Abstract

B7H3, an immune checkpoint molecule, is a highly N-glycosylated membrane protein. However, the key glycosylated asparagine residues that mediate the function of the B7H3 protein are still unclear. Here we identify that N-glycans attached to asparagine residues N91/309 and N104/322 are required for proper B7H3 localization on the cell surface membrane. We demonstrate that mutations in these two pairs of N-glycosylation sites induce ER accumulation of B7H3 by blocking its ER-to-Golgi translocation and subsequently promote its degradation via the endoplasmic reticulum-associated protein degradation pathway. Additional evidence suggests that N-glycosylation at N91/309 and N104/322 of B7H3 is essential for its inhibition of T-cell proliferation and activation. More importantly, a monoclonal antibody, Ab-82, preferentially targeting B7H3 glycosylated at N91/309 and N104/322 is developed, which exhibits the ability to elicit cytotoxic T lymphocyte-mediated antitumor immunity via B7H3 internalization. Together, these findings offer a rationale for targeting glycosylated B7H3 as a potential strategy for immunotherapy.