BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 226, 59–69 (1996) ARTICLE NO. 1311 ErbB Receptor Activation, Cell Morphology Changes, and Apoptosis Induced by Anti-Her2 Monoclonal Antibodies Yoshiko Kita,* ,1 Julia Tseng,* Thomas Horan,² Jie Wen,² John Philo,² David Chang,* Barry Ratzkin,‡ Robert Pacifici,‡ David Brankow,§ Sylvia Hu,§ Yi Luo,§ Duanzhi Wen,§ Tsutomu Arakawa,² and Margery Nicolson* *Department of Immunology, ² Department of Protein Chemistry, ‡Department of Molecular Biology, and §Department of Mammalian Cell Molecular Biology, Amgen Inc., Amgen Center, Thousand Oaks, California 91320 Received July 12, 1996 A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells. These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphor- ylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb, mAb74, induced cell morphology changes and apoptosis.  1996 Academic Press, Inc. There have been numerous studies showing that high expression of erbB2/Her2 tyrosine kinase receptor correlates with poor prognosis in patients with breast cancer (1,2,3,4). Her2 is a member of the epidermal growth factor (EGF) receptor subfamily, which includes EGF receptor, and Her3 and Her4 receptors (5,6). EGF, transforming growth factor-a, amphiregulin, heparin binding EGF and betacellulin are known as ligands of the EGF receptor. Neu differenti- ation factor (NDF) or heregulin and other structurally related ligands including p25 from MDP- activated macrophage conditioned media, NAF, p75 from SKBR3 conditioned media, NEL- GF, ARIA and GGF all have been shown to increase tyrosine phosphorylation of the Her2 receptor and, therefore, were initially assumed to be ligands for the Her2 receptor (7,8,9,10,11,12,13,14). There is now convincing evidence that NDF neither binds directly to Her2 nor stimulates its kinase activity (15) but rather binds to Her3 or Her4 and stimulates tyrosine phosphorylation of these receptors (6,16,17). The conventional approach to circumvent the absence of ligand is to generate a ligand-like monoclonal antibody (mAb). In fact, several groups have generated anti-Her2 mAbs using cells expressing high levels of p185 Her2 for immunizations (18,19,20,21). The p185 Her2 overex- pressing cells possibly coexpress p180 Her3 and /or p180 Her4 , and therefore the mAbs using those cells as an antigen may cross-react to p180 Her3 and/or p180 Her4 . In addition, Her2 expressed on 1 To whom correspondence should be addressed. Abbreviations used: EGF, epidermal growth factor; NDF, neu differentiation factor; MDP, muramyl dipeptide; NAF, neu protein-specific activating factor; NEL-GF, neu erbB2 ligand-growth factor; ARIA, acetylcholine receptor inducing activity; GGF, Glial growth factor; mAb, monoclonal antibody; CHO, Chinese hamster ovary; sHer2, soluble Her2 receptor; HEG, a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and intracellular domains of EGF; EGFR, EGF receptor; PBS, Dulbecco’s phosphate-buffered saline; HRP, horseradish peroxidase; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 59