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Updated: Jan 11, 2026
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Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
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Framework humanization enhances GM3(Neu5Gc)-targeting CAR-T cell function by reducing tonic signaling.
Jiaxin Tu1, Xinyu Li1, Yuge Zhu2
1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Laboratory of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing, China.
Frontiers in Immunology
|November 10, 2025
Summary
Humanizing chimeric antigen receptor (CAR)-T cells targeting GM3(Neu5Gc) gangliosides improved their anti-tumor activity. This antibody framework optimization reduced tonic signaling, enhancing CAR-T cell persistence and efficacy against solid tumors.
Area of Science:
- Immunotherapy
- Oncology
- Biotechnology
Background:
- GM3(Neu5Gc) is a tumor-associated ganglioside absent in normal human tissues, making it a promising target for solid tumor immunotherapy.
- Chimeric antigen receptor (CAR)-T cell therapy offers a potential treatment strategy for GM3(Neu5Gc)-positive solid tumors.
Purpose of the Study:
- To evaluate antibody humanization strategies for enhancing GM3(Neu5Gc)-targeted CAR-T cells.
- To optimize CAR design by comparing different hinge domains and assessing the impact of scFv humanization on CAR-T cell functionality.
Main Methods:
- Systematic evaluation of CARs based on murine (14F7) and humanized (14F7hT) antibodies with varying hinge domains (CD8α, CD28, IgG4).
- In vitro cytotoxicity assays and in vivo xenograft models to assess CAR-T cell performance.
- Repeated tumor stimulation assays to evaluate functional durability under chronic antigen exposure.
- CAR-Toner analysis to predict and experimental validation of tonic signaling levels.
Main Results:
- Humanized 14F7hT-based CARs consistently outperformed murine counterparts in vivo.
- Optimized 14F7hT-CD28 CAR-T cells showed superior expansion, persistence, tumor infiltration, and anti-tumor efficacy.
- Humanized CAR-T cells exhibited enhanced cytotoxicity, degranulation, proliferation, and functional durability.
- Reduced tonic signaling in humanized CAR-T cells correlated with improved functionality.
Conclusions:
- Framework humanization of CARs, specifically the 14F7hT-CD28 construct, is a key strategy to improve CAR-T cell performance against solid tumors.
- Optimized CAR design through antibody humanization and hinge selection can overcome limitations of excessive tonic signaling, leading to enhanced therapeutic efficacy.
