Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the VL domain of the scFv-0 (VL-VH) molecule is primarily responsible for formation of a tetramer–trimer equilibrium

Olan Dolezal1,3, Ross De Gori1, Mark Walter2, Larissa Doughty2, Meghan Hattarki1, Peter J. Hudson1 and Alexander A. Kortt1

1 CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville 3052, Victoria, Australia 2 Present address: Saint Vincent’s Institute of Medical Research, Fitzroy 3065, Victoria, Australia

3 To whom correspondence should be addressed. E-mail: olan.dolezal{at}csiro.au


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the VL to VH orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the VH-VL isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between VL-VH trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55–L59 and L13–L17 from the two adjacent VL domains within the VL-VH trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer–trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

Keywords: multimerization/scFv/tetrabody/tetramer/trimer equilibrium/triabody


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recombinant single-chain Fv (scFv) antibody fragments can be engineered to assemble into stable multimeric forms of high avidity that retain parent IgG specificity to target antigens and haptens [reviewed by Kortt et al. (Kortt et al., 2001Go)]. The multimeric size of an scFv functional unit can be controlled by selection of the linker length and orientation of V-domains (Kortt et al., 1997aGo; Arndt et al., 1998Go; LeGall et al., 1999Go; Dolezal et al., 2000Go). In constructs with linker lengths of 3–10 residues, the VH domain is unable to associate with its attached VL domain to generate a monomeric Fv fragment (~25 kDa) and instead VH and VL domains from one scFv molecule associate with those from a second scFv molecule to form a non-covalent bivalent dimer (~50 kDa), termed a diabody (Holliger et al., 1993Go; Perisic et al., 1994Go). In our model scFv of the murine anti-neuraminidase antibody (NC10), assembled in the conventional VH to VL orientation (Malby et al., 1993Go), we found that when the linker length is reduced to less than three residues or when VH and VL domains are directly ligated to each other, three scFv molecules associate to form a triabody (~75 kDa) (Kortt et al., 1997aGo; Atwell et al., 1999Go). By comparison, linking of V-domains in the reverse VL-VH orientation resulted in a significantly different oligomeric profile of the multimers formed by this scFv (Dolezal et al., 2000Go). As was the case for the VH-VL orientation, diabodies were the predominant conformation for three-, four- and five-residue linkers. However, a tetramer–trimer equilibrium mixture with the tetrabody (~100 kDa) as the preferred multimeric conformation was found for VL-VH domains joined directly (scFv-0). Furthermore, the transition between diabodies and tria/tetrabodies was not as distinct as for the VH-VL orientation on reducing the linker length to less than three residues. Instead, scFv proteins containing either two or one residue linkers formed equilibrium mixtures consisting of dimers, trimers, tetramers and higher molecular mass multimers.

Multimeric scFvs offer an improvement over the parent IgG in tumour imaging and in vivo targeting since these smaller molecules allow for faster blood clearance and for rapid tumour uptake and retention at the tumour site (Adams et al., 1998Go; Wu et al., 1999Go; Tahtis et al., 2001Go). However, for successful application of scFvs and their multimers in vivo it is necessary that these molecules have a defined multimeric state. Our experience with NC10 and other scFvs has shown that obtaining highly stable scFv trimers may not always be achieved by simply modifying the linker length (Kortt et al., 2001Go). In order to understand some of the factors that influence the assembly and transition between scFv multimers, the refined crystal structure for NC10 Fv fragment (Malby et al., 1994Go, 1998Go) was used to construct models of dimeric, trimeric and tetrameric scFvs (Atwell et al., 1999Go; Dolezal et al., 2000Go). In the studies presented here we utilized these models to evaluate the hypothesis, which predicts that steric restrictions within the NC10 scFv-0 (VL-VH) trimer may be causing the preference for four scFv-0 molecules to assemble into tetramers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence numbering

Antibody residues were numbered according to Kabat et al. (Kabat et al., 1991Go) and for NC10 correspond exactly to Malby et al. (Malby et al., 1993Go). Residues in the light and heavy chains of the scFv are superscripted and prefixed L and H. Mutant scFvs are described using the following nomenclature: GluL57->GlyL57 (or EL57->GL57) denotes that glutamate residue 57 of the VL domain (variable region from antibody light chain), in the wild-type NC10 scFv, was replaced with glycine.

Molecular modelling

Computer models of NC10 scFv triabodies and tetrabodies were generated using Fv modules that corresponded to the coordinates of the NC10 Fv domain (PDB reference code 1NMB) (Malby et al., 1994Go, 1998Go). Fv modules were initially manipulated as rigid bodies with the O molecular graphics package (Jones et al., 1991Go) such that the diabody structure and gross alignment of linkers corresponded to the crystal structure described by Perisic et al.(1994)Go and the triabodies corresponded to the model described by Kortt et al.(1997a)Go. Modeling of the NC10 scFv multimer complexes involved visual analysis of the protein model to find the most appropriate amino acid to mutate and then optimize its approximate location and orientation on the molecule surface where the side chain rotamers could make favourable interactions. The Biopolymer module within the SYBYL software package (version 6.7; Tripos, St. Louis, MO) was used for mutating, manipulating and building the protein. After a short initial minimization of 1000 cycles, 5 ps of molecular dynamics simulation was performed at 1500 K using a time integration step of 0.5 fs. The peptide was annealed to 300 K by reducing the temperature of the heat bath by 10 K every 5 ps followed by energy minimization. The molecular mechanics calculations were carried out using the Kollman parameter set (Weiner et al., 1986Go). The protein complex was again initially optimized for 1000 iterations using the Steepest Descent method to relax the geometry of the side chains and thus remove any bad contacts. Then, with all hydrogen atoms removed, the structure was optimized for a further 10 000 steps using the Conjugate Gradient method with a cutoff at 0.05 kcal/mol. A validation of the model stereochemistry was performed on the final output structure using the PROCHECK program (Laskowski et al., 1993Go).

Construction of NC10 scFv mutants

General cloning procedures and protocols used for the construction, identification and expression of pGC/NC10scFv (VL-VH) mutant clones were described previously (Dolezal et al., 2000Go). The starting templates for mutagenesis were the ‘single-FLAG’ versions of the pGC/NC10 scFv constructs. The second FLAG encoding DNA sequence was removed by digesting the ‘double-FLAG’ wild-type constructs with SacII restriction enzyme and subsequently ligating the agarose gel-purified linear vectors to generate ‘single-FLAG’ pGC/NC10 scFv wild-type plasmids (Figure 1Go). All pGC/NC10 scFv-0 mutant clones were constructed using a QuikChange site-directed mutagenesis kit (Stratagene) in accordance with the protocol supplied by the manufacturer. A single pair of complementary oligonucleotides (Table IaGo) was used to mutate the GAG codon encoding GluL57 in the pGC/NC10 scFv-0wt construct to the GGC codon encoding GlyL57 to generate pGC/NC10 scFv-0M1 mutant plasmids. Ten pairs of complementary nucleotides (Table IbGo ) were used to mutate the CTG codon encoding LeuL15, also in the pGC/NC10 scFv-0wt construct, to generate following pGC/NC10 mutant clones: scFv-0M2, scFv-0M3, scFv-0M4, scFv-0M5, scFv-0M6, scFv-0M7, scFv-0M8, scFv-0M9, scFv-0M10 and scFv-0M11. Similarly, the QuikChange site-directed mutagenesis kit and M5S and M5AS primers (Table IbGo) were used to construct the pGC/NC10 scFv-1M5 and pGC/NC10 scFv-2M5 plasmid clones (Figure 1Go). Finally, the pGC/NC10 scFv-15M1, scFv-15M2, scFv-15M4 and scFv-15M5 clones were constructed by excising the NcoI/XhoI NC10 VL gene fragments from the corresponding pGC/NC10 scFv-0M(1, 2, 4, 5)constructs and inserting them into the pGC/NC10 scFv-15wt vector to generate the pGC/NC10 scFv-15M(1, 2, 4, 5) plasmids (Figure 1Go).



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Fig. 1. (a) Schematic diagram of the NC10 scFv-0 constructs in pGC. (b) Amino acid sequences of the C-terminus of the VL domain, the peptide linker (in bold) and the N-terminus of the VH domain used in each of the mutant constructs.

 

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Table I. Oligonucleotide pairs used for site-directed mutagenesis of pGC/NC10 scFv clones at positions L57 (a) and L15 (b)
 
Expression and purification of NC10 scFvs

All pGC/NC10 scFv constructs were expressed using Escherichia coli TOP 10F' strain (Invitrogen) according to the previously published protocol (Dolezal et al., 1995Go). Expression experiments were terminated 3 h post-induction and proteins were isolated from the E.coli periplasm as described (Dolezal et al., 2000Go). Each scFv was isolated from the periplasmic fraction by affinity chromatography using either an anti-FLAG IgG column (Brizzard et al., 1994Go) or an NC10 anti-idiotype 3-2G12 IgG column (Kortt et al., 1997aGo). The affinity columns were prepared by immobilizing antibodies (anti-FLAG or 3-2G12 IgG) on to Mini-Leak Low gels (Kem-En-Tec, Denmark) according to manufacturer’s instructions. The scFv protein in the periplasmic fraction was bound to the affinity gels by a batch procedure (Power et al., 2001Go). Routinely, 200–1000 ml of periplasmic extract (from 1–5 l of shake flask expression culture) was mixed with 20 ml of affinity gel and gently mixed at 4°C for 1 h to absorb the FLAG tagged or 3-2G12 IgG binding proteins. The affinity gel was then transferred on to a sintered funnel, washed with PBS, pH 7.4, to remove unbound proteins and finally transferred to a column. After further washing with PBS, the bound protein was eluted with 100 mM glycine, pH 3.0, and the collected solution (~13.5 ml) was neutralized with 1.5 ml of 1 M Tris–HCl, pH 8.

Biochemical characterization of NC10 scFv proteins

The purity of the NC10 scFvs was monitored by SDS–PAGE and western blot analysis as described previously (Kortt et al., 1994Go). The concentrations of the scFv fragments were determined spectrophotometrically using values for the extinction coefficient ({varepsilon}0.1%) at 280 nm of 1.65 for NC10 scFvs calculated as described by Gill and von Hippel (Gill and von Hippel, 1989Go). The relative molecular mass and oligomeric status of each affinity-purified NC10 scFv were compared by size-exclusion chromatography using a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) at 21°C in PBS, pH 7.4, calibrated with Bio-Rad Gel Filtration Standard proteins. The flow rate was 0.5 ml/min and the absorbance of the effluent stream was monitored both at 214 and 280 nm.

Standard protocol for analysis of NC10 scFv proteins

Affinity-purified proteins were analysed by size-exclusion chromatography immediately after elution and neutralization (t = 0 days). Following this initial analysis, proteins were concentrated to 1 mg/ml using a MICROSEP micro-concentrator (10 kDa cut-off; PALL GelmanSciences) and dialysed against 2x2 l of PBS–0.02% (w/v) NaN3 for 16 h. Proteins were dispensed in 200 µl batches and stored at room temperature (21°C), 4°C and -20°C. Stored samples were re-analysed by size-exclusion chromatography after the dialysis (t = 1 day) and then after a set period of storage (t = 30 days).

Kinetic binding experiments

A BIAcore 1000 instrument (Pharmacia Biosensor, Uppsala, Sweden) was utilized to measure the binding interactions of various NC10 scFv-15 mutant proteins. In one series of experiments, tern N9 neuraminidase was immobilized on a CM5 sensor chip in 10 mM sodium acetate buffer, pH 4.0, via amine groups using the amine coupling kit (Pharmacia Biosensor) as described previously (Gruen et al., 1993Go). In a second series of experiments, NC10 anti-idiotype 3-2G12 IgG was also immobilized at pH 4.0 via amine groups. Binding analyses were performed in HBS buffer at constant flow rates of either 5 or 20 µl/min under conditions previously demonstrated to minimize mass transport and rebinding phenomena for these interactions (Kortt et al., 1997bGo, 1999Go). Neuraminidase, a homotetramer of 190 kDa, was not stable to acidic conditions and the surface was regenerated by running the dissociation reaction to completion (2000 s) before starting a new binding experiment. Immobilized 3-2G12 IgG was regenerated with 10 mM sodium acetate, pH 3.0, with negligible loss of binding activity. NC10 scFv protein samples for binding analyses were prepared prior to each experiment by size-exclusion chromatography on Superdex 200 to remove any lower or higher molecular mass species which may have formed upon storage (Kortt et al., 1994Go). The binding data were evaluated with BIAevaluation 3.0.2 software as described previously (Kortt et al., 1999Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Modeling studies and design of scFv-0 mutants

The initial structures used for modelling of the NC10 scFv-0 (VL-VH) multimers were single Fv units, which were manipulated as rigid bodies into a trimer with threefold symmetry (scFv3-0; Figure 2aGo) and a tetramer with fourfold (scFv4-0; Figure 2bGo) symmetry. These models assume that the symmetric Fv heads are assembled in a cyclic head-to-tail fashion compatible with scFv-0 linkage. The trimer model in particular, is based on the Fv fragment with a direct ligation of the VH and VL domains that formed an scFv-0 (VH-VL) multimer with three antigen-combining sites (Atwell et al., 1999Go). The scFv-0 multimers were then examined for potential clash sites in each of the triabody and tetrabody configurations. These examinations revealed that the loops L13–L17 and L55–L59 from the two adjacent VL domains might be sterically compromised within the scFv-0 trimer model. In particular, it was observed that the LeuL15 and GluL57 side chains from opposing loops protrude away from their parental C{alpha} backbone and towards each other, suggesting the potential for several clashes and close contacts (Figure 2cGo). Of the two possible side chain rotamers for leucine, the rotamer 1 model was chosen in the final model since it did not significantly clash with its parental VL residues. In contrast, the LeuL15 rotamer 2 model was discarded since the C{delta}1 atom interacts too closely with LeuL78 from the same VL framework (1.7 Å). Three different side chain conformations for glutamate were examined and shortest atomic distances with respect to leucine rotamer 1 were measured (Table IIGo). This analysis revealed that the glutamate rotamer 1 conformation was not plausible as the distances between interacting atoms within LeuL15 and GluL57 side chains fell below van der Waals forces threshold (~2.6 Å) and consequently these would be expected to interfere with each other in an unfavourable manner. In the case of GluL57 rotamer 2 and rotamer 3 (shown in Figure 2cGo), the relevant atomic distances appeared to satisfy the acceptable van der Waals forces threshold and consequently would not be expected to disrupt the trimeric arrangement (Table IIGo). However, since no other unfavourable interactions within the modelled trimer were clearly evident, it was suggested that rationally designed mutations at one or both of L15 and L57 sites might alleviate the apparent steric clashes and subsequently lead to the stabilization of the scFv-0 trimer. As discussed previously, the visual inspection of NC10 Fv crystal structures revealed that GluL57 and LeuL15 side chains appear to protrude away from their parental C{alpha} backbone. Consequently, neither of these two side chains is required for the stabilization of the V-domain ß-strand framework, for example through hydrogen bonding, nor is expected to play an important role in the VL to VH association. The substitutions of either of these two amino acids with alternative residues were, therefore, not expected to disrupt significantly the folding and association of the VL domain.



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Fig. 2. Molecular models (C{alpha} trace) of NC10 scFv-0 (VL-VH) trimer (a) and tetramer (b). The VL domains are shaded light grey and the VH domains are dark grey. To highlight the interactions across the Fv interface and the steric clash in a triabody configuration, loops L13–L17 and L55–L59 are shown in green and magenta, respectively. Bold arrows indicate the position of the binding sites. Numbers #1 to #4 designate individual scFv-0 monomer subunits that assemble into trimer and tetramer. (c) Stereo representation of the amino acids in the loop junction (L13–L17 and L55–L59) of the wild-type NC10 scFv-0 trimer model (GluL57 = rotamer 3 in Table IIGo). Black, red and blue spheres represent carbon, oxygen and nitrogen atoms, respectively. The figure was prepared using MOLSCRIPT (Kraulis, 1991Go) and RASTER3 D (Merrit and Bacon, 1997Go).

 

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Table II. Side chain rotamers of the LeuL15 and GluL57 residues and their interacting atomic distances (Å)
 
Construction and expression of NC10 scFv-0 mutant clones

The pGC/NC10 scFv-0 construct, in which the VL domain was linked directly to the VH domain, was used as a template to generate several constructs with mutations in codons encoding L15 and L57 residues. Thus, the glutamate residue at position L57 was replaced with glycine (M1 mutation) while 10 different amino acids were substituted for leucine at position L15. These LeuL15 mutations included substitutions with hydrophobic residues (alanine–M2, valine–M4, phenylalanine–M8 and isoleucine–M11), strongly charged residues (lysine–M3 and aspartate–M6) and polar residues (asparagine–M5, glutamine–M7, threonine–M9 and glycine–M10). All of the above-described pGC/NC10 scFv-0 mutants were constructed using site-directed mutagenesis strategy and the resulting constructs, and also the original wild-type scFv-0 construct, were expressed in E.coli TOP 10F' cells. The soluble NC10 scFv fragments were purified from periplasmic extracts by affinity chromatography. Typical yields of affinity-isolated scFv-0 fragments, migrating as single bands at 25 kDa on a reducing SDS–PAGE gel, were ~0.5 mg per litre of shake flask culture (data not shown).

Biochemical analysis of NC10 scFv-0 proteins

Size-exclusion chromatography on a calibrated Superdex 200 column was used to analyse the multimeric status of affinity-purified NC10 scFv-0 wild-type and mutant proteins. Analysis performed immediately after elution from the FLAG column (t = 0 days) revealed a large amount of trimer in all of the NC10 scFv-0 protein preparations (Figure 3Go). When compared with the wild-type scFv-0, which contained similar amounts of trimer and tetramer, the replacement of glutamate residue at position L57 in the wild-type with a glycine residue (M1 mutant), however, resulted in a substantial reduction in the amount of tetramer formed (Figure 3aGo). Similarly, the replacement of leucine residue at L15 with 10 other amino acids resulted in a decrease in the amount of tetramer formed (Figure 3b–dGo). Not surprisingly, the reduction in the amount of tetramer was less obvious when large hydrophobic residues, such as phenylalanine and isoleucine, were introduced (Figure 3bGo); however, it became fairly significant when smaller hydrophobic (valine and alanine; Figure 3bGo) or strongly charged (Figure 3cGo) and polar (Figure 3dGo) residues were substituted at L15. These results not only supported our modelled prediction that the LeuL15 and GluL57 residues are sterically compromised within the scFv-0 trimer molecule, but also indicated that this trimer can be potentially stabilized by introducing specific amino acid substitutions at these positions.



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Fig. 3. Size-exclusion chromatographic profiles of affinity-purified NC10 scFv-0 proteins on a calibrated Superdex200 HR10/30 column. All NC10 scFv-0 proteins were analysed immediately after purification on an anti-FLAG IgG affinity gel by loading 100 µl aliquots at a protein concentration of ~100 µg/ml. Superimposed on the same scales are profiles for (a) wild-type (LL15/EL57, solid line) and M1 mutant (LL15/GL57, dashed line), (b) hydrophobic residue substitutions at LL15: M2 mutant (AL15/EL57, solid line); M4 mutant (VL15/EL57, dashed line); M8 mutant (FL15/EL57, dotted line); M11 mutant; (IL15/EL57, short dashed line), (c) charged residue substitutions at LL15: M3 mutant (KL15/EL57, solid line); M6 mutant (DL15/EL57, dashed line), (d) polar residues substitutions at LL15: M5 mutant (NL15/EL57, solid line); M7 mutant (QL15/EL57, dashed line); M9 mutant (TL15/EL57, dotted line) and M10 mutant (GL15/EL57, short dashed line).

 
Interestingly, the Figure 3Go profiles also revealed a protein species that eluted at approximately same time as the wild-type NC10 scFv-15 monomer. When this species was peak-purified using size-exclusion chromatography and analysed on a reducing SDS–PAGE gel, it gave a single protein band at 25 kDa that co-migrated with the peak-purified scFv tetramer and trimer bands (Figure 4Go). The protein sequencing of the first 15 residues of tetramer, trimer and monomer proteins not only revealed their expected N-terminal sequence (DIELTQTTSSLSASK) but also confirmed that the correct mutation was introduced at L15. The amount of scFv-0 monomer species produced was variable for different mutants expressed, ranging from small amounts for the wild-type protein to >50% of total purified protein in case of the M3 mutant (Figure 3Go). As the V-domains in an scFv-0 monomer cannot associate to form an active Fv domain (owing to absence of a flexible linker sequence), this monomer was not expected to bind to the NC10 anti-idiotype 3-2G12 antibody. Consequently this monomer species was not recovered from periplasmic extracts when employing 3-2G12 IgG affinity gel for purification (data not shown). BIAcore experiments further confirmed that the scFv-0 monomer did not bind to either the 3-2G12 antibody or its original antigen, tern N9 neuraminidase (data not shown). Furthermore, re-chromatography experiments on a Superdex 200 column showed no evidence of conversion of the peak-purified monomer species into the tri–tetramer mixture and vice versa (data not shown). These findings suggested that the scFv-0 monomer species was irreversibly misfolded and therefore inactive.



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Fig. 4. Reducing SDS–PAGE of gel filtration-purified NC10 scFv-0 (M3, KL15/EL57) oligomeric forms stained with Coomassie Brilliant Blue G-250. Proteins were purified using anti-FLAG affinity chromatography, concentrated to 1 mg/ml, dialysed into PBS + 0.02% (w/v) azide and subjected to size-exclusion chromatography on a calibrated Superdex 200 column. Peaks corresponding to tetramer, trimer and monomer were collected individually and loaded on to 4–20% Tris–glycine gradient SDS–PAGE gel under reducing conditions. Lanes: 1, NC10 scFv-0 tetramer; 2, NC10 scFv-0 trimer; 3, NC10 scFv-0 monomer.

 
Stability of NC10 scFv-0 multimeric proteins

Previous observations showed a significant variability in the tetramer to trimer ratio in different NC10 scFv-0 wild-type protein preparations (Dolezal et al., 2000Go). The storage conditions and protein concentration affected the state of this equilibrium. Since it was not possible to purify and analyse various NC10 scFv-0 proteins simultaneously under identical conditions, a standard purification and analysis protocol (see Materials and methods) was adopted to account for any variability that may have resulted from this constraint. Consequently, all NC10 scFv-0 protein preparations shown in Figure 3Go were concentrated to 1 mg/ml and re-analysed by size-exclusion chromatography immediately after dialysis against PBS (t = 1 day) and then after one month of storage (t = 30 days). Typical size-exclusion chromatography profiles obtained for NC10 scFv-0 wild-type, M2 and M5 mutant proteins stored at different temperatures for 30 days are shown in Figure 5Go. The amount of tetramer in the scFv-0 wild-type protein sample increased significantly upon dialysis into PBS and subsequent sample concentration (compare Figures 3aGo and 5aGo). Small but significant variations in the amounts of wild-type tetramer species were observed after the long-term storage at different temperatures (compare Figure 5aGo with Figure 5b–dGo). The stability results obtained for the M2 and M5 mutants, however, showed no such significant increase in the amount of tetramer in either of these two protein samples (Figure 5a–dGo). Interestingly, the monomer protein disappeared from all three Superdex 200 profiles after 30 days of storage at 4 and 21°C (Figure 5c and dGo), suggesting that in contrast to tetramer and trimer, this species was highly susceptible to protein degradation.



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Fig. 5. Size-exclusion chromatograpic profiles on a calibrated Superdex 200 HR10/30 column showing affinity purified NC10 scFv-0 wild-type (LL15/EL57, solid line), M2 (AL15/EL57, dashed line) and M5 (NL15/EL57, dotted line) proteins on a calibrated Superdex 200 HR10/30 column. Following affinity-purification on an anti-FLAG IgG gel, all NC10 scFv-0 proteins were concentrated to 1 mg/ml, dialysed into PBS + 0.02% (w/v) azide and analysed by loading 10 µl aliquots: (a) immediately after dialysis (t = 1 day), (b) after 30 days of storage at -20°C, (c) after 30 days of storage at 4°C and (d) after 30 days of storage at room temperature (21°C).

 
To account for the relative differences in the tetramer–trimer equilibrium between all of the affinity-purified NC10 scFv-0 mutant proteins, extensive size-exclusion chromatographic analyses were performed at various stages of sample preparation and storage. The overall data representing three different expression experiments for each of these NC10 scFv proteins are recorded in Table IIIGo. These results clearly demonstrate that, when compared with the wild-type, the amount of tetramer formed was reduced for all of the NC10 scFv-0 mutant proteins at all stages of their analysis. The introduction of polar (Asn, Gln, Thr and Gly) and charged (Asp) residues at L15 had the most significant effect upon the tetramer–trimer equilibrium (see M5, M7, M9, M10 and M6 in Table IIIGo). In all of these proteins, virtually no tetramer species was observed immediately after the affinity purification. More importantly, no significant amount of tetramer was formed upon concentration of these samples and upon long-term storage at different temperatures, thus indicating that these mutations generated highly stable trimeric proteins. The introduction of hydrophobic residues (M2, M4, M8 and M11 mutants) at LeuL15 did not generate such a dramatic effect. Similarly to wild-type, all four of these scFv-0 mutant proteins clearly retained a tetramer–trimer equilibrium status with the only exception being the M2 (LeuL15->AlaL15) mutant for which the equilibrium mixture shifted significantly in favour of the trimer species (Table IIIGo). The long-term storage of these samples generally led to a small increase in the amount of tetramer formed and there appeared to be only minor differences between these samples when stored at different temperatures. An interesting result was observed when lysine was introduced at L15 (M3 mutant), where a relatively small amount of tetramer was observed immediately after affinity purification and following concentration to 1 mg/ml. A significant increase in the amount of tetramer was observed, however, upon long-term storage of this protein, especially when stored at -20°C.


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Table III. Size-exclusion chromatographic analysis of tetramer–trimer equilibrium for various NC10 scFv-0 proteins
 
When compared with the wild-type, the M1 mutation (GluL57->GlyL57) resulted in a reduction in the amount of tetramer formed at all stages of size-exclusion chromatographic analysis (Table IIIGo), thus suggesting that GluL57 does contribute to the steric clash within the trimer. No significant differences were observed, however, when single mutant constructs (e.g. ValL15/GluL57) and corresponding double mutant constructs (ValL15/GlyL57) were compared using the same experimental conditions as summarized in Table IIIGo (data not shown). Consequently, these findings suggested that the presence of a leucine residue at position L15 is primarily responsible for the formation of the tetramer–trimer equilibrium.

Binding analysis of NC10 scFv-0 and scFv-15 mutant proteins

To determine whether the mutations introduced at either LeuL15 or GluL57 modified the binding kinetics relative to the scFv wild-type proteins, several pGC/NC10 scFv-15 mutant monomer proteins, with mutations identical with those that were introduced into scFv-0 M1, M2, M4 and M5, were also prepared. The kinetic binding interactions of these scFv-15 mutant monomers and NC10 scFv-0 mutant trimers were evaluated using a BIAcore instrument. A comparison of the binding of NC10 scFv-15 wild-type and M5 monomers with scFv-0 M5 trimer to immobilized NC10 anti-idiotype 3-2G12 antibody showed that the scFv-15 monomers exhibited identical binding profiles (Figure 6aGo) whereas the scFv-0 M5 trimer showed a significantly slower dissociation rate (kd {approx} 6x10-5 s-1), consistent with multivalent binding (Figure 6aGo) as reported previously for scFv-0 (VH-VL) wild-type trimer (Kortt et al., 1997aGo). The other scFv-0 mutants showed similar dissociation rate constants (data not shown), confirming that the various mutations at LeuL15 and GluL57 did not affect the binding properties of the monomers and multimers. The binding kinetics of NC10 scFv-15 wild-type, M1, M2, M4 and M5 mutant monomers to immobilized tern N9 neuraminidase and 3-2G12 antibody were measured and the binding data were analysed globally using the 1:1 Langmuir model. The data for the scFv-15 wild-type (Figure 6bGo) and the mutants gave a good fit to the 1:1 model consistent with each monomer binding the antigen epitope via a 1:1 mechanism. The kinetic constants for the scFv-15 mutants were essentially the same as that for the wild-type, confirming that the mutations did not affect the binding properties of these scFvs monomers (Table IVGo).



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Fig. 6. (a) Sensorgrams showing the binding of NC10 scFv-15 (VL-VH) wild-type and M5 (NL15/EL57) mutant monomer (115 nM) and scFv-0 (VL-VH) M5 mutant trimer (230 nM) to immobilized NC10 anti-idiotype 3-2G12 antibody (1123 RU) at a constant flow rate of 5 µl/min with an injection volume of 35 µl. (b) Sensorgrams showing the binding of NC10 scFv-15 (VL-VH) wild-type monomer (14.4–230 nM) to immobilized tern N9 neuraminidase (1165 RU) at a constant flow rate of 5 µl/min with an injection volume of 35 µl. The circles are the fits to the data obtained on evaluation of each sensorgram with the 1:1 Langmuir binding model using simultaneous fitting of ka and kd as described in BIAevalution 3.0.2. For each cycle the dissociation was allowed to proceed by washing the surface with HBS until the response returned to the initial value.

 

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Table IV. Apparent kinetic rate constants and equilibrium binding constants for the interaction of NC10 scFv (VL-15-VH) wild-type and mutants with immobilized (a) tern N9 neuraminidase and (b) NC10 anti-idiotype antibody 3-2G12
 
The active multimeric status of various wild-type and mutant scFv multimers was confirmed further by preparing NC10 scFv trimer–(3-2G12 Fab)3 and NC10 scFv tetramer–(3-2G12 Fab)4 complexes and imaging them by electron microscopy according to previously described procedures (Lawrence et al., 1998Go). The results obtained were identical with those reported previously (Dolezal et al., 2000Go). Thus, for example, the NC10 scFv-0 (M4) tetramer/trimer–3-2G12 Fab complex appeared under an electron microscope as a mixture of Y- and X-shaped images consistent with one scFv trimer binding to three 3-2G12 Fab molecules and one scFv tetramer binding to four 3-2G12 Fab molecules, respectively (data not shown). These results, therefore, demonstrated that all of the mutants constructed during this study produced multimeric proteins with each binding site being fully active.

Effect of LeuL15->AsnL15 mutation within the NC10 scFv-1 and scFv-2 proteins

Having established that the substitution of the leucine residue at L15 with polar residues was primarily responsible for the stabilization of the NC10 scFv-0 in a trimeric arrangement, we sought to investigate the effects of similar mutations upon scFv-1 and scFv-2 (VL-VH) proteins, which were previously shown to form equilibrium mixtures of dimers, trimers, tetramers and higher molecular mass multimers (Dolezal et al., 2000Go). Modelling studies of scFv-2 and scFv-1 (VL-VH) dimers suggested that the same steric clashes involving LeuL15 may be responsible for the relatively instability of these two scFv multimers (data not shown). The LeuL15->AsnL15 (M5) mutation was therefore introduced into the pGC/NC10 scFv-1wt and scFv-2wt clones using the same strategy that was previously utilized for the construction of scFv-0 mutant clones. Following expression in E.coli TOP 10F' cells, the resulting NC10 scFv-1 and scFv-2 wild-type and M5 mutant proteins were affinity purified using 3-2G12 IgG gel and characterized by size-exclusion chromatography on a calibrated Superdex 200 HR 10/30 column. The elution profiles for the wild-type scFv-1 and scFv-2 protein were similar to those found previously (Dolezal et al., 2000Go), consisting of an equilibrium mixture of scFv dimers, trimers, tetramers and higher molecular mass multimers (Figure 7Go). The scFv-1 M5 protein also consisted of a similar equilibrium mixture but when compared with the scFv-1 wild-type protein it was biased towards the smaller species (Figure 7aGo). In contrast to the wild-type, the scFv-2 M5 protein yielded one major peak (Figure 7bGo) eluting at the same time as the NC10 scFv (VL-VH) dimer (Dolezal et al., 2000Go). Two minor peaks, eluting before the main dimer peak, corresponding to the trimer and tetramer species, were also observed in these elution profiles consistent with the previous results observed for scFv dimers. For all of these scFv species, no significant differences in the relative proportions of these multimers were observed upon long-term storage at different temperatures (data not shown).



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Fig. 7. Size-exclusion chromatographic analysis of NC10 scFv wild-type (LL15/EL57, solid line) and M5 (NL15/EL57, dashed line) proteins on a calibrated Superdex 200 HR10/30 column. (a) scFv-1 proteins; (b) scFv-2 proteins. Proteins were purified using NC10 anti-idiotype 3-2G12 IgG affinity gel, concentrated to 1 mg/ml, dialysed into PBS + 0.02% (w/v) azide and analysed by loading 10 µl aliquots.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In addition to the ‘in silico’ analysis of the model structures of the scFv-0 trimer and tetramer, the sequence alignment of murine kappa light chains (Kabat et al., 1991Go; Nieba et al., 1997Go) also played an important role in the design of various mutations at positions L15 and L57 within NC10 VL domain. Thus, the decision to create a GluL57->GlyL57 mutation was based on consensus sequences across all six subgroups of murine kappa light chains, which indicated that the glycine residue is highly conserved at position L57. In fact, the occurrence of GluL57 is rare and consequently it appeared that the introduction of glycine would not only allow for the insertion of a highly conserved amino acid at this position but would also result in the size reduction of side chain groups. Similarly, alanine and valine mutations at L15 were particularly suited to our mutagenesis studies not only because they occur fairly frequently at L15 (Nieba et al., 1997Go) but also because they were expected to reduce the predicted steric interference of the hydrophobic side chain group of leucine. In contrast, some of the other amino acids introduced at L15 did not necessarily fit the conserved residue or smaller size criteria during the mutagenesis design. For example, the LeuL15-> LysL15 and LeuL15->GlnL15 mutations not only introduced residues that are, from an evolutionary perspective (based on sequence alignments), extremely rare at position L15 but also produced residues with larger side groups. Introduction of polar residues such as asparagine and threonine was expected to reduce the relative hydrophobicity of the protruding L15 side chain and possibly create a potential for these side chains to participate in hydrogen bond formation between adjacent VL domains and, in so doing, stabilize the trimeric arrangement.

The size-exclusion chromatographic data obtained during this study showed that, apart from the tetramer–trimer equilibrium mixture, the affinity-purified NC10 scFv-0 (VL-VH) proteins (on a FLAG column) also contained a monomeric form. This species was initially assumed to be an Fv fragment occurring as a result of a proteolytic cleavage near the junction between VL and VH domains, as shown previously for the NC10 scFv-0 (VH-VL) protein (Kortt et al., 1997aGo). In that case, gel filtration data also revealed a presence of a 25 kDa protein species, which upon SDS–PAGE analysis dissociated into VH and VL species migrating separately as ~15 and ~14 kDa protein bands, respectively. In this study, the SDS–PAGE and N-terminal sequence analysis identified the NC10 scFv-0 (VL-VH) monomer species as intact 25 kDa protein with the expected N-terminal sequence for the NC10 VL domain. Furthermore, the lack of binding activity and no evidence for interconversion with the tetramer–trimer mixture strongly suggested that this protein species was, in fact, an incorrectly folded monomeric scFv-0. This finding contrasted with recent findings for an anti-Lewisy scFv-0 (VH-VL) trimer, which was shown to be in equilibrium with a freely interconvertible monomer species (B.E.Power and A.A.Kortt, in preparation). Misfolded monomer species might be occurring as a result of amino acid residues at the VL-VH junction acting as a ‘de facto linker and, in so doing, generating misfolded VL and/or VH domains that are unable to assemble into active Fv modules. The anti-Lewisy scFv-0 monomer, on the other hand, probably contains correctly folded VH and VL domains that assemble into an active multimer.

Previous studies of scFvs with long linkers showed that the introduction of mutations into framework regions of antibody fragments could prevent correct folding of individual V domains into their native conformations thus rendering them inactive (de Haard et al., 1998Go; Jung et al., 2001Go). The BIAcore experiments presented here demonstrated that mutations introduced into the NC10 VL domain at positions L15 and L57 did not affect the binding of various NC10 scFv-0 multimers either to neuraminidase or to 3-2G12 antibody. Furthermore, kinetic binding studies with several monomeric scFv-15 mutants showed that there were no significant difference in association and dissociation rate constants, thus suggesting that these mutations have no effect upon binding activity.

Most of the direct evidence presented to date has demonstrated that the length of the linker is the most important factor controlling the status of scFv oligomerization (Arndt et al., 1998Go; Atwell, et al., 1999Go; LeGall et al., 1999Go). The orientation of V domains in the scFv construct, however, can also play an important role (Dolezal et al., 2000Go). The results obtained in these studies have shown that a single amino acid residue (Leu at L15) is primarily responsible for the formation of NC10 scFv-0 (VL-VH) tetramer–trimer equilibrium. By substituting this leucine with an alternative residue, e.g. asparagine, a stable NC10 scFv-0 trimer was generated. Furthermore, the LeuL15->AsnL15 mutation within NC10 scFv-1 and scFv-2 proteins was also shown to affect significantly the multimeric status of these proteins. The effect of this mutation was particularly evident for the scFv-2, where this mutation resulted in conversion from a wild-type multimeric protein mixture into a protein that was mainly dimeric. This is an interesting finding, especially when considering that this mutation had a significant effect upon scFv-0 and scFv-2 while having only a limited effect upon scFv-1. Consequently, the length and the flexibility of the linker residues as well as the specific V domain sequences at the interface between adjacent Fv domains significantly influence the geometric parameters required for scFv proteins to attain particular oligomeric arrangements.

The occurrence of NC10 scFv-0, scFv-1 and scFv-2 (VL-VH) oligomeric equilibria presented an ideal opportunity to investigate how specific V domain sequences affect oligomerization states. Computer-generated models for NC10 scFv trimer and tetramer were sufficient to predict that a steric clash between LeuL15 from one VL domain and GluL57 from the adjacent VL domain within the scFv-0 trimer was responsible for its instability and reassembly to an apparently more stable tetramer. Similarly, when scFv-2 and scFv-1 (VL-VH) were modelled as dimers the same steric clashes were predicted. Further attempted modelling of various charged and polar residues at L15 within the scFv-0 trimer and scFv-2 models, however, failed to elucidate clearly the underlying principle for this stability. Since our scFv oligomer models are primarily based on the wild-type NC10 Fv structure, these models are limited, for example, in terms of knowledge regarding the effect of local sequence upon the conformational flexibility of the sterically compromised loops. Consequently, any tangible explanations for the relative stability of mutated scFv-0 trimers and scFv-2 dimers will only be possible if refined crystal structures for these multimeric proteins can be obtained. Nonetheless, we predict that the combination of the hydrophobic nature and relative size of the LeuL15 side chain in the wild-type NC10 scFv-0 (VL-VH) trimer is primarily responsible for the presumed steric interference with the L55–L59 loop from the adjacent VL domain. These predictions are supported by following findings: (1) a significant reduction of the amount of tetramer formed occurred when amino acid residues with smaller side chains were introduced at L15; (2) introduction of residues with larger side chains at L15 that were not hydrophobic also reduced the amount of tetramer formed; (3) replacement of glutamate with glycine at L57 only partially reduced the amount of tetramer formed; (4) comparisons of single mutant constructs with corresponding double mutant constructs did not result in a significant tetramer reduction, thus suggesting that the effect of a glutamate side chain at L57 upon the tetramer–trimer equilibrium is relatively minor.

Based on the results obtained with NC10 scFv multimers (VH-VL and VL-VH), we propose that as the linker length is shortened, the residues within loops that are located at the bottom of V domains come into direct contact with residues from the adjacent V domains and as a result destabilize the specific oligomeric arrangement. This destabilization may result either in the formation of an oligomeric equilibrium or in total elimination of smaller oligomeric states. These observations have important implications when aiming to engineer specific scFv multimers for in vivo use where it is necessary for these molecules to have defined oligomerization states. It may be tempting to assume that similar mutations to those made in this study will generate similar outcomes for other scFv multimers. The general applicability of this assumption is, however, difficult to predict, especially since well characterized trimers and/or tetramers have not been widely reported in the literature. Future studies will therefore need to be directed towards generating greater numbers of scFvs with very short linkers and applying the acquired knowledge for relating particular oligomeric states with specific V domain sequences.


    Acknowledgments
 
The authors thank Professors D.Metzger and R.Webster for providing the 3-2G12 and NC10 hybridoma cell lines, Drs A.McCoy and R.Malby for useful discussions and assistance with molecular modelling, P.Strike for performing N-terminal amino acid sequencing, L.Pearce for technical assistance and Drs S.Nuttall and J.Gorman for critical reviews of the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adams,G.P., Schier,R., McCall,A.M., Crawford,R.S., Wolf,E.J., Weiner,L.M. and Marks,J.D. (1998) Br. J. Cancer, 77, 1405–1412.[ISI][Medline]

Arndt,K.M., Muller,K.M. and Plückthun,A. (1998) Biochemistry, 37, 12918–12926.[CrossRef][ISI][Medline]

Atwell,J.L., Breheney,K.A., Lawrence,L.J., McCoy,A.J., Kortt,A.A. and Hudson,P.J. (1999) Protein Eng., 12, 597–604.[Abstract/Free Full Text]

Brizzard,B.L., Chubet,R.G. and Vizard,D.L. (1994) BioTechniques, 16, 730–735.[ISI][Medline]

de Haard,H.J.W., Kazemier,B., van der Bent,A., Oudshoorn,P., Boender,P., van Gemen,B., Arends,J.-W. and Hoogenboom,H.R. (1998) Protein Eng. 11, 1267–1278.[Abstract]

Dolezal,O., Coia,G., Guthrie,R.E., Lilley,G.G. and Hudson,P.J. (1995) Immunotechnology, 1, 197–205.[CrossRef][ISI][Medline]

Dolezal,O., Pearce,L.A., Lawrence,L.J., McCoy,A.J., Hudson,P.J. and Kortt,A.A. (2000) Protein Eng., 13, 565–574.[Abstract/Free Full Text]

Gill,S.C. and von Hippel,P.H. (1989) Anal. Biochem., 182, 319–326.[ISI][Medline]

Gruen,L.C., Kortt,A.A. and Nice,E. (1993) Eur. J. Biochem., 217, 319–325.[Abstract]

Holliger,P., Prospero,T. and Winter,G. (1993) Proc. Natl Acad. Sci. USA, 90, 6444–6448.[Abstract]

Jones,T.A., Zou,J.-Y., Cowan,S.W.,and Kjeldgaard,M. (1991) Acta Crystallogr., Sect. A 47, 110–119.[CrossRef][ISI][Medline]

Jung,S., Spinelli,S., Schimmele,B., Honegger,A., Pugliese,L., Cambillau,C. and Plückthun,A. (2001) J. Mol. Biol., 309, 701–716.[CrossRef][ISI][Medline]

Kabat,E.A., Wu,T.T., Perry,H.M., Gottensman,K.S. and Foeler,C. (1991) Sequences of Proteins of Immunological Interest. US Department of Health and Human Service, US Public Health Service, NIH, Bethesda, MD.

Kortt,A.A., Malby,R.L., Caldwell,J.B., Gruen,L.C., Ivancic,N.M, Lawrence,M.C., Howlett,G.J., Webster,R.G., Hudson,P.J. and Colman,P.M. (1994) Eur. J. Biochem., 221, 151–157.[Abstract]

Kortt,A.A. et al. (1997a) Protein Eng., 10, 423–433.[Abstract]

Kortt,A.A., Gruen,L.C. and Oddie,G.W. (1997b) J. Mol. Recogn., 10, 148–158.[CrossRef][ISI][Medline]

Kortt,A.A., Nice,E. and Gruen,L.C. (1999) Anal. Biochem., 273, 133–141.[CrossRef][ISI][Medline]

Kortt,A.A., Dolezal,O., Power,B.E. and Hudson,P.J. (2001) Biomol. Eng., 18, 95–108.[CrossRef][ISI][Medline]

Kraulis,P. (1991) J. Appl. Crystallogr., 24, 946–950.[CrossRef][ISI]

Laskowski,R.A, MacArthur,M.W., Moss, D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283–291.[CrossRef][ISI]

Lawrence,L.J., Kortt,A.A., Iliades,P., Tulloch,P.A. and Hudson,P.J. (1998) FEBS Lett., 425, 479–484.[CrossRef][ISI][Medline]

LeGall,F., Kipriyanov,S.M., Moldenhauer,G. and Little,M. (1999) FEBS Lett., 453, 164–168.[CrossRef][ISI][Medline]

Malby,R.L. et al. (1993) Proteins: Struct. Funct. Genet., 16, 57–63.[ISI][Medline]

Malby,R.L., Tulip,W.R., Harley,V.R., McKimm-Breschin,J.L., Laver,W.G., Webster,R.G. and Colman,P.M. (1994) Structure, 2, 733–746.[ISI][Medline]

Malby,R.L., McCoy,A.J., Kortt,A.A., Hudson,P.J. and Colman,P.M. (1998) J. Mol. Biol., 279, 901–910.[CrossRef][ISI][Medline]

Merrit,E.A. and Bacon,D.J. (1997) Methods Enzymol, 277, 505–524.[ISI]

Nieba,L., Honneger,A. Krebber,C. and Plückthun,A. (1997) Protein Eng., 10, 435–444.[Abstract]

Perisic,O., Webb,P.A., Holliger,P., Winter,G. and Williams,R.L. (1994) Structure, 2, 1217–1226.[ISI][Medline]

Power,B.E. et al. (2001) Cancer Immunol. Immunother., 50, 241–250.[CrossRef][ISI][Medline]

Tahtis,K., Lee,F.-T., Smyth,F.E., Power,B.E., Renner,C., Brechbiel,M.W., Old,L.J., Hudson,P.J. and Scott,A.M. (2001) Clin. Cancer Res., 7, 1061–1072.[Abstract/Free Full Text]

Weiner,S.J., Kollman,P.A., Nguyen,D.T. and Case,D.A. (1986) J. Comput. Chem. 7, 230–252.[ISI]

Wu,A.M., Williams,L.E., Zieran,L., Padma,A., Sherman,M., Bebb,G.G., Odom-Maryon,T., Wong,J.Y., Shively,J.E. and Raubitschek,A.A. (1999) Tumour Target., 4, 47–54.

Received June 10, 2002; revised October 21, 2002; accepted November 12, 2002.





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