Enhanced functionality of low-affinity CD19 CAR T-cells is associated with activation priming and a polyfunctional cytokine phenotype

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR, CAT) that showed enhanced expansion, cytotoxicity, and anti-tumour efficacy compared to the high-affinity (FMC63 based) CAR used in tisagenlecleucel, in pre-clinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a Phase I clinical study. To understand the molecular mechanisms behind the improved properties of CAT CAR T-cells, we performed a systematic in vitro characterization of the transcriptomic (RNA-seq) and protein (CyTOF) changes occurring in T-cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. We demonstrate that CAT CAR T-cells show enhanced activation to CD19 stimulation and a distinct transcriptomic/protein profile with increased cytokine polyfunctionality post-stimulation compared with FMC63 CAR T-cells. Our results suggest that the enhanced functionality of low-affinity CAR T-cells may be sustained by the establishment of a self-reinforcing circuit activated through cytokines polyfunctional crosstalk.