Background Chimeric antigen receptor (CAR) T cells have significantly improved our ability to treat cancer. However, these therapies suffer from metabolic and functional exhaustion, limiting their clinical utility in vivo. Finding ways to enhance their metabolic capacity and plasticity could hold the key to making this cellular therapy more effective. AMP activated protein kinase (AMPK) is a cellular energy sensor which promotes metabolic efficiency and cellular survival by manipulating cellular metabolism through a variety of pathways. We investigated the impact of increasing AMPK signaling via overexpression of its regulatory gamma domain in combination with CAR constructs utilizing two clinically distinct co-receptors – CD28 and 41BB.
Methods Healthy human donor T cells were expanded and transduced with either CD28- or 41BB- co-stimulated CARs targeting CD19. Cells were then co-transduced with either AMPKγ2 or an empty vector (EV) control. CARTs were sorted, expanded in IL2, and evaluated for metabolic and functional capacity using the Seahorse metabolic analyzer, the Incucyte co-culture incubator, and flow cytometry.
Results Co-transduction with AMPKγ2 increased the oxidative capacity of both CD28- and 41BB-costimulated CART cells (figure 1), but the functional phenotypes of these two CARTs differed. AMPKγ2 overexpression increased the inflammatory capacity of CD28-CARTs as measured by faster NALM6-leukemia cell killing in both standard and tumor-conditioned media (figure 2), and increased cytokine production evaluated by intracellular flow staining (figure 3). These changes correlated with an increased yield of effector memory CARTs in the AMPKγ2 group following IL-2 expansion, supporting their increased activation (figure 4). Meanwhile, 41BB-CARTs demonstrated a less inflammatory phenotype, with reduced intracellular cytokine production following NALM6 exposure (figure 3) and an increased yield of central memory CARTs (figure 4). 41BB CARTs also had an increased ability to expand in the presence of IL-2 (figure 5), a feature that was not replicated in CD28-CARTs.
Conclusions Metabolic reprogramming is an attractive option to improve the in vivo function of CART cells. Herein, we demonstrate that AMPKγ2 overexpression increases the metabolic capacity of both CD28- and 41BB-CARTs. However, the functional phenotype of these cells differs, with CD28-CARTs enhancing inflammatory capacity while 41BB-CARTs increase their in vitro expansion and central memory yield. With the in vivo environment posing unique challenges in solid versus liquid tumors, our next step will be to evaluate these metabolically enhanced CARTs to assess whether these in vitro phenotypic profiles predict improved tumor clearance and survival in vivo.
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