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&CHAPTER 2 Antibody Molecular Structure ROBYN L. STANFIELD and IAN A. WILSON 2.1 Introduction 51 2.2 General Structural Features 2.3 Canonical Conformations 52 56 2.4 Fab Conformational Changes 56 2.5 Human Anti-HIV-1 Antibodies 2.6 Shark and Camel Antibodies 58 61 2.7 Summary 63 Acknowledgments 63 References 63 ABSTRACT The structural features of antibodies have been studied extensively over the years by many techniques, including electron microscopy, x-ray crystallography, and NMR. Consequently, a wealth of structural information is available for antibodies, alone and in complex with antigens ranging in size from small haptens to whole viruses. The knowledge gained from these studies has greatly facilitated the engineering of antibodies for use as human therapeutics. 2.1 INTRODUCTION Antibodies are the key component of the humoral adaptive immune response against foreign pathogens. Their enormous sequence and structural diversity allows for recognition of any foreign antigen imaginable, with high affinity and specificity. While antibodies have been best studied in mice and humans, they are also found in animals as evolutionarily distant as the cartilaginous fish, such as sharks, skates, and rays. An abundance of structural information has accumulated for antibodies, including over 800 crystal structures that are predominantly Fab, Fab 0 , Fv, or occasionally VH fragments, many in complex with antigens ranging in size from small haptens, to peptide and DNA fragments, to proteins. In addition, combined cryo-electron microscope and crystallographic studies have revealed how Fab and IgG molecules interact with intact viruses. This structural information Therapeutic Monoclonal Antibodies: From the Bench to the Clinic. Edited by Zhiqiang An Copyright # 2009 John Wiley & Sons, Inc. 51 52 ANTIBODY MOLECULAR STRUCTURE has been of great value for our understanding of the antibody-antigen recognition process, and also for development of antibodies as human therapeutics. 2.2 GENERAL STRUCTURAL FEATURES Mammalian antibodies are constructed from two types of protein sequences, called the heavy and light chains. The five classes of antibodies in humans and other placental mammals differ in their heavy chain sequences, with heavy chain types m, d, g, 1, and a found in IgM, IgD, IgG, IgE, and IgA antibodies, respectively. Each heavy chain can pair with one of two types of light chain, called l or k. Most antibodies are made up of four chains; two copies of the heavy chain and two copies of the light chain (Fig. 2.1). However, the IgM class can also exist as a pentamer of these four chains, with a resultant 10 heavy and 10 light chains, and IgA can exist in a form called secretory IgA, where an additional J chain stabilizes the dimerization of two antibodies, to give a total of four heavy and four light chains. The majority of antibodies found in the serum belong to the IgG class, and most structural information has been derived for this class of antibody; thus, most of our discussion will deal with IgG antibodies. An intact IgG molecule has two heavy (55,000 Da each) and two light chains (24,000 Da each) that fold into three large domains: two Fab fragments (one light and the N-terminal half of a heavy chain) and one Fc fragment (two C-terminal heavy chain halves) (Fig. 2.1). Fab and Fc are abbreviations for fragment-antibody binding and fragment-crystallizable, so called because the Fab fragment binds antigen, and the first Fc fragments studied were easy to crystallize, although that clearly now is somewhat of a misnomer. The overall shape of the IgG can be described as a Y, with the Fc fragment forming the base of the Y, and the two Fab fragments forming the two arms (Fig. 2.2). Figure 2.1 IgG light and heavy chains. The light chains are shown in light gray, and the heavy chains in dark gray, with the different Ig domains indicated. 2.2 GENERAL STRUCTURAL FEATURES 53 Figure 2.2 IgG domain organization. The Fc and two Fab domains form an overall Y shape. Figure 2.3 Structure of IgG b12 determined by x-ray crystallography. Residues in the linker region of the heavy chain that have no visible electron density are shown as a dotted line. Carbohydrate within the Fc domain is shown in a gray CPK representation. Rather than a rigid Y shape as depicted in Figure 2.2, in a real IgG, the Fab arms are very flexible with respect to the Fc domain, allowing for greater flexibility in binding antigen. (See color insert.) 54 ANTIBODY MOLECULAR STRUCTURE Figure 2.4 Fab fragment. (Left) The light chain is on the left in light gray, and the heavy chain on the right in darker gray. The CDR loops that contact antigen are labeled. (Right) Looking down onto the Fab antigen-binding site. This view is the same as on the left, but rotated 90 degrees about a horizontal axis. (See color insert.) Figure 2.5 An IgG Fc domain. The two heavy chains are colored dark and light gray, and the carbohydrate found at the center of the Fc in an IgG is shown in a ball-and-stick representation. (See color insert.) 2.2 GENERAL STRUCTURAL FEATURES 55 However, highly flexible hinge regions join the Fc to the two Fabs, and electron microscopy (Roux, Strelets, and Michaelsen 1997) and crystal structures of IgGs with intact hinge regions (Harris et al. 1992, 1997; Saphire et al. 2001) show that the Fabs have a wide range of flexibility with respect to the Fc (Fig. 2.3). The Fab fragment can be further subdivided into smaller domains called the variable domain (the N-terminal half of the Fab; VL and VH) and constant domain (the C-terminal half of the Fab; CL and CH1) (Fig. 2.4). As suggested by their names, the variable domain contains regions with high sequence diversity, while the constant domain sequences are highly conserved. The Fc fragment also has two domains, formed by dimerization of CH2 –CH2 and CH3–CH3 regions (Fig. 2.5). The final, smallest level of domain architecture is the immunoglobulin or Ig domain. Each individual VL, VH, CL, CH1, CH2, or CH3 domain is an Ig domain, consisting of a characteristic Ig fold with either seven or nine b strands, in two b sheets, that form a Greek-key b-barrel, with a highly conserved disulfide connecting the two sheets at the core of the barrel (Fig. 2.6). A constant-type Ig domain has seven strands, three in one sheet and four in the other, while a variable-type Ig domain adds two extra strands (C0 and C00 ) to the three-stranded sheet, so that the barrel is composed of five- and four-stranded b-sheets (Fig. 2.6). While first identified in immunoglobulins, the Ig fold is found in many other types of molecules and, in addition to the variable- and constant-type Ig domain, other different but related types of Ig domains exist, some without the canonical disulfide bridge (Bork, Holm, and Sander 1994; Halaby, Poupon, and Mornon 1999). Figure 2.6 A constant-type Ig domain (left) and a variable-type Ig domain (right). The variable domain has two extra strands (C0 and C00 ) that form the CDR2 loop. This domain is from a light chain; however, a heavy chain variable domain has the same domain architecture. (See color insert.) 56 ANTIBODY MOLECULAR STRUCTURE The Fab fragment recognizes antigen at a site formed by six linear polypeptide segments called complementarity determining region (CDR) loops. Three CDRs are contributed by the VL (L1, L2, and L3) and three by the VH domain (H1, H2, and H3) (Fig. 2.4). These loops are hypervariable in sequence, and were predicted to be the interaction site for antigen recognition long before any structural information existed for the Fab fragment (Wu and Kabat 1970; Kabat and Wu 1971). However, the structure of the first Fab-hapten complex (McPC603-phosphocholine) determined by x-ray crystallography in 1973 (Padlan et al. 1973; Segal et al. 1974) verified that the antigen interacts mainly with the CDR loop regions of the Fab. While recognition of protein antigens by Fab generally, but not always, utilizes all six of the CDR loops, recognition of smaller antigens may use a more limited set of these CDRs; in fact, some isolated VH fragments containing CDRs H1-3 are sufficient for recognition of their antigen (Davies and Riechmann 1996). Typical VH-VL antibody-antigen interface sizes (the combined buried molecular surface area for both the antigen and antibody) range from ´ ´ ´ around 300 –600 Å2 for small haptens, to 800–900 Å2 for peptides, to 1500– 1800 Å2 for proteins (Wilson et al. 1991). 2.3 CANONICAL CONFORMATIONS While the CDR loops are the most variable part of the Fab (in both their sequence and structure), five of the six loops have been shown to have a limited number of conformations, termed canonical structures (Chothia and Lesk 1987). Based on their sequence, and a set of rules derived from known structures, the conformations of these loops can usually be predicted with reasonable accuracy. However, CDR H3 has been far more difficult to classify. H3 is the most variable of the CDR loops in sequence and length, and structures of many Fab fragments have shown that H3 is also the most structurally variable and flexible of the CDRs. The base of CDR H3 can usually be found in one of two different conformations that can often be predicted from the sequence (Shirai, Kidera, and Nakamura 1996; Al-Lazikani, Lesk, and Chothia 1997; Koliasnikov et al. 2006), but the remainder of the loop has no conserved structure, is frequently seen to undergo large changes in conformation upon binding of antigen, and is often poorly ordered in crystal structures of unliganded Fabs. More recent studies have emphasized important functional differences in the lengths of the CDR H3s in humans versus mice that can be exploited for antigen recognition (Collis, Brouwer, and Martin 2003). 2.4 Fab CONFORMATIONAL CHANGES In addition to hinge flexibility between the Fab and Fc fragments, flexibility also occurs between the domains that make up the Fab fragment. The variable and constant domains can move with respect with each other around what is termed the elbow angle. This angle has been seen to vary between 117 degrees and 227 degrees (Stanfield et al. 2006), with larger values seen more often with antibodies that contain l light chains (Stanfield et al. 2006). The VL and VH domains can also rotate with respect to each other by as much as 16 degrees (Stanfield et al. 1993), and these movements can serve to dramatically reconfigure the antigen binding site. Currently about 100 Fabs have been determined in both the unliganded and ligand-bound form. Comparisons of these liganded and ligand-free structures have shown that conformational changes often occur in the antigen-binding site to enable greater complementarity of fit to the antigen (Stanfield and Wilson 1994; Wilson and Stanfield 1994). These changes usually take place in the CDR loops and, as mentioned earlier, the largest changes are often seen in CDR H3. While often termed induced-fit binding, studies of antibodies in solution indicate that the conformational changes are probably not completely induced by binding of the antigen, but rather the antigen may in some cases bind to one of several preexisting antibody conformations found in solution, where small structural changes then allow the antibody to adapt and custom-fit its binding site to the antigen in question (Foote and Milstein 1994; James, Roversi, and Tawfik 2003). 2.4 Fab CONFORMATIONAL CHANGES 57 Conformational changes in antibodies include changes in side-chain rotamers, movements of the CDR loops as either a rigid unit or by more extensive structural rearrangements, and through changes in the relative disposition of their VH/VL domains. An excellent illustration of a key side-chain rearrangement is found in the anti-progesterone antibody DB3 (Arevalo et al. 1993; Arevalo, Taussig, and Wilson 1993; Arevalo et al. 1994). When the unliganded and steroid-bound structures Figure 2.7 Examples of conformational changes in Fabs after binding antigen. Top left: The anti-progesterone Fab DB3 in its unliganded form, with TrpH100 filling its antigen-binding pocket. Top right: DB3 with progesterone (ball-and-stick) bound, and TrpH100 moved away from its position in the unliganded Fab. (Middle left) The antiHIV-1 Fab 50.1 in its unliganded form. Middle right: 50.1 with bound peptide (ball-and-stick). The H3 and H1 CDRs are labeled. H3 undergoes a structural rearrangement, while H1 moves away from the binding site while maintaining its overall shape. Bottom left: The unliganded anti-HIV-1 Fab X5, with its long CDR H3 labeled. Bottom right: Fab X5 in its gp120-bound conformation, with H3 labeled. A large conformational change occurs in the CDR H3 between the unliganded and liganded forms. (See color insert.) 58 ANTIBODY MOLECULAR STRUCTURE for DB3 are compared, a substantial conformational change in the position of the TrpH100 side chain is observed (Fig. 2.7). Interestingly, this side chain fills the antigen-binding site in the unliganded Fab structure, and then moves out of the way to allow binding of steroid (Fig. 2.7). Thus, TrpH100 acts as a surrogate ligand for the antibody in the absence of steroid. One of the most extensive conformational changes has been seen for antibody 50.1, an anti-HIV-1 neutralizing antibody that recognizes the HIV-1 gp120 V3 loop (Rini et al. 1993; Stanfield et al. 1993). A structural comparison of the unliganded and V3 peptide-bound Fab showed that the H3 CDR loop undergoes a large structural ´ rearrangement, with the side chain of Tyr H97 moving by about 6 Å. The CDR H1 also moves, in a rigid body or segmental fashion, with a root-mean square deviation for the H1 main-chain atoms of ´ 1.1 to 1.4 Å (for multiple copies of both the native and peptide-bound Fab in the crystals). In addition, the VL and VH domains of 50.1 move with respect to each other by about 16 degrees (Fig. 2.7). The combination of loop rearrangement and domain rotation result in a drastically differently shaped and configured binding pocket for the peptide ligand (Fig. 2.7). Another example of an extremely large H3 CDR movement is seen in crystal structures of the anti-HIV-1 antibody X5 that recognizes the recessed CD4-binding site of gp120. The long X5 CDR loop undergoes an extensive rearrangement ´ and moves by as much as 17 Å (Darbha et al. 2004; Huang et al. 2005) between the unbound and gp120 bound states (Fig. 2.7). Unusually long H3 CDR loops are not uncommon in human antiviral antibodies, and it has been proposed that long CDR loops may help antibodies target recessed clefts in viral antigens (Burton et al. 2005). 2.5 HUMAN ANTI-HIV-1 ANTIBODIES While most of the early crystal structures for antibodies were obtained for human myeloma antibodies, the advent of the monoclonal antibody technology made it very easy to produce large amounts of mouse monoclonal antibodies to known antigens. Thus, many more crystal structures have been determined for antibody fragments from mice than from humans. However, subsequent developments, such as Epstein-Barr virus immortalized human B-cells (Steinitz et al. 1977) and human-mice heterohybridomas (Cole et al. 1984), production of human antibodies and antibody fragments in bacteria (Skerra and Pluckthun 1988), and phage-display of antibody fragments (Huse et al. 1989), have made it possible to produce human monoclonal antibodies against known antigens. In addition, techniques, such as humanization of mouse or rat antibodies (Jones et al. 1986) or the production of human/mouse chimeric antibodies (Boulianne, Hozumi, and Shulman 1984; Morrison et al. 1984; Neuberger et al. 1985; Better et al. 1988), have been made possible by now standard molecular biology techniques. Currently about 40 fully human Fab or Fv fragments have crystal structures deposited in the PDB, Q1 although many of these are for the same fragment with multiple, related antigens. Many of the human antibodies that have been studied by x-ray crystallography are against viral antigens, including the HIV-1 virus. The HIV-1 virus is able to rapidly evolve to evade the host immune system, but only a handful of antibodies have been discovered that are able to effectively neutralize a wide variety of the different strains of the virus (Burton, Stanfield, and Wilson 2005). A successful vaccine would induce similar antibodies that are able to neutralize any strain of the virus that an individual might encounter. Structures of these rare antibodies have shown that the antibodies outwit the virus by using novel structural features in their mechanisms of antigen recognition (Burton, Stanfield, and Wilson 2005). One of the most interesting of the anti-HIV-1 antibodies is 2G12. 2G12 was isolated from an HIV-1 infected patient, and is one of the most potent, broadly neutralizing antibodies known. While initial studies indicated that 2G12 recognized a carbohydrate epitope on the HIV-1 surface (Sanders et al. 2002; Scanlan et al. 2002), it was difficult to understand how the antibody might recognize carbohydrate with high affinity. The carbohydrate on the HIV-1 viral surface is transferred onto the viral coat proteins by the human host cellular machinery. Thus, these sugars should appear as self to our immune system and, hence, give rise to tolerance and not lead to a strong antigenic response. Such 2.5 HUMAN ANTI-HIV-1 ANTIBODIES 59 anti-carbohydrate antibodies should be exceedingly rare and, as for other anti-carbohydrate antibodies against non-self sugars, be of low affinity. The crystal structure of the 2G12 Fab fragment was, therefore, a huge surprise (Calarese et al. 2003), and revealed the Fab had dimerized via a domain swap of its VH domains (Fig. 2.8). This domain-swapped dimer has two closely spaced antigen-binding sites and a potential third binding region at the interface of the two newly associated VH domains (VHVH0 ). These intertwined Fab regions give rise to the unusual linear shape of the intact IgG that differs from the more typical Y configuration seen for other IgGs. Electron microscope (EM) studies have also clearly shown this unusual linear structure in the intact 2G12 IgG (Roux et al. 2004), and analytical ultracentrifugation also confirmed that the Fab is an obligate dimer in solution (Calarese et al. 2003). Crystal structures have been determined for the dimeric 2G12 Fab in complex with carbohydrates (Calarese et al. 2003, 2005) ranging in size from Mana1,2Man to Man9GlcNac2, and these structures indicate that the Fab can bind to the terminal Mana1-2Man in either the D1 or D3 arms of Man9GlcNac2. Mutagenesis and modeling studies of 2G12 have pointed to a conserved cluster of high mannose moieties, especially those linked to gp120 residues 332, 339, and 392, as being the likely epitope for this antibody (Sanders et al. 2002; Scanlan et al. 2002; Calarese et al. 2003). In addition, glycan array and solution-phase ELISA analyses have helped to define the carbohydrate specificity of 2G12 (Blixt et al. 2004; Bryan et al. 2004; Calarese et al. 2005). Modeling studies show that the distances between these groups on gp120 carbohydrates are compatible with the distance between the two antigen-binding sites on the dimeric Fab (Calarese et al. 2003) and that a third, novel antigen-binding site may exist at the VH-VH0 interface. The inherent high avidity (nM) of the dimeric 2G12 has thus given the antibody high affinity for carbohydrate on the virus due to the multivalency. Analysis of the 2G12 sequence uncovers several unusual residues that may favor the domain swap event. One rare residue is ProH113, located in the linker between VL and VH. Other unusual residues include the hydrophobic IleH19 and PheH77 that may help to stabilize the novel VH-VH0 interface, Figure 2.8 The broadly neutralizing, anti-HIV-1 Fab 2G12. 2G12 forms an unusual domain swapped dimer, with the VH domains from each heavy chain (labeled VH-VH0 ) swapping to the other Fab, resulting in a tightly linked dimer of Fabs. Bound Man9GlcNac2 is shown in a CPK representation. (See color insert.) 60 ANTIBODY MOLECULAR STRUCTURE whereas an Arg at position H39 disrupts a highly conserved GlnL38-GlnH39 interaction that is found in all antibodies, as well as in T cell receptors. Two other unusual anti-HIV-1 antibodies that recognize neighboring epitopes are 2F5 and 4E10. Both of these antibodies recognize epitopes in the membrane proximal epitope region (MPER) of gp41. The MPER region, as its name implies, is proximal to the membrane spanning region of gp41, and is thought to be only transiently exposed during the viral-cell fusion process. The 2F5 and 4E10 Fab structures include complexes with gp41 peptides (Ofek et al. 2004; Cardoso et al. 2005; Cardoso et al. 2007), with the 2F5 peptide forming a b-turn, and the 4E10 peptide forming an a-helix. Estimations of the distances of these gp41 epitopes to the membrane spanning regions of gp41 indicate that the Fabs must come very close to or even contact the host cell membrane in order to bind these epitopes. Interestingly, the Fab CDR residues surrounding the peptide-binding site include a large number of hydrophobic residues, including a number of Trp and Gly residues in 4E10 that may facilitate interaction with the membrane. An interesting class of anti-HIV-1 antibodies has been found that only bind to gp120 after it binds to its primary receptor, CD4. Thus, these antibodies are called CD4-induced, or CD4i. The binding of CD4 to gp120 induces conformational changes in the gp120 that are necessary for binding to the co-receptor (usually CCR5 or CXCR4) and, hence, required for viral entry into the cell. The CCR5 co-receptor has sulfated tyrosine residues in its N terminus, and this N-terminal region is proposed to interact with gp120 during the binding process. Surprisingly, some of the CD4i antibodies were also found to have acquired sulfated tyrosine residues in their highly acidic CDR H3 loops through posttranslational modification, suggesting that they may be structural mimics of the co-receptor (Choe et al. 2003; Huang et al. 2004, 2007). A recent structure of gp120 in complex with CD4 and sulfated antibody 412D (Fig. 2.9) shows how these two sulfated tyrosine residues in the CDR Figure 2.9 The anti-HIV-1 Fab 412D has sulfated tyrosine residues in CDR H3. Left: The complex of Fab 412d with CD4 and gp120. Right: enlargement of the sulfated tyrosine residues and their interaction with gp120. It is thought that the gp120 co-receptor (CCR5) binds to the bridging sheet and V3 regions of the gp120 molecule. CCR5 has four sulfated tyrosine residues in its N-terminal region, and at least two of these are thought to take part in the interaction with gp120. Several antibodies that recognize the same region of gp120 have also evolved to have sulfated tyrosine residues. (See color insert.) 2.6 SHARK AND CAMEL ANTIBODIES 61 Figure 2.10 Broadly neutralizing anti-HIV-1 antibody b12 interactions with gp120. Left: The CD4 (tubes) binding site on gp120 (solid surface) is shown. Right: b12 binds to the CD4-binding site, accessing a deep cleft with its long CDR loop (H2) and clasping the CD4-binding loop between CDRs H2 and H1 on one side and H3 on the other side. (See color insert.) H3 region interact with the gp120 bridging sheet/V3 regions, and mimic the interaction with CCR5 (Choe et al. 2003; Huang et al. 2004, 2007). Antibody b12 was discovered by phage-display technology using a library developed from blood marrow taken from an HIV-1 infected, long-term nonprogressing patient. Crystal structures of the intact b12 IgG (Saphire et al. 2001) and the recently determined structure of b12 Fab in complex with gp120 (Zhou et al. 2007) revealed that the Fab recognizes the highly conserved, but deeply recessed CD4-binding site (Fig. 2.10). The Fab CDR H2 loop accesses the CD4-binding site, with TyrH53 binding in the same pocket as Phe43 of CD4. The long CDR H3 unexpectedly was found to bind on the outside of the binding site, so that the CD4-binding loop is sandwiched between the H3 CDR on one side, and H1 and H2 on the other side. Interestingly, the antibody light chain makes no contact with the gp120 monomer, but may possibly contact part of the intact gp120 trimer on the viral surface. Thus, this antibody has managed to find a site of vulnerability on the virus, its Achilles’ heel, and interacts primarily with the structurally conserved outer domain that is the primary site of attachment for CD4. But unlike CD4, it can remain bound to the outer domain by itself with high affinity, whereas CD4 reorganizes the highly flexible inner domain of gp120 to decrease its off-rate and assemble the bridging sheet that constitutes a major portion of the CCR5 co-receptor binding site (Zhou et al. 2007). 2.6 SHARK AND CAMEL ANTIBODIES Crystal structures have also been determined for antibodies from rats, hamsters, camels, and sharks. While the rodent and human antibodies are very similar, camels and sharks both have, in addition to a conventional antibody repertoire, unusual antibodies that exist as dimers of heavy chains with 62 ANTIBODY MOLECULAR STRUCTURE no associated light chains. In the case of the camel (and llama), these antibodies are the result of a gene deletion of the IgG CH1 domain, resulting in a heavy chain with VH, CH2, and CH3. Light chains do not associate with the CH1-free heavy chain; however, the free heavy chains still pair up through their two associated constant domains. The camel heavy chain antibodies are closely related in sequence to their normal IgG antibodies. Sharks and other cartilaginous fish, such as skates and rays, also have heavy chain antibodies, called new antigen receptors or IgNAR, that contain two heavy chains and no light chains. However, unlike the camel heavy chain dimers, the IgNAR antibodies are not closely related by sequence to the other, more typical antibodies found in the shark (IgM and IgX). The five constant domains on each heavy chain dimerize to form a long stalk, leaving the two variable domains free to bind antigen independently. Although the shark and camel VH domains have very low sequence homology, they have co-evolved many unique structural features, including mutations in the interface that would normally be involved in the VL-VH dimerization, to make their VH domains more soluble. Both camel and shark VH domains also have long CDR3 regions that usually contain noncanonical disulfides to tether their long CDR H3s to the body of the VH domain. While the Figure 2.11 Shark IgNAR single domain antibodies bound to lysozyme. Left: a Type I IgNar (bottom) is shown bound to lysozyme (top). The Type I IgNAR is characterized by a long CDR3 region stabilized by two noncanonical disulfide bonds tethering the CDR to the IgNAr framework. Other regions that have a large number of somatic mutations and may interact with antigen are HV2 and HV4. Right: a Type II IgNAR is characterized by a long CDR3 region with one noncanonical disulfide that tethers it to the CDR1 loop. (See color insert.) REFERENCES 63 camel VH domain has the three CDR regions found in a typical VH domain (Desmyter et al. 1996; Spinelli et al. 1996), the shark IgNAR domains have only CDR1 and 3, and are missing the C0 and C00 strands that make up a typical CDR2 (Stanfield et al. 2004) (Fig. 2.11). Despite having only three (camel) or two (shark) CDR loops, these small, independent VH domains are able to bind antigen with very high affinity. The production of camel and shark VH domains has been adapted to phage-display technology (Arbabi Ghahroudi et al. 1997; Dooley, Flajnik, and Porter 2003) and the resulting molecules show great promise as small antigen-binding fragments for many different uses. Several studies have shown that the shark and camel single-domain antibodies are able to access deeply recessed clefts better than their Fab counterparts (Stanfield et al. 2007). In addition, the solubilityenhancing mutations in these single-domain fragments have been adapted into engineered, human single-domain VH fragments to enhance their solubility (Davies and Riechmann 1996). 2.7 SUMMARY An enormous wealth of structural information is available for antibodies and their fragments, making the antibody molecule one of the most highly studied in the entire protein universe. This information has proven to be invaluable for scientists working towards the development and production of antibodies and antibody fragments for human therapeutic products. Nevertheless, although we know a great deal about antibody structure, recent structures of several human antibodies have revealed surprisingly novel and diverse structural features, such as Fab domain swapping, long but relatively rigid CDR H3s, and posttranslational modifications, that were totally unexpected from the study of an enormous number of mouse antibodies. Given the tremendous diversity of the antibody immune response, we expect to continue to uncover further novel aspects of antibody-antigen recognition that will provide new ideas and inspiration for the development of antibody-based therapeutics. ACKNOWLEDGMENTS This is manuscript number MB-19299 from the Scripps Research Institute. 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