Rapidly Manufactured CAR-T with Conserved Cell Stemness and Distinctive Cytokine-Secreting Profile Shows Improved Anti-Tumor Efficacy.

The emergence of chimeric antigen receptor T-cell (CAR-T) immunotherapy holds great promise in treating hematologic malignancies. While advancements in CAR design have enhanced therapeutic efficacy, the time-consuming manufacturing process has not been improved in the commercial production of CAR-T cells. In this study, we developed a "DASH CAR-T" process to manufacture CAR-T cells in 72 h and found the excelling anti-tumor efficacy of DASH CAR-T cells over conventionally manufactured CAR-T cells. Four different CAR-T manufacturing processes were first proposed and examined by flow cytometry in regard to cell viability, T-cell purity and activation, CAR expression, and cell apoptosis. The selected two processes, 48H DASH CAR-T and 72H DASH CAR-T, were applied to the subsequent functional assessments, including T-cell differentiation, antigen-dependent cytotoxicity and expansion, cytokines secretion profile, and in vivo anti-tumor efficacy. We demonstrated that rapidly manufactured CAR-T cells generated within 48-72 h was feasible and exhibited increased naïve and memory T-cell ratios, a distinctive secretory profile, superior expansion capacity, and enhanced in vitro and in vivo anti-tumor activity compared to conventionally manufactured CAR-T cells. Our findings suggest that "DASH CAR-T" process is a valuable platform in reducing CAR-T manufacturing time and producing high-efficacy CAR-T cells for future clinical application.