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Published on: June 6, 2020
Functional epitope mapping of cell surface glucose-regulated protein 94: A combinatorial approach for therapeutic
Yea Bin Cho1, In Young Ko2, Hyun Jung Kim3
1Department of Chemistry, Kookmin University, Seoul 02707, Republic of Korea.
Abstract:
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.
Insights
The antibody K101.1 targets Glucose-regulated protein 94 (GRP94), inhibiting colorectal cancer (CRC) growth and angiogenesis by binding a specific epitope. This discovery offers new avenues for targeted cancer therapies and vaccines.
Area of Science:
- Oncology
- Immunology
- Biochemistry
Background:
- Glucose-regulated protein 94 (GRP94) overexpression is crucial for tumor cell survival in various cancers.
- The antibody K101.1 targets cell surface GRP94 and has shown efficacy in inhibiting colorectal cancer (CRC) angiogenesis.
Purpose of the Study:
- To identify the specific interaction site of K101.1 on GRP94.
- To further elucidate the role of GRP94 in tumor angiogenesis.
Main Methods:
- Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify the K101.1 binding site on GRP94.
- Enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cell (HUVEC) tube formation assays to validate binding and functional activity.
Main Results:
- K101.1 demonstrated significant reduction in tumor growth (30%) and angiogenesis (69%) in a CRC xenograft model with minimal toxicity.
- HDX-MS identified a linear epitope (Glu26-Ala34) on GRP94 as the specific binding site for K101.1.
- The identified epitope was confirmed to be functionally involved in GRP94-mediated angiogenesis.
Conclusions:
- The study identified a key epitope on GRP94 targeted by K101.1, crucial for its anti-angiogenic activity in CRC.
- This research advances the understanding of GRP94's role in CRC angiogenesis.
- Findings provide insights for developing novel targeted cancer therapies and vaccines for CRC prevention and treatment.
