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.

Conformational features and interaction mechanisms of VH H antibodies with ß-hairpin CDR3: A case of Nb8-HigB2 interaction.

The ß-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR3s are diverse, CDR3s sometimes adopt ß-hairpin conformations. However, characteristic features and interaction mechanisms of ß-hairpin CDR3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VH H antibody Nb8, which has a CDR3 that forms a ß-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate the design and optimization of single-domain antibodies.

abYpap: improvements to the prediction of antibody VH/VL packing using gradient boosted regression.

The Fv region of the antibody (comprising VH and VL domains) is the area responsible for target binding and thus the antibody's specificity. The orientation, or packing, of these two domains relative to each other influences the topography of the Fv region, and therefore can influence the antibody's binding affinity. We present abYpap, an improved method for predicting the packing angle between the VH and VL domains. With the large data set now available, we were able to expand greatly the number of features that could be used compared with our previous work. The machine-learning model was tuned for improved performance using 37 selected residues (previously 13) and also by including the lengths of the most variable 'complementarity determining regions' (CDR-L1, CDR-L2 and CDR-H3). Our method shows large improvements from the previous version, and also against other modeling approaches, when predicting the packing angle.

Evaluation of the Potential Impact of In Silico Humanization on VHH Dynamics.

Camelids have the peculiarity of having classical antibodies composed of heavy and light chains as well as single-chain antibodies. They have lost their light chains and one heavy-chain domain. This evolutionary feature means that their terminal heavy-chain domain, VH, called VHH here, has no partner and forms an independent domain. The VHH is small and easy to express alone; it retains thermodynamic and interaction properties. Consequently, VHHs have garnered significant interest from both biotechnological and pharmaceutical perspectives. However, due to their origin in camelids, they cannot be used directly on humans. A humanization step is needed before a possible use. However, changes, even in the constant parts of the antibodies, can lead to a loss of quality. A dedicated tool, Llamanade, has recently been made available to the scientific community. In a previous paper, we already showed the different types of VHH dynamics. Here, we have selected a representative VHH and tested two humanization hypotheses to accurately assess the potential impact of these changes. This example shows that despite the non-negligible change (1/10th of residues) brought about by humanization, the effect is not drastic, and the humanized VHH retains conformational properties quite similar to those of the camelid VHH.

The solution structure of the heavy chain-only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions.

Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy in vivo. Here, we have characterized the solution arrangement of the molecule. Two 1443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.

Structural, nonclinical, and clinical features of ozoralizumab: A novel tumour necrosis factor inhibitor.

Tumour necrosis factor (TNF) inhibitors are currently the most widely used biological agents to treat rheumatoid arthritis. Ozoralizumab (OZR), a novel TNF inhibitor, is an antibody using variable heavy-chain domains of heavy-chain antibody (VHHs) and became the first VHH drug approved for the treatment of rheumatoid arthritis in September 2022. VHHs isolated from camelid heavy-chain antibodies can bind antigens with a single molecule. OZR is a trivalent VHH that consists of two anti-human TNFα VHHs and one anti-human serum albumin (anti-HSA) VHH. This review summarizes OZR's unique structural characteristics and nonclinical and clinical data. The clinical data outline the pharmacokinetics, efficacy, relationship between efficacy and pharmacokinetics, and safety of OZR, focusing on a Phase II/III confirmatory study (OHZORA trial).

Molecular recognition requires dimerization of a VHH antibody.

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability.

Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for antibody affinity but its impact on stability is often neglected. Here, we present a mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between VH-K94 and VH-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (VH-K94:VH-D101:VH-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (VH-Y100E:VL-Y49) at the VH-VL interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. VH-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.

Research Progresses and Applications of Fluorescent Protein Antibodies: A Review Focusing on Nanobodies.

Since the discovery of fluorescent proteins (FPs), their rich fluorescence spectra and photochemical properties have promoted widespread biological research applications. FPs can be classified into green fluorescent protein (GFP) and its derivates, red fluorescent protein (RFP) and its derivates, and near-infrared FPs. With the continuous development of FPs, antibodies targeting FPs have emerged. The antibody, a class of immunoglobulin, is the main component of humoral immunity that explicitly recognizes and binds antigens. Monoclonal antibody, originating from a single B cell, has been widely applied in immunoassay, in vitro diagnostics, and drug development. The nanobody is a new type of antibody entirely composed of the variable domain of a heavy-chain antibody. Compared with conventional antibodies, these small and stable nanobodies can be expressed and functional in living cells. In addition, they can easily access grooves, seams, or hidden antigenic epitopes on the surface of the target. This review provides an overview of various FPs, the research progress of their antibodies, particularly nanobodies, and advanced applications of nanobodies targeting FPs. This review will be helpful for further research on nanobodies targeting FPs, making FPs more valuable in biological research.

General Trends of the Camelidae Antibody VHHs Domain Dynamics.

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen-antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design.

Quality assessment of VHH models.

Heavy Chain Only Antibodies are specific to Camelid species. Despite the lack of the light chain variable domain, their heavy chain variable domain (VH) domain, named VHH or nanobody, has promising potential applications in research and therapeutic fields. The structural study of VHH is therefore of great interest. Unfortunately, considering the huge amount of sequences that might be produced, only about one thousand of VHH experimental structures are publicly available in the Protein Data Bank, implying that structural model prediction of VHH is a necessary alternative to obtaining 3D information besides its sequence. The present study aims to assess and compare the quality of predictions from different modelling methodologies. Established comparative & homology modelling approaches to recent Deep Learning-based modelling strategies were applied, i.e. Modeller using single or multiple structural templates, ModWeb, SwissModel (with two evaluation schema), RoseTTAfold, AlphaFold 2 and NanoNet. The prediction accuracy was evaluated using RMSD, TM-score, GDT-TS, GDT-HA and Protein Blocks distance metrics. Besides the global structure assessment, we performed specific analyses of Frameworks and CDRs structures. We observed that AlphaFold 2 and especially NanoNet performed better than the other evaluated softwares. Importantly, we performed molecular dynamics simulations of an experimental structure and a NanoNet predicted model of a VHH in order to compare the global structural flexibility and local conformations using Protein Blocks. Despite rather similar structures, substantial differences in dynamical properties were observed, which underlies the complexity of the task of model evaluation.Communicated by Ramaswamy H. Sarma.

Toward real-world automated antibody design with combinatorial Bayesian optimization.

Antibodies are multimeric proteins capable of highly specific molecular recognition. The complementarity determining region 3 of the antibody variable heavy chain (CDRH3) often dominates antigen-binding specificity. Hence, it is a priority to design optimal antigen-specific CDRH3 to develop therapeutic antibodies. The combinatorial structure of CDRH3 sequences makes it impossible to query binding-affinity oracles exhaustively. Moreover, antibodies are expected to have high target specificity and developability. Here, we present AntBO, a combinatorial Bayesian optimization framework utilizing a CDRH3 trust region for an in silico design of antibodies with favorable developability scores. The in silico experiments on 159 antigens demonstrate that AntBO is a step toward practically viable in vitro antibody design. In under 200 calls to the oracle, AntBO suggests antibodies outperforming the best binding sequence from 6.9 million experimentally obtained CDRH3s. Additionally, AntBO finds very-high-affinity CDRH3 in only 38 protein designs while requiring no domain knowledge.

Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Investigating protein-excipient interactions of a multivalent VHH therapeutic protein using NMR spectroscopy.

ALPHABETICAL LIST OF ABBREVIATIONS: Fab Fragment antigen-binding; Fc Fragment crystallizable; HMW High molecular weight; ∆HMW Difference between HMW species at stress temperature and 5°C controls; IgG Immunoglobulin G; mAbs Monoclonal antibodies; MV-VHH Multivalent VHH molecule with the format aC-L1-aC-L1-aD; NMR Nuclear magnetic resonance; scFv Single-chain fragment variable; SEC Size-exclusion chromatography; VHH Variable domain of Heavy chain of Heavy chain-only antibody.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA.

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

VHH Structural Modelling Approaches: A Critical Review.

VHH, i.e., VH domains of camelid single-chain antibodies, are very promising therapeutic agents due to their significant physicochemical advantages compared to classical mammalian antibodies. The number of experimentally solved VHH structures has significantly improved recently, which is of great help, because it offers the ability to directly work on 3D structures to humanise or improve them. Unfortunately, most VHHs do not have 3D structures. Thus, it is essential to find alternative ways to get structural information. The methods of structure prediction from the primary amino acid sequence appear essential to bypass this limitation. This review presents the most extensive overview of structure prediction methods applied for the 3D modelling of a given VHH sequence (a total of 21). Besides the historical overview, it aims at showing how model software programs have been shaping the structural predictions of VHHs. A brief explanation of each methodology is supplied, and pertinent examples of their usage are provided. Finally, we present a structure prediction case study of a recently solved VHH structure. According to some recent studies and the present analysis, AlphaFold 2 and NanoNet appear to be the best tools to predict a structural model of VHH from its sequence.

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools.

Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.

Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development.

The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a two-decade long commitment in time and research effort to realize the clinical potential of single-domain antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in sharks provides an alternative small binding domain to conventional monoclonal antibodies and their fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposition to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new (neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.