Showing papers by "Serge Muyldermans published in 2021"
TL;DR: A variety of engineering technologies that have been explored to broaden the application range of Nbs are discussed and those applications where designed Nbs might offer a marked advantage over other affinity reagents are summarized.
Abstract: A nanobody (Nb) is a registered trademark of Ablynx, referring to the single antigen-binding domain of heavy chain-only antibodies (HCAbs) that are circulating in Camelidae. Nbs are produced recombinantly in micro-organisms and employed as research tools or for diagnostic and therapeutic applications. They were - and still are - also named single-domain antibodies (sdAbs) or variable domain of the heavy chain of HCAbs (VHH). A variety of methods are currently in use for the fast and efficient generation of target-specific Nbs. Such Nbs are produced at low cost and associate with high affinity to their cognate antigen. They are robust, strictly monomeric and easy to tailor into more complex entities to meet the requirements of their application. Here, we review the various sources and different strategies that have been developed to identify rapidly, target-specific Nbs. We further discuss a variety of engineering technologies that have been explored to broaden the application range of Nbs and summarise those applications where designed Nbs might offer a marked advantage over other affinity reagents.
128 citations
TL;DR: Unique, functional, homodimeric heavy chain-only antibodies, devoid of light chains, are circulating in the blood of Camelidae, and several Nanobody-based constructs have been designed to develop new therapeutic products.
Abstract: Unique, functional, homodimeric heavy chain-only antibodies, devoid of light chains, are circulating in the blood of Camelidae. These antibodies recognize their cognate antigen via one single domain, known as VHH or Nanobody. This serendipitous discovery made three decades ago has stimulated a growing number of researchers to generate highly specific Nanobodies against a myriad of targets. The small size, strict monomeric state, robustness, and easy tailoring of these Nanobodies have inspired many groups to design innovative Nanobody-based multi-domain constructs to explore novel applications. As such, Nanobodies have been employed as an exquisite research tool in structural, cell, and developmental biology. Furthermore, Nanobodies have demonstrated their benefit for more sensitive diagnostic tests. Finally, several Nanobody-based constructs have been designed to develop new therapeutic products.
98 citations
TL;DR: In this paper, the authors assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) forPET imaging of tumor-associated macrophages.
Abstract: Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications. One sentence summary Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.
68 citations
TL;DR: In this article, the authors argue that the number of animals used in immunization campaigns is dwarfed by the number sacrificed during preclinical studies, and that improving quality control of antibodies before entering in vivo studies will have a larger impact on animal consumption.
56 citations
TL;DR: In this article, an alpaca was immunized with an inactivated S. aureus strain followed by the construction of a Nb library from which four target-specific Nbs were retrieved, and a sandwich ELISA employing the Nb147 and biotinylated-Nb147 pair was established to possess a detection limit of 1.4 × 105 colony forming units (CFU)/mL.
32 citations
TL;DR: Single-domain antibodies as discussed by the authors are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum and are very stable and heat-resistant, and hence do not require cold storage.
Abstract: Antibodies have proven to be central in the development of diagnostic methods over decades, moving from polyclonal antibodies to the milestone development of monoclonal antibodies. Although monoclonal antibodies play a valuable role in diagnosis, their production is technically demanding and can be expensive. The large size of monoclonal antibodies (150 kDa) makes their re-engineering using recombinant methods a challenge. Single-domain antibodies, such as "nanobodies," are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum. The immune system of the camelid family (camels, llamas, and alpacas) has evolved uniquely to produce heavy-chain antibodies that contain a single monomeric variable antibody domain in a smaller functional unit of 12-15 kDa. Interestingly, the same biological phenomenon is observed in sharks. Since a single-domain antibody molecule is smaller than a conventional mammalian antibody, recombinant engineering and protein expression in vitro using bacterial production systems are much simpler. The entire gene encoding such an antibody can be cloned and expressed in vitro. Single-domain antibodies are very stable and heat-resistant, and hence do not require cold storage, especially when incorporated into a diagnostic kit. Their simple genetic structure allows easy re-engineering of the protein to introduce new antigen-binding characteristics or attach labels. Here, we review the applications of single-domain antibodies in laboratory diagnosis and discuss the future potential in this area.
26 citations
TL;DR: In this paper, a mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology, as well as their activation is associated with the onset and dissemination of disease.
Abstract: Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the "good guys" by controlling homeostasis, connexin hemichannels are considered as the "bad guys", as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology.
20 citations
TL;DR: In this article, a modified cysteine strategically positioned at the C-terminal end of the nanobody was used for direct immobilization of nanobodies on gold sensors.
Abstract: Single-domain antibodies, known as nanobodies, have great potential as biorecognition elements for sensors because of their small size, affinity, specificity, and robustness. However, facile and efficient methods of nanobody immobilization are sought that retain their maximum functionality. Herein, we describe the direct immobilization of nanobodies on gold sensors by exploiting a modified cysteine strategically positioned at the C-terminal end of the nanobody. The experimental data based on secondary ion mass spectrometry, circular dichroism, and surface plasmon resonance, taken together with a detailed computational work (molecular dynamics simulations), support the formation of stable and well-oriented nanobody monolayers. Furthermore, the nanobody structure and activity is preserved, wherein the nanobody is immobilized at a high density (approximately 1 nanobody per 13 nm2). The strategy for the spontaneous nanobody self-assembly is simple and effective and possesses exceptional potential to be used in numerous sensing platforms, ranging from clinical diagnosis to environmental monitoring.
13 citations
TL;DR: In this paper, an unbiased immunization and selection strategy was employed to select nanobodies (Nbs) recognizing specifically macadamia allergen, as well as to establish a detection method to unveil a protein contamination.
Abstract: Macadamia nut contains important food allergens that potentially cause allergic reactions with severe adverse effects in infants and adults. Reliable and accurate detection of macadamia is critical to avoid allergic reactions. However, knowledge on macadamia allergen is scarce and a reliable detection method has not been reported, yet. In this study, an unbiased immunization and selection strategy was employed to select nanobodies (Nbs) recognizing specifically macadamia allergen, as well as to establish a detection method to unveil a macadamia protein contamination. An alpaca was immunized with a crude protein extract of macadamia followed by construction of a Nb library from its lymphocytes. The panning and screening of this immune Nb repertoire resulted in the selection of six target-specific Nbs. Nb-mediated immuno-capturing combined with mass spectrometry allowed us to identify the target as the macadamia vicilin-like antimicrobial peptides 2-3 (MiAMP2), a novel food allergenic protein abbreviated as Mac i 1. Later on, an immunoassay of a heterologous sandwich ELISA method based on the selected Nb-pairs was established, providing a linear response in the range of 0.442-2,800 μg/mL and with a limit of detection of 27.1 ng/mL. The dedicated immunoassay has been verified by detecting the antigen spiked in food samples. Our study provided evidence for the successful application of the unprejudiced strategy to retrieve Nbs against a priori undefined macadamia allergen. These target-specific Nbs were used to design a highly reliable and effective immunoassay.
11 citations
TL;DR: In this paper, a set of nanobodies, single-domain antibody fragments derived from camelid heavy chain-only antibodies, was generated against recombinant SIRPa and characterized in terms of affinity for the recombinant antigen and binding specificity on cells.
Abstract: Glioblastoma (GBM) is the most common malignant primary brain tumor. Glioblastomas contain a large non-cancerous stromal compartment including various populations of tumor-associated macrophages and other myeloid cells, of which the presence was documented to correlate with malignancy and reduced survival. Via single-cell RNA sequencing of human GBM samples, only very low expression of PD-1, PD-L1 or PD-L2 could be detected, whereas the tumor micro-environment featured a marked expression of signal regulatory protein alpha (SIRPa), an inhibitory receptor present on myeloid cells, as well as its widely distributed counter-receptor CD47. CITE-Seq revealed that both SIRPa RNA and protein are prominently expressed on various populations of myeloid cells in GBM tumors, including both microglia- and monocyte-derived tumor-associated macrophages (TAMs). Similar findings were obtained in the mouse orthotopic GL261 GBM model, indicating that SIRPa is a potential target on GBM TAMs in mouse and human. A set of nanobodies, single-domain antibody fragments derived from camelid heavy chain-only antibodies, was generated against recombinant SIRPa and characterized in terms of affinity for the recombinant antigen and binding specificity on cells. Three selected nanobodies binding to mouse SIRPa were radiolabeled with 99mTc, injected in GL261 tumor-bearing mice and their biodistribution was evaluated using SPECT/CT imaging and radioactivity detection in dissected organs. Among these, Nb15 showed clear accumulation in peripheral organs such as spleen and liver, as well as a clear tumor uptake in comparison to a control non-targeting nanobody. A bivalent construct of Nb15 exhibited an increased accumulation in highly vascularized organs that express the target, such as spleen and liver, as compared to the monovalent format. However, penetration into the GL261 brain tumor fell back to levels detected with a non-targeting control nanobody. These results highlight the tumor penetration advantages of the small monovalent nanobody format and provide a qualitative proof-of-concept for using SIRPa-targeting nanobodies to noninvasively image myeloid cells in intracranial GBM tumors with high signal-to-noise ratios, even without blood-brain barrier permeabilization.
11 citations
06 Jan 2021
TL;DR: In this paper, the authors focus on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discuss several therapeutic nanobody directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.
Abstract: Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.
TL;DR: In this article, the authors developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT.
Abstract: Multiple myeloma (MM) is a hematological malignancy characterized by the presence of clonal plasma cells in the bone marrow niche. Despite significant therapeutic advances, MM remains incurable for the majority of patients. Targeted radionuclide therapy (TRNT) has emerged as a promising treatment option to eradicate residual cancer cells. In this study, we developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT. We first validated CS1 as potential target for TRNT. CS1 is expressed in normal and malignant plasma cells in different disease stages including progression and relapse. It is expressed in dormant as well as proliferating MM cells, while low expression could be observed in environmental cells. Biodistribution studies demonstrated the specific uptake of anti-CS1 sdAbs in tissues of 5TMM cell infiltration including bone, spleen and liver. TRNT using anti-CS1 sdAbs labeled with actinium-225 significantly prolonged survival of syngeneic, immunocompetent 5T33MM mice. In addition, we observed an increase in CD8+ T-cells and more overall PD-L1 expression on immune and non-immune cells, implying an interferon gamma signature using actinium-225 labeled CS1-directed sdAbs. In this proof-of-principle study, we highlight, for the first time, the therapeutic potential and immunomodulating effects of anti-CS1 radionuclide therapy to target residual MM cells.
TL;DR: In this article, a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine.
Abstract: Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naive mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.
TL;DR: In this article, a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens was proposed.
Abstract: Detection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens.
TL;DR: In this article, the Oxford nanopore MinION sequencing was used to generate complete genomes of PPRV isolates from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively.
Abstract: Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.
TL;DR: In this paper, the authors have immunized an alpaca with lupine protein extracts and retrieved nanobodies (Nbs) against β-conglutin and confirmed the immunoassay was confirmed by detecting the samples with spiked allergen.
Abstract: The declaration of lupine supplements is mandatory to avoid lupine allergy for sensitive individuals. However, reliable detection methods against lupine allergen remain critical to prevent the unintended consumption of allergen contaminated food. In this study, we have immunized an alpaca with lupine protein extracts and retrieved nanobodies (Nbs). Nevertheless, the target antigen has been recognized as Lup an 1, which has been classified as β-conglutin, and confirmed to connect with lupine allergy. After selection of the best Nb-pair, a sandwich enzyme-linked immunosorbent assay (ELISA) has been developed providing a linear range of 0.036–4.4 μg/mL with detection limit of 1.15 ng/mL. This immunoassay was confirmed by detecting the samples with spiked allergen, and a recovery from 86.25% to 108.45% with coefficient of variation (CV) less than 4.0% has been determined. Generally, this study demonstrated the selection of Nbs against allergen with crude protein content to develop the immunoassay for lupine surveillance in foods.
TL;DR: The isolation and characterization of llama-derived Nanobodies recognizing the major viral viroplasm component, P9-1 are described, and fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections.
Abstract: Mal de Rio Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
TL;DR: In this paper, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α.
Abstract: Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.
TL;DR: In this article, an immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobbody gene.
Abstract: Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.
TL;DR: In this article, the authors describe the generation of 38 nanobodies that can be subdivided into five CDR3 families, which are recombinant Ag-binding fragments derived from the variable part of H chain-only Abs occurring in Camelidae.
Abstract: IL-13 is a pleiotropic cytokine mainly secreted by Th2 cells. It reacts with many different types of cells involved in allergy, inflammation, and fibrosis, e.g., mastocytes, B cells, and fibroblasts. The role of IL-13 in conditions involving one or several of these phenotypes has therefore been extensively investigated. The inhibition of this cytokine in animal models for various pathologies yielded highly promising results. However, most human trials relying on anti-IL-13 conventional mAbs have failed to achieve a significant improvement of the envisaged disorders. Where some studies might have suffered from several weaknesses, the strategies themselves, such as targeting only IL-13 using conventional mAbs or employing a systemic administration, could be questioned. Nanobodies are recombinant Ag-binding fragments derived from the variable part of H chain-only Abs occurring in Camelidae. Thanks to their single-domain structure, small size (≈15 kDa), good stability, and solubility, they can be engineered into multispecific constructs for combined therapies or for use in new strategies such as formulations for local administration, e.g., pulmonary administration. In this study, we describe the generation of 38 nanobodies that can be subdivided into five CDR3 families. Nine nanobodies were found to have a good affinity profile (KD = 1-200 nM), but none were able to strongly inhibit IL-13 biological activity in vitro (IC50 > 50 µM: HEK-Blue IL-13/IL-4 cells). Multimeric constructs were therefore designed from these inhibitors and resulted in an up to 36-fold improvement in affinity and up to 300-fold enhancement of the biological activity while conserving a high specificity toward IL-13.