title: An intracellular anti-erbB-2 single-chain antibody is specifically cytotoxic to human breast carcinoma cells overexpressing erbB-2.
abstract: We previously demonstrated that delivery of a gene encoding an anti-erbB-2 intracellular single-chain antibody (sFv) resulted in down-regulation of cell surface erbB-2 levels and induction of apoptosis in erbB-2 overexpressing ovarian cancer cells. Based upon these findings, we hypothesized that human breast carcinomas overexpressing erbB-2 would be similarly affected by this genetic intervention. We evaluated the phenotypic effects resulting from intracellular expression of the anti-erbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, BT-474, MCF-7 and MDA-MB-231. Recombinant adenoviruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directed anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and fluorescent microscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quantified the endogenous erbB-2 levels of each cell line. The anti-erbB-2 sFv-encoding-adenovirus, Ad21, but not the beta-galactosidase encoding adenovirus, AdCMVLacZ, was cytotoxic to > 95% of the tumor cells in the MDA-MB-361 and SK-BR-3 lines, and > 60% of the tumor cells in the BT-474 line. In marked contrast, the MCF-7 and MDA-MB-231 cell lines showed no change in the rate of cell proliferation following this treatment. The cytotoxic effects generated in the first three lines were a consequence of the induction of apoptosis by the anti-erbB-2 sFv. An ELISA specific for erbB-2 showed that the breast cancer cell lines most susceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overexpressed the erbB-2 protein while the cell lines demonstrating no response to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowest levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accomplished in the context of breast carcinoma. Furthermore, erbB-2 levels in breast tumor cells may be predictive of their sensitivity to sFv-mediated killing. The ability to accomplish selective cytotoxicity of breast cancer cell lines overexpressing the erbB-2 tumor marker should allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.
authors.affiliation: Gene Therapy Program, University of Alabama at Birmingham 35294, USA.
authors.forename: M
authors.initials: M
authors.lastname: Wright
authors.forename: J
authors.initials: J
authors.lastname: Grim
authors.forename: J
authors.initials: J
authors.lastname: Deshane
authors.forename: M
authors.initials: M
authors.lastname: Kim
authors.forename: T V
authors.initials: TV
authors.lastname: Strong
authors.forename: G P
authors.initials: GP
authors.lastname: Siegal
authors.forename: D T
authors.initials: DT
authors.lastname: Curiel
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000906:Antibodies / Q000008:administration & dosage* / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D001943:Breast Neoplasms / Q000378:metabolism / Q000628:therapy*; D003673:Defective Viruses / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000906:Antibodies; D018719:Erb-b2 Receptor Tyrosine Kinases