Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering

Abstract

Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed “Repebody,” showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

Repeat proteins consist of varying numbers of consecutive homologous-structural modules (units) of 20–40 amino acid residues with characteristic secondary structures. Numerous repeat proteins have been identified in nature, and their modular architecture has evolved to be suitable for mediating many important biological functions, including protein–protein interactions, cell adhesion, signaling processes, neural development, bacterial pathogenicity, extracellular matrix assembly, and immune response (1). Due to their unique features, such as modular architecture and large interaction surfaces, repeat proteins have recently attracted much attention as templates for the development of alternative scaffolds to immunoglobulin antibodies (2–4). Immunoglobulin antibodies are widely used in biotechnology and biomedical fields as binding molecules and therapeutics; however, they have some drawbacks, such as requirement for an expensive mammalian cell-based manufacturing system, difficulty in rational design, large molecular mass, a tendency to aggregate, and intellectual property restrictions (5, 6). Hence, considerable effort has been made to develop the alternatives, and a variety of protein scaffolds have been reported including ankyrin repeat, fibronectins, anticalins, affibodies, and A domains, etc. (7, 8).

It was recently shown that the adaptive immune system in jawless vertebrates such as lampreys and hagfish is based on variable lymphocyte receptors (VLRs) instead of immunoglobulin antibodies (9–11). VLRs consist of highly diverse Leucine-rich repeat (LRR) modules and are characterized by an assembly of repeating 20–29 residue LRR modules, each of which has a β-strand-turn-α helix structure, in a horseshoe-shaped solenoid fold. LRR modules are known to be one of the most abundant structural motifs and have been identified in more than 2,000 proteins in nature (12). VLRs are produced in lymphocytes by somatic gene rearrangement of diverse LRR modules, giving rise to a vast repertoire of > 10¹⁷ unique receptors. Since their initial discovery in jawless vertebrates, the functional and structural aspects of VLRs have been demonstrated (13–18). Along with the unique structural feature of repeat proteins, the inherent role of VLRs as highly diverse antibodies suggests that they could be developed into alternative binding scaffolds devoid of the limitations found in immunoglobulin antibodies.

Here, we present the development of a binding scaffold based on VLRs by module engineering. A template scaffold was first constructed by joining consensus LRR repeat modules between the N- and C-capping motifs from VLRs found in nature. For ease of molecular engineering and manufacturing using bacterial expression system, the N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold was named “Repebody” because it was derived from naturally occurring antibodies composed of repeat modules. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 (MD2) and hen egg lysozyme (HEL), respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. To demonstrate general applicability of the scaffold, we constructed a phage-displayed library and selected the repebodies with different binding affinities for interleukin-6 (IL-6).

Results

Design of Consensus LRRV Module and a Template Scaffold.

Variable lymphocyte receptors (VLRs) of jawless vertebrates are composed of an N-terminal cap (LRRNT), the first LRR (LRR1), up to nine 24-residue variable LRR (LRRV), an end LRRV (LRRVe), a connecting peptide (CP), and the C-terminal cap (LRRCT) (Fig. 1A). To develop a binding scaffold based on VLRs, we first designed a consensus LRRV module consisting of the framework residues by analyzing the conserved pattern in variable LRR modules of VLRs. Consensus design of repeating modules was shown to increase the solubility and stability of various repeat proteins (19–22). Based on the sequence alignments for 1,000 LRR modules from the UniProt database (23) and 439 from the National Center for Biotechnology Information (NCBI) nonredundant protein sequences (nr) database (24), we obtained a consensus 24-residue LRRV module (Fig. 1B). The LRRV-framework residues were conserved, and a moderately conserved region was fixed with the most relevant and less-charged residues. The residues in the variable regions, which are denoted as “x,” can be rationally selected or randomized for the generation of molecular binders for various targets. Ten positions were almost perfectly conserved, and dominant amino acids were found at six positions. At positions 3, 15, and 24, three amino acid residues (N, Q, and K) were chosen as consensus residues because they were found in a large portion of both databases and have smaller charges. Another five sites showed large variations in sequences; four were in the concave region, and one was in the convex region. The residues on the most highly conserved and hypervariable regions are shown in the typical VLR structure (Fig. S1 A and B).

Fig. 1.

Design of consensus LRR module and Repebody scaffold. (A) Overall architecture of a VLR in jawless vertebrates. A typical VLR consists of an N-terminal cap (LRRNT), the first LRR (LRR1), up to nine 24-residue variable LRR (LRRV), an end LRRV (LRRVe), a connecting peptide VLR (CP), and the C-terminal cap (LRRCT). (B) Sequence of a consensus LRRV module based on the analysis of a conserved pattern in diverse LRR modules of VLRs. Perfectly conserved residues at 10 positions are indicated in black, and the residues dominantly occurring at six positions are presented in orange. X denotes arbitrary amino acid residues. (C) Module-pairs composed of two adjoining repeat modules from internalin B and the template scaffold (VLRc-5), respectively. Six and five module-pairs were generated from internalin B and the template scaffold, respectively, and the module-pair 2 from internalin-B was shown to have the highest similarity to the module-pair 1 from the template scaffold as indicated in red. (D) Schematic of the design of the Repebody scaffold. Based on the similarity scores of the module-pairs, the internalin-B domain spanning from the N-terminal cap to LRR2 (violet) was fused to the domain of the template scaffold spanning from LRRV2 to the C terminal (red), resulting in the Repebody scaffold. Hence, the newly designed Repebody scaffold was composed of the N-terminal cap, LRR1, LRRV1 from internalin B (violet), four LRRV (2–5), LRRVe, CP, and the C-terminal cap from the template scaffold (red). The model structures of the template and Repebody scaffolds were generated by homology modeling using the crystal structures of VLR (PDB ID code 2O6S) and internalin B (PDB ID code 1OTO) as the templates, respectively.

The number of LRRV modules in naturally occurring VLRs has been shown to range between 0–9 (17). By taking into account the number of LRRV modules in the known VLRs with antigen-binding affinities, we constructed a template scaffold by sealing five consensus-LRRV modules using the N- and C-capping motifs of VLRs found in nature, which was designated as VLRc-5. Amino acid residues at hypervariable regions were selected based on their frequencies for each position in LRR modules in nature. Hence, the template scaffold was composed of LRRNT, LRR1, five 24-residue LRRV, LRRVe, CP, and LRRCT, which had a molecular mass of approximately 29 kDa. The nucleotide sequence of the gene coding for the template scaffold is shown in Table S1, and the gene was synthesized after codon optimization for expression in Escherichia coli. The template scaffold was expressed in soluble form in the E coli Origami strain, showing an expression level of about 2 mg/L.

Redesign of the N-Terminal Capping Motif.

Even though the template scaffold was expressed in soluble form in E. coli, its expression level was too low for practical applications. Thus, we used a variety of methods to increase the expression level of the template scaffold, including fusion of various protein partners and expression under different conditions in the presence of chaperons etc.; however, no significant increase in expression was observed when these different approaches were used. It was recently shown that the folding of LRR proteins proceeds through an N-terminal transition state ensemble and that the α-helical cap may polarize better the folding pathway of the repeat proteins by acting as a fast-growing nucleus (25). Therefore, we attempted to redesign the N-terminal domain of the template scaffold based on the most favorable capping motif found in LRR proteins. We analyzed the N-terminal capping motifs found in LRR proteins in terms of the helical content and similarity to the consensus LRRV module, and found that the N-terminal capping motif of internalin B fits most the criteria. Internalin B also belongs to a family of LRR proteins and is composed of 22-residue LRR modules (26). Internalin B was shown to induce phagocytosis in hepatocytes in nature, and its crystal structure was determined [Protein Data Bank (PDB) ID code 1OTO].

Our strategy was to substitute the internalin-B cap for the N-terminal domain of the template scaffold using a computational approach involving two consecutive steps. The first step was to determine the site to connect the N-terminal cap of internalin B to the template scaffold by analyzing the modular similarity between the respective repeat units, and the second step was to optimize the amino acid residues at the fusion site connecting the two heterogeneous modules. For this, we obtained the model structure of the template scaffold by homology modeling (see SI Materials and Methods, Modeling of Protein Structures). To determine the optimal connecting region, six and five module-pairs composed of two adjoining LRR modules from internalin B and the template scaffold, respectively, were generated (Fig. 1C). Comparison of the modular similarity between the respective module-pairs from internalin B and the template scaffold revealed that the module-pair 2 from internalin B had the highest similarity to the module-pair 1 of the template scaffold (Table S2). Based on this analysis, we fused the internalin-B domain spanning from the N-terminal cap to LRR2 with the domain of the template scaffold spanning from LRRV2 to the LRRCT as depicted in Fig. 1D. We assumed that if the sequences of two module-pairs were most similar, the first module of internalin B would match well with the second module in the template scaffold, and the corresponding module-pairs were considered a primary candidate for connecting the two different LRR proteins. Using this approach, both LRR3 of internalin B and LRRV1 of the template scaffold were deleted. We further optimized the amino acid residues on the connected modules using the fixed backbone design protocol of the Rosetta software (27). The newly designed scaffold was termed “Repebody” because it was derived from repeat module-based antibodies. Consequently, the Repebody scaffold was composed of the N-terminal cap, LRR1, LRRV1 from internalin B, four LRRV (2–5), LRRVe, CP, and the C-terminal cap from the template scaffold; and its nucleotide sequence was shown in Table S1. We tested the expression of the Repebody scaffold in E. coli after codon optimization, and the expression level was significantly increased up to 60 mg/L culture (Fig. 2A).

Fig. 2.

Expressions of the Repebody scaffold in E. coli. (A) SDS-PAGE analyses of the expressed template and Repebody scaffolds. Lanes 1 and 2 are the supernatant fractions from the template and Repebody scaffolds, respectively. Lane 3 is the Repebody scaffold after purification over a Ni-NTA column. M represents standard size marker. (B) SDS-PAGE analysis of the expressed Repebody scaffolds with different LRRV module numbers ranging from 3–6. Lanes 1 and 2, three modules; Lanes 3 and 4, four modules; Lanes 5 and 6, five modules; Lanes 7 and 8, six modules. P indicates the insoluble pellet fraction from the protein lysate, and S the supernatant fraction from the protein lysate. M indicates standard size marker. (C) Expression levels and melting temperatures of the Repebody scaffolds with different LRRV module numbers. Expression levels of proteins were measured after purification over a Ni-NTA column. Expression level and melting temperature of the template scaffold were about 2 mg/L and 81 °C, respectively. (D) Melting temperature of the Repebody scaffold at different pH values. Melting temperatures were determined by measuring molar ellipticities at 222 nm as a function of temperature at indicated pH.

Variation in the Size of the Repebody Scaffold.

Repeat proteins exhibit the unique structural features resulting from the assembly of homologous structural repeat units (1, 12). To test the modularity of the Repebody scaffold, we varied the numbers of LRRV modules from 3–6, and investigated the biophysical properties of the resulting scaffolds in terms of expression level and stabilities against temperature and pH. The nucleotide sequences of the constructed Repebody scaffolds are listed in Table S1. All of them were well expressed in soluble form in E. coli (Fig. 2B), and their expression levels ranged from 50–80 mg/L culture (Fig. 2C). The scaffold containing the three LRRV modules (Repebody-3) displayed the highest expression level of about 80 mg/L, and the expression level decreased at the number of LRRV modules increased. The melting temperatures of the Repebody scaffold were closely related to the number of LRRV modules, and the scaffold with six LRRV modules (Repebody-6) had the highest melting temperature of 85 °C (Fig. 2C). The number of LRRV modules seems to be the major factor dictating the thermodynamic stability of the Repebody, whereas the substituted internalin-B cap had only a negligible effect. The stability of the Repebody scaffold against pH was also tested, and it was shown to be stable over pH values ranging between 3–12 (Fig. 2D). These results indicate that the Repebody scaffold retains a modular architecture and highly stable conformation.

Design of Repebodies by a Rational Approach.

To assess ease of molecular engineering, we first designed a repebody for myeloid differentiation protein-2 (MD2) by a rational approach. MD2 was chosen as a target protein because it plays a crucial role in the mammalian TLR4-mediated innate immune response (28–30), and the crystal structure of the TV3/MD2 (PDB ID code 2Z65) complex has been reported (31). TV3 is a hybrid protein comprising the LRR modules from human TLR4 (Toll-like receptor 4) and a hagfish VLR. To design a repebody for MD2, we predicted the residues that interact with MD2 by superimposing the model structure of the Repebody scaffold onto the crystal structure of TV3 in complex with MD2 (Fig. S2A). Based on the analysis, 11 residues on the scaffold were selected and changed (Fig. S2B): N91I, S93T, T94G, A115V, N117V, T118E, S139N, G141A, Y142H, R163D, and N165S. The gene coding for the molecular binder for MD2 (Table S1), designated as MD2-repebody, was synthesized and expressed in E. coli. MD2-repebody was also expressed at high level as a monomeric form (Fig. S3A), and showed a distinct binding to MD2 (Fig. S3B, C) and a negligible cross-reactivity (Fig. S3D). Binding affinity of MD2-repebody (K_D) for MD2 was estimated to be 388 nM by SPR equilibrium analysis (Fig. 3A). We then tested whether the MD2-repebody had an effect on the lipopolysaccharide (LPS)-induced immune response using a cell-based assay system. The MD2-repebody exhibited a considerable attenuating activity (Fig. S3E), implying that it may be a potential therapeutic for treatment of severe inflammation and sepsis.

Fig. 3.

Binding affinities and crystal structures of designed repebodies. (A) Binding affinity of MD2-repebody for MD2 by SPR equilibrium analysis. The fitting curve was obtained by plotting the response units (RU) against the MD2 concentrations. (B) Binding affinity of HEL-repebody for HEL by ITC (C) Superposition of the crystal structure of the MD2-repebody (blue) on the crystal structure of VLR (PDB ID code 2O6S, yellow). (D) Superposition of the crystal structure of the HEL-repebody (pink) on the crystal structure of VLR (PDB ID code 2O6S, yellow). (E) Interaction interface of the MD2-repebody. Eleven mutated residues potentially interacting with MD2 are shown in blue sticks. (F) Interaction interface of the HEL-repebody. Nine mutated residues potentially interacting with HEL are indicated in pink sticks, and the replaced C-cap region is represented in light blue.

To further test ease of molecular design, we attempted to produce a repebody for hen egg lysozyme (HEL). We chose HEL because it is easy to prepare large amounts of the protein and the crystal structure of the VLR/HEL (PDB ID code 3G3A) complex has been reported (17). Similarly, the crystal structure of the MD2-repebody was superimposed on the crystal structure of the VLR/HEL complex to determine the residues to be changed (Fig. S2C). As a result, nine residues of the MD2-repebody were selected (Fig. S2D): N139Y, A141Y, E161R, S165D, Y166N, D185Q, R187S, Y189N, and Q190D. In addition to the nine mutations, CP and LRRCT spanning from Gln-208 to Thr-266 were replaced with those of VLR in complex with HEL because the loop of LRRCT on VLR was shown to participate in the interaction with HEL (17). The designed repebody for HEL was designated as HEL-repebody, and its nucleotide sequence is listed in Table S1. HEL-repebody also was shown to be expressed at high level and form the complex with HEL in solution (Fig. S4A). HEL-repebody displayed a distinct binding to HEL (Fig. S4B) and a negligible cross-reactivity (Fig. S4C). Binding affinity of HEL-repebody for HEL was estimated to be 3.7 μM by isothermal titration calorimetry (ITC) analysis (Fig. 3B).

To gain insight into the structural features and interaction interfaces, the crystal structures of the MD2- and HEL-repebodies were determined at 1.7 Å and 1.8 Å resolutions, respectively (Table S3). The crystal structures of the two repebodies revealed that both proteins maintain a characteristic horseshoe-shaped fold, displaying well conserved backbone structures despite a number of mutations. To check if substitution of the internalin-B cap caused any changes in the conformation of the LRR domain, we superimposed the crystal structures of two designed repebodies on the crystal structure of a lamprey VLR (PDB ID code 2O6S) (Fig. 3 C and D). The backbone root-mean-square deviations of the two repebodies relative to VLR except for the substituted internalin-B cap were about 0.33 Å and 0.36 Å, respectively. This result indicates that the Repebody scaffold retained nearly the same conformation, and that fusion of the internalin-B cap had a negligible effect on the conformation of the original LRR domain. To understand the interaction interfaces, the crystal structures of the MD2- and HEL-repebodies were superimposed on the complex crystal structures of TV3/MD2 (PDB ID code 2Z65) and VLR/HEL (PDB ID code 3G3A), respectively (Fig. 3 E and F). Analysis of the binding hot spots by FoldX 3.0 beta (complex_alascan function) suggests that hydrogen bonds between the side chains play a major role in interactions. Specifically, in the case of the MD2-repebody, E118, D163, and S165 were predicted to be the binding hot spot, and E118 and S165 appeared to form hydrogen bonds with T112, E111, and R106 of MD2. In addition, D163 of the MD2-repebody was likely to interact with the positively charged residue, R106, of MD2. As for the HEL-repebody, hydrogen bonds involving R161, D165, Y241, and N243 seemed to be critical for the binding of the HEL-repebody to HEL. The charged residues, R161 and D165, were likely to have charge interactions with D48 and R73 of HEL, mediating the hydrogen bonding with P70 and R73 of HEL. The accuracy of the model structure of the Repebody scaffold was tested by superimposition on the crystal structure of the MD2-repebody (Fig. S5) (32). The model structure was well fitted into the crystal structure of the MD2-repebody with a Cα rmsd of 0.95 Å.

Selection of a Repebody by Phage Display.

In order to show general applicability of the Repebody scaffold, we attempted to generate a repebody for other target by phage display selection. As a protein target, interleukin-6 (IL-6) was employed because it was known to be involved in many diseases like inflammation and cancers (33). Two adjoining repeat modules (LRRV module 1 and 2) of the Repebody scaffold were chosen, and three hypervariable sites (positions 8, 10, and 11) on each repeat module were subjected to randomization for generating a synthetic diversity (Fig. 4A). The library was constructed by repeat module-based overlap PCR using the primers with NNK (where N stands for any of four nucleotides and K for G or T) degenerate codon, and a phage-displayed library of approximately 10⁸ clones was generated. After four rounds of standard panning process against IL-6, 96 clones were randomly chosen for the assay of the binding activity in a 96-well plate using phage ELISA. We selected 82 positive clones showing significant signals (signal to noise > 10) and determined their sequences. Selected repebodies were shown to have distinct amino acid residues at the mutation sites, and their sequence conservation is shown by sequence logos (Fig. 4B). Of 82 positive clones, we selected three repebody clones (B3, C8, and F11) showing high signals in phage ELISA. Three isolated clones were tested in terms of specificity using phage ELISA, and they were shown to be highly specific for IL-6 (Fig. 4C), displaying negligible cross-reactivities against other proteins. The selected repebodies were expressed in E. coli, and their binding affinities for IL-6 were determined by ITC (Fig. 4D). The selected repebodies displayed the binding affinities ranging from 48–117 nM, showing variation in amino acid residue between one and three (Fig. 4E). This result indicates that the Repebody scaffold can be broadly used for generating the molecular binders with high affinity and specificity for a variety of targets by phage display selection.

Fig. 4.

Selection of a repebody for IL-6 by phage display. (A) Sites for introducing mutations for the construction of a phage-displayed library. The numbers indicate the positions on the Repebody scaffold. (B) Sequence conservation of 82 repebody clones shown as sequence logos. The height of individual letters indicates the frequency of recovered amino acid at specified position. The numbers indicate the positions on each repeat module (C) Specificity of the selected repebodies by phage ELISA. Purified proteins were coated on a 96-well plate, sequentially reacted with purified phage and HRP-conjugated anti-M13 monoclonal antibody, and absorbance was measured at 450 nm using a plate reader. In the case of a competitive assay, soluble interleukin-6 (sIL-6) was added. Error bars indicate the deviation in triplicate experiments. (D) Isothermal titration calorimetry data for IL-6 binding to the selected repebodies (F) Amino acid sequences and K_D values from ITC of the selected repebodies.

Discussion

We have successfully developed the Repebody scaffold based on VLRs by module engineering. The present results demonstrate that the developed scaffold can be widely used for generating the target-specific molecular binders for applications in biotechnology and biomedical fields by a rational design and phage display selection. One of the key issues in the development of an alternative scaffold is the ease of engineering and mass production using bacterial expression system (7, 8). Our approach to redesign the N-terminal domain of the template scaffold based on the internalin-B cap successfully achieved a high-level soluble expression of the Repebody scaffold up to 80 mg/L in E. coli, enabling ease of manufacturing and engineering using bacterial expression system. This result seemed to stem from the role of the N-terminal capping motif of internalin B, which may have acted as a fast-growing nucleus to create a discrete and polarized folding pathway onto which proximal LRR modules can propagate. Approximately half of internalin B, which is composed of the N-terminal helical cap and the first three LRR modules, is likely to facilitate such folding pathway. We tested the applicability of our approach to Toll-like receptor 4 (TLR4) with different modular structure. A template scaffold was first constructed by assembling seven LRR modules of the TLR-4 ectodomain between the N- and C-terminal capping motifs from a hagfish VLR. However, this template scaffold was found not to be expressed at all in E. coli, and a variety of methods to express the scaffold had no effect. We redesigned the N-terminal domain of the scaffold based on the internalin-B cap using the same approach as described above. The resulting TLR-4 scaffold was shown to be expressed in a soluble form in E. coli, and its expression level reached about 10 mg/L (Fig. S6), which supports that our approach can effectively be used for developing binding scaffolds based on repeat proteins composed of LRR modules.

The unique structural feature of repeat proteins lies in a modular architecture stemming from an assembly of homologous structural modules (repeats) in a horseshoe-shaped solenoid fold (34). The modular architecture of the Repebody scaffold allowed for variation in the numbers of LRR modules, and all the constructed scaffolds were also well expressed at high levels in soluble form in E. coli, showing high thermodynamic and pH stabilities. It is worth noting that the target interaction surface of the Repebody scaffold can be easily controlled by adding or deleting the LRRV modules without disruption of the overall structure of the Repebody scaffold, whereas non-repeat-globular proteins have fixed sizes of interaction surfaces. Increases in the concave surface area and molecular mass of the Repebody scaffold per LRRV module were estimated to be 220 Å² (16) and 3 KDa, respectively, which indicates that the interaction surface of the Repebody scaffold can be effectively modulated. The melting temperature of the Repebody scaffold increased in proportion to the number of LRRV modules. A similar trend in thermodynamic stability was reported for the consensus-designed ankyrin-repeat proteins (35). It was shown that LRR modules with the framework residues assemble into a solenoid fold, forming a tight hydrophobic core that laterally stabilized consecutive repeat modules. Hence, the number of LRRV modules appeared to be the main factor dictating the thermodynamic stability of the Repebody scaffold, whereas the internalin-B cap had a negligible effect on thermodynamic stability. The high stability of the Repebody scaffold against pH is likely to be closely related to its high thermodynamic stability.

We have shown ease of molecular engineering and general applicability of the Repebody scaffold by creating the molecular binders for three defined targets by a rational design and phage display selection. The designed repebodies for MD2 and HEL had the binding affinities of 388 nM and 3.7 μM, respectively, displaying unique interaction interfaces for the respective targets. The crystal structures of the MD2- and HEL-repebodies indicated that they retain a characteristic horseshoe-shaped fold despite numerous mutations on the concave surface, presenting a modular architecture and rigid backbone structure. A diverse phage-displayed library could be constructed by introducing mutations specifically into six hypervariable sites on two repeat modules. The use of phage display enabled a successful selection of the repebodies for IL-6 with K_D values ranging from 48–117 nM. The selected repebodies were shown to be highly specific for IL-6, displaying negligible cross-activities, which seems to stem from the inherent role of VLRs in adaptive immune system. The modularity of the Repebody scaffold allowed variations in the number of repeat modules as well as in amino acid residues on individual modules. Thus, interacting surface of the Repebody scaffold for a target can be easily modulated by changing the number of repeat modules to be mutated for a library construction. It has been suggested that proteins with a large flat surface and rigid structure offer distinct advantage in the design of molecular binders for a variety of targets, partly because they induce the rigid body interactions and consequently a low loss of entropy upon binding (5). With a modular architecture and rigid backbone structure, the Repebody scaffold offers distinct advantages over globular proteins in creating the target-specific molecular binders by rational and library-based approaches.

In conclusion, the present results demonstrate a successful development of the Repebody scaffold based on VLRs by module engineering as an alternative to immunoglobulin antibodies. With unique biophysical and structural features, the Repebody scaffold can broadly be used for generating molecular binders for therapeutic purpose as well as for applications in diagnostics such as protein chips, bioimaging, and immuno-assays by rational design and library-based approaches. In addition, a repebody with high affinity and specificity for a target is expected to be applied to affinity purification, due to its high thermodynamic and pH stabilities.

Materials and Methods

The genes encoding various Repebody scaffolds were synthesized after codon optimization for E. coli (Genscript), and their sequences are listed in Table S1. The model structure of the template scaffold was obtained by homology modeling using Modeller9v4 software (32). The crystal structure of a VLR (PDB ID code 2O6S) was used as a template. We designed molecular binders for MD2 and hen egg lysozyme (HEL) as the protein targets based on the complex crystal structures of TV3/MD2 (PDB ID code 2Z65) and VLR/HEL (PDB ID code 3G3A), respectively. Binding affinities of repebodies were determined by surface plasmon resonance (SPR) analysis using a Biacore 3000 system (GE Healthcare) and isothermal titration calorimetry (ITC) (iTC200 system, Microcal). Cell-based assay for LPS-induced immune response was performed using the macrophage-like cell line THP-1 (TIB-202TM, ATCC). A phage display selection was carried out as described elsewhere (36). A repebody library was constructed by introducing random mutations specifically into hypervariable sites on repeat modules of the Repebody scaffold using the primers with NNK degenerate codon. Detailed experimental procedures are described in SI Materials and Methods.