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Updated: Jan 7, 2026
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy
Adam Blaisdell1, Stefanie Bachl1,2, Luis R Sandoval2,3
1Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
Abstract:
T cell exhaustion limits the efficacy of cancer immunotherapies. Here, we performed genome-wide loss-of-function screening in repetitively stimulated human T cells and identified the mulitfunctional ubiquitin-modifying protein A20/TNFAIP3 as a major negative regulator of exhausted T cell persistence. Protein large language modeling, deep base-editing mutagenesis, and studies in immunocompetent mice with domain-specific inactivating mutations revealed A20's non-enzymatic M1 ubiquitin-binding zinc finger 7 (A20ZF7) motif as critical to suppression of anti-tumor immunity. A20ZF7-deficient CD8+ tumor-infiltrating lymphocytes (TILs) resisted terminal exhaustion and circumvented an unappreciated mechanism restraining perforin degranulation in terminally exhausted cells. Human chimeric antigen receptor (CAR)-T cells engineered via base-editing to inactivate A20ZF7 via a single missense mutation also resisted exhaustion, secreted more perforin and robustly suppressed cancer in vivo. These studies pinpoint A20ZF7 as a novel T cell checkpoint and reveal precision base-editing of missense mutations as an effective approach to enhance CAR-T cell therapy.
Insights
Scientists identified A20/TNFAIP3 as a key regulator of T cell exhaustion in cancer immunotherapy. Targeting its A20ZF7 motif with base-editing enhances anti-tumor T cell function and cancer suppression.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- T cell exhaustion hinders cancer immunotherapy effectiveness.
- Identifying regulators of T cell exhaustion is crucial for improving treatments.
Purpose of the Study:
- To identify novel regulators of T cell exhaustion.
- To investigate the role of A20/TNFAIP3 in T cell persistence and anti-tumor immunity.
- To explore base-editing strategies for enhancing CAR-T cell therapy.
Main Methods:
- Genome-wide loss-of-function screening in human T cells.
- Protein large language modeling and deep base-editing mutagenesis.
- In vivo studies using immunocompetent mice and engineered CAR-T cells.
Main Results:
- A20/TNFAIP3 identified as a major negative regulator of exhausted T cell persistence.
- A20's M1 ubiquitin-binding zinc finger 7 (A20ZF7) motif is critical for suppressing anti-tumor immunity.
- A20ZF7-deficient T cells resisted exhaustion, enhanced perforin degranulation, and improved tumor suppression.
- Base-edited human CAR-T cells targeting A20ZF7 resisted exhaustion and demonstrated potent anti-cancer activity in vivo.
Conclusions:
- A20ZF7 is a novel T cell checkpoint that suppresses anti-tumor immunity.
- Precision base-editing of missense mutations offers a promising strategy to enhance CAR-T cell therapy efficacy.
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