Multivalent Nanobodies Research Articles

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering.

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants

SummaryThe spike glycoprotein of SARS-CoV-2 engages with human ACE 2 to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.

Multivalent nanobody as capture antibody-based enzyme linked immunosorbent assay for detection of 3-phenoxybenzoic acid in urine

Nanobodies (Nbs) as capture antibodies in enzyme-linked immunosorbent assays (ELISAs) is greatly hampered by their poor performance after attaching onto polystyrene microplates. Reasons behind those phenomena remain unknown. One of possible explanation is that Nbs with a single domain might lose their accessibility of paratope when adsorbed on the plates. Increasing their binding sites might improve performance in capture Nbs-based ELISA. In this study, anti-3-phenoxybenzoic acid (3-PBA) Nbs was assembled to trivalent form (Nb3) in tandem with flexible linkers (G4S)3. Direct competitive ELISA on the basis of Nb3 and 3-PBA-horseradish peroxidase was developed for detection of 3-PBA in livestock urine. The ELISA had a half-maximum (IC50) inhibition concentration of 0.51 ng/mL, with a limit of detection of 0.02 ng/mL, which was more sensitive than that of the parental Nb with a IC50 of 2.39 ng/mL. The average recoveries of 3-PBA spiked in swine, sheep and dairy cow urine samples by the assay ranged from 89.52% to 114.25% and agreed well with those of liquid chromatography mass spectrometry (LC-MS). The above results indicated that multivalent Nbs could be treated as the capture antibody in ELISA for routine screening analysis of 3-PBA residues in urine.

Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS-CoV-2 Variants.

The COVID‐19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS‐CoV‐2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS‐CoV‐2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS‐CoV‐2 cross‐reactive nanobody (VHH‐72) initially generated against SARS‐CoV. This synergy is epitope specific and is not observed for a second high‐affinity nanobody against a non‐conserved epitope in the receptor‐binding domain. Importantly, a hexavalent VHH‐72 nanobody retains binding to spike proteins from multiple highly transmissible SARS‐CoV‐2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH‐72 nanobodies also display drug‐like biophysical properties, including high stability, high solubility, and low levels of non‐specific binding. The unique neutralizing and biophysical properties of VHH‐72 multivalent nanobodies make them attractive as therapeutics against SARS‐CoV‐2 variants.

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond

Next Generation Immunotherapies – Emerging Strategies for Immune Modulation against Cancer, Infections, and Beyond

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1–3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD–ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and—to our knowledge—rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

A double punch against SARS-CoV-2

Coronavirus Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science , this issue p. [eabe6230][1]; see also p. [681][2] [1]: /lookup/doi/10.1126/science.abe6230 [2]: /lookup/doi/10.1126/science.abg2294

Development of multivalent nanobodies blocking SARS-CoV-2 infection by targeting RBD of spike protein

BackgroundThe outbreak and pandemic of coronavirus SARS-CoV-2 caused significant threaten to global public health and economic consequences. It is extremely urgent that global people must take actions to develop safe and effective preventions and therapeutics. Nanobodies, which are derived from single‑chain camelid antibodies, had shown antiviral properties in various challenge viruses. In this study, multivalent nanobodies with high affinity blocking SARS-CoV-2 spike interaction with ACE2 protein were developed.ResultsTotally, four specific nanobodies against spike protein and its RBD domain were screened from a naïve VHH library. Among them, Nb91-hFc and Nb3-hFc demonstrated antiviral activity by neutralizing spike pseudotyped viruses in vitro. Subsequently, multivalent nanobodies were constructed to improve the neutralizing capacity. As a result, heterodimer nanobody Nb91-Nb3-hFc exhibited the strongest RBD-binding affinity and neutralizing ability against SARS-CoV-2 pseudoviruses with an IC50 value at approximately 1.54 nM.ConclusionsThe present study indicated that naïve VHH library could be used as a potential resource for rapid acquisition and exploitation of antiviral nanobodies. Heterodimer nanobody Nb91-Nb3-hFc may serve as a potential therapeutic agent for the treatment of COVID-19.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Multivalent nanobody-biotin amplified enzyme-linked immunosorbent assay for the environmental detection of 3-phenoxybenzoic acid.

The 3-phenoxybenzoic acid (3-PBA) metabolized from pyrethroids is more toxic and has a longer half-life to degradation in a natural environment compared to its parent compounds. Few reports have focused on the environmental detection of 3-PBA. In this study, anti-3-PBA nanobodies in trivalent form (Nb3) were biotinylated. A sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of Nb3-biotin and streptavidin-horseradish peroxidase (SA-HRP) was developed for the environmental detection of 3-PBA. After optimization, the ELISA showed a half-maximum signal inhibition concentration (IC50) of 0.39 ng mL-1 in phosphate-buffered saline (pH 7, 20% MeOH) and a limit of detection (LOD) of 0.02 ng mL-1, which was more sensitive than the parent Nb-based ELISAs with IC50 and LOD values of 1.4 ng mL-1 and 0.1 ng mL-1, respectively. The Nb3-biotin amplified assay showed negligible cross-reactivity with its structural analogues (<0.1%). The average recoveries of 3-PBA from spiked canal water and soil samples ranged from 86.54-109.25% at 0.5-50 ng mL-1 (or ng g-1 (dw)). The 3-PBA residues in canal water and soil samples determined using this assay were in the ranges <LOD-1.46 ng mL-1 and <LOD-4.35 ng g-1 (dw), respectively, which correlated well with the results obtained using liquid chromatography tandem mass spectrometry. The results suggest that multivalent Nb-biotin-SA amplified ELISA is a promising tool for the routine environmental screening of 3-PBA.

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs.

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.

Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2.

Cost-effective, efficacious therapeutics are urgently needed to combat the COVID-19 pandemic. In this study, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD). We discovered Nbs with picomolar to femtomolar affinities that inhibit viral infection at concentrations below the nanograms-per-milliliter level, and we determined a structure of one of the most potent Nbs in complex with the RBD. Structural proteomics and integrative modeling revealed multiple distinct and nonoverlapping epitopes and indicated an array of potential neutralization mechanisms. We bioengineered multivalent Nb constructs that achieved ultrahigh neutralization potency (half-maximal inhibitory concentration as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization and aerosolization.

A capillary electrophoresis based approach for the identification of anti-drug antibodies against camelid VHH biologics (Nanobodies®)

Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space.

Construction of Immunomagnetic Particles with High Stability in Stringent Conditions by Site-Directed Immobilization of Multivalent Nanobodies onto Bacterial Magnetic Particles for the Environmental Detection of Tetrabromobisphenol-A.

Bacterial magnetic particles (BMPs) are an attractive carrier material for immunoassays because of their nanoscale size, dispersal ability, and membrane-bound structure. Antitetrabromobisphenol-A (TBBPA) nanobodies (Nbs) in the form of monovalence (Nb1), bivalence (Nb2), and trivalence (Nb3) were biotinylated and immobilized onto streptavidin (SA)-derivatized BMPs to construct the complexes of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3, respectively. An increasing order of binding capability of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3 to TBBPA was observed. These complexes showed high resilience to temperature (90 °C), methanol (100%), high pH (12), and strong ionic strength (1.37 M NaCl). A BMP-SA-Biotin-Nb3-based enzyme linked immunosorbent assay (ELISA) for TBBPA dissolved in methanol was developed, showing a half-maximum inhibition concentration (IC50) of 0.42 ng mL-1. TBBPA residues in landfill leachate, sewage, and sludge samples determined by this assay were in a range of <LOD-1.17 ng mL-1, <LOD-0.75 ng mL-1, and <LOD-3.65 ng g-1 (dw), respectively, correlating well with the results by liquid chromatography tandem mass spectrometry. The BMP-SA-Biotin-Nb3 was reusable at least three times without significant loss of the binding capability. The BMP-SA-Biotin-Nb3-based ELISA, with a total assay time of less than 30 min, is promising for the rapid monitoring of TBBPA in the environment.

Identification of New DR5 Agonistic Nanobodies and Generation of Multivalent Nanobody Constructs for Cancer Treatment.

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing “TNF-related apoptosis-inducing ligand” (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.

Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex.

The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn’s disease, rheumatoid arthritis, and psoriasis.