Identification of the B7-H3 Interaction Partners Using a Proximity Labeling Strategy

B7 homolog 3 (B7-H3) has emerged as a promising target for cancer therapy due to its high expression in various types of cancer cells. It not only regulates the activity of immune cells but also modulates the signal transduction and metabolism of cancer cells. However, the specific interaction partners of B7-H3 still remain unclear, limiting a comprehensive understanding of the precise role of B7-H3 in cancer progression. In this study, we report that B7-H3 can bind to resting Raji cells, stimulated THP-1 cells, and even PC3 prostate cancer cells through its IgV domain alone. Furthermore, to identify the potential interaction partners of B7-H3 on these cells, we adopted an ascorbate peroxidase 2 (APEX2)-based proximity labeling strategy, which revealed about 10 key potential interaction partners. Interestingly, our results suggest that CD45 could be a putative receptor for B7-H3 on Raji cells, while the epidermal growth factor receptor (EGFR) could closely interact with B7-H3 on PC3 cells. Based on further computational structure modeling studies, we show that B7-H3 can bind to the epidermal growth factor (EGF) binding pocket of EGFR—surprisingly, with a stronger affinity than EGF itself. Overall, our study provides an effective approach to identifying B7-H3 interaction partners in both immune and cancer cell lines.