Anovel anti-B7-H3 nanobody and its DM1 conjugate with marked anti-tumor activity against esophageal squamous cell...

Esophageal squamous cell carcinoma (ESCC), a highly aggressive malignancy with poor therapeutic outcomes, currently lacks effective molecularly targeted therapies. Despite emerging interest in B7-H3 as an attractive tumor-specific target, its therapeutic potential in ESCC remains unexplored. This study investigates the feasibility and efficacy of B7-H3 targeting using nanobodies and their drug conjugates as a therapeutic strategy for ESCC. Here, we report the development of BH1, a novel B7-H3-specific nanobody demonstrating subnanomolar binding affinity (EC₅₀ = 2.32 nM) to a conformational epitope spanning the IgC1-IgV2 domains. In vivo evaluation using KYSE30 xenograft models revealed rapid tumor-selective accumulation of BH1 within 4 h post-injection. BH1 monotherapy inhibits tumor cell proliferation, induces G0/G1 phase arrest and apoptosis, achieving 37.7 % tumor growth inhibition, whereas DM1 conjugation markedly enhances efficacy (71.9 % suppression, p < 0.001). Notably, BH1-DM1 maintained potent antitumor activity (59.8 % suppression, p < 0.001) in the BH1 monotherapy-insensitive but B7-H3-positive KYSE450 model. Structural analysis identified an extended CDR3 loop enabling BH1 to access sterically constrained regions within the B7-H3 IgC1-IgV2 cleft, a feature may be difficult to achieve with conventional high-molecular-weight antibodies. These findings propose B7-H3 as a viable therapeutic target for ESCC. Additionally, results from the BH1 study suggest its potential as a modular scaffold for targeted delivery applications. BH1-DM1, distinct from existing B7-H3 monoclonal antibody-based therapies, may offer a breakthrough therapeutic strategy for ESCC.