Automated radiolabelling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy

doi:10.21203/rs.3.rs-1404837/v1

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Automated radiolabelling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy

https://doi.org/10.21203/rs.3.rs-1404837/v1

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FAP-targeted radiopharmaceuticals represent a breakthrough in cancer imaging and a viable option for therapeutic applications. OncoFAP, an ultra-high affinity ligand of FAP, 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 radiolabelling procedures with FASTlab 2 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-yields. Chemical and biochemical characterization after radiolabelling confirmed excellent stability, retention of high affinity for FAP and absence of radiolysis by-products. The in vivo biodistribution of [¹⁸F]AlF-OncoFAP-NOTA, a candidate for PET imaging procedures in patients, was assessed in mice bearing FAP-positive solid tumours. The product showed rapid accumulation in solid tumours, with an average of 6.6% ID/g one hour after systemic administration and excellent tumour-to-healthy organs ratio.

We have established methodologies to efficiently produce an OncoFAP-based radiotheranostic pair in which [⁶⁸Ga]Ga-OncoFAP-DOTAGA is used as a companion diagnostic of the targeted therapeutic agent [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA

Objectives: We have developed simple, quick, safe and robust synthetic procedures for the preparation of theranostic OncoFAP-compounds based on Gallium-68, Lutetium-177 and Alluminium-Fluoride-18 using the commercially available FASTlab 2 synthesis module.

OncoFAP

theranostics

68Ga-labeled

177Lu-labeled

Al18F-labeled

FAP-radiopharmaceuticals

Small organic ligands that selectively bind to tumour-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 protease overexpressed on the surface of cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) in most epithelial malignancies [3]. FAP has recently emerged as a novel in vivo imaging biomarker with prognostic and theranostic potential. Favourable biodistribution profile and tumour-to-background ratio have been demonstrated for several FAP-based radiopharmaceuticals in development for PET/CT imaging. A wide range of cancer types have been imaged successfully with FAP-radioligands based on small molecules, particularly in those clinical conditions where imaging with [¹⁸F]FDG, the most extensively used PET radiopharmaceutical in oncology, has proved limitations [4–8].

To the best of our knowledge, OncoFAP is the FAP small organic ligand with highest affinity reported to date, with a dissociation constant of 680 pM [9]. OncoFAP-radio and OncoFAP-fluorophore conjugates exhibit rapid, selective and efficient tumour-targeting performance in murine models of cancer [9]. Extensive pre-clinical characterization of [⁶⁸Ga]Ga-OncoFAP-DOTAGA demonstrate its excellent imaging performance with similar targeting proprieties in a head-to-head comparison with [⁶⁸Ga]Ga-FAPI-46 [8]. First-in-human data have recently confirmed the pan-tumoral targeting potential of [⁶⁸Ga]Ga-OncoFAP-DOTAGA, with high uptake in primary tumours (breast cancer, colon cancer, fibrosarcoma, hepatocellular carcinoma), lymph node and distant metastases and a rapid clearance from healthy organs [8].

Given the high expression of FAP in cancer and the absence of significant uptake of FAP-targeting small molecule tracers in normal tissues, OncoFAP-based radioligands have inherent potential for theranostic applications. OncoFAP-DOTAGA can be radiolabelled with radioactive metals entailing therapeutic potential such as ¹⁷⁷Lu, the current most popular radionuclide for therapeutic applications for neuroendocrine tumours [10] and prostate cancer [11], leading to a new family of theranostic radiopharmaceuticals. In this work, we present the chemical, biological and pre-clinical characterization of a series of radiotheranostic OncoFAP derivatives, specifically OncoFAP-DOTAGA, OncoFAP-NODAGA and OncoFAP-NOTA with gallium-68 and fluorine-18 for PET/CT imaging and with lutetium-177 for therapeutic applications. Simple kit preparations were developed for [⁶⁸Ga]Ga-OncoFAP-DOTAGA and [⁶⁸Ga]Ga-OncoFAP-NODAGA radiotracers.

Chemical synthesis

Detailed chemical procedures and compound characterization for the synthesis of OncoFAP-NOTA and OncoFAP-NODAGA are reported in the Supplementary Material. OncoFAP-DOTAGA has been produced as previously described [9].

Radiochemistry

[⁶⁸Ga]Ga-OncoFAP-derivatives synthesis

[⁶⁸Ga]Ga-OncoFAP-DOTAGA/NODAGA/NOTA (15–25 µg) were synthesized in an FASTlab 2 synthesis module (GE Healthcare), the configuration of the cassette is presented in the Figure S3 of the Supplementary Material. The synthesis was carried out using ⁶⁸Ga (t_1/2 = 68 min, β⁺ = 89%, and EC = 11%) automatically eluted with 0.1 M HCl (0.36% 4.5 mL, TRASIS ALLinONE reagent kit) from a 1.85 GBq (50 mCi) ⁶⁸Ge/⁶⁸Ga radionuclide generator (Eckert & Ziegler 1850 MBq, GalliaPharm, Radiopharma GmbH) without pre-purification. About 3.5 mL of the [⁶⁸Ga]GaCl₃ eluate and the precursor, dissolved in sodium acetate (750 µL, 0.7M TRASIS ALLinONE reagent kit), were transferred into the reaction vessel. After stirring for 5 min at room temperature under gentle nitrogen flow, the reaction mixture was loaded onto the preactivated C18 cartridge (Waters, Milford, Massachusetts, 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.

For the synthesis of [⁶⁸Ga]Ga-OncoFAP-DOTAGA and [⁶⁸Ga]Ga-OncoFAP-NODAGA, a simple preformulated radiopharmaceutical kit (OncoFAP-kit) was developed. This novel kit is ‘ready-to-use’ after radiolabelling with ⁶⁸Ga eluted by commercially available ⁶⁸Ge/⁶⁸Ga generators, as in the SomaKit TOC [12]. To this aim, a 10 mL vial prefilled with 10, 30, 50 or 70 µL of a sodium formate solution of OncoFAP at a concentration of 1 mg/mL (550 µL, BioULTRA Formic acid solution 1.0 M in H₂O, Sigma Aldrich and Sodium hydroxide solution BioUltra, for molecular biology, 10 M in H₂O) at a pH of 3.3 was placed in an appropriate heating block at 98°C before starting the generator elution. The ⁶⁸Ge/⁶⁸Ga radionuclide generator (Eckert & Ziegler 1850 MBq, GalliaPharm, Radiopharma GmbH) was manually eluted with 5 mL of 0.1 M HCl solution. A 0.22 µm sterilizing filter was placed at the end of the generator line. The first 4 mL of the eluate, containing about 250–1050 MBq of ⁶⁸Ga were directly collected in the disposable vial whereas the last 1 mL remained in the line and in the sterilizing filter. The mixture was then heated to 98°C for 10 min.

[¹⁸F]AlF-OncoFAP

Radiolabelling of OncoFAP-NODAGA and OncoFAP-NOTA (200–300 µg) with ¹⁸F was performed via aluminium (Al³⁺) fluoride complex. Also, for this synthesis, we used the FASTlab 2 synthesis module. The configuration of the cassette is detailed in the 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, Massachusetts, 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 aluminium chloride solution (AlCl₃, 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 [¹⁸F]AlF, the solution of the precursor (600 µL of 350 µg/mL OncoFAP-NOTA 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 onto the preactivated C18 cartridge (Waters, Milford, Massachusetts, 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.

Fluorine-18 radiolabelling of OncoFAP-NODAGA was achieved as follow: [¹⁸F]Fluorine (647 MBq) was trapped in the cartridge (45mg PS-HCO₃-) and washed with 3 mL metal-free H₂O and dried with 7 mL air. The cartridge was then eluted with 100 µL of 0.9% NaCl solution directly in the AlCl₃ solution (2 mM; pH 4.7; 10 µL) and reacted for 5 min. 10 µL of OncoFAP-NODAGA in sodium acetate (0.5 M, pH 4, 50 µg) and 200 µL of ACN was added to the solution and the resulting reaction mixture was heated at 100°C for 20 min. After the labelling, the mixture was injected on HPLC (Synergy 4 µm Hydro-RP 80Å; 20/80 ACN/water TFA 0.1% gradient 30min). The product peak was collected at 5.8 min and diluted in 60 µL of water. The solution passed through an Oasis® HLB 1 cc/30 mg and the product was eluted with 400 µL ethanol and reconstituted with 0.9% NaCl solution (5 mL). The RCY (decay corrected) of this reaction is 3% and the RCP > 95%.

[¹⁷⁷Lu]Lu-OncoFAP

[¹⁷⁷Lu]Lu-OncoFAP-DOTAGA was synthesized in an FASTlab 2 synthesis module (GE Healthcare), at different specific activities ranging from 1.2 to 2.8 mCi/µg. The configuration of the cassette is presented in Figure S7 of the Supplementary Material. Carrier-free ¹⁷⁷Lu (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 OncoFAP-DOTAGA 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 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.

Analysis and quality control

Radiochemical, chemical and radionuclide purity, pH, half-life, residual organic solvents, filter integrity and endotoxin content were assessed for all the radiolabelled 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 x 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 mins linear gradient elution from 90–0% eluent A, 10 to 14.5 mins 0% eluent A, 14.50 to 15 mins from 0–90% eluent A. "Cold" labelled derivatives were used as reference standards. The % of colloidal gallium was determined through iTLC on silica gel with a 1:1 mixture of 1.0 M NH₄OAc/Methanol as eluant. For the determination of radionuclide purity, a sample of final product with a known activity and volume was analysed (energetic spectrum from 0 to 1800 KeV) after one day in case of ⁶⁸Ga-coumpounds (⁶⁸Ga T_decay > 20 half-life) and 1.5 day for [¹⁸F]AlF-OncoFAP derivatives (¹⁸F T_decay > 20 half-life). 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). Final activity of ⁶⁸Ga/Al¹⁸F-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 20000 column (30 m × 0.53 mm × 1 µm, Varian) following the pharmacopoeia 2.2.28 (inlet 140°C temperature, isocratic column temperature 50°C, flame ionization detection temperature 250°C during 5 mins). Retention time of 2.9 mins for ethanol and 4.75 mins 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 carried out with an Endosafe PTS Reader® rapid test (Charles River).

In vitro Stability and Lipophilicity

In vitro stability of radiopharmaceuticals preparations was tested by incubating 10 MBq of each radiolabelled compounds in 1 mL of saline solution or human plasma at 37°C. Small aliquots were analysed by HPLC at 30, 60, 90, 120, 150 and 180 mins for [⁶⁸Ga]Ga-OncoFAP-DOTAGA/NOTAGA derivatives, 50, 100, 150 and 200-mins for [¹⁸F]AlF-OncoFAP-NOTA/NOGADA or 1, 3, 6, 12, 24, 48, 72, 96 hours and 7 days for [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA, as show in Fig. 3.

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].

Characterization of OncoFAP Binding to hFAP

To assess the binding properties of radiolabelled OncoFAP-derivatives preparations to hFAP, [⁶⁸Ga]/[¹⁸F]AlF/[¹⁷⁷Lu]-OncoFAP derivatives (500 kBq) were incubated with hFAP (2 µM, 100 µL) and then loaded on PD-10 columns, pre-equilibrated with running buffer (50 mM Tris, 100 mM NaCl, and 1 mM ethylenediaminetetraacetic acid [EDTA], pH = 7.4) and eluted with running buffer. Fractions of the flow through (500 µL) were collected, and the radioactivity intensity was measured immediately on a gamma counter (λ = 511 KeV for ⁶⁸Ga and ¹⁸F and λ = 208 KeV for ¹⁷⁷Lu).

Cell binding

Cell binding assays were performed in triplicate on SK-RC-52.hFAP and SK-R-RC-52.wt cells (density of 1.5x10⁵ cells each tube), growth in RPMI-1640 medium (Gibco) containing 10% Fetal Bovine Serum (Gibco) and 1% Antibiotic-Antimycotic (Gibco) at 37°C in 5% carbon dioxide (CO₂). After adding [⁶⁸Ga]Ga-ONCOFAP-DOTAGA, [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA or [¹⁸F]AlF-OncoFAP-NOTA (10 µL, 0.1 MBq) to the culture medium, the cells were incubated at 37°C for 10, 30, 60 mins (gallium-68), 10, 30, 60, 120, 240 mins (fluorine-18) or 10, 30, 60, 120, 240, 360 mins and 24 and 48 hours (lutetium-177). As a control, nonspecific binding was carried out incubating the “cold” labelled OncoFAP conjugate (100 µM) with the same cell lines for 5 min before incubation with the radiolabelled compounds. After incubation, the cell pellets were washed twice with 1 mL of cold PBS (pH 7.4), centrifuged (400 g, 5 min) and the pellet measured by an automatic gamma counter (WIZARD2, PerkinElmer).

Animal studies

Animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under the license number ZH006/2021 granted by the Veterinäramt des Kantons Zürich.

Implantation of subcutaneous tumours

Tumour cells were grown to 80% confluence in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (Thermofisher) and 1% antibiotic-antimycotic (Gibco) and detached with Trypsin-EDTA 0.05%. HT-1080.hFAP, cells (FAP positive cells) were resuspended in Hanks’ Balanced Salt Solution medium. Aliquots of 5 × 10⁶ cells (100 µL of suspension) were injected subcutaneously in the right flanks of female athymic Balb/c AnNRj-Foxn1 mice (6 to 8 wk of age, Janvier).

Biodistribution studies in tumour bearing mice

Female athymic Balb/c AnNRj-Foxn1 mice (6 to 8 wk of age) were implanted in the right flank with HT-1080.hFAP tumours as described above. Tumours were allowed to grow to an average volume of 250 mm³. Mice were randomized (n = 5 per group) and injected intravenously with radiolabelled preparations of [¹⁸F]AlF-OncoFAP-NOTA (10 nmol/mice; 77 kBq). Mice were euthanized 1h after the injection by CO₂ asphyxiation. Organs were extracted, weighted, and radioactivity was measured with a Packard Cobra Gamma Counter. Values are expressed as percent ID/g ± SD.

OncoFAP-COOH (compound 1) was used as common precursor for the synthesis of OncoFAP-DOTAGA (compound 2), OncoFAP-NODAGA (compound 3) and OncoFAP-NOTA (compound 4). Final compounds were afforded by following standard coupling and deprotection procedures. Detailed synthetic procedures and product characterization are reported in the Supplementary Material.

Radiosynthesis for [⁶⁸Ga]Ga-OncoFAP

Production of [⁶⁸Ga]Ga-OncoFAP-DOTAGA and [⁶⁸Ga]Ga-OncoFAP-NODAGA and [⁶⁸Ga]Ga-OncoFAP-NOTA using a 5–10 mins procedure through an automatic synthesis module was highly reproducible, with purity > 99.5% and high radiochemical yields (78–83% for [⁶⁸Ga]Ga-OncoFAP-DOTAGA, 82–85% for [⁶⁸Ga]Ga-OncoFAP-NODAGA and 78–83% for [⁶⁸Ga]Ga-OncoFAP-NOTA, as reported in Tables 1–4). The afforded molar activity resulted in 20–30 GBq/µmol. Excellent labelling yields were obtained with 15 µg of precursors and a reaction time of 5 mins. No significant differences were observed increasing the amount of precursor to 20 µg and 25 µg or increasing the reaction time to 10 mins. In addition, [⁶⁸Ga]Ga-OncoFAP-NODAGA and [⁶⁸Ga]Ga-OncoFAP-DOTAGA production was achieved in high RCY and purity using a simple preformulated radiopharmaceutical kit (OncoFAP-kit) as shown in Tables 5–6. The use of formate buffer to maintain a pH of 3.3 allowed to reduce the colloidal gallium impurity below 1.5 ± 0.2%.

Table 1

Different synthetic conditions used for [⁶⁸Ga]Ga-OncoFAP-NOTA
	Batch 1	Batch 2	Batch 3
OncoFAP-NOTA (µg)	15	20	25
Reaction time (min)	10	5	5
Reaction temperature (°C)	90	90	90
Reaction pH	4.2	4.2	4.2
Starting activity (MBq)	590	635	675
Final activity (MBq)	460	515	560
RCY (%)	78	81	83
RCY decay corrected (%)	89.6	87	89.2
Final pH	5.0	5.0	5.0
Final volume (mL)	12	12	12
Purity (%)	99.7	99.8	99.8

Table 2

Different synthetic conditions used for [⁶⁸Ga]Ga-OncoFAP-NODAGA
	Batch 1	Batch 2	Batch 3
OncoFAP-NODAGA (µg)	15	20	25
Reaction time (min)	10	5	5
Reaction temperature (°C)	90	90	90
Reaction pH	4.2	4.2	4.2
Starting activity (MBq)	750	875	950
Final activity (MBq)	615	735	808
RCY (%)	82	84	85
RCY decay corrected (%)	94	90	91
Final pH	5.0	5.0	5.0
Final volume (mL)	12	12	12
Purity (%)	99.7	99.7	99.7

Table 3

Different synthetic conditions used for [⁶⁸Ga]Ga-OncoFAP-DOTAGA
	Batch 1	Batch 2	Batch 3
OncoFAP-DOTAGA (µg)	15	20	25
Reaction time (min)	10	5	5
Reaction temperature (°C)	90	90	90
Reaction pH	4.2	4.2	4.2
Starting activity (MBq)	1110	1062	1132
Final activity (MBq)	829	844	884
RCY (%)	74.0	79.5	78.1
RCY decay corrected (%)	83.0	88.9	86.8
Final pH	5.0	5.0	5.0
Final volume (mL)	12	12	12
Purity (%)	99.7	99.7	99.7

Table 4

Acceptance criteria and average result of synthesis for [⁶⁸Ga]Ga-OncoFAP-derivatives.
Parameters	Acceptance criteria	Average [⁶⁸Ga]Ga-OncoFAP-NOTA	Average [⁶⁸Ga]Ga-OncoFAP -NODAGA	Average [⁶⁸Ga]Ga-OncoFAP-DOTAGA
[⁶⁸Ga]-labelled activity (MBq)	200–1300	460–560	750–950	829–884
Volume (mL)	12	12	12	12
Aspect	Clear colourless solution	Clear colourless solution	Clear colourless solution	Clear colourless solution
Half-life (min)	62–74	66.8	66.9	67.1
- ⁶⁸Ga³⁺ - ⁶⁸Ga colloidal - Radiochemical purity	≤ 2% ≤ 3% ≥ 95%	0.2% 0.3% 99.5%	0.1% 0.2% 99.7%	0.1% 0.2% 99.7%
Ethanol concentration (v/v)	< 10%	4.9% ± 0.5%	4.8% ± 0.5%	4.9% ± 0.5%
⁶⁸Ge breakthrough	< 10^− 3%	< 10^− 4%	< 10^− 4%	< 10^− 4%
pH	4.0–8.0	5.0 ± 0.3	5.0 ± 0.3	5.0 ± 0.3
Bacterial endotoxins	< 175 EU/V	< 0.5EU/mL	< 0.5EU/mL	< 0.5EU/mL
Filter integrity (psi)	≥ 50	> 50	> 50	> 50
Sterility	Sterile	Sterile	Sterile	Sterile

Table 5

Different conditions used for the simple preparation of [⁶⁸Ga]Ga-OncoFAP-NODAGA
	Batch 1	Batch 2	Batch 3
OncoFAP-NODAGA (µg)	10	20	30
1 M Sodium Formate (µL)	500	550	550
Reaction time (min)	10	10	10
Reaction temperature (°C)	95	95	95
Reaction pH	3.0	3.2	3.2
Starting activity (MBq)	1036	980	930
Final activity (MBq)	962	908	861
Final pH	3.0	3.5	3.5
Final Volume (mL)	4.5	4.5	4.5
- ⁶⁸Ga³⁺ content - Colloidal ⁶⁸Ga content - Radiochemical purity	6.2% 1.3% 92.5%	3.0% 1.7% 95.3%	1.0% 1.5% 97.5%

Table 6

Different conditions used for the simple preparation of [⁶⁸Ga]Ga-OncoFAP-DOTAGA
	Batch 1	Batch 2	Batch 3
OncoFAP-DOTAGA (µg)	50	70	70
1 M Sodium Formate (µL)	500	550	550
Reaction time (min)	10	10	10
Reaction temperature (°C)	95	95	95
Reaction pH	3.0	3.2	3.2
Starting activity (MBq)	375	399	495
Final activity (MBq)	345	369	457
Final pH	3.0	3.5	3.5
Final Volume (mL)	4.5	4.5	4.5
- ⁶⁸Ga³⁺ content - Colloidal ⁶⁸Ga content - Radiochemical purity	13.3% 1.5% 85.2%	4.2% 1.7% 94.1%	3.3% 1.7% 95.0%

Radiosynthesis for [¹⁸F]AlF-OncoFAP-NOTA/NODAGA

For the radiosynthesis of [¹⁸F]AlF-OncoFAP-NOTA, the [¹⁸F]AlF approach allowed us to obtain 20.9% RCY with a radiochemical purity 92%. On the contrary, the synthesis of [¹⁸F]AlF-OncoFAP-NODAGA resulted in low RCY (~ 2.4%) and radiochemical purity (~ 68.4%) (Tables 7–9). To improve the radiochemical purity, the preparation was further purified by HPLC.

Table 7

Different conditions used for the synthesis of [¹⁸F]AlF-OncoFAP-NOTA
	Batch 1	Batch 2	Batch 3
OncoFAP-NOTA (µg)	200	250	300
Reaction time (min)	25	25	25
Reaction temperature (°C)	95	95	95
Reaction pH	4.5	4.5	4.5
Initial activity (MBq)	3811	9250	16280
Product activity (MBq)	644	1750	2967
RCY (%)	16.9	18.9	18.2
RCY decay corrected (%)	18.7	20.9	20.2
Final pH	5.5	5.5	5.5
Final volume (mL)	24	24	24
Purity (%)	92.0	89.6	90.5

Table 8

Different conditions used for the synthesis of [¹⁸F]AlF-OncoFAP-NODAGA
	Batch 1	Batch 2	Batch 3
OncoFAP-NODAGA (µg)	200	250	300
Reaction time (min)	25	25	25
Reaction temperature (°C)	95	95	95
Reaction pH	4.5	4.5	4.5
Initial activity (MBq)	3219	4255	6660
Product activity (MBq)	78	96	137
RCY (%)	2.4	2.3	2.1
RCY decay corrected (%)	2.6	2.5	2.3
Final pH	5.5	5.5	5.5
Final volume (mL)	24	24	24
Purity (%)	60.3	68.4	67.5

Table 9

Acceptance criteria and average result of synthesis. [¹⁸F]AlF-OncoFAP
Parameters	Acceptance criteria	Average [¹⁸F]AlF-OncoFAP-NOTA	Average [¹⁸F]AlF-OncoFAP-NODAGA
[¹⁸F]-labelled activity	200–4000 MBq	644–2967 MBq	74–130 MBq
Volume (mL)	12	12	12
Aspect	Clear colourless solution	Clear colourless solution	Clear colourless solution
Half-life (min)	105–115	110.2	110.1
- ¹⁸F⁻content - Other - Radiochemical purity	≤ 5% ≤ 10% ≥ 85%	4.3% 6.1% 89.6%	12.4% 27.3% 60.3%
Ethanol concentration(v/v)	< 10%	5.6% ±0.5%	5.4% ±0.5%
pH	4.0–8.0	5.5 ± 0.3	5.5 ± 0.3
Bacterial endotoxins	< 175 EU/V	< 0.5EU/mL	< 0.5EU/mL
Filter integrity (psi)	≥ 50	> 50	> 50
Sterility	Sterile	Sterile	Sterile

Radiosynthesis for [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA

For the radiosynthesis of [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA, high specific activities (2.7 mCi/µg) were obtained with RCY of 92.9% and radiochemical purity > 99.8% (Table 10–11). Notably, the RCY 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 of free 177Lu. No loss of [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA was observed in this process (Figure S10 in the Supplementary material).

Table 10

Different conditions used for the radiosynthesis of [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA
	Batch 1	Batch 2	Batch 3	Batch 4
OncoFAP-DOTAGA (µg)	13.3	5.1	8.2	5.3
Reaction volume (mL)	2.5	2.5	2.5	1.5
Reaction time (min)	30	30	30	30
Reaction temperature (°C)	90	90	90	90
Reaction pH	4.2	4.2	4.2	4.2
Initial activity (MBq)	592	870	1047	573
Product activity (MBq)	577	307	670	533
RCY %	97.5	35.3	54.1	92.9
Final pH	5.0	5.0	5.0	5.0
Final volume (mL)	12	12	12	12
Gentisic acid (mg)	0	20	20	20
Final specific activity (mCi/µg)	1.17	1.61	2.25	2.71
Purity (%)	> 99.8	> 99.8	> 99.8	> 99.8

Table 11

Acceptance criteria and average result of [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA radiosynthesis.
Parameters	Acceptance criteria	Average OncoFAP-DOTAGA
[¹⁷⁷Lu]-labelled activity (MBq)	200–1500	307–670
Volume (mL)	25	25
Aspect	Clear colourless solution	Clear colourless solution
Half-life (days)	6.6–6.8	6.7
- ¹⁷⁷Lu content - Other - Radiochemical purity	≤ 2% ≤ 3% ≥ 95%	0.2% < 0.1% 99.8%
Ethanol concentration (v/v)	< 10%	3.2% ±0.9%
pH	4.0–8.0	5.5 ± 0.3
Bacterial endotoxins	< 175 EU/V	< 0.5EU/mL
Filter integrity (psi)	≥ 50	> 50
Sterility	Sterile	Sterile

Characterization of OncoFAP Binding to hFAP

Co-elution experiments (Fig. 2) showed that all the radiolabelled preparations of OncoFAP derivatives described in this work retained the ability to form stable complexes with recombinant human FAP.

Assessment of in vitro lipophilicity and stability

All the radiolabelled preparations of OncoFAP derivatives exhibited high hydrophilicity. Details are given in Supplementary Material. All [⁶⁸Ga]Ga-OncoFAP and [¹⁸F]AlF-OncoFAP radio-conjugates were highly stable (> 99% of intact compound) in 0.9% saline solution and in human plasma at 37°C for over 2 hours. Contrarily, [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA exhibited partial radiolysis, that was avoided by addition of gentisic acid as a radical’s scavenger (Fig. 3b).

Cell binding

Cell binding studies showed a similar trend of binding on FAP-positive SK-RC-52 cells for all the OncoFAP radio-conjugates. Particularly, the compounds displayed an intense binding at early time point (10 min), followed by a progressive decrease over time. However, a major difference in the absolute binding value was found among [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA (20%), [⁶⁸Ga]Ga-OncoFAP-DOTAGA (2.3%) and [¹⁸F]AlF-OncoFAP-NOTA (0.25%), most probably as a consequence of the amount of cold precursors contained in the different final preparation.

Quantitative Biodistribution of [¹⁸F]AlF-OncoFAP-NOTA in Tumour-Bearing Mice

In vivo biodistribution properties of [¹⁸F]AlF-OncoFAP-NOTA (500 nmol/kg, ~ 4 MBq/Kg) were assessed in athymic Balb/c AnNRj-Foxn1 mice bearing subcutaneous HT-1080.hFAP fibrosarcoma. The compound showed selective accumulation in FAP-positive tumours (6.6% ID/g, 1 hour post intravenous injection), with excellent selectivity against healthy organs. A tumour-to-blood and tumour-to-kidney ratio of 6.5-to-1 and 4.3-to-1, respectively, were observed at the 1-hour time point (Fig. 5).

In the recent years, the development of tumor targeting ligands resulted in the discovery of new generation diagnostic and therapeutic products with high uptake in cancer lesions and low accumulation in healthy organs. Lutathera® and ¹⁷⁷Lu-PSMA-617 provide examples of radioligand therapeutics with proven clinical efficacy and limited systemic toxicity [13, 14].

Radioligand diagnostic and therapeutic products targeting Fibroblast Activation Protein (FAP) represent a promising class of compounds with pan-tumoral applicability and high selectivity for tumor lesions. FAP is an extracellular protease, highly expressed on the cell surface of cancer-associated fibroblasts (CAFs). Being among the major cell populations within the stroma of solid tumours, cancer-associated fibroblasts (CAFs) provide physical support for tumour cells and exert protumorigenic functions, modulating disease progression and resistance to therapies [15]. In this context, targeting stromal structures may represent therapeutic advantages over approaches directed against cellular antigens like PSMA and somatostatin receptors [16].

FAP-targeting strategies have gained growing relevance in nuclear medicine for the development of radiotheranostics. Although anti-FAP antibodies have been known since the 1990s [17–20], the discovery of small organic FAP ligands (the so-called “FAPI” tracers) in the last few years represented a significant improvement in the field. Among FAP small organic ligands, OncoFAP is the one with the highest affinity described so far (Kd = 680 pM) [9]. Various FAP-targeting agents coupled to different radionuclide chelators have been recently developed and characterized for their high affinity and selectivity towards FAP. These conjugates exhibit a rapid accumulation in cancer lesions and low uptake in healthy organs both in preclinical murine models and cancer patients. However, the translation of radiopharmaceutical agents from preclinical development to clinical applications requires the implementation of appropriate non-clinical procedures, including recommendations for standard and automated radiolabelling procedures.

In this work, we have described the development of robust and automated methodologies for the efficient radiolabelling of OncoFAP-derivatives (OncoFAP-DOTAGA, OncoFAP-NODAGA and OncoFAP-NOTA) with ⁶⁸Ga, Al¹⁸F and ¹⁷⁷Lu radioisotopes. Methodologies described here are novel, easy to implement, safe and robust. We based these procedures on the FASTLab2 automated module, a radiolabelling platform already broadly applied to produce other radiopharmaceuticals at the commercial scale [21–23].

With the aim to enable production of [⁶⁸Ga]-OncoFAP-DOTAGA/NOGADA radiopharmaceuticals also in small radiopharmacy sites with limited financial resources, we have created and validated a radiolabelling single-vial cold kit. This approach, already proposed for other products (Illuccix™ and NETSPOT®; SOMAKIT TOC) [12, 24, 25], renders the production of ⁶⁸Ga-based products as easy as the one of ^99mTc-based radiopharmaceuticals. The “OncoFAP-kits”, which already contains all necessary items (buffers and ligands) to be combined with the radioisotopes, allow to efficiently afford [⁶⁸Ga]Ga-OncoFAP-NODAGA and [⁶⁸Ga]Ga-OncoFAP-DOTAGA for PET imaging applications.

OncoFAP-DOTAGA can be labelled both with ⁶⁸Ga and ¹⁷⁷Lu, and thus can be used as a precursor of a “theranostic pair”. This approach has been already successfully implemented for the diagnosis and therapy of Non-Endocrine Tumours (Lutathera®/NETSPOT®) [10] and prostate cancers (⁶⁸Ga-PSMA-11/¹⁷⁷Lu-PSMA-617) [11]. [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA was obtained with specific activities of about 2.5 mCi/µg, which almost doubles the specific activities currently routinely achieved in practice with [¹⁷⁷Lu]Lu-PSMA-617 [26]. Incorporation of high loads of Lutetium-177 represents a critical parameter to obtain a product bearing therapeutic clinical doses of radioactivity (i.e., in the range of 200–400 mCi) in relatively low amounts of precursor, thus avoiding the need of injecting ligand doses that would saturate tumor structures [9]. After radiolabelling, [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA is highly stable and presents high affinity towards isolated hFAP and against FAP-positive cancer cells.

As an alternative to ⁶⁸Ga for diagnostic applications, we also developed two novel OncoFAP-conjugates bearing NODAGA and NOTA radiometal chelators, which are known to form stable complexes with Al¹⁸F [27–31]. We performed the radiochemical synthesis of both [¹⁸F]AlF-OncoFAP-NODAGA and [¹⁸F]AlF-OncoFAP-NOTA. The radiolabelling of OncoFAP-NODAGA with [¹⁸F]AlF resulted in very low radiochemical yields and radiochemical purities, probably due to the possibility of the NODAGA chelator to form a stable neutral complex with the Al³⁺ ion, thus partially preventing the incorporation of the fluoride (¹⁸F⁻) ion. On the contrary, OncoFAP-NOTA, which devoids a carboxylic acid function, was efficiently radiolabelled with [¹⁸F]AlF, with excellent purity and a radiochemical yield of approximately 20%. [¹⁸F]AlF-OncoFAP-NOTA retains affinity towards hFAP (co-elution experiments) and FAP-positive SK-RC-52 cells showing binding proprieties similar to the ones of [⁶⁸Ga]Ga-OncoFAP-DOTAGA. In vivo biodistribution studies in tumor-bearing mice confirmed favourable tumor-targeting performance of [¹⁸F]AlF-OncoFAP-NOTA. The novel radiotracer selectively accumulates in solid lesion, with high tumour-to-background ratio at early time points (i.e., 1 hour after systemic administration), thus supporting further development of the molecule as diagnostic PET radiopharmaceutical in clinical practice. The biodistribution profile of [¹⁸F]AlF-OncoFAP-NOTA in murine model is comparable with the one of the clinically validated [⁶⁸Ga]Ga-OncoFAP-DOTAGA [8]. Application of [¹⁸F]AlF-OncoFAP-NOTA as a PET/CT imaging agent may result in significant logistical and clinical advantages as a consequence of the imaging characteristic of the isotope. Fluorine-18 is characterized by ∼97% β⁺ emission, 635 keV maximum positron energy, short β⁺ trajectory with mean positron range of 0.27 mm in soft tissue. Moreover, it can be produced in large scale in cyclotrons [32, 33] 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 ¹⁸F-based imaging procedures allows an easier patient preparation (no need of fasting, no need of maintaining low glucose level) and offer an optimal imaging time window of approximately 30–180 min after tracer injection. 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 [¹⁸F]FDG, [¹⁸F]FLT, [¹⁸F]F-MCH and [¹⁸F]F-PSMA-1007 [1, 34]. Examples are represented by esophageal [35], liver [36] and pancreatic cancers [4], brain primary tumours and metastases [37] or head and neck cancers [38].

Our results provide the reference for robust and efficient radiolabelling methodologies of OncoFAP-radioligand products that are easy to implement in clinical practice in small, medium and large size hospital radiopharmacies. The different radiosynthetic strategies presented in this article for each radioisotope can be selected on the basis of the specific needs of the various clinical centers. Automated production of [⁶⁸Ga]Ga-OncoFAP-DOTAGA, a clinically validated PET tracer [8], and of [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA will facilitate the implementation of this theranostic pair in clinical practice. Efficient production of [¹⁸F]AlF-OncoFAP-NOTA represents the basis for the implementation of this novel radiotracer for PET imaging applications.

Efficient production of pharmaceutical-grade [⁶⁸Ga]Ga-OncoFAP and [¹⁸F]AlF-OncoFAP can be achieved through automatic radiolabelling procedures described in this article. [⁶⁸Ga]Ga-OncoFAP can be obtained with a simple kit-based approach, thus allowing small radiopharmacies to offer this new PET radiotracer to cancer patients with limited financial burden. Furthermore, we have established methodologies to efficiently produce an OncoFAP-based radiotheranostic pair in which [⁶⁸Ga]Ga-OncoFAP-DOTAGA is used as a companion diagnostic of the targeted therapeutic agent [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA. The procedures described in this article are being implemented in the clinical protocols of theranostic trials with OncoFAP-derivatives to enable imaging and therapy of patients with solid tumours.

Acknowledgments

Heartfelt thanks to Jacqueline Mock and Maria Kominia for their assistance in the project design and management.

Compliance with Ethical Standards:

Funding: No funding was received.

Conflict-of-interest: the authors declare the following competing financial interests related to the content of the present work

PAE, FB have contract research with Philogen

PAE is Editorial Board member of the EJNMI, EJHI, European Radiology Experimental, Clinical and Translational Imaging, QJNM and is Board member of EANM (Education Chair)

P.H. is Editor-in-Chief of EJNMI Radiopharmacy and Chemistry

RS is Associated Editor for EJNMMI and EJHI

DN is a cofounder and shareholder of Philogen (http://www.philogen.com/en/), a Swiss-Italian Biotech company that operates in the field of ligand-based pharmacodelivery.

AZ, AG, JM, FM and SC are employees of Philochem AG, the daughter company of Philogen that owns and has patented OncoFAP and its derivatives.

No other potential conflicts of interest relevant to this article exist.

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under the license number ZH006/2021 granted by the Veterinäramt des Kantons Zürich.

Authors' contributions: PAE, SC and DN conceptualized the study; PAE, FB, PH, and SC designed the study; FB, PH and LRN performed the radiopharmaceuticals synthesis and the quality controls, AZ, AG, JM, FM and SC performed the OncoFAP synthesis and the biodistribution studies; PAE, SC and RS performed data analysis; PAE, FB, PH, SC, DN and RS critically interpreted results; PAE, FB, SC, AZ and JM drafted the paper. PAE and SC coordinated and supervised the project's activities. All the authors critically revised the paper and approved the submitted version of the manuscript.

Availability of data and material: Neither this manuscript nor one with substantially similar content under the same authorship has been published or is being considered for publication elsewhere. Paola Anna Erba had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Raw data are available on specific request to the corresponding author.

AcONa	Sodium acetate
DOTAGA	2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid
E&Z	Eckert & Ziegler
EtOH	Ethanol
FAP	Fibroblast activation protein
GMP	good manufacturing practice
NODAGA	2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid
NOTA	2,2'-(7-carboxy-1,4,7-triazonane-1,4-diyl)diacetic acid
PET	Positron emission tomography
PSMA	Prostate specific membrane antigen

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Automated radiolabelling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Chemical synthesis

Radiochemistry

[⁶⁸Ga]Ga-OncoFAP-derivatives synthesis

[¹⁸F]AlF-OncoFAP

[¹⁷⁷Lu]Lu-OncoFAP

Analysis and quality control

Characterization of OncoFAP Binding to hFAP

Cell binding

Animal studies

Implantation of subcutaneous tumours

Biodistribution studies in tumour bearing mice

Results

Radiosynthesis for [⁶⁸Ga]Ga-OncoFAP

Radiosynthesis for [¹⁸F]AlF-OncoFAP-NOTA/NODAGA

Radiosynthesis for [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA

Characterization of OncoFAP Binding to hFAP

Cell binding

Quantitative Biodistribution of [¹⁸F]AlF-OncoFAP-NOTA in Tumour-Bearing Mice

Discussion

Conclusion

Declarations

Acknowledgments

Abbreviations

References

Supplementary Files

Status:

Version 1

Automated radiolabelling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Chemical synthesis

Radiochemistry

[68Ga]Ga-OncoFAP-derivatives synthesis

[18F]AlF-OncoFAP

[177Lu]Lu-OncoFAP

Analysis and quality control

Characterization of OncoFAP Binding to hFAP

Cell binding

Animal studies

Implantation of subcutaneous tumours

Biodistribution studies in tumour bearing mice

Results

Radiosynthesis for [68Ga]Ga-OncoFAP

Radiosynthesis for [18F]AlF-OncoFAP-NOTA/NODAGA

Radiosynthesis for [177Lu]Lu-OncoFAP-DOTAGA

Characterization of OncoFAP Binding to hFAP

Cell binding

Quantitative Biodistribution of [18F]AlF-OncoFAP-NOTA in Tumour-Bearing Mice

Discussion

Conclusion

Declarations

Acknowledgments

Abbreviations

References

Supplementary Files

Status:

Version 1

[⁶⁸Ga]Ga-OncoFAP-derivatives synthesis

[¹⁸F]AlF-OncoFAP

[¹⁷⁷Lu]Lu-OncoFAP

Radiosynthesis for [⁶⁸Ga]Ga-OncoFAP

Radiosynthesis for [¹⁸F]AlF-OncoFAP-NOTA/NODAGA

Radiosynthesis for [¹⁷⁷Lu]Lu-OncoFAP-DOTAGA

Quantitative Biodistribution of [¹⁸F]AlF-OncoFAP-NOTA in Tumour-Bearing Mice