Thermal Stability, Plasma Pharmacokinetics, and Tumor Response Modeling of a Cell-Penetrating Protein That Cleaves RAS Family GTPases

Abstract

RRSP–RBD–TAT is a cell-penetrating fusion protein composed of a RAS/Rap1-specific endopeptidase (RRSP), a RAS-binding domain (RBD), and a transactivator-of-transcription (TAT) cell-permeable peptide. 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–RBD–TAT retained its secondary structure and anticancer activity for 3 days at 20–37 °C, whereas temperatures above approximately 50 °C caused rapid unfolding, aggregation, and irreversible loss of activity. The Simple Western assay separated immunoreactive RRSP–RBD–TAT from the albumin-rich region of serum and plasma, enabling quantitative concentration–time 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–pharmacodynamic–tumor 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.