Recombinant herpes simplex virus having expression cassette expressing fused protein of cancer cell-targeting domain and extracellular domain of HVEM and use thereof
11421017 · 2022-08-23
Assignee
Inventors
Cpc classification
C07K2319/33
CHEMISTRY; METALLURGY
A61K39/001129
HUMAN NECESSITIES
C07K2317/14
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12N2710/16643
CHEMISTRY; METALLURGY
A61K47/65
HUMAN NECESSITIES
A61K39/001109
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to a recombinant herpes simplex virus (HSV) containing an expression cassette capable of expressing a fused protein of a cancer-cell-targeting domain and an extracellular domain of HVEM and the use thereof. When the recombinant HSV infects and enters target cells, which are cancer cells, HSV proliferates, and an adapter, which is the fused protein, is expressed in the cells and is released to the outside of the cells along with the proliferated HSV virion upon cell lysis, or is released even before the virion is released due to cell lysis when the adapter contains a leader sequence, and the fused protein released to the outside of the cells acts to induce the HSV virion to infect surrounding cancer cells expressing a target molecule recognized by the cancer-cell-targeting domain or to increase the infection efficiency thereof.
Claims
1. A recombinant herpes simplex virus (HSV), in which an adapter expression cassette expressing a fusion protein of a cancer-cell-targeting domain and an extracellular domain of HVEM is inserted into a genome of the herpes simplex virus without inhibiting proliferation of the herpes simplex virus, wherein the adapter expression cassette expressing the fusion 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, and wherein the gene encoding glycoprotein D (gD) is mutated so as to prevent the encoded gD from binding to nectin-1 while still retaining the ability of said gD to bind to HVEM.
2. The recombinant herpes simplex virus of claim 1, wherein the extracellular domain of HVEM is HveA82, comprising an amino acid sequence of SEQ ID NO: 7 or 8, HveA87, comprising an amino acid sequence of SEQ ID NO: 9 or 10, HveA102, comprising an amino acid sequence of SEQ ID NO: 11 or 12, or HveA107, comprising an amino acid sequence of SEQ ID NO: 13 or 14.
3. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured such that the cancer-cell-targeting domain and the extracellular domain of HVEM are linked via a linker peptide comprising 1 to 30 amino acids.
4. The recombinant herpes simplex virus of claim 3, wherein the linker peptide comprises at least one amino acid selected from among Ser, Gly, Ala and Thr.
5. The recombinant herpes simplex virus of claim 1, wherein the cancer-cell-targeting domain is a domain that recognizes and binds to a target molecule of a cancer cell that is a target cell, and the target molecule is an antigen or a receptor on a surface of the cancer cell, which is expressed only in the cancer cell or is overexpressed in the cancer cell compared to a normal cell.
6. The recombinant herpes simplex virus of claim 5, 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), 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 lysis-inducing ligand receptor), VEGFR2 (vascular endothelial growth factor receptor 2), HGFR (hepatocyte growth factor receptor), CD44 or CD166.
7. The recombinant herpes simplex virus of claim 1, wherein the cancer-cell-targeting domain is a domain that recognizes and binds to HER2, which is a target molecule of a cancer cell that is a target cell, and the domain is an scFv in which VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2 are linked in an order of VH, a linker peptide and VL via the linker peptide.
8. The recombinant herpes simplex virus of claim 7, wherein the linker peptide comprises an amino acid sequence of SEQ ID NO: 5.
9. The recombinant herpes simplex virus of claim 1, wherein the cancer-cell-targeting domain is a domain that recognizes and binds to CEA, which is a target molecule of a cancer cell that is a target cell, and the domain is an scFv in which VL of SEQ ID NO: 3 and VH of SEQ ID NO: 4 are linked in an order of VL, a linker peptide and VH via the linker peptide.
10. The recombinant herpes simplex virus of claim 9, wherein the linker peptide comprises an amino acid sequence of SEQ ID NO: 6.
11. 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 the amino acid sequence of gD (glycoprotein D) comprising the sequence of SEQ ID NO: 15 are substituted with asparagine (N) and isoleucine (I), respectively.
12. 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.
13. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is recombinant HSV-1 derived from an HSV-1 KOS strain.
14. The recombinant herpes simplex virus of claim 1, further including a second expression cassette comprising a gene expressing any one selected from among (i) cytokine, (ii) chemokine, (iii) an antagonist to an immune checkpoint, (iv) a co-stimulatory factor inducing activation of an immune cell, (v) an antagonist to TGFβ inhibiting an immune response to a cancer cell, (vi) heparinase degrading heparan sulfate proteoglycan for a solid tumor microenvironment, (vii) an antagonist inhibiting a function of an angiogenic receptor VEGFR-2 (VEGF receptor-2), and (viii) a prodrug-activating enzyme converting a prodrug into a drug that is toxic to a cancer cell is further inserted into the genome of the herpes simplex virus without inhibiting proliferation of the herpes simplex virus.
15. The recombinant herpes simplex virus of claim 14, wherein the cytokine is at least one selected from among interleukins (ILs), interferons (IFNs), TNFα, GM-CSF, and G-CSF, wherein the IL is selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-24, and wherein the IFN is selected from the group consisting of IFNα, IFNβ, and IFNγ, and 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), KIRs (killer-cell immunoglobulin-like receptors), 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 CD3Σ, 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.
16. The recombinant herpes simplex virus of claim 14, wherein the second 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 adapter expression cassette of the fusion protein.
17. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured in an order of NH.sub.2/cancer-cell-targeting domain/extracellular domain of HVEM/COOH or extracellular domain of HVEM/cancer-cell-targeting domain.
18. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured such that the cancer-cell-targeting domain and the extracellular domain of HVEM are linked via a linker peptide, and the fusion protein is configured in an order of NH.sub.2/cancer-cell-targeting domain/linker peptide/extracellular domain of HVEM/COOH or extracellular domain of HVEM/cancer-cell-targeting domain.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
(14) A better understanding of the present invention will be given through the following examples. However, these examples are not to be construed as limiting the scope of the present invention.
<Example 1> Production of HVEM-Restricted HSV-1
(15) An HSV-1 gene is composed of a large gene of about 152 kb, 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 introduce a mutation into a specific locus. An 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 gene, enables cloning at the bacterial level through transformation of DH10B bacteria (Invitrogen) (Gierasch W. W. et al.; J. Virol. Methods. 2006. 135:197-206). In the KOS-37 BAC, a bacterial artificial chromosome (BAC) 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 designed to remove the BAC gene using a Cre-Lox system in subsequent procedures. The schematic view thereof is shown in
(16) 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: 15) are substituted with asparagine (N) and isoleucine (I), respectively, was manufactured.
(17) 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).
(18) The genomic structure of HVEM-restricted HSV-1 is schematically shown in
(19) The 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.).
(20) 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: 16, reverse primer gD-rpsL Rev: SEQ ID NO: 17) including a locus to introduce a mutation into gD. The gD-rpsL-neo/kan cassette is composed of the rpsL gene, which is a selective marker that confers sensitivity to streptomycin, and the gD homologous region at the insertion locus, and the neo/kan gene that confers kanamycin resistance. When the gD-rpsL-neo/kan cassette is inserted, E. coli having sensitivity to streptomycin antibiotics by the rpsL gene and kanamycin resistance by 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 pRedET (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 has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli selected 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, which is an oligonucleotide encoding gD (SEQ ID NO: 18) substituted with N and I at positions 222 and 223 of gD, was performed. Based on the principle whereby streptomycin resistance blocked by rpsL is activated while 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). In the selected candidates, DNA was isolated 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.
(21) Next, for viral production, the completed KOS-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 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 DNA transfection, the formation of a plaque 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 Expressing EmGFP
(22) An expression cassette capable of expressing EmGFP (Emerald Green Fluorescent Protein) was inserted into the gene UL26/UL27 locus of the KOS-gD-R222N/F223I virus manufactured in Example 1 above. 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.
(23) The genomic structure of HVEM-restricted HSV-1 expressing UL26/27-EmGFP is schematically shown in
(24) For EmGFP expression, pCMV-EmGFP-tkpA using a gene promoter of cytomegalovirus and tkpA as a polyadenylation signal of HSV TK (herpes simplex virus thymidine kinase) was inserted into KOS-BAC-gD-R222N/F223I.
(25) All insertion methods were carried out according to the manufacturer's protocol using a counter selection BAC modification kit (GenBridges. Inc) as in Example 1 above.
(26) Specifically, an E. coli clone containing the KOS-BAC-gD-R222N/F223I genome 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: 19, reverse primer UL26/27-rpsL_Rev: SEQ ID NO: 20) including a locus to introduce a target gene between UL26 and UL27. The clone containing KOS-BAC-gD-R222N/F223I 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 was inserted into the UL26/27 locus of KOS-BAC-gD-R222N/F223I through homologous recombination. E. coli into which UL26/27-rpsL-neo/kan was inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli selected from the kanamycin medium that UL26/27-rpsL-neo/kan was inserted therein, and the final step of inserting a gene was performed.
(27) 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 the 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-tkpA_For (SEQ ID NO: 21) and a reverse primer UL27-pCMV_Rev (SEQ ID NO: 22).
(28) Based on the principle whereby streptomycin resistance blocked by rpsL is activated while 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 in UL26/27 was confirmed through EcoRI and Xhol treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified through sequencing of the PCR product.
(29) An experiment was conducted for normal expression of a fluorescent protein and production of a virus. The completed KOS-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). 3 days after transfection, the fluorescence expression of EmGFP was observed using a fluorescence microscope, and viral production was observed through the plaque formation of Cre-Vero-HVEM cells. After confirmation of the 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.
(30) 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 HVEM and nectin-1 as virus HSV-1 receptors (Petrovic B. et al., 2017. PLoS Pathog. 19; 13(4):e1006352). The 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, 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 hr, 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).
(31) The results thereof are shown in
(32) Based on the above results, it was confirmed that the infection with the KOS-EmGFP-gD-R222N/F223I virus 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, rather than nectin-1.
<Example 3> Production of KOS-EmGFP-Gd-R222N/F223I Virus Expressing HER2scFv-HveA Adapter, HveA-HER2scFv Adapter, and CEAscFv-HveA Adapter
(33) Each of a HER2scFv-HveA adapter-expressing cassette, an HveA-HER2scFv adapter-expressing cassette, and a CEAscFv-HveA adapter-expressing cassette were inserted into the gene UL3/UL4 locus of the KOS-EmGFP-gD-R222N/F223I virus having the EmGFP expression cassette (tkpA-EmGFP-pCMV) inserted therein, manufactured in Example 2.
(34) The KOS-HER2scFv-HveA-EmGFP-gD/R222N/F223I virus genome into which pCMV-HER2scFv-HveA-bGHpA as the HER2scFv-HveA adapter-expressing cassette was inserted, the KOS-HveA-HER2scFv-EmGFP-gD/R222N/F223I virus genome into which pCMV-HveA-HER2scFv-bGHpA as the HveA-HER2scFv adapter-expressing cassette was inserted, and the KOS-CEAscFv-HveA-EmGFP-gD/R222N/F223I virus genome into which pCMV-CEAscFv-HveA-bGHpA as the CEAscFv-HveA adapter-expressing cassette was inserted are schematically shown in
(35) After the scFv sequence for HER2 or CEA and the NH.sub.2-GGGGS sequence, which is the linker sequence of the HveA sequence, EF (base sequence: GAATTC), which is a restriction enzyme EcoRI site for easy cloning, is added, and after the HveA sequence and the NH.sub.2-GGGGS sequence, which is the linker sequence of the scFv sequence for Her2, GS (base sequence: GGATCC), which is a restriction enzyme BamHI site for easy cloning, is added, and is also a sequence that may be excluded from the adapter. bGHpA is a bGH-PolyA (bovine growth hormone polyadenylation) signal sequence. The amino acid sequence and gene sequence of the full length of the HER2scFv-HveA adapter used in this example are represented in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, the amino acid sequence and gene sequence of the full length of the HveA-HER2scFv adapter are represented in SEQ ID NO: 25 and SEQ ID NO: 26, respectively, and the amino acid sequence and gene sequence of the full length of the CEAscFv-HveA adapter are represented in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.
(36) The insertion of the HER2scFv-HveA adapter-expressing cassette, the HveA-HER2scFv adapter-expressing cassette, and the CEAscFv-HveA 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.
(37) Specifically, the E. coli clone containing the KOS-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 HSV-1_UL3/4-rpsL-neo_for: SEQ ID NO: 29, reverse primer HSV-1_UL3/4-rpsL-neo_rev: SEQ ID NO: 30) including a locus to introduce a target gene between UL3 and UL4. The clone containing KOS-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 above. Through such homologous recombination, the UL3/4-rpsL-neo/kan cassette was inserted into the UL3/4 locus of KOS-BAC-EmGFP-gD-R222N/F223I. E. coli into which UL3/4-rpsL-neo/kan was inserted has kanamycin resistance, but streptomycin resistance is blocked by the rpsL gene. It was inferred for E. coli selected from the kanamycin medium that UL3/4-rpsL-neo/kan was inserted therein, and the final step of inserting a target gene was performed.
(38) 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-HveA-Her2scFv-bGHpA cassette and the UL3/4-pCMV-CEAscFv-HveA-bGHpA cassette. The UL3/4-pCMV-Her2scFv-HveA-bGHpA cassette, the UL3/4-pCMV-HveA-Her2scFv-bGHpA cassette and the UL3/4-pCMV-CEAscFv-HveA-bGHpA cassette were manufactured using a forward primer HSV-1_UL3/4-HM_pCMV_For (SEQ ID NO: 31) and a reverse primer UL3/4_bGH_poly_R (SEQ ID NO: 32) using, as a template, a pCDNA3.1-HER2scFv-HveA plasmid, a pCDNA3.1-HveA-HER2scFv plasmid, and a pCDNA3.1-CEAscFv-HveA plasmid (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(39) Based on the principle whereby streptomycin resistance blocked by rpsL is activated while replacing the conventionally inserted UL3/4-rpsL-neo/kan cassette with the above inserted UL3/4-pCMV-Her2scFv-HveA-bGHpA, UL3/4-pCMV-HveA-Her2scFv-bGHpA and UL3/4-pCMV-CEAscFv-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-HveA-Her2scFv-bGHpA and UL3/4-pCMV-CEAscFv-HveA-bGHpA into 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.
(40) The completed KOS-BAC-Her2scFv-HveA-EmGFP-gD-R222N/F223I, KOS-BAC-HveA-Her2scFv-EmGFP-gD-R222N/F223 and KOS-BAC-CEAscFv-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). 3 days after transfection, the fluorescence expression of EmGFP and the plaque formation of cells were observed using a fluorescence microscope. After confirmation of the 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-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus, a KOS-HveA-Her2scFv-EmGFP-gD-R222N/F223I virus, and a KOS-CEAscFv-HveA-EmGFP-gD/R222N/F223I virus.
<Example 4> Targeting of HER2- or CEA-Expressing Cancer Cells Using Adapter-Expressing Oncolytic Virus
<Example 4-1> Targeting of HER2-Expressing Cancer Cells Using HER2scFv-HveA Adapter-Expressing Oncolytic Virus
(41) In order to evaluate whether viral infection of surrounding cancer cells or lysis after infection is induced by expressing the HER2scFv-HveA adapter using the HER2scFv-HveA adapter-expressing KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus manufactured in Example 3, the following experiment was carried out.
(42) The cell lines that were used in the experiment were cell lines (J1, CHO-K1, MDA-MB-231) not expressing HER2 and cell lines (J-HER2, CHO-HER2, SK-OV-3) expressing HER2. The Chinese hamster ovary cell lines CHO-K1 and CHO-HER2 (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), and J1 and J-HER2 (Petrovic B et al., 2017. PLoS Pathog. 19; 13(4):e1006352), a breast cancer cell line MDA-MB-231 (ATCC, HTB-26), and an ovarian cancer cell line SK-OV-3 (ATCC, HTB-77) were cultured using DMEM containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS.
(43) For HER2-specific viral infection, 1×10.sup.4 J cell lines at 10 MOI, 1.5×10.sup.4 CHO cell lines at 1 MOI, and 1×10.sup.4 SK-OV-3 and MDA-MB-231 cell lines at 0.1 MOI were infected with the HER2scFv-HveA adapter-expressing virus manufactured in Example 3. After 90 min, the medium was replaced with a fresh medium to remove the remaining early virus and the HER2scFv-HveA adapter. 3 days after infection, viral infection of each cell line was observed through fluorescence expression using a fluorescence microscope (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(44) The results thereof are shown in
(45) Also, CHO-Her2, J-Her2, and SK-OV-3, which are cell lines expressing HER2, were first infected with the virus despite the lack of the HVEM receptor. The reason for this is that the HER2scFv-HveA adapter-expressing virus manufactured in Example 3 used for infection contains a trace amount of the HER2scFv-HveA adapter bound to gD of the virus or the expressed HER2scFv-HveA adapter during viral production through Cre-Vero-HVEM cells.
(46) In order to observe lysis caused by the virus, 1.5×10.sup.4 CHO-K1 and CHO-Her2 cell lines were infected at 1 MOI with the HER2scFv-HveA adapter-expressing virus, and 1×10.sup.4 SK-OV-3 and MDA-MB-231 cell lines were infected at 2 MOI with the HER2scFv-HveA adapter-expressing virus. 3 days after infection, each cell line was observed using an optical microscope. The results thereof are shown in
(47) Also, 1×10.sup.4 SK-OV-3 cells, which are the cell line expressing HER2, were infected at 2 MOI with each of an HVEM-restricted herpes virus (gD/NI) and a HER2scFv-HveA adapter-expressing virus (Her2 Adapter), and lysis was observed on the 1.sup.st, 2.sup.nd, 3.sup.rd, 4.sup.th and 5.sup.th day through cell staining using Alamar blue (Sigma). The results thereof are shown in
<Example 4-2> Targeting of CEA-Expressing Cancer Cells Using CEAscFv-HveA Adapter-Expressing Oncolytic Virus
(48) In order to evaluate whether viral infection of surrounding cancer cells is induced by expressing CEAscFv-HveA using the CEAscFv-HveA adapter-expressing KOS-CEAscFv-HveA-EmGFP-gD/R222N/F223I virus manufactured in Example 3, the following experiment was carried out.
(49) The cell lines that were used in the experiment were cell lines (CHO-K1) not expressing CEA and cell lines (CHO-CEA, MKN45) expressing CEA. The Chinese hamster ovary cell lines CHO-K1 and CHO-CEA cell lines (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, and a stomach cancer cell line MKN45 (JCRB, JCRB0254) was cultured using an RPMI-1640 medium containing 100 U/ml penicillin/100 μg/ml streptomycin (Welgene) and 10% FBS (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(50) For CEA-specific viral infection, 1.5×10.sup.4 CHO-K1 and CHO-CEA cells were infected at 10 MOI with the virus. After 90 min, the medium was replaced with a fresh medium to remove the remaining early virus and the CEAscFv-HveA adapter. After 72 hr, the extent of viral infection of each cell line was observed using a fluorescence microscope.
(51) The results thereof are shown in
(52) The reason why the CEAscFv-HveA-expressing gD R222N/F223I mutated virus causes slight infection of the CHO-K1 cell line, lacking in HVEM and nectin-1, is that, as described above, slight infection also occurs through cell attachment of gB and gC, in addition to the function of gD (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(53) Also, in order to confirm specific infection of CEA-expressing cancer cells, 6×10.sup.4 MKN45 cell lines were infected at 1 MOI with each of the KOS-EmGFP-gD-R222N/F223I virus (gD/NI, control) manufactured in Example 2 and the KOS-CEAscFv-HveA-EmGFP-gD-R222N/F223I (scCEA-HveA) obtained in Example 3. After 90 min, the medium was replaced with a fresh medium to remove the remaining early virus and the CEAscFv-HveA adapter. After 72 hr, the extent of viral infection of each cell line was observed using a fluorescence microscope.
(54) The results thereof are shown in
<Example 5> Change in Infection Activity of Adapter-Expressing Oncolytic Virus Depending on Structural Change of HER2 Adapter
(55) In order to evaluate changes in the infection activity depending on the adapter structure by expressing the HER2scFv-HveA adapter or the HveA-HER2scFv adapter using the HER2scFv-HveA adapter-expressing KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus and the HveA-HER2scFv adapter-expressing KOS-HveA-HER2scFv-EmGFP-gD-R222N/F223I virus, having different adapter structures manufactured in Example 3, the following experiment was carried out.
(56) The cell lines that were used in the experiment were CHO-K1 cell lines not expressing HER2 and CHO-HER2 cell lines expressing HER2. The Chinese hamster ovary cell lines CHO-K1 and CHO-HER2 (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).
(57) In order to evaluate changes in the activity of viral infection depending on the adapter structure, 1.5×10.sup.4 CHO-K1 and CHO-HER2 cell lines were infected at 1 MOI with each of the HER2scFv-HveA adapter-expressing virus and the HveA-HER2scFv adapter-expressing virus manufactured in Example 3. After 90 min, the medium was replaced with a fresh medium to remove the remaining early virus and the HER2scFv-HveA and HveA-HER2scFv adapters. 3 days after infection, the cells were stained with VP16, which is a protein essential for transcription of the early gene of the virus, and the infection with the adapter-expressing viruses having different adapter structures was observed using a fluorescence microscope (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(58) With reference to the images on the right of
(59) The reason why the HER2scFv-HveA or HveA-HER2scFv-expressing gD R222N/F223I mutated virus causes slight infection of the CHO-K1 cell line, lacking in HVEM and nectin-1, is that, as described above, slight infection also occurs through cell attachment of gB and gC, in addition to the function of gD (Baek H. J. et al., Mol. Ther. 2011. 19(3):507-514).
(60) Also, CHO-Her2, which is the HER2-expressing cell line, was first infected with the virus despite the lack of the HVEM receptor. The reason for this is that the HER2scFv-HveA or HveA-HER2scFv adapter-expressing virus manufactured in Example 3 used for infection contains a trace amount of the HER2scFv-HveA adapter or HveA-HER2scFv adapter bound to gD of the virus or the expressed HER2scFv-HveA during viral production through Cre-Vero-HVEM cells.
<Example 6> Extracellular Expression of Adapter of Adapter-Expressing Virus and Observation of Viral Spreading to Surrounding Cancer Cells
(61) In order to confirm the release of the HER2scFv-HveA adapter expressed in the cells by the HER2scFv-HveA adapter-expressing KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus manufactured in Example 3 to the outside of the cells, the following experiment was carried out.
(62) The cell line that was used in the experiment was a Vero-HVEM cell line (Gierasch et al.; J. Virol. Methods. 2006. 135:197-206). The Vero-HVEM cell line was cultured 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). 2.0×10.sup.5 Vero-HVEM cells were infected at 0.1 MOI with KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I and, as a control, a KOS-EmGFP-gD-R222N/F223I virus. After 90 min, the medium was replaced with a fresh medium not containing FBS to remove the remaining early virus and the HER2scFv-HveA adapter. After 48 hr, the medium was collected. The extent of protein expression was measured through Western blotting in order to confirm the HER2scFv-HveA adapter expression of the collected medium.
(63) The results thereof are shown at the top of
(64) Based on the above results, it was confirmed that the expression of the inserted adapter and the release thereof to the outside of the cells proceeded efficiently through intracellular infection of the KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus, as intended.
(65) Moreover, in order to observe spreading of viral infection to surrounding cancer cells due to cell lysis and additionally due to the adapter released to the outside of the cells, the following experiment was carried out.
(66) The cell line that was used in the experiment was a SK-OV-3 cell line. As an ovarian cancer cell line, the SK-OV-3 cell line was cultured 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).
(67) And then, 1×10.sup.4 SK-OV-3 cell lines were diluted and infected at 0.01 MOI with a KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus. The reason for viral dilution and infection is to observe spreading of viral infection to surrounding cancer cells. The viral infection and spreading was observed using a fluorescence microscope.
(68) The results thereof are shown at the bottom of
(69) Based on the above results, as intended, through intracellular infection of the KOS-Her2scFv-HveA-EmGFP-gD-R222N/F223I virus, the inserted adapter was released to the outside of the cells and was bound to the antigen on the surface of the surrounding cancer cells, and the pattern whereby the virus released due to cell lysis targeted the adapter bound to the cancer cells or spread infection to the surrounding cancer cells expressing a target molecule using the adapter bound to the virus was confirmed.
(70) Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
(71) 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).