Abstract Text: Autologous Chimeric Antigen Receptor (CAR)-T cell therapies are effective against B cell lymphomas, yet are hindered by challenges such as suboptimal T cell quality, treatment delays, and financial constraints. Allogeneic CAR-T therapy, if successful in preventing Graft versus Host disease (GvHD) and rejection, could overcome these limitations. Prior strategies involving genome editing to eliminate T Cell receptor (TCR) and Major Histocompatibility Complex class I (MHC-I) expression have encountered issues of genotoxicity, off-target editing, and procedural complexity. This study introduces an innovative approach that sidesteps genome editing by employing a chimeric protein.
The chimeric protein, comprising a single chain variable fragment targeting CD3ε fused to US6 protein from Human Cytomegalovirus, serves as a retention motif. This construct hijacks CD3 within the endoplasmic reticulum (ER) while inhibiting the Transporter Associated with Antigen Processing (TAP), thereby abolishing class I presentation. Expression of the CD3-US6 chimeric protein in T cells successfully downregulated TCR and HLA-I expression.
Furthermore, CD3-US6 T cells demonstrated reduced reactivity against allogeneic stimuli in vitro and in vivo compared to control T cells. The rejection response elicited on CD3-US6 T cells was also attenuated. Evaluation of CAR-T cell activity in both control and CD3-US6 T cells revealed comparable efficacy in vitro and in vivo. In summary, we show that the expression of CD3-US6 and CAR in T cells achieves effective TCR and MHC-I downregulation while preserving CAR-T cell functionality. This innovative strategy avoids the pitfalls associated with genome editing, presenting a promising avenue for the development of allogeneic CAR-T cell therapies.
