INTRODUCTIONChronic lymphocytic leukemia (CLL)

is an incurable disease of unknown etiol-ogy (1,2). The blood and lymphoid tis-sues of CLL patients contain high levelsof a single clone of B lymphocytes withunique surface membrane immunoglob-ulin (smIg) components of B-cell recep-tors (BCRs) for antigen, essentially the

cell-surface version of a monoclonal anti-body (mAb). On the basis of the biaseduse of specific immunoglobulin heavychain variable (IGHV) genes by CLLB cells (3) and the not uncommon strik-ing structural restriction in amino acidsequence (4–8), however, it seems likelythat disease initiation and progressionare influenced by (auto)antigen selection

and drive of a specific set of humanB lymphocytes (1,9,10). Studies to datesuggest that this drive is more frequentand effective among CLL clones with un-mutated IGHV genes (U-CLL) (11) thatcan bind multiple antigens than amongCLL clones with mutated IGHVs (M-CLL) (12–15).

Furthermore, the categorization ofCLL BCRs according to the degree of so-matic nucleotide mutations in the ex-pressed IGHV segment (3) can quite ac-curately predict a patient’s clinical course(16,17). Specifically, patients whoseclones exhibit BCRs containing 2% orless difference from the germline (U-CLLBCRs) are much more likely to exhibitaggressive disease than those >2% differ-ence (M-CLL BCRs). Therefore, charac-terization of BCR-antigen interactions isof general importance for understandingmore and less aggressive forms of the

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Peptides That Bind Specifically to an Antibody from a ChronicLymphocytic Leukemia Clone Expressing UnmutatedImmunoglobulin Variable Region Genes

Yun Liu,1 Chelsea D Higgins,2 Cathie M Overstreet,3 Kanti R Rai,4 Nicholas Chiorazzi,1,4 and Jonathan R Lai2

1The Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, New York, United States of America;2Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America; 3Department ofChemistry, Department of Molecular Biology and Biochemistry, University of California, Irvine, California, United States of America;and 4Departments of Medicine and of Molecular Medicine, Hofstra North Shore-LIJ School of Medicine, Hofstra University,Hempstead, New York, United States of America

Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the dis-ease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) en-gage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined thebinding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable do-mains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected pep-tide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in familiesbased on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb re-vealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (mi-cromolar KD), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations(nanomolar KD) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens.This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthysubjects with monoclonal B lymphocytosis.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2013.00082

Address correspondence to Nicholas Chiorazzi, The Feinstein Institute for Medical Re-

search, 350 Community Drive, Manhasset, NY 11030, USA. Phone: 516-562-1090; Fax: 516-

562-1011; E-mail: NChizzi@NSHS.edu; or Jonathan R Lai, Department of Biochemistry, Albert

Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Phone: 718-

430-8641; Fax: 718-430-8565; E-mail: Jon.Lai@Einstein.YU.edu.

Submitted August 1, 2013; Accepted for publication August 1, 2013; Epub

(www.molmed.org) ahead of print August 2, 2013.

disease. Current evidence suggests thatU-CLL BCR combining sites can interactwith a variety of antigens (12–15). Al-though these U-CLL BCR-antigen inter-actions are presumed to be of low affin-ity, specific measurements of affinity arenot available.

Because of these observations, it istantalizing to hypothesize that multispe-cific interactions between U-CLL BCRsand antigens drive the proliferation ofleukemic B cells. Here we describe theuse of peptide phage display containinga very large number of random epitopesto study recognition by a U-CLL BCR.Our motivation for this work was two-fold. First, the identification of agentsthat can target U-CLL BCRs would be ofpotential use for diagnosis and treat-ment. Current CLL therapies are not tar-geted to BCRs with specific structuresand, therefore, the specificity of suchtreatments could be enhanced by devel-opment of U-CLL BCR-binding peptidesas targeting agents. Furthermore, suchreagents could be used in flow cytome-try assays to determine whether B lym-phocytes bearing specific BCRs are pres-ent in blood and in what abundance.This might be especially helpful, notonly in CLL, but also in healthy individ-uals with the condition known as mono-clonal B lymphocytosis (18,19) that ap-pears to be a prerequisite preamble toCLL (20). Second, the characterization ofbinding specificities offers insight intothe behavior of a disease-associatedgermline-encoded antibody, and the ex-tent that these antibodies resemble earlystages of normal B-cell and antibodyevolution. Peptides that are specific forU-CLL could serve as biological andstructural probes to decipher aspects ofU-CLL BCR recognition.

In this study, we identified a diversepanel of peptide-phage clones that bindan mAb derived from a U-CLL clone.Studies with synthetic peptides indicatedthat some were specific for this U-CLLBCR and that in general the affinities ofthese, measured by surface plasmon res-onance, were much lower than that of anM-CLL mAb.

MATERIALS AND METHODS

Expression and Purification of U-CLLmAb 068 and M-CLL mAb169

Studies were approved by the Institu-tional Review Board of North Shore–LIJHealth System (Manhasset, NY, USA) andperformed in accordance with theHelsinki agreement. In brief, ribonucleicacid (RNA) from patient’s blood mononu-clear cells was reverse-transcribed intocDNA. The variable segments of re-arranged immunoglobulin heavy (IGH)and immunoglobulin light (IGL) of U-CLL068 and M-CLL 169 were expressed ashuman IgG1 as described previously (15).

Phage Library Sorting and AnalysisPreparation of the pVIII peptide library

was reported previously (21). Wells ofCostar EIA/RIA high-binding plates(Corning, Big Flats, NY, USA) were coatedwith U-CLL mAb068 in phosphate-buffered saline (PBS, pH 7.4) for 1 h atroom temperature. The well solutionswere decanted and unbound well sitesblocked by incubation with PBS contain-ing 3% (w/v) human serum albumin(HSA) for 1 h at room temperature. Afterwashing with PBS containing 0.05 % (v/v)Tween 20 (PBS-T), freshly amplified li-brary phage at titers of ~1013 plaque- forming units (PFU)/mL in PBS/0.5 %HSA were added to wells and allowed tobind for 1 h. Next, wells were washed sixtimes with PBS-T. Bound phages wereeluted by the addition of 100 μL100 mmol/L glycine hydrochloride(Gly·HCl) pH 2.0 for 5 min. The phage so-lution was neutralized in 30 μL of 2 mol/L tris(hydroxymethyl)aminomethane (Tris)pH 8.0 and then propagated overnight at37°C in XL1-Blue E. coli (Stratagene, LaJolla, CA, USA). The cells were removedby centrifugation, the phage precipitatedby addition of 0.5 mol/L NaCl and 4%(w/v) polyethylene glycol (PEG) 8000 andused directly for the subsequent round ofselection.

Reactivity of output populations fromthe selection with U-CLL mAb068 orHSA were assessed using phage enzyme-linked immunosorbent assay (ELISA).

mAb068 or HSA was immobilized intowells of Costar EIA/RIA plates andblocked as above. Freshly-amplifiedphage populations from each round ofselection were incubated with the targetfor 1 h at room temperature, and thewells washed three times with PBS-T. Ananti-M13/horseradish peroxidase conju-gate (GE Healthcare, Piscataway, NJ,USA) was added as per the manufac-turer’s protocol and allowed to incubatefor 1 h. After washing with PBS-T, 100 μLof 3,3′,5,5′-tetramethylbenzidine sub-strate (SureBlue, Kirkegaard & PerryLaboratories, Gaithersburg, MD, USA)were added. The color was allowed todevelop for 5 min, quenched with1 mol/L HCl, and the absorbance at450 nm determined. Analyses of individ-ual clones from the R3 and R4 popula-tions were similar, except that the phageswere grown as small-scale cultures(1 mL) in 96-deep well plates. Followingcentrifugation, the culture supernatantswere applied directly to the immobilizedU-CLL mAb068 or HSA.

Peptide Synthesis and PurificationAll peptide were produced by solid-

phase peptide synthesis using standardFmoc (N-[9-fluorenyl]methoxycarbonyl)chemistry at the Tufts University Biopoly-mer Facility (Boston, MA, USA). Follow-ing synthesis, simultaneous side chaindeprotection and cleavage from resinwere achieved by treating them with 95%trifluoroacetic acid, 2.5% 1,2-ethanedithiol,and 2.5% thioanisole for 3 h. The resinwas removed by filtration, and the pep-tide precipitated by the addition of colddiethylether. The peptide was pelleted bycentrifugation, the pellet washed twicewith cold diethyl ether, and the peptidesdissolved in water/acetonitrile andlyophilized. For peptides containingdisulfide bonds, the crude peptide wasdissolved in water at low concentrations(<0.5 mg/mL) and the pH adjusted to 7to 8. Disulfide oxidation was achieved bybubbling air through this solution for 2 to3 d. Oxidation progress was monitoredby reverse-phase analytical high- performance liquid chromatography

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(HPLC) on a Vydac® C18 column (10 μm ×250 mm × 21.2 mm; Grace, Deerfield, IL,USA) with water/acetonitrile/0.1% TFAmobile phases. Once oxidation was com-plete, the solution was lyophilized. Crudepeptides were purified by reverse phase(RP)-HPLC on a Vydac preparatory-scaleC18 column (10 μm × 250 mm ×21.2 mm). Peptide purity was generally>95% as judged by analytical RP-HPLC,and the identity of all peptides was con-firmed by matrix- assisted laser desorp-tion/ionization (MALDI MS).

Surface Plasmon ResonanceAll measurements were performed on

a Biacore 3000 instrument at room tem-perature in running buffer (20 mmol/Lphosphate buffer [pH 7.4], 130 mmol/LKCl, 3.4 mmol/L EDTA, 0.005% Tween20 [v/v]). U-CLL mAb068 or M-CLLmAb169 were immobilized to CM5 chips (GE Healthcare) as specified by the manufacturer at loading levels of17,000–22,000 response units (RUs). Thepeptides were dissolved in runningbuffer and sensorgrams obtained byflowing peptide solutions at various con-centrations over the immobilized mAbsurface at a flow rate of 40 μL/min. Sensorgrams were fit to a standard 1:1Langmuir model using BIAevaluation 4.1(Biacore) software. Adequacy of data fit-ting was assessed using the parametersχ2. When possible, mAb-coated surfaceswere regenerated by 10 mmol/L KOH.Peptide and protein concentrations weredetermined by absorbance at 280 nm.

All supplementary materials are availableonline at www.molmed.org.

RESULTS

A mAb That Corresponds to a U-CLLBCR

The amino acid sequences of theIGHV-D-J and IGLVκ-Jκ rearrangementsdetermine the architecture of the antigen-binding domain of a BCR (Figure 1). Forthe following studies, the IGHV-D-J andIGKVκ-Jκ rearrangements from theleukemic cells of patient CLL068 were

linked molecularly to an IGHCγ1 seg-ment to produce an immunoglobulin G1(IgG1) mAb. Although the native CLL068 BCR/mAb is of the immunoglobulinM (IgM) isotype, structural studies withfull-length mAbs and mAb fragments(for example, antigen-binding fragments,Fabs, or single-chain variable fragments

(scFvs), indicate that overall combiningsite topology is usually independent ofconstant domains (22), although excep-tions have been noted (23). Therefore, anIgG1 mAb containing the IGHV-D-J/IGLV/J rearrangements of a U-CLL BCRwill likely be an effective mimic of thecell-surface combining site.

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Figure 1. (A) Domain architecture of an IgG1 mAb and the surface membrane IgM of aBCR. (B) Polyclonal phage ELISA of R0–R4 populations, as well as K07 control phage, againstU-CLL mAb068. The R3 and R4 populations also were tested against wells containing thenegative control protein HSA (gray). CH: immunoglobulin heavy chain constant region; CL:Immunoglobulin light chain constant region; CH1, CH2, CH3, and CH4: immunoglobulin con-stant region heavy chains 1, 2, 3, and 4, respectively; VL: immunoglobulin light chain variableregion; VH: immunoglobulin heavy chain variable region.

The design and analysis of U-CLLmAb068 expressed with a human IgG1domain has been described previously(15). The U-CLL mAb068 antigen-bindingdomain contains the IGHV1-69 germlinesegment, which is frequently selectedamong virus neutralizing antibodies andwhose combining site interactions typi-cally involve a hydrophobic second heavychain complementarity-determining re-gion (HCDR2) (24), linked to IGHD3-16and IGHJ3 segments. It is estimated thatthe IGHV1-69 germline segment com-prises less than 2% of productive re-arrangements in normal blood B lym-phocytes (25), perhaps a surprisingobservation considering its prevalence inCLL cells and viral responses. Both theIGHV-D-J and IGLVκ-Jκ rearrangementsof U-CLL mAb068 are identical togermline segments (Supplementary Fig-ure S1). In addition, these rearrange-ments exhibit characteristic junctionalamino acids (4). Finally, the antigen-binding site represented by the U-CLLmAb068 IGHV-D-J and IGLVκ-Jκ re-arrangement is shared by multiple CLLpatients (4-8). Therefore understandingthe antigen-binding characteristics of U-CLL mAb068 will be applicable to thoseof multiple patients.

Phage Display Selection of Peptidesthat Bind U-CLL mAb068

To explore the range of U-CLLmAb068 combining site reactivities, wescreened a high complexity peptide

phage display library against immobi-lized U-CLL mAb068. Previous attemptsto isolate binding peptides that targetU-CLL mAb068 from a commercially-available library containing random lin-ear peptide segments displayed on theM13 minor coat protein pIII (the “PhD li-brary”) yielded only a single sequencewith low-level binding activity (sequenceWPLWSIPPFSPR) (26). Here, we used alibrary in which random peptide seg-ments (containing both linear and disul-fide crosslinks) were expressed on themajor M13 coat protein pVIII using atwo-plasmid phagemid/helper phagesystem (27). The diversity of the dis-played peptides (Table 1) includes ran-dom segments from 7 to 20 residues, inmany cases containing cysteine residuesat various spacings that are expected to

form disulfide bonds upon phage assem-bly. Linear random sequences (X8) alsowere included, as were sequences con-taining a central reverse turn–promoting~GP~ dipeptide. The pVIII display for-mat is expected to display peptides on~50 of the 2700 pVIII copies per M13phage particle. The library complexitywas 1011 clones (21).

Four rounds of selection were per-formed against U-CLL mAb068. PhageELISA of the unselected library popula-tion (R0) and the output populationsfrom rounds 2, 3 and 4 (R2–R4) indicatethat the selection resulted in enhancedbinding activity for wells containingU-CLL mAb068. To examine the speci-ficity of the later rounds, the R3 and R4populations were tested for binding towells containing the negative control

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Table 1. Random peptide sequences inphage display library.a

CX5C X2CX4CX2 X4CX10CX4

CX5CX X2CX5CX2 X5CX8CX5

CX5CX2 X2CX6CX2 X5CX9CX4

XCX5C X2CX7CX2 X6CX6CX6

X2CX5C X2CX8CX2 X6CX7CX5

X2CX9CX2 X7CX4CX7

X2CX2CX2 X2CX10CX2 X7CX5CX6

X2CX2CX3

X2CX3CX2 X4CX2GPX4CX4 X8

aAll random segments produced asN-terminal pVIII fusions; X = any aminoacid as encoded by the combinatorialcodon NNS (N = A/T/C/G, S = C/G).

Table 2. Isolated phage clones from selection with U-CLL mAb068.

Clone(s) (Frequency)a Sequenceb [A450 (mAb068)]/[A450 (HSA)]c

B12, D7, E4, F9 (4) NMSIATVP 28C2, F3, F7, H11 (4) ASCYIDDGIRYCTG 28A6, B1, D2, E12 (4) VSCNPFSWLPRCYA 19A5, D4, D5 (3) DYCNLSVSWCYP 26F1, F5 (2) STCWLMFSMCRV 24A4, B6 (2) TPCEGSWFFCTL 23E2 LGCDVIYWSSGWGCKF 31B3 GSCSLSIVTIPCGV 30D3 VTCAAFSLFPFCYN 28E7 CHPGWPCFW 28D8 GGCNLSVLWCLD 27F10 NECILVNGDRWCIS 27E6 FPCWALPSQYLCRP 27D11 TACNLSVSWCEF 20G11 TNCGFLYLGCVI 18E9 FDCGDGSWRYYLDCVR 18B7 TSCRVLWIMTLCNA 18B10 EVCNNKLWPCFW 16A8 GGCGWLWVSCVI 14A2 CSPAWPCWW 14F2 LPCAGRSFPCYW 14C8 DRCLLWVSLCMA 13E1 CIPMWPCWW 13

aIdentified clones and observed frequencies in cases where multiples were observed.bThe underlined sequences specifically bound to U-CLL mAb068, but not an unrelatedmAb, and retained binding activity in 750 mmol/L NaCl.cRatio of ELISA signals for wells coated with U-CLL mAb068 over wells coated with HSAnegative control. In cases where a sequence was observed more than once, the averageis shown. For all others, results for a single replicate are shown. In general, data amongindependent experiments were in good agreement.

protein, human serum albumin (HSA)(Figure 1B). Although some binding ac-tivity of the R3 and R4 populations wasobserved against HSA, the higher bind-ing signals against U-CLL mAb068 atlower phage titers relative to HSA indi-cated that U-CLL mAb068-specificclones were present in the R3 and R4population. Furthermore, a negativecontrol phage bearing no peptide (K07)showed minimal binding to U-CLLmAb068. Sequence analysis indicatedthe R3 population clones were highly di-verse in sequence.

Analysis of Individual Phage ClonesIndividual clones from the R3 and R4

population were screened for reactivitywith U-CLL mAb068 using high-throughput phage ELISA. We identified36 clones that had strong positive ELISAsignals for U-CLL mAb068 (A450 >1.0)and had specificity over control wellscontaining HSA (an [A450 (mAb068)]/[A450 (HSA)] ratio of 13-fold was used as a cutoff). These clones correspondedto 23 unique amino acid sequences(Table 2). Many of the sequences con-tained the cysteine residues at variousspacings ranging from 2 to 10, althougha CX6C pattern was more commonly ob-served. Four copies of the linear se-quence NMSIATVP also were present inthe dataset. To further evaluate selectiv-ity for binding the CLL-associated mAb,we tested all 23 of these sequences forbinding to an unrelated mAb and for

their capacity to retain U-CLL mAb068binding activity under high salt condi-tions (750 mmol/L NaCl). Eighteen ofthe 23 sequences fulfilled the criteria ofbeing specific for U-CLL mAb068 and re-taining binding activity in high salt (un-derlined in Table 2 and shown in Supple-mentary Table S1). Since these clones didnot bind an unrelated IgG1 mAb, weconclude that they have specificity forU-CLL mAb068 and that the peptidesbind the variable domains of the CLLmAb. Furthermore, the ability of theseclones to bind under high salt conditionssuggests the interactions between thepeptides and U-CLL mAb068 involvespecific contacts and are not due to gen-eral electrostatic or hydrophobic binding.

To gain further insight into binding selectivity, we grouped the 18 mAb068-specific sequences into families based on amino acid composition. We foundthat the clones clustered into five dis-tinct families of varying conservation(Figure 2). Clone E6 was not closely re-lated to any other clone and thereforewas not classified according to any fam-ily. Members of families 1, 2 and 4showed high conservation, suggestingthat these clones bind in a similar man-ner. However, families 3 and 5 containedmore disparate primary sequences. Over-all, the diversity in the sequences, fromcontent of hydrophilic/hydrophobicresidues to spacing between cysteineresidues, indicates that U-CLL mAb068 isable to bind a number of sequences.

Phage display studies with the linearPhD library resulted in strong consen-sus sequences against mAbs associatedwith the less aggressive M-CLL BCR-containing B-cells (26). Previous studiessuggest that greater antigen-bindingpromiscuity of U-CLL than M-CLL BCRcombining sites may lead to the higheractivation of U-CLL clones (28) andtheir more aggressive clinical properties(16,17). Of note, selection of phage pep-tides against antibodies does not neces-sarily provide information about bind-ing promiscuity. For example, anaffinity-matured HIV-1 antibody hasbeen reported to display bindingpromiscuity for several disparate pep-tide sequences from synthetic andphage displayed libraries (29). Nonethe-less, it is interesting to note that distinctfamilies of sequences were obtainedfrom selection against U-CLL mAb068.

In Vitro Characterization of U-CLLmAb068-Binding Peptides

We synthesized several peptides corre-sponding to sequences from the U-CLLmAb068 selection isolated here: peptideD4-Btn belonging to the well conservedfamily 2; peptide D2-Btn from the lessconserved family 5 with longer length;and peptide E6-Btn with no significantsimilarity to other selectants (Table 3). Wealso produced two peptides identified inour previous study using the commer-cially available 12-mer PhD library (26):peptide PhD-068-4-Btn isolated withU-CLL mAb068 and peptide PhD-169-8-Btn, isolated by an IGHV-mutated CLLmAb with different IGH and IGL variableregions (M-CLL mAb169). All peptideswere purified by HPLC in good yield. Inall of the above cases, a C-terminal biotinwas included for detection. Disulfidebonds in D4-Btn, D2-Btn and E6-Btn wereformed by air oxidation; MALDI-MS andHPLC analysis indicated disulfide bondformation was complete in all cases.

ELISAs in which the binding of pep-tides to immobilized U-CLL 068 mAbwas detected with streptavidin-horse-radish peroxidase (SA-HRP) were per-formed (Figure 3). Peptide D2-Btn

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Figure 2. Family analysis of the 18 sequences that had specificity for U-CLL mAb068.

bound U-CLL mAb068 with the highestELISA signal and the lowest half- maximal binding titer. A drop-off in theELISA signal was evident at concentra-tions higher than 100 nmol/L, possiblydue to the prozone effect. Peptide E6-Btn also exhibited some ELISA signal,albeit weaker in comparison to D2-Btnand with less dose responsiveness. Asexpected, PhD-068-4-Btn exhibited abinding ELISA signal, but this hadhigher half-maximal binding titer thanD2-Btn. Peptide D4-Btn from conservedfamily 2, did not show any binding sig-nal. Thus, binding did not correlate di-rectly with conservation among familiesshown in Figure 2. As expected, PhD-169-8-Btn (selected against M-CLLmAb169 (26)), did not bind U-CLLmAb068.

To further explore the binding proper-ties of these peptides, we examined thebinding of these peptides to U-CLLmAb068 and/or M-CLL mAb169 by sur-face plasmon resonance (SPR). To dothis, we synthesized several variantswith a C-terminal polylysine segment(E6-3K, D2-3K, D4-3K, PhD-068-4-4K,PhD-169-8-4K). The polylysine segmentwas included to improve solubility andminimize aggregation or nonspecific hy-drophobic effects for in vitro assays.

Because many binding sensorgramsdid not obey 1:1 Langmuir binding, we performed equilibrium analysis

rather than kinetic analysis in thosecases. We found that E6-3K had a strongconcentration-dependent binding signalto U-CLL mAb068 but not M-CLLmAb169 (Figure 4A). These data areconsistent with the biotinylated peptideELISA results in Figure 3, and indicatethat E6-3K bears specificity for theU-CLL mAb068 over M-CLL mAb169.Peptide D2-3K also bound U-CLLmAb068, but the amplitude of the equi-librium signal (in response units) wasgenerally lower than E6-3K at equiva-lent peptide concentrations (Figure 4B).Furthermore, D2-3K also showed speci-

ficity for U-CLL mAb068 over M-CLLmAb169. Aggregation at high concentra-tions prevented determination of thesaturation point for binding U-CLLmAb068 for both peptides. However, fitting of the partial sigmoids led to KD

estimates in the micromolar range (~110 μmol/L for E6-3K and ~60μmol/L for D2-3K). Both PhD-068-4-4Kand PhD-068-4-Btn had robust SPRbinding signals for U-CLL mAb068,whereas peptide D4-3K did not (notshown); these were consistent with thebiotinylated peptide binding ELISAfindings. However, thermodynamic

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Figure 3. ELISA of biotinylated peptides against U-CLL mAb068.

Table 3. Peptides synthesized for in vitro analysis.a

Peptidea Sequence KDb Selection target (comment)

D4-Btn DYCNLSVSWCYP-K (Btn) ND U-CLL mAb 068 (this work; family 2)D4-3K DYCNLSVSWCYP-GSGKKK NB

D2-Btn VSCNPFSWLPRCYA-K (Btn) ND U-CLL mAb 068 (this work; family 5)D2-3K VSCNPFSWLPRCYA-GSGKKK 58 μmol/L

E6-Btn FPCWALPSQYLCRP-K (Btn) ND U-CLL mAb 068 (this work)E6-3K FPCWALPSQYLCRP-GSGKKK 111 μmol/L

PhD-068-4-Btn WPLWSIPPFSPR-K (Btn) weak U-CLL mAb 068 (reference [5])PhD-068-4-3K WPLWSIPPFSPR-KKKK weak

PhD-169-8-Btn DNYAAALAQRAR-K (Btn) ND M-CLL mAb 169 (reference [5])PhD-169-8-4K DNYAAALAQRAR-KKKK 31 nmol/L

aAll peptides synthesized as C-terminal amides.bND, not determined; NB, no binding.

analysis indicated these interactionswere weaker than E6-3K and that therewas little specificity for U-CLL mAb068over M-CLL mAb169 (Figures 4C, 4D).

Thus, U-CLL mAb068 participated in abroad range of low-affinity interactions.The disparity in the off-phage and on-phage binding behaviors may be due topolyvalency effects on the phage surface(~50 copies of each peptide are displayedfor the peptide isolated in this report; ~5 copies/phage for peptides from thePhD library in the previous report) (26).

By contrast, PhD-169-8-4K showed astrong, robust binding signal to M-CLLmAb169 (Supplementary Figure S2). Thebinding behavior of this peptide obeyed1:1 Langmuir binding kinetics, and bothkinetic and equilibrium analyses were in agreement, resulting in KD values of 32 nmol/L and 31 nmol/L. Furthermore,this interaction was highly specific be-cause no significant binding was ob-served against U-CLL mAb068 (notshown). It is interesting to note that highaffinity peptides could be readily iso-lated against M-CLL mAb 169, but not

against U-CLL mAb 068 using the PhDlibrary.

DISCUSSIONThus we have selected and character-

ized peptides that bind an antibodyfrom a CLL B-cell clone with a surfacemembrane Ig coded by germline IGHV-D-J and IGLVκ -Jκ segments, findingthat this U-CLL mAb recognizes a di-verse set of peptide sequences, some ofwhich cluster into conserved families.These U-CLL mAb-peptide interactionsare generally of modest micromolaraffinities as determined by surface plas-mon resonance and contrast with thehigh affinity (nanomolar) interaction ofan M-CLL mAb with a phage-derivedpeptide.

These findings indicate that some U-CLL mAbs bind with relative selectiv-ity to peptides of defined and discretestructure, suggesting that selection forreactivity with these epitopes occurredin normal B cells giving rise to CLL inpatients. These epitopes may be environ-mental or self in nature, depending on

the type of CLL mAb (1,9,10). Consistentwith this are several recent studies ana-lyzing the reactivities of U-CLL andM-CLL mAbs with foreign antigens, self- antigens and peptides. Hoogeboom et al.have shown that a specific stereotypedBCR expressing a mutated IGHV3-07 re-acts with β-(1,6)-glucan, a major anti-genic determinant of yeasts and filamen-tous fungi (30), and Binder et al. (31) andDühren-von Minden et al. (32) haveidentified peptides bound by an array ofCLL mAbs (both U-CLL and M-CLL)that recognize autoepitopes in humanIgs which may, after BCR binding, de-liver survival signals to CLL B cells andto their precursors. The data reportedhere provide biochemical informationabout the level of specificity and thestrength of some of these interactions.This information may be of value inidentifying and targeting B lymphocytesexpressing specific BCRs in CLL patientsand in healthy individuals with mono-clonal B lymphocytosis.

ACKNOWLEDGMENTSThis work was supported in part by

grants from the United States NationalInstitutes of Health (CA81554 to N Chio-razzi and CA155472 to JR Lai), and bystartup funds from the Albert EinsteinCollege of Medicine (to JR Lai). TheKarches Foundation, the Nash FamilyFoundation, the Marks Foundation, theJerome Levy Foundation, the Leon LevyFoundation and the Mildred and FrankFeinberg Foundation also provided sup-port (to KR Rai and N Chiorazzi). Wethank Gregory Weiss (University of Cali-fornia – Irvine) for providing the pVIII li-brary, Samantha Wilner for technical as-sistance with peptide purification andHuiyong Cheng for assistance with SPRexperiments.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

R E S E A R C H A R T I C L E

M O L M E D 1 9 : 2 4 5 - 2 5 2 , 2 0 1 3 | L I U E T A L . | 2 5 1

Figure 4. Equilibrium SPR analysis of peptide binding to U-CLL mAb068 or M-CLL mAb 169.(A) E6-3K; (B) D2-3K; (C) PhD-068-4-4K; (D) PhD-068-4-Btn.

REFERENCES1. Chiorazzi N, Rai KR, Ferrarini M. (2005) Chronic

lymphocytic leukemia. N. Engl. J. Med. 352:804–15.2. Zenz T, Mertens D, Kuppers R, Dohner H, Stil-

genbauer S. (2010) From pathogenesis to treat-ment of chronic lymphocytic leukaemia. Nat.Rev. Cancer. 10:37–50.

3. Fais F, et al. (1998) Chronic lymphocytic leukemiaB cells express restricted sets of mutated and unmutated antigen receptors. J. Clin. Invest.102:1515–25.

4. Ghiotto F, et al. (2004) Remarkably similar antigenreceptors among a subset of patients with chroniclymphocytic leukemia. J. Clin. Invest. 113:1008–16.

5. Tobin G, et al. (2004) Subsets with restricted im-munoglobulin gene rearrangement features indi-cate a role for antigen selection in the develop-ment of chronic lymphocytic leukemia. Blood.104:2879–85.

6. Stamatopoulos K, et al. (2007) Over 20% of pa-tients with chronic lymphocytic leukemia carrystereotyped receptors: Pathogenetic implicationsand clinical correlations. Blood. 109:259–70.

7. Agathangelidis A, et al. (2012) Stereotyped B-cellreceptors in one-third of chronic lymphocyticleukemia: a molecular classification with implica-tions for targeted therapies. Blood. 119:4467–75.

8. Messmer BT, et al. (2004) Multiple distinct sets of stereotyped antigen receptors indicate a rolefor antigen in promoting chronic lymphocyticleukemia. J. Exp. Med. 200:519–25.

9. Chiorazzi N, Ferrarini M. (2003) B cell chroniclymphocytic leukemia: lessons learned fromstudies of the B cell antigen receptor. Annu. Rev.Immunol. 21:841–94.

10. Stevenson FK, Caligaris-Cappio F. (2004) Chroniclymphocytic leukemia: revelations from the B-cellreceptor. Blood. 103:4389–95.

11. Caligaris-Cappio F, Hamblin TJ. (1999) B-cellchronic lymphocytic leukemia: a bird of a differ-ent feather. J. Clin. Oncol. 17:399–408.

12. Broker BM, et al. (1988) Chronic lymphocyticleukemic cells secrete multispecific autoantibod-ies. J. Autoimmun. 1:469–81.

13. Sthoeger ZM, et al. (1989) Production of autoanti-bodies by CD5-expressing B lymphocytes from pa-tients with chronic lymphocytic leukemia. J. Exp.Med. 169:255–68.

14. Borche L, Lim A, Binet JL, Dighiero G. (1990) Ev-idence that chronic lymphocytic leukemia B lym-phocytes are frequently committed to productionof natural autoantibodies. Blood. 76:562–9.

15. Herve M, et al. (2005) Unmutated and mutatedchronic lymphocytic leukemias derive from self- reactive B cell precursors despite expressing differ-ent antibody reactivity. J. Clin. Invest. 115:1636–43.

16. Damle RN, et al. (1999) Ig V gene mutation statusand CD38 expression as novel prognostic indica-tors in chronic lymphocytic leukemia. Blood.94:1840–7.

17. Hamblin TJ, Davis Z, Gardiner A, Oscier DG,Stevenson FK. (1999) Unmutated Ig VH genesare associated with a more aggressive form ofchronic lymphocytic leukemia. Blood. 94:1848–54.

18. Rawstron AC, et al. (2002) Inherited predispositionto CLL is detectable as subclinical monoclonal B-lymphocyte expansion. Blood. 100:2289–90.

19. Ghia P, et al. (2004) Monoclonal CD5+ and CD5-B-lymphocyte expansions are frequent in the pe-ripheral blood of the elderly. Blood. 103:2337–42.

20. Landgren O, et al. (2009) B-Cell clones as earlymarkers for chronic lymphocytic leukemia. N. Engl.J. Med. 360:659–67.

21. Murase K, et al. (2003) EF-Tu binding peptidesidentified, dissected, and affinity optimized byphage display. Chem. Biol. 10:161–8.

22. Tonegawa S. (1983) Somatic generation of anti-body diversity. Nature. 302:575–81.

23. Torres M, May R, Scharff MD, Casadevall A.(2005) Variable-region-identical antibodies differ-ing in isotype demonstrate differences in finespecificity and idiotype. J. Immunol. 174:2132–42.

24. Morris L, et al. (2011) Isolation of a human anti-HIV gp41 membrane proximal region neutraliz-ing antibody by antigen-specific single B cellsorting. PLoS ONE. 6:e23532.

25. Brezinschek HP, et al. (1997) Analysis of the humanVH gene repertoire. Differential effects of selectionand somatic hypermutation on human peripheralCD5(+)/IgM+ and CD5(-)/IgM+ B cells. J. Clin. In-vest. 99:2488–501.

26. Seiler T, et al. (2009) Characterization of struc-turally defined epitopes recognized by mono-clonal antibodies produced by chronic lympho-cytic leukemia B cells. Blood. 114:3615–24.

27. van Houten NE, Zwick MB, Menendez A, ScottJK. (2006) Filamentous phage as an immunogeniccarrier to elicit focused antibody responsesagainst a synthetic peptide. Vaccine. 24:4188–200.

28. Stevenson FK, Krysov S, Davies AJ, Steele AJ,Packham G. (2011) B-cell receptor signaling inchronic lymphocytic leukemia. Blood. 118:4313–20.

29. Keitel T, et al. (1997) Crystallographic analysis ofanti-p24 (HIV-1) monoclonal antibody cross-reac-tivity and polyspecificity. Cell. 91:811–20.

30. Hoogeboom R, et al. (2013) A mutated B cellchronic lymphocytic leukemia subset that recog-nizes and responds to fungi. J. Exp. Med. 210:59–70.

31. Binder M, et al. (2013) CLL B-cell receptors canrecognize themselves: alternative epitopes andstructural clues for autostimulatory mechanismsin CLL. Blood. 121:239–41.

32. Dühren-von Minden M, et al. (2012) Chroniclymphocytic leukaemia is driven by antigen- independent cell-autonomous signalling. Nature.489:309–12.

2 5 2 | L I U E T A L . | M O L M E D 1 9 : 2 4 5 - 2 5 2 , 2 0 1 3

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