Preclinical evaluation of [13xLa]La-FAP-2286 as a novel theranostic agent for tumors expressing fibroblast activation protein

A 30 MeV cyclotron (Cyclone-30, IBA, Belgium) was used to prepare ^132/135La. FAP-2286 and Diglycolamide (DGA) resin were purchased from MedChemExpress (USA) and Eichrom (USA) Companies, respectively. Barium carbonate (99.999%) and other chemicals were supplied from Sigma-Aldrich Chemical Co. (UK). The investigation of radionuclide purity and measurement of radioactivity was performed using a Silena high-purity germanium (HPGe) detector and ISOMED 1010 dose calibrator (Dresden, Germany). ICP-AES, optima 7300 DV (USA) was used to measure ions. Polymer-backed silica gel (Schleicher & Schuell, Germany) and Whatman No. 1 papers (Whatman, UK) were used to assess the radiochemical purity (RCP) while the papers read by a bioscan AR-2000 radio TLC scanner instrument (Europe Ltd Co., France). Analytical high-performance liquid chromatography (HPLC) was carried out by a JASCO 880-PU intelligent pump, equipped with a multiwavelength UV detector and a flowthrough Raytest-Gabi γ-detector (Ohio, USA), using a ZORBAX C-18 column (250 × 4.6 mm). The human serum and mice were provided by the Royan Institute (Iran). The animal experiments were carried out according to the National Institutes of Health (NIH) animal use and care guidelines. This study was approved by the NSTRI Ethical Committee (Approval No.: RA-1-MP-0212-17). All methods were reported under ARRIVE guidelines²⁰.

Production of 13xLa

^13xLa radionuclides were produced by proton irradiation of natural barium target through ^natBa(p, n)^13xLa nuclear reaction. To make the target, barium carbonate powder was placed in an aluminum tablet with a diameter of 11 mm and a thickness of 0.8 mm, and it was compressed under a pressure of 10 ton/cm², and finally was covered by a high-purity aluminum sheet, and was irradiated for 150 µAh.

After cooling, ^13xLa was separated from the ^natBa target using DGA resin, following a procedure previously published⁸, with slight modification. Briefly, the irradiated target was dissolved in 3 M nitric acid, and the solution was passed through solid-phase extraction cartridges containing 500 mg of resin preconditioned by 3 M nitric acid (10 mL). The cartridge was again washed with 3 M nitric acid (30 mL) to remove the remaining barium and other metal impurities. Finally, using 1 M hydrochloric acid, ^13xLa in chloride form was removed from the cartridge. The activity was measured by an HPGe detector.

Quality control of 13xLa

The radionuclide purity of ^13xLa was assessed by an HPGe detector. Chemical purity was studied to check the presence of metal impurities in the radioactive solution, which can cause problems in the labeling processes, using the Inductively Coupled Plasma Mass Spectrometry (ICP-MS) method. The RCP of the product was evaluated using the RTLC method, with a mobile phase consisting of a methanol: saline (5:1) mixture and 20 mM citric acid, while the Whatman No. 2 paper was used as the stationary phase.

Preparation of [13xLa]La-FAPI-2286

To optimize the labeling reaction, several parameters were varied, including pH (2–6), peptide concentration (5–20 nmol), reaction time (15–60 min), and temperature (65–95 °C), while keeping the lanthanum activity constant. Briefly, the specified activity of [^13xLa]LaCl₃ and various amounts of the peptide were spilled into a borosilicate reaction vial. Sodium acetate buffer was used to adjust the reaction pH. The reaction was carried out at different temperature, while the RCP was monitored at various intervals.

Quality control of the radiolabeled compound

The RCP of the final radiolabeled compound was evaluated using RTLC and HPLC methods. Whatman No.2 paper and methanol: saline (5:1) mixture were used as the appropriate stationary and mobile phases, respectively. HPLC was performed using the two different solvent systems (A = Ultrapure water + 1% TFA, B = Acetonitrile). A flow rate of 1.5 mL/min was considered to wash the column for 30 min according to the following gradient: 100% A: 0% B for 0–10 min, 50% A: 50% B for 10–20 min, 0% A: 100% B for 20–30 min.

Stability tests

The stability of ^132/135La-FAPI-2286 was studied in human serum (37 °C) and PBS buffer (4 °C). The complex was stored in PBS buffer for 24 h, and the RCP of the complex was assessed at different time points using the RTLC method. For the stability assessment in human blood serum, 37 MBq of the compound was added to 500 µL of human serum and the mixture incubated at 37 °C for 24 h, after which the RCP was analyzed using the RTLC method.

Distribution coefficient (log D)

The distribution coefficient (log D) of [^13xLa]La-FAPI-2286 was studied according to other literature by the shake-flask method²¹. Briefly, 1-octanol (500 µL) and PBS (500 µL) at pH 7.4 was added to a pre-lubricated Eppendorf tube. The radiolabeled compound (10 pmol) was added to the tube, and the mixture was shaken and centrifuged at 3000 g, for 30 and 10 min, respectively. 100-µL samples were taken from each phase and measured using a well counter. The distribution coefficient was provided as the average log ratio value of the radioactivity in the organic and PBS fractions.

Cellular studies

For binding affinity study, approximately one million HEK FAP+, and MCF7 and CHO FAP- cells were seeded into the wells of a 6-well plate along containing a complete culture medium and incubated for 24 h. Then, the culture medium was removed, the cells were washed, and incubated with fresh culture medium for 1 h at 37 °C. The plates were then placed on ice for 30 min, and different concentrations of [^13xLa]La-FAPI-2286 (1-100 nM) were added. The experiment was repeated by adding unlabeled peptide (1 µM) to the half of wells to assess non-specific binding. After incubation, the cells were washed with ice-cold PBS, harvested, and the bound radioactivity was measured using a gamma counter. Finally, half-maximal inhibitory concentration (IC50) was calculated by IDBS ActivityBase software. For internalization studies, the radiolabeled compound was incubated with cells for 30, 60, 120, 240, and 360 min at 37 ˚C. Surface-bound radiolabeled peptide was removed by incubation with 1 M glycine buffer for 5 min, and the radioactivity was measured using a gamma counter.

Biodistribution studies in normal and tumor-bearing mice

[^13xLa]LaCl₃ and [^13xLa]La-FAPI-2286 (200 µL; 7.4 MBq) were intravenously (i.v.) injected into both normal and tumor-bearing mice through their tail vein. 8–10-week-old NOD/SCID tumor-bearing mice with an average weight of 21.4 ± 1.7 gr were used for biodistribution studies. Tumor induction was achieved by injecting approximately 1 million HEK FAP + cells subcutaneously into the mice’s flanks. The mice were sacrificed using CO₂ gas, and the biodistribution in different tissues was determined at the specified time intervals up to 48 h post-injection. Blood samples were collected, and tissues such as kidneys, lungs, liver, brain, heart, intestines, stomach, skin, muscles and bones were weighed, washed, and their activity was measured by an HPGe detector. The percentage of injected activity per tissue mass (ID/g%) was calculated according to Eq. (1) for different body organs.

Where the total activity is the time-corrected injected activity.

Imaging studies

To study the biodistribution of the labeled peptide via imaging, [^13xLa]LaCl3 and [^13xLa]La-FAPI-2286 (3.7 MBq) were injected into tumor-bearing mice. The animals were anesthetized by combining ketamine–xylazine–PBS (2:1:1), and images were captured using a dual-head SPECT system (MSV, France).

Statistical analysis

For statistical analysis, three mice were considered for each interval (n = 3). All values were presented as mean ± standard deviation. The Student’s T-test was used to compare the data.