Bachiller, MireiaBarceló-Genestar, NinaRodriguez-Garcia, AlbaAlserawan, LeticiaDobaño-López, CèliaGiménez-Alejandre, MartaCastellsagué, JoanColell, SalutOtero-Mateo, MarcAntoñana-Vildosola, AsierEspañol-Rego, MartaFerruz, NoeliaPascal, MarionaMartín-Antonio, BeatrizAnguela, Xavier MFillat, CristinaOlesti, EulàliaCalvo, GonzaloJuan, ManelDelgado, JulioPérez-Galán, PatriciaUrbano-Ispizua, ÁlvaroGuedan, Sonia2025-01-172025-01-172025-01-08Bachiller M, Barceló-Genestar N, Rodriguez-Garcia A, Alserawan L, Dobaño-López C, Giménez-Alejandre M, Castellsagué J, Colell S, Otero-Mateo M, Antoñana-Vildosola A, Español-Rego M, Ferruz N, Pascal M, Martín-Antonio B, Anguela XM, Fillat C, Olesti E, Calvo G, Juan M, Delgado J, Pérez-Galán P, Urbano-Ispizua Á, Guedan S. ARI0003: Co-transduced CD19/BCMA dual-targeting CAR-T cells for the treatment of non-Hodgkin lymphoma. Mol Ther. 2025 Jan 8;33(1):317-335.1525-0016https://hdl.handle.net/20.500.12105/26052CD19 CAR-T therapy has achieved remarkable responses in relapsed/refractory non-Hodgkin lymphoma (NHL). However, challenges persist, with refractory responses or relapses after CAR-T administration linked to CD19 loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual targeting could enhance long-term efficacy. We optimized different dual-targeting approaches, including co-transduction of two lentiviral vectors, bicistronic, tandem, and loop and pool strategies, based on our academic anti-CD19 (ARI0001) and anti-BCMA (ARI0002h) CAR-T cells. Comparison with anti-CD19/CD20 or anti-CD19/CD22 dual targeting was also performed. We demonstrate that anti-CD19/BCMA CAR-T cells can be effectively generated through the co-transduction of two lentiviral vectors after optimization to minimize competition for cellular resources. Co-transduced T cells, called ARI0003, effectively targeted NHL tumor cells with high avidity, outperforming anti-CD19 CAR-T cells and other dual-targeting approaches both in vitro and in vivo, particularly in low CD19 antigen density models. ARI0003 maintained effectiveness post-CD19 CAR-T treatment in xenograft models and in spheroids from relapsed CART-treated patients. ARI0003 CAR-T cells were effectively manufactured under Good Manufacturing Practice conditions, with a reduced risk of genotoxicity compared to other dual-targeting approaches. A first-in-human phase 1 clinical trial (CARTD-BG-01; this study was registered at ClinicalTrials.gov [NCT06097455]) has been initiated to evaluate the safety and efficacy of ARI0003 in NHL.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/CAR-T cellsCo-transductionDual targetingLymphomaAnimalsAntigens, CD19Cell Line, TumorDisease Models, AnimalGenetic VectorsHumansImmunotherapy, AdoptiveLentivirusLymphoma, Non-HodgkinMiceReceptors, Chimeric AntigenT-LymphocytesTransduction, GeneticXenograft Model Antitumor AssaysAttribution-NonCommercial-NoDerivatives 4.0 International39563035331317-33510.1016/j.ymthe.2024.11.0281525-0024Molecular therapy : the journal of the American Society of Gene Therapyopen access