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

Selection of a CXCR4 antagonist from a human heavy
chain CDR3-derived phage library
Andy Chevigne
´
1
, Aure
´
lie Fischer
1
, Julie Mathu
1
, Manuel Counson
1
, Nadia Beaupain
1
,
Jean-Marc Plesse
´
ria
1
, Jean-Claude Schmit
1,2
and Sabrina Deroo
1
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:
(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 l
M and acted as a CXCR4
antagonist (IC
50
=23lM). 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
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
receptors, to identify target-specific peptides [1–8].
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; V
H
, heavy
chain variable region fragment.
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS 2867
fragments (V
H
). 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].
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
life of the B-cell repertoire [16,17]. In the mouse
model, the HCDR3 repertoire evolved during develop-
ment to eliminate outliers in terms of 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 V
H
gene families [20,21].
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
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].
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
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
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
HCDR3-derived CXCR4 antagonists A. Chevigne
´
et al.

2868 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
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. Journal compilation ª 2011 FEBS
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).
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 (ECL2
lin
⁄ HA ratio,

23; ECL2
cycl
⁄ HA ratio, 9.8) (Fig. 1).
Characterization of the antagonistic properties of
ECL2-specific clones
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 IC
50
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

Table 1. Sequence, length, isolation frequency and specificity (ECL2
lin
⁄ HA ratio) of heavy chain complementarity determining region 3
(HCDR3) clones isolated from the IgMCys library. Residues presented in bold are identical in the different sequences. CAR- and -WC
sequences are part of the framework 3 and 4 residues, respectively. ECL2, second extracellular loop; HA, hemagglutinin.
Name Sequence Length
Frequency
(n) Elution
ECL2
lin
⁄
HA ratio
Cl 1 CAR-DR-GRYDST- - - - LRY-WC 11-mer 4 Dithiothreitol 5.1
Cl 43 CAR-GS-G-WDST- - - - GNY-WC 10-mer 1 pH 1.2
Cl 26 CAR-GRPGTGTTN- - - - LNY-WC 12-mer 1 pH 8.2
Cl 29 CAK-DGKGQYGSG- - -Y- - - - -INY-WC 13-mer 1 pH 8.0
Cl 6 CAR-DRRGWYCSGGSCY- - - - -LNY-WC 16-mer 4 Dithiothreitol 4.5
Cl 2 CAK-DRR-RDSSG WYY-WC 11-mer 1 Dithiothreitol 1.8
Cl 28 CAR-DRG-SGSYG VGY-WC 11-mer 1 pH 8.1
Cl 3 CAR-DRGGTYPGR- - - - —Y-WC 10-mer 29 Dithiothreitol ⁄ pH 23
Cl 11 CAR-DRGWGAVAG
PP- -IRY-WC 14-mer 2 Dithiothreitol 1.8
Cl 4 CAR-DRG AG PP GY-WC 9-mer 1 Dithiothreitol 1.5
Cl 18 CAR-GGGIAAPGG PP- -PVY-WC 14-mer 5 pH 1.2
Cl 24 CAR-PAGG VRGRVQQQPPGTVVY-WC 19-mer 1 pH 8.2
Cl 40 CAR-RGDG SRGWAD- Y-WC 11-mer 1 pH 1.4
Cl 7 CAR-DGTM- -VRGVPG- Y-WC 11-mer 7 Dithiothreitol 1.4
Cl 9 CAR-DGTG- -YGGNSGG- - - - - -VY-WC 14-mer 1 Dithiothreitol 1
Cl 13 CAR-EA- - - - -RAGPY- Y-WC 8-mer 1 Dithiothreitol 1.2
Cl 38 CAR-DPTLGYSGG

GPLS- - - - - - -Y-WC 14-mer 1 pH 2.1
Cl 31 CAR-SGPRS-S FD Y-WC 9-mer 1 pH 3.7
Cl 39 CAR-APPVSGSQVGYPY- - - - - - -Y-WC 14-mer 1 pH 5.7
Cl 8 CAR-DNPRSYSSSSGAV- - - - - - -Y-WC 14-mer 1 Dithiothreitol 1.5
Cl 30 CAR-DGTYSGY–EYPR- Y-WC 12-mer 1 pH 9.6
A. Chevigne
´
et al. HCDR3-derived CXCR4 antagonists
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS 2869
confirmed in a similar analysis using CCR5-expressing
cells (CEM.NK
R
-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 K
D
value of 5.6 lm for peptide 3
(Fig. 3A).
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.
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
Fig. 1. Specificity of the most 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 · 10
12
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.

Fig. 2. Antagonistic properties of the second extracellular 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 · 10
12
particlesÆmL
)1
) was incubated
with forskolin (FSK) and the chemokine CXCL12 (30 n
M). Variation
in the CXCL12-induced cAMP inhibition was detected using time-
resolved fluorescence resonance energy transfer technology.
(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.
HCDR3-derived CXCR4 antagonists A. Chevigne
´
et al.
2870 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS
the complexities of the HCDR3 libraries in terms of
length, amino acid content and position variability.
To better characterize the HCDR3 length diversity,

a set of forward and backward primers based on the
nucleotide alignment of 1182 human V
H
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
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 after cloning 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.
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 efficient interaction with the 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
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).
Analysis of the individual amino acid frequency
as a function of the HCDR3 length
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).
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 n
M).
Red sensorgrams represent the fitted curves on a two-state inter-
action model (v
2
= 0.276) using the BIAEVALUATION 4.1 program.
SPR, surface plasmon resonance. (B) Ala scanning of the interac-
tion between phage clone 3 (2 · 10
12
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.
A. Chevigne
´
et al. HCDR3-derived CXCR4 antagonists
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS 2871
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, respectively; two-tailed P < 0.6). The fre-
quency of Ile tended to increase with increasing
HCDR3 length (Pearson’s r = 0.5884; two-tailed
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
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
160 nt150 nt 180 nt 190 nt
10 aa
120 nt 140 nt 160 nt 180 nt
11 aa
A
120 nt 160 nt150 nt 180 nt 190 n
t
200 nt 120 nt 140 nt 160 nt 180 nt 200 nt
BC D
11 aa
9 aa
120 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.
K
Q
C
H
Y 15%
M
E
N
P 3%%
I 3%
W 4%
D 12%
T 4%
V 5%
G 12%
R 5%
%
A 5%
S 9%
L 5%
F 6%
E
I
Q
M

C
W
N
L
G 25%
A 4%
T 5%
V 6%
S 7%
Y 14%
D 8%
R 11%
P 10%
A
B
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.
HCDR3-derived CXCR4 antagonists A. Chevigne
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2872 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
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. Journal compilation ª 2011 FEBS
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
of the G-protein-coupled receptor CXCR4. Peptide
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].
Eleven selection campaigns with five conventional
fully randomized phage-displayed peptide libraries,
each displaying single size peptides on ECL2, were not
successful. These results illustrate the difficulty in
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 clone isolated at the highest
frequency acted as an antagonist (IC
50
=23lm) 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.
To explain the difference in efficiency between bio-
logical (HCDR3) and fully randomized synthetic pep-
tide libraries, the properties of the HCDR3 phage
library were analyzed in terms of length, amino acid
composition and sequence variability.
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-

ertoires; ideal versus skewed Gaussian-like profiles
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].
The overall amino acid distribution in our set of
HCDR3 sequences revealed a biased content in the
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
Cys 0.864 0.059
Lys 0.922 0.026
Asn 0.534 0.354
Ala 0.522 0.335
Gln 0.424 0.477
Thr 0.496 0.395
Gly )0.711 0.178
Pro )0.532 0.356
Phe )0.678 0.209
Leu )0.522 0.367

Tyr )0.350 0.564
Asp )0.349 0.565
Ile 0.329 0.588
His 0.201 0.745
Arg 0.215 0.729
Glu 0.171 0.784
Met 0.238 0.701
Trp )0.029 0.964
Ser 0.020 0.974
Val )0.032 0.960
A. Chevigne
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et al. HCDR3-derived CXCR4 antagonists
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS 2873
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 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
positive correlation as a function of the HCDR3
length, as observed by Zemlin et al. [35]. For Phe,
a negative correlation was identified.
In addition, HCDR3 repertoires displayed position-
dependent biased sequence diversity. Clear limited
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–
102. The variability of the C-terminal end of the
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.
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
flexible linkers to separate ‘hot spots’ for binding.
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-
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].
In addition to the advantages inherent 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.
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.
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
HCDR3-derived CXCR4 antagonists A. Chevigne
´
et al.
2874 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS
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].
In summary, we have demonstrated the value of nat-
urally size and sequence randomized human HCDR3
libraries, in comparison with fully synthetic peptide
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.
Materials and methods
Chemicals and cell lines

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
(NVSEADDRYICDRFYPNDLWVVFQFQ) and cyclic
(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)
(VYYCARDRGGTYPGRYWCQG), elongated with six
(VYYCAR) and four (WCQG) amino acids from the
framework 3 and 4 residues, respectively, was purchased
from Bachem.
The cell lines MT-4 expressing CXCR4 and CEM.NK
R
-
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.
Fully randomized peptide and HCDR3 phage
libraries
Five different fully randomized phage-displayed peptide
libraries were screened. For minor coat protein (pIII) dis-
played libraries, linear dodecapeptide (12-mer) (PhD-12)
and disulfide bridge-constrained heptapeptide (7-mer)

(CX
7
C) libraries were purchased from New England Biol-
abs (Ipswich, MA, USA). In the CX
7
C 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 (X
5
CXCX
5
) and tetradeca-
peptide Cys4 (X
4
CX
4
CX
4
) libraries, were kindly provided
by G. P. Smith (University of Missouri, CO, USA).
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 V
H
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 · 10
8
clones [12].
Affinity selection of fully randomized peptide and
HCDR3 phage libraries
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).
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 · 10
12
phage particles
for 2 h in 2% milk. After washing the beads five times with
NaCl ⁄ P
i
⁄ 0.5% Tween 20, the target-bound phage was first
eluted with 500 lLof75mm 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
buffer, pH 8.0. Eluates were used to infect log phase
Escherichia coli TG1 cells. Phage was rescued with
M13K07 helper phage and employed for three additional
selection rounds using 1 · 10
12
,1· 10
11
and 1 · 10
10
phage
particles and 10, 20 and 40 washing steps in rounds 2, 3
and 4, respectively.
Screening of positive clones
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
A. Chevigne
´
et al. HCDR3-derived CXCR4 antagonists
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS 2875
streptavidin. After blocking plates with NaCl ⁄ P
i
⁄ 2% milk
for 1 h, plates were washed with NaCl ⁄ P

i
⁄ 0.05% Tween 80
and incubated with culture supernatants in NaCl ⁄ P
i
⁄ 2%
milk for 2 h. After washing with NaCl ⁄ P
i
⁄ 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.
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.
Length distribution analysis of the amplified
HCDR3 repertoires
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 lLof
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.
Analysis of amino acid sequences
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
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.NK
R
.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
Ò
647-labeled antibodies (1 : 100 dilution). Cells (20 000 per
well) were incubated for 30 min at room temperature with
2 · forskolin CXCL12 per phage or RANTES per phage
(30 nm and 5 · 10
12
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
standard curves (10
)6
–10
)11
m). Experiments were per-
formed in triplicate.
Surface plasmon resonance
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
(10 min) 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)ata
flow rate of 70 lLÆmin
)1
. Regeneration of the surface was
performed by a single injection of 15 lLof10mm 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.
Ala scanning
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.
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 · 10
12
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.

HCDR3-derived CXCR4 antagonists A. Chevigne
´
et al.
2876 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS
References
1 Koivunen E, Wang B & Ruoslahti E (1995) Phage
libraries displaying cyclic peptides with different ring
sizes: ligand specificities of the RGD-directed integrins.
Biotechnology (NY) 13, 265–270.
2 Miceli RM, DeGraaf ME & Fischer HD (1994) Two-
stage selection of sequences from a random phage dis-
play library delineates both core residues and permitted
structural range within an epitope. J Immunol Methods
167, 279–287.
3 Parhami-Seren B, Keel T & Reed GL (1997) Sequences
of antigenic epitopes of streptokinase identified via ran-
dom peptide libraries displayed on phage. J Mol Biol
271, 333–341.
4 Pelletier J & Sidhu S (2001) Mapping protein–protein
interactions with combinatorial biology methods. Curr
Opin Biotechnol 12, 340–347.
5 Scott JK & Smith GP (1990) Searching for peptide
ligands with an epitope library. Science 249, 386–
390.
6 Smith GP (1985) Filamentous fusion phage: novel
expression vectors that display cloned antigens on the
virion surface. Science 228, 1315–1317.
7 Chevigne A, Yilmaz N, Gaspard G, Giannotta F,

Francois JM, Frere JM, Galleni M & Filee P (2007)
Use of bifunctional hybrid beta-lactamases for epitope
mapping and immunoassay development. J Immunol
Methods 320, 81–93.
8 Deroo S, El Kasmi KC, Fournier P, Theisen D, Brons
NH, Herrmann M, Desmet J & Muller CP (1998)
Enhanced antigenicity of a four-contact-residue epitope
of the measles virus hemagglutinin protein by phage
display libraries: evidence of a helical structure in the
putative active site. Mol Immunol 35, 435–443.
9 Hoogenboom HR (1997) Designing and optimizing
library selection strategies for generating high-affinity
antibodies. Trends Biotechnol 15, 62–70.
10 Ward ES, Gussow D, Griffiths AD, Jones PT & Winter
G (1989) Binding activities of a repertoire of single
immunoglobulin variable domains secreted from Escher-
ichia coli. Nature 341, 544–546.
11 Winter G, Griffiths AD, Hawkins RE & Hoogenboom
HR (1994) Making antibodies by phage display technol-
ogy. Annu Rev Immunol 12, 433–455.
12 Deroo S, Fischer A, Beaupain N, Counson M, Bouton-
net N, Pletinckx J, Loverix S, Beirnaert E, De Haard
H, Schmit JC et al. (2008) Non-immunized natural
human heavy chain CDR3 repertoires allow the isola-
tion of high affinity peptides mimicking a human
influenza hemagglutinin epitope. Mol Immunol 45,
1366–1373.
13 Kabat EA & Wu TT (1991) Identical V region amino
acid sequences and segments of sequences in antibodies
of different specificities. Relative contributions of VH

and VL genes, minigenes, and complementarity-deter-
mining regions to binding of antibody-combining sites.
J Immunol 147, 1709–1719.
14 Knappik A, Ge L, Honegger A, Pack P, Fischer M,
Wellnhofer G, Hoess A, Wolle J, Pluckthun A & Virne-
kas B (2000) Fully synthetic human combinatorial anti-
body libraries (HuCAL) based on modular consensus
frameworks and CDRs randomized with trinucleotides.
J Mol Biol 296, 57–86.
15 Morea V, Tramontano A, Rustici M, Chothia C &
Lesk AM (1998) Conformations of the third hypervari-
able region in the VH domain of immunoglobulins.
J Mol Biol 275, 269–294.
16 Souto-Carneiro MM, Sims GP, Girschik H, Lee J &
Lipsky PE (2005) Developmental changes in the human
heavy chain CDR3. J Immunol 175, 7425–7436.
17 Schroeder HW Jr, Zhang L & Philips JB III (2001)
Slow, programmed maturation of the immunoglobulin
HCDR3 repertoire during the third trimester of fetal
life. Blood 98, 2745–2751.
18 Ivanov II, Schelonka RL, Zhuang Y, Gartland GL,
Zemlin M & Schroeder HW Jr (2005) Development of
the expressed Ig CDR-H3 repertoire is marked by
focusing of constraints in length, amino acid use, and
charge that are first established in early B cell progeni-
tors. J Immunol 174
, 7773–7780.
19 Baranzini SE, Jeong MC, Butunoi C, Murray RS,
Bernard CC & Oksenberg JR (1999) B cell repertoire
diversity and clonal expansion in multiple sclerosis

brain lesions. J Immunol 163, 5133–5144.
20 Foreman AL, Lemercier B, Lim A, Kourlisky P, Kenny
T, Gershwin ME & Gougeon ML (2008) VH gene usage
and CDR3 analysis of B cell receptor in the peripheral
blood of patients with PBC. Autoimmunity 41, 80–86.
21 Voswinkel J, Weisgerber K, Pfreundschuh M & Gause
A (2001) B lymphocyte involvement in ankylosing spon-
dylitis: the heavy chain variable segment gene repertoire
of B lymphocytes from germinal center-like foci in the
synovial membrane indicates antigen selection. Arthritis
Res 3, 189–195.
22 Saphire EO, Parren PW, Pantophlet R, Zwick MB,
Morris GM, Rudd PM, Dwek RA, Stanfield RL,
Burton DR & Wilson IA (2001) Crystal structure of a
neutralizing human IGG against HIV-1: a template for
vaccine design. Science 293, 1155–1159.
23 Manoutcharian K, Acero G, Munguia ME, Becerril B,
Massieu L, Govezensky T, Ortiz E, Marks JD, Cao C,
Ugen K et al. (2004) Human single chain Fv antibodies
and a complementarity determining region-derived
peptide binding to amyloid-beta 1-42. Neurobiol Dis
17, 114–121.
24 Smith JW, Tachias K & Madison EL (1995) Protein
loop grafting to construct a variant of tissue-type
plasminogen activator that binds platelet integrin alpha
IIb beta 3. J Biol Chem 270, 30486–30490.
A. Chevigne
´
et al. HCDR3-derived CXCR4 antagonists
FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante

´
. Journal compilation ª 2011 FEBS 2877
25 Brelot A, Heveker N, Montes M & Alizon M (2000)
Identification of residues of CXCR4 critical for human
immunodeficiency virus coreceptor and chemokine
receptor activities. J Biol Chem 275, 23736–23744.
26 Zhou N, Luo Z, Luo J, Liu D, Hall JW, Pomerantz RJ
& Huang Z (2001) Structural and functional character-
ization of human CXCR4 as a chemokine receptor and
HIV-1 co-receptor by mutagenesis and molecular mod-
eling studies. J Biol Chem 276, 42826–42833.
27 Jahnichen S, Blanchetot C, Maussang D, Gonzalez-Pa-
juelo M, Chow KY, Bosch L, De Vrieze S, Serruys B,
Ulrichts H, Vandevelde W et al. (2010) CXCR4 nano-
bodies (VHH-based single variable domains) potently
inhibit chemotaxis and HIV-1 replication and mobilize
stem cells. Proc Natl Acad Sci USA 107, 20565–20570.
28 Baribaud F, Edwards TG, Sharron M, Brelot A, Heve-
ker N, Price K, Mortari F, Alizon M, Tsang M &
Doms RW (2001) Antigenically distinct conformations
of CXCR4. J Virol 75, 8957–8967.
29 Strizki JM, Turner JD, Collman RG, Hoxie J & Gonz-
alez-Scarano F (1997) A monoclonal antibody (12G5)
directed against CXCR-4 inhibits infection with the
dual-tropic human immunodeficiency virus type 1
isolate HIV-1(89.6) but not the T-tropic isolate
HIV-1(HxB). J Virol 71, 5678–5683.
30 Johnson G & Wu TT (2004) The Kabat database and a
bioinformatics example. Methods Mol Biol 248, 11–25.
31 Ramirez-Benitez Mdel C, Moreno-Hagelsieb G &

Almagro JC (2001) VIR.II: a new interface with the
antibody sequences in the Kabat database. Biosystems
61, 125–131.
32 Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch
V, Abagyan R, Brooun A, Wells P, Bi FC et al. (2010)
Structures of the CXCR4 chemokine GPCR with
small-molecule and cyclic peptide antagonists. Science
330, 1066–1071.
33 Zheng HM, Jiang Y, Wang JR, Gong XL & Guo BY
(2011) Mimic peptides bonding specifically with the first
and second extracellular loops of the CC chemokine
receptor 5 derived from a phage display peptide library
are potent inhibitors of experimental autoimmune
encephalomyelitis. Inflamm Res, doi:10.1007/s00011-011-
0331-8.
34 Johnson G & Wu TT (1998) Preferred CDRH3 lengths
for antibodies with defined specificities. Int Immunol 10,
1801–1805.
35 Zemlin M, Klinger M, Link J, Zemlin C, Bauer K,
Engler JA, Schroeder HW Jr & Kirkham PM (2003)
Expressed murine and human CDR-H3 intervals of
equal length exhibit distinct repertoires that differ in
their amino acid composition and predicted range
of structures. J Mol Biol 334, 733–749.
36 Raaphorst FM, Raman CS, Tami J, Fischbach M &
Sanz I (1997) Human Ig heavy chain CDR3 regions in
adult bone marrow pre-B cells display an adult
phenotype of diversity: evidence for structural selection
of DH amino acid sequences. Int Immunol 9, 1503–
1515.

37 Miqueu P, Guillet M, Degauque N, Dore JC, Soulillou
JP & Brouard S (2007) Statistical analysis of CDR3
length distributions for the assessment of T and
B cell repertoire biases. Mol Immunol 44, 1057–1064.
38 Koide S & Sidhu SS (2009) The importance of being
tyrosine: lessons in molecular recognition from minimal-
ist synthetic binding proteins. ACS Chem Biol 4,
325–334.
39 Evers TH, van Dongen EM, Faesen AC, Meijer EW &
Merkx M (2006) Quantitative understanding of the
energy transfer between fluorescent proteins connected
via flexible peptide linkers. Biochemistry 45, 13183–
13192.
40 Robinson CR & Sauer RT (1998) Optimizing the
stability of single-chain proteins by linker length and
composition mutagenesis. Proc Natl Acad Sci USA 95,
5929–5934.
41 Fellouse FA, Barthelemy PA, Kelley RF & Sidhu SS
(2006) Tyrosine plays a dominant functional role in
the paratope of a synthetic antibody derived from a
four amino acid code. J Mol Biol 357, 100–114.
42 Lapidot A, Peled A, Berchanski A, Pal B, Kollet O,
Lapidot T & Borkow G (2008) NeoR6 inhibits
HIV-1–CXCR4 interaction without affecting CXCL12
chemotaxis activity. Biochim Biophys Acta 1780,
914–920.
43 Tamamura H, Imai M, Ishihara T, Masuda M,
Funakoshi H, Oyake H, Murakami T, Arakaki R,
Nakashima H, Otaka A et al. (1998) Pharmacophore
identification of a chemokine receptor (CXCR4) antag-

onist, T22 ([Tyr(5,12),Lys7]-polyphemusin II), which
specifically blocks T cell-line-tropic HIV-1 infection.
Bioorg Med Chem 6, 1033–1041.
44 Chevigne
´
A, Delhalle S, Verschueren C, Beaupain N,
Counson M, Plesse
´
ria JM, Devaux C, Schmit JC &
Deroo S (2011) Identification of a HIV-1 neutralizing
antibody fragment from the heavy chain CDR3 reper-
toire of a long-term non-progressor. In: 18th Conference
on Retrovirus and Opportunutsic Infections (CROI 2011,
eds), Poster 380. Boston, MA.
45 Siciliano SJ, Kuhmann SE, Weng Y, Madani N,
Springer MS, Lineberger JE, Danzeisen R, Miller MD,
Kavanaugh MP, DeMartino JA et al. (1999) A critical
site in the core of the CCR5 chemokine receptor
required for binding and infectivity of human
immunodeficiency virus type 1. J Biol Chem 274,
1905–1913.
46 Parmley SF & Smith GP (1988) Antibody-selectable
filamentous fd phage vectors: affinity purification of
target genes. Gene 73, 305–318.
HCDR3-derived CXCR4 antagonists A. Chevigne
´
et al.
2878 FEBS Journal 278 (2011) 2867–2878 ª 2011 CRP - Sante
´
. Journal compilation ª 2011 FEBS