A Fast and Low-Cost Approach for Binding Mode Validation of AI-Designed Therapeutics

Abstract

High-resolution binding site mapping is important for in-depth activity assessment of new therapeutics including AI-designed antibodies. However, complex protein targets such as glycosylated antigens are challenging for many methods including crystallography. PD1 is a highly glycosylated antigen, and with the traditional HDX-MS method, only 51% sequence coverage could be obtained with multiple epitope residues undetected for Pembrolizumab. By implementing glyco-peptide detection, subzero temperature LC-MS and electron based MSMS fragmentation, the new HDX FineMapping methodology enabled 100% sequence coverage and complete epitope characterization for the Pembrolizumab-PD1 system, with amino acid level resolution. Furthermore, HDX FineMapping detects binding epitopes directly in solution, without any mutation or modification to either the antigen or the antibody. The amino acid level resolution combined with low cost, minimal sample consumption, fast turnaround time, and no need of mutant library or crystallization makes it a competitive methodology for binding mode validation of AI-designed therapeutics.