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Journal of Virology, February 2003, p. 2640-2650, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2640-2650.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Developing Novel Oncolytic Adenoviruses through Bioselection

Wen Yan, Galila Kitzes, Farid Dormishian, Lynda Hawkins,{dagger} Adam Sampson-Johannes, Josh Watanabe, Jenny Holt, Vivian Lee, Thomas Dubensky,{ddagger} Ali Fattaey, Terry Hermiston,§ Allan Balmain,|| and Yuqiao Shen*

ONYX Pharmaceuticals, Inc., Richmond, California 94806

Received 23 August 2002/ Accepted 12 November 2002


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ABSTRACT
 
Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed.


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INTRODUCTION
 
Conditionally replicating viruses represent a promising new class of anticancer agents (2, 8, 21, 23, 24, 29). Derivatives of human adenovirus type 5 (Ad5) have been developed that selectively replicate in and kill cancer cells. The prototype of such viruses, ONYX-015, has demonstrated clinical benefit for cancer patients, particularly when combined with chemotherapy, in several phase I and phase II clinical trials to treat recurrent cancers of the head and neck or liver metastatic disease (7, 16, 20, 22, 31, 34, 35). ONYX-015 is currently being tested in placebo-controlled phase III clinical trials.

ONYX-015 replication has been demonstrated to occur selectively in tumor tissues from patient biopsy samples, and the virus has been well tolerated when directly injected into the tumor mass, administered via the hepatic artery, or injected intravenously. Based on the experience from clinical trials with ONYX-015, enhanced replication and/or spread is highly desired of adenovirus cancer therapy strategies (14, 16, 20, 22, 33-35). While no dose-limiting toxicities have been observed in clinical trials with ONYX-015, and the response rates have been encouraging, the extent of virus replication and spread was often limited in tumor tissues. A more-potent virus would presumably increase the efficacy of the oncolytic adenoviral therapy and/or the durability of the responses. One strategy to enhance the clinical efficacy is to combine ONYX-015 with other therapies, e.g., standard chemotherapy (16, 20). An extension of such combination therapies is to arm the oncolytic adenoviruses with anticancer genes (17), such as antiangiogenesis factors, cytotoxic agents, prodrug converting enzymes, or cytokines with antitumor activity. Selective replication of the viruses in tumor cells allows targeted expression of these gene products within the cancer environment. Meanwhile, it would be highly desirable to enhance the therapeutic virus' inherent replication efficacy such that the virus would replicate faster or produce more viral progeny per unit of time. Conceivably, this would allow the therapeutic viruses to kill cancer cells more rapidly and spread more extensively within tumors, leading to efficient eradication of the cancer. Another highly desired feature of cancer therapeutic viruses is enhanced tissue and/or cell type specificity; such features would, potentially, confer systemic efficacy on viruses, allowing for specific tissue and/or cell targeting after systemic administration.

Two general approaches have been undertaken to develop novel adenoviruses with desired features. The first is targeted genetic manipulation, in which designated viral genes have been deleted or modified, therapeutic transgenes have been inserted, or strong, tissue-type selective promoters have been substituted for endogenous viral regulatory sequences. Although these approaches have been successfully utilized to construct many novel viruses (for examples, see references 13, 18, 19, 30, 42, and 51), their application has been limited by the requirement for a complete understanding of the biology of the virus life cycle. Even in the case of Ad5, one of the most extensively studied viruses, such information is not always available or complete. Thus, the consequences of targeted genetic manipulations are in many cases unpredictable. The second general approach to generate viruses with desired properties is genetic selection under carefully controlled conditions. Viruses selected in this fashion preferentially replicate and reproduce under a predefined set of conditions (for examples, see references 4-6, 9, 38, and 46). In essence, this is an evolution process occurring under carefully controlled conditions in the laboratory.

In this study, we exploited a genetic selection approach to isolate mutants of Ad5 that replicate more efficiently in tumor cells than the parental strain. Two such viruses, ONYX-201 and -203, were derived, and their genetic makeup was analyzed in detail. This study provided a clear proof of concept for the genetic selection approach, which we have termed bioselection, for developing novel oncolytic viruses with enhanced potency and resulted in two mutant viruses that may have important clinical implications as oncolytic agents.


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MATERIALS AND METHODS
 
Cells and viruses. All human cancer cell lines were obtained from the American Type Culture Collection and were propagated as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 µg of nonessential amino acids (NEAA)/ml, 10 U of penicillin/ml, and 10 µg of streptomycin/ml. Primary human normal cells were obtained from Clonetics Corp. and were propagated under conditions recommended by the manufacturer. Wild-type Ad5 was obtained from the American Type Culture Collection and propagated in 293 cells. ONYX-201 and -203 and their derivatives were propagated in HT29 cells until the last proliferation step, for which they were grown in 293 cells. All viruses were purified by the CsCl gradient banding method and titrated by plaque assay on 293 cells. In several cases plaque assays were performed on HT29 cells as well as on 293 cells, and the results from both were consistent. Infections of cancer cells were performed in DMEM supplemented with 2% FBS, 2 mM L-glutamine, 100 µg of NEAA/ml, 10 U of penicillin/ml, and 10 µg of streptomycin/ml. Infections of normal cells were performed in their recommended growth media.

Random mutagenesis. Random mutagenesis of Ad5 with nitrous acid was performed as described previously (12, 25, 39, 50). Briefly, wild-type Ad5 was treated with 0.7 M NaNO2 in 1 M acetate buffer, pH 4.6. The reaction was terminated at various time points by the addition of 4 volumes of ice-cold 1 M Tris-Cl (pH 7.9). The virus was then dialyzed overnight against 20 mM phosphate-buffered saline (PBS) (pH 7.2)-10% glycerol and stored at -80°C. The infectivity of the treated virus was examined by plaque assay on 293 cells.

Bioselection. Mutagenized Ad5 was passaged repeatedly on selected human cancer cell lines representing various human cancers. In all cases, infections were carried out in T-185 tissue culture flasks containing approximately 107 adherent cells in 25 ml of culture medium (2% FBS). For the first round of passaging, cells were infected at a multiplicity of infection (MOI) of 1. Tissue culture media were harvested at the very initial sign of visible cytopathic effect (CPE). In subsequent passages, 1, 0.1, or 0.01 ml of the harvested media from the previous passage was used as the inoculum. Cultures that began to reveal CPE at 3 to 5 days postinoculation were considered effective, and media were collected at the initial sign of CPE. This strategy allowed us to avoid infection with too many virus particles, which may reduce the effectiveness of bioselection, or too little virus, which would reduce the complexity of the viral population. Passaging was carried out for 6 to 20 rounds, depending on the cell lines used for selection.

Cytolytic assay. Viral cytolytic activities were examined by using the MTT assay as described previously (43). Briefly, cells were seeded into 96-well plates at a density of 3,000 cells per well in the appropriate growth media. Infections were performed at 24 h after seeding. In most cases, infections were carried out in quadruplicate with serial threefold dilutions of the viruses. A total of 10 dilutions were used for each virus, starting at an MOI of 30 and ending at an MOI of 1.5 x 10-3. Some of the MTT assays with primary human cells (see Fig. 6) had a starting MOI of 10 and an ending point at 5 x 10-4. Cytolytic assays described in the legend to Fig. 3 were conducted in triplicate at MOIs of 10, 1, 0.1, and 0.01. Infected cells were incubated at 37°C, and colorimetric reactions were performed at the indicated time points by using the CellTiter 96 nonradioactive cell proliferation assay (Promega) according to the manufacturer's instructions. Cells that were mock infected were used as negative controls and set as the reference (100% survival).



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  		FIG. 6. Cytolytic activity in primary normal human cells. Cells were grown in 96-well dishes and were infected with Ad5 and ONYX-201 and -203 in serial threefold dilutions. (A) Quiescent MEC. (B) Quiescent SAEC. (C) Proliferating MVEC. Similar experiments were carried out multiple times in various primary normal human cells (proliferating or nonproliferating). Similar results were obtained; only three representative experiments are shown here. (D). Relative cytotoxicity in matched tumor and normal cells. MTT assays were performed on primary MEC and a mammary cancer cell line, MB468, with Ad5 and ONYX-201, -203, and -015. The 50% inhibitory concentration (IC50) was defined as that MOI which resulted in 50% cell killing. These values were then plotted relative to that of Ad5 for each virus as follows: IC50 of Ad5/IC50 of test virus. Therefore, the relative activity of Ad5 in normal and tumor cells is 1. The height of the bars represents the cytotoxicity compared to Ad5, and the difference between the striped bar and the stippled bar indicates the selectivity.



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  		FIG. 3. Kinetics of HT29 cytotoxicity. HT29 cells were infected with Ad5, ONYX-201, and ONYX-203 at various MOIs. At different time points postinfection the percentage of viable cells was assessed by MTT assay and plotted versus time.

Virus production assays. HT29 cells were seeded in 24-well dishes at 4 x 104 cells per well. After attachment, cells were infected with Ad5 and ONYX-201 and -203 at the indicated MOIs. After an incubation period of 90 min, nonabsorbed viruses were removed by aspiration and cells were washed once with PBS. Infected cells were then incubated at 37°C in DMEM supplemented with 2 mM L-glutamine, 100 µg of NEAA/ml, 10 U of penicillin/ml, 10 µg of streptomycin/ml, and 2% FBS. At the indicated time points after infection, cells and culture media were harvested separately. Cell fractions were freeze-thawed three times to release virus particles, and lysates were cleared by centrifugation. The total virus yield (cell and medium combined) was determined by plaque assay on 293 cell monolayers.

Viral DNA replication. HT29 cells were infected with Ad5 and ONYX-201 and -203 at various MOIs. At the indicated times postinfection, cells and culture media were harvested and DNAs were extracted from combined cell and medium fractions with the Blood DNA Extraction kit (Qiagen). DNAs were digested to completion with HindIII, and digested DNAs were resolved on 0.8% agarose gels. After Southern transfer, DNA blots were hybridized with probes prepared with the digoxigenin High Prime DNA labeling kit (Roche Biochemicals). Purified Ad5 genomic DNA was used as a template for probe synthesis.

Western blot analysis. HT29 cells were either mock infected or infected with Ad5 and ONYX-201 and -203 at various MOIs. At indicated times postinfection, cells were harvested and lysed in sodium dodecyl sulfate (SDS) gel loading buffer (100 mM Tris-Cl [pH 6.8], 5 mM EDTA, 1% SDS, 5% ß-mercaptoethanol). Proteins were fractionated by electrophoresis on 4 to 20% protein gels (Bio-Rad). After electrophoresis, the proteins were electrophoretically transferred to nylon membranes. Blots were then incubated with antibodies diluted in PBS containing 1% dry milk and 0.1% Tween 20 and visualized by enhanced chemiluminescence (Amersham). Anti-E1A antibody M73 (Calbiochem) was diluted 1:500; a polyclonal rabbit anti-Ad5 (structural protein) antibody was used at a dilution of 1:10,000. A polyclonal antibody against the i-leader protein (a generous gift from M. Green, St. Louis University) was used at a dilution of 1:1,000.

DNA sequencing. Genomic DNAs of ONYX-201 and -203 were purified from CsCl gradient-banded virus particles. Briefly, virus particles were lysed by incubation at 37°C in 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.6% SDS, and 1.5 mg of pronase (Sigma)/ml. Lysed particles were extracted twice with phenol-chloroform, and DNA was precipitated with ethanol. The genome of ONYX-201 was sequenced by Lark Technologies, Inc., Houston, Tex. The genomic DNA of ONYX-203 was sequenced at Onyx by using an automated sequencer (CEQ2000XL; Beckman).

Construction of recombinant viruses. Genomic DNAs of Ad5 and ONYX-201 and -203 were purified from CsCl gradient-banded virus particles. For construction of ONYX-211 and -212, genomic DNAs from Ad5 and ONYX-201 were both digested to completion with SpeI, which cuts only once (Ad5 nucleotide no. 27075) within the viral genome. Digested DNAs were mixed in equal amounts and ligated in the presence of T4 DNA ligase at room temperature overnight. This ligation mixture was then transfected into 293 cells with the FuGene reagent (Promega). Plaques derived from this transfection were isolated and screened by DNA sequencing. Proper clones were isolated by an additional round of plaque purification. ONYX-231 through -236 were constructed in a similar fashion, except that DNAs from Ad5 and ONYX-203 were digested with PmeI, BamHI, or SpeI, respectively (see Fig. 7). All recombinant viruses were confirmed by sequencing the relevant regions surrounding the mutation sites in ONYX-201 and -203.



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  		FIG. 7. (A) Recombination schemes. Various recombinant viruses were constructed as described in Materials and Methods. The exclamation marks indicate mutations present in each recombinant virus. Restriction sites for PmeI, BamHI, and SpeI are indicated on the viralgenomes. (B) Cytolytic activity of the recombinant viruses was examined in HT29 cells by using MTT assays. (C) ONYX-201 and -203 encode the truncated i-leader protein. HT29 cells were infected at an MOI of 10. Expression of the i-leader proteins was examined by immunoblot analysis. Mock, mock-infected cells. (D) Accumulation of the i-leader protein. HT29 cells grown on chamber slides were infected with Ad5 and ONYX-201 and -203 at an MOI of 10. At 24 h postinfection, the cells were fixed, permeabilized, and analyzed by dual immunofluorescent staining with M73 (a monoclonal antibody against E1A) (green) and a polyclonal antibody against the i-leader protein (red). Representative fields are shown.

Dual immunofluorescent staining. HT29 cells were seeded on Lab-Tek II chamber slides the day before infection. Cells were infected at an MOI of 10 with ONYX-201 or -203 or wild-type Ad5. Twenty-four hours postinfection, cells were washed with PBS and fixed for 30 min at room temperature with 4% formaldehyde in PBS. Cells were permeabilized and blocked with PBS supplemented with 0.1% Triton X-100, 0.05% Tween 20, and 10% goat serum for 30 min at room temperature. Samples were incubated with the anti-E1A monoclonal antibody M73 (1:1,000; Calbiochem) and the anti-i-leader protein polyclonal antibody (1:1,000), followed by Alexa 594-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse secondary antibodies (Molecular Probes).


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RESULTS
 
Adenovirus mutagenesis. To generate a genetically diverse virus population for bioselection, wild-type Ad5 was mutagenized by treatment with the chemical mutagen nitrous acid (NaNO2). Incubation with NaNO2 for 6 min at room temperature dramatically reduced the viability of Ad5 by a factor of 2 x 103 (Fig. 1A), which was consistent with earlier observations (12, 50). To estimate the mutation frequency resulting from this treatment, we isolated 22 individual plaques formed on A549 cell monolayers infected with this virus population, purified the viral DNAs, and amplified a 340-bp fragment within the E1A region by PCR for sequence analysis. Of the 22 individual virus isolates analyzed, two contained a single-base-pair mutation within the amplified region. We therefore estimated that mutations occurred at a frequency of 2.7 x 10-4 (2 mutations/22 x 340 nucleotides sequenced), or an average of 10 single point mutations per 36-kb viral genome. This calculation was based on the assumption that mutations occurred evenly along the virus genome. This number might have been an underestimation as the region analyzed (E1A) is essential for virus replication. Nevertheless, it appears that this estimation was consistent with the actual number of mutations identified in the two viral genomes we have fully deciphered through sequence analysis (see below).



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  		FIG. 1. (A) Wild-type Ad5 was mutagenized by treatment with NaNO2. Infectivity of the treated virus was examined by plaque assay on 293 cells and plotted as a function of incubation time with NaNO2. (B) Representative plaque morphology on an HT29 cell monolayer 5 days postinfection with wild-type (Wt) Ad5 or bioselection virus. (C) Microscopic view (magnification, x40) of representative plaques formed on HT29 cells by Ad5 or ONYX-201.

Bioselection. To isolate viral variants with enhanced oncolytic activity, mutagenized Ad5 was serially passaged in parallel in a number of human cancer cell lines of diverse tissue origin, as described in detail in Materials and Methods. During each passage cycle, medium from infected monolayer cultures was harvested at the first sign of CPE and used to inoculate the next fresh monolayer culture of the same cell line. This strategy was adopted to enrich the selection of viruses with enhanced replication and spread phenotypes. Depending upon the cell line used, the passaging procedure was carried out between 6 and 20 cycles.

To test the effectiveness of this bioselection protocol, we performed a proof-of-concept experiment. Two viruses, wild-type Ad5 and LGM, a derivative of Ad5 that contains the green fluorescent protein gene in place of the E1B-55,000-molecular-weight protein (E1B-55K protein) gene, were mixed at a PFU ratio of 1:1 and passaged on monolayers of U2OS human osteosarcoma cells by using the protocol described above. It has been shown previously that adenovirus mutants defective in the E1B-55K gene grow poorly in U2OS cells relative to wild-type Ad5 (7, 15, 41). After each passage cycle, culture medium was harvested and the relative abundance of the two viruses was measured by Southern blot analysis (data not shown). After 3 passages on U2OS cells, Ad5 clearly became the dominant species, constituting >95% of the total viral DNA detected. By passage 5 there was no detectable LGM in the culture medium. This experiment demonstrated that the passaging protocol used in this study could enrich for viruses with a selective advantage for replication and spread.

Characterization of bioselected viruses. In our studies, the human colorectal cancer cell line HT29 was relatively resistant to infection by Ad5, usually taking more than 4 days to develop significant CPE, even at an MOI of 10. In contrast, with each cycle of serial passage in HT29 cells, the harvested virus pool progressively demonstrated an improvement in its cytolytic capacity. Due to the enhanced cytolytic phenotype, this virus population, henceforth referred to as VHT29, was characterized following 19 serial passages in HT29 cells. The VHT29 pool was first analyzed by plaque assay on eight tumor cell lines: HT29 (colon), A549 and H2009 (lung), DU145 and PC-3 (prostate), MB231 (breast), Panc-1 (pancreas), and Hlac (head and neck) as well as HEK-293 cells. Wild-type Ad5 was used as a control. Approximately 50% of the total plaques formed by VHT29 on the HT29 monolayers were exceptionally large (3 to 5 mm in diameter after 7 days) compared to the plaques formed by Ad5 (<2 mm in diameter).

Twenty large plaques from the VHT29 pool were isolated for further characterization. Plaque preparations were propagated in HT29 cells and reexamined by plaque assay on HT29 cells. Two of the plaque-purified virus isolates, ONYX-201 and -203, were selected for further analysis. Each virus produced uniformly large plaques on an HT29 cell monolayer compared to Ad5 (Fig. 1B and C). To further characterize the relative replication efficacy of ONYX-201 and -203, HT29 cells were infected either with these viruses or with Ad5 at an MOI of 10. Cells infected with ONYX-201 and -203 showed CPE much more rapidly than cells infected with Ad5 (Fig. 2A), with ONYX-201 consistently demonstrating higher cytolytic activity than ONYX-203. Additionally, the morphology of HT29 cells infected with ONYX-201 and -203 was distinct from that of cells infected with Ad5. ONYX-201- and -203-infected cells were well separated from one another and cells were swollen and had a smooth surface, whereas Ad5-infected cells clumped together, displaying a classical grapevine-like shape characteristic of Ad5 infection.



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  		FIG. 2. (A) CPE of HT29 cells either mock infected (Mock) or infected (at an MOI of 10) with wild-type Ad5 and ONYX-201 and -203. Pictures were taken 3 days postinfection. (B) Cytolytic activity in HT29 cells was examined by using MTT assays. HT29 cells were infected with serial threefold dilutions of various viruses, with MOIs ranging from 30 to 1.5 x 10-3. MTT assays were performed 5 days after infection as described. Results from one of the many independent experiments are shown here. Similar results were obtained in other experiments.

The cytolytic activities of ONYX-201 and -203 and Ad5 were compared by a cell viability assay (MTT assay) (Fig. 2B). As defined by the MOI producing 50% cytolysis of the culture at 5 days postinfection, ONYX-201 was consistently 300- to 1,000-fold more active than Ad5, whereas ONYX-203 was approximately 30- to 50-fold more active than Ad5. The cytotoxicity of the VHT29 virus pool was between those of ONYX-201 and ONYX-203 (Fig. 2B), consistent with the fact that VHT29 was a mixture of viral mutants with various degrees of cytotoxicity.

The difference in cytolysis between bioselected viruses and the wild-type Ad5 was unlikely to be due to inaccurate assessment of virus titers. Ad5, ONYX-201 and -203 and VHT29 were titrated both in HT29 cells and in 293 cells, and the results were consistent in both cell lines with all viruses tested (data not shown). In addition, by fluorescence-activated cell sorter analyses and immunofluorescent staining studies of cells that were infected at a low MOI (0.01), the fraction of cells positive for the viral early protein E1A at early times postinfection was similar for Ad5 and ONYX-201 and -203 (data not shown). The particle-to-infectious unit (particle:PFU) ratios for these viruses were similar and ranged from 10 to 50 (data not shown).

Kinetics of cell lysis and viral replication. We next examined the progression of HT29 cytolysis as a function of time and MOI. At an MOI of 10, ONYX-201 lysed cells more rapidly than ONYX-203, which killed cells more efficiently than Ad5 (Fig. 3). The cytolytic differences between the viruses were even more pronounced at lower MOIs. At MOIs of 0.1 and 0.01, cells infected with Ad5 showed no clear signs of CPE after 6 days with minimal change in cell viability. In distinct contrast, cells infected with ONYX-201 and -203 at the same MOIs displayed unambiguous CPE between days 3 and 4, and cell viability dropped dramatically (Fig. 3).

The enhanced cytolysis observed with ONYX-201 and -203 may result from a number of possible mechanisms, including more-efficient cell entry, faster rate of replication, larger progeny yield per cell, and more-efficient release and spread. To begin to explore the mechanism(s) for the distinct phenotypes of ONYX-201 and -203, we examined the kinetics of viral progeny production, DNA replication, and early as well as late gene expression at MOIs ranging from 0.01 to 10. Immunofluorescent staining studies indicated that Ad5 and ONYX-201 and -203 were indistinguishable with respect to infectivity, as the number of E1A-expressing cells at early times postinfection was similar after infection with equivalent MOIs of each virus (data not shown). When analyzed after infection at an MOI of 10, at which the majority of the cells were infected, the total yields of ONYX-201 and -203 and Ad5 virus progeny were similar (Fig. 4A). However, it took Ad5 considerably longer to reach the maximum virus progeny yield (5 versus 3 days for ONYX-201 and -203) (Fig. 4A). This result suggested that the total number of virus progeny produced per cell was similar but that the rates of production were significantly different. Indeed, viral DNA replication occurred more rapidly (Fig. 4B) and viral gene products accumulated faster (Fig. 4C) for ONYX-201 and -203 than for Ad5. These observations supported the hypothesis that ONYX-201 and -203 replicate more rapidly than wild-type Ad5. It is unclear why E1A disappeared in the ONYX-201- and -203-infected cells at 3 and 4 days postinfection. We speculate that, due to highly efficient virus DNA replication, these cells rapidly entered the late phase, at which time E1A ceased to be expressed. At lower MOIs, the differences between ONYX-201 and -203 and Ad5 with respect to progeny production, DNA replication, and accumulation of viral proteins was more pronounced (Fig. 4), consistent with the cytolysis results (Fig. 3). This can be attributed to the fact that at low MOIs (MOI < 1) multiple rounds of infection occurred, and the difference in the replication rates between the viruses was amplified through each virus production cycle.



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  		FIG. 4. HT29 cells were infected at MOIs of 10, 1, 0.1, and 0.01. At different time points after infection, cells and culture media were collected. (A) Virus yields. HT29 cells (4 x 104) were infected. Total virus yields (cells and culture media combined) were determined by plaque assay on 293 cells. (B) Viral DNA replication. DNAs were extracted by using the Blood DNA Extraction kit (Qiagen), digested with HindIII, and resolved on 0.8% agarose gels. After Southern transfer, the blots were hybridized with probes prepared with the digoxigenin High Prime DNA labeling kit (Roche Biochemicals). Ad5 genomic DNA served as a template for probe synthesis. (C) Viral gene expression. Cell extracts were prepared at various days postinfection (dpi) and separated by SDS-polyacrylamide gel electrophoresis. The expression of E1A and viral late proteins (structural protein) was examined by Western blot analysis. M, mock infected.

Cytotoxicity in diverse human cancer cells. To assess whether the enhanced cytolytic activity of ONYX-201 and -203 was restricted to HT29 cells, we examined their cell killing activity in a number of human cancer cell lines. Six tumor cell lines derived from human colorectal cancers, HT29, HCT116, CCL221, RKO, SW480, and SW620, and 6 cell lines of diverse tissue origins, A549 (lung), DU145 (prostate), MB231 (breast), Panc-1 (pancreas), U2OS (osteosarcoma), and 293 (transformed human embryonic kidney cells), were tested by MTT assay. Results from a representative experiment are shown in Fig. 5. ONYX-201 and -203 and VHT29 displayed a significantly higher cytolytic activity than Ad5 in HT29 cells, consistent with the results shown in Fig. 2. Additionally, the bioselected viruses showed substantially higher cytolytic activity than Ad5 in a number of, but not all, other cancer cell lines. As shown in Fig. 5, in A549 and in HCT116 cells, ONYX-201 and -203 and VHT29 were significantly more potent in cell killing than Ad5, whereas in DU145 and Panc-1 cells, their differences were marginal. It should be noted that in all cell lines tested, ONYX-201 was more active than Ad5. Interestingly, VHT29 was significantly more active in killing some cell types (for example, SW480 and SW620) than Ad5 or even ONYX-201 and -203. This observation suggests that the VHT29 pool may contain viral variants that replicate efficiently in these tumor cells. We conclude that the viruses selected on HT29 cells following chemical mutagenesis and serial passage accumulated mutations that promoted efficient replication in cancer cell lines of diverse tissue origin as well as in other cell lines derived from colon cancers.



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  		FIG. 5. Cytolytic activity in various tumor cells. Tumor cells were seeded in 96-well dishes at a density of 3,000 cells/well. Twenty-four hours after seeding, they were infected with Ad5, ONYX-201, ONYX-203, or the virus pool that was passaged in HT29 cells (VHT29). Infections were conducted by using serial threefold dilutions of each virus, starting from an MOI of 30. Results from one of the three independent experiments are shown here. Similar results were obtained in other experiments. MOIs are indicated on the x axes. Percentages of cell survival are indicated on the y axes.

Cytolytic activity in normal cells. Our goal is to generate and develop selectively replicating viruses as cancer therapeutic agents. One measure of the therapeutic index of oncolytic adenoviruses is the ratio of productive replication in cancer cells compared to normal cells. To determine whether the bioselection procedure affected virus replication selectivity for cancer versus normal cells, the cytotoxicity of ONYX-201 and -203 in primary human normal cells was examined by MTT assay. In all of the primary cell types tested, which included small airway epithelial cells (SAEC), prostate epithelial cells, mammary epithelial cells (MEC), renal epithelial cells, human umbilical vascular endothelial cells, and microvascular endothelial cells (MVEC), whether proliferating or contact inhibited, the replication properties of ONYX-201 and -203 were similar to those of Ad5. In general, ONYX-201 was only slightly more active than Ad5, and ONYX-203 was slightly attenuated compared to Ad5. Representative results from SAEC, MEC, and MVEC are shown in Fig. 6. The selectivity of each virus was assessed by comparing its cytotoxicity in normal primary MEC with that in MB468, a mammary cancer cell line. The cytolytic activity of each virus in MB468 cells and MEC was normalized to that of Ad5 and plotted as represented in Fig. 6D. ONYX-201 and -203 demonstrated a tumor-to-normal selectivity similar to that of ONYX-015, indicating that all three viruses preferentially killed tumor cells rather than normal cells. Importantly, ONYX-201 and -203 were substantially more potent in tumor cells than either ONYX-015 or Ad5. In conclusion, these bioselected viruses obtained increased tumor cell selectivity as well as enhanced cytotoxicity compared to their parent, Ad5.

Mutation mapping. To understand the underlying genetic basis for the increased cytotoxicity of ONYX-201 and -203 in tumor cells, their entire genomes were sequenced. Table 1 lists the identified mutations along with their possible consequences. ONYX-201 and -203 each contained seven single point mutations, four of which were shared by both viruses and the remaining three were unique to each virus.


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  		TABLE 1. Summary of mutations in ONYX-201 and -203

To delineate which point mutation(s) was responsible for the observed phenotypes, we constructed a series of recombinant viruses harboring particular mutations identified in ONYX-201 and -203. An illustration of these recombinant viruses and their construction strategy is shown in Fig. 7A. The cytolytic activity of these recombinant viruses was compared by MTT assay on HT29 cells. Results from the MTT assay (Fig. 7B), combined with analysis of infected HT29 cell morphology, indicated that all recombinant viruses containing the mutation at nucleotide 8350 (C to T) displayed the enhanced-cytotoxicity phenotype. ONYX-212, -232, -234, and -236 all had activities similar to that of ONYX-203, and they produced the characteristic morphology after infection of HT29 cells. In contrast, ONYX-231, -233, and -235 were indistinguishable from wild-type Ad5. Therefore, the C-to-T transition at nucleotide 8350 appeared to be both necessary and sufficient for the increased cytolytic activity of ONYX-203. In contrast, while this mutation was necessary to account for the superior cytolytic activity of ONYX-201, it was not sufficient. An additional mutation(s) appears to be required to achieve full cytolytic activity of ONYX-201. We are currently engaged in an effort to further characterize such mutation(s).

The C-to-T mutation at nucleotide 8350 is located in the i-leader region of the major late transcription unit of Ad5 (1, 11, 26, 47, 48). The i-leader sequence is spliced to a subset of L1 mRNA, which predominantly encodes the 52/55K protein, and may modulate expression of the 52/55K protein (1, 28, 36, 45). The i-leader itself contains an open reading frame encoding the 145-amino-acid i-leader protein (1, 11, 26, 47, 48). The exact role of the i-leader protein in adenovirus replication has not been clearly defined. However, this protein may play an important role in the initiation of viral DNA replication or switch to the late phase of the viral life cycle (1, 27, 28, 47). The C-to-T mutation at nucleotide 8350 changes the Gln codon (CAG) at amino acid 125 to a stop codon UAG, thus eliminating the last 21 amino acids of the i-leader protein. The size difference between the truncated i-leader protein and the wild-type protein is clearly demonstrated in Fig. 7C. Our results indicate that the truncated protein accumulated more rapidly in the infected cells than the wild-type protein (Fig. 7D). In cells infected with ONYX-201 and -203, most of the infected cells expressed high levels of the i-leader protein at 24 h postinfection. In contrast, the full-length i-leader protein was undetectable in the vast majority of cells infected with wild-type Ad5 at 24 h postinfection. The subcellular localization of the i-leader protein did not seem to be affected by this mutation (data not shown). Given the reported function of the i-leader protein in viral DNA replication, it is possible that the rapid accumulation of the truncated i-leader protein may play a role in the early onset of viral DNA replication, accelerating the viral life cycle.


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DISCUSSION
 
Random mutagenesis of human adenovirus followed by repeated passaging in certain cell lines or under defined culture conditions has been used to isolate temperature-sensitive (10, 12, 32, 39, 50) and host range (3, 25) mutants. Presumably, these viruses have accumulated mutations that facilitate their productive growth in particular host environments or culture conditions. We suggest that, by using the same concept, adenoviruses can also be adapted to express highly potent, tumor-selective phenotypes. The work presented here describes for the first time this biological selection approach, by which we have isolated adenoviral variants that may specifically and efficiently target certain tumor cells for infection and killing.

In this paper, we report an in vitro bioselection procedure to isolate two mutants of Ad5, ONYX-201 and -203, that have significantly accelerated viral DNA replication and protein synthesis, leading to more-efficient tumor cell killing and virus spread. Both viruses were isolated by serial passage in the HT29 human colorectal cancer cell line. ONYX-201 and -203 formed larger plaques in HT29 cells (Fig. 1), replicated more rapidly, and killed HT29 cells up to 1,000-fold faster (Fig. 2 and 4) than the parental virus Ad5. The difference was more pronounced when cells were infected at lower MOIs, presumably due to the compounding effects of multiround infections (Fig. 3 and 4). More importantly, the superior cytolytic activity of ONYX-201 and -203 was not limited to HT29 cells but was observed in a number of, but not all, other human cancer cell lines of diverse tissue origin tested (Fig. 5). In contrast, in a number of primary human cells tested, their cytotoxicity was similar to that of wild-type Ad5, effectively enhancing the tumor cell selectivity of these oncolytic agents (Fig. 6) compared to wild-type virus. The genomic DNAs of ONYX-201 and -203 were sequenced, and at least one mutation was identified as responsible for the observed phenotype (Table 1 and Fig. 7).

Adenovirus mutants that induce apoptosis also display enhanced CPE and the large-plaque phenotype (37, 40, 49). However, three lines of evidence suggested that ONYX-201 and -203 do not induce apoptosis in infected cells. First, there was no evidence for DNA fragmentation (data not shown), a hallmark for apoptosis. Second, the morphology of the infected cells (Fig. 2A) suggested that they were not dying of apoptosis, as apoptosis is characterized by cell shrinking and membrane blebbing and cells infected by ONYX-201 and -203 were swollen with a smooth surface. Third, viral progeny yields often reduce dramatically when apoptosis is induced (44), and this was clearly not the case for ONYX-201 and -203 in infected tumor cells (Fig. 4A).

We suggest that one of the main reasons for the enhanced cytolysis by ONYX-201 and -203 is their ability to replicate more rapidly. Figures 2 and 4 clearly indicate that cell lysis, viral macromolecular synthesis, and virus production were significantly more rapid in ONYX-201 and -203 during one-step infections (i.e., MOI = 10). The vast majority of the cells were infected at this MOI; therefore, the release and spread of the virus progenies were unlikely to be important factors. Nevertheless, it is entirely possible that these viruses have also acquired the capacity to spread more efficiently in cell culture, further enhancing their cytolytic activity in the low MOI settings. It is intriguing to speculate that one of the mutations in ONYX-201 may act in this fashion, complementing the mutation in the i-leader region.

The molecular mechanism of enhanced cytolysis for ONYX-201 and -203 is unclear. We confirmed that all cells were infected prior to cytolysis (data not shown), suggesting that they were killed directly as a consequence of a full viral life cycle rather than by a diffusible factor secreted by adjacent infected cells. We identified the mutation at nucleotide 8350 as necessary and sufficient for the enhanced oncolytic activity of ONYX-203. This mutation also exists in ONYX-201 and was required, but not sufficient, for the superior activity of ONYX-201. Additional mutation(s) may be required to achieve the full activity of ONYX-201. The C-to-T mutation at nucleotide 8350 is located in the i-leader of the major late transcription unit of Ad5 (1, 11, 26, 47, 48). The i-leader sequence is spliced to a subset of L1 mRNA, which predominantly encodes the 52/55K protein, and may modulate expression of the 52/55K protein (1, 28, 36, 45). The i-leader itself contains an open reading frame encoding the 145-amino-acid i-leader protein (1, 11, 26, 47, 48). The exact roles of the 52/55K and the i-leader protein in adenovirus replication are not clearly defined. They are the only proteins encoded in the major late transcription unit that are expressed prior to viral DNA replication, suggesting that these two proteins may have an important role in the initiation of viral DNA replication or switch to the late phase of viral replication (1, 27, 28, 47). It is possible that early accumulation of the truncated i-leader protein can accelerate viral DNA replication through an unknown mechanism. Further analysis of the i-leader protein's function during the viral life cycle is needed in order to address this question.

ONYX-201 and -203 share 4 mutations (Table 1), suggesting that some or all 4 of the mutations are critical for the enhanced killing phenotype. Whether these mutations were independently selected, or whether the two viruses were derived from a common ancestor through differential recombination events, is not clear. The fact that ONYX-201 and -203 share more than one mutation favors the latter possibility.

ONYX-015, an adenovirus with E1B-55K deleted, efficiently replicates in and lyses tumor cells with a defective p53 pathway. In a number of human tumor cells, the loss of p53 function compensates for the loss of E1B-55K function during the viral life cycle. We may speculate that specific common genetic alterations are present in HT29 and other tumor cell lines in which ONYX-201 and -203 replicate with high efficiency and provide a cellular environment that selectively supports expression of their rapid replication phenotype. We are currently devising strategies to decipher the genetic basis for such possible mechanisms. In all of the normal human cells tested, the cytotoxicity of ONYX-201 or -203 was similar to that of wild-type Ad5. This not only lends support to the cellular genetic basis model for the ONYX-201 and -203 phenotype but also indicates that, as oncolytic agents, these viruses have an enhanced overall therapeutic index compared to wild-type virus.

Our observation that ONYX-201 and -203 replicated in, and killed, tumor cells drastically more rapidly than Ad5 at low MOIs may have profound clinical impact. Human solid tumors at clinical presentation are commonly large masses of densely populated cells. Even in the case of a direct intratumoral injection, infection by therapeutic viruses occurs at a very low multiplicity. Thus, the infection conditions in viral oncolytic therapy regimens are not optimal. Furthermore, following systemic administration, the amount of virus that reaches the tumor is estimated to be a very small fraction of the injected dose, so the multiplicity of infection would be even lower than intratumoral administration. Therefore, the rapid replication phenotype observed under low MOI described here is likely relevant to the clinical setting, for both local-regional and systemic administration. The ability to quickly replicate should give these viruses a greater chance to eliminate solid tumor masses.

The work presented here provides groundwork for using bioselection to develop highly potent, tumor-selective oncolytic viruses. To the best of our knowledge, this is the first case demonstrated for human adenovirus. ONYX-201 and -203 and their recombinant derivatives are being examined in animal models carrying human tumors. Additionally, the key mutations identified in the present study are being incorporated with other genetically engineered adenoviruses, such as ONYX-015 and -411 (19), to create new generations of cancer therapeutic adenoviruses.


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ACKNOWLEDGMENTS
 
We thank Jaina Sumortin for DNA sequencing; Farah Fawaz, Sandy McCoy, and Amy Aspelund for excellent technical assistance; and M. Green (St. Louis University) for the anti-i-leader protein antibody. We also thank Leisa Johnson, Sylvie Laquerre, Ed Harlow, and Frank McCormick for helpful discussions and Aleida Perez, Gaston Habets, and Leisa Johnson for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Onyx Pharmaceuticals, Inc., 3031 Research Dr., Richmond, CA 94806. Phone: (510) 243-3679. Fax: (510) 222-9758. E-mail: jshen{at}onyx-pharm.com. Back

{dagger} Present address: Genetic Therapy, Inc., Gaithersburg, MD 20878. Back

{ddagger} Present address: Cerus Corporation, Concord, CA 94520. Back

§ Present address: Berlex Laboratory, Inc., Richmond, CA 94804. Back

|| Present address: UCSF Comprehensive Cancer Center, San Francisco, CA 94115. Back


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Journal of Virology, February 2003, p. 2640-2650, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2640-2650.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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