title: Assembly of targeting complexes driven by a single-chain antibody.
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.
authors.affiliation: SibTech, Inc., Newington, CT 06111, USA. mbacker@sibtech.com
authors.forename: Marina V
authors.initials: MV
authors.lastname: Backer
authors.forename: Jennifer
authors.initials: J
authors.lastname: Elliot
authors.forename: Timur I
authors.initials: TI
authors.lastname: Gaynutdinov
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004337:Drug Carriers / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D018014:Gene Transfer Techniques; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004337:Drug Carriers; C512004:Hu-P4G7 antibody; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D040301:Vascular Endothelial Growth Factor Receptor-2; C074799:ribonuclease A (21-124); D012259:Ribonuclease, Pancreatic