Abstract #2763: Binding properties of the anti-EGFR monoclonal antibody, nimotuzumab, limit interaction with the EGFR in renal and epidermal cells
Authors: Greta Garrido, Ailem Rabasa, Elias Gracia, Ilia Tikhomirov, Rolando Pérez
Journal: Cancer Research (2009)

Nimotuzumab (Kd=10-8 M) has demonstrated anti-tumor activity in preclinical and clinical trials in absence of severe side-effects commonly observed with other anti-EGFR mAbs, such as cetuximab (Kd=10-10M). The severe toxicities associated the other anti-EGFR agents (acneiform rash and hypomagnesaemia), are believed to be caused by interaction of the mAbs with EGFR in normal tissues. Binding of the nimotuzumab to cancer cell lines has been previously demonstrated to require bivalent binding (1), which is dependent on EGFR density. Healthy cells are thought to express low EGFR levels, therefore it is postulated that the bivalent binding properties of nimotuzumab may offer a mechanistic explanation for the low incidence and severity of toxicities seen in clinical trials. Here we characterize EGFR expression of human renal cortical epithelial cells (HRCE) and human epidermal cells (HEC) and investigate whether differences in monovalent/bivalent binding profiles may be responsible for nimotuzumab and cetuximab exhibiting different toxicity profiles. HRCE were obtained from Lonza Group (Switzerland). HEC were prepared from normal human skin (breast) by trypsinization, as previously described (2). The cell line A431 (106 EGFR/cell) was the positive control. EGFR expression of cell lines was characterized by protein immunoblot. The cells were incubated with increasing concentrations of nimotuzumab, cetuximab (0-1mM) and Fab fragments (0-2mM) of the mAbs and analyzed by FACS. FITC-conjugated anti-human antibodies were used (IgG and Fab specific); data expressed as fluorescence mean intensity (FMI). HRCE and HEC expressed low EGFR levels compared to A431. Binding of cetuximab to HRCE and HEC was significant and concentration dependent. Binding of nimotuzumab to the cell lines was lower by a factor of 2-3 compared to cetuximab. Cetuximab Fab bound to all cell lines and binding was similar to that observed with the whole mAb (equimolar concentrations). Nimotuzumab Fab binding to HRCE and HEC was undetectable regardless of the Fab concentrations. Binding patterns of both mAbs to A431 were similar at all concentrations. Only marginal nimotuzumab Fab binding to A431 was detected at the highest concentrations. To the best of our knowledge, this is the first report to characterize binding of nimotuzumab and cetuximab to HRCE and HEC. This translational research demonstrates that, unlike cetuximab, attachment of nimotuzumab requires bivalent binding. This leads to the nimotuzumab discriminating between cells that over-express EGFR, while minimizing interaction with healthy tissue, such as renal and skin cells, that express normal EGFR levels. This intrinsic property of nimotuzumab may be responsible for sparing of healthy tissues by the mAb observed in clinical studies. 1. Tikhomirov et al, AACR Meeting Abstracts (2008) 2008: A36 2. Eisinger et al., PNAS 1979, 76:5340-5344.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2763.