T cell receptor knockout efficiency utilizing an engineered meganuclease is influenced by stimulation conditions.

Recent reports describing allogeneic chimeric antigen receptor (CAR) T cell therapies have generated tremendous excitement over the possibility of a universal CAR T platform. We are utilizing a highly engineered meganuclease designed to target the T cell receptor (TCR) alpha chain to eliminate expression of the TCR, preventing graft versus host disease following adoptive transfer. Meganuclease cleavage efficiency is highly dependent on stimulation of T cells prior to delivery of the meganuclease. To determine optimal activation conditions for TCR knockout and cell yield, T cells were activated with Dynabeads® Human T cell Activator CD3/CD28, Immunocult™ CD3/CD28 and CD3/CD28/CD2 T Cell Activators, and MACS® GMP TransAct CD3/CD28 reagent prior to nucleofection. T cells were stained prior to and following nucleofection to identify cell activation markers and cytokine receptors (CD69, IL2R, IL7R, IL15R, IL21R). Knockout efficiency was determined by staining for CD3 expression. Our results indicate that Dynabead-stimulated T cells expressed the highest levels of activation markers at 18 hours post-stimulation, and that Dynabeads® and Immunocult™ CD3/CD28/CD2 reagents both promote robust T cell proliferation and support the highest TCR knockout efficiency following our protocol. Furthermore, expression of cytokine receptors varies both during stimulation and post-nucleofection, suggesting an optimal cocktail of cytokines may promote T cell proliferation. Future studies will focus on further optimization of T cell stimulation conditions that may promote TCR knockout and expansion of T cells post-nucleofection.