Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer

Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer | 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 Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer Seul-Gi Lee, Myungjee Lee, Hye-Min Lee, Ga-Hyun Son, Sang-Rok Yoon, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8589580/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted 13 You are reading this latest preprint version Abstract Background Pancreatic ductal adenocarcinoma (PDAC) often escapes T cell–mediated immunity through impaired major histocompatibility complex class I (MHC-I) antigen presentation, rendering it largely refractory to current immunotherapies. Because natural killer (NK) cells are capable of eliminating MHC-I–low tumor cells, redirecting NK cytotoxicity represents a promising strategy for these immune-cold tumors. Among activating NK receptors, NKG2D recognizes stress-induced ligands (MICA/B and ULBP family members) that are broadly upregulated across malignancies, including PDAC. However, the impact of NKG2D ligand (NKG2DL) identity and binding affinity on NK-cell engager potency has not been quantitatively defined. Results We engineered a panel of epidermal growth factor receptor (EGFR)–targeted, heterodimeric IgG1 Fc–based αEGFR×NKG2DL immune cell engagers (ICEs), each pairing a panitumumab-derived EGFR arm with one of six human NKG2DL ectodomains (MICA, MICB, ULBP1, ULBP2, and the ULBP6 allelic variants ULBP0601 and ULBP0602). All ICEs bound specifically to EGFR and NKG2D, and NKG2D binding affinity correlated directly with NK-cell cytotoxic potency across PDAC lines expressing different EGFR densities. Among native ligands, ULBP0602 exhibited the strongest NKG2D binding and highest killing activity. Yeast display–based affinity maturation of ULBP0602 yielded the variant ULBP6#2, which showed approximately a 13-fold improvement in affinity due primarily to slower dissociation kinetics. The affinity-matured αEGFR×ULBP6#2 induced up to an 11-fold reduction in EC₅₀ and greater maximal lysis than the parental construct, along with enhanced IFN-γ and TNF-α secretion by NK cells. In NK-humanized NSG mice bearing PDAC xenografts, αEGFR×ULBP6#2 achieved superior tumor growth inhibition compared with αEGFR×ULBP0602 in both PANC-1 (89% versus 57%) and BxPC-3 (60% versus 25%) models, without observable toxicity or weight loss. Conclusions These findings establish a quantitative affinity–activity relationship for NKG2D-engaging ICEs and demonstrate that affinity maturation of ULBP6 effectively translates stronger NKG2D binding into enhanced NK-cell effector function and in vivo antitumor efficacy. This work provides a generalizable design framework for engineering high-potency, tumor-tethered NKG2D engagers for the immunotherapy of PDAC and other MHC-I–deficient solid tumors. αEGFR×NKG2DL immune cell engager (ICE) NKG2D ULBP6 affinity maturation Natural killer cells Pancreatic ductal adenocarcinoma (PDAC) Full Text Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.eps SupplementaryEGFRNKG2DL.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted Editorial decision: Revision requested 10 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviews received at journal 04 Feb, 2026 Reviews received at journal 28 Jan, 2026 Reviews received at journal 25 Jan, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers agreed at journal 16 Jan, 2026 Reviewers invited by journal 16 Jan, 2026 Editor assigned by journal 14 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 13 Jan, 2026 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-8589580","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576822479,"identity":"796b28e6-cc42-477d-bc6e-c474a0af4627","order_by":0,"name":"Seul-Gi Lee","email":"","orcid":"","institution":"Ajou University","correspondingAuthor":false,"prefix":"","firstName":"Seul-Gi","middleName":"","lastName":"Lee","suffix":""},{"id":576822481,"identity":"b66d215f-00ea-4bc2-bdf7-c576f85c8f8c","order_by":1,"name":"Myungjee Lee","email":"","orcid":"","institution":"Ajou 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cancer","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"αEGFR×NKG2DL immune cell engager (ICE), NKG2D, ULBP6 affinity maturation, Natural killer cells, Pancreatic ductal adenocarcinoma (PDAC)","lastPublishedDoi":"10.21203/rs.3.rs-8589580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8589580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) often escapes T cell–mediated immunity through impaired major histocompatibility complex class I (MHC-I) antigen presentation, rendering it largely refractory to current immunotherapies. Because natural killer (NK) cells are capable of eliminating MHC-I–low tumor cells, redirecting NK cytotoxicity represents a promising strategy for these immune-cold tumors. Among activating NK receptors, NKG2D recognizes stress-induced ligands (MICA/B and ULBP family members) that are broadly upregulated across malignancies, including PDAC. However, the impact of NKG2D ligand (NKG2DL) identity and binding affinity on NK-cell engager potency has not been quantitatively defined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe engineered a panel of epidermal growth factor receptor (EGFR)–targeted, heterodimeric IgG1 Fc–based αEGFR×NKG2DL immune cell engagers (ICEs), each pairing a panitumumab-derived EGFR arm with one of six human NKG2DL ectodomains (MICA, MICB, ULBP1, ULBP2, and the ULBP6 allelic variants ULBP0601 and ULBP0602). All ICEs bound specifically to EGFR and NKG2D, and NKG2D binding affinity correlated directly with NK-cell cytotoxic potency across PDAC lines expressing different EGFR densities. Among native ligands, ULBP0602 exhibited the strongest NKG2D binding and highest killing activity. Yeast display–based affinity maturation of ULBP0602 yielded the variant ULBP6#2, which showed approximately a 13-fold improvement in affinity due primarily to slower dissociation kinetics. The affinity-matured αEGFR×ULBP6#2 induced up to an 11-fold reduction in EC₅₀ and greater maximal lysis than the parental construct, along with enhanced IFN-γ and TNF-α secretion by NK cells. In NK-humanized NSG mice bearing PDAC xenografts, αEGFR×ULBP6#2 achieved superior tumor growth inhibition compared with αEGFR×ULBP0602 in both PANC-1 (89% versus 57%) and BxPC-3 (60% versus 25%) models, without observable toxicity or weight loss.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings establish a quantitative affinity–activity relationship for NKG2D-engaging ICEs and demonstrate that affinity maturation of ULBP6 effectively translates stronger NKG2D binding into enhanced NK-cell effector function and in vivo antitumor efficacy. This work provides a generalizable design framework for engineering high-potency, tumor-tethered NKG2D engagers for the immunotherapy of PDAC and other MHC-I–deficient solid tumors.\u003c/p\u003e","manuscriptTitle":"Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 13:40:26","doi":"10.21203/rs.3.rs-8589580/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-10T15:14:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T22:24:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T10:43:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T14:52:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T03:32:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238489149649843004283463507129636967258","date":"2026-01-21T08:36:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164049142373156569359350593855017083365","date":"2026-01-20T18:29:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"339549576203102330668997438728442502865","date":"2026-01-19T09:55:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1407258382359201841358335014826890823","date":"2026-01-17T02:20:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-16T21:22:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T16:04:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T16:04:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Biological Engineering","date":"2026-01-13T08:42:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c18480d1-dae1-4325-a51f-a3e6d6e52f26","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:05:46+00:00","versionOfRecord":{"articleIdentity":"rs-8589580","link":"https://doi.org/10.1186/s13036-026-00651-y","journal":{"identity":"journal-of-biological-engineering","isVorOnly":false,"title":"Journal of Biological Engineering"},"publishedOn":"2026-03-10 15:59:49","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2026-01-20 13:40:26","video":"","vorDoi":"10.1186/s13036-026-00651-y","vorDoiUrl":"https://doi.org/10.1186/s13036-026-00651-y","workflowStages":[]},"version":"v1","identity":"rs-8589580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8589580","identity":"rs-8589580","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}