RECOMBINANT HERPES SIMPLEX VIRUS HAVING MODIFIED GLYCOPROTEIN GH FOR RETARGETING AND USE THEREOF

Abstract

Proposed are a recombinant herpes simplex virus having a modified glycoprotein gH for retargeting and the use thereof. Particularly, the recombinant herpes simplex virus is capable of infecting a target cell having a target molecule to which a cell-targeting domain specifically recognizes and binds due to the presence of the cell-targeting domain in the glycoprotein gH thereof, and is thus useful for anticancer therapy or gene therapy.

Claims

1. A recombinant herpes simplex virus, configured such that a cell-targeting domain, which specifically recognizes and binds to a target molecule of a target cell, is inserted and fused into a glycoprotein gH.

2. (canceled)

3. The recombinant herpes simplex virus of claim 1, wherein the cell-targeting domain is inserted and fused into an N-terminus position, a position within a region of amino acids 12 to 49, or a position within a region of amino acids 116 to 137 in an amino acid sequence of a gH glycoprotein of SEQ ID NO: 1.

4. The recombinant herpes simplex virus of claim 1, wherein the cell-targeting domain is inserted and fused into an N-terminus position, 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 in an amino acid sequence of a gH glycoprotein of SEQ ID NO: 1.

5. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is configured such that an additional cell-targeting domain is inserted into gB, gC or gD.

6. The recombinant herpes simplex virus of claim 1, wherein, when the cell-targeting domain is inserted and fused, a linker peptide is present at an N-terminus and a C-terminus of the cell-targeting domain, and the linker peptide comprises at least one amino acid selected from among Ser, Gly, Ala, and Thr.

7. The recombinant herpes simplex virus of claim 1, wherein the target cell is a diseased cell, and the target molecule is an antigen or a receptor present on a surface of the diseased cell.

8. The recombinant herpes simplex virus of claim 1, wherein the target cell is a cancer cell, and the target molecule is an antigen or a receptor present on a surface of the cancer cell.

9. The recombinant herpes simplex virus of claim 8, wherein the antigen or the receptor is EGFRvIII, 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.

10. The recombinant herpes simplex virus of claim 1, wherein the target molecule is HER2, and the targeting domain is scFv for HER2, 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.

11. The recombinant herpes simplex virus of claim 1, wherein the target molecule is EpCAM, and the targeting domain is scFv for EpCAM, 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.

12. The recombinant herpes simplex virus of claim 1, wherein the target molecule is CEA, and the targeting domain is scFv for CEA, configured such that VL of SEQ ID NO: 8 and VH of SEQ ID NO: 9 are linked in an order of VL, a linker peptide, and VH via the linker peptide.

13. The recombinant herpes simplex virus of claim 10, wherein a linker peptide of SEQ ID NO: 19 is linked to an N-terminus of the scFv, and a linker peptide of SEQ ID NO: 20 is linked to a C-terminus of the scFv.

14. The recombinant herpes simplex virus of claim 11, wherein a linker peptide of SEQ ID NO: 22 is linked to an N-terminus of the scFv, and a linker peptide of SEQ ID NO: 23 is linked to a C-terminus of the scFv.

15. The recombinant herpes simplex virus of claim 12, wherein a linker peptide of SEQ ID NO: 25 is linked to an N-terminus of the scFv, and a linker peptide of SEQ ID NO: 26 is linked to a C-terminus of the scFv.

16. The recombinant herpes simplex virus of claim 1, 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.

17. The recombinant herpes simplex virus of claim 1, 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.

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

19. The recombinant herpes simplex virus of claim 1, 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 inserted into a genome of a herpes simplex virus without inhibiting propagation of the herpes simplex virus.

20. The recombinant herpes simplex virus of claim 19, 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.

21. A pharmaceutical composition for treating cancer, comprising the recombinant herpes simplex virus of claim 1 as an active ingredient.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0076] FIG. 1 schematically shows the genomic structure of KOS-37 BAC;

[0077] FIG. 2 schematically shows the genomic structure of an HVEM-restricted HSV-1 virus;

[0078] FIG. 3 schematically shows the genomic structure of an HVEM-restricted HSV-1 virus expressing EmGFP;

[0079] 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;

[0080] FIG. 5 schematically shows the genomic structure of each of HSV-1 having a modified glycoprotein gH for targeting HER2, HSV-1 having a modified glycoprotein gH for targeting EpCAM, and HSV-1 having a modified glycoprotein gH for targeting CEA;

[0081] FIG. 6 shows the entire amino acid sequence of the HER2scFv, gH-EpCAM2scFv and gH-CEAscFv ligands inserted into the gH glycoprotein and the configuration of the corresponding sequence;

[0082] FIG. 7 shows the results of specific infection and cell death of a HER2-expressing cancer cell by the HSV-1 virus having a modified glycoprotein gH for targeting HER2;

[0083] FIG. 8 shows the results of specific infection and cell death of an EpCAM-expressing cancer cell by the HSV-1 virus having a modified glycoprotein gH for targeting EpCAM; and

[0084] FIG. 9 shows the results of specific infection of a CEA-expressing cancer cell by the HSV-1 virus having a modified glycoprotein gH for targeting CEA.

DETAILED DESCRIPTION

[0085] 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

[0086] 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., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, 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 K O. 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 DH10B bacteria (Invitrogen) (Gierasch W. W. et al., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, 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.

[0087] 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: 10) were substituted with asparagine (N) and isoleucine (I), respectively, was manufactured.

[0088] 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., Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition, J. Virol. 2009. 83(7):2951-2961), and is thus advantageous from the aspect of safety because it cannot infect normal cells having the nectin-1 receptor.

[0089] The genomic structure of the HVEM-restricted KOS-gD-R222N/F223I virus is schematically shown in FIG. 2.

[0090] 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.).

[0091] 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., Rapid modification of bacterial artificial chromosomes by ET-recombination, 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: 11, reverse primer gD-rpsL Rev: SEQ ID NO: 12) 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., Rapid modification of bacterial artificial chromosomes by ET-recombination, 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: 13), 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., Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination. Microb. Cell Fact. 7, 14, 2008). DNA was isolated from the selected candidates using a DNA preparation method (Horsburgh B. C. et al., Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes, 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.

[0092] 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 W. W. et al., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, 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., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, 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

[0093] 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., Engineering herpes simplex viruses by infection-transfection methods including recombination site targeting by CRISPR/Cas9 nucleases, 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.

[0094] The genomic structure of KOS-EmGFP-gD-R222N/F223I expressing EmGFP is schematically shown in FIG. 3.

[0095] 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.

[0096] 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.

[0097] 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., Rapid modification of bacterial artificial chromosomes by ET-recombination, 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: 14, reverse primer UL26/27-rpsL_Rev: SEQ ID NO: 15) 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.

[0098] 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: 16), and a reverse primer UL26/27-pCMV_Rev (SEQ ID NO: 17).

[0099] 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., Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination. Microb. Cell Fact. 7, 14, 2008). DNA was isolated from the selected candidates using a DNA preparation method (Horsburgh B. C. et al., Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes, 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.

[0100] 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., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, J. Virol. Methods. 2006. 135:197-206), and sonicated, thus obtaining a KOS-EmGFP-gD-R222N/F223I virus (gDm).

[0101] 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., Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins. 2017. PLoS Pathog. 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., Insertion of a ligand to HER2 in gB retargets HSV tropism and obviates the need for activation of the other entry glycoproteins. 2017. PLOS Pathog. 13(4):e1006352). Each cell line was cultured in DMEM (Welgene) containing 100 U/ml penicillin/100 pg/ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene). 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., Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells, Mol. Ther. 2011. 19(3):507-514).

[0102] 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.

[0103] 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 entry receptor, without nectin-1.

<Example 3> Production of HSV-1 Having Modified Glycoprotein gH for Targeting HER2, HSV-1 Having Modified Glycoprotein gH for Targeting EpCAM, and HSV-1 Having Modified Glycoprotein gH for Targeting CEA

[0104] For the production of a retargeting HSV capable of targeting a target molecule expressed in specific cancer, a ligand (scFv) that recognizes HER2, EpCAM or CEA 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: 1), which is a glycoprotein of HSV-1. A gene capable of expressing HER2scFv, EpCAMscFv, or CEAscFv was inserted between amino acids 29 and 30 of the glycoprotein gH in the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA into which the EmGFP-expressing cassette (pCMV-EmGFP-tkpA) was inserted, manufactured in Example 2.

[0105] The genomic structures of the KOS-gH/HER2scFv-EmGFP-gD/R222N/F223I virus, the KOS-gH/EpCAMscFv-EmGFP-gD/R222N/F223I virus, and the KOS-gH/CEAscFv-EmGFP-gD/R222N/F223I virus, in which a HER2scFv ligand, an EpCAMscFv ligand, and a CEAscFv ligand, respectively, was inserted into gH of HSV-1, are schematically shown in FIG. 5, and the entire sequence of the HER2scFv ligand, the gH-EpCAM2scFv ligand or the gH-CEAscFv ligand inserted into the gH glycoprotein and the configuration of the corresponding sequence are shown in FIG. 6.

[0106] 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: 18, and the linker peptide of SEQ ID NO: 19 and the linker peptide of SEQ ID NO: 20 are linked to the N-terminus and the C-terminus of the HER2 scFv, respectively. Also, 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: 21, and the linker peptide of SEQ ID NO: 22 and the linker peptide of SEQ ID NO: 23 are linked to the N-terminus and the C-terminus of the EpCAM scFv, respectively. Also, scFv for CEA is configured such that VL of SEQ ID NO: 8 and VH of SEQ ID NO: 9 are linked via a linker peptide of SEQ ID NO: 24, and the linker peptide of SEQ ID NO: 25 and the linker peptide of SEQ ID NO: 26 are linked to the N-terminus and the C-terminus of the CEA scFv, respectively.

[0107] Used in the present example, the amino acid sequence and the gene sequence of the full length of the HER2scFv are represented in SEQ ID NO: 27 and SEQ ID NO: 28, respectively, the amino acid sequence and the gene sequence of the full length of the EpCAMscFv are represented in SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and the amino acid sequence and the gene sequence of the full length of the CEAscFv are represented in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0108] The insertion of the HER2scFv ligand, the EpCAMscFv ligand or the CEAscFv ligand into gH was performed according to the manufacturer's protocol using a counter-selection BAC modification kit (GeneBridges Inc.), as in Examples 1 and 2.

[0109] Specifically, the E. coli clone containing the KOS-37/BAC-EmGFP-gD-R222N/F223I DNA 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., Rapid modification of bacterial artificial chromosomes by ET-recombination, 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: 33, reverse primer gH29/30-rpsL-neo_rev: SEQ ID NO: 34) including a locus at which to introduce a target gene between amino acids 29 and 30 of gH. The clone containing KOS37-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 of KOS-37/BAC-EmGFP-gD-R222N/F223I DNA. 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.

[0110] 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 each of a gH29/30-HER2scFv ligand, a gH29/30-EpCAMscFv ligand and a gH29/30-CEAscFv ligand. The gH29/30-HER2scFv ligand, the gH29/30-EpCAMscFv ligand, and the gH29/30-CEAscFv ligand were manufactured using a forward primer gH29/30-scFv_For (SEQ ID NO: 35) and a reverse primer gH29/30-scFv_Rev (SEQ ID NO: 36) using, as respective templates, a pCAGGSMCS-gH-HER2scFv plasmid, a pCAGGSMCS-gH-EpCAMscFv plasmid, and a pCAGGSMCS-gH-CEAscFv plasmid. The pCAGGSMCS-gH-HER2scFv plasmid, the pCAGGSMCS-gH-EpCAMscFv plasmid, and the pCAGGSMCS-gH-CEAscFv plasmid were manufactured by inserting respective scFv genes into MCS of a pCAGGSMCS plasmid (Atanasiu D. et al., Dual split protein-based fusion assay reveals that mutations to herpes simplex virus (HSV) glycoprotein gB alter the kinetics of cell-cell fusion induced by HSV entry glycoproteins. J. Virol. 2013, November 87(21):11332-11345).

[0111] 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-target scFv, candidates were selected in a streptomycin medium (Heermann R. et al., Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination. Microb. Cell Fact. 7, 14, 2008). DNA was isolated from the selected candidates using a DNA preparation method (Horsburgh B. C. et al., Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes, Methods Enzymol. 1999. 306:337-352). The introduction of each scFv 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.

[0112] The completed KOS-37/BAC-gH/HER2scFv-EmGFP-gD-R222N/F223I DNA, KOS-37/BAC-gH/EpCAMscFv-EmGFP-gD-R222N/F223I DNA, and KOS-37/BAC-gH/CEAscFv-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., Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS, J. Virol. Methods. 2006. 135:197-206), and sonicated, ultimately obtaining a KOS-gH/HER2scFv-EmGFP-gD-R222N/F223I virus (gH-scHER2), a KOS-gH/EpCAMscFv-EmGFP-gD-R222N/F223I virus (gH-scEpCAM), and a KOS-gH/CEAscFv-EmGFP-gD-R222N/F223I virus (gH-scCEA).

<Example 4> Infection and Cytotoxicity of HER2-Expressing Cancer Cells Using HSV-1 Virus Having Modified Glycoprotein gH for Targeting HER2

[0113] In order to confirm whether the KOS-gH/HER2scFv-EmGFP-gD-R222N/F223I virus (gH-scHER2) manufactured in Example 3 induces viral infection into surrounding cancer cells using the HER2scFv ligand expressed in the glycoprotein gH and whether it induces cell death after infection, the following experiment was conducted.

[0114] 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, MCF7, and MDA-MB-231). For breast cancer cell lines MDA-MB-231 (ATCC, HTB-26) and MCF-7(ATCC, HTB-22) and an ovarian cancer cell line SK-OV-3 (ATCC, HTB-77), culture was performed using DMEM containing 100 U/ml penicillin/100 pg/ml streptomycin (Welgene) and 10% FBS. For a breast cancer cell line MDA-MB-453 (ATCC, HTB-131), culture was performed using an RPMI 1640 medium (Welgene) containing 100 U/ml penicillin/100 pg/ml streptomycin (Welgene) and 10% FBS.

[0115] For HER2-specific viral infection, 1×10.sup.4 SK-OV-3 and MDA-MB-231 cell lines were used at 2 MOI and infected with the virus (gH-scHER2) expressing the HER2scFv ligand in gH manufactured in Example 3 and the virus (gDm) not expressing the HER2scFv ligand as a control. 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 fluorescence expression in each cell line. In order to measure cell death for 5 days, the cytotoxicity of cancer cell lines due to each virus after treatment with an EZ-Cytox (DoGenBio) reagent was observed using a fluorescence microscope. 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.

[0116] The results thereof are shown in FIG. 7. As is apparent from FIG. 7, in MDA-MB-231 not expressing HER2, both the gH-scHER2 virus expressing the HER2scFv ligand in gH and the gDm virus not expressing the HER2scFv ligand were uninfected, so fluorescence was not observed. However, it can be confirmed that the HER2-expressing cell lines SK-OV-3, MDA-MB-453, and MCF-7 were specifically infected with the gH-scHER2 virus expressing the HER2scFv ligand, and thus fluorescence was observed.

[0117] In addition, FIG. 7 shows the results of observation of cytotoxicity of cancer cells due to the virus for 5 days after infection. In MDA-MB-231 not expressing HER2, cell death did not occur due to the gH-scHER2 virus expressing the HER2scFv ligand or due to the gDm virus not expressing the HER2scFv ligand. However, SK-OV-3, MDA-MB-453, and MCF-7, which are cell lines expressing HER2, were observed to have cell viability values of 41%, 61%, and 49%, respectively, due only to the gH-scHER2 virus expressing the HER2scFv ligand in gH. In conclusion, it was confirmed that the gH-scHER2 virus expressing the HER2scFv ligand in gH effectively induces specific infection and cytotoxicity of cancer cells expressing HER2.

<Example 5> Infection and Cytotoxicity of EpCAM-Expressing Cancer Cells Using HSV-1 Virus Having Modified Glycoprotein gH for Targeting EpCAM

[0118] In order to confirm whether the KOS-gH/EpCAMscFv-EmGFP-gD-R222N/F223I virus (gH-scEpCAM) manufactured in Example 3 induces viral infection into surrounding cancer cells due to the EpCAMscFv ligand expressed in the glycoprotein gH and whether it induces cell death after infection, the following experiment was conducted.

[0119] 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 pg/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 pg/ml streptomycin (Welgene)) and 10% FBS.

[0120] For EpCAM-specific viral infection, 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 the virus (gH-scEpCAM) expressing the EpCAMscFv ligand in gH manufactured in Example 3 and the virus (gDm) not expressing the EpCAMscFv ligand as a control. 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 fluorescence expression in each cell line (Baek H. J. et al., Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells, 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.

[0121] The results thereof are shown in FIG. 8. As is apparent from FIG. 8, in Mia-PaCa-2 not expressing EpCAM, both the virus (gH-scEpCAM) expressing the EpCAMscFv ligand and the virus (gDm) not expressing the EpCAMscFv ligand, which is a control, were uninfected, so fluorescence was not observed. However, it can be confirmed that the EpCAM-expressing cell lines SK-OV-3, MDA-MB-453, and MCF-7 were specifically infected only with the virus (gH-scEpCAM) expressing the EpCAMscFv ligand, and thus fluorescence was observed.

[0122] In addition, FIG. 8 shows the results of observation of cytotoxicity of cancer cells due to the virus for 5 days after infection. In Mia-PaCa-2 not expressing EpCAM, cell death did not occur due to the virus (gH-scEpCAM) expressing the EpCAMscFv ligand or due to the virus (gDm) not expressing the EpCAMscFv ligand, which is a control. However, the cell lines expressing EpCAM, for example, BT-474, MDA-MB-453, and MCF-7, were observed to have cell viability values of 35%, 22% and 36%, respectively, due only to the virus (gH-scEpCAM) expressing the EpCAMscFv ligand. In conclusion, it was confirmed that the virus expressing the EpCAMscFv ligand in gH effectively induces specific infection and cell death of cancer cells expressing EpCAM.

<Example 6> Infection and Cytotoxicity of CEA-Expressing Cancer Cells Using HSV-1 Virus Having Modified Glycoprotein gH for Targeting CEA

[0123] In order to confirm whether the KOS-gH/CEAscFv-EmGFP-gD-R222N/F223I virus (gH-scCEA) manufactured in Example 3 induces viral infection into surrounding cancer cells due to the CEAscFv ligand expressed in the glycoprotein gH, the following experiment was conducted.

[0124] The cell lines that were used in the experiment were a cell line (CHO-K1) not expressing CEA and a cell line (CHO-CEA) expressing CEA. The Chinese hamster ovary cell lines CHO-K1 and CHO-CEA (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).

[0125] For EpCAM-specific viral infection, 2.5×10.sup.4 CHO-K1 and CHO-CEA cell lines were used at 20 MOI and infected with the virus (gH-scCEA) expressing the CEAscFv ligand in gH manufactured in Example 3 and the virus (gDm) not expressing the EpCAMscFv ligand as a control. 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 fluorescence expression in each cell line (Baek H. J. et al., Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells, Mol. Ther. 2011. 19(3):507-514).

[0126] The results thereof are shown in FIG. 9. As is apparent from FIG. 9, in CHO-K1 not expressing CEA, both the virus (gH-scCEA) expressing the CEAscFv ligand in gH and the virus (gDm) not expressing the EpCAMscFv ligand as a control were uninfected, so fluorescence was not observed. However, in CHO-CEA, which is a cell line expressing CEA, it can be confirmed that fluorescence was observed due to specific infection only with the virus (gH-scCEA) expressing the CEAscFv ligand. In conclusion, it was confirmed that the virus expressing the CEAscFv ligand in gH effectively induces specific infection of CEA-expressing cells.

[0127] 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).