A Novel Promiscuous Class of Camelid

Single-Domain Antibody Contributes to the

This information is current as
of June 20, 2022.
Antigen-Binding Repertoire
Nick Deschacht, Kurt De Groeve, Cécile Vincke, Geert
Raes, Patrick De Baetselier and Serge Muyldermans
J Immunol 2010; 184:5696-5704; Prepublished online 19
April 2010;
doi: 10.4049/jimmunol.0903722
http://www.jimmunol.org/content/184/10/5696

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Supplementary http://www.jimmunol.org/content/suppl/2010/04/19/jimmunol.090372
Material 2.DC1
References This article cites 47 articles, 11 of which you can access for free at:
http://www.jimmunol.org/content/184/10/5696.full#ref-list-1

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 The Journal of Immunology

A Novel Promiscuous Class of Camelid Single-Domain

Antibody Contributes to the Antigen-Binding Repertoire

Nick Deschacht,*,†,1 Kurt De Groeve,*,†,1 Cécile Vincke,*,† Geert Raes,*,†

Patrick De Baetselier,*,† and Serge Muyldermans*,†
It is well established that, in addition to conventional Abs, camelids (such as Camelus dromedarius and Lama glama) possess unique
homodimeric H chain Abs (HCAbs) devoid of L chains. The Ag-binding site of these HCAbs consists of a single variable domain, referred
to as VHH. It is widely accepted that these VHHs, with distinct framework-2 imprints evolved within the V(H) clan III-family 3, are
exclusively present on HCAbs. In this study, we report the finding of a distinct leader signal sequence linked to variable genes displaying
a high degree of homology to the clan II, human VH(4) family that contributes to the HCAb Ag-binding diversity. Although the VHH
framework-2 imprints are clearly absent, their VH(4)-D-JH recombination products can be rearranged to the H chains of both classical
and HCAbs. This suggests that for these V domains the presence of a L chain to constitute the Ag-binding site is entirely optional. As such,
the capacity of this promiscuous VH(4) family to participate in two distinct Ab formats significantly contributes to the breadth of the

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camelid Ag-binding repertoire. This was illustrated by the isolation of stable, dendritic cell-specific VH(4) single domains from a VH(4)-
HCAb phage display library. The high degree of homology with human VH(4) sequences is promising in that it may circumvent the need
for “humanization” of such single-domain Abs in therapeutic applications. The Journal of Immunology, 2010, 184: 5696–5704.

C
amelidae are known to possess a dichotomous immune
system that enables them to generate 1) classical Abs
composed of two H and two L chains and 2) Abs composed
of two H chains and no L chains. The latter Abs are referred to as H
chain Abs (HCAbs) (1, 2), and their H chain also lacks the first
constant domain (CH1), which in a classical Ab interacts with the
constant domain of the L chain. The Ag-binding fragment of these
HCAbs is reduced in size to a single variable domain (referred to as
VHH) and is generated from a V-D-JH gene rearrangement for
which a distinct set of V genes, the VHH germline genes, is available
in the camelid genome (3–5). Different subfamilies have been dis-
tinguished for these VHH genes, but all are closely related to the
human VH(3) family of clan III (4, 6). A VHH(3) domain of drom-
edary or llama HCAbs differs from the VH(3) domain of classical
Abs only by a few crucial substitutions of amino acids normally
involved in VL pairing (7, 8). These framework region 2 (FR2)
mutations, Val37Phe/Tyr, Gly44Glu, Leu45Arg, and Trp47Gly [Kabat
numbering system (9)], are encoded in a dedicated subset of V
germline genes of camelids and are therefore not the result of so-
*Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel; and
†
Department of Molecular and Cellular Interactions, VIB, Brussels, Belgium
1
N.D. and K.D.G. contributed equally to this work.
Received for publication November 19, 2009. Accepted for publication March 8,
2010.
This work was supported by the Instituut Voor de Aanmoediging van Innovatie door
Wetenschap en Technologie in Vlaanderen (to N.D.). K.D.G. is a Ph.D. fellow of the
Horizontale Onderzoeks Actie of the Vrije Universiteit Brussel.
Address correspondence and reprint requests to Dr. Serge Muyldermans, Laboratory
of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Building E, Plein-
laan 2, 1050 Brussels, Belgium. E-mail address: svmuylde@vub.ac.be
The online version of this article contains supplemental material.
Abbreviations used in this paper: BM, bone marrow; BMDC, bone marrow-derived
dendritic cell; CD, circular dichroism; CH1, first constant domain; DC, dendritic cell;
FR, framework region; HCAb, H chain Ab; HIS, histidine; IMAC, immobilized metal
ion affinity chromatography; LB, Luria-Bertani; LS, leader signal; rmGM-CSF, re-
combinant murine granulocyte macrophage colony-stimulating factor; sdAb, single-
domain Ab; SEC, size exclusion chromatography.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
matic hypermutation (2). Obviously, such hallmark mutations in the
FR2 will reshape the VL interface and abrogate a possible VH–VL
association. These VHH hallmark residues are also considered to be
important for the solubility of the autonomous VHH(3) domain (10,
11) and were demonstrated to affect its stability (12, 13). However,
classical Ag-binding single-domain Ab (sdAb) fragments of family
VH(3) (i.e., lacking the VHH characteristic hallmark residues in FR2)
have been isolated occasionally from immune V libraries, although
some controversy remains about the exact IgG isotype (HCAbs or
classical) at its origin (14–17). Apart from the absence of the VH(3)
hallmark residues, these VH(3) sdAbs also lack the typical interloop
disulfide bridge and the long CDR3 loops, yet they remain highly
soluble, stable, and functional. So far, it was assumed that all of these
variable domains of HCAbs are part of the same clan III VH(3) family.
In this study, we report the presence of novel variable domains of
clan II and describe their ability to be part of classical as well as HCAbs.
Indeed, although all of the V domains of this clan were classical Ab VH
sequences, they are able to generate functional HCAbs, so-called VH-
HCAbs. Analysis of these novel clan II VH-HCAbs and sequence
comparison with human V sequences indicate a strong resemblance
with the human VH(4) family. We also report that the same VH(4)-D-
JH recombination products can be rearranged to both camelid classical
and HCAbs. Therefore, the variable domain obtained after a VH(4)-D-
JH recombination is functional as an Ag-binding entity as a single
domain or after combining with a VL as a VH–VL pair. Hence, the use
of a L chain is entirely superfluous to generate Abs with these VH(4)
genes. That these V domains are functional in Ag binding was dem-
onstrated by the retrieval of dendritic cell-specific VH(4) sdAbs from
a llama sdAb library derived from the VH(4)-HCAbs. These llama
sdAbs exhibit good solubility and are the first autonomous single-
domain Ab fragments reported from the VH(4)-HCAbs repertoire.
Materials and Methods
Animals
Mice. Female C57BL/6J mice (6–8 wk old) were used from the in house
breeding facility. Animal care and treatment were conducted in conformity
with institutional guidelines of the Belgian Council for Laboratory Animal

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903722
The Journal of Immunology
Science as inspired by the Federation of European Laboratory Animal
Science Associations recommendations.
Llama. The Lama glama were kept at the premises of the veterinary school
of the University of Ghent. All of the animal experiments were conducted
with the approval of the Ethical Committee of the Faculty of Veterinary
Medicine (University of Ghent, Ghent, Belgium) (18).
Dromedary. The Camelus dromedarius was housed at the Central Veteri-
nary Research Laboratory (Dubai, United Arab Emirates).
Cells
Splenic CD11c+ dendritic cells. Four female C57BL/6J mice were infected
with 5000 Trypanosoma brucei brucei parasites (administered i.p. to recruit
dendritic cells [DCs] to the spleen) and sacrificed 7 d postinfection. Spleens
were taken and treated with 400 U/ml collagenase III (Roche, Basel,
Switzerland) for 30 min at 37˚C in HBSS (Gibco, Invitrogen, Paisley, U.K.)
and neutralized by adding 2% heat-inactivated FCS (Hyclone, Logan, UT)
and 2 mM EDTA (Invitrogen). Cells were treated with Tris-buffered am-
monium chloride to lyse erythrocytes and passed through a nylon mesh to
remove undigested material. The splenic myeloid cell fraction was enriched
by centrifugation (2000 rpm for 25 min at room temperature) on a Nyco-
denz gradient (OD360nm = 0.660–0.670). The cellular fraction of the in-
terphase was purified by positive selection on magnetic separation columns
(MACS) with CD11c+ beads (Miltenyi Biotec, Gladbach, Germany) ac-
cording to manufacturers’ recommendations.
Murine bone marrow-derived CD11c+ DCs. Femurs and tibias were removed
and mechanically purified from surrounding tissues. Subsequently, epiphyses
were removed, and bone marrow (BM) was flushed using RPMI 1640 (Invi-
trogen). Erythrocytes in the single-cell suspensions were lysed as mentioned
above. Fresh BM cells were cultured in 10 ml R10 medium consisting of RPMI
1640 (Invitrogen) supplemented with penicillin (100 U/ml; Invitrogen),
streptomycin (100 mg/ml; Invitrogen), 2-ME (50 mM; Sigma-Aldrich, St.
Louis, MO), and 5% heat-inactivated FCS (Hyclone). At day 0, BM cells were
seeded at 2 3 106 cells per 100-mm diameter bacteriological Petri dish (Becton
Dickinson) in 10 ml R10 medium containing 200 U/ml (20 ng/ml) recombinant
murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) (gift
from laboratory of Prof. K. Thielemans). At day 3, another 10 ml R10 medium
containing 200 U/ml rmGM-CSF was added to the plates. At day 6, half of the
culture supernatant was collected and centrifuged, and the cell pellet was re-
suspended in 10 ml fresh R10 medium containing 100 U/ml rmGM-CSF and
poured into the original plate. Reducing the concentration of the rmGM-CSF
during culture diminishes the granulocyte contamination. Harvesting the cells
1 d later yielded the immature DC population. For complete maturation of DCs,
the 7-d-old nonadherent cells were collected by gentle pipetting, centrifuged at
1500 rpm for 5 min at room temperature, and resuspended in 6 ml fresh R10
medium into a fresh six-well plate containing 100 U/ml rmGM-CSF and 1 mg/
ml LPS (Sigma-Aldrich). Cells were then cultured for an additional 1 or 2 d to
obtain the mature DC population (19).
RACE of dromedary IgG
PBLs were isolated from dromedary or llama using Lymphoprep (Axis-Shield,
Oslo, Norway), and mRNA was extracted (20). Then, with a SMART RACE
cDNA amplification kit (BD Clontech, Heidelberg, Germany), cDNA was gen-
erated, and 59 RACE PCR products were amplified by using a FR4-specific prim-
er (59-GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-39). These 59
RACE products were cloned into the pCR2.1-TOPO vector using the TOPO TA
cloning kit (Invitrogen), and insert-positive clones were identified for sequencing
by blue–white selection. Subsequently the 59 ends encoding our dromedary and
llama leader signal (LS) sequences and V elements were aligned to the corre-
sponding sequences of the functional human IgV germline genes (21), as the
human IgH locus is well established (22, 23).
cDNA analysis and phylogenetic study
On the basis of the 59 RACE-positive clones, a novel primer (CALL5) for
the new LS sequence was designed (59-TGGTGGCAGGTCCCCAAGGT-
39). Standard PCR with CALL5 and a CH2-IgG–specific primer (59-
GGTACGTGCTGTTGAACTGTTCC-39) was performed on llama cDNA
(PBLs). The two amplification products (6700 and 1000 bp) were gel-
purified, cloned separately into TOPO TA, and identified for sequencing as
above. Alignments were calculated with CLUSTAL X (version 1.8) using
the GONNET series protein matrices. From this alignment, rooted phy-
logenetic trees were constructed by using the neighbor-joining method of
MEGA 4.0 (24). The amino acid sequences of FR1–FR3 as defined by the
Kabat numbering system (9) were used to generate the tree. From the
amino acid sequences of the VH(4)s, the isoelectric point (pI) and
weighted hydrophobicity were calculated by Expasy proteomics (25).
5697
Llama immunization and VH(4) library construction
A llama (L. glama) was injected s.c. with 108 immature mouse BM-derived
DCs (BMDCs) without adjuvant. The immunization was repeated six times
at weekly intervals. After the last boost, anticoagulated peripheral blood
was taken from the jugular vein to prepare an immune library. Gene
fragments from the V region leader sequence up until the CH2 domain of
the IgGs were first amplified as described above. The PCR fragments
containing the variable gene fragments of the IgG HCAbs were purified
from agarose gels. Subsequently, the gene fragments encoding the entire
V region were amplified with one additional PCR with nested primers
annealing at FR1 (59-CCAGCCGGCCATGGCTCAGGTGCAGCTGGT-
CCAGTCTGG-39) and FR4 (59-GGACTAGTGCGGCCGCTGGAGAC-
GGTGACCTGGGT-39). The final PCR fragments were ligated into the
phagemid vector pHEN4 (20), using the restriction sites NcoI and NotI
(underlined). Ligated material was transformed in Escherichia coli cells
(TG1) and plated on selective medium (16). The colonies were scraped
from the plates and washed, and this cell library was stored at 280˚C in
Luria-Bertani (LB) medium supplemented with glycerol (50% final con-
centration).
Selection of Ag-specific sdAb fragments
A representative aliquot of the library was cultured in LB medium with
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ampicillin and infected with M13K07 helper phages (16) to express the
cloned V region at the tip of phage particles as fusion products with the
gene III bacteriophage coat protein. DC-specific V domains from this li-
brary were enriched using whole-cell panning on an in vivo cell population
of CD11c+ monocytes from the spleens of mice (26). Freshly harvested
splenic DCs (1 ml, 4 3 107 cells) were incubated with phage particles
(1.5 3 1011) for 1 h at room temperature. Cells were then pelleted by low-
speed centrifugation and resuspended in 2 ml MACS buffer, and anti-
CD11c beads (Miltenyi Biotec) were added to the suspension. Once mixed
with the beads, cells were then isolated via CD11c+ selection by MACS
according to the manufacturer’s protocol (Miltenyi Biotec). After the final
wash, the column was removed from the magnet, and the bead-coated/
phage-coated/CD11c+ cells were eluted from the column with 5 ml sterile
PBS. The CD11c+ cells were immediately centrifuged at 1800 rpm for 10
min and resuspended in 200 ml triethylamine (pH 10) to elute cell-bound
phages and lyse cells. After 10 min incubation at room temperature, the
solution of phage particle eluate and cellular debris was neutralized with
200 ml Tris-HCl (1 M, pH 7.4) and used to infect exponentially growing
E. coli TG1 cells that were further cultured for the second round of se-
lection. An aliquot of the neutralized phage particle eluate was also taken
to infect bacteria and plated on LB agar plates supplemented with ampi-
cillin. After each round of panning, individual colonies were picked ran-
domly from the titration plate, and their sdAb gene was sequenced.
Purification and identification of DC-specific sdAb fragments
Purification of the sdAb. The variable regions of the clones that appeared
multiple times after panning in our sequence analysis were recloned into the
pHEN6 expression vector (27) using the restriction enzymes NcoI and
Eco91I (Fermentas) and transformed into E. coli WK6 cells. The V gene,
under control of the lac promoter, is preceded by a PelB sequence to direct
the translation product to the periplasm and followed by a His6 tag to
facilitate the purification of the sdAb protein by immobilized metal ion
affinity chromatography (IMAC). Liter-scale production and purification
of these sdAbs by IMAC and gel filtration (Sephadex 75 column) in PBS
were performed as described previously (16).
Biophysical stability. To analyze the oligomeric state of the sdAbs, the VH(4)
sdAbs and two (family 3) sdAbs (28) were expressed and subsequently purified
(16). The purified monomeric fractions of the sdAbs in PBS at two different
concentrations (35 mM [0.5 mg/ml] and 175 mM [2.5 mg/ml]) were incubated
at 37 and 4˚C for up to 14 d. Samples (0.5 mg of each sdAb) were taken at
different time points (day 0, 1, 7, and 14), and their integrity was analyzed by
size exclusion chromatography (SEC) on an ÄKTA Explorer (GE Healthcare,
Munich, Germany) using Sephadex 75 in PBS and by circular dichroism (CD)
measurement. CD spectra were obtained with a Jasco J715 spectropolarimeter
in the UV region (200–350 nm), using a protein concentration of 0.2 mg/ml
and a 0.1-cm cell path length. Data were acquired with a reading frequency of
1/20 s21, a 1 s integration time, and a 2 nm bandwidth. Data analysis was
performed according to Dumoulin et al. (13).
Temperature-induced denaturation. Thermal unfolding was followed by CD
with a Jasco J715 spectropolarimeter. A total volume of 300 ml of each
sample was heated in 50 mM sodium phosphate buffer (pH 7). The tem-
perature was increased from 35 to 95˚C at a rate of 1˚C/min, and the signal
intensity at 205 nm was recorded as a function of temperature.
5698
Flow cytometry. For flow cytometric analysis, the DC population was stained
with Abs (from BD Biosciences [San Jose, CA], unless otherwise stated) for
20 min at 4˚C using standard protocols. Cells (106 per 100 ml) were incubated
with 1 mg anti-FcgR Ab (clone 2.4G2) before adding allophycocyanin-
conjugated CD11c (HL3) Ab and 1 mg FITC-conjugated MHC II (clone M5/
114.15.2), FITC-conjugated CD86 (Gl-1), or FITC-conjugated CD40 Ab (clone
3/23). Cell recognition of different sdAbs (at 1 mg) was monitored using 0.1 mg
FITC-conjugated anti-histidine (HIS) (Serotec, Oxford, U.K.) as a secondary
Ab. Multiparameter acquisition was performed on an FACSCanto II (BD Bio-
sciences), followed by analysis with FlowJo (TreeStar, Ashland, OR).
Alexa Fluor 488 labeling of sdAb. The sdAbs were labeled using the Alexa
Fluor 488 protein labeling kit (Molecular Probes, Eugene, OR). Briefly, 100 ml
of 1 M sodium bicarbonate buffer was added to 1 ml sdAb solution at a con-
centration of 1 mg/ml. The sdAb solution (either sdAb_31 or sdAb_32) was
subsequently transferred to a vial of reactive dye (Alexa Fluor 488 carboxylic
acid, tetrafluorophenyl ester) and incubated overnight at 4˚C under mild agi-
tation. Labeled sdAb_31 and sdAb_32 were separated from unreacted dye by
SEC on Sephadex 75 in PBS. The protein concentration and the degree of
Alexa Fluor 488 labeling of the sdAb was determined via spectrophotometry
(Nanodrop ND-1000; Isogen Life Sciences, De Meern, The Netherlands).
In vitro competition study. Each BMDC population (106 cells in 100 ml)
was preincubated with 40 mg unlabeled sdAb (sdAb-BCII10, sdAb_31 or
sdAb_32) for 20 min at 4˚C. The cells with bound sdAb were recovered in
a pellet by low-speed centrifugation (supernatant containing the unbound
sdAb was removed), resuspended in PBS, stained with 1 mg Alexa Fluor
488-labeled sdAb_31 or sdAb_32, and incubated for 20 min at 4˚C.
Multiparameter acquisition was performed on an FACSCanto II (Becton
Dickinson), followed by analysis with FlowJo (Tree Star).
Results
Identification in camelids of a distinct LS linked to clan II
variable genes
A 59 RACE on cDNA of llama or dromedary PBLs with a JH-
specific primer (i.e., FR4 of VH) and a 59 universal primer was used to
investigate the V repertoire. This 59 RACE resulted in a PCR fragment
of ∼500 bp that was cloned into TOPO TA. Sequence analysis of these
cloned inserts revealed a few clones encoding a LS sequence that
deviates from the established LS sequence of camelid VH(3) and
VHH(3) genes (3) (Supplemental Fig. 1). The V region downstream
of the novel LS was aligned to the human IgV genes (23). Whereas
previous reports assigned the dromedary V genes to the VH(3) family
of clan III (3, 4), the International ImMunoGeneTics information
system amino acid numbering/V-QUEST (www.imgt.org) and Ig-
Blast (www.ncbi.nlm.nih.gov/igblast) search allocated the novel LS
and its V sequences to the human V genes of clan II.
Note that on previous occasions we always amplified the VHH genes
withLS-specificprimersoftheVHH(3)familythatwouldfailtoamplify
these new V genes, and therefore these V genes were eliminated from all
of our immune V libraries (15). Hence, a new primer (CALL5) based on
this novel LS (Supplemental Fig. 1) was designed. Interestingly, the
PCR amplification with CALL5 and a CH2-IgG–specific primer gen-
erated two distinct amplicons (Fig. 1). From their sizes, it can be in-
ferred that the longest fragment (61000 bp) corresponds to the H chain
fragment of classical IgG Abs, whereas the shorter fragment (6700 bp)
probably encodes a fragment of the H chain of the HCAbs. The dif-
ference in size accounts for the absence or presence of the CH1 region
that occurs only in H chains of classical IgGs (29). Fragments of both,
the HCAbs and the classical Abs, were gel-purified, cloned in-
dependently in TOPO TA vector, and sequenced. These particular clan
II variable genes occurred in the HCAb of all camelid samples tested,
although the band intensity varied and the fraction in the classical H
chain was always more pronounced (Fig. 1). Surprisingly, a 59 RACE
IgG analysis of the classical H chains demonstrated that the VH(4)
genes contributed up to 50% of the classical Ab repertoire.
Phylogenetic sequence analysis
A total of ∼150 individual clones, randomly chosen over both
clonings (i.e., classical and HCAb-derived), were sequenced. After
NOVEL CAMELID FAMILY 4 SINGLE-DOMAIN Abs
FIGURE 1. H chain amplification of IgG of two different L. glama.
Transcripts encoding the classical IgG and the H chain IgGs were am-
plified by PCR with primers specific to the clan II leader sequence and the
CH2 exon. A m.w. size marker was applied in the left lane.
removing ambiguous sequence readings, nonfunctional sequences
(containing a reading frame shift), or incomplete sequences, we
assembled two sets of 58 V gene sequences for the H chain of the
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classical and HCAb IgGs, respectively (Supplemental Figs. 2, 3). Se-
quence alignment demonstrated that the V regions of both Ab classes
belong to the same V gene family and both groups of V regions en-
coded classical VH elements (not VHH). Furthermore, a neighbor-
joining analysis was performed with the V-encoding cDNA sequences
and the functional human IgV germlines. Analysis of these llama V
elements (of classical Abs and HCAbs) showed that they all cluster in
one branch that is most closely related to the branch of the human VH
(4) family, which forms part of clan II (Fig. 2). The llama VH(4) and
human Ig VH(4) domains display high sequence similarity, yet some
framework residues (Thr30, Ile48, Thr66, Arg71, Gln81, Pro84, and Glu85)
deviate regularly from the human VH(4) sequences (23). The impact of
these deviations is visualized by the hydrophobicity plot (Fig. 3). The
decreased hydrophobicity profile at the outer framework-3 (asterisks in
Fig. 3) for the llama VH(4)s compared with that of human VH(4) is
suggestive for a reduced aggregation-prone behavior for llama VH(4)
relative to that reported for human VH(4) (30).
VH(4) enlarges both the classical and the HCAb diversity
Analysis of VH(4) sequences from both isotypes (i.e., classical Abs
and HCAbs) showed that sequence variation was the highest in the
CDRs of the V regions (Supplemental Figs. 2, 3). Variability was also
noticed in the FR3 regions, although to a lesser extent compared with
that of the CDRs. No obvious differences in CDR1 sequence are
observed between VH(4)s from classical Abs and HCAbs (Sup-
plemental Figs. 2, 3). With the notable exception of three clones
(conv12, 32, and HCAb28), both VH(4) groups contain, similar to the
human VH(4), 7 aa in their CDR1. Such a CDR1 loop of 7 aa is
predicted to adopt canonical structure type-3 (31). The HCAb28
clone possesses an enlarged CDR1 and forms potentially a novel
loop structure, whereas the other two classical VH(4) clones (clones
12 and 32) probably adopt canonical loop structure type-1 and type-
2, respectively. Likewise, no major differences are detected for
CDR2 between VH(4)s of both groups (Supplemental Figs. 2, 3).
The vast majority of the CDR2 loops display a length of 16 aa,
except clones conv56 and conv57 (Supplemental Fig. 2). Therefore,
with the exception of these clones, the CDR2 regions of llama VH(4)
are predicted to fold into the known canonical loop structures of the
human VH(4) domains (31). In summary, the CDR1 and CDR2 Ag-
binding loops of llama VH(4) apparently adopt the canonical loop
structures observed for human VH(4) domains. In sharp contrast,
these loops are routinely deviating from the canonical confor-
mations in the case of VHH of V family 3 (11, 32). Nonetheless it is
obvious that the mere presence of these VH(4) domains within the
The Journal of Immunology
FIGURE 2. Neighbor-joining phylogenetic tree of human IgV germline
genes and llama VH sequences. The tree is based on the amino acid se-
quences of FR1–FR3 as defined by the Kabat annotation (9). Sequences of
llama are represented by gray branches; human sequences in black rep-
resent human IgV germline genes as specified by the gene family anno-
tations. The three designated clans are represented by Roman numbers.
HCAb type enlarges the known structural repertoire of these unique
camelid Abs (4). In contrast to CDR1 and CDR2 that is imprinted in
the V germline gene, CDR3 arises from an imprecise V-D-JH re-
combination, which creates a much larger variability in loop length
and sequence. Within the VH(4) dataset, CDR3 ranges from 7 to 22
aa (Supplemental Figs. 2, 3), although the average length of CDR3
from classical Abs (12.8 aa) does not deviate significantly from that
of HCAbs (13.5 aa). In contrast, for dromedary VH(3) and VHH(3),
a net difference in CDR3 length is observed (9 and 15 aa, re-
spectively) (33), although this difference is less pronounced in llama
(9 and 13 aa) (8).
From inspection of the CDR3 sequences, it appears that the VH
(4) sequences of classical Abs and HCAbs are recombined to a
common set of D and JH genes, although such analysis is com-
plicated by imprecise recombination and somatic hypermutation. A
minor difference is that VH(4) of the classical Ab group lacks inter-
or intraloop disulfide bridges (Supplemental Figs. 2, 3). In contrast,
for the HCAb VH(4) sequences, clones 36 and 12 possess two
cysteine residues in their CDR3 loop that probably form an
intraloop disulfide bond.
In conjunction, no major difference in sequence or loop structurewas
discerned between the VH(4) from classical Abs and HCAbs (Sup-
plemental Figs. 2, 3). Interestingly, three different VH(4) sequences
5699
FIGURE 3. Weighted Kyte-Doolittle hydrophobicity plot for VH(4)s.
Weighted hydrophobicities (y-axis) were calculated for the distribution of
amino acids observed at each position (x-axis) of the human VH(4)
germline genes (gray line) and the llama rearranged conventional and
HCAb VH(4)s (black line). The asterisks depict the areas in FR3 with the
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most notable differences. In the Kyte-Doolittle scale, the higher positive
values correspond to the more hydrophobic amino acid stretches.
were found twice, once in the classical Ab group and once in the HCAb
group (clones HCAb10/conv46, HCAb8/conv19, and HCAb52/
conv7). When aligned pairwise (Fig. 4A), these VH(4)s contained
a minimal amount of point mutations and a homologous CDR3 se-
quence, indicating that each pair probably originates from an identical
V-D-JH recombination. One sequence of each pair was linked with (or
properly spliced to) an intact CH1 region and will therefore be part of
an Ab with an Ag-binding fragment comprising a VH and VL domain.
The twin sequence partner definitely lacked the CH1 region, and the V
region was immediately followed by the hinge sequence, which dem-
onstrates that this V domain was derived from a HCAb devoid of L
chains. Therefore, this latter VH(4) domain will function as a single-
domain Ag-binding fragment in a HCAb. This indicates that a VH(4)
gene involved in a V-D-JH recombination in a pre-B cell generates
a VH domain that might function either independent from a VL in
a HCAb or paired with a VL domain in a classical Ab.
Generation of VH(4) library and selection of Ag-specific
single-domain Abs
To verify that the isolated VH(4) domains from HCAbs (i.e., in the
absence of a VL partner) are indeed functional in Ag binding, we
generated a VH(4)-HCAb–derived single-domain phage display
library of a llama immunized with immature BMDCs. Two rounds
of panning were performed on purified splenic CD11c+ cells (26).
After the second round of panning, the number of phage particles
eluted from CD11c+ cells increased 100-fold relative to the first
round using the same amount of input phage particles and iden-
tical washing steps. Individual bacterial colonies (6100) from the
second round were randomly picked and sequenced. Two se-
quences (referred to as sdAb_31 and sdAb_32) were represented
repeatedly, although several point mutations throughout the frame-
work or CDR occurred in the homologous clones of the domi-
nating sdAb_31 sequence. The alignment of the deduced amino
acid sequences, against the human VH consensus sequence (Fig.
4B) confirmed their VH(4) signature. The CDR3 lengths of 14 and
15 aa for sdAb_31 and sdAb_32, respectively, contrast with the
shorter average CDR3 length of llama VH(3) (8). In addition,
cysteine residues were absent in the CDR, and therefore these long
loops cannot be tethered by an inter- or intraloop disulfide bond
that might assist in the stabilization of the sdAb (34).
5700
FIGURE 4. Amino acid sequence alignment VH(4) variable domains, numbered according to Kabat (above) and International ImMunoGeneTics in-
formation system amino acid numbering (below), with framework regions and CDR indicated (9, 21). A, Sequence alignment of three variable domains
[clan II, family VH(4)] present on llama classical IgG and H chain IgG. Differences between the sequences are underlined and in bold. B, sdAb_31 and
sdAb_32 are aligned with human IgHV4_30-4p01 (Z14238) and human IgHV4_39p03 (305715), respectively, because these sequences display the highest
nucleotide identity according to the IgBlast analysis. Differences in sequence from the reference human sequence are underlined and in bold.
Biochemical properties of Ag-specific VH(4) sdAb
The two VH(4) sdAbs were recloned in the pHEN6 expression vector.
After induction, the periplasmic proteins were extracted, and the
recombinant protein was purified by IMAC and SEC (Supplemental
Fig. 4). A yield of 5–7 mg protein per liter of culture was obtained for
these sdAbs, which is within the range (5–10 mg/l culture) normally
obtained for sdAbs of the VHH(3) family (15). The sdAb_31 eluted
from a Superdex 75 gel filtration column as a single symmetrical peak
as expected for a soluble, monomeric protein, whereas the elution
profile of the sdAb_32 showed an additional minor fraction of di-
merized material. The thermal stability of the sdAbs was measured by
CD, and Tm values of 62.4 and 63.5˚C were recorded for sdAb_31 and
sdAb_32, respectively (Supplemental Fig. 5). Although these Tm
values are similar to the values measured for VHH(3) sdAbs (13), they
are high for sdAbs belonging to the VH(4) family, because V domains
of the human VH(4) family form consistently less stable domains
(30). Earlier studies proposed for sdAbs a direct correlation between
their net acidic charge and aggregation resistance (35, 36). Therefore,
we calculated the theoretical pI of VH(4)s of both Ab groups. The
distribution of the individual pIs of VH(4)s from classical Abs or
HCAbs as well as the pI ranges are indistinguishable (Supplemental
Fig. 6A). Remarkably, in this current dataset of VH(4)s of unknown
specificity, no pI values between 7.0 and 7.5 were recorded. However,
the DC-enriched sdAb_32 and sdAb_31 display a near neutral and
basic pI, respectively, and are soluble and monomeric entities, in
sharp contrast to predictions from previous work (35, 36).
We next analyzed the integrity and aggregation tendency of our
two DC-specific VH(4) sdAbs over time, relative to the properties of
VHH(3)-derived sdAbs and DC-specific VH(3) sdAbs (28). These
four sdAbs at two different concentrations (35 and 175 mM) were
incubated at 4 and 37˚C for different time intervals up to 14 d. Gel
filtration was used to profile the quaternary structure of the sdAbs,
and the CD signal was taken to ascertain the maintenance of their
structural integrity. The VH(4) sdAbs eluted as a single symmetric
peak at all incubation times, and their chromatographic profiles
were indistinguishable from those of sdAbs of family 3 (Supple-
mental Fig. 7A–F). The prolonged incubation of the sdAbs at 37˚C
did not provoke oligomerization because their profiles were iden-
tical to those of the sdAbs kept at 4˚C. The VH(4)s at different
concentrations also yielded identical profiles, and even the VH(4)
sdAbs at the highest concentration (10 mg/ml) remained mono-
meric (data not shown). In addition and in agreement with the gel
filtration results, the incubation condition (concentration, time, and
NOVEL CAMELID FAMILY 4 SINGLE-DOMAIN Abs
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temperature) had no effect on the CD profile of the sdAb (Sup-
plemental Fig. 7G, 7H). We observe that under these conditions no
prominent changes to the secondary structure are seen and that
camelid VH(3), VHH(3), and VH(4) sdAbs appear robust.
The Ag-binding profiles of sdAb_31 and sdAb_32 on mature and
immature BMDCs were analyzed by flow cytometry (Fig. 5A). The
results of this in vitro binding study demonstrate that sdAb_31 and
sdAb_32 are functional in Ag binding, and as expected from the
results above, even after 2 wk incubation at 37˚C, our sdAbs re-
mained functional (data not shown). Their epitope is present on
CD11c+ cells, including both mature and immature DCs (Fig. 5A).
The observed signals are due to the sdAbs recognizing specifically
their cognate cell surface markers, because control experiments of
BMDCs with sdAb_BCII10, a sdAb with specificity for b-lactamase,
gave background signals that were indistinguishable from those
obtained with cells mixed with FITC-labeled anti-HIS secondary Ab.
In addition, the experiments with other non-DC-specific VH(4)
sdAbs all failed to give a fluorescence intensity shift (Fig. 5A). This
precludes that the signal originates from a nonspecific adsorption of
VH(4) sdAbs on the BM-DCs. Furthermore, the flow cytometry
signal intensity with the sdAbs directed against the immature DCs is
slightly higher than that for mature DCs, and the relative shift of the
fluorescence intensity peak is more pronounced for sdAb_32 than
that for sdAb_31. Finally, experiments with Alexa Fluor 488-labeled
sdAb tested in competition with an excess of unlabeled sdAb con-
firmed the specific recognition of their cognate epitope and indicated
that sdAb_31 and sdAb_32 recognize different (i.e., noncompeting)
epitopes on their target cells (Fig. 5B).
Discussion
The Ab generation process in Camelidae should deviate, at least in
some details, from the general scheme in other vertebrates because
camelids are actively producing unique, functional HCAbs. For the
production of HCAbs, the camelids use dedicated Cg germline genes
with a truncated 59 splicing site at the “CH1 hinge” intron so that
splicing of the RNA transcript leads to a direct connection of the V
exon to the hinge exon (Fig. 6) (3, 5, 37). In addition, the camelid
genome contains two sets of V germline genes (VH and VHH), pro-
bably interspersed in their H locus (5, 33), and downstream clusters of
D genes and JH genes (4, 5, 37). In our previous reports, we mentioned
only the presence of VHHs belonging to the V clan III, family 3 (4).
During Ab generation the rearranged VHH(3)-D-JH is eventu-
ally coexpressed with either the dedicated Cg2 or Cg3 and folds in
The Journal of Immunology 5701

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FIGURE 5. Binding profile of VH(4) sdAb_31 and sdAb_32 on BMDCs. The activation state of the DC populations was evaluated using different cell
surface markers (CD40, CD86, and MHC II). A, DC populations were stained with 1 mg sdAb-BCII10 [VHH(3); negative control], a nonspecific VH(4) sdAb,
or VH(4) sdAb_31 or sdAb_32 visualized by 0.1 mg anti–HIS-FITC Ab gated on CD11c+ BMDCs (solid black line and open profiles). The profiles in dashed
lines and gray filling are from cells treated with 0.1 mg anti–HIS-FITC only. B, Immature DCs preincubated with 40 mg unlabeled sdAb-BCII10, sdAb_31, or
sdAb_32 (gray-filled, dashed-lined contoured profiles) followed by staining with 1 mg sdAb_32-Alexa Fluor 488 (open, solid-line contoured profiles).

a homodimeric Ig. In contrast, VH(3)-D-JH is cotranslated with
the classical Cg1a or Cg1b, and the polypeptide associates with a
L chain to form a classical Ig (Fig. 6). However, it has been re-
ported that occasionally VH(3)D-JH products lacking the CH1
exon can be cloned from cDNA, indicating that they probably are
part of the HCAb pool (14, 16). The majority of such classical V
domains of a HCAb are (most often) generated after an unusual D-
JH gene recombination whereby the codon for the conserved
Trp103 (a residue that is essential for VL pairing in classical Abs)
is substituted mostly by an arginine codon (6, 14, 16). In this
study, we demonstrate that the V repertoire of camelids also
contains functional V genes that belong to clan II. These V genes
cluster with the Ig VH(4) family in the phylogenetic tree and the
IgBlast search (Fig. 2). The occurrence in alpaca of such genes
was briefly mentioned recently (5). Interestingly, in llama, the VH
(4) gene is transcribed in nearly 50% of the expressed classical
IgGs (possessing CH1 and by default also the L chain). This
percentage comes from a 59 RACE cloning of the H chain of
classical Abs and shotgun sequencing that was not biased for any
particular V element. The abundant usage of VH(4) within this
pool suggests that these V genes fulfill a central role in forming
the Ag-binding repertoire of classical Abs in llama. The reason
why their presence was overlooked until now is dual: first, most
work on camelid Abs focuses on the HCAbs and neglects the
classical Abs (14, 15). Secondly, the V genes are normally am-
plified after PCR with primers designed to anneal at the LS se-
quence or at the FR1, and these primers fail to hybridize to the
corresponding regions in the VH(4) family. In this study, we
demonstrate that this VH(4) family contributes significantly to the
classical Ig Ab repertoire and, more importantly, is numerously
present as part of HCAbs as well.
Nonetheless, the frequency of the VH(4) occurrence within the
HCAb pool is lower than that in the classical Ab group. Interestingly,
the very same VH(4)-D-JH rearrangement can occasionally be
found within both classical Abs and HCAbs. In the former case, it is
coexpressed with a CH1 domain, and probably the VH domain will
pair with a VL domain to constitute a functional Ag-binding site. In
the latter case, the CH1 exon is definitely removed during
the splicing reaction within the mRNA of the HCAb-specific Cg2
or Cg3 genes and as such impeding a possible association with
5702 NOVEL CAMELID FAMILY 4 SINGLE-DOMAIN Abs

FIGURE 6. Schematic representation of the H locus with V genes of VH(4) and VH(3) family followed by D and JH gene clusters and constant genes

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(only Cg genes are shown). Note that the current organization of V genes (clustered or interspersed) is currently unknown, and therefore the order of VH(4),
VHH(3), and VH(3) is entirely tentative. The dedicated HCAb Cg2 and Cg3 have a deficient CH1 splicing site denoted by a star. A, (i) Generation of
classical IgG is obtained after rearrangement of a classical VH(3) gene and coexpression with classical Cg(1a,1b) genes, (ii) generation of HCAbs is
possible when a classical VH(3) gene recombines with a D-JH that encodes an Arg103 mutation (although sometimes Arg103 is absent) and coexpresses with
the dedicated HCAb Cg2 and Cg3 genes, and (iii) the majority of HCAbs are formed when a VHH(3) gene that differs from the VH(3) genes in its hallmark
FR2 amino acids is linked after a D-JH rearrangement to the dedicated Cg2, Cg3 constant genes. B, Generation of HCAbs with a VH(4) gene without the
Trp103 substitution can be joined to both sets of Cg genes to produce classical Abs or to produce HCAbs.

a L chain. Because the VH(4)-D-JH translation product seems to be
competent to function on both types of Abs, the role of the L chain
(and the VL in particular) appears to be superfluous for the ex-
pression of the BCR and for proper Ag recognition. Consequently,
the VH(4) domain is forced to act as an autonomous, single-domain
Ag-binding fragment in a HCAb molecule. Hereto, the stability and
solubility of the VH(4) domain and its secretion from the B cell
should be assured, and the Ag recognition of the autonomous do-
main should be guaranteed.
The dual usage of VH(4) raises two questions. First, is VH(4) of
the HCAbs a particular subset of the VH(4)-D-J rearrangement
products or are all of the VH(4) domains capable to appear on either
Ab type? Second, what makes these camelid VH(4) different from
the human VH(4) so that they can function as an isolated sdAb
whereas a human VH(4) is much less adapted to fulfill this role (30)?
With regard to the first question, VH(4) on HCAbs and classical
Abs seems to be indistinguishable. There is apparently no differ-
ence in usage of the VH(4) or JH genes. In both cases, they re-
arrange with the D gene to generate CDR3s of equal lengths. Also,
the amino acid content and hydrophobicity of CDR3 within the VH
(4) domains of both Abs reveal no significant bias (Supplemental
Fig. 6). Additionally, the distribution of the pI of the VH(4)
domains from HCAbs or classical Abs is identical. Remarkably,
few domains are formed with a pI between 7.0 and 7.5, which might
reflect a counterselection against such domains because these
would be more prone to aggregation at a physiological pH. We also
analyzed areas on the VH(4) domain, such as FR2 and Trp103 (first
amino acid of FR4), where VH(3)s and VHH(3)s of HCAbs and
classical Abs are distinct. Yet again, FR2 of VH(4)s from HCAbs
and classical Abs are identical. Although a few sequences from
the HCAb pool show some amino acid substitutions in this con-
served VL interface region, similar substitutions were noted for
the classical Abs as well (Supplemental Figs. 2, 3; HCAb23,
HCAb24, and conv33, respectively). The Trp103 (encoded by the
59 end of the JH gene) is another major component of the VL-
contacting side of a VH domain. Indeed, for the VH(3)-derived
Ag-binding fragments of HCAbs, we observe regularly an argi-
nine at this position, which will abrogate the VL pairing (7, 38).
Remarkably, the Trp103Arg substitution is highly employed in
VH(3)-HCAbs having a short CDR3, whereas a small number of
VH(3)-HCAbs have a long CDR3 loop but lack this Trp103Arg
substitution. This indicates that the CDR3 amino acid content in
conjunction with the long sequence that collapses on the erstwhile
VL interface mimics the Trp103Arg effect. The situation for VH
(4) is different. The VH(4) of HCAbs and classical Abs possesses
a CDR3 loop of equal average length, and in contrast with the VH
(3)-HCAbs the majority of the VH(4)-HCAbs do normally not use
Arg103 substitution to impede the VH–VL association.
Concerning the second question, we searched for possible
determinants that enhance the solubility and good biophysical
properties of the isolated camelid VH(4) domain compared with the
poor behavior of the isolated human VH(4) (30). These determinants
should belong to the CDRs or to the framework, and in the latter case
preferably the VL binding interface (39–42). Although the sub-
stitution of Trp103, Leu45, or both of FR2 by a charged residue (ar-
ginine) may be favorable for the solubility of sdAbs, previous
studies clearly demonstrate that it is not a prerequisite (17, 36, 41,
43). Despite the high sequence similarity between human and llama
VH(4), profound sequence analysis of the camelid VH(4)s high-
lighted a few residues that deviate subtly but consistently from the
human VH(4) framework. Remarkably, these residues are mainly
present in FR3 (Supplemental Fig. 4) outside the VL binding face of
the domain and render this area more hydrophilic than the human
VH(4)s. Because framework residues influence the biochemical
properties of sdAbs (44–46), it is conceivable that the unfavorable
properties described for human single domains of family VH(4) (30)
are attenuated through these substitutions. However, it would be
surprising that these differences are the sole determinant for the
autonomous character of the camelid VH(4)-HCAbs. Additional
key residues are probably located in the CDR loops because these
regions are reported to participate in the soluble behavior of au-
tonomous human VH(3) domains (36, 41, 43, 47). Furthermore, the
The Journal of Immunology
nature of the CDR3 loop has been shown to contribute to the sta-
bilization and solubility of sdAbs (30, 40). However, the inherent
hypervariable character (in length and sequence) of the CDR re-
gions precludes the identification of the critical residues to convert
VH–VL domains into functional monomeric sdAbs.
That the VH(4) sdAbs are indeed functional in Ag binding was
shown by immunizing a llama with DCs, cloning the VH(4) rep-
ertoire from the HCAb-specific pool, and retrieving DC-specific
sdAbs after phage display. Of note, in the absence of any information
on a possible Ag preference of the VH(4) of HCAbs, we preferred to
immunize with cells as opposed to individual proteins, because
theoretically these harbor an infinite number and complex type of
epitopes, which maximizes our chances to retrieve binders after-
ward. In addition, the retrieved VH(4) sdAbs produce readily in
bacterial expression systems, display good solubility, thermal sta-
bility, and shelf life, and are functional in the recognition of CD11c+
cells as measured by flow cytometry. Furthermore, the VH(4)s of
camelids are closely related and share a high degree of identity with
human VH(4)s. It is therefore conceivable that for human therapy
one would prefer to select specifically VH(4)-derived sdAbs instead
of VHH(3)-based sdAbs because the latter might require more
drastic humanization efforts to minimize immunogenicity (12).
We also foresee two extra possible developments whereby our
insights on camelid autonomous VH(4)s could become practical in
the future. In one example, we propose to remediate an “unstable”
human VH(4) that was identified (either as an autonomous sdAb or
paired with L domains in a scFv or Fab format) by substituting its
FR3 beginning and ending sequences with the more hydrophilic
amino acids as those of the llama VH(4). For the second possibility,
a strategy could be developed whereby a synthetic library would be
constructed by randomizing the CDR3 sequence on a llama VH(4)
sequence that occurred in a HCAb and a classical Ab. After re-
trieving Ag binders from this sdAb library, one could supply this
particular sdAb with a library of (human) VLs and search for VH–
VL Ag-binding molecules of enhanced affinity or specificity,
whereby the VH–VL pair would function as a “clamp” like the
tethered fibronectin 3 and PDZ domains (48).
In summary, we demonstrated the presence within camelids of
a novel family of V elements, closely related to human VH(4). The
members of this family contribute abundantly to the classical Ab
repertoire but surprisingly also participate, albeit at a lower frequency,
to the VH(4)-HCAb repertoire. No apparent sequence difference is
noted between the VH(4)-derived domains from HCAb or classical
Ab repertoires. Remarkably, it thus appears that the same VH(4)-D-JH
recombination product from the pre-B cell can end up in both types of
Abs. Therefore, it looks as if the L chain is superfluous in llama Abs
harboring VH(4) elements. Hence, the VH(4)s enlarge the currently
known Ag-binding repertoire of camelid HCAbs (4) and can be ex-
ploited in a sdAb format because they produce soluble, stable re-
combinant Ag-specific recognition units with a high degree of
sequence identity to human VHs.
Acknowledgments
We thank C. Heirman and K. Thielemans of the Laboratory for Molecular
and Cellular Therapy for providing the GM-CSF and R. Van den Bergh of
the Laboratory for Molecular and Cellular Therapy for invaluable com-
ments on this paper.
Disclosures
The authors have no financial conflicts of interest.
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