Structure-guided disulfide engineering restricts antibody conformation and flexibility to elicit TNFR agonism

Structure-guided disulfide engineering restricts antibody conformation and flexibility to elicit TNFR agonism | 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 Structure-guided disulfide engineering restricts antibody conformation and flexibility to elicit TNFR agonism Ivo Tews, Isabel Elliott, Jonathan Essex, Julie Herniman, Hayden Fisher, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4790930/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract A promising strategy in cancer immunotherapy is activation of immune signalling pathways through antibodies that target co-stimulatory receptors. hIgG2, one of four human antibody isotypes, is known to deliver strong agonistic activity, and modification of hIgG2 hinge disulfides can influence immune-stimulating activity. This was shown for antibodies directed against the hCD40 receptor, where cysteine-to-serine exchange mutations caused changes in antibody conformational dynamics and flexibility. Here we demonstrate that the principles of increasing agonism by restricting antibody conformation through hinge disulfide modification can be translated to the co-stimulatory receptor h4-1BB, another member of the tumour necrosis factor receptor superfamily. Furthermore, we explore structure-guided design of the anti-hCD40 antibody ChiLob7/4 and show that engineering additional disulfides can elicit even greater conformational restriction, concomitant with enhanced agonism. These results reveal the impact of modulating conformational flexibility to tune immunostimulatory activity and provide strategies for the rational design of more powerful antibody therapeutics. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Structural biology/X-ray crystallography Biological sciences/Structural biology/SAXS Biological sciences/Structural biology/Molecular modelling Full Text Additional Declarations Yes there is potential Competing Interest. MSC acts as a retained consultant for BioInvent International, consults for several other biotech companies and receives institutional payments and royalties from antibody licenses. He has received research funding from BioInvent, GSK, iTeos, UCB, Surrozen and Roche. This work is related to patent Family WO 2015/145360 protecting antibodies containing modified hIgG2 domains which elicit agonist or antagonistic properties. JWE receives funding from: GSK, AZ, AstexPharmaceuticals, Sygnature, A*STAR, UCB, dstl, DTRA, Diamond Light Source and exScientia. All other authors declare that they have no competing interests. Supplementary Files ElliottSI240723.pdf Cite Share Download PDF Status: Published Journal Publication published 12 Apr, 2025 Read the published version in Nature Communications → 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4790930","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334918261,"identity":"d3de4c00-b7bc-478c-aa0c-75c5ccaccc3c","order_by":0,"name":"Ivo Tews","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBAC+QYGBuY/QAY/kiAzXi1sICIBiCUbGBgbSNNicIBoLRLJBxgSKg7bG58/fPzBxx0M8vwNPMYG+LWkJTAknDmcuO1GWmLjzDMMhjMO8Bgn4NXCc8aAIbHtdoLZDR7DZt42BsYNDDzGBwhr+Xfb3rj/DFiLPWEt7D1ALQ23gYbngLUkgrTgdxh7W8KBhGP/E2cA/TJzZptE8ozDbMV4vS/fzHzwQUJNmj1//+EDHz622dj2tzdvlsCnBQSQXS5BKFZGwSgYBaNgFBADAEhARMTayJyDAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4704-1139","institution":"University of Southampton","correspondingAuthor":true,"prefix":"","firstName":"Ivo","middleName":"","lastName":"Tews","suffix":""},{"id":334918262,"identity":"23dd53d6-bf3a-4209-be61-e620ecf31ea0","order_by":1,"name":"Isabel Elliott","email":"","orcid":"https://orcid.org/0000-0003-4535-5932","institution":"University of Southampton","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Elliott","suffix":""},{"id":334918263,"identity":"a54f51cf-b2d1-4fc7-b9f3-9ae777ae5278","order_by":2,"name":"Jonathan Essex","email":"","orcid":"https://orcid.org/0000-0003-2639-2746","institution":"University of Southampton","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Essex","suffix":""},{"id":334918264,"identity":"ff3cbf2a-6483-40ba-aa8f-8f758223ef0f","order_by":3,"name":"Julie Herniman","email":"","orcid":"","institution":"University of Southampton","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Herniman","suffix":""},{"id":334918265,"identity":"38e57679-52f2-4a59-bde7-fd124fcb45ba","order_by":4,"name":"Hayden Fisher","email":"","orcid":"https://orcid.org/0000-0003-0093-0921","institution":"European Synchrotron Radiation Facility","correspondingAuthor":false,"prefix":"","firstName":"Hayden","middleName":"","lastName":"Fisher","suffix":""},{"id":334918266,"identity":"aa2174f2-8a83-4860-8726-6ff050d0fe85","order_by":5,"name":"H.T. 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