Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

Chie Miyazaki, Yoshitaka Iba1, Yukio Yamada, Haruo Takahashi, Jun-ichi Sawada2 and Yoshikazu Kurosawa1,3

Toyota Central R&D Laboratories Inc., Nagakute, Aichi 480-1192, 1 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192 and 2 Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyogo, Setagaya, Tokyo 158, Japan


    Abstract
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The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.

Keywords: antibodies/error-prone PCR/phage-display antibody/steroid/structural models


    Introduction
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 Abstract
 Introduction
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Antibodies can specifically bind to antigens. The capacity of antibodies for such a specific binding is achieved in vivo by the generation of a large repertoire of antibodies. The considerable sequence diversity found in the VH and VL domains of antibodies is generated, to a major extent, by DNA rearrangements, such as VH–D–JH and VL–JL, at the loci of genes for immunoglobulins (Igs) (Tonegawa, 1983Go). After stimulation by specific antigens, somatic mutations introduced into the V-coding region of the relevant clones enhance the diversity of the V-region sequences and the affinity maturation of antibodies is achieved by selection at the cell level (Berek and Milstein, 1987Go). Although rearrangements of DNA can generate large numbers of antibodies, the number of germline V genes appears to be rather limited (Tomlinson et al., 1992Go; Cox et al., 1994Go; Kawasaki et al., 1997Go). Therefore, the size of a `naive repertoire' that is generated by rearrangements of somatic DNA is not very large, except in the case of CDR3 of the VH domain (Ichihara et al., 1988Go). The diversity of sequences found in CDR1 and CDR2 in the naive repertoire is limited by the number of germline V genes. To overcome these limitations in vivo, introduction of mutations with subsequent selection allows generation of antibodies with high specificity and high affinity (Berek and Milstein, 1987Go).

Since the development of PCR technology and phage-display systems, several groups have reported the construction of libraries of phage antibodies (Griffiths et al., 1994Go; de Kruif et al., 1995Go; Vaughan et al., 1996Go). Although these groups succeeded in isolating antibodies with high specificity and high affinity for several antigens just by screening their libraries, such libraries should be considered to correspond to a naive repertoire in view of the absence of stimulation with antigens, with the exception of the antibodies for some certain specific antigens. Introduction of random mutations in vivo appeared to be an efficient method for increasing both specificity and affinity in the presence of strong selective pressure. In the present study, we used error-prone PCR followed by competition panning to mimic the mechanism in vivo and we attempted to introduce changes in the specificity for steroid antigens.

In a previous study (Iba et al., 1998Go), we attempted to change the specificity of antibodies by introducing mutations at restricted residues. A monoclonal Ab, 1E9, specific for 17{alpha}-hydroxyprogesterone (17-OHP), was used as the starting Ab. Using a model that had been generated by a computer-driven model-building system, we constructed a phage-display library of antibodies in which 16 residues were mutated in three CDRs of the heavy chain that appeared to form the steroid-binding pocket. Although the clones that we isolated by screening the library with cortisol (CS) as antigen exhibited newly developed CS-binding ability, they still retained strong 17-OHP-binding activity (Iba et al., 1998Go). Therefore, in the present study, we examined the effectiveness of random mutation by error-prone PCR that was followed by competition panning (Hawkins et al., 1992Go).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antigens

11-Deoxycortisol (11-DOC), cortisol 3-(O-carboxymethyl) oxime (3-CMO) and ovalbumin (OVA) were purchased from Sigma. 11-Deoxycortisol was reacted with an equimolar amount of carboxymethoxy-HCl (Wako, Japan) to yield 11-DOC 3-CMO. Cortisol 3-CMO and 11-DOC 3-CMO were conjugated to OVA, in 0.1 M potassium phosphate buffer (pH 7.5), with N-dicyclohexylcarbodiimide and N-hydroxysuccinimide. The average stoichiometry for 11-DOC and CS conjugated with OVA was estimated to be 51 molecules and 27 molecules/one molecule of OVA, respectively.

Oligonucleotide primers

The following oligonucleotides were synthesized chemically and used as primers for construction of plasmid DNAs.


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Construction of plasmid DNA encoding mAb SCET

Plasmid pAALFab (Iba et al., 1997Go) was modified as follows. The CAA codon, corresponding to the fifth residue of the VH domain, was changed from CTGCAA to CTGCAG with the generation of a PstI site. The VH and V{kappa} genes encoding mAb SCET were amplified from SCET-VH cDNA and SCET-V{kappa} cDNA (Sawada et al., 1991Go) by PCR with two sets of primers, SHPst plus SHBst and SLSpe plus SLXho, respectively. The PstI- and BstPI-digested VH gene-containing fragment and also the SpeI- and XhoI-digested V{kappa} gene-containing fragment were inserted into the modified pAALFab vector. The resulting plasmid, designated pFCA-SCHL, was used in subsequent experiments. The amino acid sequences of VH and V{kappa} domains encoded by pFCA-SCHL were slightly different from those of the authentic SCET mAb as follows: the N-terminus of the VH domain, QIQLV->QVQLQ; the N-terminus of the V{kappa} domain, SIVMTQTPKFLHVSVGDRVTITCKA->DIELTQ-SPASLSASVGETVTITCRT; and amino acid residues 96 and 97 of the V{kappa} domain, YT ->RA.

Construction of a large set of mutant antibodies

A large set of mutant antibodies, in which a variety of amino acid residues were introduced at 14 positions in the three CDRs of the VH domain, was constructed as follows. As indicated schematically in Figure 1Go, introduction of mutations at three residues in CDR1 was performed by PCR with pFCA-SCHL DNA as template and primers HISa and HISb. Diversification at six and five residues in CDR2 and CDR3, respectively, was achieved by a similar PCR-based strategy. In this case, however, since we planned to introduce codons for Trp and Tyr at position 50, as well as codons for Arg and Tyr at position 98, we synthesized two similar but different primers to avoid the presence of termination codons in the degenerate primer sequences. Thus, we performed four separate PCRs with four kinds of primer combination: HIISa-1 plus HIISb-1, HIISa-1 plus HIISb-2, HIISa-2 plus HIISb-1 and HIISa-2 plus HIISb-2. After PCR, the four products were mixed together. Finally, we connected the three CDR-containing fragments by PCR, using a mixture of the product of the first PCR and the pooled products of the second PCR as templates, with primers HIIISa and SHBst. In order to remove products whose CDR2 sequences had not been diversified, we digested the products with NdeI and PstI. The recognition sequence CATATG for NdeI was present in the original template DNA but not in the primer sequences used in the present study. Conversely, the recognition sequence CTGCAG for PstI was present in primer HISb but not in the template DNA. The final products were digested with SnaI and BstPI and they were used to replace the corresponding region in pFCA-SCHL, sandwiched between SnaI and BstPI sites. The ligated DNAs were used to transfect Escherichia coli DH12S cells by electroporation, and a library, designated pFCA-SCHL-Lib1, was generated. While the number of clones with different sequences should have been 5x107, we estimated the number of independent clones in this first library was 1x108.



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Fig. 1. Schematic representation of the method used for introduction of mutations into three CDRs of the VH gene. DNA of plasmid pFCA-SCHL, which encodes the mAb SCET, was used as the template. Two PCR reactions were performed with primers HISa plus HISb and HIISa-1 or 2 plus HIISb-1 or 2. Wavy portions of respective primers indicate the presence of degenerate codons. After the products in the first PCRs had been combined, they were subjected to the second PCR with primers HIIISa and SHBst. Since the products of the second PCR included fragments in which diversification of CDR2 had not occurred, they were removed by digestion with NdeI and PstI. Circles indicate the position of NdeI and PstI sites. Abbreviations: Ps, PstI; Sn, SnaI; Nd, NdeI; Bs, BstPI.

 
Screening of the library by panning

Clones with newly developed CS-binding activity were isolated from pFCA-SCHL-Lib1 by panning. The method used for panning was essentially the same as described previously (Iba et al., 1998Go). The surface of an immunotube was coated with 10 µg/ml CS-OVA in phosphate-buffered saline (PBS) for 2 h at room temperature. After panning processes had been repeated five times in all, phagemid DNAs were prepared, digested with SalI, self-ligated and transduced into E.coli DH12S. The characteristics of the Fab–PP forms of antibodies encoded by this plasmid DNA have been described in previous work (Ito and Kurosawa, 1993Go; Iba et al., 1998Go; Ib et al., 1998). In brief, Fab–PP is an abbreviation that refers to the artificial form of Ab composed of Fab fragment fused to two Fc-binding domains of protein A.

Enzyme-linked immunosorbent assay (ELISA)

Two kinds of antibodies were subjected to ELISA. The wells of immunoplates were coated with 100 µl aliquots of solutions of CS-OVA or 11-DOC-OVA at various concentrations. In the case of phage antibodies, wells were blocked with 2% skimmed milk in PBS. Then, 1x1010 colony forming units (c.f.u.) of phages in 2% skimmed milk in PBS that contained 0.1% Tween 20 were added to wells. Phages that bound to the wells were detected with rabbit antibodies against M13, alkaline phosphate-conjugated antibodies against rabbit IgG and p-nitrophenyl phosphate. In the case of Fab–PP antibodies, the wells were blocked with 0.5% OVA in PBS. Fab–PP antibodies were prepared from periplasmic fractions as previously described (Iba et al., 1997Go, 1998Go). The periplasmic fractions, each of which had been diluted 10-fold with PBS that contained 0.1% CHAPS, were added to the wells. The bonding of Fab–PP to CS or 11-DOC was detected with rabbit antibodies against mouse F(ab')2 with the addition of alkaline phosphatase-conjugated Ab against rabbit IgG and p-nitrophenyl phosphate. In the case of the competition ELISA for which results are shown in Figure 6Go, the 10 ng aliquots of purified Fab–PPs were first preincubated with various amounts of 11-DOC in 100 µl PBS that contained 0.1% Tween 20 at room temperature for 30 min and then the mixtures were added to the wells that had been coated with CS-OVA.



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Fig. 6. Results of the competition ELISA of Fab–PP. Fab–PPs of DcC16, cc53, cc96 and cc118 were purified. After 10 ng Fab–PPs had been incubated with various concentrations of 11-DOC, they were added to plates coated with CS-OVA. The symbols refer to samples as follows; •, DoC16; {blacksquare}, cc53; {blacktriangleup}, cc96; and {blacklozenge}, cc118.

 
Introduction of random mutations into the entire VH-coding region by error-prone PCR

The reagents for error-prone PCR were 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 7.5 mM MgCl2, 0.5 mM MnCl2, 0.1% Triton X-100, 0.2 mM dATP, 0.2 mM dGTP, 1.0 mM dCTP, 1.0 mM TTP, 0.3 µM primers SHPst and SHBst, 100 ng of plasmid DcC16 and 2.5 U Taq DNA polymerase (Promega Corp., Madison, WI) in 100 µl. The reaction was allowed to proceed for 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 4 min. Then reaction mixtures were treated with phenol and chloroform and DNA was precipitated with ethanol. After digestion with PstI and BstPI, products were inserted into the corresponding region of pFCA-DcC16.

Isolation of CS-specific clones by competition panning

The phage library obtained by error-prone PCR, designated pFCA-SCHL-Lib2, was dissolved in PBS. Then 7.2x1011 phages were mixed with various amounts of 11-DOC in 3.8 ml PBS containing 2% skimmed milk and 0.1% Tween 20 and the mixtures allowed to stand at room temperature for 30 min. Each mixture was transferred to an immunotube that had been coated with 5 µg/ml CS-OVA and incubated at room temperature for 2 h. Other conditions were the same as those for standard panning. This competition panning was repeated four times in all.

Kinetic analysis by measurements of surface plasmon resonance (SPR)

The Fab–PP forms of representative antibodies were prepared from 2 liter cultures. Proteins in supernatants of overnight culture were precipitated with 70% of ammonium sulfate. Pellets were suspended in 20 ml PBS and dialyzed against PBS. Soluble proteins were loaded onto a column of IgG-linked Sepharose 6 Fast Flow (Pharmacia), bound proteins were eluted with 0.1 M glycine, pH 3.0, and eluates were immediately neutralized. Then proteins were loaded onto a column of CS-OVA-linked Sepharose 4B that had been prepared by immobilizing CS-OVA on CNBr-activated Sepharose 4B (Pharmacia). In the case of SCET mAb, 11-DOC-OVA-linked Separose was used instead of CS-OVA-linked Separose 4B. The purified Fab–PPs were analyzed by SDS–PAGE and no unanticipated bands were detected in all cases. Protein concentration was determined from the absorbance at 280 nm (for Fab': E280 = 1.48 g–1·l·cm–1). The purified Fab–PPs were used for subsequent kinetic analysis.

Association and dissociation rate constants of the purified Fab–PPs for CS-OVA and 11-DOC-OVA were measured with a BIAcore instrument (Johnson et al., 1991Go; Chaiken et al., 1992Go). We coupled the ligands to a CM5 sensor chip using the amino coupling kit supplied by the manufacturer. First, OVA of 1 mg/ml was immobilized in 10 mM sodium acetate, pH 3.5. Next, cortisol 3-CMO or 11-DOC 3-CMO, which had been pretreated with N-hydroxysucciimide, was conjugated with OVA. Then purified Fab–PPs at 10, 20, 50, 100 and 200 nM were injected into cells. Measurements of surface plasmon resonance (SRP) were made at a flow rate of 5 µl/min in 10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.05% BIAcore surfactant P20 at 25°C. Surfaces were regenerated by treatment with 10 mM HCl. We calculated the `on' and `off' rate constants using the BIA evaluation program 2.1 (Pharmacia Biosensor AB).

Structural modeling of steroid-binding pockets

We constructed models using the method of homology modeling using the LOOK & SegMod module of GeneMine (Levitte, 1992Go). On the basis of sequence alignment, we chose the Fv fragment of 1DBB, whose crystal structure had been obtained from the Brookhaven Protein DataBank (Arevalo et al., 1993aGo,bGo), as the backbone of our starting structure. Identity of the amino acid sequences of VH domain between SCET and DB3 was calculated to be 80.7%. The addition of the necessary hydrogen atoms to the antibody structure and the building of the antigen structure were performed by the biopolymer module of Insight II 97.0.

In the first attempt to simulate the complexes, cortisol and 11-DOC were placed at the same position as progesterone, which is bound to 1DBB in the crystal structure (Arevalo et al., 1993aGo). Simulations were performed by the Discover 3.0.0. program (Molecular Simulations Inc., San Diego, CA) with energy minimization and molecular dynamic calculations. Nonbonded parameters were obtained by Cell Multipole method (Greengard and Rokhlin, 1987Go; Schmnidt and Lee 1991Go; Ding et al., 1992Go) under the CVFF force field (Dauber-Osguthorpe et al., 1988Go). Molecular dynamic calculation were performed in NVT ensemble at 296 K.


    Results and discussion
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Strategy for changing the specificity of antibodies

The molecular structures of 11-DOC and CS are almost identical, with the absence and presence, respectively, of a hydroxy group at the eleventh carbon of the steroid ring being the only difference between them. The original SCET mAb was isolated after immunization with 11-DOC that had been conjugated with BSA and it exhibited weak cross-reactivity with CS (0.2%; Hosoda et al., 1987Go). Since the three-dimensional structure of a progesterone-specific mAb, DB3, had been reported (Arevalo et al., 1993aGo,bGo) and since the VH gene of the SCET mAb belongs to the same family as that of DB3, we were able to predict the structure of the steroid-binding pocket of SCET with some confidence. In the present study, we attempted to isolate clones that had gained CS-binding activity and had lost some or all of their 11-DOC-binding activity. In a previous study (Iba et al., 1998Go), we attempted a similar feat. An mAb, designated 1E9, that was specific for 17-OHP was mutated and the resulting clones had newly developed CS-binding ability. However, they retained 17-OHP-binding activity. In our earlier attempt, we introduced mutations at 16 residues that seemed likely to form the steroid-binding pocket. In the present study, therefore, we adopted a two-step procedure in an attempt to change the Ag specificity entirely. During the first step, we introduced mutations at specific positions in three CDRs of the VH domain that supposedly formed the steroid-binding pocket, generating an initial mutated library. This step was the same as that in our previous study. During the second step, we introduced mutations randomly into the entire VH gene of an isolated clone that had already developed CS-binding ability. This step can be performed by error-prone PCR followed by a competition panning.

Isolation of a clone with newly developed CS-binding activity

We chose to mutate residues at 14 positions, at which from two to seven possible amino acids are located, as summarized in Table IGo. The principles that governed the choice of positions and amino acids were essentially the same as those described in a previous paper (Iba et al., 1998Go). In brief, since we could predict that the steroid ring should be sandwiched between TrpH50 and TyrH100 (Arevalo et al., 1993aGo,bGo), we chose 14 positions that would be located in the region surrounding the steroid. Since the CDRs of the VL domain should form a binding pocket for the A ring of the steroid, we did not mutate this domain. The first library, pFCA-SCHL-Lib1, was panned for selection of CS-binding clones. Although neither unselected clones nor pools of eluted phages after one to four rounds of panning exhibited CS-binding activity, a pool of phages after the fifth panning appeared to show evidence of CS-binding activity in a phage ELISA with CS-OVA-coated wells (data not shown). Phagemid DNA was prepared from the final pool of eluted phages, digested with SalI, self-ligated and used to transfect E.coli DH12S. Twenty clones were picked up and examined for CS-binding activity. One of them, designated DcC16, had high CS-binding activity, as shown in Figure 2aGo. As had been the case in our previous study (Iba et al., 1998Go), this clone had newly developed CS-binding activity but it retained strong 11-DOC-binding activity (Figure 2bGo). The nucleotide sequence of DcC16 revealed substitution of amino acids at five positions in CDR2, as indicated in Table IGo.


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Table I. Amino acid sequences of VH CDRs

 


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Fig. 2. Results of the ELISA of the Fab-PP form of Ab. Immunoplates were coated with (a) CS-OVA and with (b) 11-DOC-OVA at various concentrations (0 to 1 µg/ml). Samples were Fab–PP of SCET(•) and that of DcC16({blacksquare}).

 
Isolation of clones with increased CS-binding activity and decreased 11-DOC-binding activity

As noted above, the DcC16 clone had newly developed CS-binding activity but retained strong 11-DOC-binding activity. As a second step, therefore, we introduced random mutations into the VH gene of DcC16, by error-prone PCR, in which the presence of Mn2+ ions and limited amounts of dATP and dGTP enhanced the frequency of incorporation of mismatched nucleotides (Leung et al., 1989Go). The number of clones in the second library, constructed by error-prone PCR, was estimated to be 5x106. We selected 20 clones and determined their nucleotide sequences. We found that all the mutations were nucleotide substitutions and the frequency of such mutations was 3.1 bases per 312 nucleotides.

We used the competition panning method to select clones that had increased CS-binding activity and little or no 11-DOC-binding activity. The appropriate concentration of free 11-DOC for use as a competitor was determined as follows. After incubation of the mutated phage library with free 11-DOC at various concentrations, the mixtures were subjected to ELISA in immunoplates that had been coated with 5 µg/ml CS-OVA. As Figure 3Go shows, 50, 70 and 90% inhibition was observed in the presence of 11-DOC at 0.16, 0.32 and 1 µg/ml, respectively. Using 11-DOC at these three concentrations as the competitor, we eluted phage particles that had bound to CS-OVA-coated tubes. The panning procedure, namely, the binding, elution and growth of phages, was repeated four times in all. After each cycle, we examined the ability of the eluted phages to bind to CS and 11-DOC. In the presence of 11-DOC at 1.0 µg/ml, the eluted phages did not bind to CS. By contrast, at the other concentrations of 11-DOC, after two rounds of panning, the ratio of CS-binding activity to 11-DOC-binding activity exceeded unity, as shown in Figure 4Go. Judging from these data, 0.32 µg/ml appeared to be the best of the tested concentrations. The following experiments were performed using the sample that was eluted after four rounds of panning in the presence of 11-DOC at 0.32 µg/ml.



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Fig. 3. Competition ELISA of phage antibodies. 1.9x1010 phages in pFCA-SCHL-Lib2 were mixed with various concentrations of 11-DOC in 100 µl PBS and allowed to stand at room temperature for 30 min. The mixtures were added to ELISA wells coated with CS-OVA. The other conditions for the ELISA were the same as those for the ordinary ELISA of phage antibodies.

 


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Fig. 4. Improvements of relative affinities of phage antibodies to CS versus 11-DOC in fractions selected by competition panning. pFCA-SCHL-Lib2 was subjected to competition panning. 11-DOC at three concentrations, namely, 1, 0.32 and 0.16 µg/ml, was used as the competitor. Since 11-DOC at 1 µg/ml did not yield any positive results, only results for 11-DOC at 0.32 µg/ml (a) and 0.16 µg/ml (b) are shown. The competitive panning was repeated four times in all and, after each cycle, the eluted phages were subjected to the phage ELISA, in which binding to CS and to 11-DOC was examined. The binding ratio was indicated as follows:

 
We isolated 24 clones from the eluted phages and determined nucleotide sequences. We also measured CS-binding activity and 11-DOC-binding activity by ELISA. The results are shown in Figure 5Go. We calculated the ratio of the CS-binding to 11-DOC-binding activity of each clone relative to the ratio of the CS-binding to 11-DOC-binding activity of DcC16. Thus, values greater than unity indicated an improvement in relative affinity for CS but did not indicate an improvement in the absolute affinity for CS. The clones were classified into six groups. The nucleotide sequences of two clones were the same as that of DcC16. Although cc10 and cc20 included mutations, the improvements in affinity were not significant. The other 20 clones that were classified into three groups showed significant improvements in terms of relative affinity for CS.



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Fig. 5. Relative affinities for CS versus 11-DOC of representative clones. Each of 24 clones isolated after the fourth panning with CS at 0.32 µg/ml 11-DOC as competitor was converted to the Fab–PP form and each periplasmic extract was subjected to ELISA in which the CS-binding and 11-DOC binding activities were examined. The binding ratio was defined as follows:

Each number in parentheses indicates the number of isolated clones of the particular type. Standard deviations are indicated by bars. Samples 1–6 correspond to the following clones: 1, DcC16-type; 2, cc118-type; 3, cc96-type; 4, cc53-type; 5, cc10; 6, cc20.

 
Kinetic analysis of binding of antibodies to CS and 11-DOC

In order to estimate the changes in the absolute values of the affinity for CS and 11-DOC, we prepared Fab–PP forms of representative clones and subjected them to kinetic analysis with a BIAcore instrument. The results are shown in Table III. Although the SCET mAb showed 0.2% cross-reactivity to CS in a previous study (Hosoda et al., 1987Go), it did not give any evidence of binding to CS in this experiment. This apparent discrepancy might have reflected a difference of valency. We used a monovalent form of the Ab in the present experiment and a divalent form in our previous experiment. As shown by the ELISA results, DcC16 had newly developed CS-binding activity but had not lost 11-DOC-binding activity. The other three clones isolated by competition panning exhibited 1.6–3.8-fold increases in CS-binding activity, with a loss of 10–60% of the 11-DOC-binding activity of DcC16. While the ELISA results indicate only 1.3–1.5-fold improvements in the relative affinity for CS versus 11-DOC, the results obtained by BIAcore analysis indicated 4.1–4.5-fold improvements in the relative affinity for CS versus 11-DOC, as well as 1.6–3.8-fold improvements in the absolute affinity for CS. Since the results obtained by the ELISA might not have been strictly quantitative, the values from the BIAcore analysis should be considered to be more accurate.

We examined the improvements in the relative affinity for CS versus 11-DOC in further detail by competition ELISA, as shown in Figure 6Go. In this experiment, purified Fab–PPs were first mixed with different amounts of free 11-DOC in solution. Then the mixtures were added to wells that had been coated with CS-OVA. In the case of DcC16, 1 µg/ml 11-DOC gave 90% inhibition. By contrast, in the case of cc-53, even with 11-DOC at 10 µg/ml, only 80% inhibition was observed.

Theoretical considerations related to the competition panning method

Under the conditions adopted in the present study, the frequency of mutations introduced by error-prone PCR was estimated to be 3.1 per 312 base pairs. In general, more mutations might provide more opportunities to generate clones with the desired affinity, but they would simultaneously increase the likelihood of destruction of the authentic antigen-binding activity. The number of mutations introduced into each clone should correspond to Poisson's distribution. Poisson's law (applied to the present case) states that

where Pr is the fraction of a large number of clones that contains r mutations each, if an average of {chi} mutations per clone is distributed at random over the entire ensemble of clones. Thus, in the library constructed by error-prone PCR, the percentage of clones with no mutations should be 4.5%; with one mutation, 14.0%; with two mutations, 21.6%; with three mutations, 22.4%; with four mutations, 17.3%; with five mutations, 10.7%; with six mutations, 5.6% and so on. We analyzed 24 clones that we had picked up independently. Since cc118-type clones and cc53-type clones had the same mutations at four and six residues, respectively, each of them should have been derived from the same clone generated during error-prone PCR. Although the numbers of cc118-type phages and cc53-type phages present in the constructed library could not be accurately estimated, the number might not have been very large. Nevertheless, eight clones of the cc118-type and five clones of the cc53-type were identified among the 24 clones that we isolated. In the case of cc96-type clones, although each had only one mutation, since the sequences were identical, it is likely that they too were derived from a single clone. By contrast, only two non-mutated clones were identified among the 24 clones examined, even though there should have been 2.3x105 non-mutated clones (4.5%) in the library. The other two clones, cc10 and cc20, did not show any significant improvement in CS-binding activity. These results allowed us to draw the following conclusions. (i) The competition panning worked efficiently to enrich for clones with relatively higher affinity for CS than for 11-DOC. (ii) The degree of enrichment at each panning might have been several dozen-fold. (iii) There might have been only a few clones in the entire library with improved CS-binding activity and decreased 11-DOC-binding activity. (iv) The majority of clones in the library had at least somewhat decreased CS-binding and 11-DOC-binding activity. Casson and Manster (1995) reported that a majority of mutants in libraries generated by error-prone PCR failed to express detectable Fab.

While the results of the competition ELISA shown in Figure 6Go indicated that preincubation of the Fab-PP antibodies of cc53, cc118 and cc96 with 11-DOC at 1 µg/ml resulted in only 40–60% inhibition of the binding of CS in the ELISA, the phage antibodies eluted from the immunotubes when this concentration of 11-DOC was used as competitor did not have any CS-binding activity. Inclusion of 11-DOC at 0.32 µg/ml resulted in efficient enrichment for clones with enhanced relative affinity for CS as compared with 11-DOC. This apparent discrepancy might have resulted from the difference in the form of the Ab, namely, the phage form versus the Fab–PP form, or from a difference in population, namely, a very heterogeneous versus a homogeneous population.

During competition panning and during the ELISA, phage antibodies first formed complexes with free 11-DOC. Since the concentration of antigens were much higher than those of antibodies, the concentration of antigens can be considered to have remained constant during formation of antigen–antibody complexes. Thus, the following relationship might be valid.

Then, since KA for values of 11-DOC ranged from 6.70 to 17.0x106 M–1, 6–14.3% of phage antibodies were not in a complexed form in the solution that contained 0.32 µg/ml (0.88 µM) 11-DOC. After mixtures had been added to immunotubes coated with CS-OVA, dissociation from and association with CS or 11-DOC occurred. Although it is very difficult to describe these processes accurately, two factors appeared to determine the final results. One was the relative affinity for CS versus 11-DOC. Three clones, cc53, cc96 and cc118, gave similar values, which ranged from 1.52 to 1.71. The other factor was the absolute affinity for CS. In this respect, cc53 was the best clone. The results shown in Figure 6Go indicate that the extent of inhibition of the binding to CS of cc53 by 11-DOC was the smallest among these three clones. During the competition panning for isolation of improved clones, a very heterogeneous mixture was used. Therefore, the actual number of each type of phage antibody was probably very low during panning. It is likely that 11-DOC at 1 µg/ml 11-DOC was insufficient for enrichment of improved clones because a critical (and higher) concentration might be required for the recovery of such clones. Therefore, in general, for competition panning, a careful examination should be made to determine the appropriate concentration of antigens that are used as competitors.

Comparison of models predicted by computer modeling

The three-dimensional structure of a progesterone-specific antibody, DB3, has been well characterized by X-ray crystallography (Arevalo et al., 1993aGo,bGo). Eight amino acid residues in the VH domain and six residues in the VL domain are involved in direct contacts with progesterone. In particular, the indole side chains of TrpH50 and TrpH100 sandwich the steroid ring. Methyl groups at positions 18 and 19 of progesterone are directed toward TrpH50. Although Tyr is located at position 100 in the mAb SCET instead of Trp, the model constructed by computer simulation suggested that the overall arrangement of the binding pocket in SCET should be similar to that in DB3. This predicted similarity might have been achieved by the identity of the amino acids, such as AsnH35, TrpH47 and TrpH50, which are direct contact residues to steroid in CDR1 and CDR2 in addition to high identity of the overall amino acid sequences (80.7%). As shown in Figure 7aGo, the steroid ring was stacked between the aromatic rings of TrpH50 and TyrH100. The 20-keto group of progesterone makes a hydrogen bond with AsnH35 in DB3, while not only did the 20-keto group of 11-DOC make a hydrogen bond with AsnH35 but also the 21-hydroxy group appeared to form a hydrogen bond with TrpH50 (Figure 7bGo).



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Fig. 7. Structural models of antigen-binding pockets complexed with 11-DOC. Models of complexes of (a, b) SCET with 11-DOC and (c, d) DcC16 with 11-DOC. The residues around the pocket were 50 and 100 (yellow), 33 (green), 35 (cyan), 52 (orange), 56 (light green), 58 (blue) and others (52a, 53, 54, 97; light pink) of the H chain; 91, 96 (magenta) of the L chain in the CPK (Corey–Pouling–Keltun) space-filling (a, c), and in backbone and side chain atoms with hydrogen-bond interactions (b, d). 11-DOC was shown with C atom (light green), O atom (red) and H atom (white). The hydrogen-bond interactions between antigen-binding pocket and 11-DOC are shown in pink along with distance (Å) representations, and also interactions among residues are shown in pink.

 
In DcC16, five amino acids in the CDR2 were different from those in SCET. While SCET bound 11-DOC but not CS, DcC16 bound both 11-DOC and CS. When we compared the models of complexes of SCET with 11-DOC (Figure 7a and bGo) and of DcC16 with 11-DOC (Figure 7c and dGo), we found that TyrH50 in DcC16 instead of TrpH50 in SCET seemed to have a major effect on the width of the steroid binding cleft. While the 21-hydroxy group of 11-DOC could form a hydrogen bond with TyrH50, the 20-keto group appeared to make a hydrogen bond with GlyH33 instead of AsnH35. The change from Thr to Tyr at position 58 might also have had an effect on the local arrangement of the amino acid at position 50. The model for the complex of DcC16 with CS (Figure 8a and bGo) suggested that DcC16 bound CS in a slightly different manner. Although the 20-keto group formed a hydrogen bond with GlyH33, the 21-hydroxy group appeared to make a hydrogen bond with AsnH31 instead of TyrH50. According to this model, TyrH50 was not involved in formation of a hydrogen bond with CS. Since, in the case of SCET, TrpH50 was tightly packed with respect to the steroid ring, SCET might not exhibit the flexibility required for accommodation of CS. The addition of a hydroxy group at position 11 would not be permitted because of steric hindrance. These considerations might explain why DcC16 had newly developed CS-binding activity.



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Fig. 8. Structural models of antigen-binding pockets complexed with CS. Models of complexes of (a, b) DcC16 with CS and (c, d) cc53 with CS. The residues around the pocket were 50 and 100 (yellow), 31 (gray), 33 (green), 35 (cyan), 52 (orange), 56 (light green), 58 (blue) and others (52a, 53, 54, 97; light pink) of the H chain; 91, 96 (magenta) of the L chain in the CPK (Corey–Pouling–Keltun) space-filling (a, c), and in backbone and side chain atoms with hydrogen-bond interactions (b, d). CS was shown with C atom (light green), O atom (red) and H atom (white). The hydrogen-bond interactions between antigen-binding pocket and CS are shown in pink along with distance (Å) representations, and also interactions among residues were shown in pink.

 
Using error-prone PCR followed by competition panning, we obtained three kinds of clone with increased CS-binding activity and decreased 11-DOC-binding activity. None of the substituted amino acids found in these three clones were directly involved as an antigen-binding residue. In these three kinds of clone, a common characteristic was a change in the amino acid at position 56. Although this particular amino acid was not directly involved in formation of the antigen-binding pocket, however, it seemed to play an important role to fine tune the pocket to bind CS. Since the models of the complexes of these three mutant antibodies with CS appeared to be similar to each other, only the cc53–CS complex is shown in Figure 8c and dGo. TyrH50 in cc53 seemed to form a hydrogen bond with the 11-hydroxy group of CS. The appearance of this hydrogen bond was predicted for these three mutant antibodies. Moreover, the 20-keto group appeared to form a hydrogen bond with AsnH35 instead of GlyH33. Such a change in the local arrangement of the TyrH50 in the each of three mutant antibodies might be due to the structural constraint caused by the change in the amino acid at position 56. While the changes in cc53 were quite different from those in cc96 and cc118, whose changes were similar to each other, the structures predicted by computer simulation turned out to have similar characteristics. Thus, the appearance of a hydrogen bond between the 11-hydroxy group of CS and TyrH50 might provide the molecular basis for the increase in the CS-binding activity.

Perspective

In order to change the antigen specificity of antibodies from 11-DOC to CS, we adopted two kinds of mutagenesis in the present study. During the first step, mutations were introduced at restricted positions in CDRs. Although, in the present study, we isolated only one clone from the resulting library that newly developed a CS-binding activity, we analyzed many such clones in the previous study (Iba et al., 1998Go). The respective characteristics of the clones analyzed previously were similar to each other. In all cases, binding to the original antigen was unaffected. During the second step, mutations were introduced randomly into the entire VH-coding region by error-prone PCR. The clones with enhanced binding to CS and decreased binding to 11-DOC had mutations at positions located outside of the steroid-binding pocket. It has been reported that a majority of clones in libraries with random mutations in the entire V-coding regions failed to express detectable Fab (Casson and Manser, 1995Go). These observations suggest how variations in sequences might be achieved for construction of a large Ig repertoire. During V(D)J rearrangement in Ig loci, random sequences can be generated only in CDR3 of the VH domain. The sequences in the other regions are predetermined in the germline V and J genes. We have been making libraries using human Ig genes. More than 70% of clones in the libraries, each of which has a random combination of VH and VL genes, can be expressed as an Fab form in E.coli (to be published elsewhere). Thus, the majority of Ig genes encoded on the germline genome appeared to be faithfully expressed even in E.coli. In general, appropriate sets of germline V genes should have been selected during evolution. Introduction of random mutations both in vivo and in vitro into the entire regions might destroy the integrity of the Ig-fold structure of V domains at high frequency. Therefore, somatic mutations in vivo can contribute only to the affinity maturation of antibodies in the presence of strong selection systems. This principle could also be applicable to in vitro systems. While introduction of random mutations into the entire V-coding regions might generate a large set of mutants, only minor fractions can be expressed as forms with antigen-binding ability. However, fine tuning for accommodation by the antigen-binding pocket of the target antigen would require a random process. Moreover, similar to the selection system in vivo, in the present study competition panning adopted in the second screening process should have facilitated selection of clones that changed the specificity.


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Table II. Amino acid sequences of clones isolated from the randomly mutated library

 

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Table III. Kinetic parameters for the association and dissociation of FAB–PPs with CS (a) and 11-DOC (b)

 

    Acknowledgments
 
We thank Ms A.Suzuoki for preparing the manuscript. This work was supported in part by a Grant-in-Aid for Research on Advanced Medical Technology from the Ministry of Health and Welfare, and by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received November 16, 1998; revised December 25, 1998; accepted January 28, 1999.