Epithelial cell adhesion moleculeを介して感染する受容体標的化腫瘍溶解性HSVの開発

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2 1 1-1 1-2 1-3 1-4 oHSV

Epithelial cell adhesion molecule EpCAM

4 2 2-1 2-2 2-3 2-4 2-5 2-1-1 2-1-2 2-3-1 2-3-2 2-4-1 2-4-2 2-5-1 2-5-2 2-5-3 2-5-4 EpCAM gD EpCAM gD EpCAM gD EpCAM EpCAM oHSV EpCAM oHSV

EpCAM oHSV EpCAM

EpCAM oHSV

EpCAM oHSV EpCAM

BxPC-3 EpCAM EpCAM

oHSV

EpCAM oHSV

EpCAM oHSV EpCAM HSV EpCAM oHSV EpCAM EpCAM oHSV EpCAM EpCAM oHSV EpCAM

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1

1-1

HSV Miest and

Cattaneo, 2014; Russell et al., 2012; Lichty et al., 2014

HSV oncolytic HSV; oHSV infected cell polypeptide ICP 34.5 ICP6 Chou et al., 1990; Mineta et al., 1995; MacLean et al., 1991; Kemeny et al., 2006; Hu et al., 2006; Todo et al., 2001

oHSV

Markert et al., 2000 HSV nectin-1

HSV Huang et al., 2007; Yu et

al., 2007 oHSV

1-2 oHSV

HSV

Cai et al., 1988; Ligas et al., 1988; Desai et al., 1988; Hutchinson et al., 1992 B glycoprotein B; gB C gC

Shukla and Spear, 2001

D gD herpesvirus entry mediator HVEM nectin-1 3-O-sulfated heparin sulfate Montgomery et al., 1996; Geraghty et al., 1998; Shukla et al., 1999 gD gD

gB H gH / L gL Carfi et al,

2001; Fusco et al., 2005; Krummenacher et al., 2005

gD gD

epidermal growth factor receptor EGFR carcinoembryonic antigen CEA

HSV Uchida et al., 2013 gD 2-24

HVEM 38

nectin-1 1 25 EGFR

CEA scFv

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Uchida et al., 2012 oHSV oHSV

1-3 Epithelial cell adhesion molecule EpCAM

EpCAM 1 1979

Herlyn et al., 1979

Patriarca et al., 2012

EpCAM ×

EpCAM Gires et al., 2009

EpCAM

Armstrong et al., 2003; Chaudry et al., 2007; Liljefors et al., 20005; Riethmuller et al., 1998; Weiner et al., 1988; Marme et al., 2002 EpCAM CD3

Heiss et al., 2010

1-4

oHSV

EpCAM oHSV

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2

2-1 EpCAM gD

EpCAM EpCAM scFv gD EpCAM

2-1-1 EpCAM gD

EpCAM scFv EpCAM MY24 Yamaguchi

et al., 2014 RNA cDNA cDNA PCR

VL VH VL VH 15 scFv gD 1 25 EpCAM gD 3 2-1-2 EpCAM gD EpCAM EpCAM gD HSV EpCAM 4 gD gD HSV gD CHO-nectin-1 5 gD:d2-24/Y38C gD HSV

CHO-nectin-1 EGFP Y38C nectin-1

5 EGFR gD gD

HSV CHO-EGFR 5 CHO-nectin-1 EGFP

EpCAM gD CHO-EpCAM

5 EpCAM MY24 scFv gD

EpCAM gD EpCAM HSV

2-2 EpCAM oHSV

EpCAM gD HSV EpCAM

oHSV bacterial artificial chromosome BAC

EpCAM oHSV BAC pKB3G-gB:N/T-gDscEpCAM

6 UL 3 UL 4 EGFP

UL 27 gB gB:D285N/A549T Uchida et al., 2010

UL 37 UL 38 loxP BAC

pKB3G-gB:N/T Uchida and Glorioso et al., unpublished US 6 gD EpCAM

gD gD:d2-24/Y38C-scEpCAM BAC Hind III

BstB I BamH I Xho I

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loxP BAC 6 EpCAM oHSV 2

EpCAM gD HSV HSV EpCAM gD

EpCAM oHSV EpCAM

HSV

EpCAM oHSV

2-3-1 EpCAM oHSV EpCAM

gD EGFR EpCAM

CHO-K1 CHO-nectin-1 CHO-HVEM CHO-EGFR CHO-EpCAM

HSV EGFR oHSV EpCAM oHSV

HSV gD CHO-K1 CHO-EGFR CHO-EpCAM

gD CHO-HVEM CHO-nectin-1

7 A EGFR oHSV CHO-K1 CHO-HVEM CHO-nectin-1

EGFR CHO-EGFR

7A EpCAM oHSV CHO-K1 CHO-HVEM CHO-nectin-1

CHO-EGFR EpCAM CHO-EpCAM

7A

B78H1 EpCAM

oHSV EpCAM B78-EpCAM 7B

EpCAM oHSV gD HVEM nectin-1

EpCAM gD EpCAM

EpCAM oHSV EpCAM

EpCAM EpCAM oHSV

Vero/EpCAM IgG1 MY24 1

HSV EpCAM oHSV 8A HSV MY24 EpCAM oHSV 10 µg/mL 100 µg/mL MY24 EGFP 10 µg/mL 100 µg/mL EpCAM BxPC-3 12

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EpCAM oHSV EpCAM 2-4 EpCAM oHSV HSV EpCAM oHSV EpCAM EpCAM EpCAM EpCAM oHSV

HSV EpCAM oHSV gD EpCAM 9 HSV gD B78H1 B78-EpCAM EGFP gD B78-HVEM B78-nectin-1 10

EpCAM oHSV B78H1 B78-HVEM B78-nectin-1 EGFP

EpCAM B78-EpCAM 10

EpCAM oHSV EpCAM

2-4-2 BxPC-3 EpCAM EpCAM oHSV

EpCAM EpCAM oHSV

EpCAM BxPC-3

BxPC-3 EpCAM

short interfering RNA siRNA

EpCAM siRNA 4 11A BxPC-3

EpCAM

siRNA siEpCAM EpCAM HVEM nectin-1 EGFR

11B EpCAM

oHSV

2-4-1 HSV EGFR oHSV EpCAM oHSV

HSV EGFR oHSV siEpCAM

BxPC-3 siRNA BxPC-3

11C EpCAM oHSV siEpCAM BxPC-3

11C

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EpCAM oHSV BxPC-3 EpCAM

EpCAM oHSV EpCAM

HSV

EpCAM oHSV

2-5-1 EpCAM oHSV EpCAM HSV

7 HVEM nectin-1 EGFR EpCAM

12 7 EpCAM

EGFR 4

12

HSV 7 13 EGFR

oHSV EGFR EGFR

HSV 13 EpCAM

oHSV EpCAM EpCAM HSV

13

EpCAM oHSV EpCAM

HSV 7 14 EGFR

oHSV EGFR U87

HSV 14 EpCAM oHSV EpCAM

HuCCT1 HepG2 SK-OV-3 HSV

14 EpCAM oHSV EpCAM

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HSV EGFR oHSV EpCAM oHSV HSV 7

EGFR oHSV EpCAM oHSV MOI 3

15 EpCAM oHSV EpCAM

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3

HSV gD oHSV

oHSV scFv

EpCAM scFv gD EpCAM

oHSV EpCAM oHSV

3-1 oHSV

HSV

Mineta et al., 1995 oHSV

interleukin-13 receptor α2

Roizman Zhou and Roizman, 2006 human epidermal growth factor receptor 2 HER2 Campadelli-Fiume Menotti et al., 2008; Menotti et al., 2009

EGFR CEA Uchida et al., 2013

gD EGFR CEA

EpCAM

EpCAM Patriarca et al., 2012 EpCAM

Winter et al., 2003 EpCAM

micro RNA

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3-3 oHSV oHSV HSV gB gD gH gL gD Rauch gD gD Rauch et al., 2000 gD

Babic et al., 1993; Peeters et al., 1992; Peeters et al., 1993; Rauh et al., 1991 gE gI HSV-1

Johnson and Huber, 2002,

gD gB:N/T

gH:N753K/A778V Uchida et al., 2013

gB gH oHSV EpCAM oHSV

EpCAM oHSV HepG2 HuCCT1 SK-OV-3 HSV

EGFR CEA

Uchida et al., 2013 HSV EpCAM oHSV

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4

4-1

Vero ATCC CCL-81 Vero/Cre DMEM Nacalai

Tesque 5 % FBS 1 % 37

5 % CO2

Vero/Cre-EpCAM Cre/EpCAM Vero PLAT-A

pMXc-puro-hEpCAM pSYM-4 Lipofectamin 2000 Life Technologies

hEpCAM Vero/Cre

David Leib 4 µg/mL puromycin

Vero/Cre-EpCAM DMEM 5 % FBS 1 % 4 µg/mL puromycin 37 5 % CO2

Vero/EpCAM EpCAM Vero PLAT-A

pSYM-4 Lipofectamin 2000 Life Technologies

hEpCAM Vero 4

µg/mL puromycin Vero/EpCAM

DMEM 5 % FBS 1 % 4 µg/mL puromycin

37 5 % CO2

CHO-K1 ATCC CCL-61 CHO-nectin-1 nectin-1 CHO-K1 Geraghty et al., 1998 CHO-HVEM HVEM CHO-K1

Montgomery et al., 1996 CHO-EGFR EGFR CHO-K1 Nakamura et al., 2004

F12K Gibco 10 % FBS 1 % 1 %

37 5 % CO2

CHO-EpCAM EpCAM CHO-K1 CHO-K1

pTARGET-hEpCAM Lipofectamine LTX & Plus Reagent Life Technologies 800 µg/mL G418 Wako

CHO-EpCAM F12K Gibco 10 % FBS 1 %

1 % 37 5 % CO2

B78H1 Gary Cohen B78-nectin-1 nectin-1 B78H1 Uchida et al., 2009 B78-HVEM HVEM B78H1 Uchida et al., 2009 DMEM 5 % FBS 1%

37 5 % CO2

B78-EpCAM EpCAM B78H1 PLAT-A

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hEPCAM Vero

4 µg/mL puromycin B78-EpCAM

DMEM 5 % FBS 1 % 4 µg/mL

puromycin 37 5 % CO2

SW620 ATCC CCL-227 BxPC-3 ATCC CRL-1469

HuCCT1 RBRC-RCB1960 RPMI1640 Nacalai Tesque 10 % FBS

1 % 1 % 37 5 %

CO2

HepG2 ATCC HB-8065 SK-OV-3 ATCC HTB-77

U87 ATCC HTB-14 A2058 ATCC CRL-11147

PLAT-A Platinum-amphotropic gag pol 293T DMEM 10 % FBS 1 % 37 5 % CO2 pTAC-2 TA pSP72 pTARGET-hEpCAM CMV hEpCAM

PC3 mRNA cDNA cDNA hEpCAM

open reading frame ORF EpCAM #2231EpCAM-158~180 5’-TCTTCTCGGCGCGCGCGCAGCAT-3’/ #2232EpCAM-1170~1144 5’-GTGTCCATTTGCTATTTC CCTTCTTCT-3’ NM_002354 PCR PCR pTARGET

TA

pMXc-puro-hEpCAM pSYM-4 hEpCAM

pTarget-hEpCAM Xba I/ Sal I hEpCAM pSP72 Nhe I/ Sal I pSP72-hEpCAM pSYM-3 pSYM-3 BamH I/ Hind III hEpCAM

pYO-14*1 _{Hind III/ Avr II Avr II/ BamH I} *1_{pYO-14 pMXc-puro-IL13Rp2}

A375 mRNA cDNA cDNA IL13Rα2

ORF IL13Rα2 #2275 h IL13Rα2 F

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TGGAAAGTCTTC-3’ PCR PCR pTARGET TA pTARGET-h IL13Rα2 pTARGET-h IL13Rα2

Nhe I/ Sal I IL13Rα2 T4 DNA ligase

pMXc-puro

BamH I T4 DNA ligase

pMXc-puro IL13Rα2 ORF pMXc-puro- IL13Rα2 pMXc-puro- IL13Rα2 pYO-14

pgD:d2-24/Y38-scMYa24 pSBT-21

HSV-1 gD gD:d2-24/Y38C-scEpCAM

EpCAM MY24 Yamaguchi et al., 2014 RNA cDNA

cDNA VL hu255 5’-GGAAGATCTGAYA

TTGTGMTSACMCARWCTMCA-3’/ hu256 5’-CCGAATTCGGATACAGTTGGTGCAGCATC-3’ Mixed base code: R=a,g; Y=c,t; M=a,c; K=g,t; S=c,g; W=a,t; V=a,c,g; N=a,c,g,t. VH

hu257 5’-GGAAGATCTSARGTNMAGCTGSAGSAGTC-3’/ hu259 5’-GGAAGATCTSARGTNMAGCTGSAGSAGTCWGG-3’/ hu270 5’-CCGAATTCATAGACAGATG GGGGTGTCGTTTTGGC-3’ PCR Wang et al., 2000 PCR VL pTAC-2

TA pTAC2-MYa24VL pSBT-7 VH

BgI II/ EcoR I pSP72 Bgl II/ EcoR I pSP72-MYa24VH pSBT-5

pSBT-7 pSBT-5 VL pSBT-7

VL hu323 5’-CCAGATCTGACATTGTGAT GACCCAGTCTCACAAATTTATGTCCACATCAGTAGGA-3’/ hu324 5’-CCGAATTCGCTAGCAG CCCGTTTTATCTCCAGCTTGGT-3’ PCR pTAC-2 TA

pTAC2-scMYa24VL pSBT-13 VH pSBT-5 7 GGGGSGS hu259/ hu322 5’-CCGGATCCTGAACCTCCAC CACCTGAGGAGACGGTGACCGTGGTC-3’ PCR PCR Bgl II/ BamH I

pSP72 Bgl II/ BamH I pSP72-MYa24VH-FX pSBT-24 pSBT-13

pSBT-24 VL

VH pgD:d2-24/Y38C-scEGFR hu253

5’-CCCTCGAGGAGCTCAGATCTGACATTCTAATGACCCAATCTCCAC-3’ / hu254 5’-CCGAATT CGGATCCGCCACCGCCCGACCCGCCACCGCCGCTACCGCCCCCGCCGCTAGCGGCCGCACG TTTGATTTCCAGCT-3’ PCR scEGFR VL 15

PCR Sac I/ EcoR I pCR -TOPO Invitrogen

TA pCR -TOPO-scEGFRVL pTS-9 Bgl II

pSP-72 Sca I/ EcoR I

MCS gD

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Bgl II MCS pSP72dBgl pTS-19 pTS-9 Sac I/ Xba I scEGFR

VL pTS-19 Sac I/ Xba I

pSP72-scEGFRVL pTS-20 pTS-20 MCSP VL Bgl II/ Nhe I

pSP72-SHM48VL-LL pTS-24 pSBT-13 Bgl II/ Nhe I MY24VL pTS-24 SHM48VL pSP72-MYa24VL-LL pSBT-19 pSBT-24 Bgl II/ BamH I

MY24VH pSBT-19 BamH I

pSP72-scMYa24 pSBT-20 pSBT-20 Bgl II/ BamH I

scMY24 pgD:d2-24/Y38C pHU174 BamH I gDscMY24 pgD:d2-24/Y38C-scMYa24 pSBT-21

pgDSac-FX pHU66 Tsvitov et al., 2007

HSV-1 gD gD

pgD:d2-24/Y38C pHU174 Uchida et al,. 2013

HSV-1 gD gD:d2-24/Y38C

pgD:d2-24/Y38C-scEGFR pHU165 Uchida et al., 2013 HSV-1 gD gD:d2-24/Y38C-scEGFR

pgD:d2-24/Y38C-scMY24-AflKan pSBT-26

gD ORF gD 50bp I-Sce

gD:d2-24/Y38CscEpCAM gD

pEP-Kan-S2 Dr. Klaus Osterrieder gD 50bp hu101 5’-CCGAATTCTTAAGGTCTCTTTTGTGTGGTGCGTTCCGGTATGGGGGGGGCTGCCGCCAGG ATGACGACGATAAGTAGGG-3’/ hu1025’-CCGGATCCTTAAGCTACAACCAATTAACCAATTC

TGATTAG-3’ I-Sce

Afl II pHU174 gD ORF Afl II

pgD:d2-24/Y38C-Aflkan pHU196

pSBT-20 Bgl II/ BamH I scMY24 pgD:d2-24/Y38C-Afl Kan BamH I

pRed/ET GENE BRIDGES

BAD L-arabinose Red/ET

pBAD-I-Sce I Tischer et al., 2006

BAD I-Sce

KgB:N/T-gDnull gD HSV

KOS UL 27 gB gB:D285N/A549T

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2010

KdB3G-gB:N/T HSV

KOS UL 3 UL 4 EGFP

UL 27 gB gB:D285N/A549T

pKB3G-gB:N/T

pEP-Kan-S2 EGFP 50bp hu245 5’-CCGAATTCGGATCCACCGGA TCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTAGGATGACGACGATAAG TAGGG-3’/ hu246 5’-CCTCTAGAGGATCCCTACAACCAATTAACCAATTCTGATTAG-3’

PCR I-Sce

EcoR I/ Xba I pSP72 pSP72-EGFP-Kan pYO-10 pYO-10 BamH I EGFP

I-Sce pEGFP-C1 Clontech

BamH I pEGFP-KAN pYO-11 pYO-11

UL 3 UL 4 50bp hu249 5’-CCTCACTGCCCGTCGCGCGTGTTTGATGTTAATA AATAACACATAAATTTTAGTTATTAATAGTAATCAATTACGGGG-3’/ hu250 5’-CCGACACTG AAATGCCCCCCCCCCCTTGCGGGCGGTCCATTAAAGACAACACGCGTTAAGATACATTGAT

GAGTTTG-3’ PCR pKB-gB:N/T Uchida and

Glorioso, unpublished pRed/ET

EGFP UL 3 UL 4 BAC Bgl II Not I KdB3G-gB:N/T HSV BAC pKB3G-gB:N/T Vero 2 Vero/Cre BAC KdB3G-gB:N/T HSV

KdB3G-gB:N/T-gDscEGFR EGFR oHSV

KOS UL 3 UL 4 EGFP

US 6 gD 2-24 HVEM

EGFR 528 scEGFR nectin-1

38 Y38C

pEP-Kan-S2 hu101/ 102 PCR Afl II

Afl II pHU165 pgD:d2-24/Y38C-scEGFRAflKAN pHU195 pHU195 hu15 5’-AAGCAGGGGTTAGGGAGTTG-3’/ hu4ANL 5’-TCCGGACGTCTTCGGAGGCCCC-3’

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pRed/ET

US 6 gD EGFR gD gD:d2-24/Y38C-scEGFR

BAC EcoR I Bgl II BstB I

BAC pKB3G-gB:N/T-gDscEGFR Vero 2

Vero/Cre BAC KdB3G-gB:N/T-gDscEGFR EGFR oHSV KdB3G-gB:N/T-gDscMY24 EpCAM oHSV

KOS UL 3 UL 4 EGFP

US 6 gD 2-24 HVEM

EpCAM MY24 scEpCAM nectin-1

38 Y38C

pSBT-26 hu15/ hu4ANL PCR gD:d2-24/Y38C-scEpCAM pKB3G-gB:N/T pRed/ET

US 6 gD EpCAM gD

gD:d2-24/Y38C-scEpCAM BAC Hind

BstB I BamH I Xho I

BAC pKB3G-gB:N/T-gDscEpCAM Vero/Cre-EpCAM

EpCAM oHSV 2

KdB3G-gB:N/T-gDscEpCAM EpCAM oHSV

siRNA

Stealth RNAi Negative Control Duplexes Low GC duplex 12935-200 Invirogen

TACSTD1HSS106226 3_RNAI Invitrogen CCUACUGGAUCAUCAUUGAACUAAA UUUAGUUCAAUGAUGATCCAGUAGG

TACSTD1HSS180961 3_RNAI Invitrogen CCGUAAACUGCUUUGUGAAUAAUAA UUAUUAUUCACAAAGCAGUUUACGG

4-2

gD

Vero 5.0 105_cells/well ₆ ₃₇

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Technologies Vero gD HSV MOI 5 Vero 4 gD HSV 0.1 M [pH 3.0] 3 DMEM 5 % FBS 2 DMEM 5 % FBS 37 CHO-K1 1.0 105 cells/well 48 37 24 3,000 rpm 15 CHO-K1 100 µL/well 37 2 2 F12K 10 % FBS 37 24 4 % EGFP EpCAM oHSV

pRed/ET DH10B Invitrogen BAC

pKB3G-gB:N/T

pSBT-26 hu15/ hu4ANL N I-Sce gD:d2-24/Y38C-scEpCAM Red/ET pKB3G-gB:N/T-gDscEpCAMAflkan BAC pRed/ET pBAD-I-Sce I pKB3G-gB:N/T-gDscEpCAM

loxP BAC BAC

pKB3G-gB:N/T-gDscEpCAM Vero/Cre-EpCAM Lipofectamine LTX & Plus Reagent Life

Technologies 37 72

KdB3G-gB:N/T-gDscEpCAM Vero/Cre-EpCAM 2 EpCAM oHSV

in vitro 5 10 cm dish Thermo

Vero/EpCAM KdB3G-gB:N/T-gDscEpCAM MOI 0.03/mL 37 2

DMEM 5 % FBS 96 3,000 rpm

15

gD KdB3G-gB:N/T EGFR gD KdB3G-gB:N/T-gDscEGFR Vero/EpCAM MOI 0.03/mL 72

in vivo 10 175 cm2

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DMEM 5 % FBS 10 mL 120 5 M NaCl 0.5 M 45 50 mL 15 3,000 rpm 0.8 µm 4 30 15,000 rpm PBS 1 4 30 15,000 rpm 1 mL PBS 48 CHO-K1 B78H1 37 HSV EGFR oHSV

EpCAM oHSV MOI 3/100 µL

100 µL/well 15 37

2 2 150 µL

16 4 % EGFP

OLYMPUS IX70 OLYMPUS

BZ-X700 KEYENCE

48 Vero/EpCAM BxPC-3 37

HSV EpCAM oHSV MOI 3/50 µL Vero/EpCAM MOI 5/50µL BxPC-3 Elution Buffer IgG elution buffer 1M Tris-HCl [pH 9.0] 10:1 0 2 20 200 µg/100 µL

DMEM 100 µL 0 1 10 100 µg/100 µL PBS 300 µL 100 µL/well 1 15 1 50 µL/well 15 37 2 2 0.1M [pH 3.0] 300 µL 2 250 µL 37 6 8 4 % EGFP

OLYMPUS IX70 OLYMPUS BZ-X700

KEYENCE

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20 µM siRNA Lipofectamine RNAiMAX Transfection reagent Invitrogen Opti-MEM Life Technologies BxPC-3 5.0 106_cells _{10 cm dish}

siRNA 20 nM

*2 _PBS _{2 µg/100 µL in PBS} _FACS _{100 µL/}

FACS 2.0 105_cells/

1,300 rpm 3 PBS 10 mL 1,300

rpm 3 2 1 100 µL

PBS FACS 100 µL/tube 15 FACS

4 30 4 1,500 rpm 5

PBS 2 mL 4

1,500 rpm 5 2 PBS Alexa Fluor 488 Goat

Anti-Mouse IgG (H+L) Antibody Life Technology , A-11001 2 µg/100 µL 100 µL/

15 FACS 4

30 4 1,500 rpm 5

PBS 2 mL 4 1,500 rpm 5

2 FACS Calibur BD

*2

LEAF Purified Mouse IgG1, κisotype control antibody MG1-45 401405 Biolegend

LEAF Purified mouse IgG2a, κisotype control antibody MOPC-173 400224 Biolegend

mouse anti-HVEM monoclonal antibody CW10 sc21718 Santa Cruz Biotechnology

mouse anti-nectin1 monoclonal antibody R1.302.12 sc69718 Santa Cruz Biotechnology

mouse anti-EGFR monoclonal antibody 528 sc120 Santa Cruz Biotechnology

mouse anti-EpCAM monoclonal antibody MY24

EpCAM MY24 Yamaguchi et al., 2014

Infectious center

48 Vero/EpCAM B78H1

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HSV EpCAM oHSV MOI 5/100 µL 100 µL/well 15 37 2 2 0.1 M [pH 3.0] DMEM 5 % FBS 2 DMEM 5 % FBS 250 µL 37 1 1 3 30 100 300 cells/300 µL 37 1 1 4 1 % 8 % FBS DMEM 48 4 %

EGFP OLYMPUS IX70

OLYMPUS BxPC siRNA 48 6 37 RPMI1640 10 % FBS 1 % 8 % FBS RPMI1640 72 4 % EGFP BZ-X700 KEYENCE

Image J n=15 Schneider et al., 2012 siRNA

48 6 37

30 300 pfu/100 µL 30 300 pfu/1 mL

48 100 µL 6 1 mL

15 37 2

2 1 % 8 % FBS DMEM RPMI1640

96 4 % EGFP

OLYMPUS IX70 OLYMPUS

Image J n=15 HepG2 student t-test HuCCT1

SK-OV-3 Welch T

96 2.5 104_cells/well ₃₇

HSV EGFR oHSV EpCAM oHSV MOI 0.0003 0.003

0.03 0.3 3 37 4 MTT

Mosmann., 1983 PBS 0.5 mg/mL MTT

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SH-9000 Lab 570 nm 630 nm OD570 – OD630 6 n=18 / 100 SW620 26G 1 mL SW620 1.0 107_{cells in} PBS/100 µL/ 0.5 2 _{Tomayko et al.,} 1989 160 200 mm3 _PBS _EpCAM oHSV 26G 1 mL 7.5 mg/kg 29G

PBS PBS 50 µL EpCAM oHSV 1.0 108 _{pfu in PBS/}

50 µL 0 0.5

2 ₂ _{analysis of variant} _P _0.05

In vitro 2 Student t-test Welch T P 0.05

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5

Armstrong A, Eck SL. EpCAM: A new therapeutic target for an old cancer antigen. Cancer Biol Ther 2003; 2: 320-6.

Babic N, Mettenleiter TC, Flamand A, Ugolini G. Role of essential glycoproteins gII and gp50 in transneuronal transfer of pseudorabies virus from the hypoglossal nerves of mice. J Virol 1993; 67: 4421-6. Cai WH, Gu B, Person S. Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J Virol 1988; 62: 2596-604.

Carfi A, Willis SH, Whitbeck JC, Krummenacher C, Cohen GH, Eisenberg RJ et al. Herpes simplex virus glycoprotein D bound to the human receptor HveA. Mol Cell 2001; 8: 169-79.

Chaudry MA, Sales K, Ruf P, Lindhofer H, Winslet MC. EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges. Br J Cancer 2007; 96: 1013-9. Chou J, Kern ER, Whitley RJ, Roizman B. Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture. Science 1990; 250: 1262-6.

Desai PJ, Schaffer PA, Minson AC. Excretion of non-infectious virus particles lacking glycoprotein H by a temperature-sensitive mutant of herpes simplex virus type 1: evidence that gH is essential for virion infectivity. J Gen Virol 1988; 69: 1147-56.

Fusco D, Forghieri C, Campadelli-Fiume G. The pro-fusion domain of herpes simplex virus glycoprotein D (gD) interacts with the gD N terminus and is displaced by soluble forms of viral receptors. Proc Natl Acad Sci U S A 2005; 102: 9323-8.

Geraghty RJ, Krummenacher C, Cohen GH, Eisenberg RJ, Spear PG. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 1998; 280: 1618-20. Gires O, Klein CA, Baeuerle PA. On the abundance of EpCAM on cancer stem cells. Nat Rev Cancer 2009; 9: 143; author reply 143.

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Herlyn M, Steplewski Z, Herlyn D, Koprowski H. Colorectal carcinoma-specific antigen: detection by means of monoclonal antibodies. Proc Natl Acad Sci U S A 1979; 76: 1438-42.

Huang YY, Yu Z, Lin SF, Li S, Fong Y, Wong RJ. Nectin-1 is a marker of thyroid cancer sensitivity to herpes oncolytic therapy. J Clin Endocrinol Metab 2007; 92: 1965-70.

Hu JC, Coffin RS, Davis CJ, Graham NJ, Groves N, Guest PJ et al. A phase I study of OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor. Clin Cancer Res 2006; 12: 6737-47.

Hutchinson L, Browne H, Wargent V, Davis-Poynter N, Primorac S, Goldsmith K et al. A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH. J Virol 1992; 66: 2240-50.

Johnson DC, Huber MT. Directed egress of animal viruses promotes cell-to-cell spread. J Virol 2002; 76: 1-8.

Kemeny N, Brown K, Covey A, Kim T, Bhargava A, Brody L et al. Phase I, open-label, dose-escalating study of a genetically engineered herpes simplex virus, NV1020, in subjects with metastatic colorectal carcinoma to the liver. Hum Gene Ther 2006; 17: 1214-24.

Krummenacher C, Supekar VM, Whitbeck JC, Lazear E, Connolly SA, Eisenberg RJ et al. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J 2005;

24: 4144-53.

Lichty BD, Breitbach CJ, Stojdl DF, Bell JC. Going viral with cancer immunotherapy. Nat Rev Cancer 2014; 14: 559-67.

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MacLean AR, ul-Fareed M, Robertson L, Harland J, Brown SM. Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the 'a' sequence. J Gen Virol 1991; 72: 631-9.

Marme A, Strauss G, Bastert G, Grischke EM, Moldenhauer G. Intraperitoneal bispecific antibody (HEA125xOKT3) therapy inhibits malignant ascites production in advanced ovarian carcinoma. Int J Cancer 2002; 101: 183-9.

Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther 2000; 7: 867-74.

Mazzacurati L, Marzulli M, Reinhart B, Miyagawa Y, Uchida H, Goins WF et al. Use of miRNA response sequences to block off-target replication and increase the safety of an unattenuated, glioblastoma-targeted oncolytic HSV. Mol Ther 2015; 23: 99-107.

Menotti L, Cerretani A, Hengel H, Campadelli-Fiume G. Construction of a fully retargeted herpes simplex virus 1 recombinant capable of entering cells solely via human epidermal growth factor receptor 2. J Virol 2008; 82: 10153-61.

Menotti L, Nicoletti G, Gatta V, Croci S, Landuzzi L, De Giovanni C et al. Inhibition of human tumor growth in mice by an oncolytic herpes simplex virus designed to target solely HER-2-positive cells. Proc Natl Acad Sci U S A 2009; 106: 9039-44.

Miest TS, Cattaneo R. New viruses for cancer therapy: meeting clinical need. Nat Rev Microbiol 2014; 12: 23-24.

Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med 1995; 1: 938-43.

Montgomery RI, Warner MS, Lum BJ, Spear PG. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 1996; 87: 427-36.

(28)

Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 2012; 38: 68-75.

Peeters B, de Wind N, Hooisma M, Wagenaar F, Gielkens A, Moormann R. Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion. J Virol 1992; 66: 894-905.

Peeters B, Pol J, Gielkens A, Moormann R. Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. J Virol 1993; 67: 170-7.

Rauch DA, Rodriguez N, Roller RJ. Mutations in herpes simplex virus glycoprotein D distinguish entry of free virus from cell-cell spread. J Virol 2000; 74: 11437-46.

Rauh I, Mettenleiter TC. Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration. J Virol 1991; 65: 5348-56.

Riethmuller G, Holz E, Schlimok G, Schmiegel W, Raab R, Hoffken K et al. Monoclonal antibody therapy for resected Dukes' C colorectal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol 1998; 16: 1788-94.

Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol 2012; 30: 658-70.

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 9: 671-5.

Shukla D, Liu J, Blaiklock P, Shworak NW, Bai X, Esko JD et al. A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 1999; 99: 13-22.

Shukla D, Spear PG. Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. J Clin Invest 2001; 108: 503-10.

(29)

Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989; 24: 148-54.

Tsvitov M, Frampton AR Jr., Shah WA, Wendell SK, Ozuer A, Kapacee Z, Goins WF, Cohen JB, Glorioso JC. 2007. Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection. Virology 360:477-491

Uchida H, Shah WA, Ozuer A, Frampton AR, Jr., Goins WF, Grandi P 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: 2951-61.

Uchida H, Chan J, Goins WF, Grandi P, Kumagai I, Cohen JB et al. A double mutation in glycoprotein gB compensates for ineffective gD-dependent initiation of herpes simplex virus type 1 infection. J Virol 2010;

84: 12200-9.

Uchida H, Marzulli M, Nakano K, Goins WF, Chan J, Hong CS et al. Effective treatment of an orthotopic xenograft model of human glioblastoma using an EGFR-retargeted oncolytic herpes simplex virus. Mol Ther 2013; 21: 561-9.

Uchida H, Chan J, Shrivastava I, Reinhart B, Grandi P, Glorioso JC et al. Novel mutations in gB and gH circumvent the requirement for known gD Receptors in herpes simplex virus 1 entry and cell-to-cell spread. J Virol 2013; 87: 1430-42.

Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R et al. Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity. J Immunol Methods 2000; 233: 167-77.

Weiner LM, Moldofsky PJ, Gatenby RA, O'Dwyer J, O'Brien J, Litwin S et al. Antibody delivery and effector cell activation in a phase II trial of recombinant gamma-interferon and the murine monoclonal antibody CO17-1A in advanced colorectal carcinoma. Cancer Res 1988; 48: 2568-73.

Winter MJ, Nagtegaal ID, van Krieken JH, Litvinov SV. The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology. Am J Pathol 2003; 163: 2139-48.

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Yu Z, Adusumilli PS, Eisenberg DP, Darr E, Ghossein RA, Li S et al. Nectin-1 expression by squamous cell carcinoma is a predictor of herpes oncolytic sensitivity. Mol Ther 2007; 15: 103-13.

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2 gD 2-24

38 gD

scFv

oHSV

epithelial cell adhesion molecule EpCAM

EpCAM

oHSV

SP: signal

peptide

TM: transmembrane domain

CT: cytosolic

tail

EpCAM

_標的化

gD

VH VL

N

α-EpCAM scFv

C

1 317 339 369

SP

TM CT

N

C

Δ2-24

野生型 gD

EpCAM

_{標的化 gD}

Y38C

HVEM

nectin-1

(33)

EpCAM

&

MY24

RNA

&

PCR

&

cDNA

V

_L

V

_H V_L V_H

Δ

2-24

Y38C

(GGGGS)

3

GGGGS

gD

3 gD

EpCAM

MY24

RNA

V

_L

, V

_H

V

_L

V

_H

scFv

gD

EpCAM

gD

RNA

cDNA

G U C A U _{T C A} _{G T A}

(34)

(35)

5 EpCAM

gD EpCAM

48 CHO-K1

gD

HSV

24 4%

EGFP

500µm

gD

gD :

d2-24/Y38C

EGFR

gD

EpCAM

gD

(36)

6 HSV

HSV

U

_L

: long unique sequences U

_S

:

short unique sequences

TR

_L

: terminal repeat long IR

_L

: internal repeat short

IR

_S

: internal repeat short TR

_S

: terminal repeat short

CMVp:

EGFP:

gDscEGFR:

EGFR

gD gDscEpCAM:

EpCAM

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EpCAM

oHSV

EpCAM

48 A CHO-K1

B B78H1

HSV EGFR

oHSV

EpCAM

oHSV MOI 3

16 4%

OLYMPUS IX70

BZ-X700

EGFP

&

HSV

EGFR

oHSV

EpCAM

oHSV

CHO-K1

CHO-HVEM CHO-nectin-1

CHO-EGFR

CHO-EpCAM

B78H1

B78-HVEM

B78-nectin-1 B78-EpCAM

(38)

(39)

EpCAM

oHSV

EpCAM

48 A Vero/EpCAM

B BxPC-3

IgG1

MY24

1 HSV

EpCAM

HSV

MOI 3 Vero/EpCAM

MOI 5 BxPC-3

2

8 4%

OLYMPUS IX70

BZ-X700

EGFP

(40)

9 infectious center

48 gD

EGFR EpCAM

48 HSV MOI 5 gD

EGFR EpCAM

(41)

10 EpCAM

oHSV

EpCAM

48 Vero/EpCAM

HSV

EpCAM

oHSV MOI 5

2 HSV

Vero/EpCAM 48

1.0 × 10

5

_cells/well

B78H1

1%

48 4%

OLYMPUS IX70

EGFP

40 HSV

B78H1

B78-HVEM

B78-nectin-1

B78-EpCAM

(42)

(43)

11 BxPC-3

EpCAM

siEpCAM

BxPC-3

EpCAM

A

siEpCAM

8 EpCAM

B

NC: Negative control LowGC

7 MY24 528 R1.302.12 CW10

C EpCAM

B x P C - 3

6 4 8

BxPC-3

HSV EGFR

oHSV

EpCAM

oHSV

MOI 5

2 HSV

BxPC-3 6

EpCAM

BxPC-3

1%

72 4%

BZ-X700

EGFP

n=15

Image J

NC

NC: Negative control Low GC

:

EpCAM

oHSV

(44)

12

7

8 EpCAM

MY24

nectin-1

R1.302.12

HVEM

CW10

EGFR

528

7 MY24 528 R1.302.12

CW10

EGFR

EpCAM

nectin-1

HVEM

100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells 100 101 102 103 104 FL1-H 0 100 200 300 400 # Cells