Structural mechanism of biased activation of T cell receptor by a high-affinity antibody

Antibodies targeting the TCR–CD3 complex are widely used in cancer therapy but limited by cytokine release syndrome (CRS). Here we identified a TCR–CD3-specific antibody, 4B1, with high binding affinity. Compared to the well-known anti-TCR–CD3 antibody OKT3, 4B1 leads to enhanced T cell activation but significantly reduced cytokine release. 4B1-based bispecific antibodies (BsAbs) demonstrate superior tumor elimination capability with lower cytokine-inducing activity in humanized mice, suggesting that 4B1 uncouples T cell cytotoxicity from inflammatory cytokine release. Cryo-EM analysis reveals that 4B1 Fab binds specifically to a single CD3ε subunit, forming a 1:1 stoichiometry with the TCR–CD3 complex, while OKT3 Fab binds to both CD3ε subunits, resulting in a 2:1 stoichiometry. Confocal microscopy reveals that 4B1 induces mild TCR clustering, while OKT3 leads to robust clustering of TCR–CD3 complexes. In addition, 4B1 triggers delayed and milder CD3ζ phosphorylation and intracellular calcium signals in T cells compared to rapid, potent responses triggered by OKT3. Collectively, these data indicate that CRS depends on the extent of TCR clustering induced by TCR–CD3 antibodies rather than on their binding affinity. Our study identifies a novel TCR–CD3-specific antibody and reveals its biased mechanism for T cell activation, opening new opportunities for safer and more effective TCR-targeting therapeutics.