Structural and functional insights into CD3 bispecific antibodies targeting DLL3 in cancer immunotherapy.

CD3 bispecific antibodies (BsAbs) targeting delta-like ligand 3 (DLL3) have emerged as promising candidates in cancer immunotherapy by enabling T-cell-mediated tumor lysis. A critical unmet need is to establish structural correlates of their preclinical activity and clinical efficacy to guide rational drug optimization. This study systematically characterized three clinical-stage CD3×DLL3 BsAbs, Tarlatamab (AMG757), BI764532 and HPN328, via structural modeling, T-cell activation assays, cytokine profiling, and tumor cytotoxicity tests. Preclinical data were further correlated with clinical trial outcomes to verify the predictive value of in vitro models. Preclinical data demonstrate a strong correlation with clinical outcomes, confirming that in vitro assays can reliably predict therapeutic response. Notably, structural features including immune synapse (IS) distance and epitope binding are tightly correlated with efficacy and safety. HPN328 exhibited the most potent T-cell activation and tumor killing activity, which aligned with its clinical confirmed complete response rate (cORR) of 50% in small cell lung cancer. BI764532 demonstrated a balanceed efficacy (ORR 18% in SCLC), while Tarlatamab achieved an ORR range of 13-40% across clinical trials. IS distance and epitope binding are strongly correlated with efficacy and safety of DLL3-targeted BsAbs, providing a critical framework for optimizing T-cell engager design in cancer immunotherapy.