Analytical Tools to Evaluate CAR-T Cell Signaling & Activation

In this App Note, we demonstrate a streamlined workflow to engineer, identify, and characterize CAR-T cell signaling and activation status, revealing the kinetics of CAR-T cell activation and signal transduction with reproducible quantification. Specifically, we leveraged Bio-Techne’s non-viral gene editing TcBuster™ platform to edit human primary T cells with a CD19 CAR. Following activation of the CAR using CD19 protein, we implemented automated protein analytical solutions to characterize cytokine secretion using the microfluidic ELISA Simple Plex™ platform and intracellular signaling events using the capillary immunoassay Simple Western™ platform with high-quality antibodies from CST.

The results presented here include granular time course information on CAR activation and stimulation of downstream signaling events using only 1-2 μg of lysate per capillary with hands-free automation, which would be difficult, if not impossible, to achieve with traditional immunoassays. Combining flow cytometry and Simple Western analyses, we observed CAR-T cells downregulate CAR expression in less than 10 minutes following antigen engagement. However, CAR presence at the cell surface may linger for 15 minutes or longer. Furthermore, the Simple Western results indicate CAR expression increases again at 4 hours, suggesting a ‘recycling’ of the CAR in T cells.

We anticipate these solutions will become standard methods in CAR-T cell therapy development, with applications including, but not limited to, CAR activation, such mechanism of action studies, and monitoring tonic signaling, a pivotal event controlling CAR-T cell efficacy.