Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab

Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab | 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 Article Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab David Klenerman, Emre Fertan, Jeff Lam, Giulia Albertini, Maarten Dewilde, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5290983/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab have been developed for treatment of Alzheimer’s disease. Here, we have used single-molecule detection techniques and super-resolution imaging to characterise the binding of these antibodies to beta-amyloid aggregates including human post-mortem brain samples. Lecanemab is the best antibody in terms of binding to the small-soluble beta-amyloid aggregates, affinity, aggregate coating, and the ability to bind to post-translationally modified species, explaining its therapeutic success. Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Alzheimer's disease Biological sciences/Biophysics/Intrinsically disordered proteins Alzheimer’s disease monoclonal antibody beta-amyloid soluble aggregate super-resolution microscopy single-molecule detection therapeutic success. Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations Yes there is potential Competing Interest. Professor Bart De Strooper has been a consultant for Eli Lilly, Biogen, Janssen Pharmaceutica, Eisai, AbbVie and other companies, but not on their antibody programs. He is consultant to Muna Therapeutics, a scientific founder of Augustine Therapeutics, and a scientific founder and stockholder of Muna Therapeutics. Supplementary Files 111024MAbSuppTable.pdf Cite Share Download PDF Status: Under Review Version 1 posted 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-5290983","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":371364465,"identity":"47b9cf52-27e5-4eb5-9bd6-c67a1bda4c54","order_by":0,"name":"David Klenerman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYDCCM4wNBxIMbOTgAhKEtTA3PvhQkGZMihb2ZsMZHw4nNhCthe/MwTZpHgPm9P720wkMP2oYEmc2ENAiebYRpIUtd8aZ3A2MPccYEmcTssXgPCNIC0/uBgYg4m1gSJxHpBaJdAP+txsY/xKl5Wwj0PsGBgkGErkbmEG2EHSY5JmDwEA2SDCccePthsMyxySMCXqf70z6gwMJf/7L8/fnbnz4psZGdsYBQtYggwPEROQoGAWjYBSMAiIAAO/GRUoiyDoGAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7116-6954","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Klenerman","suffix":""},{"id":371364466,"identity":"4b3a4802-1367-4f29-9e1a-11077e081d89","order_by":1,"name":"Emre Fertan","email":"","orcid":"https://orcid.org/0000-0002-0060-5806","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Emre","middleName":"","lastName":"Fertan","suffix":""},{"id":371364467,"identity":"47a0d281-1288-4844-b1bc-896935a0dc0b","order_by":2,"name":"Jeff Lam","email":"","orcid":"https://orcid.org/0000-0001-7408-2342","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Jeff","middleName":"","lastName":"Lam","suffix":""},{"id":371364468,"identity":"3c2baf3a-7204-4799-abf1-3fd071db5396","order_by":3,"name":"Giulia Albertini","email":"","orcid":"https://orcid.org/0000-0002-8215-2432","institution":"VIB/KU Leuven Center for Brain \u0026 Disease Research","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Albertini","suffix":""},{"id":371364469,"identity":"979f8335-4989-4b31-99ef-2162e5ad06a5","order_by":4,"name":"Maarten Dewilde","email":"","orcid":"https://orcid.org/0000-0002-3138-281X","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Maarten","middleName":"","lastName":"Dewilde","suffix":""},{"id":371364470,"identity":"4d7df200-0774-4ca6-a6e2-73f3f3a04c71","order_by":5,"name":"Yunzhao Wu","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Yunzhao","middleName":"","lastName":"Wu","suffix":""},{"id":371364471,"identity":"46e1491c-ab6a-48ff-96b0-ab7a6c4da965","order_by":6,"name":"Oluwatomi Akingbade","email":"","orcid":"https://orcid.org/0000-0002-2510-2471","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Oluwatomi","middleName":"","lastName":"Akingbade","suffix":""},{"id":371364472,"identity":"55b44d31-900a-4a4a-9232-9251af25c38d","order_by":7,"name":"Dorothea Böken","email":"","orcid":"https://orcid.org/0009-0008-8443-4469","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Dorothea","middleName":"","lastName":"Böken","suffix":""},{"id":371364473,"identity":"ac72f7d2-9c74-443e-abcb-c6bed0f83d01","order_by":8,"name":"Elizabeth English","email":"","orcid":"https://orcid.org/0000-0003-0348-9318","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"English","suffix":""},{"id":371364474,"identity":"c7ff63c8-809d-4257-bf88-396eb75262bb","order_by":9,"name":"Bart De Strooper","email":"","orcid":"https://orcid.org/0000-0001-5455-5819","institution":"VIB Centre for Brain \u0026 Disease Research, Department of Neurosciences, Leuven Brain Institute, UK Dementia Research Institute at UCL, The Francis Crick Institute","correspondingAuthor":false,"prefix":"","firstName":"Bart","middleName":"","lastName":"De Strooper","suffix":""}],"badges":[],"createdAt":"2024-10-18 17:15:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5290983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5290983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68384691,"identity":"cb0c32fe-617b-4ee0-89aa-26d06e384cf0","added_by":"auto","created_at":"2024-11-06 17:20:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":633510,"visible":true,"origin":"","legend":"\u003cp\u003eVisual summary of the experiments. Monoclonal antibodies were produced in-house from \u0026nbsp;publicly available sequences and tested with silica nanoparticles coated with beta-amyloid, in-vitro aggregates, and human post-mortem Alzheimer’s disease brain homogenates using SiMoA, single-molecule pulldown, and dSTORM. The SiMoA platform utilises paramagnetic beads coated with \u0026nbsp;capture antibodies, binding to targets of interest at ultra-low concentrations, forming a small number of \u0026nbsp;immunocomplexes on the beads, which are then detected using streptavidin beta-galactosidase and \u0026nbsp;resorufin beta-D-galactopyranoside interactions, providing a binary (digital) readout for aggregate \u0026nbsp;quantification. Direct stochastic optical reconstruction microscopy (dSTORM) along with single-molecule pulldown (SiMPull) uses a glass surface optimised to capture targets of interest with high \u0026nbsp;sensitivity, which are then imaged with a resolution limit of 30 nm (under the diffraction limit of light), \u0026nbsp;allowing the morphological characterisation of soluble aggregates (scale bars are 50 nm).\u003c/p\u003e","description":"","filename":"111024MAbFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5290983/v1/21eb623e9c53f85f6ed144d3.png"},{"id":68384251,"identity":"7e767161-b8ce-45a6-a2fc-d3bf8ac4f7b2","added_by":"auto","created_at":"2024-11-06 17:12:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143180,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of the monoclonal antibodies using synthetic aggregates. Signal on \u0026nbsp;SiMoA was compared between Aducanumab, Lecanemab, Gantenerumab, and Donanemab using beta \u0026nbsp;amyloid 42 (A) and pyroglutamate beta-amyloid (B) coated glass nanoparticles at different \u0026nbsp;concentrations (x-axis shows concentration in nM). Each antibody-bead concentration combination was \u0026nbsp;tested at least on four independent wells. For beta-amyloid 42 coated beads, a significant antibody by \u0026nbsp;concentration interaction was determined (AIC = 470.4, F = 306.6, p \u0026lt; 0.001, ηp2 = 0.855). While \u0026nbsp;Donanemab did not show a dose dependent increase in signal, Aducanumab, Lecanemab, and \u0026nbsp;Gantenerumab all showed a significant increase with increased bead concentration, with the greatest \u0026nbsp;increase seen in Lecanemab (A). For pyroglutamate beta-amyloid coated beads, once again a significant \u0026nbsp;antibody by concentration interaction was present (AIC = 101.3, F = 264.4, p \u0026lt; 0.001, ηp2 = 0.860). All \u0026nbsp;four antibodies showed a concentration-dependent increase in signal, with the greatest signal acquired \u0026nbsp;from Lecanemab and Donanemab, while the weakest signal was measured from Gantenerumab (B). In \u0026nbsp;addition to coated silica-nanoparticles, in-vitro aggregates of beta-amyloid were also prepared by \u0026nbsp;incubating beta-amyloid 42 and pyroglutamate beta-amyloid for different durations. A thioflavin T \u0026nbsp;assay was performed to characterise the aggregation dynamics of different species, revealing the \u0026nbsp;formation of thioflavin T-positive aggregates over time in the samples containing pure beta-amyloid 42 \u0026nbsp;and a mixture of beta-amyloid 42 and pyroglutamate beta-amyloid, however the signal did not change \u0026nbsp;for the pure pyroglutamate beta-amyloid signal, showing a lack of aggregation (C; x-axis shows time \u0026nbsp;in minutes). Then these samples were tested on SiMoA on three independent wells, revealing a three-way interaction between the antibody, aggregate type, and concentration (AIC = 584.2, F = 23.7, p \u0026lt; \u0026nbsp;0.001, ηp2 = 0.797). Overall, the signal with the mixed aggregates was higher for all antibodies than the \u0026nbsp;pure beta-amyloid aggregates and the signal increased with aggregate concentration. The highest signal \u0026nbsp;was detected by Lecanemab, followed by Aducanumab, Gantenerumab, and Donanemab (D; x-axis \u0026nbsp;shows concentration in nM). The limit of detection was in parallel to the signal at high-concentrations \u0026nbsp;(E). The performance of Aducanumab, Lecanemab, and Gantenerumab with in-vitro aggregates at \u0026nbsp;different sizes were also compared using single-molecule pulldown (F-G), with imaging the aggregates \u0026nbsp;on two independent wells for at least nine fields of view per well. For the number of aggregates detected, \u0026nbsp;there was an antibody by aggregate type interaction (AIC = 800.0, F = 4.9, p = 0.002, ηp2 = 0.214). The \u0026nbsp;greatest number of aggregates were detected with Gantenerumab, followed by Aducanumab, and \u0026nbsp;Lecanemab. While Aducanumab and Lecanemab showed a strong preference for early and late \u0026nbsp;oligomers, the difference was smaller for Gantenerumab (F). Aggregate brightness was also measured, \u0026nbsp;with controlling for the number of dyes per antibody, providing a measure for number of antibodies \u0026nbsp;binding to unit area of each aggregate (i.e. the aggregate coating ability). Again, an antibody by \u0026nbsp;aggregate type interaction (AIC = 703317.0, F = 91.8, p \u0026lt; 0.001, ηp2 = 0.006) was found, Lecanemab achieved the highest brightness of all antibodies when tested with early oligomers, which was \u0026nbsp;significantly higher than the signal it provided with fibrils and least with late oligomers. On the other \u0026nbsp;hand, Gantenerumab and Aducanumab provided the brightest signal with fibrils, with the signal from \u0026nbsp;Aducanumab being significantly dimmer than Gantenerumab, collectively showing a strong preference \u0026nbsp;for early oligomers by Lecanemab, and a preference for fibrillar aggregates by Gantenerumab, and to a \u0026nbsp;lesser extent Aducanumab (G).\u003c/p\u003e","description":"","filename":"111024MAbFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5290983/v1/0e982c1a803f17916616dd2a.png"},{"id":68384692,"identity":"fb5d4779-51ec-4692-8d83-62bc6d22729d","added_by":"auto","created_at":"2024-11-06 17:20:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221441,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of the monoclonal antibodies using post-mortem human Alzheimer’s \u0026nbsp;disease brain homogenates. Signal on SiMoA was compared between Aducanumab, Lecanemab, \u0026nbsp;Gantenerumab, and Donanemab using human post-mortem brain homogenates from patients with Braak \u0026nbsp;stage 0, 3, or 5 Alzheimer's disease, with two independent brain samples tested in two independent wells \u0026nbsp;per condition (A). A significant antibody by Braak stage interaction was observed (AIC = 63.19, F = \u0026nbsp;128.1, p \u0026lt; 0.001, ηp2 = 0.964); while Aducanumab and Gantenerumab signal positively correlated with \u0026nbsp;the Braak stage, Lecanemab showed the highest detection with the Stage 3 samples. Meanwhile, \u0026nbsp;Donanemab signal was at the baseline for the brain homogenates containing soluble beta-amyloid \u0026nbsp;aggregates. Brain homogenates were also tested on single-molecule pulldown with dSTORM, to \u0026nbsp;measure the length (B) and area (C) of the aggregates detected by the antibodies on two independent \u0026nbsp;wells with at least two fields of view each. There was an antibody by Braak stage interaction for both \u0026nbsp;aggregate length (AIC = 231669.0, F = 54.9, p \u0026lt; 0.001, ηp2 = 0.008) and area (AIC = 497262.0, F = \u0026nbsp;61.7, p \u0026lt; 0.001, ηp2 = 0.008). All antibodies showed a size preference and mostly bound to the \u0026nbsp;aggregates 90 nanometres in length, yet the average length of the detected aggregates increased at Braak \u0026nbsp;stage 5 for Aducanumab and Gantenerumab, while Lecanemab remained preferentially binding to the \u0026nbsp;smaller aggregates at this stage. Average aggregate brightness as also measured during diffraction-limited imaging (same brain samples imaged in two independent wells with nine fields of view), with \u0026nbsp;controlling for the number of dyes per antibody, providing a measure for number of antibodies binding \u0026nbsp;to unit area of each aggregate (D). While the brightness signal was provided by Lecanemab overall and \u0026nbsp;on average the Stage 5 brains were brighter, Lecanemab actually provided the brightest signal with the \u0026nbsp;Stage 0 samples, followed by Stage 3, showing a preference for aggregates formed at early disease \u0026nbsp;stages, unlike Gantenerumab and Aducanumab, which had a brighter signal with the Stage 5 samples, \u0026nbsp;resulting in an antibody by Braak stage interaction (AIC = 466760.0, F = 98.6, p \u0026lt; 0.001, ηp2 = 0.007). \u0026nbsp;Lastly, an immuno-pulldown was performed using the antibodies to identify the amount of available \u0026nbsp;binding regions on the aggregates they detect (E). The greatest signal reduction was seen in \u0026nbsp;Aducanumab, followed by Gantenerumab, and Lecanemab, once again showing the superior aggregate \u0026nbsp;coating ability of Lecanemab.\u003c/p\u003e","description":"","filename":"111024MAbFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5290983/v1/946c5d63571e9143588c8853.png"},{"id":68385669,"identity":"745b6aef-9f2a-416b-be48-44ab7ef0c668","added_by":"auto","created_at":"2024-11-06 17:29:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1064475,"visible":true,"origin":"","legend":"","description":"","filename":"111024MAbManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5290983/v1_covered_cc60677d-79a3-4ee4-a064-396b7be1f4e0.pdf"},{"id":68384250,"identity":"b9512ab5-c78d-4fae-bdd8-fa19cef72509","added_by":"auto","created_at":"2024-11-06 17:12:58","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12272,"visible":true,"origin":"","legend":"","description":"","filename":"111024MAbSuppTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5290983/v1/d77de1fa22a5eaa0da4900d2.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nProfessor Bart De Strooper has been a consultant for Eli Lilly, Biogen, Janssen Pharmaceutica, Eisai, AbbVie and other companies, but not on their antibody programs. He is consultant to Muna Therapeutics, a scientific founder of Augustine Therapeutics, and a scientific founder and stockholder of Muna Therapeutics.","formattedTitle":"Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alzheimer’s disease, monoclonal antibody, beta-amyloid, soluble aggregate, super-resolution microscopy, single-molecule detection, therapeutic success.","lastPublishedDoi":"10.21203/rs.3.rs-5290983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5290983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab have been developed for treatment of Alzheimer’s disease. Here, we have used single-molecule detection techniques and super-resolution imaging to characterise the binding of these antibodies to beta-amyloid aggregates including human post-mortem brain samples. Lecanemab is the best antibody in terms of binding to the small-soluble beta-amyloid aggregates, affinity, aggregate coating, and the ability to bind to post-translationally modified species, explaining its therapeutic success.","manuscriptTitle":"Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 17:12:54","doi":"10.21203/rs.3.rs-5290983/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eb590c38-9856-42b2-a020-ae608005b265","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":39519211,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Alzheimer's disease"},{"id":39519212,"name":"Biological sciences/Biophysics/Intrinsically disordered proteins"}],"tags":[],"updatedAt":"2024-11-06T17:12:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-06 17:12:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5290983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5290983","identity":"rs-5290983","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}