# Ameliorating off-target toxicities of CAR T cells by engineering NOT gates

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $442,900

## Abstract

Project Summary/Abstract
Engineered therapeutic T cells have shown transformative success in treating B cell cancers but applying this
approach to solid tumors has proven far more difficult. There do not appear to be absolutely tumor-specific single
antigen targets for solid cancers, and thus, CAR T cells that attack most tumor-associated antigens have led to
toxic cross-reaction with normal organs that also express the antigen. If we are to successfully and safely treat
solid tumors with CAR T cells, it will be essential to mitigate toxic cross-reaction with normal tissues.
To prevent off-tumor toxicity of therapeutic T cells, we propose to engineer multi-receptor T cell circuits that can
recognize a tumor based on a multi-antigen profile. In this proposal, we focus specifically on engineering NOT
gate circuits -- circuits that can override and inhibit CAR T cell activation and killing upon detecting an antigen
that is uniquely indicative of a cross-reactive normal tissue (i.e., antigen is absent in the tumor). Our recently
published bioinformatic analysis shows that there are numerous tissue-specific antigens that could be used as
signals to induce T cell inactivation in common cross-reactive tissues like the brain and lung. Nonetheless, there
is currently a lack of robust NOT-gate circuits demonstrated to work well in tumor models. Thus, we will develop
and test new NOT circuits that can inactivate a CAR T cell in an antigen-induced manner. Our specific aims are:
Aim 1. Engineer, prototype and optimize new NOT gate circuits that use diverse mechanisms to block
therapeutic T cell activation in antigen-induced manner
 Aim 1.1. T cell NOT gates using transcriptional repressors of CAR expression.
Aim 1.2. T cell NOT gates that inhibit T cell proliferation by antigen-induction of cell death effectors.
Aim 1.3. T cell NOT gates that locally induce production of secreted immunosuppressive factors (paracrine)
Aim 2. Applying NOT gate circuits to prevent anti-GD2 CAR T cross-reaction with brain/CNS tissue.
 Aim 2.1. in vitro prototyping of NOT gate circuit targeting the brain antigen MOG to turn off anti-GD2 CAR
Aim 2.2. Test if brain NOT gates block CNS toxicity of anti-GD2 CAR T cells in vivo, while maintaining
efficacy against murine neuroblastoma xenograft models (GD2+).
Aim 2.3. Test in vivo safety & efficacy of NOT gate CAR T cells in an immunocompetent model of
neuroblastoma.
This work should provide important general capabilities for engineering CAR T cells that selectively turn
themselves OFF when they are in the wrong, cross-reactive tissue. These are much needed tools that are
currently missing in the toolbox for T cell engineering, but which will be critical for engineering T cells that safely
treat solid cancers.

## Key facts

- **NIH application ID:** 10657356
- **Project number:** 5R01CA258789-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** WENDELL A LIM
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $442,900
- **Award type:** 5
- **Project period:** 2022-08-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10657356

## Citation

> US National Institutes of Health, RePORTER application 10657356, Ameliorating off-target toxicities of CAR T cells by engineering NOT gates (5R01CA258789-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10657356. Licensed CC0.

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