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 PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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
Description
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 |
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 | / |
Claims (20)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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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KR20230002564A (ko) | 2023-01-05 |
TW202142687A (zh) | 2021-11-16 |
JP2023524915A (ja) | 2023-06-13 |
CA3178631A1 (en) | 2021-11-18 |
EP4141111A1 (en) | 2023-03-01 |
EP4141111A4 (en) | 2023-12-20 |
CN111286493A (zh) | 2020-06-16 |
JP7468958B2 (ja) | 2024-04-16 |
CN111286493B (zh) | 2020-10-27 |
AU2021271961A1 (en) | 2022-12-15 |
US20230256079A1 (en) | 2023-08-17 |
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