Human epidermal growth factor receptor 2 (HER2/neu) is an important target for 
the treatment of the breast cancers in which it is overexpressed. However, no 
approved anti-HER2/neu therapy is available for the majority of breast cancer 
patients, who express HER2/neu at low levels (with scores of 1+ or 
2+/fluorescence in situ hybridization-negative). The trifunctional antibody 
ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence 
of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and 
accessory cells form to cause tumor cell lysis. In a phase I trial with 
metastatic breast cancer patients, ertumaxomab could be applied safely and 
resulted in radiographically confirmed clinical responses. In this study, we 
compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that 
express HER2/neu at low and high levels. Under optimal conditions for 
trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only 
ertumaxomab was able to mediate the elimination of tumor cell lines that express 
HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a 
Th1-based cytokine release, a unique mode of action of trifunctional antibodies. 
Competitive binding studies with trastuzumab and 520C9 mapped the binding site 
of ertumaxomab to the extracellular regions II and III of the HER2/neu 
ectodomain. This site is distinct from the binding site of trastuzumab, so that 
HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence 
of high amounts of trastuzumab. The ability of ertumaxomab to induce 
cytotoxicity against various tumor cell lines, including those with low HER2/neu 
antigen density, may provide a novel therapeutic option for breast cancer 
patients who are not eligible for trastuzumab treatment.