Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of Fibroblast Activation Protein-α or Folate Receptor-α overexpression in cancer

Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of Fibroblast Activation Protein-α or Folate Receptor-α overexpression in cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of Fibroblast Activation Protein-α or Folate Receptor-α overexpression in cancer Herlinde Dierick, Laurent Navarro, Hannelore Ceuppens, Thomas Ertveldt, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4523820/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jul, 2024 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background Radiofluorination of single domain antibodies (sdAbs) via N -succinimidyl-4-[ 18 F]fluorobenzoate ([ 18 F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (1012). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, can provide a detailed picture of the characteristics of both the tumour and its environment, which is critical for the success of cancer treatments. Results [ 18 F]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 minutes and results in [ 18 F]SFB with a RCP > 90% and a RCY d.c. of 44 ± 4% (n = 13). The conjugation reaction after purification produced [ 18 F]FB-sdAbs with a RCP > 95%, an end of synthesis activity > 600 MBq and an apparent molar activity > 10 GBq/µmol. Overall RCY d.c. were 9% and 5 ± 2% (n = 3) for [ 18 F]FB-1012 and [ 18 F]FB-4AH29, respectively. Conclusion [ 18 F]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging. Fluorine-18 Single domain Antibodies automation biomolecules Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Both the alpha isoforms of the Folate Receptor (FR) and Fibroblast Activation Protein (FAP) are established tumour markers. FR-α has an elevated expression in epithelial cancers, such as ovarian, cervical, and head and neck cancer [ 1 ]. At the same time, this isoform has a minimal physiological role in healthy tissue (except during embryogenesis), making it an interesting anticancer target. FR-α also shows a high affinity for both physiological and non-physiological substrates, which further cements its relevance for diagnostic and theranostic purposes [ 2 , 3 ] Only one FR-α targeting therapy, Mirvetuximab, Soravtansine [ 4 ], has been approved for use in patients [ 5 ]. Other promising agents, such as Farletuzumab [ 6 ] and Vintafolide [ 7 ], failed to meet their primary endpoints. A positron emission tomography (PET) tracer that specifically targets FR-α has the potential to be a companion diagnostic for FR-α targeting therapies and can help in patient stratification [ 5 , 8 ] While a large number of folate tracers, however not specific to FR-⍺, labelled with fluorine-18 ( 18 F) have been developed over the last decades, to our knowledge, only two have made it to clinical trials, namely [ 18 F]-AzaFol [ 9 ] and [ 18 F]fluoro-PEG-folate [ 10 ]. FAP is known to be overexpressed in breast, colorectal, ovarian, and other cancers, more specifically in the stroma. This indicates the presence of cancer-associated fibroblasts, while their expression in normal tissues is low or undetectable[ 11 ]. Due to this attractive expression pattern, anti-FAP radiopharmaceuticals have been a hot topic for diagnostic and therapeutic applications. Several anti-FAP small molecule-based, for example OncoFAP [ 12 ], FAPI-04 [ 13 , 14 ], FAPI-46 [ 15 ], FAPI-74 [ 16 ] and PNT6555 [ 17 ] and peptide-based radiopharmaceuticals, such as FAP-2286 [ 18 ], have been developed in recent years and are currently being tested in the clinic [ 19 – 22 ]. Different targeting moieties have been used to develop PET tracers for established tumour markers. Immune-derived vectors such as monoclonal antibodies (mAbs), minibodies, single-domain antibodies (sdAbs), allow to combine their highly specific targeting with the sensitivity and resolution of PET [ 23 ]. SdAbs have gained quite some interest as targeting molecules for PET imaging. Their key characteristics, such as their small size (around 15 kDa), high affinity, high specificity, low off-target accumulation, high (thermo)stability and solubility [ 24 ] allowed them to be successfully translated to the clinic as diagnostic [ 25 , 26 ] and therapeutic [ 27 ] radiopharmaceuticals. Compared to mAb-based diagnostics, their most notable advantages that their short biological half-life and fast tumour penetration allow for their labelling with short-lived radionuclides such as gallium-68 ( 68 Ga) and 18 F [ 24 ]. From a diagnostic standpoint, 18 F is an ideal radionuclide for PET imaging with its high positron (β + ) yield of 97%, relatively low energy (max 0.634 MeV) of the emitted β + and thus short trajectory (mean positron range in soft tissue: 0.27 mm) resulting in high-resolution images. Its half-life of 109.8 min is long enough to allow shipment of the radiopharmaceutical to other centres but still short enough to avoid unnecessary extended irradiation of the patients. The ease of producing large amounts with a cyclotron cements its place as the favourite radionuclide in PET imaging [ 23 , 28 ]. The direct 18 F-labelling of sdAbs and other biomolecules is prevented by the harsh reaction conditions, elevated temperatures, organic solvents, and high pH needed for radiofluorination. The development of prosthetic groups (PG) like N -succinimidyl 4-[ 18 F]Fluorobenzoate ([ 18 F]SFB), [ 18 F]Fluorobenzaldehyde ([ 18 F]FBA) and N -[2-(4-[ 18 F]-Fluorobenzamido)ethyl]maleimide ([ 18 F]FBEM), makes radiofluorination of proteins possible in aqueous medium under mild conditions. [ 18 F]SFB is a popular PG thanks to its reactivity with lysins, amino acid group naturally present on the surface of proteins, including sdAbs. The resulting [ 18 F]FB-bioconjugate has demonstrated good in vivo stability [ 29 – 31 ]. Distribution and commercialization of highly specific PET radiofluorinated radiopharmaceuticals becomes possible, while the centralized production of 68 Ga-labeled products is more difficult to organize. [ 23 , 31 ] This study aims to develop a generic semi-automated radiofluorination strategy for sdAbs as a platform for the radiofluorination of two sdAb with high interest targets, namely FR-α and FAP-α. The production of the PG, [ 18 F]SFB was optimized and automated on the AllInOne (AiO) module (Trasis), while the conjugation reaction to the sdAbs was achieved manually using an optimized protocol. Methods The cell lines used in this study were generated for this purpose. The methodologies for their generation, culture conditions and validation by flow cytometry (supplemental Fig. 1) can be found in the Supplementary Information (SI). 3.1. sdAbs An anti-FAP-α sdAb, cross-reactive for mouse/human FAP-α and an anti-FR-α sdAb, reactive to human FR-α were kindly provided by Precirix. The anti-FAP-α sdAb (4AH29)[ 32 ], the FR-α sdAb (1012) and the non-targeting control sdAb (R3B23) [ 33 ] were produced and characterized as previously described [ 34 ]. All sdAbs in this study were free of tags. 3.2. Radiochemistry 3.2.1. Automated [ 18 F]SFB synthesis N -succinimidyl-4-[ 18 F] fluorobenzoate ([ 18 F]SFB) was synthesized using a three-step, one-pot reaction (Fig. 1 a.). The complete production process of [ 18 F]SFB, including the purification, was automated with an AiO module (Trasis) using disposable cassettes. [ 18 F]F − was produced by irradiation of enriched [ 18 O]water (Rotem medical and Campro) in Niobium targets with a Cyclone KIUBE cyclotron (IBA) via the 18 O(p,n) 18 F nuclear reaction. The [ 18 F]fluoride aqueous solution was passed through a Sep-Pak Light Accell Plus QMA anion exchange cartridge (Waters) to trap [ 18 F]fluoride and recover the enriched water. The [ 18 F]fluoride was eluted from the cartridge with 600 µL of Cryptant Solution (4.2 mg of K 2 CO 3 and 22.6 mg of Cryptand (K 222 ) in acetonitrile/water (1:1)) (ABX). The solvent was evaporated to form anhydrous Kryptofix K 222 /K[ 18 F]F complex (60–70 GBq). A solution of 0.8 mg (0.002 mmol) of ethyl-4-(trimethylammonium)benzoate (ABX) in 2 mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to the dried [ 18 F]F − complex in the reactor and heated to 110°C for 15 min to produce ethyl-4-[ 18 F]fluorobenzoate. This compound was hydrolysed at 95°C for 5 min by a 0.38 M (0.76 mmol) tetrapropylammonium hydroxide (TPAOH) aqueous solution diluted in DMSO. The subsequent activation was performed with 26 mg (0.072 mmol) of N , N , N ′, N ′-tetramethyl-O-( N -succinimidyl)uronium hexafluorophosphate (HSTU, Sigma-Aldrich) in 1 mL of acetonitrile at 110°C for 5 min to form [ 18 F]SFB. The reaction mixture (RM) was diluted with 12 mL of an acetic acid solution (1.7% acetic acid/ NaCl 0.6%) before trapping on an HLB prime Plus Light solid-phase extraction (SPE) cartridge (Waters). The cartridge was washed with 1 mL of aqueous EtOH solution (5%) and reverse eluted with 0.8 mL of EtOH (Emsure, VWR). The purity of the [ 18 F]SFB was determined by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Detailed information on the chromatographic analysis can be found in the SI. The PG production described above and its automation was optimized based on the work of Xavier et al. [ 35 ]. Detailed insights into the optimization procedures are available in the SI. 3.2.2. Manual conjugation of [ 18 F]SFB to sdAbs At this point in the production of the tracers, the conjugation step was optimised and performed manually. The different sdAbs in phosphate buffered saline (PBS) pH 7.4 ± 0.1 (Table 1 ) are diluted with 0.5 M CHES buffer pH 8.7 ± 0.1 and PBS. This mixture is added to 200 µL of the ethanolic [ 18 F]SFB (3–5 GBq) and left to incubate for at least 15 minutes at room temperature (Fig. 1 b.). The radiolabelled sdAb was purified using two disposable desalting Hitraps (Cytiva) placed in series (pre-equilibrated with NaCl 0.9% with 5 mg/mL ascorbic acid, pH 5.9–6.2) using a peristaltic pump (Ismatec Reglo ICC, Masterflex) with a flow rate of 5 mL.min − 1 . The final product was passed through a 0.22 µm filter (Millipore) and analysed by RP-HPLC and Size-Exclusion (SE) -HPLC (see SI). Detailed insights into the optimization procedures, starting from the work of Xavier et al . [ 35 ] are available in the SI. Table 1 molecular weight and mass of sdAb used in conjugation reaction sdAb Molecular weight (g/mol) Amount of sdAb 4AH29 12350.8 8.1 x 10 − 8 mol, 1000 µg, 100 µL 1012 13042.4 7.7 x 10 − 8 mol, 1000 µg, 100 µL R3B23 13913.3 7.2 x 10 − 8 mol, 1000 µg, 100 µL 3.3. Animal models hFR-α knock-in female mice (kindly provided by Precirix) and wildtype C57BL/6 female mice (Charles River) were used to evaluate biodistribution and tumour uptake of [ 18 F]FB-1012 and [ 18 F]FB-4AH29 respectively. They were subcutaneously inoculated at the tail base, under the control of 2.5% isoflurane in oxygen (Abbott), with TC-1-hFR-α cells (5 x 10 4 ) suspended in PBS in the case of hFR-α knock-in mice and with TC-1-hFAP-α cells (5 x 10 4 ) suspended in PBS in the case of the wildtype C57BL/6 mice. The tumours were allowed to grow for up to two weeks (100–300 mm 3 ). 3.4. Biodistribution & PET/CT imaging hFR-α knock-in female mice bearing TC-1-hFR-α tumours (n = 4 per group) were i.v. injected (25 µg; 15 MBq) with [ 18 F]FB-1012 or [ 18 F]FB-R3B23. Wildtype C57BL/6 female mice bearing TC-1-hFAP-α tumours were i.v. injected (25 µg; 15 MBq) with [ 18 F]FB-4AH29 (n = 4) or [ 18 F]FB-R3B23 (n = 3). One hour after injection, micro-PET/CT images were acquired (detailed information in SI), followed by dissections 1h10 or 1h30 post injection in mice bearing TC-1-hFR-α tumours and mice bearing TC-1-hFAP-α tumours, respectively. The timepoint discrepancies are due to differences in the preclinical study design of both tracers. Animals were dissected, and organ and tissue activities were counted against a standard of known activity with an automated gamma counter (Wizard 2 2480, PerkinElmer) and expressed as a percentage of injected activity per gram (%IA/g), corrected for decay. In vitro characterization of the tracers, affinity measurement by cell saturation assay (supplemental Fig. 2) and in vitro stability in plasma (supplemental table 3), can be found in the SI. 3.5. Statistical analysis Data were expressed as average ± SD. The statistical analysis used GraphPad Prism 10. One-way ANOVA, two-way ANOVA with multiple comparison tests, or unpaired t-test were used to evaluate statistical significance. Results 4.1. Radiolabelling [ 18 F]SFB was synthesized using a three-step, one-pot reaction, which was fully automated. The total time of the procedure was 54 minutes and allowed to obtain [ 18 F]SFB (23.31 ± 6.28 GBq, n = 13) with a RCP > 90% and a radiochemical yield (RCY) decay corrected (d.c.) of 44 ± 4% (n = 13). A schematic representation of the automated radiosynthesis procedure is shown in Fig. 2 . The [ 18 F]F − enters the module via the syringe in the 6th position (P6) in the layout. The cyclotron-produced [ 18 F]F − is separated from the 18 O-enriched water by the QMA cartridge on P5. Then, [ 18 F]F is eluted with the Cryptand solution (P2), with the help of a syringe located in P3. The mixture is transferred to the 6 mL reactor (P7), after which the azeotropic drying of the [ 18 F]fluoride is started. To the dried [ 18 F]K 222 -fluoride, 0.8 mg of FB-precursor, dissolved in DMSO (P8), is added. The reactor is heated to 110ºC for 15 minutes to obtain the ethyl-4-[ 18 F]fluorobenzoate and cooled down afterwards. Next, the product is hydrolysed by adding the 0.38M TPAOH DMSO solution (vial P10) to the reactor. The reactor is heated to 95ºC for 5 minutes to obtain the 4-[ 18 F]fluorobenzoic acid and cooled down again. For the third and last step, 26 mg of HSTU dissolved in anhydrous acetonitrile (P11) is transferred to the reactor. The reactor is heated to 110ºC for 5 minutes, obtaining the crude [ 18 F]SFB, and cooled down again. The RM inside the reactor is diluted with a mixture of 4 mL of 4.8% acetic acid solution (P17) and 8 mL of 0.9% NaCl (P13), prepared by the module by mixing both components within the 20 mL syringe (P9) in the layout. The same syringe applies the RM to the HLB light cartridge (P33). Next, the cartridge and lines are rinsed with 5% EtOH/water solution (P35). To complete the purification, the final product is reverse eluted with EtOH (vial P14), using the 3 mL syringe (P15) and collected in a final vial. The manual conjugation reaction produced [ 18 F]FB-sdAbs with a RCY of 22 ± 4% (n = 2), 19 ± 7% (n = 3) and 19 ± 1% (n = 2) d.c. starting from the added [ 18 F]SFB for [ 18 F]FB-1012, [ 18 F]FB-4AH29 and [ 18 F]FB-R3B23 respectively. The purified [ 18 F]FB-sdAbs were obtained with a RCP > 95%, and the end of synthesis activity amounted to 783 ± 8.50 MBq (n = 2) for [ 18 F]FB-1012, 694 ± 80 MBq (n = 2) [ 18 F]FB-4AH29, and 907 ± 227 MBq (n = 2) for [ 18 F]FB-R3B23. The apparent molar activity was 12.55 ± 0.21 GBq/µmol (n = 2), 10.42 ± 1.28 GBq/µmol (n = 2), and 15.58 ± 3.90 GBq/µmol (n = 2) respectively. Overall RCY d.c. were 9% (n = 1), 5 ± 2% (n = 3) and 8 ± 1% (n = 2) for [ 18 F]FB-1012, [ 18 F]FB-4AH29 and [ 18 F]FB-R3B23 respectively. 4.2. Biodistribution studies and PET/CT imaging hFR-α knock-in female mice bearing TC-1-hFR-α tumours (n = 4 per group) were i.v. injected with [ 18 F]FB-1012 (28 ± 2 µg; 14.69 ± 0.36 MBq, 6.95 ± 1.19 GBq/µmol) or [ 18 F]FB-R3B23 (non-targeting control sdAb conjugate) (28 ± 2 µg; 16.08 ± 0.30 MBq, 8.13 ± 1.68 GBq/µmol). Wildtype C57BL/6 female mice bearing TC-1-hFAP-α tumours were i.v. injected with [ 18 F]FB-4AH29 (26 ± 3 µg; 14.53 ± 1.33 MBq, 7.53 ± 0.88 GBq/µmol, n = 4) or [ 18 F]FB-R3B23 (non-targeting control sdAb conjugate) (20 ± 0 µg; 12.75 ± 1.68 MBq, 8.68 ± 1 .20 GBq/µmol, n = 3). Injected and apparent molar-specific activities are reported at the time of injection. Tumour uptake of [ 18 F]FB-1012 was visible on the PET image (1h p.i., Fig. 3 a). It was confirmed by quantification of dissection data (1h10 p.i.) (Fig. 4 a), showing statistically significant (p < 0.0001) higher tumour uptake (8.13 ± 1.15 IA/g) for the FR-targeting sdAb compared to the non-targeting sdAb (0.27 ± 0.09 IA/g). Furthermore, the dissection studies evaluating [ 18 F]FB-1012 displayed about 2-fold higher kidney accumulation (25.37 ± 2.61 vs 14.06 ± 3.70 IA/g; p < 0.01), 3-fold higher accumulation in the ovaries (1.28 ± 0.27 vs 0.46 ± 0.21 IA/g; p < 0.01) and 3-fold higher accumulation in the brain (0.13 ± 0.02 vs 0.04 ± 0.01 IA/g; p < 0.0001) compared to the non-targeting sdAb. The in vivo profile of the anti-FAP-α sdAb, [ 18 F]FB-4AH29, was investigated in TC-1-hFAP-α tumour bearing mice by a similar protocol, including micro-PET/CT imaging at 1h p.i. (Fig. 3 b) and dissection analysis at 1.5h p.i. (Fig. 4 c), and compared to [ 18 F]FB-R3B23. Ex vivo biodistribution studies indicated specific tumour uptake (2.46 ± 0.50 IA/g) compared to the non-targeting sdAb (0.40 ± 0.34 IA/g), no unspecific organ accumulation except in the joints (1.58 ± 0.09 vs 0.44 ± 0.51 IA/g; p < 0.005), pancreas (0.55 ± 0.08 vs 0.13 ± 0.07 IA/g; p < 0.001), skin (1.53 ± 0.38 vs 0.48 ± 0.26 IA/g; p < 0.05), blood (0.60 ± 0.16 vs 0.26 ± 0.14 IA/g; p < 0.05) and uterus (1.28 ± 0.31 vs 0.40 ± 0.27 IA/g; p < 0.05) compared to the non-targeting sdAb. For both tracers, fast excretion of the unbound tracer was observed via the kidneys ([ 18 F]FB-4AH29: 9.97 ± 1.25% IA/g; [ 18 F]FB-R3B23: 6.82 ± 1.34% IA/g). The tumour-to-blood (T/B) ratios were calculated for both tracers. T/B ratios for [ 18 F]FB-1012 and [ 18 F]FB-4AH29 were significantly higher compared to their respective control sdAb (Fig. 4 b&d). Discussion The radiofluorination strategy of sdAbs described herein uses the well-established PG [ 18 F]SFB. This PG is widely used for labelling peptides and proteins and its radio-synthesis has been continuously refined and optimized. In this study, the three-step, one-pot reaction was automated on a Trasis AiO. Automation of the PG production has been successfully implemented on in-house developed automation synthesis equipment[ 36 ] and commercial automated synthesis modules such as the IBA Syntera module[ 35 , 37 , 38 ], TRACERlab FX FN synthesizer[ 39 , 40 ] (GE Healthcare) and the Ora-Neptis synthesizer [ 41 ]. We first optimized the automated production process by five times reducing the mass of the commercially available precursor[ 35 , 37 , 42 ] without negatively impacting the RCY of the reaction (see SI, Table 1 ). We hypothesized that this reduction would also reduce the formation of potential process-related impurities and help increase specific activity. A second optimization was the purification of the PG. In the literature, different strategies can be found, such as HPLC methods, SPE using one single cartridge [ 35 , 37 , 39 ], multiple cartridges in series[ 40 ] or strategies combining both HPLC and SPE[ 36 , 41 ]. The automated synthesis procedure described in this study uses a single SPE cartridge for purification, reducing time spent on purification compared to HPLC purification strategies. By opting for reverse elution of the cartridge, it was possible to reduce the elution volume to 800 µL. When comparing the SPE strategy used here to the other SPE strategies in literature[ 35 , 37 , 39 , 40 , 43 ], the final formulation of the PG in a small volume (0.8 mL) of ethanol, avoiding a reformulation step or time-consuming evaporation step before starting the subsequent conjugation reaction, is a significant advantage to reduce the time of the whole production process. The conjugation reaction described in this study was optimized with sdAbs in mind and included a 20% V/V content of ethanol. This ethanol concentration showed no negative impact on the conjugation reaction (see SI table 2) and is in line with the results of several studies[ 44 , 45 ] that showed denaturation of proteins caused by alcohols occurs at concentrations above 20%. The change of final solvent to ethanol was facilitated by replacing the previously used tC18[ 35 , 37 , 42 ] with an HLB cartridge. A slight reduction in RCP, > 90% compared to the previously reported[ 31 , 35 , 39 , 40 , 46 , 47 ] > 95%, could be observed, with [ 18 F]FBA as the identified radioactive impurity. Most likely, this reduction in RCP is caused by a combination of radiolysis, increasing amount of radioactive impurity with increased volumetric activity concentration (up to more than 25 GBq/mL), and hydrolysis, as the impurity increases over time as well. However, as the impurity does not compete with the PG in the following conjugation reaction, the slight decrease in RCP was deemed insignificant. For optimization of the conjugation reaction, we opted for CHES as a coupling buffer due to the superior stability of the PG in this buffer compared to the conventional borate buffer [ 35 , 38 , 42 ]. Nagachinta et al .[ 41 ] performed the coupling of sdAbs to the PG using a phosphate buffer at pH 8.4, we prefer the use of CHES as its buffering range (pH 8.6 to 10 compared to 5.8 to 7.4 for a phosphate buffer) is more in range with the optimal reactivity of the sdAbs’ amino groups towards acylation (pH < 8.5). The higher buffer capacity and, thus fewer fluctuations in pH of CHES compared to phosphate also allows for a more robust coupling reaction. Detailed insights into the buffer selection are available in the SI, supplemental Fig. 3. The purification of the radiolabelled sdAbs was performed using SE resins HiTrap desalting cartridges instead of the PD-10 desalting column, with the latter being the most described option in literature [ 35 , 38 , 41 – 43 ]. The conjugation of the sdAbs to the PG resulted in reasonable decay-corrected conjugation yields (20–25%, starting from [ 18 F]SFB) with high RCP and reasonable apparent molar activity. The conjugation yield was comparable to or higher than others reported for sdAbs and proteins [ 35 , 39 , 41 , 42 , 47 , 48 ]. The comparable conjugation results (similar RCY d.c., apparent molar activities. and final activities) for all three sdAbs show that this strategy could also be used as a generic radiolabelling strategy for sdAbs, similar to the generic 68 Ga-chelator approach currently used [ 25 , 26 , 32 , 43 , 49 ]. This generic 68 Ga-chelator approach has already been successfully used to introduce sdAb-based tracers in the clinic, as shown by the clinical translation of sdAbs targeting HER2 [ 25 , 26 ] and CD206 [ 43 , 49 ]. The advantages of this method compared to radiofluorination are the ease of its chemistry, higher RCYs and its lower initial financial investment, as there is no need for a cyclotron or automation modules. On the other hand, by developing a radiolabelling method with 18 F for sdAbs, we can take advantage of the superior imaging quality of 18 F. At the same time, its longer half-life allows for easier radiopharmaceutical distribution and still matches the biological half-life of sdAbs. Because of the ease of production of high amounts of the radionuclide with a cyclotron, upscaling the obtained activity will allow for multi-patient preparations produced in PET radiopharmacies or centralized production sites. The biodistribution and imaging studies for both tracers showed excellent targeting properties and specificity for FR-α or FAP-α, fast excretion via the kidneys of both [ 18 F]FB-1012 and [ 18 F]FB-4AH29, respectively. The known FR-α expression in the fallopian tubes, proximal tubule cells of the kidneys, and choroid plexus in the brain, might explain the observed elevated uptake in these organs [ 2 , 3 , 50 ] Besides specific uptake of [ 18 F]FB-4AH29 in the tumour, elevated accumulation was seen in pancreas, skin and uterus. This is in line with previous findings [ 51 , 52 ] in mice, showing an interspecies difference in FAP expression compared to humans. The elevated uptake in blood and joints could be attributed to the increased shedding of FAP protein in mice [ 52 ]. Conclusion Using a Trasis AiO, [ 18 F]SFB synthesis was successfully automated and upscaled, yielding consistently around 20 GBq of pure product. The anti-hFAP-α, anti-hFR-α and non-targeting control sdAbs were successfully radiofluorinated, yielding similar DC-RCYs and RCPs. The herein presented semi-automated radiofluorination approach could be used as a generic radiofluorination method for sdAbs, allowing for faster preclinical validation of sdAbs as PET tracers and opens opportunities for further development towards clinical production. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging. Abbreviations [ 18 F]FBA [ 18 F]Fluorobenzaldehyde [ 18 F]FBEM N -[2-(4-[ 18 F]-Fluorobenzamido)ethyl]maleimide [ 18 F]SFB N -succinimidyl 4-[ 18 F]Fluorobenzoate 18 F fluorine-18 68 Ga gallium-68 AiO AllInOne CHES 2-(Cyclohexylamino)ethane-1-sulfonic acid d.c. Decay corrected DMSO dimethyl sulfoxide EtOH Ethanol FAP fibroblast activation protein FR Folate receptor HSTU N,N,N′,N′- tetramethyl- O -( N- succinimidyl)uronium hexafluorophosphate mAbs monoclonal antibodies PBS phosphate buffered saline PG Prostethic group RCP Radiochemical purity RCY Radiochemical yield RM reaction mixture RP-HPLC Reverse Phase High Performance Liquid Chromatography sdAbs single-domain antibodies SE Size-Exclusion SI Supplementary Information SPE solid-phase extraction T/B tumour-to-blood TPAOH tetrapropylammonium hydroxide Declarations Ethics approval and consent to participate The ethical committee for animal experiments at the Vrije Universiteit Brussel approved the in vivo study protocols (22-272-12 & 19-272-17). All mouse experiments were executed in accordance with the European guidelines for animal experimentation. Written informed consent was not required for this study. Consent for publication Not applicable Availability of data and material The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests K.B., N.D., M.D., M.K., T.L and J.B. have patents on using sdAbs for imaging and therapy. M.K is an editor in EJNMMI. J.B is an unpaid board member of eSRR. T.L., N.D. and M.K. have ownership in AbScint. M.K. received research funding from Precirix. N.D. and M.D are resp. consultant and employee for and hold ownership in Precirix. L.N. and A.R.P.A. are employees of Precirix. Funding This research was performed with the financial support of Strategic Research Programs (SRP50, SRP95 and SRP62) and the Industrial Research Fund (IOF3018 and IOF3009) of the VUB Research Council and is part of the joint R&D project IMPACT, financially supported by Innoviris and Precirix (2020-RDIR-1). T.E., J.B., M.K. and M.D. were, respectively, pre-doctoral researcher (1S06622N), postdoctoral fellow (1230824N), senior clinical investigator (1801619N) and postdoctoral fellow (12H3619N) of the Research Foundation Flanders (FWO-V) during the execution of this work. This research was partly performed at the Virus Production Unit, Molecular Biology Facility, and In vivo Cellular and Molecular Imaging Core facility, core facilities financially supported by the University Medical Center Onderzoeksraad. The BD Celesta flow cytometer and Molecubes β-CUBE PET/CT system were funded via FWO-Hercules grants (I001618N and I005622N). Author contributions M.K, N.D, K.B., M.D. and T.L contributed to the study conception. J.B, V.C., A.R.P.A, M.D. N.D. and K.B. contributed to the study design. Material preparation, data collection and analysis were performed by H.D., L.N., H.C., T.E. and A.R.P.A. The first draft of the manuscript was written by H.D. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Elsy Vaeremans and Petra Roman for technical support during the cloning of the lentiviral transfer plasmids and generation of lentiviral particles, Kevin De Jonghe, Melissa Lucero, and Maxime Deladrière for handling the animals and performing the PET/CT-imaging. We thank Yana Dekempeneer for enabling the in vitro and in vivo experiments. References Sega EI, Low PS. Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Rev. 2008;27:655–64. Scaranti M, Cojocaru E, Banerjee S, Banerji U. Exploiting the folate receptor α in oncology. Nat Rev Clin Oncol Nat Res; 2020. p. 349–59. Boss SD, Ametamey SM. Development of Folate Receptor – Targeted PET Radiopharmaceuticals for Tumor Imaging—A Bench-to-Bedside Journey. Cancers 2020, Vol 12, Page 1508. 2020;12:1508. Moore KN, Angelergues A, Konecny GE, García Y, Banerjee S, Lorusso D, et al. Mirvetuximab Soravtansine in FRα-Positive, Platinum-Resistant Ovarian Cancer. N Engl J Med. 2023;389:2162–74. Harada H, Gonzalez T, Muminovic M, Nano O, Vulfovich M. Folate Receptor Alpha—A Novel Approach to Cancer Therapy. Int J Mol Sci 2024. 2024;25:25:1046. Herzog TJ, Pignata S, Ghamande SA, Rubio MJ, Fujiwara K, Vulsteke C, et al. Randomized phase II trial of farletuzumab plus chemotherapy versus placebo plus chemotherapy in low CA-125 platinum-sensitive ovarian cancer. Gynecol Oncol. 2023;170:300–8. Vynfinit | European Medicines Agency [Internet]. [cited 2024 Jan 29]. https://www.ema.europa.eu/en/medicines/human/EPAR/vynfinit . Guzik P, Fang HY, Deberle LM, Benešová M, Cohrs S, Boss SD, et al. Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy. J Nucl Med. 2021;62:1475–81. Gnesin S, Müller J, Burger IA, Meisel A, Siano M, Früh M et al. Radiation dosimetry of 18F-AzaFol: A first in-human use of a folate receptor PET tracer. EJNMMI Res. 2020;10. Verweij NJF, Yaqub M, Bruijnen STG, Pieplenbosch S, ter Wee MM, Jansen G et al. First in man study of [18F]fluoro-PEG-folate PET: a novel macrophage imaging technique to visualize rheumatoid arthritis. Sci Rep. 2020;10. Fitzgerald AA, Weiner LM. The role of fibroblast activation protein in health and malignancy. Cancer Metastasis Rev. 2020;39:783–803. Backhaus P, Gierse F, Burg MC, Büther F, Asmus I, Dorten P, et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA. Eur J Nucl Med Mol Imaging. 2022;49:1822–32. Wang S, Zhou X, Xu X, Ding J, Liu T, Jiang J et al. Dynamic PET/CT Imaging of 68Ga-FAPI-04 in Chinese Subjects. Front Oncol. 2021;11. Wang H, Zhu W, Ren S, Kong Y, Huang Q, Zhao J, et al. 68Ga-FAPI-04 Versus 18F-FDG PET/CT in the Detection of Hepatocellular Carcinoma. Front Oncol. 2021;11:693640. Meyer C, Dahlbom M, Lindner T, Vauclin S, Mona C, Slavik R, et al. Radiation Dosimetry and Biodistribution of 68Ga-FAPI-46 PET Imaging in Cancer Patients. J Nucl Med. 2020;61:1171–7. Giesel FL, Adeberg S, Syed M, Lindner T, Jiménez-Franco LD, Mavriopoulou E, et al. FAPI-74 PET/CT Using Either 18F-AlF or Cold-Kit 68Ga Labeling: Biodistribution, Radiation Dosimetry, and Tumor Delineation in Lung Cancer Patients. J Nucl Med. 2021;62:201–7. Poplawski SE, Hallett RM, Dornan MH, Novakowski KE, Pan S, Belanger AP et al. Preclinical Development of PNT6555, a Boronic Acid–Based, Fibroblast Activation Protein-α (FAP)–Targeted Radiotheranostic for Imaging and Treatment of FAP-Positive Tumors. Journal of Nuclear Medicine. 2023;00:jnumed.123.266345. Zboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging [Internet]. 2022 [cited 2024 Apr 29];49:3651–67. https://pubmed.ncbi.nlm.nih.gov/35608703/ . Millul J, Bassi G, Mock J, Elsayed A, Pellegrino C, Zana A, et al. An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications. Proc Natl Acad Sci U S A. 2021;118:e2101852118–2101852118. Zhao L, Niu B, Fang J, Pang Y, Li S, Xie C et al. Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of 68Ga-Labeled FAPI Dimer. J Nucl Med. 2022;63. Zboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E, et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 2022;49:3651. Toyohara J, Al-Qahtani M, Huang YY, Cazzola E, Todde S, Furumoto S, et al. Highlight selection of radiochemistry and radiopharmacy developments by editorial board. EJNMMI Radiopharm Chem. Springer Science and Business Media Deutschland GmbH; 2022. Wei W, Rosenkrans ZT, Liu J, Huang G, Luo QY, Cai W, ImmunoPET. Concept, Design, and Applications [Internet]. Chem Rev. 2020 [cited 2020 Sep 7]. pp. 3787–851. https://dx.doi.org/10.1021/acs.chemrev.9b00738 . De Pauw T, De Mey L, Debacker JM, Raes G, Van Ginderachter JA, De Groof TWM, et al. Current status and future expectations of nanobodies in oncology trials. Expert Opin Investig Drugs. 2023;32:705–21. Keyaerts M, Xavier C, Everaert H, Vaneycken I, Fontaine C, Decoster L, et al. Phase II trial of HER2-PET/CT using 68Ga-anti-HER2 VHH1 for characterization of HER2 presence in brain metastases of breast cancer patients. Ann Oncol. 2019;30:iii25–6. Keyaerts M, Xavier C, Heemskerk J, Devoogdt N, Everaert H, Ackaert C, et al. Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma. J Nucl Med. 2016;57:27–33. D’Huyvetter M, Vos J, De, Caveliers V, Vaneycken I, Heemskerk J, Duhoux FP, et al. Phase I Trial of 131I-GMIB-Anti-HER2-VHH1, a New Promising Candidate for HER2-Targeted Radionuclide Therapy in Breast Cancer Patients. J Nucl Med. 2021;62:1097–105. Cleeren F, Lecina J, Bridoux J, Devoogdt N, Tshibangu T, Xavier C et al. Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al 18 F-RESCA method. Nat Protoc [Internet]. 2018 [cited 2020 Sep 8];13:2330–47. https://doi.org/10.1038/s41596-018-0040-7 . Kim HK, Javed MR, Chen S, Zettlitz KA, Collins J, Wu AM, et al. On-demand radiosynthesis of: N -succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) on an electrowetting-on-dielectric microfluidic chip for 18F-labeling of protein. RSC Adv. 2019;9:32175–83. Jacobson O, Kiesewetter DO, Chen X. Fluorine-18 radiochemistry, labeling strategies and synthetic routes. Bioconjug Chem. American Chemical Society; 2015. pp. 1–18. Vaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F. Nat Protoc [Internet]. 2006 [cited 2020 Sep 8];1:1655–61. https://www.nature.com/articles/nprot.2006.264 . Dekempeneer Y, Massa S, Santens F, Navarro L, Berdal M, Lucero MM, et al. Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α. J Nucl Med. 2023;64:1941–8. Lemaire M, D’Huyvetter M, Lahoutte T, Van Valckenborgh E, Menu E, De Bruyne E, et al. Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic Nanobodies. Leukemia. 2014;28:444–7. Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res. 2012;110:927–37. Xavier C, Blykers A, Vaneycken I, D’Huyvetter M, Heemskerk J, Lahoutte T et al. 18F-nanobody for PET imaging of HER2 overexpressing tumors. Nucl Med Biol [Internet]. 2016 [cited 2020 Sep 7];43:247–52. http://dx.doi.org/10.1016/j.nucmedbio.2016.01.002 . Fujimoto H, Fujita N, Hamamatsu K, Murakami T, Nakamoto Y, Saga T, et al. First-in-Human Evaluation of Positron Emission Tomography/Computed Tomography With [18F]FB(ePEG12)12-Exendin-4: A Phase 1 Clinical Study Targeting GLP-1 Receptor Expression Cells in Pancreas. Front Endocrinol (Lausanne). 2021;12:717101. Ackermann U, Yeoh SD, Sachinidis JI, Poniger SS, Scott AM, Tochon-Danguy HJ. A simplified protocol for the automated production of succinimidyl 4-[ 18F]fluorobenzoate on an IBA Synthera module. J Label Comp Radiopharm. 2011;54:671–3. Blykers A, Schoonooghe S, Xavier C, D’Hoe K, Laoui D, D’Huyvetter M, et al. PET imaging of macrophage mannose receptor-expressing macrophages in tumor stroma using 18F-radiolabeled camelid single-domain antibody fragments. J Nucl Med. 2015;56:1265–71. Scott PJH, Shao X. Fully automated, high yielding production of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), and its use in microwave-enhanced radiochemical coupling reactions. J Label Comp Radiopharm. 2010;53:586–91. Tang G, Tang X, Wang X. A facile automated synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) for 18F-labeled cell-penetrating peptide as PET tracer. J Label Comp Radiopharm. 2010;53:543–7. Nagachinta S, Novelli P, Joyard Y, Maindron N, Riss P, Dammicco S. Fully automated 18F-fluorination of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for indirect labelling of nanobodies. Sci Rep 2022. 2022;12(1):12:1–8. Bala G, Blykers A, Xavier C, Descamps B, Broisat A, Ghezzi C, et al. Targeting of vascular cell adhesion molecule-1 by 18F-labelled nanobodies for PET/CT imaging of inflamed atherosclerotic plaques. Eur Heart J Cardiovasc Imaging. 2016;17:1001–8. Xavier C, Blykers A, Laoui D, Bolli E, Vaneyken I, Bridoux J, et al. Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages. Mol Imaging Biol. 2019;21:898–906. Nikolaidis A, Moschakis T. On the reversibility of ethanol-induced whey protein denaturation. Food Hydrocoll. 2018;84:389–95. Nikolaidis A, Andreadis M, Moschakis T. Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra. Food Chem. 2017;232:425–33. Tang G, Zeng W, Yu M, Kabalka G. Facile synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([ 18F]SFB) for protein labeling. J Label Comp Radiopharm. 2008;51:68–71. Thonon D, Goblet D, Goukens E, Kaisin G, Paris J, Aerts J, et al. Fully automated preparation and conjugation of N-Succinimidyl 4-[ 18F]fluorobenzoate ([ 18F]SFB) with RGD peptide using a GE FASTlab™ synthesizer. Mol Imaging Biol. 2011;13:1088–95. Davis RA, Drake C, Ippisch RC, Moore M, Sutcliffe JL. Fully automated peptide radiolabeling from [ 18 F] fluoride †. 2019. Gondry O, Xavier C, Raes L, Heemskerk J, Devoogdt N, Everaert H, et al. Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I). J Nucl Med. 2023;64:1378–84. Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem. 2005;338:284–93. Li J, Chen K, Liu H, Cheng K, Yang M, Zhang J, et al. An Activatable Near Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha. Bioconjug Chem. 2012;23:1704. Keane FM, Yao TW, Seelk S, Gall MG, Chowdhury S, Poplawski SE, et al. Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs. FEBS Open Bio. 2014;4:43–54. Supplementary Files supplementarydataV2.docx Cite Share Download PDF Status: Published Journal Publication published 23 Jul, 2024 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Editorial decision: Minor revision 30 Jun, 2024 Reviewers agreed at journal 12 Jun, 2024 Reviewers invited by journal 12 Jun, 2024 Editor assigned by journal 12 Jun, 2024 First submitted to journal 12 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4523820","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":313560016,"identity":"9c012f54-dde8-4d30-a9b3-06fb68c5ad66","order_by":0,"name":"Herlinde Dierick","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PMYvCMBTA8ReEdjlwrcv5FV4JRETpZ2ko2MXhtrvNHEJuqfhtpGNAuC75AN1sEdwdBcFLcyA4pN54Q/5TEvjxXgB8vn8YAhCVwsAcueiuOAQIgIheAo9kJP5CwJL09wHVEzIJN41qy9krhNnn8eMtobResoaUcyeZFhUqrnMKL+061pgxVi8nSPTCvVi9AMXlnouIy5FANWdas4jIvZscTpasDPm6dIQWltx6pgSWpGCmEEMYhkVHlJtou1geS/MXs1hGo0q+I9eZm1TfpL3I2XgY5u1ZXJN4ux7smnOZOMm94OGWPgc+n8/n6+kH0ztXxAHIxokAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8558-2569","institution":"VUB: Vrije Universiteit Brussel","correspondingAuthor":true,"prefix":"","firstName":"Herlinde","middleName":"","lastName":"Dierick","suffix":""},{"id":313560017,"identity":"19508695-b994-426d-8f1a-5ab37a3b2fac","order_by":1,"name":"Laurent Navarro","email":"","orcid":"","institution":"Precirix","correspondingAuthor":false,"prefix":"","firstName":"Laurent","middleName":"","lastName":"Navarro","suffix":""},{"id":313560018,"identity":"eba78087-e8fd-4f24-804c-f9ea7c99188d","order_by":2,"name":"Hannelore Ceuppens","email":"","orcid":"","institution":"VUB: Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Hannelore","middleName":"","lastName":"Ceuppens","suffix":""},{"id":313560019,"identity":"b70f5ad2-3f9e-4674-90a0-ad135d113455","order_by":3,"name":"Thomas Ertveldt","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Ertveldt","suffix":""},{"id":313560020,"identity":"3b26c958-7254-4cb7-9fab-a136bb542146","order_by":4,"name":"Ana Rita Pombo Antunes","email":"","orcid":"","institution":"precirix","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Rita Pombo","lastName":"Antunes","suffix":""},{"id":313560021,"identity":"1fefcd1f-67b4-42ea-bcd6-4d071a7e41df","order_by":5,"name":"Marleen Keyaerts","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Marleen","middleName":"","lastName":"Keyaerts","suffix":""},{"id":313560022,"identity":"c84ad77b-4329-4119-a6eb-4625330a5059","order_by":6,"name":"Nick Devoogdt","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Nick","middleName":"","lastName":"Devoogdt","suffix":""},{"id":313560023,"identity":"db598413-624c-4ac0-a5e2-a72b85160a50","order_by":7,"name":"Karine Breckpot","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Karine","middleName":"","lastName":"Breckpot","suffix":""},{"id":313560024,"identity":"7b2f0d65-4a84-46e9-8ad2-f58ad1872a66","order_by":8,"name":"Matthias D’Huyvetter","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"D’Huyvetter","suffix":""},{"id":313560025,"identity":"fb21442e-5337-45ce-a15d-4fee2e0120c3","order_by":9,"name":"Tony Lahoutte","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Tony","middleName":"","lastName":"Lahoutte","suffix":""},{"id":313560026,"identity":"3533542a-34dc-4c0d-ac64-a18166ebd17a","order_by":10,"name":"Vicky Caveliers","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Vicky","middleName":"","lastName":"Caveliers","suffix":""},{"id":313560027,"identity":"54f5ce55-8d4a-40e7-b6f2-867099ebb900","order_by":11,"name":"Jessica Bridoux","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Bridoux","suffix":""}],"badges":[],"createdAt":"2024-06-03 19:44:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4523820/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4523820/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-024-00286-8","type":"published","date":"2024-07-24T00:35:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59871369,"identity":"ff95f68e-08c5-4583-8aa6-d4ba251f4af2","added_by":"auto","created_at":"2024-07-08 17:05:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31261,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of [\u003csup\u003e18\u003c/sup\u003eF]FB-sdAb: a. Synthesis of [\u003csup\u003e18\u003c/sup\u003eF]SFB in a three-step, one-pot reaction; b. conjugation of [\u003csup\u003e18\u003c/sup\u003eF]SFB to sdAb. RT = room temperature\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/d41baff73476e19a7c2e89ba.png"},{"id":59872515,"identity":"aeaabb8a-9d22-47a1-930e-88b0cb4dd212","added_by":"auto","created_at":"2024-07-08 17:13:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1132429,"visible":true,"origin":"","legend":"\u003cp\u003eLayout of the automated radiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]SFB on a Trasis AiO. The 3-step one-pot procedure (upper row, rotors 1à17), as well as the purification of the PG (lower row and vial P14 and syringe P15), is included on the module.\u003c/p\u003e","description":"","filename":"Figure2Updatedtrasislayoutwithkeyparts.png","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/9549128855844a02f6373bba.png"},{"id":59871373,"identity":"0da0f9c7-4bd2-4ab9-a7f0-c2c718fe85aa","added_by":"auto","created_at":"2024-07-08 17:05:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1661148,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum intensity projection PET/CT imaging of (a) [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 hFR-α knock-in mouse bearing TC-1-hFR-α tumours and (b) [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 C57BL/6 mouse bearing TC-1-hFAP-α tumours 1h p.i..\u003c/p\u003e","description":"","filename":"Figure3PETCT.png","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/44b6d538ad77c2a2f680be14.png"},{"id":59871370,"identity":"4c0a346a-19da-45a3-8edc-6c88278bcbe9","added_by":"auto","created_at":"2024-07-08 17:05:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":346795,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo biodistribution results and T/B ratios of (i) [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 compared to [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 (A and B), 1h10 post injection; (ii) [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 compared to [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 (C and D) at 1h30 post injection. Two-way ANOVA or unpaired student t-test was used to calculate statistical significance. Statistical significance was set at p\u0026lt;0.05 (ns, not significant, * p\u0026lt;0.05; ** p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure4exvivos.png","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/12d9efa0c642b4058e67fb04.png"},{"id":61039322,"identity":"9bc016f3-7859-4783-bf97-a6fc7166faca","added_by":"auto","created_at":"2024-07-25 00:35:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3701036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/3a67c198-6263-42cc-9777-f4c4eb111e9b.pdf"},{"id":59871374,"identity":"56a805e2-47fd-4521-ad0a-5d6f170fe880","added_by":"auto","created_at":"2024-07-08 17:05:34","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":621040,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydataV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4523820/v1/a02dd1412f4fbf9bd841c731.docx"}],"financialInterests":"","formattedTitle":"Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of Fibroblast Activation Protein-α or Folate Receptor-α overexpression in cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eBoth the alpha isoforms of the Folate Receptor (FR) and Fibroblast Activation Protein (FAP) are established tumour markers. FR-α has an elevated expression in epithelial cancers, such as ovarian, cervical, and head and neck cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At the same time, this isoform has a minimal physiological role in healthy tissue (except during embryogenesis), making it an interesting anticancer target. FR-α also shows a high affinity for both physiological and non-physiological substrates, which further cements its relevance for diagnostic and theranostic purposes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eOnly one FR-α targeting therapy, Mirvetuximab, Soravtansine [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], has been approved for use in patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Other promising agents, such as Farletuzumab [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and Vintafolide [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], failed to meet their primary endpoints. A positron emission tomography (PET) tracer that specifically targets FR-α has the potential to be a companion diagnostic for FR-α targeting therapies and can help in patient stratification [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] While a large number of folate tracers, however not specific to FR-⍺, labelled with fluorine-18 (\u003csup\u003e18\u003c/sup\u003eF) have been developed over the last decades, to our knowledge, only two have made it to clinical trials, namely [\u003csup\u003e18\u003c/sup\u003eF]-AzaFol [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and [\u003csup\u003e18\u003c/sup\u003eF]fluoro-PEG-folate [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFAP is known to be overexpressed in breast, colorectal, ovarian, and other cancers, more specifically in the stroma. This indicates the presence of cancer-associated fibroblasts, while their expression in normal tissues is low or undetectable[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Due to this attractive expression pattern, anti-FAP radiopharmaceuticals have been a hot topic for diagnostic and therapeutic applications. Several anti-FAP small molecule-based, for example OncoFAP [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], FAPI-04 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], FAPI-46 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], FAPI-74 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and PNT6555 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and peptide-based radiopharmaceuticals, such as FAP-2286 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], have been developed in recent years and are currently being tested in the clinic [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent targeting moieties have been used to develop PET tracers for established tumour markers. Immune-derived vectors such as monoclonal antibodies (mAbs), minibodies, single-domain antibodies (sdAbs), allow to combine their highly specific targeting with the sensitivity and resolution of PET [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. SdAbs have gained quite some interest as targeting molecules for PET imaging. Their key characteristics, such as their small size (around 15 kDa), high affinity, high specificity, low off-target accumulation, high (thermo)stability and solubility [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] allowed them to be successfully translated to the clinic as diagnostic [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and therapeutic [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] radiopharmaceuticals. Compared to mAb-based diagnostics, their most notable advantages that their short biological half-life and fast tumour penetration allow for their labelling with short-lived radionuclides such as gallium-68 (\u003csup\u003e68\u003c/sup\u003eGa) and \u003csup\u003e18\u003c/sup\u003eF [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a diagnostic standpoint, \u003csup\u003e18\u003c/sup\u003eF is an ideal radionuclide for PET imaging with its high positron (β\u003csup\u003e+\u003c/sup\u003e) yield of 97%, relatively low energy (max 0.634 MeV) of the emitted β\u003csup\u003e+\u003c/sup\u003e and thus short trajectory (mean positron range in soft tissue: 0.27 mm) resulting in high-resolution images. Its half-life of 109.8 min is long enough to allow shipment of the radiopharmaceutical to other centres but still short enough to avoid unnecessary extended irradiation of the patients. The ease of producing large amounts with a cyclotron cements its place as the favourite radionuclide in PET imaging [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The direct \u003csup\u003e18\u003c/sup\u003eF-labelling of sdAbs and other biomolecules is prevented by the harsh reaction conditions, elevated temperatures, organic solvents, and high pH needed for radiofluorination. The development of prosthetic groups (PG) like \u003cem\u003eN\u003c/em\u003e-succinimidyl 4-[\u003csup\u003e18\u003c/sup\u003eF]Fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]SFB), [\u003csup\u003e18\u003c/sup\u003eF]Fluorobenzaldehyde ([\u003csup\u003e18\u003c/sup\u003eF]FBA) and \u003cem\u003eN\u003c/em\u003e-[2-(4-[\u003csup\u003e18\u003c/sup\u003eF]-Fluorobenzamido)ethyl]maleimide ([\u003csup\u003e18\u003c/sup\u003eF]FBEM), makes radiofluorination of proteins possible in aqueous medium under mild conditions. [\u003csup\u003e18\u003c/sup\u003eF]SFB is a popular PG thanks to its reactivity with lysins, amino acid group naturally present on the surface of proteins, including sdAbs. The resulting [\u003csup\u003e18\u003c/sup\u003eF]FB-bioconjugate has demonstrated good \u003cem\u003ein vivo\u003c/em\u003e stability [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDistribution and commercialization of highly specific PET radiofluorinated radiopharmaceuticals becomes possible, while the centralized production of \u003csup\u003e68\u003c/sup\u003eGa-labeled products is more difficult to organize. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThis study aims to develop a generic semi-automated radiofluorination strategy for sdAbs as a platform for the radiofluorination of two sdAb with high interest targets, namely FR-α and FAP-α. The production of the PG, [\u003csup\u003e18\u003c/sup\u003eF]SFB was optimized and automated on the AllInOne (AiO) module (Trasis), while the conjugation reaction to the sdAbs was achieved manually using an optimized protocol.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe cell lines used in this study were generated for this purpose. The methodologies for their generation, culture conditions and validation by flow cytometry (supplemental Fig.\u0026nbsp;1) can be found in the Supplementary Information (SI).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3.1. sdAbs\u003c/h2\u003e \u003cp\u003eAn anti-FAP-α sdAb, cross-reactive for mouse/human FAP-α and an anti-FR-α sdAb, reactive to human FR-α were kindly provided by Precirix. The anti-FAP-α sdAb (4AH29)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the FR-α sdAb (1012) and the non-targeting control sdAb (R3B23) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] were produced and characterized as previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. All sdAbs in this study were free of tags.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Radiochemistry\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Automated [\u003csup\u003e18\u003c/sup\u003eF]SFB synthesis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-succinimidyl-4-[\u003csup\u003e18\u003c/sup\u003eF] fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]SFB) was synthesized using a three-step, one-pot reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.). The complete production process of [\u003csup\u003e18\u003c/sup\u003eF]SFB, including the purification, was automated with an AiO module (Trasis) using disposable cassettes. [\u003csup\u003e18\u003c/sup\u003eF]F\u003csup\u003e\u0026minus;\u003c/sup\u003e was produced by irradiation of enriched [\u003csup\u003e18\u003c/sup\u003eO]water (Rotem medical and Campro) in Niobium targets with a Cyclone KIUBE cyclotron (IBA) via the \u003csup\u003e18\u003c/sup\u003eO(p,n)\u003csup\u003e18\u003c/sup\u003eF nuclear reaction. The [\u003csup\u003e18\u003c/sup\u003eF]fluoride aqueous solution was passed through a Sep-Pak Light Accell Plus QMA anion exchange cartridge (Waters) to trap [\u003csup\u003e18\u003c/sup\u003eF]fluoride and recover the enriched water. The [\u003csup\u003e18\u003c/sup\u003eF]fluoride was eluted from the cartridge with 600 \u0026micro;L of Cryptant Solution (4.2 mg of K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and 22.6 mg of Cryptand (K\u003csub\u003e222\u003c/sub\u003e) in acetonitrile/water (1:1)) (ABX). The solvent was evaporated to form anhydrous Kryptofix K\u003csub\u003e222\u003c/sub\u003e/K[\u003csup\u003e18\u003c/sup\u003eF]F complex (60\u0026ndash;70 GBq). A solution of 0.8 mg (0.002 mmol) of ethyl-4-(trimethylammonium)benzoate (ABX) in 2 mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to the dried [\u003csup\u003e18\u003c/sup\u003eF]F\u003csup\u003e\u0026minus;\u003c/sup\u003e complex in the reactor and heated to 110\u0026deg;C for 15 min to produce ethyl-4-[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoate. This compound was hydrolysed at 95\u0026deg;C for 5 min by a 0.38 M (0.76 mmol) tetrapropylammonium hydroxide (TPAOH) aqueous solution diluted in DMSO. The subsequent activation was performed with 26 mg (0.072 mmol) of \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026prime;,\u003cem\u003eN\u003c/em\u003e\u0026prime;-tetramethyl-O-(\u003cem\u003eN\u003c/em\u003e-succinimidyl)uronium hexafluorophosphate (HSTU, Sigma-Aldrich) in 1 mL of acetonitrile at 110\u0026deg;C for 5 min to form [\u003csup\u003e18\u003c/sup\u003eF]SFB. The reaction mixture (RM) was diluted with 12 mL of an acetic acid solution (1.7% acetic acid/ NaCl 0.6%) before trapping on an HLB prime Plus Light solid-phase extraction (SPE) cartridge (Waters). The cartridge was washed with 1 mL of aqueous EtOH solution (5%) and reverse eluted with 0.8 mL of EtOH (Emsure, VWR). The purity of the [\u003csup\u003e18\u003c/sup\u003eF]SFB was determined by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Detailed information on the chromatographic analysis can be found in the SI.\u003c/p\u003e \u003cp\u003eThe PG production described above and its automation was optimized based on the work of Xavier \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Detailed insights into the optimization procedures are available in the SI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Manual conjugation of [\u003csup\u003e18\u003c/sup\u003eF]SFB to sdAbs\u003c/h2\u003e \u003cp\u003eAt this point in the production of the tracers, the conjugation step was optimised and performed manually. The different sdAbs in phosphate buffered saline (PBS) pH 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are diluted with 0.5 M CHES buffer pH 8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 and PBS. This mixture is added to 200 \u0026micro;L of the ethanolic [\u003csup\u003e18\u003c/sup\u003eF]SFB (3\u0026ndash;5 GBq) and left to incubate for at least 15 minutes at room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb.). The radiolabelled sdAb was purified using two disposable desalting Hitraps (Cytiva) placed in series (pre-equilibrated with NaCl 0.9% with 5 mg/mL ascorbic acid, pH 5.9\u0026ndash;6.2) using a peristaltic pump (Ismatec Reglo ICC, Masterflex) with a flow rate of 5 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The final product was passed through a 0.22 \u0026micro;m filter (Millipore) and analysed by RP-HPLC and Size-Exclusion (SE) -HPLC (see SI). Detailed insights into the optimization procedures, starting from the work of Xavier \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] are available in the SI.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003emolecular weight and mass of sdAb used in conjugation reaction\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esdAb\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular weight (g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmount of sdAb\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4AH29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12350.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.1 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mol, 1000 \u0026micro;g, 100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13042.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.7 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mol, 1000 \u0026micro;g, 100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR3B23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13913.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mol, 1000 \u0026micro;g, 100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Animal models\u003c/h2\u003e \u003cp\u003ehFR-α knock-in female mice (kindly provided by Precirix) and wildtype C57BL/6 female mice (Charles River) were used to evaluate biodistribution and tumour uptake of [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 and [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 respectively. They were subcutaneously inoculated at the tail base, under the control of 2.5% isoflurane in oxygen (Abbott), with TC-1-hFR-α cells (5 x 10\u003csup\u003e4\u003c/sup\u003e) suspended in PBS in the case of hFR-α knock-in mice and with TC-1-hFAP-α cells (5 x 10\u003csup\u003e4\u003c/sup\u003e) suspended in PBS in the case of the wildtype C57BL/6 mice. The tumours were allowed to grow for up to two weeks (100\u0026ndash;300 mm\u003csup\u003e3\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Biodistribution \u0026amp; PET/CT imaging\u003c/h2\u003e \u003cp\u003ehFR-α knock-in female mice bearing TC-1-hFR-α tumours (n\u0026thinsp;=\u0026thinsp;4 per group) were i.v. injected (25 \u0026micro;g; 15 MBq) with [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 or [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23. Wildtype C57BL/6 female mice bearing TC-1-hFAP-α tumours were i.v. injected (25 \u0026micro;g; 15 MBq) with [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 (n\u0026thinsp;=\u0026thinsp;4) or [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 (n\u0026thinsp;=\u0026thinsp;3). One hour after injection, micro-PET/CT images were acquired (detailed information in SI), followed by dissections 1h10 or 1h30 post injection in mice bearing TC-1-hFR-α tumours and mice bearing TC-1-hFAP-α tumours, respectively. The timepoint discrepancies are due to differences in the preclinical study design of both tracers. Animals were dissected, and organ and tissue activities were counted against a standard of known activity with an automated gamma counter (Wizard 2 2480, PerkinElmer) and expressed as a percentage of injected activity per gram (%IA/g), corrected for decay. \u003cem\u003eIn vitro\u003c/em\u003e characterization of the tracers, affinity measurement by cell saturation assay (supplemental Fig.\u0026nbsp;2) and \u003cem\u003ein vitro\u003c/em\u003e stability in plasma (supplemental table 3), can be found in the SI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical analysis used GraphPad Prism 10. One-way ANOVA, two-way ANOVA with multiple comparison tests, or unpaired t-test were used to evaluate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Radiolabelling\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SFB was synthesized using a three-step, one-pot reaction, which was fully automated. The total time of the procedure was 54 minutes and allowed to obtain [\u003csup\u003e18\u003c/sup\u003eF]SFB (23.31\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28 GBq, n\u0026thinsp;=\u0026thinsp;13) with a RCP\u0026thinsp;\u0026gt;\u0026thinsp;90% and a radiochemical yield (RCY) decay corrected (d.c.) of 44\u0026thinsp;\u0026plusmn;\u0026thinsp;4% (n\u0026thinsp;=\u0026thinsp;13).\u003c/p\u003e \u003cp\u003eA schematic representation of the automated radiosynthesis procedure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The [\u003csup\u003e18\u003c/sup\u003eF]F\u003csup\u003e\u0026minus;\u003c/sup\u003e enters the module via the syringe in the 6th position (P6) in the layout. The cyclotron-produced [\u003csup\u003e18\u003c/sup\u003eF]F\u003csup\u003e\u0026minus;\u003c/sup\u003e is separated from the \u003csup\u003e18\u003c/sup\u003eO-enriched water by the QMA cartridge on P5. Then, [\u003csup\u003e18\u003c/sup\u003eF]F is eluted with the Cryptand solution (P2), with the help of a syringe located in P3. The mixture is transferred to the 6 mL reactor (P7), after which the azeotropic drying of the [\u003csup\u003e18\u003c/sup\u003eF]fluoride is started. To the dried [\u003csup\u003e18\u003c/sup\u003eF]K\u003csub\u003e222\u003c/sub\u003e-fluoride, 0.8 mg of FB-precursor, dissolved in DMSO (P8), is added. The reactor is heated to 110\u0026ordm;C for 15 minutes to obtain the ethyl-4-[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoate and cooled down afterwards. Next, the product is hydrolysed by adding the 0.38M TPAOH DMSO solution (vial P10) to the reactor. The reactor is heated to 95\u0026ordm;C for 5 minutes to obtain the 4-[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoic acid and cooled down again. For the third and last step, 26 mg of HSTU dissolved in anhydrous acetonitrile (P11) is transferred to the reactor. The reactor is heated to 110\u0026ordm;C for 5 minutes, obtaining the crude [\u003csup\u003e18\u003c/sup\u003eF]SFB, and cooled down again. The RM inside the reactor is diluted with a mixture of 4 mL of 4.8% acetic acid solution (P17) and 8 mL of 0.9% NaCl (P13), prepared by the module by mixing both components within the 20 mL syringe (P9) in the layout. The same syringe applies the RM to the HLB light cartridge (P33). Next, the cartridge and lines are rinsed with 5% EtOH/water solution (P35). To complete the purification, the final product is reverse eluted with EtOH (vial P14), using the 3 mL syringe (P15) and collected in a final vial.\u003c/p\u003e \u003cp\u003eThe manual conjugation reaction produced [\u003csup\u003e18\u003c/sup\u003eF]FB-sdAbs with a RCY of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;4% (n\u0026thinsp;=\u0026thinsp;2), 19\u0026thinsp;\u0026plusmn;\u0026thinsp;7% (n\u0026thinsp;=\u0026thinsp;3) and 19\u0026thinsp;\u0026plusmn;\u0026thinsp;1% (n\u0026thinsp;=\u0026thinsp;2) d.c. starting from the added [\u003csup\u003e18\u003c/sup\u003eF]SFB for [\u003csup\u003e18\u003c/sup\u003eF]FB-1012, [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 and [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 respectively. The purified [\u003csup\u003e18\u003c/sup\u003eF]FB-sdAbs were obtained with a RCP\u0026thinsp;\u0026gt;\u0026thinsp;95%, and the end of synthesis activity amounted to 783\u0026thinsp;\u0026plusmn;\u0026thinsp;8.50 MBq (n\u0026thinsp;=\u0026thinsp;2) for [\u003csup\u003e18\u003c/sup\u003eF]FB-1012, 694\u0026thinsp;\u0026plusmn;\u0026thinsp;80 MBq (n\u0026thinsp;=\u0026thinsp;2) [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29, and 907\u0026thinsp;\u0026plusmn;\u0026thinsp;227 MBq (n\u0026thinsp;=\u0026thinsp;2) for [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23. The apparent molar activity was 12.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 GBq/\u0026micro;mol (n\u0026thinsp;=\u0026thinsp;2), 10.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28 GBq/\u0026micro;mol (n\u0026thinsp;=\u0026thinsp;2), and 15.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90 GBq/\u0026micro;mol (n\u0026thinsp;=\u0026thinsp;2) respectively. Overall RCY d.c. were 9% (n\u0026thinsp;=\u0026thinsp;1), 5\u0026thinsp;\u0026plusmn;\u0026thinsp;2% (n\u0026thinsp;=\u0026thinsp;3) and 8\u0026thinsp;\u0026plusmn;\u0026thinsp;1% (n\u0026thinsp;=\u0026thinsp;2) for [\u003csup\u003e18\u003c/sup\u003eF]FB-1012, [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 and [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Biodistribution studies and PET/CT imaging\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ehFR-α knock-in female mice bearing TC-1-hFR-α tumours (n\u0026thinsp;=\u0026thinsp;4 per group) were i.v. injected with [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;g; 14.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 MBq, 6.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 GBq/\u0026micro;mol) or [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 (non-targeting control sdAb conjugate) (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;g; 16.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 MBq, 8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 GBq/\u0026micro;mol). Wildtype C57BL/6 female mice bearing TC-1-hFAP-α tumours were i.v. injected with [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 (26\u0026thinsp;\u0026plusmn;\u0026thinsp;3 \u0026micro;g; 14.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 MBq, 7.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 GBq/\u0026micro;mol, n\u0026thinsp;=\u0026thinsp;4) or [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23 (non-targeting control sdAb conjugate) (20\u0026thinsp;\u0026plusmn;\u0026thinsp;0 \u0026micro;g; 12.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 MBq, 8.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1 .20 GBq/\u0026micro;mol, n\u0026thinsp;=\u0026thinsp;3). Injected and apparent molar-specific activities are reported at the time of injection.\u003c/p\u003e \u003cp\u003eTumour uptake of [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 was visible on the PET image (1h p.i., Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). It was confirmed by quantification of dissection data (1h10 p.i.) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), showing statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) higher tumour uptake (8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 IA/g) for the FR-targeting sdAb compared to the non-targeting sdAb (0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 IA/g). Furthermore, the dissection studies evaluating [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 displayed about 2-fold higher kidney accumulation (25.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61 vs 14.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 3-fold higher accumulation in the ovaries (1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 vs 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 3-fold higher accumulation in the brain (0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to the non-targeting sdAb.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e profile of the anti-FAP-α sdAb, [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29, was investigated in TC-1-hFAP-α tumour bearing mice by a similar protocol, including micro-PET/CT imaging at 1h p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and dissection analysis at 1.5h p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), and compared to [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23. \u003cem\u003eEx vivo\u003c/em\u003e biodistribution studies indicated specific tumour uptake (2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 IA/g) compared to the non-targeting sdAb (0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 IA/g), no unspecific organ accumulation except in the joints (1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 vs 0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), pancreas (0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), skin (1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 vs 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), blood (0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 vs 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and uterus (1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 vs 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 IA/g; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the non-targeting sdAb. For both tracers, fast excretion of the unbound tracer was observed via the kidneys ([\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29: 9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25% IA/g; [\u003csup\u003e18\u003c/sup\u003eF]FB-R3B23: 6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34% IA/g).\u003c/p\u003e \u003cp\u003eThe tumour-to-blood (T/B) ratios were calculated for both tracers. T/B ratios for [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 and [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 were significantly higher compared to their respective control sdAb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026amp;d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe radiofluorination strategy of sdAbs described herein uses the well-established PG [\u003csup\u003e18\u003c/sup\u003eF]SFB. This PG is widely used for labelling peptides and proteins and its radio-synthesis has been continuously refined and optimized. In this study, the three-step, one-pot reaction was automated on a Trasis AiO. Automation of the PG production has been successfully implemented on in-house developed automation synthesis equipment[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and commercial automated synthesis modules such as the IBA Syntera module[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], TRACERlab FX\u003csub\u003eFN\u003c/sub\u003e synthesizer[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] (GE Healthcare) and the Ora-Neptis synthesizer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We first optimized the automated production process by five times reducing the mass of the commercially available precursor[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] without negatively impacting the RCY of the reaction (see SI, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We hypothesized that this reduction would also reduce the formation of potential process-related impurities and help increase specific activity. A second optimization was the purification of the PG. In the literature, different strategies can be found, such as HPLC methods, SPE using one single cartridge [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], multiple cartridges in series[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] or strategies combining both HPLC and SPE[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The automated synthesis procedure described in this study uses a single SPE cartridge for purification, reducing time spent on purification compared to HPLC purification strategies. By opting for reverse elution of the cartridge, it was possible to reduce the elution volume to 800 \u0026micro;L. When comparing the SPE strategy used here to the other SPE strategies in literature[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], the final formulation of the PG in a small volume (0.8 mL) of ethanol, avoiding a reformulation step or time-consuming evaporation step before starting the subsequent conjugation reaction, is a significant advantage to reduce the time of the whole production process. The conjugation reaction described in this study was optimized with sdAbs in mind and included a 20% V/V content of ethanol. This ethanol concentration showed no negative impact on the conjugation reaction (see SI table 2) and is in line with the results of several studies[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] that showed denaturation of proteins caused by alcohols occurs at concentrations above 20%. The change of final solvent to ethanol was facilitated by replacing the previously used tC18[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] with an HLB cartridge. A slight reduction in RCP, \u0026gt;\u0026thinsp;90% compared to the previously reported[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u0026thinsp;\u0026gt;\u0026thinsp;95%, could be observed, with [\u003csup\u003e18\u003c/sup\u003eF]FBA as the identified radioactive impurity. Most likely, this reduction in RCP is caused by a combination of radiolysis, increasing amount of radioactive impurity with increased volumetric activity concentration (up to more than 25 GBq/mL), and hydrolysis, as the impurity increases over time as well. However, as the impurity does not compete with the PG in the following conjugation reaction, the slight decrease in RCP was deemed insignificant.\u003c/p\u003e \u003cp\u003eFor optimization of the conjugation reaction, we opted for CHES as a coupling buffer due to the superior stability of the PG in this buffer compared to the conventional borate buffer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Nagachinta \u003cem\u003eet al\u003c/em\u003e.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] performed the coupling of sdAbs to the PG using a phosphate buffer at pH 8.4, we prefer the use of CHES as its buffering range (pH 8.6 to 10 compared to 5.8 to 7.4 for a phosphate buffer) is more in range with the optimal reactivity of the sdAbs\u0026rsquo; amino groups towards acylation (pH\u0026thinsp;\u0026lt;\u0026thinsp;8.5). The higher buffer capacity and, thus fewer fluctuations in pH of CHES compared to phosphate also allows for a more robust coupling reaction. Detailed insights into the buffer selection are available in the SI, supplemental Fig.\u0026nbsp;3. The purification of the radiolabelled sdAbs was performed using SE resins HiTrap desalting cartridges instead of the PD-10 desalting column, with the latter being the most described option in literature [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The conjugation of the sdAbs to the PG resulted in reasonable decay-corrected conjugation yields (20\u0026ndash;25%, starting from [\u003csup\u003e18\u003c/sup\u003eF]SFB) with high RCP and reasonable apparent molar activity. The conjugation yield was comparable to or higher than others reported for sdAbs and proteins [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The comparable conjugation results (similar RCY d.c., apparent molar activities. and final activities) for all three sdAbs show that this strategy could also be used as a generic radiolabelling strategy for sdAbs, similar to the generic \u003csup\u003e68\u003c/sup\u003eGa-chelator approach currently used [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This generic \u003csup\u003e68\u003c/sup\u003eGa-chelator approach has already been successfully used to introduce sdAb-based tracers in the clinic, as shown by the clinical translation of sdAbs targeting HER2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and CD206 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The advantages of this method compared to radiofluorination are the ease of its chemistry, higher RCYs and its lower initial financial investment, as there is no need for a cyclotron or automation modules. On the other hand, by developing a radiolabelling method with \u003csup\u003e18\u003c/sup\u003eF for sdAbs, we can take advantage of the superior imaging quality of \u003csup\u003e18\u003c/sup\u003eF. At the same time, its longer half-life allows for easier radiopharmaceutical distribution and still matches the biological half-life of sdAbs. Because of the ease of production of high amounts of the radionuclide with a cyclotron, upscaling the obtained activity will allow for multi-patient preparations produced in PET radiopharmacies or centralized production sites.\u003c/p\u003e \u003cp\u003eThe biodistribution and imaging studies for both tracers showed excellent targeting properties and specificity for FR-α or FAP-α, fast excretion via the kidneys of both [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 and [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29, respectively. The known FR-α expression in the fallopian tubes, proximal tubule cells of the kidneys, and choroid plexus in the brain, might explain the observed elevated uptake in these organs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBesides specific uptake of [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29 in the tumour, elevated accumulation was seen in pancreas, skin and uterus. This is in line with previous findings [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] in mice, showing an interspecies difference in FAP expression compared to humans. The elevated uptake in blood and joints could be attributed to the increased shedding of FAP protein in mice [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUsing a Trasis AiO, [\u003csup\u003e18\u003c/sup\u003eF]SFB synthesis was successfully automated and upscaled, yielding consistently around 20 GBq of pure product. The anti-hFAP-α, anti-hFR-α and non-targeting control sdAbs were successfully radiofluorinated, yielding similar DC-RCYs and RCPs. The herein presented semi-automated radiofluorination approach could be used as a generic radiofluorination method for sdAbs, allowing for faster preclinical validation of sdAbs as PET tracers and opens opportunities for further development towards clinical production. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]Fluorobenzaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]FBEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-[2-(4-[\u003csup\u003e18\u003c/sup\u003eF]-Fluorobenzamido)ethyl]maleimide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SFB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-succinimidyl 4-[\u003csup\u003e18\u003c/sup\u003eF]Fluorobenzoate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003e\u003csup\u003e18\u003c/sup\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003efluorine-18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003egallium-68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eAiO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eAllInOne\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eCHES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003e2-(Cyclohexylamino)ethane-1-sulfonic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003ed.c.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eDecay corrected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003edimethyl sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eEtOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eFAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003efibroblast activation protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eFR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eFolate receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eHSTU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eN,N,N\u0026prime;,N\u0026prime;-\u003c/em\u003etetramethyl-\u003cem\u003eO\u003c/em\u003e-(\u003cem\u003eN-\u003c/em\u003esuccinimidyl)uronium hexafluorophosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003emAbs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003emonoclonal antibodies\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003ephosphate buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003ePG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eProstethic group\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eRCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eRadiochemical purity\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eRCY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eRadiochemical yield\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eRM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003ereaction mixture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eRP-HPLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eReverse Phase\u0026nbsp;High Performance Liquid Chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003esdAbs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003esingle-domain antibodies\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eSize-Exclusion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003eSupplementary Information\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eSPE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003esolid-phase extraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eT/B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003etumour-to-blood\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.218543046357617%\" valign=\"top\"\u003e\n \u003cp\u003eTPAOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"82.78145695364239%\" valign=\"top\"\u003e\n \u003cp\u003etetrapropylammonium hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThe ethical committee for animal experiments at the Vrije Universiteit Brussel approved the \u003cem\u003ein vivo\u003c/em\u003e study protocols (22-272-12 \u0026amp; 19-272-17). All mouse experiments were executed in accordance with the European guidelines for animal experimentation. Written informed consent was not required for this study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eK.B., N.D., M.D., M.K., T.L and J.B. have patents on using sdAbs for imaging and therapy. M.K is an editor in EJNMMI. J.B is an unpaid board member of eSRR. T.L., N.D. and M.K. have ownership in AbScint. M.K. received research funding from Precirix. N.D. and M.D are resp. consultant and employee for and hold ownership in Precirix. L.N. and A.R.P.A. are employees of Precirix.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was performed with the financial support of Strategic Research Programs (SRP50, SRP95 and SRP62) and the Industrial Research Fund (IOF3018 and IOF3009) of the VUB Research Council and is part of the joint R\u0026amp;D project IMPACT, financially supported by Innoviris and Precirix (2020-RDIR-1). T.E., J.B., M.K. and M.D. were, respectively, pre-doctoral researcher (1S06622N), postdoctoral fellow (1230824N), senior clinical investigator (1801619N) and postdoctoral fellow (12H3619N) of the Research Foundation Flanders (FWO-V) during the execution of this work. This research was partly performed at the Virus Production Unit, Molecular Biology Facility, and In vivo Cellular and Molecular Imaging Core facility, core facilities financially supported by the University Medical Center Onderzoeksraad. The BD Celesta flow cytometer and Molecubes \u0026beta;-CUBE PET/CT system were funded via FWO-Hercules grants (I001618N and I005622N).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eM.K, N.D, K.B., M.D. and T.L contributed to the study conception. J.B, V.C., A.R.P.A, M.D. N.D. and K.B. contributed to the study design. Material preparation, data collection and analysis were performed by H.D., L.N., H.C., T.E. and\u0026nbsp;A.R.P.A.\u0026nbsp;The first draft of the manuscript was written by H.D. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Elsy Vaeremans and Petra Roman for technical support during the cloning of the lentiviral transfer plasmids and generation of lentiviral particles, Kevin De Jonghe, Melissa Lucero, and Maxime Deladri\u0026egrave;re for handling the animals and performing the PET/CT-imaging. We thank Yana Dekempeneer for enabling the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSega EI, Low PS. Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Rev. 2008;27:655\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScaranti M, Cojocaru E, Banerjee S, Banerji U. Exploiting the folate receptor α in oncology. Nat Rev Clin Oncol Nat Res; 2020. p. 349\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoss SD, Ametamey SM. Development of Folate Receptor\u0026thinsp;\u0026ndash;\u0026thinsp;Targeted PET Radiopharmaceuticals for Tumor Imaging\u0026mdash;A Bench-to-Bedside Journey. Cancers 2020, Vol 12, Page 1508. 2020;12:1508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoore KN, Angelergues A, Konecny GE, Garc\u0026iacute;a Y, Banerjee S, Lorusso D, et al. Mirvetuximab Soravtansine in FRα-Positive, Platinum-Resistant Ovarian Cancer. N Engl J Med. 2023;389:2162\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarada H, Gonzalez T, Muminovic M, Nano O, Vulfovich M. Folate Receptor Alpha\u0026mdash;A Novel Approach to Cancer Therapy. Int J Mol Sci 2024. 2024;25:25:1046.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerzog TJ, Pignata S, Ghamande SA, Rubio MJ, Fujiwara K, Vulsteke C, et al. Randomized phase II trial of farletuzumab plus chemotherapy versus placebo plus chemotherapy in low CA-125 platinum-sensitive ovarian cancer. Gynecol Oncol. 2023;170:300\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVynfinit | European Medicines Agency [Internet]. [cited 2024 Jan 29]. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ema.europa.eu/en/medicines/human/EPAR/vynfinit\u003c/span\u003e\u003cspan address=\"https://www.ema.europa.eu/en/medicines/human/EPAR/vynfinit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzik P, Fang HY, Deberle LM, Benešov\u0026aacute; M, Cohrs S, Boss SD, et al. Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy. J Nucl Med. 2021;62:1475\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGnesin S, M\u0026uuml;ller J, Burger IA, Meisel A, Siano M, Fr\u0026uuml;h M et al. Radiation dosimetry of 18F-AzaFol: A first in-human use of a folate receptor PET tracer. EJNMMI Res. 2020;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerweij NJF, Yaqub M, Bruijnen STG, Pieplenbosch S, ter Wee MM, Jansen G et al. First in man study of [18F]fluoro-PEG-folate PET: a novel macrophage imaging technique to visualize rheumatoid arthritis. Sci Rep. 2020;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzgerald AA, Weiner LM. The role of fibroblast activation protein in health and malignancy. Cancer Metastasis Rev. 2020;39:783\u0026ndash;803.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBackhaus P, Gierse F, Burg MC, B\u0026uuml;ther F, Asmus I, Dorten P, et al. Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA. Eur J Nucl Med Mol Imaging. 2022;49:1822\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Zhou X, Xu X, Ding J, Liu T, Jiang J et al. Dynamic PET/CT Imaging of 68Ga-FAPI-04 in Chinese Subjects. Front Oncol. 2021;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Zhu W, Ren S, Kong Y, Huang Q, Zhao J, et al. 68Ga-FAPI-04 Versus 18F-FDG PET/CT in the Detection of Hepatocellular Carcinoma. Front Oncol. 2021;11:693640.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer C, Dahlbom M, Lindner T, Vauclin S, Mona C, Slavik R, et al. Radiation Dosimetry and Biodistribution of 68Ga-FAPI-46 PET Imaging in Cancer Patients. J Nucl Med. 2020;61:1171\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiesel FL, Adeberg S, Syed M, Lindner T, Jim\u0026eacute;nez-Franco LD, Mavriopoulou E, et al. FAPI-74 PET/CT Using Either 18F-AlF or Cold-Kit 68Ga Labeling: Biodistribution, Radiation Dosimetry, and Tumor Delineation in Lung Cancer Patients. J Nucl Med. 2021;62:201\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoplawski SE, Hallett RM, Dornan MH, Novakowski KE, Pan S, Belanger AP et al. Preclinical Development of PNT6555, a Boronic Acid\u0026ndash;Based, Fibroblast Activation Protein-α (FAP)\u0026ndash;Targeted Radiotheranostic for Imaging and Treatment of FAP-Positive Tumors. Journal of Nuclear Medicine. 2023;00:jnumed.123.266345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging [Internet]. 2022 [cited 2024 Apr 29];49:3651\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/35608703/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/35608703/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMillul J, Bassi G, Mock J, Elsayed A, Pellegrino C, Zana A, et al. An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications. Proc Natl Acad Sci U S A. 2021;118:e2101852118\u0026ndash;2101852118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L, Niu B, Fang J, Pang Y, Li S, Xie C et al. Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of 68Ga-Labeled FAPI Dimer. J Nucl Med. 2022;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E, et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 2022;49:3651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyohara J, Al-Qahtani M, Huang YY, Cazzola E, Todde S, Furumoto S, et al. Highlight selection of radiochemistry and radiopharmacy developments by editorial board. EJNMMI Radiopharm Chem. Springer Science and Business Media Deutschland GmbH; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei W, Rosenkrans ZT, Liu J, Huang G, Luo QY, Cai W, ImmunoPET. Concept, Design, and Applications [Internet]. Chem Rev. 2020 [cited 2020 Sep 7]. pp. 3787\u0026ndash;851. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dx.doi.org/10.1021/acs.chemrev.9b00738\u003c/span\u003e\u003cspan address=\"10.1021/acs.chemrev.9b00738\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Pauw T, De Mey L, Debacker JM, Raes G, Van Ginderachter JA, De Groof TWM, et al. Current status and future expectations of nanobodies in oncology trials. Expert Opin Investig Drugs. 2023;32:705\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeyaerts M, Xavier C, Everaert H, Vaneycken I, Fontaine C, Decoster L, et al. Phase II trial of HER2-PET/CT using 68Ga-anti-HER2 VHH1 for characterization of HER2 presence in brain metastases of breast cancer patients. Ann Oncol. 2019;30:iii25\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeyaerts M, Xavier C, Heemskerk J, Devoogdt N, Everaert H, Ackaert C, et al. Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma. J Nucl Med. 2016;57:27\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Huyvetter M, Vos J, De, Caveliers V, Vaneycken I, Heemskerk J, Duhoux FP, et al. Phase I Trial of 131I-GMIB-Anti-HER2-VHH1, a New Promising Candidate for HER2-Targeted Radionuclide Therapy in Breast Cancer Patients. J Nucl Med. 2021;62:1097\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCleeren F, Lecina J, Bridoux J, Devoogdt N, Tshibangu T, Xavier C et al. Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al 18 F-RESCA method. Nat Protoc [Internet]. 2018 [cited 2020 Sep 8];13:2330\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41596-018-0040-7\u003c/span\u003e\u003cspan address=\"10.1038/s41596-018-0040-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HK, Javed MR, Chen S, Zettlitz KA, Collins J, Wu AM, et al. On-demand radiosynthesis of: N -succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) on an electrowetting-on-dielectric microfluidic chip for 18F-labeling of protein. RSC Adv. 2019;9:32175\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacobson O, Kiesewetter DO, Chen X. Fluorine-18 radiochemistry, labeling strategies and synthetic routes. Bioconjug Chem. American Chemical Society; 2015. pp. 1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F. Nat Protoc [Internet]. 2006 [cited 2020 Sep 8];1:1655\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nature.com/articles/nprot.2006.264\u003c/span\u003e\u003cspan address=\"https://www.nature.com/articles/nprot.2006.264\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDekempeneer Y, Massa S, Santens F, Navarro L, Berdal M, Lucero MM, et al. Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α. J Nucl Med. 2023;64:1941\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLemaire M, D\u0026rsquo;Huyvetter M, Lahoutte T, Van Valckenborgh E, Menu E, De Bruyne E, et al. Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic Nanobodies. Leukemia. 2014;28:444\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res. 2012;110:927\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXavier C, Blykers A, Vaneycken I, D\u0026rsquo;Huyvetter M, Heemskerk J, Lahoutte T et al. 18F-nanobody for PET imaging of HER2 overexpressing tumors. Nucl Med Biol [Internet]. 2016 [cited 2020 Sep 7];43:247\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.nucmedbio.2016.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.nucmedbio.2016.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujimoto H, Fujita N, Hamamatsu K, Murakami T, Nakamoto Y, Saga T, et al. First-in-Human Evaluation of Positron Emission Tomography/Computed Tomography With [18F]FB(ePEG12)12-Exendin-4: A Phase 1 Clinical Study Targeting GLP-1 Receptor Expression Cells in Pancreas. Front Endocrinol (Lausanne). 2021;12:717101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAckermann U, Yeoh SD, Sachinidis JI, Poniger SS, Scott AM, Tochon-Danguy HJ. A simplified protocol for the automated production of succinimidyl 4-[ 18F]fluorobenzoate on an IBA Synthera module. J Label Comp Radiopharm. 2011;54:671\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlykers A, Schoonooghe S, Xavier C, D\u0026rsquo;Hoe K, Laoui D, D\u0026rsquo;Huyvetter M, et al. PET imaging of macrophage mannose receptor-expressing macrophages in tumor stroma using 18F-radiolabeled camelid single-domain antibody fragments. J Nucl Med. 2015;56:1265\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott PJH, Shao X. Fully automated, high yielding production of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), and its use in microwave-enhanced radiochemical coupling reactions. J Label Comp Radiopharm. 2010;53:586\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang G, Tang X, Wang X. A facile automated synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) for 18F-labeled cell-penetrating peptide as PET tracer. J Label Comp Radiopharm. 2010;53:543\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagachinta S, Novelli P, Joyard Y, Maindron N, Riss P, Dammicco S. Fully automated 18F-fluorination of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for indirect labelling of nanobodies. Sci Rep 2022. 2022;12(1):12:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBala G, Blykers A, Xavier C, Descamps B, Broisat A, Ghezzi C, et al. Targeting of vascular cell adhesion molecule-1 by 18F-labelled nanobodies for PET/CT imaging of inflamed atherosclerotic plaques. Eur Heart J Cardiovasc Imaging. 2016;17:1001\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXavier C, Blykers A, Laoui D, Bolli E, Vaneyken I, Bridoux J, et al. Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages. Mol Imaging Biol. 2019;21:898\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikolaidis A, Moschakis T. On the reversibility of ethanol-induced whey protein denaturation. Food Hydrocoll. 2018;84:389\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikolaidis A, Andreadis M, Moschakis T. Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra. Food Chem. 2017;232:425\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang G, Zeng W, Yu M, Kabalka G. Facile synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([ 18F]SFB) for protein labeling. J Label Comp Radiopharm. 2008;51:68\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThonon D, Goblet D, Goukens E, Kaisin G, Paris J, Aerts J, et al. Fully automated preparation and conjugation of N-Succinimidyl 4-[ 18F]fluorobenzoate ([ 18F]SFB) with RGD peptide using a GE FASTlab\u0026trade; synthesizer. Mol Imaging Biol. 2011;13:1088\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis RA, Drake C, Ippisch RC, Moore M, Sutcliffe JL. Fully automated peptide radiolabeling from [ 18 F] fluoride \u0026dagger;. 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGondry O, Xavier C, Raes L, Heemskerk J, Devoogdt N, Everaert H, et al. Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I). J Nucl Med. 2023;64:1378\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem. 2005;338:284\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Chen K, Liu H, Cheng K, Yang M, Zhang J, et al. An Activatable Near Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha. Bioconjug Chem. 2012;23:1704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeane FM, Yao TW, Seelk S, Gall MG, Chowdhury S, Poplawski SE, et al. Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs. FEBS Open Bio. 2014;4:43\u0026ndash;54.\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":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fluorine-18, Single domain Antibodies, automation, biomolecules","lastPublishedDoi":"10.21203/rs.3.rs-4523820/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4523820/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRadiofluorination of single domain antibodies (sdAbs) via \u003cem\u003eN\u003c/em\u003e-succinimidyl-4-[\u003csup\u003e18\u003c/sup\u003eF]fluorobenzoate ([\u003csup\u003e18\u003c/sup\u003eF]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (1012). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, can provide a detailed picture of the characteristics of both the tumour and its environment, which is critical for the success of cancer treatments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 minutes and results in [\u003csup\u003e18\u003c/sup\u003eF]SFB with a RCP\u0026thinsp;\u0026gt;\u0026thinsp;90% and a RCY d.c. of 44\u0026thinsp;\u0026plusmn;\u0026thinsp;4% (n\u0026thinsp;=\u0026thinsp;13). The conjugation reaction after purification produced [\u003csup\u003e18\u003c/sup\u003eF]FB-sdAbs with a RCP\u0026thinsp;\u0026gt;\u0026thinsp;95%, an end of synthesis activity\u0026thinsp;\u0026gt;\u0026thinsp;600 MBq and an apparent molar activity\u0026thinsp;\u0026gt;\u0026thinsp;10 GBq/\u0026micro;mol. Overall RCY d.c. were 9% and 5\u0026thinsp;\u0026plusmn;\u0026thinsp;2% (n\u0026thinsp;=\u0026thinsp;3) for [\u003csup\u003e18\u003c/sup\u003eF]FB-1012 and [\u003csup\u003e18\u003c/sup\u003eF]FB-4AH29, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.\u003c/p\u003e","manuscriptTitle":"Generic semi-automated radiofluorination strategy for single domain antibodies: [18F]FB-labelled single domain antibodies for PET imaging of Fibroblast Activation Protein-α or Folate Receptor-α overexpression in cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 17:05:29","doi":"10.21203/rs.3.rs-4523820/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2024-07-01T02:38:42+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-12T13:19:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-12T10:54:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-12T09:08:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2024-06-12T04:46:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8dfcf95b-1dc7-43d7-b00c-94e97e069156","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-25T00:35:36+00:00","versionOfRecord":{"articleIdentity":"rs-4523820","link":"https://doi.org/10.1186/s41181-024-00286-8","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2024-07-24 00:35:36","publishedOnDateReadable":"July 24th, 2024"},"versionCreatedAt":"2024-07-08 17:05:29","video":"","vorDoi":"10.1186/s41181-024-00286-8","vorDoiUrl":"https://doi.org/10.1186/s41181-024-00286-8","workflowStages":[]},"version":"v1","identity":"rs-4523820","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4523820","identity":"rs-4523820","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}