Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction

Abstract

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Rational protein engineering to enhance MHC-independent T cell receptors

“…Chronic stimulation with Nalm6 WT or Nalm6 CD80/CD86 demonstrated poor mut035 expansion ( Supplementary figure 8e ) and early loss of tumor control at ∼day 13 of chronic stimulation ( Figures 4e ). Consistent with our previous findings 2 , 19/BBσ CAR T cells expanded and contracted rapidly, losing tumor control ∼day 17. Chronic stimulation of mut035 T cells with Nalm6 41BBL or Nalm6 triple , however, resulted in a more modest but more durable T cell expansion and a ∼9-day delay in T cell contraction compared to CAR T cells ( Supplementary figure 8e ).…”

“…Both acute and re-challenge studies demonstrated equivalent cytotoxicity between 19/BBσ CAR T cells and mut035 T cells ( Supplementary figures 8b-c ), and thus we moved to a more stressful in vitro model. We previously developed a protocol that reliably induces 19/BBσ CAR T cell dysfunction following 12-17 days of chronic antigen stimulation 2,24 . We established co-cultures of 19/BBσ CAR T cells with Nalm6 WT and mut035 T cells with Nalm6 41BBL , as well as Nalm6 WT , Nalm6 CD80/86 and Nalm6 triple , and replenished these with fresh tumor every other day to maintain persistent stimulation.…”

“…Consistently, mut035 T cells demonstrated more durable control of Nalm6 41BBL and Nalm6 triple , losing control ∼day 22 ( Figure 4e ). To further test the durability of mut035 T cell functionality, we employed an in vivo stress model in which immunodeficient (NOD/SCID/ψc -/- ) mice were engrafted with Nalm6 WT or Nalm6 41BBL and given a low dose (2.5x10 5 ) of engineered T cells seven days later 2,25 . Tracking of disease using bioluminescent imaging confirmed identical growth kinetics of these two tumors in vivo and demonstrated that mut035 T cells significantly delayed disease progression as compared to 19/BBσ CAR T cells ( Figure 4f ).…”

TOP CAR with TMIGD2 as a safe and effective costimulatory domain in CAR cells treating human solid tumors

“…Persistent exposure to antigens can lead to dysfunctional phenotypes and suboptimal effector responses in CAR-T cells ( 32 ). In this setting, different CAR constructions can greatly influence the expression of a variety of cell surface markers ( 33 , 34 ). To examine whether our TMIGD2 and CD28.4-1BB costimulatory domains would also differentially alter cell surface protein expression, we adapted an in vitro model of chronic antigen exposure (CAE) wherein CAR-T cells were continuously cultured with sufficient HCC827 tumor cells so that the tumor cells were always present in the coculture ( 32 ).…”

The CD4/CD8 ratio of infused CD19‐CAR‐T is a prognostic factor for efficacy and toxicity

“…The CD4/CD8 ratio of infused CD19-CAR-T is a prognostic factor for efficacy and toxicity Eugenio Galli 1 | Silvia Bellesi 1 | Ilaria Pansini 2 | Giacomo Di Cesare 1 |…”