Selection of monoclonal antibodies which induce internalization and phosphorylation of P185HER2 and growth inhibition of cells with HER2/neu gene amplification

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Abstract
In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu‐3), which demonstrated an over‐expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14‐amino‐acid carboxy‐terminal‐HER2/neu. Two MAbs, designated MGR2 (IgG,) and MGR3 (IgG2), selected for reactivity on Calu‐3 and negativity on A431 live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185‐kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross‐competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the pl85HES2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.