RECOMBINANT HERPES SIMPLEX VIRUS FOR MULTIPLE TARGETING AND USE THEREOF

Abstract

Proposed are a recombinant herpes simplex virus for multiple targeting and the use thereof. Particularly, a recombinant HSV capable of multiple targeting through multiple expression of an adapter, which is a fused protein of a cancer-cell-targeting domain and an extracellular domain of HVEM, a recombinant HSV capable of multiple targeting by having a modified glycoprotein so as to enable retargeting, in addition to being capable of expressing the adapter that is the fused protein of the cancer-cell-targeting domain and the extracellular domain of HVEM, and the use of the virus for anti-inflammatory therapy are disclosed.

Claims

1.-19. (canceled)

20. A recombinant herpes simplex virus for multiple targeting, in which (i) at least one expression cassette capable of expressing an adapter, which is a fused protein of a targeting domain specifically binding to a cancer cell target molecule and an extracellular domain of HVEM, is inserted into a genome of a herpes simplex virus without inhibiting propagation of the herpes simplex virus, and (ii) a targeting domain specifically binding to a cancer cell target molecule is inserted and fused into a glycoprotein qB, qC, qD or qH thereof.

21.-25. (canceled)

26. The recombinant herpes simplex virus of claim 20, wherein the glycoprotein is gH, and the targeting domain that is inserted and fused into the glycoprotein is inserted and fused into an N-terminus, a position within a region of amino acids 12 to 88, a position within a region of amino acids 116 to 137, or a position within a region of amino acids 209 to 839 in an amino acid sequence of gH of SEQ ID NO: 3.

27. The recombinant herpes simplex virus of claim 20, wherein the glycoprotein is gH, and the targeting domain that is inserted and fused into the glycoprotein is a position after amino acid 12, a position after amino acid 22, a position after amino acid 23, a position after amino acid 29, a position after amino acid 83, a position after amino acid 116, a position after amino acid 209, a position after amino acid 215, a position after amino acid 225, a position after amino acid 277, a position after amino acid 386, a position after amino acid 437, a position after amino acid 447, a position after amino acid 472, a position after amino acid 636, a position after amino acid 637, a position after amino acid 666, a position after amino acid 731, a position after amino acid 763, a position after amino acid 764, a position after amino acid 775, a position after amino acid 806, a position after amino acid 824, or a position after amino acid 838.

28. The recombinant herpes simplex virus of claim 20, wherein the targeting domain of the fused protein and the targeting domain that is inserted and fused have (i) targeting domains specifically binding to an identical target molecule, or (ii) different targeting domains specifically binding to different target molecules.

29. The recombinant herpes simplex virus of claim 20, wherein the extracellular domain of HVEM is HveA82 comprising an amino acid sequence of SEQ ID NO: 8 or 9, HveA87 comprising an amino acid sequence of SEQ ID NO: 10 or 11, HveA102 comprising an amino acid sequence of SEQ ID NO: 12 or 13, or HveA107 comprising an amino acid sequence of SEQ ID NO: 14 or 15.

30. The recombinant herpes simplex virus of claim 20, wherein the fused protein is a fused protein in which a 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 among Ser, Gly, Ala, and Thr.

31. The recombinant herpes simplex virus of claim 20, wherein the target molecule is an antigen or a receptor on a surface of a cancer cell that is expressed only in a cancer cell or is overexpressed in a cancer cell compared to a normal cell.

32. The recombinant herpes simplex virus of claim 31, wherein the antigen or the receptor is EGFRvIll, EGFR, a metastin receptor, a receptor tyrosine kinase, HER2 (human epidermal growth factor receptor 2), a tyrosine kinase-18-receptor (c-Kit), HGF receptor c-Met, CXCR4, CCR7, an 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 (Glypican 3), PSMA (prostate-specific membrane antigen), TAG-72 (tumor-associated glycoprotein 72), GD3 (disialoganglioside), HLA-DR (human leukocyte antigen-DR), MUC1 (Mucin 1), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), LMP1 (latent membrane protein 1), TRAILR2 (tumor-necrosis factor-related apoptosis-inducing ligand receptor), VEGFR2 (vascular endothelial growth factor receptor 2), HGFR (hepatocyte growth factor receptor), CD44, or CD166.

33. The recombinant herpes simplex virus of claim 20, wherein the target molecule to which the targeting domain that is inserted and fused into the glycoprotein specifically binds is HER2, and the targeting domain that is inserted and fused into the glycoprotein is scFv, configured such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are linked in an order of VH, a linker peptide, and VL via the linker peptide.

34. The recombinant herpes simplex virus of claim 20, wherein the target molecule to which the targeting domain that is inserted and fused into the glycoprotein specifically binds is EpCAM, and the targeting domain is scFv, configured such that VL of SEQ ID NO: 6 and VH of SEQ ID NO: 7 are linked in an order of VL, a linker peptide, and VH via the linker peptide.

35. The recombinant herpes simplex virus of claim 20, wherein the recombinant herpes simplex virus is configured such that arginine (R) at position 222 and phenylalanine (F) at position 223 of an amino acid sequence of gD (glycoprotein D) of SEQ ID NO: 16 are substituted with asparagine (N) and isoleucine (I), respectively.

36. The recombinant herpes simplex virus of claim 20, wherein the recombinant herpes simplex virus is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or an HSV-1 and HSV-2 chimeric virus.

37. The recombinant herpes simplex virus of claim 20, wherein the recombinant herpes simplex virus is a recombinant HSV-1 derived from an HSV-1 KOS strain.

38. The recombinant herpes simplex virus of claim 20, wherein the recombinant herpes simplex virus is configured such that an expression cassette expressing any one selected from among (i) cytokine, (ii) chemokine, (iii) an antagonist to an immune checkpoint, (iv) a co-stimulatory factor, which induces activation of an immune cell, (v) an antagonist to TGFβ, which inhibits an immune response to a cancer cell, (vi) heparanase, which degrades heparan sulfate proteoglycan for a solid tumor microenvironment, (vii) an antagonist, which inhibits a function of an angiogenesis factor receptor VEGFR-2 (VEGF receptor-2), and (viii) a prodrug-activating enzyme, which converts a prodrug into a drug that exhibits toxicity to a cancer cell, is further inserted into the genome of the herpes simplex virus without inhibiting propagation of the herpes simplex virus.

39. The recombinant herpes simplex virus of claim 38, wherein the cytokine is at least one selected from among 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 among CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, and XCL-1, the immune checkpoint is at least one selected from among PD-1 (programmed cell death 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), CD27 (cluster of differentiation 27), CD28 (cluster of differentiation 28), CD70 (cluster of differentiation 70), CD80 (cluster of differentiation 80), CD86 (cluster of differentiation 86), CD137 (cluster of differentiation 137), CD276 (cluster of differentiation 276), 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 among 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 among cytosine deaminase, rat cytochrome P450 (CYP2B1), carboxylesterase, bacterial nitroreductase, and PNP (purine nucleoside phosphorylase) isolated from E. coli.

40. The recombinant herpes simplex virus of claim 20, wherein the expression cassette of the fused protein is inserted between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, between UL48 and UL49 genes, between UL53 and UL54 genes, or between US1 and US2 genes in the genome of the virus.

41. The recombinant herpes simplex virus of claim 38, wherein the expression cassette is inserted between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, between UL48 and UL49 genes, between UL53 and UL54 genes, or between US1 and US2 genes in the genome of the virus, in which an insertion locus thereof is different from that of the expression cassette of the fused protein.

42. The recombinant herpes simplex virus of claim 20, wherein the fused protein is configured in an order of NH2/cancer-cell-targeting domain/extracellular domain of HVEM/COOH or in a reverse order thereof.

43. The recombinant herpes simplex virus of claim 20, wherein the fused protein is configured such that a cancer-cell-targeting domain and the extracellular domain of HVEM are linked via a linker peptide, and the fused protein is configured in an order of NH2/cancer-cell-targeting domain/linker peptide/extracellular domain of HVEM/COOH or in a reverse order thereof.

44. The recombinant herpes simplex virus of claim 20, wherein the expression cassette of the fused protein has a polycistronic configuration in which at least two fused protein genes are contained, a nucleic acid sequence encoding an RES (internal ribosome entry site) or a 2A peptide is located between the genes, and one expression cassette is inserted.

45.-46. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0088] FIG. 1 schematically shows the genomic structure of KOS-37 BAC;

[0089] FIG. 2 schematically shows the genomic structure of an HVEM-restricted HSV-1 virus;

[0090] FIG. 3 schematically shows the genomic structure of an HVEM-restricted HSV-1 virus expressing EmGFP;

[0091] FIG. 4 shows the results of fluorescence expression of an HVEM-restricted HSV-1 virus expressing EmGFP and specific infection of cells having an HVEM receptor therewith;

[0092] FIG. 5 schematically shows the genomic structure of each of an HSV-1 virus expressing a HER2-targeting adapter, an HSV-1 virus expressing an EpCAM-targeting adapter, and an HSV-1 virus expressing a HER2/EpCAM dual-targeting adapter;

[0093] FIG. 6 shows the entire sequence of the HER2scFv-HveA adapter and the EpCAMscFv-HveA adapter and the configuration of the corresponding sequence;

[0094] FIG. 7 schematically shows the genomic 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 adapter;

[0095] FIG. 8 shows the entire amino acid sequence of the HER2scFv ligand inserted and fused into gH and the configuration of the corresponding sequence;

[0096] FIG. 9 schematically shows the genomic structure of each of (i) a single-targeting virus having an EpCAM-targeting modified glycoprotein gH and (ii) an EpCAM dual-targeting HSV-1 virus having an EpCAM-targeting modified glycoprotein gH and expressing an EpCAM-targeting adapter;

[0097] FIG. 10 shows the entire amino acid sequence of the EpCAMscFv ligand inserted and fused into gH and the configuration of the corresponding sequence;

[0098] FIG. 11 shows the results of measurement of the proliferation, in Vero-HVEM cells and SK-OV-3 cells, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (HADa-S) expressing a HER2scFv-HveA adapter, a virus (HgH-S) having a HER2scFv ligand in gH, and a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter;

[0099] FIG. 12 shows the results of measurement of the amplification, in Vero-HVEM cells, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (HADa-S) expressing a HER2scFv-HveA adapter, a virus (HgH-S) having a HER2scFv ligand in gH, and a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter;

[0100] FIG. 13 shows the results of measurement of the expression level of the HER2scFv-HveA adapter of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (HADa-S) expressing a HER2scFv-HveA adapter, a virus (HgH-S) having a HER2scFv ligand in gH, and a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter, in Vero-HVEM cells and SK-OV-3 cells;

[0101] FIG. 14 shows the results of specific infection depending on the expression of HER2 in SK-OV-3 cells and the like, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (HADa-S) expressing a HER2scFv-HveA adapter, a virus (HgH-S) having a HER2scFv ligand in gH, and a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter;

[0102] FIG. 15 shows the results of specific cell death depending on the expression of HER2 in SK-OV-3 cells and the like, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (HADa-S) expressing a HER2scFv-HveA adapter, a virus (HgH-S) having a HER2scFv ligand in gH, and a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter;

[0103] FIG. 16 shows the results of tumor suppression by a dual-targeting virus (HADa-HgH-D) having a HER2scFv ligand in gH and expressing a HER2scFv-HveA adapter in an animal experiment;

[0104] FIG. 17 shows the results of specific infection depending on the expression of EpCAM in BT-474 cells and the like, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (EADa-S) expressing an EpCAMscFv-HveA adapter, a virus (EgH-S) having an EpCAMscFv ligand in gH, and a dual-targeting virus (EADa-EgH-D) having an EpCAMscFv ligand in gH and expressing an EpCAMscFv-HveA adapter;

[0105] FIG. 18 shows the results of specific cell death depending on the expression of EpCAM in BT-474 cells and the like, of a virus (gDm) expressing a fluorescent protein EmGFP and enabling cell entry using only HVEM as a cell receptor, a virus (EADa-S) expressing an EpCAMscFv-HveA adapter, a virus (EgH-S) having an EpCAMscFv ligand in gH, and a dual-targeting virus (EADa-EgH-D) having an EpCAMscFv ligand in gH and expressing an EpCAMscFv-HveA adapter;

[0106] FIG. 19 schematically shows the genomic structure of a dual-targeting HSV-1 virus (EADa-HgH-D) having a HER2-targeting modified glycoprotein gH and expressing an EpCAM-targeting adapter;

[0107] FIG. 20 shows the results of dual targeting depending on the expression of HER2 and EpCAM in CHO-K1 cells and the like, of a dual-targeting HSV-1 virus (EADa-HgH-D) having a HER2-targeting modified glycoprotein gH and expressing an EpCAM-targeting adapter; and

[0108] FIG. 21 shows the results of dual targeting depending on the expression of HER2 and EpCAM in CHO-K1 cells and the like, of an HSV-1 virus (EADa-HADa-D) expressing a HER2/EpCAM dual-targeting adapter.

DETAILED DESCRIPTION

[0109] A better understanding of the present disclosure will be given through the following examples. However, these examples are not to be construed as limiting the scope of the present disclosure.

EXAMPLE 1

Production of HVEM-Restricted HSV-1 Virus

[0110] An HSV-1 gene is composed of a large gene about 152 kb in size, and thus KOS-37/BAC (GenBank Accession No. MF156583) (Gierasch W. W. et al. J. Virol. Methods. 2006. 135:197-206) was used to insert a foreign gene or a mutation at a specific locus. The HSV-1 KOS strain is a kind of HSV-1 strain mainly used in laboratories because of the well-known characteristics thereof and the usefulness thereof for investigation of gene function and etiology (Smith KO. Proc. Soc. Exp. Biol. Med. 1964. 115:814-816). KOS-37/BAC, manufactured by inserting a BAC plasmid into a KOS genome, enables cloning at the bacterial level through transformation of DH1OB bacteria (Invitrogen) (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206). In the KOS-37/BAC, BAC (bacterial artificial chromosome) is inserted along with a LoxP site at both sides thereof into a locus between UL37 and UL38 of the HSV-1 KOS genome. This is intended to remove the BAC gene using a Cre-Lox system in subsequent procedures. The schematic view thereof is shown in FIG. 1.

[0111] In order to manufacture HVEM-restricted HSV-1, which enters cells only through the HVEM cell receptor, a gD-R222N/F223I HSV-1 virus, in which arginine (R) at position 222 and phenylalanine (F) at position 223 of the HSV-1 gD amino acid sequence (GenBank Accession No. ASM47818, SEQ ID NO: 16) were substituted with asparagine (N) and isoleucine (I), respectively, was manufactured.

[0112] The gD-R222N/F223I HSV-1 virus manufactured through mutation is able to infect host cells only through HVEM (HveA) rather than nectin-1 as the cell entry receptor (Uchida H et al., J. Virol. 2009. 83(7):2951-2961), and is thus advantageous from the aspect of safety because it cannot infect normal cells having the nectin-1 receptor.

[0113] The genomic structure of the HVEM-restricted KOS-gD-R222N/F223I virus is schematically shown in FIG. 2.

[0114] The KOS-gD-R222N/F223I HSV-1 virus was manufactured by introducing R222N/F223I mutations into the gD site of KOS-37/BAC according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.).

[0115] Specifically, an E. coli clone containing KOS-37/BAC was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A gD-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer gD-rpsL For: SEQ ID NO: 17, reverse primer gD-rpsL Rev: SEQ ID NO: 18) including a locus at which to introduce a mutation into gD. The gD-rpsL-neo/kan cassette is composed of the gD homologous region at the insertion locus, the rpsL gene, which is a selective marker for conferring sensitivity to streptomycin, and the neo/kan gene, which confers kanamycin resistance. When the gD-rpsL-neo/kan cassette is inserted, E. coli having sensitivity to streptomycin antibiotics due to the rpsL gene and kanamycin resistance due to the neo/kan gene is manufactured. After inducing the expression of RecE and RecT so as to enable homologous recombination by activating the function of pRed/ET by adding L-arabinose (Sigma-Aldrich) to the E. coli clone containing KOS-37/BAC and pRed/ET (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557), transformation with 200 ng of the manufactured gD-rpsL-neo/kan cassette was performed. Through homologous recombination, the gD-rpsL-neo/kan cassette is inserted into the gD locus of KOS-37/BAC. E. coli in which gD-rpsL-neo/kan is inserted into KOS-37/BAC exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that gD-rpsL-neo/kan was inserted therein, and the final step of inserting a gene was performed. After inducing the expression of RecE and RecT so as to enable homologous recombination by activating the function of pRed/ET by adding L-arabinose (Sigma-Aldrich) to E. coli containing the KOS 37-BAC gD-rpsL-neo/kan clone, transformation with 100 pmol of R222N_F223I_mutant (SEQ ID NO: 19), which is an oligonucleotide in which R and F at respective positions 222 and 223 of gD were substituted with N and I, was performed. Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the existing gD-rpsL-neo/kan cassette with the inserted oligonucleotide, candidates were selected in a 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 B. C. et al., Methods Enzymol. 1999. 306:337-352), and the substitution of N and I at respective positions 222 and 223 of gD was confirmed through PCR (polymerase chain reaction) and DNA sequencing.

[0116] Next, for viral production, the completed KOS-37/BAC-gD-R222N/F223I DNA was extracted using a large-construct DNA purification kit (Macherey-Nagel), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen). Then, cell culture was carried out using DMEM (Dulbecco's Modified Eagle's Medium) (Welgene) containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene). The Cre-Vero-HVEM cell line is a cell line inducing HVEM protein expression by inserting the HVEM gene into the Cre-Vero cell line (Gierasch 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 may be removed using Cre recombinase of the cells and also that infection with the KOS-gD-R222N/F223I virus due to HVEM overexpression is effective, and thus mass production of viruses becomes easy. 3-4 days after gene introduction, the formation of cell plaques was confirmed, after which the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206), and sonicated, ultimately obtaining a KOS-gD-R222N/F223I virus.

EXAMPLE 2

Production of HVEM-Restricted HSV-1 Virus Expressing EmGFP

[0117] For the production of HVEM-restricted HSV-1 expressing EmGFP, an expression cassette capable of expressing EmGFP (emerald green fluorescent protein) was inserted into the UL26/UL27 locus of the KOS-37/BAC-gD-R222N/F223I DNA manufactured in Example 1 (Tiffany A. et al., J. Virol Methods. 2015. 231:18-25). This is to facilitate the observation of viral production and infection using EmGFP as a marker. A pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) was used to manufacture the EmGFP cassette.

[0118] The genomic structure of KOS-EmGFP-gD-R222N/F223I expressing EmGFP is schematically shown in FIG. 3.

[0119] 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.

[0120] All insertion methods were carried out according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Example 1.

[0121] Specifically, a clone containing KOS-37/BAC-gD-R222N/F223I was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J.P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A UL26/27-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer UL26/27-rpsL_For: SEQ ID NO: 20, reverse primer UL26/27-rpsL_Rev: SEQ ID NO: 21) including a locus at which to introduce a target gene between UL26 and UL27. The clone containing KOS-37/BAC-gD-R222N/F223I DNA and pRed/ET was added with L-arabinose (Sigma-Aldrich) to thus induce homologous recombination, followed by transformation with 200 ng of the manufactured UL26/27-rpsL-neo/kan cassette. The UL26/27-rpsL-neo/kan cassette is inserted into the UL26/27 locus of KOS-37/BAC through homologous recombination. E. coli into which UL26/27-rpsL-neo/kan is inserted exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that UL26/27-rpsL-neo/kan was inserted therein, and the final step of inserting a gene was performed.

[0122] E. coli containing the UL26/27-rpsL-neo/kan cassette was added with L-arabinose (Sigma-Aldrich) activating the function of pRed/ET to thus induce homologous recombination, followed by transformation with 200 ng of a UL26/27-tkpA-EmGFP-pCMV cassette. The UL26/27-tkpA-EmGFP-pCMV cassette was manufactured using a pCDNA6.2-GW/EmGFP-miR plasmid (Invitrogen) as a template, a forward primer UL26/27-tkpA_For (SEQ ID NO: 22), and a reverse primer UL26/27-pCMV_Rev (SEQ ID NO: 23).

[0123] Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the existing UL26/27-rpsL-neo/kan cassette with the inserted UL26/27-tkpA-EmGFP-pCMV, candidates were selected in a 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 B. C. et al., Methods Enzymol. 1999. 306:337-352). The introduction of tkpA-EmGFP-pCMV at UL26/27 was confirmed through restriction enzyme EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.

[0124] An experiment was conducted for normal expression of a fluorescent protein and production of a virus. The completed KOS-37/BAC-EmGFP-gD-R222N/F223I DNA was extracted using a large-construct DNA purification kit (Macherey-Nagel), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen) to remove the BAC gene using Cre recombinase. 3 days after transfection, expression of the EmGFP protein was observed using a fluorescence microscope, and viral production was observed through the formation of Cre-Vero-HVEM cell plaques. After confirmation of plaque formation, the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W.W. et al.; J. Virol. Methods. 2006. 135:197-206), and sonicated, thus obtaining a KOS-EmGFP-gD-R222N/F223I virus (gDm).

[0125] For infection with the KOS-EmGFP-gD-R222N/F223I virus and fluorescence expression thereof, HVEM-free cell lines (J1 and J-Nectin) and cell lines expressing HVEM (J-HVEM) were used. J1 cells are young hamster kidney cell lines that are deficient in the virus HSV-1 receptors HVEM and nectin-1 (Petrovic B. et al., 2017. PLOS Pathog. 19; 13(4):e1006352). J-Nectin and J-HVEM cell lines are cell lines that overexpress nectin-1 and HVEM respectively in J1 cells (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). 1×10.sup.4 cells were infected at 10 MOI (multiplicity of infection) with the KOS-EmGFP-gD-R222N/F223I virus obtained above, and after 24 hours, the fluorescent protein expression and viral infection were observed using a fluorescence microscope (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).

[0126] The results thereof are shown in FIG. 4, upper and lower images of which were taken using a fluorescence microscope and an optical microscope, respectively. With reference to the upper fluorescence microscope images of FIG. 4, 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.

[0127] Based on the above results, it was confirmed that the propagation of the KOS-EmGFP-gD-R222N/F223I virus (gDm) was easily observed through the expression of the fluorescent protein, as intended, and cell entry became possible using only HVEM as the cell entry receptor, without nectin-1.

EXAMPLE 3

Production of HSV-1 Virus Expressing HER2-Targeting Adapter, HSV-1 Virus Expressing EpCAM-Targeting Adapter, and HSV-1 Virus Expressing HER2/EpCAM Dual-Targeting Adapter

[0128] Each of an adapter-expressing cassette expressing HER2scFv-HveA, an adapter-expressing cassette expressing EpCAM-HveA, and an adapter-expressing cassette expressing both HER2scFv-HveA and EpCAM-HveA was inserted into the UL3/UL4 locus of the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA into which the EmGFP expression cassette (pCMV-EmGFP-tkpA) was inserted, manufactured in Example 2.

[0129] The genomic structure of a KOS-UL3/4-HER2scFv-HveA-EmGFP-gD/R222N/F223I virus into which pCMV-HER2scFv-HveA-bGHpA as the adapter-expressing cassette expressing HER2scFv-HveA was inserted, the genomic structure of a KOS-UL3/4-EpCAMscFv-HveA-EmGFP-gD/R222N/F223I virus into which pCMV-EpCAMscFv-HveA-bGHpA as the adapter-expressing cassette expressing EpCAMscFv-HveA was inserted, and the genomic structure of a KOS-UL3/4-EpCAMscFv-HveA-HER2scFv-HveA-EmGFP-gD/R222N/F223I virus into which pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA as the adapter-expressing cassette expressing both EpCAM-HveA and HER2scFv-HveA was inserted are schematically shown in FIG. 5, and the entire sequence of the HER2scFv-HveA adapter and the EpCAMscFv-HveA adapter and the configuration of the corresponding sequence are shown in FIG. 6.

[0130] Here, scFv for HER2 is configured such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are linked via a linker peptide of SEQ ID NO: 24, scFv for EpCAM is configured such that VL of SEQ ID NO: 6 and VH of SEQ ID NO: 7 are linked via a linker peptide of SEQ ID NO: 25, and HveA is HveA82 of SEQ ID NO: 8 in the HER2scFv-HveA adapter and the EpCAMscFv-HveA adapter. In addition, in the HER2scFv-HveA adapter and the EpCAMscFv-HveA adapter, the leader sequence of SEQ ID NO: 26 is included at the N-terminus thereof, particularly before VH of HER2scFv and before VL of EpCAMscFv.

[0131] EF (base sequence: GAATTC), which is a restriction enzyme EcoRI site for easy cloning, is added after the scFv sequence for HER2 or EpCAM and the NH.sub.2-GGGGS sequence, which is the linker sequence of the HveA sequence. Also, pCMV is the gene promoter of cytomegalovirus, bGH-pA is the bGH-PolyA (bovine growth hormone polyadenylation) signal sequence, and P2A in the adapter-expressing cassette pCMV-HER2scFv-HveA-P2A-EpCAMscFv-HveA-bGHpA expressing both HER2scFv-HveA and EpCAM-HveA is 2A of porcine teschovirus-1 (P2A).

[0132] Used in the present example, the amino acid sequence and the gene sequence of the full length of the HER2scFv-HveA adapter including the leader sequence are represented in SEQ ID NO: 27 and SEQ ID NO: 28, respectively, and the amino acid sequence and the gene sequence of the full length of the EpCAMscFv-HveA adapter including the leader sequence are represented in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0133] The insertion of the HER2scFv-HveA adapter-expressing cassette, the EpCAMscFv-HveA adapter-expressing cassette, and the EpCAM-HveA-HER2scFv-HveA dual adapter-expressing cassette was performed according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Examples 1 and 2.

[0134] Specifically, the E. coli clone containing the KOS-37/BAC-EmGFP-gD-R222N/F223I genome manufactured in Example 2 was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A UL3/4-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer UL3/4-rpsL-neo_for: SEQ ID NO: 31, reverse primer UL3/4-rpsL-neo_rev: SEQ ID NO: 32) including a locus at which to introduce a target gene between UL3 and UL4. The clone containing KOS-37/BAC-EmGFP-gD-R222N/F223I and pRedET was added with L-arabinose (Sigma-Aldrich) to thus induce homologous recombination, followed by transformation with 200 ng of the UL3/4-rpsL-neo/kan cassette manufactured as described above. Through such homologous recombination, the UL3/4-rpsL-neo/kan cassette is inserted into the UL3/4 locus of KOS-37/BAC-EmGFP-gD-R222N/F223I. E. coli into which UL3/4-rpsL-neo/kan is inserted exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that UL3/4-rpsL-neo/kan was inserted therein, and the final step of inserting a target gene was performed.

[0135] E. coli containing the UL3/4-rpsL-neo/kan cassette was added with L-arabinose (Sigma-Aldrich) activating the function of pRed/ET to thus induce homologous recombination, followed by transformation with 200 ng of each of the UL3/4-pCMV-HER2scFv-HveA-bGHpA cassette, the UL3/4-pCMV-EpCAMscFv-HveA-bGHpA cassette, and the UL3/4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA cassette. The UL3/4-pCMV-HER2scFv-HveA-bGHpA cassette, the UL3/4-pCMV-EpCAMscFv-HveA-bGHpA cassette, and the UL3/4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA were manufactured using a forward primer UL3/4_pCMV_For (SEQ ID NO: 33) and a reverse primer UL3/4_bGH_poly_R (SEQ ID NO: 34) using, as respective templates, a pCDNA3.1-HER2scFv-HveA plasmid, a pCDNA3.1-EpCAMscFv-HveA plasmid, and pCDNA3.1-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA (Baek H. J. 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).

[0136] Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the conventionally inserted UL3/4-rpsL-neo/kan cassette with the above inserted UL3/4-pCMV-HER2scFv-HveA-bGHpA, UL3/4-pCMV-EpCAMscFv-HveA-bGHpA, and UL3/4-pCMV-EpCAMscFv-HveA-P2A-HER2scFv-HveA-bGHpA, candidates were selected in a 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 B.C. et al., Methods Enzymol. 1999. 306:337-352). The introduction of the 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 through restriction enzyme EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.

[0137] The completed 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 DNA were extracted using a large-construct DNA purification kit (Macherey-Nagel), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen) to remove the BAC gene using Cre recombinase. 3 days after transfection, the fluorescence expression of the EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirmation of plaque formation, the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206), and sonicated, ultimately obtaining a KOS-UL3/4_HER2scFv-HveA-EmGFP-gD-R222N/F223I virus (HADa-S) expressing the HER2-targeting adapter, a KOS-UL3/4_EpCAMscFv-HveA-EmGFP-gD/R222N/F223I virus (EADa-S) expressing the EpCAM-targeting adapter, and a KOS-UL3/4_EpCAMscFv-HveA-P2A-HER2scFv-HveA-EmGFP-gD/R222N/F223I virus (EADa-HADa-D) expressing the HER2/EpCAM dual-targeting adapter.

EXAMPLE 4

Production of HSV-1 Virus having HER2-Targeting Modified Glycoprotein gH and HSV-1 Virus having HER2-Targeting Modified Glycoprotein gH and Expressing HER2-Targeting Adapter

[0138] For the production of a retargeting HSV capable of targeting a target molecule expressed in specific cancer, a ligand (HER2 scFv) that recognizes HER2 specifically expressed in cancer cells was inserted between amino acids 29 and 30 of the amino acid sequence of gH (GenBank Accession No. ASM47773, SEQ ID NO: 3), which is a glycoprotein of HSV-1. A gene capable of expressing HER2scFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA and the KOS-37/BAC-UL3/4_HER2scFv-HveA-EmGFP-gD/R222N/F223I DNA manufactured in Examples 2 and 3.

[0139] The genome structure of each of the KOS-gH/HER2scFv-EmGFP-gD/R222N/F223 I virus (HgH-S) and the KOS-UL3/4_HER2scFv-HveA-gH/HER2scFv-EmGFP-gD/R222N/F223I virus (HADa-HgH-D), in which the HER2scFv ligand was inserted into the gH of HSV-1, is shown in FIG. 7, and the entire sequence of the gH-HER2scFv ligand and the construction of the corresponding sequence are shown in FIG. 8. Here, scFv for HER2 is configured such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are connected via a linker peptide of SEQ ID NO: 24, and the linker peptide of SEQ ID NO: 35 is linked to the N-terminus of the scFv, and the linker peptide of SEQ ID NO: 36 is linked to the C-terminus thereof.

[0140] The amino acid sequence and the gene sequence of the full length of HER2scFv used in the present example are represented in SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0141] The insertion of the gH-HER2scFv ligand was performed according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Examples 1, 2 and 3.

[0142] Specifically, the E. coli clone containing the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA and the KOS-37/BAC-UL3/4_HER2scFv-HveA-EmGFP-gD-R222N/F223I DNA manufactured in Examples 2 and 3 was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A gH29/30-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer gH29/30-rpsL-neo_for: SEQ ID NO: 39, reverse primer gH29/30-rpsL-neo_rev: SEQ ID NO: 40) including a locus at which to introduce a target gene between amino acids 29 and 30 of gH. The clone containing each of KOS-37/BAC-EmG FP-gD-R222 N/F223 I DNA and KOS-37/BAC-UL3/4_HER2scFv-HveA-EmGFP-gD-R222N/F223I DNA and pRed/ET was added with L-arabinose (Sigma-Aldrich) to thus induce homologous recombination, followed by transformation with 200 ng of the gH29/30-rpsL-neo/kan cassette manufactured as described above. Through such homologous recombination, the gH29/30-rpsL-neo/kan cassette is inserted at the position between amino acids 29 and 30 of gH. E. coli into which gH29/30-rpsL-neo/kan is inserted exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that gH29/30-rpsL-neo/kan was inserted therein, and the final step of inserting a target gene was performed.

[0143] E. coli containing the gH29/30-rpsL-neo/kan cassette was added with L-arabinose (Sigma-Aldrich) activating the function of pRed/ET to thus induce homologous recombination, followed by transformation with 200 ng of a gH29/30-HER2scFv ligand. The gH29/30-HER2scFv ligand was manufactured using a forward primer gH29/30-scFv_For (SEQ ID NO: 41) and a reverse primer gH29/30-scFv_Rev (SEQ ID NO: 42) using, as a template, a pCAGGSMCS-gH-HER2scFv plasmid. The pCAGGSMCS-gH-HER2scFv plasmid was manufactured by inserting HER2scFv into a pCAGGSMCS plasmid (Atanasiu D. et al., J. Virol. 2013, Nov. 87(21):11332-11345), and, in detail, was manufactured by treating a pCAGGSMCS plasmid and HER2scFv amplified via PCR (Carter P. et al., Proc. Natl. Acad. Sci. USA. 1992, 15;89(10):4285-9) with a Notl restriction enzyme (NEB, R3189) and joining the pCAGGSMCS plasmid and the HER2scFv, which were cleaved by Notl, using T4 DNA ligase (NEB, M0202).

[0144] Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the conventionally inserted gH29/30-rpsL-neo/kan cassette with the above inserted gH29/30-HER2scFv, candidates were selected in a 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 B. C. et al., Methods Enzymol. 1999. 306:337-352), the introduction of HER2scFv at gH29/30 was confirmed through restriction enzyme EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.

[0145] The completed KOS-37/BAC-g H/HER2scFv-EmG FP-gD-R222N/F223 I 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), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen) to remove the BAC gene using Cre recombinase. 3 days after transfection, the fluorescence expression of the EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirmation of plaque formation, the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206), and sonicated, ultimately obtaining a KOS-gH/HER2scFv-EmGFP-gD-R222N/F223I virus (HgH-S) and a KOS-UL3/4_HER2scFv-HveA-gH/HER2scFv-EmGFP-gD-R222N/F223I virus (HADa-HgH-D).

EXAMPLE 5

Production of HSV-1 Virus having EpCAM-Targeting Modified Glycoprotein gH and HSV-1 Virus having EpCAM-Targeting Modified Glycoprotein gH and Expressing EpCAM-Targeting Adapter

[0146] For the production of a retargeting HSV capable of targeting a target molecule expressed in specific cancer, a ligand (EpCAM scFv) that recognizes EpCAM specifically expressed in cancer cells was inserted between amino acids 29 and 30 of the amino acid sequence of gH (GenBank Accession No. ASM47773, SEQ ID NO: 3), which is a glycoprotein of HSV-1. A gene capable of expressing EpCAMscFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA and the KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD/R222N/F223I (EADa-S) DNA manufactured in Examples 2 and 3.

[0147] The genomic structure of each of the KOS-gH/EpCAMscFv-EmGFP-gD/R222N/F223I virus (EgH-S) and the KOS-UL3/4_EpCAMscFv-HveA-gH/EpCAMscFv-EmGFP-gD/R222N/F223I virus (EADa-EgH-D), in which the EpCAMscFv ligand was inserted into the gH of HSV-1, is shown in FIG. 9, and the entire sequence of the gH-EpCAMscFv ligand and the construction of the corresponding sequence are shown in FIG. 10. Here, scFv for EpCAM is configured such that VL of SEQ ID NO: 6 and VH of SEQ ID NO: 7 are linked via a linker peptide of SEQ ID NO: 25, and the linker peptide of SEQ ID NO: 43 is linked to the N-terminus of this scFv, and the linker peptide of SEQ ID NO: 44 is linked to the C-terminus thereof.

[0148] The amino acid sequence and the gene sequence of EpCAMscFv used in the present example are represented in SEQ ID NO: 45 and SEQ ID NO: 46, respectively.

[0149] The insertion of the gH-EpCAMscFv ligand was performed according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Example 4.

[0150] Specifically, the E. coli clone containing the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA and the KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I DNA manufactured in Examples 2 and 3 was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A gH29/30-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer gH29/30-rpsL-neo_for: SEQ ID NO: 39, reverse primer gH29/30-rpsL-neo_rev: SEQ ID NO: 40) including a locus at which to introduce a target gene between amino acids 29 and 30 of gH. The clone containing each of KOS-37/BAC-EmGFP-gD-R222N/F223I DNA and KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I DNA and pRed/ET was added with L-arabinose (Sigma-Aldrich) to thus induce homologous recombination, followed by transformation with 200 ng of the gH29/30-rpsL-neo/kan cassette manufactured as described above. Through such homologous recombination, the gH29/30-rpsL-neo/kan cassette is inserted between amino acids 29 and 30 of gH. E. coli into which gH29/30-rpsL-neo/kan is inserted exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that gH29/30-rpsL-neo/kan was inserted therein, and the final step of inserting a target gene was performed.

[0151] E. coli containing the gH29/30-rpsL-neo/kan cassette was added with L-arabinose (Sigma-Aldrich) activating the function of pRed/ET to thus induce homologous recombination, followed by transformation with 200 ng of a gH29/30-EpCAMscFv ligand. The gH29/30-EpCAMscFv ligand was manufactured using a forward primer gH29/30-scFv_For (SEQ ID NO: 41) and a reverse primer gH29/30_scFv_Rev (SEQ ID NO: 42) using, as a template, a pCAGGSMCS-gH-EpCAMscFv plasmid. The pCAGGSMCS-gH-EpCAMscFv plasmid was manufactured by inserting EpCAMscFv into a pCAGGSMCS plasmid (Atanasiu D. et al., J. Virol. 2013, Nov. 87(21):11332-11345), and, in detail, was manufactured by treating a pCAGGSMCS plasmid and EpCAMscFv amplified via PCR (Willuda J. et al., Cancer Res. 1999, 15;59(22):5758-67) with a Notl restriction enzyme (NEB, R3189) and joining the pCAGGSMCS plasmid and the EpCAMscFv, which were cleaved by Notl, using T4 DNA ligase (NEB, M0202).

[0152] Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the conventionally inserted gH29/30-rpsL-neo/kan cassette with the above inserted gH29/30-EpCAMscFv, candidates were selected in a 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 B.C. et al., Methods Enzymol. 1999. 306:337-352). The introduction of EpCAMscFv at gH29/30 was confirmed through restriction enzyme EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.

[0153] The completed KOS-37/BAC-g H_EpCAMscFv-EmG FP-gD-R222 N/F223 I and KOS-37/BAC-U L3/4_EpCAMscFv-HveA-g H/EpCAMscFv-EmG FP-gD-R222 N/F223 I DNA were extracted using a large-construct DNA purification kit (Macherey-Nagel), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen) to remove the BAC gene using Cre recombinase. 2 days after transfection, the fluorescence expression of the EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirmation of plaque formation, the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W. W. et al.; J. Virol. Methods. 2006.135:197-206), and sonicated, ultimately obtaining a KOS-gH/EpCAMscFv-EmGFP-gD-R222N/F223I virus (EgH-S) and a KOS-gH/EpCAMscFv-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I virus (EADa-EgH-D).

EXAMPLE 6

Measurement of Activity of HER2 Dual-Targeting Oncolytic Virus

[0154] In order to confirm the expression level of the HER2scFv-HveA adapter as well as the viral proliferation and amplification of the KOS-UL3/4_HER2scFv-HveA-EmGFP-gD-R222N/F223I virus (HADa-S) expressing the HER2scFv-HveA adapter, the KOS-gH/HER2scFv-EmGFP-gD-R222N/F223I virus (HgH-S) expressing HER2scFv in gH, and the KOS-UL3/4_HER2scFv-HveA-gH/HER2scFv-EmGFP-gD-R222N/F223I (HADa-HgH-D) dual-targeting virus expressing the HER2scFv-HveA adapter and HER2scFv in gH, as manufactured in Examples 3 and 4, the following experiment was performed.

[0155] In order to perform the viral proliferation experiment, 2.0×10.sup.5 Vero-HVEM cells and SK-OV-3 cells were applied on a 12 well plate.

[0156] An HSV-1 wild-type virus (KOS), the virus gDm expressing the fluorescent protein EmGFP manufactured in Example 2 and enabling cell entry using only HVEM as a cell receptor, the virus HADa-S expressing the HER2scFv-HveA adapter manufactured in Example 3, the virus HgH-S having the HER2scFv ligand in gH manufactured in Example 4, and the dual-targeting virus HADa-HgH-D having the HER2scFv ligand in gH and expressing the HER2scFv-HveA adapter manufactured in Example 4 were diluted and used for infection so that 20-50 viruses were contained in a single well. After 90 minutes, in order to remove the remaining initial virus and prevent the viral proliferation, the medium that was used was replaced with a medium containing 0.2% methylcellulose. After 3 days, viral proliferation was measured through the size of a virus plaque using a fluorescence microscope.

[0157] The results thereof are shown in FIG. 11. As is apparent from FIG. 11, 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, respectively, than that of the wild-type virus (KOS). In SK-OV-3 cells as the HER2-expressing cell line, the plaque size of HgH-S was reduced by 53% compared to the wild-type virus (KOS), but HADa-S and HADa-HgH-D viruses were increased by 20% and 19%, respectively. The reason why the plaque size of HgH-S was decreased is deemed to be that, when gD binds to the entry receptor, gH plays a role in inducing cell fusion by transmitting an activation signal to gB through such binding, and also that endocytosis is induced through binding to integrins, and thus it is judged that viral proliferation or replication is inhibited by affecting such signal transmission or endocytosis due to structural changes through insertion of scFv into gH.

[0158] In order to perform the virus replication experiment, 1.0×10.sup.4 Vero-HVEM cells were applied on a 96 well plate. The gDm, HgH-S expressing the HER2scFv ligand in gH, HADa-S expressing the HER2scFv-HveA adapter, and dual-targeting HADa-HgH-D viruses were used for infection at 0.1 MOI. After 90 minutes, the medium that was used was replaced with a fresh medium in order to remove the remaining initial virus. A virus culture solution was obtained at 3, 24, and 48 hours after infection, and the number of viruses in the culture solution was measured.

[0159] The results thereof are shown in FIG. 12. As is apparent from FIG. 12, although the virus propagation activity of gDm, HADa-S and HADa-HgH-D was similar in the Vero-HVEM cell line, it was confirmed that the virus propagation activity in HgH-S was reduced due to the decreased virus proliferation ability, as in the results of FIG. 11.

[0160] In order to perform the experiment to measure the expression level of the adapter, 2.0×10.sup.5 Vero-HVEM and SK-OV-3 cells were applied on a 12-well plate. Then, gDm, HgH-S expressing the HER2scFv ligand in gH, HADa-S expressing the HER2scFv-HveA adapter, and the dual-targeting HADa-HgH-D virus were used for infection at 0.1 MOI. After 90 minutes, the medium that was used was replaced with a fresh medium without FBS in order to remove the remaining initial virus. 48 hours after infection, a virus culture solution was obtained, and protein expression levels were measured through Western blotting in order to measure the adapter in the culture solution.

[0161] The results thereof are shown in FIG. 13. As is apparent from FIG. 13, in the Vero-HVEM cell line, the HADa-HgH-D dual-targeting virus expressed the adapter in an amount as large as at least 3 times compared to the HADa-S virus expressing only the adapter. However, the adapter was not measured in gDm and HgH-S virus culture solutions having no adapter. In SK-OV-3 cells expressing HER2, only the adapter of the HADa-HgH-D dual-targeting virus was detected. This is because the HADa-HgH-D dual-targeting virus exhibits a higher infection rate in the cell line expressing HER2 compared to the HADa-S virus, so the expression of the adapter is proportionally higher.

[0162] Consequently, the HADa-HgH-D dual-targeting virus was confirmed to be relatively improved in view of the viral proliferation and amplification and the adapter expression level compared to other viruses.

EXAMPLE 7

Infection and Cytotoxicity of HER2-Expressing Cancer Cells using HER2 Dual-Targeting Oncolytic Virus

[0163] An experiment was performed in a HER2-expressing cancer cell line using gDm, HgH-S expressing the HER2scFv ligand in gH, HADa-S expressing the HER2scFv-HveA adapter, and the dual-targeting HADa-HgH-D virus expressing the HER2scFv-HveA adapter and the HER2scFv ligand in gH, manufactured in Examples 2, 3 and 4. In order 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 adapter and whether it induces cytotoxicity after infection, the following experiment was conducted.

[0164] The cell lines that were used in the experiment were a cell line not expressing HER2 (MDA-MB-231) and cell lines expressing 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 an ovarian cancer cell line SK-OV-3 (ATCC, HTB-77), culture was performed using DMEM containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS, and for a breast cancer cell line MDA-MB-453 (ATCC, HTB-131), culture was performed using an RPMI 1640 medium containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS.

[0165] For a HER2-specific viral infection experiment, 8×10.sup.3 SK-OV-3 and MDA-MB-231, 4.0×10.sup.4 MCF-7, 8.0×10.sup.4 MDA-MB-453, and 7.0×10.sup.4 BT-474 cell lines were used at 2 MOI and infected with HgH-S expressing the HER2scFv ligand in gH, HADa-S expressing the HER2scFv-HveA adapter, the dual-targeting HADa-HgH-D virus, and as a control, HER2-non-targeting gDm virus. After 90 minutes, the medium that was used was replaced with a fresh medium in order to remove the remaining initial virus. 2 days after infection, viral infection was confirmed through EmGFP fluorescence expression in each cell line (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514). Also, in order to measure cytotoxicity for 4 days after infection, the extent of color development of formazan, which is a color-developing material formed only in living cells using an EZ-Cytox (DoGenBio) reagent, was measured at 450 nm using an ELISA reader. Absorbance was quantified to determine the cytotoxicity of cancer cell lines due to each virus.

[0166] The results thereof are shown in FIG. 14. As is apparent from FIG. 14, the HADa-S virus expressing the adapter had a high infection rate in SK-OV-3 and MCF7 cells, but a low infection rate was observed in MDA-MB-453 and BT-474. The HgH-S virus expressing the HER2scFv ligand in gH had a high infection rate in SK-OV-3, MCF7, and MDA-MB-453 cells, but a low infection rate was observed in BT-474. Unlike the virus that expresses each of the ligand and the adapter, 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 was not targeted to HER2, and infection with all viruses was not observed in MDA-MB-231 cells not expressing HER2, as a control. The low infection rate in MDA-MB-453 and BT-474 is deemed to be due to the cell morphology and characteristics of MDA-MB-453 and BT-474 and the initial low infection with the HADa-S virus.

[0167] In addition, FIG. 15 shows the results of observation of the cytotoxicity of cancer cells due to the virus for 4 days after infection. The HgH-S, HADa-S, and HADa-HgH-D viruses exhibited respective cell viability values of 41%, 27%, and 25% in SK-OV-3, of 61%, 52%, and 39% in MCF-7, and of 49%, 100%, and 23% in MDA-MB-453. In the three cell lines expressing HER2, it was observed that the cytotoxicity due to infection with the dual-targeting HADa-HgH-D virus was the highest. However, since the gDm virus was not targeted to HER2, cytotoxicity was not observed, and MDA-MB-231 not expressing HER2 was not capable of infection, so it was observed that the three viruses were not involved in cytotoxicity. The reason why there was no effect on the viability of MDA-MB-453 cells seems to be that the initial infection with the HADa-S virus was very low compared to the other viruses.

EXAMPLE 8

Inhibition of Growth of Tumor Cells due to HER2 Dual-Targeting Oncolytic Virus in Mice

[0168] In order to confirm whether the dual-targeting HADa-HgH-D virus manufactured in Example 4 induces inhibition of growth of cancer cells expressing HER2 in mice, the following experiment was conducted.

[0169] After subcutaneous injection of SK-OV-3 at 5×10.sup.6 cells/mouse into 5-week-old Balb/c nude mice (Orient Bio), the tumors were observed until the size thereof became 100 mm.sup.3. Intratumoral injection of the HER2 dual-targeting HADa-HgH-D virus at 2×10.sup.7 pfu/mouse into 5 mice having tumors was performed, and PBS was injected into 5 mice as a control. After virus injection, the size of the tumor generated in mice was observed for 28 days.

[0170] The results thereof are shown in a graph of the tumor size for 28 days in FIG. 16. In the control using PBS, the tumor grew to 815.28±141.36 mm.sup.3 from 116.46±11.21 mm.sup.3 in the initial stage, but in the mice injected with the HER2 dual-targeting HADa-HgH-D virus, tumor growth was observed from 108.85±15.54 mm.sup.3 to 110.02±55.44 mm.sup.3, which was regarded as inhibited compared to the control.

EXAMPLE 9

Infection and Cytotoxicity of EpCAM-Expressing Cancer Cells using EpCAM Dual-Targeting Oncolytic Virus

[0171] An experiment was performed in cancer cell lines using the EADa-S virus expressing the EpCAMscFv-HveA adapter, the EgH-S virus expressing the EpCAMscFv ligand in gH, and the dual-targeting EADa-EgH-D virus expressing both the EpCAMscFv-HveA adapter and the EpCAMscFv ligand in gH, manufactured in Examples 3 and 5.

[0172] In order to confirm whether each virus induces viral infection into surrounding cancer cells due to the EpCAMscFv ligand expressed in the glycoprotein gH or due to the adapter and whether it induces cytotoxicity after infection, the following experiment was conducted.

[0173] The cell lines that were used in the experiment were a cell line not expressing EpCAM (Mia-PaCa-2) and cell lines expressing EpCAM (MCF-7, MDA-MB-453, and BT-474). For breast cancer cell lines MCF-7 (ATCC, HTB-22) and BT-474 (ATCC, HTB-20) and a pancreatic cancer cell line Mia-PaCa-2 (ATCC, CRL-1420), culture was performed using DMEM containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS, and for a breast cancer cell line MDA-MB-453 (ATCC, HTB-131), culture was performed using an RPMI medium containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS.

[0174] For an EpCAM-specific viral infection experiment, 4.0×10.sup.4 MCF-7, 8.0×10.sup.4 MDA-MB-453, and 7.0×10.sup.4 BT-474 cell lines were used at 1 MOI and infected with EADa-S expressing the EpCAMscFv-HveA adapter manufactured in Example 3, the EgH-S virus expressing the EpCAMscFv ligand in gH manufactured in Example 4, the dual-targeting EADa-EgH-D virus expressing both the adapter and the ligand, and, as a control, the gDm virus not targeting EpCAM manufactured in Example 2. After 90 minutes, the medium that was used was replaced with a fresh medium in order to remove the remaining initial virus. 2 days after infection, viral infection was confirmed through EmGFP fluorescence expression in each cell line (Baek H.J. et al., Mol. Ther. 2011. 19(3):507-514). Also, in order to measure cytotoxicity for 5 days after infection, the extent of color development of formazan, which is a color-developing material formed only in living cells using an EZ-Cytox (DoGenBio) reagent, was measured at 450 nm using an ELISA reader. Absorbance was quantified to determine the cytotoxicity of cancer cell lines due to each virus.

[0175] The results thereof are shown in FIG. 17. As is apparent from FIG. 17, the infection of BT-474, MDA-MB-453 and MCF7 cells with all of the EADa-S virus expressing only the EpCAM-HveA adapter, the EgH-S virus expressing the EpCAMscFv ligand in gH, and the dual-targeting EADa-EgH-D virus expressing both the adapter and the ligand was observed through fluorescence, and the dual-targeting EADa-EgH-D virus was observed to exhibit a high infection rate compared to the EADa-S and EgH-S viruses. Since the gDm virus was not targeted to EpCAM, it was observed that the cancer cell lines were not infected therewith, and also that there was no viral infection in Mia-PaCa-2 cells not expressing EpCAM.

[0176] In addition, FIG. 18 shows the results of observation of the cytotoxicity of cancer cells due to the virus for 5 days after infection. The EgH-S, EADa-S, and EADa-EgH-D viruses exhibited respective cell viability values of 35%, 34%, and 26% in BT-474, of 22%, 19%, and 17% in MDA-MB-453, and of 36%, 31%, and 20% in MCF-7. In the three cell lines expressing EpCAM, it was observed that the cytotoxicity due to infection with the EADa-EgH-D dual-targeting virus was the highest. However, since the gDm virus was not targeted to EpCAM, cytotoxicity was not observed, and Mia-PaCa-2 not expressing EpCAM was not infected, so any virus was not involved in cytotoxicity.

EXAMPLE 10

Production of HSV-1 having HER2-Targeting Modified Glycoprotein gH and Expressing EpCAM-Targeting Adapter

[0177] For the production of HSV capable of dual targeting of two target molecules (HER2/EpCAM) expressed in specific cancer, a ligand (HER2 scFv) that recognizes HER2 specifically expressed in cancer cells was inserted between amino acids 29 and 30 of the amino acid sequence of gH (GenBank Accession No. ASM47773, SEQ ID NO: 3), which is a glycoprotein of the KOS-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I (EADa-S) virus expressing the EpCAMscFv-HveA adapter.

[0178] A gene capable of expressing HER2scFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD/R222N/F223I (EADa-S) DNA manufactured in Example 3.

[0179] The genomic structure of the KOS-UL3/4_EpCAMscFv-HveA-gH/HER2scFv-EmGFP-gD/R222N/F223I virus in which the HER2scFv ligand was inserted into the gH of HSV-1 is shown in FIG. 19, and the entire sequence of the gH-HER2scFv ligand and the construction of the corresponding sequence are shown in FIG. 8. Here, scFv for HER2 is configured such that VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 are linked via a linker peptide of SEQ ID NO: 24, and the linker peptide of SEQ ID NO: 35 is linked to the N-terminus of this scFv, and the linker peptide of SEQ ID NO: 36 is linked to the C-terminus thereof.

[0180] The amino acid sequence and the gene sequence of the full length of HER2scFv used in the present example are represented in SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0181] Insertion of the gH-HER2scFv ligand was performed according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Example 4.

[0182] Specifically, the E. coli clone containing the KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I DNA manufactured in Example 3 was transformed with a pRed/ET plasmid expressing RecE and RecT capable of performing the function of homologous recombination (Muyrers J. P. et al.; Nucleic Acids Res. 1999. 27(6):1555-1557). A gH29/30-rpsL-neo/kan cassette was manufactured using a set of homologous region primers (forward primer gH29/30-rpsL-neo_for: SEQ ID NO: 39, reverse primer gH29/30-rpsL-neo_rev: SEQ ID NO: 40) including a locus at which to introduce a target gene between amino acids 29 and 30 of gH.

[0183] The clone containing KOS-37/BAC-UL3/4_EpCAMscFv-HveA-EmGFP-gD-R222N/F223I DNA and pRed/ET was added with L-arabinose (Sigma-Aldrich) to thus induce homologous recombination, followed by transformation with 200 ng of the gH29/30-rpsL-neo/kan cassette manufactured as described above. Through such homologous recombination, the gH29/30-rpsL-neo/kan cassette is inserted between amino acids 29 and 30 of gH. E. coli into which gH29/30-rpsL-neo/kan is inserted exhibits kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli obtained from the kanamycin medium that gH29/30-rpsL-neo/kan was inserted therein, and the final step of inserting a target gene was performed.

[0184] E. coli containing the gH29/30-rpsL-neo/kan cassette was added with L-arabinose (Sigma-Aldrich) activating the function of pRed/ET to thus induce homologous recombination, followed by transformation with 200 ng of the gH29/30-HER2scFv ligand. The gH29/30-HER2scFv ligand was manufactured using a forward primer gH29/30-scFv_For (SEQ ID NO: 41) and a reverse primer gH29/30scFv_Rev (SEQ ID NO: 42) using a pCAGGSMCS-gH-HER2scFv plasmid as a template.

[0185] Based on the principle whereby streptomycin resistance blocked by rpsL is activated upon replacing the conventionally inserted gH29/30-rpsL-neo/kan cassette with the above inserted gH29/30-HER2scFv, candidates were selected in a 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 B.C. et al., Methods Enzymol. 1999. 306:337-352). The introduction of HER2scFv at gH29/30 was confirmed through restriction enzyme EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.

[0186] The completed 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), after which 2×10.sup.5 Cre-Vero-HVEM cells were transfected with 1 μg of DNA using a Lipofectamine 2000 reagent (Invitrogen) to remove the BAC gene using Cre recombinase. 3 days after transfection, the fluorescence expression of the EmGFP protein and the formation of cell plaques were observed using a fluorescence microscope. After confirmation of plaque formation, the virus-containing cells were collected, subjected three times to a freeze-thaw process (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206), and sonicated, ultimately obtaining a KOS-UL3/4_EpCAMscFv-HveA-gH_HER2scFv-EmGFP-gD-R222N/F223I dual-targeting virus (EADa-HgH-D) expressing the EpCAMscFv-HveA adapter and the HER2scFv ligand in gH.

EXAMPLE 11

Experiment of Dual-Targeting Effect of HSV-1 having HER2-Targeting Modified Glycoprotein gH and Expressing EpCAM-Targeting Adapter

[0187] In order to confirm induction of infection of cells expressing HER2 and EpCAM proteins with the KOS-UL3/4_EpCAMscFv-HveA-gH/HER2scFv-EmGFP-gD-R222N/F223I dual-targeting virus (EADa-HgH-D) expressing the EpCAMscFv-HveA adapter and the HER2scFv ligand in gH manufactured in Example 10, the following experiment was conducted.

[0188] The cell lines that were used in the experiment were a cell line (CHO-K1) not expressing HER2 and EpCAM, a cell line (CHO-HER2) expressing HER2, and a cell line (CHO-EpCAM) expressing EpCAM. The Chinese hamster ovary cell lines CHO-K1, CHO-HER2, and CHO-EpCAM (Kuroki M et al., J. Biol. Chem. 1991. 74:10132-10141) were cultured using a HaM's F-12K medium (Welgene) containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS (fetal bovine serum).

[0189] For specific viral infection, 2.5×10.sup.4 CHO-K1, CHO-HER2, and CHO-EpCAM cell lines were applied on a 96-well plate. After 24 hours, the HER2 dual-targeting virus (HADa-HgH-D) manufactured in Example 4, the EpCAM dual-targeting virus (EADa-EgH-D) manufactured in Example 5, and the HER2/EpCAM dual-targeting virus (EADa-HgH-D) manufactured in Example 10 were used at 5 MOI for infection. After 90 minutes, the medium that was used was replaced with a fresh medium in order to remove the remaining initial virus and adapter. 2 days after infection, viral infection was observed through fluorescence expression in each cell line (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).

[0190] The results thereof are shown in FIG. 20. As is apparent from FIG. 20, fluorescence microscope images of the cell lines infected with the viruses are shown. It was observed that no viruses infected the CHO-K1 cell line not expressing HER2 and EpCAM. The HER2 dual-targeting virus (HADa-HgH-D) infected only CHO-HER2, and the EpCAM dual-targeting virus (EADa-EgH-D) infected only CHO-EpCAM. However, it was confirmed that the HER2/EpCAM dual-targeting virus (EADa-HgH-D) infected all of CHO-HER2 and CHO-EpCAM cells. Based on the above results, it was possible to confirm the possibility of a strategy to target at least two target molecules using the virus capable of targeting two target molecules together.

EXAMPLE 12

Experiment of Dual-Targeting Effect of HSV-1 Expressing HER2/EpCAM Dual-Targeting Adapter

[0191] In order to confirm induction of infection of cells expressing HER2 and EpCAM proteins with the HER2 and EpCAM dual-targeting virus (EADa-HADa-D) expressing both the EpCAMscFv-HveA adapter and the HER2scFv-HveA adapter manufactured in Example 3, the following experiment was conducted.

[0192] The cell lines that were used in the experiment were a cell line (CHO-K1) not expressing HER2 and EpCAM, a cell line (CHO-HER2) expressing HER2, and a cell line (CHO-EpCAM) expressing EpCAM. The Chinese hamster ovary cell lines CHO-K1, CHO-HER2, and CHO-EpCAM (Kuroki M et al., J. Biol. Chem. 1991. 74:10132-10141) were cultured using a HaM's F-12K medium (Welgene) containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS (fetal bovine serum).

[0193] For specific viral infection, 2.5×10.sup.4 CHO-K1, CHO-HER2, and CHO-EpCAM cell lines were applied on a 96-well plate. After 24 hours, the virus (HADa-S) expressing only the HER2scFv-HveA adapter, the virus (EADa-S) expressing only the EpCAMscFv-HveA adapter, and the dual-targeting virus (EADa-HADa-D) expressing the EpCAMscFv-HveA adapter and the HER2scFv-HveA adapter, manufactured in Example 3, were used at 5 MOI for infection. After 90 minutes, the medium that was used was replaced with a fresh medium in order to remove the remaining initial virus and adapter. 2 days after infection, viral infection was observed through fluorescence expression in each cell line using a fluorescence microscope (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).

[0194] As a result, FIG. 21 shows fluorescence microscope images of the cell lines that were infected with individual viruses. It was observed that no viruses infected the CHO-K1 cell line not expressing HER2 and EpCAM. The virus (HADa-S) expressing only the HER2scFv-HveA adapter infected only CHO-HER2, and the virus (EADa-S) expressing only the EpCAMscFv-HveA adapter infected only CHO-EpCAM. However, it was confirmed that the dual-targeting virus (EADa-HADa-D) expressing both the EpCAMscFv-HveA adapter and the HER2scFv-HveA adapter infected all of CHO-HER2 and CHO-EpCAM cells.

[0195] Based on the above results, it was possible to confirm the possibility of a strategy to target at least two target molecules using the virus expressing the adapter capable of targeting two target molecules together.

[0196] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e).