Epitope mapping

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] Epitope characterization can also help elucidate the mechanism of binding for an antibody[7] and can strength intellectual property (patent) protection.[8][9][10] Experimental epitope mapping data are incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data.[11]

Epitopes are generally divided into two classes: linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but brought together upon three-dimensional protein folding. Most antigen-antibody interactions rely on binding to conformational epitopes.

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 antibodies target conformational epitopes that are only present when the protein is in its native (properly folded) state, which can make epitope mapping challenging.[12] Epitope mapping has been crucial to the development of vaccines against prevalent or deadly viral pathogens, such as chikagunya,[13] dengue,[14] Ebola,[5][15][16] and Zika viruses,[17] by determining the antigenic elements (epitopes) that confer long-lasting immunization effects.[18] 

Complex target antigens, such as membrane proteins (e.g., G protein-coupled receptors [GPCRs]) 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 only fold correctly in the context of a lipid bilayer. As a result, mAb epitopes on these membrane proteins are often conformational and, therefore, more difficult to map.[12]

Importance for intellectual property (IP) protection

Epitope mapping has become prevalent in protecting the intellectual property (IP) 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).[8][9][19] The ability to differentiate accurately 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).[20] 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.[9][10]

Epitope data have been central to several high-profile legal cases involving disputes over the specific protein regions targeted by therapeutic antibodies.[19] 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.[21]

Methods

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

  • X-ray co-crystallography. This technique 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. However, this approach is technically challenging, time-consuming, and expensive, and it requires large amounts of purified protein. Moreover, not all proteins are amenable to crystallization.[22]
  • 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.[18][23] The epitope mapping resolution depends on the number of overlapping peptides that are used. The main disadvantage of this approach is that it cannot generally be used to obtain conformational epitopes, which are the most relevant epitope type for human therapeutic mAbs. However, one study[24] rarely mapped discontinuous epitopes on CD20 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[25]).
  • Site-directed mutagenesis mapping. The molecular biological technique of site-directed mutagenesis (SDM) can be used to enable epitope mapping. In SDM, systematic 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 mapping.[2][8] High-throughput shotgun mutagenesis is a well-validated and widely used approach for mapping epitopes of mAbs at single-amino-acid resolution. As of Oct 2018, shotgun mutagenesis had been used to map >1,000 mAbs, with epitopes being successfully obtained in >95% of cases.[26] These mapping data have revealed mechanistic information for protein-drug interactions,[27] the functional regions of complex membrane proteins, including GPCRs,[28] target binding information for small molecules,[29][30] and epitope information for intellectual property (patent) protection purposes.[9][31] 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-to-serine substitution). Hundreds of plasmid clones from the library are individually arrayed in 384-well micro plates, expressed in mammalian 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 mutagenesis mapping include: 1) the ability to identify both linear and conformational epitopes, 2) the shorter assay time compared to 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)[32][33].
  • Hydrogen–deuterium exchange. This method, which is growing in popularity, gives information about the solvent accessibility of various parts of the antigen and antibody, demonstrating reduced solvent accessibility in regions of protein-protein interactions.[34]
  • Cross-linking-coupled mass spectrometry.[35] Antibody and antigen are bound to a labeled cross-linker, and complex formation is confirmed by high-mass MALDI detection. The binding location of the antibody to the antigen can then be identified by mass spectrometry (MS). The cross-linked complex is highly stable and can be exposed to various enzymatic and digestion conditions, allowing many different peptide options for detection. MS or MS/MS techniques are used to detect the amino-acid locations of the labelled cross-linkers and the bound peptides (both epitope and paratope 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 phage display and limited proteolysis, provide high-throughput monitoring of antibody binding but lack reliability, especially for conformational epitopes.[36]

See also

References

  1. ↑ DeLisser, HM. "Epitope mapping". Methods Mol Bio. 96: 11–20. doi:10.1385/1-59259-258-9:11. PMID 10098119.
  2. 1 2 3 Davidson, E; Doranz, B. "A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes" (PDF). Immunology. 143: 13–20. doi:10.1111/imm.12323.
  3. ↑ 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.
  4. ↑ Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael (2007). "Epitope mapping: the first step in developing epitope-based vaccines". BioDrugs. 21 (3): 145–56. doi:10.2165/00063030-200721030-00002. PMID 17516710.
  5. 1 2 Saphire, EO. et al. "Systematic analysis of monoclonal antibodies against Ebola virus GP defines features that contribute to protection". Cell. 174: P938–952. doi:10.1016/j.cell.2018.07.033.
  6. ↑ Dutton, G (January 1, 2016). "Integral Molecular sizes up Ebola: Membrane protein specialist maps Ebola's binding sites to advance vaccine discovery". Genetic Engineering & Biotechnology News. 36 (1).
  7. ↑ Davidson, E; et al. "Mechanism of binding to Ebola virus glycoprotein by the ZMapp, ZMAb, and MB-003 cocktail antibodies". Journal of Virology. 89: 10982–10992. doi:10.1128/JVI.01490-15.
  8. 1 2 3 Banik, S; Deng, X; Doranz, B. "Using Epitope Mapping to Derive More Value from mAbs". Genetic Engineering & Biotechnology News. 37 (15).
  9. 1 2 3 4 Deng, X; Storz, U; Doranz, BJ (2018). "Enhancing antibody patent protection using epitope mapping information". mAbs. 10(2): 204–9.
  10. 1 2 Ledford, H. "Rush to protect lucrative antibody patents kicks into gear". Nature. 557: 623–624.
  11. ↑ Potocnakova L, Bhide M, Pulzova LB (2017). "An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction". Journal of Immunology Research. 2016: 6760830.
  12. 1 2 Banik, SSR; Doranz, BJ (2010). "Mapping complex antibody epitopes". Genetic Engineering & Biotechnology News. 3 (2): 25–8.
  13. ↑ Zhang, R; et al. (2018). "Mxra8 is a receptor for multiple arthritogenic alphaviruses". Nature. 557: 570–4.
  14. ↑ Nivarthi, UK; et al. (2017). "Mapping the human memory B cell and serum neutralizing antibody responses to dengue virus serotype 4 infection and vaccination". Journal of Virology. 91(5): e02041–16. doi:10.1128/JVI.02041-16.
  15. ↑ Flyak AI; et al. (2018). "Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2–MPER region". Nature Microbiology. 3: 670–677.
  16. ↑ Zhao, X; et al. (2017). "Immunization-elicited broadly protective antibody reveals ebolavirus fusion loop as a site of vulnerability". Cell. 169: 891–904. doi:10.1016/j.cell.2017.04.038.
  17. ↑ Sapparapu, G; et al. (2016). "Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice". Nature. 540: 443–7. doi:10.1038/nature20564.
  18. 1 2 Gaseitsiwe, S.; et al. (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. doi:10.1128/CVI.00208-09. PMC 2812096. PMID 19864486.
  19. 1 2 Sandercock CG, Storz U (2012). "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.
  20. ↑ Teeling, TJ; et al. (2006). "The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20". Journal of Immunology. 177 (1): 362–71. ISSN 0022-1767. PMID 16785532.
  21. ↑ "Amgen Inc. et al v. Sanofi et al". Retrieved 2017-07-23.
  22. ↑ Abbott WM, Damschroder MM, Lowe DC (2014). "Current approaches to fine mapping of antigen–antibody interactions". Immunology. 142 (4): 526–535. doi:10.1111/imm.12284. PMC 4107663. PMID 24635566.
  23. ↑ Linnebacher, M; et al. (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. doi:10.1007/s00216-012-5781-5. PMID 22349330.
  24. ↑ Cragg, M. S. (2011). "CD20 antibodies: doing the time warp". Blood. 118 (2): 219–20. doi:10.1182/blood-2011-04-346700. PMID 21757627.
  25. ↑ Timmerman, P; et al. (2009). "Functional reconstruction of structurally complex epitopes using CLIPS™ technology". The Open Vaccine Journal. 2 (1): 56–67. doi:10.2174/1875035400902010056. hdl:11245/1.309707.
  26. ↑ "Epitope Mapping Services". Integral Molecular. Retrieved September 21, 2018.
  27. ↑ Paes C, Ingalls J, Kampani K, Sulli C, Kakkar E, Murray M, Kotelnikov V, Greene TA, Rucker JB, Doranz BJ (2009). "Atomic-Level Mapping of Antibody Epitopes on a GPCR". Journal of the American Chemical Society. 131 (20): 6952–6954. doi:10.1021/ja900186n. PMC 2943208. PMID 19453194.
  28. ↑ Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PAS, Rucker JB (2011). "Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin". PLOS One. 6 (5): e20123. doi:10.1371/journal.pone.0020123. PMC 3101243. PMID 21629661.
  29. ↑ Wescott MP, Kufareva I, Paes C, Goodman JR, Thaker Y, Puffer BA, Berdougo E, Rucker JB, Handel TM, Doranz BJ (2016). "Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices". PNAS. 113 (35): 9928–9933. doi:10.1073/pnas.1601278113. PMC 5024644. PMID 27543332.
  30. ↑ Thomas A, Sulli C, Davidson E, Berdougo E, Phillips M, Puffer BA, Paes C, Doranz BJ, Rucker JB (2017). "The Bitter Taste Receptor TAS2R16 Achieves High Specificity and Accommodates Diverse Glycoside Ligands by using a Two-faced Binding Pocket". Scientific Reports. 7 (1): 7753. doi:10.1038/s41598-017-07256-y. PMC 5552880. PMID 28798468.
  31. ↑ Ledford H (2018). "Rush to protect lucrative antibody patents kicks into gear". Nature. 557: 623–624.
  32. ↑ Bogan AA, Thorn KS (1998). "Anatomy of hot spots in protein interfaces". Journal of Molecular Biology. 280 (1): 1–9. doi:10.1006/jmbi.1998.1843. PMID 9653027.
  33. ↑ Lo Conte L, Chothia C, Janin J (1999). "The atomic structure of protein-protein recognition sites". Journal of Molecular Biology. 285: 2177–2198.
  34. ↑ Casina VC, Hu W, Hanby HA, Pickens B, Mayne L, Ostertag E, Kacir S, Siegel DL, Englander SW, Zheng XL (2014). "Autoantibody Epitope Mapping By Hydrogen-Deuterium Exchange Mass Spectrometry at Nearly Single Amino Acid Residue Resolution Reveals Novel Exosites on ADAMTS13 Critical for Substrate Recognition and Mechanism of Autoimmune Thrombotic Thrombocytopenic Purpura". Blood. 124: 108.
  35. ↑ "Epitope Mapping". www.covalx.com/epitope2. Retrieved 2017-02-23.
  36. ↑ 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).
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