High Resolution NMR-based Model for the Structure of a scFv-IL-1β Complex

Abstract

Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1β. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.

Introduction

The ability of antibodies to bind to an almost unlimited number of target proteins with high specificity makes them one of the fastest growing classes of therapeutics in the biological drugs market (1). Since the first description of monoclonal antibodies (2), dramatic progress has been made in the expression, engineering, humanization, and applications of antibodies as therapeutics. A wide variety of antibody fragments have been evaluated as potential therapeutics including the well characterized antigen binding fragment (Fab), which contains the light chain (V_L and C_L domains) and N-terminal portion of the heavy chain (V_H and C_H domains). The smallest fragment to retain full binding activity has also attracted considerable interest, with the so-called single chain variable fragment (scFv)³ (3) consisting of the two variable domains joined by a short peptide.

A detailed understanding of the interactions between candidate therapeutic antibodies and target proteins is key to further progress in rational design and humanization. Currently, identification of the binding sites for antibodies on target proteins is achieved via one or a combination of indirect methods such as protease protection, peptide scanning, site-directed mutagenesis, or analysis of backbone amide exchange (4–6). Although providing valuable information, each of these approaches has drawbacks; in particular, they may not detect discontinuous epitopes and do not provide information on the spatial organization of epitopes. For such an important area of biotherapeutics, relatively few crystal structures have been determined for potential therapeutic antibody-target protein complexes, which probably reflects the inherent flexibility and solubility of antibodies, resulting in limited success in crystallization trials.

Continued developments in NMR spectroscopy mean that it is now possible to obtain detailed structural information for proteins and complexes of up to 80 kDa in solution (7), which makes this an attractive approach for determining the structures of isolated scFvs (28 kDa) and Fabs (50 kDa), as well as complexes formed with target proteins. NMR spectroscopy is an ideal tool for mapping the precise interaction sites on both the antibodies and also target proteins. To date, only a few limited NMR studies of functional antibody fragments have been reported, including scFv, Fv, and isolated V_L domains (8, 9), with broad line widths limiting the experiments possible. In the case of scFvs, the formation of domain-swapped dimers at even relatively low concentrations is now well documented (10, 11) and presumably accounts for the line width problems encountered in previous attempts to obtain detailed structural information for scFvs using NMR-based methods.

In this study, we report the successful use of NMR spectroscopy to determine a reliable model for the scFv-IL-1β complex, which reveals details of the scFv residues involved in IL-1β recognition, as well as the binding site on IL-1β. We also provide direct evidence that a scFv binds to a target protein in the same manner as an equivalent Fab, indicating that high resolution models for scFv-target protein complexes can be used as reliable guides for the rational design and development of therapeutic antibodies.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

The scFv, Fab, and IL-1β were expressed as soluble proteins in Escherichia coli and purified using a combination of affinity and size-exclusion chromatography. Full details of the expression vectors used and purification protocols are given in the supplemental materials.

Analysis of the Monomer to Domain-swapped Multimer Equilibrium for the scFv

The concentration dependence (28–395 μm) of the monomer to the domain-swapped dimer ratio for the isolated scFv was determined by analytical gel filtration on a Superdex 75 16/60 column using a 25 mm sodium phosphate, 100 mm sodium chloride, 0.02% sodium azide buffer at pH 6.5. The column was calibrated using a range of molecular mass protein standards (6.5, 13.7, 29.0, 43.0, and 75.0 kDa) supplied by GE Healthcare.

NMR Spectroscopy

NMR spectra were acquired from 0.35-ml samples of 0.1–0.5 mm free scFv and 0.2-0.7 mm scFv-IL-1β complex in a 25 mm sodium phosphate, 100 mm sodium chloride, 10 μm EDTA, 100 μm 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride, 0.02% sodium azide buffer at pH 6.5, containing 10% D₂O, 90% H₂O. All NMR data were acquired at 35 or 40 °C, on 600-MHz Bruker DRX or 800-MHz Bruker Avance spectrometers equipped with triple-resonance (¹⁵N/¹³C/¹H) cryoprobes.

The two-dimensional and TROSY-based three-dimensional spectra recorded to obtain sequence specific backbone assignments for the scFv-IL-1β complex were: ¹⁵N/¹H HSQC, ¹⁵N/¹³C/¹H HNCACB, HN(CO)CACB, HNCA, HN(CO)CA, and HNCO (12). Typical acquisition times/spectral widths for three-dimensional experiments were 8–9 ms/24–65 ppm in F₁ (¹³C) (except for the HNCO, which was 24 ms/11 ppm), 21–24 ms/36 ppm in F₂ (¹⁵N), and 70 ms/14 ppm in F₃ (¹H). The three-dimensional spectra were collected for between 64 and 80 h, and the ¹⁵N/¹H HSQC spectra were collected for between 0.5 and 2 h, with acquisition times/spectral widths of 60 ms/14 ppm in F₂ (¹H) and 50 ms/36 ppm in F₁ (¹⁵N). NOE data were obtained using ¹⁵N/¹H NOESY-HSQC experiments, with a mixing time of 450 ms, and collected over 90 h. ¹⁵N/¹H HSQC spectra for comparison of ¹⁵N/²H-labeled IL-1β complexed with either unlabeled scFv or unlabeled Fab were collected for ∼2 h.

All triple-resonance experiments were acquired on samples of the complex consisting of ²H/¹³C/¹⁵N-labeled scFv bound to unlabeled IL-1β and vice versa with concentrations of 0.5–0.6 mm. Similarly, ¹⁵N/¹H NOESY-HSQC experiments were recorded using samples with one component ²H/¹⁵N labeled and the other unlabeled with concentrations of 0.51–0.55 mm.

Residual dipolar coupling (RDC) data were collected using 4 mg/ml Pf1 phage (ASLA BIOTECH Ltd.) in samples of 0.2–0.3 mm scFv-IL-1β complex with either the scFv or IL-1β ²H/¹⁵N labeled. Backbone amide RDC values were derived from the differences between the ¹⁵N-¹H scalar couplings for isotropic and partially aligned samples using ¹⁵N/¹H HSQC and TROSY spectra (13), with acquisition times/spectral widths of 60 ms/14 ppm in F₂ (¹H) and 50 ms/36 ppm in F₁ (¹⁵N), and collected for ∼10 h. Digital resolution in the spectra used for RDC measurements was 1.1 Hz. All NMR data were processed using TopSpin (Bruker Biospin Ltd.) and analyzed using Sparky (University of California, San Francisco). Backbone amide proton line widths were measured for a selection of well resolved cross peaks in two-dimensional ¹⁵N/¹H HSQC spectra of several ¹⁵N-labeled proteins (Pdcd4 MA-3 domains, ESAT-6-CFP-10 complex, scFv, Fab, and scFv-IL-1β complex; 15–45 kDa (14, 15)), with the line width determined at half-height.

Sequence-specific resonance assignments (N, H^N, C^α, C^β, and C′) were obtained for the scFv-IL-1β complex from the identification of intra- and inter-residue connectivities in TROSY-based three-dimensional triple-resonance spectra, with additional supporting evidence provided by sequential NOEs observed in ¹⁵N/¹H NOESY-HSQC spectra. The chemical shift index (16) and TALOS (17) programs were used to determine the positions of elements of regular secondary structure from the chemical shift data.

The minimal shift approach, as described previously (18, 19), was used to identify scFv residues involved in IL-1β binding. The minimal shift values were obtained from the combined chemical shift difference in ¹⁵N and ¹H for each assigned peak in the ¹⁵N/¹H HSQC spectrum of the ¹⁵N-labeled scFv bound to unlabeled IL-1β when compared with all peaks observed in the ¹⁵N/¹H HSQC spectrum of the free ¹⁵N-labeled scFv. A histogram of minimal combined shift versus the protein sequence was used to reveal residues from the scFv with significantly perturbed backbone amide signals. IL-1β residues involved in scFv binding were determined in a similar manner by the determination of actual combined backbone amide ¹⁵N and ¹H shifts from assigned ¹⁵N/¹H HSQC spectra of free and scFv-bound IL-1β.

Homology Modeling of the scFv

Initially, two BLAST (20) searches were completed against the Protein Data Bank (PDB), one with the V_H domain of the scFv and the other with the V_L domain. The closest antibody structures were retained and aligned to the scFv using FUGUE (21). Each of the pair-wise alignments was scored based on sequence similarity within the framework region, the conservation of solvent-inaccessible residues, the similarity in complementarity-determining region (CDR) length, and the resolution of the template structure. The alignment with the best score (PDB code 1L7I (22)) was used as the input to MODELLER (23) to build a homology model of the Fv region. The model was then subject to manual refinement. CDR H2 was identified by HARMONY3 as a potentially problematic region, and following manual examination, it was replaced by that from the deposited structure 1KNO (24). The resulting model of the Fv region was converted to a scFv by adding the flexible linker.

Calculation of a Reliable Model for the scFv-IL-1β Complex

The structure of the scFv-IL-1β complex was determined by NMR restraint-driven docking of IL-1β and the scFv using HADDOCK (25), in which residues involved in interaction sites were defined as semiflexible. A homology model of the scFv and a crystal structure of free IL-1β (PDB code 2I1B (26)) were used as starting points for the docking calculations. To dock the scFv and IL-1β, ambiguous interaction restraints were selected to define the protein-protein interaction surface using either active or passive residues. Active residues are ones that have been experimentally identified as being involved in the interaction and are solvent-exposed, with passive residues being all solvent-accessible neighbors of active residues. Analysis of the chemical shift perturbation data and of solvent accessibilities using NACCESS (27) resulted in the identification of active residues as scFv residues with a minimal shift of over 0.075 ppm and 20% solvent accessibility and IL-1β residues with a combined shift of over 0.1 ppm and 20% solvent accessibility. The active and passive residues selected are summarized in supplemental Table 1. The interacting regions of IL-1β (residues 1–17, 30–58, 103–111, and 143–153) and both the IL-1β and the V_L–V_H interfaces for the scFv (23–37, 43–53, 65–69, 86–101, 155–167, 172–197, and 223–241) were defined as semiflexible during the simulated annealing phase of the docking, with the scFv linker defined as fully flexible.

The axial tensor (D_a) and rhombicity (R) components of the alignment tensor for partially aligned samples of the scFv-IL-1β complex were calculated using PALES (28), and together with the backbone amide RDC data, they were used to incorporate restraints defining the orientation of the scFv and IL-1β. In addition, intervector projection angle restraints (29) were derived from the RDC data. A substantial number of long range, intramolecular H^N to H^N NOEs identified were also included as distance restraints during the docking calculations (387 for the scFv and 240 for IL-1β). The NOEs were calibrated on the basis of peak intensity and determined to correspond to ¹H–¹H distance restraints of <5, 5–6.5, or 6.5–8 Å.

In the first stage of the docking calculations, 1000 initial complex structures were generated by rigid body energy minimization. The 200 complexes with the lowest overall energy were then selected and refined in an explicit water shell (25). After refinement, the complexes were initially sorted into structurally related groups using the default clustering cut-off of 7.5 Å, which placed all the structures in one group with an overall backbone r.m.s.d. of 2.2 Å. The complexes were therefore reclustered using a more stringent clustering cut-off of 2.2 Å, which yielded one dominant cluster and eight sparsely populated ones. To verify the scFv-IL-1β complex obtained, backbone amide RDCs calculated from the complex structures were compared with the experimentally determined RDCs using PALES. Analysis of the calculated structures was carried out using the programs MOLMOL (30) and PyMOL (DeLano Scientific LLC).

RESULTS

Domain-swapped Dimer Formation

A number of previous studies have reported the formation of domain-swapped dimers for purified scFv proteins at relatively modest concentrations (20 μm) (10, 11, 31). We have investigated the behavior of two distinct scFvs selected for specific and high affinity binding to IL-1β. Analysis by gel filtration revealed that they were predominantly monomeric at concentrations below 10 μm, but at concentrations required for detailed NMR structural analysis (>200 μm), they approached nearly 50% domain-swapped dimer, as illustrated in Fig. 1a.

FIGURE 1.

Analysis of the monomer to domain-swapped multimer equilibrium for the free and IL-1β-bound scFv. a, gel filtration analysis of the free scFv over a range of protein concentrations, illustrating the high proportion of domain-swapped dimer present at the concentrations required for NMR spectroscopy. mAU, milliabsorbance units. b, a typical profile obtained for the separation of monomeric and dimeric scFv-IL-1β complex by gel filtration, with SDS-PAGE analysis of the fractions corresponding to dimeric complex (lane 1), monomeric complex (lane 2), and free IL-1β (lane 3) shown in c. Further gel filtration analysis showed that the monomeric scFv-IL-1β complex obtained by this approach remained completely monomeric after many weeks at concentrations of up to 0.7 mm.

¹⁵N/¹H HSQC spectra obtained from samples of the scFvs containing nearly 50% domain-swapped dimer were surprisingly good (Fig. 2a), which in part reflects the conservation of the V_L—V_H domain interface in the dimer (supplemental Fig. 1) and results in only a few signals being shifted on dimer formation. In contrast, dimer formation has a marked influence on the signal line width, resulting in significantly reduced sensitivity and resolution, with average backbone amide proton line widths of 31.5 ± 5.5 Hz when compared with less than 25 Hz expected for a monomeric scFv. This predicted value was based on extrapolation from the experimentally determined amide proton line widths for a selection of proteins (15–45 kDa).

FIGURE 2.

A comparison of ¹⁵N/¹H HSQC spectra for free and bound scFv. a and b, ¹⁵N/¹H HSQC spectra for free ¹⁵N-labeled scFv (a) and ¹⁵N-labeled scFv-unlabeled IL-1β complex (b). The resolution and sensitivity in the spectrum of the free scFv are reduced due to the presence of almost 50% domain-swapped dimer.

NMR Spectroscopy

We reasoned that the formation of a monomeric scFv-IL-1β complex at low concentration may stabilize the bound scFv and allow subsequent concentration of the complex for detailed NMR analysis without the formation of domain-swapped scFv dimers. This approach proved very successful and enabled the preparation of 200–700 μm samples of a scFv-IL-1β complex that contained no detectable dimer (Fig. 1b). ¹⁵N/¹H HSQC spectra of these samples showed significantly reduced backbone amide proton line widths (27.5 ± 3.0 Hz) when compared with the scFv alone (31.5 ± 5.5 Hz), resulting in increased resolution and sensitivity, as illustrated in Fig. 2. Complexes were prepared with either IL-1β or the scFv appropriately labeled (¹⁵N/²H or ¹⁵N/¹³C/²H), and a series of double and triple resonance spectra was used to determine comprehensive sequence-specific resonance assignments for both proteins in the complex. For the scFv essentially complete backbone (H^N, N, C^α, C^β, and C′), assignments were obtained for all residues excluding the linker and C-terminal His tag apart from Asp-1, Pro-8, Ser-9, Asn-28, Pro-40, Gly-41, Lys-42, Ala-43, Tyr-87, Cys-88, Gln-89, Arg-108, Thr-109, Asp-157, Val-177, Gln-211, and Cys-225 (92%), and for IL-1β, assignments were obtained for all residues except Ala-1, Gln-34, Asn-53, Lys-63, Glu-64, Lys-65, Asn-66, Asp-75, Gly-139, and Gln-141 (93%).

Mapping of Binding Sites

Residues involved in the interaction between the scFv and IL-1β were identified by the comparison of ¹⁵N/¹H HSQC spectra acquired for the free and bound proteins (Figs. 3a and 4a), as described previously (18, 19). It was not possible to obtain assignments for the free scFv (see above), and so significantly perturbed backbone amide signals were identified by minimal shift analysis (18, 19). Assignments have been previously reported for IL-1β (32), and it proved relatively straightforward to obtain nearly complete backbone resonance assignments for both the free and the scFv-bound protein under our experimental conditions, which allowed the actual combined shifts in amide ¹⁵N and ¹H to be determined. The observed changes in backbone amide signals for both proteins on complex formation are summarized in the histograms shown in Figs. 3b and 4b, which clearly indicate an extensive and specific interaction surface. The interaction surfaces are roughly equal in size on both proteins with 29 residues (Arg-24, Gly-27, Asn-31, Tyr-32, Phe-91, Trp-92, Ser-93, Phe-96, Arg-160, Tyr-161, Asp-162, Trp-176, Gly-183, Gly-184, Gly-185, Ser-186, Thr-187, Tyr-188, Phe-189, Asp-191, Gly-195, Thr-198, Ser-200, Asn-203, Lys-230, Lys-231, Leu-232, Thr-233, and Phe-234) from the scFv showing significant shifts on complex formation (Fig. 3) and 27 residues (Arg-4, Ser-5, Leu-6, Asn-7, Thr-9, Asp-12, Gln-15, Ser-17, Leu-18, Gln-39, Val-47, Glu-50, Glu-51, Ser-52, Asp-54, Ile-56, Val-58, Glu-105, Ile-106, Asn-107, Lys-109, Leu-110, Thr-147, Gln-149, Val-151, Ser-152, and Ser-153) from IL-1β (Fig. 4). For the scFv, residues with significantly perturbed backbone amides mainly lie in the CDRs (Fig. 3b), as expected for antibody binding.

FIGURE 3.

Mapping of the IL-1β binding site on the scFv by minimal shift analysis. a, a small region from the ¹⁵N/¹H HSQC spectra of ¹⁵N/²H-labeled scFv-unlabeled IL-1β complex in red and free ¹⁵N/²H-labeled scFv in blue, with the assignments for the complex indicated. It is clear that Arg-61 and Gln-70 show no significant shifts, whereas the backbone amide peaks for Phe-189 and Lys-230 show substantially changes. b, the histogram shows the backbone amide minimal shifts seen for the scFv upon complex formation with IL-1β. The positions of the CDRs are indicated above the histogram by green bars, whereas regions of regular secondary structure are indicated by red bars for helices and blue arrows for β-sheets. c, the minimal shift data are mapped onto a space-filled view of the scFv with significantly perturbed residues (shift >0.04 ppm) colored with a gradient from white to red. Residues shown in yellow correspond to ones for which no minimal shift data are available. d, a ribbon representation of the scFv structure in the same orientation as c.

FIGURE 4.

Mapping of the scFv binding site on IL-1β by combined backbone amide shift analysis. a, a small region from the ¹⁵N/¹H HSQC spectra of ¹⁵N/²H-labeled IL-1β-unlabeled scFv complex in red and free ¹⁵N-labeled IL-1β in blue, with assignments indicated for both. Signals from a number of residues, such as Cys-8 and Val-19, clearly undergo significant shifts on complex formation, whereas others, such as Val-72, remain unperturbed. b, the histogram shows the combined backbone amide shifts seen for IL-1β on binding to the scFv. Regions of regular secondary structure are indicated by red bars for helices and blue arrows for β-sheets. c, the shift data are mapped onto a space-filled view of IL-1β, with significantly perturbed residues (shift >0.1 ppm) colored on a gradient from white to red. Residues highlighted in yellow are ones for which no chemical shift perturbation data were obtained. d, a ribbon representation of the IL-1β structure in the same orientation as c.

Restraint-driven Docking

Analysis of the C^α, C^β, and C′ assignments obtained for the scFv-IL-1β complex revealed that both proteins have an unchanged secondary structure in the complex (supplemental Fig. 2), which strongly suggests that neither protein undergoes a significant conformational change on complex formation. This clearly indicates that the backbone amide signal perturbations seen on complex formation reflect changes at the interaction sites and supports a restraint-driven docking approach to determine the structure of the scFv-IL-1β complex.

Backbone amide chemical shift perturbation data were used to identify the scFv-IL-1β interface and to define ambiguous interaction restraints for docking, which resulted in 17 active and 25 passive residues being selected for the scFv and 26 active and 19 passive residues being selected for IL-1β. Information concerning the relative orientation of the two proteins was obtained from backbone amide RDC measurements for the complex (supplemental Fig. 3), with partial alignment achieved using Pf1 phage. RDC values were obtained for 107 residues of IL-1β (70%) and 166 residues of the scFv (65%), with data for the remaining residues missing due to overlap in the ¹⁵N/¹H HSQC and TROSY spectra. For the scFv, 387 long range, intramolecular H^N to H^N NOE-derived distance restraints were also included in the docking calculations (119 sequential (i, i + 1), 102 medium (i, i ≤ 4), and 166 long range (i, i ≥ 5)), and for IL-1β, 240 long range, intramolecular restraints were included, (78 sequential (i, i + 1), 58 medium (i, i ≤ 4), and 104 long range (i, i ≥ 5)).

The docking calculations using HADDOCK (25) produced one main cluster for the scFv-IL-1β complex, which contained 77 of the 200 calculated structures and is shown in Fig. 5a. A best fit superposition of the structures for the backbone atoms of residues in elements of regular secondary structure in both IL-1β and the scFv gives backbone atom (N, C^α, and C′) r.m.s.d.s to the mean of 0.7 ± 0.1 Å for IL-1β and 0.9 ± 0.2 Å for the scFv. The remaining structures were grouped into eight sparsely populated clusters, which all showed poorer agreement with the NMR data and higher energies.

FIGURE 5.

Solution structure of the scFv-IL-1β complex. a, a best fit superposition of the 77 converged structures obtained for the scFv-IL-1β complex. The structures were superimposed using the backbone atoms in elements of regular secondary structure in both proteins, which gives a backbone atom r.m.s.d. to the mean of 0.74 ± 0.2 Å for the complex. b, a ribbon representation of the structure closest to the mean in the same orientation as a. c, an expanded view of the scFv-IL-1β interface, with the contact surface of IL-1β shown in gray and the backbones of the scFv CDRs shown as colored loops. The side chains of the scFv residues involved in intermolecular interactions are shown as stick models, with interacting framework residues highlighted (F1, F2, and F3).

The agreement between the measured RDC data and the two proteins in the scFv-IL-1β complex was greatly improved after docking and refinement, with Cornilescu quality factor (Q) values (33) reducing from 0.31 to 0.11 for IL-1β and from 0.48 to 0.11 for the scFv. The final set of converged scFv-IL-1β complexes is entirely consistent with the NMR-derived constraints used for docking, with no significant or consistent violations. The family of structures obtained was also validated by comparison of the backbone dihedral angle ranges indicated by TALOS analysis of the resonance assignments (276 pairs of ϕ and ψ angle ranges for the scFv and 166 pairs for IL-1β) with those observed in the converged complex structures. Consistent differences were found for only 2 residues in IL-1β (Glu-96 and Ile-106) and 6 residues in the scFv (Ala-51, Thr-69, Phe-91, Gly-184, Asn-229, and Lys-230), which probably reflects the known 2% error rate for TALOS predictions (15). The excellent agreement between the substantial NMR data available and the complex structures obtained clearly indicates that the approach reported here can produce reliable, high resolution models for scFv-target protein complexes. The scFv-IL-1β complex structures, together with the NMR constraints, have been deposited in the Protein Data Bank under accession number 2KH2.

ScFv-IL-1β Complex Structure

The structure obtained for the scFv-IL-1β complex is shown in Fig. 5 and is typical of that expected for an antibody-target protein complex, with residues in the CDR loops responsible for most of the contacts with IL-1β. The complex features a fairly large interface between the two proteins, with the buried surface area upon complex formation corresponding to 1930 ± 130 Ų. Residues were considered to be involved in intermolecular contacts at the interface if the distance between neighboring atoms corresponded to less than the sum of their van der Waals radii plus 0.5 Å (34). On this basis, 18 residues from both the scFv and IL-1β contribute to the protein-protein interface, as summarized in Table 1. For the scFv, four of the six available CDRs (L1, L3, H2, and H3) make significant contacts with IL-1β, and interestingly, 3 framework residues (Asp-1, Asp-191, and Lys-194) are also found at the interface.

TABLE 1.

Summary of the interface residues and interactions made in the scFv-IL-1β complex

In addition to the expected contacts from CDR loops in the scFv, three framework (FR) residues also interact with IL-1β. Residues highlighted with an asterisk are involved in hydrogen bonds across the interface.

Chain	scFv	IL-1β
VL
FR L1	Asp-1*	Ser-152, Ser-153*
CDR L1	Asn-28	Ala-1
	Ile-29*	Arg-4*
	His-30	Arg-4
	Asn-31*	Arg-4*
	Tyr-32	Arg-4
CDR L3	Trp-92	Arg-4, Ser-5, Pro-46
	Ser-93	Leu-6
	Leu-94	Pro-150, Val-151, Ser-152
VH
CDR H2	Ser-181	Asn-108
	Gly-183	Asn-108
	Gly-185	Gln-15
	Ser-186*	Asn-108*, Thr-147
	Thr-187*	Arg-11*
	Tyr-188	Phe-150
FR H3	Asp-191*	Val-151, Ser-152*
	Lys-194*	Glu-37*, Gln-39*
CDR H3	Lys-231*	Asn-53*, Asp-54, Glu-105*

Docking Protocol Robustness

The robustness of the docking procedure and in particular its dependence on the completeness of the backbone amide RDC data were evaluated by repeating the docking calculations with subsets of the RDC data randomly removed. The full set of backbone amide RDC data obtained covered 65% of the scFv residues and 70% of the IL-1β residues. Removal of up to 30% of the RDC data obtained, resulting in coverage of 46% of the scFv residues and 48% of the IL-1β residues, led to the production of several equally populated clusters from the docking calculations. However, the cluster of complex structures with the lowest overall energy and best agreement with the NMR data were closest to the scFv-IL-1β complex obtained with the full data set (backbone r.m.s.d.s of 1.5 ± 0.15 Å for all residues). As expected, removal of over 50% of the RDC data collected resulted in significant variability between the clusters of scFv-IL-1β complexes produced by docking, with no reliable criteria on which to select the correct complex structure. This analysis clearly illustrates the importance of obtaining a good coverage of RDC measurements to obtain a reliable complex structure by NMR restraint-driven docking. For the scFv-IL-1β complex reported here, this corresponds to backbone amide RDC data for over 60% of the residues in each protein, which is likely to reflect the requirement for tight protein complexes in general.

The impact of the long range, intramolecular H^N to H^N NOE-derived distance restraints on the docking calculations was assessed by comparing the structures obtained for the scFv-IL-1β complex with and without these restraints included. The removal of the NOE-derived restraints had no significant effect on the complex structures obtained. This probably reflects the initial correctness of the input homology model for the scFv and input crystal structure for IL-1β, which both fully satisfied all relevant NOE restraints. However, the inclusion of NOE data would be very important in correcting any significant errors in homology models for scFvs or in interpreting large conformational changes in the proteins on complex formation.

ScFvs as Models for Fabs

To the best of our knowledge, no direct evidence has been previously presented to show that a scFv and Fab with identical V_L and V_H domains bind in an essentially identical manner to a target protein. A comparison of the ¹⁵N/¹H HSQC spectra for ¹⁵N-labeled IL-1β bound to an equivalent scFv and Fab is shown in Fig. 6. The spectra of IL-1β bound to the scFv and Fab look nearly identical in terms of the positions of signals, which provides direct evidence that scFvs bind in a very similar manner to equivalent Fabs. Very subtle chemical shift differences can be seen in the N- and C-terminal regions of IL-1β, which are involved in interactions with the antibody fragments (Fig. 6 and supplemental Fig. 4). However, these are approximately one-tenth of those induced by scFv or Fab binding (Fig. 3b) and must reflect only very subtle variations in the interactions with the Fab and scFv, which are unlikely to be detectable by other NMR measurements, such as intermolecular NOEs, or by x-ray crystallography.

FIGURE 6.

Comparison of IL-1β binding to equivalent scFv and Fab antibody fragments. Shown are overlaid ¹⁵N/¹H HSQC spectra for ¹⁵N-labeled IL-1β bound to unlabeled scFv in red and unlabeled Fab in green. The spectra overlay extremely closely, showing that the interactions with IL-1β are essentially identical when bound to either the scFv or the Fab.

DISCUSSION

The work reported here clearly demonstrates that an NMR restraint-driven docking approach can be successfully used to determine a reliable and informative model for the structure of a scFv-target protein complex. One limitation of the approach described is the lack of direct experimental data to define the conformations of amino acid side chains in scFv-target protein interfaces, which in the model reported here are simply a best computational solution to optimize the interactions of side chains involved in the interface. The precise scFv-IL-1β interactions involving side chains should therefore be viewed as reasonable possibilities, rather than confirmed interactions, but provide a good basis for the assessment of potentially key interactions by further experimental work, such as site-directed mutagenesis. Detailed analysis of the scFv-IL-1β interface and comparison with other antibody-antigen structures are summarized in Table 1 and supplemental Table 2. The nature of the interface, in particular the high content of aromatic and non-polar residues and the size of the buried surface area, are consistent with a representative sample of previously reported antibody-target protein complexes (35–42).

Of particular importance for the design and humanization of therapeutic antibodies is a knowledge of which residues from the antibody make interactions with the target protein. In the scFv-IL-1β complex, 18 of the scFv residues make contacts with IL-1β and are distributed equally between the variable heavy and light domains, as summarized in Table 1. Residues in CDRs L1 and H2 make the most interactions with IL-1β with CDRs L2 and H1 playing no role in binding. Interestingly, of the eight representative antibody complexes selected for comparison from the Protein Data Bank (43), three (2GHW, 1LK3, and 1DZB) had interactions with the target protein involving framework residues as well as CDR loops (defined using the International ImMunoGeneTics (IMGT) information system (44)). This feature is also seen in the scFv-IL-1β structure reported here, with 3 scFv framework residues contacting IL-1β (Table 1). A comparison of the interface characteristics of antibodies selected as therapeutics with ones not selected specifically for this purpose (supplemental Table 3) reveals no obvious differences in the size of the contact surface, the types of interactions, or the locations of residues involved in interactions.

The strikingly similar spectra seen for IL-1β bound to equivalent scFv and Fab antibody fragments (Fig. 6) provide direct evidence that scFvs bind target proteins in an essentially identical manner to Fabs, which makes scFv-target protein complexes an attractive target for structural studies directed at a knowledge-based approach to rational design and humanization of antibodies. In addition to scFvs and Fabs targeted at IL-1β, we have obtained equivalent quality NMR data for complexes formed with a number of secreted proteins, including sclerostin (45). This strongly suggests that NMR spectroscopy will provide a reliable and fairly widely applicable approach for determining high resolution models of the structures of scFv-target protein complexes, which has the potential to have a major impact on the rational design, optimization, and humanization of therapeutic antibodies.