Purpose: A monoclonal antibody (TRA-8) has been developed that binds to death receptor 5 (DR5), one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand. The purpose of this study was to evaluate in vitro the binding and cytotoxicity of TRA-8 to human breast cancer cell lines. The antitumor efficacy of TRA-8 was evaluated in a xenograft human breast cancer murine model, as a single agent and in combination with chemotherapy or radiation therapy. Experimental Design: The binding of TRA-8 to a panel of nine human breast cancer cell lines was evaluated by indirect immunofluorescence and flow cytometry. Cytotoxicity of TRA-8 alone and in the presence of Adriamycin or paclitaxel was measured in vitro using the ATP-lite assay. Antitumor efficacy was determined by treatment of nude mice bearing well-established s.c. DR5-positive 2LMP human breast cancer xenografts with TRA-8 alone or in combination with Adriamycin of paclitaxel. Tumor size and regression rates were determined. In addition, a study was carried out with TRA-8 and Adriamycin in combination with 3 Gy 60Co irradiation of 2LMP xenografts on days 9 and 17. Results: All nine human breast cancer cell lines expressed DR5 with TRA-8 reactivity varying from strongly to weakly positive. Four cell lines were sensitive to TRA-8 cytotoxicity with IC 50 of 17-299 ng/ml, whereas other cell lines had weak cytotoxicity or were resistant. In vivo studies demonstrated significant inhibition of growth of 2LMP xenografts by TRA-8 treatment alone. The combination of TRA-8 + Adriamycin or paclitaxel produced significant inhibition of tumor growth as compared with controls of either agent alone. An aggregate analysis of all 166 animals studied demonstrated that TRA-8 alone or in combination with Adriamycin, paclitaxel, or radiation produced a significant increase in tumor doubling time compared with any modality alone with mean doubling time in days of 12 (untreated), 14 (radiation), 17 (Adriamycin), 25 (paclitaxel), 39 (Adriamycin + radiation), 47 (TRA-8), 65 (TRA-8 + radiation), 71 (TRA-8 + paclitaxel), 81 (TRA8 + Adriamycin), and >140 (TRA-8 + Adriamycin and radiation). Complete tumor regressions occurred in 1 of 42 untreated animals, 1 of 54 animals receiving chemotherapy and/or radiation, and 28 of 68 animals receiving TRA-8 alone or TRA-8 combination regimens. Fourteen of those 28 complete regressions did not relapse over periods of follow-up between 99 and 171 days, with a mean of 146 ± 24 days. Conclusions: The TRA-8 anti-DR5 antibody alone or in combination with chemotherapy and/or radiation has striking antitumor efficacy in breast cancer xenograft models. Additional studies with other tumor types and chemotherapy agents are warranted. These studies support the generation of a humanized TRA-8 for introduction into early clinical trials.