Sequential Antigen-loss and Branching Evolution in Lymphoma after CD19- and CD20-Targeted T-cell Redirecting Therapy.

CD19 CAR T-cells and CD20 targeting T-cell engaging bispecific antibodies have been approved in B-cell Non-Hodgkin lymphoma lately, heralding a new clinical setting where patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19 and CD20 directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline on 28 longitudinally collected specimen from seven patients, we identified truncating mutations in the gene encoding CD20 conferring antigen-loss in 80% of patients relapsing from CD20 bispecs. Pronounced T-cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19-loss after CAR T-cell therapy and report the case of sequential CD19- and CD20-loss. We observed branching evolution with re-emergence of CD20-positive subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as an evolutionary bottleneck selecting for antigen-loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies.