On and off-target editing efficiencies of sgRNAs used for generation of antigen loss RAJI cell line models for CAR-T cell evaluation.

Antigen loss/escape remains a significant mechanism of resistance to chimeric antigen receptor (CAR)-T cell therapy, leading to relapse in patients with B-cell malignancies and representing a major clinical challenge. Antigen loss triggers relapse in more than 40% of patients undergoing CD19 CAR-T cell therapy. To rigorously validate antigen loss, it is essential to develop robust in vitro models that faithfully mimic the dynamic process of antigen escape. The current absence of these models hampers our ability to fully evaluate and optimize treatment strategies. To model this clinically relevant phenomenon, we generated single (sKO), double (dKO), and triple (tKO) knockout Raji cell lines targeting tumor-associated antigens CD19, CD20, and CD22 using CRISPR/Cas9 genome editing, which were validated at the genomic, transcript, and protein levels. To facilitate quantitative assessment of CAR-T cell-mediated cytotoxicity, we first generated a clonal, luciferase-expressing Raji cell line, ensuring a uniform luminescence background across all knockout models. This enabled high-throughput in vitro cytotoxicity assays and non-invasive in vivo tumor tracking using bioluminescence imaging (BLI). Functional co-culture assays demonstrated that antigen-deficient Raji cells exhibited resistance to antigen-specific CAR-T cell killing, mirroring clinical relapse scenarios. The triple knockout (tKO) model, in particular, provided a superior system compared to commonly used K562 models, as it retains the same lymphoma background while eliminating key antigenic targets. These antigen-loss models serve as valuable tools for studying mechanisms of CAR-T cell resistance and evaluating next-generation CAR designs, including dual-targeting and combination therapies.