[CANCER RESEARCH 59, 2608 –2614, June 1, 1999] Fibroblast Growth Factor 2 Retargeted Adenovirus Has Redirected Cellular Tropism: Evidence for Reduced Toxicity and Enhanced Antitumor Activity in Mice Dan-ling Gu, 1 Ana Maria Gonzalez, Marie A. Printz, John Doukas, Wenbin Ying, Mark D’Andrea, Diana K. Hoganson, David T. Curiel, Joanne T. Douglas, Barbara A. Sosnowski, Andrew Baird, Sharon Lea Aukerman, 2 Glenn F. Pierce Selective Genetics, Inc., San Diego, California 92121 [D. G., A. M. G., M. A. P., J. D., W. Y., M. D., D. K. H., B. A. S., A. B., S. E. A., G. F. P.], and Gene Therapy Program, University of Alabama at Birmingham, Birmingham, Alabama 35294 [D. T. C., J. T. D.] ABSTRACT Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. De- spite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447–1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient speci- ficity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR  ) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR  tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180 –260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages. INTRODUCTION Replication-deficient human Ad, 3 serotypes 2 and 5, have been used as vectors for gene delivery in numerous preclinical models and clinical indications. Despite achieving high expression levels using adenoviral vectors, the toxicity, short-term transgene expression, and immunogenicity limit their usefulness and have prevented demonstra- tion of meaningful clinical efficacy (1– 6). The lack of specificity precludes systemic and, in many instances, locoregional application. Several approaches are under investigation to block the native tropism of Ad, decrease its immunogenicity via deletion of parts of its ge- nome, or target the virus to cell types of interest. To date, these studies have resulted in mixed but minimal therapeutic success (7–18). In rodent models, the majority of Ad vectors delivered i.v. is cleared rapidly (within the first 24 h) through the liver (19, 20). Concomitantly, there is considerable transduction of hepatocytes and associated transgene expression (8 –10). This is in part due to a high concentration of the Ad cellular receptor, CAR, in the rodent liver (21). Ad transgene expression rapidly declines over the first 7 days after Ad vector administration, but Ad transduction of hepatocytes is associated with significant liver toxicity as manifest by increased serum transaminases, hepatocellular necrosis, and inflammation (8, 10, 19). Retargeting of Ad away from its native tropism for CAR may abrogate this liver toxicity. We have developed a broadly useful method that retargets Ad by using a neutralizing Fab to the knob domain of the Ad fiber protein (12, 22). The fiber protein is used by Ad for binding to its receptor, CAR. By attaching FGF2 as a targeting ligand to this Fab, this bifunctional molecule targets and redirects Ad cellular entry via high-affinity FGFRs and, additionally, blocks uptake through CAR (23). FGF2 binds FGFRs with extraordinarily high affinity (Kd, 10 -12 M) compared with most other ligand-receptor interactions. The increased affinity is due to the initial binding of the FGF ligand to cell surface heparan sulfate proteoglycans (low-affinity receptors: Kd, 10 -9 M), followed by binding to, and dimerization of, the high-affinity tyrosine kinase receptors. In contrast, most other ligands used for targeting, including antibodies, bind at 3– 4 logs lower affinity to their receptors. Importantly, FGFRs are up-regulated in a number of diseases characterized by unwanted cellular proliferation, and many human malignancies contain elevated levels of one or more of the four recognized FGFRs (24 –29). Although there are about 20 FGF ligands identified, most have specificity for specific splice variants of a subset of FGFRs (30). FGF2, in contrast, is an injury-response molecule and can bind most splice variants of the FGFRs (30), making it a more useful ligand for targeting FGFR-bearing malig- nant or injured cells. We have previously established that FGF2 targets condensed DNA in vitro (31) and have observed a greater than 10-fold increase in gene expression when FGF2-retargeted Ad was compared with Ad in delivering reporter genes or the TK gene to human Kaposi’s sarcoma, pancreatic cancer, and ovarian cancer cell lines in vitro (22, 32). We have also demonstrated enhanced antitumor activity when FGF-retargeted Ad was locally delivered to ovarian carcinoma in the peritoneal cavity (32). Because this unexpectedly enhanced in vitro and in vivo potency implied greater selectivity and therapeutic benefit, we assessed whether the altered tropism of FGF2-Ad would show diminished toxicity in vivo. To establish whether the increased potency observed in vitro would translate into in vivo therapeutic benefit, FGF2-redirected Ad encoding the TK gene (AdTK) was also evaluated in mice chal- lenged with tumor cells resistant to native Ad infection. Received 12/18/98; accepted 4/5/99. 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. 1 To whom requests for reprints should be addressed, at Selective Genetics, Inc., 11035 Roselle Street, San Diego, CA 92121. Phone: (619) 625-0100; Fax: (619) 625-0222; E-mail: dlgu@selectivegenetics.com. 2 Present address: Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608. 3 The abbreviations used are: Ad, adenovirus; ALT, alanine transaminase; AST, aspartate transaminase; Alk Phos, alkaline phosphatase; -Gal, -galactosidase; CAR, Coxsackie and Ad receptor; FGF, fibroblast growth factor; FGF2, basic FGF; FGFR, FGF receptor; FGFR + , FGFR positive; pfu, plaque-forming unit(s); TK, herpes simplex virus thymidine kinase; X-Gal, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside; MOI, mul- tiplicity of infection; FI, fluorescence intensity; GCV, ganciclovir. 2608 Research. on February 9, 2016. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from