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Clinical Cancer Research Vol. 11, 8845-8855, December 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Dual Promoter–Controlled Oncolytic Adenovirus CG5757 Has Strong Tumor Selectivity and Significant Antitumor Efficacy in Preclinical Models

Yuanhao Li1, Neeraja Idamakanti1, Trini Arroyo1, Steve Thorne2, Tony Reid2, Scott Nichols2, Melinda VanRoey1, Gail Colbern1, Natalie Nguyen1, Orlena Tam1, Peter Working1 and De-Chao Yu1

Authors' Affiliations: 1 Cell Genesys, Inc., South San Francisco, California and 2 Stanford University, Palo Alto, California

Requests for reprints: Yuanhao Li, Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080. Phone: 650-714-4380; E-mail: yuanhaoli88{at}gmail.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential.

Experimental Design: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent.

Results: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens.

Conclusions: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.

Conditionally replicating oncolytic adenoviruses are being actively evaluated for treatment of a variety of cancers (1, 2). One strategy to achieve tumor specificity is the use of tumor-selective transcriptional response elements to control the essential early E1 genes, which are required to transactivate the other adenoviral genes (3, 4). Examples of these are the oncolytic adenoviruses CG8840, CG7870, OV798, and OV890, in which tumor- or tissue-selective transcriptional response elements, such as uroplakin, prostate-specific antigen, carcinoembryonic antigen, and {alpha}-fetoprotein, respectively, were used to control the early adenovirus genes (5–8). These oncolytic adenoviruses are each specific for a certain tissue type, such as CG8840 for the urothelium.

To create an oncolytic adenovirus with broad antitumor activity, an oncolytic adenovirus, CG5757, in which the tumor-selective expression of both E1a and E1b genes is accomplished by replacing the nonselective viral promoters with tumor-selective promoters derived from the human E2F-1 and human telomerase reverse transcriptase (hTERT) genes, respectively, was constructed. The E2F-1 promoter is expected to be active in tumor cells that have a defective retinoblastoma (Rb) pathway, ~85% of all tumor types. The hTERT promoter is expected to be active in tumors with up-regulated telomerase expression, ~90% of all human cancers.

The Rb pathway plays a complex role in cell proliferation, primarily by Rb-repressive interaction with the transcription factor E2F (9, 10). Rb suppression of E2F is regulated by phosphorylation through cyclin-dependent kinases (11, 12). Numerous studies have confirmed the loss of Rb function in many different cancer types (13–16). A direct indication of Rb deficiency in these tumor cells is the release of uncontrolled E2F transcription factor. The released E2F can lead to a loss of control over cell cycle progression, a critical step in neoplastic transformation. Because E2F regulates its own promoter, the E2F-1 promoter is preferentially active in tumor cells in which the Rb pathway is deregulated due to mutations in the Rb protein or in other gene products that regulate Rb protein, such as the cyclin-dependent kinase p16 (17, 18).

Like E2F, telomerase is involved at a critical point in the cell cycle and is aberrantly expressed in a large percentage of human tumors. Telomerase is largely silenced in normal adult human somatic tissue, essentially placing a cap on the number of replication cycles normal cells can undergo. Its reactivation, however, can allow a cell to replicate an indefinite number of times, another hallmark of neoplastic transformation. Telomerase activity has been found in almost all tumor samples and has become a broad tumor marker (19).

Using E2F-1 and hTERT promoters as control elements, CG5757 is expected to replicate in and kill cancer cells that have a defect in the Rb pathway and are positive for hTERT activity. In this communication, we have evaluated tumor selectivity, cytotoxicity, and antitumor efficacy of CG5757 in a variety of preclinical models.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture. Unless otherwise indicated, tumor cell lines were obtained from the American Type Culture Collection (Manassas, VA). They have been shown previously by others to have defective Rb pathway and hTERT expression. These tumor cells include hepatocellular carcinoma Hep3B (20, 21), lung carcinoma A549 (22, 23), colorectal carcinoma LoVo (24, 25), pancreatic carcinoma Panc1 (26, 27), prostate carcinoma LNCaP (28, 29), and cervical cancer HeLa (30, 31). The bladder transitional cell carcinoma cell line 253J B-V was kindly provided by Dr. Colin Dinney (M.D. Anderson Cancer Center, Houston, TX) and was originally derived from the 253J cell line, which is also Rb defective and hTERT positive (32–35). WI-38 cells are normal human diploid embryonic lung fibroblasts, and WI-38 VA-13 cells (also known as VA-13) are SV40-transformed WI-38 cells. This isogenic pair has different Rb status in that WI-38 has a normal Rb, whereas VA-13 has a defective Rb pathway due to expression of the SV40 large T antigen (4, 36). Both WI-38 and VA-13 cells have no detectable telomerase activity (17, 37). ARPE-19 cells are diploid normal human retinal pigmented epithelial cells with normal Rb and no hTERT expression (38, 39). The human embryonic kidney 293 cell line was used for virus titer determination. All these cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 2.05 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Tissue culture reagents were obtained from JRH Biosciences, Inc. (Lenexa, KS). FBS was purchased from Irvine Scientific, Inc. (Irvine, CA). Furthermore, different primary human cells were purchased from Clonetics (San Diego, CA) and cultured in accordance with vendor instructions. These primary cells are derived from normal human tissues and therefore have wild-type (WT) Rb pathway and no hTERT expression and used previously as normal cell controls for viral characterization (38, 39).

Promoter cloning and tumor specificity. The human E2F-1 promoter was PCR cloned from human genomic DNA as a DNA fragment containing nucleotides –216 to +55 corresponding to the E2F-1 transcription initiation site (GenBank S74230) with flanking AgeI (PinAI) restriction sites at the 5' and 3' ends. The hTERT promoter was cloned from plasmid pGRN-316 obtained from Geron, Inc. (Menlo Park, CA; ref. 40). The hTERT promoter fragment corresponding to nucleotides –187 to +40 relative to the transcription initiation site (GenBank AF325900; ref. 41) was connected to flanking SalI sites at the 5' and 3' ends to facilitate further cloning. Each tumor-specific promoter was inserted into a plasmid to regulate expression of the luciferase reporter gene (E2F-1-Luc and hTERT-Luc) for promoter activity assays following the methods described previously (7).

Viruses. The recombinant adenoviruses were generated following the procedure as described previously (6). All cloning steps to modify the E1 region in adenoviral genome were done on plasmid pXC.1 (purchased from Microbix Biosystems, Inc., Toronto, Ontario, Canada). The E1b 19-kDa coding region from nucleotides 1,714 to 1,975 was deleted (5). E2F-1 and hTERT promoters were inserted into the adenoviral genome by conventional molecular cloning techniques to control E1a and E1b expression, respectively. The AgeI sites in the E2F-1 promoter DNA fragment and the SalI sites in the hTERT promoter DNA fragment were used to clone the promoters in front of the adenoviral E1a or E1b regions, respectively, as described previously (5, 6). The resulting plasmid CP1509 has the E2F-1 to drive E1a expression and hTERT for E1b 55 kDa gene expression. This E1-containing plasmid and pBHGE3, a pBR322-based plasmid carrying an adenovirus type 5 (Ad5) genome truncated in the E1 region (42), were used to rescue recombinant virus CG5757 by cotransfection in 293 cells.

Western blot analysis. Confluent cells in six-well plates were infected at a multiplicity of infection of 10 plaque-forming units per cell with either Ad5 or CG5757 and harvested 24 hours after infection. Cells were washed with cold PBS and lysed for 30 minutes on ice in a lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% Igepal CA360 (Sigma Chemical Co., St. Louis, MO), 0.5% sodium deoxycholate, and protease inhibitor mixture (Roche, Indianapolis, IN). After centrifugation in a benchtop centrifuge at maximum speed for 30 minutes at 4°C, the supernatant was harvested and the protein concentration was determined with the Protein Assay ESL kit (Roche). Protein (50 µg) per lane was separated on 8% to 16% SDS-PAGE and electroblotted onto Hybond enhanced chemiluminescence membrane (Amersham Pharmacia, Piscataway, NJ). The membrane was blocked overnight in PBS with 0.1% Tween 20 supplemented with 5% nonfat dry milk. Primary antibody incubation was done at room temperature for 2 to 3 hours in PBS with 0.1% Tween 20/1% nonfat milk followed by wash in PBS with 0.1% Tween 20 and 1-hour incubation with diluted horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Enhanced chemiluminescence was used for signal detection. Anti-E1A antibody (clone M58) was purchased from NeoMarkers (Fremont, CA) and anti-E1B 55-kDa antibody was purchased from Oncogene (Cambridge, MA).

Virus yield assay and TCID50. Six-well dishes were seeded with 5 x 105 cells per well in complete medium (RPMI 1640 with 10% FBS). Twenty-four hours later, cells were infected at a multiplicity of infection of 2 plaque-forming units per cell for 3 hours in serum-free medium. After infection, the virus-containing medium was removed, cell monolayers were washed with PBS, and complete medium (4 mL) was added to each well. For normal cells, including primary cells, the special vendor-recommend medium with 4% FBS was used for each individual cell type. Seventy-two hours after infection, cells were scraped into the culture medium and lysed by three cycles of freeze-thaw. Virus titer was determined by either plaque assay or TCID50 assay as described previously (6, 43).

Cytotoxicity assay. Tumor or normal cells were seeded in medium with 8% FBS at 104 per well in 96-well plates. Twenty-four or 48 hours later, equal volume of serum-free medium diluted viruses were added to the tumor and normal cells, respectively, to reach desired multiplicity of infection in medium with 4% FBS. Eight or 10 days postinfection, cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt cytotoxicity assay using CellTiter 96 AQueous Nonradioactive Cell Proliferation Assay kit from Promega (Madison, WI). Following the addition of the colorimetric tetrazolium reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, cells were incubated for 3 hours for color development. The absorbance values were measured at 490 nm using a microplate reader from Molecular Dynamics (Sunnyvale, CA). Absorbance values were expressed as a percentage of uninfected controls and plotted versus dose on a logarithmic scale. A sigmoidal dose-response curve was fit to the data and a LD50 value was calculated for each replicate using GraphPad Prism software version 3.0.

Clinical tissue biopsy ex vivo culture and viral infectivity analysis. An ex vivo culture system using clinically derived tumor or normal tissue biopsies was developed to evaluate tumor specificity of oncolytic adenoviruses. These tissue specimens were collected and processed by our collaborators at the Veterans Affairs Palo Alto Hospital under the institutional review board–approved procedure. All the clinical tissues were obtained with approval of the research ethics committees and with informed consent. The tissue specimens obtained from surgery were quickly placed on ice in Iscove's modified Dulbecco's medium with 10% FBS and 100 units/mL penicillin/streptomycin. Tissue samples were dissected on ice and homogeneous tissue slices were selected for the experiment. Tumor or normal sections (1-mm3 cubes) were rinsed and placed in six-well plates in Iscove's modified Dulbecco's medium with 5% FBS. For each tissue sample, CG5757 or Ad5 (1 x 109 plaque-forming units) was added to the well. The samples were incubated for 2 hours and the culture medium was replaced with fresh Iscove's modified Dulbecco's medium containing 5% FBS, 10 µmol/L insulin, and 1 µmol/L hydrocortisone. Tissue specimens were placed on Millicell membrane culture inserts (0.45-µm pore size) from Millipore (Billerica, MA) in six-well plates and incubated at 37°C with 5% CO2. The culture samples were subjected to immunohistochemical examination at 24 hours postinfection or the tissue specimen was harvested at day 5 postinfection for TCID50 determination after three freeze-thaw cycles.

Immunohistochemistry staining. For ex vivo cultured samples, viral infection was determined by immunohistochemical staining for adenoviral E1A proteins. In brief, the tissue sections were dewaxed in xylene and rehydrated in ethanol of decreasing concentrations. The sections were unmasked with target retrieval solution from DAKO (Ely, United Kingdom) followed by blocking of nonspecific binding with normal rabbit serum for 30 minutes. A rabbit monoclonal anti-E1A antibody purchased (Santa Cruz Biotechnology) was used at a dilution of 1:50 in PBS and incubated for 1 hour at 25°C. The sections were washed in PBS and then incubated with a biotin-conjugated rabbit anti-mouse antibody (DAKO) for 30 minutes and washed again. Tissue sections were treated with 0.03% hydrogen peroxide in methanol for 20 minutes, washed, and incubated with streptavidin-horseradish peroxidase complex (DAKO) for 30 minutes. After washing in PBS, the slides were developed using the substrate 3,3'-diaminobenzidine (DAKO) as a chromogen. After color development, the sections were counterstained with hematoxylin, dehydrated in increasing concentrations of ethanol, histocleared with xylene, and mounted for light microscopy analysis.

For xenograft tumors, virus-infected cells were determined by immunohistochemical staining for adenoviral hexon protein following a similar procedure with in-house developed mouse monoclonal antibody 13G9. These xenograft samples were also used to determine cell apoptosis using In situ Cell Death Detection kit (Roche) following the manufacturer's instructions.

In vivo studies. For preliminary toxicity evaluation, male CB-17 severe combined immunodeficient mice, at least 20 g in body weight, were grouped and dosed to achieve 8.5 x 1011 viral particles/kg based on weight. Viruses were i.v. given into the lateral tail vein. The vehicle control was PBS/10% glycerol. Clinical observations and evaluations of selected clinical pathology variables, including liver enzymes and blood count, were done on the collected samples.

Antitumor efficacy studies were conducted in nude mice with human tumor xenografts. Six- to 8-week-old NCR-nude mice were obtained from Simonson Laboratories (Gilroy, CA) and acclimated to laboratory conditions for 1 week before tumor implantation. Xenografts were established by s.c. injecting 1 million to 5 million A549, 253J B-V, or LNCaP cells suspended in 100 µL RPMI 1640 and an equal volume of Matrigel. When tumors reached 100 to 200 mm3, mice were randomized and dosed with 50 µL test article or vehicle control (PBS with 10% glycerol) by i.t. injection or at a volume of 100 µL by tail vein injection. Tumors were measured in two dimensions using digital calipers, and volumes were estimated by the formula: 0.35 x (length x width)1.5 (mm3; ref. 44). Animals were humanely killed when their tumor burden became excessive (>1,500 mm3). All animal studies were approved by the Institutional Animal Care and Use Committee.

Statistical analysis. Statistical analysis was done using unpaired, two-tailed Student's t test. In vivo tumor growth rate was also compared by linear regression analysis using the Prism GraphPad software.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Tumor-specific expression of viral genes in vitro. The promoters from the E2F-1 and hTERT genes were cloned by PCR and confirmed by sequencing. Tumor specificity of these promoters was validated using the luciferase reporter gene in a cell-based transient transfection system as described previously (7). Both E2F-1 and hTERT promoters drive a high level of luciferase expression in Rb-defective and hTERT-positive cancer cells but are silent in WT Rb- and hTERT-negative cells, including primary cells (data not shown), thereby showing that these promoters have strong tumor selectivity.

CG5757 was constructed by inserting the E2F-1 and hTERT promoters upstream of the E1a and E1b genes, respectively, and deleting the coding region of the E1b 19-kDa gene (Fig. 1A). To examine the tumor-specific expression of viral genes that are under the control of the E2F-1 and hTERT promoters, a Western blot was done to visualize E1A and E1B protein expression in a panel of human cells with different Rb and hTERT status. Among them, the isogenic WI-38 and VA-13 cells that have different Rb status were used to evaluate the Rb status–dependent expression of E1A during CG5757 infection. WI-38 is a normal human diploid fibroblast cell line with a WT Rb pathway, whereas VA-13 is a Rb-defective isogenic cell line due to constitutive SV40 large T antigen expression (4). Under the current experimental conditions, there was no difference in E1A expression between WI-38 and VA-13 cells infected with WT Ad5 by Western blot analysis (Fig. 1B), whereas only VA-13 cells specifically support the production of CG5757 E1A protein. Previously published data show that VA-13 cells up-regulate E2F-1 expression (4); therefore, activation of the E2F-1 promoter in VA-13 cells controls the expression of CG5757 E1a. This Rb status–dependent expression of E1a gene in CG5757-infected cells was further extended into a broad panel of cancerous and normal cells (Fig. 1D).



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  		Fig. 1. Tumor-selective expression of oncolytic adenovirus CG5757 E1 products evaluated by Western blots. A, schematic genome structure of WT Ad5 and recombinant CG5757 (not drawn to scale). Key modifications are illustrated and other areas remain unchanged from Ad5. ITR, adenoviral inverted terminal repeat; {Psi}, viral packaging signal; pE1a and pE1a, endogenous WT promoters for E1a and E1b genes, respectively. E1b 19 kDa was deleted in CG5757. CG5757 was compared with WT Ad5 for E1A (B) or E1B 55 kDa (C) expression in cells with different Rb and telomerase status. A broad panel of tumor and normal cells was also used to compare E1A expression from nonselective Ad5 and the tumor-specific CG5757 (D). C, uninfected cell lysate control; OV, oncolytic virus CG5757.

 
Similarly, expression of E1B 55 kDa by CG5757 was detectable only in hTERT-positive bladder transitional cell carcinoma 253J B-V cells but not in hTERT-negative human primary renal epithelial HRE cells (Fig. 1C). These data indicate that the E2F-1 and hTERT promoters remain tumor selective when inserted into adenoviral genome and that expression of the E1 genes in CG5757 is highly tumor specific.

Tumor-selective viral replication and cytotoxicity in vitro. During adenovirus infection, expression of the E1 genes is essential to activate other viral genes and for viral replication. Therefore, tumor-specific expression of E1 genes will consequently make the entire viral replication cycle tumor selective. To evaluate the replication specificity of CG5757, a panel of human tumor cells (Rb-defective and hTERT-positive) and normal cells (WT Rb pathway and hTERT-negative) were infected with either CG5757 or WT adenovirus and the yield of progeny virus was measured by plaque assay at 72 hours after infection. CG5757 replicated efficiently, producing comparable levels of progeny virus to Ad5 in the Rb pathway–defective and hTERT-positive tumor cells, including Hep3B, A549, LoVo, Panc1, HeLa, LNCaP, and 253J B-V (Fig. 2A). It replicated inefficiently, however, in normal cells with the WT Rb pathway and no hTERT expression, producing 100- to 10,000-fold less progeny virus than WT Ad5 (P < 0.05). The overall comparison of CG5757 production between tumor and normal cells also suggested that CG5757 had ~100- to 10,000-fold higher replication in tumor cells.



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  		Fig. 2. Selective replication and dose-response cytotoxicity curves of CG5757 in different cell types. A, after 3 days of infection, the mean ± SD virus yield of CG5757 is compared with WT Ad5 in a panel of tumor and normal cells. B, the dose-response cytotoxicity of CG5757 was compared with WT Ad5, Addl1520, and Addl312 in representative tumor and normal cells (Hep3B and HRE, respectively). 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay was done on day 8 and cell viability is expressed as a percentage of the uninfected control and fit to a sigmoidal dose-response curve using Prism GraphPad software. Points, mean of three replicates; bars, SD. PPC, viral particles per cell.

 
Cytotoxicity is a measure of the potency and tumor selectivity of oncolytic adenoviruses. The ability of CG5757 to kill both tumor cells and normal cells was evaluated using a quantitative 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay. Viral-induced cytotoxicity was determined as a percentage of the infected cells compared with uninfected control using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt data. The cytotoxicity profile of CG5757 was compared with that of the positive control WT Ad5, the E1b 55-kDa deleted mutant Addl1520, and the negative control replication-defective Addl312. The dose-response curves indicate the viability change in a hepatocellular carcinoma cell line, Hep3B, and a human primary renal epithelial cell, HRE, after infection with the different viruses (Fig. 2B). In Hep3B cells, CG5757 was slightly less cytotoxic than Ad5 but was much more cytotoxic than Addl1520, showing the increased cytolytic potency of the former. In human primary HRE cells, the cytotoxicity of CG5757 was much more attenuated than WT Ad5 or Addl1520. In fact, the LD50 of CG5757 shows that it is at least 1,000-fold less cytotoxic than Ad5 in HRE cells (Table 1) compared with a <10-fold difference between Addl1520 and Ad5 in these primary human cells.


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  		Table 1. LD50 values of CG5757 and WT Ad5 in tumor and normal cells (viral particles per cell)

 
LD50 values for CG5757 cytotoxicity were determined in a panel of human cells in comparison with Ad5 within the same cells to control for differences in infectivity. For most tumor cell lines tested, CG5757 has a higher LD50 value, suggesting that it is less potent than WT Ad5 (Table 1). However, the tumor cell selectivity of CG5757 is evident in the relative LD50 values (LD50 of Ad5 / LD50 of CG5757), in that the LD50 in all tumor cells was consistently higher than in primary cells. Consequently, the selectivity indices (SIpro) of cytotoxicity (relative LD50 in tumor cells / relative LD50 in normal cells) are always >1.

Tumor-selective gene expression and virus production in ex vivo primary tumor cultures. To further evaluate the tumor selectivity of CG5757, a newly developed ex vivo primary tumor culture system was used to determine viral gene expression and virus production. Human tumor or normal tissues were collected from patients who underwent surgical resection for colorectal or pancreatic cancer, providing biopsy specimens of colon cancer and normal colon, pancreas, and spleen tissue for use in the ex vivo culture and viral infection experiment. In general, the ex vivo cultures remained viable for at least 2 days as shown by H&E staining (data not shown). Selected tissue samples were processed 24 hours postinfection for immunohistochemical staining of viral E1a gene expression and others were used to determine viral productivity via TCID50 titration at 5 days postinfection.

The analysis of adenovirus E1A protein expression by immunohistochemical staining with anti-E1A antibody is shown in Fig. 3A. In CG5757-infected tissue, E1A is detectable only in colon tumor tissues but not in normal tissues, suggesting selective activity of CG5757 in primary colon tumors. However, E1A was detected in both tumor and normal tissues following WT Ad5 infection (data not shown).



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  		Fig. 3. Selective infection and virus production of CG5757 in clinically derived tissue samples. Viral-infected tumor or normal tissues samples were immunochemically stained for adenoviral E1A at 1 day postinfection (A) or titrated for viral production at 5 days postinfection (B). A, immunohistochemcial staining with rabbit monoclonal anti-E1A antibody for E1A expression. B, production of CG5757 and WT Ad5 in paired colon cancer and normal colon tissues from several patients. SIpro was calculated as indicated in the text; a SIpro above 1 indicates tumor selectivity.

 
Preferential production of CG5757 in tumor tissue was observed in paired tissue samples collected from five colon cancer patients (Fig. 3B). All of the paired tissue specimens contained tumor and normal tissues from the same patient. On average, the yield of CG5757 was at least 10-fold higher in tumor tissue than in adjacent normal tissue from the same patient. For example, tumor tissue from patient 39 yielded ~10,000-fold more infectious CG5757 than the normal tissue from the same patient. In contrast, there was no clear difference in virus yield between tumor and normal tissues from the same patient following infection with WT Ad5.

To determine tumor selectivity of virus production, the TCID50 of CG5757 was compared with that of Ad5 within the same pair of tissues to control for differences in infectivity and viral production. The SIpro was calculated as follows:

In this index, the nonselective WT Ad5 is used to normalize the infection efficiency for the oncolytic virus CG5757 in each paired patient sample. A value above 1 indicates selective production of CG5757 in tumor tissues. Data presented in Fig. 3B show that the SIpro values are all above 1. Collectively, both in vitro studies with human cells and ex vivo culture experiments with primary tissues show the high selectivity of CG5757 for tumor cells.

Toxicity in severe combined immunodeficient mice. The relative toxicity of i.v. given CG5757 was compared with that of WT Ad5 and a replication-defective Addl312 in a pilot study in severe combined immunodeficient mice. In this study, male mice were separately injected with CG5757, WT Ad5, or Addl312 at a dose level of 8.5 x 1011 viral particles/kg. No animal death was observed in the CG5757, Addl312, or vehicle treatment groups. In contrast, all of the mice treated with WT Ad5 were moribund by day 5 postinfection and were euthanized. Relative to day 1, there was no body weight gain in any treatment group between days 3 and 8 (Fig. 4A). Ad5-treated animals lost weight steadily between days 1 and 5, when they were euthanized. In contrast, the CG5757-treated group gained body weight after an initial loss between days 3 and 8. The difference in mean body weight for this group was not significant compared with the vehicle- or Addl312-treated groups (P > 0.6 on day 8 by t test).



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  		Fig. 4. Comparison of toxicity in severe combined immunodeficient mice following systemic administration of viruses. Severe combined immunodeficient mice were injected with a single i.v. dose (8.5 x 1011 viral particles/kg) of the indicated virus on day 1. A, change in body weight. Points, mean change in body weight expressed as a percentage of the day 1 body weight; bars, SD. B, serum chemistry at day 4 postadministration. Columns, mean alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK; n = 3) on day 4 in each treatment group; bars, SD. *, P < 0.05, compared with PBS-treated controls.

 
Selected clinical chemistry variables were monitored on days 2, 4, 14, and 28. During the study, creatine kinase levels were not different between any treatment group at any time point, suggesting no significant gross toxicity in skeletal muscle, cardiac muscle, or brain (data not shown). No differences in serum levels of alanine aminotransferase and aspartate aminotransferase were evident on day 2 between any treatment group (data not shown). However, significantly elevated aspartate aminotransferase and alanine aminotransferase levels (P < 0.05) were seen on day 4 in mice treated with WT Ad5 compared with control or Addl312 treatment groups (Fig. 4B). The CG5757 treatment group had a slight elevation of both alanine aminotransferase and aspartate aminotransferase on day 4, but the elevations were not significantly different than in vehicle- or Addl312-treated mice. By day 14, their serum enzymes returned to normal levels in the CG5757 treatment group (data not shown). Taken together, these observations suggest that CG5757 may be less toxic than WT Ad5.

Antitumor efficacy in animal models. The antitumor efficacy of i.t. administration of CG5757 was studied in the bladder transitional cell carcinoma 253J B-V and lung cancer A549 xenograft models in NCR-nude mice. In 253J B-V xenograft model, animals received i.t. injections of either vehicle or CG5757 (4 x 108 viral particles/mm3 of tumor) every 3 days for four injections. By 4 weeks after the first treatment, tumors in control animals had increased ~9.4-fold in size, whereas tumors in animals treated with CG5757 regressed to 72% of the original baseline size (Fig. 5A). Furthermore, 50% of the treated animals had complete regression of the 253J B-V tumor xenografts.



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  		Fig. 5. Antitumor efficacy of CG5757 in xenograft tumor models following local and systemic administration. Human bladder transitional cell carcinoma 253J B-V (A) and lung cancer A549 (B) cells were s.c. implanted into the flanks of female nude mice and treatment initiated on day 19 when tumors were ~100 and 160 mm3, respectively (n = 10). Four doses of virus (4 x 108 viral particles/mm3) or vehicle control (PBS with 10% glycerol) was i.t. injected into the tumor using the indicated regimen. In the human prostate cancer LNCaP model (C), treatment was started on day 30 when the s.c. tumors reached ~170 mm3 (n = 8). CG5757 was injected at a dose of 4 x 1010 viral particles per mouse through the tail vein (i.v.) twice with a 3-day interval. Points, mean tumor volume; bars, SE. D, CG5757-treated A549 tumors were harvested 60 days after the first treatment and processed for H&E and immunochemical staining for either adenoviral hexon or terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL). The areas in the box are further examined at higher magnification.

 
In A549 xenograft model, a different i.t. delivery regimen was applied in which vehicle or CG5757 (4 x 108 viral particles/mm3 of tumor) was injected daily for 4 consecutive days (Fig. 5B). Significant antitumor efficacy was shown after viral treatment (P < 0.0001). Additionally, in vivo viral replication and viral-induced apoptosis were observed in CG5757-treated A549 xenografts. In this study, A549 tumors were harvested 60 days following four consecutive CG5757 treatments and subjected to immunohistochemical staining for Ad5 hexon as a marker for viral replication and for terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining to detect apoptotic cells. The data shown in Fig. 5D are indicative of viral replication within the tumor at sites where active necrosis or apoptosis is under way. The close colocalization of viral replication, necrosis, and apoptosis further supports the rationale that replication of oncolytic viruses leads to tumor oncolysis.

The systemic antitumor efficacy of CG5757 was also evaluated in the LNCaP prostate cancer tumor xenograft model following i.v. administration (Fig. 5C). NCR-nude mice bearing s.c. LNCaP xenografts (average volume, 170 mm3) on the back were injected i.v. via the tail vein with either vehicle (PBS with 10% glycerol) or with the same volume of CG5757 (4 x 1010 viral particles per mouse) on days 1 and 4. Six weeks after treatment, tumors in CG5757-treated animals increased in volume at a rate of 7.6 mm3/d compared with 25.3 mm3/d for the vehicle-treated animals, representing a reduction in tumor growth rate of ~72% (P < 0.05).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we describe the use of tumor-selective promoters from the E2F-1 and hTERT genes to control the adenoviral E1a and E1b genes, respectively, and create a potentially broad-spectrum anticancer virus, CG5757. Both of these promoters have been used as single controlling elements in other oncolytic adenoviruses (4, 30, 45) and also in two dual promoter–controlled viruses, one in which E2F-1 controls the E1a gene and the hTERT promoter drives the E4 gene (18) and the other in which dual E2F-1 promoters independently control the E1 and E4 transcription units (46). Both of these engineered adenoviruses have proven to be genetically unstable, leading to the formation of recombinant viruses of differing selectivity and potency during viral replication (47, 48). To address the problem of genetic instability, these two promoters were used to control E1a and E1b genes, as in CG7870 (8), a prostate-specific oncolytic adenovirus using two heterologous promoters control these two genes, which have been proven to be genetically stable (49). In our study, CG5757 also exhibits acceptable stability.3

Another advantage of dual heterologous promoters in CG5757 is the improvement of anticancer selectivity. The incorporation of a second prostate-specific promoter in CG7870 was shown to result in a significant enhancement in target cell specificity relative to a single transcriptional response element variant (8). Similarly, when hTERT is used as a secondary control on E1b gene, the derived virus also shows higher tumor selectivity compared with the virus with only one transcriptional response element control on E1a gene (data not shown). Therefore, the dual promoter–controlled oncolytic adenovirus, CG5757, is expected to have improved tumor specificity in the majority of human cancers that have a defect in the Rb pathway and up-regulated telomerase expression.

During adenovirus infection, expression of the E1 genes is essential to activate other viral genes and, by extension, viral replication. Therefore, transcriptional control of the E1 genes by tumor-specific promoters will consequently cause viral replication to be tumor specific. However, the amount of virus produced reflects numerous processes, including the ability of a particular cell type to be infected, to transactivate promoters, and to allow replication of the virus, and thus provides a good measure of the selectivity of a targeted adenovirus. The promoters engineered into CG5757 control E1 expression and are highly tumor selective, and E1 expression correlates closely with the status of both cellular Rb pathway and hTERT expression in the cells. In our study, high levels of E1A expression are detected in tumor cells that have a defect in the Rb pathway, and CG5757 produces a similar amount of progeny virus in these cells as WT Ad5. As expected from the specificity of its promoters, CG5757 produces 100- to 10,000-fold more progeny virus in tumor cells than in normal human cells. To evaluate its specificity by viral cytotoxicity result, CG5757 kills more tumor cells than normal cells. The relative LD50 values in the tumor cells are consistently higher than in the normal cells, suggesting that the SIpro of CG5757 are all above 1. The SIpro measures the ratio of the toxic effect to the desired effect of a specific agent. For oncolytic virus, SIpro above 1 indicates viral selectivity to the tumor cells compared with normal cells.

Evaluation of CG5757 in an ex vivo culture model of a primary tumor tissue similar to one recently used to evaluate nontranscriptionally controlled adenoviruses (50) confirmed the selectivity of CG5757 for tumor tissue. This model avoids the limitations inherent in using serial-passaged tumor cell lines to evaluate the virus by using primary human tumor cells. The ex vivo model also preserves the characteristics of a three-dimensional tumor with its native tumor matrix and structure, and it may be the closest model of in vivo tumor conditions available. In this study, only tumor cells were infected by CG5757, with no evident infection of normal colon, pancreas, or spleen tissue samples from the same patient. It is possible that this tumor specificity was mediated by a higher relative infectivity of cancer cells by CG5757 than WT Ad5 as reported previously in a study comparing tumor infectivity by WT Ad5 and an E1A-CR2 oncolytic mutant dl922-947 (50). However, more progeny CG5757 virus was produced in colon tumor tissue than in patient-matched normal colon tissue, whereas no significant difference in the amount of progeny WT Ad5 was detected in the tumor and normal tissue in the same samples, showing the preferential replication of the oncolytic adenovirus in tumors.

The selective tumor targeting of CG5757 was also evident in vivo. The virus induced significant less systemic and hepatic toxicity than WT Ad5 in severe combined immunodeficient mice, a species well established as a tool for evaluating the toxicity of oncolytic adenoviruses (4, 18). Therefore, the use of tumor-specific promoters to control E1 expression is essential to reduce virus-related toxicity in vivo. In addition, CG5757 had a good safety profile in tumor-bearing nude mice as measured by body weight change after i.t. doses as high as 2 x 1013 viral particles/kg and i.v. doses as high as 4 x 1012 viral particles/kg. CG5757 also exhibited significantly improved antitumor activity when given by different administration routes in several mouse xenograft tumor systems, including bladder cancer, lung cancer, and prostate cancer models. We also compared the in vivo efficacy profiles between single promoter (E2F-1) and dual promoter–controlled viruses in different tumor models; no significant difference can be identified (data not shown). However, dual promoter–controlled virus is expected to have higher selectivity to tumor cells and less toxicity to normal cells.

In conclusion, CG5757, which uses the E2F-1 and hTERT promoters to control E1a and E1b gene expression, shows enhanced tumor specificity and reduced toxicity both in vitro and in vivo. CG5757 may therefore have the potential to be an efficacious therapeutic for the treatment of a broad range of human cancers.


   Acknowledgments
 
We thank Drs. Seshidhar Police, Nagarajan Ramesh, Michael Robinson, and Gary Lee for critical discussion and review of this article, Ginny Rojas and Melissa Gonzalez for histology study, Amanda Koehne, Sheila Ulafatu, Tammy Langer, and Jason Ho for in vivo studies, and Joseph Wypych and Mebratu Gebreyesus for virus production.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: T. Reid is currently at the University of California, San Diego, CA.

3 N. Idamakanti and Y. Li, unpublished data. Back

Received 8/10/05; revised 9/12/05; accepted 9/30/05.


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