Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target expressing cells

Abstract Recently, a rare type of relapse was reported upon treating a B-ALL patient with anti-CD19 CAR-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cell). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs) inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LV containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMC and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 d. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.