Rapid manufactured CAR-T cells enriched for CD45RA-negative T cells exhibit superior persistence compared to conventional CAR-T therapy

CD19 CAR-T has achieved high remission rates in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, rapid generation with durable in vivo persistence remains a major challenge. CD45RA-negative T cells exhibit sustained antileukemic activity and preserved immune memory. Here, we developed a rapid protocol (InstanCAR-T) enriched for CD45RA-negative T cells, and systematically compared it with conventional CAR-T across in vitro functional assays, murine B-ALL xenograft models, and a Phase I clinical trial. InstanCAR-T shortened ex vivo culture to 3 days while enriching CD45RA-negative subsets, demonstrating enhanced viability, proliferation, and cytotoxicity in vitro. In vivo, InstanCAR-T achieved durable tumor control with improved expansion and long-term persistence. Clinically, high-dimensional immune profiling revealed sustained stemness, reduced exhaustion, and stable phenotypic differentiation of InstanCAR-T cells. Moreover, InstanCAR-T was well tolerated and achieved comparable overall response rates, with a numerically higher proportion of minimal residual disease-negative remission compared with ConvenCAR-T. In conclusion, InstanCAR-T enriched for CD45RA-negative T cells represents a rapid and effective CAR-T strategy with sustained proliferative capacity and superior in vivo persistence. Integrated high-dimensional profiling further highlights its enhanced immunological fitness, supporting its potential to improve clinical outcomes in B-ALL. TRIAL REGISTRATION: This trial was registered at www.clinicaltrials.gov as (NCT05309213) and (NCT06209671).