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Updated: Mar 30, 2026
Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells
Published on: February 14, 2025
NOT-gated chimeric antigen receptor circuits in T and NK cells
Seunghee Lee1, Jingyao Chen1, Menna Y Siddiqui1
1Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA.
Abstract:
Chimeric antigen receptor (CAR) T cells are powerful cancer immunotherapies, but the lack of tumor-specific single antigens for most cancers limits the therapeutic window. A NOT-gated CAR circuit, consisting of an activating CAR (aCAR) and an inhibitory CAR (iCAR), is particularly powerful because it enables new sets of antigens to be used in tumor targeting. We systematically evaluated more than 60 pairs of NOT-gated CAR circuits. Different inhibitory dose-response characteristics were observed depending on the CD3ζ signaling domain on the aCAR. Furthermore, LIR1-iCAR-expressing T cells display fewer exhaustion phenotypes. Mechanistic studies have shown that SHP-1 is the major intracellular regulator of LIR1-iCAR and the iCAR function in cis. Several of the NOT-gated circuits developed in T cells were functionally transferred into primary natural killer (NK) cells. Our NOT-gated CAR circuits provide a precise targeting strategy for safer and more effective cancer immunotherapies.
Insights
New NOT-gated CAR circuits enhance cancer immunotherapy by enabling precise targeting of tumor antigens. This approach improves safety and efficacy by combining activating and inhibitory CARs, overcoming limitations of current therapies.
Area of Science:
- Immunology
- Oncology
- Cell Therapy
Background:
- Chimeric antigen receptor (CAR) T cells are effective cancer immunotherapies.
- Limited tumor-specific antigens restrict the therapeutic window for most cancers.
- NOT-gated CAR circuits offer a solution by combining activating (aCAR) and inhibitory (iCAR) components for dual antigen targeting.
Purpose of the Study:
- To systematically evaluate NOT-gated CAR circuits for improved cancer immunotherapy.
- To investigate the impact of different CD3ζ signaling domains on aCAR function.
- To assess the potential of NOT-gated CAR circuits in T cells and natural killer (NK) cells.
Main Methods:
- Systematic evaluation of over 60 pairs of NOT-gated CAR circuits.
- Analysis of inhibitory dose-response characteristics based on CD3ζ signaling.
- Assessment of T cell exhaustion phenotypes in LIR1-iCAR-expressing cells.
- Mechanistic studies to identify intracellular regulators (e.g., SHP-1).
- Functional transfer of NOT-gated circuits into primary NK cells.
Main Results:
- Diverse inhibitory dose-response profiles were observed, influenced by the aCAR's CD3ζ domain.
- T cells expressing LIR1-iCAR exhibited reduced exhaustion phenotypes.
- SHP-1 was identified as a key regulator of LIR1-iCAR and cis-iCAR function.
- NOT-gated CAR circuits were successfully transferred and functional in NK cells.
Conclusions:
- NOT-gated CAR circuits provide a versatile platform for precise tumor targeting in cancer immunotherapy.
- This strategy enhances safety and efficacy by overcoming single-antigen limitations.
- The developed circuits hold promise for next-generation T cell and NK cell-based therapies.
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