Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two

M.H. Parker1,2, Y. Chen1,3, F. Danehy1,4, K. Dufu1,5, J. Ekstrom1,6, E. Getmanova1,7, J. Gokemeijer1, L. Xu1,2 and D. Lipovsek1,8,9

1Phylos, Inc., succeeded by Compound Therapeutics, 100 Beaver Street, Waltham, MA 02453, USA 2Present address: Merrimack Pharmaceuticals, Inc., Cambridge, MA 02142, USA 3Present address: Novartis Institute for Biomedical Research, Cambridge, MA 02139, USA 4Present address: Idenix, Cambridge, MA 02138, USA 5Present address: Harvard Medical School, Boston, MA 02115, USA 6Present address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA 7Present address: Massachusetts General Hospital, Boston, MA 02114, USA 8Present address: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

9 To whom correspondence should be addressed. E-mail: dlipovsek{at}ml1.net


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The tenth human fibronectin type three domain (10Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from 10Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related 10Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (Kd) of 13 nM, is highly stable [melting temperature (Tm) = 62°C] and soluble, whereas the second, which binds VEGF-R2 with 40x higher affinity, is less stable (Tm < 40°C) and relatively insoluble. We used our understanding of these two 10Fn3 derivatives and of wild-type 10Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (Kd = 0.59 nM), stability (Tm = 53°C) and solubility. Our findings illustrate that 10Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type 10Fn3 sequence is mutated in order to satisfy target-binding requirements.

Keywords: antibody mimic/fibronectin/Fn3/VEGF-R2


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The ability of the immune system to produce binding proteins specific to almost any macromolecular target has led to the widespread use of antibodies as research reagents and as drugs. In addition to full-length polyclonal and monoclonal antibodies made in experimental animals (Koehler and Milstein, 1975Go), antibody fragments with high affinity for their antigens have been developed as in vitro and in vivo reagents (Dall'Acqua and Carter, 1998Go; Hudson and Souriau, 2003Go). The advantages of antibody fragments over full-length antibodies are that the fragments are easier to manipulate by molecular biology techniques and to express in microbial systems and that their three-dimensional structures are easier to determine and to interpret. In addition, selections from antibody-fragment libraries with diverse complementarity-determining regions (CDRs) can be performed in vitro, under well-defined and controlled conditions.

Currently, the smallest widely used antibody fragments are single-chain antibodies [covalently linked variable heavy (VH) and light (VL) domains; ~260 residues] (Clackson et al., 1991Go; Marks et al., 1991Go; Barbas et al., 1992Go) and single-domain antibodies (monomeric variable domains; ~120 residues) (Ward et al., 1989Go; Davies and Riechmann, 1995Go; Martin et al., 1997Go; Reiter et al., 1999Go; Nuttall et al., 2000Go; Conrath et al., 2001Go; Holt et al., 2003Go; Revets et al., 2005Go). An attempt to work with even smaller antibody fragments led to the selection of ‘minibodies’, 61-residue subdomains of the antibody heavy-chain variable domain (Pessi et al., 1993Go) that bound IL-6 (Martin et al., 1994Go, 1996Go). Those minibodies characterized in detail (Pessi et al., 1993Go; Bianchi et al., 1994Go) were found to be less stable and considerably less soluble than natural variable domains, suggesting that VH and VL domains may be the smallest antibody fragments with a stable fold.

An alternative approach to specific target-binding reagents with properties favorable for in vitro manipulation uses proteins that are not directly related to antibodies. The goal of such scaffolds (Skerra, 2003Go; Mathonet and Fastrez, 2004Go; Nygren and Skerra, 2004Go), also known as antibody mimics, is to imitate as many of the favorable properties of antibodies and antibody fragments as possible, such as tight, specific binding and low toxicity. At the same time, antibody mimics aim to avoid some of the limitations of antibodies, such the requirement of most natural antibody domains for intradomain disulfide bonds and the tendency of less stable antibody fragments to aggregate. We chose to focus on the tenth human fibronectin type III domain (10Fn3) (Koide et al., 1998Go; Xu et al., 2002Go) due to its small size, structural similarity to antibody variable domains and human origin.

Like an antibody variable domain, 10Fn3 is a sandwich of two anti-parallel ß-sheets, with solvent-accessible loops near the two ends of the polypeptide chain (Figure 1) (Main et al., 1992Go; Dickinson et al., 1994Go). The three loops near the N-terminus of 10Fn3, named BC, DE and FG, can be considered structurally analogous to the VH complementarity-determining regions CDR1, CDR2 and CDR3, respectively. The fact that the sequences of loops BC, DE and FG vary among Fn3 domains (Bork and Doolittle, 1992Go) suggests that the Fn3 fold may be able to tolerate artificially introduced loop-sequence diversity. This was confirmed by Koide and co-workers, who showed that derivatives of human 10Fn3 with four-glycine insertions in the BC, DE and FG loops are folded proteins, albeit destabilized by 1.2, 2.3 and 0.4 kcal/mol, respectively (Batori et al., 2002Go).



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Fig. 1. NMR solution structure of human 10Fn3 domain (Main et al., 1992Go). The loops randomized in the antibody-mimic library, BC (23–29), DE (52–55) and FG (78–87), and also the heavy atoms of their side chains, are shown in light blue, green and red, respectively, except for FG residues 78, 81, 82 and 86, which differ between VR28 and 159 and are colored white. The two residues outside the randomized loops that mutated in 159, 8 and 56, are colored magenta and orange, respectively. The N-terminus is dark blue and the C-terminus dark red. All molecular graphics figures generated by PyMOL (www.pymol.org, DeLano Scientific).

 
Despite the fact that wild-type 10Fn3 contains no cysteines and hence no disulfide bonds, it is extremely stable, with a melting temperature above 80°C and reported free energy of unfolding between 6.1 and 9.4 kcal/mol (Plaxco et al., 1997Go; Cota et al., 2000Go; Batori et al., 2002Go). The absence of cysteines makes 10Fn3 straightforward to produce in Escherichia coli and to refold, if necessary. Finally, the presence of Fn3 domains in normal human serum at a high concentration of 0.1 mg/ml (Cenbrowski and Mosherb, 1984Go) raises the possibility of using Fn3-based antibody mimics as protein drugs.

Several different antibody-mimic libraries have been constructed by randomizing the CDR-like loops of the human 10Fn3 domain. Selections using phage display (Koide et al., 1998Go; Richards et al., 2003Go; Karatan et al., 2004Go), mRNA display (Xu et al., 2002Go) and yeast two-hybrid system (Koide et al., 2002Go) have generated target-binding Fn3 with dissociation constants as low as 20 pM (Xu et al., 2002Go). In addition to binding purified target protein in solution, selected Fn3 variants have been shown to recognize their target when they are immobilized on an array (Xu et al., 2002Go), perform similarly to monoclonal antibodies in western blotting and immunoprecipitation experiments (Karatan et al., 2004Go) and have biological activity in cell culture. For example, an {alpha}vß3-integrin-binding Fn3 variant recognizes the integrin which is expressed on cell surface and inhibits {alpha}vß3-dependent cell adhesion (Richards et al., 2003Go).

To date there have been few reports of the biophysical properties of selected Fn3 domains. The ubiquitin-binding variant analyzed in the most detail was shown to be destabilized by 2 kcal/mol and was relatively insoluble (Koide et al., 1998Go). Yet robust biophysical properties will be as important as high affinity in determining the usefulness of new Fn3-derived proteins. Therapeutic applications, in particular, require a long serum half-life in patients, which, in turn, depends on high thermodynamic and kinetic stability and on high solubility (Willuda et al., 1999Go). In order to evaluate the biophysical properties of Fn3-like proteins isolated from a heavily randomized population, we focused on Fn3 variants that bind human VEGF-R2 (also known as KDR and Flk-1), an attractive target for anti-vascular and anti-angiogenic cancer therapy (Brekken and Thorpe, 2001Go; Zhu et al., 2002Go; Glade-Bender et al., 2003Go).

This study is based on two VEGF-R2-binding antibody mimics that were previously selected and affinity-matured from an Fn3-based library with 21 randomized residues (Xu et al., 2002Go) using mRNA display (Roberts and Szostak, 1997Go; Lipovsek and Pluckthun, 2004Go). We found that the increase in affinity for VEGF-R2 during affinity maturation was associated with a significant loss of stability and solubility. Using structure-based, site-directed mutagenesis, we then engineered antibody mimics that combine high affinity for VEGF-R2 with high stability and solubility. Affinity and specificity of the engineered Fn3-based antibody mimics for their target are similar to those of monoclonal antibodies and their biophysical properties are comparable to those of antibody variable domains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antibody-mimic clones VR28 and 159

The sequences of the starting VEGF-R2-binding antibody mimics, VR28 and 159, are shown in Figure 2.



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Fig. 2. Sequences of VR28, 159 and 159 derivatives. BC, DE and FG loops are shown in italics in the wild-type sequence. Residues identical with the corresponding residues of wild-type 10Fn3 are shown as ‘.’; mutated residues that are identical with the corresponding residues in VR28 are underlined.

 
Site-directed mutagenesis

Derivatives of 159 were made using the QuikChange site-directed mutagenesis kit (Stratagene). In order to obtain numerous substitutions at position 56, 159 was mutagenized using an oligonucleotide with the codon NNS (where S is an equimolar mixture of G and C) instead of the codon for Ala56. The mutated DNA was transformed into XL1-Blue (Stratagene) E.coli, subcloned and identified by sequencing. Clones with every possible substitution at position 56 except for Cys, Met, Trp and Tyr were expressed in high-throughput mode and scored for their tendency to aggregate upon prolonged heating.

The three clones where the selected 159 loop BC, DE or FG was replaced by the equivalent wild-type 10Fn3 loop were constructed essentially as described by Xu et al. (Xu et al., 2002Go), except that single clones instead of a library were used as the template and that wild-type fragments of 10Fn3 instead of randomized fragments were introduced into the construct. To make 159(wt DE), residues 52–56 (i.e. loop DE plus the adjacent position 56) were exchanged with the corresponding wild-type residues (Figure 2).

Radioassay for VEGF-R2 binding

35S-labeled VR28, 159 and 159(Q8L,A56E) domains were produced and purified as described previously (Xu et al., 2002Go). A 100 µl amount of each domain at 0.2 nM was incubated for 1 h at 30°C with 100 nM human VEGF-R2, murine VEGF-R2 or human IgG (all from R&D Systems) or without target. The complex between the target and the radiolabeled Fn3 domains was captured by incubating the mixture with 75 µl of Protein A magnetic beads (Dynal) in 50 mM HEPES, 150 mM NaCl, 0.02% Triton X-100 and 1% BSA. A 10 min incubation at room temperature was followed by four 1 min washes in the same buffer. The protein was eluted from washed beads with 100 µl of 0.1 M KOH. Volumes of 50 µl of the mixture applied to Protein A magnetic beads, of the elution and of stripped beads were dried on a LumaPlate-96 (Packard). The amount of 35S on the plate was measured using a TopCount NXT instrument (Packard).

Expression and purification

All clones were expressed in E.coli BL21(DE3) pLysS (Invitrogen), in a construct that encoded residues 1–101 of each Fn3 derivative followed by His6. A 500 ml amount of LB with 50 µg/ml kanamycin and 34 µg/ml chloramphenicol was inoculated with 10 ml of an overnight culture, then grown at 37°C, with shaking at 225 r.p.m., to an A600 of 0.5–0.8. The culture was induced with 1 mM IPTG, shaken for another 12–18 h at 25°C and harvested by centrifugation at 3000 g. The cells were resuspended in 50 ml of 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 5 mM CHAPS, 25 mM imidazole and 1x Complete EDTA-free Protease Inhibitor Cocktail (Roche), then sonicated on ice at 80 W for four 15 s pulses separated by 10 s pauses. The soluble and the insoluble fractions were separated by a 30 min centrifugation at 30 000 g, 4°C.

To purify the protein from the soluble fraction (for wild-type 10Fn3 and for VR28), the supernatant was tumbled for 1 h, at 4°C, with 10 ml of TALON Superflow Metal Affinity Resin (Clontech) pre-equilibrated with wash buffer (50 ml of 50 mM phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 25 mM imidazole). The resin was then washed with 250 ml of wash buffer, followed by 250 ml of PBS, pH 7.4, 25 mM imidazole.

To purify the protein from the insoluble fraction (for 159 and all its derivatives), the pellet was dissolved in 15 ml of 7 M guanidine hydrochloride, 125 mM Tris, pH 8.0, and centrifuged for 10 min at 2000 g; the supernatant was then incubated with 10 ml of TALON Superflow Metal Affinity Resin and the resin was washed as described above for the soluble fraction.

For both the soluble and the insoluble fractions, purified protein was eluted with 100–150 ml of PBS, 500 mM imidazole and dialyzed extensively, at 4°C, against PBS. Any precipitate was removed by filtering at 0.22 µm (Millipore). When required, the protein was concentrated using UltraFree concentrators with a molecular weight cut-off of 5 kDa (Millipore). Each sample was analyzed by size-exclusion chromatography in PBS as described below. Those purified proteins which appeared as a mixture of monomer and dimer, were further purified on a Superdex 75 Hi-Load 16/60 size-exclusion column controlled by an AKTA Prime chromatography system (Amersham Biosciences). The peak corresponding to the monomer was collected, concentrated to 0.5–5 mg/ml and frozen at –80°C. The final yields of all the Fn3 domains purified for this study were between 5 and 20 mg per liter of culture.

To screen the 16 substitutions at position 56, the purification procedure was adapted to a high-throughput format. Each mutant was expressed in 5 ml of LB medium, 50 µg/ml kanamycin; after induction with 1.0 mM IPTG, the cultures were grown for 3 h at 30°C, 350 r.p.m. and harvested by centrifugation. Each cell pellet was resuspended in 0.5 ml of 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM CHAPS, 40 mM imidazole, 1x Complete EDTA-free Protease Inhibitor Cocktail with 1 mM PMSF (Roche) and 2 µg/ml aprotinin (Roche), by shaking at room temperature and 250 r.p.m., for 30 min. The lysates were centrifuged for 45 min at 3200 g, 4°C. The pellets were solubilized in 6 M GdnHCl, 50 mM sodium phosphate, 40 mM imidazole, pH 8.0, then loaded on to 96-well Swellgel nickel chelating plates (Pierce) pre-equilibrated with 6 M GdnHCl, 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM CHAPS, 40 mM imidazole. The mixture of resin and sample was incubated at room temperature for 5 min, then the plate was subjected to vacuum to remove the flow-through. Each well was washed with >40 column volumes of 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM CHAPS, 40 mM imidazole. The histidine-tagged protein was eluted at 4°C with 100 µl of 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 10% glycerol, 10 mM CHAPS, 40 mM imidazole, 50 mM EDTA.

Thermal stability/solubility screen

Our high-throughput screen for protein stability and solubility is based on the observation that aggregated particles, but not soluble protein, absorb light at 340 nm (Winder and Gent, 1971Go) and that unfolded proteins in the absence of denaturant are prone to aggregation. The sensitivity of the assay to aggregate can be adjusted by using a higher wavelength (for lower sensitivity) or using a fluorescence instead of a UV spectrophotometer, with both the emission and the excitation wavelength set to 340 nm (for higher sensitivity).

The A56X mutants purified by the high-throughput method were dialyzed into PBS and filtered at 0.22 µm. The concentration of each sample was determined by UV spectrophotometry (Pace et al., 1995Go) and adjusted to 0.10 mg/ml. For each sample, 200 µl were dispensed into a 96-well, flat-bottomed plate. The samples were overlaid with 70 µl of mineral oil per well and absorbance at 340 nm was measured. The plate was incubated at 60°C for 24 h, then allowed to cool to room temperature for 15 min before A340 was measured again. An increase in A340 after the 60°C incubation was taken as a measure of heat-induced aggregation. When the experiment was performed on VR28-derived clones with known calorimetric melting temperatures (Tm), A340 was found to correlate inversely with thermal stability (data not shown), validating the use of this method as a first screen for protein stability.

Determination of dissociation constants by surface plasmon resonance

All measurements were performed at 25°C on a BIAcore 2000 instrument. Target protein was immobilized on a CM5 chip (BIAcore) using amine coupling. Human VEGF-R2-Fc fusion (R&D Systems) was immobilized at 1000–1100 RU, human IgG (R&D Systems) was immobilized at 2000 RU and murine VEGF-R2 (R&D Systems) was immobilized at 600 RU. VEGF-R2-binding antibody mimics were analyzed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20 at eight concentrations between 10 and 70 nM and the VEGF-A control (R&D Systems) was analyzed at 0.1–2 nM. The flow-rate was 30 µl/min; association and dissociation times were 2 and 10 min, respectively. The chip was regenerated after each run by a 30 s pulse of 10 mM glycine.HCl, pH 1.5. Each concentration was run in duplicate and the response from an empty flow cell and from buffer injections was subtracted from each data set.

To test the specificity of antibody mimics to human VEGF-R2, chips with immobilized human VEGF-R2, human IgG and murine VEGF-R2 were mounted in series and the same antibody mimic [VR28, 159 or 159(Q8L,A56E)] flowed through all three cells at concentrations between 250 nM and 5 µM.

The data were analyzed using BIAeval (BIAcore) software, with a global fitting to the 1:1 (Langmuir) binding model. There were no indications of mass transport-limited kinetics or of other complications.

Analytical size-exclusion chromatography

Samples were filtered at 0.1 µm (Millipore) and injected on to a Superdex 75 HR 10/30 column controlled by an AKTA Purifier 10 system (Amersham Biosciences). Elution was monitored at 215 nm. The correlation between the retention volume and molecular weight was established using bovine serum albumin (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen (25.0 kDa), RNase A (13.7 kDa) and aprotinin (6.5 kDa) standards.

Prediction of pI values

The pI values of Fn3 mutants with different charged residues in position 56 were predicted using the program SEDNTERP (http://www.rasmb.bbri.org/rasmb/windows/sednterp-philo/).

Differential scanning calorimetry

Purified antibody-mimic samples at 0.3–0.8 mg/ml were analyzed in a Nano II differential scanning calorimeter (Calorimetry Sciences). The samples were scanned at 2°C/min from 5 to 100°C. The data were fitted to a non-two-state model using Origin software (OriginLab).

Determination of the free energy of unfolding using guanidine denaturation

Antibody-mimic samples at 0.1 mg/ml were incubated in 0–6.25 M guanidine hydrochloride (GdnHCl) for at least 6 h in PBS, at room temperature. Fluorescence was measured with a Hitachi F-2500 spectrofluorimeter in a 10 mm cuvette at 25°C, with excitation at 280 nm and emission at 350 nm; both slit widths were 2.5 nm. The equilibrium constant of unfolding, Kunf, was calculated from the fluorescence intensity (F) at each concentration of GdnHCl using the relationship Kunf = (FfF)/(Ff Fu), where Ff is the fluorescence intensity of folded protein and Fu is the fluorescence intensity of folded protein. In the case of 159, the lowest observed fluorescence was assumed to be Ff. The free energy of unfolding ({Delta}Gunf) at each concentration of GdnHCl was calculated by {Delta}Gunf = –RTlnKunf. {Delta}Gunf was plotted against concentration of GdnHCl and fitted, using Origin, to the linear equation {Delta}Gunf = {Delta}Gunf(H2O)m[GdnHCl] (Kellis et al., 1988Go), where {Delta}Gunf(H2O) is the free energy of unfolding in the absence of denaturant. The difference between {Delta}Gunf of two different Fn3-like domains was calculated using the equation {Delta}{Delta}Gunf (50)AB =mav([GdnHCl]50 B – [GdnHCl]50 A), where mav is the average slope for the two domains and [GdnHCl]50 is the concentration of GdnHCl at which half of the molecules in a sample are folded and the other half unfolded (Sali et al., 1991Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Characterization of VEGF-R2-binding antibody mimics VR28 and 159

None of the VEGF-R2-binding antibody mimics discussed here have a significant sequence homology with VEGF. The sequence of the VEGF-R2-binding domain VR28 differs from that of wild-type 10Fn3 in 20 of the 21 positions randomized in the Fn3-based library, preserving only the wild-type Pro25. Not surprisingly, the mutation of a fifth of the wild-type sequence resulted in a significant destabilization of the Fn3 domain: the VR28 Tm is lower than that of wild-type 10Fn3 by 22°C and its free energy of unfolding is lower by 3.5 kcal/mol (Table I). Still, VR28 is completely folded at 37°C; it can be purified either from the soluble fraction of E.coli lysate or from inclusion bodies with yields above 20 mg/l and it remains soluble in PBS at 4°C when concentrated to 25 mg/ml. Unlike the wild-type 10Fn3, metal chelate-purified VR28 protein is a mixture of monomers and dimers that interconvert slowly at room temperature (Table I). Whereas VR28 monomers bind VEGF-R2 with a Kd of 13 nM, isolated VR28 dimers show no detectable binding to VEGF-R2 immobilized on a BIAcore chip (data not shown).


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Table I. Properties of wild-type 10Fn3 and of VEGF-R2-binding antibody mimics

 
An effort to improve the affinity of VR28 for VEGF-R2 had led to the development of a related clone, 159. Clones VR28 and 159 have identical BC and DE loops, but they differ in four of the 10 residues in the FG loop (Figures 1 and 2), which has the sequence VAQNDHELIT in VR28 and MAQSGHELFT in 159. Of the FG residues that differ between the two clones, two hydrophobic residues of VR28 have been replaced by two other hydrophobic residues in 159 (V78 M and I85F) and one polar residue has been replaced by another polar residue (N81S); less conservatively, one negatively charged residue in VR28 has been replaced by the uncharged, flexible glycine (D82G).

The sequence that was not randomized intentionally during library construction, i.e. the residues outside loops BC, DE and FG, is identical between VR28 and wild-type 10Fn3. In contrast, two 159 residues outside the randomized loops mutated during affinity maturation. One of these mutations occurred near the flexible N-terminus (L8Q) and the other occurred immediately adjacent to the DE loop (T56A). Residues 8 and 56 are far apart in the sequence, but they are relatively close in space in folded, wild-type 10Fn3 domain (Figures 1 and 3B).



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Fig. 3. GdnHCl-induced denaturation of VR28 and 159 monitored by fluorescence. (A) Fluorescence of VR28 (filled triangles) changed from ~0.1 to 2 (arbitrary units) on addition of denaturant. Denaturation curves similar to VR28 were observed for all the other clones discussed here, with the exception of 159 (open triangles), which had a higher fluorescence in 0 M GdnHCl and a lower fluorescence in high-concentration GdnHCl. One possible reason for this discrepancy is illustrated in (B), which shows the position of the single Trp (white) in 10Fn3 in relation to the two scaffold positions that were mutated in 159, 8 (magenta) and 56 (orange). Light blue, residues in loop BC; green, residues in loop DE; dark blue, N-terminus.

 
As was the case for VR28 and for wild-type 10Fn3, the mutation of six of the 101 residues led to significant differences in target binding and in biophysical properties between VR28 and 159 (Table I). Most importantly, 159 binds VEGF-R2 nearly two orders of magnitude more tightly than does VR28; the Kd values of 159 and VR28 are 0.34 and 13 nM, respectively (Table I). The 159 is found exclusively in inclusion bodies; once purified, it is at least 50 times less soluble than VR28, with a solubility in PBS at 4°C of 0.4 mg/ml. Surprisingly, 159 is entirely monomeric, which suggests that, in this family of Fn3 domains, solubility and the tendency to dimerize are not directly related. That the difference in dimerization is not an artifact of different expression levels or of variations in the purification procedure, but is an equilibrium property associated with the two different sequences, was demonstrated by a simple size-exclusion chromatography (SEC) experiment. When VR28 monomer was purified by gel filtration, then incubated at room temperature for 18 h, the sample re-equilibrated to ~55% monomer and 45% dimer; in contrast, the 159 sample remained 100% monomeric (data not shown). Nevertheless, 159 is less stable than VR28 (Table I, Figure 3). In addition to being the least stable of the Fn3 domains studied, 159 showed unusual behavior both in thermal- and guanidine-denaturation experiments. First, in differential scanning calorimetry (DSC), 159 showed several overlapping lower-temperature transitions in addition to the major transition at 52°C (Figure 4). This behavior may mean that the denaturation of 159 is not a simple two-state transition or, alternatively, that another endothermic transition, such as dissociation of soluble aggregates, takes place at between 32 and 42°C. Second, the GdnHCl denaturation curve for 159 is qualitatively different from all the other domains studied, including its derivatives (Figure 3A). The most striking difference is the relatively high fluorescence shown by the 159 sample in denaturant-free buffer. Again, this may mean that a subpopulation of 159 protein is unfolded even in the absence of denaturant; on the other hand, the native structure of 159 may be sufficiently different from those of all other clones that the fluorescence of Trp 22 is not quenched as efficiently as in other Fn3 domains. Figure 3B, which shows the position of Trp 22 between Leu8 and Thr56, the two scaffold positions mutated in 159, supports the latter explanation.



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Fig. 4. Stability of VR28, 159 and 159 derivatives measured by DSC and by GdnHCl-induced denaturation. (A) and (C) show the dependence of heat capacity on temperature as determined by DSC. The peak of each transition occurs at the Tm of the protein and the area under the curve is the calorimetric enthalpy of unfolding ({Delta}Hcal). (B) and (D) show the dependence of free energy of unfolding on concentration of GdnHCl. The intercept of each linear fit at 0 M GdnHCl yields an estimate of {Delta}Gunf under native conditions and the distance between the lines in the area of the denaturation transition is the {Delta}{Delta}Gunf between the two proteins. (A) and (B): wild-type 10Fn3, solid line in (A), bullets in (B); 159(wt DE), dash-dotted line in (A), open circles in (B); VR28, dashed line in (A), solid triangles in (B); 159, dotted line in (A), open triangles in (B). (C) and (D): 159(Q8L,A56E), solid line in (C), crosses in (D); 159(Q8L), dash-dotted line in (C), open squares in (D); 159(A56E), dashed line in (C), solid squares in (D); 159, dotted line in (C), open triangles in (D).

 
A preliminary observation that the VR28 family of antibody mimics compete with VEGF for binding to VEGF-R2 in cell assays and that lower IC50 corresponds to lower Kd values (S.Shamah, personal communication), makes the comparison of VEGF-R2 binding in vitro between these antibody mimics and the natural ligand, VEGF, particularly interesting. The BIAcore-determined koff of VR28 from human VEGF-R2 is 1.3 times that of VEGF, whereas the koff of 159 and 159 derivatives [159(Q8L), 159(A56E) and 159(Q8L,A56E)] range from 0.16 to 0.38 times the koff of VEGF (Table I, Figure 5). The kon of VR28 is only 0.07 times the kon of VEGF and the kon values of the rest of the family range from 0.13 to 0.32 times that of VEGF (Table I).



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Fig. 5. Binding of 159(A56E) and VEGF to VEGF-R2. VEGF-R2-Fc was immobilized on a BIAcore CM5 chip. 159(A56E) at concentrations between 10 and 70 nM (A) and VEGF at concentrations between 0.1 and 1.2 nM (B) were introduced into the mobile phase. The change in refractive index due to binding (upper panel) and the difference between the signal and the mathematical fit (lower panel) are shown.

 
Replacement of selected sequences with wild-type sequences

In the comparison of wild-type 10Fn3, VR28 and 159, the sequences most similar to the wild-type were also the most stable and soluble. This suggested that the biophysical properties of 159 could be improved by replacing as much of its mutated sequence as possible with wild-type 10Fn3 residues. An earlier selection of Fn3-like antibody mimics for binding to TNF-{alpha} (Xu et al., 2002Go) yielded several clones that bound their target when one of their three CDR-like loops had the wild-type 10Fn3 sequence. Moreover, not all CDR loops in every antibody interact with the antigen: for example, VHH domains appear to interact with their antigens primarily through the CDR3 (Muyldermans, 2001Go). Consequently, it is plausible that not all 22 non-wild-type residues in clone 159 are required for VEGF-R2 binding and that some of those residues can be replaced with wild-type 10Fn3 residues to improve the biophysical properties of the antibody mimic without abolishing its affinity for VEGF-R2. The first two strategies attempted here were to replace single selected loops BC, DE or FG with the analogous wild-type 10Fn3 loops and to reverse the L8Q mutation.

Each of the three loops was replaced by the corresponding wild-type 10Fn3 sequence. The properties of the resulting clones, 159(wt BC), 159(wt DE) and 159(wt FG), are listed in Table I. None of the three clones showed detectable binding to VEGF-R2. Replacement of either the BC or the FG loop with the wild-type sequence led to a modest improvement in stability and solubility of 159, but in both cases a significant proportion of the protein was dimeric. In contrast, replacement of the 159 DE loop and of the adjacent residue 56 (LQPPA) with the corresponding wild-type residues (GSKST) led to a dramatic improvement in stability and solubility and preserved the 100% monomeric nature of 159. 159(wt DE) shows a single thermal transition with a Tm 18°C higher than that of 159. In addition, the {Delta}Gunf of 159(wt DE) is 4.5 kcal/mol higher than that of 159 and only 2.5 kcal/mol lower than that of wild-type 10Fn3 (Figure 4).

When a single selected residue rather than an entire loops was reverted to wild-type, we observed a similar pattern of improved biophysical properties; for example, the reversion of position 8 in clone 159 to leucine increased the Tm by 4°C and eliminated the lower temperature transitions. Its stability at 25°C increased by 1.0 kcal/mol (Table I). In contrast to substitutions of entire loops, the single-residue reversion diminished rather than eliminated affinity for VEGF-R2. For example, 159(Q8L) had a Kd 6-fold higher than 159.

Engineered variants of 159 with substitutions at positions 8 and 56

Spontaneous mutations in position 56 occurred in approximately half of the clones recovered in an earlier selection against TNF-{alpha} (Xu et al., 2002Go), and also in other clones affinity-matured for VEGF-R2 binding (E.Getmanova, personal communication). Such mutations were selected despite a relatively low rate of PCR mutagenesis in the ‘constant’ regions of Fn3-based libraries, suggesting that position 56 may contribute to binding of at least two different targets. In other words, there might be an advantage to randomizing position 56 together with the adjacent stretch 52–55 that makes up the DE loop. Since not all possible residues at position 56 are likely to have been sampled by accidental PCR mutagenesis, a residue not yet observed at this position might confer upon 159 a better combination of affinity and stability than the Ala56 of clone 159. Consequently, we made 15 of the 19 possible amino acid substitutions at that position, excluding only the sulfur-containing residues (Cys, Met) and two of the three aromatic residues (Trp, Tyr). We screened the resulting 15 clones and the original clone, 159, for solubility after an extended incubation at 60°C.

The 159(A56X) derivatives varied widely in their tendency to aggregate after being incubated at an elevated temperature (Figure 6). Negatively charged residues at position 56 caused little or no aggregation, whereas positively charged residues resulted in the most aggregation. Of the two acidic substitutions, A56E preserved the 100% monomeric nature of 159, but A56D led to a 50:50 mixture of monomer and dimer; consequently, 159(A56E) was selected for further study. The differences in predicted pI values for 159(A56X) derivatives were relatively small: 6.7 for 159 and other mutants with an uncharged side chain at position 56, 6.5 for 159(A56D) and 159(A56E) and 7.0 for 159(A56K) and 159(A56R.)



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Fig. 6. Aggregation of 159(A56X) derivatives after 24 h of incubation at 60°C. A large increase in A340 after heating is assumed to correlate with lower solubility, lower thermal stability or a combination of the two.

 
The details of biophysical and binding properties of 159(A56E) and also of 159(Q8L,A56E), its derivative with the wild-type Leu at position 8, are shown in Table I and in Figure 4C and D. The replacement of Ala56 with Glu increased the stability of 159 by 0.5 kcal/mol and slightly increased its Kd for VEGF-R2 from 0.34 to 0.59 nM. The additional Q8L reversion stabilized the protein by another 0.9 kcal/mol, but also increased its Kd further, to 2.1 nM. Double-mutant-cycle analysis (Figure 7) (Horovitz et al., 1990Go) shows that the stabilizing effects of mutations Q8L and A56E are nearly additive, with a {Delta}{Delta}{Delta}Gunf of only 0.1 kcal/mol. We conclude that amino acid residues at positions 8 and 56 modulate Fn3 stability independently of each other.



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Fig. 7. Additivity of the effects of Q8L and A56E mutations on 159 stability. Double-mutant cycle analysis highlights that mutation Q8L makes a similar contribution to stability whether or not mutation A56E is present and vice versa. {Delta}{Delta}Gunf1, {Delta}{Delta}Gunf2, {Delta}{Delta}Gunf3 and {Delta}{Delta}Gunf4 are the differences between free energy of unfolding between two adjacent mutants. {Delta}{Delta}{Delta}Gunf is the difference between {Delta}{Delta}Gunf2 and {Delta}{Delta}Gunf1 and between {Delta}{Delta}Gunf4 and {Delta}{Delta}Gunf3.

 
Specificity of VR28, 159 and 159(Q8L,A56E)

To evaluate the specificity of antibody-mimic binding to VEGF-R2, we measured the affinity of VR28, 159 and 159(Q8L,A56E) for murine VEGF-R2, which is 85% identical in sequence with human VEGF-R2, and for human IgG, which contains the Fc region also present in the human VEGF-R2-Fc chimera used in the mRNA-display selection of VR28 and 159. In an assay using in vitro-produced, 35S-labeled VR28, 159 and 159(Q8L,A56E), none of the three clones tested bound the surrogate targets above background level (Figure 8). When the affinities were measured by BIAcore, using immobilized targets, we detected no binding of any clone to the human IgG and no binding of VR28 to murine VEGF-R2. The 159 and 159(Q8L,A56E) both bound murine VEGF-R2 with a Kd of ~3 µM, i.e. with affinity 3–4 orders of magnitude lower than their affinity for human VEGF-R2.



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Fig. 8. Specificity of binding of VR28, 159 and 159(Q8L,A56E). 35S-labeled, in vitro-translated antibody mimics were incubated with different targets, which were then captured on magnetic beads with immobilized Protein A. The amount of radioactivity associated with the beads is a measure of binding: h, binding to human VEGF-R2; m, binding to murine VEGF-R2; i, binding to human IgG; b, background binding with no target.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
How well do antibody mimics based on the human 10Fn3 domain mimic antibodies? In particular, are they suitable for use as in vitro affinity reagents or therapeutics? We addressed these questions by studying a set of related antibody mimics directed against VEGF-R2, a potential target for anti-angiogenesis therapy (Brekken and Thorpe, 2001Go; Zhu et al., 2002Go; Glade-Bender et al., 2003Go). This family comprises an Fn3 domain selected by mRNA display (VR28), its affinity-matured derivative (159) and seven site-directed mutants designed to modulate their function and biophysical properties.

Antibody-mimic function was followed by measuring their affinity for VEGF-R2 and their biophysical properties were analyzed by measuring their stability, solubility and tendency to dimerize. Both tight binding and robust biophysical properties are essential in affinity reagents and, especially, in therapeutic proteins. For example, high affinity for the target reduces the therapeutic dose and thus the probability of side-effects. At the same time, highly stable and soluble proteins are more resistant to proteases and less likely to aggregate, leading to a longer life in the bloodstream.

The Fn3-based antibody mimics examined here show many similarities to antibodies and to antibody fragments. Their Kd for VEGF-R2 range from 0.34 to 13 nM, well within the 10–10–10–8 M range of therapeutic antibodies approved to date (http://www.fda.gov/cber/products.htm) (Reichert, 2002Go) and similar to the affinity of in vitro-selected antibody fragments against the same target (Lu et al., 2003Go; Zhu et al., 2003Go). Like antibodies, Fn3-based antibody mimics show a high specificity for their target: all the variants tested showed at least a 1000-fold lower affinity for murine than for human VEGF-R2, despite the fact that the sequences of these two receptors differ by only 15%. Moreover, the loss of affinity for VEGF-R2 when any one of the three selected loops is replaced by the wild-type sequence suggests that at least two loops are involved in target binding, similarly to multiple CDRs participating in antigen binding. An alternative explanation would invoke indirect effects of one or more loops on the conformation of the actual target-binding sequences. Indirect structural effects are also a likely explanation for the accumulation of ‘scaffold mutations’, substitutions in the ß-sheet of Fn3 during in vitro affinity maturation. These mutations are reminiscent of framework mutations in affinity-matured antibodies, where such mutations mediate the improved affinity without the mutated residues being in direct contact with the target (Foote and Winter, 1992Go; Hawkins et al., 1993Go; Arkin and Wells, 1998Go; Boder et al., 2000Go). Three-dimensional structures of complexes between antibody mimics and their targets will be needed to distinguish between direct target binding and indirect structural effects of the Fn3 residues that are critical for function. In contrast with the reports that most of the improvement in the affinity of affinity-matured antibody fragments is due to a slower koff (Yang et al., 1995Go; Pini et al., 1998Go), our affinity-matured Fn3-based antibody mimic (159) has both a koff that is 0.12 times that of the parent clone (VR28) and a kon that is 4.8 times that of the parent clone.

A major difference between Fn3-based antibody mimics and natural antibodies is their size. The low molecular weight of Fn3, ~10 kDa, suggests that a single Fn3 domain would be removed from the bloodstream by rapid renal clearance. Residence time in plasma, and also the ability to penetrate solid tumors, for Fn3-based antibody mimics will need to be manipulated by engineering their size, e.g. by conjugation with polyethylene glycol (Clark et al., 1996Go) or by head-to-tail multimerization of multiple Fn3 domains in an arrangement similar to natural fibronectin (Leahy et al., 1994Go).

Since the complex, multi-domain structure of full-length antibodies makes it difficult to quantify their stability, we compared the stability of antibody mimics with that previously reported for isolated antibody domains. Table II shows that the free energy of unfolding of the VEGF-R2-binding Fn3 domains, 3.8–5.1 kcal/mol, falls within the ranges reported for VH and VL domains, which are 3.3–13 and 3.6–8.2 kcal/mol, respectively, and overlaps the range determined for camel VHH domains, 5.0–8.4 kcal/mol (Ewert et al., 2002Go, 2003Go). In addition, thermal denaturation experiments (Figure 4) revealed that, with the exception of variant 159, none of these antibody mimics shows detectable unfolding in PBS below 40°C. The likelihood that these antibody mimics would be completely folded under physiological conditions must be confirmed by further stability studies in sera and in live animals.


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Table II. Thermodynamic stability of VEGF-R2-binding, Fn3-derived antibody mimics compared with the published stability of antibody variable domains

 
It appears that the stability of VEGF-R2-binding Fn3 domains is limited by the selected sequence in the DE loop, LQPP. The replacement of residues 52–56, LQPPA, with the equivalent wild-type 10Fn3 sequence, GSKST, stabilizes the protein by a {Delta}{Delta}Gunf of 4.5 kcal/mol and by a {Delta}Tm of at least 18°C. Most likely this is due to the difficulty of accommodating a constrained sequence with two adjacent prolines in a tight turn (Figure 1). In the future, such destabilizing sequences can be avoided by alternative library design or by selection for stability in addition to affinity (Shusta et al., 2000Go; Matsuura et al., 2002Go; Pedersen et al., 2002Go). Nevertheless, this example illustrates how the unusually high stability of wild-type 10Fn3 ensures workable biophysical properties even after some of the stability is ‘traded’ for binding affinity.

Several VEGF-R2-binding antibody mimics described here tend to dimerize or aggregate. Two lines of evidence suggest that the non-wild-type, selected residues in or adjacent to the three CDR-like loops are likely to be involved in this dimerization. First, variants that differ in sequence only at four positions in these regions can have dramatically different tendencies to dimerize or aggregate; and second, dimers of VR28 do not bind VEGF-R2, suggesting that the target-binding residues are occluded by the Fn3:Fn3 interface. The DE region appears to play a critical role in solubility in addition to its role in stability; for example, the replacement of Ala 56 in clone 159 with Glu improved the solubility ~10-fold. Since the wild-type residue (Thr56) does not appear to impart the best stability and solubility (Figure 4), position 56 is a logical candidate to randomize in the construction of future Fn3-based libraries.

In conclusion, the properties of Fn3-based, VEGF-R2-binding antibody mimics presented here support the notion that such binding proteins could be developed into useful affinity reagents and drugs. The most promising features of this family of proteins are their high affinity for VEGF-R2, high thermostability and ease of production in bacterial expression systems. Further studies will be required to determine whether similar properties are shared by Fn3-like domains that bind targets other than VEGF-R2 and to explore the pharmacokinetics and in vivo biological effects of Fn3-based antibody mimics.


    Notes
 
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Steve Shamah, Michael Wittekind and Martin Wright for helpful discussions and Edward Fritsch for his support of the project. Funding to pay the Open Access publication charges for this article was provided by Compound Therapeutics.


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Received April 21, 2005; accepted July 4, 2005.

Edited by Elizabeth Meiering