Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling

Abstract Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR signaling caused the intrinsic instability mAb single-chain variable fragment (scFv) promote self-aggregation via CD3? chain incorporated into construct. This phenomenon was detected in encoding either CD28 or 4-1BB costimulatory endodomains. Instability scFv specific amino acids within framework regions (FWR) can be identified computational modeling. Substitutions causing instability, humanization FWRs, corrected CAR, without modifying specificity, enhanced effects cells. Overall, demonstrated cells determined molecular analyses implemented correct with costimulation.