Abstract The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1514.