Fluorescence-Guided Surgery of Rhabdomyosarcoma Using a B7-H3 Targeted Antibody–Fluorophore Conjugate

Resection of sarcomas is often limited by infiltrative tumor growth and microscopic residual disease, underscoring the need for targeted intraoperative imaging strategies that enhance visualization of primary lesions and metastatic deposits. Here, a clinical-grade anti-B7-H3 antibody (MGA271) was conjugated to IRDye 800CW to generate MGA271-IRDye800, with an isotype control IgG-IRDye800 and unconjugated IRDye800 included as comparators. The resulting antibody–dye conjugates, MGA271-IRDye800 and IgG-IRDye800, exhibited an average dye-to-protein ratio of approximately 1. In vitro studies confirmed specific binding and strong fluorescence of MGA271-IRDye800 in six B7-H3-expressing sarcoma cell lines, with only minimal nonspecific uptake observed for control probes and in a B7-H3-negative control cell line. Based on these in vitro results, RH30 cells were selected to establish an orthotopic rhabdomyosarcoma xenograft model, in which in vivo near-infrared fluorescent imaging (NIRF) demonstrated substantially improved tumor visualization with MGA271-IRDye800 compared with IgG-IRDye800 and free IRDye800. Quantitative analysis confirmed significantly higher tumor-to-background ratios (TBRs) for MGA271-IRDye800 at 144 h (5.6 ± 2.2) compared with IgG-IRDye800 (2.7 ± 1.1) and unconjugated IRDye800 (1.0 ± 0.1), [ p < 0.0001] supporting B7-H3-mediated tumor enrichment and superior contrast suitable for surgical navigation. During fluorescence-guided surgery, MGA271-IRDye800 enabled clear delineation of tumor margins, distinction of tumor from surrounding normal tissue and relevant background organs, and detection of otherwise occult micrometastatic lesions. Collectively, these findings demonstrate that B7-H3-targeted NIRF imaging with MGA271-IRDye800 offers sensitive and specific intraoperative visualization of sarcoma. This probe demonstrates strong potential for clinical translation as a molecular imaging agent to guide surgical resection, reduce residual disease, and ultimately improve outcomes for patients with B7-H3-expressing sarcomas.