RETARGETING OF ADENOVIRAL INFECTION TO MELANOMA: COMBINING GENETIC ABLATION OF NATIVE TROPISM WITH A RECOMBINANT BISPECIFIC SINGLE-CHAIN DIABODY (scDb) ADAPTER THAT BINDS TO FIBER KNOB AND HMWMAA Dirk M. NETTELBECK 1,* , Angel A. RIVERA 1 , Jo ¨rg KUPSCH 2 , Detlef DIECKMANN 3 , Joanne T. DOUGLAS 1 , Roland E. KONTERMANN 4 , Ramon ALEMANY 5 and David T. CURIEL 1 1 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA 2 RAFT Institute, Mount Vernon Hospital, Northwood, United Kingdom 3 Department of Dermatology, University of Erlangen-Nuremberg, Erlangen, Germany 4 Institute of Molecular Biology and Tumor Research, Philipps-University of Marburg, Marburg, Germany 5 Gene Therapy Unit, Institut Catala ` d’Oncologia, L’Hospitalet Barcelona, Spain Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the develop- ment of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-medi- ated gene transfer to melanoma cells. This strategy com- bines genetic ablation of native adenoviral tropism with re- directed viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain dia- body, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associ- ated antigen (HMWMAA). This antigen is widely and specif- ically expressed on the surface of melanoma cells and its expression is associated with tumor development and pro- gression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection ex- periments, we demonstrated i) substantially (>50-fold) re- duced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA- mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of trans- gene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild- type fibers. We confirmed the utility of this targeting strat- egy with human primary melanoma cells that represent clin- ically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold in- creased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tro- pism exhibits both enhanced specificity and augmented in- fectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma. © 2003 Wiley-Liss, Inc. Key words: adenovirus targeting; melanoma, single-chain diabody; high molecular weight melanoma-associated antigen; CAR/integrin- binding ablation Gene therapy is a promising new strategy for treatment of cancer where conventional therapeutic regimens are often in- adequate. Proof-of-principle has been clearly established for therapeutic gene transfer aiming at direct tumor cell killing, prodrug-activation by tumor cells, mutation compensation, im- munopotentiation and viral oncolysis following the transfer of genomes of replication competent viruses to tumor cells. 1,2 How- ever, the application of gene therapy in patients is hampered by insufficient gene transfer efficiencies and by toxic effects mediated by gene transfer to normal tissues. 3 Thus, the development of efficient and targeted gene transfer vectors is key to the successful clinical translation of gene therapy. 4,5 In this regard, adenoviral vectors possess critical properties required for this endeavor. These include a highly evolved gene transfer mechanism, the stability of virus particles and the ease of virus production at high titers. 6 Most importantly, the capsid structure, genome and replication cycle of adenoviruses, particularly of the most commonly employed sero- type 5, have been extensively characterized, which allows for the molecular modifications required for their utilization as gene trans- fer vectors. The necessity of such modifications is predicated by the observation that the primary receptor for Ad5 and other ade- novirus serotypes, CAR is widely expressed on normal tissues resulting in nonspecific susceptibility to adenoviral infection. In addition, reduced or absent expression of CAR has been reported previously for several tumor types, including melanoma, indicat- ing resistance to adenoviral infection by tumor cells in situ. 7–9 . These considerations of adenoviral biology are paralleled by the observation of limited efficacy and vector-related toxicity in pre- clinical and clinical adenoviral gene therapy studies. Therefore, the development of tropism-modified, tumor-targeted adenoviral gene transfer vectors is a key endeavor in current gene therapy research. Towards this goal, the native tropism of adenoviruses needs to be ablated (infectivity ablation) and a new, tumor-specific tropism needs to be engineered into viral particles (retargeting). Abbreviations: Ad5, adenovirus serotype 5; bp, base pairs; CAR, cox- sackie-adenovirus receptor; EGF, epidermal growth factor; FGF, fibroblast growth factor; HMWMAA, high molecular weight melanoma-associated antigen; IGF, insulin-like growth factor; IMAC, immobilized metal affinity chromatography; MAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; pfu, plaque-forming units; scDb, single-chain diabody; scFv single-chain Fv antibody fragment; sCAR, extracellular domain of CAR; TNF, tumor necrosis factor alpha; vp, viral particles. Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers; NE832/1; Grant sponsor: National Institutes of Health; Grant numbers: R01 HL67962, P50 CA89019, R01 CA86881, U19 DK57958; Grant spon- sor: CFAR AIDS; Grant number: P30AI27767; Grant sponsor: UAB Center for AIDS Research; Grant number: P30 A1-27767 *Correspondence to: Department of Dermatology, University of Erlangen-Nuremberg, Hartmannstrasse 14, 91052 Erlangen, Germany. Fax: +49-9131-853-6417. E-mail: dirk.nettelbeck@derma.imed.uni-erlangen.de Received 31 March 2003; Revised 30 May 2003; Accepted 4 August 2003 DOI 10.1002/ijc.11563 Published online 7 October 2003 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 108, 136 –145 (2004) © 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer