Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Mapping and Characterizing Antibody Binding Epitopes on the RAGE V-Domain Using Recombinant Loop-Variants

The receptor for advanced glycation end-products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobin superfamily. RAGE consists of a variable-like (V) domain, two constant-type domains (C1 and C2) with the V domain appearing to be the predominant ligand binding site. RAGE has emerged as a key receptor in many conditions, such as cancer, diabetes, and Alzheimer’s disease. The ability to block RAGE activation by its ligands could have many therapeutic benefits. The aim of this study is to define the binding epitopes on the RAGE V-domain of a panel of monoclonal antibodies. The experimental approach chosen utilizes the genetic engineering of the recombinant RAGE-VC1 domain and the systematic mutation of four potential epitope loops. In the first step, we used structural information to define four loop regions of interest within the V-domain. We then developed a strategy to assemble five recombinant gene constructs encoding wild type (WT) RAGE VC1 sequence and the loop variants with an additional tag for in-vivo biotinylation. The biotinylation will allow the immobilization of the folded protein on streptavidin coated surfaces. Recombinant RAGE VC1 is being expressed in E. coli, isolated and purified by chromatography. Two assay formats will be used to map antibody binding to WT RAGE and the loop variants. The long-term goal of the project is to identify ligand specific monoclonal antibodies that block the activation of RAGE by specific ligands (e.g., HMGB1), while not interfering with RAGE signaling involving other RAGE ligands.