# What fuels CAR T-cell serial killing

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2024 · $294,032

## Abstract

Project summary
For
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cellular immunotherapies, clinical outcomes depend on he proliferative potency and metabolic
of the therapeutic product. For their most successful indications, CAR T cells are effective
killers,' each T cell recognizing and eliminating many target cells. Certain metabolic
( e.g. glycolytic capacity) are associated with T cell cytotoxic potential. However, the
underpinnings for these relationships are under-defined and have been studied using a
ex-vivo expansion process followed by a standard cytotoxicity assay in nutrient-rich
Two critical events define the efficiency of T cell serial killing: migration and immune
 formation/cytolysis. Importantly, each event is influenced by the local metabolic milieu.
a specialized CAR T cell conditioning regimen, the goal of this research to is determine the
energy cost of migration versus cytolysis using innovative eSIGHT RTCA technology. Our
will reveal how the spare respiratory capacity (SRC), supports CAR T cell migration and/or
founded on the hypothesis that cells that can replenish their SRC in repetitive antigen
models, are more efficient serial killers. We also use multi-omic approaches to identify
synthase 1 (ASS1), a gene distinguishable at the metabolic (Seahorse),
(RNAseq), epigenetic l evel (ATAC seq), and functional (tumor clearance in vivo) in
T cells. Supporting the premise of our work, we show that ASS1 supports high SRC levels
frequent antigen encounter in repetitive stimulation models in vitro. In parallel work, we
data t hat reductive glutamine metabolism is enhanced in 28  CARTs, suggesting
for why 28  CARTs outperform BB  CARTs in some hypoxic tumor models. We also
that expressing i soforms of the GOT family of amino transaminases enhance CAR T cell anti-
function. Given the prior link connecting reductive glutamine metabolism and GOT1 in Jurkat
we hypothesize a fundamental link, involving reductive glutamine metabolism, GOT1-mediated
replenishment, and fumarate production via ASS1, to support mitochondrial function and
killing in CAR T cells. Finally, we will test the hypothesis that fructose can be repurposed as
important fuel for CD123 CAR T cell cytotoxic function in acute myeloid leukemia (AML). AML is
in that fructose levels can increase from 20  M up to 8mM in the bone marrow. To better
the i mmune-suppressive microenvironment in AML, we will expand on our existing
cell RNA sequencing data to i dentify the source of ructose-producing cells in the AML
bone marrow. Our findings will have translational relevance in improving cellular
for advanced cancers.
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## Key facts

- **NIH application ID:** 10806151
- **Project number:** 5R01CA278837-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Roderick O'Connor
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $294,032
- **Award type:** 5
- **Project period:** 2023-03-09 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10806151

## Citation

> US National Institutes of Health, RePORTER application 10806151, What fuels CAR T-cell serial killing (5R01CA278837-02). Retrieved via AI Analytics 2026-05-29 from https://api.ai-analytics.org/grant/nih/10806151. Licensed CC0.

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