Targeted Gene Transfer to Hepatocellular Carcinoma Cells In Vitro Using A Novel Monoclonal Antibody-Based Gene Delivery System

Abstract

Gene therapy approaches for the treatment of malignant tumors will require high–level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor–specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor–reactive monoclonal antibody (mAb), designated AF–20, coupled to a DNA–binding cationic amphiphile, cholesteryl–spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high–affinity mAb, AF–20, recognizes a rapidly internalized 180–kd cell–surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF–20 mAb and an isotype–matched control antibody (C7–57) were covalently coupled to cholesteryl–spermine. Binding and internalization of AF–20-cholesteryl–spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)–labeled anti–mouse IgG antibody. Following transfection of FITC–labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or β–galactosidase reporter genes complexed to AF–20-cholesteryl–spermine resulted in high levels of gene expression in AF–20 antigen–positive tumor cells. Very low levels of gene expression were observed using the control compound (C7–57–cholesteryl–spermine), which does not recognize the AF–20 tumor antigen or when AF–20 antigen–negative NIH 3T3 cells were transfected with AF–20-cholesteryl–spermine. Thus, covalent coupling of the AF–20 mAb to cholesteryl–spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF–20 antigen–positive HCC cells.