Oncolytic adenovirus CG7870 in combination with radiation ...

Cancer Gene Therapy (2005) 12, 715–722 All rights reserved 0929-1903/05 $30.00

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Oncolytic adenovirus CG7870 in combination with radiation demonstrates synergistic enhancements of antitumor efficacy without loss of specificity Jeanette Dilley,1 Seshidhar Reddy,1 Derek Ko,1 Natalie Nguyen,1 Ginny Rojas,1 Peter Working,1 and De-Chao Yu1 1

Cell Genesys, Inc., South San Francisco, California 94080, USA.

Conditionally replicating adenoviruses that selectively replicate in tumor cells, but not in normal cells, are being explored as virotherapeutic agents for cancer. A prostate-specific oncolytic adenovirus, CG7870 is currently being evaluated in phase 1/2 clinical trials for the treatment of prostate cancer. To decrease the effective dose and further increase the therapeutic efficacy of CG7870, the combination of virotherapy with radiation therapy was explored in this study. CG7870 is an oncolytic adenovirus in which tumor-specific promoters are driving the expression of E1A and E1B proteins. The effects of combined treatment with CG7870 and radiation on cultured cells were determined in cytotoxicity and virus yield assays. The antitumor efficacy of CG7870 (1 107 particles/mm3 of tumor), 10 Gy of local radiation or both was evaluated in established subcutaneous LNCaP xenografts in nude mice. In vitro, the dual agent treatment resulted in synergistically enhanced potency at suboptimal doses of radiation and virus. Virus yield in irradiated cells increased relative to yield in nonirradiated cells without compromising the specificity of the vector for its target cell types. In vivo, CG7870 treatment alone suppressed tumor growth and extended tumor nonprogression time. The average tumor-volume of the groups treated with CG7870 only and radiation only was 121 and 126% of baseline, respectively, 39 days after treatment. The average tumor-volume of the combination group was 34% of baseline 39 days after a single dose of treatment. No significant body weight loss was observed in any treatment group. There was a significant drop in serum level of prostate-specific antigen (PSA) in the combination group compared to the group treated with either agent alone. In mice treated with CG7870 only or radiation only, serum PSA levels changed to 26 and 383% of baseline, respectively, by study day 46. In contrast, PSA levels in mice treated with CG7870 plus radiation decreased to less than 11% of baseline by study day 46. Histological analysis of tumor sections collected from the combination group revealed enhanced necrosis and more apoptotic cells. Combination of CG7870 with radiotherapy significantly increased antitumor efficacy compared to either agent alone. These results suggest that CG7870 in combination with radiation has improved antitumor efficacy at lower doses and with no additional side effects. Cancer Gene Therapy (2005) 12, 715–722. doi:10.1038/sj.cgt.7700835; published online 15 April 2005 Keywords: prostate cancer; oncolytic adenovirus; radiation; combination therapy

urgery, radiation, and chemotherapy are conventional S therapies for cancer. Although improvements have been made to radiation and chemotherapies, their use is often associated with significant toxicity and limited efficacy. In addition, the therapeutic efficacy of these methods is limited when cancer cells develop resistance to radiation and chemotherapy. This underscores an urgent need for the development of more effective and less toxic novel cancer treatments. Gene therapy represents one such modality, in which genes are delivered into cancer cells to destroy the tumor or to enhance the immune Received October 13, 2004.

Address correspondence and reprint requests to: Dr De-Chao Yu, Cell Genesys, Inc., 500 Forbes Blvd, South San Francisco, CA 94080, USA. E-mail: [email protected]

response against the cancer. Adenoviral vectors are frequently used as gene transfer vectors because they have efficient mechanisms for delivery of genes. In addition, conditionally replication-competent adenoviruses, which are modified to replicate preferentially in cancer cells, can directly damage and mediate lysis of transduced cells. Although monotherapy using oncolytic adenoviral vectors is effective in xenograft tumor models in mice, only limited efficacy has been seen to date in clinical trials.1 A narrow therapeutic window, coupled with dose-limiting toxicities and the immune status of cancer patients has restricted the effectiveness of virotherapy. One approach to increase therapeutic efficacy and potentially decrease toxic side effects is combination therapy. In combination therapy, two or more types of treatment modalities are combined. The mode of action of radiation, chemotherapy, and

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virotherapeutic agents differ, and thus would not be expected to lead to development of cross-resistance. Synergistic effects between oncolytic adenoviral vectors (ONYX-015, CG7870, formerly known as CV787) and chemotherapeutic drugs (e.g. paclitaxel, docetaxel, doxorubicin, cisplatin, and 5-fluorouracil) have been reported in animal models and in clinical trials.2–4 Combination with radiation therapy allowed a 50-fold decrease in the dose of the oncolytic adenovirus CG7060 (formerly CV706) in a prostate xenograft model.5,6 CG7870, a prostate tumor-specific replication-competent adenoviral vector, is currently in phase 1/2 clinical trials for the treatment of prostate cancer. This vector features a number of alterations in design demonstrated to improve efficacy, relative to CG7060, in single-agent experiments. Namely, in CG7870 expression of the viral E1A gene is under the control of the rat probasin promoter, and E1B expression is under the control of the human PSA promoter.7 Additionally, the Adenovirus 5 (Ad5) wild-type E3 region, deleted in many previous adenovirus-based vectors, has been retained. We previously demonstrated that a single intratumoral administration of CG7870 at 5 108 virus particles/mm3 of tumor eliminates subcutaneous xenograft tumors in 6 weeks.7 We have also demonstrated that CG7870 in combination with chemotherapeutic agents in vitro gives a synergistic response in cell killing and virus production assays.4 In the present study, the combination of CG7870 with radiation has been evaluated in vitro and in nude mice bearing subcutaneous implants of human prostate cancer LNCaP xenografts.

Materials and methods

Cells and viruses The human embryonic kidney cell line 293, which expresses the adenovirus E1A and E1B gene products, was purchased from Microbix Biosystem, Inc. (Toronto, Ontario, Canada). The tumor cell lines LNCaP (prostate cancer), 253J B-V and RT4 (bladder cancer), OVCAR-3 (ovarian cancer), and human normal cells (HBL-100, normal breast epithelium and hBSM, human primary bladder smooth muscle cells) were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained at 371C with 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone; Logan, UT), penicillin (100 U/ml) and streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA). For in vitro studies, cells were irradiated with gamma-radiation (6 Gy) using the Mark 1 Research Irradiator (Model #30, JH Sheperd Associates, Cesium 137 Source; San Fernando, CA). Construction of CG7870 has been described previously.7 The vector was amplified in 293 cells, purified and concentration was determined spectrophotometrically, assuming 1A260 ¼ 1.1 1012 virus particles.8

using the Promega CellTiter 96s Aqueous Non-Radioactive Cell Proliferation Assay kit, a measure of cell viability that has been described, previously.4,7 Briefly, cells were seeded at 2 104 cells/well in 96-well plates 24 hours prior to adenoviral infection at multiplicities of infection (MOI) ranging from 0.0001 to 1. Some of the infected wells were irradiated (0–40 Gy) at 24-hour postinfection using the Mark 1 Research Irradiator. Cells were treated with virus for 7 days and then the MTT cytotoxicity assay was performed at 7 days postinfection to determine cell viability. To evaluate the specificity of the combination of CG7870 and radiation, cytotoxicity assays were also carried out on several nonprostate cancer cell lines and normal primary bladder smooth muscle cells.

Statistical analysis The dose–response interactions between CG7870 and radiation at the point of IC50 were evaluated by the isobologram method described previously.4 The dose– response curves were plotted with CurveExpert (Version 1.34) on a semilog scale as a percentage of the control, the absorbance of which was obtained from the samples not exposed to the drugs. Based upon the dose–response curves of CG7870 alone and radiation alone, isobolograms (three isoeffect curves, mode 1 and mode 2 lines) were computed. The envelope of additivity, surrounded by mode 1 and mode 2 isobologram lines, was constructed from the dose–response curves of CG7870 alone and radiation alone. The observed data were compared with the predicted maximum and minimum data for the presence of synergism, additivity, or antagonism by a statistical analysis using the Stat View 4.01 software program (Abacus Concepts, Berkeley, CA). When the data points of the drug combination fall within the area surrounded by mode 1 and/or mode 2 lines (i.e. within the envelope of additivity), the combination is described as additive. A combination that gives data points to the left of the envelope of additivity can be described as supraadditive (synergism) and a combination that gives data points to the right of the envelope of additivity, can be described as subadditive (antagonistic).4

Virus yield assay The virus yield assay used in these studies has been described previously.6,7 Briefly, LNCaP cells were plated in six-well tissue culture plates at 4 104 cells/well. After 24 hours, the cells were infected with CG7870 at an MOI of 0.01, 0.1, or 1 PFU/cell. Some of the infected wells were irradiated (6 Gy) at 24-hour postinfection using the Mark 1 Research Irradiator. At predetermined time points, cells scraped into the medium and subjected to three cycles of freeze/thaw. Virus titer in the lysate was determined in a standard plaque assay using 293 cells.7

MTT assay

Animal studies

The MTT (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2Htertazolium bromide) cytotoxicity assay was performed

All animal procedures were performed in the animal facility at Cell Genesys Inc. in accordance with applicable

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animal welfare regulations under an approved Institutional Animal Care and Use (IACUC) protocol and study design. Athymic Balb/c nu/nu mice (6–8 weeks old) (Simonson Laboratories; Gilroy, CA) were implanted subcutaneously in the right flank with 1 106 LNCaP cells mixed with an equal volume of Matrigel (BD Matrigelt Matrix, BD Biosciences; San Jose, CA) using manual restraint. When tumors reached between 300 and 500 mm3, mice were randomly sorted into groups of 13. CG7870 was administered intratumorally on study day 1; radiation therapy occurred 24 hours after vector administration. Mice in vector only and combination therapy groups were treated one time intratumorally with CG7870 at a dose of 1 107 particles/mm3 of tumor. The tumors of mice in radiation only and in combination therapy groups were irradiated at 10 Gy/tumor using the Mark 1 137Cs Irradiator. For radiation, animals were immobilized with ketamine and xylazine anesthesia and secured in lucite chambers. The whole body was shielded with lead except for tumor-bearing sites on the back. All animals were observed daily, throughout the study, for food and water consumption, appearance and body condition. Body weights were monitored twice a week. Blood (150 ml) was collected by retro-orbital bleeding on study day 3, 18, 32, and 46 and used to measure serum levels of PSA by ELISA (Medicorp; Montreal, Quebec, Canada). Tumors were measured twice a week in three dimensions by external caliper, and the volumes estimated by the formula {length (mm) width (mm) height (mm)}/2. Mean tumor volume for each treatment group7SE of the mean was plotted versus day after vector injection. The difference in relative tumor volume between groups was compared for statistical significance using the type 2 (two-sample equal variance), two-tailed t-test.

Histology During course of the study, a limited number of mice from each group were euthanized and the tumors were collected (days 14 and 42). The tumor samples were embedded in paraffin blocks, and 4 m sections were cut and stained with H&E. Tumor sections were also stained for the presence of adenoviral hexon protein using hexonspecific monoclonal antibody 13G9 developed at Calydon Inc. Histology methods for detection of hexon protein were described previously.6 The necrotic cells were scored on tumor sections by light microscopy at 400 magnification. The extent of necrosis was based on scoring 500 cells/section as either necrotic or non-necrotic. The average necrosis score was calculated based on counting 10 fields distributed evenly across the area of the tumor section. To assess the effect of CG7870, radiation, or combination treatment on tumor vascularization, the number of blood vessels was counted at a magnification of 400, and the average number of blood vessels was calculated from 10 fields distributed evenly across the area of the whole tumor section.9 Anti-CD31 antibody (BD Pharmingen; San Diego, CA) based immunohistochemical staining was used for detection of vascular endothelial

cells as described previously.10 Apoptotic cells were detected using the TUNNEL assay (Roche Molecular Biochemicals; Indianapolis, IN), as suggested by the manufacturer. The morphological features used to identify apoptosis in the tumor sections have been described previously.11 The apoptotic cells were scored on tumor sections at 400 magnification, and the average score of apoptotic cells was calculated from 10 fields of nonnecrotic areas detected randomly across each tumor section.

Results

Combination of CG7870 with radiation increases cytotoxicity To determine the effects of combined treatment with CG7870 and radiation on cultured cells, LNCaP cells were first infected with varying MOI of CG7870 alone or treated with varying doses of radiation alone and the cell viability assessed. Suboptimal doses of CG7870 or radiation causing minimal cell killing were determined from these single-agent studies. As shown in Figure 1a, infection of LNCaP cells with CG7870 at an MOI of 0.1 resulted in B40% cell death (B60% viability) at 7 days postinfection. Similarly, B65% of LNCaP cells survived following exposure to 6 Gy radiation at 7 days postinfection. When LNCaP cells were infected with CG7870 at an MOI of 0.1 and exposed to 6 Gy of radiation only 5% of the cells were viable at 7 days postinfection. Viability of LNCaP cells in the combination group dropped further to 0% at 9 days postinfection. Using an isobologram, the cytotoxicity resulting from combination treatment was compared to that resulting from treatment with either CG7870 or radiation alone to determine if the effect was synergistic, additive or antagonistic (Fig 1b). Analysis of the dose– response curves revealed that responses were below the theoretical envelope of additivity and indicates a synergistic increase in cell killing as a result of combination treatment.

Combination of CG7870 with radiation increases viral burst size To determine the impact of radiation on viral replication, viral burst size (yield) assay was performed on LNCaP cells. The cells were infected with CG7870, exposed to radiation and harvested at various time points postinfection. As shown in Figure 2, at day 2 postinfection, the viral yields were similar in irradiated and nonirradiated CG7870 infected cells. However, at later time points the virus yields were much higher in cells that were irradiated (3000 PFU) than in cells that were not irradiated (500 PFU). Replication of CG7870 reached a plateau at day 6 in irradiated LNCaP cells and at day 8 in nonirradiated cells (Fig 2). These results suggest that treating the infected cells with radiation enhances the replication of CG7870.

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Figure 2 One-step growth curve of CG7870 (MOI, 0.1) in human prostate cancer LNCaP cells with (filled circle) or without (filled square) radiation (6 Gy).

Figure 1 Viability of prostate cancer LNCaP cells treated with mock, CG7870, radiation, or CG7870 plus radiation. (a) LNCaP cells were infected with CG7870 (moi ¼ 0.1) 24 hours before exposure to radiation. (b) Isobologram analysis of the observed data for the combination of CG7870 and radiation. The concentration that produced 50% cell growth inhibition (IC50) is expressed as 1.0 in the ordinate and abscissa of the isobologram. Y-axis, radiation (IC50); X-axis, CG7870 (IC50).

Combination of CG7870 with radiation does not alter specificity of virus-mediated cytotoxicity To determine if radiation alters the specificity of CG7870, cytotoxicity assay was carried out in selected cancer cell lines and normal cells. The combination of CG7870 at an MOI of 0.1 and radiation at a dose of 6 Gy did not lead to a significant change in specificity relative to CG7870 in single-agent experiments. As in previous experiments, the combination treatment decreased LNCaP cell viability to

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Figure 3 Effect of radiation on the virus-mediated cytotoxicity of CG7870. Cells were infected with CG7870 (MOI, 0.1) 24 hours before exposure to radiation (6 Gy). Cell viability was determined by MTT assay.

less than 20% at 7 days postinfection and to almost 0% viability at 9 days postinfection (Fig 3). In nontarget hBSM, 253J B-V, OVCAR-3, RT4, and HBL-100 cells, CG7870 in combination with radiation did not induce significant levels of cytotoxicity (Fig 3).

Antitumor efficacy of CG7870 in combination with radiation in vivo The effects of CG7870 in combination with radiotherapy were assessed in nude mice bearing subcutaneous LNCaP xenografts. To demonstrate additive or synergistic effects,


a 50-fold lower dose of CG7870 than was effective in the single treatment experiment was used. Mice bearing LNCaP tumors received either intratumoral injection of 1 107 CG7870 particles/mm3 of tumor volume, 10 Gy of radiation or a combination of both. A significant decrease in tumor volume between controls and all treatment groups was observed (Fig 4). Although a single treatment with either CG7870 or radiation was effective in inhibiting tumor growth, the combination of the two resulted in significant tumor regression. The average tumor-volume of the group treated with CG7870 was 121% of baseline at 38 days after treatment, and the tumor volume of the group treated with 10 Gy radiation was 126% of baseline at this time point. However, when the identical dose of CG7870 was followed at 24 hours by the identical dose of radiation, a statistically significant drop in the relative

tumor volume to 34% of baseline was observed (Po.001; Fig 4). At day 52, average tumor volume in the CG7870 treatment group and in the radiation group was about 100% of baseline, whereas average tumor volume in the combination group was only 20% of baseline. In addition, the majority of animals (80%) in the combination group became tumor-free by study week 5 and remained tumor-free until the end of the study.

Serum PSA levels The sera samples were collected on study day 3, 18, 32, and 46 and tested for PSA. The relative level of PSA increased steadily in the vehicle group (Fig 5). In mice treated with CG7870 only, serum PSA decreased to 26% of baseline by study day 46. In the radiation group, the level of PSA increased after day 18 and reached 383% of baseline by study day 46. In contrast, PSA level in mice treated with CG7870 plus radiation decreased substantially and declined to less than 11% of baseline by study day 46 (Fig 5).

Immunohistochemical analysis of tumor sections

Figure 4 Antitumor efficacy of CG7870 in combination with radiation therapy in LNCaP xenografts. LNCaP tumor volumes in mice treated either with vehicle, CG7870 (1 107 particles/mm3 of tumor volume, delivered intratumorally), radiation (10 Gy/tumor), or a combination of CG7870 plus radiation are shown.

Histological analysis of the tumors collected at day 14 and day 42 post-treatment documented the synergistic effect of the combination of CG7870 with radiation. At day 14 in the combination treatment group, large areas of tumor tissue that stained positive for hexon were evident, thereby indicating that the cells were infected with CG7870. In tissues from animals treated with CG7870 alone, fewer cells stained positively for hexon (Table 1). Tumors from mice treated with vehicle or radiation had no hexon-positive cells. At day 42, hexon-positive cells were detected in tumor sections of mice treated with CG7870 only. However, tumors that received the combination of CG7870 and radiation showed no evidence of hexon-positive cells, but were full of necrotic cells and scar tissue.

Figure 5 Serum PSA levels in LNCaP tumor-bearing mice treated with CG7870, radiation, or combination of the two. Serum collected at indicated time points from treated mice was used to determine the levels of PSA by ELISA as described in Material and methods.

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Table 1 Effect of CG7870, radiation, or both on necrosis, vascularization, apoptosis, and hexon positive cells in LNCaP tumor xenografts Study group Vehicle (SD14) CG7870 (SD14) Radiation (SD14) CG7870+radiation (SD14) Vehicle (SD42) CG7870 (SD42) Radiation (SD42) CG7870+radiation (SD42)

Necrosis (%)

Blood vessels

Hexon

CD31+ cells

Apoptotic cells

0 0 0 5 5 75 40 100

80 70 42.5 40 20 2.5 45 0

0 9 0 81 0 26 0 0

20 20 15 4 25 13 21 0

7 0 9 204 5 252 8 0

LNCaP tumor-bearing mice were treated intratumorally with CG7870, radiation, or both as described in Materials and methods. Tumor samples were harvested on study days (SD) 14 and 42 and analyzed by histological staining (H&E for necrosis and blood vessels and immunohistochemical staining with antihexon, anti-CD31 antibodies) as described in Materials and methods.

The number of apoptotic cells detected at day 14 using the TUNNEL assay in the tumors treated with CG7870 in combination with radiation was 23-fold higher than with the group treated with radiation alone, 20-fold higher than in the group with CG7870 alone, and 29-fold higher than in the group treated with vehicle. At day 42, the tumors treated with CG7870 in combination with radiation were reduced to necrotic and scar tissue. CD31 is expressed constitutively on the surface of adult and embryonic endothelial cells and has been used as a marker to detect angiogenesis. A mouse monoclonal antibody to human CD31 was used to stain tissue from all groups at day 14 and day 42. The xenografts collected from all four groups at 14 days post-treatment contained blood vessels. However, the xenografts collected from the combination group had fewer blood vessels in the areas that had more hexon-positive cells, and the blood vessels in these areas had a disrupted appearance. On study day 42, the xenograft sections of mice treated with virus alone had fewer blood vessels compared to mice treated with vehicle or radiation alone, tumor sections of mice treated with combination therapy had no obvious blood vessels. The H&E stained sections of xenografts from mice treated with the combination of CG7870 and radiation revealed necrotic areas throughout and without any obvious blood vessels. The sections of xenografts obtained from mice treated with the virus or radiation alone had variable amounts of both necrotic and tumor cells distributed throughout and with several blood vessels. At day 14, more necrotic cells were observed in the tumors treated with CG7870 and radiation than those treated with either agent alone. The amount of necrosis in tumors treated with CG7870 alone was higher than that seen in the control tumors or in the tumors treated with radiation alone. At day 42 after treatment, the synergistic response was very evident from the anti-CD31 staining pattern: necrotic tissue had increased to 100% in the combination treatment group tissue, 75% in the CG7870 treated tissue, 40% in the radiation treated tissue, and 5% in the vehicle control tissue.

Body weight Tolerability of the virus and/or radiation treatment was monitored by body weight measurements (Fig 6). Animals

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Figure 6 Relative body weight of tumor bearing animals treated either with vehicle, CG7870, radiation, or combination of CG7870 and radiation.

treated with both CG7870 and radiation gained 14% more weight than untreated control animals (Fig 6). The combination treatment was also found to protect the animals from the transient weight loss that was observed in animals treated with CG7870 or radiation alone (Fig 6).

Discussion

Currently, prostate cancer is treated with ‘‘watchful waiting’’, radical prosatectomy, radiation therapy, or hormonal therapy. Surgery and radiation therapy are the two most commonly used therapeutic modalities for localized prostate cancers. However, a narrow therapeutic window coupled with limiting high-dose toxicity of radiation therapy limits its effectiveness. As a result, there is an urgent need for more effective and less toxic therapies. Gene therapy approaches using adenoviral


vectors have received wide attention for treatment of prostate cancer. Although promising results have been obtained in preclinical studies with gene therapy approaches, clinical trials to date have shown limited efficacy. Gene therapy and radiation approaches can be made more effective by combining these single agents because each has a different mode of action. In fact, the evidence from several preclinical and clinical studies suggests that combination of gene therapy with standard cancer therapies such as radiation and chemotherapy results in improved therapeutic benefit. Augmentation of efficacy has been observed with ONYX-015 when combined with cisplatin or 5-fluorouracil in mouse xenograft tumor models12 and in human clinical trials for head and neck cancers.2 The combination of gene therapy and radiation has also demonstrated synergistic antitumor effects both in vitro and in vivo in several tumor models.6,13 The combination of an oncolytic adenoviral vector with Taxol produces an additive antitumor effect in preclinical studies in mouse xenograft tumor models.4 Thus, combination treatment has great potential for translation into effective clinic therapy in the immediate future.14–16 In the case of CG7870, the alterations in vector design (relative to earlier adenovirus-based therapeutic agents) presented an unknown material impact on the efficacy of a treatment regimen combining the vector and radiation. Namely, CG7870 is transcriptionally regulated by dual, heterologous promoters and retains the Ad5, wild-type E3 region. Transcriptional regulation of oncolytic adenoviruses by dual, heterologous promoters has previously been demonstrated to increase the specificity of vectors relative to similar vectors regulated by a single, heterologous promoter.17 As such, the potential translation of this increased specificity to a combined treatment regimen may further augment the therapeutic window. Retention of the Ad5, wild-type E3 region has previously been demonstrated to enhance the ability of the vector to eliminate distant tumor xenografts via intravenous administration.7 This is thought to be due in varying parts to the immunomodulatory and lytic proteins encoded by the E3 region. Given the disparate mechanisms of action of oncolytic adenovirus vectors and radiation, the effects of such proteins on the action of a combined treatment regimen are difficult to predict. Therefore, to investigate the therapeutic benefits of CG7870 and radiation, we established and evaluated combination treatments in vitro and in vivo in the LNCaP xenograft tumor model. The combination of CG7870 and radiation resulted in a synergistic increase in cell killing activity in vitro compared to that resulting from treatment with either agent alone. The combination of CG7870 and radiation did not lead to a significant change in specificity and selectivity of the oncolytic virus relative to CG7870 in single-agent experiments. Moreover, the virus yields were much higher from LNCaP cells that were irradiated than from cells that were not irradiated. The synergistic cytotoxicity noted in vitro with CG7870 and radiation was also observed in the in vivo LNCaP

xenograft model. It has previously been shown that a single intratumoral administration of CG7870 at 5 108 virus particles/mm3 of tumor eliminated pre-established tumors in the mouse LNCaP xenograft tumor model within 6 weeks after treatment, but a single dose at 1 107 CG7870 particles/mm3 of tumor yielded just modest antitumor effects.7 It has also been reported that a single radiation dose of 10 Gy/tumor delayed tumor growth in this model.6 In the present study, combination of 1 107 CG7870 particles/mm3 of tumor with radiation therapy significantly increased antitumor efficacy compared to either agent alone. Moreover, in the combination group, no significant body weight loss was observed. In addition, serum PSA levels in the combination treatment group were lower than those of the vehicle and single-agent treatment groups indicating a greater stabilization and decline in viable tumor tissue over time. Histological analyses of tumor sections collected from the combination group revealed enhanced necrosis and increased apoptosis compared to tumors collected from mice treated with either agent alone corroborating the biochemical characterization of tumor progression, stabilization, and regression. These results show that CG7870 in combination with radiation has an improved oncolytic activity at lower doses and with no added toxicity. Radiation is known to kill mammalian cells by activating apoptotic pathways and breaking DNA strands. Most radiation-induced DNA double-stranded breaks are rapidly repaired by constitutively expressed DNA repair mechanisms. DNA repair becomes more active in irradiated cells, potentially allowing for greater replication/multiplication of the episomal adenoviral DNA. Owing to its small target size, the adenoviral genome (B36 kb) is far less likely to sustain radiationinduced damage than is human DNA (3 106 kb). Recently, Zhang et al12 reported enhanced adenovirus infection of tumor cells both in vitro and in vivo following ionizing radiation. Currently, the mechanisms involved in enhanced uptake of adenovirus following ionizing radiation are not fully understood. Changes caused by radiation both at the cell surface and in the cytoplasm and nucleus may all contribute to improved virus uptake by tumor cells. Oncolytic viruses also are believed to be more effective when tumor growth rates are reduced by certain chemo- or radiotherapeutic regimens.18 In humans, inflammation of the prostate due to expression of viral antigens and radiation followed by removal of necrotic tissue may contribute to a reduction of tumor burden.19 In addition, CG7870 may also augment the antitumor activity of radiation. Malignant tumors that express adenoviral proteins may become more sensitive to treatment with DNA-damaging agents such as radiation in vivo. Toth et al20 have recently reported that the combination of radiation plus oncolytic adenovirus suppresses the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than either radiation alone or oncolytic vector alone. Our current findings with CG7870 clearly show that the virotherapy

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and radiation combination significantly improves both cytotoxicity and virus burst size in vitro as well as efficacy in the xenograft tumor model in vivo, but without a concomitant increase in toxicity. From a clinical point of view, the combination therapy produces additional therapeutic benefit over either individual modality without additive adverse toxicity. These findings will help us to design improved combination treatment regimens for prostate cancer using CG7870 in ongoing phase 1/2 clinical trials.

Acknowledgments

We thank Gail Colbern, Melinda Vanroey and Yu Chen and the staff of the animal facility at Cell Genesys Inc., for excellent help in animal studies.

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