Engineering CD3 Subunits with Endoplasmic Reticulum Retention Signal Facilitates Allogeneic CAR T Cell Production

The success of autologous CAR T cell therapies has driven interest in developing off-the-shelf allogeneic CAR T cells as a scalable and readily available option for broader patient access. Most of the current approaches involve the knockout of T cell receptor (TCR) subunits via genome editing for preventing graft-versus-host disease (GvHD). However, clinical translation of these methods faces challenges due to manufacturing complexities and emerging safety concerns like unintended long deletions and chromosomal loss. In this study, we explored an alternative approach by engineering synthetic CD3 subunits containing an endoplasmic reticulum retention (ERR) signal to suppress TCR surface expression by disrupting its trafficking to the plasma membrane. We screened multiple CD3-ERR candidate designs to identify the construct with the highest efficacy in TCR downregulation. The selected candidate, CD3ζ-ERR, was further characterized, demonstrating its ability to minimize TCR-mediated activation and alloreactivity without affecting T cell phenotype, cell cycle and cytokine-induced expansion. Subsequent assays revealed that CD3ζ-ERR CD19 CAR T cells retained their CAR-mediated cytotoxic function against CD19 ⁺ malignant cells. This study presents an alternative approach for TCR downregulation that circumvents genome editing. By using a transgene compatible with conventional viral vector delivery, this approach holds promise for scalable clinical-grade manufacturing of allogeneic CAR T cell therapies.