Nanobody Research Articles

Recombinant Antibody Fragments for Neurological Disorders: An Update.

Recombinant antibody fragments are promising alternatives to full-length immunoglobulins, creating big opportunities for the pharmaceutical industry. Nowadays, antibody fragments such as antigen-binding fragments (Fab), single-chain fragment variable (scFv), single-domain antibodies (sdAbs), and bispecific antibodies (bsAbs) are being evaluated as diagnostics or therapeutics in preclinical models and in clinical trials. Immunotherapy approaches, including passive transfer of protective antibodies, have shown therapeutic efficacy in several animal models of Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), Huntington's disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). There are various antibodies approved by the Food and Drug Administration (FDA) for treating multiple sclerosis and two amyloid beta-specific humanized antibodies, Aducanumab and Lecanemab, for AD. Our previous review summarized data on recombinant antibodies evaluated in pre-clinical models for immunotherapy of neurodegenerative diseases. Here, we explore recent studies in this fascinating research field, give an update on new preventive and therapeutic applications of recombinant antibody fragments for neurological disorders and discuss the potential of antibody fragments for developing novel approaches for crossing the blood-brain barrier (BBB) and targeting cells and molecules of interest in the brain.

Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of thecamelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G4S)2. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×102 CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detectedafter 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16.

Cervical cancer ranks as the third most prevalent malignancy in women worldwide. The persistence of Human papillomavirus (HPV) infection stands out as the foremost risk factor for cervical cancer development. Among the numerous HPV subtypes, HPV16 infection emerges as the primary pathogenic determinant of cervical cancer. To date, no specific drugs have been approved. In this study, we engineered two high-affinity fusion protein targeting HPV16 L1 protein based on the alpaca-derived single-domain antibody 2C12 previously obtained in our laboratory. These two fusion proteins exhibited potent neutralizing activity against HPV16 pseudovirus with IC50 values of 7.8 nM and 6.5 nM, respectively. Molecular docking analysis revealed that 2C12 formed ten pairs of hydrogen bonds with HPV16 L1, among which Arg39 and Thr100 established multiple pairs of hydrogen bonds with HPV16 L1, indicating their crucial roles in antigen-antibody binding process. These structural and biological findings underscore the effective binding capacity of these fusion proteins to HPV16, leading to reduced viral load and providing valuable insights into therapeutic antibody and vaccine development against HPV 16 infection.

Generation of nanobodies with conformational specificity for tau oligomers that recognize tau aggregates from human Alzheimer's disease samples.

Tauopathies are neurodegenerative diseases that involve tau misfolding and aggregation in the brain. These diseases, including Alzheimer's disease (AD), are some of the least understood and most difficult to treat neurodegenerative disorders. Antibodies and antibody fragments that target tau oligomers, which are especially toxic forms of tau, are promising options for immunotherapies and diagnostic tools for tauopathies. In this study, we have developed conformational, tau oligomer-specific nanobodies, or single-domain antibodies. We demonstrate that these nanobodies, OT2.4 and OT2.6, are highly specific for tau oligomers relative to tau monomers and fibrils. We used epitope mapping to verify that these nanobodies bind to discontinuous epitopes on tau and to support the idea that they interact with a conformation present in the oligomeric, and not monomeric or fibrillar, forms of tau. We show that these nanobodies interact with tau oligomers in brain samples from AD patients and from healthy older adults with primary age-related tauopathy. Our results demonstrate the potential of these nanobodies as tau oligomer-specific binding reagents and future tauopathy therapeutics and diagnostics.

Vδ2 T-cell engagers bivalent for Vδ2-TCR binding provide anti-tumor immunity and support robust Vγ9Vδ2 T-cell expansion

BackgroundVγ9Vδ2 T-cells are antitumor immune effector cells that can detect metabolic dysregulation in cancer cells through phosphoantigen-induced conformational changes in the butyrophilin (BTN) 2A1/3A1 complex. In order to clinically exploit the anticancer properties of Vγ9Vδ2 T-cells, various approaches have been studied including phosphoantigen stimulation, agonistic BTN3A-specific antibodies, adoptive transfer of expanded Vγ9Vδ2 T-cells, and more recently bispecific antibodies. While Vγ9Vδ2 T-cells constitute a sizeable population, typically making up ~1-10% of the total T cell population, lower numbers have been observed with increasing age and in the context of disease. MethodsWe evaluated whether bivalent single domain antibodies (VHHs) that link Vδ2-TCR specific VHHs with different affinities could support Vγ9Vδ2 T-cell expansion and could be incorporated in a bispecific engager format when additionally linked to a tumor antigen specific VHH. ResultsBivalent VHHs that link a high and low affinity Vδ2-TCR specific VHH can support Vγ9Vδ2 T-cell expansion. The majority of Vγ9Vδ2 T-cells that expanded following exposure to these bivalent VHHs had an effector or central memory phenotype and expressed relatively low levels of PD-1. Bispecific engagers that incorporated the bivalent Vδ2-TCR specific VHH as well as a tumor antigen specific VHH triggered antitumor effector functions and supported expansion of Vγ9Vδ2 T-cells in vitro and in an in vivo model in NOG-hIL-15 mice.ConclusionBy enhancing the number of Vγ9Vδ2 T-cells available to exert antitumor effector functions, these novel Vδ2-bivalent bispecific T cell engagers may promote the overall efficacy of bispecific Vγ9Vδ2 T-cell engagement, particularly in patients with relatively low levels of Vγ9Vδ2 T-cells.

Safety Assessment of Camelid-Derived Single-Domain Antibody as Feed Additive for Juvenile Whiteleg Shrimp (Litopenaeus vannamei) Against White Spot Syndrome Virus.

A six-week feeding trial was conducted to assess the safety of single-domain antibodies (sdAbs) derived from camelids against the white spot syndrome virus (WSSV) (WSSVvp28 was used as the antigen), focusing on the whole-organism responses and molecular-level changes in juvenile whiteleg shrimp (Litopenaeus vannamei). Five experimental diets with varying levels of sdAbs were formulated: CON (no sdAb supplementation); SDA8.2 (8.20% of sdAbs); SDA16.4 (16.40% of sdAbs); SDA24.6 (24.60% of sdAbs); and SDA32.8 (32.80% of sdAbs). In the CON diet, 450 mL of water per kg of diet (45%) was used to form a feed dough, while sdAbs were used to replace the water in the treatment diets. A total of 450 shrimp, with an initial body weight of 3.27 ± 0.02 g (mean ± SEM), were randomly distributed in 15 tanks (30 shrimp per tank; three tanks per treatment). Each tank was filled with 30 L of seawater (77 L capacity) in an indoor semi-recirculating system with a constant water flow rate of 1.2 L min-1. The photoperiod was maintained at 12 h of light and 12 h of dark. The water temperature, pH, salinity, and dissolved oxygen were 27.3 ± 0.1 °C, 7.61 ± 0.01, 34 ± 1 ppt, and 5.94 ± 0.04 mg L-1, respectively. During the feeding trial, the shrimp were fed the experimental diet (40% protein and 11% lipid) three times a day for six weeks. Following the feeding trial, an acute cold-water-temperature stress test was conducted by abruptly exposing the shrimp from each treatment to 15 °C for 4 h, down from 27 °C. The results showed no significant differences in the growth performance (weight gain, feed utilization efficiency, survival, etc.), plasma metabolites (aspartate aminotransferase activity, alanine aminotransferase activity, total protein, and glucose), or antioxidant enzymes (superoxide dismutase and glutathione peroxidase) among all the experimental diets (p > 0.05). In the acute cold-temperature stress test, there was no significant interaction between sdAb supplementation and temperature stress, nor any main effect from either factor, except for the main effect of temperature stress on the glucose levels, which was significantly higher in shrimp exposed to cold-temperature stress (p < 0.05). The next-generation sequencing of differentially expressed genes (DEGs) in the hepatopancreases of shrimp fed the CON, SDA16.4, and SDA32.8 diets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, indicated that DEGs were significantly enriched in signaling pathways associated with growth, cold stress, and antioxidant systems. Overall, the results from conventional measurements suggest that the use of sdAbs against the WSSV may be safe for juvenile whiteleg shrimp. However, findings from the sophisticated analysis indicate that further research is needed to understand the molecular mechanisms underlying the observed changes, and to evaluate the long-term effects of sdAb supplementation in shrimp diets.

Acetaminophen production in the edible, filamentous cyanobacterium Arthrospira platensis.

Spirulina is the common name for the edible, nonheterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A. platensis, including single-domain antibodies for oral delivery, there remains a need for a modular, characterized genetic toolkit. Here, we independently establish a reproducible method for the transformation of A. platensis and engineer this bacterium to produce acetaminophen as proof-of-concept for small molecule production in an edible host. This work opens A. platensis to the wider scientific community for future engineering as a functional food for nutritional enhancement, modification of organoleptic traits, and production of pharmaceuticals for oral delivery.

PirA- or PirB-binding nanobodies can protect whiteleg shrimp from the acute hepatopancreatic necrosis disease toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a devastating shrimp disease caused by a binary toxin, PirAB, produced by Vibrio parahaemolyticus and other closely related bacteria. To address AHPND, over 300 unique single-domain antibodies (also known as nanobodies) derived from the VHH domains of Lama glama heavy-chain-only antibodies were raised against either PirA or PirB and characterized. Nanobodies were shortlisted based on their affinities for either PirA or PirB, their relative stability in intestinal fluids, and their ability to reduce PirAB-induced death in brine shrimp Artemia salina. From these data, a subset of nanobodies was tested for their ability to reduce AHPND in whiteleg shrimp Penaeus vannamei, and nanobodies targeting either PirA or PirB provided significant disease protection to whiteleg shrimp. These results show that nanobodies can be a new option for shrimp farmers to reduce or eliminate the impact of AHPND on their operations.

Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail

Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD.

The development of single-domain VHH nanobodies that target the Candida albicans cell surface.

Candida albicans causes life-threatening invasive infections that are hard to diagnose and treat, with drug resistance leading to treatment failure. The goal of this study was to develop VHH (single variable domain on a heavy chain) nanobodies to detect drug-resistant infections. Llamas were immunized with a mixture of heat killed and fixed C. albicans cells of different morphologies. Llama lymphocyte RNA was used to generate phage display libraries that were tested for binding to C. albicans cells or cell wall fractions, and single antibody domains were isolated. The libraries were panned against echinocandin-resistant C. albicans isolates and counter-selected against echinocandin-susceptible isolates with the aim of isolating binding domains specific for antigens on drug-resistant cells. Thirty diverse VHH nanobodies were selected, and binding characteristics were assessed via dose-response ELISA. Binding was tested against a variety of C. albicans isolates and other Candida species, indicating that the VHHs were specific for C. albicans. The VHH nanobodies were sorted into four distinct groups based on their binding patterns. Two of the groups bound preferentially to the yeast cell poles and hyphae, respectively. Nanobody binding to C. albicans deletion mutants was tested by fluorescence microscopy and ELISA to identify the antigen targets. VHH19 nanobody, belonging to the largest group, recognized the Als4 adhesin. VHH14 antibody in the hyphae-specific group recognized Als3. None of the isolated VHH nanobodies was selective for drug-resistant clinical isolates. Our data indicate that this approach can generate valuable single-domain antibodies specific to C. albicans proteins.IMPORTANCEThe human fungal pathogen Candida albicans causes a range of diseases from superficial mucosal infections such as oral and vaginal thrush to life-threatening, systemic infections. Accurate and rapid diagnosis of these infections remains challenging, and currently, there are no rapid ways to diagnose drug-resistant infections without performing drug susceptibility testing from blood culture, which can take several days. In this proof-of-concept study, we have generated a diverse set of single domain VHH antibodies (nanobodies) from llamas that recognize and bind specifically to C. albicans cell surface. The nanobodies were classified into four groups based on their binding patterns, for example, cell poles or hyphae. Specific nanobodies were verified as recognizing the important adhesin Als4 or the hyphae associated invasin Als3, respectively. The data validate the approach that small VHH antibody domains hold future promise for diagnostic applications and as probes to study the fungal cell surface.

Nanobodies: from high-throughput identification to therapeutic development.

The camelid single-domain antibody fragment, commonly referred to as a nanobody, achieves the targeting power of conventional monoclonal antibodies (mAbs) at only a fraction of their size. Isolated from camelid species (including llamas, alpacas, and camels), their small size at ∼15 kDa, low structural complexity and high stability compared with conventional antibodies have propelled nanobody technology into the limelight of biologic development. Nanobodies are proving themselves to be a potent complement to traditional mAb therapies, showing success in the treatment of e.g. autoimmune diseases and cancer, and more recently as therapeutic options to treat infectious diseases caused by rapidly evolving biological targets such as the SARS-CoV-2 virus. This review highlights the benefits of applying a proteomic approach to identify diverse nanobody sequences against a single antigen. This proteomic approach coupled with conventional yeast/phage display methods enables the production of highly diverse repertoires of nanobodies able to bind the vast epitope landscape of an antigen, with epitope sampling surpassing that of mAbs. Additionally, we aim to highlight recent findings illuminating the structural attributes of nanobodies that make them particularly amenable to comprehensive antigen sampling and to synergistic activity - underscoring the powerful advantage of acquiring a large, diverse nanobody repertoire against a single antigen. Lastly, we highlight the efforts being made in the clinical development of nanobodies, which have great potential as powerful diagnostic reagents and treatment options, especially when targeting infectious disease agents.

Determination of 3-Phenoxybenzoic Acid by an Improved Enzyme-Linked Immunosorbent Assay (ELISA) by the Immobilization of Nanobodies with Streptavidin and Biotin

Nanobodies (Nbs) are single domain antibodies that are frequently utilized in enzyme-linked immunosorbent assays (ELISAs). However, a major limitation of nanobodies is their low sensitivity in capture assays. To address this issue, the combination of streptavidin and biotin can be employed to immobilize the nanobodies on plates in an oriented manner, thereby exposing their binding sites. In this study, nanobodies specific to 3-phenoxybenzoic acid (3-PBA) were biotinylated at their C-termini and immobilized onto streptavidin-coated microplates. A direct competitive ELISA was developed for the determination of 3-phenoxybenzoic acid in ovine and bovine urine. Compared to directly coating the nanobodies, the streptavidin-bridged nanobody capture-based ELISA demonstrated improved sensitivity, with a 16.6% enhancement, and a half-maximum inhibition concentration (IC50) of 2.05 ng/mL. The developed assay provided a linear range from 0.27 to 20.2 ng/mL (IC20-IC80) and a limit of detection (LOD) of 0.1 ng/mL. The average recoveries of 3-phenoxybenzoic acid spiked in the urine ranged from 82.57% to 102.75% and were consistent with values obtained by liquid chromatography – mass spectrometry (LC-MS). These findings demonstrate that the streptavidin-bridged capture nanobody-based ELISA significantly enhanced the sensitivity for the determination of 3-phenoxybenzoic acid residues in animal urine.

The 1H, 15N, and 13C resonance assignments of a single-domain antibody against immunoglobulin G.

Research on camelid-derived single-domain antibodies (sdAbs) has demonstrated their significant utility in diverse biotechnological applications, including therapy and diagnostic. This is largely due to their relative simplicity as monomeric proteins, ranging from 12 to 15kDa, in contrast to immunoglobulin G (IgG) antibodies, which are glycosylated heterotetramers of 150-160kDa. Single-domain antibodies exhibit high conformational stability and adopt the typical immunoglobulin domain fold, consisting of a two-layer sandwich of 7-9 antiparallel beta-strands. They contain three loops, known as complementary-determining regions (CDRs), which are assembled on the sdAb surface and are responsible for antigen recognition. The single-domain antibody examined in this study, sdAb-mrh-IgG, was engineered to recognize IgG from rats, mice, but it also weakly recognizes IgG from humans (Pleiner et al. 2018). A search of the Protein Data Bank revealed only one NMR structure of a single-domain antibody, which is unrelated to sdAb-mrh-IgG. The NMR chemical shift assignments of sdAb-mrh-IgG will be utilized to study its molecular dynamics and interactions with antigens in solution, which is fundamental for the rational design of novel single-domain antibodies.

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

A single-domain antibody targeting factor XII inhibits both thrombosis and inflammation

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders.

Erratum to the article “Development of technology for culturing a cell line producing a single-domain antibody fused with the Fc fragment of human IgG1”

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Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

ImmunoPET imaging of EpCAM in solid tumours with nanobody tracers: a preclinical study.

Epithelial cell adhesion molecule (EpCAM) is a potential therapeutic target and anchoring molecule for circulating and disseminated tumour cells (CTC/DTC) in liquid biopsy. In this study, we aimed to construct EpCAM-specific immuno-positron emission tomography (immunoPET) imaging probes and assess the diagnostic abilities in preclinical cancer models. By engineering six single-domain antibodies (e.g., EPCD1 - 6) targeting EpCAM of different binding properties and labelling with 68Ga (T1/2 = 1.1 h) and 18F (T1/2 = 110 min), we developed a series of EpCAM-targeted immunoPET imaging probes. The probes' pharmacokinetics and diagnostic accuracies were investigated in cell-derived human colorectal (LS174T) and esophageal cancer (OE19) tumour models. Based on in vitro binding affinities and in vivo pharmacokinetics of the first three tracers ([68Ga]Ga-NOTA-EPCD1, [68Ga]Ga-NOTA-EPCD2, and [68Ga]Ga-NOTA-EPCD3), we selected [68Ga]Ga-NOTA-EPCD3 for tumour imaging which showed an average tumour uptake of 2.06 ± 0.124%ID/g (n = 3) in LS174T cell-derived tumour model. Development and characterisation of [18F]AIF-RESCA-EPCD3 showed comparable tumour uptake of 1.73 ± 0.0471%ID/g (n = 3) in the same tumour model. Further validation of [68Ga]Ga-NOTA-EPCD3 in OE19 cell-derived tumour model showed an average tumour uptake of 4.27 ± 1.16%ID/g and liver uptake of 13.5 ± 1.30%ID/g (n = 3). Near-infrared fluorescence imaging with Cy7-EPCD3 confirmed the in vivo pharmacokinetics and relatively high liver accumulation. We further synthesized another three 18F-labeled nanobody tracers ([18F]AIF-RESCA-EPCD4, [18F]AIF-RESCA-EPCD5, and [18F]AIF-RESCA-EPCD6) and found that [18F]AIF-RESCA-EPCD6 had the best pharmacokinetics with low background. [18F]AIF-RESCA-EPCD6 showed explicit uptake in the subcutaneously inoculated OE19 tumour model with an average uptake of 4.70 ± 0.26%ID/g (n = 3). In comparison, the corresponding tumour uptake (0.17 ± 0.25%ID/g, n = 3) in the EPCD6 blocking group was substantially lower (P < 0.001), indicating the targeting specificity of the tracer. We developed a series of 68Ga/18F-labeled nanobody tracers targeting human EpCAM. ImmunoPET imaging with [18F]AIF-RESCA-EPCD6 may facilitate better use of EpCAM-targeted therapeutics by noninvasively displaying the target's expression dynamics.

A Nanobody of PEDV S1 Protein: Screening and Expression in Escherichia coli.

Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses to the pig farming industry in various countries for a long time. Currently, there are no highly effective preventive or control measures available. Research into the pathogenic mechanism of PEDV has shown that it primarily causes infection by binding the S protein to the CD13 (APN) receptor on the membrane of porcine intestinal epithelial cells. The S1 region contains three neutralization epitopes and multiple receptor-binding domains, which are closely related to viral antigenicity and ad-sorption invasion. Nanobodies are a type of single-domain antibody that have been discovered in recent years. They can be expressed on a large scale through prokaryotic expression systems, which makes them cost-effective, stable, and less immunogenic. This study used a phage display library of nanobodies against the PEDV S1 protein. After three rounds of selection and enrichment, the DNA sequence of the highly specific nanobody S1Nb1 was successfully obtained. To obtain soluble nanobody S1Nb1, its DNA sequence was inserted into the vector Pcold and a solubility-enhancing SUMO tag was added. The resulting recombinant vector, Pcold-SUMO-S1Nb1, was then transformed into E. coli BL21(DE3) to determine the optimal expression conditions for the nanobody. Following purification using Ni-column affinity chromatography, Western blot analysis confirmed the successful purification of S1Nb1 carrying the solubility-enhancing tag. ELISA results demonstrated a strong affinity between the S1Nb1 nanobody and PEDV S1 protein.