H60 is a murine minor histocompatibility antigen that binds to NKG2D and activates an effector phenotype in NK and T cells. In the present study, H60 was genetically fused to the tumor-targeting murine MAb TNT-3. The resultant fusion protein, named H60/TNT-3, was produced in NS0 cells and determined by ELISA to possess an H60 epitope. The Ka of H60/TNT-3 (2.43 x 10(9) M(-1)) was nearly identical to that of the parental Ab (2.22 x 10(9) M(-1)), demonstrating that addition of the H60 moiety to the N-terminus of TNT-3 heavy chain did not affect antigen affinity. In vitro, H60/TNT-3 bound and activated murine NK cells, eliciting IFN-gamma production in a higher percentage of cells than the activating NKG2D Ab A10. In vivo, H60/TNT-3 possessed a half-life of approximately 12 hours and effectively targeted tumor tissue versus control organs, with nearly 2% injected dose per gram of tumor retained after 48 hours. Finally, H60/TNT-3 was tested for antitumor efficacy in BALB/c and C57BL/6 mice bearing subcutaneous syngeneic carcinomas. Tumor volume reduction was observed in both CT26 and Lewis Lung models (53% and 52%, respectively) relative to untreated control mice. Further, Lewis Lung carcinoma-bearing mice treated with H60/TNT-3 experienced a statistically significant survival advantage. Taken together, these data characterize a new immunotherapeutic MAb with antitumor efficacy that prolonged overall survival in a resistant solid tumor model.