A single-domain antibody (sdAb), also known as a Nanobody, is an
antibody fragment consisting of a single
monomericvariable antibody domain. Like a whole antibody, it is able to bind selectively to a specific
antigen. With a molecular weight of only 12–15
kDa, single-domain antibodies are much smaller than common antibodies (150–160 kDa) which are composed of two
heavy protein chains and two
light chains, and even smaller than
Fab fragments (~50 kDa, one light chain and half a heavy chain) and
single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).[1]
The first single-domain antibodies were engineered from
heavy-chain antibodies found in
camelids; these are called VHH fragments.
Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained.[2] An alternative approach is to split the dimeric variable domains from common
immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, Nanobodies derived from light chains have also been shown to bind specifically to target
epitopes.[3]
Camelid Nanobodies have been shown to be just as specific as antibodies, and in some cases they are more robust. They are easily isolated using the same phage panning procedure used for antibodies, allowing them to be cultured in vitro in large concentrations. The smaller size and single domain make these antibodies easier to transform into bacterial cells for bulk production, making them ideal for research purposes.[4]
A single-domain antibody is a
peptide chain of about 110
amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG. These peptides have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards
detergents and high concentrations of
urea. Those derived from camelid and fish antibodies are less
lipophilic and more
soluble in water, owing to their
complementarity-determining region 3 (CDR3), which forms an extended loop (coloured orange in the ribbon diagram above) covering the lipophilic site that normally binds to a light chain.[10][11] In contrast to common antibodies, two out of six single-domain antibodies survived a temperature of 90 °C (194 °F) without losing their ability to bind antigens in a 1999 study.[12] Stability towards
gastric acid and
proteases depends on the amino acid sequence. Some species have been shown to be active in the
intestine after oral application,[13][14] but their low
absorption from the gut impedes the development of systemically active orally administered single-domain antibodies.
The comparatively low
molecular mass leads to a better permeability in tissues, and to a short plasma
half-life since they are eliminated
renally.[1] Unlike whole antibodies, they do not show
complement system triggered
cytotoxicity because they lack an
Fc region. Camelid and fish derived sdAbs are able to bind to hidden antigens that are not accessible to whole antibodies, for example to the active sites of
enzymes.[15] This property has been shown to result from their extended CDR3 loop, which is able to penetrate such buried sites.[11][16][15]
Production
From heavy-chain antibodies
A single-domain antibody can be obtained by immunization of
dromedaries,
camels,
llamas,
alpacas or
sharks with the desired antigen and subsequent isolation of the
mRNA coding for the variable region (VNAR and VHH) of heavy-chain antibodies. Large phage displayed VNAR and VHH single domain libraries were established from nurse sharks[17] and dromedary camels.[18][19] Screening techniques like
phage display and
ribosome display help to identify the clones binding the antigen.[20][17][21][18][22][8][19] The single domain antibodies including VNARs can be humanized for clinical applications.[23]
From conventional antibodies
Alternatively, single-domain antibodies can be made from common
murine,[24] rabbit[25] or human IgG[26] with four chains.[27] The process is similar, comprising gene libraries from immunized or naïve donors and display techniques for identification of the most specific antigens. A problem with this approach is that the binding region of common IgG consists of two domains (VH and VL), which tend to
dimerize or aggregate because of their lipophilicity. Monomerization is usually accomplished by replacing lipophilic by hydrophilic amino acids, but often results in a loss of affinity to the antigen.[28] If affinity can be retained, the single-domain antibodies can likewise be produced in E. coli,[25][26][29]S. cerevisiae or other organisms.
From human single-domain antibodies
Humans occasionally produce single domain antibodies by the random creation of a stop codon in the light chain. Human single-domain antibodies targeting various tumor antigens including mesothelin,[29] GPC2[30] and GPC3[26][31] were isolated by phage display. The HN3 human single-domain antibodies have been used to create immunotoxins [31][32][33] and chimeric antigen receptor (CAR) T cells[34] for treating liver cancer. Blocking the Wnt binding domain of GPC3 by the HN3 human single-domain antibody inhibits Wnt activation in liver cancer cells.[35]
Potential applications
Single-domain antibodies allow a broad range of applications in biotechnical as well as therapeutic use due to their small size, simple production and high affinity.[36][37][15]
Biotechnological and diagnostic
The fusion of a fluorescent protein to a Nanobody generates a so-called
chromobody. Chromobodies can be used to recognize and trace targets in different compartments of living cells. They can therefore increase the possibilities of live cell microscopy and will enable novel functional studies.[38] The coupling of an anti-
GFP Nanobody to a monovalent matrix, called GFP-nanotrap, allows the isolation of GFP-fusion proteins and their interacting partners for further biochemical analyses.[39] Single molecule localization with super-resolution imaging techniques requires the specific delivery of
fluorophores into close proximity with a target protein. Due to their large size the use of antibodies coupled to organic dyes can often lead to a misleading signal owing to the distance between the fluorophore and the target protein. The fusion of organic dyes to anti-GFP Nanobodies targeting GFP-tagged proteins allows nanometer spatial resolution and minimal linkage error because of the small size and high affinity.[40] The size dividend of Nanobodies also benefits the
correlative light-electron microscopy study. Without any permeabilization agent, the cytoplasm of the chemically fixed cells are readily accessible to the fluorophore tagged Nanobodies. Their small size also allows them to penetrate deeper into volumetric samples than regular antibodies. High ultrastructural quality is preserved in the tissue that is imaged by fluorescence microscope and then electron microscope. This is especially useful for the neuroscience research that requires both molecular labeling and electron microscopic imaging.[41]
In diagnostic
biosensor applications Nanobodies may be used prospectively as a tool. Due to their small size, they can be coupled more densely on biosensor surfaces. In addition to their advantage in targeting less accessible epitopes, their conformational stability also leads to higher resistance to surface regeneration conditions. After immobilizing single-domain antibodies on sensor surfaces sensing human
prostate-specific antigen (hPSA) were tested. The Nanobodies outperformed the classical antibodies in detecting clinical significant concentrations of hPSA.[42]
To increase the
crystallization probability of a target molecule, Nanobodies can be used as crystallization
chaperones. As auxiliary proteins, they can reduce the conformational heterogeneity by binding and stabilizing just a subset of conformational states. They also can mask surfaces interfering with the crystallization while extending regions that form crystal contacts.[43][37]
Therapeutic
Single-domain antibodies have been tested as a new therapeutic tool against multiple targets. In mice infected with
influenza A virus subtype H5N1, Nanobodies directed against
hemaglutinin suppressed replication of the H5N1 virus in vivo and reduced morbidity and mortality.[44] Nanobodies targeting the cell receptor binding domain of the
virulence factors toxin A and toxin B of Clostridium difficile were shown to neutralize cytopathic effects in
fibroblastsin vitro.[45] Nanobody conjugates recognizing antigen presenting cells have been successfully used for tumor detection[46] or targeted antigen delivery to generate strong immune response.[47]
Orally available single-domain antibodies against E. coli-induced diarrhoea in piglets have been developed and successfully tested.[14] Other diseases of the
gastrointestinal tract, such as
inflammatory bowel disease and
colon cancer, are also possible targets for orally available single-domain antibodies.[48]
Detergent-stable species targeting a surface protein of Malassezia furfur have been engineered for use in anti-
dandruff shampoos.[10]
As an approach for
photothermal therapy Nanobodies binding to the
HER2 antigen, which is overexpressed in breast and ovarian cancer cells, were conjugated to branched gold nanoparticles (see figure). Tumor cells were destroyed photothermally using a laser in a test environment.[49]
Ablynx expects that their Nanobodies might cross the
blood–brain barrier and permeate into large solid tumours more easily than whole antibodies, which would allow for the development of drugs against
brain cancers.[48]
Nanobodies that tightly bind to the RBD domain of the spike protein of betacoronaviruses (including
SARS-CoV-2 which causes
COVID-19) and blocks interactions of spike with the cell receptor ACE2, has been recently identified[52][18]
Application of various single domain antibodies (Nanobodies) for the prevention and treatment of infection by various highly pathogenic human coronaviruses (HPhCoVs) has been reported. The prospects, potency and challenges of deploying Nanobodies to bind and neutralize SARS-CoV-2 and akin have been recently highlighted.[53]
One of the most common causes of
nagana – Trypanosoma brucei brucei – can be targeted by sdAbs. Stijlemans et al. 2004 succeeded in inducing effective sdAbs from
rabbit and Camelus dromedarius by displaying a
variable surface glycoprotein antigen to the vertebrates' immune systems using a phage. In the future, these therapies will surpass natural antibodies by reaching locations currently unreachable due to natural antibodies' larger size.[54]
^Ghannam A, Kumari S, Muyldermans S, Abbady AQ (March 2015). "Camelid nanobodies with high affinity for broad bean mottle virus: a possible promising tool to immunomodulate plant resistance against viruses". Plant Molecular Biology. 87 (4–5): 355–369.
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^van der Linden RH, Frenken LG, de Geus B, Harmsen MM, Ruuls RC, Stok W, et al. (April 1999). "Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1431 (1): 37–46.
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^Harmsen MM, van Solt CB, Hoogendoorn A, van Zijderveld FG, Niewold TA, van der Meulen J (November 2005). "Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea". Veterinary Microbiology. 111 (1–2): 89–98.
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abHarmsen MM, van Solt CB, van Zijderveld-van Bemmel AM, Niewold TA, van Zijderveld FG (September 2006). "Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy". Applied Microbiology and Biotechnology. 72 (3): 544–551.
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^Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, et al. (November 2006). "Targeting and tracing antigens in live cells with fluorescent nanobodies". Nature Methods. 3 (11): 887–889.
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^Clinical trial number NCT01020383 for "Comparative Study of ALX-0081 Versus GPIIb/IIIa Inhibitor in High Risk Percutaneous Coronary Intervention (PCI) Patients" at
ClinicalTrials.gov