Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR α/β chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA–resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients. [Cancer Res 2009;69(23):9003–11]