THE LEVEL OF ERBB2 EXPRESSION PREDICTS SENSITIVITY TO THE CYTOTOXIC EFFECTS OF AN INTRACELLULAR ANTI-ERBB2 SFV

We have previously demonstrated that an intracellular antibody (sFv) d irected against erbB2 can achieve a specific cytotoxicity in erbB2 ove rexpressing cancer cells of varying histogenesis. In order to further delineate the mechanistic basis of the induced apoptosis, transient an d stable cotransfections were performed. Transient cotransfection of e rbB2 mutant and chimeric molecules demonstrated that the cytoplasmic d omain of erbB2, or the homologous cytoplasmic domain of the epidermal growth factor receptor, is required for apoptosis induction. These res ults were confirmed in assays utilizing differential derivation of sta ble clones. To examine the effects of varying ratios of the anti-erbB2 sFv and its target erbB2 we performed additional cotransfection exper iments in erbB2 negative target cells, When erbB2 levels are held cons tant, observed cytotoxicity is proportional to the amount of sFv added . In addition, when sFv levels are held constant, increasing levels of cotransfected erbB2 can overcome the apoptotic response. These result s indicate that a minimal threshold level of the sFv and its target ar e required to induce cytotoxicity. To examine this phenomenon in an er bB2 positive cell line, SKOV3 ovarian carcinoma cells were utilized to derive a stable clone expressing low levels of sFv. When this cell li ne was compared to the parental SKOV3 cell line, it was shown that les s exogenous sFv was needed to induce cytotoxicity in the clone already expressing low levels of sFv, indicating that endogenous and exogenou s levels of sFv are additive. In summary, the results presented hero i ndicate that the carboxy-terminus of the intracellular domain of the e rbB2 molecule is involved in the induction of apoptosis. Furthermore, the expression levels of the sFv and its target protein need to overco me a threshold level in order to achieve a cytotoxic response.