1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:Unidad de Terapia Celular IMIBIC-HURS, Hospital Reina Sofía; 3:Hospital Clínic de Barcelona

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as medicaments against several type B malignancies and multiple myeloma. In spite of the impressive benefit, there are still important limitations. A main drawback comes from its autologous nature that impedes that all the selected patients can be treated and that also precludes the standardization as a medicament. Allogeneic off-the-shelf-CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated the feasibility of generating TCRKO ARI-0001 CAR-T cells using CRISPR/Cas9 as ribonucleoprotein to eliminate the TCRα-chain and lentiviral vectors expressing the CAR through the EFI1-a promoter. We first analysed the efficacy and potential safety issues that arise during disruption of the TCR gene. We found that efficient TCRKO leads to on-target large deletions mainly mediated by microhomology repair mechanism (alt-EJ), indicating a potential safety risk of this procedure that need monitoring. Importantly, TCRKO-ARI-0001 CAR-T cells maintain similar phenotype and antitumor efficacy compared to ARI-0001 CAR-T cells, with an unexpected increase in mitochondrial fitness. In summary, we describe a feasible platform for generating allogenic CAR T cells, a closer step in the roadway to generate a CAR-T therapy for patients with no possibility to obtain clinical approved ARI-0001 CAR T cells.