Chimeric switch scaffold protein augments CAR synapse formation and signaling networks

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has achieved clinical success in hematologic malignancies, but remains limited in solid tumors due to immune checkpoint-mediated suppression and intrinsic signaling constraints. Unlike T-cell receptors (TCRs), CARs fail to efficiently recruit the linker for activation of T cells (LAT) signalosome, resulting in suboptimal signal propagation and unstable immunological synapse organization. METHODS: To address this limitation, we engineered a programmed cell death protein 1 (PD-1)-LAT (PLAT) chimeric switch scaffold that couples PD-1 engagement to LAT-dependent proximal signaling. We compared the signaling kinetics, synaptic architecture, and antitumor efficacy of PLAT-expressing HER2 CAR T cells against those expressing conventional PD-1-CD28 switch receptors or dominant-negative PD-1 using biochemical signaling assays, quantitative imaging analysis, chronic antigen stimulation models, and in vivo xenograft validation. RESULTS: PLAT enhanced LAT phosphorylation and nucleated LAT-associated signaling complexes on programmed death-ligand 1 engagement. This reorganized the CAR immune synapse into a concentric, TCR-like architecture, restoring proximal signaling strength. This signaling reprogramming resulted in increased calcium flux and NFAT/NF-κB activation, driving enhanced cytotoxicity, sustained proliferation, and resistance to functional exhaustion under chronic antigen exposure. In direct comparisons, PLAT outperformed conventional PD-1-CD28 switch receptors in early functional responses and demonstrated superior functional durability and antitumor activity in vivo compared with dominant-negative PD-1 strategies. CONCLUSIONS: These findings establish PLAT as a scaffold-based strategy that directly addresses intrinsic CAR signaling deficiencies by converting immune checkpoint engagement into LAT-dependent proximal signaling restoration. This work provides a new framework for engineering CAR T cells with improved function and persistence in immunosuppressive tumor environments.