A comprehensive comparison between camelid nanobodies and single chain variable fragments

Abstract

By the emergence of recombinant DNA technology, many antibody fragments have been developed devoid of undesired properties of natural immunoglobulins. Among them, camelid heavy-chain variable domains (VHHs) and single-chain variable fragments (scFvs) are the most favored ones. While scFv is used widely in various applications, camelid antibodies (VHHs) can serve as an alternative because of their superior chemical and physical properties such as higher solubility, stability, smaller size, and lower production cost. Here, these two counterparts are compared in structure and properties to identify which one is more suitable for each of their various therapeutic, diagnosis, and research applications.

Full text

R E V I E W Open Access
A comprehensive comparison between
camelid nanobodies and single chain
variable fragments
Yasaman Asaadi
1
, Fatemeh Fazlollahi Jouneghani
2
, Sara Janani
2
and Fatemeh Rahbarizadeh
3,4*
Abstract
By the emergence of recombinant DNA technology, many antibody fragments have been developed devoid of
undesired properties of natural immunoglobulins. Among them, camelid heavy-chain variable domains (VHHs) and
single-chain variable fragments (scFvs) are the most favored ones. While scFv is used widely in various applications,
camelid antibodies (VHHs) can serve as an alternative because of their superior chemical and physical properties
such as higher solubility, stability, smaller size, and lower production cost. Here, these two counterparts are
compared in structure and properties to identify which one is more suitable for each of their various therapeutic,
diagnosis, and research applications.
Keywords: Single-chain variable fragment (scFv), camelid VHH, nanobody, single-domain antibody
Background
Antibodies (Abs) are distinguished binding tools for tar-
geting almost any biomarker specifically. Although the
inherent high affinity and specificity of immunoglobulins
(Ig) is achieved by series of somatic hyper mutations and
affinity maturation processes in the B cells, the recom-
binant DNA technology facilitates in-vitro production of
various antibodies for a diverse set of targets [1]. Up to
date, with about 100 FDA-approved antibodies in the
market [2], the monoclonal antibody is a 145 billion dol-
lar industry with 11% growth rate [3].
In structure, immunoglobulin (Ig) consists of two sep-
arate regions that can be dissociated by proteolytic
cleavage with papain and pepsin; namely Fragment
antigen-binding (Fab) domain and fragment
crystallizable (Fc) region. While Fc region initiates bio-
logical processes upon antigen binding, Fab is
responsible for antigen recognition, and the binding spe-
cificity of the whole Ig molecule is solely dependent on
this domain, especially the two variable domains on the
top-variable heavy chain (VH) and variable light chain
(VL) [4]. This modular structure of immunoglobulin en-
abled scientists to introduce many structural modifica-
tions on the structure of Abs to improve their
performance, by means of protein engineering and re-
combinant DNA technology. Smaller antibody fragments
(such as Fab, scFv, diabodies, triabodies, mini bodies,
and single-domain antibodies) are among these modified
structures, designed to be reliable alternatives for con-
ventional antibodies. Their smaller size, superior proper-
ties, and ease of manufacturing while retaining the
targeting specificity of the whole Ig molecule make them
perfect tools for diagnosis and clinical applications [5,6].
Among all the engineered and recombinant antibody
formats, single-chain variable fragments (scFvs) and
camelid heavy-chain variable domains (VHHs) - also
known as nanobodies- are the most popular ones. Previ-
ously scientists considered single-chain variable frag-
ment (scFv) -composed of VH and VL- as the smallest
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: rahbarif@modares.ac.ir
3
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat
Modares University, Tehran, Iran
4
Research and Development Center of Biotechnology, Tarbiat Modares
University, Tehran, Iran
Full list of author information is available at the end of the article
Asaadi et al. Biomarker Research (2021) 9:87
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antibody fragment with the same antigen-binding speci-
ficity to the whole Ig molecule. However, the discovery
of camelid VHH [7] and shark variable new antigen re-
ceptor (VNAR) [8] demonstrated that a single V-like do-
main can retain the affinity of a whole antibody
molecule [9]. Due to the broad and similar applications
of scFv and VHH, this article aims to review the differ-
ences of these two antibody fragments in structure and
function to illustrate whether the superior properties of
nanobodies can make them a capable alternative for
scFvs or not.
Nanobody and scFv in structure
As stated previously, the variable domains of Fab are re-
sponsible for the binding specificity of the whole anti-
body. Therefore, the smallest unit of Ig with antigen-
binding activity is the fragment variable or Fv in which
the two variable domains (VH and VL) connect with a
disulfide bond. ScFv is an engineered form of Fv that, in-
stead of a disulfide bond, the two variable domains are
joined together by a flexible linker (Fig. 1). The length
and amino acid composition of this linker play an im-
portant role in correct folding of the protein [10], and it
is typically 10-25 amino acid long with Glu Lys stretches
to increase the solubility and Gly Ser stretches for the
flexibility of the final protein [11,12]. Within each of the
two variable domains of the scFv, there are three hyper
variable domains or complementary determining regions
(CDRs) that are linked together with framework regions
(FRs). While the CDRs are responsible for antigen bind-
ing, and their structure is complementary to the epitope,
the remainder of the variable domains (FRs) acts as a
scaffold and has inconsiderable variability compared to
CDRs. Interestingly, the contribution of each CDR in
antigen binding is different. For instance, the CDR3 in
the heavy chain has a critical role by 29% contribution
in binding specificity while the involvement of CDR2L is
just 4% [13].
The higher contribution of the VH in antigen binding
raised a research hypothesis that whether a single heavy
chain can retain the parent Ab's binding affinity. In sem-
inal studies, mouse single variable domains were investi-
gated for their functional activity [14]. However, their
troublesome properties, such as low affinity, poor solu-
bility, and higher production cost, hampered their
broader development. The discovery of heavy chain only
antibodies (HCAbs) in camelids [7] and immunoglobulin
new antigen receptor (IgNAR) in cartilaginous fish [15]
was a new beginning in single domain antibody develop-
ment. The antigen-binding domain of these specific im-
munoglobulins (VHH and V-NAR) is a high affinity
single V-like domain that has evolved to be devoid of
the disadvantages of previous single domain fragments.
These superior properties result from major adaptations
in sequence and structure [16–19]. As camelid nanobo-
dies have easier handling, more robust antibody
Fig. 1 The differences of scFv and nanobody in structure. Aconventional IgG structure, scFv structure composed of VH and VL of antibody
joined with a flexible linker, the detailed structure of VH in scFv. Bcamel HCAb structure, nanobody that is derived from camelid VHH, the
detailed structure of VHH
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responses [17,20], and higher yield in recombinant ex-
pression [21] than shark V-NARs, VHH fragments are
more frequently used and will be the focus of this
review.
Similar to VH, camelid VHHs comprise nine beta-
strands forming a typical IgV fold; however, VL loss
caused notable differences between these two fragments,
especially in FR2 and hypervariable loops. In the conven-
tional VH region, the FR2 consist of four highly con-
served hydrophobic amino acids (Val37, Gly44, Leu45,
and Trp47) that in contribution with Gln39, Gly44,
Tyr91, and Trp103 form a conserved hydrophobic inter-
face of ~700 Å
2
to facilitate VL joining [22]. With the
absence of VL in nanobodies, these four hydrophobic
residues are substituted for more hydrophilic amino
acids (Phe37, Glu44, Arg45, and Gly47) [23–25]to
avoid the exposure of such a sizeable hydrophobic re-
gion to solvent (Fig. 1). In addition to this substitu-
tion, residues adjacent to this interface have rotated
their side chains without deforming the Cαbackbone
to increase the VHH surface's hydrophilicity. Further-
more, the CDR3 domain of VHH folds over this
interface to shield the amino acids formerly covered
by the VL partner [26]. These alterations elucidate
the augmented solubility of VHHs in comparison to
thesingleVHdomainandscFvs[27].
In VHHs, extension in hypervariable loops repays the
loss of three VL CDRs and VH–VL combinatorial diver-
sity. Extension of CDR1 and CDR3 provides a 600–800
Å2 antigen-interacting surface as offered by six loops
from the VH–VL domain [24,28]. Furthermore, the
elongated CDR1, with mutational hotspots imprinted in
the VHH germline, compensates for the VL partner's
variability [29] while the somatic mutations in 28 and 30
residues of CDR1 are selected during the affinity matur-
ation process to participate in antigen binding directly
[30,31]. Although the elongated CDR3 can extend into
epitopes that are almost inaccessible for specific anti-
bodies, the enlarged loop suggests broader flexibility, im-
peding antigen-binding entropically [24,28]. To solve
this issue, camellia VHHs evolved with an extra disulfide
bond toward either the CDR1, CDR2, or FR2 [24,28,
32]. All these structural features increase the paratope
diversity and allow for a wide variety of geometrical loop
structures that deviate fundamentally from the canonical
loop structures defined for conventional antibodies and
facilitate the orientation of the CDR3 toward the anti-
gens [33,34].
Nanobody and scFv in properties
As a result of significant structural differences, scFv and
Nb display distinct properties in vitro and in vivo. These
different characterizations are investigated below in
more detail (Table 1).
Size
First and foremost, these two fragments have notable
dissimilarity in their size, while scFv is almost twice the
Nb size by about 30 kDa weight [35]. This smaller size
facilitates VHHs genetic manipulation [36], and the
presence of only three antigen-binding loops allows for
easy enhancement of their intrinsic tendency to antigen
[37]. Due to the renal filtration and degradation, the
smaller size of VHHs also results in their short half-life
in blood [38]. This feature can be helpful since it results
in high tissue permeability but unfavorable because their
molecular weight is below glomerular filtration cutoff
size (65kDa) and makes problems in some clinical ther-
apies requiring antibody circulation over extended
periods.
This limitation has led to the development of half-life
extension strategies that combine VHHs with additional
molecules. One of the most popular ones is addition of
stabilizing groups such as Poly-ethylene glycol (PEG)
molecules that slow down blood clearance rate with high
tumor or any other target site accumulation. Fusion with
long-circulating serum proteins like albumin or albu-
min's specific binders, effectively increase the VHH half-
life in the blood. Fc fusion can also stabilize them in
blood while provoking the immune system to the target
site. Furthermore, Fc or albumin fusion makes the anti-
body fragment size larger and implements FcRn-
mediated recycling to increase the protein half-life in the
blood [39].
Solubility and Stability
As described previously, the substitution of four highly
conserved hydrophobic amino acids for more hydro-
philic residues in VHH leads to significant differences in
properties between Nbs and scFvs. In scFvs, these four
residues (V37, G44, L45, and W47) in FR2 form a hydro-
phobic interface to facilitate VH-VL joining. However,
on the downside, this hydrophobic region lowers scFvs’
solubility, resulting in their high tendency for aggrega-
tion. The substitution of polar and smaller amino acid
residues in this position (F37or Y37, E44, R45, and G47)
makes them more hydrophilic and, consequently, more
soluble than scFvs. Furthermore, this nonpolar to polar
transition leads to the molecular and thermodynamic
stability of VHHs in comparison to scFvs. Therefore,
Nbs are more resistant to chemical denaturants and pro-
tease enzymes [40] and have higher stability under harsh
PH or ionic strength [41]. This higher conformational
stability also stems from the presence of an extra disul-
fide bond, which lowers the probability of heat-induced
aggregation and limits VHHs flexibility [42–47]. Because
of higher stability, they show high refolding efficiency,
which means raising or lowering the sample temperature
does not affect Nb conformation, i.e., it de-binds and
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binds to the target, respectively, without any aggregation
or denaturation [48].
This rigidity in structure is a favorite property in the
clinic since non-native protein aggregation is a common
downside of antibody treatment, raising the immune re-
sponse in severe cases [49,50]. However, although in
scFvs the hydrophobic interface between VL and VH
dampens their stability, this two domain structures make
them more flexible and more advantageous for some
applications.
Production process
Both scFvs and Nbs are generated from immune or
naive libraries that will be screened to discover high-
affinity fragments against our desired target. Despite sig-
nificant similarities in library screening, the mechanism
of library construction is less challenging for Nbs. VHH
libraries are created from blood serum of either naive or
immunized camelids. After mRNA isolation, cDNA is
reverse transcribed by reverse transcription-polymerase
chain reaction (RT-PCR). This pool of VHH sequences
is applied to construct a library screened by versatile dis-
play technologies such as phage display to discover a
specific VHH for any potential antigen [51]. Although
the inaccessibility of a camel, dromedary, llama, or al-
paca to immunize is a bottleneck, HCAbs generating
transgenic mice, or commercial naïve or synthetic Nb li-
braries can circumvent this issue.
For scFvs, libraries are typically created from either
naive or immunized murine or human. Similar to VHH,
VH, and VL genes obtain from RT-PCR [52–55], but in
contrast to Nbs, an extra step is required to connect VH
and VL cDNAs through SOE-PCR. Pairing these frag-
ments is a challenging step in the library construction of
scFvs due to the low efficiency of this technique [56,57].
Since the mispaired VH and VL developed by this
method may not detect and bind to the target antigen
[58].
After isolation of a specific scFv or nanobody, attaining
a high expression yield is challenging, especially for
scFvs. In scFvs, as discussed earlier, the intrinsic hydro-
phobic interaction between VH and VL domains leads
to their higher tendency to aggregate. Therefore it is
complicated to express them in various expression sys-
tems appropriately. Many methods should be employed
to improve these fragments’stability, such as loop graft-
ing, altering specific positions in their structure, and ran-
dom mutagenesis [59]. Nevertheless, the superior
properties of VHHs, such as their high hydrophilicity
lead to a less demanding production cycle with fewer
steps [60].
Bacterial expression of scFvs can occur in the either
cytoplasmic or periplasmic environment. Each of these
systems has its own limitations for the proper formation
of scFv. On the one hand, while chaperones and disul-
fide isomerases in periplasm are preferable for appropri-
ate folding of scFvs [10], they lead to a lower production
yield [61]. Furthermore, unpaired cysteine residues of
scFvs can form covalent bonds with the periplasmic pro-
tein's cysteine, which results in aggregation [62,63].
Adding a signal peptide such as Pel B should also be
considered to guide scFv into the periplasm [61]. On the
Table 1 A comparison between scFv and Nanobody in properties
Physiochemical properties scFv Nanobody
size 30-35 KDa
2×3 nm
12-15 KDa
1×2.5 nm
Half-life in blood <1h <<1h
Preferred expression system Bacteria yeast
Water solubility + +++
Aggregation ++ −
Stability under harsh condition + +++
Tissue penetration + ++
Paratope diversity + ++
Cavity binding −+
High affinity + +
Ease of manipulation for enhance affinity + ++
Complexity of library construction techniques ++ +
Nonspecific background binding ++ +
Intracellular functionality + ++
Ease of expression + +++
Ability to concatenate + +++
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other hand, the reducing environment of the cytoplasm
hampers disulfide bond formation, which leads to expos-
ure of hydrophobic VH-VL patches to the solvent and
production of insoluble aggregate forms of scFvs called
inclusion bodies. These aggregated products have to be
re-folded in one additional time-consuming, costly, and
ineffective step with the need of denaturing agents like
urea [64].
Another approach to lower scFv aggregation promoted
by inter-domain hydrophobic interaction is the
humanization of scFvs by replacing hydrophobic amino
acids with hydrophilic residues to prevent accumulation.
Although protein solubility has improved by this
method, these replacements can also have minor effects
on the antigen-binding affinity of the final scFv [65]. The
single entity nature of VHH makes their production
much easier besides scFvs. In VHH cytoplasmic expres-
sion, there are no such hydrophobic- interaction-related
issues [66], which lower production costs [10].
scFvs are not efficiently expressed in the yeast expres-
sion system as well. Due to the higher hydrophobicity of
scFvs, they cannot be produced with proper folding in
Saccharomyces cerevisiae's Endoplasmic Reticulum (ER)
[67]. Some extra refolding steps, such as co- or over-
expression of chaperons, should be applied to overcome
these limitations [68]. In contrast, S. cerevisiae is the
best expression system for VHH [69], as organelles like
ER or Golgi ensure proper disulfide bonds and glycosyla-
tion [70]. Because of these difficulties, E.coli remains the
best host for scFv expression [10,64,68].
Immunogenicity
One of the significant drawbacks of scFvs is their rodent
origin, as the Hybridoma technique is only well devel-
oped for mice and rats and not for humans [71]. Murine
VL and VH exhibit only 53% and 51% sequence identity,
respectively, with corresponding regions in humans [72],
while nanobodies display high sequence similarity with
human VH (VH3 gene family) with ~75–90% identity
correlated with their Low immunogenicity in clinical ap-
plications [73,74]. As a result, the humanization process
is more straightforward in VHHs. Even after the
humanization of murine-derived scFv, the variable re-
gions of scFv can elicit an anti-idiotypic response be-
cause eliminating critical residues in this region may
affect antigen binding [75–77]. Also, scFvs’engineering
for reducing human anti-mouse antibody (HAMA) re-
sponses [78] will inactivate [79] injected scFvs and lessen
their clinical effectiveness [80,81], and allergic reactions
will arise in repeated administration [78,79]. Further-
more, humanization reduces the binding affinity of these
fragments [10,65], and CDR grafting may represent new
immunogenic epitopes [75–77,82]. In general, the
humanization of murine-derived scFvs can overcome
these immunogenicity problems to some extent but not
entirely [59].
Affinity
Although scFvs and Nbs provide similar affinity, they
show a distinct preference for epitopes. Nbs have better
access for grooves and clefts on the surface of antigens
like ion channels [83], viral glycoproteins [84], or im-
mune synapses [85], but scFvs prefer flat linear epitopes.
These differences result from the longer CDR3 loop in
Nb, allowing for a highly convex shape to access concave
epitopes. Nbs also show a good affinity for flat epitopes
suggesting that these fragments can form various inter-
face complexes. Furthermore, nonspecific background
binding is lower for nanobodies in comparison to scFvs
[37].
Nanobody and scFv in application
In recent years, by the rapid progress in antibody frag-
ments engineering, the smaller size of scFv and VHH
makes them suited for a broad range of applications ran-
ging from therapy and diagnosis to research and explor-
ation. Here the various utilizations of these prominent
antibody fragments are discussed in detail to demon-
strate which counterpart is more appropriate for each
application.
Therapeutic applications of Nb and scFv in various
formats
Development in targeted medicine has expanded treat-
ment options, particularly in cancer therapy [45]. ScFv
and VHH as high-affinity antibody fragments play an
important role in targeted medicine [86] and are utilized
in various formats (Fig. 2) as therapeutic options for sev-
eral conditions.
Unconjugated neutralizing agent
ScFv and VHH can be utilized as neutralizing agents
through direct binding and inactivation of foreign parti-
cles such as toxins and viruses [87] and cancer antigens
or disease mediating cytokines and growth factors. These
antibody fragments are administered naked in monova-
lent or multivalent format to block the function of their
target or fused to an Fc region to increase serum half-
life and trigger the immune system.
Nb is mostly a preferable candidate for this concern
because of five main reasons. a) Their smaller size,
resulting in perfect diffusion in tissues and extending
neutralizing performance beyond the vessels. b) Higher
flexibility and binding tendency enabling the bivalent
and trivalent conjugation of Nanobodies and improving
neutralization [88]. c) Their lower immunogenicity and
more straightforward humanization process [89]. d)
High stability that makes VHH production easier. e)
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Structural properties of VHHs that provide the power to
target inaccessible epitopes [90].
Fatal toxins are a neglected but vital health problem
all around the world. Plasma anti-venom serum (PAS)
therapy is passive immunotherapy against toxins, with a
rapid, effective response of direct antibody injection.
Toxin neutralizers need to have rapid diffusion and
clearance to provide enough power to identify and
neutralize toxins with high tissue penetration [89] and
then uptaken by kidneys in a shortened period. There-
fore, antibody fragments such as nanobody and scFv are
suitable for toxin inactivation, and scFvs were success-
fully applied in vitro. In a study done by Miethe and his
colleagues, a neutralizing scFv-Fc inhibited the endopep-
tidase activity of botulinum neurotoxin [91]. In another
paper, scFv was utilized to neutralize scorpion toxin Cn2
[88]. However, these fragments were not effective in vivo
because of their high serum half-life [92]. In contrast,
Nbs, with their smaller size and stronger binding affinity,
have shown promising results in toxin inactivation
in vivo [89]. Anti-scorpions toxin nanobody was able to
protect mice more quickly than PAS therapy from
Androctonus australis hector (Aah) scorpions [88]. In
another study, despite the nanobody mixture failed to
stop the venom lethality, the results proved the efficacy
and usefulness of VHH developed against Bothrops
atrox snake venom [93], which highlights the potential
of VHH as anti-toxin agents.
Antibody fragments can neutralize viruses as well. In
the last decades, one of the most successful approaches
against viral agents was neutralizing monoclonal anti-
bodies, which can suppress viral load partly through
humoral immunity [94]. Nanobodies derived from Cam-
elid and murine scFv are considered candidates for
Fig. 2 Different Formats of Nanobody and scFv in therapeutic applications
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neutralizing viral agents because of their smaller size,
higher solubility, and stability. Virus-neutralizing nano-
bodies have been developed against several animal and
human virus families such as HIV-1 [95], human respira-
tory syncytial virus (hRSV) [96], and H5N1 Influenza
[97]. The glycoprotein envelope of the virus canyons is
filled with VHH, therefore the virus cannot conjugate
with a co-receptor on the cell surface [98]. Bivalent and
trivalent fragments are more efficient in identifying simi-
lar or different epitopes on the envelope glycoproteins,
e.g., trivalent nanobodies against HIV [95] and hRSV
[96] extended the neutralization breadth compared to
the monovalent format. ScFvs are also utilized as viral
neutralizing agents. Recombinant scFv has been utilized
for targeting many viruses such as HIV [99], influenza
[100], Porcine epidemic diarrhea virus (PEDV) [101],
and HPV [102]. However, nanobody is more creditable
because its smaller size provides the power to match and
fill virus canyons more efficiently.
During the current SARS-Co-2 pandemic, anti-viral
agents are one of the considerable therapeutic candi-
dates. The receptor-binding domain (RBD) of the spike
protein of the virus and the human angiotensin-
converting enzyme 2 (ACE2) receptor on the cell surface
are the key components in the viral entry that various
forms of antibody fragments can target. Nanobodies
[103] and scFvs are both appropriate candidates for RBD
blocking, but Nbs are more common. The theranostic
potential of camelid nanobodies in covid-19 infections is
reviewed recently [104,105]. Although a handful of sci-
entific groups are working on finding the best nanobody
set for SARS-cov2 neutralization [106–109], attempts
are still enduring to discover a Recombinant scFv for
this mission [110,111]. Nevertheless, the superior prop-
erties of nanobodies for viral neutralization, such as
lower production cost, the potential for aerosol delivery
because of their high stability, and ease of multimeriza-
tion put them forward in this race.
In terms of cancer immunotherapy, antibody frag-
ments can inhibit tumor evasion by binding to the
tumor itself or blocking the vital component for tumor
growth and invasion. As growth factors play a critical
role in angiogenesis, especially in the tumor microenvir-
onment, blocking them or their cognate receptor can en-
hance tumor regression. Both scFv and VHH are utilized
to bind and neutralize Vascular endothelial growth fac-
tor receptor 2 (VEGFR-2) [112,113]. It is also demon-
strated that the epidermal growth factor receptor
(EGFR) blocked by a monovalent and bivalent nanobody
called CONAN-1 could be a potential anticancer therapy
[114].
Cytokine and chemokine inhibition is a potential
therapeutic option for cancer and autoimmune disease
[115]. Camelid VHHs and scFvs have been utilized for
blocking several inflammatory and immunomodulatory
cytokines as a treatment for rheumatoid arthritis [116]
and chronic inflammation [117,118], autoimmune in-
flammatory diseases [119] as well as cancer [86,120],
and stroke [121]. Cytokine- nanobody complex performs
a sufficient neutralizing capacity when its half-life in-
creases using albumin binding. It may act as a superior
option in reduction of neuroinflammatory response in
brain ischemia by high penetration, even crossing the
brain-blood barrier [121]. Moreover, they can inhibit the
enzymatic activity of their target protein, such as car-
bonic anhydrase (CAIX), by blocking their active site
[122]. However, the efficiency of antibody fragment me-
diated immunotherapy can be further augmented by
their conjugation with other therapeutic payloads dis-
cussed in the next section.
Conjugated to therapeutic payloads
Antibody fragments can be chemically or genetically
conjugated to effector domains to deliver therapeutic
payloads to a specific target, thereby reducing nonspe-
cific toxicity to normal cells. Furthermore, this conjuga-
tion makes them larger, which results in increased
circulation time in the blood [123].
One of the familiar domains that are genetically
fused to these antibody fragments is toxins. Upon
binding to the target cell, the complex is taken in
through endocytosis, and finally, the toxin kills the
desired cell- usually tumor or virally infected tissue
[124]. Both scFv and nanobody have been utilized as
an immunotoxin by conjugation with bacterial toxins
like Enterobacter cloacae β-Lactamase [125]and
Pseudomonas Exotoxin A [126,127], or less immuno-
genic human origin cytotoxic elements such as TRAIL
[128–130]andgranzymeB[131].
Immunocytokines are another group of antibody frag-
ment conjugates that can induce immune cell prolifera-
tion and anti-tumor activity in cancer immune therapy.
Many studies demonstrated the effective function of
these conjugates in specific anti-tumor activity [132–
135]. The most popular cytokines delivered by antibody
fragments are IL-2, IL-12, and TNF, whose systemic ad-
ministration may lead to serious side effects [136].
Although scFv and nanobody can both serve as target-
ing agents for immunotoxins and immune cytokines,
since tissue penetration is critical, especially in cancer,
VHH is a better option for conjugation with larger frag-
ments. However, the larger size of scFvs makes it suit-
able for conjugation with a smaller domain such as
siRNA [137,138]. Nevertheless, the superior physico-
chemical properties, easier humanization and better
antigen recognition properties, and higher stability make
camelid VHH preferable even in siRNA delivery [139].
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Conjugated to immune cell engaging antibody fragments
Due to the critical role of the immune system in cancer,
dual-specific antibody fragments can link between tumor
and immune system components. Bite or bispecific T
cell engagers are the most familiar form of these bispeci-
fic conjugates. By binding to CD3 with one of their rec-
ognition domains, they can activate T cell-mediated
targeted tumor lysis. Bites are historically constructed
from scFvs and scFv based the FDA now approves CD3
× CD19 BiTE to treat B-cell acute lymphoblastic
leukemia [140]. However, camelid VHH can perform in
each binding domain with a smaller size and higher
modularity. Nevertheless, in recent studies, these two
counterparts are mixed. For example, Harwood et al.
created a novel form of T cell engagers named ATTACK
by linking three anti-EGFR nanobodies with an anti-
CD3 scFv as an ideal format for developing the next
generation of T cell-redirecting bispecific antibodies
[141].
Other immune system components such as NK cells
γδTcells and APC can be redirected to the tumor site
similarly by targeting their cognate marker. CD16 for
engaging NK cells [142–144], T cell receptor (TCR) of
Vγ9Vδ2 cells [145] and CD11b for attracting γδTcells,
MHC-II or other specific surface proteins for targeting
APCs [146,147].
Targeting moiety of nanocarriers
Because of the binding specificity of these targeting
agents, nanobodies and scFvs are broadly used in several
drug delivery platforms to deliver their cargo to its spe-
cific location. High penetration, stability, pH-
temperature resistance, and low aggregation are factors
that shed light on VHH importance as a mediating deliv-
ery agent [148]. These delivery systems can overcome
limitations of drug conjugate systems, such as poor
chemical and enzymatic stability, solubility, rapid blood
clearance, and adverse side effects to normal tissues.
These nanocarriers can also release their cargo in more
extended periods to decrease the frequency of drug ad-
ministration [149]. Both VHH and scFv have been
employed as a targeting moiety of nanocarriers such as
liposomes [150–152], micelles [153,154], albumin-based
nanoparticles (NANAPs) [155,156], and polymer-based
NPS [157,158]. But studies demonstrate the superiority
of Nb in this regard [151]. In a study conducted by Oli-
veira et al., anti-EGFR-nanobody liposomes downregu-
late EGFR expression while its scFv based counterpart
was unable to do so. The reason behind this effect is
probably the sensitive structure of scFv that loses its
proper folding in the acidic condition of lysosome after
liposome internalization [151]. Furthermore, as these
nanocarriers’primary target is surface receptors that me-
diate cellular internalization [148], Nb, with its smaller
size, is preferable to act as an antagonist for these
receptors.
Targeting moiety of viruses
Viruses are eminent gene therapy agents applied for the
treatment of various diseases and as a vaccine to induce
an immune response against any desired protein. How-
ever, these highly efficient gene delivery systems cannot
perform specifically by only infecting their target cells
[159]. The binding specificity of antibody fragments is
helpful to circumvent this issue. Many lentiviral and
adenoviral vectors have been modified by either Nb or
scFv [159–164], but the smaller size and high stability
make it a better option in this regard. The superiority of
Nb as a binding domain of adenovirus is perfectly inves-
tigated by Poulin et al. the result of this study demon-
strate that the single domain antibody construct was
efficiently incorporated into the Ad capsid and enhanced
virus infection of cells expressing the targeted receptor
while the scFv construct incorporated into the capsid at
a very low level, insufficient to retarget virus infection
[164].
Intrabody
Although most studies investigate the potential of anti-
body fragments in targeting extracellular antigens, most
signaling pathways occur intracellularly [165]. Therefore
targeting these intracellular factors may be effective in
the treatment of various diseases, including cancer [166]
and neurodegenerative conditions [167] such as Hun-
tington's disease [168]. Intrabodies have been developed
to express and operate within the cell and bind to an
intracellular protein specifically. Because the reducing
environment of cytoplasm is the main playground of
intrabodies, stability under harsh conditions is the factor
that makes Nbs outshine all the other antibody frag-
ments as well as scFvs. However, the therapeutic poten-
tial of antibodies is hindered by the lack of an effective
delivery system [165]. Viral delivery of intrabody coding
sequence to the target cell is the most eminent method
[167]. However, bacterial systems such as the type III se-
cretion system (T3SS) of E. coli have been utilized to
translocate the translated form of intrabody into their
eukaryotic target.
Antigen binding domain in synthetic Receptors
Today with the impressive advancement in synthetic
biology, a handful of synthetic receptors have been de-
signed to sense and respond to extracellular signals in a
programmable way [169,170]. CAR (Chimeric antigen
receptor) is the most popular form of these receptors
that recently entered the clinic [171]. Similar to native
receptors, the extracellular domain of these proteins is
responsible for sensing extracellular factors. To improve
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the system's modularity and enable recognizing any de-
sired factor, the extracellular part of these receptors is
mainly composed of an antibody-derived fragment, such
as scFv or nanobody. Although scFv is the most widely
used form of the extracellular domain [169,172], the su-
perior properties of nanobody and its smaller size make
it a preferable option in this position [172,173]. In sev-
eral studies, camelid VHHs have proven their efficiency
as the antigen-binding moiety of CAR T cells [172–179].
Furthermore, the utilization of a bispecific nanobody-
based construct in the receptor's extracellular domain
broadens CAR T cell therapy specificity by recognizing
two distinct antigens simultaneously [180].
Camellia VHH can serve as antigen binding domain in
other less popular receptors as well. For instance, nano-
bodies have been utilized in the recently designed Syn-
Notch receptor [181], GEMA [182], and C-STAR [183].
These new studies suggest that maybe in the future,
nanobodies can take the position of scFvs in the extra-
cellular domain of synthetic receptors due to their favor-
able properties such as high stability, smaller size, and
low immunogenicity.
Diagnostic applications of Nb and scFv
Although antibody fragments are well known for their
therapeutic application, the binding specificity of these
targeting agents suit them for several diagnosis applica-
tions. Not only in medical diagnosis but also environ-
mental and food analysis applications. Their broad
utilization in diagnosis is summarized in the following
paragraphs.
Molecular imaging
Antibody fragments can serve as specific molecular
probes for the detection of various disease-related anti-
gens. Especially in cancer treatment, molecular imaging
is critical for the early detection of tumor progression or
monitoring the success or failure of the therapy in can-
cer patients. mAbs were the first high-affinity probes in
molecular imaging, but their weak tumor penetration
and high serum half-life lead to low contrast images with
a low tumor to background ratio [184]. Smaller antibody
fragments could circumvent this issue; however, Nb, as
the smallest antibody-derived targeting domain, is super-
ior in this regard. Compared with scFv and Fab, Nbs
were shown to be promising probes for molecular im-
aging due to high tumor uptake, rapid blood clearance,
low liver uptake, and high stability [185]. However, the
rapid renal clearance of Nbs result in high signals in the
kidneys and bladder, therefore they are not favorable for
imaging at their nearby sites [37].
To date, several imaging techniques have been devel-
oped and applied for clinical application, and each of the
antibody fragments is labeled with different agents to be
visualized in that specific imaging technique [186–188].
Positron emission tomography (PET), single-photon
emission computed tomography (SPECT) are the most
popular imaging technique with many radionucleotides
labeled nanobodies in clinical trials [189].
Immunoassays
ecause of the antigen-binding activity of immunoglob-
ulins, the various format of the antibody-based immune
assay, such as enzyme-linked immunosorbent assay
(ELISA) and lateral flow immune assays (LFA) is de-
signed for detection of various factors in medical diagno-
sis and environmental and food analysis. The mAb is the
widely used targeting agent in various immunoassays,
but high stability, lower production cost, and the ability
to recognize the epitopes out of reach for larger frag-
ments make Nbs a favorable immurement [184].
Nobody-based immune assays have shown promising re-
sults in detecting T. solium [190] and Trypanosoma
[191], with better outcomes than their whole Ab
counterparts.
To date, several formats of nanobody and scFv based
immunoreagents have been utilized for detecting micro-
organisms, natural proteins, and chemicals in various
specimens [192–201]. These detection systems are also
packages as a point of care detection device known as a
biosensor to enable portability, easier handling, and de-
crease production costs [202].
Research applications of Nb and scFv
The application of antibody fragments is not limited to
theranostics. These high-affinity binders are a proficient
tool in research applications to understand the structure
and function of proteins. Therefore, in this part, we
mainly mention the use of Nbs and scFvs in fundamen-
tal research.
Protein visualization studies
Due to inefficient folding and chain assembly of scFvs,
Nbs are better candidates to be stably expressed and
then trace proteins in living cells [48,203].i.e., reducing
intracellular environment hampers proper folding and
formation of disulfide bonds [204] in scFvs, results in
their poor function and stability [205,206], .therefore
their use as intrabodies is restricted, and just a few of
them have been used in fundamental biology researches.
Nevertheless, Nbs do not show this limitation and can
fold accurately inside the cells, which favors their use as
a research tool for versatile applications over scFvs. ,
Furthermore adding a tag to label and visualize cytoplas-
mic proteins by vhhs is less challenging than scFvs as
they show higher stability. Because of the single domain
nature of Nbs, adding a tag does not influence their
Asaadi et al. Biomarker Research (2021) 9:87 Page 9 of 20
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

binding activity [207], while in scFvs, it is harder to add
a tag without affecting the binding affinity
The first method for tracing any desired protein in de-
velopmental biology was fusing it with a fluorescent
protein-like GFP. However, the intracellular expression
of a protein and a fluorescent protein may influence the
correct formation and function of the protein. To over-
come such challenges, chromobodies were developed by
fusing nanobodies with a fluorescent tag like RFP. Al-
though target-specific chrome bodies can circumvent
many challenges in this regard, the production of a spe-
cific nanobody for each target is labor-intensive. Hence
targeting the fusion domains by chrome bodies enables
the tracing of various proteins by a single nanobody. To
date, many tracer nanobodies against small peptide fu-
sions like SunTag and PTM have been developed; how-
ever, anti-GFP VHHs, termed (GFP binding proteins)
GBPs, are extensively employed against GFP fusion pro-
teins [208].
Another method of protein visualization used in fluor-
escence microscopy is imaging in a sandwich format
utilizing a fluorescently labeled secondary antibody to
detect the target bound Nb [209,210]. However, in
super-resolution microscopy, since it is more efficient to
have a less distance between the target molecule and
fluorescent label [211,212], coupling organic dyes to
GBPs can directly visualize any GFP labeled structure
[213,214]. In addition, chromobodies expressed in living
cells can trace endogenous targets, therefore they are
also employed in super-resolution imaging techniques
[215]. In general, compared to scFvs, the smaller size of
VHHs allows for a more accurate determination of the
target's location in microcopy [216].
Protein function studies
Understanding the interactions of a protein to its surround-
ing environment is a critical step in studying the function
of the desired protein. Antibody fragments as high-affinity
binders can serve as a professional tool in studying protein-
protein or even RNA-protein interactions. A study done by
Sheetz et al. evaluated the function of human scFv for un-
derstanding the biogenesis of a subset of oncogenic micro-
RNAs by using an anti-NCL scFv, they demonstrated that
NCL is a critical protein in cancer biogenesis as it reacts
with oncogenic microRNA. These antibody fragments can
also serve as primary antibodies in ELISA [217,218].
Another approach for study protein-protein interaction is
GBP-based fluorescent-three-hybrid. In this approach, GBP
is first coupled with an intracellular anchoring protein
resulting in its fixation in the predetermined subcellular
compartment. Then two target proteins which each one is
fused with a fluorescent tag, interact with the central GBP
binding protein. Therefore a GFP–RFP colocalization signal
is produced at the location of GBP, which can be
monitored to improve our understanding of protein-
protein interaction [219].
Another method to understand the function of a pro-
tein is inactivation by degradation or interfering in the
function, therefore the role of knockout protein in the
pathway can be revealed in its absence. Unlike RNAi,
Nanobodies, by their smaller size, can bind to the ef-
fector domain of desired proteins to defunctionalize that
particular domain and enable the fundraising of its role
in the process. In contrast, by RNA interference, the
function of the whole protein would be disrupted, and
the role of each domain will remain unclear [220].
Nbs can knock out their target protein by inducing
protein degradation [221–224]. For instance, GBP nano-
bodies were combined with the F-box domain (which is
a component of the SKP1–CUL1–F-box (SCF) ubiquitin
E3 ligases complex) and so initiated polyubiquitination,
which results in degradation of intracellular GFP-fused
protein through proteasome [225]. Another approach is
adding a PEST motif to Nb so that the target antigen
will undergo a proteasomal degradation pathway [226].
Another step to studying a protein function is under-
standing its location of function. GBP nanobodies com-
bined with monomeric RFP can localize GFP fusion
proteins as they bind to GFP, and the resulting GFP–
chromobody shows the location of GFP fusion proteins
in nuclei, cytoplasm, or even membrane [227]. Nb is also
a valuable tool to impose a new location for a protein of
interest inside a cell. Nb fusion to localization domains
redirect the POI to a new cellular compartment leading
to its relocalization. In this manner, the role of location
on protein function during animal development can be
understood. If POI is tagged with a fluorescent molecule,
this relocalization can also be monitored by fluorescent
microscopy [219]. For instance, Nb coupled to KDEL
peptide keeps its antigen inside the endoplasmic
reticulum and coupling Nb with lamin molecule locate
the antigen to the nuclear membrane [228]. Adding a
tag to Nb can also immobilize the antigen is side the cell
membrane and prevent its diffusion outside the cell
[215,229,230]. By preventing the spread of secreted
proteins, their effects on neighbor cells can be identified
in developmental biology [231].
Nanobodies can also act as a scaffold that binds to two or
more different targets in a non-overlapping manner. These
target proteins can be DNA binding domains, and tran-
scriptionfactorsasa‘transcription device dependent on
GFP’(T-DDOG) [232] controls gene expression. This tool
utilizestwoGBPs,oneofthesenanobodiesisequipped
with a DNA-binding domain, and the other joined with the
activation domain of the viral protein VP16. In GFP ex-
pressing living cells, these two Nbs come together in the
presence of the GFP, leading to the desired gene
expression.
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Protein structure studies
Protein crystallography is one of the standard methods
for understanding the structure of any desired protein.
However, because proteins are highly dynamic, they are
bound to crystallization chaperons for reduced conform-
ational heterogeneity. Antibody fragments are superior
among various binding partners since they can be engi-
neered to target almost any desired protein [233]. In
scFvs, the hydrophobic interface between VL and VH
dampens their stability, and this two domain-structure
make them more flexible than single-domain fragments
like VHHs. This feature hampers their utility as a
crystallization chaperon to understand an unknown and
challenging protein [37].
In contrast, Nbs lower intrinsic flexibility show effect-
ive crystal formation, and it is easier to identify the con-
formation and mechanism of epitope binding by them
rather than scFvs. VHHs can reveal protein conforma-
tions in each folding step, so transient intermediates can
be determined even of highly dynamic protein [234]
without inducing any out-of-native structure. To date,
the structure of many proteins has been determined by
VHHs, including high-value G protein-coupled receptor
[235] and amyloid proteins in different pathological con-
ditions [236,237].
Affinity purification and Immunoprecipitation
Due to Nbs superior properties, including small size, mono-
valent mode, and easy directional immobilization to solid
substrates, they can purify an increased amount of biomole-
cules in chromatography [24,28,238]. De Genest et al. de-
veloped VHHs against Glu–Pro–Glu–Ala (EPEA) in highly
efficient affinity chromatography for any EPEA-tagged pro-
tein purification [239]. Nbs show lower nonspecific
Table 2 Some examples of various formats of scfvs in the market or clinical trails
Format Antibody
fragment
Target Disease Clinical
trials
Phase Status Last
update /
approval
date
Sponser
Unconjugated
neutralizing agent
Brolucizumab Vascular
endothelial
growth factor A
(VEGFA)
neovascular age-
related macular de-
generation (AMD)
NCT03386474 3 FDA
approved
Oct 2019 Novartis
Emicizumab Factor VIII Hemophilia A NCT02795767 3 FDA
approved
Oct 2018 Hoffmann-La
Roche
Dinutuximab GD2 neuroblastoma NCT03098030 2&3 FDA
approved
Apr 2015 United
Therapeutics
Conjugated to
immune cell
engaging antibody
fragments
Blinatumomab CD19 Lymphoblastic
Leukemia
NCT02393859 3 FDA
approved
Mar 2018 Amgen
Duvortuxizumab CD19, CD3E B-cell Malignancies NCT02454270 1 Terminated Dec 2018 Janssen
Research &
Development
Targeting moiety of
nanocarriers
SGT 53 transferrin
receptor (TfR)
Solid Tumors NCT02340156 2 terminated Mar 2021 SynerGene
Therapeutics
SGT-94 transferrin
receptor (TfR)
Solid Tumors NCT01517464 1 completed Apr 2017 SynerGene
Therapeutics
MM-302 HER2 Breast Cancer NCT02213744 2&3 terminated Jan 2017 Merrimack
Pharmaceuticals
Antigen binding
domain in synthetic
Receptors
Ciloleucel
Yescarta
CD19 Diffuse large B-cell
lymphoma (DLBCL)
NCT03391466 3 FDA
approved
Oct 2017 Gilead
Tisagenlecleucel
Kymirah
CD19 Non-Hodgkin
Lymphoma
NCT03570892 3 FDA
approved
Aug 2017 Novartis
Brexucabtagene CD19 Relapsed/Refractory
Mantle Cell
Lymphoma
NCT02601313 2 FDA
approved
Jul 2020 Gilead
Lisocabtagene
Maraleucel
CD19 Non-Hodgkin, B-
Cell, Large B-Cell
lymphoma
NCT03483103 2 FDA
approved
Feb 2021 Celgene
idecabtagene
vicleucel
CD19 Multiple Myeloma NCT03361748 2 FDA
approved
Aug 2021 Celgene
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Table 3 Different format of Nbs in clinical trials or market
Format Antibody
fragment
Target Disease Clinical
trials
Phase Status Last update
/ approval
date
Sponser
Unconjugated
neutralizing agent
ALX-0081
Caplacizumab
von
Willebrand
Factor
Thrombocytopenic
Purpura
NCT02553317 3 FDA
approved
Feb 2019 Ablynx
ALX-0061
Vobarilizumab
Interleukin 6
receptor
(IL6R)
Systemic lupus
erythematosus
NCT02437890 2 Completed Feb 2019 Ablynx
Rheumatoid
Arthritis
NCT02287922 2 Completed Aug 2019
ALX-0651 CXCR4 Healthy Volunteers NCT01374503 1 Terminated Apr 2012 Ablynx
PF-05230905 CXCR4 Healthy NCT01284036 1 Completed Jan 2013 Ablynx
ALX-0171 respiratory
syncytial
virus (RSV)
RSV infection NCT02979431 2 Completed Oct 2019 Ablynx
ALX-0761 IL17A, IL17F
and IL17A/F
Psoriasis NCT03384745 2 Completed Aug 2021 Avillion
MSB0010841 IL17A/F Psoriasis NCT02156466 1 Completed Jan 2017 Merck
ATN-103
Ozoralizumab
TNF Rheumatoid
Arthritis
NCT01063803 2 Completed Apr 2016 Ablynx
KN035 PD-L1 Breast cancer
Hepatocellular
carcinoma
NCT04034823 2 Not yet
Recruiting
July 2019 3D Medicines
(Sichuan)
KN044 CTLA-4 Advanced Solid
Tumors
NCT04126590 1 Recruiting July 2021 Changchun
Intellicrown
Pharmaceutical
M6495 ADAMTS-5 Osteoarthritis, Knee NCT03583346 1 Completed Jan 2020 Merck
LMN-101 Flagellin
filament
protein
Campylobacter
Infections
NCT04182490 2 Not yet
recruiting
Aug 2020 Lumen Bioscience
VHH batch
203027
Rotavirus Rotavirus Diarrhoea NCT01259765 2 Completed Aug 2011 International Centre
for Diarrhoeal Disease
Bangladesh
n3088
n3130
Colchicine
SARS Cov2 COVID-19 NCT04322682 3 Completed Jan 2021 Montreal Heart
Institute
TAS266 DR5 Solid tumors NCT01529307 1 Terminated Dec 2020 Novartis
BI 836880 VEGF/Ang2 Neoplasm
Metastasis
NCT03697304 2 Recruiting Aug 2021 Boehringer Ingelheim
Conjugated to
therapeutic
payloads
L-DOS47 CEACAM6 Pancreas cancer
Lung
adenocarcinoma
NCT04203641
NCT03891173
2 Recruiting Apr 2021 Helix BioPharma
Antigen binding
domain of Synthetic
receptors
TC-210 T Cells Mesothelin Mesothelioma
Cholangiocarcinoma
Ovarian Cancer
Non Small Cell Lung
Cancer
NCT03907852 1&2 Recruiting June 2021 TCR2 Therapeutics
BCMA CAR T
cell
BCMA Myeloma NCT03664661 1 Unknown Sep 2018 Henan Cancer Hospital
CD19/20
bispecific CAR
T cell
CD19 and
CD20
B-Cell Lymphoma NCT03881761 1 Recruiting Mar 2019 Henan Cancer Hospital
αPD1-MSLN
CAR T cell
MSLN Colorectal cancer
Ovarian cancer
NCT04503980 1 Recruiting Aug 2020 Shanghai Cell Therapy
Group
Molecular imaging 131I-SGMIB
Anyi-HER2
VHH1
HER2 Breast cancer NCT02683083 1 Completed Aug 2019 Precirix
Asaadi et al. Biomarker Research (2021) 9:87 Page 12 of 20
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background binding than larger antibody formats like scFvs;
besides, their high refolding efficiency and denaturation re-
sistance allows for repeated column regeneration with only
milder elution buffer, which later matters for sensitive tar-
gets [240]. Nanobodies can precipitate serum immunoglo-
bins like IgG, so with their help, we can purify different
types of antibodies from the blood. For example, Klooster
et al. could purify human protein HSA and IgG from blood
with the help of Nbs [241]. Furthermore, they are also per-
fect candidates for antibody-based slide, bead arrays [238],
and chromatin immunoprecipitation with DNA microarray
(chIP-on-chip) to discover new transcription factor-binding
sites [242].
Conclusion and future perspective
Antibodies as well-known targeting moieties are utilized
for a handful of applications. The emergence of recom-
binant DNA technology enabled the design and con-
struction of various antibody fragments, including
Camelid nanobody and scFv. These two Ab fragments
are the most widespread ones with a broad range of ap-
plications. Here we compared these fragments in struc-
ture and properties to investigate which of these
antigen-binding domains is preferable for each applica-
tion. In General, the higher stability, solubility, and lower
production cost of Nb make it favorable in almost all ap-
plications, while its smaller size acts as a double-edged
sword.
In applications that favor rapid clearance of targeting
agents, such as molecular imaging or anti-venom ther-
apy, the smaller size of nanobody is advantageous. While
in other therapeutic utilizations that require persistence
in the body, including neutralizing foreign invaders or
targeting cancer, half-life extension strategies should be
employed to increase the size of the targeting agent. On
the downside, this size increment is against tissue per-
meability which is a critical property in cancer treat-
ment. Therefore balancing the tradeoff between serum
half-life and tissue penetration is necessary to achieve an
optimized size for cancer antibody-based therapeutics.
Another issue that makes choosing between these two
counterparts controversial is their preferred antigenic
site. In other words, because of the structural difference
between scFv and nanobodies, they prefer different epi-
topes while binding to the same antigen. Nbs are high-
affinity binders for grooves and clefts, while scFvs prefer
flat linear epitopes. Therefore, the antigen structure is
an essential factor to decide which one is a better candi-
date for targeting the desired protein.
In spite of the superior properties of Nbs, scFvs are
still dominant in the clinic with about ten FDA ap-
proved products and more than 80 ongoing clinical
trials (Table 2). However, in less than 30 years since
the discovery of HCAb, the superior properties of this
single domain targeting agent make it a pioneer in
the field of recombinant Ab engineering. In 2019 the
first Nanobody entered the clinic and there are more
than 27 Nb based drug candidate in clinical trials,
waiting for FDA approval (Table 3). Nevertheless, be-
cause of the long history behind scFvs and other frag-
ments derived from conventional Ab, nanobodies still
have a long way to become a dominant player in re-
combinant antibody market.
Abbreviation
Ab: Antibody; Nb: Nanobody; scFv: Single-chain variable fragment;
Ig: Immunoglobulin; Fab: Fragment antigen-binding; Fc: Fragment
crystallizable; VH: Variable heavy chain; VL: Variable light chain; V-NAR: Shark
variable new antigen receptor; CDR: Complementary determining regions;
FR: Framework region; HCAb: Heavy chain only antibody;
IgNAR: Immunoglobulin new antigen receptor; FcRn: Neonatal Fc receptor
Table 3 Different format of Nbs in clinical trials or market (Continued)
Format Antibody
fragment
Target Disease Clinical
trials
Phase Status Last update
/ approval
date
Sponser
68GaNOTA
Anti-HER2
VHH1
HER2 Breast cancer NCT03331601 2 Recruiting Feb 2021 Universitair Ziekenhuis
Brussel
99mTc-NM-02 HER2 Breast cancer NCT04040686 1 Recruiting Dec 2020 Shanghai General
Hospital
99mTc-Anti-
PD-L1
PD-L1 Non-small cell lung
cancer
NCT02978196 1 Completed Aug 2020 Shanghai General
Hospital
99mTc
MIRC208
HER2 Cancer NCT04591652 NA Recruiting Oct 2020 Beijing Cancer Hospital
68GaNOTA
Anti-MMR
VHH2
Macrophage
Mannose
Receptor
(MMR)
Breast cancer
Head and Neck
Cancer
Melanoma
Carotid Stenosis
Atherosclerosis of
Artery
NCT04758650 2 Recruiting Feb 2021 Universitair Ziekenhuis
Brussel
Asaadi et al. Biomarker Research (2021) 9:87 Page 13 of 20
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Acknowledgements
Not applicable.
Authors’contributions
Yasaman Asaadi: Conceptualization, Investigation, Writing-original draft,
Writing-review & editing, Validation, Supervision. Fatemeh Fazlollahi: Writing-
original draft, Writing-review & editing, Validation Sara Janani: Writing original
draft, Writing-review & editing, Validation. Fatemeh Rahbarizadeh: Writing-
review & editing, Validation, Supervision. The author(s) read and approved
the final manuscript.
Funding
Not applicable.
Availability of data and materials
Not applicable.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Biotechnology, College of Science, University of Tehran,
Tehran, Iran.
2
Department of Cell & Molecular Biology, Faculty of Life
Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
3
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat
Modares University, Tehran, Iran.
4
Research and Development Center of
Biotechnology, Tarbiat Modares University, Tehran, Iran.
Received: 14 July 2021 Accepted: 22 September 2021
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