Further improvement of broad specificity hapten recognition with protein engineering

Teemu Korpimäki1,3, Jaana Rosenberg2, Pekka Virtanen1, Urpo Lamminmäki1, Mika Tuomola1 and Petri Saviranta1

1 Department of Biotechnology, and 2 Department of Bio-Organic Chemistry, University of Turku, FIN-20520 Turku, Finland

3 To whom correspondence should be addressed. E-mail: teemu.korpimaki{at}utu.fi


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Sulfa-antibiotics (sulfonamides) are widely used in veterinary medicine. Meat and milk from treated animals can be contaminated with sulfa residues. Current sulfonamide assays are unfit for screening of food, because they are either too laborious, insensitive or specific for a few sulfa compounds only. An immunoassay for detection of all sulfas in a single reaction would be useful for screening. Previously we have improved the broad specificity sulfa binding of antibody 27G3 with random mutagenesis and phage display. In order to improve the properties of this antibody further, mutants from the previous study were recombined and more mutations introduced. These new libraries were enriched with phage display and several different mutant antibodies were isolated. The cross-reaction profile of the best mutant was better than that of the wild-type antibody and the mutants of the previous study: it was capable of binding 10 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas 5- to 11-fold better than the mutants of the previous study.

Keywords: DNA shuffling/drug residues/group specificity/phage display/sulfonamides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Sulfa-antibiotics (sulfonamides) are used in veterinary and human medicine for the treatment and prevention of microbial infections. Sulfas are also used as additives to animal feed, because of their growth hastening properties. If proper care is not observed after their use, foodstuffs (f. ex. meat and milk) derived from treated animals may well be contaminated with residual sulfonamides. These residues can cause unwanted reactions in some humans. It has been estimated that approximately 5% of the patients undergoing sulfonamide therapy have some kind of unwanted effects from the drugs (Sheth and Sporns, 1991Go). Thus, a maximum residue limit (MRL) for sulfonamides has been set to 100 µg/kg in the United States and the European Union. In Japan, it has been set as low as 20 µg/kg (Haasnoot et al., 2000Go; Muldoon et al., 2000Go).

Current sulfonamide detection methods are based on bacteriological growth inhibition (Murphy et al., 1986Go; Korsrud et al., 1998Go) or chromatography (Jennings and Landgraf, 1977Go; Abian et al., 1993Go; Volmer, 1996Go; Abjean, 1997Go; Le Boulaire et al., 1997Go; Dost et al., 2000Go). Single sulfonamide drugs have also been measured with immunochemical assays (Heering et al., 1998Go; Elliott et al., 1999Go; Akkoyun et al., 2000Go; Lee et al., 2001Go). The listed methods are either laborious or slow for mass screening or capable of detecting only a single analyte per each assay reaction, as is the case with the immunoassays.

Different sulfa-antibiotics (Figure 1Go) are structurally related, since they are N1-substituted derivatives of p-aminobenzenesulfonamide (sulfanilamide). Therefore, a rapid immunoassay capable of detecting the whole group of sulfonamides in one reaction should be possible. This kind of assay would be very useful for the mass screening of foodstuffs. The development of such a tool has proven to be difficult; despite several efforts (Sheth and Sporns, 1991Go; Spinks et al., 1999Go; Muldoon et al., 1999Go, 2000Go; Haasnoot et al., 2000Go; Korpimäki et al., 2002Go), a completely generic assay is still not available. The main obstacle seems to be the development of an antibody capable of binding all different sulfonamides with an affinity yielding sufficient assay sensitivity. It seems that all the antibodies generated so far bind the original immunogen or the phage display collection antigen and the structurally similar sulfonamides with greater affinity than the sulfonamides which are structurally divergent from these.



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Fig. 1. The structures of selected sulfonamides.

 
We previously used protein engineering to modify monoclonal antibody (Mab) 27G3A9B10 to recognize a wider range of structurally different sulfonamides with similar affinities (Korpimäki et al., 2002Go). Several of the obtained mutants had significantly altered binding properties and improved broad specificity. Unfortunately, none of the mutants were still not completely generic binders. In this study we generated new antibody libraries by recombining the enriched libraries of the previous study with DNA shuffling and introducing new mutations with error-prone PCR and oligonucleotide-directed mutagenesis (Stemmer, 1994Go; Lorimer and Pastan, 1995Go; Boder et al., 2000Go). The libraries were screened with phage display (for a recent review, see Wittrup, 1999Go) using three different selection schemes. The binding properties of the new mutants were evaluated in a competitive time-resolved fluoroimmunoassay.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Strains, plasmids, reagents and instruments

The bacterial host used throughout the work was Escherichia coli K12 strain XL1-Blue (Stratagene, La Jolla, CA). The vectors used (Figure 2Go) either belonged to or were derived from the pAK series of vectors (Krebber et al., 1997Go) and they were obtained as gifts from the lab of Andreas Plückthun (Biochemisches Institut, Universität Zürich, Switzerland). The helper phage used in the phage production was VCS-M13 (KanR, Stratagene). Wild-type antibody 27G3A9B10 (Haasnoot et al., 2000Go) was a gift from the lab of Willem Haasnoot (RIKILT-DLO, Wageningen, The Netherlands). The recombinant scFv27G3 antibody was cloned and the phage libraries displaying mutants of this antibody (Library 1 and 4) were constructed and enriched previously in our lab (Korpimäki et al., 2002Go).



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Fig. 2. The plasmid vectors used. Two different plasmids of the pAK series (Krebber et al., 1997Go) were used as the gene carrying vectors in this work. Marked in the maps are the E.coli propagation signal ColE1, the M13 phage packing signal f1-IG and chloramphenicol resistance selection marker Cam(R). The scFv (VL, VH and linker domains are marked) expression from both plasmids is controlled by the lac promoter (lac PO) repressed by the lac inhibitor (lacI). The signal sequence (pel B) activates transport of scFv to periplasm for folding.

 
All reagents used in the organic synthesis were commercially available and were of reagent grade or better. Rabbit anti-mouse IgG-coated microtitre plates, DELFIA assay buffer, DELFIA wash solution and DELFIA enhancement solution were obtained from Perkin-Elmer Life Sciences (Turku, Finland). The biotinylation reagent Biotin-XX-NHS (cat. 203114) was purchased from Calbiochem (San Diego, CA). LB agar plates, SB medium and SOC medium were prepared as described previously (Sambrook et al., 1989Go). Antibiotics used in the cultures were obtained from Sigma–Aldrich (Helsinki, Finland). The concentrations of the antibiotics were: chloramphenicol 25 mg/L, tetracycline 5 mg/L and kanamycin 50 mg/L. Isopropyl-ß-D-thiogalactopyranoside (IPTG) was purchased from Promega (Madison, WI). Various sulfa-antibiotics (Figure 1Go) and fraction V bovine serum albumin (BSA) were purchased from Sigma–Aldrich. The biotinylated sulfamerazine derivative and the europium chelate labeled sulfamerazine derivative (Eu-sulfamerazine) were synthesized in our lab as described earlier (Korpimäki et al., 2002Go). The concentration of the biotinylated derivative could not be determined due to low synthesis yields, so the dilution used is given in the text instead.

All sequencing was done using ABI PRISM 377 dye terminator cycle sequencer (Applied Biosystems, Foster City, CA). 1H NMR spectra were recorded at 400 MHz on a JNM-GX-400 spectrometer (JEOL, Peabody, MA) or at 200 MHz on a AM200 spectrometer (Bruker, Täby, Sweden). The chemical shifts are given in p.p.m. from internal TMS. LC/ESI-MS analyses were performed on a Sciex API 365 LC/MS/MS triple quadruple mass spectrometer (Perkin-Elmer, Boston, MA). Time-resolved fluorescence was measured with a Victor 1420 Multilabel Counter (Perkin-Elmer Life Sciences).

N1-(4-carboxyphenyl)sulfanilamide (A)

A was synthesized as previously described (Sheth and Sporns, 1991Go): 1H NMR (400 MHz, DMSO) {delta} 6.54 (d, 2H, ArH), 7.15 (d, 2H, ArH), 7.45 (d, 2H, ArH), 7.77 (d, 2H, ArH), 10.47 (br s, 1H, NH).

4-(4-amino-benzenesulfonylamino)-N-(6-amino-hexyl)-benzamide (E)

A (0.87 g) was first activated with N-hydroxysuccinimide (0.51 g) and N,N '-dicyclohexylcarbodiimide (0.92 g) in dioxane (20 ml) by stirring overnight at room temperature followed by filtration and evaporation. The product (B) was used without further purification.

4,4'-dimethoxytritylchloride (0.79 g) was dissolved in dioxane (10 ml) and then added slowly to a mixture of 1,6-diaminohexane (0.54 g) in dioxane (15 ml). The mixture was stirred for 2 h at room temperature after which 10 ml of MeOH was added. Solvents were evaporated and the precipitated products were dissolved in dichloromethane (30 ml). The organic phase was washed twice with 20 ml of aqueous NaHCO3, dried (Na2SO4) and evaporated to dryness. The product (C) was purified with flash chromatography (FC) using silica gel and 5/10/85 = triethylamine/MeOH/dichloromethane. The yield was 43%.

The activated product B (0.2 g) was allowed to react with C (0.22 g) in dry dioxane (4 ml) by stirring overnight at room temperature. The reaction mixture was evaporated and the product (D) was purified with FC (siliga gel, 0.25/2.5/97.25 = triethylamine/MeOH/dichloromethane). The yield was 32%.

D (0.11 mg) was dissolved in a 3% solution of dichloroacetic acid in dichloromethane (20 ml) and stirred at room temperature for 2 h. The product (E) was evaporated and used without any further purification. 1H NMR (200 MHz, DMSO) {delta} 7.98 (t, 1H, NH), 7.66 (d, 2H, ArH), 7.47 (d, 2H, ArH), 7.12 (d, 2H, ArH), 6.56 (d, 2H, ArH), 5.47 (s, 2H, ArNH2) 3.30 (s, 2H, NHCH2-), 1.93–0.84 (m, 10H, -CH2-).

Biotinylation of the sulfacarboxyphenyl derivative

Biotin-XX-NHS (6.6 mg) in dry DMF was added to the solution of sulfonamide derivative E (5 mg) in dry DMF (0.68 ml). Diisopropylethylsulfanilamide (18.5 µl) was added and the reaction mixture was stirred overnight at room temperature. The product was purified with RP-HPLC using a Hybersil 150 x 4.6 mm, 5 µm HyPurity Elite C18 Column, a gradient from aqueous 0.1% TFA to acetonitrile in 30 min, a flow rate of 1.0 ml/min and detection at 265 nm. The peaks were analyzed using LC/ESI-MS. The peak at 15.75 min found 843.2 [M + H]+, C41H62N8O7S2 + H requires 843.42609. The purified product (Figure 3Go) was dissolved in 1 ml of DMF and was from hereon referred to as biotinylated sulfacarboxyphenyl. The concentration of the biotinylated derivative could not be determined due to low synthesis yield, so the dilution used is given in the text instead.



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Fig. 3. Biotinylated sulfacarboxyphenyl derivative synthesized for use in phage display.

 
DNA shuffling libraries

The new libraries were constructed with DNA shuffling combined with error-prone PCR (Stemmer, 1994Go; Lorimer and Pastan, 1995Go; Boder et al., 2000Go). Fresh batches of XL-1 cells in the logarithmic growth phase were infected either with phage Library 1 or Library 4 or with four times enriched phage Library 1 or Library 4 (Korpimäki et al., 2002Go). After overnight growth, plasmid DNA was isolated from each of the four cultures. About 50 µg of each plasmid was digested with ClaI and KpnI restriction endonucleases to cut loose the scFv genes (Figure 2Go). The scFv fragments were purified with agarose gel electrophoresis.

About 1 µg of the unpanned Library 1 and Library 4 gene fragments each and 4 µg of the four times panned Library 1 and Library 4 gene fragments each were mixed together in a total volume of 143 µl of DNase I digestion buffer (50 mM Tris–HCl pH 7.6, 10 mM MnCl2, 1 mg/ml BSA, Lorimer and Pastan, 1995Go). DNase I (0.4 U, Sigma–Aldrich) was added and the mixture was incubated for 20 min at 15 °C. The digestion reaction was stopped with the addition of 48 µl of 50 mM EDTA. A sample of the digested DNA was analyzed with agarose gel electrophoresis to ascertain that the DNase I had digested the scFv genes to random fragments averaging 50 bp in length. The fragmented DNA was purified with a DNA grade Sephadex G-50 NICK spin column (Amersham Biosciences, Uppsala, Sweden) as instructed by the manufacturer.

The DNA fragments were reassembled in PCR reactions containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2 mM MgCl2, 200 µM each dNTP, 10 µl scFv DNA fragments and 0.1 U/µl Taq polymerase in a total reaction volume of 50 µl to generate Library A. The PCR program used was 95 °C for 2 min, 40 cycles of 95 °C for 30 sec, 60 °C for 1 min and 72 °C for 1 min + 2 sec per cycle and the final extension of 72 °C for 6 min. Library B was generated in the same way, except that oligonucleotide MK23 (Table IGo) containing degenerate bases was added to the PCR mix to a final concentration of 200 nM in order to introduce more mutations to certain sequence positions.


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Table I. Sequences of the PCR primers
 
Shuffled full-length scFv genes were amplified in PCR reactions containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.25 mM MgCl2, 200 µM each dNTP, 1 µl reassembled Library A or B DNA, 0.375 mM MnCl2, 100 nM SfiI site-containing primers scFor and scRev (Table IGo) and 0.1 U/µl Taq polymerase in a total reaction volume of 50 µl in order to introduce additional mutations to the recombined genes. The PCR program was 95 °C for 2 min, 30 cycles of 93 °C for 30 sec, 58 °C for 1 min and 72 °C for 2 min.

The PCR products (about 1.5 µg per library) were cloned to pAK100 phagemid vector (Figure 2Go) using the SfiI sites. The ligation reactions were precipitated with ethanol and transformed to electrocompetent E.coli XL1-Blue cells with electroporation in five separate aliquots each, yielding approximately 8 x 107 transformants per library (determined with platings on LB/chloramphenicol agar). Electroporated cells were diluted to 10 ml total volume in SOC medium and they were incubated in shake flasks at 37 °C and 200 r.p.m. for 1 h. Cultures were diluted to 100 ml with SB medium containing tetracycline, chloramphenicol and 0.2% glucose. Growth was continued at 37 °C and 240 r.p.m., until the optical density (OD600 nm) of the cultures reached 0.5. The cultures were then infected with 2.5 x 1011 p.f.u. of VCS-M13 helper phage each and recombinant phage preparations were produced as before (Korpimäki et al., 2002Go).

Enrichment of the libraries

Libraries A and B each were enriched with three different panning schemes. The first scheme was as follows (rounds 1–3). Phage libraries were diluted in TBT-0.05 buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% BSA, 0.05% Tween 20) to final concentration of 1 x 1012 p.f.u./ml. Each diluted library was divided to two 200 µl aliquots. Appropriate dilution of the selected biotinylated panning antigen (Table IIGo) was added to one of the replicas. Tubes were agitated in a rotamix for 1 h at 25 °C. Dynabeads M-280 magnetic streptavidin particles (Dynal Biotech, Oslo, Norway) were pre-washed three times with 200 µl of TBT-0.05 and about 7000 beads was transferred to each phage dilution tube in a volume of 10 µl. The tubes were incubated in a rotamix for 30 min at room temperature. Afterwards, the beads were washed five times in 200 µl aliquots of TBT-0.5 buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% BSA, 0.5% Tween 20) using a magnetic concentrator. The particles were finally suspended in 192 µl aliquots of 0.1 M glycine buffer (pH 2.2) and incubated in a rotamix for 15 min at room temperature. The particles were removed and the eluate was neutralized with 2 M Tris–HCl buffer pH 9.


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Table II. The type and dilution of the panning antigen and the concentration of competing sulfonamides used during each panning rounda
 
1 ml of fresh XL1-Blue cells were mixed with each eluate batch. Tubes were incubated for 30 min at 37 °C without agitation. Serial dilutions of each tube were plated on LB/chloramphenicol agar. Shake flasks containing 20 ml of SB medium with tetracycline, chloramphenicol and 0.2% glucose were inoculated with 1 ml of infected cell suspension from the replicas that had the biotinylated antigen added to them. The flasks were shaken at 37 °C and 300 r.p.m., until the OD600 nm of the cultures reached 0.4. The cells were then infected with 3.1 x 1011 p.f.u. of VCS-M13 helper phage and the recombinant phage preparations were produced as before (Korpimäki et al., 2002Go) to be utilized in the next panning round.

The second panning scheme (rounds I–III) was essentially the same as the first one, except that different type and concentration of panning antigen was used in each round of the enrichment (Table IIGo). The third scheme (rounds a–d) also had different type and concentration of panning antigen present in each round, but it also differed from the first panning scheme in such a way that varying concentrations of sulfonamides (sulfamethizole, sulfathiazole, sulfachloropyridazine, sulfamethoxypyridazine and sulfapyridine) were added to the panning reactions together with the biotinylated panning antigen (Table IIGo).

Isolation of active clones

Cell lysates of a selection of clones from the enriched libraries were prepared for activity measurements as previously described (Korpimäki et al., 2002Go). Binding activity was determined using a time-resolved, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) (Hemmilä et al., 1984Go). Rabbit anti-mouse IgG-coated microtitre plates were pre-washed once with wash solution. A 100 µl aliquot of assay buffer was applied to each well followed by 100 µl of the supernatant sample. The plates were incubated for 1 h at room temperature with gentle shaking and washed four times as above. To each well, 200 µl of assay buffer containing 30 ng/ml of Eu-sulfamerazine was applied. The plates were incubated for 1 h at room temperature with gentle shaking and washed four times. A 200 µl aliquot of enhancement solution was added to each well. After 5 min incubation in gentle shaking, time-resolved fluorescence was measured.

Characterization of the active clones

Active clones were produced in varying scales (5–100 ml) to be used in competitive assays, as previously described (Korpimäki et al., 2002Go). The cross-reaction profiles of the active antibody mutants for different sulfonamide antibiotics were determined. Rabbit anti-mouse IgG-coated microtitre plates were pre-washed once with wash solution and aliquots of the cell lysate samples (200 µl) were applied to the wells. The strips were incubated for 1 h at room temperature with gentle shaking and washed four times as above. To each well, 100 µl of assay buffer containing an appropriate concentration of Eu-sulfamerazine (Tables IIIGo and IVGo) selected to give a good signal was applied. An aliquot of 100 µl of assay buffer containing varying concentrations of different sulfonamides (Figure 1Go) was added to each well. The plates were incubated for 1 h at room temperature with gentle shaking and washed four times. A 200 µl aliquot of enhancement solution was added to each well. After 5 min incubation in gentle shaking, time-resolved fluorescence was measured. Six different concentrations were measured for each sulfonamide in order to obtain data points for the whole range of inhibition and sigmoidal curves were fitted to this data. The R2 values obtained from the comparison of the fitted curves to the raw data varied between 1 and 0.97, being almost always better than 0.98.


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Table III. The performance of the mutants in a competitive sulfonamide assay for three different sulfonamides
 

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Table IV. The performance of selected clones in a competitive sulfonamide assay for 13 different sulfonamides
 
Selected clones were characterized also with respect to their binding affinity towards the Eu-sulfamerazine tracer as previously described (Korpimäki et al., 2002Go).

Modeling of antibody 27G3

The computer program suite used for the homology modeling of antibody 27G3 was InsightII (Molecular Simulations, San Diego, CA). The templates used in the modeling of the VL and VH domains were the structures of antibody {alpha}gp41 (PDB code 1NLD) and (minus the H-CDR3-loop) of antibody TE33 (PDB code 1TET), respectively. The distance and angle of the VL and VH domains with respect to each other were set using the structure of TE33 as a guide.

For the modeling of the H-CDR3-loop, structure of antibody D44.1 was used (PDB code 1MLB) as the template. The model H-CDR3-loop conformation was then further refined by locking the rest of the model and running the Steepest Descent-minimizing algorithm of the DISCOVER-module for 40 iterations. Finally the whole model was energy minimized by running the Steepest Descent-algorithm for 40 iterations and Conjugate Gradients-algorithm for 100 iterations.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
DNA shuffling libraries

The mutants with improved broad specificity recognition of sulfonamides constructed in our earlier study (Korpimäki et al., 2002Go) were recombined with each other using DNA shuffling combined with error-prone PCR (Stemmer, 1994Go; Lorimer and Pastan, 1995Go; Boder et al., 2000Go). ScFv genes from unpanned Libraries 1 and 4 and four times enriched Libraries 1 and 4 (Korpimäki et al., 2002Go) were randomly digested with DNase I to fragments averaging 50 bp in length. The digested fragments were mixed together in such a way that there were four times more fragments from the panned libraries in order to favor the mutations selected by the enrichment. An aliquot of the fragment mix was randomly reassembled in a PCR reaction to yield Library A. Another aliquot was reassembled in the presence of a partly degenerated oligonucleotide MK23 (Table IGo) homologous to L-CDR1 of 27G3 in order to introduce a lot of mutations to this area. L-CDR1 was chosen for intensive mutagenesis, since it was the CDR closest to the most common mutation (L:L36M) in the previous study (Korpimäki et al., 2002Go). The L-CDR1 residues targeted by the degenerate oligonucleotide were L:H27d and L:K30, because mutations were previously found at these locations (Korpimäki et al., 2002Go), and L:D28 and L:Y32, because it was thought that the large side-chains of these residues might reach towards the bound antigen. The reassembly with the oligonucleotide yielded Library B. The sequencing chromatogram of Library B DNA as compared to that of Library A DNA indicated a concentration of garbled sequence at the locations MK23 was supposed to mutagenize (results not shown), ascertaining that MK23 had incorporated into some of the reassembled scFv genes.

The reshuffled scFv genes of Library A and Library B were amplified with PCR in the presence of Mn2+ (Boder et al., 2000Go) in order to introduce additional mutations to the genes. Both PCR products were cloned to a pAK100 phagemid vector (Figure 2Go). The scFv mutants were produced as filamentous phage coat protein III fusions from this vector, enabling phage display. After transformation to E.coli XL1-Blue cells, both libraries contained approximately 8 x 107 independent clones.

Enrichment of the libraries

Libraries A and B were panned using three different kinds of panning schemes. In all of the schemes, sulfa binders were repeatedly enriched by collecting phages with biotinylated sulfonamides attached to streptavidin-coated paramagnetic beads followed by phage amplification in vivo. The schemes differed with respect to the type and dilution of the collection antigen and the concentration of competing sulfonamides each round. These parameters for each round are shown in Table IIGo. In the first panning scheme (rounds 1–3), the collection antigen was switched between rounds to supposedly enrich mutants which are less sensitive to the structural variation in the side-chains of different sulfonamides. The second panning scheme (rounds I–III) utilized decreasing concentrations of the same collection antigen. The purpose of this protocol was to ascertain whether this kind of approach could enrich mutants with better broad specificity. In the third panning scheme (rounds a–d), competing sulfonamides (those for which the affinity of wild-type 27G3 is good) were added to the panning reactions in addition to the collection antigen. This approach was to enrich mutants which would bind the collection antigen better, but the competing antigens poorer than 27G3.

Figure 4Go shows how many phages were collected from each library during each round of panning and how many of those were collected by unspecific background binding. It was clear that in the cases of both libraries and all three panning schemes (1–3, I–III, a–d), the number of specifically collected phages stayed over the background level as the rounds proceeded, even when the amount of collection antigen was rapidly decreased and the amount of competing sulfonamides was increased between each round. This indicated that all three schemes enriched mutants capable of sulfa binding from both libraries.



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Fig. 4. Number of phages collected during each panning round. Shown for Libraries A and B is the number of phages collected from the input pool of about 2 x 1011 phages with and without (background) the panning antigen during each round of panning. The rounds of the first, second and third panning schemes are referred to as 1–3, I–III and a–d, respectively.

 
Isolation of active clones

In order to isolate and characterize active clones, scFv genes from the panned libraries were cut out with SfiI digestion and cloned to pAK100CL vector (Figure 2Go). From this vector, scFv antibodies were produced as mouse IgG constant light domain fusions, which enabled immobilization on anti-mouse IgG-coated surfaces. From each enriched library, 20 individual clones were grown in minicultures and sulfonamide binding activity was measured. Of the clones isolated from enriched libraries A.3, A.III and A.d (Library A enriched with each of the three different schemes), 6, 3 and 1 showed good (S/N > 5) sulfonamide binding activity, as well as 4, 7 and 3 of the clones isolated from enriched libraries B.3, B.III and B.d (Library B enriched with each of the three different schemes) respectively. Several other clones from all enriched libraries displayed measurable binding activity (S/N > 2), but these were judged to be binders of low affinity and were not chosen for further study. It can be assumed that a random mutagenesis of a whole scFv gene with DNA shuffling and error-prone PCR produces a library containing mostly inactive mutants. The presence of a number of highly and weakly active clones among the random 20 picked from each enriched library made it likely that the panning procedure had resulted in enrichment of active binders.

Characterization of active clones

Each clone showing good sulfonamide binding activity was grown in cultures of varying scale. Cell lysates were tested with a time-resolved competitive sulfa fluoroimmunoassay. Table IIIGo shows the concentration of selected sulfonamides needed to inhibit 50% of the tracer binding for each active mutant antibody (IC50). Most of the IC50 values determined for the mutants were improved over those of the wild-type antibody. The first panning scheme seemed to enrich a few mutants with a better broad specificity than the wild-type (IC50 values for the three different sulfas had closed on each other), but also mutants for which the reverse was true. The second panning scheme however enriched almost exclusively mutants with better broad specificity than the wild-type. The third panning scheme seemed to select for mutants with almost unchanged broad specificity.

Six clones (A.3.5, B.3.2, B.3.12, A.III.3, B.III.8 and B.III.10) were selected for further characterization on the basis of them giving the best signals in the competitive assay and having the best improvement in the broad specificity binding of the three sulfas. The performance of the selected mutants in a competitive sulfa assay for 13 different sulfonamides is shown in Table IVGo. The results show that the mutants mostly recognized different sulfonamides within a narrower range of affinities as compared to the wild-type antibody. In this respect their performance was comparable to that of the two best mutants (Lib1.9 and Lib4.11) isolated in the previous study (Korpimäki et al., 2002Go). But in addition to that, these new mutants gave a lot better signals in the competitive assays than Lib1.9 or Lib4.11. Thus 10–100 fold lower tracer concentrations could be used. However, the sensitivities of the competitive assay for different sulfas obtained with each of the new mutants as binder were not significantly better than those obtained with Lib1.9 or Lib4.11 as binder. An exception to this was mutant A.3.5, which seemed to combine the best qualities of Lib1.9 and Lib4.11. This mutant recognized 10 of the 13 sulfas tested with very similar affinities like Lib4.11 did, but also had 5- to 11-fold improved recognition of sulfamethazine, sulfaquinoxaline and sulfadimethoxine over wild-type, like Lib1.9.

The mutations in the sequence of the six clones and also their affinity constants for the Eu-sulfamerazine tracer are shown in Figure 5Go. The sequence data shows that the DNA shuffling did recombine the mutations present in the clones of the enriched libraries of the previous study (Korpimäki et al., 2002Go). The concentration of mutations at the L-CDR1 area of the clones isolated from enriched libraries originating from Library B demonstrates that the degenerated oligonucleotide was incorporated into the recombined clones by the DNA shuffling procedure as it was supposed to. The affinity of the mutants for the Eu-sulfamerazine tracer shows significant improvement over the wild-type antibody and over the clones of the previous study (Korpimäki et al., 2002Go). This could be expected, since a sulfamerazine derivative was one of the collection antigens used in the enrichments. The best mutant B.d.2, enriched with the third panning scheme, had 60-fold improvement in affinity over the wild-type antibody.



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Fig. 5. Isolated mutant sequences. The mutations present in light chain framework (LF1–LF4) and CDR (LC1–LC3) regions, in heavy chain framework (HF1–HF4) and CDR (HC1–HC3) regions and in the linker region are illustrated in the figure. The numbering of the residues follows that devised by Kabat (Kabat et al., 1991Go). The affinity for the sulfamerazine derivative used as a tracer in the immunoassays is also shown for some clones.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
The libraries and enrichment

There has been a lot of work towards the development of generic sulfa binders (Sheth and Sporns, 1991Go; Muldoon et al., 1999Go, 2000Go; Spinks et al., 1999Go; Haasnoot et al., 2000Go; Korpimäki et al., 2002Go). Some of these studies modeled the minimum energy conformations of different sulfonamides and pointed out that the steric and electrostatic properties of these conformations differ from each other (Spinks et al., 1999Go; Muldoon et al., 2000Go). We demonstrated earlier that almost generic sulfa binding is still possible, plausibly by binding of higher energy conformations of the sulfonamides (Korpimäki et al., 2002Go). In this previous study, some sulfas stood out as being more problematic to bind than the others. These were the sulfonamides with the bulkiest N1-rings, and it was hypothesized that the reason for lower affinity might be that these sulfas do not fit properly to the binding cavity of the antibodies.

In the previous study (Korpimäki et al., 2002Go) we speculated whether recombining the isolated mutants might yield better binders in respect with binding the problematic sulfas, so in this study we tested this hypothesis by not only recombining the enriched libraries of the previous study, but also by introducing further mutations with error-prone PCR and degenerate oligonucleotides as suggested by others (Stemmer, 1994Go; Boder et al., 2000Go). We also tested three different schemes to enrich desirable mutants from the new libraries.

Sequencing data (Figure 5Go) indicated that we succeeded in achieving our desired goals of library generation with the DNA shuffling method. We got recombination of the mutations from the previous libraries, but also new mutations throughout the scFv genes and a mutational hotspot around the L-CDR1, which was the CDR-loop closest to the most common mutation L:L36M in the clones of the previous study (Korpimäki et al., 2002Go).

The three enrichment schemes also worked quite well. The first scheme, which was based on switching the collection antigen between rounds in order to stress the recognition of the generic area of sulfas in the selection (as in Korpimäki et al., 2002Go), isolated clones of varying properties (Table IIIGo). We got mutants with better, but also worse, generic binding capabilities than the wild-type antibody. However, the best mutant of this study, A.3.5, was enriched by this scheme. The second scheme was an increasing stringency enrichment with the same type of collection antigen used each round. This protocol enriched almost exclusively clones that had improved generic binding of sulfas over wild-type (Table IIIGo). Unfortunately, there were not many mutants with exceptional improvement in generic binding. Most of the improvement seemed to go towards recognition of the Eu-sulfamerazine tracer, since less tracer could be used in the competitive assays to get adequate signal (Table IIIGo). The third enrichment protocol was intended to enrich mutants with binding preference towards the problematic sulfas, by blocking the binding of mutants which bound certain sulfonamides (Table IIGo). But this scheme only seemed to enrich the mutants with the highest affinities for the Eu-sulfamerazine tracer (Figure 5Go), but lacking in affinity for the sulfonamides (Table IIIGo).

Observed mutations

The sequencing results demonstrate how DNA shuffling in combination with error-prone PCR and enrichment is capable of recombining favorable mutations together to form better binders as suggested before (Figure 5Go, Boder et al., 2000Go). The best generic binder mutant A.3.5 is obviously formed by the recombination of Lib4.11 (mutations H:N35T, H:K46Q and H:T52aA), Lib1.9 (at least mutations H:N82aD and H:L82cI), some other mutants and possibly point mutations caused by the error-prone PCR or the DNA shuffling process. This could explain why the properties of A.3.5 seem to be a mix between Lib4.11 and Lib 1.9.

According to our homology model of 27G3, most of the amino acid changes present in mutant A.3.5 are located on the surface of the antibody and are not in proximity of the probable binding cavity (Figure 6AGo). Thus these mutations probably affect the binding properties of the mutant in only a minor way at the best. However, mutation H:N35T, which A.3.5 inherited, affects an amino acid residue located in the bottom of the probable binding cavity (Figure 6AGo). This residue H:35 has been shown to closely interact with the ligand in several hapten–antibody complexes, most typically via a hydrogen bond (Alzari et al., 1990Go; Arevalo et al., 1993Go; Schildbach et al., 1994Go; Jeffrey et al., 1995Go; Lamminmäki and Kankare, 2001Go). The cross-reaction data (Table IVGo) shows that all mutants (especially Lib4.11) with mutation H:N35T have significantly different binding profiles from the wild-type antibody. Hence it is likely that this residue forms a close interaction with the ligand in the case of antibody 27G3 as well, maybe even through a hydrogen bond. The H:N35T mutation thus probably causes a significant shift in the orientation of the bound sulfa in the binding cavity.



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Fig. 6. Location of mutated residues in the homology model of 27G3. Illustrated in the figure are the residues mutated in clone A.3.5 (A) and the cluster of mutated residues in the tip of L-CDR1 present in the highest affinity clones (B). Shown on the left is a view from the side of the scFv antibody. On the right is a view from the top. The numbering of the residues follows that devised by Kabat (Kabat et al., 1991Go).

 
Mutation L:L36M, which A.3.5 also inherited, affects an amino acid residue located under the bottom of the binding cavity in the interface between the VL and VH domains (Figure 6AGo). The cross-reaction data of our previous study (Korpimäki et al., 2002Go) shows that antibodies with this mutation generally have a higher affinity for sulfonamides than 27G3, so the effect of the mutation seems beneficial for sulfa binding. It is hard to predict how this mutation achieves this effect, but a change of leucine to a slightly bulkier methionine in the antibody domain interface might affect the orientation of the antibody domains with respect to each other and so provide a better binding cavity.

The other mutations of A.3.5 located in the CDR-loops where they might affect binding are L:L50Q, L:V51G and H:T52aA (Figure 6AGo). Mutation H:T52aA was an inherited one, which implies that this mutation might be of importance. However, this residue of the H-CDR2-loop probably does not make any direct contact with the bound ligand, since it is not in proximity of the binding site. Thus, if this mutation has any effect on sulfa binding, it presumably works by changing the conformation of the amino acid residues around it, such as those of the H-CDR2-loop. Mutations L:L50Q and L:V51G in the L-CDR2-loop emerged during the DNA shuffling procedure. Their importance is hard to predict, but since L:L50Q is present in every clone sequenced in this study, it might be the more important of the two. If these mutations have any effect on sulfa binding, they presumably operate by changing the conformation of their neighbors, since neither residue is in proximity of the binding site. The emergence of the highest affinity Eu-sulfamerazine binders (B.d.2 and B.d.15) from Library B demonstrates the feasibility of oligonucleotide-directed mutagenesis in conjunction with DNA shuffling. The big cluster of mutations present in the L-CDR1 of these mutants (L:H27dR, L:S27eR, L:D28E, L:K30V and L:Y32L) is likely at least partially caused by the mutagenizing oligonucleotide. These mutations are located in the tip of the L-CDR1-loop (Figure 6BGo), which protrudes into the solvent, so some of these amino acid residues might make contacts with the linker part of the Eu-sulfamerazine molecule. Therefore these five mutations are probably at least partially responsible for the best affinity of mutants B.d.2 and B.d.15 for the Eu-sulfamerazine tracer. Although the oligonucleotide mutagenesis did not help the formation of good broad specificity binders in this study, this is more likely due to a suboptimal choice in the mutagenesis positions than a fault in the basic technique.

None of the mutants had any mutations in the area of the L-CDR3-loop. This implies that this CDR area of scFv 27G3 is so well adapted to sulfonamide binding that a single substitution already causes a significant loss of affinity. There was also only a single position in the root of the H-CDR3-loop which had any mutations, possibly meaning that the sequence of this loop is also important for the sulfa binding. Similar rarity of mutations in H-CDR3-loop has been observed in the case of engineering of an estradiol antibody (Saviranta et al., 1998Go).

Often mutations found frequently among the clones of an enriched population of mutants can be considered of importance for the property which the library is being enriched for. However, mutation H:K46Q, which is among the most frequent ones found in the characterized clones of this study, seems to be a puzzling exception. According to our model, residue H:46 is located on the surface of the antibody (Figure 6AGo), far away from the binding site, where it probably forms salt bridges with neighboring residues like H:D62. However, since even the neighbors are distant from the ligand binding site, it is very hard to imagine how the mutation H:K46Q could have any effect on sulfa binding and so the high frequency in the characterized clones is more likely accidental.

All in all, a lot of mutation data was gathered during the course of this study. Although the presence of multiple mutations in each clone prevented a more thorough analysis of the importance of each single mutation, this mutation data could nevertheless be useful if further oligonucleotide-directed mutagenesis was to be carried out in order to optimize the broad specificity binding properties of A.3.5.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
To this day, hybridoma technology has been unable to produce a broad specificity antibody capable of binding all sulfonamides with sufficient affinity for the purpose of developing a group-specific immunoassay (Sheth and Sporns, 1991Go; Muldoon et al., 1999Go, 2000Go; Spinks et al., 1999Go; Haasnoot et al., 2000Go). We demonstrated in our earlier study that a protein engineering approach can be used to solve this problem, as suggested before (Muldoon et al., 2000Go), by improving the broad specificity sulfa binding capabilities of antibody 27G3 with this technology (Korpimäki et al., 2002Go). In this study we improved the antibody even further by combining the powerful techniques of DNA shuffling, error-prone PCR, oligonucleotide-directed mutagenesis and various enrichment schemes, thus producing more proof that protein engineering is a valid approach for the generation of broad specificity antibodies. With our best mutant, even sulfamethazine, the most problematic sulfonamide for the wild-type monoclonal antibody 27G3, can now be measured with an IC50 very close to the MRL of 100 µg/L in a buffer system. It should be remembered, however, that the cross-reaction profiles of antibodies can change, when used in real sample matrices. Thus it will be necessary to test the performance of our mutants in sample matrix environments in the future.


    Acknowledgments
 
We thank Willem Haasnoot for providing the antibody 27G3. This work has been supported by a grant from the Finnish National Technology Agency (TEKES).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
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Received July 18, 2002; revised November 6, 2002; accepted December 3, 2002.





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