CD19 exon 2 skipping is a potential prognostic correlate of anti-CD19 CAR-T therapy relapse

Relapse following anti-CD19 chimeric antigen receptor (CAR) T cell therapy remains a concern in the treatment of refractory B-cell malignancies. Although the CD19<b>Δ</b>exon2 splice variant has been linked to treatment failure, reliable pre-treatment biomarkers for relapse risk are lacking. Here, we analyzed RNA-sequencing data from a small publicly available cohort of four anti-CD19 CAR-T-treated B-cell acute lymphoblastic leukemia patients, including one responder, one non-responder, and two who relapsed after initial response. We quantified the percent spliced in (PSI) of CD19 exon 2, as a proxy for CD19<b>Δ</b>exon2 abundance before and after treatment. The patient with the lowest pre-treatment exon 2 PSI (i.e., highest estimated abundance of CD19<b>Δ</b>exon2) experienced the earliest relapse, whereas the complete responder showed no detectable exon 2 skipping. In silico protein structure modeling indicated reduced structural stability of the FMC63 epitope region in the CD19<b>Δ</b>exon2 variant, supporting a potential mechanistic link between exon 2 exclusion and antigen escape. Analysis of larger RNA-sequencing datasets from CAR-T treatment-naïve B-cell malignancies and healthy tissues revealed low-level exon 2 skipping in some individuals across both malignant and normal B cells. These findings suggest that CD19 exon 2 skipping may correlate with relapse after CAR-T therapy, and its presence in treatment-naïve individuals highlights its potential for evaluation as an RNA- or qPCR-based biomarker in future studies.