Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status

Abstract

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.

Figures

Figure 1

Expression of EGFR on the surface of NSCLC cells. Surface expression of EGFR on H460 (A), H292 (B), H1299 (C), Ma-1 (D), and H1975 (E) cells was determined by flow cytometry. Representative histograms of cells stained with an anti-EGFR mAb (red peak) or with an isotype-matched control mAb (black peak) are shown.

Figure 2

Effect of nimotuzumab on EGFR phosphorylation in NSCLC cells. H460 (A), H292 (B), H1299 (C), Ma-1 (D), and H1975 (E) cells were deprived of serum overnight and then incubated first for 15 min in the absence or presence of the indicated concentrations of nimotuzumab or gefitinib (10 μM) and then for an additional 15 min in the additional absence or presence of EGF (100 ng ml−1). Cell lysates were then subjected to immunoblot analysis with antibodies to the Tyr1068-phosphorylated form of EGFR (pEGFR) as well as with those to total EGFR.

Figure 3

Effect of nimotuzumab on the response of NSCLC cells to radiation in vitro. H460 (A), H292 (B), H1299 (C), Ma-1 (D), and H1975 (E) cells were incubated with or without 700 nM nimotuzumab in medium supplemented with 1% fetal bovine serum for 24 h, exposed to the indicated doses of γ-radiation, and then incubated in drug-free medium supplemented with 10% serum for 10–14 days for determination of colony-forming ability. Survival curves were generated after correction of colony formation observed for combined treatment with ionising radiation (IR) and nimotuzumab by that apparent for treatment with nimotuzumab alone. Data are means±s.d. of triplicates from a representative experiment.

Figure 4

Effect of nimotuzumab on the response of NSCLC cells to radiation in vivo. H460 (A), H292 (B), or Ma-1 (C) cells were injected subcutaneously in athymic nude mice. Treatment was initiated when tumours in each group achieved an average volume of approximately 170–200 mm3. Mice were treated with a single dose of nimotuzumab (1.0 mg per mouse) intraperitoneally, a single dose of γ-radiation (10 Gy), or neither (control) or both modalities, and tumour volume was determined at the indicated time points thereafter. Data are means±s.d. for seven to eight mice per group.