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Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment., (© 2024. The Author(s).)

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Matrix metalloproteinase-2 (MMP-2) is an endopeptidase involved in cardiovascular disease and cancer. To date, no highly selective MMP-2 inhibitors have been identified for potential use in humans. Aim of our work was to apply the nanobody technology to the generation of highly selective inhibitors of human MMP-2 and to assess their effects on platelet function and their applicability as conjugated nanobodies. We constructed a nanobody library after immunising an alpaca with human active MMP-2 and identified, after phage display and screening, one MMP-2 inhibitory nanobody (VHH-29), able to hinder the effects of MMP-2 on platelet activation, and one nanobody not inhibiting MMP-2 activity (VHH-136) which, chemically conjugated to a fluorescent probe, allowed the detection of human MMP-2 by flow-cytometry and immune-cytochemistry. In conclusion, we have generated and characterized two new nanotechnological molecular tools for human MMP-2 which represent promising agents for the study of MMP-2 in cardiovascular pathophysiology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human toxocariasis is a zoonosis resulting from the migration of larval stages of the dog parasite Toxocara canis into the human paratenic host. Despite its well-known limitations, serology remains the most important tool to diagnose the disease. Our objective was to employ camelid single domain antibody fragments also known as nanobodies (Nbs) for a specific and sensitive detection of Toxocara canis excretory/secretory (TES) antigens. From an alpaca immune Nb library, we retrieved different Nbs with specificity for TES antigens. Based on ELISA experiments, these Nbs did not show any cross-reactivity with Ascaris lumbricoides, Ascaris suum, Pseudoterranova decipiens, Anisakis simplex and Angiostrongylus cantonensis larval antigens. Western blot and immunocapturing revealed that Nbs 1TCE39, 1TCE52 and 2TCE49 recognise shared epitopes on different components of TES antigen. The presence of disulphide bonds in the target antigen seems to be essential for recognition of the epitopes by these three Nbs. Three separate sandwich ELISA formats, using monovalent and bivalent Nbs, were assessed to maximise the detection of TES antigens in solution. The combination of biotinylated, bivalent Nb 2TCE49 on a streptavidin pre-coated plate to capture TES antigens, and Nb 1TCE39 chemically coupled to horseradish peroxidase for detection of the captured TES antigens, yielded the most sensitive ELISA with a limit of detection of 0.650 ng/ml of TES antigen, spiked in serum. Moreover, the assay was able to detect TES antigens in sera from mice, taken 3 days after the animals were experimentally infected with T. canis. The specific characteristics of Nbs make this ELISA not only a promising tool for the detection of TES antigens in clinical samples, but also for a detailed structural and functional study of TES antigens., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds., Competing Interests: The authors have declared that no competing interests exist.

Campylobacter infections are among the most prevalent foodborne infections in humans, resulting in a massive disease burden worldwide. Broilers have been identified as the major source of campylobacteriosis and reducing Campylobacter loads in the broiler caeca has been proposed as an effective measure to decrease the number of infections in humans. Failure of current methods to control Campylobacter in broilers stresses the urgency to develop novel mitigation measures. We obtained six nanobodies with a broad specificity, that recognize strains belonging to the two most relevant species, Campylobacter jejuni and Campylobacter coli. The target of the nanobodies was identified as the major outer membrane protein, a porin that contributes to bacterial virulence and viability. Multimerization of the nanobodies led to agglutination of C. jejuni cells, which may affect colonization in the chicken gut. These Campylobacter-specific nanobodies may be useful to develop a strategy for preserving chickens from Campylobacter colonization.

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.

Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×10 7 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC 50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC 50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds 'structural' mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hemiscorpius lepturus scorpionism poses one of the most dangerous health problems in many parts of the world. The common therapy consists of using antivenom antibody fragments derived from a polyclonal immune response raised in horses. However, this immunotherapy creates serious side effects, including anaphylactic shock sometimes even leading to death. Thus, many efforts have been made to introduce new replacement therapeutics that cause less adverse reactions. One of the most attractive approaches to replacing the available therapy is offered by single-domain antibody fragments, or nanobodies (Nbs). We immunized dromedaries with H. lepturus toxin and identified a functional recombinant Nb (referred to as F7Nb) against heminecrolysin (HNc), the major known hemolytic and dermonecrotic fraction of H. lepturus venom. This Nb was retrieved from the immune library by phage display selection. The in vitro neutralization tests indicated that 17.5 nmol of the F7Nb can inhibit 45% of the hemolytic activity of 1 EC100 (7.5 μg/ml) of HNc. The in vivo neutralization tests demonstrated that F7Nb had good antihemolytic and antidermonecrotic effects against HNc in all tested mice. Surprisingly, F7Nb (8.75 nmol) neutralized 1 LD100 of HNc (10 μg) via an intracerebroventricular route or 1 LD100 (80 μg) via a subcutaneous route. All of the control mice died. Hence, this Nb is a potential leading novel candidate for treating H. lepturus scorpionism in the near future., (© FASEB.)

One of the main disadvantages of current t-PA thrombolytic treatment is the increased bleeding risk. Upon activation, thrombin activatable fibrinolysis inhibitor (TAFI) is a very powerful antifibrinolytic enzyme. Therefore, co-administration of a TAFI inhibitor during thrombolysis could reduce the required t-PA dose without compromising the thrombolytic efficacy. In this study we generated and characterised a nanobody that is inhibitory towards rat TAFI and evaluated its profibrinolytic property in vitro and in vivo. Nanobody VHH-rTAFI-i81 inhibits (at a 16-fold molar ratio nanobody over TAFI) the thrombin/thrombomodulin (T/TM)-mediated activation of rat TAFI (rTAFI) by 83 ± 1.8% with an IC50 of 0.46 (molar ratio nanobody over TAFI). The affinity (KA) of VHH-rTAFI-i81 for rTAFI, as determined by surface plasmon resonance (Biacore®), is 2.5 ± 0.2 x 10(10) M(-1) and illustrates a very strong binding. In an in vitro clot lysis assay, administration of VHH-rTAFI-i81 strongly enhances the degree of lysis and reduces time to reach full lysis of t-PA-mediated clot lysis. Epitope mapping discloses that Lys392 is of primary importance for the nanobody/rTAFI interaction besides minor contributions of Tyr175 and Glu183. In vivo application of VHH-rTAFI-i81 in a tissue factor-induced mouse thromboembolism model significantly decreases fibrin deposition in the lungs in the absence of exogenous administered t-PA. Nanobody VHH-rTAFI-i81 is a very potent inhibitor of T/TM-mediated TAFI activation. Co-administration of this nanobody and t-PA enhances the fibrinolytic efficacy. In an in vivo mouse thromboembolism model, VHH-rTAFI-i81 reduces fibrin deposition in the lungs.

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.

Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit., Methods: We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading., Results: With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS)., Conclusions: CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.

Background: Gene regulatory processes are largely resulting from binding of transcription factors to specific genomic targets. Leucine-responsive Regulatory Protein (Lrp) is a prevalent transcription factor family in prokaryotes, however, little information is available on biological functions of these proteins in archaea. Here, we study genome-wide binding of the Lrp-like transcription factor Ss-LrpB from Sulfolobus solfataricus., Results: Chromatin immunoprecipitation in combination with DNA microarray analysis (ChIP-chip) has revealed that Ss-LrpB interacts with 36 additional loci besides the four previously identified local targets. Only a subset of the newly identified binding targets, concentrated in a highly variable IS-dense genomic region, is also bound in vitro by pure Ss-LrpB. There is no clear relationship between the in vitro measured DNA-binding specificity of Ss-LrpB and the in vivo association suggesting a limited permissivity of the crenarchaeal chromatin for transcription factor binding. Of 37 identified binding regions, 29 are co-bound by LysM, another Lrp-like transcription factor in S. solfataricus. Comparative gene expression analysis in an Ss-lrpB mutant strain shows no significant Ss-LrpB-mediated regulation for most targeted genes, with exception of the CRISPR B cluster, which is activated by Ss-LrpB through binding to a specific motif in the leader region., Conclusions: The genome-wide binding profile presented here implies that Ss-LrpB is associated at additional genomic binding sites besides the local gene targets, but acts as a specific transcription regulator in the tested growth conditions. Moreover, we have provided evidence that two Lrp-like transcription factors in S. solfataricus, Ss-LrpB and LysM, interact in vivo.

The preparation of a V(H)H (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this V(H)H. IH4, representing 67% of the retrieved V(H)H sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100 mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y52PPE55 sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4-GPA equilibrium is 33 nM. IH4 is a stable protein with a transition melting temperature of 75.8 °C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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

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.

Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen-VHH complexes. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

VHHs or Nanobodies are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1. Interestingly, VHHs showed a potent antipolio activity with EC50 values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs., (Copyright 2010 Elsevier B.V. All rights reserved.)

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Tumors may escape from immune control by the induction of CD11b(+)Gr-1(+) myeloid suppressor cells in the spleen. In this study, we demonstrate that this cell population can be subdivided into a CD11b(hi)Gr-1(int)SSC(lo)Ly6G(neg)M-CSFR(int) immature monocytic fraction and a CD11b(hi+)Gr-1(hi)SSC(hi)Ly6G(hi)M-CSFR(neg) granulocytic fraction. Upon in vitro culture, the monocytic CD11b(+)Gr-1(+) cell fraction is sufficient for cytotoxic T lymphocyte (CTL) suppression, which is linked to the gradual differentiation of these monocytic cells into mature F4/80(+) CD68(+) macrophages. These CTL-suppressive macrophages are alternatively activated (M2), as demonstrated by the expression of known and novel M2 signature genes. In search of M2-associated genes involved in the suppressive activity, it is shown that stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and inhibition of phospholipase A(2) (PLA(2)) activity cooperate to alleviate CTL suppression. Of importance, purified tumor-associated macrophages display a similar M2 phenotype and are suppressive for antitumor CTLs, via a mechanism that can be almost completely reversed by PPARgamma ligands. Overall, our data identify PLA(2) and especially PPARgamma as new potential therapeutic targets to subvert macrophage-mediated CTL suppression in cancer.

Mononuclear phagocytes often function as control switches of the immune system, securing the balance between pro- and anti-inflammatory reactions. For this purpose and depending on the activating stimuli, these cells can develop into different subsets: classically (M1) or alternatively (M2) activated mononuclear phagocytes, the molecular and functional characterization of which is a current topic of investigation. Accumulating evidence suggests that cells of the monocyte/macrophage lineage can be hijacked by tumors for their own benefit. Either as immature cells in the periphery, or as mature macrophages at the tumor site, mononuclear phagocytes are able to influence the behavior of cancer cells, shape the tumor microenvironment and subvert anti-tumor immunity, thereby contributing to tumor growth and progression. This review focuses on the mechanisms behind monocyte/macrophage-mediated tumor promotion and interprets the available data within the M1/M2 conceptual frame.

Depending on the cytokine environment, macrophages can differentiate into distinct subsets that perform specific immunological roles. In this regard, the functions of macrophages activated by interferon gamma, referred to as classically activated macrophages, have been extensively documented, particularly during immune responses to infection. Recently, it was recognized that macrophages exposed to cytokines generated by T helper cell type 2 (Th2) cells exert an alternative activation program. However, the nature and functions of alternatively activated macrophages are ill defined. Evidence for the presence of alternatively activated macrophages and their possible influence in the outcome of several parasite diseases are discussed here.

Because of their ability to inhibit proteases, protease inhibitors have generally been considered to counteract tumor progression and metastasis. However, expression of serine protease inhibitors (SPIs) in tumors is often associated with poor prognosis of cancer patients. Moreover, there is growing evidence that SPIs may even promote malignancy of cancer cells, opening new avenues for their use as biomarkers in malignancy. To isolate cancer promoting genes, we applied the suppression subtractive hybridization method to low-malignant Lewis Lung Carcinoma 3LL-S versus high-malignant 3LL-S-sc cells. This resulted in the identification of the SPI secretory leukocyte protease inhibitor (SLPI), as one of the genes whose expression was higher in 3LL-S-sc than in 3LL-S cells. By stable transfection of 3LL-S cells with mouse or human SLPI, we demonstrated that elevated levels of SLPI expression increased both the tumorigenicity and lung-colonizing potential of 3LL-S cells. Moreover, we showed that this function of SLPI depended on its protease inhibitory capacity. Our results also reveal that although SLPI enhanced the proliferation of 3LL-S cells in vitro, its promalignant activity in vivo was not solely due to its effect on cell proliferation. In this study, we report a causal role for SLPI in the malignant behavior of cancer cells, underscoring the potential malignancy-promoting activities of SPIs.