Epitopes are pivotal for research scientists investigating the immune system and drug discovery teams exploring new potential vaccines, but what is an epitope? An epitope is a specific part of an antigen recognized by an antibody produced by the immune system in response to an invading pathogen or a small part of an antigen presented by cells for cellular immune surveillance. Antigens may contain several epitopes at different positions throughout their sequence, depending on the host’s immune response. This widens their applications, particularly in research and diagnostic assays. For antibody assays, either monoclonals or polyclonals are available; a monoclonal antibody recognizes one specific single epitope, whereas a polyclonal (containing several antibody species) can recognize multiple sites across the antigen. In drug development, mostly monoclonal antibodies or derivatives are used to bind specific epitopes on their target. The location of the epitope often determines the functionality of the therapeutic antibody drug. The type of antibody is selected to provide optimal performance in the chosen application.
Understanding this interaction between an antibody and the antigen’s epitope sequence is of great interest in the scientific field. It can aid in developing vital vaccines and immunotherapies and help design synthetic antibodies to capture pathogens in diagnostic assays. It can also be of interest in the study of autoimmune diseases and other disorders in which the immune system attacks normal cells and tissues. Thus, epitopes and their definitive characterizations are valuable entities in scientific and medical advancements.
That is why Biosynth is proud to offer an outstanding peptide-based epitope mapping service. The technology was invented by the founder of Pepscan, now part of Biosynth, more than 35 years ago to identify linear epitopes of important therapeutic and diagnostic antibodies. This technology has since been perfected by our peptide experts to broaden its applications, and deliver unparalleled sensitivity and reproducibility for epitope mapping of all sample types. Our technology can successfully identify the binding site of a given antibody to its target by creating 3D structured CLIPSTM peptide constructs to mimic the native conformational elements of the target protein. Our team uses high-throughput synthesis to generate microarrays with overlapping linear and structurally constrained CLIPSTM peptides. An applied antibody can then specifically bind either linear or conformational peptides, as they may be present in the epitope.
More details about our methods are available on our epitope mapping webpage, and in our epitope mapping brochure. Read below to find out more about epitopes and the significance of epitope mapping.
Different Types of Epitope
Epitopes can be classified as linear, conformational, or discontinuous. Each has characteristics that make them more suitable for certain scientific applications. Each epitope type is depicted below on a ribbon diagram of ubiquitin.
Linear epitopes consist of a continuous stretch of amino acid residues in a particular region of a protein.
Antibodies raised against a peptide or antibodies used in immunoassays, such as immunohistochemistry, western blotting and immunofluorescence-based confocal microscopy, often recognize a linear epitope type. In most cases, the prevalence of linear epitopes for such antibodies is due to the protein having undergone denaturation through various sample preparation steps in these types of assays. This may be down to pH changes, reduction or fixation.
So, how can we better understand these epitope and antibody interactions? One answer is to use epitope mapping.
What is Epitope Mapping?
Epitope mapping is a pioneering technology that allows for the identification of the structure and sequence of an epitope. This is the basis of therapeutic antibody and diagnostic assay development. It enables scientists to understand antibodies functionally and enables the selection of the best candidate antibodies with preferred specificity. Epitope mapping allows:
- Two antibodies with a similar epitope to be compared
- Different antibodies to be classified into separate binding bins
- Essential amino acid positions within an epitope to be identified
- An understanding of the amino acid requirements in a cross-species or multi-strain reactivity analysis]
The therapeutic and diagnostic sectors are carefully regulated. The inclusion of specific binding site information is necessary for regulatory dossiers for novel antibodies for FDA/EMA approval. Epitope mapping can provide vital data for regulatory filings, protecting intellectual property (IP), and novel patent claims. It can also support the freedom to operate if analysis shows that a granted antibody claim is directed to an epitope different to the one upon which you are working. Differences in the epitopes of one single amino acid may already be sufficient to claim a novel invention. An example of this is exemplified by the below case study.
Case Study: CLIPSTM Precision Epitope Mapping Differentiates anti-CD20 Monoclonal Antibodies
Both ofatumumab and rituximab are monoclonal antibodies that bind to CD20, which is a targeted transmembrane cellular protein in the treatment of B-cell malignancies. In order to ensure freedom to operate for ofatumumab and to gain market differentiation, it was necessary to discriminate the distinct binding sites of ofatumumab and rituximab on CD20. To achieve this, CLIPSTM Epitope Mapping was used. As shown by image A, CLIPSTM Epitope Mapping successfully demonstrated that ofatumumab (Arzerra®) binds a unique discontinuous epitope different to that of rituximab (Rituxan®).
CLIPSTM Epitope Mapping results for ofatumumab and rituximab (A). The epitopes of the antibodies map to distinct regions of the extracellular domain of CD20 (Teeling et al. 2006). (B) 14 years after the identification of the epitope of rituximab using CLIPSTM Epitope Mapping, a structure of the full CD20 protein with rituximab was finally published (Rougé et al. 2020), confirming the CLIPSTM peptide mapping approach
The knowledge and regulatory evidence that epitope mapping offers confirms its use as an essential tool in vaccine development. This is exemplified by the original search for the influenza A vaccine. Epitope mapping was used to uncover a conserved epitope on the influenza surface protein haemagglutinin (HA), which allowed neutralizing antibodies targeting this epitope to be designed.
Similarly, epitope mapping is useful to identify epitopes involved in the excessive activation of autoreactive T-cells, which are prevalent in autoimmune diseases such as psoriasis, immune bowel diseases and asthma. It may also be beneficial in monitoring disease progression and for the development of targeted therapies against the respective immune disorder. The mapping of T-cell epitopes can pave the way to designing novel treatments for patients suffering from conditions with limited T-cell response. This is apparent in conditions such as in ageing, HIV, patients with effector or regulatory T-cell repertoire abnormalities and other immune deficiencies. Novel cancer immunotherapies, which include cancer vaccines and adoptive T-cell therapy, exploit the mechanism by which T cells recognize ‘foreign’ peptides. T-cells can thus be stimulated or modified to recognize new (=neo) or tumor-associated (TA) antigens and target the body’s immune system against them, allowing the elimination of cancerous cells. An example of such a therapy is Chimeric Antigen Receptor (CAR) T-cell therapy, which is largely used to treat blood cancers. There are currently several FDA approved CAR T-cell therapies, such as Tisagenlecleucel (for the treatment of B-cell acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma) and Idecabtagene vicleucel (for the treatment of multiple myeloma).
For more information about T-cell activity screening methods, visit our T-Cell Activating Peptide Libraries webpage.
Peptide Based Epitope Mapping at Biosynth
Peptide-based epitope mapping has several advantages over other mapping techniques (X-ray Crystallography, Nuclear Magnetic Resonance and Full Protein Mutagenesis), including identifying linear, as well as conformational and discontinuous parts of epitopes. It is the only viable technique to evaluate binding of polyclonal antibodies and sera, as competition due to overlap of epitopes of individual antibodies is limited on small peptides. However, peptide mapping can be limited by the availability of high-quality samples.
Biosynth’s peptide-based epitope mapping platform uses a solid support with a proprietary hydrogel matrix. Peptide sequences are directly synthesized on this matrix at a high density using robust Fmoc peptide synthesis. This results in highly sensitive arrays and reliable detection of even the weakest binding signals, making the platform suitable not only for epitope mapping but also for mapping other protein-protein interactions. The reusability of the arrays permits the screening of a series of antibodies or sera on a single array, making peptide epitope mapping a cost-effective option for best-candidate selection, antibody characterization, antibody profiling and further development. Biosynth’s epitope mapping service is also enhanced through the use of our CLIPS™ technology which allows the creation of 3D structured CLIPS™ peptides to mimic the conformational elements of the target protein.
Key Benefits of Biosynth’s CLIPS™ Epitope Mapping
- Broad Applicability – linear, conformational, discontinuous, and complex multimeric protein epitopes
- Target Diversity – soluble targets, exodomains, membrane proteins, viruses
- Sample Diversity – antisera, polyclonal Abs, purified mAbs, nanobodies, tagged proteins or complexes
- Due to the high sensitivity of our platform, even the weakest interactions can be characterized
- Reusable Arrays – dozens of samples on a single array, cost-effective and direct comparison of Abs
Contact our team today to begin your epitope mapping project
References
Björn Forsström et al. (2015) Dissecting Antibodies with Regards to Linear and Conformational Epitopes. PLOS ONE.
10(3): e0121673.
Irene M. Francino-Urdaniz and Timothy A. Whitehead (2021) An overview of methods for the structural and
functional mapping of epitopes recognized by anti-SARS-CoV-2 antibodies. RSC Chemical Biology. 2(6): 1580-1589.
Jonathan M. Gershoni et al. (2012) Epitope Mapping: The First Step in Developing Epitope-Based Vaccines. Biodrugs.
21: 145-156.
Jessica L. Teeling et al. (2006) The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique
Epitopes on CD201. The Journal of Immunology. 177(1):362-371.
Lionel Rougé et al. (2020) Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.
Science. 367(6483): 1224-1230.
An Introduction to Antibodies: Antigens, Epitopes and Antibodies. MERCK. Available from :
https://www.sigmaaldrich.com/CH/de/technical-documents/technical-article/protein-biology/elisa/antigensepitopes-antibodies
CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers. National Cancer Institute. 2022. Available
from: https://www.cancer.gov/about-cancer/treatment/research/car-t-cell
Keyword: Peptide Research Chemicals Powder