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Nanobodies as novel agents for cancer therapy

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 Review
Vaccines & Antibodies

Nanobodies as novel agents for

1. Introduction
2. What is a nanobody?
3. Generation and production of
recombinant nanobodies
4. Unique features of nanobodies
cancer therapy
Hilde Revets†, Patrick De Baetselier & Serge Muyldermans
†VrijeUniversiteit Brussel, Department of Molecular and Cellular Interactions, Laboratory of Cellular
and Molecular Immunology, Vlaams Interuniversitair Instituut voor Biotechnologie, Pleinlaan 2,
Building E8, B-1050 Brussels, Belgium
Nanobodies are the smallest fragments of naturally occurring heavy-chain
antibodies that have evolved to be fully functional in the absence of a light

f
5. Achievements and medical chain. As such, the cloning and selection of antigen-specific nanobodies
opportunities for nanobodies obviate the need for construction and screening of large libraries, and for
lengthy and unpredictable in vitro affinity maturation steps. The unique and

o
6. Nanobodies in cancer diagnosis
and therapy well-characterised properties enable nanobodies to excel conventional ther-
apeutic antibodies in terms of recognising uncommon or hidden epitopes,

o
7. Expert opinion and conclusions
binding into cavities or active sites of protein targets, tailoring of half-life,

r
drug format flexibility, low immunogenic potential and ease of manufac-
ture. Moreover, the favourable biophysical and pharmacological properties
of nanobodies, together with the ease of formatting them into multifunc-

r P
tional protein therapeutics, leaves them ideally placed as a new generation
of antibody-based therapeutics. This review describes the state of the art on
nanobodies and illustrates their potential as cancer therapeutic agents.

Keywords: camels, cancer therapy, heavy-chain antibodies, llama, nanobody,

o
single-domain antibody, VHH

h
Expert Opin. Biol. Ther. (2005) 5(1):xxx–xxx

t
1. Introduction

u
The mouse hybridoma technology described by Köhler and Milstein in 1975 [1] was an
important step in the development of antibody technology and paved the way for the
emergence of therapeutic monoclonal antibodies (mAbs). Today, several antibodies have

A
been approved as therapeutic agents in a wide range of indications, including transplant
rejection [2-4], inflammation [5-7], infectious diseases [8-10] and oncology [11-12] (Table 1).
With many more candidates in late-phase clinical trials, including oncology trials,
antibodies have become well-established therapeutics [13-14].
Unmodified mouse mAbs were the first FDA-approved immunotherapeutic
agents (Table 1), but their in vivo applications in humans were limited because their
xenogeneic nature provoked human antimouse antibody (HAMA) responses when
repetitively injected into patients [15-16]. To mitigate potential immunogenicity, ther-
apeutic mAb technology transitioned from creating completely murine mAbs to
exchanging the murine constant domains for human constant domains to create
‘chimeric’ antibodies [17]. This was followed by ‘implanting’ the complementarity-
determining regions (CDRs) of the mouse antibody into the variable regions of a
human IgG, thus creating humanised antibodies [18]. Modern strategies now allow
the selection of fully human antibodies directly from natural or synthetic repertoires,
including transgenic mice producing purely human antibodies [19-20].
A remaining impediment to the development of new antibody therapeutics is the
cost of producing antibodies in mammalian expression systems. Moreover, a rising
Ashley Publications awareness of antibody effector functions stimulates the efforts towards the modula-
www.ashley-pub.com
tion of Fc-associated functions, such as Fc-receptor activity and complement

10.1517/14712598.5.1.xxx © 2005 Ashley Publications Ltd ISSN 1471-2598 1

Nanobodies as novel agents for cancer therapy

Table 1. Approved monoclonal antibodies.

Product* Specificity Type of molecule Indication Year

OKT®3 (muromonab-CD3; Ortho Biotech) CD3 Murine Transplant rejection 1986
ReoPro® (abciximab; Centocor Inc.) Platelet gpIIb/IIIa Chimeric Cardiovascular disease 1994
Panorex® (edrecolomab; Centocor) EpCAM Murine Colorectal cancer 1995
Rituxan® (rituximab; IDEC) CD20 Chimeric Non-Hodgkin’s lymphoma 1997
Zenapax® (daclizumab; Roche) IL-2 receptor Humanised Transplant rejection 1997
Herceptin® (trastuzumab; Genentech, Inc.) Her-2 Humanised Metastatic breast cancer 1998
Remicade® (infliximab; Centocor) TNF-α Chimeric Crohn’s disease 1998
Simulect® (basiliximab; Novartis AG) IL-2 receptor Chimeric Kidney transplant rejection 1998
Synagis® (palivizumab; MedImmune, Inc.)
Mylotarg® (gemtuzumab
Ozogamicin; Wyeth Research)
CroFab™ (Protherics)
RSV (anti-F)
CD33

Snake venom
Humanised
Humanised

Ovine Fab
RSV

f
Acute myeloid leukaemia

o
Rattlesnake antidote
1998
2000

2000

o
DigiFab® (Protherics) Digoxin Ovine Fab Digoxin overdose 2001

r
Campath® (alemtuzumab; Berlex, Inc.) CD52 Humanised B cell chronic lymphocytic 2001
leukaemia
Zevalin® (ibritumomab tiuxetan; IDEC) CD20 (90yttrium Murine B cell non-Hodgkin’s 2002

P
conjugate) lymphoma
Xolair® (omalizumab; Genentech) IgE-Fc Humanised Allergy 2002

r
Bexxar® (tositumomab; Corixa) CD20 Murine Non-Hodgkin’s lymphoma 2003
(radiolabelled)

o
Humira® (adalimumab; Abbott Laboratories) TNF-α Human Reactive arthritis 2003
Raptiva® (efalizumab; Genentech) CD11a Humanised Psoriasis 2003

h
Avastin® (bevacizumab; Genentech) VEGF Humanised Colorectal cancer 2004

t
Erbitux® (cetuximab; ImClone Systems Inc.) EGFR Chimeric Solid tumours 2004
*Product names are registered trademarks.
EGFR: Epidermal growth factor receptor; EpCAM: Epithelial cell adhesion molecule; RSV: Respiratory syncytial virus; TNF: Tumour necrosis factor;

u
VEGF: Vascular endothelial growth factor.

A
activation [21-23]. Consequently, there has been a growing
interest in new small therapeutic antibody formats that can
be tailored ‘at will’ and manufactured by high-volume liquid
bacterial or yeast cell culture. Gene technology has succeeded
in the cloning and engineering of smaller fragments of anti-
body genes [24-25]. The scaffold chosen for recombinant anti-
bodies has been a simple choice between Fab or single-chain
variable fragment (scFv) molecules, with most groups favour-
ing scFv due to the higher expression levels compared with Fab
in prokaryotic and lower eukaryotic microorganisms [26-27]. In
comparison with whole antibodies, small antibody frag-
ments, such as Fab or scFv, exhibit better pharmacokinetics
for tissue penetration, but are monovalent and suffer from
fast off-rates and poor retention time on the target [28-29]. To
overcome these obstacles, various methods have been devised
to tailor the functionality and physicochemical properties of
these fragments, leading to improved stability, functional
affinity, tumour penetration and biodistribution when used
in vivo [30-34]. Despite their beneficial properties, scFvs and
conjugates thereof are still amenable for improvement in
terms of stability [35], expression yield, protease resistance and
aggregation caused by synthetic linkers [36].
Minimising the size of antigen-binding proteins to a single
immunoglobulin domain with high affinity to a target anti-
gen has been one of the major goals of antibody engineering
over the past decade. Antigen-binding fragments comprising
the single variable domain of the conventional heavy chains
(variable heavy [VH]) were generated in the late 1980s [37]
and are referred to as single domain antibodies or dAbs. These
dAbs, however, exhibit some serious shortcomings. The anti-
gen-binding site is matured during the immunisation as a
combination of VH and variable light (VL), so that isolated
VHs will have a lower affinity for their antigen, as compared
with the original, matured antibody [38]. Moreover, the hydro-
phobic interface becomes exposed to the aqueous environ-
ment following removal of the VL domain and this renders
dAbs ‘sticky’. The poor solubility and reduced affinity for the
antigen indicated that additional protein engineering would
be required to successfully generate functional single-domain
antibody fragments. A recent paper by Jespers et al. [39]

2 Expert Opin. Biol. Ther. (2005) 5(1)

 Revets, De Baetselier & Muyldermans

1 VH
VL
CL CH
Fab

Light chain Fv
CH2 Antigen-binding
Fc fragments
scFv
CH3
VH CDR1 CDR2 CDR3

W47
VH

G44
V37

L45
VHH CDR1 CDR2 CDR3
CH2

f
G47
R45
E44
F37
Fc

VHH

o
CH3
nanobody

o
Figure 1. Schematic representation of the conventional (top) and heavy-chain IgG antibodies (bottom) present in camelid
serum. The conventional IgG contains two variable regions (each composed of a VH and VL domain) that confer antigen-binding

r
specificity of the antibody, and an Fc fragment in the constant region that recruits effector functions of the immune system. The entire
light chain and CH1 domain are absent in the HCAb. The antigen-binding domains of conventional antibodies after proteolysis (Fab) or
after cloning and expression of the VH and VL fragments are shown. scFvs are generated by introducing a synthetic linker between the

P
VH and VL domain. The recombinant VHH or nanobody of a heavy-chain of HCAb is obtained after cloning and expression. The
differences between VH and nanobody are also shown. The CDR1 and CDR3 of nanobodies is longer than in VH genes, and they are
often connected by an intersulfide bond (thick line). The hallmark amino acid substitutions in framework-2 and their position according
to the Kabat numbering [57] are given.

r
CDR: Complementarity-determining regions; HCAb: Heavy-chain antibody; scFv: Single-chain variable fragment; VH: Variable heavy; VHH: Camelid variable heavy;

o
VL: Variable light.

described how the aggregation-prone dAbs could be removed The HCAbs of Camelidae have a unique structure consist-

t h
from a repertoire displayed at the tip of filamentous phages by
introducing a heating step. However, by serendipity, it was
discovered that a similar selection for soluble dAbs was already
ing of a single variable domain (VHH), a hinge region and
two constant domains (CH2 and CH3) (Figure 1). These
HCAbs lack the first domain of the constant region (CH1),

u
performed much earlier in nature. Part of the humoral which is present in the genome, but is spliced out during
response of camels, dromedaries and llamas is based on a mRNA processing [42-43]. The absence of the CH1 domain
unique repertoire of fully functional antibodies composed explains the absence of the light chain in the HCAbs, as this

A
solely of heavy chains (Figure 1) [40]. This finding, together
with the concurrent improvement of technologies to select
antigen-binding fragments from large libraries and readily
express them in bacteria [25,41], led to the development of a
new antibody technology platform based on the single varia-
ble domain of these camelid heavy chain antibodies (VHH).
The crystal structure of an isolated VHH indicated that it is
a problate particle of 2.5 nm in diameter and ∼ 4 nm high,
and has been referred to as a nanobody. This review focuses
on the unique biophysical and pharmacological properties
of nanobodies and will illustrate their potential as cancer
therapeutic agents.
2. What is a nanobody?
A nanobody is the smallest available intact antigen-binding
fragment (15 kDa) harbouring the full antigen-binding
capacity of the original heavy chain of naturally occurring
heavy-chain antibodies (HCAbs) that have evolved to be fully
functional in the absence of a light chain.
domain is the anchoring place for the constant domain of the
light chain [44]. Consequently, HCAbs naturally evolved to
confer antigen-binding specificity and high affinity by three
CDRs only, instead of the six naturally occurring CDRs from
conventional antibodies or fragments thereof.
The crystal [45-47] and solution [48-49] structures of several
nanobodies have been solved and show that their scaffolds
consist of two α-sheeted structures similar to a VH immu-
noglobulin fold in a conventional antibody. By contrast, the
structures of the antigen-binding loops of nanobodies deviate
importantly from the sets found in mouse and human anti-
bodies [50]. This finding provides evidence that the antigen-
binding loops of nanobodies exhibit a much larger structural
repertoire than observed for conventional VH [51-53]. In addi-
tion, the CDR3 regions of nanobodies are on average longer
than those of VH. The average CDR3 length in human and
mouse VH is 12 and 9 amino acids, respectively [54], whereas
in dromedary-derived nanobodies, a length of 16 – 18 amino
acids is frequently observed, although in the llama, a consider-
able fraction of the nanobodies seem to have a much shorter

Expert Opin. Biol. Ther. (2005) 5(1) 3

Nanobodies as novel agents for cancer therapy
F37,E44 V37,G44
R45,G47 L45,W47
Nanobody Human VH
Figure 2. The immunoglobulin fold of a nanobody (VHH)
compared with a human VH. The CDR3 is shown in black and
the location of camel mutations on the antibody interface surface
are indicated. See text for additional explanation.
CDR: Complementarity-determining regions; VH: Variable heavy;
VHH: Camelid variable heavy.
CDR of ∼ 6 amino acids [51,53,55]. Nanobody sequences also
contain amino acid substitutions in the framework regions
that are not observed in VH domains that pair with VL
o
domains [56]. Hence, these nanobody hallmark substitutions
might have evolved to compensate for the absence of the VL
domain in the antigen binding site, and they might accom-
modate more flexible loops than a VH.
t
The hydrophobic to hydrophilic amino acid substitutions
(V37F or V37Y, G44E, L45R and W47G) within the frame-
h amplified by a single set of primers. A secondary polymerase
u
work-2 region (the residues in this region of the VH normally
interact with the VL domain and are well conserved throughout
evolution [57]) are solvent accessible in the nanobodies (Figure 2).
A
Consequently, they have been allocated to improve the solubility
of nanobodies [56,58]. This led to the idea of ‘camelisation’ of
human VH domains, a technique which involved the replace-
ment of the residues at positions 44, 45 and 47 in human VH
domains with those found in nanobodies [59]. This process of
camelisation resulted in stable entities where the tendency to
aggregate was eliminated [60-63]. Unfortunately, the expression
level was sometimes reduced and the original antigen-binding
activity was lost [61,64]. Further work indicated that besides the
nanobody ‘tetrad’, other mutations together with CDR length
and diversity, contribute to the stability and solubility of the
engineered antibody domains [65-68].
Apart from the Camelidae, wobbegong and nurse shark also
contain HCAbs [69]. Their variable domain can be cloned and
expressed in bacterial systems, and their structure has been
solved recently [70,71]. Their overall size is similar to the nano-
bodies from camelids; however, the primary structure, as well
as the α-strand organisation, is much more divergent to that of
human or mouse VH or nanobodies. In addition, other scaf-
folds of similar size as camelid nanobodies have been employed
4 Expert Opin. Biol. Ther. (2005) 5(1)
to generate single domain antigen binders, such as the FN3
domain or the cytotoxic T lymphocyte antigen-4 [72-74]. All of
these single domain variants are stable and monomeric entities
that are well expressed in bacterial expression systems. The
overall dimensions of these soluble particles are similar to those
of a camelid nanobody and, therefore, in this respect, they can
be considered as nanobodies.
3. Generation and production of recombinant
nanobodies
Cloning the repertoire of antigen-binding fragments from
an immunised animal into a phage display vector and selec-
f
tion of antigen-specific clones by panning has become in the
past decade a routine method for selecting antigen-specific
o
molecules [25,75]. This method is straightforward to adapt to
nanobodies, whereby the single-domain nature of nanobod-
o
ies simplifies the method considerably [76]. Indeed, tapping
the antigen-binding repertoire of the HCAbs of an immu-
r
nised dromedary or llama is less complicated with respect to
the repertoire cloning of conventional antibodies, for exam-
P
ple, in the form of scFv, as the intact antigen-binding site is
encoded by a single gene fragment (the nanobody). Gener-
ally, cDNA is prepared from peripheral blood lymphocytes,
r
isolated from an immunised dromedary or llama (Figure 3).
As all nanobodies belong to one single gene family, they are
encoded by a single exon with homologous border
sequences. Consequently, the complete in vivo matured
nanobody repertoire of a single immunised animal can be
chain reaction with nested primers is then performed to pro-
duce more material and to include restriction enzyme sites
for cloning purposes. Following cloning of the amplified
nanobody gene fragments in the appropriate expression vec-
tor, a nanobody library containing the repertoire of the
intact in vivo matured antigen-binding sites is obtained [76].
Because of the in vivo maturation of the HCAbs, relatively
small libraries of only 106 – 107 individual nanobody genes
have routinely resulted in the isolation of nanobodies with
nanomolar affinity for their antigen [76-78]. Nanobody librar-
ies can be screened for the presence of antigen-specific bind-
ers either by direct colony screening or by panning. Retrieval
of binders by panning is the preferred method, as panning
allows selection for binders with the highest affinities and
those that express better in bacteria. Soluble nanobodies can
then efficiently be produced in bacteria or lower
eukaryotes [79]. Despite the success of retrieving high-affinity
lead compounds in a short period of time, the method
sometimes suffers from the requirement of sufficient
amounts of target antigen for immunisation, although good
immunisations have been obtained with as little as 100 µg of
antigen (for entire immunisation). The availability of syn-
thetic nanobody libraries should offer a solution to identify
antigen binders in cases where difficulties to immunise are
encountered (lack of antigen, low immunogenic or toxic

Collect Isolate lymphocytes
blood
Extract mRNA
RT-PCR
Immunise camel/llama
with immunogens
Generate library
of ∼ 107
transformants
for additional explanation.
Figure 3. Schematic overview of the cloning and selection of nanobodies from an immunised dromedary (or llama). See text
antigen). Synthetic phage or phagemid libraries, containing
a randomised CDR3 on a ‘camelised’ human VH or on a
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mouse VH, have been constructed [59,80]. From such a reper-
toire, camelised VH domains specific for hapten, peptide
and protein antigens have been selected [61,62]. However, as
r
the binders obtained from this repertoire are not matured
against the antigen, the affinity of these camelised human
o
VH domains were only in the submicromolar range. Like-
wise, the availability of a naive, llama nanobody library
seems to be a good source to retrieve antigen-specific binders
t h
against all possible targets [81]. However, here as well, the
affinities seem to be inferior to those of nanobodies retrieved
from ‘immune’ libraries. Several methods have been devel-
u
oped for the in vitro affinity maturation of recombinant
antibody fragments from synthetic or naive libraries to
improve the affinity of these weakly binding antibody frag-
A
ments [82-84]. This approach involves, again, the randomisa-
tion of some residues of the selected camelised VH domain
and the construction of a second library that should be as
large as possible to have a fair chance of success. Alterna-
tively, the ribosome display method has been employed for
the same purpose of affinity improvement [81]. Considering
the time that is spent to increase the first generation binders
from synthetic and naive libraries to bring their affinity to
the required level, it might in the end be much faster to
retrieve high-affinity nanobodies from an immunised drom-
edary or llama. This strategy obviates the need for construc-
tion and screening of large libraries, and for lengthy and
unpredictable affinity maturation steps. Starting from
immune animals, one can also apply an alternative, even more
simple method, avoiding library construction altogether. This
involves the direct cloning of VHH genes from purified, anti-
gen-specific B cells. Combined with the ease of manufacture,
high-affinity nanobodies can be generated within a few
weeks from accessing the target, although some purified
antigen is required for the immunisation (and selections).
Expert Opin. Biol. Ther. (2005) 5(1)
Revets, De Baetselier & Muyldermans
Select specific
VHHs by panning
Produce soluble
antigen-specific VHH
f
o o
4. Unique
r
features of nanobodies
The single domain nature of nanobodies endows them with
several unique features as compared with antigen-binding
derivatives of conventional antibodies. Besides the easy cloning
and selection of high-affinity binders from an in vivo matured
library, nanobodies have other technological and biophysical
advantages that enable them to outperform conventional
antibodies in several areas and are discussed below.
4.1 Ease of manufacture and formulation
The expression of the recombinant nanobodies produces sin-
gle-domain proteins of 15 kDa. In contrast to VH domains of
conventional antibodies, nanobodies are efficiently expressed
as soluble, non-aggregating recombinant proteins due to their
framework substitutions in framework-2: V37Y or V37F;
G44E; L45R or L45C and W47, most often substituted by
G. Without optimisation, recombinant nanobodies are rou-
tinely obtained at levels up to 10 mg/l when expressed in
E. coli grown in shake-flask cultures [76]. The production
process is scalable and expression systems other than bacteria
can be used. The high-level secretion of nanobodies from a
range of fungal and yeast species has been shown [85], with
secretion by Saccharomyces cerevisiae at levels > 100 mg l-1
from shake-flask cultures and > 1 g l-1 from a 10 l fed-batch
fermentation [86]. Scaled-up yields of > 1 kg of nanobody were
obtained from a 15-m3 fermentation [87]. Performing the final
round of nanobody selection in the production organism, for
example, by yeast surface display, further improves maximum
yields [88]. Due to their superior stability compared to conven-
tional antibodies, nanobodies can be formulated as a long
shelf-life, ready-to-use solution.
4.2 Nanobodies are highly soluble and stable
Nanobodies are naturally soluble in aqueous solution, proba-
bly due to the substitution of hydrophobic by hydrophilic
5
Nanobodies as novel agents for cancer therapy
residues in the framework-2 region. VH from conventional
antibodies interact through hydrophobic areas with the CH1
and VL domains. As a consequence, the separate expression
of the VH domain only leads to the formation of inclusion
bodies or to folded domains exposing hydrophobic patches
which render them sticky. Replacement of the hydrophobic
framework-2 residues in these VH domains by the nano-
body-specific sequences results in increased solubility and
lack of aggregation [61].
A striking property of several nanobodies is their high ther-
mal and conformational stability. It has been shown that
nanobodies retain > 80% of their binding activity after
1 week of incubation at 37°C [76], indicating that nanobodies
have a very good shelf-life. In addition, nanobodies regain
antigen-binding specificity after prolonged incubation at
temperatures up to 90°C, a property that has been attributed
to a reversible unfolding behaviour [89-91]. Melting points of
nanobodies are in the range of 60 – 78°C [66,91], whereas anti-
bodies and their fragments derived from other species often
aggregate irreversibly following thermal denaturation [89].
Several approaches, such as molecular evolution and site-
directed mutagenesis, have been developed to optimise the
thermostability of antibody fragments [92-93].
Besides their thermal resistance, nanobodies were shown to
be stable against the denaturing effect of chaotropic
agents [91], in the presence of proteases and to extremes of pH
o
(SM, unpublished results). Therefore, nanobodies are
expected to be able to survive harsh conditions, such as those
found in the stomach, and remain biologically active in the
to treat gastrointestinal diseases.
t h
gut, creating opportunities for the oral delivery of nanobodies
u
4.3 Nanobodies recognise unique conformational
epitopes
The nanobody repertoire contains only three of the six natu-
A
rally occurring CDRs from an antibody. Although surprising
at first glance that three CDRs are sufficient to confer anti-
gen-binding specificity and high affinity, evolution has by
itself arrived at that very solution in camelids and some shark
species. Therefore, irrespective of the precise driving force
behind the emergence of HCAbs in camelids, their variable
domains bind a wide range of antigens, ranging from small
molecules, such as haptens [94-95] and peptides [96], to large
antigens, such as proteins [45-46] or viruses [97].
The small size of the nanobody allows the epitope recogni-
tion even when the target proteins are densely packed so that
the epitopes become cryptic for conventional antibodies [98].
Thus, it can be argued that the nanobodies have access to a
wider range of epitopes than conventional antibodies. This
was also concluded from a study where the immune responses
of heavy-chain and conventional antibodies were compared
for enzymatic interactions [77].
Clefts and cavities play a critical role in multiple biologi-
cal activities, as these often form the interaction site
between two molecules, for example, enzyme–substrate and
6 Expert Opin. Biol. Ther. (2005) 5(1)
receptor–ligand [99]. The antigen-binding surface of conven-
tional antibodies forms a concave or flat surface, depending
on the antigen [100]. Large protruding flexible loops are not
encountered frequently at the antigen-binding surface of
conventional antibodies because these would be immobi-
lised following antigen interaction and would have a nega-
tive influence (entropic effect) on the binding energy [101].
In nanobodies, the long CDR3 loop usually accounts for
most of the binding interaction with the antigen. The crys-
tallographic structure of a recombinant dromedary nanobody
in complex with lysozyme illustrated how the N-terminal part
of the long CDR3 loop forms a loop that extends from the
remaining antigen-binding site and inserts into the active site
o f
of the enzyme [45]. Biochemical analysis further demonstrated
that the antibody fragment inhibits the enzymatic activity of
lysozyme in a competitive manner [102]. From the cloned
nanobody repertoire of a dromedary, immunised with small
o
quantities of enzymes, several inhibitory binders were
retrieved against the different enzyme targets. Interestingly,
r
the inhibitory nanobodies for each given enzyme target used
totally different CDR sequences, indicating that the drome-
P
dary finds different solutions to interact with the same
epitope [77]. The careful reader might question how the
camelid nanobodies with a long CDR3 loop avoid the nega-
r
tive influence on the binding energy following fixation of the
loop during antigen pairing. The crystal structures of nano-
bodies reveal the solution to this apparent paradox. Appar-
ently, a large part of the long CDR3 loop folds over the
framework-2 region, where it shields the residual hydrophobic
patches of this area from contact with the aqueous environ-
ment [45]. In addition, the long CDR3 loops seem to contain
a cysteine that forms a disulfide bond with a cysteine in the
CDR1 (or at position 45) [53,103].
5. Achievements and medical opportunities for
nanobodies
Nanobodies are distinguished from other conventional
antibody formats by their unique properties of size, solubil-
ity, intrinsic stability, easy tailoring into pluripotent con-
structs, recognition of uncommon or hidden epitopes,
binding into cavities or active sites of enzyme targets, ease
and speed of drug discovery, and ease of manufacture.
These features should lead to a number of biotechnological
and medical applications in which nanobodies should excel
other antibody formats.
5.1 Nanobodies as modular building blocks for
manifold constructs
Bispecific and bifunctional antibodies have many practical
applications in immunodiagnosis and therapy. The structural
requirement for multispecificity is to fuse two or more bind-
ing domains together, with sufficient flexibility to allow simul-
taneous binding to different target epitopes. The simplest
bispecific antibody is one that binds two adjacent epitopes on

a single target antigen, thereby gaining an avidity advantage to
the antigen [104]. However, most bispecific antibodies are
designed as crosslinking reagents that bind to different target
antigens. This has been a major focus in cancer therapy, par-
ticularly for the recruitment of cytotoxic T cells to tumour
cells [13,105]. Besides bispecific antibodies, bifunctional anti-
bodies are particularly attractive, as they can deliver a ‘toxic
payload’, such as radionuclides, cytotoxic drugs, peptides or
enzymes, to the target antigen, including cancer cells [13,106].
The ideal characteristics of such a multispecific molecule
are: small size, absence of linkers prone to aggregation or pro-
teolytic cleavage, high solubility, high stability and good
expression yields. The construction of bispecific and bifunc-
tional scFvs as well as innovative alternatives, including dia-
bodies [107], seemed a promising approach, but large-scale
clinical development can be hampered by low expression lev-
els, sensitivity to aggregation and proteolytic degradation at
the level of the linkers, or unproductive association between
the VH and VL chains in the case of diabodies (due to
promiscuous bindings between various VHs and VLs).
The strict single-domain nature of nanobodies promotes
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them as ideal molecular recognition units into more complex
entities, such as bispecific, multivalent and bifunctional con-
structs. Moreover, nanobodies are highly soluble and remarka-
r
bly robust, allowing a high versatility as building blocks for
manifold constructs. Nanobodies have been tandemly cloned,
o
spaced by the natural hinge of camel HCAbs [108]. The result-
ing bifunctional or bivalent proteins were highly expressed in
bacteria in shake-flasks and retained fully the original func-
t h
tionality. Immunotoxins and other immunoconjugates have
also been generated by fusing the gene of a nanobody to that
of a toxic protein or of an enzyme [77,98,108]. Recently, pentam-
u
erisation of single-domain antibodies was possible by fusion of
a single-domain monomer to the B-subunit of Escherichia coli
verotoxin, or shiga-like toxin, which self-assembles into a
A
homopentamer. A strong avidity effect was noticed for the
pentavalent nanobodies, and from structural considerations, it
should be feasible to generate a bispecific pentavalent con-
struct by cloning one nanobody at the N terminal end and
another at the C terminal end of the verotoxin scaffold [109].
The genetic fusion between the genes for the S-layer pro-
tein and a nanobody offers the possibility to produce fusion
proteins that crystallise spontaneously into a monomeric pro-
tein lattice that can be used as a nanopatterned sensing layer
to detect low concentrations of antigen [110-111].
5.2 Nanobodies as a format to stabilise intrinsically
flexible or unstable proteins
Amyloid diseases are characterised by an aberrant assembly
of specific proteins or protein fragments into fibrils and
plaques that are deposited in various organs and tissues,
often with serious pathological consequences. A nanobody
with specificity for human lysozyme was shown to inhibit
dramatically the in vitro aggregation of D67H variant, a nat-
urally occurring mutational variant that is responsible for
Expert Opin. Biol. Ther. (2005) 5(1)
Revets, De Baetselier & Muyldermans
systemic non-neuropathic amyloid disease. These results
provide compelling evidence that reducing the ability of an
amyloidogenic protein to form partially unfolded species
can be a highly effective method of preventing its aggrega-
tion, and suggest new and potentially general approaches to
the rational design of therapeutic agents directed against
protein deposition diseases [112].
In a similar application, a nanobody against an intrinsi-
cally flexible protein, the addiction antidote MazE, was
shown to stabilise the antigen and to keep it in a structured
form for crystallisation [113].
Finally, a llama nanobody binding to the poly-A binding
protein nuclear 1, containing an expanded alanine region
5.3 Nanobodies
o f
causing a protein aggregation disorder, was retrieved from an
‘immune’ library and used for diagnostic screenings [114].
as intrabodies
o
Intracellular expression of recombinant antibodies (intrabod-
ies) is a powerful tool to inhibit in vivo functions of selected
r
molecules. The expression level, correct folding and appropri-
ate subcellular targeting of the antibody are important factors
for successful immunomodulation. Indeed, most antibodies
do not function intracellularly because critical disulfide bonds
cannot form in the reducing environment of the cytoplasm or
because of difficulties in targeting to subcellular compart-
ments. Moreover, few antibodies bind to the active site of
enzymes and, thus, they generally do not neutralise the cata-
lytic activity of enzymes. Jobling and co-workers [105] demon-
strated that nanobodies can be correctly targeted to
subcellular organelles and inhibit enzyme function in plants
more efficiently than antisense approaches. Because nanobod-
ies are in general more stable than scFvs, they better preserve
their antigen-binding capacity even in the reducing environ-
ment of the cytoplasm. For this approach, it might be prefera-
ble to use llama nanobodies, as these possess much less
frequent interloop disulfide bonds compared with nanobodies
from dromedaries [116]. In a recent publication, it has been
indicated that the intracellularly expressed llama nanobody
against the p15 matrix protein of the porcine endogenous ret-
rovirus Gag polyprotein prevents the production of porcine
retroviruses [117]. This approach may be further developed to
combat viral infections of human organs after gene therapy
with such intrabodies. Alternatively, intrabodies may be used
to block intracellular signalling pathways in cells [118]. Inter-
estingly, efforts are being made at present to increase the sta-
bility of scFv antibodies and to try to endow them with
properties of nanobodies [119,120].
6. Nanobodies in cancer diagnosis and therapy
Based on their unique biophysical and pharmacological prop-
erties, nanobodies should be ideally placed to become a new
class of cancer therapeutics. In addition to therapy, nanobod-
ies are also expected to have a future as a tool for the diagnosis
of cancer.
7
Nanobodies as novel agents for cancer therapy
6.1 Nanobodies as a cancer diagnostic tool
Fast and reliable in vivo diagnosis at an early stage of the disease,
or monitoring treatment efficiency, remains a major challenge
in cancer diagnosis. The ideal cancer-imaging agent should
deliver an amount of label sufficient to detect the smallest
metastases against a low-level of nonspecific noise background.
In addition, it should localise promptly by penetrating the
tumour, and unbound conjugate should clear rapidly from the
system to reduce target-to-background ratios. Prolonged clear-
ance kinetics have hampered the development of intact anti-
bodies as imaging agents, despite their ability to effectively
deliver radionuclides to tumour targets in vivo [121-123]. Small
antibody fragments have potential for good tumour targeting,
as they penetrate rapidly and give high tumour-to-background
ratios at very early time points after administration [30].
The superior penetration potential of nanobodies, due to
their high-affinity target binding and fast clearance from the
circulation of the excess of non-targeted nanobodies, repre-
sents an ideal basis for imaging purposes. A nanobody against
lysozyme, which was previously shown to inhibit lysozyme
activity in vitro [76,108], effectively targeted bulky tumours and
metastatic lesions transgenic for hen egg white lysozyme,
whereas excess of this nanobody was rapidly cleared from the
circulation. A few hours after administration, tumour-to-
blood ratios of up to 10 could be demonstrated with radiola-
belled nanobodies [124]. Nanobody uptake by tumours
o
matches well with the half-life of radionuclides, including
99mTc and 123I. Recently, a stable one-step 99mTc labelling of
His-tagged recombinant proteins has been developed [125].
t h
Application of this technology to His-tagged nanobodies may
lead to the development of a new class of radioimmunophar-
maceuticals for scintigraphic imaging of tumours. Initial
u
experiments have indicated that nanobodies are stably labelled
with 99mTc using this one-step labelling method. The radiola-
belled nanobodies are stable in vitro and in vivo, and allowed
A
clear-cut imaging of tumours at 5-h postadministration with
tumour-to-background ratios > 9:1 (Cortez-Retamozo,
unpublished results).
Besides their potential as imaging agents, nanobodies may
offer added value to cancer diagnostic tests used at present. For
example, early detection and staging of prostate cancer is based
on the detection of prostate-specific antigen (PSA) in the blood
circulation. However, different isoforms of PSA are present in
the blood, of which some correlate better with prostatic disorders
than others [126]. Recently, nanobodies have been generated that
can discriminate between different isoforms of PSA. Remarkably,
these nanobodies seem to sense or induce conformational
changes on different PSA isoforms, a feature that may be
exploited to discriminate different stages of prostate cancer [127].
A fusion of one of these PSA-specific binders with the S-layer
protein was used for direct detection of low PSA concentrations
by surface plasmon resonance [110]. Moreover, llama-derived
nanobodies were shown to be excellent tools in immunocyto-
chemical, immunohistochemical and immunoblot analysis to
detect oculopharyngeal muscular dystrophy [114].
8 Expert Opin. Biol. Ther. (2005) 5(1)
6.2 Nanobodies in cancer therapeutic tools
Given their high affinity and specificity, the small size of
nanobodies makes them particularly suitable for targeting
antigen in obstructed locations, such as tumours, where pene-
tration into poorly vascularised tissue is crucial to the success
of the drug [124]. In addition, nanobodies have an extremely
low immunogenic potential. In animal studies, repetitive
administration of nanobodies does not yield any detectable
humoral or cellular immune response [124].
As a versatile building block for manifold constructs, nano-
bodies can easily be used as a delivery vehicle for a range of
molecules that provide ancillary functions after target binding.
This ‘magic bullet’ would be delivering the toxic payload to the
o f
tumour while minimising the length of time that the toxic
compound could cause damage to healthy cells. In a separate
experiment, a bifunctional fusion was successfully constructed
between a nanobody directed against carcinoembryonic anti-
gen and a bacterial β-lactamase. Use of this conjugate in an
o
antibody-dependent enzyme prodrug therapy setting led to a
r
complete cure of established LS 174T human adenocarcinoma
xenografts without any adverse effect [78].
P
Today, with the discovery of bacterial strains that specifi-
cally target tumours, and aided by genetic engineering, there
is a new interest in the use of bacteria as tumour vectors.
r
Clostridium, Salmonella and Bifidobacterium have been shown
to preferentially replicate in solid tumours, and can be manip-
ulated to transport and amplify genes encoding factors such as
toxins, angiogenesis inhibitors and cytokines [128]. To improve
the tropism of these bacteria for tumours, retargeting mole-
cules, such as antibodies, may be expressed on their surface. In
this context, it has been shown that nanobody-based manifold
constructs can be efficiently translocated to the bacterial cell
surface, whereas scFv constructs failed to do so, probably
reflecting the high stability of nanobodies and their low
tendency to form aggregates [129].
Besides targeting to obstructed lesions, the delivery of thera-
peutics across the blood–brain barrier (BBB) remains a major
challenge in developing treatments for neurological diseases
and cancers of the brain. Macromolecule delivery across the
BBB using antibodies takes advantage of transporters or recep-
tors selectively expressed on brain capillary endothelium, such
as transferring-receptor, that undergo receptor-mediated trans-
cytosis. Nanobodies have been isolated that target the brain
in vivo and transmigrate across human BBB endothelium [130].
The potential of nanobodies as intrabodies provides an
attractive means for manipulating antitumour triggering
agents when expressed in tumour cells. Such antibodies can
neutralise or modify the activity of oncogenic molecules when
addressed in specific subcellular compartments and/or they
can be used to trigger antitumour immunity when expressed
on the tumour cell surface.
Besides applications where rapid clearance of the therapeu-
tic agent is wanted, there are therapeutic situations where a
prolonged serum half-life is desirable to increase the time that
the antibody can interact with its target and to reduce the fre-
 Revets, De Baetselier & Muyldermans

quency of dosing. Several methods have been used to increase
the half-life of antibody fragments, including PEGylation
[131], conjugation or fusion to albumin [132], and fusion to the
Fc fragment of an antibody [133]. It is known that the neonatal
receptor FcRn regulates the serum half-lifes of both IgG [134]
and albumin [135]. Hence, fusion of antibody fragments to
albumin or Fc probably confers a prolonged serum half-life
and also reduces kidney clearance (due to an increase in
molecular size). Alternatively, peptides with high affinity for
serum albumin can be fused to an antibody fragment and
extend the half-life [136]. All of these strategies can also be eas-
ily applied to increase the serum half-life of nanobodies. Con-
necting the antigen-specific nanobody with a serum albumin
binding entity or with the Fc of human IgG increases the
serum half-life of a nanobody in a mouse from 45 min to 2
weeks. This versatility will obviously increase the range of
therapeutic options available for nanobodies.
includes protruding loops. As a consequence, the surface of the
paratope of nanobodies can be as large as that of conventional
antibodies. However, the footprint it leaves on the antigen is
smaller due to the large concave paratope architecture. There-
fore, hidden or convex epitopes can still be targeted by nano-
bodies. As such, nanobodies can interact with receptor clefts
and the active site of enzymes where they actually act as inhibi-
tors. Hence, nanobodies may represent an interesting lead to
design small enzyme inhibitors from which peptide mimetic
drugs can eventually be derived.
The immunogenicity of nanobodies is a concern that
should be thoroughly investigated. However, the large degree
of sequence identity between a nanobody and human VH of

o f
family III is a good sign. The lack of any B or T cell response
against nanobodies in mice is another. Furthermore, the
retrieval of nanobodies with the framework-2 imprint of a
human VH [137] might be the ultimate solution to obtain

o
non-immunogenic nanobodies.
7. Expert opinion and conclusions The nanobody technology platform that has been devel-

r
oped can quickly deliver therapeutic leads for a wide range of
Since their discovery a decade ago, nanobodies have progressed targets within a few weeks. Based on their heavy-chain-only

P
from a ‘Darwinian evolution curiosity’ to a highly versatile origin, nanobodies obviate the need for construction and
molecule with a wide variety of potential biotechnological and screening of large libraries and for subsequent affinity matura-
medical applications. Nanobodies are the smallest functional tion techniques. Moreover, nanobodies are endowed with

r
antigen-binding fragments derived from in vivo matured camel unique properties such as a high stability against extremes of
or llama HCAbs. These entities are extremely stable and bind temperature and pH, and the possibility to produce large

o
antigen with nanomolar affinity. The subtle substitutions and quantities at low cost. Because of these unique properties and
structural rearrangements that occur between the VL-interact- the ease of generating manifold constructs, it is clear that
ing surface of a VH domain and the corresponding site of a nanobodies possess advantageous characteristics for therapy

t h
nanobody provide an example of natural protein engineering
converting a heterodimeric molecule into a monomeric mole-
cule. The absence of a VL domain is compensated in nanobod-
and diagnostics. The possibility of engineering multiple and
robust antibody-based formats positions them far ahead of
conventional antibodies. Future studies in humans will be

u
ies by an extended CDR1 and a more exposed CDR3 that required to confirm their performance, benefits and their effi-
often folds over the framework-2 region. The structural reper- cacy in cancer immunodiagnosis and/or therapy. Bringing to
toire of nanobodies is thus sufficiently diverse. Therefore, the the field nanobodies with specificities to other targets of bio-

A
CDR grafting from an antigen-binding human or mouse
VH domain to a nanobody scaffold has little chance for
success. Besides the standard architecture of planar surfaces
and cavities, the antigen-binding site of nanobodies also
logical relevance will be another future challenge, not only to
address fundamental understandings about their molecular
characteristics and structural stabilities, but also to position
them as clinically useful pharmaceuticals.

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Affiliation
Hilde Revets†, Patrick De Baetselier &
Serge Muyldermans
†Author for correspondence
Vrije Universiteit Brussel, Department of
Molecular and Cellular Interactions, Laboratory
of Cellular and Molecular Immunology, Vlaams
Interuniversitair Instituut voor Biotechnologie,
Pleinlaan 2, Building E8, B-1050 Brussels,
Belgium
Tel: +32 2 629 19 76; Fax: +32 2 629 19 81;
E-mail: harevets@vub.ac.be
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Expert Opin. Biol. Ther. (2005) 5(1)