Post on 08-Jan-2023
doi:10.1016/j.jmb.2009.03.019 J. Mol. Biol. (2009) 388, 541–558
Available online at www.sciencedirect.com
Affinity Maturation of a Humanized Rat Antibody forAnti-RAGE Therapy: Comprehensive MutagenesisReveals a High Level of Mutational Plasticity Both Insideand Outside the Complementarity-Determining Regions
William J. Finlay1†, Orla Cunningham1†, Matthew A. Lambert1,Alfredo Darmanin-Sheehan1, Xuemei Liu1, Brian J. Fennell1,Ciara M. Mahon1, Emma Cummins1, Jason M. Wade1,Cliona M. O'Sullivan1, Xiang Yang Tan2, Nicole Piche2,Debra D. Pittman2, Janet Paulsen2, Lioudmila Tchistiakova2,Sreekumar Kodangattil2, Davinder Gill2 and Simon E. Hufton1⁎
1Wyeth Research Ireland,Conway Institute, UniversityCollege Dublin, Belfield,Dublin 4, Ireland2Wyeth Research, 87 CambridgePark Drive, Massachusetts,MA 02140, USA
Received 22 January 2009;received in revised form6 March 2009;accepted 7 March 2009Available online13 March 2009
*Corresponding author. E-mail addr† W.J.J.F. and O.C. contributed eqAbbreviations used: RAGE, recep
mRAGE, mouse RAGE ectodomain;high-mobility group box-1 chromosfusion protein; CDR, complementarreaction; SEC, size-exclusion chromathe heavy chain; CLP, cecal ligationhorseradish peroxidase; PBS, phosp
0022-2836/$ - see front matter © 2009 E
Antibodies that neutralize RAGE (receptor for advanced glycation endproducts)–ligand interactions have potential therapeutic applications inboth acute and chronic diseases. We generated XT-M4, a rat anti-RAGEmonoclonal antibody that has in vivo efficacy in an acute sepsis model. Thisantibody was subsequently humanized. To improve the affinity of thisantibody for the treatment of chronic indications, we used random andtargeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants.We identified a panel of single-chain Fv antibody fragments (scFv's) thatwas improved up to 110-fold in a homogeneous time-resolved fluorescencecompetition assay against parental XT-M4 immunoglobulin G (IgG). Afterreformatting to bivalent scFv–Fc fusions and IgGs, we observed similargains in potency in the same assay. Further analysis of binding kinetics asIgG revealed multiple variants with subnanomolar apparent affinity thatwas dictated primarily by improvements in the off-rate. All variants alsohad improved binding to cell surface-expressed human RAGE, and allretained, or had improved, apparent affinity for mouse RAGE. F100bL inVH (variable region of the heavy chain) complementarity-determiningregion 3 (CDR3) was one of a number of key mutations that correlated withaffinity improvements and was independently identified by both mutagen-esis strategies. Random mutagenesis coupled with ribosome display andhigh-throughput screening revealed an unexpectedly high level of muta-tional plasticity across the whole length of the humanized scFv, suggestinggreater scope for structural optimization outside of the primary antigen-combining site defined by VH CDR3 and Vκ CDR3. In summary, ourcomprehensive mutagenesis approach not only achieved the desiredaffinity maturation of XT-M4 but also defined multiple mutational hotspots
ess: huftons@wyeth.com.ually to this study.tor for advanced glycation end products; hRAGE, human RAGE ectodomain;hRAGE–Fc, hRAGE Fc fusion; mRAGE–Fc, mRAGE Fc fusion; HMGB1,omal protein; scFv, single-chain Fv antibody fragment; scFv–Fc, single-chain Fv–Fcity-determining region; SOE-PCR, splice overlap extension polymerase chaintography; HTRF, homogeneous time-resolved fluorescence; VH, variable region ofand puncture; VL, variable region of the light chain; IgG, immunoglobulin G; HRP,hate-buffered saline; BSA, bovine serum albumin; RU, response units.
lsevier Ltd. All rights reserved.
Fig. 1. Reformatting of humanizpurified using Ni2+-affinity chromatpurified material: lane 1, prestainedM4 scFv with reducing agent; lanes 4purified M4 scFv run at a concentratof M4 scFv binding to hRAGE and mbackground binding to BSA-coatehighlighted in blue.
542 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additionalsites within the CDR loops where human germ-line amino acids may beintroduced without affecting function.
© 2009 Elsevier Ltd. All rights reserved.
Keywords: affinity maturation; phage display; ribosome display; antibodyhumanization; RAGE
Edited by I. WilsonIntroduction
RAGE (receptor for advanced glycation endproducts) is emerging as an important clinical targetfor the treatment of multiple inflammatory diseases.It is a multiligand member of the immunoglobulinsuperfamily1–3 consisting of an extracellular domain,a single membrane-spanning domain, and a cyto-solic tail. Expression of the receptor is observed onmultiple cell types, although constitutive expressionlevels are generally very low outside the lungendothelium. Ligation of RAGE has been shown tosignificantly increase expression levels of the recep-tor in both acute and chronic disease states, such asrheumatoid arthritis4,5 and diabetic nephropathy.6
To date, a large number of ligands have beenidentified for RAGE, including AGEs, HMGB1(high-mobility group box-1 chromosomal protein),7
the S100/calgranulin family,8 and peptides contain-ing β-sheet fibrils, such as β-amyloid.9 Interestingly,RAGE has also been identified as a counter-receptorfor the β2-integrins and is thought to be involved inthe regulation of cell adhesion and migration duringinflammatory cell recruitment.10 Recent evidencealso suggests a role for RAGE in the damage-associated molecular pattern system of the innateimmune system through its regulation of HMGB1interaction with toll-like receptors 2 and 4.11
HMGB1 is a DNA-binding protein that can alsoact as an inflammatory cytokine upon its releasefrom cells via necrosis. It is a late-stage mediator oflethality in the murine cecal ligation and puncture(CLP) model of polymicrobial sepsis and has beenshown to be elevated 1 to 2 days after CLP, with thiselevation beingmaintained throughout the course ofthe disease. The critical role of HMGB1 in this modelof sepsis was highlighted by the decrease inmortality rates upon administration of an antag-onistic anti-HMGB1 antibody.12 Lutterloh et al.demonstrated that RAGE−/− mice have a signifi-cantly decreased septic response to CLP and thatexogenous administration of anti-RAGE IgG to
ed anti-RAGE antibody (Xography followed by gel fmolecular weight markersand 5, purified M4 scFv w
ion of 100 μg/ml in 50 mMRAGE both immobilized
d plates). (c) M4 scFv s
block RAGE ligands in wild-type mice can alsoimprove survival rates after CLP.13
We previously isolated XT-M4, a rat monoclonalanti-RAGE antibody that antagonizes RAGE interac-tion with multiple ligands, including HMGB1. Thisantibody cross-reacts with both mouse RAGE(mRAGE ectodomain) and human RAGE (hRAGEectodomain) and was shown to have a potent protec-tive effect in the CLP model.13 XT-M4 was subse-quently humanized in order to develop its potential asa therapeutic for the treatment of sepsis. While XT-M4has potent activity in the acute treatment of sepsis in invivo murine models, it was characterized as having afaster off-rate and a lower affinity for hRAGE than formRAGE. As binding affinity can influence the bio-logical activity of a therapeutic antibody,14 we soughtto affinity mature XT-M4 while maintaining itsfunctional epitope on hRAGE.Many studies have reported the affinity optimiza-
tion of fully human antibodies derived from phage-display libraries. However, few studies have sought toimprove the affinity of a humanized rat monoclonalantibody that has previously shown efficacy in in vivopreclinical models.13 Several mutagenesis strategieshave been used successfully in antibody affinityoptimization, ranging from random mutagenesisacross the full protein sequence via error-pronepolymerase chain reaction (PCR)15 to targetedapproaches where specific positions in the comple-mentarity-determining region (CDR) loops arediversified.16 In the case of humanized mouseantibodies, most molecular evolution strategies haveappliedmutagenesis targeted to the CDR loops17–20 oracross the VH (variable region of the heavy chain)domain.21 In a single additional case, error-prone PCRmutagenesis has also been applied across both V-genes in yeast-display libraries.22 We subjected XT-M4to targeted and random mutageneses using phagedisplay and ribosome display, respectively. We identi-fied a number of variants that had improved affinityfor hRAGE while maintaining both the functionallyrelevant epitope and the required cross-reactivity to
T-M4) as an scFv in the VL–VH format. (a) M4 scFv wasiltration into PBS, pH 7.2. The SDS-PAGE inset shows the(the arrowmarks the 30-kDa band); lanes 2 and 3, purifiedithout reducing agent. The analytical SEC trace representssodium phosphate and 30 mMNaCl, pH 7.4. (b) Analysisat 1 μg/ml by ELISA (all values have been subtracted forequence with Kabat numbering.23 Back mutations are
544 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
mRAGE. Our broad mutational scanning approachhas given us some insight into key residues thatmediate XT-M4 interaction with RAGE and identifiedmultiple mutational hotspots within the CDRs andframework regions. These allow us to predict addi-tional residues within our humanized molecule thatmay be converted to the corresponding human germ-line residue without affecting function.
Results
Reformatting of parental humanized XT-M4 tosingle-chain Fv antibody fragment
The CDRs of XT-M4 rat monoclonal antibodywere grafted onto DP54/DPK9 human germ-line Vgene frameworks. A series of back mutations wererequired to restore full binding activity to thehumanized antibody (unpublished results). Huma-nized XT-M4 was then converted to a single-chainFv antibody fragment (scFv) (M4) in the VL (variableregion of the light chain)–VH format, incorporating a16-aa flexible linker (Fig. 1c). M4 was expressed andpurified from Escherichia coli containing the phage-mid pWRIL-1-M4. SDS-PAGE analysis revealed asingle band of approximately 30 kDa after affinitychromatography and size-exclusion chromatogra-phy (SEC). Analytical SEC of M4 showed a 65% to35% split of dimeric to monomeric species (Fig. 1a).Purified M4 retained binding to hRAGE and boundto mRAGE with a lower EC50 in ELISA (Fig. 1b),consistent with previous data for the parental ratimmunoglobulin G (IgG). Functional display of M4on phage was proven by anti-RAGE phage ELISA,and model enrichment experiments with biotiny-lated RAGE also confirmed the ability of M4 to forma functionally folded protein in complex withribosomes (data not shown).
Mutagenesis of parental scFv antibody
Mutagenized scFv libraries were generated bysplice overlap extension (SOE)-PCR and cloned intothe phage-display vector pWRIL-1. Final library sizes(total transformants) for the VH-CDR3 and Vκ-CDR3libraries were 1.2×109 and 5.0×108, respectively. Thefrequency and distribution of mutations in bothCDR3 libraries were consistent with those predictedby the oligonucleotide design (data not shown). In aparallel strategy, error-prone PCR with a predictedmutation rate of 4.5–9 mutations per kilobase wasused to introduce mutations across the full length ofthe scFv. This mutation rate was confirmed by thesequencing of randomly chosen clones.
Selection of targeted mutagenesis librariesby phage display
A series of model selections of M4 phagedetermined that the maximal and minimal biotin-hRAGE–Fc concentrations for M4 selection were 25and 0.5 nM, respectively. The higher affinity of M4
for mRAGE was reflected in maximal and minimalconcentrations of 5 and 0.05 nM, respectively. Basedon these observations, the antigen concentration ofbiotin-hRAGE–Fc used in round 1 of selections was5 nM and the concentration was then lowered inlog10 increments in successive selection rounds. Aselection on biotin-mRAGE–Fc at 0.05 nM was alsoincluded in round 4 to maintain mouse reactivity inselected clones. All libraries were selected separatelyin initial rounds in order to encourage effectivesampling of the diversity present in both the VH-CDR3 and Vκ-CDR3 libraries. In round 3, selectedheavy- or light-chain populations were then pooledand maintained as separate pools for all subsequentselection rounds, to allow comparison of themutagenesis of each CDR3.
Selection of random mutagenesis librariesby ribosome display
Model enrichment experiments with M4 in thepresence of decreasing concentrations of biotiny-lated hRAGE–Fc determined that antigen-specificenrichment occurred at a minimum concentration of5 nM (as determined by reverse transcriptase PCR),and this was subsequently used as the startingantigen concentration for library selection. As in thephage-display library selections, antigen concentra-tion was reduced in log10 increments in consecutiverounds. Analyses of sequence diversity and bindingfunction of selected outputs necessitated PCRamplification and cloning of cDNAs from consecu-tive selection rounds into pWRIL-1.
Identification and analysis ofaffinity-matured variants
Analysis of selected populations was performedby randomly picking a total of 92 clones from eachround and performing a homogeneous time-resolved fluorescence (HTRF) competition assay.The HTRF assay measures the decrease in fluores-cence observed upon binding of europium cryptate-labeled parental XT-M4 IgG to hRAGE in thepresence of competing scFv antibodies. This allowsthe identification of clones that compete strongly forbinding to the original epitope of XT-M4, which hasbeen shown to mediate biological potency in vivo.13
An increase was observed in the number of highlycompeting clones retrieved after round 3 of selectionof targetedmutagenesis libraries (Fig. 2a), indicatingthat selection for clones with higher affinity wasoccurring. Based on sequence information, selectionoutputs from the VH-CDR3 and Vκ-CDR3 librariesfrom rounds 3 and 6 were chosen for furtheranalysis. A total of 32 clones (17 heavy-chain and15 light-chain mutants) were purified as scFv forranking in titration HTRF assays on hRAGE. Light-chain mutants exhibited a maximum of 5-foldimprovement in the titration HTRF assay, whileheavy-chain mutants were improved up to 70-fold,suggesting a greater importance of VH CDR3 inantigen binding. HTRF titration analyses for the 10
Fig. 2. Screening of selected clones. (a) Ninety-six clones were picked from rounds 3–6 of selection using phagedisplay. Changes in output fluorescence for mutated clones (gray squares) were measured relative to parental M4 (whitesquares) and a negative control scFv (black squares). M4 variants in the boxed population were purified and compared intitration HTRF analysis. (b) The 10 highest performing scFv's from this analysis are shown relative to parental M4 and anegative control scFv. (c) A high-throughput HTRF screen was carried out on 5000 randomly picked clones fromribosome-display selections. Output fluorescence for mutated clones (gray squares) was compared with parental M4(white squares) and a negative control scFv (black squares). Clones in the boxed population were purified and comparedin titration HTRF analysis using hRAGE. (d) The 7 highest performing clones from the HTRF analysis.
545Affinity Maturation of XT-M4 for Anti-RAGE Therapy
VH-CDR3 clones with greatest improvements areshown in Fig. 2b, and associated IC50 values areshown in Table 1.Sequence analysis of selection outputs from
ribosome display showed a broad diversity ofmutations distributed over the full length of thescFv sequence (with an average of 2.5 amino acidmutations per selected scFv). Although some dupli-cate clones were found in consecutive rounds ofselection, sequence diversity remained high. Inorder to further analyze this diverse population ofmutated variants, we performed a high-throughputscreen of 5000 clones using the HTRF competitionassay (Fig. 2c). Confirmatory HTRF analysis on bothhRAGE and mRAGE identified 123 leading clonesthat were subsequently sequenced to assess muta-tional diversity. Within this population, 44 uniqueclones with strong neutralization activity werepurified and ranked in titration HTRF assays. Thispanel was reduced to 7 clones with significantlyimproved IC50 values (Fig. 2d; Table 1).The greatest affinity improvements were observed
most consistently in clones derived from VH CDR3-targeted mutagenesis (Table 1). These clones werereformatted to scFv–Fc fusion proteins, expressedtransiently in COS cells, purified on protein A, andtitrated in the HTRF assay to rapidly gauge theirbehavior in a bivalent format. Further gains were
achieved upon conversion to a bivalent format, withimprovements over M4 scFv–Fc fusion ranging from6.2- to 62.7-fold against hRAGE and from 2.2- to11.3-fold against mRAGE (Table 2), as measured inthe HTRF assay.The selected scFv molecules were also reformatted
to whole IgG, expressed transiently in COS cells,and purified on protein A. Purified proteins wereanalyzed by SDS-PAGE and analytical SEC and hadless than 1% high-molecular-weight species in allcases (data not shown). All variants were shown tobe functional as IgGs when tested in the competitionHTRF assay (Fig. 3a). The fold gains for variantsover parental M4 IgG ranged from 14.3 to 172.0(Table 2). The IgGs were further characterized byBIAcore global-fit analysis for each IgG, at multipleconcentrations ranging from 0 to 10 nM. The rateconstants (defined as “apparent Kd” values to reflectpotentially avid binding) are summarized in Table 2,demonstrating that the apparent affinity of the M4IgG was 4.45 nM, while improved clones reached ashigh as 150 pM. These affinity improvements weredetermined primarily by changes in the off-rate.Cell-based ELISAs measuring IgG binding to CHOcells stably overexpressing hRAGE demonstratedapproximately 4- to 10-fold improvements in EC50values for the variants over the parental XT-M4 IgG(Fig. 3b; Table 2).
Table 1. Locations of mutations in selected variants with improved potency in HTRF competition assay
aM4 is the parental anti-RAGE scFv.bShaded residues represent mutations from parental anti-RAGE scFv within VH CDR3.cIC50 values (in nanomolar) of purified scFv variants for hRAGE in HTRF assay.
546Affinity
Maturation
ofXT-M
4for
Anti-R
AGE
Therapy
Fig. 3. Characterization of reformatted affinity-improved clones. (a) Selected phage-display scFv cloneswere reformatted to full-length human IgG1. These weretransiently expressed in COS cells, purified, and titrated inHTRF assay. (b) A cell-binding ELISAwas carried out withthe purified IgGs to ensure that all M4 variants alsoexhibited improved binding to cell-surface RAGE. Ninety-six-well plates were coated with a parental CHO cell lineor a CHO cell line stably overexpressing hRAGE. Resultsare presented as binding to CHO-RAGE cells afterbackground subtraction of binding to parental cell line.Relative EC50 values are reported in Table 2.
Table 2. HTRF IC50 and apparent Kd values for improved M4 variants in the scFv–Fc and IgG formats
Clone IDscFv–Fc huIC50(fold gain)a
scFv–Fc muIC50(fold gain)b
IgG huIC50(fold gain)
IgG Ka[(M−1 s−1)×106]c
IgG Kd[(s−1)×10−2]c
IgG Kd(nM)c
IgG EC50CHO-RAGE (nM)
M4 1.0 1.0 1.0 1.14 0.510 4.45 1.5973A6 9.9 3.7 172.0 1.44 0.049 0.34 0.1663B2 34.2 7.6 19.8 0.37 0.024 0.64 0.2113B4 25.0 11.3 56.0 1.10 0.029 0.27 0.2253D2 31.3 8.4 22.9 1.66 0.097 0.59 0.2533G5 62.7 6.8 27.0 1.53 0.023 0.15 0.2526B2 6.2 2.2 14.3 9.45 0.029 0.03 0.2506B6 7.6 3.4 41.0 1.00 0.043 0.43 0.2786C2 9.4 3.7 22.6 3.21 0.110 0.35 0.2976C3 38.6 5.2 14.4 1.52 0.047 0.31 0.351
a Improvements in potency in the HTRF assay for hRAGE are reported as fold gain over parental M4 scFv–Fc.b Improvements in potency in the HTRF assay for mRAGE are reported as fold gain over parental M4 scFv–Fc.c Surface plasmon resonance kinetic analysis was carried out for the purified IgG variants. Affinity constants were determined by 1:1
global-fit analysis of binding curves for multiple IgG concentrations (0–10 nM) binding to directly immobilized hRAGE extracellulardomain (coated at ∼100 RU). These values are therefore potentially affected by avidity and should be regarded as “apparent Kd.”However, all sensorgram data fit well to 1:1 interaction models and χ2 values for all analyses were b0.5.
547Affinity Maturation of XT-M4 for Anti-RAGE Therapy
Sequence analysis of selected clones fromtargeted mutagenesis
Analysis of the sequence diversity in 88 Vκ-CDR3mutants from selection rounds 3–6 (Fig. 4a) demon-strated that mutations can be tolerated at allpositions, but only positions 90 and 93 toleratedmutation at high frequency. Additionally, whileclones that carried mutations in positions 95–97 hadbeen selected in round 3, these mutations were notassociated with increased affinity and were nolonger observed in the selected population byround 4 (selection on mRAGE). Only 5 uniqueclones exhibiting significant improvements in affi-nity were identified, and these clones carriedcombinations of mutations in positions 90, 91, and93 (Fig. 4b). Several dominant mutations wereidentified, including E90Q, which replaces the ratresidue at this position with the most commonresidue observed in human germ-line Vκ-CDR3sequences (Fig. 4b).Sequence analysis of 201 VH CDR3-targeted
mutants from selection rounds 3–6 (Fig. 4c) showedthat the GGDI motif at positions 95–98 of the VH-CDR3 sequence was not tolerant of mutation (anobservation further confirmed using our error-pronePCR strategy; see Fig. 4f and Table 1). In contrast,residues 99–102 were all shown to be highly tolerantof mutation, with multiple substitutions beingpermitted. F100bL, a key substitution, was identi-fied in the VH CDR3 that was present in the majorityof affinity-improved selected variants (Fig. 4d).Clearly, this F100bL substitution was stronglypreferred at this position, as representative clonescontained all leucine codons possible under the NNScodon randomization system (CTG, CTC, and TTG).F100bI and F100bM mutations were also observedin several improved clones from round 3 and round6 pools (Fig. 4c), but these mutations were asso-ciated with lesser gains in affinity than thoseobserved in combination with the F100bL mutation.Sequence analysis of improved variants from VH-
CDR3mutagenesis identified several distinct familiesof clones. A large family of cloneswas found to have a
motif in the center of the VH-CDR3 loop (positions 99,100, and 100a), predominantly composed of K/R–V–G/S sequences. A second family of improved clones
548 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
had a different motif at positions 99–100a composedof L/V–D–S/G or L–V–G/S sequences. The T99K/R/L and T100V/D mutations represent significantchanges in side-chain chemistry at these positions. Inalmost all clones sequenced, there was a preferencefor a small amino acid (S, G) at position 100a. This
Fig. 4 (legend o
represents conservation of the parental amino acidchemistry at this position. The vast majority ofimproved clones exhibit a preference for a negativelycharged residue (D) at position 101. The predisposi-tion for D at position 101 of VH CDR3 is observed atvery high frequency in the human repertoire and is
n next page)
Table 3. Characterization of I31 and F100b variants
Clone IDKa
[(M−1 s−1)×106]Kd
[(s−1)×10−2]Kd
(nM)
XT-M4 1.14 0.506 4.45I31F Vκ and M4 VH 0.64 0.027 0.43I31F Vκ and T104 VH (1G6) 0.55 0.008 0.15M4 Vκ and T104 VH 1.51 0.137 0.91M4 Vκ and F100bI VH 1.71 0.275 1.61I31FVκ and F100bI VH (3B3) 1.45 0.142 0.98M4 Vκ and 3B4 VH (3B4) 1.10 0.029 0.27I31F Vκ and 3B4 VH 0.70 0.012 0.17
Surface plasmon resonance kinetic analysis was carried out for thepurified IgG I31F and F100b variants. Affinity constants weredetermined by 1:1 global-fit analysis of binding curves formultiple IgG concentrations from 0 to 10 nM binding to directlyimmobilized hRAGE extracellular domain (coated at ∼100 RU).All values were compared with those obtained for parental XT-M4 IgGs. These values may reflect avid binding and should beregarded as “apparent Kd.”
549Affinity Maturation of XT-M4 for Anti-RAGE Therapy
associated with stabilizing the CDR3 loop via a saltbridge with R94.24,25 It is of interest, however, thatone of the higher affinity clones identified (clone 3G5)carries a D101P mutation (Table 1). Proline is rarelyobserved in this position in the natural repertoire, andthis substitution may be related to subtle optimiza-tion of CDR–framework interactions unique to thisCDR-grafted antibody. The last position in the CDR3(Y102) was highly variable among the total selectedpopulation (Fig. 4c and d) but in the highest affinityclones was maintained primarily as one of the largearomatic and/or hydrophobic residues (Y, F, V, I)typically found at this position in natural antibodies(Table 1).In 6 of 10 improved clones from VH-CDR3
mutagenesis (Table 1), positions 101 and 102 of VHCDR3 (encoded asDYinM4, JH4 germ line) exhibitedsequence motifs frequently observed in naturallyderived human antibodies, such as DV (encoded byJH6) and DY (encoded by JH4). These motifs, incombination with A93 and R94, are in accordancewith the maintenance of the classic “kinked-base”style of the CDR3 loop,24 which is observed in theparental antibody. Other selected motifs (LPY, LRY,LHF) that may still form the kinked base but couldalso indicate subtle changes in secondary structure atthe base of the VH-CDR3 loop were also observed.
Sequence analysis of selected clones fromrandom mutagenesis
High-throughput screening identified a diverse setof 123 variants with improved potency in the HTRFassay. Sequence analysis of this population showed abroad spectrum of mutations distributed over thescFv length (Fig. 4e and f). Seventeen mutationalhotspots were identified (defined as having afrequency ≥5 in a population of 123 clones), with 8in the Vκ domain (R18, I31, R45, R50, F71, E81, F83,and K103) and 9 in the VH domain (P14, N30, N52a,D55, S62, F67, A93, F100b, and S113). Of 17 hotspots,only 6 were within the CDRs (35%) (Fig. 4e and f). Inour study, we observed no clear mutational biastoward the CDR loops, in contrast to what had beenreported during the affinity maturation of a fullyhuman antibody via ribosome display26 and duringsomatic hypermutation in vivo.27,28The VH-CDR3 F100bL/I mutation was a domi-
nant change recovered from both random andtargeted VH-CDR3 mutageneses (Fig. 4d and f),reflecting the role of this residue in mediating
Fig. 4. Sequence analysis of M4 variants generated via tarwas performed on selected functional mutants, and amino accolor coded and expressed as the percentage of usage per posfrequency at each position for random mutagenesis in plotsderived from the Vκ CDR3-targeted libraries (positions 89–improved IC50 values by HTRF titration assay. (c) Two hundCDR3-targeted libraries (positions 95–102). (d) Seventeen mutimproved IC50 values by HTRF titration assay. (e) Frequencymutants). CDR residues are shown in red. (f) Frequency ofmutants). CDR residues are shown in red. Black arrows in plo≥5/123). Residues are numbered according to Kabat.23
improved binding to both hRAGE and mRAGE. Asecond dominant mutation (I31F) was identified inVκ CDR1, being recovered by random mutagenesisalone. This mutation was retrieved in 16 of 123clones (Fig. 4e) and was also associated with asignificant improvement in potency in the HTRFassay (Table 1). In order to investigate these hotspotsfurther, we prepared a series of variant IgGs,derived from clones 1G6, 3B3, and 3B4, containingI31F and F100bI alone or in combination with otherpotentially synergistic mutations (summarized inTable 3). BIAcore analysis revealed that clone 1G6(I31F and T104N) exhibited the most improved off-rate. I31F alone can reduce the off-rate, leading to a10-fold improvement in apparent Kd value whencombined with the wild-type M4 VH domain, but anF100bI single mutant exhibits only 2.8-fold improve-ment. The improvements in apparent Kd for clone1G6 can therefore be accredited primarily to the I31Fmutation, with T104N providing a synergistic role.However, a combination of I31F with F100bI (clone3B3) negates the beneficial properties of I31F,suggesting that these two mutations representalternative, noncomplementary solutions to increasebinding affinity for hRAGE. The I31F mutationshows a moderate synergistic effect when combinedwith the VH-CDR3 mutations found in clone 3B4(Table 1), raising the apparent Kd value by approxi-mately 2-fold (Table 3). Combined mutations of bothI31 and F100b can therefore be beneficial in thecontext of the 3B4 CDR3 sequence, although not as
geted and random mutageneses. DNA sequence analysisid variability plots were generated. Amino acid usage isition for targeted mutagenesis in plots (a)–(d) and in total(e) and (f). (a) Eighty-eight functional binding mutants
97). (b) Five binding mutants that were shown to havered one functional binding mutants derived from the VHants derived from the H1 library that were shown to haveof observed mutations per position in the light chain (123observed mutations per position in the heavy chain (123ts (e) and (f) indicate hotspots of mutagenesis (frequency
550 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
significantly as I31F in combination with T104Nalone, as observed for clone 1G6.Random mutagenesis of XT-M4 and analysis of
functionally selected antibody variants have alsoallowed retrospective analysis of the humanizationprocess of XT-M4.During the initial humanization, 13rat framework residues were analyzed for theirfunctional importance by back mutation—11 resi-dues in Vκ (Y36, A43, K45, L46, L47, G64, G66, T69,D70, F71, and Y87) and 2 in VH (N52 and A88). Ofthese residues, 4 were retained in the construct usedas the starting point for this study (Vκ Y36F, K45R,L46R, and G66R). This study confirms the choicesmade during the initial humanization process as 10 ofthese 13 residues were also mutated in our study andshown not to affect functional binding. We were alsoable to identify that the back mutation Vκ K45Rincorporated into the parental humanized XT-M4was not essential for function as variants carrying thehuman germ-line lysine residue had improvedpotency in the high-throughput HTRF assay. Ourstrategy has also suggested that an additional backmutation of Vκ F71Y within framework region 3(Fig. 4e) may be appropriate. A preference for thetyrosine (rat residue) was seen at this position and ispredicted to play a role in CDRorientationwithin XT-M4.29,30 In the 123 clones with improved potency, thetyrosine residue present at position 71 in the originalrat sequence was selected in 5 clones (Fig. 4e).Another interesting outcome of the error-prone PCRapproachwas the identification of residueswithin theCDRs that may be converted to human germ linewithout affecting potency. Several clones wereidentified with mutations back to the human germ-line sequence within the CDRs [N30S and T35S (VHCDR1), D65G (VH CDR2), and T52S (Vκ CDR2)].A significant number of the affinity-matured
clones identified had mutations within the flexiblelinker, with a distinct bias for hydrophilic aminoacids (D1N, G6D, G7N, G7D, G9E, S10F, G12R,G12E, G13R, S15G, and S15N). In the final panel ofseven scFv clones derived by random mutagenesis,10H6, 10D8, and 2E6 all carried a mutation to anasparagine residue (D1N, G7N, and S15N, respec-tively), suggesting strong selective pressure for thisamino acid change (Table 1).
Discussion
Antibodies represent a unique class of therapeuticsdue to their high specificity. We previously identifiedXT-M4, a ratmonoclonal antibody toRAGE,whichhasa protective effect in the CLP model of acute sepsis13
and broad potential in the treatment of chronic andacute inflammatory conditions. The use of murinemonoclonal antibodies to treat human disease islimited due to their short half-life, their inability totrigger human effector function, and the induction of ahuman anti-murine immune response.31,32 Humani-zation of murine monoclonal antibodies can greatlyimprove their therapeutic potential, and this is oftenachieved using CDR-grafting technology.33 XT-M4
was humanized using this approach, resulting in ahumanized antibodywith only four backmutations inthe framework regions. Both the parental IgG andhumanized versions of XT-M4 were characterized ashaving not only a fast on-rate (Ka=1.14×10
6 M−1 s−1)in binding to hRAGE but also a relatively fast off-rate(Kd=5.05×10
−3 s−1). To facilitate the evaluation of XT-M4 in preclinical and clinical treatments of chronicinflammatory diseases, we sought to increase theaffinity of the antibody for hRAGE without affectingthe therapeutically relevant epitope.Antibody potency in vivo is frequently governed by
affinity, and molecular display technologies, such asphage display,34 ribosome display,35 and yeastdisplay,36 are commonly used to achieve affinitymaturation in vitro. A number of candidate therapeu-tic antibodies, including anti-αvβ3 integrin,17 anti-RSV,37 anti-VEGF receptor 2,38 and anti-CEA,36 havebeen successfully affinity matured. However, there isnot always a direct correlation between the affinity ofa matured antibody in vitro and subsequent potencyin vivo. This can be due to reduced pharmacokineticsin virus neutralization,18,37 decreased tissue penetra-tion in tumor targeting,39 and reduced efficacy inreceptor agonism/antagonism.40 In this study, wegenerated a large panel of in vitro evolved antibodyvariants in differentmolecular formats thatwill allowus to determine the optimal affinity and structuralscaffold for a biotherapeutic that can effectively blockRAGE function in vivo.In cases where affinity maturation of a clinical lead
is required, strict criteria must be applied to maintainthe epitope correlatedwith biological potency in vivo.Mutagenesis of the antibody-combining site can leadnot only to the isolation of variants with higher targetaffinity but also to alterations in the epitope. Loss ofepitope specificity may decrease the potency of theresulting antibody in vivo or lead to decreases inspecies cross-reactivity, affecting subsequent precli-nical studies. Our selection and screening assays forimproved affinity were designed to maintain thefunctional neutralizing epitope of the parental anti-body and retain cross-reactivity to the mouse target.In the absence of structure–function information forXT-M4, we applied two complementary mutagenesisstrategies in combination with high-throughputscreening in order to sample the maximum sequencespace possible. VH CDR3 is the most highly variableloop in natural antibodies, and our primary strategyaggressively targeted this region via overlappingstretches of six randomized codons.41 VL CDR3 canalso make a significant contribution to antigenbinding; however, analysis of amino acid usage inN7000 human VH and Vκ sequences from publicdatabases demonstrated that the Vκ-CDR3 loopexhibits greater restriction in natural diversity thanthat observed in the VH-CDR3 loop (data not shown).We therefore decided to use a parsimonious codonmutagenesis strategy42 to minimize the introductionof amino acids at positions where they are rarelyfound in human Vκ-CDR3 sequences. This approachreduces the mutational load compared with NNSrandomization, but it also reduces the occurrence of
551Affinity Maturation of XT-M4 for Anti-RAGE Therapy
stop codons and potentially improves the proportionof correctly folded antibodies in the library.XT-M4 is a humanized CDR-grafted antibody and
as such comprises a “nonnatural” combination of ratCDRs and human framework regions. Our second-ary strategy aimed to evaluate the potential forimprovements in affinity outside theCDR3 loops.Weused error-prone PCR in combination with ribosomedisplay to introduce diversity over the entire lengthof the molecule. A major advantage of this approachis the potential to identify synergistic mutations atdistal sites within the antibody sequence.Our solution-phase HTRF competition assays
confirmed that both targeted and random muta-genesis strategies yielded scFv clones with N100-fold improvement in IC50 over parental M4. Thiswas achieved while maintaining, and in somecases improving, mRAGE cross-reactivity. In theIgG format, analyses of variants derived fromtargeted and error-prone mutation identified sev-eral clones with HTRF IC50 and BIAcore-derivedapparent Kd values in the subnanomolar range.Improved EC50 values were also observed for allclones tested in binding to native, cell surface-expressed RAGE, when compared with parentalXT-M4 IgG. These EC50 values were derived usinga clonal CHO cell line overexpressing RAGE;however, full confirmation that this correlateswith improved therapeutic potential is the subjectof ongoing in vivo studies.High-throughput screening of our ribosome-dis-
playM4 library has allowed us tomap the functionalparatope of XT-M4 and to identify beneficial muta-tions across the entire antibody sequence. Sequenceanalysis of 384 functionally selected clones identifieda number of invariant residues in VH CDR3 (G95,G96, D97), Vκ CDR3 (E90, P95), Vκ CDR1 (A25, S26),and VH CDR2 (Y58), and these are likely to have adirect role in antigen binding (Fig. 5a and b). Theidentification of the VH-CDR3 (G95, G96, D97)sequence as a key determinant of binding was alsoconfirmed with our VH-CDR3 mutagenesis strategy.However, the identification of VκCDR3 (E90) as partof the functional paratope (Fig. 5a) by ribosomedisplay was not confirmed by phage display, andthis illustrates that even in studies that sampleenormous sequence diversity, differences in themutagenesis and display approaches used can leadto subtle changes in the data set.In addition, we have defined key residues within
the frameworks that are intimately associated withantibody function. Several residues located in theframework regions of Vκ (F36, P44) and VH (V37,Q39) could not be mutated without affectingfunction. These residues are often found at the VH–Vκ interface,
30 supporting a role in the stabilizationof VH and Vκ interaction in XT-M4. In our model ofXT-M4, these residues are positioned at the VH–Vκinterface and in close proximity to the key GGDmotif of VH CDR3 (Fig. 5c and d). Similarly, VH A24(FR1) has been reported to play an important role inVH-CDR1 conformation,32,33,37 and the finding thatthis residue cannot be mutated without disrupting
function suggests a similar role for this residue inCDR1 conformation within XT-M4.Of 17 mutational hotspots identified in XT-M4, 11
are found within the framework regions (Fig. 4e andf). This finding is in contrast to that of a previousstudy on the affinity maturation of a fully humanantibody in vitro26 and towhat is observed during thenatural process of somatic hypermutation in vivo,27,28
which both demonstrate a strong preference formutation within the CDR loops. This unexpectedlyhigh level of mutational plasticity outside the CDRswas further confirmed by sequence analysis of 384functionally selected variants that retain binding tohRAGE. Of this population, only 33% of mutationswere foundwithin theCDRs (Fig. 5a andb). This highlevel of tolerance to mutation outside the CDR loopsmay be associated with more subtle alterations in theinteractions between rat CDRs and human frame-work residues or optimization of VH–Vκ domaininteractions, rather than direct optimization of resi-dues that interactwith antigen.Whether the increasedpropensity for mutational hotspots and mutationaltolerance outside of the CDRs can be extrapolated toother humanized antibodies remains to be seen.In general, affinity gains were higher in clones
derived from VH CDR3-targeted mutagenesis, sug-gesting that the heavy-chain CDR3 of XT-M4 mayhave been only partially matured in vivo and is stillhighly amenable to mutation outside the criticalGGDI motif (Fig. 4c and d). Dominant mutationswere identified using both targeted and error-proneapproaches, with the VH-CDR3 F100bL mutationbeing derived independently using both strategies.This residue is clearly a key determinant of affinityimprovement. Interestingly, this mutation is imme-diately N-terminal to D101 of VH CDR3, which is oneof the six residues constituting the core of theinterface between the VH and Vκ domains.30 In themouse, the preference is for F or M at this position,while in humans, L is also frequently observed. Thesestrongly suggest that a component of the affinityimprovement we see with the F100bL mutation isassociated with adjustments to the interface betweenthe VH and Vκ domains. Modeling of this mutationsuggests that F100b inVHCDR3 has interactionswithVκ residues N34 (CDR1), L89 (CDR3), F91 (CDR3),and L96 (CDR3) of Vκ (Fig. 5f). However, L100b in theheavy chain of the high-affinity variants only inter-acts with N34 (CDR1) and L89 (CDR3) (Fig. 5e and f).The exact structural significance of this observation isnot clear; however, we hypothesize that the fewercontacts entertained by L100b may translate intofewer structural constraints, leading to greater flex-ibility of Vκ CDR1, Vκ CDR3, and VH CDR3 andimproved VH–Vκ interactions. How these structuraladaptations associated with the F100bL mutationrelate to the epitope of XT-M4 on RAGE is not yetknown and is the subject of further studies. Theidentification of the I31F mutation in the Vκ-CDR1loop as a dominant mutation correlated withimproved affinity either independently of or incombination with the F100bL/I mutation. Theidentification of this mutation highlights the value
553Affinity Maturation of XT-M4 for Anti-RAGE Therapy
of a random mutagenesis approach as it could nothave been predicted a priori that the Vκ-CDR1 loopwould carry beneficial mutations.While humanized antibodies are likely to be
minimally immunogenic in patients, they still retainxenogenic CDRs that can potentially elicit anti-idiotypic responses in humans.43–45 As all residuesin amurineCDRare not essential for antigen binding,studies that successfully used a rational design tofurther humanize grafted CDRs to carry only thosemurine residues that directly relate to antigenbinding and specificity have been described.46,47
The study presented here shows that our approachto molecular evolution not only defines key residuesthat must be retained to preserve antibody specificitybut also predicts sites within CDRs that can beconverted back to the human germ line withoutaffecting antigen binding [N30S and T35S (VHCDR1), D65G (VH CDR2), and T52S (Vκ CDR2)],and in the case of E90Q in Vκ CDR3, it may even leadto a subtle improvement in affinity. These findingslead us to speculate that ribosome display incombination with error-prone PCR may be anefficient method for further humanization withinCDR loops. Conventional humanization and in vitroaffinity maturation are often performed in discretesteps; however, this sequential approach may over-look potentially beneficial combinations of frame-work and CDR mutations. Both affinity maturationand humanization can be performed simultaneously,resulting in antibodies with murine sequence limitedto only those residues that are absolutely essential foraffinity and specificity.During the sequence analysis of affinity-matured
clones derived from ribosome display, it wasobserved that several mutations had accumulatedin the flexible linker, with most mutations inserting acharged residue (N, D, E, or R). These linkermutations are, for the most part, different fromthose previously reported,48 where random muta-tion and reselection of scFv's resulted in linkermutations that were not biased toward chargedresidues (predominantly GNS, GNC, and GNP). Ithas been described that the linker sequence can haveeither a direct or an indirect effect on multiplebiochemical properties of scFv's, such as affinity,conformational stability, extent of multimerization,proteolytic stability, refolding kinetics, and expres-sion level.49–52 It is possible that the charged
Fig. 5. Structure–function correlation from mutational ssubstitutions in the VH (a) and Vκ (b) chains of XT-M4 (n=38Residues intolerant of amino acid substitution are indicated. (cof humanized XT-M4 with the light chain in light gray andhighlighted in red are the mutational hotspots, and the amantibody paratope/invariant residues. The antibody-combininbold line. Panel (d) represents a view of the end of the Fv struafter rotation through 180°. (e and f) Molecular models of anand humanized XT-M4 (f). The ribbon representation of the hblue. The CDRs of the heavy and light chains are shown in reshown with their van der Waals surface in the background. Thin the heavy chain of the XT-M4 has interactions with N34 (CDchain. L100b in the heavy chain of the high-affinity derivative
residues selected in this study play an indirect rolein improving scFv binding to RAGE throughenhanced solubility, optimized VH–Vκ orientation,improved folding, and/or masking of hydrophobicsurfaces exposed during expression.In conclusion, by using a systematic approach to
antibody affinity maturation, we have been able togenerate significant affinity improvements in atherapeutically relevant humanized anti-RAGE ratantibody. Furthermore, our mutational scanningapproach, facilitated by ribosome display and high-throughput screening, has revealed that humanizedXT-M4 tolerates levels of mutation both inside andoutside the CDR loops higher than those previouslyreported for similar studies on fully human anti-bodies. Finally, this study suggests a possible strategyto both affinity optimize and humanize murinemonoclonals simultaneously, generating antibodieswith reducedmurine sequencewithin theCDR loops.
Materials and Methods
Generation of display vectors
The phagemid vector pWRIL-1 was constructed viarestriction–ligation from two separate synthetic genefragments (GeneArt). Briefly, a backbone mini-plasmidwas generated containing the required functional ele-ments for a functional phagemid. These included DNAsequences encoding a bacterial origin of replication (pUCori), a resistance cassette (AmpR), and an F1 bacteriophageorigin for phage packaging. A secondary “insert con-struct”was also created, which contained DNA sequencesencoding the expression and display elements of thevector: a lac promoter–operator sequence, OmpA leaderpeptide, dual SfiI restriction sites containing a stufferfragment, hexa-histidine and c-Myc peptide tags, anamber stop codon, and a truncated M13 g3 protein(encoding residues 250–406).The ribosome-display vector pWRIL-3 was also con-
structed from synthetic gene fragments (GeneArt). Keyfunctional elements were incorporated, including anorigin of replication (pUC ori), a resistance cassette(AmpR), a T7 promoter and ribosome binding site for invitro transcription and translation in prokaryotic (E. coli)extracts, 5′ and 3′ stem–loop structures, a spacer (residues249–318 of M13 g3 protein), and a Flag tag at the N-terminus. A key additional feature of pWRIL-3 was theincorporation of SfiI restriction sites that are fullycompatible with those in pWRIL-1.
canning of XT-M4. (a and b) Frequency of amino acid4 binding mutants). The CDRs are marked by black bars.and d) Space-filling representations of a molecular modelthe heavy chain in dark gray. The amino acid residuesino acid residues highlighted in green are the predictedg site comprising the six CDR loops is marked in (c) with acture, diametrically opposite to the antigen-combining siteaffinity-matured clone containing the F100bL mutation (e)eavy chain is in dark blue; that of the light chain, in lightd and pink, respectively, and the highlighted residues aree residue at Kabat position 100b is shown in purple. F100bR1), L89 (CDR3), F91 (CDR3), and L96 (CDR3) of the lightonly interacts with N34 (CDR1) and L89 (CDR3).
554 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
Synthesis and characterization of the anti-RAGEM4-V2.11 scFv
Humanized XT-M4 scFv (designatedM4 hereinafter) wassynthesized (GeneArt) in the VL–VH formatwith the flexiblelinker amino acid sequence DGGGSGGGGSGGGGSS.Additionally, the M4 scFv was constructed to contain extrarestriction sites at both the N-terminus of the Vκ domain(BssHII) and the C-terminus of the VH domain (BclI) tofacilitate rapid cloning to the scFv–Fc fusion format.M4 scFvwas restriction digested with SfiI, ligated into pWRIL-1(hereinafter designated as pWRIL-1-M4), and transformedinto E. coli TG1 (Stratagene). M4 scFv was examined forfunction on phage and when produced in non-p3-linkedsoluble form in the periplasm by anti-mRAGE–Fc ELISA.53
Production of functional, full-length soluble scFv in theperiplasm of E. coli was further confirmed by Western blotanalysis of periplasmic extracts, using anti-c-Myc 9E10-HRP(horseradish peroxidase) (Roche) for scFv detection.
Mutagenesis and construction of phage libraries
M4scFv librarieswere constructed bySOE-PCR to containa mutagenized sequence in either the heavy-chain CDR3 orthe light-chain CDR3. To construct two separate sublibrariesof heavy-chain CDR3 mutants (M4-H1 and M4-H2), weperformed saturation mutagenesis by SOE-PCR using NNSdegenerate codons, essentially as described previously.41
Primary and secondary (SOE) PCRs were performed usingPhusion polymerase (Finnzymes), according to the manu-facturer's recommendations. Two separate sublibraries(M4-L1 and M4-L2) were also generated with mutagenesisspanning the light-chain CDR3 using a mix of NNS andparsimonious codons that encode for a more restrictedset of amino acids and using no stop codon in the caseof the NNT codon.34,42 Primary PCRs were performedusing the mutagenic primer M4-L3-mut-LS1 (TTCGCC-ACCTACTACTGCNNGNAKNNTNNTNNTNNSCCTCT-GACCTTTGGCGGCGGAACAAAG) or M4-L3-mut-LS2(TTCGCCACCTACTACTGCCTGGAGTTCNNTNN-TNNSNNTNNSNNTTTTGGCGGCGGAACAAAG).SOE-PCR products were restriction digested, purified,
and cloned into the pWRIL-1 vector in E. coli TG1 asdescribed previously.53 Total library sizes were calculatedby plating serial dilutions of transformations onto LBagar/100 μg per milliliter of carbenicillin/2% (v/v)glucose (LB-CG). Total cell populations from each electro-poration were plated onto 22-cm bioassay dishes (Genetix)containing 2YT agar/100 μg per milliliter of carbenicillin/2% (v/v) glucose (2YT-CG), incubated overnight at 30 °C,and finally harvested by scraping and resuspension in 2YTbroth/20% (v/v) glycerol. Library aliquots were thenfrozen at −80 °C.
Definition of effective concentration range for solubleantigen in phage selections
E. coli TG1 cells containing pWRIL-1-M4 were grown tomid-log phase in 5 ml of 2YT-CG medium, infected withM13KO7 helper phage (1.0×1010 pfu/ml), and incubatedfor 1 h at 37 °C/shaking at 80 rpm toperformM4 scFvphagerescue. Infected cells were then harvested by centrifugationand resuspended in 50ml of 2YT broth/100 μg permilliliterof carbenicillin/50 μg per milliliter of kanamycin (2YT-CK)before culture overnight at 30 °C/shaking at 300 rpm. M4-phage particles were purified from the culture supernatantusing double precipitation with PEG (polyethylene glycol)and sodiumchloride (froma 5× stock: 200 g of PEG8000 and
150 g of NaCl per liter in H2O) and centrifugation. Twice-precipitated phage was then resuspended in phosphate-buffered saline (PBS)/3% (w/v) driedmilk protein (M-PBS)at a concentration of ∼1×1012 cfu/ml. A solution-phaseselection method54 was applied, with selection on thebiotinylated forms of either hRAGE–Fc or mRAGE–Fc orselection on an irrelevant antigen [bovine serum albumin(BSA)] at multiple concentrations (25, 5, 0.5, and 0.05 nM).Antigen-specific pull down was performed using streptavi-din-conjugated magnetic beads (M280, Invitrogen), and thebeads were washed 10 times with 1ml of PBS/0.1% Tween-20 (PBS-T), followed by 5 times with 1 ml of PBS. Phageelution was performed using triethanolamine (100 mM) atroom temperature for 30 min, followed by neutralizationwith Tris–HCl, pH 7.4. Recovered phageswere infected intolog-phase E. coli TG1 cultures, and infection rates wereenumerated by titration plating on 2YT-CG agar plates.
Ribosome-display library construction
M4 scFv was digested with SfiI and cloned into pWRIL-3 (hereinafter designated as pWRIL-3-M4). For theconstruction of the random mutagenesis (error-pronePCR) library for ribosome display, the pWRIL-3-M4DNA was used as a starting template and amplifiedusing a GeneMorphII random mutagenesis kit (Strata-gene). The reaction was performed according to themanufacturer's instructions at a predicted mutation rateof 4.5–9 mutations per kilobase. Library construction wasperformed essentially as described previously.55
The assembled scFv library with gene-3 tethersequences was transcribed using a Ribomax kit (Promega)with a total DNA input of 5 μg. Transcribed RNA waspurified using Illustra Probe Quant G-50 microcolumns(GE Healthcare) according to the manufacturer's proto-cols. RNA quality was estimated using OD260/OD280 andagarose gel electrophoresis. Translation was performedwith an input of 10 μg of RNA using a PureSystem S–S kit(Wako) as per the manufacturer's instructions with theaddition of 0.1 μg/μl of protein disulfide isomerase(Sigma-Aldrich).
Phage library rescue and selections
Cell density in each of the mutagenized library stockswas estimated by OD600, and inoculum volumes werecalculated not only to begin growth of starter cultures forlibrary rescue at anOD600b0.1 but also to overrepresent thetotal theoretical diversity of each library 10-fold. Libraryrescue was then performed as described previously.56
The solution-phase selection method54 was applied toselect for affinity-improved binders from each library. Inbrief, a progressively lower concentration of biotinylatedantigen was applied in each round beginning at 5 nM (theapproximate on-phage EC50 for M4 scFv on biotin-hRAGE–Fc) in round 1 and then dropping in log10increments per round. Increasing numbers of washingsteps and off-rate competition with 1000-fold excess ofnonbiotinylated antigen were also applied in rounds 4–6.All four libraries were kept separate in round 1, withprogressive pooling of selected libraries L1+2 and H1+2in rounds 2–6.
Ribosome-display library selections
Ribosome display was carried out essentially asdescribed previously.55 Equilibrium selections in solution54
555Affinity Maturation of XT-M4 for Anti-RAGE Therapy
were performed for 1 h at 4 °C using biotinylated hRAGE–Fc. The starting antigen concentration in round 1 ofselections was 5 nM. Antigen concentrations in subsequentrounds were progressively decreased from 5 nM to 0.5 pMin log10 increments. The eluted RNAwas purified using aHigh Pure DNA Isolation kit (Roche) as per the manufac-turer's recommendations and immediately denatured andconverted to cDNA using a Superscript III first-strandsynthesis kit (Invitrogen).The progression of selections was monitored by the
analysis of cDNA samples.55 Outputs from sequentialrounds of selection were amplified and cloned intopWRIL-1, as an SfiI-digested fragment, for sequenceanalysis and binding activity in ELISA and HTRF assays.In total, three rounds of selection were performedinterspersed with low-fidelity PCR to introduce furthersequence diversity.
ScFv expression and purification
For high-throughput screening, E. coli clones wereplated on 2YT-CG agar in 22-cm bioassay trays (Genetix),picked into standard sterile 96-well plates containing 2YT-CG broth using a QPix II colony picker (Genetix), andgrown in a Multitron multiplate incubator (Infors AG) at600 rpm, 37 °C, and 80% humidity overnight. Followinggrowth, glycerol was added to a final concentration of 30%(v/v) and plates were stored at −80 °C or used toimmediately inoculate 96-well deep-well plates containing900 μl of 2YT-CG broth. These were grown at 37 °C, 80%humidity, and 600 rpm for 5–6 h; expression was inducedby addition of IPTG to a final concentration of 0.02 mMand incubation at 30 °C overnight. Cells were pelleted bycentrifugation at 1260g and resuspended in 150 μl of ice-cold periplasmic buffer [50 mM Hepes, 0.5 mM ethylene-diaminetetraacetic acid, and 20% sucrose (w/v), pH 7.5].Osmotic shock was induced by addition of 150 μl/well ofa 1:5 dilution of periplasmic buffer, and samples wereplaced on ice for 30 min. This was followed bycentrifugation at 3220g for 20 min, and the supernatant,consisting of the periplasmic fraction containing expressedscFv's, was recovered. High-throughput screening byELISA or HTRF assay was carried out using crudeperiplasmic extracts in single-point analyses.Small-scale, single-step scFv purifications were carried
out for more detailed HTRF titration analysis. E. coli clonesof interest were selected on the basis of performance in theHTRF screening assay and inoculated for small-scaleprotein expression and purification as previouslydescribed.57 In cases where a more detailed biochemicalanalysis was required, clones were prepared for larger-scale purifications by overnight induction in 500ml of 2YT-C shaking culture, and 20 ml of each PeriPrep buffer wasthen used to resuspend the resulting pellet. ScFv proteinswere subsequently purified in a two-step protocol using anAKTA Explorer system as previously described.57
SDS-PAGE, protein quantification, and SEC analysis
Protein samples were run under reducing conditionsusing a NuPAGE Novex system (Invitrogen). Gels werestained with Instant Blue (Novexin) and visualized usingthe Chemidoc XRS gel documentation system (Bio-Rad).Protein concentration was determined using a MicroBCAprotein assay kit (Pierce) and read in an Envision Multi-plate Reader (Perkin Elmer) at 595 nm. Analytical SECwascarried out on purified proteins to test for the presence ofimpurities and high-molecular-weight species or aggre-
gates. Due to the limiting quantities of protein availablefrom small-scale preparations, protocols were optimizedusing an Agilent 1200 series HPLC system with anautosampler that can load from standard 96-well plates.57
Binding ELISA
Maxisorp plates (Nunc) were coated with 1 μg/ml ofhuman or murine RAGE–Fc in PBS overnight at 4 °C.Wells were washed three times with 300 μl of PBScontaining 0.05% (v/v) Tween-20, using a Zoomwasherliquid handling robot (Titertek), and blocked in 200 μl ofPBS/3% (w/v) dried milk protein with 1% BSA for 1 h.Crude periplasmic extracts (25%, v/v) or serial dilutionsof purified proteins (100 μl) were added to the plate andincubated for 1 h. Following five washing cycles on theZoomwasher, detection was achieved using an HRP-conjugated anti-human or anti-murine antibody for scFv–Fc and IgG analyses (Pierce) and an HRP-conjugated anti-c-myc antibody for scFv (Roche). The reaction wasdeveloped using UltraTMB (Pierce) and stopped by a 1:1addition of 0.18 M phosphoric acid. The plate was read inan EnVision Multiplate Reader at 450 nm. Data wereplotted using Prism 5 software (GraphPad).
High-throughput HTRF screening assay
A high-throughput competition HTRF assay wasestablished in order to allow identification of affinity-improved clones. The parental humanized M4 antibodywas labeled with europium cryptate using a cryptatelabeling kit (CisBio) according to the manufacturer'sinstructions. The final reaction mix contained 1.5 nMbiotinylated human or murine RAGE–Fc, 1:1600 dilutionof SA-XL665 (CisBio), 1:1000 dilution of the europiumcryptate-labeled parental XT-M4, and 0.5% (v/v) periplas-mic extract containing scFv's of interest, prepared asdescribed above, in a total reaction volume of 20 μl in 1×assay buffer [50 mM sodium phosphate, pH 7.5, 400 mMpotassium fluoride, and 0.1% BSA (w/v)]. Reagents wereadded sequentially on a MiniTrak Liquid HandlingPlatform (Perkin-Elmer) into 384-well low-volume blackplates (Nunc). Reactions were allowed to proceed for 3 hat room temperature, and plates were subsequently readon the EnVision Multilabel Plate Reader (Perkin-Elmer)with excitation at 340 nm and two emission readings at615 nm (measuring input donor fluorescence from XT-M4-europium cryptate) and 665 nm (measuring outputacceptor fluorescence from SAXL665). All readings wereexpressed as the percentage of change in fluorescence,%ΔF, where:
kDF=ðF665 Sample=F615 SampleÞ�ðF665 Control=F615 ControlÞ
ðF665 Control=F615 ControlÞ� �
� 100
“Control” represents the background fluorescence energytransfer in wells containing 1:1000 labeled XT-M4, in assaybuffer, alone. All data were plotted using Decision Site 8(Spotfire) and Prism 5 software (GraphPad).
scFv–Fc and IgG expression and purification
Fc fusion proteins and IgGs were transiently expressedin COS-1 cells after transfection using Lipofectamine 2000(Invitrogen). Conditioned medium was harvested after72 h, and cells were removed by centrifugation. The
556 Affinity Maturation of XT-M4 for Anti-RAGE Therapy
resulting supernatant was filtered and purification wascarried out using Protein A MultiTrap (GE Healthcare) aspreviously described.57 Purified proteins were pooledbefore buffer exchange into PBS.
BIAcore analysis of IgGs
BIAcore analysis was performed using T-100 biosensor,series S CM5 chips, an amine-coupling kit, 10 mM sodiumacetate immobilization buffer at pH levels of 4, 4.5, 5.0,and 5.5, 10× HBS-P running buffer, and 50 mMNaOH (GEHealthcare). Assay conditions were established to mini-mize the influence of mass transfer, avidity, and rebindingevents, as described below. A targeted ligand immobiliza-tion programwas set to immobilize approximately 100 RU(response units) of purified hRAGE at pH 5.0. A blankimmobilization was carried out on a separate flow cell forreference subtraction. The purified IgGs were diluted inHBS-P running buffer to a range of final concentrations (0–10 nM for calculation of IgG kinetic constants usingglobal-fit analysis). Each concentration was injected for3 min at a fast flow rate of 50 μl/min and allowed todissociate for 10 min, followed by a 5-s regeneration pulsewith 50 mMNaOH. Reference-subtracted sensorgrams foreach concentration were analyzed using BIAcore T-100evaluation software (1.1.1).
Modeling XT-M4 anti-RAGE scFv
Molecular models of the humanized anti-RAGE XT-M4antibody heavy chain were built with Modeler 8v2 using1MHP (anti-alpha1 beta1 antibody), 1IGT (anti-caninelymphoma monoclonal antibody), 8FAB (anti-p-azophe-nylarsonate antibody), 1MQK (anti-cytochrome C oxidaseantibody), and 1H0D (anti-angiogenin antibody) astemplates. From 100 initial models, the model with thelowest restraint violations, as defined by the molecularprobability density function, was subjected to an energy-minimization cascade consisting of steepest descent,conjugate gradient, and adopted-basis Newton–Raphsonmethods until an RMS gradient of 0.01 was satisfied usingCharmm 27 force field (Accelrys Software, Inc.) andgeneralized Born implicit solvation as implemented inDiscovery Studio 1.6 (Accelrys Software, Inc.). Duringenergy minimization, the movement of backbone atomswas restrained using a harmonic constraint of 10 massforce. A similar procedure was followed for modeling thehumanized (CDR grafted) anti-RAGE XT-M4 antibodylight chain, using 1B6D, 1FGV (anti-CD18 antibody), 1UJ3(anti-tissue factor antibody), and 1WTL as templates.Mutation F106L was modeled into the model of RAGE XT-M4 by using the “model.mutate” routine of Modeler 8v2,followed by a similar energy-minimization cascade asmentioned for the parent molecule, except that all atoms ofthe mutated residue, atoms within 5-Å radius of themutated residue, and all atoms in the loops were notsubject to the harmonic restraint.
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