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Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To circumvent the lack of specificity of the anti-steroid Abs, steroids can be extracted with an organic solvent and separated by chromatography to determine their concentrations by immunoassay (5, 6, 7). However, this method is laborious, and direct immunoassay from plasma or urine extracts is much more desirable for multiple determinations.
A different approach may be to modify the characteristics of an available anti-steroid Ab to decrease its cross-reactions with analogues. Much progress has been made recently in Ab engineering. The development of methods for the cloning and heterologous expression of Ab variable gene sequences, leading to the synthesis of functional Ab fragments, together with the application of methods for site-directed mutagenesis has greatly facilitated structure-function studies of Ab-combining sites (8, 9, 10). The value of these methods is further enhanced by the use of molecular modeling, allowing the prediction of Ab-combining site structure. The use of these techniques combined with powerful phage display technology (11) has led to the production of several Abs with improved affinity (12, 13, 14, 15, 16).
We have used site-specific randomization to build a library of mutant Ab fragments and then phage display to select from this library anti-cortisol Abs with the required high specificity and affinity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Escherichia coli strain TG1
(
(lac-pro), supE, thi, hsdD5/F', traD36, proAB,
LacIq, lac ZM15) was used as the
bacterial host for the preparation of phagemids and as the host for
bacteriophage M13KO7. E. coli HB2151 ((lac-pro),
ara, nalr, thi/F' proAB,
LacIq, lacZM15) was used
for expression of soluble scFv.
Construction of the wild-type anti-cortisol scFv4
To construct the 5A4 scFv, VH and VL genes were amplified by PCR from hybridoma cDNA prepared as previously described (17). The 3' primer used to amplify VH contained a part of the coding sequence for the linker GSTSGSGKPGSGEGSTKG (18), and the 5' primer used to amplify the VL contained the rest of the sequence (with 15 overlapping bases). The scFv was assembled by splice overlap extension PCR (19). The primers used resulted in two restriction enzyme sites (NcoI and EagI) being introduced at the 5' and 3' ends of the scFv gene. These sites were used to ligate the fragments into the pHEN1 phagemid vector (20) to give pHEN-5A4.
Production of scFv and phAb
HB2151 cells harboring the phagemid pHEN-5A4, PHEN1, or pHEN
coding for scFv mutants were grown at 30°C to an OD600 of
0.8 in 2YT containing 100 µg/ml ampicillin and 2% glucose (2YTAG).
The culture was centrifuged for 5 min at 3000 x g and
25°C, and the cells were resuspended in the same volume of 2YT
containing 100 µg/ml ampicillin and 100 µM IPTG to induce
expression. The culture was incubated at 30°C for 180 min. Cell
fractionation was performed as previously described (21) (Fig. 1
A). For equilibrium dialysis,
periplasmic extracts were dialyzed 12 h against 0.1 M sodium
phosphate (pH 7.2), 10 mM azide, and 0.1% gelatin (phosphogel buffer)
just before use.
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Construction of the mutant library
Overlap PCR was used to build the library (Fig. 2). Four PCR were performed with pHEN-5A4
DNA as the template and with primer pairs 5PelB and PMH3, RPMH3 and
LINKINF, LINKSUP and PML3, and RPML3 and 3CMYC (Eurogentec, Seraing,
Belgium; see below for primer sequences). The PCR products were
purified on polyacrylamide gels, mixed, and used as template in a final
PCR using primers 5PelB and 3CMYC. The final PCR product (5 µg) was
inserted into the pHEN1 phagemid (2 µg). Fifty electroporations
allowed us to obtain a library of 2.5 x 107 clones.
Some of the clones were tested both by DNA Miniprep and restriction for
the presence of an insert of expected size and also for the production
of a 30-kDa product using the anti-tag Ab as previously described
(20). The library was rescued as previously described (20) using helper
phage M13KO7.
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The primer sequences were: 5PelB, CTC GCK GCS CAG CCG GCC ATG GC (K = G/T, S = G/C; the NcoI site is in italic); PMH3, GCC ATC TAT TAC TGT GCA AGA 123 AGT GTC TAT GGT AGC AGC 453 CCC 663 GAT TCC TGG GGC CAA GG; RPMH3, CTT GCA AGT AAT AGA TGG C; LINKINF, TC ACC TGA ACC AGG TTT ACC AGA ACC TGA GGT AGA ACC TGA GGA GAC GGT GAC; LINKSUP, CT GGT AAA CCT GGT TCA GGT GAA GGT AGT ACT AAA GGT GAC ATT GTG CTG AC; PML3, GCC ACT TAT TAC TGC CAG CAG TGG AGT AGT 223 CCA 553 ACG TTC GGT GCT GGG ACC; RPML3, GCT GGC AGT AAT AAG TGG C; and 3CMYC, C AAG CTT ACT AGT TTA TGC GGC CCC ATT CAG ATC C (1: A 7%, C 79%, G 7%, T 7%; 2: A 79%, C 7%, G 7%, T 7%; 3: G 21%, T 79%; 4: A 70%, C 10%, G 10%, T, 10%; 5: A 10%, C 70%, G 10%, T 10%; 6: A 7%, C 7%, G 7%, T 79%).
Selection of a phAb library
The phAb were selected by binding to Ag-coated immunotubes (Maxisorp, Nunc, Naperville, IL). The Ag cortisol-3-CMO-BSA (Sigma, St. Louis, MO) was used to coat the tubes overnight at 4°C at 10 µg/ml in PBS and was then saturated with 4% milk PBS (MPBS) for 1 h at 37°C. For the first round of selection, 1013 titrated units of the phAb library in a total volume of 2 ml of MPBS was used per immunotube. Before binding phAb particles were also saturated for 2 h at 25°C in MPBS. Immunotubes were washed, and bound phAb were eluted by incubation for 10 min with 1 ml of 100 mM triethylamine (and immediately neutralized with 0.5 ml of 1 M Tris-HCl, pH 8.0) or by 50 µM free cortisol in PBS for 120 min at 25°C on a rotator. After each round of enrichment, E. coli TG1 were reinfected with eluted phAb, and the phAb were rescued and used for the next round of panning. Selection was stopped when the recovery of eluted phAb increased significantly. The cortisol concentration used for elution was decreased to 10 µM for the second round and to 1 µM for subsequent steps as indicated in the text. For competitive binding protocols, the analogue (prednisolone or dexamethasone) was added to the MPBS buffer used to saturate the phAb particles. Analog concentrations used during first, second, and subsequent rounds were 1, 10, and 50 µM, respectively.
ELISA screening of clones
Single ampicillin-resistant colonies resulting from infection of E. coli TG1 with eluted phAb were used to inoculate 150 µl of 2YT medium containing 100 µg/ml ampicillin and 2% glucose in 96-well plates. The phAb was prepared as previously described (20). Supernatants containing phAb were tested for binding by ELISA in Falcon 96-well plates (Becton Dickinson, Oxnard, CA) coated overnight with cortisol-3-CMO-BSA at 10 µg/ml in PBS and saturated with MPBS. PhAb binding was detected with a horseradish peroxidase anti-M13 Ab conjugate (Amersham Pharmacia Biotech, Uppsala, Sweden).
For a first approximation of the affinities and specificities of phAb in solution, the ELISA method of Friguet et al. (22) was used. Free cortisol at concentrations between 100 nM and 1 µM was mixed with a fixed amount of phAb (determined by ELISA titration of a polyethylene glycol-purified preparation) in MPBS. After incubation for 20 h at 25°C, the free phAb titer was measured by ELISA as described above. The Kd value was calculated using the following equation: A0/(A0 - A1) = Kd/at1 + 1, where at1 is the given concentration of Ag, A0 is the ELISA signal obtained without cortisol in the preincubation, and A1 is the ELISA signal obtained following preincubation of phAb with cortisol at at1. Each condition was performed in duplicate. The absorbance of each well of the ELISA plates was measured when the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) reaction was still in the linear range, a fact that was confirmed by taking several time points per plate. To determine the percentage of cross-reaction, a fixed amount of phAb was incubated in ELISA wells (coated with 10 µg/ml cortisol-3-CMO-BSA) in the presence of cortisol or an analogue at concentrations ranging from 10 nM to 1 µM for 4 h at 25°C in MPBS. After washing, bound phAb was revealed as described above. From the inhibition curve, concentrations allowing 50% inhibition (IC50) were determined. The percentage of cross-reaction was given by the equation: (IC50 cortisol/IC50 analogue) x 100. The absorbance of each well of the ELISA plates was measured when the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) reaction was still in the linear range (confirmed by taking several time points per plate).
Equilibrium dialysis of scFv
The phagemid corresponding to the phAb that showed interesting affinity measurements by the ELISA method were sequenced, and those that were mutated were used to transform HB2151 cells to prepare scFv extracts. To determine affinity and percentage of cross-reactions of scFv, equilibrium dialysis was performed in a noncommercial equilibrium microvolume dialyzer (Immunotech Coulter Beckman, Marseille, France) in which paired chambers of 150 µl each were separated by a 6000–8000 m.w. cut-off Spectra/Por membrane (Spectrum Medical Industries, Houston, TX). For standardization of the scFv concentration, periplasmic extracts containing scFv were diluted (1 up to 64) in phosphogel buffer. One hundred microliters of dilution was mixed with 50 µl of phosphogel and loaded into chambers on one side of the membrane; on the other side, 50 µl of [1,2,6,7-3H]cortisol (2.9 TBq/mmol; New England Nuclear, Boston, MA) diluted in phosphogel (0.18 pmol final) was mixed with 100 µl of periplasmic extract from HB2151 cells harboring the pHEN1 vector at the same dilution as that used for the periplasmic extracts containing scFv. Dialysis cells were incubated for 24 h at 4°C on rotator. The radioactivity in each cell was determined by liquid scintillation counting (PCS liquid scintillation, Amersham Pharmacia Biotech). For each periplasmic extract, the dilution corresponding to 50% of the disintegrations per minute bound was chosen to calibrate the scFv concentration for the following experiment.
One hundred microliters of the appropriate concentration of periplasmic extract was mixed with 50 µl of different amounts (0.0625 up to 250 pmol in phosphogel) of steroid (cortisol, dexamethasone, and prednisolone) and loaded into chambers on one side of the membrane; on the other side, 150 µl of the mixture described above ([1,2,6,7-3H]cortisol mixed with periplasmic extract) was added. Specific binding was determined by subtracting the amount of radioactivity in control chambers lacking unlabeled steroid from that obtained in the chambers containing steroid. Bound and free cortisol concentrations were calculated, and the equilibrium constant for binding was determined by Scatchard analysis. From the inhibition curves, concentrations allowing 50% inhibition (IC50) were determined. The percentage of cross-reaction was given by the equation: (IC50 cortisol/IC50 analogue) x 100. All cells were used in triplicate with three different periplasmic extracts for each scFv.
Molecular modeling
The 5A4 three-dimensional model has been described previously (17). The refined Ab variable region structures were predicted using the model-building computer program INSIGHT II (Biosym Technologies, San Diego, CA). The relating structures were energy minimized using the DISCOVER computer program (Biosym Technologies) with its supplied energy parameters.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We cloned the anti-cortisol 5A4 mAb (obtained by immunization
with cortisol-3-CMO-BSA conjugate) in the scFv format (VH
and VL domains linked by a flexible peptide linker) to
display on the surface of filamentous phage. This system allows
powerful selection of Ab libraries (20). Oligonucleotides able to
amplify murine VH or VL genes were first
designed using the Kabat database (23). The VH and
VL genes of the 5A4 mAb were amplified using appropriate
primers and assembled by overlap PCR. The resulting gene was inserted
into the pHEN1 phagemid vector (20) to give pHEN-5A4, which was
introduced into E. coli by electroporation. The pHEN-5A4
allows fusion of the PelB signal peptide to the scFv to send the
fragment in the oxidizing environment of the periplasm, leading to
disulfide bond formation and proper folding. The c-Myc tag is also
fused to the C-terminus of the scFv to facilitate detection. Cell
fractionation and Western blot analysis revealed that only about 20%
of the scFv was produced as a soluble periplasmic protein in HB2151
cells (Fig. 1
A). The rest was probably aggregated as
periplasmic inclusion bodies. One of the more useful features of the
pHEN1 is the presence of an amber codon at the junction between the Ab
sequence and that part of the gene encoding the mature capsin protein
III of M13 filamentous phage. In supE cells, suppression was
approximately 20% efficient, such that both soluble and pIII-linked
scFv were produced (Fig. 1B). Infection of these cells
harboring pHEN-5A4 by a helper phage allowed production of phage
displaying the Ab fragment on their surface (called phAb). For a first
approximation, affinity measurements and competitive binding assay of
phAb were performed in solution by ELISA (22). In this condition, the
relative affinities of wild-type 5A4 phAb toward cortisol,
prednisolone, and dexamethasone were 335, 800, and 840 nM (data not
shown). The relative affinities of wild-type 5A4 scFv determined by
equilibrium dialysis toward the same steroids were 70, 230, and 320 nM,
respectively. The large difference observed could be due to the method
used and/or the nature of the Ab (phAb or scFv). For the following
experiments, we decided to use the scFv format and equilibrium
dialysis, which is a more rigorous method in this case. By this method
the affinity of 5A4 wild-type scFv to cortisol was 7.0 nM (Fig. 3A), whereas the affinity of
5A4 Fab was 6.6 nM. Cross-reactions of the wild-type scFv were similar
to those of the 5A4 Fab (prednisolone: 30% for Fab, 29% for wild-type
scFv; dexamethasone: 22% for Fab, 21% for wild-type scFv; see Table IV).
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The next step of this work was to create a library of scFv mutants
from which those with a higher specificity for the cortisol could be
selected. We chose to degenerate paratope residues in contact with or
close to the hapten. The amounts of soluble and active scFv 5A4
produced by E. coli are very low, so crystallographic
studies are not feasible. We therefore tried to obtain structural
information from molecular models of the scFv complexed with cortisol.
A computer molecular model of the scFv 5A4 was constructed as
previously described (17). The position of cortisol inside the paratope
was modified according to new energy minimizations and biochemical
data; the six CDR loops in the molecular model form a pocket containing
several aromatic amino acids (TrpH50, TrpH47,
TrpL91, HisH95, TyrH33,
PheH100d) that provide a hydrophobic environment around the
cortisol (Fig. 4A). The
structure of this pocket was similar to the three-dimensional structure
of the anti-progesterone DB3 Fab fragment as determined by x-ray
crystallography (24). The stacking interactions between
TrpH50 and TrpH100 located on either side of
the hydrophobic steroid nucleus in the DB3 paratope appear to
correspond to TrpH50 and TrpL91 in our model.
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-face interacting with TrpL47 and
TrpL50 and the ß-face, which possesses the two methyl
groups, interacting with TrpL91. The 21-hydroxyl group of
cortisol might form a hydrogen bond with HisH95 or
ThrH100b, and the 3-ketone and 11-hydroxyl group might
interact with AsnL94 and ProL96, respectively
(Fig. 4A). The model predicts that residues
TyrH33, SerH35, AsnH58, and
PheH100d are also close enough to cortisol to interact.
We compared these results with published findings following mutagenesis
of some of the paratope residues and affinity measurements to identify
positions involved in hapten recognition. These studies are consistent
with our model. Many of the contact positions from our model have been
shown to be essential for high affinity binding in other Abs (Table I). Our prediction of contact amino acids
was also strongly supported by the study of MacCallum et al. (25),
which described positions of contact residues from 10 x-ray Ab fragment
structures complexed with their hapten (Table II). Indeed, almost all the positions we
chose are described as highly frequent contact positions, with the
exception of position H100d, which is only present in long CDR H3.
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For practical reasons, it is difficult to obtain libraries of
>108 clones (transformation efficiency being the major
limiting factor), which means that the number of degenerate positions
(where an amino acid is substituted by one of the 20 other residues)
must be limited to 6 (206 = 6.4 x 107).
Eleven positions in mAb 5A4 were predicted to be potentially in
interaction with the hapten (Table II). We thus had to choose five or
six mutagenesis targets among these residues. TrpH50 and
TrpL91 are thought to interact by hydrophobic stacking (4, 26) with the steroid nucleus. TrpH47 is also involved in
interactions between VH and VL domains. These
three residues were thus conserved. Among the eight remaining residues,
we decided to restrict the mutagenesis to residues belonging to CDR H3
and L3 (HisH95, ThrH100b, PheH100d,
AsnL94, and ProL96) because these CDRs form the
center of the paratope and contain positions that have most frequently
been shown to be in contact with the Ag, especially in the case of
haptens (25).
The nature of these targeted residues is probably very important
for the binding activity, and multiple mutations per Ab would probably
create a large number of functionally useless Abs. To avoid this
problem, we used parsimonious mutagenesis (27, 28). The principle of
this method is to use mutagenesis codons leading to about 50% amino
acids at each degenerated positions being wild type. This has several
advantages. First, the total number of substitutions per Ab is reduced,
resulting in a larger number of well-folded and potentially active
binders in the library. Secondly, if a residue chosen as mutagenesis
target is essential for binding, classical mutagenesis methods will
frequently substitute it for one of the 20 others, leading to a library
in which 95% of the clones are inactive. If two crucial residues are
chosen, 99.75% of the library clones will be inactive. In the same
case, parsimonious mutagenesis allows the conservation of crucial amino
acid in about 50% of clones for one mutation and 25% for two
mutations. Nevertheless, each substitution at each mutagenesis position
is well represented: the least represented of all substitutions (0.1%)
will still be present at 104 copies in a 107
clone library (Table III). This favors
the selection of substitutions leading to a stronger affinity or
specificity.
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). Ligation
of large amounts of DNA and 50 electroporations allowed the
construction of 2.5 x 107 clones. The library quality
was then checked using DNA restriction analysis and Western blot
analysis of whole cell extract after induction, using the
anti-c-Myc tag Ab. Ninety-six percent (46 of 48) of the clones had
an insert of the right size, and 29% (14 of 48) gave a detectable
signal by Western blots (data not shown). Selection of clones with improved characteristics
As a control, a first selection was performed in immunotubes
coated with cortisol-3-CMO-BSA. After binding and washing, the bound
phAb were eluted using a classical elution method by pH shock (100 mM
triethylamine, pH 12.0). After the second round of amplification and
selection, we observed a 10-fold amplification of the ratio of output
phAb to input phAb. Forty-eight clones were tested for binding to
cortisol-3-CMO-BSA, and 43 of 48 were positive. Sequencing of 18 clones
indicated that two kinds of phAb had been selected. The first type (11
of 18) had the amino acid sequence of the wild-type Ab. The third base
of each of the relevant codons was different from that of the wild-type
as expected from the sequence of primers used for the mutagenesis. The
other sequences contained a deletion in the scFv gene, leading to the
display of truncated VH domains. By ELISA we showed that
these clones were able to bind any steroid conjugated to the BSA
(probably by a nonspecific hydrophobic interactions) and could not be
displaced by free steroid in a competitive binding ELISA (data not
shown). To avoid the selection of such clones, we subsequently eluted
the specifically bound phAb with a buffer containing free cortisol.
Thus, phAb bound by nonspecific interaction are less likely to be
recovered and thus amplified. We used lower concentrations of cortisol
during the successive rounds of amplification/selection/elution to
favor the recovery of phAb with high affinity for free cortisol (see
Materials and Methods). Two new clones emerged from this
selection. Both of them had a single mutation at codon L94 (CDR L3).
The wild-type asparagine was replaced by a serine or a threonine. Both
of these clones had a higher affinity for cortisol (Table IV). The affinities determined by
equilibrium dialysis and Scatchard analysis for the wild-type scFv and
the AsnL94Ser scFv mutant were 7.0 and 2.8 nM, respectively
(Fig. 3A). Interestingly, the relative affinity for
analogues was also higher but to a lesser extent, leading to a
reduction of cross-reaction by a factor of 2 (Table IV). The relative
affinity determined by equilibrium dialysis of the
AsnL94Ser scFv mutant for analogues (prednisolone and
dexamethasone) is shown in Fig. 3B.
Use of competitive binding and pre-elution in presence of analogues
We thought that we could select more specific binders by blocking
cross-reacting phAb with analogues before the selection with cortisol.
The specific elution was conserved in the following selections, but
before selection on a solid surface coated with cortisol-3 CMO-BSA, the
phAb library was incubated in a buffer containing the analogue. Analog
concentrations were increased at each round from 1 to 50 µM (see
Materials and Methods). After binding to the solid surface
(in the presence of analogues) and washing, a pre-elution was performed
with 50 µM analogue in washing buffer to remove all phAb still able
to bind the analogue efficiently. The bound phAb was then recovered by
specific elution with 50 µM free cortisol. After four rounds of
amplification/selection, the use of prednisolone as analogue led to the
isolation of a single clone, as shown by sequencing. This clone had one
mutation in the CDR H3 (ThrH100bAla). The affinity of this
scFv for cortisol (6.8 nM) was similar to the affinity of the wild-type
scFv (7.0 nM). However, its relative affinity for prednisolone was
reduced (Table IV). We obtained similar results with selection in the
presence of dexamethasone. A single clone was obtained with a mutation
in the CDR H3 (PheH100dTyr). The affinity of the scFv for
cortisol (7.2 nM) was not significantly different from that of the
wild-type Ab, but the relative affinity for dexamethasone was nearly
halved, leading to a 13% cross-reaction instead of 21% in the case of
the wild-type scFv (Table IV). For both mutants, the relative affinity
for the second analogue was also reduced.
Double mutants
To determine whether the observed effects of the mutations were
additive, we combined the mutation AsnL94Thr independently
with ThrH100bAla or PheH100dTyr. As a control
the mutation AsnL94Thr was also introduced into the
wild-type scFv, and the results obtained were undistinguishable from
those obtained with binders selected by competitive elution (data not
shown). Both clones Asn L94Thr/ThrH100bAla and
Asn L94Thr/PheH100dTyr had a very high
affinities for cortisol (0.9 and 1.6 nM) and were more specific than
the single mutant AsnL94Thr for prednisolone and
dexamethasone (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B). Obviously it is very difficult, if not
impossible, to obtain by classical hybridoma methods and a simple
screen an mAb capable of discriminating between these two molecules.
Consequently, we investigated whether it was possible to tailor the
paratope of an Ab to suppress the unwanted cross-reactions. In the
absence of an x-ray structure of the Ab, we decided to use a strategy
to improve its characteristics by randomization of few positions chosen
with the help of molecular modeling (29, 30) and a powerful selection
by phage display. Ab structures are very conserved, and even
hypervariable loop folding can be efficiently predicted from the
primary sequence (31). Schiweck et al. (32) succeeded in designing a
completely new paratope using a molecular model, and subsequent x-ray
crystallographic analysis confirmed their model. It should be
emphasized that the aim of our model was only to identify amino acid
positions close to the steroid. Determining the side chain orientation,
the most difficult task in molecular modeling, was not very important
in our case. Furthermore, the choice of mutagenesis targets was
strongly supported by an analysis of biochemical and structural data
for Ab-hapten complexes. We decided to substitute only five amino acids
of the paratope to limit the theoretical diversity. After cloning and
electroporation we obtained a library of 2.5 x 107
clones. The theoretical diversity (205 = 3.2 x
106) was thus less than the number of clones. Only about
30% of clones produced an scFv as assessed by Western blot analysis.
Similar results have already been obtained (15, 33). This low yield of
expressing clones may be due to the successive PCR amplifications
leading to incorporation of deletions and stop codons, for example.
Nevertheless, the number of producing clones was still higher than the
diversity. Selecting for anti-cortisol Abs in this library in the absence of any selection pressure, we obtained clones that have conserved the original amino acid sequence. This is not surprising, since in our library, 3% of clones are theoretically wild type and can obviously bind to the cortisol. However, we also selected clones lacking the VL domain plus a part of the VH domain. These clones are expected to present a hydrophobic interface that is usually packed with the VL interface. This could be responsible for the nonspecific binding observed with these clones, leading to their selection. Surprisingly, these deleted clones are very well expressed in E. coli and thus have a selective advantage over the entire scFv-producing clones during each amplification (data not shown). To avoid the selection of such "sticky" clones, we eluted phAb with a buffer containing free cortisol (50 µM). We thereby isolated mutant phAb with a higher affinity for cortisol (2.5-fold for the mutation AsnL94Ser and 7.8-fold for the mutation AsnL94Thr). These clones carried mutations at the position L94, a residue close to the ketone function on position C3 of both cortisol and analogues in the molecular model. These clones may interact with this ketone group, available only on the free cortisol (but absent in the cortisol-3-CMO-BSA used for immunization and selection). Indeed, both selected residues (Ser and Thr) have a hydroxyl group able to form a strong hydrogen bond. Interestingly, the affinities of the different clones (wild-type < AsnL94Ser < AsnL94Thr) seem to correlate with the strength of this putative bond (amide-ketone < primary alcohol-ketone < secondary alcohol-ketone). This interaction can be also found in the analogues, but in these steroids, the electrons of the ketone group are delocalized by a double bond between carbons 1 and 2 of the A ring, leading to a weaker putative interaction with the Ser or Thr residue. This could explain why these mutants cross-reacted up to twice less strongly with the analogues. It should be emphasized that these clones were isolated by competitive elution with free cortisol. This elution step is not involved in protocols allowing the recovery of mAbs, and, conversely to phAb, selection of mAbs interacting with the group used to link the carrier protein is highly improbable, thereby leading to Abs with lower affinities and specificities.
Selection in the presence of analogues led to the isolation of two
mutants, ThrH100bAla and PheH100dTyr, with
prednisolone or dexamethasone, respectively. Both these clones have a
affinity for cortisol similar to that of the wild-type scFv. However,
the relative affinity of these clones for analogues is lower than that
of the wild-type Ab (Table IV). Note that competitive elution,
selecting good cortisol binders, leads effectively to clones with a
higher affinity for cortisol, whereas competitive selection, selecting
clones unable to bind analogues efficiently, leads to clones with a
wild-type affinity for cortisol but with a lower relative affinity for
the analogues. The mutation PheH100dTyr reduces only
slightly the affinity for cortisol. Nevertheless, it seems that this
mutation causes a steric hindrance that has a larger effect on analogue
binding than on cortisol binding. When this mutation was combined with
the mutation AsnL94Thr, we observed the same effect; the
affinity for cortisol was sightly modified (from 1.3 to 1.6 nM), and
the relative affinity for analogues was reduced by a factor of >2
(from 130 to 320 nM for prednisolone and from 210 to 450 nM for
dexamethasone). The effects of mutations AsnL94Thr and
PheH100dTyr were identical in both single- and
double-mutant contexts, and their effects were additive. Consequently,
the cross-reactions of this double mutant for prednisolone and
dexamethasone were fivefold lower than that for the wild-type Ab.
Mutation ThrH100bAla also reduced the relative affinity for
analogues but more weakly than mutation PheH100dTyr.
However in this case, the effect of this mutation was completely
different when combined with the mutation AsnL94Thr. The
double mutant AsnL94Thr/ThrH100bAla had higher
relative affinities for analogues than the single mutant
AsnL94Thr. The affinity for cortisol was also increased.
Thus, in this case it seems that the presence of mutation
ThrH100bAla increases the effect of mutation
AsnL94Thr. The effects of these mutations were not
additive, but there was nevertheless an interaction between the two
mutations. Interestingly, substitution ThrH100bAla alone
reduced the relative affinity for both analogues but to a lesser extent
in the case of dexamethasone (6% instead of 10% for prednisolone),
and this was also the case for the double mutant
AsnL94Thr/ThrH100bAla (3% for dexamethasone
instead of 6% for prednisolone; Table IV). Although this effect is
difficult to explain (because the only difference between prednisolone
and cortisol, the double bond between the carbons 1 and 2, is also
present in dexamethasone), it is interesting to note that this clone
was selected for a lower relative affinity toward prednisolone but not
toward dexamethasone. From this point of view, these observations are
logical.
Is it possible to describe a model explaining these findings? The results for mutations AsnL94Ser or AsnL94Thr suggest the creation of a new hydrogen bond between the ketone groups on the A ring of steroids and the substituted amino acids. The effects of mutations selected in the presence of analogues are more difficult to explain. Both mutations decrease relative affinity for prednisolone and dexamethasone. In our model, positions H100b and H100d are near the steroid D ring. The D ring of prednisolone is identical with that in cortisol. Therefore, a new direct interaction involving this D ring is improbable. However, the tyrosine hydroxyl group available on the mutant may interact with another paratope residue, inducing a small rearrangement or maintaining CDR loop conformations through this atomic interaction. The hindrance caused by this change may be stronger for the analogues and thereby lead to reduced cross-reactions. This kind of paratope modification has been demonstrated in the case of an anti-hapten affinity maturation (34, 35, 36). Wedemayer et al. (36) demonstrated by crystallographic studies that none of nine somatic mutations responsible for a large increase of affinity (30,000-fold) was situated in the hapten binding site; the mutations are all responsible for rearrangements and stabilization of CDR positions.
The case of the mutation ThrH100bAla is different because this mutation affects the mutation AsnL94Thr effect. This result may be explain by a modification of the steroid position in the binding site, due to the local modification of the paratope near the steroid D ring, induced by the substitution. The new position of the hapten may cause steric hindrance for the analogues but not for the cortisol. However, in the double mutant the new position adopted by analogues may allow a stronger interaction between the Thr residue and the 3-ketone group on the steroid A ring, enhancing the effect of the AsnL94Thr mutation. It would be very interesting to compare the crystallographic structures of the two double mutants complexed with cortisol as this may help elucidate the effects of the mutations at a molecular level.
Interestingly, all the selected clones have wild-type amino acids at positions L96 (Pro) and H95 (His). These residues are probably essential for cortisol binding or efficient folding of the Ab. In our model, the proline L96, a position always found in interaction with the hapten in 10 x-ray structures of Ab-hapten complexes (25), may interact with the 11-hydroxyl function of cortisol and its analogues. This interaction is very important, since the cortisol analogue lacking this function (11-deoxycortisol) is not recognized by mAb 5A4. This would explain the conservation of this residue in this study. Similarly, the conservation of the HisH95 residue may have been due to an essential interaction with the cortisol D ring. The conservation of these two residues in all selected clones emphasizes the importance of using parsimonious mutagenesis for this kind of study.
Which of these new anti-cortisol Ab fragments is the best for
diagnostic purposes? In normal humans, cortisol levels exhibit a
diurnal rhythm, and the plasma concentration varies between 130–650 nM
(1). For the cortisol immunoassay, affinities between 1–10 nM are well
suited. The best Ab is thus the most specific, which is Ab
AsnL94Thr/PheH100dTyr. In conclusion, we show
that using guided randomization for diversity generation and phage
display for selection, it is possible to obtain anti-hapten Abs
with improved affinity and specificity even if there is only a very
small difference between the Ag and its analogues (Fig. 4B).
The specificity could probably be further increased by conserving these
mutations and creating secondary libraries with randomization of other
contact positions. An alternative solution is to mutate amino acids
responsible for the position of entire CDR, thereby allowing small
changes in the paratope cavity, which could be useful for the fine
tuning of specificity. Finally, this work demonstrates that the
development of recombinant Abs may be a solution of choice for
obtaining the highly specific Abs required for steroid immunoassays.
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Acknowledgments |
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Footnotes |
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2 Current address: University Hospital Maastricht, P. Debyelaan 25, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
3 Address correspondence and reprint requests to Dr. Daniel Baty, UPR 9027, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. E-mail address:
4 Abbreviations used in this paper: scFv, single-chain variable fragment; VH, immunoglobulin heavy chain variable region; VL, immunoglobulin light chain variable region; phAb, phage antibodies; IPTG, isopropyl-thio-ß-D-galactopyranoside; cortisol-3-CMO-BSA, cortisol 3-(O-carboxymethyl)oxime-bovine serum albumin; MPBS, 4% skimmed milk powder in phosphate-buffered saline; supE, strain of Escherichia coli that carries a glutamine-inserting amber (UAG) suppressor transfer ribonucleic acid; CDR, complementarity-determining region; H1, complementarity-determining region 1 of the heavy chain V region; H2, complementarity-determining region 2 of the heavy chain V region; H3, complementarity-determining region 3 of the heavy chain V region; L3, complementarity-determining region 3 of the light chain V region.
Received for publication December 8, 1998. Accepted for publication July 9, 1998.
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-Hydroxyprogesterone, 4-androstenedione, and testosterone profiled by routine stable isotope dilution/gas chromatography-mass spectrometry in plasma of children. Pediatr. Res. 38:76.[Medline]
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,
wild-type; 
, AsnL94Ser). B, The relative
affinities of scFv were determined by equilibrium dialysis in which
binding to cortisol was inhibited by the analogues (
, and •, AsnL94Ser with cortisol,
prednisolone, and dexamethasone, respectively). The IC50 is
the concentration of steroid necessary to obtain 50% of the control
signal (that without any competition).