Targeting Interferon-α Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus

original article

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Targeting Interferon-a Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus Elena V Shashkova1,3, Jacqueline F Spencer2, William SM Wold2 and Konstantin Doronin2,3 1 VirRx Inc., St. Louis, Missouri, USA; 2Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, USA; 3Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota, USA

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-a) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-a pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-a in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-a in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-a-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-a-expressing replication-competent Ads. Received 30 August 2006; accepted 30 October 2006; published online 26 December 2006. doi:10.1038/sj.mt.6300064

INTRODUCTION Oncolytic adenoviruses (Ad) replicate in and kill cancer cells as a result of virus replication. A conditionally replicative Ad carrying a deletion of the e1b55k gene (Onyx-015) was evaluated in clinical trials and has not shown significant antitumor activity when used as a monotherapy.1 New generations of oncolytic Ad

vectors are being developed that provide higher levels of efficacy and specificity, with the goal of achieving a therapeutic response as a monotherapy and/or an improved response in combination regimens.2–8 In this study, we explored the hypothesis that genetic defects in the interferon-alpha (IFN-a) pathway can be exploited to improve the efficacy and safety of oncolytic Ad. An additional hypothesis was that IFN-a expression can be combined with the oncolytic activity of an Ad vector overexpressing the adenovirus death protein (ADP; E3-11.6K) without compromising the ADPoverexpression phenotype. We have previously described the conditionally replicative Ad vector KD3, which has enhanced spread within tumors due to the overexpression of the ADP.9 Cancer-specific replication of KD3 was achieved by the dl1101/ 1107 mutation in the e1a gene of Ad serotype 5 (Ad5), resulting in the inability of mutated E1A proteins to bind the cellular p300/CREB-binding protein (CBP) and pRb proteins and restricting the replication of the vector to dividing cells.9,10 To test our hypotheses, we inserted the gene for human IFN-a2b into the E3 region of KD3 downstream of the adp gene to retain ADP overexpression and to achieve cancer-specific expression of IFN-a. Recombinant IFN-a protein is used in oncology to treat carcinoid tumors, chronic myelogenous leukemia, metastatic bladder cancer, renal cell carcinoma, hairy cell leukemia, malignant melanoma, and Kaposi’s sarcoma.11 Several studies explored the anticancer activities of replication-defective Ad vectors expressing human or murine IFN-a, while one study described a nonselective replication-competent Ad expressing human IFN-acon1.12–17 Ads counteract IFN-a antiviral activity by two different mechanisms, including disruption of IFN-ainduced gene expression by the Ad E1A proteins, and prevention of double-stranded RNA-dependent protein kinase (PKR) activation by a small Ad RNA named VA-RNAI, and therefore Ads are not sensitive to IFN-a.18,19 As one of the mutations in the E1A proteins (dl1101) is known to prevent binding of E1A to p300/CBP, rendering the mutated E1A unable to block IFN-astimulated gene expression,20 we hypothesized that oncolytic Ad containing this mutation would be sensitive to IFN-a in normal

Correspondence: Konstantin Doronin, Mayo Clinic, Guggenheim 5-16, 200 First Street SW, Rochester, Minnesota 55902, USA. E-mail: [email protected]

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cells. In contrast, this Ad should retain oncolytic activity in a cancer cells, because many cancer cells are known to have a deregulated IFN-a pathway.21,22 We expected that the new vector, KD3-IFN, would have greater antitumor activity and that it also would be sensitive to IFN-a in normal cells, thereby improving the therapeutic window.

RESULTS Vector design The dl1101/1107 mutation restricts Ad replication to actively proliferating cells,9,10 and it was found to be more versatile than other e1a mutations by providing both cancer selectivity and the ability to drive efficiently the replication of Ad in cancer cells23 (Figure 1a). Wild type (wt) Ad5 virus release is controlled by ADP, which is expressed at late stages of infection.24,25 Deletions of immunomodulatory genes from the E3 region of the KD3 vector result in overexpression of ADP leading to earlier release of the vector from infected cells and improved vector spread9,26,27 (Figure 1b). Transgenes replacing the E3B region of Ad were shown to be expressed at strictly late stages of Ad infection.28 We explored whether both overexpression of ADP and strictly late expression of transgenes from the KD3 vector backbone could be achieved from the major late promoter (MLP) via the endogenous Ad splicing signals (Figure 1b). KD3-IFN was constructed by inserting the human IFN-a2b gene into the E3 region downstream of the adp gene.9 The E3 intron (12.5K gene) is not essential for antitumor activity,9 and was deleted to increase the cloning capacity. Vectors KD3-DsRed and KD3-SEAP carrying reporter genes were also constructed to visualize (DsRed) or quantify (SEAP) expression levels in cell culture and in animal models.

IFN-a Effect on Tumor-specific Oncolytic Ad

Expression of functional transgenes at the late stage of Ad infection in human cancer cells We compared the level of ADP expression by KD3-IFN and KD3 by Western blot analysis. The expression level, kinetics of expression, and molecular weight of ADP produce by KD3-IF were indistinguishable from those produce by KD3 (Figure 2a, left). The biological activity of IFN-a produced by KD3-IFN in A549 cells and estimated in a vesicular stomatitis virus (VSV) replication inhibition assay (Figure 2a, right) was 25,600 IU/ 1
106 cells at 48 h postinfection. KD3-IFN produced 109 ng of IFN-a/1
106 A549 cells at 48 h postinfection as measured by enzyme-linked immunosorbent assay (ELISA). Owing to overexpression of ADP, the KD3 vector has higher anticancer activity in human cancer cells in culture as compared to dl1101/1107, which expresses wt levels of ADP.9,26 We compared the anticancer activity of dl1101/1107, KD3, and KD3-IFN in A549 and Hep3B cells. Both KD3 and KD3-IFN were statistically significantly different from dl1101/1107 (Po0.001) in that they killed the cells at a lower multiplicity of infection (MOI) than did dl1101/1107 (Figure 2b). KD3 and KD3-IFN were not different from each other (P ¼ 1.00 for A549; P ¼ 0.793 for Hep3B) indicating that the functional activity of IFN-a does not compromise the manifestation of the ADP overexpression phenotype. Strictly late expression of transgenes from a conditionally replicative Ad vector is advantageous because it allows for linking transgene expression to cancer-specific replication, thereby increasing the concentration of the therapeutic protein in the target tumor tissue. We studied the expression of reporter genes from KD3-DsRed and KD3-SEAP in human cancer cells in the presence or absence of the DNA replication inhibitor cytosine arabinoside (Ara-C) (Figure 2c and d). KD3-DsRed in

Figure 1 Schematic representation of Ad vector genomes. C, encapsidation signal. Arrows, Ad promoters. MLP, major late promoter. pA, polyadenylation signals. Boxes, Ad open reading frames. m.u., map units (percent of wt Ad5 genome). (a) Mutations in the E1 region. Early region E1 of wt Ad5 contains two transcription units, E1A and E1B. dl1101/1107 and vectors derived from this deletion mutant carry mutations in the e1a gene that prevent binding of E1A proteins to p300/CBP and pRb. (b) Mutations in the E3 region. Early region E3 of wt Ad5 contains two subregions, E3A and E3B, derived from the single E3 promoter and defined by the E3A and E3B polyadenylation signals. The adp gene is expressed early from the E3 promoter and abundantly at the late stages of infection from the MLP. KD3 carries deletions in E3A and E3B regions resulting in the absence of immune-modulating genes and overexpression of the ADP (E3-11.6K). Transgenes were inserted into E3 region downstream of the adp gene. The E3 intron (12.5K gene) was deleted to increase the cloning capacity of the vector.

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Figure 2 Vectors express functional transgenes in human cancer cells at the late stage of infection. (a, left) ADP overexpression is retained in the KD3-IFN vector. A549 cells were infected with the indicated viruses at an MOI of 20 PFU/cell, and the levels of ADP expression at 24 and 48 h postinfection were detected by Western blotting. (a, right) KD3-IFN produces biologically active IFN-a. Supernatants from KD3- or KD3-IFN–infected A549 cells, collected at 48 and 72 h postinfection, were analyzed in a VSV replication inhibition assay on HeLa cells. Test supernatants or a human IFNa standard were 2-fold serially diluted and added to the cells. The initial and final concentrations of the IFN-a standard in the wells were 125 and 6.1
102 IU/well. The range of test supernatant dilutions was from 50 to 2.4
104 ml/well. After 24 h of incubation, the cells were infected or mockinfected with VSV at an MOI of 1 PFU/cell. The cells were stained with crystal violet at 24 h postinfection. (b) Functional activity of ADP overexpression is retained in KD3-IFN. A549 or Hep3B cells were infected in triplicate with dl1101/1107, KD3, or KD3-IFN at the indicated MOI. The viability of the cells was quantified at day 7 postinfection. (c) KD3-DsRed expresses DsRed at the late stage of infection. A549 cells were infected with KD3-DsRed at an MOI of 20 PFU/cell. Ara-C was added where indicated to maintain the early stage of infection. The morphology of the DsRed-expressing cells is indicated by arrows. Original magnification:
100. (d) KD3-SEAP produces high levels of SEAP activity strictly at the late stage of infection. A549 or Hep3B cells were infected in triplicate with KD3-SEAP at an MOI of 100 PFU/cell. Ara-C was used to maintain the early stage of infection.

the absence of Ara-C produced cytopathic effect (CPE) in A549 cells beginning at 48 h postinfection. DsRed expression was detectable at 24 h postinfection. In the presence of Ara-C, both CPE and expression of DsRed were not pronounced during the course of the experiment (Figure 2c). Expression of SEAP by KD3-SEAP in A549 and Hep3B cells was blocked to undetectable levels by the addition of Ara-C (Figure 2d). In the absence of Ara-C, SEAP was produced to high levels in both cell lines after 24 h postinfection. The cumulative level of SEAP activity at 74 h postininfection in A549 cells was 3.47 times higher than in Hep3B cells (Figure 2d). The addition of Ara-C blocks Ad replication; therefore, the sensitivity of transgene expression to Ara-C indicates its linkage to viral genome replication. 600

Productive replication, spread, and expression of transgenes at the late stage of infection in Syrian hamster cancer cells A new immunocompetent animal model permissive for oncolytic Ad was recently reported.29 It was shown that Syrian hamster cells support the replication of Ad in vitro and in vivo. To assess the ability of conditionally replicative Ad to replicate productively in hamster cancer cells, we compared progeny virus yields in HaK cells infected with Ad5, dl1101/1107, KD3, or KD3-IFN (Figure 3a). Ad5 and KD3 produced high yields typical for permissive cells (P ¼ 0.412 for Ad5 versus KD3). dl1101/1107 and KD3-IFN (P ¼ 0.995 for dl1101/1107 versus www.moleculartherapy.org vol. 15 no. 3, march. 2007

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KD3-IFN) produced about one order of magnitude reduced yields as compared with Ad5 and KD3 (Po0.001). dl1101/1107 is known to produce lower yields in human cancer cells as compared to wt Ad5, which might be explained by the attenuating effect of the dl1101/1107 mutation on Ad replication.9 Lower yields of KD3-IFN in HaK cells could be plausibly explained by induction of apoptosis by human IFN-a in these cells (data not shown). When the anticancer activities of dl1101/ 1107, KD3, and KD3-IFN were studied in HaK cells at varying MOI (Figure 3b), it was found that all vectors were statistically significantly different from one another (P ¼ 0.012 for KD3-IFN versus dl1101/1107; P ¼ 0.035 for KD3-IFN versus KD3; Po0.001 for KD3 versus dl1101/1107). KD3-IFN in this assay had an activity intermediate between dl1101/1107 and KD3. To study whether transgene expression from conditionally replicative Ad vectors in the hamster cells is restricted to the late stages of infection, we assayed the expression of reporter genes in HaK cells. KD3-DsRed in the absence of Ara-C produced CPE beginning at 48 h postinfection. DsRed expression was detectable at 24 h postinfection. In the presence of Ara-C, neither CPE nor expression of DsRed were detected during the course of the experiment (Figure 3c). Expression of SEAP by KD3-SEAP in HaK cells remained at a very low level in the presence of Ara-C. In the absence of Ara-C, SEAP was produced to a lower level

IFN-a Effect on Tumor-specific Oncolytic Ad

relative to human cancer cell lines (Figure 3d). The cumulative level of SEAP activity in HaK cells at 74 h postinfection was 11.6 and 3.34 times lower than in A549 and Hep3B cells, respectively (Figure 2d). The addition of Ara-C blocked both CPE and transgene expression implying that virus genome replication and transgene expression were linked in hamster cells. IFN-a anticancer activity in vivo is known to be dependent on the immune system;30 therefore, the use of an immunocompetent model to evaluate the anticancer activity of the vector expressing INF-a requires that the cytokine be functionally active in this model. We evaluated the biological activity of human IFN-a in hamster HaK cells. HaK cell sensitivity to VSV was similar to that of human HeLa cells (data not shown). The IFN-a expressed by KD3-IFN was able to inhibit the replication of VSV in HaK cells, thereby indicating functional activity of IFN-a; however, the activity was reduced 62.5-fold as compared with HeLa cells.

IFN-a expression improves the antitumor potency of oncolytic Ad in large established tumors Replication-deficient Ad vectors expressing human IFN-a2b demonstrated anticancer activity in xenografts of human tumor cells (including Hep3B cells) in nude mice.12,15 We used large, established, vascularized Hep3B subcutaneous tumor xenografts to evaluate the efficacy of KD3-IFN. First, to visualize the spread

Figure 3 Vectors productively replicate, spread, and express transgenes at the late stage of infection in Syrian hamster HaK kidney cancer cells. (a) Vectors productively replicate in hamster HaK cells. Cells were infected in triplicate with the indicated viruses at an MOI of 100 or 1,000 PFU/ cell and incubated for 7 days. Virus yields were determined by titering on A549 cells. (b) Functional activity of ADP overexpression in hamster HaK cells. Cells were infected in triplicate with dl1101/1107, KD3, or KD3-IFN at the indicated MOI. The viability of the cells was quantified at day 7 postinfection. (c) Strictly late expression of DsRed in HaK cells from the KD3-DsRed vector. HaK cells were infected with KD3-DsRed at an MOI of 100 PFU/cell. Ara-C was added to cells where indicated to maintain the early stage of infection. The morphology of the DsRed-expressing cells is indicated by arrows. Original magnification:
100. (d) SEAP is expressed from KD3-SEAP in HaK cells strictly at the late stage of infection. HaK cells were infected in triplicate with KD3-SEAP at an MOI of 100 PFU/cell. Ara-C was used to maintain the early stage of infection.

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and replication of the vector, mice were injected intratumorally with KD3-DsRed, and reporter gene expression was detected by fluorescence microscopy of tumor sections at day 9 after virus injection (Figure 4a). As this vector expresses the transgene only at late stages of infection, and the vector life cycle is shorter than 9 days, we conclude that the expression of DsRed results from vector replication and spreads within the tumor. We previously found that KD3 was able to suppress Hep3B tumor growth and that the efficacy was improved by increasing the vector dose and applying multiple injections.9,31 In addition, prolonged sustained levels of IFN-a were shown to be crucial for antitumor activity in human cancer patients and were achieved by frequent administrations of the recombinant protein.11 KD3-IFN, KD3, or buffer were injected intratumorally every second day for a total of 10 injections (5
1010 plaque forming units (PFU) per tumor) (Figure 4b). In the buffer-treated group, some animals

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had tumors exceeding 2,000 mm3 at day 4 after the start of treatment and had to be euthanized. In the groups treated with KD3-IFN or KD3, all tumors were suppressed up to day 20 after the start of treatment. The KD3-IFN group had significantly smaller tumors as compared with KD3 (P ¼ 0.046). After 10 additional injections, both vectors significantly prolonged survival compared with the group treated with the buffer (Po0.0001) (Figure 4c). Animals treated with KD3-IFN survived significantly longer than did the group treated with KD3 (P ¼ 0.035). We also tested the antitumor efficacy of KD3-IFN in the immunocompetent Syrian hamster model.29 Established subcutaneous allografts of HaK tumors in Syrian hamsters were treated with multiple intratumoral injections of KD3-IFN, KD3, or buffer to a total of 5
1010 PFU per tumor (Figure 4d). In the group treated with buffer, tumors started to exceed 3,000 mm3 at

Figure 4 Inclusion of the human IFN-a gene into the Ad vector genome increases the anticancer efficacy of the oncolytic vector in immunodeficient and immunocompetent animal cancer models. (a) KD3-DsRed replicates and expresses the transgene in established Hep3B xenografts in nude mice. Established subcutaneous Hep3B tumors in nude mice were injected intratumorally with 1
109 PFU of KD3-DsRed (top) or with buffer (bottom). Tumor sections were analyzed with fluorescence microscopy at day 9 postinjection. Arrows indicate borders of DsRed-expressing area of the tumor. Original magnification:
100. (b) Expression of IFN-a increases the antitumor efficacy of the oncolytic Ad vector in a xenograft animal model. Established subcutaneous Hep3B tumors were injected intratumorally with 5
109 PFU of the indicated vectors (n ¼ 12 for buffer, n ¼ 13 for KD3, n ¼ 12 for KD3-IFN). The injections were repeated every second day for a total of 10 injections per tumor. Boxes represent the interquartile range (25–75%) of tumor volumes; the whiskers show complete range, excluding outliers (open circles, 1.5–3 box lengths from the box edge) and extremes (*, 43 box lengths from the box edge). The horizontal line inside each box depicts the median tumor volume. (c) Expression of IFN-a by KD3-IFN prolongs the survival of nude mice with Hep3B tumors. Animals from the experiment in b received additional 10 injections of the vectors (5
109 PFU/day every second day); animals were euthanized when the tumor volume reached 2,000 mm3, and the survival curves were plotted. (d) Expression of IFN-a increases the antitumor efficacy of the oncolytic Ad vector in an immunocompetent hamster cancer model. Established subcutaneous HaK tumors were injected intratumorally with PBS or 5
109 PFU of virus vectors (n ¼ 10 for buffer, n ¼ 11 for KD3, n ¼ 12 for KD3-IFN). The injections were repeated every second day for a total of 10 injections per tumor. Boxplots of tumor volumes are shown.

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IFN-a Effect on Tumor-specific Oncolytic Ad

day 24 after the start of treatment, and animals bearing these tumors were euthanized. All tumors treated with KD3-IFN or KD3 were suppressed up to day 32 after the start of treatment. The KD3-IFN group had significantly smaller tumors as compared with KD3 (P ¼ 0.042), indicating that inclusion of the IFN-a gene into this oncolytic vector was advantageous in this animal tumor model. In addition, the Ad vectors were able to transduce and decrease the number of A549 lung metastases in an orthotropic lung tumor model (Supplementary Figure S1). KD3-IFN produced improved results in this model as compared with KD3 but this difference did not reach statistical significance.

Inclusion of the IFN-a gene reduces hepatotoxicity of conditionally replicative Ad Wt Ad5 has been reported to replicate abortively in the mouse liver at doses over 1
109 PFU after intravenous administration and to produce severe hepatotoxicity.32 The dl1101/1107 mutation reduces Ad replication in normal cells,9,23 and also reduces liver toxicity in mice upon intravenous administration.33 It was reported previously that recombinant human IFN-a2b protein could induce protection against viral infections in murine animal models.34,35 We studied hepatotoxicity in C57BL/ 6 mice injected intravenously with buffer, replication-deficient vector AdCMVpA, Ad5, KD3, or KD3-IFN (Figure 5a). Ad5 was the most toxic among all studied viruses (Po0.002). KD3 was significantly more toxic than KD3-IFN (P ¼ 0.001) and AdCMVpA (Po0.001). KD3-IFN and AdCMVpA were the least toxic. Although the overall difference in toxicity of KD3-IFN and AdCMVpA was not significant (P ¼ 0.4), KD3-IFN at the highest dose did cause severe liver toxicity in mice, whereas AdCMVpA at the same dose level was well tolerated. In a separate experiment, we found that the maximum tolerated dose (MTD) for KD3 and KD3-IFN in C57BL/6 mice was five times higher than for wt Ad5 (1
1010 versus 2
109 PFU). Serum alanine aminotransferase (ALT) levels in mice injected with Ad5, KD3, or KD3-IFN at tolerated doses remained elevated at day 7 and returned to the background level by day 14. Mice injected with KD3-IFN gained normal body weight faster than those injected with KD3 (data not shown). Additionally, we studied hepatotoxicity of the vectors in Syrian hamsters (Figure 5b). Only injections of Ad5 produced elevated ALT serum levels in this model. Within the studied dose range, C57BL/6 mice showed higher toxicity after Ad5 intravenous administration at similar doses (PFU/kg of body weight) as compared with Syrian hamsters (P ¼ 0.001). The dl1101/1107 mutation confers Ad5 replication sensitivity to IFN-a in normal cells in vitro and in vivo We have reported previously that KD3 was restricted in growtharrested but was able to replicate in growing normal human HEL299 lung fibroblasts.9 To investigate how expression of IFNa influences replication of the vector in normal human cells, we infected growing HEL299 cells with KD3-IFN, KD3, or dl1101/ 1107 (Figure 6a). As we observed previously,9 KD3 produced Molecular Therapy vol. 15 no. 3, march. 2007

Figure 5 The dl1101/1107 E1A mutation reduces the hepatotoxicity of the replication-competent vector KD3; expression of IFN-a further reduces the toxicity of the vector. (a) Vector toxicity in C57BL/6 mice. C57BL/6 mice were injected intravenously through the tail vein with the indicated viruses (3.125
109, 6.25
109, 1.25
1010, 2.5
1010, or 5
1010 PFU/animal; n ¼ 3). At day 3 after injection, blood was collected and analyzed for serum ALT levels. The dashed line represents serum ALT level in mock-infected animals. (b) Vector toxicity in Syrian hamsters. Syrian hamsters received a single intravenous injection of the indicated viruses through jugular vein (1.25
1010, 2.5
1010, or 5
1010 PFU/animal; n ¼ 3). Blood was collected at day 3 after injection and analyzed for serum ALT levels. The dashed line represents the serum ALT levels in mock-infected animals.

CPE earlier as compared with dl1101/1107 (P ¼ 0.005). There was no significant difference between KD3-IFN and dl1101/1107 in this assay (P ¼ 1.00). Interestingly, KD3-IFN was significantly less toxic than KD3 (P ¼ 0.006). To study the selective effect of the dl1101/1107 mutation on IFN-a sensitivity in normal as opposed to cancer cells, we studied replication of wt Ad5 and KD3 in cancer (A549 and Hep3B) and normal (HEL299) cells pretreated with IFN-a (Figure 6b). In cancer cells, IFN-a pretreatment did not affect replication of either virus (P40.66 for A549; P40.21 for Hep3B). As we observed previously,9 KD3 was less efficient than Ad5 in Hep3B cells (Po0.019). In growing normal HEL299 fibroblasts, the replication of Ad5 was not affected by IFN-a pretreatment (P ¼ 0.98). In contrast, the replication of KD3, which carries the dl1101/1107 mutation, was significantly impaired by IFN-a (Po0.001). The replication of KD3 in proliferating HEL299 cells was delayed compared with Ad5 (P ¼ 0.02). We conclude that replication of wt Ad5 in vitro is not affected by IFN-a, whereas the dl1101/1107 mutation confers Ad sensitivity to IFN-a selectively in normal cells. 603

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Figure 6 IFN-a attenuates the replication of vectors carrying the dl1101/1107 mutation in normal cells in vitro; induction of IFN-a reduces the liver toxicity in vivo. (a) Expression of IFN-a impairs the replication of KD3-IFN in HEL299 normal human fibroblasts. (left) HEL299 cells were infected in triplicate with the indicated vectors at various MOI. Cell viability was quantified at day 20 postinfection. (right) HEL299 cells were infected with the indicated vectors at an MOI of 1,000 PFU/cell. Phase contrast microphotographs of the monolayers were taken at day 7 postinfection. Arrows indicate CPE produced by KD3. Original magnification:
200. (b) Pretreatment of human cancer cells with IFN-a does not affect replication of wt Ad5 or vector KD3 carrying the dl1101/1107 mutation; pretreatment of normal human cells with IFN-a does not affect the replication of wt Ad5 but reduces the replication of KD3. Human cancer cells (A549 or Hep3B) and growing normal human fibroblasts HEL299 were pretreated with IFN-a and infected in triplicate with virus vectors at the indicated MOI. The viability of the cells was quantified at day 7 for cancer cells and at day 12 for HEL299 cells. (c, left) KD3-SEAP expresses SEAP in C57BL/6 mice after intravenous injection. C57BL/6 mice (n ¼ 3) were injected intravenously with 1
1010 PFU of KD3-SEAP. On day 3 postinjection, blood samples were collected and SEAP enzymatic activity in the serum was quantified. (c, right) Induction of endogenous IFN by poly(I:C) reduces the toxicity of vector KD3 carrying the dl1101/1107 mutation. C57BL/6 mice (n ¼ 3) received single intravenous injection of 1
1010 PFU/injection of KD3. Poly(I:C) was injected intraperitoneally. Blood was collected at days 3 and 7 and analyzed for serum ALT levels.

To elucidate the mechanism of reduced toxicity of KD3-IFN in vivo, we carried out additional experiments in C57BL/6 mice. We detected reporter gene expression in the serum of the animals 3 days after intravenous administration of KD3-SEAP (Figure 6c, left). This result indicates that some transgene expression from this type of vector occurs in normal mouse tissues despite restricted vector replication. To confirm that alleviated hepatotoxicity of KD3-IFN is mediated by IFN-a, we studied the influence of endogenous IFN-a on the hepatotoxicity of KD3 carrying the dl1101/1107 mutation. Endogenous IFN-a was induced by injection of polyinosinic:polycytidylic acid (poly(I:C)). On day 7 after vector administration, ALT levels 604

were significantly reduced in the serum of mice treated with KD3 and poly(I:C) as compared with mice treated with KD3 and buffer (P ¼ 0.001) (Figure 6c, right). Control mice injected with an equal dose of wt Ad5 and treated with buffer or poly(I:C) became moribund and were euthanized on day 3.

DISCUSSION At the current stage of development, oncolytic Ads are not efficacious as a monotherapy at the doses used in clinical trials. Therefore, higher doses of vectors will be required to achieve a therapeutic response. However, with the increase in dose it is likely that toxic levels might be reached. We sought to solve this www.moleculartherapy.org vol. 15 no. 3, march. 2007

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problem by arming an oncolytic Ad with a gene that could both increase antitumor efficacy and decrease the toxicity associated with high vector doses. We hypothesized that inclusion of the IFN-a gene into an oncolytic Ad should result in improved antitumor efficacy of the vector because IFN-a has demonstrated immune-stimulating, apoptosis-inducing, antiangiogenic, and antiproliferative anticancer effects, and has been used in clinical oncology since 1986.11,36 Delivery of IFN-a gene using an Ad vector could be advantageous as compared with the use of recombinant protein because it allows the production of IFN-a in situ and solves the problem of low protein stability.13 It was shown that expression of human hybrid IFN-a2a1 under a constitutive promoter from replication-deficient Ad vector can cause IFN-mediated toxicity in mice.14 Replication-linked expression of IFN-a from a cancerspecific vector results in IFN-a synthesis in the tumor site, thus decreasing the toxicity in nontarget tissues. We constructed the KD3-IFN vector by inserting the human IFN-a2b gene into the previously described oncolytic vector KD3. KD3-IFN retained high level and functional activity of ADP that is characteristic for KD3 and produced functional IFNa. Reporter genes inserted into the same site were expressed strictly at the late stage of virus infection. We studied the antitumor activity of KD3-IFN in two different animal cancer models. In subcutaneous xenografts of Hep3B cells in immunodeficient mice, KD3-IFN suppressed the growth of the tumors and prolonged the survival significantly better than KD3. An immunocompetent animal model, the Syrian hamster, permissive for replication of Ad vectors with a wt E1 region was recently reported.29 In the current study, we demonstrated productive replication of the vectors carrying the dl1101/1107 mutation and retention of the functional activity of IFN-a and ADP in hamster HaK cells. The in vivo antitumor efficacy of KD3-IFN in subcutaneous allografts of HaK cells in Syrian hamsters was superior to that of KD3. Although oncolytic Ads replicate in tumors, high doses of initial vector injections are required to produce significant antitumor activity in animal models.37 However, high doses of replicative Ads have been associated with liver toxicity when administered intravascularly. The level of toxicity was correlated with the doses and cancer-specific features of the vectors.33,38–40 Intratumoral injections limit the initial vector dose to the tumor site, thereby decreasing systemic toxicity, although some leakage into the bloodstream occurs.41–43 The Ad E1A proteins bind to transcription adapters p300/ CBP preventing activation of IFN-stimulated gene factor 3 (ISGF3), a transcription factor required for transcription of antiviral genes.18,20,44–46 We hypothesized that the dl1101/1107 mutation, which abolishes the binding of E1A proteins to p300/ CBP,20 would result in sensitivity of Ad replication to IFN-a in normal cells thus reducing the toxicity of the KD3-IFN vector. The IFN pathway is deregulated in many cancers; therefore, we expected that KD3-IFN replication would be attenuated in normal cells but not in most cancer cells. Ad VA-RNAI blocks double-stranded RNA-induced activation of PKR,19,47 however, this mechanism may not be sufficient to completely block the IFN-a-induced antiviral response.18,46 Molecular Therapy vol. 15 no. 3, march. 2007

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Whereas E1A blocks activation of IFN-a-induced genes early in infection, VA-RNAs function at the late stages of infection. Consequently, mutations in the VA-RNA genes provided cancer specificity to Ad vectors in cell culture but the hepatotoxicity profile remained unaffected as compared with the wt Ad5.48 We studied the toxicity of KD3-IFN in two animal models, C57BL/6 mice and Syrian hamsters. Wt Ad5 replicates in mice upon systemic administration of high virus doses and causes lethal hepatotoxicity.32 We found that the MTD for KD3 in mice exceeded the MTD for wt Ad5 by a factor of five. The dose causing overt toxicity for KD3-IFN was further increased as compared with KD3 by a factor of two. The dose–response curve for KD3-IFN had a shape different from that of KD3 and wt Ad5, implying that IFN-a influenced the toxicity of the vector. In Syrian hamsters, only wt Ad5 produced hepatotoxicity at the studied dose range while KD3 and KD3-IFN were well tolerated. Expression of IFN-a from the Ad vector or pretreatment of the cells with IFN-a protein reduced the replication of Ad carrying the dl1101/1107 mutation in normal cells but not in the cancer cell lines that were examined. This phenomenon was not observed with wt Ad5. Induction of endogenous IFN-a reduced the hepatotoxicity of KD3 in vivo. Interestingly, it was recently reported that Ad induced type I IFN response in mice and humans, and that this did not lead to a deleterious effect on wt Ad replication.49 Our data imply that reduced hepatotoxicity of vectors with the dl1101/1107 mutation in the e1a gene could be at least partially explained by their increased sensitivity to the antiviral effect of endogenous IFN-a. Exogenous IFN-a was used in animal experiments and clinical studies to increase the therapeutic window of type I IFN-sensitive viruses. Expression of the gene for another type I IFN, IFN-b, was reported to improve the therapeutic window of oncolytic VSV.50 We show here for the first time that oncolytic Ad can be targeted to the type I IFN pathway. Human IFN-a is known to display species specificity, therefore, we expect that it might have a more pronounced influence on the therapeutic window of KD3 in human patients when expressed from the vector or administered as recombinant protein. In summary, we conclude that the inclusion of the human IFN-a gene into an oncolytic Ad carrying the dl1101/1107 mutation leads to an increase in antitumor efficacy and a decrease in toxicity resulting in an improved therapeutic window. The combination of Ad oncolysis, ADP overexpression, and IFN-a-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads.

MATERIALS AND METHODS Cell culture. Human cancer cell lines A549 (lung adenocarcinoma),

Hep3B (hepatocellular carcinoma), HeLa (cervical carcinoma), Syrian hamster kidney cancer HaK, and normal human embryonic lung fibroblast strain HEL299 were from the American type culture collection (ATCC) (Manassas, VA). HEK 293 cells were from Microbix (Toronto, ON). All cell cultures were maintained in DMEM (JRH Biosciences, Lenexa, KS)/10% FBS (HyClone, Logan, UT). For Ad infection experiments HaK cells were maintained in Dulbecco’s modified Eagle’s

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medium (DMEM)/2% fetal bovine serum (FBS). Suspension KB cells used for Ad propagation were maintained in Joklik-modified minimum essential medium (MEM)/5% equine serum (HyClone). Virus vectors. Wt human Ad5, dl1101/1107, and KD3 were described previously.9 AdCMVpA is a replication-defective Ad carrying the HCMV-BGH polyA cassette without a transgene in place of E1 region deletion. Vectors KD3-IFN, KD3-DsRed, and KD3-SEAP carry a version of the E3 region that is a hybrid between the Ad vectors KD1 and KD39 (deletion of Ad5 bp 27,858 to 27,860 and an insert of TAA; deletion of Ad5 bp 27,982 to 28,134; deletion of Ad5 bp 28,395 to 29,397 and insert of CCTTAATTAAA; deletions of Ad5 bp 29,783 to 30,469) with a SwaI cloning site inserted downstream of the adp. Human IFN-a2b gene (Invivogen, San Diego, CA), the red fluorescent protein DsRed-Express gene (BD Biosciences, San Jose, CA), or the secreted placental alkaline phosphatase (SEAP) gene (Invivogen) were cloned downstream of the adp gene. Ad plaques were obtained in 293 cells, plaque-purified in A549 cells, grown up in KB cells, and purified on CsCl gradient. The vectors were titered by limiting dilution assay in A549 and 293 cells. Western blotting. A549 cells were infected with the viruses at the MOI

of 20 PFU/cell and collected at 24 and 48 h postinfection. Ten micrograms of protein per sample were electrophoresed on 15% sodium dodecyl sulfate–polyacrylamide gel, transferred onto an Immobilon polyvinylidene fluoride (PVDF) membrane, incubated overnight at 41C in TBST/10% nonfat dry milk, and probed with a rabbit polyclonal antibody against residues 63–77 of ADP. Secondary antibody was goat antirabbit horseradish peroxidase (HRPO; Cappel Organon Teknika, Durham, NC). Bands were visualized using the ECL system (Amersham Biosciences, Piscataway, NJ). IFN-a assays. Test supernatants were collected from KD3- or KD3 IFN–infected A549 cells (MOI ¼ 50 PFU/cell) at 48 and 72 h postinfection, filtered through 0.2 mm filters, incubated 30 min at 561C for virus inactivation, and frozen at 801C. HeLa or HaK cells were seeded at 1
104 cells/well into 96-well plates. After 24 h incubation, 100 ml of test supernatants, or 250 IU of a human IFN-a standard (Namalwa/Sendai; Reference Reagent Repository, NIAID, Gaithersburg, MD) were 2-fold serially diluted and added to the plate. After 24 h of incubation, the cells were infected with VSV (Indiana strain) at an MOI ¼ 1 PFU/cell. The cells were stained with crystal violet at 24 h postinfection. IFN-a production was quantified by ELISA (Amersham Biosciences). In vitro cell viability assays. A549, Hep3B, HaK, or HEL299 cells

grown in 96-well plates were infected with virus vectors at the indicated MOI at day 0. At day 7, 12, or 20 after infection, the viability of the cells was quantified using the MTT assay (Sigma-Aldrich, St Louis, MO). To study the effect of exogenous IFN-a on virus replication in vitro, A549, Hep3B, or HEL299 cells grown in 96-well plates were pretreated overnight with heat-inactivated supernatants from KD3- or KD3 IFN–infected A549 cells (70 ml/well), and then were infected with Ad5 or KD3 at the indicated MOI. At day 7 (A549, Hep3B) or day 12 (HEL299) after infection, the cell viability was quantified by MTT assay. The viability of mock-infected cells at the end of the experiment was considered 100%. Reporter gene expression. For visualization of expression, A549 or

HaK cells were infected with KD3-DsRed at the indicated MOI and microphotographed. To quantify the levels of expression, A549, Hep3B, or HaK cells were infected with KD3-SEAP at 100 PFU/cell. SEAP activity was quantified by OD405 measurement of p-nitrophenylphosphate (Sigma-Aldrich) conversion. Ara-C (Sigma-Aldrich) was used at 20 mg/ml to maintain the early stages of infection and was replenished every 12 h. To study transgene expression in vivo, 1
1010 PFU of KD3-

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SEAP in 100 ml of PBS were injected into 4 to 6 week–old female C57BL/ 6 mice (Harlan Sprague Dawley, Indianapolis, IN) via the tail vein. At day 3 postinjection, SEAP activity was assayed in the serum. Established (day 21, ca. 500 mm3) subcutaneous Hep3B tumors were injected intratumorally with 1
109 PFU of KD3-DsRed or PBS. Tumor sections were analyzed under fluorescence microscopy at day 9 postinjection. In vivo antitumor efficacy and toxicity studies. Hep3B tumors were

established subcutaneously in the hind flank of 4 to 6 week–old female nude mice (Harlan Sprague Dawley) by injection of 5
106 cells in 100 ml of DMEM/50% Matrigel (BD Biosciences). Established tumors (day 12–15, mean volume 398 mm3) were injected intratumorally with PBS or 5
109 PFU of the vectors in 100 ml of PBS. The injections were repeated every second day for a total of 20 injections per tumor. HaK tumors were established subcutaneously in the hind flank of 4 to 5 week–old female Syrian hamsters (Harlan Sprague Dawley) by injection of 4
107 cells in 100 ml of DMEM. At day 31, the tumors (mean volume 214 mm3) were injected intratumorally with PBS or 5
109 PFU of the vectors in 100 ml of PBS. The injections were repeated every second day for a total of 10 injections per tumor. Tumor volumes were calculated as width2
length
0.5. Animals were euthanized when the tumor volume reached 2,000 mm3 (mice) or 3,000 mm3 (hamsters). Hepatotoxicity was estimated by measuring the serum levels of the liver enzyme ALT. C57BL/6 mice or Syrian hamsters received a single intravenous injection of Ad5, KD3, KD3-IFN, or AdCMVpA at various doses at day 0. At day 3 after injection, serum was analyzed for ALT levels. To induce endogenous IFN-a, C57BL/6 female mice were injected intraperitoneally with 150 mg/animal of poly(I:C) (Calbiochem, La Jolla, CA) immediately after the single tail vein injection of 1
1010 PFU of Ad5 or KD3 at day 0. Blood was analyzed for serum ALT levels at days 3 and 7. Statistical analysis. The data are presented as mean7SD. Sigmoid curves were fitted to the data using MicroCal Origin software (OriginLab, Northampton, MA). Tumor volumes are shown as boxplots. Survival data are plotted as Kaplan–Meyer curves. The data were analyzed using SPSS version 10 (SPSS, Chicago, IL). The statistical significance was assessed with factorial ANOVA (in vitro data and in vivo toxicity data) or repeated measures ANOVA (tumor volume data) followed by Tukey’s HSD test for pairwise comparisons between groups. Survival rates were compared with the log-rank test. Po0.05 was considered significant.

ACKNOWLEDGMENTS We thank RM Buller for providing VSV, Reference Reagent Repository (NIAID) for providing IFN-a standard, and M Maske for technical assistance with blood biochemistry. The work was supported by NIH Grants CA108335, CA118022 to WSMW, and CA105841 to KD.

SUPPLEMENTARY MATERIAL Figure S1. Intravenous delivery of transgenes to the lung metastases of A549 cells in nude mice.

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