Recombinant oncolytic virus targeting tumor hypoxic microenvironment and application thereof

Abstract

The application provides a recombinant oncolytic virus targeting a tumor hypoxic microenvironment and an application thereof. A genome of the recombinant oncolytic virus comprises at least one nucleic acid fragment selected from a first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment; wherein the first nucleic acid fragment comprises genes encoding an immunosuppression relief and T cell local activation unit and genes encoding a T cell function enhancement and chemotactic expansion unit; the second nucleic acid fragment comprises genes encoding a hypoxia sensing unit and genes encoding a T cell activation antibody expression unit; and the third nucleic acid fragment comprises at least one gene encoding an immunomodulatory unit. The recombinant oncolytic virus has three action mechanisms of a high remodeling of a solid tumor microenvironment, a universal in vivo T cell adapter and a virus conditional replication regulation, and can be effectively used for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a universal recombinant oncolytic virus that targets the hypoxic microenvironment of tumors and has both killing and safety properties, as well as its applications. Background Technology

[0002] Oncolytic virus therapy is an emerging treatment method for cancer. Oncolytic viruses can selectively replicate in and destroy tumor cells while stimulating an anti-tumor immune response in the body, without harming normal cells. Since the beginning of the 21st century, the field of oncolytic viruses has gained considerable attention following numerous positive results from clinical trials. To date, four oncolytic virus drugs have been approved globally.

[0003] Oncolytic virus therapy was introduced into cancer treatment strategies at the beginning of the last century. After continuous development, oncolytic viruses are undergoing their third iteration—using genetic engineering to upgrade the viral backbone, carrying a greater number and diversity of effective exogenous genes to enhance oncolytic activity and activate a stronger immune response. These iterative oncolytic viruses often offer improved safety while retaining their direct tumor-lysing activity. Simultaneously, the modified oncolytic viruses can carry exogenous genes such as tumor suppressor genes, pro-apoptotic genes, anti-angiogenic genes, suicide genes, and immunomodulatory genes, exerting far-reaching anti-tumor effects through different mechanisms. Early modifications focused on improving target specificity, selective replication, and oncolytic efficacy. Subsequently, it was discovered that tumor lysis can trigger antigen-specific anti-tumor immune responses, thereby activating distant anti-tumor effects—another advantage of oncolytic viruses as immunotherapy. In recent years, strategies have gradually shifted towards developing viral vectors that can enhance intratumoral immune responses or regulate tumor angiogenesis and metabolism to combat the malignant tumor microenvironment (TME). Modifying oncolytic viruses in the tumor microenvironment has advantages such as high efficiency in killing tumor cells, good targeting, few side effects, and low cost; at the same time, the inherent genes of some viruses can enhance the effect of combined drug therapy and improve the sensitivity of tumor cells to radiotherapy and chemotherapy.

[0004] However, the killing efficiency and safety of oncolytic viruses still need to be further improved. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a universal recombinant oncolytic virus that targets the hypoxic tumor microenvironment and possesses both high lethality and high safety, as well as its applications. This invention utilizes an oncolytic virus platform to develop a tumor immunotherapy vector technology that combines a high degree of solid tumor microenvironment remodeling, a universal in vivo T-cell connective, and a triple mechanism of action involving conditional viral replication regulation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a recombinant oncolytic virus whose genome comprises at least one nucleic acid fragment selected from a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein:

[0008] The first nucleic acid fragment contains a gene encoding an immunosuppression relief and T cell local activation unit and a gene encoding a T cell function enhancement and chemotactic amplification unit. The gene encoding the immunosuppression relief and T cell local activation unit sequentially contains a gene encoding a first viral transcription promoter, a gene encoding a T cell connector targeting immune checkpoints, a gene encoding a first autosplitting peptide, and a gene encoding a first chemokine. The gene encoding the T cell function enhancement and chemotactic amplification unit sequentially contains a gene encoding a first viral transcription promoter, a gene encoding an immune-enhancing cytokine, a gene encoding a second autosplitting peptide, and a gene encoding a second chemokine.

[0009] The second nucleic acid fragment includes a gene encoding a hypoxia-sensing unit and a gene encoding a T-cell activation antibody expression unit, wherein the gene encoding the hypoxia-sensing unit includes a gene encoding a hypoxia response element and a gene encoding an artificial transcription factor connected via a gene encoding a second viral transcription promoter, and the gene encoding the T-cell activation antibody expression unit includes a gene encoding an artificial transcription factor recognition element and a gene encoding a T-cell activation antibody connected via a gene encoding a second viral transcription promoter.

[0010] The third nucleic acid fragment contains at least one gene encoding an immunomodulatory unit, wherein the gene encoding the immunomodulatory unit contains a gene encoding a hypoxia response element and a gene encoding an immunomodulator, which are linked via a gene encoding a third viral transcription promoter.

[0011] It should be noted that two adjacent coding genes may contain genes such as those encoding adapters, and the appropriate selection can be made by those skilled in the art as needed.

[0012] According to some embodiments of the present invention, the T-cell connector targeting immune checkpoints is a bispecific T-cell connector targeting CTLA4, preferably having an amino acid sequence as shown in SEQ ID NO: 5; more preferably, the nucleotide sequence of the gene encoding the T-cell connector targeting immune checkpoints is shown in SEQ ID NO: 4.

[0013] According to some embodiments of the present invention, the first chemokine and the second chemokine are each independently selected from one of CXCL9, CXCL10 and CXCL11; preferably, the first chemokine is CXCL9 and the second chemokine is CXCL10.

[0014] According to some embodiments of the present invention, the immune-enhancing cytokine is an interleukin; preferably, the immune-enhancing cytokine is selected from one or more of IL-2, IL-7, IL-12, IL-15, IL-21 and IL-27; more preferably, the immune-enhancing cytokine is IL-21.

[0015] According to some embodiments of the present invention, the first self-cleaving peptide and the second self-cleaving peptide are each independently selected from P2A and T2A.

[0016] According to some embodiments of the present invention, the nucleotide sequence of the gene encoding the hypoxia response element (HRE) is as shown in SEQ ID NO: 31, or is a repeating nucleotide sequence of more than one, preferably three, more preferably five, nucleotide sequences as shown in SEQ ID NO: 31.

[0017] According to some embodiments of the present invention, the gene encoding the artificial transcription factor is selected from one or more of the following: the CRE gene with nucleus-deposition signal, the CRE-VP64 gene with nucleus-deposition signal, the CRE-VP64-ODD gene with nucleus-deposition signal, the CRE-RAP94 gene with nucleus-deposition signal, the CRE-RAP94-ODD gene with nucleus-deposition signal, or the CRE-VP160 gene with nucleus-deposition signal.

[0018] Preferably, the gene encoding the artificial transcription factor is the CRE-VP64 gene with a denuclearized localization signal.

[0019] More preferably, the nucleotide sequence of the CRE-VP64 gene of the denucleated localization signal is shown in SEQ ID NO: 22.

[0020] According to some embodiments of the present invention, the gene encoding the artificial transcription factor recognition element is one or more loxp genes.

[0021] Preferably, the nucleotide sequence of the gene encoding the artificial transcription factor recognition element is a repeating gene sequence of more than one, preferably three, and more preferably five loxp genes. For example, the nucleotide sequence of the gene encoding the artificial transcription factor recognition element is as shown in SEQ ID NO: 29.

[0022] According to some embodiments of the present invention, the T-cell activating antibody is an antibody targeting CD3, CD5, CD7 or CD28; preferably, the T-cell activating antibody is an antibody targeting CD3.

[0023] According to some embodiments of the present invention, the first viral transcription promoter, the second viral transcription promoter and the third viral transcription promoter are each independently selected from one or more of PE / L, PE / LL, truncated PE / L, further truncated PE / L, truncated P7.5, further truncated P7.5 or P11.

[0024] Preferably, the nucleotide sequence of PE / L is shown in SEQ ID NO: 1; the nucleotide sequence of P11 is shown in SEQ ID NO: 24; and the nucleotide sequence of PE / LL is shown in SEQ ID NO: 32.

[0025] According to some embodiments of the present invention, the immunomodulator is interferon, preferably selected from one or more of IFNα, IFNβ and IFNγ.

[0026] According to some embodiments of the present invention, the third nucleic acid fragment contains two genes encoding an immunomodulatory unit, wherein the immunomodulator in one gene is IFNβ and the immunomodulator in the other gene is IFNγ.

[0027] According to some embodiments of the present invention, the virus is selected from one or more of the following: vaccinia virus, adenovirus, herpes simplex virus type I, herpes simplex virus type II, vesicular stomatitis virus, echovirus, reovirus, alphavirus, yellow fever virus, Coxsackie virus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus, or retrovirus.

[0028] According to some embodiments of the present invention, the first nucleic acid fragment is located in the TK region of the genome of the recombinant oncolytic virus.

[0029] According to some embodiments of the present invention, the second nucleic acid fragment is located in the C16L-VGF1 region of the genome of the recombinant oncolytic virus.

[0030] According to some embodiments of the present invention, the third nucleic acid fragment is located in the C16R-VGF2 region of the genome of the recombinant oncolytic virus.

[0031] In this invention, "TK region" and "TK gene" have the same meaning, both referring to the thymidine kinase gene.

[0032] In this invention, "C16L-VGF1 region", "C16L-VGF1 gene" and "C16L-VGF1 module" have the same meaning, all referring to the left inverted terminal repeat (ITR) region of vaccinia virus, which contains the viral growth factor gene VGF1 and the immunosuppressive protein gene C16L. The two encode the viral growth factor VGF1 and the immunosuppressive protein C16L, respectively, and synergistically promote viral replication and immune escape.

[0033] In this invention, "C16R-VGF2 region", "C16R-VGF2 gene" and "C16R-VGF2 module" have the same meaning. They all refer to a diploid gene module located in the right ITR region of vaccinia virus, which is homologous and symmetrical with the left VGF1-C16L module, contains the viral growth factor gene VGF2 and the immunosuppressive protein gene C16R, and forms a functionally redundant diploid gene module with the left module.

[0034] In a preferred embodiment, the present invention provides a recombinant vaccinia virus, wherein the recombinant vaccinia virus is obtained by knocking out the TK gene, VGF1-C16L gene and VGF2-C16R gene of the wild-type vaccinia virus genome, and then inserting a first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment having the following structures into the knockout sites, namely the TK region, the VGF1-C16L region and the VGF2-C16R region, respectively.

[0035] A first nucleic acid fragment is inserted into the TK region. The gene encoding the immunosuppression relief and T cell local activation unit of the first nucleic acid fragment sequentially includes genes encoding the following elements: viral transcription promoter PE / L, fusion protein αCTLA4-BiTE, autosplicing peptide P2A, and chemokine CXCL9; the gene encoding the T cell function enhancement and chemokine amplification unit of the first nucleic acid fragment sequentially includes genes encoding the following elements: viral transcription promoter PE / L, interleukin IL-21, autosplicing peptide T2A, and chemokine CXCL10.

[0036] A second nucleic acid fragment is inserted into the VGF1-C16L region. The gene encoding the hypoxia-sensing unit of the second nucleic acid fragment contains genes encoding the following elements in sequence: hypoxia response element HRE, viral transcription promoter P11, and CRE-VP64 (i.e., dCRE); the gene encoding the T cell activation antibody expression unit of the second nucleic acid fragment contains genes encoding the following elements in sequence: artificial transcription factor recognition element 5*loxp, viral transcription promoter P11, and αCD3.

[0037] A third nucleic acid fragment is inserted into the VGF2-C16R region. The third nucleic acid fragment contains two genes encoding immune regulatory units. The first gene encoding an immune regulatory unit contains genes encoding the following elements in sequence: hypoxia response element (HRE), viral transcription promoter PE / LL, and interferon IFNβ. The second gene encoding an immune regulatory unit contains genes encoding the following elements in sequence: hypoxia response element (HRE), viral transcription promoter PE / LL, and interferon IFNγ.

[0038] Particularly preferably, the nucleotide sequence of the first nucleic acid fragment inserted into the TK region of the recombinant vaccinia virus is shown in SEQ ID NO: 18, the nucleotide sequence of the second nucleic acid fragment inserted into the VGF1-C16L region is shown in SEQ ID NO: 30, and the nucleotide sequence of the third nucleic acid fragment inserted into the VGF2-C16R region is shown in SEQ ID NO: 40.

[0039] in:

[0040] The nucleotide sequence of PE / L is shown in SEQ ID NO: 1.

[0041] The amino acid sequence of αCTLA4-BiTE is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding αCTLA4-BiTE is shown in SEQ ID NO: 4.

[0042] The amino acid sequence of P2A is shown in SEQ ID NO: 7, and the nucleotide sequence of the gene encoding P2A is shown in SEQ ID NO: 6.

[0043] The amino acid sequence of CXCL9 is shown in SEQ ID NO: 9, and the nucleotide sequence of the gene encoding CXCL9 is shown in SEQ ID NO: 8.

[0044] The nucleotide sequence of the gene encoding IL-21 is shown in SEQ ID NO: 10.

[0045] The amino acid sequence of T2A is shown in SEQ ID NO: 12, and the nucleotide sequence of the gene encoding T2A is shown in SEQ ID NO: 11.

[0046] The amino acid sequence of CXCL10 is shown in SEQ ID NO: 14, and the nucleotide sequence of the gene encoding CXCL10 is shown in SEQ ID NO: 13.

[0047] The nucleotide sequence of the gene encoding HRE is shown in SEQ ID NO: 31.

[0048] The nucleotide sequence of P11 is shown in SEQ ID NO: 24.

[0049] The amino acid sequence of CRE-VP64 is shown in SEQ ID NO: 23, and the nucleotide sequence of the gene encoding CRE-VP64 is shown in SEQ ID NO: 22.

[0050] The nucleotide sequence of the gene encoding loxp is shown in SEQ ID NO: 29.

[0051] The amino acid sequence of αCD3 is shown in SEQ ID NO: 28, and the nucleotide sequence of the gene encoding αCD3 is shown in SEQ ID NO: 27.

[0052] The nucleotide sequence of PE / LL is shown in SEQ ID NO: 32.

[0053] The amino acid sequence of IFNβ is shown in SEQ ID NO: 34, and the nucleotide sequence of the gene encoding IFNβ is shown in SEQ ID NO: 33.

[0054] The amino acid sequence of IFNγ is shown in SEQ ID NO: 36, and the nucleotide sequence of the gene encoding IFNγ is shown in SEQ ID NO: 35.

[0055] The amino acid sequence of the first nucleic acid fragment is shown in SEQ ID NO: 18.

[0056] The amino acid sequence of the second nucleic acid fragment is shown in SEQ ID NO: 30.

[0057] The amino acid sequence of the third nucleic acid fragment is shown in SEQ ID NO: 40.

[0058] The preparation method of the recombinant oncolytic virus of the present invention can refer to the relevant schemes in the prior art, and will not be described in detail here.

[0059] In a second aspect, the present invention provides a pharmaceutical composition for treating cancer, comprising a recombinant oncolytic virus according to the first aspect of the invention, and optionally pharmaceutically acceptable excipients.

[0060] According to some embodiments of the present invention, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.

[0061] Thirdly, the present invention provides the use of the recombinant oncolytic virus according to the first aspect of the present invention in the preparation of a medicament for treating cancer.

[0062] According to some embodiments of the present invention, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.

[0063] Fourthly, the present invention provides a method for treating cancer, comprising: administering a therapeutically effective amount of the recombinant oncolytic virus according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention to a subject in need.

[0064] According to some embodiments of the present invention, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.

[0065] According to some embodiments of the present invention, the method further includes administering additional chemotherapy drugs, radiotherapy, surgical treatment, immune cell drugs (including but not limited to CAR-T, NK, NKT, iNKT, CAR-NK, CAR-NKT, CAR-iNKT, etc.), and other oncolytic viruses to subjects in need.

[0066] According to some embodiments of the present invention, the method is intravenous injection or intratumoral injection.

[0067] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0068] Through in-depth research, the inventors of this invention selected and knocked out the TK gene, VGF1-C16L gene, and VGF2-C16R gene of vaccinia virus to reduce the virulence of recombinant vaccinia virus and stabilize it, ensuring it does not change during replication. Furthermore, this invention inserts exogenous genes with specific functions at the knockout sites, thereby achieving the following triple mechanism of action:

[0069] (1) Highly remodeling the tumor microenvironment: The expression of the T cell binder targeting the CTLA4 target of highly efficient Treg cells in the tumor microenvironment can effectively clear Tregs; combined with the previously inserted T cell costimulatory factor IL-21, it forms a combination that remodels the tumor microenvironment in a synergistic manner. This combination molecule is inserted into the TK region of vaccinia virus.

[0070] (2) Multi-target killing of tumor cells, preventing escape and improving effectiveness: It can regulate the expression of T cell connectives in vivo under hypoxia and anchor them on the surface of tumor cell membranes to activate T cells to kill tumors. This combination molecule is inserted into the C16L-VGF1 region of vaccinia virus.

[0071] (3) Enhanced safety by carrying a conditional inhibition mechanism for viral replication: By designing and expressing a set of molecular elements, the virus can self-limit its replication in normal tissues and replicate preferentially in the hypoxic environment of tumors, thereby further enhancing safety. This combination of molecules is inserted into the C16R-VGF2 region of vaccinia virus.

[0072] Experimental verification showed that the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 of this invention exhibits highly efficient killing activity against VERO cells overexpressing CTLA4 and can strongly induce the expression of IL-21, CXCL-9, and CXCL10. The recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 of this invention specifically responds to hypoxic environments, exhibiting significantly higher tumor cell killing activity under hypoxic conditions than under normoxic conditions, significantly lower interferon expression levels under hypoxic conditions than under normoxic conditions, and superior replication capacity under hypoxic conditions compared to normoxic conditions.

[0073] Furthermore, animal experiments have demonstrated that the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 of this invention has excellent anti-tumor effects and tumor clearance rate, and also exhibits high safety. Attached Figure Description

[0074] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0075] Figure 1 The image shows the recombinant plasmids containing PE / L-αCTLA4-BiTE-P2A-CXCL9~PE / L-IL21-T2A-CXCL10 at the TK gene locus of vaccinia virus constructed in Example 1 of this invention.

[0076] Figure 2 The image shows the recombinant plasmid loaded with HRE-P11-dCRE~5*loxp-P11-αCD3-TM at the C16L-VGF1 gene locus of the vaccinia virus constructed in Example 1 of this invention.

[0077] Figure 3 The image shows the recombinant plasmid map of the vaccinia virus C16R-VGF2 gene locus loaded with HRE-PE / LL-IFNβ~HRE-PE / LL-IFNγ constructed in Example 1 of the present invention.

[0078] Figure 4The diagram shows the structure and plaques of the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 constructed in Example 2 of this invention. Figure 4 A shows the structure of inserting three sets of genes at the TK, C16L-VGF1 and C16R-VGF2 sites of vaccinia virus; Figure 4 B shows that the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 emits red, green and blue light simultaneously.

[0079] Figure 5 The results of the killing effect detection on VERO cells overexpressing CTLA4 in Example 3 of the present invention are shown, and the results are values ​​after deducting the baseline of the control.

[0080] Figure 6 The results of detecting IL-21 expression levels in Example 4 of this invention are shown.

[0081] Figure 7 The results of detecting the expression level of CXCL-9 in Example 5 of the present invention are shown.

[0082] Figure 8 The results of detecting the expression level of CXCL-10 in Example 6 of the present invention are shown.

[0083] Figure 9 This demonstrates the killing effect of vaccinia virus on Huh7 liver cancer cells in Example 7 of the present invention.

[0084] Figure 10 The levels of interferon secreted by the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 in Example 8 of this invention are shown under normoxic and hypoxic conditions. Figure 10 A shows the levels of IFN-β secreted by recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 under normoxic and hypoxic conditions; Figure 10 B shows the levels of IFN-γ secreted by recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 under normoxic and hypoxic conditions.

[0085] Figure 11 The replication compatibility curves of five vaccinia viruses in Example 9 of this invention are shown. Among them, Figure 11 A shows the replication curve of vaccinia virus under hypoxic conditions; Figure 11 B shows the replication curve of vaccinia virus under normoxic conditions.

[0086] Figure 12 The experimental protocol for the mouse experiment of Example 10 of the present invention is shown.

[0087] Figure 13The survival percentage of mice in each group in Example 10 of this invention is shown, where TTV represents recombinant Tiantan strain vaccinia virus and UTD represents untransduced T cells.

[0088] Figure 14 This illustrates the changes in tumor volume over time in mice inoculated with NCI-H292 lung cancer cells in Example 10 of this invention under the intervention of different vaccinia viruses. Figure 14 A, 14B, 14C, 14D, 14E, and 14F correspond to mouse experimental groups that received PBS, wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus, respectively.

[0089] Figure 15 This illustrates the changes in tumor volume over time in mice inoculated with Huh7 liver cancer cells under different vaccinia virus interventions, as shown in Example 10 of this invention. Figure 15 A, 15B, 15C, 15D, 15E, and 15F correspond to mouse experimental groups that received PBS, wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus, respectively.

[0090] Figure 16 The comparison analysis of tumor growth curves under different vaccinia virus interventions in Example 10 of the present invention is shown. Figure 16 A shows a comparative analysis of the growth curves of NCI-H292 lung cancer cells under different vaccinia virus interventions; Figure 16 B shows a comparative analysis of the growth curves of Huh7 liver cancer cells under different vaccinia virus interventions.

[0091] Figure 17 The changes in body weight over time in mice inoculated with NCI-H292 and Huh7 tumor cells in Example 10 of this invention are shown under different vaccinia virus interventions. Figure 17 A, 17B, 17C, 17D, 17E, and 17F correspond to mouse experimental groups that received PBS, wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus, respectively.

[0092] Figure 18 The comparison analysis of body weight change curves of tumor-bearing mice under different vaccinia virus interventions is shown in Example 10 of the present invention. Detailed Implementation

[0093] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0094] Unless otherwise specified, the experimental methods used in the following embodiments are conventional experimental methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are conventional biochemical reagents purchased from sales companies. Among them:

[0095] DMEM culture medium was purchased from Corning.

[0096] Fetal bovine serum was purchased from BI Company.

[0097] The LIPOFECTAMINE 3000 transfection kit was purchased from Thermo Fisher Scientific.

[0098] The gene synthesis was completed by Beijing Qingke Biotechnology Co., Ltd.

[0099] Stabl3 chemocompetent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0100] The endotoxin-free plasmid miniprep kit was purchased from OMEGA.

[0101] VERO cells, TK143 - Cells, NCI-H292 and Huh7 cancer cells were purchased from ATCC in the United States.

[0102] NCI-H292 and Huh7 cancer cells that stably integrate the firefly luciferase gene were obtained by infecting tumor cells with a lentiviral vector loaded with the luciferase gene and then selecting cells that stably express the luciferase protein through monoclonal screening.

[0103] The fluorescence microscope was purchased from Nikon, Japan.

[0104] The luciferase substrate was purchased from Promega Biotechnology Co., Ltd.

[0105] The GloMax96 microplate luminescence detector was purchased from Promega Biotechnology Co., Ltd.

[0106] Example 1: Construction of recombinant plasmid for vaccinia virus

[0107] The nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 (i.e., the nucleotide sequences of the gene encoding the signal peptide, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3), SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13) were synthesized by Beijing Qingke Biotechnology Co., Ltd., and cloned into the corresponding TK gene recombinant plasmids to obtain the PE / L-αCTLA4-BiTE-P2A-CXCL9~PE / L-IL21-T2A-CXCL10 recombinant plasmids carrying the nucleic acid sequence SEQ ID NO: 18. The recombinant plasmids used blue fluorescence as the recombinant selection signal. The nucleotide sequence encoding blue fluorescent protein (BFP) is shown in SEQ ID NO: 16, the amino acid sequence of BFP is shown in SEQ ID NO: 17, and the blue fluorescent protein promoter is the nucleotide sequence shown in SEQ ID NO: 15. The plasmid map is shown in [image missing]. Figure 1 As shown.

[0108] The nucleotide sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25 (i.e., the nucleotide sequences encoding the CD8 hinge region and CD8 transmembrane region (cd8hinger+Cd8tm), the amino acid sequence of cd8hinger+Cd8tm is shown in SEQ ID NO: 26), SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31 were synthesized by Beijing Qingke Biotechnology Co., Ltd. These sequences were assembled and cloned into the corresponding C16L-VGF1 gene recombinant plasmid using molecular cloning techniques, yielding SEQ ID NO: 30, HRE-P11-dCRE~5*loxp-P11-αCD3-TM (αCD3-TM is an abbreviation, specifically referring to the αCD3 antibody fusion with cd8hinger+Cd8tm). The recombinant plasmid used a green fluorescent signal as the recombinant selection signal. The nucleotide sequence encoding green fluorescent protein (GFP) is shown in SEQ ID NO: 26. As shown in Figure 20, the amino acid sequence of GFP is shown in SEQ ID NO: 21, the green fluorescent protein promoter is the nucleotide sequence shown in SEQ ID NO: 19, and the plasmid map is shown in Figure 20. Figure 2 As shown.

[0109] The nucleotide sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38 were synthesized by Beijing Qingke Biotechnology Co., Ltd., and cloned into the corresponding C16R-VGF2 gene recombinant plasmids to obtain the HRE-PE / LL-IFNβ~HRE-PE / LL-IFNγ recombinant plasmids of SEQ ID NO: 40. The recombinant plasmids used red fluorescence as the recombinant selection signal. The nucleotide sequence encoding red fluorescent protein (mCherry) is shown in SEQ ID NO: 38, the amino acid sequence of mCherry is shown in SEQ ID NO: 39, and the red fluorescent protein promoter is the sequence shown in SEQ ID NO: 37. The plasmid map is shown below. Figure 3 As shown.

[0110] Example 2: Recombination, purification and titer determination of vaccinia virus

[0111] The symbols are explained as follows:

[0112] WT: Wild-type vaccinia virus Tiantan strain (Shanghai Xinwan Biotechnology Co., Ltd.);

[0113] ΔTK: Knock out the TK gene and insert the nucleotide sequence shown in SEQ ID NO: 18 at the corresponding position;

[0114] ΔTKΔVGF1: Simultaneously knock out the TK gene and the C16L-VGF1 gene, and insert the nucleotide sequences shown in SEQ ID NO: 18 and SEQ ID NO: 30 at the corresponding positions, respectively;

[0115] ΔTKΔVGF2: Simultaneously knock out the TK gene and the C16R-VGF2 gene, and insert the nucleotide sequences shown in SEQ ID NO: 18 and SEQ ID NO: 40 at the corresponding positions, respectively;

[0116] ΔTKΔVGF1ΔVGF2: Simultaneous knockout of the TK, C16L-VGF1, and C16R-VGF2 genes, followed by insertion of the nucleotide sequences shown in SEQ ID NO: 18, SEQ ID NO: 30, and SEQ ID NO: 40 at their respective sites. The structures of the three gene sets inserted into the TK, C16L-VGF1, and C16R-VGF2 sites of the vaccinia virus are shown below. Figure 4 As shown in A; the vaccinia virus with three sets of exogenous genes inserted can emit red, green and blue light simultaneously. Figure 4 B).

[0117] The specific method of this embodiment is as follows:

[0118] 1. Recombination of vaccinia virus using vaccinia gene recombinant plasmids.

[0119] 1) Cell preparation: Prepare TK143 cells. - Cells were seeded in 6-well plates, approximately 1 × 10⁶ cells per well. 6 After culturing for approximately 24 hours, when the cells have adhered to the wall and covered the entire bottom surface, proceed to the next step.

[0120] 2) Vaccine virus incubation: Infect cells with wild-type vaccinia virus Tian Tan strain at 0.0125 / 3 PFU (PFU: plaque forming units, virus titer) / cell. After incubation at 37℃ for 1 hour, remove the cells, aspirate the supernatant, wash once with 1 mL PBS, and then add 1 mL DMEM complete medium (DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)).

[0121] 3) Plasmid transfection: Transfect TK143 with the vaccinia virus gene recombinant plasmid. - Cells. Incubate at 37°C for approximately 48 hours, the exact time depending on the cytopathic effect.

[0122] 4) Prepare 2×DMEM maintenance medium (containing 1% PS and 2% FBS) for virus plaque preparation, and add 2% preheated low melting point agarose.

[0123] 5) Remove the supernatant from the 6-well plate and add 4 mL of the speckle-laying mixture to each well (300 μL). Then carefully place the plate in a 4°C freezer to promote solidification. Once the low-melting-point agarose has solidified, transfer it to a 37°C incubator for further incubation.

[0124] 6) Pick recombinant viral plaques under a fluorescence microscope and add 500 μL of DMEM complete culture medium. Repeat the freeze-thaw cycle at -80℃ at least three times to release as much virus as possible. This purification process needs to be performed at least 5 times.

[0125] 7) Then perform small-scale amplification of recombinant vaccinia virus, and plate it with TK143. - Cells were cultured in 6-well plates, 1 × 10⁶ cells per well. 6 Each cell is used when the area of ​​the well plate is approximately 100% of the bottom area of ​​the plate.

[0126] 8) Before inoculating with the virus, replace the medium in the wells with 2 mL of DMEM maintenance medium (DMEM medium + 2% FBS + 1% PS). Repeatedly pipette the purified, fluorescent virus solution until it disperses. Add approximately 100 μL of virus solution to each well. Incubate at 37°C for approximately 48 hours, and collect the samples based on the formation of viral plaques.

[0127] 9) Sample collection: Carefully aspirate 1 mL of the culture medium supernatant from the well. Use the remaining 1 mL of culture medium to thoroughly blow off the cells and collect them in an EP tube. This can be used for subsequent genome extraction and as a seed for amplification.

[0128] 2. Amplification and purification of recombinant vaccinia virus

[0129] 1) VERO cell plate making: Take 10 cm dishes, approximately 5 × 10 cells per dish. 6 It is advisable to ensure that the cell density reaches 100% when inoculating with the vaccinia virus the next day.

[0130] 2) Before virus inoculation, replace the DMEM complete medium with 8 mL of maintenance medium (DMEM medium + 2% FBS + 1% PS). Inoculate the cells in the maintenance medium with the virus at an inoculation volume of approximately 0.02 MOI (MOI = virus PFU / cell number). Continue culturing in an incubator at 37°C and 5% CO2 for approximately 48 hours. Collect samples based on the formation of viral plaques.

[0131] 3) Collecting vaccinia virus: Discard 8 mL of culture medium in the dish, take 2 mL of maintenance culture medium, blow off the remaining cells, and collect them in a 15 mL centrifuge tube.

[0132] 4) After freezing for 24 hours, the collected virus solution was repeatedly frozen and thawed twice. It was then subjected to density gradient centrifugation with 36% sucrose solution at 16000 g and 4℃ for 90 min. The supernatant was carefully discarded, and the virus precipitate in the centrifuge tube was dissolved with PBS buffer. The tubes were then aliquoted and stored at -80℃ until the virus titer was determined.

[0133] 3. Titer determination of recombinant vaccinia virus

[0134] 1) TK143 - Cell preparation: TK143 - Cells were seeded in 24-well plates, approximately 2 × 10⁶ cells per well. 5 Each cell should be used in a 24-well plate with a cell density of 100% of the bottom area.

[0135] 2) Dilute the virus. Dilute the vaccinia virus solution with maintenance medium, starting from 1:100 and performing 10-fold serial dilutions until the final volume is 1100 μL.

[0136] 3) Discard the complete culture medium in the 24-well plate, add 500 μL of diluted virus solution to each well, and make two replicates. Incubate at 37°C and 5% CO2 for about 48 hours, depending on the formation of viral plaques.

[0137] 4) Viral plaque counting: First, observe whether the number of viral plaques decreases by a factor of ten. Then, count the number of single-digit plaques in the two replicates of the seed virus. The sum of the plaque values ​​in the two wells, multiplied by the reciprocal of the dilution value of that well, is the virus titer in 1 mL.

[0138] Example 3: Detection of the killing effect of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus on VERO cells overexpressing CTLA4

[0139] This embodiment validates the in vitro CTLA4-VERO cell killing experiment mediated by the control oncolytic vaccinia virus ΔTKΔVGF1ΔVGF2-Ipilimumab (constructed using the same method as in Example 2) and the experimental group ΔTKΔVGF1ΔVGF2. The control oncolytic vaccinia virus targets CTLA4 with Ipilimumab, which is achieved by replacing the fragment corresponding to SEQ ID NO: 4 of the experimental group ΔTKΔVGF1ΔVGF2 with the encoding nucleic acid of Ipilimumab (as shown in SEQ ID NO. 43).

[0140] This example uses the CCK-8 kit (Cell Counting Kit-8) (Novizan, catalog number A311-02), a rapid and highly sensitive colorimetric assay kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), widely used for cell proliferation and cytotoxicity. The detection principle is as follows: In the presence of the electron coupling agent 1-Methoxy PMS (1-methoxy-5-methylphenolazinium sulfate methyl ester), WST-8 can be reduced by certain dehydrogenases in mitochondria to generate an orange-yellow, water-soluble formazan. The intensity of the formazan color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at 450 nm using a microplate reader to indirectly reflect the number of viable cells.

[0141] The specific method of this embodiment is as follows:

[0142] Preparing the cell plate: CTLA4-VERO target cells were cultured at a concentration of 1×10⁻⁶. 4 The cells were seeded at a density of cells / well in 96-well flat-bottomed transparent plates and incubated overnight at 37°C until they adhered to the plate.

[0143] Twenty-four hours after plating the cells, 250 PFU of control oncolytic vaccinia virus ΔTKΔVGF1ΔVGF2-Ipilimumab and experimental ΔTKΔVGF1ΔVGF2 were added to each well, with a volume of 100 μL. Then, 50 μL of activated T cells were added, with effector-to-target ratios (E:T) of 1:1 and 2:1. Controls were set up with supernatant and T cells alone, no T cells, and no cells. Cells were incubated overnight at 37°C for cell killing.

[0144] Cell killing assay: Perform the cell killing assay at approximately 22 h (cell killing can be observed under a microscope). Discard the supernatant in the 96-well plate, tap it a few times on absorbent paper, add the prepared 10% CCK-8 solution (10 μL CCK-8 solution + 90 μL culture medium) at 100 μL / well, and incubate the plate in the dark for 2 hours. Measure the absorbance at 450 nm using a microplate reader (BioTek, model 800TS).

[0145] The results are as follows Figure 5 As shown. From Figure 5 It can be seen that both the control oncolytic vaccinia virus ΔTKΔVGF1ΔVGF2-Ipilimumab and the experimental group ΔTKΔVGF1ΔVGF2 mediate the activity of T cells in efficiently killing CTLA4-VERO target cells, and the killing activity is significantly enhanced with the increase of effector-to-target ratio. Comparing the control oncolytic vaccinia virus ΔTKΔVGF1ΔVGF2-Ipilimumab and the experimental group ΔTKΔVGF1ΔVGF2, it can be seen that at the same effector-to-target ratio, the experimental group showed significantly higher activity in killing CTLA4-VERO cell lines than the control group, with p-values ​​of 0.0028 (effector-to-target ratio of 1:1) and 0.0093 (effector-to-target ratio of 2:1), respectively. This indicates that the CTLA4 T cell adjuvant expressed in the recombinant vaccinia virus ΔTKΔVGF1ΔVGF2 of the present invention has a potent activity in mediating the killing of CTLA4-VERO cells in vitro.

[0146] Example 4: Detection of the ability of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus to express IL-21

[0147] TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates and infected the next day after cell adhesion. The multiplicity of infection (MOI) was 0.025, and the cells were infected with wild-type vaccinia virus and ΔTKΔVGF1ΔVGF2 vaccinia virus. After infection, the cells were incubated at 37°C for 24 h. After incubation, the supernatant was collected to detect IL-21 expression using an IL-21 detection kit (Biolegend, catalog number: 433804).

[0148] The day before, dilute the capture antibody (IL-21 antibody) (Biolegend, catalog number: 433804) with 1× coating buffer B. Add 100 μL of the capture antibody solution to the corresponding wells of a 96-well plate. Seal the plate and incubate overnight (16–18 hours) between 2°C and 8°C. Equilibrate all reagents at room temperature before use. The next day, add 300 μL of wash buffer (Biolegend, catalog number: 421601) to each well and wash the plate four times, then tap the plate firmly to remove residual buffer. To block nonspecific binding and reduce background, add 200 μL of 1× assay diluent A (Biolegend, catalog number: 433804) to each well, seal the plate, incubate at room temperature for 1 hour, and shake on a plate shaker. Wash the plate four times with wash buffer. Add 100 μL of sample per well to the corresponding well. Seal the plate and incubate at room temperature with shaking for 2 hours. Wash the plate four times with wash buffer. Add 100 μL of diluted detection antibody solution to each well, seal the plate, and incubate with shaking at room temperature for 1 hour. Wash the plate 4 times with wash buffer. Add 100 μL of diluted avidin-HRP solution to each well. Seal the plate and incubate with shaking at room temperature for 30 minutes. Wash the plate 5 times with wash buffer, 30 seconds to 1 minute each time. Add 100 μL of freshly mixed TMB substrate (A:B ratio 1:1) and incubate in the dark for 15 minutes. Positive wells should turn blue. Sealing the plate is not required in this step. Add 100 μL of stop solution (Biolegend, catalog number: 423001) to each well to stop the reaction. Positive wells should change from blue to yellow. Immediately place the plate in a microplate reader (BioTek, Gen5) and measure the OD value of each well at 450 nm within 5 minutes.

[0149] The results are as follows Figure 6 As shown. From Figure 6 It can be seen that the OD450 absorbance value of the WT group is close to 0, indicating that wild-type vaccinia virus hardly expresses IL-21. In contrast, the OD450 absorbance value of the experimental group is about 2.5, indicating that the ΔTKΔVGF1ΔVGF2 recombinant vaccinia virus can strongly induce IL-21 expression, with a level approximately 26 times that of the WT group, which is significantly higher than that of wild-type vaccinia virus.

[0150] Example 5: Detection of the ability of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus to express CXCL9

[0151] TK143 - Cells at 2×10 5Cells were seeded per well into 12-well plates and infected the next day after cell adhesion. The multiplicity of infection (MOI) was 0.025, and the cells were infected with wild-type vaccinia virus and ΔTKΔVGF1ΔVGF2 vaccinia virus. After infection, the cells were incubated at 37°C for 24 h. After incubation, the supernatant was collected to detect CXCL9 expression using a CXCL9 detection kit (Abclonal, catalog number: RK04210).

[0152] Remove the unused microplate strips from the plate frame, and place the remaining strips back into the aluminum foil bag containing desiccant, then reseal and store. Add 350 μL of 1× wash buffer (Abclonal, catalog number: RM00026) to each well, let stand for 40 seconds, then discard the liquid. Repeat this washing step 3 times. Add 100 μL of sample diluent (Abclonal, catalog number: RM00023) to the blank wells. Add 100 μL of infection supernatant to each of the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 hours. Prepare the biotinylated antibody (30×) working solution (Abclonal, catalog number: RM00024) 15 minutes before use. Discard the liquid in the wells, and add 350 μL of 1× wash buffer (Abclonal, catalog number: RM00026) to each well for washing 3 times. Add 100 μL of prepared 1× biotinylated antibody working solution to each well, cover with a new sealing film, and incubate at 37°C for 1 h. Prepare streptavidin-HRP (30×) working solution 15 minutes before use. Discard the liquid in the wells, add 350 μL of 1× wash buffer to each well and wash 3 times. Add 100 μL of 1× streptavidin-HRP working solution to each well, cover with a new sealing film, and incubate at 37°C for 30 min. Discard the liquid in the wells and repeat the washing steps. Add 100 μL of TMB substrate (Abclonal, catalog number: RM00027) to each well. Incubate at 37°C in the dark for 15–20 min. Add stop solution (Abclonal, catalog number: RM00028) (50 μL / well) and immediately place the well in a microplate reader (BioTek, instrument model Gen5) and measure the OD value at 450 nm for each well within 5 min.

[0153] The results are as follows Figure 7 As shown. From Figure 7 It can be seen that the OD450 absorbance value of the WT group is close to 0, indicating that wild-type vaccinia virus hardly expresses CXCL-9. In contrast, the OD450 absorbance value of the experimental group is about 3.3, indicating that the ΔTKΔVGF1ΔVGF2 recombinant vaccinia virus can strongly induce the expression of CXCL-9, at a level about 50 times that of the WT group, which is significantly higher than that of wild-type vaccinia virus.

[0154] Example 6: Detection of the ability of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus to express CXCL10

[0155] TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates and infected the next day after cell adhesion. The multiplicity of infection (MOI) was 0.025, and the cells were infected with wild-type vaccinia virus and ΔTKΔVGF1ΔVGF2 vaccinia virus. After infection, the cells were incubated at 37°C for 24 h. After incubation, the supernatant was collected to detect CXCL10 expression using a CXCL10 detection kit (Abclonal, catalog number: RK00054).

[0156] Remove the unused microplate strips from the plate frame, and place the remaining strips back into the aluminum foil bag containing desiccant, then reseal and store. Add 350 μL of 1× wash buffer (Abclonal, catalog number: RM00026) to each well, let stand for 40 seconds, then discard the liquid. Repeat this washing step 3 times. Add 100 μL of sample diluent (Abclonal, catalog number: RM00023) to the blank wells. Add 100 μL of infection supernatant to each of the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 hours. Prepare the biotinylated antibody (30×) working solution (Abclonal, catalog number: RM00024) 15 minutes before use. Discard the liquid in the wells, and wash 3 times with 350 μL of 1× wash buffer per well. Add 100 μL of the prepared 1× biotinylated antibody working solution to each well, cover with a new sealing film, and incubate at 37°C for 1 hour. Prepare streptavidin-HRP (30x) working solution (Abclonal, catalog number: RM00025) 15 minutes before use. Discard the liquid in the wells and wash three times with 350 μL of 1× wash buffer per well. Add 100 μL of 1× streptavidin-HRP working solution to each well, cover with a new sealing film, and incubate at 37°C for 30 min. Discard the liquid in the wells and repeat the washing steps. Add TMB substrate (Abclonal, catalog number: RM00027) (100 μL / well) to the wells. Incubate at 37°C in the dark for 15–20 min. Add stop solution (Abclonal, catalog number: RM00028) (50 μL / well) and immediately place the plate in a microplate reader (BioTek, model Gen5). Measure the OD value at 450 nm for each well within 5 min.

[0157] The results are as follows Figure 8 As shown. From Figure 8It can be seen that the OD450 absorbance value of the WT group is close to 0, indicating that wild-type vaccinia virus hardly expresses CXCL-10. In contrast, the OD450 absorbance value of the experimental group is about 3.5, indicating that the ΔTKΔVGF1ΔVGF2 recombinant vaccinia virus can strongly induce the expression of CXCL-10, with a level approximately 53 times that of the WT group, which is significantly higher than that of wild-type vaccinia virus.

[0158] Example 7: Detection of Differential Killing of Tumor Cells by Recombinant Oncolytic Vaccine Virus

[0159] Tumor cell killing efficiency was assessed using a luciferase-based cytotoxicity assay. First, 1×10⁻⁶ cells were used... 4 One Huh7-Luc cell line (a liver cancer cell line modified with the firefly luciferase gene) was seeded into each well of a 96-well plate with 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum (FBS). The cells were incubated at 37°C in a 5% CO2 incubator for 18 h. On the second day, the multiplicity of infection (MOI) was 0.025. Wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus were added to the wells containing the target cells. The cells were then incubated under either normoxic (21% O2) or hypoxic (with 16 μM molidustat) conditions for 22 h. After co-culture, the luciferase activity of the target cells was measured using a GloMax® 96 microplate luminescence detector.

[0160] The formula for calculating cell killing rate is shown below:

[0161] Cell killing rate (%) = (Luciferase activity value of the uninfected vaccinia virus group - Luciferase activity value of the experimental group) / Luciferase activity value of the uninfected vaccinia virus group × 100

[0162] The results are as follows Figure 9 As shown. From Figure 9It can be seen that neither wild-type vaccinia virus nor ΔTK vaccinia virus can effectively kill Huh7 tumor cells under normoxic or hypoxic conditions, and they lack selective killing characteristics. ΔTKΔVGF2 vaccinia virus without an in vivo T-cell adjuvant exhibits low tumor-killing activity under both normoxic and hypoxic conditions, with a kill rate of 11.05% under normoxic conditions and 2.2% under hypoxic conditions. In contrast, ΔTKΔVGF1 and ΔTKΔVGF1ΔVGF2, equipped with in vivo T-cell adjuvants, can selectively and efficiently kill Huh7 tumor cells under hypoxic conditions, with kill rates of 30% and 29.3%, respectively. Their specific tumor-killing activity under hypoxic conditions is 1.4 times and 3.3 times that under normoxic conditions, respectively.

[0163] Example 8: Detection of the ability of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus to express interferon

[0164] TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates and infected the next day after cell adhesion. The multiplicity of infection (MOI) was 0.025, and wild-type vaccinia virus and ΔTKΔVGF1ΔVGF2 vaccinia virus were used for infection. After infection, cells were incubated at 37°C for 24 h under normoxic (21% O2) or hypoxic (16 μM modostat) conditions. After incubation, the supernatant was collected to detect the expression of IFN-β and IFN-γ using an IFN-β detection kit (Abclonal, catalog number: RK01630) and an IFN-γ detection kit (Abclonal, catalog number: RK00015), respectively.

[0165] Remove the unused microplate strips from the plate frame, and place the remaining strips back into the aluminum foil bag containing desiccant, then reseal and store. Add 350 μL of 1× wash buffer (Abclonal, catalog number: RM00026) to each well, let stand for 40 seconds, then discard the liquid. Repeat this washing step 3 times. Add 100 μL of sample diluent (Abclonal, catalog number: RM00023) to the blank wells. Add 100 μL of infection supernatant to each of the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 hours. Prepare the biotinylated antibody (30×) working solution (Abclonal, catalog number: RM00024) 15 minutes before use. Discard the liquid in the wells, and wash 3 times with 350 μL of 1× wash buffer per well. Add 100 μL of the prepared 1× biotinylated antibody working solution to each well, cover with a new sealing film, and incubate at 37°C for 1 hour. Prepare streptavidin-HRP (30×) working solution (Abclonal, catalog number: RM00025) 15 minutes before use. Discard the liquid in the wells and wash three times with 350 μL of 1× wash buffer per well. Add 100 μL of 1× streptavidin-HRP working solution to each well, cover with a new sealing film, and incubate at 37°C for 30 min. Discard the liquid in the wells and repeat the washing steps. Add TMB substrate (Abclonal, catalog number: RM00027) (100 μL / well) to the wells. Incubate at 37°C in the dark for 15–20 min. Add stop solution (Abclonal, catalog number: RM00028) (50 μL / well) and immediately place the plate in a microplate reader (BioTek, model Gen5). Measure the OD value of each well at 450 nm within 5 min.

[0166] The results are as follows Figure 10 As shown. Figure 10 As shown in Figure A, under normoxic conditions, the OD450 absorbance is approximately 2.6, indicating a high expression level of INF-β. However, under hypoxic conditions, the OD450 absorbance is approximately 0.6, indicating a significant decrease in the expression level of INF-β. Figure 10 A showed a 4-fold difference in INF-β expression levels between normoxic and hypoxic conditions in ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus. Figure 10 As shown in Figure B, under normoxic conditions, the OD450 absorbance is approximately 2.6, indicating a high expression level of INF-γ. However, under hypoxic conditions, the OD450 absorbance is approximately 1.3, indicating a significant decrease in the expression level of INF-γ. Figure 10 B showed a two-fold difference in the expression level of INF-γ between ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus and normoxic and hypoxic conditions.

[0167] Figure 10The results indicate that ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus can differentially express vaccinia virus inhibitory factors INF-β and INF-γ under normoxic and hypoxic conditions. The expression levels of INF-β and INF-γ under hypoxic conditions are significantly lower than those under normoxic conditions, thus enabling ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus to function specifically in hypoxic environments, such as tumor environments.

[0168] Example 9: Detection of Recombinant Oncolytic Vaccine Virus Replication Capacity

[0169] The liver cancer cells Huh7 were treated at a rate of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well into 12-well plates, with 1 mL of cell culture medium (DMEM medium containing 10% (v / v) fetal bovine serum (FBS)) added to each well. The plates were incubated overnight, and wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus were added to each well. Two hours after infection, the cells were washed twice with 1 mL of PBS, and then replaced with 1 mL of DMEM medium containing 2% (v / v) fetal bovine serum (FBS). The plates were then incubated at 37°C in a 5% CO2 incubator. Cells and culture medium were harvested at 12, 24, 48, and 72 hours, and viral copy numbers of the five vaccinia viruses were measured at each time point.

[0170] The detection method in this embodiment is as follows:

[0171] (1) Cell seeding: Huh7 cells were seeded into 96-well plates at a density of 1.5 × 10⁶ cells per well. 5 Each cell.

[0172] (2) Virus inoculation: Five types of vaccinia virus were inoculated at a rate of 1×10⁻⁶ per well. 4 Copy the inoculation into Huh7 cells; wash twice with 1×PBS 2 hours after infection, replace with 1 mL of new DMEM medium containing 2% (v / v) FBS (with 16 μM modostat), and incubate normally in a 37℃, 5% CO2 incubator.

[0173] (3) Collecting the cell-virus solution: Add 10 μL of proteinase K solution to the corresponding well. After the cells detach from the wall of the 12-well plate, collect them together with the culture medium into an EP tube, and then add 50 μL of 20× lysis buffer (1% SDS + 2 mg / ml proteinase K solution). Mix well and incubate in a metal bath at 58°C for 1 hour. Take 50 μL of the sample and add 50 μL of 1×TE buffer. Mix well and place in a PCR instrument at 98°C for 10 min to inactivate PK enzyme.

[0174] (4) Detection of sample copy number: Take 2 μL as template and perform qPCR reaction. The reaction conditions are set as follows: 95℃ pre-denaturation for 2 min; then enter the cyclic reaction, including 95℃ denaturation for 10 s; 60℃ annealing and extension for 30 s, 40 cycles; the instrument defaults to 1 cycle. The primer sequences used are as follows: forward primer TTV-qF: 5'-CCATTTAATGCGAGGCCGTT-3' (SEQ ID NO: 41); reverse primer TTV-qR: 5'-CAACCACATATCTGCGACGG-3' (SEQ ID NO: 42).

[0175] (5) Calculation method: The standard quality plasmid was puc18-E10L (stored at Shanghai Xinwan Biotechnology Co., Ltd.). The standard was serially diluted 10-fold using nuclease-free TE buffer to create three concentration gradients: 1.205 × 10⁻⁶. 8 Copies / μL, 1.205 × 10 7 Copy / μL and 1.205×10 6 Copy number / μL. Mix thoroughly after each dilution to avoid cross-contamination. Then, take 2 μL as a template. Obtain the cycle threshold (Cq value) and the logarithm of the copy number (Log value) to plot a linear equation. Then, substitute the Cq value of the sample into the constructed linear equation to calculate the copy number of the original sample solution, and multiply by the sample volume to obtain the total copy number of the sample.

[0176] The results are as follows Figure 11 As shown. Figure 11 The replication curves of wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus are shown. Under hypoxic conditions, ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus showed a 3.8-fold increase compared to normal oxygen conditions; while wild-type vaccinia virus showed a 1.2-fold decrease, ΔTK vaccinia virus showed a 1.2-fold increase, ΔTKΔVGF1 vaccinia virus showed a 5-fold decrease, and ΔTKΔVGF2 vaccinia virus showed a 3.2-fold decrease. Under hypoxic conditions, the replication capacity of ΔTKΔVGF1ΔVGF2 oncolytic vaccinia virus was not weakened, but rather increased.

[0177] Example 10: Antitumor effect, tumor clearance rate, and body weight change of oncolytic vaccinia virus in mouse models.

[0178] One day prior to the inoculation, NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) housed in sterile isolators underwent localized hair removal on their backs. Hair removal cream or an animal shaving tool could be used to expose the skin at the tumor cell inoculation site. The mouse was held firmly with the left hand, simultaneously grasping its head, neck, and back skin, turning it to the left to fully expose the shaved area on the right side of its back. The right hand was then used to disinfect the area with an alcohol swab. 1 mL of the prepared NCI-H292 or Huh7 tumor cells were mixed by pipetting with a 1 mL insulin syringe, and then 125 μL of the cell suspension (5 × 10⁻⁶ cells / mL) was aspirated. 6 (125 μL of tumor cells). Insert the needle subcutaneously into the mouse at a 30°-40° angle to the skin and slowly inject the cell suspension, avoiding cell leakage. After injecting 125 μL of cell suspension, leave the needle in place for 2-3 seconds and then quickly withdraw it. A clearly visible small bulge will appear under the skin at the injection site.

[0179] After cell inoculation, the tumor formation and health status of mice were observed every 2-3 days. After tumor formation, the baseline tumor volume was measured with calipers and subsequent experiments were conducted.

[0180] 1×10⁻⁶ cells were infused intratumorally. 4 PFU wild-type vaccinia virus, ΔTK vaccinia virus, ΔTKΔVGF1 vaccinia virus, ΔTKΔVGF2 vaccinia virus, and ΔTKΔVGF1ΔVGF2 vaccinia virus. T cells were intravenously infused on the third day after intratumoral injection, and tumor size and mouse weight were measured every 2-3 days. Figure 12 The major and minor axes of the tumor were measured using vernier calipers. The formula for calculating the tumor volume is as follows:

[0181] Volume = (major axis × minor axis 2) / 2

[0182] The results showed that ΔTKΔVGF1 and ΔTKΔVGF1ΔVGF2 vaccinia virus (with an in vivo T-cell adaptor) could effectively inhibit the growth of NCI-H292 (lung cancer) and Huh7 (liver cancer). Among them, ΔTKΔVGF1 oncolytic virus showed a 100% inhibition rate against NCI-H292 cells 63 days after injection. Figure 14 D and Figure 16 The inhibition rate of ΔTKΔVGF1ΔVGF2 vaccinia virus against NCI-H292 cancer was also 100%. Figure 14 F and Figure 16 ).

[0183] Compared to the wild-type control group vaccinia virus, the recombinant oncolytic virus ΔTKΔVGF1ΔVGF2 significantly prolonged the survival time of mice. All mice in the wild-type control group died within 40 days, while all mice in the ΔTKΔVGF1ΔVGF2 vaccinia virus group survived for 60 days. Figure 13).

[0184] The results of weight change in mice indicate that the ΔTKΔVGF1ΔVGF2 vaccinia virus has a high degree of safety. Figure 17 and Figure 18 In the ΔTKΔVGF1ΔVGF2 vaccinia virus group, only one mouse experienced a decrease in body weight on day 59, while the rest steadily increased their weight. Figure 17 F). Half of the control mice experienced a decrease in body weight. Figure 17 A), all mice in the wild-type vaccinia virus group experienced a decrease in body weight. Figure 17 B), the body weight of all mice in the ΔTK vaccinia virus group decreased ( Figure 17 C), ΔTKΔVGF1 vaccinia virus caused a decrease in body weight in 3 / 8 of mice ( Figure 17 D), 5 / 8 mice in the ΔTKΔVGF2 vaccinia virus group experienced a decrease in body weight ( Figure 17 E).

[0185] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

[0186] The specific sequences corresponding to each serial number in this invention are as follows:

[0187] SEQ ID NO: 1:

[0188] aaaaattgaaattttattttttttttttggaatataaata

[0189] SEQ ID NO: 2:

[0190] atgggctggtcttgcattattctgttcctcgtggccaccgccacaGGCGTGCATAGT

[0191] SEQ ID NO: 3:

[0192] MGWSCIILFLVATATGVHS

[0193] SEQ ID NO: 4:

[0194]

[0195] SEQ ID NO: 5:

[0196] DIQLTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADSFPLTFGPGTKVDIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSNPFTFGQGTKVEIKRGGGGSGGGGSGGGGSEVQLLESGGGLVQPGESLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNILYLQMNSLRAEDTAVYYCAKGPVGESVLYYYYGMDVWGQGTPVTVSS

[0197] SEQ ID NO: 6:

[0198] GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG

[0199] SEQ ID NO: 7:

[0200] ATNFSLLKQAGDVEENPGP

[0201] SEQ ID NO: 8:

[0202] AAGAAGTCTGGAGTGCTGTTCCTCCTCGGAATTATTCTCCTCGTGCTCATCGGCGTGCAGGCACACCAGTGGTGCGCAAGGGACGCTGCTCTTGCATTTCTACCAACCAGGGCACAATTCACCTCCAGTCCCTCAAGGACCTCAGCAAGTCGCCCCTAGCCCATCTTGCGAGAGAGATTGATTGATT ATCGCCACACTCAAGAACGGGGTGCAGACATGCCTGAACCCCGACTCCGCCGACGTGAAGGAGCTGATTAAGAAGTGGGAGAAGCAGGTGTCCCAGAAAGAAGCAGAAACGGAAAGAAGCACCAGAAGAAGGTGCTCAAGGTGCCAAGTCCCAGCGGTCCCGGAGAGAGAGAGCAACAAAGA

[0203] SEQ ID NO: 9:

[0204] KKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKVSQKKKKNGKKHQKKKVLKVRKSQRSRQKKTT

[0205] SEQ ID NO: 10:

[0206] atgagatccagtcctggcaacatggagaggattgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgccacatgattagaatgcgtcaacttatagatattgttgatcagctgaaaaattatgtgaatgacttggtccctgaatttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgTtttcagaaggcccaactaaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccaccttccacaaatgcagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacccaaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatctgtccAGtagaacacacggaagtgaagattcc

[0207] SEQ ID NO: 11:

[0208] gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca

[0209] SEQ ID NO: 12:

[0210] EGRGSLLTCGDVEENPGP

[0211] SEQ ID NO: 13:

[0212] AACCAGACCGCCATCCTGATTTGCTGCCTGATCTTCCTGACACTGTCCGGCATTCAGGGCGTGCCACTGAGCCGCACAGTGAGATGCACATGCATTTCCATTTCTAACCAGCCCGTGAACCCAAGGAGTTTGGAAAAGTTGGAGATCATTCCAGCCTCGCAGTTCTGCCCACGCGTGGAGATCATCGCTACAATGAAGAAGAAGGGCGAGAAGCGCTGCCTCAACCCCGAGTCTAAGGCTATTAAGAACCTGCTCAAGGCCGTGTCTAAGGAGCGGTCTAAGCGAAGTCCA

[0213] SEQ ID NO: 14:

[0214] NQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP

[0215] SEQ ID NO: 15:

[0216] taaataataaatacaataattaatttctcgtaaaagtagaaaatatattctaatttattgcacggtaaggaagtagatcataactcgagc

[0217] SEQ ID NO: 16:

[0218] ATGagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagacgggggcgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcaccgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacatcaagaccacatatagatccaagaaacccgctaagaacctcaagatgcctggcgtctactatgtggactacagactggaaagaatcaaggaggccaacaacgagacctacgtcgagcagcacgaggtggcagtggccagatactgcgacctccctagcaaactggggcacaagcttaattGa

[0219] SEQ ID NO: 17:

[0220] MSELIKENMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN-

[0221] seq Id No: 18:

[0222]

[0223] SEQ ID NO: 19:

[0224] gctcgagttatgatctacttccttaccgtgcaataaattagaatatattttctacttttacgagaaattaattattgtatttattatttatgggtgaaaaac

[0225] SEQ ID NO: 20:

[0226] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0227] SEQ ID NO: 21:

[0228] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK-

[0229] SEQ ID NO: 22:

[0230]

[0231] SEQ ID NO: 23:

[0232] MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNFIRNLDSETGAMVRLLEDGDSRAAA

[0233] SEQ ID NO: 24:

[0234] ATTTAGAATATATGTATGTAAAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAA

[0235] SEQ ID NO: 25:

[0236] GCCCCAACAATCGCTTCTCAGCCACTGTCTCTCAGACCCGAGGCTTGCCGGCCTGCCGCTGGCGGGGCCGTGCACACACGGGGACTCGACTTCGCTTGCGACATTTACATTTGGGCCCCACTCGCTGGAACATGCGGCGTGCTCCTGCTGTCTCTGGTGATCACACTGTACGTA

[0237] SEQ ID NO: 26:

[0238] APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYV

[0239] SEQ ID NO: 27:

[0240] CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGTCCTGTAAGGCCTCCGGCTACACCTTTACCAGGTACACAATGCACTGGGTCAGGCAGGCTCCTGGCCAGGGCCTGGAGTGGATGGGCTACATCAATCCTAGCAGGGGCTACACAAATTACAACCAGAAGTTTAAGGACAGAGTGACAATCACAGCCGATAAGAGCACCAGCACTGCTTATATGGAACTGTCCAGCCTGAGGTCCGAGGACACCGCCGTGTACTACTGTGCCAGGTACTACGATGACCACTACTGCCTGGATTACTGGGGCCAGGGCACCACCGTGACAGTGAGCAGCGGCGGCGGCGGAAGCGGAGGCGGAGGAAGCGGCGGAGGAGGATCTGGCAGCGAGATCGTGCTGACCCAGAGCCCTGCCACCCTGAGCCTGAGCCCTGGCGAGAGGGCCACCCTGTCCTGCTCCGCTTCCAGCAGCGTGTCCTACATGAACTGGTATCAACAAAAGCCTGGCAAAGCTCCTAAGAGACTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCATCCAGATTCTCCGGCTCCGGCAGCGGCACCGATTTTACCCTGACCATCAACAGCCTCGAAGCCGAGGACGCCGCCACATACTACTGCCAGCAGTGGAGCTCCAACCCTTTTACCTTCGGCCAGGGCACAAAGGTGGAGATCAAGAGG

[0241] SEQ ID NO: 28:

[0242] QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSNPFTFGQGTKVEIKR

[0243] SEQ ID NO: 29:

[0244] ATAACTTCGTATAATGTATGCTATACGAAGTTAT

[0245] SEQ ID NO: 30:

[0246]

[0247] SEQ ID NO: 31:

[0248] CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT

[0249] SEQ ID NO: 32:

[0250] ttttttttttttggaatataaata

[0251] SEQ ID NO: 33:

[0252] ATGACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTACAGCTCTTTCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAACtaa

[0253] SEQ ID NO: 34:

[0254] MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN-

[0255] SEQ ID NO: 35:

[0256] ATGAAATATACAAGTTATATCTTGGCTTTTCAGCTCTGCATCGTTTTGGGTTCTCTTGGCTGTTACTGCCAGGACCCATATGTAAAAGAAGCAGAAAACCTTAAGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAATGGAACTCTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAGAAAAATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTTAAAAACTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCATCAAGGAAGACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAAACGAGATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTTGAATGTCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAACTGTCGCCAGCAGCTAAAACAGGGAAGCGAAAAAGGAGTCAGATGCTGTTTCGAGGTCGAAGAGCATCCCAGTAA

[0257] SEQ ID NO: 36:

[0258] MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ-

[0259] SEQ ID NO: 37:

[0260] GAATATATGTATGTAaaaatatagtagaatttcATTTTGTTTTTTTCTATGCTATAA

[0261] SEQ ID NO: 38:

[0262] atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtag

[0263] SEQ ID NO: 39:

[0264] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK-

[0265] SEQ ID NO: 40:

[0266]

[0267] SEQ ID NO: 43:

[0268]

Claims

1. A recombinant oncolytic virus, the genome of which comprises at least one nucleic acid fragment selected from a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein: The first nucleic acid fragment contains a gene encoding an immunosuppression relief and T cell local activation unit and a gene encoding a T cell function enhancement and chemotactic amplification unit. The gene encoding the immunosuppression relief and T cell local activation unit sequentially contains a gene encoding a first viral transcription promoter, a gene encoding a T cell connector targeting immune checkpoints, a gene encoding a first autosplitting peptide, and a gene encoding a first chemokine. The gene encoding the T cell function enhancement and chemotactic amplification unit sequentially contains a gene encoding a first viral transcription promoter, a gene encoding an immune-enhancing cytokine, a gene encoding a second autosplitting peptide, and a gene encoding a second chemokine. The second nucleic acid fragment includes a gene encoding a hypoxia-sensing unit and a gene encoding a T-cell activation antibody expression unit, wherein the gene encoding the hypoxia-sensing unit includes a gene encoding a hypoxia response element and a gene encoding an artificial transcription factor connected via a gene encoding a second viral transcription promoter, and the gene encoding the T-cell activation antibody expression unit includes a gene encoding an artificial transcription factor recognition element and a gene encoding a T-cell activation antibody connected via a gene encoding a second viral transcription promoter. The third nucleic acid fragment contains at least one gene encoding an immunomodulatory unit, wherein the gene encoding the immunomodulatory unit contains a gene encoding a hypoxia response element and a gene encoding an immunomodulator, which are linked via a gene encoding a third viral transcription promoter.

2. The recombinant oncolytic virus according to claim 1, wherein, The T-cell connector targeting immune checkpoints is a bispecific T-cell connector targeting CTLA4, preferably with the amino acid sequence shown in SEQ ID NO: 5; More preferably, the nucleotide sequence of the gene encoding the T-cell connector targeting immune checkpoints is shown in SEQ ID NO:

4.

3. The recombinant oncolytic virus according to claim 1, wherein, The first chemokine and the second chemokine are each independently selected from one of CXCL9, CXCL10 and CXCL11; preferably, the first chemokine is CXCL9 and the second chemokine is CXCL10. Optionally, the immune-enhancing cytokine is an interleukin; preferably, the immune-enhancing cytokine is selected from one or more of IL-2, IL-7, IL-12, IL-15, IL-21, and IL-27; more preferably, the immune-enhancing cytokine is IL-21. Optionally, the first self-cleaving peptide and the second self-cleaving peptide are each independently selected from P2A and T2A.

4. The recombinant oncolytic virus according to claim 1, wherein, The nucleotide sequence of the gene encoding the hypoxia response element is as shown in SEQ ID NO: 31, or is a repeating nucleotide sequence of more than one, preferably three, more preferably five, nucleotide sequences as shown in SEQ ID NO: 31; Optionally, the gene encoding the artificial transcription factor is selected from one or more of the following: the CRE gene with a nucleus-deposition signal, the CRE-VP64 gene with a nucleus-deposition signal, the CRE-VP64-ODD gene with a nucleus-deposition signal, the CRE-RAP94 gene with a nucleus-deposition signal, the CRE-RAP94-ODD gene with a nucleus-deposition signal, or the CRE-VP160 gene with a nucleus-deposition signal; preferably, the gene encoding the artificial transcription factor is the CRE-VP64 gene with a nucleus-deposition signal; more preferably, the nucleotide sequence of the CRE-VP64 gene with a nucleus-deposition signal is as shown in SEQ ID NO:

22.

5. The recombinant oncolytic virus according to claim 1, wherein, The gene encoding the artificial transcription factor recognition element is one or more loxp genes; preferably, the nucleotide sequence of the gene encoding the artificial transcription factor recognition element is a repeating gene sequence of more than one, preferably three, more preferably five loxp genes, such as the nucleotide sequence shown in SEQ ID NO:

29. Optionally, the T-cell activating antibody is an antibody targeting CD3, CD5, CD7, or CD28; preferably, the T-cell activating antibody is an antibody targeting CD3. Optionally, the first viral transcription promoter, the second viral transcription promoter, and the third viral transcription promoter are each independently selected from one or more of PE / L, PE / LL, truncated PE / L, further truncated PE / L, P7.5, truncated P7.5, further truncated P7.5, LTRminiP, or P11; preferably, the nucleotide sequence of PE / L is as shown in SEQ ID NO: 1; the nucleotide sequence of P11 is as shown in SEQ ID NO: 24; and the nucleotide sequence of PE / LL is as shown in SEQ ID NO:

32.

6. The recombinant oncolytic virus according to claim 1, wherein, The immunomodulator is interferon, and preferably, the immunomodulator is selected from one or more of IFNα, IFNβ and IFNγ; Preferably, the third nucleic acid fragment contains two genes encoding an immunomodulatory unit, wherein the immunomodulator in one gene is IFNβ and the immunomodulator in the other gene is IFNγ.

7. The recombinant oncolytic virus according to any one of claims 1 to 6, wherein, The virus is selected from one or more of the following: vaccinia virus, adenovirus, herpes simplex virus type I, herpes simplex virus type II, vesicular stomatitis virus, echovirus, reovirus, alpha virus, yellow fever virus, Coxsackie virus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus, or retrovirus. Preferably, the recombinant oncolytic virus is a recombinant vaccinia virus.

8. The recombinant oncolytic virus according to any one of claims 1 to 6, wherein, The first nucleic acid fragment is located in the TK region of the genome of the recombinant oncolytic virus; And / or, the second nucleic acid fragment is located in the C16L-VGF1 region of the genome of the recombinant oncolytic virus; And / or, the third nucleic acid fragment is located in the C16R-VGF2 region of the genome of the recombinant oncolytic virus; Preferably, the nucleotide sequence of the first nucleic acid fragment is shown in SEQ ID NO: 18; Preferably, the nucleotide sequence of the second nucleic acid fragment is as shown in SEQ ID NO: 30; Preferably, the nucleotide sequence of the third nucleic acid fragment is as shown in SEQ ID NO:

40.

9. A pharmaceutical composition for treating cancer, comprising a recombinant oncolytic virus according to any one of claims 1 to 8, and optionally pharmaceutically acceptable excipients; Preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.

10. Use of the recombinant oncolytic virus according to any one of claims 1 to 8 in the preparation of a medicament for treating cancer; Preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, or brain cancer.

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