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Nanobodies and their potential applications

Article  in  Nanomedicine · June 2013

DOI: 10.2217/nnm.13.86 · Source: PubMed

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 Perspective

Nanobodies and their potential applications

Nanobodies are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy chain-
only antibodies. The innate supremacy of nanobodies as a renewable source of affinity reagents, together
with their high production yield in a broad variety of expression systems, minimal size, great stability,
reversible refolding and outstanding solubility in aqueous solutions, and ability to specifically recognize
unique epitopes with subnanomolar affinity, have combined to make them a useful class of biomolecules
for research and various medical diagnostic and therapeutic applications. This review speculates on a
number of technological innovations that might be introduced in the nanobody identification platform

f
to streamline the generation of more potent nanobodies and to expand their application range.

oo
Keywords: Camelidae n crystallization chaperone n deep sequencing n enzyme Gholamreza
inhibitor n heavy chain-only antibody n immune repertoire n intracellular delivery Hassanzadeh-
n nanobody n single-domain antibody
Ghassabeh1,2,
Nick Devoogdt1,3,
From heavy chain-only antibodies to Therefore, antigen-specific scFvs are preferably Pieter De Pauw1,4,
nanobodies
Pr
obtained in a two-step procedure: first, a large
Cécile Vincke1,4
The natural immune system receives much inter- single-pot repertoire of naive or synthetic VH
& Serge Muyldermans*1,4
est because of its importance in various funda- and VL genes are cloned in an scFv format; 1
Vrije Universiteit Brussel, Research
mental and applied sciences. The soluble IgG second, the antigen-specific scFvs are retrieved group Cellular & Molecular
glycoproteins circulating in the blood play an from this repertoire after phage display. It is well Immunology, Pleinlaan 2,
1050 Brussels, Belgium
essential role in the humoral immune system of established that phage display panning selects for
or

mammals. These antibodies, produced by B lym-
phocytes, recognize with high specificity the
foreign biological or chemical substance against
which they are affinity matured, and might alter
h
binders with the highest specificity and affinity
to the target, in addition to the better producers
and more stable constructs. With the objective
of making even smaller antigen binders, similar
2
VIB, Nanobody Service Facility, Vrije
Universiteit Brussel, Brussels, Belgium
3
Vrije Universiteit Brussel, Research
group ICMIC, Laarbeeklaan 109,
1090 Jette, Belgium
4
VIB, Department of Structural Biology,

Vrije Universiteit Brussel, Brussels,

the characteristics (e.g., function) of this cognate
antigen. The basic structure of an IgG molecule
comprising two identical heavy (H) polypeptide
ut
single-pot libraries have been constructed based Belgium„
on soluble, stable, VHs with good expression, *Author for correspondence:
Tel.: +32 2 629 19 69
where codons for amino acids within antigen-

Fax: +32 2 629 19 81

chains and two identical light (L) polypeptide
chains is highly conserved among mammals.
The molecular weight of an IgG is approxi-
mately 160,000 Da, and it is a complex mol-
A
binding loops were randomized. While such svmuylde@vub.ac.be
synthetic VH libraries seem to be a good source
to identify single-domain antibody fragments,
they can be substituted equally well by other

ecule in which H and L chains fold into four and
two domains, respectively (Figure 1) . Compared
with smaller antigen-binding fragments, such
as Fab (molecular weight: 50,000 Da) or scFv
(molecular weight: 30,000 Da), only low yields
of functional recombinant IgG are obtained
from microorganisms. The scFv is a manmade
product composed of an N‑terminal, variable
domain of the H chain (VH) and an N‑terminal,
variable domain of the L chain (VL), joined by
a short synthetic peptide linker (Figure 1) . The
scFv is the smallest intact antigen-binding frag-
ment that can be derived from a conventional
IgG molecule. Unfortunately, bacterial expres-
sion of scFvs cloned from previously identi-
fied hybridomas often suffers from low yields.
immunoglobulin folds such as VL or fibronectin
domains, as well as by engineered nonimmu-
noglobulin scaffolds, including ankyrin repeat
proteins (DARPins), lipocalins (anticalins) or
protein A (affibodies), to name just a few [1] .
Each of these scaffolds has its benefits and prob-
ably limitations as well.
At the time that first successes were experi-
enced in retrieving antigen-specific scFvs or Fabs
from large synthetic or naive libraries displayed
on a phage, it was discovered serendipitously
that dromedaries incorporate a unique class of
antibodies devoid of L chains in their sera, in
addition to the conventional H 2L 2‑type IgGs
[2] . Thus, these homodimeric H2‑type antibod-
ies were called H chain-only antibodies (HCAbs) part of

10.2217/NNM.13.86 © 2013 Future Medicine Ltd Nanomedicine (2013) 8(6), 1–14 ISSN 1743-5889 1
Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans

bactrianus, Lama glama, Lama pacos, Lama gua-

ScFv
Nb nicoe and Lama vicugna). However, similar func-

VH
tional antibodies devoid of L chains are also pres-

VL
ent in an assortment of non-mammals, such as

VH
H
sharks (Orectolobus maculates and Ginglymostoma
VH

VH
cirratum) and ratfish. In these cartilaginous fish,

VH

H
VL
VL

VH
these antibodies, commonly known as Ig‑NARs,
CH

H
1
H
C
1

L
have an architecture fairly different from that of
CL

Fab
C

camelid HCAbs: an Ig‑NAR is a homodimeric

CH2

CH2
H chain, each chain consisting of one single vari-
CH2

CH2

Fc able antigen-binding domain (V‑NAR) and five

Fc
constant domains (Figure 1) [3] .

CH3

CH3
Inspection of the molecular organization of
CH3

CH3

the H2L2 antibodies, the camelid HCAbs and

sdAb Ig‑NARs, teaches us that the H2‑type antibod-
ies recognize their cognate antigen by one single
AR

f
N domain, the VHH or the V‑NAR, respectively
VH
VH

V-

(Figure 1) . Crystal structures of VHHs reveal a

oo
VL
VL

AR
V-

prolate (rugby ball) shape of approximately

C1

N
C

N
AR

V-
2.5 nm in diameter and 4.2 nm in length [4] .
L
CL

Hence, these ‘nanometer-sized antibody frag-

ments’ are referred to as ‘nanobodies’ (Nbs)
C2

C2

C2

C2

[5] . Although V‑NARs and VHHs have similar

Pr sizes, the term Nb is only used for (recombinant)
camelid single-domain antibody fragments.
C3

C3

C3

C3

The fascination of nanotechnology originates

from altered mechanical, thermal and catalytic
C4

C4

C4

C4

properties of materials as their size decreases

from the micro- to nano-meter scale, enabling
or

novel applications. Likewise, the size reduction

C5

C5

C5

C5

of an HCAb into an Nb (and the concomitant

reduction in valency from bivalent to monova-
lent) can cause a dramatic change in biologi-
h
C6

C6

C6

C6

cal activity. Indeed, while a monoclonal HCAb

with specificity to trypanosomes is harmless to
the parasite (in the absence of complement), the
ut

Figure 1. Mammalian and shark antibodies. (A) Conventional mammalian IgG

antibodies, (B) camelid heavy chain-only antibodies, (C) cartilaginous fish IgW and isolation of Nbs from these HCAbs makes these
(D) shark Ig‑NARs. The smallest practical antigen-binding entity of IgG, the scFv, is antigen-binding entities highly trypanolytic
also shown, as well as the single-domain antigen-binding fragments of the camelid by blocking a very early endocytosis step via a
heavy chain-only antibodies and shark Ig‑NAR, known as nanobody and V‑NAR
mechanism that has not been elucidated yet [6] .
A

(sdAb), respectively.
C: Constant domain; CH: Constant domain of the heavy chain; CL: constant
domain of the light chain; Nb: Nanobody; SdAb: Single-domain antibody;
V-NAR: Variable antigen-binding domain; VH: Variable domain of the heavy chain;
VHH: N‑terminal variable domain of the heavy chain-only antibody; VL: Variable
domain of the light chain.
(Figure 1) . It was also noticed that the H chain
of HCAbs is folded into three domains: an
N‑terminal domain that is variable in sequence
(VHH), followed by a hinge region and two
constant domains. Therefore, the equivalent of
the first constant domain of the H chain of a
conventional antibody is missing in H chains of
H2 antibodies. The occurrence of such functional
HCAbs within mammals as a component of their
humoral immune system seems to be limited to
Camelidae species (Camelus dromarius, Camelus
Biochemical & biophysical properties
of Nbs
After vaccinating a camelid (or a cartilaginous
shark) to raise an H2‑type antibody response,
the VHH (or V‑NAR) gene fragments from
lymphocytes are amplified by PCR, ligated in
phage display vectors and transformed in bac-
teria to construct an immune VHH library.
Expression of the cloned VHHs in fusion with
the phage coat protein pIII, its assembly at the
tip of phage particles and enrichment of phage-
displayed, antigen-specific VHHs on immobi-
lized antigens in two or three rounds of panning
allows the identification, with a high success
rate, of Nbs targeting a wide variety of antigens

2 Nanomedicine (2013) 8(6) future science group


with nanomolar to even picomolar affinities
within weeks of the first antigen injection [7] .
The retrieved Nbs are successfully expressed
in microorganisms, thereby providing an easy
route to obtain large amounts of in vivo affinity-
matured, antigen-specific single-domain anti-
bodies. This is an important advantage over scFv
or manmade scaffolds where in vivo affinity mat-
uration cannot be accomplished easily by immu-
nization. In cases where vaccination is difficult
(e.g., chemical and nonimmunogenic targets), it
is possible to construct large single-pot synthetic
VHH (or V‑NAR) libraries to identify antigen
binders [8] . However, the antigen-specific binders
retrieved from synthetic libraries are not affin-
ity matured and might benefit from additional
sequence diversification steps and subsequent
selections to generate more potent binders [9] .
The crystal structures of VHHs in complex
with their antigen [10] confirmed that the side of
the domain (known as the framework‑2 region)
that contacts a VL in an H2L2‑type antibody is
resurfaced from a hydrophobic region in VH to a
more hydrophilic region in a VHH. This frame-
work‑2 resurfacing by amino acid substitutions
contributes to the strict monomeric behavior and
the high solubility of VHH domains. Moreover,
it explains the stickiness of many VHs in the
absence of a VL partner. However, the reduced
or
solubility of isolated VHs in aqueous solution
can be remediated by ‘camelizing’ the mouse
or human VH, whereby hallmark amino acids
of the framework‑2 region of a camelid VHH
h
are imprinted in the VH sequence [11] . Con-
versely, although Nbs are poorly immunogenic
in humans, due to their high sequence identity
ut
to human VHs, it is probably good practice
for prolonged therapeutic applications involv-
ing multiple administrations to first ‘human-
ize’ the framework‑2 region of Nbs to imprint
A
an amino acid sequence closer to human VHs
in that region [12] . The humanization of Nbs,
like any other site-directed mutagenesis on Nbs,
is straightforward as the gene is only approxi-
mately 380 base pairs.
The crystal structure of VHHs further
revealed new structural organizations of the
antigen-binding loops, so Nbs are more suitable
than scFv paratopes for interacting with grooves
on the surface of the antigen, such as the cata-
lytic site of enzymes [10] . Indeed, it is well estab-
lished that many Nbs act as enzyme inhibitors or
modulate the function of the target and that the
preferred antigenic sites for scFvs and VHHs are
distinct. Of note, the V‑NARs and VHHs seem
to share this preferential binding to clefts [3] .
future science group
Nanobodies & their potential applications Perspective
Streamlining the Nb identification
platform
Successful identification of antigen-specific Nbs
is straightforward after immunizing a camelid
(or shark for V‑NAR) with purified, soluble,
properly folded proteinaceous antigens and
phage display [7] . However, immunization with
more difficult targets, such as multipass mem-
brane proteins, receptor complexes, intrinsically
disordered proteins or unstructured peptides and
the subsequent selections against such targets
remain critical steps.
„„ Immunization of camelids or
transgenic mice
The procedures to raise HCAbs in camelids are
f
similar to those used to elicit conventional anti-
bodies in other animals. Various adjuvants as
oo
well as different immunization routes and boost
intervals successfully induce a HCAb response
in camelids. Camelid immunization simultane-
ously elicits conventional and HCAb responses,
with the former often being dominant. Any opti-
Pr
mized immunization protocol (e.g., a particular
adjuvant and/or immunization route) favoring
the HCAb response is highly desired. Taking
into account that camelids are large and out-
bred animals, this immunization optimization
is challenging, but feasible.
Most antibody targets are membrane pro-
teins, which are difficult to express and purify
as functional recombinant proteins. The avail-
ability of such proteins in sufficient quantities for
immunization and selection is, therefore, a hur-
dle in all antibody technology platforms. Since
the vast majority of HCAbs and Nbs recognize
conformational epitopes, the immunization of
camelids with peptides (even coupled to larger
protein carriers) is not recommended as it raises
poor H2‑type antibody responses.
DNA immunization, cell immunization and
DNA prime/cell boost are potential alternatives
to protein immunization. Both DNA prime fol-
lowed by protein boost or cell immunization
have been used to generate Nbs [13,14] . Never-
theless, the former method still requires proteins,
whereas cell boost or cell immunization results
in an undesired antibody response to nontarget
cellular antigens, thereby complicating panning
and screening steps. To optimize DNA immuni-
zation protocols without the need for protein or
cell boosts, it will be necessary to systematically
analyze the target-specific Nbs identified after
such immunization.
As testified by the large number of publica-
tions, immunization of camelids – or better to
www.futuremedicine.com 3
Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans
say, the size and exotic nature of camelids – has
not been prohibitive in generating Nbs and in
exploiting these Nbs in various applications.
However, immunizing transgenic mice, with the
exon for the first constant domain in H chain
gene(s) deleted, might streamline the Nb iden-
tification platform. The proof of principle for
such mice as a source of Nbs has already been
given [15,101] , but the concept of transgenic mice
as substitutes for camelids to rapidly generate
potent HCAbs and/or high-affinity Nbs still
remains to be demonstrated.
„„ Nb selection
Current Nb identification strategies involve
enrichment of antigen-specific binders from
immune or naive libraries by phage display,
bacterial two-hybrid or bacterial surface dis-
oo
play [7,8,16] , and screening of individual colonies
from enriched phage populations, primarily by
ELISA. However, advanced approaches are in
sight, and examples of such approaches are pro-
posed below.
Pr
Deep sequencing to avoid repertoire
cloning & selection
Recently, next-generation sequencing (NGS)
was used to calculate the relative frequencies of
genes encoding VH and VL in a cDNA pool
or
of plasma cells from immunized mice. Subse-
quently, the most abundant VH and VL genes
were synthesized and paired to obtain antigen-
specific scFvs [17] . This study provides an exam-
h
ple of bypassing tedious library construction,
biopanning and ELISA screening steps. Moreo-
ver, the NGS screening identified antibodies that
ut
were lost during panning or went undetected by
ELISA [18] . Interestingly, the recombinant anti-
bodies identified by NGS, but not by standard
panning and ELISA screening, were expressed
A
in Escherichia coli at relatively high levels under
optimized conditions.
We anticipate that implementing the NGS
approach will be extremely favorable to rapidly
discover antigen-specific Nb sequences. Indeed,
the antigen-binding fragment of HCAbs involves
only one single exon (VHH) instead of two (VH
and VL) in conventional antibodies. Therefore,
the most abundant VHH sequences in a pool of
lymphocytes should correspond to those from
HCAbs on B cells that maximally proliferated
during immunization. Moreover, the single-
domain Nb format immediately corresponds to
the intact antigen-binding site of HCAbs, and
avoids the requirement to combine all abundant
VHs and VLs in all possible combinations to
4 Nanomedicine (2013) 8(6)
find the best pairs as they were affinity-matured
in vivo during immunization.
Selection of Nbs against proteome
fractions
It is common practice to immunize camelids
with a mixture of five to ten antigens and to sort
by panning antigen-specific binders to each com-
ponent afterwards. With this in mind, it should
be possible to immunize with a crude extract of
any particular cell, possibly containing a cur-
rently unknown ‘active’ protein. The camelid
will raise HCAbs to immunogenic proteins and
the cloned VHH library can be panned subse-
quently on the same proteome extract to enrich
for Nbs against any component within the crude
f
extract. After the last round of panning, indi-
vidual clones can be screened by ELISA [19] or
by a functional assay to identify Nbs that bind
or modulate the ‘activity’ of a particular compo-
nent of the mixture (see ‘Nb-based phenotypic
screening’ section). Once such Nbs are isolated,
they can be purified and immobilized to cap-
ture the antigen from the mixture. The captured
antigen can then be identified, for example, by
mass spectrometry [20] .
If the biomarker is known, it is possible to
use a minimal component fraction (i.e., a cell
fraction enriched for the biomarker and depleted
of irrelevant proteins) for immunization and
subsequent phage selections. However, in cases
where the biomarker of interest is unknown,
the possibility to use crude, unfractionated
cell extracts has an advantage over the use of
minimal component fractions because the iden-
tification of the fraction containing the (unde-
termined) biomarker causing the phenotype of
interest can be very tedious (especially when it
is a multicomponent biomarker).
Nb-based phenotypic screening
The potential of antibodies for high-throughput
phenotypic screening to discover new targets
of therapeutic value still remains unexplored.
This is partly due to the inability of antibod-
ies to access intracellular targets and the low
throughput of antibody discovery platforms.
Despite these shortcomings, antibodies repre-
sent an interesting class of molecules for high-
throughput phenotypic screening, because of
their high affinity, fine specificity and almost
limitless repertoire diversity. Indeed, the feasibil-
ity to use antibodies for the discovery of novel
drugs or drug targets by phenotypic screening
was recently demonstrated by using both naive
and immune scFv libraries [21] . In this work,
future science group

individual scFvs, from affinity selections on
Pseudomonas aeruginosa whole cells, were tested
by ELISA for binding to heterologous serotype
strains. The scFvs exhibiting serotype-inde-
pendent binding to P. aeruginosa were recon-
stituted into an IgG1 format and assayed for
opsonophagocytic killing of P. aeruginosa. The
target antigens of scFvs with such activity were
then identified by assaying the binding of scFvs
to P. aeruginosa mutant strains [21] .
The robustness of the Nb identification tech-
nology and the possibility to generate and iden-
tify Nbs on the proteome scale [19] , together with
numerous data on the potent and target-specific
effects of Nbs, suggest that Nbs can serve as
alternatives to small chemical compounds for
phenotypic screening aimed at the discovery of
both novel drugs and drug targets. However, for
this concept to be fully realized, future develop-
ments are vital such as incorporation of NGS
into the Nb identification platform to increase
its throughput and preferably the ability to tar-
get intracellular molecules in a high-throughput
manner.
Selection on cells
Purification of properly folded, multipass mem-
brane receptors (e.g., pharmacologically impor-
tant G protein-coupled receptors) or receptor
or
complexes (e.g., integrins) for immunization and
selection is a major bottleneck. For such targets,
there are two solutions: either, immunization
is avoided and non-affinity-matured Nbs are
h
retrieved from naive or synthetic libraries [8] ,
or immune libraries will be constructed after
immunizing with DNA only [14] or transgenic
ut
cells that overexpress such receptors. For the lat-
ter, transgenic DUBCA cell lines might be very
useful as these dromedary-derived cells are sup-
posedly poorly immunogenic in camelids. Dur-
A
ing the subsequent Nb selection process, special
measures need to be taken to retrieve binders
targeting the (perhaps less immunodominant)
receptor of interest from a vast pool of poten-
tial binders that target unwanted membrane
proteins. It is imperative to eliminate the Nbs
to these irrelevant antigens. This is achieved to
some extent by the biopanning and rapid ana­
lysis of selective interactive ligands, the ‘BRA-
SIL’ method [22] , or by panning on two different
transgenic cell-lines with very different origins.
Otherwise, multiple rounds of selections can
be performed on a cell mixture, in which fluo-
rescently or magnetically labeled transfected
cells are diluted into unlabeled, untransfected
cells and then subjected to flow cytometric or
future science group
Nanobodies & their potential applications Perspective
magnetic sorting. Nbs against common antigens
(present on both transfected and nontransfected
cells) will bind mainly to the untransfected cells
due to their higher abundancy, and, therefore,
those Nbs will be eliminated by sorting the
labeled transfected cells.
Nbs with improved properties
„„
In vitro affinity improvement of Nbs
In some instances, the immunization of a
camelid is impossible (owing to highly toxic
or pathogenic immunogens, lack of purified
properly folded antigen and nonimmunogenic
compounds) and, thus, alternatives to in vivo
maturation might be necessary to obtain
high‑affinity antigen-specific binders.
f
Generating bivalent Nb constructs might
increase the functional affinity (i.e., avidity)
oo
[23] , but does not change the intrinsic affin-
ity between Nbs and antigens. In principle,
improvement of the intrinsic affinity of Nbs
for their antigen should be easier than that
with scFvs because of Nbs’ smaller size, single-
Pr
domain nature, easy folding and the presence of
only three antigen-binding loops. These char-
acteristics make the construction of second-
generation libraries on a previously selected
Nb scaffold less complex and allow more robust
phage or yeast display selections instead of more
demanding selection techniques, such as ribo-
some display. Libraries of Nb variants with
either localized or unbiased random mutations
need to be generated. The targeted options use
site-specific randomization of the codons for
the amino acids of the antigen-binding loops.
The availability of structural information of the
antigen-binding loops of the Nb, preferably in
complex with its antigen, is helpful, if not criti-
cal, to pinpoint the mutagenesis hot spots. Such
antigen-binding hotspots can also be identified
by Ala-scanning mutagenesis [9] . Apart from
these techniques based on evolution, rational
approaches might also be successful. Indeed, it
has been shown that mutating a limited num-
ber of amino acids, carefully selected at the
periphery of the paratope, guided by param-
eterized quantitative descriptors (using a mul-
tivariate experimental design), and measuring
the effect of these mutations on the antigen-
binding kinetics could be used to construct
an algorithm that predicts the kinetic binding
parameters as well as the equilibrium binding
constant of other possible mutations at those
positions [24] . Conversely, the unbiased, rand-
omized Nb libraries are obtained via error prone
PCR or the use of mutator strains. These fully
www.futuremedicine.com 5
Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans
random approaches have the disadvantage that
a vast number of dysfunctional Nbs are formed
and the advantage that occasionally a mutant
might emerge with an amino acid substitution
outside the antigen-binding loops with indirect
favorable binding properties.
Finally, the implementation of Nbs in the
phage-assisted continuous evolution technol-
ogy, an elegant technique in which phage fitness
constitutes the driving force to select perma-
nently novel mutants evolving spontaneously,
rapidly and continuously [25] , is expected to
produce Nbs of greatly improved potency.
Cell-penetrating Nbs
The availability of transducing Nbs (i.e., Nbs
with the capacity to penetrate the membrane of
their specific target cell) that deliver functional
oo
molecules to the cytoplasm would offer an ele-
gant solution to overcome the impermeability of
cells to most macromolecules [26] . These trans-
ducing Nbs could be used to produce ‘Trojan
horses’, directing proteins, DNA or siRNA spe-
Pr
cifically to diseased cells to provoke cytotoxicity,
to restore a lacking function in deficient cells,
or to induce production of complementary pro-
teins. To achieve transduction, one could take
advantage of the existing internalizing proteins
on the target cell surface (e.g., by inducing
or
receptor dimerization and internalization with
a bivalent or bispecific Nb). Another approach
would be the addition of protein transduction
domains, such as the one derived from the HIV
h
Tat protein or penetratin, or by increasing the
Nbs’ arginine content, a condition that facilitates
internalization [27] . Finally, a promising tech-
ut
nology was developed whereby Nbs equipped
with a short peptide are delivered into human
cells, directly from E. coli via its type III protein
secretion system [28] . It is surmised that expres-
A
sion of a cell type-specific Nb on the surface
of attenuated, invasive bacterial strains might
direct these engineered cells to specific organs
or tissues of the host. Colonization of the target
organ (e.g., solid tumors, infected or diseased
cells) might then allow prolonged delivery of a
protein therapy by a relevant second Nb (i.e.,
intrabody), injected to interfere or reprogram
the diseased cells.
Nbs in research
„„ Nbs as affinity capture reagents
Nbs are suitable affinity capture reagents [29]
because their small size and single-domain for-
mat provide higher capacity binding surfaces
and lower nonspecific background binding, as
6 Nanomedicine (2013) 8(6)
compared with larger antibody formats. Their
monovalent mode of binding allows mild elution
conditions, which is particularly important for
sensitive molecules. Moreover, their high stabil-
ity and refolding capacity permits repeated and
stringent regeneration conditions.
Nb-based affinity capture has been used to
study protein–protein interactions [30] , ana-
lyze in vivo DNA–protein interactions at the
genomic scale after chromatin immunoprecipita-
tion, track protein conformations, trace bacterial
infections and purify recombinant proteins. Nbs
have been employed for scavenging toxin from
plasma [31] and for depletion of abundant serum
proteins, a step required for proteomic ana­lysis
of blood samples [32] . The use of KDEL-specific
f
Nbs to capture endoplasmic reticulum-resident
proteins [23] raises the possibility to use signal
peptide-specific Nbs as a generic tool for high-
throughput mapping of protein locations in
physiological and pathophysiological conditions
by combining Nb-based affinity capture with,
for example, anti-proteome Nb arrays. Unfortu-
nately, generating Nbs against other signal pep-
tides (e.g., palmytoylation, sumoylation, phos-
phorylation, S-nitrosylation and Y-nitration)
might be very challenging as these sites are less
well conserved and/or located in a flexible region
of the protein (i.e., poorly antigenic). Neverthe-
less, the KDEL and chromatin immunoprecipi-
tation examples enforce the idea that Nb-based
affinity capture, alone or in combination with
other proteomics tools, might serve many pur-
poses of modern proteomics.
„„ Nbs as crystallization chaperones
Nbs exhibit a good track record as chaperones
to crystallize challenging proteins [33–35] . Indeed,
these robust, single-domain antibody fragments
often lock proteins in a particular conformation
[36] . The ability of Nbs to stabilize intrinsically
flexible regions or shield aggregating surfaces
from contact with solvents are key features to
allow the crystallization process [37] . DARPins
and anticalins appear to serve the same purpose,
whereas crystallization with scFvs appear to be
less successful, possibly because of the intrinsic
flexibility of VH and VL interactions, or the
linker. The favorable properties of Nbs allowed
an impressive list of structures of highly dynamic
proteins to be elucidated and identification of
unknown conformations, providing insight into
the mechanisms of action of their antigens. In
addition, Nbs might become an essential tool to
obtain valuable structural information on par-
tially disordered or amyloid proteins [38,39] . For
future science group

example, conformation-sensitive Nbs stabilize
Amyloidb protofibrils and prevent the forma-
tion of mature amyloid fibrils [40] . Structural
data of such trapped amyloid intermediates
might unravel the aggregation process of these
tangled targets and improve our understanding
of the mechanisms leading to their pathological
conversion.
„„ Intracellular target imaging &
immunomodulation
Antibodies acting inside living cells are known
as ‘intrabodies’. Nbs fold surprisingly well into
functional entities, even in the reducing intra-
cellular environment. Therefore, intracellular
expression of Nbs fused to fluorescent proteins
produces useful chromobodies or fluobod-
ies that can be used to trace their antigen in
living cells [41,42] . This technology has been
adapted to advanced super-resolution imaging
techniques [43] .
The intracellular robustness, together with
the target-modulating activity of Nbs, makes
them an ideal tool to impair antigen activity
inside the cells, producing functional knockouts
[41,44–46] . Apart from being a target validation
tool, such Nbs might become valuable as leads
to investigate molecules currently considered to
be ‘undruggable’ (by definition, an undruggable
or
target cannot be modulated appropriately by any
currently known, available and safe molecule) .
Nbs fused with leader signal peptides can be
directed to specific subcellular compartments.
h
The expression of Nbs, even those without
intrinsic target inhibitory activity, at these sites
might sequester their targets from their normal
ut
subcellular compartments, thereby inhibit-
ing the function of the antigen. One example
involves the intracellular retention of otherwise
secreted proteins such as viral coat proteins [47] .
A
Nbs, fused to a secretory signal peptide and a
C-terminal KDEL peptide, sequester both the
Nb and its antigen in the endoplasmic reticu-
lum. In another setting, Nb-based intrabodies
containing a PEST motif redirected the target
antigen to the proteasomal degradation pathway
[48] . Likewise, green fluorescent protein-tagged
proteins could be depleted in vivo via the ubiq-
uitin depletion pathway by the intracellular
expression of a green fluorescent protein-specific
Nb fused to the F‑box domain [49] . Despite these
successful examples, the broad applicability of
Nb-based PEST and F‑box-mediated protein
knockout awaits independent confirmation.
In summary, because of their extraordinary
intracellular functionality, Nbs have a clear
future science group
Nanobodies & their potential applications Perspective
advantage over other antibody fragments, and
they will probably become a valuable research
tool. However, the progression of Nb-based
intrabodies towards clinical application is less
certain and relies on the development of spe-
cific and efficient transduction methods. In this
respect, RNAi methodologies are certainly more
advanced. However, we believe that Nbs might
find a niche in cases where post-translationally
modified protein variants or isoforms that
cannot be discriminated via RNAi need to be
targeted.
Nbs in diagnosis
„„ Nbs as probes in novel biosensors
Biosensors development is a popular area of his-
f
tory because sensitive analyte detecting devices
are required for on-site application in many
oo
major fields, such as medical diagnosis, environ-
mental and food ana­lysis. Many of these innova-
tive biosensors rely on small, robust, highly spe-
cific affinity probes, where size and directional
immobilization matter. Nbs are particularly
Pr
suited for controlled and reproducible coupling
because site-specific functional groups are eas-
ily introduced, allowing covalent and oriented
binding with minimal loss of specificity and
affinity [50] . The Nbs’ small size provides high
binding capacity surfaces resulting in higher
sensitivity, and the high stability and refolding
competence of Nbs allow stringent washing and
regeneration conditions.
Moreover, Nbs directionally immobilized
onto solid sensor surfaces allow immunoreac-
tions to occur very close to the sensor–solu-
tion interface, resulting in increased sensitivity.
Alternatively, the analyte-capturing Nbs can
be joined spontaneously to magnetic particles
[51] , which are suspended in the sample solution
to bind analyte before being attracted to the
electrode by magnetic fields. The concentra-
tion enhancement of the analyte at the surface
of the electrode then leads to amplification of
the signal.
„„ Nbs for noninvasive in vivo imaging
Noninvasive molecular imaging uses labeled
tracers to visualize molecules and cellular bio-
markers in the whole body without disturbing
the host. This technology is very useful to inves-
tigate biological processes in animal models and
is expected to become a major tool in nuclear
medicine departments of hospitals to diagnose
and stratify patients following PET and SPECT
scans. An ideal (clinical) tracer should be sta-
ble in vivo, with the ability to reach all sites in
www.futuremedicine.com 7
Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans
the body, generate signals over a long range of
biomarker concentrations, interact with the bio-
marker with high affinity and specificity, easy
to label with dyes or radionuclides, inexpensive,
and nontoxic. In clinical imaging, the generation
of contrast at the site of biomarker expression
within hours of tracer administration is essential.
An Nb meets most, but not all, requirements to
become a generic imaging tracer [52] . Nbs are
usually stable, easily labeled, have low immuno-
genicity and have affinities in the low nanomolar
to high picomolar range. Their small size assures
optimal tissue penetration and fast blood clear-
ance, so that high contrast is attained within
hours of administration. Their target specific-
ity has been demonstrated using gene-deficient
oo
Pr
Liver
or
Kidneys
h
Tumor
ut
Bladder
A
Injection site
Figure 2. Maximal Intensity projection of
fused pinhole SPECT/micro-computed
tomography image of a mouse bearing a
TSA tumor implanted in the mammary fat
pad. The image was taken 3 h after injection
with a 99mTc radio-labeled anti‑macrophage
mannose receptor nanobody together with an
excess of bivalent, unlabeled macrophage
mannose receptor nanobody to reduce tracer
uptake in organs other than the tumor. Note the
accumulation of the tracer inside the tumor,
visualizing mannose receptor-positive
tumor‑infiltrating macrophages [53] .
8 Nanomedicine (2013) 8(6)
animals [53] or via coinjection of an unlabeled
competitor [54] . In preclinical animal models,
we have shown focal uptake of radiolabeled Nbs
in tumors targeting markers of cancer cells [52]
or tumor-infiltrating macrophages (Figure 2) [53] ,
in arthritic lesions, atherosclerotic plaques [54]
and immune organs [53] . A Phase I study with
an anti-HER2 Nb for PET imaging of breast
cancer patients is currently ongoing.
Nb-based imaging also has some limitations.
First, their fast renal excretion leads to high
signals in the kidneys and bladder, and imag-
ing of specific tracer uptake at nearby sites is
obstructed. Second, Nbs rarely penetrate the
blood–brain barrier [55] and, therefore, might be
suboptimal in the field of neuroimaging. Third,
f
while Nb-based imaging of cell surface receptors
is straightforward, tracing intracellular or solu-
ble targets is more challenging. Finally, GMP
production of Nbs is far more expensive than
other useful tracers that are based on peptides
or chemical compounds.
Nbs for therapy
„„ Serum half-life extension
Antibody fragments, including Nbs, display
short serum half-life profiles. Although this is
highly desirable for certain applications, such as
imaging with radiolabeled tracers, many thera-
peutic applications require a slow drug clearance
rate to avoid high doses and frequent administra-
tion. PEGylation or modification with a serum
albumin-specific small chemical compound, and
genetic fusion with conformationally disordered
polypeptides (e.g., HAPylation and PASylation),
with a protein having an inherently long serum
half-life (e.g., serum albumin) or with a mol-
ecule binding to an abundant serum protein
are among the many different approaches used
to extend the serum half-life of therapeutics
[56] . However, these modifications might have
adverse effects on antibody functionality [57] .
Nbs specific for abundant serum proteins,
such as albumin and IgG, have a long serum half-
life [58,59] , and fusion of such Nbs to other thera-
peutic compounds provides a generic approach
to extend the plasma half-life of this therapeutic.
Other studies described the extension of Nbs’
serum half-life by PEGylation [60] , pentameriza-
tion and fusion with IgFc [61] . The small amount
of data on Nbs’ serum half-life extension makes
it difficult to speculate on the preferred method
for each application. However, it is evident that
methods that adversely affect Nbs’ advantages
should be avoided. For instance, fusion of Nbs to
albumin or IgFc may complicate the expression
future science group

of Nbs in microorganisms, and pentamerization
(by fusion to verotoxin) may render Nbs immu-
nogenic. It is also worth noting that fusion with
IgG-specific Nbs for serum half-life extension
may provoke Ig-mediated immune effector cell
activation at undesired locations.
„„ Nbs against envenoming
The preferred serotherapeutic application of
Nbs over other antibody formats is illustrated
in the field of envenoming caused by scorpions or
snakes [62,63] . Current antivenoms are polyclonal
immunoglobulin fragments purified from the
blood of venom-immunized horses and sheep.
However, these antivenoms are often associated
with low potency, variable efficacy and severe
adverse effects (e.g., serum sickness). This low
potency is mainly attributed to a poor immune
response of host animals because most toxic
compounds within venom are small and poor
immunogens. Owing to their small size and
the absence of an Fc region, Nbs diffuse rapidly
through the body and reach a tissue biodistribu-
tion that closely matches that of the small venom
toxins. In addition, even after capturing and
neutralizing the toxin, the toxin–Nb complex
remains sufficiently small to be eliminated rap-
idly via the kidneys. As a result, a bispecific Nb
against the Androctonus australis hector AahI and
or
AahII toxin – the most toxic molecules in scor-
pion venom – was shown to possess, in mouse
models, a higher neutralization capacity than
the current anti-scorpion toxin immunotherapy
h
[64] . Recently, in vivo echocardiography in rats
demonstrated the ability of this 30‑kDa con-
struct to successfully prevent the fatal hemody-
ut
namic disturbances provoked by a lethal dose of
venom [65] . Intact camelid IgG antibodies and,
in particular, their Nb derivatives are equally or
more potent than the conventional antivenom in
A
neutralizing the lethal, hemorrhagic and coagu-
lopathic effects of west African viper (Echis ocel-
latus) venom [62] . Further research to define the
pathology-inducing compounds of the complex
snake venoms might lead to the identification
of Nbs against conserved epitopes, which may
eventually result in the development of polyreac-
tive toxin-specific antivenoms. Finally, the pro-
duction of sustainable Nb-based recombinant
antivenoms is expected to contribute favorably
to the reproducibility, safety and treatment cost
of envenomed victims.
„„ Nbs against infections
Antibodies are natural defense molecules against
bacterial, viral and parasite infections. It is well
future science group
Nanobodies & their potential applications Perspective
established that polyclonal antibodies are more
effective at combatting such infections than
monoclonal antibodies. However, the possibil-
ity to screen thousands of antigen-specific Nbs
from immune VHH libraries, in conjunction
with their intrinsic capacity to target epitopes,
which are cryptic for conventional antibodies,
has provided access to a number of neutralizing
Nbs against a myriad of pathogens [66,67] . A suc-
cessful Phase I clinical trial with Nbs targeting
respiratory syncytial virus has demonstrated
the potential of Nbs to combat infections [102] .
Since Nbs are devoid of any Fc region, they do
not have Fc-mediated effector functions and are
unable to neutralize and eliminate the patho-
gen. However, several Nbs have an inherent
f
neutralizing effect that is masked in HCAbs
because pathogens have evolved to escape this
oo
antibody-mediated threat [6] . Alternatively, the
strict monomeric nature of Nbs facilitates their
fusion with molecules with innovative effector
functions, such as enzymes or toxic molecules,
in order to eradicate the pathogen. Additional
Pr
success with Nb therapies will come from more
patient-friendly administration (topical, inhala-
tion or oral delivery, and colonizing bacteria-
releasing therapeutic Nbs) and from the variety
of strategies used to obtain improved blood levels
over a prolonged time.
„„ Nbs against amyloidosis
The search for lasting treatments that target the
underlying causes and alter the natural course
of amyloidosis disorders (e.g., Alzheimer’s,
Parkinson’s and Huntington’s diseases), linked
with misfolding or aggregation of proteins into
fibrillar deposits, is an extremely active area of
clinical investigation. Regarding Alzheimer’s
disease, active and passive immunotherapy
have been suggested for more than a decade
as potential strategies to retard or even reverse
disease progression. The form of the amyloid‑b
peptide that leads to synaptic, neurotic and
behavioral abnormalities has been difficult to
define, although growing evidence suggests that
oligomeric amyloid‑b intermediates are prob-
ably more toxic than the polymeric fibrils [68] .
Antibody-mediated interference must prevent
the conformational transitions that lead to the
assembly of oligomers, protofibrils and fibrils,
which ultimately lead to plaque deposition. The
clearance should, therefore, preferably be per-
formed before the proteins cluster into the toxic
oligomers, by only targeting a specific subset
of amyloid‑b precursors. The single-domain
nature of Nbs and their availability to work
www.futuremedicine.com 9
Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans
with immune repertoires greatly facilitates their
isolation as highly specific probes for patho-
logical target structures with excellent affinity
[69,70] . In addition, Nbs are readily available for
large-scale expression, and if penetration in the
CNS appears to be necessary for efficacy, some
Nbs that recognize unique epitopes on epithelia
have been described to transmigrate through
the human blood–brain barrier in vitro and
in vivo via active transport [71,72] .
„„ Nbs against pathologies & cancer
Therapeutic antibodies often rely on activation
of immune effector mechanisms for optimal
efficacy. Due to the lack of Fc, which medi-
ates immune effector cell activation, Nbs in
therapeutic intervention are entirely depend-
ent on intrinsic antagonistic antigen targeting,
oo
and possibly on agonistic signaling. Conversely,
lack of Fc in Nbs may result in fewer unwanted
immune-mediated side effects.
The application of Nbs to combat diseases
and pathologies mainly focuses on the inhibi-
Pr
tion of ligand–receptor interactions. Examples
include antagonizing anti-von Willebrand fac-
tor Nbs to block thrombosis initiation [73] , anti-
TNF‑a Nbs to treat arthritis [74] and EGF–
EGFR blocking Nbs to treat cancer [75] . Other
examples include Nbs that keep antigens in an
or
inactive receptor state [76] or provoke receptor
activation (agonist action) for apoptosis induc-
tion of cancer cells. One advantage of Nbs is
their small size, which means that dense tissues
h
can be penetrated by bivalent or trivalent Nb
complexes [59] . Hence, they are effective even
at occluded sites. Since Nbs are usually rapidly
ut
excreted from the body, their serum half-life
needs to be controlled (see ‘Serum half-life
extension’ section).
Recently, Nbs have been employed as a cell
A
(re-)targeting moiety in larger theranostic
vehicles, such as lentiviruses [77] , microbub-
bles [78] , branched nanogold particles [79] and
albumin nanoparticles [80] , bio-nanocapsules
[81] , and polymeric micelles [82] . The toxicologi-
cal profile of these relatively large particles in
clinical settings still needs to be evaluated, as
well as their targeting capacity after systemic
administration.
„„ Bispecific Nbs & Nb-based T-body
Complex diseases are often multifactorial in
nature; therefore, necessitating simultaneous
targeting of multiple factors or pathways for
maximal therapeutic effect. This can be achieved
by combination therapy (i.e., simultaneous
10 Nanomedicine (2013) 8(6)
administration of different antibodies) or,
alternatively, by using multispecific antibodies.
Compared with combination therapy, multispe-
cific antibodies provide the possibility to reduce
the costs of production, preclinical testing and
clinical trials [83] . Bispecific antibodies hold
great promise as next-generation therapeutics,
especially for cancer treatment [83–85] .
The interesting properties of Nbs, in par-
ticular their strict monomeric behavior, single-
domain nature and minimal size, make them
attractive candidates for the development of
manifold constructs [86,87] . Based on the pub-
licly available data, this notion has surprisingly
largely stayed in the explorative phase. This
might be, at least in part, due to the restrictions
f
resulting from intellectual property rights asso-
ciated with the use of Nbs, taking into account
that the major driving force behind bispecific
antibodies is the desire to finally bring them to
clinics. With some of these patents coming to
an end soon, we anticipate much faster progress
in the field of bispecific Nbs in the near future.
Bispecific antibodies have often been used to
activate immune effector cells and their rerout-
ing to cancer cells [83] . An alternative approach
to the use of bispecific antibodies for rerouting
and activation of T cells is the T‑body approach
(i.e., generation of T cells expressing chimeric
receptors consisting of an antibody directed
to a tumor-specific antigen) [88,89] . The small
size and high stability/folding capacity of Nbs
suggest that they could offer advantages over
other antibody formats for the generation of a
T‑body. Proof of concept for Nb-based T‑bodies
has been provided [90] , but further studies are
required to catalyze the translation of this con-
cept into effective therapies.
Conclusion & future perspective
The possibility to rapidly obtain in vivo affin-
ity matured Nbs, combined with their strict
monomeric behavior, and favorable biochemi-
cal and physical properties, make Nbs unique
among multitarget recognition molecules that
are currently available. Therefore, it is expected
that Nbs will make a substantial difference in
therapy, diagnostic screenings and research.
Although no Nb-based product is currently
approved as a therapeutic agent, the fast identi-
fication of a Nb-based lead gives Ablynx‑NV a
substantial advantage, as shown by the various
Nb-based products that are already in the pipe-
line and/or in advanced clinical phases. Besides
Nbs developed to combat cancer, diseases
such as amyloidoses, viral infections or toxin
future science group

envenoming could be treated with future Nb-
based therapeutics. Evidently, the development
of Nbs into therapeutics remains a high-risk,
high-reward business, and commercialization of
Nbs will be faster in the diagnostic field and as
a research tool. For diagnostic applications, Nbs
developed for noninvasive in vivo imaging of
tumors and lesions have definitely a great poten-
tial. In addition, their application as a highly
specific probe on µ‑arrays or in novel biosen-
sors will grow steadily in the very near future.
Finally, as a research tool, Nbs are already quite
useful as crystallization chaperones or when
fluorescently labeled as tracers of antigen traf-
ficking inside living cells at the highest possible
resolution. The Nbs with an intrinsic antigen-
Nanobodies & their potential applications Perspective
Acknowledgements
The authors wish to thank all personnel from our laboratory
and collaborators all over the world for sharing their excit-
ing data and for their much-appreciated contributions in
the past 20 years.
Financial & competing interests disclosure
The authors give special thanks to granting authorities
from the EU (AFFINOMICS project 241481), NATO
(Science for Peace), Belgium (Ministry of Health),
Flanders (IWT and FWO), VIB (Technology funds) and
OZR of Vrije Universiteit Brussel, ‘Vlaamse Liga tegen
Kanker’ and ‘Stichting tegen kanker’ for their financial
support. The authors have no other relevant affiliations or
financial involvement with any organization or entity with
a financial interest in or financial conflict with the subject

f
modulating activity (e.g., enzyme activation matter or materials discussed in the manuscript apart from
or inhibition), sometimes even leading to the those disclosed.

oo
production of functional antigen knockouts will No writing assistance was utilized in the production of
be a valuable research tool for target validation. this manuscript.

Executive summary
Biochemical & biophysical properties of nanobodies
Pr
ƒƒ An optimized technology platform has been developed to identify nanobodies (Nbs) against soluble, properly folded, immunogenic
proteins from immune variable domain of a heavy chain-only antibody phage display libraries.
ƒƒ Such Nbs are naturally endowed with good biochemical and biophysical properties.
Streamlining the Nb identification platform
or

ƒƒ Development of transgenic mice that can be immunized to elicit heavy chain-only antibodies and used to generate Nbs may further
streamline the Nb technology.
ƒƒ Novel immunization protocols to maximally stimulate the heavy chain-only antibodies’ immune response needs to be developed.
ƒƒ Further streamlining of the Nb identification platform for difficult targets (transmembrane biomarkers) is envisaged by DNA
immunization or cell immunization, and panning on cells.
h

ƒƒ Next-generation sequencing technologies should be introduced for rapid target-specific Nb identification obviating the need to
construct immune libraries and panning.
ut

ƒƒ Immune Nb libraries against (sub-)proteomes are predicted to constitute a valuable source for novel target identification, especially
when linked with phenotypic screening technologies that are still in their infancy.
ƒƒ Affinity maturation technologies are available, but introducing Nbs in the phage-assisted continuous evolution technology might be
developed to generate Nbs with novel innate effector functions.
A

ƒƒ Tools need to be developed for obtaining cell-specific and cell-penetrating Nbs for intracellular delivery of therapeutics.
Nbs in research
ƒƒ Nbs are excellent capturing agents to purify targets from complex mixtures or chromatin immunoprecipitation.
ƒƒ Nbs are key molecules as crystallization chaperones.
ƒƒ Nbs are useful tools for tracing and immunomodulation of intracellular antigens at the highest resolution.
Nbs in diagnosis
ƒƒ Nbs have a future as minimal-sized antigen-specific probes in µ-arrays and innovative biosensors.
ƒƒ Nbs are readily labeled and their fast renal clearance makes them ideal tools for noninvasive in vivo imaging.
Nbs for therapy
ƒƒ The very fast blood clearance of Nbs emphasizes the need for technologies to increase the blood half-life of Nbs for therapeutic
purposes.
ƒƒ The small, monomeric format of Nbs makes them a perfect plug-and-play tool to generate bispecific Nbs or larger pluripotent
constructs with an adaptable serum half-life, that are able to combat envenoming with small toxins, viral, bacterial or parasite
infections, and various pathologies.
ƒƒ Generation of Nbs that cross the blood–brain barrier, the attachment of Nbs to lentiviral particles or even on the surface of bacteria, or
creation of Nbs that are secreted by bacteria will create novel therapeutic opportunities.

future science group www.futuremedicine.com 11

Perspective Hassanzadeh-Ghassabeh, Devoogdt, De Pauw, Vincke, & Muyldermans
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