The structural basis of antibody-antigen recognition
`
`Inbal Sela-Culang†, Vered Kunik † and Yanay Ofran*
`The Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel
`
`REVIEW ARTICLE
`published: 08 October 2013
`doi: 10.3389/fimmu.2013.00302
`
`Edited by:
`Michal Or-Guil, Humboldt University
`Berlin, Germany
`Reviewed by:
`Gur Yaari, Yale University, USA
`Chaim Putterman, Albert Einstein
`College of Medicine, USA
`*Correspondence:
`Yanay Ofran, The Goodman Faculty of
`Life Sciences, Bar Ilan University,
`Ramat-Gan 52900, Israel
`e-mail: yanay@ofranlab.org
`†Inbal Sela-Culang and Vered Kunik
`have contributed equally to this work.
`
`The function of antibodies (Abs) involves specific binding to antigens (Ags) and activa-
`tion of other components of the immune system to fight pathogens. The six hypervariable
`loops within the variable domains of Abs, commonly termed complementarity determining
`regions (CDRs), are widely assumed to be responsible for Ag recognition, while the con-
`stant domains are believed to mediate effector activation. Recent studies and analyses of
`the growing number of available Ab structures, indicate that this clear functional separation
`between the two regions may be an oversimplification. Some positions within the CDRs
`have been shown to never participate in Ag binding and some off-CDRs residues often con-
`tribute critically to the interaction with the Ag. Moreover, there is now growing evidence
`for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects
`the constant region and vice versa. This review summarizes and discusses the structural
`basis of Ag recognition, elaborating on the contribution of different structural determinants
`of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches
`for their identification and their relationship to the Ag interface. We also review what is
`currently known about the contribution of non-CDRs regions to Ag recognition, namely the
`framework regions (FRs) and the constant domains. The suggested mechanisms by which
`these regions contribute to Ag binding are discussed. On the Ag side of the interaction,
`we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab
`information into B-cell epitope prediction schemes. Beyond improving the understanding
`of immunity, characterization of the functional role of different parts of the Ab molecule
`may help in Ab engineering, design of CDR-derived peptides, and epitope prediction.
`
`Keywords: antibody, CDRs, antigen, paratope, epitope, framework, constant domain
`
`INTRODUCTION
`Antibodies (Abs) have two distinct functions: one is to bind specif-
`ically to their target antigens (Ags); the other is to elicit an immune
`response against the bound Ag by recruiting other cells and mol-
`ecules. The association between an Ab and an Ag involves myriad
`of non-covalent interactions between the epitope – the binding
`site on the Ag, and the paratopes – the binding site on the Ab. The
`ability of Abs to bind virtually any non-self surface with exquisite
`specificity and high affinity is not only the key to immunity but has
`also made Abs an enormously valuable tool in experimental biol-
`ogy, biomedical research, diagnostics and therapy. The diversity
`of their binding capabilities is particularly striking given the high
`structural similarity between all Abs. The availability of increas-
`ing amounts of structural data in recent years now allows for a
`much better understanding of the structural basis of Ab function
`in general, and of Ag recognition in particular. This review sur-
`veys the recent developments and the current gaps and challenges
`in this field. We focus specifically on the current understanding
`of the determinants within the Ab structure that contribute to
`Ag binding. We first discuss the motivations for, and applications
`of, the study of the structural basis of Ag recognition. Then we
`describe and discuss the Ab-Ag interface, with specific focus on the
`paratopes and the complementarity determining regions (CDRs),
`and their role in Ag binding. The last part focuses on the contri-
`bution of the non-CDRs parts of the Ab [i.e., framework regions
`
`(FRs) and the constant domains] to Ag binding and on the recent
`suggestions regarding non-local and allosteric effects in Ab func-
`tion. Over the last few years numerous reviews have addressed
`issues that are related or tangential to the topics we review here.
`This includes reviews of the engineering of Abs (1), their stability
`(2), affinity maturation (3), and isotype selection (4). While these
`important topics are relevant to the findings and ideas we review
`here, they are beyond the scope of this review.
`
`THE MOTIVATIONS FOR, AND APPLICATIONS OF, THE STUDY
`OF Ab-Ag RECOGNITION
`UNDERSTANDING IMMUNITY AND AUTOIMMUNITY
`The adaptive immune response involves two types of lymphocytes:
`T cells, which recognizes Ags that have been processed and their
`fragments are presented by MHC molecules, and B cells which pro-
`duce soluble Abs that can identify also the intact Ag in its native
`form. While the way in which T cells recognize their epitopes
`has been extensively studied to a level that enables the successful
`prediction of T-cell epitopes (5, 6), the rules that govern Ab-Ag
`recognition, including which parts of the Ab structure underlie
`Ag recognition and how and why certain determinants on the Ag
`are selected as epitopes, are not as well characterized. Understand-
`ing the mechanisms that underlie Ab-Ag recognition, therefore, is
`crucial for understanding immunity.
`
`www.frontiersin.org
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`Sela-Culang et al.
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`Structural basis of antibody-antigen recognition
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`The immune system enables Abs to distinguish between foreign
`and self molecules (7). Autoimmune diseases are characterized by
`the inappropriate response to self-Ags. It is not always clear what
`role is played by Abs and what role is played by other components
`of the immune system in autoimmunity. A variety of molecu-
`lar mechanisms have been proposed, including sequestered Ags,
`molecular mimicry, and polyclonal B-cell activation (8). Better
`understanding of the underpinnings of Ab-Ag recognition may
`also shed light on these questions.
`
`A MODEL FOR STUDYING BIO-MOLECULAR RECOGNITION
`A fundamental characteristic of the immune system is its ability
`to continuously generate novel protein recognition sites. Ab-Ag
`interfaces, therefore, are often considered a model system for eluci-
`dating the principles governing biomolecular recognition (9–13).
`For example, Keskin (14) and McCoy et al. (15) used X-ray crystal-
`lographic structures of Ab-Ag complexes to elucidate principles of
`the molecular architecture of protein–protein interfaces. Other
`studies, however, view Ab-Ag interfaces as a specific case that
`may not allow for generalization to all types of protein–protein
`interfaces (16). Thus, large scale studies of protein–protein inter-
`actions often exclude Ab-Ag complexes from the dataset analyzed
`(16–19). It is, therefore, important to determine to what extent Ab-
`Ag complexes could serve as a general model for protein–protein
`interactions.
`
`ANTIBODY ENGINEERING
`The specificity of the Ab molecule to its cognate Ag has been
`exploited for the development of a variety of immunoassays, vacci-
`nations, and therapeutics. Ab engineering may offer to expand the
`application of Abs by permitting improvements of affinity (20, 21)
`and specificity (22, 23). Understanding of the role each structural
`element in the Ab plays in Ag recognition is essential for success-
`ful engineering of better binders. The engineering of Abs is also
`important for the clinical use of Abs from non-human sources.
`Early studies on the use of rodent Abs in humans determined
`that they can be immunogenic (24). Humanization by grafting
`of the CDRs from a mouse Ab to a human FR is a commonly
`used engineering strategy for reducing immunogenicity (25, 26).
`In most cases, the successful design of high-affinity, CDR-grafted,
`Abs requires that key residues in the human acceptor FRs that are
`crucial for preserving the functional conformation of the CDRs
`will be back-mutated to the amino acids of the original murine Ab
`(26, 27). Several groups (28–30) used the experimentally deter-
`mined 3-D structures of Ab-Ag complexes in the Protein Data
`Bank (PDB) (31) to determine which residues participate in Ag
`recognition and binding. Such knowledge can be exploited to
`identify residues that are important for the function of the Ab
`in general and for Ag recognition in particular and may guide Ab
`engineering (32, 33). Residues that help maintain the functional
`conformation of the CDRs, for example, can be used to improve
`Ab humanization efforts by CDR-grafting.
`
`Ab EPITOPE PREDICTION
`Antibody epitopes (sometimes referred to as B-cell epitopes) are
`the molecular structures within an Ag that make specific contacts
`with the Ab paratope. B-cell epitopes are used in the development
`
`of vaccines and in immunodiagnostics. Correct identification of
`B-cell epitopes within an antigenic protein, may open the door for
`the design of molecules (biologic or synthetic) that mimic poten-
`tially protective epitopes and could be used to raise specific Abs
`or be used as a prophylactic or therapeutic vaccines. Identification
`of B-cell epitopes could promote protective immunity in the con-
`text of emerging and re-emerging infectious diseases and potential
`bioterrorist threats. This may be achieved by choosing from among
`the putative epitopes those that may provide immunity (e.g., by
`eliciting Abs that hamper the molecular function of pathogenic
`Ags). The choice of such epitopes is believed to be relevant for
`understanding and controlling protective immunity. In the case of
`the vaccinia virus, for example, which was used as smallpox vac-
`cine and is the only vaccine that has led to the complete eradication
`of an infectious disease from the human population, individuals
`possessing a high frequency of memory B-cells specific for major
`neutralizing Ags of the vaccinia virus are better protected from
`smallpox than individuals with a memory B-cell pool dominated
`by specificities for non-protective Ags (34). Thus, understand-
`ing the way in which an Ab recognizes its cognate epitope is
`of particular interest for vaccine design and disease prevention
`(35). Existing tools for identification of Ab epitopes (such as X-
`ray crystallography, pepscan, phage display, expressed fragments,
`partial proteolysis, mass spectrometry, and mutagenesis analysis)
`are not only expensive, laborious, and time consuming but also
`fail to identify many epitopes (36). When talking about protein
`Ags, most of these methods typically identify linear stretches as
`epitopes, while, arguably, most of the epitopes on protein Ags
`are conformational and even discontinuous. As for computational
`approaches, despite more than 30 years of efforts (37), existing B-
`cell epitope prediction methods are not accurate enough (38, 39)
`and are, therefore, not widely used. This is exemplified in Figure 1,
`in which the structure of hen egg lysozyme (HEL) Ag and three
`Abs that bind it are shown (Figures 1A,B), as well as the epitopes
`predicted by three different methods (Figure 1C).
`In general, current methods are trying to identify epitopic
`residues based on the presence of features associated with residues
`that bind the Ab (40–50). One possible explanation for the failure
`of these methods is that the differences between epitopes and other
`residues are not substantial. Indeed, several analyses (51–53) have
`shown that the amino-acid composition of epitopes is essentially
`indistinguishable from that of other surface-exposed non-epitopic
`residues.
`intrinsic properties that clearly differentiate
`This lack of
`between epitopic and non-epitopic residues and the fact (demon-
`strated in Figure 1) that most of the Ag surface may become a
`part of an epitope under some circumstances (54–57) suggest that
`epitopes depend, to a great extent, on the Abs that recognize them.
`This is exemplified in Figure 1: most of the HEL surface residues
`are part of an epitope of at least one Ab (Figures 1A,B), even
`though this figure shows only three Abs (out of dozens known to
`bind HEL). Almost all the residues predicted to be epitopic may be
`considered as correct predictions as they bind some Ab (Figure 1C)
`but also as false predictions as they don’t bind the others. Similarly,
`predicting that a residue is not in an epitope may be either a true
`negative or a false negative, depending on the Ab considered. It
`has recently been suggested by us (Sela-Culang et al., submitted)
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`Frontiers in Immunology | B Cell Biology
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`Sela-Culang et al.
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`Structural basis of antibody-antigen recognition
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`FIGURE 1 | Predicted epitopes vs. the actual epitopes of HEL. (A) The 3-D
`structure of HEL (CPK representation) together with three Abs (ribbon
`representation). PDB IDs 1JHL, 3D9A, and 1MLC were superimposed
`according to HEL structure. Epitope residues are colored blue, green, and red
`according to the corresponding Ab. Residues that are common to two
`
`epitopes are colored orange. (B) The structure of HEL colored according to
`the same three epitopes as in (A), presented in a different orientation. (C) The
`structure of HEL colored according to the epitopes predicted by Discotope
`(light blue), ellipro (purple), and seppa (pink). Note, not all predicted residues
`of Discotope and ellipro are observable in the presented orientation.
`
`and by others (58–60) that predicting epitopes should be done
`for a certain Ab. A similar concept was successfully applied in the
`case of T-cell epitope prediction methods: these methods do not
`examine the Ag for general features. Rather, different predictions
`are made, dependent on the specific MHC molecule binding and
`presenting the epitope to T cells.
`
`THE ROLE OF CDRs AND THEIR DEFINITION
`As shown in Figure 2, Abs are all-beta proteins consisting of four
`polypeptide chains: two identical heavy (H) chains and two iden-
`tical light (L) chains (61). The light and heavy chains are linked
`by disulfide bonds to form the arms of a Y-shaped structure, each
`arm is known as a Fab (61). The Fab is composed of two vari-
`able domains (VH in the heavy chain and VL in the light chain)
`and two constant domains (CH1 and CL) (62). In the pairing of
`light and heavy chains, the two variable domains dimerize to form
`the Fv fragment which contains the Ag binding site. Within each
`variable domain lie six hypervariable loops (63), three in the light
`chain (L1, L2, and L3) and three in the heavy chain (H1, H2, and
`H3), supported by a conserved FR of β-sheets. The light and heavy
`variable domains fold in a manner that brings the hypervariable
`loops together to create the Ag binding site or paratope. Two addi-
`tional domains of the heavy chain, CH2, and CH3, compose the
`Fc region which is responsible for mediating the biological activity
`of the Ab molecule.
`
`CDRs IDENTIFICATION
`As indicated by their names, CDRs are believed to account for
`the recognition of the Ag. Therefore, a major focus in analyz-
`ing the structural basis for Ag recognition has been in identifying
`the exact boundaries of the CDRs in a given Ab. It is a common
`practice to identify paratopes through the identification of CDRs.
`Kabat and co-authors (63, 64) were the first to introduce a sys-
`tematic approach to identify CDRs in newly sequenced Abs. It was
`based on the assumption that CDRs are the most variable regions
`between Abs. Therefore, they aligned the (fairly limited) set of Ab
`sequences available at that time and identified the most variable
`positions. Based on the alignment, they introduced a numbering
`
`scheme for the residues in the hypervariable regions and deter-
`mined which positions mark the beginning and the end of each
`CDR. As structural data became available, Chothia and Lesk (65,
`66) manually analyzed a small number of experimentally solved
`3-D structures and determined the structural location of the loop
`regions. The boundaries of the FRs and CDRs were determined
`and the latter have been shown to adopt a restricted set of confor-
`mations, based on the presence of certain residues at key positions
`in the CDRs and the flanking FRs. Their finding that Kabat’s defini-
`tions of L1 and H1 are structurally incorrect led to the introduction
`of the Chothia numbering scheme. With the increase of available
`structural data, they ran their analysis anew and introduced a new
`definition of L1 (66) in 1989. In 1997 (67), however, they con-
`cluded that this correction was erroneous, and reverted to their
`original 1987 numbering scheme. While the Kabat and Chothia
`schemes treated separately the different families of immunoglob-
`ulin domains, Lefranc and colleagues (68, 69) proposed a unified
`numbering scheme (referred to as IMGT numbering scheme) for
`immunoglobulin variable domain genomic sequences, including
`Ab light and heavy variable domains, as well as T-cell receptor vari-
`able domains. To correlate between the sequence, structure, and
`domain folding behavior of all immunoglobulin variable domains,
`the Aho numbering scheme spatially aligned known 3-D structures
`of immunoglobulins and unified their numbering (70).
`A drawback of the Kabat, Chothia, and IMGT numbering
`schemes is that CDRs length variability takes into account only
`the most common loop lengths; While both Kabat and Chothia
`schemes accommodate insertions with insertion letters (e.g., 27A),
`the IMGT scheme avoids the use of insertion codes for all but the
`least common very long loops, and the Aho numbering scheme
`places insertions and deletions symmetrically around a key posi-
`tion. However, Abs with unusually long insertions may be hard
`to annotate using these methods and, as a result, their CDRs may
`not be identified correctly. For instance, the recently determined
`3-D crystal structure of two bovine Abs (71) reveal exceptionally
`long H3 CDRs (>60 residues), with long insertions which these
`methods cannot accommodate and thus cannot identify the CDRs
`of these Abs.
`
`www.frontiersin.org
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`Sela-Culang et al.
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`Structural basis of antibody-antigen recognition
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`FIGURE 2 | The structure of an Ab molecule. (A) The 3-D structure of an Ab molecule (PDB ID: 1IGT). (B) A schematic representation of the Ab scaffold.
`
`ARE CDRs GOOD PROXIES FOR THE PARATOPE?
`While identification of paratopes is often done through identifica-
`tion of CDRs, not all the residues within the CDRs bind the Ag. In
`fact, an early analysis of the 3-D structures of Abs suggested that
`only 20–33% of the residues within the CDRs participate in Ag
`binding (72). In 1996, MacCallum and colleagues (73) performed
`a detailed residue-level analysis of Ag contacts. They suggested that
`contacting residues are more common at CDRs residues which
`are located at the center of the Ag combining site, and that non-
`contacting residues within the CDRs correspond with residues that
`are important for maintaining the structural conformations of the
`hypervariable loops and not necessarily for recognition of the Ag.
`Thus, they introduced a mapping of Ag-contacting propensities
`for each Ab position and proposed a new definition for CDRs
`based on these propensities. Padlan and co-workers (28) utilized
`Abs sequence and structure data to perform a by-position sum-
`mary of Ag contacts. They found that the residues that are directly
`involved in the interaction with the Ag are also, in general, the
`most variable ones. They suggested that the residues that inter-
`act with the Ag should be called Specificity Determining Residues
`(SDRs).
`The number of publicly available structures of Ab-Ag com-
`plexes increased in recent years to a level that enabled large-scale
`analyses. In a recent analysis (29) we utilized all available protein-
`Ab complexes in the PDB to identify the structural regions in which
`Ag binding actually occurs. This approach was implemented into
`a method dubbed Paratome (30, 74) that is based on a multi-
`ple structure alignment (MSTA) of all available Ab-Ag complexes
`in the PDB. The MSTA revealed regions of structural consen-
`sus where the pattern of structural positions that bind the Ag is
`highly similar among all Abs. These regions of structural binding
`
`consensus were termed antigen binding regions (ABRs). While
`CDRs, as identified by methods such as Kabat (63), Chothia (65),
`and IMGT (69), may miss ∼20% of the Ag binding residues, ABRs
`cover ∼96% of the residues that actually bind the Ag (30). To avoid
`confusions and cumbersome nomenclature, herein we generically
`refer to CDRs, SDRs, and ABRs as “CDRs” unless otherwise spec-
`ified. Figure 3 shows an example of CDRs as identified by Kabat,
`Chothia, IMGT, and Paratome for one Ab (anti-IL-15, PDB ID:
`2XQB), compared to the actual Ag binding residues. It can be seen
`that in this example, some of the CDRs (e.g., L3, H3) identified
`by the four methods are almost identical, while in other CDRs
`(e.g., L2, H1, and H2) there are substantial differences between the
`methods. The MSTA of Abs with known 3-D structure also con-
`firmed previous observations that there are structural positions
`within the CDRs in which none, or only a small percentage of the
`Abs contact the Ag. This is shown in Figure 4 where an example
`of such a position is marked by a green arrow.
`
`INTEGRALITY VS. MODULARITY
`Designed systems are often characterized as either modular or
`integral. In a modular system different components, or mod-
`ules, function independent of the function of other modules. The
`generation of Abs in the immune system is based on combining
`different elements, in a way that may be considered modular where
`each component is capable of binding the Ag regardless of the
`others. However, some analyses suggest that Ag binding warrants
`a more integrative view of the relationships between the different
`components of the Ab.
`The binding-sites of interacting proteins are usually com-
`posed of surface patches that have good shape and electrosta-
`tic complementary (15, 75, 76). It has been shown that CDRs
`
`Frontiers in Immunology | B Cell Biology
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`Sela-Culang et al.
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`Structural basis of antibody-antigen recognition
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`FIGURE 3 | Comparison of different CDR identification methods. The light
`(A) and heavy (B) chains of PDB ID 2XQB were numbered according to Kabat
`(colored green) and Chothia (colored red) using the Abnum tool
`(www.bioinf.org.uk/abs/abnum) and CDRs were extracted according to the
`CDR definitions table (www.bioinf.org.uk/abs/#cdrs). CDRs according to
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`IMGT (colored orange) were identified using the IMGT-gap tool
`(www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi). ABRs according to
`Paratome (colored blue) were identified using the Paratome server
`(www.ofranlab.org/paratome). Contacts (colored purple) between the Ab and
`IL-15 were defined using a 6-Å cutoff value.
`
`are characterized by an amino-acid composition that is different
`from that of other protein loops (77) and also from other types
`of protein–protein interfaces (58). Thus, one would expect that
`epitopes, just like paratopes, should have a distinct amino-acid
`composition. However, several recent analyses (51, 53) have shown
`that this is not the case: while epitopes differ from other types of
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`interfaces (10, 29, 60), their amino-acid composition is virtually
`the same as that of non-epitopic surface residues.
`Several studies have shown that each CDR has its own unique
`amino-acid composition, different from the composition of the
`other CDRs (52, 58, 78). Additionally, we have shown that each
`CDR has a unique set of contact preferences, therefore, favoring
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`FIGURE 4 | Ab positions that contact the Ag. (A,B) The lower graphs show
`the percentage of Abs with known 3-D structure that have a residue in a given
`position (i.e., in other Abs there is a gap in the MSTA in that position). The
`upper graphs show the percentage of Abs that contact the Ag out of those
`Abs that have a residue in that position. (A) Depicts the heavy chain and
`(B) depicts the light chain. In the upper graphs, the ABRs are colored red and
`the FRs are colored blue. An example of a position within an ABR that is not
`in contact with the Ag in any of the Abs, is marked by a green arrow. An
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`example of a position in the FRs that is in contact with the Ag in many (8%) of
`the Abs is marked by an orange arrow. (C) The Ab Fv domain (PDB ID: 1QFU)
`is colored according to the percentage of all Abs with known 3-D structure in
`which the residue in that position is in contact with the Ag: from red (100% of
`the Abs) to blue (0%). ABR residues are presented as lines. The definition of
`the ABRs is according to the Paratome server. A 6-Å cutoff value was used to
`define residues in contact. Percentages of contacts were calculated based on
`an MSTA of all protein Ab-Ag complexes in the PDB (30).
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`certain amino-acids over others (52). Dividing epitope residues
`into six subsets according to the CDR they bind, we found that
`each of the subsets has a distinct amino-acid composition, distin-
`guishable from non-epitope surface (52). In other words, when the
`six subsets of epitope residues are considered together the unique
`composition of each subset disappears so that the overall amino-
`acid composition of the entire epitope is indistinguishable from
`the rest of the surface. Pathogenic epitopes may have evolved to
`resemble Ag surface to escape recognition. On the other hand, the
`integration of the six CDRs together, each with its own unique
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`amino-acid composition and contact preferences, could be the
`evolutionary response of the immune system that enables Abs to
`recognize virtually any surface patch on the Ag.
`Despite this integrated effect of the CDRs, Abs can be also con-
`sidered as a modular system, composed of different elements (such
`as the Fab, VH and VL, or the six CDRs), which may bind the Ag
`on their own. Such smaller Ab fragments that retain Ag binding
`affinity and specificity, hold a great potential for drug design (79–
`81) as they have improved pharmacokinetics, tissue and tumor
`penetration, and can be produced more economically (80, 81).
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`They may also be combined with other fragments to yield better
`binders. Although such smaller fragments cannot induce effector
`function such as complement activation (due to the lack of the
`constant domains), they may neutralize the targeted Ag. Fab and
`single-chain variable (scFv) fragments usually maintain specific
`binding to the Ag (82). VH and VL fragments usually show sticky
`behavior, low solubility, and reduced Ag binding affinity (83–85),
`although, they sometime retain specificity to the Ag (83, 85–87).
`The CDRs may provide additional level of modularity. Accord-
`ing to the commonly accepted hotspot hypothesis, the binding
`energy of two proteins is largely determined by a very small num-
`ber of critical interface residues (12, 88–90). Thus, one may wonder
`whether an individual CDR could bind the Ag on its own provided
`that it harbors hotspots. Several linear peptides containing one or
`more of the CDRs that retained Ag specificity have been reported
`(91–98). Although their affinity was usually in the micromolar
`range, it could be significantly improved by introducing relatively
`minor modifications (91, 99). However, many attempts to isolate
`and design such CDR derived peptides failed (100, 101). One pos-
`sible reason is that a CDR, on its own, may not fold to the same
`conformation as in the context of the entire Fab, which may be cru-
`cial for binding. Cyclizing the CDR by adding Cys residues at its
`edges was suggested as a solution for this problem (96, 102–104).
`Another reason might lie in the fact that many attempts for the
`design of CDR-derived peptides are made based on CDR-H3, as it
`is considered to be the most important CDR for Ag binding (67,
`105–107). However, the median length of ABR-H2 is substantially
`longer than that of H3, and both typically form the same number
`of interactions with the Ag (52). In addition, while ABR-H3 was
`shown to have the highest contribution to Ag binding energy on
`average (52), there are individual cases in which other CDRs are the
`dominant ones (52, 102). It is also possible that in some cases the
`binding depends on specific contacts from residues in different
`CDRs, which may preclude the design of CDR-derived peptides
`that maintain specificity. We have shown (102) that CDRs that are
`able to bind the Ag on their own have unique characteristics and,
`thus, can be computationally identified given the Ab-Ag complex
`structure. This may enhance the design of CDR-derived peptides
`that are not necessarily based on CDR-H3.
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`NON-CDR DETERMINANTS THAT HAVE A ROLE IN Ag
`BINDING
`FR RESIDUES
`Within the variable domain, the CDRs are believed to be respon-
`sible for Ag recognition, while the FR residues are considered a
`scaffold for the CDRs. However, it is now well established that
`some of the FR residues may play an important role in Ag binding
`(32, 108). As mentioned above, many such FR residues were iden-
`tified during the process of Ab humanization by CDR grafting.
`While grafting only the CDRs usually results in a significant drop
`or a complete loss of binding, the binding affinity can be retained
`by back mutating some of the FR residues to the original murine
`sequence, emphasizing their role in Ag binding (26, 109–115).
`Framework region residues that affect Ag binding can be
`divided into two categories. The first are FR residues that contact
`the Ag, thus are part of the binding-site (108, 109, 111, 116–123).
`Some of these residues are close in sequence to the CDRs (in fact
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`they may be within the boundaries of CDRs according to some
`CDR identification methods, but not according to others, as shown
`in Figure 3). Other residues are those that are far from the CDRs
`in sequence, but are in close proximity to it in the 3-D structure. In
`particular, a loop in the heavy chain FR-3, sometimes referred to as
`CDR-H4, accounts for 1.3% of human Ab-Ag contacts (78, 124).
`This CDR-H4 is also enriched (in human Abs) in somatic hyper-
`mutations (Burkovitz et al., submitted). Figure 4 shows positions
`that are not in the CDRs but are in contact with the Ag in many Abs
`[e.g., the one marked by an orange arrow (4A), which corresponds
`to CDR-H4].
`In the second category of FR residues that affect Ag bind-
`ing, are residues that are not in contact with the Ag, but affect
`Ag binding indirectly (108, 109, 120, 121). These residues can
`be further divided to those that are in spatial proximity to the
`CDRs, and those that are not. The former are assumed to affect
`binding by providing a structural support to the CDRs, enabling
`them to adopt the right conformation and orientation, shaping
`the binding-site required for Ag binding (32). For example, it has
`been suggested that a certain position in heavy chain FR-3, close
`in structure but not in sequence to CDR-H1 and CDR-H2, affects
`the orientation of CDR-H2 relative to CDR-H1 in such a way that
`a large side-chain packs between them and separates them while a
`small side-chain allows them to be closer to each other (109, 120).
`Nevertheless, this is not always true, as was shown in the case of
`the anti-lysozyme D1.3 Ab: while mutating Lys in this position to
`either Val, Ala, or Arg resulted in affinit