NECTIN4×PD-L1双特异性抗体药物偶联物整合检查点阻断与靶向细胞毒性用于尿路上皮癌和肺鳞状细胞癌

Background: The first-line approval of enfortumab vedotin plus pembrolizumab validates the clinical rationale for combining ADCs with ICIs. However, administering two separate agents poses challenges in cost, compliance, toxicity management, and in achieving comprehensive coverage of target and immune heterogeneity. To address these limitations, we engineered a NECTIN4×PD-L1 bispecific ADC using Dong-A ST BsAb platform with an enhanced knob-into-hole (eKiH) interface that integrates NECTIN4-directed cytotoxic delivery with PD-L1 axis blockade in a single molecule. The molecule was designed to overcome resistance and spatial heterogeneity in locally advanced/metastatic urothelial carcinoma (la/mUC) and LUSC. Methods: High-affinity NECTIN4 and PD-L1 antibody leads were identified and assembled into Dong-A ST BsAb platform. Site-selective conjugation was applied to generate ADCs bearing MMAE, exatecan, or dual-payload configurations. Functional characterization included: (i) binding affinity and competition to both targets, (ii) internalization and payload release in NECTIN4-positive cells, and (iii) PD-1/PD-L1 reporter assays and T-cell co-culture cytotoxicity. To support clinical translation, scRNA-seq and tissue microarray-based mIHC were performed on bladder cancer and LUSC cohorts to evaluate NECTIN4/PD-L1 co-expression, spatial heterogeneity, and correlations with immune infiltration and checkpoint pathways. Results: scRNA-seq revealed intra-patient coexistence of NECTIN4-only, PD-L1-only, and co-expressing tumor subpopulations, with notable inter-patient variability. Multiplex IHC confirmed mosaic and cluster-type spatial heterogeneity. The bispecific ADC maintained high-affinity binding to both targets, exhibited efficient internalization and payload release in NECTIN4-positive cells, and preserved potent PD-1/PD-L1 blockade activity in reporter assays. ADCs carrying MMAE, exatecan, or dual payloads demonstrated consistent in vitro cytotoxicity across NECTIN4-high, PD-L1-high, and co-expressing tumor models, along with enhanced T cell-mediated tumor killing in co-culture. Preliminary in vivo studies showed dose-dependent tumor growth inhibition in bladder cancer and LUSC xenograft models with acceptable tolerability (detailed results pending final analysis). Conclusions: This NECTIN4×PD-L1 bispecific ADC integrates targeted cytotoxicity (NECTIN4) and checkpoint blockade (PD-L1) into a single therapeutic agent, rationally designed to provide catch-all activity against target and immune microenvironment heterogeneity in la/mUC and LUSC. The preclinical data support evaluation as an early-line therapeutic strategy that could complement or substitute separate ADC+ICI combinations.