Engineering disulfide bonds within an antibody.

Antibodies have evolved to function in oxidative, extracellular environments. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Immunoglobulin G (IgG) includes several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG sub-classes. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. Ala-Ala, Ala-Val, Val-Ala, and Val-Ala) pairs were preferred for cysteine replacement in the Ig-fold domain. As such, these mutations may be useful for the intracellular use of antibodies. Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. In heavy chain variable or light chain variable domains, the introduction of additional disulfide bonds into the framework region did not reduce antigen-binding affinity, suggesting that generating disulfide bonds may be a method for stabilizing IgG and antibody fragments, such as the antigen-binding fragment, and single-chain and single-domain antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Dual beneficial effect of interloop disulfide bond for single domain antibody fragments.

The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.

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.

A bispecific nanobody to provide full protection against lethal scorpion envenoming.

Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.

Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venom.

Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.

Single-domain antibodies as building blocks for novel therapeutics.

Antibodies are large and complex molecules, with two identical parts that bind independently of each other onto the antigen and the third part of the molecule that dictates the effector function(s). To improve the therapeutic value of antibodies, protein-engineering endeavors reduced the size of the antigen-binding moiety to a single-domain unit. Occasionally, it was demonstrated that the single-domain antigen-binding derivatives of antibodies can have--on their own--an agonistic (or antagonistic) effect on their target. The small size and strict monomeric behavior, in combination with other biochemical properties such as high solubility and high specificity and affinity for the cognate antigen, make single-domain antibodies ideal to design novel man-made conjugates harnessed with innovative effector functions outside the reach of classical antibodies.

VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI'.

Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion envenoming. The most attractive approach consists in generating single-chain antibody fragments (scFv) as their pharmaco-kinetic properties should match closely those of the scorpion toxins. Although high affinity scFv reagents have been generated in the past, their production level, stability, and toxin neutralizing capacity remain disappointingly poor. In the current study, we identified one Nanobody (Nb), a single-domain antigen-binding fragment of a dromedary Heavy-chain antibody (HCAb) that recognizes specifically the Androctonus australis hector AahI' toxin. This Nb has excellent production, stability and solubility characteristics. With this Nb we further manufactured a tandem linked bivalent construct and assembled a HCAb with improved antigen binding due to avidity effects. All these constructs were shown in mouse models to possess a scorpion toxin neutralization capacity that exceeds by far all previous attempts with scFv-based materials, even when used at lower doses. It is therefore clear that in the near future Nanobodies will be at the core of novel serotherapeutics as they combine multiple benefits over other reagents to treat scorpion envenomed patients.

Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils.

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.

Antibody technology in proteomics.

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in proteomics. In the post-genomic era and with high-throughput techniques available, the goal is to discriminate between all individual proteins from the proteome including their splice variants and post-translationally modified derivatives. Aided by advances in generation, selection and engineering of antibody-based recognition units, antibody fragments provide tools for detection of high- as well as low-abundant analytes even in complex, non-fractionated proteomes in conjunction with usage of small amounts of samples and reagents. In addition, large consortia aim at generating vast numbers of antibody-based recognition units suitable for future diagnostics and therapeutics.

Parallel selection of multiple anti-infectome Nanobodies without access to purified antigens.

A strategy was developed to isolate Nanobodies, camelid-derived single-domain antibody fragments, against the parasite infectome without a priori knowledge of the antigens nor having access to the purified antigens. From a dromedary, infected with T. evansi, we cloned a pool of Nanobodies and selected after phage display 16 different Nanobodies specific for a single antigen, i.e. variant surface glycoprotein of T. evansi. Moreover 14 Nanobodies were isolated by panning on different total parasite lysates. Thus, this anti-infectome experiment generated Nanobodies, monospecific for one Trypanosoma species, whereas others were pan-reactive to various Trypanosoma species. Several Nanobodies could label specifically the coat of a set of Trypanozoon species. The recognized target(s) are present in GPI-linked membrane fractions of bloodstream- and fly-form parasites. Due to the omnipresence of these targets on different parasite species and forms, these antibody fragments are a valuable source for validation of novel, not yet identified targets to design new diagnostics and therapeutics.

Flow cytometric enumeration of parasitaemia and haematologic changes in trypanosoma-infected mice.

African trypanosomiasis is a severe parasitic disease affecting both man and livestock. It is crucial to expand our fundamental knowledge of the intimate interactions between trypanosomes and their vertebrate hosts in order to develop new and efficient control strategies. The mouse model of trypanosomiasis is the most popular for research purposes because of all the logistic advantages of using this species. Studies of any aspect of trypanosomiases in the mouse systematically require the quantification of some phenotypic traits which translate its degree of resistance/susceptibility to the disease, as blood cell counts. The present study presents a methodological approach combining everyday microsampling of tail blood and its analysis by flow cytometry. The technical options and conditions permitting a fast, reliable and reproducible daily quantification of erythrocyte, reticulocyte, leucocyte and trypanosome counts in the inoculated mouse were established. The protocol proposed allows the multiplication of blood samplings without being exposed to the time-consuming constraint of visual countings, without causing iatrogenic blood cell alterations in the mouse and without requiring specific anti-trypanosome antibodies.

Antibody Fragments as Probe in Biosensor Development.

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies.

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDR-H loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJmol(-1)), good expression level (5 mgl(-1) in E.coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity.

Antibody repertoire development in camelids.

The humoral immune response of the Camelidae is unique as these animals are the only known mammals that seem to possess functional homodimeric heavy-chain antibodies besides the classical heteromeric antibodies composed of heavy (H) and light (L) chains. By definition, the heavy-chain antibodies lack the L-chain, and it was noticed that their H-chain is devoid of the typical first constant domain (CH1) and contains a dedicated variable domain, referred to as VHH. The VHH exon is assembled from separate V-D-J gene segments. The recombined VHH region is subjected to somatic hypermutations; however, the timing and actual mechanism of the class switch from mu to the dedicated gamma-isotype remains elusive. Interestingly, antigen-specific VHHs are easily retrieved after panning of a phage-displayed rearranged V-gene pool cloned from an immunised camelid. These single-domain antigen binding entities possess a number of biophysical properties that offer particular advantages in various medical and biotechnological applications.

Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays.

Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28 fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10 ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realizes the PSA detection at clinical relevant concentrations.

Improvement of single domain antibody stability by disulfide bond introduction.

The successful medical application of single domain antibodies largely depends on their functionality. This feature is partly determined by the intrinsic stability of the single domain. Therefore a lot of research has gone into the elucidation of rules to uniformly increase stability of antibodies. Recently, a novel intra-domain disulfide bond was independently discovered by two research groups, after either rational design or careful investigation of the naturally occurring camelid antibody repertoire. By introducing this particular disulfide bond within a single domain antibody, the conformational stability can be increased in general. In this chapter it is described how to introduce this extra intra-domain disulfide bond and how to estimate the biophysical and biochemical impact of this cystine on the domain.

Nanobody-based chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP), followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq), is becoming a widely used powerful method for the analysis of the in vivo DNA-protein interactions at genomic scale.The success of ChIP largely depends on the quality of antibodies. Although polyclonal antibodies have been successfully used for ChIP, their production requires regular immunization and they exhibit high aspecificity and batch to batch variability. These problems can be circumvented by generating monoclonal antibodies (mAbs) via hybridoma technology. However, such mAbs do not often capture DNA-protein complexes and are not amenable to engineering. Nanobodies are recombinant single domain antibody fragments derived from camelid Heavy-Chain antibodies. Nanobodies exhibit high affinity and specificity towards their cognate antigens and often capture their target antigens in solution. Moreover, the Nanobody genes can be easily tailored to streamline ChIP.Here, we describe a Nanobody-based ChIP protocol which we have successfully used for genome-wide identification of the binding sites of the low-abundant transcription factor Ss-LrpB from the hyperthermoacidophilic archaeon Sulfolobus solfataricus.

Affinity is an important determinant of the anti-trypanosome activity of nanobodies.

BACKGROUND: The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket. METHODOLOGY/PRINCIPAL FINDINGS: Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (K(D)), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the K(D) remained unaffected. CONCLUSIONS/SIGNIFICANCE: This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.

Generation of anti-infectome/anti-proteome nanobodies.

The immunization of an animal with a whole proteome or the infection of an animal and the screening of the resulting antibody repertoire on either the same or different proteome(s) or the infecting agent(s), omits the laborious steps of recombinant protein expression and purification to obtain multiple antigen binders. This procedure allows the identification of antibodies that are specific to unique or common signatures of different proteomes without prior knowledge of these signatures.Nanobodies are the smallest (15 kDa, 2.2 nm diameter, 4 nm height) in vivo affinity-matured functional antigen-binding entities that are derived from camelid heavy-chain antibodies. Due to their small size, recognition of unique epitopes, high affinity, and easy tailoring, nanobodies are attractive affinity reagents for various applications, including diagnosis and therapy.We detail a protocol to generate, isolate, express, and purify anti-infectome/anti-proteome nanobodies.