The anti-ErbB2 humanized antibody trastuzumab was approved for ErbB2-positive metastatic gastric and gastro-esophageal junction cancer in 2010. Despite the effectiveness of trastuzumab, its efficacy remains variable and often modest. Thus, there is an urgent need to improve ErbB2-targeting therapy. Down-regulation of surface receptors induced by monoclonal antibody (mAb) contributes to its antitumor efficacy. Previous studies have demonstrated that if two anti-ErbB2 mAbs did not compete with each other for binding to ErbB2, the combination of them can enhance ErbB2 internalization. In the present study, we investigated ErbB2 internalization-inducing ability of non-competitive anti-ErbB2 mAb combinations and surprisingly found that most of the mAb combinations tested did not down regulate ErbB2. Only 4 of 18 non-competitive mAb pairs efficiently induced ErbB2 internalization. Interestingly, although the non-competitive anti-ErbB2 mAbs trastuzumab and pertuzumab, either alone or in combination, were ineffective at inducing ErbB2 internalization, TPL, a bispecific antibody engineered from trastuzumab and pertuzumab, potently down-regulated the ErbB2 molecule. Importantly, TPL exhibited a far greater antitumor effect on ErbB2-overexpressing gastric cancer cell line than trastuzumab plus pertuzumab, suggesting that it may be a promising agent for the treatment of gastric cancer. (C) 2016 Elsevier Inc. All rights reserved.