Application of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies

Application of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies Adam Leach, Ami Miller, Emma Bentley, Giada Mattiuzzo, Jemima Thomas, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-259484/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed Quads, that retain efficient binding to the SARS-CoV-2 spike protein and show up to two orders of magnitude enhancement in neutralization of pseudovirus infection. The tetramerization method is simple and general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation. Infectious Diseases Scientific Communication Health Economics & Outcomes Research Avidity antibody valency SARS-CoV-2 COVID-19 coronavirus ELISA spike antigen tetramerization Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Additional Declarations Competing interest reported. Terence Rabbitts is a stake holder in Quadrucept Ltd. None of the other authors have any conflicts of interest to declare. Supplementary Files Supplementaryinformationcombinedtitlev12.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 05 Apr, 2021 Reviews received at journal 09 Mar, 2021 Reviewers agreed at journal 07 Mar, 2021 Reviewers invited by journal 07 Mar, 2021 Editor assigned by journal 07 Mar, 2021 Editor invited by journal 07 Mar, 2021 Submission checks completed at journal 07 Mar, 2021 First submitted to journal 20 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-259484","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":15294967,"identity":"93a3e4e4-5811-43b8-98dc-8413d0263020","order_by":0,"name":"Adam Leach","email":"","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Leach","suffix":""},{"id":15294968,"identity":"fe84b044-ac73-4cf6-84c0-1c92120c983e","order_by":1,"name":"Ami Miller","email":"","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ami","middleName":"","lastName":"Miller","suffix":""},{"id":15294969,"identity":"1b72f51d-96d9-41b7-a771-3179341d3158","order_by":2,"name":"Emma Bentley","email":"","orcid":"","institution":"National Institute for Biological Standards and Control","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Bentley","suffix":""},{"id":15294970,"identity":"9e7969c2-fba4-46da-93be-dd199c8ad18f","order_by":3,"name":"Giada Mattiuzzo","email":"","orcid":"","institution":"National Institute for Biological Standards and Control","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Giada","middleName":"","lastName":"Mattiuzzo","suffix":""},{"id":15294971,"identity":"970d4cc5-2345-465f-a99f-c824e80365e3","order_by":4,"name":"Jemima Thomas","email":"","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jemima","middleName":"","lastName":"Thomas","suffix":""},{"id":15294972,"identity":"93d2e505-e0e6-4505-9b1f-7b07ebec2453","order_by":5,"name":"Craig McAndrew","email":"","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Craig","middleName":"","lastName":"McAndrew","suffix":""},{"id":15294973,"identity":"1b033e5f-1939-4ec7-9046-a6095086133f","order_by":6,"name":"Rob Montfort","email":"","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rob","middleName":"","lastName":"Montfort","suffix":""},{"id":15294974,"identity":"bb97a673-7d16-4731-b25b-624bc53a5d60","order_by":7,"name":"Terence Rabbitts","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYDADAwnGBiBlwwMXwKUSoUKCsbHhAEMaSVoYGIFaDiOsxaXFnr332Wcehnvy5tLN7Y8//Dkvwz/7AOOHHwyHjXHawnPceDYPQ7HhzjkHGxsOtt3mkTiXwCzZw3DYDKcWiTRmZh6GBMYNNxKBWhpu8xgA3SrNwHDYBqcW+WdgLfZgLQf+nANpYf6NV4sEG1hLIkQL2wGQFjaQLbgddiaNmXGOQUIyyC8zzrYl80icYWyz7DFIx+l99vZjzAxvKhJst0u3P/hQ8cfOnr+H+fCNHxXWhg249AABEw9qHICSAc5YgSr5gV9+FIyCUTAKRjoAALItT3khMwOBAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Cancer Research","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Terence","middleName":"","lastName":"Rabbitts","suffix":""}],"badges":[],"createdAt":"2021-02-20 16:59:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-259484/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-259484/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6799930,"identity":"ec2554e2-3136-4b21-a9f9-44870f56060d","added_by":"auto","created_at":"2021-03-10 15:03:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":542844,"visible":true,"origin":"","legend":"Characterization of anti-SARS-Cov-2 antibodies with different valencies by surface plasmon resonance \nThe binding potencies of the anti-SARS-Cov-2 tetrameric Quad proteins was determined using surface plasmon resonance. Purified proteins were captured using immobilized SARS-Cov-2 RBD, carried out with a Biacore T200 instrument. (A) Anti-SARS-Cov-2 antibodies were expressed by transfection and secretion from Expi293 suspension cells and characterized by SDS-PAGE following purification by Niaffinity chromatography and gel filtration. Gels were stained with InstantBlue Ultrafast Protein Stain and Mw size markers included (left hand lanes). (C-I) Antibody binding to SARS-Cov-2 RBD was evaluated using SPR. Biotinylated SARSCoV-2 RBD was captured on a streptavidin chip and antibodies flowed over the surface at different concentrations. CR3022 and CR3014 antibody concentrations were 10, 5, 2.5, 1.25, 0.625 and 0.312 nM, while those for H4 and B38 antibodies were 12.5, 6.25, 3.125, 1.56 and 0.78 nM. Sensograms representative of two independent experiments are shown for (C) CR3022-Fab-TD, (D) CR3022-IgG, (E) CR3022-Fab, (F) H4-Fab (G) H4-Fab-TD (H) H4-scFv-TD and (I) B38-Fab-TD. Kinetic parameters are shown in Supplementary Table S1. Tetramerization decreases dissociation rates for the multimeric species compared to the monomeric proteins. This results in increased affinity for SARS-Cov-2 RBD for CR3022-Fab-TD compared to CR3022-Fab, and for H4-Fab-TD and H4-scFv-TD compared to H4-Fab. ","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-259484/v1/9f07d80bbdbd96db42b98ea7.jpg"},{"id":6800373,"identity":"53f368f0-99a2-48d0-8489-4f18e8c5d361","added_by":"auto","created_at":"2021-03-10 15:06:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":433428,"visible":true,"origin":"","legend":"Direct ELISA immunoassays with anti-SARS-CoV-2 antibodies detecting viral spike proteins The ability and potency of engineered anti-SARS-CoV-2 antibodies to bind to the viral spike proteins was compared using Enzyme-linked immunosorbent assays (ELISAs). SARS-CoV-2 spike protein as either S1 (panel A and C) or RBD (panel B and D) was adsorbed onto immunoassay plates at 2 µg per µL for 16 hours at 4 0C. Wells were \nthoroughly washed, blocked, and various concentrations of the indicated His-tagged antibodies were added to each well and incubated for 16 hours at 40C, followed by washing and incubation with anti-His-HRP antibody for 2 hours at room temperature. \nColour was developed using TMB and absorbance read at 450l. All samples were run in triplicate. \n","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-259484/v1/e7bc744c1323d5e6f0b24238.jpg"},{"id":6799928,"identity":"1f3ddf4c-013b-4f4c-b842-9392fb5fcd1b","added_by":"auto","created_at":"2021-03-10 15:03:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":370518,"visible":true,"origin":"","legend":"Tetramerized H4 and B38 anti-SARS-CoV-2 antibodies are potent inhibitors of virus infection The viral infection neutralizing potency of tetrameric versions of the mAbs H4 and B38 was examined in SARS-CoV-2 pseudovirus infection assays. Increasing concentrations of H4-scFv-TD, H4-Fab-TD or B38-Fab-TD were incubated with HEK 293T/17-A2-T2 cells after 1 hour. The cells were incubated for 60 hours before luciferase activity, indicative of viral entry, was determined. The efficacy was compared to the neutralization effects of a commercial anti-SARS-CoV-2 mAb SAD-S35 (AcroBiosystems) and a tetramerized \nACE2-Fc-TD. Data are represented in µg/mL (panel A) and nM (panel B). Calculated IC50 \nvalues are tabulated below each set of curves. The assays were performed twice, in duplicate with error bars indicating the standard error. ","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-259484/v1/9ef2b7a5e77e64f3cd6e035c.jpg"},{"id":6800535,"identity":"6c470a05-e0a7-4da4-aa6c-b471c5c8da63","added_by":"auto","created_at":"2021-03-10 15:09:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727312,"visible":true,"origin":"","legend":"Neutralization of pseudovirus infection by tetramerized clinical stage SARS-CoV-2 antibodies The potency of clinical stage mAbs that were engineered as tetramer formats was measured in SARS-CoV-2 pseudovirus infection assays. Increasing concentrations of antibodies were incubated with HEK 293T/1A2-T2 cells after 1 hour. The cells were incubated for 60 hours before luciferase activity was determined to show viral entry. The data are split to different panels for clarity. Panel A: The neutralization potency of H4-scFTD Quad was compared to clinical stage REGN10987, REGN10933 and CB6/Junshi IgG1. Panel B: The neutralization potency of REGN10987, REGN10933 and CB6/Junshi IgG1 was compared to REGN10987 Fab-TD, CB6/Junshi Fab-TD and REGN10933 FabTD. Panel C: The neutralization potency of the REGN10987, REGN10933 and CB6/Junshi IgG1 was compared to REGN10987 mIg-TD, REGN10987 mIg-TD and CB6/Junshi mIgTD. Panel D: The neutralization potency of the REGN10987, REGN10933 and CB6/Junshi IgG1 was compared to REGN10987 Ig-TD, CB6/Junshi Ig-TD and REGN10933 Ig-TD. The assays were performed twice and each point in duplicate. The error bars indicate the standard errors. Computed IC50 data (nM and µg/ml) and fold change based on nM are \nshown in panel E. 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Terence Rabbitts is a stake holder in Quadrucept Ltd. None of the other authors have any conflicts of interest to declare.","formattedTitle":"\u003cp\u003eApplication of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. 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Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed Quads, that retain efficient binding to the SARS-CoV-2 spike protein and show up to two orders of magnitude enhancement in neutralization of pseudovirus infection. The tetramerization method is simple and general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.","manuscriptTitle":"Application of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-10 15:03:45","doi":"10.21203/rs.3.rs-259484/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-04-05T08:47:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-10T01:04:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103c1165-dc92-470c-95c4-844946bfb3fa","date":"2021-03-08T03:12:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-03-07T07:27:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-03-07T06:52:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-03-07T06:12:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-03-07T06:09:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-02-20T16:57:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36a6087f-5327-4c5a-b3b1-0dabaa5bde38","owner":[],"postedDate":"March 10th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2876538,"name":"Infectious Diseases"},{"id":2876539,"name":"Scientific Communication"},{"id":2876540,"name":"Health Economics \u0026 Outcomes Research"}],"tags":[],"updatedAt":"2021-04-29T10:59:11+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-10 15:03:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-259484","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-259484","identity":"rs-259484","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}