Chimeric antigen receptor with novel intracellular modules improves antitumor performance of T cells

The excessive cytokine release and limited persistence represent major obstacles to successful chimeric antigen receptor T (CAR-T) cell therapy in solid tumors. Conventional CARs employ an intracellular domain (ICD) from the ζ chain of CD3 as a signaling module, and it is largely unknown how alternative CD3 chains potentially contribute to CAR design. Here, we obtained a series of CAR-T cells against HER2 and mesothelin using a domain containing one immunoreceptor tyrosine-based activation motif from different CD3 subunits as the ICD of CARs. While these reconstituted CARs conferred sufficient antigen-specific cytolytic activity on equipped T cells, they elicited low tonic signal, ameliorated CAR-T cell exhaustion and facilitated memory differentiation. Intriguingly, the CD3ε-derived ICD outperformed others in generation of CAR-T cells that produced minimized cytokines. Mechanistically, CD3ε-based CARs displayed a restrained cytomembrane expression on engineered T cells, which was ascribed to endoplasmic reticulum retention mediated by the carboxyl terminal basic residues. The present study demonstrated the applicability of CAR reconstitution using signaling modules from different CD3 subunits, and depicted a novel pattern of CAR expression that reduces cytokine release, thus paving a way for generation of CAR-T cells with improved safety and persistence against diverse tumor antigens.