CD155 overexpression in cancers functions as an emerging immune checkpoint that enables tumor cells to evade immune surveillance, underscoring the necessity for novel therapeutic strategies. In this study, four fully human monoclonal antibodies (mAbs) specific to human CD155 (hCD155) were generated using phage display technology to determine the mechanisms underlying CD155-mediated immune regulation. Of these, the K116.4 clone exhibited high-affinity binding to hCD155 (KD = 0.98 nM) and potently inhibited its interactions with TIGIT and CD96, as demonstrated by sub-nanomolar IC50 values in competitive binding assays. Functional T cell activation assays revealed that K116.4 enhances T cell activation, indicating its ability to counteract CD155-mediated immune suppression. Epitope mapping and functional assays identified Pro231-Arg300 as a key region essential for receptor binding and antibody-mediated immune modulation. Notably, treatment with K116.4 resulted in the internalization of CD155 and its subsequent downregulation on tumor cell surfaces, suggesting a dual mechanism of action involving both immune checkpoint blockade and depletion of cell surface CD155. Collectively, our data provide novel molecular insights into the immunoregulatory role of CD155 and establish epitope-specific mAbs as valuable tools for studying immune checkpoint pathways. This study provides a framework for developing CD155-targeted immunotherapies and enhances the understanding of immune escape mechanisms in cancers.
