Maldonado-Perez, NoeliaTristan-Manzano, MariaJusticia-Lirio, PedroMartinez-Planes, ElenaMuñoz, PilarPavlovic, KristinaCortijo-Gutierrez, MarinaBlanco-Benitez, CarlosCastella, MariaJuan, ManelWenes, MathiasRomero, PedroMolina-Estevez, Francisco JMarañon, ConcepcionHerrera, ConchaBenabdellah, KarimMartin, Francisco2023-05-032023-05-032022-09-22Maldonado-Pérez N, Tristán-Manzano M, Justicia-Lirio P, Martínez-Planes E, Muñoz P, Pavlovic K, et al. Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma. Front Immunol. 2022 Oct 6;13:1011858http://hdl.handle.net/10668/20594Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.enAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/CAR-T cellsCRISPR/Cas9TCRKOLarge deletionsLymphomaOff-the-shelfSafetyHumansReceptors, Chimeric AntigenImmunotherapy, AdoptiveT-LymphocytesPrecursor Cell Lymphoblastic Leukemia-LymphomaLymphoma, B-Cellresearch article36275777open accessInmunoterapia adoptivaLeucemia-linfoma linfoblástico de células PrecursorasLinfocitos TLinfoma de células BReceptores quiméricos de antígenos10.3389/fimmu.2022.10118581664-3224PMC9585383https://www.frontiersin.org/articles/10.3389/fimmu.2022.1011858/pdfhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585383/pdf