Cross-Species Quantitative Benchmarking of SSTR2 Radioligands in Fresh-Frozen Brain Sections: An Adaptable Framework for Radiopharmaceutical Development

Cross-Species Quantitative Benchmarking of SSTR2 Radioligands in Fresh-Frozen Brain Sections: An Adaptable Framework for Radiopharmaceutical Development | 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 Cross-Species Quantitative Benchmarking of SSTR2 Radioligands in Fresh-Frozen Brain Sections: An Adaptable Framework for Radiopharmaceutical Development Caitlin McCutcheon, Zach Lawrence, Marie James, Taryn Palluccio, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8981508/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Rationale Somatostatin receptor subtype 2 (SSTR2) is a G-protein coupled receptor overexpressed in multiple cancers, including neuroendocrine tumors, small cell lung cancer, and hepatocellular carcinoma. Clinical validation of SSTR2-targeted radioligand therapy (Lutathera™) has driven development of next-generation ligands. Progress in next-generation SSTR2 ligand development is strengthened by quantitative platforms that preserve native receptor context and enable quantitative, cross-species binding data to drive translation and radiotherapeutic advancement. Methods Fresh-frozen tissue sections from mouse, rat, cynomolgus monkey and human brain were assessed for SSTR2 binding using the high affinity antagonist [177Lu]-DOTA-LM3. Non-equilibrium kinetics, and equilibrium binding methods were used to derive apparent affinity, target density, and affinity measurements. Section-wipe/gamma counting enabled absolute quantification without requiring isotope standards. Autoradiography and SSTR2 immunohistochemistry confirmed anatomic localization. Characterization of unlabeled DOTA-LM3 and its unlabeled lutetium- and gallium-complexed derivatives were compared to assess the effects of radiometal conjugation. Results Across all species, [177Lu]-DOTA-LM3 exhibited specific, saturable binding characteristic of a single class of high-affinity sites. Autoradiography signals co-localized with SSTR2-rich regions identified by immunohistochemistry, confirming expected distribution patterns. Affinities derived from unlabeled and radiolabeled DOTA-LM3 were consistent, validating assay reproducibility. Whilst affinities of agonists and antagonists were conserved across species, radiometal substitution altered ligand properties: the lutetium-complexed form preserved affinity comparable to the chelator-only ligand, while the gallium-complexed variant showed markedly reduced affinity. Conclusions This native-tissue platform provides efficient, translatable, reproducible and absolute quantification of SSTR2 ligand binding without tissue standards, supporting early selection of chemotypes, radiometals, and species-bridging expectations before in vivo studies. Somatostatin receptor subtype2 (SSTR2) SSTR2 radioligand therapy Novel candidate screening Radioligand Binding Radiolabeled DOTA-LM3 Full Text Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revisions 16 Apr, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers invited by journal 12 Mar, 2026 Editor assigned by journal 08 Mar, 2026 First submitted to journal 06 Mar, 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. 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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-8981508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605229125,"identity":"c589d78c-31db-4376-98f4-67583f00fce2","order_by":0,"name":"Caitlin McCutcheon","email":"","orcid":"","institution":"Ratio Therapeutics, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Caitlin","middleName":"","lastName":"McCutcheon","suffix":""},{"id":605229126,"identity":"6af0147d-ea53-4a25-8b7e-3aaaf3ab94a6","order_by":1,"name":"Zach Lawrence","email":"","orcid":"","institution":"Ratio Therpeutics, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Zach","middleName":"","lastName":"Lawrence","suffix":""},{"id":605229127,"identity":"703087cf-29e7-47a0-89a2-ee7ac1c48694","order_by":2,"name":"Marie James","email":"","orcid":"","institution":"Ratio Therapeutics, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"James","suffix":""},{"id":605229128,"identity":"9c593c5f-154d-46cc-831b-97cc202da634","order_by":3,"name":"Taryn Palluccio","email":"","orcid":"","institution":"Ratio Therapeutics, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Taryn","middleName":"","lastName":"Palluccio","suffix":""},{"id":605229129,"identity":"2843eb2d-238e-422c-95d6-8fd1b888e849","order_by":4,"name":"Maha Chowdhury","email":"","orcid":"","institution":"Ratio Therapeutics, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Maha","middleName":"","lastName":"Chowdhury","suffix":""},{"id":605229130,"identity":"9b60fd74-70b1-468a-8c2a-5eb42bee4964","order_by":5,"name":"Pradeep K. 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Clinical validation of SSTR2-targeted radioligand therapy (Lutathera™) has driven development of next-generation ligands. Progress in next-generation SSTR2 ligand development is strengthened by quantitative platforms that preserve native receptor context and enable quantitative, cross-species binding data to drive translation and radiotherapeutic advancement.\n\nMethods\n\nFresh-frozen tissue sections from mouse, rat, cynomolgus monkey and human brain were assessed for SSTR2 binding using the high affinity antagonist [177Lu]-DOTA-LM3. Non-equilibrium kinetics, and equilibrium binding methods were used to derive apparent affinity, target density, and affinity measurements. Section-wipe/gamma counting enabled absolute quantification without requiring isotope standards. Autoradiography and SSTR2 immunohistochemistry confirmed anatomic localization. Characterization of unlabeled DOTA-LM3 and its unlabeled lutetium- and gallium-complexed derivatives were compared to assess the effects of radiometal conjugation.\n\nResults\n\nAcross all species, [177Lu]-DOTA-LM3 exhibited specific, saturable binding characteristic of a single class of high-affinity sites. Autoradiography signals co-localized with SSTR2-rich regions identified by immunohistochemistry, confirming expected distribution patterns. Affinities derived from unlabeled and radiolabeled DOTA-LM3 were consistent, validating assay reproducibility. Whilst affinities of agonists and antagonists were conserved across species, radiometal substitution altered ligand properties: the lutetium-complexed form preserved affinity comparable to the chelator-only ligand, while the gallium-complexed variant showed markedly reduced affinity.\n\nConclusions\n\nThis native-tissue platform provides efficient, translatable, reproducible and absolute quantification of SSTR2 ligand binding without tissue standards, supporting early selection of chemotypes, radiometals, and species-bridging expectations before in vivo studies.","manuscriptTitle":"Cross-Species Quantitative Benchmarking of SSTR2 Radioligands in Fresh-Frozen Brain Sections: An Adaptable Framework for Radiopharmaceutical Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 10:42:37","doi":"10.21203/rs.3.rs-8981508/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-04-16T07:39:58+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-03-15T14:41:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-12T17:14:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-08T16:35:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Imaging and Biology","date":"2026-03-06T07:52:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1670150d-5e5b-49bb-9625-1c7a7ab778a3","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T18:18:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 10:42:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8981508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8981508","identity":"rs-8981508","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}