Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478

Abstract

Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2-7 (de2-7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2-7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2-7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.

Fig. 1.

Biodistribution of soluble AG1478. Mice with U87MG.Δ2–7 xenografts (n = 4) were injected i.p. with AG1478 at the doses indicated and killed 30 min later. Xenografts and tissues were removed and analyzed for levels of AG1478. Data are expressed as mean AG1478 concentration. (Bars = SD.)

Fig. 2.

Immunohistochemistry analysis of U87MG.Δ2–7 xenografts treated with AG1478. U87MG.Δ2–7 xenografts were collected 30 min after injection of vehicle (Left) or AG1478 (1,000 μg) (Right) as described in Fig. 1. Frozen sections were cut and stained for expression of total EGFR, phosphorylated EGFR, and phosphorylated Akt.

Fig. 3.

Treatment of A431 (A) and U87MG.Δ2–7(B) xenografts with AG1478 in a preventative model. A431 or U87MG.Δ2–7 cells were injected s.c. into both flanks of nude mice (n = 4) at day 0. Mice were then injected i.p. three times per week for 2 weeks with the indicated dose of AG1478 on days 0, 2, 4, 7, 9, and 11 post tumor inoculation. Data are expressed as mean tumor volume. (Bars = SE.)

Fig. 4.

Treatment of established U87MG.Δ2–7 (A) and A431 (B) xenografts with AG1478 and cisplatin in combination. U87MG.Δ2–7 or A431 cells were injected s.c. into both flanks of nude mice (n = 5) at day 0. Once xenografts were established, mice were injected i.p. three times with AG1478 (400 μg) or cisplatin (1.0 and 1.5 mg/kg for U87MG.Δ2–7 and A431 xenografts, respectively) as single agents, or in combination, on days 13, 15, and 17 for U87MG.Δ2–7 xenografts or days 7, 9, and 11 for A431 xenografts. Data are expressed as mean tumor volume. (Bars = SE.)

Fig. 5.

Treatment of established U87MG.Δ2–7 xenografts with AG1478 and temozolomide in combination. U87MG.Δ2–7 were injected s.c. into both flanks of nude mice (n = 5) at day 0. Treatment of mice commenced once xenografts were established. In A, mice were injected i.p. three times with AG1478 (400 μg) or temozolomide (2.5 mg/kg) as single agents, or in combination, on days 13, 15, and 17. In B, mice were injected with AG1478 (500 μg) on days 8, 10, 12, 15, 17, and 19 or temozolomide (5 mg/kg) on days 8 and 15 as single agents or in combination. Data are expressed as mean tumor volume. (Bars = SE.)

Fig. 6.

Combination treatment of A431 (A) and U87MG.Δ2–7 (B) xenografts with AG1478 and mAb 806 in a preventative model. A431 or U87MG.Δ2–7 cells were injected s.c. into both flanks of nude mice (n = 5) at day 0. Mice were then injected i.p. three times per week for 2 weeks with mAb 806 (0.1 mg) on days -1, 1, 3, 6, 8, and 10 or AG1478 (400 μg) on days 0, 2, 4, 7, 9, and 11 as single agents or in combination. Data are expressed as mean tumor volume. (Bars = SE.)

Fig. 7.

Combination treatment of A431 (A), U87MG.Δ2–7 (B), and HN5 (C) xenografts with AG1478 and mAb 806 in an established model. A431, U87MG.Δ2–7, or HN5 cells were injected s.c. into both flanks of nude mice (n = 6) at day 0. Treatment of mice commenced when xenografts were established. (A) Mice with A431 xenografts were then injected i.p. three times per week for 2 weeks with mAb 806 (0.1 mg) on days 5, 7, 9, 12, 14, and 16 or AG1478 (400 μg) on days 6, 8, 10, 13, 15, and 17 as single agents or in combination. (B) Mice with U87MG.Δ2–7 xenografts were injected (as for A) with mAb 806 on days 12, 14, 16, 19, 21, and 23 or AG1478 on days 13, 15, 17, 20, 22, and 24. (C) Mice with HN5 xenografts were injected (as for A) with mAb 806 on days 13, 15, 17, 20, 22, and 24 or AG1478 on days 14, 16, 18, 21, 23, and 25. Data are expressed as mean tumor volume. (Bars = SE.)

Fig. 8.

In vitro effects of AG1478 and mAb 806. (A) Antiproliferative activity of AG1478 and mAb 806. A431 and U87MG.Δ2–7 cells were treated in vitro with AG1478 (6 μM) or mAb 806 (10 μg/ml) alone or in combination, and the data were expressed as percentage inhibition of cell growth. (Bars = SD.) (B) Effect of AG1478 treatment on antibody reactivity. A431 cells were treated for 10 min with AG1478 at the doses indicated, after which cells were lysed and the amount of EGFR was recognized by mAb 806 or 528 quantified by ELISA. Data are expressed as the percentage increase in antibody reactivity. (Bars = SD.)