Disulfide-directed multicyclic peptides for chimeric antigen receptors targeting solid tumors

The clinical application of chimeric antigen receptor (CAR) T cell therapy in solid tumors remains limited due to significant safety concerns, particularly “on-target, off-tumor” toxicity and cytokine release syndrome (CRS). Here, we describe a class of CARs that employ disulfide-directed multicyclic peptides (DDMPs) as compact antigen-recognition domains targeting the tumor-associated antigens HER2 and TROP2. DDMP-based CAR T cells exhibited antigen density-dependent cytotoxicity in vitro and in vivo, efficiently eliminating cells with high antigen expression while sparing cells with low antigen levels, thereby mitigating on-target, off-tumor toxicity. In addition, DDMP-based CAR T cells secreted markedly lower levels of pro-inflammatory cytokines upon targeted killing, reducing the CRS risk. Mechanistic analyses revealed that this favorable combination of restrained cytokine release and density-gated killing is associated with distinct T cell signaling pathway engagement and reduced cell avidity relative to conventional single-chain variable fragment (scFv)-based CAR T cells. Collectively, these findings establish DDMP-based CARs as a promising framework for engineering safer, yet efficacious, CAR T cell therapies for solid tumors.