WO2021246815A1 - Recombinant herpes simplex virus having expression cassette capable of expressing fusion protein of extracellular domain of hvec and cancer cell-targeting domain, and use thereof - Google Patents

Recombinant herpes simplex virus having expression cassette capable of expressing fusion protein of extracellular domain of hvec and cancer cell-targeting domain, and use thereof Download PDF

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WO2021246815A1
WO2021246815A1 PCT/KR2021/006983 KR2021006983W WO2021246815A1 WO 2021246815 A1 WO2021246815 A1 WO 2021246815A1 KR 2021006983 W KR2021006983 W KR 2021006983W WO 2021246815 A1 WO2021246815 A1 WO 2021246815A1
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herpes simplex
simplex virus
hvec
cancer
hsv
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Korean (ko)
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권희충
백현정
주현유
박은란
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주식회사 젠셀메드
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an extracellular domain of HveC and a cancer cell targeting region, and uses thereof.
  • Surgical therapy, chemotherapy, radiation therapy, etc. have been widely used for cancer treatment, but most of them have side effects, incomplete therapeutic effects, and problems such as cancer recurrence and metastasis. Therefore, there has been a continuous demand for the development of new and effective cancer treatment methods, and in recent years, there has been rapid development in anticancer immunotherapy such as anticancer viruses and CAR-T cell therapy (chimeric antigen receptor T cell therapy).
  • anticancer immunotherapy such as anticancer viruses and CAR-T cell therapy (chimeric antigen receptor T cell therapy).
  • anticancer virus is a virus with the property of lysing cancer cells by selectively proliferating in cancer cells by manipulating the genes of living viruses, and proliferation in normal cells is limited. Viruses released by dissolving cancer cells continuously infect surrounding cancer cells, thereby producing a lasting and synergistic therapeutic effect.
  • anticancer viruses can increase the anticancer effect by stimulating the body's immune response by releasing tumor antigens with immunogenicity in the process of dissolving cancer cells. may be enhanced.
  • Anticancer viruses currently being developed can be divided into more than 10 types including adenovirus, herpes simplex virus (HSV), and vaccinia virus.
  • HSV herpes simplex virus
  • vaccinia virus As an enveloped icosahedral virion, it is divided into HSV-1 type and HSV-2 type.
  • HSV has many non-essential genes and has a large genome, so it is easy to manipulate or transport foreign genes, has a short replication cycle, and has high infection efficiency. It has the advantage of being able to improve the targeting to cancer cells by easy manipulation of the protein.
  • T-VEC (Talimogene Laherparepvec, product name: Imrisic), approved by the US FDA in October 2015, is an anticancer drug for malignant melanoma using HSV-1.
  • T-VEC is a recombinant HSV-1 type virus in which ICP34.5 and ICP47 genes are deleted to attenuate pathogenicity, and which expresses GM-CSF (granulocyte-macrophage colony stimulating factor) to promote human immune response.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • HSV is a virus with an envelope (envelope), and HSV cell entry is achieved through the complex interaction of the glycoproteins gD, gB, gH/gL and gC present in the envelope.
  • gB and gC attach to 3-O- S HS (3- O- sulfated heparan sulfate) on the cell surface, and gD is a cell receptor, herpesvirus entry mediator (HVEM), and nectin-1 (HveC).
  • HVEM herpesvirus entry mediator
  • HveC nectin-1
  • HSV enters the cell by inducing fusion between the virus and the cell membrane by binding to at least one receptor of nectin-2 (HveB) (Hiroaki Uchida et al., Generation of Herpesvirus Entry Mediator (HVEM)- restricted Herpes Simplex Virus Type 1 Mutant Viruses: Resistance of HVEM-expressing Cells and Identification of Mutations That Rescue nectin-1 Recognition. J Virol. 2009 Apr;83(7):2951-61).
  • HVEM Herpesvirus Entry Mediator
  • HveC(nectin-1) consists of four cystein-rich domains (CRDs), among which, two CRDs at the amino terminus bind to gD of HSV, thereby reducing the levels of HSV-1 and HSV-2. It has been reported to induce cell entry (Sarah A Connolly et al., Structure-based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM). J Virol. 2002 Nov;76(21): 10894-904. ).
  • CEAscFv-HA a fusion protein of a single-chain variable fragment (scFv) for CEA (carcinoembryonal antigen) and an extracellular domain of HVEM, one of the HSV cell surface receptors, and the fusion protein was used for HSV It has been previously reported that, when treated with a cell line expressing CEA with and US Patent No. 8318662).
  • the fusion proteins HveC-CEA and HveC-Her2scFv which are adapters of different configurations, are inserted between UL3 and UL4 genes of the herpes virus genome so that the fusion protein is expressed in infected cells. This was completed by confirming that it targets and infects cell lines expressing CEA or Her2.
  • the present invention relates to a cancer cell that specifically recognizes and binds the target molecule of HveC to the extracellular domain, particularly the V domain (SEQ ID NO: 11, 31 to 144 amino acid sequence region from which 1 to 30 amino acid sequences are excluded from SEQ ID NO: 12) It relates to a recombinant Herpes Simplex Virus (HSV) capable of expressing an adapter that is a fusion protein of a targeting region.
  • HSV Herpes Simplex Virus
  • the recombinant HSV of the present invention infects a target cell, which is a cancer cell, and enters the target cell, the HSV proliferates and an adapter, which is a fusion protein, is expressed in the cell, and is released together with the proliferated HSV virion through cell lysis to the outside of the cell.
  • the adapter released to the outside of the cell induces infection with the HSV virions released together with surrounding cancer cells expressing a target molecule recognized by the cancer cell targeting region of the adapter or increases the infection efficiency.
  • recombinant HSV is compared to wild-type HSV virus by introducing artificial mutations (ie, deletion, substitution, or insertion of some nucleic acid sequences) resulting in loss or alteration of certain functions, or HSV genetically engineered to express the intended protein of interest.
  • recombinant HSV is in the HSV genome, without inhibiting the proliferation of HSV, an adapter expression cassette (ie, a promoter sequence and a polyadenylation signal enabling the adapter gene to be expressed)
  • an adapter expression cassette ie, a promoter sequence and a polyadenylation signal enabling the adapter gene to be expressed
  • the recombinant HSV refers to an HSV capable of expressing an adapter in an infected cancer cell.
  • Recombinant virus production techniques such as genetic manipulation of viruses and production of virions, are very well known in the art, and specifically, described in Sandri-Goldin RM et al, Alpha Herpesviruses: Molecular and Cellular Biology, Caister Academic Press, 2006 ], Robin H Lachmann, Herpes simplex virus-based vectors, Int J Exp Pathol. 2004 Aug; 85(4): 177-190]. All documents cited in this specification, including the above documents, are considered a part of this specification.
  • the recombinant HSV of the present invention may be engineered to enable additional targeting through modification of HSV glycoprotein in addition to targeting through an adapter.
  • glycoproteins that can be used for additional targeting gB, gC, gD, gH, etc. may be used, and some or all of the glycoprotein gene is deleted or not deleted, and is located in the targeting region of cancer cells that specifically binds to the target molecule of the cancer cell. gene can be inserted.
  • the targeting region is integrated into the envelope of the virion when recombinant HSV is produced intracellularly in a form fused (or inserted) to a glycoprotein or not. do.
  • HSV-1 When a cancer cell targeting region is inserted into a glycoprotein gene, preferred positions in HSV-1 are amino acid sequence standards, gB position 43, position 81, position 95, position 100, position 137, position 241, 304, 334, 361, 419, 430, 458, 470, 481, 546, 608, 630, 664, gH It may be position 22, position 23, position 116, and the like. More specific with regard to the insertion site, see JOHN R.
  • non-essential genes include UL3 gene (see, eg, Genbank Accession No. AFE62830.1), UL4 gene (see, eg, Genbank Accession No. AFE62831.1), UL14 gene (eg, see Genbank Accession No. AFE62841.1).
  • UL16 gene see, eg, Genbank Accession No. AFE62843.1
  • UL21 gene see, eg, Genbank Accession No.
  • AFE62848.1 UL24 gene (see, eg, Genbank Accession No. AFE62851.1), UL31 gene (eg, see Genbank Accession No. AFE62851.1) , Genbank Accession No. AFE62859.1), UL32 gene (see, eg, Genbank Accession No. AFE62860.1), US3 gene (see, eg, Genbank Accession No. AFE62891.1), UL51 gene (eg, see Genbank Accession No. AFE62891.1).
  • AFE62880.1 UL55 gene (see eg Genbank Accession No. AFE62884.1), UL56 gene (see eg Genbank Accession No. AFE62885.1), US2 gene (eg Genbank Accession No.
  • AFE62890.1 US12 gene (eg Genbank Accession No. AFE62901.1; ie see ICP47 gene), LAT gene (see eg Genbank Accession No. JQ673480.1), gB gene (eg Genbank Accession No. GU734771.1 with 52996 of 55710), gL gene (see eg Genbank Accession No. AFE62828.1), gH gene (see eg Genbank Accession No. AFE62849.1), gD gene (eg Genbank Accession N o. Refer to AFE62894.1) 1) and the like.
  • the recombinant HSV of the present invention is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or an HSV-1/HSV-2 chimeric virus (ie, both DNA whose genome is derived from HSV-1 and DNA derived from HSV-2). ), preferably a recombinant HSV-1 virus, more preferably a recombinant HSV-1 derived from an HSV-1 KOS strain.
  • the HSV-1 KOS strain can be purchased from ATCC (Cat No VR-1493TM), and the genome sequence of the strain has been completely sequenced, and GenBank Accession No. JQ673480.1 (Stuart J Macdonald et al. Genome Sequence of Herpes Simplex Virus 1 Strain KOS. J Virol. 2012 Jun;86(11):6371-2).
  • the genome of HSV-1 virus is composed of 152 kb double-stranded linear DNA encoding a total of 84 genes, which is composed of two linked fragments, a long fragment (L region) and a short fragment (S region).
  • the long fragment (L region) occupies about 82% of the genome, and the short fragment (S region) occupies about 18% of the genome.
  • the L region (UL) 56 UL1-UL56 genes and 10 genes (UL8.5, 9.5, 10.5, 12.5, 15.5, 20.5) are present at the end of each fragment.
  • the adapter expression cassette is constructed by operably linking an adapter gene with a promoter sequence for enabling its expression and a polyadenylation signal sequence, which is a transcription termination signal sequence.
  • operably linked means that it is linked to enable transcription and/or translation of the expressed adapter gene. For example, if a promoter affects the transcription of an adapter gene linked thereto, the promoter is operably linked to the adapter gene.
  • a promoter is located upstream (5' side) with respect to the transcription start point of a gene, and includes a binding site for DNA-dependent RNA polymerase, a transcription initiation point, a transcription factor binding site, etc. of one or more genes. It refers to a nucleic acid sequence having a function of controlling transcription.
  • These promoters when derived from eukaryotes, have a TATA box (usually located at positions -20 to -30 of the transcription initiation point (+1)), a CAAT box (usually located at approximately -75 positions relative to the transcription initiation site), which is above the transcription initiation site. present), enhancers, transcription factor binding sites, and the like.
  • a promoter is a promoter capable of expressing a target gene linked to it, a constitutive promoter (a promoter that constantly induces gene expression), an inducible promoter (a promoter that induces expression of a target gene in response to a specific external stimulus), tissue specific promoter (promoter that induces expression of a gene in a characteristic tissue or cell), tissue non-specific promoter (promoter that induces expression of a gene in any tissue or cell), endogenous promoter (promoter derived from the cell in which the virus is infected); Any exogenous promoter (a promoter derived from a cell other than the virus-infected cell) can be used.
  • CMV cytomegalovirus
  • RSV rous sarcoma virus
  • TK thymidine kinase
  • adenovirus late promoter vaccinia virus 75K promoter
  • SV40 promoter CD45 promoter (Hematopoietic stem cell-specific promoter), CD14 promoter (monocyte cell-specific promoter), metallothionein promoter, etc.
  • CD45 promoter Hematopoietic stem cell-specific promoter
  • CD14 promoter monocyte cell-specific promoter
  • metallothionein promoter etc.
  • the adapter expression cassette is configured to include a transcription termination signal sequence in addition to the promoter.
  • the transcription termination signal sequence is a sequence that acts as a poly(A) addition signal (polyadenylation signal) to increase transcriptional integrity and efficiency.
  • Many transcription termination signal sequences are known in the art, and an appropriate one, such as SV40 transcription termination signal sequence, and a transcription termination signal sequence of Herpes simplex virus thymidine kinase (HSV TK), may be selected and used.
  • the adapter expression cassette is inserted so as to be expressible into the HSV genome without inhibiting the proliferation of HSV, such insertion is inserted without deletion of the HSV genome, or a portion or all of a non-essential gene is deleted in the HSV genome and the deleted position can be inserted into
  • the adapter expression cassette is inserted without deletion of the HSV genome, it can be inserted between each gene.
  • Preferred positions for insertion are, for example, between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, UL48 and UL49. Between genes, between UL53 and UL54 genes, etc. are mentioned.
  • the deleted non-essential gene may be any non-essential gene as exemplified above.
  • the cancer cell targeting region of the adapter specifically recognizes and binds to a target molecule of a cancer cell that is a target cell, and the target molecule recognized by the cancer cell targeting region is any antigen present on the surface of the cancer cell. or any receptor.
  • Such antigen or receptor preferably refers to an antigen or receptor that is expressed only in cancer cells or is overexpressed in cancer cells compared to normal cells.
  • epidermal growth factor receptor variant III EGFRvIII
  • EGFRvIII epidermal growth factor receptor variant III
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • anaplastic thyroid cancer breast cancer, lung cancer, glioma, etc., papillary thyroid cancer
  • metastin receptor ErbB receptor tyrosine kinases overexpressed in breast cancer, breast cancer, bladder cancer, gallbladder cancers, cholangiocarcinomas, esophagogastric HER2 (Human epidermal growth factor receptor 2) overexpressed in junction cancers, etc.
  • tyrosine kinase-18-receptor c-Kit
  • HGF receptor c-Met overexpressed in esophageal adenocarcinoma
  • PPAR- ⁇ peroxisome pro
  • TAG-72 tumor-associated glycoprotein 72
  • GD3 disialoganglioside
  • HLA-DR overexpressed in blood cancer, solid cancer, etc.
  • nasopharynx LMP1 (Latent membrane protein 1) overexpressed in tumors (Nasopharyngeal neoplasms), lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colon cancer, colon cancer, pancreatic cancer, etc.
  • TRAILR2 tumor-necrosis factor-related apoptosis-inducing ligand receptor
  • blood vessels Angiogenic factor receptor VEGFR2 vascular endothelial growth factor receptor 2
  • HGFR hepatocyte growth factor receptor
  • target molecules such as CD44 and CD166
  • CD44 and CD166 surface antigens of cancer stem cells, such as CD44 and CD166
  • CD44 and CD166 are also target molecules.
  • target molecules are known in the art that are overexpressed in cancer cells compared to normal cells, and for more specific target molecules other than those exemplified above, see Anne T Collins et al. Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res.
  • the target molecule is preferably CEA, HER2 or EpCAM.
  • target cells targeted by the recombinant HSV of the present invention by the adapter are cancer cells, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, and prostate cancer Cancer, testicular cancer, melanoma, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, multiple myeloma, blood cancer, etc.
  • the recombinant HSV of the present invention is a gene encoding ICP34.5 (Protein preventing cellular inhibition of protein synthesis), a gene encoding ICP6 (ribonucleotide reductase), a gene encoding ICP47 (Protein inhibiting TAP/MHC class Ipresentation), ICP4 (Protein repressing expression of viral immediate early genes and activating expression of viral early and late genes), TK (thymidine kinase), UNG (Uracil DNA glycosylase), dUTPase etc. can be deleted or mutated so that they do not function (ie that transcription or translation is interrupted or that translation does not show its original activity).
  • the cell targeting region of the adapter may be an antibody derivative or an antibody analog in addition to a complete antibody having specific binding ability with a target molecule.
  • An antibody derivative refers to a fragment or modified antibody of a complete antibody comprising at least one antibody variable region having specific binding ability with a target molecule. Examples of such antibody derivatives include antibody fragments such as Fab, scFv, Fv, VhH, VH, VL, and polyvalent or multispecific modified antibodies such as Fab2, Fab3, minibody, diabody, tribody, tetrabody, bis-scFv, etc.
  • Antibody analog refers to an artificial peptide or polypeptide that has specific binding ability with a target molecule like an antibody, but is different from an antibody in structure and generally has a lower molecular weight than an antibody.
  • ABD adhron, affibody, affilin, affimer, alphabody, anticalin, armadillo repeat protein, centimeter Lin (centyrin), dalpin (DARPin), phynomer (fynomer), Kunitz region, pronectin (pronectin), repeat body (repebody) and the like are mentioned.
  • the cell targeting region of the adapter is preferably a single-chain variable fragment (scFv).
  • scFv refers to a single-chain antibody linked via a short linker peptide between the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of immunoglobulin.
  • VH variable region of the heavy chain
  • VL variable region of the light chain
  • the N-terminus of VH is linked to the C-terminus of the VL, or the N-terminus of the VL is linked to the C-terminus of VH.
  • the linker peptide is a linker of any length and any sequence as long as the heavy and light chains are spatially adjacent to each other and have specific binding ability with the target molecule without interfering with the intrinsic three-dimensional structure of the heavy and light chains. It may be a peptide.
  • the linker is preferably composed of one or more amino acids among amino acids such as Ser, Gly, Asn, and Thr, and the length is 1 ⁇ 50 amino acids.
  • the scFv target molecule is CEA, HER2 or EpCAM
  • the scFv for CEA, HER2 or EpCAM is the sequence shown in FIGS. 4 to 6 .
  • scFv has a target molecule CEA as a target molecule
  • scFv for CEA is one in which VL of SEQ ID NO: 1 and VH of SEQ ID NO: 2 are linked via a linker peptide, VL, and linker peptide VH in that order (that is, the C-terminus of VL is linked to the N-terminus of VH via a linker peptide) is preferable
  • the scFv for HER2 is VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are VH via a linker peptide, Peptide linkers, those joined in VL order (that is, the C-terminus of VH is connected to the
  • the linker peptide of scFv to CEA preferably has the amino acid sequence of SEQ ID NO: 3
  • the linker peptide of scFv to HER2 preferably has the amino acid sequence of SEQ ID NO: 6
  • the linker peptide of scFv to EpCAM preferably has the amino acid sequence of SEQ ID NO: 9.
  • the extracellular domain of HveC is HveC114 (1 to 114 amino acid sequence) of SEQ ID NO: 12 including a leader sequence (1 to 30 amino acid sequence) or a leader sequence (1 to 30 amino acid sequence) used in the examples below ) from which HveC has been removed (31-114 amino acid sequence, SEQ ID NO: 11) may be used.
  • a linker sequence may be placed between the HveC extracellular domain and the cancer cell targeting region, and the linker sequence may be a linker of any length and any sequence as long as the function of each domain of these adapters is not inhibited.
  • the linker may consist of one or more amino acids among the four amino acids Ser, Gly, Asn, and Thr, and have a length of 1 to 30 amino acids, preferably 3 to 25 amino acids, more preferably 8 to 20 amino acids. It may be a dog amino acid.
  • the adapter of the present invention may be in the order of NH 2 -cancer cell targeting domain-HveC extracellular domain-COOH or the reverse order.
  • NH 2 -cancer cell targeting region-linker peptide-HveC extracellular domain-COOH may be in the order or vice versa.
  • the adapter of the present invention is in the order of NH 2 -cancer cell targeting domain-HveC extracellular domain-COOH or NH 2 -cancer cell targeting region-linker peptide-HveC extracellular domain-COOH In the order, the leader sequence of the HveC extracellular domain is excluded and used It is preferable to be
  • an amino acid corresponding to an arbitrary restriction enzyme action site may be placed between scFv and linker and between linker and HveC to facilitate cloning.
  • GS ((base sequence: GGATCC) that BamHI acts
  • EF base sequence: GAATTC) that restriction enzyme EcoRI acts on may be published.
  • the gene for the recombinant HSV may be inserted into the HSV genome so that the factors for inducing or enhancing an immune response against cancer cells are expressed alone or in any combination.
  • factors include cytokines, chemokines, antagonists of immune checkpoints (eg antibodies, antibody derivatives or antibody analogs, particularly scFv), costimulatory factors capable of inducing activation of immune cells (T cells or NK cells) (co-stimulatory factor), an antagonist capable of inhibiting the function of TGF ⁇ to suppress the immune response to cancer cells (such as antibodies, antibody derivatives or antibody analogs, especially scFv), heparan sulfate proteoglycans that constitute the solid tumor microenvironment ( Heparanase capable of degrading heparan sulfate proteoglycan, and antagonists capable of inhibiting the function of VEGF receptor-2 (VEGFR-2), an angiogenesis factor receptor (eg, antibody, antibody derivative or antibody analog, particularly scF
  • Cytokines include, for example, interleukins such as IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, and IL-24, interferons such as IFN ⁇ , IFN ⁇ , and IFN ⁇ , and tumor necrosis factor such as TNF ⁇ , GM-CSF, colony stimulating factors such as G-CSF, etc. may be used alone or in any combination of two or more to be expressed in recombinant HSV.
  • interleukins such as IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, and IL-24
  • interferons such as IFN ⁇ , IFN ⁇ , and IFN ⁇
  • tumor necrosis factor such as TNF ⁇
  • GM-CSF GM-CSF
  • colony stimulating factors such as G-CSF, etc.
  • Chemokines for example, CCL2 (CC Motif Chemokine Ligand 2), CCL5 (RANTES), CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1 ((XC Motif Chemokine Ligand 1)) alone or It can be used to be expressed in recombinant HSV in combination.
  • Antibodies against immune checkpoints are PD-1 (programmed cell death-1), PD-L1 (programmed cell deathligand 1), PD-L2 (programmed cell death-ligand 2), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD70 (cluster of differentiation 70), CD80 (cluster of differentiation 80), CD86 (cluster of differentiation 86), CD137 (cluster of differentiation 137), CD276 (cluster of differentiation 276), KIRs (killer-cell) immunoglobulin-like receptors), lymphocyte-activation gene 3 (LAG3), glucocorticoid-induced TNFR-related protein (GITR), glucocorticoid-induced TNFR-related protein ligand (GITRL), cytolytic T lymphocyte associated antigen-4 (CTLA-4) antagonists can be used alone or in combination to be expressed in recombinant HSV.
  • PD-1 programmed cell death-1
  • PD-L1 programmed cell deathligand 1
  • Co-stimulatory factors are CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T-cell co-stimulatory factor), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , etc., alone or in combination, can be used to be expressed in recombinant HSV.
  • recombinant HSV can be engineered to express prodrug-activating enzymes that convert a prodrug into a drug that is toxic to cancer cells.
  • a prodrug activating enzyme cytosine deaminase, which converts the prodrug 5-FC (5-fluorocytosine) into a drug that is 5-FU (5-fluorouracil)
  • the prodrug CPA cyclophosphamide
  • Carboxylesterase rat cytochrome P450, CYP2B1, which converts the phosphoramide mustard (PM) drug, and the prodrug irinotecan (SN-38150) to the SN-38 drug carboxylesterase
  • bacterial nitroreductase that converts prodrug BC1954 to DNA crosslinking agent 4-hydroxylamine151, prodrug 6-methylpurine-2'-deoxyriboside and PNP (purine nucleoside phosphorylase) isolated from E. coli, which converts (6-met), 5-
  • recombinant HSV may be engineered to express TRAIL (TNF-Related Apoptosis Inducing Ligand).
  • TRAIL TNF-Related Apoptosis Inducing Ligand
  • TRAIL is known to induce apoptosis by binding to its receptor overexpressed in cancer cells (Kaoru Tamura et al. Multimechanistic Tumor Targeted Oncolytic Virus Overcomes Resistance in Brain Tumors. Mol Ther. 2013 Jan;21(1):68- 77).
  • factors or prodrug activating enzymes for inducing or enhancing an immune response are the expression cassettes of the gene (that is, the promoter sequence and the polyade a construct operably linked to a nylation signal sequence) is inserted into the HSV genome without inhibiting the proliferation of HSV, such insertion without deletion of the HSV genome, or in which some or all of the non-essential genes in the HSV genome are deleted and the deletion can be inserted into position.
  • insertion positions are, for example, between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL37 and UL38 genes, between the UL48 and UL49 genes, and between the UL53 and UL54 genes. between, and the like.
  • a non-essential gene is deleted and inserted at the deleted position or inserted into the gene without deletion of the non-essential gene, such non-essential gene may be selected from any of the non-essential genes described above.
  • the present invention relates to an anticancer pharmaceutical composition
  • an anticancer pharmaceutical composition comprising the above-mentioned recombinant HSV as an active ingredient.
  • the pharmaceutical composition of the present invention has anticancer use against carcinoma expressing a target molecule targeted by a targeting region of an adapter expressed by recombinant HSV.
  • carcinoma is the same as described above with respect to the target molecule.
  • the composition of the present invention has anticancer use against carcinoma having tumor cells expressing CEA or HER2.
  • the tumor cells expressing CEA include colorectal cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rectal cancer cells, colon cancer cells, liver cancer cells, and the like. , gastric cancer cells, lung cancer cells, head and neck cancer cells, osteosarcoma cells, glioblastoma multiforme, salivary gland tumor cells, and the like.
  • anticancer includes apoptosis of cancer cells, reduction in viability of cancer cells, inhibition or delay of pathological symptoms of cancer due to inhibition of proliferation of cancer cells, inhibition or delay of onset of such pathological symptoms, inhibition of cancer metastasis, inhibition of cancer recurrence it means to
  • the pharmaceutical composition of the present invention may be used in combination or mixed with an approved anticancer agent.
  • anticancer agents include, any anticancer agents that are cytotoxic to cancer cells, such as metabolic antagonists, alkylating agents, topoisomerase antagonists, microtubule antagonists, and plant-derived alkaloids, any cytokine drug, any antibody drug, any immune checkpoint inhibitor drug , any cell therapy (car-T cell therapy, car-NK cell therapy) medicines, and the like.
  • the pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form by a conventional method known in the art according to the route of administration, including a pharmaceutically acceptable carrier or excipient.
  • Such pharmaceutically acceptable carriers or excipients do not inhibit the activity or properties of drugs without being particularly toxic to the human body, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, algae nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (eg, saline and sterile water), syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc , magnesium stearate, mineral oil, Ringer's solution, buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof.
  • a suitable carrier or excipient is one of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like.
  • the above components may be used alone or in combination, and if necessary, other conventional pharmaceutical additives such as antioxidants, buffers, and bacteriostats may be added.
  • the pharmaceutical composition of the present invention when formulated for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. It can be prepared in multiple dosage forms.
  • the pharmaceutical composition of the present invention may be prepared in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation, and the like.
  • the pharmaceutical composition of the present invention is formulated in a dosage form of a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and an oral administration route using an administration method commonly used in the art, or dermal, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, intravaginal, It may be administered by a parenteral route, such as a rectal route.
  • the dosage (effective amount) of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate, and response sensitivity. may be prescribed, and a person skilled in the art can appropriately determine the dosage in consideration of these factors.
  • the pharmaceutical composition of the present invention is prepared as an injection in the form of a unit dose, and when prepared as an injection in the form of a unit dose, the amount of recombinant HSV virus contained per unit dose of the pharmaceutical composition of the present invention is 10 2 -10 in the range of 14 pfu, in particular in the range of 10 4 -10 11 pfu.
  • the present invention relates to a method for treating or preventing cancer (ie, a method for treating or preventing a tumor) comprising administering to a subject, such as a patient, an effective amount of a pharmaceutical composition comprising the recombinant HSV as described above. .
  • the treatment method of the present invention can be applied to any carcinoma having such a target molecule.
  • the treatment method of the present invention is preferably applied to a carcinoma expressing CEA or HER2.
  • the treatment method of the present invention may be used in combination with the above other cancer treatment methods without limitation.
  • the aforementioned cytotoxic anticancer drugs, cytokine drugs, antibody drugs, immune checkpoint inhibitor drugs, cell therapy drugs (car-T cell therapy, car-NK cell therapy) drugs, radiation therapy, surgical therapy, etc. are the pharmaceuticals of the present invention. It may be used in combination before and after administration of the composition or in a simultaneous administration mode.
  • an effective amount is the present invention that can exhibit intended medical effects, such as cancer treatment or prevention effect, when the pharmaceutical composition of the present invention is administered during the administration period recommended by a medical professional, etc.
  • an effective amount can be appropriately determined by a person skilled in the art, such as a medical professional, according to the patient's age, weight, sex, pathological condition, and the like.
  • the pharmaceutical composition is preferably administered to a patient or the like as an injection, for example, intralesional (intralesional, intratumoral), intravenous, intramuscular, intraarterial, etc. can be administered by parenteral administration route.
  • a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an HveC extracellular domain and a cell targeting region, and uses thereof.
  • the recombinant HSV When the recombinant HSV infects a target cell, which is a cancer cell, and enters the target cell, it is expressed in the cell along with the proliferation of HSV, and is released to the outside of the cell together with the proliferated HSV virion by cell lysis.
  • the fusion protein released to the outside induces infection with the HSV virion released together and the surrounding cancer cells expressing a target molecule recognized by the cancer cell targeting region, or increases the infection efficiency.
  • 1 is a schematic diagram of the structure of the KOS-37 BAC genome.
  • FIG. 2 is a genomic schematic diagram of HSV-1 KOS-EmGFP expressing EmGFP.
  • FIG. 3 is a schematic diagram of HSV-1 KOS-CEAscFv-HveC-EmGFP, KOS-HER2scFv-HveC-EmGFP and KOS-EpCAMscFv-HveC-EmGFP virus genomes.
  • HSV-1 gene consists of a large gene of about 152 kb, and KOS-37/BAC (Genbank Accession No. MF156583) (Gierasch WW et al. J) to insert a foreign gene or insert a mutation at a specific position. Virol Methods. 2006. 135:197-206) was used.
  • HSV-1 KOS strain (HSV-1 KOS strain) is a kind of HSV-1 strain mainly used in laboratories because its characteristics are well known and useful for investigation of gene function and etiology (Smith KO. Proc. Soc. Exp) (Biol. Med. 1964. 115:814-816).
  • KOS-37/BAC constructed by inserting the BAC plasmid into the KOS genome, enables cloning at the bacterial level through transformation into DH10B bacteria (Invitrogen) (Gierasch WW et al.; J. Virol Methods. 2006. 135:197-206).
  • BAC bacterial artificial chromosomes
  • FIG. 1 A schematic diagram thereof is shown in FIG. 1 .
  • EmGFP HSV-1 expressing EmGFP
  • an expression cassette capable of expressing EmGFP was inserted (Tiffany A et al., J Virol Methods. 2015. 231:18). -25). This is to facilitate the observation of virus production and infection level using EmGFP as a marker.
  • the pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) was used to construct the EmGFP cassette.
  • FIG. 2 A schematic diagram of the genome of KOS-EmGFP expressing EmGFP is shown in FIG. 2 .
  • pCMV a gene promoter of cytomegalovirus
  • tkpA a polyadenylation signal of Herpes simplex virus thymidine kinase (HSV TK)
  • HSV TK Herpes simplex virus thymidine kinase
  • a clone containing KOS-37/BAC was transformed with a pRed/ET plasmid capable of expressing RecE and RecT capable of performing homologous recombination functions (Muyrers JP et al.; Nucleic Acids Res). 1999. 27(6):1555-1557).
  • a set of homologous region primers between UL26 and UL27 including the position to introduce the target gene (forward primer UL26/27-rpsL_For: SEQ ID NO: 13, reverse primer UL26/27-rpsL_Rev: SEQ ID NO: 14) was used to prepare a UL26/27-rpsL-neo/kan cassette.
  • UL26/27 prepared after inducing homologous recombination by adding L-arabinose (Sigma-Aldrich) to the clone containing KOS-37/BAC DNA and pRed/ET -rpsL-neo/kan cassette 200 ng was transformed.
  • L-arabinose Sigma-Aldrich
  • the UL26/27-rpsL-neo/kan cassette is inserted into the UL26/27 position of KOS-37/BAC.
  • E. coli into which UL26/27-rpsL-neo/kan is inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene.
  • E. coli selected in kanamycin medium was judged to have been inserted into UL26/27-rpsL-neo/kan, and the final step, the step of inserting the target gene, proceeded.
  • the UL26/27-tkpA-EmGFP-pCMV cassette uses the pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) as a template, and the forward primer UL26/27-tkpA_For (SEQ ID NO: 15) and the reverse primer UL26/27 It was produced using -pCMV_Rev (SEQ ID NO: 16).
  • Candidate groups were selected in streptomycin medium using the principle that the existing UL26/27-rpsL-neo/kan cassette and the inserted UL26/27-tkpA-EmGFP-pCMV were replaced and streptomycin resistance blocked by rpsL was activated ( Heermann R et al., Microb Cell Fact. 2008. 14:. doi: 10.1186). The selected candidate group was subjected to DNA isolation using the DNA prep method (Horsburgh BC et al., Methods enzymol. 1999.
  • virus-containing cells were picked up and KOS-EmGFP was subjected to three freeze-thaw methods (Gierasch WW et al.; J. Virol Methods. 2006. 135:197-206) and sonication. virus was obtained.
  • Example 2 Construction of KOS 37-BAC-EmGFP expressing HER2scFv-HveC, CEAscFv-HveC, and EpCAMscFv-HveC adapters
  • the scFv for CEA is a configuration in which the VL of SEQ ID NO: 1 and the VH of SEQ ID NO: 2 are linked via the linker peptide of SEQ ID NO: 3, and the scFv for HER2 is the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 is SEQ ID NO: It is a structure linked through the linker peptide of 6.
  • the scFv for EpCAM has a configuration in which the VL of SEQ ID NO: 7 and the VH of SEQ ID NO: 8 are linked via the linker peptide of SEQ ID NO: 9.
  • CEAscFv-HveC and HER2scFv-HveC adapter and HveC 144 of SEQ ID NO: 11 were used for HveC in EpCAMscFv-HveC adapter.
  • the leader sequence of SEQ ID NO: 10 is included at the N-terminus, that is, the VL front part of CEAscFv, the VH front part of HER2scFv, and the VL front part of EpCAMscFv.
  • GS base sequence: GGATCC
  • pCMV is a gene promoter of the cytomegalovirus
  • bGH-pA is a bovine growth hormone polyadenylation (bGH-PolyA) signal sequence.
  • the amino acid sequence of the full-length CEAscFv-HveC adapter including the leader sequence used in this Example is shown in SEQ ID NO: 17, and the amino acid sequence of the full-length HER2scFv-HveC adapter including the leader sequence is shown in SEQ ID NO: 18, respectively,
  • the amino acid sequence of the full length of the EpCAMscFv-HveC adapter including the leader sequence is shown in SEQ ID NO: 19, respectively.
  • Insertion of the CEAscFv-HveC adapter expression cassette, the HER2scFv-HveC adapter expression cassette, and the EpCAMscFv-HveC adapter expression cassette was performed by the manufacturer using a counter selection BAC modification kit (GenBridges. Inc) as in Example 1 above. proceeded according to the protocol.
  • pRed/ET plasmids expressing RecE and RecT performing homologous recombination in the E. coli clone containing the KOS-37/BAC-EmGFP genome prepared in Example 2 were transformed ( Muyrers JP et al .; Nucleic Acids Res. 1999. 27(6):1555-1557).
  • a homologous region primer set (forward primer UL3/4-rpsL-neo_for: SEQ ID NO: 20, reverse primer UL3/4-rpsL-neo_rev: SEQ ID NO: 21) was used to construct a UL3/4-rpsL-neo/kan cassette.
  • the prepared UL3/4 Transform 200 ng of -rpsL-neo/kan cassette is inserted into the UL3/4 position of KOS-37/BAC-EmGFP.
  • E. coli into which UL3/4-rpsL-neo/kan is inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene.
  • E. coli selected in kanamycin medium was judged to have been inserted into UL3/4-rpsL-neo/kan, and the final step, the step of inserting the target gene, proceeded.
  • Example 3 Targeting of CEA-expressing cells using HveC-CEAscFv adapter expressing anticancer virus
  • Example 2 In order to confirm whether the KOS-UL3/4_CEAscFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding CEA-expressing cells by expressing the CEAscFv-HveC adapter, the experiment was conducted as follows.
  • the cell line used in the experiment was CHO-CEA, a cell expressing CEA (Kuroki M et al., J Biol Chem. 1991. 74:10132-10141).
  • CHO-CEA was cultured using 100 U/ml penicillin/100 ⁇ g/ml streptomycin (Wellgen) and 10% FBS in HaM's F-12K medium (Wellgen).
  • CHO-CEA cells were infected with the wild-type HSV-1 KOS virus and the adapter-expressing KOS-UL3/4_CEAscFv-HveC-EmGFP virus at MOI of 5, respectively.
  • the results are shown by quantifying the virus infection in the cells using a fluorescence microscope in FIG. 7 . It can be seen that the infective effect of CEA-expressing cells by the CEAscFv-HveC adapter expressing virus is significantly higher than that of the general herpes virus.
  • Example 2 In order to confirm whether the KOS-UL3/4_HER2scFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding HER2-expressing cells by expressing the HER2scFv-HveC adapter, the experiment was conducted as follows.
  • CHO-K1 CHO-Nectin expressing Netin
  • CHO-HER2 CHO-HER2 expressing HER2 (Kuroki M et al., J Biol Chem. 1991. 74:10132-10141).
  • CHO cell lines were cultured in HaM's F-12K medium (Wellgen) with 100 U/ml penicillin/100 ⁇ g/ml streptomycin (Wellgen) and 10% FBS.
  • KOS-UL3/4_CEAscFv-HveC-EmGFP virus expressing wild-type HSV-1 KOS virus and adapter was applied to 1.5 ⁇ 10 4 CHO-K1, CHO-Nectin, and CHO-HER2 cells, respectively. 5 MOIs were infected.
  • FIG. 8 The results of the virus infection in the cells using a fluorescence microscope are shown in FIG. 8 .
  • CHO-K1 cells which do not express both HER2 and Nectin-1 (HveC)
  • HveC wild-type virus
  • HBV-1 KOS wild-type virus
  • HSV-1 KOS wild-type virus
  • HSV-1 KOS virus expressing the HER2scFv-HveC adapter
  • HER2-expressing CHO-HER2 cells only the virus expressing the HER2scFv-HveC adapter was infected, and in CHO-Nectin cells expressing HER2 and Nectin-1, both the wild-type virus and the virus expressing the HER2scFv-HveC adapter were infected.
  • Example 2 In order to confirm whether the KOS-UL3/4_EpCAMscFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding EpCAM-expressing cells by expressing the EpCAMscFv-HveC adapter, an experiment was conducted as follows.
  • Cell lines used in the experiment were CHO-K1, CHO-Nectin expressing Netin, and CHO-EpCAM expressing EpCAM cells.
  • CHO cell lines were cultured in HaM's F-12K medium (Wellgen) with 100 U/ml penicillin/100 ⁇ g/ml streptomycin (Wellgen) and 10% FBS.
  • KOS-UL3/4_EpCAMscFv-HveC-EmGFP virus expressing wild-type HSV-1 KOS virus and adapter was applied to 1.5 ⁇ 10 4 CHO-K1, CHO-Nectin, and CHO-EpCAM cells, respectively. 5 MOIs were infected.
  • the results show the virus infection in the cells using a fluorescence microscope in FIG.

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Abstract

Disclosed are: a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an extracellular domain of HveC and a cancer cell-targeting domain; and use thereof. When the recombinant HSV infects and enters target cells, which are cancer cells, the fusion protein is expressed in the cells together with the proliferation of HSV and released by cell lysis to the outside of the cells along with the proliferated HSV virions, and the fusion protein released to the outside of the cells acts to induce the HSV virions released along therewith to infect surrounding cancer cells that express a target molecule recognized by the cancer cell-targeting domain or to increase the infection efficiency thereof.

Description

HVEC의 세포외 도메인과 암세포 표적화 영역의 융합 단백질을 발현할 수 있는 발현 카세트를 가지는 재조합 헤르페스 심플렉스 바이러스 및 그 용도Recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an extracellular domain of HVEC and a cancer cell targeting region, and uses thereof
본 발명은 HveC의 세포외 도메인과 암세포 표적화 영역의 융합 단백질을 발현할 수 있는 발현 카세트를 가지는 재조합 헤르페스 심플렉스 바이러스 및 그 용도에 관한 것이다.The present invention relates to a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an extracellular domain of HveC and a cancer cell targeting region, and uses thereof.
암 치료에는 현재까지 수술 요법, 항암 화학 요법, 방사선 요법 등이 널리 이용되어 있으나 대부분이 부작용을 수반하고 불완전한 치료 효과, 암 재발과 전이 등의 문제점을 가지고 있다. 따라서 새롭고 효과적인 암 치료법 개발이 지속적으로 요구되어 왔으며, 최근 몇년 사이 항암 바이러스, CAR-T 세포 치료법(chimeric antigen receptor T cell therapy) 등 항암 면역 요법에 있어 급속한 발전이 있었다.Surgical therapy, chemotherapy, radiation therapy, etc. have been widely used for cancer treatment, but most of them have side effects, incomplete therapeutic effects, and problems such as cancer recurrence and metastasis. Therefore, there has been a continuous demand for the development of new and effective cancer treatment methods, and in recent years, there has been rapid development in anticancer immunotherapy such as anticancer viruses and CAR-T cell therapy (chimeric antigen receptor T cell therapy).
항암 면역 요법 중 항암 바이러스는 살아있는 바이러스의 유전자를 조작해 암세포에서 선택적으로 증식하여 암세포를 용해하는 특성을 가진 바이러스로서, 정상세포에서의 증식은 제한적이다. 암세포를 용해하여 방출된 바이러스는 주변 암세포를 계속 감염시킴으로써 지속적이고 상승적인 치료 효과를 낼 수 있다. 또한 항암 바이러스는 암세포를 용해하는 과정에서 면역원성을 가진 종양 항원이 방출되어 인체의 면역반응을 자극시킴으로써 항암 효과를 높일 수 있으며, 또 이러한 항암 효과는 사이토카인, 케모카인 등을 발현하도록 인위적으로 조작함으로써 증진될 수도 있다. Among anticancer immunotherapy, anticancer virus is a virus with the property of lysing cancer cells by selectively proliferating in cancer cells by manipulating the genes of living viruses, and proliferation in normal cells is limited. Viruses released by dissolving cancer cells continuously infect surrounding cancer cells, thereby producing a lasting and synergistic therapeutic effect. In addition, anticancer viruses can increase the anticancer effect by stimulating the body's immune response by releasing tumor antigens with immunogenicity in the process of dissolving cancer cells. may be enhanced.
현재의 개발되는 항암 바이러스는 아데노 바이러스와 헤르페스 심플렉스 바이러스(Herpes Simplex Virus, HSV), 백시니아 바이러스를 포함하여 10종 이상으로 나눌 수 있는데, 이중 HSV는 150kb 크기의 선상의 이중가닥 DNA를 포함하는 다면체성 바이러스(enveloped icosahedral virion)로서, HSV-1형과 HSV-2형으로 나뉜다. HSV는 많은 비필수 유전자(non-essential genes)를 가지고 있고 게놈의 크기가 커서 외부 유전자의 조작이나 운반이 용이하고, 복제 주기가 짧으며, 또 감염 효율이 높고, 세포 부착과 감염에 관여하는 당단백질의 조작이 용이하여 암세포에 대한 표적화를 개선시킬 수 있는 장점을 가진다. Anticancer viruses currently being developed can be divided into more than 10 types including adenovirus, herpes simplex virus (HSV), and vaccinia virus. As an enveloped icosahedral virion, it is divided into HSV-1 type and HSV-2 type. HSV has many non-essential genes and has a large genome, so it is easy to manipulate or transport foreign genes, has a short replication cycle, and has high infection efficiency. It has the advantage of being able to improve the targeting to cancer cells by easy manipulation of the protein.
2015년 10월 미국 FDA의 승인을 받은 T-VEC(Talimogene Laherparepvec, 제품명: 임리직)은 HSV-1을 이용한 악성 흑색종에 대한 항암제이다. T-VEC은 병원성을 약화시키기 위하여 ICP34.5와 ICP47 유전자가 결실되어 있고, 인체 면역 반응을 촉진시키기 위한 GM-CSF(granulocyte-macrophage colony stimulating factor)를 발현하는 재조합 HSV-1형 바이러스이다. 그러나 T-VEC은 일부 유전자가 소실됨에 따라 바이러스 증식이 제한되어 치료 효능이 낮은 한계점을 갖고 있다. T-VEC (Talimogene Laherparepvec, product name: Imrisic), approved by the US FDA in October 2015, is an anticancer drug for malignant melanoma using HSV-1. T-VEC is a recombinant HSV-1 type virus in which ICP34.5 and ICP47 genes are deleted to attenuate pathogenicity, and which expresses GM-CSF (granulocyte-macrophage colony stimulating factor) to promote human immune response. However, T-VEC has a low limit of therapeutic efficacy because virus proliferation is limited due to the loss of some genes.
HSV는 외피(envelope)를 가진 바이러스로, HSV의 세포 진입은 외피에 존재하는 당단백질 gD, gB, gH/gL 및 gC의 복잡한 상호 작용에 의해 이루어진다. 먼저 gB와 gC가 세포 표면의 3-O-S HS(3-O-sulfated heparan sulfate)에 부착하면, gD가 세포 수용체인 HVEM(herpesvirus entry mediator, HveA), 넥틴-1(nectin-1, HveC), 넥틴-2(nectin-2, HveB) 중 적어도 하나의 수용체 결합하여 바이러스와 세포막 사이의 융합을 유도함으로써 HSV가 세포로 진입한다(Hiroaki Uchida et al., Generation of Herpesvirus Entry Mediator (HVEM)-restricted Herpes Simplex Virus Type 1 Mutant Viruses: Resistance of HVEM-expressing Cells and Identification of Mutations That Rescue nectin-1 Recognition. J Virol. 2009 Apr;83(7):2951-61).HSV is a virus with an envelope (envelope), and HSV cell entry is achieved through the complex interaction of the glycoproteins gD, gB, gH/gL and gC present in the envelope. First, gB and gC attach to 3-O- S HS (3- O- sulfated heparan sulfate) on the cell surface, and gD is a cell receptor, herpesvirus entry mediator (HVEM), and nectin-1 (HveC). ), HSV enters the cell by inducing fusion between the virus and the cell membrane by binding to at least one receptor of nectin-2 (HveB) (Hiroaki Uchida et al., Generation of Herpesvirus Entry Mediator (HVEM)- restricted Herpes Simplex Virus Type 1 Mutant Viruses: Resistance of HVEM-expressing Cells and Identification of Mutations That Rescue nectin-1 Recognition. J Virol. 2009 Apr;83(7):2951-61).
HSV의 세포 수용체 중 HveC(nectin-1)은 4 개의 cystein-rich domain(CRD)으로 구성되어 있으며, 그 중 아미노 말단 부위의 2 개 CRD가 HSV의 gD와 결합하여 HSV-1과 HSV-2의 세포 진입을 유도하는 것으로 보고 있다(Sarah A Connolly et al., Structure-based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM). J Virol. 2002 Nov;76(21):10894-904. ).Among HSV cell receptors, HveC(nectin-1) consists of four cystein-rich domains (CRDs), among which, two CRDs at the amino terminus bind to gD of HSV, thereby reducing the levels of HSV-1 and HSV-2. It has been reported to induce cell entry (Sarah A Connolly et al., Structure-based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM). J Virol. 2002 Nov;76(21): 10894-904. ).
본 발명자들은 이전에 CEA(carcinoembryonal antigen)에 대한 scFv(single-chain variable fragment)와 HSV 세포 표면 수용체의 하나인 HVEM의 세포외 도메인의 융합 단백질(CEAscFv-HA)을 제작하여, 그 융합 단백질을 HSV와 함께 CEA을 발현하는 세포주에 처리할 경우, 그 융합 단백질이 어탭터(adapter)로 작용하여 HSV가 해당 세포주를 표적하여 감염시키도록 유도함을 이전에 보고한 바 있다(한국 등록특허 제10-0937774호 및 미국 등록특허 제8318662호 참조).The present inventors previously produced a fusion protein (CEAscFv-HA) of a single-chain variable fragment (scFv) for CEA (carcinoembryonal antigen) and an extracellular domain of HVEM, one of the HSV cell surface receptors, and the fusion protein was used for HSV It has been previously reported that, when treated with a cell line expressing CEA with and US Patent No. 8318662).
본 발명은 다른 구성의 어댑터인 융합 단백질인 HveC-CEA와 HveC-Her2scFv를 코팅하는 유전자가 헤르페스바이러스의 지놈 UL3 및 UL4 유전자 사이에서 삽입되어 상기 융합 단백질이 감염된 세포내에서 발현되도록 할 경우 그 헤르페스바이러스가 CEA나 Her2을 발현하는 세포주를 표적하여 감염시킴을 확인함으로써 완성된 것이다.According to the present invention, the fusion proteins HveC-CEA and HveC-Her2scFv, which are adapters of different configurations, are inserted between UL3 and UL4 genes of the herpes virus genome so that the fusion protein is expressed in infected cells. This was completed by confirming that it targets and infects cell lines expressing CEA or Her2.
전술한 바를 고려할 때, 본 발명의 목적은 HveC의 세포외 도메인과 암세포 표적화 영역의 융합 단백질인 어뎁터를 발현할 수 있는, 그 융합 단백질의 발현 카세트를 가지는 재조합 HSV를 제공하는 데 있다. In view of the above, it is an object of the present invention to provide a recombinant HSV having an expression cassette of the fusion protein capable of expressing an adapter that is a fusion protein between the extracellular domain of HveC and a cancer cell targeting region.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other objects or specific objects of the present invention will be set forth below.
본 발명은 HveC의 세포외 도메인 특히 V 도메인(서열번호 11, 서열번호 12에서 1~30 아미노산 서열이 제외된 31~144 아미노선 서열 영역)과 암세포의 표적분자를 특이적으로 인식하여 결합하는 암세포 표적화 영역의 융합 단백질인 어뎁터를 발현할 수 있는 재조합 헤르페스 심플렉스 바이러스(Herpes Simplex Virus, HSV)에 관한 것이다. The present invention relates to a cancer cell that specifically recognizes and binds the target molecule of HveC to the extracellular domain, particularly the V domain (SEQ ID NO: 11, 31 to 144 amino acid sequence region from which 1 to 30 amino acid sequences are excluded from SEQ ID NO: 12) It relates to a recombinant Herpes Simplex Virus (HSV) capable of expressing an adapter that is a fusion protein of a targeting region.
본 발명의 재조합 HSV가 암세포인 표적세포를 감염시켜 표적세포 내로 들어가게 되면, HSV가 증식됨과 함께 융합 단백질인 어뎁터가 세포 내에서 발현되어 세포 용해에 의하여 그 증식된 HSV 비리온과 함께 세포 외부로 방출되며, 이 세포 외부로 방출된 어뎁터는 함께 방출된 HSV 비리온을, 어뎁터의 암세포 표적화 영역이 인식하는 표적분자를 발현하는 주변 암세포로 감염을 유도하거나 감염 효율을 높이는 작용을 하게 된다.When the recombinant HSV of the present invention infects a target cell, which is a cancer cell, and enters the target cell, the HSV proliferates and an adapter, which is a fusion protein, is expressed in the cell, and is released together with the proliferated HSV virion through cell lysis to the outside of the cell. The adapter released to the outside of the cell induces infection with the HSV virions released together with surrounding cancer cells expressing a target molecule recognized by the cancer cell targeting region of the adapter or increases the infection efficiency.
일반적으로 재조합 HSV는 야생형 HSV 바이러스와 비교할 때 인위적인 돌연변이가 도입됨으로써(즉 일부 핵산 서열이 결실, 치환 또는 삽입됨으로써) 일정 기능이 상실 또는 변경되거나 의도한 목적 단백질을 발현할 수 있도록 유전적으로 조작된 HSV를 의미하는데, 본 발명에서 재조합 HSV는 HSV 게놈에, HSV의 증식을 저해하지 않으면서, 어댑터의 발현 카세트(adapter expressionl cassette)(즉 어뎁터 유전자가 이의 발현을 가능하게하는 프로모터 서열 및 폴리아데닐화 시그널 서열과 작동가능하게 연결된 구성체)를 도입(즉 삽입)함으로써 그 재조합 HSV가 감염된 암세포 내에서 어뎁터를 발현할수 있는 HSV를 의미한다. 이러한 바이러스의 유전자 조작과 비리온의 생산 등 재조합 바이러스 제조 기술은 당업계에 매우 잘 공지되어 있으며, 구체적으로는 문헌[Sandri-Goldin RM et al, Alpha Herpesviruses: Molecular and Cellular Biology, Caister Academic Press, 2006], 문헌[Robin H Lachmann, Herpes simplex virus-based vectors, Int J Exp Pathol. 2004 Aug; 85(4): 177-190] 등을 참조할 수 있다. 상기 문헌을 포함하여 본 명세서에서 인용되는 문헌들은 모두 본 명세서의 일부로 간주된다. In general, recombinant HSV is compared to wild-type HSV virus by introducing artificial mutations (ie, deletion, substitution, or insertion of some nucleic acid sequences) resulting in loss or alteration of certain functions, or HSV genetically engineered to express the intended protein of interest. In the present invention, recombinant HSV is in the HSV genome, without inhibiting the proliferation of HSV, an adapter expression cassette (ie, a promoter sequence and a polyadenylation signal enabling the adapter gene to be expressed) By introducing (ie, inserting) a construct operably linked to the sequence), the recombinant HSV refers to an HSV capable of expressing an adapter in an infected cancer cell. Recombinant virus production techniques, such as genetic manipulation of viruses and production of virions, are very well known in the art, and specifically, described in Sandri-Goldin RM et al, Alpha Herpesviruses: Molecular and Cellular Biology, Caister Academic Press, 2006 ], Robin H Lachmann, Herpes simplex virus-based vectors, Int J Exp Pathol. 2004 Aug; 85(4): 177-190]. All documents cited in this specification, including the above documents, are considered a part of this specification.
본 발명의 재조합 HSV는 어뎁터를 통한 표적화 이외에 HSV의 당단백질(glycoprotein)의 변형을 통해 추가적인 표적화가 가능하도록 조작될 수 있다. 추가적인 표적화에 사용될 수 있는 당단백질로서는 gB, gC, gD, gH 등이 사용될 수 있으며, 당단백질 유전자가 일부 또는 전부 결실되거나 결실되지 않고, 암세포의 표적분자에 특이적으로 결합하는 암세포의 표적화 영역에 대한 유전자가 삽입될 수 있다. 이러한 당단백질 유전자에 암세포 표적화 영역의 유전자가 삽입될 경우 표적화 영역은, 당단백질에 융합(또는 삽입)된 형태나 융합되지 않은 형태로 재조합 HSV가 세포내에서 생산될 때 비리온의 외피에 통합되게 된다. 당단백질 유전자에 암세포 표적화 영역을 삽입될 경우, HSV-1에 있어서 바람직한 위치는 아미노산 서열 기준, gB의 43번 위치, 81번 위치, 95번 위치, 100번 위치, 137번 위치, 241번 위치, 304번 위치, 334번 위치, 361번 위치, 419번 위치, 430번 위치, 458번 위치, 470번 위치, 481번 위치, 546번 위치, 608번 위치, 630번 위치, 664번 위치, gH의 22번 위치, 23번 위치, 116번 위치 등일 수 있다. 삽입되는 위치와 관련해서 더 구체적인 것은 문헌[JOHN R. GALLAGHER et al., Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion, PLoS Pathog, 2014, Sep 18, 10(9):e1004373], 문헌[Gatta V et al, The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors, PLoS Pathog. 2015 May 21;11(5):e1004907], 문헌[Tina M. Cairns et al., Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras, JOURNAL OF VIROLOGY, June 2003, p.6731-6742], 문헌[E.U. Lorentzen et al., Replication-Competent Herpes simplex Virus Type 1 Mutant Expressing an Autofluorescent Glycoprotein H Fusion Protein, Intervirology 2001;44:232-242] 등을 참조할 수 있다.The recombinant HSV of the present invention may be engineered to enable additional targeting through modification of HSV glycoprotein in addition to targeting through an adapter. As glycoproteins that can be used for additional targeting, gB, gC, gD, gH, etc. may be used, and some or all of the glycoprotein gene is deleted or not deleted, and is located in the targeting region of cancer cells that specifically binds to the target molecule of the cancer cell. gene can be inserted. When a gene of a cancer cell targeting region is inserted into this glycoprotein gene, the targeting region is integrated into the envelope of the virion when recombinant HSV is produced intracellularly in a form fused (or inserted) to a glycoprotein or not. do. When a cancer cell targeting region is inserted into a glycoprotein gene, preferred positions in HSV-1 are amino acid sequence standards, gB position 43, position 81, position 95, position 100, position 137, position 241, 304, 334, 361, 419, 430, 458, 470, 481, 546, 608, 630, 664, gH It may be position 22, position 23, position 116, and the like. More specific with regard to the insertion site, see JOHN R. GALLAGHER et al., Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion, PLoS Pathog, 2014, Sep 18, 10 ( 9):e1004373], Gatta V et al, The Engineering of a Novel Ligand in gH Confers to HSV an Expanded Tropism Independent of gD Activation by Its Receptors, PLoS Pathog. 2015 May 21;11(5):e1004907], Tina M. Cairns et al., Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras, JOURNAL OF VIROLOGY, June 2003, p.6731-6742], [EU] Lorentzen et al., Replication-Competent Herpes simplex Virus Type 1 Mutant Expressing an Autofluorescent Glycoprotein H Fusion Protein, Intervirology 2001;44:232-242] and the like.
또한 본 발명의 재조합 HSV는 HSV의 증식(즉 생존과 복제)에 필요하지 않은 비필수 유전자(non-essential genes)가 결실되거나 기능을 발휘하지 않도록(즉 전사나 번역이 방해를 받도록) 돌연변이(mutation) 될 수도 있다. 구체적으로 그러한 비필수 유전자는 UL3 유전자(예컨대, Genbank Accession No. AFE62830.1 참조), UL4 유전자(예컨대, Genbank Accession No. AFE62831.1 참조), UL14 유전자(예컨대, Genbank Accession No. AFE62841.1), UL16 유전자(예컨대, Genbank Accession No. AFE62843.1 참조), UL21 유전자(예컨대, Genbank Accession No. AFE62848.1 참조), UL24 유전자(예컨대, Genbank Accession No. AFE62851.1 참조), UL31 유전자(예컨대, Genbank Accession No. AFE62859.1 참조), UL32 유전자(예컨대, Genbank Accession No. AFE62860.1 참조), US3 유전자(예컨대, Genbank Accession No. AFE62891.1 참조), UL51 유전자(예컨대, Genbank Accession No. AFE62880.1 참조), UL55 유전자(예컨대, Genbank Accession No. AFE62884.1 참조), UL56 유전자(예컨대, Genbank Accession No. AFE62885.1 참조), US2 유전자(예컨대, Genbank Accession No. AFE62890.1), US12 유전자(예컨대, Genbank Accession No. AFE62901.1; 즉, ICP47 유전자 참조), LAT 유전자(예컨대, Genbank Accession No. JQ673480.1 참조), gB 유전자(예컨대, Genbank Accession No. GU734771.1의 52996과 55710 사이 서열), gL 유전자(예컨대, Genbank Accession No. AFE62828.1 참조), gH 유전자(예컨대, Genbank Accession No. AFE62849.1 참조), gD 유전자(예컨대, Genbank Accession No. AFE62894.1 참조) 1) 등을 들 수 있다.In addition, the recombinant HSV of the present invention is mutated so that non-essential genes that are not necessary for the proliferation (ie, survival and replication) of HSV are deleted or do not function (ie, transcription or translation is disturbed). ) may be Specifically, such non-essential genes include UL3 gene (see, eg, Genbank Accession No. AFE62830.1), UL4 gene (see, eg, Genbank Accession No. AFE62831.1), UL14 gene (eg, see Genbank Accession No. AFE62841.1). , UL16 gene (see, eg, Genbank Accession No. AFE62843.1), UL21 gene (see, eg, Genbank Accession No. AFE62848.1), UL24 gene (see, eg, Genbank Accession No. AFE62851.1), UL31 gene (eg, see Genbank Accession No. AFE62851.1) , Genbank Accession No. AFE62859.1), UL32 gene (see, eg, Genbank Accession No. AFE62860.1), US3 gene (see, eg, Genbank Accession No. AFE62891.1), UL51 gene (eg, see Genbank Accession No. AFE62891.1). AFE62880.1), UL55 gene (see eg Genbank Accession No. AFE62884.1), UL56 gene (see eg Genbank Accession No. AFE62885.1), US2 gene (eg Genbank Accession No. AFE62890.1), US12 gene (eg Genbank Accession No. AFE62901.1; ie see ICP47 gene), LAT gene (see eg Genbank Accession No. JQ673480.1), gB gene (eg Genbank Accession No. GU734771.1 with 52996 of 55710), gL gene (see eg Genbank Accession No. AFE62828.1), gH gene (see eg Genbank Accession No. AFE62849.1), gD gene (eg Genbank Accession N o. Refer to AFE62894.1) 1) and the like.
HSV 바이러스의 비필수 유전자에 대해 더 구체적인 것은 문헌[DM Knipe and PM Howley(ed.) Fields virology(vol.2) Lippincot Williams & Wilkins, Philadelphia, Pa. 2001. p.2399-2460)], 문헌[Subak-Sharpe J H, Dargan D J HSV molecular biology: general aspects of herpes simplex virus molecular biology Virus Genes, 1998, 16(3): 239-251], 문헌[Travis J. Taylor 및 David M. Knipe, Proteomics of Herpes Simplex Virus Replication Compartments: Association of Cellular DNA Replication, Repair, Recombination, and Chromatin Remodeling Proteins with ICP8, J Virol. 2004 Jun; 78(11): 5856-5866] 등을 참조할 수 있다. For further details on the non-essential genes of the HSV virus, see DM Knipe and PM Howley (ed.) Fields virology (vol. 2) Lippincot Williams & Wilkins, Philadelphia, Pa. 2001. p.2399-2460), Subak-Sharpe JH, Dargan DJ HSV molecular biology: general aspects of herpes simplex virus molecular biology Virus Genes, 1998, 16(3): 239-251, Travis J Taylor and David M. Knipe, Proteomics of Herpes Simplex Virus Replication Compartments: Association of Cellular DNA Replication, Repair, Recombination, and Chromatin Remodeling Proteins with ICP8, J Virol. 2004 Jun; 78(11): 5856-5866].
본 발명의 재조합 HSV는 재조합 HSV-1 바이러스, 재조합 HSV-2 바이러스, 또는 HSV-1/HSV-2 키메라 바이러스(즉, 게놈이 HSV-1로부터 유래된 DNA 및 HSV-2로부터 유래된 DNA 둘 모두를 포함하는 재조합 HSV 바이러스임)일 수 있으며, 바람직하게는 재조합 HSV-1 바이러스, 더 바람직하게는 HSV-1 KOS 균주로부터 유래된 재조합 HSV-1이다. HSV-1 KOS 균주는 ATCC(Cat No VR-1493TM)로부터 구입로부터 구입이 가능하고 그 균주의 게놈 서열은 전체 서열 분석이 완료되어 GenBank Accession No. JQ673480.1에 제시되어 있다(Stuart J Macdonald et al. Genome Sequence of Herpes Simplex Virus 1 Strain KOS. J Virol. 2012 Jun;86(11):6371-2). The recombinant HSV of the present invention is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or an HSV-1/HSV-2 chimeric virus (ie, both DNA whose genome is derived from HSV-1 and DNA derived from HSV-2). ), preferably a recombinant HSV-1 virus, more preferably a recombinant HSV-1 derived from an HSV-1 KOS strain. The HSV-1 KOS strain can be purchased from ATCC (Cat No VR-1493TM), and the genome sequence of the strain has been completely sequenced, and GenBank Accession No. JQ673480.1 (Stuart J Macdonald et al. Genome Sequence of Herpes Simplex Virus 1 Strain KOS. J Virol. 2012 Jun;86(11):6371-2).
HSV-1 바이러스의 게놈은 총 84개의 유전자를 암호화하는 152 kb의 이중가닥 선형 DNA로 구성되어 있으며, 이는 서로 연결된 두 개의 단편 즉 긴 단편(L 영역)와 짧은 단편(S 영역)으로 구성된다. 긴 단편(L 영역)은 게놈의 약 82%를 차지하고, 짧은 단편(S 영역)은 게놈의 약 18%를 차지하며, 긴 단편과 짧은 단편은 접합 영역인 2개의 IRL((intermediate inverted repeat sequence) 의해 결합되며, 각 단편의 말단에는 TRL(terminal inverted repeat segment)이 존재한다. L 영역(UL)에는 56개의 UL1~UL56 유전자와 10개의 유전자(UL8.5, 9.5, 10.5, 12.5, 15.5, 20.5, 26.5, 27.5, 43.5, 49.5)가 존재하고, S 영역(US)에는 12개의 US1~US12 유전자와 2개의 유전자(US1.5, 8.5)가 존재하며, 접합 영역인 2개의 IRL에 4개의 유전자(ICP4, ICP34.5, ICP0 및 LAT)가 포함되어 있다. The genome of HSV-1 virus is composed of 152 kb double-stranded linear DNA encoding a total of 84 genes, which is composed of two linked fragments, a long fragment (L region) and a short fragment (S region). The long fragment (L region) occupies about 82% of the genome, and the short fragment (S region) occupies about 18% of the genome. In the L region (UL), 56 UL1-UL56 genes and 10 genes (UL8.5, 9.5, 10.5, 12.5, 15.5, 20.5) are present at the end of each fragment. , 26.5, 27.5, 43.5, 49.5) exist, and there are 12 US1-US12 genes and 2 genes (US1.5, 8.5) in the S region (US), and 4 genes in the two IRL regions that are junctional regions. (ICP4, ICP34.5, ICP0 and LAT) are included.
본 발명에서, 어댑터 발현 카세트는 어댑터 유전자가 그것의 발현을 가능하게 하기 위한 프로모터 서열과 전사 종결 신호 서열인 폴리아데닐화 시그널(polyadenylation signal) 서열과 작동 가능하게 연결되어 구성된다. 여기서 "작동 가능하게 연결된다"는 것은 발현되는 어뎁터 유전자의 전사 및/또는 번역이 가능하도록 연결된다는 의미이다. 예컨대 어떠한 프로모터가 그것에 연결된 어댑터 유전자의 전사에 영향을 준다면 그 프로모터는 그 어뎁터 유전자에 작동 가능하게 연결된 것이다.In the present invention, the adapter expression cassette is constructed by operably linking an adapter gene with a promoter sequence for enabling its expression and a polyadenylation signal sequence, which is a transcription termination signal sequence. Herein, "operably linked" means that it is linked to enable transcription and/or translation of the expressed adapter gene. For example, if a promoter affects the transcription of an adapter gene linked thereto, the promoter is operably linked to the adapter gene.
일반적으로 프로모터는 어떤 유전자의 전사 개시점을 기준으로 상위(5'쪽)에 위치하고, DNA-의존 RNA 중합효소에 대한 결합 부위, 전사 개시점, 전사 인자 결합 부위 등을 포함하는, 하나 이상의 유전자의 전사를 제어하는 기능을 갖는 핵산 서열을 의미한다. 이러한 프로모터는 진핵생물 유래일 경우 전사 개시점 상위에 있는 TATA 박스(통상 전사 개시점(+1) -20 내지 -30 위치에 존재), CAAT 박스(통상 전사 개시 부위와 비교하여 대략 -75 위치에 존재), 인핸서, 전사 인자 결합 부위 등을 포함한다.In general, a promoter is located upstream (5' side) with respect to the transcription start point of a gene, and includes a binding site for DNA-dependent RNA polymerase, a transcription initiation point, a transcription factor binding site, etc. of one or more genes. It refers to a nucleic acid sequence having a function of controlling transcription. These promoters, when derived from eukaryotes, have a TATA box (usually located at positions -20 to -30 of the transcription initiation point (+1)), a CAAT box (usually located at approximately -75 positions relative to the transcription initiation site), which is above the transcription initiation site. present), enhancers, transcription factor binding sites, and the like.
프로모터는 그것에 연결된 목적유전자를 발현시킬 수 있는 프로모터라면 구성적 프로모터(상시적으로 유전자의 발현을 유도하는 프로모터), 유도성 프로모터(특정 외부 자극에 반응하여 목적 유전자의 발현을 유도하는 프로모터), 조직 특이성 프로모터(특징 조직이나 세포에서 유전자의 발현을 유도하는 프로모터), 조직 비특이적 프로모터(모든 조직이나 세포에서 유전자의 발현을 유도하는 프모모터), 내인성 프로모터(바이러스가 감염되는 세포에서 유래된 프로모터), 외인성 프로모터(바이러스가 감염되는 세포 이외의 세포에서 유래된 프로모터) 모두 사용이 가능하다. 이러한 프로모터는 당업계에 많은 것이 공지되어 있으며 공지된 것 중 적절한 것을 선택하여 사용할 수 있다. 예컨대 CMV(cytomegalovirus) 프로모터 promotor), 라우스 육종 바이러스(RSV, rous sarcoma virus) 프로모터, HSV(herpes simplex virus) TK(thymidine kinase) 프로모터, 아데노바이러스 후기 프로모터, 백시니아 바이러스 75K 프로모터, SV40 프로모터, CD45 프로모터(조혈모세포 특이적 프로모터), CD14 프로모터(단핵구세포 특이적 프로모터), 메탈로티오닌(metallothionein) 프로모터 등을 사용할 수 있다.A promoter is a promoter capable of expressing a target gene linked to it, a constitutive promoter (a promoter that constantly induces gene expression), an inducible promoter (a promoter that induces expression of a target gene in response to a specific external stimulus), tissue specific promoter (promoter that induces expression of a gene in a characteristic tissue or cell), tissue non-specific promoter (promoter that induces expression of a gene in any tissue or cell), endogenous promoter (promoter derived from the cell in which the virus is infected); Any exogenous promoter (a promoter derived from a cell other than the virus-infected cell) can be used. Many of these promoters are known in the art, and an appropriate one of the known promoters may be selected and used. For example, CMV (cytomegalovirus) promoter promoter), rous sarcoma virus (RSV) promoter, HSV (herpes simplex virus) thymidine kinase (TK) promoter, adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, CD45 promoter (Hematopoietic stem cell-specific promoter), CD14 promoter (monocyte cell-specific promoter), metallothionein promoter, etc. can be used.
상기 어댑터 발현 카세트는 프로모터 이외에 전사 종결 신호 서열을 포함하여 구성되는데, 전사 종결 신호 서열은 poly(A) 첨가 신호(polyadenylation signal)로 작용하는 서열로서 전사의 완결성 및 효율성을 높이기 위한 것이다. 많은 전사 종결 신호 서열이 당업계에 공지되어 있으며, 이중에서 적절한 것 예컨대 SV40 전사 종결 신호 서열, HSV TK(Herpes simplex virus thymidine kinase)의 전사 종결 신호 서열 등을 선택하여 사용할 수 있다.The adapter expression cassette is configured to include a transcription termination signal sequence in addition to the promoter. The transcription termination signal sequence is a sequence that acts as a poly(A) addition signal (polyadenylation signal) to increase transcriptional integrity and efficiency. Many transcription termination signal sequences are known in the art, and an appropriate one, such as SV40 transcription termination signal sequence, and a transcription termination signal sequence of Herpes simplex virus thymidine kinase (HSV TK), may be selected and used.
상기 어댑터 발현 카세트는 HSV의 증식을 저해하지 않으면서 HSV 게놈에 발현 가능하도록 삽입되는데, 그러한 삽입은 HSV 게놈의 결실 없이 삽입되거나, 또는 HSV 게놈 중 비필수 유전자가 일부 또는 전부 결실되고 그 결실된 위치에 삽입될 수 있다. 어댑터 발현 카세트가 HSV 게놈의 결실 없이 삽입될 경우 각 유전자 사이에 삽입될 수 있는데, 삽입되는 위치로서 바람직한 위치는 예컨대 UL3과 UL4 유전자 사이, UL26과 UL27 유전자 사이, UL37과 UL38 유전자 사이, UL48과 UL49 유전자 사이, UL53과 UL54 유전자 사이 등을 들 수 있다.The adapter expression cassette is inserted so as to be expressible into the HSV genome without inhibiting the proliferation of HSV, such insertion is inserted without deletion of the HSV genome, or a portion or all of a non-essential gene is deleted in the HSV genome and the deleted position can be inserted into When the adapter expression cassette is inserted without deletion of the HSV genome, it can be inserted between each gene. Preferred positions for insertion are, for example, between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, UL48 and UL49. Between genes, between UL53 and UL54 genes, etc. are mentioned.
어댑터 발현 카세트가 HSV 게놈에 비필수 유전자가 일부 또는 전부 결실되고 그 결실된 위치에 삽입될 경우, 결실되는 비필수 유전자는 앞서 예시한 바의 임의의 비필수 유전자일 수 있다.When the adapter expression cassette has a part or all of a non-essential gene deleted in the HSV genome and inserted at the deleted position, the deleted non-essential gene may be any non-essential gene as exemplified above.
본 발명의 재조합 HSV에서, 어뎁터의 암세포 표적화 영역은 표적세포인 암세포의 표적분자를 특이적으로 인식하여 결합하는 부분으로서, 이러한 암세포 표적화 영역이 인식하는 표적분자는 암세포의 표면에 존재하는 임의의 항원 또는 임의의 수용체이다. In the recombinant HSV of the present invention, the cancer cell targeting region of the adapter specifically recognizes and binds to a target molecule of a cancer cell that is a target cell, and the target molecule recognized by the cancer cell targeting region is any antigen present on the surface of the cancer cell. or any receptor.
이러한 항원 또는 수용체는 바람직하게는 암세포에서만 발현되거나 정상세포에 비해 암세포에서 과발현되는 항원 또는 수용체를 말한다. 예컨대 교모세포종(glioblastoma)에서 발현되는 EGFRvⅢ(epidermal growth factor receptor variantⅢ), 역형성 갑상선암이나 유방암, 폐암, 신경교종(glioma) 등에 과발현되는 EGFR(Epidermal growth factor receptor), 유두성 갑상선 암(papillary thyroid cancer) 등에서 과발현 메타스틴 수용체(Metastin receptor), 유방암 등에서 과발현되는 ErbB 계열의 수용체 타이로신 카이나제(Receptor tyrosine kinases), 유방암, 방광암, 담낭암(Gallbladder cancers), 담관암(Cholangiocarcinomas), 위식도접합부암(esophagogastric junction cancers) 등에서 과발현되는 HER2(Human epidermal growth factor receptor 2), 육두성 신암종 등에서 과발현되는 타이로신 카이나제-18-수용체(c-Kit), 식도 선암 등에 과발현되는 HGF 수용체 c-Met, 유방암 등에서 과발현되는 CXCR4 또는 CCR7, 전림선암에서 과발현되는 엔도테린-A 수용체, 직장암 등에서 과발현되는 PPAR-δ(peroxisome proliferator activated receptor δ), 난소암 등에서 과발현되는 PDGFR-α(Platelet-derived growth factor receptor α), 간암, 다발성 골수종 등에서 과발현되는 CD133, 폐암, 대장암, 위암, 췌장암, 유방암, 직장암, 결장암, 갑상선 수질암 등에서 과발현되는 CEA(carcinoembryonic antigen), 간암, 위암 등에서 과발현되는 EpCAM(Epithelial cell adhesion molecule), 신경모세포종 등에서 과발현되는 GD2(disialoganglioside), 간세포암 등에서 과발현되는 GPC3(Glypican 3), 전립선암 등에서 과발현되는 PSMA(Prostate Specific Membrane Antigen), 난소암, 유방암, 결장암, 폐암, 췌장암 등에서 과발현되는 TAG-72(tumor-associated glycoprotein 72), 흑색종 등에서 과발현되는 GD3(disialoganglioside), 혈액암, 고형암 등에서 과발현되는 HLA-DR(human leukocyte antigen-DR), 진행성 고형암 등에서 과발현되는 MUC1(Mucin 1), 진행성 폐암(advanced non-small-cell lung cancer) 등에서 과발현되는 NY-ESO-1(New York esophageal squamous cell carcinoma 1), 비인두종양(Nasopharyngeal neoplasms) 등에서 과발현되는 LMP1(Latent membrane protein 1), 폐암, 비호지킨 림프종, 난소암, 결장암, 대장암, 췌장암 등에서 과발현되는 TRAILR2(tumor-necrosis factor-related apoptosis-inducing ligand receptor), 혈관 신생 인자 수용체인 VEGFR2(vascular endothelial growth factor receptor 2), 간세포암 등에서 과발현되는 HGFR(hepatocyte growth factor receptor) 등이 표적분자일 수 있으며, 또한 암 줄기세포의 표면 항원인 CD44, CD166 등이 또한 표적 분자일 수 있다. 당업계에는 정상세포에 비해 암세포에서 과발현되는 많은 표적분자가 알려져 있으며, 상기 예시된 것 이외의 기타의 표적분자와 관련해서 더 구체적인 것은 문헌[Anne T Collins et al. Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 2005 Dec 1;65(23):10946-51], 문헌[Chenwei Li et al. Identification of Pancreatic Cancer Stem Cells. Cancer Res. 2007 Feb 1;67(3):1030-7], 문헌[Shuo Ma et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019 Sep 7;15(12):2548-2560], 문헌[Dhaval S Sanchala et al. Oncolytic Herpes Simplex Viral Therapy: A Stride Toward Selective Targeting of Cancer Cells. Front Pharmacol. 2017 May 16;8:270] 등을 참조할 수 있다. Such antigen or receptor preferably refers to an antigen or receptor that is expressed only in cancer cells or is overexpressed in cancer cells compared to normal cells. For example, epidermal growth factor receptor variant III (EGFRvIII) expressed in glioblastoma, epidermal growth factor receptor (EGFR) overexpressed in anaplastic thyroid cancer, breast cancer, lung cancer, glioma, etc., papillary thyroid cancer ) overexpressed metastin receptor, ErbB receptor tyrosine kinases overexpressed in breast cancer, breast cancer, bladder cancer, gallbladder cancers, cholangiocarcinomas, esophagogastric HER2 (Human epidermal growth factor receptor 2) overexpressed in junction cancers, etc., tyrosine kinase-18-receptor (c-Kit) overexpressed in nutritive renal carcinoma, etc., HGF receptor c-Met overexpressed in esophageal adenocarcinoma, etc., in breast cancer, etc. CXCR4 or CCR7 overexpressed, endothelin-A receptor overexpressed in prostate cancer, peroxisome proliferator activated receptor δ (PPAR-δ) overexpressed in rectal cancer, platelet-derived growth factor receptor α (PDGFR-α) overexpressed in ovarian cancer, etc.; CD133 overexpressed in liver cancer, multiple myeloma, etc.; carcinoembryonic antigen (CEA) overexpressed in lung cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, rectal cancer, colon cancer, medullary thyroid cancer, etc.; Epithelial cell adhesion molecule (EpCAM) overexpressed in liver cancer and gastric cancer; GD2 (disialoganglioside) overexpressed in neuroblastoma, etc., GPC3 (Glypican 3) overexpressed in hepatocellular carcinoma, etc., PSMA (Prostate S) overexpressed in prostate cancer, etc. pecific membrane antigen), TAG-72 (tumor-associated glycoprotein 72) overexpressed in ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc., GD3 (disialoganglioside) overexpressed in melanoma, HLA-DR overexpressed in blood cancer, solid cancer, etc. Human leukocyte antigen-DR), MUC1 (Mucin 1) overexpressed in advanced solid cancer, etc., NY-ESO-1 (New York esophageal squamous cell carcinoma 1) overexpressed in advanced non-small-cell lung cancer, nasopharynx LMP1 (Latent membrane protein 1) overexpressed in tumors (Nasopharyngeal neoplasms), lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colon cancer, colon cancer, pancreatic cancer, etc. TRAILR2 (tumor-necrosis factor-related apoptosis-inducing ligand receptor), blood vessels Angiogenic factor receptor VEGFR2 (vascular endothelial growth factor receptor 2), HGFR (hepatocyte growth factor receptor) overexpressed in hepatocellular carcinoma, etc. may be target molecules, and surface antigens of cancer stem cells, such as CD44 and CD166, are also target molecules. can be Many target molecules are known in the art that are overexpressed in cancer cells compared to normal cells, and for more specific target molecules other than those exemplified above, see Anne T Collins et al. Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 2005 Dec 1;65(23):10946-51], Chenwei Li et al. Identification of Pancreatic Cancer Stem Cells. Cancer Res. 2007 Feb 1;67(3):1030-7], Shuo Ma et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019 Sep 7;15(12):2548-2560], Dhaval S Sanchala et al. Oncolytic Herpes Simplex Viral Therapy: A Stride Toward Selective Targeting of Cancer Cells. Front Pharmacol. 2017 May 16;8:270] and the like.
특히 본 발명에서 표적분자는 바람직하게는 CEA, HER2 또는 EpCAM이다. In particular, in the present invention, the target molecule is preferably CEA, HER2 or EpCAM.
본 발명의 재조합 HSV가 어뎁터에 의해 표적화하는 표적세포는 암세포일 경우 식도암, 위암, 대장암, 직장암, 구강암, 인두암, 후두암, 폐암, 결장암, 유방암, 자궁 경부암, 자궁 내막체암, 난소암, 전립선암, 고환암, 흑색종, 방광암, 신장암, 간암, 췌장암, 골암, 결합 조직암, 피부암, 뇌암, 갑상선암, 백혈병, 호지킨(Hodgkin) 질환, 림프종, 다발성 골수종, 혈액암 등 암종을 불문한다. If the target cells targeted by the recombinant HSV of the present invention by the adapter are cancer cells, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, and prostate cancer Cancer, testicular cancer, melanoma, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, multiple myeloma, blood cancer, etc.
또 본 발명의 재조합 HSV가 ICP34.5(Protein preventing cellular inhibition of protein synthesis)를 암호화하는 유전자, ICP6(ribonucleotide reductase)을 암호화하는 유전자, ICP47(Protein inhibiting TAP/MHC class Ipresentation)을 암호화하는 유전자, ICP4(Protein repressing expression of viral immediate early genes and activating expression of viral early and late genes)을 암호화는 유전자, TK(thymidine kinase)을 암호화하는 유전자, UNG(Uracil DNA glycosylase)를 암호화하는 유전자, dUTPase를 암호화하는 유전자 등은 결실되거나 기능을 발휘하지 않도록(즉 전사나 번역이 방해를 받거나 번역되더라도 본래 가지는 활성이 나타나지 않도록) 돌연변이(mutation)될 수 있다.In addition, the recombinant HSV of the present invention is a gene encoding ICP34.5 (Protein preventing cellular inhibition of protein synthesis), a gene encoding ICP6 (ribonucleotide reductase), a gene encoding ICP47 (Protein inhibiting TAP/MHC class Ipresentation), ICP4 (Protein repressing expression of viral immediate early genes and activating expression of viral early and late genes), TK (thymidine kinase), UNG (Uracil DNA glycosylase), dUTPase etc. can be deleted or mutated so that they do not function (ie that transcription or translation is interrupted or that translation does not show its original activity).
이러한 유전자들은 바이러스에서 결실되거나 기능을 발휘하지 않도록 돌연변이 되더라도 세포분열을 계속하는 암세포(dividing cells)에서는 기능적 유사 유전자가 존재하여 바이러스의 복제, 증식, 세포 용해성이 여전히 가능한 유전자들이다. 그런데 이러한 유전자들은 그것이 결실되거나 기능을 발휘하지 않도록 돌연변이될 경우 세포분열을 멈춘 정상세포(nondividing cells)에서는 바이러스의 복제, 증식, 세포 용해성을 저해한다(Annu. Rev. Cancer Biol. 2018. 2:155-73).Even if these genes are deleted from the virus or mutated so that they do not function, there are functionally similar genes in dividing cells that continue to divide, so they are genes that are still capable of virus replication, proliferation, and cell lysis. However, when these genes are deleted or mutated so that they do not exert their functions, virus replication, proliferation, and cell lysis are inhibited in nondividing cells that have stopped dividing (Annu. Rev. Cancer Biol. 2018. 2:155). -73).
본 발명에서, 어뎁터의 세포 표적화 영역은 표적분자와 특이적 결합능을 가지는 완전한 항체 이외에도 항체 유도체, 항체 유사체일 수 있다. 항체 유도체는 표적분자와 특이적 결합능을 가지는 항체 가변 영역을 적어도 하나 이상 포함하는 완전한 항체의 단편 또는 변형 항체을 의미한다. 이러한 항체 유도체로서는 Fab, scFv, Fv, VhH, VH, VL 등의 항체 단편, Fab2, Fab3, 미니바디, 디아바디, 트리바디, 테트라바디, 비스-scFv 등의 다가성 또는 다중특이적 변형 항체 등을 들 수 있다. 항체 유사체는 항체와 마찬가지로 표적분자와 특이적 결합능을 갖지만 구조상 항체와는 다르고 일반적으로 항체보다 낮은 분자량을 갖는 인위적 펩티드 또는 폴리펩티드를 의미한다. 이러한 항체 유사체로서 ABD, 애드히론 (adhiron), 애피바디(affibody), 애필린(affilin), 애피머(affimer), 알파바디(alphabody), 안티칼린(anticalin), 아르마딜로(armadillo) 반복 단백질, 센티린 (centyrin), 달핀(DARPin), 피노머(fynomer), Kunitz 영역, 프로넥틴 (pronectin), 리피바디(repebody) 등을 들 수 있다.In the present invention, the cell targeting region of the adapter may be an antibody derivative or an antibody analog in addition to a complete antibody having specific binding ability with a target molecule. An antibody derivative refers to a fragment or modified antibody of a complete antibody comprising at least one antibody variable region having specific binding ability with a target molecule. Examples of such antibody derivatives include antibody fragments such as Fab, scFv, Fv, VhH, VH, VL, and polyvalent or multispecific modified antibodies such as Fab2, Fab3, minibody, diabody, tribody, tetrabody, bis-scFv, etc. can be heard Antibody analog refers to an artificial peptide or polypeptide that has specific binding ability with a target molecule like an antibody, but is different from an antibody in structure and generally has a lower molecular weight than an antibody. As such antibody analogues, ABD, adhron, affibody, affilin, affimer, alphabody, anticalin, armadillo repeat protein, centimeter Lin (centyrin), dalpin (DARPin), phynomer (fynomer), Kunitz region, pronectin (pronectin), repeat body (repebody) and the like are mentioned.
이러한 항체, 항체 유도체, 항체 유사체, 그 제조와 관련해서는 당업계에 상당히 많은 문헌이 축적되어 있으며, 그러한 문헌으로서는 예컨대 문헌[Renate Kunert & David Reinhart, Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. 2016 Apr;100(8):3451-61], 문헌[Holliger P1, Hudson PJ., Engineered antibody fragments and the rise of single domains, Nat Biotechnol. 2005 Sep;23(9):1126-36], 문헌[Xiaowen Yu et al., Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis, Annual Review of Analytical Chemistry, 2017, 10:293-320], 문헌[Abdul Rasheed Baloch et al., Antibody mimetics: promising complementary agents to animal-sourced antibodies, Critical Reviews in Biotechnology, 2016, 36:268-275] 등을 들 수 있다.A considerable amount of literature has been accumulated in the art with respect to such antibodies, antibody derivatives, antibody analogs, and their manufacture, and as such literature, for example, Renate Kunert & David Reinhart, Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. 2016 Apr; 100(8):3451-61], Holliger P1, Hudson PJ., Engineered antibody fragments and the rise of single domains, Nat Biotechnol. 2005 Sep;23(9):1126-36], Xiaowen Yu et al., Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis, Annual Review of Analytical Chemistry, 2017, 10:293-320, Abdul Rasheed Baloch et al., Antibody mimetics: promising complementary agents to animal-sourced antibodies, Critical Reviews in Biotechnology, 2016, 36:268-275, and the like.
본 발명에서, 어뎁터의 세포 표적화 영역은 바람직하게는 scFv(Single-chain variable fragment)이다. scFv는 면역글로불린의 중쇄(VH)의 가변 영역과 경쇄(VL)의 가변 영역의 짧은 링커 펩티드를 매개로 연결된 단일사슬항체를 말한다. scFv에서 VH의 N-말단은 VL의 C-말단과 연결되거나, 또는 VL의 N-말단은 VH의 C-말단과 연결된다. scFv에서 링커 펩티드는 중쇄와 경쇄의 고유 3차원 구조를 방해하지 않으면서 이들 중쇄와 경쇄가 공간적으로 인접하여 표적분자와의 특이적인 결합 능력을 가지도록 하는 한, 임의의 길이, 임의의 서열의 링커 펩티드일 수 있다. 바람직하게는 링커는 유연성(flexibility), 용해성(solubility), 단백분해에 대한 저항성 등을 고려할 때, Ser, Gly, Asn, Thr 등의 아미노산 중 한 종류 이상의 아미노산으로 이루어지는 것이 바람직하며, 길이는 1~50개 아미노산일 수 있다.In the present invention, the cell targeting region of the adapter is preferably a single-chain variable fragment (scFv). scFv refers to a single-chain antibody linked via a short linker peptide between the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of immunoglobulin. In scFv, the N-terminus of VH is linked to the C-terminus of the VL, or the N-terminus of the VL is linked to the C-terminus of VH. In scFv, the linker peptide is a linker of any length and any sequence as long as the heavy and light chains are spatially adjacent to each other and have specific binding ability with the target molecule without interfering with the intrinsic three-dimensional structure of the heavy and light chains. It may be a peptide. Preferably, in consideration of flexibility, solubility, resistance to proteolysis, etc., the linker is preferably composed of one or more amino acids among amino acids such as Ser, Gly, Asn, and Thr, and the length is 1~ 50 amino acids.
본 발명에서 특히 scFv는 표적분자를 CEA, HER2 또는 EpCAM를 표적분자로 하고, CEA, HER2 또는 EpCAM에 대한 scFv는 도 4 내지 6에 개시된 서열인 것이 바람직하다. 더 구체적으로 본 발명에서 scFv는 표적분자를 CEA를 표적분자로 하고, CEA에 대한 scFv는 서열번호 1의 VL와 서열번호 2의 VH가 링커 펩티드를 매개로, VL, 링커 펩티드 VH 순서로 연결된 것(즉 VL의 C-말단이 VH의 N-말단과 링커 펩티드를 매개로 연결된 것)이 바람직하고, HER2에 대한 scFv는 서열번호 4의 VH와 서열번호 5의 VL이 링커 펩티드를 매개로 VH, 펩티드 링커, VL 순서로 결합된 것(즉 VH의 C-말단이 VL의 N-말단과 링커 펩티드를 매개로 연결된 것)이 바람직하며, EpCAM에 대한 scFv는 서열번호 7의 VL과 서열번호 8의 VH가 링커 펩티드를 매개로 VL, 펩티드 링커, VH 순서로 결합된 것(즉 VL의 C-말단이 VH의 N-말단과 링커 펩티드를 매개로 연결된 것)이 바람직하다. 여기서 CEA에 대한 scFv의 링커 펩티드는 서열번호 3의 아미노산 서열을 갖는 것이 바람직하고, 상기 HER2에 대한 scFv의 링커 펩티드는 서열번호 6의 아미노산 서열을 갖는 것이 바람직하며, 상기 EpCAM에 대한 scFv의 링커 펩티드는 서열번호 9의 아미노산 서열을 갖는 것이 바람직하다.In particular, in the present invention, it is preferable that the scFv target molecule is CEA, HER2 or EpCAM, and the scFv for CEA, HER2 or EpCAM is the sequence shown in FIGS. 4 to 6 . More specifically, in the present invention, scFv has a target molecule CEA as a target molecule, and scFv for CEA is one in which VL of SEQ ID NO: 1 and VH of SEQ ID NO: 2 are linked via a linker peptide, VL, and linker peptide VH in that order (that is, the C-terminus of VL is linked to the N-terminus of VH via a linker peptide) is preferable, and the scFv for HER2 is VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are VH via a linker peptide, Peptide linkers, those joined in VL order (that is, the C-terminus of VH is connected to the N-terminus of VL via a linker peptide) is preferable, and the scFv for EpCAM is the VL of SEQ ID NO: 7 and SEQ ID NO: 8 Preferably, VH is linked in the order of VL, peptide linker, and VH via a linker peptide (ie, C-terminus of VL is linked to N-terminus of VH via linker peptide). Here, the linker peptide of scFv to CEA preferably has the amino acid sequence of SEQ ID NO: 3, and the linker peptide of scFv to HER2 preferably has the amino acid sequence of SEQ ID NO: 6, and the linker peptide of scFv to EpCAM. preferably has the amino acid sequence of SEQ ID NO: 9.
본 발명에서 HveC의 세포외 도메인은 리더 서열(1~30 아미노산 서열)이 포함된 서열번호 12의 HveC114(1~114 아미노산 서열)나 아래의 실시예에서 사용된, 리더 서열(1~30 아미노산 서열)이 제거된 HveC(31~114 아미노산 서열, 서열번호 11)가 사용될 수 있다. In the present invention, the extracellular domain of HveC is HveC114 (1 to 114 amino acid sequence) of SEQ ID NO: 12 including a leader sequence (1 to 30 amino acid sequence) or a leader sequence (1 to 30 amino acid sequence) used in the examples below ) from which HveC has been removed (31-114 amino acid sequence, SEQ ID NO: 11) may be used.
또 본 발명에서 HveC 세포외 도메인과 암세포 표적화 영역 사이에 링커 서열이 게재될 수 있는데, 이 링커 서열은 이들 어뎁터의 각 도메인의 기능을 저해 하지 않는 한 임의의 길이, 임의의 서열의 링커일 수 있다. 바람직하게는 링커는 Ser, Gly, Asn, Thr 4가지 아미노산 중 한 종류 이상의 아미노산으로 이루어질 수 있으며, 길이는 1~30개, 아미노산, 바람직하게는 3~25개 아미노산, 더 바람직하게는 8~20개 아미노산일 수 있다.Also, in the present invention, a linker sequence may be placed between the HveC extracellular domain and the cancer cell targeting region, and the linker sequence may be a linker of any length and any sequence as long as the function of each domain of these adapters is not inhibited. . Preferably, the linker may consist of one or more amino acids among the four amino acids Ser, Gly, Asn, and Thr, and have a length of 1 to 30 amino acids, preferably 3 to 25 amino acids, more preferably 8 to 20 amino acids. It may be a dog amino acid.
또 본 발명의 어뎁터는 NH2-암세포 표적화 도메인-HveC 세포외 도메인-COOH 순이거나 그 역순일 수 있다. 링커 펩타이드가 매개될 경우는 NH2-암세포 표적화 영역-링커 펩타이드-HveC 세포외 도메인-COOH 순이거나 그 역순일 수 있다. 본 발명의 어뎁터는 NH2-암세포 표적화 도메인-HveC 세포외 도메인-COOH 순 또는 NH2-암세포 표적화 영역-링커 펩타이드-HveC 세포외 도메인-COOH 순일 경우, HveC 세포외 도메인의 리더 서열은 제외되고 사용되는 것이 바람직하다. In addition, the adapter of the present invention may be in the order of NH 2 -cancer cell targeting domain-HveC extracellular domain-COOH or the reverse order. When the linker peptide is mediated, NH 2 -cancer cell targeting region-linker peptide-HveC extracellular domain-COOH may be in the order or vice versa. The adapter of the present invention is in the order of NH 2 -cancer cell targeting domain-HveC extracellular domain-COOH or NH 2 -cancer cell targeting region-linker peptide-HveC extracellular domain-COOH In the order, the leader sequence of the HveC extracellular domain is excluded and used It is preferable to be
또 본 발명에서 세포 표적화 영역으로 표적분자에 대한 scFv을 사용할 경우, VH와 VL 사이에, VH와 VL 사이에 링커 펩타이드가 매개될 경우는 VH 또는 VL와 링커 펩타이드 사이, scFv와 HveC 사이, scFv와 HveC 사이에 링커 펩타이드가 매개될 경우에는 scFv와 링커 사이, 링커와 HveC 사이에, 클로닝을 용이하게 하기 위하여 임의의 제한효소 작용 부위에 해당하는 아미노산이 게재될 수 있다. 예컨대 아래와 같이 실시예에서 같이 BamHI이 작용하는 GS((염기서열: GGATCC), 제한효소 EcoRI가 작용하는 EF(염기서열: GAATTC)가 게재될 수 있다.In addition, in the present invention, when using an scFv for a target molecule as a cell targeting region, between VH and VL, between VH and VL when a linker peptide is mediated, between VH or VL and linker peptide, between scFv and HveC, between scFv and When a linker peptide is mediated between HveCs, an amino acid corresponding to an arbitrary restriction enzyme action site may be placed between scFv and linker and between linker and HveC to facilitate cloning. For example, as in the examples below, GS ((base sequence: GGATCC) that BamHI acts, EF (base sequence: GAATTC) that restriction enzyme EcoRI acts on may be published.
본 발명에서, 재조합 HSV는 암세포에 대한 면역반응을 유도 또는 증진시키기 위한 인자들을 단독으로 또는 임의의 조합으로 발현하도록 해당 인자에 대한 유전자가 HSV 게놈에 삽입될 수 있다. 그러한 인자들은 사이토카인, 케모카인, 면역관문(immune checkpoint)에 대한 길항제(예컨대 항체, 항체 유도체 또는 항체 유사체, 특히 scFv), 면역세포(T 세포 또는 NK 세포)의 활성화를 유도할 수 있는 보조 자극 인자(co-stimulatory factor), 암세포에 대한 면역반응을 억제하는 TGFβ의 기능을 저해할 수 있는 길항제(예컨대 항체, 항체 유도체 또는 항체 유사체, 특히 scFv), 고형암 종양미세환경을 구성하는 헤파란 설페이트 프로테오글리칸(heparan sulfate proteoglycan)을 분해할 수 있는 헤파라나아제(heparanase), 혈관 신생 인자 수용체인 VEGFR-2(VEGF receptor-2)의 기능을 저해할 수 있는 길항제(예컨대 항체, 항체 유도체 또는 항체 유사체, 특히 scFv) 등을 발현하도록 조작될 수 있다.In the present invention, the gene for the recombinant HSV may be inserted into the HSV genome so that the factors for inducing or enhancing an immune response against cancer cells are expressed alone or in any combination. Such factors include cytokines, chemokines, antagonists of immune checkpoints (eg antibodies, antibody derivatives or antibody analogs, particularly scFv), costimulatory factors capable of inducing activation of immune cells (T cells or NK cells) (co-stimulatory factor), an antagonist capable of inhibiting the function of TGFβ to suppress the immune response to cancer cells (such as antibodies, antibody derivatives or antibody analogs, especially scFv), heparan sulfate proteoglycans that constitute the solid tumor microenvironment ( Heparanase capable of degrading heparan sulfate proteoglycan, and antagonists capable of inhibiting the function of VEGF receptor-2 (VEGFR-2), an angiogenesis factor receptor (eg, antibody, antibody derivative or antibody analog, particularly scFv ) can be engineered to express
사이토카인은 예컨대 IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, IL-24 등의 인터류킨, IFNα, IFNβ, IFNγ 등의 인터페론, TNFα 등의 종양 괴사 인자, GM-CSF, G-CSF 등의 콜로니 자극 인자 등이 단독으로 또는 2 이상의 임의의 조합으로 재조합 HSV에서 발현되도록 사용될 수 있다.Cytokines include, for example, interleukins such as IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, and IL-24, interferons such as IFNα, IFNβ, and IFNγ, and tumor necrosis factor such as TNFα , GM-CSF, colony stimulating factors such as G-CSF, etc. may be used alone or in any combination of two or more to be expressed in recombinant HSV.
케모카인은 예컨대 CCL2(C-C Motif Chemokine Ligand 2), CCL5(RANTES), CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1( (X-C Motif Chemokine Ligand 1)) 등이 단독으로 또는 조합합으로 재조합 HSV에서 발현되도록 사용될 수 있다.Chemokines, for example, CCL2 (CC Motif Chemokine Ligand 2), CCL5 (RANTES), CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1 ((XC Motif Chemokine Ligand 1)) alone or It can be used to be expressed in recombinant HSV in combination.
면역관문에 대한 항체는 PD-1(programmed cell death-1), PD-L1(programmed cell deathligand 1), PD-L2(programmed cell death-ligand 2), CD27(cluster of differentiation 27), CD28(cluster of differentiation 28), CD70(cluster of differentiation 70), CD80(cluster of differentiation 80), CD86(cluster of differentiation 86), CD137(cluster of differentiation 137), CD276(cluster of differentiation 276), KIRs(killer-cell immunoglobulin-like receptors), LAG3(lymphocyte-activation gene 3), GITR(glucocorticoid-induced TNFR-related protein), GITRL(glucocorticoid-induced TNFR-related protein ligand), CTLA-4(cytolytic T lymphocyte associated antign-4) 등에 대한 길항제가 단독으로 또는 조합으로 재조합 HSV에서 발현되도록 사용될 수 있다.Antibodies against immune checkpoints are PD-1 (programmed cell death-1), PD-L1 (programmed cell deathligand 1), PD-L2 (programmed cell death-ligand 2), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD70 (cluster of differentiation 70), CD80 (cluster of differentiation 80), CD86 (cluster of differentiation 86), CD137 (cluster of differentiation 137), CD276 (cluster of differentiation 276), KIRs (killer-cell) immunoglobulin-like receptors), lymphocyte-activation gene 3 (LAG3), glucocorticoid-induced TNFR-related protein (GITR), glucocorticoid-induced TNFR-related protein ligand (GITRL), cytolytic T lymphocyte associated antigen-4 (CTLA-4) antagonists can be used alone or in combination to be expressed in recombinant HSV.
보조 자극 인자는 CD2, CD7, LIGHT, NKG2C,CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1(림프구 기능 연관 항원-1), ICOS(유도성 T 세포 공동자극인자), CD3γ, CD3δ, CD3ε 등이 단독으로 또는 조합으로 재조합 HSV에서 발현되도록 사용될 수 있다.Co-stimulatory factors are CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T-cell co-stimulatory factor), CD3γ , CD3δ, CD3ε, etc., alone or in combination, can be used to be expressed in recombinant HSV.
본 발명에서, 재조합 HSV는 프로드럭(prodrug)을 암세포에 독성을 나타내는 약물(drug)으로 전환시켜주는 프로드럭 활성화 효소(prodrug-activating enzymes)를 발현하도록 조작될 수 있다. 이러한 프로드럭 활성화 효소로서는, 프로드럭인 5-FC(5-fluorocytosine)을 5-FU(5-fluorouracil)인 약물로 전환시켜 주는 시토신 디아민나아제(Cytosine deaminase), 프로드럭인 CPA(cyclophosphamide)를 PM( phosphoramide mustard)인 약물로 전환시키주는 랫드 사이토크롬 P450(rat cytochrome P450, CYP2B1), 프로드럭인 이리노테칸(irinotecan, SN-38150)을 SN-38인 약물로 전환시켜주는 카르복실에스터라제(carboxylesterase), 프로드럭인 BC1954를 DNA 교차결합제인 4-하이드록시아민151(4-hydroxylamine151)로 전환시켜주는 세균 니트로리덕타아제(bacterial nitroreductase), 프로드럭인 6-메틸퓨린-2'-디옥시리보시드(6-methypurine-2'-deoxyriboside)를 6-메틸퓨린(6-methylpurine)으로 전환시켜주는 대장균에서 분리된 PNP(purine nucleoside phosphorylase) 등을 들 수 있다.In the present invention, recombinant HSV can be engineered to express prodrug-activating enzymes that convert a prodrug into a drug that is toxic to cancer cells. As such a prodrug activating enzyme, cytosine deaminase, which converts the prodrug 5-FC (5-fluorocytosine) into a drug that is 5-FU (5-fluorouracil), and the prodrug CPA (cyclophosphamide) Carboxylesterase (rat cytochrome P450, CYP2B1), which converts the phosphoramide mustard (PM) drug, and the prodrug irinotecan (SN-38150) to the SN-38 drug carboxylesterase), bacterial nitroreductase that converts prodrug BC1954 to DNA crosslinking agent 4-hydroxylamine151, prodrug 6-methylpurine-2'-deoxyriboside and PNP (purine nucleoside phosphorylase) isolated from E. coli, which converts (6-methypurine-2'-deoxyriboside) to 6-methylpurine.
또한 본 발명에서, 재조합 HSV는 TRAIL(TNF-Related Apoptosis Inducing Ligand)를 발현하도록 조작될 수도 있다. TRAIL는 암세포에서 과발현되는 그것의 수용체에 결합하여 암세포의 사멸을 유도한다고 알려져 있다(Kaoru Tamura et al. Multimechanistic Tumor Targeted Oncolytic Virus Overcomes Resistance in Brain Tumors. Mol Ther. 2013 Jan;21(1):68-77).In addition, in the present invention, recombinant HSV may be engineered to express TRAIL (TNF-Related Apoptosis Inducing Ligand). TRAIL is known to induce apoptosis by binding to its receptor overexpressed in cancer cells (Kaoru Tamura et al. Multimechanistic Tumor Targeted Oncolytic Virus Overcomes Resistance in Brain Tumors. Mol Ther. 2013 Jan;21(1):68- 77).
이러한 면역반응을 유도 또는 증진시키기 위한 인자들이나 프로드럭 활성화 효소의 사용과 관련해서 더 구체적인 것은 문헌[Michele Ardolino et al., Cytokine treatment in cancer immunotherapy, J. Oncotarget, Oncotarget. 2015 Aug 14;6(23):], 문헌Bernhard Homey et al. Chemokines: Agents for the Immunotherapy of Cancer. Nat Rev Immunol. 2002 Mar;2(3):175-84], 문헌[Marianela Candolfi 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, 22:107713], 문헌[Danny N Khalil et al. The Future of Cancer Treatment: Immunomodulation, CARs and Combination Immunotherapy. Nat Rev Clin Oncol. 2016 May;13(5):273-90], 문헌[Paul E Hughes et al. Targeted Therapy and Checkpoint Immunotherapy Combinations for the Treatment of Cancer. Trends Immunol. 2016 Jul;37(7):462-476], 문헌[Cole Peters1, Samuel D Rabkin, Designing herpes viruses as oncolytics, Mol Ther Oncolytics. 2015;2:15010] 등을 참조할 수 있다.For more specific information regarding the use of factors or prodrug activating enzymes for inducing or enhancing this immune response, see Michele Ardolino et al., Cytokine treatment in cancer immunotherapy, J. Oncotarget, Oncotarget. 2015 Aug 14;6(23):], Bernhard Homey et al. Chemokines: Agents for the Immunotherapy of Cancer. Nat Rev Immunol. 2002 Mar;2(3):175-84], Marianela Candolfi 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, 22:107713], Danny N Khalil et al. The Future of Cancer Treatment: Immunomodulation, CARs and Combination Immunotherapy. Nat Rev Clin Oncol. 2016 May;13(5):273-90], Paul E Hughes et al. Targeted Therapy and Checkpoint Immunotherapy Combinations for the Treatment of Cancer. Trends Immunol. 2016 Jul;37(7):462-476], Cole Peters1, Samuel D Rabkin, Designing herpes viruses as oncolytics, Mol Ther Oncolytics. 2015;2:15010] and the like.
본 발명에서, 면역반응을 유도 또는 증진시키기 위한 인자들이나 프로드럭 활성화 효소는 전술한 바의 어텝터에서와 같이, 그 유전자의 발현 카세트(즉 그 유전자가 이의 발현을 가능하게 하는 프로모터 서열 및 폴리아데닐화 시그널 서열과 작동가능하게 연결된 구성체)가 HSV의 증식을 저해하지 않으면서 HSV 게놈에 삽입되는데, 그러한 삽입은 HSV 게놈의 결실 없이, 또는 HSV 게놈 중 비필수 유전자가 일부 또는 전부 결실되고 그 결실된 위치에 삽입될 수 있다. 이때 HSV 게놈의 결실 없이 삽입될 경우 각 유전자 사이에 삽입될 수 있으며 바람직한 삽입 위치는 예컨대 UL3과 UL4 유전자 사이, UL26과 UL27 유전자 사이, UL37과 UL38 유전자 사이, UL48과 UL49 유전자 사이, UL53과 UL54 유전자 사이 등을 들 수 있다. 비필수 유전자가 결실되고 그 결실된 위치에 삽입되거나 비필수 유전자의 결실 없이 그 유전자 중에 삽입될 경우, 그러한 비필수 유전자는 전술한 바의 임의의 비필수 유전자 중에서 선택될 수 있다.In the present invention, factors or prodrug activating enzymes for inducing or enhancing an immune response are the expression cassettes of the gene (that is, the promoter sequence and the polyade a construct operably linked to a nylation signal sequence) is inserted into the HSV genome without inhibiting the proliferation of HSV, such insertion without deletion of the HSV genome, or in which some or all of the non-essential genes in the HSV genome are deleted and the deletion can be inserted into position. At this time, when inserted without deletion of the HSV genome, it can be inserted between each gene, and preferred insertion positions are, for example, between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL37 and UL38 genes, between the UL48 and UL49 genes, and between the UL53 and UL54 genes. between, and the like. When a non-essential gene is deleted and inserted at the deleted position or inserted into the gene without deletion of the non-essential gene, such non-essential gene may be selected from any of the non-essential genes described above.
다른 측면에 있어서 본 발명은 전술한 바의 재조합 HSV를 유효성분으로 포함하는 항암용 약제학적 조성물에 관한 것이다.In another aspect, the present invention relates to an anticancer pharmaceutical composition comprising the above-mentioned recombinant HSV as an active ingredient.
본 발명의 약제학적 조성물은 재조합 HSV이 발현하는 어뎁터의 표적화 영역이 표적하는 표적분자를 발현하는 암종에 대해 항암 용도를 갖는다. 그러한 암종은 앞서 표적분자와 관련하여 설명한 바와 같다. The pharmaceutical composition of the present invention has anticancer use against carcinoma expressing a target molecule targeted by a targeting region of an adapter expressed by recombinant HSV. Such carcinoma is the same as described above with respect to the target molecule.
특히 본 발명의 조성물은 CEA나 HER2를 발현하는 종양세포를 갖는 암종에 대해 항암 용도를 갖는 것이 바람직하다. 예컨대 CEA를 발현하는 종양세포는 대장암 세포, 위암 세포, 폐암 세포, 유방암 세포, 직장암 세포, 결장암 세포, 간암 세포 등을 들 수 있고, 또 예컨대 HER2를 발현하는 종양세포는 유방암 세포, 난소암 세포, 위암 세포, 폐암 세포, 두경부암 세포, 골육종 세포, 다형성 신경교모종 세포(glioblastoma multiforme), 침샘 종양(salivary gland tumor) 세포 등을 들 수 있다.In particular, it is preferable that the composition of the present invention has anticancer use against carcinoma having tumor cells expressing CEA or HER2. For example, the tumor cells expressing CEA include colorectal cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rectal cancer cells, colon cancer cells, liver cancer cells, and the like. , gastric cancer cells, lung cancer cells, head and neck cancer cells, osteosarcoma cells, glioblastoma multiforme, salivary gland tumor cells, and the like.
본 발명에서 항암은 암세포의 사멸, 암세포의 생존능 저하, 암세포의 증식 억제에 따른 암이 가지는 병리적 증상의 억제나 지연, 그러한 병리적 증상의 발병 억제나 지연, 암 전이 억제, 암 재발 억제를 포함하는 의미이다. In the present invention, anticancer includes apoptosis of cancer cells, reduction in viability of cancer cells, inhibition or delay of pathological symptoms of cancer due to inhibition of proliferation of cancer cells, inhibition or delay of onset of such pathological symptoms, inhibition of cancer metastasis, inhibition of cancer recurrence it means to
또 본 발명의 약제학적 조성물은 허가된 항암제와 병용 또는 혼합되어 사용될 수 있다. 그러한 항암제로는 대사 길항제, 알킬화제, 토포아이소머라제 길항제, 미세소관 길항제, 식물유래 알칼로이드 등 암세포에 세포독성을 나타내는 임의의 항암제, 임의의 사이토카인 의약품, 임의의 항체 의약품, 임의의 면역관문 억제제 의약품, 임의의 세포 치료제(car-T cell therarpy, car-NK cell therapy) 의약품 등을 포함한다. 구체적으로 탁솔, 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 제피티닙, 보르테조밉, 수니티닙, 카보플라틴, 시스플라틴, 리툭시맙, 엘로티닙, 소라페닙, IL-2 의약품, INF-α 의약품, INF-γ 의약품, 트라스투주맙, 블리나투모맙, 이필리무맙, 펩브롤리주맙, 니볼리주맙, 아테졸리주맙(Atezolizumab), 두발루맙(Durvalumab), 베바시주맙, 세툭시맙, 티사젠렉루셀(Tisagenlecleucel, 킴리아), 액시캡타젠 실로루셀(Tisagenlecleucel Axicabtagene Ciloleucel, 예스카타) 등이 예시될 수 있으며, 이들 예시된 항암제 이외에도 당업계에 공지된 여타의 항암제도 제한없이 본 발명의 약제학적 조성물과 혼합되어 사용되거나 병용 사용될 수 있다. In addition, the pharmaceutical composition of the present invention may be used in combination or mixed with an approved anticancer agent. Such anticancer agents include, any anticancer agents that are cytotoxic to cancer cells, such as metabolic antagonists, alkylating agents, topoisomerase antagonists, microtubule antagonists, and plant-derived alkaloids, any cytokine drug, any antibody drug, any immune checkpoint inhibitor drug , any cell therapy (car-T cell therapy, car-NK cell therapy) medicines, and the like. Specifically, taxol, nitrogen mustard, imatinib, oxaliplatin, gefitinib, bortezomib, sunitinib, carboplatin, cisplatin, rituximab, erlotinib, sorafenib, IL-2 drugs, INF-α drugs, INF -γ drugs, trastuzumab, blinatumomab, ipilimumab, pepbrolizumab, nivolizumab, atezolizumab, duvalumab, bevacizumab, cetuximab, Tisagenlecleucel, Kymria), axicaptagen ciloleucel (Tisagenlecleucel Axicabtagene Ciloleucel, Yescata), etc. may be exemplified, and in addition to these exemplified anticancer agents, other anticancer agents known in the art are mixed with the pharmaceutical composition of the present invention without limitation can be used together or used in combination.
본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체 또는 부형제를 포함하여 투여 경로에 따라 당업계에 공지된 통상의 방법으로 경구용 제형 또는 비경구용 제형으로 제조될 수 있다. The pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form by a conventional method known in the art according to the route of administration, including a pharmaceutically acceptable carrier or excipient.
그러한 약제학적으로 허용가능한 담체 또는 부형제는 인체에 특별한 독성을 가지지 않으면서 약물의 활성이나 특성을 저해하지 않는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물(예를 들면, 식염수 및 멸균수), 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘, 미네랄 오일, 링거액, 완충제, 말토덱스트린 용액, 글리세롤, 에탄올, 덱스트란, 알부민, 또는 이들의 임의의 조합일 수 있다. 특히 본 발명의 약제학적 조성물이 액상 용액으로 제제화될 경우 적합한 담체 또는 부형제로서는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 등의 성분 중에서 하나 이상의 성분을 단독 또는 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 의약품 첨가제 등을 첨가하여 사용할 수 있다.Such pharmaceutically acceptable carriers or excipients do not inhibit the activity or properties of drugs without being particularly toxic to the human body, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, algae nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (eg, saline and sterile water), syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc , magnesium stearate, mineral oil, Ringer's solution, buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof. In particular, when the pharmaceutical composition of the present invention is formulated as a liquid solution, a suitable carrier or excipient is one of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. The above components may be used alone or in combination, and if necessary, other conventional pharmaceutical additives such as antioxidants, buffers, and bacteriostats may be added.
본 발명의 약제학적 조성물이 경구 투여제로 제제될 경우는 정제, 트로키, 캡슐, 엘릭서, 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있고, 비경구 투여제 특히 주사제로 제제화 경우 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 본 발명의 약제학적 조성물은 용액, 현탁액, 정제, 환약, 캡슐, 서방형 제제 등의 형태로도 제조화할 수 있다.When the pharmaceutical composition of the present invention is formulated for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. It can be prepared in multiple dosage forms. The pharmaceutical composition of the present invention may be prepared in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation, and the like.
본 발명의 약제학적 조성물은 약제학적 분야에서 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위 투여형의 제제 형태로 제형화시켜 당업계에서 통상적으로 사용하는 투여 방법을 이용하여 경구 투여 경로나, 또는 피부, 병변 내(intralesional), 정맥 내, 근육 내, 동맥 내, 골수 내, 수막강 내, 심실 내, 폐, 경피, 피하, 복 내, 비강 내, 소화관 내, 국소, 설하, 질 내, 직장 경로 등 비경구 투여 경로에 의하여 투여될 수 있다.The pharmaceutical composition of the present invention is formulated in a dosage form of a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and an oral administration route using an administration method commonly used in the art, or dermal, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, intravaginal, It may be administered by a parenteral route, such as a rectal route.
본 발명의 약제학적 조성물의 투여량(유효량)은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 결정할 수 있다. 바람직한 양태에 있어 본 발명의 약제학적 조성물은 단위 용량 형태의 주사제로 제조되며, 단위 용량 형태의 주사제로 제조될 경우 본 발명의 약제학적 조성물의 단위 용량 당 포함되는 재조합 HSV 바이러스의 양은 102-1014 pfu 범위, 특히 104-1011 pfu 범위일 수 있다.The dosage (effective amount) of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate, and response sensitivity. may be prescribed, and a person skilled in the art can appropriately determine the dosage in consideration of these factors. In a preferred embodiment, the pharmaceutical composition of the present invention is prepared as an injection in the form of a unit dose, and when prepared as an injection in the form of a unit dose, the amount of recombinant HSV virus contained per unit dose of the pharmaceutical composition of the present invention is 10 2 -10 in the range of 14 pfu, in particular in the range of 10 4 -10 11 pfu.
다른 양태에 있어서, 본 발명은 전술한 바의 재조합 HSV를 포함하는 약제학적 조성물을 유효량으로 환자 등의 대상체에 투여하는 단계를 포함하는 암 치료 또는 예방 방법(즉 종양 치료 또는 예방 방법)에 관한 것이다. In another aspect, the present invention relates to a method for treating or preventing cancer (ie, a method for treating or preventing a tumor) comprising administering to a subject, such as a patient, an effective amount of a pharmaceutical composition comprising the recombinant HSV as described above. .
이러한 암 치료 방법은 재조합 HSV가 그 어뎁터의 암세포 표적화 영역이 표적하는 표적분자를 가진 암세포를 용해하여 사멸시킴으로써 가능해진다. 따라서 본 발명의 치료 방법은 그러한 표적분자를 가진 임의의 암종에 대해서 적용이 가능하다. 특히 본 발명의 치료 방법은 CEA나 HER2를 발현하는 암종에 적용되는 것이 바람직하다. This cancer treatment method becomes possible when recombinant HSV dissolves and kills cancer cells having a target molecule targeted by the cancer cell targeting region of the adapter. Therefore, the treatment method of the present invention can be applied to any carcinoma having such a target molecule. In particular, the treatment method of the present invention is preferably applied to a carcinoma expressing CEA or HER2.
본 발명의 치료 방법은 상기 다른 암 치료법과 제한없이 병용되어 사용될 수 있다. 예컨대 앞서 예시된 세포독성 항암제, 사이토카인 의약품, 항체 의약품, 면역관문 억제제 의약품, 세포 치료제(car-T cell therarpy, car-NK cell therapy) 의약품, 방사선 치료 요법, 수술 요법 등이 본 발명의 약제학적 조성물의 투여 전·후나 동시 투여 방식으로 병용 사용될 수 있다. The treatment method of the present invention may be used in combination with the above other cancer treatment methods without limitation. For example, the aforementioned cytotoxic anticancer drugs, cytokine drugs, antibody drugs, immune checkpoint inhibitor drugs, cell therapy drugs (car-T cell therapy, car-NK cell therapy) drugs, radiation therapy, surgical therapy, etc. are the pharmaceuticals of the present invention. It may be used in combination before and after administration of the composition or in a simultaneous administration mode.
본 발명의 치료 방법에서 유효량은 그 적용 대상인 환자 등에 의료 전문가 등의 제언에 의한 투여 기간 동안 본 발명의 약제학적 조성물이 투여될 때, 암 치료 또는 예방 효과 등 의도한 의학적 효과를 나타낼 수 있는, 본 발명의 약제학적 조성물이 투여되는 양을 말한다. 이러한 유효량은 전술한 바와 같이 환자의 연령, 체중, 성별, 병적 상태 등에 따라 의료 전문가 등 당업자가 적절히 결정할 수 있다.In the treatment method of the present invention, an effective amount is the present invention that can exhibit intended medical effects, such as cancer treatment or prevention effect, when the pharmaceutical composition of the present invention is administered during the administration period recommended by a medical professional, etc. Refers to the amount to which the pharmaceutical composition of the invention is administered. As described above, such an effective amount can be appropriately determined by a person skilled in the art, such as a medical professional, according to the patient's age, weight, sex, pathological condition, and the like.
본 발명의 치료 방법에서, 그 약제학적 조성물은 바람직하게는 환자 등에 주사제로, 비경구 투여 경로에 의하여 예컨대 병변 내(intralesional, 종양 내), 정맥 내, 근육 내, 동맥 내 등에 투여될 수 있다.In the treatment method of the present invention, the pharmaceutical composition is preferably administered to a patient or the like as an injection, for example, intralesional (intralesional, intratumoral), intravenous, intramuscular, intraarterial, etc. can be administered by parenteral administration route.
전술한 바와 같이, 본 발명에 따르면 본 발명은 HveC 세포외 도메인과 세포 표적화 영역의 융합 단백질을 발현할 수 있는 발현 카세트를 가지는 재조합 헤르페스 심플렉스 바이러스 및 그 용도를 제공할 수 있다. As described above, according to the present invention, it is possible to provide a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of an HveC extracellular domain and a cell targeting region, and uses thereof.
상기 융합 단백질은 재조합 HSV가 암세포인 표적세포를 감염시켜 표적세포 내로 들어가게 되면, HSV가 증식됨과 함께 세포 내에서 발현되어 세포 용해에 의하여 그 증식된 HSV 비리온과 함께 세포 외부로 방출되며, 이 세포 외부로 방출된 융합 단백질은 함께 방출된 HSV 비리온을, 그 암세포 표적화 영역이 인식하는 표적분자를 발현하는 주변 암세포로 감염을 유도하거나 감염 효율을 높이는 작용을 하게 된다.When the recombinant HSV infects a target cell, which is a cancer cell, and enters the target cell, it is expressed in the cell along with the proliferation of HSV, and is released to the outside of the cell together with the proliferated HSV virion by cell lysis. The fusion protein released to the outside induces infection with the HSV virion released together and the surrounding cancer cells expressing a target molecule recognized by the cancer cell targeting region, or increases the infection efficiency.
도 1은 KOS-37 BAC 게놈 구조의 개요도이다.1 is a schematic diagram of the structure of the KOS-37 BAC genome.
도 2은 EmGFP를 발현하는 HSV-1 KOS-EmGFP의 지놈 개요도이다.2 is a genomic schematic diagram of HSV-1 KOS-EmGFP expressing EmGFP.
도 3는 HSV-1 KOS-CEAscFv-HveC-EmGFP와 KOS-HER2scFv-HveC-EmGFP 그리고 KOS-EpCAMscFv-HveC-EmGFP 바이러스 지놈 개요도이다.3 is a schematic diagram of HSV-1 KOS-CEAscFv-HveC-EmGFP, KOS-HER2scFv-HveC-EmGFP and KOS-EpCAMscFv-HveC-EmGFP virus genomes.
도 4 내지 도6은 CEAscFv-HveC 어뎁터와 HER2scFv-HveC 어댑터 그리고 EpCAMscFv-HveC 어댑터의 전체 서열과 해당 서열의 구성을 나타낸 것이다. 4 to 6 show the entire sequence of the CEAscFv-HveC adapter, the HER2scFv-HveC adapter, and the EpCAMscFv-HveC adapter and the configuration of the corresponding sequences.
도 7은 CEA 발현 세포주에 대한 CEAscFv-HveC 어댑터를 발현하는 HSV-1 KOS-CEAscFv-HveC-EmGFP의 특이적 감염 실험이다.7 is a specific infection experiment of HSV-1 KOS-CEAscFv-HveC-EmGFP expressing CEAscFv-HveC adapter against CEA-expressing cell lines.
도 8은 HER2 발현 세포주에 대한 HER2cFv-HveC 어댑터를 발현하는 HSV-1 KOS-HER2scFv-HveC-EmGFP의 특이적 감염 실험이다.8 is a specific infection experiment of HSV-1 KOS-HER2scFv-HveC-EmGFP expressing HER2cFv-HveC adapter against HER2 expressing cell line.
도 9는 EpCAM 발현 세포주에 대한 EpCAMscFv-HveC 어댑터를 발현하는 HSV-1 KOS-EpCAMscFv-HveC-EmGFP의 특이적 감염 실험이다. 9 is a specific infection experiment of HSV-1 KOS-EpCAMscFv-HveC-EmGFP expressing EpCAMscFv-HveC adapter against EpCAM-expressing cell lines.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.
<실시예 1> EmGFP를 발현하는<Example 1> Expression of EmGFP HSV-1 제작 HSV-1 production
HSV-1 유전자는 약 152kb에 달하는 커다란 유전자로 구성이 되어 있어, 외래 유전자를 삽입하거나 특정 위치에서의 변이를 삽입하기 위해서 KOS-37/BAC(Genbank Accession No. MF156583)(Gierasch WW et al. J. Virol Methods. 2006. 135:197~206)을 사용하였다. HSV-1 KOS 스트레인(HSV-1 KOS strain)은 그 특성이 잘 알려져 있고, 유전자의 기능 및 병인 조사에 유용하여 실험실에서 주로 사용되는 HSV-1 스트레인의 일종이다(Smith KO. Proc. Soc. Exp. Biol. Med. 1964. 115:814-816). KOS 게놈에 BAC 플라스미드 삽입을 통해서 제작된 KOS-37/BAC은 DH10B 박테리아(Invitrogen)에 형질 전환(transformation)을 통해 박테리아 수준에서 클로닝을 가능케 한다(Gierasch WW et al.; J. Virol Methods. 2006. 135:197~206). KOS-37/BAC는 HSV-1 KOS 게놈의 UL37과 UL38 사이의 위치에 BAC(bacterial artificial chromosomes)가 양쪽에 LoxP 사이트와 함께 삽입되어 있다. 차후 Cre-Lox system을 이용하여 BAC 유전자를 제거할 수 있게 제작됐다. 그 개요도를 도 1에 나타내었다. The HSV-1 gene consists of a large gene of about 152 kb, and KOS-37/BAC (Genbank Accession No. MF156583) (Gierasch WW et al. J) to insert a foreign gene or insert a mutation at a specific position. Virol Methods. 2006. 135:197-206) was used. HSV-1 KOS strain (HSV-1 KOS strain) is a kind of HSV-1 strain mainly used in laboratories because its characteristics are well known and useful for investigation of gene function and etiology (Smith KO. Proc. Soc. Exp) (Biol. Med. 1964. 115:814-816). KOS-37/BAC, constructed by inserting the BAC plasmid into the KOS genome, enables cloning at the bacterial level through transformation into DH10B bacteria (Invitrogen) (Gierasch WW et al.; J. Virol Methods. 2006. 135:197-206). In KOS-37/BAC, BAC (bacterial artificial chromosomes) is inserted at the position between UL37 and UL38 of the HSV-1 KOS genome together with LoxP sites on both sides. It was made so that the BAC gene could be removed later using the Cre-Lox system. A schematic diagram thereof is shown in FIG. 1 .
EmGFP를 발현하는 HSV-1 제작을 위하여 KOS-37/BAC DNA의 UL26/UL27 위치에, EmGFP(Emerald Green Fluorescent Protein)를 발현할 수 있는 발현 카세트(expression cassette)를 삽입하였다(Tiffany A et al., J Virol Methods. 2015. 231:18-25). 이는 마커로서 EmGFP를 이용하여 바이러스의 생산과 감염 정도의 관찰을 용이하게 하기 위한 것이다. EmGFP 카세트 제작에는 pCDNA6.2-GW/EmGFP-miR 플라스미드(Invitrogen)를 이용하였다. For the construction of HSV-1 expressing EmGFP In the UL26/UL27 position of KOS-37/BAC DNA, an expression cassette capable of expressing EmGFP (Emerald Green Fluorescent Protein) was inserted (Tiffany A et al., J Virol Methods. 2015. 231:18). -25). This is to facilitate the observation of virus production and infection level using EmGFP as a marker. The pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) was used to construct the EmGFP cassette.
EmGFP를 발현하는 KOS-EmGFP의 지놈 개요도는 도 2에 나타내었다.A schematic diagram of the genome of KOS-EmGFP expressing EmGFP is shown in FIG. 2 .
EmGFP 발현을 위하여 사이토메갈로바이러스(cytomegalovirus)의 유전자 프로모터인 pCMV와 HSV TK(Herpes simplex virus thymidine kinase)의 폴리아데닐레이션 시그널(polyadenylation signal)인 tkpA를 이용하여 pCMV-EmGFP-tkpA의 구조로 KOS-37/BAC DNA에 삽입하였다.For EmGFP expression, pCMV, a gene promoter of cytomegalovirus, and tkpA, a polyadenylation signal of Herpes simplex virus thymidine kinase (HSV TK), are used to form pCMV-EmGFP-tkpA, KOS-37 /BAC was inserted into the DNA.
모든 삽입 방법은 카운터 셀렉션 BAC 변형 키트(counter selection BAC modification kit; GeneBridges. Inc)를 사용하여 제조사의 프로토콜에 따라 진행하였다.All insertion methods were performed according to the manufacturer's protocol using a counter selection BAC modification kit (GenBridges. Inc).
구체적으로 KOS-37/BAC이 들어있는 클론에 상동 재조합(homologous recombination) 기능을 수행할 수 있는 RecE와 RecT를 발현할 수 있는 pRed/ET 플라스미드를 형질 전환하였다(Muyrers JP et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). UL26와 UL27 사이에 타겟 유전자를 도입하고자 하는 위치를 포함하는 상동 영역의 프라이머(homologous region primer) 세트(정방향 프라이머 UL26/27-rpsL_For: 서열번호 13, 역방향 프라이머 UL26/27-rpsL_Rev: 서열번호 14)를 사용하여 UL26/27-rpsL-neo/kan 카세트(cassette)를 제작하였다. KOS-37/BAC DNA와 pRed/ET가 들어있는 클론에 L-아라비노오스(L-arabinose; Sigma-Aldrich)를 첨가하여 상동 재조합(homologous recombination)이 가능하도록 유도한 후, 제작한 UL26/27-rpsL-neo/kan 카세트 200 ng을 형질 전환하였다. 이러한 상동 재조합에 의하여 UL26/27-rpsL-neo/kan 카세트가 KOS-37/BAC의 UL26/27 위치에 삽입되게 된다. UL26/27-rpsL-neo/kan가 삽입된 E.coli는 카나마이신 저항성을 갖고 있지만, 스트렙토마이신 저항성은 rpsL 유전자로 인해 차단된 상태이다. 카나마이신 배지에서 선별된 E.coli는 UL26/27-rpsL-neo/kan가 삽입되었다고 판단하여, 마지막 단계인 타겟 유전자를 삽입하는 단계로 진행하였다.Specifically, a clone containing KOS-37/BAC was transformed with a pRed/ET plasmid capable of expressing RecE and RecT capable of performing homologous recombination functions (Muyrers JP et al.; Nucleic Acids Res). 1999. 27(6):1555-1557). A set of homologous region primers between UL26 and UL27 including the position to introduce the target gene (forward primer UL26/27-rpsL_For: SEQ ID NO: 13, reverse primer UL26/27-rpsL_Rev: SEQ ID NO: 14) was used to prepare a UL26/27-rpsL-neo/kan cassette. UL26/27 prepared after inducing homologous recombination by adding L-arabinose (Sigma-Aldrich) to the clone containing KOS-37/BAC DNA and pRed/ET -rpsL-neo/kan cassette 200 ng was transformed. By this homologous recombination, the UL26/27-rpsL-neo/kan cassette is inserted into the UL26/27 position of KOS-37/BAC. E. coli into which UL26/27-rpsL-neo/kan is inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. E. coli selected in kanamycin medium was judged to have been inserted into UL26/27-rpsL-neo/kan, and the final step, the step of inserting the target gene, proceeded.
UL26/27-rpsL-neo/kan 카세트가 있는 E.coli에 pRed/ET의 기능을 활성화시키는 L-아라비노오스(L-arabinose; Sigma-Aldrich)를 첨가하여 상동 재조합(homologous recombination)이 가능하도록 유도하고, 그 다음 UL26/27-tkpA-EmGFP-pCMV 카세트 200 ng을 형질 전환하였다. UL26/27-tkpA-EmGFP-pCMV 카세트는 pCDNA6.2-GW/EmGFP-miR 플라스미드(Invitrogen)를 주형(Template)으로 사용하고 정방향 프라이머 UL26/27-tkpA_For(서열번호 15)와 역방향 프라이머 UL26/27-pCMV_Rev(서열번호 16)를 사용하여 제작한 것이다. To enable homologous recombination by adding L-arabinose (Sigma-Aldrich) that activates the function of pRed/ET to E. coli with UL26/27-rpsL-neo/kan cassette 200 ng of the UL26/27-tkpA-EmGFP-pCMV cassette was then transfected. The UL26/27-tkpA-EmGFP-pCMV cassette uses the pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) as a template, and the forward primer UL26/27-tkpA_For (SEQ ID NO: 15) and the reverse primer UL26/27 It was produced using -pCMV_Rev (SEQ ID NO: 16).
기존 UL26/27-rpsL-neo/kan 카세트와 삽입된 UL26/27-tkpA-EmGFP-pCMV가 교체되면서 rpsL에 의해 차단된 스트렙토마이신 저항성이 활성화되는 원리를 이용하여 후보군을 스트렙토마이신 배지에서 선별하였다(Heermann R et al., Microb Cell Fact. 2008. 14:. doi: 10.1186). 선별된 후보군은 DNA prep 방법을 이용하여 DNA 분리를 하였고(Horsburgh BC et al., Methods enzymol. 1999. 306:337-352), UL26/27에서 tkpA-EmGFP-pCMV의 도입 여부를 제한효소 EcoRI, XhoI 처리와 PCR (ploymerase chain recation)를 수행으로 확인했고, PCR 산물을 시퀀싱하여 정확한 유전자 서열을 확인하였다.Candidate groups were selected in streptomycin medium using the principle that the existing UL26/27-rpsL-neo/kan cassette and the inserted UL26/27-tkpA-EmGFP-pCMV were replaced and streptomycin resistance blocked by rpsL was activated ( Heermann R et al., Microb Cell Fact. 2008. 14:. doi: 10.1186). The selected candidate group was subjected to DNA isolation using the DNA prep method (Horsburgh BC et al., Methods enzymol. 1999. 306:337-352), and the introduction of tkpA-EmGFP-pCMV in UL26/27 was determined by restriction enzyme EcoRI, It was confirmed by XhoI treatment and PCR (ploymerase chain recation), and the correct gene sequence was confirmed by sequencing the PCR product.
형광단백질의 정상적인 발현과 바이러스의 생산을 위하여 실험을 진행하였다. 완성된 KOS-37/BAC-EmGFP DNA를 라지 컨스트럭트 DNA 분리 키트(Large construct DNA purification kit, Macherey-Nagel)를 이용하여 추출한 후, Cre 재조합 효소(recombinase)를 이용하여 BAC 유전자를 제거하기 위하여 2×105의 Cre-Vero 세포(Gierasch et al.; J. Virol Methods. 2006. 135:197~206)에 리포펙타민 2000 시약(Lipofectamine 2000 reagent, Invitrogen)을 이용하여 DNA 1 ug을 형질주입하였다. 형질주입 3일 후, EmGFP 단백질의 발현을 형광현미경을 통하여 관찰하고, Cre-Vero-HVEM 세포의 플라크(plaqe) 형성을 통해 바이러스 생산을 관찰하였다. 플라크의 형성을 확인한 다음 바이러스가 포함된 세포를 걷어서 3회의 freeze-thaw 방법(Gierasch WW et al.; J. Virol Methods. 2006. 135:197~206)과 초음파 파쇄(sonication)을 통해 KOS-EmGFP 바이러스를 획득하였다.Experiments were carried out for normal expression of fluorescent protein and production of virus. After extracting the completed KOS-37/BAC-EmGFP DNA using a large construct DNA purification kit (Macherey-Nagel), Cre recombinase was used to remove the BAC gene. 2×10 5 Cre-Vero cells (Gierasch et al.; J. Virol Methods. 2006. 135:197-206) were transfected with 1 ug of DNA using Lipofectamine 2000 reagent (Invitrogen). did. Three days after transfection, the expression of EmGFP protein was observed through a fluorescence microscope, and virus production was observed through the formation of plaques in Cre-Vero-HVEM cells. After confirming the formation of plaques, virus-containing cells were picked up and KOS-EmGFP was subjected to three freeze-thaw methods (Gierasch WW et al.; J. Virol Methods. 2006. 135:197-206) and sonication. virus was obtained.
<실시예 2> HER2scFv-HveC, CEAscFv-HveC, EpCAMscFv-HveC 어댑터를 발현하는 KOS 37-BAC-EmGFP 제작<Example 2> Construction of KOS 37-BAC-EmGFP expressing HER2scFv-HveC, CEAscFv-HveC, and EpCAMscFv-HveC adapters
상기 실시예 1에서 제작된, EmGFP 발현 카세트(pCMV-EmGFP-tkpA)가 삽입된 KOS-37/BAC-EmGFP DNA의 UL3/UL4 위치에, CEAscFv-HveC 어댑터 발현 카세트와 HER2scFv-HveC 어댑터 발현 카세트 그리고 EpCAMscFv-HveC어댑터 발현 카세트 삽입하였다.CEAscFv-HveC adapter expression cassette and HER2scFv-HveC adapter expression cassette and An EpCAMscFv-HveC adapter expression cassette was inserted.
CEAscFv-HveC 어댑터 발현 카세트인 pCMV-CEAscFv-HveC-bGHpA가 삽입된 KOS-UL3/4-CEAscFv-HveC-EmGFP 바이러스 게놈 개요도와 HER2scFv-HveC 어댑터 발현 카세트인 pCMV-HER2scFv-HveC-bGHpA이 삽입된 KOS-UL3/4-HER2scFv-HveC-EmGFP 바이러스 게놈 개요도 그리고 EpCAM-HveC 어댑터 발현 카세트 pCMV-EpCAMscFv-HveC-bGHpA가 삽입된 KOS-UL3/4-EpCAMscFv-HveC-EmGFP 바이러스 게놈 개요도를 도 3에 나타내었으며, 또 도 4 내지 도6에 CEAscFv-HveC와 HER2scFv-HveC 어댑터 그리고 EpCAMscFv-HveC 어댑터의 전체 서열과 해당 서열의 구성을 나타내었다. KOS-UL3/4-CEAscFv-HveC-EmGFP virus genome schematic into which pCMV-CEAscFv-HveC-bGHpA, a CEAscFv-HveC adapter expression cassette, and pCMV-HER2scFv-HveC-bGHpA, an HER2scFv-HveC adapter expression cassette are inserted -UL3/4-HER2scFv-HveC-EmGFP virus genome schematic and KOS-UL3/4-EpCAMscFv-HveC-EmGFP virus genome schematic into which the EpCAM-HveC adapter expression cassette pCMV-EpCAMscFv-HveC-bGHpA is inserted is shown in FIG. and, the entire sequence of the CEAscFv-HveC, HER2scFv-HveC adapter, and EpCAMscFv-HveC adapter and the configuration of the sequences are shown in FIGS.
여기서 CEA에 대한 scFv는 서열번호 1의 VL와 서열번호 2의 VH이 서열번호 3의 링커 펩티드를 매개로 연결된 구성이고, HER2에 대한 scFv는 서열번호 4의 VH와 서열번호 5의 VL이 서열번호 6의 링커 펩티드를 매개로 연결된 구성이다. 그리고 EpCAM에 대한 scFv는 서열번호 7의 VL과 서열번호 8의 VH가 서열번호 9의 링커 펩티드를 매개로 연결된 구성이다. HveC는 CEAscFv-HveC와 HER2scFv-HveC 어댑터 그리고 EpCAMscFv-HveC 어댑터에서 HveC은 서열번호 11의 HveC144가 사용되었다. 또 CEAscFv-HveC와 HER2scFv-HveC 어댑터 그리고 EpCAMscFv-HveC 어댑터에 있어서는 그 N 말단 즉 CEAscFv의 VL 앞부분과 HER2scFv의 VH의 앞부분 그리고 EpCAMscFv 의 VL 앞부분에 서열번호 10의 리더 서열이 포함되어 있다. Here, the scFv for CEA is a configuration in which the VL of SEQ ID NO: 1 and the VH of SEQ ID NO: 2 are linked via the linker peptide of SEQ ID NO: 3, and the scFv for HER2 is the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 is SEQ ID NO: It is a structure linked through the linker peptide of 6. And the scFv for EpCAM has a configuration in which the VL of SEQ ID NO: 7 and the VH of SEQ ID NO: 8 are linked via the linker peptide of SEQ ID NO: 9. For HveC, CEAscFv-HveC and HER2scFv-HveC adapter and HveC 144 of SEQ ID NO: 11 were used for HveC in EpCAMscFv-HveC adapter. In addition, in the CEAscFv-HveC, HER2scFv-HveC adapter and EpCAMscFv-HveC adapter, the leader sequence of SEQ ID NO: 10 is included at the N-terminus, that is, the VL front part of CEAscFv, the VH front part of HER2scFv, and the VL front part of EpCAMscFv.
CEA와 HER2 그리고 EpCAM에 대한 scFv 서열과 HveC 서열의 링커 서열인 NH2-GGGGS 서열 다음에는 클로닝을 용이하게 하기 위한 제한효소 BamHI 작용 부위인 GS(염기서열: GGATCC) 추가되어 있다. 그리고 pCMV는 상기 사이토메갈로바이러스(cytomegalovirus)의 유전자 프로모터이고, bGH-pA는 bGH-PolyA(bovine growth hormone polyadenylation) 시그널 서열이다. After the NH 2 -GGGGS sequence, which is the linker sequence of the scFv sequence for CEA, HER2, and EpCAM, and the HveC sequence, GS (base sequence: GGATCC), which is a restriction enzyme BamHI action site for easy cloning, is added. And pCMV is a gene promoter of the cytomegalovirus, and bGH-pA is a bovine growth hormone polyadenylation (bGH-PolyA) signal sequence.
본 실시예에서 사용된, 리더 서열까지 포함한 CEAscFv-HveC 어댑터 전장의 아미노산 서열은 서열번호 17에 개시되어 있고, 리더 서열까지 포함한 HER2scFv-HveC 어댑터 전장의 아미노산 서열은 각각 서열번호 18에 개시되어 있으며, 또 리더 서열까지 포함한 EpCAMscFv-HveC 어댑터 전장의 아미노산 서열은 각각 서열번호 19에 개시되어 있다.The amino acid sequence of the full-length CEAscFv-HveC adapter including the leader sequence used in this Example is shown in SEQ ID NO: 17, and the amino acid sequence of the full-length HER2scFv-HveC adapter including the leader sequence is shown in SEQ ID NO: 18, respectively, In addition, the amino acid sequence of the full length of the EpCAMscFv-HveC adapter including the leader sequence is shown in SEQ ID NO: 19, respectively.
CEAscFv-HveC 어댑터 발현 카세트와 HER2scFv-HveC 어댑터 발현 카세트 그리고 EpCAMscFv-HveC 어댑터 발현 카세트의 삽입은 상기 실시예 1과 마찬가지로 카운터 셀렉션 BAC 변형 키트(counter selection BAC modification kit; GeneBridges. Inc)를 사용하여 제조사의 프로토콜에 따라 진행했다. Insertion of the CEAscFv-HveC adapter expression cassette, the HER2scFv-HveC adapter expression cassette, and the EpCAMscFv-HveC adapter expression cassette was performed by the manufacturer using a counter selection BAC modification kit (GenBridges. Inc) as in Example 1 above. proceeded according to the protocol.
구체적으로 상기 실시예 2에서 제작된 KOS-37/BAC-EmGFP 유전체가 들어있는 E.coli 클론에 상동 재조합(homologous recombination) 기능을 수행하는 RecE와 RecT를 발현하는 pRed/ET 플라스미드를 형질 전환하였다(Muyrers JP et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). UL3와 UL4 사이에 타겟 유전자를 도입하고자 하는 위치를 포함하는 상동 영역의 프라이머(homologous region primer) 세트(정방향 프라이머 UL3/4-rpsL-neo_for: 서열번호 20, 역방향 프라이머 UL3/4-rpsL-neo_rev: 서열번호 21)를 사용하여 UL3/4-rpsL-neo/kan 카세트(cassette)를 제작하였다. KOS-37/BAC-EmGFP와 pRedET가 들어있는 클론에 L-아라비노오스(L-arabinose; Sigma-Aldrich)를 첨가하여 상동 재조합(homologous recombination)이 가능하도록 유도한 후, 상기 제작한 UL3/4-rpsL-neo/kan 카세트 200 ng 를 형질 전환한다. 이러한 상동 재조합에 의하여 UL3/4-rpsL-neo/kan 카세트가 KOS-37/BAC-EmGFP의 UL3/4 위치에 삽입되게 된다. UL3/4-rpsL-neo/kan가 삽입된 E.coli는 카나마이신 저항성을 갖고 있지만, 스트렙토마이신 저항성은 rpsL 유전자로 인해 차단된다. 카나마이신 배지에서 선별된 E.coli는 UL3/4-rpsL-neo/kan가 삽입되었다고 판단하여, 마지막 단계인 타겟 유전자를 삽입하는 단계로 진행하였다.Specifically, pRed/ET plasmids expressing RecE and RecT performing homologous recombination in the E. coli clone containing the KOS-37/BAC-EmGFP genome prepared in Example 2 were transformed ( Muyrers JP et al .; Nucleic Acids Res. 1999. 27(6):1555-1557). A homologous region primer set (forward primer UL3/4-rpsL-neo_for: SEQ ID NO: 20, reverse primer UL3/4-rpsL-neo_rev: SEQ ID NO: 21) was used to construct a UL3/4-rpsL-neo/kan cassette. After inducing homologous recombination by adding L-arabinose (Sigma-Aldrich) to the clone containing KOS-37/BAC-EmGFP and pRedET, the prepared UL3/4 Transform 200 ng of -rpsL-neo/kan cassette. By this homologous recombination, the UL3/4-rpsL-neo/kan cassette is inserted into the UL3/4 position of KOS-37/BAC-EmGFP. E. coli into which UL3/4-rpsL-neo/kan is inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. E. coli selected in kanamycin medium was judged to have been inserted into UL3/4-rpsL-neo/kan, and the final step, the step of inserting the target gene, proceeded.
UL3/4-rpsL-neo/kan카세트가 있는 E.coli에 pRed/ET의 기능을 활성화시키는 L-아라비노오스(L-arabinose; Sigma-Aldrich)를 첨가하여 상동 재조합(homologous recombination)이 가능하도록 유도한 후, UL3/4-pCMV-CEAscFv-HveC-bGHpA와 UL3/4-pCMV-HER2scFv-HveC-bGHpA 카세트 그리고 UL3/4-pCMV-EpCAMscFv-HveC-bGHpA 카세트 200 ng을 형질 전환 시켰다. 상기 UL3/4-pCMV-CEAscFv-HveC-bGHpA와 UL3/4-pCMV-HER2scFv-HveC-bGHpA 카세트 그리고 UL3/4-pCMV-EpCAMscFv-HveC-bGHpA 카세트는 pCDNA3.1-CEAcFv-HveC 플라스미드와 pCDNA3.1-HER2scFv-HveC 플라스미드 그리고 pCDNA3.1-pCMV-EpCAMscFv-HveC (BaeK HJ et al., Mol Ther. 2011. 19(3):507-514, Carter. p et al., Proc Natl Acad Sci U S A. 1992, 15;89(10):4285-9, Willuda J et al., Cancer Res. 1999, 15;59(22):5758-67)를 주형으로 사용하여 정방향 프라이머 UL3/4_pCMV_For(서열번호 22)와 역방향 프라이머 UL3/4_bGH_poly_R(서열번호 23)를 사용하여 제작하였다. To enable homologous recombination by adding L-arabinose (Sigma-Aldrich) that activates the function of pRed/ET to E. coli with UL3/4-rpsL-neo/kan cassette After induction, the UL3/4-pCMV-CEAscFv-HveC-bGHpA, UL3/4-pCMV-HER2scFv-HveC-bGHpA cassette and 200 ng of the UL3/4-pCMV-EpCAMscFv-HveC-bGHpA cassette were transformed . remind UL3/4-pCMV-CEAscFv-HveC-bGHpA and UL3/4-pCMV-HER2scFv-HveC-bGHpA cassette and UL3/4-pCMV-EpCAMscFv-HveC-bGHpA cassette are pCDNA3.1-CEAcFv-HveC plasmid and pCDNA3.1 -HER2scFv-HveC plasmid and pCDNA3.1-pCMV-EpCAMscFv-HveC (BaeK HJ et al ., Mol Ther. 2011. 19(3):507-514, Carter. p et al., Proc Natl Acad Sci US A. 1992, 15;89(10):4285-9, Willuda J et al., Cancer Res. 1999, 15;59(22):5758-67) as a template, the forward primer UL3/4_pCMV_For (SEQ ID NO: 22) and reverse primer UL3/4_bGH_poly_R (SEQ ID NO: 23).
기존 삽입된 UL3/4-rpsL-neo/kan 카세트와 앞서 삽입된 UL3/4-pCMV-CEAscFv-HveC-bGHpA와 UL3/4-pCMV-HER2scFv-HveC-bGHpA 그리고 UL3/4-pCMV-EpCAMscFv-HveC가 교체되면서 rpsL에 의해 차단된 스트렙토마이신 저항성이 활성화되는 원리를 이용하여 후보군을 스트렙토마이신 배지에서 선별하였다(Heermann R et al., Microb Cell Fact. 2008. 14:. doi: 10.1186). 선별된 후보군은 DNA prep 방법을 이용하여 DNA 분리를 하였고(Horsburgh BC et al., Methods enzymol. 1999. 306:337-352), UL3/4에서 UL3/4-pCMV-CEAscFv-HveC-bGHpA와 UL3/4-pCMV-HER2scFv-HveC-bGHpA 그리고 UL3/4-pCMV-EpCAMscFv-HveC의 도입 여부를 도입 여부를 제한효소 EcoRI, XhoI 처리와 PCR (ploymerase chain recation)를 수행으로 확인했고, PCR 산물을 시퀀싱하여 정확한 유전자 서열을 확인하였다.The previously inserted UL3/4-rpsL-neo/kan cassette and the previously inserted UL3/4-pCMV-CEAscFv-HveC-bGHpA and UL3/4-pCMV-HER2scFv-HveC-bGHpA and UL3/4-pCMV-EpCAMscFv-HveC Candidate groups were selected in streptomycin medium using the principle that streptomycin resistance blocked by rpsL is activated while being replaced (Heermann R et al., Microb Cell Fact. 2008. 14:. doi: 10.1186). The selected candidate group was subjected to DNA isolation using the DNA prep method (Horsburgh BC et al., Methods enzymol. 1999. 306:337-352), and UL3/4 to UL3/4-pCMV-CEAscFv-HveC-bGHpA and UL3 The introduction of /4-pCMV-HER2scFv-HveC-bGHpA and UL3/4-pCMV-EpCAMscFv-HveC was confirmed by treatment with restriction enzymes EcoRI and XhoI and PCR (ploymerase chain recation), and the PCR product was sequenced. to confirm the correct gene sequence.
완성된 KOS-37/BAC-UL3/4_CEAscFv-HveC-EmGFP와 KOS-37/BAC-UL3/4_HER2scFv-HveC-EmGFP 그리고 KOS-37/BAC-UL3/4_EpCAMscFv-HveCEmGFP DNA를 라지 컨스트럭트 DNA 분리 키트(Large construct DNA purification kit, Macherey-Nagel)를 이용하여 추출한 후, Cre recombinase를 이용하여 BAC 유전자를 제거하기 위하여 2×105의 Cre-Vero-HVEM 세포에 리포펙타민 2000 시약(Lipofectamine 2000 reagent, Invitrogen)을 이용하여 DNA 1ug을 형질주입 하였다. 형질주입 3일 후, 형광현미경을 이용하여 EmGFP 단백질의 형광발현과 세포의 플락(plaque) 형성을 관찰하였다. 플라크의 형성을 확인한 다음 바이러스가 포함된 세포를 걷어서 3회의 freeze-thaw 방법(Gierasch WW et al.; J. Virol Methods. 2006. 135:197~206)과 초음파 파쇄(sonication)을 통해, CEA 표적화 어댑터를 발현하는 KOS-UL3/4_CEAscFv-HveC-EmGFP 바이러스와 HER2 표적화 어댑터를 발현하는 KOS-UL3/4_HER2scFv-HveC-EmGFP 바이러스 그리고 EpCAM 표적화 어댑터를 발현하는 KOS-UL3/4_EpCAMscFv-HveC-EmGFP 바이러스를 획득하였다.Completed KOS-37/BAC-UL3/4_CEAscFv-HveC-EmGFP, KOS-37/BAC-UL3/4_HER2scFv-HveC-EmGFP, and KOS-37/BAC-UL3/4_EpCAMscFv-HveCEmGFP DNAs were analyzed using a large construct DNA isolation kit. After extraction using (Large construct DNA purification kit, Macherey-Nagel), 2 × 10 5 Cre-Vero-HVEM cells in order to remove the BAC gene using Cre recombinase with Lipofectamine 2000 reagent (Lipofectamine 2000 reagent, Invitrogen) was used to transfect 1 ug of DNA. Three days after transfection, the fluorescence expression of EmGFP protein and the formation of plaques in cells were observed using a fluorescence microscope. After confirming the formation of plaque, the cells containing the virus were picked up and subjected to three freeze-thaw methods (Gierasch WW et al.; J. Virol Methods. 2006. 135:197-206) and sonication to target CEA. Obtain the KOS-UL3/4_CEAscFv-HveC-EmGFP virus expressing the adapter, the KOS-UL3/4_HER2scFv-HveC-EmGFP virus expressing the HER2 targeting adapter and the KOS-UL3/4_EpCAMscFv-HveC-EmGFP virus expressing the EpCAM targeting adapter. did.
<실시예 3> HveC-CEAscFv 어댑터 발현 항암 바이러스를 이용한 CEA 발현 세포의 타겟팅<Example 3> Targeting of CEA-expressing cells using HveC-CEAscFv adapter expressing anticancer virus
상기 실시예 2에서 제작된 KOS-UL3/4_CEAscFv-HveC-EmGFP 바이러스가 CEAscFv-HveC 어댑터를 발현함으로써 주변의 CEA 발현세포로의 바이러스 감염을 유도하는지 확인하기 위하여 다음과 같이 실험을 진행했다.In order to confirm whether the KOS-UL3/4_CEAscFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding CEA-expressing cells by expressing the CEAscFv-HveC adapter, the experiment was conducted as follows.
실험에 사용된 세포주는 CEA가 발현되는 세포인 CHO-CEA(Kuroki M et al., J Biol Chem. 1991. 74:10132-10141)를 사용하였다. CHO-CEA는 HaM's F-12K 배지(Wellgen)에 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen)와 10% FBS를 사용하여 배양하였다. The cell line used in the experiment was CHO-CEA, a cell expressing CEA (Kuroki M et al., J Biol Chem. 1991. 74:10132-10141). CHO-CEA was cultured using 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen) and 10% FBS in HaM's F-12K medium (Wellgen).
CEA 특이적 바이러스 감염을 확인하기 위하여 1.5×104의 CHO-CEA 세포에 야생형 HSV-1 KOS 바이러스와 어댑터를 발현하는 KOS-UL3/4_CEAscFv-HveC-EmGFP 바이러스를 각각 5 MOI를 감염시켰다. In order to confirm the CEA-specific virus infection, 1.5×10 4 CHO-CEA cells were infected with the wild-type HSV-1 KOS virus and the adapter-expressing KOS-UL3/4_CEAscFv-HveC-EmGFP virus at MOI of 5, respectively.
90분 후, 잔류하는 초기 바이러스 및 CEAscFv-HveC 어댑터를 제거하기 위하여 새로운 배지로 교체했다. 감염 1일 후, 형광 발현으로 각각의 바이러스에 대한 세포 감염을 형광현미경으로 관찰했다(BaeK HJ et al., Mol Ther. 2011. 19(3):507-514). After 90 minutes, the medium was replaced with fresh medium to remove residual nascent virus and CEAscFv-HveC adapter. One day after infection, cell infection for each virus was observed under a fluorescence microscope by fluorescence expression (BaeK HJ et al ., Mol Ther. 2011. 19(3):507-514).
결과를 도 7에 형광현미경를 이용하여 세포에 바이러스 감염을 수치화하여 나타내었다. CEAscFv-HveC 어댑터 발현 바이러스에 의한 CEA 발현 세포의 감염효과가 일반 헤르페스 바이러스의 감염효과에 비하여 뚜렷하게 높음을 알 수 있다.The results are shown by quantifying the virus infection in the cells using a fluorescence microscope in FIG. 7 . It can be seen that the infective effect of CEA-expressing cells by the CEAscFv-HveC adapter expressing virus is significantly higher than that of the general herpes virus.
<실시예 4> HER2scFv-HveC 어댑터 발현 항암 바이러스를 이용한 HER2 발현 세포의 타겟팅<Example 4> Targeting of HER2 expressing cells using HER2scFv-HveC adapter expressing anticancer virus
상기 실시예 2에서 제작된 KOS-UL3/4_HER2scFv-HveC-EmGFP 바이러스가 HER2scFv-HveC 어댑터를 발현함으로써 주변의 HER2 발현세포로의 바이러스 감염을 유도하는지 확인하기 위하여 다음과 같이 실험을 진행했다.In order to confirm whether the KOS-UL3/4_HER2scFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding HER2-expressing cells by expressing the HER2scFv-HveC adapter, the experiment was conducted as follows.
실험에 사용된 세포주는 CHO-K1, Netin이 발현되는 CHO-Nectin, HER2이 발현되는 세포인 CHO-HER2 (Kuroki M et al., J Biol Chem. 1991. 74:10132-10141)을 사용하였다. CHO 세포주들은 HaM's F-12K 배지(Wellgen)에 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen)와 10% FBS를 사용하여 배양하였다.Cell lines used for the experiment were CHO-K1, CHO-Nectin expressing Netin, and CHO-HER2 expressing HER2 (Kuroki M et al., J Biol Chem. 1991. 74:10132-10141). CHO cell lines were cultured in HaM's F-12K medium (Wellgen) with 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen) and 10% FBS.
HER2 특이적 바이러스 감염을 확인하기 위하여 1.5×104의 CHO-K1, CHO-Nectin, CHO-HER2 세포에 야생형 HSV-1 KOS 바이러스와 어댑터를 발현하는 KOS-UL3/4_CEAscFv-HveC-EmGFP 바이러스를 각각 5 MOI를 감염시켰다. In order to confirm HER2-specific viral infection, KOS-UL3/4_CEAscFv-HveC-EmGFP virus expressing wild-type HSV-1 KOS virus and adapter was applied to 1.5×10 4 CHO-K1, CHO-Nectin, and CHO-HER2 cells, respectively. 5 MOIs were infected.
90분 후, 잔류하는 초기 바이러스 및 HER2scFv-HveC 어댑터를 제거하기 위하여 새로운 배지로 교체했다. 감염 1일 후, 형광 발현으로 각각의 바이러스에 대한 세포 감염을 형광현미경으로 관찰했다(BaeK HJ et al., Mol Ther. 2011. 19(3):507-514). After 90 minutes, the medium was replaced with fresh medium to remove residual nascent virus and HER2scFv-HveC adapter. One day after infection, cell infection for each virus was observed under a fluorescence microscope by fluorescence expression (BaeK HJ et al ., Mol Ther. 2011. 19(3):507-514).
결과를 도 8에 형광현미경를 이용하여 세포에 바이러스 감염 결과를 나타내었다. HER2와 Nectin-1(HveC)을 모두 발현하지 않는 CHO-K1 세포에서는 야생형 바이러스(HSV-1 KOS)와 HER2scFv-HveC 어댑터를 발현하는 바이러스(HSV-1 KOS HERscFv-HveC)가 모두 감염되지 않았지만, HER2를 발현하는 CHO-HER2 세포에서는 HER2scFv-HveC 어댑터를 발현하는 바이러스만이 감염되었고 HER2와 Nectin-1을 발현하는 CHO-Nectin 세포에서는 야생형 바이러스와 HER2scFv-HveC 어댑터를 발현하는 바이러스가 모두 감염되었다. The results of the virus infection in the cells using a fluorescence microscope are shown in FIG. 8 . In CHO-K1 cells, which do not express both HER2 and Nectin-1 (HveC), neither the wild-type virus (HSV-1 KOS) nor the virus expressing the HER2scFv-HveC adapter (HSV-1 KOS HERscFv-HveC) were infected. In HER2-expressing CHO-HER2 cells, only the virus expressing the HER2scFv-HveC adapter was infected, and in CHO-Nectin cells expressing HER2 and Nectin-1, both the wild-type virus and the virus expressing the HER2scFv-HveC adapter were infected.
<실시예 5> EpCAMscFv-HveC 어댑터 발현 항암 바이러스를 이용한 EpCAM 발현 세포의 타겟팅<Example 5> Targeting of EpCAM-expressing cells using EpCAMscFv-HveC adapter expressing anticancer virus
상기 실시예 2에서 제작된 KOS-UL3/4_EpCAMscFv-HveC-EmGFP 바이러스가 EpCAMscFv-HveC 어댑터를 발현함으로써 주변의 EpCAM 발현세포로의 바이러스 감염을 유도하는지 확인하기 위하여 다음과 같이 실험을 진행했다.In order to confirm whether the KOS-UL3/4_EpCAMscFv-HveC-EmGFP virus prepared in Example 2 induces virus infection into surrounding EpCAM-expressing cells by expressing the EpCAMscFv-HveC adapter, an experiment was conducted as follows.
실험에 사용된 세포주는 CHO-K1, Netin이 발현되는 CHO-Nectin, EpCAM이 발현되는 세포인 CHO-EpCAM을 사용하였다. CHO 세포주들은 HaM's F-12K 배지(Wellgen)에 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen)와 10% FBS를 사용하여 배양하였다.Cell lines used in the experiment were CHO-K1, CHO-Nectin expressing Netin, and CHO-EpCAM expressing EpCAM cells. CHO cell lines were cultured in HaM's F-12K medium (Wellgen) with 100 U/ml penicillin/100 μg/ml streptomycin (Wellgen) and 10% FBS.
EpCAM 특이적 바이러스 감염을 확인하기 위하여 1.5×104의 CHO-K1, CHO-Nectin, CHO-EpCAM 세포에 야생형 HSV-1 KOS 바이러스와 어댑터를 발현하는 KOS-UL3/4_EpCAMscFv-HveC-EmGFP 바이러스를 각각 5 MOI를 감염시켰다. To confirm EpCAM-specific virus infection, KOS-UL3/4_EpCAMscFv-HveC-EmGFP virus expressing wild-type HSV-1 KOS virus and adapter was applied to 1.5×10 4 CHO-K1, CHO-Nectin, and CHO-EpCAM cells, respectively. 5 MOIs were infected.
90분 후, 잔류하는 초기 바이러스 및 EpCAMscFv어댑터를 제거하기 위하여 새로운 배지로 교체했다. 감염 1일 후, 형광 발현으로 각각의 바이러스에 대한 세포 감염을 형광현미경으로 관찰했다(BaeK HJ et al., Mol Ther. 2011. 19(3):507-514). After 90 minutes, the medium was replaced with fresh medium to remove residual initial virus and EpCAMscFv adapter. One day after infection, cell infection for each virus was observed under a fluorescence microscope by fluorescence expression (BaeK HJ et al ., Mol Ther. 2011. 19(3):507-514).
결과를 도 9에 형광현미경를 이용하여 세포에 바이러스 감염을 나타내었다. The results show the virus infection in the cells using a fluorescence microscope in FIG.
EpCAM과 Nectin-1(HveC)을 모두 발현하지 않는 CHO-K1 세포에서는 야생형 바이러스(HSV-1 KOS)와 EpCAMscFv-HveC 어댑터를 발현하는 바이러스(HSV-1 KOS EpCAMscFv-HveC)가 모두 감염되지 않았지만, EpCAM를 발현하는 CHO-EpCAM 세포에서는 EpCAMscFv-HveC 어댑터를 발현하는 바이러스만이 감염되었고 EpCAM과 Nectin을 발현하는 CHO-Nectin 세포에서는 야생형 바이러스와 EpCAMscFv-HveC 어댑터를 발현하는 바이러스가 모두 감염되었다. In CHO-K1 cells, which do not express both EpCAM and Nectin-1 (HveC), neither the wild-type virus (HSV-1 KOS) nor the virus expressing the EpCAMscFv-HveC adapter (HSV-1 KOS EpCAMscFv-HveC) were infected, but In CHO-EpCAM cells expressing EpCAM, only the virus expressing the EpCAMscFv-HveC adapter was infected, and in CHO-Nectin cells expressing EpCAM and Nectin, both the wild-type virus and the virus expressing the EpCAMscFv-HveC adapter were infected.

Claims (16)

  1. 헤르페스 심플렉스 바이러스의 증식을 저해하지 않으면서 그 게놈에, HveC의 세포외 도메인과 암세포의 표적분자를 특이적으로 인식하여 결합하는 암세포 표적화 영역의 융합 단백질을 발현할 수 있는 발현 카세트가 삽입되어 있는, 재조합 헤르페스 심플렉스 바이러스.An expression cassette capable of expressing a fusion protein of a cancer cell targeting region that specifically recognizes and binds to the extracellular domain of HveC and a target molecule of cancer cells is inserted into its genome without inhibiting the proliferation of herpes simplex virus. , Recombinant herpes simplex virus.
  2. 제1항에 있어서,According to claim 1,
    상기 HveC의 세포외 도메인은 리더서열이 제외된 V 도메인인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus, characterized in that the extracellular domain of the HveC is the V domain excluding the leader sequence.
  3. 제1항에 있어서,According to claim 1,
    상기 융합단백질은 HveC의 세포외 도메인과 암 표적화 영역이 1 내지 30개 아미노산으로 이루어진 링커 펩티드에 의해 연결되어 있는 융합단백질인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The fusion protein is a recombinant herpes simplex virus, characterized in that it is a fusion protein in which the extracellular domain of HveC and the cancer targeting region are linked by a linker peptide consisting of 1 to 30 amino acids.
  4. 제1항에 있어서, According to claim 1,
    상기 암세포 표적화 영역은 표적세포인 암세포의 표적분자를 특이적으로 인식하여 결합하는 영역이며,The cancer cell targeting region is a region that specifically recognizes and binds to a target molecule of a cancer cell, which is a target cell,
    상기 표적분자는 암세포에서만 발현되거나 정상세포에 비해 암세포에서 과발현되는 암세포 표면의 항원 또는 수용체인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus, characterized in that the target molecule is an antigen or receptor on the surface of cancer cells that is expressed only in cancer cells or overexpressed in cancer cells compared to normal cells.
  5. 제4항에 있어서,5. The method of claim 4,
    상기 항원 또는 수용체는 EGFRvⅢ, EGFR, 메타스틴 수용체(Metastin receptor), 수용체 타이로신 카이나제(Receptor tyrosine kinases), HER2(Human epidermal growth factor receptor 2), 타이로신 카이나제-18-수용체(c-Kit), HGF 수용체 c-Met, CXCR4, CCR7, 엔도테린-A 수용체, PPAR-δ(peroxisome proliferator activated receptor δ), PDGFR-α(Platelet-derived growth factor receptor α), CD133, CEA(carcinoembryonic antigen), EpCAM(Epithelial cell adhesion molecule), GD2(disialoganglioside), GPC3(Glypican 3), PSMA(Prostate Specific Membrane Antigen), TAG-72(tumor-associated glycoprotein 72), GD3(disialoganglioside), HLA-DR(human leukocyte antigen-DR), MUC1(Mucin 1), NY-ESO-1(New York esophageal squamous cell carcinoma 1), LMP1(Latent membrane protein 1), TRAILR2(tumor-necrosis factor-related apoptosis-inducing ligand receptor), VEGFR2(vascular endothelial growth factor receptor 2), HGFR(hepatocyte growth factor receptor), CD44 또는 CD166인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The antigen or receptor is EGFRvIII, EGFR, metastin receptor (Metastin receptor), receptor tyrosine kinases (Receptor tyrosine kinases), HER2 (Human epidermal growth factor receptor 2), tyrosine kinase-18- receptor (c-Kit) ), HGF receptor c-Met, CXCR4, CCR7, endothelin-A receptor, PPAR-δ (peroxisome proliferator activated receptor δ), PDGFR-α (Platelet-derived growth factor receptor α), CD133, CEA (carcinoembryonic antigen), EpCAM (Epithelial cell adhesion molecule), GD2 (disialoganglioside), GPC3 (Glypican 3), PSMA (Prostate Specific Membrane Antigen), TAG-72 (tumor-associated glycoprotein 72), GD3 (disialoganglioside), HLA-DR (human leukocyte antigen) -DR), MUC1 (Mucin 1), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), LMP1 (Latent membrane protein 1), TRAILR2 (tumor-necrosis factor-related apoptosis-inducing ligand receptor), VEGFR2 ( Recombinant herpes simplex virus, characterized in that vascular endothelial growth factor receptor 2), HGFR (hepatocyte growth factor receptor), CD44 or CD166.
  6. 제1항에 있어서,According to claim 1,
    상기 표적분자는 CEA, HER2 또는 EpCAM인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus, characterized in that the target molecule is CEA, HER2 or EpCAM.
  7. 제1항에 있어서,According to claim 1,
    재조합 헤르페스 심플렉스 바이러스는 그 당단백질 gB, gC, gD 또는 gH에 암 세포의 표적화 영역이 삽입되어 있는 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus is a recombinant herpes simplex virus characterized in that the glycoprotein gB, gC, gD or gH has a cancer cell targeting region inserted therein.
  8. 제1항에 있어서,According to claim 1,
    상기 재조합 헤르페스 심플렉스 바이러스는 재조합 HSV-1 바이러스, 재조합 HSV-2 바이러스, 또는 HSV-1와 HSV-2 키메라 바이러스인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus according to claim 1, wherein the recombinant herpes simplex virus is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or a chimeric HSV-1 and HSV-2 virus.
  9. 제1항에 있어서,According to claim 1,
    상기 재조합 헤르페스 심플렉스 바이러스는 HSV-1 KOS 균주로부터 유래된 재조합 HSV-1인 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus is a recombinant herpes simplex virus, characterized in that the recombinant HSV-1 derived from the HSV-1 KOS strain.
  10. 제1항에 있어서,According to claim 1,
    상기 재조합 헤르페스 심플렉스 바이러스에는 헤르페스 심플렉스 바이러스의 증식을 저해하지 않으면서 그 게놈에, (i) 사이토카인, (ii) 케모카인, (iii) 면역관문(immune checkpoint)에 대한 길항제, (iv) 면역세포의 활성화를 유도할 수 있는 보조 자극 인자(co-stimulatory factor), (v) 암세포에 대한 면역반응을 억제하는 TGFβ에 대항 길항제, (vi) 고형암 종양미세환경을 구성하는 헤파란 설페이트 프로테오글리칸(heparan sulfate proteoglycan)을 분해할 수 있는 헤파라나아제(heparanase), (vii) 혈관 신생 인자 수용체인 VEGFR-2(VEGF receptor-2)의 기능을 저해할 수 있는 길항제, 및 (viii) 프로드럭(prodrug)을 암세포에 독성을 나타내는 약물(drug)으로 전환시켜주는 프로드럭 활성화 효소(prodrug-activating enzymes) 중에 선택된 것을 발현하는 발현 카세트가 추가로 삽입되어 있는 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant herpes simplex virus has its genome without inhibiting proliferation of the herpes simplex virus, (i) cytokines, (ii) chemokines, (iii) immune checkpoint antagonists, (iv) immunity A co-stimulatory factor that can induce cell activation, (v) an antagonist to TGFβ that suppresses the immune response to cancer cells, (vi) heparan sulfate proteoglycan constituting the solid tumor microenvironment heparanase capable of degrading sulfate proteoglycan, (vii) an antagonist capable of inhibiting the function of VEGFR-2 (VEGF receptor-2), an angiogenesis factor receptor, and (viii) prodrug Recombinant herpes simplex virus, characterized in that the expression cassette expressing a selected one of prodrug-activating enzymes that convert the drug into a drug that is toxic to cancer cells is additionally inserted.
  11. 제10항에 있어서,11. The method of claim 10,
    상기 사이토카인은 IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, IL-24 등의 인터류킨, IFNα, IFNβ, IFNγ 등의 인터페론, TNFα 등의 종양 괴사 인자, GM-CSF 및 G-CSF 중 하나 이상이고,The cytokines include interleukins such as IL-2, IL-4, IL-10, IL-12, IL-15, IL-18, and IL-24, interferons such as IFNα, IFNβ, and IFNγ, and tumor necrosis factor such as TNFα. , at least one of GM-CSF and G-CSF,
    상기 케모카인은 CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 및 XCL-1 중 하나 이상이며,The chemokine is at least one of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 and XCL-1;
    상기 면역관문은 PD-1(programmed cell death-1), PD-L1(programmed cell deathligand 1), PD-L2(programmed cell death-ligand 2), CD27(cluster of differentiation 27), CD28(cluster of differentiation 28), CD70(cluster of differentiation 70), CD80(cluster of differentiation 80), CD86(cluster of differentiation 86), CD137(cluster of differentiation 137), CD276(cluster of differentiation 276), KIRs(killer-cell immunoglobulin-like receptors), LAG3(lymphocyte-activation gene 3), GITR(glucocorticoid-induced TNFR-related protein), GITRL(glucocorticoid-induced TNFR-related protein ligand) 및 CTLA-4(cytolytic T lymphocyte associated antign-4) 중 하나 이상이며,The immune checkpoint is PD-1 (programmed cell death-1), PD-L1 (programmed cell deathligand 1), PD-L2 (programmed cell death-ligand 2), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation) 28), CD70 (cluster of differentiation 70), CD80 (cluster of differentiation 80), CD86 (cluster of differentiation 86), CD137 (cluster of differentiation 137), CD276 (cluster of differentiation 276), KIRs (killer-cell immunoglobulin- like receptors), lymphocyte-activation gene 3 (LAG3), glucocorticoid-induced TNFR-related protein (GITR), glucocorticoid-induced TNFR-related protein ligand (GITRL), and cytolytic T lymphocyte associated antigen-4 (CTLA-4). more than,
    상기 보조 자극 인자는 CD2, CD7, LIGHT, NKG2C,CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1(림프구 기능 연관 항원-1), ICOS(유도성 T 세포 공동자극인자), CD3γ, CD3δ 및 CD3ε 중 하나 이상이며,The co-stimulatory factors include CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T-cell co-stimulatory factor), one or more of CD3γ, CD3δ and CD3ε,
    상기 프로드럭 활성화 효소(prodrug-activating enzymes)는 시토신 디아민나아제(Cytosine deaminase), 랫드 사이토크롬 P450(rat cytochrome P450, CYP2B1), 카르복실에스터라제(carboxylesterase), 세균 니트로리덕타아제(bacterial nitroreductase) 및 대장균에서 분리된 PNP(purine nucleoside phosphorylase) 중 하나 이상인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The prodrug-activating enzymes are cytosine deaminase, rat cytochrome P450 (CYP2B1), carboxylesterase, bacterial nitroreductase ) and a recombinant herpes simplex virus, characterized in that at least one of PNP (purine nucleoside phosphorylase) isolated from E. coli.
  12. 제1항에 있어서,According to claim 1,
    상기 융합 단백질의 발현 카세트는 상기 바이러스 게놈에, UL3와 UL4 유전자 사이, UL26과 UL27 유전자 사이, UL37과 UL38 유전자 사이, UL48과 UL49 유전자 사이, UL53과 UL54 유전자 사이에 삽입되어 있는 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The expression cassette of the fusion protein is inserted into the viral genome, between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL37 and UL38 genes, between the UL48 and UL49 genes, and between the UL53 and UL54 genes. Herpes simplex virus.
  13. 제10항에 있어서,11. The method of claim 10,
    상기 발현 카세트가 상기 바이러스 게놈에, UL3와 UL4 유전자 사이, UL26과 UL27 유전자 사이, UL37과 UL38 유전자 사이, UL48과 UL49 유전자 사이, UL53과 UL54 유전자 사이에 삽입되어 있되, 상기 융합 단백질의 발현 카세트와는 다른 위치에 삽입되어 있는 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The expression cassette is inserted into the viral genome, between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL37 and UL38 genes, between the UL48 and UL49 genes, and between the UL53 and UL54 genes, wherein the expression cassette of the fusion protein and is a recombinant herpes simplex virus, characterized in that it is inserted at a different position.
  14. 제1항에 있어서,The method of claim 1,
    상기 재조합 바이러스는 ICP34.5를 암호화하는 유전자, ICP6을 암호화하는 유전자, ICP47을 암호화하는 유전자, ICP4을 암호화는 유전자, TK을 암호화하는 유전자, UNG를 암호화하는 유전자 및 dUTPase를 암호화하는 유전자 중에서 어느 하나 이상의 유전자가 결실되거나 그 기능을 발휘하지 않도록 돌연변이된 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The recombinant virus is any one of a gene encoding ICP34.5, a gene encoding ICP6, a gene encoding ICP47, a gene encoding ICP4, a gene encoding TK, a gene encoding UNG, and a gene encoding dUTPase. Recombinant herpes simplex virus, characterized in that the above gene is deleted or mutated so as not to exert its function.
  15. 제1항에 있어서,The method of claim 1,
    상기 융합 단백질은 NH2-암세포 표적화 도메인-HveC 세포외 도메인-COOH 순이거나 그 역순인 것을 특징으로 하는 재조합 헤르페스 심플렉스 바이러스.The fusion protein is NH 2 -cancer cell targeting domain-HveC extracellular domain-COOH or the reverse order of the recombinant herpes simplex virus, characterized in that.
  16. 제1항 내지 제15항 중 어느 한 항의 재조합 헤르페스 심플렉스 바이러스를 유효성분으로 포함하는 항암용 약제학적 조성물.A pharmaceutical composition for anticancer comprising the recombinant herpes simplex virus of any one of claims 1 to 15 as an active ingredient.
PCT/KR2021/006983 2020-06-03 2021-06-03 Recombinant herpes simplex virus having expression cassette capable of expressing fusion protein of extracellular domain of hvec and cancer cell-targeting domain, and use thereof WO2021246815A1 (en)

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