos, then incubate at 37° C., 5% CO 2The allantoic fluid was obtained by culturing the cells in a 100-mL incubator for 72 hours. A hemagglutination titer test was performed (e.g., the method described in Ling ZHOU, Yanfang LI, Xiali MA, Isolation and Identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary Medicine, 2015, 40(03):8-10). The positive allantoic fluid was frozen and stored in a -80°C refrigerator, and the successfully rescued recombinant Newcastle disease virus was named rNDV-VEGF-Trap.

[0059] 3. Recombinant viral RNA was extracted from the recombinant Newcastle disease virus obtained in step 2 according to the instructions of QIAamp Viral RNA Mini Kit (50), and cDNA samples were obtained by random primers. RT-PCR amplification (Thermo Fisher RT-PCR kit, the first strand of cDNA was synthesized, and PCR was performed according to the instructions of the kit) was performed for the inserted VEGF-Trap gene using P / M site primers P / MF (whose sequence was the same as SEQ ID NO: 3) and P / MR (whose sequence was the same as SEQ ID NO: 4). The amplified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were aligned using sequence analysis software DNAMAN. After sequencing, the sequenced sequences were consistent with the target sequences.

[0060] 4. The virus with the exogenous gene sequenced as correct in step 3 was taken and inoculated into the allantoic cavity of 9-11 day old SPF grade chicken embryos and cultured at 37°C for 72 hours. The chicken embryo allantoic fluid was collected for HA detection (for example, by referring to the related method recorded in Ling ZHOU, Yanfang LI, Xiali MA, Isolation and Identification of a chicken-derived Newcastle disease virus [J], Zhejiang Animal Husbandry and Veterinary Medicine, 2015, 40(03):8-10). The allantoic fluid with HA titer higher than 29 was selected for mixing and then packaged for use.

[0061] 5. The allantoic fluid of each group was collected and used for Western blot (for example, see 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) to detect the expression of each exogenous gene. The results showed that the recombinant Newcastle disease virus rNDV-VEGF-Trap can stably express the exogenous gene VEGF-Trap (see FIG. 1).

[0062] Similarly, recombinant Newcastle Disease Virus rNDV-angiostatin was prepared according to the method described above.

[0063] In the following description, unless otherwise specified, rNDV-VEGF-Trap and rNDV-Angiostatin refer to the recombinant viruses prepared by the above method using the recombinant plasmids pBrNDV-VEGF-Trap and pBrNDV-Angiostatin, respectively.

[0064] The parent virus in the following Examples 2-3 refers to a virus obtained through the following modification: Starting from Newcastle disease virus LaSota (purchased from Harbin Veterinary Epidemic Prevention Station), the F gene of the LaSota strain is replaced with the F gene of the highly pathogenic strain F48E9 of Newcastle disease virus (GenBank accession number: AY508514.1) according to the "gene replacement" engineering method described in Yong WANG et al. (Evaluation of Newcastle disease virus with derived Hemagglutinin-Neuraminidase gene of mesogenic strain, Acta Microbiologica Sinica, 2008, 48(5): 638-643). The parent strain is named rNDV in this specification.

[0065] [Example 2] Detection of the growth stability of recombinant viruses TCID50 was detected according to the following method. 1. 10,000 DF-1 cells were seeded in a 96-well microplate in DMEM medium supplemented with 10% FBS and 1% antibiotics and incubated at 37°C, 5% CO 2 The cells were cultured overnight in an incubator. 2. Before inoculation with each recombinant Newcastle Disease Virus (rNDV-VEGF-Trap and rNDV-Angiostatin) and parental virus, the original cell culture medium in the plate from step 1 was discarded and 180 μL of fresh DMEM culture medium supplemented with 10% allantoic fluid and 1% antibiotics was added thereto. 3. 20 μL of each recombinant Newcastle disease virus and parent virus were inoculated into the top two rows of wells, respectively. After mixing by pipetting, 20 μL of each mixed solution was dispensed into the lower wells for serial 10-fold gradient dilutions. Each virus was made up of triplicates. 4. Inoculated recombinant Newcastle disease viruses and parental viruses were incubated at 37°C and 5% CO 2 After incubation in a 30-well incubator for 1 h, the culture medium was discarded and washed once with 0.2 mL of 1×PBS buffer, to which 200 μL of fresh DMEM culture medium supplemented with 10% allantoic fluid and 1% antibiotics was added. 5. Culture at 37°C and 5% CO 2 The cells were incubated in an incubator for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and the number of lesion holes was observed under an inverted optical microscope and recorded. The virus titer was calculated using the Reed and Muench method, and a growth curve was plotted.

[0066] The results are shown in Figure 2. The growth trends of the recombinant Newcastle disease viruses rNDV-VEGF-Trap and rNDV-Angiostatin were not significantly different from those of the parent viruses, indicating that the insertion of the exogenous gene did not affect viral growth.

[0067] [Example 3] Therapeutic effect of recombinant Newcastle disease virus on tumors 1. Establishment of a Balb / c mouse colon cancer tumor-bearing animal model Mouse colon cancer cells CT26 were taken and stained with trypan blue to determine that the cell viability was 95% or higher, and then diluted with saline at 1 × 10 6 The cells were diluted to 1000 μg / mL of cell suspension, and the cell suspension was injected subcutaneously into the right flank at a dose of 0.1 mL per Balb / c mouse. After 8 to 12 days, the diameter of the solid tumor reached 5 to 8 mm, indicating that the model was successfully constructed and subsequent experiments could be performed. Individuals with large differences in tumor shape and size were eliminated, and mice with tumors with a diameter of 5 to 8 mm were selected as model mice.

[0068] 2. Therapeutic effect of recombinant viruses on tumors Model mice were randomly divided into 4 groups with 10 mice per group and treated as follows: rNDV-VEGF-Trap group: 0.2 mL of PBS suspension of rNDV-VEGF-Trap virus prepared in Example 1 (prepared with 1x PBS buffer; 10 7 pfu of virus) was injected into the tumors of model mice daily for 14 days; rNDV-angiostatin group: 0.2 mL of PBS suspension of rNDV-angiostatin virus prepared in Example 1 (prepared with 1x PBS buffer; 10 7 pfu of virus) was injected into the tumors of model mice daily for 14 days; rNDV group: 0.2 mL of PBS suspension of parent virus (prepared with 1x PBS buffer; 10 7 pfu of virus) was injected into the tumors of model mice daily for 14 days; Negative control group (model group): 0.2 mL of SPF chicken embryo allantoic fluid was injected into the tumors of the model mice every day for 14 days.

[0069] From the day of treatment, the tumor volume was measured every other day, and a tumor growth curve was created based on the measurement results (Figure 3). After treatment, the mice were euthanized, the tumors were removed, and the tumor weight and size were measured (the results are shown in Figures 4 and 5). The average tumor volume in the negative control group was 1889.17 mm 3 and the mean tumor volume in the parental NDV (rNDV) treated group was 728.49 mm 3 The mean tumor volume in the rNDV-angiostatin treated group was 774.37 mm 3 The mean tumor volume in the rNDV-VEGF-Trap treated group was 350.36 mm 3 The results showed that compared with the negative control group, both the parent virus and the recombinant Newcastle disease virus had significant inhibitory effects on tumor growth, in which the antitumor effect of rNDV-angiostatin was not significantly different from that of the rNDV group, but the antitumor effect of rNDV-VEGF-Trap was significantly higher than that of the rNDV group.

[0070] 3 Observation of pathological sections of tumors To observe the inhibitory effect of recombinant Newcastle disease virus rNDV-VEGF-Trap on colon cancer and associated blood vessels, tumor tissues from each group of mice were taken and fixed with 4% paraformaldehyde. Paraffin sections of tumor tissues were prepared with a thickness of 4 μm as follows. The morphology of tumor tissues in each group was observed under a microscope after HE staining, and the expression of CD34 protein in tumor tissues in each group was detected by immunohistochemical staining. The specific procedures were as follows:

[0071] 3.1 Preparation of paraffin sections (1) Paraffin was placed in a 1L beaker, beeswax was added to it, and it was then placed in a wax box at 60°C. Once the wax was completely melted, it was filtered through filter paper, removed, cooled to room temperature, and then returned to the wax box to melt, and this was repeated 2-3 times. (2) The slides were placed one by one into the prepared cleaning solution (concentrated potassium dichromate solution: 25 g potassium dichromate, 75 mL water, 400 mL concentrated sulfuric acid), soaked for 24 hours, rinsed thoroughly with tap water, further soaked in 95% alcohol by volume for 24 hours, then dried with lens paper and sterilized by dry heating at 180°C for 6 hours. After sterilization, the slides were soaked one by one into a staining vat containing APES treatment agent (APES:acetone=1:50) for 5 minutes. After removal, the slides were rinsed twice with distilled water, placed in a slice box, and dried at 60°C for subsequent use.

[0072] (3) After the above treatment, the lung tissues of the mice in each group were taken and fixed in a pre-prepared 4% neutral formaldehyde fixative for 48 hours. The tissues were removed and the tissue blocks were trimmed with a blade to a size of about 1 cm. (4) Dehydration and clearing: 30% ethanol by volume for 30 minutes, 50% ethanol by volume for 30 minutes, 70% ethanol by volume for overnight at 4°C, 80% ethanol by volume for 30 minutes the next day, 90% ethanol by volume for 30 minutes, 100% ethanol by volume for 30 minutes (twice). In a fume hood, the dehydrated tissue was immersed in a staining vat with xylene:anhydrous ethanol=1:1, rinsed for 20 minutes, then rinsed in pure xylene for 20 minutes, and repeated twice. After this, the color of the tissue block became dark and transparent, and the xylene solution became transparent. (5) Wax dipping: The transparent tissue was transferred to completely melted paraffin in (1) and placed in a 60°C incubator for 120 minutes. (6) Embedding: The paraffin in the wax box was poured into a paper box, and the tissue blocks were placed in order using small tweezers, ensuring that the cut surface was facing down. (7) Paraffin block trimming: After the paraffin in the paper box solidified, the paraffin block was taken out and trimmed into a trapezoid shape using a blade. The distance between the edge of the tissue and the edge of the paraffin block should not be less than 2 mm. (8) Sectioning: Sectioning was performed continuously at a thickness of about 4 μm. The sections were spread in a water bath of a Water Bath-Slide Drier at about 40° C. Using the slides processed in (2), remove the sections from the water bath and place them in the frame of the Water Bath-Slide Drier. After drying slightly, they were immediately placed in a 37° C. oven overnight to bring the sections closer to the slides and make them transparent. After leaving them overnight, the slides were placed in a slide box and prepared for use.

[0073] 3.2 HE staining (1) Dewaxing: After preheating the wax box to a temperature of 60°C, the prepared tumor tissue slices were placed in a 60°C oven for 1 hour, and after the paraffin on the slices melted, they were immersed in xylene for 5 minutes (3 times): absolute ethanol-2 minutes; 90% ethanol-5 minutes; 80% ethanol-5 minutes; 70% ethanol-5 minutes; 50% ethanol-5 minutes; water-5 minutes (3 times). (2) Staining: The sections were stained with hematoxylin for 30 seconds, then rinsed with distilled water for 1 minute, and inserted into an eosin staining tray, immediately removed, and immersed in distilled water for 1 minute (3 times). (3) Dehydration: 50 volume l% ethanol - 1 min; 70 volume l% ethanol - 1 min; 80 volume l% ethanol - 1 min; 90 volume l% ethanol - 1 min; 100 volume l% ethanol - 5 min (twice); xylene 1 to 5 min (twice). (4) Neutral gum sealing: A cover glass was placed on a flat surface after drying, 1-2 drops of neutral gum were added to its center, the tissue side of the slide was gently covered with the cover glass, the neutral gum was spread completely along the cover glass, and then the slide was tilted and the excess xylene was absorbed using filter paper, carefully to avoid the generation of air bubbles. The slides were dried and stored in a slide box, and the pathological changes of the tumor tissues in each group of mice were observed and compared under a microscope.

[0074] 3.3 Immunohistochemical staining 1. Dewaxing and hydration: The prepared paraffin sections were immersed in xylene twice for 5 minutes each time for dewaxing, then placed in various levels of alcohol solutions of 100%, 95%, 90%, 80% and 70% by volume for 5 minutes each, and then rinsed twice with distilled water for 3 minutes each time. 2. Antigen retrieval: 0.01M sodium citrate buffer (pH=6.0) was heated to 95°C in a water bath, then the slide was placed in it and heated for 10 minutes. It was rinsed with 1x PBS buffer three times, 5 minutes each time. 3. Inactivation of endogenous peroxidase: Drop an appropriate amount of endogenous peroxidase blocking buffer (purchased from Beyotime, Cat. No. P0100A) onto the sample to completely cover it, and incubate it at room temperature for 10 minutes. Wash it with 1×PBS buffer three times, 3 minutes each time.

[0075] 4. Blocking: Blocking buffer (purchased from Beyotime, Cat. No. P0260 QuickBlock) was dropped onto the tissue sections to block them for 10 minutes. 5. Primary antibody incubation: Using blocking buffer, CD34 primary antibody working solution (purchased from Abcam) was prepared according to the dilution ratio in the antibody's instructions. The primary antibody working solution was dropped onto each tissue section and incubated overnight at 4°C. After incubation with primary antibody (anti-CD34 antibody, purchased from Abcam), the tissue sections were washed with 1xPBST buffer three times, 5 minutes each time. 6. Secondary antibody incubation: Using blocking buffer, a working solution of goat anti-rabbit secondary antibody (purchased from Abcam) was prepared according to the dilution ratio in the antibody's instructions. The secondary antibody working solution was dropped onto each tissue section and incubated at room temperature for 1 hour. After incubation with the secondary antibody, the tissue sections were washed with 1xPBST buffer three times, 5 minutes each time. 7. Color development: 100 μL of DAB color development working solution (purchased from Beyotime) was dropped onto the sample to completely cover it. Incubation was carried out at room temperature in the dark for 15 minutes. After color development, the DAB color development working solution was removed and washed with distilled water 1-2 times to stop the color development reaction.

[0076] The results showed that after treatment, the tumor tissue structure of the mice in the negative control group (model group) was dense, with intact cell morphology and vigorous proliferation; the tumor lesions of the mice in the rNDV group were collapsed, and the structure of the tumor cells was relatively loose; the tumor structure of the mice in the rNDV-angiostatin group was not significantly different from that of the rNDV group, but the tumor tissue lesions of the mice in the rNDV-VEGF-Trap group were widely collapsed, the tumor cell structure was very loose, immune cells were infiltrated in many places, and tumor cells were scattered individually (see Figure 6). The immunohistochemical staining results showed that the expression of CD34 in the mice in the model group was abundant, and the rNDV group was similar to the negative control group, while the expression of CD34 in the mice in the rNDV-VEGF-Trap group was significantly reduced. It was shown that rNDV-VEGF-Trap had a therapeutic effect on inhibiting the proliferation of vascular endothelial cells (see Figure 7).

[0077] [Example 4] Safety testing of recombinant Newcastle disease viruses Healthy 6-week-old SPF grade Balb / c mice were selected and divided into groups of 10 mice per group. Mice in the control group were fed normally. Each mouse in the test group was given 5 × 10 8 Recombinant Newcastle Disease Virus rNDV-VEGF-Trap was intraperitoneally injected at pfu (10 times the therapeutic dose) and then observed for 30 days. Mice with obvious adverse reactions, such as lethargy, ruffled fur, and death, were considered positive.

[0078] The results showed that on the second day after injection, the fur of the three mice in the test group stood up, and their food and water intake was not affected. After one week of continuous injection of the recombinant Newcastle Disease Virus rNDV-VEGF-Trap, the fur of the mice in the test group returned to normal. After one month of continuous observation, the mice in the test group did not show any obvious adverse reactions (including lethargy and ruffled fur), and no mice died.

[0079] Therefore, the recombinant Newcastle disease virus rNDV-VEGF-Trap prepared in this application had good safety.

[0080] [Example 5] The oncolytic virus rClone30-Anh-(F) as the parental strain provided in the following examples was obtained through the following modification: starting from the Newcastle disease virus LaSota strain (purchased from Harbin Veterinary Epidemic Prevention Station), the F gene of the LaSota strain was replaced with the F gene of the Newcastle disease virus medium pathogenic strain Anhinga (GenBank accession number: EF065682.1) according to the "gene replacement" engineering method described in Yong WANG et al. (Acta Microbiologica Sinica, 2008, 48(5): 638-643, supra). In this example, the genome of the recombinant Newcastle disease virus expressing VEGF-Trap is shown in SEQ ID NO: 5 (see FIG. 10), and the nucleic acid sequence of VEGF-Trap is shown in SEQ ID NO: 1.

[0081] First, according to the method described in Example 1, VEGF-Trap gene (SEQ ID NO: 1) and angiostatin gene (NG_016200.1) were used to construct the corresponding recombinant Newcastle disease virus plasmid and recombinant Newcastle disease virus, respectively, and the recombinant Newcastle disease virus was successfully rescued. The correct recombinant Newcastle disease virus successfully rescued and identified was named rClone30-Anh-(F)-VEGF-Trap and rClone30-Anh-(F)-angiostatin, respectively.

[0082] Then, H22 subcutaneous tumor-bearing model (i.e., mouse liver cancer model) was established using H22 cells (purchased from Nanjing CoBioer) according to the method described in Example 4. When the tumors were approximately 100 mm 3Once the tumors had grown to 100 μL, 100 μL of PBS suspension (prepared using 1× PBS buffer) of each of the oncolytic viruses mentioned above (rClone30-Anh-(F)-Angiostatin and rClone30-Anh-(F)-VEGF-Trap) and the parental strain rClone30-Anh-(F) was injected into the tumors. In each treatment group, 6 animals per group were injected with 1×10 100 μL of each oncolytic virus (rClone30-Anh-(F)-Angiostatin, rClone30-Anh-(F)-VEGF-Trap, and rClone30-Anh-(F)) once a day for a total of 14 days. 7 PFU injection into the tumors, and 1x PBS buffer (without oncolytic virus) was injected into the tumors of a mouse liver cancer model as a negative control group (also named the "PBS-treated group"), and the tumor tissues were dissected to observe the therapeutic effect of each recombinant virus.

[0083] As shown in Figure 8, after treatment, the mean tumor volume in the negative control group was 1421.77 mm 3 and the mean tumor volume in the parental rClone30-Anh-(F) treated group was 807.30 mm 3 The mean tumor volume in the rClone30-Anh-(F)-angiostatin treated group was 668.60 mm 3 The mean tumor volume in the rClone30-Anh-(F)-VEGF-Trap treated group was 326.05 mm 3 The results showed that compared with the negative control group, the parental rClone30-Anh-(F)-treated group, the rClone30-Anh-(F)-angiostatin-treated group and the rClone30-Anh-(F)-VEGF-Trap-treated group were all able to inhibit tumor growth, and in particular, the rClone30-Anh-(F)-VEGF-Trap-treated group had the smallest mean tumor volume.

[0084] The above description is merely a preferred example of the present invention, and is not intended to limit the present invention. For those skilled in the art, the present invention can be modified and changed in various ways. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0085] For purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of this application. Any reference to the dates of these documents or representations of the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the dates of these documents or the accuracy of the contents of these documents. In addition, in any country, any reference to these publications in this specification does not constitute an admission that the publications form part of the general knowledge in the art.

Claims

1. A recombinant Newcastle disease virus genome, the genome comprising a VEGF-Trap coding gene, the VEGF-Trap coding gene being located between the P gene and the M gene of the Newcastle disease virus genome, the VEGF-Trap coding gene having a sequence as set forth in SEQ ID NO:1 or a sequence having at least 90% identity thereto.

2. 2. The recombinant Newcastle Disease Virus genome of claim 1, wherein the sequence of the recombinant Newcastle Disease Virus genome is set forth in SEQ ID NO: 2 or SEQ ID NO:

5.

3. 2. A recombinant Newcastle disease virus, the virus comprising the recombinant Newcastle disease virus genome of claim 1.

4. The recombinant Newcastle disease virus of claim 3, wherein the starting strain of Newcastle disease virus is selected from low pathogenicity strains LaSota, Hitchner B1 or V4; moderate pathogenicity strains Mukteswar or Anhinga; high pathogenicity strains F48E9, JS / 7 / 05 / Ch, Italian, Herts / 33 or NDV-BJ; and any chimeric strain constructed by genetic engineering means based on the starting strain.

5. A DNA molecule encoding the recombinant Newcastle Disease Virus genome of claim 1.

6. A pharmaceutical composition comprising a recombinant Newcastle Disease Virus genome according to claim 1 or 2, a recombinant Newcastle Disease Virus according to claim 3 or 4, and / or a DNA molecule according to claim 5.

7. 7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, wherein the pharma- ceutically acceptable excipient is selected from a solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, disintegrant, filler, lubricant, wetting agent, osmotic regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, antioxidant, permeation enhancer, pH adjuster, surfactant or diluent.

9. A method for preparing the recombinant Newcastle disease virus of claim 3, comprising: (1) Enzymatically cleaving a cloning vector containing a DNA sequence of a VEGF-Trap coding gene and an NDV viral vector, respectively, and ligating the resulting DNA sequence of the VEGF-Trap coding gene with the NDV viral vector to obtain a recombinant Newcastle disease virus plasmid; (2) transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain a recombinant Newcastle disease virus. A method comprising:

10. 10. The method of claim 9, wherein 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, and pBluescript II KS(+ / -).

11. The NDV viral vector is a full-length cDNA sequence of the genome of an NDV virus selected from the following: low pathogenic strains LaSota, Hitchner B1 or V4; medium pathogenic strains Mukteswar or Anhinga; high pathogenic strains F48E9, JS / 7 / 05 / Ch, Italien, Herts / 33 or NDV-BJ; The method according to claim 9 or 10.

12. The method described in claim 11, wherein the NDV viral vector is pBluescript II KS(+ / -)-NDV (pBrNDV), pCI-neo-NDV or pOLTV5-NDV vector.

13. The method of claim 9 or 10, wherein the recombinant Newcastle Disease Virus plasmid is co-transfected into the cell together with a helper plasmid selected from 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.

14. 11. The method of claim 9 or 10, wherein the cell is selected from a BHK-21 cell, a BSR-T7 / 5 cell, a VERO cell, a DF-1 cell, a 293 cell or an MDCK cell.

15. 7. The pharmaceutical composition of claim 6 for use in the treatment or amelioration of cancer.

16. The pharmaceutical composition of claim 15, wherein the cancer is selected from colon cancer, liver cancer, lung cancer, gastric cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial carcinoma, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine cancer, adrenal cancer, pituitary cancer, skin cancer, soft tissue cancer, hemangioma, brain cancer, nerve cancer, eye cancer, meningioma, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma, neuroblastoma, kidney cancer, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

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