Preclinical Evaluation of B7−H3-Targeted ADC-Based Molecular Imaging in Lung and Triple-Negative Breast Cancer

B7−H3 is widely expressed in multiple solid tumors but shows minimal expression in normal tissues, making it an ideal target for molecular imaging; however, its systematic investigation remains limited. In this study, we utilized the clinically investigated B7−H3-targeted antibody−drug conjugate (ADC) HS-20093 to develop and evaluate a novel immuno-positron emission tomography (immuno-PET) tracer, [89Zr]Zr-DFO-HS-20093, for accurate imaging of B7−H3 expression in lung cancer and triple-negative breast cancer models. [89Zr]Zr-DFO-HS-20093 was synthesized with high radiochemical purity (98.2 ± 0.6%). Cellular uptake assays revealed significantly higher tracer uptake in A549 cells than in 4T1 cells at 1, 2, and 4 h (all P < 0.0001), which was markedly reduced by blocking with excess unlabeled antibody. Immunofluorescence revealed predominant membrane localization of B7−H3 in 4T1 cells, whereas A549 cells exhibited pronounced intracellular distribution, suggesting enhanced internalization. In tumor-bearing mice, [89Zr]Zr-DFO-HS-20093 positron emission tomography (PET)/CT enabled clear tumor visualization, with peak uptake at 24 h post-injection in both A549 and 4T1 xenografts (9.63 ± 1.97 and 7.15 ± 3.14 μCi/mL, respectively), supporting its potential for tumor imaging. Ex vivo biodistribution data corroborated the PET findings, showing significantly higher tracer uptake in A549 tumors (5.01 ± 1.19 % ID/g) than in blocked tumors (2.89 ± 0.43 % ID/g) and 4T1 tumors (1.89 ± 0.47 % ID/g) (P = 0.034 and P = 0.003, respectively). Immunohistochemical analysis of A549 xenografts showed strong B7−H3 expression (3+), whereas 4T1 tumors exhibited lower expression levels (1−2+), further confirming that B7−H3 expression patterns were consistent with the imaging results. [89Zr]Zr-DFO-HS-20093 enables sensitive immuno-PET imaging of B7−H3 and reveals tumor-type-dependent differences in B7−H3 expression and intracellular behavior.