CRISPR engineering of armored CAR T cells enables tumor restricted payload delivery with enhanced efficacy and safety

CRISPR engineering of armored CAR T cells enables tumor restricted payload delivery with enhanced efficacy and safety | 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 Biological Sciences - Article CRISPR engineering of armored CAR T cells enables tumor restricted payload delivery with enhanced efficacy and safety Amanda Chen, Kah Min Yap, Joelle Kim, Kevin Sek, Yu-Kuan Huang, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3752711/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Abstract The efficacy of chimeric antig en receptor (CAR) T cell therapy in solid tumors is limited by immunosuppression and antigen heterogeneity. To overcome these barriers, “armored” CAR T cells, which secrete proinflammatory cytokines, have been developed. However, their clinical application has been limited due to toxicities related to peripheral expression of the armoring transgene. Here, we developed a novel CRISPR knock-in strategy that uses endogenous gene regulatory mechanisms to drive transgene expression in a tumor-localized manner. By screening endogenous genes with tumor-restricted expression, the NR4A2 and RGS16 promoters were identified to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumor site, leading to enhanced anti-tumor efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This was concomitant with improved CAR T cell polyfunctionality, activation of endogenous anti-tumor immunity, a favorable safety profile, and was applicable using CAR T cells from patients. Biological sciences/Immunology/Tumour immunology Biological sciences/Immunology/Immunotherapy Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTables1and2.xlsx Supplementary Tables 1 and 2 dPD1GFPOTIE0771ova.mp4 Supplementary Material 2d eNR4A2GFPparentalE0771.mp4 Supplementary Material 2e bMockOTIE0771ova.mp4 Supplementary Material 2b fNR4A2GFPE0771ova.mp4 Supplementary Material 2f aMockOTIparentalE0771.mp4 Supplementary Material 2a cPD1GFPOTIparentalE0771.mp4 Supplementary Material 2c bPD1IL12OTI.mp4 Supplementary Material 1b aMockOTI.mp4 Supplementary Material 1a cNR4A2IL12OTI.mp4 Supplementary Material 1c dPD1IL12humanCART.mp4 Supplementary Material 3d fNR4A2IL12humanCART.mp4 Supplementary Material 3f cPD1KOhumanCART.mp4 Supplementary Material 3c bMockhumanCART.mp4 Supplementary Material 3b eNR4A2KOhumanCART.mp4 Supplementary Material 3e aNontreatedhumanCART.mp4 Supplementary Material 3a Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Nature → 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-3752711","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":260006260,"identity":"9f0717f0-facf-4edd-8b24-1efeacd5832e","order_by":0,"name":"Amanda Chen","email":"","orcid":"","institution":"Peter MacCallum Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Chen","suffix":""},{"id":260006261,"identity":"68f6ae33-41ef-4357-8d6c-ea3fbe708b24","order_by":1,"name":"Kah Min Yap","email":"","orcid":"https://orcid.org/0000-0001-5009-9895","institution":"Peter MacCallum 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safety","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature 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To overcome these barriers, “armored” CAR T cells, which secrete proinflammatory cytokines, have been developed. However, their clinical application has been limited due to toxicities related to peripheral expression of the armoring transgene. Here, we developed a novel CRISPR knock-in strategy that uses endogenous gene regulatory mechanisms to drive transgene expression in a tumor-localized manner. By screening endogenous genes with tumor-restricted expression, the NR4A2 and RGS16 promoters were identified to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumor site, leading to enhanced anti-tumor efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This was concomitant with improved CAR T cell polyfunctionality, activation of endogenous anti-tumor immunity, a favorable safety profile, and was applicable using CAR T cells from patients.","manuscriptTitle":"CRISPR engineering of armored CAR T cells enables tumor restricted payload delivery with enhanced efficacy and safety","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 19:43:55","doi":"10.21203/rs.3.rs-3752711/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"nature","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nature","sideBox":"Learn more about [Nature](http://www.nature.com/nature/)","snPcode":"","submissionUrl":"","title":"Nature","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec97f64f-1c57-4918-8f9f-ae936686b3d4","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27460891,"name":"Biological sciences/Immunology/Tumour immunology"},{"id":27460892,"name":"Biological sciences/Immunology/Immunotherapy"}],"tags":[],"updatedAt":"2025-07-03T07:09:36+00:00","versionOfRecord":{"articleIdentity":"rs-3752711","link":"https://doi.org/10.1038/s41586-025-09212-7","journal":{"identity":"nature","isVorOnly":false,"title":"Nature"},"publishedOn":"2025-07-02 04:00:00","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-04-28 19:43:55","video":"","vorDoi":"10.1038/s41586-025-09212-7","vorDoiUrl":"https://doi.org/10.1038/s41586-025-09212-7","workflowStages":[]},"version":"v1","identity":"rs-3752711","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3752711","identity":"rs-3752711","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}