APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy

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.

Keywords

• MT: Regular Issue
• CAR-T cells
• immunotherapy
• melanoma
• APOBEC3
• APOBEC3B