# Role of NOTCH1 Signaling in Engineering CD4+ T Cells for Cancer Immunotherapy

> **NIH NIH F30** · UNIVERSITY OF WASHINGTON · 2022 · $51,752

## Abstract

PROJECT SUMMARY
Current methods for engineering T cells for cancer immunotherapy use agonistic anti-CD3 and -CD28
monoclonal antibodies (mAb) to activate T cells, which induce their proliferation but do not recapitulate other
fate-determining signals delivered by antigen-presenting cells. One such signaling axis, the NOTCH pathway,
controls CD4+ T cell effector function acquisition and strongly influences behavior. To study NOTCH signaling
during chimeric antigen receptor (CAR) T cell production, we developed a culture system using anti-CD3/CD28
mAb-coated beads and plate-coated agonistic NOTCH1-specific mAb to induce simultaneous T cell activation
and NOTCH signaling. When transferred into NSG mice bearing CD19+ Raji lymphoma, CD19-specific
NOTCH1-agonized (N1) CD4+ CAR T cells displayed a marked proliferative advantage over control (IgG)
cells. Tumor-bearing mice given both IgG CD8+ CAR T cells and N1 CD4+ CAR T cells demonstrated
superior expansion of both subsets compared to mice given IgG CD8+ and IgG CD4+ CAR T cells,
resulting in rapid tumor clearance and protection from tumor re-challenge. These data demonstrate that
NOTCH1 agonism could represent a significant improvement to adoptive T cell therapy, but the mechanisms
by which NOTCH signaling improves T cell anti-tumor function are currently not understood. NOTCH is known
to induce aryl hydrocarbon receptor (AhR) transcriptional activity. Pharmacologic inhibition of AhR activity
during N1 CD4+ CAR T cell culture reduced characteristic differences between N1 and IgG cells in surface
phenotype, cytokine production and proliferation upon in vitro restimulation. I hypothesize that NOTCH1
agonism organizes AhR-dependent transcriptomic changes that augment proliferative cytokine
production in CD4+ CAR T cells, promoting CD4-dependent proliferation that improves CAR T cell
efficacy. To test this hypothesis, I will assess the functional effects of AhR inhibition and activation in N1 and
IgG CD4+ CAR T cells in vitro and in vivo, and characterize fate-determining transcriptional events in N1, IgG
and AhR-inhibited N1 CD4+ CAR T cells using bulk and single-cell RNA sequencing. Collectively, these
experiments will investigate the utility of NOTCH and AhR signaling in the generation of superior CAR T cell
products and establish the mechanisms by which NOTCH1 agonism improves CAR T cell therapeutic efficacy,
laying the groundwork for translation of this promising biomedical advance into immunotherapy clinics.

## Key facts

- **NIH application ID:** 10348150
- **Project number:** 5F30CA257088-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Alec Baker Wilkens
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $51,752
- **Award type:** 5
- **Project period:** 2021-03-16 → 2024-03-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10348150

## Citation

> US National Institutes of Health, RePORTER application 10348150, Role of NOTCH1 Signaling in Engineering CD4+ T Cells for Cancer Immunotherapy (5F30CA257088-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10348150. Licensed CC0.

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