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. 2011 Nov 15;20(2):306–316. doi: 10.1038/mt.2011.242

Modification of the Early Gene Enhancer-promoter Improves the Oncolytic Potency of Adenovirus 11

Han Hsi Wong 1, Guozhong Jiang 1, Rathi Gangeswaran 1, Pengju Wang 2, Jiwei Wang 2, Ming Yuan 1, Hexiao Wang 1, Vipul Bhakta 1, Heike Müller 1, Nicholas R Lemoine 1,2, Yaohe Wang 1,2
PMCID: PMC3272483  EMSID: UKMS36897  PMID: 22086234

Abstract

Oncolytic adenoviruses based on serotype 5 (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5. Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity. Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cell's sensitivity to Ad11 killing. Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production. To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5-sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11-Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found in both the Ad5-sensitive and Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP.

Introduction

Cancer virotherapy has expanded rapidly in recent years, particularly with the use of adenoviruses. In 2005, an adenovirus with E1B 55K and E3B gene deletion (H101; Sunway Biotech, Shanghai, China) was approved in China as the world's first oncolytic virus for head and neck cancer therapy. However, a similar virus, ONYX-015 (Onyx Pharmaceuticals, Emeryville, CA), has failed to show significant activity in malignancies such as pancreatic cancer, a disease with high morbidity and mortality that is notoriously difficult to treat.1 This failure is due to the complex interactions between the virus, tumor cells, and the host immunity.2 One important factor limiting the efficacy of Ad5-based therapy has been shown to be the paucity of expression of the Coxsackie and adenovirus receptor (CAR) on some tumors. CAR is ubiquitously expressed in epithelial cells, but it is often downregulated in many cancer types due to activation of the Raf-MAPK pathway.3 The expression of the leucine-rich repeat-containing 15 (LRRC15) protein, frequently upregulated in tumor cells, can also result in the redistribution of CAR away from cell surfaces.4 Furthermore, the efficacy of intravenously delivered adenoviruses can be hindered by neutralizing antibodies and complement activation. Importantly, the liver is a predominant site of adenovirus sequestration with resulting hepatotoxicity. Adenoviruses are taken up by Kupffer cells5 and excess viruses are able to transduce liver hepatocytes, mediated by the binding of adenoviral hexon protein to the blood coagulation factor X.6

A total of 57 serotypes of human adenoviruses have been identified so far, which are divided into subgroups A to G. In contrast to other species of adenoviruses which use CAR as the primary attachment receptor, most subgroup B adenoviruses (such as Ad11) use CD46.7,8 It is expressed in all human nucleated cells and is upregulated in a number of tumor types.9,10,11 Several chimeric oncolytic Ad5 viruses have been developed (with fibers derived from subgroup B adenoviruses but the remainder of the particle from Ad5) to target CD46, although the use of intact subgroup B adenoviruses as oncolytic agents is still underexplored. They have different tropism and infectivity compared to the chimeric viruses,12 and are more beneficial in terms of a reduced propensity for neutralization by antibodies that are mainly directed against the hexon protein.13

Ad11 can be classified into the prototype strain Ad11p and the less common Ad11a, with the former having better binding affinities than Ad11a in several human cell lines.14 It has several distinct advantages over Ad5 for development as an oncolytic virus. The prevalence of neutralizing antibodies within the human population is lower for Ad11 (10–31%; 45–90% for Ad5), with no crossreactivity between them.15,16,17,18 When injected intravenously into CD46-transgenic mice, there was an almost complete absence of liver transduction15 and toxicity.19 Evidence also suggests that Ad11 attaches to other receptor(s).15,20 Tentatively named “receptor X,” Tuve et al. reported that Ad11 is the only subgroup B adenovirus that uses both CD46 and receptor X, suggesting that it could potentially infect a wider range of tumor cells and overcome the problem of receptor downregulation that confounds the use of Ad5.8 Strauss et al. showed that adenoviruses that utilize CAR or CD46 as attachment receptors failed to infect and lyse ovarian cancer cells of the epithelial phenotype, which are found in in situ tumors and tumor xenografts.21 These receptors are trapped in the tight junctions and are not accessible to the virus. However, adenoviruses that use receptor X could induce epithelial-mesenchymal transition and result in efficient oncolysis. It was just reported that desmoglein-2 is a receptor for adenovirus serotypes 3, 7, 11, and 14, which has been proposed as a marker for epithelial tumors.22 Finally, Ad11 can transduce dendritic cells with higher efficiency. This is beneficial in terms of cancer immunotherapy whereby a stronger immune response could be elicited against an encoded tumor-specific antigen.15,16,23

Here, we explored in detail the ability of Ad11 to infect and kill cancer cells in comparison to the widely-used Ad5. Consistent with cellular CD46 expression, we found that Ad11 has much higher infectivity than Ad5. However, its replicative and cell-killing capability did not correlate with its infectivity, but rather with its E1A gene transcription. Furthermore, we demonstrated that a novel Ad11 mutant with the Ad5 E1A (instead of wild-type Ad11 E1A) enhancer-promoter is a promising backbone for the future development of more potent and tumor-selective oncolytic viruses.

Results

CD46 and desmoglein-2 expression does not correlate with Ad11-mediated cytotoxicity in human cancer cell lines

Having confirmed that the levels of CD46 expression in cancer cells were higher than those of CAR (Supplementary Figure S1a) by FACS as described in Supplementary Materials and Methods, we compared the oncolytic potencies of Ad11 and Ad5 in a panel of human cancer cell lines in vitro (Figure 1a,b). Surprisingly, only nine of the 25 cell lines tested were more sensitive to Ad11—Hs766T, Capan-1, Capan-2, PaTu 8988s (pancreas), PC-3 (prostate), MCF7, MDA-MB-468, SK-BR-3 (breast), and HT-29 (colon). There was no significant difference in 22Rv1 (prostate) (P = 0.0602). Ad5 was superior to Ad11 in inducing cell death in the other lines. Given that desmoglein-2 has recently been reported as a receptor for adenovirus serotypes 3, 7, 11, and 14,22 we investigated the expression of desmoglein-2 in 18 human tumor cell lines used above by FACS as described in Supplementary Materials and Methods. As shown in Supplementary Figure S1b, the expression of desmoglein-2 varied between the Ad11-sensitive and -insensitive cell lines. These results suggest that CD46 and desmoglein-2 expression does not correlate with Ad11-mediated cytotoxicity in human cancer cell lines. We focused our attention on four of the cell lines, namely MIA PaCa-2, LNCaP (both Ad5-sensitive), Capan-2, and PC-3 (both Ad11-sensitive), to dissect the mechanisms behind the differential sensitivities. As expected, oncolytic potencies were associated with increased production of infectious virus particles (Figure 1c).

Figure 1.

Figure 1

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Oncolytic potencies and replication of adenovirus serotype 5 (Ad5) and Ad11 in human cancer cell lines. (a, b) Cells were infected in 96-well plates and MTS assay was performed on day 6 after infection. Graphs show the mean EC50 values ± SEM, with a lower value indicating better cell killing. (c) Production of infectious Ad5 and Ad11 after infection with 100 particles/cell, determined by the limiting dilution assay using JH-293 as indicator cells. Results are presented as mean plaque-forming units (PFU)/cell ± SEM.

In vivo anti-tumor efficacy by oncolytic Ad11 and Ad5 in human cancer xenografts

The potent in vitro activity of Ad11 against the Ad5-less sensitive PC-3 prostate cancer cell line prompted further evaluation of this virus in vivo. Significant growth suppression of tumors formed of subcutaneously implanted PC-3 cells was observed in the Ad11-treated group compared to Ad5 (day 24, P = 0.0016), together with improved progression-free rates of the animals (log rank P = 0.0193) (Figure 2). Unsurprisingly, Ad11 was not as effective as Ad5 in treating the Ad11-less sensitive MIA PaCa-2 pancreatic cancer xenografts (day 35, P = 0.0483; log rank P = 0.0267 between Ad5 and Ad11). The better efficacy of Ad11 or Ad5 was associated with higher levels of viral DNA replication and virus production (Figure 3).

Figure 2.

Figure 2

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Antitumoral efficacies of adenovirus serotype 5 (Ad5) and Ad11 in PC-3 and MIA PaCa-2 subcutaneous xenograft models. (a) Mean tumor volumes ± SEM in BALB/c nude mice (n = 8/group) after three intratumoral injections of phosphate-buffered saline (PBS), Ad5 or Ad11 (1 × 1010 particles/injection), measured until the first mouse in each group has a tumor dimension of >1.44 cm2. (b) Percentage of progression-free mice using the Kaplan–Meier method.

Figure 3.

Figure 3

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Virus replication after injection of 1 × 1010 particles intratumorally in PC-3 and MIA PaCa-2 subcutaneous xenograft models. (a) Viral DNA by quantitative-PCR, shown in mean arbitrary units normalized against total DNA ± SEM. (b) Production of infectious virus particles, shown in mean plaque-forming units (PFU)/ng of total DNA ± SEM.

Comparison of the infectivity of Ad5 and Ad11 in human cancer cell lines

To determine the mechanisms involved in Ad11's attenuated potency in cancer cell lines less sensitive to Ad11, we carried out a stepwise investigation of the adenovirus infectious cycle: attachment, internalization, trafficking to the nucleus, expression of early gene products, viral DNA replication, structural protein synthesis, and release of progeny viruses. Using two separate approaches, we demonstrated that significantly more Ad11 than Ad5 attached to the cell membranes of human cancer cell lines regardless of their sensitivities to Ad11 (Figure 4a). This is consistent with cellular receptor expression. The attached Ad11 particles were trafficked to the nuclei effectively, showing much higher levels of nuclear entry in the early phase of infection compared to Ad5.

Figure 4.

Figure 4

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Mechanisms involved in the attenuated oncolytic potency of adenovirus serotype 11 (Ad11). (a) Representative confocal microscopy images showing virus attachment (1 hour at 4 °C), and trafficking 60 minutes after unbound viruses were washed off (37 °C). Adenoviral capsids were labeled with Alexa Fluor 555 before infection (red); nuclei and cytoplasm were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and α-tubulin immunofluorescence (green), respectively. Graphs show the quantitative-PCR results of viral DNA for viruses attached to the cell membrane (4 °C) and those in the nuclei at 30, 60, and 120 minutes post-attachment (37 °C). Cells were infected with viruses at 1,000 particles/cell. (b) E1A mRNA levels after virus infection at 100 particles/cell, normalized against 18S rRNA. (c) Viral DNA replication after virus infection at 100 particles/cell. (d) Western blots of adenoviral hexon proteins after virus infection at 200 particles/cell. Graph results are shown in mean arbitrary units ± SEM, with the highest value in each graph set to 100.

E1A mRNA expression in human cancer cell lines showing differential sensitivities to Ad11 and Ad5

Next, we investigated the early gene expression of Ad11 and Ad5 in cancer cells. We attempted to analyze the expression of Ad11 E1A proteins by western blotting but there is no specific antibody available and the proteins could not to be detected by antibodies raised against Ad2 or Ad5 E1A. Instead, we analyzed E1A mRNA levels by quantitative reverse transcription -PCR using serotype-specific primers and probes (Figure 4b). The higher infectivity of Ad11 in all these cell lines resulted in higher levels of Ad11 E1A mRNA at 2 hours postinfection. After this, its levels appear to decrease relative to those of Ad5 E1A in cells less sensitive to Ad11 (MIA PaCa-2 and LNCaP). Not unexpectedly, the levels of Ad11 E1A mRNA were greater in the Ad11-sensitive Capan-2 and PC-3 cells.

DNA replication and hexon synthesis are related to attenuated E1A transcription

Downregulation of Ad11 E1A transcription in MIA PaCa-2 and LNCaP caused reductions in viral DNA replication and subsequent hexon protein synthesis (Figure 4c,d), and this correlates with the lower levels of progeny virus production and cytotoxicity observed earlier (Figure 1a,c). DNA replication and hexon expression from the Ad11 virus were greater in the Ad11-sensitive Capan-2 compared to Ad5 (Figure 4c,d). In PC-3, Ad5 hexon appears to be expressed at a higher level than that of Ad11 at 48 hours, although they became similar at 72 hours. This could be due to a delay in Ad11 protein synthesis despite the high amount of viral DNA.

Construction of recombinant Ad11 with Ad5 E1A enhancer and/or promoter

Having found that the levels of E1A mRNA appear to determine the replicative ability and potency of Ad11 in cancer cell lines, we hypothesized that increasing the transcription of Ad11 E1A by replacing its enhancer and/or promoter with that of Ad5 could result in better oncolytic potency in Ad11-less sensitive cells. To this end, two recombinant Ad11 mutants were constructed (Figure 5a). Ad11-Ad5-P was created by replacing the Ad11 E1A promoter with the corresponding sequence from Ad5. Ad11-Ad5-EP was made by replacing the corresponding region in Ad11 with the region from 195 bp up to the E1A-coding sequence of Ad5 (including the enhancer and promoter of Ad5 E1A). These two viruses could be produced at a high infectious titer. We did not construct a mutant Ad11 with just the Ad5 E1A enhancer because the transcriptional activity of Ad5 E1A promoter was found to be higher than that of Ad11 by luciferase reporter assay, even in the Ad11-sensitive Capan-2 and PC-3 (data not shown).

Figure 5.

Figure 5

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Recombinant Ad11 with Ad5 E1A enhancer and/or promoter and their oncolytic potencies. (a) Ad11-Ad5-P has the region between Ad11's packaging signal (PS) and the start of E1A-coding sequence replaced by the corresponding region of Ad5. The Ad5 E1A enhancer region up to the start of the E1A-coding sequence was used to replace the corresponding region in Ad11 to produce Ad11-Ad5-EP. (b, c) Oncolytic potencies of Ad5, Ad11, Ad11-Ad5-P and Ad11-Ad5-EP in human cancer cell lines sensitive and less sensitive to Ad11, respectively. Graphs show the mean EC50 values ± SEM.

Cytotoxicities of Ad11-Ad5-P and Ad11-Ad5-EP in human cancer cell lines

The in vitro potencies of the recombinant viruses were compared to those of Ad5 and Ad11 in cancer cell lines showing different sensitivities to Ad5 and Ad11. In five Ad11-sensitive cell lines, both Ad11 mutants still showed better cell killing than Ad5, although surprisingly Ad11-Ad5-EP demonstrated an even stronger cytotoxicity than Ad11 (Figure 5b). In five Ad11-less sensitive cell lines, the potency of Ad11-Ad5-EP was dramatically improved compared to that of Ad11 (Figure 5c). It was comparable to Ad5 in three cancer cell lines (MIA PaCa-2, MDA-MB-231, and HCT 116), with only one cell line (LNCaP) still more sensitive to Ad5 (P < 0.05). Strikingly, the lung cancer cell line A549 was most sensitive to Ad11-Ad5-EP, with its EC50 nearly 30 times less than that of Ad5 (P < 0.01). The potency of Ad11-Ad5-P was largely similar to that of Ad11, except that it was significantly less cytotoxic to Capan-2 and MCF7. To confirm further the potential of Ad11-Ad5-EP as a potential backbone for the development of new oncolytic adenoviruses, we compared the cytotoxicities of Ad5, Ad11, and Ad11-Ad5-EP in other nine cancer cell lines that were sensitive to Ad5 (less sensitive to Ad11) by MTS assay. As shown in Supplementary Figure S2, the cytotoxicity of Ad11-Ad5-EP was significantly increased compared to Ad11 in all the nine human cancer cell lines tested (P < 0.05), four of them (PaTu 8988t, DU 145, DLD-1, and Calu-1) showed an equivalent cytotoxicity to Ad5 (P > 0.05), with five of them (SUIT-2, PANC-1, SW620, OVCAR-3, and IGROV1) still more sensitive to Ad5 (P < 0.05). Overall, Ad11-Ad5-EP is significantly more potent than Ad11 in all the 19 cancer cell lines tested, and effective in killing 68% (13/19) of the cancer cell lines tested in Figure 5b,c as well as Supplementary Figure S2, showing equivalent or even better efficacy than Ad5 and Ad11.

Ad11-Ad5-EP has increased E1A transcription and virus replication compared to Ad11

As expected, the improved potency of Ad11-Ad5-EP correlated with E1A gene expression. In MIA PaCa-2 (less sensitive to Ad11 than to Ad5), E1A mRNA levels from Ad11-Ad5-EP were much higher than those of Ad11 and Ad11-Ad5-P, although they were lower than those of Ad5 (Figure 6a). This could be the result of the higher transcription-enhancing activity of Ad5 E1A enhancer. The Ad5 E1A promoter alone did not enhance E1A transcription, as shown by the lower levels of E1A mRNA expressed from Ad11-Ad5-P. Unexpectedly, Ad11-Ad5-EP DNA amplified at the highest level, even greater than that of Ad5 to which MIA PaCa-2 cells were most sensitive. Despite this, hexon protein synthesis and the number of infectious Ad11-Ad5-EP produced were less than Ad5, leading to the observed cytotoxicity result (Figure 5c).

Figure 6.

Figure 6

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E1A mRNA levels, viral DNA replication, hexon expression, and production of infectious particles. (a) MIA PaCa-2 and (b) PC-3 after adenovirus serotype 5 (Ad5), Ad11, Ad11-Ad5-P, or Ad11-Ad5-EP infection. Experiments and analyses were performed as described in Figures 1 and 4. Graph results represent means ± SEM.

In PC-3 (more sensitive to Ad11), E1A mRNA levels were highest for Ad11-Ad5-EP, followed by Ad11 (Figure 6b). Although the high E1A mRNA levels from Ad11-Ad5-EP and Ad11 corresponded to the levels of subsequent DNA replication, this did not apply to Ad11-Ad5-P. Its E1A mRNA levels were similar to or lower than that of Ad5, yet significantly more Ad11-Ad5-P DNA and infectious particles were produced.

Ad11-Ad5-EP is as effective as Ad5 in treating MIA PaCa-2 human pancreatic cancer xenografts

Given the potential for broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP in vitro, the in vivo efficacies of Ad11-Ad5-P and Ad11-Ad5-EP were compared to those of Ad5 and Ad11 in a MIA PaCa-2 subcutaneous xenograft model (chosen because MIA PaCa-2 was less sensitive to wild-type Ad11 than to Ad5). As shown in Figure 7a,b, Ad11-Ad5-EP was as effective as Ad5 in reducing tumor growth (day 35, P = 0.0449 compared to Ad11), culminating in similar progression-free percentages that were significantly better than the Ad11-treated group (log rank P = 0.0462). The efficacy of Ad11-Ad5-P was similar to Ad11. In vivo DNA replication and virus replication were reflective of the in vitro findings, with Ad11-Ad5-EP showing the highest amount of viral DNA but was second to Ad5 in terms of virus production (Figure 7c,d).

Figure 7.

Figure 7

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Antitumoral efficacies of adenovirus serotype 5 (Ad5), Ad11, Ad11-Ad5-P, and Ad11-Ad5-EP in a MIA PaCa-2 subcutaneous xenograft model. (a) Mean tumor volumes ± SEM in BALB/c nude mice (n = 8/group) after three intratumoral injections of phosphate-buffered saline (PBS) or viruses (1 × 1010 particles/injection), measured until the first mouse in each group has a tumor dimension of >1.44 cm2. (b) Percentage of progression-free mice using the Kaplan–Meier method. (c) Viral DNA replication in tumor tissues after one injection of 1 × 1010 particles. Results are shown in mean arbitrary units normalized against total DNA ± SEM. (d) Production of infectious virus particles, shown in mean plaque-forming units (PFU)/ng of total DNA ± SEM.

Ad11-Ad5-EP is more potent than Ad5 in treating PC-3 and A549 human xenografts

To demonstrate the superiority of Ad11-Ad5-EP in vivo, Ad11-Ad5-EP was compared to Ad5 in vivo using the Ad11-sensitive human cancer cell line PC-3 and Ad5-senstitive cell line A549 (Figure 8). In both of these subcutaneous xenograft models, Ad11-Ad5-EP demonstrated superior efficacy compared to Ad5, resulting in significant reductions in tumor growth (PC-3 on day 24, P = 0.0009; A549 on day 37, P = 0.0406) and progression-free rates (PC-3, log rank P = 0.0193; A549, log rank P = 0.0221). These results suggest that Ad11-Ad5-EP is a promising backbone for the future development of replication-selective oncolytic adenoviruses targeting a wide spectrum of human cancers.

Figure 8.

Figure 8

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Comparison of antitumoral efficacies between adenovirus serotype 5 (Ad5) and Ad11-Ad5-EP in PC-3 and A549 subcutaneous xenograft models. (a) Mean tumor volumes ± SEM in BALB/c nude mice (n = 8/group) after three intratumoral injections of phosphate-buffered saline (PBS) or viruses (1 × 1010 particles/injection), measured until the first mouse in each group has a tumor dimension of >1.44 cm2. (b) Percentage of progression-free mice using the Kaplan–Meier method.

Discussion

Inconsistent clinical trial results and the shortcomings of Ad5 prompted us to evaluate and develop Ad11 as an alternative oncolytic virus, which has several benefits over Ad5, including the overexpression of its receptors in cancer cells, lower prevalence of neutralizing antibodies and reduced hepatotoxicity. Sandberg et al. have recently shown that Ad11 could effectively transduce, replicate in and lyse the prostate cancer cell line PC-3 in vitro and in vivo, although no comparison was made with the commonly used Ad5.24 Shashkova et al. compared the oncolytic efficiencies of Ad5, -6, -11, and -35 in a panel of human cancer cell lines in vitro as well as the human prostate cancer cell line DU 145 in vivo.19 In the latter, Ad5, -6, and -11 have similar anticancer activities whereas Ad35 was not efficacious. Importantly, hepatotoxicity only developed with Ad5 but not the other serotypes.

In this study, we showed that the oncolytic potency of Ad11 was not related to the receptor CD46, nor simply to the infectivity of the virus. In fact, Ad11 was significantly more infective than Ad5 even in cells that were less sensitive to Ad11 cytotoxicity. In these cells, however, Ad11 E1A mRNA levels were much lower than that of Ad5, producing a negative effect on viral DNA replication, structural protein synthesis, progeny production and cell killing. Previous work has shown that low levels of E1A are sufficient to initiate Ad5 replication in HeLa cells, and it was suggested that Ad5 normally produces E1A in excess of that required.25 In an attempt to clarify this, Zheng et al. found that high E1A levels produced by an E1B 55K-mutated Ad5 with its E1A driven by the strong cytomegalovirus promoter resulted in increased virus replication compared to wild-type Ad5 in some cancer cells, although this did not always enhance cytotoxicity.26 Furthermore, this work was only done on viruses lacking the E1B 55K gene, which has a major role in virus replication. Our results demonstrated that it is possible that in cancer cells less sensitive to Ad11, Ad11 E1A expression does not reach its critical level and that increasing this, combined with the higher infectivity of Ad11, could result in better virus replication and killing of cells less sensitive to Ad11.

To test this hypothesis, we constructed two Ad11 mutants. Ad11-Ad5-P is a recombinant Ad11 with its E1A promoter region replaced by that of Ad5, conserving its own packaging signal and enhancer region. Ad11-Ad5-EP on the other hand, has the whole Ad5 E1A enhancer (including the packaging signal) and promoter substituting the corresponding region of Ad11. Ad5's packaging sequence appears to work as well as its Ad11 counterpart when placed in the Ad11 backbone. The high level of homology of L1 52/55K between Ad5 and Ad11 may be the reason behind this,27 as the serotype specificity of adenoviral DNA packaging is mediated by this protein.28 Infectivities of the mutant viruses were also unaltered by these genetic alterations (data not shown). The universally strong cytomegalovirus promoter was not used to drive Ad11 E1A expression for a number of reasons. Firstly, Ad11-Ad5-P and Ad11-Ad5-EP were constructed to test the hypothesis that these Ad5 E1A regulatory regions were more active in the Ad5-sensitive cancer cell lines, given the higher levels of E1A mRNA observed after Ad5 infection. Secondly the cytomegalovirus promoter, unlike the E1A enhancer region, would not be able to modulate the expression of other early genes needed for effective virus replication. Thirdly, combining the genetic materials of two very different viruses would have safety concerns when used in future clinical studies.

The E1A enhancer region originally described by Hearing and Shenk contains two elements.29,30 The repeated element I specifically enhances E1A transcription whilst element II modulates in cis all early viral transcriptional units. The oncolytic potency of Ad11-Ad5-EP was significantly better than that of Ad11 in all the cancer cell lines tested, whereas Ad11-Ad5-P was mostly comparable to Ad11. In MIA PaCa-2 cells (less sensitive to Ad11 than to Ad5), E1A mRNA levels are Ad5 > Ad11-Ad5-EP > Ad11 > Ad11-Ad5-P (identical ranking to the oncolytic potencies). The reason behind the higher levels of E1A mRNA with Ad5 compared to that of Ad11-Ad5-EP, despite the better infectivity of the latter, is unknown. Perhaps this is due to the stronger transactivating activity of Ad5 E1A proteins on their own enhancer as well as regions further upstream. However, despite this, Ad11-Ad5-EP DNA amplified much more efficiently. It is possible that the small increase in Ad11 E1A proteins (which are needed for the expression of other early genes), together with the strong enhancing activity of Ad5's enhancer element II, led to a significant elevation of E1B, E2, and E4 proteins, all of which are important for viral DNA replication. The production of infectious Ad11-Ad5-EP, and therefore its oncolytic potency, was however limited by events post-DNA replication. Western blots showed that the amount of hexon protein was significantly reduced. The exact mechanisms, whether secondary to viral mRNA export, translation, or protein degradation, are unknown at present. Nonetheless this virus was as effective as Ad5, but significantly better than Ad11, in treating MIA PaCa-2 tumors in vivo.

The higher E1A mRNA levels and enhanced oncolytic potency of Ad11-Ad5-EP compared to Ad11 in the Ad11-sensitive cell lines, such as PC-3, were unexpected. The Ad11 E1A enhancer should theoretically be stronger given the mRNA levels observed after Ad11 infection. It is possible that the Ad5 E1A enhancer was indeed more active in PC-3, but Ad5 E1A mRNA was degraded more rapidly than Ad11's. Possibly by a combination of good infectivity, more stable Ad11 E1A mRNA, the strong Ad5 E1A enhancer coupled with the higher transactivating activity of Ad11 E1A proteins, Ad11-Ad5-EP managed to achieve the highest DNA and virus replication. The reason behind the higher replication of Ad11-Ad5-P compared to Ad11, despite its lower E1A mRNA levels, is unknown. It is possible that in PC-3, a moderate amount of Ad11 E1A was sufficient for maximum virus replication, and that the Ad5 E1A promoter has transcription-enhancing activity of other early viral genes. However, mRNA levels do not always reflect the amount of proteins produced, and the translation of E1A mRNA can be downregulated by the E3 10.4K and 14.5K proteins.31 As such it is essential to develop an Ad11 E1A-specific antibody to analyze the protein levels.

The potential of Ad11 as an effective oncolytic virus cannot be ignored, especially using our new mutant Ad11-Ad5-EP. The much better infectivity of Ad11 compared to Ad5 means that inserted therapeutic genes would have much higher levels of expression for a given input dose of virus. Deletion of Ad11 genes such as E3 18.5K (equivalent to E3 gp19K of Ad5), 20.3K and 20.6K (possibly dispensable) could provide additional space for transgene insertion.27,32,33 Given that Ad11-Ad5-EP replicates in normal epithelial cells at a similar level to wild-type adenovirus (data not shown), it is necessary to further modulate Ad11-Ad5-EP for development of new oncolytic adenoviruses for cancer treatment. Approaches to improve its tumor selectivity include: specific gene deletion—various genes are crucial for the survival of viruses in normal cells but expendable in cancer, such as the pRb-binding region of E1A, E1B 55K or 21K (equivalent to Ad5 E1B 19K);27 insertion of a tumor-specific promoter driving the expression of the critical adenoviral E1A gene; more recently, gene silencing by RNA interference technology has been utilized to confer tumor selectivity.34 Its advantages over Ad5 in terms of receptor availability, lower prevalence of neutralizing antibodies and minimal hepatotoxicity mean that after achieving tumor selectivity, Ad11 would be a much safer and effective alternative to Ad5 when given intravenously to treat local and metastatic diseases.

In summary, Ad11 is likely to have a crucial role to play in the advancement of oncolytic virotherapy as it offers solutions to many of the problems with Ad5. Our results have demonstrated that Ad11-mediated cytotoxicity is regulated by its early gene transcription and not by its infectivity or cellular CD46 expression, which are significantly higher than Ad5 and CAR in cancer cells. This has important implications for the optimization of Ad11 for cancer therapy. The recombinant Ad11-Ad5-EP has not only shown superior antitumoral activity compared to Ad11 against Ad5-less sensitive cancer cell lines, but also demonstrated similar or better efficacy than Ad5 against Ad11-less sensitive cancer in vivo. Taken together, we believe that Ad11 with Ad5 E1A enhancer-promoter should be used as a backbone for the future development of potent and tumor-specific oncolytic Ad11 mutants.

Materials and Methods

Cell lines and adenoviruses. Cells were grown in media (RPMI-1640 for 22Rv1, LNCaP, DLD-1 and A2780; MEM EBSS for Calu-1; Dulbecco's modified Eagle's medium for the others) supplemented with 10% fetal calf serum, at 37 °C and 5% CO2. Human cancer cell lines used: Capan-1, Capan-2, Hs766T, MIA PaCa-2, PANC-1, PaTu 8988s, PaTu 8988t, SUIT-2 (exocrine pancreas); 22Rv1, DU 145, LNCaP, PC-3 (prostate); MCF7, MDA-MB-231, MDA-MB-468, SK-BR-3 (breast); DLD-1, HCT 116, HT-29, SW620 (colon); A2780, IGROV1, OVCAR-3 (ovary); A549, Calu-1 (nonsmall cell lung). The human embryonic kidney HEK-293 and its subclone JH-293 were also used. All cells were obtained from the Cancer Research UK Cell Services, Hertfordshire, UK, except for Capan-2, PC-3 (ATCC, Manassas, VA) and LNCaP (DSMZ, Braunschweig, Germany). Ad5 is routinely produced in our laboratory.35 Ad11p was a kind gift from Daniel Stone and André Lieber (University of Washington, Seattle, WA). Viruses were produced in HEK-293 cells and titered as described previously.35

Cell-killing assay. Cells were seeded in 96-well plates and viruses were added 18 hours later at nine 1:10 serial dilutions. Six days later, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays (Promega, Madison, WI) were performed. EC50 (dose required for 50% destruction of the target cells) was determined using GraphPad Prism (GraphPad Software, San Diego, CA). Assays were performed twice in duplicates with each virus concentration in sextuplicates.

Virus replication assay. Cells were seeded in 6-well plates and infected with viruses (100 particles/cell) in serum-free medium. After 2 hours, these were replaced by medium with 5% fetal calf serum. Cells and media were collected, then frozen, and thawed three times. Virus replication was detected by the TCID50 assay as previously described.35 Experiments were performed in triplicates.

In vivo studies. Aliquots of 1 × 107 cells in 100 µl of phosphate-buffered saline were injected subcutaneously into the flanks of BALB/c nude mice (Harlan Laboratories, Indianapolis, IN) and tumors were allowed to grow to 6–8 mm in diameter. For efficacy experiments, phosphate-buffered saline or adenoviruses (1 × 1010 particles) were injected intratumorally on days 0, 2, and 4. Animals were euthanized when tumor dimension was >1.44 cm2. For biological endpoint studies, single doses of viruses were injected and tumors were harvested in triplicates. Each tumor was homogenized in 2 ml of Dulbecco's modified Eagle's medium.

Quantitative PCR. E1A primers used: Ad5-F (5′-TGCCAAACCTTG TACCGGA-3′), Ad5-R (5′-CGTCGTCACTGGGTGGAAA-3′), Ad11-F (5′-GAAGGCTGCCAATGTTGGTT-3′), and Ad11-R (5′-ACAGCCAT GTCCAGGAAGCT-3′). Fluorescein amidite probes (Applied Biosystems, Foster City, CA) used: 5′-TTACCTGCCACGAGGCTGGC-3′ and 5′-TCAGTTGGATTGCCC-3′ for Ad5 and Ad11 E1A, respectively. DNA was obtained using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). PCR conditions: [95 °C (15 seconds), 60 °C (1 minute)] for 40 cycles. Ct values were calculated using Sequence Detection System Software (Applied Biosystems). Experiments were done in triplicates. For virus infectivity, 2 × 105 cells were incubated with buffer (1% bovine serum albumin in phosphate-buffered saline) on ice for an hour before virus infection (1,000 particles/cell). Cells were incubated at 4 °C for an hour for virus attachment, then washed to remove unbound viruses. For nuclear entry, cells were resuspended in buffer and incubated at 37 °C. Nuclear DNA was obtained for quantitative-PCR. For viral DNA replication and E1A mRNA, cells were infected as for the virus replication assay. RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). cDNA was obtained using TaqMan Reverse Transcription Reagents (Applied Biosystems). Quantitative-PCR was done together with Eukaryotic 18S rRNA Endogenous Control (Applied Biosystems).

Immunofluorescence. Conjugation of Ad5 and Ad11 with the fluorophore Alexa Fluor 555 carboxylic acid, succinimidyl ester (Invitrogen) was performed as described previously.36 2 × 104 cells per well were seeded in 4-well Lab-Tek II Chamber Slide Systems (Thermo Fisher Scientific, Waltham, MA). After 48 hours they were incubated at 4 °C for an hour before virus infection, diluted to 1:10 in 300 µl/well of serum-free medium. Viruses were allowed to attach at 4 °C for an hour, then cells were washed with phosphate-buffered saline. Virus internalization and trafficking were allowed to continue at 37 °C for an hour. Cells were stained using anti-α-tubulin (Sigma-Aldrich, St Louis, MO) and Alexa Fluor 488 (Invitrogen) antibodies. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (Vector Laboratories, Burlingame, CA) and slides were mounted with VECTASHIELD (Vector Laboratories).

Western blot. Cells were infected as for the virus replication assay except with 200 particles/cell. Primary antibodies used: adenovirus antibody (for Ad5 hexon; Abcam, Cambridge, UK), Ad11 hexon antibody (GenScript, Piscataway, NJ) and proliferating cell nuclear antigen antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Recombinant Ad11 construction. pBGwtAd11 was provided by Daniel Stone and André Lieber (University of Washington). pSS was developed by Daniel öberg in our laboratory. Primers used to clone regions of Ad5 and Ad11 for recombinant Ad11 construction are: pA1 (5′-ATCaagcttagatctGGAGA CGGTCACAGCTTGTCTG-3′), pA2 (5′-ATCtctagagcggccgcTAATT AAGAATTCGAATTAATTAATTC-3′), pA3 (5′-ATCaagcttgcggccgcATC ATCAATAATATACCTTATAG-3′), pA4 (5′-ATCggatccgatatcAGCCTTT TTATGCGTCAC-3′), pA5 (5′-AAAAATGAGAGATTTGCGATTTCTGC-3′), pA6 (5′-ATCggatccctcgagCAAAGCGAACATAACAGTTC-3′), pA7 (5′-ATCaagctttacgtaACACAGGAAGTGACAATTTTCGCGC-3′), pB1 (5′-ATCggatccgatatcAAACCTCCACGTAATGGGTCAAAGTC-3′), pB2 (5′-ATCaagctttacgtaTGGAGACTCGCCCAGGTGTTTTTCTC-3′) and pLinker (5′-GCAGAAATCGCAAATCTCTCATTTTCAGTCCCGGTGTCGGAGC-3′). Fragments A1A2, A3B1, A3A4, A5A6 were cloned from pBGwtAd11; B2Linker and A7Linker from Ad5 DNA. Fragment B2A6 (or A7A6) was obtained by PCR using B2Linker (or A7Linker) and A5A6, followed by amplification using pB2 (or pA7) and pA6. Fragment insertion into pUC18 generates pUCA1A2, pUCA3B1, pUCA3A4, pUCB2A6 and pUCA7A6 (pUCA1A2 at HindIII/XbaI, others at HindIII/BamHI). These plasmids were verified by sequencing. A3B1 or A3A4 was ligated to NotI- and BamHI-digested pUCA1A2 to produce pUCA1B1 and pUCA1A4. To construct the shuttle vector for Ad11-Ad5-P (or Ad11-Ad5-EP), A1B1 (or A1A4) was ligated to BglII- and EcoRV-digested pSS, producing pSSA1B1 (or pSSA1A4). B2A6 (or A7A6) was ligated to SnaBI- and XhoI-digested pSSA1B1 (or pSSA1A4) to produce pSSA1B1B2A6 (or pSSA1A4A7A6). These were linearized with PmeI before homologous recombination with pBGwtAd11 in BJ5183 (Stratagene, San Diego, CA). The chloramphenicol resistance gene was removed by SwaI digestion and religation. Plasmids were linearized by NotI before transfection of HEK-293 cells. Virus production was described above.

Statistical analysis. Pair-wise comparison was done using unpaired, two-tailed t-test. Progression-free percentages were analyzed by the Kaplan–Meier method and log rank test.

SUPPLEMENTARY MATERIAL Figure S1. Surface receptor expression in human cancer cell lines. Figure S2. Cytotoxicity of recombinant adenovirus serotype 11 (Ad11) with Ad5 E1A enhancer-promoter (Ad11-Ad5-EP) in nine human cancer cell lines that are sensitive to Ad5. Materials and Methods.

Acknowledgments

We thank Daniel Stone and André Lieber (University of Washington, Seattle, WA) for providing the Ad11p virus and pBGwtAd11. We also thank Daniel öberg and Gunnel Halldèn in our laboratory for their pSS plasmid. This project was supported by Cancer Research UK (C633-A6253/A6251 program grant) and the Medical Research Council (Clinical Research Training Fellowship to H.H.W).

Supplementary Material

Figure S1.

Surface receptor expression in human cancer cell lines.

Click here for additional data file. (786.2KB, tiff)
Figure S2.

Cytotoxicity of recombinant adenovirus serotype 11 (Ad11) with Ad5 E1A enhancer-promoter (Ad11-Ad5-EP) in nine human cancer cell lines that are sensitive to Ad5.

Click here for additional data file. (1.3MB, tiff)
Materials and Methods.
Click here for additional data file. (29.5KB, doc)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Surface receptor expression in human cancer cell lines.

Click here for additional data file. (786.2KB, tiff)
Figure S2.

Cytotoxicity of recombinant adenovirus serotype 11 (Ad11) with Ad5 E1A enhancer-promoter (Ad11-Ad5-EP) in nine human cancer cell lines that are sensitive to Ad5.

Click here for additional data file. (1.3MB, tiff)
Materials and Methods.
Click here for additional data file. (29.5KB, doc)

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