Cancer Gene Therapy (2007) 14, 918–926 r 2007 Nature Publishing Group All rights reserved 0929-1903/07 $30.00 www.nature.com/cgt

ORIGINAL ARTICLE Oncolytic with an HSV amplicon vector expressing granulocyte–macrophage colony-stimulating factor using the replication-competent HSV type 1 mutant HF10 as a helper S-i Kohno1,2, C Luo1,2, A Nawa3, Y Fujimoto4, D Watanabe5, F Goshima1, T Tsurumi6 and Y Nishiyama1 1Department of Virology, Graduate School of Medicine, Nagoya University, Nagoya, Japan; 2Research Division, M’s Science Corporation, Kobe, Japan; 3Department of Obstetrics and Genecology, Graduate School of Medicine, Nagoya University, Nagoya, Japan; 4Department of Otorhinolaryngology, Graduate School of Medicine, Nagoya University, Nagoya, Japan; 5Department of Dermatology, Aichi Medical University, Aichi, Japan and 6Division of Virology, Aichi Cancer Center Research Institute, Nagoya, Japan

Direct viral infection of solid tumors can cause tumor cell death, but these techniques offer the opportunity to express exogenous factors to enhance the antitumor response. We investigated the antitumor effects of a (HSV) amplicon expressing mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) using the replication-competent HSV type 1 mutant HF10 as a helper virus. HF10-packaged mGM-CSF-expressing amplicon (mGM-CSF amplicon) was used to infect subcutaneously inoculated murine colorectal tumor cells (CT26 cells) and the antitumor effects were compared to tumors treated with only HF10. The mGM-CSF amplicon efficiently replicated in CT26 cells with similar oncolytic activity to HF10 in vitro. However, when mice subcutaneously inoculated with CT26 cells were intratumorally injected with HF10 or mGM-CSF amplicon, greater tumor regression was seen in mGM-CSF amplicon-treated animals. Furthermore, mGM-CSF amplicon treatment prolonged mouse survival. Immunohistochemical analysis revealed increased inflammatory cell infiltration in the solid tumor in the mGM-CSF amplicon-treated animals. These results suggest that expression of GM-CSF enhances the antitumor effects of HF10, and HF10- packaged GM-CSF-expressing amplicon is a promising agent for the treatment of subcutaneous tumors. Cancer Gene Therapy (2007) 14, 918–926; doi:10.1038/sj.cgt.7701070; published online 10 August 2007 Keywords: HSV amplicon; HF10; oncolytic virotherapy; GM-CSF

Introduction more, the HSV-encoded thymidine kinase can enhance the antitumor effect of virotherapy using a ‘suicide gene Genetically altered, replication-competent have therapy’ approach by treating with the thymidine kinase recently been developed and used as novel anticancer inhibitor ganciclovir. Currently, a number of oncolytic agents.1,2 The use of highly attenuated viruses that HSV strains have been developed for oncolytic viro- 4 5–8 9 selectively replicate in and kill tumor cells is called therapy. Among these, G207 1716 and NV1020 have ‘oncolytic viral therapy’ or ‘oncolytic virotherapy’. been extensively investigated and used in clinical trials. Although several different viruses have been used for The combination of oncolytic HSV with chemo- 10–13 14–17 oncolytic virotherapy, most studies have used herpes therapeutic agents, radiation or immunostimula- 18 simplex virus (HSV) or adenovirus as oncolytic viruses.3 tory cytokines has recently been studied in attempts to HSV is particularly appealing as an . increase the efficacy of replication-competent oncolytic HSV infects a wide range of cells causing degenerative HSV. Other approaches using HSV as an antitumor agent changes and cell death. The virus preferentially replicates have been undertaken including the expression of in dividing cells, and recombinant virus expressing immunostimulatory cytokines using disabled infectious 19,20 exogenous genes is relatively easy to produce. Further- single cycle (DISC) HSV. Additionally, a mutant HSV encoding granulocyte–macrophage colony-stimulat- ing factor (GM-CSF),21 and HSV amplicons expressing Correspondence: Professor Y Nishiyama, Department of Virology, 22–25 Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, exogenous genes have also been described. HSV Showa-ku, Nagoya 466-8550, Japan. amplicon vectors are plasmids containing an HSV origin E-mail: [email protected] of replication and packaging sequence. These vectors Received 15 September 2006; revised 5 April 2007; accepted 19 April replicate in the presence of HSV gene products and are 2007; published online 10 August 2007 packed together in the HSV virion.26,27 Thus, the Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 919 resultant virus (HSV amplicon) consists of HSV’s between EcoRI and BamHI sites. The mGM-CSF cDNA envelope, tegument and capsid surrounding the viral was isolated using the HindIII and BamHI sites and DNA and recombinant DNA plasmid. cloned into the pHGCX vector as a transgene driven GM-CSF is a potent immune stimulator promoting the by the cytomegalovirus promoter. The mGM-CSF-expres- differentiation of progenitor cells to dendritic cells (DCs), sing HSV amplicon vector was designated as the central antigen-presenting cell responsible for indu- pHGCXmGM-CSF. Packaging of the HSV-1 amplicon cing an adaptive immune response.28,29 Thus, exogenous vector into HSV-1 HF10 virions was performed as expression of GM-CSF by an HSV amplicon could follows. COS-7 cells (3 Â 105) were seeded in a 35 mm potentially promote the induction of an antitumor dish, and, after an overnight incubation, cells were immune response to enhance the oncolytic effects of transfected with 4 mg of pHGCXmGM-CSF or pHGCX HSV. We previously reported that HF10, a highly using Lipofectamine 2000 (Invitrogen, Carisbad, CA), attenuated replication-competent HSV mutant, has a according to the manufacturer’s instructions. Two hours potent antitumor effect in various tumor models.30–34 after transfection, the transfected cells were infected with Moreover, a phase I/II clinical study was performed HF10 at MOI 0.3. The HF10-packaged pHGCXmGM- examining the oncolytic effects of HF10 on recurrent CSF (termed as mGM-CSF amplicon) or HF10-packaged breast cancer.35 Therefore, we investigated the ability of pHGCX (empty amplicon) were harvested when a an HSV amplicon expressing mouse GM-CSF (mGM- complete cytopathic effect occurred. After freezing and CSF) using replication-competent HF10 as a helper virus thawing three times and the elimination of cell debris, the to control the growth of mouse colorectal tumor cells in supernatants were collected and stored at À801C. Titers both in vitro and in vivo models. of helper virus particles were determined by plaque assay using Vero cells and expressed as plaque forming units (PFU)/ml. Titers of the mGM-CSF amplicon or empty amplicon were determined by infecting Vero cells with Materials and methods serial dilutions of the amplicon stockin the presence of Cell lines and virus 10 m ganciclovir. After 24 h, the number of green CT26 cells were obtained from American Type Culture fluorescent cells was counted using a fluorescence micro- Collection (Rockville, MD). African green monkey scope and expressed as transduction units. kidney cells (Vero and COS-7) were obtained from Riken Cell Bank(Tsukuba,Ibaragi, Japan). CT26 is a colon Immunoblotting epithelial tumor induced by intrarectal injections of CT26 cells were seeded in 35 mm dishes and infected with N-nitroso-N-methylurethane in BALB/c mice. CT26 cells HF10, empty amplicon and mGM-CSF amplicon at MOI were maintained in RPMI 1640 containing 10% fetal 1. Cells were incubated for 72 h and supernatants were bovine serum, 100 U/ml penicillin and 100 mg/ml strepto- harvested. After 20 ml of supernatant was mixed with 20 ml mycin. Vero cells were grown in Eagle’s minimum of sample buffer containing sodium dodecyl sulfate essential medium (MEM) containing 5% calf serum and (SDS), the mixture was heated at 1001C for 2 min and 60 mg/ml kanamycin. COS-7 cells were maintained in then cooled on ice. A total of 40 ml supernatants were Dulbecco’s MEM containing 10% fetal bovine serum, separated by 15% SDS-polyacrylamide gel electropho- 100 U/ml penicillin and 100 mg/ml streptomycin. All cells resis and transferred onto a nitrocellulose membrane. were maintained in a 5% CO2 humidified incubator at Nonspecific binding was blocked overnight at 41C with 371C. phosphate-buffered saline with 0.05% Tween 20 (PBS-T) HF10 is a non-selected clone derived from the containing 5% skim milk. The membrane was incubated 30 laboratory HSV type 1 (HSV-1) strain HF. The with rabbit anti-mGM-CSF antiserum (Sigma-Aldrich, properties of HF10 have been previously described.30 Saint Louis, MI) for 1 h followed by washing three times Briefly, the HF10 genome is characterized by a 3.9 kb in PBS-T. The membrane was then incubated with deletion in the right end of the unique sequence L (UL)/ horseradish peroxidase-conjugated goat anti-rabbit internal repeat L (IRL) junction, and a 2.3 kb deletion secondary antiserum (Biosource International, Camarillo, and extensive gene rearrangements at the left end of the L CA) for 1 h at room temperature. Following three washes segment. HF10 was propagated in Vero cells at a with PBS-T, the protein bands were visualized with ECL multiplicity of infection (MOI) of 0.03 at 371C. Virus Western Blotting Analysis System (Amersham Bios- titers were determined using plaque assays on Vero cells. ciences UK, Little Chalfont, UK) and exposed to Fuji film (Fuji Photo Film, Tokyo, Japan). mGM-CSF-expressing HSV-1 amplicon vector and amplicon production In vitro experiments The mGM-CSF-expressing amplicon vector was con- CT26 cells were infected with HF10, empty amplicon structed as follows. The HSV amplicon plasmid, pHGCX, (packaged into HF10 virions) or mGM-CSF amplicon was obtained from Dr Yoshinaga Saeki (Massachusetts (packaged into HF10 virions) at MOI 1 in 35 mm dishes. General Hospital, Charlestown, MA). pBluescript Transduction units of empty amplicon or mGM-CSF SKII þ mGM-CSF vector obtained from Dr Hirofumi amplicon were 6.01 Â 104 and 7.42 Â 104, respectively. Hamada (Sapporo Medical University School of After a 1 h-adsorption, the cells were washed with PBS Medicine, Sapporo, Japan) contains the mGM-CSF cDNA three times and incubated with medium at 371C. CT26

Cancer Gene Therapy Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 920 cells were harvested at 3, 8, 24, 48 and 72 h post infection. and this was considered a spontaneous rejection and The concentration of mGM-CSF in culture medium was eliminated from analysis. The tumor volume data were measured by mGM-CSF enzyme-linked immunosorbent analyzed with one-way analysis of variance (ANOVA) assay (ELISA) (Pierce Biotechnology, Rockford, IL). followed by Scheffe’s test for multiple comparisons Virus titers were measured by plaque assay using Vero among groups. The survival data were analyzed with the cells. Cell viability was determined by counting the Kaplan–Meier method and the log-ranktest. P-values less number of viable cells using trypan blue exclusion and than 0.05 were considered significant. The present studies calculated as the percentage of the mock-infected control. were conducted in accordance with guidelines issued by All assays were performed in duplicate. the Animal Center at Nagoya University School of Medicine.

Animal studies Histologic and immunohistochemistry analysis Female BALB/c mice (6 weeks old) purchased from Japan Mice were killed at indicated times and tumors were fixed SLC (Shizuoka, Japan) were used in this study. CT26 6 in 10% formalin in PBS. Hematoxylin–eosin staining and cells (1  10 cells in 100 ml of medium) were inoculated immunostaining for HSV-1 antigens were performed, as subcutaneously into the lumbar region of the mice. After described previously.34 3 or 4 days when the size of subcutaneous tumor reached approximately 5 mm, the mice were intratumorally injected with either HF10 (1  107 PFU), mGM-CSF amplicon (virus titer; 1  107 PFU, transduction unit; Results 8.13  104) or medium control (Eagle’s MEM containing Construction of mGM-CSF amplicon and expression of 5% calf serum) for 5 consecutive days. Tumor growth mGM-CSF was determined by measuring the tumor volume (p/6  We generated the HSV amplicon vector pHGCXmGM- length  width  height) until the time of animal CSF by inserting the cDNA for mGM-CSF downstream death. Survival rates were observed until 90 days post of the cytomegalovirus promoter contained in pHGCX CT26 cell inoculation. Incidentally, the tumor disap- (Figure 1a). The complete vector, pHGCXmGM-CSF, peared in one individual within 14 days post inoculation, was packaged into HF10, and Vero cells were infected

a ColEl ori oris b

IE4/5 promoter Amp r

GFP pHGCXmGM-CSF Mock HF10 Empty AmpliconmGM-CSF Amplicon

SV40 pA mGM-CSF 16.1 kDa pac CMV promoter

mGM-CSF BGH pA

c 5000 Mock 4000 HF10 Empty Amplicon mGM-CSF Amplicon 3000

2000

1000 GM-CSF (pg /mL) GM-CSF (pg

0 38 244872 hours post infection

Figure 1 (a) Schematic diagram of the pHGCXmGM-CSF amplicon vector. The two cis-acting sequences from HSV-1, ‘pac’ in which the HSV packaging signal is located, and ‘oris’ the HSV-1 origin of DNA replication, are required for amplicon genome amplification and packaging. The procaryotic sequences ColE1 oris and ampicillin resistance gene (Ampr) in the vector allow for growth and selection in bacteria. The GFP cassette driven by the viral immediate-early promoter IE4/5 is inserted into the amplicon vector as a transfection marker. mGM-CSF transgene expression is controlled by the CMV promoter. (b) Expression of mGM-CSF in CT26 cells. CT26 cells were infected with HF10, empty amplicon or mGM-CSF amplicon. After 72 h, supernatant was harvested and subjected to western blotting with an anti-mGM-CSF antiserum. (c) Quantitation of expressed mGM-CSF. CT26 cells were infected with HF10, empty amplicon or mGM-CSF amplicon. At 3, 8, 24, 48 and 72 h post infection, supernatant was analyzed by ELISA assay. All assays were performed in duplicate. Each column and bar represent the mean and standard error. CMV, cytomegalovirus; GFP, green fluorescent protein; HSV, herpes simplex virus; mGM-CSF, mouse granulocyte–macrophage colony-stimulating factor.

Cancer Gene Therapy Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 921 with the HF10-packaged mGM-CSF amplicon (mGM- was no difference in the in vitro oncolytic effects of the CSF amplicon). pHGCXmGM-CSF contains a green three viruses. fluorescent protein (GFP) expression cassette driven by the immediate-early promoter IE4/5. After 24 h, green The therapeutic efficacy of mGM-CSF amplicon in fluorescent cells were easily detected by fluorescent syngeneic subcutaneous tumor models using mouse microscopy, indicating the efficient expression of the colorectal tumor cells HSV amplicon. The fluorescence was also detected in the We next examined the ability of the mGM-CSF amplicon presence of 10 mM ganciclovir. to control tumor cell growth in vivo. Two independent We next infected CT26 cells with HF10, empty series of experiments were performed. In the first series amplicon or mGM-CSF amplicon and examined mGM- of experiments, syngeneic BALB/c mice were subcuta- CSF expression by both western blotting and ELISA. neously inoculated with 1 Â 106 CT26 cells. The tumors In mGM-CSF amplicon-infected cells, we detected a were then injected with HF10 (1 Â 106 PFU), empty 16.1 kDa band consistent in size with mGM-CSF, 72 h amplicon expressing only GFP (virus titer: 1 Â 106 PFU; after infection (Figure 1b). No mGM-CSF expression was amplicon: 6.58 Â 104 transduction unit), mGM-CSF seen in mock-, HF10- and empty amplicon-treated cells. amplicon (virus titer: 1 Â 106 PFU; amplicon: 8.13 Â 104 Additionally, mGM-CSF was detected in the super- transduction unit) or an equal volume of MEM contain- natants of mGM-CSF amplicon-infected cells as early as ing 5% of calf serum seven times at 3, 5, 7, 9, 11, 13 and 3 h post infection; mGM-CSF levels reached 3000 pg/ml 15 days after tumor inoculation. As shown in Figure 3a, by 72 h (Figure 1c). Negligible amounts of mGM-CSF the mean volumes of tumors in mice treated with GM- were detected in mock-, HF10- or empty amplicon- CSF amplicon were significantly smaller than those in infected cell supernatants (Figure 1c). other groups of mice, and the differences were statistically significant. In the second series of experiments, titers of Replication and oncolytic activity of mGM-CSF HF10 were increased by 10-fold without changing the amplicon in mouse colorectal tumor cells titers of GM-CFS amplicon (Figure 3b). Mice were We next examined the ability of the mGM-CSF amplicon inoculated with 1 Â 106 CT26 cells, treated with either to replicate in the mouse colorectal tumor cell line CT26. HF10 (1 Â 107 PFU) or mGM-CSF amplicon (virus titer: At early time points after infection, the virus titers 1 Â 107 PFU; amplicon: 8.13 Â 104 transduction unit) five of both empty- and mGM-CSF amplicon-infected cells times at 3, 4, 5, 6 and 7 days after tumor inoculation. were higher than that of HF10-infected cells. However, Seven days after CT26 inoculation (after four treatments), 24 h post infection, the virus titers reached a plateau, and both HF10- and mGM-CSF amplicon-injected tumors there was little difference between each treatment were significantly smaller than non-injected controls (Figure 2a). (Po0.01). These antitumor effects continued throughout Consistent with the viral titer data, the viability of the observation period. At 18 days after tumor inocula- CT26 cells infected with HF10, empty amplicon and tion, mice injected with mGM-CSF amplicon had mGM-CSF amplicon declined equally with time significantly smaller tumors than those injected with (Figure 2b). Indeed, under all conditions, the number of HF10 (Po0.05). Thus, our results showed that the mGM- surviving cells was less than 10% the number of mock- CSF amplicon inhibited tumor growth to a greater extent treated cells by 72 h post infection (Figure 2b). Thus, there than HF10.

a 108 b 120 100 107 80 106 60 Mock 105 HF10 HF10 40 Empty Amplicon Empty Amplicon mGM-CSF Amplicon Virus titer (pfu/mL) 4 10 mGM-CSF Amplicon 20 Cell viability (% of control) 1000 0 012 2436486072 38 24 48 72 hours post infection hours post infection

Figure 2 Replication and oncolytic activity of mGM-CSF amplicon in mouse colorectal tumor cells. CT26 cells were infected with HF10, empty amplicon or mGM-CSF amplicon. At 3, 8, 24, 48 and 72 h post infection, the infected cells were harvested. All experiments were performed in duplicate. (a) Replication of mGM-CSF amplicon. Virus titers were determined by plaque assay using Vero cells. The data represent the mean PFU. (b) Oncolytic activity of mGM-CSF amplicon. Viable cells were counted by trypan blue exclusion. Each column represents the mean percent of cell survival compared to the uninfected control. Bars represent standard errors. mGM-CSF, mouse granulocyte–macrophage colony- stimulating factor; PFU, plaque forming unit.

Cancer Gene Therapy Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 922 a Control 1.0 HF10 Empty Amplicon 0.8 6000 500 mGM-CSF Amplicon

) 0.6 3 375 5000 0.4

250 Survival rate 0.2 ) 3 4000 125 0.0

Tumor volume (mm volume Tumor 20 30 40 50 60 70 80 90 100 3000 0 0 2 4 6 8 10 12 14 16 Days post CT26 cells inoculation Days post CT26 cells 2000 injection Figure 4 mGM-CSF amplicon treatment prolonged survival of mice inoculated with mouse colorectal tumor cells. Mice were subcuta- neously inoculated with CT26 cells, followed by intratumoral injection 1000 with control medium (open circle), HF10 (closed circle) or mGM-CSF amplicon (closed square) (n ¼ 10–11). The survival data were 0 analyzed with Kaplan–Meier survival estimates and log-rank test. 0 10 20 30 40 50 P ¼ 0.0738; control group vs HF10 treatment group. Po0.05; control Days post CT26 cells injection group vs mGM-CSF amplicon treatment group. mGM-CSF, mouse granulocyte–macrophage colony-stimulating factor. b Control 5000 500 HF10 All control-treated mice died within 70 days after CT26

) mGM-CSF Amplicon 3 375 cell inoculation (Figure 4), and this corresponded to a 4000 50% survival rate of 49 days by Kaplan–Meier analysis. 250 The comparable 50% survival rate in the HF10 and ) (mm volume Tumor 3 mGM-CSF amplicon treatment groups were 53 and 58 125 3000 days, respectively. Although the HF10 treatment failed

Tumor volume (mm volume Tumor to prolong animal survival (control group vs HF10, 0 024681012 ** P ¼ 0.0738), mGM-CSF amplicon treatment significantly 2000 Days post CT26 cells prolonged the survival period (control group vs mGM- injection ** Tumor volume (mm volume Tumor CSF amplicon, Po0.05). One HF10-treated mouse and # two mGM-CSF amplicon-treated mice survived through- 1000 ** # ** ** out the 90-day observation period (Figure 4). During the # ** ** # observation period, no mice developed signs of neurologic ** ** 0 ** dysfunction after HF10 or mGM-CSF amplicon treat- 0 5 10 15 20 25 30 ment. Days post CT26 cells injection

Figure 3 mGM-CSF amplicon treatment enhanced the inhibitory Histopathologic and immunohistochemical analysis 6 effect of HF10 on tumor growth. CT26 cells (1 Â 106 cells) were Mice were subcutaneously inoculated with 1 Â 10 CT26 subcutaneously inoculated into BALB/c mice. (a) Tumors were cells, and tumors were injected with HF10 (1 Â 107 PFU), injected with HF10 (closed circle: 1 Â 106 PFU), empty amplicon mGM-CSF amplicon (virus titer: 1 Â 107 PFU; amplicon: 6 expressing only GFP (open square: virus titer, 1 Â 10 PFU; 8.13 Â 104 transduction unit) or an equal volume of MEM amplicon, 6.58 Â 104 transduction unit), mGM-CSF amplicon (closed 6 4 containing 5% of calf serum at 4, 5, 6, 7 and 8 days after square: virus titer, 1 Â 10 PFU; amplicon, 8.13 Â 10 transduction inoculation of tumor cells. At 6 and 11 days after tumor unit) or an equal volume of MEM containing 5% of calf serum (open circle) seven times at 3, 5, 7, 9, 11, 13 and 15 days after tumor inoculation, two mice from each group were killed and inoculation (n ¼ 7–8). (b) Tumors were treated with HF10 (closed their tumors were examined histopathologically. As circle: 1 Â 107 PFU), mGM-CSF amplicon (closed square: virus titer, shown in Figures 5a and b, a number of HSV antigen- 1 Â 107 PFU; amplicon, 8.13 Â 104 transduction unit) or an equal positive cells were detected in tumors from HF10-treated volume of medium (open circle) five times at 3, 4, 5, 6 and 7 days mice. At day 11, viral antigen-positive cells were detected after tumor inoculation (n ¼ 10–11). Tumor size was assessed with around degenerative and necrotic areas of tumors micrometer calipers twice per week until the first death occurred. (Figure 5b). In mGM-CSF amplicon-treated mice, viral Each symbol and bar represents the mean7s.e. The inset figure antigen-positive cells were detected at day 6, but not at provides greater detail of the early time points. The tumor volume day 11 (data not shown). As expected, we observed data were analyzed with one-way ANOVA followed by Scheffe’s test extensive tumor cell death and fibrosis in both HF10- and for multiple comparisons among groups. **P 0.01; control vs HF10 o mGM-CSF-treated mice compared to mock-treated mice and control vs mGM-CSF amplicon. *Po0.05; control vs mGM-CSF amplicon. #Po0.05; HF10 vs mGM-CSF amplicon. ANOVA, analysis (Figures 5c–h). Interestingly, there was a greater degree of of variance; GFP, green fluorescent protein; mGM-CSF, mouse inflammatory cell infiltration in and around the tumors granulocyte–macrophage colony-stimulating factor; MEM, minimum injected with the mGM-CSF amplicon compared to those essential medium; PFU, plaque forming unit. treated with HF10 (Figures 5c–h). Such enhancement of

Cancer Gene Therapy Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 923

Figure 5 Histologic and immunohistochemistry analysis following intratumoral injection of HF10 or mGM-CSF amplicon. CT26 cells (1 Â 106 cells) were subcutaneously inoculated into BALB/c mice. Mice were intratumorally injected with 1 Â 107 PFU/head of HF10 (a, b, d and g), mGM- CSF amplicon (e and h; virus titer, 1 Â 107 PFU; amplicon, 8.13 Â 104 transduction unit) or an equal volume of medium (c and f)at4,5,6,7and8 days after inoculation of cells. At 6 days (a, c, d and e) and 11 days (b, f, g and h) after tumor inoculation, mice were killed and tumors were processed for immunohistochemical and histopathological examination. Immunoperoxidase-ABC staining for HSV-1 antigens (a and b); Â 200 magnification. Hematoxylin–eosin staining; Â 40 magnification (c–e); Â 200 magnification (f–h). mGM-CSF, mouse granulocyte–macrophage colony-stimulating factor; PFU, plaque forming unit.

Cancer Gene Therapy Oncolytic virotherapy with GM-CSF HSV amplicon S-i Kohno et al 924 inflammatory cell infiltration was already observed at day HF10 was derived from an in vitro-passaged laboratory 6 (Figures 5d and e). strain of HSV-1.30 The mechanism causing HF10 attenuation is unclear, but HF10 lacks the UL56 gene. UL56 associates with the kinesin motor protein KIF1A, Discussion and the absence of UL56 causes decreased pathogenicity without affecting viral replication in tissue culture.44–46 A number of cytokines have been investigated for their Thus, the lackof UL56 may be responsible for the ability to promote tumor rejection, and GM-CSF is one attenuated nature of HF10. of the most effective cytokines examined to date.23,36–39 In this study, HF10 was clearly capable of replicating in GM-CSF has profound effects on the functions of various mouse colorectal tumor cells, and infection with HF10 leukocytes and can recruit neutrophils, monocytes and led to lysis of these cells. Additionally, there were no lymphocytes to enhance their functions in the host differences in viral replication or tumor cell lysis following immune responses. GM-CSF can also enhance the CT26 infection with HF10, empty amplicon and mGM- adaptive antitumor response by recruiting and activating CSF amplicon. Thus, mGM-CSF does not affect in vitro DCs within the tumor. In the present study, we oncolytic activity. However, when we examined the demonstrate that the expression of mGM-CSF using an antitumor effects in vivo, mGM-CSF amplicon treatment HSV amplicon in CT26 tumors enhances the antitumor caused greater tumor regression than treatment with potential of a replication-competent oncolytic virus HF10. Thus, the intratumoral expression of mGM-CSF HF10. The enhanced suppression of tumor growth as enhanced the antitumor effects of HF10 in vivo. well as enhanced local inflammation was observed even Multiple mechanisms are thought to mediate the at the very early stage of treatment with the mGM-CSF antitumor effects seen in oncolytic virotherapy using amplicon, suggesting that the activation of nonspecific HSV. First, HSV replication is directly oncolytic leading antitumor immunity induced by mGM-CSF may be to some tumor cell death. Additionally, HSV infection predominantly important for this enhanced suppression induces an early innate immune response characterized by of tumor growth at the early stage of treatment. natural killer cells, macrophages and/or the production of Two methods are typically used to cause exogenous interferon. Finally, tumor-specific immunity is induced gene expression, liposome-mediated transfection and viral leading to tumor cell killing by cytotoxic T lymphocytes transduction. The efficiency of liposome transfection (CTL). Treatment of mice with HF10 significantly varies greatly from cell line to cell line, and it is difficult prolonged their survival in models of both peritoneal to induce stable, high-level gene expression using many metastasis31 and superficial bladder cancer.32 Interest- viral systems. The HSV amplicon system has many ingly, in the peritoneal metastasis model using Colon26 advantages including high efficiency, safety and great cells, all surviving mice were resistant to rechallenge by flexibility. In the amplicon system, a helper virus is the same tumor cells. Thus, HF10 treatment appears to usually used to package the amplicons. An efficient helper promote the generation of an adaptive immune response virus-free amplicon system was recently reported using with associated immunologic memory. CT26 cells process bacterial artificial chromosomes encoding the HSV and present the H2Ld-restricted gp70 tumor antigen genome without the packaging sequences.40 Such a helper peptide AH-1,19 and AH-1 is a dominant epitope for virus-free system could potentially eliminate the numerous CD8 þ CTL. Toda et al.47 reported that intratumoral HSV-encoded immunomodulatory genes such as ICP47. inoculation with oncolytic HSV induces a tumor-specific However, the viral yield is relatively low (less than T-cell response and generates AH-1 peptide-specific CTL 107 PFU/ml) using this system, and low viral titers may activity. In addition, CT26 tumor regression can be not support a full antitumor effect. On the other hand, in induced by intratumoral injection of DISC/mGM-CSF virotherapy using replication-competent HSV, intratumor virus.19 When considered with the present results, it is virus production and oncolysis occur, and low viral titers likely that the intratumor expression of GM-CSF can are less of a concern. enhance the subsequent, specific antitumor immune A number of oncolytic HSV strains have been response to promote tumor killing and regression. investigated for cancer therapy. Among these, G207,4 Recently, a phase I/II study examining the efficacy of 17165–7 and NV10209 have already been used for clinical HF10 in six patients with recurrent breast cancer was trials. In mouse models, HSV strains are classified into undertaken.35 Patients were treated with intratumoral three pathogenic phenotypes.41,42 Class I strains, includ- injection with HF10 containing no more than ing wild-type virus and clinical isolates, cause lethal 5 Â 105 PFU once a day for 3 days. Adverse events were encephalitis both by the intracerebral and the peripheral not observed, and anti-HSV-1 antibody titers did not routes of infection. Class II strains, including HF10, are change during the observation period. One patient having highly virulent following intracerebral, but not peripheral, received 5 Â 105 PFU per injection experienced nearly infection.43 Class III strains, including G207 or NV1020, 100% cancer cell death in the injected lesion. The present are not virulent by both routes of infection. Class III HSV study demonstrated that tumor treatment with the strains are suited for the treatment of tumors in the mGM-CSF amplicon significantly prolonged animal central nervous system, but the oncolytic activity of these survival in a model of subcutaneous tumor growth. strains is substantially reduced limiting their application Taken together with the phase I/II study of HF10, our as a viral vector. results suggest that HF10-packaged GM-CSF-expressing

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