Construction and Molecular Characterization of Human Chimeric T-Cell Antigen Receptors Specific for Carcinoembryonic Antigen

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. Adoptive immunotherapy involving the transfer of lymphokine-activated killer (LAK) cells or tumor infiltrating lymphocytes (TILs) has attracted significant attention (1, 2). However, the clinical application of these therapies has not necessarily worked well due to the weak trafficking ability of LAK cells to the tumor site and difficulties in isolating the effector populations and their expansion with high-dose interleukin (IL) -2 (3, 4). In addition, the effectiveness of such therapies and other HLA-dependent therapy, such as cancer vaccination, cannot always be expected, since HLA- peptide complexes are frequently lost or down-regulated in many types of human tumors (5, 6).