Camelid Heavy-chain Antibodies Research Articles

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination

A chip format sandwich-type immunoassay based on Nanobodies® (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200µg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized.

Inhibition of biofilm formation by Camelid single-domain antibodies against the flagellum of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell–cell and cell–surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.

Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin

Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000μgL−1 with a detection limit of 0.01μgL−1. The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings.

Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility.

Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

Retroviral transduction of fluonanobody and the variable domain of camelid heavy-chain antibodies to chicken embryonic cells.

Single domain antibodies from camel heavy chain antibodies (VHH or nanobody), are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecular weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals. To study the development of transgenic chicken using a recombinant retrovirus containing fluonanobody. The retrovirus constructs containing nanobody genes along with secretory signals and GFP gene were established and packed. The virus particle containing the obtained fusion gene was injected into the eggs in stage X. Molecular detection and protein analysis was done in the G0 chickens. The rate of hatched chicken after gene manipulation was estimated to be about 33%. Real-Time PCR assay showed that the nanobody along with GFP gene were integrated in cells of 1.2% of chickens. We conclude that although the rate of gene transfer by recombinant viruses in chickens is low, it would be possible to transfect the target camel immunoglobulin gene into chicken genome.

Single‐Domain Antibodies and Their Utility

Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid heavy-chain antibodies. These nature-made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In this unit, we will present and discuss comprehensive and step-by-step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage-display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere.

Novel Therapy Based on Camelid Nanobodies

Nanobodies (Nbs) are small antibody fragments derived from camelid heavy chain antibodies through recombinant gene technology. Their exceptional physicochemical properties, possibility of humanization and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components. Several different therapeutic approaches based on the novel camelid Nbs have been developed to treat a wide range of diseases ranging from immune, bone, blood and neurological disorders; infectious diseases and cancer. This review provides a comprehensive overview of the current state of the use of camelid-derived Nbs as novel therapeutic agents against multiple diseases.

Generation of single-domain antibody multimers with three different self-associating peptides

Conventional Y-shaped antibodies have been widely used in research, diagnostics and therapeutics. Their large size result in disadvantages in certain applications, which makes production difficult. Different parts of an antibody have been used to replace the whole antibody to make it smaller. Single-domain antibodies (sdAbs) derived from the camelid heavy chain antibodies are among the smallest antibody fragments engineered for various applications. To improve the affinity of these single- sdAbs for correspondent antigens and provide suitable size of reagents for various applications, we fused an anti-epidermal growth factor receptor sdAb EG2 to self-associating peptides, RHCC derived from a right-handed coiled-coil peptide of an archaebacterium, COMPcc from human cartilage oligomeric matrix protein and C4bpα derived from human plasma C4-binding protein α-chain, respectively, to make multimeric antibodies. The multimeric antibodies were expressed as soluble cytoplasmic proteins to spontaneously form tetramers, pentamers or heptamers and were purified by affinity chromatography. The avidity of multimeric forms of sdAbs compared with that of the monomeric form of sdAbs was increased without altering binding specificity.

Design and Selection of a Camelid Single-Chain Antibody Yeast Two-Hybrid Library Produced De Novo for the Cap Protein of Porcine Circovirus Type 2 (PCV2)

Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×106 cfu/3 µg and 2×109 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation.

CHARACTERIZATION AND ENZYME-CONJUGATION OF A SPECIFIC ANTI-L1 NANOBODY

Persistent infection of the human papillomaviruses (HPV) has been shown to result in cervical cancer and intraepithelial neoplasia. Early detection and screening programs are essential strategies against cervical cancer. A nanobody is the smallest antigen-binding fragment known and is derived from a camelid heavy-chain antibody. This tiny protein shows high solubility and stability. It can be produced cost-effectively with high yield production. In this study, we enriched a nanobody library against the L1 protein of HPV. Several colons were selected from this enriched library using monoclonal phage-enzyme linked immunosorbent assay (phage-ELISA) and analyzed for identification of nanobody genes. The expression of nanobody fragments was performed in Rosetta gami2. The C74 nanobody that showed strong binding to the L1 protein of HPV16 was selected, purified, and characterized by Western blotting and ELISA. The selected nanobody was tested for sensitivity, specificity, and affinity. A nanobody conjugated to horseradish peroxidase (HRP) was selected and used for detection of L1 protein of HPV16. This study demonstrates that the C74-HRP, due to its specificity and good binding affinity for a specific viral antigen, is a potential diagnostic tool that can be used as a promising reagent for the new generation of HPV diagnosis approaches.

Disulfide linkage engineering for improving biophysical properties of human VH domains

To enhance their therapeutic potential, human antibody heavy chain variable domains (V(H)s) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of V(H) domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (V(H)Hs). Using four model domains we demonstrate that this strategy is also applicable to human V(H) domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of V(H)s by 14-18°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced V(H) aggregation in domains with the Cys54-Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the V(H)s to protein A. This indicates that it may be prudent to use the synthetic V(H) libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders.

Isolation and characterisation of deoxynivalenol affinity binders from a phage display library based on single-domain camelid heavy chain antibodies (VHHs)

Deoxynivalenol (DON), or vomitoxin, is a mycotoxin of the trichothecene group produced by Fusarium species. The present work described construction of a phage display library based on variable domain of heavy chain of HCAbs (VHHs) and panning for binders of DON. Two pair PCR primers designed based on the VHHs conserved domains were used for cloning coding sequences of complementary DNA (cDNA). After total RNA extraction, semi-nested PCR, subcloning and electro transformation, the primary library was generated containing 1.9×107 independent clones. Phage display library was rescued by infection of the primary library with helper phage KM13, and the titre of the library was 5.6×1012 CFU/ml. Three clones with specific DON-binding affinity were obtained after three rounds of panning. Since the library was derived from non-immune animal, it has potential to be used for panning binders of other mycotoxins or low molecular weight haptens. The binders with specific affinity provided new recourses for developing detection methods or tools of mycotoxins in foodstuffs.

High Affinity Nanobodies against the Trypanosome brucei VSG Are Potent Trypanolytic Agents that Block Endocytosis

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood–brain barrier

Previously selected amyloid beta recognizing heavy chain antibody fragments (VHH) affinity binders derived from the Camelid heavy chain antibody repertoire were tested for their propensity to cross the blood-brain barrier (BBB) using an established in vitro BBB co-culture system. Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated by a three amino acid substitutions in its N-terminal domain. Additional studies indicated that the mechanism of transcellular passage of ni3A is by active transport. As VHH ni3A combines the ability to recognize amyloid beta and to cross the BBB, it has potential as a tool for non-invasive in vivo imaging and as efficient local drug targeting moiety in patients suffering from cerebral amyloidosis such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA).

Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel

Brucella are invasive gram-negative bacteria that multiply and survive within eukaryotic cells causing brucellosis. Syrian (and Middle East) health and economy sectors are still affected by this disease causing a serious national problem that needs to be solved. Here, a strategy was developed to introduce a new generation of binders, known as Nanobodies (Nbs) in our combat against Brucella. These Nbs, recombinant single-domain variable fragments derived from camelid heavy-chain antibodies are very stable and highly soluble, making them a useful tool in numerous biotechnological and medical applications. In this work and without having access to purified antigens (Ags), a camel was immunised successfully with heat-killed Brucella melitensis strain Riv1 as demonstrated by the high titer of Ag-specific heavy-chain antibodies in the serum. Lymphocytes of the immunised camel were isolated and their Nb genes were cloned in a relatively large library of 10 8 individual transformants, of which 81% contained an insert with the proper size of a Nb gene. Phage display expression of the Nbs from this library and pannings on the Brucella lysate resulted in a clear enrichment of three distinct Nb-displaying phages (phage-Nbs), referred to as NbBruc01, 02 and 03, with specificity for Brucella. Producing these binders in a pure, soluble form, as well as identifying their specific targets, which are likely to be immunodominant Ags in Brucella, is expected to open wide perspectives for following the vaccination, diagnosis and treatment of brucellosis.

Inherent Anti-amyloidogenic Activity of Human Immunoglobulin γ Heavy Chains

We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of Abeta, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig gamma heavy chains. A gamma(1) heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloid-laden tissue sections. F1 did not bind to native Abeta monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig gamma heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble Abeta oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig gamma heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules.

Effector Functions of Camelid Heavy-Chain Antibodies in Immunity to West Nile Virus

Three classes of IgG have been described for camelids. IgG1 has a conventional four-chain structure, while IgG2 and IgG3 do not incorporate light chains. The structures and antigen-binding affinities of the so-called heavy-chain classes have been studied in detail; however, their regulation and effector functions are largely undefined. The aim of this study was to examine the participation of conventional and heavy-chain IgG antibodies in the camelid immune defense directed against West Nile virus (WNV). We found that natural infection or vaccination with killed WNV induced IgG1 and IgG3. Vaccination also induced IgG1 and IgG3; IgG2 was produced during the anamnestic response to vaccination. When purified IgGs were tested in plaque-reduction neutralization titer (PRNT) tests, IgG3 demonstrated PRNT activities comparable to those of conventional IgG1. In contrast, IgG2 demonstrated only suboptimal activity at the highest concentrations tested. Flow cytometric analysis revealed that macrophages bound IgG1, IgG2, and IgG3. Furthermore, subneutralizing concentrations of all three isotypes enhanced WNV infection of cultured macrophages. Our results document distinctions in regulation and function between camelid heavy-chain isotypes. The reduced size and distinct structure of IgG3 did not negatively impact its capacity to neutralize virus. In contrast, IgG2 appeared to be less efficient in neutralization. This information advances our understanding of these unusual antibodies in ways that can be applied in the development of effective vaccines for camelids.