Spotlight on Epitope & Paratope Mapping
Mapping of TNFα
Researchers conduct epitope mapping studies to pinpoint the specific amino acid residues on TNFα that are protected from hydroxyl radical oxidation upon antibody binding, revealing the physical interaction interface with Adalimumab.This detailed structural information enables safer and more efficient design and implementation of new antibodies by guiding rational drug development, supporting biosimilarity assessments, and clarifying mechanisms of action.
Figure legend: The epitope of TNFα was identified using the Fox Technology. A crystal structure (PDB: 3WD5) of the TNFα tetramer was annotated to show residues with decreased solvent accessibility (dark blue, fold change >2; light blue, fold change <2). Residues implicated in adalimumab binding are marked as spheres (substitutions causing >10-fold loss of binding) or dots (substitutions without effect). Most protected residues overlap with those critical for adalimumab binding, with additional protections observed in the trimer core consistent with condensation upon antibody binding. This information supports rational antibody design, biosimilarity assessment, and mechanistic insight.
Comparing an Epitope Identified from a Crystal Structure with In-Solution Mass Spectrometry
Researchers use epitope mapping techniques like HRPF and XL-MS to identify binding interfaces between HLA proteins and antibody Fab domains in solution, complementing crystal structure data and revealing conformational flexibility not captured in static crystal forms. This information helps ensure that new therapeutic antibodies are designed with greater precision and safety, improving efficacy, minimizing off-target effects, and supporting rational development and biosimilarity assessments.
Figure legend: Crystal structure of the Fab domain, HLA, B2M, and peptide annotated with HRPF and XL-MS data. Peptides with decreased solvent accessibility are shown in blue; those with increased accessibility are in red. Crosslinks are represented by green bars (30 Å). HRPF identified significant changes in 6 of 8 peptides containing epitopic residues defined by the crystal structure. XL-MS identified 2 of 3 crosslinked HLA regions as part of the epitope. Together, HRPF and XL-MS reveal complementary features of the HLA–Fab interaction and highlight conformational flexibility in solution not evident in the crystal state, underscoring the value of integrating multiple techniques for comprehensive protein characterization.
