An E1B-19 kDa gene deletion mutant adenovirus demonstrates tumor necrosis factor-enhanced cancer selectivity and enhanced oncolytic potency

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doi:10.1016/j.ymthe.2004.03.017

An E1B-19 kDa Gene Deletion Mutant Adenovirus Demonstrates Tumor Necrosis Factor-Enhanced Cancer Selectivity and Enhanced Oncolytic Potency Ta-Chiang Liu, Gunnel Hallden, Yaohe Wang, Gabriel Brooks, Jennelle Francis, Nick Lemoine, and David Kirny,* Viral and Genetic Therapy Program, Cancer Research U.K. and Imperial College Faculty of Medicine, London, UK *

Current affiliations: Department of Clinical Pharmacology, Oxford University Medical School, Oxford, UK, and Jennerex Biotherapeutics, Inc. y

To whom correspondence and reprint requests should be addressed. E-mail: [email protected].

Oncolytic adenoviruses hold promise as a new treatment platform for cancer, but limitations have been identified, including limited spread and potency. The adenoviral protein E1B-19 kDa is a Bcl2 homologue that blocks apoptosis induction via the intrinsic and extrinsic pathways, specifically including tumor necrosis factor-mediated cell death. We demonstrate that an E1B-19 kDa gene deletion mutant had tumor necrosis factor-enhanced cancer selectivity, in vitro and in vivo, due to genetic blocks in apoptosis pathways in cancer cells. In addition, this mutant demonstrated significantly enhanced viral spread and antitumoral potency relative to dl1520 (aka Onyx-015) and wild-type adenovirus in vitro. Significant antitumoral efficacy was demonstrated in vivo by intratumoral and intravenous routes of administration. E1B-19 kDa deletion should be considered as a feature of oncolytic adenoviruses to enhance their safety, spread, and efficacy. Key Words: oncolytic, adenovirus, cancer, immunology, virology, experimental therapeutics

INTRODUCTION Replication-selective oncolytic viruses hold promise as therapeutic agents for cancer [1 – 5]. Adenovirus and herpesvirus deletion mutants, for example, have been engineered and developed for replication-selective antitumoral treatment [6 – 9]. Selective replication of a viral agent within tumors may lead to improved efficacy over nonreplicating agents due to the self-perpetuating nature of the treatment with virus multiplication, lysis of the infected cancer cells, and spread to adjacent cells. To date, the most common approach used to engineer tumor selectivity is the gene-deletion approach, described initially by Martuza [8]. The approach is to delete a gene product whose function is necessary for replication and cytolysis in normal cells but is expendable in cancer cells due to genetic inactivation of the cellular target protein(s). Unfortunately, due to the multifunctional nature of many viral proteins, the currently utilized gene deletions conferring selectivity also frequently result in significantly reduced potency of the virus in tumors [3,10,11]. dl1520 (Onyx-015), for example, has demon-

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strated tumor-selective replication and necrosis induction within head and neck carcinomas in patients; normal tissues have not been adversely affected clinically [12 – 15]. However, although selective tumor necrosis has been demonstrated in several clinical trials, objective responses with this virus as a single agent have been uncommon (V10%) [15]. Similar results have been reported with gene-deleted herpesviruses (e.g., G207, 1716) in Phase I trials [16 – 18]. It would therefore be desirable to maintain the selectivity but enhance the efficacy of these oncolytic viruses. Potency improvement will presumably require identification of mutations resulting in selectivity but not attenuation in cancer cells. Of note, mathematical models [19] and experimental data [20] both demonstrate that the rate of viral spread from cell to cell in a tumor will strongly correlate with efficacy; mutations that accelerate spread are therefore highly desirable. Apoptosis is a critical cellular defense mechanism to protect against inappropriate S-phase entry and abnormal DNA synthesis [21]. Apoptosis mechanisms must therefore be blocked for efficient DNA virus replication or

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carcinogenesis to occur [22,23]. It is generally believed that two apoptosis induction mechanisms, namely the death-receptor (extrinsic) signaling pathway and the mitochondria (intrinsic) signaling pathway, control the apoptosis process [24]. Apoptosis is inhibited in the majority of human cancers through overexpression of antiapoptotic proteins (e.g., Bcl-2) or decreased expression of proapoptotic proteins (e.g., p53, Bax) [25,26]. Bcl-2 over-

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expression, for example, occurs in low-grade follicular lymphomas due to the chromosome 14:18 translocation. Bcl-2 overexpression also occurs in a majority of nonsmall-cell lung carcinoma and colon and breast cancers [27,28]. Finally, loss of p53 function is another critical mechanism leading to apoptosis inhibition in cancer [29]. The p53 pathway appears to be blocked in the vast majority of human tumors through numerous mecha-

FIG. 1. Total viral replication in tumor cells is increased by E1B-19 kDa gene deletion. Cells were infected with 100 or 1000 ppc and harvested at indicated time points. Replication was assessed in the human tumor cells (A) PT45, (B) A2780, (C) A2780/CP, and (D) Hep3B; in murine tumor cells (E) CMT-167, (F) CMT-64, and (G) JC; and in human normal cells (H) NHBE. The resulting quantity of infectious virus was determined as described and values were normalized on an infectious unit produced per cell basis. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment.

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nisms. It is therefore believed that apoptosis pathways are dysregulated in virtually all human cancers [30,31]. Human adenoviruses use a similar strategy to block apoptosis of infected cells following E1A expression and induction of viral DNA replication [32 – 35]. It has been shown that after the initiation of apoptosis by the deathsignaling molecule TNF-a, caspase-8 is activated, which leads to Bid truncation. The truncated Bid is then translocated into mitochondria, where it binds Bax, and leads to cytochrome c release and caspase-9 activation [36].

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Adenoviral E1B-19 kDa, a functional homologue of Bcl-2, is able to bind Bax and/or Bak and inhibit the downstream apoptosis process [37 – 39]. It has also been shown that E1B-19 kDa prevents Fas-mediated apoptosis by disrupting FADD oligomerization [40]. Hence, in terms of antiapoptotic function, E1B-19 kDa is capable of inhibiting apoptosis both from receptor-mediated death-signaling pathways and via the mitochondrial pathway [41]. E1B-19 kDa gene expression and inhibition of apoptosis may therefore be necessary for efficient adenovirus repli-

FIG. 2. Virus release is increased by E1B-19 kDa gene deletion. Cells were infected as described, and culture media were collected and titered at indicated time points. Virus release was assessed in the human tumor cells (A) PT45, (B) A2780, and (C) A2780/CP; in murine tumor cells (D) CMT-167, (E) CMT-64, and (F) JC; and in human normal cells (G) NHBE. The resulting quantity of infectious virus was determined as described and values were normalized on an infectious unit produced per cell basis. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment.

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cation in cells with intact apoptosis pathways. Since the vast majority of tumor cells already have blocked apoptosis pathways genetically, we hypothesized that the replication of E1B-19 kDa viral mutants would be abrogated in normal cells but not in tumor cells. In addition, as TNF-a is

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one of the major antiadenoviral cytokines [34,42 – 44], we hypothesized that the selectivity of the E1B-19 kDa viral mutant would be amplified in a system in which TNF-a is present, as it would be in vivo in patients. This would translate into more rapid clearance of E1B-19 kDa viral

FIG. 3. EC50 value comparison between dl250 and Ad2wt; potency is increased by E1B-19 kDa gene deletion. The dose – response curves of Ad2wt, dl250, and inactivated dl312 in different cell lines were obtained by MTS assay as described. The EC50 values of each virus were then determined and compared in human cancer cells (A) PT45, (B) A2780, (C) A2780CP, and (D) Hep3B; in murine cancer cells (E) CMT-167, (F) CMT64, and (G) JC; and in normal human cells (H) NHBE. Representative data from one experiment are shown here. Red curves, dl250; green curves, Ad2wt; blue curves, PUV-inactivated dl312.

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mutants in normal tissues but not in cancerous tissues in vivo. In addition, it has been proposed that E1B-19 kDa is able to counteract the function of the E3 protein ADP in preventing premature viral release [45]; therefore, its deletion should lead to more rapid viral release and might result in enhancement of viral spread in tumor tissues. Since cell-to-cell spread has been a limiting factor for the efficacy with oncolytic adenoviruses [19,20], we hypothesized that the more rapid cytopathic effect induction and spread by E1B-19 kDa-deleted viruses would lead to enhanced potency in tumor cells in vitro and superior intratumoral spread and efficacy in vivo. Several recent publications have described the antitumoral effects of E1B-19 kDa mutants for the treatment of cancers [46 – 48]. Although these data are of clear interest, a number of critical questions were left unanswered. The E1B-19 kDa mutant used in these studies also contains a deletion in the E3B gene region; this is of clear importance since this region itself has antiapoptotic functions and has effects on antitumoral efficacy (T. Liu, submitted for publication). In addition, no normal cells were tested in vitro and no normal tissue was analyzed in vivo; hence, selectivity was not explored. Although in vivo efficacy studies were performed in nude mice with one tumor cell line, the relative antitumoral efficacy of the E1B-19 kDa/ E3B gene deletion mutant versus a wild-type control was not evaluated. Of note, previous studies in our laboratory demonstrated that the deletion of E3B results in decreased viral persistence in tumors in immunocompetent mice in vivo [49]. Thus, the antitumoral efficacy of an adenoviral mutant with E1B-19 kDa deletion in isolation is still unknown. In addition, the roles of TNF-a and/or other immune system components were not investigated in these studies. Therefore, additional efficacy and selectivity data with E1B-19 kDa gene-deletion viruses were eagerly awaited, particularly from in vivo model systems. We therefore compared the selectivity and potency of adenoviral mutants with deletions in one or both gene regions that antagonize TNF-mediated apoptosis: E1B-19 kDa, E3B, and E1B-19 kDa/E3B mutants (T. Liu et al., submitted for publication). The adenoviral E1B-19 kDa/ E3B mutant demonstrated the greatest TNF-mediated attenuation in normal cells; the single-gene-deletion mutants demonstrated similar but less pronounced attenuation. However, the E3B gene deletion also led to reductions in efficacy and increased in vivo clearance from tumors. In contrast, the antitumoral potency of oncolytic adenovirus was significantly enhanced when E1B-19 kDa was deleted while maintaining the E3B region intact. Here we report on data from an E1B-19 kDa mutant adenovirus (dl250) on ovarian, breast, hepatic, lung, laryngeal, pancreatic, and murine lung and mammary tumors in vitro and/or in vivo, in addition to primary, nonimmortalized normal cells of epithelial origin. Importantly, the impact of TNF-a on selectivity was clearly demonstrated. Enhanced tumor selectivity was

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also demonstrated in a recently described novel immunocompetent tumor model for oncolytic adenovirus. In addition, improved antitumoral efficacy due to the E1B-19 kDa deletion was demonstrated following intratumoral and intravenous administration.

RESULTS Adenoviral E1B-19 kDa Deletion Enhances Viral Replication in Tumor Cells but Not in Normal Cells We infected human tumor cells (PT45, A2780, A2780/CP, Hep3B), human normal cells (NHBE), and murine tumor cells (CMT-167) with either dl250 or Ad2wt as described. We harvested culture media and cell lysates 48, 72, and, in some cases, 96 h postinfection (p.i.) and titered as described. In tumor cell lines, the E1B-19 kDa mutant virus dl250 consistently showed greater replication (up to sevenfold higher) than Ad2wt at both 48 and 72 h p.i. (Figs. 1A – 1G; P < 0.05 in all cell lines). The difference between dl250 and Ad2wt was more significant at 72 and 96 h p.i. in all but one tumor cell line (Hep3B), presumably due to the amplification during the viral replication cycles. A similar pattern of replication enhancement was observed in PT45 and CMT-167 cells at 96 h p.i. Of note, the titer of dl250 in Hep3B cells at 72 h p.i. remained at a level similar to that at 48 h p.i. This is due to rapid cell death caused by dl250; the monolayer was almost completely destroyed at 48 h p.i. (data not shown) and hence no surviving cells were able to produce viral progeny. Normal NHBE cells were also able to support the replication of dl250. In contrast to most cancer cell line results, however, dl250 replication decreased over time relative to Ad2wt (Fig. 1H). At 48 h p.i. the titer ratio of dl250 to Ad2wt was 2.58, and at 72 h the ratio was 1.58 ( P value between Ad2wt and dl250 at 72 h p.i. was 0.2013). The titer obtained from samples collected 96 h p.i. also showed no difference between dl250 and Ad2wt. Furthermore, in contrast to cancer cell lines, the titer of dl250 did not increase significantly from

TABLE 1: EC50 ratio comparison EC50 ratio (mut/wt) Cell line

dl250

dl1520

H1299 MDA-MB-231 HLaC MiaPaCa PT45 A2780 A2780CP Hep3B

0.022 0.13 0.182 0.00029 0.45 0.51 0.36 0.26

36.84 19.25 29.25 59.33

The E1B-19 kDa mutant dl250 and E1B-55 kDa mutant dl1520 were tested on a panel of tumor cell lines and the EC50 values were determined. The EC50 ratios of each mutant virus and wild-type control were then obtained and compared. Representative data from one experiment are shown here. dl250 is more potent than dl1520 in the cell lines tested.

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FIG. 4. Effects of TNF-a on viral replication, cytopathic potency, and apoptosis induction. Viral replication, release, EC50, and apoptosis induction were measured with and without TNF treatment as described under Materials and Methods. Total viral replication was assessed in (A) PT45 cancer cells and (B) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Viral release was assessed in (C) PT45 cancer cells and (D) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *TNF-a treatment; +level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment. The percentage change in EC50 values of (E) Ad2wt and (F) dl250 after TNF treatment was studied. Error bars represent standard errors between different experiments (n = 4). In situ caspase-3 activation was determined in (G) NHBE and (H) PT45 cells as an indication of apoptosis induction. Cells were untreated or treated with viruses and/or TNF-a as described. At 24, 48, and 96 h p.i., cells were fixed and stained for active form caspase-3 as mentioned (n = 4). Open bars, data from 48 h p.i.; filled bars, data from 72 h p.i.; gray bars, data from 96 h p.i. Error bars represent standard errors between different experiments.

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FIG. 4 (continued ).

48 to 96 h p.i. although the monolayer was intact at 96 h (data not shown). Taken together, dl250-infected tumor cells supported enhanced viral replication compared to that of Ad2wt, whereas in normal cells the replication of dl250 was similar to that of Ad2wt. This is likely due to apoptosis induction and virus degradation in normal cells infected with dl250, which lead to protection of uninfected cells (see below). Adenoviral E1B-19 kDa Deletion Results in Enhanced Viral Release from Infected Cells We tested tumor cell lines (PT45, A2780, A2780/CP, CMT-167, CMT-64 and JC) and normal cells (NHBE)

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for viral release into the media over time. We infected and harvested the cells as mentioned above and compared viral titers in the culture media between dl250 and Ad2wt. Not only did dl250 cause earlier viral release compared with Ad2wt (up to 9-fold higher at 48 h p.i.) in tumor cell lines, but also the absolute titer released continued to increase over time (Figs. 2A – 2F; P value < 0.05). Therefore, the E1B-19 kDa deletion resulted in enhanced speed and magnitude of viral release from tumor cells. Normal NHBE cells infected with dl250 also showed earlier viral release. The viral titers in culture media of dl250-infected samples were higher than those of Ad2wt-infected samples at all time points tested

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(Fig. 2G; P < 0.05). However, while the titer of Ad2wtinfected samples increased 16-fold from 48 to 96 h p.i. ( P < 0.05), no significant change was found in the titer of dl250-infected samples after 48 h p.i. This is in marked contrast to results with dl250 in cancer cell lines. Adenoviral E1B-19 kDa Mutant Demonstrates Potency Superior to That of Ad2wt in Tumor Cell Lines We tested dl250 and Ad2wt on a panel of cell lines for their potency. We measured cytotoxicity by determining the EC50 value for the viruses on each cell line. We compared the EC50 value of dl250 and Ad2wt in each cell line. In parallel, we also determined the EC50 value for Onyx-015 (dl1520) and compared it. The EC50 value for dl250 was lower (i.e., more potent) than that for Ad2wt in all cell lines tested, including normal cells (Fig. 3). In addition, dl250 was more potent than Onyx-015 in the tumor cell lines tested (Table 1).

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TNF-a Treatment Inhibits Viral Replication and Release of dl250 in Normal Cells through Apoptosis Induction Previous published data on dl250 suggested that E1B-19 kDa played a critical role in protecting infected cells from TNF-a-mediated killing [56,57]. We hypothesized that the behavior of dl250 might differ in the presence of TNF-a. We therefore explored the effects of TNF-a on viral infection and release in human (PT45) or murine (CMT-167, CMT-64, and JC) tumor cell lines and normal human (NHBE) cells. Six hours postinfection, we added TNF-a to the culture medium (0.5 ng/ml). At multiple time points p.i., we harvested samples and titered them as described above. dl250 was still associated with enhanced viral replication compared to Ad2wt in PT45 tumor cells (Fig. 4A). Of note, the titer of both viruses did not differ from those of the samples without TNF-a treatment. Similar results were obtained from CMT-167, CMT-64, and JC cells (data not shown). TNF-a therefore had no

FIG. 5. Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10  20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10  20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10  20).

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FIG. 5 (continued ).

demonstrable effect on viral replication in these tumor cells. In contrast, in normal NHBE cells the replication of dl250, but not of Ad2wt, was significantly decreased when TNF-a was added (Fig. 4B; P < 0.05 for dl250 with and without TNF-a treatment at 72 and 96 h p.i.). Whereas the titer of Ad2wt was similar to samples without TNFa treatment at all time points tested, the titer of dl250 was significantly reduced at 72 and 96 h p.i. in the presence of TNF-a to a level even lower than at 48 h p.i. ( P < 0.05). The titers obtained at 72 and 96 h p.i. were also lower than those of Ad2wt ( P < 0.05). This decrease in titer over time suggests that apoptosis caused degradation of both cellular and viral DNA. We also compared viral release into the culture medium after TNF-a treatment. In PT45 cancer cells, the titers of

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both dl250 and Ad2wt remained similar in the medium with and without TNF-a treatment (Fig. 4C). Similar results were obtained from murine tumor cells (data not shown). In NHBE cells, we found no significant change in the titer of Ad2wt with and without TNF-a treatment (Fig. 4D). In contrast, the titer of dl250 was significantly decreased in the presence of TNF-a (decreased to 62, 42, and 36% at 48, 72, and 96 h p.i., respectively; P < 0.05 at 72 and 96 h p.i.). We hypothesized that the decrease in replication and release of dl250 in normal cells was due to TNF-mediated apoptosis. To assess this possibility, we determined the EC50 values for dl250 and Ad2wt with and without TNF-a added at 6 h p.i. The EC50 value for Ad2wt did not significantly change in any of these cell lines after

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FIG. 5 (continued ).

TNF-a treatment (Fig. 4E). Likewise, the EC50 value for dl250 in the tumor cell lines did not significantly change in the presence of TNF-a. In contrast, the EC50 value for dl250 in NHBE showed a 20% decrease (Fig. 4F; P < 0.05). Therefore, only dl250-infected NHBE cells were sensitized to TNF-a. We determined immunoreactivity for active caspase3, an index for apoptosis, for apoptosis induction in normal cells (NHBE) and cancer cells (PT45). TNF-a alone did not increase apoptosis proportion in these cells. dl250 caused a markedly higher level of apoptosis than Ad2wt as early as 24 h p.i. (Fig. 4G; P < 0.05); dl250 + TNF-a showed significantly higher level of apoptosis compared to dl250 alone or Ad2wt + TNF-a in NHBE cells ( P < 0.05). In contrast, no significant differences

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between virus and virus + TNF-a treatment groups were found in PT45 cells (Fig. 4H). Therefore, the decrease in EC50 value and viral replication/release after TNF-a treatment in dl250-infected NHBE correlated with induction of apoptosis. In Vivo Cancer-Selectivity Enhancement The results shown above indicated that the selectivity of the E1B-19 kDa mutant adenovirus might be enhanced in vivo, especially in animals with an intact immune system. We therefore tested these viruses on immunocompetent tumor-bearing mice, which allowed us to compare the replication status of these viruses in tumors in vivo. We implanted CMT-167 and JC cells in the flanks of C57BL/6 or Balb/c mice, respectively, and injected viruses intrave-

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FIG. 5 (continued ).

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nously once tumors reached 30 – 60 mm3. Since the majority of intravenously injected virus is cleared by the liver [58], we compared the virus replication and pathology in both liver and tumor tissues. At various time points posttreatment, we harvested livers and tumors and analyzed them for viral replication (viral gene expression and plaque-forming unit (PFU) recovery), general histopathology, and apoptosis induction. Of note, murine liver is semipermissive for adenoviral replication [59]. In addition to the intravenous in vivo selectivity studies, we also injected viruses intratumorally into JC tumors to determine the intratumoral viral replication status of dl250 vs Ad2wt. dl250 gene expression and replication were significantly lower than with Ad2wt in normal livers at late time points (48 h); in contrast, in tumor tissue dl250 replication significantly exceeded that of Ad2wt. While at 24 h postinjection the viral gene expression levels were similar between Ad2wt and dl250 treatment groups, by 48 h posttreatment the adenoviral E1A levels were significantly higher in livers treated with Ad2wt than in those treated with dl250 (Figs. 5A and 5B; P < 0.05). Similar results were obtained for hexon expression level (data not shown). We confirmed this by infectious PFU recovery: dl250 showed levels of PFU titer similar to those of Ad2wt at 24 h postinjection

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but they were significantly reduced at 48 h postinjection (Fig. 5C; P < 0.05). In contrast, the dl250 treatment group showed higher E1A and hexon expression within JC or CMT-167 tumors at all time points studied (Figs. 6A and 6C; P < 0.05). We confirmed this result by the infectious PFU recovery from tumor samples at similar time points. In the intratumoral (i.t.) injection study, the dl250 treatment group showed 1800-fold higher PFU titer compared to the Ad2wt treatment group at day 6 (Fig. 6B; P < 0.01) and 6-fold higher than the Ad2wt group at day 10 ( P < 0.05). When the viruses were given intravenously, the dl250 group showed 7-fold higher titer in tumors than the Ad2wt treatment group at 48 h postinjection (Fig. 6D; P < 0.01). We scored the cytopathic effect (CPE) severity induced in virus-treated liver tissues from 1 to 4; higher scores represented more severe CPE. In C57BL/6 mice, the CPE severity following dl250 treatment did not increase throughout 120 h posttreatment, while the CPE severity in Ad2wt-treated mice increased significantly between 24 and 120 h posttreatment ( P values between Ad2wt and dl250 at 48 and 120 h both <0.05; Fig. 5D). We also compared the acute inflammation severity in the periportal area since virus delivered by the intravenous route localized mainly within this

FIG. 6. Adenoviral gene expression and replication/persistence in replication-semipermissive murine tumor tissues in vivo. Viruses or PBS were injected on day 1 (109 particles) into JC tumors in immunocompetent mice. (A) E1A score (day 6 postinjection). Error bars represent standard errors between different animals. *Undetectable. (B) Plaque-forming unit (PFU) recovery data are shown on a per gram of tumor basis. Open bars, data from day 6; closed bars, day 10. Error bars represent standard errors between different animals. *Undetectable. Viruses (1  1010 particles) were injected on day 1 via tail vein into immunocompetent mice bearing CMT-167 tumors in the flank as described. (C) Hexon intensity score at various time points. Open bars, data from 24 h postinjection; filled bars, 48 h postinjection; gray bars, 120 h postinjection. Error bars represent standard errors between different animals. (D) PFU recovery data are shown on a per gram of tumor basis. Open bars, data from 24 h postinjection; filled bars, data from 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable.

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area. The Ad2wt treatment group showed a significantly higher degree of periportal inflammation than the dl250 treatment group at both 24 and 48 h posttreatment (Fig. 5E; P < 0.05). Fig. 5F represents examples of the pathology of liver. In Balb/c mice bearing JC tumors, Ad2wt also induced more severe liver damage than did dl250 (data not shown). In contrast, in tumor tissue there was no difference in cytopathic effect between the two treatment groups. Finally, we evaluated active caspase-3 over time posttreatment in both liver and tumor tissue. As predicted, dl250-treated livers had significantly greater apoptosis scores at 24 h posttreatment (Figs. 5G and 5H); this finding was consistent with the proposed mechanism of selectivity. By 48 h posttreatment, however, the dl250 treatment group showed a marked decrease in active caspase-3 (P < 0.05). In contrast, the active form caspase-3 in the Ad2wt-treated livers continued to increase from 24 to 48 h posttreatment, exceeding that of the dl250 treatment group. Thus, the decreased hepatotoxicity of dl250 was associated with acute apoptosis induction and viral clearance. In contrast, as predicted by previous in vitro experiments, the active form caspase-3 in tumors was equivalent between the two treatment groups (data not shown). Taken together, these results suggest that dl250 not only replicates preferentially in tumors compared to normal tissue, it also is associated with enhanced replication/release and/or delayed clearance in tumors compared to Ad2wt. The Antitumoral Efficacy of the E1B-19 kDa Mutant in Vivo Was Equivalent or Superior to That of Wild-Type Adenovirus We tested the viruses in a human pancreatic cancer (PT45) xenograft model. We gave intratumoral injections of viruses as described under Materials and Methods. At 90 days posttreatment, all animals treated with PBS showed progressive disease, while both dl250 and Ad2wt viruses resulted in 25 – 30% complete responses (Fig. 7A). We also observed delayed tumor growth in dl250 and Ad2wt treatment groups compared to the control group (Fig. 7B). dl250 showed comparable efficacy to Ad2wt in this treatment model. We have previously shown the efficacy of both viruses in an orthotopic human breast cancer MDAMB-231 xenograft model. Following intravenous treatment for 5 consecutive days, the dl250 treatment group showed a 50% (6/12) complete response rate, compared to none in the Ad2wt treatment group (P < 0.05 versus dl250). Because more patients have been treated with dl1520 (aka Onyx-015 E1B-55 kDa-deleted adenovirus) than any other oncolytic adenovirus, we also evaluated dl1520 in this model; only one of nine tumors responded completely (P < 0.05 versus dl250). The dl250-treated group also had a significantly greater

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FIG. 7. Antitumoral efficacy of E1B-19 kDa deletion mutant dl250 in vivo. PT45 human pancreatic cancer xenograft was implanted into C57BL/6 nu/nu mice as described. PBS or viruses (1  1010 particles) were injected intratumorally on days 1, 3, and 5. (A) Animal survival was monitored and compared. Both dl250 and Ad2wt treatment showed similar survival rates and both were higher than control group. (B) Tumor growth curve. Both dl250 and Ad2wt treatment groups showed significant delays in tumor growth compared with the PBS group.

survival than all other treatment groups, as well (P < 0.05). Therefore, dl250 was associated with superior efficacy compared to Ad2wt and dl1520 in a nude mouse – human tumor xenograft model of intravenous treatment.

DISCUSSION In this paper we report data gathered to test two hypotheses relating to an adenoviral mutant with a deletion in the E1B-19 kDa gene. First, we hypothesized that this virus might demonstrate enhanced viral release from tumor cells compared to wild-type adenovirus; we predicted that this phenotype would be associated with increased efficacy. In addition, we predicted that replication of the E1B-19 kDa mutant in normal cells would be reduced secondary to rapid apoptosis induction, particularly in the presence of TNF-a, since the apoptosis pathway activated by TNF-a would no longer

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be inhibited by E1B-19 kDa. In contrast, due to blocks at multiple levels in apoptosis pathways in tumor cells (e.g., p53 mutation, Bcl-2 overexpression), TNF-a was predicted to have limited effects on E1B-19 kDa mutant adenovirus replication in tumor cells. Thus, we predicted that the E1B-19 kDa deletion mutant would be cancer-selective in vivo. Indeed, the E1B-19 kDa mutant dl250 showed enhanced viral replication and release in a broad range of cancer cells regardless of TNF-a treatment. In normal cells, in contrast, TNF-a significantly reduced the replication of dl250, but not of Ad2wt; apoptosis-associated degradation of dl250 DNA was suggested by a decrease in infectious units and an increase in caspase-3 over time. However, only one normal cell type was tested. Therefore, dl250 demonstrated TNF-a-dependent selectivity in vitro in addition to increased antitumoral potency. dl250 also demonstrated clear cancer selectivity in vivo in immunocompetent tumor-bearing mice. While viral titers decreased in the livers of immunocompetent mice by >90% over 48 h, intratumoral viral titers were significantly higher than those of the Ad2wt group at 48 h posttreatment. Moreover, the intratumoral spread of dl250 was dramatically increased versus Ad2wt. Finally, the efficacy of dl250 was equal to or superior to that of Ad2wt in the efficacy models tested. These efficacy findings were consistent between intravenous and intratumoral dosing routes. Therefore, the E1B-19 kDa mutant dl250 is both a broadly potent and a highly selective oncolytic adenovirus. Previous studies have demonstrated that adenoviral E1B-19 kDa deletion/deficiency mutants have larger plaques on infected cells (lp phenotype), have enhanced cytopathic effect (cyt phenotype), and can cause viral and cellular DNA degradation (deg phenotype) in human cells in vitro [50,60,61]. This is believed due to protection against E1A-induced apoptosis by E1B-19 kDa in vitro [62,63]. In addition, it has been proposed that E1B-19 kDa counters the adenovirus death protein (ADP) indirectly, hence delaying cell death and subsequent viral release. E1B-19 kDa expressed in isolation from the viral genome was also shown to protect infected cells from TNF-a-induced apoptosis [36]. However, these studies were largely based on one or two immortalized cell lines (e.g., A549, KB, HeLa cells), and in some cases the cell lines were found to contain gene integration from other viral species (e.g., HPV) [57,64,65]. Thus, the role of this viral gene in a broad range of cancer cells, and in the context of the adenovirus life cycle, was unknown. Moreover, the effects of dl250 on normal primary cells had never been explored. Finally, the impact of TNF-a treatment on E1B-19 kDadeleted adenoviruses in cancer versus normal cells had not been described, this despite the critical role of TNF-a family members on adenovirus biology in vivo. Our study is therefore the first to report that an E1B-19

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ARTICLE kDa mutant has reduced cytopathic effects and replication in normal cells, specifically in the presence of TNF-a in vitro. This report is also the first to study these endpoints with intact E3B (see Discussion below). Apoptosis of dl250-infected normal cells leads to degradation of infectious viruses, thereby limiting further virus spread and toxicity. The cancer selectivity of E1B-19 kDa mutant adenoviruses is affected by other adenovirus gene products. Of note, previous publications describing E1B-19 kDa mutant viruses as oncolytic agents also tested a virus that also had a deletion in the E3B region [47,48,65]. The genes deleted from this region include the RID complex (E3-10.4/14.5k) and E3-14.7k. These proteins block death-receptor signaling pathways at a level upstream of mitochondria [42,66 – 69]. We have shown that an adenoviral E1B-19 kDa/E3B mutant had enhanced selectivity and antitumoral potency compared to the E3B mutant (T. Liu et al., submitted for publication). Of note, however, the E1B-19 kDa mutant with intact E3B region had similar selectivity plus enhanced efficacy compared to the E1B-19 kDa/E3B double-mutant virus. E3B deletion also appears to accelerate clearance from tumors and normal tissue in vivo [49]. Therefore, although both gene regions appear to play important roles in protecting against apoptosis, the E1B-19 kDa gene deletion appears uniquely to increase both selectivity and efficacy. In addition, several other cell-death-promoting adenoviral gene products have been shown to be promising in causing efficient oncolysis (e.g., ADP, E4ORF4) [70 – 73]. It will be worth exploring whether the potency and selectivity of E1B-19 kDa deletion mutants are impacted when combined with manipulations in these genes. Although these in vitro findings were encouraging, we still needed to test the hypothesis that the in vivo selectivity of this virus would be further enhanced with an intact immune system and that its antitumoral potency would be maintained or even enhanced. We took advantage of our recently identified immunocompetent murine tumor efficacy models in which adenovirus gene expression and/or replication is supported by tumor cells; these models may therefore more closely approximate the immunocompetent clinical setting. Our study is the first to describe the use of an E1B-19 kDa mutant in an immunocompetent and replication-semipermissive murine tumor model. We demonstrated that this mutant virus had a markedly increased therapeutic index relative to wild-type adenovirus, both with an enhanced safety profile and with increased antitumoral effects. Of note, a publication by Duncan et al. had previously demonstrated adenovirus replication within Balb/c murine liver (in contrast to murine lung, for example) [59]. In our studies of both Balb/c and C57BL/6 mouse strain models, dl250 consistently induced less viral gene expression, liver damage, and local inflammation than

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Ad2wt. As predicted, dl250-infected hepatocytes underwent rapid apoptosis, and spread of the cytopathic effects was extremely limited. Finally, infectious viral titers of dl250 were lower and cleared more readily from the liver compared with Ad2wt. These findings mirrored the in vitro data in the presence of TNF-a and indicated that E1B-19 kDa mutant adenoviruses could also be cleared through accelerated apoptosis induction in normal tissue in vivo; apoptosis occurred prior to significant viral gene expression or tissue damage. In contrast, despite more rapid clearance from normal liver, viral gene expression and infectious unit production in treated tumors was significantly greater following dl250 than Ad2wt treatment. Thus, the in vivo cancer selectivity of the E1B-19 kDa-deleted mutant was magnified by both reduced toxicity to normal tissue and enhanced replication and efficacy in tumor tissue. These results have important clinical implications for oncolytic adenoviruses. Systemic toxicity, especially hepatotoxicity, could be potentially reduced through E1B-19 kDa deletion, with a concomitant increase in intratumoral replication. This is critical since most replication-selective adenoviruses (e.g., dl1520, E2F-promoter-driven adenovirus) have significant attenuation in cancer cells compared with wild-type virus [3,74,75]. Human carcinomas have multiple blocks within apoptosis pathways, demonstrating the critical role of apoptosis in protecting the host from carcinogenesis. Likewise, apoptotic pathways must be inactivated by adenoviruses to prevent premature viral clearance. Apoptosis can be inhibited in cancers through loss of tumor suppressor genes (e.g., p53, bax, PTEN) or overexpression of oncogenes (e.g., bcl-2). Recent publications have pointed out the interactions between E1B-19 kDa and Bcl-2 family members (e.g., Bax, Bak) [39,76]. The status of these proteins in cancer cells might therefore affect the potency and replication efficiency of E1B-19 kDa mutants. The human cancer cell lines reported in this publication have a variety of genetic alterations, suggesting that the virus is able to take advantage of a wide range of antiapoptotic mechanisms. For example, these lines varied in their p53, bax, and bcl-2 status, but all lines were highly permissive for replication and cytopathic effects. We do not yet know which blocks in apoptosis will be ideally complemented by E1B-19 kDa gene deletion, although data indicate that bax might be crucial [77]. Matched A2780 cell lines demonstrated that induction of cisplatin resistance was associated with a generalized decrease in adenoviral permissiveness. Studies are planned with matched cell lines plus/minus overexpression of specific Bcl-family member proteins. It will be important to explore the impact of these cellular proteins on the phenotype of the infection when infected with an E1B-19 kDa mutant and wild-type adenovirus. Elucidation of the interaction of the virus

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and these cellular proteins might help to identify cancer types and patients that are most likely to benefit from E1B-19 kDa virotherapy. In addition, E1A can sensitize Ad-infected cells to other TNF superfamily cytokines, which can also be inhibited by E1B19 kDa [78,79]. One potential approach might be to engineer E1B-19 kDa deletion mutants to express these cytokines. Great efforts have been made in recent years to reveal the mechanisms of adenovirus clearance and toxicity in vivo. Although TNF-a is known to be one of the major antiadenoviral cytokines, studies have shown that depleting TNF-a alone does not completely ameliorate the toxicity and clearance of micro-organisms such as human adenovirus, as the redundancy of LT-a compensates for TNF-a deletion [80]. In contrast, another group reported that the status of TNFR (both TNFR1 and TNFR2), but not the cytokines LT-a and/or TNF-a themselves, determined the toxicity and clearance of adenovirus [81]. As in vitro selectivity was markedly increased by TNF-a, it will be interesting to see whether the toxicity and clearance of E1B-19 kDa mutants will be altered in TNF and/or TNFR knockout mice. The potential roles of other acute inflammatory cytokines that are known to be induced by adenovirus will also be studied (e.g., TRAIL, interferon-a/h or -g, interleukins-4 and -6). In summary, we report that deletion of the E1B-19 kDa gene from adenovirus resulted in marked selectivity in the presence of TNF-a and significantly improved replication and potency in tumor lines in vitro; potency was markedly greater than with the lead clinical adenovirus Onyx-015. in vivo, the E1B-19 kDa deletion not only reduced toxicity to normal tissue, it also enhanced viral replication and spread in tumor tissue, leading to significant antitumoral efficacy. This deletion also results in additional space being available for the insertion of a therapeutic transgene(s) and/or heterologous regulatory elements (e.g., tissue-specific promoter – enhancer elements). This additional space in the genome will be critical for next-generation adenoviruses. Oncolytic viruses can be armed with genes to enhance their efficacy and safety [4,82], but space is limiting since packaging becomes extremely inefficient once the viral genome increases by more than 5% [83]. This is particularly important for replication-competent adenoviruses since E1 genes such as E1A must be maintained, and deletion of E1B-55 kDa leads to significant attenuation even in some p53deficient cancer cells [10,84]. Likewise, we have recently demonstrated that E3 immunomodulatory genes have a significant impact on efficacy in immunocompetent tumor-bearing mouse models [49]. Finally, deletion of the E3-ADP also appears to reduce antitumoral potency [85]. It may therefore be preferable to keep the majority of the E3 region intact. Other gene regions

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such as E1B-19 kDa will need to be deleted to allow space for therapeutic transgene insertion (aka ‘‘armed viruses’’). Therefore, the E1B-19 kDa deletion should be considered for incorporation into multimutated oncolytic adenoviruses. One future direction for oncolytic adenovirus development, for example, will be to combine the E1A-CR2 and E1B-19 kDa deletions, as these two deletions both lead to selectivity and enhanced antitumoral potency [86]. Further studies will be necessary to determine whether other oncolytic viruses may also benefit from the deletion of their antiapoptotic genes.

MATERIALS AND METHODS Cell lines and cell cultures. Human pancreatic carcinoma cell line PT45, human hepatocellular carcinoma cell line Hep3B, human ovarian carcinoma cell lines A2780 and A2780/CP, and murine lung adenocarcinoma cell lines CMT-64 and CMT-167 were obtained from the Cancer Research UK Central Cell Service (Cancer Research UK, London, UK). Human embryonic kidney cell line HEK 293, human breast carcinoma cell line MDA-MB-231, human laryngeal carcinoma cell line HLaC, human pancreatic carcinoma cell line MiaPaCa, human non-small-cell lung cancer cell line H1299, and murine mammary adenocarcinoma cell line JC were obtained from ATCC. PT45, Hep3B, MDA-MB-231, HLaC, MiaPaCa, HEK 293, CMT-167, and JC were maintained in DMEM supplemented with 10% FCS. H1299, A2780, and A2780/CP were maintained in RPMI supplemented with 10% FCS. Normal human bronchial epithelial cells NHBE were obtained from Clonetics Corp. (CA, USA) and maintained following the instructions of the provider. Viruses. Wild-type Ad2 and the E1B-19 kDa-deleted Ad2 mutant dl250 [50] were provided by Dr. G. Chinnadurai. The E1B-55 kDa-deleted mutant dl1520 was provided by Dr. A. Berk [51]. The nonreplicating Ad5 mutant dl312 [52] was inactivated by psoralen – UV as previously described [53] to serve as a nonreplicating particle control for in vitro cell survival and in vivo efficacy studies. All adenoviruses were grown on the human embryonic kidney cell line HEK293 as previously described [54]. In vitro viral replication assay. Cells were seeded in six-well plates (3  105 cells/well) and infected with different viruses at 100 or 1000 particles/ cell (ppc) for 1 h. Replication assays were repeated at least three times with each condition in triplicate. Cells were harvested and media collected 48, 72, and/or 96 h p.i., either with or without medium and cell lysate separation. For NHBE, PT45, A2780, A2780/CP, CMT-167, CMT-64, and JC, the samples were collected and media and cell lysates were analyzed separately. For Hep3B, media and cell lysates were analyzed without separation. The collected samples were freeze/thawed three times, and replicating virus was determined by the limiting dilution assay as described before [55]. To determine the effect of TNF-a on infected cells, NHBE, PT45, CMT-167, and JC cells were infected as described above and 0.5 ng/ml TNF-a (Oncogene, CA, USA) was added to the cells at 6 or 24 h p.i. Samples were harvested at 48, 72, and/or 96 h p.i. In vitro cell survival assay. For cell survival assays, 1 – 2  104 cells were seeded in 96-well plates and 24 h later infected with different viruses at various concentrations. Cell survival was determined by CellTiter 96 AQueous nonradioactive cell proliferation assay (MTS assay; Promega, CA, USA) 3 – 7 days p.i. and the EC50 values of different adenoviruses were obtained from the dose – response curve. For NHBE and Hep3B cells, assays were terminated at 3 days p.i.. For PT45 cells, assays were terminated at 4 days p.i. For all other cell lines, assays were terminated at 6 days p.i. All assays were performed at least three times with each condition in quadruplicate. To determine the effect of TNF-a on infected cells, cells were infected as described above and TNF-a was added 6 h later (0.5 ng/

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ml). Adenoviruses and TNF-a remained in the culture medium during the remaining incubation period. In situ caspase-3 activity detection. For in vitro studies, cells were grown in chamber slides 24 h before treatment. Viruses and TNF-a were added according to the schedule used for the viral replication assays. At various time points p.i., slides were washed with cold PBS and fixed with methanol. The active form caspase-3 was detected by immunohistochemistry (IHC) using rabbit anti-active caspase-3 antibody (R&D Systems, Abingdon, UK; diluted 1:1000). The Envision+ System (DAKO, Cambridgeshire, UK) was subsequently used according to the instructions of the provider. Active form caspase-3 was defined by brownish color and ‘‘ground glass’’ appearance in cytoplasm. For in vitro studies, the percentage of caspase-3-positive cells was determined by counting a total of 1000 cells for each condition in triplicate. Three independent experiments were performed and the average percentage of apoptotic cells was reported. Similar staining procedures were used to analyze in vivo samples on slides. The in vivo samples were scored as follows: for liver tissues, average number of cells showing positive active caspase-3 in each high-power field (HPF; 10  20) was obtained by observing 10 – 15 random HPFs. For tumor tissues, the percentage of cells showing positive signals was scored as follows: 0, no positive cells; 1, less than 10% positive cells; 2, 10 – 20% positive cells; 3, >20% positive cells. Animal viral toxicity and biodistribution study. Murine lung adenocarcinoma cell line CMT-167 cells (5  106 cells) were injected subcutaneously into both flanks of immunocompetent C57BL/6 mice (obtained from Cancer Research UK Biological Resources). The animals were then randomized into three treatment groups (n = 9 per group): PBS, Ad2wt, and dl250. Intravenous injection of viruses was initiated once the tumor size reached 30 – 60 mm3 and 1  1011 viral particles were injected via the tail vein in a volume of 100 Al. At 24 and 48 h postinjection, mice were sacrificed (three mice per time point), and tumor and liver samples were harvested and processed for general histopathology, in situ apoptosis detection, viral gene expression, and replication assays. Serum samples were analyzed for liver function tests. Similar experiments were done with immunocompetent Balb/c mice bearing JC tumors. General histopathology. The harvested tissue samples were immediately dipped into precooled isopentane for 3 min, frozen in liquid nitrogen, and stored at 70jC. Each sample was cut into cryostat sections (5 – 7 Am) serially. The first three and the last three sections were processed by staining the sample slides with hematoxylin and eosin, followed by light microscopy. CPE in liver tissue was defined as either necrosis or one of the following: enlarged nucleus, eosinophilic cytoplasm, or intranuclear inclusion bodies. The severity of the cytopathic effects was scored as follows: 1, <25% of cross section demonstrating CPE; 2, 25 – 49%; 3, 50 – 74%; 4, z75%. The extent of periportal infiltration was assessed by scoring the numbers of acute inflammatory cells (neutrophils and eosinophils) surrounding the periportal area and quantitating as follows: 0, 1 – 3 inflammatory cells; 1, 4 – 10 inflammatory cells; 2, 11 – 15 cells; 3, 16 – 30 cells; 4, >30 cells. The perivascular inflammation severity in tumors was analyzed by determining the average number of intratumoral vessels surrounded by acute inflammatory cells (neutrophils, eosinophils) per square centimeter of tissue. IHC studies. IHC staining was used for adenoviral E1A and hexon detection in tissue samples. Samples were frozen in liquid nitrogen, sectioned, and fixed with 4% formaldehyde prior to staining. These sections were first blocked with porcine serum for E1A staining and horse serum for hexon staining (DAKO; diluted 1:20) and incubated with either rabbit anti-E1A (Santa Cruz Biotechnology, CA, USA; diluted 1:50) or goat anti-hexon (Accurate Chemicals, NY, USA; diluted 1:150). After repeated washing in PBS, the biotinylated secondary antibodies (porcine anti-rabbit antibody for E1A, DAKO, diluted 1:300, and horse anti-goat antibody for hexon, Vector, CA, USA, diluted 1:150) were then

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added. The endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. The slides were incubated with streptavidin – horseradish peroxidase complex (DAKO; diluted 1:300), followed by development in diaminobenzidine solution (DAKO) supplied with 0.03% hydrogen peroxide. Once the desired staining intensity was achieved, the slides were washed, counterstained with hematoxylin, dehydrated, mounted, and analyzed by light microscopy. For liver tissues, the expression of E1A was analyzed by obtaining the number of E1A-positive cells with nuclear staining from 10 random HPFs, and the average number of E1A-positive cells per HPF is shown. The expression of hexon was scored as in tumor tissues (described below). For tumor tissues, the percentage of E1A- or hexon-positive cells was analyzed as follows: 0, no positive cells; 1, less than 10% positive cells; 2, 10 – 20% positive cells; 3, 20 – 30% positive cells; 4, more than 30% positive cells. In vivo viral PFU recovery. For in vivo viral PFU recovery assays, tissue samples were first homogenized in DMEM into suspension by tissue grinder (Kendal, MA, USA). The suspension was then freeze/thawed three times in DMEM. Following centrifugation at 4000 rpm for 5 min, the supernatant was used to infect HEK293 cells with the limiting dilution assay mentioned above. Intratumoral viral replication studies. JC cells were implanted in both flanks of immunocompetent Balb/c mice. Tumor growth was monitored twice weekly. Treatment was started when tumor sizes reached 30 – 50 mm3. PBS, Ad2wt, dl250, or UV-inactivated dl312 were given i.t. (1  1010 viral particles) in a volume of 80 Al. Injections were given at days 1, 2, and 3. At various time points postinjection, animals were sacrificed and tumors harvested for PFU recovery and/or viral gene expression analysis. Efficacy studies. The antitumoral efficacy of dl250 compared to Ad2wt was tested in vivo. PT45 human pancreatic cancer cells were implanted subcutaneously into one flank (5  106 cells) of 6- to 8-week-old ICRF C57BL/6 nu/nu mice (from Cancer Research UK Biological Resources). Once the tumors reached 30 – 50 mm3, the mice were randomized as described above (n = 12 per group). PBS or viruses were injected intratumorally at days 1, 3, and 5 at a dose of 3  1010 particles. Tumor size and animal survival were monitored and animals were sacrificed when tumor size reached 1 cm3 or after 3 months. MDA-MB-231 human breast cancer cells were implanted subcutaneously into the mammary fat pads (1  106 cells) of ICRF C57BL/6 nu/nu mice (from Cancer Research UK Biological Resources). Once the tumors reached 30 – 50 mm3, the mice were randomized into four groups (n = 12 per group): PBS, dl250, Ad2wt, and dl1520. Viral particles (1  1010) were injected via the tail vein for 5 consecutive days. Tumor size and animal survival were monitored as above. Statistical analysis. Unpaired t test or one-way ANOVA were used for all in vitro studies and in vivo toxicity and replication studies. Kaplan – Meier survival curves were compared using log-rank test. RECEIVED FOR PUBLICATION FEBRUARY 15, 2004; ACCEPTED MARCH 22, 2004.

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