In immunology, epitope mapping is the process of experimentally identifying the binding site, or epitope, of an antibody on its target antigen (usually, on a protein). Identification and characterization of antibody binding sites aid in the discovery and development of new therapeutics, vaccines, and diagnostics. Epitope characterization can also help elucidate the binding mechanism of an antibody and can strengthen intellectual property (patent) protection. Experimental epitope mapping data can be incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data. Epitopes are generally divided into two classes: linear and conformational/discontinuous. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes epitopes are formed by amino acids that are nearby in the folded 3D structure but distant in the protein sequence. Note that conformational epitopes can include some linear segments. B-cell epitope mapping studies suggest that most interactions between antigens and antibodies, particularly autoantibodies and protective antibodies (e.g., in vaccines), rely on binding to discontinuous epitopes.

Importance for antibody characterization

By providing information on mechanism of action, epitope mapping is a critical component in therapeutic monoclonal antibody (mAb) development. Epitope mapping can reveal how a mAb exerts its functional effects - for instance, by blocking the binding of a ligand or by trapping a protein in a non-functional state. Many therapeutic mAbs target conformational epitopes that are only present when the protein is in its native (properly folded) state, which can make epitope mapping challenging. Epitope mapping has been crucial to the development of vaccines against prevalent or deadly viral pathogens, such as chikungunya, dengue, Ebola, and Zika viruses, by determining the antigenic elements (epitopes) that confer long-lasting immunization effects. Complex target antigens, such as membrane proteins (e.g., [G protein-coupled receptors GPCRs]) and multi-subunit proteins (e.g., ion channels) are key targets of drug discovery. Mapping epitopes on these targets can be challenging because of the difficulty in expressing and purifying these complex proteins. Membrane proteins frequently have short antigenic regions (epitopes) that fold correctly only when in the context of a lipid bilayer. As a result, mAb epitopes on these membrane proteins are often conformational and, therefore, are more difficult to map.

Importance for intellectual property (IP) protection

Epitope mapping has become prevalent in protecting the intellectual property (IP) of therapeutic mAbs. Knowledge of the specific binding sites of antibodies strengthens patents and regulatory submissions by distinguishing between current and prior art (existing) antibodies. The ability to differentiate between antibodies is particularly important when patenting antibodies against well-validated therapeutic targets (e.g., PD1 and CD20) that can be drugged by multiple competing antibodies. In addition to verifying antibody patentability, epitope mapping data have been used to support broad antibody claims submitted to the United States Patent and Trademark Office. Epitope data have been central to several high-profile legal cases involving disputes over the specific protein regions targeted by therapeutic antibodies. In this regard, the Amgen v. Sanofi/Regeneron Pharmaceuticals PCSK9 inhibitor case hinged on the ability to show that both the Amgen and Sanofi/Regeneron therapeutic antibodies bound to overlapping amino acids on the surface of PCSK9.

Methods

There are several methods available for mapping antibody epitopes on target antigens: Other methods, such as yeast display, phage display, and limited proteolysis, provide high-throughput monitoring of antibody binding but lack resolution, especially for conformational epitopes.