TY - JOUR T1 - Construction and Molecular Characterization of Human Chimeric T-Cell Antigen Receptors Specific for Carcinoembryonic Antigen JF - Anticancer Research JO - Anticancer Res SP - 2731 LP - 2738 VL - 30 IS - 7 AU - NAOTO SHIRASU AU - HIROTOMO SHIBAGUCI AU - MOTOMU KUROKI AU - HIROMI YAMADA AU - MASAHIDE KUROKI Y1 - 2010/07/01 UR - http://ar.iiarjournals.org/content/30/7/2731.abstract N2 - Background: Chimeric T-cell antigen receptors (CAR) provide a promising approach for adoptive T-cell immunotherapy of cancer. Extensive studies on CARs have been conducted, but the detailed molecular mechanisms of the activation of a CAR-grafted T-cell remain ambiguous. This study constructed a CAR bearing anti-carcinoembryonic antigen (CEA) derived from a human monoclonal antibody (clone C2-45), and investigated the molecular basis of the CAR-mediated activation in Jurkat T-cells. Materials and Methods: A gene of a single chain fragment variable (scFv) specific for CEA was functionally cloned by the phage display method. The scFv gene was fused to human cDNAs coding for transmembrane and cytoplasmic domains of CD28 and an intracellular domain of CD3ζ. The resultant CAR45-28ζ was transiently expressed in Jurkat cells, and T-cell activation was examined by Western blotting and a cytokine production assay. A fluorescent protein-tagged ZAP-70 was used to determine whether CAR45-28ζ and ZAP-70 were co-localized at the cell surface by confocal microscopy. Results: A Western blot analysis showed CAR45-28ζ activated the ERK JNK, and p38 pathways in a CEA-dependent manner. An immunofluorescent analysis revealed the CEA-dependent formation of the signaling clusters at the antigen-CAR interface. Conclusion: CAR45-28ζ induced a wild-type T-cell receptor-like molecular event upon CEA binding, suggesting that this CAR fused gene may be useful for cancer therapy. ER -