Tonic 4-1BB signaling from chimeric antigen receptors (CARs) impairs expansion of T cells due to Fas-mediated apoptosis

Abstract

Abstract High expression of CARs in T cells or the presence of CAR ligands in cell culture results in tonic CAR signaling that enhances differentiation and exhaustion of T cells and compromises anti-tumor activity. A recent study demonstrated tonic signaling negatively affected T cells expressing a CD28.zeta GD2-specific CAR, indicating 4-1BB costimulation may improve their function. However, whether 4-1BB is a uniformly optimal costimulatory signal for CAR-transduced T cells is unclear. We found that retroviral expression of several 2nd generation CARs with a 4-1BB domain (4-1BB.zeta) unexpectedly impaired T cell expansion. High expression of 4-1BB.zeta CD19 and kappa CARs led to upregulation of Fas and FasL and enhanced caspase 8-dependent apoptosis. Decreasing CAR expression in retro- or lentiviral systems normalized Fas levels and restored T cell expansion. However, a similar approach did not rescue expansion of 4-1BB.zeta CD5 CAR T cells, likely because the presence of the target antigen CD5 on T cells sustains high tonic signaling. 4-1BB.zeta CD5 CAR T cells recruit Fas and FasL to form stable fratricidal immunologic synapses. Disrupting TRAF2 binding site in the 4-1BB domain prevented Fas upregulation and restored T cell function but compromised costimulation. Therefore, we developed a regulated retroviral expression system that uses a small molecule to reversibly inhibit CAR expression, preventing tonic signaling and the resulting fratricide and differentiation. Withdrawing the inhibitor restores CAR expression and the anti-tumor function of T cells. Hence, we have described a mechanism by which chronic 4-1BB signaling may undermine CAR T cell function and provide a solution that curtails detrimental tonic CAR signaling.