Epitope mapping is the process of experimentally identifying the binding sites, or ‘epitopes’, of antibodies on their target antigens. Identification and characterization of the binding sites of antibodies can aid in the discovery and development of new therapeutics, vaccines, and diagnostics. Characterization of epitopes can also help elucidate the mechanism of binding for an antibody and facilitate the prediction of B cell epitopes using robust algorithms. Epitopes can be generally divided into 2 main classes-linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding. The vast majority of antigen-antibody interactions have conformational epitopes.
The importance of epitope mapping
Many therapeutically important mAbs target conformational epitopes formed only in the native structure of a protein, emphasizing the importance for epitope mapping of this type of epitope. Technological advances including B cell cloning, deep sequencing of human genomes and the widespread use of phage display have greatly increased the ability to obtain large numbers of mAbs. In contrast to the large increase in mAbs being isolated, high-throughput mAb characterization techniques have not kept pace.
Epitope mapping of complex target antigens, such as integral membrane proteins or multi-subunit proteins, is often challenging because of the difficulty in expressing and purifying these types of antigens. Human membrane proteins and receptors, key targets for therapeutic antibodies, often have short antigenic regions that fold correctly only in the context of a lipid bilayer. Furthermore, membrane proteins contain complex disulfide bonding patterns and post translational modifications (glycosylation), many of which cannot be fully recapitulated in bacterial and yeast expression systems.
Methods for epitope mapping
There are several methods available for mapping antibody epitopes on target antigens:
X-ray co-crystallography: The gold standard approach which allows direct visualization of the interaction between the antigen and antibody. This approach is technically challenging, requires large amounts of purified protein, and can be time-consuming and expensive.
Array-based oligo-peptide scanning (sometimes called overlapping peptide scan or pepscan analysis): This technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein and tests for their ability to bind the antibody of interest. This method is fast and relatively inexpensive, and specifically suited to profile epitopes for large number of candidate antibodies against a defined target. By combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (such as by using CLIPS scaffolds), discontinuous epitopes can be mapping with very high reliability and precision.
Site-directed mutagenesis: Using this approach, systematic mutations of amino acids are introduced into a protein sequence followed by measurement of antibody binding in order to identify amino acids that comprise an epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and slow, typically limiting analysis to a small number of amino acid residues.
High Throughput Mutagenesis Mapping. This approach utilizes a comprehensive mutation library, with each clone containing a unique amino acid mutation (conservative, non-conservative, or alanine) and the entire library covering every amino acid in the target protein. Hundreds of plasmid clones from the mutation library are individually arrayed in 384-well micro plates, expressed in mammalian cells and tested for antibody binding. Amino acids that are required for antibody binding can be identified by a loss of fluorescent reactivity and mapped onto protein structures to visualize epitopes. A customized database enables all mutagenesis information to be managed in a systematic, accurate and efficient manner. The database also performs the statistical calculations used throughout data analysis and epitope refinement, including patented algorithms to derive the final epitopes.
To date this approach, coined "Shotgun Mutagenesis" has been used to construct mutation libraries totaling over 10,000 individual point mutations, representing viral envelope proteins [dengue virus-3 (DENV-3) prM/E, DENV-4 prM/E, chikungunya virus E2/E1, hepatitis C E1/E2, hepatitis B virus surface antigen, respiratory syncytial virus F protein and HIV gp160], GPCR proteins (CCR5, CXCR2, CXCR4 and TAS2R16), 4TM proteins (claudin-1 and claudin-4) and other membrane proteins (MCAM-1 and Her-2). The Shotgun mutagenesis approach has been used to epitope map hundreds of mAbs targeting DENV (representing one of the largest collections of epitope information against a viral protein), chikungunya virus and hepatitis C virus, with additional mAb epitopes mapped on hepatitis B virus, respiratory syncytial virus and HIV. It has also been used to define atomic-level mAb epitopes on the GPCRs CCR5  and CXCR4, the identification of cancer biomarker epitopes on the 4TM proteins claudin-1 and claudin-4, an atomic-level model describing the intramolecular signal transduction pathway of CXCR4, a proposed mechanism for the ligand specificity and sensitivity of the GPCR TAS2R16, mapping of inhibitor-binding sites on TAS2R16, and mapping of paratope residues on a clinical antibody against respiratory syncytial virus.
Hydrogen–deuterium exchange: A method growing in popularity which gives information about the solvent accessibility of various parts of the antigen and the antibody, demonstrating reduced solvent accessibility where protein to protein interactions occur.
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