A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs

M.K. Short1,, P.D. Jeffrey2,, A. Demirjian and M.N. Margolies3,

Antibody Engineering Laboratory, Department of Surgery, Massachusetts General Hospital, MGH East, 149 13th Street, Box 31, Charlestown,MA 02129 and 2 Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

Keywords: antibody specificity/bacteriophage display/digoxin/immunoglobulin fragments/protein structure

Abbreviations: Ab, antibody • mAb, monoclonal antibody • Fab, antigen-binding fragment of antibody • Ag, antigen • V, variable region • Fv, antibody fragment including heavy- and light-chain variable regions only • sFv, single-chain Fv • Ig, immunoglobulin • CDR, complementarity determining region • PCR, polymerase chain reaction • bp, nucleotide base pair • BSA, bovine serum albumin • ELISA, enzyme-linked immunosorbent assay • LB, Luria–Bertani medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, pH 7.0) • SB, superbroth (3% tryptone, 2% yeast extract, 1% 3-(N-morpholino)propane- sulfonic acid, pH 7.0) • PBSA, 0.14 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, pH 7.4, with 0.02% NaN3 • wt, wild type


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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The increase in average antibody (Ab) affinity appearing temporally following immunization occurs via antigen-driven selection of somatic mutants (Möller, 1987Go). Affinity maturation in vivo is constrained by the intrinsic affinity of a given germline variable (V) region and the structural adaptability of that sequence to sustain somatic mutation with improvement in binding. The extent of this adaptability is related to germline gene codon bias and the existence of `hot spots' of somatic mutation (Winter and Milstein, 1991Go). Maturation of specificity and affinity is not so constrained ex vivo, where unique variants may be constructed that are not observed in vivo.

`Instructional' approaches to engineering Ab combining sites use, as a starting point, three-dimensional X-ray crystallographic models of Fab or Fv in complex with antigen. Based on inspection and computational modeling procedures which incorporate estimates of the energetics of interaction, the effects of binding site amino acid substitutions upon affinity and specificity are predicted and appropriate mutants are constructed and analyzed. Functional results of the engineered mutations and models or crystal structures of the mutants provide tools to alter Ab affinity and specificity. The utility of instructional approaches depends on the accuracy and reliability of computational modeling and the atomic resolution of the crystal structures. Despite success in increasing affinity by mutagenesis based on examination of crystal structures (Riechmann et al., 1992Go; Wong et al., 1995Go), precise predictions that result in the desired effect are not the rule; most mutations designed at residues in Abs which were `optimized' by affinity maturation in vivo, in particular contact residues, decrease affinity (Denzin et al., 1991Go; Glockshüber et al., 1991Go; Schildbach et al., 1993aGo, bGo, 1994Go). Increasing affinity in unmutated germline-encoded Abs is possible by inspection of crystal structures (Wong et al., 1995Go) or by recapitulation of mutations observed in vivo to be recurrent in Abs encoded by the same V regions (Kocks and Rajewsky, 1988Go; Sharon et al., 1989Go).

An instructive approach could also be used to target mutations to Ab residues likely to facilitate a specificity switch. A single residue change in Ab 36-71 causes a specificity switch from p-azophenylarsonate to p-azophenylsulfonate (Kussie et al. 1994Go; Wong et al., 1998Go). However, this result was not based on analysis of the crystal structure, but on mutations observed in antigen-elicited monoclonal antibodies in vivo (Ellenberger et al., 1993Go).

`Selective' methods of Ab engineering involve cloning genes encoding Fab or sFv into filamentous phage vectors (Parmley and Smith, 1988Go) which incorporate foreign DNA fused to phage coat proteins III or VIII and are functionally displayed on the phage surface (McCafferty et al., 1990Go; Barbas et al., 1991Go; Clackson et al., 1991Go; Kang et al., 1991Go; Burton and Barbas, 1994Go; Winter et al., 1994Go). Rare phage carrying V genes encoding a desired specificity are selected directly with antigen by `panning' on plates, affinity columns or magnetic beads and enriched via several rounds of affinity selection. The sequence of the cloned DNA associated with the target specificity is determined directly on phagemid DNA. This approach is used principally to select Abs from naive combinatorial libraries of V region gene repertoires. The affinity of Abs of desired specificity recruited from libraries could be increased by CDR mutagenesis as, for example, in H:CDR3 by `chain shuffling', or by random V region mutagenesis (Burton and Barbas, 1994Go; Winter et al., 1994Go).

Phage display methodology permits a direct assessment of the differentiative potential of particular Ab V domains, not subject to the biases of the in vivo hypermutation and selection process. Thus, mutations affecting specificity or removing unwanted specificities may be revealed that are not detectable in vivo. Using phage display methodology, the affinity of an anti-protein Fab was increased in the absence of a three-dimensional structure (Jackson et al., 1995Go). A similar approach resulted in improved affinity and broadened specificity of an anti-HIV Ab (Thompson et al., 1996Go). Saturation mutagenesis of the entire H:CDR3 of an anti-tetanus Ab permitted selection of mutants which bound fluorescein (Barbas et al., 1992Go).

The model system used here to study antigen–Ab complementarity for purposes of engineering binding changes is the anti-digoxin murine monoclonal Ab 26-10, elicited by immunization with a digoxin–protein conjugate (Mudgett-Hunter et al., 1982Go). Digoxin is a relatively large and rigid hydrophobic hapten lacking charge groups, with mobility limited to rotation about the C3–sugar bonds, sugar–sugar bonds and steroid–lactone bond (Figure 1Go). There are hundreds of structurally related natural and synthetic digoxin congeners of known stereochemistry (summarized in Thomas et al., 1974), which differ from each other by steroid ring substitutions, alterations of the lactone and the nature and number of attached sugars, thus permitting measurement of `fine' specificity. The X-ray crystal structure of Fab 26-10 in complex with digoxin and that of the uncomplexed 26-10 Fab was determined (Jeffrey et al., 1993Go).



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Fig. 1. Schematic representation of the digoxin (digoxigenin tridigitoxoside) numbering system and steroid ring nomenclature. The digoxin analogs used in panning are substituted at position 16 in the D ring (16-H, digoxin; 16-OH, gitoxin; 16-OCHO, 16-formylgitoxin; 16-OCOCH3, 16-acetylgitoxin).

 
The H:CDR3 loop of 26-10 contains three contiguous residues, each of which contacts the hapten digoxin at the base of the binding pocket. The present study was undertaken to determine the extent to which sequence diversification in this region is limited by structural constraints required for binding. In particular, can mutants with increased affinity or altered specificity be recruited?


    Materials and methods
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Vector construction

Nucleotide sequences encoding the 26-10 Fab, from H chain position Glu-H1 to Arg-H228 (Kabat et al., 1991Go) in the hinge region (Near et al., 1991Go; Schildbach et al., 1991Go) and the entire L chain (Asp-L1 to Cys-L214) were introduced into the pComb3 vector (Barbas et al., 1991Go) as described (Short et al., 1995Go). 26-10 Fab was detected on the surface of bacteriophage M13 as a fusion protein with M13 gene 3. Excision of the gene 3 portion resulted in secretion of soluble 26-10 Fab into the culture supernatant (Short et al., 1995Go) as described (Barbas et al., 1991Go). A new vector, here designated pComb3-26-10-H3, was constructed with H:CDR3 alterations designed to prevent parental 26-10 contamination of 26-10 libraries randomized in H:CDR3 and consequent competition in biopanning. The altered H:CDR3 was generated by oligonucleotide-directed mutagenesis employing the method of Eckstein (Olsen et al., 1993Go) (Kit RPN1523, Amersham, Arlington Heights, IL) using oligonucleotide No. 1, GTCTATTACTGTGCAAAGCTTCCGGGGAATAAGTGGGC. The new nucleotide sequence encodes a unique HindIII site (Figure 2Go) and changes H chain amino acid residues 94–96 from wt G–S–S to K–L–P. The new restriction enzyme site allows linearization and elimination of unwanted parental plasmids after ligation of insert. Plasmids with inserts containing randomized library sequences are preserved; the altered region 94–96 is thereby replaced (see below). The amino acid changes were predicted to reduce significantly Ag binding in this template and this further reduced the probability of selection of a contaminating parental plasmid.



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Fig. 2. Diagram of the 26-10 H chain V region portion of vector pComb3-26-10 AHN. The oligonucleotides used for randomization of H:CDR3 are shown. Nucleotide numbering is in italics.

 
To prepare pComb3-26-10H3 for insertion of cassettes containing randomized H:CDR3 sequences, first a unique NcoI site was removed from the DNA 3' of H:CDR1 (using oligonucleotide No. 2, 5'-ATC AAG GCT CTT TCC GTG GCT CTG CCT CAC C-3') and inserted 3' of H:CDR3 (using oligonucleotide No. 3, 5'-GGT GAC TGA GGC TCC ATG GCC CCA GTA GTC CAT AG-3'). The resulting vector was designated pComb3-26-10AHN (the order of restriction sites being 5'-AccI-HindIII-NcoI-3').

Randomization of H:CDR3

Saturation mutagenesis of H:CDR3 included either five or 10 contiguous codons at positions 99–101 or 95–102, respectively [the numbering system of Kabat et al. (Kabat et al., 1991Go) is used throughout].

Oligonucleotide primers No. 4, 5'-GTGGTGTTACTGGCTACAACC-3', and No. 5, 5'-GC TCC ATG GCC CCA GTA (SNN)5 ATT CCC CGA CGA TCC TGC ACA G-3', or No. 6, 5'-GC TCC ATG GCC CCA (SNN)10 TCC TGC ACA GTA ATA GAC TGC-3', where N represents an equimolar mixture of all four dNTPs and S represents an equimolar mixture of dCTP and dGTP, were used in a polymerase chain reaction (PCR) (Taq polymerase from Perkin-Elmer, Norwalk, CT) with the HindIII altered template or a single-stranded 26-10 template. The 175 bp product of this reaction extends from 5' of the AccI site in the V region of 26-10 H chain through CDR3 and the adjacent NcoI site at nucleotide position 714 (corresponding to amino acids Ser72–Gly106 (Figure 2Go) (Short et al., 1995Go).

This 175 bp fragment was digested with AccI and NcoI and the resulting 115 bp fragment band was excised from a 4% NuSieve agarose gel (FMC, Rockland, ME) and purified by binding to and elution from glass milk beads (Mermaid kit, Bio 101, Vista, CA). The fragment was ligated to an NcoI–AccI-digested p26-10AHN vector and introduced by electroporation into Escherichia coli XL1-Blue F' cells. Infection of each XL1-Blue library of H:CDR3 mutants with VCSM13 helper phage (XL1-Blue F' and VCSM13 were obtained from Stratagene, La Jolla, CA) generated two libraries of phage with surface Fab containing either five or 10 substituted positions in CDR3.

Phage were recovered and concentrated by polyethylene glycol/NaCl precipitation from bacterial supernatants (Barbas et al., 1991Go). Bacteriophage yield was quantitated by titration on lawns of XL1-Blue F' and phagemid library complexity was quantitated by post-infection XL1-Blue F' colony formation on LB/agar/carbenicillin plates (Sambrook et al., 1990Go; Barbas and Lerner, 1991Go).

Site-directed mutagenesis

The site-directed mutant M100bL was constructed by subcloning the PCR product directed by oligonucleotides No. 7, 5'-GC TCC ATG GCC CCA GTA GTC CAG AGC CCA CTT ATT CCC-3', and No. 4 into the NcoI–AccI sites of pComb3-26-10AHN. The site-directed mutant W100R was constructed in an analogous manner, using oligonucleotide No. 8, 5'-GC TCC ATG GCC CCA GTA GTC CAT AGC CCG CTT ATT CCC GAC GAT CCT GGA-3'.

Biopanning

Enrichment of bacteriophage expressing specifically bound Fab mutants was achieved by successive rounds of binding to glycoside–BSA-coated microtiter wells (Costar 3690, Cambridge, MA), followed by washing, elution, reinfection and growth according to the procedure of Barbas and Lerner (1991). Cardiac glycoside–BSA coupling was described previously (Short et al., 1995Go). Phage from each panning round was grown in E.coli without VCSM13 helper and phagemid DNA was isolated from a 50 ml culture using a Plasmid Midi Kit (Qiagen, Chatsworth, CA). An aliquot was sequenced to estimate the minimum complexity of the sequences in the mutated CDR3 region. Phage were analyzed after four (5-mer library) or seven (10-mer library) successive rounds of biopanning. A 1 µg sample of pooled phagemid DNA from the final panning round was cut with NheI and SpeI and religated to remove gene 3 (Barbas and Lerner, 1991Go). After religation, the DNA was transformed into XL1-Blue cells and plated on Luria–Bertani medium (LB)/ carbenicillin plates. Bacterial colonies were isolated and screened for Fab production and antigen binding. The DNA sequences of cardiac glycoside ELISA-positive clones were determined using the dideoxy sequencing method. Fab was produced as described (Short et al., 1995Go).

ELISA

Bacterial supernatants were tested in a direct binding ELISA in a 96-well microtiter plate coated with BSA alone, cardiac glycoside–BSA or goat anti-mouse Fab antibody (ICN, Costa Mesa, CA) (Short et al., 1995Go). Briefly, wells were coated with antigen and blocked with 3% BSA in PBSA. Bacterial supernatant was added and incubated for 3 h at room temperature and detected after a 2 h room temperature incubation with peroxidase-labeled F(ab')2 fragment of goat anti-mouse IgG and IgM using the TMB microwell peroxidase substrate system and neutralization with 1.0 M phosphoric acid. Color development was measured at 450 nM in a Bio-Tek ELISA reader.

Affinity and specificity determination

Affinities for digoxin were measured on dilute bacterial supernatants with a saturation equilibrium assay (Short et al., 1995Go), using filtration through glass-fiber filters to separate bound and free [3H]digoxin (New England Nuclear, Boston, MA). Each tube contained a constant amount of Fab, one of 12 serial dilutions of [3H]digoxin (1.1x10–7–3.0x10–11 M, depending on preliminary screening) and 0.5 µg/ml goat anti-mouse Fab antibody. Scatchard analysis was used to determine affinities.

The specificity of Fab for different cardiac glycosides was determined using a competition radioimmunoassay based on the affinity assay described above (Short et al., 1995Go). The values reported are ratios of molar concentrations of inhibitor required to give 50% inhibition relative to the molar concentration of digoxin that gave 50% inhibition.

Association and dissociation rate constants were obtained using a variation of the same filter assay. Association rates were determined by adding [3H]digoxin to equilibrated Fab–G{alpha}M mixtures and stopping the reaction at various time points by filtration. Dissociation rates were determined by adding a 1000-fold excess of cold digoxin to tubes in which association had reached equilibrium and stopping the reaction by filtration.

Statistical distribution of mutations in cardiac glycoside binding clones

In order to analyze sets of sequences obtained from cardiac glycoside binding clones, the statistical distribution of amino acids was determined for each NNS-substituted position by the method of Matthews et al. (1994). Gln derived from the native codon or from the suppressed TAG stop codon in XL1Blue E.coli was included in the same group. {Delta}{sigma} is the difference between the observed frequency of an amino acid and its expected frequency as standard deviation (Matthews et al., 1994Go).

Each mutant with a unique nucleotide sequence was counted as a new mutant regardless of whether the amino acid sequence was selected more than once. Two or more mutants having the same nucleotide sequence were counted only once.


    Results
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 Materials and methods
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One hundred and four antigen-binding clones were selected and analyzed from a 5-mer library of 26-10 randomized at HCDR3 positions 99, 100, 100a, 100b and 101. Based on titration of post-transformational colonies, the libraries generated contained (1–2)x108 clones each.

Phage aliquots were panned 4–6 times against each of four BSA-conjugated cardiac glycosides differing at position 16 (Figure 1Go) on the cardenolide moiety (16-H, digoxin; 16-OH, gitoxin; 16-OCHO, formylgitoxin (gitaloxin); and 16-OCOCH3, 16-acetylgitoxin. Clones were isolated after successive panning following analysis by batch DNA sequence determination. The secreted Fab were tested for binding to digoxin–BSA, the cardiac glycoside–BSA used in selection and goat anti-mouse Fab antibody.

Of 106 clones selected from the 5-mer library, 104 bound antigen. Of these, 40 were selected after panning on digoxin–BSA, 25 using gitoxin–BSA, 19 using 16-formylgitoxin–BSA and 20 using 16-acetylgitoxin–BSA (Tables I–IVGoGoGoGo). Mutants are designated by a single initial for the congener used in panning: digoxin (D), gitoxin (G), 16-formylgitoxin (F) or 16-acetylgitoxin (A), followed by the clone number. The secreted Fab from all clones bound digoxin and the antigen used for selection.


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Table I. Amino acid sequencesa, affinity for digoxin and specificity of binding to digoxin congeners of clones selected by digoxin–BSA from a 26-10 Fab phage library randomized at H chain CDR3 positions 99–101
 

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Table II. Amino acid sequences, affinity for digoxin and specificity of binding to digoxin congeners of clones selected by gitoxin–BSA from a 26-10 Fab phage library randomized at H chain CDR3 positions 99–101
 

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Table III. Amino acid sequences, affinity for digoxin and specificity of binding to digoxin congeners of clones selected by 16-formylgitoxin–BSA from a 26-10 Fab phage library randomized at H chain CDR3 positions 99–101
 

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Table IV. Amino acid sequences, affinity for digoxin and specificity of binding to digoxin congeners of clones selected by 16-acetylgitoxin–BSA from a 26-10 Fab phage library randomized at H chain CDR3 positions 99–101
 
Continuous DNA sequences were obtained for each mutant clone for the region including the PCR fragment and the flanking AccI–NcoI insertion site in the H chain V region of each mutant Fab (Figure 2Go). There were no sequence alterations outside the NNS-substituted region and all selected clones had G or C in the third position of the NNS-substituted codons. There were 83 unique nucleotide sequences in the randomized region among the 104 antigen-binding clones.

In several instances, clones were selected which had different nucleotide sequences encoding the same amino acids, suggesting that the library size was sufficient to assess the sequence space of a 5-mer randomized segment. Striking sequence restriction was found among the 16-formylgitoxin-selected clones. Eleven clones had the sequence RRALD, encoded by five different codon combinations (Table IIIGo).

Certain clones with identical DNA sequences were observed repeatedly, owing to a growth and/or affinity selection advantage. Some mutants selected using one cardiac glycoside were selected in panning by other cardiac glycosides, often with a different nucleotide sequence encoding the mutant amino acids.

The digoxin-selected 26-10 H:CDR3 mutant clones exhibited distinct consensus sequences and could be readily separated into four groups, depending upon the residues occurring at positions 100 and 100a (Tables I and VGoGo), i.e. wt 100:W–100a:A or 100:H–100a:A, 100:R–100a:A or 100:R–100a:X, with X being F, H, S or W. The mutants selected by 16-formylgitoxin and 16-acetylgitoxin all contained the residues 100:R–100a:A, with the exception of clones A19 and F1 (Tables III and IVGoGo). The gitoxin-selected group also included predominantly 100:R100a:A-containing clones (Table IIGo). None of the clones selected by 16-substituted analogs of digoxin exhibit the wt residue Trp at position 100; Arg instead predominates. Of 95 clones selected by the four different cardiac glycosides with affinities >107 for digoxin, 93 had a Leu residue at position 100b. The exceptions, clones D22 and A11 (Tables I and IVGoGo), had the wt Met. The residues observed at positions 99 and 101 were more diverse, irrespective of the antigen used for panning.


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Table V. Comparison of affinity and specificity of sets of Fab 26-10 phage-displayed mutants, selected by digoxin–BSA and three congeners, with differing consensus sequence at H chain CDR3 positions 99–101
 
The statistical distribution of amino acids at positions 99–101 is shown in Figure 3Go (Matthews et al., 1994Go). These were calculated from the combined sets of mutants with unique nucleotide sequences selected by all four cardiac glycosides. All mutant clones bound the Ag used in selection and also bound digoxin, the immunizing antigen for the parental 26-10 Ab. At positions 99 and 101 (Figure 3Go), few amino acids appear with a frequency which differs from the expected frequency by >=2{Delta}{sigma}. The most prevalent amino acid at position 99 in unique clones selected by all four analogs is the wt K. Proline is the only amino acid that occurs significantly less often than random. The most prevalent amino acids at position 101 in unique clones selected by all four analogs are D, K, N or Q. In contrast to positions 99 and 101 noted above, there is a remarkable restriction observed at the other randomized positions. The significant amino acids at positions 100 (H, R, W), 100a (A) and 100b (L) (Figure 2Go) are also apparent from the data in Tables I–IVGoGoGoGo.



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Fig. 3. Statistical distribution of amino acids at H chain positions 99–101 among a set of unique digoxin binding clones of Ab 26-10 recovered after panning using four different cardiac glycosides. The corresponding amino acid sequences are listed in Tables I–IVGoGoGoGo. Position numbers are at the upper right for each panel. {Delta}{sigma} is the standard deviation of the difference between the observed frequency and the expected frequency (Matthews et al., 1994Go).

 
Binding constants for H:CDR3 mutants

Affinities for digoxin of the 26-10 H:CDR3 mutant Fabs were measured and compared with that of wt Fab. Affinities (Ka) ranged from 8x107 to 2.2x1010 (Table IGo). The highest affinities were associated with the group of digoxin-selected clones containing the wt W at position 100 (Table IGo). Every mutant Fab in this group had an affinity for digoxin greater than 2x109. Three mutants (D22, D32 and D36) (Table IGo) bound digoxin with affinities up to 3-fold greater than wt. Among clones containing 100:H, affinities ranged from 5x108 to 2.2x109. The set of mutants which contained 100:R–100a:A demonstrated lower affinities (average 0.9x109) (Table VGo). Of the 36 mutants with unique amino acid sequences in the 100:R–100a:A group (Tables I–IVGoGoGoGo), all had affinities between 8x107 and 1.6x109. A distinct correlation between observed affinity for digoxin and the identity of residue 100 is shown in Table VGo. Affinities for digoxin of mutants with residues other than W, H or R at position 100 were all <6x108 (Tables II and IVGoGo).

The wt sequence KWAMD was not identified among selected mutants. The mutant sequence most homologous to wt (K W A L E) has an affinity (Ka = 8.3x109) indistinguishable from wt (Ka = 8.0x109) (Table IGo). In order to investigate whether the preference (93 of 95) among mutants for Leu at position 100b over the wt Met was due to differences in digoxin affinity, the mutant KWALD was produced by site-directed mutagenesis. The affinity of KWALD did not differ significantly from that of the wt KWAMD. Since there was also no significant difference in the affinity of another pair of selected mutants, D22 (AWAMQ) and D32 (AWALQ) (Table IGo), the on and off rates for these were measured to ascertain whether differences in the association or dissociation rates were responsible for the preponderance of Leu versus the wt Met at position 100b. There was no difference in these rates (data not shown).

Specificity of mutant Fabs for digoxin and 16-substituted analogs

The specificity of mutant Fab for cardiac glycosides that differ at the C16 position was compared using a competition assay. The results are presented as the ratio of concentration of congener to digoxin that inhibits 50% of the binding of [3H]digoxin to Fab where the IC50 of digoxin equals one. The data are given in Tables I–IVGoGoGoGo. The specificity differences among selected mutants (Table VGo) can be correlated with the occurrence of consensus residues at positions 100 and 100a. All mutants containing 100:W–100a:A at these positions demonstrated patterns of specificity indistinguishable from wt (Tables I and VGoGo). These mutant Fabs bind gitoxin ~5-fold less than digoxin, and 16-formylgitoxin and 16-acetylgitoxin ~25- and 150-fold less than digoxin, respectively. Those mutants containing 100:H demonstrated decreased relative binding to the 16-substituted congeners (Tables I and VGoGo) and are thus more specific for digoxin than wt 26-10.

Competition assays on mutants containing 100:R–100a:A displayed a specificity shift. Most 100:R–100a:A mutants bound gitoxin to a greater extent than digoxin. In addition, the binding to 16-formylgitoxin and 16-acetylgitoxin was substantially improved compared with that seen for the wt or the 100:W–100a:A group (Tables I–VGoGoGoGoGo). A comparison of the competition curves for mutants A5 (sequence KRALN) and 26-10 (sequence KWAMD) shows that the inhibitory concentration for digoxin is 5-fold lower than for gitoxin for the wt, whereas that for gitoxin of mutant A5 is 0.4 compared with the wt 26-10 (Table IVGo). The inhibitory ratios for the 16-formyl- and 16-acetyl-substituted congeners are also decreased for the mutant A5 compared with wt 26-10. The estimated affinities of clone A5 for all 16-substituted congeners, based on the data in Table IVGo, are slightly higher than those of the wt 26-10. The clone RRALD (Tables I–IVGoGoGoGo), which was repetitively selected, had inhibitory ratios of 1:0.8:3:45, typical of the 100:R–100a:A group. The RRALD and the GRALN mutants exhibit the highest affinity for digoxin (Ka = 1.6x109) of this group. Although there is a remarkable correlation among the large number of arginine mutants at H:100 with heteroclitic binding to gitoxin, the mutants also contained other substitutions. We therefore constructed, by site-directed mutagenesis, a mutant of 26-10 containing a single replacement of H:Trp100 by Arg. The affinity of this KRAMD mutant was 6-fold lower than the wt 26-10 (KWAMD) and bound gitoxin with a 7-fold increase in relative affinity based on competition assay. These results are consistent with the contention that the single H:100 substitution of Arg for Trp is responsible for the observed specificity shifts detected among the randomized mutants.

The shifts in specificity observed for the 100:H and 100:R groups are accompanied by 6.7- and 11.5-fold reductions in affinity, respectively, on average (Table VGo).

The 100:R–100a:X group demonstrated an eclectic specificity pattern, with some mutants having a wt pattern and others having a pattern more like that seen for the 100:R–100a:A group (Tables I and VGoGo).

H:CDR3 10-residue randomly mutated library

Mutants were also selected from a 26-10 phage library where DNA encoding all 10 amino acids of H:CDR3 (residues 95–101) were replaced by NNS-randomized codons. After seven rounds of panning against digoxin-BSA, 21 of 40 clones were antigen positive. These yielded nine unique amino acid sequences, several selected more than once. All mutants had the residues 100a:A–100b:L that predominated in clones selected from the 5-mer CDR3 library. H:Ser95, a contact residue to hapten in the crystal structure of Fab 26-10:digoxin (Jeffrey et al., 1993Go), was conserved in seven of nine sequences.

The 10-mer 26-10 CDR3 library was also panned against 16-acetylgitoxin-BSA, the analog with the bulkiest C16 substitution. Only four unique mutants were found among 10 antigen-positive clones of the 30 clones screened. As with the digoxin-selected clones, residues 100a:A–100b:L were present in every mutant and H:Ser95 was found in three of four mutants.

Only a few mutants in the 10-mer library had affinities for digoxin greater than 107, and these did not reveal any specificity differences. The size of the 10-residue-substituted library (2x108) was insufficient to assess the entire potential library of nucleotide sequences (1.1x1015).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The anti-digoxin monoclonal Ab 26-10 binds digoxin with high affinity (Ka = 1010 M–1) (Schildbach et al., 1993aGo). The three-dimensional structure of Fab 26-10 complexed with digoxin (digoxigenin tridigitoxoside, Figure 1Go), as well as the structure of the uncomplexed Fab, was determined by X-ray crystallography (Jeffrey et al., 1993Go). In this discussion, structural analysis is based on PDB entry 1IGJ. The first digitoxose moiety (attached to the cardenolide A ring at position 3) is exposed to solvent, consistent with the fact that digoxin was coupled to protein carrier through the terminal digitoxose for purposes of immunization (Mudgett-Hunter et al., 1982Go). Digoxin binds 26-10 in a deep elliptical pocket that is predominantly hydrophobic. The lactone ring of digoxin is buried at the bottom of the pocket. The steroid rings are sandwiched between residues lining each side of the binding pocket, on one side by residues from CDRs H1 and H2 and on the other side by residues from CDRs H3 and L1. In particular, the residue H:Trp100 from H:CDR3 makes extensive interactions with steroid rings B, C and D of digoxin. There are no significant conformational changes detected in comparing the complexed and uncomplexed Fab. The specificity and affinity of Fab 26-10 derive solely from shape complementarity between the site and the relatively rigid hydrophobic digoxin hapten. In this report we examined structural constraints on the binding of the 26-10 anti-digoxin Fab to digoxin and digoxin congeners by selection of phage-displayed Fab randomly mutagenized at five or 10 contiguous amino acid residues in the H:CDR3 region.

Of the five HCDR3 residues randomized (positions 99–101 inclusive), three (W100, A100a, M100b) contact digoxin (Figure 4Go). The library size (2x108) was sufficient to assess the entire potential library of nucleotide sequences (3.4x107). The library was panned against BSA conjugates of digoxin and congeners substituted at the C16 position (see Figure 1Go). Among 104 Ag-binding clones, there were 83 independent nucleotide sequences encoding 66 different amino acid sequences. The sequence data showed a distinct consensus pattern and the mutants could be separated into homology sets (Tables I and IVGoGo).



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Fig. 4. Stereo view of digoxin (digoxigenin tridigitoxoside) with portions of H:CDR1 and H:CDR3 from the X-ray crystal structure of the 26-10 Fab:digoxin complex. Digoxin is at the center with the lactone at the bottom. At the top is shown a single attached sugar (digoxigenin monodigitoxoside); H:CDR3 (positions 99–101) is on the left; the residues that contact digoxin are labeled explicitly (100, 100a, 100b). H:CDR1 (positions 33–35) is on the right; Tyr33 and Asn35 contact the hapten.

 
Positions 99 (wt:K) and 101 (wt:D) are residues which do not contact the hapten and not surprisingly showed a large number of amino acid substitutions consistent with digoxin and gitoxin binding. The side chains at 99 and 101 project away from the binding site, are solvent-exposed and hydrophilic.

Residue H:Met100b occupies a site at the base of the binding pocket immediately adjacent to the steroid D ring and the lactone ring. Virtually all of the digoxin binding clones (93 of 95) encode leucine at position 100b, rather than the wt Met, which is found only once. This preponderance is not entirely accounted for by codon frequency (there are three Leu codons for every Met codon in an NNS library) in the mutagenesis strategy. Neither is it explained by theoretical preferential E.coli codon usage (Makrides, 1996Go) nor actual codon usage in the XL1Blue strain used in these experiments, since Met and Leu were found at NNS-substituted positions 99 and 101 at about the expected frequency (Tables I–IVGoGoGoGo). Differences in the association or dissociation rates measured by a filter assay (data not shown) did not account for the observed dominance of Leu at position 100b.

Inspection of the 26-10:digoxin complex crystal structure suggests that Met should be preferred over Leu at position 100b. Introduction of a Leu residue into this site gives rise to potential steric clashes between the side chain and neighboring groups: the side chains of H:Ser95, H:Asn35 and the lactone ring of digoxin. Although Met and Leu have similar hydrophobic properties and side chain volume, the branching of the side chain at C{gamma} in Leu should introduce unfavorable non-bonded contacts. We constructed, by site-directed mutagenesis, Met100bLeu in 26-10 wt; this mutation, however, did not significantly change the affinity. Mutant clones D22 (AWAMQ) and D32 (AWALQ) (Table IGo), selected during panning with digoxin, also have similar affinities for digoxin. The observation that the affinity is essentially unchanged for the Met–Leu substitution suggests that the structure can rearrange sufficiently in this locality to relieve these energetically unfavorable interactions. Thus the preference for Leu is not affinity-based and may be caused by relative repression of Met100b-containing Fab clones on phage.

Substitutions at H:Ala100a and H:Trp100 are likely to produce much more profound effects on affinity, however, as compared with substitutions at H:Met100b. The side chain of H:Ala100a is buried and points into a pocket of small volume; it makes contact with the cardiac glycoside lactone ring through the polypeptide backbone (Figure 4Go). The introduction of larger amino acids at this position would necessarily entail structural rearrangement and possible movement of the polypeptide backbone in H:CDR3. Thus, Ala at position 100a is a dominant residue, together with any of three residues at position 100. However, this is not the case for the fourth set of mutants, shown in Table IGo. In all clones not containing Ala at position H:100a, Arg occurs at position H:100. In order to substitute larger side chains (Phe, His, Ser, Gln and Trp) at 100a, there must be a backbone rearrangement in this region, perhaps allowed because of the substitution of the more flexible associated mutation W:100R. The majority of mutants with the motif WAL, RAL or HAL at positions 100, 100a and 100b exhibit affinities >=109 for digoxin (Tables I and IVGoGo).

Similarly, H:Trp100 is so tightly sandwiched between the bound hapten digoxin and the VL domain of 26-10 that there is little capacity to move the side chain. The nature of the binding site also dictates that the aromatic ring is oriented such that the Chi1 angle is zero. This `eclipsed' arrangement in side chain conformation is energetically unfavorable and may explain why mutations to Tyr and Phe are not found amongst high-affinity mutants of 26-10 H:CDR3, i.e. the arrangements of the larger aromatic CH1 group (Tyr, Phe) next to the main chain peptide may be significantly more unfavorable than the placement of an aromatic NH1 group (Trp, His). Thus, at position 100 the wt residue W, and also R and H, are permissible and frequent (Tables I and IVGoGo). The residues R and H can be substituted for W here, but the buried surface area is thereby reduced, which may account for the decrease in affinity (3–40-fold) of these mutants relative to wt. Phage display has been shown to be a tractable method for increasing affinity for hapten, even when the affinity is very high to begin with. Thus, only mutants containing Trp100 were among the clones with higher digoxin affinity than wt 26-10 (Tables I and VGoGo), consistent with the crystal structure data.

In contrast to the distribution of sequences recruited by panning against digoxin–BSA (Table IGo), panning with C16-substituted analogs resulted in a preponderance of sequences exhibiting the motif RAL at positions 100–100b (Table IIGo). This change in a consensus hapten contact residue indicates the exquisite sensitivity of panning to small differences in hapten structure and its potential as a method for engineering fine specificity changes in Ab combining sites.

Specificity analyses (Tables I and IVGoGo) showed that several of these clones had affinities for gitoxin and 16-formylgitoxin that were significantly increased relative to wt; in some cases, heteroclicity for gitoxin was observed (i.e. the mutants bind gitoxin with higher affinity than digoxin). In addition, binding to 16-acetylgitoxin improved 3–5-fold. These results were unexpected, as C16 lies on the opposite side of the cardenolide from this segment of H:CDR3 (Figure 4Go). Substitutions of Arg for Trp at position H:100 may not appear to be a conservative change, but the charged end of the arginine side chain would be able to project into bulk solvent, eliminating any unsatisfied electrostatic interactions. The extensive hydrophobic body of the arginine side chain can in turn act as a partial replacement for the more extensive hydrophobic surface of the wt Trp side chain. Arginine is inherently a much more flexible side chain than any of the aromatic groups, which may allow it to move within the binding pocket much more readily than the wt Trp or His mutant side chains. The results suggest that the increased flexibility of Arg is an important factor in two observed effects, the accommodation of 16-substituted haptens and mutations at H:Ala100a.

The 16-position of digoxin projects toward part of the structure at the opposite side of the binding site to H:Trp100 and in particular toward the side chains of H:CDR1 and residue H:100b of CDR3. Introduction of a larger group than hydrogen at the 16-position of the cardenolide pushes on that surface and in turn pushes the antigen against the residue at position H:100 on the opposite side of the binding site. There is a clear correlation between the binding of 16-substituted digoxin analogs and the presence of Arg at position H:100, which we propose is a direct result of the ability of the side chain to move to accommodate the shift of the hapten within the binding site.

As stated above, substitution of larger side chains at H:Ala100a will also produce structural rearrangements of the antibody, probably involving backbone rearrangements. The location of side chains is critically dependent on the direction of the C{alpha}–Cß bond, in turn dependent on backbone conformation. Arg at H:100 is the only side chain that is compatible with both hapten binding and the presence of larger H:100a mutations. It can still maintain an acceptable affinity for the antigen via hydrophobic interactions, while changing local conformation in response to altering backbone conformation in H:CDR3.

We previously examined sequence constraints for digoxin binding in 26-10 by constructing a bacteriophage library randomized in H:CDR1 (Short et al., 1995Go). Phage were selected by digoxin and three C16-substituted analogs. Although diverse sequences were consistent with high-affinity binding, H:Asn35 was highly conserved and no mutants with significant improvement in relative binding for C16-substituted analogs were recruited, even though this portion of H:CDR1 contacts the D ring of digoxin at C16. The finding in the work reported here, that instead, mutations on the opposite side of the cardenolide involving H:CDR3 resulted in specificity shifts for C16-substituted analogs, is counterintuitive and was not anticipated (Figure 4Go). An alternative hypothesis is that the C16-substituted analogs are rotated 180° in the mutant binding sites, so that the flipped C16 substitutions would point toward Arg100 and potentially form new hydrogen bonds to H:Arg100. In such a model the lactone ring would remain in place, consistent with the high affinities of these analogs for 26-10 H:Arg100 mutants and the extensive contact between the lactone and the base of the Ab binding cavity in the co-crystal structure. However, because the cardenolide is canted in relation to the lactone ring, there is no room for the rotated cardenolide in the site and the polar end of Arg would be too far removed from the 16-substituents to form new hydrogen bonds.

The results reported here and previously (Short et al., 1995Go) indicate that the construction of mutants with altered specificity based on inspection of the Fab:26-10-digoxin crystal complex alone, without the selective approach of phage display of antibodies containing randomly mutated segments, would be unlikely to be successful. Clearly, combined `instructive' and `selective' approaches are useful for engineering binding changes.


    Notes
 
1 Present address: Department of Hematology/Oncology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA Back

3 To whom correspondence should be addressed Back


    Acknowledgments
 
We are grateful to Drs Carlos Barbas,III and Dennis Burton for providing the pComb3 vector and to Rou-Fun Kwong for technical assistance. This work was supported by National Institute of Heart, Lung and Blood grant RO1 HL47415 to M.N.M.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received August 23, 2000; revised November 29, 2000; accepted December 29, 2000.