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.
Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of ...SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBD
. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.
Visualization of ion transport in electrolytes provides fundamental understandings of electrolyte dynamics and electrolyte-electrode interactions. However, this is challenging because existing ...techniques are hard to capture low ionic concentrations and fast electrolyte dynamics. Here we show that stimulated Raman scattering microscopy offers required resolutions to address a long-lasting question: how does the lithium-ion concentration correlate to uneven lithium deposition? In this study, anions are used to represent lithium ions since their concentrations should not deviate for more than 0.1 mM, even near nanoelectrodes. A three-stage lithium deposition process is uncovered, corresponding to no depletion, partial depletion, and full depletion of lithium ions. Further analysis reveals a feedback mechanism between the lithium dendrite growth and heterogeneity of local ionic concentration, which can be suppressed by artificial solid electrolyte interphase. This study shows that stimulated Raman scattering microscopy is a powerful tool for the materials and energy field.
Globally, there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric ...Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss after infection, decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology, and prevents viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.
The antibody immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the antibody repertoire. Here, we developed a proteomic strategy to survey, ...at an unprecedented scale, the landscape of antigen-engaged, circulating camelid heavy-chain antibodies, whose minimal binding fragments are called VHH antibodies or nanobodies. The sensitivity and robustness of this approach were validated with three antigens spanning orders of magnitude in immune responses; thousands of distinct, high-affinity nanobody families were reliably identified and quantified. Using high-throughput structural modeling, cross-linking mass spectrometry, mutagenesis, and deep learning, we mapped and analyzed the epitopes of >100,000 antigen-nanobody complexes. Our results revealed a surprising diversity of ultrahigh-affinity camelid nanobodies for specific antigen binding on various dominant epitope clusters. Nanobodies utilize both shape and charge complementarity to enable highly selective antigen binding. Interestingly, we found that nanobody-antigen binding can mimic conserved intracellular protein-protein interactions.
A record of this paper’s Transparent Peer Review process is included in the Supplemental information.
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•Proteomic pipeline for identification and quantification of camelid nanobody repertoires•Discovery of thousands of distinct and high-affinity antigen-specific nanobody families•Global epitope mapping by integrative structural proteomics•Mechanisms underlying high-affinity antigen-nanobody binding
Xiang et al. developed a strategy to systematically investigate the repertories of camelid nanobodies for antigen binding. Thousands of diverse and high-affinity Nb families have been identified and affinity-classified. The authors employed hybrid structural proteomics to map epitopes of >100,000 antigen-nanobody complexes to understand the mechanisms underlying mammalian humoral immunity.
Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman ...serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs “Duraleukin” and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.
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•We provide a resource of high-affinity and versatile albumin nanobodies for drug delivery•We systematically map albumin nanobody epitopes by hybrid structural approaches•We parallelly measure the pharmacokinetics of nanobodies in a humanized mouse model•We develop nanobody-cytokine conjugates “Duraleukin” for cancer immunotherapy
Drug delivery system; Biochemical engineering; Biotechnology; Structural biology; Proteomics
Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational ...potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimize solutions to humanize Nbs. Finally, we used Llamanade to successfully humanize a cohort of structurally diverse and potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a server to support the development of therapeutic Nbs into safe and effective trials.
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•Large-scale analysis of Nbs and IgGs reveals unique features of Nbs•Humanization is guided by big data analysis•A robust software "Llamanade" is developed and verified on SARS-CoV-2 Nbs•Llamanade rapidly analyzes Nb features and optimizes humanization
Sang et al. systematically analyze the sequence and structural properties that distinguish nanobodies (Nbs) from human IgGs. Guided by big data, they develop an open-source software, Llamanade, that facilitates rational Nb humanization. By humanizing a set of structurally diverse SARS-CoV-2 neutralizing Nbs, they further validate the robustness of Llamanade.
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Variable domains of heavy chain-only antibodies (VHH), also known as nanobodies (Nbs), are monomeric antigen-binding domains derived from the camelid heavy chain-only antibodies. Nbs ...are characterized by small size, high target selectivity, and marked solubility and stability, which collectively facilitate high-quality drug development. In addition, Nbs are readily expressed from various expression systems, including E. coli and yeast cells. For these reasons, Nbs have emerged as preferred antibody fragments for protein engineering, disease diagnosis, and treatment. To date, two Nb-based therapies have been approved by the U.S. Food and Drug Administration (FDA). Numerous candidates spanning a wide spectrum of diseases such as cancer, immune disorders, infectious diseases, and neurodegenerative disorders are under preclinical and clinical investigation. Here, we discuss the structural features of Nbs that allow for specific, versatile, and strong target binding. We also summarize emerging technologies for identification, structural analysis, and humanization of Nbs. Our main focus is to review recent advances in using Nbs as a modular scaffold to facilitate the engineering of multivalent polymers for cutting-edge applications. Finally, we discuss remaining challenges for Nb development and envision new opportunities in Nb-based research.
The development of a highly active and stable hydrogen evolution photocatalyst is a key issue for achieving efficient and comprehensive water splitting, and it is still challenging for the scientific ...community and practical applications. Herein, we build up a novel visible-light-driven S-scheme in CdS/ZnTHPP binary nanosystem by simple sintering process ensuring the fast transfer of the photo-generated carriers, showing dark green upon simulated sunlight irradiation. Under an optimal condition, the highest hydrogen evolution turnover number (TON) reaches around 22,264 during 128 h irradiation, and the H2 evolution rates of the best CdS/ZnTHPP binary nanosystem was boosted up to a 400 mmol/g under irradiation for 8 hours with a conversion rate over 10-fold-higher than that of pristine CdS NRs. The macrocyclic compound serves as a region of electron absorption assisting the fast and continuous consumption of photo-generated electrons in this system, thereby concurrently alleviating the recombination of electron-hole pairs. Theoretical analyses reveal that the active sites and the generation of H2 are controlled by the thermodynamic process. This new finding provides a new approach to rationally designing an efficient solar-to-chemical conversion system.
A novel visible-light-driven Z-scheme in CdS/ZnTHPP binary nanosystem was built up by a simple sintering process ensuring the fast transfer of the photo-generated carriers, showing a robust hydrogen evolution performance. Display omitted
•A novel visible-light-driven Z-scheme in CdS/ZnTHPP binary nanosystem was built.•The best H2 evolution rate of CdS/ZnTHPP was 400 mmol/g under irradiation for 8 hours.•Theoretical analyses reveal the active sites and the generation of H2 are controlled by the thermodynamic process.
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