All at once: a sstR2-targeted radiohybrid theranostic agent for PET imaging and β- therapy with excellent preclinical performance

All at once: a sstR2-targeted radiohybrid theranostic agent for PET imaging and β- therapy with excellent preclinical performance | 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 Article All at once: a sstR2-targeted radiohybrid theranostic agent for PET imaging and β - therapy with excellent preclinical performance Sandra Deiser, Sebastian Fenzl, Victor König, Shigeyoshi Inoue, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8090442/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The radiohybrid (rh) design of radiopharmaceuticals has recently produced new theranostics suitable for both positron emission tomography (PET) imaging and peptide receptor radionuclide therapy (PRRT). This approach aims to address the limitations of current medical radionuclides by offering a new strategy for combining radionuclides that previously lacked both therapeutic and diagnostic applications. Here, we report on a somatostatin receptor subtype 2 (sstR2)-targeted radiohybrid compound, rhTATE4 , which features a bifunctional silicon-based fluoride acceptor (SiFA) - named (SiFA) SeFe - for 18 F labelling, along with a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelator for 177 Lu coordination. The rh-theranostic agent demonstrates similar in vitro behaviour compared to the gold standards [ 177 Lu]Lu-DOTA-TATE and SiFA lin -TATE, along with an exceptionally high tumor uptake (53.58 ± 5.51% ID/g for the radiofluorinated version) after 1 h post-injection in AR42J tumor-bearing mice, making it ideal for imaging. Moreover, clearance from normal tissues and considerable tumor retention (10.32 ± 7.04%ID/g) for [ 177 Lu]Lu-TATE4 were observed at 24 p.i., suggesting good therapeutic applicability. Biological sciences/Cancer Physical sciences/Chemistry Biological sciences/Drug discovery Health sciences/Oncology Fluorine-18 Lutetium-177 theranostics somatostatin receptor radiopharmaceuticals radiohybrid Figures Figure 1 Figure 2 Figure 3 Introduction The term "theranostics" refers to the integration of two medical procedures that enable both diagnostic imaging and therapeutic treatment. In the context of nuclear medicine, this involves the use of identical or similar radiopharmaceutical compounds that contain radioactive isotopes. 1 – 3 A classical categorization includes the following main strategies for theranostic design: isotopically matched pairs, the use of ‘true’ theranostic nuclides, and matched radiopharmaceuticals. The iodine radioisotopes serve as a notable example of isotopically matched pairs , being the first radiopharmaceutical that laid the groundwork for theranostic applications. 4, 5 Thus, iodine-123 ( γ -emitter) is used for diagnostics in single-photon emission computed tomography (SPECT), and iodine-131 ( γ - and β - -emitter) for the treatment of thyroid cancer. 6 The radiopharmaceuticals of this group contain either a therapeutic or a diagnostic radionuclide of the same element, whereby both compounds behave chemically and biologically identically, enabling precise dosimetry and therapy control. 7 In the case of a ‘ true’ theranostic approach, the incorporated radionuclide emits either γ -rays or positrons that can be used for imaging, and α or β - radiations for therapy. A prominent example of this approach is the nuclide lutetium-177, which is already used in the clinic within the high-affinity somatostatin receptor subtype 2 (sstR2)-targeted [ 177 Lu]Lu-DOTA-TATE (TATE = (Tyr 3 )-octreotate, DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Lutathera ® ) for the therapy of neuroendocrine tumors. 8 , 9 The γ-rays from lutetium-177 can be utilized for SPECT imaging, allowing dosimetry calculations to be performed after the first therapy cycle. 10 However, 177 Lu-based SPECT scans are not used in clinical practice for therapy planning due to their lower resolution. 11 To overcome this limitation, the matched radiopharmaceuticals approach provides a combination of imaging and therapeutic nuclides. In the case of neuroendocrine tumors, [ 68 Ga]Ga-DOTA-TATE (NETSPOT ® ) is used for positron emission tomography (PET) imaging as complementary to the therapeutic [ 177 Lu]Lu-DOTA-TATE. 12 – 15 However, the differing radiometal complexes of Ga 3+ and Lu 3+ result in distinct pharmacokinetic profiles, leading to diverse pharmacological properties. 16 – 21 In addition to the aforementioned classical design approaches, the radiohybrid strategy is emerging, whereby a single molecule features two separate binding sites for different radionuclides (Fig. 1 A). 22 This aims to establish a more consistent pharmacological profile for the radioconjugate, resulting in more personalized and effective treatment plans. In detail, as shown in Fig. 1 A, the concept typically includes a silicon-based fluoride acceptor (SiFA) for fast and efficient 18 F-fluorination via an 18 F/ 19 F isotope exchange reaction, as well as the chelator DOTAGA (2-[1,4,7,10-Tetraazacyclododecane]-pentanedioic acid) for radiometalation (e.g. with 177 Lu). 22 This creates a chemically identical compound pair (either 19 F/radiometal or 18 F/non-radioactive metal) that enables precise dosimetry and therapy monitoring. Moreover, it provides economic and strategic advantages, as the approval for the therapeutic radiohybrid can be streamlined following the approval of the diagnostic counterpart. 23 , 24 The first example of this approach was reported by Wurzer et al. et al. , 23 and successfully reached FDA approval with the compound [ 18 F]Ga-rhPSMA-7.3 (POSLUMA ® , Fig. 1 B) for PET imaging of prostate cancer. 25 The therapeutic counterpart [ 177 Lu]Lu-rhPSMA7.3 and its DOTA-analogue [ 177 Lu]Lu-rhPSMA10.1 showed promising preclinical results. 26 , 27 A primary concern in the classical design of radiohybrid tracers is the high lipophilicity of the SiFA building block (SiFA)BA (4-(di-tert-butylfluorosilyl)benzoic acid), which affects the biodistribution of the resulting radiotracers and may lead to unfavourable in vivo performance. 28 – 30 In a recent attempt to generate an SSTR2-targeted radiohybrid compound, based on the more hydrophilic yet para -functionalized SiFA lin moiety, Wendlinger et al. could not exceed the performance of the benchmark SiFA lin -TATE. 31 To address this limitation, we have recently reported on a new bifunctional SiFA synthon 3-amino-5-(di-tert-butylfluorosilyl)benzoic acid (SiFA) SeFe ), that includes a carboxylic acid and an amine group located at the two meta-positions relative to the Si moiety. 32 This feature not only decreases the lipophilic nature of the SiFA synthon but also enables its bridged incorporation into a radiotracer, akin to a classical amino acid moiety. (SiFA) SeFe has been recently used to develop three different rh-compounds ( rhTATE1-3 ) that showed high accumulation in AR42J tumor-bearing female CD1-nu/nu mice (up to 27%ID/g 1 h post injection (p.i.)), but also in the kidneys (up to 99%ID/g 1 h p.i.). 32 Although total tumor uptake was increased compared to [ 18 F]SiFA lin -TATE (18.51 ± 4.89%ID/g 1 h p.i.), the tumor-to-background (T/B) ratios for most organs were comparable or lower. Additionally, the rapid renal excretion of [ 18 F]Lu-rhTATE3 likely prevents its therapeutic application. Here, we report on an optimized theranostic radiohybrid ligand rhTATE4 , which can be quickly and efficiently labelled with fluorine-18 using the (SiFA) SeFe moiety, as well as stably labelled with lutetium-177 via a DOTA chelator (Fig. 1 B). In this instance, the bifunctional (SiFA) SeFe is incorporated bridging the sstR2 targeting TATE and the chelator. The compound was characterized by RP-HPLC (reverse-phase high-performance liquid chromatography) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). Further, the in vitro properties of the ligand, including lipophilicity, human serum albumin (HSA) binding, sstR2 receptor binding affinity, as well as stability in human plasma were evaluated and compared to the FDA and EMA approved benchmark for PRRT [ nat/177 Lu]Lu-DOTA-TATE, as well as to the clinically tested PET tracer [ nat/18 F]SiFA lin -TATE. 33 Additionally, the biodistribution of [ 18 F]Lu-rhTATE4 was investigated in vivo in AR42J tumor-bearing CD1-nu/nu mice at 1 h p.i.. Similarly, rhTATE4 was labelled with lutetium-177 and its biodistribution studied at 1 h, 6 h, and 24 h p.i. in the same model. Design and Synthesis To generate a theranostic platform, the rh-concept was applied to the TATE analogue rhTATE4 , which contains the SiFA building block (SiFA) SeFe for 18 F labelling and a terminal DOTA chelator for 177 Lu labelling (Fig. 1 B). For this purpose, the linker unit Fmoc-O 2 Oc-OH (8-amino-3,6-dioxaoctanoic acid) was first placed between the pharmacophore TATE and the SiFA synthon in order to maintain a good affinity to sstR2 binding, analogous to SiFA lin -TATE. A d -Asp was introduced as a linker between the DOTA chelator and the SiFA moiety. The metal-free ligand and the corresponding references were synthesized by fluorenylmethoxycarbonyl standard peptide synthesis on solid phase (Fmoc SPPS) using a 2-chlorotrityl chloride (2-CTC) resin (see SI for details, Table S1 ), yielding 3–19% RP-HPLC purified precursors (chemical purity > 95%, determined by RP-HPLC and mass spectrometry, Figures S1 - S1 0). Non-radioactive lutetium labelling was performed quantitatively with a 2.5-fold excess of LuCl 3 at 80°C for 15 min (Figures S4 and S9). Radiolabelling The 18 F-labelling of the SiFA moiety was carried out manually according to a procedure described in the literature. 34 The radiofluorination of TATE analogues was completed within 30 minutes, resulting in radiochemical yields (RCY) > 38%, and radiochemical purities (RCP) > 98%. The confirmation of peptide integrity and the quality controls are shown in the supplementary materials (Figures S11-S19, Table S3). Radiolabelling of rhTATE4 with lutetium-177 was achieved within 5 min at 70°C with RCY and RCP > 97% (radio-RP-HPLC and radio-TLC, Fig. S14 and S19). After radiolabelling, all peptides were used without further purification. In vitro evaluation The compound rhTATE4 was evaluated in vitro and compared with the clinical standards [ nat/177 Lu]Lu-DOTA-TATE and [ nat/18 F]SiFA lin -TATE, including determination of lipophilicity, human serum albumin (HSA) binding, receptor binding affinity to sstR2-expressing CHO sstR2 cells, and human plasma stability (see SI for details, Table S4). The lipophilicity was determined using the 1-octanol-PBS partition coefficient at pH = 7.4 (log D pH=7.4 ) by the shake flask method. Both 18 F- and 177 Lu-labelled rhTATE4 were investigated to estimate their chemical equality. Minor differences were observed for the 18 F- and 177 Lu-labelled species with log D pH = 7.4 : -1.42 ± 0.05 and − 1.70 ± 0.06, respectively. These differences are likely attributed to the different labelling conditions (see Experimental section for details). Overall, these log D pH=7.4 values were comparable to those of SiFA lin -TATE (Fig. 2 A); while the reference [ 177 Lu]Lu-DOTA-TATE was the most hydrophilic compound (log D pH=7.4 : -3.70 ± 0.05). The binding to HSA was determined using high-performance affinity chromatography (HPAC, Figure S20, Table S2). Lu-rhTATE4 showed high binding to HSA of > 92%, comparable to SiFA lin -TATE (Fig. 2 B). Instead, markedly reduced HSA binding was observed for the more lipophilic Lu-DOTA-TATE (51%). 35, 36 To evaluate the binding affinity to the sstR2 receptor, the half-maximal inhibitory concentration ( IC 50 ) of Lu-rhTATE4 was examined in a competitive binding assay ([ 125 I]TOC as competitor) using CHO sstR2 cells (Chinese hamster ovary (CHO) cells stably transfected with human sstR2 (epitope-tagged at the N -terminal side)) (Fig. 2 C). When compared to the references, Lu-DOTA-TATE ( IC 50 = 7.24 ± 0.9 nM) and SiFA lin -TATE ( IC 50 = 7.46 ± 1.40 nM), Lu-rhTATE4 demonstrated a similar receptor binding affinity with an IC 50 value of 9.32 ± 0.49 nM. This similarity suggests that the linker sequence Fmoc-O2Oc-OH used in Lu-rhTATE4 effectively retains receptor affinity. Stability studies in human plasma were conducted by incubating the ligands for 1 h ( 18 F-labelled) and 24 h ( 177 Lu-labelled) at 37°C (Fig. 2 D). [ 18 F]Lu-rhTATE4 , as well as the reference [ 18 F]SiFA lin -TATE, 37 showed no degradation after 1 h incubation in human serum (≥ 98% intact tracer). This confirms the stable fluorination of the bridged (SiFA) SeFe building block. With regard to therapeutic applications, the reference ligand [ 177 Lu]Lu-DOTA-TATE and the radiohybrid [ 177 Lu]Lu-rhTATE4 also revealed a high stability after 24 h incubation in human plasma (> 90% intact tracer). Ex vivo biodistribution To evaluate the imaging capabilities of rhTATE4 , the 18 F-labelled compound was first analysed in AR42J tumor-bearing CD1 nu/nu mice after 1 h post injection (p.i.). Additional competition studies with Ga-DOTA-TATE (40 nmol, 851x excess with respect to [ 18 F]Lu-rhTATE4 ) were carried out to determine the specificity to sstR2 (Fig. 3 A, see SI for details, Table S5). After 1 h p.i., extremely high activity levels of 53.58 ± 5.51%ID/g were detected for [ 18 F]Lu-rhTATE4 in the AR42J tumor, while uptake in non-target tissues (heart, spleen, intestine, muscle, bone with and without bone marrow: 0.34-2.0 %ID/g) was low. A slightly increased accumulation of activity was observed in the blood (4.88 ± 0.3 %ID/g) and in the well-perfused lung (6.87 ± 0.1 %ID/g), as well as in the liver (5.47 ± 0.7 %ID/g). In addition, increased activity levels were observed in the pancreas (11.65 ± 0.1 %ID/g) and stomach (10.63 ± 0.9 %ID/g), which was expected due to endogenous sstR2 expression in these organs. 38 The observed accumulation in the kidneys (26.88 ± 1.9 %ID/g) indicates primarily renal excretion. In a competition study with co-injected excess Ga-DOTA-TATE, an sstR2-specific uptake of [ 18 F]Lu-rhTATE4 was verified in the tumor (1.96%ID/g), pancreas (0.75%ID/g) and stomach (2.50%ID/g). In contrast, no reduction in uptake was noted in the lungs, intestine and adrenal glands, suggesting that the binding in these areas is primarily non-sstR2-mediated. The comparatively high accumulation of renal activity during the competition experiment can be attributed to receptor saturation by the cold competitor, resulting in an increased excretion rate of the radioligand. For a more precise assessment of the imaging quality, the T/B ratios were determined (Fig. 3 B, for more details see SI, Table S6). [ 18 F]Lu -rhTATE4 exhibits high T/B ratios for the lung (7.37 ± 0.97), the spleen (40.13 ± 4.45), the pancreas (4.54 ± 0.54), intestine (21.31 ± 3.54), adrenal glands (17.33 ± 1.73) and the bones with (31.61 ± 6.12) and without marrow (77.5 ± 27.54). Only for kidneys (1.85 ± 0.26) and blood (10.87 ± 0.78), lower T/B ratios were reached. One mouse of each biodistribution study was chosen for representative µ SPECT/CT scans of [ 18 F]Lu-rhTATE4 and the competition study of [ 18 F]Lu-rhTATE4 with Ga-DOTA-TATE after euthanization. The resulting µ SPECT/CT images recorded 1 h p.i. are shown in Fig. 3 C and are in agreement with the ex vivo biodistribution studies, displaying favorable imaging characteristics for [ 18 F]Lu-rhTATE4 , with high activity uptake in the sstR2-overexpressing tumor. Furthermore, low accumulation in non-target tissues and preferential elimination by the kidneys were observed. Since no bone uptake could be observed in both images, the in vivo stability against defluorination was further confirmed. In order to investigate long-term in vivo behaviour of the compound, the 177 Lu-labelled rhTATE4 was evaluated at 1, 6, and 24 h p.i. in AR42J tumor-bearing CD1 nu/nu mice (Fig. 3 D, Table S5). The biodistribution data of [ 177 Lu]Lu-rhTATE4 are in line with to those of [ 18 F]Lu-rhTATE4 at 1 h p.i. indicating comparable in vivo performance; although, different absolute values of 18 F- and 177 Lu-labelled rhTATE4 are recorded in the kidney (26.88 ± 1.4 %ID/g vs. 14.80 ± 1.9 %ID/g, respectively) and in the tumor (53.58 ± 5.5 %ID/g vs. 32.15 ± 8.5 %ID/g, respectively). In all cases, the tumor uptake exceeds that achieved with the previously reported theranostic [ 177 Lu]Lu-rhTATE-3 compound (27.32 ± 8.8 %ID/g). 32 Most importantly, the compound accumulates more in the tumor than what has been reported for [ 177 Lu]Lu-DOTA-TATE (21.35 ± 5.9 %ID/g); although a different mouse model was used to grow the AR42J tumor in this case. 39 After 24 h p.i., a considerable amount of activity could still be observed in the tumor (10.32 ± 7.04%ID/g), which is only slightly lower than the therapeutic benchmark [ 177 Lu]Lu-DOTA-TATE in an AR42J xenograft model (16.14 ± 2.07%ID/g 24 h p.i.). 39 Of note, significantly lower off-target uptake of [ 177 Lu]Lu-rhTATE4 compared to [ 177 Lu]Lu-DOTA-TATE was observed in certain organs (lung: 1.30 ± 0.68%ID/g vs 11.89%ID/g, pancreas: 0.26 ± 0.18%ID/g vs 3.99 ± 0.47%ID/g, adrenal gland: 0.22 ± 0.14%ID/g vs 4.70 ± 1.07%ID/g, kidney: 0.55 ± 0.26%ID/g vs. 1.76 ± 0.35%ID/g respectively), favourable for future therapeutic application of the new rh-compound. The T/B ratios of [ 177 Lu]Lu-rhTATE4 were directly compared over time (Figure S21, Table S6), showing a steady increase in all organs relative to the benchmark compound. One mouse of each biodistribution study was chosen for representative µ SPECT/CT scans of [ 177 Lu]Lu-rhTATE4 after euthanization. Images were acquired at 1 h and 24 h p.i. (Fig. 3 E). µ SPECT/CT images showed activity accumulation in the AR42J xenografts, and slightly elevated renal levels at 1 h p.i. (Fig. 3 F, left), in line with what was observed for [ 18 F]Lu-rhTATE4 . In agreement with quantitative data from biodistribution studies, the activity was efficiently cleared through the kidneys over time and almost entirely excreted after 24 h (Fig. 3 F, right). In addition, good excretion from non-cancerous tissues was also confirmed after 24 h p.i.. CONCLUSIONS AND PERSPECTIVES Molecular theranostic approaches have gained significant importance in modern medicine over the past years. To date, only a limited number of radiohybrid compounds have been identified as next-generation theranostic agents, in which the PET nuclide 18 F is primarily introduced through an isotopic exchange reaction via the highly lipophilic silicon-fluoride acceptor (SiFA) moiety. 22 Different strategies have been employed to enhance the radiohybrid hydrophilicity, including the introduction of polar auxiliaries, as seen in SiFA lin -TATE, or the use of tetrafluoroborate for 18 F exchange. 40 In our work, the rh-compound rhTATE4 features the bifunctional synthon (SiFA) SeFe , bridging the DOTA chelator to the TATE peptide. Notably, the compound exhibited reduced lipophilicity with respect to previously developed SiFA-based radiohybrids, and was comparable to that of the optimized [ 18 F]SiFA lin -TATE. 32 The labelling of rhTATE4 with fluorine-18 and lutetium-177 could be efficiently implemented, with high RCYs and RCPs. The derivative [ nat/18 F][ nat/177 Lu]Lu-rhTATE4 showed high HSA binding and stability in serum, as well as comparable sstR2 binding affinity to the benchmark Lu-DOTA-TATE. Initial in vivo evaluation of [ 18 F]Lu-rhTATE4 demonstrated noticeable imaging properties at 1 h p.i.; moreover, receptor specificity was confirmed by the lack of uptake in the tumor and other sstR2-positive organs in competition experiments. Furthermore, biodistribution studies of [ 177 Lu]Lu-rhTATE4 revealed high tumor accumulation even at 24 hours p.i., comparable to the benchmark [ 177 Lu]Lu-DOTA-TATE. Overall, our study further demonstrates the potential of the radiohybrid approach for the development of targeted theranostics. Additionally, it highlights the advantages of the (SiFA) SeFe moiety, enabling a more versatile design. Further work will be devoted to incorporating this synthon into rh-tracers targeting different receptors, such as the chemokine receptor 4 (CXCR4) or the gastrin-releasing peptide receptor (GRPR), to further broaden the scope of peptide-based theranostics. 41 EXPERIMENTAL SECTION Synthesis Descriptions of the syntheses and the corresponding labelling, as well as the characterization of all products, can be found in the Supporting Information. Complexation of the DOTA chelator with non-radioactive lutetium For the incorporation of lutetium, LuCl 3 (20 m m in H 2 O, 3.0 eq.) was added to a 2 m m solution of the compound in DMSO and diluted to 1 m m by addition of DMSO. The obtained solution was incubated at 70 ⁰C for 15 min. Lipophilicity (logD) For the determination of the octanol-PBS partition coefficient (log D 7.4 values), 500 µ L of 1-octanol and 500 µ L of PBS were added to a 1.5 mL reaction tube (Eppendorf Tube ® ) (n = 6). Thereafter, 1 MBq of each 18 F- or 177 Lu-labelled compound was added and vortexed for 3 min at room temperature. After centrifugation (9.000 rpm, 5 min, room temperature), 200 µ L of each layer was taken separately, and the activity was quantified by a WIZARD 2480 automatic γ -counter ( Perkin Elmer , Waltham, USA). Binding to human serum albumin (HSA) HSA binding studies were performed according to a previously published procedure, using RP-HPLC and HSA which is solid-phase fixed on a Chiralpak HSA column (50 × 3 mm, 5 µ m, H13 h-2433, Daicel , Tokyo, Japan). 42 A flow rate of 0.5 mL/min was used at room temperature. A freshly prepared 50 m m aqueous solution of NH 4 OAc (pH = 6.9) was used as mobile phase A, and isopropanol (HPLC grade, VWR , Germany) was used as mobile phase B. A gradient of 100% A (0 to 3 min) followed by 80% A (3 to 40 min) was used for the experiments. Before the analysis of all compounds, a calibration curve with nine reference substances with literature known HSA binding in the range of 13 to 99% was determined (Figure S20, Table S2). 42 , 43 All compounds, were prepared in a 1/1 mixture ( v / v ) of isopropanol and a 50 m m aqueous solution of NH 4 OAc (pH = 6.9) at a final concentration of 0.5 mg/ml. Nonlinear regression was performed using OriginPro 2016G software (Northampton, United States). Cells and cell culture maintenance The adherent sstR2-transfected CHO sstR2 cells (Chinese hamster ovary (CHO) cells stably transfected with human sst2R (epitope-tagged at the N -terminal end, provided by Dr. Jenny Koenig, University of Cambridge, Cambridge, United Kingdom) were cultured in DMEM/F12 GlutaMax medium (plus 10% FBS v / v ) at 37°C in a humidified 5% CO 2 atmosphere. To ensure uniform cell growth, cells were passaged at approximately 80% confluence (2–4 days). The spent medium is removed and the remaining cell lawn washed with PBS (10 mL, 37°C). By treatment with 5 mL trypsin/EDTA (0.05%/0.02% in PBS without Ca 2+ /Mg 2+ ) for 5 min at 37°C, the cells were detached and suspended adding 5 mL DMEM/F12 GlutaMax medium (plus 10% FBS v / v ). The suspension was centrifuged (1.300 rpm, 3 min, RT) and the cell pellet resuspended in fresh DMEM/F12 GlutaMax medium (20 mL, plus 10% FBS v / v , 37°C). A portion of the suspension was transferred to new culture flasks and the volume was adjusted to 25 mL with DMEM/F12 GlutaMax medium (plus 10% FBS v / v ). Cell density was checked regularly under an inverted microscope. AR42J cells ( CLS GmbH , Eppelheim, Germany and Sigma Aldrich , Gillingham, UK) for ex vivo studies were cultivated in RPMI medium (10% FBS + 2.5 vol% l -Gln solution (200 m m ) + 1 vol% MEM non-essential amino acid solution, v / v ) at 37°C in a humidified 5% CO 2 atmosphere. To ensure uniform cell growth, they were passaged at approximately 80% confluence (2–4 days). The medium was removed and the remaining cell lawn washed with PBS (6 mL, 37°C). By treatment with EDTA (0.1%) in PBS (5 mL, 5 min, 37°C), the cells were detached and suspended in 5 mL RPMI medium (10% FBS + 2.5 vol% l -Gln solution (200 mM) + 1 vol% MEM non-essential amino acid solution, v / v ). The suspension was centrifuged (1.300 rpm, 3 min, RT) and the cell pellet resuspended in fresh RPMI medium (10% FBS + 2.5 vol% l -Gln solution (200 m m ) + 1 vol% MEM non-essential amino acid solution, v / v ). A portion of the suspension was transferred to new culture flasks and the volume adjusted to 25 mL with RPMI medium (10% FBS + 2.5 vol% l -Gln solution (200 m m ) + 1 vol% MEM non-essential amino acid solution, v / v ). Cell density was checked regularly under an inverted microscope. Receptor binding affinity determinations (IC 50 ) In vitro competition studies were performed on CHO sstR2 cells (Chinese hamster ovary (CHO) cells stably transfected with human sstR2 (epitope-tagged at the N -terminal end, provided by Dr. Jenny Koenig, University of Cambridge, Cambridge, United Kingdom), which were seeded (24-well plates, 1.0 × 10 5 cells/well, DMEM/F12 GlutaMax plus 10% FBS) and incubated at 37°C for 24 ± 2 h before the experiment. On the day of the experiment, the medium was removed and each well was washed with 300 µ L of HBSS (supplemented with 1 vol% of bovine serum albumin = HBSA). After the addition of 200 µ L of HBSA, 25 µ L/well of HBSA (control, n = 3) or the respective ligand in concentrations ranging from 10 − 10 to 10 − 4 M (n = 3) was added. Subsequently, 25 µ L of the radiolabelled reference [ 125 I]TOC (1 n m in HBSA, for synthesis and characterization see SI) was added to each well. After incubation at RT for 1 h, the supernatant was removed, washed with ice-cold PBS (300 µ L), and the washing solutions were combined with the supernatants. The cells were lysed by adding NaOH (300 µ L, 1 m ). The cell lysate is removed after incubation at RT for 20 min and washed with NaOH (300 µ L, 1 M), while both NaOH-containing fractions were combined. Subsequently, the activities of both the supernatant and the lysate were measured separately in a γ -counter, and the IC 50 values (concentration that is needed to replace 50% of the reference competitor from the receptor) were calculated using GraphPad Prism software ( GraphPad Prism 4.0 Software Inc ., La Jolla, California, USA). Data were considered valid when the R² fit was > 0.95. Each experiment was performed in triplicate. Stability studies in human serum 5 MBq of the respective 18 F- or 177 Lu-labelled compound were added to 200 µ L of human serum (from a healthy volunteer) and incubated at 37°C for 1 h. After the addition of 50 vol% of cold ethanol and 150 vol% of cold MeCN, centrifugation was performed at 13.000 rpm for 20 min. The supernatant was decanted and centrifuged at 13.000 rpm for 10 min in a centrifuge tube with a 0.45 µ m cellulose acetate filter. Approximately 0.2 MBq of the remaining filtrate was injected into RP-HPLC and the amount of intact radioligand was quantified. Ex vivo biodistribution studies Animal experiments were performed by certified personnel following a previously published method. 34 Experiments were performed in agreement with the general animal welfare regulations in Germany (German Animal Welfare Act, as published on May 18, 2006, as amended by Article 280 of June 19, 2020, permit no. ROB-55.2-2532.Vet_02-18-109 by the General Directorate of Upper Bavaria ) and institutional guidelines for the care and use of animals. Specifically, female CD1-nu/nu mice aged 5–6 weeks ( Charles River Laboratories International Inc ., Sulzfeld, Germany) were acclimated in the in-house animal facility for one week prior to inoculation. Tumor xenografts were generated using AR42J cells (7.0 × 10 6 cells per 200 µ L) suspended in a 1/1 mixture ( v / v ) of RPMI 1640 medium and Cultrex® Basement Membrane Matrix Type 3 ( Trevigen , Gaithersburg, MD, USA). This suspension was inoculated subcutaneously onto the right shoulder, and animals were used when the tumor volume was > 100 mm 3 (1–2 weeks after inoculation). Exclusion criteria for animals from an experiment were either weight loss greater than 20%, tumor size greater than 1500 mm 3 , tumor ulceration, respiratory distress, or behavioural change. None of these criteria applied to any of the animals from the trial. No randomized or blinded approach was used in the allocation of the experiments. Health status is SPF according to the FELASA recommendation. Biodistribution studies (n = 3) were performed after 1 h, 6 h, or 24 h p.i., and approximately 2–3 MBq (300 pmol) were administered. Collected data were statistically analyzed using Excel ( Microsoft Corporation , Redmond, WA, USA) and OriginPro software (version 9.7) from OriginLab Corporation (Northampton, MA, USA). In the case of [ 177 Lu]Lu-rhTATE4 , the study was carried out using a different gamma counter with lower sensitivity. For this reason, measurement limits were determined experimentally and then evaluated graphically, yielding a minimum measurement limit of 97.0 cpm/30 s (see SI, Table S5). Declarations Competing interests The authors declare no competing financial or non-financial interests. Funding statement The authors acknowledge the Technical University of Munich for financial support. Author Contribution Conceptualization and supervision: A.C. and S.I.; synthesis and characterization of SiFA synthon: S.F.; design, synthesis and *in vitro* evaluation of sstR2-based compounds: S.D., V.K; *ex vivo* evaluation: S.D., S.F.; data analysis and interpretation: S.D., S.F., A.C.; writing of the main manuscript text: S.D., S.F., A.C.. All authors contributed to the drafting or revision of the manuscript. All authors have given approval to the final version of the manuscript. Acknowledgement We thank Dr. Nadine Holzleitner for the help regarding human serum studies and Dr. Mara Parzinger for providing the Reference TOC for affinity studies and the SiFA-building block SiFAlin-Aldehyde. The authors acknowledge the Technical University of Munich for financial support. Data Availability The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. References Baum, R. P.; Kulkarni, H. 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10:49:33","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144568,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/b9a15d726d9523af769da3c0.html"},{"id":97433594,"identity":"5acd380e-db31-43c3-a948-23260ef084b0","added_by":"auto","created_at":"2025-12-04 10:49:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788952,"visible":true,"origin":"","legend":"\u003cp\u003eA) Schematic representation of the radiohybrid (rh) strategy targeted to sstR2 (somatostatine receptor subtype 2). The rh design enables labelling the same compound with either \u003csup\u003e18\u003c/sup\u003eF for diagnostic imaging or with \u003csup\u003e177\u003c/sup\u003eLu for radioligand therapy. Typically, a SiFA synthon is used for \u003csup\u003e18\u003c/sup\u003eF-fluorination, while a chelator is applied for radiometalation (e.g. with \u003csup\u003e177\u003c/sup\u003eLu). B) Structure of the FDA-approved [\u003csup\u003e18\u003c/sup\u003eF]Ga-rhPSMA‑7.3 (POSLUMA\u003csup\u003e®\u003c/sup\u003e) for PET imaging of prostate cancer. C) Structure of the herewith reported sstR2-targeted rh-compound \u003cstrong\u003eLu-rhTATE4\u003c/strong\u003e for both PET imaging and b\u003csup\u003e- \u003c/sup\u003etherapy.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/85928e50f16f20ad37fb091f.png"},{"id":97433596,"identity":"19959774-3a10-4e45-9fa4-9d5ffddfee01","added_by":"auto","created_at":"2025-12-04 10:49:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn\u0026nbsp;vitro\u003c/em\u003e evaluation, including: determination of A) lipophilicity (logD\u003csub\u003epH=7.4\u003c/sub\u003e); B) human serum albumin (HSA) binding (HPAC method, n = 2); C) sstR2 binding affinity (IC\u003csub\u003e50\u003c/sub\u003e) in a competitive binding assay with the competitor [\u003csup\u003e125\u003c/sup\u003eI]I-TOC in the AR42J cell line; and D) human plasma stability of the \u003csup\u003e18\u003c/sup\u003eF- or \u003csup\u003e177\u003c/sup\u003eLu-labelled theranostic radiohybrid compound \u003cstrong\u003erhTATE4\u003c/strong\u003e in comparison to benchmarks [\u003csup\u003enat/177\u003c/sup\u003eLu]Lu‑DOTA‑TATE and [\u003csup\u003enat/18\u003c/sup\u003eF]SiFAlin-TATE. The value for the stability of [\u003csup\u003e18\u003c/sup\u003eF]SiFAlin-TATE was taken from the literature.\u003csup\u003e37\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/42862c5eac5c0634cfcc68ca.png"},{"id":97667212,"identity":"a9fa9aaa-bb67-4d04-88eb-9c22fc342636","added_by":"auto","created_at":"2025-12-08 09:23:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1127381,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo biodistributions and µSPECT/CT scans performed in AR42J tumor-bearing female CD1 nu/nu mice. A) Ex vivo biodistribution data of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Lu‑rhTATE4 \u003c/strong\u003e(n\u0026nbsp;=\u0026nbsp;3, 300\u0026nbsp;pmol each) and competition study (47\u0026nbsp;pmol + 40\u0026nbsp;nmol Ga-DOTA-TATE, n\u0026nbsp;=\u0026nbsp;1) in selected organs 1 h post injection (p.i.) (exact values Table S5). B) Tumor to background (T/B) ratios of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Lu‑rhTATE4\u003c/strong\u003e in selected organs after 1 h p.i (300\u0026nbsp;pmol each). C)\u003cstrong\u003e \u003c/strong\u003eMaximum intensity projections (MIPs) of µSPECT/CT scans acquired 1\u0026nbsp;h p.i. of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Lu‑rhTATE4\u003c/strong\u003e (left, 40\u0026nbsp;pmol) and competition study (right, 47\u0026nbsp;pmol \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Lu‑rhTATE4\u003c/strong\u003e + 40\u0026nbsp;nmol Ga‑DOTA-TATE). Tumors are indicated with white arrows. D) Ex vivo\u003cstrong\u003e \u003c/strong\u003ebiodistribution data of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e177\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eLu]Lu‑rhTATE4\u003c/strong\u003e in selected organs at 1 h, 6\u0026nbsp;h and 24\u0026nbsp;h p.i. (n = 5, 300\u0026nbsp;pmol each). E) MIPs of µSPECT/CT scans acquired 1 h p.i. (left) and 24 h p.i. (right) of \u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e177\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eLu]Lu-rhTATE4\u003c/strong\u003e (300\u0026nbsp;pmol each). Tumors are indicated with white arrows. Static µSPECT/CT images in C) and E) were acquired post mortem (CO\u003csub\u003e2\u003c/sub\u003e asphyxiation and cervical dislocation) and after cardiac puncture with an acquisition time of 45\u0026nbsp;min.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/f74f3d332914f07633bd0f6f.png"},{"id":97677656,"identity":"15c6a5e0-d760-4c8f-a0bb-415fedc35597","added_by":"auto","created_at":"2025-12-08 09:53:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3015428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/170b1d94-f8e3-4808-ab00-c3680dec9352.pdf"},{"id":97667702,"identity":"c2958666-e232-493c-b936-38aecbaf6288","added_by":"auto","created_at":"2025-12-08 09:24:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1913892,"visible":true,"origin":"","legend":"","description":"","filename":"npjImagingSIrhTATE4submitted.docx","url":"https://assets-eu.researchsquare.com/files/rs-8090442/v1/56040b535fb773ca530a30ab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAll at once: a sstR2-targeted radiohybrid theranostic agent for PET imaging and β\u003csup\u003e-\u003c/sup\u003e therapy with excellent preclinical performance\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe term \"theranostics\" refers to the integration of two medical procedures that enable both diagnostic imaging and therapeutic treatment. In the context of nuclear medicine, this involves the use of identical or similar radiopharmaceutical compounds that contain radioactive isotopes.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e A classical categorization includes the following main strategies for theranostic design: isotopically matched pairs, the use of \u0026lsquo;true\u0026rsquo; theranostic nuclides, and matched radiopharmaceuticals.\u003c/p\u003e\u003cp\u003eThe iodine radioisotopes serve as a notable example of \u003cem\u003eisotopically matched pairs\u003c/em\u003e, being the first radiopharmaceutical that laid the groundwork for theranostic applications. \u003csup\u003e4, 5\u003c/sup\u003e Thus, iodine-123 (\u003cem\u003eγ\u003c/em\u003e-emitter) is used for diagnostics in single-photon emission computed tomography (SPECT), and iodine-131 (\u003cem\u003eγ\u003c/em\u003e- and \u003cem\u003eβ\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e-emitter) for the treatment of thyroid cancer.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The radiopharmaceuticals of this group contain either a therapeutic or a diagnostic radionuclide of the same element, whereby both compounds behave chemically and biologically identically, enabling precise dosimetry and therapy control.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn the case of a \u0026lsquo;\u003cem\u003etrue\u0026rsquo; theranostic\u003c/em\u003e approach, the incorporated radionuclide emits either \u003cem\u003eγ\u003c/em\u003e-rays or positrons that can be used for imaging, and \u003cem\u003eα\u003c/em\u003e or \u003cem\u003eβ\u003c/em\u003e\u003csup\u003e-\u003c/sup\u003e radiations for therapy. A prominent example of this approach is the nuclide lutetium-177, which is already used in the clinic within the high-affinity somatostatin receptor subtype 2 (sstR2)-targeted [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE (TATE = (Tyr\u003csup\u003e3\u003c/sup\u003e)-octreotate, DOTA\u0026thinsp;=\u0026thinsp;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (Lutathera\u003csup\u003e\u0026reg;\u003c/sup\u003e) for the therapy of neuroendocrine tumors.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The γ-rays from lutetium-177 can be utilized for SPECT imaging, allowing dosimetry calculations to be performed after the first therapy cycle.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, \u003csup\u003e177\u003c/sup\u003eLu-based SPECT scans are not used in clinical practice for therapy planning due to their lower resolution.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e To overcome this limitation, the \u003cem\u003ematched radiopharmaceuticals\u003c/em\u003e approach provides a combination of imaging and therapeutic nuclides. In the case of neuroendocrine tumors, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-TATE (NETSPOT\u003csup\u003e\u0026reg;\u003c/sup\u003e) is used for positron emission tomography (PET) imaging as complementary to the therapeutic [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE.\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, the differing radiometal complexes of Ga\u003csup\u003e3+\u003c/sup\u003e and Lu\u003csup\u003e3+\u003c/sup\u003e result in distinct pharmacokinetic profiles, leading to diverse pharmacological properties.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn addition to the aforementioned classical design approaches, the \u003cem\u003eradiohybrid\u003c/em\u003e strategy is emerging, whereby a single molecule features two separate binding sites for different radionuclides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e This aims to establish a more consistent pharmacological profile for the radioconjugate, resulting in more personalized and effective treatment plans. In detail, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the concept typically includes a silicon-based fluoride acceptor (SiFA) for fast and efficient \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-fluorination \u003cem\u003evia\u003c/em\u003e an \u003csup\u003e18\u003c/sup\u003eF/\u003csup\u003e19\u003c/sup\u003eF isotope exchange reaction, as well as the chelator DOTAGA (2-[1,4,7,10-Tetraazacyclododecane]-pentanedioic acid) for radiometalation (e.g. with \u003csup\u003e177\u003c/sup\u003eLu).\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e This creates a chemically identical compound pair (either \u003csup\u003e19\u003c/sup\u003eF/radiometal or \u003csup\u003e18\u003c/sup\u003eF/non-radioactive metal) that enables precise dosimetry and therapy monitoring. Moreover, it provides economic and strategic advantages, as the approval for the therapeutic radiohybrid can be streamlined following the approval of the diagnostic counterpart.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The first example of this approach was reported by Wurzer et al. \u003cem\u003eet al.\u003c/em\u003e,\u003csup\u003e23\u003c/sup\u003e and successfully reached FDA approval with the compound [\u003csup\u003e18\u003c/sup\u003eF]Ga-rhPSMA-7.3 (POSLUMA\u003csup\u003e\u0026reg;\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) for PET imaging of prostate cancer.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The therapeutic counterpart [\u003csup\u003e177\u003c/sup\u003eLu]Lu-rhPSMA7.3 and its DOTA-analogue [\u003csup\u003e177\u003c/sup\u003eLu]Lu-rhPSMA10.1 showed promising preclinical results.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eA primary concern in the classical design of radiohybrid tracers is the high lipophilicity of the SiFA building block (SiFA)BA (4-(di-tert-butylfluorosilyl)benzoic acid), which affects the biodistribution of the resulting radiotracers and may lead to unfavourable \u003cem\u003ein vivo\u003c/em\u003e performance.\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In a recent attempt to generate an SSTR2-targeted radiohybrid compound, based on the more hydrophilic yet \u003cem\u003epara\u003c/em\u003e-functionalized SiFA\u003cem\u003elin\u003c/em\u003e moiety, Wendlinger \u003cem\u003eet al.\u003c/em\u003e could not exceed the performance of the benchmark SiFA\u003cem\u003elin\u003c/em\u003e-TATE.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e To address this limitation, we have recently reported on a new bifunctional SiFA synthon 3-amino-5-(di-tert-butylfluorosilyl)benzoic acid (SiFA)\u003cem\u003eSeFe\u003c/em\u003e), that includes a carboxylic acid and an amine group located at the two meta-positions relative to the Si moiety.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e This feature not only decreases the lipophilic nature of the SiFA synthon but also enables its bridged incorporation into a radiotracer, akin to a classical amino acid moiety. (SiFA)\u003cem\u003eSeFe\u003c/em\u003e has been recently used to develop three different rh-compounds (\u003cb\u003erhTATE1-3\u003c/b\u003e) that showed high accumulation in AR42J tumor-bearing female CD1-nu/nu mice (up to 27%ID/g 1 h post injection (p.i.)), but also in the kidneys (up to 99%ID/g 1 h p.i.).\u003csup\u003e32\u003c/sup\u003e Although total tumor uptake was increased compared to [\u003csup\u003e18\u003c/sup\u003eF]SiFA\u003cem\u003elin\u003c/em\u003e-TATE (18.51\u0026thinsp;\u0026plusmn;\u0026thinsp;4.89%ID/g 1 h p.i.), the tumor-to-background (T/B) ratios for most organs were comparable or lower. Additionally, the rapid renal excretion of [\u003csup\u003e18\u003c/sup\u003eF]Lu-rhTATE3 likely prevents its therapeutic application.\u003c/p\u003e\u003cp\u003eHere, we report on an optimized theranostic radiohybrid ligand \u003cb\u003erhTATE4\u003c/b\u003e, which can be quickly and efficiently labelled with fluorine-18 using the (SiFA)\u003cem\u003eSeFe\u003c/em\u003e moiety, as well as stably labelled with lutetium-177 via a DOTA chelator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In this instance, the bifunctional (SiFA)\u003cem\u003eSeFe\u003c/em\u003e is incorporated bridging the sstR2 targeting TATE and the chelator. The compound was characterized by RP-HPLC (reverse-phase high-performance liquid chromatography) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). Further, the \u003cem\u003ein vitro\u003c/em\u003e properties of the ligand, including lipophilicity, human serum albumin (HSA) binding, sstR2 receptor binding affinity, as well as stability in human plasma were evaluated and compared to the FDA and EMA approved benchmark for PRRT [\u003csup\u003enat/177\u003c/sup\u003eLu]Lu-DOTA-TATE, as well as to the clinically tested PET tracer [\u003csup\u003enat/18\u003c/sup\u003eF]SiFA\u003cem\u003elin\u003c/em\u003e-TATE.\u003csup\u003e33\u003c/sup\u003e Additionally, the biodistribution of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e was investigated \u003cem\u003ein vivo\u003c/em\u003e in AR42J tumor-bearing CD1-nu/nu mice at 1 h p.i.. Similarly, \u003cb\u003erhTATE4\u003c/b\u003e was labelled with lutetium-177 and its biodistribution studied at 1 h, 6 h, and 24 h p.i. in the same model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDesign and Synthesis\u003c/h3\u003e\n\u003cp\u003eTo generate a theranostic platform, the rh-concept was applied to the TATE analogue \u003cb\u003erhTATE4\u003c/b\u003e, which contains the SiFA building block (SiFA)\u003cem\u003eSeFe\u003c/em\u003e for \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF labelling and a terminal DOTA chelator for \u003csup\u003e177\u003c/sup\u003eLu labelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For this purpose, the linker unit Fmoc-O\u003csub\u003e2\u003c/sub\u003eOc-OH (8-amino-3,6-dioxaoctanoic acid) was first placed between the pharmacophore TATE and the SiFA synthon in order to maintain a good affinity to sstR2 binding, analogous to SiFA\u003cem\u003elin\u003c/em\u003e-TATE. A \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-Asp was introduced as a linker between the DOTA chelator and the SiFA moiety. The metal-free ligand and the corresponding references were synthesized by fluorenylmethoxycarbonyl standard peptide synthesis on solid phase (Fmoc SPPS) using a 2-chlorotrityl chloride (2-CTC) resin (see SI for details, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), yielding 3\u0026ndash;19% RP-HPLC purified precursors (chemical purity\u0026thinsp;\u0026gt;\u0026thinsp;95%, determined by RP-HPLC and mass spectrometry, Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e0). Non-radioactive lutetium labelling was performed quantitatively with a 2.5-fold excess of LuCl\u003csub\u003e3\u003c/sub\u003e at 80\u0026deg;C for 15 min (Figures S4 and S9).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRadiolabelling\u003c/h2\u003e\u003cp\u003eThe \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labelling of the SiFA moiety was carried out manually according to a procedure described in the literature.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e The radiofluorination of TATE analogues was completed within 30 minutes, resulting in radiochemical yields (RCY)\u0026thinsp;\u0026gt;\u0026thinsp;38%, and radiochemical purities (RCP)\u0026thinsp;\u0026gt;\u0026thinsp;98%. The confirmation of peptide integrity and the quality controls are shown in the supplementary materials (Figures S11-S19, Table S3). Radiolabelling of \u003cb\u003erhTATE4\u003c/b\u003e with lutetium-177 was achieved within 5 min at 70\u0026deg;C with RCY and RCP\u0026thinsp;\u0026gt;\u0026thinsp;97% (radio-RP-HPLC and radio-TLC, Fig. S14 and S19). After radiolabelling, all peptides were used without further purification.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn vitro evaluation\u003c/h3\u003e\n\u003cp\u003eThe compound \u003cb\u003erhTATE4\u003c/b\u003e was evaluated \u003cem\u003ein vitro\u003c/em\u003e and compared with the clinical standards [\u003csup\u003enat/177\u003c/sup\u003eLu]Lu-DOTA-TATE and [\u003csup\u003enat/18\u003c/sup\u003eF]SiFA\u003cem\u003elin\u003c/em\u003e-TATE, including determination of lipophilicity, human serum albumin (HSA) binding, receptor binding affinity to sstR2-expressing CHO\u003csub\u003esstR2\u003c/sub\u003e cells, and human plasma stability (see SI for details, Table S4). The lipophilicity was determined using the 1-octanol-PBS partition coefficient at pH\u0026thinsp;=\u0026thinsp;7.4 (log\u003cem\u003eD\u003c/em\u003e\u003csub\u003epH=7.4\u003c/sub\u003e) by the shake flask method. Both \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF- and \u003csup\u003e177\u003c/sup\u003eLu-labelled \u003cb\u003erhTATE4\u003c/b\u003e were investigated to estimate their chemical equality. Minor differences were observed for the \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF- and \u003csup\u003e177\u003c/sup\u003eLu-labelled species with log\u003cem\u003eD\u003c/em\u003e\u003csub\u003epH = 7.4\u003c/sub\u003e: -1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and \u0026minus;\u0026thinsp;1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, respectively. These differences are likely attributed to the different labelling conditions (see Experimental section for details). Overall, these log\u003cem\u003eD\u003c/em\u003e\u003csub\u003epH=7.4\u003c/sub\u003e values were comparable to those of SiFA\u003cem\u003elin\u003c/em\u003e-TATE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); while the reference [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE was the most hydrophilic compound (log\u003cem\u003eD\u003c/em\u003e\u003csub\u003epH=7.4\u003c/sub\u003e: -3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eThe binding to HSA was determined using high-performance affinity chromatography (HPAC, Figure S20, Table S2). \u003cb\u003eLu-rhTATE4\u003c/b\u003e showed high binding to HSA of \u0026gt;\u0026thinsp;92%, comparable to SiFA\u003cem\u003elin\u003c/em\u003e-TATE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Instead, markedly reduced HSA binding was observed for the more lipophilic Lu-DOTA-TATE (51%).\u003csup\u003e35, 36\u003c/sup\u003e To evaluate the binding affinity to the sstR2 receptor, the half-maximal inhibitory concentration (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e50\u003c/sub\u003e) of \u003cb\u003eLu-rhTATE4\u003c/b\u003e was examined in a competitive binding assay ([\u003csup\u003e125\u003c/sup\u003eI]TOC as competitor) using CHO\u003csub\u003esstR2\u003c/sub\u003e cells (Chinese hamster ovary (CHO) cells stably transfected with human sstR2 (epitope-tagged at the \u003cem\u003eN\u003c/em\u003e-terminal side)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). When compared to the references, Lu-DOTA-TATE (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nM) and SiFA\u003cem\u003elin\u003c/em\u003e-TATE (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 nM), \u003cb\u003eLu-rhTATE4\u003c/b\u003e demonstrated a similar receptor binding affinity with an \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e value of 9.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 nM. This similarity suggests that the linker sequence Fmoc-O2Oc-OH used in \u003cb\u003eLu-rhTATE4\u003c/b\u003e effectively retains receptor affinity.\u003c/p\u003e\u003cp\u003eStability studies in human plasma were conducted by incubating the ligands for 1 h (\u003csup\u003e18\u003c/sup\u003eF-labelled) and 24 h (\u003csup\u003e177\u003c/sup\u003eLu-labelled) at 37\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e, as well as the reference [\u003csup\u003e18\u003c/sup\u003eF]SiFA\u003cem\u003elin\u003c/em\u003e-TATE,\u003csup\u003e37\u003c/sup\u003e showed no degradation after 1 h incubation in human serum (\u0026ge;\u0026thinsp;98% intact tracer). This confirms the stable fluorination of the bridged (SiFA)\u003cem\u003eSeFe\u003c/em\u003e building block. With regard to therapeutic applications, the reference ligand [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE and the radiohybrid \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e also revealed a high stability after 24 h incubation in human plasma (\u0026gt;\u0026thinsp;90% intact tracer).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEx vivo biodistribution\u003c/h3\u003e\n\u003cp\u003eTo evaluate the imaging capabilities of \u003cb\u003erhTATE4\u003c/b\u003e, the \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labelled compound was first analysed in AR42J tumor-bearing CD1 nu/nu mice after 1 h post injection (p.i.). Additional competition studies with Ga-DOTA-TATE (40 nmol, 851x excess with respect to \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e) were carried out to determine the specificity to sstR2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, see SI for details, Table S5). After 1 h p.i., extremely high activity levels of 53.58\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51%ID/g were detected for \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e in the AR42J tumor, while uptake in non-target tissues (heart, spleen, intestine, muscle, bone with and without bone marrow: 0.34-2.0 %ID/g) was low. A slightly increased accumulation of activity was observed in the blood (4.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 %ID/g) and in the well-perfused lung (6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 %ID/g), as well as in the liver (5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 %ID/g). In addition, increased activity levels were observed in the pancreas (11.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 %ID/g) and stomach (10.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 %ID/g), which was expected due to endogenous sstR2 expression in these organs.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e The observed accumulation in the kidneys (26.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 %ID/g) indicates primarily renal excretion.\u003c/p\u003e\u003cp\u003eIn a competition study with co-injected excess Ga-DOTA-TATE, an sstR2-specific uptake of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e was verified in the tumor (1.96%ID/g), pancreas (0.75%ID/g) and stomach (2.50%ID/g). In contrast, no reduction in uptake was noted in the lungs, intestine and adrenal glands, suggesting that the binding in these areas is primarily non-sstR2-mediated. The comparatively high accumulation of renal activity during the competition experiment can be attributed to receptor saturation by the cold competitor, resulting in an increased excretion rate of the radioligand. For a more precise assessment of the imaging quality, the T/B ratios were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, for more details see SI, Table S6). \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu -rhTATE4\u003c/b\u003e exhibits high T/B ratios for the lung (7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97), the spleen (40.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45), the pancreas (4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54), intestine (21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54), adrenal glands (17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73) and the bones with (31.61\u0026thinsp;\u0026plusmn;\u0026thinsp;6.12) and without marrow (77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;27.54). Only for kidneys (1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26) and blood (10.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78), lower T/B ratios were reached.\u003c/p\u003e\u003cp\u003eOne mouse of each biodistribution study was chosen for representative \u003cem\u003e\u0026micro;\u003c/em\u003eSPECT/CT scans of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e and the competition study of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e with Ga-DOTA-TATE after euthanization. The resulting \u003cem\u003e\u0026micro;\u003c/em\u003eSPECT/CT images recorded 1 h p.i. are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and are in agreement with the \u003cem\u003eex vivo\u003c/em\u003e biodistribution studies, displaying favorable imaging characteristics for \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e, with high activity uptake in the sstR2-overexpressing tumor. Furthermore, low accumulation in non-target tissues and preferential elimination by the kidneys were observed. Since no bone uptake could be observed in both images, the \u003cem\u003ein vivo\u003c/em\u003e stability against defluorination was further confirmed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to investigate long-term \u003cem\u003ein vivo\u003c/em\u003e behaviour of the compound, the \u003csup\u003e177\u003c/sup\u003eLu-labelled \u003cb\u003erhTATE4\u003c/b\u003e was evaluated at 1, 6, and 24 h p.i. in AR42J tumor-bearing CD1 nu/nu mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Table S5). The biodistribution data of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e are in line with to those of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e at 1 h p.i. indicating comparable \u003cem\u003ein vivo\u003c/em\u003e performance; although, different absolute values of \u003csup\u003e18\u003c/sup\u003eF- and \u003csup\u003e177\u003c/sup\u003eLu-labelled \u003cb\u003erhTATE4\u003c/b\u003e are recorded in the kidney (26.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 %ID/g vs. 14.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 %ID/g, respectively) and in the tumor (53.58\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 %ID/g vs. 32.15\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 %ID/g, respectively). In all cases, the tumor uptake exceeds that achieved with the previously reported theranostic [\u003csup\u003e177\u003c/sup\u003eLu]Lu-rhTATE-3 compound (27.32\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 %ID/g).\u003csup\u003e32\u003c/sup\u003e Most importantly, the compound accumulates more in the tumor than what has been reported for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE (21.35\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 %ID/g); although a different mouse model was used to grow the AR42J tumor in this case.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAfter 24 h p.i., a considerable amount of activity could still be observed in the tumor (10.32\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04%ID/g), which is only slightly lower than the therapeutic benchmark [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE in an AR42J xenograft model (16.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07%ID/g 24 h p.i.).\u003csup\u003e39\u003c/sup\u003e Of note, significantly lower off-target uptake of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE was observed in certain organs (lung: 1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68%ID/g vs 11.89%ID/g, pancreas: 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18%ID/g vs 3.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47%ID/g, adrenal gland: 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%ID/g vs 4.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07%ID/g, kidney: 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26%ID/g vs. 1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35%ID/g respectively), favourable for future therapeutic application of the new rh-compound. The T/B ratios of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e were directly compared over time (Figure S21, Table S6), showing a steady increase in all organs relative to the benchmark compound.\u003c/p\u003e\u003cp\u003eOne mouse of each biodistribution study was chosen for representative \u003cem\u003e\u0026micro;\u003c/em\u003eSPECT/CT scans of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e after euthanization. Images were acquired at 1 h and 24 h p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). \u003cem\u003e\u0026micro;\u003c/em\u003eSPECT/CT images showed activity accumulation in the AR42J xenografts, and slightly elevated renal levels at 1 h p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, left), in line with what was observed for \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e. In agreement with quantitative data from biodistribution studies, the activity was efficiently cleared through the kidneys over time and almost entirely excreted after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, right). In addition, good excretion from non-cancerous tissues was also confirmed after 24 h p.i..\u003c/p\u003e"},{"header":"CONCLUSIONS AND PERSPECTIVES","content":"\u003cp\u003eMolecular theranostic approaches have gained significant importance in modern medicine over the past years. To date, only a limited number of radiohybrid compounds have been identified as next-generation theranostic agents, in which the PET nuclide \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF is primarily introduced through an isotopic exchange reaction via the highly lipophilic silicon-fluoride acceptor (SiFA) moiety.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Different strategies have been employed to enhance the radiohybrid hydrophilicity, including the introduction of polar auxiliaries, as seen in SiFA\u003cem\u003elin\u003c/em\u003e-TATE, or the use of tetrafluoroborate for \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF exchange.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e In our work, the rh-compound \u003cb\u003erhTATE4\u003c/b\u003e features the bifunctional synthon (SiFA)\u003cem\u003eSeFe\u003c/em\u003e, bridging the DOTA chelator to the TATE peptide. Notably, the compound exhibited reduced lipophilicity with respect to previously developed SiFA-based radiohybrids, and was comparable to that of the optimized [\u003csup\u003e18\u003c/sup\u003eF]SiFA\u003cem\u003elin\u003c/em\u003e-TATE.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The labelling of \u003cb\u003erhTATE4\u003c/b\u003e with fluorine-18 and lutetium-177 could be efficiently implemented, with high RCYs and RCPs. The derivative \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003enat/18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF][\u003c/b\u003e\u003csup\u003e\u003cb\u003enat/177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e showed high HSA binding and stability in serum, as well as comparable sstR2 binding affinity to the benchmark Lu-DOTA-TATE. Initial \u003cem\u003ein vivo\u003c/em\u003e evaluation of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e18\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eF]Lu-rhTATE4\u003c/b\u003e demonstrated noticeable imaging properties at 1 h p.i.; moreover, receptor specificity was confirmed by the lack of uptake in the tumor and other sstR2-positive organs in competition experiments. Furthermore, biodistribution studies of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e revealed high tumor accumulation even at 24 hours p.i., comparable to the benchmark [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE.\u003c/p\u003e\u003cp\u003eOverall, our study further demonstrates the potential of the radiohybrid approach for the development of targeted theranostics. Additionally, it highlights the advantages of the (SiFA)\u003cem\u003eSeFe\u003c/em\u003e moiety, enabling a more versatile design. Further work will be devoted to incorporating this synthon into rh-tracers targeting different receptors, such as the chemokine receptor 4 (CXCR4) or the gastrin-releasing peptide receptor (GRPR), to further broaden the scope of peptide-based theranostics.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"EXPERIMENTAL SECTION","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis\u003c/h2\u003e\u003cp\u003eDescriptions of the syntheses and the corresponding labelling, as well as the characterization of all products, can be found in the Supporting Information.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eComplexation of the DOTA chelator with non-radioactive lutetium\u003c/h3\u003e\n\u003cp\u003eFor the incorporation of lutetium, LuCl\u003csub\u003e3\u003c/sub\u003e (20 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e in H\u003csub\u003e2\u003c/sub\u003eO, 3.0 eq.) was added to a 2 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e solution of the compound in DMSO and diluted to 1 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e by addition of DMSO. The obtained solution was incubated at 70 ⁰C for 15 min.\u003c/p\u003e\n\u003ch3\u003eLipophilicity (logD)\u003c/h3\u003e\n\u003cp\u003eFor the determination of the octanol-PBS partition coefficient (log\u003cem\u003eD\u003c/em\u003e\u003csub\u003e\u003cem\u003e7.4\u003c/em\u003e\u003c/sub\u003e values), 500 \u003cem\u003e\u0026micro;\u003c/em\u003eL of 1-octanol and 500 \u003cem\u003e\u0026micro;\u003c/em\u003eL of PBS were added to a 1.5 mL reaction tube (Eppendorf Tube\u003csup\u003e\u0026reg;\u003c/sup\u003e) (n\u0026thinsp;=\u0026thinsp;6). Thereafter, 1 MBq of each \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF- or \u003csup\u003e177\u003c/sup\u003eLu-labelled compound was added and vortexed for 3 min at room temperature. After centrifugation (9.000 rpm, 5 min, room temperature), 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL of each layer was taken separately, and the activity was quantified by a WIZARD 2480 automatic \u003cem\u003eγ\u003c/em\u003e-counter (\u003cem\u003ePerkin Elmer\u003c/em\u003e, Waltham, USA).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBinding to human serum albumin (HSA)\u003c/h2\u003e\u003cp\u003eHSA binding studies were performed according to a previously published procedure, using RP-HPLC and HSA which is solid-phase fixed on a Chiralpak HSA column (50 \u0026times; 3 mm, 5 \u003cem\u003e\u0026micro;\u003c/em\u003em, H13 h-2433, \u003cem\u003eDaicel\u003c/em\u003e, Tokyo, Japan).\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e A flow rate of 0.5 mL/min was used at room temperature. A freshly prepared 50 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e aqueous solution of NH\u003csub\u003e4\u003c/sub\u003eOAc (pH\u0026thinsp;=\u0026thinsp;6.9) was used as mobile phase A, and isopropanol (HPLC grade, \u003cem\u003eVWR\u003c/em\u003e, Germany) was used as mobile phase B. A gradient of 100% A (0 to 3 min) followed by 80% A (3 to 40 min) was used for the experiments. Before the analysis of all compounds, a calibration curve with nine reference substances with literature known HSA binding in the range of 13 to 99% was determined (Figure S20, Table S2).\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e All compounds, were prepared in a 1/1 mixture (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) of isopropanol and a 50 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e aqueous solution of NH\u003csub\u003e4\u003c/sub\u003eOAc (pH\u0026thinsp;=\u0026thinsp;6.9) at a final concentration of 0.5 mg/ml. Nonlinear regression was performed using \u003cem\u003eOriginPro 2016G\u003c/em\u003e software (Northampton, United States).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCells and cell culture maintenance\u003c/h2\u003e\u003cp\u003eThe adherent sstR2-transfected CHO\u003csub\u003esstR2\u003c/sub\u003e cells (Chinese hamster ovary (CHO) cells stably transfected with human sst2R (epitope-tagged at the \u003cem\u003eN\u003c/em\u003e-terminal end, provided by Dr. Jenny Koenig, University of Cambridge, Cambridge, United Kingdom) were cultured in DMEM/F12 GlutaMax medium (plus 10% FBS \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. To ensure uniform cell growth, cells were passaged at approximately 80% confluence (2\u0026ndash;4 days). The spent medium is removed and the remaining cell lawn washed with PBS (10 mL, 37\u0026deg;C). By treatment with 5 mL trypsin/EDTA (0.05%/0.02% in PBS without Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e) for 5 min at 37\u0026deg;C, the cells were detached and suspended adding 5 mL DMEM/F12 GlutaMax medium (plus 10% FBS \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e). The suspension was centrifuged (1.300 rpm, 3 min, RT) and the cell pellet resuspended in fresh DMEM/F12 GlutaMax medium (20 mL, plus 10% FBS \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e, 37\u0026deg;C). A portion of the suspension was transferred to new culture flasks and the volume was adjusted to 25 mL with DMEM/F12 GlutaMax medium (plus 10% FBS \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e). Cell density was checked regularly under an inverted microscope.\u003c/p\u003e\u003cp\u003eAR42J cells (\u003cem\u003eCLS GmbH\u003c/em\u003e, Eppelheim, Germany and \u003cem\u003eSigma Aldrich\u003c/em\u003e, Gillingham, UK) for \u003cem\u003eex vivo\u003c/em\u003e studies were cultivated in RPMI medium (10% FBS\u0026thinsp;+\u0026thinsp;2.5 vol% \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gln solution (200 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e)\u0026thinsp;+\u0026thinsp;1 vol% MEM non-essential amino acid solution, \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. To ensure uniform cell growth, they were passaged at approximately 80% confluence (2\u0026ndash;4 days). The medium was removed and the remaining cell lawn washed with PBS (6 mL, 37\u0026deg;C). By treatment with EDTA (0.1%) in PBS (5 mL, 5 min, 37\u0026deg;C), the cells were detached and suspended in 5 mL RPMI medium (10% FBS\u0026thinsp;+\u0026thinsp;2.5 vol% \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gln solution (200 mM)\u0026thinsp;+\u0026thinsp;1 vol% MEM non-essential amino acid solution, \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e). The suspension was centrifuged (1.300 rpm, 3 min, RT) and the cell pellet resuspended in fresh RPMI medium (10% FBS\u0026thinsp;+\u0026thinsp;2.5 vol% \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gln solution (200 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e)\u0026thinsp;+\u0026thinsp;1 vol% MEM non-essential amino acid solution, \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e). A portion of the suspension was transferred to new culture flasks and the volume adjusted to 25 mL with RPMI medium (10% FBS\u0026thinsp;+\u0026thinsp;2.5 vol% \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gln solution (200 m\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e)\u0026thinsp;+\u0026thinsp;1 vol% MEM non-essential amino acid solution, \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e). Cell density was checked regularly under an inverted microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eReceptor binding affinity determinations (IC\u003csub\u003e50\u003c/sub\u003e)\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e competition studies were performed on CHO\u003csub\u003esstR2\u003c/sub\u003e cells (Chinese hamster ovary (CHO) cells stably transfected with human sstR2 (epitope-tagged at the \u003cem\u003eN\u003c/em\u003e-terminal end, provided by Dr. Jenny Koenig, University of Cambridge, Cambridge, United Kingdom), which were seeded (24-well plates, 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well, DMEM/F12 GlutaMax plus 10% FBS) and incubated at 37\u0026deg;C for 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2 h before the experiment. On the day of the experiment, the medium was removed and each well was washed with 300 \u003cem\u003e\u0026micro;\u003c/em\u003eL of HBSS (supplemented with 1 vol% of bovine serum albumin\u0026thinsp;=\u0026thinsp;HBSA). After the addition of 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL of HBSA, 25 \u003cem\u003e\u0026micro;\u003c/em\u003eL/well of HBSA (control, n\u0026thinsp;=\u0026thinsp;3) or the respective ligand in concentrations ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M (n\u0026thinsp;=\u0026thinsp;3) was added. Subsequently, 25 \u003cem\u003e\u0026micro;\u003c/em\u003eL of the radiolabelled reference [\u003csup\u003e125\u003c/sup\u003eI]TOC (1 n\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e in HBSA, for synthesis and characterization see SI) was added to each well. After incubation at RT for 1 h, the supernatant was removed, washed with ice-cold PBS (300 \u003cem\u003e\u0026micro;\u003c/em\u003eL), and the washing solutions were combined with the supernatants. The cells were lysed by adding NaOH (300 \u003cem\u003e\u0026micro;\u003c/em\u003eL, 1 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e). The cell lysate is removed after incubation at RT for 20 min and washed with NaOH (300 \u003cem\u003e\u0026micro;\u003c/em\u003eL, 1 M), while both NaOH-containing fractions were combined. Subsequently, the activities of both the supernatant and the lysate were measured separately in a \u003cem\u003eγ\u003c/em\u003e-counter, and the \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e50\u003c/sub\u003e values (concentration that is needed to replace 50% of the reference competitor from the receptor) were calculated using GraphPad Prism software (\u003cem\u003eGraphPad Prism 4.0 Software Inc\u003c/em\u003e., La Jolla, California, USA). Data were considered valid when the R\u0026sup2; fit was \u0026gt;\u0026thinsp;0.95. Each experiment was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStability studies in human serum\u003c/h2\u003e\u003cp\u003e5 MBq of the respective \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF- or \u003csup\u003e177\u003c/sup\u003eLu-labelled compound were added to 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL of human serum (from a healthy volunteer) and incubated at 37\u0026deg;C for 1 h. After the addition of 50 vol% of cold ethanol and 150 vol% of cold MeCN, centrifugation was performed at 13.000 rpm for 20 min. The supernatant was decanted and centrifuged at 13.000 rpm for 10 min in a centrifuge tube with a 0.45 \u003cem\u003e\u0026micro;\u003c/em\u003em cellulose acetate filter. Approximately 0.2 MBq of the remaining filtrate was injected into RP-HPLC and the amount of intact radioligand was quantified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003ebiodistribution studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnimal experiments were performed by certified personnel following a previously published method.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Experiments were performed in agreement with the general animal welfare regulations in Germany (German Animal Welfare Act, as published on May 18, 2006, as amended by Article 280 of June 19, 2020, permit no. ROB-55.2-2532.Vet_02-18-109 by the \u003cem\u003eGeneral Directorate of Upper Bavaria\u003c/em\u003e) and institutional guidelines for the care and use of animals. Specifically, female CD1-nu/nu mice aged 5\u0026ndash;6 weeks (\u003cem\u003eCharles River Laboratories International Inc\u003c/em\u003e., Sulzfeld, Germany) were acclimated in the in-house animal facility for one week prior to inoculation. Tumor xenografts were generated using AR42J cells (7.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL) suspended in a 1/1 mixture (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) of RPMI 1640 medium and Cultrex\u0026reg; Basement Membrane Matrix Type 3 (\u003cem\u003eTrevigen\u003c/em\u003e, Gaithersburg, MD, USA). This suspension was inoculated subcutaneously onto the right shoulder, and animals were used when the tumor volume was \u0026gt;\u0026thinsp;100 mm\u003csup\u003e3\u003c/sup\u003e (1\u0026ndash;2 weeks after inoculation). Exclusion criteria for animals from an experiment were either weight loss greater than 20%, tumor size greater than 1500 mm\u003csup\u003e3\u003c/sup\u003e, tumor ulceration, respiratory distress, or behavioural change. None of these criteria applied to any of the animals from the trial. No randomized or blinded approach was used in the allocation of the experiments. Health status is SPF according to the FELASA recommendation. Biodistribution studies (n\u0026thinsp;=\u0026thinsp;3) were performed after 1 h, 6 h, or 24 h p.i., and approximately 2\u0026ndash;3 MBq (300 pmol) were administered. Collected data were statistically analyzed using Excel (\u003cem\u003eMicrosoft Corporation\u003c/em\u003e, Redmond, WA, USA) and OriginPro software (version 9.7) from \u003cem\u003eOriginLab Corporation\u003c/em\u003e (Northampton, MA, USA). In the case of \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-rhTATE4\u003c/b\u003e, the study was carried out using a different gamma counter with lower sensitivity. For this reason, measurement limits were determined experimentally and then evaluated graphically, yielding a minimum measurement limit of 97.0 cpm/30 s (see SI, Table S5).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing financial or non-financial interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding statement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the Technical University of Munich for financial support.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization and supervision: A.C. and S.I.; synthesis and characterization of SiFA synthon: S.F.; design, synthesis and *in vitro* evaluation of sstR2-based compounds: S.D., V.K; *ex vivo* evaluation: S.D., S.F.; data analysis and interpretation: S.D., S.F., A.C.; writing of the main manuscript text: S.D., S.F., A.C.. All authors contributed to the drafting or revision of the manuscript. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Nadine Holzleitner for the help regarding human serum studies and Dr. Mara Parzinger for providing the Reference TOC for affinity studies and the SiFA-building block SiFAlin-Aldehyde. The authors acknowledge the Technical University of Munich for financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaum, R. P.; Kulkarni, H. R., THERANOSTICS: From Molecular Imaging Using Ga-68 Labeled Tracers and PET/CT to Personalized Radionuclide Therapy - The Bad Berka Experience. \u003cem\u003eTheranostics\u003c/em\u003e 2012, \u003cem\u003e2\u003c/em\u003e (5), 437\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBowden, G. D.; Scott, P. J. H.; Boros, E., Radiochemistry: A Hot Field with Opportunities for Cool Chemistry. \u003cem\u003eACS Central Science\u003c/em\u003e 2023.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, S.; Wang, X.; Gao, X.; Chen, X.; Li, L.; Li, G.; Liu, C.; Miao, Y.; Wang, R.; Hu, K., Radiopharmaceuticals and their applications in medicine. \u003cem\u003eSignal transduction and targeted therapy\u003c/em\u003e 2025, \u003cem\u003e10\u003c/em\u003e (1), 1.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoesch, F.; Martin, M., Radiometal-theranostics: the first 20 years*. \u003cem\u003eJournal of Radioanalytical and Nuclear Chemistry\u003c/em\u003e 2023, \u003cem\u003e332\u003c/em\u003e (5), 1557\u0026ndash;1576.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVahidfar, N.; Eppard, E.; Farzanehfar, S.; Yordanova, A.; Fallahpoor, M.; Ahmadzadehfar, H., An impressive approach in nuclear medicine: theranostics. \u003cem\u003ePET Clin.\u003c/em\u003e 2021, \u003cem\u003e16\u003c/em\u003e (3), 327\u0026ndash;340.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMettler, F. 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Sci.\u003c/em\u003e 2003, \u003cem\u003e92\u003c/em\u003e (11), 2236\u0026ndash;2248.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamazaki, K.; Kanaoka, M., Computational prediction of the plasma protein-binding percent of diverse pharmaceutical compounds. \u003cem\u003eJournal of Pharmaceutical Sciences\u003c/em\u003e 2004, \u003cem\u003e93\u003c/em\u003e (6), 1480\u0026ndash;1494.\u003c/span\u003e\u003c/li\u003e\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":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fluorine-18, Lutetium-177, theranostics, somatostatin receptor, radiopharmaceuticals, radiohybrid","lastPublishedDoi":"10.21203/rs.3.rs-8090442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8090442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe radiohybrid (rh) design of radiopharmaceuticals has recently produced new theranostics suitable for both positron emission tomography (PET) imaging and peptide receptor radionuclide therapy (PRRT). This approach aims to address the limitations of current medical radionuclides by offering a new strategy for combining radionuclides that previously lacked both therapeutic and diagnostic applications. Here, we report on a somatostatin receptor subtype 2 (sstR2)-targeted radiohybrid compound, \u003cb\u003erhTATE4\u003c/b\u003e, which features a bifunctional silicon-based fluoride acceptor (SiFA) - named (SiFA)\u003cem\u003eSeFe\u003c/em\u003e - for \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF labelling, along with a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelator for \u003csup\u003e177\u003c/sup\u003eLu coordination. The rh-theranostic agent demonstrates similar \u003cem\u003ein vitro\u003c/em\u003e behaviour compared to the gold standards [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-TATE and SiFA\u003cem\u003elin\u003c/em\u003e-TATE, along with an exceptionally high tumor uptake (53.58\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51% ID/g for the radiofluorinated version) after 1 h post-injection in AR42J tumor-bearing mice, making it ideal for imaging. Moreover, clearance from normal tissues and considerable tumor retention (10.32\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04%ID/g) for \u003cb\u003e[\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu]Lu-TATE4\u003c/b\u003e were observed at 24 p.i., suggesting good therapeutic applicability.\u003c/p\u003e","manuscriptTitle":"All at once: a sstR2-targeted radiohybrid theranostic agent for PET imaging and β- therapy with excellent preclinical performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 10:49:28","doi":"10.21203/rs.3.rs-8090442/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T15:01:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T20:44:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-21T17:44:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-21T01:19:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228549320928220183505770498149163763909","date":"2025-12-11T13:42:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317575296608309710811368752746763939559","date":"2025-12-10T12:57:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69437852528457233979065221625790480796","date":"2025-12-02T09:17:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T09:05:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-24T17:01:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T17:59:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2025-11-11T22:12:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0488c002-c85f-44a6-80ae-6bb58b044493","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59075575,"name":"Biological sciences/Cancer"},{"id":59075576,"name":"Physical sciences/Chemistry"},{"id":59075577,"name":"Biological sciences/Drug discovery"},{"id":59075578,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-16T07:58:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 10:49:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8090442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8090442","identity":"rs-8090442","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}