# Label-free imaging of CAR T cell metabolism

> **NIH NIH R01** · MORGRIDGE INSTITUTE FOR RESEARCH, INC. · 2024 · $615,789

## Abstract

PROJECT SUMMARY / ABSTRACT
The goal of this proposal is to develop non-invasive single-cell technologies to improve the potency of T cell
therapies against cancer. The first 6 chimeric antigen receptor (CAR) T cell therapies were recently approved
and >800 CAR and T cell therapies are in clinical trials. However, barriers remain in achieving durable remissions
(>1 year) for ~50% of patients who receive CAR T cell therapy. Due to the rapid development of these therapies
and a great need for process optimization, we focus on improving three translational roadblocks to effective CAR
T cell therapy: (1) screening patients whose T cells are unfit for CAR T cell manufacturing, (2) optimizing in vitro
CAR T cell production for higher potency, and (3) identifying metabolic features of potent CAR T cells in vivo.
CAR T cell therapy could be improved by enriching for naïve and stem cell memory (SCM) T cells in starting
materials and final products. Deficiencies in naïve and SCM T cells occurs in ~50% of untreated cancer patients,
and manufacturing autologous CAR T cell products from these sources has been unsuccessful. Even if SCM T
cells can be isolated, after CAR incorporation, the expansion process typically diminishes potency through T cell
exhaustion. After infusion, the presence of memory-like phenotypes in vivo correlate with better responses. To
date, there are no robust, non-destructive technologies to monitor CAR T cell manufacturing to optimize
production and assess potency in vivo at a single-cell level. These issues limit the impact of CAR T cell therapy.
Current approaches to measure T cell function are labor-intensive, destructive, or lack single-cell resolution,
which limits the frequency or specificity of these measurements. For CAR T cell therapy to realize its clinical
potential, new methods are needed to monitor T cells for optimal potency throughout manufacturing and post-
infusion. Changes in cell metabolism provide an attractive yet under-explored assay to track T cell potency.
Previous studies, including our own, show that T cells undergo drastic metabolic changes with activation, and
that naïve, exhausted, and memory-like T cells have distinct metabolic features. Our preliminary data shows that
non-invasive single-cell imaging of the fluorescence intensity and lifetime of NAD(P)H and FAD (optical
metabolic imaging, or OMI) can predict CAR T cell manufacturing conditions that produce a more vs. less
potent anti-tumor response in vivo. Given these metabolic features of CAR T cell potency, we propose to
determine whether label-free OMI of T cell autofluorescence and multivariate models can identify patient T cell
fitness, optimal in vitro expansion conditions, and in vivo cell biomarkers of potent and persistent CAR T cell
response. Overall, these technologies will streamline processes and interventions for consistently potent T cell
therapy and increase our knowledge of CAR T cell metabolism in vitro and in vivo.

## Key facts

- **NIH application ID:** 10895431
- **Project number:** 5R01CA278051-02
- **Recipient organization:** MORGRIDGE INSTITUTE FOR RESEARCH, INC.
- **Principal Investigator:** Christian Capitini
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $615,789
- **Award type:** 5
- **Project period:** 2023-08-01 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10895431

## Citation

> US National Institutes of Health, RePORTER application 10895431, Label-free imaging of CAR T cell metabolism (5R01CA278051-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10895431. Licensed CC0.

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