EpCAM (CD326) is one of the most frequently and highly expressed tumor-associated antigens known and recently has also been found on cancer stem cells derived from human breast, colon, prostate, and pancreas tumors. However, like many other tumor-associated antigens used for antibodybased immunotherapeutic approaches, EpCAM is expressed on normal tissues including epithelia of pancreas, colon, lung, bile ducts, and breast. To assess the therapeutic window of an EpCAM/CD3-bispecific single-chain antibody construct of the bispecific T-cell engager (BiTE) class, we constructed murine surrogate of MT110 (muS110) from single-chain antibodies specific for murine EpCAM and CD3 antigens. Immunhistochemical analysis showed that, with minor differences, the expression of EpCAM protein on a large variety of tissues from man and mouse was similar with respect to distribution and level. MuS110 exhibited significant antitumor activity at as low as 5 Mg/kg in both syngeneic 4T1 orthotopic breast cancer and CT-26 lung cancer mouse models. Dosing of muS110 for several weeks up to 400 Mg/kg by intraanimal dose escalation was still tolerated, indicating existence of a significant therapeutic window for an EpCAM-specific BiTE antibody in mice. MuS110 was found to have similar in vitro characteristics and in vivo antitumor activity as MT110, a human EpCAM/human CD3-bispecific BiTE antibody that currently is in formal preclinical development. [Cancer Res 2008;68(1):143-51]