Antibody and Antigen Contact Residues Define Epitope and Paratope Size and Structure This information is current as James W. Stave and Klaus Lindpaintner of September 26, 2021. J Immunol published online 24 June 2013 http://www.jimmunol.org/content/early/2013/06/22/jimmun ol.1203198 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/06/24/jimmunol.120319 Material 8.DC1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 24, 2013, doi:10.4049/jimmunol.1203198 The Journal of Immunology Antibody and Antigen Contact Residues Define Epitope and Paratope Size and Structure James W. Stave and Klaus Lindpaintner1 A total of 111 Ag–Ab x-ray crystal structures of large protein Ag epitopes and paratopes were analyzed to inform the process of eliciting or selecting functional and therapeutic Abs. These analyses illustrate that Ab contact residues (CR) are distributed in three prominent CR regions (CRR) on L and H chains that overlap but do not coincide with Ab CDR. The number of Ag and Ab CRs per structure are overlapping and centered around 18 and 19, respectively. The CR span (CRS), a novel measure introduced in this article, is defined as the minimum contiguous amino acid sequence containing all CRs of an Ag or Ab and represents the size of a complete structural epitope or paratope, inclusive of CR and the minimum set of supporting residues required for proper conformation. The most frequent size of epitope CRS is 50–79 aa, which is similar in size to L (60–69) and H chain (70–79) CRS. ∼ The size distribution of epitope CRS analyzed in this study ranges from 20 to 400 aa, similar to the distribution of independent Downloaded from protein domain sizes reported in the literature. Together, the number of CRs and the size of the CRS demonstrate that, on average, complete structural epitopes and paratopes are equal in size to each other and similar in size to intact protein domains. Thus, independent protein domains inclusive of biologically relevant sites represent the fundamental structural unit bound by, and useful for eliciting or selecting, functional and therapeutic Abs. The Journal of Immunology, 2013, 191: 000–000. onoclonal Abs (mAbs) are important tools, diagnostic Purified, full-length native and recombinant proteins are useful http://www.jimmunol.org/ reagents, and therapeutic drugs. High-value commercial for eliciting Abs that bind native proteins, but are often difficult M diagnostic, functional, and therapeutic mAbs must rec- to prepare in quantities and qualities sufficient for immunization ognize full-length native proteins folded in native conformations as and selection of mAbs. In addition, immunization with full-length they exist in patients or patient samples. Given their potential value, proteins results in a polyclonal response where the majority of strategies for developing mAbs to native protein Ags is of intense Abs are directed to a limited number of immunodominant sites of interest; however, the underlying biology dictating performance the protein (5, 6). In cases where the predefined site of interest makes the task of developing such Abs both complicated and costly is nonimmunodominant, or even nonimmunogenic, it is very diffi- (1). For an Ab to exert a desired biological effect, it is usually the cult to isolate mAbs with the required performance specifications. by guest on September 26, 2021 case that it must bind to a specific, predefined site. In such cases, Thus, there is a critical need for a means of directing Ab develop- B cell epitope prediction algorithms are of limited value because ment to predefined functional epitopes of native protein Ags re- the Ab must bind to the predefined site regardless of its inherent gardless of their inherent immunogenicity. immunogenicity. Of paramount importance is binding at the target In the case of therapeutics, once candidate mAbs have been site to the three-dimensional conformation the protein exists in isolated with the requisite binding characteristics, the CDR can be under the conditions where the Ab must function. taken from the isolated mAb (7) and inserted into engineered Ab The most common method for developing Abs is to inject purified constructs having desired effecter function and characterized im- protein into animals and isolate mAbs that bind to native proteins. A munogenicity and toxicity profiles. It has been reported for six difficulty with this approach is the preparation of protein of suitable protein Ags that only a fraction of the CDR residues participate in purity and functional conformation. For decades, researchers have binding (8), and that Ab contact residues (CRs) in nine crystal attempted to use peptides as surrogates of full-length native proteins structures do not precisely correspond with residues modified by with little success (2). Anti-peptide Abs react well to their cognate somatic hypermutation in Vk (9). It is critical to identify specific peptides and may be useful for detecting linear epitopes in techniques residues involved in contacting Ag and understand how they are like Western blot, but Abs raised to unstructured peptides more often organized within CDR to engineer Ab reactivity. Thus, the native than not do not recognize the same sequence in native protein (3, 4). three-dimensional structure of both Ag epitope and Ab paratope are fundamental to the success of developing and engineering high- Antibody Discovery Research and Development, Strategic Diagnostics Inc., Newark, performance functional and therapeutic mAbs. DE 19702 With these considerations in mind, we undertook the analysis of 1Current address: Thermo Fisher Scientific Inc., Analytical Technologies, Waltham, MA. a large number of protein–Ab x-ray crystal structures to better Received for publication November 19, 2012. Accepted for publication May 23, understand both epitope and paratope size, structure, and interac- 2013. tion. A number of researchers have reported identifying CR of Abs Address correspondence and reprint requests to Dr. James W. Stave, Strategic Diag- and Ags by analysis of publicly available crystal structures (10–12). nostics Inc., 111 Pencader Drive, Newark, DE 19702. E-mail address: jstave@ A large body of work in this field is aimed at identifying CR as a sdix.com means of developing algorithms for predicting B cell epitopes (10, The online version of this article contains supplemental material. 13–17), whereas others have focused on analyzing or predicting Abbreviations used in this article: CR, contact residue; CRR, contact residue region; CR in CDR of Abs (8, 18–20). The majority of the existing studies CRS, contact residue span; PDB, Protein Data Bank. define two residues to be in contact when they are within a specified Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 distance of each other. Many researchers use 4 A˚ as the definition of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203198 2 EPITOPE AND PARATOPE STRUCTURE CR (10, 14–17), and the Molecular Modeling Database (21), at the of predefined functional sites represent the basic structural unit National Center for Biotechnology Information, defines protein in- useful for immunization, screening, and selection of functional teractions using a 4-A˚ cutoff. One group has published several and therapeutic mAbs. works using 6 A˚ (13, 20, 22) and reported that their conclusions were unaffected by cutoffs ranging from 4 to 6 A˚ (20). Materials and Methods ˚ Haste Andersen et al. (14) identified epitope CR using a 4-A cutoff A list of 110 unique Ab–Ag x-ray crystal structures representing 111 unique in 76 crystal structures composed of Ags .25 aa (29 of the structures Abs (structure 3LOH contains 2 different Abs bound to the same Ag at were variants of lysozyme). Using this approach, these researchers different sites) was manually curated from the Protein Data Bank (PDB) were able to determine the number of CRs per epitope, the maximum (24). To provide information regarding the full extent of Ag–Ab interaction, $ continuous sequence of CR within each epitope, and the distribution only structures containing 85 Ag aa were included in the analysis. Each Ab in the data set occurs only once. The resolution of all structures is #3.8 A˚ . of continuous CR per segment. These results highlighted the dis- The amino acid sequence for each Ab was numbered using Abysis and the continuous nature of protein Ag epitopes. Kabat convention (25) (http://www.bioinf.org.uk/abysis). CRs are defined as Padlan et al. (8) determined Ab CR in six crystal structures with Ag and Ab amino acid residues within 4 A˚ of each other (10). CRs were protein Ags and compared the sequence positions with CDR defined identified from PDB x-ray crystal structures using the Cn3D tool and Mo- lecular Modeling Database (21), found at the National Center for Biotech- by nucleic acid sequence variability (23). MacCallum et al. (12) nology Information Web site (http://www.ncbi.nlm.nih.gov/Structure/CN3D/ determined Ab CR sequence positions for 7 protein–Ab structures cn3d.shtml). In some analyses, CR at other distances are also presented.