Biological Activity In Vitro of Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies with Different Affinities

Abstract

Epidermal growth factor receptor (EGFR) is frequently overexpressed in epithelial tumors and is associated with a poor prognosis. An increasing interest in developing anti-EGFR therapies has resulted in the evaluation of monoclonal antibodies with the capacity to bind to the EGFR, inhibiting EGFR-dependent cellular transformation. A differential toxicity and therapeutic effect in vivo are associated with the affinity and isotype of the molecule. In this study, we examined the biological activities of three monoclonal antibodies (MAbs) -- Ior egf/r3 (mouse IgG2a, 10(-9) M), Nimotuzumab (humanized IgG1, 10(-9) M), and Cetuximab (human/mouse chimeric IgG1, 10(-10) M) -- considering inhibition of cell proliferation, apoptosis, and complement-mediated cell death in squamous cell carcinoma A431 in vitro. All the antibodies bound to the EGFR on these cells, inhibiting the receptor phosphorylation, as measured by flow cytometry, inmunocytochemistry, and Western blot. Exposure to the different antibodies inhibited cell proliferation in culture in a range from 50 to 80% compared to controls. Furthermore, similar capabilities to induce either complement-mediated cytotoxicity (ranging between 70 and 90%) or a two-fold increase in the rate of apoptotic cells were found when tumor cells were exposed to the antibodies. These results suggest that the affinity between specific anti-EGFR antibodies and its receptor could affect, but not determine their biological activity at least in those cell lines that exhibit high sensitivity to withheld EGFR. Our findings also confirm previous evidences that blocking EGFR in A431 cells by means of antibodies significantly changes tumor cell biology by promoting apoptosis while decreasing tumor cell proliferation.

Radiosensitisation of U87MG brain tumours by anti-epidermal growth factor receptor monoclonal antibodies

“…On the other hand, tumours in mice treated with cetuximab exhibited a complete abrogation in the levels of phosphorylation of EGFR and ERK even when the antibody was administered as a single agent. These results indicate a higher capacity to block EGFR signalling in tumour cells for cetuximab, compared with nimotuzumab, accordingly with earlier findings (Diaz Miqueli et al, 2007). Moreover, the different Targeting of CD133 þ radioresistant cancer stem cell in U87MG tumours in vivo by anti-EGFR mAb…”

Pharmacodynamic Trial of Nimotuzumab in Unresectable Squamous Cell Carcinoma of the Head and Neck: A SENDO Foundation Study

“…In addition, several reports have shown similar antitumor effect between nimotuzumab and other anti-EGFR antibodies in tumors overexpressing EGFR. In human A431 squamous cell carcinoma model, both cetuximab and nimotuzumab showed similar capacity to inhibit EGFR phosphorylation and to induce either complement-mediated cytotoxicity or apoptosis (38). In A431 xenografts, the response was total tumor regression with either nimotuzumab or cetuximab (39).…”

Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling

“…After 24 h, the medium was changed to 1% (v/v) FCS for serum starvation in the presence of 100 nM cetuximab or DARPin for another 24 h. As a control, cells were incubated with serum starvation medium alone. After this treatment, cells were stimulated with 10 ng/ml (1.6 nM) EGF for 15 min at 37°C and subsequently washed with PBS and incubated in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) SDS, 1 mM sodium orthovanadate, and 1 tablet of PhosSTOP (Roche Applied Science)) (43)(44)(45). Total protein concentration in the lysates was determined by a BCA assay (Sigma), and 20 g of total protein/ sample was separated by 10% SDS-PAGE and transferred to an Immobilon FL membrane (Millipore) by Western wet blotting.…”