Overexpression of Rorγt in CAR T Cells Improves Persistence and Reduces Exhaustion
Authors: Cimons Jennifer , Kohler Mark Eric , Yarnell Michael , Fry Terry J. 
Published: November 2021

Abstract
Although T cells modified with a chimeric antigen receptor (CAR) are highly effective, durability of remissions may be limited by the poor persistence of adoptively transferred CAR T cells in patients. Growing evidence supports the idea that the pre-infusion phenotype of CAR T cells affects the efficacy of CAR T cell therapies, in part due to the increased ability of certain subsets of CAR T cells to persist long-term. Studies using conventional T cells have suggested that adoptive transfer of T cells programmed to have Th17/Tc17 effector functions can increase the anti-tumor efficacy and persistence of transferred cells when compared to adoptive transfer of non-Th17/Tc17 cells. However, standard CAR formats currently in clinical use that contain either a CD28 or 4-1BB costimulatory domain fail to generate substantial numbers of Th17/Tc17-like cells. Thus, few studies have investigated whether Th17/Tc17 CAR T cells could enhance the efficacy of CAR T cell therapies.
We generated a multi-cistronic lentiviral construct encoding ROR-related Orphan Receptor gamma (RORγt), a key lineage-specifying transcription factor involved in Th17/Tc17 differentiation, followed by a murine CAR targeting CD19 that contains a CD28 costimulatory domain (1928z). RORγt expression did not hinder degranulation in response to CAR antigen upon co-culture with E2aPbx, a murine acute lymphoblastic leukemia cell line. Significantly higher numbers of RORγt-1928z CAR T cells produce IL-17 compared to 1928z CAR T cells (p&lt;0.0001) suggesting that RORγt expression is sufficient to program type-17 effector functions in CAR T cells.
To determine the impact of RORγt overexpression on CAR T cell phenotype and persistence in vivo, we adoptively transferred 1x10 6 1928z or RORγt-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at days 4, 11, and 31. As early as day 4 post-transfer, RORγt-1928z CAR T cells were present at significantly higher numbers than 1928z CAR T cells, and remained higher through Day 31 (0.16% of the bone marrow for 1928z CAR T cells vs 0.84% of the bone marrow for RORγt-1928z CARs; p= 0.0012, n=5 mice per group. The RORγt-1928z CAR T cells were enriched for an effector/effector memory phenotype (CD62L-) compared to 1928z CAR T cells, which were mostly of a central memory phenotype (CD62L+). Interestingly, the RORγt-1928z CAR T cells were enriched for IL-7Rα+ cells and expressed more IL7Rα than their 1928z CAR T cell counterparts (p=0.0012), suggesting an increased capacity for self-renewal in RORγt-1928z CAR T cells.
Previous work from our lab has demonstrated that CAR T cells that persist after leukemia clearance have an exhausted phenotype, with increased expression of PD1 and a reduced functionality 1. After leukemia clearance, RORγt-1928z CAR T cells expressed lower levels of PD1 than their 1928z counterparts (p&lt;0.0001). Finally, 1928z CAR T cells form a population of Eomes hiPD1 hi cells, associated with terminal exhaustion, that is absent in the RORγt-1928z CAR T cells.
Taken together, our preliminary data demonstrates that overexpression of RORγt is sufficient to program type-17 effector functions in CAR T cells and suggests that RORγt overexpression can enhance the ability of CAR T cells to persist and self-renew, and prevent terminal exhaustion. RORγt overexpression may enhance persistence of CAR T cells and durability of remissions in hematologic malignancies. Furthermore, RORγt overexpression may also be a strategy to enhance CAR T cell efficacy against solid tumors, where an immunosuppressive microenvironment contributes to T cell dysfunction.
Yinmeng Yang, Mark Eric Kohler, Terry J. Fry; Effect of Chronic Endogenous Antigen Stimulation on CAR T Cell Persistence and Memory Formation. Blood2017; 130 (Supplement 1): 166. doi: https://doi.org/10.1182/blood.V130.Suppl_1.166.16

Disclosures
Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.