Báo cáo khoa học: Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library

Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library Andy Chevigne´ 1, Aure´ lie Fischer1, Julie Mathu1, Manuel Counson1, Nadia Beaupain1, Jean-Marc Plesse´ ria1, Jean-Claude Schmit1,2 and Sabrina Deroo1

1 Laboratoire de Re´ trovirologie, Centre de Recherche Public-Sante´ , Luxembourg, Luxembourg 2 Service National des Maladies Infectieuses, Centre Hospitalier Luxembourg, Luxembourg

Keywords CXCR4 antagonist; HCDR3; natural sequence randomization; peptide repertoire; phage display

Correspondence A. Chevigne´ , Laboratoire de Re´ trovirologie, Centre de Recherche Public-Sante´ , 84, Val Fleuri, L-1526 Luxembourg, Luxembourg Fax: +352 26970 221 Tel: +352 26970 336 E-mail: andy.chevigne@crp-sante.lu

(Received 16 March 2011, revised 27 May 2011, accepted 6 June 2011)

doi:10.1111/j.1742-4658.2011.08208.x

Phage display technology is a powerful selection approach to identify strong and speciﬁc binders to a large variety of targets. In this study, we compared the efﬁcacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identiﬁcation of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any speciﬁc ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine speciﬁc peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an afﬁnity of 5.6 lM and acted as a CXCR4 antagonist (IC50 = 23 lM). To comprehend the basis of the highly efﬁcient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efﬁcacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difﬁcult targets.

Introduction

to identify target-speciﬁc peptides

Phage display technology allows the handling of large molecular repertoires in a small and suitable format with a direct link between the DNA information and the peptide displayed at the surface of the phage. This technology has become a standard approach for the identiﬁcation of strong and speciﬁc peptide binders, as well as for the study of protein–protein interactions. To date, phage libraries displaying fully randomized peptides of different sizes, structurally constrained or

not, have been successfully applied to a wide variety of targets, such as antibodies, proteins, enzymes and [1–8]. receptors, In the last decade, engineering of the human antibody repertoire has greatly facilitated the development of therapeutic and diagnostic antibodies. The size of the antibodies expressed as libraries on phage has evolved from Fab fragments to single-chain antibody fragments and, ﬁnally, to heavy chain variable region

Abbreviations ECL, extracellular loop; HA, hemagglutinin; HCDR3, heavy chain complementarity determining region 3; UPA, undecapeptidyl arch; VH, heavy chain variable region fragment.

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the surface of the receptor, as shown in the recently resolved three-dimensional structure, and has been reported to be a critical feature for the interaction between the receptor and both of its natural ligands, the chemokine CXCL12 and the HIV-1 envelope pro- tein [25,26]. Moreover, epitopes of several CXCR4 neutralizing antibodies are located in this loop, fur- ther emphasizing its important role in ligand binding [27–29].

fragments (VH). However, drawbacks in terms of poor solubility and limited access of these large antibody fragments to some targets have prompted a further size reduction of the displayed antibody fragments [9–11]. Recently, we have demonstrated the feasibility of fur- ther reducing the size of the antibody to the peptide level by engineering phage libraries displaying heavy chain complementarity determining region 3 (HCDR3) repertoires representing human ‘biologically random- ized peptide collections’. These phage libraries have been used successfully to identify HCDR3 peptides displaying nanomolar afﬁnity for the anti-human inﬂu- enza hemagglutinin (HA) IgG [12].

In parallel with biopanning, we investigated the bio- chemical properties of the natural IgM HCDR3 reper- toire in terms of length and sequence diversity to acquire better insights into the efﬁciency and advanta- ges of these libraries.

Results

Selection with fully randomized peptide phage libraries on ECL2

to eliminate outliers in terms of

These HCDR3 phage libraries reﬂect the natural HCDR3 diversity mainly achieved by variable, diver- sity and joining gene segment rearrangements and ran- dom nucleotide addition. This vast potential of HCDR3 diversity is also associated with the central role of HCDR3 in the determination of antigen speci- ﬁcity [13–15]. The HCDR3 repertoire complexity also reﬂects the developmental changes from fetal to adult the B-cell repertoire [16,17]. In the mouse life of model, the HCDR3 repertoire evolved during develop- ment length and amino acid composition to reach an average hydro- phobicity [18]. Finally, skewed HCDR3 repertoires reﬂect perturbed B-cell repertoires associated with cer- tain pathologies. The HCDR3 length distribution of patients with multiple sclerosis displayed a reduced complexity compared with the distribution of healthy donors [19]. For other autoimmune diseases, such as primary biliary cirrhosis and rheumatoid arthritis, skewed HCDR3 length distributions were observed for particular VH gene families [20,21].

A total of nine selection campaigns was performed on linear biotinylated ECL2 peptide using two linear (12- and 15-mer) and three constrained (7-, 13- and 14-mer) fully randomized peptide phage libraries. Selec- tion strategies were performed on immobilized peptide (plastic support or beads), as well as on soluble ECL2 peptide. Increasing stringency was applied in all selec- tion campaigns, i.e. decreasing input phage titers and increasing washing steps in consecutive selection cycles. Three different elution approaches were tested: acid elution, dithiothreitol elution to recover phage binding to ECL2 in a constrained format and competitive elu- tion with an undecapeptide corresponding to the N-terminal arch of ECL2 (undecapeptidyl arch, UPA). None of the selection campaigns resulted in positive phage clones, except for the campaign performed with the multivalent f88 Cys1 library on ECL2 peptide immobilized on beads for which four positive clones were obtained. Speciﬁcity ELISA with these four puri- ﬁed clones on ECL2 and irrelevant peptides revealed that none of these phages was target speciﬁc. Finally, two selection campaigns with the constrained f88 Cys1 library were performed on cyclic biotinylated ECL2 peptide without success.

Selection with an IgM-derived HCDR3 phage library on ECL2

The importance of HCDR3 loops in antibodies and their multiple characteristics make these fragments attractive for repertoire display: (a) HCDR3 is the smallest part of an antibody retaining antigen-binding ability [22–24]; (b) HCDR3 is the most diverse region in length, shape and sequence; (c) HCDR3 is ﬂanked by conserved framework residues allowing speciﬁc ampliﬁcation and cloning in different vectors; and (d) naive and immune HCDR3 repertoires can be easily isolated from peripheral blood mononuclear cells from different species. In this report, we compared the efﬁ- ciency of a human HCDR3 library engineered from a nonimmunized donor with a set of conventional con- strained and nonconstrained libraries displaying pep- tides of different size. These libraries were screened against a peptide derived from the second extracellular loop (ECL2) of the CXCR4 receptor implicated in cancer and HIV infection. This loop (residues 176– 202) adopts a short antiparallel b-strand structure at

A library displaying constrained HCDR3 loops from the IgM repertoire of a nonimmunized donor, previ- ously used to isolate nanomolar binders to an anti-HA antibody, was screened on ECL2 peptide immobilized on beads [12]. Four selection rounds with increased

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Characterization of the antagonistic properties of ECL2-speciﬁc clones

stringency, equivalent to the conditions applied to the fully randomized peptide libraries, were performed. The supernatant of 260 individual clones of the fourth round was tested by ELISA on ECL2 and on an irrele- vant peptide (HA mimotope). Analysis revealed 66 (25%) positive clones, 22 of which displayed a signal on ECL2 peptide higher than 0.5 and an ECL2 ⁄ HA ratio > 10. The other 44 clones displayed signals higher than 0.2 on ECL2 peptide with an ECL2 ⁄ HA ratio between 3 and 10. Although, in the ﬁrst set of 22 clones, a unique sequence 95-DRGGTYPGRY-102 (Kabat numbering) was identiﬁed, the second set com- prised 21 different sequences including 95-DRGGTYP- GRY-102 (Table 1).

Binding of the nine target-speciﬁc phage clones (1, 3, 6, 24, 26, 28, 29, 30 and 39) to ECL2, as presented in the native CXCR4 receptor, was analyzed in a receptor activation assay. Agonistic properties were ﬁrst evalu- ated by measuring the effect of the phage on cAMP production. None of the ECL2-speciﬁc phage clones displayed agonistic properties. The antagonistic prop- erties of the selected HCDR3 phage were evaluated by monitoring cAMP production in the presence of the chemokine CXCL12. Four clones (1, 3, 28 and 29) restored the initial forskolin-induced cAMP production (Fig. 2A), indicating their recognition and interaction with native receptor. Clone 3 (95-DRGGTYPGRY- 102), isolated at the highest frequency (n = 29), was further analyzed in free peptide format. Analysis of the cyclic peptide corresponding to the HCDR3 sequence of clone 3 extended with framework 3 and 4 residues (89-VYYCAR-DRGGTYPGRY-WCQG-106) (peptide 3) in cAMP assays demonstrated an antagonistic activ- ity towards CXCR4, characterized by an IC50 of 23 lm (Fig. 2B). No inhibition was observed with the irrelevant HA mimotope. The speciﬁcity for CXCR4 of this sequence in peptide and phage format was

Speciﬁcity analysis of the 21 puriﬁed positive phage clones revealed that nine (clones 1, 3, 6, 24, 26, 28, 29, 30 and 39) were clearly speciﬁc for linear ECL2 and displayed at least a four-fold higher interaction with the target peptide than with the HA mimotope (aver- age fold of 5.8) (Table 1). None of the speciﬁc phage interacted with a peptide corresponding to ECL2 of another chemokine receptor CCR5. The phage clone isolated at the highest frequency (clone 3) (n = 29) displayed the strongest interaction with peptide ECL2 in both linear and cyclic format (ECL2lin ⁄ HA ratio, 23; ECL2cycl ⁄ HA ratio, 9.8) (Fig. 1).

isolation frequency and speciﬁcity (ECL2lin ⁄ HA ratio) of heavy chain complementarity determining region 3 in the different sequences. CAR- and -WC

Table 1. Sequence, length, (HCDR3) clones isolated from the IgMCys library. Residues presented in bold are identical sequences are part of the framework 3 and 4 residues, respectively. ECL2, second extracellular loop; HA, hemagglutinin.

Name

Sequence

Frequency (n)

Length

Elution

ECL2lin ⁄ HA ratio

Cl 1 Cl 43 Cl 26 Cl 29 Cl 6 Cl 2 Cl 28 Cl 3 Cl 11 Cl 4 Cl 18 Cl 24 Cl 40 Cl 7 Cl 9 Cl 13 Cl 38 Cl 31 Cl 39 Cl 8 Cl 30

11-mer 10-mer 12-mer 13-mer 16-mer 11-mer 11-mer 10-mer 14-mer 9-mer 14-mer 19-mer 11-mer 11-mer 14-mer 8-mer 14-mer 9-mer 14-mer 14-mer 12-mer

4 1 1 1 4 1 1 29 2 1 5 1 1 7 1 1 1 1 1 1 1

Dithiothreitol pH pH pH Dithiothreitol Dithiothreitol pH Dithiothreitol ⁄ pH Dithiothreitol Dithiothreitol pH pH pH Dithiothreitol Dithiothreitol Dithiothreitol pH pH pH Dithiothreitol pH

5.1 1.2 8.2 8.0 4.5 1.8 8.1 23 1.8 1.5 1.2 8.2 1.4 1.4 1 1.2 2.1 3.7 5.7 1.5 9.6

CAR-DR-GRYDST- - - - - - - - -LRY-WC CAR-GS-G-WDST- - - - - - - - -GNY-WC CAR-GRPGTGTTN- - - - - - - - -LNY-WC CAK-DGKGQYGSG- - -Y- - - - -INY-WC CAR-DRRGWYCSGGSCY- - - - -LNY-WC CAK-DRR-RDSSG- - - - - - - - -WYY-WC CAR-DRG-SGSYG- - - - - - - - -VGY-WC CAR-DRGGTYPGR- - - - - - - - —Y-WC CAR-DRGWGAVAG- - - - -PP- -IRY-WC CAR-DRG- - - -AG- - - - -PP- - -GY-WC CAR-GGGIAAPGG- - - - -PP- -PVY-WC CAR-PAGG- -VRGRVQQQPPGTVVY-WC CAR-RGDG- -SRGWAD- - - - - - - -Y-WC CAR-DGTM- -VRGVPG- - - - - - - -Y-WC CAR-DGTG- -YGGNSGG- - - - - -VY-WC CAR-EA- - - - -RAGPY- - - - - - - -Y-WC CAR-DPTLGYSGGGPLS- - - - - - -Y-WC CAR-SGPRS-S- - - FD- - - - - - - -Y-WC CAR-APPVSGSQVGYPY- - - - - - -Y-WC CAR-DNPRSYSSSSGAV- - - - - - -Y-WC CAR-DGTYSGY–EYPR- - - - - - -Y-WC

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the most

Fig. 1. Speciﬁcity of frequently isolated phage clone (clone 3) for the ECL2 peptide. Speciﬁcity was determined by ELISA on immobilized linear (ECL2X4lin) and cyclic (ECL2X4cycl) ECL2 peptide, linear ECL2 peptide derived from the chemokine receptor CCR5 (ECL2R5) and three irrelevant peptides (Ctrl HA, Ctrl 2 and Ctrl 3). Two-fold dilutions of phage (starting at 2 · 1012 phage per well per 100 lL) were added. Phage binding to peptides was detected using an anti-M13 IgG conjugated to horseradish per- oxidase. The experiment was performed three times and resulted in equivalent proﬁles. ECL2, second extracellular loop; HA, hemag- glutinin.

the second extracellular

conﬁrmed in a similar analysis using CCR5-expressing cells (CEM.NKR-CCR5) and the chemokine CCL5. The binding afﬁnity of peptide 3 was analyzed by sur- face plasmon resonance. A clear binding was observed with immobilized ECL2 peptide, whereas no binding to a truncated ECL2 peptide corresponding to the ﬁrst 11 residues of ECL2 (i.e. UPA) was measured. These data suggest that full-length ECL2 peptide is required for binding. Kinetic analysis using the two-state reac- tion model revealed a KD value of 5.6 lm for peptide 3 (Fig. 3A).

Fig. 2. Antagonistic properties of loop (ECL2)-speciﬁc phage ⁄ peptide monitored by cAMP modulation assay. (A) Antagonistic properties of the target-speciﬁc phage were determined by monitoring the inhibition of the CXCL12-induced cAMP suppression. Phage (5 · 1012 particlesÆmL)1) was incubated with forskolin (FSK) and the chemokine CXCL12 (30 nM). Variation in the CXCL12-induced cAMP inhibition was detected using time- technology. resolved ﬂuorescence resonance energy transfer (B) Antagonistic properties of free peptide derived from clone 3 towards CXCR4–CXCL12 and the CCR5–CCL5 activation pathways. Inhibitory properties were expressed as the percentage of inhibition of the initial CXCL12-induced cAMP signal measured in the absence of peptide. Results were compared with those recorded with an irrelevant peptide (hemagglutinin, HA). The data shown here represent the mean of triplicate measurements ± standard deviation.

Length distribution analysis of human IgM HCDR3 repertoires

To unravel the biochemical features of the HCDR3 libraries resulting in very efﬁcient biopanning when com- pared with conventional peptide libraries, we analyzed

To determine the residues of clone 3 critical for binding to the target, Ala scanning was performed on the region spanning the HCDR3 sequence including Cys92 and Cys104 (Fig. 3B). The replacement of Cys92 and Cys104 resulted in 60% and 90% decreases in binding, respectively. In addition, mutation of the three positively charged residues Arg94, Arg96 and Arg101, present in the HCDR3 sequence, reduced the binding by 40%, 39% and 53%, respectively. Interest- ingly, mutation of the residues included in the cluster between Tyr100 and Trp103 decreased the interaction with the target by 32–60%. Together, these results indicate the importance of the disulﬁde bridge, the positive charges and the variable sequence following the DRGG ⁄ R motif in the selected HCDR3 sequences for target binding.

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cloning

after

was Gaussian-like, with one major peak at 11 amino acids, and the length varied between 3 and 21 amino acids. To analyze the quality of the IgM-derived HCDR3 fragments when displayed on phage, the same length analysis was performed on the HCDR3 PCR products in the phagemid vector (= library) (Fig. 4B). The same Gaussian-like pattern, consisting of 19 peaks with the major peak at 11 amino acids, was observed for the HCDR3 repertoire ligated in the phagemid. The smallest and longest frag- ments corresponded to 3 and 21 amino acids, respec- tively.

interaction with the

efﬁcient

The HCDR3 length distribution of the subpopula- tion of phage isolated after each selection round was monitored and compared with the length distribution of the initial library. Analysis of the phage population isolated in the fourth selection round clearly showed a selection for the HCDR3 fragments with a length between nine and 14 amino acids and an enrichment of HCDR3 loops of 18 residues. HCDR3 fragments with a length between ﬁve and eight were less repre- sented, suggesting that a minimal size of nine is required for target (Fig. 4C). Indeed, analysis of the individual length of the ECL2-positive HCDR3 loops revealed a length ranging from eight to 19 residues, with an average value of 12 ± 2.4.

Analysis of the global amino acid frequency

Fig. 3. Characterization of the interaction between the heavy chain complementarity determining region 3 (HCDR3) clone 3 and target second extracellular loop (ECL2). (A) Kinetic analysis of the binding of peptide 3 on immobilized biotinylated ECL2 peptide. Black sen- sorgrams represent the experimental data obtained on the ECL2 surface subtracted from the signal recorded on an irrelevant pep- tide using different concentrations of peptide 3 (31.2–2000 nM). Red sensorgrams represent the ﬁtted curves on a two-state inter- action model (v2 = 0.276) using the BIAEVALUATION 4.1 program. SPR, surface plasmon resonance. (B) Ala scanning of the interac- tion between phage clone 3 (2 · 1012 phage per well) and the ECL2 target peptide. Data are represented as the percentage of the initial binding signal recorded with wild-type (WT) phage (clone 3) and correspond to an average of three measurements ± stan- dard deviation.

To further characterize the overall amino acid content of the IgM HCDR3 library, a set of 128 randomly iso- lated clones from the library was sequenced. Analysis of the global frequency of the 20 individual amino acids of the IgM HCDR3 sequences revealed that Tyr, Asp, Gly and Ser represented more than 45% of the total content (47.6%). The second class of residues for which each amino acid represented 5% of the overall content comprised Ala, Arg, Leu, Phe, Thr and Val. The residues Cys, Gln, His and Met were less abun- dant and represented maximally 2.5% of the total con- tent. The residue present at the lowest frequency (1%) in the analyzed set of HCDR3 sequences was Lys (Fig. 5A).

the complexities of the HCDR3 libraries in terms of length, amino acid content and position variability.

Analysis of the individual amino acid frequency as a function of the HCDR3 length

To better characterize the HCDR3 length diversity, a set of forward and backward primers based on the nucleotide alignment of 1182 human VH sequences was used to amplify the complete diversity of the HCDR3 loops of the donor [12,30,31]. The PCR products cor- responding to the ampliﬁed IgM HCDR3 repertoire were separated as a function of their length by electro- phoresis (Fig. 4A). The IgM length distribution proﬁle

The frequency of each individual amino acid was calcu- lated as a function of the HCDR3 length (Table 2). The frequency of Cys and Lys increased with increasing HCDR3 length (Pearson’s r = 0.864 and 0.922 with two-tailed P values of 0.059 and 0.0259, respectively).

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9 aa

11 aa

10 aa

11 aa

A

B

C

D

120 nt

150 nt

160 nt

180 nt 190 nt

120 nt

140 nt

160 nt

180 nt

200 nt 120 nt

140 nt

160 nt

180 nt

200 nt

120 nt

150 nt

160 nt

180 nt 190 nt

Fig. 4. Length distribution of human IgM- and IgG-derived heavy chain complementarity determining region 3 (HCDR3) repertoires. (A) IgM-derived HCDR3 distribution displaying one major peak at 11 amino acids (aa). (B) Initial IgM HCDR3 distribution displayed on phage (= phage library). (C) Distribution of the HCDR3 fragments selected at the end of the fourth round of the selection campaign (dithiothreitol elution). (D) IgG-derived HCDR3 distribution displaying a skewed Gaussian proﬁle. The red proﬁle corresponds to the ROX-labeled (A, D) and DS33-labeled (B, C) size standards expressed in nucleotides (nt) used to calculate the length of the HCDR3 fragments of the proﬁle.

E

I QMC

KQ C

B

H

A

M

Y 15%

WN

E

L

N

G 25%

A 4%

P 3%%

I 3%

T 5%

W 4%

D 12%

V 6%

T 4%

V 5%

S 7%

Y 14%

G 12%

R 5%%

D 8%

A 5%

S 9%

L 5%

P 10%

R 11%

F 6%

Fig. 5. Frequency of individual amino acids in a set of IgM-derived heavy chain complementarity determining region 3 (HCDR3) sequences. (A) Relative amino acid frequency in a set of IgM-derived HCDR3 sequences (n = 128) randomly selected from the initial phage library. Tyr, Gly, Ser and Asp represented more than 45% of the total amino acid content. (B) Amino acid distribution of the 21 target-positive HCDR3 sequences. The predominant amino acids were Gly, Tyr, Arg, Pro and Asp.

P < 0.6). The residues His, Arg, Glu and Met displayed a very weak positive correlation (Pearson’s r = 0.201, 0.217, 0.171 and 0.238, respectively; two- tailed P < 0.8). The frequency of Trp, Ser and Val displayed no correlation with the HCDR3 length (Pearson’s r < 0.05; two-tailed P > 0.9) (Table 2).

Analysis of the position-dependent amino acid variability in the HCDR3 loops

respectively;

The position-dependent amino acid distribution and variability were analyzed as a function of the HCDR3 loop length (11–15 residues). The last three positions

Although less signiﬁcant, a positive correlation was observed between frequency and HCDR3 length for Asn, Ala, Gln and Thr (Pearson’s r = 0.534, 0.552, 0.424 and 0.496, respectively; two-tailed P < 0.5). The frequency of the amino acids Gly, Pro, Phe and Leu tended to decrease with increasing HCDR3 length (Pearson’s r = )0.711, )0.532, )0.678 and )0.522, respectively; two-tailed P < 0.5). A weak negative cor- relation between frequency and HCDR3 length was observed for Tyr and Asp (Pearson’s r = )0.350 and )0.349, two-tailed P < 0.6). The fre- Ile tended to increase with increasing quency of two-tailed HCDR3 length (Pearson’s

r = 0.5884;

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Table 2. Analysis of the frequency of individual amino acids as a function of the heavy chain complementarity determining region 3 (HCDR3) length.

Amino acid

Pearson’s r

Two tailed P value

isolated at

clone

screening random phage-displayed peptide libraries on a peptide target. Indeed, only rare examples have been published on the successful isolation of peptides on a peptide target [33]. In contrast, only one selection cam- paign with a constrained human IgM-derived HCDR3 library from a nonimmunized donor was required to isolate four HCDR3 sequences of different lengths (10, 11 and 13 residues) recognizing the native CXCR4 receptor and interfering with the binding of the chemo- kine. Among these sequences, at least one peptide cor- responding to the the highest frequency acted as an antagonist (IC50 = 23 lm) and displayed an afﬁnity of 5.6 lm. A discrepancy in the potency of clone 3 in phage format (nanomolar range activity) and in peptide format (micromolar range activity) was observed. These data suggest an impor- tant contribution of the phage scaffold to the binding. Avidity effects in the phage format are less probable, as a phagemid system resulting mainly in a monova- lent display was used.

Cys Lys Asn Ala Gln Thr Gly Pro Phe Leu Tyr Asp Ile His Arg Glu Met Trp Ser Val

0.864 0.922 0.534 0.522 0.424 0.496 )0.711 )0.532 )0.678 )0.522 )0.350 )0.349 0.329 0.201 0.215 0.171 0.238 )0.029 0.020 )0.032

0.059 0.026 0.354 0.335 0.477 0.395 0.178 0.356 0.209 0.367 0.564 0.565 0.588 0.745 0.729 0.784 0.701 0.964 0.974 0.960

the properties of

To explain the difference in efﬁciency between bio- logical (HCDR3) and fully randomized synthetic pep- tide libraries, the HCDR3 phage library were analyzed in terms of length, amino acid composition and sequence variability.

at the C-terminal base of the HCDR3 loop (residues 100x to 102) showed limited variability compared with the other positions. The motif FDY was most com- monly identiﬁed at these positions for the IgM-derived HCDR3 loops varying from 11 to 15 residues. When analyzing the evolution of the amino acid composition of the last three C-terminal positions as a function of HCDR3 length, their variability tended to decrease with increasing HCDR3 length (Pearson’s r = )0.575, )0.606 and )0.875 with two-tailed P values of 0.310, 0.278 and 0.05, respectively).

Discussion

In this study, we have demonstrated that HCDR3 rep- ertoires displayed on phage can be efﬁciently used to identify speciﬁc bioactive sequences by targeting ECL2 the G-protein-coupled receptor CXCR4. Peptide of ECL2 was initially selected as target for its critical role in the viral envelope protein (gp120) and chemokine CXCL12 interactions [25]. As demonstrated recently, the binding pocket of CXCR4 is smaller and involves the extracellular surface to a larger extent when com- pared with other G-protein-coupled receptors [32].

Length analysis of ampliﬁed IgM HCDR3 loops revealed a Gaussian-like proﬁle with lengths from three to 21 amino acids. The same length proﬁle was obtained for the repertoire of phage-displayed HCDR3 fragments, indicating that no bias occurred on cloning. The observed length proﬁles were in agreement with those reported in the literature on very large sequence panels [34,35]. In comparison with HCDR3 sequences of adult mature B cells (n = 42), varying between six and 23 amino acids, our sequence set also comprised smaller HCDR3 loops [36]. HCDR3 length proﬁles were markedly different between the IgM and IgG rep- ideal versus skewed Gaussian-like proﬁles ertoires; were observed for IgM and IgG repertoires, respec- tively (Fig. 4A, D). In addition, the IgG proﬁle dis- played a reduced range of HCDR3 lengths (6–18 residues) compared with the IgM distribution (3–21 residues) (Fig. 4A, D). These ﬁndings correlate with the afﬁnity maturation process of the IgM and IgG isotypes. IgM repertoires reﬂect a polyclonal response, whereas IgG repertoires represent oligoclonal expan- sions corresponding to the development of high-afﬁnity antibodies [37]. These observations, together with pre- viously reported successful isolation of nanomolar binders from the IgM library, prompted us to use the IgM-derived HCDR3 library [12].

Eleven selection campaigns with ﬁve conventional fully randomized phage-displayed peptide libraries, each displaying single size peptides on ECL2, were not illustrate the difﬁculty in successful. These results

The overall amino acid distribution in our set of HCDR3 sequences revealed a biased content in the

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afﬁnity antigen binding. As an illustration, minimal Tyr, Ser, Ala and Asp phage libraries were engineered in vitro and were successfully screened on globular pro- teins [41].

inherent

In addition to the advantages

IgM sequences. Tyr, Gly, Ser and Asp were over-rep- resented (total of 50%), whereas Glu, Lys and Cys were under-represented (total of 3%) (Fig. 5). These results were in agreement with the analysis on a large set of HCDR3 sequences retrieved from antibody data- bases by Zemlin et al. [35].

Analysis of

to the biochemical properties of the HCDR3 loops, peptides corresponding to HCDR3 sequences selected from phage libraries can be easily produced in large amounts by solid phase synthesis. The results observed with the synthetic peptide VYYCARDRGGTYPGRYWCQG (peptide 3) indicate that this HCDR3 sequence retains binding and antagonistic properties outside the phage format.

individual amino acid contents as a function of HCDR3 length demonstrated that the Gly content decreased with increasing HCDR3 length, whereas the Cys content increased with longer HCDR3 loops, in agreement with data obtained on large sets of sequences [35]. The absence of Cys and the higher con- tent of Gly in the shorter HCDR3 loops render these loops more ﬂexible to adapt to a variety of antigens, whereas an increase in Cys content in the longer HCDR3 loops could favor the formation of structural constraints. In our dataset, Lys displayed a signiﬁcant the HCDR3 positive correlation as a function of length, as observed by Zemlin et al. [35]. For Phe, a negative correlation was identiﬁed.

the C-terminal end of

In addition, HCDR3 repertoires displayed position- limited dependent biased sequence diversity. Clear sequence diversity was observed at the C-terminal end of the HCDR3 loops. The majority of the HCDR3 sequences displayed the motif FDY at positions 100x– the 102. The variability of HCDR3 loops was negatively correlated with the loop length. Longer HCDR3 loops displayed less variability at these positions, and these data suggest that the loss of sequence variability is compensated for by the longer loop lengths, providing a higher degree of struc- tural freedom.

Together, the efﬁcacy of HCDR3 libraries compared with conventional peptide libraries can be explained by the natural bias selected during evolution, resulting in four levels of complexity: (a) a large variety of peptide lengths; (b) a biased sequence diversity towards amino acids crucial for high afﬁnity and speciﬁc binding (Tyr, Gly and Ser); (c) length- and position-dependent sequence bias; and (d) repetitive motifs of Ser, Gly and Tyr. In conventional peptide libraries, diversity is mainly achieved by introducing NNN or NNK codons, resulting in an equal representation of the 20 amino acids at each position. However, the natural HCDR3 sequence bias leading to high-efﬁcacy peptide libraries cannot be achieved with this approach. In addition to this natural HCDR3 variability, a supple- mentary level of complexity was added in vitro by engi- neering a disulﬁde bridge between Cys92 in the framework 3 residues and a Gly to Cys mutation at position 104 in the framework 4 residues, mimicking more closely the parental antibody context.

The isolation of four HCDR3 sequences acting as antagonists in phage format, with at least one sequence retaining its biological properties in peptide format, indicates that soluble ECL2 peptide represents a valu- able alternative to native receptor screening on living cells when targeting the extracellular surface implicated in ligand binding. Nevertheless, further optimization and afﬁnity maturation of the four HCDR3 sequences on complete receptor are required to develop nanomo- lar afﬁnity CXCR4 antagonists.

Remarkably, the majority of HCDR3 sequences dis- played repetitive motifs of particular amino acids. Doublets of Ser, Gly and Tyr were identiﬁed in the majority of the sequences. Higher order repetitions were only observed with Tyr (quadruplets and quintu- plets). Studies on protein–protein interactions have demonstrated the importance of Tyr to obtain high afﬁnity and speciﬁc protein–protein interactions [38]. Gly- and Ser-rich clusters were also frequently observed in HCDR3 sequences, and probably act as for binding. ﬂexible linkers to separate ‘hot spots’ Indeed, these Gly ⁄ Ser linkers are commonly used in protein engineering to separate functional domains in proteins [39,40].

Our HCDR3 sequence analysis clearly demonstrates that paratopes of antibodies have evolved to yield biased amino acid contents enriched for Tyr, Gly and Ser. Enrichment of these residues and their differential repartition in IgM and IgG repertoires indicate that HCDR3 fragments display remarkable biochemical properties, particularly suited and efﬁcient for high-

Interestingly, analysis of the amino acid content of the isolated HCDR3 sequences binding to the ECL2 peptide revealed changes, in particular for Arg, Gly, Pro, Asp and Phe, in comparison with the initial reper- toire. Arg and Gly contents underwent a two-fold increase, whereas the Pro content was four-fold higher. In contrast, Asp decreased from 12.3% to 7.6%, and Phe was almost absent in positive clones (Fig. 5B). The enrichment for Arg (11%) is correlated with the biochemical properties of the target peptide, displaying

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(7-mer) and disulﬁde bridge-constrained heptapeptide (CX7C) libraries were purchased from New England Biol- abs (Ipswich, MA, USA). In the CX7C library, the random- ized peptides are ﬂanked by a pair of Cys residues which, by oxidation during phage assembly, result in the formation of a disulﬁde bridge. The major coat protein (pVIII) dis- played libraries, linear pentadecapeptide 15-mer (f88) and constrained tridecapeptide Cys1 (X5CXCX5) and tetradeca- peptide Cys4 (X4CX4CX4) libraries, were kindly provided by G. P. Smith (University of Missouri, CO, USA).

a net negative charge of four in its N-terminal part (four Asp, one Glu and one Arg). In addition, muta- tion of the three Arg residues in the most frequently isolated HCDR3 sequence resulted in a loss of binding, conﬁrming their importance for target interaction. This Arg-rich content is in strong agreement with previous studies reporting the importance of positive charges for interaction with CXCR4 extracellular surface and receptor antagonists, as observed in T22 and NeoR6 [42,43].

Engineering of the phage library displaying constrained human IgM-derived HCDR3 fragments derived from a nonimmunized donor has been described previously by Deroo et al. [12]. Brieﬂy, HCDR3 fragments were ampliﬁed from VH from a nonimmunized donor. In this library, Gly at position 104 was substituted by a Cys, allowing the for- mation of a disulﬁde bridge with Cys92. The complexity of the IgMCys library corresponded to 3 · 108 clones [12].

In summary, we have demonstrated the value of nat- urally size and sequence randomized human HCDR3 in comparison with fully synthetic peptide libraries, libraries, in particular for difﬁcult targets. The efﬁcacy of HCDR3 phage libraries was also observed in bio- panning with more complex targets, such as enzymes and viral gp120 protein. In addition, HCDR3 libraries derived from immunized donors were successfully explored [44]. This proof of concept opens up a very interesting ﬁeld for drug discovery.

Afﬁnity selection of fully randomized peptide and HCDR3 phage libraries

Materials and methods

Chemicals and cell lines

and

Biopanning with the fully randomized peptide phage libraries was performed according to the manufacturer (New England Biolabs) and as described previously [46]. Linear or cyclic biotinylated ECL2 peptide (5 nmol) was immobilized on either magnetic Dynabeads or plastic wells (0.5 nmol) coated with streptavidin. The elution of speciﬁc phage was performed with 75 mm dithiothreitol for the phage libraries displaying cyclic peptides, glycine-HCl solu- tion at pH 2.2 (acid) or by competition with free ECL2 peptide or a peptide comprising a 12-mer sequence of ECL2 (UPA).

Biotinylated peptides were purchased from JPT (Berlin, Germany) or Bachem (Bubendorf, Switzerland). The bio- tinylated peptide corresponding to the predicted sequence of ECL2 of human CXCR4 (176–201) was synthesized in linear cyclic (NVSEADDRYICDRFYPNDLWVVFQFQ) (C-NVSEADDRYICDRFYPNDLWVVFQFQ-C) formats [25]. Biotinylated peptides corresponding to ECL2 of human CCR5 (167–198) (GGGTRSQKEGLHYTCSSHF- PYSQYQFWKNFQTLKI) and a non-G-protein-coupled receptor-related peptide corresponding to a mimotope of the HA epitope (GGGSPAPERRGYSGYDVPDY) were used as negative controls [12,45]. Cyclic peptide corre- sponding to the most frequently isolated HCDR3 (clone 3) elongated with six (VYYCARDRGGTYPGRYWCQG), (VYYCAR) and four (WCQG) amino acids from the framework 3 and 4 residues, respectively, was purchased from Bachem.

cells. Phage was

The cell lines MT-4 expressing CXCR4 and CEM.NKR- CCR5 were obtained through the AIDS Research and Ref- erence Reagent Program, Division of AIDS, NIAID, National Institutes of Health, from D. Richman and A. Trkola, respectively. Biopanning with the IgMCys HCDR3 library was per- formed on linear biotinylated ECL2 peptide (5 nmol) immobilized on magnetic Dynabeads (Invitrogen, Mere- lbeke, Belgium) coated with streptavidin. For the ﬁrst round, beads were incubated with 2 · 1012 phage particles for 2 h in 2% milk. After washing the beads ﬁve times with NaCl ⁄ Pi ⁄ 0.5% Tween 20, the target-bound phage was ﬁrst eluted with 500 lL of 75 mm dithiothreitol, and the remain- ing fraction of bound phage was eluted with glycine ⁄ HCl buffer, pH 2.2, for 10 min and neutralized with Tris ⁄ HCl log phase buffer, pH 8.0. Eluates were used to infect Escherichia coli TG1 rescued with M13K07 helper phage and employed for three additional selection rounds using 1 · 1012, 1 · 1011 and 1 · 1010 phage particles and 10, 20 and 40 washing steps in rounds 2, 3 and 4, respectively.

Fully randomized peptide and HCDR3 phage libraries

Screening of positive clones

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fully randomized phage-displayed peptide Five different libraries were screened. For minor coat protein (pIII) dis- (PhD-12) played libraries, linear dodecapeptide (12-mer) Culture supernatants of phage rescued from the fourth selection round were tested by ELISA on biotinylated ECL2 and a control peptide (10 lgÆmL)1) immobilized via

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HCDR3-derived CXCR4 antagonists

well) were incubated for 30 min at room temperature with 2 · forskolin CXCL12 per phage or RANTES per phage (30 nm and 5 · 1012 phageÆmL)1). Substrate containing Eu-W8044-labeled streptavidin and biotin–cAMP was added and incubated for 1 h at room temperature. The LANCE signal was recorded at 665 nm and compared with cAMP (10)6–10)11 m). Experiments were per- standard curves formed in triplicate. streptavidin. After blocking plates with NaCl ⁄ Pi ⁄ 2% milk for 1 h, plates were washed with NaCl ⁄ Pi ⁄ 0.05% Tween 80 and incubated with culture supernatants in NaCl ⁄ Pi ⁄ 2% milk for 2 h. After washing with NaCl ⁄ Pi ⁄ 0.05% Tween 80, phage binding was detected with an anti-M13 IgG conju- gated to horseradish peroxidase (GE Healthcare, Diegem, Belgium). The plates were developed with ortho-phenyl- enediamine (Sigma-Aldrich, St Louis, MO, USA) and read at 492 nm.

Surface plasmon resonance

Puriﬁed phage was prepared by PEG ⁄ NaCl precipitation from the individual positive supernatant cultures and tested by ELISA on a panel of biotinylated immobilized peptides (linear and cyclic ECL2 of CXCR4, linear ECL2 of CCR5, HA epitope) as described above to conﬁrm target speciﬁcity. (10 min)

Length distribution analysis of the ampliﬁed HCDR3 repertoires

Biotinylated ECL2 and UPA were immobilized on a strep- tavidin chip (GE Healthcare) by injecting peptide (1 lm) at a ﬂow rate of 5 lLÆmin)1 in 0.01 m Hepes, pH 7.4, containing 0.15 m NaCl, 3 mm ethylenediaminetet- raacetic acid (EDTA) and 0.005% (v ⁄ v) surfactant P20 (HBS-EP) on a BIAcore 3000 (GE Healthcare, Diegem, Belgium). Typically, a signal ranging from 1000 to 1500 RU was obtained. Kinetic analysis was performed by inject- ing peptide 3 at various concentrations (30–2000 nm) at a ﬂow rate of 70 lLÆmin)1. Regeneration of the surface was performed by a single injection of 15 lL of 10 mm glycine, pH 2. For all sensorgrams, the signal obtained on a control surface (irrelevant peptide) was subtracted from the signal obtained on the relevant surface. The presence of mass transfer phenomena was excluded by performing the con- trol assays, as recommended by BIAcore. Kinetic data anal- ysis was performed using biaevaluation 4.1 software employing a two-state reaction model in agreement with the presence of linked reactions. HCDR3 fragments corresponding to the IgM and IgG rep- ertoires of a nonimmunized donor were PCR ampliﬁed with the 6-carboxyﬂuorescein (FAM)-labeled pool of backward primers and a pool of forward primers, as described previ- ously [12]. A total of 1 lL of labeled PCR product was mixed with 12 lL of HiDi formamide (Applied Biosystems, Nieuwerkerk a ⁄ d Ijssel, the Netherlands) and 0.5 lL of ROX and DS33 size standards (Applied Biosystems). The ﬂuorescent HCDR3 fragments were separated by electro- phoresis on an ABI3100 capillary sequencer, and their lengths (Kabat positions 95–102) were calculated using the ROX and DS33 size standards.

Ala scanning

Analysis of amino acid sequences

Ala mutations were introduced at each position of the HCDR3 sequence of clone 3 (positions 92–104) by overlap- ping PCR using a set of speciﬁc primers. Mutated frag- ments were digested by BglI and NotI and cloned into the phagemid vector. HCDR3 loops of randomly picked colonies of the ampliﬁed IgM HCDR3 repertoire of the nonimmunized donor were sequenced on an ABI3100 capillary sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems).

cAMP assay

Sequence analysis was performed to ensure the presence of the mutation, and all individual Ala mutated clones were ampliﬁed and tested at a single concentration (2 · 1012 phage per well) in phage-ELISA on ECL2 peptide. The sig- nal recorded with each Ala mutant was compared with the signal recorded with wild-type phage clone 3 and irrelevant phage (HA). Experiments were performed in triplicate.

Acknowledgements

This study was ﬁnancially supported by the ‘Fonds National de la Recherche’, Luxembourg [BIOSAN ⁄ 07 ⁄ 19 (PEPSAR project) and C09 ⁄ BM ⁄ 20 (MIMOKINE project)] and the ‘Centre de Recherche Public-Sante´ ’, Luxembourg (grant SAN ⁄ 03 ⁄ 00, 20070115). The authors thank Charle` ne Verschueren for technical assistance.

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The inhibition of primary intracellular cAMP production induced by binding of the chemokine CXCL12 to CXCR4 was evaluated in the presence of the ECL2-speciﬁc HCDR3 phage clones using time-resolved ﬂuorescence resonance energy transfer LANCE cAMP assay (Perkin-Elmer, Waltham, MA, USA) adapted for a 96-well plate format. MT-4 cells expressing CXCR4 and CEM.NKR.CCR5 cells (National Institutes of Health AIDS Program) were har- vested, washed with simulation buffer [Hank’s buffered salt solution (1 · ) containing 5 mm Hepes, 0.1% BSA, 0.5 mm 3-Isobutyl-1-methylxanthine (IBMX), pH 7.4] and resus- pended in simulation buffer containing Alexa Fluor(cid:2) 647-labeled antibodies (1 : 100 dilution). Cells (20 000 per

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HCDR3-derived CXCR4 antagonists

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