Physiological lentiviral vectors for the generation of improved CAR-T cells Tristán Manzano, María Maldonado Pérez, Noelia Justicia Lirio, Pedro Muñoz Fernández, Pilar Cortijo Gutiérrez, Marina Pavlovic, Kristina Sánchez Hernández, Sabina Aguilar González, Araceli Marañón, Concepción Marchal Corrales, Juan Antonio Benabdellah, Karim Martín Molina, Francisco Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products. 2022-09-13T10:49:33Z 2022-09-13T10:49:33Z 2022-05-18 journal article María Tristán-Manzano... [et al.]. Physiological lentiviral vectors for the generation of improved CAR-T cells, Molecular Therapy - Oncolytics, Volume 25, 2022, Pages 335-349, ISSN 2372-7705, [https://doi.org/10.1016/j.omto.2022.05.003] http://hdl.handle.net/10481/76676 10.1016/j.omto.2022.05.003 eng http://creativecommons.org/licenses/by/4.0/ open access Atribución 4.0 Internacional Cell Press