The costimulatory domain influences CD19 CAR-T cell resistance development in B-cell malignancies.

CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro. Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells.