WO2022033573A1 - 一种突变型羊传染性脓疱皮炎病毒及其用途 - Google Patents

一种突变型羊传染性脓疱皮炎病毒及其用途 Download PDF

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WO2022033573A1
WO2022033573A1 PCT/CN2021/112408 CN2021112408W WO2022033573A1 WO 2022033573 A1 WO2022033573 A1 WO 2022033573A1 CN 2021112408 W CN2021112408 W CN 2021112408W WO 2022033573 A1 WO2022033573 A1 WO 2022033573A1
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gene
virus
cancer
orfv112
orfv111
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French (fr)
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胡敏杰
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苏州般若生物科技有限公司
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Priority to EP21855623.1A priority Critical patent/EP4206327A1/en
Priority to CA3192853A priority patent/CA3192853A1/en
Priority to CN202180056444.5A priority patent/CN116744952A/zh
Priority to US18/041,234 priority patent/US20230340424A1/en
Priority to JP2023511666A priority patent/JP2023537643A/ja
Publication of WO2022033573A1 publication Critical patent/WO2022033573A1/zh

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    • C12N2710/24271—Demonstrated in vivo effect

Definitions

  • the invention relates to the field of biotechnology, in particular to the use of a mutant ovine infectious pustular dermatitis virus (ORFV) and a pharmaceutical composition thereof in cancer treatment.
  • ORFV ovine infectious pustular dermatitis virus
  • Sheep infectious pustular dermatitis virus belongs to the genus Parapoxvirus in the Poxviridae family.
  • the genus also includes bovine papular stomatitis virus, pseudovaccinia virus, parapox virus of red deer in New Zealand, and Sealpox virus.
  • Patent CN 104017776 B discloses an attenuated ORFV obtained after separation and cell subculture, which is used for vaccine development.
  • Patent CN 108026542 A describes the use of virus strain D1701 to construct a recombinant ORFV vector to express viral antigens, tumor antigens, etc. for the preparation of vaccines.
  • Oncolytic viral therapy is a systemic therapy that uses naturally occurring or engineered viruses to treat tumors. Those specific gene mutations that originally promote the proliferation and survival of tumor cells can also promote the growth of viruses with lytic function in them, which is one of the reasons why oncolytic viruses precisely attack tumor cells [1, 2, 3] .
  • Oncolytic viruses after infecting cancer cells can promote the lysis and death of cancer cells at appropriate time points in the virus life cycle, and release infectious virus particles to further infect surrounding uninfected cancer cells.
  • the neoantigen (neoantigen) released by the lysis of cancer cells activates the immune system, thereby making a follow-up attack on cancer [4] .
  • ORFV virus is a double-stranded DNA virus with a length of about 134-139kb. Virus particles are 230-280nm long and 150-200nm wide, and are elliptical coil-like [6] .
  • ORFV virus has the following characteristics: 1) Similar to members of the Poxviridae family of the same family, ORFV virus only replicates in the cytoplasm of the host cell, does not enter the nucleus, and thus will not integrate into the genome of the host cell, so it is safe, The probability of carcinogenicity is extremely low [7, 10] ; 2) It can infect humans through contact with damaged skin and animal infection areas, and produce clinically mild pustules at the infection site (usually fingers), and there are basically no other adverse side effects.
  • ORFV strains typically encode about 130-134 genes.
  • the currently 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) and so on [11] .
  • the proteins encoded by these genes can also regulate the body's immune response [13] .
  • ORFV002 is a late viral gene, which is located in the nucleus after protein synthesis and inhibits the NF- ⁇ B pathway in the nucleus. It was identified as the first NF- ⁇ B nuclear inhibitor produced by ORFV virus [19] .
  • the gene ORFV005 is a hypothetical protein gene, and its mechanism of action is still unclear.
  • the gene ORFV007 encodes deoxyuridine pyrophosphatase (duTPase) [15] .
  • This protein is an important enzyme in the synthesis of dNTPs (deoxyribonucleoside triphosphates).
  • dNTPs deoxyribonucleoside triphosphates
  • concentration of dNTPs in normal cells is tightly regulated and thus detrimental to viral replication.
  • cancer cells there are higher concentrations of dNTPs, which can promote virus replication [16] .
  • the deletion of the ORFV007 gene blocks the replication of the virus in normal cells, but does not affect its amplification in tumor cells, so that the ORFV virus can selectively replicate in cancer cells.
  • the gene ORFV111 is a hypothetical protein gene, and its mechanism of action is still unclear.
  • CBP protein ORFV112 (chemokine-binding protein, chemokine-binding protein), is about 864 bp long, and its function is to inhibit the antiviral mechanism of immune cells [14] .
  • This protein is similar in structure and function to CBP-II proteins of other poxviruses, which can not only inhibit the migration of DC cells to the inflammatory site, but also inhibit the activation of T cells by DC cells.
  • NZ-2, NZ-7, D1701, NA1/11 and so on have been published and used for the research of oncolytic virus ORFV virus strains.
  • ORFV virus can insert relatively large exogenous DNA fragments, it is generally used as a vector to insert a certain tumor-specific antigen or viral antigen, cytokine, etc., to obtain a genetically recombinant virus for research.
  • Patent WO 2012122649A1 discloses a recombinant OFRV virus (insert vaccinia virus E3L gene) that infects tumor cells containing specific host range genes (SPI-1, K1L, C7L, B5R, p28/N1R, E3L, etc.) for cancer drug development.
  • Patent CN 108220251 A discloses a recombinant infectious pustular oncolytic virus and its preparation method and application, which mainly include knocking out the ORFV132 (VEGF) gene of the NA1/11 strain and inserting a P53-EGFP fusion protein at the same position Genes for cancer drug development.
  • the strains described in the above two patents have neither reported the deletion of the ORFV112 gene nor the deletion of the ORFV007 gene.
  • a mutant sheep transmissible pustular dermatitis virus characterized by the deletion of the functional expression product of the gene ORFV112 and/or the gene ORFV111, wherein the deletion of the functional expression product is caused by the gene ORFV112 and/or the gene encoding CBP.
  • the inventors unexpectedly found that the complete deletion or partial deletion of the ORFV 112 gene and/or the ORFV 111 gene can significantly enhance the anti-tumor effect of the virus.
  • the genome sequence of the mutant sheep infectious pustular dermatitis virus was compared with the genome sequences of all other published ORFV virus strains, and it was found that the ORFV 007 dUTPase gene of the mutant sheep infectious pustular dermatitis virus appeared. Completely missing. Further, the above comparison also confirmed that the ORFV002 and ORFV005 genes of the ovine infectious pustular dermatitis virus were completely deleted.
  • the mutant ovine infectious pustular dermatitis virus of the present disclosure has a guiding role in the development of anti-tumor drugs for this type of virus.
  • the inventors unexpectedly discovered that the complete deletion or partial deletion of the gene ORFV112 (encoding the CBP protein) and/or the gene ORFV111 (encoding a hypothetical protein, hereinafter referred to as the hypothetical protein 111) (especially the 5' partial deletion of the gene ORFV112 and the gene ORFV111) Deletion of the 3' part of ovine infectious pustular dermatitis virus) enhances the antitumor activity of ovine infectious pustular dermatitis virus. Further, the inventors also unexpectedly found that the gene ORFV007 of the mutant ovine infectious pustular dermatitis virus is completely deleted.
  • Infectious pustular dermatitis virus of sheep with functional CBP protein and/or hypothetical protein 111 deletion can be detected by conventional molecular biology methods (eg molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing etc.) or emerging molecular biology methods (such as gene editing, etc.).
  • conventional molecular biology methods eg molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing etc.
  • emerging molecular biology methods such as gene editing, etc.
  • the present invention provides a mutant sheep transmissible pustular dermatitis virus characterized by deletion of the functional expression product of gene ORFV112 and/or gene ORFV111.
  • the deletion of the functional expression product of the gene ORFV112 results from a complete or partial (e.g., 5' part or 3' part, especially the 5' part) deletion of the gene ORFV112.
  • the mature sequence of the expression product of the gene ORFV112 is shown in SEQ ID NO:2.
  • the complete sequence of the gene ORFV112 is shown in SEQ ID NO:3.
  • SEQ ID NO:3 is completely deleted.
  • SEQ ID NO:3 is partially deleted, eg, 1-1161, 1-1140, 1-538, 539-1139, or 1141-1160 5' bases are deleted.
  • the partially deleted gene ORFV112 is shown in SEQ ID NO:4 or SEQ ID NO:57.
  • the deletion of the functional expression product of the gene ORFV111 results from a complete or partial (e.g., 5' part or 3' part, especially 3' part) deletion of the gene ORFV111.
  • the sequence of the expression product of the gene ORFV111 is shown in SEQ ID NO:58.
  • the complete sequence of gene ORFV111 is set forth in SEQ ID NO:59.
  • SEQ ID NO:59 is completely deleted. In one embodiment, SEQ ID NO:59 is partially deleted, eg, 1-793, 1-309, or 310-792 3' bases are deleted. In one embodiment, the partially deleted gene ORFV111 is shown in SEQ ID NO:60. In one embodiment, SEQ ID NO:3 and SEQ ID NO:59 are completely deleted. In one embodiment, the expression product is a protein and/or a nucleic acid (especially a functional nucleic acid). In one embodiment, the present invention provides the genome of the virus.
  • the present invention provides a method for transforming ovine infectious pustular dermatitis virus, which comprises reducing or eliminating the expression and/or activity of the expression product of gene ORFV112 and/or gene ORFV111.
  • the method comprises deletion of the gene ORFV112 in whole or in part (e.g., the 5' part or the 3' part, especially the 5' part).
  • the mature sequence of the expression product of the gene ORFV112 is shown in SEQ ID NO:2.
  • the complete sequence of the gene ORFV112 is shown in SEQ ID NO:3.
  • the method comprises deleting SEQ ID NO:3 completely.
  • the method comprises a partial deletion of SEQ ID NO: 3, such as deletion of 1-1161, 1-1140, 1-538, 539-1139 or 1141-1160 5' bases.
  • the method produces the partially deleted gene ORFV112 as set forth in SEQ ID NO:4 or SEQ ID NO:57.
  • the method comprises deletion of the gene ORFV111 in whole or in part (e.g. in the 5' part or the 3' part, especially the 3' part).
  • the sequence of the expression product of the gene ORFV111 is shown in SEQ ID NO:58.
  • the complete sequence of gene ORFV111 is set forth in SEQ ID NO:59.
  • the method comprises deleting SEQ ID NO:59 completely.
  • the method comprises a partial deletion of SEQ ID NO: 59, such as deletion of 1-793, 1-309 or 310-792 3' bases.
  • the method produces a partially deleted gene ORFV111 as set forth in SEQ ID NO:60.
  • the method comprises the complete deletion of SEQ ID NO:3 and SEQ ID NO:59.
  • expression is transcription and/or translation.
  • the present invention provides a virus obtained by the method. In one embodiment, the present invention provides the genome of the virus.
  • the methods of the present invention can be performed by conventional molecular biology techniques (eg molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.) or emerging molecular biology techniques (eg gene editing, etc.) .
  • conventional molecular biology techniques eg molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.
  • emerging molecular biology techniques eg gene editing, etc.
  • the present invention provides a method for identifying the mutant ovine infectious pustular dermatitis virus and/or its genome, in particular for detecting the (presence or sequence) of gene ORFV112 and/or gene ORFV111 and/or its expression Methods of product (presence and/or activity).
  • the methods of the present invention can be performed by conventional molecular biology techniques (eg, activity assays, hybridization (eg, Southern, Northern, or Western)), which detect the presence and/or activity of proteins and/or nucleic acids and/or sequences, Restriction endonucleases, PCR, electrophoresis (eg, gel electrophoresis, including protein electrophoresis and nucleic acid electrophoresis, including agarose electrophoresis and PAGE electrophoresis, including reducing and non-reducing electrophoresis), sequencing (including protein sequencing and nucleic acid sequencing), etc. ) or emerging molecular biology techniques (such as next-generation sequencing, etc.).
  • conventional molecular biology techniques eg, activity assays, hybridization (eg, Southern, Northern, or Western)
  • electrophoresis eg, gel electrophoresis, including protein electrophoresis and nucleic acid electrophoresis, including agarose electrophoresis and PAGE electrophor
  • the present invention provides methods for detecting the integrity of the gene ORFV112 and/or the gene ORFV111.
  • the method is performed by PCR and gel electrophoresis.
  • the method can be performed by hybridization or sequencing.
  • the present invention provides a method of treating cancer in a subject using the mutant ovine infectious pustular dermatitis virus and/or its genome.
  • the present disclosure provides use of the mutant ovine infectious pustular dermatitis virus and/or its genome for treating cancer in a subject.
  • the present disclosure provides use of the mutant ovine infectious pustular dermatitis virus and/or its genome to prepare a medicament for treating cancer in a subject.
  • the present disclosure provides the mutant ovine infectious pustular dermatitis virus and/or its genome for use in the treatment of cancer in a subject.
  • the present disclosure provides the mutant ovine infectious pustular dermatitis virus and/or its genome for use in the manufacture of a medicament for treating cancer in a subject.
  • the cancer is a solid tumor.
  • the solid tumor is cervical cancer, bladder cancer, liver cancer, ovarian cancer, melanoma, colorectal cancer, lung cancer, breast cancer, gastric cancer, uterine cancer, head and neck cancer, thyroid cancer, esophageal cancer, prostate cancer , pancreatic cancer, sarcoma, brain tumor, etc.
  • the subject is a mammal, including rodents (eg, mice and rats), non-human primates (eg, cynomolgus monkeys), and humans.
  • Figure 1 shows the results of agarose gel electrophoresis of PCR products after PCR amplification using ORFV112 gene-specific forward primers and reverse primers (SEQ ID NOs: 5 and 6).
  • Sample No. 1 in the figure is the ORFV112 gene deletion virus strain POV-601-1A1
  • sample No. 2 is the ORFV112 gene intact virus strain POV-601-3F8, and M is the DNA size marker.
  • Figure 2 shows the tumor suppressor effect of the ORFV112 gene deletion virus strains POV-601-1A1 and POV-604-1D1 on the MB49 murine bladder cancer model.
  • Figure 3 shows the tumor suppressor effect of ORFV112 gene deletion virus strains POV-601-1A1 and POV-604-1D1, and ORFV112 gene intact virus strain POV-601-3F8 in B16-F10 murine melanoma tumor model.
  • Figure 4 shows the effect of POV-601-1A1 virus on the body weight of mice under different doses and administration routes.
  • Figure 5 shows the changes of the mouse immune system in the human C-33A cervical cancer bilateral tumor model after administration of POV-601-1A1 virus strain.
  • the square represents the result of intratumoral administration on the right side
  • the circle represents the result of no administration on the left side.
  • intratumoral administration the activation ratio of CD45 + and NK cells in the tumor was increased.
  • Fig. 5C shows the activation of NK cells in blood, and the ratio of activation of NK cells was increased by intravenous administration.
  • Figure 6 shows the alignment of the complete nucleotide sequence of the gene ORFV112 of the present invention with an example of a partially deleted sequence.
  • Figure 7 shows the alignment of the complete sequence of the CBP protein of the present invention with the complete sequence of the CBP protein of other parapoxvirus strains.
  • 3F8 means POV-601-3F8Strain
  • B029 means ORFV Strain B029
  • GO means ORFV Strain GO
  • NA11 means ORFV Strain NA1/11
  • NZ2 means ORFV Strain NZ2
  • NA17 means ORFV Strain NA17
  • OV-SA00 means ORFV Strain OV-SA00
  • O-IA82 means ORFV Strain OV-IA82
  • SJ1 means ORFV Strain SJ1
  • SY17 means ORFV Strain SY17
  • OV-NH3_12 means ORFV Strain OV-HN3/12
  • NP means ORFV Strain NP
  • YX means ORFV Strain YX.
  • Fig. 8 shows the alignment of the complete sequence of ORFV112 gene of the present invention with the complete sequence of ORFV112 gene of other parapoxvirus strains.
  • 3F8 means POV-601-3F8Strain
  • B029 means ORFV Strain B029
  • GO means ORFV Strain GO
  • NA11 means ORFV Strain NA1/11
  • NZ2 means ORFV Strain NZ2
  • NA17 means ORFV Strain NA17
  • OV-SA00 means ORFV Strain OV-SA00
  • O-IA82 means ORFV Strain OV-IA82
  • SJ1 means ORFV Strain SJ1
  • SY17 means ORFV Strain SY17
  • OV-NH3_12 means ORFV Strain OV-HN3/12
  • NP means ORFV Strain NP
  • YX means ORFV Strain YX.
  • Figure 9 shows the alignment of the complete nucleotide sequence of the gene ORFV111 of the present invention with an example of a partially deleted sequence.
  • Figure 10 shows the alignment of the hypothetical protein 111 complete sequence of the present invention with the hypothetical protein 111 complete sequence of other parapoxvirus strains.
  • POV-601-3F8 means POV-601-3F8Strain
  • B029 means ORFV Strain B029
  • GO means ORFV Strain GO
  • NA11 means ORFV Strain NA1/11
  • NZ2 means ORFV Strain NZ2
  • NA17 means ORFV Strain NA17
  • OV-SA00 means ORFV Strain OV-SA00
  • “OV-IA82” means ORFV Strain OV-IA82
  • SJ1 means ORFV Strain SJ1
  • SY17 means ORFV Strain SY17
  • OV- NH3_12 means ORFV Strain OV-HN3/12
  • NP means ORFV Strain NP
  • YX means ORFV Strain YX.
  • Figure 11 shows the alignment of the complete sequence of ORFV111 gene of the present invention with the complete sequence of ORFV111 gene of other parapoxvirus strains.
  • POV-601-3F8 means POV-601-3F8 Strain
  • B029 means ORFV Strain B029
  • GO means ORFV Strain GO
  • NA11 means ORFV Strain NA1/11
  • NZ2 means ORFV Strain NZ2
  • NA17 means ORFV Strain NA17
  • OV-SA00 means ORFV Strain OV-SA00
  • O-IA82 means ORFV Strain OV-IA82
  • SJ1 means ORFV Strain SJ1
  • SY17 means ORFV Strain SY17
  • OV -NH3_12
  • NP means ORFV Strain OV-HN3/12
  • NP means ORFV Strain NP
  • YX means ORFV Strain YX.
  • Fig. 12 shows the alignment of the complete amino acid sequence of the gene ORFV111 of the present invention and the example of the partial deletion sequence
  • Fig. 13 shows the alignment of the complete amino acid sequence of the gene ORFV112 of the present invention and the example of the partial deletion sequence
  • Figure 14 shows the tumor suppressor effect of the ORFV112 gene deletion virus strain POV-601-1A1 and the ORFV112 gene complete virus strain POV-601-3F8 in the CT-26 mouse colon cancer tumor model.
  • Figure 15 shows the tumor suppressive effect of POV-601-1A1 and construct v611a in the B16-F10 murine melanoma tumor model.
  • Figure 16 shows the tumor suppressive effect of POV-601-1A1 and constructs v615a and v616a in a B16-F10 murine melanoma tumor model.
  • Figure 17 shows the tumor suppressive effect of POV-601-1A1 and constructs v617a and v618a in a B16-F10 murine melanoma tumor model.
  • the invention discloses the use of a mutant sheep infectious pustular dermatitis virus (ORFV) and its medicinal composition in cancer treatment.
  • ORFV sheep infectious pustular dermatitis virus
  • the virulence genes of wild-type aphthous goat infectious pustular dermatitis virus mainly include OVIFNR (orf virus interferon resistance gene, interferon inhibitory protein), CBP (chemokine-binding protein, chemokine-binding protein), GIF (GM-CSF/IL-2 inhibitory protein, GM-CSF/IL-2 inhibitory protein), vIL-10 (viral interleukin 10, viral interleukin 10) and VEGF-like protein (vascular endothelial growth factor like protein, vascular Endothelial growth factor-like protein) and so on [11,13] .
  • OVIFNR orf virus interferon resistance gene, interferon inhibitory protein
  • CBP chemokine-binding protein, chemokine-binding protein
  • GIF GM-CSF/IL-2 inhibitory protein
  • vIL-10 viral interleukin 10
  • VEGF-like protein vascular endothelial growth factor like protein, vascular Endothelial growth
  • Attenuated strains can generally be obtained by removing a certain virulence factor gene of the virus using molecular and/or cellular biological methods.
  • a method for obtaining an attenuated strain is disclosed by deleting the virulence gene OVIFNR of the aphtha virus strain SHZ1 to achieve rapid reduction of its virulence, which is used for preparing attenuated vaccines.
  • the mutant sheep infectious pustular dermatitis virus disclosed in the present invention is obtained by modifying and screening the original POV-601 virus strain deposited in the China Center for Type Culture Collection (CCTCC) (accession number: V201713).
  • the inventor designed specific primers for the ORFV112 gene according to the literature [17] .
  • the forward primer (SEQ ID NO:5) was designed on the coding region of the ORFV111 gene
  • the reverse primer (SEQ ID NO:6) was designed on the coding region of the ORFV112 gene, thereby establishing a PCR-based molecule Biological identification technology to identify the integrity of the ORFV112 gene of the POV-601 attenuated strain.
  • African green monkey kidney cells CV-1 were infected with the original POV-601 virus strain, and the virus-infected cells were collected, and single-cell sorting and culture expansion were performed by flow cytometry.
  • the viral genomic DNA was extracted from the amplified, virus-infected cells, and the ORFV virus attenuated strain with the partial deletion of the ORFV112 gene was obtained by the established molecular biology identification technology, Designated POV-601-1A1.
  • the ORFV112 gene coding region sequence of this strain 312 bases are missing at the 5' end, 552 bases remain, and 72 bases remain in the non-coding region at the 3' end, for a total of 624 bases, see SEQ ID NO: 4.
  • a virus strain POV-601-3F8 with a complete ORFV112 gene was also obtained, and its coding region and non-coding region totaled 1162 bases.
  • the complete sequence of the ORFV112 gene is shown in SEQ ID NO:3.
  • the inventors further artificially used gene editing means for the POV-601-1A1 virus strain, trying to knock out all the remaining coding regions of the gene ORFV112 of the virus strain.
  • the remaining coding region of the gene ORFV112 was completely knocked out (only 22 bases of the 3'-end non-coding region remained, see sequence SEQ ID NO: 57), and confirmed by DNA sequencing.
  • the virus strain thus obtained was named POV-604-1D1.
  • the attenuated strain also had better anti-tumor effect than the virus strain POV-601-3F8 with complete ORFV112 gene.
  • any method that leads to the inability to express CBP protein such as knocking out or changing certain sequences in the coding region and non-coding region of the gene ORFV112, can also achieve the same effect.
  • the POV-601-1A1 virus strain was deposited in the China Collection of Type Cultures (CCTCC) (Wuhan) on May 19, 2020 in accordance with the provisions of the Budapest Treaty, and the CCTCC deposit number is V202029.
  • CTCC China Collection of Type Cultures
  • the mutated aphthous transmissible pustular dermatitis virus of the present disclosure can selectively infect melanoma cells (B16-F10), bladder cancer cells (MB49), liver cancer cells (Hepa1-6), colon cancer cells cells (CT26), human cervical cancer cells (C-33A), human ovarian cancer cells (SK-OV-3), etc., and replicate in them.
  • the gene ORFV007 of the mutant aphthous guinea pig infectious dermatitis virus of the present disclosure was unexpectedly found to be completely deleted, while all the aphthous guinea pig infectious pustular dermatitis virus strains that have been published in NCBI, such as NZ -2, NZ-7, D1701, NA1/11 strains, etc., the gene ORFV007 is complete.
  • the ORFV007 gene encodes deoxyuridine pyrophosphatase (duTPase). This protein is an important enzyme in dNTP synthesis. In general, the concentration of dNTPs in normal cells is tightly regulated and thus detrimental to viral replication.
  • Wild-type ORFV virus can generally be cultured in primary cells of bovine and ovine animal tissue (CN 103952377 A).
  • Patent WO2012122649A1 discloses for the first time that the virus can be propagated and expanded in human cervical cancer cell Hela.
  • the present invention discloses a preparation method of the mutant aphthous goat infectious pustular dermatitis virus, using a mammalian cell line as the host cell, preferably, using the African green monkey kidney cell CV-1 as the host cell, For viral infection amplification.
  • the cancer is any solid tumor cancer.
  • the solid tumor cancer types 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, pancreas cancer Carcinoma, sarcoma, brain tumor, etc.
  • the individual is a mammal, including rodents and humans.
  • the mechanism by which oncolytic virus inhibits tumor growth can generally be divided into: 1) replicating and expanding in infected tumor cells, lysing cancer cells, and achieving the purpose of oncolysis; 2) after oncolysis, the specific Messages, such as tumor neoantigens, are released, activating the immune system to systematically attack the remaining cancer cells.
  • the mutant aphthous ovine infectious pustular dermatitis virus of the present disclosure can effectively activate the autonomous immune system represented by NK cells.
  • intratumoral and/or intravenous injection of virus increased the proportion of CD45 + and NK cell activation in tumors and/or blood.
  • OV Oncolytic virus, which is an oncolytic virus.
  • solid tumor refers to a tumor entity composed of multiple cells to distinguish hematological tumors; it can be 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 early or late stages.
  • continuous cell line a group of cells with special genetic properties, biochemical properties or specific markers obtained from a primary culture or cell line is called a cell line, and a cell line that can be serially passaged is called a continuous cell line cell line.
  • CPE cytopathic effect, ie cellular degeneration following infection of cultured cells by a virus.
  • CPE cytopathic effect
  • sheep infectious pustular dermatitis virus also known as sheep aphthous virus, aphthous virus, ORFV, orf virus, etc., is a pox virus that can cause contagious, epitheliophilic diseases of sheep and goats.
  • an effective amount refers to an amount effective at the dose and for the time necessary to achieve the desired therapeutic or prophylactic effect.
  • a therapeutically effective amount also refers to an amount in which any toxic or detrimental consequences of the therapeutic agent are outweighed by the therapeutically beneficial effects.
  • PBS is phosphate buffer saline, the abbreviation of phosphate buffered saline.
  • MOI Multiplicity of Infection, which refers to the ratio (pfu/cell) between the number of virus particles and the total number of target cells during virus infection of cells.
  • POV-601-1A1 virus strain “POV” is the abbreviation of "Prajna Oncolytic Virus", which means that the virus strain originated from Suzhou Prajna Biotechnology Co., Ltd.; in the internal experimental records of Suzhou Prajna Biotechnology Co., Ltd. , POV-601-1A1, v601-1A1, v601-p0-1A1 and 1A1, all represent the same strain of virus.
  • the virus strain is named "mutant type of ovine infectious pustular dermatitis virus POV-601-1A1" in the CCTCC's deposit registration table, which belongs to the ovine infectious pustular dermatitis virus in the genus Parapoxvirus of the Pox family.
  • POV-601-3F8 virus strain “POV” stands for "Prajna Oncolytic Virus", which means that the virus strain originated from Suzhou Prajna Biotechnology Co., Ltd.; in the internal records of Suzhou Prajna Biotechnology Co., Ltd., POV-601- 3F8, v601-3F8, v601-p0-3F8 and 3F8 all represent the same strain of virus.
  • POV-604-1D1 virus strain “POV” stands for "Prajna Oncolytic Virus", which means that the virus strain originated from Suzhou Prajna Biotechnology Co., Ltd.; in the internal records of Suzhou Prajna Biotechnology Co., Ltd., POV-604- 1D1, v604-1D1 and 1D1 all represent the same strain of virus.
  • virus storage buffer the preparation method is to draw 500 mL of PBS (ie, phosphate buffer, CORNING, product number: 21-040-CVR), add 1.25 mL of 1M MgCl 2 ⁇ 6H 2 O (Sangon Bioengineering (Shanghai) Co., Ltd.
  • the present invention relates to a (native) CBP protein.
  • the (native) CBP protein has or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical full-length sequence or having or comprising as shown in SEQ ID NO: 2 or from the same or similar organism origin (eg, strain, species, genus, family) and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to it the mature sequence of, or (essentially) consist of.
  • the (native) CBP protein has or comprises as set forth in SEQ ID NOs: 31-42 or is derived from the same or similar biological source (e.g. strain, species, genus, family) and has at least 90%, Full-length sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, and corresponding mature sequences.
  • the present invention also relates to a mutant CBP protein.
  • a mutation is an addition, deletion or substitution of one or more amino acid residues, or any combination thereof, relative to the native sequence.
  • the function of the mutant CBP protein is reduced or absent.
  • the present invention also relates to the reduction or deletion of the expression of CBP proteins (eg, native CBP proteins or mutant CBP proteins).
  • CBP proteins eg, native CBP proteins or mutant CBP proteins.
  • a reduction is at least a 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% reduction compared to native function and/or expression , 97%, 98%, 99%, up to 100%.
  • the present invention relates to a (native) ORFV112 gene.
  • the (native) ORFV112 gene encoding has or comprises at least 90%, 91% as set forth in SEQ ID NO: 1 or from the same or similar biological source (e.g.
  • the (native) CBP protein has or comprises as set forth in SEQ ID NOs: 31-42 or is derived from the same or similar biological source (e.g. strain, species, genus, family) and has at least 90%, Full-length sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, and corresponding mature sequences.
  • the present invention relates to a (native) ORFV112 gene
  • the (native) ORFV112 gene has or comprises as shown in SEQ ID NO: 3 (or its coding region), encodes the same amino acid sequence, or is derived from the same or Approximate biological origin (eg, strain, species, genus, family) with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% therewith , 98%, 99% or 100% identical nucleotide sequences, or (substantially) consist of them.
  • the (native) ORFV112 gene has or comprises, encodes the same amino acid sequence as set forth in SEQ ID NOs: 43-54 (or coding regions thereof), or is derived from the same or similar biological source (e.g., strain, species, genus, family) with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % identity of nucleotide sequences.
  • the present invention also relates to a mutant ORFV112 gene.
  • a mutation is an addition, deletion or substitution of one or more bases, or any combination thereof, relative to the native sequence.
  • the mutant ORFV112 gene is a completely deleted ORFV112 gene.
  • the complete deletion type ORFV112 gene lacks the entire ORFV112 gene.
  • the mutant ORFV112 gene is a partially deleted ORFV112 gene.
  • the partially deleted ORFV112 gene lacks one or more but not all bases of the ORFV112 gene.
  • the partially deleted ORFV112 gene lacks one or more bases of the 5' untranslated region, the coding region and/or the 3' untranslated region, or any combination thereof.
  • the partially deleted ORFV112 gene is a 5'-deleted ORFV112 gene, that is, one or more bases at the 5' end are deleted, eg, the entire or part of the 5' untranslated region is deleted, the entire 5' untranslated region is deleted and all or part of the coding region, or deletion of the entire 5' untranslated region, the entire coding region and all or part of the 3' untranslated region.
  • the ORFV112 gene is deleted upstream (into the ORFV111/112 intergenic region, if present, and/or the ORFV111 gene, particularly the 3' end) and/or downstream (into the ORFV112/113 intergenic region, if If present, and/or the ORFV113 gene, especially the 5' end) extension.
  • the partially deleted ORFV112 gene lacks SEQ ID NO: 3 One or more bases of the sequence shown in ID NO: 3, eg, 1-1161 bases.
  • the native ORFV112 gene has or comprises or (essentially) consists of the nucleotide sequence shown in SEQ ID NO: 3 (or its coding region)
  • the 5'-terminal deletion ORFV112 gene Deletion of one or more bases at the 5' end of the sequence shown in SEQ ID NO: 3, particularly 1-1161, 1-1140, 1-538, 539-1139 or 1141-1160 bases at the 5' end base.
  • the 5'-deleted ORFV112 gene has or comprises, or (essentially) consists of, the nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:57 (or its coding region) .
  • the native ORFV112 gene has or comprises or (essentially) consists of the nucleotide sequence set forth in SEQ ID NOs: 43-54 (or its coding region)
  • the 5'-terminal deletion type The ORFV112 gene deletes one or more bases at the 5' end of the sequences shown in SEQ ID NOs: 43-54, especially one or more of the segments corresponding to 1140 or 538 bases at the 5' end of SEQ ID NO: 3 multiple bases. These deleted genes were unable to express protein due to deletion of the 5' untranslated region and/or start codon.
  • the mutant ORFV112 gene of the invention results in reduced or absent functional CBP protein, including reduced or absent expression and/or activity of CBP protein.
  • a reduction is at least a 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% reduction compared to native function and/or expression , 97%, 98%, 99%, up to 100%.
  • the present invention relates to a (native) hypothetical protein 111.
  • the (native) hypothetical protein 111 has or comprises at least 90%, 91% as set forth in SEQ ID NO: 58 or from the same or similar biological source (e.g. strain, species, genus, family) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, or consist of (essentially).
  • the (native) hypothetical protein 111 has or comprises at least 90% as set forth in SEQ ID NOs: 61-72 or from the same or similar biological source (eg strain, species, genus, family) , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • the present invention also relates to a mutant hypothetical protein 111.
  • a mutation is an addition, deletion or substitution of one or more amino acid residues, or any combination thereof, relative to the native sequence.
  • the function of the mutant hypothetical protein 111 is reduced or absent.
  • the invention also relates to the reduction or deletion of the expression of a hypothetical protein 111 (eg, a native hypothetical protein 111 or a mutant hypothetical protein 111).
  • a hypothetical protein 111 eg, a native hypothetical protein 111 or a mutant hypothetical protein 111.
  • a reduction is at least a 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% reduction compared to native function and/or expression , 97%, 98%, 99%, up to 100%.
  • the present invention relates to a (native) ORFV111 gene.
  • the (native) ORFV111 gene encoding has or comprises as shown in SEQ ID NO: 58 or is from the same or similar biological source (eg strain, species, genus, family) and has at least 90%, 91% therewith , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences, or (essentially) a hypothetical protein 111 consisting of them.
  • the (native) hypothetical protein 111 has or comprises at least 90% as set forth in SEQ ID NOs: 61-72 or from the same or similar biological source (eg strain, species, genus, family) , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • the present invention relates to a (native) ORFV111 gene
  • the (native) ORFV111 gene has or comprises as shown in SEQ ID NO: 59 (or its coding region), encodes the same amino acid sequence, or is derived from the same or Approximate biological origin (eg, strain, species, genus, family) with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% therewith , 98%, 99% or 100% identical nucleotide sequences, or (substantially) consist of them.
  • SEQ ID NO:59 has a full length of 794 bases, wherein the 1-28th base is the 5' non-coding region, the 29-568th base is the protein coding region, and the 569-794th base is the 3' non-coding region. coding area.
  • the (native) ORFV111 gene has or comprises, encodes the same amino acid sequence as set forth in SEQ ID NOs: 73-84 (or coding regions thereof), or is derived from the same or similar biological source (e.g. strain, species, genus, family) with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % identity of nucleotide sequences.
  • the present invention also relates to a mutant ORFV111 gene.
  • a mutation is an addition, deletion or substitution of one or more bases, or any combination thereof, relative to the native sequence.
  • the mutant ORFV111 gene is a complete deletion of the ORFV111 gene.
  • the complete deletion type ORFV111 gene lacks the entire ORFV111 gene.
  • the mutant ORFV111 gene is a partially deleted ORFV111 gene.
  • the partially deleted ORFV111 gene lacks one or more but not all bases of the ORFV111 gene.
  • the partially deleted ORFV111 gene lacks one or more bases of the 3' untranslated region, the coding region and/or the 5' untranslated region, or any combination thereof.
  • the partially deleted ORFV111 gene is a 3'-deleted ORFV111 gene, that is, one or more bases at the 3' end are deleted, eg, the entire or part of the 3' untranslated region is deleted, the entire 3' untranslated region is deleted and all or part of the coding region, or deletion of the entire 3' untranslated region, the entire coding region and all or part of the 5' untranslated region.
  • the ORFV111 gene is deleted downstream (into the ORFV111/112 intergenic region, if present, and/or the ORFV112 gene, particularly the 5' end) and/or upstream (into the ORFV110/111 intergenic region, if If present, and/or the ORFV110 gene, especially the 3' end) extension.
  • the partially deleted ORFV111 gene lacks SEQ ID NO: 59 One or more bases of the sequence set forth in ID NO: 59, eg, 1-793 bases.
  • the native ORFV111 gene has or comprises or (essentially) consists of the nucleotide sequence shown in SEQ ID NO: 59 (or its coding region)
  • the 3'-deleted ORFV111 gene One or more bases at the 3' end of the sequence shown in SEQ ID NO: 59 are deleted, particularly 1-793, 1-309 or 310-792 bases at the 3' end.
  • the 3'-deleted ORFV111 gene has or comprises, or (essentially) consists of, the nucleotide sequence set forth in SEQ ID NO:60 (or its coding region).
  • the native ORFV111 gene has or comprises or (essentially) consists of the nucleotide sequence shown in SEQ ID NO: 73-84 (or its coding region)
  • the 3'-terminal deletion type The ORFV111 gene deletes one or more bases at the 3' end of the sequences shown in SEQ ID NOs: 73-84, especially one or more bases in the segment corresponding to the 309 bases at the 3' end of SEQ ID NO: 59 base. These deleted genes may not express protein due to the deletion of the 3' untranslated region.
  • the mutant ORFV111 gene of the invention results in reduced or absent functional hypothetical protein 111, including reduced or absent expression and/or activity of hypothetical protein 111.
  • a reduction is at least a 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% reduction compared to native function and/or expression , 97%, 98%, 99%, up to 100%.
  • the present invention relates to an ORFV virus genome.
  • the ORFV viral genome has the ORFV112 gene and/or ORFV111 gene described above, including native ORFV112 gene and/or ORFV111 gene and mutant ORFV112 gene and/or ORFV111 gene.
  • the present invention relates to an ORFV virus genome deficient in the ORFV112 gene and/or the ORFV111 gene.
  • the ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome comprises the mutant ORFV112 gene and/or ORFV111 gene described above.
  • the ORFV viral genome (eg, ORFV112 gene and/or ORFV111 gene-deficient ORFV viral genome) is completely deficient in the ORFV007 gene.
  • the 3' non-coding region of the ORFV111 gene coincides with the 5' non-coding region of the ORFV112 gene.
  • the present invention relates to an ORFV virus.
  • the ORFV virus has the CBP protein and/or hypothetical protein 111 described above, including native CBP protein and/or hypothetical protein 111 and mutant CBP protein and/or hypothetical protein 111.
  • the present invention relates to a CBP protein and/or hypothetical protein 111-deficient ORFV virus.
  • the CBP protein and/or the hypothetical protein 111 deficient ORFV virus has reduced or deleted functional CBP protein and/or the hypothetical protein 111.
  • the reduction or deletion of functional CBP protein and/or hypothetical protein 111 can be a reduction or deletion of the expression and/or activity of the CBP protein and/or hypothetical protein 111.
  • the present invention relates to an ORFV virus.
  • the ORFV virus has the ORFV112 gene and/or ORFV111 gene described above, including native ORFV112 gene and/or ORFV111 gene and mutant ORFV112 gene and/or ORFV111 gene.
  • the ORFV virus has the ORFV virus genome described above, including the ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome.
  • the present invention relates to a CBP protein and/or hypothetical protein 111-deficient ORFV virus comprising the above-mentioned mutant ORFV112 gene and/or ORFV111 gene or the above-mentioned ORFV112 gene and/or ORFV111 gene-deficient ORFV virus Genome.
  • an ORFV virus eg, a CBP protein and/or a hypothetical protein 111-deficient ORFV virus
  • the present invention relates to a method for modifying ORFV virus genome or ORFV virus.
  • the method comprises mutating the ORFV112 gene and/or the ORFV111 gene in the ORFV viral genome.
  • the method comprises addition, deletion or substitution, in particular deletion of one or more bases of the ORFV112 gene and/or ORFV111 gene, in particular one or more bases of the 5' end of the ORFV112 gene and/or ORFV111 One or more bases at the 3' end of a gene.
  • the method comprises deleting SEQ ID NO: : One or more bases of the sequence shown in 3, eg, 1-1162 bases.
  • the method comprises deleting SEQ ID NO: : one or more bases at the 5' end of the sequence shown in 3, especially 1-1161, 1-1140, 1-538, 539-1139 or 1141-1160 bases at the 5' end, such as 538 or 1140 bases.
  • the method comprises deleting SEQ ID in its entirety NO:3 or delete the 1140 or 538 5' end bases of SEQ ID NO:3.
  • the method comprises deleting said nucleotide sequences entirely or deleting all The segment corresponding to the 1140 or 538 5' end bases of SEQ ID NO: 3 in the nucleotide sequence.
  • the method comprises deleting SEQ ID NO: 59 : one or more bases of the sequence shown in 59, eg, 1-794 bases.
  • the method comprises deleting SEQ ID NO: 59 : one or more bases at the 3' end of the sequence shown in 59, particularly 1-793, 1-309 or 310-792 bases, eg, 309 bases, at the 3' end.
  • the method comprises deleting SEQ ID in its entirety NO:59 or delete the 309 3' terminal bases of SEQ ID NO:59.
  • the method comprises deleting said nucleotide sequences entirely or deleting all The segment corresponding to the 309 3'-terminal bases of SEQ ID NO: 59 in the nucleotide sequence.
  • the method comprises the complete deletion of SEQ ID NO: 3 and SEQ ID NO:59. In one embodiment, the method further comprises mutating (eg, deletion, including complete deletion or partial deletion) of the ORFV007 gene in the ORFV virus genome, particularly the ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome. In one embodiment, the 3' non-coding region of the ORFV111 gene coincides with the 5' non-coding region of the ORFV112 gene.
  • the methods of the present invention can be performed by conventional molecular biology techniques (eg molecular cloning, DNA recombination, homologous recombination, PCR, restriction nucleases, gene knockout, silencing, etc.) or emerging molecular biology techniques (eg gene editing, etc.) .
  • the present invention relates to ORFV virus genomes (eg ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genomes) and ORFV viruses (eg CBP protein and/or hypothetical protein 111-deficient ORFV virus) obtained by the method.
  • the present invention relates to a method for identifying ORFV virus genome or ORFV virus, in particular for detecting (presence or activity) and/or genes of CBP protein and/or hypothetical protein 111 (especially mutant CBP protein and/or hypothetical protein 111) Methods for the (presence or sequence) of ORFV112 and/or ORFV111 genes, especially mutant ORFV112 genes and/or ORFV111 genes.
  • the methods of the present invention can be performed by conventional molecular biology techniques (eg, activity assays, hybridization (eg, Southern, Northern, or Western)), which detect the presence and/or activity of proteins and/or nucleic acids and/or sequences, Restriction nucleases, PCR, electrophoresis (eg, gel electrophoresis, including protein electrophoresis and nucleic acid electrophoresis, including agarose electrophoresis and PAGE electrophoresis, including reducing and non-reducing electrophoresis), sequencing (including protein sequencing and nucleic acid sequencing), etc.) or Emerging molecular biology techniques such as next-generation sequencing, etc.
  • molecular biology techniques eg, activity assays, hybridization (eg, Southern, Northern, or Western)
  • electrophoresis eg, gel electrophoresis, including protein electrophoresis and nucleic acid electrophoresis, including agarose electrophoresis and PAGE electrophoresis, including
  • the present invention provides methods for detecting the integrity (or length) of the genes ORFV112 and/or ORFV111.
  • the method is performed by PCR and gel electrophoresis.
  • the method can be performed by nucleic acid hybridization or sequencing.
  • the target region of the upstream primer can be, but is not limited to, be positioned at the 5' end of the gene ORFV112 or extend upstream, such as into the ORFV111/112 intergenic region, if any, or
  • the gene ORFV111 (especially the 3' end of the gene ORFV111, such as the 3' untranslated region);
  • the target region of the downstream primer can be set at the 3' end of the gene ORFV112 or extended downstream, such as into the ORFV112/113 intergene region, if any, or the gene ORFV113 (especially the 5' end of the gene ORFV113, eg, the 5' untranslated region).
  • primer/probe targets can be designed based on similar principles.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a certain amount, particularly an effective amount, such as a therapeutically effective amount or a prophylactically effective amount, of the ORFV virus genome of the present invention (for example, ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome) and /or ORFV virus (eg CBP protein and/or hypothetical protein 111 deficient ORFV virus).
  • the composition comprises a pharmaceutically acceptable carrier.
  • the composition is in the form of a powder, solution, transdermal patch, ointment, or suppository.
  • the composition is administered by intravenous, intratumoral, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal routes.
  • the present invention relates to a method of treating a disease or delaying disease progression in a subject, comprising administering an amount, particularly an effective amount, such as a therapeutically effective amount or a prophylactically effective amount, of an ORFV viral genome of the present invention (such as the ORFV112 gene and /or ORFV111 gene-deficient ORFV virus genome) and/or ORFV virus (eg CBP protein and/or hypothetical protein 111-deficient ORFV virus).
  • an ORFV viral genome of the present invention such as the ORFV112 gene and /or ORFV111 gene-deficient ORFV virus genome
  • ORFV virus eg CBP protein and/or hypothetical protein 111-deficient ORFV virus
  • the present invention relates to an amount, in particular an effective amount, such as a therapeutically effective amount or a prophylactically effective amount, of an ORFV virus genome (eg ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome) and/or ORFV virus (eg CBP protein) of the present invention and/or hypothetical protein 111-deficient ORFV virus) in the preparation of medicaments.
  • an ORFV virus genome eg ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome
  • ORFV virus eg CBP protein
  • the present invention relates to an amount, in particular an effective amount, such as a therapeutically effective amount or a prophylactically effective amount, of an ORFV virus genome (eg ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome) and/or ORFV virus (eg CBP protein) of the present invention and/or hypothetical protein 111-deficient ORFV virus) for use in treating disease or delaying disease progression in a subject.
  • an ORFV virus genome eg ORFV112 gene and/or ORFV111 gene-deficient ORFV virus genome
  • ORFV virus eg CBP protein
  • the disease is cancer.
  • the cancer is a solid tumor.
  • 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.
  • the subject is a mammal, including rodents (eg, mice and rats), non-human primates (eg, cynomolgus monkeys), and humans.
  • rodents eg, mice and rats
  • non-human primates eg, cynomolgus monkeys
  • Example 1 Screening and purification method of mutant sheep infectious pustular dermatitis virus
  • the sorted 96-well plate was transferred to a carbon dioxide incubator (Thermo, 160i) at 37°C with a concentration of 5% CO 2 for cultivation. Observe the virus infection every day;
  • the PCR product was subjected to Sanger Sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.), and it was confirmed that the ORFV112 gene encoding of POV-601-1A1 has 538 bases missing at the 5' end, including 226 non-coding 5' ends. bases and 312 bases of the 5' end of the ORFV112 protein coding region (see SEQ ID NO:4); while POV-601-3F8 has the complete ORFV112 gene sequence (see SEQ ID NO:3).
  • ⁇ PCR program parameters pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 62°C for 30s, extension at 68°C for 1 min, 30 cycles; final extension at 68°C for 7min.
  • Example 2 Species confirmation of mutant ovine infectious pustular dermatitis virus
  • the virus genome was extracted from the POV-601-1A1 strain (Mouse Tail Genomic DNA Kit, Cat#: CW2094S), and sent to a third-party sequencing company for second-generation sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.). Assemble the sequencing results. After the full sequence of the B2L gene [21] (ORFV011 gene) of the virus strain was searched by BLAST nucleic acid sequence, the one with high similarity to the sequencing result was OV/HLJ/04, with a similarity of 99.91%, confirming the isolated virus strain is ORFV.
  • Source of host cell African green monkey kidney cell CV-1 (ATCC No.CCL-70 TM )
  • the construction method of recombinant sheep infectious pustular oncolytic virus mainly includes the following steps:
  • flanking sequences of the ORFV112 gene (left homology arm and right homology) were cloned by PCR method. arm) and EGFP reporter gene;
  • step 3 connecting the PCR product in step 1) with the pUC19 plasmid after digestion to obtain a CBP shuttle plasmid;
  • step 4) Take the CBP shuttle plasmid ligation product obtained in step 3) as a template, adopt specific primers (SEQ ID NOs: 13-14) with EcoRI and AgeI restriction endonuclease sites at the ends respectively, and amplify by PCR method , to obtain a linear CBP shuttle vector;
  • step 5 Use EcoRI and AgeI to digest the new px330 plasmid vector and px330 plasmid vector backbone (px330 backbone) in step 5), and cut the product by T4 DNA ligase ligase; transform the ligated product into the screening host bacterium DH5 ⁇ to obtain a positive single. Cloning, and expanding the culture, and extracting the plasmid. Sanger sequencing was performed to identify the recombinant plasmid (px330- ⁇ NLS) without nuclear localization signal (NLS);
  • step (4) Transfect the obtained px330- ⁇ NLS-CBP gRNA expression plasmid into the selected parapoxvirus host cell CV-1, and then infect the transfected CV-1 cells with POV-601-1A1 virus strain.
  • the linear CBP shuttle vector obtained in step (4) was transfected into the px330- ⁇ NLS-CBP gRNA plasmid and the CV-1 cells infected with POV-601-1A1 virus;
  • the target cells with EGFP fluorescent virus are collected by enrichment culture, and then sorted and inoculated into a 96-well plate by flow cytometer, and 1 cell is inoculated in each well, and the culture is expanded.
  • the target cells were collected from the wells rich in EGFP fluorescent virus, and the viral genomic DNA was extracted and identified by PCR (primers SEQ ID NO: 19-30), and the virus with the target band was re-infected CV-1 cells for expansion culture. ;
  • Example 4 Amplification and culture of mutant sheep infectious pustular dermatitis virus
  • Source of host cell African green monkey kidney cell CV-1 (ATCC No.CCL-70 TM )
  • the virus can be harvested when more than 90% of the virus-infected cells have CPE;
  • Example 5 Evaluation of antitumor activity of mutant ovine infectious pustular dermatitis virus in murine bladder cancer tumor model
  • mice male C57BL/6 mice, 6W, from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice Male C57BL/6 mice were subcutaneously inoculated with MB49 cells resuspended in PBS (Guangzhou Genio Biotechnology Co., Ltd.) at a cell density of 2 ⁇ 10 6 /ml and an inoculation volume of 0.1ml/mice. The day of cell seeding was defined as Day 0. When the average tumor volume in the control group reached about 100 mm 3 , the mice were randomly divided into groups according to the tumor size, and administered according to the description in Table 1. All mice were euthanized on Day 30 after inoculation with cells.
  • mice were weighed and tumors were measured twice a week for the entire study.
  • the tumor volume was measured using a vernier caliper (model: 16ER; Mahr GmbH). The results are shown in Figure 2.
  • Tumor inhibition rate TGI% (1-T/C) ⁇ 100%.
  • Example 6 Evaluation of antitumor activity of mutant sheep infectious pustular dermatitis virus in murine melanoma model
  • mice Female C57BL/6 mice were subcutaneously inoculated with B16-F10 cells resuspended in PBS (Cell Bank of the Type Culture Collection, Chinese Academy of Sciences) at a cell density of 5 ⁇ 10 6 /ml and an inoculation volume of 0.1ml/ml only. The day of cell injection was defined as Day 0. When the average tumor volume in the control group reached about 100 mm 3 , the mice were randomly divided into groups according to the tumor size, and administered according to the description in Table 3. All mice were comforted on Day 15 after inoculation with cells.
  • PBS Cell Bank of the Type Culture Collection, Chinese Academy of Sciences
  • mice were weighed and tumors were measured twice a week for the entire study.
  • the tumor volume was measured using a vernier caliper (model: 16ER; Mahr GmbH). The results are shown in Figure 3.
  • Tumor inhibition rate TGI% (1-T/C) ⁇ 100%.
  • s.c. subcutaneous injection
  • i.v. tail vein injection
  • Test and control articles were injected subcutaneously or tail vein on the right back of female C57BL/6 mice. The date of the first injection was defined as Day 0. Mice were completely randomized and treated according to the description in Table 5. All mice were observed on the 15th day after administration. Mice were weighed daily for the first week and every two to three days thereafter for the entire study.
  • mice Body weight changes were monitored in mice. Animal body weights were measured using an ML1602T electronic balance (Mettler). The relative body weight change rate was calculated using the following equation and the results are shown in Figure 4.
  • Relative body weight change (%) [weight day new / weight day 0 ] x 100.
  • Example 8 Modulating effect of mutant sheep infectious pustular dermatitis virus on immune system
  • i.t. intratumoral injection
  • i.v. tail vein injection
  • mice Female Balb/c-nude mice, 6W, Beijing Huafukang Biotechnology Co., Ltd.
  • mice Female Balb/c-nude mice were subcutaneously inoculated with C-33A cells (Cell Bank of the Type Culture Collection, Chinese Academy of Sciences) resuspended in PBS at a cell density of 1 ⁇ 10 8 /ml and an inoculum volume of 0.1 ml/only. The day of cell injection was defined as Day 0. When the average tumor volume reached about 100 mm 3 , the drug treatment was performed according to the description in Table 6. The experiment was terminated 24 hours after the last administration.
  • C-33A cells Cell Bank of the Type Culture Collection, Chinese Academy of Sciences
  • Example 9 Infectious effect of mutant sheep infectious pustular dermatitis virus on cells in vitro
  • Example 8 Evaluation of antitumor activity of mutant ovine infectious pustular dermatitis virus in murine colon cancer model
  • CT-26 cells resuspended in PBS were subcutaneously inoculated on the right back of female Balb/c mice at a cell density of 5 ⁇ 10 6 /ml and the inoculum volume was 0.1ml/ml only.
  • the day of cell injection was defined as Day 0.
  • the mice were randomly divided into groups according to the tumor size, and administered according to the description in Table 8. All mice were comforted on Day 25 after inoculation with cells.
  • Tumor inhibition rate TGI% (1-T/C) ⁇ 100%.
  • Example 9 Evaluation of antitumor activity of recombinant mutant sheep infectious pustular dermatitis virus in murine melanoma model
  • Transmissible pustular dermatitis virus of sheep with complete or partial deletion of the ORFV111 gene and/or ORFV112 gene coding region and/or non-coding region was constructed by recombinant method, and the effect was tested by the method described in Example 6, unless otherwise specified.
  • v611a Complete deletion of the ORFV112 gene.
  • v615a delete the ORFV111 and ORFV112 coding regions
  • v616a Completely remove ORFV111 and ORFV112.
  • CTCC China Collection of Type Cultures
  • T-VEC talimogene Laherparepvec

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Abstract

提供了一种突变型羊传染性脓疱皮炎病毒(ORFV)及其药用组合物在癌症治疗方面的用途。

Description

一种突变型羊传染性脓疱皮炎病毒及其用途
相关申请
本申请要求2020年8月13日提交、申请号202010813096.9的中国申请的优先权。
技术领域
本发明涉及生物技术领域，具体涉及一种突变型羊传染性脓疱皮炎病毒(ORFV)及其药用组合物在癌症治疗方面的用途。
发明背景
羊传染性脓疱皮炎病毒(简称ORFV)隶属于痘病毒科(Poxviridae)中副痘病毒属(Parapoxvirus)。该属还有牛丘疹性口炎病毒(Bovine papular stomatitis virus)、伪牛痘病毒(pseudocowpox virus)、新西兰红鹿副痘病毒(parapox virus of red deer in New Zealand)、海豹痘病毒(Sealpox)等多种病毒，能引起绵羊和山羊的接触传染性、嗜上皮性的疾病。病毒感染羊后，其病程为首先在羊的口唇、舌、鼻、乳房等部位开始出现红疹，进而发展为丘疹、水疱、脓疱，最后形成结痂，以增生性炎症为典型特征。
专利CN 104017776 B，公开了一种经分离和细胞传代培养后获得的弱毒ORFV，用于疫苗研制。专利CN 108026542 A描述使用病毒株D1701进行重组ORFV载体构建，表达病毒抗原、肿瘤抗原等，用于制备疫苗。
溶瘤病毒疗法(oncolytic viral therapy)是一种选用天然存在或经改造的病毒对肿瘤进行治疗的系统性疗法。那些原本促进肿瘤细胞增殖和存活的特定基因突变，恰恰也可促进具有裂解功能的病毒在其内生长，这即是溶瘤病毒精准攻击肿瘤细胞的原因之一 [1、2、3]。感染癌细胞后的溶瘤病毒能够在病毒生命周期内的适当时间点促使癌细胞裂解、死亡，并释放出感染性病毒颗粒，进一步感染周围尚未感染的癌细胞。同时，癌细胞裂解释放出的新抗原(neoantigen)，激活免疫系统，从而对癌症进行后续打击 [4]。目前全球已有90个以上的临床试验使用溶瘤病毒治疗人类恶性肿瘤，其中T-Vec溶瘤病毒 药物已成功获得FDA批准 [5]。
ORFV病毒为双链DNA病毒，长约134-139kb。病毒粒子长230-280nm，宽150-200nm，呈椭圆形的线团样 [6]。ORFV病毒具有下列特征：1)与同家族的痘病毒科成员相似，ORFV病毒只在宿主细胞的胞质中复制，不进入细胞核，从而不会整合到宿主细胞的基因组中，因此安全性高，致癌性概率极低 [7、10]；2)通过破损皮肤与动物感染区接触，可感染人，在感染部位(一般为手指)产生临床上较轻症状的脓疱，基本无其它不良副反应，一般不会引发致命疾病，6-8周后可自愈 [8、11]；3)感染时不受细胞表面相关受体限制 [9、10]，有望为克服实体瘤的高异质性提供一条途径；4)基因组庞大，有利于进行外源基因的插入和高效表达；5)病毒基因组稳定，复制的保真度高；6)在宿主体内很少或基本不产生中和抗体，可反复多次感染 [11]，实现静脉反复注射；7)可以诱导机体的先天性(Innate)和适应性(Adaptive)抗肿瘤免疫反应 [12]；8)同家族中的痘苗病毒作为抗天花疫苗，已在全球几十亿人中使用，临床应用风险低；9)基于其自身的特性，有将冷(Cold)肿瘤转化为热(Hot)肿瘤的能力 [18]。基于以上的特点，该种属病毒可望作为新型溶瘤病毒，用于实体瘤治疗。
ORFV病毒株一般编码约130-134个基因。目前已确认的毒力因子主要有病毒干扰素抑制蛋白(OVIFNR,ORFV020)、趋化因子结合蛋白(CBP,ORFV112)、GM-CSF/IL-2抑制蛋白(GIF,ORFV117)、病毒白细胞介素10(vIL-10,ORFV127)和血管内皮生长因子类似蛋白(VEGF-like protein,ORFV132)等 [11]。这些基因编码的蛋白也可调节机体的免疫反应 [13]。
基因ORFV002是一种病毒晚期基因，蛋白合成后定位于细胞核内，在细胞核内抑制NF-κB途径，它被鉴定为由ORFV病毒产生的第一个NF-κB核抑制剂 [19]。
基因ORFV005为假设蛋白(Hypothetical protein)基因，作用机制目前尚不明确。
基因ORFV007，长约483bp，编码脱氧尿苷焦磷酸酶(duTPase) [15]。该蛋白是dNTP(脱氧核糖核苷三磷酸)合成中一种重要的酶。一般情况下，正常细胞中的dNTP浓度受到严格调节，因而不利于病毒复制。而在癌细胞中，有较高浓度的dNTP，可促使病毒复制 [16]。ORFV007基因的缺失，使该病毒在正常细胞中的复制受阻，但并不影响其在肿瘤细胞中的扩增，以达到 ORFV病毒选择性地在癌细胞中复制的目的。
基因ORFV111为假设蛋白(Hypothetical protein)基因，作用机制目前尚不明确。
编码CBP蛋白的基因ORFV112(chemokine-binding protein,趋化因子结合蛋白)，长约864bp，功能是抑制免疫细胞的抗病毒机制 [14]。该蛋白在结构和功能上与其它痘病毒的CBP-II蛋白相似，其既能抑制DC细胞向炎症部位的迁移，又能抑制DC细胞激活T细胞。
目前已公开并用于溶瘤病毒研究的ORFV病毒株有NZ-2、NZ-7、D1701、NA1/11等。现有技术中，因ORFV病毒可插入较为庞大的外源DNA片断，一般将其作为载体，插入某种肿瘤特异性抗原或病毒抗原、细胞因子等，获得基因重组病毒，以供研究。
专利WO 2012122649A1中，公开一种重组OFRV病毒(插入牛痘病毒E3L基因)，感染含特定宿主范围基因(SPI-1、K1L、C7L、B5R、p28/N1R、E3L等)的肿瘤细胞，用于癌症药物开发。专利CN 108220251 A中，公开一种重组传染性脓疱溶瘤病毒及其制备方法与应用，主要为敲除NA1/11病毒株的ORFV132(VEGF)基因后，在同位置插入P53-EGFP融合蛋白基因，用于癌症药物开发。以上两专利所陈述的毒株，均未有报道ORFV112基因缺失，也没有报道ORFV007基因缺失。
本文公开一种突变型羊传染性脓疱皮炎病毒，其特征在于基因ORFV112和/或基因ORFV111的功能性表达产物的缺失，其中功能性表达产物的缺失是由编码CBP的基因ORFV 112和/或编码假设蛋白111的基因ORFV111完全缺失造成或部分缺失造成的。本发明人意外发现所述ORFV 112基因和/或ORFV111基因的完全缺失或部分缺失，对病毒的抗肿瘤效果有明显增强的作用。进一步的，所述突变型羊传染性脓疱皮炎病毒的基因组序列与其它所有已公开的ORFV病毒株的基因组序列进行比较，发现所述突变型羊传染性脓疱皮炎病毒的ORFV 007 dUTPase基因出现完全缺失。进一步的，上述比较也证实所述羊传染性脓疱皮炎病毒的ORFV002及ORFV005基因出现完全缺失。
因此，本公开的突变型羊传染性脓疱皮炎病毒对于该种类病毒在抗肿瘤药物开发有指导作用。
发明概述
发明人意外发现，基因ORFV112(编码CBP蛋白)和/或基因ORFV111(编码一种假设蛋白，以下称作假设蛋白111)的完全缺失或部分缺失(特别是基因ORFV112的5’部分缺失和基因ORFV111的3’部分缺失)可增强羊传染性脓疱皮炎病毒的抗肿瘤活性。进一步地，发明人还意外发现，所述突变型羊传染性脓疱皮炎病毒的基因ORFV007完全缺失。功能性CBP蛋白和/或假设蛋白111缺失的羊传染性脓疱皮炎病毒可通过常规分子生物学方法(例如分子克隆、DNA重组、同源重组、PCR、限制性核酸酶、基因敲除、沉默等)或新兴分子生物学方法(例如基因编辑等)获得。
一方面，本发明提供一种突变型羊传染性脓疱皮炎病毒，其特征在于基因ORFV112和/或基因ORFV111的功能性表达产物的缺失。在一个实施方案中，基因ORFV112的功能性表达产物的缺失是由基因ORFV112的完全或部分(例如5’部分或3’部分，尤其是5’部分)缺失造成的。在一个实施方案中，基因ORFV112的表达产物的成熟序列如SEQ ID NO:2所示。在一个实施方案中，基因ORFV112的完整序列如SEQ ID NO:3所示。在一个实施方案中，SEQ ID NO:3完全缺失。在一个实施方案中，SEQ ID NO:3部分缺失，例如缺失1-1161个，1-1140个，1-538个，539-1139个或1141-1160个5’端碱基。在一个实施方案中，部分缺失型基因ORFV112如SEQ ID NO:4或SEQ ID NO:57所示。在一个实施方案中，基因ORFV111的功能性表达产物的缺失是由基因ORFV111的完全或部分(例如5’部分或3’部分，尤其是3’部分)缺失造成的。在一个实施方案中，基因ORFV111的表达产物的序列如SEQ ID NO:58所示。在一个实施方案中，基因ORFV111的完整序列如SEQ ID NO:59所示。在一个实施方案中，SEQ ID NO:59完全缺失。在一个实施方案中，SEQ ID NO:59部分缺失，例如缺失1-793个，1-309个或310-792个3’端碱基。在一个实施方案中，部分缺失型基因ORFV111如SEQ ID NO:60所示。在一个实施方案中，SEQ ID NO:3和SEQ ID NO:59完全缺失。在一个实施方案中，表达产物是蛋白质和/或核酸(特别是功能性核酸)。在一个实施方案中，本发明提供所述病毒的基因组。
一方面，本发明提供一种改造羊传染性脓疱皮炎病毒的方法，其包括降低或消除基因ORFV112和/或基因ORFV111的表达产物的表达和/或活性。在一个实施方案中，该方法包括完全或部分(例如5’部分或3’部分，尤其是5’ 部分)删除基因ORFV112。在一个实施方案中，基因ORFV112的表达产物的成熟序列如SEQ ID NO:2所示。在一个实施方案中，基因ORFV112的完整序列如SEQ ID NO:3所示。在一个实施方案中，该方法包括完全删除SEQ ID NO:3。在一个实施方案中，该方法包括部分删除SEQ ID NO:3，例如删除1-1161个，1-1140个，1-538个，539-1139个或1141-1160个5’端碱基。在一个实施方案中，该方法产生如SEQ ID NO:4或SEQ ID NO:57所示的部分缺失型基因ORFV112。在一个实施方案中，该方法包括完全或部分(例如5’部分或3’部分，尤其是3’部分)删除基因ORFV111。在一个实施方案中，基因ORFV111的表达产物的序列如SEQ ID NO:58所示。在一个实施方案中，基因ORFV111的完整序列如SEQ ID NO:59所示。在一个实施方案中，该方法包括完全删除SEQ ID NO:59。在一个实施方案中，该方法包括部分删除SEQ ID NO:59，例如删除1-793个，1-309个或310-792个3’端碱基。在一个实施方案中，该方法产生如SEQ ID NO:60所示的部分缺失型基因ORFV111。在一个实施方案中，该方法包括完全删除SEQ ID NO:3和SEQ ID NO:59。在一个实施方案中，表达是转录和/或翻译。在一个方面，本发明提供通过所述方法得到的病毒。在一个实施方案中，本发明提供所述病毒的基因组。本发明的方法可通过常规分子生物学技术(例如分子克隆、DNA重组、同源重组、PCR、限制性核酸酶、基因敲除、沉默等)或新兴分子生物学技术(例如基因编辑等)进行。
另一方面，本发明提供一种鉴定所述突变型羊传染性脓疱皮炎病毒和/或其基因组的方法，特别是检测基因ORFV112和/或基因ORFV111(的存在或序列)和/或其表达产物(的存在和/或活性)的方法。本发明的方法可通过检测蛋白质的存在和/或活性和/或核酸的存在和/或序列的常规分子生物学技术(例如活性测定法、杂交(例如Southern杂交、Northern杂交、或Western杂交)、限制性核酸内切酶、PCR、电泳(例如凝胶电泳，包括蛋白质电泳和核酸电泳，包括琼脂糖电泳和PAGE电泳，包括还原电泳和非还原电泳)、测序(包括蛋白质测序和核酸测序)等)或新兴分子生物学技术(例如下一代测序等)进行。本发明尤其提供检测基因ORFV112和/或基因ORFV111完整性的方法。在一个实施方案中，该方法通过PCR和凝胶电泳进行。在一个实施方案中，该方法可通过杂交或测序进行。
另一方面，本发明提供一种使用所述突变型羊传染性脓疱皮炎病毒和/ 或其基因组在受试者中治疗癌症的方法。另一方面，本公开提供所述突变型羊传染性脓疱皮炎病毒和/或其基因组用于在受试者中治疗癌症的用途。另一方面，本公开提供一种所述突变型羊传染性脓疱皮炎病毒和/或其基因组制备用于在受试者中治疗癌症的药物的用途。另一方面，本公开提供所述突变型羊传染性脓疱皮炎病毒和/或其基因组，其用于在受试者中治疗癌症。另一方面，本公开提供所述突变型羊传染性脓疱皮炎病毒和/或其基因组，其用于制备用于在受试者中治疗癌症的药物。在一个实施方案中，所述癌症是实体瘤。在一个实施方案中，所述实体瘤为宫颈癌、膀胱癌、肝癌、卵巢癌、黑色素瘤、结直肠癌、肺癌、乳腺癌、胃癌、子宫癌、头颈癌、甲状腺癌、食道癌、前列腺癌、胰腺癌、肉瘤、脑瘤等。在一个实施方案中，所述受试者是哺乳动物，包括啮齿动物(例如小鼠和大鼠)、非人灵长动物(例如食蟹猴)和人。
附图简述
图1示出使用ORFV112基因特异性正向引物及反向引物(SEQ ID NO:5和6)进行PCR扩增后，PCR产物的琼脂糖凝胶电泳结果。图中1号样品为ORFV112基因缺失病毒株POV-601-1A1，2号样品为ORFV112基因完整病毒株POV-601-3F8，M为DNA大小marker。
图2示出ORFV112基因缺失病毒株POV-601-1A1和POV-604-1D1在MB49鼠源膀胱癌模型上的抑瘤效果。
图3示出ORFV112基因缺失病毒株POV-601-1A1和POV-604-1D1，以及ORFV112基因完整病毒株POV-601-3F8在B16-F10鼠源黑色素瘤肿瘤模型中的抑瘤效果。
图4示出POV-601-1A1病毒在不同剂量及给药途径条件下对小鼠体重的影响。
图5示出POV-601-1A1病毒株给药后，人源C-33A宫颈癌双侧接瘤模型中小鼠免疫系统的变化情况。图5A及图5B中，正方形表示为右侧瘤内给药结果，圆圈表示左侧未给药结果。通过瘤内给药，肿瘤内的CD45 +及NK细胞的活化比例有所提高。图5C示出血液中NK细胞的活化情况，通过静脉给药，NK细胞的活化比例有所提高。
图6示出本发明的基因ORFV112核苷酸完整序列与部分缺失序列实施例 的比对。
图7示出本发明的CBP蛋白完整序列与其它副痘病毒株的CBP蛋白完整序列比对。其中“3F8”表示POV-601-3F8Strain，“B029”表示ORFV Strain B029，“GO”表示ORFV Strain GO，“NA11”表示ORFV Strain NA1/11，“NZ2”表示ORFV Strain NZ2，“NA17”表示ORFV Strain NA17，“OV-SA00”表示ORFV Strain OV-SA00，“OV-IA82”表示ORFV Strain OV-IA82，“SJ1”表示ORFV Strain SJ1，“SY17”表示ORFV Strain SY17，“OV-NH3_12”表示ORFV Strain OV-HN3/12，“NP”表示ORFV Strain NP，“YX”表示ORFV Strain YX。
图8示出本发明的ORFV112基因完整序列与其它副痘病毒株的ORFV112基因完整序列比对。其中“3F8”表示POV-601-3F8Strain，“B029”表示ORFV Strain B029，“GO”表示ORFV Strain GO，“NA11”表示ORFV Strain NA1/11，“NZ2”表示ORFV Strain NZ2，“NA17”表示ORFV Strain NA17，“OV-SA00”表示ORFV Strain OV-SA00，“OV-IA82”表示ORFV Strain OV-IA82，“SJ1”表示ORFV Strain SJ1，“SY17”表示ORFV Strain SY17，“OV-NH3_12”表示ORFV Strain OV-HN3/12，“NP”表示ORFV Strain NP，“YX”表示ORFV Strain YX。
图9示出本发明的基因ORFV111核苷酸完整序列与部分缺失序列实施例的比对。
图10示出本发明的假设蛋白111完整序列与其它副痘病毒株的假设蛋白111完整序列比对。其中“POV-601-3F8”表示POV-601-3F8Strain，“B029”表示ORFV Strain B029，“GO”表示ORFV Strain GO，“NA11”表示ORFV Strain NA1/11，“NZ2”表示ORFV Strain NZ2，“NA17”表示ORFV Strain NA17，“OV-SA00”表示ORFV Strain OV-SA00，“OV-IA82”表示ORFV Strain OV-IA82，“SJ1”表示ORFV Strain SJ1，“SY17”表示ORFV Strain SY17，“OV-NH3_12”表示ORFV Strain OV-HN3/12，“NP”表示ORFV Strain NP，“YX”表示ORFV Strain YX。
图11示出本发明的ORFV111基因完整序列与其它副痘病毒株的ORFV111基因完整序列比对。其中“POV-601-3F8”表示POV-601-3F8 Strain，“B029”表示ORFV Strain B029，“GO”表示ORFV Strain GO，“NA11”表示ORFV Strain NA1/11，“NZ2”表示ORFV Strain NZ2，“NA17”表示ORFV Strain NA17，“OV-SA00”表示ORFV Strain OV-SA00，“OV-IA82”表示ORFV Strain OV-IA82，“SJ1”表示ORFV Strain SJ1，“SY17”表示ORFV Strain SY17， “OV-NH3_12”表示ORFV Strain OV-HN3/12，“NP”表示ORFV Strain NP，“YX”表示ORFV Strain YX。
图12示出本发明的基因ORFV111氨基酸完整序列与部分缺失序列实施例的比对
图13示出本发明的基因ORFV112氨基酸完整序列与部分缺失序列实施例的比对
图14示出ORFV112基因缺失病毒株POV-601-1A1以及ORFV112基因完整病毒株POV-601-3F8在CT-26鼠源结肠癌肿瘤模型中的抑瘤效果。
图15示出POV-601-1A1以及构建体v611a在B16-F10鼠源黑色素瘤肿瘤模型中的抑瘤效果。
图16示出POV-601-1A1以及构建体v615a和v616a在B16-F10鼠源黑色素瘤肿瘤模型中的抑瘤效果。
图17示出POV-601-1A1以及构建体v617a和v618a在B16-F10鼠源黑色素瘤肿瘤模型中的抑瘤效果。
发明详述
本发明公开的是一种突变型羊传染性脓疱皮炎病毒(ORFV)及其药用组合物在癌症治疗方面的用途。
根据文献报道，野生型羊口疮羊传染性脓疱皮炎病毒的毒力基因主要有OVIFNR(orf virus interferon resistance gene，干扰素抑制蛋白)、CBP(chemokine-binding protein，趋化因子结合蛋白)、GIF(GM-CSF/IL-2 inhibitory protein，GM-CSF/IL-2抑制蛋白)、vIL-10(viral interleukin 10，病毒白细胞介素10)和VEGF-like protein(vascular endothelial growth factor like protein，血管内皮生长因子类似蛋白)等 [11,13]。一般可通过使用分子和/或细胞生物学方法，除去该病毒的某个毒力因子基因，从而获得弱毒株。如在专利CN 104878043 B中，公开一种缺失掉羊口疮病毒株SHZ1的毒力基因OVIFNR，以实现其毒力快速降低，从而获得弱毒株的方法，用于制备弱毒疫苗。
本发明公开的突变型羊传染性脓疱皮炎病毒，通过对在中国典型培养物保藏中心(CCTCC)保藏(保藏号：V201713)的原始POV-601病毒株进行改性筛选而获得。
发明人根据文献 [17]，设计了ORFV112基因的特异性引物。正向引物(SEQ ID NO:5)设计在ORFV111号基因编码区上，反向引物(SEQ ID NO:6)设计在ORFV112号基因编码区上，以此建立了一种以PCR为基础的分子生物学鉴定技术，以鉴定POV-601弱毒株ORFV112基因的完整性。随后，将原始POV-601病毒株感染非洲绿猴肾细胞CV-1，收集病毒感染后的细胞，采用流式细胞仪进行单细胞分选及培养扩增。使用上述ORFV112基因特异性引物对，从已扩增的、经病毒感染的细胞中提取病毒基因组DNA，通过已建立的分子生物学鉴定技术，获得了所述ORFV112基因部分缺失的ORFV病毒弱毒株，命名为POV-601-1A1。该毒株的ORFV112基因编码区序列中5’端缺失了312个碱基，尚存留552个碱基，3’端非编码区72个碱基存留，共计存留624个碱基，见SEQ ID NO:4。同时，也获得了ORFV112基因完整的病毒株POV-601-3F8，其编码区及非编码区共计1162个碱基，其ORFV112基因完整序列见SEQ ID NO:3。
对所述POV-601-1A1病毒弱毒株进行动物肿瘤药效模型评价时，意外发现所述病毒株具有较ORFV112基因完整病毒株POV-601-3F8更好的抗肿瘤效果。
另一方面，本发明人对所述POV-601-1A1病毒株进一步人为使用基因编辑手段，试图将病毒株的基因ORFV112剩余编码区全部敲除。在实施例中，基因ORFV112的剩余编码区被全部敲除(仅保留3’端非编码区22个碱基，见序列SEQ ID NO:57)，并用DNA测序加以证实。以此获得的病毒株被命名为POV-604-1D1。在进行动物肿瘤药效模型评价时，意外发现该弱毒株也较ORFV112基因完整的病毒株POV-601-3F8有更好的抗肿瘤效果。进一步地，任何导致CBP蛋白无法表达的方法，例如将基因ORFV112的编码区及非编码区的某些序列敲除或改变，也可获得同样的效果。
POV-601-1A1病毒株于2020年05月19日在中国典型培养物保藏中心(CCTCC)(武汉)按照布达佩斯条约规定进行保藏，CCTCC保藏编号为V202029。
在实施例中，本公开的突变型羊口疮羊传染性脓疱皮炎病毒能够选择性地感染黑色素瘤细胞(B16-F10)、膀胱癌细胞(MB49)、肝癌细胞(Hepa1-6)、结肠癌细胞(CT26)、人源宫颈癌细胞(C-33A)、人源卵巢癌细胞(SK-OV-3)等，并在其中进行复制。
在实施例中，本公开的突变型羊口疮羊传染性脓疱皮炎病毒的基因ORFV007被意外发现完全缺失，而所有已在NCBI中公布的羊口疮羊传染性脓疱皮炎病毒毒株，比如NZ-2、NZ-7、D1701、NA1/11株等，基因ORFV007都是完整的。ORFV007基因编码脱氧尿苷焦磷酸酶(duTPase)。该蛋白是dNTP合成中一种重要的酶。一般情况下，正常细胞中的dNTP浓度受到严格调节，因而不利于病毒复制。而在癌细胞中，有较高浓度的dNTP，可促使病毒复制 [16]。ORFV007基因的缺失，使该病毒在正常细胞中的复制受阻，但并不影响其在肿瘤细胞中的扩增，以达到ORFV病毒选择性地在癌细胞中复制的目的。
野生型ORFV病毒一般可在牛、羊类动物组织原代细胞(CN 103952377 A)中进行培养，在专利WO2012122649A1中首次公开该病毒可在人宫颈癌细胞Hela中进行增殖扩大培养。进一步地，本发明公开一种所述突变型羊口疮羊传染性脓疱皮炎病毒的制备方法，使用哺乳类细胞系作为宿主细胞，优选地，使用非洲绿猴肾细胞CV-1作为宿主细胞，用于病毒的感染扩增。
进一步地，提供一种上述病毒在个体癌症治疗方面的应用。所述癌症是任何实体瘤癌症。所述的实体瘤癌症类型包含：宫颈癌、膀胱癌、肝癌、卵巢癌、黑色素瘤、结直肠癌、肺癌、乳腺癌、胃癌、子宫癌、头颈癌、甲状腺癌、食道癌、前列腺癌、胰腺癌、肉瘤、脑瘤等。更进一步地，所述个体为哺乳类动物，包括啮齿类动物和人类。
溶瘤病毒抑制肿瘤生长的机制，一般可分为：1)在感染后的肿瘤细胞内复制扩增，裂解癌细胞，达到溶瘤的目的；2)溶瘤后，将癌细胞中储存的特异信息(比如肿瘤新抗原)释放出来，激活免疫系统，从而对余留的癌细胞进行系统攻击。
在实施例中，本公开的突变型羊口疮羊传染性脓疱皮炎病毒可以有效地激活以NK细胞为代表的自主免疫系统。在双侧接种肿瘤的人源C-33A宫颈癌细胞模型中，通过瘤内注射和/或静脉注射病毒，可增加肿瘤和/或血液内的CD45 +和NK细胞活化比例。
I.通用技术
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、病毒学、微生物学、细胞生物学、生物化学、和免疫学的常规技术，这些都在本领域的技术范围内。
II.定义
●术语“OV”是Oncolytic virus，即溶瘤病毒的简称。
●术语“实体瘤”。是指由多个细胞组成的肿瘤实体，用以区分血液瘤；可以是宫颈癌、膀胱癌、肝癌、卵巢癌、黑色素瘤、结直肠癌、肺癌、乳腺癌、胃癌、子宫癌、头颈癌、甲状腺癌、食道癌、前列腺癌、胰腺癌、肉瘤、脑瘤等。癌症可以是早期或晚期阶段。
●术语“连续细胞株”：从原代培养物或细胞系中获得的具有特殊遗传特性、生化性质或特异标记的细胞群称为细胞株(Cell line)，可以连续传代的细胞株称为连续细胞株。
●术语“CPE”：为细胞病变效应(cytopathic effect)，即病毒对培养细胞侵染后产生的细胞变性。在体外实验中，通过细胞培养和接种杀细胞性病毒，经过一定时间后，可用显微镜观察到细胞变圆，坏死，从瓶壁脱落等现象，称之细胞病变效应。
●术语“羊传染性脓疱皮炎病毒”：又称羊口疮病毒、口疮病毒、ORFV、orf virus等，是一种能引起绵羊和山羊的接触传染性、嗜上皮性疾病的痘科病毒。
●术语“有效量”：指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。治疗有效量还指治疗剂的治疗有益效果胜过任何有毒或有害后果的量。
●术语“PBS”：是phosphate buffer saline，即磷酸盐缓冲盐水的简称。
●术语“MOI”：为感染复数(Multiplicity of Infection)，指在病毒感染细胞过程中，病毒颗粒数和靶细胞总细胞数之间的比率(pfu/cell)。
●术语：“POV-601-1A1病毒株”：其中“POV”是“Prajna Oncolytic Virus”的缩写，代表该病毒株源自苏州般若生物科技有限公司；在苏州般若生物科技有限公司内部实验记录中，POV-601-1A1、v601-1A1、v601-p0-1A1和1A1，均代表这同一株病毒。该病毒株在CCTCC的保藏登记表中名称为“突变型羊传染性脓疱皮炎病毒POV-601-1A1”，属于痘科病毒副痘病毒属中的羊传染性脓疱皮炎病毒。
●术语“POV-601-3F8病毒株”：“POV”代表“Prajna Oncolytic Virus”，代表该病毒株源自苏州般若生物科技有限公司；在苏州般若生物科技有限公司内部记录中，POV-601-3F8、v601-3F8、v601-p0-3F8和3F8，均代表这同一 株病毒。
●术语“POV-604-1D1病毒株”：“POV”代表“Prajna Oncolytic Virus”，代表该病毒株源自苏州般若生物科技有限公司；在苏州般若生物科技有限公司内部记录中，POV-604-1D1、v604-1D1和1D1，均代表这同一株病毒。
●术语“病毒储存缓冲液”：配制方法为吸取500mL PBS(即磷酸盐缓冲液，CORNING，货号：21-040-CVR)，加入1.25mL 1M MgCl 2·6H 2O(生工生物工程(上海)股份有限公司，货号：A610328-0500)，使MgCl 2·6H 2O终浓度为2.48mM，接着加入2.5mL 1M Tris-HCl(pH9.0)(生工生物工程(上海)股份有限公司，货号：B548128-0500)，使Tris-HCl终浓度为4.96mM，混合均匀，即得病毒储存缓冲液(MgCl 2·6H 2O理论终浓度为2.5mM，Tris-HCl理论终浓度为5mM)。
III.组合物和方法
1.ORFV112基因的表达产物，CBP蛋白
本发明涉及一种(天然)CBP蛋白。在一个实施方案中，(天然)CBP蛋白具有或包含如SEQ ID NO:1所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的全长序列或具有或包含如SEQ ID NO:2所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的成熟序列，或(本质上)由其组成。在另一个实施方案中，(天然)CBP蛋白具有或包含如SEQ ID NO:31-42所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的全长序列，以及相应的成熟序列。
本发明还涉及一种突变型CBP蛋白。在一个实施方案中，突变是相对于天然序列而言一个或多个氨基酸残基的添加、删除或替代，或其任意组合。在一个实施方案中，突变型CBP蛋白的功能降低或缺失。
本发明还涉及CBP蛋白(例如天然CBP蛋白或突变型CBP蛋白)表达的降低或缺失。
例如，降低是与天然功能和/或表达相比降低至少50％、60％、70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％，直至100％。
2.ORFV112基因
本发明涉及一种(天然)ORFV112基因。在一个实施方案中，(天然)ORFV112基因编码具有或包含如SEQ ID NO:1所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的全长序列或具有或包含如SEQ ID NO:2所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的成熟序列，或(本质上)由其组成的CBP蛋白。在另一个实施方案中，(天然)CBP蛋白具有或包含如SEQ ID NO:31-42所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的全长序列，以及相应的成熟序列。
本发明涉及一种(天然)ORFV112基因，在一个实施方案中，(天然)ORFV112基因具有或包含如SEQ ID NO:3所示(或其编码区)、与其编码相同氨基酸序列、或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的核苷酸序列，或(本质上)由其组成。SEQ ID NO:3全长1162个碱基，其中第1-226位碱基为5’非编码区，第227-1090位碱基为蛋白编码区，第1091-1162位碱基为3’非编码区。在另一个实施方案中，(天然)ORFV112基因具有或包含如SEQ ID NO:43-54所示(或其编码区)、与其编码相同氨基酸序列、或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的核苷酸序列。
本发明还涉及一种突变型ORFV112基因。在一个实施方案中，突变是相对于天然序列而言一个或多个碱基的添加、删除或替代，或其任意组合。在一个实施方案中，突变型ORFV112基因是完全缺失型ORFV112基因。完全缺失型ORFV112基因缺失整个ORFV112基因。在一个实施方案中，突变型ORFV112基因是部分缺失型ORFV112基因。部分缺失型ORFV112基因缺失ORFV112基因的一个或多个但非所有碱基。在一个实施方案中，部分缺失型ORFV112基因缺失5’非翻译区、编码区和/或3’非翻译区的一个或多个碱基，或其任意组合。在一个实施方案中，部分缺失型ORFV112基因是5’端缺失型 ORFV112基因，即缺失5’端的一个或多个碱基，例如缺失整个或部分5’非翻译区、缺失整个5’非翻译区和整个或部分编码区、或缺失整个5’非翻译区、整个编码区和整个或部分3’非翻译区。在一个实施方案中，ORFV112基因缺失向上游(进入ORFV111/112基因间区，如果存在的话，和/或ORFV111基因，特别是3’端)和/或下游(进入ORFV112/113基因间区，如果存在的话，和/或ORFV113基因，特别是5’端)延伸。
在天然ORFV112基因具有或包含如SEQ ID NO:3所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，部分缺失型ORFV112基因缺失SEQ ID NO:3所示序列的一个或多个碱基，例如1-1161个碱基。在天然ORFV112基因具有或包含如SEQ ID NO:3所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，5’端缺失型ORFV112基因缺失SEQ ID NO:3所示序列的5’端的一个或多个碱基，特别是5’端的1-1161个，1-1140个，1-538个，539-1139个或1141-1160个碱基。在一个实施方案中，5’端缺失型ORFV112基因具有或包含如SEQ ID NO:4或SEQ ID NO:57所示的核苷酸序列(或其编码区)，或(本质上)由其组成。在天然ORFV112基因具有或包含如SEQ ID NO:43-54所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，5’端缺失型ORFV112基因缺失SEQ ID NO:43-54所示序列的5’端的一个或多个碱基，特别是与SEQ ID NO:3的5’端1140个或538个碱基对应的区段的一个或多个碱基。由于缺失5’非翻译区和/或起始密码子，这些缺失型基因无法表达蛋白。
在一个实施方案中，本发明的突变型ORFV112基因导致功能性CBP蛋白的降低或缺失，包括CBP蛋白的表达和/或活性的降低或缺失。例如，降低是与天然功能和/或表达相比降低至少50％、60％、70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％，直至100％。
3.ORFV111基因的表达产物，假设蛋白111
本发明涉及一种(天然)假设蛋白111。在一个实施方案中，(天然)假设蛋白111具有或包含如SEQ ID NO:58所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列，或(本质上)由其组成。在另一个实施方案中，(天然)假设蛋白111具有或包含如SEQ ID NO:61-72所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、 91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
本发明还涉及一种突变型假设蛋白111。在一个实施方案中，突变是相对于天然序列而言一个或多个氨基酸残基的添加、删除或替代，或其任意组合。在一个实施方案中，突变型假设蛋白111的功能降低或缺失。
本发明还涉及假设蛋白111(例如天然假设蛋白111或突变型假设蛋白111)表达的降低或缺失。
例如，降低是与天然功能和/或表达相比降低至少50％、60％、70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％，直至100％。
4.ORFV111基因
本发明涉及一种(天然)ORFV111基因。在一个实施方案中，(天然)ORFV111基因编码具有或包含如SEQ ID NO:58所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列，或(本质上)由其组成的假设蛋白111。在另一个实施方案中，(天然)假设蛋白111具有或包含如SEQ ID NO:61-72所示或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
本发明涉及一种(天然)ORFV111基因，在一个实施方案中，(天然)ORFV111基因具有或包含如SEQ ID NO:59所示(或其编码区)、与其编码相同氨基酸序列、或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的核苷酸序列，或(本质上)由其组成。SEQ ID NO:59全长794个碱基，其中第1-28位碱基为5’非编码区，第29-568位碱基为蛋白编码区，第569-794位碱基为3’非编码区。在另一个实施方案中，(天然)ORFV111基因具有或包含如SEQ ID NO:73-84所示(或其编码区)、与其编码相同氨基酸序列、或来自相同或近似生物学来源(例如株、种、属、科)且与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的核苷酸序列。
本发明还涉及一种突变型ORFV111基因。在一个实施方案中，突变是相 对于天然序列而言一个或多个碱基的添加、删除或替代，或其任意组合。在一个实施方案中，突变型ORFV111基因是完全缺失型ORFV111基因。完全缺失型ORFV111基因缺失整个ORFV111基因。在一个实施方案中，突变型ORFV111基因是部分缺失型ORFV111基因。部分缺失型ORFV111基因缺失ORFV111基因的一个或多个但非所有碱基。在一个实施方案中，部分缺失型ORFV111基因缺失3’非翻译区、编码区和/或5’非翻译区的一个或多个碱基，或其任意组合。在一个实施方案中，部分缺失型ORFV111基因是3’端缺失型ORFV111基因，即缺失3’端的一个或多个碱基，例如缺失整个或部分3’非翻译区、缺失整个3’非翻译区和整个或部分编码区、或缺失整个3’非翻译区、整个编码区和整个或部分5’非翻译区。在一个实施方案中，ORFV111基因缺失向下游(进入ORFV111/112基因间区，如果存在的话，和/或ORFV112基因，特别是5’端)和/或上游(进入ORFV110/111基因间区，如果存在的话，和/或ORFV110基因，特别是3’端)延伸。
在天然ORFV111基因具有或包含如SEQ ID NO:59所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，部分缺失型ORFV111基因缺失SEQ ID NO:59所示序列的一个或多个碱基，例如1-793个碱基。在天然ORFV111基因具有或包含如SEQ ID NO:59所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，3’端缺失型ORFV111基因缺失SEQ ID NO:59所示序列的3’端的一个或多个碱基，特别是3’端的1-793个，1-309个或310-792个碱基。在一个实施方案中，3’端缺失型ORFV111基因具有或包含如SEQ ID NO:60所示的核苷酸序列(或其编码区)，或(本质上)由其组成。在天然ORFV111基因具有或包含如SEQ ID NO:73-84所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，3’端缺失型ORFV111基因缺失SEQ ID NO:73-84所示序列的3’端的一个或多个碱基，特别是与SEQ ID NO:59的3’端309个碱基对应的区段的一个或多个碱基。由于缺失3’非翻译区，这些缺失型基因可能无法表达蛋白。
在一个实施方案中，本发明的突变型ORFV111基因导致功能性假设蛋白111的降低或缺失，包括假设蛋白111的表达和/或活性的降低或缺失。例如，降低是与天然功能和/或表达相比降低至少50％、60％、70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％，直至100％。
5.ORFV病毒基因组
本发明涉及一种ORFV病毒基因组。在一个实施方案中，ORFV病毒基因组具有上文所述ORFV112基因和/或ORFV111基因，包括天然ORFV112基因和/或ORFV111基因和突变型ORFV112基因和/或ORFV111基因。本发明尤其涉及一种ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组。在一个实施方案中，ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组包含上文所述突变型ORFV112基因和/或ORFV111基因。在一个实施方案中，ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)完全缺失ORFV007基因。在一个实施方案中，ORFV111基因的3’非编码区与ORFV112基因的5’非编码区重合。
6.ORFV病毒
本发明涉及一种ORFV病毒。在一个实施方案中，ORFV病毒具有上文所述CBP蛋白和/或假设蛋白111，包括天然CBP蛋白和/或假设蛋白111和突变型CBP蛋白和/或假设蛋白111。本发明尤其涉及一种CBP蛋白和/或假设蛋白111缺陷型ORFV病毒。在一个实施方案中，CBP蛋白和/或假设蛋白111缺陷型ORFV病毒降低或缺失功能性CBP蛋白和/或假设蛋白111。功能性CBP蛋白和/或假设蛋白111的降低或缺失可以是CBP蛋白和/或假设蛋白111的表达和/或活性的降低或缺失。
本发明涉及一种ORFV病毒。在一个实施方案中，ORFV病毒具有上文所述ORFV112基因和/或ORFV111基因，包括天然ORFV112基因和/或ORFV111基因和突变型ORFV112基因和/或ORFV111基因。在一个实施方案中，ORFV病毒具有上文所述ORFV病毒基因组，包括ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组。本发明尤其涉及一种CBP蛋白和/或假设蛋白111缺陷型ORFV病毒，其包含上文所述突变型ORFV112基因和/或ORFV111基因或上文所述ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组。
在一个实施方案中，ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)缺失基因ORFV007的功能性转录和/或翻译产物，包括表达和/或活性的缺失。
7.改造ORFV病毒的方法
本发明涉及一种改造ORFV病毒基因组或ORFV病毒的方法。在一个实 施方案中，该方法包括突变ORFV病毒基因组中的ORFV112基因和/或ORFV111基因。在一个实施方案，该方法包括添加、删除或替代，特别是删除ORFV112基因和/或ORFV111基因的一个或多个碱基，特别是ORFV112基因的5’端的一个或多个碱基和/或ORFV111基因的3’端的一个或多个碱基。在天然ORFV112基因具有或包含如SEQ ID NO:3所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括删除SEQ ID NO:3所示序列的一个或多个碱基，例如1-1162个碱基。在天然ORFV112基因具有或包含如SEQ ID NO:3所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括删除SEQ ID NO:3所示序列的5’端的一个或多个碱基，特别是5’端的1-1161个，1-1140个，1-538个，539-1139个或1141-1160个碱基，例如538个或1140个碱基。在天然ORFV112基因具有或包含如SEQ ID NO:3所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括完整删除SEQ ID NO:3或删除SEQ ID NO:3的1140个或538个5’端碱基。在天然ORFV112基因具有或包含其它核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括完整删除所述核苷酸序列或删除所述核苷酸序列中与SEQ ID NO:3的1140个或538个5’端碱基对应的区段。在天然ORFV111基因具有或包含如SEQ ID NO:59所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括删除SEQ ID NO:59所示序列的一个或多个碱基，例如1-794碱基。在天然ORFV111基因具有或包含如SEQ ID NO:59所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括删除SEQ ID NO:59所示序列的3’端的一个或多个碱基，特别是3’端的1-793个，1-309个或310-792个碱基，例如309个碱基。在天然ORFV111基因具有或包含如SEQ ID NO:59所示核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括完整删除SEQ ID NO:59或删除SEQ ID NO:59的309个3’端碱基。在天然ORFV111基因具有或包含其它核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中，该方法包括完整删除所述核苷酸序列或删除所述核苷酸序列中与SEQ ID NO:59的309个3’端碱基对应的区段。在天然ORFV111基因和天然ORFV112基因具有或包含其它核苷酸序列(或其编码区)或(本质上)由其组成的情况中，在一个实施方案中， 该方法包括完整删除SEQ ID NO:3和SEQ ID NO:59。在一个实施方案中，该方法还包括突变(例如删除，包括完全删除或部分删除)ORFV病毒基因组(特别是ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)中的ORFV007基因。在一个实施方案中，ORFV111基因的3’非编码区与ORFV112基因的5’非编码区重合。本发明的方法可通过常规分子生物学技术(例如分子克隆、DNA重组、同源重组、PCR、限制性核酸酶、基因敲除、沉默等)或新兴分子生物学技术(例如基因编辑等)进行。本发明涉及通过所述方法得到的ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)和ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)。
8.鉴定ORFV病毒基因组和ORFV病毒的方法
本发明涉及一种鉴定ORFV病毒基因组或ORFV病毒的方法，特别是检测CBP蛋白和/或假设蛋白111(尤其是突变型CBP蛋白和/或假设蛋白111)(的存在或活性)和/或基因ORFV112和/或ORFV111基因(尤其是突变型ORFV112基因和/或ORFV111基因)(的存在或序列)的方法。本发明的方法可通过检测蛋白质的存在和/或活性和/或核酸的存在和/或序列的常规分子生物学技术(例如活性测定法、杂交(例如Southern杂交、Northern杂交、或Western杂交)、限制性核酸酶、PCR、电泳(例如凝胶电泳，包括蛋白质电泳和核酸电泳，包括琼脂糖电泳和PAGE电泳，包括还原电泳和非还原电泳)、测序(包括蛋白质测序和核酸测序)等)或新兴分子生物学技术(例如下一代测序等)进行。本发明尤其提供检测基因ORFV112和/或ORFV111完整性(或长度)的方法。在一个实施方案中，该方法通过PCR和凝胶电泳进行。在一个实施方案中，该方法可通过核酸杂交或测序进行。
引物和探针的设计在本领域技术人员能力范围之内。本领域技术人员清楚如何选择引物和探针在目标序列上的靶区。例如，为了检测基因ORFV112的完整性，一方面，上游引物的靶区可以但不限于设置在基因ORFV112的5’端或向上游延伸，例如进入ORFV111/112基因间区，如果有的话，或者基因ORFV111(特别是基因ORFV111的3’端，例如3’非翻译区)；另一方面，下游引物的靶区可以设置在基因ORFV112的3’端或向下游延伸，例如进入ORFV112/113基因间区，如果有的话，或者基因ORFV113(特别是基因ORFV113的5’端，例如5’非翻译区)。类似地，可以基于相似的原则设计引物 /探针的靶物。
9.ORFV病毒基因组和ORFV病毒的用途
本发明涉及一种药物组合物，其包含一定量，特别是有效量，例如治疗有效量或预防有效量的本发明的ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)和/或ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)。在一个实施方案中，所述组合物包含药学上可接受的载剂。
在一个实施方案中，所述组合物为粉剂、溶液、透皮贴剂、膏剂、或栓剂的形式。在一个实施方案中，所述组合物通过静脉内、瘤内、肌内、皮下、经直肠、经阴道或腹腔路径来施用。
本发明涉及一种在受试者中治疗疾病或延缓疾病进展的方法，其包括施用一定量，特别是有效量，例如治疗有效量或预防有效量的本发明的ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)和/或ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)。
本发明涉及一定量，特别是有效量，例如治疗有效量或预防有效量的本发明的ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)和/或ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)在制备药物中的用途。在一个实施方案中，所述药物用于在受试者中治疗疾病或延缓疾病进展。
本发明涉及一定量，特别是有效量，例如治疗有效量或预防有效量的本发明的ORFV病毒基因组(例如ORFV112基因和/或ORFV111基因缺陷型ORFV病毒基因组)和/或ORFV病毒(例如CBP蛋白和/或假设蛋白111缺陷型ORFV病毒)，其用于在受试者中治疗疾病或延缓疾病进展。
在一个实施方案中，所述疾病是癌症。在一个实施方案中，所述癌症是实体瘤。在一个实施方案中，所述实体瘤是宫颈癌、膀胱癌、肝癌、卵巢癌、黑色素瘤、结直肠癌、肺癌、乳腺癌、胃癌、子宫癌、头颈癌、甲状腺癌、食道癌、前列腺癌、胰腺癌、肉瘤、脑瘤等。
在一个实施方案中，所述受试者是哺乳动物，包括啮齿动物(例如小鼠和大鼠)、非人灵长动物(例如食蟹猴)和人。
实施例
实施例1：突变型羊传染性脓疱皮炎病毒筛选纯化方法
●病毒株来源：POV-601病毒株(中国典型培养物保藏中心(CCTCC)，保藏号：V201713)
宿主细胞来源：非洲绿猴肾细胞CV-1(中国科学院典型培养物保藏委员会细胞库/中国科学院上海生命科学研究院)
●流式分选及单克隆挑选方法：
1)将POV-601病毒株感染非洲绿猴肾细胞CV-1后，用流式细胞仪(BD)对感染病毒的细胞进行分选，分选后细胞接种到96孔板中，每个孔接种1个细胞；
2)将分选后的96孔板转移至37℃，5％CO 2浓度的二氧化碳培养箱(Thermo，160i)中进行培养。每天观察病毒感染情况；
3)待病毒感染的细胞出现足够CPE时，收集、冻存病毒；
4)收集后的单克隆病毒，使用QuickExtract TM DNA Extraction Solution(cat#：QE09050，Lucigen)，分别提取病毒基因组DNA；
5)用设计的ORFV112基因特异性正、反向引物(SEQ ID NO:5和6)，以从单克隆病毒提取的基因组DNA为模版，分别进行目的片段扩增及琼脂糖凝胶电泳；图1显示单克隆病毒株POV-601-1A1和POV-601-3F8电泳结果，其条带大小分别约为500bp和1000bp。同时，分别用ORFV112基因特异性正、反向引物(SEQ ID NO:5和6)及POV-601-3F8正、反向引物(SEQ ID NO:55和56)，进行PCR扩增，并对PCR产物进行桑格测序(Sanger Sequencing)(苏州金维智生物科技有限公司)，确认POV-601-1A1的ORFV112基因编码在5’端缺失了538个碱基，其中含5’端非编码的226个碱基和5’端ORFV112蛋白编码区的312个碱基(见SEQ ID NO:4)；而POV-601-3F8具有完整的ORFV112基因序列(见SEQ ID NO:3)。
●PCR程序参数：94℃预变性5min；94℃变性30s，62℃退火30s，68℃延伸1min，30个循环；68℃终延伸7min。
实施例2：突变型羊传染性脓疱皮炎病毒种属确认
对POV-601-1A1病毒株进行病毒基因组提取(Mouse Tail Genomic DNA Kit，Cat#：CW2094S)，送第三方测序公司进行二代测序(苏州金维智生物科技有限公司)。对测序结果进行组装拼接。将该病毒株的B2L基因 [21] (ORFV011基因)全序列结果经BLAST核酸序列搜索后，与测序结果相似性较高的为OV/HLJ/04，相似性为99.91％，确认分离的病毒株为ORFV。
Figure PCTCN2021112408-appb-000001
实施例3：突变型羊传染性脓疱皮炎病毒的构建
●病毒株来源：POV-601-1A1病毒株(中国典型培养物保藏中心(CCTCC)，保藏号：V202029)
宿主细胞来源：非洲绿猴肾细胞CV-1(ATCC No.CCL-70 TM)
●重组羊传染性脓疱溶瘤病毒构建方法，主要有以下步骤：
1)采用特定设计、末端分别带有HindIII和EcoRI限制性内切酶位点的引物(SEQ ID NO：7-12)，通过PCR方法克隆ORFV112基因的侧翼序列(左同源臂和右同源臂)及EGFP报告基因；
2)使用HindIII和EcoRI酶切pUC19质粒；
3)将步骤1)中的PCR产物与酶切后的pUC19质粒连接，获得CBP穿梭质粒；
4)以步骤3)中获得的CBP穿梭质粒连接产物为模版，采用末端分别带有EcoRI和AgeI限制性内切酶位点的特定引物(SEQ ID NO：13-14)，通过PCR方法扩增，得到线性CBP穿梭载体；
5)以px330质粒载体为模板，采用末端分别带有EcoRI和AgeI限制性内切酶位点的特定引物(SEQ ID NO：15-18)，通过PCR方法扩增不含核定位信号(NLS)Cas9的新px330质粒载体和px330质粒载体骨架(px330 backbone)
6)使用EcoRI和AgeI酶切步骤5)中的新px330质粒载体和px330质粒载体骨架(px330 backbone)，通过T4 DNA ligase连接酶切产物；将连接产物转化到筛选宿主菌DH5α中，得到阳性单克隆，并扩大培养，提取质粒。进行Sanger测序鉴定，获得没有核定位信号(NLS)的重组质粒(px330-ΔNLS)；
7)合成CBP gRNAs，使用BbsI酶切重组质粒(px330-ΔNLS)，将CBP gRNAs与酶切重组质粒(px330-ΔNLS)进行连接，将其转化到宿主菌DH5α中(上海生工，B528413-0020)，得到阳性单克隆，并扩大培养，提取质粒。获得px330-ΔNLS-CBP gRNA表达质粒；
8)将得到的px330-ΔNLS-CBP gRNA表达质粒转染特选的副痘病毒宿主细胞CV-1，随后用POV-601-1A1病毒株感染转染后的CV-1细胞。将步骤(4)得到的线性CBP穿梭载体转染到已转染px330-ΔNLS-CBP gRNA质粒和感染了POV-601-1A1病毒的CV-1细胞中；
9)富集培养并收集带EGFP荧光病毒的目标细胞，随后用流式细胞仪分选接种到96孔板中，每个孔接种1个细胞，培养扩增。从富含EGFP荧光病毒的孔收集目标细胞，提取病毒基因组DNA，使用PCR方法进行鉴定(引物SEQ ID NO：19-30)，将出现目标条带的病毒再次感染CV-1细胞，进行扩大培养；
10)经过多轮接病毒、分选、收病毒、PCR鉴定，最终得到ORFV112基因完全缺失的纯化单克隆病毒，命名为POV-604-1D1。
实施例4：突变型羊传染性脓疱皮炎病毒的扩增培养
宿主细胞来源：非洲绿猴肾细胞CV-1(ATCC No.CCL-70 TM)
病毒株来源：POV-601、POV-601-1A1、POV-604-1D1和POV-601-3F8病毒株
●病毒感染细胞：
1)根据需要量进行CV-1细胞传代培养，当扩增达到一定量，培养瓶中细胞饱和度达到90％左右可进行病毒感染；
2)病毒液用2％FBS的DMEM完全培养基适当稀释；
3)弃掉培养瓶中旧的培养基，并补足适量新鲜的2％FBS的DMEM完全培养基；
4)按照MOI 0.5加入稀释好的病毒液，轻轻摇晃培养瓶使病毒液均匀分布，标识培养瓶，放入37℃，5％CO 2培养箱中培养。
●受病毒感染的细胞冻存
1)受病毒感染的细胞有90％以上出现CPE时，即可收获病毒；
2)从细胞培养箱中取出病毒感染的细胞，收集上清液，将仍贴壁的细胞消化后合并至上清液中；
3)于3000rpm，4℃下离心10min；
4)PBS润洗一次；
5)使用适量PBS再次重悬细胞；
6)转移至-80℃储存。
●病毒裂解释放：
1)将受病毒感染的细胞从-80℃转移至37℃水浴锅中解冻；
2)超声处理(型号：FB120,Thermo)：“APLITUDE”为100％，“Time”为1min，共3次；
3)加入全能核酸酶，每毫升细胞裂解液悬液加入25个单位的酶，混合均匀，37℃下处理30min；
4)1300rpm，离心10min；
5)收集病毒上清液，转移至-80℃储存。
实施例5：突变型羊传染性脓疱皮炎病毒在鼠源膀胱癌肿瘤模型中抗肿瘤活性评估
A.实验设计
Figure PCTCN2021112408-appb-000002
B.实验动物：雄性C57BL/6小鼠，6W，来自浙江维通利华实验动物技术有限公司
C.实验过程
在雄性C57BL/6小鼠右侧背部皮下接种重悬于PBS中的MB49细胞(广州吉妮欧生物科技有限公司)，细胞密度为2×10 6/ml，接种量为0.1ml/只。细胞接种日期定义为Day 0。等对照组肿瘤体积均值达到100mm 3左右，根据肿瘤大小随机分组，根据表1描述进行给药处理，所有小鼠在接种细胞后Day30安乐。
每周对小鼠称重和测量肿瘤2次，持续整个研究。使用游标卡尺(型号：16ER；Mahr GmbH)测量肿瘤体积，结果见附图2。
通过下列公式计算肿瘤体积：肿瘤体积(mm 3)＝a*b 2/2，其中a为肿瘤 长度(mm)，b为肿瘤宽度(mm)。垂直测量长度和宽度。
通过下列公式计算相对平均肿瘤体积：RTV(Relative Tumor Volume)＝V t/V 0。其中V 0为分组给药时测量所得平均肿瘤体积，V t为每一次测量时的平均肿瘤体积。相对肿瘤增殖率T/C(％)＝T RTV/C RTV*100％。(T RTV：治疗组RTV；C RTV：对照组RTV)；
肿瘤抑制率TGI％＝(1-T/C)×100％。
使用ANOVA统计分析数据：用ANOVA检验方法比较治疗组肿瘤体积与对照组肿瘤体积比有无显著性差异。所有的数据均用SPSS 17进行分析。P<0.05为具有显著性差异。
D.结果
Figure PCTCN2021112408-appb-000003
实施例6：突变型羊传染性脓疱皮炎病毒在鼠源黑色素瘤模型中抗肿瘤活性评估
A.实验设计
Figure PCTCN2021112408-appb-000004
B.实验动物-雌性C57BL/6小鼠，6-8W，来自浙江维通利华实验动物技术有限公司
C.实验过程
在雌性C57BL/6小鼠右侧背部皮下接种重悬于PBS中的B16-F10细胞(中 国科学院典型培养物保藏委员会细胞库)，细胞密度为5×10 6/ml，接种量为0.1ml/只。细胞注射日期定义为Day 0。等对照组肿瘤体积均值达到100mm 3左右，根据肿瘤大小随机分组，根据表3描述进行给药处理，所有小鼠在接种细胞后Day15安乐。
每周对小鼠称重和测量肿瘤2次，持续整个研究。使用游标卡尺(型号：16ER；Mahr GmbH)测量肿瘤体积，结果见附图3。
通过下列公式计算肿瘤体积：肿瘤体积(mm 3)＝a*b 2/2，其中a为肿瘤长度(mm)，b为肿瘤宽度(mm)。垂直测量长度和宽度。
通过下列公式计算相对平均肿瘤体积：RTV(Relative Tumor Volume)＝V t/V 0。其中V 0为分组给药时测量所得平均肿瘤体积，V t为每一次测量时的平均肿瘤体积。相对肿瘤增殖率T/C(％)＝T RTV/C RTV*100％。(T RTV：治疗组RTV；C RTV：对照组RTV)；
肿瘤抑制率TGI％＝(1-T/C)×100％。
使用ANOVA统计分析数据：用ANOVA检验方法比较治疗组肿瘤体积与对照组肿瘤体积比有无显著性差异。所有的数据均用SPSS 17进行分析。P<0.05为具有显著性差异。
D.结果
Figure PCTCN2021112408-appb-000005
实施例7：突变型羊传染性脓疱皮炎病毒安全性评估
A.实验设计
Figure PCTCN2021112408-appb-000006
Figure PCTCN2021112408-appb-000007
s.c.＝皮下注射；i.v.＝尾静脉注射
B.测试品和对照品
测试品-溶瘤病毒POV-601-1A1；对照品-磷酸盐缓冲液
C.实验动物：雌性C57BL/6小鼠，6-8W，浙江维通利华实验动物技术有限公司
D.实验操作
在雌性C57BL/6小鼠右侧背部皮下或者尾静脉注射测试品和对照品。第一次注射日期定义为Day 0。小鼠完全随机分组，根据表5描述进行给药处理，所有小鼠在给药后第15天结束观察。第一周每天对小鼠称重，之后每两到三天对小鼠称重，持续整个研究。
对小鼠监测体重变化。使用ML1602T电子天平(Mettler)测量动物体重。使用下面的等式计算相对体重变化率，结果见附图4。
相对体重变化(％)＝[重量 日新/重量 日0]x 100。
实施例8：突变型羊传染性脓疱皮炎病毒对免疫系统调节作用
A.实验设计
Figure PCTCN2021112408-appb-000008
Figure PCTCN2021112408-appb-000009
i.t.＝瘤内注射；i.v.＝尾静脉注射
B.测试品和对照品：
测试品-溶瘤病毒POV-601-1A1
对照品-磷酸盐缓冲液
C.实验动物：雌性Balb/c-nude小鼠，6W，北京华阜康生物科技股份有限公司
D.实验方法
在雌性Balb/c-nude小鼠双侧背部皮下接种重悬于PBS中的C-33A细胞(中国科学院典型培养物保藏委员会细胞库)，细胞密度为1×10 8/ml，接种量为0.1ml/只。细胞注射日期定义为Day 0。待肿瘤体积均值达到100mm 3左右，根据表6描述进行给药处理。末次给药后24h结束实验，收集小鼠全血、肿瘤，将组织剪碎消化，制备成单细胞悬液，进行荧光抗体标记(Anti-mouse CD45 APC-eFluor 780、Anti-mouse CD49b-PE、Anti-mouse CD69 APC)，然后进行流式检测(AFC2，Thermo)，见附图5(小图A-C)。
实施例9：突变型羊传染性脓疱皮炎病毒对体外细胞的感染作用
病毒株：POV-601-1A1
1)将培养好的各肿瘤细胞消化后收集计数，按照1.5x10 4cell/孔接种至96孔板，培养24h后接毒；
2)用2％FBS的完全培养基将病毒液分别稀释至1.5x10 8pfu/ml、1.5x10 7pfu/ml、1.5x10 6pfu/ml、1.5x10 5pfu/ml，四个浓度；
3)将稀释好的病毒液加入96孔细胞板中，每孔加入100ul，使最终加入到每孔的病毒MOI分别为1000，100，10，1，每个浓度设置3个复孔；
4)设置3个孔，加入无病毒液的2％FBS的完全培养基，作为阴性对照；
5)轻微摇晃使病毒液均匀覆盖在单层细胞表面，确保每个角落都覆盖到；
6)将接种对照或病毒的96孔板放回37℃，5％CO 2培养箱中培养72h；
7)从培养箱中取出96孔板，每孔加入10ul Alamar Blue Cell Viability Reagent(Invitrogen，Cat#：2072060)，轻轻摇晃培养板使染料均匀分散，用锡箔纸覆盖96孔板于37℃培养箱中避光孵育；
8)孵育结束后去掉板盖，将96孔板放入酶标仪(SPECTRAMAX M4，Molecular Devices)中，设置激发波长为560nm，发射波长为590nm，进 行吸光度检测，并进行细胞死亡率换算，换算公式为：
Figure PCTCN2021112408-appb-000010
对结果进行分析；
9)结果显示，POV-601-1A1病毒对黑色素瘤细胞(B16-F10)、膀胱癌细胞(MB49)、肝癌细胞(Hepa1-6)、结肠癌细胞(CT26)、人源宫颈癌细胞(C-33A)、人源卵巢癌细胞(SK-OV-3)等细胞具有感染性，并随着MOI值增加，病毒感染性增加，但对vero细胞基本不感染。
Figure PCTCN2021112408-appb-000011
实施例8：突变型羊传染性脓疱皮炎病毒在鼠源结肠癌模型中抗肿瘤活性评估
A.实验设计
Figure PCTCN2021112408-appb-000012
B.实验动物-雌性Balb/c小鼠，6-8W，来自浙江维通利华实验动物技术有限公司
C.实验过程
在雌性Balb/c小鼠右侧背部皮下接种重悬于PBS中的CT-26细胞(中国科学院典型培养物保藏委员会细胞库)，细胞密度为5×10 6/ml，接种量为0.1ml/ 只。细胞注射日期定义为Day 0。等对照组肿瘤体积均值达到100mm 3左右，根据肿瘤大小随机分组，根据表8描述进行给药处理，所有小鼠在接种细胞后Day25安乐。
每周对小鼠称重和测量肿瘤2次，持续整个研究。使用游标卡尺(型号：16ER；Mahr GmbH)测量肿瘤体积，结果见附图14。
通过下列公式计算肿瘤体积：肿瘤体积(mm 3)＝a*b 2/2，其中a为肿瘤长度(mm)，b为肿瘤宽度(mm)。垂直测量长度和宽度。
通过下列公式计算相对平均肿瘤体积：RTV(Relative Tumor Volume)＝V t/V 0。其中V 0为分组给药时测量所得平均肿瘤体积，V t为每一次测量时的平均肿瘤体积。相对肿瘤增殖率T/C(％)＝T RTV/C RTV*100％。(T RTV：治疗组RTV；C RTV：对照组RTV)；
肿瘤抑制率TGI％＝(1-T/C)×100％。
使用ANOVA统计分析数据：比较治疗组肿瘤体积与对照组肿瘤体积比有无显著性差异。所有的数据均用SPSS 17进行分析。P<0.05为具有显著性差异。
D.结果：
Figure PCTCN2021112408-appb-000013
实施例9：重组突变型羊传染性脓疱皮炎病毒在鼠源黑色素瘤模型中抗肿瘤活性评估
通过重组方法构建了完全或部分删除ORFV111基因和/或ORFV112基因编码区和/或非编码区的羊传染性脓疱皮炎病毒，并以实施例6记载的方法测试效果，除非另外指明。
Figure PCTCN2021112408-appb-000014
Figure PCTCN2021112408-appb-000015
v611a：完全删除ORFV112基因。
Figure PCTCN2021112408-appb-000016
v615a：删除ORFV111和ORFV112编码区；
v616a：完全删除ORFV111和ORFV112。
Figure PCTCN2021112408-appb-000017
Figure PCTCN2021112408-appb-000018
v617a：完全删除ORFV111；
v618a：删除ORFV111编码区。
材料保藏
以下材料已经按照布达佩斯条约规定保藏于中国典型培养物保藏中心(CCTCC)(中国，武汉，武汉大学，430072)：
Figure PCTCN2021112408-appb-000019
引用文献
1.Hanahan,D.,Weinberg,R.A.(2011).Hallmarks of cancer:the next generation.Cell,144(5):646-74.
2.Miest,T.S.,Cattaneo,R.(2014).New viruses for cancer therapy:meeting clinical needs.Nat Rev Microbiol,12(1):23-34.
3.Burke,J.,Nieva,J.,Borad,M.J.,Breitbach,C.J.(2015).Oncolytic viruses:perspectives on clinical development.Curr Opin Virol,13:55-60.
4.Seymour,L.W.,Fisher,K.D.(2016).Oncolytic viruses:finally delivering.Br J Cancer,Feb 16；114(4):357-361.
5.Harrington,K.J.,Puzanov,I.,Hecht,J.R.,Hodi,F.S.,Szabo,Z.,Murugappan,S.,Kaufman,H.L.(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.殷震,刘景华(1997).动物病毒学(第二版),科学出版社,977-978.
7.王志军(2018).生物技术药物研究开发和质量控制(第三版),科学出版社.
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,B.T.,McCaughan,C.A.,Handel,T.M.,Mercer,A.,Brunetti,C.,McFadden,G.,Fleming,S.B.(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.刘伟,杨侃侃,殷冬冬,王元红,俞赵荣,蒋书东,李传峰,李永东,王勇(2018).口疮病毒dUTPase基因的原核表达及亚细胞定位,中国兽医科学,7:818-823.
16.Irwin,C.R.,Hitt,M.M.,Evans,D.H.(2017).Targeting Nucleotide Biosynthesis:A Strategy for Improving the Oncolytic Potential of DNA Viruses.Front Oncol,7:229.
17.Fleming,S.B.,McCaughan,C.,Lateef,Z.,Dunn,A.,Wise,I.M.,Real,N.C.,Mercer,A.A.(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,L.P.,Eunice Kwok,Y.Y.,John,C.B.,Nelson, B.H.(2018).Oncolytic viruses as engineering platforms for combination immunotherapy.Nat Rev Cancer,18:419-432.
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21.王光祥,尚佑军,陈江涛,吕占禄,张克山,刘湘涛(2012).湖北省羊口疮病毒的分离鉴定,动物医学进展,033(011):37-40.
序列
Figure PCTCN2021112408-appb-000020
Figure PCTCN2021112408-appb-000021
Figure PCTCN2021112408-appb-000022
Figure PCTCN2021112408-appb-000023
Figure PCTCN2021112408-appb-000024
Figure PCTCN2021112408-appb-000025
Figure PCTCN2021112408-appb-000026
Figure PCTCN2021112408-appb-000027
Figure PCTCN2021112408-appb-000028
Figure PCTCN2021112408-appb-000029
Figure PCTCN2021112408-appb-000030
Figure PCTCN2021112408-appb-000031

Claims (21)

  1. 一种突变型羊传染性脓疱皮炎病毒，其特征在于基因ORFV112和/或基因ORFV111的功能性表达产物的缺失。
  2. 如权利要求1所述的病毒，其中基因ORFV112的功能性表达产物的缺失是由基因ORFV112的完全或部分缺失造成的。
  3. 如权利要求1所述的病毒，其中基因ORFV111的功能性表达产物的缺失是由基因ORFV111的完全或部分缺失造成的。
  4. 如权利要求1-3任一项所述的病毒，其中表达产物为蛋白质和/或核酸。
  5. 一种突变型羊传染性脓疱皮炎病毒，其特征在于基因ORFV112的完全或部分缺失和/或基因ORFV111的完全或部分缺失。
  6. 一种改造羊传染性脓疱皮炎病毒的方法，其包括降低或消除基因ORFV112的表达产物的表达和/或活性和/或降低或消除基因ORFV111的表达产物的表达和/或活性。
  7. 如权利要求6所述的方法，其包括完全或部分删除基因ORFV112。
  8. 如权利要求6所述的方法，其包括完全或部分删除基因ORFV111。
  9. 如权利要求6-8任一项所述的方法，其中表达为转录和/或翻译。
  10. 一种改造羊传染性脓疱皮炎病毒的方法，其包括完全或部分删除基因ORFV112和/或完全或部分删除基因ORFV111。
  11. 通过权利要求6-10任一项的方法得到的病毒。
  12. 如权利要求1-5和11任一所述的病毒，其中基因ORFV002(NF-κB核抑制剂)、ORFV005(Hypothetical protein)和ORFV007(dUTPase)完全缺失。
  13. 如权利要求1-5、11和12任一项所述的病毒，其具有复制和溶瘤能力。
  14. 以CCTCC保藏编号V202029保藏的羊传染性脓疱皮炎病毒。
  15. 如权利要求1-5和11-14任一项所述的病毒的基因组。
  16. 一种组合物，其包含如权利要求1-5和11-14任一项所述的病毒和药学上可接受的载剂。
  17. 如权利要求16所述的组合物，其为粉剂、溶液、透皮贴剂、膏剂、或栓剂的形式。
  18. 如权利要求16所述的组合物，其通过静脉内、瘤内、肌内、皮下、 经直肠、经阴道、或腹腔路径来施用。
  19. 如权利要求1-5和11-14任一项所述的病毒制备用于治疗癌症的药物的用途。
  20. 如权利要求19所述的用途，其中所述癌症是实体瘤。
  21. 如权利要求20所述的用途，其中所述实体瘤为宫颈癌、膀胱癌、肝癌、卵巢癌、黑色素瘤、结直肠癌、肺癌、乳腺癌、胃癌、子宫癌、头颈癌、甲状腺癌、食道癌、前列腺癌、胰腺癌、肉瘤或脑瘤。
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