coupling protein design andin vitroselection strategies: improving specificity and affinity of a...

J. Mol. Biol. (1996) 255, 86–97

Coupling Protein Design and in Vitro SelectionStrategies: Improving Specificity and Affinityof a Designed b-protein IL-6 Antagonist

Franck Martin 1, Carlo Toniatti 2, Anna Laura Salvati 2

Gennaro Ciliberto 2, Riccardo Cortese 1 andMaurizio Sollazzo 1*

1Department of Biotechnology The minibody is a designed small b-protein conceived to enable theIRBM P. Angeletti construction of large libraries of minimal discontinuous epitopes displayed

on the surface of filamentous phage. The 61 residue molecule consists ofVia Pontina Km 30,600three strands from each of the two b-sheets of the variable domain of00040 Pomezia (Rome), Italyimmunoglobulins packed face to face, along with the exposed H1 and H22Department of Genetics hypervariable regions. We have previously shown that from a minibody

IRBM P. Angeletti repertoire of more than 50 million molecules displayed on phage, we wereVia Pontina Km 30,600 able to select a minibody with micromolar affinity for human interleukin-600040 Pomezia (Rome), Italy that behaves as a selective cytokine antagonist. The minibody exposes a

surface composed of two constrained loops, which provides the possibilityof improving IL-6 binding and specificity by swapping the hypervariableregions, followed by further selection. We established experimentalconditions for ‘‘stringent’’ selection such as monovalent phage display,competitive selection and epitope masking. Here, we show that by virtueof the optimization/selection process, we have isolated a minibody withimproved antagonistic potency and greater specificity. Furthermore, usinghIL-6 mutants carrying amino acid substitutions in distinct surface sites itwas possible to carefully define the cytokine region that binds theminibody.

7 1996 Academic Press Limited

Keywords: phage display; protein engineering; affinity selection;*Corresponding author interleukin-6 antagonist; hypervariable regions

Introduction

Proteins with tailored functions can be designedeither by modifying existing molecules or, in

principle, by inventing entirely new structures andsequences that do not exist in nature. Some progresstoward de novo design has been achieved, but theseproteins still lack some of the features typical ofnative polypeptide structures (Richardson et al.,1992). Additional power can be harnessed when thedesign of partially degenerate sequences is com-bined with biological selection and amplification.Given our present limited understanding of therules governing protein folding, the most promisingavenue for developing proteins with useful biologi-cal functions combines theoretical design withexperimental screening and genetic selection sys-tems (Sander, 1994).

Our approach to developing b-proteins with novelactivities is that of combining the rational design ofa convenient framework (minibody) with mutagen-esis of targeted regions to generate large repertoires

Abbreviations used: MB, minibody; V, immuno-globulin variable domain; CDR, complementaritydetermining region; H1 and H2 regions are definedaccording to Chothia et al. (1989); mAb, monoclonalantibody; hIL-6, human interleukin-6; hIL-6Ra, humaninterleukin-6 receptor a; hCNTF, human ciliaryneurotrophic factor; hOM, human oncostatin M; ELISA,enzyme-linked immunosorbent assay; UV CD, ultraviolet circular dichroism; FPLC, flow-performanceliquid chromatography; AP, alkaline phosphatase;TES buffer, 50 mM Tris-HCl, pH 8.0, 10 mMEDTA, 50 mM NaCl; TBST buffer, 50 mM Tris-HCl,pH 7.5, 150 mM NaCl; 0.05% Tween-20; BSA, bovineserum albumin.

0022–2836/96/010086–12 $12.00/0 7 1996 Academic Press Limited

Optimization of a Minibody IL-6 Inhibitor 87

of b-polypeptides with variable surfaces composedof b-turns and possibly with predictable main-chainconformation based on ‘‘canonical structures’’(Chothia et al., 1989). The engineered minibodymolecule consists of three strands from each of thetwo b-sheets of the variable domain of im-munoglobulins, along with the exposed H1 and H2regions (Pessi et al., 1993; Tramontano et al., 1994),thus providing the means for the engineering ofminimal discontinuous binding surfaces.

As a first step toward creating a minibody looprepertoire, we constructed a phage displayedlibrary of minibodies (150 million members) withdifferent surface residues by randomizing theregions corresponding to hypervariable regions H1and H2. We reported the affinity-selection from alibrary of minibodies of MB02, which binds withmicromolar affinity to hIL-6, and is an inhibitor ofthe cytokine biological activity (Martin et al., 1994).The potency of MB02, albeit moderate, is wellwithin the range of that of antibody fragments(Winter et al., 1994; Davies & Riechmann, 1995) orpeptide ligands (Cortese et al., 1995 and referencestherein) selected from phage displayed repertoiresof comparable sizes.

Strategies for selecting phage that expresspolypeptide mutants have been applied to manydifferent ligand/target pairs. Phage display canimprove on nature in unexpected ways: mutants ofthe growth hormone CNTF and neutrophil elastaseinhibitor have been constructed whose affinities fortheir receptors range from ten- to 100-fold higherthan the unmutagenized natural ligands (Robertset al., 1992; Wells & Lowman, 1992; Saggio et al.,1995). Repertoires of small peptides have beendisplayed on phage and screened with antibodies aswell as with non-antibody molecules, leading to theidentification of new ligands that do not necessarilyresemble their natural counterparts, yet displaysimilar binding specificity (reviewed by Corteseet al., 1995). Large collections of antibody fragments(from immunized or naive sources) have beenexpressed on phage surface and then successfullyscreened with various antigens (reviewed byWinter et al., 1994). In all cases, the binding affinitiesfor the affinity-selected molecules have ranged frommicromolar to nanomolar, depending on the librarysize; in general, the bigger the library, the better thebinding affinity. A variety of strategies to increaseaffinity of antibody fragments for their targetantigens while minimizing cross-reactivity havebeen developed, including error-prone PCR muta-genesis (Gram et al., 1992), chain shuffling (Markset al., 1992), codon-based mutagenesis (Glaser et al.,1992), and CDR walking (Barbas et al., 1994). Theoverall complexity of a library, mainly limited bybacterial transformation efficiency, has been im-proved dramatically by exploiting in vivo recombi-nation between two distinct libraries, one for eachantibody chain, utilizing the lox P/Cre system.Using this strategy, a combinatorial library ap-proaching 1011 members has been constructedand sampled, yielding a large panel of antibody

fragments specific for many different antigens withaffinities in the nanomolar range and lower(Griffiths et al., 1994).

Recent findings suggest that ex vivo enhancementof antibody affinity by random mutagenesis maylead to a reduction in specificity that couldcompromise their efficacy in the various appli-cations (Casson & Manser, 1995). This potentiallimitation could be more dramatic when dealingwith a designed polypeptide framework whosesurface is smaller than that of antibody bindingsites. We wanted to explore to what extent it waspossible to further improve the biological activity(both in terms of binding affinity and specificity) ofminibody-like molecules. To this end, we undertookthe optimization of MB02 binding affinity for hIL-6,while minimizing cross-reactivity for moleculesstructurally related to the target. Because theminibody exposes two constrained peptide loops,we were able to rapidly sample a large area of theprotein’s surface in a stepwise fashion by loopswapping, hence overcoming the limits posedby transformation efficiency (Martin et al., 1994;Stemmer, 1995). To raise the output of our screeningwe established experimental conditions for astringent and selective affinity-isolation by takingadvantage of certain properties of the target hIL-6molecule.

Here, we show that both the affinity and thespecificity of the minibody for its target can beeasily improved by rapid sequence optimization.The resulting optimized minibody (MBk) is a morepotent and specific hIL-6 antagonist in vitro and intissue culture assays than the parental MB02. Byusing mutants of hIL-6, we were able to establishthat the cytokine binding site recognized by MBkencompasses the C-terminal region of the IL-6putative D-helix that is also part of the hIL-6Rarecognition site (Leebeek et al., 1992; Savino et al.,1993). This finding substantiates, at the molecularlevel, the antagonistic property of the surfaceoptimized minibody, also emphasizing that byestablishing careful experimental conditions it ispossible to rescue ligands with pre-defined specifi-city.

Results

Library design and selection

The scheme in Figure 1 describes the rationale foroptimizing the binding affinity and specificity of thehIL-6 antagonist MB02 by loop swapping. Startingfrom a library of minibodies of 15 × 107 sequences,we recently reported the isolation of MB02, aminibody inhibitor of hIL-6 activity with a bindingaffinity in the micromolar range (Martin et al., 1994).Given the fact that the MB02 displays a singlerecombinant region (H2), we decided to explore theconformational space of H1 in an attempt tooptimize its affinity and specificity for the cytokine.To this end, we constructed a virtually completecombinatorial repertoire of all natural amino acids

Optimization of a Minibody IL-6 Inhibitor88

Figure 1. A scheme illustrating the rationale for the optimization of the minibody surface by loop swapping. Thewild-type H1 sequence of MB02 was replaced with all the possible combinations of the 20 natural amino acids usingrandom oligonucleotides. The minibody structural model is also shown. N (amino), C (carboxy) termini, H1 and H2regions (Chothia et al., 1989) are indicated.

in the H1 region of MB02. We replaced the H1wild-type sequence (GFTFSDF), except for thecentral F residue, which was maintained as aninvariant residue for its stabilizing role as discussedby Tramontano et al. (1994), by inverse PCR(Hemsley et al., 1989).

In order to obtain molecules with a better bindingactivity and superior specificity profile than theparent MB02, we opted for a monovalent displayscheme using the pHEN1 phagemid vector(Hoogenboom et al., 1991), shown in Figure 2, andthe selection strategy described below. The ration-ale for such a selection procedure is twofold: first,we exploited the fact that IL-6 embodies at leastthree distinct receptor binding sites (Figure 3). Site 1defines the IL-6Ra binding region, while sites 2 and3 are the regions recognized by two distinct gp130molecules (Paonessa et al., 1995). Site 3 alsoembodies the epitope recognized by the monoclonalantibody mAb16 (Brakenhoff et al., 1994). Previousresults have established that MB02 can bind hIL-6without interfering with the binding of mAb16(unpublished results). On the basis of this findingwe established the procedure that follows. Magneticbeads coated with mAb16 were used to immobilizehIL-6 in a spatially oriented fashion and in a nativeconformation with respect to sites 1 and 2 asestablished before (Paonessa et al., 1995). Thisexperimental scheme also has the advantage of‘‘masking’’ one of the gp130 binding sites (site 3),hence focusing the selection of ligands toward theaccessible surface of the target molecule. Second, we

used hOM, a cytokine structurally related to IL-6(Mott & Campbell, 1995), reasoning that an excessof soluble hOM during the selection process wouldcounter-select for those minibodies ‘‘cross-reactive’’with hOM and possibly related cytokines.

After three rounds of affinity-selection underthe experimental conditions described above, wepurified and sequenced 24 phagemid clones(Table 1). No phagemids displaying MB02 sequencewere present among these isolates, providingevidence that re-selection of wild-type clones wasprevented. Before testing their ability to interactwith hIL-6 coated on polystyrene plates using arabbit polyclonal anti-f1 Ab (phage-ELISA), theamount of minibody-pIII fusion expressed by theselected phagemids was normalized by Westernblotting using a rabbit anti-minibody. The phagemidthat displayed the highest binding value inphage-ELISA (not shown) was further characterizedas a soluble polypeptide named MBk. Interestingly,the same clone was isolated at high frequency(4/24). To study this molecule further, the geneencoding the FLAG-tagged polypeptide was ex-pressed in Escherichia coli and its product waspurified and re-folded as described in Materials andMethods.

Characterization of the optimized minibody

The secondary structure of MBk tagged with theFLAG epitope was probed by far-UV CD spec-troscopy as previously shown and compared with


Figure 2. A diagram of the pHEN1-minibody (pHMB) construct. PelB is the leader sequence; MB is the minibody;pIII is the f1 minor coat protein. The minibody gene and its corresponding amino acid sequence (one-letter code) arerepresented. Regions corresponding to H1 and H2 regions are within shaded boxes. The peptide linker betweenminibody and pIII, containing the factor Xa (FXa) protease recognition site (IEGR), is also indicated. In bold is shownthe constant central phenylalanine (F) residue in H1. The MB-pIII fusion is under the control of the lacZ promoter; Ampis the b-lactamase gene; M13 ori is the phage origin of replication; ColE1 is the plasmid replicon.

the spectrum of MB02 FLAG-tagged molecule. TheCD pattern shown in Figure 4 confirms that theoverall b-stranded fold of MBk is maintained,despite having numerous amino acid residueschanged (12 loop residues in addition to theC-terminal FLAG epitope) compared with the firstgeneration MB1 molecule (Pessi et al., 1993; Bianchiet al., 1994). The tagged MB02 polypeptide showsthe presence of a minimum in the ‘‘random coil’’region of the spectrum (198 to 200 nm), which is notobserved in the spectrum recorded for the samemolecule devoid of the C-terminal FLAG sequence(Martin et al., 1994).

The purified MBk polypeptide binds to immobi-lized hIL-6 with higher affinity than MB02, asshown by solid-phase direct binding assay: theminibody bound to the immobilized cytokine isrevealed using the anti-FLAG mAb M2 andAP-conjugated anti-mouse Ab (Figure 5). The KD ofbinding to hIL-6 at equilibrium, estimated by IAsys(see Materials and Methods), is 220 nM consideringthe experimental kinetic values of kass and kdiss

shown in Table 2, whereas under the same con-ditions, MB02 has a KD at equilibrium of 730 nM.This improvement in the affinity of MBk appears tobe due mostly to an increased association rate(5.4-fold better than that of MB02). In contrast, the

dissociation rate appears to be slightly faster thanthat estimated for MB02, resulting in an overall3.3-fold increase in the KD value at equilibrium. Thisconclusion is supported by the results of bindingexperiments carried out in solution phase (Figure 6).In this experiment, hIL-6 is immobilized onto ELISAplates and the binding of MBk to the solid phaseis measured in the presence of increasing amountsof soluble hIL-6 as a competitor. The residualminibody bound to the immobilized cytokine isrevealed as described above. By comparing theconcentration of soluble hIL-6 that yields 50% ofbinding inhibition, we can estimate the relative IC50

for MBk (150 nM) and MB02 (690 nm) in solutionphase. These values are well in agreement with theequilibrium KD acquired by IAsys solid-phaseexperiments suggesting a four to fivefold improve-ment in binding affinity for the target.

As with MB02, we wanted to test if MBk could actas an IL-6Ra inhibitor in vitro. To this end, weperformed an in vitro receptor binding competitionassay in which minibodies and hIL-6 were co-incubated under the experimental conditions de-scribed in Materials and Methods, with a recombi-nant version of the hIL-6Ra immobilized on thesolid phase. Ultimately, the receptor-bound cyto-kine is revealed with mAb16 and AP-conjugated


Figure 3. Location of putative receptor binding sites onthe three-dimensional model of hIL-6. The diagramsummarizes previous findings about structural andfunctional features of the cytokine. Site 1 (blue) is theregion of interaction with IL-6Ra including D-helix andAB loop. Site 2 (red), helix A and C is presumed to be thesite of interaction with one subunit of gp130. Site 3(green), centered around the N-terminal end of D-helix,is the site of interaction for a second molecule of gp130and for the mAb16. Side-chains of mutants defining therelevant binding sites are shown (AB loop mutationswithin site 1 are not displayed; for details see Materialsand Methods). The Figure was drawn using RIBBONS(Carson, 1987).

concentration does not inhibit the binding of MBkto hIL-6, whereas the same amount of solublehIL-6 results in a 78% inhibition (Figure 8(a)).Even at higher molar excess (20-fold) of hOMand hCNTF, we could not detect any significantbinding competition. From these data we con-cluded that MBk binds selectively to hIL-6. Similarresults are obtained for MB02, where the percent-age of binding inhibition (58%) is consistentlylower than that observed for MBk.

The receptor inhibition experiment suggests thatMBk binds to a region of hIL-6 that coincides, orat least overlaps, with the site recognized by theIL-6Ra (site 1) as this prevents binding to the lattermolecule. The structural model of hIL-6 (Figure 3)shows the localization of the cytokine binding sitesas established by mutagenesis and functionalstudies (Savino et al., 1994; Paonessa et al., 1995).In order to define more precisely the site ofinteraction of MBk with hIL-6, we used availablehIL-6 variants having amino acid substitutionslocated within distinct binding sites. At the sametime, these experiments allow us to probe thefine-specificity of minibodies for the target’sanalogs. It has been shown that site 1 embodiesresidues in the C-terminal region of the putativeD-helix and AB loop (Leebeek et al., 1992; Savinoet al., 1993). We therefore used the followingmutants: for site 1 a single point mutant mappingin the C terminus of D-helix hereinafter called R,a triple residue substitution in the same regioncalled D and a double residue mutant in the ABloop called AB; for site 2 a four-residue variantdefined as C (for further details see Materials andMethods). Figure 8(b) shows the result of bindingcompetition of hIL-6 mutants to MBk that clearlydefine the binding of MBk as located in thecarboxy terminus of the cytokine putative D-helix:mutants R and D prevent binding to MBk,whereas mutations in the AB loop (AB) and insite 2 (C) do not prevent binding. Thus MBk actsas a competitor of hIL-6Ra because this mini-body binds the C terminus of the predicted hIL-6D-helix that partially overlaps with the IL-6Rabinding site (site 1). It is worthy of note that thehIL-6 mutants used in this experiment have beenshown to be properly folded (Fontaine et al., 1993;Savino et al., 1994; Cabibbo et al., 1995). Since MBkand MB02 can bind hIL-6 in the presence ofmAb16, we did not include any site 3 mutantin our analysis. Both MB02 and MBk are selectivefor hIL-6, as demonstrated by the lack of bindingto hCNTF and hOM, and it is interesting toobserve that our selection procedure has led toa fine-specificity ligand. MBk can discriminateamong hIL-6 analogs (R and D) that differ in theC terminus region, whereas MB02 is a ‘‘cross-reactive’’ ligand as it recognizes all cytokineanalogs with affinities similar to that of wild-typehIL-6. A small difference in binding activity isobserved for the triple substitution mutant D,supporting the view that MB02 also binds to theD-helix.

anti-mouse Ab. The results, shown in Figure 7,indicate that MBk inhibits binding of hIL-6 tohIL-6Ra immobilized onto the solid phase, in adose-dependent manner with an IC50 of 625 nM, avalue approximately fourfold lower than that ofMB02 (2.5 mM), the negative control MBns isinactive.

Specificity of the optimized minibody

The selectivity of MBk was tested in directbinding (solid phase) and by competition (liquidphase) assays, using hOM and hCNTF, twocytokines structurally related to IL-6 (Mott &Campbell, 1995) as reagents. In the solid phasebinding experiment the two cytokines were coated(at 10 mg/ml) on ELISA plates and the assaycarried out as for Figure 5. No signal wasdetectable at the highest concentration of MBktested (5 mM). In the competition assay, a fourfoldmolar excess of these cytokines over the ligand


Table 1. Amino acid sequences of the H1 and H2 loops of affinity-selected phage clonesCLONE H1 H2 CLONE H1 H2

MBa RKGFCRF GKEEVD MBl WSGFNGR GKEEVDMBb RNLFSWN GKEEVD MBm VWLFGMR GKEEVDMBc LGGFKGE GKEEVD MBn NRKFTGC GKEEVDMBd GLGFSPA GKEEVD MBo RGRFRQG GKEEVDMBe GRQFKRV GKEEVD MBp WRQFGQR GKEEVDMBf TQVFEKV GKEEVD MBq RYAFDNG GKEEVDMBg PQCFKER GKEEVD MBr SDSFEAH GKEEVDMBh RNLFTTW GKEEVD MBs DEQFGDE GKEEVDMBi GARFWRD GKEEVD MB02b GFTFSDF GKEEVDMBj TGQFMIQ GKEEVD MBnsc GFTFSDF DLHSNDMBka RLRFSWN GKEEVD

The central phenylalanine (F) residue in the middle of H1 region is an invariantposition in the library.

a Isolated four times.b Non-optimized parental minibody (Martin et al., 1994).c Non-selected minibody (Venturini et al., 1994).

The affinity-selected MBk is a potentIL-6 antagonist

Having established that MBk is a competitor ofhIL-6Ra in vitro, we decided to test its activity intissue culture. It is well established that hIL-6Raassociates with the signal-transducer moleculegp130 only upon binding to hIL-6, and that gp130does not show any binding activity for hIL-6 alone(Kishimoto et al., 1994). By binding to the putativeD-helix of hIL-6 (site 1), MBk inhibits interactionwith hIL-6Ra, hence it should also interfere with

formation of the ternary complex cytokine/recep-tor/gp130 and in turn with biological activity. Toverify this and determine the relative potency ofMBk with respect to MB02, we carried out a cellgrowth stimulation assay. We used a clone of theBAF cell line that expresses the mouse membrane-bound form of gp130 molecule and whose growthis dependent upon treatment with the hIL-6/hIL-6Ra soluble complex. Cell growth can be monitoredeasily by [3H]thymidine incorporation over 20 hourstreatment with the bioactive cytokine/receptorcomplex. Figure 9 shows that by co-incubation ofBAF cells with the hIL-6, hIL-6Ra and increasingamounts of inhibitor (up to the maximal concen-tration of MBk testable of 1 mM), we achieveddose-dependent inhibition of [3H]thymidine incor-poration with an IC50 of 770 nM. By contrast,co-incubation with MB02 yielded only 40% growthinhibition at maximal concentration. As a conse-quence of its binding activity for hIL-6, MBkinterferes with the formation of the complex withIL-6Ra, thereby blocking the signal transduction

Figure 4. Far-UV CD spectroscopy. Secondary structureof MB02 and MBk bearing the FLAG epitope wasmonitored by far UV CD spectroscopy. Spectra wererecorded as described in Materials and Methods. Theresults are expressed as mean residue ellipticity, [U]defined as 100Uobs/lc, where Uobs is the observedellipticity in degrees, c the concentration in residue molesper liter and l the light path-length in centimetres.

Figure 5. Direct binding of MB02 and MBk tosolid-phase immobilized hIL-6. The minibody-boundmolecules were detected using the anti-FLAG mAb M2and AP-conjugated antimouse as described in Materialsand Methods. Data are the average of triplicate samples.


Table 2. Kinetic values estimated by IAsys as describedin Materials and Methods

kass (M−1 s−1) kdiss (s−1)

MB02 1.0 × 103 2 120 7.3 × 10−4 2 3.0 × 10−5

MBk 5.4 × 103 2 315 1.2 × 10−3 2 7.8 × 10−5

Figure 7. Inhibition of hIL-6 binding to immobilizedhIL-6Ra in vitro. Engineered soluble hIL-6Ra was coatedonto ELISA wells and the cytokine binding wasdetermined in the presence of increasing amounts ofminibodies using mAb16. Data are the average of threeexperiments. MBns is the negative control.

mediated by gp130 engagement, and appears to bemore potent than MB02.

Discussion

Our choice to engineer biologically activeminibody-like proteins stems from the profoundunderstanding of the antibody V domain structure-function relationship accumulated over the past25 years. Antibody V domains are unrivalled in-struments for generating molecular diversity. Asimplified representation of these domains is sixpartially randomized constrained loops built into arigid b-pleated framework. By combining the rightamount of sequence randomization of the loopswith a reasonable degree of constraint to allow for‘‘induced fit’’ and alternative packing geometry oftheir V domains, antibodies optimize structuraldiversity and generate virtually all possible surfacesfor binding any antigen (Wilson & Stanfield, 1994).We felt that the reproduction of this extraordinarystructural diversity in a repertoire of smallerdesigned polypeptides such as minibodies wouldbe an important accomplishment. Previous bio-chemical characterization of minibodies provided

support for the structural model and suggested thatthe b-sheet framework could effectively serve asa scaffold for engineering minimal discontinuoussurfaces both by rational design (Pessi et al., 1993)and combinatorial strategies using phage display(Venturini et al., 1994).

From a phage displayed library of minibodies(5 × 107) in which the loop regions were random-ized we recently reported the affinity-selection ofMB02, a micromolar inhibitor of hIL-6 biological

Figure 6. Relative IC50 in solution phase. Microtiterwells coated with hIL-6 were incubated with 500 nM ofMB02 and MBk in the presence of increasing amount ofhIL-6 as soluble competitor. The minibody bound to theimmobilized ligand was revealed using the anti-FLAGmAb M2. Data are the average of triplicate samples.

Figure 8. Specificity and binding site mapping. (a) Thespecificity of minibodies was assessed by competitionassay carried out as for Figure 6 in the presence of afourfold molar excess of competitor proteins hCNTF andhOM. (b) Mapping of the binding site was achieved asdescribed above using a set of purified hIL-6 mutants insite 1 and site 2. Data are the average of triplicate samples.


Figure 9. Inhibition of hIL-6 biological activity in cellculture. hIL-6 growth-dependent BAF cells were treatedwith increasing concentration of minibodies for 20 hours.The cell-associated [3H]thymidine (incorporated countsper minute) are plotted on the y axis (average of triplicatesamples) as a function of inhibitor concentration.

signal transduction. These findings indicate that byoptimizing the ligand we have also considerablysharpened the binding specificity. One possibleexplanation for the improved specificity is thatMB02 primarily makes main-chain contacts with thetarget, whereas the optimized MBk recognizes moreside-chain atoms. Recent findings suggest thatin vitro affinity maturation of antibodies may leadto cross-reactive molecules (Casson & Manser,1995). Our results emphasize that careful choice ofthe selection scheme is required to overcome thesepotential drawbacks of in vitro affinity maturationapproaches. The data previously reported in studieson antibody repertoires on phage (reviewed byWinter et al., 1994) cannot be surmised as directlyapplicable to the minibody, given the designernature of the molecule and that three-dimensionalstructural data are not available.

Attempts to produce active antibody fragmentssmaller than Fv(s), such as functional single-domainantibodies, have been reported (Ward et al., 1989).The idea that single variable domains may besufficient for adequate binding affinity acquiredfurther support with the discovery that camelscan mount effective immune responses using anti-bodies consisting solely of heavy chains (Hamers-Casterman et al., 1993; Muyldermans et al., 1994).Davies & Riechmann (1994) have succeeded in im-proving the solubility of human VH domains bymutagenizing the interface of this region that isusually buried with the equivalent surface of the VL

domain, so as to mimic camel antibodies (camelizedantibodies), which have subsequently been used toconstruct a library by randomization of the CDR3.This repertoire has proved a good source of bindingsites for both haptens and large protein antigenswith affinities in the low micromolar range (Davies& Riechmann, 1995). Although the surface of theminibody is relatively small, it appears capable ofachieving both high-affinity binding and specificity,comparable with that of single-domain antibodyfragments.

Both MB02 (single-loop recombinant) and MBk(double-loop recombinant) and other loop mutants(unpublished results), show far-UV CD spectracompatible with b-pleated structure. This resultsupports our expectation that the minibodyframework is tolerant to sequence changes in theloop regions, as is its counterpart in the im-munoglobulin V domain. In addition to F29, anotherresidue, G26 (Kabat numbering) of the H1 loop,within VH domains is often conserved (Chothiaet al., 1989). We decided to maintain only F11(minibody numbering) invariant in the center of theputative loop because of its stabilizing role(Tramontano et al., 1994), and to change G8 in orderto sample alternative main-chain conformations.Careful thermodynamic studies will be required todetermine the effect of loop replacements on thestability of selected minibodies.

Target-specific minibodies could be very useful asresearch reagents and possibly for clinical purposes(Martin et al., 1994; Sollazzo, 1995): it is possible

activity (Martin et al., 1994). Being able to swap‘‘complete’’ repertoires of mutants allows for therapid optimization of the protein’s surface, a clearadvantage over combinatorial libraries consistingsolely of linear peptides (reviewed by Stemmer,1995). Here, we report the combination of loopswapping and selection strategies to carry throughthe sequence optimization of this minibodyantagonist of hIL-6 biological activity.

Because of our understanding of the targetmolecule structural-functional relationship (Savinoet al., 1994; Paonessa et al., 1995), we could establishexperimental conditions for high-stringency, epi-tope-targeted affinity-selection. We switched to amonovalent phagemid display scheme to avoidavidity effects as discussed before (Lowman et al.,1991). By using a mAb for immobilizing the targetmolecule we sought to reduce the selection ofbinders directed to non-native forms of hIL-6. At thesame time, the mAb16 ‘‘masks’’ one of the cytokinebinding sites for gp130 (Brakenhoff et al., 1994). Asimilar masking procedure was reported in theselection of epitope-specific antibody fragments(Ditzel et al., 1995). We used recombinant hOMduring the selection process to minimize cross-reac-tivity with molecules structurally related to thetarget. Conceptually equivalent schemes of ‘‘com-petitive phage selection’’ have been appliedsuccessfully by others (Dennis & Lazarus, 1994;deKruif et al., 1995).

The binding site mapping experiments suggestthat MBk binds to the C terminus of hIL-6, a regionthat embodies the site recognized by the IL-6Ra(site 1) as shown by mutagenesis and functionalstudies of hIL-6 (Leebeek et al., 1992; Savino et al.,1993). This result explains the antagonistic proper-ties of MBk, because by competing with IL-6Ra,this minibody interferes with the formation of thehigh-affinity complex with gp130 and thus with


to envisage applications such as targeting moietiesfor assembly of therapeutics like immunotoxins(Brinkmann et al., 1993), comtoxins (Varshavsky,1995) etc., although it is likely that the affinitiesachieved to date will not be sufficient for thesepurposes. There are several possibilities for im-proving the potency of these molecules: harnessingthe chelate effect (Neri et al., 1995) either bymultimerization or by conjugation with a secondpolypeptide moiety or a small organic molecule (i.e.hormones, synthetic ligands, etc.) that recognizeadjacent non-overlapping sites on the targetmolecule, could be one possibility. Another is tofurther optimize the surface sequence by targetinga third putative b-turn centered around S54 andQ55, or to extend each hypervariable loop by one ortwo residues. Recently, other groups have describedthe successful use of natural protein frameworkswith a similar intent of producing novel non-anti-body binding molecules suitable for therapy orbiotechnology applications (Bianchi et al., 1995; Ku& Schultz, 1995; McConnell & Hoess, 1995).

Materials and Methods

Microbiological and recombinant DNA techniques

Microbiological and recombinant DNA techniqueswere carried out according to standard protocols(Ausubel et al., 1995) or as recommended by suppliers.Electroporation of E. coli host cells was performed asdescribed (Dower et al., 1988). Oligonucleotides weresynthesised using an Applied Biosystems (Foster City,CA) 380B synthesizer. The PCR primers used were thefollowing from 5' to 3':

MB02up: GAGGTTGACACCACCGAGTACTMB02dwn: CTCTTTACCTCGCGAAGCAGCMB02f/1: AAACAACCATGGCTGCTAACTCCCAGGC-

GACMBH2DN: ATACATATGGCTAACTCCCAGGCGNotPIII: TATTAGCGGCCGCACGACCTTCGATGG.

Phage manipulations were carried out as described(Hoogenboom et al., 1991; Martin et al., 1994). Nucleotidesequences were determined using SequenaseTM (UnitedStates Biochemical Corp., Cleveland, OH) according to thesupplier’s recommendations. The gene encoding MBkwas retrieved from pHEN1 using the primers MBH2DNand NotPIII. The amplified fragment was digested withNdeI and PstI, then subcloned into the pT7 expressionvector (Moffat & Studier, 1987). High levels of minibodyexpression were achieved as described (Bianchi et al.,1994). Inclusion bodies were dissolved in TES buffercontaining 8 M urea and chromatographed underdenaturing conditions using the Pharmacia Superdex-75FPLC column. Fractions containing the minibody werediluted to 30 mg/ml protein concentration with 6 M ureaand re-folded by extensive dialysis against TBST buffer at21°C. After dialysis, the soluble material was concen-trated using Centriprep (Amicon, USA). Protein concen-tration was determined by amino acid analysis.

Library construction and affinity-selection

To construct the MB02-derived phagemid library weused a plasmid-borne minibody library in pUC8 con-

sisting of 7 × 107 H1 random mutants (Martin et al., 1994).Using inverse PCR (Hemsley et al., 1989) with the primersMB02up and MB02dwn, the wild-type H2 loop sequence(NKGNKY) was replaced with the GKEEVD peptidesequence derived from MB02. We obtained 2 × 108

independent colonies after electroporation of E. coliMC1061 cells. The plasmid pool was extracted and asecond PCR was performed to introduce NcoI and NotIrestriction sites at the 5' and 3' ends of the gene usingMB02f/1 and NotPIII primers. After enzymatic digestionof the PCR product the fragment pool was ligated intopHEN1 vector (linearized with NcoI/NotI) and electropo-rated. The complexity of the library was of 8 × 107 clones.Prior to selection, the phagemid library was transformedin TG1 (F') E. coli strain and super-infected with M13K07DgIII helper phage (a kind gift from Dr D. Chiswell,Cambridge Antibody Technology, U.K.) leading to theproduction of a multivalently displayed library. Thisphage library was used to infect TG1 cells which, in turn,were super-infected with M13K07 helper phage toproduce monovalently displayed phage clones thatpurged our library from those clones unable to expressintegral MB-pIII fusions. Dynabeads M-280 sheepanti-mouse IgG (Dynal) were coated with 90 pmol ofmAb16 (CLB) according to the supplier’s instructions. Asaturating amount of hIL-6 (400 pmol in 50 mM Trisbuffer, pH 7.4) was used for immobilization onto themAb16. After two hours incubation at room temperaturethe unbound hIL-6 was eliminated by five washes withPBS buffer. The beads were then blocked with 2% (w/v)BSA in TBST buffer and 4 × 1011 TU (transducting units)of library (in 100 ml of TBST containing 1% BSA) wereaffinity-selected for five hours at 23°C in the presenceof 900 pmol of hOM as a competitor. Subsequently, thebeads were washed ten times with TBST buffer andresuspended in 100 ml buffer. The suspension was loadedonto 700 ml of a 30% (w/v) sucrose cushion and the beadswere collected by applying a magnetic field (Dynal MPC).This step was repeated a second time before acidic elutionof the bound phage with 0.1 M Tris-glycine buffer(pH 2.2). Following neutralization and amplification inTG1 cells, the selection was repeated twice using the sameinput of CsCl-purified phage as in the first round. Adepletion step onto mAb16 coated beads was carried outafter each enrichment cycle to counter-select for phageinteracting with the matrix devoid of hIL-6. For thephage-ELISA experiment, the relative amount of mini-body expressed on the phage surface was normalized byWestern blotting using a rabbit anti-serum raised againstthe minibody molecule previously described (Bianchiet al., 1994).

ELISA direct binding

Microtiter wells coated with recombinant hIL-6(5 mg/ml) in 200 mM Tris (pH 8.0) were blocked with 3%non-fat milk in TBST buffer. Increasing amounts ofminibody were added on the plate in TBST/1% BSA andincubated for one hour at 23°C. To facilitate the detectionof minibody we genetically fused a double-strandedoligonucleotide encoding the peptide DYKDDDDK(FLAGTM) to the 3' end of the minibody gene; for this taga specific mAb (M2) is commercially available (IBI, USA).After washing, plates were incubated with anti-FLAGTM

mAb-M2 (1:3000) and the complex revealed withAP-conjugated anti-mouse Ab (1:7000). The AP activitywas determined with the soluble chromogenic substrateand the absorbance at 405 nm was recorded after 15 to 60minutes of incubation at 23°C.


ELISA binding competition

The ELISA competition experiments were performed asdescribed above except that the concentration ofminibodies was kept constant at 500 nM. Competitor wasadded in TBST/1% BSA buffer in a final volume of 100 ml.For the hIL-6 competition, increasing amounts of solublecytokine were added as shown in the legend to Figure 5.For the specificity assay, purified hCNTF, a gift from DrR. Laufer (IRBM), and hOM, a gift from Dr G. Paonessa(IRBM), were used at 2 mM concentration. The hIL-6mutants herein identified as: R (R179N) has been described(Fontaine et al., 1993; Savino et al., 1993); C was describedas Y31D/G35F/S118R/V121D by Savino et al. (1994); AB(Q75Y/S76I, Toniatti, unpublished result) and D (Q175I/S176R/Q183A) will be described elsewhere (Cabibbo et al.,1995). MBns is a non-selected minibody mutant (Table 1).Mutants were added at 2 mM concentration for themapping experiment. The results presented are theaverage of triplicate experiments.

Real-time interaction analysis

The determination of kass and kdiss for MBk and MB02was carried out using IAsysTM (FISONS, Cambridge,U.K.). One thousand arcs of hIL-6 (22 kDa) wereimmobilized onto the cuvette system using the NHScoupling kit supplied by the manufacturer. Fiveconcentrations of minibody (determined by amino acidanalysis) were used in duplicate for each bindingexperiment. Kinetic data were collected for association(five minutes) and for dissociation (15 minutes) at 21°C.The Fastfit (FISONS, U.K.) software (version 1.01) wasused to process the experimental kinetic data followingthe guidelines reviewed by O’Shannessy (1994).

CD spectroscopy

CD spectra were recorded using a Jasco J-710spectropolarimeter with a 16 second time-constant and a2 nm/minute scan speed using a 0.1 cm cell at 21°C anda peptide concentration of 24 mM and 5 mM for MB02 andMBk, respectively, in 10 mM Mops (pH 7) as described(Bianchi et al., 1994).

Receptor binding inhibition assay

Microtiter wells were coated with 100 ml of 50 mMNa2CO3 containing 5 to 10 mg/ml of soluble hIL-6Raproduced from a transfected Chinese hamster ovary cellline as described (Sporeno et al., 1994). Increasingconcentrations of competitors and 10 ng of hIL-6 in 100 mlof TBST buffer were directly added to the immobilizedreceptor and left for 45 minutes at room temperature.After washing, mAb16 was added (one hour at 23°C,1:3000) to detect the amount of IL-6 bound to the receptor.Anti-mouse AP-conjugated Ab (1:7000) was used fordetecting the bound mAb16.

IL-6 dependent cell-growth assay

BAFm130, a stable transfectant of mouse BAF cellsoverexpressing mouse gp130, was a generous gift fromDrs T. Kishimoto and T. Taga (Osaka University, Japan).Cells were maintained in RPMI 1640 medium sup-plemented with 10% (v/v) fetal calf serum and 10%WEHI-3B cells supernatant. To test the inhibitory activityof antibodies, cells were cultured in 96-well microtiterplates for 20 hours in the presence of 10 ng/ml of both

hIL-6 and shIL-6Ra, and of increasing concentrations ofminibodies. After incubation, cells were pulse-labelledwith [3H]thymidine (1.2 m Ci/well) for six hours andcollected onto glass filters. Cell-associated (incorporated)radioactivity was measured using a TopCount MicroplateScintillation Counter (Packard). The results were normal-ized using the same concentration of negative control,MBns (this value was considered as 100% of incorpor-ation).

AcknowledgementsThe authors are grateful to Drs G. Paonessa, R. Laufer,

A. Lahm and A. Tramontano for their comments. Wethank A. Lahm for helping with the molecular graphics;P. Neuner for the synthesis of oligonucleotides; E. Bianchiand S. Venturini for their help with the CD spectroscopy;C. Volpari for technical assistance; and J. Clench andB. McManus for reviewing the manuscript.

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Edited by J. Karn

(Received 7 August 1995; accepted 10 October 1995)

coupling protein design andin vitroselection strategies: improving specificity and affinity of a...

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