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 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 beta-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.
Mohr
L
L
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown 02129, MA, USA.
Schauer
J I
JI
Boutin
R H
RH
Moradpour
D
D
Wands
J R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D019084:Fluorescent Antibody Technique, Indirect; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000235:genetics* / Q000276:immunology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D008156:Luciferases; D001616:beta-Galactosidase