Recombinant Human Anti-EGFR Antibody (Nimotuzumab)

Figure 1 Binding of nimotuzumab, cetuximab and their monovalent fragments to human tumor cell lines with different EGFR expression.

(A) Immunoblot for the total EGFR and actin were performed for the indicated tumor cells.

Garrido, G., Tikhomirov, I. A., Rabasa, A., Yang, E., Gracia, E., Iznaga, N., ... & Pérez, R. (2011). Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile. Cancer biology & therapy, 11(4), 373-382.

Figure 2 Binding of nimotuzumab, cetuximab and their monovalent fragments to human tumor cell lines with different EGFR expression.

(B) Tumor cells (grey histograms) were stained with an anti-EGFR antibody (black histograms), followed by incubation with FITC-conjugated rabbit anti-rat IgG-H&L.

Garrido, G., Tikhomirov, I. A., Rabasa, A., Yang, E., Gracia, E., Iznaga, N., ... & Pérez, R. (2011). Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile. Cancer biology & therapy, 11(4), 373-382.

Figure 3 Examination of cetuximab and nimotuzumab binding kinetics using surface plasmon resonance.

(B) Under bivalent binding conditions (intended to represent binding to cells with high EGFR expression), antibodies behaved similarly and accumulated to the equivalent levels; ligand-FcEGFR chimera (dimer); analyte-100 nM anti-EGFR antibodies. Experiments were performed three times with similar results. Representative data are shown.

Garrido, G., Tikhomirov, I. A., Rabasa, A., Yang, E., Gracia, E., Iznaga, N., ... & Pérez, R. (2011). Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile. Cancer biology & therapy, 11(4), 373-382.

Figure 4 In vitro effects of nimotuzumab and cetuximab on cells with different EGFR expression levels.

(B) Cells treated with antibodies (70 nM) or their monovalent Fab fragments (140 nM) for 24 h were fixed with ice-cold methanol/acetone and stained with PI (400 μg/ml). The percentage of cells with DNA fragmentation (apoptotic cells) was analyzed by flow cytometry. For both experiments the U1906 cell line was used as a negative control for EGFR expression and the fenritenide was used as positive control in the apoptosis assay (data not shown). One representative experiment out of three is shown in each case.

Garrido, G., Tikhomirov, I. A., Rabasa, A., Yang, E., Gracia, E., Iznaga, N., ... & Pérez, R. (2011). Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile. Cancer biology & therapy, 11(4), 373-382.

Figure 5 Competition experiments performed for Nimotuzumab.

A, ELISA plates were coated with 5 μg/mL eEGFR and incubated with different mixtures containing a constant concentration of biotinylated Nimotuzumab Fab fragment and decreasing concentrations of the Cetuximab Fab fragment. OD, optical density.

Talavera, A., Friemann, R., Gómez-Puerta, S., Martinez-Fleites, C., Garrido, G., Rabasa, A., ... & Krengel, U. (2009). Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation. Cancer research, 69(14), 5851-5859.