Affinity and costimulatory architecture-associated toxicity in mesothelin-targeted TCR-mimetic CAR-T cells.

Introduction: TCR-mimetic CAR-T combines antibody-based targeting with physiological TCR signaling, yet suboptimal co-stimulation and safety risks remain key barriers for solid tumor therapy. The prevailing assumption that stronger co-stimulation yields superior efficacy has not been systematically tested in TCR-mimetic CAR-T architectures. Methods: We developed a Mesothelin-targeted scFv-CD3ϵ TCR-mimetic CAR-T platform and systematically compared co-stimulatory modules-4-1BB, BRM-truncated 4-1BB Results: The optimized SS1-ϵζ-80BB TCR-mimetic CAR-T exhibited potent anti-tumor activity but unexpectedly induced dose-dependent lethal toxicity. Systematic controls identified the intact 80BB chimera as the construct associated with lethality. Deleting the 4-1BB intracellular domain, rerouting the 4-1BB intracellular domain to an antigen-dependent format, or replacing SS1 with MOR each abrogated toxicity while preserving anti-tumor efficacy. The SS1-ϵζ-80BB architecture was associated with excessive T-cell expansion and multi-organ infiltration while the MOR-ϵζ-80BB architecture was moderate. Discussion: These observations suggest that the mode of co-stimulatory configuration, rather than its mere presence, influences safety outcomes in TCR-mimetic CAR-T cells. Modifying either the affinity-related properties or the 80BB-based architecture preserved efficacy while reducing toxicity, providing potential design considerations for affinity selection and co-stimulator design in the mesothelin-targeted TCR-mimetic CAR-T platform.