Abstract LB-112: Engineering AP1 to combat CAR T cell exhaustion
Authors: Lynn Rachel C. , Weber Evan W. , Gennert David , Sotillo Elena , Jones Robert , Xu Peng , Satpathy Ansuman , Chang Howard Y. , Mackall Crystal L. 
Published: August 2018

Abstract
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell leukemia. However, application of CAR T cell therapy in solid tumors has been disappointing. We believe the development of T cell exhaustion, a phenomenon well described in chronic viral infection, is a major barrier to widespread success of CAR T cell therapy in other types of cancer. Therefore, understanding mechanisms of and engineering solutions to avoid the development of CAR T cell exhaustion is highly warranted.
Here, we utilize a high affinity (HA) GD2-directed CAR model in which T cells undergo chronic activation via spontaneous clustering of surface CAR molecules. Tonically signaling HA-28Z CAR T cells demonstrate phenotypic and functional characteristics of exhaustion with high expression of inhibitory receptors (PD-1, TIM-3, LAG-3, and CD39), poor cytokine production, enhanced effector differentiation, and failure to eradicate disease upon transfer into tumor-bearing mice. To interrogate the transcriptional and epigenetic programs driving CAR T cell exhaustion, we performed total RNA-seq and ATAC-seq using HA-28Z or control CD19-28Z CAR T cells.
The most significantly enriched transcription factor motifs in HA-specific open chromatin peaks belonged to the AP1 family, suggesting AP1 factors may play an integral role in mediating exhaustion. RNA-seq and western blot confirmed increased expression of AP1 factors in exhausted CAR T cells. Interestingly, exhausted HA-28Z CAR T cells demonstrate insufficient IL2 production, a gene classically regulated by AP1 Fos/cJun heterodimers, although Fos and cJun are present in CAR T cells. However, other potentially inhibitory AP1 family members (including JunB, BATF, ATF, and IRF family members) are overexpressed to a greater degree. We hypothesize that the relative balance of classical and inhibitory AP1 family members may contribute to exhaustion.
To disrupt this balance, we constitutively overexpressed Fos and cJun in exhausted HA-28Z CAR T cells and observed that JUN+ CAR T cells demonstrated increased IL2 and IFNg production and reduced inhibitory receptor expression. Furthermore, adoptively transferred JUN-HA-28Z CAR T cells resulted in rapid tumor clearance and long term survival of NSG mice bearing GD2+ leukemia whereas control HA-28Z CAR T cells could not overcome tumor growth. Co-immunoprecipitation experiments show high levels of JunB/BATF3/IRF4 complexed with cJun in exhausted HA-28Z CAR T cells. Preliminary data using CRISPR-Cas9 gene disruption suggest that knocking out inhibitory AP1 factors in HA-28Z CAR T cells can also enhance functional activity. Together, our data highlight the AP1 family in regulating T cell dysfunction upon chronic antigen encounter. Finally, we demonstrate that AP1 factors can be engineered to produce superior T cells for adoptive cell therapy.
Citation Format: Rachel C. Lynn, Evan W. Weber, David Gennert, Elena Sotillo, Robert Jones, Peng Xu, Ansuman Satpathy, Howard Y. Chang, Crystal L. Mackall. Engineering AP1 to combat 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-112.