Related Experiment Video
Updated: Jun 10, 2025
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR
Jack Swanson1,2, Jason Tonne2, Thanich Sangsuwannukul2
1Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
Abstract:
Chimeric antigen receptor (CAR) T cells are a clinically approved therapy for blood cancers. To produce clinical-grade CAR T cells, a retroviral or lentiviral vector is used to deliver the CAR and associated genes to patient T cells. Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) enzymes are known to be upregulated after transfection and retroviral infection and to deaminate cytidine to uracil in nucleic acids, resulting in cytidine-to-thymine mutations in DNA. Here, we hypothesized that APOBEC3 enzymes, induced during the production of CAR T cells, impact the efficacy of the resulting CAR T cells. We demonstrated that APOBEC3 family member APOBEC3B was upregulated at the RNA and protein levels after transfection of HEK293T cells with plasmids to make lentivirus, and that APOBEC3 signature mutations were present in the CAR construct. APOBEC3B overexpression in HEK293T cells led to further mutations in the resulting CAR T cells, and significantly decreased CAR T cell killing. APOBEC3B knockout in HEK293T cells led to reduced mutations in the CAR construct and significantly increased in CAR T cell killing. These results suggest that generation of CAR-expressing viruses from producer cell lines deficient in genome-modifying proteins such as APOBEC3B could enhance the quality of CAR T cell production.
Insights
APOBEC3B enzyme activity during CAR T-cell production causes mutations, reducing cancer-killing efficacy. Eliminating APOBEC3B enhances CAR T-cell quality and therapeutic potential.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunotherapy
Background:
- Chimeric antigen receptor (CAR) T-cells are an approved therapy for blood cancers.
- CAR T-cell production involves viral vectors to deliver genes into patient T-cells.
- APOBEC3 enzymes can cause mutations in nucleic acids during viral production.
Purpose of the Study:
- To investigate the impact of APOBEC3 enzymes on CAR T-cell production and efficacy.
- To determine if APOBEC3B upregulation affects CAR construct integrity and CAR T-cell function.
Main Methods:
- Assessed APOBEC3B expression in HEK293T cells during lentivirus production.
- Analyzed CAR constructs for APOBEC3-induced mutations.
- Quantified CAR T-cell killing activity after manipulating APOBEC3B levels.
Main Results:
- APOBEC3B was upregulated post-transfection, with APOBEC3-signature mutations found in the CAR construct.
- Overexpression of APOBEC3B increased CAR construct mutations and decreased CAR T-cell killing efficacy.
- APOBEC3B knockout reduced mutations and significantly enhanced CAR T-cell killing.
Conclusions:
- APOBEC3B contributes to CAR construct mutations during lentiviral vector production.
- Reducing APOBEC3B activity in producer cell lines can improve CAR T-cell quality and therapeutic effectiveness.
