Abstract 4702: 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells
Authors: Long Adrienne H. , Haso Waleed M. , Smith Jillian P. , Walker Alec J. , Fry Terry J. , Orentas Rimas J. , Mackall Crystal L. 
Published: August 2015

Abstract
Published reports have demonstrated high response rates of CD19 chimeric antigen receptor (CAR) modified T cells against acute lymphoblastic leukemia. CARs incorporating either CD28 or 4-1BB costimulatory domains mediated significant anti-leukemic effects. However CD19-41BBz CAR T cells could be detected for months and induced prolonged B cell depletion, whereas most patients treated with CD19-28z CAR T cells had no detectable CAR+ T cells by day 28, and B cell recovery was routinely observed. We report that CAR T cells incorporating the CD28 versus 4-1BB costimulatory domains show differential susceptibilities to T cell exhaustion. Using the tonically signaling 14g2a-based GD2 CAR as a model system, we identify that GD2-28z CARs rapidly acquire an exhaustion phenotype, characterized by high level expression of PD1, TIM3 and LAG3, high expression of exhaustion related transcription factors Blimp1 and T-bet, poor proliferative capacity, poor cytokine production, and poor anti-tumor efficacy in vivo. Interestingly, second generation GD2-28z CARs induce greater exhaustion than first generation GD2-z CARs, implicating CD28 costimulation in the induction of exhaustion. In contrast, GD2-41BBz CARs express lower levels of PD1 and TIM3, show increased cytokine production, improved persistence in vivo, and improved anti-tumor efficacy against both osteosarcoma and neuroblastoma xenografts. Similarly, CD19-41BBz CARs have improved persistence and lower expression of exhaustion markers compared to CD19-28z CARs following leukemia challenge. Finally, CD22-BBz CARs show lower exhaustion marker expression, improved persistence, and improved anti-leukemia efficacy in immunodeficient mice compared to CD22-28z CARs. Gene expression experiments are underway to further delineate the mechanism by which 4-1BB signaling ameliorates exhaustion in the setting of chronic CAR signaling. Together, this data demonstrates that exhaustion is a critical factor limiting the efficacy of CAR expressing T cells. CD28 and 4-1BB have opposing effects on exhaustion in CAR T cells, with CD28 signaling augmenting the phenotype while 4-1BB signaling mitigating exhaustion. This data is the first to demonstrate differential effects of costimulatory pathways on T cell exhaustion, likely explains differential persistence observed in the clinical trials of CAR therapeutics, and provides important insights for CAR design for future clinical applications.
Citation Format: Adrienne H. Long, Waleed M. Haso, Jillian P. Smith, Alec J. Walker, Terry J. Fry, Rimas J. Orentas, Crystal L. Mackall. 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2015-4702