CDR Walking Mutagenesis for the Affinity …. Mol. Biol.(1995) 254, 392–403 CDR Walking...

J. Mol. Biol. (1995) 254, 392–403

CDR Walking Mutagenesis for the Affinity Maturationof a Potent Human Anti-HIV-1 Antibody into thePicomolar Range

Wei-Ping Yang 1, Kimberly Green 1, Sally Pinz-Sweeney 1

Amelia T. Briones 1, Dennis R. Burton 1,2 and Carlos F. Barbas III 1*

We describe the investigation of methodologies for the creation of very high1Department of Molecularaffinity human antibodies. The high affinity human antibody b4/12 wasBiology, The Scripps Researchoptimized for its affinity to the human envelope glycoprotein gp120 ofInstitute, 10666 North Torrey

Pines Road, La Jolla, CA human immunodeficiency virus type 1 (HIV-1). Five libraries of b4/1292037, USA were constructed by saturation mutagenesis of complementarity-determin-

ing regions (CDRs). Libraries of antibody Fab fragments were displayed on2Department of Immunology the surface of filamentous phage and selected in vitro for binding toThe Scripps Research immobilized gp120. Sequential and parallel optimization strategies ofInstitute, 10666 North Torrey CDRs were examined. The sequential CDR walking strategy consistentlyPines Road, La Jolla, CA yielded b4/12 variants of improved affinity in each of the four different92037, USA optimization sequences examined. This resulted in a 96-fold improvement

in affinity. Additivity effects in the antibody combining site were exploredby combining independently optimized CDRs in the parallel optimizationstrategy. Six variants containing optimized CDRs were constructed.Improvement of affinity based on additivity effects proved to beunpredictable but did lead to a modest improvement in affinity. Indeed,only one of the six combinations demonstrated additivity. The highestaffinity Fab prepared using this strategy was improved 420-fold in affinity.The affinity of this Fab was 15 pM as compared to 6.3 nM for b4/12.Examination of the kinetics of Fab binding to gp120 revealed thatimprovements in affinity were dominated by a slowing of the off-rate ofthe Fab. The methodology presented here provides a route for theimprovement of the affinities of antibodies typical of tertiary immuneresponses into the picomolar range. Such improvements may haveprofound effects on the utility of antibodies as therapeutic and prophylaticagents.

7 1995 Academic Press Limited

Keywords: phage display; human immunodeficiency virus;structure/function; passive immunization; additivity effects*Corresponding author

Introduction

Antibodies represent the most molecularly di-verse and structurally studied family of proteins(Padlan, 1994). Their therapeutic use in humans hasbeen studied for over a hundred years (Behring,1893) and their role as adaptable binding moleculesof defined specificities has aided the flowering ofmolecular biology in the latter half of this century.

Hence, antibodies play a central role in basic scienceand in our own survival. Our interest has been todevelop a more thorough understanding of anti-bodies through the purposeful manipulation oftheir affinity and specificity.

The current HIV-1 pandemic demands thedevelopment of potent anti-viral molecules. In thisregard we have utilized phage display of combina-torial antibody Fab fragment libraries to select alarge number of human anti-HIV-1 antibodies(Barbas et al., 1993a; Burton & Barbas, 1994). Themost promising of these is antibody b4/12, whichwhen prepared as an IgG1 has been demonstratedto efficiently neutralize a large number of primary

Abbreviations used: CDR, complementarity-determining region; HIV-1, human immunodeficiencyvirus type 1; gp, glycoprotein; sCD4, soluble CD4,PCR, polymerase chain reaction; Ig, immunoglobulin.

0022–2836/95/480392–12 $12.00/0 7 1995 Academic Press Limited

Affinity Maturation of a Human Antibody 393

isolates of HIV-1 (Burton et al., 1994). To improve thelikelihood that this molecule could succeed inprophylactic and therapeutic application we havedeveloped methods for the improvement of itsaffinity. A number of methods and reports havebeen published which are devoted to the improve-ment of antibody affinity (for a review, see Burton& Barbas, 1994). We have sought methods for thedevelopment of very high affinity antibodieswherein modifications to the parental antibody areconstrained to the complementarity determiningregions. Changes in primary sequence in theseregions are less likely to generate immunogenicantibodies than changes in the more sequence-con-strained framework regions. In a previous reportwe improved the affinity of b4/12 for gp120 abouteightfold and demonstrated that affinity wascorrelated with neutralization potency (Barbas et al.,1994).

In this report we extend and develop ourpreliminary examination of the CDR walkingmutagenesis strategy. Sequential and paralleloptimization strategies have been explored andthe general applicability of additivity effects in thecombining site examined. We have improved theaffinity of Fab b4/12 420-fold in its affinity forthe HIV-1 envelope protein gp120. This studysuggests a route to the creation of very high affinityantibodies.

Results

Design of antibody libraries

No specific structural information on the anti-body b4/12 or its antigen gp120 was available toguide the design of Fab libraries. Antibodies,however, constitute one of the most structurallystudied classes of proteins (Padlan, 1994; Wilson &Stanfield, 1993). General information regarding thechemistry of this class of proteins, together withprevious results obtained by chain shufflingexperiments with this antibody, has served to guidethe experiments under consideration here (Barbaset al., 1993b). Five segments within four CDRs weretargeted for saturation mutagenesis and affinityoptimization using phage display. Within the heavychain, the entire CDR1-H region consisting ofresidues H31 to H35 was targeted in library H1(numbering convention of Kabat et al. (1991)). TheCDR3-H region of b4/12, 18 amino acid residueslong, is significantly longer than the averageobserved in human antibodies, which is approxi-mately 12 residues (Wu et al., 1993). Due to technicallimitations in the size of libraries which may beconstructed and surveyed with confidence using thephage display approach, six codons or less aregenerally targeted for saturation mutagenesis usingNNK or NNS doping strategies (Lowman & Wells,1993; Barbas & Barbas, 1994). For this reason, thenine residues at the core of the VDJ fusion regionwere targeted in a two-step sequential optimization

strategy; H97-100 and H100A-E. Chain shufflingexperiments suggested this region to be a naturalhotspot in the somatic evolution of this antibody(Barbas et al., 1993b). In previous studies, asequential selection of library H1 followed byrandomization and selection of residues H97-100 ofCDR3-H yielded a clone designated 3B3 with a8.2-fold improvement in affinity (Barbas et al., 1994).As detailed below, the increase in affinity of the 3B3clone was due almost exclusively to optimization ofthe H97-100 region. In this report, the adjacentresidues in CDR3-H, H100A-E, were chosen foroptimization in the context of the clone 3B3; libraryH3B3. In the light chain, CDRs 1 and 3 weretargeted for random mutagenesis. Interaction ofCDR2-L with antigen is, in general, less frequentand was not considered for optimization here(Wilson & Stanfield, 1993). In CDR1-L six residuespredicted to be solvent exposed, L27A to 32, weretargeted for random mutagenesis in library L1. InCDR3-L residues L90, and L92 to 95 were targetedfor randomization. Residue L91 which is a highlyconserved tyrosine in light chains in general (Wuet al., 1991) and in b4/12 variants obtained by chainshuffling and was not targeted. The CDRs whichhave been targeted are four of the five most heavilyutilized contact regions observed in structures ofantibody–antigen complexes (Padlan, 1994; Wilson& Stanfield, 1993). These regions are predicted toform a significant portion of the core of the bindingsurface of the antibody. This is demonstrated inFigure 1 by highlighting the regions underconsideration on the combining site of the humanantibody Kol, for which a crystal structure has beendetermined (Marquart et al., 1980).

Sequential optimization of CDRs

To assess the sequential CDR walking strategy,four sequential CDR optimization experiments wereperformed. Selected clones were characterized bysequencing of the entire variable domain anddetermination of affinity of purified Fab for antigenusing the BIAcore biosensor from Pharmacia(Karlsson et al., 1991). The first of these experimentsextends the previous two-step sequential walkwhich targeted CDR1-H in the H1 library andresidues H97 to 100 of CDR3-H (Barbas et al., 1994).The highest affinity clone that resulted from thesestudies was clone 3B3. The subsequent step was toCDR1-L in library L1. Library L1 consisted 5 × 108

independent variants in this region built within thecontext of the 3B3 heavy chain. Following fourrounds of selection, eight clones were sequencedfollowing verification of binding activity with thesoluble protein. Five clones were identical to thestarting clone at the nucleic acid level and resultedfrom contamination of the original sequence in theconstruction of the library. Contamination was at alow level since sequencing of ten unselectedvariants revealed ten unique sequences. Theaffinities of the three remaining unique clones

Affinity Maturation of a Human Antibody394

Figure 1. The tube structure of aFab on which the regions targetedfor optimization have been high-lighted. The antibody Kol has beenutilized as a model here because ofits extended CDR3-H region whichis similar in length to antibodyb4/12 under consideration here. Thelight chain variable region is shownon the left in pink and the heavychain variable region on the right inblue. The individual regionstargeted within the CDRs have beencolor coded with the key given inthe Figure. This view highlights therelative disposition of the CDRregions targeted for optimization.Molecular models were constructedby Mike Pique using AVS software.

3B3/L1.1-3 were characterized. The clones ascompared to their parent 3B3 were found to be oflower or similar affinity (Table 1). Selection was thencontinued for an additional four rounds resulting inthe isolation of a single clone following thesequencing of ten clones, 3B3/L1.4. This clone wasimproved 29-fold in affinity with respect to b4/12and 3.5-fold with respect to 3B3 (see Table 1). In thenext step, five residues within the CDR3-L of3B3/L1.4 were mutagenized and a library of 2 × 108

variants was constructed; library L3A. Followingfive rounds of selection, nine clones were character-ized by sequence analysis and affinity determi-nation (Table 2). Each clone contained a uniqueCDR3-L sequence and an affinity for antigen thatwas generally lower than the parent clone of thisseries, 3B3/L1.4. To improve the selection of higher

affinity variants in the sub-nanomolar range usinga solid phase selection format, an off-rate biasedselection was developed. In five subsequent roundsof selection, 8 mM 3B3 was added to the selectionwell following the washing step. The selection platewas then placed at 37°C for three hours, the wellwashed, and bound phage eluted at low pH. Thisselection resulted in the isolation of clones3B3/L.14L3.10 to 12. These clones differ in sequenceonly at position L95. Each clone was improved inaffinity for antigen. The highest affinity clone3B3/L1.4L3.11 was improved 3.3-fold in affinityabove 3B3/L1.4 and 96-fold above b4/12. This cloneis the product of a four-step sequential CDR walk.A parallel experiment without off-rate selectionwith antibody revealed a multitude of clones similarto that seen following the initial five rounds of

Table 1. Light chain CDR1 mutants isolated from the L1 library and by chain shufflingResidue position Kd (parent)

Kd (mutant)Kd (b4/12)Kd (mutant)Clone 27A 28 29 30 31 32

b4/12 S I R S R R 1 —3B3 S I R S R R 8.2 8.23B3/L1.1 K E F G R R 0.16 1.33B3/L1.2 T V Y R D R 0.7 5.73B3/L1.3 P L H R A R 1.1 9.03B3/L1.4 Q L D G S R 3.5 29CS N I R S R R NA NDCS R I S S R R NA NDCS R I G S R R NA NDCS N I W S R R NA ND

The clone designated 3B3 has been described. 3B3 contains mutations in CDR1-H and CDR3-Honly. Clones isolated from the L1 library contain the L1 designation in their name and in theexamples are paired with the heavy chain from 3B3. Dissociation constants were determined withthe BIAcore instrument from Pharmacia. The ratio of parent and mutant Kd values gives the foldincrease in affinity for this mutagenesis step which is attributed to changes in this region. The ratioof Kd b4/12 and mutant Kd gives the overall improvement in affinity from the starting Fab b4/12.Clones isolated from chain shuffling experiments have been described and characterized (Barbaset al., 1993b) and are given the CS designation. The CS clones contain multiple mutations in thelight chain variable region paired with the original heavy chain of b4/12 and are of an affinitysimilar to b4/12. For these CS clones ratios of Kd values were not applicable (NA) and were notdetermined (ND) with the BIAcore instrument.


Table 2. Light chain CDR3 mutants isolated from L3 libraries and by chain shufflingResidue position Kd (parent)

Kd (mutant)Kd (b4/12)Kd (mutant)Clone 89 90 91 92 93 94 95 96 97

b4/12 Q V Y G A S S Y T 1 13B3/L1.4 Q V Y G A S S Y T 3.5 293B3/L1.4L3.1 Q V Y G W S Q Y T 0.43 123B3/L1.4L3.2 Q L Y G R G N Y T 0.52 153B3/L1.4L3.3 Q T Y G R G V Y T 0.18 5.23B3/L1.4L3.4 Q T Y G W S G Y T 0.49 143B3/L1.4L3.5 Q S Y G G R D Y T 0.44 133B3/L1.4L3.6 Q K Y G D S F Y T 1.0 293B3/L1.4L3.7 Q M Y G G R D Y T 0.05 1.53B3/L1.4L3.8 Q M Y G G F T Y T 0.36 103B3/L1.4L3.9 Q Q Y G D S L Y T 0.50 153B3/L1.4L3.10 Q T Y G R G S Y T 3.0 873B3/L1.4L3.11 Q T Y G R G H Y T 3.3 963B3/L1.4L3.12 Q T Y G R G I Y T 1.7 493B3/L3.13 Q V Y G A S S Y T 1 8.23B3/L3.14 Q Q Y G W P F Y T 5.6 463B3/L3.15 Q V Y G G S A Y T 0.23 1.93B3/L3.16 Q K Y G G G T Y T 3.2 26CS Q T Y G G S S Y T NA NDCS Q T Y G G A T Y T NA NDCS Q T Y G G S S Y S NA ND

Clones containing CDR1-L mutations contain the L1 designation in their name and in the examples above are paired with the heavychain from 3B3. Clones containing CDR3-L mutations contain the L3 designation in their name. Dissociation constants were determinedwith the BIAcore instrument from Pharmacia. The ratio of parent and mutant Kd values gives the fold increase in affinity for thismutagenesis step which is attributed to changes in this region. The ratio of Kd b4/12 and mutant Kd gives the overall improvementin affinity from the starting Fab b4/12. Clones isolated from chain shuffling experiments have been described and characterized (Barbaset al., 1993b) and are given the CS designation. The CS clones contain multiple mutations in the light chain variable region pairedwith the original heavy chain from b4/12 and are of an affinity similar to b4/12. For these CS clones ratios of Kd values were notapplicable (NA) and were not determined (ND) with the BIAcore instrument.

selection, indicating the increased efficacy of theoff-rate selection procedure. Previously, an antigen-based off-rate selection was reported by Hawkinset al. (1992). This technique was not considered heredue to the very high cost of the gp120 antigen.

The second sequential walk involved CDR1-H toCDR3-H to CDR3-L steps. In this experiment,4 × 108 variants in CDR3-L were produced, pairedwith the heavy chain from 3B3; library L3B.Following the selective protocol utilized for the L3Alibrary, four unique clones were characterizedfollowing sequencing. Clone 3B3/L3.14 was im-proved 5.6-fold in affinity as compared to 3B3 and46-fold as compared to b4.12. This clone was thepredominant clone obtained after selection asrevealed by sequencing.

The third sequential CDR walk involved CDR1-Hto CDR3-H97-100 to CDR3-H100A-E. This involvedthe construction of library H3B3 wherein 1 × 108

variants in the H100A-E region were generated inthe context of clone 3B3. Following a selectiveregime involving antibody assisted off-rate selec-tion, seven clones of highly related sequence wereisolated and characterized (Table 3). All but oneclone was improved in affinity for antigen, with thehighest affinity clone h1.3B/h3.33 being improved7.7-fold as compared to 3B3 and 63-fold ascompared to b4/12.

The last CDR walk involved a re-investigation ofthe single step walk to CDR1-H. Previously, libraryH1 had been generated and selected to yield asub-library of functional CDR1-H variants of b4/12.

The individual clones were not characterized andwere utilized as a collection in subsequentmutagenesis of CDR3-H generating among others,clone 3B3 (Barbas et al., 1994). The sequence atposition H31 to H33 of 3B3 was Asn-Phe-Thr. Thissequence generates a potential glycosylation site inCDR1-H. To circumvent this sequence, as well as toidentify a more optimal sequence in CDR1-H,library H1 was selected for an additional six roundsof panning beyond that previously reported. Theresults of this selection are given in Table 4. Cloneslacking the potential glycosylation site werecharacterized. Clone h1.1 exhibited a 3.9-foldimprovement in affinity for antigen.

Analysis of selected CDRs

Analysis of CDR1-H variants shown in Table 4demonstrated a strong selection towards a consen-sus sequence. H31 was exclusively His or Asn,suggesting a functional role for the conservation ofthe d-amino group, which is a shared element in theside chains of these residues. H32 was exclusivelyan aromatic residue, most frequently Phe. H33 wasexclusively Thr. CDR1-H of b4/12 is predicted tohave a class 1 canonical structure. It contains the keyresidues which define this class: Ala at 24, Gly at 26,Tyr at 27, Phe at 29, Ile at 34, and Arg at 94 (Chothiaet al., 1989, 1992). Interestingly, selection at H34revealed Ile, Leu, or Val, all of which fall withinthose described for a class 1 structure. Position H35revealed a selection towards the parental residue


Table 3. Heavy chain CDR3 mutants isolated from the H3B3 library and by chain shufflingResidue position Kd (parent)

Kd (mutant)Kd (b4/12)Kd (mutant)95 96 97 98 99 100 A B C D E F G I J K 101 102

b4/12 V G P Y S W D D S P Q D N Y Y M D V 1 1h1.3B/h3.3(3B3) V G E W G W D D S P Q D N Y Y M D V ND 8.2h3.3 V G E W G W D D S P Q D N Y Y M D V 7.9 7.9h1.3B/h3.32 V G E W G W E Q F R F D N Y Y M D V 1.8 15h1.3B/h3.33 V G E W G W E M F R Y D N Y Y M D V 7.7 63h1.3B/h3.34 V G E W G W E M R R F D N Y Y M D V 5.9 48h1.3B/h3.35 V G E W G W H Q R R Y D N Y Y M D V 5.6 46h1.3B/h3.36 V G E W G W D Q R R Y D N Y Y M D V 0.43 3.5h1.3B/h3.38 V G E W G W T Q R R F D N Y Y M D V 4.4 36h1.3B/h3.39 V G E W G W D Q V R Y D N Y Y M D V 5.5 45CS V G E W T W D D S P Q D N Y Y M D V NA NDCS V G E W T W D D F P Q D N Y Y M D V NA NDCS V G E W T W D M D P Q A N Y Y M D V NA NDCS V G P Y T W D D S P Q D N Y Y M D V NA ND

Heavy chain CDR1 mutants contain the h1 designation in their name and in the examples above all Fabs contain the original lightchain from b4/12. Clones containing CDR3-H mutations contain the h3 designation in their name. Clone h3.3 was created by swappingthe mutant h1.3B CDR1-H region of 3B3 with the original CDR1-H region of b4/12. Dissociation constants were determined with theBIAcore instrument from Pharmacia. The ratio of parent and mutant Kd values gives the fold increase in affinity for this mutagenesisstep which is attributed to changes in this region. The ratio of Kd b4/12 and mutant Kd gives the overall improvement in affinity fromthe starting Fab b4/12. Clones isolated from chain shuffling experiments have been described and characterized (Barbas et al., 1993b)and are given the CS designation. The CS clones contain multiple mutations in the heavy chain variable region paired with the originalb4/12 light chain and are of an affinity similar to b4/12. For these CS clones ratios of Kd were not applicable (NA) and were notdetermined (ND) with the BIAcore instrument.

His at this position. Correlation of affinity withsequence suggests that His at H31 is a key elementin the increased affinities of clones h1.1-4. CDR1-Hof h1.1 and h1.3B differ in sequence only at positionH31 but differ by approximately fourfold in affinity.Variation in the aliphatic side-chains of the residueat H34 had little effect on the affinity of these clones.

Previous analysis of CDR3-H variants derived byselection of the H97 to 100 region suggested the Pro

to Glu change to be associated with the higheraffinity of clone 3B3. The CDR1-H segment of 3B3contributes little to the increase in affinity observedin 3B3. This is demonstrated by swapping theCDR1-H region of b4/12 for that of 3B3. Theresulting clone h3.3 bound with approximatelythe same affinity as 3B3 (Table 3). Sequence analysisof the H100A to E region selected in library H3B3reveals a set of highly related clones. PositionH100A was predominately Glu or Asp. H100B wasGln or Met. Met at this position was observed in aclone derived by chain shuffling. H100C waspredominately Arg, but Phe and Val were alsoobserved. Phe had been observed at H100C in aclone derived by chain shuffling. Position H100Dwas exclusively Arg and H100E was either Phe orTyr. Within this set of clones are several related bysingle point changes. Clone h1.3B/h3.35 andh1.3B/h3.36 differ only at H100A, which is His andAsp in the respective clones. They differ in affinity,however, by 13-fold. The affinity can be completelyrestored as shown in clone h1.3B/h3.39 with themutation of Arg at H100C to Val. This resulthighlights the interdependence of residues in thisregion.

CDR1-L sequences shown in Table 1 indicate thata number of the targeted positions accept a widerange of residues with little effect on affinity.Compare 3B3/L1.2 and 3B3/L1.3. Conservation ofArg at L32 is noted in b4/12 and all variants. Chainshuffling had produced variants with Asn or Arg atL27A and Ser, Gly, or Trp at L29. None of thesemutations is noted in the variants obtained bysaturation mutagenesis. The highest affinity variantdiffers from the parental sequence in five of the sixselected regions with a significant change in the netcharge of this region from +3 to 0. CDR1-L of b4/12and selected variants do not possess sequences

Table 4. Heavy Chain CDR1 mutants isolated from the H1library and by chain shuffling

Residue position Kd (b4/12)Kd (mutant)Clone 31 32 33 34 35

b4/I2 N F V I H 1h1.1 H F T V H 3.9h1.3 H F T L H 3.2h1.4 H F T I M 2.6h1.2 N Y T L Q NDh1.5 N F T L I NDh1.6 N W T I M NDh1.3B/h3.3(3B3) N F T V H 8.2CS N F T V H NDCS N F T V H ND

Heavy chain CDR1 mutants contain the h1 designation in theirname and in the examples above all Fabs contain the originallight chain from b4/12. Clones containing CDR3-H mutationscontain the h3 designation in their name. Dissociation constantswere determined with the BIAcore instrument from Pharmacia.The ratio of Kd b4/12 and mutant Kd gives the overallimprovement in affinity from the starting Fab b4/12 and in allcases except clone 3B3 gives the fold increase in affinity for thismutagenesis step which is attributed to changes in this region.Clones isolated from chain shuffling experiments have beendescribed and characterized (Barbas et al., 1993b) and are giventhe CS designation. The CS clones contain multiple mutations inthe heavy chain variable region paired with the original b4/12light chain and are of an affinity similar to b4/12. For these CSclones ratios of Kd were not determined (ND) with the BIAcoreinstrument. Clones containing the potential glycosylation siteAsn-X-Thr were not characterized with the exception of 3B3.


Table 5. Binding kinetics of b4/12, b4/12 mutants and sCD4kon/104 koff/10−4 Kd (b4/12)

Kd (mutant)Clone (M−1 s−1) (s−1) Kd (nm) DDGI (kcal/mol)

sCD4 1.7 2 0.03 1.0 2 0.007 5.9 NAb4/12 7.6 2 0.02 4.8 2 0.02 6.3 1h1.1 14 2 1.0 2.3 2 0.03 1.6 3.9h1.3Bh3.33 7.0 2 0.8 0.071 2 0.006 0.10 63h1.1h3.33 15 2 2.6 0.034 2 0.001 0.022 286 0.094h1.1h3.33/L1.4L3.14 7.8 2 0.7 0.012 2 0.002 0.015 420 −1.54h1.1h3.33/L1.4L3.11 9.2 2 0.9 0.090 2 0.007 0.097 65 −2.33h1.1h3.33/L3.14 7.0 2 0.1 0.083 2 0.005 0.12 53 −2.02L1.4L3.14 12 2 1.1 4.1 2 0.02 3.4 1.9 −1.39L1.4L3.11 14 2 1.2 4.2 2 0.02 3.9 1.6 −1.17

Binding kinetics were determined using the BIAcore instrument from Pharmacia. The envelopeglycoprotein gp120 was immobilized on the biosensor chip. The Kd value was calculated from koff/kon. Theratio of Kd values indicates the fold increase in binding affinity as compared to the Fab b4/12. DDGI wascalculated using equation (1) and DDG as + RT ln(Kdb4/12/Kdmutant).

which fall within those described for classificationof this region into a canonical structure class(Chothia et al., 1989).

CDR3-L sequences demonstrated a strikingselection for Gly at L92 in all 16 characterizedmutants (Table 2). This position was conserved in allmutants derived by chain shuffling. L93 waspredominately Gly or Arg. Gly at this position wasthe predominant mutant obtained by chain shuf-fling. Other positions were more permissive. Fourclones are related by amino acid changes at a singleposition, L95. Clone 3B3/L1.4L3.3, which has Val atthis position binds gp120 with an affinity 18-foldlower than 3B3/L1.4L3.11, which has His at thisposition. Conversion of His to either Ser or Ile in3B3/L1.4L3.10 and 3B3.L1.4L3.12 has little effect onaffinity. This highly related set has Thr at positionL90, which was the only mutant at this positionobtained by chain shuffling. With respect tocanonical structures predicted for this region, theparental antibody and all variants except 3B3/L3.14do not contain conformation defining residues of theappropriate identity required for classification.Selection for Gln at L90 and Pro at L94 in this cloneclassifies this as a class 2 canonical structure(Chothia et al., 1989). This was the highest affinityclone selected in the CDR1-H to CDR3-H toCDR3-L walk and differs radically in sequence from3B3/L1.4L3.11, which was the optimal cloneselected in the context of the optimized CDR1-Lregion L1.4.

Investigation of additivity effects in theantibody combining site

An alternative CDR walking strategy involves aparallel optimization strategy wherein CDRs areindependently optimized and recombined in asingle clone. This strategy is based on theassumption that in most cases additivity will beobserved when non-interacting mutations arecombined (Wells, 1990). To assess the applicabilityof additivity principles to the antibody combining

site, six new antibodies were created by shufflingoptimized CDR regions. The resulting clones wereexpressed, purified, and the affinity for antigendetermined. The results are shown in Table 5.Combination of the most optimal CDR3-H regionwith the most optimal CDR1-H region producedclone h1.1h3.33, which was improved 286-fold in itsaffinity for antigen as compared to b4/12. In thiscase the measured affinity is in good agreementwith that predicted assuming additivity; 286-fold ascompared to 246. Recruitment of CDR1-L andCDR3-L segments L1.4 and L3.14 into this cloneresulted in only a 1.5-fold increase in affinity, seeclone h1.1h3.33/L1.4L3.14. These segments yielded3.5 and 5.6-fold increases in affinity, respectively, inthe context of the antibody in which they wereoriginally selected. If simple additivity had beenoperative, a 19.6-fold improvement in affinityrelative to h1.1h3.3 would have been observed andan antibody with a 5600-fold improvement inaffinity would have resulted. These two light chainCDRs were the products of independent selections.To investigate the source of the discrepancybetween the observed and predicted results, cloneh1.1h3.3/L3.14 was constructed. This clone intro-duces only a single optimized CDR3-L region. A5.5-fold decrease in affinity was observed ascompared to clone h1.1h3.33. This result demon-strates an incompatibility of the L3.14 CDR3-Lregion with the h1.1h3.33 heavy chain. In cloneh1.1h3.33/L1.4L3.11, the light chain containing thesequentially optimized CDR1 and CDR3 regionsL1.4 and L3.11 was combined with the h1.1h3.33heavy chain. The resulting clone demonstrated anaffinity 4.4-fold lower than that observed forh1.1h3.3 paired with the original light chain derivedfrom b4/12. Simple additivity would have pro-duced a 12-fold increase in affinity. The resultsobtained by combining optimized light chainregions with heavy chain variants are unpre-dictable. Combination of L1.4L3.14 and L1.4L3.11light chains with the original heavy chain of b4/12resulted in approximately a twofold increase inaffinity in each case.


Changes in the binding kinetics of improvedantibodies

Insight into the mechanism of affinity improve-ment is provided by determination of the bindingkinetics using the BIAcore instrument. As shown inTable 5, improvements in affinity are dominated bydecreases in off-rates. Only a twofold variation inon-rate was observed compared to a 400-foldvariation in off-rate. This trend was maintained inthe 34 additional clones reported in Tables 1 to 4(data not shown). Off-rate is expected to be mostaffected if phage selections are performed underconditions approaching equilibrium (Lowman &Wells, 1993). The kinetics of soluble CD4 (sCD4)binding to gp120 were determined for comparativepurposes. CD4 is the natural ligand for gp120. Theaffinity determined by BIAcore is in close agree-ment with the reported value of 3 nM (Ryu et al.,1990) This measurement serves to correlate theaffinities reported here with those determined byother methods. The apparent Kd value of Fab b4/12as determined by competition ELISA is 9 nM(Barbas et al., 1992b). Affinity improvementsdetermined with the BIAcore instrument aregenerally well correlated with those determinedwith other methods (Lowman & Wells, 1993; Kelley& O’Connell, 1993). Antibody binding kineticscompare favorably with sCD4. Fab on-rate wasfaster in all cases than that observed for sCD4. Theoff-rate of sCD4 is 4.8-fold slower than b4/12 and83-fold faster than the highest affinity Fabh1.1h3.33/L1.1L3.14. Since small quantities ofaggregated protein can lead to artifactually slowoff-rates (van der Merwe et al., 1993), thedissociation of Fabs b4/12 and h1.1h3.33 werestudied following gel filtration chromatography. Inboth cases the off-rates determined for theseproteins were within the values determined for theproteins that had not been subjected to thisadditional purification step. In an additional studythe binding kinetics of Fab h1.1h3.33 binding togp120 were determined in the reverse orientation,i.e. with Fab immobilized and gp120 as the solubleligand. The on-rate of this interaction was 2.8-foldslower and the off-rate was 1.3-fold faster than thatdetermined with gp120 immobilized. This variationis within the range expected since it is difficult tomatch the densities and orientations of the proteinswhich are immobilized. Thus, the differences inoff-rates reported herein are not artifacts resultingfrom protein aggregation.

Discussion

Antibody b4/12 represents the most potent andbroadly neutralizing human anti-HIV-1 antibodyyet described (Burton et al., 1994). This antibody hasbeen demonstrated to neutralize 75% of 36 primaryisolates of HIV-1 tested at concentrations whichcould be achieved by passive immunization. Thisextends earlier work that demonstrated potentneutralization of laboratory adapted isolates of

HIV-1 with monovalent Fab of this antibody (Barbaset al., 1992b). The Fab fragment of b4/12 wasoriginally isolated from a phage display libraryprepared from an individual who had been HIV-1positive for six years but had no symptoms ofdisease. The antibody binds a highly confor-mational epitope in the CD4-binding region of theHIV-1 envelope glycoprotein gp120. The epitopewithin the CD4 binding region of gp120 recognizedby b4/12 is unique as compared to five otherantibodies directed against this region and isolatedfrom the same individual (Roben et al., 1994).

To potentially improve the therapeutic or prophy-lactic efficacy of this antibody, we investigatedstrategies for the evolution of its affinity for antigen.Phage display provides a convenient format for therefinement of the contact between receptor andligand or antibody and antigen which may resultfrom unpredictable sequence changes in the regionof interest. The limitations of the phage displayapproach have been considered elsewhere (Low-man & Wells, 1993; Barbas & Barbas, 1994). We haveproposed that repeated introduction of diversityinto CDRs followed by stringent selections shouldallow for the refinement of human antibodies tolevels of affinity far beyond those generated by theimmune response (Barbas et al., 1994). CDRtargeted mutagenesis is advantageous since optim-ization of these regions is most likely to improveaffinity and least likely to create problems ofimmunogenicity. CDR walking involves targetedmutagenesis of CDR regions and selection forfitness with monovalent phage display (Barbas et al.,1994).

The free energy changes that result from thecombination of sets of mutations may be expressedby the equation:

DDGAB = DDGA + DDGB + DDGI (1)

where DDGA and DDGB are changes in the freeenergy of mutants A and B, DDGAB is the change infree energy for the protein that combines themutations of A and B, and DDGI is the change in theinteraction energy between the two sets of residues(after Lowman & Wells, 1993). In accord withequation (1), two strategies are evident for theapplication of CDR walking, either sequential orparallel optimization of CDRs. The most appropri-ate route to the optimization of antibody affinity isdependent on the term DDGI. In this study we haveinvestigated these two CDR walking strategies.

Sequential CDR walking takes into account thatDDGI may not always be negligible and that optimalbinding may result from the interdependence ofCDR loops. Such interdependence could result fromcoordinated structural changes on binding antigenand is supported by recent evidence which suggestsinduced-fit mechanisms may best describe thebinding observed in some antibody–antigen com-plexes (Wilson & Stanfield, 1993). Together with aprevious study we have performed five sequentialCDR walking experiments. In the previous study alibrary of CDR1-H variants, H1 library, with five


residues randomized was selected by panningagainst gp120. The collection of clones whichresulted was then used in the construction of aCDR3-H library where four additional residueswere randomized. The resulting library wasselected by an additional six rounds of panning. Avariety of clones with improved binding wereproduced. Correlations within this set of selectedprogeny were examined to establish functionaltrends which may be predictive of the activity ofsubsequent generations of b4/12 (Barbas et al.,1994). With the gp120 envelope protein derivedfrom the MN isolate of HIV-1, affinity was wellcorrelated with neutralizing ability. Correlation ofaffinity with neutralizing ability in the HIV-1system is very dependent on the epitope recognized(Roben et al., 1994) and has been observedelsewhere (Nakamura et al., 1993). A 54-foldimprovement in neutralizing potency was deter-mined for the highest affinity clone 3B3. The abilityof these evolved monovalent Fabs to neutralize withpotencies equivalent or better than sCD4 is unique.In a recent multi-center study of human and mouseanti-HIV-1 antibodies, no bivalent antibody hasdemonstrated such potency (D’Souza et al., 1994).Furthermore, four primary clinical isolates of HIV-1not neutralized by the parent b4/12 were neutral-ized by the highest affinity clone selected, 3B3.Clone 3B3 was improved 8.2-fold in binding gp120for a final affinity of 0.77 nM. These experimentssuggest that improvement in the affinity of b4/12should improve its therapeutic potency. To dissectthe relative contribution of the mutant CDR1-H andCDR3-H of 3B3, CDR1-H was converted towild-type sequence in clone h3.3. This cloneretained the binding affinity of 3B3. This exper-iment suggested that the use of the pool of selectedCDR1-H variants in this experiment was not thebest starting point for further optimization. Withstringent selection higher affinity antibodies fromthe original H1 library were selected as shown inTable 4. Based on this result all subsequent CDRwalks reported in this study use a single optimizedclone on which diversity is incorporated, asopposed to a collection of clones of selected butill-defined binding characteristics. In every caseexamined, saturation mutagenesis within the CDRsfollowed by selection using phage display resultedin the improvement of affinity. Selection of cloneswith sub-nanomolar binding affinities was facili-tated by incorporating an off-rate biased selection inthe panning step (vida supra). Affinity gains at eachstep of the walks were generally in smallincrements, about fourfold, with the exception of thetwo steps in CDR3-H which produced about aneightfold improvement in each case. Three differentwalks yielded clones of similarly high affinity137-65 pM; 3B3/L1.4L3.11, 3B3/L3.14, and h1.3/h3.33. The net improvement in affinity of theseclones was 46 to 96-fold.

If the free energy change of a multiple mutant (inthis case several optimized CDRs combined) isnearly equal to the sum of the free energy changes

observed in the point mutants (or singly optimizedCDRs) the free energy changes are said to beadditive; DDGI is negligible. The reader should notethat additivity in free energy changes results inmultiplicative changes in Ka; a fourfold improvedaffinity in CDR1 combined with a fourfoldimproved affinity in CDR2 results in a clone with a16-fold improvement in affinity. In protein engin-eering studies this is generally the case unlessresidues interact with each other by direct contact orindirectly through electrostatic or structural pertur-bations (Wells, 1990). This (interaction) will cer-tainly be the case for optimization within the longCDR loops of CDR1-L, CDR2-H and CDR3-Hwhich require optimization of more than fiveresidues and at least two sequential optimizationswithin the given CDR. Parallel CDR walking makesthe assumption that the optimized loops will exhibitadditivity in free energy changes when theindividually optimized loops are combined. Theadvantage of a parallel approach is the speed withwhich the final desired clone might be obtained. Ina study guided by a high resolution X-ray structureand extensive alanine scanning mutagenesis anadditivity-based approach has been convincinglydemonstrated in the human growth hormonesystem with the generation of a hormone with a380-fold improvement in affinity for its receptor(Lowman et al., 1991; Lowman & Wells, 1993).Within the present system we are guided by thedata base of available antibody structures and hintsat somatic hotspots observed in chain shufflingexperiments. Within antibodies of known structure,CDR-CDR contacts are well documented. However,from crystallographic studies it is not clear howsignificant these interactions are or to what extentchange in sequence in interacting regions will affectaffinity for antigen. Examples of the utilization ofadditivity in the affinity optimization with anti-bodies have been reported (Foote & Winter, 1992;Hawkins et al., 1992, 1993; Riechmann & Weill,1993). The extent of mutation in the combinedsegments in these reports is generally much lowerthan that reported here.

Encouraged by these results, a parallel CDRwalking mutagenesis strategy was examined whereindependently optimized CDRs were combined.Table 5 summarizes the binding data for sixcombinations of CDRs. Only in the combination ofCDR1-H with CDR3-H was additivity observed.This result could not be rationalized by knowledgeof structures of antibodies, since these CDRs areknown to form many van der Waals’ contacts.Changes within the CDR1-H in h1.1 are, however,modest in comparison to selected changes in otherCDRs. Recently interchain CDR-CDR contacts inantibodies of known structure have been tabulated(Padlan, 1994). This study revealed that CDR3-Lgenerally forms van der Waals’ contacts with all theCDRs of the heavy chain whereas CDR3-Hgenerally interacts with all the CDRs of the lightchain. CDR1-L, however, contacts predominatelyCDR3-H. Overall the results of combinations were


unpredictable but did lead to some modestimprovements over the clones derived fromsequential selection. The large negative interactionenergies observed for most combinations given inTable 5 are sufficient to undermine the small0.82 kcal/mol increases which would accompanythe about fourfold increases in affinity of theindividual optimized CDRs. The failure of theadditivity-based strategy is most likely due to directinteractions though long-range non-additive effectsare also possible (LiCata & Ackers, 1995). The−2.02 kcal/mol interaction energy observed in theconstruction of h1.1h3.33/L3.14 suggests that to alarge degree non-additivity is observed in thesecases due to the incompatibility, perhaps due tosteric interaction, directly or through antigen, of theoptimized CDR3-L regions with independentlyoptimized CDR3-H region. The highest affinityclone produced by parallel optimization, h1.1h3.3/L1.4L3.1 was improved 420-fold in affinity forantigen. This is only a 4.4-fold improvement over3B3/L1.4L3.11, the highest affinity clone obtainedin the sequential approach, and would likely havebeen achieved in the next sequential optimizationstep. The highest affinity Fab generated in thesestudies, h1.1h3.3/L1.4L3.14, bound gp120 with a15 pM dissociation constant. Since most of the CDRsequence of this antibody remains to be optimized,the limit to which one may improve affinity remainsto be reached.

Examination of the kinetics of binding using theBIAcore instrument revealed that improvements inaffinity were dominated by slowing of the off-rate.The highest affinity clone demonstrated a 400-folddecrease in off-rate as compared to b4/12. TheBIAcore sensorgrams for Fabs b4/12 and h1.1h3.3/L1.4L3.14 are given in Figure 2. The slowing inoff-rate observed here is in agreement with thatobserved in the affinity maturation of humangrowth hormone (Lowman & Wells, 1993) and isexpected since experimentally selective pressure isprovided to enrich for the most stable Fab/gp120

complex. In comparison to the natural ligand forCD4, the Fab fragments bound with a four toeightfold faster on-rate than sCD4 and in the bestcase, with an off-rate 83-fold slower than sCD4.Further improvements in off-rates cannot bemeasured with the BIAcore instrument which islimited to off-rates faster than 10−7 s−1

This study demonstrates that the sequential CDRwalking strategy is an effective route to thegeneration of very high affinity antibodies. Thelarge affinity increases observed in the optimizationof the CDR3-H region suggest that the mostexpeditious route to improve affinity may beoptimization of this region. This is likely to beparticularly true for an antibody such as b4/12,which has an extended CDR3-H of 18 residues.Since human and murine CDR3-H regions havemean lengths of approximately 12 and 9 residues,respectively (Wu et al., 1993), which are generatedby mechanisms most analogous to saturationmutagenesis (Sanz, 1991), it should not be surpris-ing that the humoral response has not selected thecombinatorially most optimized segment in thisregion.

The ability to create very high affinity antibodieshas implications for their use as catalysts, sinceoptimization of affinity for the transition-state of areaction is directly correlated with enhancedcatalytic efficiency (Lerner et al., 1991). Fortherapeutic and prophylactic application, very highaffinity antibodies have important theoreticaladvantages if potency, for example in viralneutralization assays, is strictly correlated withaffinity. In therapeutic applications the quantity ofprotein required could be drastically reduced. Forexample, if a serum level of 25 mg/ml were requiredfor a given indication, a 420-fold improvementwould reduce the quantity of protein required to0.06 mg/ml. In prophylactic applications, increasedpotency could dramatically extend the time inwhich the individual is protected from disease.Given the 21 day half-life of an antibody in the body

Figure 2. Comparison of BIAcoresensorgrams obtained for the bind-ing of Fabs b4/12 and h1.1h3.33/L1.4L3.14 to immobilized gp120.RU, resonance units.


(Capon et al., 1989), an antibody which providedprotection through a single half-life would, ifimproved 420-fold and given in the same quantity,provide protection for six months.

Materials and Methods

Reagents, strains and vectors

Oligonucleotides were from Operon Technologies(Alameda, CA). Escherichia coli, phage, and the phagemidvector pComb3H are as described (Barbas et al., 1991;Barbas & Wagner, 1995)). Restriction enzymes wereobtained from Boehringer Mannheim. Taq polymeraseand DNA ligase were obtained from Promega andGibco-BRL, respectively. The recombinant protein gp120IIIB was purchased from Intracel Corp. (Cambridge, MA).Reagents for surface plasmon resonance experimentswere obtained from Pharmacia.

Construction and selection of libraries

The construction of library H1 has been described(Barbas et al., 1994). This library was selected for gp120binding by an additional six panning cycles beyond thefour cycles performed in the previous report. Forselections, gp120 IIIB was immobilized on Costar 3690microtiter wells. One well, coated with 1 ag of gp120overnight, was used for each round of selection. Toconstruct library H3B3 clone 3B3 was utilized as atemplate for overlap PCR mutagenesis as described(Barbas et al., 1994). The two fragments required for thisprocedure were obtained with oligonucleotide primerpairs FTX3 (Barbas et al., 1992a) and 3B3C350B(5' - CCAGACGTCCATATAATAATTGTCMNNMNNMN -NMNNMNNCCAACCCCACTCCCCCACTCT - 3') andwith R3B (Barbas et al., 1993b) and H4H3OF (5'-GACAAT-TATTATATGGACGTCTGG-3'), respectively. N = A, C, G,or T,: M = A or C which gives K = T or G in thecomplementary strand (NNK doping strategy). Followingfusion and gel purification, the fragment was digestedwith restriction enzymes XhoI and SpeI, purified andligated into the phagemid vector pComb3H containing theb4/12 light chain from which a 300 base-pair stufferfragment in the heavy chain cloning sites XhoI and SpeIhad been removed. The ligation products were intro-duced into XL1-Blue cells by electrotransformation toyield 1 × 108 independent transformants. The library wasselected by panning against gp120 IIIB for four cycles. Inthe fifth through the tenth cycles of selection, 8 mM Fab3B3 was added to the well following removal of unboundphage by washing. The plate was then incubated with theFab for three hours at 37°C (off-rate selection). The wellwas then washed five times and bound phage were elutedand propagated as usual. Library L1 was prepared in asimilar procedure using 3B3 as a template for overlap PCRmutagenesis with oligonucleotide primer pairs KEF(Barbas et al., 1993b) and HIVCDR1-OV-B (5'-AGGTTTG-TGCTGGTACCAGGCTACMNNMNNMNNMNNMNN-MNNGTGACTGGACCTACAGGAGAAGGT-3') and T7B(Barbas et al., 1993b) and HIVCDR1-FO (5'-GTAGCCTG-GTACCAGCACAAACCT-3'). Following fusion and gelpurification, the fragment was digested with restrictionenzymes SacI and XbaI, purified and ligated into thephagemid vector pComb3H containing the 3B3 heavychain wherein a 1.2 kb stuffer fragment between SacI andXbaI had been removed. The ligation products wereintroduced into XL1-Blue cells by electrotransformation to

yield 5 × 108 independent transformants. Libraries L3Aand L3B were prepared with primer pairs KEF andH4KCDR3-B0 (5'-CAGTTTGGTCCCCTGGCCAAAAGT-GTAMNNMNNMNNMNNATAMNNCTGACAGTAGT-ACAGTGCAAAGTC-3') and T7B and HVKFR4-FO (5'-TACACTTTTGGCCAGGGGACCAAACTG-3'). LibraryL3A was prepared with 3B3/L1.4 as the PCR templateand library L3B was prepared with 3B3 as the template.Library sizes were 2 × 108 and 4 × 108, respectively.Library L3A was selected for binding to gp120 with fivecycles of panning followed by five cycles of panning with3B3 used in an off-rate selection as described above.Library L3B was selected with three panning cyclesfollowed by four cycles with Fab 3B3 off-rate selection.

Expression of soluble Fab

Following selection, phagemid DNA was isolated anddigested with SfiI and the approximately 1500 bp cassettewas ligated with SfiI-digested pPhoA. pPhoA wasconstructed by insertion of the phoA promoter andantibody encoding cassette between the AatII and HindIIIsites of pBR322. The phoA promoter was isolated fromgenomic E. coli DNA by PCR. The antibody encodingexpression cassette was derived from pComb3H. ThepPhoA vector was designed to be compatible withpComb3H and is based on the previously reportedpAK19 expression vector by Carter et al. (1991). Thisvector is designed for use in fermentation, though for theexperiments reported here fermentation was not necess-ary. Expression of Fab in shaker flasks with this vector wasachieved by phosphate starvation as described (Carteret al., 1992). In some cases transfer to pPhoA was notperformed. In these cases NheI/SpeI restriction enzymedigestion and religation of the pComb3H vector leads toefficient expression of soluble Fab as described (Barbaset al., 1991). Following screening for binding activity in anenzyme linked immunosorbant assay (ELISA), thecomplete amino acid sequences of the variable regions ofELISA positive clones were deduced by dideoxysequencing as described. Fab was purified by affinitychromatography as described (Barbas et al., 1992b). Toexamine if protein aggregates are isolated using thisprotocol, an additional gel filtration purification step wasperformed on Fabs b4/12 and h1.1h3.33. Gel filtrationchromatography was performed using a standardizedHiPrep 16/60 Sephacryl S-100 column equilibrated andrun (0.5 ml/min) in PBS buffer. Fabs eluted at 50 kDa andwere collected and used for BIAcore studies.

Recruitment of optimized complementaritydetermining regions

Shuffling of optimized CDR regions was facilitated byan encoded SacII site in framework three of the heavychain and a KpnI site in framework two of the light chain.The heavy chain h1.1h3.33 was constructed from the210 bp XhoI/SacII fragment of h1.1 and the 470 bpSacII/SpeI fragment of h1.3B/h3.33. Light chain L14L3.11was constructed from the 100 bp SacI/KpnI fragment ofL1.4 and the 550 bp KpnI/XbaI fragment of L3.14.Combinations of mutant light and heavy chains wereconstructed in the pPhoA vector.

Surface plasmon resonance

The kinetic constants for the binding of Fab to gp120IIIB were determined by surface plasmon resonance-


based measurements using the BIAcore instrument fromPharmacia. The sensor chip was activated for immobiliz-ation with N-hydroxysuccinimide and N-ethyl-N '-(3-di-ethyl aminopropyl) carbodiimide. The protein, gp120, wascoupled to the surface by injection of 50 ml of a 50 mg/mlsample. Excess activated esters were quenched with 15 mlof 1 M ethanolamine (pH 8.5). Typically 4000 resonanceunits were immobilized. Binding of Fab fragments toimmobilized gp120 was studied by injection of Fab in arange of concentrations (0.5 to 10 mg/ml) at a flow rate of5 ml/min. The binding surface was regenerated with HCl,1 M NaCl (pH 3) and remained active for 030measurements. Each immobilization surface was stan-dardized by determination of the binding kinetics of clone3B3 so that relative values could be determined. Theassociation rate (kon) was determined by studying the rateof binding of the protein to the surface at five differentprotein concentrations ranging from 10 mg/ml to 0.5 mg/ml. The dissociation rate (koff) was determined byincreasing flow rate to 50 ml/min after the associationphase. The koff value is the average of three measurements.The kon and koff values were calculated using BIAcoreTM

kinetics evaluation software. The derived equilibriumdissociation constants (Kd) were calculated from the rateconstants. To exclude protein aggregation as a cause forthe slowing of the off-rates of mutant Fabs, the off-ratesof Fabs b4/12 and h1.1h3.33 were determined followinggel filtration purification to remove any aggregatedprotein. The binding of gp120 to immobilized Fab b4/12was also studied. Fab was immobilized and kineticsdetermined as described above using five concentrationsof gp120 ranging from 20 mg/ml to 1.25 mg/ml todetermine association rate and 50 mg/ml injections todetermine off-rate.

AcknowledgementsWe thank Mike Picque for molecular graphics, Richard

Lerner and Robyn L. Stanfield for helpful discussions,and Ray Sweete for sCD4. We thank a reviewer for callingthe van der Merwe et al. reference to our attention. C.F.B.is a Scholar of the American Foundation for AIDSResearch and recipient of an Investigator Award from TheCancer Research Institute. This work was supported bythe National Institutes of Health grant RO1 AI 37470 toC.F.B.

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Edited by F. E. Cohen

(Received 10 April 1995; accepted in revised form 5 September 1995)

CDR Walking Mutagenesis for the Affinity …. Mol. Biol.(1995) 254, 392–403 CDR Walking...

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