Epitope mapping
In
Epitopes are generally divided into two classes: linear and conformational/discontinuous.
Importance for antibody characterization
By providing information on
Complex target antigens, such as
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.[10][11][22] 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.[23] 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.[11][12]
Epitope data have been central to several high-profile legal cases involving disputes over the specific protein regions targeted by therapeutic antibodies.
Methods
This article may require cleanup to meet Wikipedia's quality standards. The specific problem is: Most methods mentioned are only for antibodies, not B- or T-cells. (March 2024) |
There are several methods available for mapping antibody epitopes on target antigens:
- X-ray co-crystallography and cryogenic electron microscopy (cryo-EM). X-ray co-crystallography has historically been regarded as the gold-standard approach for epitope mapping because it allows direct visualization of the interaction between the antigen and antibody. Cryo-EM can similarly provide high-resolution maps of antibody-antigen interactions.[25] However, both approaches are technically challenging, time-consuming, and expensive, and not all proteins are amenable to crystallization. Moreover, these techniques are not always feasible due to the difficulty in obtaining sufficient quantities of correctly folded and processed protein. Finally, neither technique can distinguish key epitope residues (energetic "hot spots")[26] for mAbs that bind to the same group of amino acids.
- Array-based oligo-peptide scanning. Also known as 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, relatively inexpensive, and specifically suited to profile epitopes for large numbers of candidate antibodies against a defined target.[20][27] The epitope mapping resolution depends on the number of overlapping peptides that are used. The main disadvantage of this approach is that discontinuous epitopes are deconstructed into smaller peptides, which can cause lower binding affinities. However, advances have been made with technologies such as constrained peptides, which can be used to mimic conformational as well as discontinuous epitopes. For example, an antibody against CD20 was mapped in a study[28] using array-based oligo-peptide scanning, by combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (e.g., by using CLIPS scaffolds[29]). Replacement analysis on peptides also allows single amino acid resolution, and can therefore pinpoint key epitope residues.
- mutations of amino acids are introduced into the sequence of the target protein. Binding of an antibody to each mutated protein is tested to identify the amino acids that comprise the epitope. This technique can be used to map both linear and conformational epitopes but is labor-intensive and time-consuming, typically limiting analysis to a small number of amino-acid residues.[2]
- High-throughput shotgun mutagenesis epitope mapping.[2][10][30] Shotgun mutagenesis is a high-throughput approach for mapping the epitopes of mAbs.[30] The shotgun mutagenesis technique begins with the creation of a mutation library of the entire target antigen, with each clone containing a unique amino acid mutation (typically an alanine substitution). Hundreds of plasmid clones from the library are individually arrayed in 384-well microplates, expressed in human cells, and tested for antibody binding. Amino acids of the target required for antibody binding are identified by a loss of immunoreactivity. These residues are mapped onto structures of the target protein to visualize the epitope. Benefits of high-throughput shotgun mutagenesis epitope mapping include: 1) the ability to identify both linear and conformational epitopes, 2) a shorter assay time than other methods, 3) the presentation of properly folded and post-translationally modified proteins, and 4) the ability to identify key amino acids that drive the energetic interactions (energetic "hot spots" of the epitope).[26][31]
- Hydrogen–deuterium exchange (HDX). This method gives information about the solvent accessibility of various parts of the antigen and antibody, demonstrating reduced solvent accessibility in regions of protein-protein interactions.[32] One of its advantages is that it determines the interaction site of the antigen-antibody complex in its native solution, and does not introduce any modifications (e.g. mutation) to either the antigen or the antibody. HDX epitope mapping has also been demonstrated to be the effective method to rapidly supply complete information for epitope structure.[33] It does not usually provide data at the level of amino acid, but this limitation is being improved by new technology advancements.[34] It has recently been recommended as a fast and cost-effective epitope mapping approach,[35] using the complex protein system influenza hemagglutinin as an example.
- Cross-linking-coupled mass spectrometry. are determined in one experiment). The key advantage of this technique is the high sensitivity of MS detection, which means that very little material (hundreds of micrograms or less) is needed.
Other methods, such as yeast display, phage display,[37] and limited proteolysis, provide high-throughput monitoring of antibody binding but lack resolution, especially for conformational epitopes.[38]
See also
References
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- ^ a b c Banik, S; Deng, X; Doranz, B (2017). "Using epitope mapping to derive more value from mAbs". Genetic Engineering & Biotechnology News. 37 (15).
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- ^ a b Banik, SSR; Doranz, BJ (2010). "Mapping complex antibody epitopes". Genetic Engineering & Biotechnology News. 3 (2): 25–8.
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{{cite journal}}
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External links
- Epitope+mapping at the U.S. National Library of Medicine Medical Subject Headings (MeSH)