Background : A variety of human tumors frequently express high levels of epidermal growth factor (EGF) receptor and its ligand, transforming growth factor α (TGF-α), which in some tumors is associated with poor prognosis. Monoclonal antibodies (MAbs) that block the binding of TGF-α or EGF to the receptor can inhibit proliferation of tumor cells that express the receptor. Studies suggest that these MAbs may enhance the antitumor effects of chemotherapy. Purpose : Our purpose was to study, in vitro and in vivo, the antitumor effects of doxorubicin in combination with anti-EGF receptor MAbs against tumor cells expressing high levels of EGF receptor. Our goal was to achieve maximum initial cytoreduction with high-dose doxorubicin in association with prolonged blockade of EGF receptor with MAbs. Methods : Anti-EGF receptor MAbs 528 (isotype IgG2a) and 225 (isotype IgG1) were used in combination with doxorubicin against cells from human A431 squamous cell carcinoma and human MDA-468 breast adenocarcinoma. Both A431 and MDA-468 cells express high levels of EGF receptors and TGF-α. Cultured cells were treated with doxorubicin (range, 0–10 n M ) in the presence or absence of MAb 528 or 225 (range, 0–30 n M ). At 48 hours, doxorubicin-containing medium was removed, and treatment with antibody was continued for 5 days, when cell proliferation assays were performed. The activity of the agents and the combinations against well-established xenografts in BALB/c nude mice was also studied. In nude mice, doxorubicin was given at doses of 50–100 μg/20 g body weight on 2 successive days, and MAbs 528 and 225 were given at a dose range of 0–2 mg intraperitoneally twice a week. Results : MAbs 528 and 225 both enhanced the antitumor effects of doxorubicin against A431 and MDA-468 tumor cells, producing additive growth suppression in cell cultures. MAb 528 increased the antitumor effects of doxorubicin by 32%–42%, and similar results were obtained with MAb 225. In BALB/c athymic mice, the treatment of well-established xenografts with either doxorubicin or anti-EGF receptor MAb alone temporarily inhibited growth, but the combination of both agents substantially enhanced antitumor activity over that of doxorubicin alone in A431 and MDA-468 cell xenografts. The combination treatment of mice bearing A431 xenografts resulted in tumor eradication of 40%–100% in the surviving mice in several independent experiments. The enhanced antitumor activity was dose dependent. Conclusions : Our results suggest that anti-EGF receptor MAbs substantially enhance the effects of doxorubicin against well-established xenografts of tumor cells expressing high levels of EGF receptors. Implications : Clinical trials with anti-EGF receptor MAbs are being conducted, and trials with anti-EGF receptor MAbs combined with doxorubicin are planned. [J Natl Cancer Inst 85: 1327–1333, 1993]