Abstract LB-111: Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion
Authors: Weber Evan W. , Lynn Rachel C. , Malipatlolla Meena , Sotillo Elena , Xu Peng , Mackall Crystal L. 
Published: August 2018

Abstract
In patients with chronic viral infection or cancer, continuous antigen exposure results in T cell exhaustion, which is characterized by sustained co-expression of multiple inhibitory receptors (ex. PD-1, TIM-3, LAG-3) and a hierarchical loss of effector function. Human T cells expressing a high-affinity anti-GD2 chimeric antigen receptor (CAR, HA.28z) develop phenotypic and functional hallmarks of exhaustion due to clustering of surface CAR and tonic signaling in the absence of antigen. In the present study, we exploited the HA.28z CAR as a model to characterize human T cell exhaustion and assess its potential for reversibility. CyTOF analyses of dysfunctional HA.28z CAR T cells demonstrated a distinct phenotype in which canonical exhaustion markers, T cell differentiation markers, and effector transcription factors were differentially expressed compared to CD19.28z CAR T cells that do not tonically signal.
To interrogate the reversibility of T cell exhaustion, we engineered a regulatable HA.28z CAR by fusing a destabilization domain to the C-terminus of the CAR that rapidly induces CAR protein degradation. Addition of a small molecule stabilizes the protein and results in CAR surface expression. Using this model, we modulated the duration and intensity of tonic signaling by precisely modulating CAR surface expression. We hypothesized that transient cessation of tonic signaling would allow exhausted CAR T cells to recover and regain effector function.
Following onset of exhaustion, elimination of CAR surface expression for 4 days resulted in a profound reversal of the exhausted phenotype. CyTOF analyses of reinvigorated cells indicated enhanced T cell memory formation, diminished surface marker expression of multiple exhaustion markers (ex. PD-1, TIM-3, LAG-3, CTLA-4, CD39) and reduced expression of transcription factors T-bet and Blimp-1. Furthermore, RNA sequencing of exhausted and reinvigorated HA.28z CAR T cells revealed a global molecular reprogramming upon removal of tonic CAR signaling, suggesting that CAR T cell exhaustion is functionally reversible. Upon CAR re-expression and co-culture with tumor antigen, reinvigorated HA.28z CAR T cells exhibited augmented killing and cytokine secretion compared to exhausted cells that continuously expressed surface CAR during expansion. In vivo studies assessing CAR T cell exhaustion reversibility using this model are currently being evaluated.
In summary, tuning CAR surface expression offers a novel strategy to augment both the safety and efficacy of CAR T cell therapy. Moreover, these studies suggest that transient “rest” in T cells experiencing chronic antigen stimulation may be one mechanism by which exhaustion is prevented or reversed.
Citation Format: Evan W. Weber, Rachel C. Lynn, Meena Malipatlolla, Elena Sotillo, Peng Xu, Crystal L. Mackall. Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-111.