ObjectiveTo identify HLA-G-binding proteins (HGBPs) by screening targeting ankyrin sequences from a phage display-based ankyrin protein library using human leukocyte antigen G (HLA-G) as the target, and to evaluate their functions.MethodsThe expression of HLA-G in tumor tissues and its correlation with clinical prognosis and immune infiltration were analyzed using bioinformatics tools such as TCGA and GTEx databses. The extracellular domain of HLA-G was subjected to biopanning with a phage-displayed ankyrin protein library, followed by random selection and sequencing of monoclonal phage clones. The functional properties of dominant phage clones were validated using ELISA and immunofluorescence staining. HGBPs were produced and purified using a prokaryotic expression system, and their affinity and tumor-specific binding ability were evaluated using ELISA, surface plasmon resonance (SPR), and immunofluorescence staining.ResultsBioinformatics analysis revealed that HLA-G is widely overexpressed in tumor tissues and is correlated with overall survival (OS) and immune cell infiltration (P < 0.05). After five rounds of biopanning, dominant clones were obtained. Both ELISA and immunofluorescence staining results showed that these dominant phages had a significantly higher affinity to HLA-G positive cells compared to HLA-G negative cells (P < 0.05, P < 0.001). The purified HGBPs exhibited an affinity of up to 17 nmol/L for HLA-G. ELISA results showed significant binding of HGBP to HLA-G (P < 0.05), and immunofluorescence staining confirmed that HGBP could specifically bind to HLA-G-positive cells (P < 0.01).ConclusionThe HGBPs identified via phage display exhibit high affinity and specificity to HLA-G on tumor cells.