In this study, human T cells were provided with a reactivity against the Lewis-Y (Le^Y) carbohydrate antigen, which is overexpressed on 70% of epithelial-derived tumors, but not normally recognized by T cells. Antitumor reactivity was achieved by transduction of T cells with a gene encoding a cell-surface chimeric receptor composed of single-chain anti-Le^Y antibody linked to an enhanced cytoplasmic signaling domain made up of CD28 and CD3-ζ. Importantly, the single-chain antibody was humanized to try to reduce potential problems of human anti-mouse antibody responses in patients receiving chimeric receptor-modified T cells in future clinical trials. T cells expressing the chimeric receptor were demonstrated to secrete cytokines and proliferate in response to receptor ligation and lysed Le^Y+ tumors in vitro. Another aspect of this study was the finding that no activity was observed against normal tissue, as represented by autologous neutrophils that express low levels of Le^Y. Significantly, systemic delivery of anti-Le^Y T cells dramatically inhibited established s.c. human ovarian OVCAR-3 tumors (a recognized difficult model to treat) in mice. Finally, we demonstrated that anti-Le^Y T cells preferentially expanded or accumulated in the tumor compared with control empty vector T cells, thereby providing mechanistic insight into the specific antitumor response. This study supports the use of humanized gene-modified T cells as a potential therapy for Le^Y+ malignancies.