392. Super-Resolution (STED) Imaging Reveals Simultaneous Co-Docking of Tandem Chimeric Antigen Receptors to Two Target Antigens Enhancing T Cell Functionality and Mitigating Antigen Escape

Background: Tandem chimeric antigen receptors (CARs) are bispecific CAR molecules with two different target recognition exodomains joined in tandem. Tandem CARs mediate distinct T cell activation to either target antigen and super-additive functionality upon encounter of both targets simultaneously. Tandem CAR T cells successfully mitigate antigen escape and exhibit enhanced antitumor activity compared to their unispecific counterparts. Stimulated emission depletion (STED) microscopy can achieve a sub-diffractive lateral resolution of ~ 50 nm enabling imaging of the immunological synapse at near single molecule levels.Hypothesis: Super resolution STED Imaging can differentiate heterodimers created by Tandem CAR molecules simultaneously co-docking to both target antigens at the immunological synapse (IS).Methods: Human T cells were retrovirally transduced to express a Tandem CAR molecule specific for HER2(ErbB2) and IL13Rα2. Conjugates of Tandem CAR T cells and the human glioblastoma cell line U373 were fixed after 30 minutes of incubation then labelled with primary unconjugated monoclonal antibodies against HER2 and IL13Rα2 tumor ligands. The IS was examined at the T cell/ U373 interface using confocal and STED microscopy then confirmed with in situ proximity ligation assay (PLA). Unispecific CAR T cells, T cells co-expressing HER2 and IL13Rα2 CARs and non-transduced T cells were used as experimental controls.Results: Confocal microscopy showed co-clustering of HER2 and IL13Rα2 at the IS in contrast to only HER2 or IL13Rα2 localized to the IS for the IL13Rα2 CAR and HER2 CAR T cell/GBM conjugates, respectively. Using a fixed intensity threshold for all conjugates analyzed, quantification of receptor accumulation at the IS revealed that there was increased collective clustering of target molecules at the IS (p= 0.0002). Super-resolution STED microscopy was used to interrogate 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 “co-docked” 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.