Antibody-dependent cellular cytotoxicity of cetuximab against tumor cells with wild-type or mutant epidermal growth factor receptor

Abstract

Cetuximab (Erbitux, IMC-C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody-dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild-type or mutant EGFR. ADCC activity and complement-dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement-dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild-type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose-dependent manner, but not in 293M cells (<10%). These results indicate that ADCC-dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface.

Figure 1

Epidermal growth factor receptor (EGFR) expression and cetuximab‐mediated antibody‐dependent cellular cytotoxicity (ADCC) activity in the tumor cell lines. (a) Correlation between the expression of cetuximab‐combined EGFR and R‐1‐combined EGFR. The values for cetuximab‐combined EGFR expression are shown for a concentration of 1 µg/mL. The correlation coefficient between the results of these assays was 0.999. (b) Cetuximab‐mediated ADCC activity in tumor cell lines at concentrations ranging from 0.001 to 10 µg/mL was determined using the CytoTox 96 Non‐Radioactive Cytotoxicity Assay. (c) Correlation between expression values of cetuximab‐combined EGFR and ADCC activity in the seven tumor cell lines. The values for cetuximab‐combined EGFR expression and cetuximab‐mediated ADCC activity are shown for a concentration of 1 µg/mL. The correlation coefficient between the results of these assays was 0.924. (d) Correlation between expression values of cetuximab‐combined EGFR and ADCC activity in transfected HEK293 cell lines. The correlation coefficient between the results of these assays was 0.952.

Figure 2

Growth inhibitory effect of cetuximab on non‐small cell lung cancer cell lines: (a) A431; (b) PC‐9; (c) PC‐14; (d) A549; (e) Ma‐1; (f) 11_18; and (g) K562. Cell growth was not inhibited at any concentration, even a high concentration (10 µg/mL). The figure shows the dose‐dependent growth inhibitory effect of gefitinib with various concentrations of cetuximab (0–10 µg/mL). Results are expressed as percentages of the untreated control value. The data shown are the mean + SD values from triplicate experiments.

Figure 3

Growth inhibitory effects of combining gefitinib and cetuximab‐mediated antibody‐dependent cellular cytotoxicity (ADCC). The figure shows dose‐dependent growth inhibitory effects of gefitinib with various concentrations of cetuximab (solid circle, 0 µg/mL; solid square, 0.1 µg/mL; open circle, 1.0 µg/mL; solid triangle, 10 µg/mL). Results are expressed as a percentage of the untreated control value. The data shown represent the median values of triplicate experiments.

Figure 4

Effects of cetuximab on phosphorylation of epidermal growth factor receptor (EGFR), Akt and p44/42 MAPK in non‐small cell lung cancer cell lines. (a) EGFR mutant cell line PC‐9 (with the E746_A750del mutation). (b) EGFR wild‐type cell line PC‐14. (c) EGFR wild‐type cell line A549. Cells were treated with cetuximab at the indicated concentrations for 24 h. Immunoblots of cellular protein were analyzed for phosphorylated and total EGFR, p44/42 MAPK and Akt. The experiments were repeated at least twice.