762. A Quantitative Imaging Tool Box for the Functional Analysis of Chimeric Antigen Receptor - T Cell Immune Synapse Helps Identify Novel Structural and Functional Features of CAR T Cells

Introduction: The cytolytic immunological synapse (IS) in T cells is a discrete structural entity formed after the ligation of specific activation receptors and leading to the destruction of a cancerous cell through the targeted release of the contents of T cell lytic granules. Although the “native” T cell IS formation where the TCR binds to antigenic peptide-MHC complex has been intensely studied, the “engineered” CAR T cell synapse and its variations based on single chain variable fragment (scFv) design and co-stimulatory domain arrangements, still remains a black box. Our work focuses on the quantitative evaluation of the CAR T cell lytic IS using highly quantitative high and super resolution imaging techniques. Specifically, by combining quantitative interrogations of CAR synapse formation kinetics and dynamics at the cellular level, we are able to obtain an unprecedented level of insight into the effectiveness of therapy cells relative to the traditional measures of cytotoxicity and effector assays like chromium release, ELISPOT and flow cytometry. Super-resolution microscopy enables us to further “see” subtle variations in therapy cell quality at a near molecular level and can be used as a very precise functional readout of CAR T cell rational design. Methods: High and super resolution fluorescence microscopy platforms are used to image CAR T cell-target conjugates in the x, y and z dimensions. Imaging parameters include direct evaluation of CAR engagement at the IS by measuring mean fluorescence intensity (MFI) and volume of synaptic aggregates of CAR specific ligands as well as their relative distribution at the synapse. Results: We report here the quantitative measurement and comparison of single and dual CAR targeted ligand aggregation at the IS, lytic machinery re-arrangement and time to target cell killing. Our studies using this predictive imaging tool box establishes the superiority of T cells expressing a bispecific (tandem) CAR to those expressing one or two monospecific CARs by the presence of significantly increased accumulation of F-Actin (> five fold), greater convergence and polarization of the lytic granule machinery. Dual engagement of the targeted ligands by a tandem CAR also significantly offsets antigen escape variants in tumors predicting reduced tumor relapse. Furthermore, application of our imaging tool box reveals that tonic signaling of CARs containing a 4-1BB co-stimulatory domain can result in an almost three fold upregulation of Fas on plasma membrane and co-localization with Fas L leading to enhanced apoptosis and lower persistence of CAR T cells. Conclusions: Using our quantitative imaging tool box we are able to define a specific list of parameters to measure CAR engagement and lytic function at the IS. These imaging parameters can be directly applied to interrogate and predict functionality outcomes upon CAR design variations. By doing this we hope to provide crucial early insight in CAR T cell design that can guide improvements in the efficacy, safety and, ultimately, clinical effectiveness of CAR T cell therapies.