Mutant Orff virus and its use

Abstract

The present invention provides the use of mutant orf viruses (ORFVs) and pharmaceutical compositions thereof for treating cancer.

Description

[Technical Field]

[0001] Related applications This application claims priority to the Chinese application filed on 13 August 2020, application number 202010813096.9.

[0002] This invention relates to the field of biotechnology, and more specifically to the use of mutant ORFV (ORFV) and its pharmaceutical composition for the treatment of cancer. [Background technology]

[0003] ORFV (abbreviated as ORFV) belongs to the genus Parapoxvirus within the family Poxviridae. This genus also includes Bovine papular stomatitis virus, pseudobovine poxvirus, and New Zealand red deer parapoxvirus. It contains several types of viruses, including red deer in New Zealand and seal box virus (Sealpox), and causes contact-transmitted, epitheliotropic diseases in sheep and goats. After the virus infects sheep, the disease typically progresses to erythema appearing on areas such as the lips, tongue, nose, and mammary glands of goats and sheep, which then develop into papules, vesicles, and pustules, finally crusting and exhibiting hyperplastic inflammation.

[0004] Chinese patent CN104017776B discloses the use of isolated and cell-passaged attenuated ORFV in vaccine creation. Chinese patent publication CN108026542A describes the construction of recombinant ORFV vectors using virus strain D1701, expressing viral antigens, tumor antigens, etc., and using them in vaccine production.

[0005] Oncolytic viral therapy is a systemic therapy that uses selected, naturally occurring or engineered viruses to treat tumors. Certain gene mutations that promote the proliferation and survival of tumor cells are precisely what viruses with lytic properties can use to promote growth within them; this is one reason why oncolytic viruses can precisely attack tumor cells. [1、2、3] Oncolytic viruses that infect cancer cells can promote the lysis and death of cancer cells at the appropriate point in the viral lifecycle, releasing infectious viral particles that can infect surrounding uninfected cancer cells. Furthermore, neoantigens released by the lysis of cancer cells activate the immune system, leading to subsequent attacks against cancer. [4] Currently, there are more than 90 clinical trials worldwide using oncolytic viruses to treat human malignancies, and among them, the T-Vec oncolytic virus drug has received FDA approval. [5] .

[0006] ORFV viruses are double-stranded DNA viruses with a length of approximately 134–139 ​​kb. The virus particles are 230–280 nm long and 150–200 nm wide, and have an oval, coiled shape. [6] The ORFV virus has the following characteristics: 1) Similar to other members of the Poxviridae family, the ORFV virus replicates only in the cytoplasm of the host cell and does not enter the cell nucleus. Therefore, it does not become incorporated into the host cell genome, making it highly safe and extremely low in terms of carcinogenicity. [7、10] 2) Humans can be infected by contact with the infected area of ​​an animal through damaged skin. Clinically, mild pustules develop at the site of infection (generally the fingers), with few other undesirable side effects, and it generally does not cause fatal disease. It can heal on its own after 6-8 weeks. [8、11] 3) Not restricted by the relevant receptors on the cell surface during infection [9、10] High heterogeneity of solid tumors There is a prospect of providing a route to overcome - 4) The genome is huge, which is advantageous for the insertion of foreign genes and high - efficiency expression. 5) The viral genome is stable and has a high replication fidelity. 6) Almost no or no neutralizing antibodies are generated in the host body, and infection can be repeated multiple times

[11] , enabling repeated intravenous injection. 7) It can induce the innate and adaptive anti - tumor immune responses of the organism

[12] . 8) Vaccinia virus in the same family has already been used in billions of people worldwide as an anti - smallpox vaccine, and the risk of clinical application is low. 9) Based on its own characteristics, it has the ability to transform cold tumors into hot tumors

[18] . Due to the above characteristics, the virus of this genus has the prospect of being used in the treatment of solid tumors as a new oncolytic virus.

[0007] The ORFV virus strain generally encodes about 130 - 134 genes. Currently, the already identified virulence factors mainly include viral interferon - inhibitory protein (OVIFNR, ORFV020), chemokine - binding protein (CBP, ORFV112), GM - CSF / IL - 2 inhibitory protein (GIF, ORFV117), viral interleukin 10 (vIL - 10, ORFV127), and vascular endothelial growth factor - like protein (VEGF - like protein, ORFV132), etc.

[11] . The proteins encoded by these genes can also regulate the immune response of the organism

[13] .

[0008] The gene ORFV002 is a late - stage viral gene that localizes in the cell nucleus after protein synthesis and inhibits the NF - κB pathway in the cell nucleus, and has been identified as the first NF - κB nuclear inhibitor produced by the ORFV virus.

[19] .

[0009] The gene ORFV005 is a hypothetical protein gene, and its mechanism of action has not yet been clarified.

[0010] The gene ORFV007 is approximately 483 bp long and encodes deoxyuridine triphosphatase (duTPase).

[15] This protein is an important enzyme in the synthesis of dNTPs (deoxyribonucleoside triphosphate). Normally, the concentration of dNTPs in healthy cells is strictly regulated, which is detrimental to viral replication. However, cancer cells have relatively high concentrations of dNTPs, which can promote viral replication.

[16] By deleting the ORFV007 gene, the replication of the virus in normal cells is inhibited, but its amplification in tumor cells is not affected, thus achieving the objective of selective replication of the ORFV virus in cancer cells.

[0011] The ORFV111 gene is a hypothetical protein gene, and its mechanism of action has not yet been elucidated.

[0012] The gene ORFV112 (chemokine-binding protein), which encodes the CBP protein, is approximately 864 bp long, and its function is to inhibit the antiviral mechanisms of immune cells.

[14] This protein is structurally and functionally similar to the CBP-II protein of other poxviruses, and can suppress the migration of DC cells to inflammatory sites, as well as inhibit DC cells from activating T cells.

[0013] Currently, publicly available ORFV virus strains used in oncolytic virus research include NZ-2, NZ-7, D1701, and NA1 / 11. In conventional techniques, ORFV viruses can accommodate a relatively large amount of exogenous DNA fragments, and are typically used as vectors to insert into certain tumor-specific antigens or viral antigens, cytokines, etc., to obtain genetically modified viruses for research.

[0014] International Publication WO2012122649A1 discloses recombinant OFRV viruses (with the vaccinia virus E3L gene inserted) used to infect tumor cells containing specific host range genes (SPI-1, K1L, C7L, B5R, p28 / N1R, E3L, etc.) for the creation of cancer drugs. Chinese Patent Publication CN108220251A discloses recombinant infectious pustular oncolytic virus, its manufacturing method, and use, mainly involving knocking out the ORFV132(VEGF) gene of an NA1 / 11 virus strain, inserting a P53-EGFP fusion protein gene at the same position, and using it for the creation of cancer drugs. Neither of the virus strains described in the above two patent documents have been reported to have deletions of the ORFV112 gene, nor have any reported deletions of the ORFV007 gene.

[0015] This specification provides a mutant Orff virus characterized by the deletion of the functional expression product of the gene ORFV112 and / or the gene ORFV111, the deletion of which is caused by the complete or partial deletion of the gene ORFV112 encoding CBP and / or the gene ORFV111 encoding virtual protein 111. Surprisingly, the inventors have found that the complete or partial deletion of the ORFV112 gene and / or the ORFV111 gene has a clear enhancing effect on the antitumor effect of the virus. Furthermore, when the genome sequence of the mutant Orff virus was compared with the genome sequences of all other disclosed ORFV virus strains, it was found that the mutant Orff virus completely deletes the ORFV007 dUTPase gene. Furthermore, the above comparison has also demonstrated that the Orff virus completely deletes the ORFV002 and ORFV005 genes.

[0016] Therefore, the mutated Orff virus disclosed here has a valuable leadership role to play in the use of such viruses in the creation of antitumor drugs. [Overview of the project]

[0017] The inventors have surprisingly found that complete or partial deletions of the gene ORFV112 (encoding the CBP protein) and / or the gene ORFV111 (encoding a virtual protein, hereinafter referred to as virtual protein 111) (in particular, deletion of the 5' portion of gene ORFV112 and deletion of the 3' portion of gene ORFV111) can enhance the antitumor activity of the Orff virus. Furthermore, the inventors have also surprisingly found that the mutant Orff virus completely deletes the gene ORFV007. Orff viruses lacking the functional CBP protein and / or virtual protein 111 can be obtained by conventional molecular biological methods (e.g., molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.) or emerging molecular biological methods (e.g., gene editing, etc.).

[0018] In one embodiment, the present invention provides a mutant Orff virus characterized by the deletion of the functional expression product of the gene ORFV112 and / or the gene ORFV111. In one embodiment, the deletion of the functional expression product of the gene ORFV112 is caused by the deletion of all or part of the gene ORFV112 (e.g., the 5' portion or the 3' portion, in particular the 5' portion). In one embodiment, the mature sequence of the expression product of the gene ORFV112 is as shown by SEQ ID NO:2. In one embodiment, the complete sequence of the gene ORFV112 is as shown by SEQ ID NO:3. In one embodiment, SEQ ID NO:3 is completely deleted. In one embodiment, SEQ ID NO:3 is partially deleted, for example, by deleting 1 to 1161, 1 to 1140, 1 to 538, 539 to 1139, or 1141 to 1160 5' end bases. In one embodiment, the partially deleted gene ORFV112 is as indicated by SEQ ID NO:4 or SEQ ID NO:57. In one embodiment, the gene ORFV The deletion of the functional expression product of ORFV111 is caused by the deletion of all or part of the gene ORFV111 (e.g., the 5' or 3' portion, especially the 3' portion). In one embodiment, the sequence of the expression product of the gene ORFV111 is as shown by SEQ ID NO: 58. In one embodiment, the complete sequence of the gene ORFV111 is as shown by SEQ ID NO: 59. In one embodiment, SEQ ID NO: 59 is completely deleted. In one embodiment, SEQ ID NO: 59 is partially deleted, for example, by deleting 1-793, 1-309, or 310-792 3' end bases. In one embodiment, the partially deleted gene ORFV111 is as shown by SEQ ID NO: 60. In one embodiment, SEQ ID NO: 3 and SEQ ID NO:59 is completely deleted. In one embodiment, the expression product is a protein and / or nucleic acid (in particular, a functional nucleic acid). In one embodiment, the present invention provides the genome of the virus.

[0019] In one embodiment, the present invention provides a method for manipulating Orff viruses, comprising reducing or removing the expression and / or activity of the expression products of the gene ORFV112 and / or the gene ORFV111. In one embodiment, the method comprises deleting the gene ORFV112 completely or partially (e.g., the 5' portion or the 3' portion, in particular the 5' portion). In one embodiment, the mature sequence of the expression product of the gene ORFV112 is as shown by SEQ ID NO:2. In one embodiment, the complete sequence of the gene ORFV112 is as shown by SEQ ID NO:3. In one embodiment, the method comprises completely deleting SEQ ID NO:3. In one embodiment, the method comprises partially deleting SEQ ID NO:3, for example, deleting 1 to 1161, 1 to 1140, 1 to 538, 539 to 1139, or 1141 to 1160 5' end bases. In one embodiment, the method generates a partially deleted gene ORFV112, indicated by SEQ ID NO:4 or SEQ ID NO:57. In one embodiment, the method includes deleting the gene ORFV111 completely or partially (e.g., the 5' portion or the 3' portion, particularly the 3' portion). In one embodiment, the sequence of the expression product of the gene ORFV111 is as indicated by SEQ ID NO:58. In one embodiment, the complete sequence of the gene ORFV111 is as indicated by SEQ ID NO:59. In one embodiment, the method includes completely deleting SEQ ID NO:59. In one embodiment, the method includes SEQ The method includes partially deleting ID NO:59, for example, deleting 1 to 793, 1 to 309, or 310 to 792 3' terminal bases. In one embodiment, the method generates the partially deleted gene ORFV111, indicated by SEQ ID NO:60. In one embodiment, the method includes completely deleting SEQ ID NO:3 and SEQ ID NO:59. In one embodiment, expression is transcription and / or translation. In one embodiment, the present invention provides a virus obtained by the method described above. In one embodiment, the present invention provides the genome of the virus described above. The methods of the present invention may be carried out by common molecular biological techniques (e.g., molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.) or emerging molecular biological techniques (e.g., gene editing, etc.).

[0020] In another embodiment, the present invention provides a method for identifying the mutant ORF virus and / or its genome, in particular a method for detecting the presence or sequence of the gene ORFV112 and / or the gene ORFV111 and / or the presence and / or activity of its expression product. The method of the present invention utilizes common molecular biological techniques for detecting the presence and / or activity of proteins and / or the presence and / or sequence of nucleic acids (e.g., activity assays, hybridization (e.g., Southern hybridization, Northern hybridization, or Western hybridization), restriction endonucleases, PCR, electrophoresis (e.g., gel electrophoresis, protein electrophoresis, and nucleic acid electrophoresis). The method may be carried out by pneumatophoresis (including agarose electrophoresis and PAGE electrophoresis, including reductive and non-reductive electrophoresis), sequencing (including protein sequencing and nucleic acid sequencing), or emerging molecular biological techniques (e.g., next-generation sequencing). The present invention provides a method for detecting the integrity of the gene ORFV112 and / or the gene ORFV111. In one embodiment, the method is carried out by PCR and gel electrophoresis. In one embodiment, the method may be carried out by hybridization or sequencing.

[0021] In another embodiment, the present invention provides a method for treating cancer in a subject using the mutant Orff virus and / or its genome. In another embodiment, this publication provides the use of the mutant Orff virus and / or its genome for the treatment of cancer in a subject. In another embodiment, this publication provides the use of the mutant Orff virus and / or its genome for the manufacture of a drug for the treatment of cancer in a subject. In another embodiment, this publication provides the mutant Orff virus and / or its genome for the treatment of cancer in a subject. In another embodiment, this publication provides the use of the mutant Orff virus and / or its genome for the manufacture of a drug for the treatment of cancer in a subject. In one embodiment, the cancer is a solid tumor. In one embodiment, the solid tumor is cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, brain tumor, etc. In one embodiment, the subject is a mammal, and is a rodent (e.g., mouse and rat), a non-human primate (e.g., cynomolgus macaque), or a human. [Brief explanation of the drawing]

[0022] [Figure 1]Figure 1 shows the agarose gel electrophoresis results of PCR products after PCR amplification using ORFV112 gene-specific forward and reverse primers (SEQ ID NO: 5 and 6). In the figure, sample number 1 is the virus strain POV-601-1A1, which lacks the ORFV112 gene, and sample number 2 is the virus strain POV-601-3F8, which has the complete ORFV112 gene, with M being the DNA size marker. [Figure 2] Figure 2 shows the tumor-suppressing effects of the ORFV112 gene deletion virus strains POV-601-1A1 and POV-604-1D1 in a bladder cancer model derived from MB49 mice. [Figure 3] Figure 3 shows the tumor-suppressing effects of the virus strains POV-601-1A1 and POV-604-1D1, which lack the ORFV112 gene, and the virus strain POV-601-3F8, which has a complete ORFV112 gene, in a B16-F10 mouse-derived melanoma tumor model. [Figure 4] Figure 4 shows the effects of POV-601-1A1 virus on mouse body weight under different dose and administration route conditions. [Figure 5] Figure 5 shows the changes in the mouse immune system in a human C-33A cervical cancer bilateral tumor dissemination model after administration of the POV-601-1A1 virus strain. In Figures 5A and 5B, the squares on the right show the results of intratumoral administration, and the circles on the left show the results without administration. Intratumoral administration improved the activation rate of CD45+ and NK cells within the tumor. Figure 5C shows the activation status of NK cells in the blood, and intravenous administration improved the activation rate of NK cells. [Figure 6] Figure 6 shows a comparison of the complete sequence and partially deleted sequence of the ORFV112 nucleotide gene of the present invention in examples. [Figure 7]Figure 7 shows a comparison of the complete sequence of the CBP protein of the present invention with the complete sequence of the CBP protein of other parapoxvirus strains. In the diagram, "3F8" represents POV-601-3F8 Strain, "B029" represents ORFV Strain B029, "GO" represents ORFV Strain GO, "NA11" represents ORFV Strain NA1 / 11, "NZ2" represents ORFV Strain NZ2, "NA17" represents ORFV Strain NA17, "OV-SA00" represents ORFV Strain OV-SA00, "OV-IA82" represents ORFV Strain OV-IA82, "SJ1" represents ORFV Strain SJ1, "SY17" represents ORFV Strain SY17, "OV-NH3_12" represents ORFV Strain OV-HN3 / 12, "NP" represents ORFV Strain NP, and "YX" represents ORFV Strain YX. [Figure 8-1] Figure 8 shows a comparison of the complete sequence of the ORFV112 gene of the present invention with the complete sequence of the ORFV112 gene of other parapoxvirus strains. In the diagram, "3F8" represents POV-601-3F8 Strain, "B029" represents ORFV Strain B029, "GO" represents ORFV Strain GO, "NA11" represents ORFV Strain NA1 / 11, "NZ2" represents ORFV Strain NZ2, "NA17" represents ORFV Strain NA17, "OV-SA00" represents ORFV Strain OV-SA00, "OV-IA82" represents ORFV Strain OV-IA82, "SJ1" represents ORFV Strain SJ1, "SY17" represents ORFV Strain SY17, "OV-NH3_12" represents ORFV Strain OV-HN3 / 12, "NP" represents ORFV Strain NP, and "YX" represents ORFV Strain YX. [Figure 8-2] Figure 8 (continued) [Figure 9] Figure 9 shows a comparison of the complete sequence and partially deleted sequence of the ORFV111 nucleotide gene of the present invention in examples. [Figure 10]Figure 10 shows a comparison of the complete sequence of the virtual protein 111 of the present invention with the complete sequence of the virtual protein 111 of other parapoxvirus strains. In the diagram, "POV-601-3F8" represents POV-601-3F8 Strain, "B029" represents ORFV Strain B029, "GO" represents ORFV Strain GO, "NA11" represents ORFV Strain NA1 / 11, "NZ2" represents ORFV Strain NZ2, "NA17" represents ORFV Strain NA17, "OV-SA00" represents ORFV Strain OV-SA00, "OV-IA82" represents ORFV Strain OV-IA82, "SJ1" represents ORFV Strain SJ1, "SY17" represents ORFV Strain SY17, "OV-NH3_12" represents ORFV Strain OV-HN3 / 12, "NP" represents ORFV Strain NP, and "YX" represents ORFV Represents Strain YX [Figure 11-1] Figure 11 shows a comparison of the complete sequence of the ORFV111 gene of the present invention with the complete sequence of the ORFV111 gene of another parapoxvirus strain. In the diagram, "POV-601-3F8" represents POV-601-3F8 Strain, "B029" represents ORFV Strain B029, "GO" represents ORFV Strain GO, "NA11" represents ORFV Strain NA1 / 11, "NZ2" represents ORFV Strain NZ2, "NA17" represents ORFV Strain NA17, "OV-SA00" represents ORFV Strain OV-SA00, "OV-IA82" represents ORFV Strain OV-IA82, "SJ1" represents ORFV Strain SJ1, "SY17" represents ORFV Strain SY17, "OV-NH3_12" represents ORFV Strain OV-HN3 / 12, "NP" represents ORFV Strain NP, and "YX" represents ORFV Represents Strain YX [Figure 11-2] Figure 11 (continued) [Figure 12] Figure 12 shows a comparison of the complete sequence and partially deleted sequence of the ORFV111 amino acid gene of the present invention in examples. [Figure 13] Figure 13 shows a comparison of the complete sequence and partially deleted sequence of the ORFV112 amino acid gene of the present invention in examples. [Figure 14] Figure 14 shows the tumor-suppressing effects of the virus strain POV-601-1A1, which lacks the ORFV112 gene, and the virus strain POV-601-3F8, which has a complete ORFV112 gene, in a CT-26 mouse-derived colon cancer tumor model. [Figure 15] Figure 15 shows the tumor-suppressing effects of POV-601-1A1 and construct v611a in a B16-F10 mouse-derived melanoma tumor model. [Figure 16] Figure 16 shows the tumor-suppressing effects of POV-601-1A1 and constructs v615a and v616a in a B16-F10 mouse-derived melanoma tumor model. [Figure 17] Figure 17 shows the tumor-suppressing effects of POV-601-1A1 and constructs v617a and v618a in a B16-F10 mouse-derived melanoma tumor model. [Modes for carrying out the invention]

[0023] This invention provides for the use of mutant ORFV (ORFV) and its pharmaceutical composition in the treatment of cancer.

[0024] According to literature reports, the virulence genes of wild-type Orf virus are mainly OVIFNR (Orf virus interferon resistance gene), CBP (chemokine-binding protein), GIF (GM-CSF / IL-2 inhibitory protein), vIL-10 (viral interleukin 10), and VEGF-like protein (vascular endothelial growth factor-like protein). [11,13]Typically, attenuated strains of the virus can be obtained by deleting a certain toxic factor gene using molecular and / or cell biological techniques. For example, Chinese patent CN104878043B discloses a method for obtaining an attenuated strain of Orff virus strain SHZ1 by deleting the toxic gene OVIFNR, thereby achieving a rapid reduction in its toxicity, and its use in the manufacture of attenuated vaccines.

[0025] The mutant Orff virus disclosed in this invention is obtained by modifying and screening the original POV-601 virus strain, which was commissioned (accession number: V201713) by the China Center for Typical Cultures (CCTCC).

[0026] The inventor,

[17] Based on this, specific primers for the ORFV112 gene were designed. A forward primer (SEQ ID NO: 5) was designed for the gene coding region of ORFV111, and a reverse primer (SEQ ID NO: 6) was designed for the gene coding region of ORFV112. This allowed for the construction of a PCR-based molecular biological identification technique to identify the integrity of the ORFV112 gene in the attenuated POV-601 strain. Subsequently, African green monkey kidney cells CV-1 were infected with the original POV-601 virus strain, and virus-infected cells were collected. Single-cell screening and culture amplification were performed using flow cytometry. Using the above ORFV112 gene-specific primer pair, viral genomic DNA was extracted from the amplified, virus-infected cells. An attenuated ORFV virus strain with a partial deletion of the ORFV112 gene was obtained using the constructed molecular biological identification technique and named POV-601-1A1. This virus strain has a deletion of 312 bases at the 5' end of the ORFV112 gene coding region, leaving 552 bases, and 72 bases remaining in the non-coding region at the 3' end, for a total of 624 bases remaining, as shown in SEQ ID NO:4. In addition, a virus strain POV-601-3F8 with a complete ORFV112 gene has also been obtained, and its coding and non-coding regions have a total of 1162 bases, and the complete sequence of its ORFV112 gene is shown in SEQ ID NO:3.

[0027] The aforementioned POV-601-1A1 virus attenuated strain was evaluated using an animal oncological pharmacodynamic model. Surprisingly, we found that the aforementioned virus strain exhibited superior antitumor effects compared to the virus strain POV-601-3F8, which has a complete ORFV112 gene.

[0028] In another embodiment, the inventors further artificially used gene editing means on the POV-601-1A1 virus strain to knock out the remaining coding region of the ORFV112 gene. In this example, the remaining coding region of the ORFV112 gene was completely knocked out (leaving only 22 bases in the non-coding region at the 3' end, see sequence SEQ ID NO: 57), as confirmed by DNA sequencing. The resulting virus strain was named POV-604-1D1. Surprisingly, during animal oncological pharmacodynamic model evaluation, it was found that this attenuated strain also exhibited superior antitumor effects compared to the virus strain POV-601-3F8, in which the ORFV112 gene was complete. Furthermore, similar effects can be obtained by any method that does not express the CBP protein (for example, by knocking out or modifying some sequences in the coding and non-coding regions of the ORFV112 gene).

[0029] The POV-601-1A1 virus strain was deposited with the China Center for Typical Cell Cultures (CCTCC) (Wuhan) on May 19, 2020, in accordance with the provisions of the Budapest Convention, and its CCTCC accession number was V202029.

[0030] In the examples, the mutant Orff virus disclosed herein can selectively infect and replicate within melanoma cells (B16-F10), bladder cancer cells (MB49), liver cancer cells (Hepa1-6), colon cancer cells (CT26), human cervical cancer cells (C-33A), human ovarian cancer cells (SK-OV-3), and other cells.

[0031] In the examples, the mutant Orff virus published here was surprisingly found to have a complete deletion of the ORFV007 gene, but all Orff virus strains published by NCBI (e.g., NZ-2, NZ-7, D1701, NA1 / 11 strain, etc.) also have a complete deletion of the ORFV007 gene. The ORFV007 gene encodes deoxyuridine triphosphatase (duTPase). This protein is an important enzyme in dNTP synthesis. Normally, dNTP concentrations in normal cells are tightly regulated, which is detrimental to viral replication. However, cancer cells have relatively high concentrations of dNTPs, which can promote viral replication.

[16] Deletion of the ORFV007 gene disrupts the replication of the virus in normal cells, but does not affect its amplification in tumor cells, thus achieving the objective of selective replication of the ORFV virus in cancer cells.

[0032] Wild-type ORFV virus can usually be cultured in primary cells of bovine and sheep animal tissues (CN103952377A), and the International Publication WO2012122649A1 first disclosed that the virus can be grown and cultured in human cervical cancer cells (Hela). Furthermore, the present invention provides a method for producing the mutant ORFV virus using a mammalian cell line as a host cell, preferably African green monkey kidney cells (CV-1), for viral infection and amplification.

[0033] Furthermore, the invention provides the use of the aforementioned virus for the treatment of individual cancers. The cancer is any solid tumor cancer. The types of solid tumor cancers include cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, brain tumor, etc. Furthermore, the individual is a mammal, including rodents and humans.

[0034] The mechanisms by which oncolytic viruses inhibit tumor growth are typically: 1) replicating and amplifying within infected tumor cells, lysing cancer cells, and achieving the purpose of oncolysis; and 2) tumor lysis. Afterward, the cancer cells release specific information stored within them (e.g., tumor neoantigens), activating the immune system and launching a systemic attack against any remaining cancer cells.

[0035] In the examples, the disclosed mutant Orff virus can effectively activate the autoimmune system, including NK cells. In a human C-33A cervical cancer cell model with bilateral tumor dissemination, intratumoral and / or intravenous injection of the virus activated CD45 cells in the tumor and / or blood. + Furthermore, it can increase the activation rate of NK cells.

[0036] I. General-purpose technologies Unless otherwise stated, the implementation of this invention employs general techniques of molecular biology (including recombinant techniques), virology, microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art.

[0037] II. Definition ● The term "OV" is an abbreviation for Oncolytic virus, which means a virus that causes tumor lysis.

[0038] ● The term "solid tumor" is used to distinguish it from hematological tumors and refers to a tumor entity composed of multiple cells, and may include cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, brain tumor, etc. Cancer can be in its early or late stages.

[0039] ●Regarding the term "continuous cell line," a cell line is a population of cells that possess special genetic characteristics, biochemical characteristics, or specific markers obtained from a primary culture or cell line, and a continuous cell line is a cell line that is continuously passed through generations.

[0040] ● The term "CPE" refers to the cytopathic effect, which is the cellular degeneration that occurs after infecting cultured cells with a virus. In in vitro tests, when a cytotoxic virus is cultured and seeded into cells, after a certain period of time, phenomena such as the cells becoming round, necrotic, and detaching from the flask wall are observed under a microscope, and this is called the cytopathic effect.

[0041] ● The term "Orf virus (Sheep Infectious Pustular Dermatitis Virus)" is also known as sheep mouth ulcer virus, mouth ulcer virus, ORFV, orf virus, etc., and is a pox family virus that causes contact-transmitted, epitheliotropic disease in sheep and goats.

[0042] ● The term "effective dose" refers to the amount that achieves the desired therapeutic or preventive effect in the required dosage and time. The therapeutic effective dose further refers to the amount of therapeutic agent in which the therapeutic benefits outweigh any toxicity or adverse consequences.

[0043] ● The term "PBS" is an abbreviation for phosphate buffer saline.

[0044] ● The term "MOI" stands for Multiplicity of Infection, and refers to the ratio (pfu / cell) of the number of viral particles to the total number of target cells during the process of a virus infecting cells.

[0045] ● In the term "POV-601-1A1 virus strain," "POV" is an abbreviation for "Prajna Oncolytic Virus," and it means that the virus strain was obtained from Suzhou Prajna Biotechnology Co., Ltd., and refers to the internal test records of Suzhou Prajna Biotechnology Co., Ltd. Therefore, POV-601-1A1, v601-1A1, v601-p0-1A1, and 1A1 all represent the same virus strain. The name of this virus strain in the CCTCC registry is "Orff virus POV-601-1A1," and it belongs to the Orff virus genus of the Poxviridae family.

[0046] ●In the term "POV-601-3F8 virus strain," "POV" stands for "Prajna Oncolytic Virus," and it indicates that the virus strain was obtained from Suzhou Prajna Biotechnology Co., Ltd. In the internal test records of Suzhou Prajna Biotechnology Co., Ltd., POV-601-3F8, v601-3F8, v601-p0-3F8, and 3F8 all refer to the same virus strain.

[0047] ●In the term "POV-604-1D1 virus strain," "POV" stands for "Prajna Oncolytic Virus," indicating that the virus strain was obtained from Suzhou Prajna Biotechnology Co., Ltd., and in Suzhou Prajna Biotechnology Co., Ltd.'s internal test records, POV-604-1D1, v604-1D1, and 1D1 all refer to the same virus strain.

[0048] ●Regarding the term "virus storage buffer," its preparation method is as follows: 500 mL of PBS (i.e., phosphate buffer, CORNING, catalog number: 21-040-CVR) is drawn up, 1.25 mL of 1 M mgCl2·6H2O (Shanghai Biotechnology Co., Ltd., catalog number: A610328-0500) is added to bring the final concentration of MgCl2·6H2O to 2.48 mM, then 2.5 mL of 1 M Tris-HCl (pH 9.0) (Shanghai Biotechnology Co., Ltd., catalog number: B548128-0500) is added to bring the final concentration of Tris-HCl to 4.96 mM, and the mixture is homogeneously mixed to obtain the virus storage buffer (the theoretical final concentration of MgCl2·6H2O is 2.5 mM, and the theoretical final concentration of Tris-HCl is 5 mM).

[0049] III. Compositions and Methods 1. Expression product of the ORFV112 gene, CBP protein The present invention relates to (natural) CBP protein. In one embodiment, the (natural) CBP protein contains (or has) a sequence derived from the one indicated by SEQ ID NO:1 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto, or contains (or has) a mature sequence derived from the one indicated by SEQ ID NO:2 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto, or is (substantially) composed of such. In another embodiment, the (natural) CBP protein comprises (or has) a complete sequence and a corresponding mature sequence derived from a biological source (e.g., strain, species, genus, family) indicated by SEQ ID NO:31-42 or the same or similar thereto, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0050] The present invention further relates to mutant CBP proteins. In one embodiment, the mutation is the addition, deletion, or replacement of one or more amino acid residues from the native sequence, or any combination thereof. In one embodiment, the function of the mutant CBP protein is reduced or deleted.

[0051] The present invention further relates to the reduction or deletion of CBP protein expression (e.g., native CBP protein or mutant CBP protein).

[0052] For example, a decrease means a decrease of at least 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the natural function and / or expression, and continuing until it reaches 100%.

[0053] 2. ORFV112 gene The present invention relates to the (natural) ORFV112 gene. In one embodiment, the (natural) ORFV112 gene comprises (or has) a complete sequence derived from the one indicated by SEQ ID NO:1 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto, or comprises (or has) a mature sequence derived from the one indicated by SEQ ID NO:2 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto, or encodes a CBP protein substantially composed of such a mature sequence. In another embodiment, the (natural) CBP protein is SEQ ID A complete sequence and corresponding mature sequence derived from a biological source (e.g., strain, species, genus, family) indicated in NO:31-42 or the same or similar thereto, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0054] The present invention relates to the (natural) ORFV112 gene, and in one embodiment, the (natural) ORFV112 gene is represented by SEQ ID NO:3 (or its coding region) and comprises (or has) or (substantially) a nucleotide sequence that codes for the same amino acid sequence as or is derived from the same or similar biological source (e.g., strain, species, genus, family) and has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to it. The full length of SEQ ID NO:3 is 1162 bases, of which the bases from positions 1 to 226 constitute the 5' non-coding region, the bases from positions 227 to 1090 constitute the protein coding region, and the bases from positions 1091 to 1162 constitute the 3' non-coding region. In another embodiment, the (natural) ORFV112 gene is indicated by SEQ ID NO: 43-54 (or its coding region) and comprises (or has) a nucleotide sequence that codes for the same amino acid sequence as it or is derived from the same or similar biological source (e.g., strain, species, genus, family) and has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with it.

[0055] The present invention further relates to mutant ORFV112 genes. In one embodiment, the mutant is the addition, deletion, or substitution of one or more bases to the native sequence, or any combination thereof. In one embodiment, the mutant ORFV112 gene is a complete deletion ORFV112 gene. A complete deletion ORFV112 gene is a complete deletion of the ORFV112 gene. In one embodiment, the mutant ORFV112 gene is a partial deletion ORFV112 gene. A partial deletion ORFV112 gene is a deletion of one or more bases (but not all bases) of the ORFV112 gene. In one embodiment, a partial deletion ORFV112 gene is a deletion of one or more bases, or any combination thereof, in the 5' untranslated region, coding region, and / or 3' untranslated region. In one embodiment, the partially deleted ORFV112 gene is a 5'-end deleted ORFV112 gene, that is, it has one or more bases deleted at the 5' end, for example, deleting all or part of the 5' untranslated region, or deleting all of the 5' untranslated region and all or part of the coding region, or deleting all of the 5' untranslated region, all of the coding region and all or part of the 3' untranslated region. In this state, the deletion of the ORFV112 gene extends upstream (into the inter-ORFV111 / 112 gene region, and / or to the ORFV111 gene, especially the 3' end, if present) and / or downstream (into the inter-ORFV112 / 113 gene region, and / or to the ORFV113 gene, especially the 5' end, if present).

[0056] Where the native ORFV112 gene contains (or has) or (substantially) the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO:3, in one embodiment, the partially deleted ORFV112 gene deletes one or more bases, for example, 1 to 1161 bases, from the sequence indicated by SEQ ID NO:3. Where the native ORFV112 gene contains (or has) or (substantially) the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO:3, in one embodiment, the 5'-end deleted ORFV112 gene deletes one or more bases at the 5' end of the sequence indicated by SEQ ID NO:3, in particular, 1 to 1161, 1 to 1140, 1 to 538, 539 to 1139, or 1141 to 1160 bases at the 5' end. In one embodiment, the 5'-end deletion type ORFV112 gene contains (or has) or (substantially) the nucleotide sequence (or its coding region) indicated by SEQ ID NO:4 or SEQ ID NO:57. The native ORFV112 gene is SEQ In one embodiment, where a nucleotide sequence (or coding region thereof) represented by ID NO:43-54 is included (or has) or (substantially) composed thereof, a 5'-end deletion type ORFV112 gene deletes one or more bases at the 5' end of the sequence represented by SEQ ID NO:43-54, in particular one or more bases in the segment corresponding to the 1140 or 538 bases at the 5' end of SEQ ID NO:3. Because the 5' untranslated region and / or start codon are deleted, these deletion types of genes cannot express the protein.

[0057] In one embodiment, the mutant ORFV112 gene of the present invention causes a decrease or deletion of functional CBP protein (including a decrease or deletion of CBP protein expression and / or activity). For example, decrease means a decrease of at least 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the native function and / or expression, up to a decrease of 100%.

[0058] 3. Expression product of the ORFV111 gene, virtual protein 111 The present invention relates to a (natural) virtual protein 111. In one embodiment, the (natural) virtual protein 111 contains (or has) or (substantially) a sequence derived from the one indicated by SEQ ID NO: 58 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto. In another embodiment, the (natural) virtual protein 111 contains (or has) a sequence derived from the one indicated by SEQ ID NO: 61-72 or the same or similar biological source (e.g., strain, species, genus, family) and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0059] The present invention further relates to a mutant virtual protein 111. In one embodiment, the mutation is the addition, deletion, or replacement of one or more amino acid residues from the native sequence, or any combination thereof. In one embodiment, the function of the mutant virtual protein 111 is reduced or deleted.

[0060] The present invention further relates to the reduction or deletion of the expression of virtual protein 111 (for example, native virtual protein 111 or mutant virtual protein 111).

[0061] For example, a decrease means a decrease of at least 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the natural function and / or expression, up to a decrease of 100%.

[0062] 4. ORFV111 gene The present invention relates to the (natural) ORFV111 gene. In one embodiment, the (natural) ORFV111 gene encodes a virtual protein 111 that contains (or has) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the sequence indicated by SEQ ID NO: 58 or the same or similar biological source (e.g., strain, species, genus, family). In another embodiment, the (natural) virtual protein 111 contains (or has) a sequence that is derived from or is the same or similar biological source (e.g., strain, species, genus, family) indicated by SEQ ID NO: 61-72 and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0063] The present invention relates to the (natural) ORFV111 gene, and in one embodiment, the (natural) ORFV111 gene is represented by SEQ ID NO:59 (or its coding region) and comprises (or has) or (substantially) a nucleotide sequence that codes for the same amino acid sequence as thereto, or that originates from the same or similar biological source (e.g., strain, species, genus, family) and has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto. The full length of SEQ ID NO:59 is 794 bases, of which the bases at positions 1-28 constitute the 5' non-coding region, the bases at positions 29-568 constitute the protein coding region, and the bases at positions 569-794 constitute the 3' non-coding region. In another embodiment, the (natural) ORFV111 gene is indicated by SEQ ID NO: 73-84 (or its coding region) and comprises (or has) a nucleotide sequence that codes for the same amino acid sequence as therefor, or that originates from the same or similar biological source (e.g., strain, species, genus, family) and has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to therefor.

[0064] The present invention further relates to mutant ORFV111 genes. In one embodiment, the mutant is the addition, deletion, or substitution of one or more bases to the native sequence, or any combination thereof. In one embodiment, the mutant ORFV111 gene is a complete deletion ORFV111 gene. A complete deletion ORFV111 gene is a complete deletion of the ORFV111 gene. In one embodiment, the mutant ORFV111 gene is a partial deletion ORFV111 gene. A partial deletion ORFV111 gene is a deletion of one or more bases (but not all bases) of the ORFV111 gene. In one embodiment, a partial deletion ORFV111 gene is a deletion of one or more bases, or any combination thereof, in the 3' untranslated region, coding region, and / or 5' untranslated region. In one embodiment, a partially deleted ORFV111 gene is a 3'-end deleted ORFV111 gene, that is, it has one or more bases deleted at the 3' end, for example, deleting all or part of the 3' untranslated region, or deleting all of the 3' untranslated region and all or part of the coding region, or deleting all of the 3' untranslated region, all of the coding region and all or part of the 5' untranslated region. In one embodiment, the ORFV111 gene deletion is downstream (between ORFV111 / 112 genes). It enters the region and, if present, extends to the ORFV112 gene, particularly its 5' end, and / or upstream (the inter-ORFV110 / 111 gene region, and, if present, to the ORFV110 gene, particularly its 3' end).

[0065] In one embodiment, where the native ORFV111 gene contains (or has) or (substantially) the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO: 59, the partially deleted ORFV111 gene deletes one or more bases, for example, 1 to 793 bases, from the sequence indicated by SEQ ID NO: 59. In one embodiment, where the native ORFV111 gene contains (or has) or (substantially) the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO: 59, the 3'-end deleted ORFV111 gene deletes one or more bases at the 3' end of the sequence indicated by SEQ ID NO: 59, particularly 1 to 793, 1 to 309, or 310 to 792 bases at the 3' end. In one embodiment, the 3'-end deletion type ORFV111 gene contains (or has) or (substantially) consists of the nucleotide sequence (or its coding region) indicated by SEQ ID NO: 60. In the case where the native ORFV111 gene contains (or has) or (substantially) consists of the nucleotide sequence (or its coding region) indicated by SEQ ID NO: 73-84, in one embodiment, the 3'-end deletion type ORFV111 gene contains one or more bases at the 3' end of the sequence indicated by SEQ ID NO: 73-84, in particular, SEQ ID NO:59 has one or more deletions in the segment corresponding to the 309 bases at the 3' end. Because the 3' untranslated region is deleted, these deletion-type genes may not be able to express the protein.

[0066] In one embodiment, the mutant ORFV111 gene of the present invention causes a reduction or deletion of functional virtual protein 111 (including a reduction or deletion of the expression and / or activity of virtual protein 111). For example, reduction means a reduction of at least 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the native function and / or expression, up to a reduction of 100%.

[0067] 5. ORFV virus genome The present invention relates to the ORFV virus genome. In one embodiment, the ORFV virus genome has the ORFV112 gene and / or ORFV111 gene (including the native ORFV112 gene and / or ORFV111 gene and the mutant ORFV112 gene and / or ORFV111 gene). The present invention particularly relates to the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome. In one embodiment, the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome includes the mutant ORFV112 gene and / or ORFV111 gene described above. In one embodiment, the ORFV virus genome (e.g., the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome) completely deletes the ORFV007 gene. In one embodiment, the 3' non-coding region of the ORFV111 gene overlaps with the 5' non-coding region of the ORFV112 gene.

[0068] 6. ORFV virus The present invention relates to the ORFV virus. In one embodiment, the ORFV virus has the CBP protein and / or virtual protein 111 described above (including native CBP protein and / or virtual protein 111 and mutant CBP protein and / or virtual protein 111). The present invention relates in particular to the CBP protein and / or virtual protein 111 deletion type ORFV virus. In one embodiment, the CBP protein ORFV viruses with and / or virtual protein 111 deletion cause a decrease or deletion of functional CBP protein and / or virtual protein 111. The decrease or deletion of functional CBP protein and / or virtual protein 111 may also be a decrease or deletion of the expression and / or activity of CBP protein and / or virtual protein 111.

[0069] The present invention relates to the ORFV virus. In one embodiment, the ORFV virus has the ORFV112 gene and / or ORFV111 gene (including the native ORFV112 gene and / or ORFV111 gene and the mutant ORFV112 gene and / or ORFV111 gene). In one embodiment, the ORFV virus has the ORFV virus genome (including the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome). The present invention particularly relates to the CBP protein and / or virtual protein 111 deletion type ORFV virus, which includes the mutant ORFV112 gene and / or ORFV111 gene or the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome.

[0070] In one embodiment, the functional transcription and / or translation product of the ORFV007 deletion gene of an ORFV virus (e.g., CBP protein and / or virtual protein 111 deletion ORFV virus) comprises a deletion of expression and / or activity.

[0071] 7. How to deal with the ORFV virus The present invention relates to a method for manipulating the ORFV virus genome or the ORFV virus. In one embodiment, the method comprises mutantizing the ORFV112 gene and / or the ORFV111 gene in the ORFV virus genome. In one embodiment, the method comprises addition, deletion or substitution, and in particular, deletion of one or more bases of the ORFV112 gene and / or the ORFV111 gene, in particular, one or more bases at the 5' end of the ORFV112 gene and / or one or more bases at the 3' end of the ORFV111 gene. Where the native ORFV112 gene contains (or has) or (substantially) consists of the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO:3, in one embodiment, the method comprises deletion of one or more bases of the sequence indicated by SEQ ID NO:3, for example, 1 to 1162 bases. Where the native ORFV112 gene contains (or has) or (substantially) consists of the nucleotide sequence (or coding region) indicated by SEQ ID NO:3, in one embodiment, the method includes deleting one or more bases at the 5' end of the sequence indicated by SEQ ID NO:3, in particular 1 to 1161, 1 to 1140, 1 to 538, 539 to 1139, or 1141 to 1160 bases at the 5' end, for example, 538 or 1140 bases. Where the native ORFV112 gene contains (or has) or (substantially) consists of the nucleotide sequence (or coding region) indicated by SEQ ID NO:3, in one embodiment, the method includes completely deleting SEQ ID NO:3, or deleting 1140 or 538 5' end bases of SEQ ID NO:3. Where the native ORFV112 gene contains (or has) or is (substantially) composed of other nucleotide sequences (or coding regions thereof), in one embodiment, the method includes completely deleting the nucleotide sequence or deleting the segments of the nucleotide sequence corresponding to 1140 or 538 5' end bases of SEQ ID NO:3.Where the native ORFV111 gene contains (or has) or (substantially) consists of the nucleotide sequence (or coding region thereof) indicated by SEQ ID NO: 59, in one embodiment, the method comprises deleting one or more bases, e.g., 1 to 794 bases, from the sequence indicated by SEQ ID NO: 59. The native ORFV111 gene is the nucleo indicated by SEQ ID NO: 59. Where the sequence includes (or has) or is substantially composed of a nucleotide sequence (or its coding region), in one embodiment, the method includes deleting one or more bases at the 3' end of the sequence indicated by SEQ ID NO: 59, particularly 1 to 793, 1 to 309, or 310 to 792 bases at the 3' end, for example, 309 bases. Where the native ORFV111 gene includes (or has) or is substantially composed of a nucleotide sequence (or its coding region) indicated by SEQ ID NO: 59, in one embodiment, the method includes completely deleting SEQ ID NO: 59 or deleting 309 3' end bases of SEQ ID NO: 59. Where the native ORFV111 gene contains (or has) or is (substantially) composed of other nucleotide sequences (or their coding regions), in one embodiment, the method includes completely deleting the nucleotide sequence or deleting the segment of the nucleotide sequence corresponding to the 309 3' end bases of SEQ ID NO:59. Where the native ORFV111 gene and the native ORFV112 gene contain (or has) or is (substantially) composed of other nucleotide sequences (or their coding regions), in one embodiment, the method includes SEQ ID NO:3 and SEQ ID The method includes completely deleting NO:59. In one embodiment, the method further includes mutating (e.g., deleting (including complete or partial deletion)) the ORFV007 gene in the ORFV virus genome (particularly the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome). In one embodiment, the 3' non-coding region of the ORFV111 gene overlaps with the 5' non-coding region of the ORFV112 gene. The method of the present invention can be carried out by common molecular biological techniques (e.g., molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.) or emerging molecular biological techniques (e.g., gene editing, etc.). The present invention relates to the ORFV virus genome (e.g., the ORFV112 gene and / or ORFV111 gene deletion type ORFV virus genome) and the ORFV virus (e.g., the CBP protein and / or virtual protein 111 deletion type ORFV virus) obtained by the method described above.

[0072] 8. Method for identifying the ORFV virus genome and ORFV virus The present invention relates to a method for identifying the ORFV virus genome or ORFV virus, and more particularly to a method for detecting the presence or activity of the CBP protein and / or virtual protein 111 (particularly mutant CBP protein and / or virtual protein 111) and / or the gene ORFV112 and / or ORFV111 gene (particularly mutant ORFV112 gene and / or ORFV111 gene) (particularly the presence or sequence). The methods of the present invention are carried out by common molecular biological techniques (e.g., activity assays, hybridization (e.g., Southern hybridization, Northern hybridization, or Western hybridization), restriction nucleases, PCR, electrophoresis (e.g., gel electrophoresis (including protein electrophoresis and nucleic acid electrophoresis, including agarose electrophoresis and PAGE electrophoresis, including reductive and non-reductive electrophoresis), sequencing (including protein sequencing and nucleic acid sequencing), etc.) or emerging molecular biological techniques (e.g., next-generation sequencing, etc.). The present invention provides a method for detecting the integrity (or length) of the gene ORFV112 and / or ORFV111. In one embodiment, the method is carried out by PCR and gel electrophoresis. In one embodiment, the method may be carried out by nucleic acid hybridization or sequencing.

[0073] The design of primers and probes is within the capabilities of those skilled in the art. Those skilled in the art will clearly understand how to select target regions in the target sequences of primers and probes. For example, to detect the integrity of the gene ORFV112, in one embodiment, The target region of the downstream primer may be located at the 5' end of the ORFV112 gene, or extended upstream, but is not limited to these locations. For example, it may enter the intergeneric region of ORFV111 / 112, if present, or enter the ORFV111 gene (particularly the 3' end of ORFV111, e.g., the 3' untranslated region). In another embodiment, the target region of the downstream primer may be located at the 3' end of the ORFV112 gene, or extended downstream, for example, entering the intergeneric region of ORFV112 / 113, if present, or enter the ORFV113 gene (particularly the 5' end of ORFV113, e.g., the 5' untranslated region). Similarly, primer / probe targets can be designed based on similar principles.

[0074] 9. Uses of ORFV virus genome and ORFV virus The present invention provides a drug composition comprising a certain amount, particularly an effective amount, for example, a therapeutically effective amount or a prophylactically effective amount, of the ORFV virus genome (e.g., ORFV112 gene and / or ORFV111 gene deletion type ORFV virus) and / or ORFV virus (e.g., CBP protein and / or virtual protein 111 deletion type ORFV virus) of the present invention. In one embodiment, the composition comprises a pharmaceutically acceptable vector.

[0075] In one embodiment, the composition is in the form of a powder, liquid, transdermal patch, ointment, or suppository. In one embodiment, the composition is administered via an intravenous, intratumoral, intramuscular, subcutaneous, intrarectal, vaginal, or intraperitoneal route.

[0076] The present invention relates to a method for treating a disease or delaying the progression of a disease in a subject, the method comprising administering a certain amount, in particular an effective amount, for example, a therapeutic effective amount or a prophylactic effective amount, of the ORFV virus genome (e.g., ORFV112 gene and / or ORFV111 gene deletion type ORFV virus) and / or ORFV virus (e.g., CBP protein and / or virtual protein 111 deletion type ORFV virus) of the present invention.

[0077] The present invention relates to the use of a certain amount, particularly an effective amount, for example, a therapeutic or prophylactic effective amount, of the ORFV virus genome (e.g., ORFV112 gene and / or ORFV111 gene deletion type ORFV virus) and / or ORFV virus (e.g., CBP protein and / or virtual protein 111 deletion type ORFV virus) of the present invention for the manufacture of a drug. In one embodiment, the drug is used to treat a disease or delay the development of a disease in a subject.

[0078] The present invention relates to a certain amount, particularly an effective amount, such as a therapeutic effective amount or a preventive effective amount, of the ORFV virus genome (e.g., ORFV112 gene and / or ORFV111 gene deletion type ORFV virus) and / or ORFV virus (e.g., CBP protein and / or virtual protein 111 deletion type ORFV virus) used to treat a disease or delay the progression of a disease in a subject.

[0079] In one embodiment, the disease is cancer. In one embodiment, the cancer is a solid tumor. In one embodiment, the solid tumor is cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, brain tumor, etc.

[0080] In one embodiment, the subject is a mammal and includes rodents (e.g., mice and rats), non-human primates (e.g., cynomolgus macaques), and humans.

[0081] Examples Example 1: Method for screening and purifying mutant Orff virus ● Virus strain origin: POV-601 virus strain (China Center for Typical Cultures (CCTCC), accession number: V201713) Host cell origin: African green monkey kidney cells CV-1 (Cell collection of the Chinese Academy of Sciences Committee on Typical Cultures / Shanghai Institute of Biosciences, Chinese Academy of Sciences)

[0082] ● Flow cytometry and monoclonal screening methods 1) African green monkey kidney cells CV-1 were infected with the POV-601 virus strain. The virus-infected cells were then screened by flow cytometry (BD), and the screened cells were seeded into 96-well plates, with one cell inoculated per well.

[0083] 2) The screened 96-well plates were transferred to a carbon dioxide incubator (Thermo, 160i) at 37°C and 5% CO2 concentration for incubation. The viral infection status was observed daily.

[0084] 3) When sufficient CPE appeared in cells infected with the virus, the virus was collected and cryopreserved.

[0085] 4) For the collected monoclonal viruses, QuickExtract TM Viral genomic DNA was extracted using DNA Extraction Solution (cat#:QE09050, Lucigen).

[0086] 5) Using the designed ORFV112 gene-specific forward and reverse primers (SEQ ID NO: 5 and 6), genomic DNA extracted from monoclonal viruses was used as a template for amplification of the target fragments and agarose gel electrophoresis, respectively. Figure 1 shows the electrophoresis results for monoclonal virus strains POV-601-1A1 and POV-601-3F8, with band sizes of approximately 500 bp and 1000 bp, respectively. Furthermore, PCR amplification was performed using ORFV112 gene-specific forward and reverse primers (SEQ ID NO: 5 and 6) and POV-601-3F8 forward and reverse primers (SEQ ID NO: 55 and 56), respectively. Sanger sequencing was then performed on the PCR products (Suzhou Jinweizhi Biotechnology Co., Ltd.), confirming that the ORFV112 gene coding region of POV-601-1A1 had a deletion of 538 bases at the 5' end (including 226 bases in the non-coding region at the 5' end and 312 bases in the ORFV112 protein coding region at the 5' end) (see SEQ ID NO: 4). In addition, POV-601-3F8 has the complete ORFV112 gene sequence (see SEQ ID NO: 3).

[0087] ●PCR program parameters: Pre-denaturation was performed at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 68°C for 1 minute. This process was repeated 30 times, followed by final extension at 68°C for 7 minutes.

[0088] Example 2: Identification of the species and genus of the mutant Orff virus. The viral genome was extracted from the POV-601-1A1 virus strain (Mouse Tail Genomic DNA Kit, Cat#:CW2094S), and sent to a third-party sequencing company for next-generation sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.). The resulting sequence was then assembled. The B2L gene of the virus strain was analyzed.

[21] After searching the complete sequence result of the (ORFV011 gene) using BLAST nucleic acid sequencing, the one with the higher similarity to the sequence determination result was OV / HLJ / 04, and the similarity was 99.91%, confirming that the isolated virus strain was ORFV.

[0089] [Table 1]

[0090] Example 3: Construction of a mutant Orff virus ● Virus strain origin: POV-601-1A1 virus strain (China Center for Typical Cultures (CCTCC), accession number: V202029) Host cell origin: African green monkey kidney cells CV-1 (ATCC No. CCL-70) TM )

[0091] ●The method for constructing recombinant Orff virus mainly involves the following steps. 1) Using specially designed primers (SEQ ID NO: 7-12) each having HindIII and EcoRI restriction endonuclease sites at their terminals, the flanking sequences (left homology arm and right homology arm) of the ORFV112 gene and the EGFP reporter gene were cloned by PCR. 2) Enzymatically cleave the pUC19 plasmid using HindIII and EcoRI. 3) The PCR product from step 1) and the pUC19 plasmid after enzymatic cleavage are ligated together to obtain the CBP shuttle plasmid. 4) Using the CBP shuttle plasmid ligated product obtained in step 3) as a template, amplification is performed by PCR using specific primers (SEQ ID NO: 13-14) having EcoRI and AgeI restriction endonuclease sites at their respective ends, respectively, to obtain a linear CBP shuttle vector. 5) Using the px330 plasmid vector as a template, specific primers (SEQ ID NO: 15-18) having EcoRI and AgeI restriction endonuclease sites at their terminals, respectively, are employed, and a novel px330 plasmid vector and px330 plasmid vector backbone without nuclear localization signal (NLS) Cas9 are amplified by PCR. 6) The new px330 plasmid vector and px330 plasmid vector backbone from step 5) are enzymatically cleaved using EcoRI and AgeI, the cleavage products are ligated with T4 DNA ligase, and the ligated products are transformed into the screening host bacterium DH5α to obtain a positive monoclonal. Further expansion culture is performed to extract the plasmid. Sanger sequencing is performed to identify the plasmid and obtain a recombinant plasmid (px330-ΔNLS) that does not have a nuclear localization signal (NLS). 7) Synthesize CBP gRNAs, enzymatically cleave the recombinant plasmid (px330-ΔNLS) using BbsI, ligate the CBP gRNAs with the enzymatically cleaved recombinant plasmid (px330-ΔNLS), transform the host bacterium DH5α (Shanghai Biotechnology, B528413-0020) to obtain a positive monoclonal, perform further expansion culture, and extract the plasmid. Obtain the px330-ΔNLS-CBP gRNA expression plasmid. 8) Using the obtained px330-ΔNLS-CBP gRNA expression plasmid, specific parapogenetic XV virus host cells CV-1 are transfected, and then the transfected CV-1 cells are infected with the POV-601-1A1 virus strain. The linear CBP shuttle vector obtained in step (4) is transfected into the transfected px330-ΔNLS-CBP gRNA plasmid and the CV-1 cells infected with the POV-601-1A1 virus. 9) Target cells containing EGFP fluorescent virus are concentrated and collected, then screened by flow cytometry and seeded into 96-well plates, with one cell seeded per well, followed by culture and amplification. Target cells are collected from the EGFP fluorescent virus-rich wells, viral genomic DNA is extracted, and identification is performed using PCR (primer SEQ ID NO: 19-30). CV-1 cells are then reinfected with the virus that shows the target band, and expanded culture is performed. 10) Virus seeding, screening, virus collection, and PCR identification were performed multiple times, and finally, a purified monoclonal virus with a complete deletion of the ORFV112 gene was obtained and named POV-604-1D1.

[0092] Example 4: Amplification and culture of mutant Orff virus Host cell origin: African green monkey kidney cells CV-1 (ATCC No. CCL-70) TM ) Virus strain origins: POV-601, POV-601-1A1, POV-604-1D1, and POV-601-3F8 virus strains

[0093] ●Virus-infected cells: 1) Passaging of CV-1 cells is performed according to the required amount, and once a certain quantity has been amplified, viral infection can be performed when the cell saturation in the culture bottle reaches approximately 90%. 2) The virus solution was appropriately diluted in 2% FBS DMEM complete medium. 3) The old culture medium in the culture bottle was discarded and replaced with an appropriate amount of fresh 2% FBS DMEM complete medium. 4) Diluted virus solution was added according to an MOI of 0.5, the culture bottle was gently shaken to distribute the virus solution evenly, the culture bottle was marked, and the samples were incubated in an incubator at 37°C and 5% CO2.

[0094] ● Cryopreservation of virus-infected cells 1) In more than 90% of cells infected with the virus, the virus can be collected if CPE (Chronic Pediatric Epithelial Phenomenon) occurs. 2) Virus-infected cells were removed from the cell culture incubator, the supernatant was collected, and any cells still attached to the cell wall were digested and then added to the supernatant. 3) Centrifuged at 3000 rpm and 4°C for 10 minutes. 4) Rinse once with PBS. 5) The cells were resuspended in an appropriate amount of PBS. 6) The product was moved to -80°C for storage.

[0095] ● Dissolution and release by viruses: 1) Virus-infected cells were thawed by moving them to a water bath pot at -80°C to 37°C. 2) Ultrasonic treatment (model number: FB120, Thermo): The "APLITUDE" was set to 100%, the "Time" was 1 minute, and the treatment was performed a total of 3 times. 3) Benzoase (25 units per 1 ml of cell lysate suspension) was added, mixed uniformly, and treated at 37°C for 30 minutes. 4) Centrifuged at 1300 rpm for 10 minutes. 5) The viral supernatant was collected and stored at -80°C.

[0096] Example 5: Evaluation of the antitumor activity of mutant Orff virus in a mouse-derived bladder cancer tumor model.

[0097] A. Test Design [Table 2]

[0098] B. Test animals: Male C57BL / 6 mice, 6W, obtained from Zhejiang Weitong Lihua Test Animal Technology Co., Ltd.

[0099] C. Examination Process MB49 cells (Guangzhou Jiniou Biotechnology Co., Ltd.), resuspended in PBS, were subcutaneously seeded into the right dorsal side of male C57BL / 6 mice, resulting in a cell density of 2 × 10⁶. 6 The ratio was / ml, and the seeding rate was 0.1ml / cell. The day on which cells were seeded was defined as Day 0. The mean tumor volume in the control group was 100 mm².3 When the condition worsened, the mice were randomly divided into groups according to tumor size, administered treatments as described in Table 1, and all mice were euthanized on Day 30 after cell seeding.

[0100] Throughout the study, mouse body weight and tumor measurements were performed twice a week. Tumor volume was measured using a caliper (model: 16ER; Mahr GmbH), and the results are shown in Figure 2.

[0101] The tumor volume was calculated using the following formula. Length and width were measured vertically. Tumor volume (mm 3 ) = a × b 2 / 2 (In the formula, a is the length of the tumor (mm), and b is the width of the tumor (mm).)

[0102] The relative mean tumor volume was measured using the following formula. RTV (Relative Tumor Volume) = V t / V0 (In the formula, V0 is the average tumor volume measured when the doses were administered to different groups, V t This is the average tumor volume measured each time. Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% (T RTV This is the treatment group RTV, and C RTV This is the RTV of the control group. Tumor inhibition rate TGI%=(1-T / C)×100%.

[0103] Analyze data using ANOVA statistics Using the ANOVA test method, is there a significant difference between the tumor volume in the treatment group and the tumor volume in the control group? We compared whether or not the case occurred. All data was analyzed using SPSS 17. A P<0.05 indicates a statistically significant difference.

[0104] D. Results [Table 3]

[0105] Example 6: Evaluation of the antitumor activity of mutant Orff virus in a mouse-derived melanoma model. A. Test Design [Table 4]

[0106] B. Test animals - Female C57BL / 6 mice, 6-8 weeks old, obtained from Zhejiang Weitong Lihua Test Animal Technology Co., Ltd.

[0107] C. Examination Process B16-F10 cells (from the Cell Collection of the Chinese Academy of Sciences' Committee for the Preservation of Typical Cultures), resuspended in PBS, were subcutaneously seeded on the right dorsal side of female C57BL / 6 mice, resulting in a cell density of 5 × 10⁶. 6 The sample size was 0.1 ml / cell, and the seeding rate was 0.1 ml / cell. Day 0 was defined as the day the cells were injected. The mean tumor volume in the control group was 100 mm². 3 When the condition worsened, the mice were randomly divided into groups according to tumor size, administered treatments as described in Table 3, and all mice were euthanized on Day 15 after cell seeding.

[0108] Throughout the study, mouse weight was measured and tumor size was measured twice a week. (Caliper model number) Tumor volume was measured using 16ER (Mahr GmbH), and the results are shown in Figure 3.

[0109] The tumor volume was calculated using the following formula. Length and width were measured vertically. Tumor volume (mm 3 ) = a × b 2 / 2 (In the formula, a is the length of the tumor (mm), and b is the width of the tumor (mm).)

[0110] The relative mean tumor volume was calculated using the following formula. RTV (Relative Tumor Volume) = V t / V0 (In the formula, V0 is the average tumor volume measured when the doses were administered to different groups, V t This is the average tumor volume measured each time. Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% (T RTV This is the treatment group RTV, and C RTV This is the RTV of the control group. Tumor inhibition rate TGI%=(1-T / C)×100%.

[0111] Analyze data using ANOVA statistics The ANOVA test was used to compare whether there was a significant difference between the tumor volume of the treatment group and the tumor volume of the control group. All data were analyzed using SPSS 17. A p-value of < 0.05 indicates a statistically significant difference.

[0112] D. Results [Table 5]

[0113] Example 7: Safety evaluation of mutant Orff virus A. Test Design [Table 6]

[0114] B. Measured specimens and control specimens Test sample: Oncolytic virus POV-601-1A1, Control sample: Phosphate buffer

[0115] C. Test animal: female C57BL / 6 mouse, 6-8W, Zhejiang Wetong Lihua Test Animal Technology Co., Ltd.

[0116] D. Test Procedure Female C57BL / 6 mice were injected subcutaneously or intravein into the right dorsal region with the test product and the control product. The day of the first injection was defined as Day 0. Mice were completely randomly assigned to groups and administered according to the instructions in Table 5. Observation of all mice was completed on day 15 after administration. Throughout the entire study period, the body weight of the mice was weighed daily during the first week, and then every 2-3 days thereafter.

[0117] We monitored the weight changes of mice. The animals' weight was measured using an ML1602T electronic balance (Mettler). The relative weight change rate was calculated using the following formula, and the results are shown in Figure 4. Relative weight change (%) = [weight 日新 / weight 日0 ] × 100.

[0118] Example 8: Modulatory effect of mutant Orff virus on the immune system A. Test Design [Table 7]

[0119] B. Measured specimens and control specimens: Measured item - Oncolytic virus POV-601-1A1 Control product - Phosphate buffer solution

[0120] C. Test animal: female Balb / c-nude mouse, 6W, Beijing Huafukang Biological Technology Co., Ltd.

[0121] D. Test Method Female Balb / c-nude mice were subcutaneously seeded with C-33A cells (from the Cell Collection of the Chinese Academy of Sciences' Committee for the Preservation of Typical Cultures) resuspended in PBS on both sides of the dorsal surface, resulting in a cell density of 1 × 10⁶. 8 The sample size was 0.1 ml / milli, and the seeding rate was 0.1 ml / milli. The day on which cells were injected was defined as Day 0. The average tumor volume was 100 mm². 3When the condition was met, the administration procedure was carried out according to the explanation in Table 6. 24 hours after the final administration, the experiment was terminated, whole mouse blood and tumors were collected, tissues were dissected and digested, and single-cell suspensions were prepared. These were then labeled with fluorescent antibodies (Anti-mouse CD45 APC-eFluor 780, Anti-mouse CD49b-PE, Anti-mouse CD69 APC), followed by flow detection (AFC2, Thermo), as shown in Figure 5 (A-C in the same figure).

[0122] Example 9: Infectious effect of mutant Orff virus on in vitro cells Virus strain: POV-601-1A1 1) After digesting each cultured tumor cell, collect and count them, 1.5 x 10⁻⁶ 4 Cells were seeded in a 96-well plate at a cell / well rate, cultured for 24 hours, and then seeded again. 2) Dissolve the virus in 1.5 × 10⁶ of 2% FBS complete medium. 8 pfu / ml, 1.5 × 10 7 pfu / ml, 1.5 × 10 6 pfu / ml, 1.5 × 10 5 The sample was diluted to four different concentrations of pfu / ml. 3) The diluted virus solution was added to a 96-well cell plate, and 100 μl was added to each well until the MOI of the virus in each well was 1000, 100, 10, and 1, respectively, with three duplicate wells set up for each concentration. 4) Three wells were prepared and 2% FBS complete medium without the virus solution was added to serve as a negative control. 5) Gently shake the cells to evenly coat the surface of the monolayer cells with the virus solution, ensuring that every corner is covered. 6) The control sample or the 96-well plate inoculated with the virus was returned to an incubator at 37°C and 5% CO2 and incubated for 72 hours. 7) Remove the 96-well plate from the incubator, add 10 μl of Alamar Blue Cell Viability Reagent (Invitrogen, Cat#:2072060) per well, gently shake the incubator to evenly disperse the dye, cover the 96-well plate with aluminum foil, and incubate in a light-shielded incubator at 37°C. 8) After the culture was complete, the cover was removed, and the 96-well plate was placed in an ELISA (SPECTRAMAX M4, Molecular Devices). The excitation wavelength was set to 560 nm and the emission wavelength to 590 nm, and absorbance detection was performed. The cell mortality rate was converted using the following conversion formula, and the results were analyzed.

number

[0123] [Table 8]

[0124] Example 8: Evaluation of the antitumor activity of mutant Orff virus in a mouse-derived colon cancer model. A. Test Design [Table 9]

[0125] B. Test animals - Female Balb / c mice, 6-8 weeks old, obtained from Zhejiang Weitong Lihua Test Animal Technology Co., Ltd.

[0126] C. Examination Process CT-26 cells (from the Cell Collection of the Chinese Academy of Sciences' Committee for the Preservation of Typical Cultures), resuspended in PBS, were subcutaneously seeded into the right dorsal side of female Balb / c mice, resulting in a cell density of 5 × 10⁶. 6 The sample size was / ml, and the seeding rate was 0.1ml / animal. The day on which cells were injected was designated as Day 0. The mean tumor volume of the control group was 100 mm². 3 In that case, the mice were randomly divided into groups according to tumor size, administered according to the explanation in Table 8, and all mice were euthanized on Day 25 after cell seeding.

[0127] Throughout the study, mouse body weight and tumor measurements were performed twice a week. Tumor volume was measured using a caliper (model: 16ER; Mahr GmbH), and the results are shown in Figure 14.

[0128] The tumor volume was calculated using the following formula. Length and width were measured vertically. Tumor volume (mm 3 ) = a × b 2 / 2 (In the formula, a is the length of the tumor (mm), and b is the width of the tumor (mm).)

[0129] The relative mean tumor volume was calculated using the following formula. RTV (Relative Tumor Volume) = V t / V0 (In the formula, V0 is the average tumor volume measured when the doses were administered to different groups, V t This is the average tumor volume measured each time. Relative tumor growth rate T / C(%)=T RTV / C RTV ×100% (T RTV This is the treatment group RTV, and C RTV This is the RTV of the control group. Tumor inhibition rate TGI%=(1-T / C)×100%.

[0130] ANOVA statistics were used to analyze the data and compare whether there was a significant difference in the ratio of tumor volume between the treatment group and the control group. All data were analyzed using SPSS 17. A P<0.05 indicates a statistically significant difference.

[0131] D. Results [Table 10]

[0132] Example 9: Evaluation of the antitumor activity of recombinant mutant Orff virus in a mouse-derived melanoma model. By using a recombinant method, an ORFV virus is constructed in which the coding region and / or non-coding region of the ORFV111 gene and / or the ORFV112 gene are completely or partially deleted. Unless otherwise specified, the effects were measured using the method described in Example 6.

[0133] [Table 11]

[0134] [Table 12]

[0135] [Table 13]

[0136] Material contracting The following materials are being held at the China Center for Typical Cultures (CCTCC) (Wuhan University, Wuhan, China, 430072) in accordance with the provisions of the Budapest Convention.

[0137] [Table 14]

[0138] References 1. Hanahan, D., Weinberg, RA(2011). Hallmarks of cancer: the next generation. Cell, 144(5): 646-74. 2. Miest, TS, Cattaneo, R. (2014). New viruses for cancer therapy: meeting clinical needs. Nat Rev Microbiol, 12(1): 23-34. 3. Burke, J., Nieva, J., Borad, MJ, Breitbach, CJ(2015). Oncolytic viruses: perspectives on clinical development. Curr Opin Virol, 13: 55-60. 4. Seymour, LW, Fisher, KD(2016). Oncolytic viruses: finally delivering.Br J Cancer, Feb 16; 114(4): 357-361. 5. Harrington, KJ, Puzanov, I., Hecht, JR, Hodi, FS, Szabo, Z., Murugappan, S., Kaufman, HL(2015). Clinical development of talimogene Laherparepvec(T-VEC): a modified herpes simplex virus type-1-derived oncolytic immunotherapy. Expert Rev Anticancer Ther, 15(12): 1389-1403. 6. Yin Zhen, Liu Jinghua (1997). Animal Science (Second Edition), Science Press, 977-978. 7. Wang Zhijun (2018). Biotechnology Research, Creation and Quality Control (Third Edition), Science Press. 8. CDC(2006). Orf Virus Infection in Humans-New York, Illinois, California, and Tennessee, 2004-2005.Morbidity and Mortality Weekly Report, 55(3): 65-68. 9. Kirn, D.H., Thorne, S.H.(2009). Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer.Nat Rev Cancer, 9: 64-71. 10. McFadden, G.(2005). Poxvirus tropism.Nat Rev Microbiol, 3(3): 201-213. 11. Wang, R., Wang, Y., Liu, F., Luo, S.(2018). Orf virus: A promising new therapeutic agent.Rev Med Virol, e2013. 12. Rintoul, J.L., Lemay, C.G., Tai, L.H., Stanford, M.M., Falls, T.J., Bridle, B.W., Souza, C.T., Daneshmand, M., Ohashi, P.S., Wan, Y., Lichty, B.D., Mercer, A.A., Auer, R.C., Atkins, H.L., Bell, J.C.(2012). ORFV: a novel oncolytic and immune stimulating parapoxvirus therapeutic.Mol Ther, 20(6): 1148-1157. 13. Seet, BT, McCaughan, CA, Handel, TM, Mercer, A., Brunetti, C., McFadden, G., Fleming, SB(2003). Analysis of an orf virus chemokine-binding protein: shifting ligand specificities among a family of poxvirus viroceptors. Proceedings of the National Academy of Sciences, 100(25): 15137-15142. 14. Bergqvist, C., Kurban, M., Abbas, O. (2017). Orf virus infection. Rev Med Virol, 27(4). 15. Liu Wei, Yang Kankan, Yin Dongdong, Wang Yuanhong, Yu Zhaoying, Jiang Shudong, Li Chuanfeng, Li Yongdong, Wang Yong (2018). Prokaryotic manifestation and intracellular localization of orofacid dUTPase progeny. Chinese Journal of Veterinary Science, 7: 818-823. 16. Irwin, CR, Hitt, MM, Evans, DH(2017). Targeting Nucleotide Biosynthesis: A Strategy for Improving the Oncolytic Potential of DNA Viruses. Front Oncol, 7: 229. 17. Fleming, SB, McCaughan, C., Lateef, Z., Dunn, A., Wise, IM, Real, NC, Mercer, AA(2017). Deletion of chemokine binding protein gene from the parapoxvirus orf virus reduces virulence and pathogenesis in sheep. Front Microbiol, 8: 46. 18. Twumasi-Boateng, K., Jessica, LP, Eunice Kwok, YY, John, CB, Nelson, BH(2018). Oncolytic viruses as engineering platforms for combination immunotherapy. Nat Rev Cancer, 18: 419-432. 19. Diel, DG, Lou, S., Delhon, G., Peng, Y., Flores, EF, Rock, DL (2011). A nuclear inhibitor of NF-kappaB encoded by a poxvirus. J Virol, 85(1): 264-275. 20. Son, SJ, Harris, PW, Squire, CJ, Baker, EN, Kent, SB, Brimble, MA (2014). Total Chemical Synthesis of an Orf Virus Protein, ORFV002, an Inhibitor of the Master Gene Regulator NF-κB. Biopolymers (Peptid Science), 102(2): 137-144. 21. Wang Guangxiang, Shang Youjun, Chen Jiangtao, Lu Zhanlu, Zhang Keshan, Liu Xiangtao (2012). Separation and determination of オルフウイルスの in Hubei Province, Journal of Veterinary Medicine, 033(011): 37-40.

[0139] Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30

Claims

1. A mutant Orff virus characterized by the deletion of the functional expression products of genes ORFV112 and ORFV111.

2. The virus according to claim 1, wherein the expression product is a protein and / or nucleic acid.

3. A mutant Orff virus characterized by complete or partial deletion of genes ORFV112 and ORFV111, wherein the partial deletion causes the expression products of each gene to lose their function.

4. To reduce or remove the expression and / or activity of the expression product of the gene ORFV112, and to reduce or remove the expression and / or activity of the expression product of the gene ORFV111. Instructions for handling Orff viruses, including those mentioned.

5. The method according to claim 4, wherein the expression is transcription and / or translation.

6. A method for manipulating Orff virus, comprising completely or partially deleting gene ORFV112 and completely or partially deleting gene ORFV111, wherein the partial deletion causes the expression product of each of the genes to lose its function.

7. A virus obtained by the method described in any one of claims 4 to 6.

8. The virus according to any one of claims 1 to 3 and 7, wherein the genes ORFV002 (NF-κB nuclear inhibitor), ORFV005 (hypothetical protein), and ORFV007 (dUTPase) are completely deleted.

9. A virus according to any one of claims 1 to 3, 7, and 8, which has oncolytic ability and therapeutic ability.

10. The Orff virus was accepted under CCTCC accession number V202029.

11. The viral genome according to any one of claims 1 to 3 and 7 to 10.

12. A composition comprising the virus described in any one of claims 1 to 3 and 7 to 10 and a pharmaceutically acceptable vector.

13. The composition according to claim 12, which is in the form of a powder, liquid, transdermal patch, ointment, or suppository.

14. The composition according to claim 12 or 13, administered via an intravenous, intratumor, intramuscular, subcutaneous, intrarectal, vaginal, or intraperitoneal route.

15. Use of the virus according to any one of claims 1 to 3 and 7 to 10 in the manufacture of a drug for treating cancer, wherein the virus is formulated for administration via an intravenous, intratumoral, intramuscular, subcutaneous, intrarectal, vaginal, or intraperitoneal route.

16. The use according to claim 15, wherein the cancer is a solid tumor.

17. The use according to claim 16, wherein the solid tumor is cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, or brain tumor.

18. The composition according to claim 12 for treating cancer.

19. The composition according to claim 18, wherein the cancer is a solid tumor.

20. The composition according to claim 19, wherein the solid tumor is cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, stomach cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer, pancreatic cancer, sarcoma, or brain tumor.

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