Directed evolution-based discovery of ligands for in vivo restimulation of chimeric antigen receptor T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B cell malignancies, but many patients relapse owing to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (amph-vax). Here we demonstrate a general strategy to discover and optimize peptide mimotopes enabling amph-vax generation for any CAR. We use yeast surface display to identify peptide binders to FMC63 (the scFv used in clinical CD19 CARs), which are then subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-acute lymphoblastic leukaemia/lymphoma, and enhanced control of disease progression compared with CD19 CAR-T-only-treated mice. This approach enables amph-vax boosting to be applied to any clinically relevant CAR-T cell product.