WO2021228105A1 - 一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物 - Google Patents

一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物 Download PDF

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WO2021228105A1
WO2021228105A1 PCT/CN2021/093142 CN2021093142W WO2021228105A1 WO 2021228105 A1 WO2021228105 A1 WO 2021228105A1 CN 2021093142 W CN2021093142 W CN 2021093142W WO 2021228105 A1 WO2021228105 A1 WO 2021228105A1
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oncolytic virus
cells
strain
attenuated
cancer
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PCT/CN2021/093142
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English (en)
French (fr)
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周国庆
杨何
张凡
张苏宏
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上海荣瑞医药科技有限公司
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Priority to AU2021271961A priority Critical patent/AU2021271961A1/en
Priority to KR1020227038518A priority patent/KR20230002564A/ko
Priority to JP2023509871A priority patent/JP7468958B2/ja
Priority to CA3178631A priority patent/CA3178631A1/en
Priority to EP21803001.3A priority patent/EP4141111A4/en
Publication of WO2021228105A1 publication Critical patent/WO2021228105A1/zh
Priority to US18/054,597 priority patent/US20230256079A1/en

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Definitions

  • This application relates to the field of biomedicine, in particular to an oncolytic virus vaccine and a drug for treating tumors in combination with immune cells.
  • VSV Vesicular Stomatitis Virus
  • McMaster University in Canada showed that VSV can be used as a new tumor vaccine carrier to promote immune response.
  • the related research of VSV has been paid more and more attention by researchers. From a safety point of view, VSV is relatively safe for humans, and there is no case of VSV infecting humans.
  • VSV is a prototype non-segmented negative-strand RNA virus. Its 11kb genome encodes 5 proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large aggregate Enzyme protein (L). VSV can express a variety of cell surface molecules including low-density lipoprotein receptor, phosphatidylserine, sialolipid and heparan sulfate, and can attach to the cell surface through such molecules. It has the characteristics of fast replication and cross-synaptic speed, and super high expression of foreign genes.
  • N nucleocapsid protein
  • P phosphoprotein
  • M matrix protein
  • G glycoprotein
  • L large aggregate Enzyme protein
  • VSV can express a variety of cell surface molecules including low-density lipoprotein receptor, phosphatidylserine, sialolipid and heparan sulfate, and can attach to the cell surface through such molecules. It has the characteristics of fast replication and
  • VSV genome is small and easy to operate; the replication time is shorter; it has an independent cell cycle; it can grow rapidly in a wide range of cell lines and has a higher titer, allowing Large-scale production; cytoplasmic replication of host cells has no risk of transformation.
  • This oncolytic virus will not be integrated into DNA, and after attenuation, it can avoid the nervous system inflammation caused by wild-type virus. Therefore, VSV has great potential in tumor immunotherapy.
  • VSV can significantly eliminate brain tumors, and also has a significant inhibitory effect on breast cancer and osteosarcoma.
  • researchers have studied the anti-tumor function and toxic side effects of VSV on liver cancer and found that the survival time of liver cancer tumor-bearing mice is significantly increased without significant toxic side effects.
  • VSV-GP treatment the subcutaneous tumors and bone metastases of the prostate cancer mice were significantly reduced; the reduction of the in situ tumors and lung metastases of the melanoma tumor-bearing mice was also significantly improved.
  • M51R VSV can directly induce apoptosis of colorectal cancer cells.
  • VSV can also further affect the development of tumors by regulating innate immunity or acquired immunity.
  • VSV reduces the infiltration of immunosuppressive cells MDSC and macrophages in colorectal cancer tissues, and increases the infiltration of CD4 + T cells, thereby reducing the formation of malignant ascites.
  • VSV can induce the immune response of CD8-specific T cells and reduce the effects of other immunosuppressive cells, thereby enhancing the efficacy of tumor vaccines.
  • the above studies show that VSV has a strong anti-tumor effect and has good safety.
  • T cell receptor genetically engineered T cell (T cell receptor gene engineered T cells, TCR-T) therapy is based on modified T cells and is applied to adoptive cellular immunotherapy for malignant tumors.
  • TCR mediates T cells to recognize MHC Molecule-presented antigens, so that antigen-specific T cells exert immune effects on tumor target cells.
  • Existing research makes it possible to use VSV and TCR-T in combination to treat tumors.
  • VSV can lyse tumor cells through selective replication in tumor cells, and the lysed tumor cells can induce tumor-specific immune responses, promote the activation, expansion, and recruitment of T cells, and activated T cells can pass through the tumor. Kill tumor cells by regulating immune suppression and other methods. The combined use of the two can theoretically exert a better effect than VSV therapy or TCR-T therapy alone.
  • VSV and TCR-T are used in combination for tumor immunotherapy, there are still at least the following problems: (1) The direct use of VSV wild strain or attenuated strain and TCR-T in combination with TCR-T has a low cure rate. Compared with the treatment, the effect is not improved; (2) Wild-type VSV still has certain safety risks. It is currently known to have strong neurotoxicity to rodents. For clinical use, it needs to be genetically modified to further Reduce the risk of disease; (3) Random genetic modification may result in poor oncolytic effect, or may not be successfully packaged, and it may not be possible to produce recombinant virus.
  • VSV recombinant virus with good safety and high cure rate, and to use it in combination with TCR-T and other immune cells as drugs, has important scientific research value and application significance in the field of tumor gene therapy.
  • the purpose of the present invention is to provide an oncolytic virus vaccine and its combination with immune cells to treat tumors.
  • the technical solution adopted by the present invention is: an attenuated strain of oncolytic virus, the gene sequence of the matrix protein (M) is shown in SEQ ID NO 3.
  • an oncolytic virus attenuated strain is based on the VSV MuddSummer subtype strain, and is obtained after at least the following site-specific gene mutations: the 51st methionine (M) is mutated to arginine (R); Leucine (L) coding base at position 111 is knocked out; Valine (V) at position 221 is mutated to phenylalanine (F); Serine (S) at position 226 is mutated to arginine (R).
  • the oncolytic virus attenuated strain is used as a carrier in the medical field.
  • the oncolytic virus attenuated strain is used in the preparation of medicines or vaccines.
  • an oncolytic virus vaccine is prepared after inserting an antigen into the attenuated strain.
  • an oncolytic virus vaccine is prepared after inserting a tumor antigen into the attenuated strain.
  • the antigen is NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1 , Col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE protein, GAGE protein, survivin, tyrosine Enzymes, SSX2, Cyclin-A1, KIF20A, MUC5AC, Meloe, Lengsin, Kallikrein 4, IGF2B3, Glypican 3 and other tumor antigens.
  • the medicine includes both the oncolytic virus vaccine and immune cells, and the immune cells are T cells, NK cells, macrophages or other immune cells.
  • the immune cells are T cells, NK cells, macrophages or other immune cells.
  • the T cells are any one of TCR-T cells, CAR-T cells, and ⁇ / ⁇ -T cells; when the T cells are TCR-T cells, The TCR-T cell is a TCR-T cell transfected with a lentivirus, or a TCR-T cell isolated from blood; when the immune cell is a NK cell, the NK cell is any of CAR-NK One; when the immune cell is a macrophage, the macrophage is any one of CAR-M cells.
  • the tumor or cancer is head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, round cell Any type of liposarcoma.
  • the present invention has the following beneficial effects: the present invention provides a brand-new attenuated strain of oncolytic virus by site-directed mutation of the matrix protein M of VSV wild-type virus.
  • the attenuated strain can be used alone as a medicine to treat tumors. The cure rate is better than wild-type virus and other attenuated strains.
  • the attenuated strain can also be used as a carrier (skeleton) to be connected to antigens or cytokines, so as to deliver antigens or cytokines and other substances to the desired location to become vaccines or drugs; the specific types of antigens or cytokines to be connected can be based on The actual need for treatment depends on the type of tumor or other diseases, and it is highly adaptable.
  • the present invention On the basis of the attenuated strain of the oncolytic virus, the present invention also provides a vaccine that can be applied to tumor treatment by inserting the exogenous gene NY-ESO-1 into the attenuated strain.
  • the vaccine has a high cure rate and high biological safety.
  • the present invention also combines the vaccine with TCR-T cells to provide a drug that can effectively treat multiple types of tumors; in the mouse lung cancer model, the cure rate can reach an astonishing 95% .
  • the present application provides an oncolytic virus attenuated strain, which is characterized in that: compared with the VSV MuddSummer subtype strain, the matrix protein M gene of the oncolytic virus attenuated strain has been modified, and the modification includes an amino acid site The 111th Leucine encoding base was knocked out.
  • the matrix protein M of the attenuated strain of the oncolytic virus is modified by knocking out the 111th leucine-encoding base of the amino acid position.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain further includes the mutation of methionine at position 51 of the amino acid position to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain is modified by knocking out the 111th leucine encoding base at the amino acid position and mutating the 51st methionine to arginine.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain further includes the mutation of valine at position 221 to phenylalanine.
  • the matrix protein M of the attenuated strain of oncolytic virus is modified by knocking out the 111th leucine encoding base and mutating the 221st valine to phenylalanine.
  • the modification of the matrix protein M of the attenuated strain of oncolytic virus further includes an amino acid mutation in which the 226th serine is mutated to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain is modified by knocking out the 111th leucine encoding base at the amino acid position and mutating the 226th serine to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain is modified by knocking out the 111th leucine encoding base at the amino acid position, mutating the 221st valine to phenylalanine and Serine at position 226 was mutated to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain is modified by mutating methionine at position 51 to arginine; knocking out the encoding base of leucine at position 111; Valine was mutated to phenylalanine and the 226th serine was mutated to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain has any one of the following amino acid sequences: SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO :9, SEQ ID NO: 10 and SEQ ID NO: 11 are shown in the amino acid sequence.
  • this application also provides the application of the oncolytic virus attenuated strain as a carrier in the field of medicine.
  • the application of the oncolytic virus attenuated strain as a carrier in the medical field is characterized by the application of the oncolytic virus attenuated strain in the preparation of medicines or vaccines.
  • this application provides an oncolytic virus vaccine, which is characterized in that it is prepared after inserting an antigen into the oncolytic virus attenuated strain.
  • the oncolytic virus vaccine is characterized in that the antigen is a specific tumor antigen.
  • the oncolytic virus vaccine is characterized in that the antigen is NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3 , MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopa pigment tautomerism Among the enzymes, BAGE protein, GAGE protein, Survivin, Tyrosinase, SSX2, Cyclin-A1, KIF20A, MUC5AC, Meloe, Lengsin, Kallikrein 4, IGF2B3, Glypican 3 Any kind.
  • the enzymes BAGE protein, GAGE protein, Survivin, Tyrosinase, SSX2, Cyclin-A1, KIF20A, MUC5
  • this application also provides an anti-tumor drug or a drug for treating cancer prepared by the oncolytic virus vaccine.
  • the anti-tumor drug or the drug for treating cancer includes both the oncolytic virus vaccine and immune cells.
  • the anti-tumor drug or the drug for treating cancer is characterized in that the immune cells are any one of T cells, NK cells, macrophages, DC cells, and TIL cells:
  • the T cells are any one of TCR-T cells, CAR-T cells, ⁇ / ⁇ -T cells, and gene-edited T cells;
  • the T cells are TCR-T cells,
  • the TCR-T cell is a TCR-T cell transfected by lentivirus or mRNA technology, a TCR-T cell isolated from blood, or a TCR-T obtained by any technique;
  • the immune cell is a NK cell
  • the NK cells are either NK or CAR-NK
  • the macrophages are either macrophages or CAR-M cells.
  • the anti-tumor drug or the drug for treating cancer is characterized in that: the tumor or cancer is head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder Any of cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.
  • Figures 1A-1B are schematic diagrams of the replication ability of each attenuated strain in LLC cells and MEF cells in vitro;
  • 2A-2B are schematic diagrams of the killing ability of each attenuated strain on LLC cells and Hela cells in vitro;
  • Figure 3 is a schematic diagram of the killing ability of each attenuated strain on MEF cells in vitro
  • Figures 4A-4B are schematic diagrams showing the effect of each attenuated strain on the expression level of IFN- ⁇ in LLC cells and MEF cells in vitro;
  • Figure 5 is a schematic diagram of the construction of an oncolytic virus vaccine
  • Figure 6 is a schematic diagram of the influence of each attenuated strain on the size of non-small cell lung cancer (transplanted tumor) in mice;
  • Figure 7 is a schematic diagram showing the effect of each vaccine on the volume of non-small cell lung cancer (transplanted tumor) in mice;
  • Figure 8 is a schematic diagram of the size of non-small cell lung cancer (transplanted tumor) in mice treated with each attenuated strain and vaccine at the end of the experiment;
  • Figure 9 is a schematic diagram of the effects of various attenuated strains and vaccines on the metastasis of non-small cell lung cancer cells in mice;
  • Figure 10 is a schematic diagram of the effect of each vaccine on the size of fibrosarcoma (transplanted tumor) in mice;
  • Figure 11 is a schematic diagram of the size of fibrosarcoma (transplanted tumor) in mice treated with each vaccine at the end of the experiment;
  • Figure 12 is a schematic diagram of the effect of each vaccine on the volume of melanoma (transplanted tumor) in mice;
  • Figure 13 is a schematic diagram of the volume of melanoma (transplanted tumor) in mice treated with each vaccine at the end of the experiment;
  • Figure 14 is a schematic diagram showing the effect of different doses of JBS004 on the volume of non-small cell lung cancer (transplanted tumor) in mice;
  • Figure 15 is a schematic diagram of the size of non-small cell lung cancer (transplanted tumor) in mice treated with different doses of JBS004 at the end of the experiment;
  • Figure 16 is a schematic diagram showing the effect of different doses of JBS004 on the metastasis of non-small cell lung cancer cells in mice;
  • Figure 17 is a schematic diagram of the effect of different doses of JBS004 on the body weight of lung cancer mice;
  • Figure 18 is a schematic diagram of the effect of different doses of JBS004 on the body temperature of lung cancer mice;
  • Figure 19 is the quantitative standard curve of PCR detection method
  • Figure 20 is a schematic diagram of the nucleic acid copy number of JBS004 in the tumor at different time points in the LLC xenograft tumor model;
  • Figure 21 is a schematic diagram showing the effect of different doses of JBS004 on the body temperature of healthy female mice at different time points;
  • Figure 22 is a schematic diagram of the effect of different doses of JBS004 on the body temperature of healthy male mice at different time points;
  • Figure 23 is a schematic diagram showing the effect of different doses of JBS004 on the body weight of healthy female mice at different time points;
  • Figure 24 is a schematic diagram of the effect of different doses of JBS004 on the body weight of healthy male mice at different time points;
  • Figure 25 is a schematic diagram of the impact of each vaccine on the size of lung cancer (transplanted tumor) when treated alone or in combination with JBS-NY TCR-T;
  • Figure 26 is a schematic diagram of the size of lung cancer (transplanted tumor) when each vaccine is treated alone or in combination with JBS-NY TCR-T at the end of the experiment;
  • Figure 27 is a schematic diagram of the effect of each vaccine alone or in combination with JBS-NY TCR-T on lung cancer cell metastasis.
  • modified generally refers to the modification of the structure and/or properties of naturally occurring organisms/molecules by artificial means.
  • the way of modification can be, for example, modification, mutation, synthesis, and/or insertion of foreign molecules to the molecule.
  • Modified can be distinguished from naturally occurring. For example: If a cell or organism is manipulated to change its genetic information (such as introducing new genetic material that did not exist before, for example, through transformation, matching, somatic hybridization, transfection, transduction, or other mechanisms, or changing or removing the previous Genetic material that exists, for example by substitution or deletion mutation), is then considered “engineered”.
  • the modified oncolytic virus can mutate the gene encoding the oncolytic virus protein, insert an exogenous gene into the gene of the oncolytic virus, or mutate the amino acid of the oncolytic virus protein.
  • matrix protein M can be used interchangeably with “M protein” and generally refers to the matrix protein of vesicular stomatitis virus.
  • Matrix protein M is an important virulence factor of VSV and a protein known to interfere with the natural immune response of mice in vesicular stomatitis virus.
  • the term “matrix protein M” also includes its homologs, orthologs, variants, functionally active fragments and the like.
  • the matrix protein M of the wild-type vesicular stomatitis virus MuddSummer subtype Indiana strain may include the amino acid sequence shown in SEQ ID NO: 2.
  • the expression of protein mutation site is usually expressed by "amino acid + amino acid number + (mutated amino acid)".
  • the mutation may include, but is not limited to, addition, substitution, deletion and/or deletion of amino acids.
  • M51R generally refers to the mutation of methionine M at position 51 to arginine R.
  • mutation generally refers to altering the nucleotide or amino acid sequence of a wild-type molecule. Mutations in DNA can change the codons, resulting in changes in the amino acid sequence.
  • Nucleotide changes may include nucleotide substitutions, deletions, insertions, alternative splicing and/or truncation of nucleic acid sequences.
  • Amino acid changes can include amino acid substitutions, deletions, deletions, insertions, additions, truncations, or protein processing or cleavage.
  • tumor generally refers to any new pathological tissue proliferation.
  • the tumor may be benign or malignant.
  • the tumor may be a solid tumor and/or hematoma.
  • An attenuated strain of an oncolytic virus characterized in that the matrix protein M of the oncolytic virus is modified, and the gene sequence of the modified matrix protein M is shown in SEQ ID NO 3.
  • an oncolytic virus attenuated strain characterized in that: the attenuated strain is based on the VSV MuddSummer subtype strain, and at least the M protein gene of the VSV MuddSummer subtype strain is obtained after the following site-specific gene mutations: No. 51 The methionine position was mutated to arginine; the 111th leucine encoding base was knocked out; the 221st valine was mutated to phenylalanine; the 226th serine was mutated to arginine.
  • An oncolytic virus vaccine characterized in that it is prepared after inserting an antigen into the attenuated strain described in embodiment 1 or 2.
  • the antigen is NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A
  • the anti-tumor drug or the drug for treating cancer prepared according to the oncolytic virus vaccine of embodiment 9, characterized in that: the immune cells are any one of T cells, NK cells, and macrophages;
  • the T cells are any one of TCR-T cells, CAR-T cells, and ⁇ / ⁇ -T cells; when the T cells are TCR-T cells, the TCR -T cells are TCR-T cells transfected by lentivirus or mRNA technology, or TCR-T cells isolated from blood; when the immune cells are NK cells, the NK cells are any of CAR-NK One; when the immune cells are macrophages, the macrophages are any of CAR-M cells.
  • the anti-tumor drug or the drug for treating cancer prepared according to the oncolytic virus vaccine of embodiment 9 or 10, characterized in that: the tumor or cancer is head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, Any of esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.
  • the present invention provides a brand new attenuated strain of oncolytic virus prepared by precisely modifying oncolytic virus.
  • the oncolytic virus is vesicular stomatitis virus (Vesicular stomatitis virus, VSV), specifically selected from the Indiana strain of vesicular stomatitis virus, VSV MuddSummer subtype strain, and its M protein gene sequence is shown in SEQ ID NO 1, M The amino acid sequence of the protein is shown in SEQ ID NO 2.
  • the present invention modifies the vesicular stomatitis virus as follows to obtain an oncolytic virus attenuated strain: site-directed gene mutation is performed on the M protein gene of the vesicular stomatitis virus to obtain an attenuated strain.
  • the mutation sites include: (1) the 51st methionine (M) is mutated to arginine (R); (2) the 111th leucine (L) coding base is knocked out; (3) ) Valine (V) at position 221 was mutated to phenylalanine (F); (4) Serine (S) at position 226 was mutated to arginine (R).
  • the vesicular stomatitis virus after the mutation is numbered: JBS003; named: XN2-M51R- ⁇ L111-V221F-S226R; the gene sequence of the M protein is shown in SEQ ID NO 3, and the amino acid sequence of the M protein is SEQ ID NO 4 is shown.
  • the JBS003 is safer and can be used as a carrier (skeleton) for antigens, cytokines and other substances, and can be used as a vaccine after being combined with antigens and cytokines. Or use drugs.
  • JBS003 can also be used directly as an oncolytic virus in tumor immunotherapy without combining with other substances, and its effect is better than that of wild-type VSV and other attenuated strains of VSV.
  • the present invention provides an oncolytic virus vaccine based on the attenuated strain of oncolytic virus.
  • the attenuated strain provided by the present invention can be combined with an antigen to form a vaccine.
  • the present invention inserts a gene that can express NY-ESO-1 between the G protein and the L protein of JBS003 to construct an oncolytic virus vaccine, numbered: JBS004.
  • NY-ESO-1 (New York esophageal squamous cell carcinoma 1) belongs to the Cancer-Testis Antigen (CTA) family. It is expressed in the testis, ovary and many tumor tissues, but not in other normal tissues. It is currently known to be the most immunogenic tumor-specific antigen discovered. NY-ESO-1 is expressed differently in different tumor tissues. The highest protein expression is myxoid round cell liposarcoma (89%-100%), neuroblastoma (82%), synovial sarcoma (90%). ), melanoma (46%), ovarian cancer (43%). NY-ESO-1 antigen has immunogenicity and safety, and is a clinically dominant antigen for immunotherapy.
  • CTA Cancer-Testis Antigen
  • the JBS004 oncolytic virus vaccine constructed after the introduction of NY-ESO-1 can efficiently induce the body's specific anti-tumor immune response in the peripheral lymphatic system and tumor tissues. Tests have shown that in anti-tumor immunotherapy, especially in the treatment of the above-mentioned cancers and tumors, oncolytic virus vaccines have obvious advantages in immunogenicity, effectiveness, targeting, safety and tolerability.
  • the present invention also provides a drug that can treat tumors in a targeted manner.
  • the drug includes the oncolytic virus or oncolytic virus vaccine.
  • the method of use is: intratumor injection or intravenous injection of the JBS003 oncolytic virus or JBS004 oncolytic virus vaccine. Take a small number of injections.
  • the drug also includes TCR-T cells.
  • the TCR-T cells are T lymphocytes transfected with NY-ESO-1 receptor, and the specific preparation method is as follows: (1) Separating T cells from the peripheral blood of NCG-HLA-A2.1/Gpt humanized mice Lymphocytes; (2) Artificially synthesize the target gene NY-ESO-1 receptor sequence and perform gene sequencing, and recombine it with the lentiviral vector to obtain the NY-ESO-1 receptor recombinant lentivirus; (3) Use the described The NY-ESO-1 receptor recombinant lentivirus was used to transfect T lymphocytes to obtain T lymphocytes transfected with NY-ESO-1 receptor, named: JBS-NY TCR-T.
  • the constructed JBS-NY TCR-T cells were expanded in vitro, and the expression of NY-ESO-1 in JBS-NY TCR-T cells was detected by Western Blot method to confirm that the construction was successful.
  • the method of using oncolytic virus or oncolytic virus vaccine in combination with JBS-NY TCR-T is as follows: first intravenous injection of JBS-NY TCR-T, followed by a small number of intratumoral injections or intravenous injections of oncolytic virus or oncolytic virus vaccine.
  • the present application provides an oncolytic virus attenuated strain, which is characterized in that, compared with the VSV MuddSummer subtype strain, the matrix protein M gene of the oncolytic virus attenuated strain has been modified.
  • the matrix protein M of the VSV MuddSummer subtype strain comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the matrix protein M of the VSV MuddSummer subtype strain comprises the nucleic acid sequence shown in SEQ ID NO:1.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain may include the knockout of the 111th leucine-encoding base of the amino acid position.
  • the matrix protein M of the oncolytic virus attenuated strain is compared with the matrix protein M of the VSV MuddSummer subtype strain, and the 111th leucine encoding base is knocked out.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO:7.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain may also include knocking out the 111th leucine encoding base of the amino acid position and mutating the 51st methionine to arginine.
  • the matrix protein M of the attenuated strain of the oncolytic virus is compared with the matrix protein M of the VSV MuddSummer subtype strain, the 111th leucine encoding base is knocked out and the 51st methionine The acid is mutated to arginine.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO: 8.
  • the modification of the matrix protein M of the attenuated strain of oncolytic virus may include the knockout of the 111th leucine-encoding base of the amino acid position and the mutation of the 221st valine to phenylalanine.
  • the matrix protein M of the oncolytic virus attenuated strain is compared with the matrix protein M of the VSV MuddSummer subtype strain, the 111th leucine encoding base is knocked out and the 221st valine Mutation to phenylalanine.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO: 9.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain may include the knockout of the 111th leucine coding base of the amino acid position and the mutation of the 226th serine to arginine.
  • the matrix protein M of the attenuated strain of the oncolytic virus is compared with the matrix protein M of the VSV MuddSummer subtype strain.
  • the 111th leucine encoding base is knocked out and the 226th serine is mutated to Arginine.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO: 10.
  • the modification of the matrix protein M of the oncolytic virus attenuated strain may include the knockout of the 111th leucine coding base of the amino acid position, the mutation of the 221st valine to phenylalanine and the Serine 226 was mutated to arginine.
  • the matrix protein M of the oncolytic virus attenuated strain is compared with the matrix protein M of the VSV MuddSummer subtype strain, the 111th leucine encoding base is knocked out, and the 221st valine Mutation to phenylalanine and mutation of serine at position 226 to arginine.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO: 11.
  • the modification of the matrix protein M of the attenuated strain of the oncolytic virus may include the knockout of the 111th leucine coding base at the amino acid position, the mutation of the 51st methionine to arginine, and the The 221th valine was mutated to phenylalanine and the 226th serine was mutated to arginine.
  • the matrix protein M of the attenuated strain of the oncolytic virus is compared with the matrix protein M of the VSV MuddSummer subtype strain.
  • the acid mutation was arginine
  • the 221st valine was mutated to phenylalanine
  • the 226th serine was mutated to arginine.
  • the amino acid sequence of the matrix protein M of the oncolytic virus attenuated strain is shown in SEQ ID NO: 4.
  • this application also provides a nucleic acid molecule, which can encode the matrix protein M of the oncolytic virus.
  • the nucleic acid sequence encoding the matrix protein M of the oncolytic virus may be as shown in SEQ ID NO: 3.
  • this application also provides the application of the oncolytic virus attenuated strain as a carrier in the field of medicine.
  • the application of the oncolytic virus attenuated strain as a carrier in the medical field may include the application of the oncolytic virus attenuated strain in the preparation of medicines or vaccines.
  • this application also provides an oncolytic virus vaccine, which can be prepared after inserting an antigen into the attenuated strain of the oncolytic virus.
  • an antigen for example, a specific tumor antigen can be inserted into the attenuated strain of the oncolytic virus to obtain the oncolytic virus vaccine.
  • the antigen can be selected from any one of the following: NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX/Y, HPV, alpha-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE protein, GAGE protein, Survivin, Tyrosinase, SSX2, Cyclin-A1, KIF20A, MUC5AC, Meloe, Lengsin, Kallikrein 4, IGF2B3 and Glypican 3.
  • the constructed oncolytic virus attenuated strain is used as a vector and introduced into NY-ESO-1 to obtain the oncolytic virus vaccine.
  • a preparation method of the oncolytic virus vaccine includes: constructing an oncolytic virus attenuated strain plasmid, artificially synthesizing a linking sequence with restriction enzyme cutting sites, using biological technology and Gene recombination technology, insert it into the non-coding region between the G protein and L protein of the oncolytic virus attenuated strain, insert the foreign gene, and obtain the recombinant plasmid of the attenuated strain carrying the foreign gene.
  • Oncolytic virus vaccine includes: constructing an oncolytic virus attenuated strain plasmid, artificially synthesizing a linking sequence with restriction enzyme cutting sites, using biological technology and Gene recombination technology, insert it into the non-coding region between the G protein and L protein of the oncolytic virus attenuated strain, insert the foreign gene, and obtain the recombinant plasmid of the attenuated strain carrying the foreign gene.
  • Oncolytic virus vaccine includes: constructing an oncolytic virus attenuated strain plasmid, artificially synthe
  • this application also provides an anti-tumor drug or a drug for treating cancer prepared by the oncolytic virus vaccine.
  • the anti-tumor drug or the drug for treating cancer includes both the oncolytic virus vaccine and immune cells.
  • the immune cells may include any one of T cells, NK cells, macrophages, DC cells, and TIL cells: when the immune cells are T cells, the T cells may include TCR-T cells.
  • the TCR-T cells may include lentivirus or mRNA technology Transfected TCR-T cells, or TCR-T cells isolated from blood, or TCR-T cells obtained by any technique;
  • the immune cells when the immune cells are NK cells, the NK cells may include NK or CAR -Any one of NK;
  • the immune cells when the immune cells are macrophages, the macrophages may include any one of macrophages or CAR-M cells.
  • the tumor or cancer may include head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma And/or round cell liposarcoma.
  • the medicament may also include an optionally pharmaceutically acceptable carrier.
  • Plasmid transfection was carried out according to the method described in lipofectamine 2000 instruction manual. After 4 hours, it was replaced with a fresh DMEM complete medium containing 10% fetal bovine serum. After 48 hours, the supernatant was aspirated and the poxvirus was filtered with a 0.22 ⁇ m filter. Add the filtrate to fresh BHK-21 cells; observe the cytopathic conditions every day, collect the supernatant when the cells are pathologically pathological, and use the virus plaque experiment to purify the virus after the success of the RTPCR identification. The attenuated strain is obtained.
  • M protein gene sequencing Trizol kit was used to extract viral genome RNA, random primers were used to perform reverse transcription reaction, and primers designed for the M protein gene sequence were used to perform PCR on the reverse transcribed cDNA.
  • the primer sequence is: 5'-AAAAAAGTAACAGATATCAC-3' (SEQ ID NO: 5); 5'-ACATTTTTCCAGTTTCCTTTTTGG-3' (SEQ ID NO: 6).
  • the product was recovered after 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • step (2) Dilute the supernatant harvested in step (2) continuously by 10 times in a 1.5 mL EP tube, from 10 -1 to 10 -11 , for a total of 11 titers.
  • step (3) Inoculate the diluted supernatant into the 96-well culture plate of step (3), inoculate one row for each dilution (total 8 wells), and inoculate 100 ⁇ L per well. Set up a column of normal cell control group.
  • Test for the ease of elimination of different attenuated strains in cells Test with IFN- ⁇ index.
  • Test with IFN- ⁇ index follow the steps (1) and (2) of step 3 above to culture the cells and add the attenuated strain. Then break each group of cells, extract total RNA from each cell with TRIzol (Invitrogen), use PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit to reverse transcription into cDNA, and use LightCycler 480SYBR Green I Master (Roche) dye Perform staining, and detect the Ct value of each gene on the LightCycler 480 quantitative PCR machine. The ⁇ Ct method was used to calculate the relative expression of target genes IFN- ⁇ and VSV-G.
  • Example 1 On the basis of the attenuated strains and wild-type viruses prepared in Example 1, the NY-ESO-1 gene was inserted to construct and obtain an oncolytic virus vaccine. The schematic diagram of the construction is shown in FIG. 5. Table 2 shows the situation of inserts in each group.
  • Vaccine number Vaccine naming Corresponding attenuated strain JBS004 XN2-M51R- ⁇ L111-V221F-S226R—NY-ESO-1 JBS003+NY-ESO-1 JBS005 XN2-M51R- ⁇ L111-NY-ESO-1 JBS002+NY-ESO-1 JBS006 XN2-M51R-NY-ESO-1 JBS001+NY-ESO-1 JBS007 XN2-WT-NY-ESO-1 JBS000+NY-ESO-1 JBS011 XN2- ⁇ L111-NY-ESO-1 JBS008+NY-ESO-1 JBS012 XN2- ⁇ L111-V221F-NY-ESO-1 JBS009+NY-ESO-1 JBS013 XN2- ⁇ L111-S226R-NY-ESO-1 JBS010+NY-ESO-1 JBS015 XN2- ⁇ L111-V221F-S226R-NY
  • JBS004-JBS007, JBS011-JBS013, and JBS015 are conventional techniques in the field, and are briefly described as follows. It should be noted that the following description does not limit JBS004-JBS007, JBS011-JBS013, JBS015 to the following methods, but gives examples.
  • Attenuated strain plasmid construction Artificially synthesize the linking sequence with restriction enzyme sites Xho I and Mlu I, using biological technology and gene recombination technology, insert them into the non-coding between the G protein and L protein of each attenuated strain prepared in Example 1. Region, obtain the plasmids of each attenuated strain.
  • mice 136 C57BL/6 mice with insignificant differences were selected, and 2 ⁇ 10 5 LLC cells (mouse lung cancer cells) were subcutaneously inoculated.
  • the control group PBS group
  • the remaining 16 groups were the treatment groups, respectively Carry out intratumoral inoculation of JBS000, JBS001, JBS002, JBS003, JBS004, JBS005, JBS006, JBS007, JBS008, JBS009, JBS010, JBS011, JBS012, JBS013, JBS014, JBS015, once every 2 days, for a total of 3 doses.
  • the detection method is as follows: LLC cells have red fluorescent protein, which will show yellow fluorescence under a green fluorescence microscope; when cancer cells have transferred to the lung tissue, place the lung tissue under the microscope and take a fluorescence picture. Analyze the gray value of the picture through Image J to analyze the proportion of lung cancer cells, that is, the proportion of cancer cells that have metastasized.
  • mice were treated according to the method of "Treatment of LLC-NY-ESO-1 Non-Small Cell Lung Cancer Xenograft", and 10 6 MCA-205-NY-ESO-1 fibrosarcoma cells were subcutaneously inoculated, and the tumor volume to be transplanted would grow to 100mm Process at around 3 hours.
  • intratumor injection of 50 ⁇ L of PBS was used as the control group.
  • intratumoral inoculation of JBS004, JBS005, JBS006, JBS007, JBS011, JBS012, JBS013, JBS015 were carried out, with 6 animals in each group, once every 2 days.
  • mice were treated according to the treatment method of the transplanted tumor test, and 2 ⁇ 10 6 B16-F10-NY-ESO-1 melanoma cells were subcutaneously inoculated, and the transplanted tumor was treated when the volume of the transplanted tumor grew to about 100 mm 3.
  • intratumor injection of 50 ⁇ L of PBS was used as the control group.
  • intratumoral inoculation of JBS004, JBS005, JBS006, JBS007, JBS011, JBS012, JBS013, JBS015 were carried out, with 6 animals in each group, once every 2 days. were administered three times, a single inoculation were 10 8 pfu / only.
  • mice with a body weight of about 18 g and 6-8 weeks old were selected, and 2 ⁇ 10 5 LLC cells (mouse lung cancer cells) were subcutaneously inoculated.
  • the control group PBS group
  • the remaining 4 groups are the treatment groups.
  • Acute toxicity test Choose 40 C57BL/6 mice, half male and half female. Divided into 3 administration groups, a single intramuscular injection of JBS004 solution was given, and the doses of each group were: 10 3 pfu/ mouse, 10 6 pfu/ mouse, and 10 9 pfu/mouse.
  • the blank control group was given a single dose of vehicle control (single intramuscular injection of PBS), and the administration volume was 100 ⁇ L.
  • the animal administration day was set as the first day of observation of the group of animals. The animals were observed for 14 days after administration and dissected on the 15th day after administration.
  • mice were observed by cage in detail before administration and 30min, 1h, 2h, 4h and 10h after administration. In subsequent experiments, cage observation was carried out at least once a day.
  • the peripheral blood of the mouse was taken for hematology and blood biochemical testing (blood sugar, creatinine, urea nitrogen, blood urea nitrogen/creatinine, phosphorus ions, calcium ions, total protein, albumin, globulin, etc.), and Necropsy collects the main organs, including the heart, liver, spleen, lung, kidney, brain, testis/ovary, and weighs the tissues, and calculates the organ coefficient. Due to space limitations, all index data for hematology and blood biochemistry testing are not included here. The test results showed that there were no abnormal deaths of mice, and no clinical symptoms related to JBS004.
  • JBS-NY TCR-T is a T cell obtained after transfection of T lymphocytes with NY-ESO-1 receptor recombinant lentivirus.
  • the specific construction method is a conventional technology in the field, and is briefly described as follows:
  • ESO TCR-R1 5'-ATAGTTTAGCGGCCGCCTAGCCTCTGGAA-3' (SEQ ID NO: 13).
  • the cloned ESO TCR cDNA was used as a template for PCR in vitro amplification.
  • the PCR reaction conditions were: 94°C pre-denaturation for 3 minutes, 94°C, 30s, 55°C, 30s, 72°C, 45s, and 72°C extension for 5 minutes after 35 cycles. 1.2% agarose gel electrophoresis to separate specific fragments (1824bp) amplified by PCR.
  • the PCR product and pCL20c-MSCV-GFP plasmid were digested with EcoR I and Not I, respectively, and the digested product was recovered by glass milk gel.
  • the two digested products were ligated with T4DNA ligase at 16°C overnight. Then the ligation product was transformed into competent DH5 ⁇ bacteria, cultured and amplified, and then the plasmid was extracted with the B-type small plasmid kit.
  • T293 cells were routinely cultured in a DMEM medium containing 10% fetal bovine serum, in a 95% humidity, 5% CO 2 , 37°C incubator, and subcultured 3 to 4 times a week.
  • DMEM medium containing 10% fetal bovine serum
  • the transfection solution was prepared 1h before transfection, and the transfection solution was composed of A solution and B solution.
  • Solution A pCL20c-HIV-gp plasmid 6 ⁇ g, pCAG4-RTR2 plasmid 2 ⁇ g, CAG-VSV-G plasmid 2 ⁇ g, pCL20c-MSCV-ESOTCR plasmid 10 ⁇ g, 50 ⁇ L 2.5mol/L CaCl 2 , make up to 500 ⁇ L volume with deionized water , Flick and mix well, and place at room temperature for 5 minutes.
  • T cells in peripheral blood Isolate T lymphocytes from the peripheral blood of humanized NCG-HLA-A2.1/Gpt humanized mice, add cell separation solution, centrifuge at 1500g/min for 15 minutes, take the second layer of ring-shaped milky white lymphocytes, and add 5 mL of cell washing solution After mixing thoroughly, centrifuge at 1800g/min for 20 minutes, discard the supernatant, and resuspend the precipitated lymphocytes.
  • Table 3 Display table of treatment situation of each group

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Abstract

提供了一种溶瘤病毒减毒株、溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物。通过对VSV野生型病毒基质蛋白M进行定点突变，提供了一种新的溶瘤病毒减毒株。在所述溶瘤病毒减毒株的基础上，还提供了一种可应用在肿瘤治疗中的疫苗。在所述疫苗的基础上，还将疫苗与免疫细胞联合应用，提供了一种可高效治疗多类肿瘤的药物。

Description

一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物 技术领域
本申请涉及生物医药领域，具体的涉及一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物。
背景技术
根据国家癌症中心2019年1月发布的全国癌症统计数据显示：2015年恶性肿瘤发病约392.9万人，死亡约233.8万人。平均每天超过1万人被确诊为癌症，每分钟有7.5个人被确诊为癌症。肝癌、结直肠癌、女性乳腺癌等实体瘤依然是我国主要的恶性肿瘤。恶性肿瘤(癌症)已经成为严重威胁中国人群健康的主要公共卫生问题之一。尽管目前癌症治疗在手术、化疗、放疗及分子靶向治疗等多学科综合治疗方面取得很大进展，但肿瘤的复发和转移迄今仍缺乏有效治疗手段。因此，新型治疗手段，即肿瘤免疫治疗逐渐受到人们的关注。
2003年，Giedlin MA提出水疱性口炎病毒(Vesicular stomatitis virus，VSV)可以作为治疗肿瘤的溶瘤病毒。其原理是它不能与正常细胞中内源性的IFN-β相互作用，只能选择性地在肿瘤细胞中扩增、生长。2009年，加拿大麦克马斯特大学研究表明：VSV可以作为新的肿瘤疫苗载体促进免疫应答。近年来，VSV的相关研究已越来越受到研究者们的重视。从安全的角度而言，VSV对人类相对安全，目前未见VSV感染人类的案例。
VSV是一种原型非节段负链RNA病毒，其大小11kb基因组编码5种蛋白:核衣壳蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和大聚合酶蛋白(L)。VSV可表达包括低密度脂蛋白受体、磷脂酰丝氨酸、唾液糖脂和硫酸类肝素在内的多种细胞表面分子，并可通过此类分子附着细胞表面。具有复制及跨突触速度快，外源基因表达量超高的特点。与目前正在研发的其他溶瘤细胞病毒平台相比，VSV基因组小，容易操作；复制时间更短；有独立的细胞周期；在广泛的细胞系中可快速增长，且具有较高滴度，允许大规模生产；宿主细胞的细胞质复制无转化风险。这种溶瘤病毒不会集成到DNA中，并且经过减毒后可以避免野生型病毒引起的神经系统炎症。因此，VSV在肿瘤免疫治疗上具有很大的潜力。
在肿瘤模式动物中，研究发现VSV能显著消除脑部肿瘤，对乳腺癌和骨肉瘤也有明显的抑制作用。研究者通过对VSV对肝癌的抗肿瘤功能及毒副作用的研究发现肝癌荷瘤小鼠的生存期显著增加，且并未产生明显的毒副作用。经VSV-GP处理后前列腺癌小鼠的皮下肿瘤及骨转移瘤明显减小；减小黑色素瘤荷瘤小鼠的原位肿瘤及肺转移瘤也得到显著改善。M51R VSV可直接诱导结直肠癌细胞的凋亡。同时，VSV也可通过调控固有免疫或者获得性免疫进 一步影响肿瘤的发展。M51R VSV降低结直肠癌组织中的免疫抑制细胞MDSC、巨噬细胞的浸润，增加CD4 +T细胞的浸润，从而减少恶性腹水的形成。VSV能够诱导CD8特异性T细胞的免疫应答，降低其它免疫抑制细胞的作用，从而增强肿瘤疫苗的疗效。以上研究可见VSV具有较强的抗肿瘤作用，并具有很好的安全性。
目前的研究表明，单独使用VSV进行肿瘤免疫治疗时，治疗反应率存在一定的瓶颈，这主要与其特异性不足及肿瘤内微环境的抑制作用有关。因此，关于VSV与其他治疗的联合应用也越来越多。在乳头瘤小鼠模型的研究中发现，VSV联合肿瘤疫苗明显提高了抗肿瘤效应。来自明尼苏达大学医学院的Manish R.Patel等人发表了使用JAK/STAT抑制剂(Ruxolitinib)联合VSV-IFNβ对肺癌进行治疗，结果显示，Ruxolitinib联合VSV-IFNβ取得了较好的溶瘤治疗效果。多种细胞因子武装的溶瘤病毒也被用于与CAR-T细胞疗法联合；在异种移植瘤模型中，这种联合策略增强了抗肿瘤活性。
T细胞受体基因工程改造的T细胞(T cell receptor gene engineered T cells,TCR-T)疗法是以修饰T细胞为基础的，应用于恶性肿瘤的过继细胞免疫治疗，TCR介导T细胞识别MHC分子递呈的抗原，从而使得抗原特异性的T细胞对肿瘤靶细胞发挥免疫效应。现有研究使得联合使用VSV和TCR-T治疗肿瘤成为可能。VSV可通过在肿瘤细胞内的选择性复制从而溶解肿瘤细胞，而溶解的肿瘤细胞会诱导肿瘤特异性免疫应答，促进T细胞的活化、扩增、募集，活化的T细胞又可在肿瘤内通过调控免疫抑制等方式杀伤肿瘤细胞。两者联合使用，理论上可以发挥优于单独使用VSV疗法或TCR-T疗法的效果。
但在联合使用VSV和TCR-T进行肿瘤免疫治疗时，仍然至少存在如下问题：(1)直接将VSV野生株或减毒株与TCR-T联合使用，治愈率不高，与单独使用一种疗法相比，效果提升不明显；(2)野生型VSV仍然存在一定的安全风险，目前已知对啮齿类动物具有较强的神经毒性，为临床使用考虑，需要对其进行基因修饰，以进一步降低致病风险；(3)随机进行基因修饰，可能导致溶瘤效果不佳，也可能无法成功包装，根本无法制备出重组病毒。
因此，提供一种安全性良好、治愈率高的VSV重组病毒，并将其与TCR-T及其他免疫细胞作为药物联合使用，对肿瘤基因治疗领域具有重要的科研价值和应用意义。
发明内容
本发明的目的是提供一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物。
为实现上述发明目的，本发明所采用的技术方案是：一株溶瘤病毒减毒株，其基质蛋白(M)的基因序列如SEQ ID NO 3所示。
相应的，一株溶瘤病毒减毒株，所述减毒株以VSV MuddSummer亚型株为基础，至少进 行如下定点基因突变后获得：第51位甲硫氨酸(M)突变为精氨酸(R)；第111位亮氨酸(L)编码碱基敲除；第221位缬氨酸(V)突变为苯丙氨酸(F)；第226位丝氨酸(S)突变为精氨酸(R)。
相应的，所述的溶瘤病毒减毒株作为载体在医药领域中的应用。
优选的，所述溶瘤病毒减毒株在制备药物或疫苗中的应用。
相应的，一种溶瘤病毒疫苗，在所述减毒株中插入抗原后制备得到。
相应的，一种溶瘤病毒疫苗，在所述减毒株中插入肿瘤抗原后制备得到。
优选的，所述抗原为NY-ESO-1、gp33、gp100、TX103、Mucin-1、WT-1、MART-1、MAGE A1、MAGE A3、MAGE A4、MAGE B2、PRAME、SURVIVIN、MART-1、col6A3、tyrosinase、T antigen、SLC45A2、VCX/Y、HPV、甲胎蛋白、癌胚抗原、CA 125、Her2、多巴色素互变异构酶、BAGE蛋白、GAGE蛋白、存活蛋白、酪氨酸酶、SSX2、细胞周期蛋白-A1、KIF20A、MUC5AC、Meloe、Lengsin、激肽释放酶4、IGF2B3、磷脂酰肌醇蛋白聚糖3及其他肿瘤抗原中的任意一种。
相应的，所述溶瘤病毒疫苗在制备肿瘤免疫治疗药物中的应用。
优选的，所述药物同时包括所述溶瘤病毒疫苗和免疫细胞，所述免疫细胞为T细胞、NK细胞、巨噬细胞(macrophage)或其他免疫细胞。
优选的，所述免疫细胞为T细胞时，所述T细胞为TCR-T细胞、CAR-T细胞、γ/δ-T细胞中的任意一种；所述T细胞为TCR-T细胞时，所述TCR-T细胞为经慢病毒转染的TCR-T细胞，或从血液中分离出的TCR-T细胞；所述免疫细胞为NK细胞时,所述NK细胞为CAR-NK中的任意一种；所述免疫细胞为巨噬细胞(macrophage)时,所述巨噬细胞(macrophage)为CAR-M细胞中的任意一种。
优选的，所述肿瘤或癌症为头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤、圆细胞型脂肪肉瘤中的任意一种。
本发明具有以下有益效果：本发明通过对VSV野生型病毒的基质蛋白M进行定点突变，提供了一种全新的溶瘤病毒减毒株，该减毒株可单独作为药物治疗肿瘤，安全性与治愈率均优于野生型病毒及其它减毒株。该减毒株还可作为载体(骨架)，与抗原或细胞因子等连接，从而将抗原或细胞因子等物质定点输送到所需位置，成为疫苗或药物；具体连接的抗原或细胞因子种类可根据实际需要治疗的肿瘤或其它疾病种类而定，适应性强。本发明在所述溶瘤病毒减毒株的基础上，通过在减毒株内插入外源基因NY-ESO-1，还提供了一种可应用在肿瘤 治疗中的疫苗。所述疫苗治愈率高，且生物安全性高。本发明在所述疫苗的基础上，还将疫苗与TCR-T细胞联合应用，提供了一种可高效治疗多类肿瘤的药物；在小鼠的肺癌模型上，治愈率可达到惊人的95％。
一方面，本申请提供了溶瘤病毒减毒株，其特征在于：与VSV MuddSummer亚型株相比，所述溶瘤病毒减毒株的基质蛋白M基因经过改造，所述改造包含氨基酸位点第111位亮氨酸编码碱基敲除。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造还包含氨基酸位点第51位甲硫氨酸突变为精氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除和第51位甲硫氨酸突变为精氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造还包含第221位缬氨酸突变为苯丙氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为第111位亮氨酸编码碱基敲除和第221位缬氨酸突变为苯丙氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造还包含第226位丝氨酸突变为精氨酸的氨基酸突变。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除和第226位丝氨酸突变为精氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除、第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M的改造为第51位甲硫氨酸突变为精氨酸；第111位亮氨酸编码碱基敲除；第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。
在某些实施方式中，所述溶瘤病毒减毒株的基质蛋白M具有下组中任一种氨基酸序列：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的氨基酸序列。
另一方面，本申请还提供了所述溶瘤病毒减毒株作为载体在医药领域中的应用。
在某些实施方式中，所述溶瘤病毒减毒株作为载体在医药领域中的应用的特征在于：所 述溶瘤病毒减毒株在制备药物或疫苗中的应用。
另一方面，本申请提供了一种溶瘤病毒疫苗，其特征在于：在所述的溶瘤病毒减毒株中插入抗原后制备得到。
在某些实施方式中，所述溶瘤病毒疫苗的特征在于：所述抗原为特异性肿瘤抗原。
在某些实施方式中，所述溶瘤病毒疫苗的特征在于：所述抗原为NY-ESO-1、gp33、gp100、TX103、Mucin-1、WT-1、MART-1、MAGE A1、MAGE A3、MAGE A4、MAGE B2、PRAME、SURVIVIN、MART-1、col6A3、tyrosinase、T antigen、SLC45A2、VCX/Y、HPV、甲胎蛋白、癌胚抗原、CA 125、Her2、多巴色素互变异构酶、BAGE蛋白、GAGE蛋白、存活蛋白、酪氨酸酶、SSX2、细胞周期蛋白-A1、KIF20A、MUC5AC、Meloe、Lengsin、激肽释放酶4、IGF2B3、磷脂酰肌醇蛋白聚糖3中的任意一种。
另一方面，本申请还提供了所述溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物。
在某些实施方式中，所述抗肿瘤药物或治疗癌症的药物同时包括所述溶瘤病毒疫苗和免疫细胞。
在某些实施方式中，所述抗肿瘤药物或治疗癌症的药物的特征在于：所述免疫细胞为T细胞、NK细胞、巨噬细胞、DC细胞、TIL细胞中的任意一种：所述免疫细胞为T细胞时，所述T细胞为TCR-T细胞、CAR-T细胞、γ/δ-T细胞、基因编辑的T细胞中的任意一种；所述T细胞为TCR-T细胞时，所述TCR-T细胞为经慢病毒或mRNA技术转染的TCR-T细胞，从血液中分离出的TCR-T细胞，或任意一种技术得到的TCR-T；所述免疫细胞为NK细胞时，所述NK细胞为NK或CAR-NK中的任意一种；所述免疫细胞为巨噬细胞时，所述巨噬细胞为巨噬细胞或CAR-M细胞中的任意一种。
在某些实施方式中，所述抗肿瘤药物或治疗癌症的药物的特征在于：所述肿瘤或癌症为头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤、圆细胞型脂肪肉瘤中的任意一种。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的 示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A-1B为各减毒株在体外的LLC细胞和MEF细胞内复制能力示意图；
图2A-2B为各减毒株对体外的LLC细胞和Hela细胞杀伤能力示意图；
图3为各减毒株对体外的MEF细胞杀伤能力示意图；
图4A-4B为各减毒株对体外LLC细胞和MEF细胞内IFN-β表达水平影响示意图；
图5为溶瘤病毒疫苗构建示意图；
图6为各减毒株对小鼠体内非小细胞肺癌(移植瘤)体积大小影响示意图；
图7为各疫苗对小鼠体内非小细胞肺癌(移植瘤)体积大小影响示意图；
图8为实验终点时，各减毒株和疫苗处理下小鼠体内非小细胞肺癌(移植瘤)体积大小示意图；
图9为各减毒株和疫苗对小鼠体内非小细胞肺癌细胞转移情况影响示意图；
图10为各疫苗对小鼠体内纤维肉瘤(移植瘤)体积大小影响示意图；
图11为实验终点时，各疫苗处理下小鼠体内纤维肉瘤(移植瘤)体积大小示意图；
图12为各疫苗对小鼠体内黑色素瘤(移植瘤)体积大小影响示意图；
图13为实验终点时，各疫苗处理下小鼠体内黑色素瘤(移植瘤)体积大小示意图；
图14为不同剂量的JBS004对小鼠体内非小细胞肺癌(移植瘤)体积大小影响示意图；
图15为实验终点时，不同剂量的JBS004处理下小鼠体内非小细胞肺癌(移植瘤)体积大小示意图；
图16为不同剂量的JBS004对小鼠体内非小细胞肺癌细胞转移情况影响示意图；
图17为不同剂量的JBS004对肺癌小鼠的体重影响示意图；
图18为不同剂量的JBS004对肺癌小鼠的体温影响示意图；
图19为PCR检测方法定量标准曲线；
图20为LLC移植瘤模型中，不同时间点时肿瘤内JBS004的核酸拷贝数示意图；
图21为不同时间点时不同剂量的JBS004对健康雌性小鼠体温影响示意图；
图22为不同时间点时不同剂量的JBS004对健康雄性小鼠体温影响示意图；
图23为不同时间点时不同剂量的JBS004对健康雌性小鼠体重影响示意图；
图24为不同时间点时不同剂量的JBS004对健康雄性小鼠体重影响示意图；
图25为各疫苗单独治疗或与JBS-NY TCR-T联合治疗时，对肺癌(移植瘤)体积大小影响示意图；
图26为实验终点时，各疫苗单独治疗或与JBS-NY TCR-T联合治疗时肺癌(移植瘤)体积大小示意图；
图27为各疫苗单独治疗或与JBS-NY TCR-T联合治疗对肺癌细胞转移情况影响示意图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“经改造的”通常指通过人工手段对天然存在的生物体/分子的结构和/或性能加以改变。改变的方式可以是，例如对所述分子进行修饰、突变、合成和/或插入外源分子等。“经改造的”可以区别于天然存在的。例如：如果细胞或生物体经操作使得其基因信息改变(例如引入先前不存在的新基因材料，例如通过转型、匹配、体细胞杂交、转染、转导或其它机制，或改变或移除先前存在的基因材料，例如通过取代或缺失突变)，那么其被视为“经改造的”。例如：经改造的溶瘤病毒可以是对编码溶瘤病毒蛋白的基因进行突变，也可以是在溶瘤病毒的基因中插入外源基因，也可以是对溶瘤病毒蛋白的氨基酸进行突变。
在本申请中，术语“基质蛋白M”可以与“M蛋白”互换使用，通常指水疱性口炎病毒基质蛋白。基质蛋白M是VSV重要的毒力因子，也是水疱性口炎病毒中已知可干扰小鼠天然免疫应答的蛋白。术语“基质蛋白M”还包括其同源物，直系同源物、变体、功能活性片段等。例如，野生型水疱性口炎病毒MuddSummer亚型印第安纳株的基质蛋白M可以包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，蛋白突变位点的表述通常由“氨基酸+氨基酸位数+(突变后的氨基酸)”来表述。在本申请中，所述突变可以包括但不限于氨基酸的增加、替换、缺失和/或删除。例如，术语“M51R”通常指第51位甲硫氨酸M突变为精氨酸R。
在本申请中，术语“突变”通常是指改变野生型分子的核苷酸或氨基酸序列。DNA中的突变可以改变密码子，从而导致氨基酸序列中的变化。核苷酸变化可以包括核苷酸的置换、缺失、插入、对核酸序列的可变剪接和/或截短。氨基酸变化可以包括氨基酸的置换、删除、缺失、插入、添加、截短、或蛋白质的加工或切割。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。肿瘤可能是良性的，也可能是恶性的。在本申请中，所述肿瘤可以是实体瘤和/或血液瘤。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
发明详述
1.一种溶瘤病毒减毒株，其特征在于：对所述溶瘤病毒的基质蛋白M进行改造，改造后的基质蛋白M的基因序列如SEQ ID NO 3所示。
2.一种溶瘤病毒减毒株，其特征在于：所述减毒株以VSV MuddSummer亚型株为基础，至少对VSV MuddSummer亚型株的M蛋白基因进行如下定点基因突变后获得：第51位甲硫氨酸突变为精氨酸；第111位亮氨酸编码碱基敲除；第221位缬氨酸突变为苯丙氨酸；第226位丝氨酸突变为精氨酸。
3.实施方案1或2所述的溶瘤病毒减毒株作为载体在医药领域中的应用。
4.根据实施方案3所述的溶瘤病毒减毒株作为载体在医药领域中的应用，其特征在于：所述溶瘤病毒减毒株在制备药物或疫苗中的应用。
5.一种溶瘤病毒疫苗，其特征在于：在实施方案1或2所述减毒株中插入抗原后制备得到。
6.根据实施方案5所述的溶瘤病毒疫苗，其特征在于：所述抗原为特异性肿瘤抗原。
7.根据实施方案6所述的溶瘤病毒疫苗，其特征在于：所述抗原为NY-ESO-1、gp33、gp100、TX103、Mucin-1、WT-1、MART-1、MAGE A1、MAGE A3、MAGE A4、MAGE B2、PRAME、SURVIVIN、MART-1、col6A3、tyrosinase、T antigen、SLC45A2、VCX/Y、HPV、甲胎蛋白、癌胚抗原、CA 125、Her2、多巴色素互变异构酶、BAGE蛋白、GAGE蛋白、存活蛋白、酪氨酸酶、SSX2、细胞周期蛋白-A1、KIF20A、MUC5AC、Meloe、Lengsin、激肽释放酶4、IGF2B3、磷脂酰肌醇蛋白聚糖3中的任意一种。
8.实施方案5～7任意一项所述溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物。
9.根据实施方案8所述的溶瘤病毒疫苗的抗肿瘤药物或治疗癌症的药物，其特征在于：所述药物同时包括所述溶瘤病毒疫苗和免疫细胞。
10.根据实施方案9所述的溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物，其特征在于：所述免疫细胞为T细胞、NK细胞、巨噬细胞中的任意一种；
所述免疫细胞为T细胞时，所述T细胞为TCR-T细胞、CAR-T细胞、γ/δ-T细胞中的任意一种；所述T细胞为TCR-T细胞时，所述TCR-T细胞为经慢病毒或mRNA技术转染的TCR-T细胞，或从血液中分离出的TCR-T细胞；所述免疫细胞为NK细胞时,所述NK细胞为CAR-NK中的任意一种；所述免疫细胞为巨噬细胞时,所述巨噬细胞为CAR-M细胞中的任意一种。
11.根据实施方案9或10所述的溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物，其 特征在于：所述肿瘤或癌症为头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤、圆细胞型脂肪肉瘤中的任意一种。
一、本发明提供了一种全新的精确改造溶瘤病毒制备的溶瘤病毒减毒株。所述溶瘤病毒为水疱性口炎病毒(Vesicular stomatitis virus，VSV)，具体选自水疱性口炎病毒印第安纳株，VSV MuddSummer亚型株，其M蛋白基因序列如SEQ ID NO 1所示，M蛋白的氨基酸序列为SEQ ID NO 2所示。本发明对所述水疱性口炎病毒进行了如下修饰，以获得溶瘤病毒减毒株：对所述水疱性口炎病毒的M蛋白基因进行了定点基因突变，获得减毒株。所述突变的位点包括：(1)第51位甲硫氨酸(M)突变为精氨酸(R)；(2)第111位亮氨酸(L)编码碱基敲除；(3)第221位缬氨酸(V)突变为苯丙氨酸(F)；(4)第226位丝氨酸(S)突变为精氨酸(R)。将完成突变后的水疱性口炎病毒编号为：JBS003；命名为：XN2-M51R-△L111-V221F-S226R；其M蛋白的基因序列为SEQ ID NO 3所示，M蛋白的氨基酸序列为SEQ ID NO 4所示。
与野生型VSV及其它已知的VSV减毒株相比，所述JBS003的安全性更高，可作为抗原、细胞因子等物质的载体(骨架)，与抗原、细胞因子等结合后，作为疫苗或药物进行使用。同时，JBS003也可不与其它物质结合，直接作为溶瘤病毒应用在肿瘤免疫治疗中，其效果也比野生型VSV及其它VSV减毒株的治疗效果更佳。
二、本发明在溶瘤病毒减毒株的基础上，提供了一种溶瘤病毒疫苗。如上所述，本发明提供的减毒株可以与抗原结合构成疫苗。本发明在JBS003的G蛋白和L蛋白间插入可表达NY-ESO-1的基因，构建了一种溶瘤病毒疫苗，编号为：JBS004。
NY-ESO-1(New York esophageal squamous cell carcinoma 1)属于肿瘤-睾丸抗原(Cancer-Testis Antigen,CTA)家族，在睾丸、卵巢及多种肿瘤组织中表达，而在其他正常组织中不表达，是目前已知发现的免疫原性最强的肿瘤特异性抗原。NY-ESO-1在不同肿瘤组织中表达丰度不同，蛋白表达最高的是粘液样圆细胞型脂肪肉瘤(89％～100％)、神经母细胞瘤(82％)、滑膜肉瘤(90％)、黑色素瘤(46％)、卵巢癌(43％)。NY-ESO-1抗原具有免疫原性和安全性，是一种用于免疫治疗的临床优势抗原。目前，复发、转移性的头颈部鳞癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤等仍无法得到有效治疗。引入NY-ESO-1后构建的JBS004溶瘤病毒疫苗能高效诱导机体在外周淋巴系统和肿瘤组织中特异性抗肿瘤免疫反应。试验表明，在抗肿瘤免疫治疗、尤其是针对上述癌症和肿瘤的治疗中，溶瘤病毒疫苗的免疫原性、有效性、靶向性、安全性和耐受性优势明显。
三、本发明在所述溶瘤病毒疫苗的基础上，还提供了一种可针对性治疗肿瘤的药物。所 述药物包括所述溶瘤病毒或溶瘤病毒疫苗。使用方法为：将所述JBS003溶瘤病毒或JBS004溶瘤病毒疫苗进行瘤内注射或者静脉注射。采取少量多次的方法进行注射。
为提高治愈率，优选的方案为：所述药物还包括TCR-T细胞。所述TCR-T细胞为转染了NY-ESO-1受体的T淋巴细胞，具体制备方法为：(1)从NCG-HLA-A2.1/Gpt人源化小鼠外周血中分离T淋巴细胞；(2)人工合成目的基因NY-ESO-1受体序列并进行基因测序，将其与慢病毒载体进行重组，获得NY-ESO-1受体重组慢病毒；(3)利用所述NY-ESO-1受体重组慢病毒对T淋巴细胞进行转染，从而获得转染了NY-ESO-1受体的T淋巴细胞，命名为：JBS-NY TCR-T。将构建好的JBS-NY TCR-T细胞在体外进行扩增，通过Western Blot方法对JBS-NY TCR-T细胞中的NY-ESO-1表达量进行检测，确定构建成功。
溶瘤病毒或溶瘤病毒疫苗和JBS-NY TCR-T联合使用的方法为：先静脉注射1次JBS-NY TCR-T，随后少量多次瘤内注射或静脉注射溶瘤病毒或溶瘤病毒疫苗。
一方面，本申请提供了一种溶瘤病毒减毒株，其特征在于：与VSV MuddSummer亚型株相比，所述溶瘤病毒减毒株的基质蛋白M基因经过改造。在某些实施方案中，所述VSV MuddSummer亚型株的基质蛋白M包含如SEQ ID NO:2所示的氨基酸序列。在某些实施方案中，所述VSV MuddSummer亚型株的基质蛋白M包含如SEQ ID NO:1所示的核酸序列。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造可以包含氨基酸位点第111位亮氨酸编码碱基敲除。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:7所示。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造还可以包含氨基酸位点第111位亮氨酸编码碱基敲除和第51位甲硫氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除且第51位甲硫氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:8所示。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造可以包含氨基酸位点第111位亮氨酸编码碱基敲除和第221位缬氨酸突变为苯丙氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除且第221位缬氨酸突变为苯丙氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:9所示。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造可以包含氨基酸位点第111位亮 氨酸编码碱基敲除和第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除且第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:10所示。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造可以包含氨基酸位点第111位亮氨酸编码碱基敲除，第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除，第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:11所示。
在本申请中，所述溶瘤病毒减毒株的基质蛋白M的改造可以包含氨基酸位点第111位亮氨酸编码碱基敲除，第51位甲硫氨酸突变为精氨酸，第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M与VSV MuddSummer亚型株的基质蛋白M相比，第111位亮氨酸编码碱基敲除，第51位甲硫氨酸突变为精氨酸，第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。在某些实施方案中，所述溶瘤病毒减毒株的基质蛋白M的氨基酸序列如SEQ ID NO:4所示。
另一方面，本申请还提供了核酸分子，其能编码所述溶瘤病毒的基质蛋白M。例如，编码所述溶瘤病毒的基质蛋白M的核酸序列可以如SEQ ID NO:3所示。
另一方面，本申请还提供了所述溶瘤病毒减毒株作为载体在医药领域中的应用。
在本申请中，所述溶瘤病毒减毒株作为载体在医药领域中的应用可以包括所述溶瘤病毒减毒株在制备药物或疫苗中的应用。
另一方面，本申请还提供了溶瘤病毒疫苗，所述溶瘤病毒疫苗可以在所述溶瘤病毒减毒株中插入抗原后制备得到。例如，可以在所述溶瘤病毒减毒株中插入特异性肿瘤抗原得到所述溶瘤病毒疫苗。在本申请中，所述抗原可以选自以下任意一种：NY-ESO-1、gp33、gp100、TX103、Mucin-1、WT-1、MART-1、MAGE A1、MAGE A3、MAGE A4、MAGE B2、PRAME、SURVIVIN、MART-1、col6A3、tyrosinase、T antigen、SLC45A2、VCX/Y、HPV、甲胎蛋白、癌胚抗原、CA 125、Her2、多巴色素互变异构酶、BAGE蛋白、GAGE蛋白、存活蛋白、酪氨酸酶、SSX2、细胞周期蛋白-A1、KIF20A、MUC5AC、Meloe、Lengsin、激肽释放酶4、IGF2B3和磷脂酰肌醇蛋白聚糖3。例如，在本申请中，将构建的溶瘤病毒减毒株作为载体，引入NY-ESO-1，获得所述溶瘤病毒疫苗。
在本申请中，还提供了所述溶瘤病毒疫苗的制备方法，所述方法包括：构建溶瘤病毒减毒株质粒，人工合成带限制性酶切位点的链接序列，利用生物学技术和基因重组技术，将其插入溶瘤病毒减毒株的G蛋白和L蛋白之间的非编码区，插入外源基因，获得携带外源基因的减毒株重组质粒，通过疫苗拯救步骤构建所述溶瘤病毒疫苗。
另一方面，本申请还提供了所述溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物。
在本申请中，所述抗肿瘤药物或治疗癌症的药物同时包括所述溶瘤病毒疫苗和免疫细胞。在本申请中，所述免疫细胞可以包括T细胞、NK细胞、巨噬细胞、DC细胞、TIL细胞中的任意一种：所述免疫细胞为T细胞时，所述T细胞可以包括TCR-T细胞、CAR-T细胞、γ/δ-T细胞、基因编辑的T细胞中的任意一种；所述T细胞为TCR-T细胞时，所述TCR-T细胞可以包括经慢病毒或mRNA技术转染的TCR-T细胞，或从血液中分离出的TCR-T细胞，或任意一种技术得到的TCR-T细胞；所述免疫细胞为NK细胞时，所述NK细胞可以包括NK或CAR-NK中的任意一种；所述免疫细胞为巨噬细胞时，所述巨噬细胞可以包括巨噬细胞或CAR-M细胞中的任意一种。在本申请中，所述肿瘤或癌症可以包括头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤和/或圆细胞型脂肪肉瘤。在本申请中，所述药物还可以包含任选地药学上可接受的载剂。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1
1、按表1的方式，对所述水疱性口炎病毒印第安纳株进行定点突变，获得7组突变后的减毒株。未进行基因突变的组别编号为：JBS000，作为对照。
表1各组突变情况展示表
Figure PCTCN2021093142-appb-000001
Figure PCTCN2021093142-appb-000002
减毒株的具体构建方法为本领域的常规技术，简述如下：
(1)构建质粒。以pVSV-XN2质粒为模板，利用PCR方法引入如表1所述的不同的突变位点。将质粒与各突变位点的引物共同进行PCR，再将PCR产物进行1％琼脂糖凝胶电泳，随后通过凝胶回收试剂盒进行割胶回收，获得M基质蛋白不同突变的质粒。
(2)病毒拯救。按MOI＝5，用表达T7RNA聚合酶的痘病毒vTF7-3感染接种BHK-21细胞，感染1h后，使用DPBS缓冲液漂洗一次BHK-21细胞。随后制备质粒转染预混液，具体包括:pBS-N、pBS-P、pBS-L、步骤(1)制备的突变质粒。其中pBS-N、pBS-P、pBS-L分别指克隆了VSV N、VSV P、VSV L蛋白基因的表达质粒，分别表达病毒挽救所需的N、P、和L蛋白。按lipofectamine 2000使用说明书所述的方法进行质粒转染，4h后更换新鲜的含10％胎牛血清的DMEM完全培养基，48h后吸取上清，用0.22μm的滤膜滤去除痘病毒。将滤液加入到新鲜的BHK-21细胞中；每天观察细胞病变情况，待细胞出现病变情况时收取上清，经RTPCR鉴定确认成功后，用病毒空斑实验纯化病毒。即获得减毒株。
(3)M蛋白基因测序。利用Trizol试剂盒抽提病毒基因组RNA，用随机引物进行逆转录反应，用针对M蛋白基因序列设计的引物对逆转录出来的cDNA进行PCR。引物序列为:5'-AAAAAAGTAACAGATATCAC-3'(SEQ ID NO:5)；5'-ACATTTTTCCAGTTTCCTTTTTGG-3'(SEQ ID NO:6)。产物经1％琼脂糖凝胶电泳后回收，并送往测序公司进行测序。
2、不同减毒株对细胞的体外侵染能力展示。分别在MEF细胞(人的成纤维细胞)培养 液、LLC细胞(鼠源非小细胞肺癌细胞)培养液中，分别加入JBS000、JBS001、JBS002、JBS003、JBS008、JBS009、JBS010、JBS014各200pfu，检测各组减毒株产生的半数组织培养感染剂量(Tissue culture infective dose,TCID50)。具体测试方法为：
(1)在6孔培养板中每孔加入细胞悬液3mL，使细胞量达到4×10 5个/孔，共6个孔，37℃、5％CO 2条件下培养16h。
(2)在各孔中分别加入病毒JBS000、JBS001、JBS002、JBS003、JBS008、JBS009、JBS010、JBS014各200pfu，设正常细胞对照2个孔。在24h时，取细胞上清100μL。
(3)在96孔培养板中，每孔加入Vero细胞悬液100μL，使细胞量达到1×10 4个/mL，37℃、5％CO 2条件下培养16h。
(4)在1.5mL EP管中将步骤(2)中收获的上清作连续10倍的稀释，10 -1～10 -11，共11个滴度。
(5)将稀释好的上清接种到步骤(3)的96孔培养板中，每一稀释度接种一列(共8孔)，每孔接种100μL。设正常细胞对照组一列。
(6)48h后观察每孔细胞荧光情况，有荧光标记为此孔被感染。
(7)按Karber法计算TCID50。
结果如图1A和图1B所示。构建的各溶瘤病毒减毒株在体外肺癌细胞(LLC)中的复制扩增能力比在正常的成纤维细胞(MEF)内各溶瘤病毒减毒株的复制扩增能力强，其中JBS003在体外肺癌细胞(LLC)中的复制扩增能力较强，感染24h后产生的病毒粒子数接近野生型病毒。而在正常的成纤维细胞(MEF)内各溶瘤病毒减毒株的复制感染能力均有所下降。因此：JBS003载体对肿瘤细胞具有较强的特异性侵染能力。
3、不同减毒株对细胞的体外杀伤能力展示。使用等量各减毒株分别体外感染不同细胞，24h后MTT法检测细胞活力。具体方法如下：
(1)在96孔培养板中每孔加入细胞悬液100μL，使细胞量达到1×10 4个/孔，37℃，5％CO 2条件下培养16h。测试的细胞种类为：LLC、MEF、Hela(人肿瘤细胞)。
(2)分别将JBS000、JBS001、JBS002、JBS003、JBS008、JBS009、JBS010、JBS014稀释到MOI(multiplicity of infection，感染复数)分别为0.001、0.01、0.1、1.0，每一稀释梯度接种4个孔，每孔接种100μL，在37℃，5％CO 2条件下培养40h。
(3)弃去96孔培养板中的上清，加入新鲜的DMED培养基，再加入5mg/mL的MTT溶液，20μL/孔。在37℃，5％CO 2条件下培养4h。
(4)将96孔板离心，设置转速2500g/min，室温离心5分钟。随后使用1mL一次性无 菌注射器，轻轻吸掉上清。
(5)再向每孔中加入DMSO，100μL/孔，37℃放置10分钟。
(6)使用多功能酶标仪，震荡2分钟，在570nm波长下，测定各孔的OD值。
结果如图2A、图2B、和图3所示。结果表明各溶瘤病毒减毒株都展现了良好的肿瘤细胞杀伤的能力，除JBS000外，均对MEF细胞无显著的杀伤。即，体外状态下，除JBS000外，各减毒株均对肿瘤细胞具有特异性杀伤，对正常细胞无显著影响。
4、不同减毒株在细胞内清除难易度测试。以IFN-β指标进行测试。按上述步骤3的步骤(1)、(2)培养细胞并加入减毒株。随后破碎各组细胞，用TRIzol(Invitrogen)从各细胞中提取总RNA，利用PrimeScript RT Reagent Kit with DNA Eraser(Takara)反转录试剂盒逆转录成cDNA，并用LightCycler 480SYBR Green I Master(Roche)染料进行染色，在LightCycler 480定量PCR仪上检测各个基因的Ct值。用ΔΔCt法计算目的基因IFN-β、VSV-G相对表达量。结果如图4A和4B所示。在LLC细胞系中，除JBS000外，所有减毒株均可引起IFN-β表达水平的提高，其中JBS003载体的调节能力最低；而在MEF细胞中，所有病毒均可上调IFN-β的表达水平，其中JBS003的水平最高，为野生型病毒载体(JBS000)的3倍。即：JBS003在肿瘤细胞中难以被清除，而在正常细胞中则容易被清除。
实施例2溶瘤病毒疫苗的构建及效果展示
1、在实施例一制备的各减毒株及野生型病毒的基础上，插入NY-ESO-1基因，构建获得溶瘤病毒疫苗，构建示意图如图5所示。各组插入片段情况如表2所示。
表2各组插入片段情况展示表
疫苗编号 疫苗命名 对应的减毒株
JBS004 XN2-M51R-△L111-V221F-S226R—NY-ESO-1 JBS003+NY-ESO-1
JBS005 XN2-M51R-△L111-NY-ESO-1 JBS002+NY-ESO-1
JBS006 XN2-M51R-NY-ESO-1 JBS001+NY-ESO-1
JBS007 XN2-WT-NY-ESO-1 JBS000+NY-ESO-1
JBS011 XN2-△L111-NY-ESO-1 JBS008+NY-ESO-1
JBS012 XN2-△L111-V221F-NY-ESO-1 JBS009+NY-ESO-1
JBS013 XN2-△L111-S226R-NY-ESO-1 JBS010+NY-ESO-1
JBS015 XN2-△L111-V221F-S226R-NY-ESO-1 JBS014+NY-ESO-1
所述JBS004-JBS007、JBS011-JBS013、JBS015的具体制备方法为本领域的常规技术，简述如下。需要说明的是，下述记载并非限定JBS004-JBS007、JBS011-JBS013、JBS015只能按照如下方法进行，而是给出示例。
(1)减毒株质粒构建。人工合成带限制性酶切位点Xho I和Mlu I的链接序列，利用生物学技术和基因重组技术，将其插入实施例一制备的各减毒株的G蛋白和L蛋白之间的非编码区，获得各减毒株质粒。
(2)外源基因插入。将各减毒株质粒用Xho I和Mlu I进行双酶切，随后插入NY-ESO-1外源基因，获得携带NY-ESO-1的减毒株重组质粒。
(3)疫苗拯救。参照实施例一中“病毒拯救”的方法对各减毒株重组质粒所对应的疫苗进行拯救，从而构建各溶瘤病毒疫苗。
2、治疗LLC-NY-ESO-1非小细胞肺癌(移植瘤)效果展示。
选择差异不显著的136只C57BL/6小鼠，分别皮下接种2×10 5个LLC细胞(小鼠肺癌细胞)。接种第9天，待移植瘤体积长至100mm 3左右时，将所有小鼠分为17组(n＝8)：对照组(PBS组)瘤内注射50μL PBS，其余16组为治疗组，分别进行瘤内接种JBS000、JBS001、JBS002、JBS003、JBS004、JBS005、JBS006、JBS007、JBS008、JBS009、JBS010、JBS011、JBS012、JBS013、JBS014、JBS015，每2天给药1次，共给药3次，单次接种量均为10 7pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积，体积(mm 3)＝(长径×短径 2)/2。检测癌细胞的转移比例，检测方法为：LLC细胞带有红色荧光蛋白，在绿色荧光显微镜下会呈现黄色荧光；当癌细胞转移至肺组织后，将肺组织置于显微镜下，拍摄荧光图片，通过Image J分析图片灰度值，从而分析肺癌细胞的比例，即得癌细胞的转移比例。
肿瘤体积变化情况如图6～8所示。结果显示：各治疗组均对移植瘤有一定的抑制作用。其中JBS003组有1只被完全治愈。JBS004对移植瘤的治愈率达37.5％。癌细胞转移情况如图9所示。从图6～9可以看出：移植瘤体积大小与肺部转移多少存在一定的相关性。JBS003对肺转移灶的治疗要优于JBS000、JBS001组；JBS004抑制或防止肺癌细胞转移的能力优于其他组。
3、治疗MCA-205-NY-ESO-1纤维肉瘤(移植瘤)的效果展示。
按“治疗LLC-NY-ESO-1非小细胞肺癌移植瘤”的方法对小鼠进行处理，皮下接种10 6个MCA-205-NY-ESO-1纤维肉瘤细胞，待移植瘤体积长至100mm 3左右时进行处理。同样以瘤内注射50μL PBS作为对照组，治疗组中，分别进行瘤内接种JBS004、JBS005、JBS006、JBS007、JBS011、JBS012、JBS013、JBS015，每组6只动物，每2天给药1次，共给药3次， 单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积。结果如图10、11所示。结果显示：各治疗组都能够在一定程度上减少肿瘤体积。经JBS004治疗后，其中2只小鼠的移植瘤完全消除(占33.33％)，剩余小鼠的肿瘤体积也得到很好地控制，与其余组别差异显著。JBS004治疗纤维肉瘤的总反应率为100％。
4、治疗B16-F10-NY-ESO-1黑色素瘤(移植瘤)的效果展示。
按上述移植瘤试验的处理方法对小鼠进行处理，皮下接种2×10 6个B16-F10-NY-ESO-1黑色素瘤细胞，待移植瘤体积长至100mm 3左右时进行处理。同样以瘤内注射50μL PBS作为对照组，治疗组中，分别进行瘤内接种JBS004、JBS005、JBS006、JBS007、JBS011、JBS012、JBS013、JBS015，每组6只动物，每2天给药1次，共给药3次，单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积。结果如图12、13所示。结果显示：各治疗组对黑色素瘤均有一定的治疗作用，JBS004组效果尤佳。
5、不同剂量的JBS004的效果展示。
选择6～8周龄，体重在18g左右的C57BL/6小鼠，分别皮下接种2×10 5个LLC细胞(小鼠肺癌细胞)。接种第9天，待移植瘤体积长至100mm 3左右时，将所有小鼠分为5组，每组6只动物：对照组(PBS组)瘤内注射50μL PBS，其余4组为治疗组，分别进行瘤内接种10 6pfu/只、10 7pfu/只、10 8pfu/只、10 9pfu/只JBS004，每2天给药1次，共给药3次。自开始给药至实验终点，每2天记录一次移植瘤体积，结果如图14、15所示。并于实验结束时，对安乐死的小鼠进行剖检，取小鼠肺组织，检测癌细胞的转移比例，结果如图16所示。结果显示：不同剂量的JBS004均对肺癌小鼠有一定治疗效果，其中10 8pfu/只剂量水平下，治愈率达33.33％，有效控制率达33.33％，未发现肺部癌细胞转移率为66.67％，显著优于其他剂量组。
另外，如图17、18所示，在整个实验过程中，小鼠体温、体重均维持在正常范围内，未出现体温、体重异常情况，说明不同剂量JBS004对肺癌小鼠的体温、体重无显著影响。就体重而言，PBS组中小鼠体重稳步增长，其余治疗组的小鼠出现增长迟缓的情况，其原因应该与移植瘤的减小有关，实验终点时，各组小鼠的体重无显著差异。证明JBS004安全性良好。
实施例3 JBS004的药代动力学及急性毒性实验结果展示
1、药代动力学实验。选择C57BL/6小鼠，皮下接种2×10 5个LLC细胞，接种9天左右后，移植瘤体积长至100mm 3左右，建立LLC移植瘤模型。单次瘤内注射10 8pfu/只的JBS004，分别于0min(+15min)、6h、12h、48h、96h、120h及14天进行肿瘤组织采样(重复3份)，用全自动研磨仪进行组织破碎，通过Trizol提取肿瘤组织的总RNA，最终利用定量PCR(荧光探针法)进行病毒核酸拷贝数的分析。结果如图19、20所示。结果显示：感染6h肿瘤内 的病毒量到达峰值，较初始计量，约复制了500倍；感染48h后，病毒量开始低于初始注射量；14天后，未检测到病毒核酸拷贝数。因此，JBS004可快速、高效在肿瘤内进行复制；14天后，并未检测到JBS004，证明其不会在体内长时间蓄积，造成潜在的后续损伤，安全性良好。
2、急性毒性实验。选择40只C57BL/6小鼠，雌雄各半。分为3个给药组，单次肌肉注射给予供JBS004溶液，各组给药量分别为：10 3pfu/只、10 6pfu/只、10 9pfu/只。空白对照组单次给予溶媒对照(单次肌肉注射PBS)，给药体积均为100μL。动物给药日定为该组动物观察的第1天。动物在给药后观察14天，于给药后第15天解剖。
实验过程中，每2天记录一次小鼠的体温和体重，结果如图21～24所示。给药前及给药后的30min、1h、2h、4h及10h对小鼠进行详细笼边观察，后续实验中每天至少进行一次笼边观察。实验结束时，取小鼠外周血用于血液学和血液生化的检测(血糖、肌酐、尿素氮、血尿素氮/肌酐、磷离子、钙离子、总蛋白、白蛋白、球蛋白等)，并剖检采集主要脏器，包括心、肝、脾、肺、肾、脑、睾丸/卵巢进行组织称重，计算脏器系数。受篇幅限制，此处未体现血液学和血液生化学检测的全部指标数据。检测结果表明：未见小鼠异常死亡的情况，未出现与JBS004相关的临床症状。注射不同剂量的JBS004对小鼠脏器重量无显著影响；对小鼠血液学和血液生化指标无显著影响。本实验条件下，最大耐受剂量(MTD)至少为10 9pfu/只。故上述最佳剂量(10 8pfu/只)在安全剂量内。
实施例4 JBS-NY TCR-T的构建
JBS-NY TCR-T为利用NY-ESO-1受体重组慢病毒对T淋巴细胞进行转染后获得的T细胞，具体构建方法为本领域的常规技术，简述如下：
(1)根据已公布的NY-ESO-1的受体基因序列人工合成NY-ESO-1受体基因。
(2)构建重组慢病毒载体。PCR扩增NY-ESO-1TCR基因片段，引物为：ESO TCR-F1:5’-GGAATTCATGGAGACCCTCT-3’(SEQ ID NO:12)；
ESO TCR-R1:5’-ATAGTTTAGCGGCCGCCTAGCCTCTGGAA-3’(SEQ ID NO:13)。
以已克隆的ESO TCR cDNA作为模板进行PCR体外扩增，PCR反应条件为:94℃预变性3min，94℃、30s，55℃、30s，72℃、45s，35个循环后72℃延伸5min。1.2％琼脂糖凝胶电泳分离PCR扩增的特异性片段(1824bp)。用EcoR I和Not I分别双酶切PCR产物及pCL20c-MSCV-GFP质粒，玻璃奶凝胶回收酶切产物。将两种酶切产物用T4DNA连接酶连接，16℃过夜。然后将连接产物转化感受态DH5α菌，培养扩增后用B型小提质粒试剂盒提取质粒。
(3)pCL20c-MSCV-ESO TCR重组慢病毒载体的鉴定。pCL20c-MSCV-ESO TCR质粒经 EcoR I和Not I双酶切、PCR后送往测序。
(4)pCL20c-MSCV-ESO TCR重组慢病毒的包装。将T293细胞在含10％胎牛血清的DMEM培养基中、在95％湿度、5％CO 2、37℃孵箱中常规培养，每周传代3～4次。转染前1d，将5×10 6～6×10 6个T293细胞种于D＝10cm的细胞培养皿中。转染前2h，换10mL新鲜10％DMEM培养基。
转染前1h开始配置转染液，转染液由A液和B液组成。A液：pCL20c-HIV-gp质粒6μg、pCAG4-RTR2质粒2μg、CAG-VSV-G质粒2μg、pCL20c-MSCV-ESOTCR质粒10μg、50μL2.5mol/L的CaCl 2，用去离子水补至500μL体积，轻弹混匀，室温放置5min。B液：2×HBSS(280mmol/L的NaCl、50mmol/L HEPES、1.5mmol/L Na 2HPO 4，pH＝7.02)500μL。将A液滴入B液中，边加边震荡，室温放置20min，即得转染液。从孵箱内取出培养皿，将其向身体倾斜15°，将转染液小心滴加在培养皿的低位一侧，边加边左右摇动混匀，孵箱中培养16h或过夜。随后换10mL新鲜生长培养基。转染48h后收集上清，-80℃保存。取出包装上清100mL,室温融化，2000×g离心30min，收集上清，用0.45μm的PVDF膜过滤，12000×g超速离心3h，加入1mL无血清的DMEM，重悬病毒沉淀，100μL/管分装，-80℃保存。
(5)外周血中T细胞分离。从NCG-HLA-A2.1/Gpt人源化小鼠外周血中分离T淋巴细胞，加入细胞分离液，1500g/min离心15min，取第二层环状乳白色淋巴细胞层，加入细胞洗涤液5mL，充分混匀后，1800g/min离心20分钟，弃去上清后，将沉淀的淋巴细胞重悬。
(6)重组慢病毒与T细胞共转染。将重悬的淋巴细胞调整为2×10 5个/mL,加入6μg/mL的polybrene和适量10 4pfu病毒，重复混匀，37℃孵育，继续培养3～4天后，即可获得JBS-NY TCR-T。
实施例5溶瘤病毒疫苗与JBS-NY TCR-T联合使用的效果展示
选择6-8周龄，体重18-20g的NCG-HLA-A2.1/Gpt人源化小鼠，分别皮下接种2×10 5个非小细胞肺癌A549细胞，相同、适宜条件下培养至移植瘤体积为100mm 3左右时，开始处理。各组处理条件如表3所示。表3中，JBS NY TCR-T细胞的接种量为10 6个，采取单次静脉注射的方式进行。如果联合使用JBS-NY TCR-T细胞和溶瘤病毒疫苗，则在静脉注射JBS-NY TCR-T细胞后24h瘤内注射对应溶瘤病毒疫苗。每2天注射1次溶瘤病毒疫苗，共给药3次，单次接种量均为10 8pfu/只。
表3各组处理情况展示表
组别 是否接种TCR-T TCR-T的种类 接种的溶瘤病毒种类 其它
对照组 否 / / 等量PBS
组1 否 / JBS004 /
组2 否 / JBS005 /
组3 否 / JBS006 /
组4 否 / JBS007 /
组5 是 JBS-NY TCR-T / /
组6 是 JBS-NY TCR-T JBS004 /
组7 是 JBS-NY TCR-T JBS005 /
组8 是 JBS-NY TCR-T JBS006 /
组9 是 JBS-NY TCR-T JBS007 /
自开始给药至实验终点，每2天记录一次移植瘤体积。结果如图25、26所示。并检测各组肺癌细胞转移的情况，结果如图27所示。结果显示：除对照组外，各组均对移植瘤体积有一定的抑制作用，并且从图中可惊喜地发现，JBS004联合JBS-NY TCR-T后治疗效果提升显著，从原本25％治愈率提升至92％～95％的治愈率，显示了联合治疗的优势；对抑制肺癌细胞的转移也体现了优越的效果。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (20)

  1. 溶瘤病毒减毒株，其特征在于：与VSV MuddSummer亚型株相比，所述溶瘤病毒减毒株的基质蛋白M经过改造，所述改造包含氨基酸位点第111位亮氨酸编码碱基敲除。
  2. 根据权利要求1所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除。
  3. 根据权利要求1-2中任一项所述的溶瘤病毒减毒株，其基质蛋白M的改造还包含氨基酸位点第51位甲硫氨酸突变为精氨酸。
  4. 根据权利要求3所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除和第51位甲硫氨酸突变为精氨酸。
  5. 根据权利要求1-4中任一项所述的溶瘤病毒减毒株，其基质蛋白M的改造还包含第221位缬氨酸突变为苯丙氨酸。
  6. 根据权利要求5所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为第111位亮氨酸编码碱基敲除和第221位缬氨酸突变为苯丙氨酸。
  7. 根据权利要求1-6中任一项所述的溶瘤病毒减毒株，其基质蛋白M的改造还包含氨基酸位点第226位丝氨酸突变为精氨酸的氨基酸突变。
  8. 根据权利要求7所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除和第226位丝氨酸突变为精氨酸。
  9. 根据权利要求7所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为氨基酸位点第111位亮氨酸编码碱基敲除、第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。
  10. 根据权利要求9所述的溶瘤病毒减毒株，所述溶瘤病毒减毒株的基质蛋白M的改造为第51位甲硫氨酸突变为精氨酸；第111位亮氨酸编码碱基敲除；第221位缬氨酸突变为苯丙氨酸和第226位丝氨酸突变为精氨酸。
  11. 根据权利要求1-10中任一项所述的溶瘤病毒减毒株，其基质蛋白M具有下组中任一种氨基酸序列：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的氨基酸序列。
  12. 权利要求1-11中任一项所述的溶瘤病毒减毒株作为载体在医药领域中的应用。
  13. 根据权利要求12所述的应用，其特征在于：所述溶瘤病毒减毒株在制备药物或疫苗中的应用。
  14. 溶瘤病毒疫苗，其特征在于：在权利要求1-11中任一项所述的溶瘤病毒减毒株中插入抗原后制备得到。
  15. 根据权利要求14所述的溶瘤病毒疫苗，其特征在于：所述抗原为特异性肿瘤抗原。
  16. 根据权利要求14-15中任一项所述的溶瘤病毒疫苗，其特征在于：所述抗原为NY-ESO-1、gp33、gp100、TX103、Mucin-1、WT-1、MART-1、MAGE A1、MAGE A3、MAGE A4、MAGE B2、PRAME、SURVIVIN、MART-1、col6A3、tyrosinase、T antigen、SLC45A2、VCX/Y、HPV、甲胎蛋白、癌胚抗原、CA 125、Her2、多巴色素互变异构酶、BAGE蛋白、GAGE蛋白、存活蛋白、酪氨酸酶、SSX2、细胞周期蛋白-A1、KIF20A、MUC5AC、Meloe、Lengsin、激肽释放酶4、IGF2B3、磷脂酰肌醇蛋白聚糖3中的任意一种。
  17. 权利要求14-16中任一项所述溶瘤病毒疫苗制备的抗肿瘤药物或治疗癌症的药物。
  18. 根据权利要求17所述的抗肿瘤药物或治疗癌症的药物，其特征在于：所述药物同时包括所述溶瘤病毒疫苗和免疫细胞。
  19. 根据权利要求17-18中任一项所述的抗肿瘤药物或治疗癌症的药物，其特征在于：所述免疫细胞为T细胞、NK细胞、巨噬细胞、DC细胞、TIL细胞中的任意一种：所述免疫细胞为T细胞时，所述T细胞为TCR-T细胞、CAR-T细胞、γ/δ-T、基因编辑的T细胞中的任意一种；所述T细胞为TCR-T细胞时，所述TCR-T细胞为经慢病毒或mRNA技术转染的TCR-T细胞，或从血液中分离出的TCR-T细胞，或任意一种技术得到的TCR-T细胞；所述免疫细胞为NK细胞时，所述NK细胞为NK细胞或CAR-NK中的任意一种；所述免疫细胞为巨噬细胞时，所述巨噬细胞为巨噬细胞或CAR-M细胞中的任意一种。
  20. 根据权利要求17-19中任一项所述的抗肿瘤药物或治疗癌症的药物，其特征在于：所述肿瘤或癌症为头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤、圆细胞型脂肪肉瘤中的任意一种。
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