Modified vaccinia virus and use thereof

Abstract

The present application relates to a modified vaccinia virus and a use thereof. Compared with an unmodified vaccinia virus, the modified vaccinia virus comprises an amino acid mutation in L109 protein, and / or the expression and / or activity of L006 protein and L007 protein are inhibited.

Description

Modified vaccinia viruses and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a modified vaccinia virus and its use. Background Art

[0002] Vaccinia virus (VV or VACV) is a double-stranded DNA virus with many characteristics that make it an oncolytic virus with therapeutic advantages. (1) VV has demonstrated good safety in the process of being used as a vaccine to prevent the highly contagious disease smallpox, so its safety as an oncolytic virus can be guaranteed, and its safety has been further confirmed in clinical trials conducted as an oncolytic virus. (2) It can rapidly replicate itself in cells, producing new virus particles in about 6-8 hours, and thus can quickly lyse infected cells. (3) The genome of VV is approximately 190K, and this large genome has a great ability to carry foreign genes. (4) Vaccinia virus does not require specific receptors to infect cells, which makes it suitable for treating more types of tumors. (5) It can be administered through multiple routes, including local intratumoral injection, intraperitoneal injection, intrathoracic injection, and systemic intravenous injection, so it can be used to treat tumors in any part of the body. (6) In addition, the hypoxic microenvironment commonly found in solid tumors affects the replication and efficacy of many types of oncolytic viruses, and VV can replicate effectively in this environment (Hiley et al., Gene Therapy 17, 281-287, 2010). (7) In addition, there are a variety of natural and synthetic promoters that can be applied to VV, which makes it an ideal choice as a vector for carrying exogenous nucleic acid sequences. In recent years, clinical results using vaccinia virus have demonstrated that VV has good anti-tumor effects and is safe (Park et al., Lancet Oncol 9: 533-542, 2008; Breitbach et al., Nature 477: 99-102, 2011).

[0003] Although progress has been made in oncolytic virus research over the past decade, vaccinia virus therapeutic products have not yet entered the clinic and are on the market. The difficulty lies in how to scientifically select and modify vaccinia viruses to make them more efficient in production and have better anti-tumor effects. Summary of the Invention

[0004] The present application provides an oncolytic virus, and more specifically, relates to a modified vaccinia virus. The vaccinia virus of the present application can selectively infect cancer cells, which can be beneficially used in the treatment of cancer.

[0005] The modified vaccinia virus described in this application is obtained by continuous passage. During the continuous passage of vaccinia virus, many meaningless random mutations occur. These mutations do not have much impact on the vaccinia virus with stronger replication and tumor-killing ability studied in this application. However, some meaningful mutations will also appear during the viral passage process. Compared with meaningless mutations, these meaningful mutations will gradually accumulate during the passage process. The proportion of vaccinia viruses with these mutations in the virus cluster gradually increases, becoming the dominant strain. After analysis, such meaningful mutations are the replacement of amino acids A15 and / or H67 on the L109 protein in the vaccinia virus. According to the experimental results of this application, vaccinia viruses containing a single mutation of the L109 protein A15V have significantly enhanced tumor-killing ability. At the same time, vaccinia viruses containing a double mutation of the L109 protein A15V and H67R have an anti-tumor effect that is better than vaccinia viruses with single mutations. This shows that the A15 mutation and the H67 mutation in the L109 protein are both important for enhancing the anti-tumor effect of vaccinia viruses.

[0006] The present applicants also discovered that deletion of the List006 and List007 genes (also referred to herein as Del2 genes) in vaccinia viruses resulted in superior in vitro cell-killing and in vivo anti-tumor efficacy compared to strains without these genes. Furthermore, vaccinia viruses lacking the List006 and List007 genes exhibited higher yields. However, there are currently no reports in the literature on the functions of the List006 and List007 genes.

[0007] The present application provides a modified vaccinia virus, which comprises one or more of the following modifications compared to an unmodified vaccinia virus: (1) the expression and / or activity of L006 protein and L007 protein is inhibited, and (2) an amino acid mutation of L109 protein.

[0008] In certain embodiments, the expression and / or activity of the L006 protein and the L007 protein are inhibited by one or more methods selected from the following: (1) deleting all or part of the genes encoding the L006 protein and the L007 protein; and (2) inserting exogenous genes into the genes encoding the L006 protein and the L007 protein; wherein the partial gene encoding the L006 protein includes a gene encoding a functional domain of the L006 protein, and the partial gene encoding the L007 protein includes a gene encoding a functional domain of the L007 protein.

[0009] In certain embodiments, the expression and / or activity of the L006 protein and the L007 protein is inhibited by completely deleting the L006 protein and the L007 protein.

[0010] In certain embodiments, the L006 protein is encoded by the List006 gene, and the L007 protein is encoded by the List007 gene.

[0011] In certain embodiments, the expression and / or activity of the L006 and L007 proteins are inhibited by one or more of the following methods: (1) deleting all or part of the List006 gene and the List007 gene; and (2) inserting exogenous genes into the genes of the List006 gene and the List007 gene; wherein the List006 gene contains a gene encoding the functional domain of the L006 protein, and the List007 gene contains a gene encoding the functional domain of the L007 protein.

[0012] In certain embodiments, the partial deletion of the List006 and List007 genes comprises deletion of promoters or premature terminators. In certain embodiments, the expression and / or activity of the L006 and L007 proteins is inhibited by complete deletion of the List006 and List007 genes.

[0013] In certain embodiments, the amino acid mutation of the L109 protein includes a substitution of the amino acid at position H67 on the L109 protein. In certain embodiments, the amino acid substituted at position H67 on the L109 protein is an amino acid with an isoelectric point greater than 7.5. In certain embodiments, the amino acid substituted at position H67 on the L109 protein is an amino acid without a cyclic structure on the side chain. In certain embodiments, the amino acid substituted at position H67 on the L109 protein is a basic amino acid. In certain embodiments, the amino acid substituted at position H67 on the L109 protein is R.

[0014] In certain embodiments, the amino acid mutation of the L109 protein further comprises a substitution of the amino acid at position A15 of the L109 protein. In certain embodiments, the amino acid substituted at position A15 of the L109 protein is an amino acid with a molecular weight greater than 90. In certain embodiments, the amino acid substituted at position A15 of the L109 protein is a hydrophobic amino acid. In certain embodiments, the amino acid substituted at position A15 of the L109 protein is V.

[0015] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0016] (1) The amino acid at position H67 on the L109 protein was substituted;

[0017] (2) The amino acid at position A15 on the L109 protein was substituted;

[0018] (3) complete or partial deletion of List006 and List007 genes;

[0019] (4) L006 protein and L007 protein are completely or partially missing; and

[0020] (5) Insert foreign genes into the genes of List006 and List007.

[0021] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0022] (1) The amino acid at position H67 on the L109 protein is substituted with R;

[0023] (2) The amino acid at position A15 on the L109 protein is substituted with V;

[0024] (3) List006 and List007 genes are completely missing;

[0025] (4) L006 and L007 proteins are completely absent;

[0026] (5) List006 and List007 genes are expressed with promoter deletion or premature terminator; and

[0027] (6) Insert foreign genes into the genes of List006 and List007.

[0028] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0029] (1) The L109 protein comprises the amino acid sequence shown in SEQ ID NO: 12;

[0030] (2) the L109 protein comprises the amino acid sequence shown in SEQ ID NO: 14;

[0031] (3) the L109 protein comprises the amino acid sequence shown in SEQ ID NO: 16; and

[0032] (4) The L006 protein and the L007 protein are completely missing, the L006 protein comprises the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein comprises the amino acid sequence shown in SEQ ID NO: 8.

[0033] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0034] (1) L109 protein has the amino acid sequence shown in SEQ ID NO: 12;

[0035] (2) L109 protein has the amino acid sequence shown in SEQ ID NO: 14;

[0036] (3) the L109 protein has the amino acid sequence shown in SEQ ID NO: 16; and

[0037] (4) The L006 protein and the L007 protein are completely missing, the L006 protein has the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein has the amino acid sequence shown in SEQ ID NO: 8.

[0038] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0039] (1) The List109 gene comprises the amino acid sequence shown in SEQ ID NO: 11;

[0040] (2) the List109 gene comprises the amino acid sequence shown in SEQ ID NO: 13;

[0041] (3) the List109 gene comprises the amino acid sequence shown in SEQ ID NO: 15; and

[0042] (4) The List006 gene and the List007 gene are completely deleted, the List006 gene comprises the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene comprises the amino acid sequence shown in SEQ ID NO: 7.

[0043] In certain embodiments, the modified vaccinia virus is selected from one or more of the following groups:

[0044] (1) The List109 gene has the amino acid sequence shown in SEQ ID NO: 11;

[0045] (2) the List109 gene has the amino acid sequence shown in SEQ ID NO: 13;

[0046] (3) the List109 gene has the amino acid sequence shown in SEQ ID NO: 15; and

[0047] (4) The List006 gene and the List007 gene are completely deleted, the List006 gene has the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene has the amino acid sequence shown in SEQ ID NO: 7.

[0048] In certain embodiments, the modified vaccinia virus is selected from one or more of the following: Lister, Western Reserve, Copenhagen, Paris, Tashkent, Tiantan, Wyeth, Brighton, Ankara, Dairen I, LIVP, Connaught, New York City Health Department, and variants thereof. In certain embodiments, the vaccinia virus is a Lister strain.

[0049] In certain embodiments, the modified vaccinia virus further comprises mutations in one or more genes selected from the group consisting of: (1) backbone genes: TK, A34R, A35R, A36R, A46R, A56R, L025, K3L, and F14.5L, and / or (2) genes encoding one or more proteins selected from the group consisting of: B5R, B8R, B15R, B18R, C12L, H3L, A27L, and L1R.

[0050] In certain embodiments, the modified vaccinia virus expresses an exogenous therapeutic gene. In certain embodiments, the exogenous therapeutic gene is an immune-related gene. In certain embodiments, the immune-related gene encodes a chemokine, an immune cell growth factor, and / or an immune cell activating factor.

[0051] On the other hand, the present application also provides a recombinant protein, the recombinant protein comprising L109 recombinant protein.

[0052] In certain embodiments, the L109 recombinant protein has a substitution at amino acid position H67. In certain embodiments, the amino acid substituted at position H67 in the L109 recombinant protein is R. In certain embodiments, the L109 recombinant protein has a substitution at amino acid position A15. In certain embodiments, the amino acid substituted at position A15 in the L109 recombinant protein is V.

[0053] In certain embodiments, the L109 recombinant protein is selected from one of the following groups:

[0054] (1) The L109 protein comprises the amino acid sequence shown in SEQ ID NO: 12;

[0055] (2) the L109 protein comprises the amino acid sequence shown in SEQ ID NO: 14; and

[0056] (3) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:16.

[0057] In certain embodiments, the L109 recombinant protein is selected from one of the following groups:

[0058] (1) L109 protein has the amino acid sequence shown in SEQ ID NO: 12;

[0059] (2) the L109 protein has the amino acid sequence shown in SEQ ID NO: 14; and

[0060] (3) The L109 protein has the amino acid sequence shown in SEQ ID NO:16.

[0061] In certain embodiments, the L109 recombinant protein is encoded by a List109 gene, and the List109 gene is selected from one of the following groups:

[0062] (1) The List109 gene comprises the amino acid sequence shown in SEQ ID NO: 11;

[0063] (2) the List109 gene comprises the amino acid sequence shown in SEQ ID NO: 13; and

[0064] (3) The List109 gene comprises the amino acid sequence shown in SEQ ID NO:15.

[0065] In certain embodiments, the L109 recombinant protein is encoded by a List109 gene, and the List109 gene is selected from one of the following groups:

[0066] (1) The List109 gene has the amino acid sequence shown in SEQ ID NO: 11;

[0067] (2) the List109 gene has the amino acid sequence shown in SEQ ID NO: 13; and

[0068] (3) The List109 gene has the amino acid sequence shown in SEQ ID NO:15.

[0069] On the other hand, the present application also provides a kit comprising the L109 recombinant protein.

[0070] In certain embodiments, the kit is used to determine the content of chondroitin sulfate in a mixture.

[0071] The present application also provides an oncolytic virus comprising the L109 recombinant protein.

[0072] In certain embodiments, the oncolytic virus is selected from one or more of the following: adenovirus, reovirus, herpes virus, poxvirus, paramyxovirus, rhabdovirus, picornavirus, influenza virus, parvovirus, and variants of these viruses.

[0073] On the other hand, the present application also provides one or more nucleic acid molecules encoding the modified vaccinia virus, the recombinant protein, and / or the oncolytic virus.

[0074] On the other hand, the present application also provides a vector comprising the nucleic acid molecule. In certain embodiments, the vector comprises an expression vector. In certain embodiments, the vector comprises a DNA vector and an RNA vector.

[0075] On the other hand, the present application also provides one or more cells comprising the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, and / or the vector.

[0076] In certain embodiments, the cell comprises a host cell. In certain embodiments, the cell comprises a tumor cell.

[0077] On the other hand, the present application also provides a pharmaceutical composition comprising the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, the vector, any one of the cells, and / or optionally a pharmaceutically acceptable carrier.

[0078] In certain embodiments, the vaccinia virus is used alone as a monotherapy. In certain embodiments, the vaccinia virus is used in combination with a small molecule targeted anticancer agent, an antibody drug, adoptive cell therapy, and / or an oncolytic virus enhancer.

[0079] On the other hand, the present application also provides a drug kit comprising the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0080] On the other hand, the present application also provides the use of the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the kit in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0081] In certain embodiments, the disease and / or condition comprises a tumor. In certain embodiments, the tumor comprises a solid tumor and / or a non-solid tumor. In certain embodiments, the non-solid tumor is a hematologic tumor. In certain embodiments, the hematologic tumor is a leukemia or a lymphoma. In certain embodiments, the hematologic tumor is selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, myelodysplastic syndrome or plasmacytoma. In certain embodiments, the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, bile duct cancer, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma and endometrial cancer.

[0082] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which comprises administering the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the drug kit to a subject in need.

[0083] In certain embodiments, the disease and / or condition comprises a tumor. In certain embodiments, the tumor comprises a solid tumor and / or a non-solid tumor. In certain embodiments, the non-solid tumor is a hematologic tumor. In certain embodiments, the hematologic tumor is a leukemia or a lymphoma. In certain embodiments, the hematologic tumor is selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, myelodysplastic syndrome or plasmacytoma. In certain embodiments, the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, bile duct cancer, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma and endometrial cancer.

[0084] The present application provides the vaccinia virus, the recombinant protein, the oncolytic virus, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the drug kit, which are used to prevent and / or treat diseases and / or conditions.

[0085] In certain embodiments, the disease and / or condition comprises a tumor. In certain embodiments, the tumor comprises a solid tumor and / or a non-solid tumor. In certain embodiments, the non-solid tumor is a hematologic tumor. In certain embodiments, the hematologic tumor is a leukemia or a lymphoma. In certain embodiments, the hematologic tumor is selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin's lymphoma, myeloma, myelodysplastic syndrome or plasmacytoma. In certain embodiments, the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, bile duct cancer, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma and endometrial cancer.

[0086] The modified vaccinia virus provided in the present application has the following advantages: 1) excellent replication ability; 2) stronger in vitro cell killing ability; 3) better in vivo anti-tumor efficacy than unmodified vaccinia virus; 4) generally and significantly inhibits tumors, especially solid tumors; 5) higher yield; 6) no obvious toxicity to normal cells and good tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0088] Figure 1 shows the PCR identification results of vaccinia viruses described herein that contain and do not contain a double deletion of the List006 and List007 genes. Del2 represents the amplified List006 and List007 genes, VV Lister represents the control Lister virus, L09 represents the amplified control gene fragment, M represents the molecular weight marker, VACV 109 refers to a vaccinia virus containing a double deletion of the List006 and List007 genes (double deletion strain), and VACV 108 refers to a vaccinia virus that does not contain a double deletion of the List006 and List007 genes (non-deletion strain). Lane M (far left) indicates the molecular weight of the standard protein. Lane 1 is VV lister L09, which is the Lister strain that amplifies the control fragment; lane 2 is VV Lister Del2, which is the Lister strain that amplifies the List006 and List007 genes; lane 3 is VACV 109 L09, which is the double-deletion strain that amplifies the control fragment; lane 4 is VACV 109 Del2, which is the double-deletion strain that amplifies the List006 and List007 genes; lane 5 is VACV 108 L09, which is the non-deletion strain that amplifies the control fragment; lane 6 is VACV 108 Del2, which is the non-deletion strain that amplifies the List006 and List007 genes.

[0089] Figure 2 shows the amplification curves of vaccinia viruses containing and not containing the double deletion of List006 and List007 genes described in this application. MOI is the multiplicity of infection, that is, the average number of viruses infected per cell, which here represents 0.01 pfu of virus per cell.

[0090] FIG3 is a schematic diagram showing the ability of vaccinia viruses containing or not containing double deletions of List006 and List007 genes described in the present application to kill tumor cells in vitro.

[0091] Figure 4 shows the in vivo anti-tumor effects of vaccinia viruses described herein with and without a double deletion of the List006 and List007 genes. (* indicates statistically significant differences between the VACV108 / VACV109 and PBS groups; the top row indicates the difference between the VACV109 and PBS groups, and the bottom row indicates the difference between the VACV108 and PBS groups; ***P < 0.001, ****P < 0.0001.)

[0092] FIG5 is a schematic diagram showing the mutation sites on the L109 protein in the vaccinia virus described in the present application.

[0093] Figure 6 shows an in vitro replication experiment of vaccinia viruses containing different L109 protein mutations described herein. Lis-con represents an unmutated L109 protein strain, Lis-single represents a single A15V mutant in L109 protein, and Lis-double represents a double A15V and H67R mutant in L109 protein.

[0094] FIG7 is a schematic diagram showing the ability of vaccinia viruses containing different L109 protein mutations described in the present application to kill different tumor cells in vitro.

[0095] Figure 8 shows the antitumor effects of vaccinia viruses containing different L109 protein mutations described herein in a subcutaneous pancreatic cancer model in C57 / BL6 mice. Figure 8A shows tumor volume, with data presented as mean tumor volume ± SD. (**, p < 0.01, ****, p < 0.0001 vs. Control; ##, p < 0.01, ###, p < 0.001 vs. Lis-con. Figure 8B shows the relative change in tumor volume over 15 days. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 vs. Control; #, p < 0.05, ##, p < 0.01 vs. Lis-con.) Figure 8C shows photographs of resected tumors 15 days after treatment. Figure 8D shows a bar graph of changes in tumor weight. (**, p < 0.01, ***, p < 0.001 vs. Control.) Figure 8E shows the change in mean body weight over time for animals in the control and treatment groups.

[0096] Figure 9 shows a schematic diagram of the therapeutic effect of the vaccinia virus containing a double mutation in the L109 protein described in the present application on subcutaneous rhabdomyosarcoma in nude mice. Figure 9a represents the change in tumor volume during treatment. Figure 9b represents the weight of the tumor removed after dissection after the observation period. Figure 9c represents the change in body weight of nude mice during treatment. (* represents the statistical difference between the double mutant strain / positive control group and the negative control group; when * is a double row, the upper row represents the difference between the double mutant strain and the negative control group, and the lower row represents the difference between the positive control group and the negative control group; when * is a single row, Figure 9a represents the difference between the double mutant strain and the negative control group, and Figure 9c represents the difference between the positive control group and the negative control group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The positive control is a vaccinia virus that does not contain a mutation in the L109 protein.)

[0097] Figure 10 shows a schematic diagram of the therapeutic effect of the vaccinia virus containing a double mutation in the L109 protein described in this application on subcutaneous melanoma in golden hamsters. Figure 10a represents the change in tumor volume during treatment. Figure 10b represents the change in weight of golden hamsters during treatment. (* represents the statistical difference between the double mutant strain / positive control group and the negative control group; when * is a double row, the upper row represents the difference between the double mutant strain and the negative control group, and the lower row represents the difference between the positive control group and the negative control group; when * is a single row, it represents the difference between the double mutant strain and the negative control group; **P<0.01, ****P<0.0001. # Represents the statistical difference between the double mutant strain and the positive control group, # P < 0.05. The positive control was a vaccinia virus that did not contain the L109 protein mutation.

[0098] Figure 11 shows a schematic diagram of the therapeutic effect of the vaccinia virus containing a double mutation of the L109 protein described in the present application on subcutaneous pancreatic cancer in golden hamsters. Figure 11a represents the change in tumor volume during treatment. Figure 11b represents the weight of the tumor removed after dissection at the end of the observation period. Figure 11c represents the proportion of tumors cleared in each group at the end of the observation period, and from left to right refers to the negative control group, positive control group and double mutant strain. Figure 11d represents the change in weight of golden hamsters during treatment. (* represents the statistical difference between the double mutant strain / positive control group and the negative control group; when * is double row, the upper row represents the difference between the double mutant strain and the negative control group, and the lower row represents the difference between the positive control group and the negative control group; *P<0.05, ***P<0.001, ****P<0.0001. The positive control is a vaccinia virus that does not contain the L109 protein mutation.) DETAILED DESCRIPTION

[0099] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0100] Before further describing the present application, it is to be understood that the present application is not limited to the particular embodiments illustrated herein, and it is to be further understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0101] Definition of terms

[0102] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally, the concepts of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and proteins and nucleic acids described herein are concepts generally recognized by those skilled in the art.

[0103] As used herein, the term "comprising," or similar terms such as "including," "containing," or "having" should be understood to include the listed elements but not to exclude the presence of other elements. These terms also include situations where the composition consists solely of the listed elements. The term "consisting of..." does not exclude elements that do not significantly affect the solution in question.

[0104] The numerical ranges described in the terms, such as temperature ranges, time ranges, composition or concentration ranges, or other numerical ranges, include the end values, all intermediate ranges, subranges (e.g., ranges between a certain intermediate value and a certain end value), and all individual values ​​within the range, especially intermediate ranges, subranges, and individual integer values ​​with integer values ​​as end values. Furthermore, any intermediate ranges, subranges, and all individual values ​​described in the numerical range may be excluded from the numerical range.

[0105] The “and / or” described herein should be understood as any one element or a combination of any several elements used for connection.

[0106] The term "oncolytic virus" refers to a virus that can selectively replicate in and lyse tumor cells. Typically, oncolytic viruses do not affect the growth and proliferation of normal cells. Oncolytic viruses can include adenoviruses, reoviruses, herpes viruses, poxviruses, paramyxoviruses, rhabdoviruses, picornaviruses, influenza viruses, and parvoviruses.

[0107] The term "poxvirus" or "Poxviridae" refers to the largest and most complex viruses that infect mammals. Its genetic material is double-stranded DNA, and the virus has a diameter of about 140-260nm. "Poxvirus" includes the subfamily Chordopoxvirinae and the subfamily Entomopoxvirinae. Among them, the subfamily Chordopoxvirinae has 8 genera, including the genus Orthopoxvirus and the genus Parapoxvirus. There is a wide range of cross-immune reactions between viruses of the same genus, so poxviruses have complex antigenicity and can induce specific antibodies and cross-reactive antibodies. Poxviruses can include but are not limited to Vaccinia virus (VACV / VV), monkeypox virus (Mpox virus), myxoma virus (Myxoma virus) and variola virus (Variola virus).

[0108] The term "vaccinia virus" or "VACV" or "VV" generally refers to a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome of approximately 190 kbp in length that encodes approximately 200 proteins. The vaccinia virus strain can be selected from one or more of the following: Lister, Western Reserve, Copenhagen, Paris, Tashkent, Tiantan, Wyeth, Brighton, Ankara, Dairen I, LIVP, Connaught, New York City Health Department, and variants thereof.

[0109] The term "modified vaccinia virus" refers to the modification of natural vaccinia virus through artificial intervention, so that the genome of vaccinia virus differs from that of wild type. The modification can be obtained through multiple passage screening, or it can be obtained through genetic engineering methods such as homologous recombination and CRISPR. Compared with unmodified vaccinia virus, modified vaccinia virus can also include backbone modification and / or introduction of therapeutic genes. The backbone modification can be a modification of the vaccinia virus vector genome, and the modification can include deletion, mutation, insertion of genes to affect viral genome expression or the combination with other molecules to affect viral genome expression. The therapeutic gene can be an immune-related gene or a tumor antigen-related gene. The immune-related gene can be a gene encoding an immune cell epitope, a gene encoding a chemokine, a gene encoding an immune cell growth factor, or a gene encoding an immune cell activating factor. The tumor antigen-related gene can be a gene related to cell proliferation and differentiation, and can include EGFR, RAS and MYC; it can be a gene expressing a tumor cell-specific protein.

[0110] The term "L109 protein" refers to the major immunogenic protein on mature intracellular viral particles (IMVs), primarily involved in anchoring to host cells and mediating endocytosis. The L109 protein is encoded by the List109 gene. The IMV is the most abundant virion produced during vaccinia virus infection and has only a single membrane. L109 protein names may also include ACAM3000_MVA_104, ACAM3000_MVA_105, ACAM3000_MVA_116, A5R, A19L, D7R, D8L, J4R, J6R, MVA090R, MVA104R, MVA105L, MVA116R, OPG103, OPG105, OPG119, OPG146, RPO18, RPO19, RPO22, RPO147, TD8L, VACWR096, VACWR098, VACWR112, VACWR113, VACWR124 and VACWR139.

[0111] The term "L006 protein" refers to the protein encoded by the List006 gene in vaccinia virus, and its names may also include SynVACV_010, SynVACV_232, VACAC2_010, VACAC2_232, VACli_010 and VACli_232.

[0112] The term "L007 protein" refers to a protein in vaccinia virus that contains a chemokine receptor domain and belongs to the SCP-1 protein class. SCP-1 is a protein associated with spermatocyte meiotic prophase, primarily expressed in tumor cells, and can trigger an immune response. Other names for L007 protein include SynVACV_012, SynVACV_230, VACAC2_012, VACAC2_230, VACV-DUKE-012, VACV-DUKE-214, VACV191, VACV192, VACli_012, and Virion core protein.

[0113] The term "List006 gene" refers to a gene present in vaccinia virus, the nucleotide sequence of which is shown in SEQ ID NO: 5. The List006 gene encodes the L006 protein. List006 gene names can also include 44.1rMVA_006, 44.1rMVA_188, 44 / 47.1_rMVA_006, 44 / 47.1_rMVA_194, 51.2rMVA_006, 51.2rMVA_202, m8002L, mO002L, synVACV_010, synVACV_232, VAC_DPP16_010, VAC_DPP16_232, VAC_DPP19_010, VAC_DPP19_232, VAC_DPP21_010, VAC_DPP21_232, VACAC2_010, VACAC2_232, VACli_010 and VACli_232.

[0114] The term "List007 gene" refers to the SCP-1 gene, which encodes a protein comprising a chemokine receptor domain. The SCP-1 gene encodes the SCP-1 protein. List007 gene names can also include C13L, 44.1rMVA_007, 44 / 47.1_rMVA_007, 51.2rMVA_007, synVACV_012, synVACV_230, VAC_DPP10_012, VAC_DPP10_230, VAC_DPP11_012, VAC_DPP11_230, VAC_DPP12_012, VAC_DPP12_230, VAC_DPP15_012, VAC_DPP15_230, VAC_DPP16_012, VAC_DPP16_230, VAC_DPP17_012, VAC_DPP17_230, VAC_DPP19_012, VAC_DPP19_230, 230, VAC_DPP20_012, VAC_DPP20_230, VAC_DPP21_012, VAC_DPP21_230, VAC_DPP25_ 012, VAC_DPP25_230, VAC_DPP9_012, VAC_DPP9_230, VAC_IHDW1_010, VAC_IHDW1_20 5, VAC_TKT3_197, VAC_TKT4_197, VAC_TP5_009, VAC_TP5_211, VACAC2_012, VACAC2_230, VACli_012, VACli_230, VACV-DUKE-012, VACV-DUKE-214, VACV_191 and VACV_192.

[0115] The term "protein expression and / or activity is inhibited" means that the protein is unable to produce physiological activity due to non-expression, partial expression, or insertion of an inhibitory peptide. Non-expression or partial expression of a protein can be achieved by inhibiting the expression of the gene encoding the protein. Methods for inhibiting gene expression or inserting an inhibitory peptide can be performed by methods well known in the art.

[0116] The term "domain" refers to a component of a protein (eg, a protein subunit) that can assemble with other molecules to form a protein complex.

[0117] The term "functional domain" refers to a region within a protein structure that possesses a function and is the smallest structural unit that enables a protein to possess physiological activity. Functional domains can be arranged in series, in parallel, or a combination of both.

[0118] The term "gene expression is inhibited" means that the gene is not expressed, is deleted, or is inhibited by insertion of an exogenous gene, so that the protein encoded by the gene is inactive. Gene deletion may be partial or complete. Gene deletion or exogenous gene insertion can be performed by methods well known in the art.

[0119] The term "deletion" refers to an incomplete nucleotide sequence of a gene in the viral genome, resulting in inactivation or non-expression of the protein encoded by the gene. The genomic deletion can be a deletion of a gene encoding a functional protein domain, a knockout of a gene promoter, one or more nucleotide deletions, a complete deletion of the gene sequence, or premature expression of a stop codon.

[0120] The term "mutation" refers to a change in the structure or amino acid sequence of a specific protein compared to the wild-type protein, which results in a change in the function of the specific protein, but it still has physiological activity. The change in protein structure can be caused by the replacement, knockout or insertion of nucleotides at one or more sites on the nucleic acid (DNA or RNA) encoding the protein. The change in protein structure can be a change in the primary structure of the protein, a change in the secondary structure of the protein, a change in the tertiary structure of the protein, or a change in the quaternary structure of the protein. The change in the amino acid sequence can be the replacement of the amino acid at one or more sites with another amino acid, the knockout of the amino acid at one or more sites, or the insertion of one or more amino acids into one or more sites.

[0121] The term "substitution" refers to the replacement of one or more amino acid positions in an amino acid sequence with an amino acid other than the one at that position. This means that the original amino acid disappears from the sequence and a different amino acid appears at the same position. In contrast to "knockout" and "insertion," the total number of amino acids before and after a "substitution" remains unchanged.

[0122] The "amino acid at position 15" or "amino acid at position 67" described herein may be arranged in the order shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, where M is the first amino acid and they are arranged in order from left to right.

[0123] The term "standard amino acids" refers to the 20 amino acids used to synthesize eukaryotic proteins. Compared with other amino acids, "standard amino acids" may have corresponding genetic codes. The term "backbone gene" refers to the gene present in the unmodified vaccinia virus genome. The purpose of modifying the backbone gene may generally be to reduce viral toxicity, improve replication defects, enhance oncolytic activity or improve production efficiency. The method of modifying the backbone gene may be to delete the backbone gene, to mutate the backbone gene nucleotides, to insert an exogenous gene to silence the backbone gene, or to replace the backbone gene with an exogenous gene.

[0124] The term "exogenous therapeutic gene" refers to an exogenous gene with a specific physiological function, which can typically be inserted into the vaccinia virus genome and expressed to exert a therapeutic effect. The physiological function may include enhancing tumor targeting, enhancing oncolysis, enhancing tumor killing, enhancing immunogenicity, altering the tumor microenvironment, or enhancing tumor lysis. The therapeutic effect may include reducing or alleviating the progression, severity, and / or duration of a proliferative disease, or ameliorating one or more symptoms of a proliferative disease.

[0125] The term "immune-related gene" refers to an exogenous gene whose physiological function is related to immune diseases, and may include genes encoding chemokines, immune cell growth factors, and immune cell activating factors.

[0126] The term "chemokine" refers to a signaling protein secreted by a cell that has the ability to induce directional chemotaxis of nearby responding cells.

[0127] The term "immune cell growth factor" generally refers to proteins or polypeptides that regulate cell growth. They are a class of signaling molecules secreted by specific cells of the immune system that mediate and regulate immunity, production, and hematopoiesis. The immune cells can be T cells, B cells, NK cells, or lymphocytes.

[0128] The term "immune cell activating factor" refers to a class of cytokines that can stimulate the activation of immune cells and assist immune cells in releasing cell growth factors.

[0129] The term "L109 recombinant protein" refers to an L109 protein that has one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type L109 protein. The substituted sites may include an A15 mutation, an H67 mutation, or both an A15 and H67 mutation. The recombinant protein can be prepared by any method known in the art, including chemical synthesis and recombinant expression techniques. The L109 recombinant protein can be isolated from any L109 protein-containing vector, such as vaccinia virus, or synthesized in vitro.

[0130] The term "vector" refers to a tool that enables gene expression inside or outside cells. Vectors can be DNA or RNA vectors, and more specifically, plasmids. Vectors can be used to introduce specific genes into target cells and express the protein encoded by the gene using the protein production system within the target cells.

[0131] The term "pharmaceutical composition" means that in addition to the ingredients mentioned herein, it may also contain one or more pharmaceutically acceptable carriers. The pharmaceutical composition may exist in the final pharmaceutical dosage form or may be an intermediate for preparing a dosage form. The pharmaceutically acceptable carrier refers to a molecular entity and composition that does not produce an allergic or similar adverse reaction when administered to a subject (e.g., a human being), and may be selected from excipients, lubricants, wetting agents, sweeteners, fragrances, and preservatives. The composition can be formulated according to conventional methods. The pharmaceutical composition of the present application can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient, particularly after administration to a mammal. According to the formulation, the pharmaceutical composition of the present application can be appropriately administered to an individual. Such administration may include intra-cancer tissue, intradermal, intramuscular, intraperitoneal, intravenous, intra-arterial, subcutaneous, intranasal, parenteral, epidural, and oral routes.

[0132] The term "kit" refers to an administration device for providing the modified vaccinia virus described herein, which may contain the aforementioned modified vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, or a pharmaceutical composition containing the aforementioned.

[0133] The term "kit" refers to a device for administering the L109 recombinant protein described herein, and may further comprise one or more components, such as instructions for use, devices, and other reagents and components, such as tubes, containers, or syringes, that can be used to perform the methods described herein.

[0134] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer and tumor are used interchangeably in this application. Cancer can be a solid cancer or a non-solid cancer. Tumors can be disseminated or circulating.

[0135] Detailed Description of the Invention

[0136] A. Modified vaccinia virus

[0137] In one aspect, the present application provides a modified vaccinia virus comprising a protein mutation. The mutation comprises a substitution, deletion, or insertion in the protein amino acid sequence. The protein mutation can be achieved by mutating a gene encoding the protein. The gene mutation comprises a substitution, deletion, or insertion.

[0138] In the present application, the vaccinia virus can be selected from one or more of the following: Lister strain, Western Reserve strain, Copenhagen strain, Paris strain, Tashkent strain, Tiantan strain, Wyeth strain, Brighton strain, Ankara strain, Dairen I strain, LIVP strain, Connaught strain, New York City Health Department strain, and variants thereof. For example, the vaccinia virus can be a Lister strain.

[0139] In the present application, the modified vaccinia virus further comprises mutations in one or more genes selected from the following: (1) backbone genes: TK, A34R, A35R, A36R, A46R, A56R, L025, K3L and F14.5L; and / or; (2) genes encoding one or more proteins selected from the following: B5R, B8R, B15R, B18R, C12L, H3L, A27L and L1R.

[0140] In the present application, the protein mutation may include inhibition of protein expression and / or activity. In certain embodiments, the protein whose expression and / or activity is inhibited may include a protein associated with complement-mediated immune responses. In certain embodiments, the protein whose expression and / or activity is inhibited may include a protein associated with reducing immune cell-mediated immune responses. For example, the protein associated with reducing immune cell-mediated immune responses may include a protein that inhibits the expression of the SCP-1 protein. In another example, the protein that inhibits the expression of the SCP-1 protein may be the L007 protein.

[0141] In the present application, the protein mutation may include inhibition of expression and / or activity of the L006 protein. The L006 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 6. For example, the L006 protein may comprise the amino acid sequence of SEQ ID NO: 6.

[0142] In certain embodiments, the protein mutation may include inhibition of the expression and / or activity of the L006 protein. The methods for inhibiting the expression and / or activity of the L006 protein may include: (1) deleting all or part of the gene encoding the L006 protein; and / or (2) inserting the aforementioned exogenous gene and / or exogenous nucleic acid into the gene encoding the L006 protein. Methods for deleting all or part of a gene include, but are not limited to, all methods described herein. The partial gene encoding the L006 protein may include a gene encoding a functional domain of the L006 protein.

[0143] In certain embodiments, the protein mutation may include inhibiting the expression and / or activity of the L006 protein. The L006 protein may be encoded by the List006 gene. For example, the sequence of the List006 gene may be at least 80%, 85%, 90%, 95%, or 100% identical to the sequence set forth in SEQ ID NO:5. For another example, the sequence of the List006 gene is set forth in SEQ ID NO:5.

[0144] In certain embodiments, the protein mutation may include inhibiting the expression and / or activity of the L006 protein. Methods for inhibiting the expression and / or activity of the L006 protein may include: (1) deleting all or part of the List006 gene; and / or (2) inserting the aforementioned exogenous gene and / or exogenous nucleic acid into the List006 gene. Methods for deleting all or part of a gene include, but are not limited to, all methods described herein. The List006 gene may include a gene encoding a functional domain of the L006 protein.

[0145] In certain embodiments, the protein mutation may include suppressing the expression and / or activity of the L006 protein. The method for suppressing the expression and / or activity of the L006 protein may include partial deletion of the List006 gene. The partial deletion of the List006 gene may include deletion of the promoter and / or premature terminator expression.

[0146] In certain embodiments, the protein mutation may include inhibition of L006 protein expression and / or activity. The method for inhibiting L006 protein expression and / or activity may include complete deletion of the List006 gene. Methods for complete deletion include, but are not limited to, all methods described herein.

[0147] In certain embodiments, the protein mutation may include inhibition of expression and / or activity of the L007 protein. The L007 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8. For example, the L007 protein may comprise the amino acid sequence set forth in SEQ ID NO: 8.

[0148] In certain embodiments, the protein mutation may include inhibition of the expression and / or activity of the L007 protein. The methods for inhibiting the expression and / or activity of the L007 protein may include: (1) deleting all or part of the gene encoding the L007 protein; and / or (2) inserting the aforementioned exogenous gene and / or exogenous nucleic acid into the gene encoding the L007 protein. Methods for deleting all or part of a gene include, but are not limited to, all methods described herein. The partial gene encoding the L007 protein may include a gene encoding a functional domain of the L007 protein.

[0149] In certain embodiments, the protein mutation may include inhibiting the expression and / or activity of the L007 protein. The L007 protein may be encoded by the List007 gene. For example, the sequence of the List007 gene may be at least 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:7. For another example, the sequence of the List007 gene is set forth in SEQ ID NO:7.

[0150] In certain embodiments, the protein mutation may include inhibiting the expression and / or activity of the L007 protein. Methods for inhibiting the expression and / or activity of the L007 protein may include: (1) deleting all or part of the List007 gene; and / or (2) inserting the aforementioned exogenous gene and / or exogenous nucleic acid into the List007 gene. Methods for deleting all or part of a gene include, but are not limited to, all methods described herein. The List007 gene may include a gene encoding a functional domain of the L007 protein.

[0151] In certain embodiments, the protein mutation may include inhibition of L007 protein expression and / or activity. The inhibition of L007 protein expression and / or activity may include partial deletion of the List007 gene. The partial deletion of the List007 gene may include deletion of the promoter and / or premature terminator expression.

[0152] In certain embodiments, the protein mutation may include inhibition of L007 protein expression and / or activity. The method for inhibiting L007 protein expression and / or activity may include complete deletion of the L007 gene. Methods for complete deletion include, but are not limited to, all methods described herein.

[0153] In certain embodiments, the protein mutation may include inhibiting the expression and / or activity of L006 and L007 proteins. The L006 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6. The L007 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8. For example, the L006 protein may comprise the amino acid sequence of SEQ ID NO:6. The L007 protein may comprise the amino acid sequence of SEQ ID NO:8.

[0154] In certain embodiments, the protein mutation may include inhibition of the expression and / or activity of L006 and L007 proteins. The methods for inhibiting the expression and / or activity of L006 and L007 proteins may include: (1) deleting all or part of the genes encoding the L006 and L007 proteins; and / or (2) inserting the aforementioned exogenous genes and / or the aforementioned exogenous nucleic acids into the genes encoding the L006 and L007 proteins. Methods for deleting all or part of a gene include, but are not limited to, all methods mentioned herein. The partial gene encoding the L006 protein may include a gene encoding a functional domain of the L006 protein. The partial gene encoding the L007 protein may include a gene encoding a functional domain of the L007 protein.

[0155] In certain embodiments, the protein mutation may include inhibition of the expression and / or activity of L006 protein and L007 protein. The L006 protein may be encoded by the List006 gene. The L007 protein may be encoded by the List007 gene.

[0156] For example, the sequence of the List006 gene may be at least 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5. The sequence of the List007 gene may be at least 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. For another example, the sequence of the List006 gene is as shown in SEQ ID NO: 5. The sequence of the List007 gene is as shown in SEQ ID NO: 7.

[0157] In some embodiments, the method of inhibiting the expression and / or activity of L006 protein and L007 protein may include partial deletion of the List006 gene and List007 gene. For example, the partial deletion of the List006 gene and List007 gene may include deletion of promoter and / or premature terminator expression.

[0158] In some embodiments, the method of inhibiting the expression and / or activity of L006 protein and L007 protein may include complete deletion of the List006 gene and List007 gene.

[0159] In the present application, the protein mutation may include a change in the amino acid sequence of the protein, thereby changing or not changing the higher-order structure of the protein, and ultimately changing the function of the protein, but the mutated protein still retains its pre-mutation function. For example, the mutated protein can be selected from one or more of the 81 viral proteins in the mature intracellular virus particle (IMV). The 81 viral proteins of IMV are: A2.5L, A3L, A4L, A5R, A6L, A7L, A9L, A10L, A12L, A13L, A14L, A14.5L, A15L, A16L, A17L, A18R, A21L, A22R, A24R, A25L, A26L, A27L, A28L, A29L, A30L, A31R, A32L, A42R, A45R, A46R, B1R, C6L, D1R, D2R, D6R, D 7R, D8L, D11L, D12L, D13L, E1L, E4L, E6R, E8R, E10R, E11L, F8L, F9L, F10L, F17R, G1L, G3L, G4L, G5R, G5.5R, G7L, G9R, H1L, H2R, H3L, H4L, H5R, H6R, I1L, I2L, I3L, I5L, I6L, I7L, I8R, J1R, J3R, J4R, K4L, L1R, L3L, L4R, L5R or O2L. For another example, the mutated protein can be selected from one or more of the following: viral intracellular proteins, membrane proteins and viral secretory proteins.

[0160] In the present application, the protein mutation may comprise an amino acid mutation in a viral protein. The viral protein may comprise an enzyme. For example, the enzyme may be TK. In the present application, the protein mutation may comprise an amino acid mutation in a viral secretory protein. The viral secretory protein may be VGF. In the present application, the protein mutation may comprise an amino acid mutation in a viral membrane protein. In some embodiments, the membrane protein may comprise a viral membrane protein and / or an outer membrane protein. In some embodiments, the viral membrane may be an envelope synthesized by the virus itself. In some embodiments, the outer membrane may be an outer layer of the virus obtained from a cell membrane and / or an organelle membrane during infection and migration. In some embodiments, the viral membrane protein may comprise a class of proteins associated with anchoring to host cells and mediating endocytosis. For example, the proteins associated with anchoring to host cells and mediating endocytosis may be selected from one or more of the following: A27L, H3L, and L109.

[0161] In the present application, the protein mutation can include an amino acid mutation of the L109 protein. The mutation can include the replacement of one or more amino acids on the L109 protein. The substituted site can be selected from one or more of the following sites: N9, A15, E30, T34, T35, R44, N46, F47, K48, G49, G50, Y51, N59, E60, L63, S64, H67, D75, Y76, H95, W96, N97, K98, K99, Y101, S102, S103, Y104, E105, E106, K108, H110, D112, Q122, L124, D126, K163, T187, P188, N190, and R220. For example, the substituted site can be A15. For another example, the substituted site may be H67. For another example, the substituted site may be A15 and H67.

[0162] In the present application, the protein mutation may include an amino acid mutation in the L109 protein, wherein the amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid may include any standard amino acid except H. In certain embodiments, the substituted amino acid includes an amino acid with an isoelectric point greater than 7.5. For example, the amino acid with an isoelectric point greater than 7.5 is R or K. In another example, the substituted amino acid is R. In certain embodiments, the substituted amino acid includes an amino acid without a cyclic structure on the side chain. For example, the amino acid without a cyclic structure on the side chain is A, I, L, V, N, C, Q, M, S, T, R, K, D, E, or G. In another example, the amino acid without a cyclic structure on the side chain is R or K. In another example, the substituted amino acid is R. In certain embodiments, the substituted amino acid includes a basic amino acid. For example, the basic amino acid is R or K. For example, the substituted amino acid is R. In certain embodiments, the substituted amino acid may include an amino acid capable of forming multiple hydrogen bonds. For example, the substituted amino acid may be R. In certain embodiments, the substituted amino acid may include an amino acid with a molecular weight greater than histidine. For example, the substituted amino acid may be F, W, Y, or R. For another example, the substituted amino acid may be R. In certain embodiments, the mutated L109 protein has the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the L109 protein is encoded by the List109 gene, the sequence of which is set forth in SEQ ID NO:11.

[0163] In the present application, the protein mutation may include an amino acid mutation of the L109 protein, wherein the amino acid at position A15 of the L109 protein is substituted, and the substituted amino acid may include any standard amino acid except A. In certain embodiments, the substituted amino acid may include an amino acid with a molecular weight greater than 90. For example, the substituted amino acid may be I, L, V, F, W, Y, N, C, Q, M, S, T, R, H, K, D, E, or P. In another example, the substituted amino acid may be V or L. In another example, the substituted amino acid may be V. In certain embodiments, the substituted amino acid may include a hydrophobic amino acid. For example, the substituted amino acid may be W, F, V, L, I, P, or M. In another example, the substituted amino acid may be V or L. In another example, the substituted amino acid may be V. In certain embodiments, the mutated L109 protein comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the L109 protein is encoded by the List109 gene, and the sequence of the mutated List109 gene is shown in SEQ ID NO: 15.

[0164] In the present application, the protein mutation may comprise an amino acid mutation of the L109 protein, wherein the amino acids at positions A15 and H67 on the L109 protein are substituted, wherein the amino acid after the substitution at position A15 may comprise any standard amino acid other than A, and the amino acid after the substitution at position H67 may comprise any standard amino acid other than H. In certain embodiments, the amino acid after the substitution at position A15 may be V or L, and the amino acid after the substitution at position H67 may be R or K. In certain embodiments, the amino acid after the substitution at position A15 may be V, and the amino acid after the substitution at position H67 may be R. In certain embodiments, the mutated L109 protein may comprise the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the L109 protein is encoded by the List109 gene, and the sequence of the List109 gene is shown in SEQ ID NO: 13.

[0165] In the present application, the protein mutation may be an amino acid mutation of the L109 protein and the expression and / or activity of the L006 protein and / or the L007 protein may be inhibited. The amino acid mutation of the L109 protein may include any of the aforementioned mutation modes, and the expression and / or activity of the L006 protein and / or the L007 protein may be inhibited including any of the aforementioned modes of expression and / or activity inhibition.

[0166] In certain embodiments, the amino acid mutation of the L109 protein may include amino acid substitutions at positions A15 and / or H67, wherein the amino acid substituted at position A15 may include any standard amino acid other than A, and the amino acid substituted at position H67 may include any standard amino acid other than H. For example, the amino acid substituted at position A15 may be V or L, and the amino acid substituted at position H67 may be R or K. For another example, the substituted amino acids may be A15V and H67R, respectively. For another example, the mutated L109 protein may include the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 14. For another example, the L109 protein is encoded by the List109 gene, and the sequence of the List109 gene is shown in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 13. In certain embodiments, the L006 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, and / or the L007 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. For example, the L006 protein may comprise the amino acid sequence of SEQ ID NO: 6, and / or the L007 protein may comprise the amino acid sequence of SEQ ID NO: 8. In some embodiments, the L006 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, and the L007 protein may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. For example, the L006 protein may comprise the amino acid sequence shown in SEQ ID NO:6, and the L007 protein may comprise the amino acid sequence shown in SEQ ID NO:8.

[0167] In certain embodiments, the method for inhibiting the expression and / or activity of L006 protein and / or L007 protein may include: (1) deleting all or part of the gene encoding the L006 protein and / or L007 protein; and / or (2) inserting the aforementioned exogenous gene and / or the aforementioned exogenous nucleic acid into the gene encoding the L006 protein and / or L007 protein; methods for deleting all or part of a gene include, but are not limited to, all methods mentioned herein. The L006 protein may be encoded by the List006 gene. The L007 protein may be encoded by the List007 gene, and the partial gene encoding the L006 protein may include a gene encoding a functional domain of the L006 protein, and the partial gene encoding the L007 protein may include a gene encoding a functional domain of the L007 protein. For example, the sequence of the List006 gene may have at least 80%, 85%, 90%, 95%, or 100% identity to the nucleotide sequence shown in SEQ ID NO: 5. The sequence of the List007 gene may be at least 80%, 85%, 90%, 95% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 7. For another example, the sequence of the List006 gene is shown in SEQ ID NO: 5. The sequence of the List007 gene is shown in SEQ ID NO: 7.

[0168] In certain embodiments, the protein mutation may be an amino acid mutation in the L109 protein, and the expression and / or activity of the L006 protein and / or the L007 protein may be inhibited. The method for inhibiting the expression and / or activity of the L006 protein and / or the L007 protein may include partial deletion of the List006 gene and / or the List007 gene. The partial deletion of the List006 gene and / or the List007 gene may include deletion of a promoter and / or premature terminator expression.

[0169] In certain embodiments, the protein mutation may be an amino acid mutation in the L109 protein, and the expression and / or activity of the L006 protein and / or the L007 protein may be inhibited. The protein mutation may include inhibiting the expression and / or activity of the L006 protein and / or the L007 protein. The method for inhibiting the expression and / or activity of the L006 protein and / or the L007 protein may include completely deleting the List006 gene and / or the List007 gene.

[0170] In certain embodiments, the protein mutation may be an amino acid mutation of the L109 protein and the expression and / or activity of the L006 protein and the L007 protein may be inhibited. The protein mutation may include the inhibition of the expression and / or activity of the L006 protein and the L007 protein. The method of inhibiting the expression and / or activity of the L006 protein and the L007 protein may include: (1) deleting the entire List006 gene and the List007 gene; and / or (2) inserting the aforementioned exogenous gene and / or the aforementioned exogenous nucleic acid into the genes of the List006 gene and the List007 gene.

[0171] In certain embodiments, the protein mutation may be an amino acid mutation in the L109 protein, resulting in inhibition of the expression and / or activity of the L006 and / or L007 proteins. The amino acid mutation in the L109 protein may include an amino acid substitution at position A15 and / or position H67. The amino acid substituted at position A15 may include any standard amino acid except A, and the amino acid substituted at position H67 may include any standard amino acid except H. The method for inhibiting the expression and / or activity of the L006 and / or L007 proteins may include complete deletion of the List006 and / or List007 genes. The method for complete deletion may be selected from one or more of the following: promoter knockout, introduction of a stop codon, and deletion of the entire coding gene. For example, the amino acid substituted at position A15 may be V or L. The amino acid substituted at position H67 may be R or K. In another example, the substituted amino acids may be A15V and H67R, respectively. For another example, the mutated L109 protein may comprise the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 14. For another example, the L109 protein may be encoded by the List109 gene, the sequence of which is shown in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 13. For example, the L006 protein may comprise the amino acid sequence shown in SEQ ID NO: 6. The L007 protein may comprise the amino acid sequence shown in SEQ ID NO: 8. For another example, the L006 protein may be encoded by the List006 gene, the sequence of which is shown in SEQ ID NO: 5. The L007 protein may be encoded by the List007 gene, the sequence of which is shown in SEQ ID NO: 7.

[0172] Specifically, the mutated protein in the modified vaccinia virus described in the present application can be selected from one of the following:

[0173] (1) The amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid can be R or K;

[0174] (2) The amino acid at position A15 on the L109 protein is substituted, and the substituted amino acid can be V or L;

[0175] (3) The amino acids at positions A15 and H67 of the L109 protein were substituted. The amino acid at position A15 could be V or L, and the amino acid at position H67 could be R or K.

[0176] (4) L006 and L007 proteins are completely absent;

[0177] (5) missing promoters and / or premature terminator expression on the coding genes List006 and List007;

[0178] (6) the coding genes List006 and List007 are completely missing;

[0179] (7) Insertion of foreign genes into the coding genes List006 and List007;

[0180] (8) The amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid can be R or K; and the L006 and L007 proteins are completely missing;

[0181] (9) The amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid can be R or K; and the promoter and / or premature terminator expression of the coding genes List006 and List007 are missing;

[0182] (10) The amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid can be R or K; and the encoding genes List006 and List007 are completely missing;

[0183] (11) The amino acid at position H67 on the L109 protein is substituted, and the substituted amino acid can be R or K; and foreign genes are inserted into the coding genes List006 and List007;

[0184] (12) The amino acid at position A15 on the L109 protein is substituted, and the substituted amino acid can be V or L; and the L006 protein and L007 protein are completely missing;

[0185] (13) The amino acid at position A15 of the L109 protein is substituted, and the substituted amino acid can be V or L; and the promoter and / or premature terminator of the coding genes List006 and List007 are missing;

[0186] (14) The amino acid at position A15 on the L109 protein is substituted, and the substituted amino acid can be V or L; and the encoding genes List006 and List007 are completely missing;

[0187] (15) The amino acid at position A15 on the L109 protein is substituted, and the substituted amino acid can be V or L; and foreign genes are inserted into the coding genes List006 and List007;

[0188] (16) The amino acids at positions A15 and H67 of the L109 protein were substituted. The amino acid at position A15 can be V or L, and the amino acid at position H67 can be R or K. The L006 and L007 proteins are completely missing.

[0189] (17) The amino acids at positions A15 and H67 of the L109 protein are substituted. The amino acid substituted at position A15 can be V or L, and the amino acid substituted at position H67 can be R or K. The promoter and / or premature terminator are missing from the genes encoding List006 and List007.

[0190] (18) The amino acids at positions A15 and H67 of the L109 protein are substituted, the amino acid at position A15 can be V or L, and the amino acid at position H67 can be R or K; and the coding genes List006 and List007 are completely deleted; and

[0191] (19) Amino acids at positions A15 and H67 of the L109 protein were substituted. The amino acid substituted at position A15 can be V or L, and the amino acid substituted at position H67 can be R or K. Exogenous genes were inserted into the coding genes List006 and List007.

[0192] More specifically, the mutated protein in the modified vaccinia virus described herein can be selected from one of the following:

[0193] (1) The L109 protein has the amino acid sequence shown in SEQ ID NO: 12, and the L006 and L007 proteins are completely missing. The L006 protein has the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein has the amino acid sequence shown in SEQ ID NO: 8;

[0194] (2) The L109 protein has the amino acid sequence shown in SEQ ID NO: 12, and the List006 gene and the List007 gene are completely deleted, the List006 gene comprises the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene comprises the amino acid sequence shown in SEQ ID NO: 7;

[0195] (3) The L109 protein has the amino acid sequence shown in SEQ ID NO: 14, and the L006 and L007 proteins are completely missing. The L006 protein has the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein has the amino acid sequence shown in SEQ ID NO: 8;

[0196] (4) The L109 protein has the amino acid sequence shown in SEQ ID NO: 14, and the List006 gene and the List007 gene are completely deleted, the List006 gene contains the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene contains the amino acid sequence shown in SEQ ID NO: 7;

[0197] (5) the L109 protein has the amino acid sequence shown in SEQ ID NO: 16, and the L006 and L007 proteins are completely absent, the L006 protein has the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein has the amino acid sequence shown in SEQ ID NO: 8; and

[0198] (6) The L109 protein has the amino acid sequence shown in SEQ ID NO: 16, and the List006 gene and the List007 gene are completely deleted. The List006 gene contains the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene contains the amino acid sequence shown in SEQ ID NO: 7.

[0199] In the present application, the protein mutation may include inhibition of protein expression and / or activity. In certain embodiments, inhibition of protein expression and / or activity may include deletion of the entire protein amino acid sequence. In certain embodiments, inhibition of protein expression and / or activity may include silencing of the coding gene. For example, silencing of the coding gene may include targeted knockout of the entire DNA sequence. The targeted knockout techniques described herein may be methods well known in the art, including but not limited to CRISPR technology and / or homologous recombination technology. The homologous recombination may be accomplished via a shuttle vector. As another example, silencing of the coding gene may include targeted knockout of a promoter. In certain embodiments, inhibition of protein expression and / or activity may include silencing of the coding gene. For example, silencing of the coding gene may include the premature appearance of a stop codon. The stop codon may occur at the initiation of replication. In certain embodiments, inhibition of protein expression and / or activity may include partial deletion of the protein amino acid sequence. For example, the partial deletion may be deletion of a functional domain of the protein caused by deletion of a large segment of DNA sequence. The deletion of a large segment of DNA sequence may be caused by targeted knockout. In certain embodiments, the inhibition of protein expression and / or activity may include a partial deletion of the protein's amino acid sequence. For example, the partial deletion may be caused by the premature appearance of a stop codon. The premature appearance of a stop codon may lead to premature termination of protein translation, and the amino acids following this site are no longer attached to the translated amino acid chain. In certain embodiments, the inhibition of protein expression and / or activity may include the insertion of an exogenous gene and / or exogenous nucleic acid into the genome, resulting in the absence of expression or reduced expression in the cell. For example, the exogenous gene may include an element that inhibits gene expression. For example, the element may be an operon, including but not limited to the lactose operon and / or the tryptophan operon. In another example, the element may be a silencer. In certain embodiments, the inhibition of protein expression and / or activity may include the insertion of an exogenous gene and / or exogenous nucleic acid into the genome, resulting in the absence of expression or reduced expression in the cell. For example, the exogenous gene may include a regulatory gene. In another example, the regulatory gene may include a repressor protein and / or a specific factor. In certain embodiments, the inhibition of protein expression and / or activity may include the insertion of exogenous genes and / or exogenous nucleic acids into the genome, resulting in the cell failing to express the protein or reducing its expression. In one embodiment, the exogenous nucleic acids may comprise in vitro synthesized polynucleotides and / or oligonucleotides that inhibit gene expression. For example, the exogenous nucleic acids may be selected from one or more of the following: shRNA, siRNA, and miRNA. In one embodiment, the exogenous nucleic acids may comprise products of in vivo gene expression. For example, the exogenous nucleic acids may be selected from one or more of the following: antisense RNA, snRNA, snoRNA, miRNA, and siRNA.

[0200] In certain embodiments, the inhibition of protein expression and / or activity may include modification of the gene promoter region. For example, the modification may be DNA methylation. In certain embodiments, the inhibition of protein expression and / or activity may include inhibition at the translational level. For example, the inhibition at the translational level may include inhibition of translation initiation by phosphorylation of the translation initiation factor eIF-2α.

[0201] B. Therapeutic genes

[0202] In another aspect, the present application provides a vaccinia virus into which an exogenous therapeutic gene is inserted.

[0203] In certain embodiments, the vaccinia virus can carry an exogenous gene, and the exogenous gene can have one or more of the following functions: enhancing its replication ability, enhancing its tumor killing ability, enhancing its tumor targeting ability, and other related functions.

[0204] In certain embodiments, the exogenous gene may be a therapeutic gene.

[0205] In certain embodiments, the therapeutic gene may be an immune-related gene.

[0206] In certain embodiments, the immune-related gene may be a gene encoding an immune cell epitope.

[0207] In certain embodiments, the gene encoding the immune cell epitope can cause the virus to produce humoral and cellular immune responses. In certain embodiments, the immune cell can be selected from one or more of the following: T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes and mast cells. The granulocyte can be selected from one or more of the following: neutrophils, eosinophils and basophils.

[0208] In certain embodiments, the immune-related gene may be a gene encoding an immune cell growth factor.

[0209] In certain embodiments, the immune cell growth factor can be selected from one or more of the following cytokines: IL-12, IL-21, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, etc.

[0210] In certain embodiments, the immune-related gene may be a gene encoding an immune cell activating factor.

[0211] In some embodiments, the immune cell activating factor can be selected from one or more of the following: interleukin, interferon, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor and variants thereof. In some embodiments, the interleukin can be selected from one or more of the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and IL-36. In certain embodiments, the interferon can be selected from one or more of the following: IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-γ and IFN-λ. In certain embodiments, granulocyte colony stimulating factor (G-CSF) can be produced by macrophages activated by endotoxin, monocytes activated by TNF-α and IFNγ, fibroblasts / endothelial cells activated by IL-1 or TNF-α, and / or bone marrow stromal cells activated by IL-1 or endotoxin. In certain embodiments, granulocyte-macrophage colony stimulating factor (GM-CSF) can be a monomeric glycoprotein cytokine secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and / or fibroblasts. In certain embodiments, the gene encoding the immune cell activating factor can encode one or more immune cell activating factors. In certain embodiments, the one or more gene sequences encoding the immune cell activating factor can be multiple copies or multiple genes encoding the immune cell activating factor that are different from each other.

[0212] In certain embodiments, the tumor antigen-associated gene may be a gene that expresses a tumor cell-specific protein. The tumor cell-specific protein may be a protein expressed by tumor cells but not by normal cells, or a protein expressed at a higher level in tumor cells than in normal cells.

[0213] In certain embodiments, the tumor antigen can be selected from one or more of the following: Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII, BCMA and MSLN.

[0214] In certain embodiments, the tumor antigen may be other exemplary antigens present in the extracellular matrix of a tumor, such as fibronectin, tenascin, and / or oncofetal variants of necrotic areas of a tumor.

[0215] C. Recombinant protein

[0216] In another aspect, the present application provides an L109 recombinant protein, which may have one or more amino acid modifications compared to the wild-type protein.

[0217] In certain embodiments, the modification may comprise an amino acid mutation of the L109 protein. The mutation may comprise a substitution, deletion, or insertion in the amino acid sequence of the L109 protein. For example, the mutation is a substitution in the amino acid sequence of the L109 protein.

[0218] In certain embodiments, the modification can include amino acid mutations of the L109 protein. The mutation can include substitutions at one or more sites in the L109 protein amino acid sequence. The substituted sites can be selected from one or more of the following sites: N9, A15, E30, T34, T35, R44, N46, F47, K48, G49, G50, Y51, N59, E60, L63, S64, H67, D75, Y76, H95, W96, N97, K98, K99, Y101, S102, S103, Y104, E105, E106, K108, H110, D112, Q122, L124, D126, K163, T187, P188, N190, and R220. For example, the substituted site can be A15. For another example, the substituted site may be H67. For another example, the substituted site may be A15 and H67.

[0219] In the present application, the amino acid mutation of the L109 protein may be a substitution of the amino acid at position H67, and the substituted amino acid may include any standard amino acid except H. In certain embodiments, the substituted amino acid includes an amino acid with an isoelectric point higher than 7.5. For example, the amino acid with an isoelectric point higher than 7.5 is R or K. In another example, the substituted amino acid is R. In certain embodiments, the substituted amino acid includes an amino acid without a cyclic structure on the side chain. For example, the amino acid without a cyclic structure on the side chain is A, I, L, V, N, C, Q, M, S, T, R, K, D, E, or G. In another example, the amino acid without a cyclic structure on the side chain is R or K. In another example, the substituted amino acid is R. In certain embodiments, the substituted amino acid includes a basic amino acid. For example, the basic amino acid is R or K. For example, the substituted amino acid is R. In certain embodiments, the substituted amino acid may include an amino acid capable of forming multiple hydrogen bonds. For example, the substituted amino acid may be R. In certain embodiments, the substituted amino acid may include an amino acid with a molecular weight greater than histidine. For example, the substituted amino acid may be F, W, Y, or R. For another example, the substituted amino acid may be R. In certain embodiments, the mutated L109 protein has the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the L109 protein is encoded by the List109 gene, the sequence of which is set forth in SEQ ID NO:11.

[0220] In the present application, the amino acid mutation of the L109 protein may be a substitution of the amino acid at position A15, and the substituted amino acid may include any standard amino acid except A. In certain embodiments, the substituted amino acid may include an amino acid with a molecular weight greater than 90. For example, the substituted amino acid may be I, L, V, F, W, Y, N, C, Q, M, S, T, R, H, K, D, E, or P. In another example, the substituted amino acid may be V or L. In another example, the substituted amino acid may be V. In certain embodiments, the substituted amino acid may include a hydrophobic amino acid. For example, the substituted amino acid may be W, F, V, L, I, P, or M. In another example, the substituted amino acid may be V or L. In another example, the substituted amino acid may be V. In certain embodiments, the mutated L109 protein comprises the amino acid sequence set forth in SEQ ID NO:16. In certain embodiments, the L109 protein is encoded by the List109 gene, and the sequence of the mutated List109 gene is set forth in SEQ ID NO:15.

[0221] In the present application, the amino acid mutation of the L109 protein may be a substitution of amino acids at positions A15 and H67, wherein the amino acid substituted at position A15 may include any standard amino acid other than A, and the amino acid substituted at position H67 may include any standard amino acid other than H. In certain embodiments, the amino acid substituted at position A15 may be V or L, and the amino acid substituted at position H67 may be R or K. In certain embodiments, the amino acid substituted at position A15 may be V, and the amino acid substituted at position H67 may be R. In certain embodiments, the mutated L109 protein may include the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the L109 protein is encoded by the List109 gene, and the sequence of the List109 gene is shown in SEQ ID NO: 13.

[0222] In certain embodiments, the L109 recombinant protein can be isolated from any vector containing the L109 protein or synthesized in vitro. For example, it can be produced or synthesized by the following methods: (1) in vitro amplification, such as by polymerase chain reaction (PCR), (2) cloning and recombination, (3) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (4) synthesis, such as by chemical synthesis.

[0223] In certain embodiments, the L109 recombinant protein can be isolated from any vector containing the L109 protein. The vector can be an oncolytic virus. Oncolytic viruses include, but are not limited to, vaccinia virus and other examples that may express the L109 protein.

[0224] On the other hand, the present application provides a kit comprising any one of the aforementioned L109 recombinant proteins.

[0225] In certain embodiments, the kit can be used to detect the content of chondroitin sulfate in a mixture. The mixture can be an in vitro sample, including but not limited to blood, urine, feces, sputum and / or cavitary effusion. Detecting the content of chondroitin sulfate in the mixture can help understand the body's skeletal state, thereby diagnosing and treating diseases. In certain embodiments, the kit may also include other containers or reagents that may be used in the detection process. For example, the reagent may include an adsorption liquid and / or an eluent. The container may be selected from one or more of the following: a centrifuge tube, a syringe, and an adsorption column.

[0226] On the other hand, the present application provides an oncolytic virus comprising any of the aforementioned L109 recombinant proteins. The oncolytic virus may be the vaccinia virus mentioned in the present application; may be other poxviruses, such as monkeypox virus, myxoma virus and / or smallpox virus; or may be other different types of oncolytic viruses, the different types of oncolytic viruses may be selected from one or more of the following: adenovirus, reovirus, herpes virus, poxvirus, paramyxovirus, rhabdovirus, picornavirus, influenza virus and parvovirus.

[0227] In certain embodiments, the oncolytic virus may comprise the L109 recombinant protein. The oncolytic virus comprising the L109 recombinant protein has a higher replication ability and / or the ability to target and kill tumors than other oncolytic viruses. In certain embodiments, the oncolytic virus may comprise the L109 recombinant protein. The L109 recombinant protein may be added to an oncolytic virus and / or a composition containing an oncolytic virus. In certain embodiments, the L109 recombinant protein may be expressed by an oncolytic virus. For example, an expression vector carrying the L109 recombinant protein is integrated into the oncolytic virus genome by stable transfection. The transfection method may be selected from one or more of the following: viral transfection, liposome transfection, calcium transfection, electroporation, laser perforation, and ultrasonic microwave perforation. For another example, an expression vector carrying the L109 recombinant protein is transferred into an oncolytic virus for transient expression by transient transfection.

[0228] D. Nucleic acid

[0229] In another aspect, the present application provides a nucleic acid molecule that can encode the aforementioned vaccinia virus, the aforementioned recombinant protein, and / or the aforementioned oncolytic virus comprising the L109 recombinant protein.

[0230] In certain embodiments, the nucleic acid molecule can be purified from the oncolytic virus genome. For example, the nucleic acid molecule can be isolated from the oncolytic virus by a DNA extraction kit. For another example, the nucleic acid molecule can be obtained by extracting RNA from the oncolytic virus by an RNA extraction kit and then reversely transcribing it, and the RNA can be mRNA (messenger RNA).

[0231] In certain embodiments, the nucleic acid molecule can be produced or synthesized by: (1) amplified in vitro, such as by polymerase chain reaction (PCR), (2) produced by cloning and recombination, (3) purified, such as by enzyme digestion and gel electrophoresis fractionation, or (4) synthesized, such as by chemical synthesis. In certain embodiments, the nucleic acid molecule can also include a nucleic acid molecule prepared by recombinant DNA technology.

[0232] On the other hand, the present application provides a vector, which may contain the aforementioned nucleic acid molecule.

[0233] In certain embodiments, the vector may comprise an expression vector. In certain embodiments, the vector may be selected from one or more of the following: a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector.

[0234] E. cells

[0235] In another aspect, the present application provides a cell, which may comprise the aforementioned vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, and / or the aforementioned vector.

[0236] In certain embodiments, the cell may comprise a host cell.

[0237] In certain embodiments, the cell may comprise a tumor cell. The tumor cell may be present in vivo.

[0238] For example, the tumor cells may be tumor cell clusters that are aggregated in a certain organ and / or tissue in the body. In another example, the tumor cells may be single or multiple tumor cells that are dispersed in a certain organ and / or tissue in the body. In another example, the tumor cells may be circulating tumor cells in the body's circulatory system. The circulatory system may include blood vessels and / or lymphatic vessels.

[0239] In certain embodiments, the cells may comprise tumor cells. The tumor cells may comprise cell lines isolated and cultured in vitro.

[0240] For example, the tumor cells can be primary cell lines isolated from in vivo tumor tissue. In another example, the tumor cells can be continuous cell lines. The continuous cell lines can be commercial or non-commercial.

[0241] In certain embodiments, the cells may comprise tumor cells. The tumor cells may be derived from any organism.

[0242] For example, the tumor cells can be of human or non-human origin. The non-human origin can be of mammalian or non-mammalian origin. Examples of the mammals can be selected from one or more of the following: mouse, rat, guinea pig, rabbit, and monkey.

[0243] F. Pharmaceutical Compositions

[0244] In another aspect, the present application provides a composition comprising the aforementioned vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, and / or the aforementioned cell.

[0245] In certain embodiments, the composition may comprise a pharmaceutical composition. The pharmaceutical composition may comprise the aforementioned vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or, optionally, a pharmaceutically acceptable carrier. The optionally pharmaceutically acceptable carrier may comprise saline, buffer, glucose, water, glycerol, ethanol, and any combination thereof. The pharmaceutical formulation should match the mode of administration.

[0246] In certain embodiments, the pharmaceutical composition can be used alone to treat tumors. In certain embodiments, the pharmaceutical composition can be used in combination with other drugs to treat tumors. The other drugs can be selected from one or more of the following: small molecule targeted anticancer agents, antibody drugs, adoptive cell therapy, oncolytic virus enhancers, and other types of oncolytic viruses.

[0247] In some embodiments, the pharmaceutical composition can be combined with a small molecule targeted anticancer agent. For example, the small molecule targeted anticancer agent can include a tyrosine kinase inhibitor, and / or a serine / threonine kinase inhibitor. The tyrosine kinase can be selected from one or more of the following: epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor receptor (FGFR), Janus kinase (JAK) and focal adhesion kinase (FAK). The serine / threonine kinase can be selected from one or more of the following: cyclin-dependent kinase (CDK), mitogen-activated protein kinase (MAPK), protein kinase D (PKD), DNA-dependent protein kinase (DNA-PK), Aurora protein kinase and pancreatic kininogenase. For example, the target of the small molecule targeted anticancer agent can be selected from one or more of the following: cell membrane target, cytoplasmic target and nuclear target. The cell membrane target may be selected from one or more of the following: EGFR, EGFR-TK, ALK, MET, KRAS, NRAS, HER-1, HER-2, and BCR-ABL. The cytoplasmic target may be selected from one or more of the following: mTOR, VEGF, BRAF, and 26S proteasome. The nuclear target may include a nucleotide methyltransferase and / or a histone deacetylase. For example, the target of the small molecule targeted anticancer agent may include a target for angiogenesis. The target for angiogenesis may be selected from one or more of the following: TNF-α, VEGF, VEGFR, COX-2, RAF, PDGFR, FLT, c-Kit, and KIT.

[0248] In some embodiments, the pharmaceutical composition can be combined with an antibody drug. The target of the antibody drug may include immune checkpoints and / or cytokines. For example, the immune checkpoints may be selected from one or more of the following: PD-1, PD-L1, PD-L2, CTLA4, VISTA, CD155, TIGIT, CD28, ICOS, GITR, GITRL, TIM-3, CD137, and LAG-3. The cytokines may be selected from one or more of the following: IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, and IFN-γ.

[0249] In some embodiments, the pharmaceutical composition can be combined with adoptive cell therapy to treat tumors. The adoptive cell therapy may include extracting the host immune components from the body, modifying their immune activity and then infusing them back into the same host. For example, the host immune components may include immune cells, and the immune cells may be selected from one or more of the following: T lymphocytes, tumor infiltrating lymphocytes (TIL), natural killer cells (NK cells), macrophages and B cells. The modification method may include chimeric antigen receptor (CAR) modification and / or engineered T cell receptor (TCR) modification. For example, examples of the adoptive cell therapy may be selected from one or more of the following: CAR-T, CAR-NK, CAR-M, TCR-T, TCR-NK and TCR-M.

[0250] In some embodiments, the pharmaceutical composition can be combined with an oncolytic virus enhancer to treat tumors.

[0251] In some embodiments, the pharmaceutical composition can be combined with other types of oncolytic viruses to treat tumors, and the other types of oncolytic viruses include but are not limited to the oncolytic viruses mentioned in this application and / or other possible examples.

[0252] In some embodiments, the pharmaceutical composition can be used alone to treat tumors.

[0253] In some embodiments, the composition may comprise a viral stock solution of an oncolytic virus or vaccinia virus as described herein. In certain embodiments, the viral stock solution may be a homogeneous stock solution. The preparation and analysis of viral stock solutions may be well known in the art. For example, viral stock solutions may be made in roller bottles containing cells transduced with a viral vector. Subsequently, the viral stock solution may be purified using a continuous nycodenze gradient and divided into several aliquots and stored for later use. The titer of viral stock solutions varies considerably, depending largely on the viral genotype and the protocol and cell line used to prepare these stock solutions.

[0254] In some embodiments, the titer of a viral stock encompassed herein (e.g., a Lister-based vector viral stock) can be at least 10 5 plaque forming units (pfu), for example, at least 10 6 pfu, at least 10 7 pfu, at least 10 8 pfu, at least 10 9 pfu, at least 10 10 pfu or at least 10 11 pfu.

[0255] G.Medicine box

[0256] On the other hand, the present application provides a drug kit for treating tumors and / or cancer, which comprises the aforementioned modified vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned pharmaceutical composition.

[0257] In certain embodiments, it may comprise the aforementioned modified vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned pharmaceutical composition. In certain embodiments, it may further comprise one or more components, such as instructions for use, devices and / or other reagents and components, such as tubes, containers and / or syringes for performing the methods described herein. In certain embodiments, it may further comprise one or more reagents administered in combination with the modified vaccinia virus, for example, anticancer agents and / or immunomodulators. In certain embodiments, it may comprise a device for administering the vaccinia virus to a subject. Any of a variety of devices known in the art for administering drugs and pharmaceutical compositions may be included in the kit provided herein. For example, but not limited to, the device includes a hypodermic needle, an intravenous needle, a catheter, a needle-free injection device, an inhaler and / or a liquid dispenser, for example, an eye dropper. In certain embodiments, the recombinant vaccinia virus to be delivered systemically (e.g., by intravenous injection) may be included in the kit together with a hypodermic needle and a syringe.

[0258] In certain embodiments, the kit may further comprise a plurality of inactivated tumor cells. In certain embodiments, the kit may further comprise instructions for mixing the modified vaccinia virus with the inactivated tumor cells prior to use. In certain embodiments, the kit may further comprise administering the modified vaccinia virus, the immunomodulator (including combinations of immunomodulators), and / or the plurality of inactivated tumor cells simultaneously to the tumor site.

[0259] In another aspect, the present application provides a drug kit for treating tumors and / or cancers with a synergistic combination drug. The kit may include: a first container containing a vaccinia virus and a second container containing immune cells, wherein the first container and the second container are independent; and instructions specifying the timing and method of administration; wherein the vaccinia virus can selectively replicate in tumor cells; and wherein the surface of the immune cells can be modified with a chimeric antigen receptor, wherein the chimeric antigen receptor can include an operably linked, sequentially connected antigen-binding domain, a spacer, a transmembrane region, and an intracellular domain.

[0260] H. Application

[0261] On the other hand, the present application provides a method for preventing or treating cancer, which method comprises exposing the cancer cell to a vaccinia virus or a composition thereof as described herein under conditions sufficient to allow the modified vaccinia virus to infect the cancer cell and replicate in the cancer cell, and wherein the replication of the oncolytic virus in the cancer cell causes cell death. In certain embodiments, the expression of tumor suppressive miRs in cancer cells is reduced compared to non-cancer cells. In certain embodiments, the cancer cells killed by this method can be performed in vivo. In certain embodiments, the cancer cells killed by this method can be performed in a tumor.

[0262] The present application relates to a method for treating cancer in a subject in need thereof, which method may comprise administering to the subject a prophylactically effective amount or a therapeutically effective amount of a vaccinia virus, viral stock solution, or composition as described herein. As used herein, a "subject" may include any animal that exhibits symptoms of a disease, disorder, or condition that can be treated with the recombinant viral vectors, compositions, and methods disclosed herein. Suitable subjects (e.g., patients) may include non-human primates and / or human patients. Non-human primates may be selected from one or more of the following: laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses or cows), livestock, and pets (e.g., cats or dogs).

[0263] The "administration" described in this application refers to introducing the aforementioned modified vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell and / or the aforementioned pharmaceutical composition into the subject, or contacting the aforementioned modified vaccinia virus, the aforementioned recombinant protein, the aforementioned oncolytic virus, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell and / or the aforementioned pharmaceutical composition with cells and / or tissues. Administration can be carried out by injection, flushing, inhalation, consumption, electroosmosis, hemodialysis, iontophoresis and / or other methods known in the art. The route of administration may vary with the location and nature of the disease being treated. The route of administration may include an administration site and a mode of administration. The administration site can be selected from one or more of the following: auris, buccal, conjunctival, transdermal, dental, intracervical, endosinusial, intratracheal, intestinal, epidural, interstitial, intraarticular, intraarterial, intraabdominal, intraauricular, intrabiliary, intrabronchial, intrabursal, intracavernous, intracerebral, intracisternal, intracorneal, intracoronary, intracoronary, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intraduodenal, intradural, intraepicardial, intraepidermal, intraesophageal, intragastric, intragingival, intrahepatic, intraileal, intralesional, intralingual, intraluminal, intralymphatic

[0013] The invention relates to the administration of intravenous, intrathecal, intrathoracic, intracanalicular, intraventricular, intraventricular, intraventricular, intravesical ... The administration mode may be selected from one or more of the following: perfusion, lavage and direct injection.

[0264] As used herein, "treating" or "treatment" refers to administering a therapeutically effective amount of a vaccinia virus or a composition thereof as described herein to a subject so that the subject's disease or disorder, or symptoms of the disease or disorder, are improved. The improvement is any improvement or cure of the disease or disorder, or symptoms of the disease or disorder. The improvement can be an observable or measurable improvement, or it can be an overall sense of the subject's health. Therefore, those skilled in the art recognize that treatment can improve the disease condition, but may not be a complete cure for the disease. A "prophylactically effective amount" refers to an amount of virus, viral stock, or composition that is effective to achieve the desired preventive result. As used herein, "prevention" can refer to the complete prevention of disease symptoms, the delay of the onset of disease symptoms, or the reduction in the severity of subsequent disease symptoms. Typically, but not necessarily, the prophylactic effective amount is less than the therapeutically effective amount because the prophylactic dose can be used for the subject before or early in the course of the disease.

[0265] "Cancer" as described herein can be solid cancer and / or hematologic tumors. In certain embodiments, the solid tumor can be selected from one or more of the following: lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, fibrosarcoma, esophageal cancer, skin cancer, thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, and pancreatic cancer. In addition, the hematologic tumor can be selected from one or more of the following: lymphoma, acute leukemia, and multiple myeloma. The tumor can be a disseminated tumor or a circulating tumor.

[0266] The dosage of the modified vaccinia virus of the present application may vary according to the individual's condition and / or weight, severity of the disease, drug form, route of administration and / or cycle, and may be appropriately selected by those skilled in the art. Specifically, the dosage may be such that the patient receives 1×10 5 to 1×10 18 , for example 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , 5×10 9 , 1×10 10 , 5×1010 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 The dosage of the virus can be 0.1 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL or more, and can include all values ​​and ranges therebetween.

[0267] On the other hand, the present application also provides the following implementation methods:

[0268] 1. A modified vaccinia virus, comprising L006 protein and L007 protein whose expression and / or activity is inhibited compared to an unmodified vaccinia virus.

[0269] 2. The modified vaccinia virus according to embodiment 1, wherein the expression and / or activity of the L006 protein and the L007 protein are inhibited by one or more means selected from the following:

[0270] (1) deleting all or part of the genes encoding the L006 protein and the L007 protein; and

[0271] (2) inserting foreign genes into the genes encoding the L006 protein and the L007 protein;

[0272] The partial gene encoding the L006 protein includes a gene encoding the functional domain of the L006 protein, and the partial gene encoding the L007 protein includes a gene encoding the functional domain of the L007 protein.

[0273] 3. The modified vaccinia virus according to any one of embodiments 1-2, wherein the expression and / or activity of the L006 protein and the L007 protein are inhibited by completely deleting the L006 protein and the L007 protein.

[0274] 4. The modified vaccinia virus of any one of embodiments 1-5, wherein the L006 protein is encoded by the List006 gene and the L007 protein is encoded by the List007 gene.

[0275] 5. The modified vaccinia virus according to any one of embodiments 1 to 6, wherein the expression and / or activity of the L006 and L007 proteins are inhibited by one or more of the following:

[0276] (1) Delete all or part of the List006 gene and List007 gene; and

[0277] (2) inserting foreign genes into the genes of List006 and List007;

[0278] Among them, the List006 gene includes a gene encoding the functional domain of the L006 protein, and the List007 gene includes a gene encoding the functional domain of the L007 protein.

[0279] 6. The modified vaccinia virus according to embodiment 5, wherein the partial deletion of the List006 and List007 genes comprises a deletion of a promoter or premature terminator expression.

[0280] 7. The modified vaccinia virus according to any one of embodiments 1 to 6, wherein the expression and / or activity of the L006 and L007 proteins are inhibited by complete deletion of the List006 and List007 genes.

[0281] 8. A modified vaccinia virus comprising an amino acid mutation in the L109 protein compared to an unmodified vaccinia virus.

[0282] 9. According to the modified vaccinia virus described in embodiment 8, the amino acid mutation of the L109 protein includes a substitution of the amino acid at position H67 on the L109 protein.

[0283] 10. The modified vaccinia virus according to embodiment 9, wherein the amino acid substituted at position H67 on the L109 protein is an amino acid with an isoelectric point higher than 7.5.

[0284] 11. The modified vaccinia virus according to embodiment 9, wherein the amino acid substituted at position H67 on the L109 protein is an amino acid without a cyclic structure on the side chain.

[0285] 12. The modified vaccinia virus according to embodiment 9, wherein the amino acid substituted at position H67 on the L109 protein is a basic amino acid.

[0286] 13. The modified vaccinia virus according to any one of embodiments 8-12, wherein the amino acid substituted at position H67 on the L109 protein is R.

[0287] 14. The modified vaccinia virus according to any one of embodiments 8-13, wherein the amino acid mutation of the L109 protein further comprises a substitution of the amino acid at position A15 on the L109 protein.

[0288] 15. The modified vaccinia virus according to embodiment 14, wherein the amino acid substituted at position A15 on the L109 protein is an amino acid with a molecular weight greater than 90.

[0289] 16. The modified vaccinia virus according to embodiment 15, wherein the amino acid substituted at position A15 on the L109 protein is a hydrophobic amino acid.

[0290] 17. The modified vaccinia virus of any one of embodiments 14-16, wherein the amino acid substituted at position A15 on the L109 protein is V.

[0291] 18. The modified vaccinia virus of any one of embodiments 1-17, selected from one or more of the following groups:

[0292] (1) The amino acid at position H67 on the L109 protein was substituted;

[0293] (2) The amino acid at position A15 on the L109 protein was substituted;

[0294] (3) complete or partial deletion of List006 and List007 genes;

[0295] (4) L006 protein and L007 protein are completely or partially missing; and

[0296] (5) Insert foreign genes into the genes of List006 and List007.

[0297] 19. The modified vaccinia virus of any one of embodiments 1-18, selected from one or more of the following groups:

[0298] (1) The amino acid at position H67 on the L109 protein is substituted with R;

[0299] (2) The amino acid at position A15 on the L109 protein is substituted with V;

[0300] (3) List006 and List007 genes are completely missing;

[0301] (4) L006 and L007 proteins are completely absent;

[0302] (5) List006 and List007 genes are expressed with promoter deletion or premature terminator; and

[0303] (6) Insert foreign genes into the genes of List006 and List007.

[0304] 20. The modified vaccinia virus of any one of embodiments 1-19, selected from one or more of the following groups:

[0305] (1) The L109 protein comprises the amino acid sequence shown in SEQ ID NO: 12;

[0306] (2) the L109 protein comprises the amino acid sequence shown in SEQ ID NO: 14;

[0307] (3) the L109 protein comprises the amino acid sequence shown in SEQ ID NO: 16; and

[0308] (4) The L006 protein and the L007 protein are completely missing, the L006 protein comprises the amino acid sequence shown in SEQ ID NO: 6, and the L007 protein comprises the amino acid sequence shown in SEQ ID NO: 8.

[0309] 21. The modified vaccinia virus of any one of embodiments 1-20, selected from one or more of the following groups:

[0310] (1) The List109 gene comprises the amino acid sequence shown in SEQ ID NO: 11;

[0311] (2) the List109 gene comprises the amino acid sequence shown in SEQ ID NO: 13;

[0312] (3) the List109 gene comprises the amino acid sequence shown in SEQ ID NO: 15; and

[0313] (4) The List006 gene and the List007 gene are completely deleted, the List006 gene comprises the amino acid sequence shown in SEQ ID NO: 5, and the List007 gene comprises the amino acid sequence shown in SEQ ID NO: 7.

[0314] 22. The modified vaccinia virus of any one of embodiments 1-21, wherein the modified vaccinia virus is selected from one or more of the following: Lister, Western Reserve, Copenhagen, Paris, Tashkent, Tiantan, Wyeth, Brighton, Ankara, Dairen I, LIVP, Connaught, New York City Health Department, and variants thereof.

[0315] 23. The modified vaccinia virus of any one of embodiments 1-22, wherein the vaccinia virus is a Lister strain.

[0316] 24. The modified vaccinia virus of any one of embodiments 1-23, further comprising a mutation in one or more genes selected from:

[0317] (1) Backbone genes: TK, A34R, A35R, A36R, A46R, A56R, L025, K3L, and F14.5L, and / or

[0318] (2) Genes encoding one or more proteins selected from the group consisting of B5R, B8R, B15R, B18R, C12L, H3L, A27L and L1R.

[0319] 25. The modified vaccinia virus of any one of embodiments 1-24, wherein an exogenous therapeutic gene is expressed on the modified vaccinia virus.

[0320] 26. The modified vaccinia virus of embodiment 25, wherein the exogenous therapeutic gene is an immune-related gene.

[0321] 27. The modified vaccinia virus of embodiment 26, wherein the immune-related gene encodes a chemokine, an immune cell growth factor, and / or an immune cell activating factor.

[0322] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0323] Example

[0324] Materials and methods

[0325] Cell lines: The following murine tumor cell lines were used in this study: C57B / 6 mouse ovarian cancer cell line ID8 was obtained from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., catalog number BNCC339488; the murine pancreatic ductal adenocarcinoma cell line DT6606 was a gift from Queen Mary University, United Kingdom; the golden hamster melanoma cell line RPMI-1846 was obtained from ATCC, catalog number CTL-49; and the golden hamster pancreatic cancer cell line HPD-1NR was obtained from Shanghai Baiye Biotechnology Center, catalog number HPD-1NR. The African green monkey "normal" kidney cell line CV1 was obtained from ATCC, catalog number CCL-70, and was used as the stock cell line to facilitate large-scale virus production and all viral titration assays. Human tumor cell lines include: human rhabdomyosarcoma cell line A673, obtained from Wuhan Prosai Biotechnology Co., Ltd., Catalog No. CL-0017; human ovarian cancer cell line SK-OV-3, obtained from Wuhan Prosai Biotechnology Co., Ltd., Catalog No. CL-0215; human lung adenocarcinoma cell line HCC827, obtained from Wuhan Prosai Biotechnology Co., Ltd., Catalog No. CL-0094; and human embryonic kidney cell line HEK293, obtained from Guangzhou Geneo Biotechnology Co., Ltd., Catalog No. HEK293.

[0326] Virus: Wild-type vaccinia virus Listeria monocytogenes was obtained from ATCC, ATCC No. VR-1549.

[0327] Statistical methods: Unless otherwise stated, all data involved in this application were analyzed using Graphpad Prism for comparative statistics. Tumor volume was calculated as (length × width 2 × π) / 6. Unpaired t-tests were used for double-condition comparisons. For additional variables with more than one condition, 1- or 2-way ANOVAs were performed. Survival data were presented as Kaplan-Meier plots with log-rank analysis to depict whether any differences between groups were statistically significant. However, the data processing platform involved in this application is not limited to Graphpad Prism.

[0328] Example 1: Obtaining Vaccinia Virus with Double Deleted List006 and List007 Genes

[0329] Example 1.1 Screening of monoclonal virus strains

[0330] 1. Dilute wild-type vaccinia virus (Listeria monocytogenes) to an MOI of 0.0001 pfu / cell and infect a six-well plate containing CV1 cells grown to 80-90% confluence. The MOI is calculated based on the number of cells at the time of infection.

[0331] 2. Mark the location of the single clone under a microscope 24 hours after infection. Pick out the single clone and place it in a cryovial containing cell culture medium.

[0332] 3. After one freeze-thaw cycle, add 200 μL of the virus solution to a new 6-well plate containing CV1 cells. After 48 hours of infection, scrape the infected cells and centrifuge at 4°C, 2000 rpm, for 5 minutes to obtain a cell pellet.

[0333] Example 1.2 Verification that the monoclonal virus is a vaccinia virus with double deletions of the List006 and List007 genes

[0334] 1. Take part of the cell pellet in Example 1.1 to extract viral DNA.

[0335] 2. Use the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 to construct forward and reverse primers for verifying the double deletion; use the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 to construct forward and reverse primers for verifying the control gene without deletion.

[0336] 3. The target gene in the viral DNA was amplified by PCR to obtain the corresponding DNA fragment, and the PCR products were analyzed by 1% agarose gel electrophoresis. As shown in Figure 1, it was verified that the VACV 109 virus strain was a virus strain with a double deletion of the List006 and List007 genes, and VACV 108 was a strain without a double deletion.

[0337] Example 1.3 Amplification and large-scale production of target virus strains

[0338] 1. Primary Amplification: Lyse VACV 108 and VACV 109 obtained in Example 1.2 using a single rapid freeze-thaw cycle at -80°C. Grow CV1 cells to 80-90% confluence in a T175 flask containing approximately 30 mL of cell culture medium. Add 50 μL of viral lysate. After 48 hours, scrape the cells and culture medium and store.

[0339] 2. Large-Scale Production: The primary virus amplification obtained in step 2 was rapidly freeze-thawed once and diluted to the volume required to infect 36 T175 culture flasks containing CV1 cells (80-90% confluence). After 48 hours, the infected CV1 cells were scraped and centrifuged once at 4°C, 2,000 rpm, for 5 minutes to collect the cell pellet. The pellet was washed in PBS, lysed in 12 mL of 10 mM Tris-HCl (pH 9) buffer, and stored at -80°C.

[0340] Example 1.4 Purification of target virus

[0341] 1. Freeze-thaw the viral lysate suspension obtained in Example 1.3 once and vortex for a few seconds. Centrifuge at 2,000 rpm for 5 minutes at 4°C. Collect the supernatant and dilute to a total volume of 30 mL with 10 mM Tris-HCl (pH 9) buffer.

[0342] 2. Dispense 30 mL of virus dilution into four Beckman ultracentrifuge tubes, add 17 mL of 36% sucrose solution, and centrifuge at 13,500 rpm for 80 minutes at 4°C.

[0343] 3. The final pellet was resuspended in 1 mL of virus resuspension buffer (PBS, 10% glycerol, 138 mM NaCl, pH 7.4) and stored at -80°C.

[0344] Example 2 Replication Capacity of Vaccinia Virus with Double Deletion of List006 and List007 Genes

[0345] 1. In a 6-well plate containing cell culture medium, 6 × 10 5 The cells were inoculated and infected with vaccinia virus at an MOI of 0.01 pfu / cell on the next day. The MOI was calculated based on the number of cells at the time of infection.

[0346] 2. Infected cells and their culture medium were harvested 24, 48, and 72 hours after infection. Virus concentrations were measured at different time points, and vaccinia virus amplification curves were generated. As shown in Figure 2, VACV 109 demonstrated superior replication capacity compared to VACV 108. At 48 and 72 hours after infection, the viral load of VACV 109 was 4.3 and 6.6 times that of VACV 108, respectively.

[0347] Example 3 Tumor Killing Ability of Vaccinia Virus with Double Deletion of List006 and List007 Genes

[0348] Example 3.1 Ability of List006 and List007 gene double-deleted vaccinia virus to kill tumor cells in vitro

[0349] 1. In a 96-well plate, 1×10 3 CV1 cells were inoculated into 10 cells / well and infected with virus 16 hours later. The initial virus amount was MOI = 100 pfu / cell. Each row of cells was infected in sequence according to a 1:10 dilution ratio. MOI was calculated according to the number of cells at the time of infection.

[0350] 2. Cell viability on day 6 after virus infection was determined by crystal violet staining, and the EC50 value (the viral dose required to kill 50% of tumor cells) was calculated. All assays were performed at least three times. As shown in Figure 3, VACV 109 had a stronger ability to kill tumor cells in vitro than VACV 108.

[0351] Example 3.2 In vivo antitumor effect of vaccinia virus with double deletion of List006 and List007 genes

[0352] 1. Human rhabdomyosarcoma cell line A673 was inoculated unilaterally subcutaneously on the back with an inoculum of 5×10 6 The tumor mouse model was established with 100 μL of cells per mouse. The mice used were 5-6 week old female BALB / c nude mice.

[0353] 2. Tumor volume reaches 100mm 3 The mice were then divided into three groups based on tumor size: PBS, VACV 108, and VACV 109, which received PBS, VACV 108, and VACV 109, respectively. Each group consisted of four mice.

[0354] 3. The drug was administered by intratumoral injection on D1, D5 and D9, with a dosage of 1x 10 6 pfu / 50μL / mouse, and the PBS group was injected with 50μL / mouse of PBS.

[0355] 4. The day of the first administration was recorded as D1. Tumor volume was measured twice a week until the tumor volume reached 2000 mm 3 The mice were killed.

[0356] As shown in Figure 4, VACV 109 demonstrated superior in vivo antitumor activity compared to VACV 108. At days 9 and 13, tumor volumes in the VACV 109 and VACV 108 groups were significantly different from those in the PBS group, indicating that both strains significantly inhibited tumor growth. However, the tumor volume in the VACV 109 group was smaller than that in the VACV 108 group, indicating a stronger antitumor effect.

[0357] Example 4 Obtaining Vaccinia Virus with L109 Protein Mutation

[0358] Example 4.1 Screening of monoclonal virus strains

[0359] 1. Virus passage: HEK293 cells were infected with the vaccinia virus containing double deletions of the List006 and List007 genes obtained in Example 1. After 2 days, the virus-infected cell suspension was collected. 0.01 mL of the suspension was used to reinfect the HEK293 cells, and the obtained suspension was collected again. This operation was repeated 25 times.

[0360] 2. Re-plating: Lyse the suspension after 25 passages in step 1 by freeze-thawing and store at -80°C. After thawing, use 0.5 μL of this lysate to infect all 6 wells of a 6-well plate containing CV1 cells grown to 80-90% confluence.

[0361] 3. Monoclonal strain selection: 48 hours after infection, monoclonal strains were selected using a fluorescence microscope. The selected monoclonal strains were immersed in a cryovial containing cell culture medium (the cell culture medium was the culture medium for culturing HEK293 cells).

[0362] 4. Repeat the selection to obtain a purified monoclonal strain: After one freeze-thaw cycle, add 5 μL of the virus solution to each well of a new 6-well plate containing CV1 cells and repeat step 3. Repeat this process 4 to 5 times to obtain a pure monoclonal virus strain.

[0363] Example 4.2 Sequencing of monoclonal viruses

[0364] 1. Obtain viral DNA: Extract viral DNA using Qiagen's DNA extraction kit.

[0365] 2. Viral Sequencing: Forward and reverse primers for List109 gene amplification were constructed using the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 to amplify the List109 gene from viral DNA. The amplified fragment was then subjected to next-generation sequencing to determine if any amino acid mutations occurred in the L109 protein. The original sequence region encoding the L109 gene is shown in SEQ ID NO:9.

[0366] Example 4.3 Amplification and large-scale production of target virus strains

[0367] 1. After selecting a vaccinia virus strain containing the L109 protein A15V mutation or the A15V / H67R double mutation as described in Example 4.1, the mutation sites are shown in Figure 5, and the virus strain is subjected to primary amplification and large-scale production.

[0368] 2. Primary Amplification: Grow CV1 cells to 80-90% confluence in a T175 flask containing approximately 30 mL of cell culture medium. Add 50 μL of viral lysate. After 48 hours, scrape the cells and store.

[0369] 3. Large-Scale Production: The primary virus obtained in step 2 was amplified and rapidly frozen and thawed once, and diluted to the volume required to infect 36 T175 culture flasks containing CV1 cells (80-90% confluence). After 48 hours, the infected CV1 cells were scraped and centrifuged twice at 2,000 rpm for 5 minutes at 4°C to collect the cell pellet. The pellet was washed in PBS, lysed in 12 mL of 10 mM Tris-HCl (pH 9) buffer, and stored at -80°C.

[0370] Example 4.4 Purification of target virus

[0371] 1. Freeze-thaw the viral lysate suspension from Example 4.3 once and vortex for a few seconds. Centrifuge at 2,000 rpm for 5 minutes at 4°C, collect the supernatant, and dilute to a total volume of 30 mL with 10 mM Tris-HCl (pH 9) buffer.

[0372] 2. Dispense 30 mL of virus dilution into four Beckman ultracentrifuge tubes, add 17 mL of 36% sucrose solution, and centrifuge at 13,500 rpm for 80 minutes at 4°C.

[0373] 3. The final pellet was resuspended in 1 mL of virus resuspension buffer (PBS, 10% glycerol, 138 mM NaCl, pH 7.4) and stored at -80°C. The purified target viruses were subsequently referred to as vaccinia virus mutants (A15V single mutant and A15V / H67R double mutant).

[0374] Example 5 Replication Capacity of Vaccinia Virus Mutants

[0375] 1. Human rhabdomyosarcoma cell line A673 and African green monkey kidney normal cell line CV1 were used for testing. 6×10 cells / well were cultured in a 6-well plate containing cell culture medium. 5 Cells were inoculated with vaccinia virus at an MOI of 0.01 pfu / cell. The strains screened for infection included strains containing mutations at position A15 of the L109 protein, strains containing mutations at positions A15 and H67 of the L109 protein, and strains without mutations at positions A15 and H67 of the L109 protein. The titers were determined on CV1 cells using the TCID50 assay. The MOI was calculated based on the number of cells at the time of infection.

[0376] 2. Collect infected cells and their culture medium 24, 48, and 72 hours after infection. Measure viral concentrations at different time points and generate vaccinia virus amplification curves.

[0377] 3. The results are shown in Figure 6. Compared with the unmutated virus (Lis-con), both the L109 protein A15V single mutation (Lis-single) and the A15V / H67R double mutation (Lis-double) can significantly improve the virus replication ability in both cancer cells and normal cells.

[0378] Example 6 Tumor Killing Ability of Vaccinia Virus Mutants

[0379] Example 6.1 Ability of Vaccinia Virus Mutants to Kill Tumor Cells in Vitro

[0380] 1. In a 96-well plate, 1×10 3 Cells were seeded at 100 cells / well and infected with virus 16 hours after seeding.

[0381] 2. The cytotoxicity of the viral mutants in human (human rhabdomyosarcoma cell line A673, human ovarian cancer cell line SK-OV-3, human lung adenocarcinoma cell line HCC827), mouse (mouse ovarian epithelial cancer cell line ID8, mouse pancreatic ductal adenocarcinoma cell line DT6606) and golden hamster (golden hamster pancreatic cancer cell line HPD-1NR) cell lines was detected. The cell viability on the 6th day after virus infection was determined by crystal violet staining, and the EC50 value (virus dose that kills 50% of tumor cells) was calculated. All assays were performed at least three times. The results are shown in Figure 7. Compared with the strain control without mutation (Lis-con), the EC50 of various tumor cells was significantly decreased after the administration of vaccinia viruses with L109 protein A15V single site mutation (Lis-single) and A15V / H67R double site mutation (Lis-double), indicating that both the A15V single mutation and the A15V / H67R double mutation on the L109 protein significantly enhanced the ability of the virus to kill tumor cells in vitro.

[0382] Example 6.2 In vivo anti-tumor effects of vaccinia virus mutants

[0383] Example 6.2.1 Treatment of subcutaneous pancreatic cancer in mice with vaccinia virus mutants

[0384] 1. Inoculate 5×10 cells of pancreatic cancer cell line DT6606 subcutaneously on the back of C57 / BL6 mice. 6 cells / 100 μL / mouse to establish a tumor mouse model.

[0385] 2. Tumor volume reaches 120 mm 3 The mice were randomly divided into 4 different drug administration groups: negative control group, single mutation group, double mutation group and non-mutation group, and injected with PBS (control), Lis-single strain, Lis-double and Lis-con strain respectively, with a sample size of 6 in each group.

[0386] 3. The administration frequency is once every 2 days, and the administration is done by intratumoral injection. The dosage of different Lister strains is 5×10 6 pfu / 50μL / mouse, the drug was given for 5 consecutive times, and the negative control group was injected with 50μL / mouse of PBS.

[0387] 4. The day of drug treatment was recorded as D1, and the experiment ended on D15.

[0388] The results are shown in Figure 8. The average tumor volume of the control group continued to increase during the experiment, and different Lister strains showed good inhibitory effects on tumor growth, among which the Lis-single and Lis-double groups showed stronger inhibitory effects. Figure 8D shows that compared with the Control, the tumor weight of the Lister-single treatment group was significantly reduced, and the tumor weight of the Lis-double treatment group was reduced the most. Figure 8E shows that during the entire experimental administration process, C57 / BL6 mice showed good tolerance to the tested virus strains, without obvious toxicity, and no significant decrease in body weight. Therefore, both the single and double mutant strains of the L109 protein have better in vivo anti-tumor effects than the unmutated strains in the C57 / BL6 mouse subcutaneous pancreatic cancer model, among which the double mutant strain is better than the single mutant strain.

[0389] Example 6.2.2 Treatment of subcutaneous human rhabdomyosarcoma in nude mice with vaccinia virus mutants

[0390] 1. Human rhabdomyosarcoma cell line A673 was inoculated unilaterally subcutaneously on the back with an inoculum of 5×10 6 The tumor mouse model was established with 100 μL of cells per mouse. The mice used were 4-5 week old female BALB / c nude mice.

[0391] 2. Tumor volume reaches 100mm 3 The above were then randomly divided into three different drug administration groups: negative control group, positive control group, and double mutant group, which were injected with PBS, positive control strain, and double mutant strain, respectively, with a sample size of 5 in each group.

[0392] 3. The administration frequency is once every 3 days, and the dosage is 1×10 6 pfu / 50μL / mouse, the drug was given for 5 consecutive times, and the negative control group was injected with 50μL / mouse of PBS.

[0393] 4. The day of drug treatment was recorded as D1, and the experiment ended on D31.

[0394] As shown in Figure 9, the average weight of the animals in the negative control group continued to increase as the tumor volume increased. The BALB / c nude mice showed good tolerance to the test drugs, without obvious toxicity, and no significant decrease in body weight. At the end of the experiment, the tumor weight of the negative control group at D19 was 2.5826±1.2582g, the tumor weight of the double mutation group at D31 was 0.085±0.092g, and the tumor weight of the positive control group at D31 was 0.276±0.474g. Both the double mutation group and the positive control group showed good inhibition of tumor growth (Figure 9b), and the double mutation group had a better inhibitory effect than the positive control group.

[0395] Example 6.2.3 Treatment of subcutaneous melanoma in golden hamsters with vaccinia virus mutants

[0396] 1. Inoculate RPMI-1846, a hamster melanoma cell line, subcutaneously on one side of the back with a seeding volume of 2×10 6 The tumor mouse model was established with 100 μL of cells per mouse. The golden hamsters used were 5-week-old female mice.

[0397] 2. Tumor volume reaches 100mm 3 The above were then randomly divided into three different drug administration groups: negative control group, positive control group, and double mutant group, which were injected with PBS, positive control strain, and double mutant strain, respectively, with a sample size of 6 in each group.

[0398] 3. The administration frequency is once every 2 days, and the dosage is 3×10 7 pfu / 50μL / mouse, the drug was given for 5 consecutive times, and the negative control group was injected with 50μL / mouse of PBS.

[0399] 4. The day of drug treatment was recorded as D1, and the experiment ended on D19.

[0400] As shown in Figure 10 , at D1, the average tumor volumes of the negative control group, positive control group, and double mutation group were 119.1767±16.8446mm 3 、116.1183±13.5940mm 3 、122.5117±11.9393mm 3 There was no statistical difference between the groups (P>0.05). During the experiment, the tumors of animals in the negative control group continued to grow. When observed to D12, the tumor volumes of the positive control group and the double mutation group were significantly lower than those of the negative control group (P<0.0001). When observed to D16, there was a significant difference in tumor volume between the double mutation group and the positive control group (P<0.05), and the difference was more obvious compared with the negative control group (P<0.0001). The average tumor volumes of the negative control group, positive control group, and double mutation group were 2346.5433±814.0831mm, respectively.3 、1387.1783±1706.2908mm 3 、201.15±319.9380mm 3 (Figure 10a). Throughout the experimental administration process, golden hamsters showed good tolerance to the tested virus strains, without obvious toxicity or significant weight loss (Figure 10b). Therefore, the double mutant strain has a better in vivo anti-tumor effect than the positive control strain in the golden hamster subcutaneous melanoma model.

[0401] Example 6.2.4 Treatment of subcutaneous pancreatic cancer in golden hamsters with vaccinia virus mutants

[0402] 1. Inoculate 2×10 6 The tumor mouse model was established with 100 μL of cells per mouse. The golden hamsters used were 5-week-old female mice.

[0403] 2. Tumor volume reaches 300mm 3 The above were then randomly divided into three different drug administration groups: negative control group, positive control group, and double mutant group, which were injected with PBS, positive control strain, and double mutant strain, respectively, with a sample size of 4 in each group.

[0404] 3. The administration frequency is once every 2 days, and the dosage is 3×10 7 pfu / 100 μL / mouse, and the drug was given for 5 consecutive times. The negative control group was injected with 100 μL of PBS / mouse.

[0405] 4. The day of drug treatment was recorded as D1, and the experiment ended on D21.

[0406] As shown in Figure 11 , the average tumor volumes of the negative control group, positive control group, and double mutation group were 323.332 ± 23.236 mm 3 、326.783±20.084mm 3 、314.138±8.594mm 3 There was no statistical difference between the groups (P>0.05). During the experiment, the tumors in the negative control group continued to grow. At the end of the experiment, D21, the tumor volumes in the positive control group and the double mutation group were significantly lower than those in the negative control group. The average tumor volumes in the negative control group, the positive control group, and the double mutation group were 1616.063±435.288mm, respectively. 3 、215.543±61.418mm 3 、119.273±141.364mm 3(Figure 11a). When observed until D17, the tumors of two golden hamsters in the double mutation group disappeared. By the end of the experiment, two of the four animals in the double mutation group had zero tumors (50% tumor clearance rate), while there were none in the positive control group (Figure 11c). At the end of the experiment, the tumor weights of the negative control group, the positive control group, and the double mutation group were 1.060±0.241g, 0.168±0.043g, and 0.168±0.043g, respectively (Figure 11b). Throughout the experimental administration process, the golden hamsters showed good tolerance to the tested virus strains, without obvious toxicity, and no significant decrease in body weight (Figure 11d). Therefore, the double mutant strain has a better in vivo anti-tumor effect than the positive control strain in the golden hamster subcutaneous pancreatic cancer model.

[0407] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A modified vaccinia virus, compared with the unmodified vaccinia virus, comprises one or more of the following modifications: (1) The expression and / or activity of L006 protein and L007 protein are inhibited, and (2) An amino acid mutation of L109 protein.

2. The modified vaccinia virus according to claim 1, wherein the expression and / or activity of the L006 protein and the L007 protein are inhibited by one or more of the following methods: (1) Deleting all or part of the genes encoding the L006 protein and the L007 protein; and (2) Inserting a foreign gene at the genes encoding the L006 protein and the L007 protein; Among them, The partial gene encoding the L006 protein contains the gene encoding the functional domain of the L006 protein, and the partial gene encoding the L007 protein contains the gene encoding the functional domain of the L007 protein.

3. The modified vaccinia virus according to any one of claims 1-2, wherein the expression and / or activity of the L006 protein and L007 protein are inhibited by the complete deletion of the L006 protein and L007 protein.

4. The modified vaccinia virus according to any one of claims 1-5, wherein the L006 protein is encoded by the List006 gene, and the L007 protein is encoded by the List007 gene.

5. The modified vaccinia virus according to any one of claims 1-6, wherein the expression and / or activity of the L006 and L007 proteins are inhibited by one or more of the following methods: (1) Deleting all or part of the List006 gene and List007 gene; and (2) Inserting a foreign gene at the genes of the List006 gene and List007 gene; Among them, The List006 gene contains the gene encoding the functional domain of the L006 protein, and the List007 gene contains the gene encoding the functional domain of the L007 protein.

6. The modified vaccinia virus according to claim 5, wherein the partial deletion of the List006 and List007 genes includes deleting the promoter or premature terminator expression.

7. The modified vaccinia virus according to any one of claims 1-6, wherein the expression and / or activity of the L006 and L007 proteins are inhibited by the complete deletion of the List006 and List007 genes.

8. The modified vaccinia virus according to claim 1, wherein the amino acid mutation of the L109 protein includes the substitution of the amino acid at position H67 on the L109 protein.

9. The modified vaccinia virus according to claim 8, wherein the amino acid after the substitution at position H67 on the L109 protein is an amino acid with an isoelectric point higher than 7.

5.

10. The modified vaccinia virus according to claim 8, wherein the amino acid after the substitution at position H67 on the L109 protein is an amino acid without a cyclic structure on the side chain.

11. The modified vaccinia virus according to claim 8, wherein the amino acid after substitution at position H67 of the L109 protein is a basic amino acid.

12. The modified vaccinia virus according to any one of claims 8-11, wherein the amino acid after substitution at position H67 of the L109 protein is R.

13. The modified vaccinia virus according to any one of claims 1-12, wherein the amino acid mutation of the L109 protein further comprises substitution of the amino acid at position A15 of the L109 protein.

14. The modified vaccinia virus according to claim 13, wherein the amino acid after substitution at position A15 of the L109 protein is an amino acid with a molecular weight greater than 90.

15. The modified vaccinia virus according to claim 13, wherein the amino acid after substitution at position A15 of the L109 protein is a hydrophobic amino acid.

16. The modified vaccinia virus according to any one of claims 13-15, wherein the amino acid after substitution at position A15 of the L109 protein is V.

17. The modified vaccinia virus according to any one of claims 1-16, which is selected from one or more of the following groups: (1) The amino acid at position H67 of the L109 protein is substituted; (2) The amino acid at position A15 of the L109 protein is substituted; (3) The List006 gene and the List007 gene are completely or partially deleted; (4) The L006 protein and the L007 protein are completely or partially deleted; and (5) An exogenous gene is inserted at the gene locus of the List006 gene and the List007 gene.

18. The modified vaccinia virus according to any one of claims 1-17, which is selected from one or more of the following groups: (1) The amino acid at position H67 of the L109 protein is substituted, and the substituted amino acid is R; (2) The amino acid at position A15 of the L109 protein is substituted, and the substituted amino acid is V; (3) The List006 gene and the List007 gene are completely deleted; (4) The L006 protein and the L007 protein are completely deleted; (5) The List006 and List007 genes lack a promoter or have premature termination of expression; and (6) An exogenous gene is inserted at the gene locus of the List006 gene and the List007 gene.

19. The modified vaccinia virus according to any one of claims 1-18, which is selected from one or more of the following groups: (1) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:12; (2) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:14; (3) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:16; and (4) The L006 protein and the L007 protein are completely deleted, the L006 protein comprises the amino acid sequence shown in SEQ ID NO:6, and the L007 protein comprises the amino acid sequence shown in SEQ ID NO:

8.

20. The modified vaccinia virus according to any one of claims 1-19, which is selected from one or more of the following groups: (1) The List109 gene contains the amino acid sequence shown in SEQ ID NO:11; (2) The List109 gene contains the amino acid sequence shown in SEQ ID NO:13; (3) The List109 gene contains the amino acid sequence shown in SEQ ID NO:15; and (4) The List006 gene and the List007 gene are completely deleted, the List006 gene contains the amino acid sequence shown in SEQ ID NO:5, and the List007 gene contains the amino acid sequence shown in SEQ ID NO:

7.

21. The modified vaccinia virus according to any one of claims 1-20, wherein the modified vaccinia virus is selected from one or more of the following: Lister strain, Western Reserve strain, Copenhagen strain, Paris strain, Tashkent strain, Tian Tan strain, Wyeth strain, Brighton strain, Ankara strain, Dairen I strain, LIVP strain, Connaught strain, New York City Health Department strain, and variants of the above strains.

22. The modified vaccinia virus according to any one of claims 1-21, wherein the vaccinia virus is the Lister strain.

23. The modified vaccinia virus according to any one of claims 1-22, wherein the modified vaccinia virus further contains mutations in one or more genes selected from the following: (1) Skeleton genes: TK, A34R, A35R, A36R, A46R, A56R, L025, K3L, and F14.5L, and / or (2) Genes encoding one or more proteins selected from the following: B5R, B8R, B15R, B18R, C12L, H3L, A27L, and L1R.

24. The modified vaccinia virus according to any one of claims 1-23, wherein an exogenous therapeutic gene is expressed on the modified vaccinia virus.

25. The modified vaccinia virus according to claim 24, wherein the exogenous therapeutic gene is an immune-related gene.

26. The modified vaccinia virus according to claim 25, wherein the immune-related gene encodes a chemokine, an immune cell growth factor, and / or an immune cell activation factor.

27. A recombinant protein, which contains the L109 recombinant protein.

28. The L109 recombinant protein according to claim 27, wherein the L109 recombinant protein has a substitution at the amino acid position 67 (H67).

29. The L109 recombinant protein according to claim 28, wherein the amino acid after substitution at the H67 position of the L109 recombinant protein is R.

30. The L109 recombinant protein according to any one of claims 27-29, wherein the L109 recombinant protein has a substitution at the amino acid position 15 (A15).

31. The L109 recombinant protein according to claim 30, wherein the amino acid after substitution at position A15 on the L109 recombinant protein is V.

32. The L109 recombinant protein according to any one of claims 27-31, wherein the L109 recombinant protein is selected from one of the following groups: (1) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:12; (2) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:14; and (3) The L109 protein comprises the amino acid sequence shown in SEQ ID NO:

16.

33. The L109 recombinant protein according to any one of claims 27-32, wherein the L109 recombinant protein is encoded by the List109 gene, and the List109 gene is selected from one of the following groups: (1) The List109 gene comprises the amino acid sequence shown in SEQ ID NO:11; (2) The List109 gene comprises the amino acid sequence shown in SEQ ID NO:13; and (3) The List109 gene comprises the amino acid sequence shown in SEQ ID NO:

15.

34. A kit, which comprises the L109 recombinant protein according to any one of claims 27-33.

35. The kit according to claim 34, wherein the kit is used for measuring the content of chondroitin sulfate in a mixture.

36. An oncolytic virus, which comprises the L109 recombinant protein according to any one of claims 27-33.

37. The oncolytic virus according to claim 36, wherein the oncolytic virus is selected from one or more of the following: adenovirus, reovirus, herpes virus, poxvirus, paramyxovirus, rhabdovirus, picornavirus, influenza virus, parvovirus, and variants of these viruses.

38. A nucleic acid molecule, which encodes the modified vaccinia virus according to any one of claims 1-26, the recombinant protein according to any one of claims 27-33, and / or the oncolytic virus according to any one of claims 36-37.

39. A vector, which comprises the nucleic acid molecule according to claim 38.

40. The vector according to claim 39, wherein the vector comprises an expression vector.

41. The vector according to claim 39 or 40, wherein the vector comprises a DNA vector and an RNA vector.

42. A cell, which comprises the vaccinia virus according to any one of claims 1-26, the recombinant protein according to any one of claims 27-33, the oncolytic virus according to any one of claims 36-37, the nucleic acid molecule according to claim 38, and / or the vector according to any one of claims 39-41.

43. The cell according to claim 42, wherein the cell comprises a host cell.

44. The cell according to claim 42 or 43, wherein the cell comprises a tumor cell.

45. A pharmaceutical composition comprising the vaccinia virus according to any one of claims 1 - 26, the recombinant protein according to any one of claims 27 - 33, the oncolytic virus according to any one of claims 36 - 37, the nucleic acid molecule according to claim 38, the vector according to any one of claims 39 - 41, the cell according to any one of claims 42 - 44, and / or optionally a pharmaceutically acceptable carrier.

46. The pharmaceutical composition according to claim 45, wherein the vaccinia virus is used alone as a single therapy.

47. The pharmaceutical composition according to claim 45, wherein the vaccinia virus is used in combination with a small molecule targeted anti - cancer agent, an antibody drug, adoptive cell therapy, and / or an oncolytic virus enhancer.

48. A kit comprising the vaccinia virus according to any one of claims 1 - 26, the recombinant protein according to any one of claims 27 - 33, the oncolytic virus according to any one of claims 36 - 37, the nucleic acid molecule according to claim 38, the vector according to any one of claims 39 - 41, the cell according to any one of claims 42 - 44, and / or the pharmaceutical composition according to any one of claims 45 - 47.

49. Use of the vaccinia virus according to any one of claims 1 - 26, the recombinant protein according to any one of claims 27 - 33, the oncolytic virus according to any one of claims 36 - 37, the nucleic acid molecule according to claim 38, the vector according to any one of claims 39 - 41, the cell according to any one of claims 42 - 44, the pharmaceutical composition according to any one of claims 45 - 47, and / or the kit according to claim 48 in the preparation of a drug for preventing and / or treating a disease and / or disorder.

50. The use according to claim 49, wherein the disease and / or disorder includes a tumor.

51. The use according to claim 50, wherein the tumor includes a solid tumor and / or a non - solid tumor.

52. The use according to claim 51, wherein the non - solid tumor is a hematological tumor.

53. The use according to claim 52, wherein the hematological tumor is leukemia or lymphoma.

54. The use according to claim 52 or 53, wherein the hematological tumor is selected from one or more of the following: B - cell lymphoma, T - cell lymphoma, leukemia, Hodgkin lymphoma, myeloma, myelodysplastic syndrome, or plasmacytoma.

55. The use according to claim 51, wherein the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma, and endometrial cancer.

56. A method for preventing and / or treating a disease and / or disorder, comprising administering to a subject in need thereof a vaccinia virus as described in any one of claims 1-26, a recombinant protein as described in any one of claims 27-33, an oncolytic virus as described in any one of claims 36-37, a nucleic acid molecule as described in claim 38, a vector as described in any one of claims 39-41, a cell as described in any one of claims 42-44, a pharmaceutical composition as described in any one of claims 45-47, and / or a kit as described in claim 48.

57. The method according to claim 56, wherein the disease and / or disorder includes a tumor.

58. The method according to claim 57, wherein the tumor includes a solid tumor and / or a non-solid tumor.

59. The method according to claim 58, wherein the non-solid tumor is a hematological tumor.

60. The method according to claim 59, wherein the hematological tumor is leukemia or lymphoma.

61. The method according to claim 59 or 60, wherein the hematological tumor is selected from one or more of the following: B-cell lymphoma, T-cell lymphoma, leukemia, Hodgkin lymphoma, myeloma, myelodysplastic syndrome, or plasmacytoma.

62. The method according to claim 58, wherein the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma, and endometrial cancer.

63. The vaccinia virus as described in any one of claims 1-26, the recombinant protein as described in any one of claims 27-33, the oncolytic virus as described in any one of claims 36-37, the nucleic acid molecule as described in claim 38, the vector as described in any one of claims 39-41, the cell as described in any one of claims 42-44, the pharmaceutical composition as described in any one of claims 45-47, and / or the kit as described in claim 48, for preventing and / or treating a disease and / or disorder.

64. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition, and / or kit according to claim 63, wherein the disease and / or disorder includes a tumor.

65. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition, and / or kit according to claim 64, wherein the tumor includes a solid tumor and / or a non-solid tumor.

66. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition, and / or kit according to claim 65, wherein the non-solid tumor is a hematological tumor.

67. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition, and / or kit according to claim 66, wherein the hematological tumor is leukemia or lymphoma.

68. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition and / or kit according to claim 66 or 67, wherein the hematologic tumor is selected from one or more of the following: B cell lymphoma, T cell lymphoma, leukemia, Hodgkin lymphoma, myeloma, myelodysplastic syndrome or plasmacytoma.

69. The vaccinia virus, recombinant protein, oncolytic virus, nucleic acid molecule, vector, cell, pharmaceutical composition and / or kit according to claim 65, wherein the solid tumor is selected from one or more of the following: adrenocortical carcinoma, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, lymphoma, esophageal cancer, glioma, head and neck squamous cell carcinoma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric cancer, thymic carcinoma and endometrial cancer.

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