Background & Aim Chimeric antigen receptors (CARs) are engineered proteins that consist of an extracellular antigen binding domain that is linked, via a hinge region and transmembrane domain, to intracellular activation domains that drive T cell activation, proliferation, and target cell killing. A few decades of CAR engineering efforts have made it clear that merely linking these protein domains together is insufficient for generating an effective CAR; subtle differences in how the domains are linked to each other can greatly impact functionality. During CAR optimization biochemical assays can be used to assess critical quality attributes such as potency. Although these reductionistic approaches are useful, their results don't necessarily correlate with cancer killing efficacy within a more physiologically relevant context. To rigorously compare different CAR constructs and identify unwanted behavior such as tonic signaling, it is widely acknowledged that CAR function should be evaluated within the complex milieu of the immune synapse, using bona fide cancer cell killing as the readout. For this purpose, release assays (51Cr, LDH) have historically been the gold standard, but suffer from the fact that they require significant “hands on” time and only provide end point data. Requiring just target cancer cell seeding and a subsequent CAR T cell addition step, herein we use the Agilent xCELLigence RTCA eSight to quantitatively evaluate CAR T cell-mediated killing of cancer cells in real-time over multiple days and at physiologically relevant low E:T ratios. Methods, Results & Conclusion Providing a direct and objective assessment of target cancer cell number, cell size, and cell-substrate attachment strength, impedance biosensors embedded within the base of eSight microplates are shown to quantitatively track the continuum of target cell killing, spanning from early (reduced cell-substrate adhesion strength) to late (lysis) events. Concurrently, eSight is shown to capture live cell images in brightfield and three fluorescence channels, providing an orthogonal readout of the killing process – including real-time assessment of T cell activation and exhaustion markers such as CD69, CD107a, CTLA4, and LAG-3. By combining the strengths of real-time impedance monitoring (simplicity, analytical sensitivity, and objectivity) with that of live cell imaging (specificity of the readout), eSight is shown to increase the information richness of the CAR T cell killing assay without increasing the workload.
