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Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical fields due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purification. In order to determine the optimal purification strategy for VHH to improve the yield, a method to monitor purification at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purification steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD·mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9-106.0% and 100.7-108.0%. The method was applied to a variety of real samples for the detection of different VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purification protocols especially for nanobodies.

Biotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as heterogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs possess better biodistribution coefficient due to lower molecular weight. They preserve the functional features of mAbs, such as antigen specificity and binding, while at the same time, ensuring much better tissue penetration. An additional benefit of AbFs is the possibility of their production in bacterial and yeast cells due to the small size, more robust structure, and lack of posttranslational modifications. In this review, we described current approaches to the AbF production with recent examples of AbF synthesis in bacterial and yeast expression systems and methods for the production optimization.

Biotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as heterogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs possess better biodistribution coefficient due to lower molecular weight. They preserve the functional features of mAbs, such as antigen specificity and binding, while at the same time, ensuring much better tissue penetration. An additional benefit of AbFs is the possibility of their production in bacterial and yeast cells due to the small size, more robust structure, and lack of posttranslational modifications. In this review, we described current approaches to the AbF production with recent examples of AbF synthesis in bacterial and yeast expression systems and methods for the production optimization.

Biotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as heterogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs possess better biodistribution coefficient due to lower molecular weight. They preserve the functional features of mAbs, such as antigen specificity and binding, while at the same time, ensuring much better tissue penetration. An additional benefit of AbFs is the possibility of their production in bacterial and yeast cells due to the small size, more robust structure, and lack of posttranslational modifications. In this review, we described current approaches to the AbF production with recent examples of AbF synthesis in bacterial and yeast expression systems and methods for the production optimization.

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapp

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a critical role in transducing information into a cell, and as such are important drug targets. Biophysical characterisation and drug discovery against GPCRs is challenging as they express at low levels in recombinant systems and demonstrate low stability when removed from the native lipid bilayer environment, which together, can mean that the routine generation of purified receptor protein is difficult. The inability to routinely generate purified GPCR protein is a bottleneck to the application of many biophysical techniques. In particular, this thesis focused on the application of anti-GPCR biologics discovery. The oxytocin receptor (OTR) and vasopressin 1A receptor (V1AR) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of selective compounds targeting OTR and V1AR, limitted understanding about how these peptides bind and activate their receptors, and a lack of delineation regarding their expression patterns in the brain. There is therefore a need for selective compounds for OTR and V1AR. I propose that OTR and V1AR binding antibodies, and nanobodies are an interesting modality to explore to these ends. Antibodies are an emerging method of targeting GPCRs, both therapeutically, and as tools for biomedical research. Theoretically, antibodies should bind to GPCR subtypes with improved selectivity compared to small molecules, while also providing benefits such as restricted biodistribution, an extended half-life, and additional functionality via the design of antibody-drug conjugates. However, the identification of antibodies against GPCRs has been hindered by the challenges associated with generating GPCR antigens that represent the 3D conformation of the receptor. The ability to generate a sufficient yiel

Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.

Aflatoxin B1 (AFB1) is the most potent genotoxic and carcinogenic aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus. AFB1 is considered a major concern in food safety because of its high toxicity and possible contamination of a variety of food commodities such as cereals, groundnuts, tree nuts, and milk. To guarantee the food safety and address health concerns of consumers, it is important to monitor the contamination of the food chain with this toxin. Screening and regular testing for aflatoxin are mainly done by reference laboratories from the European Union (EU) and the United States (US), using sensitive but low-throughput technologies, thus limiting the frequency of product testing. The problem is still more serious in developing countries, where contamination is often high and rapid field methods are needed. New technologies are needed to provide broader, more thorough and efficient screening for these mycotoxins. In this context, we present the development of a lateral flow immunoassay (LFIA) for the detection of AFB1 as a semiquantitative approach. We have combined the advantages of engineered nanobodies specific to AFB1 with an easy fluorescence read-out system of green fluorescence protein (GFP) fused as a label to a specific nanobody. Optimized LFIA reached a limit of detection (LOD) of 4.80 ng/mL in undiluted almond milk. Because of lifestyle changes, there is an increasing demand for almond milk compared to dairy products. Almond milk is susceptible to contamination with aflatoxin and thus was selected as a food matrix for AFB1 monitoring. The U.S. Food and Drug Administration (FDA) and the European Commission established an action level or maximum residue level of 20 and 8 ng/mL for almond milk, respectively. The developed LFIA has been optimized to distinguish between compliant/noncompliant samples taking into consideration the signal provided by the control line (CL). The CL signal has been set as a threshold of the maximum residue

Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD < 1 nM), covering different antigenic domains as revealed by epitope binning, and including 3 monomers (T6, T15 and T16) that inhibited TeNT binding to neuron gangliosides, were chosen as building blocks to generate 11 VHH multimers. These multimers contained either 1 or 2 different TeNT binding VHHs fused to 1 VHH binding to either albumin (A12) or immunoglobulin (G13) to extend serum half-life in animals. Multimers consisting of 2 TeNT binding VHHs showed more than a 10-fold increase in affinity (KD of 4–23 pM) when compared to multimers containing only one TeNT binding VHH. The T6 and T16 VHHs showed synergistic in vivo TeNT neutralization and, when incorporated into a single VHH trimer (T6T16A12), they showed a very high TeNT neutralizing capacity (1,510 IU/mg).

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Vowel hypo- and hyperarticulation (VHH) was investigated in Swedish infant-directed speech (IDS) to Swedish 12-month-olds using a measure that normalizes across speakers and vowels: the vhh-index. The vhh-index gives the degree of VHH for each individual vowel token, which allows for analysis of the dynamics of VHH within a conversation. Using both the vhh-index and traditional measures of VHH, the degree of VHH was compared between Swedish IDS and ADS. The vowel space area was larger in IDS than in ADS, and the average vhh-index as well as the modal value was higher in IDS than in ADS. Further, the proportion of vowel tokens that were highly hyperarticulated (vhh-index > 75th percentile) were fewer in ADS than in IDS. Vowels in Swedish IDS to 12-month-olds are thus concluded to be hyperarticulated compared to vowels in Swedish ADS, both in terms of degree and frequency. Findings are in line with previous reports on VHH in Swedish IDS as well as on VHH in IDS to infants around 12 months in other languages. The study considers the importance of robust formant estimation, highlights the need for replication of studies on VHH in IDS on previously studied languages and ages, and discusses the benefits of the vhh-index. Those benefits include that it normalizes across speakers and vowels, can be used for dynamic measures within speech samples, and permits analyses on token-level.

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.

BackgroundThe variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health.ResultsVHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL-1 and a limit of detection (LOD) of 0.3 ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively.ConclusionThe VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Lactic acid bacteria (LAB) are among the most widely used microorganisms in food fermentation. However, some LAB species can also be used as live vehicles for the in situ delivery of therapeutic molecules to the mucosa of the human gastrointestinal tract (GIT). Many LAB species have ‘qualified presumption of safety’ status and survive passage through the GIT. Indeed, some are part of the usual GIT microbiota. These are appropriate candidates for the in situ production of recombinant prophylactic and therapeutic proteins. Live recombinant LAB that produce microbial antigens have been shown to elicit an immune response that confers protection against the corresponding pathogens; these LAB could therefore be used as oral vaccines. In addition, some LAB have been genetically engineered to produce therapeutic, neutralizing antibodies. The variable domain of heavy-chain-only antibodies from camelids – known as VHH antibodies or nanobodies – has peculiar properties (nanoscale size, robust structure, acid resistance, high affinity and specificity, easily produced in bacteria, etc.) that make them ideal choices as LAB-produced immunotherapeutic agents. The present review examines the advantages offered by LAB for the in situ production of therapeutic proteins in the human GIT, discusses the use of in situ produced VHH antibody fragments, and assesses the usefulness of this strategy in the treatment of infectious and non-infectious gastrointestinal diseases.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Antibodies display high selectivity and affinity and have been the preferred platform for antigen targeting. Despite the development of antigen-delivery systems that enable T cell activation, targeting approaches that enhance antibody responses need improvement. This need specially applies to poorly antigenic compounds that facilitate pathogen and tumor cells to evade the immune system. Single-domain antibody fragments (VHHs) have been used extensively as neutralizing antibodies in the manner of conventional antibodies, but not yet as an antigen delivery system. The present collection of works investigated whether targeting broadly distinct antigen-presenting cell (APC) populations using VHHs could be used as an antigen-delivery platform to trigger immune responses, deliver drugs to specific cells, and to label cell in vivo for imaging and diagnostics.

Tremendous effort has been expended over the past two and a half decades to understand many aspects of camelid heavy chain antibodies, from their biology, evolution, and immunogenetics to their potential applications in various fields of research and medicine. In this article, I present a historical perspective on the development of camelid single-domain antibodies (sdAbs or VHHs, also widely known as nanobodies) since their discovery and discuss the advantages and disadvantages of these unique molecules in various areas of research, industry, and medicine. Commercialization of camelid sdAbs exploded in 2001 with a flurry of patents issued to the Vrije Universiteit Brussel (VUB) and later taken on by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) and, after 2002, the VIB-founded spin-off company, Ablynx. While entrepreneurial spirit has certainly catalyzed the exploration of nanobodies as marketable products, IP restrictions may be partially responsible for the relatively long time span between the discovery of these biomolecules and their entry into the pharmaceutical market. It is now anticipated that the first VHH-based antibody drug, Caplacizumab, a bivalent anti-vWF antibody for treating rare blood clotting disorders, may be approved and commercialized in 2018 or shortly thereafter. This elusive first approval, along with the expiry of key patents, may substantially alter the scientific and biomedical landscape surrounding camelid sdAbs and pave the way for their emergence as mainstream biotherapeutics.

Nanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain-only antibodies, known as VHH. For selection, libraries of VHH gene segments from naïve, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence-activated cell sorting (FACS; yeast display). This review briefly describes these conventional approaches and focuses on the distinct properties of an E. coli display system developed in our laboratory, which combines the benefits of both phage display and yeast display systems. We demonstrate that E. coli display using an N-terminal domain of intimin is an effective platform for the surface display of VHH libraries enabling selection of high-affinity Nbs by magnetic cell sorting and direct selection on live mammalian cells displaying the target antigen on their surface. Flow cytometry analysis of E. coli bacteria displaying the Nbs on their surface allows monitoring of the selection process, facilitates screening, characterization of antigen-binding clones, specificity, ligand competition and estimation of the equilibrium dissociation constant (KD).

PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer.PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS).RESULTS: Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR.CONCLUSIONS: These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.

[ES] Los venenos contienen mezclas complejas de toxinas cuyo tratamiento efectivo requiere la administración de anticuerpos policlonales. En este trabajo se han producido colecciones de anticuerpos VHH fusionados a HSA y D3-HSA en Nicotiana benthamiana mediante un sistema de expresión transitoria basado en la agroinfiltración de replicones virales, [EN] Venoms comprise complex mixtures of toxins whose efficient neutralization requires the administration of polyclonal antibodies as antidotes. Here we describe the production of anti-venom VHH antibody libraries fused to HSA or D3-HSA in Nicotiana benthamiana plants using a transient expression system based on agroinfiltration of viral replicons.

Este proyecto se encuentra enmarcado en el campo del molecular farming, en el que las plantas se convierten en biofactorias de moléculas específicas, en este caso de mini-anticuerpos de llama (nanobodies o VHHs). Este trabajo busca la expresión en N. benthamiana de diferentes variantes de VHHs frente a la proteína VP6 de rotavirus, usando para ello el sistema de expresión transitoria Magnicon de Icon Genetics. En primer lugar se ensayó la expresión de una VHH denominada 2B3, probando diferentes localizaciones subcelulares. Los resultados se analizaron mediante Western-blot y Coomassie. Viendo que la expresión es posible, se pasó a la búsqueda de nuevas VHHs para su expresión en una misma planta. Se obtuvieron 3 nuevas VHHs, denominadas a1, a3 y b4, que se clonaron y se agroinfiltraron en N. benthamiana, tanto por separado como en conjunto. Para comprobar si tenía lugar la co-expresión simultánea de las 4 VHHs, y dado que poseen diferentes pIs, el resultado se analizó mediante un gel bidimensional. Los resultados obtenidos parecen indicar que es posible co-expresar en una misma planta varios nanobodies de forma simultánea. Además en este punto se procedió a la cuantificación de las VHH, siendo esta de una cantidad de VHH purificada de 41micrag/gFW. Para comprobar la actividad de las diferentes VHHs frente al antígeno VP6 se realizó un test antígeno-ELISA, en el que se obtuvo un resultado positivo con las 4 VHHs. Estos resultados indican que es posible utilizar el sistema Magnifection para la producción de combinaciones de nanobodies en hojas de N. benthamiana.

Este proyecto se encuentra enmarcado en el campo del molecular farming, en el que las plantas se convierten en biofactorias de moléculas específicas, en este caso de mini-anticuerpos de llama (nanobodies o VHHs). Este trabajo busca la expresión en N. benthamiana de diferentes variantes de VHHs frente a la proteína VP6 de rotavirus, usando para ello el sistema de expresión transitoria Magnicon de Icon Genetics. En primer lugar se ensayó la expresión de una VHH denominada 2B3, probando diferentes localizaciones subcelulares. Los resultados se analizaron mediante Western-blot y Coomassie. Viendo que la expresión es posible, se pasó a la búsqueda de nuevas VHHs para su expresión en una misma planta. Se obtuvieron 3 nuevas VHHs, denominadas a1, a3 y b4, que se clonaron y se agroinfiltraron en N. benthamiana, tanto por separado como en conjunto. Para comprobar si tenía lugar la co-expresión simultánea de las 4 VHHs, y dado que poseen diferentes pIs, el resultado se analizó mediante un gel bidimensional. Los resultados obtenidos parecen indicar que es posible co-expresar en una misma planta varios nanobodies de forma simultánea. Además en este punto se procedió a la cuantificación de las VHH, siendo esta de una cantidad de VHH purificada de 41micrag/gFW. Para comprobar la actividad de las diferentes VHHs frente al antígeno VP6 se realizó un test antígeno-ELISA, en el que se obtuvo un resultado positivo con las 4 VHHs. Estos resultados indican que es posible utilizar el sistema Magnifection para la producción de combinaciones de nanobodies en hojas de N. benthamiana.

Este proyecto se encuentra enmarcado en el campo del molecular farming, en el que las plantas se convierten en biofactorias de moléculas específicas, en este caso de mini-anticuerpos de llama (nanobodies o VHHs). Este trabajo busca la expresión en N. benthamiana de diferentes variantes de VHHs frente a la proteína VP6 de rotavirus, usando para ello el sistema de expresión transitoria Magnicon de Icon Genetics. En primer lugar se ensayó la expresión de una VHH denominada 2B3, probando diferentes localizaciones subcelulares. Los resultados se analizaron mediante Western-blot y Coomassie. Viendo que la expresión es posible, se pasó a la búsqueda de nuevas VHHs para su expresión en una misma planta. Se obtuvieron 3 nuevas VHHs, denominadas a1, a3 y b4, que se clonaron y se agroinfiltraron en N. benthamiana, tanto por separado como en conjunto. Para comprobar si tenía lugar la co-expresión simultánea de las 4 VHHs, y dado que poseen diferentes pIs, el resultado se analizó mediante un gel bidimensional. Los resultados obtenidos parecen indicar que es posible co-expresar en una misma planta varios nanobodies de forma simultánea. Además en este punto se procedió a la cuantificación de las VHH, siendo esta de una cantidad de VHH purificada de 41micrag/gFW. Para comprobar la actividad de las diferentes VHHs frente al antígeno VP6 se realizó un test antígeno-ELISA, en el que se obtuvo un resultado positivo con las 4 VHHs. Estos resultados indican que es posible utilizar el sistema Magnifection para la producción de combinaciones de nanobodies en hojas de N. benthamiana.

Diabetes on merkittävä maailmanlaajuinen terveysongelma. On arvioitu, että maailmassa on tällä hetkellä yli 220 miljoonaa diabeetikkoa, joista yli 90 % sairastaa tyypin 2 diabetesta. Tämän sairauden peruspiirre on veren kroonisesti kohonnut glukoosipitoisuus eli hyperglykemia. Tyypin 2 diabetes johtuu pääasiassa insuliiniresistenssistä ja epänormaalista insuliinin erittymisestä. Insuliiniresistenssillä tarkoitetaan tilaa, jossa verenkierron normaali insuliinipitoisuus ei lisää glukoosin kuljetusta solujen sisään insuliinin tärkeimmissä kohdekudoksissa. Lukuisissa tutkimuksissa on osoitettu vapaiden rasvahappojen vaikuttavan insuliinin tehon heikkenemiseen ja siten tyypin 2 diabeteksen muodostumiseen. Proteiinikinaasi C (PKC) isoentsyymit kuuluvat seriini/treoniinikinaasien suurperheeseen, jolla on keskeinen rooli useissa solutason tapahtumissa. PKCε-isoentsyymin aktiivisuuden on todettu vaikuttavan myös insuliiniresistenssiin ja sitä kautta tyypin 2 diabetekseen. Vapaiden rasvahappojen aikaansaaman insuliinireseptorin geenitoiminnan estymisen on yhteydessä PKCε-isoentsyymin fosforylaatioon insuliinin tärkeimmissä kohdesoluissa. Fosforyloitunut PKCε-isoentsyymi saa aikaan insuliiniresepterorin geeniekspression estymisen. Tämä erikoistyö on osa VHH-vasta-aineisiin liittyvää tutkimusta, jonka tarkoituksena on karakterisoida VHH-vasta-aineita ja selvittää niiden vaikutuksia PKC:hen. VHH-vasta-aineet ovat laamoista eristettyjen vasta-aineiden raskaan ketjun vaihtelevia osia ja ne pystyvät sitomaan antigeenin vain yhden vaihtelevan osansa avulla. Erikoistyössä tutkittiin mihin PKCε-isoentsyymin toiminnallisiin domeeneihin VHH-vasta-aineet sitoutuvat. Lisäksi tutkittiin VHH-vasta-aineiden sitoutumista koko PKCε-isoentsyymiin. PKCε-isoentsyymin koko proteiinia ja muita domeeneja tuotettiin Sf9-hyönteissoluissa. Sitoutumista tutkittiin Western blottauksen ja immunopresipitaation avulla. Radioligandisitoutumiskokeissa tutkittiin yhteensä 368 VHH:n sitoutumista domeeni 2-osaan, The burden of diabetes is increasing globally as the number of people with diabetes reaches over 220 million. Over 90 per cent of these people are suffering from type 2 diabetes. This condition is primarily defined by the chronic increase in blood glucose level or hyperglycemia. Type 2 diabetes is characterized by insulin resistance and is usually associated with abnormal insulin secretion. Insulin resistance is a state where normal amount of blood insulin is inadequate to increase glucose uptake in the most important target tissues of insulin. Numerous reports demonstrate that oversupply of lipids leads to loss of insulin activity and the formation of type 2 diabetes. Protein kinase C (PKC) isozymes comprise a family of serine/threoninekinases, which have a regulatory role in a multiple cellular processes. PKC!-isozyme activity is known to play a role in insulin resistance and therefore in type 2 diabetes. Free fatty acid (FFA) induced insulin gene function inhibition is associated with phosphoinositide dependent kinase1 (PDK1) independent phosphorylation of PKC!-isozyme in the most important insulin target tissues. Phosphorylated PKC!-isozyme causes insulinreceptor gene expression inhibition. Present study is part of a VHH-antibodies related research where the goal is to characterize these antibodies and to find out their effects on protein kinase C. VHH-antibodies are Ilama derived antibodies which contain a single heavy-chain variable domain, that is fully capable of antigen binding. In this work, we studied VHH-antibodies binding to PKC!-isozyme and its functional domains. PKC!-isozyme and its domains were produced in Sf9-insect cells. The binding was studied using Western blot and immunoprecipitation assays. In addition, the binding of 368 VHHantibodies to PKCε-isozyme´s domain 2 were studied. With Western blot, it was discovered that E7-VHH-antibody binds to PKCε-isozyme full length and to domain 3. Other VHHantibodies tested in Western blot did not bind to P

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca2+-activated gelsolin (K d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca 2+ (K d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated. © 2010 Birkhäuser Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca2+-activated gelsolin (K d ~10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca 2+ (K d ~5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated. © 2010 Birkhäuser Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published