Automated Radiosynthesis, Preliminary In Vitro/In Vivo Characterization of OncoFAP-Based Radiopharmaceuticals for Cancer Imaging and Therapy

FAP-targeted radiopharmaceuticals represent a breakthrough in cancer imaging and a viable option for therapeutic applications. OncoFAP is an ultra-high-affinity ligand of FAP with a dissociation constant of 680 pM. OncoFAP has been recently discovered and clinically validated for PET imaging procedures in patients with solid malignancies. While more and more clinical validation is becoming available, the need for scalable and robust procedures for the preparation of this new class of radiopharmaceuticals continues to increase. In this article, we present the development of automated radiolabeling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy. A new series of [68Ga]Ga-OncoFAP, [177Lu]Lu-OncoFAP and [18F]AlF-OncoFAP was produced with high radiochemical yields. Chemical and biochemical characterization after radiolabeling confirmed its excellent stability, retention of high affinity for FAP and absence of radiolysis by-products. The in vivo biodistribution of [18F]AlF-NOTA-OncoFAP, a candidate for PET imaging procedures in patients, was assessed in mice bearing FAP-positive solid tumors. The product showed rapid accumulation in solid tumors, with an average of 6.6% ID/g one hour after systemic administration and excellent tumor-to-healthy organs ratio. We have developed simple, quick, safe and robust synthetic procedures for the preparation of theranostic OncoFAP-compounds based on Gallium-68, Lutetium-177 and Fluorine-18 using the commercially available FASTlab synthesis module.


Introduction
Small organic ligands that selectively bind to tumor-associated antigens are increasingly applied as targeting delivery vehicles of small bioactive payloads such as radionuclides [1,2]. Fibroblast activation protein (FAP, FAP-α) is a type-II transmembrane serine

[ 68 Ga]Ga-OncoFAP-Derivatives
The production of [ 68 Ga]Ga-DOTAGA-OncoFAP, [ 68 Ga]Ga-NODAGA-OncoFAP and [ 68 Ga]Ga-NOTA-OncoFAP using a 5-10 min automatic synthesis procedure at a reaction temperature of 95 °C was highly reproducible, with high radiochemical purity (RCP) and high radiochemical yields (RCY, Tables 1-4). The molar activity was 20-30 GBq/μmol with excellent RCY, exceeding 80% for >15 μg precursors amount. The best RCY were obtained using 25 μg of OncoFAP-derivates, a reaction time of 5 min and a reaction pH of 4.2. No significant differences were observed increasing the amount of precursor to 20 μg and 25 μg or increasing the reaction time to 10 min. The validation data of our synthesis conditions are reported in Table 5.
For the OncoFAP kit, the best result was obtained with 10 min radiolabeling at a reaction temperature of 95 °C with 40 μg of precursor and pH of 3.2 (Tables S2 and S3). The reduction of the pH to 3 using formate buffer was necessary to reduce the colloidal gallium impurity below 1.5 ± 0.2%. In fact, the use of the same condition of the automatic synthesis procedure (acetate buffer at pH 4.2) resulted in RCP of 88 ± 2.4% with significant amount of both free gallium-68 and colloidal gallium-68 (Tables 5 and 6).

[ 68 Ga]Ga-OncoFAP-Derivatives
The production of [ 68 Ga]Ga-DOTAGA-OncoFAP, [ 68 Ga]Ga-NODAGA-OncoFAP and [ 68 Ga]Ga-NOTA-OncoFAP using a 5-10 min automatic synthesis procedure at a reaction temperature of 95 • C was highly reproducible, with high radiochemical purity (RCP) and high radiochemical yields (RCY, Tables 1-4). The molar activity was 20-30 GBq/µmol with excellent RCY, exceeding 80% for >15 µg precursors amount. The best RCY were obtained using 25 µg of OncoFAP-derivates, a reaction time of 5 min and a reaction pH of 4.2. No significant differences were observed increasing the amount of precursor to 20 µg and 25 µg or increasing the reaction time to 10 min. The validation data of our synthesis conditions are reported in Table 5.   For the OncoFAP kit, the best result was obtained with 10 min radiolabeling at a reaction temperature of 95 • C with 40 µg of precursor and pH of 3.2 (Tables S2 and S3). The reduction of the pH to 3 using formate buffer was necessary to reduce the colloidal gallium impurity below 1.5 ± 0.2%. In fact, the use of the same condition of the automatic synthesis procedure (acetate buffer at pH 4.2) resulted in RCP of 88 ± 2.4% with significant amount of both free gallium-68 and colloidal gallium-68 (Tables 5 and 6).  (Table 7). The synthesis of [ 18 F]AlF-NODAGA-OncoFAP resulted in very low RCY and low RCP ( Table 8). The best result for the NOTA-precursor was obtained using 250 µg of precursor with a reaction time of 25 min and a reaction temperature of 95 • C (Table 9).   (Tables 10 and 11) using a reaction time of 30 min and a reaction temperature of 95 • C at a pH 4. We validated the radiolabeling method repeating the synthesis 3 times at the highest molar activity, simulating a typical production of a batch for PRRT i.e., 9.2 GBq of Lutetium-177 (Table 11). The RCY of [ 177 Lu]Lu-DOTAGA-OncoFAP strictly depends on the reaction volume and on the amount of precursor used in synthesis. The preparation was easily purified through C18 cartridge to remove the excess free Lutetium-177. No loss of [ 177 Lu]Lu-DOTAGA-OncoFAP was observed in this process (Figure S10 in the Supplementary Material).

Preliminary In Vitro/In Vivo Characterization of OncoFA-Derivatives
The co-elution experiments showed that all the OncoFAP derivatives described in this work retained the ability to form stable complexes with recombinant hFAP (Figure 2 and Figure S10).
All [ 68 Ga]Ga-OncoFAP and [ 18 F]AlF-OncoFAP radio-conjugates were highly stable (>99% of intact compound) in 0.9% saline solution and in human plasma at 37 • C for 2 h. On the other hand, a stability test of [ 177 Lu]Lu-DOTAGA-OncoFAP radiolabeled batch showed the presence of radiolysis of about 10% every 24 h, which was significantly reduced to about 2% within 8 days by the addition of 20 mg of gentisic acid as a radical scavenger (Figure 3b) [15,16]. All the radiolabeled preparations of OncoFAP derivatives exhibited high hydrophilicity. Details are given in Supplementary Material.
Cell binding studies showed a similar trend of binding on FAP-positive SK-RC-52 cells for all the OncoFAP radio-conjugates. All the compounds displayed an intense binding at early time point (10 min), followed by a progressive decrease over time. As shown in Figure 4, a major difference in the absolute binding value was found among 20% for [ 177 Lu]Lu-DOTAGA-OncoFAP, 2.3% for [ 68   Cell binding studies showed a similar trend of binding on FAP-positive SK-RC-52 cells for all the OncoFAP radio-conjugates. All the compounds displayed an intense binding at early time point (10 min), followed by a progressive decrease over time. As shown in Figure 4, a major difference in the absolute binding value was found among 20% for [ 177 Lu]Lu-DOTAGA-OncoFAP, 2.3% for [ 68      Cell binding studies showed a similar trend of binding on FAP-positive SK-RC-52 cells for all the OncoFAP radio-conjugates. All the compounds displayed an intense binding at early time point (10 min), followed by a progressive decrease over time. As shown in Figure 4, a major difference in the absolute binding value was found among 20% for [ 177 Lu]Lu-DOTAGA-OncoFAP, 2.3% for [ 68    In vivo biodistribution of [ 18 F]AlF-NOTA-OncoFAP (500 nmol/kg, ~4 MBq/Kg, RCP 89%) in athymic Balb/c AnNRj-Foxn1 mice bearing subcutaneous HT-1080.hFAP fibrosarcoma showed selective accumulation in FAP-positive tumors (6.6% ID/g, 1 h post intravenous injection), with excellent selectivity against healthy organs. Tumor-to-blood and tumor-to-kidney ratios of 6.5-to-1 and 4.3-to-1, respectively, were observed at the 1-h time point (Figure 5). In vivo biodistribution of [ 18 F]AlF-NOTA-OncoFAP (500 nmol/kg,~4 MBq/Kg, RCP 89%) in athymic Balb/c AnNRj-Foxn1 mice bearing subcutaneous HT-1080.hFAP fibrosarcoma showed selective accumulation in FAP-positive tumors (6.6% ID/g, 1 h post intravenous injection), with excellent selectivity against healthy organs. Tumor-to-blood and tumor-to-kidney ratios of 6.5-to-1 and 4.3-to-1, respectively, were observed at the 1-h time point ( Figure 5).

Discussion
In recent years, the development of tumor-targeting ligands resulted in the discovery of a new generation of diagnostic and therapeutic products with high uptake in cancer lesions and low accumulation in healthy organs. Lutathera ® and [ 177 Lu]Lu-PSMA-617 provide examples of radioligand therapeutics with proven clinical efficacy and limited systemic toxicity [17,18]. Radioligand diagnostic and therapeutic products targeting FAP in the tumor microenvironment represent a promising class of compounds with pan-tumoral applicability and high selectivity for tumor lesions [19,20].
FAP-targeting strategies have gained growing relevance in nuclear medicine for the development of radiolabeling. Although anti-FAP antibodies have been known since the 1990s [21][22][23][24], the discovery of small organic FAP ligands in the last few years represented

Discussion
In recent years, the development of tumor-targeting ligands resulted in the discovery of a new generation of diagnostic and therapeutic products with high uptake in cancer lesions and low accumulation in healthy organs. Lutathera ® and [ 177 Lu]Lu-PSMA-617 provide examples of radioligand therapeutics with proven clinical efficacy and limited systemic toxicity [17,18]. Radioligand diagnostic and therapeutic products targeting FAP in the tumor microenvironment represent a promising class of compounds with pan-tumoral applicability and high selectivity for tumor lesions [19,20].
FAP-targeting strategies have gained growing relevance in nuclear medicine for the development of radiolabeling. Although anti-FAP antibodies have been known since the 1990s [21][22][23][24], the discovery of small organic FAP ligands in the last few years represented a revolution in the field of nuclear medicine. Various FAP-targeting agents coupled to different radionuclide chelators have been recently developed and characterized for their high affinity and selectivity towards FAP-positive tumors. These conjugates exhibit rapid accumulation in cancer lesions and low uptake in healthy organs both in preclinical murine models and cancer patients. Among FAP-specific small organic ligands, OncoFAP is the compound with the highest affinity described so far (Kd = 680 pM) [9]. Preliminary data with [ 68 Ga]Ga-DOTAGA-OncoFAP have demonstrated the feasibility of 68 Ga-radiolabeling and highly favorable targeting properties in both small animal and patients with cancer, validating [ 68 Ga]Ga-OncoFAP as a new powerful alternative to clinically established PET tracers [8]. BiOncoFAP, a bivalent derivative of OncoFAP, has been recently described as a novel compound with high and prolonged tumor uptake in murine models of cancer [25]. With the aim to identify a clinical [ 18 F]-OncoFAP candidate, we generated NODAGA-OncoFAP and NOTA-OncoFAP as two preclinical prototypes that could be labeled both with Fluorine-18 and with Gallium-68. NODAGA-OncoFAP rapidly complexes Gallium-68 with excellent product stability, thus opening promising avenues to the development of simple kit preparations The translation of radiopharmaceutical agents from preclinical development to clinical applications requires the implementation of standard synthetic and radiolabeling procedures [26]. In order to facilitate clinical development of novel FAP-radiopharmaceuticals, we have developed highly reproducible automated methodologies for the efficient radiolabeling of OncoFAP-derivatives (DOTAGA-OncoFAP, NODAGA-OncoFAP and NOTA-OncoFAP) with Gallium-68, Fluorine-18 and Lutetium-177 radioisotopes. The procedures described in this article are easy to implement, safe and robust. We based these procedures on the FASTLab automated module, a radiolabeling platform already broadly applied to produce other radiopharmaceuticals at the commercial scale [27][28][29]. With the aim to enable production of [ 68 Ga]-DOTAGA/NODAGA-OncoFAP radiopharmaceuticals also in small radiopharmacy sites with limited financial resources, we have also generated a radiolabeling single-vial cold kit. This approach, already proposed for other products (Illuccix™ and NETSPOT ® ; SOMAKIT TOC) [30][31][32], renders the production of 68 Ga-based products as easy as the one of 99m Tc-based radiopharmaceuticals. The "OncoFAP-kit", which already contains all necessary items (buffers, ligands and excipients) to be combined with the radioisotopes, allows efficient production of [ 68 Ga]Ga-NODAGA-OncoFAP and [ 68 Ga]Ga-DOTAGA-OncoFAP for PET imaging applications. The reasons for the development of such a strategy are related to the need to simplify and facilitate, while maintaining high standard of quality, efficiency and reproducibility the preparation of 68 Ga imaging agents. Indeed, the traditional approach of automatic synthesis of 68 Ga-based radiopharmaceuticals generates substantial investments, i.e., hot cells and synthesis modules, quality control equipment, and highly qualified personnel who may not be easily accessible to small-scale radiopharmacies, thus hampering the widespread use of 68 Ga-PET imaging agents and its equal accessibility for patients throughout the world. Consequently, interest has grown on the development of cold kits for PET tracers. The main advantages of a kit-based process are lower investments, ease of use, absence of purification steps (only a 0.22 µm sterilizing filter is required at the generator outlet to ensure the sterility of the final formulation), absence of EtOH and the lower final volume at the cost of higher amounts of precursor required, thus resulting in lower molar activity. Further, the lack of purification requires high-quality radioactive eluate.
Very few data are currently available on the direct comparisons of the operator's radiation exposure when producing 68 Ga-radiopharmaceuticals using on automatic synthesis modules and simple kits. Frinde et al., by measuring the dose at the extremities via proximity meters fixed to the first phalanx of each middle finger for a 1.85 GBq generator at the date of calibration, reported 70 µSv for the left hand and 132 µSv for the right hand for labeling with an automatic synthesis module but 179 µSv and 152 µSv for kit-based preparation of [ 68 [34] as a result of the different radiolabeling environment in which the procedures were performed: a hot cell with 50 mm lead shield for the automated synthesis as compared to a class II biosecurity cabinet shielded with a 3 mm lead layer, with a 10 mm lead shielded desktop screen for the kit-based preparation. When the same 50 mm lead hot cell was used, the whole-body exposure dropped to 2 ± 0.5 µSv also for the kit-based preparation [35] with a dose at the extremities of about 1.5 ± 0.4 mSv. Therefore, reducing the exposure for simple kit labeling is possible, but dependent on the environment of the radiolabeling and on the experience of the operator.
As an alternative to Gallium-68 for diagnostic applications, we also developed two novel OncoFAP-conjugates bearing NODAGA and NOTA radiometal chelators, which are known to form stable complexes with Alluminum-Fluorine-18 [36][37][38][39][40]. We performed the radiochemical synthesis of both [ 18 F]AlF-NODAGA-OncoFAP and [ 18 F]AlF-NOTA-OncoFAP. The radiolabeling of NODAGA-OncoFAP with Alluminum-Fluorine-18 resulted in very low RCY and RCP, probably due to the ability of the NODAGA chelator to form a stable neutral complex with the Al 3+ ion, thus partially preventing the incorporation of the fluorine-18 as also reported by Liu et al. [41]. On the contrary, NOTA-OncoFAP, which devoid a carboxylic acid function, was efficiently radiolabeled with Aluminium-Fluorine-18, with a good purity and a RCY of approximately 20%. DOTAGA-OncoFAP can be labeled both with Gallium-68 and Lutetium-177, and thus can represent a "theranostic pair". This approach has been already successfully implemented for the diagnosis and therapy of Neuro-Endocrine Tumors (Lutathera ® /NETSPOT ® ) [10] and prostate cancers ([ 68 Ga]Ga-PSMA-11/[ 177 Lu]Lu-PSMA-617) [11]. Our data prove the feasibility of producing high-quality [ 177 Lu]Lu-DOTAGA-OncoFAP with molar activities of 100 GBq/µmol, which is almost double the molar activities currently routinely achieved with [ 177 Lu]Lu-PSMA-617 [42]. Incorporating the high activity of Lutetium-177 represents a critical parameter to obtain a therapeutic radiopharmaceutical (i.e., in the range of 7.4-14.8 GBq) containing relatively low amounts of precursor, thus avoiding saturation of targets at the tumor site [9]. Such results serve as the basis to provide a fast track for the clinical application of therapeutic OncoFAP derivatives such as [ 177 Lu]Lu-DOTAGA-OncoFAP, retaining selective uptake in the FAP-positive tumor already 10 min after injection (32% injected dose [ID]/g), with a sustained uptake (i.e., higher than 20% ID/g) in the lesions over the first 6 h time window and the more recently developed [ 177 Lu]Lu-DOTAGA-BiOncoFAP, exhibiting a more stable and prolonged tumor uptake than [ 177 Lu]Lu-DOTAGA-OncoFAP (~20% ID/g vs.~4% ID/g, at 24 h p.i., respectively) favorable tumor-to-organ ratios with low kidney uptake as well potent anti-tumor efficacy when administered at therapeutic doses in tumor-bearing mice [25].
All OncoFAP derivatives based on Gallium-68 and Aluminum-fluorine-18 studied in this work show excellent stability without the need of a stabilizer if not the small percentage of ethanol found in the preparation coming from purification process. On the contrary [ 177 Lu]Lu-DOTAGA-OncoFAP-shows an intrinsic instability when there are high radioactive concentrations, therefore necessitating the addition of gentisic acid as a radical scavenger normally formed due to the β − decay of Lutetium-177. All the OncoFAP derivatives also retained high affinity towards hFAP as shown from the coelution experiment of [ 68 [44,45] with the possibility of a subsequent delivery to satellite sites other than the production site, as the half-life of the isotope is long enough to allow this strategy (t 1/2 = 109.8 min). Translation in the clinical setting will be of significant impact also in terms of daily organization of the PET/CT schedule. In particular, the use of fluorine-18-based FAPI-derivatives allow an easier patient preparation (no need of fasting and maintaining low glucose level respect the use of FDG) and offer a longer imaging time window of approximately 30-180 min after tracer injection as compared to gallium-68 FAPI derivatives. In addition, OncoFAP-derivatives can be exploited as PET/CT imaging agents for a broader window of malignancies, which are not efficiently detected by other marketed radiopharmaceuticals, such as [ 18 46]. Examples are represented by oesophageal [47], liver [48] and pancreatic cancers [4], brain primary tumors and metastases [49] or head and neck cancers [50]. However, the results from the synthesis using the Aluminum-Fluorine-18 approach still warrant further development and optimization before the method can be efficiently translated into clinical use. In fact, progress is needed to increase the labeling yield and molar activities while minimizing the precursor, thus avoiding a post-radiolabeling purification step to obtain a product with high molar activity.
Our results provide the reference for a robust and efficient radiolabeling method for OncoFAP-radioligand products that are easy to implement in clinical practice in small-, medium-and large-sized hospital radiopharmacy. The different radiosynthetic strategies presented in this article for each radioisotope can be chosen on the basis of the specific needs of the various clinical centers. Automated production of [ 68 Ga]Ga-DOTAGA-OncoFAP, a clinically validated PET tracer [8], and of [ 177 Lu]Lu-DOTAGA-OncoFAP will facilitate the implementation of this theranostic pair in clinical practice. Efficient production of [ 18 F]AlF-NOTA-OncoFAP will represent the basis for the implementation of this novel radiotracer for large-scale PET imaging applications.

Chemical Synthesis
Detailed chemical procedures and compound characterization for the synthesis of NOTA-OncoFAP and NODAGA-OncoFAP are reported in the Supplementary Material. DOTAGA-OncoFAP has been produced as previously described [9].  Figure S3 of the Supplementary Material. The synthesis was carried out using 68 Ga (t 1/2 = 68 min, β + = 89%, and EC = 11%) automatically eluted with 0.1 M HCl (4.5 mL, TRASIS ALLinONE reagent kit) from a 1.85 GBq (50 mCi) 68 Ge/ 68 Ga radionuclide generator (Eckert & Ziegler 1850 MBq, GalliaPharm, Radiopharma GmbH) without pre-purification. About 3.5 mL of the [ 68 Ga]GaCl 3 eluate and the precursor, dissolved in sodium acetate (750 µL, 0.7 M TRASIS ALLinONE reagent kit), were transferred into the reaction vessel. After stirring for 5 min at 95 • C under gentle nitrogen flow, the reaction mixture was loaded for purification onto the preactivated C18 cartridge (Waters, Milford, MA, USA), washed with 0.9% NaCl solution (5 mL, TRASIS ALLinONE reagent kit) and eluted with a mixture of 700 µL of absolute ethanol (TRASIS ALLinONE reagent kit) and 800 µL of water for injection (WFI, Fresenius kabi). Then, the product was diluted with 0.9% NaCl solution to obtain the final formulation obtaining a volume of 12 mL.

[ 68 Ga]GaOncoFAP-Derivatives Synthesis
For the synthesis of [ 68 Ga]Ga-DOTAGA-OncoFAP and [ 68 Ga]Ga-NODAGA-OncoFAP, a simple preformulated radiopharmaceutical kit (OncoFAP-kit) was developed. This novel kit is 'ready-to-use' for radiolabeling with 68 Ga eluted by commercially available 68 Ge/ 68 Ga generators, as in the SomaKit TOC [32]. To this aim, we develop an approach consisting of generator elution with HCl (5 mL, 0.1 M TRASIS ALLinONE reagent kit) directly in the vial containing the precursor; at the end of the elution either sodium acetate (900 µL, 0.7 M TRASIS ALLinONE reagent kit) to have a reaction pH of 4.5 or sodium formate (600 µL, 1 M BioULTRA Formic acid solution 1.0 M in H 2 O, Sigma Aldrich Taufkirchen, Germany and Sodium hydroxide solution BioUltra, for molecular biology, 10 M in H 2 O) to have a reaction pH of 3 is added. Once the generator has been eluted into the vial containing the precursor and the appropriate buffer has been added, the vial is then placed in a thermoblock preheated to 98 • C and heated for 10 min. The synthesis was repeated in different conditions, varying the amount of precursor (10-40 µg) and the reaction pH (3.2-4.5).

[ 18 F]AlF-OncoFAP
Radiolabeling of NODAGA-OncoFAP and NOTA-OncoFAP (200-300 µg) with 18 F was performed via aluminum (Al 3+ ) [ 18 F]fluoride complex. In addition, for this synthesis, we used the FASTlab synthesis module. The configuration of the cassette is detailed in Figure S5 of the Supplementary Material.
Fluorine-18 was transferred to the module and trapped on a preactivated Sep-Pak light Accel plus QMA cartridge (Cl − form: Waters, Milford, MA, USA). The cartridge was washed with 6 mL of water (HPCE grade, Sigma Aldrich) and subsequently eluted with 500 µL of the eluent solution, composed of 250 µL of 0.9% NaCl solution (99.999% trace metals basis NaCl, Sigma Aldrich) in water for injection (WFI, Fresenius kabi) and 250 µL absolute ethanol (TRASIS ALLinONE reagent kit), into a 5 mL reactor vial prefilled with 25 µL of 2 mM aluminum chloride solution (AlCl 3 , anhydrous, powder, 99.999% trace metals basis, Sigma-Aldrich) in sodium acetate buffer (0.1 M, pH 4.1). After stirring for 5 min at room temperature under gentle nitrogen flow to allow the formation of [ 18 F]AlF, the solution of the precursor (600 µL of 350 µg/mL NOTA-OncoFAP or NODAGA-OncoFAP in sodium acetate 0.1 M pH 4.5) was added to the reactor vial, which was sealed and heated for 10 min at 95 • C. Next, the mixture was cooled to 40 • C. The reaction was diluted with 3.5 mL of 0.9% NaCl solution, loaded for purification onto the preactivated C18 cartridge (Waters, Milford, MA, USA), washed with 5 mL of 0.9% NaCl solution and eluted with 1.5 mL of absolute EtOH (TRASIS ALLinONE reagent kit). The product is diluted with 0.9% NaCl solution to obtain the final formulation obtaining a final volume of 12 mL. A method using semi-preparative HPLC purification is given in the supplementary information.

[ 177 Lu]Lu-OncoFAP
[ 177 Lu]Lu-DOTAGA-OncoFAP was synthesized in an FASTlab synthesis module (GE Healthcare), at different molar activities ranging from 44 to 105 GBq/µmol. The configuration of the cassette is presented in Figure S7 of the Supplementary Material. Carrier-free Lutetium-177 (EndolucinBeta ® 40 GBq/mL-pharmaceutical precursor, solution) with a concentration activity of 37 MBq/µL in a volume of 0.5 mL acetate buffer at pH 4.5 is transferred to the reactor vial. The reaction was scaled down to an amount of DOTAGA-OncoFAP ranging from 5.1 to 13.3 µg, which was dissolved in acetate buffer (1 mL, 0.1 M pH 4.5 TRASIS ALLinONE reagent kit) and aspirated into the reactor vessel. The solution was stirred for 30 min at 90 • C under gentle nitrogen flow, then loaded for purification onto the preactivated C18 cartridge, washed with 5 mL of 0.9% NaCl solution and eluted with eluent solution made of 700 µL of absolute ethanol (TRASIS ALLinONE reagent kit) and 800 µL of water for injection (WFI, Fresenius kabi). The product was diluted with 0.9% NaCl solution to obtain the final formulation. Further, the radiolabeling was also performed by adding 20 mg of gentisic acid [16] to assess its effects on the radiolytic stability as radical scavenger.

Reproducibility of the Method
To test the robustness of the synthesis method, we varied the reaction time and the amount of precursor used in the tests. We repeated each experiment at least three times to test the reproducibility.

Quality Controls
Radiochemical, chemical and radionuclide purity, pH, half-life, residual organic solvents, filter integrity and endotoxin content were assessed for all the radiolabeled preparations of OncoFAP derivatives.
Radiochemical and chemical purity were determined by HPLC (Berthold HERM LB500 radio detector with Jasco MD-2010 DAD at 250 nm) using a RP-18 column (Phenomenex Luna ® column 150 × 3 mm 3 µm 100 Å) with a flow of 0.6 mL/min at 40 • C. As mobile phase, water + TFA 0.1% (v/v, phase A) and acetonitrile + TFA 0.1% (v/v, phase B) were used with a gradient 0 to 0.5 min 90% eluent A, 0.5 to 10 min linear gradient elution from 90% to 0% eluent A, 10 to 14.5 min 0% eluent A, 14.50 to 15 min from 0% to 90% eluent A. The OncoFAP derivatives complex with the non-radioactive isotopes (Gallium-69, Fluorine-19 and Lutetium-175) were used as reference standards. iTLC on silica gel using citrate as a buffer eluent (0.1 M, pH 5) was used to quantify gallium-68 hydroxide with (Rf of 0-0.1), the labeled product (Rf between 0.3 and 0.5) and free gallium-68 (Rf between 0.6 and 0.8) both for the automated synthesis and the simple kit formulation.
For the determination of radionuclide purity, a sample of final product with a known activity and volume was analyzed (energetic spectrum from 0 to 1800 KeV) after one day in case of 68 Ga-compounds ( 68 Ga T decay > 20 half-lives) and 1.5 days for [ 18 F]AlF-OncoFAP derivatives ( 18 F T decay > 20 half-lives). To this aim, we selected only radioactive impurities with a half-life longer than 2 h. The pH of the final product was measured with pH indicator strips (pH range 2.0 to 9.0, MColorpHast™, Merck, Milano, Italy). Final activity of 68 Ga/Al 18 F-OncoFAP was measured with an appropriate interval between the three measurements of 5 min. Residual organic solvents were measured using a gas chromatography system with a macrogol 20,000 column (30 m × 0.53 mm × 1 µm, Varian, Santa Clara, CA, U.S.) following the pharmacopoeia 2.2.28 (inlet 140 • C temperature, isocratic column temperature 50 • C, flame ionization detection temperature 250 • C during 5 min). Retention time of 2.9 min for ethanol and 4.75 min for n-propanol were used as reference standard.
Bubble point test of the Millex-GS vented filter was performed closing the vented portion of the filter and gradually pushing down the plunger inside a syringe filled with air to increase the pressure on the pressure gauge. The pressure when a continuous stream of air bubbles that appeared out of the 0.22 µm membrane filter was measured as the product-wetted bubble point value.
Endotoxin determination was conducted with a Endosafe PTS Reader ® rapid test (Charles River).

In Vitro Stability and Lipophilicity
The in vitro stability of the radiopharmaceuticals preparations in saline solution was evaluated on the bulk solution i.e., with the highest radioactive concentration. In addition, the in vitro stability of radiopharmaceutical preparations was tested by incubating 10 MBq of each radiolabeled compounds in 1 mL of human plasma at 37 • C. Small aliquots of bulk solution were analyzed by HPLC and iTLC and 10 µL of plasma solution were analyzed by iTLC at 30, 60, 90, 120, 150 and 180 min for [ 68   Lipophilicity was determined by partitioning between n-octanol and saline solution (LogP) or PBS (LogD 7.4 ) at room temperature using the conventional shake-flask method. An aliquot of the formulated solution containing~500 kBq of radiopharmaceuticals was added to a tube containing 6 mL of n-octanol/saline solution or n-octanol/PBS (1:1 v/v, LogP or LogD 7.4 , respectively). The tubes were shaken for 20 min, followed by centrifugation (5000× g for 5 min) and separation of the phases. Aliquots of 1 mL of the organic and the aqueous phases were taken, and the activity was measured using an automated gamma counter. The LogP or LogD 7.4 were calculated as the decadic logarithm of [activity (cpm/mL) in n-octanol]/[(activity (cpm/mL) in saline solution or PBS].