Background For cellular immunotherapies, clinical outcomes depend on the proliferative potency and metabolic fitness of the therapeutic product. For their most successful indications, CAR T cells are effective ‘serial killers,’ each T cell recognizing and eliminating many target cells. What fuels CAR T cell serial killing is unknown. Two critical events define the efficiency of T cell serial killing: migration and immune synapse formation/cytolysis. Importantly, each event is influenced by the local metabolic milieu.

Methods Using a specialized CAR T cell conditioning regimen, the goal of this research to is determine the relative energy cost of migration versus cytolysis using innovative eSIGHT RTCA technology. Our project will reveal how the spare respiratory capacity (SRC), supports CAR T cell migration and/or cytolysis, founded on the hypothesis that cells that can replenish their SRC in repetitive antigen stimulation models, are more efficient serial killers.

Results Our data sheds light on critical metabolic states that impair CAR T cell cytolytic activity. We also use multi-omic approaches to identify arginosuccinate synthase 1 (ASS1), a gene distinguishable at the metabolic (Seahorse), transcriptional (RNAseq), epigenetic level (ATAC seq), and functional (tumor clearance in vivo) in

CAR T cells. Supporting the premise of our work, we show that ASS1 supports high SRC levels despite frequent antigen encounter in repetitive stimulation models in vitro. In parallel work, we provide data that reductive glutamine metabolism is enhanced in 28zCARTs, suggesting mechanisms for why 28zCARTs outperform BBz CARTs in some hypoxic tumor models (figure1B). We also show that expressing isoforms of the GOT family of amino transaminases enhance CAR T cell anti-tumor function (figure 1A&C). Given the prior link connecting reductive glutamine metabolism and GOT1 in Jurkat cells we hypothesize a fundamental link, involving reductive glutamine metabolism, GOT1-mediated aspartate replenishment, and fumarate production via ASS1, to support mitochondrial function and serial killing in CAR T cells (figure 1D).

Conclusions Our findings reveal unique conditioning and genetic strategies to arm CAR T cells with unique metabolic attributes against solid tumors.

• Download figure
• Open in new tab
• Download powerpoint

Abstract 1238 Figure 1

A model for enhancing CART metabolic activity(A) Serial quantification of tumor burden in Nalm6 tumor-bearing mice treated with CAR T cells. CARTs with a superior ability to replenish aspartate (GOT2) demonstrate superior tumor control, even after tumor cell re-challenge (day 31). Data are means ± SEM from starting cohorts (n=5 mice per treatment arm), from a single experiment. (B) Lentivirally transduced CAR T cells (SS1-CAR-28ζ vs SS1-CAR-BBζ) were expanded for 9 days until restdown. These cells were restimulated with mesothelin-fc, that was previously conjugated to dynabeads. At 5 of re-stimulation, the cells were treated with [1-13C] glutamine that distinguishes reductive gln metabolism overnight at 20% O2 or 1% O2. Citrate labeling (m+1 isotopologue) is accentuated in 28ζ CARTs. Data are means ± SEM from two independent studies with separate donors. (C) Aspartate replenishment via GOT1 enhances CAR T cell cytolytic potency as assessed by eSIGHT impedance assay. SY5Y-CD19 target cells were adhered to the well. 24 hr later, CARTs were added at various E:T ratios as indicated. Data are means ± SEM from a single experiment. (D) A novel model proposing how 28ζ signaling drives the vectorial flux of glutamine to citrate, which replenishes OAA, is converted to aspartate which supports TCA anaplerosis (fumarate) by ASS1.