# Tuning CAR-T cell function

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2021 · $496,990

## Abstract

Abstract
Chimeric antigen receptors (CAR) expressed by T cells recognize tumor cells via single chains antibodies and
activate T cell cytotoxic machinery and costimulation. In clinical studies, costimulation mediated by CD28 and
4-1BB endodomains integrated into the CD19-specific CAR has been shown to be equally effective in causing
tumor regression. However, CD28 and 4-1BB costimulation differentially modulates the kinetic, metabolism
and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. In
this study, we have identified that LCK recruited by co-receptors into the synapse of the CAR encoding CD28
leads to antigen-independent CAR-CD3ζ endodomain phosphorylation and imprints T cell activation upon
antigen engagement. In contrast, the synapse formed by the CAR encoding 4-1BB recruits the THEMIS-SHP1
phosphatase complex that attenuates CAR-CD3ζ endodomain phosphorylation and T cell activation. We have
also proved that the CAR synapse can be engineered to tune down the activity of CD28 costimulation or to
tune up the activity of the 4-1BB costimulation. This discovery has been recently published in Cancer Cell.
Remarkably, we observed that LCK mediated constitutive phosphorylation of CAR-CD3ζ in 4-1BB-
costimulated CAR-Ts does not lead to premature exhaustion of CAR-Ts in xenotransplant models. Therefore,
we hypothesize that the LCK-mediated imprinting in 4-1BB costimulated CAR-Ts leads to unique and critical
signaling pathways in CAR-Ts.
Furthermore, in addition to proximal signaling, CARs profoundly affect downstream T cell signaling. We found
that NF-κB activity is influenced by the type of CAR costimulation. Precisely, 4-1BB induces more pronounced
NF-κB activity than CD28 in CAR-Ts. NF-κB hyperactivity in 4-1BB is not caused by NF-κB overexpression,
but rather by reduced A20 activity. Therefore, we hypothesize that 4-1BB sequesters A20 reducing its
inhibitory effects on NF-κB. Furthermore, since NF-κB/A20 interplay is critical in controlling T cell function at
multiple levels, we hypothesize that regulating NF-κB/A20 may enhance efficacy, persistence and safety of
CAR-Ts. We will develop two specific aims:
Aim 1: To mechanistically assess how LCK-mediated imprinting of 4-1BB costimulated CAR-Ts promotes rapid
antitumor activity without causing T cell exhaustion. We will assess whether LCK overexpression in the 4-1BB
CAR activates unique phosphorylation, transcriptome and metabolic pathways.
Aim 2: To mechanistically assess how 4-1BB affect and how the NF-κB/A20 interplay can be manipulated to
modulate CAR-T cell functions. Since A20/NF-κB interplay is differentially regulated in 4-1BB vs. CD28
costimulated CAR-Ts, we propose to understand how this interplay functions and to develop pharmacologic
and genetic interventions to transiently or permanently modulate NF-κB activity to enhance safety, persistence
and efficacy of CAR-Ts.

## Key facts

- **NIH application ID:** 10139312
- **Project number:** 1R01CA247436-01A1
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Gianpietro Dotti
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $496,990
- **Award type:** 1
- **Project period:** 2021-01-01 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10139312

## Citation

> US National Institutes of Health, RePORTER application 10139312, Tuning CAR-T cell function (1R01CA247436-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10139312. Licensed CC0.

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