394. Generation of a Novel Allogeneic CAR T Cell Platform Utilizing an Engineered Meganuclease and AAV Donor Template to Achieve Efficient Disruption of T Cell Receptor Expression and Simultaneous Homology-Directed Insertion of a CD19 CAR

Abstract

S156interrogate the quality and quantity of co-localized Tan CAR target ligands at the immune synapse. Measurement of co-localized HER2 and IL13Rα2 aggregate diameters showed a distribution of 80-200 nm aggregate size. The aggregate size has high correlation to the predicted size of CAR-tumor ligand-antibody-antigen complex, indicating increased probability of co-docking of the target ligands at the Tan CAR synapse. Co-localization of the two tumor antigens was present with a very low frequency in the bi-specific T cell -tumor synapse with their average aggregate size ranging from 300-500 nm, suggesting that these ligands formed independent and not "codocked" conjugates. Co-localization was absent in the single CAR -tumor synapse. In situ PLA with a maximum distance of 30-40 nm showed accumulation of HER2/IL13Rα2 heterodimer signals at the immunological synapse of Tandem CAR Tcell/ GBM IS.Conclusion: Tandem CAR molecules simultaneously engage two target antigens mediating significantly enhances T cell activation through a bifunctional immunological synapse.