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234 Metabolic enhancement of alternately co-stimulated CART cells through AMPK creates functionally distinct phenotypes
  1. Erica Braverman,
  2. Mengtao Qin,
  3. Herbert Schuler and
  4. Craig Byersdorfer
  1. University of Pittsburgh, Pittsburgh, PA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.

Abstract 234 Figure 1

ANPKy2 overexpression increases CART oxidative metabolism. Human CD28-CART (A) or 4 1BB-CART (B) cells co-transduced with AMPKy2 (y2) or Empty (EV) vectors, expanded, and stimulated overnight against NALM6 targets prior to assessment with the Seahorse Metabolic Analyzer mitostress kit. ***p<0.0001, ****p<0.0001.

Abstract 234 Figure 2

AMPKy2-CD28-CAR T cells Demonstrate Increased Cytotoxicity in vitro. AMPKy2-CAR T cells and EV-CAR T cells were co-cultured with ZsGreen + NALM6 cells (E:T=1:3) NALM6 death was measured with IncuCyte, demonstrated by loss of green fluorescence. (A) low-glucose (5.5mM) RPMI. (B) NALM6 conditioned RPMI. ****p<0.0001.

Abstract 234 Figure 3

AMPKy2 overexpression increases cytokine production in CD28-CARTs in vitro. CD28-CART cells and 41BB-CART cells were co- transduced with either AMPKy2 or EV control and expanded. CARTs were then co-cultured with NALM6 targets for 16hrs in the presence of brefeldinA. Cells were subsequently intracellularly stained for cytokines, and the median flouresence intensity (MFI) compared via flow cytometry. Each experiment was normalized to the EV-control. *p<0.05, **p<0.01, ***p<0.001

Abstract 234 Figure 4

AMPKy2 overexpression increases central memory yield of 41BB-CARTs and effector memory yield of CD28-CARTs. Human 41BB-CART (A) cells or CD28-CART (B) cells were co-transduced with AMPKy2 (y2) or Empty (EV) vectors. After expansion, cells were co-stained with CD62L and CD45RO to identify central memory (CD62L+CD45RO+) or effector memory (CD62L-CD45RO+) populations

Abstract 234 Figure 5

AMPKy2 overexpression increases IL2 mediated expansion of 41BB-CARTs but not CD28-CARTs. Human 41BB-CART (A) or CD28-CART (B) cells were co-transduced with AMPKy2 (y2) or Empty (EV) vectors. Cells were cultured with or without IL2, and confluence was assessed by the Incucyte as a measure of expansion. ***p<0.001

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