Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

T.K. Nevanen1,2, M.-L. Hellman1, N. Munck1, G. Wohlfahrt1,3, A. Koivula1 and H. Söderlund1

1VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland 3Present address: Orion Pharma, PO Box 65, FIN-02101 Espoo, Finland

2 To whom correspondence should be addressed. e-mail: tarja.nevanen@vtt.fi


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

Keywords: canonical/enantioselective antibody/molecular modelling/recombinant Fab/site-directed mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The legislation for the pharmaceutical industry requires that the properties of pure enantiomers are studied individually even if the drug can eventually be used as a racemate. No single universal method for enantiomeric separation is yet available. Chromatographic methods, e.g. chiral protein columns, are widely used, especially in early phases of drug development, where the amounts of pure enantiomers needed are limited. Many of the chiral protein columns are made of unspecific, easily available proteins (e.g. ovomucoid). The separation is based on low-affinity partitioning and enantiomers must be collected from dilute fractions.

Immobilized monoclonal or polyclonal antibodies have been used to separate enantiomers (Mertens et al., 1982Go; Takasaki and Tanaka, 1992Go; Knox and Galfre, 1986Go; Hofstetter et al., 1998Go), but they have not been economically feasible. Antibodies are protein molecules created by the immune system and are able to bind with high specificity many different kinds of molecules from large protein antigens to low molecular weight haptens. Even minor differences in composition or configuration of haptens can be distinguished by antibodies (Hemminki et al., 1998Go; Hofstetter et al., 1998Go; Nevanen et al., 2001Go; Lua and Chou, 2002Go). With the modern methods of molecular biology, it is possible to produce only the antigen binding part of an antibody in bacterial hosts, such as Escherichia coli, thus making the production of antibody affinity columns economically more feasible. In addition, the binding properties of antibody fragments can be adjusted to better fit the application.

Recently, we reported the cloning of two enantioselective antibody fragments (ENA5His and ENA11His) and showed that they can be used to fractionate two enantiomers of a racemic drug candidate, Finrozole (4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzonitrile) (Nevanen et al., 2001Go; Lee et al., 2002Go; Mitchell et al., 2002Go). These cloned antibody fragments (Fab fragments) consist of the intact light chain (VLCL) and the variable (VH) and the first constant domain (CH1) of the heavy chain of the antibody. The drug Finrozole contains two chiral atoms, thus the synthesis product is a mixture of four stereoisomers. Diastereomers can be easily separated from the mixture by crystallization whereas the fractionation of the enantiomers is difficult with conventional methods. In the case of Finrozole, it is important to purify the d-enantiomer (named according to its retention order in chiral chromatography based on ovomucoid column) which is more potent as a drug than either the a-enantiomer or the b- and c-diastereomers. The two cloned antibody fragments, ENA5His and ENA11His, have different specificities and both of them efficiently separate the enantiomers. Immunoaffinity columns made of ENA5His bind specifically the d-enantiomer from the racemic mixture of a- and d-enantiomers. This particular anti body fragment has such a high affinity that problems arise in elution of the bound enantiomer. Harsh and partially denaturing elution conditions were needed for quantitative recovery. However, stability and reusability of the affinity column is required for the productive use of such a separation method.

In the present study, we have used molecular modelling based on homologous antibody structures and fragments to design mutations—aiming for reduction of binding affinity without dramatically impairing the specificity. Antibodies are a special class of proteins where the framework regions are very conserved among different antibodies, whereas the loop regions responsible for antigen binding are variable. The hypervariable loops (also called complementarity determining regions, CDR) can be grouped in canonical structural classes, which share common structural features (Chothia and Lesk, 1987Go). Over 400 crystal structures of antibodies or antibody fragments are available in the Protein Data Bank (Berman et al., 2000Go) providing templates for homology modelling in most cases.

Five single mutants and, after their preliminary characterization, one combined double mutant were constructed. One single mutant and the double mutant were used to build immunoaffinity columns for enantioseparation of the enantiomers of the drug. We show that using homology modelling-based mutant design it is possible to fine-tune the binding properties of the antibody and, in this case, to develop a reusable and continuous separation system for the a- and d-enantiomers of the Finrozole drug.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibody modelling

The search for templates in the PDB (Bernstein et al., 1977Go; Berman et al., 2000Go) was carried out with BLAST (Altschul, 1991Go) using the Blosum62 matrix. Alignments were made separately for heavy and light chains in order to find the best template structures for the framework. The crystal structures of idiotypic Fab 1iai_703.1.4 (IgG2a) and anti-idiotypic Fab 1iai_409.5.3 (IgG1) (Ban et al., 1994Go) were chosen as templates for ENA5His light and heavy chain framework. ENA5His light chain (VL + CL) has an overall sequence identity of 81% with 1iai_730.1.4 and the heavy chain (VH + CH1) shows 77% overall sequence identity to 1iai_409.5.3. As the selected template structures for the light and the heavy chain framework come from different immunoglobulins, they have been combined by least-square superposition of the main chain atoms of the whole Fab fragments using the homology module of InsightII (Accelrys). The template candidates for the hypervariable loops in the light chain (CDR-L1,2,3) and in the heavy chain (CDR-H1,2) were chosen according to the rules of Chothia et al. (Chothia and Lesk, 1987Go; Chothia et al., 1989Go). For modelling of the most variable loop (CDR-H3), the rules derived by Shirai et al. (1996Go) and Morea et al. (1998Go) were used. The final choice for the CDR template structures was based on considerations on maximal sequence identity, antigen/hapten size and similarity with Finrozole, and on finding best structural spatial combination of loops.

Two models were built based on the crystallographically determined structures of antibodies

The template structures used in the model building are presented in the Table I. The Chothia canonical class assignments (Chothia and Lesk, 1987Go; Chothia et al., 1989Go; Martin and Thornton, 1996Go) for CDRs of ENA5His and the template structures are also shown in Table I. Sequence alignments of ENA5His and the chosen CDRs for models 1 and 2 are presented in Figure 1a and b, respectively. Both models for the variable and constant domains of the Fab fragment were constructed with the InsightII homology module (Accelrys). The backbone co-ordinates were taken directly from the template structures and, if the aligned residues were identical, the side-chain conformation was maintained also. The initial models were energy minimized (Case et al., 1997Go) and the quality of the models generated was evaluated with PROCHECK (Laskowski et al., 1993Go) and ERRAT (Colovos and Yeates, 1993Go).


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Table I. Crystallographic structures (PDB codes) used as templates for the CDR loops of the two ENA5His models
 


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Fig. 1. The amino acid sequences of ENA5His and of the antibodies used for model building. CDR loops important for antigen recognition are underlined. Fragments of other antibodies used for model building are printed in bold under the corresponding ENA5His sequence. Mutagenized amino acids are marked with an asterisk. (a) and (b) contain sequence comparisons for model 1 and 2, respectively.

 
Hapten modelling, docking and refinement of the complexes

A search for low energy conformers of the hapten (4-[3-(4- fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzo nitrile) (Figure 2) was carried out using the AM1 method (Stewart, 1990Go), included in InsightII (Accelrys). The lowest energy conformations were used to derive atomic point charges for the hapten by ab initio calculations (Frisch et al., 1995Go) at the B3LYP/6–31G* level.



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Fig. 2. The structure of 4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzonitrile. It has two chiral centres C1 and C2. A linker was connected to the nitrile group to allow attachment of the hapten to the protein (keyhole limpet haemocyanin) used for immunization.

 
For the immunization, the hapten was conjugated to a carrier protein via a 2-carboxyethane linker synthesized to its nitrile group (Nevanen et al., 2001Go; Södervall et al., 2002)Go. Hence, it can be concluded that its opposite part is buried in the antigen-binding site of ENA5His. Initial hapten orientations were obtained by manual docking of different low energy conformations of the two diastereomers S,S and S,R to the proposed binding regions of the antibody in the variable domains (VH and VL). In the molecular dynamics simulations, we concentrated on the S,S-enantiomer, based on knowledge of hapten configurations at the time of the modelling (Nevanen et al., 2001Go). The molecular mechanics and molecular dynamics simulations (Case et al., 1997Go) for the hapten were performed with a cut-off distance of 11 Å for non-bonded interactions with explicit water. After initial minimization of the complex structure, a sphere with a radius of 16 Å with water molecules was defined and centred at the binding pocket. Minimizations were run for 2000 cycles, first using the steepest descent method and then continuing with the conjugate gradient method until a convergence criterion of 0.2 kcal/mol Å was met. All hypervariable loops, with one anchor residue at both ends, the hapten and all water molecules were free to move with bond lengths constrained using the SHAKE algorithm for all the bonds, whilst the rest of the protein was frozen during MD. A time step of 1 fs was used for the 200–300 ps molecular dynamics simulations at 298 K. The procedure was applied to all the starting complex structures having different initial conformations and orientations of the hapten.

Design of mutants

Two mutants were prepared based on model 1. The choice for template loop structures of model 1 was made on the basis of canonical class and maximal overall sequence identity. The numbering of all mutants is according to Kabat et al. (1991Go). The mutant Trp100JLeu (CDR-H3) was designed to introduce more space in the binding site, while the mutation Tyr94Leu (CDR-L3) was intended to reduce possible hydrophobic interactions between residue 94 and the fluorobenzene moiety of the hapten.

The choice for template loop structures in the second model (model 2) was based on canonical class and maximal overall sequence identity, but also on structural similarity of the antigens. Three mutants were made based on this model 2. In the first mutant, Trp33 (CDR-H1), which is located quite deep in the pocket, was replaced by alanine to evaluate the destabilizing effect of introducing a cavity in the antibody–antigen interface and to disable possible stacking with the fluorobenzene group. The Tyr96Val mutant (CDR-L3) was also made in order to disable potential stacking interactions of fluorobenzene with residues at the bottom of the binding cavity. The third mutation Thr97Glu (CDR-H3) was made to determine the effect of removing or changing potential hydrogen bonds. By mutating Tyr94, Tyr96 and Trp33, conservation of canonical classes was maintained, as these residue positions are expected to tolerate larger variation based on the canonical class definition by Martin and Thornton (1996Go).

Site-directed mutagenesis and cloning of the double mutant

The wild-type antibody was cloned into a production vector pKKTac (Takkinen et al., 1991Go) as a Fab fragment which has the intact light chain (VHCL) and variable (VH) and first constant domain (CH1) of the heavy chain. This new construct, pENA5His, was used as a template for PCR mutagenesis as well as a production vector. DNA work was performed using standard recombinant DNA protocols (Sambrook et al., 1990Go). Oligonucleotide primers were used in PCR to introduce mutations to pENA5His template. Amino acids to be changed have been marked on the amino acid sequence of ENA5His in Figure 1a (model 1) and b (model 2). Mutations were confirmed by nucleotide sequence analysis. The resulting plasmids were designated as pENA5HisTyr94 Leu, pENA5HisTyr96Val, pENA5HisTrp33Ala, pENA5 HisTrp100JLeu and pENA5HisThr97Glu according to the mutated residue. To construct the double mutant, the light chain of pENA5HisTyr96Val was digested by SacI and XbaI and ligated to SacI–XbaI-digested vector pENA5HisTrp33Ala. The double mutant was designated as pENA5HisTrp33Ala/Tyr96Val. The change of the light chain was verified by nucleotide sequencing. All constructions have codons for six histidines at the 3' end of the light chain. This histidine tag was used for purification and immobilization of antibody fragments.

Production and purification of mutant antibody fragments

Expression vectors containing mutated antibody genes were transformed into production host E.coli RV308 (ATCC 31608). Best producing colonies were screened by cultivating 4–16 single transformants in 2 ml cultures. The supernatants were then analysed by standard SDS–PAGE (Laemmli, 1970Go) followed by western blotting (Towbin et al., 1979Go) using alkaline phosphatase conjugated anti-mouse (Fab-specific) antibody (Sigma) as a detection antibody. The ability of the mutant Fab fragments to recognize antigen was checked by ELISA. 96-well EIA-plates (Dynatech Laboratories, Chantilly, VA, USA) were coated with 0.5–1 µg of d-BSA conjugate in 100 µl 0.1 M bicarbonate buffer pH 9.8 overnight at 4°C. The coated plates were washed three times with PBS pH 7.4 (20 mM Na–phosphate pH 7.4, 150 mM NaCl) without incubation and blocked with 200 µl 0.5% BSA–PBS pH 7.4 for 1 h at ambient temperature. Culture supernatants (100 µl) were applied and incubated for 1 h, washed as previously mentioned and the bound Fab fragment detected using the same antibody as in western blotting. As a substrate for alkaline phosphatase PNPP (p-nitrophenylphosphate; Orion, Finland), 2 mg in 1 ml of diethanolamine–MgCl2 buffer (Reagena, Finland), was used and the absorbance at 405 nm was measured after 5–30 min depending on colour development.

High density cell cultivations, purification with metal affinity chromatography followed by protein G–affinity chromatography were performed in a similar manner as for the wild-type Fab fragment ENA5His (Nevanen et al., 2001Go). Purity, assembly and functionality of mutated Fab fragments were tested with SDS–PAGE, western blotting and ELISA, as described earlier.

Determination of IC50 values using surface plasmon resonance

BIAcore (Pharmacia Biosensor, Uppsala, Sweden) was used to compare the binding properties of the five single mutants to the wild-type ENA5His (Karlsson et al., 1991Go). All the assays were performed in HBS buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.05% surfactant P20, Amersham Pharmacia Biotech). The d-BSA was coupled to CM5 sensor chip using the amine coupling kit provided by the manufacturer. In order to compare the overall association and dissociation behaviour of mutants to the wild-type ENA5His, 300 nM of every Fab was injected at a flow rate of 5 µl/min over a BIAcore flow cell coated with 1200 RU (resonance unit) of d-BSA. After the sample application and association phase, the Fab fragments were allowed to dissociate in HBS buffer for 10 min. Aliquots of the same samples were also injected over the BSA-coated flow cell (500–1100 RU) for the determination of potential non-specific binding to BSA alone. Between the sample applications the d-BSA and the BSA surfaces were regenerated with 100 mM NaOH (7 µl). Sensorgrams were overlaid for preliminary comparison of the mutants to the wild type.

The relative affinities (i.e. IC50 values) of the Fab fragments were measured by a competition assay on a BIAcore chip. 1200 RU of d-BSA was immobilized onto the sensorchip surface. Serial dilutions of the a- and d-enantiomers at final concentrations of 10–4–10–10 M were allowed to react for at least 10 min with 100 nM Fab solution and then run over the d-BSA surface at a constant flow rate of 5 µl/min. Fab samples without any free enantiomer were used as controls. Injected sample volume was 25 µl. After 250 s of association, RU values were collected for each run and plotted against concentrations of free enantiomers to determine the IC50 values.

Immunoaffinity chromatography for enantiospecific separation

The enantiomeric separation properties of the Fab fragments were examined in small-scale immunoaffinity chromatography experiments as reported before (Nevanen et al., 2001Go). Two milligrams of Fab fragments were immobilized via His6 tag to 200 µl metal affinity matrix loaded with copper as according to the manufacturers’ instructions (Pharmacia Amersham Biotech). The maximum amount of the racemate, containing a- and d-enantiomers in 1:1 ratio in PBS pH 7.4 with 2% DMSO, was applied to the column and unbound a-enantiomer was washed away with PBS pH 7.4 and 2% DMSO. The bound d-enantiomer was eluted using PBS pH 7.4 supplemented with methanol. A range of 25–50% methanol in eluent was used to examine the effective solvent concentration for each mutant. Immobilized ENA5His was used as a reference. The possible binding of a-enantiomer to the mutant Fab fragments, the efficiency of elution of the bound d-enantiomer and the reusability of the affinity matrices were characterized with these small-scale columns.

Based on the results obtained with the small-scale affinity columns described above, the mutant Tyr96Val and the double mutant Tyr96Val/Trp33Ala were further studied in detail. Mutant Tyr96Val (28 mg) and the double mutant Tyr96Val/Trp33Ala (34 mg) were immobilized via the histidine tag (according to the manufacturers’ instructions) to HR5/5 columns containing 1.1 ml Chelating Sepharose Fast flow (Amersham Pharmacia Biotech, Sweden). Separation of enantiomers was done with 10S ÄKTA-system (Amersham Pharmacia Biotech, Sweden) having a UV-Vis detector. In all these runs, the following chromatography conditions were used: flow rate 1 ml/min, injection volume 10 ml, room temperature. The columns were equilibrated with 10 bed volumes of PBS pH 7.4 before loading the racemate in PBS pH 7.4 containing 2% DMSO. Initially, the columns were overloaded with the racemate in order to determine the experimental capacity of the column in efficient enantioseparation. After racemate application, the unbound a-enantiomer was washed out from the column with PBS pH 8.5 containing 2–10% DMSO followed by elution of the bound d-enantiomer with 15 bed volumes of 40% methanol–PBS pH 7.4. After this, the columns were further washed with 10 bed volumes of 40% methanol–PBS pH 7.4 to ensure the release of any residual d-enantiomer and then equilibrated with 10 bed volumes of PBS pH 7.4 before the next cycle of separation. The amount of the enantiomers in the fractions was analysed by a HPLC system (Waters, Milford, MA) as described earlier (Nevanen et al., 2001Go).


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 Materials and methods
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Quality and description of the models

In the present study, the models of the antigen-binding site of ENA5His in complexed form were used to determine the residues in contact with the hapten and to specify which ones of these could be candidates for mutations. Due to the large conformational variation of the CDR-H3 loop, a univocal choice for its template structure was difficult to make. This was the main reason why two models were built, based on different antibody template sequences (Figure 1).

The superposition of the template structures of the two Fab fragments (1iai_703.1.4 and 1iai_409.5.3) used for heavy and light chain construction demonstrated that the packing angle is similar between the template structures. Thus, a good fit of interfaces between the light and heavy chains could be obtained even when the chains were combined from these two different structures. The stereochemical quality of both ENA5His model structures according to PROCHECK is good. The MD simulations even improved the distribution of residues occupying favourable regions in the Ramachandran plot (data not shown). One amino acid residue in model 1 and two in model 2 lie outside the favoured regions and are located in the hypervariable loops, CDRs, forming the antigen-binding site. The six CDR loops in both models of ENA5His antibody form a deep binding cavity and they contain altogether 10 tyrosine and three tryptophan residues. The overall structure of the binding domain of the antibody and one of the enantiomers, based on model 2, is shown in Figure 3.



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Fig. 3. An overall representation of the ENA5His antibody model with the hapten in its binding site.

 
Most differences between the binding sites of the two complex models are due to different conformations of the hypervariable loop 3 in the heavy chain (CDR-H3). In both models, the hapten is orientated so that its fluorine is pointing to the bottom of the cavity. In model 2, the binding site geometry enables deeper binding of the hapten, whilst model 1 does not properly explain the recognition of the triazole ring. Five point mutations were originally suggested to reduce the affinity; two of them are based on model 1 and three on model 2. A detailed view of the arrangement of mutagenised residues is given in Figure 4 and a more precise description of the binding sites in the Discussion.




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Fig. 4. (a) The hapten-binding site of model 1. Positions of the residues subjected to mutation as based on model 1 are indicated by their side chains. (b) The binding site of model 2. Positions of the residues subjected to mutation as based on model 2 are indicated by their side chains.

 
Site-directed mutagenesis, production and purification of mutant antibody fragments

Nucleotide sequence analysis of the mutated Fab fragments revealed no changes in amino acid composition of the variable regions other than the intended ones. All the mutants were expressed in E.coli cells and produced in a soluble form to the culture supernatant. The relative amounts of covalently assembled heavy and light chains of the mutant Fabs varied only slightly compared to wild type according to western blot analysis in non-reducing conditions (data not shown). Final yields from one litre fermentor cultivations after the two purification steps were in all cases 20–50 mg of mutant Fabs. The ability of the mutants to bind d-enantiomer was first verified by ELISA before BIAcore analysis and column assays. All single mutants and the double mutant gave detectable signal in ELISA (data not shown).

Determination of IC50 values of the single mutants using surface plasmon resonance

Association and dissociation behaviour of the single mutants was compared to the wild-type ENA5His by BIAcore analysis. The same amount (300 nM) of all single mutants and of the parent ENA5His were subjected to the d-enantiomer-BSA immobilized surface. BSA alone was used as a control surface in the BIAcore and no response was observed either for the wild type or any of the mutants. From the overlay plot shown in Figure 5, differences can be seen in the association and dissociation phases of the sensorgrams of the mutants. The mutants Trp33Ala, Trp100JLeu and Tyr96Val showed faster dissociation as compared to the wild-type ENA5His. The association phase of mutant Tyr96Val was divergent from the others, its saturation level was reached earlier. This low-level saturation of binding was probably due to the dissociation of the low affinity mutant Tyr96Val from the d-BSA surface during the sample injection. The double mutant was shown to give a response in the BIAcore (data not shown) and displayed a sensorgram comparable to the mutant Tyr96Val, but had a slightly faster dissociation phase.



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Fig. 5. Comparison of the association and dissociation behaviour of the ENA5His wild type and five single mutants by BIAcore. A 300 nM solution of each Fab fragment was passed on the sensorchip surface (d-BSA) and the resulting sensorgrams, displaying the association and beginning of the dissociation phases, are presented as an overlay plot. 1 = Tyr94Leu; 2 = ENA5His, wild-type; 3 = Trp33Ala; 4 = Trp100JLeu; 5 = Thr97Glu; 6 = Tyr96Val.

 
The affinities and specificities of the single mutants were also compared to the wild-type ENA5His antibody fragment by competitive BIAcore analysis (Table II). d-BSA was used as an immobilized antigen in competition studies for both a- and d-enantiomer. Results of the competitive assay with d-enantiomer supported the overlay plot of the mutants. Concentrations of the free d-enantiomer needed to halve the response was less for the mutant Tyr94Leu than for the wild type, indicating that the affinity of this mutant could be slightly higher than that of ENA5His. Other mutants have higher IC50 values than wild type, indicating lower affinities. The IC50 values for the a-enantiomer were extrapolated from the inhibition curve for the higher concentrations as final concentrations >0.1 mM were not possible due to the poor solubility of the hapten. Therefore the IC50 values for the a-enantiomer are only indicative. By introducing one single mutation to a selected location, the affinity for the d-enantiomer was decreased up to 5- to 10-fold.


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Table II. Relative affinities of Fab fragments for enantiomers
 
BIAcore measurements were used to rank the mutants for the decision on which mutant Fab would be studied in continuous affinity chromatography experiments and which two mutations were to be combined to construct the double mutant. Absolute values for IC50 determination varied depending on data handling and conditions used for measurements, such as the amount of Fab and immobilized d-BSA. Therefore only those values obtained from the same set of measurements were used for comparison of mutants.

Enantiospecific separation in columns

All single mutants were studied for their enantioseparation properties first in small scale (200 µl) columns. Different concentrations (25–50%) of methanol were used as eluent to determine efficient elution conditions. All columns except for the Tyr94Leu column released the bound enantiomer in 40% methanol–PBS pH 7.4. Therefore, the elution efficiency of the single mutant affinity columns were compared to the ENA5His column using 40% methanol–PBS pH 7.4 (Table III). Two milligrams (40 nmol) of each mutant was immobilized to the matrix and the maximal amount of the enantiomers (80 nmol racemate having both enantiomers in 1:1 ratio) was applied to the columns. The amounts of enantiomers in flow through, washes and elution were determined by chiral HPLC. The a-enantiomer was washed away from all the mutant columns before elution. Elution efficiencies were calculated from the input/output ratio of the enantiomers. Accuracy of the analytical method was affected by possible methanol evaporation during sample collection and treatment, leading sometimes to recoveries >100%. The affinity column with the mutant Tyr94Leu performed in a similar manner as the wild-type column, possibly having a slightly decreased elution efficiency, thus supporting the BIAcore data of increased affinity (Table II). The four other mutant affinity columns shifted towards the desired elution properties (Table III). Columns containing either Trp33Ala, Trp100JLeu or Tyr96Val mutant protein were also capable for several rounds of enantioseparation without loss of binding capacity when eluted with 40% methanol–PBS. The double mutant column could use a 35% methanol solvent concentration for the quantitative elution of the d-enantiomer, whilst with 30% methanol, only 92% of the bound enantiomer was eluted.


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Table III. Efficiency of elution of bound d-enantiomer with 40% methanol–PBS pH 7.4
 
The mutant Tyr96Val and the double mutant Trp33Ala/Tyr96Val containing affinity matrices were subjected to automated, continuous enantioseparation on a bigger scale, where repetitions of enantioseparation could be analysed in a more quantitative manner. The elution conditions for continuous enantioseparation was 40% methanol–PBS pH 7.4 for both mutant affinity columns. Both mutants were separating enantiomers efficiently even after 20 cycles (Figure 6). After 10 and 20 rounds of enantioseparation, columns were overloaded with racemate and the experimental binding capacity was compared to the theoretical binding capacity for the d-enantiomer. After 10 rounds of enantioseparation, the experimental binding capacity was 85% of the theoretical maximum for the mutant Tyr96Val column and 86% for the double mutant Trp33Ala/Tyr96Val column, and after 20 rounds, 88% and 79%, respectively. The amount of the bound a-enantiomer was 1% and it was eluted out of the column in the first fraction of elution.




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Fig. 6. Chromatograms of the affinity columns of ENA5His mutants Tyr96Val and Trp33Ala/Tyr96Val separating a- and d-enantiomers of the Finrozole drug. (a) A chromatogram of Tyr96Val affinity column showing the appearance of a-enantiomer in fractions (1–20) of sample application and washes (–) and d-enantiomer in fractions (21–38) of elution (o). (b) Corresponding chromatogram of Trp33Ala/Tyr96Val affinity column.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
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 Discussion
 References
 
Homology modelling has previously been used to increase affinity or to change the binding properties of antibodies by identifying those amino acids, which are important for binding the hapten. Based on modelling, either site-directed or random mutagenesis has then been applied and the mutants screened for improved properties (Roberts et al., 1987Go; Casipit et al., 1998Go; Hemminki et al., 1998Go; Iba et al., 1998Go; Miyazaki et al., 1999Go). In order to perform more rational improvement of the binding properties, high-resolution structures of both free and complexed forms of the antibody are needed. Some studies have demonstrated the involvement of conformational changes upon hapten binding and these are difficult to predict by computational methods (Arevalo et al., 1994Go; Valjakka et al., 2002Go). In this study, modelling of a hapten-antibody complex was used to identify amino acid residues in the antigen-binding pocket of ENA5His contributing to the enantioselective recognition and binding of the hapten. Modelling-based design of single mutations proved to be a quick and effective way to adjust the affinity of the antibody.

The configuration of the d-enantiomer was S,S according to previous preliminary determination. Later, when the modelling and the mutagenesis work was done, more precise structure determination revealed that the configurations of the racemic mixture of a- and d-enantiomers are S,R and R,S (Södervall et al., 2002Go). However, the changes in affinity for four of the mutants designed according to S,S-enantiomer–ENA5His complex were successful, despite the uncertainty of the absolute configuration of the hapten. This could be due to the fact that ENA5His has relatively high affinity for b- and d-stereoisomers and also residual affinity for a- and c- stereoisomers. Furthermore, in most of the successful mutants, the altered residues have interactions with the fluorobenzene, which is common for all configurations of the hapten.

Antigen-binding sites are, in general, rich in tyrosine residues (Padlan, 1990Go; Dougan et al., 1998Go; Collins et al., 2003Go). These tyrosines have a dual nature as they can act as hydrophilic residues when the antibody is in the free water exposed form, and as hydrophobic residues in the bound form (Tsumoto et al., 1995Go). High proportions of tyrosine and tryptophan residues are also found in the six CDRs of the ENA5His antibody (10 tyrosines and 3 tryptophans). A major part of the binding forces most probably comes from these aromatic residues, which show stacking interactions with the buried part of the hapten during our MD simulations.

For the designed mutations, it was considered important to maintain the canonical structure of the CDRs and the physico-chemical character of the residues to be mutated. Five single point mutations were made to the ENA5His antibody fragment. Tyr94 and Tyr96 are located in the hypervariable loop 3 of the light chain (CDR-L3). Both of these positions are known to tolerate moderately conservative mutations without changing the canonical structure of the loop (Martin and Thornton, 1996Go). The experimental studies (Table II) show that the binding of the Tyr94Leu mutant is similar to the wild type or even slightly improved. In both our models, this residue is located at the edge of the cavity, close to the solvent. In model 2, where the hapten is bound deeper in the pocket, Tyr94 has a stacking interaction with the phenyl ring connected to the linker contributing to higher affinity. Another mutation in CDR-L3, Tyr96Val, was designed based on model 2 where Tyr96 is located near the bottom of the cavity and has stacking interaction with the fluorobenzene (Figure 4b). Mutating Tyr96 to Val lowers the affinity of the Fab fragment over 10-fold (Table II). According to MacCallum et al. (1996Go) and Jirholt et al. (2001Go), the corresponding residue often interacts with the antigen and Kusharyoto et al. (2002Go) have reported that the change of Tyr to Phe in this position of an anti-atrazine antibody decreased the affinity over 2-fold.

The third mutation Trp33Ala is situated in the heavy chain, in the CDR-H1 loop, and this mutant antibody fragment has 5-fold lower affinity for the d-enantiomer compared to the wild type. The Trp33Ala mutation in CDR-H1 generates additional space in the binding cleft, but should have only a minor effect on the backbone conformation of the loop. Trp33 has been previously reported as an important residue for binding (MacCallum et al., 1996Go; Chen et al., 1999Go; Jirholt et al., 2001Go; Kusharyoto et al., 2002Go).

The mutations Thr97Glu and Trp100JLeu are also situated in the heavy chain, in the CDR-H3 loop, which is generally known to be the most variable loop and usually has many contacts with the antigen. The Thr97Glu mutant shows an ~10-fold reduced affinity for the d-enantiomer (Table II). The Thr97Glu mutation is based on model 2 and it was intended as a control, which should create a strong effect in order to verify the models. Our experimental results show a clearly reduced binding affinity of this mutant confirming the better predictive quality of model 2 for the complex, at least for the geometry between the CDR-H3 loop and the hapten. The other mutation in the CDR-H3, Trp100JLeu, which is near the surface in both models, is more conservative in its nature. It lowers the affinity for the d-enantiomer 6-fold (Table II). The association rate is similar to the wild type (Figure 5), but the dissociation is faster, which most probably results from loss of favourable aromatic interactions with the hapten in the 97 position (Figure 4b).

It is important that antibody affinity columns for preparative scale are stable over multiple rounds of fractionation without dramatic loss of their binding capacity. We have shown that, by adjusting the affinity of immobilized antibody fragments, it is possible to use them for efficient enantioseparation continuously. Decreasing the affinity by one or more orders of magnitude (Tyr96Val and Tyr96Val/Trp33Ala mutants) enabled the use of a lower concentration of organic solvent for elution, which increased the lifetime of the antibody fragment columns. The feasibility of antibody affinity columns for the small-scale preparative enantioseparation of a low molecular weight compound (the molecular weight of the Finrozole drug is 322 g/mol) can be calculated using, for example, the Tyr96Val mutant as a model antibody system. From a one litre fermentor cultivation, enough Fab was obtained to fractionate at least 10 mg of a drug in 20 cycles of enantioseparation. This amount of pure enantiomers is generally enough for the early phases of drug development.


    Acknowledgements
 
We thank for Marja Södervall (Hormos Medical Corp) for providing Finrozole and Timo Lotta (Orion Pharma, Finland) for support during this work. Arja Kiema and Riitta Suihkonen are thanked for excellent technical assistance. We are grateful to Tapani Suortti for the HPLC analysis of the numerous fractions and to Markus Linder for his expertise in ÄKTA-chromatography system and comments on the manuscript. Sarah Coleman is thanked for revising the English language. The financial support of the National Technology Agency (Finland) is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received April 23, 2003; revised September 25, 2003;; accepted October 21, 2003





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