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Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

Open AccessPublished:April 30, 2013DOI:https://doi.org/10.1074/jbc.M112.435289
      The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.
      Background: Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates.
      Results: A V-NAR specific for human serum albumin was humanized, and its crystal structure in complex with the antigen was solved, revealing an unusual recognition mode.
      Conclusion: Humanization preserved antigen binding properties and activity of the parental shark antibody.
      Significance: A structural framework for humanization of shark antibodies was established.

      Introduction

      Antibody-based targeting has become an established paradigm of biologic drug development. High affinity, excellent specificity, generally good stability, and Fc-associated effector functions all make antibodies the molecules of choice for many diagnostic and therapeutic applications. At the same time, novel non-antibody scaffolds are constantly being sought by industry to allow for development of new therapeutic agents offering advantages over classical antibody platforms (
      • Binz H.K.
      • Amstutz P.
      • Plückthun A.
      Engineering novel binding proteins from nonimmunoglobulin domains.
      ,
      • Skerra A.
      Alternative non-antibody scaffolds for molecular recognition.
      ). In particular, smaller size for better tissue penetration, reduced complexity for easier production, and enhanced biological and biophysical stability are some of the properties desired for the new generation of biologics.
      Multiple formats and optimization strategies that try to incorporate these properties have been described. Some of the resulting molecules, such as scFv,
      The abbreviations used are: scFv, single chain Fv; IgNAR, immunoglobulin new antigen receptor; HSA, human serum albumin; HEL, hen egg white lysozyme; CDR, complementarity-determining region; FW, framework; HV, hypervariable region; V, variable region; VH, variable heavy chain; VL, variable light chain; VHH, variable domain of the H chain of heavy chain antibodies; hFc, human IgG1 Fc; CM, conditioned medium.
      DVD-IgTM, diabody, scFv-Fc, and others, represent novel designs or effector function variants based on traditional antibody scaffolds (
      • Carter P.J.
      Potent antibody therapeutics by design.
      ,
      • Presta L.G.
      Molecular engineering and design of therapeutic antibodies.
      ). In addition, naturally occurring single variable domain antibodies from cartilaginous fish (IgNARs) and camelids (VHH antibodies; also known as nanobodies) provide an attractive alternative (
      • Barelle C.
      • Gill D.S.
      • Charlton K.
      Shark novel antigen receptors—the next generation of biologic therapeutics?.
      ,
      • Wesolowski J.
      • Alzogaray V.
      • Reyelt J.
      • Unger M.
      • Juarez K.
      • Urrutia M.
      • Cauerhff A.
      • Danquah W.
      • Rissiek B.
      • Scheuplein F.
      • Schwarz N.
      • Adriouch S.
      • Boyer O.
      • Seman M.
      • Licea A.
      • Serreze D.V.
      • Goldbaum F.A.
      • Haag F.
      • Koch-Nolte F.
      Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.
      ). The variable domains of these antibodies can be linked in tandem to provide multispecificity and increase the size and thus the in vivo half-life of the molecules. They can also be linked to Fc domains of traditional antibodies to provide them with desired effector functions.
      IgNARs were discovered in sharks in the 1990s (
      • Greenberg A.S.
      • Avila D.
      • Hughes M.
      • Hughes A.
      • McKinney E.C.
      • Flajnik M.F.
      A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks.
      ,
      • Roux K.H.
      • Greenberg A.S.
      • Greene L.
      • Strelets L.
      • Avila D.
      • McKinney E.C.
      • Flajnik M.F.
      Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.
      ). Their variable regions (V-NARs) are small (12–13-kDa), independently folding domains that demonstrate high biophysical stability, solubility, and ability to bind to a variety of antigens including epitopes located in clefts on protein surfaces (e.g. enzyme active sites) that are non-accessible by traditional antibody variable domains (
      • Henderson K.A.
      • Streltsov V.A.
      • Coley A.M.
      • Dolezal O.
      • Hudson P.J.
      • Batchelor A.H.
      • Gupta A.
      • Bai T.
      • Murphy V.J.
      • Anders R.F.
      • Foley M.
      • Nuttall S.D.
      Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition.
      ,
      • Stanfield R.L.
      • Dooley H.
      • Verdino P.
      • Flajnik M.F.
      • Wilson I.A.
      Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.
      ). A similar preference for cleft recognition was demonstrated for camelid VHH antibodies (
      • Transue T.R.
      • De Genst E.
      • Ghahroudi M.A.
      • Wyns L.
      • Muyldermans S.
      Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate.
      • De Genst E.
      • Silence K.
      • Decanniere K.
      • Conrath K.
      • Loris R.
      • Kinne J.
      • Muyldermans S.
      • Wyns L.
      Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies.
      ,
      • Lauwereys M.
      • Arbabi Ghahroudi M.
      • Desmyter A.
      • Kinne J.
      • Hölzer W.
      • De Genst E.
      • Wyns L.
      • Muyldermans S.
      Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.
      • Desmyter A.
      • Spinelli S.
      • Payan F.
      • Lauwereys M.
      • Wyns L.
      • Muyldermans S.
      • Cambillau C.
      Three camelid VHH domains in complex with porcine pancreatic α-amylase. Inhibition and versatility of binding topology.
      ). In both cases, the key to such recognition is the structural organization of the CDR loops, in particular CDR3, which is often long (15–18 residues) and protruding from the V-NAR or VHH surface.
      V-NARs are distinct from typical Ig VH and VL domains as well as camelid VHH domains, sharing higher structural homology to immunoglobulin VL and T-cell receptor V domains than with immunoglobulin VH. The most unique feature of V-NARs is the absence of a CDR2 loop and of two β-strands, C′ and C″, associated with it. Instead, a distinct “belt” is formed around the middle of the β-sandwich structure (
      • Stanfield R.L.
      • Dooley H.
      • Verdino P.
      • Flajnik M.F.
      • Wilson I.A.
      Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.
      ,
      • Stanfield R.L.
      • Dooley H.
      • Flajnik M.F.
      • Wilson I.A.
      Crystal structure of a shark single-domain antibody V region in complex with lysozyme.
      ). This region shows an elevated rate of somatic mutations and has thus been termed hypervariable region 2 (HV2) (
      • Dooley H.
      • Stanfield R.L.
      • Brady R.A.
      • Flajnik M.F.
      First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.
      ). Another region of increased mutation frequency is located between HV2 and CDR3, comprising a loop that links β-strands D and E similar to that in T-cell receptor V chains; thus, this region was termed HV4. Structurally, HV2 is most proximal to CDR3, whereas HV4 is in proximity to CDR1.
      Several structural types of IgNAR variable domains have been classified based on the number and position of extra cysteine residues in CDRs and frameworks (FWs) in addition to the canonical cysteine pair (Cys23/Cys88 for VL; Kabat nomenclature) of the Ig fold (
      • Barelle C.
      • Gill D.S.
      • Charlton K.
      Shark novel antigen receptors—the next generation of biologic therapeutics?.
      ). Type I V-NAR, found in nurse sharks, has 2 cysteines in CDR3 and 2 more cysteines in frameworks (FW2 and FW4). The more common type II has one extra cysteine pair, which links CDR1 and CDR3. Type III, detected primarily in neonatal shark development, is similar to type II but has a conserved Trp residue in CDR1 and limited CDR3 diversity. Another structural type of V-NAR, which we have termed type IV, has only two canonical cysteine residues. So far, this type has been found primarily in dogfish sharks (Ref.
      • Liu J.L.
      • Anderson G.P.
      • Delehanty J.B.
      • Baumann R.
      • Hayhurst A.
      • Goldman E.R.
      Selection of cholera toxin specific IgNAR single-domain antibodies from a naive shark library.
      and this study) and was also isolated from semisynthetic V-NAR libraries derived from wobbegong sharks (
      • Streltsov V.A.
      • Varghese J.N.
      • Carmichael J.A.
      • Irving R.A.
      • Hudson P.J.
      • Nuttall S.D.
      Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.
      ).
      The single domain nature and the lack of CDR2 in V-NARs heighten the requirement for CDR1 and CDR3 to provide specific and high affinity binding to prospective antigens. CDR3, which is more variable in terms of sequence, length, and conformation, plays the key role in antigen recognition. The placing of cysteine residues in different V-NAR types is important for determining the conformation of CDRs. For example, CDR3 is long and extended (and tethered to CDR1) in PBLA8, a type II V-NAR, which enables it to access the active site cavity of its target, hen egg white lysozyme (HEL; Ref.
      • Stanfield R.L.
      • Dooley H.
      • Verdino P.
      • Flajnik M.F.
      • Wilson I.A.
      Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.
      ). In contrast, 5A7, a type I V-NAR also directed against lysozyme and targeting a similar surface epitope, has a long CDR3 that adopts a bent conformation and forms a rather flat binding surface that does not enter deep into the HEL active site (
      • Stanfield R.L.
      • Dooley H.
      • Verdino P.
      • Flajnik M.F.
      • Wilson I.A.
      Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.
      ,
      • Stanfield R.L.
      • Dooley H.
      • Flajnik M.F.
      • Wilson I.A.
      Crystal structure of a shark single-domain antibody V region in complex with lysozyme.
      ). Nevertheless, both HEL binders form comparable buried surface area with their target (∼700 Å2) and bind with low nanomolar affinity. The extent of the surface area is similar to values observed for the complexes of heavy chains of classical antibodies with their targets (
      • Janin J.
      • Chothia C.
      The structure of protein-protein recognition sites.
      ,
      • Lo Conte L.
      • Chothia C.
      • Janin J.
      The atomic structure of protein-protein recognition sites.
      ).
      Besides CDR1 and CDR3, a few other elements are involved in the HEL interaction by PBLA8 and 5A7. Of note is the residue Arg61 in the HV4 loop of PBLA8 that forms a hydrogen bond with Asp101 in HEL (
      • Stanfield R.L.
      • Dooley H.
      • Verdino P.
      • Flajnik M.F.
      • Wilson I.A.
      Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.
      ). Detailed mutational analysis of 5A7 by Fennell et al. (
      • Fennell B.J.
      • Darmanin-Sheehan A.
      • Hufton S.E.
      • Calabro V.
      • Wu L.
      • Müller M.R.
      • Cao W.
      • Gill D.
      • Cunningham O.
      • Finlay W.J.
      Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.
      ) revealed a high degree of mutational plasticity within the V-NAR domain and suggested that residues outside of the CDR1 and CDR3 loops may form additional contacts with the antigen. For example, mutation of Ser61 to Arg in HV4 of 5A7 increases HEL binding ∼5-fold likely due to the formation of a contact with Asp101 similar to Arg61 in PBLA8. Likewise, mutation of Ala1 to Asp in 5A7 results in increased HEL binding due to a putative ionic interaction (
      • Fennell B.J.
      • Darmanin-Sheehan A.
      • Hufton S.E.
      • Calabro V.
      • Wu L.
      • Müller M.R.
      • Cao W.
      • Gill D.
      • Cunningham O.
      • Finlay W.J.
      Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.
      ).
      In contrast to typical antibodies, the structure described in this study utilizes CDR1 only minimally for antigen binding. An atypical “sideways” binding mode is observed that relies heavily on framework residues to achieve antigen binding in addition to CDR3. This binding mode has been described previously for other single domain antibodies (
      • Desmyter A.
      • Spinelli S.
      • Payan F.
      • Lauwereys M.
      • Wyns L.
      • Muyldermans S.
      • Cambillau C.
      Three camelid VHH domains in complex with porcine pancreatic α-amylase. Inhibition and versatility of binding topology.
      ) but has not been seen previously in V-NARs.
      It is assumed that to be useful in therapeutic applications all novel non-human scaffolds, such as V-NARs or camelid VHH single domains, need to be humanized to reduce immunogenicity and/or improve thermodynamic stability, folding, and expression properties. Considerable expertise has been accumulated in this subject area, particularly with rodent mAbs (
      • Ewert S.
      • Honegger A.
      • Plückthun A.
      Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering.
      ,
      • Hwang W.Y.
      • Almagro J.C.
      • Buss T.N.
      • Tan P.
      • Foote J.
      Use of human germline genes in a CDR homology-based approach to antibody humanization.
      • Tsurushita N.
      • Hinton P.R.
      • Kumar S.
      Design of humanized antibodies: from anti-Tac to Zenapax.
      ). Typically, CDRs of a murine antibody of interest are grafted onto an appropriate human germ line framework (selected for sequence similarity, expression properties, or both), and then back-mutations are introduced at key positions responsible for particular CDR conformation and thus antigen binding. This approach has yielded many humanized antibodies with a number of them making it into the clinic.
      With camelid VHH domains, humanization has been relatively straightforward because of the overall structural similarity and high sequence homology (∼80%) between human and camel or llama sequences. In most instances, only ∼10 mutations of “non-human” surface residues toward the human germ line of the closest VH3 type need to be introduced into VHH scaffolds; in addition, two of four VHH hallmark residues in FW2 (positions 42, 49, 50, and 52) can be changed (
      • Conrath K.
      • Vincke C.
      • Stijlemans B.
      • Schymkowitz J.
      • Decanniere K.
      • Wyns L.
      • Muyldermans S.
      • Loris R.
      Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH.
      ,
      • Vincke C.
      • Loris R.
      • Saerens D.
      • Martinez-Rodriguez S.
      • Muyldermans S.
      • Conrath K.
      General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold.
      ). All those changes can result in biophysically stable, well expressed, and biologically active VHH domains with nearly 100% framework identity to human germ line sequences.
      Shark V-NARs represent an obvious challenge for humanization because of the structural differences (e.g. lack of CDR2) and low overall sequence identity (generally ∼30%) to human VH/VL sequences. However, available crystal structures of V-NAR domains demonstrate organization of key framework regions similar to that of human Ig variable domains, thus making an attempt at humanization possible. In this study, we describe the generation of humanized versions of type I and type IV V-NARs based on the human germ line VL scaffold, DPK9. We also provide detailed structural analysis of the type IV V-NAR clone, E06, in complex with its target, human serum albumin. Our analysis provides the foundation for further improvement and humanization of shark V-NARs.

      DISCUSSION

      Shark IgNARs demonstrate a surprising ability for specific and high affinity binding to diverse antigens by using just two variable domains per molecule, each carrying only two CDRs. The structural basis for this property is a result of additional recombination events in the IgNAR V-D-J cluster and the introduction of junctional diversity, which results in significant heterogeneity of CDR3 sequences. The varying length of CDR3 and the unique positioning of non-canonical cysteine residues in several structural types of V-NARs all help create a remarkable structural plasticity for antigen binding.
      Although CDR1 and CDR3 are considered the two major determinants for antigen binding by V-NAR domains, other regions, such as HV2 and HV4, show an increased frequency of somatic mutations, indicating their potential involvement in antigen recognition. However, such an involvement has not been demonstrated previously for shark single domains. In this study, we solved the crystal structure of a type IV V-NAR complexed with its target and demonstrated the remarkable role of V-NAR framework residues in antigen recognition. This structure provides an example of antigen recognition using essentially one single CDR, which is remarkable when viewed in contrast to the six CDRs available for antigen binding for classical antibodies. This is made possible by the extensive use of framework residues for antigen recognition. A significant fraction of those framework interactions come from the HV2 region, corresponding to the observation of increased frequency of somatic mutation. To achieve these contacts, antigen binding is sideways with respect to the antibody framework and the face typically contacting antigen. Although previously described in camelids (VHH in complex with pancreatic α-amylase; Protein Data Bank codes 1KXV and 1KXT; Ref.
      • Desmyter A.
      • Spinelli S.
      • Payan F.
      • Lauwereys M.
      • Wyns L.
      • Muyldermans S.
      • Cambillau C.
      Three camelid VHH domains in complex with porcine pancreatic α-amylase. Inhibition and versatility of binding topology.
      ), our structures are the first example of such binding for V-NAR domains. Interestingly, in both cases, CDR1 essentially does not participate in the antigen binding interface, and the same alternative face of the antibody domain makes up the rest of the antigen recognition surface. This alternative face is the same face as would be buried in the dimerization interface of a conventional antibody. Thus, it would seem that the sideways binding phenomenon may be exclusive to single domain antibodies as the side that provides the necessary framework residues is normally buried and not available for antigen recognition. Furthermore, this face of an Ig domain seems to be primed for protein-protein interaction despite the absence of the hydrophobic patch present in conventional antibodies that drives complex formation between heavy and light chains. It remains to be seen whether this binding mode is a more universal phenomenon for type IV V-NARs or an antigen-specific case. A strikingly similar mode of binding is seen in the interaction between the fibronectin type III domain (Fn3 monobody) and estrogen receptor α (Protein Data Bank 2OCF) that features the binding of an α-helical structure (estrogen receptor α) by a surface of β-sheets and loops corresponding to CDR3 and HV2 (
      • Koide A.
      • Abbatiello S.
      • Rothgery L.
      • Koide S.
      Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor.
      ).
      The other key aspect of our study is the design of a humanized version of shark V-NAR domain. Such an approach becomes possible due to a high degree of structural homology between V-NAR and Ig VL framework regions despite a low degree of sequence identity. We have demonstrated that replacement of most framework elements in V-NAR with those shared with a human VL scaffold can result in a functional humanized V-NAR molecule with >50% overall human content. As expected, changes in the regions specific to V-NARs (such as HV2 and HV4) result in partial loss of activity. We have provided a structural explanation for the importance of these regions in maintaining overall V-NAR structures. Humanized E06 can serve as a universal scaffold for humanization of other V-NAR binders provided that key elements involved in antigen recognition and V-NAR stability are identified and preserved in humanized molecules.

      Acknowledgments

      We thank Mark Johnson for assistance in screening and Julia Bianco and Xiaotian Zhong for protein expression. We thank the Aberdeen team for the isolation of E06.

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