The Her-2/neu oncogene, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. Antibodies directed to this molecule have been previously shown to have an antitumor effect in vivo. In an attempt to understand the mechanisms of the antitumor activity, we generated 2 monoclonal antibodies (mAbs), HRO G1 and HRT G1, that recognize different epitopes on Her-2/neu. Both of the mAbs bound HER2/neu on the tumor surface, resulting in phosphorylation of HER2/neu. We also generated IgG2a and IgG2b mAbs from these 2 mAbs, respectively. The results of in vitro studies showed that these anti-Her-2/neu mAbs could not inhibit the growth of the tumor cells that express Her-2/neu molecules by themselves. However, in an antibody-dependent cellular cytotoxicity study using mouse splenocytes as effector cells, HRT mAbs had antitumor activities superior to those of HRO mAbs, indicating that the epitope specificity may also partake in antibody-dependent cellular cytotoxicity with antibody isotype. In a complement-dependent cytotoxicity study, the IgG2a and IgG2b mAbs showed stronger effects than IgG1 isotype mAbs irrespective of the epitope specificities. The results of in vivo studies also showed that HRT mAbs had superior antitumor activity to those of HRO mAbs. The antitumor activity was most prominent in the HRT G2b isotype among HRT mAbs. HRT G1 also showed a moderate antitumor effect, while HRT G2a showed only slight inhibition effect. These data indicate that both the epitope specificity and the differences in Fc region of mAbs could play important roles in the antitumor activities. © 2002 Wiley-Liss, Inc. Key words: Her-2/neu; monoclonal antibody; isotype; epitope; antitumor effectThe Her-2/neu gene encodes a M r 185 kDa transmembrane protein that is a member of the type I family of growth factor receptors. 1,2 Amplification of this gene results in overexpression of the 185-kd encoded receptor tyrosine kinase, which is homologous to the EGF 3 receptor. 3-5 However, unlike the EGF receptor, which binds many known ligands, no direct ligand of Her-2/neu has been reported. 6 The Her-2/neu protein was found to be amplified and overexpressed in several types of human adenocarcinomas, especially in tumors of the breast and the ovary. 5,7,8 The overexpression was correlated with short time to relapse and poor survival of breast cancer patients, 9 -11 suggesting that Her-2/neu overexpression likely plays a critical role in the development of human cancers. Several lines of evidence also support a direct role of Her-2/neu in the pathogenesis and clinical aggressiveness of Her-2/neu-expressing tumors. 12 In fact, a large number of published studies demonstrated that Her-2/neu overexpression is a cause of human cancer and not just a consequence. Therefore, Her-2/neu oncogene is an excellent target for development of therapeutic agents specific for Her-2/neu overexpressing human cancers.A number of approaches have been used to therapeutically target Her-2/neu-overexpressing cancers. A common appr...