ANTISENSE AND NUCLEIC ACID DRUG DEVELOPMENT 12:311–325 (2002) © Mary Ann Liebert, Inc. Development and Effects of Immunoliposomes Carrying an Antisense Oligonucleotide Against DHFR RNA and Directed Toward Human Breast Cancer Cells Overexpressing HER2 MERCÈ RODRÍGUEZ, SÍLVIA COMA, VÉRONIQUE NOÉ, and CARLOS J. CIUDAD ABSTRACT The development and the effect of immunoliposomes directed against human breast cancer cells overexpressing p185/HER2 are described. These immunoliposomes carry an antisense oligonucleo- tide directed toward the translational start site of dihydrofalate reductase (DHFR) RNA, which causes high cytotoxicity. To prepare the immunoliposomes, we followed two methodologies based on the high affinity between streptavidin and biotin and the use of biotinylated antibodies. In the first approach, the streptavidin molecule is covalently attached to the phospholipid DOPE, which is mixed with the cationic liposome DOTAP complexed with the antisense oligonucleotide. The second approach, which is much easier to perform, involves the binding of streptavidin to antibody and oli- gonucleotide, both biotinylated, and the latter complexed with DOTAP. The formation of the inter- mediary complexes of this immunoliposome was studied sequentially by gel electrophoresis. The up- take of the oligonucleotide carried by the immunoliposome was monitored by flow cytometry and confocal microscopy. As a model, we used SKBR3 cells that overexpress p185. The full immunolipo- somes were more toxic than the antisense oligonucleotide in the absence of the antibody, thus in- creasing the sensitivity of the treatment. INTRODUCTION A NTISENSE OLIGONUCLEOTIDES (AS-ODNS) are a promising therapeutic tool to treat cancer, as they in- hibit gene expression. However, this therapy needs to be directed to specific target cells for maximum efficiency. Dihydrofolate reductase (DHFR) is a key enzyme in the de novo synthesis of thymidine, purines, and glycine, and, thus, is needed for cell proliferation. We have previ- ously shown the efficiency of AS-ODNs directed toward selected regions of the DHFR RNA, which cause cyto- toxicity in both hamster and human cell lines. The most effective oligonucleotides were those directed to the translational start region of this RNA, causing a specific decrease in mRNA expression and enzymatic activity (Rodríguez et al., 1999). These AS-ODNs may become an alternative to enzymatic inhibitors of DHFR, for ex- ample, methotrexate, which inhibits its expression (Wiedemann and Johnson, 1979; Mariani et al., 1981). AS-ODNs were internalized using the cationic liposome DOTAP through a mechanism that is thought to depend on endocytosis (Gao and Huang, 1993). Antibodies (Ab) may be used to interfere with tumor progression. For example, Abs have been directed against the p185 receptor, a transmembrane epidermal growth factor receptor (EGFR) family protein (Hes- keth, 1995) encoded by the HER2/c-erbB2/neu proto- oncogene, which contributes to the development and growth of malignant tumors (Bertram et al., 1994). p185 is a potent transforming oncogene that is overexpressed Department of Biochemistry, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain. 311