Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting

Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting | 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 Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting Valentina Lykhopiy, Valentina Lykhopiy, Laurie Rangan, Michelle Stakenborg, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6681470/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 Regulatory T cells (Tregs) are central to maintaining immune tolerance, and their selective activation via interleukin-2 (IL-2) signaling presents a promising therapeutic strategy for autoimmune diseases and transplant rejection. In this study, we introduce a novel approach to develop IL-2 receptor (IL-2R) agonists, employing obligate trispecific antibodies that simultaneously engage all three IL-2R subunits – IL-2Rα (CD25), IL-2Rβ (CD122), and the common γ chain (CD132, γc). This design preferentially activates and expands Tregs over conventional T cells and natural killer cells that express only the dimeric IL-2R (CD122 and CD132), both ex vivo and in vivo. Incorporation of a second CD25-targeting VHH domain confers enhanced specificity and potency for CD25⁺ Tregs compared to antibodies engaging only IL-2Rβ and γc. Furthermore, extensive engineering of antibody geometry was critical to maximize Treg selectivity, highlighting the importance of spatial configuration in receptor engagement. This study reports the first successful development of obligate trispecific IL-2R-targeting antibodies and significantly expands the potential of antibody-based immunomodulation. By selectively activating the high-affinity trimeric IL-2R on Tregs, this versatile platform offers a differentiated and promising strategy for the treatment of autoimmune diseases and transplant rejection. Biological sciences/Immunology/Immunological disorders Biological sciences/Drug discovery/Biologics trispecific antibodies agonist IL-2 receptor regulatory T cells antibody engineering Full Text Additional Declarations Yes there is potential Competing Interest. V.L., L.R., M.V., G.T., N.L., E.D.L. and C.B. are (former) employees/consultants of argenx and are holders of employee equity in argenx. G.T., N.L., T.T. and L.V.R. are former employees of Dualyx. V.L., G.T., T.T., C.B., L.V.R. and S.M.S. are coinventors on the related patent. The other authors declare that they have no conflicts of interest. Supplementary Files SupplementaryInformation.pdf Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting 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. 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-6681470","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":464302086,"identity":"b99050f3-bcf0-400a-be02-f29f946807d2","order_by":0,"name":"Valentina Lykhopiy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYFAC5gbGxgYGORDzwAPitDCCtRiDtSSQoiWxAcQmSovB8YNtkjN32KTPDzv8EGiLnZxuAyEtZxLbJDeeScvdeDvNAKgl2djsAAEtkg1ALQ/bDudunJ0A0nIgcRtBLf0PwVrSDWenfyBOC78EyGFthxPkpXOItIVf4mGz5cy2NMMN0jkFBxIMiPALG3/ywZu9bTby8rPTN3/4UGEnR1ALELBIgEgDsEoDwspBgPkDiJRvIE71KBgFo2AUjEAAAFUjSzKTI/e8AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2623-8888","institution":"argenx","correspondingAuthor":true,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Lykhopiy","suffix":""},{"id":464302087,"identity":"fa506549-5374-4872-9c9b-7d43a74c2255","order_by":1,"name":"Valentina Lykhopiy","email":"","orcid":"","institution":"argenx","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Lykhopiy","suffix":""},{"id":464302088,"identity":"19439264-2738-46d8-9168-a369305e3a08","order_by":2,"name":"Laurie Rangan","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Laurie","middleName":"","lastName":"Rangan","suffix":""},{"id":464302089,"identity":"76ced04e-f9a5-40fd-8809-44c5501f8d4b","order_by":3,"name":"Michelle Stakenborg","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Stakenborg","suffix":""},{"id":464302090,"identity":"4f5262dc-83e3-46f3-82c0-016a9069966f","order_by":4,"name":"Emilie Pollenus","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Emilie","middleName":"","lastName":"Pollenus","suffix":""},{"id":464302091,"identity":"5a01768b-f7f5-4f4f-9ba7-f1462e52cec8","order_by":5,"name":"Tugsan Tezil","email":"","orcid":"","institution":"Dualyx","correspondingAuthor":false,"prefix":"","firstName":"Tugsan","middleName":"","lastName":"Tezil","suffix":""},{"id":464302092,"identity":"081cfc19-919a-4bc0-9544-b51e4b0fdd83","order_by":6,"name":"Michiel Varheust","email":"","orcid":"","institution":"argenx","correspondingAuthor":false,"prefix":"","firstName":"Michiel","middleName":"","lastName":"Varheust","suffix":""},{"id":464302093,"identity":"31fccab7-b1d0-45e5-958b-c69a61459ecd","order_by":7,"name":"Giel Tanghe","email":"","orcid":"","institution":"Dualyx","correspondingAuthor":false,"prefix":"","firstName":"Giel","middleName":"","lastName":"Tanghe","suffix":""},{"id":464302094,"identity":"d02cb75f-20e5-4175-a801-be487b69802c","order_by":8,"name":"Nina Lambrechts","email":"","orcid":"https://orcid.org/0000-0001-9233-6211","institution":"Dualyx","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Lambrechts","suffix":""},{"id":464302095,"identity":"ed4706de-1903-4103-ad0a-7f287962d639","order_by":9,"name":"Eva De Langhe","email":"","orcid":"","institution":"argenx","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"De Langhe","suffix":""},{"id":464302096,"identity":"c7ca7dda-428a-48db-a1ab-508de4520b88","order_by":10,"name":"Christophe Blanchetot","email":"","orcid":"","institution":"argenx","correspondingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Blanchetot","suffix":""},{"id":464302097,"identity":"4b299b9d-e499-4625-a479-e72272611979","order_by":11,"name":"Luc Van Rompaey","email":"","orcid":"","institution":"Dualyx","correspondingAuthor":false,"prefix":"","firstName":"Luc","middleName":"Van","lastName":"Rompaey","suffix":""},{"id":464302098,"identity":"f2752d74-b653-424f-9b6c-de4c235bf617","order_by":12,"name":"Susan Schlenner","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Schlenner","suffix":""}],"badges":[],"createdAt":"2025-05-16 14:15:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6681470/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6681470/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84414720,"identity":"de819a83-d381-4714-9810-973c1b460b41","added_by":"auto","created_at":"2025-06-11 16:15:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1153865,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6681470/v1_covered_aa061ca6-3137-480d-aa15-2c5081fbb87e.pdf"},{"id":84414005,"identity":"a3520c6b-8987-424a-b2bb-e309cb426833","added_by":"auto","created_at":"2025-06-11 16:07:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2321801,"visible":true,"origin":"","legend":"Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6681470/v1/f44ff480d54f7e51f2df08a2.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nV.L., L.R., M.V., G.T., N.L., E.D.L. and C.B. are (former) employees/consultants of argenx and are holders of employee equity in argenx. G.T., N.L., T.T. and L.V.R. are former employees of Dualyx. V.L., G.T., T.T., C.B., L.V.R. and S.M.S. are coinventors on the related patent. The other authors declare that they have no conflicts of interest.","formattedTitle":"Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting","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":"trispecific antibodies, agonist, IL-2 receptor, regulatory T cells, antibody engineering","lastPublishedDoi":"10.21203/rs.3.rs-6681470/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6681470/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Regulatory T cells (Tregs) are central to maintaining immune tolerance, and their selective activation via interleukin-2 (IL-2) signaling presents a promising therapeutic strategy for autoimmune diseases and transplant rejection. In this study, we introduce a novel approach to develop IL-2 receptor (IL-2R) agonists, employing obligate trispecific antibodies that simultaneously engage all three IL-2R subunits – IL-2Rα (CD25), IL-2Rβ (CD122), and the common γ chain (CD132, γc). This design preferentially activates and expands Tregs over conventional T cells and natural killer cells that express only the dimeric IL-2R (CD122 and CD132), both ex vivo and in vivo. Incorporation of a second CD25-targeting VHH domain confers enhanced specificity and potency for CD25⁺ Tregs compared to antibodies engaging only IL-2Rβ and γc. Furthermore, extensive engineering of antibody geometry was critical to maximize Treg selectivity, highlighting the importance of spatial configuration in receptor engagement. This study reports the first successful development of obligate trispecific IL-2R-targeting antibodies and significantly expands the potential of antibody-based immunomodulation. By selectively activating the high-affinity trimeric IL-2R on Tregs, this versatile platform offers a differentiated and promising strategy for the treatment of autoimmune diseases and transplant rejection.","manuscriptTitle":"Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 16:07:26","doi":"10.21203/rs.3.rs-6681470/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":"344c5c69-c3af-4295-a7c8-10223530262b","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49303696,"name":"Biological sciences/Immunology/Immunological disorders"},{"id":49303697,"name":"Biological sciences/Drug discovery/Biologics"}],"tags":[],"updatedAt":"2026-02-16T03:05:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 16:07:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6681470","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6681470","identity":"rs-6681470","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}