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1431 Metabolically Flexible CAR T Cells (mfCAR-T), with Constitutive Expression of PGC-1α Resistant to Post Translational Modifications, Exhibit Superior Survival and Function in Vitro

Program: Oral and Poster Abstracts
Session: 703. Adoptive Immunotherapy: Poster I
Hematology Disease Topics & Pathways:
Biological, Therapies, CAR-Ts, Biological Processes, gene therapy, Technology and Procedures, gene editing, metabolomics, microenvironment
Saturday, December 5, 2020, 7:00 AM-3:30 PM

Reginald M Atkins, PhD1*, Meghan A Menges1*, Alexis Bauer, BS2*, Joel G. Turner, PhD2 and Frederick L. Locke, MD3

1Clinical Science Division, Moffitt Cancer Center, Tampa, FL
2Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center, Tampa, FL
3Department of Blood and Marrow Transplant and Cellular Immunotherapy, Moffitt Cancer Center, Tampa, FL

Background: Remarkable durable responses are seen with chimeric antigen receptor (CAR) T cell therapy in B cell lymphoma, however the majority of patients relapse (Locke et al. Lancet Oncol. 2019). Improvements enabling CAR T cells (CAR-T) to circumvent mechanisms of resistance may increase efficacy. Hypoxia, nutrient deprivation and acidosis, all common in the tumor microenvironment (TME), impair metabolic function necessary for CAR-T to kill tumor (Chang et al. Cell 2015). The metabolic response gene peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) co-activates genes that upregulate mitochondrial and glycolytic machinery for ATP synthesis from myriad carbon sources. Post translational modifications (PTM) fine tune PGC-1α activity to meet energy demands (Luo et al. IJC 2019). We hypothesized that CAR-T co-expressing full-length PGC-1α or the truncated (ie. short) NT-PGC-1α isoform, with mutations that prevent suppressive PTMs, would confer metabolic flexibility to improve function under TME conditions.

Methods: We constructed four PGC-1α encoded retroviral vectors with an IRES and DsRed fluorescent protein: full-length wild type (WT); full-length mutant (GA); wild type short isoform (NT); and mutant short isoform (mNT). GA contained T295A and S571A mutations to abrogate GSK3β and Akt mediated PTMs. mNT sequence contained K to A mutations at K78/K145/K184/K254 to prevent acetylation by GCN5, and L to A mutations of the nuclear export sequence corresponding to L29/L33/L36/L38. Human CD8 T cells were activated with αCD3/αCD28 beads + 100 IU IL-2/mL, and transduced at 48 hr. to express FMC63-CD28/CD3z CAR and non-functional truncated CD34. Cells were co-transduced with WT, or in the case of metabolically flexible CAR T cells (mfCAR-T) with a mutant and/or short isoform PGC-1α vector. After 7 days of expansion CD34+DsRed+ cells were isolated by FACS. In vitro experiments were performed within 2 weeks to characterize mitochondrial dynamics/oxidative stress (flow cytometry), cytokine secretion (ELISA), and real-time cytotoxicity (xCelligence). The effect of glucose restriction was evaluated in normal (10 mM) and low glucose (0.01 mM) medium. A Mitochondrial stress test (Seahorse) was performed 30 days after FACS. CAR-T (WT and control w/o co-transduction) and mfCAR-T were stimulated with CD19+ K562 or 3T3 cells.

Results: Representative PGC-1α metabolic fitness target genes (ERRα, TFAM, and NRF2) were increased in mfCAR T cells (p≤0.001). mfCAR-T exhibited decreased mitochondrial biomass (p≤0.01) and mitochondrial membrane potential (MMP) (p≤0.01) in both glucose conditions. However, MMP:mitochondrial biomass and autophagy were greater (p≤0.01, p≤0.001), suggesting accelerated mitochondrial quality control (MQC). Oxidative stress was generally decreased (p≤0.01) in mfCAR-T, accompanied by reduced apoptosis. All mfCAR and control CAR T cells cytolysed 100% of targets at a 1:1 ratio but differed in cytolytic rate. Relative to CAR only, WT CAR-T and GA mfCAR-T killed 1.6 and 1.9 times faster, while shorter isoforms required 1.9 times longer to lyse all targets. IFNγ and IL-2 secretion by GA-mfCAR-T was increased above control CAR-T and other mfCAR T cells (p≤0.01), while others were similar. At 30 days both WT-CAR-T and all 3 mfCAR-T had increased spare respiratory capacity (SRC) compared to control CAR-T (p≤0.05); however ATP production and OCR/ECAR was increased (p≤0.001, p≤0.0.05) in mfCAR-T above control CAR-T and WT-CAR-T.

Conclusion: Enforced expression of mutant or truncated PGC-1α in CAR-T enhanced mitochondrial quality control with commensurate function. mfCAR-T cells exhibited equivalent cytotoxicity in vitro, improved survival, and a metabolism less reliant on glucose. Stark differences in SRC, OCR/ECAR, and mitochondrial ATP production between WT and mfCAR-T suggest signaling pathways in CAR T cells may target PTM mediated suppression of PGC-1α and lead to metabolic exhaustion in the TME. mfCAR-T are a promising new strategy to improve the function of CAR-T cells in the TME. Further in vitro and in vivo experiments are needed to validate the approach.

Disclosures: Locke: Kite, a Gilead Company: Consultancy, Research Funding; Celgene/Bristol-Myers Squibb: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Wugen: Consultancy; GammaDelta Therapeutics: Consultancy; Calibr: Consultancy; Allogene: Consultancy; Novartis: Consultancy.

*signifies non-member of ASH