WO2024114576A1 - 用于治疗肿瘤的柯萨奇b组1型病毒 - Google Patents

用于治疗肿瘤的柯萨奇b组1型病毒 Download PDF

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WO2024114576A1
WO2024114576A1 PCT/CN2023/134325 CN2023134325W WO2024114576A1 WO 2024114576 A1 WO2024114576 A1 WO 2024114576A1 CN 2023134325 W CN2023134325 W CN 2023134325W WO 2024114576 A1 WO2024114576 A1 WO 2024114576A1
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cvb1
sequence
mir
tumor
seq
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French (fr)
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程通
王玮
付文锟
方昌建
赵灿阳
靳舒驰
赵欢
方木锦
夏宁邵
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厦门大学
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the present invention relates to the field of viruses and tumor treatment. Specifically, the present invention relates to a coxsackievirus B group 1 type (CVB1) containing a specific amino acid site located at a structural protein, a recombinant virus further containing an exogenous nucleic acid using such CVB1 as a virus backbone, and their use in treating tumors.
  • the present invention also relates to a method for using such CVB1 or a recombinant virus for treating tumors.
  • oncolytic viruses can replicate themselves in tumor cells, thereby killing, dissolving tumor cells, or arresting tumor cell growth.
  • the use of oncolytic viruses may bring the risk of non-specific viral infection of healthy cells, leading to the death of non-cancerous cells and tissues. Therefore, while selectively killing cancer cells, it is crucial to maintain the health and vitality of normal, non-cancerous cells to reduce or even eliminate undesirable off-target effects for the development of oncolytic virus therapy.
  • Coxsackievirus B1 The oncolytic activity of Coxsackievirus B1 (CVB1) has been reported. However, after infecting humans, Coxsackievirus B1 may cause chronic autoimmune diseases such as viral myocarditis, pancreatitis, hepatitis, aseptic meningitis, and insulin-dependent diabetes mellitus in newborns, infants, and immunocompromised adults.
  • the inventors By analyzing the key site information that determines the oncolytic activity and toxicity of the CVB1 virus, the inventors provided a class of CVB1 viruses containing specific amino acid sites located in the structural protein, which have broad and effective oncolytic activity while having significantly reduced toxicity to normal non-cancerous cells, thereby providing the following invention.
  • the present invention provides an isolated Coxsackievirus group B type 1 (CVB1), wherein the 84th amino acid of the VP1 protein of the CVB1 is lysine K, the 224th amino acid of the VP1 protein is valine V, and the 232nd amino acid of the VP3 protein is glycine G.
  • CVB1 Coxsackievirus group B type 1
  • VP1 protein of CVB1 refers to the VP1 protein encoded by the CVB1 genome.
  • the expression “VP1 gene of CVB1” refers to the nucleic acid sequence encoding the VP1 protein in the viral genome.
  • VP3 protein of CVB1 refers to the VP3 protein encoded by the CVB1 genome
  • VP3 gene of CVB1 refers to the nucleic acid sequence encoding the VP3 protein in the viral genome.
  • the codon corresponding to the 84th amino acid of the VP1 protein in the VP1 gene of CVB1 is a codon encoding lysine K
  • the codon corresponding to the 224th amino acid of the VP1 protein is a codon encoding valine V
  • the codon corresponding to the 232nd amino acid of the VP3 protein in the VP3 gene of CVB1 is a codon encoding glycine G.
  • the VP1 gene of CVB1 is located at 2453--3286bp of the viral genome (the position refers to the genomic sequence shown in SEQ ID NO:22).
  • the VP3 gene of CVB1 is located at 1739--2452bp of the viral genome (the position refers to the genomic sequence shown in SEQ ID NO:22).
  • the VP1 protein of CVB1 has an amino acid sequence as shown in SEQ ID NO: 39. In certain embodiments, the VP3 protein of CVB1 has an amino acid sequence as shown in SEQ ID NO: 41.
  • the VP1 gene of CVB1 has a nucleotide sequence as shown in SEQ ID NO: 40.
  • the VP3 gene of CVB1 has a nucleotide sequence as shown in SEQ ID NO: 42.
  • the CVB1 provided by the present invention may be a wild-type strain naturally possessing the above-mentioned VP1/VP3 protein characteristics, for example, isolated from a clinic.
  • the CVB1 provided by the present invention may also be a non-naturally occurring or engineered CVB1 strain, which has been artificially modified (eg, amino acid substitution) to possess the above-mentioned VP1/VP3 protein characteristics.
  • the genomic sequence of CVB1 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the following: the nucleotide sequence shown in SEQ ID NO:22.
  • genomic sequence of CVB1 is shown as SEQ ID NO:22.
  • the genomic sequence of CVB1 differs from the sequence shown in SEQ ID NO: 22 only in that the codon corresponding to the 362nd lysine (K) of the nonstructural protein 3D is replaced with a codon encoding glutamic acid (E).
  • the 3D gene encoding the nonstructural protein 3D is located at 5903-7288bp of the viral genome (the position refers to the genomic sequence shown in SEQ ID NO: 22).
  • the cDNA sequence of CVB1 has at least 70%, At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: the nucleotide sequence shown in SEQ ID NO:1.
  • the cDNA sequence of CVB1 is shown as SEQ ID NO:1.
  • the cDNA sequence of CVB1 differs from the sequence shown in SEQ ID NO:1 only in that the codon corresponding to lysine (K) at position 362 of non-structural protein 3D is replaced by a codon encoding glutamate (E).
  • the CVB1 provided by the present invention can be used as a viral backbone to carry exogenous nucleic acids to form recombinant viruses. Therefore, in certain embodiments, the CVB1 is a recombinant virus and contains (for example, in its genome) exogenous nucleic acids.
  • CVB1 that does not contain exogenous nucleic acids may also be referred to as a CVB1 viral backbone
  • CVB1 that contains exogenous nucleic acids may also be referred to as a CVB1 recombinant virus or a modified virus.
  • the CVB1 recombinant virus described herein can be obtained by reverse genetics technology, which is known in the art, for example, see Yang L S, Li S X, Liu Y J, et al. Virus Res, 2015, 210: 165-168; Hou W H, Yang L S, Li S X, et al. Virus Res, 2015, 205: 41-44; all of which are incorporated herein by reference.
  • the cDNA of the original CVB1 strain is usually modified (e.g., insertion of exogenous nucleic acid) to obtain the CVB1 recombinant virus.
  • the exogenous nucleic acid is selected from a nucleic acid sequence encoding a cytokine, a nucleic acid sequence encoding an anti-tumor protein or polypeptide, a target sequence of a microRNA, or any combination thereof.
  • the exogenous nucleic acid comprises a nucleic acid sequence encoding a cytokine and/or encoding an anti-tumor protein or polypeptide.
  • the cytokine is a cytokine with anti-tumor activity, such as an interleukin (e.g., IL-2, IL-12, IL-15), an interferon (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), a tumor necrosis factor (e.g., TNF- ⁇ ), or a colony stimulating factor (e.g., GM-CSF).
  • an interleukin e.g., IL-2, IL-12, IL-15
  • an interferon e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇
  • TNF- ⁇ tumor necrosis factor
  • colony stimulating factor e.g., GM-CSF
  • the cytokine is GM-CSF (e.g., human GM-CSF).
  • the nucleic acid sequence encoding hGM-CSF is shown in SEQ ID NO:13.
  • the anti-tumor protein or polypeptide is an immune checkpoint inhibitor (e.g., PD-L1 antibody, PD-1 antibody, CTLA-4 antibody).
  • antibody refers to an immunoglobulin-derived molecule that can specifically bind to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen through an antigen binding site located in its variable region.
  • the term “antibody” unless the context clearly indicates, it includes not only complete antibodies but also antigen binding fragments that can specifically bind to a target antigen.
  • the antibody is preferably It is an antigen-binding fragment, such as scFv, Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv or single domain antibody (sdAb).
  • the anti-tumor protein or polypeptide is an anti-PD-1 or PD-L1 scFv (e.g., an anti-human PD-1 or PD-L1 scFv).
  • the nucleic acid sequence encoding the anti-human PD-1 or PD-L1 scFv is as shown in SEQ ID NO: 14.
  • the insertion site of the nucleic acid sequence encoding the cytokine and/or anti-tumor protein or polypeptide is located between the 5'UTR and the VP4 gene of the viral genome, or between the VP1 gene and the 2A gene.
  • the exogenous nucleic acid comprises a target sequence for a microRNA.
  • the microRNA is a tumor suppressor microRNA.
  • tumor suppressor microRNAs It has been previously reported that the expression of certain microRNAs in tumor cells is significantly lower than that in normal cells and/or has obvious tissue specificity, and these microRNAs can be referred to as tumor suppressor microRNAs.
  • the replication of recombinant viruses containing target sequences of tumor suppressor microRNAs in non-cancerous/normal cells is reduced or attenuated compared to the replication in cancer cells.
  • microRNAs are known in the art, and detailed teachings on such microRNAs can be found, for example, Kennedy, Edward M et al.
  • examples of the microRNA include, but are not limited to, miR-1-3p, miR-126-3p, etc. specifically expressed in myocardial tissue; miR-216a-5p, miR-217-5p, etc. specifically expressed in pancreatic tissue; miR-204-5p, miR-219a-5p, etc. specifically expressed in spinal cord tissue; miR-122, miR-192, miR-483, etc. specifically expressed in liver tissue; miR216a/b, miR-217 and miR-375 specifically expressed in pancreatic tissue; miR-1, miR-133a/b, miR-208, etc. specifically expressed in heart; miR-192, miR-196a/b, miR-219a/b, miR-375, etc.
  • kidney tissue specifically expressed in kidney tissue; b, miR-204, miR-215, etc.; miR-133a/b, miR-206, etc. specifically expressed in muscle tissue; miR-204, miR219a, etc. specifically expressed in spinal cord tissue; miR-124a, miR-125a/b, miR-128a/b, miR-138, etc.
  • miR-34 specifically expressed in brain tissue; and miR-34, miR-122a, miR-26a, which are lowly expressed in liver tumor tissue; miR-107, miR-96 and miR-196, which are lowly expressed in pancreatic tumor tissue; miR-34, which is lowly expressed in kidney tumor tissue; miR-143, miR-133a/b, which are lowly expressed in bladder tumor tissue; and miR-143, miR-133a/b, which are lowly expressed in lung tumor tissue. miR-Let-7, miR-29; etc.
  • the microRNA is selected from, for example, miR-1-3p, miR-126-3p, miR-204-5p, miR-217-5p, miR-219a-5, or any combination thereof.
  • the microRNA comprises miR-1-3p and/or miR-126-3p.
  • the target sequence of the miR-1-3p is shown in SEQ ID NO:4.
  • the target sequence of the miR-126-3p is shown in SEQ ID NO:5.
  • the insertion of the target sequence of miR-1-3p and/or miR-126-3p is advantageous. This is because miR-1-3p and miR-126-3p are specifically expressed in myocardial tissue, so by inserting the target sequence of miR-1-3p and/or miR-126-3p into the modified CVB1, the tissue tropism of the oncolytic virus can be changed to reduce or avoid killing of normal myocardial tissue.
  • the microRNA comprises miR-216a-5p and/or miR-217-5p.
  • the target sequence of the miR-216a-5p is shown in SEQ ID NO:7.
  • the target sequence of the miR-217-5p is shown in SEQ ID NO:8. In some cases, the insertion of the target sequence of miR-216a-5p and/or miR-217-5p is advantageous.
  • miR-216a-5p and miR-217-5p are specifically expressed in pancreatic tissue, so by inserting the target sequence of miR-216a-5p and/or miR-217-5p into the modified CVB1, the tissue tropism of the oncolytic virus can be changed to reduce or avoid killing of normal pancreatic tissue.
  • the microRNA comprises miR-204-5p and/or miR-219a-5p.
  • the target sequence of the miR-204-5p is shown in SEQ ID NO:6.
  • the target sequence of the miR-219a-5p is shown in SEQ ID NO:9.
  • the insertion of the target sequence of miR-204-5p and/or miR-219a-5p is advantageous. This is because miR-204-5p and miR-219a-5p are specifically expressed in spinal cord tissue, so by inserting the target sequence of miR-204-5p and/or miR-219a-5p into the modified CVB1, the tissue tropism of the oncolytic virus can be changed to reduce or avoid killing of normal spinal cord tissue.
  • the recombinant virus comprises one or more (e.g., 1, 2, 3, or 4) target sequences of microRNAs.
  • the target sequence may include one or more (e.g., 1, 2, 3, or 4) copies.
  • the insertion site of the target sequence of the microRNA is in the 5' untranslated region (5'UTR) and/or the 3' untranslated region (3'UTR) of the viral genome.
  • the insertion site of the 5' untranslated region is between 737-738bp.
  • the insertion site of the 3' untranslated region is 7296-7297bp.
  • the 5'UTR and 3'UTR of the viral genome are both inserted with the target sequence of the microRNA.
  • the genome sequence of the recombinant virus has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the following: a nucleotide sequence as shown in any one of SEQ ID NOs: 25-29.
  • the genome sequence of the recombinant virus is as shown in any one of SEQ ID NOs: 25-29.
  • the genome sequence of the recombinant virus differs from the sequence shown in any one of SEQ ID NOs: 25-29 only in that the codon corresponding to lysine (K) at position 362 of nonstructural protein 3D is replaced with a codon encoding glutamic acid (E).
  • the cDNA sequence of the recombinant virus has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the following: a nucleotide sequence as shown in any one of SEQ ID NOs: 17-21.
  • the cDNA sequence of the recombinant virus is as shown in any one of SEQ ID NOs: 17-21.
  • the cDNA sequence of the recombinant virus differs from the sequence shown in any one of SEQ ID NOs: 17-21 only in that the codon corresponding to lysine (K) at position 362 of nonstructural protein 3D is replaced with a codon encoding glutamic acid (E).
  • the CVB1 described in any of the above embodiments can be pretreated to reduce or eliminate the subject's immune response to the virus, wherein the pretreatment may include: loading the CVB1 into cells (e.g., human mesenchymal stem cells, lymphocytes, etc.), liposomes or micelles, and/or using proteases (e.g., chymotrypsin or trypsin) to remove the viral capsid protein to reduce the host's humoral and/or cellular immunity to the virus.
  • cells e.g., human mesenchymal stem cells, lymphocytes, etc.
  • proteases e.g., chymotrypsin or trypsin
  • the present invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of:
  • the isolated nucleic acid molecule consists of the genomic sequence or cDNA sequence of the CVB1, or the complementary sequence of the genomic sequence or cDNA sequence. In certain embodiments, the nucleic acid molecule has the genomic sequence of the CVB1. In certain exemplary embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO:22, 25-29. In certain exemplary embodiments, the nucleotide sequence of the nucleic acid molecule differs from the sequence shown in any one of SEQ ID NO:22, 25-29 only in that the codon corresponding to the 362nd lysine (K) of the non-structural protein 3D is replaced with a codon encoding glutamic acid (E).
  • the isolated nucleic acid molecule is a vector (eg, a cloning vector or an expression vector) comprising the genomic sequence or cDNA sequence of CVB1, or a complementary sequence of the genomic sequence or cDNA sequence.
  • the nucleic acid molecule is a vector (e.g., a cloning vector or an expression vector) comprising the cDNA sequence of CVB1, or a complementary sequence of the cDNA sequence.
  • the nucleic acid molecule is a vector comprising the nucleotide sequence shown in any one of SEQ ID NOs: 1, 17-21.
  • the nucleic acid molecule is a vector comprising the following nucleotide sequence: the nucleotide sequence differs from the sequence shown in any one of SEQ ID NOs: 1, 17-21 only in that the codon corresponding to the 362nd lysine (K) of nonstructural protein 3D is replaced by a codon encoding glutamic acid (E).
  • the CVB1 (including CVB1 virus skeleton and CVB1 recombinant virus) provided by the present invention can be obtained by reverse genetics technology.
  • Reverse genetics technology based on infectious clones i.e., virus rescue
  • This technology is to construct a full-length cDNA molecular clone of the viral genome using a vector under the premise of a known viral genome sequence, and then transfect cells to obtain live viruses.
  • enterovirus is a positive-strand RNA virus
  • its RNA can directly translate viral proteins, that is, the genomic RNA of enterovirus directly has the ability to infect, so as long as the transfection of the complete full-length RNA is guaranteed, the virus can be rescued.
  • the present invention also provides a method for preparing the CVB1 described in the first aspect, which comprises: (1) providing a vector (e.g., a plasmid) containing the cDNA sequence of the CVB1 to obtain an infectious clone; (2) introducing (e.g., transfecting) the infectious clone into a host cell; (3) obtaining the virus from the culture and/or lysate of the host cell (i.e., rescuing the virus).
  • a vector e.g., a plasmid
  • step (2) comprises co-introducing (eg, transfecting) the infectious clone and the helper plasmid into a host cell.
  • the infectious clone comprises a T7 promoter.
  • the backbone vector of the infectious clone is a pSVA plasmid.
  • the helper plasmid expresses T7 RNA polymerase.
  • the helper plasmid is a pAR3126 plasmid.
  • the host cell is a eukaryotic cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated CVB1 of the first aspect or the nucleic acid molecule of the second aspect, and a pharmaceutically acceptable carrier or excipient (eg, a stabilizer).
  • a pharmaceutically acceptable carrier or excipient eg, a stabilizer
  • the pharmaceutical composition comprises an effective amount of the isolated CVB1 described in the first aspect.
  • the isolated CVB1 described in the first aspect may be used in combination. Therefore, the pharmaceutical composition may include one or more of the isolated CVB1 described in the first aspect.
  • the pharmaceutical composition comprises one or more CVB1 virus backbones provided in the first aspect. In certain embodiments, the pharmaceutical composition comprises one or more CVB1 recombinant viruses provided in the first aspect. In certain embodiments, the pharmaceutical composition comprises any combination of one or more CVB1 virus backbones and CVB1 recombinant viruses provided in the first aspect.
  • the pharmaceutical composition comprises an effective amount of the nucleic acid molecule of the second aspect.
  • the pharmaceutical composition comprises the isolated genomic sequence of CVB1 of the first aspect.
  • the pharmaceutical composition comprises a vector comprising the isolated cDNA sequence of CVB1 of the first aspect.
  • the nucleic acid molecules of the present invention can be delivered by any means known in the art, for example, by direct injection of naked nucleic acid molecules (e.g., naked RNA), or by using a non-viral delivery system.
  • the non-viral delivery system can be prepared by various materials well known in the art, wherein the materials include but are not limited to the various materials described in detail in "Yin H, et al. Nat Rev Genet. 2014 Aug; 15(8): 541-55.” and “Riley MK, Vermerris W. Nanomaterials (Basel). 2017 Apr 28; 7(5). pii: E94.”, all of which are incorporated herein by reference, such as liposomes, inorganic nanoparticles (e.g., gold nanoparticles), polymers (e.g., PEG), etc.
  • the pharmaceutical composition may comprise the nucleic acid molecule described in the second aspect and the delivery system.
  • the pharmaceutical composition may further comprise an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an additional oncolytic virus, a chemotherapeutic agent, or an immunotherapeutic agent.
  • the other oncolytic viruses include but are not limited to herpes virus, adenovirus, parvovirus, reovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus or any combination thereof.
  • the chemotherapeutic agent includes but is not limited to 5-fluorouracil, mitomycin, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclines (such as epirubicin or doxorubicin), etoposide, platinum compounds (such as carboplatin or cisplatin), taxanes (such as paclitaxel or taxotere) or any combination thereof.
  • the immunotherapeutic agent includes but is not limited to immune checkpoint inhibitors (such as PD-L1/PD-1 inhibitors or CTLA-4 inhibitors), tumor-specific targeting antibodies (such as rituximab or Herceptin) or any combination thereof.
  • the isolated CVB1 of the first aspect or the nucleic acid molecule of the second aspect and the additional pharmaceutically active agent can be provided as separate components or as mixed components.
  • the agents may be administered simultaneously, separately or sequentially.
  • the isolated CVB1 described in the first aspect, the nucleic acid molecule described in the second aspect, or the pharmaceutical composition described in the third aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use. In certain embodiments, the preferred dosage form is an injection, an injection, or a lyophilized powder.
  • the separated CVB1 described in the first aspect, the nucleic acid molecules described in the second aspect, or the pharmaceutical composition described in the third aspect can be administered in various suitable ways. In some cases, their mode of administration depends on the location and type of the tumor.
  • the virus or nucleic acid molecule is optionally administered by direct injection into the tumor (e.g., intratumoral injection); for tumors of the hematopoietic system, the virus or nucleic acid molecule can be administered intravenously or by other intravascular routes; for tumors that are not easily accessible in the body (e.g., metastatic tumors), the virus or nucleic acid molecule can be systematically administered so that it spreads throughout the body and thus reaches the tumor (e.g., intravenous or intramuscular injection).
  • the virus or nucleic acid molecule of the present invention can be administered subcutaneously, intraperitoneally, intrathecally (e.g., for brain tumors), topically (e.g., for melanoma), orally (e.g., for oral or esophageal cancer), nasally, or by inhalation spray (e.g., for lung cancer).
  • the separated CVB1 described in the first aspect, the nucleic acid molecule described in the second aspect, or the pharmaceutical composition described in the third aspect can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, and the like.
  • the pharmaceutical composition comprises a unit dose of the CVB1 of the first aspect, for example, at least 1 ⁇ 10 2 pfu, at least 1 ⁇ 10 3 pfu, at least 1 ⁇ 10 4 pfu, 1 ⁇ 10 5 pfu, 1 ⁇ 10 6 pfu, at least 1 ⁇ 10 7 pfu, at least 1 ⁇ 10 8 pfu, at least 1 ⁇ 10 9 pfu, at least 1 ⁇ 10 10 pfu, at least 1 ⁇ 10 11 pfu, at least 1 ⁇ 10 12 pfu, at least 1 ⁇ 10 13 pfu, at least 1 ⁇ 10 14 pfu, or at least 1 ⁇ 10 16 pfu of CVB1.
  • the medicament comprises 1 ⁇ 10 2 pfu to 1 ⁇ 10 17 pfu of CVB1.
  • the pharmaceutical composition contains a unit dose of the nucleic acid molecule of the second aspect, for example, the nucleic acid molecule contains 3 ⁇ 10 10 to 3 ⁇ 10 14 virus genome copies.
  • the present invention provides a method for treating a tumor in a subject (e.g., a human), the method comprising administering to the subject an effective amount of the CVB1 of the first aspect, the nucleic acid molecule of the second aspect, or the pharmaceutical composition of the third aspect.
  • the present invention also provides the CVB1 of the first aspect, the nucleic acid molecule of the second aspect, or the pharmaceutical composition of the third aspect.
  • the tumor is a solid tumor or a hematological tumor.
  • the tumor is a solid tumor.
  • the tumor is selected from colorectal cancer, gastric cancer, lung cancer, liver cancer, ovarian cancer, endometrial cancer, cervical cancer, melanoma, breast cancer, kidney cancer, pancreatic cancer, lymphoma, osteogenic sarcoma, prostate cancer, glioma, neuroblastoma, tongue cancer, nasopharyngeal carcinoma, nasal septum squamous cell carcinoma, pharyngeal squamous cell carcinoma, submandibular gland squamous cell carcinoma, laryngeal cancer, thyroid cancer, thyroid ductal carcinoma and bladder cancer.
  • the subject is a mammal, such as a human.
  • the isolated CVB1 described in the first aspect may be used in combination.
  • one or several of the isolated CVB1 described in the first aspect may be administered.
  • the isolated nucleic acid molecules described in the second aspect may be used in combination.
  • one or several of the isolated nucleic acid molecules described in the second aspect may be administered.
  • the CVB1 described in the first aspect, the nucleic acid molecule described in the second aspect, or the pharmaceutical composition described in the third aspect may be used in combination with another pharmaceutically active agent having anti-tumor activity.
  • the CVB1 described in the first aspect, the nucleic acid molecule described in the second aspect, or the pharmaceutical composition described in the third aspect can be administered in combination with a second therapeutic agent (such as an anti-tumor agent) or a treatment (such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative treatment).
  • a second therapeutic agent such as an anti-tumor agent
  • a treatment such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative treatment.
  • the second therapeutic agent or treatment can be administered before, simultaneously, or after the administration of the above-mentioned agent of the present invention.
  • the second therapeutic agent is a drug with anti-tumor activity, such as an additional oncolytic virus, a chemotherapeutic agent, or an immunotherapeutic agent.
  • the CVB1 described in the first aspect can be administered in any amount of 1 to 1 ⁇ 10 15 pfu/kg of subject body weight, for example, at least 1 ⁇ 10 3 pfu/kg, at least 1 ⁇ 10 4 pfu/kg, 1 ⁇ 10 5 pfu/kg, 1 ⁇ 10 6 pfu/kg, at least 1 ⁇ 10 7 pfu/kg, at least 1 ⁇ 10 8 pfu/kg, at least 1 ⁇ 10 9 pfu/kg, at least 1 ⁇ 10 10 pfu/kg, at least 1 ⁇ 10 11 pfu/kg, or at least 1 ⁇ 10 12 pfu/kg of subject body weight.
  • the nucleic acid molecule described in the second aspect can be administered in any amount of 3 ⁇ 10 10 to 3 ⁇ 10 14 virus genome copies/kg of subject body weight.
  • CVB1, or a nucleic acid molecule as described herein may be administered 3 times a day, 2 times a day, once a day, once every two days, or once a week, optionally with weekly or monthly repeats as appropriate.
  • Coxsackivirus B1 refers to a Coxsackievirus B group of the Picomaviridae family, the Enterovirus genus, and a Coxsackievirus B group, whose genome is a single-stranded positive-strand RNA consisting of a 5’ non-coding region (5’UTR), an open reading frame (ORF), a 3’ non-coding region (3’UTR) and a polyadenylic acid tail; its ORF encodes a precursor polyprotein, which, after self-protease hydrolysis and cleavage, can produce structural proteins VP1 to VP4 and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D; in order to more clearly describe the present invention, the nucleic acid sequences corresponding to the above proteins in the CVB1 genome are respectively referred to as VP1 gene, VP2 gene, VP3 gene, VP4 gene, 2A gene, 2B gene,
  • CVB1 can further contain exogenous nucleic acid as a virus backbone to form a recombinant virus (or modified virus). Therefore, in the present invention, the expression "Coxsackivirus B1 (CVB1)” includes the CVB1 virus backbone itself and the recombinant virus (or modified virus).
  • the term "oncolytic virus” refers to a virus that can infect tumor cells, replicate in tumor cells, cause tumor cell death, lysis, or prevent tumor cell growth. Preferably, the virus has little toxic effect on non-tumor cells.
  • tumor specific refers to selectively exhibiting biological functions or activities in tumor cells.
  • tumor specific when used to describe the killing selectivity of a virus, it means that the virus can selectively kill tumor cells without killing or substantially killing non-tumor cells, or the virus is more effective in killing tumor cells than non-tumor cells.
  • the term "oncolytic activity” mainly includes tumor killing activity.
  • the oncolytic activity of the virus can generally be measured by indicators such as its ability to infect tumor cells, its ability to replicate in tumor cells, and/or its ability to kill tumor cells.
  • the oncolytic activity of a virus can be determined by any method known in the art. For example, the ability of a virus to infect tumor cells can be evaluated by measuring the dose of virus required to infect a given percentage of tumor cells (e.g., 50% cells); the ability to replicate in tumor cells can be evaluated by measuring the growth of the virus in tumor cells; the ability to kill tumor cells can be evaluated by observing the cytopathic effect (CPE) or measuring tumor cell activity.
  • CPE cytopathic effect
  • toxicity refers to the pathogenicity of CVB1 after challenging mice, which is mainly evaluated by monitoring the survival rate, body weight and health score of mice.
  • cDNA sequence of CVB1 means a DNA representation of the viral genomic RNA sequence, which differs from the RNA sequence only in that the ribonucleotides in the RNA sequence are replaced by corresponding deoxyribonucleotides, for example, uracil ribonucleotide (UMP) is replaced by thymine deoxyribonucleotide (dTMP).
  • UMP uracil ribonucleotide
  • dTMP thymine deoxyribonucleotide
  • exogenous nucleic acid refers to an artificially introduced nucleotide sequence that is foreign to the original sequence.
  • Exogenous nucleic acids include, but are not limited to, any gene or nucleotide sequence not found in the viral genome. However, in the present invention, it is particularly preferred that the exogenous nucleic acid consists of at most 1500, such as at most 1200, at most 1000 nucleotides.
  • the exogenous nucleic acid encodes a protein or polypeptide with anti-tumor killing activity, such as a cytokine, or an anti-tumor protein or polypeptide; alternatively, the exogenous nucleic acid comprises a target sequence of a microRNA (microRNA, miRNA).
  • the microRNA is preferably a microRNA whose expression level in tumor cells is significantly lower than that in normal cells and/or has obvious tissue specificity (i.e., tumor suppressor microRNA), examples of which include but are not limited to miR-1-3p, miR-126-3p, etc. specifically expressed in myocardial tissue; miR-216a-5p, miR-217-5p, etc.
  • pancreatic tissue specifically expressed in pancreatic tissue; miR-204-5p, miR-219a-5p, etc. specifically expressed in spinal cord tissue; miR-122, miR-192, miR-483, etc. specifically expressed in liver tissue; miR-126a-5p, miR-126a-5p, miR-126a-5p, etc. specifically expressed in pancreatic tissue; ...
  • miR-34, miR-122a, and miR-26a are highly expressed in pancreatic tumor tissues; miR-107, miR-96, and miR-196 are lowly expressed in pancreatic tumor tissues; miR-34 is lowly expressed in renal tumor tissues; miR-143 and miR-133a/b are lowly expressed in bladder tumor tissues; miR-Let-7 and miR-29 are lowly expressed in lung tumor tissues, etc. (see, for example, Ruiz A J and Russell S J. MicroRNAs and oncolytic viruses. [J]. Curr Opin Virol.
  • the recombinant CVB1 when the recombinant CVB1 contains the target sequence of the above-mentioned microRNA, it is regulated by the microRNA in cells/tissues where the microRNA is highly expressed or specifically expressed, so that the replication of the oncolytic virus is weakened or even loses its killing activity, while in tumor cells/tissues where the microRNA is lowly expressed or not expressed, the replication of the oncolytic virus is normal. Replication and normal killing of tumor cells.
  • cytokine has a meaning known to those skilled in the art. However, in the present invention, when the oncolytic virus of the present invention is used to treat a tumor, it is particularly preferred that the cytokine is a cytokine that can be used for tumor treatment.
  • cytokines include, but are not limited to, interleukins (e.g., IL-2, IL-12, and IL-15), interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), tumor necrosis factors (e.g., TNF- ⁇ ), colony stimulating factors (e.g., GM-CSF), and any combination thereof (see, e.g., Ardolino M, Hsu J, Raulet D H. Cytokine treatment in cancer immunotherapy [J]. Oncotarget, 2015, 6 (23): 19346-19347).
  • interleukins e.g., IL-2, IL-12, and IL-15
  • interferons e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇
  • tumor necrosis factors e.g., TNF- ⁇
  • colony stimulating factors e.g., GM-CSF
  • anti-tumor protein or polypeptide refers to a protein or polypeptide with tumor therapeutic activity, including but not limited to: (1) a protein or polypeptide that is toxic to cells, can inhibit cell proliferation or induce cell apoptosis, examples of which include but are not limited to thymidine kinase TK (TK/GCV), TRAIL and FasL (see, for example, Candolfi M, King G D, Arabic AG, et al. Evaluation of proapototic transgenes to use in combination with Flt3L in an immune-stimulatory gene therapy approach for Glioblastoma multiforme (GBM) [J].
  • TK/GCV thymidine kinase TK
  • TRAIL thymidine kinase
  • FasL FasL
  • a protein or polypeptide with immunotherapeutic effect examples of which include but are not limited to single chain antibody (scFv) or nano antibody against cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4), anti-programmed death receptor 1 (anti-PD-1) and single chain antibody (scFv) or nano antibody against programmed death ligand 1 (anti-PDL-1) (see, for example, Nolan et al. E, Savas P, Policheni A N, et al. Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer [J].
  • proteins or polypeptides that inhibit tumor angiogenesis examples of which include but are not limited to single-chain antibodies (scFv) against vascular endothelial growth factor (anti-VEGF), VEGF-derived polypeptides (e.g., D (LPR), KSVRGKGKGQKRKRKKSRYK, etc.) and ATN-161 (see, for example, Rosca EV, Koskimaki J E, Rivera CG, et al. Anti-angiogenic peptides for cancer therapeutics [J]. Curr Pharm Biotechnol, 2011, 12 (8): 1101-1116; all of which are incorporated herein by reference).
  • scFv single-chain antibodies
  • anti-VEGF vascular endothelial growth factor
  • VEGF-derived polypeptides e.g., D (LPR), KSVRGKGKGQKRKRKKSRYK, etc.
  • ATN-161 see, for example, Rosca EV, Koskimaki J E, River
  • scFv refers to a single polypeptide chain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL and VH are connected by a linker.
  • VH heavy chain variable region
  • VL light chain variable region
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab') 2 fragment means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • Fab'fragment means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains)
  • Fv refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody
  • single-domain antibody (sdAb) refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody.
  • Single-domain antibodies are also called nanobodies.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • nucleic acid molecule comprising the genomic sequence of CVB1 or "a nucleic acid molecule comprising the genomic sequence of CVB1” has the meaning commonly understood by those skilled in the art, i.e., when the nucleic acid molecule is DNA, the nucleic acid molecule comprises the DNA representation of the genomic sequence of CVB1 (e.g., a viral backbone or a recombinant virus); when the nucleic acid molecule is RNA, the nucleic acid molecule comprises the genomic sequence of CVB1 (e.g., a viral backbone or a recombinant virus).
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like.
  • pH regulators include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, Chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity (e.g., oncolytic activity) of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc.
  • desired activity e.g., oncolytic activity
  • desired activity e.g., oncolytic activity
  • the active ingredient in the drug including but not limited to sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g.
  • treat refers to treating or curing a disease (eg, a tumor), delaying the onset of symptoms of a disease (eg, a tumor), and/or delaying the progression of a disease (eg, a tumor).
  • a disease eg, a tumor
  • delaying the onset of symptoms of a disease eg, a tumor
  • delaying the progression of a disease eg, a tumor
  • a therapeutically effective amount refers to an amount that can effectively achieve the intended purpose.
  • a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (e.g., a tumor), delay the onset of symptoms of a disease (e.g., a tumor), and/or delay the development of a disease (e.g., a tumor).
  • a disease e.g., a tumor
  • delay the onset of symptoms of a disease e.g., a tumor
  • a disease e.g., a tumor
  • delay the onset of symptoms of a disease e.g., a tumor
  • a disease e.g., a tumor
  • a disease e.g., a tumor
  • Such an effective amount can be easily determined by a person skilled in the art or a doctor, and can be related to the intended purpose (e.g., treatment), the general health of the subject, age, sex, weight, severity of the disease to be treated, complications
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, human
  • cancer and tumor are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies such as lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors.
  • the present invention provides a class of CVB1 strains containing specific amino acid sites located in structural proteins (such as VP1 and/or VP3 proteins), which have high-efficiency and broad-spectrum tumor killing activity, and have significantly reduced toxicity and significantly improved safety.
  • the CVB1 oncolytic strain provided by the present invention can be used alone for the treatment of tumors, can also be used as an adjuvant method for traditional tumor treatment, or as a treatment method when other treatment methods are lacking, and has important clinical value.
  • FIG1 shows the electrophoresis diagram of genomic RNA samples of three wild-type CVB1 viruses (including CVB1-1E10, CVB1-D6F8 and CVB1-XM) obtained by the in vitro transcription method in Example 2.
  • Figure 2 shows the killing effect of wild-type CVB1 viral genome RNA on human cervical cancer cell line Hela in Example 2. The results showed that almost all Hela cells transfected with CVB1 genome RNA lysed and died 48 hours after transfection.
  • Figure 3 shows the effect of single - injection tail vein challenge of wild-type CVB1 on the survival rate, body weight and health of adult BALB/c mice in Example 3.
  • the results showed that CVB1-XM and CVB1-D6F8 caused adult BALB/c mice to become ill (erect hair, arched back, slow movement, and continuous weight loss) and died under the three challenge dose conditions of 10 6 to 10 8 TCID 50 /mouse, while CVB1-1E10 did not cause illness or death in mice under the above challenge dose conditions.
  • Figure 4 shows the effect of single-point tail vein challenge with CVB1-D6F8 single-point mutation (CVB1-D6F8-R232G, CVB1-D6F8-E84K and CVB1-D6F8-A224V), double-point mutation (CVB1-D6F8-RG-AV, CVB1-D6F8-RG-EK and CVB1-D6F8-EK-AV) and triple-point mutation (CVB1-D6F8-GKV) strains in Example 4 on the survival rate, body weight and health status of adult BALB/c mice.
  • Figure 5 shows the specific regulatory effect of miR-1-3p, miR-126-3p, miR-204-5p, miR-216a-5p, miR-217-5p and miR-219a-5p mimics on the in vitro cell proliferation of modified CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT and CVB1-miR216a/217/204/219aT strains in Example 6.
  • Figure 6 shows the effects of single-injection tail vein challenge of the modified CVB1 strains CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT, CVB1-miR216a/217/204/219aT, CVB1-hGM-CSF and CVB1-Anti-hPD-1 in Example 8 on the survival rate, body weight and health status of adult BALB/c mice.
  • the results show that The modified CVB1 strains did not cause disease or death in mice at a challenge dose of 10 7 TCID 50 /mouse.
  • Figure 7 shows the results of in vivo anti-tumor experiments of CVB1-1E10, CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT, CVB1-miR216a/217/204/219aT, CVB1-hGM-CSF and CVB1-Anti-hPD-1 in Example 9 on (A) human lung cancer cell line A549, (B) human Burkitt's lymphoma cell line Raji, (C) human endometrial cancer cell line HEC-1-B and (D) human cervical cancer cell line Hela.
  • Figure 8 shows the in vivo anti-tumor experimental results of CVB1-1E10, CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT, CVB1-miR216a/217/204/219aT, CVB1-mGM-CSF and CVB1-Anti-mPD-1 on mouse liver cancer cell line Hepa 1-6 in Example 9.
  • the results showed that in the challenge experimental group, 10 7 TCID 50 / tumor mass of CVB1 or its modified form was injected intratumorally every 2 days, for a total of 5 injections. After 10 days of treatment, the growth of subcutaneous tumors in mice stagnated until lysis disappeared; in contrast, the tumors in the negative group (CTRL) maintained normal growth, and their tumor size was significantly larger than that of the experimental group.
  • CTRL negative group
  • the patients' throat swabs and anal swabs were obtained from Xiamen Center for Disease Control and Prevention, China; African green monkey kidney cells (Vero cells; Number: CCL-81 TM ) was stored by the National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University, China, and cultured in MEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin.
  • Vero cells were plated in 24-well plates with 1 ⁇ 10 5 cells per well.
  • the growth medium (MEM medium, 10% fetal bovine serum, glutamine, penicillin and streptomycin) was removed and replaced with 1 mL of maintenance medium (MEM medium, 2% fetal bovine serum, glutamine, penicillin and streptomycin) per well.
  • MEM medium 10% fetal bovine serum, glutamine, penicillin and streptomycin
  • maintenance medium MEM medium, 2% fetal bovine serum, glutamine, penicillin and streptomycin
  • CPE cytopathic effect
  • Viruses isolated from clinical specimens were identified by RT-PCR and ELISPOT assay based on specific antibodies. Cultures positive for Coxsackievirus group B type 1 were selected for at least three cloning experiments. The cloned strains obtained by the limiting dilution method were also identified using RT-PCR and ELISPOT, and the cloned strains positive for Coxsackievirus group B type 1 were selected for the next round of cloning; combined with the results of whole genome sequencing, three cloned strains of Coxsackievirus group B type 1 with different genome sequences were selected from clinical isolates for oncolytic virus research.
  • this example completed the construction of infectious clones for the three CVB1 strains isolated and cloned above, and obtained the CVB1 backbone strain used in the present invention by reverse genetics technology. The specific method is described as follows.
  • the cDNA sequences of the above three oncolytic viruses (SEQ ID NO: 1-3) were then sent to a gene synthesis company (Nanjing GenScript Biotechnology Co., Ltd.) for full gene synthesis and ligated into the pSVA plasmid (Hou et al., Virus Res 2015, 205: 41-44) to obtain infectious clone plasmids of three CVB1 strains (i.e., CVB1-1E10, CVB1-D6F8 and CVB1-XM).
  • CVB1 strains i.e., CVB1-1E10, CVB1-D6F8 and CVB1-XM.
  • Plasmid extraction kit and E coli.DH5 ⁇ competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Hela cells ( Number: CCL-2 TM ) was preserved by the National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University, China, and cultured with DMEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin; transfection reagents Lipofactamine 2000 and Opti-MEM were purchased from Thermo Fisher Scientific.
  • Viruses This example used CVB1-1E10 (SEQ ID NO: 22) and CVB1- D6F8 (SEQ ID NO: 23) and CVB1-XM (SEQ ID NO: 24).
  • HepaRG cells were cultured in WME medium (supplemented with 1.5% DMSO); AGS and TT used F-12K medium; SH-SY5Y used DMEM:F12 (1:1) medium; CFPAC-1 used IMDM medium; RD, C-33A, EBC-1, SK-MEL-1, J82 and DU145 used MEM medium, Raji, Daudi, 5637, 786-O, TE-1, Caski, NCI-H1299, NCI-H1703, NCI-H1975, NCI-H661, SGC7901, BGC823, SW1116, HEp-2 and LNCap used RPMI-1640 medium, and other cells used DMEM medium, which required the addition of 10% fetal bovine serum, glutamine and penicillin-streptomycin double antibody. All the above cells were cultured under standard conditions of 37°C and
  • RD cells were evenly spread on a 10 cm cell culture plate, and the culture conditions were MEM medium containing 10% fetal bovine serum, glutamine, penicillin and streptomycin, 37°C, 5% CO 2 , and saturated humidity; when the cell confluence reached more than 90%, the cell culture medium was replaced with serum-free MEM medium, and 10 7 TCID 50 of CVB1 was inoculated on each plate.
  • CVB1 proliferated in the RD cells and caused CPE in the cells; when more than 90% of the cells shrank and became rounded, the granularity increased, and fell off and lysed, the cells and their culture supernatant were harvested; after three repeated freeze-thaw cycles, the culture supernatant was collected and centrifuged to remove cell debris, and the centrifugation conditions were 4000 rpm, 10 min, and 4°C; finally, The supernatant was filtered through a 0.22 ⁇ m disposable filter (Millipore) to remove impurities such as cell debris.
  • Millipore 0.22 ⁇ m disposable filter
  • RD cells were plated in a 96-well plate at a cell density of 10 4 cells/well; after the cells adhered to the wall, the virus solution obtained in Example 2.2 was diluted in a serum-free MEM medium starting from 10 times and then 10 times in a gradient. 50 ⁇ l of the diluted virus was added to the cell wells, and the cell wells where CPE appeared were observed and marked after 7 days.
  • the TCID 50 unit calculated at this time is TCID 50 /50 ⁇ l, which needs to be converted to TCID 50 /mL.
  • the original culture medium was directly discarded for adherent cells, and the original culture medium was carefully discarded after centrifugation of suspended cells, and then replaced with 100 ⁇ L of fresh serum-free culture medium per well; 10 ⁇ L of CCK-8 solution was added to each well of the inoculated cells, and an equal amount of CCK-8 solution was also added to the blank culture medium as a negative control; incubated at 37°C in a cell culture incubator for 0.5-3 hours, and detected once at 450nm using an ELISA reader at 0.5, 1, 2, and 3 hours, and the time point with a relatively suitable absorbance range was selected as a reference for cell survival rate.
  • the cell viability was calculated as:
  • CVB1-1E10 has a killing effect on most of the tumor cells tested, especially the virus has a significant killing effect on colorectal cancer cell lines, gastric cancer cell lines, lung cancer cell lines, liver cancer cell lines, cervical cancer cell lines, endometrial cancer cell lines, pancreatic cancer cell lines, prostate cancer cell lines, nasopharyngeal cancer cell lines, tongue cancer cell lines, laryngeal cancer cell lines, glioma cell lines and neuroblastoma cell lines; while the oncolytic activity of CVB1-D6F8 is significantly weaker.
  • these viruses are basically non-tumor normal cell lines, including: human normal pancreatic ductal epithelial cell line hTERT-HPNE, differentiated human liver progenitor cell line HepaRG, human embryonic lung fibroblast cell line MRC-5, human foreskin fibroblast cell line HFF-1, human skin keratinocyte cell line HaCat, human prostate stromal cell line WPMY-1 and human umbilical vein endothelial cell line HUVEC.
  • human normal pancreatic ductal epithelial cell line hTERT-HPNE differentiated human liver progenitor cell line HepaRG
  • human embryonic lung fibroblast cell line MRC-5 human foreskin fibroblast cell line HFF-1
  • human skin keratinocyte cell line HaCat human prostate stromal cell line WPMY-1
  • human umbilical vein endothelial cell line HUVEC human umbilical vein endothelial cell line HUVEC.
  • a large amount of infectious live CVB1 virus can be produced by transfecting a certain type of tumor cells with purified CVB1 genomic RNA and kill the tumor cells.
  • the CVB1 genomic RNA is obtained by in vitro transcription.
  • the method can be found in Hadac EM, Kelly E J and Russell S J. Mol Ther, 2011, 19(6): 1041-1047.
  • the infectious clone plasmid of wild-type CVB1 obtained in Example 1 was linearized, and the linearized plasmid was used as a template to transcribe and mass-produce viral RNA in vitro using a kit MEGAscript TM T7 Transcription Kit (Thermo Fisher Scientific, AM1333), and then the obtained viral RNA was purified using a MEGAclear TM Transcription Clean-Up Kit (Thermo Fisher Scientific, AM1908) for later use.
  • the RNA electrophoresis pattern of one sample is shown in FIG1 .
  • the human cervical cancer cell line Hela was inoculated into a 24-well plate at 10 5 cells/well by the in vitro anti-tumor experiment method described in Example 2.4; after the cells adhered to the wall, each well was replaced with the corresponding serum-free cell culture medium and incubated at 37°C for 30 minutes, and then the transfection reagent was used.
  • the purified viral RNA was transfected into Hela cells at 1 ⁇ g per well using the 2000 (Thermo Fisher Scientific, 11668019), and the negative control group was transfected with irrelevant RNA nucleic acid molecules. The cells were then observed under a microscope every day to see if CPE was produced.
  • Virus This example uses CVB1-1E10 (SEQ ID NO: 22), CVB1-D6F8 (SEQ ID NO: 23) and CVB1-XM (SEQ ID NO: 24) provided in Example 1.
  • the virus culture and virus titer determination methods are shown in Examples 2.2 and 2.3, respectively.
  • mice Female BALB/c mice aged 6-8 weeks were obtained from Shanghai Slake Laboratory Animal Co., Ltd. The mice were housed under SPF conditions according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University.
  • mice Female BALB/c mice aged 6 to 8 weeks were divided into cages with 8 mice per cage. A negative control group was set up for each batch of experiments, and the challenge liquid was MEM cell culture medium.
  • the tail vein challenge method was adopted: the mice were fixed with a mouse fixator, and a needle (1mL insulin injection needle, BD) was slowly inserted into the lateral vein of the tail, and 100 ⁇ L of virus liquid was gently pushed in (each group was given a 10-fold gradient dilution dose, with a dose range of 10 6 to 10 8 TCID 50 /100 ⁇ L/mouse); a negative control group was set up for each batch of experiments, and the challenge liquid was MEM cell culture medium. The control group was challenged with 100 ⁇ L MEM cell culture medium and then returned to the cage.
  • the survival rate and weight of BALB/c mice should be recorded daily, and their physical signs should be observed. Deaths on the first day after the challenge are not counted, and the mice are observed and recorded for 7 to 20 consecutive days. The health of the mice will be strictly recorded according to the physical status score system, which is as follows:
  • Table 3 Animal health status score table
  • CVB1-XM and CVB1-D6F8 can cause adult BALB/c mice to become sick (erect hair, arched back, slow movement, and continuous weight loss) and die at three challenge doses of 10 6 to 10 8 TCID 50 /mouse, while CVB1-1E10 did not cause illness or death in mice at the above three challenge doses.
  • Example 4 Analysis of oncolytic activity and virulence-determining sites of oncolytic CVB1 backbone strains
  • the 84th position of the VP1 protein of CVB1-D6F8 is E, the 224th position of the VP1 protein is A, and the 232nd position of the VP3 protein is R;
  • the 84th position of the VP1 protein of CVB1-XM is K, the 224th position of the VP1 protein is A, and the 232nd position of the VP3 protein is R;
  • the 84th position of the VP1 protein of CVB1-1E10 is K, the 224th position of the VP1 protein is V, and the 232nd position of the VP3 protein is G.
  • CVB1-1E10 has low toxicity and safety and stronger oncolytic activity phenotype compared with CVB1-D6F8, based on the cDNA of CVB1-D6F8 strain (SEQ ID NO: 2)
  • three amino acid sites with differences in structural proteins were subjected to point-by-point mutation transformation to CVB1-1E10 strain, including single point mutations (CVB1-D6F8-R232G, CVB1-D6F8-E84K and CVB1-D6F8-A224V), double-point mutations (CVB1-D6F8-RG-AV, CVB1-D6F8-RG-EK and CVB1-D6F8-EK-AV) and three-point mutations (CVB1-D6F8-GKV), and the CVB1 strains with the above gene mutations were obtained based on infectious cloning and reverse genetics technology, as shown in Example 1.2.
  • Example 2.4 some tumor cells provided in Example 2.4 were used to verify the changes in oncolytic activity of the above gene-mutated CVB1 strains, and the method for detecting the oncolytic activity of the virus in vitro was referred to Example 2.4.
  • the results are shown in Table 4. Except for the 3-point mutated CVB1-D6F8-GKV, which exhibited a strong in vitro oncolytic activity comparable to CVB1-1E10, other point mutated CVB1 strains exhibited a poor oncolytic activity comparable to CVB1-D6F8.
  • Example 3.2 the method of Example 3.2 was used to select a challenge dose of 10 7 TCID 50 /mouse, and a single-needle intravenous injection toxicity experiment was performed on the CVB1 strain with the above gene mutation in adult BALB/c mice.
  • the single-point mutation CVB1-D6F8-R232G and CVB1-D6F8-A224V can cause adult BALB/c mice to become ill (erect hair, arched back, slow movement, continuous weight loss) and partially lethal under the challenge dose of 10 7 TCID 50 /mouse, but the single-point mutation CVB1-D6F8-E84K, the double-point mutation CVB1-D6F8-RG-AV, CVB1-D6F8-RG-EK and CVB1-D6F8-EK-AV, and the 3-point mutation CVB1-D6F8-GKV did not cause the mice to become ill or die under the challenge dose.
  • the above research results show that the 84th amino acid of VP1 protein is the key site for the difference in virulence between CVB1-1E10 and CVB1-D6F8, while the 224th amino acid of VP1 protein and the 232nd amino acid of VP3 protein also affect the difference in virulence, but the better oncolytic activity of CVB1-1E10 compared with CVB1-D6F8 is related to the above three amino acid sites.
  • the above research results also show that the above VP1/VP3 protein characteristics can be obtained by artificially modifying the CVB1 strain (such as amino acid substitution), so that it has both efficient and broad-spectrum tumor killing activity and significantly improved safety.
  • the wild-type CVB1-1E10 with excellent oncolytic activity in vitro and low toxicity and safety to mice was selected as the backbone strain, and gene insertion was performed based on its cDNA (SEQ ID NO: 1), including:
  • Modification form 1 The tandem sequence (DNA sequence is shown in SEQ ID NO: 36) of the miR-1-3p target sequence (DNA sequence is shown in SEQ ID NO: 30) and the miR-126-3p target sequence (DNA sequence is shown in SEQ ID NO: 31), and the tandem sequence (DNA sequence is shown in SEQ ID NO: 37) of the miR-216a-5p target sequence (DNA sequence is shown in SEQ ID NO: 33) and the miR-217-5p target sequence (DNA sequence is shown in SEQ ID NO: 34) are respectively inserted into the 5' untranslated region between 737-738 bp and the 3' untranslated region between 7296-7297 bp of the wild-type CVB1 cDNA (SEQ ID NO: 1), thereby obtaining the cDNA (SEQ ID NO: 17) of the recombinant virus (named CVB1-miR1/126/216a/217T), whose genomic RNA sequence is SEQ ID NO: 25;
  • Modification 2 The tandem sequence (DNA sequence is shown in SEQ ID NO: 36) of the miR-1-3p target sequence (DNA sequence is shown in SEQ ID NO: 30) and the miR-126-3p target sequence (DNA sequence is shown in SEQ ID NO: 31), and the tandem sequence (DNA sequence of which is shown in SEQ ID NO:38) of the miR-204-5p target sequence (DNA sequence of which is shown in SEQ ID NO:32) and the miR-219a-5p target sequence (DNA sequence of which is shown in SEQ ID NO:35) were respectively inserted into the 5' untranslated region between 737-738 bp and the 3' untranslated region between 7296-7297 bp of the wild-type CVB1 cDNA (SEQ ID NO:1), thereby obtaining the cDNA (SEQ ID NO:18) of the recombinant virus (named CVB1-miR1/126/204/219aT), the genomic RNA sequence of which is SEQ ID NO:26;
  • Modification form 3 The tandem sequence (DNA sequence is shown in SEQ ID NO: 37) of the miR-216a-5p target sequence (DNA sequence is shown in SEQ ID NO: 33) and the miR-217-5p target sequence (DNA sequence is shown in SEQ ID NO: 34), as well as the miR-204-5p target sequence (DNA sequence is shown in SEQ ID NO: 32) and the miR-219a-5p target sequence (DNA sequence is shown in SEQ ID NO: 35)
  • the tandem sequences of (its DNA sequence is shown in SEQ ID NO:38) were inserted into the 5' untranslated region between 737-738 bp and the 3' untranslated region between 7296-7297 bp of the wild-type CVB1 cDNA (SEQ ID NO:1), thereby obtaining the cDNA (SEQ ID NO:19) of the recombinant virus (named CVB1-miR216a/217/204/219aT), whose genomic RNA sequence is SEQ ID NO:27;
  • Modification form 4 The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene (SEQ ID NO: 13) was inserted between the VP1 and 2A genes of the wild-type CVB1 cDNA (SEQ ID NO: 1), thereby obtaining the cDNA (SEQ ID NO: 20) of the recombinant virus (named CVB1-hGM-CSF), whose genomic RNA sequence is SEQ ID NO: 28;
  • GM-CSF human granulocyte-macrophage colony-stimulating factor
  • Modification form 5 The sequence encoding anti-human programmed death receptor 1 single-chain antibody (Anti-PD-1scFv) (SEQ ID NO:14) is inserted between the VP1 and 2A genes of the wild-type CVB1 cDNA (SEQ ID NO:1) to obtain the cDNA of the recombinant virus (named CVB1-Anti-hPD-1) (SEQ ID NO:21), whose genomic RNA sequence is SEQ ID NO:29.
  • Anti-PD-1scFv anti-human programmed death receptor 1 single-chain antibody
  • Example 1.2 The above oncolytic CVB1 strains were obtained based on infectious cloning and reverse genetics techniques. The method is shown in Example 1.2
  • the miRNA mimics of miR-1-3p, miR-126-3p, miR-204-5p, miR-216a-5p, miR-217-5p and miR-219a-5p and the negative control mimics corresponding to the miRNA of Caenorhabditis elegans were purchased from Dharmacon.
  • the miRNA mimics were transfected into HeLa cells at a concentration of 200 nM using Mirus mRNA transfection reagent. After 6 hours, the cells were infected with viruses CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT and CVB1-miR216a/217/204/219aT modified by inserting miRNA target sequence, with an MOI of 0.1. 24 hours after infection, the supernatant was collected for virus titration, and the cell proliferation was detected by CCK-8 method, and the method was respectively referred to in Examples 2.3 and 2.4.
  • Example 7 In vitro anti-tumor experiment of modified oncolytic CVB1 strain
  • CVB1-1E10 (SEQ ID NO: 22) provided in Example 1 and CVB1-miR1/126/216a/217T (SEQ ID NO: 25), CVB1-miR1/126/204/219aT (SEQ ID NO: 26), CVB1-miR216a/217/204/219aT (SEQ ID NO: 27), CVB1-hGM-CSF (SEQ ID NO: 28) and CVB1-Anti-hPD-1 (SEQ ID NO: 29) provided in Example 5 were used.
  • CVB1-miR1/126/216a/217T SEQ ID NO: 25
  • CVB1-miR1/126/204/219aT SEQ ID NO: 26
  • CVB1-miR216a/217/204/219aT SEQ ID NO: 27
  • CVB1-hGM-CSF SEQ ID NO: 28
  • CVB1-Anti-hPD-1 SEQ ID NO: 29
  • Example 2.4 Cell lines: In this example, some tumor cells provided in Example 2.4 were used to verify the changes in oncolytic activity of various modified virus strains of CVB1. The method for detecting the oncolytic activity of the virus in vitro is referred to Example 2.4. The results are shown in Table 5. The oncolytic activity of the five modified CVB1 strains has significant oncolytic activity.
  • Example 8 Animal toxicity experiment of modified oncolytic CVB1 strain
  • Viruses This example uses CVB1-miR1/126/216a/217T (SEQ ID NO: 25), CVB1-miR1/126/204/219aT (SEQ ID NO: 26), CVB1-miR216a/217/204/219aT (SEQ ID NO: 27), CVB1-hGM-CSF (SEQ ID NO: 28) and CVB1-Anti-hPD-1 (SEQ ID NO: 29) provided in Example 5.
  • CVB1-miR1/126/216a/217T SEQ ID NO: 25
  • CVB1-miR1/126/204/219aT SEQ ID NO: 26
  • CVB1-miR216a/217/204/219aT SEQ ID NO: 27
  • CVB1-hGM-CSF SEQ ID NO: 28
  • CVB1-Anti-hPD-1 SEQ ID NO: 29
  • mice Female BALB/c mice aged 6-8 weeks were obtained from Shanghai Slake Laboratory Animal Co., Ltd. The mice were housed under SPF conditions according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University.
  • Toxicity experiment of virus challenge was carried out in 6-8 week old BALB/c mice according to reference example 3.2.
  • a challenge dose of 10 7 TCID 50 /mouse was selected.
  • the survival rate, body weight and health score of mice were observed and recorded. The results are shown in FIG6 .
  • All the modified CVB1 strains did not cause disease or death in mice under the challenge dose of 10 7 TCID 50 /mouse.
  • Example 9 In vivo anti-tumor experiment of modified oncolytic CVB1 strain
  • CVB1-1E10 (SEQ ID NO: 22) provided in Example 1 and CVB1-miR1/126/216a/217T (SEQ ID NO: 25) provided in Example 5, CVB1- miR1/126/204/219aT (SEQ ID NO: 26), CVB1-miR216a/217/204/219aT (SEQ ID NO: 27), CVB1-hGM-CSF (SEQ ID NO: 28) and CVB1-Anti-hPD-1 (SEQ ID NO: 29).
  • CVB1-mGM-CSF and CVB1-Anti-mPD-1 carrying mouse GM-CSF cDNA sequence, SEQ ID NO: 15
  • anti-mouse PD-1 scFv sequence cDNA sequence, SEQ ID NO: 16
  • the method is shown in Example 1.2.
  • the virus culture and virus titer determination methods are shown in Examples 2.2 and 2.3, respectively.
  • mice Female BALB/c nude mice and C57BL/6 mice aged 6-8 weeks were obtained from Shanghai Slake Laboratory Animal Co., Ltd. The mice were housed under SPF conditions according to the protocol approved by the Experimental Animal Center and Ethics Committee of Xiamen University.
  • mice The tumor cells used for subcutaneous tumor formation in mice were digested with 0.01% trypsin and then resuspended into a single cell suspension using a cell culture medium containing 10% fetal bovine serum; the cell density of the suspension was counted, and the cells were centrifuged at 1000g for 3 minutes to precipitate the cells, and then the cells were resuspended with an appropriate volume of PBS to reach about 106 cells/100 ⁇ L PBS; the tumor cells were inoculated subcutaneously on the back of the mouse with a syringe at 106 cells/100 ⁇ L PBS/point, and the tumor cells were left for about 14-21 days until the tumor cells formed a subcutaneous area of about 100mm under the mouse skin.
  • Tumor size (mm 3 ) tumor length value ⁇ (tumor width value) 2 /2.
  • Figures 7A-7D show the test results of (A) A549, (B) Raji, (C) HEC-1-B and (D) Hela tumor models treated with CVB1-1E10, CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT, CVB1-miR216a/217/204/219aT, CVB1-hGM-CSF and CVB1-Anti-hPD-1, respectively, 18 days later.
  • the results showed that after treatment, the growth of the tumors gradually slowed down and stagnated, and even lysis disappeared; in contrast, the tumors in the negative group (CTRL) maintained normal growth, and their tumor size was significantly larger than that of the experimental group.
  • CTRL negative group
  • FIG 8 shows the test results of Hepa1-6 tumor model treated with CVB1-1E10, CVB1-miR1/126/216a/217T, CVB1-miR1/126/204/219aT, CVB1-miR216a/217/204/219aT, CVB1-mGM-CSF and CVB1-Anti-mPD-1 for 18 days.
  • the results showed that the tumor volume was significantly reduced after treatment; in contrast, the tumor in the negative group (CTRL) maintained normal growth, and its tumor size was significantly larger than that in the experimental group.
  • CTRL negative group

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Abstract

提供了一种分离的柯萨奇病毒B组1型(CVB1)，该CVB1的VP1蛋白第84位氨基酸为赖氨酸(K)，VP1蛋白第224位氨基酸为缬氨酸(V)，并且VP3蛋白第232位氨基酸为甘氨酸(G)。又提供了一种以该CVB1为病毒骨架进一步包含外源核酸的重组病毒。还提供了该CVB1或该重组病毒用于治疗肿瘤的方法。

Description

用于治疗肿瘤的柯萨奇B组1型病毒 技术领域
本发明涉及病毒领域和肿瘤治疗领域。具体而言，本发明涉及包含位于结构蛋白的特定氨基酸位点的柯萨奇病毒B组1型(CVB1)，以此类CVB1为病毒骨架进一步包含外源核酸的重组病毒，以及它们用于治疗肿瘤的用途。本发明还涉及使用此类CVB1或重组病毒用于治疗肿瘤的方法。
背景技术
利用溶瘤病毒对肿瘤的治疗方法目前被认为比较有前景的肿瘤治疗手段。溶瘤病毒能够在肿瘤细胞当中自我复制，从而杀死、溶解肿瘤细胞，或者使肿瘤细胞生长停滞。然而使用溶瘤病毒可能会带来非特异性病毒感染健康细胞，从而导致非癌细胞和组织死亡的风险。因此，在选择性杀伤癌细胞的同时，维持正常、非癌细胞的健康和活力，以减少甚至消除不期望的脱靶效应，对于发展溶瘤病毒疗法是至关重要的。
目前已有报道柯萨奇病毒B组1型(Coxsackivirus B1，CVB1)的溶瘤活性，然而柯萨奇病毒B组1型在感染人体后可能导致新生儿、婴幼儿以及免疫缺陷的成人出现病毒性心肌炎、胰腺炎、肝炎、无菌性脑膜炎以及胰岛素依赖型糖尿病等慢性自身免疫性疾病。
因此，获得兼具肿瘤杀伤活性和更高的肿瘤特异性以提高安全性的病毒仍然是有必要的。
发明内容
发明人通过分析决定CVB1病毒的溶瘤活性和毒力的关键位点信息，提供了一类包含位于结构蛋白的特定氨基酸位点的CVB1病毒，其在具备广泛有效的溶瘤活性的同时，具有明显降低的对正常非癌细胞的毒力，由此提供了以下发明。
包含位于结构蛋白的特定氨基酸位点的CVB1
在第一方面，本发明提供了分离的柯萨奇病毒B组1型(CVB1)，其中，所述CVB1的VP1蛋白第84位氨基酸为赖氨酸K，VP1蛋白第224位氨基酸为缬氨酸V，并且VP3蛋白第232位氨基酸为甘氨酸G。
在本文中，表述“CVB1的VP1蛋白”是指由所述CVB1基因组编码的VP1蛋白， 表述“CVB1的VP1基因”是指病毒基因组中编码VP1蛋白的核酸序列。类似地，表述“CVB1的VP3蛋白”是指由所述CVB1基因组编码的VP3蛋白，表述“CVB1的VP3基因”是指病毒基因组中编码VP3蛋白的核酸序列。
在某些实施方案中，所述CVB1的VP1基因中对应VP1蛋白第84位氨基酸的密码子为编码赖氨酸K的密码子，对应VP1蛋白第224位氨基酸的密码子为编码缬氨酸V的密码子，并且所述CVB1的VP3基因中对应VP3蛋白第232位氨基酸的密码子为编码甘氨酸G的密码子。在某些实施方案中，所述CVB1的VP1基因位于病毒基因组2453--3286bp(所述位置参考SEQ ID NO:22所示的基因组序列)。在某些实施方案中，所述CVB1的VP3基因位于病毒基因组1739--2452bp(所述位置参考SEQ ID NO:22所示的基因组序列)。
在某些实施方案中，所述CVB1的VP1蛋白具有如SEQ ID NO:39所示的氨基酸序列。在某些实施方案中，所述CVB1的VP3蛋白具有如SEQ ID NO:41所示的氨基酸序列。
在某些实施方案中，所述CVB1的VP1基因具有如SEQ ID NO:40所示的核苷酸序列。在某些实施方案中，所述CVB1的VP3基因具有如SEQ ID NO:42所示的核苷酸序列。
在某些实施方案中，本发明所提供的CVB1可以是天然具备上述VP1/VP3蛋白特征的野生型毒株，例如从临床分离获得。
在某些实施方案中，本发明所提供的CVB1也可以是非天然存在的或工程化的CVB1毒株，其经人工改造(例如氨基酸置换)以具备上述VP1/VP3蛋白特征。
在某些实施方案中，所述CVB1的基因组序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NO:22所示的核苷酸序列。
在某些示例性实施方案中，所述CVB1的基因组序列如SEQ ID NO:22所示。
在某些示例性实施方案中，所述CVB1的基因组序列与SEQ ID NO:22所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。在某些实施方案中，编码所述非结构蛋白3D的3D基因位于病毒基因组5903-7288bp(所述位置参考SEQ ID NO:22所示的基因组序列)。
在某些实施方案中，所述CVB1的cDNA序列与选自下列的核苷酸序列具有至少70％、 至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NO:1所示的核苷酸序列。
在某些示例性实施方案中，所述CVB1的cDNA序列如SEQ ID NO:1所示。
在某些示例性实施方案中，所述CVB1的cDNA序列与SEQ ID NO:1所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
在某些实施方案中，本发明所提供的上述CVB1可以作为病毒骨架来携带外源核酸以形成重组病毒。因此，在某些实施方案中，所述CVB1为重组病毒，并且包含(例如在其基因组中包含)外源核酸。为便于区分，在本文中，不包含外源核酸的CVB1也可称为CVB1病毒骨架，包含外源核酸的CVB1也可称为CVB1重组病毒或经修饰病毒。
本文所述的CVB1重组病毒可以通过反向遗传学技术获得，所述反向遗传学技术是本领域已知的，例如可参见Yang L S，Li S X，Liu Y J，et al.Virus Res，2015,210:165-168；Hou W H,Yang L S，Li S X，et al.Virus Res，2015,205:41-44；其全部通过引用并入本文。在此类实施方案中，通常对原有CVB1毒株的cDNA进行修饰(例如，外源核酸的插入)从而获得CVB1重组病毒。
在某些实施方案中，所述外源核酸选自编码细胞因子的核酸序列、编码抗肿瘤蛋白或多肽的核酸序列、微小RNA的靶序列、或其任意组合。
在某些实施方案中，所述外源核酸包含编码细胞因子和/或编码抗肿瘤蛋白或多肽的核酸序列。
在某些实施方案中，所述细胞因子为具有抗肿瘤活性的细胞因子，例如白介素(例如IL-2、IL-12、IL-15)、干扰素(例如IFN-α、IFN-β、IFN-γ)、肿瘤坏死因子(例如TNF-α)或集落刺激因子(例如GM-CSF)。
在某些实施方案中，所述细胞因子为GM-CSF(例如人GM-CSF)。在某些示例性实施方案中，所述编码hGM-CSF的核酸序列如SEQ ID NO:13所示。
在某些实施方案中，所述抗肿瘤蛋白或多肽为免疫检查点抑制剂(例如，PD-L1抗体、PD-1抗体、CTLA-4抗体)。本文所用的术语“抗体”是指能够特异性结合靶抗原的源自免疫球蛋白的分子，所述源自免疫球蛋白的分子通过位于其可变区中的抗原结合位点来结合所述靶抗原。当提及术语“抗体”时，除非上下文明确指出，其不仅包括完整抗体也包括能够特异性结合靶抗原的抗原结合片段。在某些实施方案中，所述抗体优选 为抗原结合片段，例如scFv、Fab、Fab’、(Fab’)2、Fv、二硫键连接的Fv或单域抗体(sdAb)。
在某些实施方案中，所述抗肿瘤蛋白或多肽为抗PD-1或PD-L1的scFv(例如抗人PD-1或PD-L1的scFv)。在某些示例性实施方案中，所述编码抗人PD-1或PD-L1的scFv的核酸序列如SEQ ID NO:14所示。
在某些实施方案中，所述编码细胞因子和/或抗肿瘤蛋白或多肽的核酸序列的插入位点位于所述病毒基因组的5’UTR与VP4基因之间，或者位于VP1基因与2A基因之间。
在某些实施方案中，所述外源核酸包含微小RNA的靶序列。
在某些实施方案中，所述微小RNA为肿瘤抑制性微小RNA。
之前已报道，某些微小RNA在肿瘤细胞中的表达量显著低于正常细胞和/或具有明显的组织特异性，这些微小RNA可以被称为肿瘤抑制性微小RNA。包含肿瘤抑制性微小RNA的靶序列的重组病毒在非癌/正常细胞中的复制相比于癌细胞中的复制减少或减弱。
因此在某些情况下，将这类微小RNA的靶序列插入本发明所提供的包含特定结构蛋白的CVB1的基因组以形成重组病毒是有利的，因为在正常细胞或组织内高表达的这类微小RNA可以通过对应的靶序列减少甚至阻断重组CVB1病毒在该正常细胞或组织内的复制，从而减轻甚至避免重组病毒对非肿瘤细胞的毒副作用。这类微小RNA是本领域已知，关于这类微小RNA的详细教导可参见例如，Kennedy,Edward M et al.“Design of an Interferon-Resistant Oncolytic HSV-1 Incorporating Redundant Safety Modalities for Improved Tolerability.”Molecular therapy oncolytics vol.18 476-490.8Aug.2020，其全部通过引用并入本文。
在某些实施方案中，所述微小RNA的实例包括但不限于，心肌组织中特异性表达miR-1-3p、miR-126-3p等；胰腺组织中特异性表达的miR-216a-5p、miR-217-5p等；脊髓组织中特异性表达的miR-204-5p、miR-219a-5p等；肝组织特异性表达的miR-122，miR-192，miR-483等；胰腺组织特异性表达的miR216a/b、miR-217和miR-375；心脏特异性表达的miR-1，miR-133a/b，miR-208等；肾组织特异性表达的miR-192，miR-196a/b，miR-204，miR-215等；肌肉组织特异性表达的miR-133a/b，miR-206等；脊髓组织特异性表达的miR-204、miR219a等；脑组织特异性表达的miR-124a，miR-125a/b，miR-128a/b，miR-138等；以及在肝肿瘤组织内低表达的miR-34，miR-122a，miR-26a；在胰腺肿瘤组织内低表达的miR-107，miR-96和miR-196；在肾肿瘤组织内低表达的miR-34；在膀胱肿瘤组织内低表达的miR-143，miR-133a/b；在肺肿瘤组织内低表达的 miR-Let-7，miR-29；等等。
在某些实施方案中，所述微小RNA选自例如miR-1-3p、miR-126-3p、miR-204-5p、miR-217-5p、miR-219a-5、或其任意组合。
在某些实施方案中，所述微小RNA包含miR-1-3p和/或miR-126-3p。在某些示例性实施方案中，所述miR-1-3p的靶序列如SEQ ID NO:4所示。在某些示例性实施方案中，所述miR-126-3p的靶序列如SEQ ID NO:5所示。在某些情况下，miR-1-3p和/或miR-126-3p的靶序列的插入是有利的。这是由于miR-1-3p和miR-126-3p在心肌组织中特异性表达，因此通过将miR-1-3p和/或miR-126-3p的靶序列插入所述经修饰的CVB1中的方式可以改变该溶瘤病毒的组织嗜性，以减小或避免对正常心肌组织的杀伤。
在某些实施方案中，所述微小RNA包含miR-216a-5p和/或miR-217-5p。在某些示例性实施方案中，所述miR-216a-5p的靶序列如SEQ ID NO:7所示。在某些示例性实施方案中，所述miR-217-5p的靶序列如SEQ ID NO:8所示。在某些情况下，miR-216a-5p和/或miR-217-5p的靶序列的插入是有利的。这是由于miR-216a-5p和miR-217-5p在胰腺组织中特异性表达，因此通过将miR-216a-5p和/或miR-217-5p的靶序列插入所述经修饰的CVB1中的方式可以改变该溶瘤病毒的组织嗜性，以减小或避免对正常胰腺组织的杀伤。
在某些实施方案中，所述微小RNA包含miR-204-5p和/或miR-219a-5p。在某些示例性实施方案中，所述miR-204-5p的靶序列如SEQ ID NO:6所示。在某些示例性实施方案中，所述miR-219a-5p的靶序列如SEQ ID NO:9所示。在某些情况下，miR-204-5p和/或miR-219a-5p的靶序列的插入是有利的。这是由于miR-204-5p和miR-219a-5p在脊髓组织中特异性表达，因此通过将miR-204-5p和/或miR-219a-5p的靶序列插入所述经修饰的CVB1中的方式可以改变该溶瘤病毒的组织嗜性，以减小或避免对正常脊髓组织的杀伤。
在某些实施方案中，所述重组病毒包含一个或多个(例如1个，2个，3个或4个)微小RNA的靶序列。在某些实施方案中，所述靶序列可以包括一个或多个(例如1个，2个，3个或4个)拷贝。
在某些实施方案中，所述微小RNA的靶序列的插入位点在所述病毒基因组的5’非翻译区(5’UTR)和/或3’非翻译区(3’UTR)。例如，5’非翻译区的插入位点为737-738bp之间。例如，3’非翻译区的插入位点为7296-7297bp。在某些实施方案中，所述病毒基因组的5’UTR和3’UTR均插入有所述微小RNA的靶序列。
在某些实施方案中，所述重组病毒的基因组序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NO:25-29任一项所示的核苷酸序列。在某些示例性实施方案中，所述重组病毒的基因组序列如SEQ ID NO:25-29任一项所示。在某些示例性实施方案中，所述重组病毒的基因组序列与SEQ ID NO:25-29任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
在某些实施方案中，所述重组病毒的cDNA序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NO:17-21任一项所示的核苷酸序列。在某些示例性实施方案中，所述重组病毒的cDNA序列如SEQ ID NO:17-21任一项所示。在某些示例性实施方案中，所述重组病毒的cDNA序列与SEQ ID NO:17-21任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
在某些实施方案中，上述任意实施方案中所述的CVB1可以经预处理以减少或消除受试者对该病毒的免疫反应，其中所述预处理可以包括：将所述CVB1装载在细胞(例如：人间充质干细胞、淋巴细胞等)、脂质体或胶束中，和/或使用蛋白酶(例如，糜蛋白酶或胰蛋白酶)去除病毒的衣壳蛋白以减小宿主对病毒的体液和/或细胞免疫。
在第二方面，本发明提供了分离的核酸分子，其包含选自下列的序列：
(1)第一方面所述的分离的CVB1的基因组序列或cDNA序列；和
(2)所述基因组序列或cDNA序列的互补序列。
在某些实施方案中，所述分离的核酸分子由所述CVB1的基因组序列或cDNA序列，或所述基因组序列或cDNA序列的互补序列组成。在某些实施方案中，所述核酸分子具有所述CVB1的基因组序列。在某些示例性实施方案中，所述核酸分子具有如SEQ ID NO:22，25-29任一项所示的核苷酸序列。在某些示例性实施方案中，所述核酸分子的核苷酸序列与SEQ ID NO:22，25-29任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
在某些实施方案中，所述分离的核酸分子为包含所述CVB1的基因组序列或cDNA序列，或所述基因组序列或cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。 在某些实施方案中，所述核酸分子为包含所述CVB1的cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。在某些示例性实施方案中，所述核酸分子为包含SEQ ID NO:1，17-21任一项所示的核苷酸序列的载体。在某些示例性实施方案中，所述核酸分子为包含下述核苷酸序列的载体：所述核苷酸序列与SEQ ID NO:1，17-21任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
病毒的制备
本发明所提供的CVB1(包括CVB1病毒骨架以及CVB1重组病毒)可以通过反向遗传学技术获得。基于感染性克隆的反向遗传学技术(即，病毒拯救)是广泛应用于RNA病毒领域的分子生物学技术。该技术是在已知病毒基因组序列的前提下，利用载体构建病毒基因组全长cDNA分子克隆，之后转染细胞得到活病毒。由于肠道病毒是正链RNA病毒，其RNA能够直接翻译病毒蛋白，也即肠道病毒的基因组RNA直接具有感染能力，因此只要保证完整的全长RNA的转染即可拯救出病毒。
因此，在另一方面，本发明还提供了制备第一方面所述的CVB1的方法，其包括：(1)提供包含所述CVB1的cDNA序列的载体(例如质粒)以获得感染性克隆；(2)将所述感染性克隆引入(例如转染)宿主细胞；(3)从所述宿主细胞的培养物和/或裂解物中获得病毒(即，拯救病毒)。
在某些实施方案中，步骤(2)包括将所述感染性克隆和辅助质粒共同引入(例如转染)宿主细胞。
在某些实施方案中，所述感染性克隆包含T7启动子。在某些实施方案中，所述感染性克隆的骨架载体为pSVA质粒。
在某些实施方案中，所述辅助质粒表达T7RNA聚合酶。在某些实施方案中，所述辅助质粒为pAR3126质粒。
在某些实施方案中，所述宿主细胞为真核细胞。
药物组合物
在第三方面，本发明提供了药物组合物，其包含第一方面所述的分离的CVB1或第二方面所述的核酸分子，以及药学上可接受的载体或赋形剂(例如稳定剂)。
在某些实施方案中，所述药物组合物包含有效量的第一方面所述的分离的CVB1。在 某些实施方案中，可以组合使用第一方面所述的分离的CVB1。因此，所述药物组合物可以包括第一方面所述的分离的CVB1中的一种或数种。
在某些实施方案中，所述药物组合物包含一种或多种第一方面中提供的CVB1病毒骨架。在某些实施方案中，所述药物组合物包含一种或多种第一方面中提供的CVB1重组病毒。在某些实施方案中，所述药物组合物包含一种或多种第一方面中提供的CVB1病毒骨架和CVB1重组病毒的任意组合。
在某些实施方案中，所述药物组合物包含有效量的第二方面所述的核酸分子。在某些实施方案中，所述药物组合物包含第一方面所述的分离的CVB1的基因组序列。在某些实施方案中，所述药物组合物包含包括第一方面所述的分离的CVB1的cDNA序列的载体。
本发明的核酸分子可以通过本领域已知的任何方式进行递送，例如，直接注射裸露的核酸分子(例如，裸RNA)，或利用非病毒递送系统(non-viral delivery system)。所述非病毒递送系统可通过本领域熟知的各种材料制备获得，其中所述材料包括但不限于详细描述于“Yin H,et al.Nat Rev Genet.2014 Aug；15(8):541-55.”以及“Riley MK,Vermerris W.Nanomaterials(Basel).2017 Apr 28；7(5).pii:E94.”中的各种材料，其全部通过引用并入本文，例如脂质体、无机纳米粒子(如金纳米颗粒)、多聚物(如PEG)等等。
因此，所述药物组合物可以包含第二方面所述的核酸分子以及递送系统。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。
在本发明中，所述另外的溶瘤病毒包括但不限于疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合。所述化学治疗剂包括但不限于5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合。所述免疫治疗剂包括但不限于免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
在某些实施方案中，在所述药物组合物中，第一方面所述的分离的CVB1或第二方面所述的核酸分子与所述另外的药学活性剂可以作为分离的组分或作为混合的组分提供。因此，第一方面所述的分离的CVB1或第二方面所述的核酸分子与所述另外的药学活性 剂可以同时、分开或相继施用。
第一方面所述的分离的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。在某些实施方案中，优选的剂型是注射剂，注射液或冻干粉剂。
第一方面所述的分离的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物可以通过各种合适的方式施用。在某些情况下，它们的施用方式取决于肿瘤的位置和类型。例如，对于容易接近的实体肿瘤，任选地通过直接向肿瘤注射(例如，瘤内注射)来施用该病毒或核酸分子；对于造血系统的肿瘤，可以通过静脉内或其他血管内途径施用该病毒或核酸分子；对于体内不容易接近的肿瘤(例如转移瘤)，可以系统地施用该病毒或核酸分子以使其遍布全身并由此到达肿瘤(例如，静脉内或肌肉内注射)。任选地，可以经皮下、腹膜内、鞘内(例如对于脑部肿瘤)、局部(例如对于黑素瘤)、口服(例如对于口腔或食道癌)、经鼻或通过吸入喷雾(例如对于肺癌)等途径施用本发明的病毒或核酸分子。在某些实施方案中，可以通过皮内注射、皮下注射、肌肉注射、静脉注射、口服给予等途径施用第一方面所述的分离的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物。
在某些实施方案中，所述药物组合物包含单位剂量的第一方面所述的CVB1，例如包含至少1×102pfu、至少1×103pfu、至少1×104pfu、1×105pfu、1×106pfu、至少1×107pfu、至少1×108pfu、至少1×109pfu、至少1×1010pfu、至少1×1011pfu、至少1×1012pfu、至少1×1013pfu、至少1×1014pfu或至少1×1016pfu的CVB1。在某些实施方案中，所述药物包含1×102pfu～1×1017pfu的CVB1。
在某些实施方案中，所述药物组合物含有单位剂量的第二方面所述的核酸分子，例如含有3×1010～3×1014病毒基因组拷贝数(virus genome copies)的所述核酸分子。
抗肿瘤应用
在另一方面，本发明提供了用于在受试者(例如人)中治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的第一方面所述的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物。本发明还提供了第一方面所述的CVB1、第二方面所述的核酸分 子或第三方面所述的药物组合物用于在受试者中治疗肿瘤的用途，或者用于制备在受试者中治疗肿瘤的药物的用途。
在某些实施方案中，所述肿瘤为实体瘤或血液肿瘤。
在某些实施方案中，所述肿瘤为实体瘤。
在某些实施方案中，所述肿瘤选自结直肠癌、胃癌、肺癌、肝癌、卵巢癌、子宫内膜癌、宫颈癌、黑色素瘤、乳腺癌、肾癌、胰腺癌、淋巴瘤、成骨肉瘤、前列腺癌、神经胶质瘤、神经母细胞瘤、舌癌、鼻咽癌、鼻中隔鳞状细胞癌、咽鳞癌、颌下腺鳞癌、喉癌、甲状腺癌、甲状腺导管癌和膀胱癌。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在某些实施方案中，可以组合使用第一方面所述的分离的CVB1。因此，可以施用第一方面所述的分离的CVB1中的一种或数种。
在某些实施方案中，可以组合使用第二方面所述的分离的核酸分子。因此，可以施用第二方面所述的分离的核酸分子中的一种或数种。
在某些实施方案中，第一方面所述的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物可以与具有抗肿瘤活性的另外的药学活性剂联合使用。
在某些实施方案中，第一方面所述的CVB1、第二方面所述的核酸分子或第三方面所述的药物组合物可以与第二治疗剂(如抗肿瘤剂)或治疗术(例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗)组合施用。所述第二治疗剂或治疗术可以在施用本发明的上述试剂之前、同时或之后施用。
在某些实施方案中，所述第二治疗剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。
在某些实施方案中，可以以1～1×1015pfu/kg受试者体重的任何量施用第一方面所述的CVB1，例如以至少1×103pfu/kg、至少1×104pfu/kg、1×105pfu/kg、1×106pfu/kg、至少1×107pfu/kg、至少1×108pfu/kg、至少1×109pfu/kg、至少1×1010pfu/kg、至少1×1011pfu/kg或至少1×1012pfu/kg受试者体重的量施用CVB1。在某些实施方案中，可以以3×1010～3×1014病毒基因组拷贝数(virus genome copies)/kg受试者体重的任何量施用第二方面所述的核酸分子。在某些实施方案中，可以以每日3次、每日2次、每日1次、每两日1次或每周1次的方式施用CVB1，或如本文所述的核酸分子，任选地酌情每周或每月重复如上所述的给药方案。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、细胞培养、生物化学、细胞生物学、核酸化学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“柯萨奇病毒B组1型(Coxsackivirus B1，CVB1)”是指，小核糖核酸病毒科(Picomaviridae)、肠道病毒属(Enterovirus)、柯萨奇病毒(Coxsackivirus)B组中的一种，其基因组为单股正链RNA，由5’非编码区(5’UTR)、一个开放阅读框(ORF)、3’非编码区(3’UTR)和多聚腺苷酸尾巴组成；其ORF编码一个前体多聚蛋白，经过自身蛋白酶水解切割，可产生结构蛋白VP1～VP4和非结构蛋白2A、2B、2C、3A、3B、3C和3D；为了更加清楚地描述本发明，CVB1基因组中上述各蛋白所对应的核酸序列分别称为VP1基因、VP2基因、VP3基因、VP4基因、2A基因、2B基因、2C基因、3A基因、3B基因、3C基因和3D基因。CVB1可以作为病毒骨架进一步包含外源核酸以形成重组病毒(或经修饰病毒)。因此，在本发明中，表述“柯萨奇病毒B组1型(Coxsackivirus B1，CVB1)”即包括CVB1病毒骨架自身也包括重组病毒(或经修饰病毒)。
如本文中所使用的，术语“溶瘤病毒”是指，能够感染肿瘤细胞，在肿瘤细胞中复制，引起肿瘤细胞死亡、裂解或阻止肿瘤细胞生长的病毒。优选地，该病毒对非肿瘤细胞具有很小的毒性效应。
如本文中所使用的，术语“肿瘤特异性”是指，选择性地在肿瘤细胞内展现出生物功能或活性。例如，在本发明中，当术语“肿瘤特异性”用于描述病毒的杀伤选择性时，意指该病毒能够选择性杀伤肿瘤细胞而不杀伤或基本上不杀伤非肿瘤细胞，或者，该病毒对肿瘤细胞的杀伤比对非肿瘤细胞的杀伤更为有效。
如本文中所使用的，术语“溶瘤活性”主要包括肿瘤杀伤活性。当描述病毒的溶瘤活性时，通常可以通过其病毒感染肿瘤细胞的能力、在肿瘤细胞内复制的能力和/或杀伤肿瘤细胞的能力等指标衡量该病毒的溶瘤活性。病毒的溶瘤活性可以采用本领域已知的任何方法进行测定。例如，病毒感染肿瘤细胞的能力可以通过测量感染给定百分率的肿瘤细胞(例如50％细胞)所需要的病毒剂量进行评价；在肿瘤细胞内复制的能力可以通过测量病毒在肿瘤细胞内的生长情况进行评价；杀伤肿瘤细胞的能力可以通过观察致细胞病变效应(CPE)或测量肿瘤细胞活性进行评价。
如本文中所使用的，术语“毒性”是指，CVB1在攻毒小鼠后表现出的致病能力，主要通过监测小鼠生存率、体重和健康分数进行评价。
如本文中所使用的，表述“CVB1的cDNA序列”意指，该病毒基因组RNA序列的DNA表示形式，其与所述RNA序列相比差异仅在于所述RNA序列中的核糖核苷酸被对应的脱氧核糖核苷酸替代，例如尿嘧啶核糖核苷酸(UMP)由胸腺嘧啶脱氧核糖核苷酸(dTMP)替代。
如本文中所使用的，术语“外源核酸”是指，人工引入的核苷酸序列，其相对于原始序列而言是外来的。外源核酸包括但不限于，未在所述病毒基因组中发现的任何基因或核苷酸序列。然而，在本发明中，特别优选地，所述外源核酸由至多1500个，例如至多1200个，至多1000个核苷酸组成。在某些情况下，优选地，外源核酸编码具有抗肿瘤杀伤活性的蛋白或多肽，例如细胞因子、或抗肿瘤蛋白或多肽；或者，外源核酸包含微小RNA(microRNA，miRNA)的靶序列。在本发明中，所述微小RNA优选为那些在肿瘤细胞中的表达量显著低于正常细胞和/或具有明显的组织特异性的微小RNA(即，肿瘤抑制性微小RNA)，其实例包括但不限于，心肌组织中特异性表达miR-1-3p、miR-126-3p等；胰腺组织中特异性表达的miR-216a-5p、miR-217-5p等；脊髓组织中特异性表达的miR-204-5p、miR-219a-5p等；肝组织特异性表达的miR-122，miR-192，miR-483等；胰腺组织特异性表达的miR216a/b、miR-217和miR-375；心脏特异性表达的miR-1，miR-133a/b，miR-208等；肾组织特异性表达的miR-192，miR-196a/b，miR-204，miR-215等；肌肉组织特异性表达的miR-133a/b，miR-206等；脊髓组织特异性表达的miR-204、miR219a等；脑组织特异性表达的miR-124a，miR-125a/b，miR-128a/b，miR-138等；以及在肝肿瘤组织内低表达的miR-34，miR-122a，miR-26a；在胰腺肿瘤组织内低表达的miR-107，miR-96和miR-196；在肾肿瘤组织内低表达的miR-34；在膀胱肿瘤组织内低表达的miR-143，miR-133a/b；在肺肿瘤组织内低表达的miR-Let-7，miR-29，等等(参见，例如，Ruiz A J and Russell S J.MicroRNAs and oncolytic viruses.[J].Curr Opin Virol,2015,13:40-48；Kennedy,Edward M et al.“Design of an Interferon-Resistant Oncolytic HSV-1Incorporating Redundant Safety Modalities for Improved Tolerability.”Molecular therapy oncolytics vol.18 476-490.8Aug.2020；其全部通过引用并入本文)。
在本发明中，当重组CVB1包含上述微小RNA的靶序列时，其在该microRNA高表达或特异性表达的细胞/组织中，受到该microRNA的调控，从而可使该溶瘤病毒的复制减弱、甚至丧失杀伤活性，而在该microRNA低表达或不表达的肿瘤细胞/组织中则正常 复制、正常杀伤肿瘤细胞。
如本文中所使用的，术语“细胞因子”具有本领域技术人员公知的含义。然而，在本发明中，当使用本发明的溶瘤病毒来治疗肿瘤时，特别优选地，所述细胞因子为能够用于肿瘤治疗的细胞因子。“细胞因子”的实例包括但不限于，白介素(例如IL-2、IL-12和IL-15)、干扰素(例如IFN-α、IFN-β、IFN-γ)、肿瘤坏死因子(例如TNF-α)、集落刺激因子(例如GM-CSF)，及其任何组合(参见例如，Ardolino M,Hsu J,Raulet D H.Cytokine treatment in cancer immunotherapy[J].Oncotarget,2015,6(23):19346-19347)。
如本文中所使用的，术语“抗肿瘤蛋白或多肽”是指，具有治疗肿瘤活性的蛋白或多肽，其包括但不限于：(1)对细胞具有毒性、可抑制细胞增殖或诱导细胞凋亡的蛋白或多肽，其实例包括但不限于胸苷激酶TK(TK/GCV)、TRAIL和FasL(参见例如，Candolfi M,King G D,Muhammad A G,et al.Evaluation of proapototic transgenes to use in combination with Flt3L in an immune-stimulatory gene therapy approach for Glioblastoma multiforme(GBM)[J].FASEB J,2008,22:1077.13)；(2)具有免疫治疗作用的蛋白或多肽，其实例包括但不限于抗细胞毒T淋巴细胞相关抗原4(anti-CTLA-4)、抗程序性死亡受体1(anti-PD-1)的单链抗体(scFv)或纳米抗体和抗程序性死亡配体1(anti-PDL-1)的单链抗体(scFv)或纳米抗体(参见例如，Nolan E,Savas P,Policheni A N,et al.Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer[J].Science Trans Med,2017,9:eaal4922；其全部通过引用并入本文)；(3)抑制肿瘤血管生成的蛋白或多肽，其实例包括但不限于抗血管内皮生长因子(anti-VEGF)的单链抗体(scFv)、VEGF来源多肽(例如D(LPR)、KSVRGKGKGQKRKRKKSRYK等)和ATN-161(参见例如，Rosca E V，Koskimaki J E，Rivera C G，et al.Anti-angiogenic peptides for cancer therapeutics[J].Curr Pharm Biotechnol，2011,12(8):1101-1116；其全部通过引用并入本文)。
如本文中所使用的，术语“scFv”是指，包含重链可变区(VH)和轻链可变区(VL)的单个多肽链，其中所述VL和VH通过接头(linker)相连。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。
如本文中所使用的，术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成；术 语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“单域抗体(single-domain antibody,sdAb)”是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力，单域抗体也称为纳米抗体(nanobody)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，表述“包含CVB1的基因组序列的核酸分子”或“核酸分子包含CVB1的基因组序列”具有本领域技术人员通常理解的含义，即当所述核酸分子为DNA时，所述核酸分子包含CVB1(例如病毒骨架或重组病毒)的基因组序列的DNA表示形式；当所述核酸分子为RNA时，所述核酸分子包含CVB1(例如病毒骨架或重组病毒)的基因组序列。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯， 三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如溶瘤活性)，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“治疗”是指，治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)症状的发作，和/或延缓疾病(例如肿瘤)的发展。
如本文中所使用的，术语“有效量”是指，可以有效实现预期目的的量。例如，治疗有效量可以是有效地或足以治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)症状的发作和/或延缓疾病(例如肿瘤)发展的量。这样的有效量可以由本领域技术人员或医生容易地确定，并且可以与预期目的(例如治疗)、受试者的一般健康状况、年龄、性别、体重、待治疗的疾病的严重程度、并发症、施用方式等相关。这样的有效量的确定完全在本领域技术人员的能力范围内。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤，或者，具有患有肿瘤的风险。
如本文中使用的，术语“癌症”“肿瘤”可互换使用，其是指以体内异常细胞的不受控生长为特征的一大类疾病。不受管制的细胞分裂可能导致恶性肿瘤或侵入邻近组织的细胞的形成，并可能通过淋巴系统或血流转移到身体的远端部位。癌症包括良性和恶性癌症以及休眠肿瘤或微转移。癌症也包括血液学恶性肿瘤，例如淋巴瘤，白血病，骨髓瘤或淋巴恶性肿瘤，以及脾癌和淋巴结肿瘤。
发明的有益效果
本发明提供了包含位于结构蛋白(例如VP1和/或VP3蛋白)的特定氨基酸位点的一类CVB1毒株，这类CVB1毒株具有高效广谱的肿瘤杀伤活性，同时具备明显降低的毒性以及显著提高的安全性。本发明提供的CVB1溶瘤毒株可单独用于肿瘤的治疗，亦可用作传统肿瘤治疗的辅助方法，或作为缺少其他治疗方法时的治疗手段，具有重要的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附 图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例2中通过体外转录方法获得的3种野生型CVB1病毒(包括CVB1-1E10、CVB1-D6F8和CVB1-XM)的基因组RNA样品的电泳图。
图2显示了实施例2中野生型CVB1病毒基因组RNA对人宫颈癌细胞系Hela的杀伤效果。结果显示，转染CVB1基因组RNA的Hela细胞在转染后48小时几乎全部裂解死亡。
图3显示了实施例3中野生型CVB1单针尾静脉攻毒对成年BALB/c小鼠存活率、体重和健康程度的影响。结果显示，CVB1-XM和CVB1-D6F8在106～108TCID50/只这3个攻毒剂量条件下均使成年BALB/c小鼠发病(竖毛、弓背、运动迟缓、体重持续下降)且致死，而CVB1-1E10在以上攻毒剂量条件下均未导致小鼠发病和死亡。
图4显示了实施例4中CVB1-D6F8单点突变(CVB1-D6F8-R232G、CVB1-D6F8-E84K和CVB1-D6F8-A224V)、双点突变(CVB1-D6F8-RG-AV、CVB1-D6F8-RG-EK和CVB1-D6F8-EK-AV)和3点突变(CVB1-D6F8-GKV)毒株单针尾静脉攻毒对成年BALB/c小鼠存活率、体重和健康程度的影响。结果显示，在107TCID50/只的攻毒剂量条件下，仅单点突变的CVB1-D6F8-R232G和CVB1-D6F8-A224V使成年BALB/c小鼠发病且部分致死，而单点突变的CVB1-D6F8-E84K、双点突变的CVB1-D6F8-RG-AV、CVB1-D6F8-RG-EK和CVB1-D6F8-EK-AV、以及3点突变的CVB1-D6F8-GKV均未导致小鼠发病和死亡。
图5显示了实施例6中miR-1-3p、miR-126-3p、miR-204-5p、miR-216a-5p、miR-217-5p和miR-219a-5p的模拟物对经修饰的CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT和CVB1-miR216a/217/204/219aT毒株在体外细胞内增殖的特异性调节作用。结果显示，所有基因组插入miRNA靶点序列修饰的CVB1毒株均能够在转染秀丽隐杆线虫miRNA模拟物(阴性对照)或非对应miRNA模拟物的HeLa细胞中正常增殖，而在转入对应miRNA模拟物的HeLa细胞中病毒复制滴度显著下降。
图6显示了实施例8中经修饰的CVB1毒株CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-hGM-CSF和CVB1-Anti-hPD-1单针尾静脉攻毒对成年BALB/c小鼠存活率、体重和健康程度的影响。结果显示， 以上经修饰的CVB1毒株在107TCID50/只的攻毒剂量条件下均未导致小鼠发病和死亡。
图7显示了实施例9中CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-hGM-CSF和CVB1-Anti-hPD-1对(A)人肺癌细胞系A549、(B)人Burkitt's淋巴瘤细胞系Raji、(C)人子宫内膜癌细胞系HEC-1-B和(D)人宫颈癌细胞系Hela的体内抗肿瘤实验结果。结果显示，在攻毒实验组中，每隔2天瘤内注射107TCID50/每瘤块的CVB1或其修饰形式，共5针，在处理10天后，皮下接种了肿瘤细胞的SCID小鼠所形成的肿瘤生长停滞至裂解消失；相比之下，未经溶瘤病毒治疗的阴性组(CTRL)肿瘤则保持正常生长，其肿瘤体积显著大于攻毒实验组。
图8显示了实施例9中CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-mGM-CSF和CVB1-Anti-mPD-1对小鼠肝癌细胞系Hepa 1-6的体内抗肿瘤实验结果。结果显示，在攻毒实验组中，每隔2天瘤内注射107TCID50/每瘤块的CVB1或其修饰形式，共5针，在处理10天后，小鼠皮下形成肿瘤的生长停滞至裂解消失；相比之下阴性组(CTRL)肿瘤则保持正常生长，其肿瘤大小显著大于实验组。
序列信息
本发明涉及序列的全部信息提供于下面的表1中。
表1：序列的描述

注：根据产权组织标准ST.26，在序列表中，符号"u"不能用来表示RNA分子中的尿嘧啶，RNA序列中符号"t"被理解为尿嘧啶。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1：溶瘤CVB1骨架毒株的获得及制备
1.1从患者临床标本中分离肠道病毒CVB1
(1)患者的咽拭子和肛拭子来源于中国，厦门市疾病预防与控制中心；非洲绿猴肾细胞(Vero细胞；Number:CCL-81TM)由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存，使用添加有10％胎牛血清、谷氨酰胺、青霉素和链霉素的MEM培养基进行培养。
(2)标本处理：将患者咽拭子和肛拭子在标本保存液中充分搅动，以洗下拭子上粘附的病毒及含有病毒的细胞等，随后将标本保存液在4℃条件下4000rpm高速离心30min；
(3)接种与观察：
A.将Vero细胞铺24孔板，每孔细胞数量为1×105个，吸去生长液(MEM培养基，10％胎牛血清以及谷氨酰胺、青霉素和链霉素)，每孔换上1mL的维持液(MEM培养基，2％胎牛血清以及谷氨酰胺、青霉素和链霉素)，随后除了阴性对照孔，每孔接种50μL的标本上清液，37℃，5％CO2培养箱培养；
B.一周内每天在显微镜下观察细胞，记录接种孔内是否有特征性的细胞病变效应(CPE)的出现；
C.若7天内接种孔细胞出现肠道病毒特征性CPE时，收集细胞及上清至-80℃冻存；若7天后仍无CPE出现，将细胞进行盲传；
D.盲传6代内出现细胞CPE的，收集细胞及上清至-80℃冻存；盲传6代后仍未出现CPE的，判定为阴性；
(4)病毒的分离与克隆化：
使用RT-PCR与基于特异性抗体的酶联免疫斑点法(ELISPOT)鉴定从临床标本中分离获得的病毒，选取柯萨奇病毒B组1型阳性的培养物进行至少3次克隆化实验；对每 一轮由有限稀释法获得的克隆毒株同样使用RT-PCR与ELISPOT鉴定，选取柯萨奇病毒B组1型阳性的克隆毒株后进行下一轮克隆化；结合全基因组测序结果，从临床分离株中挑选出基因组序列不同的三种柯萨奇病毒B组1型克隆化毒株用于溶瘤病毒研究。
1.2基于感染性克隆与反向遗传学技术获得溶瘤CVB1骨架毒株
为保障候选溶瘤毒株的纯度与单一性，本实施例对以上分离与克隆化获得的三种CVB1毒株完成了感染性克隆构建，并通过反向遗传学技术获得了用于本发明的CVB1骨架毒株，具体方法如下所述。
(1)病毒感染性克隆构建：CVB1-1E10的cDNA序列见SEQ ID NO:1，其基因组RNA序列为SEQ ID NO:22；CVB1-D6F8的cDNA序列见SEQ ID NO:2，其基因组RNA序列为SEQ ID NO:23；CVB1-XM的cDNA序列见SEQ ID NO:3，其基因组RNA序列为SEQ ID NO:24；
然后将上述三种溶瘤病毒的cDNA序列(SEQ ID NO:1-3)送到基因合成公司(南京金斯瑞生物科技有限公司)进行全基因合成，并连接进入pSVA质粒(Hou等人，Virus Res 2015,205:41-44)，获得三种CVB1毒株(即，CVB1-1E10、CVB1-D6F8和CVB1-XM)的感染性克隆质粒。
(2)质粒小提试剂盒和E coli.DH5α感受态购自北京天根生化科技有限公司；Hela细胞(Number:CCL-2TM)由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存，用DMEM培养基、加10％胎牛血清以及谷氨酰胺、青霉素和链霉素培养；转染试剂Lipofactamine2000和Opti-MEM购自Thermo Fisher Scientific公司。
(3)将分别包含上述三种溶瘤病毒cDNA序列的感染性克隆质粒转化进入E coli.DH5α感受态，待克隆长出后直接挑取单克隆菌株摇菌后用质粒小提试剂盒提取质粒，随后送公司(上海生工生物工程股份有限公司)进行测序分析；
(4)将测序正确的感染性克隆质粒与辅助质粒pAR3126共转染细胞拯救病毒(Hou等人，Virus Res 2015,205:41-44)。首先转染Hela细胞，具体操作方法参见转染试剂说明书；随后在显微镜下观察，当细胞出现CPE时收获细胞及培养上清，并接种至新的Hela细胞进行传代与培养，获得的拯救病毒可作为溶瘤病毒候选毒株。
实施例2：溶瘤CVB1骨架毒株的体外抗肿瘤实验
2.1所使用的病毒与细胞系
(1)病毒：本实施例使用实施例1所提供的CVB1-1E10(SEQ ID NO:22)、CVB1- D6F8(SEQ ID NO:23)和CVB1-XM(SEQ ID NO:24)。
(2)细胞系：人横纹肌肉瘤细胞RD(Number:CCL-136TM)；人结直肠癌细胞系SW1116(Number:CCL-233TM)、SW480(Number:CCL-228TM)和HT-29(Number:HTB-38TM)；人胃癌细胞系AGS(Number:CRL-1739TM)、SGC7901(CCTCC保藏编号:GDC150)、BGC823(CCTCC保藏编号:GDC151)和NCI-N87(Number:CRL-5822TM)；人食管癌细胞系TE-1(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131C0001000700089)；人小细胞肺癌细胞系DMS114(Number:CRL-2066TM)；人非小细胞肺癌细胞系SPC-A-1(CCTCC保藏编号:GDC050)、NCI-H1975(Number:CRL-5908TM)、NCI-H1299(Number:CRL-5803TM)、A549(Number:CCL-185TM)、NCI-H661(Number:HTB-183TM)、EBC-1(Thermo Fisher Scientific,Catalog#:11875101)和NCI-H1703(Number:CRL-5889TM)；人肝癌细胞系C3A(Number:CRL-10741TM)、HepG2(Number:HB-8065TM)、SMMC7721(购自中国医学科学院基础医学研究所基础医学细胞中心，编号:3111C0001CCC000087)、BEL7402(CCTCC保藏编号:GDC035)、BEL7404(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131C0001000700064)、Huh7(CCTCC保藏编号:GDC134)和PLC/PRF/5(Number:CRL-8024TM)；人卵巢癌细胞系SKOV3(Number:HTB-77TM)和Caov3(Number:HTB-75TM)；人子宫内膜癌细胞系HEC-1-A(Number:HTB-112TM)、HEC-1-B(Number:HTB-113TM)和Ishikawa(ECACC No.99040201)；人宫颈癌细胞系Hela(Number:CCL-2TM)、Caski(Number:CRL-1550TM)和C-33A(Number:HTB-31TM)；人黑色素瘤细胞系SK-MEL-1(Number:HTB-67TM)和MeWo(Number:HTB-65TM)；人乳腺癌细胞系BcaP37(CCTCC保藏编号:GDC206)、BT-474(Number:HTB-20TM)和MDA-MB-231(Number:HTB-26TM)；人肾癌细胞系A-498(Number:HTB-44TM)和786-O(Number:CRL-1932TM)；人胰腺癌细胞系Capan-2(Number:HTB-80TM)、AsPC-1(Number:CRL-1682TM)、SW1990(Number:CRL-2172TM)、HPAF-2(Number:CRL-1997TM)和CFPAC-1(Number:CRL-1918TM)；人成骨肉瘤细胞系U2OS(Number:HTB-96TM)；人前列腺癌细胞系DU145(Number:HTB-81TM)和LNCap(Number:CRL-1740TM)；人神经胶质瘤细胞系GBM(病人肿 瘤组织分离原代肿瘤细胞系)；人神经母瘤细胞系SH-SY5Y(Number:CRL-2266TM)；人舌鳞癌细胞系CAL27(Number:CRL-2095TM)和SCC-25(Number:CRL-1628TM)；人鼻咽癌细胞系CNE(购自中国医学科学院基础医学研究所基础医学细胞中心，编号:3131C0001000700013)；人鼻中隔鳞状癌细胞系RPMI 2650(Number:CCL-30TM)；人喉癌细胞系HEp-2(Number:CCL-23TM)；人甲状腺癌细胞系SW579(由国家传染病诊断试剂与疫苗工程技术研究中心保存)和人甲状腺导管癌细胞系TT(Number:CRL-1803TM)；人膀胱癌细胞系J82(Number:HTB-1TM)和5637(Number:HTB-9TM)；人Burkitt's淋巴瘤细胞系Daudi(Number:CCL-213TM)和Raji(Number:CCL-86TM)；人正常细胞系包括：人胰腺导管上皮细胞系hTERT-HPNE(Number:CRL-4023TM)、人皮肤角化细胞系HaCat(CCTCC保藏编号:GDC106)、人胚肺成纤维细胞系MRC-5(Number:CCL-171TM)、人包皮成纤维细胞系HFF-1(Number:SCRC-1041TM)、人前列腺基质细胞系WPMY-1(Number:CRL-2854TM)、人脐静脉内皮细胞系HUVEC(Thermo Fisher Scientific,Catalog#:C01510C)以及分化后的人肝祖细胞系HepaRG(具原代肝细胞特征；Thermo Fisher Scientific,Catalog#:HPRGC10)。以上细胞均由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存。HepaRG细胞在WME培养基(添加1.5％DMSO)中培养；AGS和TT使用F-12K培养基；SH-SY5Y使用DMEM:F12(1:1)培养基；CFPAC-1使用IMDM培养基；RD、C-33A、EBC-1、SK-MEL-1、J82和DU145使用MEM培养基，Raji、Daudi、5637、786-O、TE-1、Caski、NCI-H1299、NCI-H1703、NCI-H1975、NCI-H661、SGC7901、BGC823、SW1116、HEp-2和LNCap使用RPMI-1640培养基，其他细胞都使用DMEM培养基，这些培养基均需添加10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞在37℃，5％CO2的标准条件下培养。
2.2病毒的培养
将RD细胞均匀铺于10厘米细胞培养板上，培养条件为含有10％胎牛血清以及谷氨酰胺、青霉素和链霉素的MEM培养基，37℃，5％CO2，饱和湿度；待细胞汇合度达到90％以上时，将细胞培养基更换为无血清的MEM培养基，每板接种107TCID50的CVB1，继续培养24小时后，CVB1在RD细胞内增殖并引起细胞出现CPE；当90％以上细胞出现收缩变圆、颗粒感增加以及脱落裂解时，收获细胞及其培养液上清；经3次反复冻融后，收集培养上清进行离心除去细胞碎片，离心条件为4000rpm，10min，4℃；最后， 用0.22μm的一次性滤器(Millipore公司)过滤上清，除尽细胞碎片等杂质。
2.3病毒滴度的测定
将RD细胞铺于96孔板当中，细胞密度为104个/孔；待细胞贴壁之后，对实施例2.2中获得的病毒液用无血清MEM培养基进行10倍起始的10倍梯度稀释。加50μl稀释后的病毒到细胞孔当中，7天后观察并标记出现CPE的细胞孔，利用Karber法进行计算，计算公式为lgTCID50＝L-D(S-0.5)，L：最高稀释度的对数，D：稀释度对数之间的差，S：阳性孔比率总和。此时计算出来的TCID50单位为TCID50/50μl，需换算成TCID50/mL。
2.4病毒的体外抗肿瘤实验
将人肿瘤细胞与正常细胞按照104个/孔接种至96孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，并且接种MOI为0.1的病毒；随后，在病毒感染72小时后使用Cell Counting Kit-8(CCK-8试剂盒；上海碧云天生物技术有限公司)检测细胞存活率，具体方法如下：
在96孔细胞培养板中，贴壁细胞直接弃掉原培养基，悬浮细胞经离心后小心弃掉原培养基，随后更换为每孔100μL的新鲜无血清培养基；在接种细胞的孔中每孔加入10μL CCK-8溶液，同时在空白培养液中也加入等量的CCK-8溶液作为阴性对照；在细胞培养箱内37℃孵育0.5-3小时，在0.5、1、2、3小时分别利用酶标仪在吸光度为450nm进行一次检测，选取吸光度范围比较适宜的时间点作为细胞存活率的参考。三种CVB1毒株对各细胞的CCK-8检测结果参见表2，其中，“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
细胞存活率的计算方法为：
表2：CVB1溶瘤病毒骨架毒株的体外抗肿瘤实验结果(MOI 0.1)


注：“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
由表2可知，CVB1-1E10对大部分所检测的肿瘤细胞都有杀伤作用，特别的，该病毒对结直肠癌细胞系、胃癌细胞系、肺癌细胞系、肝癌细胞系、宫颈癌细胞系、子宫内膜癌细胞系、胰腺癌细胞系、前列腺癌细胞系、鼻咽癌细胞系、舌癌细胞系、喉癌细胞系、神经胶质瘤细胞系和神经母细胞瘤细胞系等杀伤十分显著；而CVB1-D6F8的溶瘤活性明显较弱。另一方面，这些病毒对非肿瘤的正常细胞系，包括：人正常胰腺导管上皮细胞系hTERT-HPNE、分化后的人肝祖细胞系HepaRG、人胚肺成纤维细胞系MRC-5、人包皮成纤维细胞系HFF-1、人皮肤角化细胞系HaCat、人前列腺基质细胞系WPMY-1和人脐静脉内皮细胞系HUVEC基本都没有毒性。
2.5 CVB1基因组RNA的溶瘤效果评价
在本实施例中，通过将纯化的CVB1基因组RNA转染某一种肿瘤细胞可产出大量具有感染性的CVB1活病毒并对肿瘤细胞造成杀伤。
首先通过体外转录的方法以获得CVB1基因组RNA，该方法可参见例如Hadac E M, Kelly E J and Russell S J.Mol Ther，2011,19(6):1041-1047。具体而言，将实施例1中所获得的野生型CVB1的感染性克隆质粒线性化，以该线性化质粒为模板，使用试剂盒MEGAscriptTM T7 Transcription Kit(Thermo Fisher Scientific,AM1333)体外转录并大量生产病毒RNA，再使用MEGAclearTMTranscription Clean-Up Kit(Thermo Fisher Scientific,AM1908)纯化获得的病毒RNA待用，1份样品的RNA电泳图谱如图1所示。
随后，通过实施例2.4中所述的体外抗肿瘤实验的操作方法，将人宫颈癌细胞系Hela按照105个/孔接种至24孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，37℃孵育30min，再使用转染试剂2000(Thermo Fisher Scientific,11668019)将纯化好的病毒RNA按照1μg每孔转染Hela细胞，阴性对照组转染无关RNA核酸分子，随后每天在显微镜下观察细胞是否产生CPE。
结果发现，转染CVB1基因组RNA的Hela细胞在转染后8小时左右开始出现CPE，随后细胞病变逐渐加剧，48小时后使用CCK8法检测存活率，Hela细胞已经几乎全部死亡裂解，Hela细胞转染48小时后的显微镜照片如图2所示；培养上清接种到新的Hela细胞中又可很快产生CPE。该结果表明，直接施用CVB1的核酸亦具有良好的杀伤活性，可用于治疗肿瘤。
实施例3：溶瘤CVB1骨架毒株的动物毒性实验
3.1病毒与实验动物
(1)病毒：本实施例中使用实施例1所提供的CVB1-1E10(SEQ ID NO:22)、CVB1-D6F8(SEQ ID NO:23)和CVB1-XM(SEQ ID NO:24)。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。
(2)实验动物：6-8周龄的雌性BALB/c小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在SPF条件下饲养。
3.2病毒的动物毒性实验
(1)动物的分组与攻毒
将6～8周龄的雌性BALB/c小鼠按每笼8只分笼，每批次实验设立阴性对照组，攻毒液体为MEM细胞培养液。采用尾静脉攻毒方式：使用小鼠固定器固定小鼠，选择尾部侧面静脉缓慢插入针头(1mL胰岛素注射针，BD)，轻轻推入100μL病毒液(每组按10倍梯度稀释的剂量给药，剂量范围为106～108TCID50/100μL/只)；每批次实验设立阴性 对照组，攻毒100μL MEM细胞培养液，随后将其放回笼中。
(2)动物攻毒后的监测
攻毒后需每日记录BALB/c小鼠的存活率、体重，并观察其体征。攻毒后第一天死亡不计入统计，连续观察7至20天并记录。小鼠健康程度将严格按照体征状态分数制记录，其具体如下：
表3：动物健康状态评分表
结果如图3所示，CVB1-XM和CVB1-D6F8在106～108TCID50/只这3个攻毒剂量条件下均可使成年BALB/c小鼠发病(竖毛、弓背、运动迟缓、体重持续下降)且致死，而CVB1-1E10在以上3个攻毒剂量条件下均未导致小鼠发病和死亡。
实施例4：溶瘤CVB1骨架毒株的溶瘤活性和毒力决定位点分析
基于上述实验数据可知，CVB1-D6F8溶瘤活性差于CVB1-1E10，毒性强于CVB1-1E10；CVB1-XM溶瘤活性与CVB1-1E10相近，但毒性强于CVB1-1E10。通过对CVB1-1E10(SEQ ID NO:22)、CVB1-D6F8(SEQ ID NO:23)和CVB1-XM(SEQ ID NO:24)的基因组编码氨基酸序列进行了对比，发现这3个毒株主要在VP1蛋白第84位氨基酸，VP1蛋白第224位氨基酸和VP3蛋白第232位氨基酸这3个位点存在差异。其中，CVB1-1E10与CVB1-D6F8基因组编码的氨基酸序列之间仅存在4个氨基酸差异，除以上3个位点外，还包括3D蛋白的第362位氨基酸(CVB1-1E10该位点为K，CVB1-D6F8该位点是E)，但是这两个毒株在体外溶瘤活性与小鼠体内毒性上差异较大(见表2和图3)。CVB1-D6F8的VP1蛋白第84位为E，VP1蛋白第224位为A，VP3蛋白第232位为R；CVB1-XM的VP1蛋白第84位为K，VP1蛋白第224位为A，VP3蛋白第232位为R；CVB1-1E10的VP1蛋白第84位为K，VP1蛋白第224位为V，VP3蛋白第232位为G。
为探究CVB1-1E10相较于CVB1-D6F8具有低毒安全和较强溶瘤活性表型的决定性位点，在CVB1-D6F8株cDNA(SEQ ID NO:2)的基础上，对结构蛋白存在差异的3个氨基酸位点进行向CVB1-1E10株的逐点突变改造，包括单点突变(CVB1-D6F8-R232G、 CVB1-D6F8-E84K和CVB1-D6F8-A224V)、双点突变(CVB1-D6F8-RG-AV、CVB1-D6F8-RG-EK和CVB1-D6F8-EK-AV)和3点突变(CVB1-D6F8-GKV)，并基于感染性克隆与反向遗传学技术获得以上基因突变的CVB1毒株，方法参见实施例1.2。
本实施例中使用实施例2.4所提供的部分肿瘤细胞对以上基因突变的CVB1毒株进行溶瘤活性变化的验证，检测病毒体外溶瘤活性的方法参考实施例2.4。结果如表4所示，除3点突变的CVB1-D6F8-GKV表现出与CVB1-1E10相当的、较强的体外溶瘤活性，其他点突变CVB1株表现出与CVB1-D6F8相当的、较差的溶瘤活性。
表4：CVB1-D6F8点突变毒株的体外抗肿瘤实验结果(MOI 0.1)
注：“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
本实施例中参考实施例3.2方法，选择107TCID50/只的攻毒剂量，对以上基因突变的CVB1毒株进行了在成年BALB/c小鼠中的单针静脉注射毒性实验。结果如图4所示，单点突变的CVB1-D6F8-R232G和CVB1-D6F8-A224V在107TCID50/只的攻毒剂量条件下均可使成年BALB/c小鼠发病(竖毛、弓背、运动迟缓、体重持续下降)且部分致死，然而单点突变的CVB1-D6F8-E84K、双点突变的CVB1-D6F8-RG-AV、CVB1-D6F8-RG-EK和CVB1-D6F8-EK-AV、以及3点突变的CVB1-D6F8-GKV在该攻毒剂量条件下均未导致小鼠发病和死亡。
以上研究结果表明，VP1蛋白第84位氨基酸是CVB1-1E10与CVB1-D6F8毒力差异的关键位点，而VP1蛋白第224位氨基酸和VP3蛋白第232位氨基酸对其毒力差异也有影响，但是CVB1-1E10相比CVB1-D6F8具有的较好溶瘤活性则与以上3个氨基酸位点共同相关。同时，以上研究结果也说明，可以通过对CVB1毒株进行人工改造(例如氨基酸置换)以获得上述VP1/VP3蛋白特征，从而使其兼具高效广谱的肿瘤杀伤活性以及显著提高的安全性。
实施例5：经修饰溶瘤CVB1毒株的构建与制备
选取体外溶瘤活性较优且对小鼠低毒安全的野生型CVB1-1E10作为骨架毒株，在其cDNA(SEQ ID NO:1)的基础上做基因插入，包括：
修饰形式1：将miR-1-3p靶序列(其DNA序列见SEQ ID NO:30)和miR-126-3p靶序列(其DNA序列见SEQ ID NO:31)的串联序列(其DNA序列见SEQ ID NO:36)、以及miR-216a-5p靶序列(其DNA序列见SEQ ID NO:33)和miR-217-5p靶序列(其DNA序列见SEQ ID NO:34)的串联序列(其DNA序列见SEQ ID NO:37)分别插入到野生型CVB1的cDNA(SEQ ID NO:1)的5’非翻译区737-738bp之间和3’非翻译区7296-7297bp之间，从而获得重组病毒(命名为CVB1-miR1/126/216a/217T)的cDNA(SEQ ID NO:17)，其基因组RNA序列为SEQ ID NO:25；
修饰形式2：将miR-1-3p靶序列(其DNA序列见SEQ ID NO:30)和miR-126-3p靶序列(其DNA序列见SEQ ID NO:31)的串联序列(其DNA序列见SEQ ID NO:36)、 以及miR-204-5p靶序列(其DNA序列见SEQ ID NO:32)和miR-219a-5p靶序列(其DNA序列见SEQ ID NO:35)的串联序列(其DNA序列见SEQ ID NO:38)分别插入到野生型CVB1的cDNA(SEQ ID NO:1)的5’非翻译区737-738bp之间和3’非翻译区7296-7297bp之间，从而获得重组病毒(命名为CVB1-miR1/126/204/219aT)的cDNA(SEQ ID NO:18)，其基因组RNA序列为SEQ ID NO:26；
修饰形式3：将miR-216a-5p靶序列(其DNA序列见SEQ ID NO:33)和miR-217-5p靶序列(其DNA序列见SEQ ID NO:34)的串联序列(其DNA序列见SEQ ID NO:37)、以及miR-204-5p靶序列(其DNA序列见SEQ ID NO:32)和miR-219a-5p靶序列(其DNA序列见SEQ ID NO:35)的串联序列(其DNA序列见SEQ ID NO:38)分别插入到野生型CVB1的cDNA(SEQ ID NO:1)的5’非翻译区737-738bp之间和3’非翻译区7296-7297bp之间，从而获得重组病毒(命名为CVB1-miR216a/217/204/219aT)的cDNA(SEQ ID NO:19)，其基因组RNA序列为SEQ ID NO:27；
修饰形式4：将人粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因(SEQ ID NO:13)插入到野生型CVB1的cDNA(SEQ ID NO:1)的VP1与2A基因之间，从而获得重组病毒(命名为CVB1-hGM-CSF)的cDNA(SEQ ID NO:20)，其基因组RNA序列为SEQ ID NO:28；
修饰形式5：将编码抗人程序性死亡受体1单链抗体(Anti-PD-1scFv)的序列(SEQ ID NO:14)插入到野生型CVB1的cDNA(SEQ ID NO:1)的VP1与2A基因之间，从而获得该重组病毒(命名为CVB1-Anti-hPD-1)的cDNA(SEQ ID NO:21)，其基因组RNA序列为SEQ ID NO:29。
基于感染性克隆与反向遗传学技术获得以上溶瘤CVB1毒株，方法参见实施例1.2
实施例6：经修饰溶瘤CVB1携带miRNA靶向序列的功能分析
miR-1-3p、miR-126-3p、miR-204-5p、miR-216a-5p、miR-217-5p和miR-219a-5p的miRNA模拟物和对应于秀丽隐杆线虫miRNA的阴性对照模拟物均购自Dharmacon。使用Mirus mRNA转染试剂在200nM的浓度下将miRNA模拟物转染到HeLa细胞中。6小时后，细胞感染经插入miRNA靶点序列修饰的病毒CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT和CVB1-miR216a/217/204/219aT，MOI为0.1。在感染后24小时，收集上清液进行病毒滴度滴定，并用CCK-8法检测细胞增殖情况，方法分别参考实施例2.3和2.4。
结果如图5所示，所有经插入miRNA靶点序列修饰的CVB1毒株均能够在转染秀丽隐杆线虫miRNA模拟物的HeLa细胞中正常增殖，而在转入对应miRNA模拟物的HeLa细胞中的复制滴度出现显著下降。这说明插入病毒基因组的miRNA靶点序列能够被细胞内对应miRNA有效识别，导致病毒的基因组降解和复制抑制，有望实现溶瘤病毒对正常组织的去靶向从而降低毒性，提高溶瘤病毒的安全性能。
实施例7：经修饰溶瘤CVB1毒株的体外抗肿瘤实验
(1)病毒：本实施例中使用实施例1所提供的CVB1-1E10(SEQ ID NO:22)和实施例5所提供的CVB1-miR1/126/216a/217T(SEQ ID NO:25)、CVB1-miR1/126/204/219aT(SEQ ID NO:26)、CVB1-miR216a/217/204/219aT(SEQ ID NO:27)、CVB1-hGM-CSF(SEQ ID NO:28)和CVB1-Anti-hPD-1(SEQ ID NO:29)。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。
(2)细胞系：本实施例中使用实施例2.4所提供的部分肿瘤细胞对CVB1各类修饰形式病毒毒株进行溶瘤活性变化的验证，检测病毒体外溶瘤活性的方法参考实施例2.4。结果如表5所示，经修饰的5株CVB1溶瘤活性具备显著的溶瘤活性。
表5：经修饰溶瘤CVB1毒株的体外抗肿瘤实验结果(MOI 0.1)

注：“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
实施例8：经修饰溶瘤CVB1毒株的动物毒性实验
8.1病毒与实验动物
(1)病毒：本实施例中使用实施例5所提供的CVB1-miR1/126/216a/217T(SEQ ID NO:25)、CVB1-miR1/126/204/219aT(SEQ ID NO:26)、CVB1-miR216a/217/204/219aT(SEQ ID NO:27)、CVB1-hGM-CSF(SEQ ID NO:28)和CVB1-Anti-hPD-1(SEQ ID NO:29)。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。
(2)实验动物：6-8周龄的雌性BALB/c小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在SPF条件下饲养。
8.2病毒的动物毒性实验
病毒攻毒的毒性实验参考实施例3.2方法在6-8周龄BALB/c小鼠中实施，选择107TCID50/只的攻毒剂量，单针静脉攻毒后观察并记录小鼠存活率、体重与健康分数。结果如图6所示，以上所有经修饰的CVB1毒株在107TCID50/只的攻毒剂量条件下均未导致小鼠发病和死亡。
实施例9：经修饰溶瘤CVB1毒株的体内抗肿瘤实验
9.1病毒、细胞系与实验动物
(1)病毒：本实施例中使用实施例1所提供的CVB1-1E10(SEQ ID NO:22)和实施例5所提供的CVB1-miR1/126/216a/217T(SEQ ID NO:25)、CVB1- miR1/126/204/219aT(SEQ ID NO:26)、CVB1-miR216a/217/204/219aT(SEQ ID NO:27)、CVB1-hGM-CSF(SEQ ID NO:28)和CVB1-Anti-hPD-1(SEQ ID NO:29)。另外，为在免疫健全小鼠荷瘤模型评估插入表达GM-CSF和Anti-PD-1对CVB1溶瘤病毒的增效作用，构建了携带小鼠GM-CSF(cDNA序列，SEQ ID NO:15)和抗小鼠PD-1scFv序列(cDNA序列，SEQ ID NO:16)的CVB1-mGM-CSF和CVB1-Anti-mPD-1，基于感染性克隆与反向遗传学技术获得以上2个溶瘤CVB1毒株，方法参见实施例1.2。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。
(2)细胞系：人非小细胞肺癌细胞系A549(Number:CCL-185TM)、人Burkitt's淋巴瘤细胞系Raji(Number:CCL-86TM)、人子宫内膜癌细胞系HEC-1-B(Number:HTB-113TM)、人宫颈癌细胞系Hela(Number:CCL-2TM)和小鼠肝癌细胞系Hepa 1-6(Number:CRL-1830TM)。以上细胞除Raji使用RPMI-1640培养基，其他细胞均使用DMEM培养基培养，并且上述培养基均添加入10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞均在37℃，5％CO2的标准条件下培养。
(3)实验动物：6-8周龄的雌性BALB/c裸鼠和C57BL/6小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在SPF条件下饲养。
9.2病毒的体内抗肿瘤实验
将用于小鼠皮下成瘤的肿瘤细胞用0.01％胰蛋白酶消化后，再使用含10％的胎牛血清的细胞培养基重悬成单细胞悬液；计数悬液的细胞密度，1000g，3min离心沉淀细胞，再用适量体积的PBS重悬细胞，使达到约106个细胞/100μL PBS；按照106个细胞/100μL PBS/点在小鼠背部皮下用注射器接种肿瘤细胞，待14-21天左右、肿瘤细胞在小鼠皮下形成大约100mm3的瘤块的时候，将荷瘤小鼠随机分为不同实验组(裸鼠荷瘤模型分别攻毒CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-hGM-CSF和CVB1-Anti-hPD-1；免疫健全小鼠荷瘤模型分别攻毒CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-mGM-CSF和CVB1-Anti-mPD-1)与阴性对照组，每组4只(n＝4)，用107TCID50/100μL无血清培养基/每瘤块的溶瘤病毒或等量无血清培养基瘤内注射处理，每两天注射1次，共处理5次。每两天用游标卡尺测量并记录肿瘤大小变化，肿瘤大小的计算方法为：
肿瘤大小(mm3)＝肿瘤长度数值×(肿瘤宽度数值)2/2。
图7A-7D显示了CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-hGM-CSF和CVB1-Anti-hPD-1分别治疗(A)A549、(B)Raji、(C)HEC-1-B和(D)Hela肿瘤模型后18天的检测结果。结果显示，经治疗后肿瘤生长均出现逐渐变慢和停滞，甚至裂解消失；相比之下，阴性组(CTRL)肿瘤则保持正常生长，其肿瘤大小显著大于实验组。
图8显示了CVB1-1E10、CVB1-miR1/126/216a/217T、CVB1-miR1/126/204/219aT、CVB1-miR216a/217/204/219aT、CVB1-mGM-CSF和CVB1-Anti-mPD-1分别治疗Hepa1-6肿瘤模型后18天的检测结果。结果显示，经治疗后的肿瘤体积显著减小；相比之下阴性组(CTRL)肿瘤则保持正常生长，其肿瘤大小显著大于实验组。
上述结果表明经修饰的溶瘤CVB1毒株均展现出显著有利的体内抗肿瘤活性，且插入表达GM-CSF和抗PD-1scFv可有效提升溶瘤CVB1毒株的肿瘤治疗效果，同时还具备提高的体内安全性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (18)

  1. 分离的柯萨奇病毒B组1型(CVB1)，其中，所述CVB1的VP1蛋白第84位氨基酸为赖氨酸(K)，VP1蛋白第224位氨基酸为缬氨酸(V)，并且VP3蛋白第232位氨基酸为甘氨酸(G)。
  2. 权利要求1所述的CVB1，其中，所述CVB1的VP1蛋白具有如SEQ ID NO:39所示的氨基酸序列，和/或，所述VP3蛋白具有如SEQ ID NO:41所示的氨基酸序列。
  3. 权利要求1或2所述的CVB1，其中，
    1)所述CVB1的基因组序列如SEQ ID NO:22所示或者与SEQ ID NO:22所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子，和/或，
    2)所述CVB1的cDNA序列如SEQ ID NO:1所示或者与SEQ ID NO:1所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
  4. 权利要求1-3任一项所述的CVB1，其中，所述CVB1为重组病毒，并且包含外源核酸；
    优选地，所述外源核酸选自编码细胞因子的核酸序列、编码抗肿瘤蛋白或多肽的核酸序列、微小RNA的靶序列、或其任意组合。
  5. 权利要求4所述的CVB1，其中，所述外源核酸包含编码细胞因子和/或编码抗肿瘤蛋白或多肽的核酸序列；
    优选地，所述细胞因子为具有抗肿瘤活性的细胞因子，例如白介素(例如IL-2、IL-12、IL-15)、干扰素(例如IFN-α、IFN-β、IFN-γ)、肿瘤坏死因子(例如TNF-α)或集落刺激因子(例如GM-CSF)；优选地，所述细胞因子为GM-CSF(例如人GM-CSF)；
    优选地，所述抗肿瘤蛋白或多肽为免疫检查点抑制剂(例如PD-L1抗体、PD-1抗体、CTLA-4抗体)；优选地，所述抗肿瘤蛋白或多肽为抗PD-1或PD-L1的scFv(例如抗人PD-1或PD-L1的scFv)；
    优选地，所述编码细胞因子和/或抗肿瘤蛋白或多肽的核酸序列的插入位点位于所述病毒基因组的5’UTR与VP4基因之间，或者位于VP1基因与2A基因之间。
  6. 权利要求4或5所述的CVB1，其中，所述外源核酸包含微小RNA的靶序列；
    优选地，所述微小RNA为肿瘤抑制性微小RNA，例如miR-1-3p、miR-126-3p、miR-204-5p、miR-217-5p、miR-219a-5p、或其任意组合；
    优选地，所述外源核酸包含一个或多个(例如2个，3个或4个)微小RNA的靶序列；
    优选地，所述微小RNA的靶序列的插入位点在所述病毒基因组的5’非翻译区(5’UTR)和/或3’非翻译区(3’UTR)；
    优选地，所述微小RNA包含miR-1-3p和/或miR-126-3p；优选地，所述miR-1-3p的靶序列如SEQ ID NO:4所示；优选地，所述miR-126-3p的靶序列如SEQ ID NO:5所示；
    优选地，所述微小RNA包含miR-216a-5p和/或miR-217-5p；优选地，所述miR-216a-5p的靶序列如SEQ ID NO:7所示；优选地，所述miR-217-5p的靶序列如SEQ ID NO:8所示；
    优选地，所述微小RNA包含miR-204-5p和/或miR-219a-5p；优选地，所述miR-204-5p的靶序列如SEQ ID NO:6所示；优选地，所述miR-219a-5p的靶序列如SEQ ID NO:9所示。
  7. 权利要求4-6任一项所述的CVB1，其中，
    1)所述重组病毒的基因组序列如SEQ ID NO:25-29任一项所示或者与SEQ ID NO:25-29任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子；和/或，
    2)所述重组病毒的cDNA序列如SEQ ID NO：17-21任一项所示或者与SEQ ID NO:17-21任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子。
  8. 分离的核酸分子，其包含：
    (1)权利要求1-7任一项所述的分离的CVB1的基因组序列或cDNA序列；和
    (2)所述基因组序列或cDNA序列的互补序列。
  9. 权利要求8所述的核酸分子，其中：
    1)所述核酸分子由所述CVB1的基因组序列或cDNA序列，或所述基因组序列或cDNA序列的互补序列组成；优选地，所述核酸分子具有所述CVB1的基因组序列；优选地，所述核酸分子具有如SEQ ID NO:22，25-29任一项所示的核苷酸序列或者与SEQ ID NO:22，25-29任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子的核苷酸序列；
    或，
    2)所述核酸分子为包含所述CVB1的基因组序列或cDNA序列，或所述基因组序列或cDNA序列的互补序列的载体(例如，克隆载体或表达载体)；优选地，所述核酸分子为包含所述CVB1的cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)；优选地，所述核酸分子为包含SEQ ID NO:1，17-21任一项所示的核苷酸序列或者与SEQ ID NO:1，17-21任一项所示的序列相比差异仅在于，对应非结构蛋白3D第362位赖氨酸(K)的密码子被置换为编码谷氨酸(E)的密码子的核苷酸序列的载体。
  10. 药物组合物，其包含权利要求1-7任一项所述的分离的CVB1、或权利要求8或9所述的分离的核酸分子，以及药学上可接受的载体或赋形剂；
    优选地，所述药物组合物还包含具有抗肿瘤活性的另外的药学活性剂，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂；
    优选地，所述另外的溶瘤病毒选自疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合；
    优选地，所述化学治疗剂选自5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合；
    优选地，所述免疫治疗剂选自免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
  11. 权利要求1-7任一项所述的分离的CVB1、或权利要求8或9所述的分离的核酸分子、或权利要求10所述的药物组合物用于制备在受试者中治疗肿瘤的药物的用途。
  12. 权利要求11所述的用途，其中，所述肿瘤选自实体瘤或血液肿瘤，例如结直肠癌、胃癌、肺癌、肝癌、卵巢癌、子宫内膜癌、宫颈癌、黑色素瘤、乳腺癌、肾癌、胰腺癌、淋巴瘤、成骨肉瘤、前列腺癌、神经胶质瘤、神经母细胞瘤、舌癌、鼻咽癌、鼻中隔鳞状细胞癌、咽鳞癌、颌下腺鳞癌、喉癌、甲状腺癌、甲状腺导管癌和膀胱癌。
  13. 权利要求11或12所述的用途，其中，所述受试者为哺乳动物，例如人。
  14. 权利要求11-13任一项所述的用途，其中，所述分离的CVB1、分离的核酸分子、或药物组合物与具有抗肿瘤活性的另外的药学活性剂联合使用；
    优选地，所述另外的药学活性剂选自另外的溶瘤病毒、化学治疗剂或免疫治疗剂；
    优选地，所述另外的溶瘤病毒选自疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合；
    优选地，所述化学治疗剂选自5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合；
    优选地，所述免疫治疗剂选自免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
  15. 用于在受试者(例如人)中治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的权利要求1-7任一项所述的分离的CVB1、或权利要求8或9所述的分离的核酸分子、或权利要求10所述的药物组合物。
  16. 权利要求15所述的方法，其中，所述肿瘤选自实体瘤或血液肿瘤，例如结直肠癌、胃癌、肺癌、肝癌、卵巢癌、子宫内膜癌、宫颈癌、黑色素瘤、乳腺癌、肾癌、胰腺癌、淋巴瘤、成骨肉瘤、前列腺癌、神经胶质瘤、神经母细胞瘤、舌癌、鼻咽癌、鼻中隔鳞状细胞癌、咽鳞癌、颌下腺鳞癌、喉癌、甲状腺癌、甲状腺导管癌和膀胱癌。
  17. 权利要求15或16所述的方法，其中，所述受试者为哺乳动物，例如人。
  18. 权利要求15-17任一项所述的方法，其中，所述分离的CVB1、分离的核酸分子、或药物组合物与具有抗肿瘤活性的另外的药学活性剂联合使用；
    优选地，所述另外的药学活性剂选自另外的溶瘤病毒、化学治疗剂或免疫治疗剂；
    优选地，所述另外的溶瘤病毒选自疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合；
    优选地，所述化学治疗剂选自5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合；
    优选地，所述免疫治疗剂选自免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
PCT/CN2023/134325 2022-11-28 2023-11-27 用于治疗肿瘤的柯萨奇b组1型病毒 WO2024114576A1 (zh)

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