[CANCER RESEARCH 64, 4319–4327, June 15, 2004] Infectivity-Enhanced Cyclooxygenase-2-Based Conditionally Replicative Adenoviruses for Esophageal Adenocarcinoma Treatment

Julia Davydova, Long P. Le, Tatyana Gavrikova, Minghui Wang, Victor Krasnykh, and Masato Yamamoto Division of Human , Departments of Medicine, Pathology, and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama

ABSTRACT limited in their ability to infect cancer cells and spread throughout the tumor (11). To overcome these obstacles, the concept of conditionally The employment of conditionally replicative adenoviruses (CRAd) con- replicative Ads (CRAds) has been proposed (12, 13). These agents are stitutes a promising alternative for cancer treatment; however, in the case designed to replicate specifically in tumor cells, followed by the of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor- specific promoter and relative resistance to adenovirus have spread of the viral progeny to neighboring cancer cells (14). A variety hampered the construction of CRAds with clinically applicable specificity of Phase I and II clinical trials have demonstrated the tolerability and and efficacy. By combining transcriptional targeting with infectivity en- safety of CRAd administration (15–22). One such Ad (dl1520/ON- hancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) YX-015) has entered a Phase III clinical trial for recurrent head and promoter-controlled replicative viral agents for the treatment of EAC. We neck carcinoma (22). However, clinical experience has indicated that used infectivity enhancement based on incorporation of an RGD-4C motif a satisfactory balance between safety and efficacy of CRAds has not into the HI loop of the adenoviral (Ad) fiber knob domain as well as yet been achieved (23). The current-generation CRAds are safe, but replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was due to their relatively poor ability to spread within the tumor mass, highly active in EAC, whereas showing no significant activity in Cox-2- negative lines and primary cells isolated from normal mouse esoph- repeated viral injections at multiple sites and over a prolonged period agus and stomach. Evaluation of infectivity-enhanced vectors revealed of time are required (11, 16, 17, 23). that the transduction and virus-cell binding ability of Ad5/Ad3-chimera One common approach to target virus replication to a specific were significantly more efficient than that of unmodified and Arg-Gly-Asp subset of cells is through the use of tissue- or tumor-specific promot- (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replica- ers (24–32). Therefore, the design of a particular tissue- or tumor tion and subsequent oncolysis in EAC cells but not in Cox-2-negative cells type-specific virus vector depends on the availability of a relevant in vitro, thus confirming the dependence of their replication on the Cox-2 tissue- or tumor-specific promoter. In the case of EAC, the lack of an promoter activity. Ad5/Ad3 CRAds exhibited significantly improved on- appropriate tumor-specific promoter that is both selective and strong colysis and progeny production compared with unmodified and RGD- in EAC cells has hampered the construction of CRAds with clinically modified vectors without sacrificing tumor selectivity. Whereas unmodi- fied and RGD-modified CRAds showed insignificant therapeutic effect in usable specificity and efficacy. Among the promoters successfully vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of estab- used to drive CRAd replication is the cyclooxygenase-2 (Cox-2) gene lished xenografts in mice. Thus, our studies have demonstrated that promoter (31). Cox-2 is the key required for the conversion of Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and po- arachidonic acid to prostaglandins, normally undetectable in most tent agents for the treatment of EAC. normal tissues (33). Recent studies have highlighted the involvement of Cox-2 in carcinogenesis and tumor progression (34, 35). Increased levels of Cox-2 have been reported in carcinomas of the colon, INTRODUCTION stomach, breast, lung, liver, and pancreas (33, 36, 37). Most impor- The incidence of esophageal adenocarcinoma (EAC) has increased tantly, Cox-2 has been found in EAC and Barrett’s esophagus, sug- rapidly since the mid-70s and is now the fastest expanding type of gesting that the Cox-2 promoter may be applicable to target EAC cancer in Western countries (1–5). The highest rate of increase was (38–43). Additionally, our studies have clearly shown that the Cox-2 nearly 10% per year in the United States (6). Although the reason for promoter possessed strong activity in many gastrointestinal cancer this rapid rise is unclear, it is well known that 10% of patients with cells among a variety of tested promoters, whereas activity in normal gastroesophageal reflux disease eventually develop Barrett’s esopha- tissues, especially in the liver, was minimal (28–31). On the basis of gus, a metaplasia of the lower esophageal epithelium, resulting with a these findings, we explored the feasibility of the Cox-2 promoter for 1% annual risk of developing adenocarcinoma (7, 8). Improvements transcriptional targeting of replicative Ads to EAC. in the diagnosis of EAC at earlier stages as well as the treatment of EAC cells are known to be very resistant to infection by conven- late-stage disease remain insignificant, whereas the overall 5-year tional Ad vectors (44–46). The efficiency of Ad5-mediated gene survival rate is Ͻ10% (9, 10). In this regard, novel transfer largely depends on the biology of the virus-cell interaction approaches using the most advanced therapeutic vectors may offer (47). The initial high-affinity binding of Ad5 to the primary more effective treatment of EAC. coxsackie-Ad receptor (CAR) occurs via the knob domain of the Ad Most initial cancer gene therapy protocols were performed with fiber (48, 49). A subsequent step of virus internalization depends on replication-incompetent adenoviruses (Ad), which by their nature are interaction between the Arg-Gly-Asp (RGD) motifs in the Ad5 penton base and the molecules on the cell surface (50). Received 1/8/04; revised 3/9/04; accepted 4/2/04. Stemming from these considerations, efforts to improve transduction Grant support: R01DK063615 (M. Yamamoto), DAMD17-03-1-0104 (M. efficiency of Ads have included changing viral tropism by using the Yamamoto), and P20CA101955 (M. Yamamoto). The costs of publication of this article were defrayed in part by the payment of page strategy of genetic modification of the viral capsid , which charges. This article must therefore be hereby marked advertisement in accordance with would enable CAR-independent entry. Specifically, it has been re- 18 U.S.C. Section 1734 solely to indicate this fact. ported that incorporation of an RGD-4C into the HI loop of Note: V. Krasnykh is currently at the Department of Experimental Diagnostic Imaging, M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030. the fiber knob domain remarkably increased the viral infectivity in Requests for reprints: Masato Yamamoto, Division of Human Gene Therapy, De- CAR-negative cells (51). In a previous study, we demonstrated the partments of Medicine, Pathology, and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, BMR2–408, 901 19th Street South, Birmingham, AL 35294- suitability of an RGD infectivity-enhanced Cox-2 promoter-based 2172. Phone: (205) 975-0172; Fax: (205) 975-8565. CRAd for pancreatic cancer (31). This enhancement was mainly 4319

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. CRAds FOR ESOPHAGEAL ADENOCARCINOMA mediated by the binding of the RGD-modified Ad fiber protein to , which are overexpressed on the surface of target cancer cells (51, 52). Another strategy to achieve CAR-independent cell entry includes fiber-knob chimerism where the fiber knob of Ad5 is re- placed with that of another Ad type (53, 54). Notably, the Ad type 3 (Ad3) receptor, the identity of which has not yet been established (55), has been shown recently to be a better target for Ad vectors designed to treat ovarian cancer, squamous cell carcinoma of the head and neck, and renal cancer (56–59). In this study, we applied these strategies of Ad tropism modifica- tion to derive novel Cox-2 promoter-based CRAds as therapeutic Fig. 1. Schematic of cyclooxygenase (Cox)-2 conditionally replicative adenoviruses (CRAd). All of the CRAds contain the nucleotide 1–358 sequence of human adenovirus agents for EAC. Importantly, we demonstrate that the oncolytic po- (Ad)5, a Cox-2 promoter-controlled E1 expression cassette in either forward or reverse tency of the Cox-2 CRAds transcriptionally targeted to EAC can be orientation (CRAdCox2F and CRAdCox2R), a restored pIX promoter, and an intact E3 augmented significantly by directing them to the Ad3 receptor. This region. The fiber was modified by incorporating an RGD-4C motif into the HI loop of the Ad fiber knob domain (RGD fiber) or replacing the Ad5 knob with the Ad3 knob modality of combining transcriptional targeting and Ad5/Ad3 modi- (Ad5/Ad3 fiber). fication to increase the therapeutic index provides a powerful and promising approach to develop oncolytic viruses for EAC. domain of Ad3 fiber (53), into the fiber shuttle vector pMG100, which includes the Ad5 genome from nucleotide 3047 (XbaI) to the 3Ј end. The resulting MATERIALS AND METHODS plasmid pMG100.5/3F was cleaved with AatII and XhoI and recombined with a SwaI-linearized pVK50 (62). The resultant Ad backbone pMG553, contain- Cell Lines. OE19 and OE33 human EAC lines were obtained from the ing the modified fiber gene, was then used for the generation of Cox-2-based European Collection of Cell Culture (CAMR Centre for Applied Microbiology CRAds by with pShuttleCox2LE1-F and and Research, Wiltshire, United Kingdom) and were grown in DMEM (Me- pShuttleCox2LE1-R. To generate viruses, 911 cells were transfected with diatech, Herndon, VA). TE7 (human EAC; Institute of Development, Aging PacI-digested plasmids containing the viral genomes. and Cancer, Tohoku University, Sendai, Japan), A549 [Cox-2 positive lung Wild-type Ad5 (AdWt) and its RGD and Ad5/Ad3-chimeric isogenic ver- line; American Type Culture Collection (ATCC), Manassas, VA], sions (RGDWt derived from pVK503 and AdMG553 derived from pMG553, and BT474 (Cox-2 negative breast cancer cell line; ATCC) cells were culti- respectively) were used as nonselective replicative control vectors. vated in RPMI 1640 (Mediatech). The medium for BT474 was supplemented The viruses were propagated in E1-transcomplementing 911 or 293 cells, with bovine insulin (0.01 mg/ml; Life Technologies, Inc., Rockville, MD). purified by double cesium chloride density gradient ultracentrifugation, and Human ovarian adenocarcinoma cell line SKOV3.ip1 (Ad3 receptor-positive dialyzed in PBS with 10% glycerol. Viral aliquots were stored at Ϫ80°C. control, kindly provided by Dr. Janet Price, M.D. Anderson Cancer Center, Titering was performed with a plaque assay and absorbance-based measure- Houston, TX) was propagated in DMEM/F12, 50:50 (Mediatech). Chinese ment. The viral particle/plaque forming unit (vp:pfu) ratios for these vectors hamster ovary cells (Ad3 receptor-negative control) were grown in HAM’s were in the range of 20–80. F-12 medium. Human hepatoma cell line HepG2 (CAR positive control; Validation of Viral Constructs. Several critical vector components of the ATCC), transformed human embryonic retina cell line 911 (a kind gift from CRAd genomes were confirmed by PCR. Viral DNA was isolated from Dr. Alex J. van der Eb, Leiden University, Leiden, the Netherlands), and 293 purified virions using a Blood DNA kit (Qiagen), and aliquots corresponding cells (ATCC) were maintained in DMEM. Each medium was also supple- to 107 genomes were analyzed by PCR. Thermal cycling conditions were: mented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM initial denaturation (5 min at 95°C), and 25 cycles of amplification (30 s at L-glutamine and penicillin (100 IU/ml), and streptomycin (100 mg/ml). Human 95°C,30sat55°C, and 1 min at 72°C), and final extension (10 min at 72°C). umbilical vein endothelial cells (HUVEC) were obtained from ATCC and The regions detected and expected band size were as follows: wild-type Ad5 cultured in EGM-2 medium (Cambrex Biosciences, Walkersville, MD). All of (485 bp), E1a (338 bp), Cox-2 promoter (405 bp), forward direction CRAd the cells were incubated at 37°C in a humidified environment with 5% CO2. (392 bp), Ad5 fiber knob region (wild-type fiber: 247 bp and RGD fiber: 274 Adenoviral Vectors. Replication-incompetent adenoviral vectors (AdCox- bp), and Ad5/Ad3 fiber knob region (293 bp). For the Ad5/Ad3 fiber knob 2LLuc,AdCox2MLuc, and AdCMVLuc) encoding the firefly luciferase (Luc) region, the following primers were used: Ad5/3F (sense) 5ЈCCTAGAATTT- reporter gene were constructed as described previously (30). AdCox2LLuc and GATTCAAACAAGGC3Ј and Ad5/3F (antisense) 5ЈCTACATTAATG- AdCox2MLuc carry the Cox2L (Ϫ1432/ϩ59; long length) and Cox2M (Ϫ883/ GAGACATTTTTGT3Ј. Other primer sequences were already reported (31). ϩ59; medium length) promoters derived from phPES2 (provided by Drs. Analysis of Cox-2 RNA Levels. Total cellular RNA was extracted from Hiroyasu Inoue and Tadashi Tanabe at the National Cardiovascular Center semiconfluent cell cultures in 10-cm dishes using the RNeasy mini RNA Research Institute, Japan; Refs. 60, 61). AdCMVLuc is a control vector that extraction kit (Qiagen). Cox-2 and glyceraldehyde-3-phosphate dehydrogenase expresses Luc under the ubiquitous cytomegalovirus (CMV) immediate early RNAs were detected in a semiquantitative manner using the Gene Amp Gold promoter. RNA PCR Core kit (Perkin-Elmer, Branchburg, NJ) as described previously For infectivity enhancement analysis, CMV promoter-driven luciferase ex- (31). pression vectors with RGD-modified Ad5 fiber (Ad5RGDLuc1), Ad5/Ad3- Flow Cytometry. Cultured cells were trypsinized, washed with PBS, cen- chimeric fiber (Ad5/3Luc1), or wild-type Ad5 fiber (Ad5Luc1) were used. All trifuged, and resuspended in ice-cold PBS supplemented with 1% BSA and 6 of these replication-incompetent Ad vectors are identical, except for their 0.1% NaN3. Cells (10 ) were incubated for1hat4°C with mouse genetically modified fibers. Ad5RGDLuc1 contains an RGD peptide insertion RmcB to CAR (1:1000; produced using hybridoma purchased from ATCC), in the HI loop of the Ad5 fiber knob (51). Ad5/3Luc1 has a chimeric fiber, LM609 to ␣v␤3 integrin (1:1000), P1F6 to ␣v␤5 integrin (1:1000), P3G8 to composed of the Ad5 shaft and the Ad3 knob (53). ␣v integrin (1:1000), P4G11 to ␤1 integrin (1:50), or HA5 to ␣5␤1 integrin The genomes of the Cox-2 promoter-based CRAds were constructed as (1:100; Chemicon, Temecula, CA). The cells were then washed twice with described previously (Ref. 31; Fig. 1). We constructed two shuttle vectors with PBS/BSA/NaN3 buffer and incubated with secondary FITC-labeled goat anti- the E1 expression cassette in two different orientations: pShuttleCox2LE1-F mouse IgG serum (1:50; Sigma). After another PBS/BSA/NaN3 rinse, 2.5 (5Ј to 3Ј) and pShuttleCox2LE1-R (3Ј to 5Ј). The shuttle vectors were used for ␮g/ml propidium iodide (Sigma) was added to sort out dead cells from the recombination with Ad5 DNA and pVK503 (62) to generate CRAd genomes sample. The cells (104) were analyzed immediately by flow cytometry at the containing the wild-type fiber gene or RGD-modified fiber gene, respectively University of Alabama at Birmingham FACS Core Facility. (31). To generate Cox-2-based CRAds carrying an Ad5/Ad3-chimeric fiber we Promoter and Gene Delivery Analysis with Luciferase Expression Ads. incorporated the NheI-MfeI fragment of pNEB.PK.F5/3, containing the chi- EAC cells grown in 24-well plates were infected with Ad vectors at a meric fiber gene encoding the tail and shaft domains of Ad5 and the knob multiplicity of infection of 50 pfu/cell. The infection medium was replaced 4320

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. CRAds FOR ESOPHAGEAL ADENOCARCINOMA with the appropriate growth medium 2 h later. Two days after infection, the with calipers. The tumor volume was calculated using the formula: tumor cells were lysed with cell culture buffer (Promega, Madison, WI), and volume ϭ width2 ϫ length/2. In accordance with institutional approved animal Luc activity was determined with the Luciferase Assay System (Promega). experimental protocol, the mice were euthanized 16 or 21 days after viral Experiments were performed in triplicate and standardized with protein con- injection due to the overgrowth of tumors in the control groups. All of the centration quantitated by the DC protein assay (Bio-Rad, Hercules, CA). animals received humane care based on the guidelines set by the American The Cox-2 promoter selectivity was also analyzed in mouse esophageal and Veterinary Association. All of the experimental protocols involving live ani- gastric primary cells. The esophagus and stomach of a Balb/C mouse were mals were reviewed and approved by the Institutional Animal Care and Use aseptically isolated and cut by scissors into small pieces, followed by wash Committee of the University of Alabama at Birmingham. with PBS twice. The tissues were dispersed with Liver Digestion Medium Statistical Methods. Statistical analysis of CRAd efficacy in vitro and in (Life Technologies, Inc.) for3hat37°C, rocking every 15 min. After adding vivo was performed with a two-tailed t test. Data are expressed as a mean Ϯ SD 25% volume of FBS to stop digestion, the cells were centrifuged and resus- of at least three sets of results. Results were considered statistically significant pended in 20% FBS DMEM/F12 containing 1 ␮M dexamethasone. The cells when P Ͻ 0.05. were plated and infected as described above. In Vitro Analysis of Cytocidal Effect by Crystal Violet Staining. To RESULTS analyze virus-mediated cell killing, 25,000 cells/well were plated in 12-well plates and infected with the viruses in 200 ␮l of growth medium containing 5% Confirmation of CRAd Structure. All of the CRAd vector struc- FBS at an multiplicity of infection of either 0.01 or 0.1 vp/cell. After 3 h, 1 ml tures were confirmed by PCR analysis of the viral DNA. None of the of the growth medium was added. Two days later, the infection medium was vectors was contaminated with nonselective replication-competent Ad replaced with 1% FBS medium. After 12 days (18 days for TE7 cell line) of resulting from spontaneous homologous recombination during vector cultivation, the cells were fixed with 10% buffered formalin for 10 min and propagation in E1-transcomplementing cell lines (Fig. 2A). The stained with 1% crystal violet in 70% ethanol for 20 min, followed by washing with water and drying. CRAds possessed both the E1a gene and the Cox-2 promoter se- Virus Binding Assay. To analyze virus-cell binding, 50,000 cells/well quence as part of their E1 expression cassettes (Fig. 2, B and C). A cultivated in 24-well plates were infected with 2000 vp/cell in 100 ␮lof 392-bp fragment corresponding to the forward direction cassette was DMEM and incubated for1hat4°C. At this temperature, virus internalization amplified from CRAdCox2F, RGDCRAdCox2F, and 5/3CRAdCox2F would not occur. After washing with PBS three times, cells were scraped from with a sense primer recognizing the left inverted terminal repeat the plates and processed with a QIAamp Blood Mini kit (Qiagen). The isolated sequence and an antisense primer recognizing the Cox-2 promoter DNA was analyzed by real-time PCR analysis to determine the Ad DNA copy sequence (Fig. 2D). In the analysis of the fiber region, vectors with number with E4-specific primers at the University of Alabama at Birmingham wild-type Ad5 fiber (CRAdCox2F and CRAdCox2R) produced a Gene Therapy Center Correlative Laboratories as described before (31). For 247-bp signal, whereas vectors with the RGD-modified fiber the standard curve, E4 template DNA with known copy numbers (108,106, (RGDCRAdCox2F and RGDCRAdCox2R) yielded a 274-bp band 104, and 102/␮l) was used. All of the PCR reactions were carried out using the Light Cycler System (Roche Molecular Biochemicals, Indianapolis, IN) as (Fig. 2E), the increased size of which represents the 9 amino acid recommended by the manufacturer. Thermal cycling conditions were: initial insertion in the HI loop. Ad5/Ad3-chimeric CRAds (5/3CRAdCox2F denaturation for 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at and 5/3CRAdCox3R) amplified with 5/3F primers showed a 293-bp 60°C. Data were analyzed with the Light Cycler software. The primers were signal, which confirms the presence of the Ad3 knob in the fiber (Fig. designed to detect a 68 bp-long sequence within E4 region: forward primer 2F). These data validate the structure and stability of the CRAd (5ЈGGAGTGCGCCGAGACAAC3Ј, nucleotides 34007–34024), reverse primer constructs. (5ЈACTACGTCCGGCGTTCCAT3Ј, nucleotides 34074–34056) and 6-car- boxyfluorescein labeled probe (6-carboxyfluorescein-TGGCATGACACTAC- GACCAACACGATCT-6-carboxytetramethylrhodamine, nucleotides 34054– 34027; Ref. 31). Quantitative in Vitro Cytotoxicity Assay. Cells (3000/well) cultured in 96-well plates were infected with Ad vectors at 1.0 vp/cell in 100 ␮lof5% FBS medium. On the next day, 100 ␮l of the growth medium containing 1% FBS was added. The cells were incubated for 12 days (18 days for TE7), and the number of surviving cells was analyzed by a colorimetric method using the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) as described by the manufacturer. Absorbance was measured at a wavelength of 490 nm in an E-Max spectrophotometer (Molecular Device Corporation, Sunnyvale, CA), and standard curves were generated by analyzing the known number of live cells. On the basis of this curve, the number of living cells was calculated for the experimental groups using the SOFTmax computer software (Molecular Device Corporation). All of the experiments were repeated in triplicate. Fig. 2. Confirmation of conditionally replicative adenoviruses (CRAd) structure by PCR. The structure of the viral DNA was analyzed using sets of primers corresponding to Viral Progeny Production Assay. To determine production of virus prog- several important regions of the virus. A, detection of wild-type adenovirus (Ad)5 (485 eny, 250,000 cells seeded in 12-well plates were infected with Ad vectors at an bp). Primers recognizing the wild-type Ad5 left-end sequence did not amplify any multiplicity of infection of 1 vp/cell in 200 ␮l of 5% FBS medium. After 3 h, fragments from any of the six cyclooxygenase (Cox)-2 CRAds. B, detection of the E1a the infection medium was replaced with 1 ml of the appropriate growth sequence (338 bp). All six of the Cox-2 CRAds contain the E1a sequence because they possess Cox-2 promoter-driven E1 expression cassettes. C, detection of the Cox-2 pro- medium. Twelve days later, cells and medium were harvested, freeze/thawed moter sequence (405 bp). All six of the CRAds contain the Cox-2 promoter sequence to three times, and centrifuged, and the supernatant was titered by a standard drive E1 expression. D, direction of the E1 expression cassette. A set of primers plaque assay on 911 cells. All of the experiments were performed in triplicate. recognizing the Cox-2 promoter, placed in the l