1. ¹Department of Antibody Development, TRION Research GmbH, Martinsried, Germany
2. ²Clinical Cooperation Group Molecular Oncology, GSF-Research Centre for Health, and Environment and Department of Otorhinolaryngology, Ludwig-Maximilians-University, Munich, Germany
3. ³Department of Preclinical Research and Development, Fresenius Biotech GmbH, Gräfelfing, Germany
4. ⁴CEO, TRION Pharma GmbH, Munich, Germany

Correspondence: Dr P Ruf, TRION Research GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany; E-mail: peter.ruf@trionresearch.de

⁵Current address: LMU Biozentrum, Martinsried, Germany.

Received 15 March 2007; Revised 6 June 2007; Accepted 19 June 2007; Published online 10 July 2007.

Abstract

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is frequently overexpressed in a variety of carcinomas. This pan-carcinoma antigen has served as the target for a plethora of immunotherapies. Innovative therapeutic approaches include the use of trifunctional antibodies (trAbs) that recruit and activate different types of immune effector cells at the tumour site. The trAb catumaxomab has dual specificity for EpCAM and CD3. In patients with malignant ascites, catumaxomab significantly increased the paracentesis-free interval, corroborating the high efficacy of this therapeutic antibody. Here, we characterised the monoclonal antibody (mAb) HO-3, that is, the EpCAM-binding arm of catumaxomab. Peptide mapping indicated that HO-3 recognises a discontinuous epitope, having three binding sites in the extracellular region of EpCAM. Studies with glycosylation-deficient mutants showed that mAb HO-3 recognised EpCAM independently of its glycosylation status. High-affinity binding was not only detected for mAb HO-3, but also for the monovalent EpCAM-binding arm of catumaxomab with an excellent K_D of 5.6 10^-10 M. Furthermore, trAb catumaxomab was at least a 1000-fold more effective in eliciting the eradication of tumour cells by effector peripheral blood mononuclear cells compared with mAb HO-3. These findings suggest the great therapeutic potential of trAbs and clearly speak in favour of EpCAM-directed cancer immunotherapies.