[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 Gene Therapy, 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 infection 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 cell 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 enzyme 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 oncolytic virus 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 protein and the integrin 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 proteins, 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 peptide 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 integrins, 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 homologous recombination with pShuttleCox2LE1-F and and Research, Wiltshire, United Kingdom) and were grown in DMEM (Me- pShuttleCox2LE1-R.