Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating cancer. However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis. To overcome this limitation, we have developed an allogeneic based platform for large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donors. This platform utilizes Transcription Activator-Like Effector Nuclease (TALENTM) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRαβ negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the capacity to perform efficient multiplex genome editing using TALENTM offers the possibility of simultaneously rendering cells resistant to standard chemotherapy or to tumor evasion mechanisms.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). In a first step, we have screened human primary T-cells harboring CARs with different antigen recognition domains in the context of multiple CAR architectures in order to identify candidates displaying specific activity against cell lines expressing variable levels of the CD123 antigen. To provide proof of concept for the general applicability of the allogeneic approach we have manufactured a TCR-deficient CD123 CAR T-cell (UCART123) and demonstrated that this product maintains a potent anti-tumoral activity in vitro. Experiments in an orthotopic AML mouse model using UCART123 cells are currently ongoing, in order to establish the absence of alloreactivity and the anti-tumoral activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T Cells from a single healthy donor could thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients. DisclosuresGaletto:Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Smith:Cellectis SA: Employment.