Ultrapotent and multivalent nanobodies against 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.
Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses
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.
Structural and functional analyses of multiepitope anti- COVID nanobdodies
There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.
Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies
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.
High-quality nanobodies for tailored drug delivery and "Duraleukin"
Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by poor pharmacokinetics. Here we developed a repertoire of distinct and high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. Using biophysics and hybrid structural methods, we have systematically characterized the Nb repertoire, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate and multiplex pharmacokinetic analysis of dozens of diverse and high-quality NbHSA and confirmed the most stable construct has a 771-fold T1/2 improvement compared to non-albumin binding Nbs. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed stable NbHSA-cytokine conjugates “Duraleukins” and confirmed the high anticancer efficacy of a Duraluekin in vivo. This high-quality Nb resource may help advance research into novel biotherapeutics.
Superimmunity by pan-sarbecovirus nanobodies
Vaccine boosters and infection can facilitate the development of SARS-CoV-2 antibodies with improved potency and breadth. Here, we observe superimmunity in a camelid extensively immunized with the SARS-CoV-2 receptor-binding domain (RBD). We rapidly isolate a large repertoire of specific ultra-high-affinity nanobodies that bind strongly to all known sarbecovirus clades using integrative proteomics. These pan-sarbecovirus nanobodies (psNbs) are highly effective against SARS-CoV and SARS-CoV-2 variants, including Omicron, with the best median neutralization potency at single-digit nanograms per milliliter. A highly potent, inhalable, and bispecific psNb (PiN-31) is also developed. Structural determinations of 13 psNbs with the SARS-CoV-2 spike or RBD reveal five epitope classes, providing insights into the mechanisms and evolution of their broad activities. The highly evolved psNbs target small, flat, and flexible epitopes that contain over 75% of conserved RBD surface residues. Their potencies are strongly and negatively correlated with the distance of the epitopes from the receptor binding sites.
Llamanade: An computational pipeline for robust nanobody humanization
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.
44.Kim J, Sang Z, Xiang Y, Shen Z, and Shi Y. Nanobodies: robust miniprotein binders in biomedicine. (2023) Advanced Drug Delivery Reviews.
41. Mitchell A. Ellison et al. Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genes.(2023) Nucleic Acids Research.
42. Tubiana J, Xiang Y, Fan L, Wolfson H, Chen K#, Schneidman-Dohovny D#, and Shi Y#. Reduced antigenicity of Omicron lowers host serologic response. (2022) Cell Reports. (In press)
41. Zhuolun Shen#, Zhe Sang and Yi Shi#. Nanobodies as a powerful platform for biomedicine. (2022).Trends in Molecular Medicine.
40. Yufei Xiang*, Wei Huang*, Hejun Liu*, Zhe Sang*, Sham Nambulli, Jerome Tubiana, Kevin L Williams, Paul Duprex, Dian Schneidman-Duhovny, Ian A Wilson, Derek J Taylor, and Yi Shi. Super-immunity by broadly protective nanobodies to sarbecoviruses. (2022). Cell Reports.
39. M.M.Lu et al., The Magic of Linking Rings: Discovery of a Unique Photoinduced Fluorescent Protein Crosslink. (2022). J. Am. Chem. Soc.
38.Yang H, Wang Y, Xiang Y, Yadav T, Ouyang J, Phoon L, Zhu X, Shi Y, Zou L, and Lan L. (2022) FMRP Promotes transcription-coupled homologous recombination via Facilitating TET1-mediated m5C RNA modification Demethylation. Proc. Natl. Acad. Sci. U.S.A.
37. C. W. Akey et al., Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex. (2022). Cell.
36. C McKennan, Z Sang, Y Shi. (2022). A novel framework to quantify uncertainty in peptide-tandem mass spectrum matches with application to nanobody peptide identification (under review).
35. MH Cheng, et al. (2022). Impact of New Variants on SAR-CoV-2 Infectivity and Neutralization: A Molecular Assessment of the Alterations in the Spike-Host Protein Interactions. iScience.
34. Sang, Z., Xiang, Y., Barhar. I., and Shi, Y. Llamanade: an open-source computational pipeline for robust nanobody humanization. (2022).Structure.
33. Chen, A., et al. (2021). Chitinase-3-like 1 protein complexes modulate macrophage-mediated immune suppression in glioblastoma. J Clin Invest 131.
32. Dapeng Sun*, Zhe Sang*, Yong Joon Kim*, Yufei Xiang, Tomer Cohen, Anna K. Belford, Alexis Huet, James F. Conway, Ji Sun, Derek J. Taylor, Dina Schneidman-Duhovny#, Cheng Zhang#, Wei Huang#, and Yi Shi# (2021). Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes. Nature Communications.
31. Sham Nambulli*, Yufei Xiang*, Natasha L. Tilston-Lunel, Linda J. Rennick, Zhe Sang, William B. Klimstra, Douglas S. Reed, Nicholas A. Crossland, Yi Shi# and W. Paul Duprex#. (2021). Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses. Science Advances.
30. Xiang, Y., Sang, Z., Bitton, L., Xu, J., Liu, Y., Schneidman-Duhovny, D., and Shi, Y (2021). Integrative proteomics identifies thousands of distinct and high-affinity nanobodies. Cell Systems.
Cover story: https://www.cell.com/cell-systems/issue?pii=S2405-4712(20)X0007-X Cell Systems.
29. Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2. Yihu Xie, Bradley P. Clarkea, Yong Joon Kimb, Austin L. Iveya, Pate S. Hilla, Yi Shi, and Yi Ren. (2021). eLife.
28. A resource of high-quality nanobodies for drug delivery. Shen Z, Xiang Y, Vegara S, Chen A, Xiao Z, Santiago U, Jin C, Sang Z, Luo J, Chen K, Schneidman-Duhovny D, Camacho C, Calero G, Hu B, Shi Y (2021). iScience.
27. Ubiquitination-mediated degradation of TRDMT1 regulates homologous recombination and therapeutic response. Zhu, X., Wang, X., Yan, W., Yang, H., Xiang, Y., Lv, F., Shi, Y., Li, H.Y., and Lan, L. (2021). NAR Cancer 3, zcab010.
26. Xiang, Y., Nambulli, S., Xiao, Z., Liu, H., Sang, Z., Duprex, W.P., Schneidman-Duhovny, D., Zhang, C., and Shi, Y. (2020). Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2. Science. (2020)
25. Yufei Xiang, Zhuolun Shen, and Yi Shi. Chemical cross-linking and mass spectrometric (CX-MS) analysis of the endogenous yeast exosome complexes. (2020). Methods in Molecular Biology.
24. Sai J Ganesan, et al. Integrative Structure and Function of the Yeast Exocyst Complex. (2020). Protein Science.
23. Iacobucci, C., et al. (2019). First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study. Analytical Chemistry 91, 6953-6961.
22. Yaqun Teng, et al (2018). ROS-induced R loops trigger a transcription-coupled but BRCA1/2-independent homologous recombination pathway through CSB. Nature Communications 9, 4115
21. Jishage, M., Yu, X., Shi, Y., Ganesan, S.J., Chen, W.Y., Sali, A., Chait, B.T., Asturias, F.J., and Roeder, R.G. (2018). Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1. Nat Struct Mol Biol 25, 859-867.
20. Kim, S.J., et al. Structure and Functional Anatomy of the Nuclear Pore Complex (2018). Nature 555, 475-482. PMID: 29539637
“An Architectural Guide to the Nuclear Pore Complex”- Comments from the NIH director Francis Collins
Publications Prior to 2017
19. Fernandez-Martinez J (2016). Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform. Cell 167, 1–14
Preview by: Gozalo, A. & Capelson, M. A New Path through the Nuclear Pore. Cell 167, 1159-1160 2016.
18. Chait, B.T., Cardene, M., Olinares, P.D., Rout, M.P., and Shi, Y. (2016). Revealing Higher Order Protein Structure Using Mass Spectrometry. Journal of the American Society for Mass Spectrometry 27, 952-965.
17. Hunziker, M., Barandun, J., Petfalski, E., Tan, D., Delan-Forino, C., Molloy, K.R., Kim, K.H., Dunn-Davies, H., Shi, Y., Chaker-Margot, M., et al. (2016). UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly. Nature Communications 7, 12090.
16. Shi, Y., Pellarin, R., Fridy, P.C., Fernandez-Martinez, J., Thompson, M.K., Li, Y., Wang, Q.J., Sali, A., Rout, M.P., and Chait, B.T. (2015). A strategy for dissecting the architectures of native macromolecular assemblies. Nature Methods 12, 1135-1138.
15. Sun, J., Shi, Y., Georgescu, R.E., Yuan, Z., Chait, B.T., Li, H., and O'Donnell, M.E. (2015). The architecture of a eukaryotic replisome. Nat Struct Mol Biol 22, 976-982.
14. LoPiccolo, J., et al. (2015). Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85alpha Homodimer. Journal of Biological Chemistry 290, 30390-30405.
13. Morris, D.H., Yip, C.K., Shi, Y., Chait, B.T., and Wang, Q.J. (2015). Beclin 1-Vps34 Complex Architecture: Understanding the Nuts and Bolts of Therapeutic Targets. Frontiers in biology 10, 398-426.
12. Cevher, M.A., Shi, Y., Li, D., Chait, B.T., Malik, S., and Roeder, R.G. (2014). Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. Nat Struct Mol Biol 21, 1028-1034.
11. Shi, Y., et al (2014). Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex. Molecular & Cellular Proteomics 13, 2927-2943.
10. Algret, et al. (2014). Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway. Molecular & Cellular Proteomics 13, 2855-2870.
9. Kim, S.J., et al. (2014). Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex. Molecular & Cellular Proteomics 13, 2911-2926.
8. Krenciute, G., Liu, S.F., Yucer, N., Shi, Y., Ortiz, P., Liu, Q.M., Kim, B.J., Odejimi, A.O., Leng, M., Qin, J., et al. (2013). Nuclear BAG6-UBL4A-GET4 Complex Mediates DNA Damage Signaling and Cell Death. Journal of Biological Chemisty 288, 20547-20557.
7. Fan, Y., et al. (2012). Lys48-linked TAK1 polyubiquitination at lysine-72 downregulates TNFalpha-induced NF-kappaB activation via mediating TAK1 degradation. Cell Signal 24, 1381-1389.
6. Malovannaya, A.,et al. (2011). Analysis of the human endogenous coregulator complexome. Cell 145, 787-799.
5. Shi, Y., Chan, D.W., Jung, S.Y., Malovannaya, A., Wang, Y., and Qin, J. (2011a). A data set of human endogenous protein ubiquitination sites. Molecular & Cellular Proteomics 10, M110 002089.
4. Shi, Y., Xu, P., and Qin, J. (2011b). Ubiquitinated proteome: ready for global? Molecular & Cellular Proteomics 10, R110 006882.
3. Fan, Y., et al. (2010). Lysine 63-linked polyubiquitination of TAK1 at lysine 158 is required for tumor necrosis factor alpha- and interleukin-1beta-induced IKK/NF-kappaB and JNK/AP-1 activation. Journal of Biological Chemistry 285, 5347-5360.
2. Sun, W., et al. (2010). USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha. Cell Signal 22, 386-394.
1. Xu, G., et al. (2010). Ubiquitin-specific peptidase 21 inhibits tumor necrosis factor alpha-induced nuclear factor kappaB activation via binding to and deubiquitinating receptor-interacting protein 1. Journal of Biological Chemistry 285, 969-978.