Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness

Tonic signaling of chimeric antigen receptor (CAR), i.e., the spontaneous CAR activation in the absence of tumor antigen stimulation, is considered to be a pivotal event controlling CAR-T efficacy. However, the molecular mechanism underlying the spontaneous CAR signals remains elusive. Here, we unveil that positively charged patches (PCPs) on the surface of the CAR antigen-binding domain mediate CAR clustering and result in CAR tonic signaling. For CARs with high tonic signaling (e.g., GD2.CAR and CSPG4.CAR), reducing PCPs on CARs or boosting ionic strength in the culture medium during ex vivo CAR-T cell expansion minimizes spontaneous CAR activation and alleviates CAR-T cell exhaustion. In contrast, introducing PCPs into the CAR with weak tonic signaling, such as CD19.CAR, results in improved in vivo persistence and superior antitumor function. These results demonstrate that CAR tonic signaling is induced and maintained by PCP-mediated CAR clustering. Notably, the mutations we generated to alter the PCPs maintain the antigen-binding affinity and specificity of the CAR. Therefore, our findings suggest that the rational tuning of PCPs to optimize tonic signaling and in vivo fitness of CAR-T cells is a promising design strategy for the next-generation CAR.