Background Tumor-associated antigens (TAAs) have been attractive targets for immunotherapy. Current chimeric antigen receptor T cell (CAR-T) therapy can target TAA molecules expressed on the cell surface. However, a large proportion of TAA is expressed intracellularly, which cannot be recognized by conventional CAR-T cells. To overcome this obstacle, we have recently established a novel CAR utilizing a single-chain variable fragment (scFv) antibody which recognizes MAGE-A4-derived peptide/HLA-A*02:01 complex, enabling to target the intracellular tumor-specific antigen MAGE-A4. However, there is a substantial need to explore for more functional CAR structures. Therefore, we further designed a novel CAR (Hybrid-TCR) in which constant regions of TCR beta and alpha fused with VH and VL domains of the scFv antibody, respectively. In this study, we tried to investigate whether peptide/MHC-specific Hybrid-TCR cells could show further enhanced functions in vitro and in vivo compared with conventional CAR-T cells
Methods Both CAR and Hybrid-TCR T cells can specifically recognize MAGE-A4 (230–239) peptide (GVYDGREHTV) presented by HLA-A*02:01 by utilizing the same scFv antibody as an antigen receptor domain. To compare the in vitro functions, MAGE-A4 CAR or Hybrid-TCR T cells were co-cultured with SK-MEL-37 (A2+, MAGE-A4+) or HCT116(A2+, MAGE-A4-) cell line. Cytotoxic function was assessed after 3 hours co-cultivation, and cytokine secretion was evaluated after 24hours co-cultivation. For In vivo experiments, we used a tumor-bearing immunodeficient NOG mice model in which NW-MEL-38 (A2+, MAGE-A4+) cells were inoculated on their back. CAR or Hybrid-TCR cells were injected intravenously on day 4 after tumor inoculation.
Results Although the molecule expression level of CAR T cells is much higher than that of Hybrid-TCR cells, Hybrid-TCR cells show stronger efficacies of anti-tumor functions in vitro and in vivo. Particularly, adoptive transfer of Hybrid-TCR cells significantly suppressed the growth of NW-MEL-38 tumor. Of note, Hybrid-TCR cells showed minimal activation (tonic signaling) induced by antigen-independent clustering of the CAR receptor compared to the conventional CAR-T cells.
Conclusions We observed that a new form of CAR (Hybrid-TCR) has improved the efficacies of anti-tumor functions in vitro and in vivo compared with a conventional form of CAR. Further studies focused on these superior functions of Hybrid-TCR cells are under investigation.
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