Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library.

Immunoglobulin G-based monoclonal antibodies (mAbs) have been effective in treating various diseases, but their large molecular size can limit their penetration of tissue and efficacy in multifactorial diseases, necessitating the exploration of alternative forms. In this study, we constructed a phage display library comprising single-domain antibodies (sdAbs; or "VHHs"), known for their small size and remarkable stability, using a total of 1.6 × 109 lymphocytes collected from 20 different alpacas, resulting in approximately 7.16 × 1010 colonies. To assess the quality of the constructed library, next-generation sequencing-based high-throughput profiling was performed, analyzing approximately 5.65 × 106 full-length VHH sequences, revealing 92% uniqueness and confirming the library's diverse composition. Systematic characterization of the library revealed multiple sdAbs with high affinity for three therapeutically relevant antigens. In conclusion, our alpaca sdAb phage display library provides a versatile resource for diagnostics and therapeutics. Furthermore, the library's vast natural VHH antibody repertoire offers insights for generating humanized synthetic sdAb libraries, further advancing sdAb-based therapeutics.

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects.

Food-borne biotoxins from microbes, plants, or animals contaminate unclean, spoiled, and rotten foods, posing significant health risks. Neutralizing such toxins is vital for human health, especially after food poisoning. Nanobodies (Nbs), a type of single-domain antibodies derived from the genetic cloning of a variable domain of heavy chain antibodies (VHHs) in camels, offer unique advantages in toxin neutralization. Their small size, high stability, and precise binding enable effective neutralization. The use of Nbs in neutralizing food-borne biotoxins offers numerous benefits, and their genetic malleability allows tailored optimization for diverse toxins. As nanotechnology continues to evolve and improve, Nbs are poised to become increasingly efficient and safer tools for toxin neutralization, playing a pivotal role in safeguarding human health and environmental safety. This review not only highlights the efficacy of these agents in neutralizing toxins but also proposes innovative solutions to address their current challenges. It lays a solid foundation for their further development in this crucial field and propels their commercial application, thereby contributing significantly to advancements in this domain.

Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain.

Almost all the neutralizing antibodies targeting the receptor-binding domain (RBD) of spike (S) protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged or emerging variants, such as Omicron and its sub-variants. This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies. Here, we present one nanobody, N235, displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants, including the newly emerged Omicron and its sub-variants. Cryo-electron microscopy demonstrates N235 binds a novel, conserved, cryptic epitope in the N-terminal domain (NTD) of the S protein, which interferes with the RBD in the neighboring S protein. The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex. Furthermore, a nano-IgM construct (MN235), engineered by fusing N235 with the human IgM Fc region, displays prevention via inducing S1 shedding and cross-linking virus particles. Compared to N235, MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro. The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo, suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method.

BACKGROUND: Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII). METHODS: Based on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders. RESULTS: A synthetic phage-displayed VHH library with 1 × 108 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (KD) of 1 × 10-8 M, 5.8 × 10-8 M and 2.6 × 10-7 M. CONCLUSION: PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.

Generation and characterization of a nanobody against the avian influenza virus H7 subtype.

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 µg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Computational Modeling and Characterization of Peptides Derived from Nanobody Complementary-Determining Region 2 (CDR2) Targeting Active-State Conformation of the ß2-Adrenergic Receptor (ß2AR).

This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides (NDPs) targeting active-state ß2-adrenergic receptor (ß2AR) conformation. Sequences of conformationally selective Nbs favoring the agonist-occupied ß2AR were initially analyzed by the informational spectrum method (ISM). The derived NDPs in complex with ß2AR were subjected to protein-peptide docking, molecular dynamics (MD) simulations, and metadynamics-based free-energy binding calculations. Computational analyses identified a 25-amino-acid-long CDR2-NDP of Nb71, designated P4, which exhibited the following binding free-energy for the formation of the ß2AR:P4 complex (ΔG = -6.8 ± 0.8 kcal/mol or a Ki = 16.5 µM at 310 K) and mapped the ß2AR:P4 amino acid interaction network. In vitro characterization showed that P4 (i) can cross the plasma membrane, (ii) reduces the maximum isoproterenol-induced cAMP level by approximately 40% and the isoproterenol potency by up to 20-fold at micromolar concentration, (iii) has a very low affinity to interact with unstimulated ß2AR in the cAMP assay, and (iv) cannot reduce the efficacy and potency of the isoproterenol-mediated ß2AR/ß-arrestin-2 interaction in the BRET2-based recruitment assay. In summary, the CDR2-NDP, P4, binds preferentially to agonist-activated ß2AR and disrupts Gαs-mediated signaling.

Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies.

Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc.

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.

Affinity purification of mAb from serum-containing hybridoma culture supernatant through a novel nanobody that discriminates mouse IgG from bovine IgG by recognizing the mouse kappa constant region (mCK).

When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.

Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions.

The development of bispecific antibodies that bind at least two different targets relies on bringing together multiple binding domains with different binding properties and biophysical characteristics to produce a drug-like therapeutic. These building blocks play an important role in the overall quality of the molecule and can influence many important aspects from potency and specificity to stability and half-life. Single-domain antibodies, particularly camelid-derived variable heavy domain of heavy chain (VHH) antibodies, are becoming an increasingly popular choice for bispecific construction due to their single-domain modularity, favorable biophysical properties, and potential to work in multiple antibody formats. Here, we review the use of VHH domains as building blocks in the construction of multispecific antibodies and the challenges in creating optimized molecules. In addition to exploring traditional approaches to VHH development, we review the integration of machine learning techniques at various stages of the process. Specifically, the utilization of machine learning for structural prediction, lead identification, lead optimization, and humanization of VHH antibodies.

An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies.

The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2µm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Proteomics Platform Reveals Broad-Spectrum Nanobodies for SARS-CoV-2 Variant Neutralization.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of different variants of concerns with immune evasion that have been prevalent over the past three years. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, have garnered interest in developing neutralizing antibodies due to their smaller size, structural stability, ease of production, high affinity, and low immunogenicity, among other characteristics. In this work, we describe an integrated proteomics platform for the high-throughput screening of nanobodies against different SARS-CoV-2 spike variants. To demonstrate this platform, we immunized a camel with subunit 1 (S1) of the wild-type spike protein and constructed a nanobody phage library. The binding and neutralizing activities of the nanobodies against 72 spike variants were then measured, resulting in the identification of two nanobodies (C-282 and C-39) with broad neutralizing activity against six non-Omicron variants (D614G, Alpha, Beta, Gamma, Delta, Kappa) and five Omicron variants (BA.1-5). Their neutralizing capability was validated using in vitro pseudovirus-based neutralization assays. All these results demonstrate the utility of our proteomics platform to identify new nanobodies with broad neutralizing capability and to develop a treatment for patients with SARS-CoV-2 variant infection in the future.

A nanobody-mediated drug system against largemouth bass virus delivered by bacterial nanocellulose in Micropterus salmoides.

Diseases caused by pathogens severely hampered the development of aquaculture, especially largemouth bass virus (LMBV) has caused massive mortality and severe economic losses to the culture of largemouth bass (Micropterus salmoides). Considering the environmental hazards and human health, effective and environmentally friendly therapy strategy against LMBV is of vital importance and in pressing need. In the present study, a novel nanobody (NbE4) specific for LMBV was selected from a phage display nanobody library. Immunofluorescence and indirect ELISA showed that NbE4 could recognize LMBV virions and had strong binding capacity, but RT-qPCR evidenced that NBE4 did not render the virus uninfectious. Besides, antiviral drug ribavirin was used to construct a targeted drug system delivered by bacterial nanocellulose (BNC). RT-qPCR revealed that NbE4 could significantly enhance the antiviral activity of ribavirin in vitro and in vivo. The targeted drug delivery system (BNC-Ribavirin-NbE4, BRN) reduced the inflammatory response caused by LMBV infection and improved survival rate (BRN-L, 33.3 %; BRN-M, 46.7 %; BRN-H, 56.7 %)compared with control group (13.3 %), ribavirin group (RBV, 26.7 %) and BNC-ribavirin (BNC-R, 40.0 %), respectively. This research provided an effective antiviral strategy that improved the drug therapeutic effect and thus reduced the dosage.

NanoBERTa-ASP: predicting nanobody paratope based on a pretrained RoBERTa model.

BACKGROUND: Nanobodies, also known as VHH or single-domain antibodies, are unique antibody fragments derived solely from heavy chains. They offer advantages of small molecules and conventional antibodies, making them promising therapeutics. The paratope is the specific region on an antibody that binds to an antigen. Paratope prediction involves the identification and characterization of the antigen-binding site on an antibody. This process is crucial for understanding the specificity and affinity of antibody-antigen interactions. Various computational methods and experimental approaches have been developed to predict and analyze paratopes, contributing to advancements in antibody engineering, drug development, and immunotherapy. However, existing predictive models trained on traditional antibodies may not be suitable for nanobodies. Additionally, the limited availability of nanobody datasets poses challenges in constructing accurate models. METHODS: To address these challenges, we have developed a novel nanobody prediction model, named NanoBERTa-ASP (Antibody Specificity Prediction), which is specifically designed for predicting nanobody-antigen binding sites. The model adopts a training strategy more suitable for nanobodies, based on an advanced natural language processing (NLP) model called BERT (Bidirectional Encoder Representations from Transformers). To be more specific, the model utilizes a masked language modeling approach named RoBERTa (Robustly Optimized BERT Pretraining Approach) to learn the contextual information of the nanobody sequence and predict its binding site. RESULTS: NanoBERTa-ASP achieved exceptional performance in predicting nanobody binding sites, outperforming existing methods, indicating its proficiency in capturing sequence information specific to nanobodies and accurately identifying their binding sites. Furthermore, NanoBERTa-ASP provides insights into the interaction mechanisms between nanobodies and antigens, contributing to a better understanding of nanobodies and facilitating the design and development of nanobodies with therapeutic potential. CONCLUSION: NanoBERTa-ASP represents a significant advancement in nanobody paratope prediction. Its superior performance highlights the potential of deep learning approaches in nanobody research. By leveraging the increasing volume of nanobody data, NanoBERTa-ASP can further refine its predictions, enhance its performance, and contribute to the development of novel nanobody-based therapeutics. Github repository: https://github.com/WangLabforComputationalBiology/NanoBERTa-ASP.

Recombinant Production and Characterization of VHHs/Nanobodies Targeting Tau to Block Fibrillar Assembly.

Tau protein was extensively studied using nuclear magnetic resonance spectroscopy, providing a powerful way to determine interaction sites between Tau and partner proteins. Here we used this analytical tool to describe the epitopes of Tau-specific VHHs (variable domain of the heavy chain of the heavy chain-only antibodies, aka nanobodies) selected from a synthetic library. An in vitro Tau aggregation assay was subsequently used as a functional screen to check VHH efficacy as aggregation inhibitors. We have observed a correlation between the targeted epitope and the aggregation-inhibition capacity of a series of Tau-specific VHHs.

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.