WO2019196617A1 - 重组溶瘤病毒组合物及其用途 - Google Patents

重组溶瘤病毒组合物及其用途 Download PDF

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WO2019196617A1
WO2019196617A1 PCT/CN2019/078999 CN2019078999W WO2019196617A1 WO 2019196617 A1 WO2019196617 A1 WO 2019196617A1 CN 2019078999 W CN2019078999 W CN 2019078999W WO 2019196617 A1 WO2019196617 A1 WO 2019196617A1
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gene
oncolytic virus
tumor
herpes simplex
recombinant oncolytic
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PCT/CN2019/078999
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English (en)
French (fr)
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田超
李小鹏
孙春阳
周华
刘家家
赵婧姝
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北京唯源立康生物科技有限公司
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Priority to US17/044,074 priority Critical patent/US20210138008A1/en
Priority to EP19784788.2A priority patent/EP3778882A4/en
Publication of WO2019196617A1 publication Critical patent/WO2019196617A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to recombinant oncolytic virus compositions and their use in the preparation of a medicament for treating tumors.
  • Tumor gene immunotherapy is an anti-tumor method with rapid progress in recent years, and its potential is huge.
  • Top 10 Scientific Breakthroughs list selected by Science magazine tumor immunotherapy topped the list.
  • the oncolytic virus represented by HSV-1 vector plays a huge role in tumor immunotherapy.
  • Oncolytic virus refers to a type of virus that selectively infects tumor cells and replicates in target cells, ultimately leading to tumor cell lysis and death. These viruses rely on their own specificity to replicate in tumor cells to lyse tumor cells. The virus released after cell lysis can further infect surrounding tumor cells, and has no destructive effect on normal cells and tissues, or has less influence. . Oncolytic virus has multiple anti-tumor mechanisms, including 1. direct lysis of tumor cells; 2. destruction of tumor blood vessels; 3. viral protein expressed by viral replication has direct cytotoxic effect; 4. anti-tumor immune response; Sensitivity to radiotherapy and chemotherapy; 6, expression of the inserted exogenous therapeutic gene.
  • the viruses currently used for the transformation of oncolytic viruses include herpes simplex virus, adenovirus and vaccinia virus. Among them, herpes simplex virus type 1 (HSV-1) is the most promising viral vector.
  • HSV-1 is a large double-stranded DNA virus with a genome size of 152 kb and encoding more than 80 genes.
  • the ⁇ 34.5 gene product can block the synthesis of host proteins due to viral infection, and the deletion of ⁇ 34.5 makes the viral vector It is not replicable in normal cells, and the ⁇ 34.5 gene product is a neurotoxic factor.
  • the ICP6 gene in HSV-1 encodes a large subunit of ribonucleotide reductase that expresses a virus, and is an enzyme necessary for HSV-1 to synthesize viral DNA in non-dividing cells.
  • the TK gene-expressed thymidine kinase is involved in the synthesis of phosphorylated deoxynucleosides, while phosphorylating deoxycytidine and nucleoside analogs to produce viral DNA synthesis precursors.
  • the UNG gene expresses uracil glycosylase to cleave uracil from DNA to prevent mutation and signal transmission to the base cleavage repair pathway.
  • ICP47 gene product blocks antigen presentation during viral infection (Shen Y, Nemunaitis J. Herpes simplex virus 1 (HSV-1) for cancer treatment. Cancer Gene Therapy 2006; 13: 975-992; Peters C, Rabkin S D. Designing Herpes Viruses as Oncolytics. Molecular Therapy Oncolytics, 2015, 2: 15010).
  • Tumor cells have multiple immune escape mechanisms, and any single treatment strategy can not completely eliminate tumor cells, so the combined application of multiple treatment strategies will be the development trend of future cancer treatment.
  • Combination therapy with oncolytic viruses and other treatments or drugs usually takes two strategies. One is that the oncolytic virus is administered separately with other treatments or drugs using a uniform treatment regimen. The second is that the oncolytic virus carries genes that are beneficial for tumor treatment.
  • the object of the present invention is to overcome the above problems in the prior art and to provide a recombinant oncolytic virus composition capable of retaining the high-efficiency tumor killing effect of the oncolytic virus while adjusting the recombinant oncolytic virus.
  • the amount of each component in the composition maximizes the synergistic anti-tumor effect between the exogenous genes expressed by the components and between the expressed exogenous gene and the oncolytic virus, and reduces the side effects of the treatment.
  • the invention relates to:
  • a recombinant oncolytic virus composition characterized in that said composition comprises:
  • a first recombinant oncolytic virus comprising a first herpes simplex virus vector and a first foreign gene;
  • the first foreign gene encoding a gene selected from any one of the following: a cytokine, a monoclonal antibody, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, an antisense RNA or a small RNA that blocks or down-regulates a tumor over-expressed protooncogene and a metabolic gene, having a preventive and/or therapeutic effect on a tumor;
  • a second recombinant oncolytic virus comprising a second herpes simplex virus vector and a second foreign gene;
  • the second foreign gene encoding a gene selected from any one of the following: a cytokine, a monoclonal antibody, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, an antisense RNA or a small RNA that blocks or down-regulates a tumor over-expressed protooncogene and a metabolic gene, having a preventive and/or therapeutic effect on a tumor;
  • first herpes simplex virus vector and the second herpes simplex virus vector are identical or different from each other, and the selected first foreign gene is different from the selected second foreign gene.
  • composition according to Item 1, wherein the composition further comprises a third recombinant oncolytic virus, the third recombinant oncolytic virus comprising a third herpes simplex virus vector and a third foreign source a gene; the third foreign gene encoding a gene selected from any one of the following: a cytokine, a monoclonal antibody having a prophylactic and/or therapeutic effect on a tumor, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, blocking Or down-regulating antisense RNA or small RNA of tumor over-expressed protooncogenes and metabolic genes;
  • the third herpes simplex virus vector is the same as or different from the first herpes simplex virus vector, the selected third foreign gene is different from the selected first foreign gene; and the third herpes simplex
  • the viral vector and the second herpes simplex virus vector are identical or different and are selected to be a third foreign gene different from the selected second foreign gene.
  • cytokine is any one selected from the group consisting of GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL- 3.
  • the monoclonal antibody having a preventive and/or therapeutic effect on tumor is selected from any one of the following: PD-1 monoclonal antibody, PD-L1 monoclonal antibody , PD-L2 monoclonal antibody, CTLA-4
  • a regulatory sequence is expressed, the expression regulatory sequence comprising at least one of a promoter, an enhancer, and a polynucleotide.
  • the recombinant oncolytic virus composition according to any one of items 1 to 7, wherein the first herpes simplex virus vector, the second herpes simplex virus vector, and the third herpes simplex virus vector each independently encode a ICP34 for deletion.
  • the insertion positions of the first foreign gene, the second foreign gene, and/or the third foreign gene are each identical or different from each other.
  • the recombinant oncolytic virus composition according to any one of 1 to 11, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1).
  • HSV-1 herpes simplex virus type 1
  • the oncolytic virus composition according to Item 13 wherein the first foreign gene is a gene encoding GM-CSF, G-CSF, M-CSF, IL-2, IL-12 or IFN- ⁇ ,
  • the second foreign gene is a gene encoding an immunological checkpoint blocking antibody.
  • the first foreign gene is a gene encoding GM-CSF, IL-2 or IL-12
  • the second foreign gene is a PD-1 resistance.
  • the oncolytic virus composition according to any one of items 11 to 15, wherein the first recombinant oncolytic virus and the second recombinant oncolytic virus have a mixing ratio of 0.5:8, 1:8 in terms of pfu. , 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1.
  • the oncolytic virus composition according to any one of items 17 to 19, wherein the first recombinant oncolytic virus and the second recombinant oncolytic virus have a mixing ratio of 0.5:2-2:0.5 in terms of pfu. Preferably, it is from 0.5:1 to 1:0.5, most preferably 1:1.
  • the tumor is selected from the group consisting of a brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer, prostate cancer At least one of gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (eg, soft tissue sarcoma and osteosarcoma).
  • a brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer, prostate cancer At least one of gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (eg, soft tissue sarcoma and osteosarcoma).
  • a pharmaceutical composition comprising the recombinant oncolytic virus composition of any of items 1-20 and a pharmaceutically acceptable carrier.
  • An article or kit comprising a vial containing a pharmaceutical composition of item 23 and a package insert, the package insert containing information relating to the use of the pharmaceutical composition.
  • a method of treating a tumor comprising administering to a subject an effective amount of the recombinant oncolytic virus composition of any one of items 1 to 20 or the pharmaceutical composition of item 23.
  • the tumor is selected from the group consisting of a brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer, At least one of prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (eg, soft tissue sarcoma and osteosarcoma).
  • a brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer At least one of prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (eg, soft tissue sarcoma and osteosarcoma).
  • Oncolytic virus refers to a type of virus that infects and kills cancer cells. After infecting cancer cells, the virus destroys infected cancer cells by oncolytic action, and simultaneously releases new infectious virus particles or virions, destroying the remaining Cancer cells. Such viruses are named for their oncolysis.
  • Herpes simplex virus is one of the first viruses (oncolytic viruses) selected for selective attack on cancer cells because it is easy to handle and relatively harmless in its natural state.
  • Herpes simplex virus type 1 (HSV-1) mutant 1716 lacks two copies of the ICP34.5 gene and is therefore no longer able to replicate in terminally differentiated and non-dividing cells, but will infect and cause cancer in very effective conditions.
  • Cell lysis has been shown to be an effective tumor targeting strategy.
  • a variety of HSV-based oncolytic viruses have been developed and are undergoing clinical trials.
  • the virus of the present invention is herpes simplex virus
  • the virus may be derived, for example, from an HSV1 strain or an HSV2 strain or a derivative thereof, preferably HSV1.
  • Derived strains include inter-type recombinants containing DNA from HSV1 strains and HSV2 strains.
  • the sequence homology of the derivatized strain to the HSV1 genome or HSV2 genome is preferably at least 70%, more preferably at least 80%, even more preferably at least 90% or 95%. More preferably, the sequence identity of the derivatized strain to the HSV1 genome or HSV2 genome is at least 70%, more preferably at least 80% identical, even more preferably at least 90%, 9% or 98% identical.
  • Herpes simplex virus vector refers to a herpes simplex virus that carries a foreign gene.
  • a plaque forming unit refers to the number of viruses that form a plaque (plaque) on a single layer of cultured animal cells.
  • Immune checkpoint blocking (inhibiting) antibody or “immunoassay blocking (inhibiting) monoclonal antibody” refers to a monoclonal antibody that inhibits or blocks an inhibitory immunological checkpoint molecule.
  • Immune checkpoints are regulators of the immune system, and their role is to prevent the immune system from attacking cells indiscriminately, which is critical for self-tolerance.
  • the immunological checkpoint is divided into inhibitory checkpoint molecules and irritant checkpoint molecules.
  • Inhibitory checkpoint molecules include, but are not limited to, A2AR, B7-H3 (CD276), B7-H4, BTLA (CD272), CTLA-4 (CD152), IDO, KIR, LAG3, NOX2, PD1, TIM3, VISTA; Sex checkpoint molecules include, but are not limited to, CD27, CD40, OX40, GITR, CD137, CD28, ICOS. Inhibitory checkpoint molecules are targets for cancer immunotherapy because they may be used in many types of cancer.
  • composition of the present invention comprising two or more recombinant oncolytic viruses exhibits an oncolytic effect (tumor therapeutic effect) greater than that of any recombinant oncolytic virus alone.
  • Oncolytic effect tumor treatment effect
  • administering to a subject a relative tumor inhibition rate achieved by a composition comprising two or more recombinant oncolytic viruses of the invention is achieved by administering to a subject alone any recombinant oncolytic virus of the composition
  • the relative tumor inhibition rate is increased by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%
  • the relative tumor inhibition rate is increased by 10% - 50%, 15%-45%, 20%-40%, 25%-35%, 20%-30%, 20%-35%, 20%-45%.
  • the range of values herein covers both the values of the endpoints and any values between the endpoints as if they were listed here.
  • Relative tumor inhibition rate (TGI) (%) (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage of the tumor volume relative to the treatment group and the control group at a specific time point.
  • T and C are the relative tumor volumes (RTV) of the treatment group and the control group at specific time points, respectively.
  • RTV animal tumor volume after treatment / control tumor volume.
  • the synergistic effect exhibited by the recombinant oncolytic virus composition of the present invention is based on a comparison of the oncolytic effect of the composition (e.g., relative tumor inhibition rate) and the oncolytic effect (e.g., relative tumor inhibition rate) of the recombinant oncolytic virus alone. As shown in the embodiment of the present application.
  • the comparison method used in the examples of the present application is also a comparison and determination method commonly used in the art to prove whether two drugs (or treatment methods) can produce synergistic effects.
  • One aspect of the invention relates to a recombinant oncolytic virus composition, characterized in that said composition comprises:
  • a first recombinant oncolytic virus comprising a first herpes simplex virus vector and a first foreign gene;
  • the first foreign gene encoding a polynucleotide sequence selected from any one of the following: a cytokine, a monoclonal antibody having a prophylactic and/or therapeutic effect on a tumor, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, an antisense RNA or a small RNA that blocks or down-regulates a tumor overexpressing protooncogene and a metabolic gene;
  • a second recombinant oncolytic virus comprising a second herpes simplex virus vector and a second foreign gene;
  • the second foreign gene encoding a gene selected from any one of the following: a cytokine, a monoclonal antibody, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, an antisense RNA or a small RNA that blocks or down-regulates a tumor over-expressed protooncogene and a metabolic gene, having a preventive and/or therapeutic effect on a tumor;
  • first herpes simplex virus vector and the second herpes simplex virus vector are identical or different from each other, and the selected first foreign gene is different from the selected second foreign gene.
  • the above recombinant oncolytic virus compositions of the invention further comprise additional one or more recombinant oncolytic viruses.
  • the above recombinant oncolytic virus composition of the present invention further comprises a third recombinant oncolytic virus, the third recombinant oncolytic virus comprising a third herpes simplex virus vector and a third foreign gene; the third exogenous group Because a gene encoding any one of the following: a cytokine, a monoclonal antibody having a prophylactic and/or therapeutic effect on tumors, a tumor antigen, a prodrug converting enzyme, a tumor suppressor protein, a proto-oncogene that blocks or down-regulates tumor overexpression And antisense RNA or small RNA of a metabolic gene;
  • the third herpes simplex virus vector is the same as or different from the first herpes simplex virus vector, the selected third foreign gene is different from the selected first foreign gene; and the third herpes simplex
  • the viral vector and the second herpes simplex virus vector are identical or different and are selected to be a third foreign gene different from the selected second foreign gene.
  • the cytokine is any one selected from the group consisting of GM-CSF, G-CSF, M-CSF, IL-1, IL-2. , IL-3, IL-4, IL-5, IL-6, IL7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23 , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ and TNF- ⁇ .
  • the monoclonal antibody having a preventive and/or therapeutic effect on a tumor is selected from any one of the following: a PD-1 monoclonal antibody, PD-L1.
  • the tumor antigen in the recombinant oncolytic virus composition of the invention described above, is a tumor-specific antigen or a tumor-associated antigen. In some embodiments, in the recombinant oncolytic virus composition of the present invention, the tumor antigen is any one selected from the group consisting of PSA, MUC1, MAGE-1, MAGE-2, MAGE-3, MAGE-12 , BAGE, GAGE and LAGE.
  • the first exogenous gene, the second exogenous gene, and the third exogenous gene each independently comprise an expression control sequence operably linked thereto .
  • the first foreign gene and the second foreign gene of the present invention preferably each independently comprise a promoter, an enhancer, and a polynucleoside One or more of the acids (including the terminator sequence).
  • the recombinant herpes simplex virus infects a host cell and expresses a gene, it is capable of transcribed and expressing a protein molecule corresponding to the foreign gene, or an antisense RNA or a small RNA molecule.
  • the promoter may be a promoter known in the art, such as at least one of a SV40 promoter, a CMV promoter, an MSV promoter, an EF1 promoter, an MMLV promoter, a U6 promoter, and an H1 promoter;
  • the enhancer may be a promoter known in the art, such as an SV40 enhancer and/or a CMV enhancer;
  • the terminator may be known in the art, such as SV40 PolyA, TK PolyA and/or BGH PolyA.
  • the type of the promoter included in each of the first foreign gene and the second foreign gene, the type of the enhancer, and the type of the polynucleotide may be the same or different.
  • the foreign gene may further comprise a foreign gene expression regulatory element other than the above elements, for example, a polyadenylation site, a Kozak sequence. , WPRE and downstream enhancer components. These are well known to those skilled in the art, and the present invention will not be described herein.
  • the first herpes simplex virus vector, the second herpes simplex virus vector, and the third herpes simplex virus vector are each independently a deletion encoding ICP34.5.
  • Herpes simplex virus of the gene, or herpes simplex virus lacking the genes encoding ICP34.5 and ICP47, and the first herpes simplex virus vector, the second herpes simplex virus vector and/or the third herpes simplex virus vector are each identical to each other or different.
  • the first herpes simplex virus vector, the second herpes simplex virus vector, and the third herpes simplex virus vector further lack at least one of genes encoding ICP6, TK, and UNG, and
  • the first herpes simplex virus vector, the second herpes simplex virus vector and/or the third herpes simplex virus vector are each identical or different from each other.
  • the insertion site of the foreign gene is any one of positions on the herpes simplex virus vector in which the gene is deleted, and the The insertion positions of a foreign gene, a second foreign gene, and/or a third foreign gene are each identical or different from each other.
  • the insertion site of the first exogenous gene, the second exogenous gene and/or the third exogenous gene is a deletion encoding the ICP34.5 gene. s position.
  • the first foreign gene is a gene encoding a cytokine
  • the second foreign gene is a gene encoding a monoclonal antibody.
  • the first foreign gene encodes GM-CSF, G-CSF, M-CSF, IL-2, IL-12 or IFN- ⁇ .
  • the gene, the second foreign gene is a gene encoding an immunological checkpoint blocking antibody.
  • the first foreign gene is a gene encoding GM-CSF, IL-2 or IL-12
  • the second foreign gene is encoded.
  • PD-1 blocking monoclonal antibody PD-L1 blocking monoclonal antibody.
  • the concentration ratio is getting larger and larger, the oncolytic effect is getting stronger and stronger, until the ratio of their ratio is 1:3, the oncolytic effect is the highest; and the recombinant oncolytic virus combination of the recombinant oncolytic virus inserted with the cytokine foreign gene is inserted.
  • the oncolytic curve achieved the strongest oncolytic effect at a ratio of 1:1 between the two recombinant oncolytic viruses.
  • Cytokines are messengers linked to the immune system. In anti-tumor therapy, they can obtain better tumor killing effects by improving the presentation of tumor antigens, directly or indirectly activating immune effector cells.
  • cytokine pathways are generally genetically pleiotropic and redundant, and many cytokines have dual effects in immune activation and inhibition.
  • high doses of cytokines often cause serious side effects in clinical applications. How to obtain potent anti-tumor effects in a low toxic dose range is a challenge for cytokine combination therapy.
  • the recombinant oncolytic virus composition expressing different cytokines and the recombinant oncolytic virus composition expressing cytokines and antibodies of the present invention it is necessary to synergize different recombinant oncolytic viruses in the composition for the treatment of cancer. In addition, it is necessary to avoid the disorder of the body's immune system caused by the inappropriate amount of cytokines, resulting in large side effects. It is necessary to carefully study and adjust the proportion of recombinant oncolytic virus in the recombinant oncolytic virus composition to achieve The regulation of the expression of the foreign gene carried by the recombinant oncolytic virus reduces the side effects caused by the overexpression of the foreign gene or the imbalance of expression of different foreign genes.
  • the mixing ratio of the first recombinant oncolytic virus to the second recombinant oncolytic virus is about 0.5:8, 1:8, 1:7 in terms of pfu. 1,6, 1:5, 1:4, 1:3, 1:2, 1:1.
  • the first foreign gene and the second foreign gene are genes encoding a cytokine, and the first foreign gene and the second foreign gene The source gene encodes a different cytokine.
  • the cytokine in the recombinant oncolytic virus composition of the invention described above, is selected from the group consisting of: GM-CSF, G-CSF, M-CSF, IL-2, IL-12, and IFN- ⁇ . In some embodiments, in the recombinant oncolytic virus composition of the invention described above, the cytokine is selected from the group consisting of: GM-CSF, IL-2, and IL-12. In some embodiments, in the recombinant oncolytic virus composition of the present invention, the first recombinant oncolytic virus and the second recombinant oncolytic virus have a mixing ratio of about 0.5:2 to 2:0.5, preferably in terms of pfu. 0.5:1-1:0.5, most preferably 1:1.
  • any numerical values of the ranges disclosed herein are not limited to the precise ranges or values, and such ranges or values are understood to include values that are close to the ranges or values.
  • the endpoint values of the various ranges, the endpoint values of the various ranges and the individual point values, and the individual point values can be combined with one another to yield one or more new ranges of values.
  • the scope should be considered as specifically disclosed herein.
  • a gene encoding ICP34.5, a gene encoding ICP47, a gene encoding ICP6, a gene encoding TK, and a gene encoding UNG are well known to those skilled in the art, and can also be found by logging in a related database.
  • the related nucleotide sequences can be queried by logging in to the GenBank database, which are conventional technical means available to those skilled in the art, and the present invention will not be repeated herein.
  • the type of the herpes simplex virus is not particularly limited, and may be herpes simplex virus type 1 or herpes simplex virus type II, but is preferably herpes simplex virus type I.
  • the source of the herpes simplex virus is also not particularly limited in the present invention, and can be obtained by conventional commercial purchase, or can be obtained by laboratory or clinical separation.
  • the recombinant oncolytic virus composition of the present invention may be mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition; it may also be packaged in a container together with a package insert containing information on the use and method of use of the recombinant oncolytic virus composition. Prepared into a kit or article.
  • the present invention relates to an oncolytic virus composition
  • an oncolytic virus composition comprising: a first recombinant oncolytic virus, the first recombinant oncolytic virus comprising a first herpes simplex virus vector and a first foreign gene; a second recombinant oncolytic virus, said The second recombinant oncolytic virus comprises a second herpes simplex virus vector and a second foreign gene, the first herpes simplex virus vector and the second herpes simplex virus vector being identical or different from each other, and the selected first foreign gene is different from The second foreign gene was selected.
  • the first herpes simplex virus vector and the second herpes simplex virus vector of the present invention carry a foreign gene
  • the first herpes simplex virus vector carries a first foreign gene
  • the foreign gene is encoded by any one selected from the following Genes: cytokines, monoclonal antibodies that prevent and/or treat tumors, tumor antigens, prodrug-converting enzymes, tumor suppressor proteins, antisense RNAs that block or down-regulate tumor-overexpressing proto-oncogenes and metabolic genes Or a small RNA
  • the second herpes simplex virus vector carries a second foreign gene, the gene encoding a gene selected from any one of the following: a cytokine, a monoclonal antibody having a prophylactic and/or therapeutic effect on a tumor, Tumor antigens, prodrug-converting enzymes, tumor suppressor proteins, antisense RNAs or small RNAs that block or down-regulate tumor over-expressed protooncogenes and metabolic genes.
  • the coding sequences for these foreign genes optionally include 5' and/or 3' transcribed but non-translated flanking sequences that are partially or fully associated with or associated with the translated coding sequence. It may optionally also include transcriptional control sequences typically associated with the transcribed sequence, such as transcription termination signals, polyadenylation sites, and downstream enhancer elements.
  • the one or more exogenous genes can be inserted into the viral vector genome by homologous recombination of the HSV strain with, for example, a plasmid vector carrying one or more exogenous genes adjacent to the HSV sequence.
  • One or more exogenous genes can be inserted into any position of the viral genome using conventional cloning techniques known to those skilled in the art (see, for example, Sambrook et al, 1989, Molecular Cloning-A laboratory manual; Cold Spring Harbor Press). The premise is that the virus can still reproduce.
  • a heterologous gene can be inserted at multiple sites within the viral genome.
  • the oncolytic virus compositions of the invention can be used in methods of treatment.
  • the oncolytic virus compositions of the invention can be used in the treatment of cancer, for example by direct intratumoral injection.
  • the oncolytic virus compositions of the invention can be used to treat any solid tumor in a mammal, preferably a human.
  • the virus of the present invention can be administered to a subject suffering from the following diseases: brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer, Prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (such as soft tissue sarcoma and osteosarcoma).
  • diseases brain glioma, melanoma, liver cancer, lung cancer, colorectal cancer, head and neck cancer, breast cancer, renal cell carcinoma, ovarian cancer, Prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, breast cancer, endometrial cancer, lymphoma, sarcoma (such as soft tissue sarcoma and osteosarcoma).
  • the oncolytic virus composition of the present invention can be treated by injecting the composition directly into the target tissue.
  • the subject is typically injected with a pharmaceutical composition comprising the oncolytic virus composition and a pharmaceutically acceptable suitable carrier or diluent, which may be administered in an amount from 1 ⁇ l to 500 ⁇ l, for example, 500 ⁇ l, 400 ⁇ l, 300 ⁇ l, 200 ⁇ l, 100 ⁇ l, 50 ⁇ l.
  • suitable carrier or diluent which may be administered in an amount from 1 ⁇ l to 500 ⁇ l, for example, 500 ⁇ l, 400 ⁇ l, 300 ⁇ l, 200 ⁇ l, 100 ⁇ l, 50 ⁇ l.
  • larger volumes of 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml can also be used.
  • the routes of administration and dosage are by way of example only.
  • the dosage can be determined according to various parameters, particularly depending on the age, weight and condition of the patient to be treated, the severity of the disease or condition, and the route of administration.
  • This preparation example is for preparing recombinant oncolytic virus (GM-CSF gene, IL-2 gene)
  • the ICP34.5 gene and the ICP47 gene of wild-type HSV-1 virus were knocked out by the method described in the patent application No. 2004100064921, the number of the patent application No. CN1283803C, and in HSV.
  • -1 virus knockout ICP34.5 gene insertion position (1) encoding mouse GM-CSF gene (5' to 3' end including: CMV promoter, GM-CSF gene (Gene ID: 12981, below) (2) BGHPolyA sequence); (2) encoding the murine IL-2 gene (5' to 3' end including: EF1 promoter, IL-2 gene (Gene ID: 16183, the same below), TKPolyA sequence); 3) Genes encoding both mouse GM-CSF and murine IL-2 double expression cassettes (5' to 3' end include: CMV promoter, GM-CSF gene, BGHPolyA sequence, EF1 promoter, IL-2 gene, TKPolyA sequence).
  • a recombinant oncolytic virus A1-1 containing a murine GM-CSF gene containing a recombinant oncolytic virus A1-2 encoding a murine IL-2 gene containing a gene encoding a murine GM-CSF and murine IL-2 double expression cassette was obtained. Compare recombinant oncolytic virus D1.
  • the GM-CSF and IL-2 genes were correctly sequenced into the herpes simplex virus vector in Beijing Sanbo Yuanzhi Company.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C under 5% CO 2 , and the cell debris was removed after harvesting, and the virus suspension obtained after high-speed centrifugation was used for the experiment.
  • This preparation example is for preparing recombinant oncolytic virus (GM-CSF gene, PD-1 monoclonal antibody gene)
  • the ICP34.5 gene and the ICP47 gene of wild-type HSV-1 virus were knocked out by the method described in the patent application No. 2004100064921, the number of the patent application No. CN1283803C, and in HSV.
  • the antibody gene, BGHPolyA sequence was obtained to contain recombinant oncolytic virus B1-2 encoding the murine PD-1 monoclonal antibody gene.
  • the mouse GM-CSF gene was inserted at the position where the ICP47 gene was knocked out (the 5' to 3' end includes: EF1 promoter, GM-CSF gene, TK PolyA sequence), and the coding was obtained. Comparison of the genes of the murine GM-CSF and murine PD-1 monoclonal antibody double expression cassettes with recombinant oncolytic virus D2.
  • the GM-CSF and PD-1 monoclonal antibody genes were correctly inserted into the herpes simplex virus vector in Beijing Sanbo Yuanzhi Company.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C under 5% CO 2 , and the cell debris was removed after harvesting, and the virus suspension obtained after high-speed centrifugation was used for the experiment.
  • This preparation example is for preparing recombinant oncolytic virus (IL-12 gene, GM-CSF gene)
  • the ICP34.5 gene and the ICP47 gene of wild-type HSV-1 virus were knocked out by the method described in the patent application No. 2004100064921, the number of the patent application No. CN1283803C, and in HSV.
  • IL-12 gene position insertion (1) encoding mouse IL-12 gene (5' to 3' end including: EF1 promoter, IL-12 gene (Gene ID: 16159, 16160 (TK PolyA sequence); (2) a gene encoding both the murine GM-CSF and the murine IL-12 double expression cassette (the 5' to the 3' end includes: CMV promoter, GM-CSF gene, BGH PolyA sequence, EF1 promoter, IL-12 gene, TK PolyA sequence).
  • a recombinant oncolytic virus C1-1 containing the murine IL-12 gene, a comparative recombinant oncolytic virus D3 containing a gene encoding the murine GM-CSF and murine IL-12 double expression cassette was obtained.
  • the IL-12 and GM-CSF genes were correctly inserted into the herpes simplex virus vector after sequencing by Beijing Qingke New Industry Co., Ltd.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C under 5% CO 2 , and the cell debris was removed after harvesting, and the virus suspension obtained after high-speed centrifugation was used for the experiment.
  • This preparation is for the preparation of recombinant oncolytic virus (PD-1 monoclonal antibody gene, IL-2 gene and IL-12 gene)
  • B1-2 was prepared in the same manner as in Preparation Example 2.
  • the mouse IL-2 gene was inserted at the position where the ICP47 gene was knocked out (the 5' end to the 3' end were sequentially included: EF1 promoter, IL -2 gene, TK PolyA sequence) and the murine IL-12 gene (5' to 3' end including: EF1 promoter, IL-12 gene, TK PolyA sequence), respectively, containing the coding mouse PD-1 monoclonal Comparison of the antibody and the murine IL-2 double expression cassette gene with recombinant oncolytic virus D4, and a comparative recombinant oncolytic virus D5 containing the gene encoding the murine PD-1 monoclonal antibody and the murine IL-12 double expression cassette.
  • the PD-1 monoclonal antibody gene, IL-2 and IL-12 genes were correctly inserted into the herpes simplex virus vector after sequencing by Beijing Qingke New Industry Co., Ltd.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C under 5% CO 2 , and the cell debris was removed after harvesting, and the virus suspension obtained after high-speed centrifugation was used for the experiment.
  • HSV-mock is an oncolytic virus that does not insert a foreign gene and knocks out the ICP34.5 gene and ICP47 gene.
  • HSV-mock is an oncolytic virus that does not insert a foreign gene and knocks out the ICP34.5 gene and ICP47 gene.
  • An animal model was constructed by inoculating a murine melanoma B16F10 cell line subcutaneously in C57BL/6 mice. The successfully modeled mice were selected, and 9 groups, 5 mice in each group, were administered according to the method of Table 3. The total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate after 14 days of administration was used as a criterion.
  • TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage of the relative tumor volume of the treatment group and the control group at a specific time point.
  • T and C are the relative tumor volumes (RTV) of the treatment group and the control group at specific time points, respectively.
  • RTV animal tumor volume after treatment / control tumor volume.
  • HSV-mock is an oncolytic virus that does not insert a foreign gene and knocks out the ICP34.5 gene and ICP47 gene.
  • An animal model was constructed by inoculating a murine liver cancer H22 cell line subcutaneously in Balb/c mice.
  • the successfully modeled mice were selected, and 8 groups, 5 mice in each group, were administered according to Table 4, and the total pfu of the virus administration was 10 6 pfu.
  • the relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • the viral vector mixture carrying the single factor was higher than the foreign gene carrying the two-factor viral vector.
  • HSV oncolytic virus inserted with GM-CSF, IL-2 or PD-1 monoclonal antibody gene can enhance tumor killing effect, and the effect of mixing two exogenous genes More significantly, it showed a good synergy between oncolytic virus, GM-CSF and IL-2 genes, and oncolytic virus, GM-CSF and PD-1 monoclonal antibody genes.
  • An animal model was constructed by inoculating a murine melanoma B16F10 cell line subcutaneously in C57BL/6 mice. Mice with a tumor volume of about 60 mm 3 were selected, and 15 groups of 10 mice each were administered in the same manner as in Table 5, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • mice An animal model was constructed by inoculating a murine liver cancer H22 cell line subcutaneously in Balb/c mice. Mice with a tumor volume of about 60 mm 3 were selected, and 16 groups of 10 mice each were administered in the same manner as in Table 6. The total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • An animal model was constructed by inoculating a mouse lung cancer Lewis cell line subcutaneously in C57BL/6 mice. Mice with a tumor volume of about 60 mm 3 were selected, and 19 groups of 10 mice each were administered according to Table 7, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • mice Animal models were constructed by inoculating a murine sarcoma S180 cell line subcutaneously in Balb/c mice. Mice with a tumor volume of about 60 mm 3 were selected, and 19 groups of 10 mice each were administered in the same manner as in Table 8, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • the present patent further implements the following test examples to increase the tumor volume to be treated to about 200 mm3 to distinguish the therapeutic effects of each combination.
  • An animal model was constructed by inoculating a murine melanoma B16F10 cell line subcutaneously in C57BL/6 mice. Mice with a tumor volume of about 200 mm 3 were selected, and 15 groups of 10 mice each were administered in the same manner as in Table 9, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • mice An animal model was constructed by inoculating a murine liver cancer H22 cell line subcutaneously in Balb/c mice. Mice with a tumor volume of about 200 mm 3 were selected, and 15 groups of 10 mice each were administered in the same manner as in Table 10, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • mice with a tumor volume of about 200 mm 3 were selected, and 15 groups of 10 mice each were administered in the same manner as in Table 10, and the total pfu of the virus administration was 10 6 pfu.
  • the relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • mice Animal models were constructed by inoculating a murine sarcoma S180 cell line subcutaneously in Balb/c mice. Mice with a tumor volume of about 200 mm 3 were selected, and 15 groups of 10 mice each were administered in the same manner as in Table 10, and the total pfu of the virus administration was 10 6 pfu. The relative tumor inhibition rate was used as a criterion.
  • the relative tumor inhibition rate was calculated in accordance with Test Example 3.
  • the combination of viral vectors has a better tumor suppressing effect than the single virus group and the two-factor group, and A1-2: B1-2 and C1-1: 1:1-2 of B1-2
  • the combination group has better synergistic anti-tumor effect than other combinations.

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Abstract

提供了重组溶瘤病毒组合物，所述组合物包含第一重组溶瘤病毒和第二重组溶瘤病毒。第一和第二溶瘤病毒都包括单纯疱疹病毒载体和外源基因，所述外源基因编码选自细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制多肽、反义RNA或小RNA中的一种，其中，第一、第二重组溶瘤病毒中的外源基因不同。还提供了所述溶瘤病毒组合物在制备用于治疗肿瘤的药物中的应用。

Description

重组溶瘤病毒组合物及其用途 技术领域
本发明涉及基因工程领域，具体涉及重组溶瘤病毒组合物及其在制备用于治疗肿瘤的药物中的应用。
背景技术
一、溶瘤病毒在肿瘤治疗上的应用
传统肿瘤治疗包括手术、放疗和化疗，治疗有效率低、复发率高，而肿瘤基因免疫治疗是近年来进展迅速的一种抗肿瘤方法，潜力巨大。在《科学》杂志评选的2013年年度10大科学突破排行榜中，肿瘤免疫治疗高居榜首。其中，以HSV-1载体为代表的溶瘤病毒在肿瘤免疫治疗中发挥了巨大作用。
溶瘤病毒是指能选择性感染肿瘤细胞并在靶细胞内复制，最终导致肿瘤细胞裂解和死亡的一类病毒。这类病毒依靠其本身的特异性在肿瘤细胞中复制来裂解肿瘤细胞，细胞裂解后释放出来的病毒又可以进一步感染周围的肿瘤细胞，同时对正常细胞和组织则没有破坏作用，或影响较小。溶瘤病毒具有多重抗肿瘤作用机制，包括1、直接裂解肿瘤细胞；2、破坏肿瘤血管；3、病毒复制表达的病毒蛋白具有直接细胞毒性作用；4、抗肿瘤免疫反应；5、增强肿瘤细胞对放化疗的敏感性；6、表达插入的外源治疗基因。目前用于溶瘤病毒改造的病毒包括单纯疱疹病毒、腺病毒和牛痘病毒等，其中I型单纯疱疹病毒(HSV-1)是最有应用前景的病毒载体。
HSV-1是一种大的双链DNA病毒，基因组大小152kb，编码80多个基因，其中γ34.5基因产物能够阻断由于病毒侵染引起的宿主蛋白质合成，删 除γ34.5使得病毒载体在正常细胞中无法复制，同时γ34.5基因产物是神经毒性因子。HSV-1中的ICP6基因编码表达病毒的核糖核苷酸还原酶的大亚基，是HSV-1在非分裂细胞中合成病毒DNA所必需的酶。TK基因表达的胸苷激酶参与磷酸化脱氧核苷的合成，同时磷酸化脱氧胞苷和核苷类似物，产生病毒DNA合成前体。UNG基因表达尿嘧啶糖基化酶可以从DNA上剪切尿嘧啶从而防止突变并为碱基剪切修复通路传递信号。ICP47基因产物阻断病毒侵染时的抗原呈递(Shen Y,Nemunaitis J.Herpes simplex virus 1(HSV-1)for cancer treatment.Cancer Gene Therapy 2006；13:975-992；Peters C,Rabkin S D.Designing Herpes Viruses as Oncolytics.Molecular Therapy Oncolytics,2015,2:15010)。
二、溶瘤病毒的联合治疗
肿瘤细胞具有多种免疫逃逸机制，任何单一治疗策略无法完全消灭肿瘤细胞，因而多种治疗策略的联合应用将是未来肿瘤治疗的发展趋势。
溶瘤病毒与其他治疗方法或药物联合治疗通常采取两种策略。其一是溶瘤病毒与其他治疗方法或药物采用统一的治疗方案分别用药。其二是溶瘤病毒携带有利于肿瘤治疗的基因。
然而，现有的治疗方法均不能够对肿瘤进行持续高效的杀伤。
发明内容
发明概述
本发明的目的是为了克服现有技术存在的上述问题，提供一种重组溶瘤病毒组合物，该溶瘤病毒组合物既能够保留溶瘤病毒的高效肿瘤杀伤作用，同时通过调整重组溶瘤病毒组合物中各成分的量，使得各组分表达的外源基因之间以及表达的外源基因与溶瘤病毒之间的协同抗肿瘤效果最大化，并且 降低治疗的副作用。
具体地，本发明涉及：
1、一种重组溶瘤病毒组合物，其特征在于，所述组合物含有：
第一重组溶瘤病毒，所述第一重组溶瘤病毒包括第一单纯疱疹病毒载体和第一外源基因；所述第一外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
第二重组溶瘤病毒，所述第二重组溶瘤病毒包括第二单纯疱疹病毒载体和第二外源基因；所述第二外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
其中，所述第一单纯疱疹病毒载体和第二单纯疱疹病毒载体彼此相同或不同，被选定的第一外源基因不同于被选定的第二外源基因。
2、根据项1所述的重组溶瘤病毒组合物，其中，所述组合物还含有第三重组溶瘤病毒，所述第三重组溶瘤病毒包括第三单纯疱疹病毒载体和第三外源基因；所述第三外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
其中，所述第三单纯疱疹病毒载体和第一单纯疱疹病毒载体相同或不同，被选定的第三外源基因不同于被选定的第一外源基因；并且，所述第三 单纯疱疹病毒载体和第二单纯疱疹病毒载体相同或不同，被选定为第三外源基因不同于被选定的第二外源基因。
3.项1或2的重组溶瘤病毒组合物，其中所述细胞因子为选自如下的任一种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
4.项1或2的重组溶瘤病毒组合物，其中所述具有预防和/或治疗肿瘤作用的单克隆抗体选自如下的任一种：PD-1单克隆抗体、PD-L1单克隆抗体、PD-L2单克隆抗体、CTLA-4单克隆抗体、CD80单克隆抗体、CD28单克隆抗体、CD137单克隆抗体、CD137L单克隆抗体、OX40单克隆抗体、OX40L单克隆抗体、CD27单克隆抗体、CD70单克隆抗体、CD40单克隆抗体、CD40L单克隆抗体、LAG-3单克隆抗体和TIM-3单克隆抗体。
5.项1或2的重组溶瘤病毒组合物，其中所述肿瘤抗原为肿瘤特异性抗原或肿瘤相关抗原。
6.项5的重组溶瘤病毒组合物，其中所述肿瘤抗原为选自如下的任一种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
7、根据项1-6任一项所述的重组溶瘤病毒组合物，其中所述第一外源基因、第二外源基因、第三外源基因还各自独立地包含与其可操作连接的表达调控序列，所述表达调控序列包括启动子、增强子和多聚核苷酸中的至少一种。
8、根据项1-7任一项所述的重组溶瘤病毒组合物，其中所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体各自独立地为 缺失编码ICP34.5的基因的单纯疱疹病毒，或缺失编码ICP34.5和ICP47的基因的单纯疱疹病毒，并且所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体和/或第三单纯疱疹病毒载体各自彼此相同或不同。
9、根据项8的重组溶瘤病毒组合物，其中所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体还缺失编码ICP6、TK和UNG的基因中的至少一种。
10.根据项1-9任一项所述的重组溶瘤病毒组合物，其中所述外源基因的插入位点为所述单纯疱疹病毒载体上缺失编码基因的位置中的任一处，并且所述第一外源基因、第二外源基因和/或第三外源基因的插入位置各自彼此相同或不同。
11.根据项10所述的重组溶瘤病毒组合物，其中所述第一外源基因、第二外源基因和/或第三外源基因的插入位点为缺失编码ICP34.5基因的位置。
12.根据1-11任一项所述的重组溶瘤病毒组合物，其中所述单纯疱疹病毒为1型单纯疱疹病毒(HSV-1)。
13、根据项1-12任一项所述的溶瘤病毒组合物，其中所述第一外源基因为编码细胞因子的基因，所述第二外源基因为编码单克隆抗体的基因。
14.项13所述的溶瘤病毒组合物，其中所述第一外源基因为编码GM-CSF、G-CSF、M-CSF、IL-2、IL-12或IFN-γ的基因，所述第二外源基因为编码免疫检查点阻断性抗体的基因。
15.项14所述的溶瘤病毒组合物，其中所述第一外源基因为编码GM-CSF、IL-2或IL-12的基因，所述第二外源基因为编码PD-1阻断性单克隆抗体、PD-L1阻断性单克隆抗体。
16.根据项11-15任一项所述的溶瘤病毒组合物，其中，以pfu计，所述 第一重组溶瘤病毒和第二重组溶瘤病毒混合比为0.5:8、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1。
17.根据项1-11任一项所述的溶瘤病毒组合物，其中所述第一外源基因和第二外源基因为编码细胞因子的基因，并且所述第一外源基因和第二外源基因编码的细胞因子不同。
18.项17所述的溶瘤病毒组合物，其中所述的细胞因子选自：GM-CSF、G-CSF、M-CSF、IL-2、IL-12和IFN-γ。
19.项18所述的溶瘤病毒组合物，其中所述的细胞因子选自：GM-CSF、IL-2和IL-12。
20.根据项17-19任一项所述的溶瘤病毒组合物，其中，以pfu计，所述第一重组溶瘤病毒和第二重组溶瘤病毒混合比为0.5:2-2:0.5，优选0.5:1-1:0.5，最优选1:1。
21.项1-20中任意一项所述的重组溶瘤病毒组合物在制备用于治疗肿瘤的药物中的用途。
22.根据项21所述的用途，其中所述肿瘤选自脑神经胶质瘤、黑色素瘤、肝癌、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、前列腺癌、胃癌、淋巴瘤、胰腺癌、膀胱癌、乳腺癌、子宫内膜癌、淋巴瘤、肉瘤(例如软组织肉瘤和骨肉瘤)中的至少一种。
23.一种药物组合物，包含项1-20中任意一项所述的重组溶瘤病毒组合物和药学可接受的载体。
24.一种制品或试剂盒，包含内装有项23的药物组合物的小瓶和包装插页，所述包装插页记载有所述药物组合物使用的相关信息。
25.一种治疗肿瘤的方法，包括给药受试者有效量的项1-20中任意一项 所述的重组溶瘤病毒组合物或项23的药物组合物。
26.根据权利要25所述的方法，其中，所述肿瘤选自脑神经胶质瘤、黑色素瘤、肝癌、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、前列腺癌、胃癌、淋巴瘤、胰腺癌、膀胱癌、乳腺癌、子宫内膜癌、淋巴瘤、肉瘤(例如软组织肉瘤和骨肉瘤)中的至少一种。
具体实施方式
1.定义
“溶瘤病毒”是指一类感染和杀死癌细胞的病毒，该病毒感染癌细胞后通过溶瘤作用破坏受感染的癌细胞，同时释放出新的感染性病毒颗粒或病毒体，破坏剩余的癌细胞。此类病毒因其溶瘤作用而得名。
“单纯疱疹病毒(HSV)”因其易于操作并且在其天然状态下相对无害，因此是最先选择用于选择性攻击癌细胞的病毒(溶瘤病毒)之一。单纯疱疹病毒1型(HSV-1)突变体1716缺乏ICP34.5基因的两个拷贝，因此不再能够在终末分化和非分裂细胞中复制，但会在非常有效的情况下感染并导致癌细胞裂解，这已被证明是一种有效的肿瘤靶向策略。多种基于HSV的溶瘤病毒已开发出来并正在进行临床试验。
当本发明的病毒为单纯疱疹病毒时，所述病毒可以来源于例如HSV1毒株或HSV2毒株或者它们的衍生毒株，优选是HSV1。衍生毒株包括含有来自HSV1毒株和HSV2毒株的DNA的型间(inter-type)重组体。衍生毒株与HSV1基因组或HSV2基因组的序列同源性优选为至少70％，更优选至少80％，甚至更优选至少90％或95％。更优选的是，衍生毒株与HSV1基因组或HSV2基因组的序列同一性为至少70％，更优选至少80％同一性，甚至更 优选至少90％、9％或98％同一性。
“单纯疱疹病毒载体”指携带外源基因的单纯疱疹病毒。
空斑形成单位(plaque forming unit)，缩写pfu，指在单层培养的动物细胞上形成一个空斑(噬斑)的病毒数。
“免疫检查点阻断(抑制)性抗体”或“免疫检查点阻断(抑制)性单克隆抗体”指抑制或阻断抑制性免疫检查点分子的单克隆抗体。免疫检查点是免疫系统的监管者，它们的作用体现在阻止免疫系统不加选择地攻击细胞，从而对自身耐受至关重要。免疫检查点分抑制性检查点分子和刺激性检查点分子。抑制性检查点分子包括但不限于：A2AR、B7-H3(CD276)、B7-H4、BTLA(CD272)、CTLA-4(CD152)、IDO、KIR、LAG3、NOX2、PD1、TIM3、VISTA；刺激性检查点分子包括但不限于：CD27、CD40、OX40、GITR、CD137、CD28、ICOS。抑制性检查点分子是癌症免疫疗法的靶标，因为它们可能用于多种类型的癌症。
本发明所使用的术语“协同”是指本发明包含二种或二种以上的重组溶瘤病毒的组合物所呈现的溶瘤效果(肿瘤治疗效果)大于单独分开使用任一种重组溶瘤病毒的溶瘤效果(肿瘤治疗效果)。例如，给予受试者本发明包含二种或二种以上的重组溶瘤病毒的组合物所实现的相对肿瘤抑制率与单独给予受试者所述组合物中的任一重组溶瘤病毒所实现的相对肿瘤抑制率相比较，相对肿瘤抑制率增加50％、45％、40％、35％、30％、25％、20％、15％或10％，例如，相对肿瘤抑制率增加10％-50％、15％-45％、20％-40％、25％-35％、20％-30％、20％-35％、20％-45％。此处的数值范围既涵盖端点的数值，又涵盖端点之间的任何数值，就如同这些数值在此处一一列出一样。相对肿瘤抑制率(TGI)(％)＝(1-T/C)×100％。T/C％为相对肿瘤增殖 率，即特定时间点治疗组和对照组相对肿瘤体积的百分比值。T和C分别为治疗组和对照组在特定时间点的相对肿瘤体积(RTV)。RTV＝治疗后动物瘤体积/对照组瘤体积。本发明重组溶瘤病毒组合物所呈现的协同效果基于所述组合物的溶瘤效果(例如相对肿瘤抑制率)和单独的重组溶瘤病毒的溶瘤效果(例如相对肿瘤抑制率)的比较所得，如本申请实施例所示。本申请实施例中使用的比较方法也是本领域常用的、证明二种药物(或治疗方法)能否产生协同作用常用的比较和判定方法。
2.本发明重组溶瘤病毒组合物
本发明一个方面涉及重组溶瘤病毒组合物，其特征在于，所述组合物含有：
第一重组溶瘤病毒，所述第一重组溶瘤病毒包括第一单纯疱疹病毒载体和第一外源基因；所述第一外源基因为编码选自如下任一种的多核苷酸序列：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
第二重组溶瘤病毒，所述第二重组溶瘤病毒包括第二单纯疱疹病毒载体和第二外源基因；所述第二外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
其中，所述第一单纯疱疹病毒载体和第二单纯疱疹病毒载体彼此相同或不同，被选定的第一外源基因不同于被选定的第二外源基因。
在一些实施方案中，本发明的上述重组溶瘤病毒组合物还含有另外的一 种或多种重组溶瘤病毒。例如，本发明的上述重组溶瘤病毒组合物还含有第三重组溶瘤病毒，所述第三重组溶瘤病毒包括第三单纯疱疹病毒载体和第三外源基因；所述第三外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
其中，所述第三单纯疱疹病毒载体和第一单纯疱疹病毒载体相同或不同，被选定的第三外源基因不同于被选定的第一外源基因；并且，所述第三单纯疱疹病毒载体和第二单纯疱疹病毒载体相同或不同，被选定为第三外源基因不同于被选定的第二外源基因。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述细胞因子为选自如下的任一种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述具有预防和/或治疗肿瘤作用的单克隆抗体选自如下的任一种：PD-1单克隆抗体、PD-L1单克隆抗体、PD-L2单克隆抗体、CTLA-4单克隆抗体、CD80单克隆抗体、CD28单克隆抗体、CD137单克隆抗体、CD137L单克隆抗体、OX40单克隆抗体、OX40L单克隆抗体、CD27单克隆抗体、CD70单克隆抗体、CD40单克隆抗体、CD40L单克隆抗体、LAG-3单克隆抗体和TIM-3单克隆抗体。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述肿瘤抗原为肿瘤特异性抗原或肿瘤相关抗原。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述肿瘤抗原为选自如下的任一种：PSA、MUC1、 MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因、第二外源基因、第三外源基因还各自独立地包含与其可操作连接的表达调控序列。
根据本发明，为了能够表达插入到单纯疱疹病毒载体中的所述外源基因，本发明的第一外源基因和第二外源基因优选各自独立地包含启动子、增强子、多聚核苷酸(包括终止子序列)中的一个或多个。在优选的情况下，在所述重组单纯疱疹病毒感染宿主细胞并进行自身基因的表达时，能够转录并表达出外源基因对应的蛋白分子、或者反义RNA或小RNA分子。其中，所述启动子可以为本领域已知的启动子，例如SV40启动子、CMV启动子、MSV启动子、EF1启动子、MMLV启动子、U6启动子、H1启动子中的至少一种；所述增强子可以为本领域已知的启动子，例如SV40增强子和/或CMV增强子；所述终止子可以为本领域已知的，例如SV40 PolyA、TK PolyA和/或BGH PolyA。所述第一外源基因和第二外源基因各自包括的启动子的种类、增强子的种类、多聚核苷酸的种类可以相同，也可以不同。
本领域技术人员可以理解，为有助于外源基因的表达，所述外源基因还可以包含除上述元件之外其它的外源基因表达调控元件，例如，聚腺苷酸化位点、Kozak序列、WPRE和下游增强子元件。这些均为本领域技术人员所公知，本发明在此不再赘述。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体各自独立地为缺失编码ICP34.5的基因的单纯疱疹病毒，或缺失编码ICP34.5和ICP47的基因的单纯疱疹病毒，并且所述第一单纯疱疹病毒载体、第二单纯疱疹病 毒载体和/或第三单纯疱疹病毒载体各自彼此相同或不同。
在本发明的一些实施方案中，所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体还缺失编码ICP6、TK和UNG的基因中的至少一种，并且，所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体和/或第三单纯疱疹病毒载体各自彼此相同或不同。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述外源基因的插入位点为所述单纯疱疹病毒载体上缺失编码基因的位置中的任一处，并且所述第一外源基因、第二外源基因和/或第三外源基因的插入位置各自彼此相同或不同。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因、第二外源基因和/或第三外源基因的插入位点为缺失编码ICP34.5基因的位置。
在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因为编码细胞因子的基因，所述第二外源基因为编码单克隆抗体的基因。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因为编码GM-CSF、G-CSF、M-CSF、IL-2、IL-12或IFN-γ的基因，所述第二外源基因为编码免疫检查点阻断性抗体的基因。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因为编码GM-CSF、IL-2或IL-12的基因，所述第二外源基因为编码PD-1阻断性单克隆抗体、PD-L1阻断性单克隆抗体。本发明的发明人发现，重组溶瘤病毒组合物中，外源基因的选择所产生的肿瘤治疗效果是有显著差异的；而且表达不同外源基因的溶瘤病毒发挥作用的浓度也是不同的。例如，对于插入了细胞因子外源基因的重组溶瘤病毒和插入了免疫检查点阻断性抗体外源基因的重组溶瘤病毒的重组溶瘤病毒组合物而言，随着前者和后者的浓度比越来越大，溶 瘤作用越来越强，直至他们的比例在1:3时溶瘤作用达到最高；而对于插入了细胞因子外源基因的重组溶瘤病毒的重组溶瘤病毒组合物而言，溶瘤曲线在二种重组溶瘤病毒之间的比例在1:1时溶瘤效果达到最强。
细胞因子是免疫系统联系的信使，在抗肿瘤治疗中，其可以通过提高肿瘤抗原的呈递、直接或间接激活免疫效应细胞等功能获得较好的肿瘤杀伤效果。但是，细胞因子通路普遍具有基因多效性和冗余性，许多细胞因子在免疫激活和抑制过程中具有双重功效。而且，高剂量的细胞因子临床应用上通常带来严重的副反应。如何在低毒性剂量范围内获得强力的抗肿瘤效果是细胞因子组合疗法的挑战。对于本发明表达不同细胞因子的重组溶瘤病毒组合物和表达细胞因子和抗体的重组溶瘤病毒组合物而言，既要使该组合物中不同的重组溶瘤病毒对癌症的治疗起到协同作用，又要避免因细胞因子的量的不适当引发机体免疫系统紊乱，产生较大的副作用，需要对重组溶瘤病毒组合物中的重组溶瘤病毒的比例进行仔细研究和调整，从而实现对重组溶瘤病毒携带的外源基因表达的调整，降低由于外源基因的过表达或不同外源基因表达不均衡所带来的副作用。选择合适的细胞因子和抗体，或者选择不同的细胞因子，并将它们的表达基因同时插入溶瘤病毒基因组中也能对肿瘤的杀伤发挥协同作用，正如PCT申请PCT/CN2018/091530所记载的，但是将不同的外源基因同时插入溶瘤病毒基因组中则无法对不同外源基因的表达量实现调控，而通过将携带单一外源基因的溶瘤病毒按不同比例进行混合，则既可以实现对外源基因的表达比例进行调控，又可以发挥最佳的肿瘤杀伤作用，同时减少了副作用。
在一些实施方案中，本发明重组溶瘤病毒组合物中，以pfu计，所述第一重组溶瘤病毒和第二重组溶瘤病毒混合比为约0.5:8、1:8、1:7、1:6、1:5、 1:4、1:3、1:2、1:1。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述第一外源基因和第二外源基因为编码细胞因子的基因，并且所述第一外源基因和第二外源基因编码的细胞因子不同。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述的细胞因子选自：GM-CSF、G-CSF、M-CSF、IL-2、IL-12和IFN-γ。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，所述的细胞因子选自：GM-CSF、IL-2和IL-12。在一些实施方案中，本发明上述的重组溶瘤病毒组合物中，以pfu计，所述第一重组溶瘤病毒和第二重组溶瘤病毒混合比为约0.5:2-2:0.5，优选0.5:1-1:0.5，最优选1:1。
在本文中所披露的范围的端点和任何数值都不限于该精确的范围或数值，这些范围或数值应当理解为包含接近这些范围或数值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在本发明中，编码ICP34.5的基因、编码ICP47的基因、编码ICP6的基因、编码TK的基因和编码UNG的基因均为本领域技术人员所公知，并且也能够通过登录相关的数据库查到，例如，可以通过登录GenBank数据库查询到相关的核苷酸序列，这些均是本领域技术人员具有的常规技术手段，本发明在此不再赘述。
在本发明中，对所述单纯疱疹病毒的种类没有特别的限定，可以为I型单纯疱疹病毒或II型单纯疱疹病毒，但优选为I型单纯疱疹病毒。本发明对所述单纯疱疹病毒的来源也没有特别的限定，可以通过常规的商购获得，也可以通过实验室或临床自行分离获得。
本发明的重组溶瘤病毒组合物可以与药学可接受的载体混合制备药物组合物；也可以包装在容器中，和记载有该重组溶瘤病毒组合物用途和使用方法等相关信息的包装插页一起制备成试剂盒或制品。
3.外源基因、重组单纯疱疹病毒
本发明涉及溶瘤病毒组合物，含有：第一重组溶瘤病毒，所述第一重组溶瘤病毒包括第一单纯疱疹病毒载体和第一外源基因；第二重组溶瘤病毒，所述第二重组溶瘤病毒包括第二单纯疱疹病毒载体和第二外源基因，所述第一单纯疱疹病毒载体和第二单纯疱疹病毒载体彼此相同或不同，被选定的第一外源基因不同于被选定的第二外源基因。本发明的所述第一单纯疱疹病毒载体和第二单纯疱疹病毒载体携带外源基因，例如所述第一单纯疱疹病毒载体携带第一外源基因，该外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；所述第二单纯疱疹病毒载体携带第二外源基因，该外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA。这些外源基因的编码序列任选包括部分或全部与被翻译的编码序列天然相连或者与其相关的5’和/或3’转录但非翻译的侧翼序列。可以任选还包括通常与转录序列相关的转录控制序列，例如转录终止信号、聚腺苷酸化位点和下游增强子元件。
通过HSV毒株与例如携带邻接HSV序列的一种或多种外源基因的质粒载体进行同源重组，可以将所述一种或多种外源基因插入到病毒载体基因组中。采用本领域技术人员已知的常规克隆技术(参见例如Sambrook等，1989， Molecular Cloning-A laboratory manual；Cold Spring Harbor Press)可以将一种或多种外源基因插入到病毒基因组的任何位置上，前提是所述病毒仍可以繁殖。可以在所述病毒基因组内的多个位点上插入异源基因。
4.治疗应用
本发明的溶瘤病毒组合物可以用于治疗方法中。具体地说，本发明的溶瘤病毒组合物可以用于癌症的治疗中，例如通过直接肿瘤内注射。本发明的溶瘤病毒组合物可以用来治疗哺乳动物、优选人体内的任何实体瘤。例如，可以将本发明的病毒给予患有以下疾病的受试者：脑神经胶质瘤、黑色素瘤、肝癌、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、前列腺癌、胃癌、淋巴瘤、胰腺癌、膀胱癌、乳腺癌、子宫内膜癌、淋巴瘤、肉瘤(例如软组织肉瘤和骨肉瘤)。
5.给药方案
本发明的溶瘤病毒组合物可以采用将所述组合物直接注射到所述靶组织进行治疗。当注射时，通常向受试者注射由所述溶瘤病毒组合物和药学上可接受的合适载体或稀释剂组成的药用组合物，注射用量可以为1μl-500μl、例如，500μl、400μl、300μl，200μl、100μl、50μl。然而，根据所述肿瘤和接种部位，也可以使用10ml、8ml、6ml、5ml、4ml、3ml的较大体积。因为本领域技术人员可以根据具体情况容易地确定最佳给药途径和剂量，所以所述给药途径和剂量仅作为举例说明。所述剂量可以根据各种参数、尤其根据待治疗患者的年龄、体重和病症、疾病或病症的严重程度以及给药途径来确定。
实施例
制备例1
本制备例用于制备重组溶瘤病毒(GM-CSF基因、IL-2基因)
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒(其基因序列的GenBank号：NC_001806，下同)的ICP34.5基因和ICP47基因敲除，并在HSV-1病毒的敲除ICP34.5基因的位置分别插入：(1)编码鼠GM-CSF基因(5’端至3’端依次包括：CMV启动子、GM-CSF基因(Gene ID：12981，以下同)、BGHPolyA序列)；(2)编码鼠IL-2基因(5’端至3’端依次包括：EF1启动子、IL-2基因(Gene ID：16183，以下同)、TKPolyA序列)；(3)同时编码鼠GM-CSF和鼠IL-2双表达框的基因(5’端至3’端依次包括：CMV启动子、GM-CSF基因、BGHPolyA序列、EF1启动子、IL-2基因、TKPolyA序列)。得到含有编码鼠GM-CSF基因的重组溶瘤病毒A1-1，含有编码鼠IL-2基因的重组溶瘤病毒A1-2，含有编码鼠GM-CSF和鼠IL-2双表达框的基因的对比重组溶瘤病毒D1。
在北京三博远志公司测序鉴定GM-CSF和IL-2基因均正确插入到单纯疱疹病毒载体中。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2条件下增殖，收获后去除细胞碎片，经过高速离心纯化后得到的病毒悬液用于实验。
制备例2
本制备例用于制备重组溶瘤病毒(GM-CSF基因、PD-1单克隆抗体基因)
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒(其基因序列的GenBank号：NC_001806，下同)的ICP34.5基因和ICP47基因敲除，并在HSV-1病毒的敲除ICP34.5基因的位置插入编码鼠PD-1单克隆抗体(J43,BioXCell，以下同)基因(5’端至3’端依次包括：CMV启动子、PD-1单克隆抗体基因、BGHPolyA序列)，得到含有编码鼠PD-1单克隆抗体基因的重组溶瘤病毒B1-2。在B1-2的基础上， 在敲除ICP47基因的位置插入编码鼠GM-CSF基因(5’端至3’端依次包括：EF1启动子、GM-CSF基因、TK PolyA序列)，得到含有编码鼠GM-CSF和鼠PD-1单克隆抗体双表达框的基因的对比重组溶瘤病毒D2。
在北京三博远志公司测序鉴定GM-CSF和PD-1单克隆抗体基因均正确插入到单纯疱疹病毒载体中。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2条件下增殖，收获后去除细胞碎片，经过高速离心纯化后得到的病毒悬液用于实验。
制备例3
本制备例用于制备重组溶瘤病毒(IL-12基因、GM-CSF基因)
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒(其基因序列的GenBank号：NC_001806，下同)的ICP34.5基因和ICP47基因敲除，并在HSV-1病毒的敲除ICP34.5基因的位置分别插入：(1)编码鼠IL-12基因(5’端至3’端依次包括：EF1启动子、IL-12基因(Gene ID：16159,16160)、TK PolyA序列)；(2)同时编码鼠GM-CSF和鼠IL-12双表达框的基因(5’端至3’端依次包括：CMV启动子、GM-CSF基因、BGH PolyA序列、EF1启动子、IL-12基因、TK PolyA序列)。得到含有编码鼠IL-12基因的重组溶瘤病毒C1-1，含有编码鼠GM-CSF和鼠IL-12双表达框的基因的对比重组溶瘤病毒D3。
在北京擎科新业公司测序鉴定IL-12和GM-CSF基因均正确插入到单纯疱疹病毒载体中。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2条件下增殖，收获后去除细胞碎片，经过高速离心纯化后得到的病毒悬液用于实验。
制备例4
本制备例用于制备重组溶瘤病毒(PD-1单克隆抗体基因、IL-2基因和IL-12基因)
按照制备例2的方式制备B1-2，在B1-2的基础上，在敲除ICP47基因的位置分别插入编码鼠IL-2基因(5’端至3’端依次包括：EF1启动子、IL-2基因、TK PolyA序列)和编码鼠IL-12基因(5’端至3’端依次包括：EF1启动子、IL-12基因、TK PolyA序列)，分别得到含有编码鼠PD-1单克隆抗体和鼠IL-2双表达框的基因的对比重组溶瘤病毒D4，以及含有编码鼠PD-1单克隆抗体和鼠IL-12双表达框的基因的对比重组溶瘤病毒D5。
在北京擎科新业公司测序鉴定PD-1单克隆抗体基因、IL-2和IL-12基因均正确插入到单纯疱疹病毒载体中。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2条件下增殖，收获后去除细胞碎片，经过高速离心纯化后得到的病毒悬液用于实验。
测试例1
细胞实验
将HepG2细胞和A549细胞分别接种到六孔板中，37℃、5％CO 2下增殖培养，贴壁后按照下表1加入相应的相同量的重组溶瘤病毒(感染复数MOI＝0.1)，分别感染HepG2细胞和A549细胞，37℃、5％CO 2培养48h后ELISA测定细胞培养上清中GM-CSF和IL-2的浓度。结果见表1。
表1
Figure PCTCN2019078999-appb-000001
HSV-mock为不插入外源基因且敲除ICP34.5基因、ICP47基因的溶瘤病毒
测试例2
细胞实验
将Vero细胞分别接种到六孔板中，37℃、5％CO 2下增殖培养，贴壁后按照下表2加入相应的相同量的重组溶瘤病毒(感染复数MOI＝0.1)，分别感染Vero细胞，37℃、5％CO 2培养48h后ELISA测定细胞培养上清中GM-CSF和PD-1单克隆抗体的浓度。结果见表2。
表2
Figure PCTCN2019078999-appb-000002
HSV-mock为不插入外源基因且敲除ICP34.5基因、ICP47基因的溶瘤病毒
测试例3
动物实验
在C57BL/6小鼠皮下接种鼠源黑色素瘤B16F10细胞株构建动物模型。选取建模成功的小鼠，设置9组，每组5只小鼠，按照表3的方式进行给药，病毒给药总pfu均为10 6pfu。采用给药14天后的相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增殖率，即特定时间点治疗组和对照组相对肿瘤体积的百分比值。T和C分别为治疗组和对照组在特定时间点的相对肿瘤体积(RTV)。RTV＝治疗后动物瘤体积/对照组瘤体积。
表3
Figure PCTCN2019078999-appb-000003
Figure PCTCN2019078999-appb-000004
HSV-mock为不插入外源基因且敲除ICP34.5基因、ICP47基因的溶瘤病毒
测试例4
动物实验
在Balb/c小鼠皮下接种鼠源肝癌H22细胞株构建动物模型。选取建模成功的小鼠，设置8组，每组5只小鼠，按照表4的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
表4
Figure PCTCN2019078999-appb-000005
从表1和表2可以看出，在不同的细胞系中，携带单因子的病毒载体混合物比携带双因子的病毒载体的外源基因表达量均要高。
从表3-表4可以看出，插入GM-CSF、IL-2或PD-1单克隆抗体基因的HSV溶瘤病毒均能够增强肿瘤杀伤作用，而将两种外源基因混合后产生的效果更为显著，表明溶瘤病毒、GM-CSF和IL-2基因之间，以及溶瘤病毒、 GM-CSF和PD-1单克隆抗体基因之间具有良好的协同作用。
测试例5
动物实验
在C57BL/6小鼠皮下接种鼠源黑色素瘤B16F10细胞株构建动物模型。选取肿瘤体积约60mm 3的小鼠，设置15组，每组10只小鼠，按照表5的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
表5：
Figure PCTCN2019078999-appb-000006
测试例6
动物实验
在Balb/c小鼠皮下接种鼠源肝癌H22细胞株构建动物模型。选取肿瘤体积约60mm 3的小鼠，设置16组，每组10只小鼠，按照表6的方式进行给 药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000007
测试例7
动物实验
在C57BL/6小鼠皮下接种鼠源肺癌Lewis细胞株构建动物模型。选取肿瘤体积约60mm 3的小鼠，设置19组，每组10只小鼠，按照表7的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000008
Figure PCTCN2019078999-appb-000009
测试例8
动物实验
在Balb/c小鼠皮下接种鼠源肉瘤S180细胞株构建动物模型。选取肿瘤体积约60mm 3的小鼠，设置19组，每组10只小鼠，按照表8的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000010
Figure PCTCN2019078999-appb-000011
从以上测试例可以看出A1-1：B1-2、A1-2：B1-2、C1-1：B1-2比例为1:3时肿瘤抑制效果最佳。A1-1：A1-2、A1-1：C1-1比例为1:1时肿瘤抑制效果最佳。为了确定肿瘤抑制的最佳组合，本专利进一步实施以下测试例，将待治疗肿瘤体积提高至约200mm3，以区分各组合的治疗效果。
测试例9
动物实验
在C57BL/6小鼠皮下接种鼠源黑色素瘤B16F10细胞株构建动物模型。选取肿瘤体积约200mm 3的小鼠，设置15组，每组10只小鼠，按照表9的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000012
Figure PCTCN2019078999-appb-000013
测试例10
动物实验
在Balb/c小鼠皮下接种鼠源肝癌H22细胞株构建动物模型。选取肿瘤体积约200mm 3的小鼠，设置15组，每组10只小鼠，按照表10的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000014
Figure PCTCN2019078999-appb-000015
测试例11
动物实验
在C57BL/6小鼠皮下接种鼠源肺癌Lewis细胞株构建动物模型。选取肿瘤体积约200mm 3的小鼠，设置15组，每组10只小鼠，按照表10的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000016
Figure PCTCN2019078999-appb-000017
测试例12
动物实验
在Balb/c小鼠皮下接种鼠源肉瘤S180细胞株构建动物模型。选取肿瘤体积约200mm 3的小鼠，设置15组，每组10只小鼠，按照表10的方式进行给药，病毒给药总pfu均为10 6pfu。采用相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率参照测试例3进行计算。
Figure PCTCN2019078999-appb-000018
从上述测试例可以看出，病毒载体的组合组相比单病毒组和双因子组的肿瘤抑制效果要更好，并且A1-2：B1-2和C1-1：B1-2的1:3组合组相比其他组合有更好的协同抗肿瘤效果。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (26)

  1. 一种重组溶瘤病毒组合物，其特征在于，所述组合物含有：
    第一重组溶瘤病毒，所述第一重组溶瘤病毒包括第一单纯疱疹病毒载体和第一外源基因；所述第一外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
    第二重组溶瘤病毒，所述第二重组溶瘤病毒包括第二单纯疱疹病毒载体和第二外源基因；所述第二外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
    其中，所述第一单纯疱疹病毒载体和第二单纯疱疹病毒载体彼此相同或不同，被选定的第一外源基因不同于被选定的第二外源基因。
  2. 根据权利要求1所述的重组溶瘤病毒组合物，其中，所述组合物还含有第三重组溶瘤病毒，所述第三重组溶瘤病毒包括第三单纯疱疹病毒载体和第三外源基因；所述第三外源基因为编码选自如下任一种的基因：细胞因子、具有预防和/或治疗肿瘤作用的单克隆抗体、肿瘤抗原、前药转化酶、肿瘤抑制蛋白、阻断或下调肿瘤过表达的原癌基因和代谢基因的反义RNA或小RNA；
    其中，所述第三单纯疱疹病毒载体和第一单纯疱疹病毒载体相同或不同，被选定的第三外源基因不同于被选定的第一外源基因；并且，所述第三 单纯疱疹病毒载体和第二单纯疱疹病毒载体相同或不同，被选定为第三外源基因不同于被选定的第二外源基因。
  3. 权利要求1或2的重组溶瘤病毒组合物，其中所述细胞因子为选自如下的任一种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
  4. 权利要求1或2的重组溶瘤病毒组合物，其中所述具有预防和/或治疗肿瘤作用的单克隆抗体选自如下的任一种：PD-1单克隆抗体、PD-L1单克隆抗体、PD-L2单克隆抗体、CTLA-4单克隆抗体、CD80单克隆抗体、CD28单克隆抗体、CD137单克隆抗体、CD137L单克隆抗体、OX40单克隆抗体、OX40L单克隆抗体、CD27单克隆抗体、CD70单克隆抗体、CD40单克隆抗体、CD40L单克隆抗体、LAG-3单克隆抗体和TIM-3单克隆抗体。
  5. 权利要求1或2的重组溶瘤病毒组合物，其中所述肿瘤抗原为肿瘤特异性抗原或肿瘤相关抗原。
  6. 权利要求5的重组溶瘤病毒组合物，其中所述肿瘤抗原为选自如下的任一种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
  7. 根据权利要求1-6任一项所述的重组溶瘤病毒组合物，其中所述第一外源基因、第二外源基因、第三外源基因还各自独立地包含与其可操作连接的表达调控序列，所述表达调控序列包括启动子、增强子和多聚核苷酸中的至少一种。
  8. 根据权利要求1-7任一项所述的重组溶瘤病毒组合物，其中所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体各自独 立地为缺失编码ICP34.5的基因的单纯疱疹病毒，或缺失编码ICP34.5和ICP47的基因的单纯疱疹病毒，并且所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体和/或第三单纯疱疹病毒载体各自彼此相同或不同。
  9. 根据权利要求8的重组溶瘤病毒组合物，其中所述第一单纯疱疹病毒载体、第二单纯疱疹病毒载体、第三单纯疱疹病毒载体还缺失编码ICP6、TK和UNG的基因中的至少一种。
  10. 根据权利要求1-9任一项所述的重组溶瘤病毒组合物，其中所述外源基因的插入位点为所述单纯疱疹病毒载体上缺失编码基因的位置中的任一处，并且所述第一外源基因、第二外源基因和/或第三外源基因的插入位置各自彼此相同或不同。
  11. 根据权利要求10所述的重组溶瘤病毒组合物，其中所述第一外源基因、第二外源基因和/或第三外源基因的插入位点为缺失编码ICP34.5基因的位置。
  12. 根据1-11任一项所述的重组溶瘤病毒组合物，其中所述单纯疱疹病毒为1型单纯疱疹病毒(HSV-1)。
  13. 根据权利要求1-12任一项所述的溶瘤病毒组合物，其中所述第一外源基因为编码细胞因子的基因，所述第二外源基因为编码单克隆抗体的基因。
  14. 权利要求13所述的溶瘤病毒组合物，其中所述第一外源基因为编码GM-CSF、G-CSF、M-CSF、IL-2、IL-12或IFN-γ的基因，所述第二外源基因为编码免疫检查点阻断性抗体的基因。
  15. 权利要求14所述的溶瘤病毒组合物，其中所述第一外源基因为编码GM-CSF、IL-2或IL-12的基因，所述第二外源基因为编码PD-1阻断性单克 隆抗体、PD-L1阻断性单克隆抗体。
  16. 根据权利要求11-15任一项所述的溶瘤病毒组合物，其中，以pfu计，所述第一重组溶瘤病毒和第二重组溶瘤病毒混合比为0.5:8、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1。
  17. 根据权利要求1-11任一项所述的溶瘤病毒组合物，其中所述第一外源基因和第二外源基因为编码细胞因子的基因，并且所述第一外源基因和第二外源基因编码的细胞因子不同。
  18. 权利要求17所述的溶瘤病毒组合物，其中所述的细胞因子选自：GM-CSF、G-CSF、M-CSF、IL-2、IL-12和IFN-γ。
  19. 权利要求18所述的溶瘤病毒组合物，其中所述的细胞因子选自：GM-CSF、IL-2和IL-12。
  20. 根据权利要求17-19任一项所述的溶瘤病毒组合物，其中，以pfu计，所述第一重组溶瘤病毒和第二重组溶瘤病毒混合比为0.5:2-2:0.5，优选0.5:1-1:0.5，最优选1:1。
  21. 权利要求1-20中任意一项所述的重组溶瘤病毒组合物在制备用于治疗肿瘤的药物中的用途。
  22. 根据权利要求21所述的用途，其中所述肿瘤选自脑神经胶质瘤、黑色素瘤、肝癌、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、前列腺癌、胃癌、淋巴瘤、胰腺癌、膀胱癌、乳腺癌、子宫内膜癌、淋巴瘤、肉瘤(例如软组织肉瘤和骨肉瘤)中的至少一种。
  23. 一种药物组合物，包含权利要求1-20中任意一项所述的重组溶瘤病毒组合物和药学可接受的载体。
  24. 一种制品或试剂盒，包含内装有权利要求23的药物组合物的小瓶和 包装插页，所述包装插页记载有所述药物组合物使用的相关信息。
  25. 一种治疗肿瘤的方法，包括给药受试者有效量的权利要求1-20中任意一项所述的重组溶瘤病毒组合物或权利要求23的药物组合物。
  26. 根据权利要25所述的方法，其中，所述肿瘤选自脑神经胶质瘤、黑色素瘤、肝癌、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、前列腺癌、胃癌、淋巴瘤、胰腺癌、膀胱癌、乳腺癌、子宫内膜癌、淋巴瘤、肉瘤(例如软组织肉瘤和骨肉瘤)中的至少一种。
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EP3778882A4 (en) 2021-06-09
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