Epitope mapping

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High-resolution epitope maps of antibodies against Ebola glycoprotein (GP), determined using shotgun mutagenesis epitope mapping. Epitope maps provide data for determining mechanism of action (MOA). Epitope-mapping-MOA.jpg
High-resolution epitope maps of antibodies against Ebola glycoprotein (GP), determined using shotgun mutagenesis epitope mapping. Epitope maps provide data for determining mechanism of action (MOA).

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). [1] [2] [3] Identification and characterization of antibody binding sites aid in the discovery and development of new therapeutics, vaccines, and diagnostics. [4] [5] [6] [7] [8] Epitope characterization can also help elucidate the binding mechanism of an antibody [9] and can strengthen intellectual property (patent) protection. [10] [11] [12] 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. [13]

Contents

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.[ citation needed ]

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. [14] Epitope mapping has been crucial to the development of vaccines against prevalent or deadly viral pathogens, such as chikungunya, [15] dengue, [16] Ebola, [5] [17] [18] and Zika viruses, [19] by determining the antigenic elements (epitopes) that confer long-lasting immunization effects. [20]

Complex target antigens, such as membrane proteins (e.g., G protein-coupled receptors [GPCRs]) [21] 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. [14] [21]

Importance for intellectual property (IP) protection

Shotgun mutagenesis epitope mapping of antibodies against HER2 revealed a novel epitope (orange spheres). Epitope maps provide supporting data for intellectual property (patent) claims. Epitope-mapping illustration-6-copy.png
Shotgun mutagenesis epitope mapping of antibodies against HER2 revealed a novel epitope (orange spheres). Epitope maps provide supporting data for intellectual property (patent) claims.

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. [22] 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. [24]

Methods

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

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

Related Research Articles

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In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response.

<span class="mw-page-title-main">Antibody</span> Protein(s) forming a major part of an organisms immune system

An antibody (Ab) is the secreted form of a B cell receptor; the term immunoglobulin (Ig) can refer to either the membrane-bound form or the secreted form of the B cell receptor, but they are, broadly speaking, the same protein, and so the terms are often treated as synonymous. Antibodies are large, Y-shaped proteins belonging to the immunoglobulin superfamily which are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses, including those that cause disease. Antibodies can recognize virtually any size antigen with diverse chemical compositions from molecules. Each antibody recognizes one or more specific antigens. This term literally means "antibody generator", as it is the presence of an antigen that drives the formation of an antigen-specific antibody. Each tip of the "Y" of an antibody contains a paratope that specifically binds to one particular epitope on an antigen, allowing the two molecules to bind together with precision. Using this mechanism, antibodies can effectively "tag" a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

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<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

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<span class="mw-page-title-main">Linear epitope</span> Segment of a molecule which antibodies recognize by its linear structure

In immunology, a linear epitope is an epitope—a binding site on an antigen—that is recognized by antibodies by its linear sequence of amino acids. In contrast, most antibodies recognize a conformational epitope that has a specific three-dimensional shape.

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<span class="mw-page-title-main">Gp41</span> Subunit of the envelope protein complex of retroviruses

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<span class="mw-page-title-main">Conformational epitope</span> Segment of a molecule which contacts an immune receptor when folded

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References

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