Targeted gene transfer to hepatocellular carcinoma cells in vitro using a novel monoclonal antibody-based gene delivery system

Gene therapy approaches for the treatment of malignant tumors will require high-level expression of therapeutic genes in tumors compared with normal t issues. This may be achieved either by targeted gene delivery to tumor cell s 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 -sp ermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high-affinity mAb, AF-20, recognizes a rapidly internalized 180-kd cell-sur face glycoprotein that is abundantly expressed on HCC and other human tumor s. The AF-20 mAb and an isotype-matched control antibody (C7-57) were coval ently coupled to cholesteryl-spermine. Binding and internalization of AF-20 -cholesteryl-spermine was confirmed by fluorescence microscopy using fluore scein isothiocyanate (FITC)-labeled anti-mouse IgG antibody. Following tran sfection of FITC-labeled oligonucleotides and ethidium monoazide-labeled pl asmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of lucife rase or P-galactosidase reporter genes complexed to AF-20-cholesteryl-sperm ine resulted in high levels of gene expression in AF-20 antigen-positive tu mor cells. Very low levels of gene expression were observed using the contr ol compound (C7-57-cholesteryl-spermine), which does not recognize the AF-2 0 tumor antigen or when AF-20 antigen-negative NIH 3T3 cells were transfect ed with AF-20-cholesteryl-spermine. Thus, covalent coupling of the AF-20 mA b to cholesteryl-spermine generated a highly specific and efficient nonvira l vector system for targeted gene delivery to AF-20 antigen-positive HCC ce lls.