AN INTRACELLULAR ANTI-ERBB-2 SINGLE-CHAIN ANTIBODY IS SPECIFICALLY CYTOTOXIC TO HUMAN BREAST-CARCINOMA CELLS OVEREXPRESSING ERBB-2

We previously demonstrated that delivery of a gene encoding an anti-er bB-2 intracellular single-chain antibody (sFv) resulted in down-regula tion of cell surface erbB-2 levels and induction of apoptosis in erbB- 2 overexpressing ovarian cancer cells. Based upon these findings, we h ypothesized that human breast carcinomas overexpressing erbB-2 would b e similarly affected by this genetic intervention. We evaluated the ph enotypic effects resulting from intracellular expression of the anti-e rbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, B T-474, MCF-7 and MDA-MB-231. Recombinant adeno-viruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directe d anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and micr oscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quant ified 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 th e 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 show ed no change in the rate of cell proliferation following this treatmen t The cytotoxic effects generated in the first three lines were a cons equence 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 sus ceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overe xpressed the erbB-2 protein while the cell lines demonstrating no resp onse to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowes t levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accompl ished in the context of breast carcinoma. Furthermore, more erbB-2 lev els in breast tumor cells may be predictive of their sensitivity to sF v-mediated killing. The ability to accomplish selective cytotoxicity o f breast cancer cell lines overexpressing the erbB-2 tumor marker shou ld allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.