The most common pattern of treatment failure following CD19-targeting chimeric antigen receptor T-cell therapy (CAR-T) is measurable residual disease (MRD) negative remission followed by relapse. We hypothesised that relapse arises from a rare “CAR-T tolerant persister” population that survives the strong therapeutic pressure and seeds relapse. To address this, we investigated the clonal origin and transcriptional dynamics of relapsed B-cell acute lymphoblastic leukaemia (B-ALL) following CAR-T via time resolved tracing of the progeny of individual B-ALL clones, using expressed molecular barcodes in an immunocompetent mouse model of CD19-targeting CAR-T.
Our immunocompetent mouse model of murine CAR-T targeting CD19 on a syngeneic, BCR::ABL1 driven B-ALL, recapitulates MRD-negative remission followed by CD19+ relapse. We applied SPLINTR, a lineage-tracing method utilising expressed molecular barcodes, to profile the transcriptional features of single leukaemia clones prior to treatment, in remission, and following relapse.
CAR-T induced significant clonal restriction of relapsing B-ALL. Only 0.7% of clones survived CAR-T and led to relapse. In fact, >99% of post-CAR T relapsed disease was comprised by just 3 (0.3%) individual clones and in one mouse disease relapse was driven entirely a single clone. Remarkably, we found that the same clones were able to drive relapse in multiple mice, consistent with the presence of heritable cell-intrinsic properties capable of causing relapse following CAR-T.
While relapse-fated clones were transcriptionally indistinguishable prior to treatment, they underwent substantial, and clone-specific transcriptional adaptation following CAR-T. In contrast to the highly concordant adaptive responses of individual clones across different recipients, the transcriptional phenotype of the relapsed bulk leukaemia was highly heterogeneous between clones.
Remarkably however, a rare cell state (~1% of the bulk B-ALL) was shared by each relapse-fated clone, with a distinct developmental and metabolic transcriptional program, highly suggestive that these cells represent an “origin-of-relapse” state acquired by cells persisting during the MRD-negative period from which relapse emerges.
The potential for post-CAR-T relapse, and the adaptive processes that lead to resistance, are clone intrinsic, heritable properties. Whilst the acquired transcriptional states of relapsed B-ALL following CAR-T are highly heterogeneous, relapse appears to emerge from a rare cell state common to all relapse-fated clones.