Epitope mapping

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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 two 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 rely upon binding to conformational epitopes.

Video Epitope mapping

Importance for antibody characterization

Epitope mapping is an important component in the development of therapeutic monoclonal antibodies (mAbs) and vaccines. Specifically, epitope mapping can allow determination of the therapeutic mechanism of action of individual mAbs e.g. blocking ligand binding or trapping a protein in a non-functional state. However, many therapeutic antibodies target conformational epitopes that are particularly difficult to map because they are only formed in the native structure of a protein. Epitope mapping is also crucial to developing vaccines against prevalent viral diseases such as Dengue virus. Epitope mapping helps develop these challenging vaccines by determining antigenic elements (or epitopes) that confer long-lasting immunization effects. 

Epitope mapping of complex target antigens, such as 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. As a result, mAb epitopes on these types of targets are often conformational, making them difficult to map.

Maps Epitope mapping

Epitopes and intellectual property

Epitope mapping has become prevalent in protecting the intellectual property of therapeutic antibodies. 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 accurately differentiate between antibodies is particularly important when patenting antibodies against well validated therapeutic targets that can be drugged by multiple competing antibodies e.g. PD1 and CD20. In addition to verifying antibody patentability, epitope mapping data has also been used to support broad antibody claims submitted to the United States Patent and Trademark Office.

Epitope data has 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 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.

src: www.jimmunol.org

Methods

There are several methods available for mapping antibody epitopes on target antigens:

• X-ray co-crystallography: This has historically been regarded as the gold standard approach because it allows direct visualization of the interaction between the antigen and antibody. Howeer, 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. Although rare, 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 have been mapped on CD20. The epitope mapping resolution here depends on the number of overlapping peptides that are used.
• 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. Sometimes referred to as shotgun mutagenesis, this approach utilizes an mutation library of the entire target antigen, with each clone containing a unique amino acid mutation (typically alanine). 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. Target protein amino acids that are required for antibody binding can be identified by a loss of reactivity and mapped onto protein structures to visualize epitopes.  In some cases high-throughput mutagenesis mapping can provide single amino acid resolution and paratope information. However, as with pepscan analysis this technique can not reliably detect discontinuous epitope regions as the structure of the protein is not retained after mutation.
• 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.
• Crosslinking coupled Mass Spectrometry: This technique first binds the antibody and the antigen with a labeled crosslinker. After confirming complex formation with high mass MALDI detection, the binding location can then be identified. Because after crosslinking the complex is highly stable, a large degree of enzymes and digestion conditions can be applied to the complex to provide many different peptide options for detection. Detection is performed using high resolution mass spectrometry or MS/MS techniques to identify the labelled crosslinkers amino acid locations and the peptides bound (both epitope and paratope are determined in one experiment). Because of the highly sensitive mass spectrometry detection, very little material (100's of micrograms or less) of material can be utilized.

Other methods, such as phage display, and limited proteolysis, provide high throughput monitoring of antibody binding but lack reliability, especially for conformational epitopes.

src: cancerres.aacrjournals.org

See also

• Epitope binning

src: jvi.asm.org

References

src: www.biochemj.org

External links

• Epitope mapping at the US National Library of Medicine Medical Subject Headings (MeSH)

Source of the article : Wikipedia