A promising strategy for cancer treatment is adoptive immunotherapy with gene-modified lymphocytes expressing a chimeric T cell receptor (cTCR) that directs tumor targeting and stimulates T cell effector functions. In this study, the activities of two novel cTCR molecules (GA gamma and GAH gamma) were investigated. Both encode a single-chain variable fragment (scFv) derived from the monoclonal antibody (MAb) GA733.2, which binds the epithelial glycoprotein 2 (EGP-2) overexpressed on a variety of human carcinomas. In the GA gamma cTCR, the scFv is directly fused to the transmembrane/cytoplasmic portions of the immunoglobulin Fc receptor (Ig FcRI) gamma subunit, which mediates T cell signaling. GAH gamma possesses an extracellular spacer composed of the CD8 alpha immunoglobulin hingelike domain inserted between the scFv and gamma chain. Activated T cells (ATCs), stimulated ex vivo using anti-CD3 MAb, were derived from either healthy donors or patients and transduced with recombinant retrovirus encoding the respective GA cTCR molecules. After culture expansion for 14 days, GA gamma-modified ATCs demonstrated enhanced targeting and lysis of EGP-2+ colon cancer cells and increased cytokine secretion. Cells transduced with the GAH gamma cTCR displayed specific lytic activity that was about twofold greater than that of GA gamma-ATCs and produced significantly more cytokine. In addition, reactivation of GAH gamma-ATC with anti-CD3 MAb prior to addition to EGP-2+ tumor target induced a further increase in lytic activity. Because the activation status influences T cell antitumor functions, our data suggest that reactivation prior to adoptive transfer would improve the clinical efficacy of GAH gamma-modified ATCs.