Purpose:The receptor tyrosine kinase RON is critical in epithelial tumorigenesis and a drug target for cancer therapy. Here we report the development and therapeutic efficacy of a novel anti-RON antibody Zt/g4-maytansinoid (DM1) conjugates for targeted colorectal cancer (CRC) therapy. Experimental Design:Zt/g4 (IgG1a/κ) was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. Three CRC cell lines were tested in vitro to determine Zt/g4-DM1-induced RON endocytosis, cell cycle arrest, and cytotoxicity. Efficacy of Zt/g4-DM1 in vivo was evaluated in mouse xenograft CRC tumor model. Results:Zt/g4-DM1 rapidly induced RON endocytosis, arrested cell cycle at G2/M phase, reduced cell viability, and caused massive cell death within 72h. In mouse xenograft CRC models, Zt/g4-DM1 at a single dose of 20 mg/kg body weight effectively delayed CRC cell-mediated tumor growth up to 20 days. In a multiple dose-ranging study with a five injection regimen, Zt/g4-DM1 inhibited more than 90% tumor growth at doses of 7, 10, and 15 mg/kg body weight. The minimal dose achieving 50% of tumor inhibition was ~5.0 mg/kg. The prepared Zt/g4-DM1 is stable at 37°C for up to 30 days. At 60 mg/kg, Zt/g4-DM1 had a moderate toxicity in vivo with an average of 12% reduction in mouse body weight. Conclusions: Zt/g4-DM1 is highly effective in targeted inhibition of CRC cell-derived tumor growth in mouse xenograft models. This work provides the basis for development of humanized Zt/g4-DM1 for RON-targeted CRC therapy in the future.