A Conditionally Replicating Adenovirus for Nasopharyngeal Carcinoma Gene Therapy

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doi:10.1016/j.ymthe.2004.03.016

A Conditionally Replicating Adenovirus for Nasopharyngeal Carcinoma Gene Therapy Marie C. Chia,a,b Wei Shi,a Jian-Hua Li,a Otto Sanchez,a,c Craig A. Strathdee,d Dolly Huang,e Pierre Busson,f Henry J. Klamut,a,b and Fei-Fei Liua,b,g,* a

Division of Experimental Therapeutics, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9 b Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada c School of Nursing, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada d Immunex/Amgen Corp., Seattle, WA, USA e Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Shatin, Hong Kong f UMR 1598 Institut Gustave Roussy, Villejuif, France g Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9 *To whom correspondence and reprint requests should be addressed at the Department of Radiation Oncology, Princess Margaret Hospital/Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. Fax: (416) 946-4586. E-mail: [email protected]. Available online 30 April 2004

Successful attainment of tumor-specific gene expression was achieved in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein – Barr virus (EBV) genome in the cancer cells. In the current study, we have utilized an EBV-dependent transcriptional targeting strategy to construct a novel conditionally replicating adenovirus, adv.oriP.E1A. After treatment with adv.oriP.E1A, we observed extensive cell death in the EBV-positive NPC cell line C666-1. In contrast, no cytotoxicity was observed in a panel of other human EBV-negative cell lines, including fibroblasts from the nasopharynx. In vitro adenoviral replication was confirmed by the time-dependent increase in the expression of adenoviral capsid fiber protein and adenoviral DNA after C666-1 cells were infected with adv.oriP.E1A. Tumor formation was inhibited for more than 100 days after ex vivo infection of C666-1 cells with adv.oriP.E1A. Combination of local tumor radiation and adv.oriP.E1A caused complete disappearance of established tumors for at least 2 weeks in two distinct EBV-positive NPC xenograft models. Safety of this treatment was determined through the systemic delivery of adv.oriP.E1A in vivo, whereby minimal temporary perturbation of liver function was observed. We have successfully established a conditionally replicating adenovirus for EBV-positive NPC, which is both safe and efficacious, indicating a strategy that may be therapeutically applicable. Key Words: Epstein – Barr virus, nasopharyngeal cancer, gene therapy, oncolytic virotherapy

INTRODUCTION Nasopharyngeal carcinoma (NPC)1 is a malignancy of the head and neck that affects a relatively young population with an overall 5-year survival of around 65% [1]. It is endemic to certain geographic regions such as Southeast Asia and is associated with several environmental and genetic factors. These include the consump-

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Abbreviations used: CRA, conditionally replicating adenovirus; Bgal, h-galactosidase; EBV, Epstein – Barr virus; Luc, luciferase; i.t., intratumoral; NPC, nasopharyngeal carcinoma; RT, radiation therapy.

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tion of nitrosamine-containing foods such as salted fish, smoking, and occupational exposure to formaldehyde [2]. NPC is unique in that it is strongly associated with the Epstein – Barr virus (EBV), in that over 80% of cases worldwide contain the EBV genome [3]. Interestingly, EBV, as identified through in-situ hybridization, is found exclusively in the malignant cells and not in the surrounding normal tissues. The role of EBV in the transformation and progression of NPC remains unclear; however, several of the EBV gene products such as LMP-1 have been shown to have transforming properties, suggesting a significant role for EBV in NPC development [4]. In NPC, EBV exists in a latent form from which it

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expresses several gene products including EBNA-1 [5]. This protein interacts with high affinity and specificity to a 30-bp sequence in 20 tandem repeats denoted as the Family of Repeats (FR) located within the origin of replication (oriP) in the EBV genome [6 – 8]. As previously described, we constructed a transcriptional targeting system that included the oriP-FR sequence upstream of the immediate-early CMV promoter [9], achieving 1000fold selective gene expression in EBV-positive cancer cells. With the successful construction of a tumor-specific system, we can now address the issue of effective in vivo delivery. One approach to improve distribution is to utilize a preferentially replicating adenovirus. Here, the adenoviral E1A gene, which has been shown to be neces-

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sary and sufficient for adenoviral replication [10], is expressed under the control of a tumor- or treatmentspecific promoter. This allows the adenovirus to replicate, generate virions, lyse the host cell, and subsequently infect surrounding tumor cells. This approach is considered one of the most promising strategies for cancer gene therapy [11]. In this study, we report the generation of a conditionally replicating adenovirus (CRA), wherein E1A is expressed in an EBNA-1-dependent manner, and demonstrate specific expression and cytotoxicity in EBV-positive nasopharyngeal cancer cells. In addition the combination of adv.oriP.E1A with ionizing radiation (RT) is capable of causing tumor regression in vivo, associated with minimal systemic toxicity.

FIG. 1. Schematic of the construction of adv.oriP.E1A. The E1A transcriptional unit was cloned downstream of the oriP-basal CMV promoter into the pDE1SP1A shuttle vector. A novel recombinant adenovirus was generated in 293 cells after homologous recombination of the pDE1SP1A vector with pJM17. This CRA hence does not contain E1B the sequence.

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RESULTS Selective transgene expression of adv.oriP. E1A in EBV-positive cells We infected EBV-positive (C666-1) and -negative (CNE1, CNE-2Z) cells with 10 plaque-forming units (pfu)/cell of adv.oriP.E1A (the construction of which is shown in Fig. 1) and assessed them for the presence of E1A protein by Western blot analysis to determine expression of the E1A transgene (Fig. 2). This revealed a time-dependent increase in the E1A protein in the C666-1 cells, in which maximal expression was observed 48 h postinfection. In contrast, there was significantly less expression in the EBV-negative NPC cell lines. Radiation treatment of 6 Gy appeared to have little effect on E1A expression in C6661 cells after treatment with either 5 or 10 pfu/cell of adv.oriP.E1A (data not shown). The positive control was provided by 293 cells, which stably express E1A. Adv.oriP. E1A reduces cell viability in EBV-positive cells We determined the cytotoxic effect of adv.oriP.E1A in both EBV-positive and EBV-negative cells by infecting with 10 or 25 pfu/cell of adv.oriP.E1A. As observed in Fig. 3A, a panel of EBV-negative human cell lines (A549, MDA-MB-231, SaOS-2, CNE-2Z, KS-1) infected with up to 25 pfu/cell of adv.oriP.E1A exhibited no evidence of cytopathic effects, examined 4 days postinfection, and eventually all grew to confluency. We confirmed high infection efficiency by the expression of h-galactosidase after treatment with 10 pfu/cell of adv.CMV.B-gal. In contrast, when we infected EBV-positive C666-1 cells with adv.oriP.E1A at 10 pfu/cell, we observed a signifi-

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cant cytopathic effect as early as 3 days postinfection (Fig. 3B, bottom). In addition, there was a clear dose-dependent cytotoxic effect demonstrated using the MTT assay, whereby almost no cells survived at the highest dose of 50 pfu/cell of adv.oriP.E1A (Fig. 3B, top). A single RT dose of 6 Gy achieved additional cytotoxicity, but at higher viral doses, no additional cytotoxic effect of RT could be appreciated due to the low number of surviving cells (20%). Despite the detectable presence of E1A in EBVnegative NPC cell lines (Fig. 2), we observed no effect on viability of CNE-1 cells, as assessed by the MTT assay (data not shown). To determine the relative potency of adv.oriP.E1A, we compared its effect to that of wild-type adenovirus type 5. We infected C666-1 cells with 0, 2.5, 5, 10, 25, or 50 pfu/ cell and assessed their viability on days 1, 4, and 7 postinfection (Fig. 3C). As expected, there was a timeand dose-dependent decrease in cell viability for both adv.oriP.E1A and wild-type adv.5; however, the wild-type adv.5 began to elicit its cytotoxic effect at earlier time points (e.g., on day 4) than adv.oriP.E1A, consistent with the notion that E1A protein production and subsequent virion assembly would delay replication kinetics in the CRA compared to the wild-type adv.5. Adv.oriP. E1A replicates in an EBV-dependent manner We confirmed adenoviral replication by infecting C666-1 cells with 1 pfu/cell of adv.oriP.E1A, adv.oriP, or wild-type adv.5 and harvested them for total DNA at 24, 48, and 72 h postinfection. Southern blot analysis demonstrated a significant increase in the adenoviral E1A gene particularly from 24 to 48 h after

FIG. 2. Selective expression of E1A in EBV-positive cells. EBV-positive C666-1 and EBV-negative CNE-1 and CNE-2Z NPC cell lines were mock infected (lanes 1) or infected with either adv.oriP.Luc (lanes 2) or adv.oriP.E1A (lanes 3 – 7) with cell lysates analyzed for expression of the 41-kDa E1A protein product. CNE-1, CNE2Z, and control C666-1 lysates (lanes 1 and 2) were obtained 48 h postinfection. C666-1 cell lysates were prepared at 8, 24, 30, 48, and 144 h (lanes 3 – 7, respectively) postinfection with adv.oriP.E1A. Cell lysates from 293 cells, which constitutively express E1A, provided the positive control. All three NPC cell lines are easily transfectable by adv (>90% transfection efficiency with 2 pfu/cell).

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FIG. 3. Effects of adv.oriP.E1A on human cell lines. (A) Lack of cytopathic effects in EBV-negative cell lines. A549 (lung carcinoma), MDA-MB-231 (breast adenocarcinoma), SaOS-2 (osteosarcoma), CNE-2Z (nasopharyngeal carcinoma), and KS-1 (nasopharyngeal fibroblast) cells were infected with 1, 10, or 25 pfu/ cell of adv.oriP.E1A, fixed, and stained 4 days postinfection. There is no evidence of a cytopathic effect in these cells. All cells are efficiently transduced by the adenovirus as demonstrated by h-galactosidase expression after 10 pfu/cell of adv.CMV.B-gal. (B) Top: Dose-dependent decrease in cell viability as determined by MTT assay in C666-1 cells. EBV-positive C666-1 cells were infected with either adv.oriP.E1A or adv.oriP (25 pfu/cell) and assayed for cell viability 7 days postinfection. Radiation (6 Gy) was administered 24 h postinfection. Experiments were conducted three independent times and are reported as means F SEM. Bottom: Cells were infected with 10 pfu/cell of adv.oriP.E1A, fixed, and stained at 3, or 5 days postinfection. Mock-infected cells grew to confluency, in contrast to adv.oriP.E1A-infected cells, which died. (C) Wild-type adv.5 elicits cytotoxicity earlier than adv.oriP.E1A. C666-1 cells were infected with either adv.oriP.E1A or wild-type adv.5 with 2.5, 5, 10, 25, or 50 pfu/cell. Cell viability was assessed 1, 4, or 7 days postinfection.

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FIG. 3 (continued ).

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adv.oriP.E1A and from 0 to 24 h after infection with wild-type adv.5 (Fig. 4A). Consistent with other reports and our cytotoxicity studies, the extent and kinetics of adv.oriP.E1A replication appeared to be reduced and delayed compared to those of the wild-type adv.5 (Figs. 4 and 3C). We corroborated the increase in E1A expression over time using a second assay, quantitative real-time PCR for E1A. In Fig. 4B, this is recapitulated by a constant increase in E1A gene copy number from 24 to 72 h. At 48 and 72 h postinfection, E1A gene copy number increased by 125and 200-fold, respectively. This increase in gene expression over time as demonstrated in Figs. 4A and 4B is further corroborated by Western blot analysis for the fiber knob protein, a structural protein located on the outer capsid of the adenovirus (Fig. 4C). After treatment with 1 pfu/cell of adv.oriP.E1A, there was a significant timedependent increase in fiber knob protein expression,

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detectable at 48 h (Fig. 4C), which follows the increase in gene expression by 24 h (Figs. 4A and 4B). Adv.oriP. E1A inhibits tumor formation and growth in vivo To determine the effects of adv.oriP.E1A on tumor formation in vivo whereby infection efficiency could be controlled, we infected C666-1 cells ex vivo with adv.oriP.E1A (15 pfu/cell) followed by implantation into the gastrocnemius muscle of SCID mice 24 h later. As observed in Fig. 5, animals injected with mock-infected cells had to be sacrificed after 3 – 4 weeks due to tumor burden. Treatment with the control adv.oriP delayed tumor formation by approximately 1 week. However, animals injected with 50% of infected cells did not develop detectable tumors until 60 days postinjection. With 100% of cells infected with 15 pfu/cell of adv.oriP.E1A, there was complete tumor suppression for more than 100 days postinocula-

FIG. 4. Replication of adv.oriP.E1A in C666-1 cells. (A) Southern blot analysis of E1A shows time-dependent increase in adenoviral DNA. EBV-positive C666-1 cells were mock infected (lane 1) or infected with adv.oriP (lane 2) or 1 pfu/cell adv.oriP.E1A (lanes 3 – 6) or wildtype adv.5 (lanes 7 – 9). DNA was isolated at 24, 48, or 72 h postinfection, digested with EcoRI, and probed for E1A. (B) Increase in E1A expression in C666-1 cells over time. Quantitative real-time PCR was conducted to assess E1A gene expression over time. EBV-positive C666-1 cells were infected with 1 pfu/cell of either adv.oriP.E1A or the control virus adv.oriP for 1 h at 37jC. Cellular and viral DNA was extracted using the QiaAMP DNA extraction assay. Ct values were normalized to h-actin expression and compared to values obtained at time 0. PCR was performed in triplicate. (C) Western blot analysis of increasing fiber knob protein expression over time. C666-1 cells were mock infected (lane 1) or infected with either adv.oriP.E1A (lanes 2 – 7, top) or adv.oriP (lanes 2 – 7, bottom) and cell lysates were analyzed for expression of the 62-kDa fiber knob protein product. C666-1 cell lysates were prepared at 4, 8, 12, 24, 48, or 72 h (lanes 2 – 7, respectively) postinfection with adv.oriP.E1A or adv.oriP. h-Actin was used as loading control (41 kDa). Increase in fiber knob protein expression is indicative of adenoviral replication.

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FIG. 5. Lack of tumor formation after ex vivo treatment with adv.oriP.E1A. EBV-positive C666-1 cells were infected in vitro with 15 pfu/cell of either adv.oriP.E1A (50 or 100%) or adv.oriP for 1 h. After overnight incubation at 37jC, cells were injected intramuscularly into SCID mice. Tumor plus leg diameter was then followed three times weekly until 15 mm or for humane endpoints.

tion. These two sets of data (50 and 100%) from the mixing experiments suggest the presence of an in vivo bystander effect, since tumor growth was significantly delayed when only 50% of the inoculated cells were initially infected with adv.oriP.E1A. We conducted therapeutic experiments on established C666-1 and C15 EBV-positive tumors with treatment commencing when the tumor plus leg diameter reached 9 mm (0.3 – 0.4 g). As shown in Fig. 6A, the combination of adv.oriP.E1A and local tumor RT was successful in causing significant tumor regression to nonpalpable levels for at least 3 weeks. In contrast, when we treated animals with adv.oriP.E1A alone, there was a marginal effect on tumor growth suppression over that of the adv.oriP control. RT alone was able to delay tumor growth, but never led to complete tumor regression. These results were recapitulated with the second C15 xenograft model (Fig. 6B), whereby the combination treatment of adv.oriP.E1A and RT also led to significant tumor regression for 2 weeks’ duration. We performed real-time PCR on the C666-1 xenograft tumors to determine whether viral replication was evident in vivo. As observed in Fig. 6C, there was significant increase in viral DNA at 4 h postinfection, but this was limited in that it returned to control levels at 8 h postinfection. These data might explain the modest in vivo effect of adv.oriP.E1A alone (Figs. 6A and 6B), despite the impressive in vitro data. Evaluation of safety and toxicity of systemically administered adv.oriP. E1A It is critical to establish the safety and toxicity aspects of this therapy. It is well established that i.t. treatments of CRAs are well tolerated in both mice and humans [19]. However, to maximize the impact of these therapies, systemic administrations are essential as long-term objectives. Hence, we proceeded to determine the safety and toxicity of this CRA when administered systemically in tumor-bearing mice. It is important to note that the majority of systemically treated mice remained healthy;

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specifically, their coats were shiny, their weights were stable, and they retained normal activity levels. We conducted biochemical and histological examinations on day 3 postinjection and at the humane endpoint of tumor burden (day 17) and compared the results to reference ranges based on normal, healthy SCID mice (4 male, 3 female). As seen in Table 1, we assessed biochemical measures indicating liver, kidney, and pancreatic functions. Alkaline phosphatase activity remained within reference ranges, indicating normal hepatic duct function. This is in contrast to elevations in SGPT at the later time point after adv.oriP.E1A injection and SGOT on day 3 postinjection. These elevations are indicative of perturbations in hepatocyte function; however, there was no histological evidence of hepatocyte damage, as documented in Fig. 7A. Urea and creatinine levels remained in the normal range. There was a modest increase in pancreatic amylase activity at a higher dose of adv.oriP.E1A, but again, we observed no evidence of pancreatic damage upon histological examination (data not shown). At day 17, amylase values returned to normal levels; however, elevations in SGPT and SGOT in animals injected with adv.oriP.E1A were still evident. Adv.oriP.E1A administration was associated with lung inflammation, seen in Fig. 7B, compared to PBS control and adv.oriP (data not shown). The infiltration with macrophages and occlusion of the alveolar space may explain why one of the three animals evaluated at the higher dose of adv.oriP.E1A (2  109 pfu) did not survive. Together, these analyses confirm that there is minimal liver toxicity, but that 2  109 pfu of adv.oriP.E1A given systemically may be the maximum safely administered dose in the SCID model.

DISCUSSION This report describes the successful generation of a novel, EBV-dependent replicating adenovirus that is selectively cytotoxic to EBV-positive cells in vitro and the effect of which is potentiated in vivo when combined with local

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FIG. 6. Significant delay of tumor growth in vivo. EBV-positive (A) C666-1 or (B) C15 xenograft tumors were established in SCID mice, and once they reached 0.3 – 0.4 g, animals were randomized into one of four groups: (1) control, no treatment; (2) RT alone, 2  4 Gy (C666-1), 2  2 Gy (C15); (3) i.t. adv.oriP.E1A; (4) i.t. adv.oriP.E1A + RT. Treatment schedule was as indicated, with each injection comprising 1  109 pfu in 100 Al volume. Statistical significance was determined using the one-way ANOVA. (A) Control vs adv.oriP.E1A + RT, P = 0.002. (B) Control vs adv.oriP.E1A + RT, P = 0.02. At least two independent experiments were conducted for each xenograft model. (C) Change in E1A expression in C666-1 tumors over time. Quantitative real-time PCR was used to assess the increase in E1A gene expression over time. Tumor-bearing SCID mice were injected i.t. with 1.4  107 pfu of adv.oriP.E1A or PBS. Tumors were removed 4, 8, or 24 h postinjection and assessed for E1A gene copy number.

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812 Table 1: Biochemical analysis of selected enzymes and chemistries after systemic administration of Adv.oriP.E1A Day 3 Normal (n = 7)

Control PBS (n = 2)

Ad5.oriP 2  108 (n = 4)

Alkaline phosphatase (U/L) ALT (SGPT) (U/L) AST (SGOT) (U/L) Amylase (U/L) Urea (mM) Creatinine (AM)

108 61 456 6129 8 24

(93 – 140) (51 – 81) (321 – 550) (5359 – 6870) (7 – 8) (20 – 31)

95 138 564 4948 6 35

(32 – 130) (48 – 280) (296 – 1089) (3917 – 5690) (5.2 – 7) (34 – 36)

87 204 449 4883 7 26

(71 – 111) (58 – 585) (334 – 547) (4145 – 4767) (7.1 – 7.5) (22 – 33)

Ad5.oriP.E1A

2  109 (n = 2) 101 208 1011 7444 6 30

(95 – 107) (142 – 274) (899 – 1022) (7009 – 7878) (5.2 – 5.9) (29 – 30)

2  108 (n = 3) 104 83 536 5061 7 28

2  109 (n = 2)

(89 – 117) (68 – 113) (535 – 650) (3931 – 4823) (5.9 – 7.2) (25 – 30)

96 103 885 18,650 7 31

(91 – 100) (96 – 110) (769 – 1,000) (13,160 – 24,140) (5.6 – 9) (30 – 31)

Day 17 Control PBS (n = 2)

Alkaline phosphatase (U/L) ALT (SGPT) (U/L) AST (SGOT) (U/L) Amylase (U/L) Urea (mM) Creatinine (AM)

61 48 447 6295 9 25

(37 – 84) (46 – 50) (387 – 506) (5840 – 6750) (5.5 – 13.1) (21 – 28)

Ad5.oriP

Ad5.oriP.E1A

2  108 (n = 3)

2  109 (n = 3)

2  108 (n = 3)

61 88 604 4330 7 23

84 87 674 7875 11 24

69 238 848 5395 9 29

(52 – 73) (45 – 168) (356 – 995) (3758 – 5409) (5.5 – 8) (17 – 27)

(82 – 86) (55 – 118) (488 – 850) (7170 – 8180) (8.6 – 12) (21 – 27)

(58 – 80) (82 – 501) (590 – 1229) (4674 – 6280) (6.2 – 11.1) (22 – 32)

2  109 (n = 1) 79 80 750 6180 10 28

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Normal reference values were generated from 4 male and 3 female SCID mice. The numbers denote means; ranges denoted in parentheses. n indicates the number of mice for each experimental group.

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FIG. 7. Minimal systemic toxicity of adv.oriP.E1A in vivo. (A) Hematoxylin and eosin-stained sections of liver, heart, spleen, and kidney from mice treated with systemic administration of PBS control, adv.oriP, or adv.oriP.E1A. (B) Lung inflammation and occlusion of the alveolar space was observed after high doses (109 pfu) of systemic administration of adv.oriP.E1A.

RT. Our group has previously demonstrated a significant cytotoxic effect of adenoviral gene therapy using the tumor suppressor p53 in both EBV-negative and EBVpositive cell lines. However, our in vivo experiments highlighted the limited distribution of the adenovirus in the tumor [14]. In combination with a tumor-specific targeting strategy, a replicating adenovirus could begin to address this distribution challenge. Our data from the current study suggest that adv.oriP.E1A is effective in preventing tumor formation, in that 50% of infected cells were able to prevent tumor formation beyond 60 days postinjection (Fig. 5). In the therapeutic experiments, a

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significant delay in tumor growth was achieved when the CRA was administered in combination with RT. However, our studies highlight the limitations of therapeutic efficacy possibly due to suboptimal kinetics of viral replication of CRAs in vivo. Consistent with previous work, the oriP-FR promoter is able to drive expression of E1A in an EBV-dependent manner [9]. As observed in Fig. 2, there is extensive E1A protein expression in the EBV-positive C666-1 cells. This expression is significantly more pronounced over that of the EBV-negative CNE-1 and CNE-2Z NPC cells. The modest expression observed (in the latter cells) at

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48 h might be explained in part by the presence of EBV in approximately 0.1% of these cells as determined by in situ hybridization for EBER (unpublished observations). However, treatment of EBV-negative cells with 10 or 25 pfu/cell of adv.oriP.E1A had no effect, and cells formed healthy, confluent monolayers (Fig. 3A). In contrast, infection of C666-1 cells with adv.oriP.E1A results in a dose-dependent decrease in cell viability (Fig. 3B) that is significant, but its kinetics is slower than that observed for the wild-type adv.5 (Fig. 3C). The replication kinetics of adv.oriP.E1A in comparison to the wild-type adv.5 appears to be delayed (Fig. 3A). This might be explained in part by the required time for transcription of E1A from the oriP-FR promoter and translation, along with assembly of the virion. Fig. 2 demonstrates that E1A protein is detected at 8 h postinfection, reaching maximal levels at 48 h. Although only low levels of E1A are required to complement a replication-deficient adenovirus, there may be a threshold that has to be achieved before viral replication can proceed [10]. Studies in HeLa cells have estimated that the entire process from entry into the host cell to virion progeny release requires 20 – 24 h and 104 virions are produced per wild-type adenovirus [20]. Our data support this kinetics in that extensive amounts of viral DNA from the wildtype adenovirus are observed at 24 h postinfection (Fig. 4A), and yet the E1A and fiber knob proteins (Fig. 4C) are not detectable until 48 h. The kinetics of our CRA, however, is comparable to those of other CRAs such as the DF3 CRA, for which there is a dramatic increase in viral titer at 48 h postinfection in DF3/Muc-1-positive human breast cancer cells [21]. Of note, this CRA contains E1A and the entire E1B region of the adenoviral genome. The prototypic CRA, Onyx-015, which is E1B-55K deleted, has been shown to replicate in p53-abrogated systems [22 – 24], although not exclusively [25,26]. Bischoff et al. report comparable replication kinetics of the Onyx-015 and a wild-type virus in a cervical carcinoma cell line, C33A, in which a significant increase in viral titer is observed as soon as 24 h postinfection [23]. An alternative explanation for the delay in replication kinetics of our CRA may be attributed to the lack of E1B and its associated proteins, E1B-19K and E1B-55K, both of which are important for native adenoviral replication. The E1B-55K protein forms a complex with the adenoviral E4-orf6 gene product and facilitates the preferential transport of viral mRNA during the late stage of lytic adenoviral infection [27,28]. Moreover, both E1B-55K and E1B-19K are important in the regulation of the host cell cycle, by forcing entry into S phase and preventing host cell apoptosis. E1B-55K acts to stabilize the p53 protein and E1B19K prevents host cell apoptosis through its action on the proapoptotic bcl-2 family members bax and bak [29 – 31]. Interestingly, the latent EBV gene product, LMP-1, has also been shown to modulate host

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cell apoptosis through the induction of bcl-2; however, the interaction, if any, of EBV gene products in modulating the efficiency of the replication of the CRA remains to be elucidated [32]. Consistent with this delay in CRA kinetics in vitro, injection of two preestablished nasopharyngeal tumor models with the CRA alone resulted in a modest suppression of tumor growth (Fig. 6). However, the combination treatment of adv.oriP.E1A and RT in vivo appears to have a significant additive effect. Such an interaction has been reported in a human glioma model whereby the combination of an Rb-dependent CRA with RT also resulted in a supra-additive effect [33]. This interaction might be related to accelerated lysis of infected cells after irradiation, thereby allowing for earlier release of progeny virions and improved viral spread. Although it has been reported that RT may create an environment more conducive to adenoviral activity, this was not related to an increase in viral replication [34]. Studies using Onyx-015 have demonstrated an improvement in clinical outcome when delivered in conjunction with RT, but have also demonstrated no changes in virus production [24]. Our initial observations likewise detected no effect of RT on E1A expression, but future detailed studies need to be conducted to characterize better this interaction in our NPC model. Alternatively, the adenoviral E1A protein may be sensitizing the host cell to RT. The E1A protein has been reported to increase susceptibility to apoptosis under stress conditions such as serum starvation, TNF, and gradiation [35,36]. The mechanism of E1A radiosensitization might be mediated through the inactivation of NFnB, thereby reducing survival signals [37]. E1A has been demonstrated to prevent radiation-induced NF-nB activation by prolonging the half-life of InB through the inhibition InB kinase activity, resulting in the cytoplasmic sequestration of NF-nB [38]. Hence, this process might also account for the observed in vivo therapeutic interaction between adv.oriP.E1A and RT [39]. Persistence of a CRA after i.t. injection has been reported to be significantly longer than that of a replication-incompetent adenovirus. Paielli et al. demonstrated that intraprostatic injection of 1010 viral particles of a CRA resulted in approximately 1 viral copy/cell at days 8 and 29 postinjection. This is in contrast to approximately 0.003 viral copies/cell after administration of the replication-deficient counterpart adenovirus [40]. Some, but not all studies have demonstrated that viral persistence is related to viral replication [40,41]. Moreover, when a wild-type (E1A and E1B containing) adenovirus was used, despite its persistence beyond 100 days, complete tumor control was rarely achieved [42]. In our current study, a single i.t. injection of 1.4  107 pfu (approximately 10 9 viral particles) of adv.oriP.E1A resulted in a significant increase in E1A gene expression at 4 h postinjection, indicative of in vivo replication (Fig. 6C). However, this gene expression declined very rapidly

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in vivo, to almost baseline levels at 24 h. This underscores the importance of understanding the complex interaction between the tumor microenvironment and biology of CRAs. The dramatic inhibitory effect of our CRA on tumor formation demonstrates significant cytotoxicity in vivo (Fig. 5). When only 50% of injected cells were infected with adv.oriP.E1A, no tumor formed by 60 days. Contrasted to the results in established tumors (Fig. 6), the effects of limited adenoviral spread are further highlighted. The physical obstacles hindering a replicating adenovirus may include unfavorable pressure gradients or binding to extracellular matrix components, both of which have been shown to limit the spread of other macromolecules such as antibodies [43]. In the context of a xenograft model, mouse inflammatory cells, fibroblasts, necrotic region, and accumulation of connective tissue as the tumor grows may also participate in preventing viral spread [44]. Finally, gene expression profiles differ significantly when cells are in a threedimensional matrix [45]; hence, there could also be cellular signals that may inhibit viral replication. To understand the potential role of CRA in clinical management of NPC, the safety and toxicity of systemic injection was investigated. The histological and biochemical data confirm that there are no acute hepatic effects after systemic injection of 109 pfu, or approximately 1011 viral particles (Table 1, Fig. 7). The slight perturbation in amylase values is consistent with a previous report by Wildner and Morris using a semipermissive cotton rat model [46]. On day 7 after administration of five daily doses of 6  1010 viral particles of a CRA, they observed a significant increase in amylase, yet found no corroborating chemical or pathological changes to support pancreatitis. Paielli et al. conducted a study examining viral persistence and germ-line transmission after intraprostatic injection of 1  1010 viral particles [40]. They also reported acceptable vector-associated toxicity and suggested that the pathologies associated with viral replication are in fact due to increased viral load and gene expression, not necessarily to virusinduced cytolysis. Clinical experience with other CRAs has been encouraging, with reports of tumor response and limited toxicity [19]. The Onyx-015 trials have demonstrated tumor response in combination with RT in head and neck cancer patients [47]. More recently, a phase I trial evaluating a prostate-specific antigen-responsive CRA with the cytotoxic fusion gene cytosine deaminase/thymidine kinase in combination with RT has demonstrated only grade I/II related toxicities and biochemical evidence of tumor response [48]. Based on the promise of these clinical trials and the success of our CRA in combination with RT, we propose future evaluation of our CRA with attachments of therapeutic molecules, which will ultimately improve outcome for patients with NPC.

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METHODS AND MATERIALS Cells, culture conditions, and tumor models. Cells and culture conditions were utilized as previously described [9,12]. Briefly, the EBVpositive NPC cell line C666-1 provided by D.H. was maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Wisent, Inc.). The EBV-negative NPC cell lines CNE-1 and CNE-2Z were obtained from The Cancer Institute/Chinese Academy of Medical Sciences. KS-1 is a primary fibroblast cell line that we obtained from the nasopharynx of a patient with NPC [13]. A549 (lung carcinoma), SaOS-2 (osteosarcoma), and MDA-MB-231 (breast carcinoma) cell lines were used to confirm the effects of our treatment in non-NPC EBVnegative cells. These cells were all obtained from ATCC (Manassas, VA, USA). Human fetal renal 293 cells were used for viral propagation. Unless otherwise stated, all cell lines were maintained in a-MEM supplemented with 10% FBS (Wisent, Inc.), and all experiments were conducted when cells were in an exponential growth phase. To generate nasopharyngeal xenografts, approximately 106 EBV-positive C666-1 cells or C15 cells (from P.B.) were implanted into the gastrocnemius muscle of SCID mice. After treatment, tumor growth was evaluated by measuring tumor plus leg diameter, as previously described [14]. Construction of the recombinant adenovirus. The novel CRA was generated by PCR amplification of the adenovirus-5 E1A transcriptional unit (1133 bp) (Expand High Fidelity PCR System, Roche) from the pXC1 plasmid (Microbix, Hamilton, Canada), ligated into pCR2.1 TOPO (Invitrogen, Carlsbad, CA, USA), and excised as an EcoRI fragment. After its sequence was verified, the E1A transcriptional unit was cloned downstream of the 897-bp oriP – basal CMV promoter, previously inserted as a SalI – HindIII fragment within the pE1SP1A vector, to generate poriPE1A (Fig. 1). The control construct poriP-SP1A contained only the oriP – basal CMV promoter. PCMV-B-gal was generated as previously described using the nuclear-localizing h-galactosidase [12]. Recombinant viruses (adv.oriP.E1A, adv.oriP, adv.oriP.Luc, adv.CMV.Bgal) were generated by homologous recombination in 293 cells following calcium phosphate cotransfection of poriPE1A, poriP, poriP.Luc, or pCMV-B-gal with the adenoviral genome contained in pJM17 (Microbix). Individual plaques were expanded in 293 cells and purified using cesium chloride gradient ultracentrifugation [15]. Viral titers were determined based on cytopathic effect using the plaque-forming assay, expressed as pfu/volume. Viral titers ranged from 109 to 1010 pfu/ml. Wild-type adenovirus type 5 (wild-type adv.5) was generously provided by Dr. M. Hitt, McMaster University, Hamilton, Canada. Infection and irradiation of cells in vitro. C666-1 cells were seeded in culture flasks (2  106 cells/T80, 5  105 cells/T25, or 1  104 cells/well in 96-well plates) in RPMI with 10% FBS. After 3 days, cells were infected with adv.oriP.E1A, adv.oriP, adv.CMV.B-gal, or wild-type adv.5 in medium with 2% FBS for 1 h at 37jC. When the effects of radiation were characterized cells were irradiated at room temperature using a 137Cs unit (Gamma-Cell 40 Exactor; Nordion International, Inc., Canada) at 6 Gy (dose rate of 1.1 Gy/min). Cells were then harvested at appropriate time points for protein extraction, MTT assay, or DNA isolation. These C666-1 cells are very sensitive to confluency, hence the maximum duration of in vitro experiments is 7 days. Western blot analysis of E1A or fiber knob protein. Cells were treated with adv.oriP.E1A or adv.oriP and harvested at selected time points. Cell extracts were prepared in lysis buffer (0.1 M Tris – Cl, pH 8.0, 0.1% SDS, 10 mM EDTA, 2 mM DTT) and protein concentrations were determined using the BCA protein assay (Bio-Rad, Hercules, CA, USA). Immunoblotting was conducted as previously described [12]. Briefly, samples containing equal amounts of protein were loaded onto a 12% SDS – PAGE gel, electrophoresed for 120 min at 100 V using the electrophoresis cell (Bio-Rad), and transferred onto nitrocellulose membranes using a Trans-Blot semidry cell (Bio-Rad). Membranes were blocked in 5% milk in PBST (0.1% Tween 20 in PBS) for 30 min at room temperature and then probed with 0.2 Ag/ml

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mouse monoclonal E1A (Neomarkers, Fremont, CA, USA), 0.2 Ag/ml fiber knob (Neomarkers), or 1.0 Ag/ml actin (Sigma – Aldrich Canada, Inc., Mississauga, ON, Canada), all in PBST containing 5% low-fat milk. Blots were then washed with PBST and incubated with horseradish peroxidaseconjugated secondary antibodies. The specific complexes were detected using chemiluminescence (DuPont, Boston, MA, USA). Southern blot analysis of E1A. Cells were treated with adv.oriP.E1A, adv.oriP, or wild-type adv.5 and harvested at 24, 48, or 72 h postinfection. Total DNA was isolated from cells (QIAamp Mini Kit; Qiagen, Valencia, CA, USA) and 5 Ag of total DNA was digested with EcoRI (Invitrogen Canada, Inc., Burlington, ON, Canada). Fragments were resolved using a 1% agarose gel and transferred onto Hybond-N+ nylon membrane (Amersham Biosciences, Piscataway, NJ, USA) following standard protocols [16]. The specific probe was prepared by using the EcoRI fragment of E1A labeled with 32P using the RadPrime DNA labeling system (Invitrogen) and purified using Amersham S-200HR. Blots were probed overnight at 65jC using the appropriate volume of probe to achieve 106 cpm/ml of hybridization buffer. Blots were washed and visualized on a Phosphoimager (Molecular Dynamics Storm 860; Amersham Biosciences). Determination of adv.oriP. E1A using quantitative real-time PCR. A set of TaqMan PCR primers and probe were designed specific for the E1A region of adenovirus type 5 by using the PrimerExpress software (Applied Biosystems, Foster City, CA, USA). C666-1 cells were infected with 1 pfu/ cell adv.oriP.E1A or control virus adv.oriP. Cells were lysed at 24, 48, or 72 h postinfection and total DNA was isolated (QIAamp Mini Kit;, Qiagen). For in vivo analysis, C666-1 cells (f106) were injected subcutaneously into SCID mice. Once tumors reached 0.2 – 0.5 g, they were treated with a single injection of 1.4  107 pfu of adv.oriP.E1A or PBS in 100 Al. Tumors were removed at 4, 8, or 24 h postinjection and snap-frozen, and then total DNA was isolated from the entire tissue (QIAamp Mini Kit; Qiagen). All samples were analyzed in triplicate and quantitative real-time PCR was performed using a Perkin – Elmer/ABI Prism 7700 sequence detection system (PE Biosystems, Foster City, CA, USA) using the following cycling profile: 95jC for 10 min followed by 40 cycles of 95jC for 15 s, 60jC for 30 s, and 72jC for 1 min. h-Actin was used as a control, probe and primers were designed commercially (Applied Biosystems). The mean fold change in expression was calculated using the 2 DDCt method [17], by which E1A Ct values are normalized to h-actin Ct values and compared to untreated control. Effect of adv.oriP. E1A on cell viability. To evaluate the cytopathic effects of adv.oriP.E1A in both EBV-negative (CNE-1, CNE-2Z, A549, SaOS-2, MDA-231, KS-1) and EBV-positive (C666-1) systems, cells were infected with adv.oriP.E1A (10 or 25 pfu/cell) or adv.CMV.B-gal (25 pfu/cell). Four days postinfection, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with hematoxylin and cellular morphology was examined. To assess the effect of adv.oriP.E1A on viability, cells were seeded in 96-well plates and, after one doubling time, exposed to either adv.oriP.E1A (2.5, 5, 10, 25, 50 pfu/cell) or adv.oriP (25 pfu/cell) for 1 h in 20 Al of 2% FBS-containing medium. This was followed by the addition of 0.2 ml of 10% FBS-containing medium. After cells were incubated for 24 h, appropriate groups were irradiated at room temperature with 6 Gy. Cell viability was determined two doubling times postinfection (7 days) using the MTT assay as previously described [18]. Briefly, cells were exposed to 10% MTT solution (Sigma), prepared in PBS and filter sterilized for 3 h, after which acid-isopropanol with 0.04 N HCl was added to all wells and mixed to dissolve the MTT formazam crystals formed by viable cells. Plates were then read on a Bio-Rad 3350 microplate reader at 570 nm (Bio-Rad). Tumor formation and therapeutic experiments in vivo. All animal experiments used SCID (severe combined immunodeficiency) BALB/c mice obtained from the Ontario Cancer Institute, Animal Research Colony, and the experiments were conducted in accordance with the guidelines of the Animal Care Committee, Ontario Cancer Institute, University Health Network.

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Tumor formation experiments. C666-1 cells were infected in vitro using 15 pfu/cell of adv.oriP.E1A (50 or 100%) or adv.oriP (100%) or mock infected for 1 h at 37jC. After an overnight incubation at 37jC in 10% FBS medium, cells were injected intramuscularly into the left gastrocnemius of SCID mice in a total of 100 Al using a 28-gauge insulin needle fused to a 1-ml syringe (Becton – Dickinson, Oakville, ON, Canada). Tumor plus leg diameter was measured three times per week. Animals were sacrificed when tumor plus leg diameter reached 15 mm or approximately 1.7 g. An intramuscular model was chosen for these experiments to reflect the rich vascularity of the nasopharynx in human patients. Therapeutic experiments. Female SCID mice ages 6 – 8 weeks were injected intramuscularly in the left gastrocnemius with approximately 106 (C666-1 or C15) cells. After 2 – 4 weeks, tumor plus leg diameter reached 9 mm (0.3 – 0.4 g tumor), and the animals were randomized into one of following five groups: (1) control, no treatment; (2) RT alone, 4 Gy  2 (C666-1) or 2 Gy  2 (C15); (3) adv.oriP i.t.; (4) adv.oriP.E1A alone i.t.; (5) adv.oriP.E1A i.t. plus RT. Normal mouse leg diameter is approximately 7 mm. The daily dose of adenoviral injection was 2  109 pfu administered in six injections of 100 Al each on days 0, 1, 2, 5, 6, and 7. For local RT on days 1 and 6, animals were immobilized in a Lucite box with the tumor-bearing leg exposed to 100 kV at a dose rate of 10 Gy/min. Assessment of safety and toxicity. Tumor-bearing animals (tumor 0.3 – 0.4 g) were randomized into three groups with intraperitoneal injections: (1) PBS control, (2) adv.oriP virus control (2  108 or 2  109 pfu), (3) adv.oriP.E1A (2  108 or 2  109 pfu). Blood chemistries were analyzed at two different time points: day 3 and day 17 (tumor plus leg diameter reached 15 mm) postinjection. Blood biochemistry reflecting liver (alkaline phosphatase, SGPT, SGOT), pancreas (amylase), and kidney (urea, creatinine) functions were analyzed upon sacrifice by Vitatech (Toronto, ON, Canada). Organs were removed (brain, heart, lung, liver, spleen, and kidney), fixed in formalin, and paraffin-embedded and representative 6Am sections were stained with hematoxylin and eosin for histological analysis. Statistical analysis. All data are reported as means F SEM unless otherwise stated. Statistical differences between groups were determined using a one-way ANOVA test (Microsoft Excel, 2000).

ACKNOWLEDGMENTS This work was supported by funds from the Canadian Institutes of Health Research and the Elia Chair in Head & Neck Cancer Research. M.C. is supported by a CIHR Doctoral Award. RECEIVED FOR PUBLICATION DECEMBER 28, 2003; ACCEPTED MARCH 19, 2004.

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