Engineered B7-H3-targeted VHH-Fc fusion antibody demonstrates rapid tumor accumulation for infrared imaging in pancreatic cancer xenograft model.

B7 homolog 3 protein (B7-H3) represents a promising target for cancer diagnosis and therapy. Conventional B7-H3-targeting agents predominantly use IgG antibodies, which accumulate in tumors slowly due to their large molecular size (160-180 kDa, comprising a dimeric Fab-Fc structure), leading to an increased risk of peripheral side effects. In this study, a novel fusion antibody, A052, was developed, integrating the variable domain of a nanobody heavy chain (VHH) and the Fc domain of IgG1. This design preserves the fundamental structure of an antibody while reducing the molecular weight to 82 kDa. Evaluation through enzyme-linked immunosorbent assay, flow cytometry, and bio-layer interferometry revealed that A052 binds with a significantly higher affinity (KD = 77.2 pM) compared to the anti-B7-H3 IgG antibody, hBRCA84D, currently in clinical trials (KD = 8.13 nM). To compare the in vivo tumor accumulation properties, both A052 and hBRCA84D were labeled with Cy5 and assessed in a PANC-1 xenograft model using infrared imaging. A052-Cy5 showed faster tumor accumulation, reaching a visible tumor-background ratio (TBR) at 3 h post-injection, peaking at 1.97 at 24 h, and maintaining a TBR above 1.50 from 3 to 144 h. In contrast, hBRCA84D-Cy5 exhibited a TBR of 1.5 only between 48 and 110 h. These results suggest that VHH-Fc fusion antibodies, such as A052, offer a novel approach for B7-H3-targeted therapies, potentially facilitating rapid tumor distribution while minimizing peripheral toxicity risks.