{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T00:42:51Z","timestamp":1792024971967,"version":"4.4.0"},"reference-count":45,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2020,12,11]],"date-time":"2020-12-11T00:00:00Z","timestamp":1607644800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/biom10121661","type":"journal-article","created":{"date-parts":[[2020,12,13]],"date-time":"2020-12-13T23:39:36Z","timestamp":1607902776000},"page":"1661","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":48,"title":["Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8963-3063","authenticated-orcid":false,"given":"Ainhoa","family":"Moliner-Morro","sequence":"first","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"J. Sheward","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"},{"name":"Division of Virology, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, 7925 Cape Town, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7026-7622","authenticated-orcid":false,"given":"Vivien","family":"Karl","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4283-812X","authenticated-orcid":false,"given":"Laura","family":"Perez Vidakovics","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ben","family":"Murrell","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2257-7241","authenticated-orcid":false,"given":"Gerald M.","family":"McInerney","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5514-2418","authenticated-orcid":false,"given":"Leo","family":"Hanke","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17177 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,12,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Atarhouch","year":"1993","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.jim.2007.04.008","article-title":"Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)","volume":"324","author":"Maass","year":"2007","journal-title":"J. Immunol. Methods"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/S0014-5793(97)01062-4","article-title":"Selection and identification of single domain antibody fragments from camel heavy-chain antibodies","volume":"414","author":"Desmyter","year":"1997","journal-title":"FEBS Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/S0141-0229(01)00497-5","article-title":"Large-scale production of VHH antibody fragments by Saccharomyces cerevisiae","volume":"30","author":"Thomassen","year":"2002","journal-title":"Proc. Enzyme Microb. Technol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1007\/s00253-007-1142-2","article-title":"Properties, production, and applications of camelid single-domain antibody fragments","volume":"77","author":"Harmsen","year":"2007","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"G\u00f3mez-Sebasti\u00e1n, S., Nu\u00f1ez, M.C., Garaicoechea, L., Alvarado, C., Mozgovoj, M., Lasa, R., Kahl, A., Wigdorovitz, A., Parre\u00f1o, V., and Escribano, J.M. (2012). Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo. BMC Biotechnol., 12.","DOI":"10.1186\/1472-6750-12-59"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/s40259-019-00392-z","article-title":"The Therapeutic Potential of Nanobodies","volume":"34","author":"Muyldermans","year":"2020","journal-title":"BioDrugs"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/j.tibtech.2014.03.001","article-title":"Nanobody-based products as research and diagnostic tools","volume":"32","author":"Muyldermans","year":"2014","journal-title":"Trends Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6146","DOI":"10.1073\/pnas.1502609112","article-title":"Noninvasive imaging of immune responses","volume":"112","author":"Rashidian","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-018-03191-2","article-title":"A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging","volume":"9","author":"Virant","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4838","DOI":"10.4049\/jimmunol.1600692","article-title":"Generation of Immunity against Pathogens via Single-Domain Antibody\u2013Antigen Constructs","volume":"197","author":"Duarte","year":"2016","journal-title":"J. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.jmb.2003.09.034","article-title":"Pentamerization of single-domain antibodies from phage libraries: A novel strategy for the rapid generation of high-avidity antibody reagents","volume":"335","author":"Zhang","year":"2004","journal-title":"J. Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9019","DOI":"10.1038\/s41598-018-26732-7","article-title":"Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections","volume":"8","author":"Goossens","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Bernedo-Navarro, R., Rom\u00e3o, E., Yano, T., Pinto, J., De Greve, H., Sterckx, Y., and Muyldermans, S. (2018). Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment. Toxins, 10.","DOI":"10.3390\/toxins10030108"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"884","DOI":"10.3389\/fimmu.2017.00884","article-title":"Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine\u2013Nanobody Complex","volume":"8","author":"Desmyter","year":"2017","journal-title":"Front. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3912","DOI":"10.1073\/pnas.1801524115","article-title":"Anti\u2013CTLA-4 therapy requires an Fc domain for efficacy","volume":"115","author":"Ingram","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1128\/CVI.00730-15","article-title":"A tetraspecific VHH-based neutralizing antibody modifies disease outcome in three animal models of clostridium difficile infection","volume":"23","author":"Schmidt","year":"2016","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1126\/science.aaq0620","article-title":"Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin","volume":"362","author":"Laursen","year":"2018","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1038\/nchembio.2007.31","article-title":"Sortagging: A versatile method for protein labeling","volume":"3","author":"Popp","year":"2007","journal-title":"Nat. Chem. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.sbi.2016.05.021","article-title":"Recent advances in sortase-catalyzed ligation methodology","volume":"38","author":"Antos","year":"2016","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"9087","DOI":"10.1074\/jbc.M114.634287","article-title":"HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation","volume":"290","author":"Truttmann","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e201643764","DOI":"10.15252\/embr.201643764","article-title":"Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication","volume":"18","author":"Hanke","year":"2017","journal-title":"EMBO Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1002\/cmmi.1696","article-title":"Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: A versatile strategy for multiple molecular imaging modalities","volume":"11","author":"Massa","year":"2016","journal-title":"Contrast Media Mol. Imaging"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1021\/acscentsci.5b00121","article-title":"Use of 18 F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer","volume":"1","author":"Rashidian","year":"2015","journal-title":"ACS Cent. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"474005","DOI":"10.1088\/1361-6463\/aae0e2","article-title":"Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINT","volume":"51","author":"Fabricius","year":"2018","journal-title":"J. Phys. D. Appl. Phys."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Lim, S., Glasgow, J.E., Filsinger Interrante, M., Storm, E.M., and Cochran, J.R. (2017). Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions. Biotechnol. J., 12.","DOI":"10.1002\/biot.201600696"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4420","DOI":"10.1038\/s41467-020-18174-5","article-title":"An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction","volume":"11","author":"Hanke","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1272","DOI":"10.1039\/b901970g","article-title":"Cu-free click cycloaddition reactions in chemical biology","volume":"39","author":"Jewett","year":"2010","journal-title":"Chem. Soc. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10131","DOI":"10.1073\/pnas.1409861111","article-title":"Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes","volume":"111","author":"Shi","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1016\/j.cell.2020.04.004","article-title":"Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2","volume":"181","author":"Monteil","year":"2020","journal-title":"Cell"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7496","DOI":"10.1021\/jacs.6b03836","article-title":"In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant","volume":"138","author":"Glasgow","year":"2016","journal-title":"J. Am. Chem. Soc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"16820","DOI":"10.1073\/pnas.1408605111","article-title":"Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity","volume":"111","author":"Wagner","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1808","DOI":"10.1038\/nprot.2013.103","article-title":"Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry","volume":"8","author":"Witte","year":"2013","journal-title":"Nat. Protoc."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Singh, S., Gupta, K., Shukla, S., Sampathkumar, S.G., and Roy, R.P. (2019). Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0217369"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"7132","DOI":"10.1002\/anie.201203612","article-title":"Bio-Click Chemistry: Enzymatic Functionalization of PEGylated Capsules for Targeting Applications","volume":"51","author":"Leung","year":"2012","journal-title":"Angew. Chemie Int. Ed."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1128\/AAC.03818-14","article-title":"Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2","volume":"59","author":"Geoghegan","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1128\/JVI.00837-18","article-title":"A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV","volume":"92","author":"Zhao","year":"2018","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.molimm.2018.11.016","article-title":"Selection and characterization of protective anti-chikungunya virus single domain antibodies","volume":"105","author":"Liu","year":"2019","journal-title":"Mol. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1084\/jem.20112655","article-title":"Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization","volume":"209","author":"McCoy","year":"2012","journal-title":"J. Exp. Med."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Hultberg, A., Temperton, N.J., Rosseels, V., Koenders, M., Gonzalez-Pajuelo, M., Schepens, B., Iba\u00f1ez, L.I., Vanlandschoot, P., Schillemans, J., and Saunders, M. (2011). Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0017665"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1093\/infdis\/jiq168","article-title":"Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infection","volume":"203","author":"Hultberg","year":"2011","journal-title":"J. Infect. Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"8278","DOI":"10.1128\/JVI.03178-13","article-title":"Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge","volume":"88","author":"Cardoso","year":"2014","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.1016\/j.cell.2020.04.031","article-title":"Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies","volume":"181","author":"Wrapp","year":"2020","journal-title":"Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1021\/acs.molpharmaceut.6b00964","article-title":"Conjugation to 10 kDa linear PEG extends serum half-life and preserves the receptor-binding ability of mmTRAIL with minimal stimulation of PEG-specific antibodies","volume":"14","author":"Nie","year":"2017","journal-title":"Mol. Pharm."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1038\/11717","article-title":"Therapeutic antibody fragments with prolonged in vivo half-lives","volume":"17","author":"Chapman","year":"1999","journal-title":"Nat. Biotechnol."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/12\/1661\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:44:04Z","timestamp":1760179444000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/12\/1661"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,12,11]]},"references-count":45,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["biom10121661"],"URL":"https:\/\/doi.org\/10.3390\/biom10121661","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,11]]}}}