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Technologies to assess CAR T cell serial killing in real-time are severely lacking. Commonly-used killing assays including chromium release as well as luciferase-based assays provide limited snapshots of cytolytic activity.
Advances in time-lapse microscopy or bioelectrical impedance have shed light on cytolytic activity in real time but require further optimization. Current approaches are hindered by the immediate close proximity of effector to target cells when CAR T cells are added to target cells in standard co-cultures.
As CARTs kill in a cooperative manner, they may only engage one-two targets in the entire assay. At lower effector:target ratios, the assay reflects proliferative potential more than cytolytic potency.
In an innovative advance, and first-of-its kind assay, we leveraged the xCELLigence eSIGHT technology to combine imaging with impedance analyses and separate effector CAR T cells from their antigen-positive targets by a layer of matrigel.
Uncoupling migration from cytolytic activity yields enhanced translational insights into the energy cost of CAR T cell killing and a potential biomarker of CAR T cell potency prior to clinical endpoints. As Matrigel has tuneable density, its composition can be matched to reflect the physiochemical properties of distinct stromal regions across several tumor microenvironments.
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