Glucose-regulated protein 94 (GRP94) overexpression plays a critical role in tumor cell survival across various cancers. Previously, we developed K101.1, a fully human antibody targeting cell surface GRP94, which effectively inhibits tumor angiogenesis in colorectal cancer (CRC). This study aims to identify K101.1's interaction site and further elucidate GRP94's role in tumor angiogenesis. In an HT29 CRC xenograft mouse model, K101.1 reduced tumor growth and angiogenesis by 30 % and 69 %, respectively, without inducing severe toxicity. Using hydrogen‑deuterium exchange mass spectrometry, we identified the binding site of K101.1 on GRP94, analyzing 225 peptides with 94.5 % sequence coverage and a redundancy score of 4.20. This revealed a linear epitope (Glu26-Ala34) as the specific binding site. The binding was further validated via enzyme-linked immunosorbent assay, and human umbilical vein endothelial cell tube formation assays using a synthetic epitope peptide corresponding to the identified epitope. Our findings suggest that this epitope is functionally involved in GRP94-mediated angiogenesis and is integral to its proangiogenic activity. By integrating epitope profiling with complementary experimental approaches, this study advances the understanding of GRP94's role in CRC angiogenesis and provides new insights for developing targeted cancer therapies and vaccines, offering promising avenues for CRC prevention and treatment.
