Research paper Assembly of targeting complexes driven by a single-chain antibody Marina V. Backer * , Jennifer Elliot, Timur I. Gaynutdinov, Joseph M. Backer SibTech, Inc., 705 North Mountain Road, Newington, CT 06111, USA Received 7 January 2004; received in revised form 3 March 2004; accepted 21 March 2004 Available online 11 May 2004 Abstract Rapid development in design and production of recombinant antibodies and antibody fragments specific for cell surface markers opens new opportunities for targeted delivery of therapeutic or imaging agents. However, the progress in this field is slowed by inactivation of many antibodies by chemical conjugation of payloads and by lack of internalization of complexes formed on the cell surface. Here, we describe conversion of a non-internalizing single chain Fv (scFv) antibody P4G7 specific for vascular endothelial growth factor receptor 2 (VEGFR-2) into a targeting protein (Hu-P4G7) for assembly of a novel type of targeting complexes. Hu-P4G7 contains an N-terminal ‘‘docking’’ Hu-tag, a 15-aa fragment of human RNase I, capable of high affinity binding of S-protein fragment of human RNase I or bovine RNase A. Purified Hu-P4G7 and complexes of Hu-P4G7 with S-protein bind both soluble and full-length cellular VEGFR-2. To assemble targeted DNA delivery complexes, S-protein modified with a DNA condensing agent was ‘‘docked’’ to Hu-P4G7, and then loaded with luciferase plasmid DNA. As expected for a non-internalizing targeting protein, Hu-P4G7-based complexes did not deliver DNA in VEGFR-2 expressing cells. However, in the presence of vascular endothelial growth factor (VEGF), these complexes selectively delivered DNA into the cells overexpressing VEGFR-2 suggesting that even a non-internalizing scFv antibody can be used for targeted intracellular drug delivery. D 2004 Elsevier B.V. All rights reserved. Keywords: Targeted drug delivery; scFv antibody 1. Introduction Advances in antibody engineering including direct selection from phage display libraries resulted in development of several approved antibody-based an- ticancer therapeutics, and many more moving through clinical trials (see, for reviews, Neilsen and Marks, 2000; Wu and Yazaki, 1999). The utility of recombi- nant antibodies specific for cell surface markers would be increased significantly, if they could be ‘‘loaded’’ with therapeutic or imaging agents through a stan- dardized procedure without damaging their binding epitopes. Furthermore, for many applications, it would be useful if antibodies carrying a ‘‘payload’’ were internalized upon binding to the cell surface targets. We have recently proposed to link drug payloads to a standardized adapter protein that can bind to a ‘‘docking’’ tag fused to a targeting protein (Backer et al., 2002 and Fig. 1). The first adapter/docking tag system for assembly of targeting complexes was 0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.03.011 Abbreviations: scFv, single chain fragment; VEGFR-2, vascular endothelial growth factor receptor 2; VEGF, vascular endothelial growth factor; KDR-Fc, soluble VEGFR-2; SLT, Shiga-like toxin; PEI, polyethylenimine. * Corresponding author. Tel.: +1-860-953-1164; fax: +1-860- 953-1317. E-mail address: mbacker@sibtech.com (M.V. Backer). www.elsevier.com/locate/jim Journal of Immunological Methods 289 (2004) 37 – 45