BBAPAP-39367; No. of pages: 8; 4C: 2, 3 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

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Principles and engineering of antibody folding and assembly☆

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Matthias J. Feige a,⁎, Johannes Buchner b,⁎

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Article history: Received 9 April 2014 Received in revised form 4 June 2014 Accepted 6 June 2014 Available online xxxx

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Keywords: Antibody folding Antibody assembly Antibody engineering

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Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis 38105, TN, USA CIPSM at the Department of Chemistry, Technische Universität München, 85748 Garching, Germany

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Antibodies are uniquely suited to serve essential roles in the human immune defense as they combine several specific functions in one hetero-oligomeric protein. Their constant regions activate effector functions and their variable domains provide a stable framework that allows incorporation of highly diverse loop sequences. The combination of non-germline DNA recombination and mutation together with heavy and light chain assembly allows developing variable regions that specifically recognize essentially any antigen they may encounter. However, it also requires tailor-made mechanisms to guarantee that folding and association of antibodies is carefully controlled before the protein is secreted from a plasma cell. Accordingly, the generic immunoglobulin fold β-barrel structure of antibody domains has been fine-tuned during evolution to fit the different requirements. Work over the past decades has identified important aspects of the folding and assembly of antibody domains and chains revealing domain specific variations of a general scheme. The most striking is the folding of an intrinsically disordered antibody domain in the context of its partner domain as the basis for antibody assembly and its control on the molecular level in the cell. These insights have not only allowed a better understanding of the antibody folding process but also provide a wealth of opportunities for rational optimization of antibody molecules. In this review, we summarize current concepts of antibody folding and assembly and discuss how they can be utilized to engineer antibodies with improved performance for different applications. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Published by Elsevier B.V.

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1. Introduction

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The human immune defense depends on reliable and efficient discrimination of self from non-self. In all higher vertebrates, antibodies play a major role in this task and protect the organism against threats ranging from toxins to microbial pathogens [1]. Antibodies are complex glycoproteins that are secreted in large quantities by plasma cells [2,3]. They are made up of heavy chains (HCs) and light chains (LCs) with the simplest human antibodies being hetero-tetramers that comprise two HCs and two LCs each (Fig. 1). Five antibody classes exist in humans (IgA, G, D, E and M) that differ in their HCs (αHC, γHC, δHC, εHC and μHC) comprising either four (αHC, γHC, δHC) or five (εHC, μHC) immunoglobulin (Ig) domains. Only two classes of light chains (κLC and λLC) are shared between the different human antibody classes. Even though the various antibody classes differ in their biological functions and structural details, they have two important features in common that enable them to defend the organism against most infectious or toxic

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☆ This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. ⁎ Corresponding authors. E-mail addresses: [email protected] (M.J. Feige), [email protected] (J. Buchner).

challenges it may encounter: variable regions that bind to antigens and constant regions that link the antibody and its bound antigen to the cellular immune defense (Fig. 1). The constant region of antibodies incorporates multiple biological functions. It binds to Fc receptors inducing antibody-dependent cellmediated cytotoxicity (ADCC) and activates the complement system [4]. The constant region furthermore has an important impact on the biological half-life of antibodies due to its binding to the neonatal Fc receptor that induces antibody recycling after cellular uptake into endosomes [5,6]. Variable regions of one HC and one LC each form a composite antigen binding surface (paratope) (Fig. 1). Antigen binding sites of the variable regions are generated by non-germline genetic rearrangements, non-templated base pair additions and hypermutation in a microevolutionary process [7,8] that gives rise to proteins that can bind essentially any target structure with high affinity and specificity. An array of techniques has been developed, from ribosome, phage and yeast display to genetically engineered mice with a human antibody repertoire to obtain antibodies with the desired specificities [9–11]. Recently, this possible repertoire has even been further extended by generating bispecific antibodies [6,12–14]. Together, the highly specific binding of antibodies and their link to the cellular immune response have rendered them the most widespread

http://dx.doi.org/10.1016/j.bbapap.2014.06.004 1570-9639/© 2014 Published by Elsevier B.V.

Please cite this article as: M.J. Feige, J. Buchner, Principles and engineering of antibody folding and assembly, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.06.004

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Fig. 1. Schematic and structural elements of the IgG1 antibody molecule and the Ig fold. Left panel: The IgG1 antibody consists of two heavy chains (blue) and two light chains (green) that together form two identical antigen binding sites (paratopes), which are part of the Fab fragment (fragment antigen binding). The smallest complete dimeric antigen binding entity is the Fv fragment (fragment variable). Effector functions are mediated via the Fc fragment (fragment crystallizable). The CH2 domains are glycosylated (gray hexagons) and disulfide bridges (S\S) connect the two heavy chains as well as the heavy and light chains. Middle panel: The Fab fragment is connected by an interchain disulfide bond and each of the domains has one buried disulfide bond (yellow, CPK representation). Proline residues within the Fab fragment are highlighted in red. Trans prolines are shown in a stick representation, and cis prolines are shown in a CPK representation. The small helices in the CL domain, which are found in all most antibody constant domains except for e.g. CH1, are colored in orange. Right panel: As shown for the CL domain, the constant domain Ig fold consists of seven strands (a–f), two of which (b and f) are connected by the buried disulfide bond. Prolines and small helices are depicted in the same manner as in the middle panel.

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2. Engineering antibodies: stability and folding pathways

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Each of the antibody domains shows the Ig fold, the most widespread protein topology in secretory pathway proteins, with variable domains belonging to the V-set and constant domains to the C-set [23,24]. Ig domains show a Greek key β-barrel topology generally composed of either seven (C-type) or nine (V-type) β-strands [23,24] (Fig. 1). A characteristic of this topology is that β-strands that are adjacent in space are not necessarily adjacent in sequence (Fig. 1). Most of the published studies on antibody optimization have focused on the antigen binding variable regions due to their immediate role in target recognition and the possibility to design smaller binding molecules without constant domains [20,25]. The opposite, however, applies when it comes to studies that have assessed Ig folding pathways in detail. These mostly focused on antibody constant domains or other members of the Ig superfamily [26–29]. Mutational approaches combined with kinetic analyses of a protein folding process allow the structural assessment of protein folding transition states (Phi value analysis) [30–33]. This approach has revealed that Ig domains initiate their folding by a nucleus composed of Ig strands b, c, e and f around which the Ig fold forms [34–36] (Fig. 1). However, antibody domains comprise several structural features that significantly influence this general mechanism with direct implications for protein engineering.

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2.1. Disulfide bonds in antibody folding

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A defining characteristic of most antibody domains is an intramolecular buried disulfide bridge perpendicular to the two sheets of the Ig fold [37] (Fig. 1). Early on, it has been recognized that this disulfide bond plays a crucial role in the stability of antibody domains in vitro [38–40] and complete antibodies in vivo [41]. Besides, the oligomeric structure of antibodies is stabilized by disulfide bonds connecting LCs and HCs as well as inter-HC disulfide bonds [42,43] (Fig. 1). In the case of the constant domain of antibody LCs (CL domain, Fig. 1), the internal disulfide bridge accelerates folding and inhibits off-pathway misfolding reactions [44,45]. Of note, in antibody constant domains the internal disulfide bridge connects strands b and f (Fig. 1), which are part of the common Ig topology folding nucleus [34–36]. For the variable domains, the intramolecular disulfide bond, also connecting strands b and f, similarly reduces their tendency to aggregate [46]. Together this argues for an intimate link between folding of antibody domains and formation of their disulfide bond which was also found in vivo [47] where disulfide bonds in antibody domains and between antibody chains already form co-translationally [41,42]. Disulfide bond formation is aided by protein disulfide isomerases (PDIs) in vivo [48,49] and in vitro [50] (Fig. 2). The time window for the action of PDI can be extended in the presence of the Hsp70 chaperone BiP [51], a major ER-resident protein involved in the folding, assembly and quality control of antibodies in the cell [52,53] (Fig. 2), by inhibiting premature collapse of the polypeptide chains. Engineering additional disulfide bonds into the Ig fold or between Ig domains is one of the most widespread approaches to stabilize antibody domains. A significant stabilization can be achieved this way [54–58] as described in several recent reviews [59–62]. A particularly well studied example in this regard is Fv fragments, the most simple dimeric binding module derived from antibodies, which comprise only one VL and one VH domain (Fig. 1). Nowadays, Fv fragments are mainly used as single chain Fv fragments (scFv) where the C-terminus of the VL and the N-terminus of the VH domain are connected by a peptide linker [63,64]. Disulfide bonds have been shown to be particularly useful to stabilize scFv fragments either by engineering the antigen binding site [65], or by, more generally applicable, the framework regions to stabilize VL–VH heterodimers [66,67]. Indeed, dissociation of the variable domains is often an initial step preceding aggregation of scFv fragments under challenging conditions and thus disulfide bond engineering can

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diagnostic and therapeutic biomolecules that can be used for a variety of applications from detecting and marking tumors in vivo, via treating autoimmune diseases to combating cancer cells [6]. Recent developments have given rise to antibodies that are able to pass the blood brain barrier, opening up a whole new field of diseases that could potentially be targeted with tailored antibodies [15,16]. Thus, a vital interest exists in optimizing antibody production, stability and half-life in the organism as well as in inhibiting side reactions like aggregation, which can cause serious side effects including immunogenicity [17–20]. As strategies to engineer specificity have been extensively reviewed elsewhere [9–11, 21,22], in the following, we will focus on recent insights into the determinants of in vitro and in vivo antibody folding and assembly pathways, with an emphasis on how these can be used to design better antibody therapeutics.

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Please cite this article as: M.J. Feige, J. Buchner, Principles and engineering of antibody folding and assembly, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.06.004

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Please cite this article as: M.J. Feige, J. Buchner, Principles and engineering of antibody folding and assembly, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.06.004

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Fig. 2. Critical steps in antibody folding, secretion and in vivo stability. IgG antibodies are assembled from two heavy and two light chains in the endoplasmic reticulum before secretion via the Golgi occurs. The molecular chaperone BiP stably binds to the permanently unfolded CH1 domain until displaced by the LC [53,80,103,104] and also transiently to other antibody domains [166,167]. Critical molecular events occurring in each of the steps are highlighted in the boxes. These represent prime targets for antibody engineering and optimization.

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2.3. Small helices in antibody domain folding

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Generally, the Ig fold is an all-β structure where flexible loops connect the β-strands that make up the topology. For antibodies, however, by far most constant domains possess two small helices that connect

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3. Engineering antibodies: cellular quality control

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Like essentially all secreted proteins, antibodies are produced in the endoplasmic reticulum (ER), an organelle dedicated to protein folding [93,94]. Quality control measures exist in the ER to ensure that only properly folded and assembled proteins leave the ER whereas incompletely folded proteins are retained and ultimately transported back to the cytosol to be degraded by the proteasome [95]. Quality control measures are of particular relevance for antibodies due to their intrinsic variability, which might affect folding and their potent effector functions once secreted. Thus, the cell must ensure that antibodies are folded and assembled correctly prior to secretion. Indeed, only completely assembled antibodies leave the ER. Most insights into this process have been obtained for IgM and IgG. In the case of IgM, an unpaired cysteine in the C-terminal tailpiece of the μHC, which forms a disulfide bond upon IgM oligomerization, plays a key role in retaining μHCs in the cell [96]. ERp44, a member of the ER-localized thioredoxin domain containing proteins, plays a major role in this process by engaging proteins that contain free cysteines [97,98]. If these proteins leave the ER prematurely, ERp44 re-shuttles them back to the ER. This is mediated by exposure of the ERp44 ER retention sequence in the more acidic post-ER compartments [97–101]. Another major quality control step for antibodies has been discovered for IgG. The first of the γHC constant domains, CH1, is permanently unfolded and thus remains stably bound to a complex of ER chaperones, the most prominent of it being BiP (heavy chain binding protein) [48,52,53,80,102] (Fig. 2). Light chains are able to displace BiP and induce folding of the CH1 domain [80,103, 104]. This reaction is limited by prolyl-isomerization, catalyzed by the PPIase CypB and rendered irreversible by the formation of the HC-LC interchain disulfide bond [80,103,104] (Fig. 2). Thus, LC-induced folding of the CH1 domain is a critical step in antibody quality control that sets apart unassembled HCs from assembled ones and might be amenable to optimization, by either improving the initial HC-LC assembly step, the assembly-dependent folding reaction itself or by manipulating the levels or composition of the chaperone machinery involved in this process [48,105,106] (Fig. 2). Alternatively, recapitulating a similar environment as in the ER in simpler organisms is an actively pursued direction [107] even though achieving authentic antibody glycosylation, important for many biological antibody functions [108,109], presents a major obstacle. It is noteworthy that although the details of antibody assembly pathways vary for different antibody classes, i.e. formation of the HC-dimer might precede assembly with LCs or, alternatively, HCLC heterodimers form first [110,111], LC-induced folding of the CH1 domain seems to be a generic feature and thus a particularly attractive target for optimization. A variation of this theme is found in pre-B cells, which express membrane-bound HCs but LC rearrangement has not yet taken place. These cells express the so-called surrogate light chain (SLC), a protein similar to LCs but made up of two non-covalently linked polypeptides, VpreB and λ5, that interact via a β-strand exchange mechanism [112–117]. HCs and SLC pair to form the pre-B cell receptor (pre-BCR) [112–116]. Even though detailed insights are still missing, the split nature of the SLC seems to be uniquely suited to test on the one hand for correct VH rearrangement and on the other hand for CH1

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Among the 20 natural amino acids, proline is the only one for which a trans conformation of the peptide bond preceding the proline residue is only slightly more energetically favorable than the generally disfavored cis conformation [72] (Fig. 2). Thus, in the absence of structural constraints, e.g. in the unfolded state of a protein, prolines undergo cis/ trans isomerization. In the native state, however, generally only one isomer is compatible with the native structure often giving rise to slow folding reactions caused by peptidyl–prolyl isomerization reactions [73–75]. Antibody domains are rich in prolines. These make up ca. 5–10% of the primary sequence and are generally found in turns connecting the β-strands (Fig. 1). Thus, very often antibody domain folding is complicated by peptidyl–prolyl isomerization [28,76]. On the other hand, proline residues allow the formation of tight turns connecting the strands of Ig domains, which might be important in increasing protease resistance in the extracellular space, and prolines have been suggested to reduce the aggregation tendency of Ig domains [77], maybe due to their low propensity to form β-strands. Peptidyl– prolyl isomerization reactions are slow, taking seconds to minutes to complete at room temperature [75]. Thus, the cell has developed a class of enzymes, the peptidyl–prolyl isomerases (PPIases) to accelerate these reactions [78,79] (Fig. 2). For antibodies, it was shown that among those PPIases, the ER-localized PPIase cyclophilin B (CypB) plays an important role [48,80]. Its inhibition leads to reduced IgG secretion [81] and a general deficiency in protein quality control of the cell [82]. In terms of antibody engineering, the role of proline residues has not been assessed in detail yet. Cis-proline residues can be generally expected to be essential for the formation of the native antibody domain structure and a conserved cis-proline residue is found between strands b and c of most antibody constant domains [37,83]. In fact, mutation of this residue to an alanine was used to trap a partially folded antibody state [84]. Exceptions exist, however, and of note, the crystal structure of the Ig domains from shark IgNAR (immunoglobulin new antigen receptor) antibodies has revealed the absence of cis-proline residues in this otherwise conserved position between strands b and c for two particularly stable IgNAR domains [83]. Thus, the replacement of these prolines and their immediate surrounding amino acids by optimized loops might be a viable strategy to increase domain stability and simplify the folding reaction to optimize protein behavior [85]. Selectively replacing transproline residues might be an alternative option for optimization of antibody domain folding pathways as cis to trans isomerization can have a significant effect on antibody domain folding [86]. Additionally, optimizing residues in sequence proximity to proline residues might be feasible as these residues influence the cis/trans equilibrium of the peptidyl\prolyl bond and the catalysis by PPIases [87–89]. This might be used to favor either a trans or a cis peptidyl\prolyl bond, dependent on the native state of the respective residue under scrutiny, or improve catalysis of the isomerization reaction in vivo.

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strands a and b and e and f, respectively (Fig. 1). These helices are highly conserved in evolution [37,83]. Recently, they have been shown to form early on in the folding of the CL domain and reduce the tendency of this domain to misfold [84]. The second helix often contains a hydrophobic or aromatic amino acid that is properly positioned to participate in the hydrophobic core of the antibody domain [83,84]. Since the formation of α-helices is relatively well understood [90,91] optimization of these small helices in antibody domain folding might be an attractive way of increasing the folding efficiency and decreasing the misfolding tendency of antibody domains (Fig. 2) or Ig domains in general. This has already been shown to be feasible for β2-microglobulin [84], a member of the Ig superfamily with a tendency to form amyloids [29,92].

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be complemented by framework engineering [68]. Insights into the structural details of antibody domain folding/unfolding pathways can help to identify particularly well-suited positions for introducing disulfide bonds and thus may even further increase the positive impact of engineered disulfide bonds [69–71]. In particular, results from the experimental analysis of the native state dynamics of antibody domains and their unfolding pathways might be useful in this context. This is of particular interest since often aggregation following unfolding renders the unfolding process irreversible, constituting a major problem in therapy [20] (Fig. 2).

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The seminal work by Goto and Hamaguchi determined crucial elements in the structure formation of antibody domains including the identification of proline cis/trans isomerization as the basis of the slow folding phase of antibodies and the role of the buried disulfide bridge in folding and stability of antibody domains [39,40,44,76]. These experiments were possible before the advent of recombinant protein expression as some patients secreted large amounts of light chains, the so-called Bence–Jones proteins. Also, further diseases exist that involve antibody deposits, such as the light chain deposition disease (LCDD). LCDD is a severe illness during which large amounts of antibody fibrils accumulate in different organs [158,159]. Some point mutations in the VL domain seem to predispose it for fibril formation [160]. Thus, antibodies, among the most important components in the defense mechanisms of our immune system, can have detrimental effects when things go awry. Work in recent years revealed the built in mechanisms to combat these processes and control the quality of each antibody molecule [28]. Antibodies are also special because they represent a rare case where insights into folding and assembly have been gained both from in vitro and in vivo studies. Antibodies were an early model system to demonstrate co-translational folding and disulfide bond formation [41,42] and our understanding of the chaperone assistance of structure formation, oxidation and assembly of proteins in the ER has significantly been shaped by studies on the biogenesis of antibodies which provided many insights into the members, organization and functions of the ER chaperone machinery [48,52,53,98,103,104,161–163]. Engineering of antibodies is important both for basic science aiming at understanding the foundations of protein structure, especially of the

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Simpler and more stable antibodies are of particular interest for diagnostics and therapy. In a case of convergent evolution [121], nature has developed simpler than human antibodies at least twice. Camelids (e.g. camels and llamas) and cartilaginous fish (e.g. sharks, skates and rays) each possess antibodies naturally devoid of light chains, which are HC-only homodimers named HC-only antibodies (HCAbs) in camelids and IgNAR in sharks [122,123]. Normally, HC-LC assembly is controlled via assembly of the LC CL domain with the otherwise unfolded HC CH1 domain [53,80,103,104]. Accordingly, HCAbs in camelids and cartilaginous fish both are devoid of a CH1 domain thus allowing secretion of HC dimers. In camelids, the CH1 domain is still present in the genomic HCAb sequence yet absent in the HCAb transcript due to a mutation in a necessary splice site [124]. In cartilaginous fish the origin of a HC devoid of a CH1 domain is less clear [121]. That nature has developed HCAbs multiple times argues for distinct evolutionary advantages of HCAbs. Some of those are of particular interest for antibody engineering. HCAbs can be readily raised against many different antigens and due to the antigen-binding function encoded in a single domain, the underlying nucleotide sequences are relatively easy to obtain [125–127]. Their small size qualifies the variable domains of HCAbs as very attractive minimal antigen binding units by themselves, called nanobodies [125], some of which have been shown to be possible effective therapeutics tolerated by the immune system [128,129]. Of particular relevance in terms of antibody folding and assembly, the variable domains of HCAbs have undergone a long evolution towards being stable entities that function as monomers. Indeed, variable domains from HCAbs derived from both camelids and cartilaginous fish have more hydrophilic residues in parts of their framework region that would normally interact with the variable domain of the LC [130–135]. However, whereas the camelid monomeric variable domain VHH (variable domain of the heavy chain of HCAbs) has clearly developed from a conventional heavy chain variable domain [122,130,131], the shark IgNAR variable domain VNAR shows characteristics of both constant and variable antibody domains as well as T cell receptor variable domains and it might have evolved from a cell surface receptor [132,133]. Structural insights into naturally occurring monomeric variable domains have been successfully used to guide the design of monomeric human VH domains [125,136–138]. A particular attractive feature of camelid VHH domains is their ability to often refold after thermal denaturation, conditions where other mammalian dimeric variable domains tend to aggregate [139–141], even though exceptions to this general rule could be established [142–144]. This beneficial behavior qualifies VHH domains for tasks where classical variable domains would be too unstable, e.g. at elevated temperatures, or to use them as templates to optimize the biophysical properties of human VH domains by comprehensively assessing the impact of different mutations rather than by simply copying structural elements from VHH domains [145]. As an example, whereas in VHH and VNAR domains one of the antigen binding loops, the CDR3 loop (complementarity determining region 3), often contributes to shielding hydrophobic remnants of the former dimerization interface [130–133], this structural feature is not necessary to generate stable variable domains [145], and thus is not a restricting factor for the binding repertoire of monomeric variable domains. Alternatively, particularly stable human domains might act as a template for optimization, even though their CDRs seem to play an important role in stability thus maybe restricting the available repertoires of stable domains [146,147]. Besides their beneficial biophysical properties, their small size and their easy production in simple organisms [125,148], single chain variable domains from camelids and cartilaginous fish also have functional characteristics, which set them apart from dimeric variable domains

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and might explain why they have evolved [121]: Their extended CDR3 loop is often stabilized by aromatic residues and in particular additional disulfide bridges, which confer stability and increase antigen binding affinity [131–133,135,149,150]. It can thus extend away from the variable domain framework to allow recognition of buried, cryptic antigens that are not easily accessible to conventional dimeric antibodies [131,132, 151]. These cryptic antigens also include enzyme active sites and pathogen coat proteins [149], which cannot easily be mutated by a pathogen to escape immune detection. That indeed recognition of cryptic antigens is an evolutionary advantage is further underlined by the recent structural insights into bovine antibodies that can encompass extremely long CDR3 regions which form knob like structures stabilized by disulfide bonds and are present on a long stalk extruding from the VH region [152]. Extended and stabilized CDR regions, which have been developed multiple times in nature in different formats [130–133,152,153], might thus be interesting engineering options for hard to target antigens. Apart from providing insights into how simpler, monomeric variable regions can be obtained, shark IgNAR antibodies might also hold a clue for how to further stabilize human antibodies for therapy and diagnostics. Sharks enrich urea in their blood to inhibit the osmotic loss of water in a marine environment [154]. Urea is a well-known protein denaturant. Accordingly, shark antibodies are believed to be particularly stable [155,156], even though sharks also counteract the effect of urea by small osmolytes like Trimethylamine-N-oxid [157]. In a recent study it could indeed be shown that some constant domains derived from IgNAR are particularly stable against thermal and chemical denaturation and the crystal structures of those IgNAR constant domains revealed structural elements that contribute to their high stability: a salt bridge between the second of the small antibody helices and the loop connecting strands c and d as well as a slightly extended hydrophobic core [83]. Transferring these elements to a human CL domain stabilized this protein and led to increased antibody secretion from mammalian cells [83], likely since a better-behaved CL domain provides an optimized template for the CH1 domain to fold on in antibody assembly control [48,52,53,80,102].

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folding as prerequisites for further B cell development mediated by preBCR signaling [118–120].

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Funding of MJF by the Leopoldina — National Academy of Sciences, grant number LPDS 2009-32 and of JB by the Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged. We thank Julia Behnke for the helpful comments on the manuscript.

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immunoglobulin fold, but also in the context of diseases based on antibodies and in particular for facilitating the use of therapeutic antibodies. Efforts in this regard are rather recent and it is envisioned that important discoveries are still ahead of us. Of particular relevance will be a more detailed understanding of antibody dissociation and unfolding pathways as antibody aggregation poses a major problem in therapy [20,164]. Methodological advancements to understand aggregation [164] and antibody optimization to resist aggregation [20,165] are recent developments of direct relevance to antibody therapy. These, however, will have to be complemented by kinetic and mechanistic insight into antibody unfolding pathways to allow rational design of aggregation-resistant antibody molecules. A deeper understanding of the rooting of the antibody fold in evolution contributed to the identification of structural features that influence basic aspects such as stability. The large database of antibody sequences available today (abys data base) is a further asset to identify points of intervention. Based on the increasing insight into the fundamentals of antibody structure and folding, progress in the areas described above is envisioned for the near future with profound implications for antibodies as agents to prevent and treat disease that can make use of the potent immune defense mechanisms already present in humans.

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Please cite this article as: M.J. Feige, J. Buchner, Principles and engineering of antibody folding and assembly, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.06.004

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