Epitope mapping

From Wikipedia, the free encyclopedia
Jump to: navigation, search

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.[1][2] 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 2 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.[3]

Importance for Antibody Characterization[edit]

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.[4] 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.[3][5] New techniques such as high-throughput mutagenesis have been developed to address these challenging epitopes.[6] Epitope mapping is also crucial to developing key vaccines against prevalent viral diseases such as Dengue virus. Epitope mapping helps develop these challenging vaccines by determining antigenic elements (or epitopes) that confer with long-lasting immunization effects.[7] 

Epitope mapping of complex target antigens, such as integral 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.[5]

Epitopes and Intellectual Property[edit]

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 facilitating the recognition of critical distinctions between current and prior art (existing) antibodies.[8] The ability to accurately differentiate between antibodies for is particularly important when patenting antibodies against well validated therapeutic targets e.g. PD1 and CD20.[9] 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.[8] 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.[10]


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

  • X-ray co-crystallography: The gold standard approach which allows direct visualization of the interaction between the antigen and antibody. This approach is technically challenging, requires large amounts of purified protein, and can be time-consuming and expensive.[citation needed]
  • 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.[7][11] 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[12]), discontinuous epitopes can be mapped with very high reliability and precision.[13] The epitope mapping resolution here depends on the number of overlapping peptides that is used. A maximum peptide-peptide overlap, i.e. only changing one amino acid at a time, will lead to the most informative results.
  • 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.[citation needed]
  • High Throughput Mutagenesis Mapping.[14] This approach utilizes a comprehensive mutation library, with each clone containing a unique amino acid mutation (conservative, non-conservative, or alanine) and the entire library covering every amino acid in the target protein. 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. Amino acids that are required for antibody binding can be identified by a loss of fluorescent reactivity and mapped onto protein structures to visualize epitopes.[5] A customized database enables all mutagenesis information to be managed in a systematic, accurate and efficient manner. The database also performs the statistical calculations used throughout data analysis and epitope refinement, including patented algorithms to derive the final epitopes.[citation needed] To date this approach, coined "Shotgun Mutagenesis" has been used to construct mutation libraries totaling over 10,000 individual point mutations, representing viral envelope proteins [dengue virus-3 (DENV-3) prM/E, DENV-4 prM/E, chikungunya virus E2/E1, hepatitis C E1/E2, hepatitis B virus surface antigen, respiratory syncytial virus F protein and HIV gp160], GPCR proteins (CCR5, CXCR2, CXCR4 and TAS2R16), 4TM proteins (claudin-1 and claudin-4) and other membrane proteins (MCAM-1 and Her-2).[6] The Shotgun mutagenesis approach has been used to epitope map hundreds of mAbs targeting DENV (representing one of the largest collections of epitope information against a viral protein), chikungunya virus and hepatitis C virus, with additional mAb epitopes mapped on hepatitis B virus, respiratory syncytial virus and HIV.[6] It has also been used to define atomic-level mAb epitopes on the GPCRs CCR5 [15] and CXCR4,[16] the identification of cancer biomarker epitopes on the 4TM proteins claudin-1 and claudin-4, an atomic-level model describing the intramolecular signal transduction pathway of CXCR4, a proposed mechanism for the ligand specificity and sensitivity of the GPCR TAS2R16, mapping of inhibitor-binding sites on TAS2R16,[17] and mapping of paratope residues on a clinical antibody against respiratory syncytial virus.[18]
  • 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.[citation needed]
  • Crosslinking coupled Mass Spectrometry:[19] 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.[3]

See also[edit]


  1. ^ Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael (2007). "Epitope Mapping". BioDrugs. 21 (3): 145–56. PMID 17516710. doi:10.2165/00063030-200721030-00002. 
  2. ^ Westwood, Olwyn M. R.; Hay, Frank C., eds. (2001). Epitope Mapping: A Practical Approach. Oxford, Oxfordshire: Oxford University Press. ISBN 978-0-19-963652-5. [page needed]
  3. ^ a b c Flanagan, Nina (May 15, 2011). "Mapping Epitopes with H/D-Ex Mass Spec: ExSAR Expands Repertoire of Technology Platform Beyond Protein Characterization". Genetic Engineering & Biotechnology News. 31 (10). 
  4. ^ Hasan, S. Saif; Miller, Andrew; Sapparapu, Gopal; Fernandez, Estefania; Klose, Thomas; Long, Feng; Fokine, Andrei; Porta, Jason C.; Jiang, Wen (2017-03-16). "A human antibody against Zika virus crosslinks the E protein to prevent infection". Nature Communications. 8: ncomms14722. PMC 5356071Freely accessible. PMID 28300075. doi:10.1038/ncomms14722. 
  5. ^ a b c Banik, Soma S. R.; Doranz, Benjamin J. (2010). "Mapping Complex Antibody Epitopes". Genetic Engineering and Biotechnology News. 3 (2): 25–8. 
  6. ^ a b c Davidson, Edgar; Doranz, Benjamin J. (2014). "A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes". Immunology. 143 (1): 13–20. PMC 4137951Freely accessible. PMID 24854488. doi:10.1111/imm.12323. 
  7. ^ a b Gaseitsiwe, S.; Valentini, D.; Mahdavifar, S.; Reilly, M.; Ehrnst, A.; Maeurer, M. (2010). "Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401". Clinical and Vaccine Immunology. 17 (1): 168–75. PMC 2812096Freely accessible. PMID 19864486. doi:10.1128/CVI.00208-09. 
  8. ^ a b Sandercock, Colin G; Storz, Ulrich. "Antibody specification beyond the target: claiming a later-generation therapeutic antibody by its target epitope". Nature Biotechnology. 30 (7): 615–618. doi:10.1038/nbt.2291. 
  9. ^ Teeling, Jessica L.; Mackus, Wendy J. M.; Wiegman, Luus J. J. M.; van den Brakel, Jeroen H. N.; Beers, Stephen A.; French, Ruth R.; van Meerten, Tom; Ebeling, Saskia; Vink, Tom (2006-07-01). "The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20". Journal of Immunology (Baltimore, Md.: 1950). 177 (1): 362–371. ISSN 0022-1767. PMID 16785532. 
  10. ^ "Amgen Inc. et al v. Sanofi et al". Retrieved 2017-07-23. 
  11. ^ Linnebacher, Michael; Lorenz, Peter; Koy, Cornelia; Jahnke, Annika; Born, Nadine; Steinbeck, Felix; Wollbold, Johannes; Latzkow, Tobias; Thiesen, Hans-Jürgen; Glocker, Michael O. (2012). "Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples". Analytical and Bioanalytical Chemistry. 403 (1): 227–38. PMID 22349330. doi:10.1007/s00216-012-5781-5. 
  12. ^ Timmerman, P.; Puijk, W. C.; Boshuizen, R. S.; Dijken, P.van; Slootstra, J. W.; Beurskens, F. J.; Parren, P.W. H.I.; Huber, A.; Bachmann, M. F.; Meloen, R. H. (2009). "Functional Reconstruction of Structurally Complex Epitopes using CLIPS™ Technology". The Open Vaccine Journal. 2 (1): 56–67. doi:10.2174/1875035400902010056. 
  13. ^ Cragg, M. S. (2011). "CD20 antibodies: doing the time warp". Blood. 118 (2): 219–20. PMID 21757627. doi:10.1182/blood-2011-04-346700. 
  14. ^ "Shotgun Mutagenesis" (PDF). Integral Molecular. 2012. [unreliable source?]
  15. ^ Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A.; Rucker, Joseph B.; Doranz, Benjamin J. (2009). "Atomic-Level Mapping of Antibody Epitopes on a GPCR". Journal of the American Chemical Society. 131 (20): 6952–4. PMC 2943208Freely accessible. PMID 19453194. doi:10.1021/ja900186n. 
  16. ^ Jahnichen, S.; Blanchetot, C.; Maussang, D.; Gonzalez-Pajuelo, M.; Chow, K. Y.; Bosch, L.; De Vrieze, S.; Serruys, B.; Ulrichts, H.; Vandevelde, W.; Saunders, M.; De Haard, H. J.; Schols, D.; Leurs, R.; Vanlandschoot, P.; Verrips, T.; Smit, M. J. (2010). "CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells". Proceedings of the National Academy of Sciences. 107 (47): 20565–70. PMC 2996674Freely accessible. PMID 21059953. doi:10.1073/pnas.1012865107. 
  17. ^ Matsunami, Hiroaki; Greene, Tiffani A.; Alarcon, Suzanne; Thomas, Anu; Berdougo, Eli; Doranz, Benjamin J.; Breslin, Paul A. S.; Rucker, Joseph B. (2011). "Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin". PLoS ONE. 6 (5): e20123. PMC 3101243Freely accessible. PMID 21629661. doi:10.1371/journal.pone.0020123. 
  18. ^ Berdougo, Eli; Couto, Joseph R.; Doranz, Benjamin J. (August 1, 2011). "Maximal Humanization of Monoclonal Abs: Integral Molecular Uses Shotgun Mutagenesis to Germline-Humanize Monoclonal Antibodies". Genetic Engineering & Biotechnology News. 31 (14). 
  19. ^ "Epitope Mapping". www.covalx.com/epitope2. Retrieved 2017-02-23. 

External links[edit]