Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapies have achieved remarkable success in treating B-cell malignancies, including acute lymphoblastic leukemia (B-ALL). However, despite high remission rates, relapse due to antigen escape remains a significant challenge. To overcome this, designing CAR T-cells targeting multiple cancer antigens simultaneously is a promising strategy. NKG2D ligands (NKG2DL) are eight stress-induced ligands expressed by cancer cells but largely absent on healthy cells. METHODS AND RESULTS: We hypothesized that simultaneous targeting of NKG2DL (using the NKG2D extracellular domain) and CD19 can prevent CD19 antigen escape and improve long-term remission rates in B-ALL patients. We developed three tandem CARs targeting both CD19 and NKG2DL and demonstrated that two tandem candidates were highly effective against both CD19+ and CD19- cancer cell lines. Importantly, when compared to CD19 CAR T-cells, tandem CAR T-cells exhibited comparable cytokine secretion, cytolytic activity and proliferation levels when incubated with cancer cells expressing CD19 and were still effective when incubated with cancer cells lacking CD19. Moreover, T-cells transduced with the selected CD19/NKG2DL tandem CAR were functional against CD19+ primary B-ALL samples and controlled tumor growth in a highly challenging xenograft model representing a CD19- B-ALL relapse. DISCUSSION: These findings provide proof-of-concept that NKG2D-based tandem CARs offer a promising approach to overcome antigen escape and enhance anti-tumor efficacy in B-cell malignancies.