Convergence of Acquired Mutations and Alternative Splicing of <i>CD19</i> Enables Resistance to CART-19 Immunotherapy

TLDR

The CD19 antigen is expressed on most B‑cell acute lymphoblastic leukemias and can be targeted with CART‑19, yet 10–20 % of pediatric responders relapse due to epitope loss. Pull‑down/siRNA experiments identified SRSF3 as a splicing factor that promotes exon 2 retention, and genome‑editing showed that exon 2 skipping bypasses CD19 mutations, producing a truncated variant that evades CART‑19 killing. Relapse samples displayed hemizygous CD19 deletions, exon 2 mutations, and alternatively spliced transcripts lacking exon 2, and the combination of these alterations enables a truncated CD19 variant that escapes CART‑19‑mediated cytotoxicity.

Abstract

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.