Visualization of the Underlying Kinetics of LckY394F and its Applications in CAR T-cell Therapy
With the emergence of various cell therapies, including CAR T-cell therapy and TCR T- cell therapy, it has become more important than ever to elucidate the underlying mechanism of T-cell activation with a higher temporal and spatial resolution for better predication and interpretation of laboratory and clinical results. Lymphocyte-specific kinase (Lck) is one of the molecules involved in T-cell proximal signaling right after TCR engagement with MHC on antigen-presenting cells (APC). Many studies have been done to elucidate its crystal structure and kinetics. However, a majority of these observations were based on Western blots and immunostaining, which could only demonstrate the endpoint effects of certain perturbations.
Seeing is believing. Here we present a FRET-based biosensor with an optimized Lck- specific substrate to visualize Lck dynamics in living cells with high spatial and temporal resolution. With this biosensor, we also observed the unique activation dynamics of LckY394F mutant, which was reported to have only minimal or no kinase activities, under CD3/CD28 coreceptor stimulation. It was noted that LckY394F was able to lower the basal activation level of Lck in resting T-cells while capable of transducing robust signals downstream when activated. We further presented preliminary results that LckYF-incorporated CAR constructs had superior in vitro killing capacity under physiologically relevant E:T ratios and designed future experiments to fully characterize and optimize this new design. We believe that this mutant Lck has huge potentials in CAR T-cell therapy as well as in off-the-shelf therapeutic product design.