Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks.

Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks. | 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 Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks. Jessica Pougoue Ketchemen, Stephen Ahenkorah, Emmanuel Nwangele, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6813431/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background Actinium-225 ( 225 Ac) based targeted alpha therapies (TAT) have emerged as a promising strategy for the treatment of several cancer types due to its favourable decay properties, including high linear energy transfer and short particle range, which enable precise tumour targeting. However, there are limited bifunctional chelators (BFCs) available for 225 Ac. In this study, we aim to evaluate the potential of DEPA-based chelators for 225 Ac-labelling. Results The BFCs 3p- C -DEPA- NO2 , 3p- C -DEPA- NCS , and 3p- C -DEPA − TFP -PEG 4 were synthesized with high yield (≥ 86%) and purity (> 96%). Excellent radiochemical conversions (RCCs) were achieved for [ 225 Ac]Ac-3p- C -DEPA- NO2 across a range of concentrations (1–20 µM) with high RCC’s (93.7 to 96.8%) after 1 hour at room temperature. Stability studies demonstrated that over 95% of this 225 Ac-labelled complex remained intact after 6 days in human serum. The HPLC and bioanalyzer analysis of the immunoconjugates 3p- C -DEPA − TFP -PEG 4 -trastuzumab, DOTA-trastuzumab, 3p- C -DEPA-trastuzumab and macropa-trastuzumab showed 98% purity with less than 2% impurities. A RCC of 94.6% was obtained for [ 225 Ac]Ac-3p- C -DEPA-trastuzumab, 93.5% for [ 225 Ac]Ac-3p- C -DEPA − TFP -PEG 4 -trastuzumab, 80.9% for [ 225 Ac]Ac-DOTA-trastuzumab, and 96.5% for [ 225 Ac]Ac-macropa-trastuzumab after 2 h incubation at 37°C. In PBS, high stability of [ 225 Ac]Ac-3p- C -DEPA − TFP -PEG 4 -trastuzumab was observed (91.3 ± 4.3%), which is comparable to that of [ 225 Ac]Ac-macropa-trastuzumab (81.9 ± 5.6%). In contrast, [ 225 Ac]Ac-3p- C -DEPA-trastuzumab and [ 225 Ac]Ac-DOTA-trastuzumab were less stable in PBS with only 48.3 ± 1.2% and 60.1 ± 0.6% intact tracer left after 10 d. There were no major significant differences between the biodistribution profile of [ 225 Ac]Ac-3p-C-DEPA-trastuzumab, [ 225 Ac]Ac-DOTA-trastuzumab and [ 225 Ac]Ac-macropa-trastuzumab in all organs of interest (p > 0.05 for all organs). However, the liver uptake of [ 225 Ac]Ac-DOTA-trastuzumab (14.1 ± 2.9% IA/g) was higher than [ 225 Ac]Ac-3p-C-DEPA-trastuzumab (9.0 ± 3.3% IA/g) (p = 0.04). Conclusions 3p- C -DEPA − TFP -PEG₄ demonstrated excellent potential as a bifunctional chelator for ²²⁵Ac, showing high radiolabelling efficiency under mild conditions and outstanding in vitro stability of the resulting 225 Ac-labelled bioconjugate. Further preclinical studies are warranted to validate its therapeutic potential. Targeted alpha therapy Actinium-225 bifunctional chelators 3p-C-DEPA trastuzumab Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND Alpha-particle (α-particle) emitting radioisotopes have emerged as particularly promising agents for the selective eradication of cancer cells [ 1 ]. Due to their high linear energy transfer (LET) and short path length in biological tissues, α-particles are capable of depositing large amounts of energy over a limited distance. This confers high cytotoxicity specifically to targeted tumour cells while minimizing collateral damage to surrounding healthy tissue. Importantly, the high LET of α-particles is more effective in inducing lethal DNA double-strand breaks compared to the lower LET of beta-minus particles (β − -particles) [ 2 – 5 ]. Several α-emitting radioisotopes, including 225 Ac, have demonstrated superior therapeutic potential in both preclinical and clinical settings when compared with their β − -emitting counterparts such as Yttrium-90 ( 90 Y) and Lutetium-177 ( 177 Lu) [ 6 – 8 ]. Among α-emitting radioisotopes, 225 Ac (t 1/2 = 9.9 d, E α = 5.8 MeV) is particularly notable due to its decay chain, which results in the emission of four α-particles, each contributing to its potent cytotoxic effect. Moreover, its relatively long half-life of 9.9 d is well-suited to long circulating biologicals such as monoclonal antibodies, whose pharmacokinetics align with this timeframe [ 9 , 10 ]. The clinical success of targeted α-therapy in metastatic castration-resistant prostate cancer using [²²⁵Ac]Ac-PSMA-617 has significantly accelerated interest in the development of novel 225 Ac-labelled radiopharmaceuticals for a wider spectrum of malignancies, including approaches based on 225 Ac-labelled monoclonal antibodies [ 8 ]. Efficient radiolabelling of ²²⁵Ac under mild conditions, combined with high kinetic inertness and thermodynamic stability of the resulting complex, is essential for the safe and effective application of TAT. These factors are critical to prevent in vivo dissociation of the radiometal, which can lead to off-target radiation exposure and dose-limiting toxicity [ 11 , 12 ]. The increasing clinical momentum behind TAT has intensified the development of new chelating agents specifically optimized for 225 Ac, as the in vivo stability of the radiometal complex is a key determinant of therapeutic success. [ 13 ]. A variety of 225 Ac-chelators have been reported, including EDTA, PEPA, CHX-DTPA, DTPA, DOTA, TETA, DOTMP, TETPA, and macropa. Among these, DOTA and macropa have shown the greatest promise in terms of practical applicability and coordination performance [ 14 – 18 ]. DOTA, a 12-membered macrocycle ligand with four nitrogen atoms and four pendant carboxylates, offers an octadentate coordination geometry well-suited for many trivalent radiometals such as ⁶⁸Ga³⁺, ¹¹¹In³⁺, and ¹⁷⁷Lu³⁺. It has been successfully integrated into multiple FDA-approved radiopharmaceuticals, including [⁶⁸Ga]Ga-DOTATATE and [¹⁷⁷Lu]Lu-DOTATATE, for both diagnostic and therapeutic use. However, the use of DOTA for ²²⁵Ac is limited by several factors. Most notably, radiolabelling requires elevated temperatures (80–95°C) to achieve high radiochemical yields, making it incompatible with heat-sensitive biomolecules such as monoclonal antibodies [ 19 ]. From a clinical perspective, room-temperature labelling in under 20 minutes is preferred to streamline radiopharmaceutical preparation and to avoid radiolytic degradation of heat-sensitive targeting vectors [ 20 , 21 ]. Additionally, the kinetic inertness of 225 Ac-DOTA complexes has been challenged by several in vitro and in vivo studies, which report partial release of ²²⁵Ac from the chelator over time [ 16 ]. This instability highlights DOTA’s limitations as a long-term carrier for ²²⁵Ac and underscores the need for chelators tailored to the unique coordination chemistry of large, low-charge-density ions. With an ionic radius of ~ 1.22 Å, Ac³⁺ forms weak electrostatic interactions with typical donor atoms, complicating efforts to achieve both strong binding and kinetic stability [ 22 ]. Macropa (bp18c6), a 4,13-diaza-18-crown-6-macrocycle ligand with two pendant picolinate arms, has demonstrated strong chelation capabilities for 225 Ac due to its high selectivity for large trivalent ions, particularly among the lanthanide and actinide series [ 23 , 24 ]. One of its major advantages over traditional chelators such as DOTA is its ability to form stable complexes with 225 Ac within 5 minutes at room temperature, even at low micromolar concentrations [ 24 , 25 ]. Macropa has also shown excellent in vitro and in vivo stability when conjugated to both small molecules and monoclonal antibodies [ 26 ]. For example, Schatz et al. evaluated the efficacy of [ 225 Ac]Ac-macropa-pelgifatamab in various cell-derived and patient-derived prostate cancer xenografts [ 27 ]. Despite its favourable coordination properties, the macropa − NCS derivative, commonly used for bioconjugation, suffers from limited shelf-life due to rapid degradation. This has prompted efforts to develop more chemically robust analogues. Recently, Kadassery et al. reported the synthesis of a more stable bifunctional macropa derivative, H 2 BZmacropa − NCS , via a five-step procedure [ 28 ]. However, the [²²⁵Ac]Ac-BZmacropa-GC33 conjugate (antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3) showed only 55% stability in human serum, indicating that further optimization is needed to improve stability. 3p- C -DEPA − NO2 (1, 2- [(carboxymethyl)] [5-(4-nitrophenyl-1-[4, 7, 10-tris(carboxymethyl) -1, 4, 7, 10- tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), is a decadentate ligand featuring ten electron donor atoms for complex formation. Its bifunctional derivative, 3p- C -DEPA − NCS , has been reported to rapidly chelate 90 Y and 177 Lu within 1 minute, achieving radiolabelling efficiencies of 89% and 94%, respectively [ 29 ]. However, to date, no data have been reported regarding its performance with 225 Ac. In this study, we aim to evaluate the potential of DEPA-based chelators for 225 Ac-labelling. We first evaluated the radiolabelling efficiency and in vitro stability of the chelator 3p- C -DEPA- NO₂ with ²²⁵Ac. We then synthesized two BFC derivatives, 3p- C -DEPA- NCS and 3p- C -DEPA- TFP -PEG 4 and conjugated them to the monoclonal antibody trastuzumab. The radiochemical stability of the resulting ²²⁵Ac-conjugates was assessed, and finally, we conducted a biodistribution study to evaluate the in vivo behaviour. As a reference, macropa- NCS and DOTA- NCS were used in the conjugation and radiolabelling of trastuzumab to compare the stability and performance of these established chelators with the newly synthesized BFCs. MATERIALS AND METHODS General All chemicals and solvents were purchased from commercial suppliers such as Sigma-Aldrich (Bornem, Belgium), Fluka (Bornem, Belgium), Fisher (Doornik, Belgium) and Acros Organics (Geel, Belgium) and were used without further purification. DOTA − NCS was purchased from Macrocyclics, Inc (Plano, TX). Macropa − NCS was synthesized as reported by Thiele et al. [ 25 ] and analysed using Nuclear Magnetic Resonance Spectroscopy ( 1 H-NMR), High performance Liquid Chromatography (HPLC) and mass spectroscopy (MS). Refer to Kang et al. for details regarding the synthesis of 3p- C -DEPA − NO2 and 3p- C -DEPA − NCS [ 30 ]. Details of the synthesis of 3p- C -DEPA − TFP -PEG 4 are provided (Supplementary Figure S1 ). All water was deionized and passed through Millipore water purification system until a resistivity of 18 MΩ·cm was achieved. Trastuzumab (RRID: AB_3112050) was of research grade and was purchased from Ichorbio (catalogue number: ICH4013, Oxford, UK). 225 Ac for bioconjugate labelling was supplied by the Isotope Program within the Office of Nuclear Physics in the Department of Energy’s Office of Science (Oak Ridge National Laboratory, TN, USA) Female Balb/C mice (RRID: IMSR_CRL:028) (n = 3/group) were purchased from Charles River (Saint-Constant, QC, CA), were at least 6 weeks of age and were used for biodistribution studies. 1. Conjugation of bifunctional chelators to trastuzumab Trastuzumab was conjugated with BFCs following lab SOPs [ 31 – 33 ]. Briefly, a 15-mole excess of a 20 mg/mL of either, 3p- C -DEPA − NCS , 3p- C -DEPA − TFP -PEG 4 , or DOTA − NCS in DMSO solution was incubated with trastuzumab in 0.1 M Na 2 CO 3 for 90 minutes at 37°C with constant shaking. For macropa − NCS conjugation, the reaction was carried out in 0.1 M NaHCO 3 and 0.15 M NaCl for 18 h at 4°C. The unreacted BFC was removed by centrifugation using Amicon Ultra-10k (Burlington, MA) filters. 3p- C -DEPA-trastuzumab, 3p- C -DEPA − TFP -PEG 4 -trastuzumab, DOTA-trastuzumab, or macropa-trastuzumab, was buffer exchanged to PBS and concentrated with Amicon Ultra centrifugal filters which afforded > 96% purity. The purified conjugates were stored at -80°C before labelling. The purity of the respective conjugates was performed using a size-exclusion HPLC (SEC-HPLC) Waters 2487 Dual λ Absorbance Detector, XBridge® BEH 200 A SEC 3.5 µm, 7.8 x 150 nm column (Waters Corporation, Milford, MA) and an Agilent 2100 Bioanalyzer system (Agilent High Sensitivity Protein 250 Kit- catalogue# 5067 − 1575) following the manufacturer’s protocol. 2. Radiochemistry of [ 225 Ac]Ac-3p- C -DEPA -NO2 [ 225 Ac]Ac(NO 3 ) 3 (0.5 M HNO 3 ) was produced on-site at SCK CEN based on literature [ 34 – 36 ] and from Oak Ridge National Lab ORNL (Oak Ridge, TN, USA) containing trace amounts of 227 Ac. All radiolabelling buffers were treated with Chelex 100 [sodium form (50–100 mesh, Sigma Aldrich)] for 15 min to remove trace metals. All solutions were degassed and filtered before use. The radiolabelling experiments were performed by reacting 90–100 kBq of [ 225 Ac]Ac(NO 3 ) 3 at 25, 40, 55 or 95°C for 1 hour (h) with different concentrations of 3p- C -DEPA − NO2 (1, 5, 10 and 20 µM) in 0.37 M TRIS buffer, pH 8.5, V = 300 µL) to yield [ 225 Ac]Ac-3p- C -DEPA − NO2 . The % radiochemical conversion (RCC) was evaluated by instant thin-layer liquid chromatography (iTLC-SG, Varian, Diegem, Belgium). iTLC-SG papers were developed in an elution chamber using acetonitrile: water (75/25 v/v) such that bound 225 Ac and daughter radionuclides will migrate with the solvent front to the upper part of the iTLC strip, while unbound radionuclides will remain at the lower part where the mixture was originally spotted. Once the solvent front reached the top of the iTLC strip, it was removed from the mobile phase and cut into two (top and bottom). The activity of the upper and lower part of the iTLC strip was measured with a gamma counter (Wallac Wizard 1480, PerkinElmer, Waltham, MA) using the 213 Bi-peak window (380–500 keV) after a time delay of 24 h to allow 213 Bi to reach equilibrium with 225 Ac as described [ 37 ]. The %RCC is calculated as activity at the top half divided by the total activity (top + bottom) x 100. 3. Radiochemistry of immunoconjugates 3p- C -DEPA-trastuzumab, 3p- C -DEPA − TFP -PEG 4 -trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab immunoconjugates were radiolabelled using [ 225 Ac]Ac(NO 3 ) 3 (2.0 MBq) dissolved in 0.1 M Hydrochloric acid (Optima grade, Fisher Scientific) at a specific activity of 8 kBq/µg as reported [ 38 ]. About 10 µL of ascorbic acid was added to prevent radiolysis in all the reactions. The pH of the reaction was determined by spotting 1 µL of the reaction mixture onto Hydrion pH paper (range, 5.0–9.0) (Sigma-Aldrich); pH of a typical reaction was 5.8-6.0. The incubation was done at 37°C on a shaker at 700 RPM for 2 h. A small aliquot (0.8 µL) was spotted on a strip of instant thin-layer chromatography silica gel impregnated paper (iTLC-SG, Agilent Technologies) to determine the extent of incorporation of 225 Ac onto the protein using mobile phase of 20 mM sodium citrate (pH 5.2). 4. In vitro stability The in vitro stability of [ 225 Ac]Ac-3p- C -DEPA − NO2 radio-complex (150 kBq/µmol) was evaluated in PBS and human serum (HS). After radiolabelling 3p- C -DEPA − NO2 , the radio-complex was purified with a Sep-Pak C 18 Light cartridge (Waters, Eschborn, Germany). Briefly, the Sep-Pak C 18 Light cartridge was pre-conditioned with absolute ethanol (5 mL) followed by water (5 mL). The reaction mixture was loaded onto the cartridge and washed with 6–8 mL water to remove unbound 225 Ac. The pure radio-complex was eluted with 0.2 mL absolute ethanol and the volume was brought to 0.5 mL by diluting with 0.3 mL of 0.9% NaCl. 80 µL of the purified radio-complex was added to a 1 mL vial containing either 420 µL of PBS or HS, and the solution was incubated at 37°C under constant gentle shaking. To determine the percentage of intact [ 225 Ac]Ac-3p- C -DEPA − NO2 , 5 µL samples were taken for iTLC analysis at selected times points (30, 60 min, 2 d, 3 d, and 6 d). Development of iTLC and counting of activity was performed as described above. The in vitro stability of the radioimmunoconjugates [ 225 Ac]Ac-DOTA-trastuzumab, [ 225 Ac]Ac-3p- C -DEPA-trastuzumab, [ 225 Ac]Ac-3p- C -DEPA − TFP -PEG 4 -trastuzumab, and [ 225 Ac]Ac-macropa-trastuzumab was determined in PBS at 37°C for 10 days (1, 2, 3, 4, 7 and 10 d). Each radioimmunoconjugate (RIC) was incubated at the respective incubation conditions to make a final concentration of ~ 400 kBq/500 µL. To analyse the purity, aliquots of 9 µL of each radioimmunoconjugate was drawn for iTLC and analysed as described above. 5. In vivo biodistribution of radioimmunoconjugates Biodistribution of [ 225 Ac]Ac-3p- C -DEPA-trastuzumab, [ 225 Ac]Ac-DOTA-trastuzumab, and [ 225 Ac]Ac-macropa-trastuzumab was studied in healthy Balb/C mice (n = 3/group). The mice were housed under standard conditions in approved facilities with 12 h light/dark cycles and given food and water ad libitum throughout the duration of the studies following a tail vein injection of 11.1 kBq of each radiolabelled construct. Mice were sacrificed at 48 h post injection (p.i.), and the activity in organs was measured using gamma counter ( 213 Bi-peak window (380–500 keV) ,Wallac Wizard 1480, PerkinElmer) and expressed as the % injected activity per gram of the organ (%IA/g). STATISTICAL ANALYSIS All data were expressed as the mean ± standard error of mean (SEM). A two-way ANOVA with Dunnett post hoc test was used to determine the statistical significance between the different mice groups. All graphs or figures were analysed using GraphPad Prism Version 10 (RRID:SCR_002798). RESULTS Synthesis of 3p- C -DEPA, 3p-C-DEPA − NCS , and 3p- C -DEPA − TFP -PEG 4 Since 3p- C -DEPA is not commercially available, it was synthesized following a strategy involving the formation of an N,N′-bisubstituted-β-amino iodide intermediate and subsequent nucleophilic ring opening of an aziridinium ion, as previously described [ 29 ]. Briefly, β-iodoamine was subjected to intramolecular rearrangement to form the aziridinium ion, which was then regioselectively opened via an SN 2 mechanism using tri-tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate. This yielded the protected chelator 3p- C -DEPA-( t Bu) 5 in an isolated yield of 86% and a purity exceeding 95%. The tert-butyl protecting groups were subsequently removed by treatment with trifluoroacetic acid (TFA), affording deprotected 3p- C -DEPA at > 96% purity. 3p- C -DEPA − NO2 (Fig. 1 A) was used directly in initial radiolabelling and in vitro stability studies. To enable conjugation of 3p- C -DEPA to a targeting vector such as trastuzumab, the chelator was further functionalized to introduce reactive groups capable of coupling to lysine residues. Two linker strategies were explored: isothiocyanate derivatization (Fig. 1 B), as previously reported by Kang et al. [ 30 ], and PEG₄-TFP ester conjugation, a tetrafluorophenyl-activated ester linker. In the PEG₄-TFP approach, 3p- C -DEPA − TFP -PEG 4 (Fig. 1 C) was synthesized by the controlled, dropwise addition of 1.5 molar equivalents of TFP-PEG₄-TFP to 3p-C-DEPA- NH₂ (tBu)₅. The reaction mixture was purified using preparative HPLC, and the tert-butyl protecting groups were removed by treatment with TFA. The final product, 3p- C -DEPA TFP -PEG 4 , was characterized by LC-HRMS, confirming > 95% chemical purity. Also, the chemical structures of DOTA − NCS and macropa − NCS are shown in Fig. 1 D and 1 E, respectively. Radiolabelling and characterization of [ 225 Ac]Ac-3p- C -DEPA − NO2 3p-C-DEPA- NO₂ demonstrated efficient radiolabelling with ²²⁵Ac under mild conditions. At a ligand concentration of 1 µM, rapid chelation was achieved within 1 hour, with radiochemical conversion (RCC) values of 93.7 ± 1.4%, 95.1 ± 1.8%, and 98.2 ± 0.9% at 25°C, 40°C, and 55°C, respectively. Further increases in ligand concentration (5–20 µM) and reaction temperature (40–95°C) resulted in consistently high RCC values exceeding 97% under all tested conditions (Fig. 2 A). Given the favourable radiolabelling kinetics, we evaluated the in vitro stability of [²²⁵Ac]Ac-3p- C -DEPA- NO₂ over a six-day period (Fig. 2 B). The radio-complex exhibited excellent stability in human serum, retaining > 95% of intact complex throughout the six-day incubation. In PBS, stability was also maintained over the first three days, with > 95% of intact radio-complex observed. By day six, 83.0 ± 4.1% of the radio-complex remained intact in PBS, indicating partial degradation under these conditions. Conjugation and characterization of immunoconjugates Encouraged by the initial radiolabelling studies with 225 Ac using 3p- C -DEPA − NO2 , the bifunctional chelators 3p- C -DEPA − NCS and 3p- C -DEPA − TFP -PEG 4 were conjugated (non-site specifically) to trastuzumab and compared with DOTA − NCS and macropa − NCS -derivatized trastuzumab (Fig. 3 A). Conjugation of trastuzumab with 3p- C -DEPA − NCS , 3p- C -DEPA − TFP -PEG 4 , DOTA − NCS , and macropa − NCS yielded immunoconjugates 3p- C -DEPA-trastuzumab, 3p- C -DEPA − TFP -PEG 4 -trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab (Fig. 3 A), respectively. The HPLC showed that all the immunoconjugates were at least 98% pure with less than 2% degradation or aggregates (Fig. 3 B- 3 E). The bioanalyzer microfluidic electrophoresis experiment confirmed the purity of the various immunoconjugates (Fig. 3 F). Radiolabelling and characterisation of radioimmunoconjugates The immunoconjugates 3p- C -DEPA-trastuzumab, 3p- C -DEPA − TFP -PEG₄-trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab were radiolabelled with [²²⁵Ac]Ac(NO₃)₃ at a specific activity of 8 kBq/µg. After 2 hours of incubation at 37°C, the RCC was 94.6% for [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab, 93.5% for [²²⁵Ac]Ac-3p- C -DEPA − TFP -PEG₄-trastuzumab, 80.9% for [²²⁵Ac]Ac-DOTA-trastuzumab, and 96.5% for [²²⁵Ac]Ac-macropa-trastuzumab (Figs. 4 A– 4 D). The high RCCs observed for both 3p- C -DEPA-, 3p- C -DEPA − TFP -PEG₄, and macropa-conjugated antibodies obviated the need for post-labelling purification. In contrast, [²²⁵Ac]Ac-DOTA-trastuzumab required purification by centrifugal filtration, yielding a final radiochemical purity of 95.9%. The ²²⁵Ac-labelled immunoconjugates were evaluated for in vitro stability (PBS, 37°C) over a 10-day period. Remarkably, [²²⁵Ac]Ac-3p- C -DEPA − TFP -PEG₄-trastuzumab exhibited the highest stability, retaining 91.3 ± 4.3% of the intact radiocomplex after 10 days. In comparison, [²²⁵Ac]Ac-macropa-trastuzumab maintained 81.9 ± 5.6% integrity, while [²²⁵Ac]Ac-DOTA-trastuzumab showed 60.1 ± 0.6% intact complex. The lowest stability was observed for [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab, with only 48.3 ± 1.2% of the radiocomplex remaining intact at day 10 (Fig. 4 E). Biodistribution studies of [ 225 Ac]Ac-3p- C -DEPA-trastuzumab, [ 225 Ac]Ac-DOTA-trastuzumab, and [ 225 Ac]Ac-macropa-trastuzumab Despite the relatively lower in vitro stability of [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab in PBS, we proceeded to evaluate its in vivo biodistribution in healthy Balb/C mice over a 48-hour period, using [²²⁵Ac]Ac-DOTA-trastuzumab and [²²⁵Ac]Ac-macropa-trastuzumab as reference standards (Table S1 ). Overall, no significant differences were observed in the biodistribution profiles across major organs (p > 0.05 for all comparisons), with two exceptions. In the liver, [²²⁵Ac]Ac-DOTA-trastuzumab exhibited significantly higher uptake (14.1 ± 2.9%IA/g) compared to [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab (9.0 ± 3.3%IA/g, p = 0.04) whereas that for [²²⁵Ac]Ac-macropa-trastuzumab was 6.7 ± 0.8. Conversely, in the large intestine, [²²⁵Ac]Ac-macropa-trastuzumab showed significantly higher accumulation (2.2 ± 0.1%IA/g) than [²²⁵Ac]Ac-DOTA-trastuzumab (1.1 ± 0.1%IA/g, p = 0.03). Consistent with expectations for radioimmunoconjugates, the highest activity was retained in the blood at 48 hours post-injection, with values of 18.3 ± 0.5%IA/g for [²²⁵Ac]Ac-DOTA-trastuzumab, 19.1 ± 6.5%IA/g for [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab, and 19.4 ± 2.7%IA/g for [²²⁵Ac]Ac-macropa-trastuzumab (Fig. 5 ). DISCUSSION ²²⁵Ac is a highly promising radionuclide for TAT due to its favourable decay properties and potent cytotoxicity against cancer cells [ 39 – 41 ]. Despite its therapeutic potential, the widespread clinical translation of ²²⁵Ac-based radiopharmaceuticals has been hindered by two major factors: limited availability of the isotope and the challenge of identifying suitable chelators for the large trivalent actinium ion (Ac³⁺). Although chelators such as DOTA, macropa, and macrodipa have been developed and studied extensively [ 24 , 25 , 41 ], each has limitations. DOTA remains the clinical standard for ²²⁵Ac coordination, but its slow radiolabelling kinetics, especially under mild conditions, pose significant challenges for heat-sensitive biomolecules such as monoclonal antibodies [ 12 ]. In this study, we investigated 3p- C -DEPA, a hybrid decadentate ligand combining structural features of DOTA and DTPA, as a potential alternative chelator for ²²⁵Ac. We successfully synthesized 3p- C -DEPA − NO₂ in high yield and purity, using a previously reported method for 3p- C -NETA [ 19 , 30 ]. The presence of an additional acyclic iminodiacetic acid arm likely contributes to the faster complexation kinetics and improved radiolabelling performance observed with 3p- C -DEPA − NO2 , compared to DOTA − NCS . Indeed, [²²⁵Ac]Ac-3p-C-DEPA − NO₂ demonstrated excellent radiolabelling efficiency (≥ 95% RCC) at room temperature at low chelator concentration, a significant advantage for radiolabelling heat-sensitive biomolecules whereas [²²⁵Ac]Ac-DOTA-trastuzumab showed a low RCC of 80.9%. These findings align with previous work by Song et al., who noted the effectiveness of 3p- C -DEPA for large metal coordination due to its larger cavity size relative to DOTA [ 42 ]. In contrast, prior studies have shown that DOTA requires high temperatures (≥ 85°C) to achieve efficient ²²⁵Ac complexation, with RCC values of only ~ 15% at 40°C even after extended incubation [ 43 ]. The ability of 3p- C -DEPA to rapidly and efficiently bind ²²⁵Ac at lower temperatures is thus a noteworthy improvement. To assess the utility of 3p- C -DEPA in an antibody-based radiopharmaceutical, we synthesized two bifunctional derivatives: 3p- C -DEPA − NCS and a novel PEGylated tetrafluorophenol ester (3p- C -DEPA- TFP -PEG₄). 3p- C -DEPA- TFP -PEG₄ is easy to synthesize starting from the reported 3p-C-DEPA- NH₂ (tBu)₅ and demonstrate good stability when stored at -20°C. Both bifunctional chelators were successfully conjugated to trastuzumab, resulting in the corresponding radioimmunoconjugates. Radiolabelling of [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab and [²²⁵Ac]Ac-3p-C-DEPA − TFP -PEG₄-trastuzumab yielded high RCCs (94.6% and 93.5%, respectively), comparable to previous results using [²⁰⁵/²⁰⁶Bi]Bi-3p- C -DEPA-trastuzumab reported by Song et al. [ 42 ]. However, in vitro stability studies in PBS revealed significant differences between the two constructs. [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab showed notable degradation over 10 days (only 48.3 ± 1.2% remaining intact at day 10), likely due to radiolytic effects. Though this observation was surprising, it is reported in the literature that Cl − (present in PBS) can undergo radiation-induced formation of hypochlorite ions which could potentially react with the enolized thiourea unit [ 44 ]. As 3p- C -DEPA is coupled to trastuzumab via a thiourea linker, this might be a reason for the observed instability. Remarkably, this effect was less observed with the macropa − NCS -conjugate which demonstrated high stability in PBS. To address this instability in PBS of [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab, the PEGylated-TFP derivative was designed to form more stable amide bonds with lysine residues. Encouragingly, [²²⁵Ac]Ac-3p- C -DEPA − TFP -PEG₄-trastuzumab exhibited superior in vitro stability, maintaining > 90% integrity after 10 days in PBS. In vivo biodistribution studies in healthy Balb/C mice showed that [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab exhibited a comparable organ distribution profile to both [²²⁵Ac]Ac-DOTA-trastuzumab and [²²⁵Ac]Ac-macropa-trastuzumab over 48 hours. This indicates that the limited in vitro stability of [²²⁵Ac]Ac-3p- C -DEPA-trastuzumab in PBS has limited consequences for the in vivo stability. Notably, liver uptake was significantly lower for [²²⁵Ac]Ac-3p-C-DEPA-trastuzumab (9.0 ± 3.3%IA/g) and [²²⁵Ac]Ac-macropa-trastuzumab (6.7 ± 0.8%IA/g) compared to [²²⁵Ac]Ac-DOTA-trastuzumab (14.1 ± 2.9%IA/g, p = 0.04). This indicates that both the ²²⁵Ac-3p-C-DEPA and ²²⁵Ac-macropa complexes exhibit comparable in vivo kinetic inertness, superior to that of the ²²⁵Ac-DOTA complex. This distinction is particularly important given the known hepatotoxicity associated with free ²²⁵Ac and its radioactive decay products [ 16 , 20 , 45 ]. The high blood retention observed for all constructs reflects the long circulation time of intact antibody conjugates. In vivo evaluation of the second-generation [²²⁵Ac]Ac-3p- C -DEPA − TFP -PEG₄-trastuzumab has not yet been conducted. However, studies in tumour-bearing mice are planned to assess their pharmacokinetics and tumour-targeting efficiency. CONCLUSION This study demonstrates the promising potential of 3p- C -DEPA-based chelators for the development of ²²⁵Ac-labelled radiopharmaceuticals. Among the constructs evaluated, 3p- C -DEPA − TFP -PEG₄ stood out as a highly effective bifunctional chelator, achieving excellent radiolabelling efficiency under mild conditions and superior in vitro stability compared to DOTA analogues. Importantly, [²²⁵Ac]Ac-3p- C -DEPA − TFP− PEG₄-trastuzumab maintained > 90% integrity in PBS over 10 days, indicating strong resistance to radiolytic and hydrolytic degradation. Additionally, the biodistribution profile of 3p- C -DEPA-based conjugates was comparable to established chelators, with notably lower liver uptake than the 225 Ac-DOTA-conjugate, reducing concerns related to off-target hepatotoxicity. These findings highlight 3p- C -DEPA − TFP -PEG₄ as a promising alternative to current 225 Ac-bifunctional chelators, offering advantages in radiolabelling kinetics and stability. Further preclinical studies, including pharmacokinetic studies in mouse tumour models, therapeutic efficacy and long-term in vivo stability studies are warranted to confirm its utility in TAT applications. Abbreviations BFC Bifunctional chelators DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA Diethylenetriamine pentaacetic acid EDTA Ethylenediaminetetraacetic acid FDA Food and Drug Administration HPLC High performance liquid chromatography HS Human serum ITLC Instant thin-layer chromatography LC-HRMS Liquid chromatography- High resolution mass spectrometry LET Linear energy transfer MS Mass spectroscopy p.i post injection PBS Phosphate-buffered saline PSMA Prostate specific membrane antigen RCC Radiochemical conversion RPM Revolutions per minutes SEM Standard error of mean TAT Targeted alpha therapies TETA Triethylenetetramine TETPA Triethylene tetramine pentaacetic acid TFA Trifuoroacetic acid TFP Tetrafluorophenol Declarations Ethics Statement Mice were approved, supervised, and maintained following the guidelines set for by the University of Saskatchewan Animal Care Committee (UACC), protocol # 20170084. Procedures were carried out according to the laboratory animal care and use of the Canadian Council on Animal Care. These mice had at least one week of acclimatization before being assigned to the various groups. Consent for publication Not applicable Availability of data and materials’ statement The datasets used and/or analysed during the current study are available from the corresponding authors Prof. Fonge and Prof. Cleeren on reasonable request. Competing Interests The authors declare that they have no competing interests. Funding This work was funded by Canadian Institute for Health Research (CIHR) Project Grants (Grant numbers 437660 and 408132) to Humphrey Fonge and internal funding KU Leuven. Acknowledgement Authors have no additional acknowledgements to make. Authors’ Contributions Conceptualization, FC, and HF; methodology, JPK, SA, SL, FC, and HF; software, JPK, SA; validation, JPK, SA, FC, and HF; formal analysis, JPK and SA; investigation, JPK, SA, EN; resources, FC and HF; data curation, JPK and SA; writing-original draft preparation, JPK and SA; writing-review and editing, JPK, SA, EN, MO, TC, SL, FC and HF; visualization, JPK, SA, FC, and HF; supervision, FC and HF; project administration, FC and HF; funding acquisition, FC and HF. References Pougoue Ketchemen, J., et al., Complete remissions of HER2-positive trastuzumab-resistant xenografts using a potent [225Ac]Ac-labeled anti-HER2 antibody-drug radioconjugate. Clinical Cancer Research, 2024. Brechbiel, M.W. and M.W. Brechbiel, Targeted α-therapy: past, present, future? Dalton Transactions, 2007/10/29(43). Liberal, F.D.C.G., et al., Targeted Alpha Therapy: Current Clinical Applications. Cancer Biotherapy & Radiopharmaceuticals, 2020-08-13. 35 (6). 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Angew Chem Int Ed Engl, 2017. 56 (46): p. 14712-14717. King, A.P., et al., 225Ac-MACROPATATE: A Novel α-Particle Peptide Receptor Radionuclide Therapy for Neuroendocrine Tumors. Journal of Nuclear Medicine, 2023. 64 (4): p. 549-554. Schatz, C.A., et al., Preclinical Efficacy of a PSMA-Targeted Actinium-225 Conjugate (225Ac-Macropa-Pelgifatamab): A Targeted Alpha Therapy for Prostate Cancer. Clinical Cancer Research, 2024. 30 (11): p. 2531-2544. Kadassery, K.J., et al., H(2)BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy. Bioconjug Chem, 2022. 33 (6): p. 1222-1231. Chong, H.-S., et al., Synthesis and comparative biological evaluation of bifunctional ligands for radiotherapy applications of 90Y and 177Lu. Bioorganic & Medicinal Chemistry, 2015. 23 (5): p. 1169-1178. Kang, C.S., et al., Synthesis and evaluation of a new bifunctional NETA chelate for molecular targeted radiotherapy using(90)Y or(177)Lu. Nucl Med Biol, 2015. 42 (3): p. 242-249. Ketchemen, J.P., et al., Biparatopic anti-HER2 drug radioconjugates as breast cancer theranostics. Br J Cancer, 2023. 129 (1): p. 153-162. Pougoue Ketchemen, J., et al., Effectiveness of [(67)Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer. Eur J Nucl Med Mol Imaging, 2024. 51 (7): p. 2070-2084. Tikum, A.F., et al., Effectiveness of (225)Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models. J Nucl Med, 2024. Cassells, I., et al., Radiolabeling of Human Serum Albumin With Terbium-161 Using Mild Conditions and Evaluation of in vivo Stability. Front. Med., 2021. 0 : p. 1359-1359. Dekempeneer, Y., et al., The therapeutic efficacy of 213Bi-labeled sdAbs in a preclinical model of ovarian cancer. Mol. Pharmaceutics, 2020. 17 (9): p. 3553-3566. McAlister, D.R. and E.P. Horwitz, Selective separation of radium and actinium from bulk thorium target material on strong acid cation exchange resin from sulfate media. Appl Radiat Isot, 2018. 140 : p. 18-23. Miederer, M., et al., Preclinical Evaluation of the α-Particle Generator Nuclide 225Ac for Somatostatin Receptor Radiotherapy of Neuroendocrine Tumors. Clin Cancer Res., 2008. 14 (11): p. 3555-3561. Solomon, V.R., et al., Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers. Cancers, 2020. 12 (11): p. 3449. Kratochwil, C., et al., Targeted a-therapy of metastatic castration-resistant prostate cancer with 225Ac-PSMA-617: Dosimetry estimate and empiric dose finding. J. Nucl. Med., 2017. 58 (10): p. 1624-1631. Ballal, S., M. Yadav, and C. Bal, Early results of 225Ac-DOTATATE Targeted Alpha Therapy in Metastatic Gastroenteropancreatic Neuroendocrine Tumors: First Clinical Experience on Safety and Efficacy. J. Nucl. Med, 2019. 60 (supplement 1): p. 60-60. Qin, Y., et al., Evaluation of actinium-225 labeled minigastrin analogue [225ac]ac-dota-pp-f11n for targeted alpha particle therapy. Pharmaceutics, 2020. 12 (11): p. 1-12. Song, H.A., et al., Efficient Bifunctional Decadentate Ligand 3p-C-DEPA for Targeted α-Radioimmunotherapy Applications. Bioconjugate Chemistry, 2011. 22 (6): p. 1128-1135. Ramogida, C.F., et al., Evaluation of polydentate picolinic acid chelating ligands and an α-melanocyte-stimulating hormone derivative for targeted alpha therapy using ISOL-produced 225Ac. EJNMMI radiopharm. chem., 2019. 4 (1): p. 1-20. Vugts, D.J., et al., Comparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for 89Zr-immuno-PET. Eur J Nucl Med Mol Imaging, 2017. 44 (2): p. 286-295. Pruszynski, M., et al., Evaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted α-Particle Therapy of Cancer. Molecular Pharmaceutics, 2018. 15 (4): p. 1457-1466. Supplementary Files 3pCDEPAandPEG4TFPSupplementaryinformationfinal.docx Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Editorial decision: Major revision 05 Jul, 2025 Reviewers agreed at journal 13 Jun, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 12 Jun, 2025 First submitted to journal 11 Jun, 2025 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-6813431","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470767614,"identity":"27ab5216-6fc3-43da-bc08-012a9386d089","order_by":0,"name":"Jessica Pougoue Ketchemen","email":"","orcid":"","institution":"Université Laval: Universite Laval","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"Pougoue","lastName":"Ketchemen","suffix":""},{"id":470767615,"identity":"7b3b83b9-fba3-4a59-8751-6f5f644e18b1","order_by":1,"name":"Stephen Ahenkorah","email":"","orcid":"","institution":"University of Iowa Hospitals and Clinics","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Ahenkorah","suffix":""},{"id":470767616,"identity":"ace97147-8415-4755-9636-07d507587237","order_by":2,"name":"Emmanuel Nwangele","email":"","orcid":"","institution":"Université Laval: Universite Laval","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Nwangele","suffix":""},{"id":470767617,"identity":"9e397625-35b6-4669-9a1d-6edc0807e9b5","order_by":3,"name":"Maarten Ooms","email":"","orcid":"","institution":"SCK-CEN: Studiecentrum voor Kernenergie","correspondingAuthor":false,"prefix":"","firstName":"Maarten","middleName":"","lastName":"Ooms","suffix":""},{"id":470767618,"identity":"1be40ce6-3378-4bc9-b2a8-6902854565b1","order_by":4,"name":"Thomas Cardinaels","email":"","orcid":"","institution":"SCK-CEN: Studiecentrum voor Kernenergie","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Cardinaels","suffix":""},{"id":470767619,"identity":"8d7ea63d-3e82-4fd9-8739-a4c577a31de3","order_by":5,"name":"Simon Leekens","email":"","orcid":"","institution":"Katholieke Universiteit Leuven","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Leekens","suffix":""},{"id":470767620,"identity":"c78e87c6-ed61-42d6-9ec6-29bc2561ca05","order_by":6,"name":"Frederik Cleeren","email":"","orcid":"","institution":"Katholieke Universiteit Leuven","correspondingAuthor":false,"prefix":"","firstName":"Frederik","middleName":"","lastName":"Cleeren","suffix":""},{"id":470767621,"identity":"b38fda95-8f38-4515-a8ab-f7ea7d4dff45","order_by":7,"name":"Humphrey fonge","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9388-6872","institution":"Université Laval: Universite Laval","correspondingAuthor":true,"prefix":"","firstName":"Humphrey","middleName":"","lastName":"fonge","suffix":""}],"badges":[],"createdAt":"2025-06-03 16:52:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6813431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6813431/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-025-00408-w","type":"published","date":"2025-12-22T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84787532,"identity":"f018117b-17a9-42ae-a701-ff1206a39198","added_by":"auto","created_at":"2025-06-17 10:43:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37334,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of (A) 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-NO2\u003c/sub\u003e; (B) 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-NCS\u003c/sub\u003e; (C) 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003eTFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e; coordination numbers = 10. (D) DOTA\u003csub\u003e-NCS\u003c/sub\u003e; coordination number = 8; and (E) macropa\u003csub\u003e-NCS\u003c/sub\u003e; coordination number = 10.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/4aaef09f8ec08fca6955ba83.png"},{"id":84788763,"identity":"459fea37-93c9-4832-a263-f1b97dd92167","added_by":"auto","created_at":"2025-06-17 10:59:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187033,"visible":true,"origin":"","legend":"\u003cp\u003eRadiolabelling and characterization of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-NO2\u003c/sub\u003e. (A) Radiochemical conversions obtained for 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-NO2\u003c/sub\u003e with [\u003csup\u003e225\u003c/sup\u003eAc]Ac(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e. Ligand concentration of 1, 5, 10 and 20 µM (0.37 M Tris buffer, pH 8.5) with 90-100 kBq [\u003csup\u003e225\u003c/sup\u003eAc]Ac(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e for 1 h, at 25, 40, 55 and 95 °C. (B) \u003cem\u003eIn vitro\u003c/em\u003e stability studies in PBS and human serum for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-NO2\u003c/sub\u003e. Stability was studied up to 6 days at 37 °C. Blue line is inserted to indicate an intact radiocomplex of 95%.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/f92ab206b0a4586bde4d08b1.png"},{"id":84787533,"identity":"6206cb62-84ff-4080-b67c-e7ce0d524f2c","added_by":"auto","created_at":"2025-06-17 10:43:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94676,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic for the synthesis of trastuzumab conjugates and their characterization. (A) Chemical synthesis of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab. SEC-HPLC of (B) 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, (C) 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, (D) DOTA-trastuzumab, and (E) macropa-trastuzumab (F) Bioanalyzer microfluidic electrophoresis of immunoconjugates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/9004b7ac0c163e60b8cd5924.png"},{"id":84787535,"identity":"f0f072b2-5034-42f1-83ec-2562508552f9","added_by":"auto","created_at":"2025-06-17 10:43:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90932,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of radioimmunoconjugates. iTLC chromatograms of (A) [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, (B) [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, (C) [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, and (D) [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab. (D) \u003cem\u003eIn vitro\u003c/em\u003e stability of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e-TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab. (E) Stability was evaluated in PBS at 37 °C for 10 days (d) in duplicates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/8b87f2d24eaec806b866594f.png"},{"id":84787536,"identity":"2f2f5c29-9853-42f1-b66d-cce2ed5f2208","added_by":"auto","created_at":"2025-06-17 10:43:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53646,"visible":true,"origin":"","legend":"\u003cp\u003eSelected organ biodistribution of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab at 48 h after a tail vein administration of 11.1 kBq activity (n = 3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/e477c28e9a39556f9eed8a99.png"},{"id":99172350,"identity":"49f9d351-30df-4bbd-87fd-24f906f4f115","added_by":"auto","created_at":"2025-12-29 16:08:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1481134,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/a78beffc-6513-4fad-8649-024c63a92d42.pdf"},{"id":84787944,"identity":"240699e6-855d-4108-9441-d7b0bfea504f","added_by":"auto","created_at":"2025-06-17 10:51:53","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":76787,"visible":true,"origin":"","legend":"","description":"","filename":"3pCDEPAandPEG4TFPSupplementaryinformationfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-6813431/v1/434e66e419362bd33cba05c7.docx"}],"financialInterests":"","formattedTitle":"Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks.","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAlpha-particle (α-particle) emitting radioisotopes have emerged as particularly promising agents for the selective eradication of cancer cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to their high linear energy transfer (LET) and short path length in biological tissues, α-particles are capable of depositing large amounts of energy over a limited distance. This confers high cytotoxicity specifically to targeted tumour cells while minimizing collateral damage to surrounding healthy tissue. Importantly, the high LET of α-particles is more effective in inducing lethal DNA double-strand breaks compared to the lower LET of beta-minus particles (β\u003csup\u003e\u0026minus;\u003c/sup\u003e-particles) [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral α-emitting radioisotopes, including \u003csup\u003e225\u003c/sup\u003eAc, have demonstrated superior therapeutic potential in both preclinical and clinical settings when compared with their β\u003csup\u003e\u0026minus;\u003c/sup\u003e-emitting counterparts such as Yttrium-90 (\u003csup\u003e90\u003c/sup\u003eY) and Lutetium-177 (\u003csup\u003e177\u003c/sup\u003eLu) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among α-emitting radioisotopes, \u003csup\u003e225\u003c/sup\u003eAc (t\u003csub\u003e1/2\u003c/sub\u003e = 9.9 d, E\u003csub\u003eα\u003c/sub\u003e = 5.8 MeV) is particularly notable due to its decay chain, which results in the emission of four α-particles, each contributing to its potent cytotoxic effect. Moreover, its relatively long half-life of 9.9 d is well-suited to long circulating biologicals such as monoclonal antibodies, whose pharmacokinetics align with this timeframe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe clinical success of targeted α-therapy in metastatic castration-resistant prostate cancer using [\u0026sup2;\u0026sup2;⁵Ac]Ac-PSMA-617 has significantly accelerated interest in the development of novel \u003csup\u003e225\u003c/sup\u003eAc-labelled radiopharmaceuticals for a wider spectrum of malignancies, including approaches based on \u003csup\u003e225\u003c/sup\u003eAc-labelled monoclonal antibodies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEfficient radiolabelling of \u0026sup2;\u0026sup2;⁵Ac under mild conditions, combined with high kinetic inertness and thermodynamic stability of the resulting complex, is essential for the safe and effective application of TAT. These factors are critical to prevent \u003cem\u003ein vivo\u003c/em\u003e dissociation of the radiometal, which can lead to off-target radiation exposure and dose-limiting toxicity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The increasing clinical momentum behind TAT has intensified the development of new chelating agents specifically optimized for \u003csup\u003e225\u003c/sup\u003eAc, as the \u003cem\u003ein vivo\u003c/em\u003e stability of the radiometal complex is a key determinant of therapeutic success. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA variety of \u003csup\u003e225\u003c/sup\u003eAc-chelators have been reported, including EDTA, PEPA, CHX-DTPA, DTPA, DOTA, TETA, DOTMP, TETPA, and macropa. Among these, DOTA and macropa have shown the greatest promise in terms of practical applicability and coordination performance [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. DOTA, a 12-membered macrocycle ligand with four nitrogen atoms and four pendant carboxylates, offers an octadentate coordination geometry well-suited for many trivalent radiometals such as ⁶⁸Ga\u0026sup3;⁺, \u0026sup1;\u0026sup1;\u0026sup1;In\u0026sup3;⁺, and \u0026sup1;⁷⁷Lu\u0026sup3;⁺. It has been successfully integrated into multiple FDA-approved radiopharmaceuticals, including [⁶⁸Ga]Ga-DOTATATE and [\u0026sup1;⁷⁷Lu]Lu-DOTATATE, for both diagnostic and therapeutic use.\u003c/p\u003e \u003cp\u003eHowever, the use of DOTA for \u0026sup2;\u0026sup2;⁵Ac is limited by several factors. Most notably, radiolabelling requires elevated temperatures (80\u0026ndash;95\u0026deg;C) to achieve high radiochemical yields, making it incompatible with heat-sensitive biomolecules such as monoclonal antibodies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. From a clinical perspective, room-temperature labelling in under 20 minutes is preferred to streamline radiopharmaceutical preparation and to avoid radiolytic degradation of heat-sensitive targeting vectors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, the kinetic inertness of \u003csup\u003e225\u003c/sup\u003eAc-DOTA complexes has been challenged by several \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies, which report partial release of \u0026sup2;\u0026sup2;⁵Ac from the chelator over time [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This instability highlights DOTA\u0026rsquo;s limitations as a long-term carrier for \u0026sup2;\u0026sup2;⁵Ac and underscores the need for chelators tailored to the unique coordination chemistry of large, low-charge-density ions. With an ionic radius of ~\u0026thinsp;1.22 \u0026Aring;, Ac\u0026sup3;⁺ forms weak electrostatic interactions with typical donor atoms, complicating efforts to achieve both strong binding and kinetic stability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMacropa (bp18c6), a 4,13-diaza-18-crown-6-macrocycle ligand with two pendant picolinate arms, has demonstrated strong chelation capabilities for \u003csup\u003e225\u003c/sup\u003eAc due to its high selectivity for large trivalent ions, particularly among the lanthanide and actinide series [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. One of its major advantages over traditional chelators such as DOTA is its ability to form stable complexes with \u003csup\u003e225\u003c/sup\u003eAc within 5 minutes at room temperature, even at low micromolar concentrations [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Macropa has also shown excellent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e stability when conjugated to both small molecules and monoclonal antibodies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For example, Schatz et \u003cem\u003eal.\u003c/em\u003e evaluated the efficacy of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-pelgifatamab in various cell-derived and patient-derived prostate cancer xenografts [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its favourable coordination properties, the macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e derivative, commonly used for bioconjugation, suffers from limited shelf-life due to rapid degradation. This has prompted efforts to develop more chemically robust analogues. Recently, Kadassery \u003cem\u003eet al.\u003c/em\u003e reported the synthesis of a more stable bifunctional macropa derivative, H\u003csub\u003e2\u003c/sub\u003eBZmacropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e, via a five-step procedure [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the [\u0026sup2;\u0026sup2;⁵Ac]Ac-BZmacropa-GC33 conjugate (antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3) showed only 55% stability in human serum, indicating that further optimization is needed to improve stability.\u003c/p\u003e \u003cp\u003e3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e (1, 2- [(carboxymethyl)] [5-(4-nitrophenyl-1-[4, 7, 10-tris(carboxymethyl) -1, 4, 7, 10- tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), is a decadentate ligand featuring ten electron donor atoms for complex formation. Its bifunctional derivative, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e, has been reported to rapidly chelate \u003csup\u003e90\u003c/sup\u003eY and \u003csup\u003e177\u003c/sup\u003eLu within 1 minute, achieving radiolabelling efficiencies of 89% and 94%, respectively [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, to date, no data have been reported regarding its performance with \u003csup\u003e225\u003c/sup\u003eAc.\u003c/p\u003e \u003cp\u003eIn this study, we aim to evaluate the potential of DEPA-based chelators for \u003csup\u003e225\u003c/sup\u003eAc-labelling. We first evaluated the radiolabelling efficiency and \u003cem\u003ein vitro\u003c/em\u003e stability of the chelator 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNO₂\u003c/sub\u003e with \u0026sup2;\u0026sup2;⁵Ac. We then synthesized two BFC derivatives, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNCS\u003c/sub\u003e and 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eTFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e and conjugated them to the monoclonal antibody trastuzumab. The radiochemical stability of the resulting \u0026sup2;\u0026sup2;⁵Ac-conjugates was assessed, and finally, we conducted a biodistribution study to evaluate the \u003cem\u003ein vivo\u003c/em\u003e behaviour. As a reference, macropa-\u003csub\u003eNCS\u003c/sub\u003e and DOTA-\u003csub\u003eNCS\u003c/sub\u003e were used in the conjugation and radiolabelling of trastuzumab to compare the stability and performance of these established chelators with the newly synthesized BFCs.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral\u003c/h2\u003e \u003cp\u003eAll chemicals and solvents were purchased from commercial suppliers such as Sigma-Aldrich (Bornem, Belgium), Fluka (Bornem, Belgium), Fisher (Doornik, Belgium) and Acros Organics (Geel, Belgium) and were used without further purification. DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e was purchased from Macrocyclics, Inc (Plano, TX). Macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e was synthesized as reported by Thiele et \u003cem\u003eal.\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and analysed using Nuclear Magnetic Resonance Spectroscopy (\u003csup\u003e1\u003c/sup\u003eH-NMR), High performance Liquid Chromatography (HPLC) and mass spectroscopy (MS). Refer to Kang \u003cem\u003eet al.\u003c/em\u003e for details regarding the synthesis of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e and 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Details of the synthesis of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e are provided (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All water was deionized and passed through Millipore water purification system until a resistivity of 18 MΩ\u0026middot;cm was achieved. Trastuzumab (RRID: AB_3112050) was of research grade and was purchased from Ichorbio (catalogue number: ICH4013, Oxford, UK). \u003csup\u003e225\u003c/sup\u003eAc for bioconjugate labelling was supplied by the Isotope Program within the Office of Nuclear Physics in the Department of Energy\u0026rsquo;s Office of Science (Oak Ridge National Laboratory, TN, USA)\u003c/p\u003e \u003cp\u003eFemale Balb/C mice (RRID: IMSR_CRL:028) (n\u0026thinsp;=\u0026thinsp;3/group) were purchased from Charles River (Saint-Constant, QC, CA), were at least 6 weeks of age and were used for biodistribution studies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1. Conjugation of bifunctional chelators to trastuzumab\u003c/h3\u003e\n\u003cp\u003eTrastuzumab was conjugated with BFCs following lab SOPs [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, a 15-mole excess of a 20 mg/mL of either, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e, or DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e in DMSO solution was incubated with trastuzumab in 0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e for 90 minutes at 37\u0026deg;C with constant shaking. For macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e conjugation, the reaction was carried out in 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e and 0.15 M NaCl for 18 h at 4\u0026deg;C. The unreacted BFC was removed by centrifugation using Amicon Ultra-10k (Burlington, MA) filters. 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, DOTA-trastuzumab, or macropa-trastuzumab, was buffer exchanged to PBS and concentrated with Amicon Ultra centrifugal filters which afforded\u0026thinsp;\u0026gt;\u0026thinsp;96% purity. The purified conjugates were stored at -80\u0026deg;C before labelling. The purity of the respective conjugates was performed using a size-exclusion HPLC (SEC-HPLC) Waters 2487 Dual λ Absorbance Detector, XBridge\u0026reg; BEH 200 A SEC 3.5 \u0026micro;m, 7.8 x 150 nm column (Waters Corporation, Milford, MA) and an Agilent 2100 Bioanalyzer system (Agilent High Sensitivity Protein 250 Kit- catalogue# 5067\u0026thinsp;\u0026minus;\u0026thinsp;1575) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003cp\u003e \u003cb\u003e2. Radiochemistry of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e225\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eAc]Ac-3p-\u003c/b\u003e \u003cb\u003eC\u003c/b\u003e \u003cb\u003e-DEPA\u003c/b\u003e \u003csub\u003e \u003cb\u003e-NO2\u003c/b\u003e \u003c/sub\u003e \u003c/p\u003e\u003cp\u003e[\u003csup\u003e225\u003c/sup\u003eAc]Ac(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e (0.5 M HNO\u003csub\u003e3\u003c/sub\u003e) was produced on-site at SCK CEN based on literature [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and from Oak Ridge National Lab ORNL (Oak Ridge, TN, USA) containing trace amounts of \u003csup\u003e227\u003c/sup\u003eAc. All radiolabelling buffers were treated with Chelex 100 [sodium form (50\u0026ndash;100 mesh, Sigma Aldrich)] for 15 min to remove trace metals. All solutions were degassed and filtered before use.\u003c/p\u003e \u003cp\u003eThe radiolabelling experiments were performed by reacting 90\u0026ndash;100 kBq of [\u003csup\u003e225\u003c/sup\u003eAc]Ac(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e at 25, 40, 55 or 95\u0026deg;C for 1 hour (h) with different concentrations of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e (1, 5, 10 and 20 \u0026micro;M) in 0.37 M TRIS buffer, pH 8.5, V\u0026thinsp;=\u0026thinsp;300 \u0026micro;L) to yield [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e. The % radiochemical conversion (RCC) was evaluated by instant thin-layer liquid chromatography (iTLC-SG, Varian, Diegem, Belgium). iTLC-SG papers were developed in an elution chamber using acetonitrile: water (75/25 v/v) such that bound \u003csup\u003e225\u003c/sup\u003eAc and daughter radionuclides will migrate with the solvent front to the upper part of the iTLC strip, while unbound radionuclides will remain at the lower part where the mixture was originally spotted. Once the solvent front reached the top of the iTLC strip, it was removed from the mobile phase and cut into two (top and bottom). The activity of the upper and lower part of the iTLC strip was measured with a gamma counter (Wallac Wizard 1480, PerkinElmer, Waltham, MA) using the \u003csup\u003e213\u003c/sup\u003eBi-peak window (380\u0026ndash;500 keV) after a time delay of 24 h to allow \u003csup\u003e213\u003c/sup\u003eBi to reach equilibrium with \u003csup\u003e225\u003c/sup\u003eAc as described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The %RCC is calculated as activity at the top half divided by the total activity (top\u0026thinsp;+\u0026thinsp;bottom) x 100.\u003c/p\u003e\n\u003ch3\u003e3. Radiochemistry of immunoconjugates\u003c/h3\u003e\n\u003cp\u003e3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab immunoconjugates were radiolabelled using [\u003csup\u003e225\u003c/sup\u003eAc]Ac(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e (2.0 MBq) dissolved in 0.1 M Hydrochloric acid (Optima grade, Fisher Scientific) at a specific activity of 8 kBq/\u0026micro;g as reported [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. About 10 \u0026micro;L of ascorbic acid was added to prevent radiolysis in all the reactions. The pH of the reaction was determined by spotting 1 \u0026micro;L of the reaction mixture onto Hydrion pH paper (range, 5.0\u0026ndash;9.0) (Sigma-Aldrich); pH of a typical reaction was 5.8-6.0. The incubation was done at 37\u0026deg;C on a shaker at 700 RPM for 2 h. A small aliquot (0.8 \u0026micro;L) was spotted on a strip of instant thin-layer chromatography silica gel impregnated paper (iTLC-SG, Agilent Technologies) to determine the extent of incorporation of \u003csup\u003e225\u003c/sup\u003eAc onto the protein using mobile phase of 20 mM sodium citrate (pH 5.2).\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. In vitro\u003c/b\u003e \u003cb\u003estability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e stability of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e radio-complex (150 kBq/\u0026micro;mol) was evaluated in PBS and human serum (HS). After radiolabelling 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e, the radio-complex was purified with a Sep-Pak C\u003csub\u003e18\u003c/sub\u003e Light cartridge (Waters, Eschborn, Germany). Briefly, the Sep-Pak C\u003csub\u003e18\u003c/sub\u003e Light cartridge was pre-conditioned with absolute ethanol (5 mL) followed by water (5 mL). The reaction mixture was loaded onto the cartridge and washed with 6\u0026ndash;8 mL water to remove unbound \u003csup\u003e225\u003c/sup\u003eAc. The pure radio-complex was eluted with 0.2 mL absolute ethanol and the volume was brought to 0.5 mL by diluting with 0.3 mL of 0.9% NaCl. 80 \u0026micro;L of the purified radio-complex was added to a 1 mL vial containing either 420 \u0026micro;L of PBS or HS, and the solution was incubated at 37\u0026deg;C under constant gentle shaking. To determine the percentage of intact [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e, 5 \u0026micro;L samples were taken for iTLC analysis at selected times points (30, 60 min, 2 d, 3 d, and 6 d). Development of iTLC and counting of activity was performed as described above.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e stability of the radioimmunoconjugates [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab was determined in PBS at 37\u0026deg;C for 10 days (1, 2, 3, 4, 7 and 10 d). Each radioimmunoconjugate (RIC) was incubated at the respective incubation conditions to make a final concentration of ~\u0026thinsp;400 kBq/500 \u0026micro;L. To analyse the purity, aliquots of 9 \u0026micro;L of each radioimmunoconjugate was drawn for iTLC and analysed as described above.\u003c/p\u003e\u003cp\u003e \u003cb\u003e5. In vivo\u003c/b\u003e \u003cb\u003ebiodistribution of radioimmunoconjugates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBiodistribution of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab was studied in healthy Balb/C mice (n\u0026thinsp;=\u0026thinsp;3/group). The mice were housed under standard conditions in approved facilities with 12 h light/dark cycles and given food and water ad libitum throughout the duration of the studies following a tail vein injection of 11.1 kBq of each radiolabelled construct. Mice were sacrificed at 48 h post injection (p.i.), and the activity in organs was measured using gamma counter (\u003csup\u003e213\u003c/sup\u003eBi-peak window (380\u0026ndash;500 keV) ,Wallac Wizard 1480, PerkinElmer) and expressed as the % injected activity per gram of the organ (%IA/g).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). A two-way ANOVA with Dunnett post hoc test was used to determine the statistical significance between the different mice groups. All graphs or figures were analysed using GraphPad Prism Version 10 (RRID:SCR_002798).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eSynthesis of 3p-\u003c/b\u003e \u003cb\u003eC\u003c/b\u003e \u003cb\u003e-DEPA, 3p-C-DEPA\u003c/b\u003e \u003csub\u003e \u003cb\u003e\u0026minus;\u0026thinsp;NCS\u003c/b\u003e \u003c/sub\u003e, \u003cb\u003eand 3p-\u003c/b\u003e\u003cb\u003eC\u003c/b\u003e\u003cb\u003e-DEPA\u003c/b\u003e\u003csub\u003e\u003cb\u003e\u0026minus;\u0026thinsp;TFP\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-PEG\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eSince 3p-\u003cem\u003eC\u003c/em\u003e-DEPA is not commercially available, it was synthesized following a strategy involving the formation of an N,N\u0026prime;-bisubstituted-β-amino iodide intermediate and subsequent nucleophilic ring opening of an aziridinium ion, as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, β-iodoamine was subjected to intramolecular rearrangement to form the aziridinium ion, which was then regioselectively opened via an SN\u003csub\u003e2\u003c/sub\u003e mechanism using tri-tert-butyl 2,2\u0026prime;,2\u0026Prime;-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate. This yielded the protected chelator 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-(\u003cem\u003et\u003c/em\u003eBu)\u003csub\u003e5\u003c/sub\u003e in an isolated yield of 86% and a purity exceeding 95%. The tert-butyl protecting groups were subsequently removed by treatment with trifluoroacetic acid (TFA), affording deprotected 3p-\u003cem\u003eC\u003c/em\u003e-DEPA at \u0026gt;\u0026thinsp;96% purity. 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was used directly in initial radiolabelling and \u003cem\u003ein vitro\u003c/em\u003e stability studies.\u003c/p\u003e \u003cp\u003eTo enable conjugation of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA to a targeting vector such as trastuzumab, the chelator was further functionalized to introduce reactive groups capable of coupling to lysine residues. Two linker strategies were explored: isothiocyanate derivatization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), as previously reported by Kang et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and PEG₄-TFP ester conjugation, a tetrafluorophenyl-activated ester linker. In the PEG₄-TFP approach, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) was synthesized by the controlled, dropwise addition of 1.5 molar equivalents of TFP-PEG₄-TFP to 3p-C-DEPA-\u003csub\u003eNH₂\u003c/sub\u003e(tBu)₅. The reaction mixture was purified using preparative HPLC, and the tert-butyl protecting groups were removed by treatment with TFA. The final product, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003eTFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e, was characterized by LC-HRMS, confirming\u0026thinsp;\u0026gt;\u0026thinsp;95% chemical purity. Also, the chemical structures of DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e and macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRadiolabelling and characterization of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e225\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eAc]Ac-3p-\u003c/b\u003e \u003cb\u003eC\u003c/b\u003e \u003cb\u003e-DEPA\u003c/b\u003e \u003csub\u003e \u003cb\u003e\u0026minus;\u0026thinsp;NO2\u003c/b\u003e \u003c/sub\u003e \u003c/p\u003e \u003cp\u003e3p-C-DEPA-\u003csub\u003eNO₂\u003c/sub\u003e demonstrated efficient radiolabelling with \u0026sup2;\u0026sup2;⁵Ac under mild conditions. At a ligand concentration of 1 \u0026micro;M, rapid chelation was achieved within 1 hour, with radiochemical conversion (RCC) values of 93.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%, 95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%, and 98.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% at 25\u0026deg;C, 40\u0026deg;C, and 55\u0026deg;C, respectively. Further increases in ligand concentration (5\u0026ndash;20 \u0026micro;M) and reaction temperature (40\u0026ndash;95\u0026deg;C) resulted in consistently high RCC values exceeding 97% under all tested conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eGiven the favourable radiolabelling kinetics, we evaluated the \u003cem\u003ein vitro\u003c/em\u003e stability of [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNO₂\u003c/sub\u003e over a six-day period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The radio-complex exhibited excellent stability in human serum, retaining\u0026thinsp;\u0026gt;\u0026thinsp;95% of intact complex throughout the six-day incubation. In PBS, stability was also maintained over the first three days, with \u0026gt;\u0026thinsp;95% of intact radio-complex observed. By day six, 83.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% of the radio-complex remained intact in PBS, indicating partial degradation under these conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConjugation and characterization of immunoconjugates\u003c/h2\u003e \u003cp\u003eEncouraged by the initial radiolabelling studies with \u003csup\u003e225\u003c/sup\u003eAc using 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e, the bifunctional chelators 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e and 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e were conjugated (non-site specifically) to trastuzumab and compared with DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e and macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e-derivatized trastuzumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Conjugation of trastuzumab with 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e, DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e, and macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e yielded immunoconjugates 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), respectively. The HPLC showed that all the immunoconjugates were at least 98% pure with less than 2% degradation or aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The bioanalyzer microfluidic electrophoresis experiment confirmed the purity of the various immunoconjugates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRadiolabelling and characterisation of radioimmunoconjugates\u003c/h3\u003e\n\u003cp\u003eThe immunoconjugates 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab, DOTA-trastuzumab, and macropa-trastuzumab were radiolabelled with [\u0026sup2;\u0026sup2;⁵Ac]Ac(NO₃)₃ at a specific activity of 8 kBq/\u0026micro;g. After 2 hours of incubation at 37\u0026deg;C, the RCC was 94.6% for [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 93.5% for [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab, 80.9% for [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab, and 96.5% for [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe high RCCs observed for both 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄, and macropa-conjugated antibodies obviated the need for post-labelling purification. In contrast, [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab required purification by centrifugal filtration, yielding a final radiochemical purity of 95.9%.\u003c/p\u003e \u003cp\u003eThe \u0026sup2;\u0026sup2;⁵Ac-labelled immunoconjugates were evaluated for \u003cem\u003ein vitro\u003c/em\u003e stability (PBS, 37\u0026deg;C) over a 10-day period. Remarkably, [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab exhibited the highest stability, retaining 91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3% of the intact radiocomplex after 10 days. In comparison, [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab maintained 81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6% integrity, while [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab showed 60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% intact complex. The lowest stability was observed for [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, with only 48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% of the radiocomplex remaining intact at day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiodistribution studies of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e225\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eAc]Ac-3p-\u003c/b\u003e \u003cb\u003eC\u003c/b\u003e \u003cb\u003e-DEPA-trastuzumab, [\u003c/b\u003e \u003csup\u003e \u003cb\u003e225\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eAc]Ac-DOTA-trastuzumab, and [\u003c/b\u003e \u003csup\u003e \u003cb\u003e225\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eAc]Ac-macropa-trastuzumab\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDespite the relatively lower \u003cem\u003ein vitro\u003c/em\u003e stability of [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab in PBS, we proceeded to evaluate its \u003cem\u003ein vivo\u003c/em\u003e biodistribution in healthy Balb/C mice over a 48-hour period, using [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab and [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab as reference standards (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Overall, no significant differences were observed in the biodistribution profiles across major organs (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all comparisons), with two exceptions.\u003c/p\u003e \u003cp\u003eIn the liver, [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab exhibited significantly higher uptake (14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%IA/g) compared to [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%IA/g, p\u0026thinsp;=\u0026thinsp;0.04) whereas that for [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab was 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8. Conversely, in the large intestine, [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab showed significantly higher accumulation (2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%IA/g) than [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%IA/g, p\u0026thinsp;=\u0026thinsp;0.03).\u003c/p\u003e \u003cp\u003eConsistent with expectations for radioimmunoconjugates, the highest activity was retained in the blood at 48 hours post-injection, with values of 18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%IA/g for [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab, 19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5%IA/g for [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, and 19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7%IA/g for [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u0026sup2;\u0026sup2;⁵Ac is a highly promising radionuclide for TAT due to its favourable decay properties and potent cytotoxicity against cancer cells [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Despite its therapeutic potential, the widespread clinical translation of \u0026sup2;\u0026sup2;⁵Ac-based radiopharmaceuticals has been hindered by two major factors: limited availability of the isotope and the challenge of identifying suitable chelators for the large trivalent actinium ion (Ac\u0026sup3;⁺). Although chelators such as DOTA, macropa, and macrodipa have been developed and studied extensively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], each has limitations. DOTA remains the clinical standard for \u0026sup2;\u0026sup2;⁵Ac coordination, but its slow radiolabelling kinetics, especially under mild conditions, pose significant challenges for heat-sensitive biomolecules such as monoclonal antibodies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we investigated 3p-\u003cem\u003eC\u003c/em\u003e-DEPA, a hybrid decadentate ligand combining structural features of DOTA and DTPA, as a potential alternative chelator for \u0026sup2;\u0026sup2;⁵Ac. We successfully synthesized 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO₂\u003c/sub\u003e in high yield and purity, using a previously reported method for 3p-\u003cem\u003eC\u003c/em\u003e-NETA [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The presence of an additional acyclic iminodiacetic acid arm likely contributes to the faster complexation kinetics and improved radiolabelling performance observed with 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO2\u003c/sub\u003e, compared to DOTA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e. Indeed, [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-C-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NO₂\u003c/sub\u003e demonstrated excellent radiolabelling efficiency (\u0026ge;\u0026thinsp;95% RCC) at room temperature at low chelator concentration, a significant advantage for radiolabelling heat-sensitive biomolecules whereas [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab showed a low RCC of 80.9%.\u003c/p\u003e \u003cp\u003eThese findings align with previous work by Song et al., who noted the effectiveness of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA for large metal coordination due to its larger cavity size relative to DOTA [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In contrast, prior studies have shown that DOTA requires high temperatures (\u0026ge;\u0026thinsp;85\u0026deg;C) to achieve efficient \u0026sup2;\u0026sup2;⁵Ac complexation, with RCC values of only\u0026thinsp;~\u0026thinsp;15% at 40\u0026deg;C even after extended incubation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The ability of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA to rapidly and efficiently bind \u0026sup2;\u0026sup2;⁵Ac at lower temperatures is thus a noteworthy improvement.\u003c/p\u003e \u003cp\u003eTo assess the utility of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA in an antibody-based radiopharmaceutical, we synthesized two bifunctional derivatives: 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e and a novel PEGylated tetrafluorophenol ester (3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eTFP\u003c/sub\u003e-PEG₄). 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eTFP\u003c/sub\u003e-PEG₄ is easy to synthesize starting from the reported 3p-C-DEPA-\u003csub\u003eNH₂\u003c/sub\u003e(tBu)₅ and demonstrate good stability when stored at -20\u0026deg;C. Both bifunctional chelators were successfully conjugated to trastuzumab, resulting in the corresponding radioimmunoconjugates. Radiolabelling of [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab and [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-C-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab yielded high RCCs (94.6% and 93.5%, respectively), comparable to previous results using [\u0026sup2;⁰⁵/\u0026sup2;⁰⁶Bi]Bi-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab reported by Song et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, \u003cem\u003ein vitro\u003c/em\u003e stability studies in PBS revealed significant differences between the two constructs. [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab showed notable degradation over 10 days (only 48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% remaining intact at day 10), likely due to radiolytic effects. Though this observation was surprising, it is reported in the literature that Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (present in PBS) can undergo radiation-induced formation of hypochlorite ions which could potentially react with the enolized thiourea unit [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. As 3p-\u003cem\u003eC\u003c/em\u003e-DEPA is coupled to trastuzumab via a thiourea linker, this might be a reason for the observed instability. Remarkably, this effect was less observed with the macropa\u003csub\u003e\u0026minus;\u0026thinsp;NCS\u003c/sub\u003e-conjugate which demonstrated high stability in PBS. To address this instability in PBS of [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, the PEGylated-TFP derivative was designed to form more stable amide bonds with lysine residues. Encouragingly, [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab exhibited superior \u003cem\u003ein vitro\u003c/em\u003e stability, maintaining\u0026thinsp;\u0026gt;\u0026thinsp;90% integrity after 10 days in PBS.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e biodistribution studies in healthy Balb/C mice showed that [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab exhibited a comparable organ distribution profile to both [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab and [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab over 48 hours. This indicates that the limited \u003cem\u003ein vitro\u003c/em\u003e stability of [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab in PBS has limited consequences for the \u003cem\u003ein vivo\u003c/em\u003e stability.\u003c/p\u003e \u003cp\u003eNotably, liver uptake was significantly lower for [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-C-DEPA-trastuzumab (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%IA/g) and [\u0026sup2;\u0026sup2;⁵Ac]Ac-macropa-trastuzumab (6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%IA/g) compared to [\u0026sup2;\u0026sup2;⁵Ac]Ac-DOTA-trastuzumab (14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%IA/g, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). This indicates that both the \u0026sup2;\u0026sup2;⁵Ac-3p-C-DEPA and \u0026sup2;\u0026sup2;⁵Ac-macropa complexes exhibit comparable \u003cem\u003ein vivo\u003c/em\u003e kinetic inertness, superior to that of the \u0026sup2;\u0026sup2;⁵Ac-DOTA complex. This distinction is particularly important given the known hepatotoxicity associated with free \u0026sup2;\u0026sup2;⁵Ac and its radioactive decay products [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The high blood retention observed for all constructs reflects the long circulation time of intact antibody conjugates. \u003cem\u003eIn vivo\u003c/em\u003e evaluation of the second-generation [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄-trastuzumab has not yet been conducted. However, studies in tumour-bearing mice are planned to assess their pharmacokinetics and tumour-targeting efficiency.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study demonstrates the promising potential of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-based chelators for the development of \u0026sup2;\u0026sup2;⁵Ac-labelled radiopharmaceuticals. Among the constructs evaluated, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄ stood out as a highly effective bifunctional chelator, achieving excellent radiolabelling efficiency under mild conditions and superior \u003cem\u003ein vitro\u003c/em\u003e stability compared to DOTA analogues. Importantly, [\u0026sup2;\u0026sup2;⁵Ac]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u0026minus;\u003c/sub\u003ePEG₄-trastuzumab maintained\u0026thinsp;\u0026gt;\u0026thinsp;90% integrity in PBS over 10 days, indicating strong resistance to radiolytic and hydrolytic degradation. Additionally, the biodistribution profile of 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-based conjugates was comparable to established chelators, with notably lower liver uptake than the \u003csup\u003e225\u003c/sup\u003eAc-DOTA-conjugate, reducing concerns related to off-target hepatotoxicity. These findings highlight 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄ as a promising alternative to current \u003csup\u003e225\u003c/sup\u003eAc-bifunctional chelators, offering advantages in radiolabelling kinetics and stability. Further preclinical studies, including pharmacokinetic studies in mouse tumour models, therapeutic efficacy and long-term \u003cem\u003ein vivo\u003c/em\u003e stability studies are warranted to confirm its utility in TAT applications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBFC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBifunctional chelators\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDOTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiethylenetriamine pentaacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEthylenediaminetetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eITLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstant thin-layer chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-HRMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiquid chromatography- High resolution mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLinear energy transfer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMass spectroscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ep.i\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epost injection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstate specific membrane antigen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRadiochemical conversion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRevolutions per minutes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard error of mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTargeted alpha therapies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTETA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriethylenetetramine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTETPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriethylene tetramine pentaacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrifuoroacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTetrafluorophenol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were approved, supervised, and maintained following the guidelines set for by the University of Saskatchewan Animal Care Committee (UACC), protocol # 20170084. Procedures were carried out according to the laboratory animal care and use of the Canadian Council on Animal Care. These mice had at least one week of acclimatization before being assigned to the various groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials’ statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding authors Prof. Fonge and Prof. Cleeren\u0026nbsp;on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Canadian Institute for Health Research (CIHR) Project Grants (Grant numbers 437660 and 408132) to Humphrey Fonge and internal funding KU Leuven.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no additional acknowledgements to make.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, FC, and HF; methodology, JPK, SA, SL, FC, and HF; software, JPK, SA; validation, JPK, SA, FC, and HF; formal analysis, JPK and SA; investigation, JPK, SA, EN; resources, FC and HF; data curation, JPK and SA; writing-original draft preparation, JPK and SA; writing-review and editing, JPK, SA, EN, MO, TC, SL, FC and HF; visualization, JPK, SA, FC, and HF; supervision, FC and HF; project administration, FC and HF; funding acquisition, FC and HF.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePougoue Ketchemen, J., et al., \u003cem\u003eComplete remissions of HER2-positive trastuzumab-resistant xenografts using a potent [225Ac]Ac-labeled anti-HER2 antibody-drug radioconjugate.\u003c/em\u003e Clinical Cancer Research, 2024.\u003c/li\u003e\n\u003cli\u003eBrechbiel, M.W. and M.W. 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Med, 2019. \u003cstrong\u003e60\u003c/strong\u003e(supplement 1): p. 60-60.\u003c/li\u003e\n\u003cli\u003eQin, Y., et al., \u003cem\u003eEvaluation of actinium-225 labeled minigastrin analogue [225ac]ac-dota-pp-f11n for targeted alpha particle therapy.\u003c/em\u003e Pharmaceutics, 2020. \u003cstrong\u003e12\u003c/strong\u003e(11): p. 1-12.\u003c/li\u003e\n\u003cli\u003eSong, H.A., et al., \u003cem\u003eEfficient Bifunctional Decadentate Ligand 3p-C-DEPA for Targeted \u0026alpha;-Radioimmunotherapy Applications.\u003c/em\u003e Bioconjugate Chemistry, 2011. \u003cstrong\u003e22\u003c/strong\u003e(6): p. 1128-1135.\u003c/li\u003e\n\u003cli\u003eRamogida, C.F., et al., \u003cem\u003eEvaluation of polydentate picolinic acid chelating ligands and an \u0026alpha;-melanocyte-stimulating hormone derivative for targeted alpha therapy using ISOL-produced 225Ac.\u003c/em\u003e EJNMMI radiopharm. chem., 2019. \u003cstrong\u003e4\u003c/strong\u003e(1): p. 1-20.\u003c/li\u003e\n\u003cli\u003eVugts, D.J., et al., \u003cem\u003eComparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for 89Zr-immuno-PET.\u003c/em\u003e Eur J Nucl Med Mol Imaging, 2017. \u003cstrong\u003e44\u003c/strong\u003e(2): p. 286-295.\u003c/li\u003e\n\u003cli\u003ePruszynski, M., et al., \u003cem\u003eEvaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted \u0026alpha;-Particle Therapy of Cancer.\u003c/em\u003e Molecular Pharmaceutics, 2018. \u003cstrong\u003e15\u003c/strong\u003e(4): p. 1457-1466.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Targeted alpha therapy, Actinium-225, bifunctional chelators, 3p-C-DEPA, trastuzumab","lastPublishedDoi":"10.21203/rs.3.rs-6813431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6813431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eActinium-225 (\u003csup\u003e225\u003c/sup\u003eAc) based targeted alpha therapies (TAT) have emerged as a promising strategy for the treatment of several cancer types due to its favourable decay properties, including high linear energy transfer and short particle range, which enable precise tumour targeting. However, there are limited bifunctional chelators (BFCs) available for \u003csup\u003e225\u003c/sup\u003eAc. In this study, we aim to evaluate the potential of DEPA-based chelators for \u003csup\u003e225\u003c/sup\u003eAc-labelling.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe BFCs 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNO2\u003c/sub\u003e, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNCS\u003c/sub\u003e, and 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e were synthesized with high yield (\u0026ge;\u0026thinsp;86%) and purity (\u0026gt;\u0026thinsp;96%). Excellent radiochemical conversions (RCCs) were achieved for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-\u003csub\u003eNO2\u003c/sub\u003e across a range of concentrations (1\u0026ndash;20 \u0026micro;M) with high RCC\u0026rsquo;s (93.7 to 96.8%) after 1 hour at room temperature. Stability studies demonstrated that over 95% of this \u003csup\u003e225\u003c/sup\u003eAc-labelled complex remained intact after 6 days in human serum. The HPLC and bioanalyzer analysis of the immunoconjugates 3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, DOTA-trastuzumab, 3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab and macropa-trastuzumab showed 98% purity with less than 2% impurities. A RCC of 94.6% was obtained for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab, 93.5% for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab, 80.9% for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab, and 96.5% for [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab after 2 h incubation at 37\u0026deg;C. In PBS, high stability of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG\u003csub\u003e4\u003c/sub\u003e-trastuzumab was observed (91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%), which is comparable to that of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab (81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6%). In contrast, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-\u003cem\u003eC\u003c/em\u003e-DEPA-trastuzumab and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab were less stable in PBS with only 48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% and 60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% intact tracer left after 10 d. There were no major significant differences between the biodistribution profile of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-C-DEPA-trastuzumab, [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-macropa-trastuzumab in all organs of interest (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all organs). However, the liver uptake of [\u003csup\u003e225\u003c/sup\u003eAc]Ac-DOTA-trastuzumab (14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% IA/g) was higher than [\u003csup\u003e225\u003c/sup\u003eAc]Ac-3p-C-DEPA-trastuzumab (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3% IA/g) (p\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e3p-\u003cem\u003eC\u003c/em\u003e-DEPA\u003csub\u003e\u0026minus;\u0026thinsp;TFP\u003c/sub\u003e-PEG₄ demonstrated excellent potential as a bifunctional chelator for \u0026sup2;\u0026sup2;⁵Ac, showing high radiolabelling efficiency under mild conditions and outstanding \u003cem\u003ein vitro\u003c/em\u003e stability of the resulting \u003csup\u003e225\u003c/sup\u003eAc-labelled bioconjugate. Further preclinical studies are warranted to validate its therapeutic potential.\u003c/p\u003e","manuscriptTitle":"Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 10:43:48","doi":"10.21203/rs.3.rs-6813431/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-07-05T06:07:47+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-13T07:46:27+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T07:45:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T17:23:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-06-11T12:19:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d8347fe-0dcb-4556-910f-37d37860dee3","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:02:37+00:00","versionOfRecord":{"articleIdentity":"rs-6813431","link":"https://doi.org/10.1186/s41181-025-00408-w","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2025-12-22 15:58:08","publishedOnDateReadable":"December 22nd, 2025"},"versionCreatedAt":"2025-06-17 10:43:48","video":"","vorDoi":"10.1186/s41181-025-00408-w","vorDoiUrl":"https://doi.org/10.1186/s41181-025-00408-w","workflowStages":[]},"version":"v1","identity":"rs-6813431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6813431","identity":"rs-6813431","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}