Localized In Vivo Prodrug Activation Using Radionuclides

RAiDER concept. Targeted radionuclides accumulate in tumor tissues, locally delivering radiation to chemically activated caged prodrugs. Radionuclide-mediated prodrug activation occurs through reduction of phenyl azide caging moiety (orange), leading to linker self-immolation and release of active drug payload (purple) from its drug delivery vehicle (blue), which in this work is long-circulating serum albumin. Consequently, locally activated therapeutic payloads combine with ionizing radiation to maximize tumor cytotoxicity while sparing off-target tissues. Image created with BioRender.com.

Radionuclide-mediated drug release from caged MMAE. (A) Caged MMAE (10 µM) was exposed to different radionuclides with varying activities. Corresponding total radiation dose release was estimated using TOPAS-nBio (26) and compared with prodrug-activated drug release, indicating varying radionuclide efficiencies (µM/Gy). (B) Drug release efficiencies (nM/Gy) across radionuclides and external beam modalities. Highest release efficiency (^99mTc) was compared with other nuclides or modalities (Violin plots: black line, median; dotted color lines, quartiles, 1-way ANOVA with Tukey multiple comparison test). Some values for ¹³⁷Cs irradiation were previously presented (18) and reproduced here to facilitate comparison. Pearson correlation coefficient R was calculated across all data points. linac = linear particle accelerator.

RAiDER restores biologic prodrug activities in vitro. (A) Cytotoxicity of albumin (Alb)–conjugated caged MMAE; ^99mTc-activated, Alb-conjugated caged MMAE; and free MMAE on TBP3743 anaplastic thyroid cancer, measured 72 h after treatment by resazurin-based assay (n = 3, mean ± SE). (B) Representative images and quantification of TBP3743 colony formation (mean ± SE, 2-way ANOVA with Geisser–Greenhouse correction). (C) Representative images of microtubule immunofluorescence (top) and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; bottom) 24 h after treatment in TBP3743 cells (mean ± SE; 2-way ANOVA with Geisser–Greenhouse correction; scale bars, 200 µm). DAPI = 4′,6-diamidino-2-phenylindole; frac. max = fraction of maximal count/signal as observed in control conditions.

Computational modeling of radionuclide-mediated drug release using TOPAS-nBio. (A) Conceptual mechanism to explain radionuclide-dependent RAiDER efficiencies. Red shading illustrates spatial distribution and frequency of free radical–generating ionization events from given radionuclide within vial. Ionization clouds interact with prodrug molecules most efficiently for nuclides generating lower-energy electrons. (B) Correlation between observed drug release across radionuclides and their estimated number of electrons generated within 100- to 110-keV energy window. (C) Comparison of drug release efficiency with dose imparted by low-energy electrons (LEEs) across radionuclides. Pearson correlation coefficient R was calculated across all data points. LET = linear energy transfer.

In vivo RAiDER biodistribution and prodrug activation. (A) Biodistribution of fluorescent albumin (Alb)–conjugated caged MMAE, measured by tissue Cy5 fluorescence, and [^99mTc]Tc-FAPI-34, measured by γ-scintillation counting in B6129SF1/J mice bearing syngeneic TBP3743 tumors. (B) Biodistribution of activated Alb-conjugated caged MMAE (with [^99mTc]Tc-FAPI-34) compared with nonactivated Alb-conjugated caged MMAE, as measured by liquid chromatography–mass spectrometry or high-performance liquid chromatography. Data are mean ± SE (n = 3–4 mice per condition, 2-tailed t test shown). %ID/g = percentage injected dose per gram of tissue.

RAiDER enhances ability of targeted radionuclides to block tumor growth. (A and B) Mice bearing TBP3743 tumors were treated with albumin (Alb)–conjugated caged MMAE (5 mg/kg/dose) and either [^99mTc]Tc-FAPI-34 (A; 18.5 MBq/dose; n = 18 total mice, 36 total tumors) or [¹⁷⁷Lu]Lu-FAPI-34 (B; 18.5 MBq; n = 20 total mice, 40 total tumors). Caliper measurements of tumor growth are shown over time (left) and with individual tumor sizes on indicated day (middle; 2-tailed Mann–Whitney tests compare combination and pooled control or monotherapies). Mouse mass was measured during treatment time course (right). Data are displayed as mean ± SE.