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It catalytically and irreversibly cleaves RAS family proteins and has shown potent antitumor activity. To advance this unconventional intracellular protein therapeutic toward translation, we characterized its thermal stability, developed a capillary-based Simple Western immunoassay for quantitative blood analysis, measured plasma pharmacokinetics and tumor-associated exposure, and linked these measurements to intratumoral signaling and tumor response. RRSP\u2013RBD\u2013TAT retained its secondary structure and anticancer activity for 3 days at 20\u201337 \u00b0C, whereas temperatures above approximately 50 \u00b0C caused rapid unfolding, aggregation, and irreversible loss of activity. The Simple Western assay separated immunoreactive RRSP\u2013RBD\u2013TAT from the albumin-rich region of serum and plasma, enabling quantitative concentration\u2013time profiling after intravenous administration. Following a single 50 mg\/kg dose, plasma concentrations declined rapidly with a biexponential profile, whereas apparent micromolar tumor-associated concentrations were detected at 3 and 6 h. Tumors showed near-complete depletion of intact RAS and prolonged suppression of ERK phosphorylation, which persisted after tumor-associated drug levels had declined below the practical detection range. An integrated semimechanistic pharmacokinetic\u2013pharmacodynamic\u2013tumor growth inhibition model reproduced the observed dose-dependent responses. Exploratory human-scaled simulations revealed dose- and schedule-dependent effects and suggested that both improved intracellular potency and prolonged systemic persistence could lower the projected effective dose, with potency enhancement producing the larger shift under the current model assumptions.<\/jats:p>","DOI":"10.1021\/acsptsci.6c00339","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T12:01:55Z","timestamp":1787227315000},"page":"2639-2653","source":"Crossref","is-referenced-by-count":0,"title":["Thermal Stability,\nPlasma Pharmacokinetics, and Tumor\nResponse Modeling of a Cell-Penetrating Protein That Cleaves RAS Family\nGTPases"],"prefix":"10.1021","volume":"9","author":[{"given":"Takanari","family":"Sugawara","sequence":"first","affiliation":[{"name":"Gifu University , , ,","place":["Gifu, Japan, 501-1194"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teiko Komori","family":"Nomura","sequence":"additional","affiliation":[{"name":"Gifu University , , ,","place":["Gifu, Japan, 501-1194"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3698-5975","authenticated-orcid":true,"given":"Ryo","family":"Honda","sequence":"additional","affiliation":[{"name":"Gifu University , , ,","place":["Gifu, Japan, 501-1194"]},{"name":"Gifu University , , ,","place":["Gifu, Japan, 501-1193"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"2026091109131807600_cit1","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1186\/s12943-025-02364-0","article-title":"Evolution of direct\nRAS inhibitors: from undruggable target to clinical breakthroughs","volume":"24","author":"Wang","year":"2025","journal-title":"Mol. Cancer"},{"key":"2026091109131807600_cit2","doi-asserted-by":"publisher","first-page":"2803","DOI":"10.3390\/cancers17172803","article-title":"KRAS G12C inhibition\nin solid tumors: biological breakthroughs, clinical evidence, and\nopen challenges","volume":"17","author":"Vitiello","year":"2025","journal-title":"Cancers"},{"key":"2026091109131807600_cit3","doi-asserted-by":"publisher","first-page":"994","DOI":"10.1158\/2159-8290.CD-24-0027","article-title":"Translational and therapeutic evaluation of RAS-GTP\ninhibition by RMC-6236 in RAS-driven cancers","volume":"14","author":"Jiang","year":"2024","journal-title":"Cancer Discovery"},{"key":"2026091109131807600_cit4","doi-asserted-by":"publisher","first-page":"2171","DOI":"10.1038\/s41591-022-02007-7","article-title":"Anti-tumor\nefficacy of a potent and selective non-covalent\nKRASG12D inhibitor","volume":"28","author":"Hallin","year":"2022","journal-title":"Nat. Med."},{"key":"2026091109131807600_cit5","doi-asserted-by":"publisher","first-page":"254","DOI":"10.1038\/s42004-025-01662-4","article-title":"Discovery of KRAS (G12D) selective degrader\nASP3082","volume":"8","author":"Yoshinari","year":"2025","journal-title":"Commun. Chem."},{"key":"2026091109131807600_cit6","doi-asserted-by":"publisher","first-page":"2135","DOI":"10.1158\/2159-8290.CD-24-0177","article-title":"Mechanisms\nof resistance to oncogenic KRAS\ninhibition in pancreatic cancer","volume":"14","author":"Dilly","year":"2024","journal-title":"Cancer Discovery"},{"key":"2026091109131807600_cit7","doi-asserted-by":"publisher","first-page":"1581","DOI":"10.1016\/j.chembiol.2021.04.013","article-title":"Specific inhibition\nof oncogenic\nRAS using cell-permeable RAS-binding domains","volume":"28","author":"Nomura","year":"2021","journal-title":"Cell Chem. Biol."},{"issue":"1","key":"2026091109131807600_cit8","doi-asserted-by":"publisher","first-page":"6495","DOI":"10.1038\/s41467-026-73300-z","article-title":"Protein-based Pan-RAS Inhibitor Induces Tumor Regression\nin female mice via IFN\u03b3 and CD8+ T Cell-Dependent Tumor Necrosis","volume":"17","author":"Nomura","year":"2026","journal-title":"Nat. Commun."},{"key":"2026091109131807600_cit9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/ncomms8396","article-title":"Site-specific\nprocessing of Ras and Rap1 Switch I by a MARTX toxin effector domain","volume":"6","author":"Antic","year":"2015","journal-title":"Nat. Commun."},{"key":"2026091109131807600_cit10","doi-asserted-by":"publisher","first-page":"4526","DOI":"10.1074\/jbc.RA119.011025","article-title":"Conformation-specific\ninhibitors of activated Ras GTPases reveal limited Ras dependency\nof patient-derived cancer organoids","volume":"295","author":"Wiechmann","year":"2020","journal-title":"J. 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Res."},{"key":"2026091109131807600_cit28","doi-asserted-by":"publisher","first-page":"907","DOI":"10.3390\/pharmaceutics14050907","article-title":"Redesigning\nof Cell-Penetrating\nPeptides to Improve Their Efficacy as a Drug Delivery System","volume":"14","author":"Szabo\u0301","year":"2022","journal-title":"Pharmaceutics"}],"container-title":["ACS Pharmacology &amp; Translational\nScience"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsptsci.6c00339","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/aptsfn\/article-pdf\/9\/9\/2639\/66895561\/acsptsci.6c00339.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/aptsfn\/article-pdf\/9\/9\/2639\/66895561\/acsptsci.6c00339.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:13:37Z","timestamp":1789132417000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/aptsfn\/article\/9\/9\/2639\/5277639\/Thermal-Stability-Plasma-Pharmacokinetics-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":28,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,8,20]]},"published-print":{"date-parts":[[2026,9,11]]}},"URL":"https:\/\/doi.org\/10.1021\/acsptsci.6c00339","relation":{},"ISSN":["2575-9108"],"issn-type":[{"value":"2575-9108","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}