We have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein comprised of an antibody-derived binder tethered to the extracellular domain of the CD3ε signaling subunit. Upon integration of the TRuC into the T cell receptor (TCR), it targets specific tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report about engineering of off-the-shelf TRuC-T cells directed against mesothelin (MSLN). 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 comprised of antibody-derived binders fused to the TCRγ and δ constant domains. Allogeneic anti-MSLN TRuC-T cells upregulate activation markers, secrete robust cytokines, and lyse tumor cells in an antigen-specific manner without allo-reactivity. In comparison with control autologous TRuC-T cells targeting mesothelin (TC-210), allogeneic TRuC-Ts demonstrate extended tumor clearance in NSG xenograft models due to their enhanced expansion and persistence. To reduce host rejection and further boost the persistence of the allogeneic TRuC-T cells, we eliminated MHC class I surface expression by B2M gene knockout and over-expressed membrane-bound IL15 tethered to its receptor alpha. In summary we have engineered persistence enhanced, allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with improved efficacy compared to donor-matched autologous TRuC-T cells.

Citation Format: Julie Donaghey, Cecilia Kwong, Allison Powell, Troy Patterson, Ella Liberzon, Richard Decker, Julio Gomez Rodriguez, Darby Kreienberg, Jennifer Bian, Urmi Patankar, Holly Horton, Jian Ding, Robert Hofmeister, Dario Gutierrez, Robert Tighe. Engineering off-the-shelf anti mesothelin t-cell receptor fusion construct (TRuC™) t-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 557.