Recombinant herpes simplex virus for multiple targeting and uses thereof

Abstract

Disclosed are recombinant herpes simplex viruses for multi-targeting and their uses. Specifically, disclosed are a recombinant herpes simplex virus capable of multi-targeting through the multiple expression of a linker, which is a fusion protein of a cancer cell targeting domain and the extracellular domain of HVEM; and a recombinant herpes simplex virus capable of multi-targeting by having a modified glycoprotein, in addition to being capable of expressing a linker as a fusion protein of a cancer cell targeting domain and the extracellular domain of HVEM; and uses of the virus for anti-inflammatory treatment.

Description

Technical Field

[0001] The present disclosure relates to recombinant herpes simplex viruses for multiple retargeting and uses thereof. Background Art

[0002] In the treatment of cancer, surgery, anticancer chemotherapy, and radiotherapy are widely used. However, most of these therapies have side effects, incomplete therapeutic effects, and problems such as cancer recurrence and metastasis. Therefore, there is a need to continuously develop new and effective cancer therapies. In recent years, rapid progress has been made in the field of anticancer immunotherapy, with examples including oncolytic virus therapy and chimeric antigen receptor T (CAR-T) cell therapy.

[0003] In anti-cancer immunotherapy, oncolytic viruses are viruses that are endowed with the ability to lyse cancer cells by manipulating the genes of live viruses and selectively propagating them in cancer cells, and their proliferation in normal cells is limited. The viruses released by cancer cell lysis can continuously infect surrounding cancer cells, thereby providing a continuous and synergistic therapeutic effect. In addition, oncolytic viruses can stimulate the body's immune response by releasing immunogenic tumor antigens in the process of lysing cancer cells, thereby increasing the anti-cancer effect. In addition, this anti-cancer effect can be enhanced by artificial manipulation to express cytokines, chemokines, etc.

[0004] Currently developed oncolytic viruses can be divided into 10 or more types, including adenovirus, herpes simplex virus (HSV), vaccinia virus, etc. Among them, herpes simplex virus is an enveloped icosahedral virion containing a linear double-stranded DNA of 152kb in size, including HSV-1 and HSV-2 types. HSV has many non-essential genes, and its genome is large, making it easy to use for manipulation or transport of external genes, and its replication cycle is short. In addition, HSV has a high infection efficiency and is ideally able to show improved cancer cell targeting efficiency by easily manipulating glycoproteins that attach to and infect cells.

[0005] T-VEC (Talimogene laherparepvec, product name: Imlygic) was approved by the U.S. FDA in October 2015. It is an oncolytic virus therapy for malignant melanoma using HSV-1. T-VEC is an attenuated HSV-1 virus in which the ICP34.5 and ICP47 genes are deleted to reduce its pathogenicity, and it expresses GM-CSF (granulocyte-macrophage colony-stimulating factor) to promote human immune response. However, the disadvantage of T-VEC is that its limited viral proliferation leads to low therapeutic efficacy, which is due to the deletion of some genes.

[0006] HSV is an enveloped virus that enters cells through a complex interaction involving gD, gB, gH / gL, and gC glycoproteins present in its envelope. First, when gB and gC are attached to 3-OS HS (3-O-sulfated heparan sulfate) on the cell surface, gD binds to at least one of the cellular receptors, such as HVEM (herpes simplex virus entry mediator, HveA), nectin-1 (HveC), and nectin-2 (HveB), thereby inducing fusion between the virus and cell membranes, thereby allowing HSV to enter cells (Hiroaki Uchida et al., Generation of herpes simplex virus type 1 mutant viruses restricted by herpes simplex virus entry mediator (HVEM): identification of mutations that rescue HVEM-expressing cell resistance and nectin-1 recognition, J. Virol. 2009 Apr;83(7):2951-61).

[0007] T-VEC (Talimogene laherparepvec, product name: Imlygic) was approved by the U.S. FDA in October 2015. It is an oncolytic virus therapy for malignant melanoma using HSV-1. T-VEC is an attenuated HSV-1 virus in which the ICP34.5 and ICP47 genes are deleted to reduce its pathogenicity, and it expresses GM-CSF (granulocyte-macrophage colony-stimulating factor) to promote human immune response. However, the disadvantage of T-VEC is that its limited viral proliferation leads to low therapeutic efficacy, which is due to the deletion of some genes.

[0008] In order to overcome this limitation, attempts have been made to carry out the retargeting of targeted cancer cells by operating the envelope glycoprotein gD, gB, gH and gC that participate in HSV entering cells, without weakening the virus. This retargeting is that the exogenous sequence of the targeting domain encoding the cancer cell target molecule is introduced into glycoprotein gD, gB, gH or gC sequence, and uses the recombinant virus with chimeric glycoprotein, wherein the targeting domain (also referred to as ligand) of the exogenous sequence inserts glycoprotein, rather than wild-type glycoprotein. This recombinant virus can enter the cancer cell with the target molecule specifically identified and combined by the targeting domain. Targeting domain is typically scFvI (single-chain variable fragment), and the target molecule of current retargeting has EpCAM (epithelial cell adhesion molecule), HER2 (human epidermal growth factor receptor 2) etc., and as gB, gH, gC etc. of glycoprotein have been modified.

[0009] Meanwhile, among the cell receptors of HSV, HVEM belongs to the tumor necrosis factor receptor protein family (TNFR family), which is mainly expressed in T / B lymphocytes, macrophages, DC cells, sensory neurons and mucosal epithelial cells (Shui JW, Kronenberg M., 2013, Gut Microbes, 4(2):146-151), but it is known to be expressed in large quantities in various tumor tissues (such as B / T lymphoma, melanoma, colorectal cancer, hepatocellular carcinoma, breast cancer, ovarian serous adenocarcinoma, clear renal cell carcinoma and glioblastoma) (Pasero C. et al., Curr. Opin. Pharmacol., 2012. 2(4):478-85; Malissen N. et al., ONCOIMMUNOLOGY, 2019, Vol. 8(12):e1665976). HVEM has four CRDs (cysteine-rich domains), which are characteristics of the TNFR family. Two of the four CRDs are linked to the gD of HSV, thereby inducing the entry of HSV-1 and HSV-2 into cells (Sarah A Connolly et al., Structure-based analysis of the glycoprotein D binding site present on the herpes simplex virus entry mediator HveA (HVEM), J. Virol., November 2002, 76(21): 10894-904).

[0010] As the inventors of the present disclosure have reported, a fusion protein (CEAscFv-HveA) of scFv (single-chain variable fragment) against CEA (carcinoembryonic antigen) and the extracellular domain of HVEM (one of the HSV cell surface receptors) was prepared. When the fusion protein is used together with HSV to treat a cell line expressing CEA, the fusion protein acts as an adapter to induce HSV to target and infect the corresponding cell line (Korean Patent KR10-0937774 and U.S. Patent US8318662).

[0011] The inventors of the present disclosure have determined that when genes encoding fusion proteins (HER2scFv-HveA, EpCAM2scFv-HveA) (which are linkers capable of targeting HER2 (human epidermal growth factor receptor 2) or EpCAM) are inserted into the genome of HSV so that the fusion proteins are expressed in cells infected with HSV, the fusion proteins act to induce HSV to target and infect cell lines expressing HER2 or EpCAM. In addition, it was confirmed that, in order to enable the use of linkers for multiple targeting, when an expression cassette capable of expressing the fusion protein (HER2scFv-HveA) (which is a linker with a targeting function for HER2) and an expression cassette capable of expressing the fusion protein (EpCAM-HveA) (which is a linker with a targeting function for EpCAM) are doubly inserted into the HSV genome so that the fusion proteins are expressed in HSV-infected cells, it was confirmed that the fusion proteins induce HSV virions to infect cell lines expressing HER2 and / or EpCAM through multiple targeting. Furthermore, it was demonstrated that recombinant HSV, which was able to retarget HER2 and / or EpCAM by inserting an expression cassette of this linker into the genome of HSV and further modifying the glycoprotein, infected cell lines expressing HER2 and / or EpCAM through multiple targeting. Summary of the Invention

[0012] Technical issues

[0013] Therefore, the present disclosure takes into account the problems encountered in the related art, and an object of the present disclosure is to provide a recombinant HSV capable of multiple targeting through multiple expression of a linker, which is a fusion protein of a cancer cell targeting domain and an extracellular domain of HVEM.

[0014] Another object of the present disclosure is to provide a recombinant HSV capable of multiple targeting by having modified glycoproteins in addition to being able to express a linker as a fusion protein of a cancer cell targeting domain and the extracellular domain of HVEM, thereby enabling retargeting.

[0015] Another object of the present disclosure is to provide a pharmaceutical composition for treating cancer, which contains recombinant HSV as an active ingredient.

[0016] Yet another object of the present disclosure is to provide a method for preventing or treating cancer, which comprises administering an effective amount of the pharmaceutical composition to a subject (eg, a patient).

[0017] Other or specific objects of the present disclosure will be described below.

[0018] Technical Solution

[0019] One aspect of the present disclosure relates to recombinant HSV capable of multiple targeting. In an exemplary aspect, the recombinant HSV capable of multiple targeting according to the present disclosure can be constructed such that at least one expression cassette expressing a fusion protein (i.e., a linker) of a cancer cell targeting domain (i.e., a domain that specifically binds to a target molecule on the surface of a cancer cell) and an extracellular domain of HVEM is inserted into the genome of the herpes simplex virus, thereby expressing the fusion protein in two or more ways, i.e., multiple ways that do not inhibit the proliferation of the herpes simplex virus.

[0020] In the recombinant HSV for multiple targeting according to the present disclosure, the fusion protein is expressed in two or more ways, i.e., multiple ways, whereby the targeting domains of the fusion protein expressed in multiple ways can target the same target molecule in multiple ways or target different target molecules in multiple ways. The target molecule is any antigen or any receptor present on the surface of a cancer cell that is specifically recognized and bound by the targeting domain of the fusion protein. The recombinant HSV of the present disclosure can be constructed so that at least one expression cassette capable of multiple expression of fusion proteins capable of targeting a target molecule (e.g., HER2) is inserted into its genome, or at least one expression cassette capable of expressing fusion proteins capable of targeting each of different target molecules (e.g., HER2 and EpCAM) is inserted into its genome.

[0021] When the recombinant HSV of the present invention infects a target cell as a cancer cell and enters the target cell, the HSV proliferates, and the linker as a fusion protein is expressed in two or more ways, that is, it is expressed in multiple ways in the cell and is released outside the cell together with the proliferated HSV virions when the cell lyses. Alternatively, in the case where the linker has a leader sequence, it can even be released before the virions are released due to cell lysis. Then, the linker expressed in multiple ways is released outside the cell and induces the HSV virions to infect the surrounding cancer cells in multiple ways, and the cancer cells express the target molecule recognized by the cancer cell targeting domain of the linker. Thus, compared with a single-target recombinant HSV (i.e., a recombinant HSV containing only one expression cassette capable of expressing a fusion protein of the cancer cell targeting domain and the extracellular domain of HVEM), its infection efficiency is further improved, and the continuous proliferation efficiency of the surrounding cancer cells is further improved, ultimately leading to more effective cancer cell death.

[0022] In another exemplary aspect, the recombinant HSV capable of multiple targeting according to the present disclosure can be a recombinant HSV capable of multiple targeting, (i) wherein at least one expression cassette capable of expressing a linker is inserted into the genome of the herpes simplex virus without inhibiting the proliferation of the herpes simplex virus, wherein the linker is a fusion protein of a cancer cell targeting domain and an extracellular domain of HVEM, and (ii) wherein the cancer cell targeting domain is inserted and fused to its glycoprotein.

[0023] The recombinant HSV for multiple targeting according to the present disclosure has a modified glycoprotein that can be retargeted by inserting and fusing a cancer cell targeting domain, thereby enabling additional targeting through the glycoprotein in addition to the linker targeting. Like the above-mentioned recombinant HSV with an expression cassette capable of expressing the linker in multiple ways, the recombinant HSV for multiple targeting is more efficient in infecting cancer cells with virions, the continuous proliferation of virions to surrounding cancer cells, and the death of cancer cells.

[0024] Generally, a recombinant HSV refers to an HSV that, compared to a wild-type HSV, is genetically manipulated to lose or change certain functions or express a target protein of interest by introducing artificial mutations (by deleting, replacing or inserting some nucleic acid sequences). In the present disclosure, a recombinant HSV is an HSV that is capable of expressing an adapter in infected cancer cells, and by introducing (i.e., inserting) an adapter expression cassette (i.e., a construct in which an adapter gene is operably linked to a promoter sequence and a polyadenylation signal sequence capable of expressing it) into the HSV genome without inhibiting the proliferation of the HSV, thereby providing a modified glycoprotein for retargeting.

[0025] Recombinant virus production technology, such as viral genetic manipulation and virion production, is well known in the art, and reference may be made to Sandri-Goldin RM et al., [Alphaherpesviruses: Molecular and Cellular Biology, Caister Academic Press, 2006], Robin H. Lachmann [Herpes Simplex Virus-Based Vectors, International Journal of Experimental Pathology (Int. J. Exp. Pathol.), August 2004; 85(4): 177-190], etc. All documents cited in this specification, including the above-mentioned documents, are considered to be part of this specification.

[0026] In particular, in addition to being manipulated to express a linker, the recombinant HSV of the present disclosure can also be manipulated to enter cells only through the HVEM receptor as an entry receptor, rather than nectin-1. In the following examples of the present disclosure, the sequence of the HSV envelope glycoprotein gD was manipulated to allow HSV to enter cells only through the HVEM receptor. Specifically, the arginine (R) at position 222 of gD and the phenylalanine (F) at position 223 of gD were replaced by asparagine (N) and isoleucine (I), respectively, thereby changing the function of gD. Recombinant HSV with such altered gD function can enter host cells only through the HVEM (HveA) receptor (Hiroaki Uchida et al., Generation of herpes simplex virus type 1 mutant viruses that restrict herpes virus invasion mediator (HVEM): identification of mutations that rescue resistance to HVEM-expressing cells and recognition of nectin-1. Journal of Virology (J. Virol.), April 2009; 83 (7): 2951-61). The HVEM (HveA) receptor is rarely present in normal cells and only exists in lymphomas, etc., while connexin-1 is usually present in normal cells. Therefore, recombinant HSV with altered gD function (which can only enter cells through the HVEM receptor rather than the connexin-1 receptor) does not infect normal cells and is therefore advantageous in terms of safety in anti-cancer treatment.

[0027] Furthermore, the recombinant HSV of the present invention can be manipulated to achieve additional targeting, in addition to targeting via a linker, by inserting and fusing a cancer cell targeting domain into a glycoprotein. Glycoproteins that can be used for additional targeting include gB, gC, gH, and the like, and the HSV in which the cancer cell targeting domain is inserted and fused to the glycoprotein can be prepared by deleting or not deleting some of the glycoprotein's genes and inserting the gene for the cancer cell targeting domain into the open reading frame. When the gene for the cancer cell targeting domain is inserted into the gene for the glycoprotein gH, when the recombinant HSV is produced in cells in a glycoprotein-fused state, the cancer cell targeting domain is integrated into the virion's envelope.

[0028] The insertion and fusion of the cancer cell targeting domain into the glycoprotein can occur in the glycoprotein, such as gB, gC, gD, gH, etc., at any position of the amino acid sequence of the glycoprotein that has not been deleted, at a position where an amino acid sequence of a certain length (particularly 1 to 40 consecutive amino acids) has been deleted, or at any position of the amino acid sequence where an amino acid sequence of a certain length has been deleted or substituted but the amino acid sequence has not been deleted or substituted. Here, in order to inactivate the binding site of the glycoprotein to a specific cell receptor such as HVEM, connexin-1, etc., amino acids of a certain length can be deleted or substituted.

[0029] When the cancer cell targeting domain is inserted and fused to the gB glycoprotein, its position can be any position including the N-terminus, but in HSV-1, the preferred position can be any position within the region of amino acids 9 to 896 in the gB amino acid sequence (SEQ ID NO: 1 GenBank Accession No.: ASM47779). In addition, the preferred position can be any position within the region of amino acids 31 to 78, any position within the region of amino acids 80 to 363, or any position within the region of amino acids 408 to 896. Furthermore, preferred positions may be the position after amino acid 43, the position after amino acid 52, the position after amino acid 70, the position after amino acid 76, the position after amino acid 80, the position after amino acid 81, the position after amino acid 95, the position after amino acid 100, the position after amino acid 137, the position after amino acid 185, the position after amino acid 187, the position after amino acid 241, the position after amino acid 261, the position after amino acid 265, the position after amino acid 304, the position after amino acid 334, the position after amino acid 361, the position after amino acid 408, the position after amino acid 419, the position after amino acid 430, the position after amino acid 458, the position after amino acid 470, the position after amino acid 480, the position after amino acid 490, the position after amino acid 508, the position after amino acid 511, the position after amino acid 520, the position after amino acid 531, the position after amino acid 540, the position after amino acid 557, the position after amino acid 561, the position after amino acid 572, the position after amino acid 584, the position after amino acid 590, the position after amino acid 591, the position after amino acid 593, the position after amino acid 594, the position after amino acid 595, the position after amino acid 596, the position after amino acid 597, the position after amino acid 598, the position after amino acid 599, the position after amino acid 601, the position after amino acid 610 The position after amino acid 81, the position after amino acid 495, the position after amino acid 497, the position after amino acid 546, the position after amino acid 608, the position after amino acid 630, the position after amino acid 663, the position after amino acid 664, the position after amino acid 665, the position after amino acid 671, the position after amino acid 673, the position after amino acid 690, the position after amino acid 725, the position after amino acid 730, the position after amino acid 732, the position after amino acid 742, the position after amino acid 772, the position after amino acid 868, the position after amino acid 869, the position after amino acid 886, the position after amino acid 893, the position after amino acid 894, or the position after amino acid 895. Here, the position is based on the gB amino acid sequence of SEQ ID NO: 1, but in the case of a mutant strain having some differences in the gB sequence, it is based on the homologous sequence corresponding thereto.

[0030] When the cell targeting domain is additionally inserted and fused to the gC glycoprotein, its position can be anywhere including the N-terminus, but in HSV-1, the preferred position can be anywhere within the region of amino acids 1 to 442 in the gC amino acid sequence (SEQ ID NO: 2, GenBank Accession No.: ASM47796). Furthermore, the preferred position can be anywhere within the region of amino acids 33 to 154. Furthermore, the preferred position can be a position after amino acid 33, a position after amino acid 82, a position after amino acid 148, a position after amino acid 149, or a position after amino acid 153 of gC. Here, the position is based on the gC amino acid sequence of SEQ ID NO: 2, but in the case of mutant strains with some differences in the gC sequence, it is based on the corresponding homologous sequence.

[0031] When the cancer cell targeting domain is inserted and fused to the gH glycoprotein, its position can be anywhere, including the N-terminus, the N-terminus of the H1A domain, etc. However, in HSV-1, a preferred position may be within the region of amino acids 12 to 88, within the region of amino acids 116 to 137, or within the region of amino acids 209 to 839 in the gH amino acid sequence (SEQ ID NO: 3, GenBank Accession No.: ASM47773). Furthermore, a preferred position may be within the region of amino acids 12 to 49, or within the region of amino acids 116 to 137. Furthermore, preferred positions may be the position after amino acid 12, the position after amino acid 22, the position after amino acid 23, the position after amino acid 29, the position after amino acid 83, the position after amino acid 116, the position after amino acid 209, the position after amino acid 215, the position after amino acid 225, the position after amino acid 277, the position after amino acid 386, the position after amino acid 437, the position after amino acid 447, the position after amino acid 472, the position after amino acid 636, the position after amino acid 637, the position after amino acid 666, the position after amino acid 731, the position after amino acid 763, the position after amino acid 764, the position after amino acid 775, the position after amino acid 806, the position after amino acid 824, or the position after amino acid 838. Here, the position is based on the amino acid sequence of SEQ ID NO: 3 of gH, but in the case of a mutant strain having some differences in the gH sequence, it is based on the homologous sequence corresponding thereto.

[0032] For more specific information on preferred insertion sites of glycoproteins, see: JOHN R. GALLAGHER et al., [Report on the effect of functional fluorescent protein insertion in herpes simplex virus gB on gB conformation before and after membrane fusion, PLOS Pathog., 2014 Sep, 18, 10(9):e1004373]; Gatta V. et al., [Engineering of novel ligands in gH confers tropism for HSV amplification independent of gD activation of its receptor, 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 autofluorescent glycoprotein H fusion protein, Intervirology, 2001; 44: 232-242]; Qing Fan et al., [Herpes simplex virus type 1 glycoprotein B (gB) mutations have different effects on cell fusion activity depending on whether gD receptor or gB receptor is overexpressed, JOURNAL OF VIROLOGY, August 2009, 83(15): 7384-7390]; Erick Lin et al., [Random linker insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B, Proc. Natl. Acad. Sci. USA, August 2007, 104(32):13140-13145]; Julia O. Jackson et al., [Insertion mutations of herpes simplex virus type 1 glycoprotein H reduce cell surface expression, slow cell fusion rate, or abolish cell fusion and viral entry, JOURNAL OF VIROLOGY, February 2010, 84(4):2038-2046]; Guoying Zhou et al., [Engineered herpes simplex virus 1 relies on the IL13R*2 receptor for cell entry, independent of glycoprotein D receptor interaction, Proc. Natl. Acad. Sci. USA, November 2002, 99(23):15124-15129]; AR Frampton Jr.et al., [Development of herpes simplex virus delivery and gene therapy vectors and their applications in the nervous system, Gene Therapy, 2005, 12:891-901]; Paola Grandi et al., [HSV-1 virions designed to specifically bind to cell surface receptors, MOLECULAR THERAPY, March 2004, 9(3):419-427]; William F Goins et al., [Retargeting of herpes simplex virus (HSV) vectors, Curr. Opin. Virol., December 2016, 21:93-101]; Xiaodan Wang et al., [Targeted gene transfer to nigrostriatal neurons in rat brain by helper virus-free HSV-1 vector particles containing chimeric HSV-1 glycoprotein C-GDNF or gC-BDNF protein, Brain Res. Mol. Brain. Res., September 2005, 139(1):88-102], etc. .

[0033] When the cancer cell targeting domain is inserted into and fused into a glycoprotein, a linker peptide may be present at the N- and C- ends of the cancer cell targeting domain. The linker peptide is intended to ensure the distance between the cancer cell targeting domain and the glycoprotein so that the cancer cell targeting domain fused with the glycoprotein does not interfere with the formation of its inherent three-dimensional structure due to fusion with the glycoprotein. The linker peptide may be a linker peptide with any length and any sequence, as long as it has the ability to specifically bind to a target molecule. In view of flexibility, solubility and resistance to proteolysis, the linker preferably includes at least one selected from amino acids such as Ser, Gly, Ala, Thr, etc., and its length may be 1 to 30 amino acids, preferably 3 to 25 amino acids, more preferably 8 to 20 amino acids.

[0034] The recombinant HSV of the present disclosure can be mutated to delete non-essential genes that are not required for HSV proliferation (i.e., survival and replication), or to render them non-functional (i.e., transcription or translation interrupted). Specific examples of non-essential genes can include the UL3 gene (e.g., gene bank accession number: AFE62830.1), the UL4 gene (e.g., gene bank accession number: AFE62831.1), the UL14 gene (e.g., gene bank accession number: AFE62841.1), the UL16 gene (e.g., gene bank accession number: AFE62843.1), the UL21 gene (e.g., gene bank accession number: AFE62848.1), the UL24 gene (e.g., gene bank accession number: AFE62851.1), the UL31 gene (e.g., gene bank accession number: AFE62859.1), the UL32 gene (e.g., gene bank accession number: AFE62860.1), the US3 gene (e.g., gene bank accession number: AFE62891.1), the UL51 gene (e.g., gene bank accession number: AFE62864.1), the UL61 gene (e.g., gene bank accession number: AFE62870.1), the UL7 gene (e.g., gene bank accession number: AFE62871.1), the UL8 gene (e.g., gene bank accession number: AFE62880.1), the UL9 gene (e.g., gene bank accession number: AFE62891.1), the UL10 gene (e.g., gene bank accession number: AFE62892.1), the UL11 gene (e.g., gene bank accession number: AFE62893 AFE62880.1), UL55 gene (e.g., GenBank Accession No.: AFE62884.1), UL56 gene (e.g., GenBank Accession No.: AFE62885.1, US2 gene (e.g., GenBank Accession No.: AFE62890.1), US12 gene (e.g., GenBank Accession No.: AFE62901.1; ICP47 gene), LAT gene (e.g., GenBank Accession No.: JQ673480.1), gB gene (e.g., GenBank Accession No.: sequence between 52996 and 55710 of GU734771.1), gL gene (e.g., GenBank Accession No.: AFE62828.1), gH gene (e.g., GenBank Accession No.: AFE62849.1), gD gene (e.g., GenBank Accession No.: AFE62894.1), etc.

[0035] For more specific information about the non-essential genes of HSV, see: DMKnipe and PMHowley (eds.) [Fields Virology (Vol. 2) Lippincott Williams & Wilkins, Philadelphia, Pa., 2001, pp. 2399-2460]; Subak-Sharpe JH and Dargan DJ [HSV molecular biology: an overview of the viral genes of herpes simplex virus molecular biology, 1998, 16(3): 239-251]; Travis J. Taylor and David M. Knipe [Proteomics of the herpes simplex virus replication compartment: association of cellular DNA replication, repair, recombination and chromatin remodeling proteins with ICP8, J. Virol., June 2004; 78(11): 5856-5866], etc.

[0036] The recombinant HSV disclosed herein can be a recombinant HSV-1 virus, a recombinant HSV-2 virus, or an HSV-1 / HSV-2 chimeric virus (i.e., a recombinant HSV wherein the genome contains DNA derived from HSV-1 and DNA derived from HSV-2), preferably a recombinant HSV-1 virus, more preferably a recombinant HSV-1 derived from the HSV-1 KOS strain. The HSV-1 KOS strain is available from ATCC (Cat No: VR1493™), and the entire genome sequence of the strain has been completely analyzed and is represented in GenBank Accession No.: JQ673480.1 (Stuart J. Macdonald et al., Genomic sequence of herpes simplex virus type 1 Kos strain, J. Virol., June 2012, 86(11):6371-2).

[0037] The genome of the HSV-1 virus consists of 152kb of double-stranded linear DNA that encodes a total of 84 genes and includes two segments connected to each other, specifically a long segment (L region) and a short segment (S region). The long segment (L region) accounts for approximately 82% of the genome, and the short segment (S region) accounts for approximately 18% of the genome. The long and short segments are connected via two IRLs (internal inverted repeats), which are connecting regions, and TRLs (terminal inverted repeats) are present at the ends of each segment. The L region (UL) contains 56 UL1-UL56 genes and 10 genes (UL8.5, 9.5, 10.5, 12.5, 15.5, 20.5, 26.5, 27.5, 43.5 and 49.5), the S region (US) contains 12 US1-US12 genes and 2 genes (US1.5 and 8.5), and the two IRLs as connecting regions contain 4 genes (ICP4, ICP34.5, ICP0 and LAT).

[0038] In the present disclosure, the linker expression cassette is constructed so that the linker gene is operably connected to the promoter sequence and polyadenylation signal sequence of the gene capable of expressing it, and the polyadenylation signal sequence is a transcription termination signal sequence. Here, "operably connected" refers to the connection of the linker gene that can be transcribed and / or translated. For example, when any promoter affects the transcription of the linker gene to which it is connected, the promoter is considered to be operably connected to the linker gene.

[0039] Generally, a promoter is a nucleic acid sequence that controls the transcription of one or more genes, is located upstream (5' side) of the transcription start site of the gene, and includes a site for binding to DNA-dependent RNA polymerase, a transcription start site, a transcription factor binding site, etc. In the case of eukaryotic origin, the promoter includes a TATA box upstream of the transcription start site (usually located at positions -20 to -30 of the transcription start site (+1)), a CAAT box (usually located at position -75 of the transcription start site), an enhancer, a transcription factor binding site, etc.

[0040] As long as the promoter is capable of expressing the target gene to which it is connected, all constitutive promoters (which induce gene expression at all times), inducible promoters (which induce the expression of the target gene in response to a specific external stimulus), tissue-specific promoters (which induce gene expression in a specific tissue or cell), tissue-nonspecific promoters (which induce gene expression in all tissues or cells), endogenous promoters (derived from virus-infected cells), and exogenous promoters (derived from cells other than virus-infected cells) can be used. Many promoters are known in the art, and a suitable promoter can be selected and used therefrom. For example, useful are CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV (herpes simplex virus) TK (thymidine kinase) promoter, adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, metallothionein promoter, CD45 promoter (hematopoietic stem cell-specific promoter), CD14 promoter (monocyte-specific promoter), and cancer cell-specific promoters (tumor-specific promoters), such as Survivin, Midkine, TERT, CXCR4, etc. In particular, when a cancer cell-specific promoter is used, the expression of the target gene is induced only in cancer cells, thereby suppressing the expression of the target gene in normal cells, thereby increasing the safety of the recombinant HSV of the present disclosure.

[0041] The linker expression cassette is constructed to include, in addition to the promoter, a transcription termination signal sequence that serves as a poly(A) addition signal (polyadenylation signal) to increase the integrity and efficiency of transcription. Many transcription termination signal sequences are known in the art, from which suitable sequences can be selected and used, such as the SV40 transcription termination signal sequence, the HSV TK (herpes simplex virus thymidine kinase) transcription termination signal sequence, and the like.

[0042] The joint expression cassette is inserted into the HSV genome, is used for its expression and does not suppress the proliferation of HSV, and can carry out this insertion when the HSV genome is not deleted, or can be inserted into the gene locus of having deleted some or all of the nonessential genes in the HSV genome.When the joint expression cassette is inserted into the HSV genome that does not delete, it can be inserted between genes.Preferred examples of inserting gene loci include the gene locus between UL3 and UL4 genes, the gene locus between UL26 and UL27 genes, the gene locus between UL37 and UL38 genes, the gene locus between UL48 and UL49 genes, the gene locus between UL53 and UL54 genes, the gene locus between US1 and US2 genes, etc.

[0043] When the linker expression cassette is inserted into a locus where some or all of the nonessential genes in the HSV genome have been deleted, the nonessential genes deleted can be any nonessential genes, as described above.

[0044] In order to express the linker in multiple ways, at least one linker expression cassette having a polycistronic configuration can be inserted into the HSV genome, or at least two linker expression cassettes having a monocistronic configuration can be inserted into the HSV genome.

[0045] In addition to the promoter and transcription termination signal sequence, the linker expression cassette having a polycistronic configuration includes at least two linker genes therebetween, and between these genes, a nucleic acid sequence encoding an IRES (internal ribosome entry site) or a 2A peptide is positioned to enable expression of each protein. Here, the 2A peptide is recognized from picornaviruses and insect virus type C rotavirus, and preferred examples of the 2A peptide include 2A (T2A) of East Asian tsenovirus (Thosea asigna virus), 2A (P2A) of porcine teschovirus-1, 2A (E2A) of ERAV (equine rhinitis virus A), 2A (F2A) of FMDV (foot-and-mouth disease virus), and the like. For detailed information on IRES or 2A peptides, please refer to: Renaud-Gabardos et al., eds., [Internal ribosome entry site-based vectors for combined gene therapy, World J. Exp. Med., February 2015, 20; 5(1): 11-20]; Szymczak et al., [Development of 2A peptide-based strategies in the design of polycistronic vectors, (2005) Expert Opin. Biol. Ther., 5: 627-638]; Kim et al., [High cleavage efficiency of 2A peptide from porcine teschovirus type 1 in human cell lines, zebrafish and mice, (2011) PLOS One, 6(4): e18556], etc.

[0046] In order to express the linker in a variety of ways, when the linker expression cassette has a monocistronic configuration, at least two linker expression cassettes are inserted into the HSV genome. Here, at least two linker expression cassettes can be continuously inserted into the same gene site in the HSV genome, or can be inserted into different corresponding gene sites. For example, the linker expression cassette of the target molecule HER2 and the linker expression cassette of the target molecule EpCAM can be inserted into the same gene site between the UL3 and UL4 genes, or the linker of the target molecule HER2 can be inserted between the UL3 and UL4 genes, and the linker of the target molecule EpCAM can be inserted between the UL26 and UL27 genes as different gene sites. When at least two linker expression cassettes are continuously inserted in the same site, a non-coding region can be inserted between the expression cassettes. The non-coding region is used to prevent at least two expression cassettes from interfering with expression (transcription and / or translation), and the non-coding region can comprise 3-60 nucleotides.

[0047] In the present disclosure, when it is necessary to express three or more types of linkers in multiple ways, a linker expression cassette having a polycistronic configuration capable of expressing two types of linkers and a linker expression cassette having a monocistronic configuration capable of expressing the remaining linkers can be inserted together into the same site or different sites in the HSV genome.

[0048] In the recombinant HSV of the present disclosure, the cancer cell targeting domain of the linker is a site that specifically recognizes and binds to a target molecule of a cancer cell, which is a target cell, and the target molecule recognized by the cancer cell targeting domain is any antigen or any receptor present on the surface of a cancer cell.

[0049] Antigens or receptors are preferably antigens or receptors that are expressed only in cancer cells or that are overexpressed in cancer cells compared to normal cells. Examples of antigens or receptors may include target molecules, such as EGFRvIII (epidermal growth factor receptor variant III) expressed in glioblastomas; EGFR (epidermal growth factor receptor) overexpressed in undifferentiated thyroid cancer, breast cancer, lung cancer, gliomas, etc.; transferrin receptors (transferrin receptors) overexpressed in papillary thyroid cancer, etc.; ErbB-based receptor tyrosine kinases overexpressed in breast cancer, etc.; HER2 (human epidermal growth factor receptor 2) overexpressed in breast cancer, bladder cancer, gallbladder cancer, bile duct cancer, esophagogastric junction cancer, etc.; tyrosine kinase-18-receptor (tyrosine kinase-28) overexpressed in sarcomatoid renal carcinoma, etc. overexpressed in esophageal adenocarcinoma, etc.; HGF receptor c-Met overexpressed in breast cancer, etc.; CXCR4 or CCR7 overexpressed in breast cancer, etc.; endothelin-A receptor overexpressed in prostate cancer; PPAR-δ (peroxisome proliferator-activated receptor δ) overexpressed in rectal cancer, etc.; PDGFR-α (platelet-derived growth factor receptor α) overexpressed in ovarian cancer, etc.; CD4 overexpressed in liver cancer, multiple myeloma, etc. 133; CEA (carcinoembryonic antigen), overexpressed in lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, rectal cancer, colon cancer, medullary thyroid cancer, etc.; EpCAM (epithelial cell adhesion molecule), overexpressed in liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, etc.; MSLN (mesothelin), overexpressed in lung cancer, breast cancer, pancreatic cancer, ovarian cancer, etc.; GD2 (disialoganglioside), overexpressed in neuroblastoma, etc.; GPC3, overexpressed in hepatocellular carcinoma, etc. (phosphatidylinositol glycan 3); PSMA (prostate-specific membrane antigen) overexpressed in prostate cancer, etc.; TAG-72 (tumor-associated glycoprotein 72) overexpressed in ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc.; GD3 (disialoganglioside) moderately expressed in melanoma, etc.; HLA-DR (human leukocyte antigen-DR) overexpressed in blood cancer, solid cancer, etc.; MUC1 (mucin 1) overexpressed in advanced solid cancer, etc.; in advanced non-small cell lung cancer, etc. NY-ESO-1 (New York esophageal squamous cell carcinoma 1) is overexpressed in cancer, etc.; LMP1 (latent membrane protein 1) is overexpressed in nasopharyngeal tumors, etc.; TRAILR2 (tumor necrosis factor-related apoptosis-inducing ligand receptor) is overexpressed in lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colon cancer, colorectal cancer, pancreatic cancer, etc.; VEGFR2 (vascular endothelial growth factor receptor 2) and HGFR (hepatocyte growth factor receptor) are moderately expressed as angiogenic factor receptors in hepatocellular carcinoma, etc.In addition, surface antigens of cancer stem cells, such as CD44, CD166, etc., can be target molecules. Many target molecules that are overexpressed in cancer cells compared to normal cells are known in the art. For other target molecules besides the above examples, see Anne T. Collins et al., [Prospective identification of tumorigenic prostate cancer stem cells, Cancer Res., December 1, 2005, 65(23):10946-51]; Chenwei Li et al., [Identification of pancreatic cancer stem cells, Cancer Res., February 1, 2007, 67(3):1030-7]; Shuo Ma et al., [Research progress of CAR-T cell therapy for solid tumors, Int. J. Biol. Sci., September 2019, 7; 15(12):2548-2560]; Dhaval S. Sanchala et al., [Oncolytic herpes simplex virus therapy: towards selective targeting of cancer cells, Front Pharmacol., May 16, 2017, 8:270], etc.

[0050] Particularly, in the present disclosure, the target molecule is preferably HER2 or EpCAM.

[0051] The target cell targeted by the linker of the recombinant HSV of the present invention is any cancer cell having a target molecule that can be targeted by the cancer cell targeting domain of the linker of the present invention. The cancer cell can be any type of cancer, such as esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, nasopharyngeal cancer (pharyngeal cancer), laryngeal cancer (laryngeal cancer), lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate 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.

[0052] In the present disclosure, in addition to the complete antibody with the ability to specifically bind to the target molecule, the cell targeting domain of the linker can be an antibody derivative or antibody analog.Antibody derivatives are fragments of complete antibodies, which include at least one antibody variable region with the ability to specifically bind to the target molecule, or are modified antibodies.Examples of antibody derivatives can include antibody fragments, such as Fab, scFv, Fv, VhH, VH, VL, etc., multivalent or multispecific modified antibodies, such as Fab2, Fab3, miniantibodies, double antibodies, three antibodies, four antibodies, double scFv, etc.Antibody analogs are artificial peptides or polypeptides that have the ability to specifically bind to a target molecule (such as an antibody), but are structurally different from antibodies and generally have a lower molecular weight than antibodies.Examples of antibody analogs can include ABD, Adhiron, affibodies, affilins, affimers, alpha bodies (alphabodies), Anticalin, armadillo repeat proteins, centririns, DARPins, fynomers, Kunitz regions, ProNectin, repebody, etc.

[0053] A large number of documents on antibodies, antibody derivatives, antibody analogs and their production have been published in the art, examples of which include Renate Kunert & David Reinhart [Readvances in recombinant antibody manufacturing. Advances in recombinant antibody manufacturing, Appl. Microbiol. Biotechnol., April 2016, 100(8):3451-61]; Holliger P. and Hudson PJ [The emergence of engineered antibody fragments and single domains, Nat. Biotechnol., September 2005, 23(9):1126-36]; Xiaowen Yu et al., [Beyond antibodies as binding partners: the role of antibody mimics in bioanalysis, Annual Review of Analytical Chemistry, 2017, 10:293-320]; Abdul Rasheed Baloch et al., [Antibody mimics: promising complements to animal-derived antibodies, Critical Reviews in Biotechnology, 2016, 36:268-275], etc.

[0054] In the present disclosure, the cell targeting domain of the linker is preferably scFv (single-chain variable fragment). scFv is a single-chain antibody in which the heavy chain variable region (heavy-chain variable region, VH) and the light chain variable region (light-chain variable region, VL) of an immunoglobulin are connected via a short linker peptide. In scFv, the C-terminus of VH is connected to the N-terminus of VL, or the C-terminus of VL is connected to the N-terminus of VH. In scFv, the linker peptide can be a linker peptide with any length and any sequence, as long as it does not interfere with the inherent three-dimensional structure of the heavy chain and the light chain, and can make the heavy chain and the light chain adjacent to each other in space, thereby having the ability to specifically bind to the target molecule. Taking into account flexibility, solubility and resistance to proteolysis, the linker preferably includes at least one selected from amino acids such as Ser, Gly, Ala, Thr, etc., and its length can be 1 to 30 amino acids, preferably 3 to 25 amino acids, more preferably 8 to 20 amino acids.

[0055] In the present disclosure, the target molecule targeted by scFv is HER2 or EpCAM. Specifically, the scFv of HER2 is preferably constructed such that the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 are connected via a linker peptide in the order of VH, linker peptide, and VL (i.e., the C-terminus of VH is connected to the N-terminus of VL via a linker peptide), and the scFv of EpCAM is preferably constructed such that the VL of SEQ ID NO: 6 and the VH of SEQ ID NO: 7 are connected via a linker peptide in the order of VL, linker peptide, and VH (i.e., the C-terminus of VL is connected to the N-terminus of VH via a linker peptide).

[0056] In the present disclosure, the extracellular domain of HVEM may be HveA87 of SEQ ID NO: 10 (the sequence of HveA87 including the leader sequence is represented by SEQ ID NO: 11), HveA102 of SEQ ID NO: 12 (the sequence of HveA102 including the leader sequence is represented by SEQ ID NO: 13), or HveA107 of SEQ ID NO: 14 (the sequence of HveA107 including the leader sequence is represented by SEQ ID NO: 15), which are disclosed in Korean Patent KR10-0937774 and U.S. Patent No. 8,318,662 (these documents are considered part of this specification), in addition to HveA82 of SEQ ID NO: 8 (the sequence of HveA82 including the leader sequence is represented by SEQ ID NO: 9), which is used in the following examples. The leader sequence contained in the sequences of SEQ ID NOs: 9, 11, 13, and 15 is the signal peptide sequence of HveA. HveA87, HveA102, and HveA107 further contain 5, 20, and 25 more amino acids than HveA82, respectively, all of which can be used as HSV acceptors for linkers, as demonstrated in the aforementioned Korean patents and US patents.

[0057] In the present disclosure, a linker sequence can be inserted between the cancer cell targeting domain and the extracellular domain of HVEM, and the linker sequence can be a linker of any length and any sequence, as long as it does not inhibit the function of each domain of the linker. Preferably, the linker comprises at least one of the four amino acids Ser, Gly, Ala, Thr, and its length can be 1 to 30 amino acids, preferably 3 to 25 amino acids, and more preferably 8 to 20 amino acids.

[0058] In addition, the linker of the present disclosure can be constructed in the order of NH2 / cancer cell targeting domain / extracellular domain of HVEM / COOH, or in the reverse order thereof. When a linker peptide is inserted therebetween, the linker can be constructed in the order of NH2 / cancer cell targeting domain / extracellular domain of HVEM / COOH, or in the reverse order thereof.

[0059] The linker disclosed herein can be constructed so that a leader sequence (i.e., a secretion-inducing signal sequence) can be further linked to its N-terminus, particularly in a linker configuration, in the order of NH2 / cancer cell targeting domain / linker peptide / extracellular domain of HVEM / COOH, linked to the N-terminus of the cancer cell targeting domain (when using scFv, it is the N-terminus of VH or VL), or in a linker configuration, in the order of NH2 / extracellular domain of HVEM / linker peptide / cancer cell targeting domain, linked to the N-terminus of the extracellular domain of HVEM. The leader sequence is a sequence that has the function of inducing a protein expressed in the cytoplasm to be secreted outside the cell through the cell membrane, and generally comprises about 15 to 30 consecutive hydrophobic amino acid residues. The leader sequence that can be used is not particularly limited, but may be any sequence present at the N-terminus of a protein secreted outside the cell membrane, such as the leader sequence of HveA, the leader sequence of the antibody variable region VL (kaapa), the leader sequence of tissue plasminogen activator (t-PA), the leader sequence of serum albumin, the leader sequence of lactoferrin, the leader sequence of α-casein, the leader sequences of various hormones including human growth hormone, the leader sequence of polypeptides secreted by yeast or bacteria, and the like.

[0060] The leader sequence is a sequence used to induce expression of the linker in target cells and release the linker to the outside of the cell and can be omitted because the linker is only used by HSV to induce infection of adjacent target cells after target cell lysis and HSV release.

[0061] In the present disclosure, for ease of cloning, when the scFv of the target molecule is used as a cell targeting domain, amino acids corresponding to any restriction endonuclease site can be inserted between VH and VL; when the linker peptide is located between VH and VL, amino acids corresponding to any restriction endonuclease site can be inserted between VH or VL and the linker peptide; when the linker peptide is located between the scFv and HVEM, or between the linker and HVEM, amino acids corresponding to any restriction endonuclease site can be inserted between the scFv and HVEM, or between the scFv and the linker. For example, EF (base sequence: GAATTC) on which the restriction endonuclease EcoRI acts, GS (base sequence: GGATCC) on which BamHI acts, or LEE1 (base sequence: CTCGAGGAGCTC) on which XhoI acts can be inserted.

[0062] In the present disclosure, in order to express factors for inducing or enhancing the immune response to cancer cells, either alone or in any combination, recombinant HSV can be constructed so that the genes of the corresponding factors are inserted into the HSV genome. These factors can be manipulated to express cytokines, chemokines, immune checkpoint antagonists (e.g., antibodies, antibody derivatives, or antibody analogs, especially scFv); costimulatory factors that can induce activation of immune cells (T cells or NK cells); antagonists that can inhibit TGFβ function, which inhibit the immune response to cancer cells (e.g., antibodies, antibody derivatives, or antibody analogs, especially scFv); heparinase that can degrade heparan sulfate proteoglycans for solid tumor microenvironment; antagonists that can inhibit the function of angiogenic factor receptor VEGFR-2 (VEGF receptor-2) (e.g., antibodies, antibody derivatives, or antibody analogs, especially scFv), etc.

[0063] As cytokines, for example, interleukins such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-24, etc., interferons such as IFNα, IFNβ, IFNγ, etc., tumor necrosis factors such as TNFα, etc., and colony stimulating factors such as GM-CSF, G-CSF, FLT3L, etc. can be used alone or in any combination of two or more thereof for expression in recombinant HSV.

[0064] As chemokines, for example, CCL2 (C-C motif chemokine ligand 2), CCL5 (RANTES), CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 and XCL-1 (XC motif chemokine ligand 1) can be used alone or in combination for expression in recombinant HSV.

[0065] As immune checkpoint antibodies, antagonists of PD-1 (programmed cell death 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed 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), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), GITR (glucocorticoid-induced TNFR-related protein), GITRL (glucocorticoid-induced TNFR-related protein ligand), and CTLA-4 (cytolytic T lymphocyte-associated antigen-4) can be used alone or in combination for expression in recombinant HSV.

[0066] As co-stimulatory factors, CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T cell co-stimulator), CD3γ, CD3δ and CD3ε can be used alone or in combination for expression in recombinant HSV.

[0067] In the present disclosure, recombinant HSV can be manipulated to express a prodrug activating enzyme that converts a prodrug into a drug that is toxic to cancer cells. Examples of prodrug activating enzymes include cytosine deaminase, which converts 5-FC (5-fluorocytosine) as a prodrug into 5-FU (5-fluorouracil) as a drug; rat cytochrome P450 (CYP2B1), which converts CPA (cyclophosphamide) as a prodrug into PM (phosphamide mustard) as a drug; carboxylesterase, which converts irinotecan (SN-38150) as a prodrug into SN-38 as a drug; bacterial nitroreductase, which converts BC1954 as a prodrug into 4-hydroxyamine 151 as a DNA cross-linking agent; PNP (purine nucleoside phosphorylase) isolated from Escherichia coli, which converts 6-methylpurine-2'-deoxyriboside as a prodrug into 6-methylpurine as a drug, and the like.

[0068] In addition, in the present disclosure, recombinant HSV can be manipulated to express TRAIL (TNF-related apoptosis-inducing ligand). TRAIL is known to induce cancer cell death by binding to its receptor, which is overexpressed in cancer cells (Kaoru Tamura et al., Multi-mechanism tumor-targeted oncolytic viruses overcome drug resistance in brain tumors, Mol. Ther., January 2013, 21 (1): 68-77).

[0069] For more details on the use of factors or prodrug activating enzymes to induce or enhance these immune responses, see the following: Michele Ardolino et al., [Cytokine therapy in cancer immunotherapy, J. Oncotarget, Oncotarget., August 14, 2015, 6(23)]; Bernhard Homey et al., [Chemokines: Reagents for cancer immunotherapy, Nat. Rev. Immunol., March 2002, 2(3):175-84]; Marianela Candolfi et al., [Evaluation of a pro-apoptotic transgene in combination with Flt3L in an immunostimulatory gene therapy approach for glioblastoma multiforme (GBM), J. FASEB J., 2008, 22:107713]; Danny N Khalil et al., [The future of cancer therapy: immunomodulation, CARs and combination immunotherapy, Nat. Rev. Clin. Oncol., May 2016, 13(5):273-90]; Paul E. Hughes et al. [Combining targeted therapy and checkpoint immunotherapy for cancer treatment], Trends Immunol., July 2016, 37(7):462-476]; Cole Peters and Samuel D. Rabkin [Engineering herpes viruses as oncolytic agents, Mol. Ther. Oncolytics., 2015; 2:15010] et al.

[0070] In the present disclosure, as in the aforementioned linker, a factor or prodrug activating enzyme for inducing or enhancing an immune response is constructed so that an expression cassette (i.e., a construct in which its gene is operably linked to a promoter sequence (enabling it to be expressed) and a polyadenylation signal sequence) of the factor or prodrug activating enzyme is inserted into the HSV genome without inhibiting the proliferation of HSV. This insertion can be carried out without deleting the HSV genome, or can be inserted into a gene locus in which some or all of the non-essential genes in the HSV genome have been deleted. Here, when inserting without deleting the HSV genome, insertion can be carried out between genes, and preferred insertion sites are, for example, between UL3 and UL4, between UL26 and UL27, between UL37 and UL38, between UL48 and UL49, between UL53 and UL54, and between US1 and US2. When inserted into a gene locus in which a non-essential gene has been deleted or into a gene in which a non-essential gene has not been deleted, as described above, this non-essential gene can be selected from any non-essential gene.

[0071] Another aspect of the present disclosure relates to a pharmaceutical composition for treating cancer, which contains the above-mentioned recombinant HSV as an active ingredient.

[0072] The pharmaceutical composition of the present disclosure has an anti-cancer effect on cancers expressing a target molecule targeted by the targeting domain of the linker expressed by the recombinant HSV. Examples of cancer tumors are as described above with respect to the target molecule.

[0073] In particular, the compositions of the present disclosure preferably have an anti-cancer effect on cancers having tumor cells expressing HER2 or EpCAM. Examples of tumor cells expressing HER2 include breast cancer cells, ovarian cancer cells, gastric cancer cells, lung cancer cells, head and neck cancer cells, osteosarcoma cells, glioblastoma multiforme cells, salivary gland tumor cells, and the like. In addition, examples of tumor cells expressing EpCAM include liver cancer cells, prostate cancer cells, breast cancer cells, colorectal cancer cells, lung cancer cells, gallbladder cancer cells, pancreatic cancer cells, and gastric cancer cells.

[0074] In the present disclosure, anti-cancer effects include death of cancer cells, reduction in viability of cancer cells, inhibition or delay of pathological symptoms of cancer due to inhibition of cancer cell proliferation, inhibition or delay of onset of such pathological symptoms, inhibition of cancer metastasis, and inhibition of cancer recurrence.

[0075] In addition to the recombinant HSV as an active ingredient, the pharmaceutical composition of the present disclosure may also include a recombinant adapter molecule. A recombinant adapter molecule is a molecule in which a fusion protein having a cancer cell targeting domain is generated by a recombination process, such as in an adapter expressed by recombinant HSV, or more specifically, as in a fusion protein of a cancer cell targeting domain and the extracellular domain of HVEM. Here, having a cancer cell targeting domain in an adapter expressed by recombinant HSV means that, when the target molecule targeted by the cancer cell targeting domain of the adapter expressed by HSV is HER2, the target molecule targeted by the cancer cell targeting domain of the adapter molecule is also HER2. The method of producing a target protein of interest using a recombinant method generally includes preparing an expression vector capable of expressing the target protein, and transforming the expression vector into a host cell, such as Escherichia coli, yeast or animal cells (CHO cells, NSO cells, BHK cells, Sp2 cells or HEK-293 cells), followed by culturing, and then isolating the target protein. The method of producing a target protein of interest using a recombinant method is well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2001)). In particular, with regard to the production of the recombinant linker molecules used in the present disclosure, reference can be made to the following documents: Korean Patent KR10-0937774 and U.S. Patent US8318662. The pharmaceutical composition of the present disclosure also includes such a recombinant linker molecule. Therefore, when the recombinant HSV as the active ingredient of the present disclosure and the recombinant linker molecule are administered to a patient together, the initial cancer cell infection efficiency of the recombinant HSV is effectively improved.

[0076] In addition, the pharmaceutical composition of the present disclosure can be used in combination with approved anticancer agents or mixed use. The example of anticancer agent may include any anticancer agent, any cytokine drug, any antibody drug, any immune checkpoint inhibitor drug, and any cell therapy agent (for chimeric antigen receptor T cell therapy (chimeric antigen receptor T cell therapy) or CAR-NK cell therapy) that shows cytotoxicity to cancer cells, such as metabolic antagonists, alkylating agents, topoisomerase antagonists, microtubule antagonists and plant-derived alkaloids. Specific examples thereof may include paclitaxel, nitrogen mustard, imatinib, oxaliplatin, gefitinib, bortezomib, sunitinib, carboplatin, cisplatin, rituximab, erlotinib, sorafenib, IL-2 drugs, IFN-α drugs, IFN-γ drugs, trastuzumab, blinatumomab, ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, bevacizumab, cetuximab, tisagenlecleucel (Kymriah), axicabgene ciloleucel (YESCARTA), and the like. In addition to the exemplary anticancer agents, other anticancer agents known in the art can be combined or mixed with the pharmaceutical compositions of the present disclosure without limitation.

[0077] The pharmaceutical composition of the present disclosure may include pharmaceutically acceptable carriers or excipients, and thus may be prepared into the form of oral preparations or parenteral preparations by typical methods known in the art according to the administration route.

[0078] This pharmaceutically acceptable carrier or excipient can not damage the activity or the character of medicine, and itself has no toxicity to human body, its example can comprise lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water (such as saline and sterilized water), syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate, mineral oil, Ringer's solution, buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin and any combination thereof.Especially, when pharmaceutical composition of the present disclosure is mixed with the form of liquid solution, suitable carrier or excipient can comprise saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol and ethanol, and it can be used alone or in combination.If necessary, can add and use other typical pharmaceutical additives, such as antioxidant, buffer agent, antibacterial agent etc.

[0079] When the pharmaceutical composition of the present disclosure is prepared as an oral preparation, it can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. When prepared as a parenteral preparation, especially an injection, it can be prepared in the form of unit dose ampoules or multiple doses. The pharmaceutical composition of the present disclosure can also be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0080] According to typical methods in the pharmaceutical field, the pharmaceutical compositions of the present disclosure can be formulated into unit dosage forms suitable for administration to the patient's body and can be administered by oral administration routes or parenteral administration routes, such as cutaneous, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal and rectal routes, using any administration method commonly used in the art.

[0081] The dosage (effective amount) of the pharmaceutical composition of the present invention may vary according to factors such as the formulation method, administration mode, age, weight and sex of the patient, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity, and may be appropriately determined by those skilled in the art taking these factors into consideration. In a preferred embodiment, the pharmaceutical composition of the present invention is prepared as a unit dosage form injection. When prepared as a unit dosage form injection, the amount of recombinant HSV contained in each unit dose of the pharmaceutical composition of the present invention may be 10 2 to 10 14 pfu, especially 10 4 to 10 11 pfu.

[0082] Yet another aspect of the present disclosure relates to a method for treating or preventing cancer (tumor), comprising administering an effective amount of a pharmaceutical composition containing the above-mentioned recombinant HSV to a subject, such as a patient.

[0083] By lysing and killing cancer cells that have a target molecule targeted by the cancer cell-targeting domain of the recombinant HSV linker, a method for treating cancer is made possible. Therefore, the treatment methods disclosed herein can be applied to any cancer that has such a target molecule. In particular, the treatment methods disclosed herein are preferably applied to cancers that express HER2 or EpCAM.

[0084] The therapeutic methods disclosed herein can be used in combination with other cancer treatment methods described above without limitation. For example, as described above, cytotoxic anticancer agents, cytokine drugs, antibody drugs, immune checkpoint inhibitor drugs, cell therapy agents (for CAR-T cell therapy or CAR-NK cell therapy), radiotherapy, surgery, etc., can be used before or after administering the pharmaceutical composition of the present disclosure, or in a manner that is administered simultaneously with the pharmaceutical composition of the present disclosure.

[0085] In the treatment methods of the present invention, an effective amount is an amount of the pharmaceutical composition of the present invention that is administered to a subject (e.g., a patient) to achieve the desired medical effect (e.g., a cancer treatment or prevention effect) when the pharmaceutical composition of the present invention is administered to the subject (e.g., a patient) based on the advice of a medical expert during administration. As described above, such an effective amount can be appropriately determined by a person skilled in the art (e.g., a medical expert, etc.), depending on the patient's age, weight, sex, pathological condition, etc., as described above.

[0086] In the treatment methods disclosed herein, the pharmaceutical composition is preferably administered to the patient in the form of an injection by parenteral administration, such as intralesional (intratumoral), intravenous, intramuscular or intraarterial administration.

[0087] Beneficial effects

[0088] According to the present disclosure, it is possible to provide a recombinant HSV capable of multi-targeting by multi-expression of a linker that is a fusion protein of a cancer cell targeting domain and an extracellular domain of HVEM, and it is also possible to provide a recombinant HSV capable of multi-targeting by having a modified glycoprotein in addition to being capable of expressing a linker that is a fusion protein of a cancer cell targeting domain and an extracellular domain of HVEM, thereby enabling retargeting.

[0089] In addition, according to the present disclosure, a pharmaceutical composition for the treatment or prevention of cancer can be provided, which comprises a recombinant HSV capable of multiple targeting as an active ingredient, and a method for preventing or treating cancer, which comprises administering an effective amount of the pharmaceutical composition to a subject (e.g., a patient).

[0090] Compared with single-target HSV, the recombinant HSV capable of multiple targeting according to the present disclosure has high cancer cell infection efficiency and high cancer cell killing function.

[0091] BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The genome structure of KOS-37 BAC is schematically shown;

[0093] Figure 2 The genome structure of the HVEM-restricted HSV-1 virus is schematically shown;

[0094] Figure 3 The genome structure of the HVEM-restricted HSV-1 virus expressing EmGFP is schematically shown;

[0095] Figure 4 Results showing fluorescence expression of HVEM-restricted HSV-1 virus expressing EmGFP and specific infection of cells with HVEM receptors;

[0096] Figure 5 The genome structure of each of the HSV-1 virus expressing a HER2-targeting linker, the HSV-1 virus expressing an EpCAM-targeting linker, and the HSV-1 virus expressing a HER2 / EpCAM dual-targeting linker is schematically shown;

[0097] Figure 6 Shown are the complete sequences of the HER2scFv-HveA linker and the EpCAMscFv-HveA linker and the configurations of the corresponding sequences;

[0098] Figure 7 Schematically shows the genome structure of each of (i) a single-targeting virus having a HER2-targeting modified glycoprotein gH and (ii) a HER2 dual-targeting HSV-1 virus having a HER2-targeting modified glycoprotein gH and expressing a HER2-targeting linker;

[0099] Figure 8 The complete amino acid sequence of the HER2 scFv ligand inserted and fused to gH and the configuration of the corresponding sequence are shown;

[0100] Figure 9 Schematic diagram of the genome structure of each of (i) a single-targeted virus having an EpCAM-targeted modified glycoprotein gH and (ii) an EpCAM dual-targeted HSV-1 virus having an EpCAM-targeted modified glycoprotein gH and expressing an EpCAM-targeting linker;

[0101] Figure 10 The complete amino acid sequence of the EpCAMscFv ligand inserted and fused to gH and the configuration of the corresponding sequence are shown;

[0102] Figure 11 Shown are the results of measurement of proliferation of a virus expressing the fluorescent protein EmGFP and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing a HER2scFv-HveA linker (HADa-S), a virus with a HER2scFv ligand in gH (HgH-S), and a dual-targeting virus with a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker (HADa-HgH-D) in Vero-HVEM cells and SK-OV-3 cells;

[0103] Figure 12Shown are the results of measurement of proliferation in Vero-HVEM cells of a virus expressing the fluorescent protein EmGFP and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing a HER2scFv-HveA linker (HADa-S), a virus with a HER2scFv ligand in gH (HgH-S), and a dual-targeting virus with a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker (HADa-HgH-D);

[0104] Figure 13 The results of measuring the expression levels of a virus expressing the fluorescent protein EmGF and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing a HER2scFv-HveA linker (HADa-S), a virus having a HER2scFv ligand in gH (HgH-S), and a dual-targeting virus having a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker (HADa-HgH-D) in Vero-HVEM cells and SK-OV-3 cells are shown;

[0105] Figure 14 The results of specific infection depending on HER2 expression in SK-OV-3 cells and the like are shown for a virus expressing the fluorescent protein EmGF and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing a HER2scFv-HveA linker (HADa-S), a virus having a HER2scFv ligand in gH (HgH-S), and a dual-targeting virus having a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker (HADa-HgH-D);

[0106] Figure 15 Results are shown for specific cell death depending on HER2 expression in SK-OV-3 cells and the like, for a virus expressing the fluorescent protein EmGF and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing a HER2scFv-HveA linker (HADa-S), a virus having a HER2scFv ligand in gH (HgH-S), and a dual-targeting virus having a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker (HADa-HgH-D);

[0107] Figure 16 The results of tumor inhibition in animal experiments are shown by a dual-targeting virus (HADa-HgH-D) with a HER2scFv ligand in gH and expressing a HER2scFv-HveA linker;

[0108] Figure 17 Shown are the results of specific infection depending on the expression of EpCAM in BT-474 cells, etc., of a virus expressing the fluorescent protein EmGF and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing an EpCAMscFv-HveA linker (EADa-S), a virus having an EpCAMscFv ligand in gH (EgH-S), and a dual-targeting virus having an EpCAMscFv ligand in gH and expressing an EpCAMscFv-HveA linker (EADa-EgH-D);

[0109] Figure 18 Results are shown for specific cell death depending on the expression of EpCAM in BT-474 cells and the like, for a virus expressing the fluorescent protein EmGF and using only HVEM as a cell receptor for cell entry (gDm), a virus expressing an EpCAMscFv-HveA linker (EADa-S), a virus having an EpCAMscFv ligand in gH (EgH-S), and a dual-targeting virus having an EpCAMscFv ligand in gH and expressing an EpCAMscFv-HveA linker (EADa-EgH-D);

[0110] Figure 19 Schematic representation of the genome structure of a dual-targeting HSV-1 virus (EADa-HgH-D) with a HER2-targeting modified glycoprotein gH and expressing an EpCAM-targeting linker;

[0111] Figure 20 Shown are the results of dual targeting depending on the expression of HER2 and EpCAM by a dual targeting HSV-1 virus (EADa-HgH-D) having a HER2-targeting modified glycoprotein gH and expressing an EpCAM-targeting linker in CHO-K1 cells, among others; and

[0112] Figure 21 Shown are the results of dual targeting depending on the expression of HER2 and EpCAM by an HSV-1 virus (EADA-hada-D) expressing a HER2 / EpCAM dual targeting linker in CHO-K1 cells, among others.

[0113] Best Practice

[0114] The present disclosure will be better understood through the following examples. However, these examples should not be construed as limiting the scope of the present disclosure.

[0115] <Example 1> Production of HVEM-restricted HSV-1 virus

[0116] The HSV-1 genome consists of a large gene of approximately 152 kb, so KOS-37 / BAC (GenBank Accession No.: MF156583) (Gierasch WW et al., J. Virol. Methods., 2006. 135:197-206) is used to insert foreign genes or mutations into specific gene loci. The HSV-1 KOS strain is a HSV-1 strain primarily used in laboratories due to its well-known properties and its usefulness in studying gene function and etiology (Smith KO. Proc. Soc. Exp. Biol. Med., 1964. 115:814-816). KOS-37 / BAC, prepared by inserting a BAC plasmid into the KOS genome, can be cloned at the bacterial level by transforming DH10B bacteria (Invitrogen) (Gierasch WW et al., J. Virol. Methods., 2006. 135:197-206). In KOS-37 / BAC, a BAC (bacterial artificial chromosome) is inserted into the gene locus between UL37 and UL38 of the HSV-1 KOS genome along with the LoxP sites on both sides. This is to allow the BAC gene to be removed using the Cre-Lox system in the subsequent process. The schematic diagram is shown in Figure 1 Shown in.

[0117] In order to prepare an HVEM-restricted HSV-1 that enters cells only through the HVEM cell receptor, a gD-R222N / F223I HSV-1 virus is required, in which the arginine (R) at position 222 and the phenylalanine (F) at position 223 of the HSV-1 gD amino acid sequence (GenBank Accession No.: ASM47818, SEQ ID NO: 16) are replaced by asparagine (N) and isoleucine (I), respectively.

[0118] The gD-R222N / F223I HSV-1 virus prepared by mutation can only infect host cells through HVEM (HveA) rather than connexin-1 as a cell entry receptor (Uchida H. et al., J. Virol., 2009. 83(7): 2951-2961). Therefore, it is advantageous in terms of safety because it cannot infect normal cells with connexin-1 receptors.

[0119] The genome structure of the HVEM-restricted KOS-gD-R222N / F223I virus is schematically shown in Figure 2 middle.

[0120] KOS-gD-R222N / F223I HSV-1 virus was prepared by introducing the R222N / F223I mutation into the gD site of KOS-37 / BAC using a counter-selection BAC modification kit (GeneBridges Inc.) according to the manufacturer's protocol.

[0121] Specifically, an E. coli clone containing KOS-37 / BAC was transformed with a pRed / ET plasmid expressing RecE and RecT, which are capable of performing homologous recombination (Muyrers JP et al., Nucleic Acids Res., 1999. 27(6):1555-1557). A gD-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer gD-rpsL For: SEQ ID NO: 17, reverse primer gD-rpsL Rev: SEQ ID NO: 18), which included a gene site for introducing mutations into gD. The gD-rpsL-neo / kan cassette consists of a gD homology region located at the insertion locus, the rpsL gene, and the neo / kan gene. The rpsL gene is a selectable marker that confers streptomycin sensitivity, and the neo / kan gene confers kanamycin resistance. When the gD-rpsL-neo / kan cassette was inserted, E. coli was prepared that was sensitive to streptomycin antibiotics due to the rpsL gene and resistant to kanamycin due to the neo / kan gene. L-arabinose (Sigma-Aldrich) was added to the E. coli clone containing KOS37 / BAC and pRed / ET to activate the function of pRed / ET (Muyrers J.P. et al., Nucleic Acids Res., 1999. 27(6):1555-1557), thereby inducing the expression of RecE and RecT to achieve homologous recombination. Then, 200 ng of the prepared gD-rpsL-neo / kan cassette was used for transformation. The gD-rpsL-neo / kan cassette was inserted into the gD gene locus of KOS-37 / BAC E. coli through homologous recombination. KOS-37 / BAC E. coli harboring the gD-rpsL-neo / kan gene exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. E. coli cultured in kanamycin culture medium were presumed to have been inserted with the gD-rpsL-neo / kan gene, and the final gene insertion step was performed. By adding L-arabinose (Sigma-Aldrich) to Escherichia coli containing the KOS 37-BAC gD-rpsL-neo / kan clone to activate the function of pRed / ET, thereby inducing the expression of RecE and RecT to achieve homologous recombination, the cells were transformed with 100 pmol of the R222N_F2231_mutant (SEQ ID NO: 19), which is an oligonucleotide in which R and F at positions 222 and 223 of gD are replaced by N and I, respectively.Candidates were selected in streptomycin medium based on the principle that when the existing gD-rpsL-neo / kan cassette was replaced with the inserted oligonucleotide, the rpsL-blocked streptomycin resistance was activated (Heermann R. et al., Microb. Cell Fact., 2008, 14: doi: 10.1186). DNA was isolated from selected candidates using a DNA preparation method (Horsburgh BC et al., Methods Enzymol., 1999. 306: 337-352), and the substitution of N and I at the corresponding positions 222 and 223 of gD was confirmed by PCR (polymerase chain reaction) and DNA sequencing.

[0122] Next, for virus production, intact KOS-37 / BAC-gD-R222N / F223I DNA was extracted using a large construct DNA purification kit (Macherey-Nagel), and then 1 μg of DNA was used to transfect 2 × 10 5 Cre-Vero-HVEM cells. Then, DMEM (Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's Medium) (Welgene) containing 100U / ml penicillin / 100μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene) was used for cell culture. The Cre-Vero-HVEM cell line is a cell line that induces HVEM protein expression by inserting the HVEM gene into the Cre-Vero cell line (Gierasch WW et al., J.Virol.Methods., 2006.135:197-206). The reason for using Cre-Vero-HVEM is that the BAC gene of KOS-37 / BAC-gD-R222N / F223I can be removed using the Cre recombinase of the cell, and it is also effective to be infected by the KOS-gD-R222N / F223I virus due to overexpression of HVEM, so that the mass production of the virus becomes easy. 3-4 days after gene introduction, the formation of cell plaques was confirmed, and then the virus-containing cells were collected and subjected to three freeze-thaw cycles (Gierasch WW et al., J. Virol. Methods., 2006. 135: 197-206), and ultrasonic treatment was performed to finally obtain KOS-gD-R222N / F223I virus.

[0123] <Example 2> Preparation of HVEM-restricted HSV-1 virus expressing EmGFP

[0124] To produce HVEM-restricted HSV-1 expressing EmGFP, an expression cassette capable of expressing EmGFP (emerald green fluorescent protein) was inserted into the UL26 / UL27 sites of the KOS-37 / BAC-gD-R222N / F223I DNA prepared in Example 1 (Tiffany A. et al., J. Virol. Methods., 2015. 231: 18-25). This facilitates the use of EmGFP as a marker to observe viral production and infection. The EmGFP cassette was prepared using the pcDNA6.2-GW / EmGFP-miR plasmid (Invitrogen).

[0125] The genome structure of KOS-EmGFP-gD-R222N / F223I expressing EmGFP is schematically shown in Figure 3 middle.

[0126] For EmGFP expression, pCMV-EmGFP-tkpA using pCMV as a gene promoter of cytomegalovirus and tkpA as a polyadenylation signal of HSV TK (herpes simplex virus thymidine kinase) was inserted into KOS-37 / BAC-gD-R222N / F223I DNA.

[0127] All insertions were performed using the Counter Selection BAC Modification Kit (GeneBridges Inc.) according to the manufacturer's protocol, as described in Example 1.

[0128] Specifically, a clone containing KOS-37 / BAC-gD-R222N / F223I was transformed with a pRed / ET plasmid expressing RecE and RecT, which is capable of performing homologous recombination (Muyrers JP et al., Nucleic Acids Res., 1999. 27(6):1555-1557). A UL26 / 27-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer UL26 / 27-rpsL_For: SEQ ID NO: 20, reverse primer UL26 / 27-rpsL_Rev: SEQ ID NO: 21), which included a site for introducing the target gene between UL26 and UL27. L-arabinose (Sigma-Aldrich) was added to a clone containing KOS-37 / BAC-gD-R222N / F223I DNA and pRed / ET to induce homologous recombination, followed by transformation with 200 ng of the prepared UL26 / 27-rpsL-neo / kan cassette. The UL26 / 27-rpsL-neo / kan cassette was inserted into the UL26 / 27 locus of KOS-37 / BAC via homologous recombination in E. coli. Homologous recombination in E. coli harboring the UL26 / 27-rpsL-neo / kan gene demonstrated kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. E. coli obtained from kanamycin culture medium were presumed to have gD-rpsL-neo / kan inserted, and the final step of gene insertion was performed.

[0129] L-arabinose (Sigma-Aldrich), which activates pRed / ET function, was added to E. coli containing the UL26 / 27-rpsL-neo / kan cassette to induce homologous recombination, and then transformed with 200 ng of the UL26 / 27-tkpA-EmGFP-pCMV cassette. The UL26 / 27-tkpA-EmGFP-pCMV cassette was prepared using the forward primer UL26 / 27-tkpA_For (SEQ ID NO: 22) and the reverse primer UL26 / 27-pCMV_Rev (SEQ ID NO: 23) using the pcDNA6.2-GW / EmGFP-miR plasmid (Invitrogen) as a template.

[0130] When the existing UL26 / 27-rpsL-neo / kan box is replaced with the inserted UL26 / 27tkpA-EmGFP-pCMV, streptomycin resistance blocked by rpsL is activated. Based on this principle, candidates are selected in streptomycin medium (Heermann R. et al., Microb. Cell Fact., 7, 14, 2008.14: doi: 10.1186). DNA preparation methods are used to isolate DNA from selected candidates (Horsburgh BC et al., Methods Enzymol., 1999.306: 337-352). The introduction of tkpA-EmGFP-pCMV at the UL26 / 27 position is confirmed by treatment with restriction endonucleases EcoRI and XhoI and PCR (polymerase chain reaction), and the exact gene sequence is identified by sequencing of the PCR product.

[0131] The normal expression of fluorescent protein and virus production were tested. The complete KOS-37 / BAC-gD-R222N / F223I DNA was extracted using a large construct DNA purification kit (Macherey-Nagel), and then 1 μg of DNA was used to transfect 2×10 5 Cre-Vero-HVEM cells were transfected to remove the BAC gene using Cre recombinase. Three days after transfection, the expression of EmGFP protein was observed using a fluorescence microscope, and the production of virus was observed by forming Cre-Vero-HVEM cell plaques. After confirming the formation of plaques, the virus-containing cells were collected and subjected to three freeze-thaw cycles (Gierasch WW et al., J. Virol. Methods., 2006. 135: 197-206), and ultrasonic treatment was performed to finally obtain KOS-gD-R222N / F223I virus.

[0132] For infection with KOS-EmGFP-gD-R222N / F223I virus and its fluorescence expression, HVEM-free cell lines (J1 and J-Nectin) and HVEM-expressing cell lines (J-HVEM) were used. J1 cells are a baby hamster kidney cell line lacking the viral HSV-1 receptor HVEM and connexin-1 (Petrovic B. et al., 2017. PLOS Pathog. 19; 13(4): e1006352). J-Nectin and J-HVEM cell lines are cell lines that overexpress connexin-1 and HVEM in J1 cells, respectively (Petrovic B. et al., 2017. PLOS Pathog. 19; 13(4): e1006352). Each cell line was cultured in DMEM (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene). The KOS-EmGFP-gD-R222N / F223I virus obtained above was used to infect 1×10 4 After 24 hours, fluorescent protein expression and virus infection were observed using a fluorescence microscope (Baek HJ et al., Mol. Ther., 2011. 19(3): 507-514).

[0133] The results are shown in Figure 4 The upper and lower images were taken using a fluorescence microscope and an optical microscope, respectively. Figure 4 From the upper fluorescence microscopy images, it can be seen that the JI cell line and the J-Nectin cell line were not infected, and only the J-HVEM cell line was infected.

[0134] Based on the above results, it was confirmed that the proliferation of KOS-EmGFP-gD-R222N / F223I virus (gDm) was easily observed by the expression of fluorescent protein as expected, and cell entry was possible using only HVEM as a cell entry receptor without using connexin-1.

[0135] <Example 3> Production of HSV-1 virus expressing HER2-targeting linker, HSV-1 virus expressing EpCAM-targeting linker, and HSV-1 virus expressing HER2 / EpCAM dual targeting linker

[0136] Each of the linker expression cassette expressing HER2scFv-HveA, the linker expression cassette expressing EpCAM-HveA, and the linker expression cassette expressing both HER2scFv-HveA and EpCAM-HveA was inserted into the UL3 / UL4 site of KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA into which the EmGFP expression cassette (pCMV-EmGFP-tkpA) prepared in Example 2 was inserted.

[0137] Figure 5 Schematic diagram showing the genome structure of KOS-UL3 / 4-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus with pCMV-HER2scFv-HveA-bGHpA inserted as the linker expression cassette for expressing HER2scFv-HveA, KOS-UL3 / 4-EpCAMscFv-HveA-bGHpA inserted as the linker expression cassette for expressing EpCAMscFv-HveA The genome structure of the virus is: EpCAM-HveA-EmGFP-gD / R222N / F223I; the genome structure of the virus is: KOS-UL3 / 4-EpCAMscFv-HveA-HER2scFv-HveA-EmGFP-gD / R222N / F223I; the genome structure of the virus is: KOS-UL3 / 4-EpCAMscFv-HveA-HER2scFv-HveA-EmGFP-gD / R222N / F223I; the genome structure of the virus is: Figure 6 The complete sequences of the HER2scFv-HveA linker and EpCAMscFv-HveA linker and the construction of the corresponding sequences are shown.

[0138] Here, the HER2 scFv was constructed such that the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 were linked via a linker peptide of SEQ ID NO: 24, the EpCAM scFv was constructed such that the VL of SEQ ID NO: 6 and the VH of SEQ ID NO: 7 were linked via a linker peptide of SEQ ID NO: 25, and HveA was HveA82 of SEQ ID NO: 8 in the HER2 scFv-HveA linker and the EpCAM scFv-HveA linker. In addition, in the HER2 scFv-HveA linker and the EpCAM scFv-HveA linker, the leader sequence of SEQ ID NO: 26 was included at their N-termini, specifically before the VH of the HER2 scFv and before the VL of the EpCAM scFv.

[0139] EF (base sequence: GAATTC), a restriction endonuclease site for easy cloning, is added after the HER2 or EpCAM scFv sequence and the NH2-GGGGS sequence (linker sequence for the HveA sequence). Furthermore, pCMV is a cytomegalovirus gene promoter, bGH-pA is a bGH-polyA (bovine growth hormone polyadenylation) signal sequence, and the linker expression cassette pCMV-HER2scFv-HveA-P2A-EpCAMscFv-HveA-bGHpA, which expresses both HER2scFv-HveA and EpCAM-HveA, contains P2A, which is 2A of porcine Teschovirus type 1 (P2A).

[0140] In this example, the full-length amino acid sequence and gene sequence of the HER2scFv-HveA linker (including the leader sequence) are represented in SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the full-length amino acid sequence and gene sequence of the EpCAMscFv-HveA (including the leader sequence) are represented in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0141] The HER2scFv-HveA linker expression cassette, EpCAMscFv-HveA linker expression cassette, and EpCAM-HveA-HER2scFv-HveA double linker expression cassette were inserted using the Counter Selection BAC Modification Kit (GeneBridges Inc.) according to the manufacturer's protocol, as described in Examples 1 and 2.

[0142] Specifically, the E. coli clone containing the KOS-37 / BACEmGFP-gD-R222N / F223I genome prepared in Example 2 was transformed with a pRed / ET plasmid expressing RecE and RecT, which are capable of performing homologous recombination functions (Muyrers JP et al.; Nucleic Acids Res., 1999. 27(6):1555-1557). A UL3 / 4-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer UL3 / 4-rpsL-neo_for: SEQ ID NO:31, reverse primer UL3 / 4-rpsL-neo_rev: SEQ ID NO:32), which included a site for introducing the target gene between UL3 and UL4. L-arabinose (Sigma-Aldrich) was added to a clone containing KOS-37 / BAC-EmGFP-gD-R222N / F223I and pRedET to induce homologous recombination, followed by transformation with 200 ng of the UL3 / 4-rpsL-neo / kan cassette prepared as described above. This homologous recombination allowed the UL3 / 4-rpsL-neo / kan cassette to be inserted into the UL3 / 4 locus of KOS-37 / BAC-EmGFP-gD-R222N / F223I E. coli. KOS-37 / BAC-EmGFP-gD-R222N / F223I E. coli harboring the UL3 / 4-rpsL-neo / kan exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. E. coli obtained from kanamycin culture medium were presumed to have been inserted with the UL3 / 4-rpsL-neo / kan, and the final step of inserting the target gene was performed.

[0143] L-arabinose (Sigma-Aldrich), which activates the pRed / ET function, was added to Escherichia coli containing the UL3 / 4-rpsL-neo / kan cassette to induce homologous recombination, and then transformed with 200 ng of each of the UL3 / 4-pCMV-HER2scFv-HveA-bGHpA cassette, UL3 / 4-pCMV-EpCAMscFv-HveA-bGHpA cassette, and UL3 / 4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA cassette. The UL3 / 4-pCMV-HER2scFv-HveA-bGHpA cassette, UL3 / 4-pCMV-EpCAMscFv-HveA-bGHpA cassette, and UL3 / 4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA cassette were prepared using the forward primer UL3 / 4_pCMV_For (SEQ ID NO: 33) and the reverse primer UL3 / 4_bGH_poly_R (SEQ ID NO: 34) with the pCDNA3.1-HER2scFv-HveA plasmid, pCDNA3.1-EpCAMscFv-HveA plasmid, and pCDNA3.1-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA as respective templates (Baek HJ et al., Mol. Ther., 2011. 19(3):507-514; Carter P. et al., Proc. Natl. Acad. Sci. USA. 1992, 15; 89(10):4285-9; Willuda J. et al., Cancer Res. 1999, 15; 59(22):5758-67).

[0144] When the conventionally inserted UL3 / 4-rpsL-neo / kan cassette was replaced with the aforementioned inserted UL3 / 4-pCMV-HER2scFv-HveA-bGHpA, UL3 / 4-pCMV-EpCAMscFv-HveA-bGHpA, and UL3 / 4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA, streptomycin resistance blocked by rpsL was activated. Based on this principle, candidates were selected in streptomycin medium (Heermann R. et al., Microb. Cell Fact., 2008. 14: doi: 10.1186). DNA was isolated from the selected candidates using a DNA preparation method (Horsburgh BC et al., Methods Enzymol., 1999. 306: 337-352). The introduction of UL3 / 4-pCMV-HER2scFv-HveA-bGHpA, UL3 / 4-pCMV-EpCAMscFv-HveA-bGHpA and UL3 / 4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA at UL3 / 4 was confirmed by treatment with restriction endonucleases EcoRI and XhoI and PCR (polymerase chain reaction), and the exact gene sequence was identified by sequencing of the PCR products.

[0145] The complete KOS-37 / BAC-UL3 / 4_HER2scFv-HveA-EmGFP-gD-R222N / F223I, KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD / R222N / F223I and KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-P2A-HER2scFv-HveA-EmGFP-gD / R222N / F223I DNAs were extracted using a large construct DNA purification kit (Macherey-Nagel), and then 2×10 cells / well were transfected with 1 μg of DNA using Lipofectamine 2000 reagent (Invitrogen). 5Cre-Vero-HVEM cells. Three days after transfection, the fluorescence expression of EmGFP protein and the formation of cell spots were observed using a fluorescence microscope. After confirming plaque formation, virus-containing cells were collected, subjected to three freeze-thaw processes (Gierasch W.W. et al.; J. Virol. Methods., 2006. 135: 197-206), and sonicated to finally obtain KOS-UL3 / 4_HER2scFv-HveA-EmGFP-gD-R222N / F223I virus (HADa-S) expressing HER2-targeting linker, KOS-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD / R222N / F223I virus (EADa-S) expressing EpCAM-targeting linker, and KOS-UL3 / 4_EpCAMscFv-HveA-P2A-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus (EADa-HADa-D) expressing HER2 / EpCAM dual targeting linker.

[0146] <Example 4> Production of HSV-1 virus with HER2-targeted modified glycoprotein gH and HSV-1 virus with EpCAM-targeted modified glycoprotein gH and HSV-1 virus expressing HER2-targeting linker

[0147] To produce a retargeted HSV capable of targeting a target molecule expressed in a specific cancer, a ligand (HER2 scFv) that recognizes HER2, which is specifically expressed in cancer cells, was inserted between amino acids 29 and 30 of the gH amino acid sequence (GenBank Accession No.: ASM47773, SEQ ID NO: 3). GH is a glycoprotein of HSV-1. A gene capable of expressing HER2 scFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_HER2scFv-HveA-EmGFP-gD / R222N / F223I DNA prepared in Examples 2 and 3.

[0148] Figure 7 The genomic structures of the KOS-gH / HER2scFv-EmGFP-gD / R222N / F223I virus (HgH-S) and the KOS-UL3 / 4_HER2scFv-HveA-gH / HER2scFv-EmGFP-gD / R222N / F223I virus (HADa-HgH-D) are shown, in which the HER2scFv ligand is inserted into the gH of HSV-1. Figure 8The complete sequence of the gH-HER2 scFv ligand and the construction of the corresponding sequence are shown. Here, the HER2 scFv was constructed such that the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 were linked via a linker peptide of SEQ ID NO: 24, a linker peptide of SEQ ID NO: 35 was linked to the N-terminus of the scFv, and a linker peptide of SEQ ID NO: 36 was linked to its C-terminus.

[0149] The full-length amino acid sequence and gene sequence of HER2 scFv used in this example are shown in SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0150] Insertion of the gH-HER2 scFv ligand was performed using the Counter Selection BAC Modification Kit (GeneBridges Inc.) according to the manufacturer's protocol, as described in Examples 1, 2, and 3.

[0151] Specifically, the E. coli clones containing KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_HER2scFv-HveA-EmGFP-gD-R222N / F223I DNA prepared in Examples 2 and 3 were transformed with pRed / ET plasmids expressing RecE and RecT, which are capable of performing homologous recombination functions (Muyrers JP et al.; Nucleic Acids Res., 1999. 27(6):1555-1557). A gH29 / 30-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer gH29 / 30-rpsL-neo_for: SEQ ID NO: 39, reverse primer gH29 / 30-rpsL-neo_rev: SEQ ID NO: 40), which includes a site for introducing the target gene between amino acids 29 and 30 of gH. L-arabinose (Sigma-Aldrich) was added to each clone containing KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_HER2scFv-HveA-EmGFP-gD-R222N / F223I DNA and pRed / ET to induce homologous recombination, and then transformed with 200 ng of the gH29 / 30-rpsL-neo / kan cassette prepared as described above. Through this homologous recombination, the gH29 / 30-rpsL-neo / kan cassette was inserted between amino acids 29 and 30 of gH E. coli. The gH E. coli harboring the gH29 / 30-rpsL-neo / kan cassette exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. For E. coli obtained from a kanamycin culture, it was assumed that gH29 / 30-rpsL-neo / kan had been inserted, and the final step of inserting the target gene was performed.

[0152] E. coli containing the gH29 / 30-rpsL-neo / kan cassette was treated with L-arabinose (Sigma-Aldrich), which activates pRed / ET function, to induce homologous recombination, and then transformed with 200 ng of the gH29 / 30-HER2scFv ligand. The gH29 / 30-HER2scFv ligand was prepared using the forward primer gH29 / 30-scFv_For (SEQ ID NO: 41) and the reverse primer gH29 / 30-scFv_Rev (SEQ ID NO: 42) using the pCAGGSMCS-gH-HER2scFv plasmid as a template. The pCAGGSMCS-gH-HER2scFv plasmid was prepared by inserting HER2scFv into the pCAGGSMCS plasmid (Atanasiu D. et al., J. Virol., November 2013, 87(21):11332-11345), specifically, by treating the pCAGGSMCS plasmid and HER2scFv amplified by PCR (Carter P. et al., Proc. Natl. Acad. Sci. USA. 1992, 15;89(10):4285-9) with NotI restriction endonuclease (NEB, R3189), and ligating the NotI-cleaved pCAGGSMCS plasmid and HER2scFv using T4 DNA ligase (NEB, M0202).

[0153] When the conventionally inserted gH29 / 30-rpsL-neo / kan cassette was replaced with the gH29 / 30-HER2 scFv inserted as described above, streptomycin resistance blocked by rpsL was activated. Based on this principle, candidates were selected in streptomycin medium (Heermann R. et al., Microb. Cell Fact., 2008.14:doi:10.1186). DNA was isolated from selected candidates using a DNA preparation method (Horsburgh BC et al., Methods Enzymol., 1999.306:337-352). The introduction of the HER2 scFv at the gH29 / 30 site was confirmed by treatment with the restriction endonucleases EcoRI and XhoI and PCR (polymerase chain reaction), and the exact gene sequence was identified by sequencing the PCR product.

[0154] The complete KOS-37 / BAC-gH / HER2scFv-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_HER2scFv-HveA-gH / HER2scFv-EmGFP-gD-R222N / F223I DNA were extracted using a large construct DNA purification kit (Macherey-Nagel), and then 2×10 cells / ... 5 Cre-Vero-HVEM cells were used to remove the BAC gene using the Cre recombinase. After 3 days of transfection, the fluorescence expression of EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirming that plaques were formed, virus-containing cells were collected and subjected to three freeze-thaw cycles (Gierasch WW et al.; J. Virol. Methods., 2006. 135: 197-206), and ultrasonic treatment was performed to finally obtain KOS-gH / HER2scFv-EmGFP-gD-R222N / F223I virus (HgH-S) and KOS-UL3 / 4_HER2scFv-HveA-gH / HER2scFv-EmGFP-gD-R222N / F223I virus (HADa-HgH-D).

[0155] <Example 5> Production of HSV-1 virus with EpCAM-targeted modified glycoprotein gH and HSV-1 virus with EpCAM-targeted modified glycoprotein gH and HSV-1 virus expressing EpCAM-targeting linker

[0156] To produce a retargeted HSV capable of targeting a target molecule expressed in specific cancers, a ligand (EpCAM scFv) that recognizes EpCAM specifically expressed in cancer cells was inserted between amino acids 29 and 30 of the gH amino acid sequence (GenBank Accession No.: ASM47773, SEQ ID NO: 3). GH is a glycoprotein of HSV-1. The gene capable of expressing EpCAM scFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_EpCAM scFv-HveA-EmGFP-gD / R222N / F223I (EADa-S) DNA prepared in Examples 2 and 3.

[0157] Figure 9The genome structures of KOS-gH / EpCAMscFv-EmGFP-gD / R222N / F223I virus (EgH-S) and KOS-UL3 / 4_EpCAMscFv-HveA-gH / EpCAMscFv-EmGFP-gD / R222N / F223I virus (EADa-EgH-D) are shown, in which the EpCAMscFv ligand is inserted into the gH of HSV-1. Figure 10 The complete sequence of the gH-EpCAM scFv ligand and the construction of the corresponding sequence are shown. Here, the EpCAM scFv was constructed such that the VL of SEQ ID NO: 6 and the VH of SEQ ID NO: 7 were linked via a linker peptide of SEQ ID NO: 25, a linker peptide of SEQ ID NO: 43 was linked to the N-terminus of the scFv, and a linker peptide of SEQ ID NO: 44 was linked to its C-terminus.

[0158] The amino acid sequence and gene sequence of EpCAMscFv used in this example are shown in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0159] Insertion of the gH-EpCAM scFv ligand was performed using the Counter Selection BAC Modification Kit (GeneBridges Inc.) according to the manufacturer's protocol, as described in Example 4.

[0160] Specifically, the E. coli clones containing KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I DNA prepared in Examples 2 and 3 were transformed with pRed / ET plasmids expressing RecE and RecT, which are capable of performing homologous recombination functions (Muyrers JP et al.; Nucleic Acids Res., 1999. 27(6):1555-1557). A gH29 / 30-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer gH29 / 30-rpsL-neo_for: SEQ ID NO: 40, reverse primer gH29 / 30-rpsL-neo_rev: SEQ ID NO: 41), which includes a site for introducing the target gene between amino acids 29 and 30 of gH. L-arabinose (Sigma-Aldrich) was added to each clone containing KOS-37 / BAC-EmGFP-gD-R222N / F223I DNA and KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I DNA and pRed / ET to induce homologous recombination, and then transformed with 200 ng of the gH29 / 30-rpsL-neo / kan cassette prepared as described above. Through this homologous recombination, the gH29 / 30-rpsL-neo / kan cassette was inserted between amino acids 29 and 30 of gH E. coli. gH E. coli harboring the gH29 / 30-rpsL-neo / kan gene exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. For E. coli obtained from a kanamycin culture, it was assumed that gH29 / 30-rpsL-neo / kan had been inserted, and the final step of inserting the target gene was performed.

[0161] E. coli containing the gH29 / 30-rpsL-neo / kan cassette was treated with L-arabinose (Sigma-Aldrich), which activates pRed / ET function, to induce homologous recombination, and then transformed with 200 ng of gH29 / 30-EpCAMscFv ligand. The gH29 / 30-EpCAMscFv ligand was prepared using the forward primer gH29 / 30-scFv_For (SEQ ID NO: 42) and the reverse primer gH29 / 30_scFv_Rev (SEQ ID NO: 43) using the pCAGGSMCS-gH-EpCAMscFv plasmid as a template. The pCAGGSMCS-gH-EpCAMscFv plasmid was prepared by inserting EpCAMscFv into the pCAGGSMCS plasmid (Atanasiu D. et al., J. Virol., November 2013, 87(21):11332-11345), specifically, by treating the pCAGGSMCS plasmid and EpCAMscFv amplified by PCR (Willuda J. et al., Cancer Res. 1999, 15;59(22):5758-67) with NotI restriction endonuclease (NEB, R3189), and ligating the NotI-cleaved pCAGGSMCS plasmid and EpCAMscFv using T4 DNA ligase (NEB, M0202).

[0162] When the conventionally inserted gH29 / 30-rpsL-neo / kan cassette was replaced with the gH29 / 30-EpCAMscFv inserted as described above, streptomycin resistance blocked by rpsL was activated. Based on this principle, candidates were selected in streptomycin medium (Heermann R. et al., Microb. Cell Fact., 2008.14:doi:10.1186). DNA was isolated from the selected candidates using a DNA preparation method (Horsburgh BC et al., Methods Enzymol., 1999.306:337-352). The introduction of EpCAMscFv into the gH29 / 30 region was confirmed by treatment with the restriction endonucleases EcoRI and XhoI and PCR (polymerase chain reaction), and the exact gene sequence was identified by sequencing the PCR product.

[0163] The complete KOS-37 / BAC-gH_EpCAMscFv-EmGFP-gD-R222N / F223I and KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-gH / EpCAMscFv-EmGFP-gD-R222N / F223I DNAs were extracted using a large construct DNA purification kit (Macherey-Nagel), and then 2 × 10 cells / well were transfected with 1 μg of DNA using Lipofectamine 2000 reagent (Invitrogen). 5 Cre-Vero-HVEM cells were transfected to remove the BAC gene using Cre recombinase. After 2 days of transfection, the fluorescence expression of EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirming the formation of plaques, virus-containing cells were collected and subjected to three freeze-thaw cycles (Gierasch WW et al.; J. Virol. Methods., 2006. 135: 197-206), and ultrasonic treatment was performed to finally obtain KOS-gH / EpCAMscFv-EmGFP-gD-R222N / F223I virus (EgH-S) and KOS-gH / EpCAMscFv-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I virus (EADa-EgH-D).

[0164] <Example 6> Measuring the activity of HER2 dual-targeting oncolytic virus

[0165] The following experiments were performed to confirm the expression level of the HER2scFv-HveA linker, as well as the viral propagation and amplification of the KOS-UL3 / 4_HER2scFv-HveA-EmGFP-gD-R222N / F223I virus (HADa-S) expressing the HER2scFv-HveA linker, the KOS-gH / HER2scFv-EmGFP-gD-R222N / F223I virus (HgH-S) expressing the HER2scFv in gH, and the UL3 / 4_HER2scFv-HveA-gH / HER2scFv-EmGFP-gD-R222N / F223I dual-targeting virus expressing the HER2scFv-HveA linker and HER2scFv in gH (HADa-HgH-D), prepared in Examples 3 and 4.

[0166] For virus proliferation experiments, 2.0×10 5 Vero-HVEM cells and SK-OV-3 cells were plated on 12-well plates.

[0167] HSV-1 wild-type virus (KOS), the virus gDm expressing the fluorescent protein EmGFP prepared in Example 2 and using only HVEM as a cell receptor for cell entry, the virus HADa-S expressing the HER2scFv-HveA linker prepared in Example 3, the virus HgH-S with a HER2scFv ligand in gH prepared in Example 4, and the dual-targeting virus HADa-HgH-D with a HER2scFv ligand in gH and expressing the HER2scFv-HveA linker prepared in Example 4 were diluted and used for infection so that 20 to 50 viruses were contained in one well. After 90 minutes, in order to remove the remaining initial virus and prevent virus proliferation, the culture medium used was replaced with a culture medium containing 0.2% methylcellulose. After 3 days, virus proliferation was measured by the size of the virus plaque using a fluorescence microscope.

[0168] The results are as follows Figure 11 As shown. Figure 11 It was clearly shown that in the Vero-HVEM cell line, the plaque sizes of gDm, HgH-S, HADa-S, and HADa-HgH-D were 18%, 49%, 4%, and 29% smaller than those of the wild-type virus (KOS), respectively. In SK-OV-3 cells, a HER2-expressing cell line, the plaque size of HgH-S decreased by 53% compared to the wild-type virus (KOS), while that of HADa-S and HADa-HgH-D increased by 20% and 19%, respectively. The reduction in HgH-S plaque size is believed to be due to the fact that when gD binds to the entry receptor, gH transmits an activation signal to gB through this binding, thereby inducing cell fusion and inducing endocytosis by binding to integrins. Therefore, it is suspected that viral proliferation or replication is inhibited by affecting this signaling or endocytosis, which is due to the structural changes caused by the insertion of the scFv into gH.

[0169] For virus replication experiments, 1.0 × 10 4 Vero-HVEM cells were plated onto 96-well plates. gDm, HgH-S expressing a HER2 scFv ligand in gH, HADa-S expressing a HER2 scFv-HveA linker, and the dual-targeting HADa-HgH-D viruses were used for infection at an MOI of 0.1. After 90 minutes, the culture medium was replaced with fresh medium to remove any remaining initial virus. Viral cultures were harvested 3, 24, and 48 hours after infection, and the amount of virus in the culture medium was measured.

[0170] The results are shown in Figure 12 In. From Figure 12It can be clearly seen that although the virus proliferation activities of gDm, HADa-S, and HADa-HgH-D were similar in the Vero-HVEM cell line, it was confirmed that the virus proliferation activity in HgH-S was reduced due to the decreased virus proliferation ability, e.g. Figure 11 The results are shown.

[0171] To perform experiments to determine linker expression levels, 2.0 × 10 5 Vero-HVEM and SK-OV-3 cells were plated onto 12-well plates. gDm, HgH-S expressing the HER2 scFv ligand in gH, HADa-S expressing the HER2 scFv-HveA linker, and the dual-targeting HADa-HgH-D viruses were then used for infection at an MOI of 0.1. After 90 minutes, the culture medium was replaced with fresh medium without FBS to remove any remaining initial virus. 48 hours after infection, viral cultures were harvested and protein expression levels were determined by Western blotting to measure linker expression in the culture medium.

[0172] The results are as follows Figure 13 As shown. Figure 13 It is clear that in the Vero-HVEM cell line, the HADa-HgH-D dual-targeting virus expressed at least three times more linker than the HADa-S virus, which only expressed the linker. However, no linker was measured in the gDm and HgH-S virus cultures without the linker. In HER2-expressing SK-OV-3 cells, only the linker was detected for the HADa-HgH-D dual-targeting virus. This is because the HADa-HgH-D dual-targeting virus exhibits a higher infection rate in HER2-expressing cell lines than the HADa-S virus, resulting in proportionally higher linker expression.

[0173] Therefore, the HADa-HgH-D dual-targeting virus was confirmed to have relatively improved viral proliferation and amplification, as well as linker expression levels, compared with other viruses.

[0174] <Example 7> Infection and cytotoxicity of HER2-expressing cancer cells using HER2 dual-targeting oncolytic viruses

[0175] Experiments were conducted in HER2-expressing cancer cell lines using the gDm prepared in Examples 2, 3, and 4, HgH-S expressing a HER2scFv ligand in gH, HADa-S expressing a HER2scFv-HveA linker, and a dual-targeting HADa-HgH-D virus expressing a HER2scFv-HveA linker and a HER2scFv ligand in gH. To confirm whether each virus induces viral infection into surrounding cancer cells due to the HER2scFv ligand expressed in the glycoprotein gH or due to the linker, and whether it induces cytotoxicity after infection, the following experiments were performed.

[0176] The cell lines used in the experiment were a cell line that does not express HER2 (MDA-MB-231) and a cell line that expresses HER2 (SK-OV-3, MCF-7, MDA-MB-453, and BT-474). For breast cancer cell lines MDA-MB-231 (ATCC, HTB-26), MCF-7 (ATCC, HTB-22), and BT-474 (ATCC, HTB-20), and ovarian cancer cell line SK-OV-3 (ATCC, HTB-77), DMEM containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS was used for culture, and for breast cancer cell line MDA-MB-453 (ATCC, HTB-131), RPMI1640 medium containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS was used for culture.

[0177] For HER2-specific viral infection experiments, 2 MOl of 8 × 10 3 SK-0V-3 and MDA-MB-231, 4.0×10 4 MCF-7, 8.0×10 4 MDA-MB-453 and 7.0×10 4BT-474 cell lines were infected with HgH-S expressing a HER2scFv ligand in gH, HADa-S expressing a HER2scFv-HveA linker, dual-targeting HADa-HgH-D virus, and a HER2-non-targeting gDm virus as a control. After 90 minutes, the culture medium was replaced with fresh culture medium to remove the remaining initial virus. After 2 days of infection, viral infection was confirmed by EmGFP fluorescence expression in each cell line (Baek HJ et al., Mol. Ther. 2011. 19 (3): 507-514). In addition, to measure cytotoxicity 4 days after infection, an ELISA reader was used and the color development of formazan was measured at 450 nm using EZ-Cytox (DoGenBio) reagent. Formazan is a color-developing material formed only in living cells. The absorbance was quantified to determine the cytotoxicity of each virus against the cancer cell line.

[0178] The results are as follows Figure 14 As shown. Figure 14 It can be clearly seen that the HADa-S virus expressing the linker has a high infection rate in SK-OV-3 and MCF7 cells, but a low infection rate is observed in MDA-MB-453 and BT-474. The HgH-S virus expressing the HER2 scFv ligand in gH has a high infection rate in SK-OV-3, MCF7 and MDA-MB-453 cells, but a low infection rate is observed in BT-474. Unlike the viruses expressing each of the ligand and the linker, the dual-targeting HADa-HgH-D virus was observed to exhibit a high infection rate in all cells expressing HER2. However, the gDm virus did not infect cancer cell lines because it does not target HER2, and as a control, no infection of any virus was observed in MDA-MB-231 cells that do not express HER2. The low infection rates of MDA-MB-453 and BT-474 are believed to be due to the cell morphology and characteristics of MDA-MB-453 and BT-474 and the initial low infection rate of the HADa-S virus.

[0179] also, Figure 15The results of observing the cytotoxicity of cancer cells caused by the virus 4 days after infection are shown. HgH-S, HADa-S and HADa-HgH-D viruses showed cell viability values ​​of 41%, 27% and 25% in SK-OV-3, 61%, 52% and 39% in MCF-7, and 49%, 100% and 23% in MDA-MB-453. Among the three cell lines expressing HER2, the highest cytotoxicity was observed to be caused by infection with the dual-targeting HADa-HgH-D virus. However, since the gDm virus does not target HER2, no cytotoxicity was observed, and MDA-MB-231, which does not express HER2, cannot be infected, so it was observed that the three viruses were not involved in cytotoxicity. The reason for the lack of effect on the viability of MDA-MB-453 cells seems to be that the initial infection rate of the HADa-S virus is very low compared to the other viruses.

[0180] <Example 8> Inhibitory effect of HER2 dual-targeting oncolytic virus on mouse tumor cell growth

[0181] To confirm whether the dual-targeting HADa-HgH-D virus prepared in Example 4 induces inhibition of the growth of HER2-expressing cancer cells in mice, the following experiment was performed.

[0182] SK-OV-3 was added at 5×10 6 The cells / mouse were subcutaneously injected into 5-week-old Balb / c nude mice (Orient Bio), and the tumors were observed until they reached a size of 100 mm. 3 The HER2 dual-targeting HADa-HgH-D virus was injected into the 7 pfu / mouse were injected intratumorally into 5 mice bearing tumors, and PBS was injected into 5 mice as a control. The size of the tumors developed in the mice was observed for 28 days after virus injection.

[0183] The results are shown in Figure 16 Figure 28 shows the tumor size at day 28. In the PBS control group, the tumor size increased from 116.46 ± 11.21 mm at the initial stage. 3 Grow to 815.28±141.36mm 3 In mice injected with HER2 dual-targeting HADa-HgH-D virus, tumor size was observed to increase from 108.85±15.54mm 3 Grow to 110.02±55.44mm 3 , which was considered to be inhibited compared with the control.

[0184] <Example 9> Infection and cytotoxicity of EpCAM-expressing cancer cells using EpCAM dual-targeting oncolytic viruses

[0185] Experiments were performed using the EADa-S virus expressing the EpCAMscFv-HveA linker prepared in Examples 3 and 5, the EgH-S virus expressing the EpCAMscFv ligand in gH, and the dual-targeting EADa-EgH-D virus expressing the EpCAMscFv-HveA linker and the EpCAMscFv ligand in gH.

[0186] To confirm whether each virus induced viral infection into surrounding cancer cells due to the EpCAMscFv ligand expressed in glycoprotein gH or due to the linker, and its cytotoxicity after induction of infection, the following experiments were performed.

[0187] The cell lines used in the experiment were a cell line that does not express EpCAM (Mia-PaCa-2) and a cell line that expresses EpCAM (MCF-7, MDA-MB-453, and BT-474). For the breast cancer cell lines MCF-7 (ATCC, HTB-22) and BT-474 (ATCC, HTB-20) and the pancreatic cancer cell line Mia-PaCa-2 (ATCC, CRL-1420), DMEM containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS was used for culture. For the breast cancer cell line MDA-MB-453 (ATCC, HTB-131), RPMI medium containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS was used for culture.

[0188] For EpCAM-specific viral infection experiments, 4.0 × 10 4 MCF-7, 8.0×10 4 MDA-MB-453 and 7.0×10 4BT-474 cell lines were infected with the EADa-S virus expressing the EpCAMscFv-HveA linker prepared in Example 3, the EgH-S virus expressing the EpCAMscFv ligand in gH prepared in Example 4, the dual-targeting EADa-EgH-D virus expressing both the linker and the ligand, and the HER2-non-targeting gDm virus prepared in Example 2 as a control. After 90 minutes, the culture medium was replaced with fresh culture medium to remove the remaining initial virus. Two days after infection, viral infection was confirmed by the expression of EmGFP fluorescence in each cell line (Baek HJ et al., Mol. Ther., 2011. 19(3): 507-514). In addition, to measure cell death at day 5, the cytotoxicity of the cancer cell lines caused by each virus after treatment with the EZ-Cytox (DogenBio) reagent was observed using a fluorescence microscope. In addition, to measure cytotoxicity 5 days after infection, an ELISA reader was used, and the EZ-Cytox (DoGenBio) reagent was used to measure the degree of color development of formazan, a chromogenic material formed only in living cells, at 450 nm. The absorbance was quantified to determine the cytotoxicity of each virus against the cancer cell lines.

[0189] The results are shown in Figure 17 In. From Figure 17 It can be clearly seen by fluorescence that BT-474, MDA-MB-453, and MCF7 cells were infected by all of the EADa-S virus expressing only the EpCAM-HveA linker, the EgH-S virus expressing the EpCAM scFv ligand in gH, and the dual-targeting EADa-EgH-D virus expressing both the linker and the ligand. It was also observed that the dual-targeting EADa-EgH-D virus exhibited a high infection rate compared to the EADa-S and EgH-S viruses. Since the gDm virus does not target EpCAM, cancer cell lines were not infected, and no virus infection was observed in Mia-PaCa-2 cells, which do not express EpCAM.

[0190] also, Figure 18The results of observing the cytotoxicity of cancer cells caused by the virus 5 days after infection are shown. EgH-S, EADa-S, and EADa-EgH-D viruses showed cell viability values ​​of 35%, 34%, and 26% in BT-474, 22%, 19%, and 17% in MDA-MB-453, and 36%, 31%, and 20% in MCF-7, respectively. Among the three cell lines expressing EpCAM, the highest cytotoxicity was observed due to infection with the EADa-EgH-D dual-targeting virus. However, since the gDm virus does not target EpCAM, no cytotoxicity was observed, and Mia-PaCa-2, which does not express EpCAM, was not infected, so no virus was involved in cytotoxicity.

[0191] <Example 10> Production of HSV-1 with HER2-targeting modified glycoprotein gH and expression of EpCAM-targeting linker

[0192] To generate an HSV capable of dual-targeting two target molecules (HER2 / EpCAM) expressed in specific cancers, a ligand (HER2 scFv) that recognizes HER2 specifically expressed in cancer cells was inserted between amino acids 29 and 30 of the gH amino acid sequence (GenBank Accession No.: ASM47773, SEQ ID NO: 35). gH is a glycoprotein of the KOS-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I (EADa-S) virus expressing the EpCAMscFv-HveA linker.

[0193] The gene capable of expressing HER2 scFv was inserted between amino acids 29 and 30 of glycoprotein gH in the KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD / R222N / F223I(EADa-S) DNA prepared in Example 3.

[0194] Figure 19 The genome structure of the KOS-UL3 / 4_EpCAMscFv-HveA-gH / HER2scFv-EmGFP-gD / R222N / F223I virus is shown, in which the HER2scFv ligand is inserted into the gH of HSV-1. Figure 8The complete sequence of the gH-HER2 scFv ligand and the construction of the corresponding sequence are shown. Here, the HER2 scFv is constructed such that the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 are linked via a linker peptide of SEQ ID NO: 24, a linker peptide of SEQ ID NO: 36 is linked to the N-terminus of the scFv, and a linker peptide of SEQ ID NO: 37 is linked to its C-terminus.

[0195] The full-length amino acid sequence and gene sequence of HER2 scFv used in this example are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0196] Insertion of the gH-HER2 scFv ligand was performed using the Counter Selection BAC Modification Kit (GeneBridges Inc.) according to the manufacturer's protocol, as described in Example 4.

[0197] Specifically, the E. coli clone containing the KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I DNA prepared in Example 3 was transformed with a pRed / ET plasmid expressing RecE and RecT, which are capable of performing homologous recombination (Muyrers JP et al.; Nucleic Acids Res., 1999. 27(6):1555-1557). A gH29 / 30-rpsL-neo / kan cassette was prepared using a set of homology region primers (forward primer gH29 / 30-rpsL-neo_for: SEQ ID NO:40, reverse primer gH29 / 30-rpsL-neo_rev: SEQ ID NO:41), which include a site for introducing the target gene between amino acids 29 and 30 of gH.

[0198] L-arabinose (Sigma-Aldrich) was added to a clone containing KOS-37 / BAC-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I DNA and pRed / ET to induce homologous recombination, followed by transformation with 200 ng of the gH29 / 30-rpsL-neo / kan cassette prepared as described above. This homologous recombination inserted the gH29 / 30-rpsL-neo / kan cassette between amino acids 29 and 30 of the gH E. coli. E. coli harboring the gH29 / 30-rpsL-neo / kan cassette exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. For E. coli obtained from kanamycin culture, it was assumed that gH29 / 30-rpsL-neo / kan was inserted, and the final step of inserting the target gene was performed.

[0199] E. coli containing the gH29 / 30-rpsL-neo / kan cassette was treated with L-arabinose (Sigma-Aldrich), which activates pRed / ET function, to induce homologous recombination, and then transformed with 200 ng of gH29 / 30-HER2scFv ligand. The gH29 / 30-HER2scFv ligand was prepared using the forward primer gH29 / 30-scFv_For (SEQ ID NO: 42) and the reverse primer gH29 / 30scFv_Rev (SEQ ID NO: 43) using the pCAGGSMCS-gH-HER2scFv plasmid as a template.

[0200] When the conventionally inserted gH29 / 30-rpsL-neo / kan cassette was replaced with the gH29 / 30-HER2 scFv inserted as described above, streptomycin resistance blocked by rpsL was activated. Based on this principle, candidates were selected in streptomycin medium (Heermann R. et al., Microb. Cell Fact., 2008.14:doi:10.1186). DNA was isolated from selected candidates using a DNA preparation method (Horsburgh BC et al., Methods Enzymol., 1999.306:337-352). The introduction of the HER2 scFv at the gH29 / 30 site was confirmed by treatment with the restriction endonucleases EcoRI and XhoI and PCR (polymerase chain reaction), and the exact gene sequence was identified by sequencing the PCR product.

[0201] The complete KOS-37 / BAC-gH / HER2scFv-UL3 / 4_EpCAMscFv-HveA-EmGFP-gD-R222N / F223I DNA was extracted using a large construct DNA purification kit (Macherey-Nagel), and then 2×10 cells / well were transfected with 1 μg of DNA using Lipofectamine 2000 reagent (Invitrogen). 5 Cre-Vero-HVEM cells were transfected to remove the BAC gene using Cre recombinase. Three days after transfection, the fluorescence expression of the EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirming plaque formation, the virus-containing cells were collected and subjected to three freeze-thaw cycles (Gierasch W et al.; J. Virol. Methods., 2006. 135: 197-206), and ultrasonic treatment was performed to finally obtain the KOS-UL3 / 4_EpCAMscFv-HveA-gH_HER2scFv-EmGFP-gD-R222N / F223I dual-targeting virus (EADa-HgH-D) expressing the EpCAMscFv-HveA linker and the HER2scFv ligand in gH.

[0202] <Example 11> Experiment on dual targeting of HSV-1 with HER2-targeting modified glycoprotein gH and expressing EpCAM-targeting linker

[0203] In order to confirm that the KOS-UL3 / 4_EpCAMscFv-HveA-gH / HER2scFv-EmGFP-gD-R222N / F223I dual-targeting virus (EADa-HgH-D) expressing the EpCAMscFv-HveA linker and HER2scFv ligand in gH prepared in Example 10 can induce infection of cells expressing HER2 and EpCAM proteins, the following experiments were performed.

[0204] The cell lines used in the experiment were a cell line that does not express HER2 and EpCAM (CHO-K1), a cell line that expresses HER2 (CHO-HER2), and a cell line that expresses EpCAM (CHO-EpCAM). Chinese hamster ovary cell lines CHO-K1, CHO-HER2, and CHO-EpCAM were cultured in Ham's F-12K medium (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum) (Kuroki M. et al., J Biol. Chem., 1991, 74: 10132-10141).

[0205] For specific viral infection, 2.5 × 10 4 CHO-K1, CHO-HER2, and CHO-EpCAM cell lines were plated on 96-well plates. 24 hours later, the HER2 dual-targeting virus (HADa-HgH-D) prepared in Example 4, the EpCAM dual-targeting virus (EADa-EgH-D) prepared in Example 5, and the HER2 / EpCAM dual-targeting virus (EADa-HgH-D) prepared in Example 10 were used for infection at an MOI of 5. After 90 minutes, the culture medium was replaced with fresh culture medium to remove any remaining initial virus. Two days after infection, viral infection was observed by fluorescence expression in each cell line (Baek HJ et al., Mol. Ther., 2011. 19(3): 507-514).

[0206] The results are shown in Figure 20 In. From Figure 20 As can be clearly seen, fluorescence microscopy images of cell lines infected with the virus are shown. It was observed that no virus infected the CHO-K1 cell line that does not express HER2 and EpCAM. The HER2 dual-targeting virus (HADa-HgH-D) only infected CHO-HER2, and the EpCAM dual-targeting virus (EADa-EgH-D) only infected CHO-EpCAM. However, it was confirmed that the HER2 / EpCAM dual-targeting virus (EADa-HgH-D) infected all CHO-HER2 and CHO-EpCAM cells. Based on the above results, the possibility of a strategy of targeting at least two target molecules using a virus that can target two target molecules together can be confirmed.

[0207] <Example 12> Experiment on Dual Targeting of HSV-1 Expressing HER2 / EpCAM Dual Targeting Linker

[0208] To confirm that the HER2 and EpCAM dual-targeting virus (EADA-hada-D) expressing both the EpCAMscFv-HveA linker and HER2scFv-HveA prepared in Example 3 induced infection of cells expressing HER2 and EpCAM proteins, the following experiment was performed.

[0209] The cell lines used in the experiment were a cell line that does not express HER2 and EpCAM (CHO-K1), a cell line that expresses HER2 (CHO-HER2), and a cell line that expresses EpCAM (CHO-EpCAM). Chinese hamster ovary cell lines CHO-K1, CHO-HER2, and CHO-EpCAM were cultured in Ham's F-12K medium (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum) (Kuroki M. et al., J Biol. Chem., 1991, 74: 10132-10141).

[0210] For specific viral infection, 2.5 × 10 4 CHO-K1, CHO-HER2, and CHO-EpCAM cell lines were plated on 96-well plates. 24 hours later, the virus expressing only the HER2 scFv-HveA linker (HADa-S), the virus expressing only the EpCAM scFv-HveA linker (EADa-S), and the dual-targeting virus expressing both the EpCAM scFv-HveA linker and the HER2 scFv-HveA linker (EADa-HADa-D) prepared in Example 3 were used for infection at an MOI of 5. After 90 minutes, the culture medium was replaced with fresh culture medium to remove any remaining initial virus and linker. Two days after infection, viral infection was observed by fluorescence expression in each cell line using a fluorescence microscope (Baek HJ et al., Mol. Ther., 2011. 19(3):507-514).

[0211] result, Figure 21 Fluorescence microscopy images of cell lines infected with various viruses are shown. No virus was observed to infect the CHO-K1 cell line, which does not express HER2 and EpCAM. A virus expressing only the HER2 scFv-HveA linker (HADa-S) infected only CHO-HER2, while a virus expressing only the EpCAM scFv-HveA linker (EADa-S) infected only CHO-EpCAM. However, a dual-targeting virus expressing both the EpCAM scFv-HveA linker and the HER2 scFv-HveA linker (EADa-HADa-D) was confirmed to infect all CHO-HER2 and CHO-EpCAM cells.

[0212] Based on the above results, the possibility of a strategy for targeting at least two target molecules using a virus capable of targeting two target molecules together was confirmed. <110> Giselemed Co., Ltd. (KOREA, Gencellmed) <120> A fusion protein containing a tumor cell targeting domain and the extracellular domain of HVEM Recombinant herpes simplex virus expressing cassette and its use (Recombinant Herpes Simplex Virus Containing Expression Cassette Expressing a Fused Protein of Tumor Cell-targeting Domain and Extracellular Damain of HVEM and Use of the Same) <130> PP20-000 <160> 46 <170> KoPatentIn 3.0 <210> 1 <211> 904 <212> PRT <213> Artificial Sequence <220> <223> gB <400> 1 Met His Gln Gly Ala Pro Ser Trp Gly Arg Arg Trp Phe Val Val Trp 1 5 10 15 Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro 20 25 30 Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn 35 40 45 Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr 50 55 60 Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro 65 70 75 80 Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr 85 90 95 Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn 100 105 110 Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu 115 120 125 Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu 130 135 140 Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys 145 150 155 160 Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly 165 170 175 His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val 180 185 190 Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg 195 200 205 Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His 210 215 220 Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala 225 230 235 240 Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro 245 250 255 Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile 260 265 270 Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val 275 280 285 Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg 290 295 300 Glu Gly Ser His Thr Glu His Thr Ser Tyr Thr Ala Asp Arg Phe Lys 305 310 315 320 Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala 325 330 335 Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val 340 345 350 Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys 355 360 365 Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe 370 375 380 Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr 385 390 395 400 Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp 405 410 415 Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr 420 425 430 His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe 435 440 445 Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr 450 455 460 Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr 465 470 475 480 Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys 485 490 495 Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His 500 505 510 Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp 515 520 525 Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys 530 535 540 Leu Asn Pro Asn Ala Ile Ala Ser Val Thr Val Gly Arg Arg Val Ser 545 550 555 560 Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val 565 570 575 Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg 580 585 590 Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp 595 600 605 Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg 610 615 620 Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr 625 630 635 640 Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser 645 650 655 His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp 660 665 670 Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val 675 680 685 Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu 690 695 700 Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp 705 710 715 720 Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly 725 730 735 Ala Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val 740 745 750 Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser 755 760 765 Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val 770 775 780 Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg 785 790 795 800 Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu 805 810 815 Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly 820 825 830 Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Lys 850 855 860 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp Met Val 865 870 875 880 Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp 885 890 895 Gly Asp Ala Asp Glu Asp Asp Leu 900 <210> 2 <211> 511 <212> PRT <213> Artificial Sequence <220> <223> gC <400> 2 Met Ala Pro Gly Arg Val Gly Leu Ala Val Val Leu Trp Ser Leu Leu 1 5 10 15 Trp Leu Gly Ala Gly Val Ala Gly Gly Ser Glu Thr Ala Ser Thr Gly 20 25 30 Pro Thr Ile Thr Ala Gly Ala Val Thr Asn Ala Ser Glu Ala Pro Thr 35 40 45 Ser Gly Ser Pro Gly Ser Ala Ala Ser Pro Glu Val Thr Pro Thr Ser 50 55 60 Thr Pro Asn Pro Asn Asn Val Thr Gln Asn Lys Thr Thr Pro Thr Glu 65 70 75 80 Pro Ala Ser Pro Pro Thr Thr Pro Lys Pro Thr Ser Thr Pro Lys Ser 85 90 95 Pro Pro Thr Ser Thr Pro Asp Pro Lys Pro Lys Asn Asn Thr Thr Pro 100 105 110 Ala Lys Ser Gly Arg Pro Thr Lys Pro Pro Gly Pro Val Trp Cys Asp 115 120 125 Arg Arg Asp Pro Leu Ala Arg Tyr Gly Ser Arg Val Gln Ile Arg Cys 130 135 140 Arg Phe Arg Asn Ser Thr Arg Met Glu Phe Arg Leu Gln Ile Trp Arg 145 150 155 160 Tyr Ser Met Gly Pro Ser Pro Pro Ile Ala Pro Ala Pro Asp Leu Glu 165 170 175 Glu Val Leu Thr Asn Ile Thr Ala Pro Pro Gly Gly Leu Leu Val Tyr 180 185 190 Asp Ser Ala Pro Asn Leu Thr Asp Pro His Val Leu Trp Ala Glu Gly 195 200 205 Ala Gly Pro Gly Ala Asp Pro Pro Leu Tyr Ser Val Thr Gly Pro Leu 210 215 220 Pro Thr Gln Arg Leu Ile Ile Gly Glu Val Thr Pro Ala Thr Gln Gly 225 230 235 240 Met Tyr Tyr Leu Ala Trp Gly Arg Met Asp Ser Pro His Glu Tyr Gly 245 250 255 Thr Trp Val Arg Val Arg Met Phe Arg Pro Pro Ser Leu Thr Leu Gln 260 265 270 Pro His Ala Val Met Glu Gly Gln Pro Phe Lys Ala Thr Cys Thr Ala 275 280 285 Ala Ala Tyr Tyr Pro Arg Asn Pro Val Glu Phe Val Trp Phe Glu Asp 290 295 300 Asp Arg Gln Val Phe Asn Pro Gly Gln Ile Asp Thr Gln Thr His Glu 305 310 315 320 His Pro Asp Gly Phe Thr Thr Val Ser Thr Val Thr Ser Glu Ala Val 325 330 335 Gly Gly Gln Val Pro Pro Arg Thr Phe Thr Cys Gln Met Thr Trp His 340 345 350 Arg Asp Ser Val Met Phe Ser Arg Arg Asn Ala Thr Gly Leu Ala Leu 355 360 365 Val Leu Pro Arg Pro Thr Ile Thr Met Glu Phe Gly Val Arg His Val 370 375 380 Val Cys Thr Ala Gly Cys Val Pro Glu Gly Val Thr Phe Ala Trp Phe 385 390 395 400 Leu Gly Asp Asp Pro Ser Pro Ala Ala Lys Ser Ala Val Thr Ala Gln 405 410 415 Glu Ser Cys Asp His Pro Gly Leu Ala Thr Val Arg Ser Thr Leu Pro 420 425 430 Ile Ser Tyr Asp Tyr Ser Glu Tyr Ile Cys Arg Leu Thr Gly Tyr Pro 435 440 445 Ala Gly Ile Pro Val Leu Glu His His Gly Ser His Gln Pro Pro Pro 450 455 460 Arg Asp Pro Thr Glu Arg Gln Val Ile Glu Ala Ile Glu Trp Val Gly 465 470 475 480 Ile Gly Ile Gly Val Leu Ala Ala Gly Val Leu Val Val Thr Ala Ile 485 490 495 Val Tyr Val Val Arg Thr Ser Gln Ser Arg Gln Arg His Arg Arg 500 505 510 <210> 3 <211> 838 <212> PRT <213> Artificial Sequence <220> <223> gH <400> 3 Met Gly Asn Gly Leu Trp Phe Val Gly Val Ile Leu Leu Gly Val Ala 1 5 10 15 Trp Gly Gln Val His Asp Trp Thr Glu Gln Thr Asp Pro Trp Phe Leu 20 25 30 Asp Gly Leu Gly Met Asp Arg Met Tyr Trp Arg Asp Thr Asn Thr Gly 35 40 45 Arg Leu Trp Leu Pro Asn Thr Pro Asp Pro Gln Lys Pro Pro Arg Gly 50 55 60 Phe Leu Ala Pro Pro Asp Glu Leu Asn Leu Thr Thr Ala Ser Leu Pro 65 70 75 80 Leu Leu Arg Trp Tyr Glu Glu Arg Phe Cys Phe Val Leu Val Thr Thr 85 90 95 Ala Glu Phe Pro Arg Asp Pro Gly Gln Leu Leu Tyr Ile Ser Lys Thr 100 105 110 Tyr Leu Leu Gly Arg Pro Pro Asn Ala Ser Leu Pro Ala Pro Ile Thr 115 120 125 Val Glu Pro Thr Ala Gln Pro Pro Pro Ala Val Ala Pro Leu Lys Gly 130 135 140 Leu Leu His Asn Pro Thr Ala Ser Val Leu Leu Arg Ser Arg Ala Trp 145 150 155 160 Val Thr Phe Ser Ala Val Pro Asp Pro Glu Ala Leu Thr Phe Pro Arg 165 170 175 Gly Asp Asn Val Ala Thr Ala Ser His Pro Ser Gly Pro Arg Asp Thr 180 185 190 Pro Pro Pro Arg Pro Pro Val Gly Ala Arg Arg His Pro Thr Thr Glu 195 200 205 Leu Asp Ile Thr His Leu His Asn Ala Ser Thr Thr Trp Leu Ala Thr 210 215 220 Arg Gly Leu Leu Arg Ser Pro Gly Arg Tyr Val Tyr Phe Ser Pro Ser 225 230 235 240 Ala Ser Thr Trp Pro Val Gly Ile Trp Thr Thr Gly Glu Leu Val Leu 245 250 255 Gly Cys Asp Ala Ala Leu Val Arg Ala Arg Tyr Gly Arg Glu Phe Met 260 265 270 Gly Leu Val Ile Ser Met His Asp Ser Pro Pro Val Glu Val Met Val 275 280 285 Val Pro Ala Gly Gln Thr Leu Asp Arg Val Gly Asp Pro Ala Asp Glu 290 295 300 Asn Pro Pro Gly Ala Leu Pro Gly Pro Pro Gly Gly Pro Arg Tyr Arg 305 310 315 320 Val Phe Val Leu Gly Ser Leu Thr Arg Ala Asp Asn Gly Ser Ala Leu 325 330 335 Asp Ala Leu Arg Arg Val Gly Gly Tyr Pro Glu Glu Gly Thr Asn Tyr 340 345 350 Ala Gln Phe Leu Ser Arg Ala Tyr Ala Glu Phe Phe Ser Gly Asp Ala 355 360 365 Gly Ala Glu Gln Gly Pro Arg Pro Pro Leu Phe Trp Arg Leu Thr Gly 370 375 380 Leu Leu Ala Thr Ser Gly Phe Ala Phe Val Asn Ala Ala His Ala Asn 385 390 395 400 Gly Ala Val Cys Leu Ser Asp Leu Leu Gly Phe Leu Ala His Ser Arg 405 410 415 Ala Leu Ala Gly Leu Ala Ala Arg Gly Ala Ala Gly Cys Ala Ala Asp 420 425 430 Ser Val Phe Phe Asn Val Ser Val Leu Asp Pro Thr Ala Arg Leu Gln 435 440 445 Leu Glu Ala Arg Leu Gln His Leu Val Ala Glu Ile Leu Glu Arg Glu 450 455 460 Gln Ser Leu Ala Leu His Ala Leu Gly Tyr Gln Leu Ala Phe Val Leu 465 470 475 480 Asp Ser Pro Ser Ala Tyr Asp Ala Val Ala Pro Ser Ala Ala His Leu 485 490 495 Ile Asp Ala Leu Tyr Ala Glu Phe Leu Gly Gly Arg Val Leu Thr Thr 500 505 510 Pro Val Val His Arg Ala Leu Phe Tyr Ala Ser Ala Val Leu Arg Gln 515 520 525 Pro Phe Leu Ala Gly Val Pro Ser Ala Val Gln Arg Glu Arg Ala Arg 530 535 540 Arg Ser Leu Leu Ile Ala Ser Ala Leu Cys Thr Ser Asp Val Ala Ala 545 550 555 560 Ala Thr Asn Ala Asp Leu Arg Thr Ala Leu Ala Arg Ala Asp His Gln 565 570 575 Lys Thr Leu Phe Trp Leu Pro Asp His Phe Ser Pro Cys Ala Ala Ser 580 585 590 Leu Arg Phe Asp Leu Asp Glu Ser Val Phe Ile Leu Asp Ala Leu Ala 595 600 605 Gln Ala Thr Arg Ser Glu Thr Pro Val Glu Val Leu Ala Gln Gln Thr 610 615 620 His Gly Leu Ala Ser Thr Leu Thr Arg Trp Ala His Tyr Asn Ala Leu 625 630 635 640 Ile Arg Ala Phe Val Pro Glu Ala Ser His Arg Cys Gly Gly Gln Ser 645 650 655 Ala Asn Val Glu Pro Arg Ile Leu Val Pro Ile Thr His Asn Ala Ser 660 665 670 Tyr Val Val Thr His Ser Pro Leu Pro Arg Gly Ile Gly Tyr Lys Leu 675 680 685 Thr Gly Val Asp Val Arg Arg Pro Leu Phe Leu Thr Tyr Leu Thr Ala 690 695 700 Thr Cys Glu Gly Ser Thr Arg Asp Ile Glu Ser Lys Arg Leu Val Arg 705 710 715 720 Thr Gln Asn Gln Arg Asp Leu Gly Leu Val Gly Ala Val Phe Met Arg 725 730 735 Tyr Thr Pro Ala Gly Glu Val Met Ser Val Leu Leu Val Asp Thr Asp 740 745 750 Asn Thr Gln Gln Gln Ile Ala Ala Gly Pro Thr Glu Gly Ala Pro Ser 755 760 765 Val Phe Ser Ser Asp Val Pro Ser Thr Ala Leu Leu Leu Phe Pro Asn 770 775 780 Gly Thr Val Ile His Leu Leu Ala Phe Asp Thr Gln Pro Val Ala Ala 785 790 795 800 Ile Ala Pro Gly Phe Leu Ala Ala Ser Ala Leu Gly Val Val Met Ile 805 810 815 Thr Ala Ala Leu Ala Gly Ile Leu Lys Val Leu Arg Thr Ser Val Pro 820 825 830 Phe Phe Trp Arg Arg Glu 835 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> HER2 VH <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> HER2 VL <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> EpCAM VL <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Leu Lys 100 105 110 Arg <210> 7 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> EpCAM VH <400> 7 Gln Leu Val Gln Ser Gly Pro Gly Leu Val Gln Pro Gly Gly Ser Val 1 5 10 15 Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 20 25 30 Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Ser Phe Lys Gly 50 55 60 Arg Phe Thr Phe Ser Leu Asp Thr Ser Ala Ser Ala Ala Tyr Leu Gln 65 70 75 80 Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Phe Ala Ile Lys Gly Asp Tyr Trp Gly Gln Gly Thr Leu Leu Thr Val 100 105 110 Ser Ser <210> 8 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> HveA 82 <400> 8 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> leader sequence _ HveA82 <400> 9 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly 115 120 <210> 10 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> HveA 87 <400> 10 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly Cys Ser Pro Gly His 85 <210> 11 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> leader sequence - HveA 87 <400> 11 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His 115 120 125 <210> 12 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> HveA 102 <400> 12 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His 85 90 95 Cys Ala Ala Cys Arg Ala 100 <210> 13 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> leader sequence - HveA 102 <400> 13 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala 130 135 140 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> HveA 107 <400> 14 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His 85 90 95 Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser 100 105 <210> 15 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> leader sequence - HveA 107 <400> 15 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser 130 135 140 Ser 145 <210> 16 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> gD (ASM47818) <400> 16 Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro 20 25 30 Gly Val Arg Arg Val Tyr His Ile Gln Ala Gly Leu Pro Asp Pro Phe 35 40 45 Gln Pro Pro Ser Leu Pro Ile Thr Val Tyr Tyr Ala Val Leu Glu Arg 50 55 60 Ala Cys Arg Ser Val Leu Leu Asn Ala Pro Ser Glu Ala Pro Gln Ile 65 70 75 80 Val Arg Gly Ala Ser Glu Asp Val Arg Lys Gln Pro Tyr Asn Leu Thr 85 90 95 Ile Ala Trp Phe Arg Met Gly Gly Asn Cys Ala Ile Pro Ile Thr Val 100 105 110 Met Glu Tyr Thr Glu Cys Ser Tyr Asn Lys Ser Leu Gly Ala Cys Pro 115 120 125 Ile Arg Thr Gln Pro Arg Trp Asn Tyr Tyr Asp Ser Phe Ser Ala Val 130 135 140 Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe Glu Thr 145 150 155 160 Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr Glu Ile 165 170 175 Thr Gln Phe Ile Leu Glu His Arg Ala Lys Gly Ser Cys Lys Tyr Ala 180 185 190 Leu Pro Leu Arg Ile Pro Pro Ser Ala Cys Leu Ser Pro Gln Ala Tyr 195 200 205 Gln Gln Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Arg Phe Ile 210 215 220 Pro Glu Asn Gln Arg Thr Val Ala Val Tyr Ser Leu Lys Ile Ala Gly 225 230 235 240 Trp His Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu 245 250 255 Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp 260 265 270 Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr Val Ala Pro 275 280 285 Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Ala Ala Thr 290 295 300 Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met Gly Leu Ile Ala Gly 305 310 315 320 Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys Gly Ile Val 325 330 335 Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu 340 345 350 Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe 355 360 365 Tyr <210> 17 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> gD-rpsL For <400> 17 cccaggccta ccagcagggg gtgacggtgg acagcatcgg gatgctgccc ggcctggtga 60 60 <210> 18 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> gD-rpsL Rev <400> 18 ccggcgatct tcaagctgta tacggcgacg gtgcgctggt tctcggggat tcagaagaac 60 tcgtcaagaa ggcgtgatgg cgggatcg 88 <210> 19 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> gD R222N_F223I_mutant <400> 19 cccaggccta ccagcagggg gtgacggtgg acagcatcgg gatgctgccc aatatcatcc 60 ccgagaacca gcgcaccgtc gccgtataca gcttgaagat cgccgg 106 <210> 20 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> UL26 / 27-rpsL_For <400> 20 gcgtgggggg gaggaaatcg gcactgacca agggggtccg ttttgtcacg tcagaagaac 60 tcgtcaagaa ggcg 74 <210> 21 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> UL26 / 27-rpsL_Rev <400> 21 aacacataaa ctcccccggg tgtccgcggc ctgtttcctc tttcctttcc ggcctggtga 60 tgatggcggg atcg 74 <210> 22 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> UL26 / 27-tkpA_For <400> 22 gcgtgggggg gaggaaatcg gcactgacca agggggtccg ttttgtcacg gcctcagaag 60 ccatagagcc cacc 74 <210> 23 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> UL26 / 27-pCMV_Rev <400> 23 aacacataaa ctcccccggg tgtccgcggc ctgtttcctc tttcctttcc tatacgcgtt 60 gacattgatt attg 74 <210> 24 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> HER2 Linker <400> 24 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 25 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> EpCAM Linker <400> 25 Ala Thr Pro Ser His Asn Ser His Gln Val Pro Ser Ala Gly Gly Pro 1 5 10 15 Thr Ala Asn Ser Gly Thr Ser Gly Ser Glu Val 20 25 <210> 26 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> scFv leader sequence <400> 26 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys 20 <210> 27 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> Whole sequence _ HER2scFv-HveA adapter <400> 27 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 145 150 155 160 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175 Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln 180 185 190 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 195 200 205 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 210 215 220 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 225 230 235 240 Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly 245 250 255 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Phe Leu Pro Ser 260 265 270 Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys 275 280 285 Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr 290 295 300 Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly 305 310 315 320 Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu 325 330 335 Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly 340 345 350 <210> 28 <211> 1053 <212> DNA <213> Artificial Sequence <220> <223> Whole sequence _ HER2scFv-HveA adapter <400> 28 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 120 tcttgtgccg ccagcggctt caacatcaag gacacctaca tccactgggt ccgacaggcc 180 cctggcaaag gacttgaatg ggtcgccaga atctacccca ccaacggcta caccagatac 240 gccgactctg tgaagggcag attcaccatc agcgccgaca ccagcaagaa caccgcctac 300 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgttc tagatgggga 360 ggcgacggct tctacgccat ggattattgg ggccagggca ccctggtcac agtttctagc 420 ggaggcggag gttctggcgg cggaggaagt ggtggcggag gctctgatat ccagatgaca 480 cagagcccca gcagcctgtc tgcctctgtg ggagacagag tgaccatcac ctgtagagcc 540 agccaggacg tgaacacagc cgtggcttgg tatcagcaga agcctggcaa ggcccctaag 600 ctgctgatct acagcgccag ctttctgtac agcggcgtgc ccagcagatt cagcggctct 660 agaagcggca ccgacttcac cctgaccata agcagtctgc agcccgagga cttcgccacc 720 tactactgtc agcagcacta caccacacct ccaaccttcg gacagggcac caaggtggaa 780 atcaagggtg gtggcggttc agaattcctg ccgtcctgca aggaggacga gtacccagtg 840 ggctccgagt gctgccccaa gtgcagtcca ggttatcgtg tgaaggaggc ctgcggggag 900 ctgacgggca cagtgtgtga accctgccct ccaggcacct acattgccca cctcaatggc 960 ctaagcaagt gtctgcagtg ccaaatgtgt gacccagcca tgggcctgcg cgcgagccgg 1020 aactgctcca ggacagagaa cgccgtgtgt ggc 1053 <210> 29 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> Whole sequence _ EpCAMscFv-HveA adapter <400> 29 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Thr Lys Ser 35 40 45 Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Leu Lys Arg Ala Thr Pro Ser His Asn Ser His Gln Val Pro 130 135 140 Ser Ala Gly Gly Pro Thr Ala Asn Ser Gly Thr Ser Gly Ser Glu Val 145 150 155 160 Gln Leu Val Gln Ser Gly Pro Gly Leu Val Gln Pro Gly Gly Ser Val 165 170 175 Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 180 185 190 Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp 195 200 205 Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Ser Phe Lys Gly 210 215 220 Arg Phe Thr Phe Ser Leu Asp Thr Ser Ala Ser Ala Ala Tyr Leu Gln 225 230 235 240 Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 245 250 255 Phe Ala Ile Lys Gly Asp Tyr Trp Gly Gln Gly Thr Leu Leu Thr Val 260 265 270 Ser Ser Gly Gly Gly Gly Ser Glu Phe Leu Pro Ser Cys Lys Glu Asp 275 280 285 Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr 290 295 300 Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro 305 310 315 320 Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys 325 330 335 Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg 340 345 350 Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly His His His His His 355 360 365 His <210> 30 <211> 1089 <212> DNA <213> Artificial Sequence <220> <223> Whole sequence _ EpCAMscFv-HveA adapter <400> 30 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt gatatccaga tgacccagtc cccgtcctcc ctgagtgctt ctgttggtga ccgtgttacc atcacctgcc gttccaccaa atccctcctg cactccaacg gtatcaccta cctttattgg 240. tatcaacaga aaccgggtaa agctccgaaa cttctgatct accagatgtc caacctggct tccggtgttc cgtctcgttt ctccagttct ggttctggta ccgacttcac cctgaccatc 300 tcttctctgc agccggaga cttcgctacc tactactgcg ctcagaacct ggaaatcccg cgtaccttcg gtcagggtac caaagttgaa cttaagcgcg ctaccccgtc tcacaactcc 420 caccaggttc catccgcagg cggtccgact gctaactctg gaactagtgg atccgaagta cagctggttc agtccggccc gggtcttgtt caaccggggtg gttccgttcg tatctcttgc 540 gctgcttctg gttacacgtt caccaactac ggcatgaact gggtcaaaca ggctccgggt 600 aaaggcctgg aatggatggg ctggatcaac acctacaccg gtgaatccac ctacgctgac 660 tccttcaaag gtcgcttcac tttctccctc gacacaagtg ctagtgctgc atacctccaa 720 atcaactcgc tgcgtgcaga ggatacagca gtctattact gcgcccgttt cgctatcaaa 780 ggtgactact ggggtcaagg cacgctgctg accgtttcct cgggtggtgg cggttcagaa 840 ttcctgccgt cctgcaagga ggacgagtac ccagtgggct ccgagtgctg ccccaagtgc 900 agtccaggtt atcgtgtgaa ggaggcctgc ggggagctga cgggcacagt gtgtgaaccc 960 tgccctccag gcacctacat tgcccacctc aatggcctaa gcaagtgtct gcagtgccaa 1020 atgtgtgacc cagccatggg cctgcgcgcg agccggaact gctccaggac agagaacgcc 1080 gtgtgtggc 1089 <210> 31 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> UL3 / 4-rpsL-neo_for <400> 31 taaataacac ataaatttgg ctggttgttt gttgtcttta atggaccgcc cgcaaggcct 60 ggtgatgatg gcgggatcg 79 <210> 32 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> UL3 / 4-rpsL-neo_rev <400> 32 taggatcccg gccggatcgc gctcgtcacc cgacactgaa acgccccccc cccctcagaa 60 gaactcgtca agaaggcg 78 <210> 33 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> UL3 / 4-HM_pCMV_For <400> 33 taaataacac ataaatttgg ctggttgttt gttgtcttta atggaccgcc cgcaatatac 60 gcgttgacat tgattattg 79 <210> 34 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> UL3 / 4_bGH_poly_R <400> 34 taggatcccg gccggatcgc gctcgtcacc cgacactgaa acgccccccc ccccgcctca 60 gaagccatag agcccacc 78 <210> 35 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HER2scFv _ N terminus linker <400> 35 Ala Ala Ala Ser Ser Gly Gly Gly Ser Gly Ser Gly Gly 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HER2scFv _ C terminus linker <400> 36 Ser Gly Gly Gly Ser Gly Ser Gly Gly Ala Ala Ala 1 5 10 <210> 37 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Whole sequence _ HER2scFv ligand <400> 37 Ala Ala Ala Ser Ser Gly Gly Gly Ser Gly Ser Gly Gly Glu Val Gln 1 5 10 15 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 20 25 30 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 35 40 45 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile 50 55 60 Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg 65 70 75 80 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 85 90 95 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp 100 105 110 Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 145 150 155 160 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 165 170 175 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 180 185 190 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 195 200 205 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 225 230 235 240 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser 245 250 255 Gly Gly Gly Ser Gly Ser Gly Gly Ala Ala Ala 260 265 <210> 38 <211> 801 <212> DNA <213> Artificial Sequence <220> <223> Whole sequence _ HER2scFv ligand <400> 38 gcggccgcca gtagtggcgg tggctctggt tccggtggag aggtgcagct ggttgaatct 60 ggcggaggac tggttcagcc tggcggatct ctgagactgt cttgtgccgc cagcggcttc 120 aacatcaagg acacctacat ccactgggtc cgacaggccc ctggcaaagg acttgaatgg 180 gtcgccagaa tctaccccac caacggctac accagatacg ccgactctgt gaagggcaga 240 ttcaccatca gcgccgacac cagcaagaac accgcctacc tgcagatgaa cagcctgaga 300 gccgaggaca ccgccgtgta ctactgttct agatggggag gcgacggctt ctacgccatg 360 gattattggg gccagggcac cctggtcaca gtttctagcg gaggcggagg ttctggcggc 420 ggaggaagtg gtggcggagg ctctgatatc cagatgacac agagccccag cagcctgtct 480 gcctctgtgg gagacagagt gaccatcacc tgtagagcca gccaggacgt gaacacagcc 540 gtggcttggt atcagcagaa gcctggcaag gccctaagc tgctgatcta cagcgccagc 600 tttctgtaca gcggcgtgcc cagcagattc agcggctcta gaagcggcac cgacttcacc 660 ctgaccataa gcagtctgca gcccgaggac ttcgccacct actactgtca gcagcactac 720 accacacctc caaccttcgg acagggcacc aaggtggaaa tcaagggtgg aggctctggt 780 tccggtggat ccgcggccgc g 801 <210> 39 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> gH29 / 30-rpsL-neo_For <400> 39 tcgtgggggt tattcttttg ggcgttgcgt ggggtcaggt ccacgactgg ggcctggtga 60 tgatggcggg etc 73 <210> 40 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> gH29 / 30-rpsL-neo_Rev <400> 40 ttcgtgtcgc gccagtacat gcggtccatg cccaggccat ccaaaaacca tcagaagaac 60 tcgtcaagaa ggcg 74 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> gH29 / 30-scFv_For <400> 41 tcgtgggggt tattcttttg gg 22 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> gH29 / 30-scFv_Rev <400> 42 ttcgtgtcgc gccagtacat g 21 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> EpCAMscFv - N terminus linker <400> 43 Ala Ala Ala Ser Ser Gly Gly Gly Ser Gly Ser Gly 1 5 10 <210> 44 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> EpCAMscFv - C terminus linker <400> 44 Ser Ser Gly Gly Gly Ser Gly Ser Gly Gly Ser Ala Ala Ala 1 5 10 <210> 45 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> Whole sequence - EpCAMscFv ligand <400> 45 Ala Ala Ala Ser Ser Gly Gly Gly Ser Gly Ser Gly Asp Ile Gln Met 1 5 10 15 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 20 25 30 Ile Thr Cys Arg Ser Thr Lys Ser Leu Leu His Ser Asn Gly Ile Thr 35 40 45 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 50 55 60 Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 65 70 75 80 Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 85 90 95 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro 100 105 110 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Leu Lys Arg Ala Thr Pro 115 120 125 Ser His Asn Ser His Gln Val Pro Ser Ala Gly Gly Pro Thr Ala Asn 130 135 140 Ser Gly Thr Ser Gly Ser Glu Val Gln Leu Val Gln Ser Gly Pro Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Val Arg Ile Ser Cys Ala Ala Ser Gly 165 170 175 Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly 180 185 190 Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser 195 200 205 Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr 210 215 220 Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Leu Thr Val Ser Ser Ser Ser Gly Gly Gly Ser 260 265 270 Gly Ser Gly Gly Ser Ala Ala Ala 275 280 <210> 46 <211> 840 <212> DNA <213> Artificial Sequence <220> <223> Whole sequence - EpCAMscFv ligand <400> 46 gcggccgcca gtagtggcgg tggctctggt tccggtgata tccagatgac ccagtccccg 60 tcctccctga gtgcttctgt tggtgaccgt gttaccatca cctgccgttc caccaaatcc 120 ctcctgcact ccaacggtat cacctacctt tattggtatc aacagaaacc gggtaaagct 180 ccgaaacttc tgatctacca gatgtccaac ctggcttccg gtgttccgtc tcgtttctcc 240 agttctggtt ctggtaccga cttcaccctg accatctctt ctctgcagcc ggaagacttc 300 gctacctact actgcgctca gaacctggaa atcccgcgta ccttcggtca gggtaccaaa 360 gttgaactta agcgcgctac cccgtctcac aactcccacc aggttccatc cgcaggcggt 420 ccgactgcta actctggaac tagtggatcc gaagtacagc tggttcagtc cggcccgggt 480 cttgttcaac cgggtggttc cgttcgtatc tcttgcgctg cttctggtta cacgttcacc 540 aactacggca tgaactgggt caaacaggct ccgggtaaag gcctggaatg gatgggctgg 600 atcaacacct acaccggtga atccacctac gctgactcct tcaaaggtcg cttcactttc 660 tccctcgaca caagtgctag tgctgcatac ctccaaatca actcgctgcg tgcagaggat 720 acagcagtct attactgcgc ccgtttcgct atcaaaggtg actactgggg tcaaggcacg 780 ctgctgaccg tttcctcgtc ttccggtgga ggctctggtt ccggtggatc cgcggccgcg 840 840

Claims

1. A recombinant herpes simplex virus for multiple targeting, wherein at least two expression cassettes of a fusion protein of a targeting domain that specifically binds to a cancer cell target molecule and an extracellular domain of HVEM are inserted into the genome of the herpes simplex virus, thereby enabling multiple expression of the fusion protein without inhibiting the reproduction of the herpes simplex virus, The at least two expression cassettes are inserted between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL48 and UL49 genes, between the UL53 and UL54 genes, or between the US1 and US2 genes in the genome of the herpes simplex virus.

2. The recombinant herpes simplex virus according to claim 1, wherein The expression cassette of the fusion protein has a polycistronic structure, comprising at least two fusion protein genes, and a nucleic acid sequence encoding an IRES (internal ribosome entry site) or a 2A peptide is located between the genes.

3. The recombinant herpes simplex virus according to claim 1, wherein The expression cassette of the fusion protein has a monocistronic configuration, wherein at least two expression cassettes are inserted into the genome of the virus.

4. The recombinant herpes simplex virus according to claim 1, wherein The fusion proteins expressed in various ways have (i) targeting domains that specifically bind to the same target molecule, or (ii) different targeting domains that specifically bind to different target molecules.

5. The recombinant herpes simplex virus according to claim 1, wherein The extracellular domain of HVEM is HveA82 comprising the amino acid sequence of SEQ ID NO: 8 or 9, HveA87 comprising the amino acid sequence of SEQ ID NO: 10 or 11, HveA102 comprising the amino acid sequence of SEQ ID NO: 12 or 13, or HveA107 comprising the amino acid sequence of SEQ ID NO: 14 or 15.

6. The recombinant herpes simplex virus according to claim 1, wherein The fusion protein is a fusion protein in which the cancer cell targeting domain and the extracellular domain of HVEM (HveA) are linked via a linker peptide comprising 1 to 30 amino acids, and The linker peptide comprises at least one amino acid selected from the group consisting of Ser, Gly, Ala and Thr.

7. The recombinant herpes simplex virus according to claim 1, wherein The target molecule is an antigen or receptor on the surface of cancer cells that is expressed only in cancer cells or is overexpressed in cancer cells compared to normal cells.

8. The recombinant herpes simplex virus according to claim 7, wherein The antigen or the receptor is EGFRvIII, EGFR, metastin receptor, receptor tyrosine kinase, 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), MSLN (mesothelin), GD2 (disialoganglioside), GPC3 (phosphatidylinositol glycan 3), PS MA (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, or CD166.

9. The recombinant herpes simplex virus according to claim 1, wherein The target molecule is HER2, and The targeting domain is scFv, constructed such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are linked via the linker peptide in the order of VH, linker peptide, and VL.

10. The recombinant herpes simplex virus according to claim 1, wherein The target molecule is EpCAM, and The targeting domain is scFv, constructed such that VL of SEQ ID NO: 6 and VH of SEQ ID NO: 7 are linked via the linker peptide in the order of VL, linker peptide, and VH.

11. The recombinant herpes simplex virus according to claim 1, wherein The recombinant herpes simplex virus is constructed such that the arginine (R) at position 222 and the phenylalanine (F) at position 223 of the amino acid sequence of gD (glycoprotein D) of SEQ ID NO: 16 are substituted by asparagine (N) and isoleucine (I), respectively.

12. 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 virus of HSV-1 and HSV-2.

13. The recombinant herpes simplex virus according to claim 1, wherein The recombinant herpes simplex virus is a recombinant HSV-1 derived from the HSV-1 KOS strain.

14. The recombinant herpes simplex virus according to claim 1, wherein The recombinant herpes simplex virus is constructed so that an expression cassette expressing any one of the following: (i) a cytokine, (ii) a chemokine, (iii) an antagonist of an immune checkpoint, (iv) a co-stimulatory factor that induces immune cell activation, (v) an antagonist of TGFβ, which inhibits the immune response to cancer cells, (vi) heparanase, which degrades heparan sulfate proteoglycans used in the microenvironment of solid tumors, (vii) an antagonist that inhibits the function of angiogenic factor receptor VEGFR-2 (VEGF receptor-2), and (viii) a prodrug activating enzyme that converts a prodrug into a drug that is toxic to cancer cells is further inserted into the genome of the herpes simplex virus without inhibiting the proliferation of the herpes simplex virus.

15. The recombinant herpes simplex virus according to claim 14, wherein The cytokine is at least one selected from the following: interleukins, including IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-24; interferons, including IFNα, IFNβ and IFNγ; tumor necrosis factors, including TNFα, GM-CSF, G-CSF and FLT3L, The chemokine is at least one selected from CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 and XCL-1, The immune checkpoint is at least one selected from the group consisting of PD-1 (programmed death receptor 1), PD-L1 (programmed death receptor ligand 1), PD-L2 (programmed death receptor 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), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), GITR (glucocorticoid-induced TNFR-related protein), GITRL (glucocorticoid-induced TNFR-related protein ligand) and CTLA-4 (cytolytic T lymphocyte-associated antigen-4), The co-stimulatory factor is at least one selected from the group consisting of CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T cell co-stimulator), CD3γ, CD3δ, and CD3ε, and The prodrug activating enzyme is at least one selected from cytosine deaminase isolated from Escherichia coli, rat cytochrome P450 (CYP2B1) carboxylesterase, bacterial nitroreductase and PNP (purine nucleoside phosphorylase).

16. The recombinant herpes simplex virus according to claim 1, wherein The fusion protein is constructed in the order of NH2 / cancer cell targeting domain / extracellular domain of HVEM / COOH, or in the reverse order.

17. The recombinant herpes simplex virus according to claim 1, wherein The fusion protein is constructed such that the cancer cell targeting domain and the extracellular domain of HVEM are linked via a linker peptide, and the fusion protein is constructed in the order of NH2 / cancer cell targeting domain / linker peptide / extracellular domain of HVEM / COOH, or in the reverse order.

18. A recombinant herpes simplex virus for multiple targeting, wherein (i) at least two expression cassettes capable of expressing a linker that is a fusion protein of a targeting domain that specifically binds to a cancer cell target molecule and an extracellular domain of HVEM are inserted into the genome of the herpes simplex virus without inhibiting the proliferation of the herpes simplex virus, and (ii) a targeting domain that specifically binds to a cancer cell target molecule is inserted and fused to its glycoprotein, and The at least two expression cassettes are inserted between the UL3 and UL4 genes, between the UL26 and UL27 genes, between the UL48 and UL49 genes, between the UL53 and UL54 genes, or between the US1 and US2 genes in the genome of the herpes simplex virus.

19. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gB, gC, gD or gH.

20. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gB, and The targeting domain inserted and fused into the glycoprotein is inserted and fused to the N-terminus and is located at any position within the region of amino acids 31 to 78, any position within the region of amino acids 80 to 363, or any position within the region of amino acids 408 to 896 in the amino acid sequence of gB of SEQ ID NO:

1.

21. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gB, and The targeting domain inserted and fused to the glycoprotein is inserted and fused to the N-terminus, located at the position after amino acid 43, the position after amino acid 52, the position after amino acid 70, the position after amino acid 76, the position after amino acid 80, the position after amino acid 81, the position after amino acid 95, the position after amino acid 100, the position after amino acid 137, the position after amino acid 185, the position after amino acid 187, the position after amino acid 241, the position after amino acid 261, the position after amino acid 265, the position after amino acid 304, the position after amino acid 334, the position after amino acid 361, the position after amino acid 408, the position after amino acid 419, the position after amino acid 430, the position after amino acid 458, the position after amino acid 470, the position after amino acid 481, the position after amino acid 490, the position after amino acid 508, the position after amino acid 511, the position after amino acid 524, the position after amino acid 530, the position after amino acid 541, the position after amino acid 552, the position after amino acid 561, the position after amino acid 570, the position after amino acid 581, the position after amino acid 590, the position after amino acid 591, the position after amino acid 592, the position after amino acid 593, the position after amino acid 594, the position after amino acid 595 the position after amino acid 481, the position after amino acid 495, the position after amino acid 497, the position after amino acid 546, the position after amino acid 608, the position after amino acid 630, the position after amino acid 663, the position after amino acid 664, the position after amino acid 665, the position after amino acid 671, the position after amino acid 673, the position after amino acid 690, the position after amino acid 725, the position after amino acid 730, the position after amino acid 732, the position after amino acid 742, the position after amino acid 772, the position after amino acid 868, the position after amino acid 869, the position after amino acid 886, the position after amino acid 893, the position after amino acid 894, or the position after amino acid 895.

22. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gC, and The targeting domain inserted and fused to the glycoprotein is inserted and fused to a position located within the region of amino acids 33 to 154 in the amino acid sequence of gC of SEQ ID NO:

2.

23. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gC, and The targeting domain inserted and fused into the glycoprotein is inserted and fused to the position after amino acid 33, the position after amino acid 82, the position after amino acid 148, the position after amino acid 149, or the position after amino acid 153 in the amino acid sequence of gC of SEQ ID NO:

2.

24. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gH, and The targeting domain inserted and fused into the glycoprotein is inserted and fused to the N-terminus, located within the region of amino acids 12 to 88, within the region of amino acids 116 to 137, or within the region of amino acids 209 to 839 in the amino acid sequence of gD of SEQ ID NO:

3.

25. The recombinant herpes simplex virus according to claim 18, wherein The glycoprotein is gH, and The targeting domain inserted and fused into the glycoprotein is inserted and fused to the N-terminus, located at a position within the region of amino acids 12 to 49 in the amino acid sequence of gD of SEQ ID NO: 3, or a position within the region of amino acids 116 to 137, located after amino acid 12, after amino acid 22, after amino acid 23, after amino acid 29, after amino acid 83, after amino acid 116, after amino acid 209, after amino acid 215, after amino acid 225, after amino acid 277, after amino acid 386, after amino acid 437, after amino acid 447, after amino acid 472, after amino acid 636, after amino acid 637, after amino acid 666, after amino acid 731, after amino acid 763, after amino acid 764, after amino acid 775, after amino acid 806, after amino acid 824, or after amino acid 838.

26. The recombinant herpes simplex virus according to claim 18, wherein The targeting domain of the fusion protein and the inserted and fused targeting domain have (i) targeting domains that specifically bind to the same target molecule, or (ii) different targeting domains that specifically bind to different target molecules.

27. The recombinant herpes simplex virus according to claim 18, wherein The extracellular domain of HVEM is HveA82 comprising the amino acid sequence of SEQ ID NO: 8 or 9, HveA87 comprising the amino acid sequence of SEQ ID NO: 10 or 11, HveA102 comprising the amino acid sequence of SEQ ID NO: 12 or 13, or HveA107 comprising the amino acid sequence of SEQ ID NO: 14 or 15.

28. The recombinant herpes simplex virus according to claim 18, wherein The fusion protein is a fusion protein in which the cancer cell targeting domain and the extracellular domain of HVEM (HveA) are linked via a linker peptide comprising 1 to 30 amino acids, and The linker peptide comprises at least one amino acid selected from the group consisting of Ser, Gly, Ala and Thr.

29. The recombinant herpes simplex virus according to claim 18, wherein The target molecule is an antigen or receptor on the surface of cancer cells that is expressed only in cancer cells or is overexpressed in cancer cells compared to normal cells.

30. The recombinant herpes simplex virus according to claim 29, wherein The antigen or the receptor is EGFRvIII, EGFR, metastin receptor, receptor tyrosine kinase, 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), MSLN (mesothelin), GD2 (disialoside phosphatase), EG ... glycosides), GPC3 (phosphatidylinositol glycan 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, or CD166.

31. The recombinant herpes simplex virus according to claim 18, wherein The target molecule to which the targeting domain inserted and fused to the glycoprotein specifically binds is HER2, and The targeting domain inserted and fused to the glycoprotein is scFv, constructed such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are linked via the linker peptide in the order of VH, linker peptide, and VL.

32. The recombinant herpes simplex virus according to claim 18, wherein The target molecule to which the targeting domain inserted and fused into the glycoprotein specifically binds is EpCAM, and The targeting domain is scFv, constructed such that VL of SEQ ID NO: 6 and VH of SEQ ID NO: 7 are linked via the linker peptide in the order of VL, linker peptide, and VH.

33. The recombinant herpes simplex virus according to claim 18, wherein The recombinant herpes simplex virus is constructed such that the arginine (R) at position 222 and the phenylalanine (F) at position 223 of the amino acid sequence of gD (glycoprotein D) of SEQ ID NO: 16 are substituted by asparagine (N) and isoleucine (I), respectively.

34. The recombinant herpes simplex virus according to claim 18, wherein The recombinant herpes simplex virus is a recombinant HSV-1 virus, a recombinant HSV-2 virus or a chimeric virus of HSV-1 and HSV-2.

35. The recombinant herpes simplex virus according to claim 20, wherein The recombinant herpes simplex virus is a recombinant HSV-1 derived from the HSV-1 KOS strain.

36. The recombinant herpes simplex virus according to claim 18, wherein The recombinant herpes simplex virus is constructed so that an expression cassette expressing any one of the following: (i) a cytokine, (ii) a chemokine, (iii) an antagonist of an immune checkpoint, (iv) a co-stimulatory factor that induces immune cell activation, (v) an antagonist of TGFβ, which inhibits the immune response to cancer cells, (vi) heparanase, which degrades heparan sulfate proteoglycans used in the microenvironment of solid tumors, (vii) an antagonist that inhibits the function of angiogenic factor receptor VEGFR-2 (VEGF receptor-2), and (viii) a prodrug activating enzyme that converts a prodrug into a drug that is toxic to cancer cells is further inserted into the genome of the herpes simplex virus without inhibiting the proliferation of the herpes simplex virus.

37. The recombinant herpes simplex virus according to claim 36, wherein The cytokine is at least one selected from the following: interleukins, including IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-24; interferons, including IFNα, IFNβ and IFNγ; tumor necrosis factors, including TNFα, GM-CSF, G-CSF and FLT3L, The chemokine is at least one selected from CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 and XCL-1, The immune checkpoint is at least one selected from the group consisting of PD-1 (programmed death receptor 1), PD-L1 (programmed death receptor ligand 1), PD-L2 (programmed death receptor 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), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), GITR (glucocorticoid-induced TNFR-related protein), GITRL (glucocorticoid-induced TNFR-related protein ligand) and CTLA-4 (cytolytic T lymphocyte-associated antigen-4), The co-stimulatory factor is at least one selected from the group consisting of CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T cell co-stimulator), CD3γ, CD3δ, and CD3ε, and The prodrug activating enzyme is at least one selected from cytosine deaminase isolated from Escherichia coli, rat cytochrome P450 (CYP2B1) carboxylesterase, bacterial nitroreductase and PNP (purine nucleoside phosphorylase).

38. The recombinant herpes simplex virus according to claim 18, wherein The fusion protein is constructed in the order of NH2 / cancer cell targeting domain / extracellular domain of HVEM / COOH, or in the reverse order.

39. The recombinant herpes simplex virus according to claim 18, wherein The fusion protein is constructed such that the cancer cell targeting domain and the extracellular domain of HVEM are linked via a linker peptide, and the fusion protein is constructed in the order of NH2 / cancer cell targeting domain / linker peptide / extracellular domain of HVEM / COOH, or in the reverse order.

40. The recombinant herpes simplex virus according to claim 18, wherein The expression cassette of the fusion protein has a polycistronic structure, which comprises at least two fusion protein genes, a nucleic acid sequence encoding an IRES (internal ribosome entry site) or a 2A peptide is located between the genes, and an expression cassette is inserted.

41. A pharmaceutical composition for treating cancer, comprising the recombinant herpes simplex virus according to any one of claims 1 to 40 as an active ingredient.

42. The pharmaceutical composition according to claim 41, further comprising a recombinant linker molecule in which the cancer cell targeting domain and the extracellular domain of HVEM are fused.

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